Microbial biocontrol of Sclerotinia stem rot of canola

Microbial biocontrol of Sclerotinia stem rot

of canola

Mohd Mostofa Kamal

MSc (Plant Sciences)

The University of Manchester United Kingdom

MS (Plant Pathology)

Bangabandhu Sheikh Mujibur Rahman Agricultural University Bangladesh

BScAg (Honours)

Sher-E-Bangla Agricultural University Bangladesh

This thesis prepared for the degree of

Doctor of Philosophy

Charles Sturt University Australia

School of Agricultural and Wine Sciences

Faculty of Science

November 2015

i

Table of Contents

Table of contentsi

Statement of authenticityiii

Acknowledgementsiv

Abstractv

Publications arising from this researchviii

Chapter 11

General Introduction

Chapter 26

Literature Review

Biology and biocontrol of Sclerotinia sclerotiorum (Lib) de Bary in oilseed Brassicas

Chapter 343

Prevalence of sclerotinia stem rot of mustard in northern Bangladesh

Chapter 4 51

Biological control of sclerotinia stem rot of canola using antagonistic bacteria

Chapter 5 62

Bacterial biocontrol of sclerotinia diseases in Australia

Chapter 671

Elucidating the mechanism of biocontrol of Bacillus cereus SC-1 against Sclerotinia

sclerotiorum

Chapter 7101

Rapid marker-assisted selection of antifungal Bacillus species from the canola

rhizosphere

ii

Chapter 8130

General Discussion

References139

Appendices143

Certificate of Authorship

I hereby declare that this submission is my own work and that to the best of my knowledge and

belief it contains no material previously published or written by another person nor material that to

a substantial extent has been accepted for the award of any other degree or diploma at Charles Sturt

University or any other educational institution except where due acknowledgement is made in the

thesis Any contributions made to the research by colleagues with whom I have worked at Charles

Sturt University or elsewhere during my candidature is fully acknowledged

I agree that the thesis be accessible for the purpose of study and research in accordance with the

normal conditions established by the Executive Director Division of Library Services or nominee for

the care loan and reproduction of theses

Name Mohd Mostafa Kamal

Date 20 November 2015

iv

Acknowledgements

I would like to express the deepest appreciation to almighty Allah Subhanahu Wa Taala (The

God) whose continuous blessings empowered me to complete this dissertation I am cordially

thankful to my supervisory team comprised of Prof Gavin James Ash Dr Sandra Savocchia

and Dr Kurt D Lindbeck whose moral encouragement scholastic guidance and endless

support from the initial to the final stage of research has provided me valuable insights to the

technical aspects needed to work in the field of biocontrol of plant pathogens Their strongly

held convictions are brought together in such an effort that resulted in a number of pioneered

publications from this research I wish to express my regards to Dr Ben Stodart who

supported me in any respect during the completion of this project My sincere thanks go to

Charles Sturt University Australia for providing me a Faculty of Science (compact)

scholarship and United States Department of Agriculture for The Norman E Borlaug

International Agricultural Science and Technology Fellowship to continue research at the

Ohio State University USA under the guidance of Professor Brian McSpadden Gardener I

am thankful to my work place Bangladesh Agricultural Research Institute for granting the

three and half years of deputation leave to complete my PhD study without any reservations I

would also like to thank my colleagues especially Md Shamsul Haque Alo Amir Sohail

Hasan Md Zubaer Md Asaduzzaman Kylie Crampton and Nirodha Weeraratne whose

cordial cooperation and moral support inspired me to go ahead My sincere thanks go to

Kamala Anggamuthu Karryn Hann and Trent Smith for their skilful administrative

assistance Above all I am dedicating this work to my beloved dad whom I lost in 2011

during the penultimate stage of my Mastersrsquo dissertation at the University of Manchester

Mohd Mostofa Kamal

v

Abstract

Stem rot caused by Sclerotinia sclerotiorum (Lib) de Bary is a major fungal disease of

canola and other oilseed rape worldwide including Australia Prevalence of the disease was

investigated in eight major northern mustard growing districts of Bangladesh where

sclerotinia stem rot is present in all districts occurring as petal and stem infections The

existence of Sclerotinia minor Jagger was also observed but only from symptomatic petals

The management of stem rot relies primarily on strategic application of synthetic fungicides

throughout the world An alternative biocontrol approach was attempted for management of

the disease with 514 naturally occurring bacterial isolates screened for antagonism to S

sclerotiorum Three bacterial isolates demonstrated marked antifungal activity and were

identified as Bacillus cereus (SC-1 and P-1) and Bacillus subtilis (W-67) using 16S rRNA

sequencing Dual culture assessment of antagonism of these isolates toward S sclerotiorum

resulted in significant inhibition of mycelial growth and restriction of sclerotial germination

due to the production of both non-volatile and volatile metabolites The viability of sclerotia

was also significantly reduced in a glasshouse pot trial with soil collected from the field

Spray treatments of bacterial strains reduced disease incidence and yielded higher control

efficacy both on inoculated cotyledons and stems under controlled environment conditions

Three field trials with application of SC-1 and W-67 at 10 flowering stage of canola

showed high control efficacy of SC-1 against S sclerotiorum in all trials when sprayed twice

at 7-day intervals The application of SC-1 twice and a single application of the

recommended fungicide Prosaro 420SC showed greatest control of the disease When taking

into consideration both cost and environmental concern surrounding the application of

synthetic fungicides B cereus SC-1 may have potential as a biological control agent of

sclerotinia stem rot of canola in Australia

vi

As a biocontrol agent against sclerotinia stem rot disease of canola both in vitro and in vivo

B cereus SC-1 was further evaluated against lettuce drop caused by S sclerotiorum Soil

drenching with B cereus SC-1 applied at 108 CFU mL

-1 in a glasshouse trial completely

restricted infection by the pathogen and no disease was observed Sclerotial colonisation was

tested and results showed that 6-8 log CFU per sclerotia of B cereus resulted in significant

reduction of sclerotial viability compared to the control Volatile organic compounds

produced by the bacteria increased root length shoot length and seedling fresh weight of the

lettuce seedlings compared with the control B cereus SC-1 was able to enhance lettuce

growth resulting in increased root length shoot length head weight and biomass weight

compared with untreated control plants The bacteria were able to survive in the rhizosphere

of lettuce plants for up to 30 days These results indicate that B cereus SC-1 could also be

used as an effective biological control agent of lettuce drop

The biocontrol mechanism of B cereus strain SC-1 was observed to be through antibiosis

The culture filtrate of B cereus strain SC-1 significantly reduced mycelial growth

completely inhibited sclerotial germination and degraded the melanin of sclerotia indicating

the presence of antifungal metabolites in the filtrates Application of culture filtrate to canola

cotyledons resulted in significant reduction in lesion development Scanning electron

microscopy of the zone of interaction of B cereus SC-1 and S sclerotiorum demonstrated

vacuolated hyphae with loss of cytoplasm and that the sclerotial rind layer was completely

ruptured with heavy bacterial colonisation Transmission electron microscopy revealed the

cell content of fungal mycelium and the organelles in sclerotial cells to be completely

disintegrated suggesting the direct antifungal action of B cereus SC-1 metabolites The

presence of bacillomycin D iturin A surfactin fengycin chitinase and β-13-glucanase

vii

associated genes in the genome of B cereus SC-1 revealed that it can produce both

lipopeptide antibiotics and hydrolytic enzymes essential for biocontrol

The dual culture screening for bacterial antagonists is a time consuming procedure therefore a

rapid marker-assisted approach was adopted for screening of Bacillus spp from the canola

rhizosphere Bacterial strains were isolated and screened using surfactin iturin A and

bacillomycin D peptide synthetase biosynthetic gene specific primers through multiplex

polymerase chain reaction Oligonucleotide primer based screening from the 96 combined

Bacillus isolates showed the presence of all the genes in the genome of one isolate The

marker selected Bacillus strain was identified by 16S rRNA gene sequencing as Bacillus

cereus (designated strain CS-42) This strain was effective in significantly inhibiting the

growth of S sclerotiorum both in vitro and in planta Scanning electron microscopy of the

dual culture interaction region revealed that mycelial growth was exhausted in the vicinity of

bacterial metabolites and that the melanised outmost rind layer of sclerotia was completely

degraded Transmission electron microscopy of ultrathin sections of hyphae and sclerotia

challenged with SC-1 resulted in partially vacuolated hyphae and the complete degradation of

sclerotial cell organelles Genetic marker assisted selection may provide opportunities for

rapid and efficient selection of pathogen-suppressing Bacillus and other bacterial species for

the development of microbial biopesticides

viii

Publications arising from this research

Journal article

1 MM Kamal S Savocchia KD Lindbeck and GJ Ash (2015) Biology and

biocontrol of Sclerotinia sclerotiorum (Lib) de Bary in oilseed Brassicas

Australasian Plant Pathology Accepted DOI 101007s13313-015-0391-2

2 MM Kamal MK Alam S Savocchia KD Lindbeck and GJ Ash (2015)

Prevalence of sclerotinia stem rot of mustard in northern Bangladesh International

Journal of BioResearch 19(2) 13-19

3 MM Kamal KD Lindbeck S Savocchia and GJ Ash (2015) Biological control

of sclerotinia stem rot of canola using antagonistic bacteria Plant Pathology 64

1375-1384 DOI 101111ppa12369

4 MM Kamal KD Lindbeck S Savocchia and GJ Ash (2015) Bacterial

biocontrol of sclerotinia diseases in Australia Acta Horticulturae (In press)

5 MM Kamal S Savocchia KD Lindbeck and GJ Ash (2015) Elucidating the

mechanisms of biocontrol of Bacillus cereus SC-1 against Sclerotinia sclerotiorum

BioControl (Formatted)

6 MM Kamal S Savocchia KD Lindbeck and GJ Ash (2015) Rapid marker-

assisted selection of antifungal Bacillus species from the canola rhizosphere FEMS

Microbiology Ecology (Formatted)

ix

Conference abstracts

1 MM Kamal K Lindbeck S Savocchia GJ Ash and BBM Gardener 2014

Marker assisted selection of bacterial biocontrol agents for the control of sclerotinia

diseases In lsquoProceedings of 8th Australasian Soil borne Disease Symposium (ASDS

2014)rsquo 10-13 November 2014 Hobart Tasmania Pp 36

2 MM Kamal K Lindbeck S Savocchia and GJ Ash 2013 Cotyledon inoculation

A rapid technique for the evaluation of bacterial antagonists against Sclerotinia

sclerotiorum in canola under control conditions In lsquoProceedings of 19th

Australasian

Plant Pathology Congress (AUPP 2013)rsquo 25-28 November 2013 Auckland New

Zealand Pp138

3 MM Kamal GJ Ash K Lindbeck and S Savocchia 2013 Microbial biocontrol of

Sclerotinia stem rot of canola In lsquoActa Phytopalogica Sinicarsquo 43(suppl) P03017

Proceedings of the International Congress of Plant Pathology (ICPP 2013) 25-30

August 2013 Beijing China Pp 39

Newsletter articles

1 Mohd Mostofa Kamal 2013 Combating stem rot disease in canola through bacterial

beating Innovator Graham Centre for Agricultural Innovation summer edition

Pp14-15

2 Mohd Mostofa Kamal 2013 Biosecurity food security and plant pathology in a

globalised economy Innovator Graham Centre for Agricultural Innovation spring

edition Pp11-12

x

3 Mohd Mostofa Kamal 2014 Marker assisted selection of biocontrol bacteria

learning from Ohio State University Innovator Graham Centre for Agricultural

Innovation winter edition Pp10

4 Mohd Mostofa Kamal 2015 Genome based detection of antifungal bacteria A

break through towards sustainable soil borne disease management Innovator

Graham Centre for Agricultural Innovation summer edition Pp11-12

5 Mohd Mostofa Kamal 2015 Progress towards biological control of sclerotinia

diseases in Charles Sturt University Innovator Graham Centre for Agricultural

Innovation winter edition Pp11-12

1

Chapter 1 General Introduction

Canola is an oilseed crop derived from rapeseed (Brassica napus Brassica rapa (syn

Brassica campestris L)) and was developed in the 1970s through traditional plant breeding

The word ldquoCanolardquo originates from a combination of two words ldquoCanadianrdquo and ldquoOilrdquo

which contain low erucic acid and glucosinolate (Colton amp Potter 1999) Canola seeds and

meal contain more than 40 oil and 36-44 protein respectively (Kimber amp McGregor

1995) These characteristics also make it an important source of animal feeds Canola oil

bears lower saturated fatty acids compared to butter lard or margarine It is free from

cholesterol and is an important source of vitamin E and phytosterols (Paas amp Pierce 2002)

Rapeseed suffers from a number of diseases worldwide of which stem rot also referred to as

white mould has negatively impacted on crop yield over past decades (Sharma et al 2015)

The disease stem rot is caused by Sclerotinia sclerotiorum (Lib) de Bary a necrotropic

fungal plant pathogen with a reported host range of over 500 species from 75 families

(Boland amp Hall 1994 Saharan amp Mehta 2008) The major hosts include canola chickpea

lettuce mustard pea lentil flax sunflower potato sweet clover dry bean buckwheat and

faba bean (Bailey 1996) The yield quality and grade of these crops can decline gradually

and cost millions of dollars annually due to Sclerotinia infection (Purdy 1979)

In Australia production of canola has notably increased over the last decade (Anonymous

2014) Concurrent with the rapid increase in canola production has been an increase in

disease pressure In Australia the annual losses from sclerotinia stem rot in canola are

estimated to be AU$ 399 million and the cost of fungicide to control stem rot estimated to be

approximately AU$ 35 per hectare (Murray amp Brennan 2012) Yield loss as high as 24

2

(Hind-Lanoiselet et al 2003) and of 039-154 tha (Kirkegaard et al 2006) have been

reported in southern New South Wales Australia In 2013-14 higher inoculum pressure was

observed in high-rainfall zones of NSW (Kurt Lindbeck personal communication) and stem

rot outbreak in canola was reported to occur across the wheat belt region of Western

Australia (Khangura et al 2014) The disease is also prevalent in north-eastern and western

districts of Victoria and has become an emerging problem in the south-eastern regions of

South Australia In addition to stem rot of canola the pathogen also causes lettuce drop in all

states of Australia and reduces yield significantly (20-70) (Villalta amp Pung 2010)

Sclerotinia stem rot is the second most damaging disease of oilseed rape worldwide after

blackleg (Saharan and Mehta 2008) Rapeseed mustard is the most important source of edible

oil in Bangladesh occupying about 059 million hectare of area with annual production of

053 million tonnes (DAE 2014) In Bangladesh S sclerotiorum has been reported on

mustard (Hossain et al 2008) chilli aubergine cabbage (Dey et al 2008) and jackfruit

(Rahman et al 2015) Standard literature regarding the prevalence of the pathogen and

incidence of sclerotinia stem rot in a region known for intensive mustard production is

limited in Bangladesh It is extremely important to know the current status of infection in

order to minimise the potential for an outbreak of stem rot disease A rigorous investigation

to determine the magnitude of petal infestation and incidence of sclerotinia stem rot is crucial

to design appropriate control measures

There are a number of approaches which are used to control S sclerotiorum but sustainable

management of the pathogen remains a challenging task worldwide Conventional disease

management approaches are not completely effective in controlling sclerotinia stem rot

Registered chemical fungicides are undoubtedly effective in reducing the incidence of disease

3

however their efficacy in the long term is not sustainable (Fernando et al 2004) Breeding

for disease resistance is difficult because the trait is governed by multiple genes (Barbetti et

al 2014) Interest in the biocontrol of Sclerotinia diseases has increased over the last few

decades as chemical pesticides have failed to deliver adequate control of the pathogen and

there is increased concern regarding their impact on the environment (Saharan amp Mehta

2008 Agrios 2005) The reduction in the density of primary and secondary inoculum

through killing of sclerotia or restricting germination infection in the rhizosphere and

phyllosphere as well as a reduction of virulence are key strategies for potential biocontrol of

Sclerotinia diseases (Saharan amp Mehta 2008)

A wide range of micro-organisms isolated from the rhizosphere phyllosphere sclerotia and

other habitats have been screened as potential biocontrol agents against S sclerotiorum

(Saharan amp Mehta 2008) The inhibition or suppression of growth and multiplication of

harmful microorganisms by antagonistic bacteria or fungi through production of antibiotics

toxic metabolites and hydrolytic enzymes is well established (Skidmore 1976 Singh amp

Faull 1988 Solanki et al 2012) Similarly a number of bacterial strains can produce

antifungal organic volatile compounds which inhibit mycelial growth as well as restrict

ascospore and sclerotial germination (Fernando et al 2005) Currently there are no native

bacterial biological control agents commercially available for the control of sclerotinia

diseases in Australia Development of a suitable bacterial biocontrol agent with multiple

modes of action either alone or as a component of integrated disease management would pave

the way for sustainable management of sclerotinia stem rot disease of canola and other

agricultural andor horticultural cropping systems

4

The selection and evaluation of bacteria with biocontrol potential from plants and soil is

accomplished using dual culture assessment However detection of antagonistic bacteria

from bulk bacterial samples using direct dual culture techniques is time consuming and

resource intense (de Souza amp Raaijmakers 2003) A number of polymerase chain reaction

(PCR) based approaches have been adapted for screening of potential biocontrol organisms

(Raaijmakers et al 1997 Park et al 2013 Giacomodonato et al 2001 Gardener et al

2001) in an effort to increase the efficacy of selection However complex methodology and

lack of specificity are major drawbacks of these approaches (Park et al 2013) A more

efficient and affordable molecular based approach is crucial for rapid detection of bacterial

antagonists that can inhibit fungal growth and protect plants from pathogen invasion

The specific objectives of this research were mentioned below

1 Survey sclerotinia stem rot in intensive rapeseed mustard growing areas of

Bangladesh

2 Identify bacterial isolates associated with canola and with multiple modes of action

against sclerotinia stem rot of canola in in vitro glasshouse and field conditions

3 Evaluate the efficacy of bacterial isolates against sclerotinia lettuce drop in vitro and

in the glasshouse

4 Determine the mechanisms of biocontrol by the most promising bacterial strain and its

effect on the morphology and cell organelles of S sclerotiorum

5 Develop a genomic approach through marker-assisted selection to screen canola

associated bacterial biocontrol agents for the potential management of S

sclerotiorum

5

The thesis is divided into eight chapters and appendices The first chapter is a general

introduction providing background information about S sclerotiorum management strategies

for sclerotinia stem rot and the aims considered in this study Chapters two to seven have

been presented as published papers or submitted manuscripts The second chapter is a review

of the literature relevant to pathogen biology importance and management of S

sclerotiorum The third chapter presents survey results of S sclerotiorum associated with

stem rot disease of rapeseed mustard in Bangladesh with emphasis on prevalence and

incidence of the pathogen in mustard dominated regions The fourth chapter focuses on a

comprehensive investigation of the development of promising bacterial biocontrol agents for

sustainable management of sclerotinia stem rot disease of canola The fifth chapter describes

the performance of promising bacterial antagonists from the previous study to control

sclerotinia lettuce drop Chapter six describes the biocontrol mechanism of the best bacterial

strain that successfully inhibit S sclerotiorum Chapter seven presents a novel PCR-based

approach for the rapid detection of antagonistic biocontrol bacterial strains to manage S

sclerotiorum Chapter eight discusses the relevant information gained from this research a

summary and an outline for further research The appendices comprise published conference

abstracts and newsletter articles Apart from the published and submitted manuscript all

references are listed at the end of the thesis by following the lsquoPlant Pathologyrsquo journalrsquos

bibliographic style

6

Chapter 2 Literature Review

MM Kamal S Savocchia KD Lindbeck and GJ Ash (2015) Biology and biocontrol of

Sclerotinia sclerotiorum (Lib) de Bary in oilseed Brassicas Australasian Plant Pathology

(Accepted DOI 101007s13313-015-0391-2)

Chapter 2 contains a literature review of the biology of the pathogen S sclerotiorum as well

as information on stem rot disease its economic importance infection process pathogenicity

factors symptoms disease cycle epidemiology and deployment of promising microbial

biocontrol strategies for sustainable management of S sclerotiorum The main objective was

to explore major findings of pathogen biology and disease management strategies with

special emphasis on biological control and identifying future research to be addressed This

chapter is presented as an accepted manuscript in the Australasian Plant Pathology journal

7

Biology and biocontrol of Sclerotinia sclerotiorum (Lib) de Bary in oilseed Brassicas 1

Mohd Mostofa Kamala Sandra Savocchia

a Kurt D Lindbeck

b and Gavin J Ash

a2

Email mkamalcsueduau3

aGraham Centre for Agricultural Innovation (an alliance between Charles Sturt University 4

and NSW DPI) School of Agricultural and Wine Sciences Charles Sturt University 5

Boorooma Street Locked Bag 588 Wagga Wagga NSW 2678 Australia 6

bNew South Wales Department of Primary Industries Wagga Wagga Agricultural Institute 7

Pine Gully Road Wagga Wagga NSW 2650 8

9

Abstract 10

Sclerotinia sclerotiorum (Lib) de Bary is a necrotrophic plant pathogen infecting over 500 11

host species including oilseed Brassicas The fungus forms sclerotia which are the asexual 12

resting structures that can survive in the soil for several years and infect host plants by 13

producing ascospores or mycelium Therefore disease management is difficult due to the 14

long term survivability of sclerotia Biological control with antagonistic fungi including 15

Coniothyrium minitans and Trichoderma spp has been reported however efficacy of these 16

mycoparasites is not consistent in the field In contrast a number of bacterial species such as 17

Pseudomonas and Bacillus display potential antagonism against S sclerotiorum More 18

recently the sclerotia-inhabiting strain Bacillus cereus SC-1 demonstrated potential in 19

reducing stem rot disease incidence of canola both in controlled and natural field conditions 20

via antibiosis Therefore biocontrol agents based on bacteria could pave the way for 21

sustainable management of S sclerotiorum in oilseed cropping systems 22

23

Key words Biology Biocontrol Sclerotinia Oilseed Brassica 24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

Introduction

Sclerotinia sclerotiorum (Lib) de Bary is a ubiquitous and necrotrophic fungal plant

pathogen with a reported host range of over 500 plant species from 75 families (Saharan and

Mehta 2008) The fungus damages plant tissue causing cell death and appears as a soft rot or

white mould on the crop (Purdy 1979) S sclerotiorum was initially detected from infected

sunflower in 1861as reported by Gulya et al (1997) The oilseed producing crops belonging

to the genus Brassica (canola rapeseed-mustard) are major hosts of S sclerotiorum (Bailey

1996 Sharma et al 2015a) The yield and quality of these crops are reduced by the disease

which can cost millions of dollars annually (Purdy 1979 Saharan and Mehta 2008) The first

report of stem rot disease in rapeseed-mustard was published by Shaw and Ajrekar (1915)

Canola production has been threatened by this yield-limiting and difficult to control disease

in Australia (Barbetti et al 2014) Sclerotinia stem rot (SSR) of canola has been reported to

cause significant yield losses around the world including Australia (Hind-Lanoiselet 2004)

Several attempts have been made to manage sclerotinia diseases through agronomic practices

such as the use of organic soil amendments (Asirifi et al 1994) soil sterilization (Lynch and

Ebben 1986) zero tillage and crop rotation (Adams and Ayers 1979 Duncan 2003 Morrall

and Dueck 1982 Gulya et al 1997 Yexin et al 2011) tillage and irrigation (Bell et al

1998) The broad host range of the pathogen has restricted the efficacy of cultural disease

management practices to minimize sclerotinia infection (Saharan and Mehta 2008)

Efforts have also been made to search for resistant genotypes to SSR however no completely

resistant commercial crop cultivars have yet been developed (Hayes et al 2010 Alvarez et al

2012) Breeding for SSR resistance is difficult because the trait is governed by multiple genes

(Fuller et al 1984) In addition the occurrence of virulent pathotypes has hindered the

progress towards the development of complete host resistance (Barbetti et al 2014) In

Australia a limited number of fungicides have been registered however the mis-match of

8

51

9

appropriate time between ascospore liberation and fungicide application often led to failure of 52

disease management (Lindbeck et al 2014) However biological control agents have been 53

reported as an alternative means in controlling the infection of the white mould pathogen 54

(Yang et al 2009 Fernando et al 2007 Wu et al 2014 Kamal et al 2015b) These naturally 55

occurring organisms can significantly reduce disease incidence by inhibiting ascospore and 56

sclerotial germination (Fernando et al 2007) In this review we discuss the economic 57

importance infection process pathogenicity factors symptoms disease cycle epidemiology 58

and deployment of promising microbial biocontrol strategies for sustainable management of 59

S sclerotiorum60

Economic importance 61

SSR has threatened the global oilseed Brassica production by causing substantial yield losses 62

(Sharma et al 2015a) The yield losses depend on disease incidence and infection at a 63

particular plant growth stage (Saharan and Mehta 2008) Infection at pre-flowering can result 64

in up to 100 per cent yield loss in infected plants (Shukla 2005) Plants usually produce little 65

or no seed if infection occurs at the early flowering stage whereas infection at a later growth 66

stage may cause little yield reduction Premature shattering of siliquae development of small 67

sunken and chaffy seeds in rapeseed are the yield limiting factors of Ssclerotiorum infection 68

(Sharma et al 2015a Morrall and Dueck 1983) Historically the estimated yield losses were 69

reported as 28 per cent in Alberta and 111- 149 per cent in Saskatchewan Canada (Morrall 70

et al 1976) 5-13 per cent in North Dakota and 112-132 per cent in Minnesota USA 71

(Lamey et al 1998) up to 50 per cent in Germany (Pope et al 1989) 03 to 347 per cent in 72

Golestan Iran (Aghajani et al 2008) 60 percent in Rajasthan India (Ghasolia et al 2004) 73

10-80 per cent in China (Gao et al 2013) and 80 percent in Nepal (Chaudhury 1993) North74

Dakota and Minnesota faced an income loss of $245 million in canola during 2000 (Lamey 75

et al 2001) In Australia the annual losses are estimated to be AU$ 399 million and the cost 76

10

of fungicide to control SSR was reported to be approximately $35 per hectare (Murray amp 77

Brennan 2012) Yield loss was reported as high as 24 (Hind-Lanoiselet et al 2003) and 78

039-154 tha (Kirkegaard et al 2006) in southern New South Wales (NSW) Australia In 79

2013-14 a higher inoculum pressure was observed in high-rainfall zones of NSW and SSR 80

outbreak in canola was reported to occur across the wheat belt region of Western Australia 81

(Khangura et al 2014) 82

83

Plant infection 84

The asexual resting propagules of S sclerotiorum commonly known as sclerotia are capable 85

of remaining viable in the soil for five years (Bourdocirct et al 2001) Sclerotia can germinate 86

either myceliogenically or carpogenically with favourable environmental conditions 87

Saturated soil and a temperature range of 10 to 20degC can trigger the development of 88

apothecia (Abawi et al 1975a) S sclerotiorum is homothallic and produces ascospores after 89

self-fertilisation without forming any asexual spores (Bourdocirct et al 2001) Apothecia are the 90

sexual fruiting bodies which form through sclerotial germination during favourable 91

environmental conditions and liberate ascospores that can disseminate over several 92

kilometres through air currents (Clarkson et al 2004) The air-borne ascospores land on 93

petals germinate and produce mycelium by using the senescing petals as an initial source of 94

nutrients (McLean 1958) The presence of water on plant parts enhances the mycelia growth 95

and infection process (Abawi et al 1975a Hannusch and Boland 1996) The secretion of 96

oxalic acid and a battery of acidic lytic enzymes kill cells ahead of the advancing mycelium 97

and causes death of cells at the infection sites (Abawi and Grogan 1979 Adams and Ayers 98

1979) Upon nutrient shortages the fungal mycelia aggregate and turn into melanised sclerotia 99

which are retained in the stem or drop to the soil and remain viable as resting structures for 100

many years Mycelium directly arising from sclerotia at plant base is not as infective as the 101

11

primary inoculum of ascospores because sclerotial mycelium compete with other 102

saprophytes (Newton and Sequeira 1972) The mycelium can directly infect host plant tissue 103

using either enzymatic degradation or mechanical means by producing appressoria (Le 104

Tourneau 1979 Lumsden 1979) 105

Pathogenicity 106

Being a necrotrophic fungus S sclerotiorum kills cells ahead of the advancing mycelium and 107

extracts nutrition from dead plant tissue Oxalic acid plays a critical role in effective 108

pathogenicity (Cessna et al 2000) Several extracellular lytic enzymes such as cellulases 109

hemi-cellulases and pectinases (Riou et al 1991) aspartyl protease (Poussereau et al 2001) 110

endo-polygalacturonases (Cotton et al 2002) and acidic protease (Girard et al 2004) show 111

enhanced activity and degrade cell organelles under the acidic environment provided by 112

oxalic acid Oxalic acid is toxic to the host tissue and sequesters calcium in the middle 113

lamellae which disrupts the integrity of plant tissue (Bateman and Beer 1965 Godoy et al 114

1990a) The reduction of extracellular pH helps to activate the secretion of cell wall 115

degrading enzymes (Marciano et al 1983) Suppression of an oxidative burst directly limits 116

the host defence compounds (Cessna et al 2000) The fungus ramifies inter or intra-cellularly 117

colonizing tissues and kills cells ahead of the invading hyphae through enzymatic dissolution 118

Pectinolytic enzymes macerate plant tissue which cause necrosis followed by subsequent 119

plant death (Morrall et al 1972) The release of lytic enzymes and the oxalic acid from the 120

growing mycelium work synergistically to establish the infection (Fernando et al 2004) 121

Characteristic symptoms 122

SSR produces typical soft rot symptoms which first appear on the leaf axils Spores cannot 123

infect the leaves and stems directly and must first grow on dead petals or other organic 124

material adhering to leaves and stems (Saharan and Mehta 2008) The petals provide the 125

necessary food source for the spores to germinate grow and eventually penetrate the plant 126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

Infection usually occurs at the stem branching points where the airborne spores land and

droplets of water can be frequently found (Fernando et al 2007) Rainfall or heavy dew helps

to create moist conditions which may keep leaves and stems wet for two to three days

Spores can remain alive and able to penetrate the tissue up to 21 days after liberation

(Rimmer and Buchwaldt 1995) Two to three weeks after infection soft watery lesions or

areas of very light brown discolouration become obvious on the leaves main stems and

branches (Fig 1A) Lesions expand turn to greyish white and may have faint concentric

markings (Fig 1B C) Plants with girdled stems wilt prematurely ripen and become

conspicuously straw-coloured in a crop that is otherwise still green (Fig 1D E) Infected

plants may produce comparatively fewer pods per plant fewer seeds per pod or tiny

shrivelled seeds that blow out the back of the combine The extent of damage depends on

time of infection during the flowering stage as well as infection time on the main stems or

branches Severely infected crops result in lodging shattering at swathing and are difficult to

swathe The stems of infected plants eventually become bleached and tend to shred and

break When the bleached stems of diseased plants are split open a white mouldy growth and

hard black resting bodies (sclerotia) become visible (Fig 1F) (Rimmer and Buchwaldt 1995

Dueck 1977)

Life cycle

S sclerotiorum spends most of its life cycle as sclerotia in the soil The sclerotia from

infected plants can become incorporated into the soil following harvest which provides a

source of inoculum for future years Sclerotia are hard walled melanised resting structures

that are resilient to adverse conditions and remain viable in the top five centimetres of the soil

for approximately four years and up to ten years if buried deeper (Khangura and MacLeod

2012) Sclerotia can infect the base of the stem through direct mycelial germination in the

presence of an exogenous source of energy However the direct myceliogenic germination

12

151

13

of sclerotia is limited as a means of infection in oilseed Brassicas (Sharma et al 2015a) 152

Carpogenic germination of sclerotia results in the formation of apothecia which occurs after 153

ldquoconditioningrdquo for at least two weeks at 10-15oC in moist soil (Smith and Boland 1989)154

Apothecia are small mushroom-like structures that can release over two million tiny air-borne 155

ascospore during a functioning period of 5 to 10 days (Sharma et al 2015a) These 156

ascospores are mainly deposited within 100 to 150 meters from their origin but can travel 157

several kilometres through air currents (Bardin and Huang 2001) The ascospores can survive 158

on the plant surface or in the soil for two to three weeks in the absence of petals (Sharma et 159

al 2015a Rimmer and Buchwaldt 1995) The petals act as a food source for the germinating 160

spores of S sclerotiorum and to establish the infection (Rimmer and Buchwaldt 1995) 161

Infected and senescing petals then lodge on leaves leaf axils or stem branches and commence 162

infection as water soaked tan coloured lesions or areas of very light brown discolouration on 163

the leaves main stems and branches 2-3 weeks after infection Lesions turn to greyish white 164

covering most plant parts and eventually become bleached and tend to shred and break At 165

the end of growing season the fungal mycelia aggregates and develop sclerotia These 166

sclerotia then return to the soil on crop residues or after harvest overwinter and the disease 167

cycle is complete under favourable environmental conditions (Fig 2) 168

Epidemiology 169

Several studies conducted in Australia Canada China USA and Norway have demonstrated 170

the effect the environment plays in increasing the disease severity caused by S sclerotiorum 171

(Koike 2000 Kohli and Kohn 1998 Zhao and Meng 2003) Spore dispersal usually occurs in 172

windy warm and dry conditions The apothecia shrivel in dry conditions and excessive rain 173

washes out the spore bearing fruiting body into the soil (Kruumlger 1975) The frequency of 174

apothecial production was found to be similar in both saturated and unsaturated soil but 175

moisture is essential for initial infection and disease progression (Teo and Morrall 1985 176

14

Morrall and Dueck 1982) The germination of apothecia also varies with temperature for 177

example the consecutive temperature of 4 12 18 and 24oC is conducive to maximize178

germination rate (Purdy 1956) A comprehensive study demonstrated that the mean percent 179

petal infestation was comparatively higher in the morning compared to the afternoon and was 180

greatly favoured during lodging but secondary infection through plant contact was limited 181

for disease development and dissemination (Turkington et al 1991 Venette 1998) Honey 182

bees are also responsible for the spread of infected pollen grains causing pod rot of rapeseed 183

(Stelfox et al 1978) The spores can survive generally for 5 to 21 days on petals while 184

survivability was found to be higher on lower and shaded leaves (Venette 1998 Caesar and 185

Pearson 1982) 186

Management of Sclerotinia 187

Host resistance 188

S sclerotiorum has a diverse host range and a completely resistant genotype of canola against189

the pathogen have not yet been reported (Fernando et al 2007) The first report of genetic 190

resistance against Ssclerotiorum was cited a century ago observed in Phaseolus coccineus 191

(Steadman 1979 Bary et al 1887) A number of broad leaf crops are now reported to have 192

resistance to S sclerotiorum including Phaseolus vulgaris (Lyons et al 1987) and Phaseolus 193

coccineus (Abawi et al 1975b) Solanum melongena (Kapoor et al 1989) Pisum sativum 194

(Blanchette and Auld 1978) Arachis hypogaea (Coffelt and Porter 1982) Carthamus 195

tinctorius (Muumlndel et al 1985) Glycine max (Nelson et al 1991) Helianthus annuus (Sedun 196

and Brown 1989) and Ipomoea batatas (Wright et al 2003) The Australian Canadian and 197

Indian registered canola varieties possess minimal or no resistance to S sclerotiorum to date 198

and complete resistance to the pathogen has not been identified (Kharbanda and Tewari 1996 199

Sharma et al 2009 Li et al 2009) In China two canola cultivars namely Zhongyou 821 and 200

15

Zhongshuang no9 were reported to be partially resistant to S sclerotiorum in addition to 201

containing low glucosinolates and erucic acid with high yield potential (Gan et al 1999 202

Buchwaldt et al 2003 Wang et al 2004) Also a less susceptible canola cultivar was 203

developed against stem rot disease in France however yields were observed to be lower 204

under disease free conditions (Winter et al 1993 Krueger and Stoltenberg 1983) 205

206

Cultural management 207

208

A number of cultural strategies including disease avoidance pathogen exclusion and 209

eradication can be used to minimise stem rot disease in canola (Kharbanda and Tewari 1996) 210

Crop rotation is also an efficient strategy to reduce the sclerotia but 3-4 years rotation cannot 211

significantly eliminate the sclerotia from field (Williams and Stelfox 1980 Morrall and 212

Dueck 1982 Bailey 1996) Furthermore a long rotation of 5-6 years is not adequate to 213

completely eliminate sclerotia that can survive below 20 cm of soil depth (Nelson 1998) 214

Tillage is regarded as a potential method of minimising disease through burying of sclerotia 215

(Gulya et al 1997) Generally the sclerotia are not functional if residing below 2-3 cm soil 216

profile but exceptionally low carpogenic germination was observed at 5 cm soil depth (Kurle 217

et al 2001 Abawi and Grogan 1979 Duncan 2003) The survival of sclerotia is prolonged 218

when buried near the soil surface (Gracia-Garza et al 2002 Merriman et al 1979) Burying 219

of sclerotia through deep ploughing reduced germination and restricted the production of 220

apothecia (Williams and Stelfox 1980) On the other hand stem rot incidence was found to 221

be greater when fields are cultivated with a mouldboard plough compared with zero tillage 222

(Mueller et al 2002b Kurle et al 2001) However minimum or zero tillage may create a 223

more competitive and antagonistic environment as well as restrict the ability of the pathogen 224

to survive (Bailey and Lazarovits 2003) 225

226

16

Chemical control 227

The application of foliar fungicides is a widely used practice to manage SSR (Hind-228

Lanoiselet and Lewington 2004 Hind-Lanoiselet et al 2008) The efficacy of foliar 229

fungicides depends on several factors including time of application crop phenology weather 230

conditions disease cycle spraying coverage protection duration (Mueller et al 2002c 231

Hunter et al 1978 Mueller et al 2002a) The general reason behind poor management of 232

disease is poor timing of the fungicide application (Mueller et al 2002a Hunter et al 1978 233

Steadman 1983) where protectant fungicides are recommended to be applied at the pre-234

infection stage (Rimmer and Buchwaldt 1995 Steadman 1979) The early blooming stage 235

before petal fall is the best time to spray a foliar fungicide for a significant reduction in 236

disease incidence (Dueck and Sedun 1983 Morrall and Dueck 1982 Rimmer and Buchwaldt 237

1995 Dueck et al 1983) The fungicides widely used against sclerotinia in Canada are 238

Benlate (benomyl) Ronilan (vinclozolin) Rovral (iprodione) Quadris (azoxystrobin) 239

Sumisclex (procymidone) Fluazinam (shirlan) and cyprodinil plus fludioxonil (Switch) 240

Fungicides registered in Australia include Rovral Liquid Chief 250 Iprodione Liquid 250 241

Corvette Liquid (iprodione) Fortress 500 (procymidone) Sumisclex 500 Sumisclex 242

Broadacre (procymidone) and Prosaroreg (prothioconazole and tebuconazole) (Anonymous 243

2001 Hind-Lanoiselet et al 2008) The registration of Benlate was ceased in Canada due to 244

public health concerns and crop damage caused by the fungicide as claimed by canola 245

growers (Gilmour 2001) 246

Biological control 247

The use of microorganisms to suppress plant diseases was observed nearly a century ago and 248

since then plant pathologists have attempted to apply naturally occurring biocontrol agents 249

for managing important plant diseases Over 40 microbial species have been explored and 250

studied for managing S sclerotiorum (Li et al 2006) Since its first report in 1837 a total of 251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

185 studies have been reported on mycoparasitism and biocontrol of the pathogen (Sharma et

al 2015b) The interest in biocontrol of Sclerotinia diseases has increased over the last few

decades as chemical pesticides failed to properly control the pathogen and concerns of their

impact on the environment (Saharan and Mehta 2008 Agrios 2005) Key strategies for

potential biocontrol of Sclerotinia diseases include a reduction in the density of primary and

secondary inoculum by killing sclerotia or restricting germination infection in the

rhizosphere and phyllosphere as well as reduction in virulence (Saharan and Mehta 2008)

Several procedures including mycelial and sclerotial baiting as well as direct isolation from

the natural habitat have been used to explore biocontrol organisms against Sclerotinia spp

(Sandys‐Winsch et al 1994) A wide range of micro-organisms have been screened

recovered from the rhizosphere phylosphere sclerotia and other habitats to detect

antagonism that are suitable potential biocontrol agents Screening of antagonistic organisms

in soil or on plant tissue instead of artificial nutrient media have been shown to better predict

the potential of the agent as field assays are expensive and impractical for large numbers of

isolates (Sandys‐Winsch et al 1994 Andrews 1992 Whipps 1987) More than 100 species of

fungal and bacteria biocontrol agents have been identified against Sclerotinia These

biocontrol agents parasitise reduce weaken or kill sclerotia as well as protect plants from

ascospore infection (Mukerji et al 1999 Saharan and Mehta 2008)

Fungal antagonists

Several sclerotial mycoparasitic fungi have been studied to control S sclerotiorim for

example Coniothyrium minitans Trichoderma spp Gliocladium spp Sporidesmium

sclerotivorum Talaromyces flavus Epicoccum purpurescens Streptomyces sp Fusarium

Hormodendrum Mucor Penicillium Aspergillus Stachybotrys and Verticillium (Adams

1979 Saharan and Mehta 2008) The sclerotial mycoparasite C minitans was discovered by

Campbell (1947) and is considered as the most studied and widely available fungal biocontrol

17

276

18

agent against S sclerotiorum (Whipps et al 2008) Application of C minitans to the soil can 277

infect and destroy sclerotia of S sclerotiorum resulting in reduced carpogenic germination 278

and viability of sclerotia (McLaren et al 1996) Sclerotial destruction by C minitans was 279

observed when 1x109 viable conidia was applied against S sclerotiorum in different hosts280

including oilseed rape (Luth 2001) Soil incorporation of C minitans three months before 281

planting to 5 cm depth allows maximum colonisation and degradation of sclerotia (Peltier et 282

al 2012) The commercial formulation of C minitans has been registered in many countries 283

including Germany Belgium France and Russia under various trade names (De Vrije et al 284

2001) The use of C minitans to control sclerotinia diseases has been extensively reviewed 285

by Whipps et al (2008) 286

T harzianum has been reported to reduce linear growth of mycelium and apothecial287

production of S sclerotiorum as well as reducing the lesion length and disease incidence 288

when applied simultaneously or seven days prior to pathogen inoculation under glass house 289

conditions (Mehta et al 2012) Reduction of mycelia growth was also observed through 290

culture filtrates of T harzianum and T viride (Srinivasan et al 2001) The use of T 291

harzianum as a soil inoculant seed treatment and foliar spray singly or in combination 292

showed significant efficacy against S sclerotiorum in mustard (Meena et al 2014) 293

Application of T harzianum isolate GR in soil and farm yard manure infested with T 294

harzianum isolate SI-02 minimised disease incidence by 69 and 608 respectively 295

(Meena et al 2009) Seed treatment with T harzianum and foliar sprays with garlic bulb 296

extract not only significantly reduced disease incidence but also provided higher economic 297

return (Meena et al 2011) T harzianum and T viride significantly reduced disease incidence 298

when mustard seeds were treated with chemicals prior to soil treatment of microbes (Pathak 299

et al 2001) Rhizosphere inhabiting strains of T harzianum and Aspergillus sp also showed 300

inhibitory effect against the pathogen (Rodriguez and Godeas 2001) The mycoparasite 301

19

Sporidesmium sclerotivorum detected in soils of several states of the USA has been 302

considered as a promising invader of sclerotia Soil incorporation of the sclerotial parasite S 303

sclerotivorum and Teratosperma oligocladum caused 95 reduction in inoculum density 304

(Uecker et al 1978 Adams and Ayers 1981) The unique characteristics of S sclerotivorum 305

is its ability to grow from one sclerotium to another through soil producing many new 306

conidia throughout the soil mass which are able to infect surrounding sclerotia (Ayers and 307

Adams 1979) Application of 100 spores of S sclerotivorum per gram of soil caused a 308

significant decline in the survival of sclerotia (Adams and Ayers 1981) 309

310

Gliocladium virens has also been evaluated as a mycoparasite of both mycelia and sclerotia 311

of S sclerotiorum (Phillips 1986 Tu 1980 Whipps and Budge 1990) Sclerotial damage was 312

found to occur over a wide range of soil moistures and pH (5-8) but bio-activity was reduced 313

at temperatures below 15oC (Phillips 1986) The type and quality of substrates used to raise314

the G virens inoculum affected its ability to infect and reduced the viability of sclerotia 315

(Whipps and Budge 1990) The use of sand and spores as substrate and inoculum 316

respectively has provided the easiest method for screening G virens as a mycoparasite 317

(Whipps and Budge 1990) Other researchers also indicated that the ability of various strains 318

of G virens to parasitise S sclerotiorum varied and that strain selection will play an 319

important role in the mycoparasite-pathogen interaction (Phillips 1986 Tu 1980) G virens 320

has great potential as a mycoparasite due to its ability to grow and sporulate quickly spread 321

rapidly and produce metabolites such as glioviren an antibiotic with significant antagonistic 322

properties (Howell and Stipanovic 1995) Other Gliocladium spp including G roseum and G 323

catenulatum can parasitise the hyphae and sclerotia of S sclerotiorumas well as produce 324

toxins and cell wall degrading enzymes such as β-(1-3)-glucanases and chitinase (Huang 325

1978 Pachenari and Dix 1980) 326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

Bacterial antagonists

Plant growth promoting rhizobacteria have been exploited for sustainable management of

both foliar and soil borne plant pathogens Some antagonistic bacteria belonging to Bacillus

Pseudomonas Burkholderia and Agrobacterium species are commercially available for their

potential role in disease management (Fernando et al 2004) A number of bacterial isolates

have been investigated against S sclerotiorum for their potential antagonistic properties

Research on the application of antagonistic bacteria for the control of S sclerotiorum still

demands to be fully explored (Boyetchko 1999) The gram positive Bacillus spp are often

considered as potential biocontrol agents against foliar and soil borne plant diseases

(McSpadden Gardener and Driks 2004 Jacobsen et al 2004) Bacillus species have been less

studied than Pseudomonas but their ubiquity in soil greater thermal tolerance rapid

multiplication in liquid culture and easy formulation of resistant spores has made them

potential biocontrol candidates (Shoda 2000)

Bacillus cereus strain SC-1 isolated from the sclerotia of S sclerotiorum shows strong

antifungal activity against canola stem rot and lettuce drop disease caused by S sclerotiorum

(Kamal et al 2015a Kamal et al 2015b) Dual cultures have demonstrated that B cereus

SC-1 significantly suppressed hyphal growth inhibited the germination of sclerotia and was

able to protect cotyledons of canola from infection in the glasshouse Significant reduction in

the incidence of canola stem rot was observed both in glasshouse and field trials (Kamal et al

2015b) In addition B cereus SC-1 showed 100 disease protection against lettuce drop

caused by S sclerotiorum in glasshouse studies (Kamal et al 2015a) The biocontrol

mechanism of B cereus SC-1 was observed to be through antibiosis PCR amplification of

genomic DNA using gene-specific primers revealed that B cereus SC-1 contains four

antibiotic biosynthetic operons responsible for the production of bacillomycin D iturin A

surfactin and fengycin Mycolytic enzymes of chitinase and β-13-glucanase corresponding

20

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

genes were also documented Sclerotia submerged in cell free culture filtrates of B cereus

SC-1 for 10 days showed degradation of melanin the formation of pores on the surface of the

sclerotia and failure to germinate Significant reduction in lesion development caused by

S sclerotiorum was observed when culture filtrates were applied to 10-day-old

canola cotyledons Histological studies using scanning electron microscopy of the

zone of interaction of B cereus SC-1 and S sclerotiorum demonstrated restricted hyphal

growth and vacuolated hyphae with loss of cytoplasm (Figure 3A) In addition cells of the

sclerotial rind layer were ruptured and heavy colonisation of bacterial cells was observed

(Figure 3B) Transmission electron microscopy studies revealed that the cell content of

fungal mycelium and the organelles in sclerotial cells were completely disintegrated

suggesting the direct antifungal action of Bcereus SC-1 metabolites on cellular

components through lipopeptide antibiotics and mycolytic enzymes (Kamal et al

unpublished data)

An endophytic bacterium B subtilis strain EDR4 was reported to inhibit hyphal growth and

sclerotial germination of S sclerotiorum in rapeseed The maximum inhibition was observed

at the same time of inoculation either by cell suspension or cell free culture filtrates

Two applications of EDR4 at initiation of flowering and full bloom stage in the field

resulted in the best control efficiency Scanning electron microscopy showed that strain

EDR4 caused leakage vacuolization and disintegration of hyphal cytoplasm as well

as delayed the formation of infection cushion (Chen et al 2014) Another endophytic

strain of B subtilis Em7 has performed a broad antifungal spectrum on mycelium

growth and sclerotial germination invitro and significantly reduced stem rot disease

incidence in the field by 50-70 The strain Em7 caused leakage and swelling of

hyphal cytoplasm and subsequent disintegration and collapse of the cytoplasm (Gao et al

2013)

The application of B subtilis BY-2 was demonstrated to suppress S sclerotiorum on oilseed

rape in the field in China The strain BY-2 as a coated seed treatment formulation or spraysat

21

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

flowering or combined application of both treatments provided significant reduction of

disease incidence (Hu et al 2013a) In addition B subtilis Tu-100 a genetically distinct

strain also demonstrated its efficacy against SSR of oilseed rape in Wuhan China (Hu et al

2013a) Evaluation of cell suspension broth culture and cell-free filtrate derived from B

subtilis strain SB24 showed significant suppression of SSR of soybean under control glass

house conditions The strain SB24 originating from soybean root showed maximum disease

reduction at two days prior to inoculation of S sclerotiorum (Zhang and Xue 2010) The

plant-growth promoting bacterium Bacillus megaterium A6 isolated from the rhizosphere of

oilseed rape was shown to suppress S sclerotiorum in the field The isolate A6 was applied as

pellet and wrap seed treatment formulations and produced comparable reduction in disease as

the chemical control (Hu et al 2013b)

Furthermore another attempt to control white mould with B subtilis showed inconsistent

results between fields Spraying of Bacillus in soil significantly reduced the formation of

apothecia and thereby reduced yield losses in oilseed rape (Luumlth et al 1993) Members of

Bacillus species showed reduced disease severity and inhibited ascospore germination of S

sclerotiorum due to pre-colonization of petals when treated 24 hours before ascospore

inoculation of canola (Fernando et al 2004) Bacillus amyloliquefaciens (strains BS6 and

E16) performed better than other strains under the greenhouse environment in spray trials

against S sclerotiorum in canola (Zhang 2004) The results demonstrated that both strains

reduced stem rot by 60 when applied at 10-8

cfu mL-1

at 30 bloom stage In addition

HPLC analysis revealed that defence associated secondary metabolites in leaves of canola

were found to increase after inoculation which might supress the germination of ascospores

(Zhang et al 2004)

In North Dakota the sclerotia associated with Bacillus spp showed reduced germination and

the sclerotial medulla was infected by the bacterium (Wu 1988) Further studies revealed that

22

401

23

more than half of the total number of sclerotia recovered from the soil were infected by 402

Bacillus spp contributing to their degradation and inhibition of germination (Nelson et al 403

2001) In western Canada 92 canola associated bacterial strains belonging to Pseudomonas 404

Xanthomonas Burkholderia and Bacillus were selected for biocontrol activity in vitro 405

against S sclerotiorum and other canola pathogens The bacteria were able to protect the root 406

and crowns of susceptible plants from infection more efficiently than fungal antagonists by 407

inhibiting myceliogenic sclerotial germination and limiting ascospore production (Saharan 408

and Mehta 2008) The bacterial antagonist Bacillus polymixa has also been demonstrated to 409

reduce the growth of S sclerotiorum under controlled environment conditions (Godoy et al 410

1990b) 411

Biological control against SSR of canola using bacterial isolates of Pseudomonas 412

cholororaphis PA-23 and Pseudomonas sp DF41 was also demonstrated in vitro through the 413

inhibition of mycelial growth and sclerotial germination in canola (Savchuk 2002) 414

Inconsistent results were observed in the green house and field where both of the strains 415

suppressed the disease through reductions in the germination of ascospores (Savchuk 2002 416

Savchuk and Dilantha Fernando 2004) Several plant pathogens including S sclerotiorum 417

have been controlled successfully through antibiotics extracted from P chlororaphis PA23 418

which demonstrated inhibition of sclerotia and spore germination hyphal lysis vacuolation 419

and protoplast leakage (Zhang and Fernando 2004a) Synthesis of two antibiotics namely 420

phenazine and pyrrrolnitrin from the PA23 strain involved in the inhibitory action was 421

confirmed through molecular studies (Zhang 2004 Zhang and Fernando 2004b) The results 422

recommended that P chlororaphis strain PA23 might potentially be used against S 423

sclerotiorum and several other soil-borne pathogens Bacterial strains Pseudomonas 424

aurantiaca DF200 and P chlororaphis (Biotype-D) DF209 isolated from canola stubble 425

generated a number of organic volatile compounds in vitro including benzothiazole 426

24

cyclohexanol n-decanal dimethyl trisulphate 2-ethyl 1-hexanol and nonanal (Fernando et al 427

2005) These compounds inhibited the mycelial growth as well as reduced sclerotia and 428

ascospore germination both in vitro and in soil Both strains released volatiles into the soil 429

which interrupted sclerotial carpogenic germination and prevented ascospore liberation 430

(Fernando et al 2005) Pantoea agglomerans formerly known as Enterobacter agglomerans 431

is a gram negative bacterium isolated from canola petals and has demonstrated a capacity to 432

produce the enzyme oxalate oxidase which can successfully inhibit the pathogenic 433

establishment of S sclerotiorum through oxalic acid degradation (Savchuk and Dilantha 434

Fernando 2004) 435

A number of Burkholderia species have been considered as beneficial organisms in the 436

natural environment (Heungens and Parke 2000 Li et al 2002 Meyer et al 2001 Parke and 437

Gurian-Sherman 2001 McLoughlin et al 1992 Hebbar et al 1994 Bevivino 2000 Mao et 438

al 1998 Jayaswal et al 1993 Kang et al 1998 Meyers et al 1987 Pedersen et al 1999 439

Bevivino et al 2005 Chiarini et al 2006) The antimicrobial activity and plant growth 440

promoting capability of B cepacia isolates depends on a number of beneficial properties 441

such as indoleacetic acid production atmospheric nitrogen fixation and the generation of 442

various antimicrobial compounds such as cepacin cepaciamide cepacidines altericidins 443

pyrrolnitrin quinolones phenazine siderophores and alipopeptide (Parke and Gurian-444

Sherman 2001) In the early 1990s four B cepacia strains obtained registration from the 445

environmental protection agency (USA) for use as biopesticides which were later classified 446

as B ambifaria and one as B cepacia (Parke and Gurian-Sherman 2001 McLoughlin 447

1992)The bacterial antagonist Erwinia herbicola has also been demonstrated to reduce the 448

growth of S sclerotiorum under controlled environment conditions (Godoy et al 1990b) 449

450

451

25

Mycoviruses 452

As the name explains mycoviruses are viruses that inhabit and affect fungi They are either 453

pathogenic or symbiotic and differ from other viruses due to lack an extracellular stage in 454

their life cycle and harbour entire life in the fungal cytoplasm (Cantildeizares et al 2014) The 455

potential of hypovirulence-associated mycoviruses including S sclerotiorum hypovirulence-456

associated DNA virus 1 (SsHADV-1) Sclerotinia debilitation-associated RNA virus 457

(SsDRV) Sclerotinia sclerotiorum RNA virus L (SsRV-L) Sclerotinia sclerotiorum 458

hypovirus 1 (SsHV-1) Sclerotinia sclerotiorum mitoviruses 1 and 2 (SsMV-1 SsMV-2) and 459

Sclerotinia sclerotiorum partitivirus S (SsPV-S) have attracted much attention for biological 460

control of Sclerotinia induced plant diseases (Jiang et al 2013) The mixed infections of these 461

mycoviruses are commonly observed with higher infection incidence on Sclerotinia Recent 462

investigation revealed that some hypovirulence-associated mycoviruses are capable of 463

virocontrol of stem rot of oilseed rape under natural field condition (Xie and Jiang 2014) S 464

sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) was discovered as the first 465

DNA mycovirus to infect the fungus and confer hypovirulence (Yu et al 2010) Strong 466

infectivity of purified SsHADV-1 particle on healthy hyphae of S sclerotiorum was 467

observed (Yu et al 2013) Application of SsHADV-1infected hyphal fragment suspension of 468

S sclerotiorum on blooming rapeseed plants significantly reduced stem rot disease incidence469

and severity as well as increased seed yield Moreover SsHADV-1 was recovered from 470

sclerotia that were collected from previously infected hyphal fragment applied field indicated 471

that SsHADV-1might transmitted into other vegetative incompatible individuals The direct 472

applicatioin of SsHADV-1 ahead of virulent strain of S sclerotiorum on leaves of 473

Arabidopsis thaliana protected plants against the pathogen (Yu et al 2013) S sclerotiorum 474

debilitation-associated RNA virus (SsDRV) and S sclerotiorum RNA virus L (SsRV-L) was 475

shown to coinfect isolate Ep-1PN of S sclerotiorum (Xie et al 2006 Liu et al 2009) Further 476

26

intensive investigation of Sclerotinia mycovirus system could help for better understanding 477

of mycovirology and their potential application as biocontrol agent The production of 478

mycovirues could affect the economic viability of this approach to biological control 479

Conclusion 480

The advantages of biological control over other types of disease management includes 481

sustainable control of the target pathogen limited side effects selective to target pathogen 482

self-perpetuating organisms non-recurring cost and risk level of agent identified prior to 483

introduction (Pal and Gardener 2006) Biocontrol agents are more environmentally friendly 484

because they tend to be endemic in most regions No significant negative effects on the 485

environment have been reported when antagonistic microorganism have increased in the soil 486

(Cook and Baker 1983) The major limitation of biological control is that it demands more 487

technical expertise intensive management and planning sufficient time even years to 488

establish and most importantly the environmental conditions often exclude some agents 489

(Cook and Baker 1983) Biocontrol agents are likely to perform inconsistently under different 490

environment and this may explain why the performance of C minitans against S 491

sclerotiorum was not sustainable in natural field conditions (Fernando et al 2004) However 492

Bacillus spp which are less sensitive to environmental conditions are well adapted in 493

rhizosphere and are able to successfully manage soil borne pathogens including S 494

sclerotiorum Future research could be directed towards purification of antimicrobial 495

compounds released from biocontrol agents and their exploitation as fungicides 496

The cosmopolitan plant pathogen S sclerotiorum is challenging the available control 497

strategies The broad host range and prolonged survival of resting structures has has led to 498

difficulties in consistent economical management of the disease It is necessary to design an 499

innovative management strategy that can destroy sclerotia in soil and protect canola petals 500

27

leaves and stems from ascospores infection A number of mycoparasites have demonstrated 501

significant antagonism against S sclerotiorum and reduced disease incidence C minitans is 502

the pioneer mycoparasite that has been commercially available as Contans WGreg for the503

management of the white mould fungus however the commercial product has performed 504

inconsistently under field conditions 505

The use of bacterial antagonists to combat the white mould fungus is limited compared to 506

mycoparasites Very recently our lab has explored the sclerotia inhabiting bacterium B 507

cereus SC-1 which suppressed Sclerotinia infection in canola both under greenhouse and 508

field conditions (Kamal et al 2015b) The bacterium was demonstrated to produce multiple 509

antibiotics and mycolytic enzymes and also provided broad spectrum activity against 510

Sclerotinia lettuce drop (Kamal et al 2015a) Histological studies demonstrated that S 511

sclerotiorum treated with B cereus SC-1 resulted in restricted hyphal growth and vacuolated 512

hyphae void of cytoplasm Heavy proliferation of bacterial cells on sclerotia and a complete 513

destruction of the sclerotial rind layer were also observed The disintegration of mycelial cell 514

content and sclerotial cell organelles was the direct outcome of the antifungal activity of B 515

cereus SC-1 through lipopeptide antibiotics and mycolytic enzymes (Kamal et al 2015 516

unpublished work) Owing to the benefits of antagonists development of commercial 517

formulations with promising agents could pave the way for sustainable management of S 518

sclerotiorum in various cropping systems including oilseed Brassicas 519

References 520

Abawi G Grogan R (1979) Epidemiology of plant diseases caused by Sclerotinia species 521 Phytopathology 69899 522

Abawi G Polach F Molin W (1975a) Infection of bean by ascospores of Whetzelinia 523 sclerotiorum Phytopathology 65 (6)673-678 524

28

Abawi G Provvidenti R Hunter J Evaluating bean germplasm for resistance to Wetzelinia 525

sclerotiorum In Annual Proceedings of The American Phytopathological Society 526 1975b vol 2 p 50 527

Adams P (1979) Comparison of antagonists of Sclerotinia species Phytopathology 79 528 (12)1345-1347 529

Adams P Ayers W (1979) Ecology of Sclerotinia species Phytopathology 69 (8)896-899 530

Adams P Ayers W (1981) Sporidesmium sclerotivorum distribution and function in natural 531 biological control of sclerotial fungi Phytopathology 7190-93 532

Aghajani MA Safaei N Alizadeh A Sclerotinia infection situation of canola in Golestan 533 province In The 18th Iranian Plant Protection Congress Hamedan Iran 2008 p 52 534

Agrios GN (2005) Plant Pathology vol 5 Elsevier Academic New York 535

Alvarez F Castro M Priacutencipe A Borioli G Fischer S Mori G Jofreacute E (2012) The plant 536 associated Bacillus amyloliquefaciens strains MEP218 and ARP23 capable of 537 producing the cyclic lipopeptides iturin or surfactin and fengycin are effective in 538 biocontrol of sclerotinia stem rot disease J Appl Microbiol 112 (1)159-174 539

Andrews JH (1992) Biological control in the phyllosphere Annu Rev Phytopathol 30 540 (1)603-635 541

Anonymous (2001) Canola growers manual Canola council of Canada 542 httpwwwcanolacouncilorgcrop-productioncanola-growers-manual-543

contentschapter-10c-diseaseschapter-10csclerotiniastemrot Accessed 14 April 544 2015 545

Asirifi K Morgan WC Parbery D (1994) Suppression of Sclerotinia soft rot of lettuce with 546 organic soil amendments Anim Prod Sci 34 (1)131-136 547

Ayers W Adams P (1979) Mycoparasitism of sclerotia of Sclerotinia and Sclerotium species 548 by Sporidesmium sclerotivorum Can J Microbiol 25 (1)17-23 549

Bailey K Lazarovits G (2003) Suppressing soil-borne diseases with residue management and 550 organic amendments Soil Till Res 72 (2)169-180 551

Bailey KL (1996) Diseases under conservation tillage systems Can J Plant Sci 76 (4)635-552

639 553

Barbetti MJ Banga SK Fu TD Li YC Singh D Liu SY Ge XT Banga SS (2014) 554

Comparative genotype reactions to Sclerotinia sclerotiorum within breeding 555

populations of Brassica napus and B juncea from India and China Euphytica 197 556

(1)47-59 557

Bardin S Huang H (2001) Research on biology and control of Sclerotinia diseases in Canada 558 Can J Plant Pathol 23 (1)88-98 559

Bary A Garnsey HEF Balfour IB (1887) Comparative morphology and biology of the fungi 560 mycetozoa and bacteria Clarendon Press Oxford UK 561

29

Bateman D Beer S (1965) Simultaneous production and synergistic action of oxalic acid and 562

polygalacturonase during pathogenesis by Sclerotium rolfsii Phytopathology 55204-563 211 564

Bell A Liu L Reidy B Davis R Subbarao K (1998) Mechanisms of subsurface drip 565 irrigation-mediated suppression of lettuce drop caused by Sclerotinia minor 566 Phytopathology 88 (3)252-259 567

Bevivino A (2000) Efficacy of Burkholderia cepacia MCI7 in disease suppression and 568 growth promotion of maize Biol Fertil Soils 31225-231 569

Bevivino A Peggion V Chiarini L Tabacchioni S Cantale C Dalmastri C (2005) Effect of 570 Fusarium verticillioides on maize-root-associated Burkholderia cenocepacia 571 populations Res Microbiol 156 (10)974-983 572

Blanchette BL Auld DL (1978) Screening field peas for resistance to white mold Crop Sci 573

18 (6)977-978 574

Bourdocirct G Hurrell G Saville D DeJong D (2001) Risk analysis of Sclerotinia sclerotiorum 575 for biological control of Cirsium arvense in pasture ascospore dispersal Biocontrol 576 Sci Technol 11 (1)119-139 577

Boyetchko SM (1999) Biological control agents of canola and rapeseed diseases-status and 578 practical approaches Biotechnological approaches in biocontrol of plant pathogens 579 Mukerji KG Chamoacutela ВP and Upadhyay RK Eds Kluwer Academic NY51-71 580

Buchwaldt L Yu F Rimmer S Hegedus D Resistance to Sclerotinia sclerotiorum in a 581 Chinese Brassica napus cultivar In 8th International Congress of Plant Pathology 582 Christchurch New Zealand 2003 583

Caesar AJ Pearson RC (1982) Environmental factors affecting survival of ascospores of 584 Sclerotinia sclerotiorum Phytopathology Mediterranean 731024-1030 585

Campbell W (1947) A new species of Coniothyrium parasitic on sclerotia Mycologia190-586

195 587

Cantildeizares MC Peacuterez-Arteacutes E Garciacutea-Pedrajas MD (2014) The complete nucleotide sequence 588 of a novel partitivirus isolated from the plant pathogenic fungus Verticillium albo-589 atrum Arch Virol 159 (11)3141-3144 590

Cessna SG Sears VE Dickman MB Low PS (2000) Oxalic acid a pathogenicity factor for 591

Sclerotinia sclerotiorum suppresses the oxidative burst of the host plant The Plant 592 Cell 12 (11)2191-2199 593

Chaudhury B (1993) Yield loss estimation by (Sclerotinia sclerotiorum)(Lib) de Bary J 594 Agric Anim Sci 14113 595

Chen Y Gao X Chen Y Qin H Huang L Han Q (2014) Inhibitory efficacy of endophytic 596 Bacillus subtilis EDR4 against Sclerotinia sclerotiorum on rapeseed Biol Control 597

7867-76 598

30

Chiarini L Bevivino A Dalmastri C Tabacchioni S Visca P (2006) Burkholderia cepacia 599

complex species health hazards and biotechnological potential Trends Microbiol 14 600 (6)277-286 601

Clarkson JP Phelps K Whipps JM Young CS Smith JA Watling M (2004) Forecasting 602 Sclerotinia disease on lettuce toward developing a prediction model for carpogenic 603 germination of sclerotia Phytopathology 94 (3)268-279 604

Coffelt T Porter D (1982) Screening peanuts for resistance to Sclerotinia blight Plant Dis 66 605 (5)385-387 606

Cook RJ Baker KF (1983) The nature and practice of biological control of plant pathogens 607 American Phytopathological Society 608

Cotton P Rascle C Fevre M (2002) Characterization of PG2 an early endoPG produced by 609 Sclerotinia sclerotiorum expressed in yeast FEMS Microbiol Lett 213 (2)239-244 610

De Vrije T Antoine N Buitelaar R Bruckner S Dissevelt M Durand A Gerlagh M Jones 611 E Luumlth P Oostra J (2001) The fungal biocontrol agent Coniothyrium minitans 612 production by solid-state fermentation application and marketing Appl Microbiol 613 Biotechnol 56 (1)58-68 614

Dueck J (1977) Sclerotinia in rapeseed Canada Agriculture 227-9 615

Dueck J Morrall R McKenzie D (1983) Control of Sclerotinia sclerotiorum in rapeseed with 616 fungicides Can J Plant Pathol 5 (4)289-293 617

Dueck J Sedun FS (1983) Distribution of Sclerotinia sclerotiorum in western Canada as 618 indicated by sclerotial levels in rapeseed unloaded in Vancouver Can Plant Dis Surv 619 63 (1)27-29 620

Duncan RW (2003) Evaluation of host tolerance biological chemical and cultural control of 621 Sclerotinia sclerotiorum in sunflower (Helianthus annuus L) University of 622 Manitoba Manitoba Canada 623

Fernando W Ramarathnam R Krishnamoorthy AS Savchuk SC (2005) Identification and 624 use of potential bacterial organic antifungal volatiles in biocontrol Soil Biol Biochem 625 37 (5)955-964 626

Fernando WGD Nakkeeran S Zhang Y (2004) Ecofriendly methods in combating 627 Sclerotinia sclerotiorum (Lib) de Bary In Recent Research in Developmental and 628

Environmental Biology vol 1 vol 2 Research Signpost Kerala India pp 329-347 629

Fernando WGD Nakkeeran S Zhang Y Savchuk S (2007) Biological control of Sclerotinia 630

sclerotiorum (Lib) de Bary by Pseudomonas and Bacillus species on canola petals 631 Crop Protect 26 (2)100-107 632

Fuller P Coyne D Steadman J (1984) Inheritance of resistance to white mold disease in a 633 diallel cross of dry beans Crop Sci 24 (5)929-933 634

31

Gan LI Meng JL Gan L (1999) Analysis on the genetic diversity of loci homologous to 635

disease resistant genes in Brassica genus Journal of Huazhong Agricultural 636 University 18540-542 637

Gao X Han Q Chen Y Qin H Huang L Kang Z (2013) Biological control of oilseed rape 638 Sclerotinia stem rot by Bacillus subtilis strain Em7 Biocontrol Sci Technol 24 (1)39-639 52 640

Ghasolia R Shivpuri A Bhargava A (2004) Sclerotinia rot of Indian mustard (Brassica 641 juncea) in Rajasthan Indian Phytopathology 5776-79 642

Gilmour G (2001) Canola growers losing Benlate fungicide Manitoba Cooperator 581 643

Girard V Fegravevre M Bruel C (2004) Involvement of cyclic AMP in the production of the acid 644 protease Acp1 by Sclerotinia sclerotiorum FEMS Microbiol Lett 237 (2)227-233 645

Godoy G Steadman J Dickman M Dam R (1990a) Use of mutants to demonstrate the role 646 of oxalic acid in pathogenicity of Sclerotinia sclerotiorum on Phaseolus vulgaris 647 Physiol Mol Plant Pathol 37 (3)179-191 648

Godoy G Steadman J Yuen G (1990b) Bean blossom bacteria have potential for biological 649 control of white mold disease caused by Sclerotinia sclerotiorum Annu Rep Bean 650 Improv Coop 3345-46 651

Gracia-Garza J Neumann S Vyn T Boland G (2002) Influence of crop rotation and tillage 652 on production of apothecia by Sclerotinia sclerotiorum Can J Plant Pathol 24 653 (2)137-143 654

Gulya T Rashid KY Masirevic SM (1997) Sunflower Technology and Production In 655 Schneiter AA (ed) Sunflower Diseases vol 35 Madison Wisconsin pp 263-379 656

Hannusch D Boland G (1996) Influence of air temperature and relative humidity on 657 biological control of white mold of bean (Sclerotinia sclerotiorum) Phytopathology 658 86 (2)156-162 659

Hayes RJ Wu BM Pryor BM Chitrampalam P Subbarao KV (2010) Assessment of 660 resistance in lettuce (Lactuca sativa L) to mycelial and ascospore infection by 661 Sclerotinia minor Jagger and S sclerotiorum (Lib) de Bary HortScience 45 (3)333-662 341 663

Hebbar KP Martel MH Heulin T Burkholderia cepacia a plant growth promoting 664

rhizobacterial associate of maize In Ryder MH Stephens PM Bowen GD (eds) 665 Third International Workshop on Plant Growth-Promoting Rhizobacteria Adelaide 666 South Australia 1994 CSIRO pp 201-203 667

Heungens K Parke J (2000) Zoospore homing and infection events effects of the biocontrol 668 bacterium Burkholderia cepacia AMMDR1 on two oomycete pathogens of pea 669 (Pisum sativum L) Appl Environ Microbiol 66 (12)5192-5200 670

Hind-Lanoiselet T Ash GJ Murray GM (2003) Prevalence of sclerotinia stem rot of canola 671 in New South Wales Anim Prod Sci 43 (2)163-168 672

32

Hind-Lanoiselet T Lewington F (2004) Canola concepts managing sclerotinia NSW 673

Department of Primary Industries AgNote 490 674

Hind-Lanoiselet T Lewington F Lindbeck K (2008) Managing sclerotinia stem rot in canola 675 NSW Department of Primary Industries Australia AgReport 676

Howell C Stipanovic R (1995) Mechanisms in the biocontrol of Rhizoctonia solani-induced 677 cotton seedling disease by Gliocladium virens antibiosis Phytopathology 85 (4)469-678 472 679

Hu X Roberts DP Xie L Maul JE Yu C Li Y Jing M Liao X Che Z Liao X (2013a) 680 Formulations of Bacillus subtilis BY-2 suppress Sclerotinia sclerotiorum on oilseed 681 rape in the field Biol Control 7054-64 682

Hu X Roberts DP Xie L Maul JE Yu C Li Y Zhang S Liao X (2013b) Bacillus 683 megaterium A6 suppresses Sclerotinia sclerotiorum on oilseed rape in the field and 684

promotes oilseed rape growth Crop Protect 52151-158 685

Huang H (1978) Gliocladium catenulatum hyperparasite of Sclerotinia sclerotiorum and 686 Fusarium species Canadian Journal of Botany 56 (18)2243-2246 687

Hunter J Abawi G Crosier D (1978) Effects of timing coverage and spray oil on control of 688 white mold of snap bean with benomyl Plant Dis Rep 62 (7)633-637 689

Jacobsen B Zidack N Larson B (2004) The role of Bacillus-based biological control agents 690 in integrated pest management systems Plant diseases Phytopathology 94 (11)1272-691 1275 692

Jayaswal R Fernandez M Upadhyay R Visintin L Kurz M Webb J Rinehart K (1993) 693 Antagonism of Pseudomonas cepacia against phytopathogenic fungi Curr Microbiol 694

26 (1)17-22 695

Jiang D Fu Y Guoqing L Ghabrial SA (2013) Viruses of the plant pathogenic fungus 696 Sclerotinia sclerotiorum Adv Virus Res vol 86 697

Kamal MM Lindbeck K Savocchia S Ash G (2015a) Bacterial biocontrol of sclerotinia 698 diseases in Australia Acta Hort(Accepted) 699

Kamal MM Lindbeck KD Savocchia S Ash GJ (2015b) Biological control of sclerotinia 700

stem rot of canola using antagonistic bacteria Plant Pathol doi101111ppa12369 701 doi101111ppa12369 702

Kang Y Carlson R Tharpe W Schell MA (1998) Characterization of genes involved in 703 biosynthesis of a novel antibiotic from Burkholderia cepacia BC11 and their role in 704

biological control of Rhizoctonia solani Appl Environ Microbiol 64 (10)3939-3947 705

Kapoor K Sharma S Gangopadhyay S (1989) Assessment of resistance in eggplant against 706 Sclerotinia wilt with a new screening technique Capsicum Newsletter 870 707

Khangura R MacLeod WJ (2012) Managing the risk of Sclerotinia stem rot in canola Farm 708 note vol 546 Department of Agriculture and Food Western Australia 709

33

Kharbanda P Tewari J (1996) Integrated management of canola diseases using cultural 710

methods Can J Plant Pathol 18 (2)168-175 711

Kirkegaard JA Robertson MJ Hamblin P Sprague SJ (2006) Effect of blackleg and 712 sclerotinia stem rot on canola yield in the high rainfall zone of southern New South 713 Wales Australia Aust J Agric Res 57 (2)201-212 714

Kohli Y Kohn LM (1998) Random Association among Alleles in Clonal Populations of 715 Sclerotinia sclerotiorum Fungal Genet Biol 23 (2)139-149 716

Koike S (2000) Occurrence of Stem Rot of Basil Caused by Sclerotinia sclerotiorum in 717 Coastal California Plant Dis 84 (12)1342 718

Krueger W Stoltenberg J (1983) Control of rape diseases II Measures for disease reduction 719 caused by Sclerotinia sclerotiorum with consideration to economical aspects 720 Phytopath Z 108114-126 721

Kruumlger W (1975) Influence of the weather on attack of rape by Sclerotinia sclerotiorum 722 (Lib) de Bary Nachrichtenblatt Deutscher Pflanzenschutzdienst 271-6 723

Kurle JE Grau CR Oplinger ES Mengistu A (2001) Tillage crop sequence and cultivar 724 effects on Sclerotinia stem rot incidence and yield in soybean Agron J 93 (5)973-982 725

Lamey A Knodel J Endres G Andol K Ashley R Barondeau D Craig B Crary V Fore Z 726 Johnson N (2001) Canola disease survey in Minnesota and North Dakota vol 71 727 North Dakota State University North Dakota USA 728

Lamey H Nelson B Gulya T Incidence of Sclerotinia stem rot on canola in North Dakota 729 and Minnesota 1991-1997 In Proc Int Sclerotinia Workshop Fargo ND 730 September 9-12 1998 pp 7-9 731

Le Tourneau D (1979) Morphology cytology and physiology of Sclerotinia species in 732 culture Phytopathology 69 (8)887-890 733

Li C Liu S Sivasithamparam K Barbetti M (2009) New sources of resistance to Sclerotinia 734

stem rot caused by Sclerotinia sclerotiorum in Chinese and Australian Brassica napus 735 and B juncea germplasm screened under Western Australian conditions Australas 736 Plant Pathol 38 (2)149-152 737

Li GQ Huang HC Miao HJ Erickson RS Jiang DH Xiao YN (2006) Biological control of 738 sclerotinia diseases of rapeseed by aerial applications of the mycoparasite 739

Coniothyrium minitans Eur J Plant Pathol 114 (4)345-355 740

Li W Roberts D Dery P Meyer S Lohrke S Lumsden R Hebbar K (2002) Broad spectrum 741

anti-biotic activity and disease suppression by the potential biocontrol agent 742 Burkholderia ambifaria BC-F Crop Protect 21 (2)129-135 743

Lindbeck K Davidson J Khangura R (2014) Managing sclerotinia stem rot in canola 744 (Northern Southern and Western regions) Sclerotinia stem rot in canola Fact Sheet 745

Grain Research and Development Corporation Australia 746

34

Liu H Fu Y Jiang D Li G Xie J Peng Y Yi X Ghabrial SA (2009) A novel mycovirus that 747

is related to the human pathogen hepatitis E virus and rubi-like viruses J Virol 83 748 (4)1981-1991 749

Lumsden R (1979) Histology and physiology of pathogenesis in plant diseases caused by 750 Sclerotinia species Phytopathology 69 (8)890-895 751

Luth P The biological fungicide Contans WG7-A preparation on the basis of the fungus 752 Coniothyrium minitans In Proc XI International Sclerotinia Workshop Central 753 Science Laboratory York UK July 8-12 2001 pp 127-128 754

Luumlth P Schulz RR Pfeffer H (1993) The influence of bacterial antagonists on the infestation 755 of a soil as well as on the yield of winter oilseed rape affected by Sclerotinia 756 sclerotiorum Zentralbl Mikrobiol 14832-32 757

Lynch JM Ebben MH (1986) The use of microorganisms to control plant disease J Appl 758

Bacteriol Symp Suppl 61 (115S-126S) 759

Lyons M Dickson M Hunter J (1987) Recurrent selection for resistance to white mold in 760 Phaseolus species J Am Soc Hort Sci 112 (1)149-152 761

Mao W Lumsden RD Lewis JA Hebbar PK (1998) Seed treatment using pre-infiltration and 762 biocontrol agents to reduce damping-off of corn caused by species of Pythium and 763 Fusarium Plant Dis 82 (3)294-299 764

Marciano P Di Lenna P Magro P (1983) Oxalic acid cell wall-degrading enzymes and pH in 765 pathogenesis and their significance in the virulence of two Sclerotinia sclerotiorum 766 isolates on sunflower Physiol Plant Pathol 22 (3)339-345 767

McLaren D Huang H Rimmer S (1996) Control of apothecial production oiacute Sclerotinia 768

sclerotiorum by Coniothyrium minitans and Talaromyces flavus Plant Dis1373-1378 769

McLean D (1958) Role of dead flower parts in infection of certain crucifers by Sclerotinia 770 sclerotiorum (Lib) de Bary Plant Dis Rep 42663-666 771

McLoughlin TJ Quinn JP Bettermann A Bookland R (1992) Pseudomonas cepacia 772 suppression of sunflower wilt fungus and role of antifungal compounds in controlling 773 the disease Appl Environ Microbiol 581760-1763 774

McSpadden Gardener BB Driks A (2004) Overview of the nature and application of 775 biocontrol microbes Bacillus spp Phytopathology 94 (11)1244 776

Meena P Awasthi R Godika S Gupta J Kumar A Sandhu P Sharma P Rai P Singh Y 777 Rathi A (2011) Eco-friendly approaches managing major diseases of Indian mustard 778

World Appl Sci J 12 (8)1192-1195 779

Meena P Chattopadhyay C Meena P Goyal P Kumar VR (2014) Shelf life and efficacy of 780 talc-based bio-formulations of Trichoderma harzianum isolates in management of 781 Sclerotinia rot of Indian mustard (Brassica juncea) Ann Plant Protect Sci 22 (1)127-782

135 783

35

Mehta N Hieu N Sangwan M (2012) Efficacy of various antagonistic isolates and species of 784

against causing white stem rot of mustard J Mycol Plant Pathol 42 (2)244-250 785

Merriman P Pywell M Harrison G Nancarrow J (1979) Survival of sclerotia of Sclerotinia 786 sclerotiorum and effects of cultivation practices on disease Soil Biol Biochem 11 787 (6)567-570 788

Meyer SLF Roberts DP Chitwood DJ Carta LK Lumsden RD Mao W (2001) Application 789 of Burkholderia cepacia and Trichoderma virens alone and in combinations against 790 Meloidogyne incognita on bell pepper Nematropica 31 (1)75-86 791

Meyers E Bisacchi G Dean L Liu W Minassian B Slusarchyk D Sykes R Tanaka S Trejo 792 W (1987) Xylocandin a new complex of antifungal peptides I Taxonomy isolation 793 and biological activity J Antibiot 40 (11)1515 794

Morrall R Duczek L Sheard J (1972) Variations and correlations within and between 795

morphology pathogenicity and pectolytic enzyme activity in Sclerotinia from 796 Saskatchewan Can J Bot 50 (4)767-786 797

Morrall R Dueck J (1982) Epidemiology of Sclerotinia stem rot of rapeseed in 798 Saskatchewan Can J Plant Pathol 4 (2)161-168 799

Morrall R Dueck J Sclerotinia stem rot of spring rapeseed in western Canada In 800 Proceedings of 6th International Rapeseed Conference Paris France 1983 pp 17-19 801

Morrall R Dueck J McKenzie D McGee D (1976) Some aspects of Sclerotinia sclerotiorum 802 in Saskatchewan 1970-75 Can Plant Dis Surv 56 (2)56-62 803

Mueller D Dorrance A Derksen R Ozkan E Kurle J Grau C Gaska J Hartman G Bradley 804 C Pedersen W (2002a) Efficacy of fungicides on Sclerotinia sclerotiorum and their 805

potential for control of Sclerotinia stem rot on soybean Plant Dis 86 (1)26-31 806

Mueller D Pedersen W Hartman G (2002b) Effect of crop rotation and tillage system on 807 Sclerotinia stem rot on soybean Can J Plant Pathol 24 (4)450-456 808

Mueller J Barbercheck M Bell M Brownie C Creamer N Hitt A Hu S King L Linker H 809 Louws F (2002c) Development and implementation of a long-term agricultural 810 systems study challenges and opportunities HortTechnology 12 (3)362-368 811

Mukerji K Chamola B Upadhyay RK (eds) (1999) Biotechnological approaches in 812 biocontrol of plant pathogens Kluwer AcademicPlenum Press New York USA 813

Muumlndel HH Huang H Kozub G (1985) Sclerotinia head rot in safflower assessment of 814 resistance and effects on yield and oil content Can J Plant Sci 65 (2)259-265 815

Nelson B Biology of Sclerotinia In Proceedings of the sclerotinia workshop Fargo North 816 Dakota 1998 North Dakota State University pp 9-12 817

Nelson B Helms T Olson M (1991) Comparison of laboratory and field evaluations of 818 resistance in soybean to Sclerotinia sclerotiorum Plant Dis 75662-665 819

36

Nelson BD Christianson T McClean P Effects of bacteria on sclerotia of Sclerotinia 820

sclerotiorum In Proceedings of the XI international sclerotinia workshop York 821 England 2001 pp 39-40 822

Newton H Sequeira L (1972) Ascospores as the primary infective propagule of Sclerotinia 823 sclerotiorum in Wisconsin Plant Dis Rep 56 (9)798-802 824

Pachenari A Dix N (1980) Production of toxins and wall degrading enzymes by Gliocladium 825 roseum Trans Br Mycol Soc 74 (3)561-566 826

Pal KK Gardener BMS (2006) Biological control of plant pathogens Plant healt instruct 827 21117-1142 828

Parke JL Gurian-Sherman D (2001) Diversity of the Burkholderia cepacia complex and 829 implications for risk assessment of biological control strains Annu Rev Phytopathol 830 39 (1)225-258 831

Pathak A Godika S Jain P Muralia S (2001) Effect of antagonistic fungi and seed dressing 832 fungicides on the incidence of stem rot of mustard Mycol Plant Pathol 31327-329 833

Pedersen E Reddy M Chakravarty P (1999) Effect of three species of bacteria on damping 834 off root rot development and ectomycorrhizal colonization of lodgepole pine and 835 white spruce seedlings Eur J For Pathol 29 (2)123-134 836

Peltier AJ Bradley CA Chilvers MI Malvick DK Mueller DS Wise KA Esker PD (2012) 837 Biology yield loss and control of Sclerotinia stem rot of soybean J Integ Pest Mngmt 838 3 (2)1-7 839

Phillips A (1986) Factors affecting the parasitic activity of Gliocladium virens on sclerotia of 840 Sclerotinia sclerotiorum and a note on its host range J Phytopathol 116 (3)212-220 841

Pope SJ Varney PL Sweet JB (1989) Susceptibility of cultivars of oilseed rape to S 842 sclerotiorum and the effect of infection on yield Asp Appl Biol 23451-456 843

Poussereau N Creton S Billon-Grand G Rascle C Fevre M (2001) Regulation of acp1 844

encoding a non-aspartyl acid protease expressed during pathogenesis of Sclerotinia 845 sclerotiorum Microbiology 147 (3)717-726 846

Purdy L (1956) Factors affecting apothecial production by Sclerotinia sclerotiorum 847

Phytopathology 46409-410 848

Purdy LH (1979) Sclerotinia sclerotiorum history diseases and symptomatology host range 849

geographic distribution and impact Phytopathology 69 (8)875-880 850

Rimmer SR Buchwaldt L (1995) Diseases In Kimber DS McGregor DI (eds) Brassica 851

oilseeds production and utilization CAB International Wallingford UK pp 111-140 852

Riou C Freyssinet G Fevre M (1991) Production of cell wall-degrading enzymes by the 853 phytopathogenic fungus Sclerotinia sclerotiorum Appl Environ Microbiol 57 854 (5)1478-1484 855

37

Rodriguez M Godeas A Comparative study of fungal antagonist Sclerotinia sclerotiorum In 856

Proc XI International Sclerotinia Workshop York UK 2001 pp 125-126 857

Saharan GS Mehta N (2008) Sclerotinia diseases of crop plants Biology ecology and 858 disease management Springer Science Busines Media BV The Netherlands 859

Sandys‐Winsch D Whipps J Fenlon J Lynch J (1994) The validity of invitro screening 860 methods in the search for fungal antagonists of Sclerotinia sclerotiorum causing wilt 861 of sunflower Biocontrol Sci Technol 4 (3)269-277 862

Savchuk S Dilantha Fernando W (2004) Effect of timing of application and population 863 dynamics on the degree of biological control of Sclerotinia sclerotiorum by bacterial 864 antagonists FEMS Microbiol Ecol 49 (3)379-388 865

Savchuk SC (2002) Evaluation of biological control control of Sclerotinia sclerotiorum on 866 Canola (Brassica napus) in the lab in the greenhouse and in the field University of 867

Manitoba Manitiba Canada 868

Sedun F Brown J (1989) Comparison of three methods to assess resistance in sunflower to 869 basal stem rot caused by Sclerotinia sclerotiorum and S minor Plant Dis 73 (1)52-55 870

Sharma P Kumar A Meena P Goyal P Salisbury P Gurung A Fu T Wang Y Barbetti M 871 Chattopadhyay C Search for resistance to Sclerotinia sclerotiorum in exotic and 872 indigenous Brassica germplasm In Proc of 16

th Australian Research Assembly on873

Brassicas Ballarat Victoria 2009 pp 1-5 874

Sharma P Meena PD Verma PR Saharan GS Mehta N Singh D Kumar A (2015a) 875 Sclerotinia sclerotiorum (Lib) de Bary causing sclerotinia rot in oilseed brassicas A 876 review Journal of oilseed brassica 6 (Special)1-44 877

Sharma P Verma PR Meena PD Kumar V Singh D (2015b) Research progress analysis of 878 sclerotinia rot (Sclerotinia sclerotiorum) of oilseed brassicas through bibliography 879 Journal of oilseed brassica 6 (Special)45-125 880

Shaw F Ajrekar S (1915) The genus Rhizoctonia in India vol 7 Memoirs of the 881 department of agriculture India Botanical series Thacker Spink India 882

Shoda M (2000) Bacterial control of plant diseases J Biosci Bioeng 89 (6)515-521 883

Shukla A (2005) Estimation of yield losses to Indian mustard (Brassica juncea) due to 884 Sclerotinia stem rot J Phytol Res 18 (2)267-268 885

Smith E Boland G (1989) A reliable method for the production and maintenance of 886 germinated sclerotia of Sclerotinia sclerotiorum Can J Plant Pathol 11 (1)45-48 887

Srinivasan A Kang I Singh R Kaur J Evaluation of selected Trichoderma isolates against 888 Sclerotinia sclerotiorum causing white rot of Brassica napus L In Proc XI 889 International Sclerotinia Workshop York UK 2001 pp 143-144 890

Steadman J (1979) Control of plant diseases caused by Sclerotinia species Phytopathology 891 69904-907 892

38

Steadman JR (1983) White mold-A serious yield-limiting disease of bean Plant Dis 67 893

(4)346-350 894

Stelfox D Williams J Soehngen U Topping R (1978) Transport of Sclerotinia sclerotiorum 895 ascospores by rapeseed pollen in Alberta Plant Dis Rep 62576-579 896

Teo B Morrall R (1985) Influence of matric potentials on carpogenic germination of sclerotia 897 of Sclerotinia sclerotiorum II A comparison of results obtained with different 898 techniques Can J Plant Pathol 7 (4)365-369 899

Tu J (1980) Gliocladium virens a destructive mycoparasite of Sclerotinia sclerotiorum 900 Phytopathology 70 (7)670-674 901

Turkington T Morrall R Gugel R (1991) Use of petal infestation to forecast Sclerotinia stem 902 rot of canola Evaluation of early bloom sampling 1985-90 Can J Plant Pathol 13 903 (1)50-59 904

Uecker F Ayers W Adams P (1978) A new hyphomycete on sclerotia of Sclerotinia 905 sclerotiorum Mycotaxon 7275-282 906

Venette J Sclerotinia spore formation transport and infection In Proc of the XI International 907 Sclerotinia Workshop York UK 1998 pp 4-7 908

Wang H Liu G Zheng Y Wang X Yang Q (2004) Breeding of the Brassica napus cultivar 909 Zhongshuang 9 with high-resistance to Sclerotinia sclerotiorum and dynamics of its 910 important defense enzyme activity Sci Agric Sinica 11-4 911

Whipps J Budge S (1990) Screening for sclerotial mycoparasites of Sclerotinia sclerotiorum 912 Mycol Res 94 (5)607-612 913

Whipps JM (1987) Behaviour of fungi antagonistic to Sclerotinia sclerotiorum on plant tissue 914 segments J Gen Microbiol 133 (6)1495-1501 915

Whipps JM Sreenivasaprasad S Muthumeenakshi S Rogers CW Challen MP (2008) Use of 916 Coniothyrium minitans as a biocontrol agent and some molecular aspects of sclerotial 917

mycoparasitism Eur J Plant Pathol 121323-330 918

Williams JR Stelfox D (1980) Influence of farming practices in Alberta on germination and 919 apothecium production of sclerotia of Sclerotinia sclerotiorum Can J Plant Pathol 2 920

(3)169-172 921

Winter W Burkhard L Baenziger I Krebs H Gindrat D Frei P (1993) Rape diseases 922

occurrence on rape varieties effect of fungicides and preventive control measures 923 Landwirtschaft Schweiz 6 (10)589-596 924

Wright P Lewthwaite S Triggs C Broadhurst P (2003) Laboratory evaluation of sweet 925 potato (Ipomoea batatas) resistance to sclerotinia rot N Z J Crop Hortic Sci 31 926 (1)33-39 927

Wu H (1988) Effects of bacteria on germination and degradation of sclerotia of Sclerotinia 928 Sclerotiorum (Lib) de Bary North Dakota State University North Dakota USA 929

39

Wu Y Yuan J Raza W Shen Q Huang Q (2014) Biocontrol traits and antagonistic potential 930

of Bacillus amyloliquefaciens strain NJZJSB3 against Sclerotinia sclerotiorum a 931 causal agent of canola stem rot J Microbiol Biotechn 24 (10)1327ndash1336 932

Xie J Jiang D (2014) New Insights into mycoviruses and exploration for the biological 933 control of crop fungal diseases Annu Rev Phytopathol 5245-68 934

Xie J Wei D Jiang D Fu Y Li G Ghabrial S Peng Y (2006) Characterization of 935 debilitation-associated mycovirus infecting the plant-pathogenic fungus Sclerotinia 936 sclerotiorum J Gen Virol 87 (1)241-249 937

Yang D Wang B Wang J Chen Y Zhou M (2009) Activity and efficacy of Bacillus subtilis 938 strain NJ-18 against rice sheath blight and Sclerotinia stem rot of rape Biol Control 939 51 (1)61-65 940

Yexin Z Huqiang Q Fengjie N Lili H Xiaoning G Zhensheng K Qingmei H (2011) 941

Investigation of Sclerotinia stem rot in Shaanxi Province Plant Protection 37116-119 942

Yu X Li B Fu Y Jiang D Ghabrial SA Li G Peng Y Xie J Cheng J Huang J (2010) A 943 geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic 944 fungus Proceedings of the National Academy of Sciences 107 (18)8387-8392 945

Yu X Li B Fu Y Xie J Cheng J Ghabrial SA Li G Yi X Jiang D (2013) Extracellular 946 transmission of a DNA mycovirus and its use as a natural fungicide Proceedings of 947 the National Academy of Sciences 110 (4)1452-1457 948

Zhang JX Xue AG (2010) Biocontrol of sclerotinia stem rot (Sclerotinia sclerotiorum) of 949 soybean using novel Bacillus subtilis strain SB24 under control conditions Plant 950 Pathol 59 (2)382-391 951

Zhang Y (2004) Biocontrol of Sclerotinia stem rot of canola by bacterial antagonists and 952 study of biocontrol mechanisms involved University of Manitoba Manitoba Canada 953

Zhang Y Daayf F Fernando WGD Induced resistance against Sclerotinia in canola mediated 954

by bacterial biocontrol agents In International Joint Workshop on PR-Proteins and 955 Induced Resistance Copenhagen Denmark 2004 pp 8-9 956

Zhang Y Fernando W (2004a) Biological control of Sclerotinia sclerotiorum infection in 957 canola by Bacillus sp Phytopathology 9394 958

Zhang Y Fernando W (2004b) Presence of biosynthetic genes for phenazine-1-carboxylic 959

acid and 2 4-diacetylphloroglucinol and pyrrolnitrin in Pseudomonas chlororaphis 960 strain PA-23 Can J Plant Pathol 26430-431 961

Zhao J Meng J (2003) Genetic analysis of loci associated with partial resistance to 962 Sclerotinia sclerotiorum in rapeseed (Brassica napus L) Theor Appl Genet 106 963

(4)759-764 964

965

966

40

Figures 967

968

Fig 1 Typical symptoms of sclerotinia stem rot in canola (A) Initial lesion development on 969

stem (B-D) Gradual lesion expansion (E) Lesion covering the whole plant and causing plant 970

death (F) Sclerotia developed inside dead stem 971

972

973

974

975

976

977

978

979

980

981

982

983

41

984

Fig 2 Disease cycle of Sclerotinia sclerotiorum on canola 985

986

987

988

989

990

991

42

992

993

994

Fig 3 SEM observation demonstrated (A) vacuolated hyphae and (D) perforated sclerotial

rind cell of Sclerotinia sclerotiorum challenged by Bacillus cereus SC-1 Figure

produced from Kamal et al (unpublished data)995

43

Chapter 3 Research Article

MM Kamal MK Alam S Savocchia KD Lindbeck and GJ Ash (2015) Prevalence of

sclerotinia stem rot of mustard in northern Bangladesh International Journal of BioResearch

19(2) 13-19

Chapter 3 investigates the Sclerotinia spp isolated from the major mustard growing areas of

northern Bangladesh Surveys of this stem rot pathogen from Bangladesh provide information

which can be used to develop better management options for the disease This chapter

presents results of the occurrence of Sclerotinia spp both from petals and stems This chapter

published in the lsquoInternational Journal of BioResearchrsquo and describes the incidence of

sclerotinia stem rot disease and its potential threat to mustard production in Bangladesh

Intl J BioRes 19(2) 13-19 August 2015 Kamal et al

44

PREVALENCE OF SCLEROTINIA STEM ROT OF MUSTARD IN NORTHERN BANGLADESH

M M Kamal1 M K Alam2 S Savocchia1 K D Lindbeck3 and G J Ash1

1Graham Centre for Agricultural Innovation (an alliance between Charles Sturt University and NSW DPI) School of Agricultural and Wine Sciences Charles Sturt University Boorooma Street

Locked Bag 588 Wagga Wagga NSW 2678 Australia 2RARS Bangladesh Agricultural Research Institute Ishurdi Pabna Bangladesh 3NSW-Department of Primary Industries Wagga Wagga Agricultural Institute

Pine Gully Road Wagga Wagga NSW 2650 Corresponding authors email mkamalcsueduau

ABSTRACT

An intensive survey of mustard in eight northern districts of Bangladesh during 2013-14 revealed the occurrence of petal and stem infection by Sclerotinia sclerotiorum Inoculum was present in all districts and stem infection was widespread over the region Sclerotinia sclerotiorum was isolated from both symptomatic petals and stems whereas Sclerotinia minor was isolated for the first time only from symptomatic petals The incidence of petal infection ranged from 237 to 487 and stem infection ranged from 23 to 74 demonstrating that mustard is susceptible to stem rot disease however the incidence of disease is not dependent on the degree of petal infection To our knowledge this is the first report where both petal infection and the incidence of stem rot were rigorously surveyed on mustard crops in Bangladesh

Key words Sclerotinia Stem rot Mustard Bangladesh

INTRODUCTION

Mustard (Brassica juncea L) is the most important source of edible oil in Bangladesh occupying about 059 million hectares with an annual production of 053 million tonnes (DAE 2014) The crop is susceptible to a number of diseases Stem rot is generally caused by Sclerotinia sclerotiorum (Lib) de Bary a polyphagous necrotropic fungal plant pathogen with a reported host range of over 500 species from 75 families (Saharan and Mehta 2008 Boland and Hall 1994) During favourable environmental conditions sclerotia germinate and produce millions of air-borne ascospores These ascospores land on petals use the petals for nutrition and initiate stem infection (Saharan and Mehta 2008) Sclerotia are hard resting structures which form on the inside or outside of stems at the end of the cropping season The yield and quality of infected crops can decline gradually resulting in losses of millions of dollars annually (Purdy 1979)

Sclerotinia stem rot is the second most damaging disease of oilseed rape after blackleg worldwide with significant yield losses having been reported in China (Liu et al 1990) Europe (Sansford et al 1996) Australia (Hind-Lanoiselet and Lewington 2004) Canada (Turkington et al 1991) and United States (Bradley et al 2006) The disease has also been reported from India Pakistan Iran and Japan (Saharan and Mehta 2008) In India the disease is widespread particularly in mustard growing regions where incidence has been recorded up to 80 in some parts of Punjab and Haryana states (Ghasolia et al 2004) Being a neighboring country of India it would be expected that crops in Bangladesh are also vulnerable to stem rot Literature regarding the prevalence of the pathogen and incidence of Sclerotinia stem rot particularly in intensive mustard growing districts is limited in Bangladesh To our knowledge this is the first report of Sclerotinia minor in mustard petal and the first survey to determine the petal infestation and incidence of Sclerotinia stem rot of mustard in northern Bangladesh


45

METHODS AND MATERIALS

Petal infestation of mustard caused by ascospores of Sclerotinia spp was surveyed in eight districts of northern Bangladesh namely Panchagarh Gaibandha Rangpur Dinajpur Nilphamari Kurigram Thakurgaon and Lalmonirhat (Figure 1)

Figure 1 Diseased sample collection sites in the mustard growing districts of northern Bangladesh (Map from httpwikitravelorgenFileMap_of_Rangpur_Divisionpng)

These districts were located between 25deg50primeN and 89deg00primeE near the Himalayan Mountains The selected fields ranged from 01 to a maximum of 1 ha In total 200 fields were randomly selected which were from 2 to 10 km apart Sampling was conducted based on the petal testing protocol for S sclerotiorum (Morrall and Thomson 1991) An average of 20 healthy or asymptomatic petals from five peduncles with more than six open flowers were collected from five distantly located sites (300 m) in a field comprising 100 petals from each field and refrigerated at 4oC until assessment in the laboratory Within 48 hours of collection the petals were placed onto half strength potato dextrose agar (PDA Oxoid) amended with 100 ppm of streptomycin and ampicillin (Sigma-Aldrich) and incubated at room temperature After 5 days Sclerotinia species were morphologically identified and scored based on colony morphology hyphal characteristics and presence of oxalic acid crystals (Hind-Lanoiselet et al 2003) The average percentage of petal infestation was calculated for each field (Morrall and Thomson 1991)

Colonies of Sclerotinia spp arising from the petals were transferred to half strength PDA and incubated at 25oC until sclerotia had formed The species of Sclerotinia were identified by observing the size of sclerotia (Abawi and Grogan 1979) and confirmed by sequencing of internal transcribed spacers (ITS) of the ribosomal DNA with the Norgenprimes PlantFungi DNA Isolation Kit (Norgen Corporation) using the ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC) primers (White et al 1990) DNA samples were purified using a PCR Clean-up kit (Bioline) according to the manufacturerrsquos instructions The purified DNA fragments were sequenced (ICDDRB Bangladesh) and aligned using


46

MEGA v 62 (Tamura et al 2013) then species were identified through a comparative similarity search in GenBank using BLAST (Altschul et al 1997)

Pathogenicity tests were conducted in triplicate by inoculating seven healthy 50-day-old mustard plants cv BARI Sarisha11 Young 3-day-old mycelial plugs (5 mm diameter) of S sclerotiorum and S minor grown on PDA were excised from the margins of a culture and attached to the internodes of the main stem of mustard plants with Parafilmreg (Sigma-Aldrich) Control plants were exposed to non-inoculated plugs and incubated at 25oC and c150 microEm-2s-1 for seven days The incidence of stem rot was assessed before windrowing in the same fields where petal infection was determined The number of infected plants was determined by placing five quadrates (1 m2) in the same sites as where the petals were collected The incidence of stem rot was determined by investigating 100 plants per sample Stem rot symptoms were identified by observing the typical stem lesion (Rimmer and Buchwaldt 1995) and the disease incidence was calculated using the formula Disease incidence = (Mean number of diseased cotyledons or stems Mean number of all investigated cotyledon or stem) times 100

RESULTS AND DISCUSSION

Severe petal infestation and symptoms of stem infection were observed throughout northern Bangladesh Sclerotial morphology arising from petal tests demonstrated that the majority of isolates were S sclerotiorum (Lib) de Bary however S minor Jaggar was also present in a couple of samples (Figure 2)

Figure 2 Petal infection (left hand side) Sclerotinia sclerotiorum (middle) and Sclerotinia minor (right hand side) on frac12 strength potato dextrose agar at room temperature

The phylogenetic analysis of DNA sequences of two samples revealed that KM-1 was closely related to S sclerotiorum while KM-2 was identified as S minor (Figure 3)

Figure 3 Phylogenetic relationship of Sclerotinia isolates KM-1 KM-2 from mustard and closely related species based on neighbour-joining analysis of the ITS rDNA sequence data


47

The nucleotide sequences of KM-1 and KM-2 were submitted to GenBank as accession KP857998 and KP857999 respectively The isolates KM-1 and KM-2 were deposited in the Oilseed Research Centre of Bangladesh Agricultural Research Institute under the accession numbers ORC211455 and ORC211456 respectively

The pathogenicity test revealed that inoculated stems showed typical lesions of stem rot including the formation of white fluffy mycelium and tiny sclerotia on the stem surface No symptoms were observed on control plants Sclerotinia sclerotiorum and S minor was reisolated from infected plant tissue therefore fulfilling Kochs postulates S minor has previously been reported on canola in Australia (Hind-Lanoiselet et al 2001 Khangura and MacLeod 2013) and Argentina (Gaetan and Madia 2008) In Bangladesh S sclerotiorum has been reported on mustard (Hossain et al 2008) To our knowledge this is the first report of S minor on mustard in Bangladesh Although S sclerotiorum is the major causal agent of stem rot in mustardin Bangladesh S minor has the potential to also cause significant yield losses

Sclerotinia sclerotiorum is becoming an increasingly important pathogen which causes white mould disease throughout a range of host species including mustard The prevalence of Sclerotinia in mustard was observed in all surveyed areas of Bangladesh (Figure 4)

Figure 4 Typical symptoms of stem rot observed on mustard in the survey areas of Bangladesh

The incidence of petal infestation was high in Gaibandha (49) and Panchagarh (47) suggesting that sclerotinia stem rot is a major threat to mustard production in these districts (Figure 5) This result also suggests that Sclerotinia inoculum is widespread throughout the mustard growing areas of Bangladesh Previous anecdotal evidence suggested that sclerotinia stem rot caused by S sclerotiorum was present in Bangladesh however this is the first report of S minor being present Sclerotinia minor generally infects plants through mycelium as the production of ascospores is scarce (Abawi and Grogan 1979) However our investigation revealed that S minor may produce ascospores which infect the mustard petals Abundance in host range and growing other susceptible crops in crop rotation has provided Sclerotinia species with the opportunity to survive for long periods of time and therefore they can repeatedly infect available host species including mustard petals

Following petal testing 200 fields were revisited and the incidence of stem rot disease determined Typical stem rot symptoms were observed throughout the mustard field (Figure 4) Stem testing revealed the presence of S sclerotiorum only where S minor was absent in all samples The maximum stem rot incidence (7) was observed in Panchagarh while the number of infected stems was comparatively lower (4) in the highest petal infested district of Gaibandha (Figure 5) These outcomes suggest that the incidence of disease


48

Figure 5 Percent petal and stem infection by Sclerotinia spp in eight districts of Bangladesh

is not dependent on the degree of petal infestation The maximum incidence of stem rot disease in Panchagarh district may be due to its close vicinity to the Himalayan mountains where winter is comparatively prolonged and humidity is high Undesired rainfall (33 mm) was observed in this district at mid-flowering which made the environment favourable to Sclerotina infection Other districts were comparatively dry as no rainfall was recorded and the winter was shorter (BMD 2014) The wide scale of petal infestation might be due to a combination of relevant factors Substantial mustard plantings during consecutive years and tight mustard rotations have resulted in increased inoculum pressure In addition varietal susceptibility prolonged flowering flowering timed with spore dispersal and conducive environmental conditions have initiated petal infections and subsequent stem invasions

CONCLUSION

This is the first report where both petal infection and the incidence of stem rot were rigorously surveyed in Bangladesh To validate the survey results it will be necessary to continue surveying for at least the next two consecutive years However the results of this preliminary study may assist growers to identify the disease and apply appropriate management strategies In addition relevant government agencies may be able to show initiative in managing the problem before the disease becomes an epidemic The development of a reliable forecasting system by incorporating weather conditions and control measures is crucial for sustainable management of sclerotinia stem rot in mustard in Bangladesh

ACKNOWLEDGEMENTS

We thank all associated technical staff in Bangladesh Agricultural Research Institute for their time and patience in completing such a large survey within a short time period The research was funded by a Faculty of Science (Compact) scholarship Charles Sturt University Australia

REFERENCES

Abawi G and R Grogan 1979 Epidemiology of diseases caused by Sclerotinia species Phytopathology 69 899-904


49

Altschul S F T L Madden A A Schaumlffer J Zhang Z Zhang W Miller and D J Lipman1997 Gapped BLAST and PSI-BLAST a new generation of protein database search programs Nucleic Aci Res 25 3389-402

BMD (Bangladesh Meteorological Department) 2014 Met data online ministry of defence Govt of the Peoples Republic of Bangladesh Meteorological Complex Agargaon Dhaka-1207 httpwwwbmdgovbdp=Apply-for-Data Accessed on September 2014

Boland G and R Hall 1994 Index of plant hosts of Sclerotinia sclerotiorum Can J Plant Pathol 16 93-108

Bradley C R Henson P Porter D LeGare L Del Rio and S Khot 2006 Response of canola cultivars to Sclerotinia sclerotiorum in controlled and field environments Plant Dis 90 215-219

DAE (Department of Agriculture Extension) 2014 Agriculture Statistics Oil Crops A report published by information wing ministry of agriculture Govt of the Peoples Republic of Bangladesh P12

Gaetan S and M Madia 2008 Occurrence of sclerotinia stem rot on canola caused by Sclerotinia minor in Argentina Plant Dis 92 172

Ghasolia RP A Shivpuri and A K Bhargava 2004 Sclerotinia rot of Indian mustard (Brassica juncea) in Rajasthan Indian Phytopathol 57 76-79

Hind-Lanoiselet T G Ash and G Murray 2001 Sclerotinia minor on canola petals in New South Wales - a possible airborne mode of infection by ascospores Aust Plant Pathol 30 289-290

Hind-Lanoiselet T and F Lewington 2004 Canola concepts managing sclerotinia A farmnote published by NSW Department of Primary Industries Australia p4

Hind-Lanoiselet T G Ash and G Murray 2003 Prevalence of sclerotinia stem rot of canola in New South Wales Animal Prod Sci 43 163-168

Hossain M M Rahman M Islam and M Rahman 2008 White rot a new disease of mustard in Bangladesh Bangladesh J Plant pathol 24 81-82

Khangura R and W Macleod 2013 First Report of Stem Rot in Canola Caused by Sclerotinia minor in Western Australia Plant Dis 97 1660

Liu C D Du C Zou and Y Huang 1990 Initial studies on tolerance to Sclerotinia sclerotiorum (Lib) de Bary in Brassica napus L Proceedings of Symposium China Internayional Rapeseed Sciences Shanghai China p70-71

Morrall R and J Thomson 1991 Petal test manual for Sclerotinia in canola A manual published by University of Saskatchewan Canada p 45

Purdy L H 1979 Sclerotinia sclerotiorum history diseases and symptomatology host range geographic distribution and impact Phytopathology 69 875-880

Rimmer S and L Buchwaldt 1995 Diseases In Brassica oilseeds (Eds DS Kimber DI McGregor) CAB International Wallingford UK p111-140

Saharan GS and N Mehta 2008 Sclerotinia diseases of crop plants Biology ecology and disease management Springer Netherlands

Sansford C B Fitt P Gladders K Lockley and K Sutherland 1996 Oilseed rape disease development forecasting and yield loss relationships Home Grown Cereals Authority UK


50

Sharma S J L Yadav and G R Sharma 2001 Effect of various agronomic practices on the incidence of white rot of Indian mustard caused by Sclerotinia sclerotiorum J Mycol Plant Pathol 31 83-84

Tamura K D Peterson N Peterson G Stecher M Nei and S Kumar 2013 MEGA5 molecular evolutionary genetics analysis using maximum likelihood evolutionary distance and maximum parsimony methods Mol Biol Evol 28 2731-2739

Turkington T R Morrall and R Gugel 1991 Use of petal infestation to forecast Sclerotinia stem rot of canola Evaluation of early bloom sampling 1985-90 Can J Plant Pathol 13 50-59

White T J T Bruns S Lee and JTaylor 1990 Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics In Innis MA Gelfand DH Shinsky J White TJ eds PCR protocols a guide to methods and applications San Diego CA USA Academic Press pp315-322

51


M M Kamal K D Lindbeck S Savocchia and G J Ash 2015 Biological control of

sclerotinia stem rot of canola using antagonistic bacteria Plant Pathology 64 1375ndash1384

DOI 101111ppa12369

Chapter 4 investigates the isolation identification and antagonism of bacterial species

towards Sclerotinia sclerotiorum in the laboratory glass house and field This chapter

describes the discovery of potential biocontrol agents to their application in field conditions

By utilising the information from chapter 2 and 3 where Sclerotinia species were observed as

a potential threat for Brassica spp a detailed investigation was carried out to develop a

Bacillus-based biocontrol agent against sclerotinia stem rot disease of canola The results

presented in this chapter have opened the opportunity to combat sclerotinia stem rot of canola

through an eco-friendly approach This chapter has been published in Plant Pathology

journal

Biological control of sclerotinia stem rot of canola usingantagonistic bacteria

M M Kamal K D Lindbeck S Savocchia and G J Ash

Graham Centre for Agricultural Innovation (an alliance between Charles Sturt University and NSW DPI) School of Agricultural and Wine

Sciences Charles Sturt University Boorooma Street Locked Bag 588 Wagga Wagga NSW 2678 Australia

Stem rot caused by Sclerotinia sclerotiorum is a major fungal disease of canola worldwide In Australia the manage-

ment of stem rot relies primarily on strategic application of synthetic fungicides In an attempt to find alternative strate-

gies for the management of the disease 514 naturally occurring bacterial isolates were screened for antagonism to

S sclerotiorum Antifungal activity against mycelial growth of the fungus was exhibited by three isolates of bacteria

The bacteria were identified as Bacillus cereus (SC-1 and P-1) and Bacillus subtilis (W-67) via 16S rRNA sequencing

In vitro antagonism assays using these isolates resulted in significant inhibition of mycelial elongation and complete

inhibition of sclerotial germination by both non-volatile and volatile metabolites The antagonistic strains caused a sig-

nificant reduction in the viability of sclerotia when tested in a greenhouse pot trial with soil collected from the field

Spray treatments of bacterial strains reduced disease incidence and yielded higher control efficacy both on inoculated

cotyledons and stems Application of SC-1 and W-67 in the field at 10 flowering stage (growth stage 400) of canolademonstrated that control efficacy of SC-1 was significantly higher in all three trials (over 2 years) when sprayed twice

at 7-day intervals The greatest control of disease was observed with the fungicide Prosaro 420SC or with two appli-

cations of SC-1 The results demonstrated that in the light of environmental concerns and increasing cost of fungicides

B cereus SC-1 may have potential as a biological control agent of sclerotinia stem rot of canola in Australia

Keywords bacteria biocontrol canola Sclerotinia stem rot

Introduction

Stem rot is a major disease of canola (Brassica napus)and other oilseed rape caused by the ascomycete fungalpathogen Sclerotinia sclerotiorum globally (Zhao et al2004) Sclerotinia sclerotiorum infects more than 400plant species belonging to 75 families including manyeconomically important crops (Boland amp Hall 1994)Sclerotia of Sclerotinia spp have the capability ofremaining viable in the soil for several years (Merriman1976) Apothecia form through sclerotial germinationduring favourable environmental conditions and releaseascospores that can disseminate over several kilometresvia air currents (Sedun amp Brown 1987) The airborneascospores land on petals germinate using the petals as anutrient source and produce mycelia which subsequentlyinvade the canola stem (McLean 1958) Typical scleroti-nia stem rot symptoms first appear on leaf axils andinclude soft watery lesions or areas of very light browndiscolouration on the leaves main stems and branches2ndash3 weeks after infection Lesions expand turn to grey-ish white and may have faint concentric markings (Saha-

ran amp Mehta 2008) The stems of infected plantseventually become bleached and tend to shred and breakWhen the bleached stems of diseased plants are splitopen a white mouldy growth and sclerotia become evi-dent Sclerotinia stem rot is the second most damagingdisease on oilseed rape yield worldwide after black legcaused by Leptosphaeria maculansLeptosphaeria biglob-osa (Fernando et al 2004) Based on average historicaldata in Australia the annual losses are estimated to beAustralian $399 million if the current control measuresare not taken The cost of fungicide to control stem rotwas reported to be approximately Australian $35 perhectare (Murray amp Brennan 2012) Fungicide resistancein Australia has not yet been reported to Sclerotinia incanola The disease is mainly caused by S sclerotiorumbut Sclerotinia minor has also been implicated (Hindet al 2001) In surveys conducted in 1998 1999 and2000 in southeastern Australia levels of stem rot insome crops were found to exceed 30 in all years andthis was correlated to yield losses of 15ndash30 (Hindet al 2003) Estimates of losses due to Sclerotinia in1999 in New South Wales (NSW) alone exceeded Aus-tralian $170 million In 2012 and 2013 an increase ininoculum pressure was observed in high-rainfall zones ofNSW and an outbreak of stem rot in canola was alsoreported across the wheat belt region of Western Austra-lia (Khangura et al 2014)

E-mail mkamalcsueduau

Published online 24 March 2015

ordf 2015 British Society for Plant Pathology 1375

Plant Pathology (2015) 64 1375ndash1384 Doi 101111ppa12369

52

Several efforts have been made for sustainable man-agement of sclerotinia stem rot of canola including thesearch for resistant genotypes (Barbetti et al 2014) andagronomic management (Abd-Elgawad et al 2010)However completely resistant genotypes of canola arevery difficult to develop because the pathogen does notexhibit gene-for-gene specificity in its interaction withthe host (Li et al 2006) There are currently no resistantvarieties of Australian canola available and thereforemanagement of the disease relies solely on the strategicuse of fungicides combined with cultural managementpractices (Khangura amp MacLeod 2012) Chemical con-trol alone has been found to be comparatively less effec-tive because of the mismatch in spray timing andascospore releases (Bolton et al 2006) and many fungi-cides are gradually losing their efficacy due to theincreasing development of resistant strains (Zhang et al2003) Reduction in performance of fungicides over timeand the reduced costndashbenefit ratio of chemical controltogether with environmental concerns have led to thesearch for an alternative management strategy againstS sclerotiorum in canola Interest in biocontrol of Scle-rotinia has increased over the last few decades (Saharanamp Mehta 2008) A wide range of microorganisms recov-ered from the rhizosphere phyllosphere sclerotia andother habitats have been screened in the search forpotential biocontrol agents Several investigations havebeen conducted on the potential of fungal biocontrolagents against sclerotinia stem rot For example Conio-thyrium minitans has been extensively studied and regis-tered as a sclerotial mycoparasite in several countries(Whipps et al 2008) However there are few reportswhere antagonistic bacteria have been used successfully(Saharan amp Mehta 2008) Currently there are no indige-nous bacterial biological control agents commerciallyavailable for the control of sclerotinia diseases in Austra-lia In this study the selection identification and activityof three bacterial strains isolated in Australia for the bio-control of S sclerotiorum infecting canola is reported

Materials and methods

Isolation of S sclerotiorum and inoculum preparation

A single sclerotium of S sclerotiorum was isolated from a

severely diseased canola plant at the Charles Sturt University

experimental farm Wagga Wagga NSW Australia in 2012

The sclerotium was surface sterilized in 1 (vv) sodium hypo-chlorite for 2 min and 70 ethanol for 4 min followed by three

rinses in sterile distilled water for 2 min The clean sclerotium

was bisected and transferred to potato dextrose agar (PDA

Oxoid) amended with 10 g L1 peptone (Sigma Aldrich) in a90 mm diameter Petri dish and incubated in a growth chamber

at 22degC for 3 days A single mycelial plug arising from the scle-

rotium was cut from the culture and transferred to fresh PDAand incubated for 3 days Several mycelial discs of 5 mm in

diameter were cut from the culture and stored at 4degC in sterile

distilled water for further studies

A mycelial suspension was used to inoculate canola seedlings(Chen amp Wang 2005 Garg et al 2008) Ten agar plugs of

5 mm diameter were cut from the margin of actively growing

3-day-old colonies transferred to a 250 mL conical flaskcontaining sterile liquid medium (24 g L1 potato dextrose

broth with 10 g L1 peptone) and placed on a shaker at

130 rpm The inoculated medium was incubated at 22degC for

3 days Colonies of S sclerotiorum were harvested and rinsedthree times with sterile deionized water Prior to the inoculation

of plants the harvested mycelial mats were transferred to

150 mL of the liquid medium and homogenized with a blenderfor 1 min at medium speed The macerated mycelia were then

filtered using three layers of cheesecloth and suspended in the

same liquid medium Finally the mycelial concentration was

adjusted to 104 fragments mL1 based on haemocytometer(Superior) counts The pathogenicity of the Sclerotinia isolate

was tested through mycelia inoculation of wounded plants A

3-day-old PDA-grown 5 mm mycelial disc was placed into the

wounded stem of canola and wrapped with Parafilm M (SigmaAldrich) The whole plant was covered with polythene to retain

moisture After 5 days the lesion size was observed and the

pathogen reisolated from the canola stem onto half-strength

PDA to satisfy Kochrsquos postulates

Production of sclerotia

Baked bean agar (BBA) medium (200 g baked beans 10 g tech-

nical agar and 1 L distilled water) was used for producing large

numerous sclerotia from S sclerotiorum (Hind-Lanoiselet2006) A 5 mm diameter mycelial disc from a 3-day-old culture

of S sclerotiorum was used to inoculate the medium The cul-

tures were incubated in the dark at 20degC After 3 weeks fully

formed sclerotia were harvested air dried for 4 h at 25degC in alaminar flow cabinet and either preserved at room temperature

(to avoid conditioning in favour of carpogenic germination

Smith amp Boland 1989) or preserved at 4degC for longer termstorage Sclerotia preserved at room temperature were used in

all further experiments

Preparation of plant materials

The canola cultivar AV Garnet was used in all glasshouse exper-

iments Initially 140 mm diameter pots (Reko) were surfacesterilized with 6 sodium hypochlorite followed by three

washes in sterile distilled water Each pot was then filled with

pasteurized clay loam soil all-purpose potting mix (Osmocote)and vermiculite at a ratio of 211 by volume Ten seeds were

sown into each pot For cotyledon assessments the seedlings

were thinned to five plants per pot after 10 days and the cotyle-

dons were used for inoculationFor stem assessments plants were grown and maintained in a

glasshouse with temperatures of 15ndash20degC and a 16 h photope-

riod Prior to the application of antagonistic bacteria or inocula-

tion with S sclerotiorum plants were grown until 10 petalinitiation equivalent to growth stage 400 (Sylvester-Bradley

et al 1984) The plants were watered regularly and Thrive fer-

tilizer (Yates) was applied at 2-week intervals

Isolation and identification of antagonistic bacteria

Bacterial isolates (514) were collected from the phyllosphere of

canola plants from five locations [Belfrayden (35deg08000PrimeS147deg03000PrimeE) Coolamon (34deg50054PrimeS 147deg11004PrimeE) Winche-

don Vale (34deg45054PrimeS 147deg23004PrimeE) Illabo (34deg49000PrimeS147deg45000PrimeE) and Wagga Wagga (35deg07008PrimeS 147deg22008PrimeE)]

Plant Pathology (2015) 64 1375ndash1384

1376 M M Kamal et al

53

across southern NSW Australia Twenty healthy plants from

each location with more than 50 open flowers were used forisolation of bacteria The petals and young leaves of canola

plants were surface sterilized with 70 ethanol and placed into

50 mL Falcon conical centrifuge tubes (Fisher Scientific) contain-

ing 30 mL sterile distilled water To dislodge the bacteria eachsample was vortexed four times for 15 s followed by a 1 min

sonication Aliquots of 1 mL of the suspension were stored in

individual microfuge tubes Microfuge tubes containing 1 mL ofeach phyllosphere-derived suspension were incubated in a water

bath at 85degC for 15 min then allowed to cool to 40degC Using a

sterile glass rod 20 lL of the bacterial suspension was then

spread onto nutrient agar (NA Amyl Media) and incubated for24ndash48 h at room temperature Following incubation individual

colonies were inoculated into 96-well microtitre plates (Costar)

containing 200 lL nutrient broth (NB Amyl Media) and incu-

bated at 28degC for 24 h A 50 lL aliquot of each liquid culturewas mixed with an equal amount of 32 glycerol and preserved

at 80degCAdditionally sclerotia were collected from severely infected

canola plants then bacterial strains were isolated from these Asingle sclerotium was surface sterilized dried and placed into a

conical flask containing NB After shaking at 160 rpm for 24 h

at 28degC the broth was heated to 85degC for 15 min to kill unde-sired fungi and non-spore-forming bacteria An aliquot of 20 lLof broth was spread on to NA and incubated at 28degC for

3 days Single colonies were established by streaking the bacteria

onto NA for 72 h These isolates were also preserved at 80degCas described earlier

The active strains were identified by their 16S rRNA

sequences obtained by PCR amplification from purified genomic

DNA using the forward primer 8F (50-AGAGTTTGATCCTGGCTCAG-30) and reverse primer 1492R (50-GGTTACCTTGT-

TACGACTT-30) (Turner et al 1999 GeneWorks) An FTS-960

thermal sequencer (Corbett Research) was used for amplificationusing the one cycle at 95degC denaturation for 3 min then 35

cycles of 95degC for 15 s annealing temperature of 56degC for

1 min followed by a final extension step at 70degC for 8 min

Each DNA sample was purified using a PCR Clean-up kit (Axy-gen Biosciences) according to the manufacturerrsquos instructions

The purified DNA fragments were sequenced by the Australian

Genome Research Facility (AGRF Brisbane Queensland) All

sequences were aligned using MEGA v 62 (Tamura et al 2013)then species were identified through a comparative similarity

search of all publicly available GenBank entries using BLAST

(Altschul et al 1997)

Assessment of bacterial antagonism through diffusiblemetabolites

To determine the inhibition spectrum of antagonistic bacterialstrains the antifungal activity of each isolate was assessed on

PDA using a dual-culture technique Single bacterial beads from

Microbank were transferred into 3 mL NB and placed on a sha-

ker at 160 rpm at 28degC for 16 h until the mid-log phase ofgrowth The optical density of the broth culture was measured

at 600 nm using a Genesys 20 spectrophotometer (Thermo

Scientific) The bacterial suspension was adjusted to 108 CFU

mL1 which was further confirmed by dilution plating A10 lL aliquot of each 108 CFU mL1 bacterial suspension was

placed onto PDA at opposite edges of the Petri plate The plates

were sealed with Parafilm M and incubated at 28degC for 24 hMycelial plugs 5 mm in diameter from the edges of actively

growing S sclerotiorum cultures were placed in the centre of

each Petri plate and incubated at 28degC A 5 mm plug of S scle-rotiorum mycelium placed alone on PDA was used as a positivecontrol The activity of each bacterial isolate was evaluated by

measuring the diameter of the fungal colony or the zone of inhi-

bition after 7 days for both bacteria-treated and control plates

The experiment was repeated three times with five replicates ofpromising isolates To test the inhibition of sclerotial germina-

tion by bacterial isolates a similar set of experiments were con-

ducted using surface sterilized sclerotia that were dipped into108 CFU mL1 individual bacterial suspensions and placed onto

PDA prior to incubation at 28degC for 7 days The experiment

was repeated three times with five replicates The antagonistic

spectrum was calculated using the following formula

Inhibition () frac14 R1 R2

R1100

where R1 is the radial mycelial colony growth of S sclerotiorumon control plate and R2 is the radial mycelial colony growth of

S sclerotiorum on bacteria-treated plate

The three most active isolates that showed inhibition of

hyphal growth of S sclerotiorum were preserved at 80degC inMicrobank (Pro-Lab Diagnostics) according to the manufac-

turerrsquos instructions and used for further evaluation

Evaluation of bacterial antagonism via production ofvolatile compounds

The inhibition of S sclerotiorum growth via production of vola-

tile compounds by the three potential antagonistic bacterial iso-lates (SC-1 W-67 and P-1) was assessed using a divided plate

method (Fernando et al 2005) Petri plates split into two com-

partments (Sarstedt) were used with PDA on one side and NA

on the opposite side For each bacterial isolate 24-h-old culturesgrowing in NB were streaked onto the NA side and incubated

for 24 h A 5 mm actively growing mycelial plug of S sclerotio-rum was placed onto the PDA side and the plate was immedi-

ately wrapped in Parafilm M to trap the volatiles The tightlysealed plates were incubated at 25degC Although mycelium

reached the edge of plate within 4 days the radial growth of

mycelium was measured after 10 days to discriminate betweenthe antagonistic capability against mycelia growth and the sus-

tainability of suppression A Petri plate containing only mycelial

discs was used as a control Furthermore a similar experiment

was conducted to evaluate the inhibition of sclerotial germina-tion by bacterial volatiles The individual bacterial isolate was

streaked onto the NA side kept for 24 h prior to placing a sur-

face sterilized sclerotium onto the PDA side and then incubated

at 25degC Myceliogenic germination was observed after 10 daysthen all challenged hyphal plugs and sclerotia were transferred

onto new PDA plates in the absence of the bacteria to assess

viability The experiment was conducted with five replicationsand the trial was repeated three times

Antagonistic effects of bacteria on sclerotia in soil

Twenty uniform sclerotia (9 9 5 mm 40ndash50 mg each) grown

on BBA were dipped into individual antagonistic bacterial sus-

pensions (108 CFU mL1) and placed into nylon mesh bags(5 cm2) with 15 mm2 holes in the mesh Pots containing clay

loam field soil were watered to 80 field capacity prior to bury-

ing the nylon bags containing the sclerotia under 2 cm of soil

The pots were maintained at 20degC in the glasshouse for 30 daysthen the sclerotia were recovered counted and washed in sterile


Biocontrol of Sclerotinia in canola 1377

54

distilled water All sclerotia were surface sterilized (20 per

batch) by soaking for 3 min in 4 NaOCl then 70 ethanoland kept at room temperature for 24 h to allow dissipation of

remaining contaminants The surface sterilization process was

repeated once Individual air-dried sclerotia were bisected and

plated onto PDA amended with 50 mg L1 penicillin and100 mg L1 streptomycin sulphate (Sigma Aldrich) The plated

sclerotia were placed in an incubator at 25degC under a 12 h12 h

lightdark regime until the growth of mycelia was observed

sclerotia that produced mycelia were considered as viable sclero-

tia The percentage of germinating sclerotia was recorded The

experiment was established as a randomized complete block

design with five replications and repeated three times Sclerotialviability was assessed using the following formula

Viability () frac14 R1 R2

R1100

where R1 is the no of myceliogenic germinated sclerotia of

S sclerotiorum in control treated with sterilized water and R2

is the no of myceliogenic germinated sclerotia of S sclerotio-rum treated with bacteria

Cotyledon bioassay

Bacterial antagonism against S sclerotiorum on 10-day-old

canola (cv AV Garnet) cotyledons was conducted in a glass-

house Individual bacterial cell suspensions (108 CFU mL1)of SC-1 W-67 and P-1 were sprayed on three cotyledon-stage

seedlings using an inverted handheld sprayer (Hills) and

plants were then covered with transparent polythene to retain

moisture As the efficiency of control depends on the inocula-tion time of the bacterial strains and of the pathogen inocu-

lum (Gao et al 2014) all bacteria were inoculated onto

seedlings 24 h before inoculation with S sclerotiorum After24 h macerated mycelial fragments (104 fragments mL1)

were applied using a similar sprayer The mycelial suspensions

were agitated prior to inoculation to maintain a homogenous

concentration Cotyledons inoculated only with mycelial frag-ments of S sclerotiorum were used as controls Misting was

conducted over the cotyledons at 8-h intervals to create and

maintain a relative humidity of 100 The seedlings were

placed in a growth chamber and maintained at low lightintensity of c 15 lE m2 s1 for 1 day then moved to c150 lE m2 s1 in a glasshouse The temperatures were

adjusted to 1915degC (daynight) The disease incidence andseverity was assessed from five seedlingspot (two cotyledon

seedlings) at 4 days post-inoculation (dpi) where 0 = no

lesion 1 = lt10 cotyledon area covered by lesion 2 = 11ndash30 cotyledon area covered by lesion 3 = 31ndash50 cotyledonarea covered by lesion and 4 = gt51 cotyledon area covered

by lesion (Zhou amp Luo 1994) The experiment was con-

ducted in a randomized complete block design with five repli-cates and repeated three times Disease incidence disease

index and control efficiency was calculated according to the

following formulae

Disease incidence()frac14 Meanno diseased cotyledons

Meanno all investigated cotyledons100

Whole plant bioassay

The inoculation of whole canola (cv AV Garnet) plants was car-

ried out in a glasshouse at growth stage 400 The three antagonis-tic bacterial suspensions (108 CFU mL1) were amended with005 Tween 20 (Sigma Aldrich) and sprayed until run-off with a

handheld sprayer (Hills) at a rate of 5 mL per plant The plants

were covered with transparent moistened polythene bags andincubated for 24 h A homogenized mixture of mycelial fragments

(104 fragments mL1) of S sclerotiorum was sprayed at a rate of

5 mL per plant The plants were covered again and kept for 24 h

within a polythene bag to retain moisture The experiments wereconducted in a controlled environment with 16 h photoperiod at

20degC darkness for 8 h at 15degC and 100 relative humidity

Plants treated with the mycelial suspensions and Tween 20 were

used as positive controls and plants sprayed with water andTween 20 were used as negative controls The experiment was

established as a randomized complete block design with 10 repli-

cates and repeated three times Data on disease incidence andseverity was taken from 10 plants for individual treatments and

recorded at 10 dpi The percentage of disease incidence was calcu-

lated by observing disease symptoms and disease severity was

recorded using a 0ndash9 scale where 0 = no lesion 1 = lt5 stemarea covered by lesion 3 = 6ndash15 stem area covered by lesion

5 = 16ndash30 stem area covered by lesion 7 = 31ndash50 stem area

covered by lesion and 9 = gt50 stem area covered by lesion

(Yang et al 2009) Disease incidence disease index and controlefficacy on stems were calculated as above for the cotyledon

bioassay where cotyledons are replaced by stems in the formulae

Disease assessment in field

During the 2013 and 2014 seasons three experimental canolacv AV Garnet field sites (2013 one site 2014 two sites) were

selected based on the known natural high inoculum incidence

documented in the previous year Petal tests were performed to

confirm the presence of adequate inoculum An average of 20healthy or symptomless petals from five peduncles with more

than six open flowers were collected from five locations within

each trial comprising 100 petals and were refrigerated at 4degCuntil assessment in the laboratory Within 48 h of collectionthe petals were placed onto half-strength PDA (Oxoid)

Disease index () frac14XNo diseased cotyledons in each indexDisease severity index

Total no cotyledons investigatedHighest disease index100

Control efficacy () frac14 Disease index of controlDisease index of treated group

Disease index of control100



55

amended with 100 mg L1 streptomycin and 250 mg L1

ampicillin (Sigma Aldrich) and incubated at room temperatureAfter 5 days Sclerotinia spp were identified and scored based

on colony morphology hyphal characteristics and presence of

oxalic acid crystals (Hind et al 2003) The average percentage

of petal infestation was calculated for each field (Morrall ampThomson 1991) Colonies of Sclerotinia spp arising from the

petals were transferred to fresh half-strength PDA and incu-

bated at 25degC until sclerotia had formed The species of Scle-rotinia were identified by observing the size of sclerotia (Abawi

amp Grogan 1979) The field sites (10 9 50 m) were located at

the NSW Department of Primary Industries and Charles Sturt

University (Wagga Wagga NSW Australia) The seed rate of5 kg ha1 was used in each 9 9 2 m2 plot to achieve a plant

population of 50ndash70 plants m2 and guard rows were estab-

lished to limit cross contamination of pathogen inoculum The

experiments were established as a randomized complete blockdesign using six treatments with four replicates per treatment

The treatments consisted of spraying bacteria (108 CFU mL1)

or a registered fungicide Prosaro 420 SC (210 g L1 prot-

hioconazole + 210 g L1 tebuconazole 70 L water ha1 BayerCropScience) as follows (i) a single application of SC-1 (ii)

two applications of SC-1 at 7-day intervals (iii) a single appli-

cation of W-67 (iv) an application of SC-1 and W-67 mixedtogether (v) a single application of Prosaro 420 SC and (vi)

untreated control (sprayed with water) Prior to spraying the

bacteria 01 Tween 20 was added as surfactant to the bacte-

rial suspensions All initial treatments were applied at growthstage 400 by using a pressurized handheld sprayer (Hills)

Standard agronomic practices for the management of canola

were conducted when required The disease incidence was

recorded by random sampling of 200 plants per plot 4 weeksafter each final spray Disease severity was assessed using the

same scale as described for the whole plant bioassay In addi-

tion calculations of disease incidence disease index and con-trol efficacy were conducted using the same formulae as used

in the glasshouse assessment

Data analysis

For every data set analysis of variance (ANOVA) was conducted

using GENSTAT (16th edition VSN International) Arcsine trans-formation was carried out for all percentage data prior to statis-

tical analysis As there was no significant difference observed in

any of the repeated experiments the data were combined to test

the differences among treatments using Fisherrsquos least significantdifferences (LSD) at P = 005

Results


Bacteria (514 samples) were isolated from canola leavespetals stems and sclerotia of S sclerotiorum The dual-culture test demonstrated strong antagonistic ability ofthree isolates SC-1 W-67 and P-1 that produced thegreatest inhibition zones against mycelial growth ofS sclerotiorum SC-1 was isolated from sclerotia ofS sclerotiorum whereas W-67 and P-1 were collectedfrom canola petals These bacteria were identified to spe-cies level by sequence analysis of the 16S rRNA geneThe phylogenetic analysis showed that SC-1 and P-1 areclosely related to Bacillus cereus while W-67 was identi-fied as Bacillus subtilis (Fig 1) The sequence similaritywas gt98 according to sequences available at NCBI

Effect of diffusible metabolites on mycelial growth andsclerotial germination in vitro

Three bacterial isolates SC-1 W-67 and P-1 stronglyinhibited the mycelial growth of S sclerotiorum A clearinhibition zone was observed between bacterial coloniesand fungal mycelium (Fig 2a) These strains exhibitedhigh levels of antagonism in repeated tests and signifi-cantly reduced the radial mycelial growth in vitro themean hyphal growth ranged from 154 to 29 mm com-pared with 85 mm for the control with the highest meaninhibition (82) recorded for SC-1 (Table 1) The abilityof the three isolates to inhibit the germination of sclero-tia was also demonstrated (Fig 2c) only bacterialgrowth was observed around sclerotia and the growth ofmycelia from sclerotia was completely inhibited at 7 dpiby all three bacterial isolates tested whereas the myceliacontinued to grow in the controls

Bacillus subtilis (GU258545) Bacillus W-67

Bacillus vallismortis (AB021198) Bacillus tequilensis (HQ223107) Bacillus mojavensis (AB021191)

Bacillus atrophaeus (AB021181) Bacillus sonorensis (AF302118) Bacillus aerius (AJ831843)

Bacillus safensis (AF234854) Bacillus altitudinis (AJ831842)

Bacillus P-1 Bacillus cereus (KJ524513) Bacillus SC-1

Pseudomonas fluorescens (FJ605510)82100

100

100100

97

98

9769

72

43

0middot01

Figure 1 Phylogenetic tree of Bacillus

isolates SC-1 W-67 and P-1 and closely

related species based on 16S rRNA gene

sequences retrieved from NCBI following

BLAST analysis Bootstrap values derived from

1000 replicates are indicated as

percentages on branches



56

Inhibition of mycelial growth and sclerotialgermination in vitro by volatile substances

Isolates SC-1 W-67 and P-1 all produced volatilesubstances that reduced mycelial growth and sclerotialgermination of S sclerotiorum in vitro Each signifi-cantly reduced the mycelial growth where the meanmycelial growth ranged from 33 to 148 mm com-

pared with 85 mm in the control (Table 1 Fig 2b)and completely inhibited the germination of sclero-tia (Fig 2d) The inhibition capability among thestrains varied significantly (P = 005) SC-1 consis-tently showed the greatest inhibition (962) Bothmycelial plugs and sclerotia from plates treatedwith bacteria failed to grow when plated onto freshPDA

(a)

(b)

(c)

(d)

(e)

(f)

Figure 2 Antagonism assessments of Bacillus isolates SC-1 W-67 and P-1 against Sclerotinia sclerotiorum using dual-culture technique by (a)

diffusible non-volatile metabolites at 7 days post-inoculation (dpi) and (b) volatile compounds at 10 dpi (c) Inhibition of sclerotial germination by

dipping the sclerotia into 108 CFU mL1 bacteria at 10 dpi (d) Inhibition of sclerotial germination by bacterial volatile compounds at 10 dpi

Evaluation of bacterial antagonism on (e) cotyledons at 4 dpi and (f) stems at 10 dpi



57

Bacterial antagonism of sclerotial recovery and viabilityin soil

The recovery and viability of sclerotia treated with bacte-rial strains showed significant (P = 005) differencescompared to the control Sclerotia were dipped into abacterial concentration of 108 CFU mL1 and placed inpotted field soil After 30 days 825ndash945 sclerotiatreated with bacteria were recovered but viability ofthese sclerotia was reduced to lt30 in comparison with88 of the control sclerotia (Table 1) The smallestnumber of recovered (825) and viable (125) sclero-tia was observed when the sclerotia were treated withSC-1 (Table 1)

Biocontrol efficacy in glasshouse

Canola cotyledons treated with antagonistic bacterialstrains were shown to have significantly (P = 005) lowerdisease incidence and disease index than the control(Table 2 Fig 2e) The efficacy of control ranged from789 to 879 The typical symptoms of infection withS sclerotiorum began 24 h post-inoculation (hpi) in con-trol plants A steady rate of disease development wasobserved over 4 days Pretreatment with SC-1 24 h priorto inoculation with S sclerotiorum reduced disease inci-dence to 132 compared to 100 for the control(Table 2) Similarly canola stems inoculated with antag-onistic bacteria 24 h before inoculation with S sclerotio-rum showed a significantly (P = 005) lower rate ofdisease incidence (376ndash472) and disease index (242ndash338) than untreated controls Control plants showedsymptoms of infection (Fig 2f) with S sclerotiorum by24 hpi with a dramatic rise in disease incidence with

further incubation reaching 100 by 10 dpi SC-1 pro-vided a control efficacy of 736 which was signifi-cantly higher than that of W-67 and P-1 The presenceof S sclerotiorum was confirmed in tissue samples withsymptoms by plating onto PDA

Evaluation of biocontrol by antagonistic bacteria in thefield

The three field trials conducted in 2013 and 2014 dem-onstrated that all of the bacterial applications at growthstage 400 significantly reduced the disease incidence ofsclerotinia stem rot and were not significantly differentto the disease incidence in the treatment using the syn-thetic fungicide Prosaro 420 SC (Table 3) with theexception of a single application of W-67 in trial 3 Interms of disease index two applications of SC-1 at 7-dayintervals significantly improved control efficacy in all tri-als (782 802 and 709) when compared to asingle application A mixed application of SC-1 and W-67 also demonstrated higher control efficacy in all trials(768 754 and 694) in comparison to a singleindividual application (Table 3) of either strainAlthough Prosaro 420 SC was more efficient in control-ling the disease in all trials (846 813 and 737)than the single application of each bacterial strain thedifferences were not significant in comparison to twoapplications of SC-1 at 7-day intervals

Discussion

The aim of the study was to identify bacterial strainsfor the management of S sclerotiorum under field con-ditions The controlled environment assays were used

Table 2 Biocontrol of Sclerotinia sclerotiorum on canola cotyledons and stems by antagonistic Bacillus sp isolates (108 CFU mL1) in the

glasshouse

Isolate

Disease incidence () Disease index () Control efficacy ()

Cotyledon Stem Cotyledon Stem Cotyledon Stem

SC-1 132 a 376 a 116 a 242 a 879 b 736 b

W-67 184 b 424 b 173 b 298 b 819 a 675 a

P-1 218 b 472 b 202 b 338 b 789 a 633 a

Control 1000 c 1000 c 956 c 916 c ndash ndash

Values in columns followed by same letter were not significantly different according to Fisherrsquos protected LSD test (P = 005)

Table 1 Effect of Bacillus strains on Sclerotinia sclerotiorum mycelial growth in vitro and sclerotial viability in soil

Strain

Mycelial colony diameter (mm) Inhibition () Sclerotia ()

Diffusible metabolites Volatile compounds Diffusible metabolites Volatile compounds Recovered Viable

SC-1 154 a 33 a 819 c 962 c 825 a 125 a

W-67 228 b 93 b 732 b 894 b 930 b 240 b

P-1 290 c 148 c 659 a 828 a 945 b 280 b

Control 850 d 850 d ndash ndash 1000 c 880 c

Values in columns followed by the same letters were not significantly different according to Fisherrsquos protected LSD test (P = 005)



58

to evaluate the potential of these strains associated withthe canola phyllosphere and sclerotia for the manage-ment of S sclerotiorum in controlled and natural fieldconditions A total of 514 isolates were evaluated usinga dual-culture assay to find strains with the ability toinhibit hyphal growth of S sclerotiorum The screeningof antagonistic bacteria against sclerotinia stem rotusing a similar strategy has been adopted in severalstudies (Chen et al 2014 Gao et al 2014 Wu et al2014) However screening of such a large number ofisolates using direct dual-culture is a time-consumingtask In the current study two isolates of B cereus(SC-1 and P-1) and one of B subtilis (W-67) wereselected after rigorous investigation in vitro and inplanta SC-1 and W-67 were also tested under fieldconditions Among the three isolates B cereus SC-1performed with the greatest efficiency under controlledand natural field conditions Previous studies have iden-tified members of this genus as potential biocontrolagents against S sclerotiorum (Saharan amp Mehta2008)The mechanism of in vitro mycelial growth suppres-

sion and inhibition of sclerotial germination is consideredas antibiosis indicating that diffusible inhibitory antibi-otic metabolites produced by bacteria may be involvedin creating adverse conditions for the pathogen Strainsof B cereus and B subtilis reduced hyphal elongationin vitro and minimized sclerotinia stem rot disease inci-dence in sunflower (Zazzerini 1987) Members of theBacillus genus produce a wide range of bioactive lipo-peptide-type compounds that suppress plant pathogensthrough antibiosis (Leclere et al 2005 Stein 2005Chen et al 2009 Leon et al 2009) SC-1 W-67 and P-1 might produce antifungal antibiotics that result in sup-pression and inhibition of mycelial growth and sclerotialgermination by inducing chemical toxicity A previousstudy revealed that strains of Bacillus amyloliquefaciensshowed antibiosis against sclerotia-forming phytopatho-genic fungi by releasing protease-resistant and thermosta-ble chemicals in vitro (Prıncipe et al 2007) Morerecently the endophytic bacterium B subtilis strainsEDR4 and Em7 were reported to have a broad antifun-gal spectrum against several fungal species includingmycelial growth of Sclerotinia sclerotiorum through leak-age and disintegration of hyphal cytoplasm (Chen et al

2014 Gao et al 2014) EDR4 significantly inhibitedsclerotial germination (728) However in the currentstudy the mode of action of the bacterial strains was notinvestigatedBacterial organic volatiles have the potential to influ-

ence the growth of several plant pathogens (Alstreuroom2001 Wheatley 2002) In the current study the resultsof the divided plate assay clearly demonstrated that vola-tile substances produced by the bacteria were alsoresponsible for suppression of mycelial growth andrestricted sclerotial germination These volatiles may con-tain chemical compounds that affect hyphal growth andinhibit sclerotial germination even under highly favour-able conditions A similar study revealed that Pseudomo-nas spp completely inhibited mycelial growth ascosporegermination and destroyed sclerotial viability of S scle-rotiorum by consistently emitting 23 types of volatiles(Fernando et al 2005) Several volatile compounds pro-duced by B amyloliquefaciens NJN-6 also showed com-plete inhibition of growth and spore germination ofFusarium oxysporum fsp cubense (Yuan et al 2012)Although volatile compounds have received less attentionthan diffusible metabolites the current study revealedthat they can induce similar or greater effects against thegrowth of S sclerotiorum Future research will be direc-ted towards the strategic use of antifungal volatile-pro-ducing bacterial strains to minimize soilborne plantpathogens including S sclerotiorumFor sustainable management of S sclerotiorum the

regulation of sclerotial germination is a major strategy asit could prevent the formation of apothecia and mycelio-genic germination In the current study a significantreduction of sclerotial recovery was observed in compari-son to the control treatments In addition the bacterialisolate SC-1 demonstrated its ability to reduce the sclero-tial viability below 13 in the soil This indicates thatthe colonizing bacteria have the potential to disintegrateor kill the overwintering sclerotia in the soil Duncanet al (2006) reported that viability of sclerotia dependson time burial depth and bacterial population The para-sitism of sclerotia with biocontrol agents is a crucialmethod for inhibiting the germination of sclerotia anderadicating sclerotinia diseases (Saharan amp Mehta2008) The incorporation and presence of strong antago-nistic strains such as SC-1 in soil amendments may be an

Table 3 Control of sclerotinia stem rot of canola in the field following the application of antagonistic bacterial strains (108 CFU mL1) or Prosaro 420

SC fungicide at growth stage 400 in Wagga Wagga NSW Australia during 2013 (trial 1) and 2014 (trials 2 and 3)

Treatment


Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3

SC-1 (91) 65 a 78 a 93 a 55 b 79 b 123 b 538 b 623 b 540 b

SC-1 (92 at 7-day intervals) 70 a 65 a 73 a 26 a 41 a 78 a 782 c 802 c 709 c

W-67 (91) 75 a 95 a 133 b 92 c 141 c 182 c 227 a 324 a 321 a

SC-1 + W-67 (91) 58 a 75 a 85 a 28 a 51 a 82 a 768 c 754 c 694 c

Prosaro 420 SC (91) 55 a 58 a 68 a 18 a 39 a 70 a 846 c 813 c 737 c

Water (91) 200 b 225 b 298 c 119 d 209 d 268 d ndash ndash ndash




59

efficient strategy to break the disease life cycle by killingoverwintering sclerotia in soilIn the controlled glasshouse experiments the disease

control efficiency by all strains in both the cotyledonand stem study produced similar results The bacterialstrains were applied 24 h before pathogen inoculationbecause bacteria might not be able to inhibit the elonga-tion of mycelia upon establishment of infection byS sclerotiorum as reported by Fernando et al (2004)The results confirmed the in vitro assessment that thebacterial strains tested possess various degrees of antago-nism In the glasshouse SC-1 yielded the best suppres-sion over 10 days when applied 24 h before inoculationof S sclerotiorum Therefore the dispersible antifungalmetabolites and volatiles may be key weapons in protect-ing canola from S sclerotiorum Future research in thisarea may shed light on understanding the mechanism ofbiocontrolUnder natural field conditions the majority of inocu-

lum is ascosporic and a small number of germinatingsclerotia can significantly increase disease severity(Davies 1986) Application of a biocontrol agent oncanola petals is extremely important as senescing petalsare commonly infected by ascospores (Turkington ampMorrall 1993) Previous studies showed that youngerrapeseed petals are more susceptible to infection by as-cospores compared to the older flowers (Inglis amp Boland1990) Therefore the main aim of the current researchwas the protection of canola petals from early infectionof ascospores using a biocontrol agent in the field In thisstudy all field trials showed that SC-1 produced morethan 70 control efficiency against sclerotinia stem rotof canola in a naturally infested field Despite the variouslevels of S sclerotiorum infection among trials thestrains of Bacillus reduced disease incidence compared tothe controls Of the bacterial treatments tested twoapplications of B cereus SC-1 at 7-day intervals gave thehighest control efficacy (802 in trial 2) which con-firms the results obtained from the glasshouse studies Arelevant investigation on phyllosphere biological controlof Sclerotinia on canola petals using antagonistic bacteriahas been reported previously in which Pseudomonaschlororaphis (PA-23) B amyloliquefaciens (BS6) Pseu-domonas sp (DF41) and B amyloliquefaciens (E16) pro-vided biocontrol activity against ascospores on canolapetals in in vitro and field conditions (Fernando et al2007) Another study revealed that Erwinia spp andBacillus spp inhibit Sclerotinia in vitro and in vivo andreduce sclerotinia rot of bean (Tu 1997) Comparativelylower control efficiency was observed at the trial 3 sitewhich was located in a highly favourable environmentfor disease development The incidence of sclerotiniastem rot depends on environmental factors (Saharan ampMehta 2008) and stage of infection (Chen et al 2014)which may have contributed to the reduction in controlefficacy in this trial Although the commercial fungicideProsaro provided the highest control efficacy in all trialsthere was no significant difference in disease incidencebetween treatment with a single application of Prosaro

or with two applications of SC-1 at 7-day intervals Asingle application of SC-1 gave lower levels of controlthan two applications indicating that the survival of bac-teria on canola requires further research Hence theselected bacterial strains gave significant protection incontrolled conditions but field performance varied signifi-cantly among strains Thus the viability longevity andmultiplication capability of bacteria under natural condi-tions in heavily infested fields also requires further inves-tigation In addition rhizosphere competence soiltreatment as well as the plant growth promotion capabil-ity of SC-1 under various cropping systems should beexploredFinally the present study successfully showed that

native biocontrol strain B cereus SC-1 can effectivelysuppress sclerotinia stem rot disease of canola both in vi-tro and in vivo in Australia Further evaluation of thisstrain against sclerotinia diseases of other high valuecrops such as lettuce drop demands investigation Bacil-lus cereus SC-1 appears to have multiple modes of actionagainst S sclerotiorum and therefore the potential bio-control mechanisms should be explored further Com-mercialization of B cereus SC-1 upon development of asuitable formulation would enhance the armoury for thesustainable management of sclerotinia stem rot disease ofcanola in Australia

Acknowledgements

This study was funded by a Faculty of Science (Compact)Scholarship Charles Sturt University Australia Theauthors also thank the plant pathology team at theDepartment of Primary Industries NSW for their assis-tance with field trials

References

Abawi GS Grogan RG 1979 Epidemiology of plant diseases caused by

Sclerotinia species Phytopathology 69 899

Abd-Elgawad M El-Mougy N El-Gamal N Abdel-Kader M Mohamed

M 2010 Protective treatments against soilborne pathogens in citrus

orchards Journal of Plant Protection Research 50 477ndash84

Alstreuroom S 2001 Characteristics of bacteria from oilseed rape in relation

to their biocontrol activity against Verticillium dahliae Journal of

Phytopathology 149 57ndash64

Altschul SF Madden TL Scheuroaffer AA et al 1997 Gapped BLAST and PSI-

BLAST a new generation of protein database search programs Nucleic

Acids Research 25 3389ndash402

Barbetti MJ Banga SK Fu TD et al 2014 Comparative genotype

reactions to Sclerotinia sclerotiorum within breeding populations of

Brassica napus and B juncea from India and China Euphytica 197

47ndash59

Boland GJ Hall R 1994 Index of plant hosts of Sclerotinia

sclerotiorum Canadian Journal of Plant Pathology 16 93ndash108

Bolton MD Thomma BPHJ Nelson BD 2006 Sclerotinia sclerotiorum

(Lib) de Bary biology and molecular traits of a cosmopolitan

pathogen Molecular Plant Pathology 7 1ndash16

Chen Y Wang D 2005 Two convenient methods to evaluate soybean

for resistance to Sclerotinia sclerotiorum Plant Disease 89 1268ndash72

Chen XH Koumoutsi A Scholz R et al 2009 Genome analysis of

Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol

of plant pathogens Journal of Biotechnology 140 27ndash37



60

Chen Y Gao X Chen Y Qin H Huang L Han Q 2014 Inhibitory

efficacy of endophytic Bacillus subtilis EDR4 against Sclerotinia

sclerotiorum on rapeseed Biological Control 78 67ndash76

Davies JML 1986 Diseases of oilseed rape In Scarisbrick DH Daniels

RW eds Oilseed Rape London UK Collins 195ndash236

Duncan RW Fernando WGD Rashid KY 2006 Time and burial depth

influencing the viability and bacterial colonization of sclerotia of

Sclerotinia sclerotiorum Soil Biology and Biochemistry 38 275ndash84

Fernando WGD Nakkeeran S Zhang Y 2004 Ecofriendly methods in

combating Sclerotinia sclerotiorum (Lib) de Bary Recent Research in

Developmental and Environmental Biology 1 329ndash47

Fernando WGD Ramarathnam R Krishnamoorthy AS Savchuk SC

2005 Identification and use of potential bacterial organic antifungal

volatiles in biocontrol Soil Biology and Biochemistry 37 955ndash64

Fernando WGD Nakkeeran S Zhang Y Savchuk S 2007 Biological

control of Sclerotinia sclerotiorum (Lib) de Bary by Pseudomonas and

Bacillus species on canola petals Crop Protection 26 100ndash7

Gao X Han Q Chen Y Qin H Huang L Kang Z 2014 Biological

control of oilseed rape Sclerotinia stem rot by Bacillus subtilis strain

Em7 Biocontrol Science and Technology 24 39ndash52

Garg H Sivasithamparam K Banga SS Barbetti MJ 2008 Cotyledon

assay as a rapid and reliable method of screening for resistance against

Sclerotinia sclerotiorum in Brassica napus genotypes Australasian

Plant Pathology 37 106ndash11

Hind TL Ash GJ Murray GM 2001 Sclerotinia minor on canola petals

in New South Wales ndash a possible airborne mode of infection by

ascospores Australasian Plant Pathology 30 289ndash90

Hind TL Ash GJ Murray GM 2003 Prevalence of sclerotinia stem rot

of canola in New South Wales Australian Journal of Experimental

Agriculture 43 163ndash8

Hind-Lanoiselet TL 2006 Forecasting Sclerotinia stem rot in Australia

Wagga Wagga NSW Australia Charles Sturt University PhD thesis

Inglis GD Boland GJ 1990 The microflora of bean and rapeseed petals

and the influence of the microflora of bean petals on white mold

Canadian Journal of Plant Pathology 12 129ndash34

Khangura R MacLeod WJ 2012 Managing the risk of Sclerotinia stem

rot in canola Farm note 546 Western Australia Department of

Agriculture and Food [httparchiveagricwagovauPC_95264html]

Accessed 27 July 2014

Khangura R Van Burgel A Salam M Aberra M MacLeod WJ 2014

Why Sclerotinia was so bad in 2013 Understanding the disease and

management options [httpwwwgiwaorgaupdfs2014

Presented_PapersKhangura20et20al20presentation20paper

20CU201420-DRpdf] Accessed 28 July 2014

Leclere V Bechet M Adam A et al 2005 Mycosubtilin overproduction

by Bacillus subtilis BBG100 enhances the organismrsquos antagonistic and

biocontrol activities Applied and Environmental Microbiology 71

4577ndash84

Leon M Yaryura PM Montecchia MS et al 2009 Antifungal activity

of selected indigenous Pseudomonas and Bacillus from the soybean

rhizosphere International Journal of Microbiology 2009 572049

Li CX Li H Sivasithamparam K et al 2006 Expression of field

resistance under Western Australian conditions to Sclerotinia

sclerotiorum in Chinese and Australian Brassica napus and Brassica

juncea germplasm and its relation with stem diameter Australian

Journal of Agricultural Research 57 1131ndash5

McLean DM 1958 Role of dead flower parts in infection of certain

crucifers by Sclerotinia sclerotiorum (Lib) de Bary Plant Disease

Reporter 42 663ndash6

Merriman PR 1976 Survival of sclerotia of Sclerotinia sclerotiorum in

soil Soil Biology and Biochemistry 8 385ndash9

Morrall RAA Thomson JR 1991 Petal Test Manual for Sclerotinia in

Canola Saskatoon SK Canada University of Saskatchewan

Murray GM Brennan JP 2012 The Current and Potential Costs from

Diseases of Oilseed Crops in Australia Kingston ACT Australia

Grains Research amp Development Corporation

Prıncipe A Alvarez F Castro MG et al 2007 Biocontrol and PGPR

features in native strains isolated from saline soils of Argentina

Current Microbiology 55 314ndash22

Saharan GS Mehta N 2008 Sclerotinia Diseases of Crop Plants

Biology Dordrecht Netherlands Springer Ecology and Disease

Management

Sedun FS Brown JF 1987 Infection of sunflower leaves by ascospores of

Sclerotinia sclerotiorum Annals of Applied Biology 110 275ndash84

Smith EA Boland GJ 1989 A reliable method for the production and

maintenance of germinated sclerotia of Sclerotinia sclerotiorum


Stein T 2005 Bacillus subtilis antibiotics structures syntheses and

specific functions Molecular Microbiology 56 845ndash57

Sylvester-Bradley R Makepeace RJ Broad H 1984 A code for stages of

development in oilseed rape (Brassica napus L) Aspects of Applied

Biology 6 399ndash419

Tamura K Stecher G Peterson D Peterson N Filipski A Kumar S

2013 MEGA5 molecular evolutionary genetics analysis version 60

Molecular Biology and Evolution 30 2725ndash9

Tu JC 1997 An integrated control of white mold (Sclerotinia

sclerotiorum) of beans with emphasis on recent advances in biological

control Botanical Bulletin of Academia Sinica 38 73ndash6

Turkington TK Morrall RAA 1993 Use of petal infestation to

forecast sclerotinia stem rot of canola the influence of inoculum

variation over the flowering period and canopy density


Turner S Pryer KM Miao VPW Palmer JD 1999 Investigating deep

phylogenetic relationships among cyanobacteria and plastids by small

subunit rRNA sequence analysis Journal of Eukaryotic Microbiology

46 327ndash38

Wheatley RE 2002 The consequences of volatile organic compound

mediated bacterial and fungal interactions Antonie van Leeuwenhoek

81 357ndash64

Whipps JM Sreenivasaprasad S Muthumeenakshi S Rogers CW

Challen MP 2008 Use of Coniothyrium minitans as a biocontrol

agent and some molecular aspects of sclerotial mycoparasitism

European Journal of Plant Pathology 121 323ndash30

Wu Y Yuan J Raza W Shen Q Huang Q 2014 Biocontrol traits and

antagonistic potential of Bacillus amyloliquefaciens strain NJZJSB3

against Sclerotinia sclerotiorum a causal agent of canola stem rot

Journal of Microbiology and Biotechnology 24 1327ndash36

Yang D Wang B Wang J Chen Y Zhou M 2009 Activity and efficacy

of Bacillus subtilis strain NJ-18 against rice sheath blight and

Sclerotinia stem rot of rape Biological Control 51 61ndash5

Yuan J Raza W Shen Q Huang Q 2012 Antifungal activity of Bacillus

amyloliquefaciens NJN-6 volatile compounds against Fusarium

oxysporum f sp cubense Applied and Environmental Microbiology

78 5942ndash4

Zazzerini A 1987 Antagonistic effect of Bacillus spp on Sclerotinia

sclerotiorum sclerotia Phytopathologia Mediterranea 26 185ndash7

Zhang X Sun X Zhang G 2003 Preliminary report on the monitoring

of the resistance of Sclerotinia libertinia to carbendazim and its

internal management Journal of Pesticide Science Adminstration 24

18ndash22

Zhao J Peltier AJ Meng J Osborn TC Grau CR 2004 Evaluation of

sclerotinia stem rot resistance in oilseed Brassica napus using a petiole

inoculation technique under greenhouse conditions Plant Disease 88

1033ndash9

Zhou B Luo Q 1994 Rapeseed Diseases and Control Beijing China

China Commerce Publishing Co



61


MM Kamal KD Lindbeck S Savocchia and GJ Ash (2015) Bacterial biocontrol of

sclerotinia diseases in Australia Acta Horticulturae (In press)

Chapter 5 investigates the potential antagonism of bacterial isolate Bacillus cereus SC-1

towards sclerotinia lettuce drop in the laboratory and glasshouse This chapter describes the

efficacy of strain SC-1 against sclerotinia lettuce drop of lettuce a model high value crop

and to evaluate its capability in various cropping systems By using the information from

chapter 4 in vitro and glasshouse investigations were carried out to assess antagonism growth

promotion and rhizosphere competence of this bacterium The results obtained from this

chapter have opened the opportunity to apply the strain SC-1 for the management of

Sclerotinia in a freshly consumed crop such as lettuce This chapter has been accepted for

publication in the Acta Horticulturae journal

62

63


Mohd Mostofa Kamal Kurt Lindbeck Sandra Savocchia and Gavin Ash

Graham Centre for Agricultural Innovation (an alliance between Charles Sturt University

and NSW DPI) School of Agricultural and Wine Sciences Charles Sturt University

Boorooma Street Locked Bag 588 Wagga Wagga NSW 2678 Australia

Keywords Biological control Sclerotinia Lettuce drop Bacillus cereus SC-1

Abstract

Among the Sclerotinia diseases in Australia lettuce drop was considered as a

model in this study which is caused by the fungal pathogens Sclerotinia minor and

Sclerotinia sclerotiorum and posed a major threat to lettuce production in Australia

The management of this disease with synthetic fungicides is strategic and the

presence of fungicide residues in the consumable parts of lettuce is a continuing

concern to human health To address this challenge Bacillus cereus SC-1 was chosen

from a previous study for biological control of S sclerotiorum induced lettuce drop

Soil drenching with B cereus SC-1 applied at 1x108 CFU mL

-1 in a glasshouse trial

completely restricted the pathogen and no disease incidence was observed (P=005)

Sclerotial colonization was tested and found 6-8 log CFU per sclerotia of B cereus

resulted in reduction of sclerotial viability to 158 compared to the control

(P=005) Volatile organic compounds (VOC) produced by the bacteria increased

root length shoot length and seedling fresh weight of the lettuce seedlings by 466

354 and 32 respectively as compared with the control (P=005) In addition to

VOC induced growth promotion B cereus SC-1 was able to enhance lettuce growth resulting in increased root length shoot length head weight and biomass weight by

348 215 194 and 248 respectively compared with untreated control

plants (P=005) The bacteria were able to survive in the rhizosphere of lettuce

plants for up to 30 days reaching populations of 7 log CFU g-1

of root adhering soil

These results indicate that biological control of lettuce drop with B cereus SC-1

could be feasible used alone or integrated into IPM and best management practice

programs for sustainable management of lettuce drop and other sclerotinia diseases

INTRODUCTION

Sclerotinia diseases mainly caused by the fungal pathogens Sclerotinia

sclerotiorum and Sclerotinia minor are a major threat to a wide range of horticultural and

agricultural crops worldwide (Saharan and Mehta 2008) In Australia lettuce drop

caused by either S minor or S sclerotiorum can be particularly devastating to lettuce

(Lactuca sativa) production causing significant yield losses (20-70) (Villalta and Pung

2010) The symptoms of this disease from soilborne sclerotia appears as water soaked rot

on the root which progress towards the stem leaves and heads resulting in wilt (Subbarao

1998) When airborne ascospores of S sclerotiorum initiate infection watery pale brown

to grey brown lesions appear on exposed parts resulting in a mushy soft rot because of

severe tissue degradation The recalcitrant black hard sclerotia are produced on leaves

beneath the soil entire crown and tap root which then function as survival propagules for

infection of subsequent lettuce crops (Hao et al 2003) Efforts have been made to search

for resistant genotypes to sclerotinia lettuce drop however no resistant commercial lettuce

cultivars have yet been developed (Hayes et al 2010) In Australia the disease is

managed by a limited number of fungicides predominantly Sumisclex (500 gL-1

64

procymidone Sumitomo Australia) and Filan (500 gkg Boscalid Nufarm Australia) but

Sumisclex has been withdrawn due to long residual effect (Villalta and Pung 2005)

Fungicides recommended have been reported to be gradually losing their effectiveness

over time (Porter et al 2002) Furthermore the presence of fungicide residues in the

consumable parts of produce is a continuing concern to human health (Fernando et al 2004) Although several investigations have been conducted on potential of fungal

biocontrol agents against sclerotinia lettuce drop there are limited reports where

antagonistic bacteria have been used successfully (Saharan and Mehta 2008) To address

this challenge the present study was conducted in invitro and inplanta using antagonistic

bacterium Bacillus cereus SC-1 retrieved from a previous study As there are no native


lettuce drop in Australia the aim of this study was to evaluate the potential of B cereus

SC-1 for sustainable lettuce drop management and to examine capabilities for plant

growth promotion

MATERIALS AND METHODS

Production of sclerotia Sclerotia were produced and maintained according to Kamal et al (2014) A 5mm

in diameter mycelial disc from a 3-day-old culture of S sclerotorium was used to

inoculate the baked bean agar media The isolate was incubated in the dark at 20oC After

3 weeks fully formed sclerotia were harvested air dried for 4 hours at 25oC in a laminar

flow and preserved at 4oC

for use in all further experiments

Lettuce growth and incorporation of sclerotia in soil

The lettuce cultivar lsquoIcebergrsquo was used in all glasshouse experiments Seeds were

sown in trays containing seedling raising mix (Osmocote Australia) and transplanted 2

weeks after emergence into 140 mm pots containing pasteurized all purpose potting mix

(Osmocote Australia) and vermiculite at a ratio of 211 by volume The soil was

inoculated with 30 sclerotia of S sclerotiorum by placing them into the top 3 cm of soil

before transplanting the seedlings After 7 days the seedlings were thinned to a single

plant per pot The plants were maintained in a glasshouse at 15 to 20oC with a 16 hour

photoperiod The plants were watered regularly and lsquoThriversquo fertiliser (Yates Australia)

was applied at fortnightly intervals

Evaluation of biocontrol potential

The bacterial strain Bacillus cereus SC-1 previously known to be antagonist of S

sclerotiorum in canola (Kamal et al 2014) was cultured in Tryptic Soy Broth (TSB) and

placed on a shaker (150 rpm) at 28degC for 24 hours The culture was centrifuged at 3000

rpm for 10 minutes The supernatant was discarded and pellet was resuspended in sterile

distilled water The concentration was adjusted to 1x108 CFU mL

-1 then 50 mL of

suspension was evenly poured into each pot immediately before transplanting the lettuce

seedlings The control pots received the same amount of water without bacteria Incidence

and severity of lettuce drop was recorded every seven days The experiment was

established as a randomized complete block design (RCBD) with 10 replicates

Effect of bacterial volatiles on growth of lettuce seedlings

Lettuce seedlings were exposed to volatile organic compounds (VOC) of B cereus

SC-1 as described by Minerdi et al (2009) with minor modification Half strength

Murashige and Skoog (MS) salt medium (Sigma-Aldrich USA) amended with 1 agar

65

and sucrose was placed into the bottom of sterile screw cap containers (Sarstedt

Australia) The lids were filled with Tryptic Soy Agar (TSA) medium and streaked with

overnight grown bacterial suspension of 1x104 CFU mL

-1 then incubated at 28

oC for 24

hours Five 2-day-old lettuce seedlings were transferred to the MS medium of each

container before the bacteria inoculated lids were placed on top sealed with Parafilmreg

(Sigma-Aldrich USA) and incubated at 20oC under cool fluorescent light A similar

procedure was followed using a non-VOC producing isolate B cereus NS and lids

without bacteria were considered as controls After 7 days root and shoot length and

seedling fresh weight were measured The experiment was carried out in a completely

randomized design with ten replicates for each treatment

Assessment of plant growth promotion in the glasshouse

Before transplanting roots of lettuce seedlings were drenched with a cell

suspension of B cereus SC-1 at 1times108 CFU mL

-1 In the control the roots of lettuce

seedlings were similarly drenched with an equal volume of sterile water At maturity

whole lettuce plants were collected and root length shoot length and head weight

measured This experiment was established as a randomized complete block design with

10 replicates per treatment

Production of rifampicin resistant strains

Rifampicin resistant strains were generated on nutrient agar amended with

rifampicin (Sigma USA) according to West et al (2010) for reisolation of inoculated

bacteria from soil and sclerotia B cereus SC-1 was cultured on nutrient broth at 28oC for

24 hours then 100 microL of bacterial suspension was spread onto nutrient agar (NA)

amended with 1 ppm rifampicin and incubated at 28oC in the dark After 2 days the

mutant strain was re-cultured onto nutrient agar amended with 5 ppm rifampicin and

incubated for 2 days then subcultured sequentially with increased concentration of

rifampicin to a maximum of 100 ppm The individual single colony of the mutant was re-

streaked onto NA amended with 100 ppm rifampicin to check for resistance The young

growing rifampicin mutant of B cereus SC-1 was then isolated and preserved in

Microbank (Prolab diagnostic USA) at -800C for further study

Evaluation of rhizosphere and sclerotial colonization

The root adhering rhizosphere soil was collected from four individual SC-1 (1x108

CFU mL-1

) treated lettuce plants at each of the seven time points 1 5 10 20 30 50 and 70 days after inoculation with bacterial suspensions The soil samples were thoroughly

mixed then 1 g of soil was resuspended in 9 mL of sterile distilled water and vortexed for

5 min to prepare a homogenous suspension The soil sample was serially diluted and

plated onto NA amended with 100 mgL-1

of rifampcin After incubation at 28degC for 24 hours the concentration of B cereus SC-1 was measured and the colony forming unit

(CFU) determined per gram of soil (Duncan et al 2006 Maron et al 2006) B cereus

SC-1 in rhizosphere was confirmed by 16S rRNA squencing

Assessment of sclerotial viability and recovery of bacteria

Sclerotia were recovered 7 weeks after transplantation and assayed for viability of

sclerotia and survivability of colonized bacteria The sclerotia were surface sterilised by

soaking in 40 NaOCl for one minute and 70 ethanol for three minutes and kept at

room temperature for 24 hours to allow germination of remaining undesired

contaminants The surface sterilisation process was repeated once and the sclerotia air-

66

dried in the laminar air flow for 8 hours Sclerotia were cut into two pieces and plated

onto potato dextrose agar amended with 50 microlL-1

penicillin and streptomycin sulphate

The plated sclerotia were incubated at 25ordmC under a 1212 h lightdark regime until

myceliogenic germination Mycelial elongation was considered as an indicator of viability

and the percentage of germinating sclerotia recorded To recover bacteria the remaining

halves of the sclerotia were sonicated for 30 seconds in sterile distilled water All viable

and non viable sclerotia were analysed individually The suspension was vortexed for 2

minutes serially diluted and plated onto half strength NA amended with 100 ppm

Rifampicin After 3 days incubation at 28oC bacterial colonies were counted on plates

and the CFU mL-1

determined (Duncan et al 2006 Maron et al 2006) Data on mean

colony number per sclerotia was analysed The experiment was established as a RCBD

with 10 replicates

Statistical analysis

The data were subjected to analysis of variance (ANOVA) using GenStat (16th

edition VSN international UK) The differences among treatments were tested using

Fisherrsquos least significant differences (LSD) at P=005


Biocontrol efficacy under glasshouse conditions

Glasshouse experiments were conducted to assess the potential of B cereus SC-1

strain against lettuce drop Soil drenched with a bacterial suspension of 1x108 CFU mL

-1

completely inhibited (100) and entirely protected lettuce plants from S sclerotiorum

while control plants treated with sclerotia alone showed 100 disease incidence (Fig 1)

Upon sclerotial parasitisation the bacteria might be produced dispersible antifungal

metabolites and volatiles to inhibit sclerotial germination Several members of the

Bacillus genus have been reported to suppress plant pathogens including B cereus UW85

against damping-off disease of alfalfa (Handelsman et al 1990) Suppression of southern

corn leaf blight and growth promotion of maize was also observed by an induced

systemic resistance eliciting rhizobacterium B cereus C1L (Huang et al 2010) Black leg

disease of canola caused by Leptosphaeria maculans was controlled by B cereus DEF4

through antibiosis and induced systemic resistance (Ramarathnam et al 2011) In

addition with the aforementioned report our investigation also demonstrates an agreement

with the results of El-Tarabily et al (2000) who reported the use of chitinolytic

bacterium and actinomycetes reduced basal drop disease of lettuce significantly compare

to control However in this study the mode of action of bacterial isolates was not

investigated but demands to be explored in future research

The viability of sclerotia treated with bacteria showed significant differences

compared to control (P=005) Seven weeks after the soil drenching application of B

cereus SC-1 the number of viable sclerotia was significantly decreased to below 2 in

comparison with the control For sustainable management of sclerotinia lettuce drop the

inhibition of sclerotial germination is a core strategy to prevent myceliogenic

germination The parasitisation of sclerotia with biocontrol agents may be the most

effective method to kill sclerotia and eradicate disease The present investigation

demonstrated that B cereus SC-1 could inhibit sclerotial germination and reduce viability

below 2 compared to the control (100) (Data not shown) Duncan et al (2006)

reported that antagonistic bacteria might have the potential to kill or disintegrate

overwintering sclerotia by parasitisation This observation indicates that bacteria could be

67

used as soil amendments to break the lifecycle of the pathogen by killing overwintering

sclerotia in soil However the viability longevity and multiplication capability of bacteria

under natural conditions in heavily infested fields requires further investigation

Plant growth promotion by B cereus SC-1 volatiles An in vitro system was adopted to investigate the influence of B cereus SC-1

mediated volatiles on growth promotion in lettuce Seven days after seedling emergence

a significant increase in root (466) and shoot (354) length and seedling fresh weight

(32) was observed as compared with the control (Fig 2) Volatile organic compounds

(VOC) emitted from several rhizobacterial species have triggered plant growth promotion

(Farag et al 2006) Our observation concurs with Ryu et al (2003) who demonstrated

that rhizobacteria use volatiles to communicate with plants and promote plant growth

They can also protect against pathogen invasion by activating induced systemic resistance

in Arabidopsis (Ryu et al 2004) Serratia marcescens NBRI 1213 (Lavania et al 2006)

and Achromobacter xylosoxidans Ax10 (Ma et al 2009) were shown to promote plant

growth by increasing root length shoot length fresh and dry weight of target plants

Growth promoting ability of B cereus SC-1 under glasshouse conditions

B cereus SC-1 was assessed for its ability to promote lettuce growth in a

glasshouse At 7 weeks post inoculation of bacteria B cereus SC-1 treated plants

exhibited a significant increase in root length shoot length head weight and biomass

weight by 348 215 194 and 248 respectively compared with untreated control

plants (Table 1) The result of this study is consistent with previous report where

rhizobacteria were found to promote plant growth by producing phytohormones nitrogen

fixation phosphate solubilisation and antagonism (Choudhary and Johri 2009) The

growth promotion of lettuce in this study might be attributed to production of metabolites

by bacteria that trigger certain metabolic activity and enhanced plant growth

Rhizosphere and sclerotial colonization of bacteria

The rhizosphere and sclerotial colonization of lettuce plant by B cereus SC-1 was

investigated The density of B cereus SC-1 in rhizosphere was 6 to 7 log CFU g-1

of soil

while bacterial population on sclerotia ranged from 6 to 8 log CFU mL-1

over seven time

points 0 1 5 10 20 30 50 and 70 days after treatment (Fig 3) The maximum

colonization was observed at 30 days post treatment in both rhizosphere and sclerotia then

a slow reduction in bacterial density was observed Several rhizobacterial species promote

plant growth and confer protection from pathogens by rhizosphere colonization

(Lugtenberg and Kamilova 2009) Our rhizosphere competence results showed that

during lettuce growth the bacterial population reached a noteworthy level indicated the

bacteria as a good colonist of lettuce rhizosphere The population dynamics of B cereus

SC-1 on sclerotia was found higher than rhizosphere has proved again its better capability

of colonization Previous studies have shown B cereus to activate induced systemic

resistance enhance plant growth by colonizing lily (Liu et al 2008) and maize (Huang et

al 2010) roots

CONCLUSION

The present study was undertaken to determine the feasibility of bacterial

biological control in Australia where management of S sclerotiorum induced lettuce drop

was considered as model system The antagonistic bacterial isolate B cereus SC-1 was

selected from a previous study where the bacteria were demonstrated as having multiple

68

modes of action and successfully reducing the disease incidence of sclerotinia stem rot of

canola both in glasshouse and field The results of this study suggest that B cereus SC-1

is a promising biocontrol agent and its judicious application might reduce lettuce drop

disease incidence under glasshouse conditions In summary there increased demands for

vegetable crops that are pesticide-free especially those that are freshly consumed B

cereus SC-1 might be a substitute for fungicides or a component of integrated disease

management program in controlling Sclerotinia lettuce drop and other horticultural and

agricultural crops

ACKNOWLEDGEMENTS

The study was supported by a Faculty of Science (Compact) scholarship Charles

Sturt University Australia

Literature Cited

Choudhary D K And Johri BN 2009 Interactions of Bacillus spp and plants-With

special reference to induced systemic resistance (ISR) Mic res 164(5) 493-513

Duncan RW Fernando WGD and Rashid KY 2006 Time and burial depth

influencing the viability and bacterial colonization of sclerotia of Sclerotinia

sclerotiorum Soil Biol Bioc 38 275-284

El‐Tarabily K Soliman M Nassar A Al‐Hassani H Sivasithamparam K and

Mckenna F 2000 Biological control of Sclerotinia minor using a chitinolytic

bacterium and actinomycetes P Path 49 573-583

Farag MA Ryu CM Sumner LW and Pareacute PW 2006 GC-MS SPME profiling of

rhizobacterial volatiles reveals prospective inducers of growth promotion and induced

systemic resistance in plants Phytochem 67 2262-2268

Fernando WGD Nakkeeran S and Zhang Y 2004 Ecofriendly methods in combating

Sclerotinia sclerotiorum (Lib) de Bary Rec Res Dev Env Biol 1 329-347

Handelsman J Raffel S Mester EH Wunderlich L and CR Grau 1990 Biological

control of damping-off of alfalfa seedlings with Bacillus cereus UW85 Appl Env

Mic 56 713-718

Hao J Subbarao KV and Koike ST 2003 Effects of broccoli rotation on lettuce drop

caused by Sclerotinia minor and on the population density of sclerotia in soil P Dis

87 159-166

Hayes RJ Wu BM Pryor BM Chitrampalam P and Subbarao KV 2010

Assessment of resistance in lettuce (Lactuca sativa L) to mycelial and ascospore

infection by Sclerotinia minor Jagger and S sclerotiorum (Lib) de Bary Hort Sci

45 333-341

Huang CJ Yang KH Liu YH Lin YJ and Chen CY 2010 Suppression of

southern corn leaf blight by a plant growth promoting rhizobacterium Bacillus cereus

C1L Ann Appl Biol 157 45-53

Kamal MM Lindbeck K Savocchia S and Ash G 2014 Biological control of

sclerotinia stem rot of canola (caused by Sclerotinia sclerotiorum) using bacteria Aus

P Path (in press)

Lavania M Chauhan PS Chauhan S Singh HB and Nautiyal CS 2006 Induction

of plant defense enzymes and phenolics by treatment with plant growth-promoting

rhizobacteria Serratia marcescens NBRI1213 Cur Mic 52 363-368

Liu YH Huang CJ and Chen CY 2008 Evidence of induced systemic resistance

against Botrytis elliptica in lily Phytopathol 98 830-836

69

Lugtenberg B and Kamilova F 2009 Plant-growth-promoting rhizobacteria Ann Rev

mic 63 541-556

Ma Y Rajkumar M and Freitas H 2009 Inoculation of plant growth promoting

bacterium Achromobacter xylosoxidans strain Ax10 for the improvement of copper

phytoextraction by Brassica juncea J Env Man 90 831-837

Maron PA Schimann H Ranjard L Brothier E Domenach AM and Lensi R

2006 Evaluation of quantitative and qualitative recovery of bacterial communities

from different soil types by density gradient centrifugation Eu J S Bio 42 65-73

Minerdi D Bossi S Gullino ML and Garibaldi A 2009 Volatile organic

compounds a potential direct long distance mechanism for antagonistic action of

Fusarium oxysporum strain MSA 35 Env Mic 11 844-854

Porter I Pung H Villalta O Crnov R and Stewart A 2002 Development of

biological controls for Sclerotinia diseases of horticultural crops in Australasia 2nd

Australasian Lettuce Industry Conference 5-8 May Gatton Campus University of

Queensland Australia

Ramarathnam R Fernando WD and Kievit T de 2011 The role of antibiosis and

induced systemic resistance mediated by strains of Pseudomonas chlororaphis

Bacillus cereus and B amyloliquefaciens in controlling blackleg disease of canola

Bio Con 56 225-235

Ryu CM Farag MA Hu CH Reddy MS Wei HX and Pareacute PW 2003

Bacterial volatiles promote growth in Arabidopsis Pro Nat Aca Sci 100 4927-

4932

Ryu CM Farag MA Hu CH Reddy MS Kloepper JW and Pareacute PW 2004

Bacterial volatiles induce systemic resistance in Arabidopsis P Phy 134 1017-1026

Saharan GS and Mehta N 2008 Sclerotinia diseases of crop plants Biology ecology

and disease management Springer Nethelands

Subbarao KV 1998 Progress toward integrated management of lettuce drop P Dis 82

1068-1078

Villalta O and Pung H 2005 Control of Sclerotinia diseases VEGE notes Hort Aus

Primary Industries Research Victoria

Villalta O and Pung H 2010 Managing Sclerotinia Diseases in Vegetables (Report on

new management strategies for lettuce drop and white mould of beans) Department of

primary industries Victoria

West E Cother E Steel C and Ash G 2010 The characterization and diversity of

bacterial endophytes of grapevine Can J Mic 56 209-216

Table

Table 1 Growth promotion of lettuce plant treated with 1x108 CFU mL

-1 of Bacillus

cereus SC-1 cell suspension in compare to control

Treatment Root length

(cm)

Shoot length

(cm)

Head weight

(g)

Biomass

increase ()

B cereus SC-1 155 plusmn 08 a 176 plusmn 09 a 711 plusmn 37 a 248

Control 101 plusmn 10 b 138 plusmn 10 b 573 plusmn 51 b -

Representative data obtained from the means of 10 replicated plants per treatment

Different letters indicate statistically significant differences according to Fisherrsquos

protected LSD test (P=005)

70

Figures

Fig 1 Soil drenching application of Bacillus cereus SC-1 compltely inhibited the

Sclerotinia infection (top line) while 100 disease incidence was obseved in control

plants (bottom line)

Fig 2 Exposure of lettuce plant to volatiles released from Bacillus cereus SC-1 at 7 days

post inoculation (A) root and shoot length (B) seedling fresh weight Represented data

obtained and combined from three consecutive trial with 10 replicates (Plt005)

Fig 3 Rhizosphere colonization of Bacillus cereus SC-1 in the (A) rhizospheric soil and

(B) Sclerotia of lettuce plant over time Data represents the mean numbers of CFU of

bacteria per gram of rhizosphere soil and sclerotia from three repetative experiments with

standard error

BAA

A B

71


MM Kamal S Savocchia KD Lindbeck and GJ Ash (2015) Elucidating the



Chapter 6 investigates the mechanism of antagonism conferred by Bacillus cereus strain SC-

1 against S sclerotiorum The presence of lipopeptide antibiotics and hydrolytic enzymes in

the genome of B cereus SC-1 was evaluated Cell free culture filtrates of the bacterium were

evaluated in vitro and in the glasshouse to observe the efficacy of metabolites to control S

sclerotiorum Scanning and transmission electron microscopy were employed to further

investigate the effect of bacteria on morphology and cytology of hyphae and sclerotia of S

sclerotiorum The results obtained from this chapter have provided a better understanding of

the mode of action of B cereus SC-1 which should assist in developing sustainable

management practices for crop diseases caused by Sclerotinia spp This chapter has been

presented according to the formatting requirements for the journal of ldquoBioControlrdquo

72

Elucidating the mechanisms of biocontrol of Bacillus cereus SC-1 against Sclerotinia 1

sclerotiorum 2

Mohd Mostofa Kamal1 Sandra Savocchia

1 Kurt D Lindbeck

2 and Gavin J Ash

13


1Graham Centre for Agricultural Innovation (an alliance between Charles Sturt University 5



2New South Wales Department of Primary Industries Wagga Wagga Agricultural Institute 8


10

ABSTRACT 11

The mechanism by which Bacillus cereus SC-1 inhibits the germination of sclerotia of 12

Sclerotinia sclerotiorum (Lib) de Bary was observed to be through antibiosis Cell-free 13

culture filtrates of B cereus SC-1 significantly inhibited the growth of S sclerotiorum with 14

degradation of sclerotial melanin and reduced lesion development on cotyledons of canola 15

Scanning electron microscopy of the zone of interaction of B cereus SC-1 and S 16

sclerotiorum demonstrated vacuolization of hyphae with loss of cytoplasm The rind layer of 17

treated sclerotia was completely ruptured and extensive colonisation by bacteria was 18

observed Transmission electron microscopy revealed the disintegration of the cell content of 19

fungal mycelium and sclerotia PCR amplification using gene specific primers revealed the 20

presence of four lipopeptide antibiotic (bacillomycin D iturin A surfactin and fengycin) and 21

two mycolytic enzyme (chitinase and β-13-glucanase) biosynthetic operons in B cereus SC-22

1 suggesting that antibiotics and enzymes may play a role in inhibiting multiple life stages of 23

S sclerotiorum24

25

KEYWORDS Biocontrol Mechanisms Bacillus cereus Sclerotinia sclerotiorum 26

73

INTRODUCTION 27

Sclerotinia sclerotiorum (Lib) de Bary commonly known as the white mould pathogen is a 28

devastating necrotrophic and cosmopolitan phytopathogen with a reported host range of more 29

than 500 plant species (Saharan and Mehta 2008) Sclerotinia species are estimated to cause 30

annual losses of US$200 million in the USA (Bolton et al 2006) whereas in Australia they 31

can cause annual losses of up to AUD$10 million in canola production (Murray and Brennan 32

2012) The typical symptom of sclerotinia stem rot appears on leaf axils as soft watery 33

lesions Two to three weeks after infection the lesions expand and the main stems and 34

branches turn greyish white and eventually become bleached Upon splitting the bleached 35

stems of diseased plants mycelia and sclerotia are often visible (Fernando et al 2004) The 36

development of canola varieties resistant to S sclerotiorum is extremely difficult because the 37

trait is governed by multiple genes (Fuller et al 1984) Therefore management of the disease 38

often relies on the strategic application of fungicides (Mueller et al 2002) Chemical 39

fungicides have been used throughout the world to decrease the incidence of disease 40

however they have little effect on sclerotial germination and release of ascospores (Huang et 41

al 2000) In China frequent applications have led to the pathogen becoming resistant to 42

agrochemicals (Zhang et al 2003) The use of beneficial microorganisms for biological 43

control has become a desired and sustainable alternative against several plant pathogens 44

including S sclerotiorum (Pal and Gardener 2006) 45

46

A diverse number of microorganisms secrete and excrete metabolites that are able to suppress 47

the growth and activities of plant pathogens Bacillus species such as B subtilis B cereus B 48

licheniformis and B amyloliquefaciens have demonstrated antifungal activity against various 49

plant pathogens through antibiosis (Pal and Gardener 2006) Cell free culture filtrates of 50

Bacillus spp contain several bioactive molecules that suppress the growth of active pathogens 51

74

or their resting structures Wu et al (2014) reported that B amyloliquefaciens NJZJSB3 52

releases a number of bioactive metabolites in culture filtrates that are antagonistic toward the 53

growth of mycelia and sclerotial germination of S sclerotiorum Another study reported that 54

culture extracts of antibiotic producing Pseudomonas chlororaphis DF190 and PA23 B 55

cereus DFE4 and B amyloliquefaciens DFE16 significantly reduced the development of 56

blackleg lesions on canola cotyledons (Ramarathnam et al 2011) Scanning electron 57

microscopy observations of sclerotia treated with the culture extract of the ant-associated 58

actinobacterium Propionicimonas sp ENT-18 revealed severely damaged sclerotial rind 59

layers suggesting the antibiosis properties of secondary metabolites produced by the 60

bacterium (Zucchi et al 2010) The role of bioactive metabolites in cell free culture filtrates 61

on plant pathogens in vitro in planta and the ultrastructural observation of cellular organelles 62

require further investigation to elucidate the biocontrol mechanism of bacteria Studies 63

focusing on the mode of action of biocontrol organisms may lead to the identification of 64

novel molecules specifically antagonistic to target pathogens 65

66

Bacillus species have been shown to produce a range of antifungal secondary metabolites 67

(Emmert et al 2004) with diverse structures ranging from lipopeptide antibiotics to a number 68

of small antibiotic peptides (Moyne et al 2004) Iturin surfactin fengycin and plispastatin 69

are lipopeptide antibiotics consisting of hydrophilic peptides and hydrophobic fatty acids 70

(Roongsawang et al 2002) Members of the iturin family of compounds have been reported 71

to provide profound antifungal and homolytic activity but their antibacterial performance is 72

limited (Maget-Dana and Peypoux 1994) The cyclic lipodecapeptide fengycin exhibits 73

specific activity against filamentous fungi by inhibiting phospholypase A2 (Nishikori et al 74

1986) whereas surfactins have been demonstrated to have activity against viruses and 75

mycoplasmas (Vollenbroich et al 1997) Apart from these antibiotics the production and 76

75

release of hydrolytic enzymes by different microbes including Bacillus spp can sometimes 77

directly interfere with the activities of the plant pathogen (Solanki et al 2012) These 78

enzymes are known to hydrolyze a range of polymeric compounds such as chitin proteins 79

cellulose hemicelluloses and DNA Chitin an insoluble linear β-14-linked polymer of N-80

acetylglucosamine (GlcNAc) is a major structural constituent of most fungal cell walls 81

(Sahai and Manocha 1993) Bacillus species have been reported to produce chitinase which 82

can degrade the chitin in the cell walls of fungi (Solanki et al 2012) Serratia marcescens 83

was reported to control the growth of Sclerotium rolfsii which might be mediated by chitinase 84

expression (Ordentlich et al 1988) The biocontrol activities of Lysobacter enzymogenes by 85

degrading the cell walls of fungi and oomycetes appeared to be due to the presence of β-13-86

glucanase gene (Palumbo et al 2005) 87

88

B cereus SC-1 isolated from the sclerotia of S sclerotiorum has strong antifungal activity89

against canola stem rot and lettuce drop diseases caused by S sclerotiorum (Kamal et al 90

2015a Kamal et al 2015b) The strain significantly suppressed hyphal growth inhibited the 91

germination of sclerotia and was able to protect cotyledons of canola from infection in the 92

glasshouse Significant reduction in the incidence of canola stem rot was observed both in 93

glasshouse and field trials In addition B cereus SC-1 provided 100 disease protection 94

against lettuce drop caused by S sclerotiorum in glasshouse studies (Kamal et al 2015a) A 95

better understanding of the mode of action of B cereus SC-1 would assist in developing 96

sustainable biocontrol practices for the management of crop diseases caused by Sclerotinia 97

spp Therefore the present investigation was conducted in vitro and in planta to observe the 98

role of metabolites produced by B cereus SC-1 against S sclerotiorum The histology of 99

mycelia and sclerotia were studied via scanning electron microscopy to observe the 100

morphological and cellular changes induced by this bacteria In addition PCR amplification 101

76

with gene specific primers was performed to detect lipopeptide antibiotics and hydrolytic 102

enzyme biosynthesis genes in B cereus SC-1 103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125


Microbial strains and culture conditions

Bacillus cereus strain SC-1 used in this study was isolated from sclerotia of S sclerotiorum

and found to be antagonistic towards sclerotinia stem rot disease of canola and lettuce drop

(Kamal et al 2015a Kamal et al 2015b) Stock cultures of B cereus SC-1 were maintained

at -80oC in Microbank (Pro-lab diagnostic) and S sclerotiorum was preserved at 4

oC in

sterile distilled water Large and numerous sclerotia were produced using 3-day-old fresh

mycelium of S sclerotiorum on baked bean agar medium (Hind-Lanoiselet 2006) and stored

at room temperature to prevent carpogenic germination (Smith and Boland 1989)

Antagonism of B cereus SC-1 culture filtrates to mycelial growth of S sclerotiorum in

vitro

Bacillus cereus SC-1 was grown in MOLP broth centrifuged at 13000 rpm for 15 minutes at

4oC and the filtrates were passed through a Millipore membrane filter (022 microm) to remove

any suspended cells The filtrates were concentrated by using a Buchi R210215 rotary

evaporator (Sigma-Aldrich) and dissolved in sterile distilled water Antifungal evaluation was

performed by incorporating 50 (vv) of culture filtrate into potato dextrose agar (PDA) in a

90 mm diameter Petri dish The control plates received only sterile MOLP medium A 5 mm

diameter mycelial plug from a 3-day-old culture of S sclerotiorum was excised from the

actively growing edge and placed onto the centre of each medium The plates were incubated

at 4oC for 3 hours to allow the diffusion of metabolites before incubation at 25

oC for 3 days

Following incubation the colony diameter was measured for each culture and the inhibition of 126

77

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

mycelial growth calculated according to the following formula Growth Inhibition () = (R1-

R2)R1 times 100 where R1 represents the average colony diameter in the control plate and R2

indicates the average colony diameter in the filtrate treated plate The bioassay was conducted

with ten replicates and repeated three times

Antagonism of B cereus SC-1 culture filtrates on sclerotia

The concentrated filtrate prepared as detailed above for the mycelia inhibition assay was

also assessed for the inhibition of sclerotial germination by transferring 10 uniformly sized

sclerotia from baked bean agar (Hind-Lanoiselet 2006) onto PDA amended with 50 of the

culture filtrate PDA without culture filtrate was used as the control Following incubation at

25oC for 4 days all sclerotia were examined using a Nikon SMZ745T zoom

stereomicroscope (Nikon instruments) to observe sclerotial germination Sclerotia were

considered as having germinated if there was emergence of white cottony mycelia The

inhibition of sclerotial germination was calculated by comparing the number of mycelium

producing sclerotia with the control and expressed as a percentage as described earlier In

addition 10 sclerotia (one replicate) were submerged in 7-day-old concentrated culture filtrate

and incubated at 25oC for 10 days Sclerotia submerged in sterile distilled water were

considered as controls Following incubation the surface of the sclerotia was examined using

a Nikon SMZ745T zoom stereomicroscope to evaluate their morphology All treated and non-

treated sclerotia were recovered dried and subjected to germination assays on PDA The trial

was conducted with three replicates and repeated three times

Inhibition of S sclerotiorum by culture filtrates of B cereus SC-1 in planta

The antagonism of B cereus SC-1 culture filtrates against S sclerotiorum was evaluated on

10-day-old canola (cv AV Garnet) cotyledons in a controlled plant growth chamber The 151

78

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

culture filtrate was spread onto the adaxial surface of cotyledons (2 cotyledonsseedling) with

an artistrsquos wool brush and air-dried Cotyledons brushed with MOLP broth were considered

as controls After 24 hours filter paper discs (similar size to cotyledons) were soaked in 3-

day-old macerated mycelial fragments of S sclerotiorum (104

fragments mL-1

) and placed

onto the surface of cotyledons Similarly culture filtrate was also applied 24 hours after

pathogen inoculation The mycelial suspensions were agitated prior to the addition of the

filter paper discs to maintain a homogenous concentration The plant growth chamber was

maintained at a relative humidity of 100 and low light intensity of ~15microEm2s for 1 day

then increased to ~150 microEm2s and daynight temperatures of 19

oC and 15

oC

respectively The lesion diameter on each cotyledon was assessed after removing the filter

paper disc at 4 days post inoculation The experiment was conducted in a randomised

complete block design with 10 replicates and repeated twice

Scanning electron microscopy (SEM) studies

Dual culture assays with B cereus SC-1 and S sclerotiorum were performed according to

Kamal et al (2015b) Mycelia were excised from the edges of the interaction zone between

the fungi and bacterial colonies 10 days after inoculation and processed according to Gao et

al (2014) with some modification The samples of mycelia were fixed in 4 (vv)

glutaraldehyde (Electron Microscopy Sciences) at 4degC for 48 hours After rinsing with 01 M

phosphate buffer (pH 68) samples were dehydrated in graded series of ethanol and replaced

by isoamyl acetate (Sigms-Aldrich) The dehydrated samples were subjected to critical-point

drying (Balzers CPD 030) mounted on stubs and sputter-coated with gold-palladium The

morphology of the hyphal surface was micrographed using a Hitachi 4300 SEN Field

Emission-Scanning Electron Microscope (Hitachi High Technologies) at an accelerator 175

79

potential of 3thinspkV The study was conducted on 10 excised pieces of mycelium and repeated 176

twice 177

A co-culture assay to evaluate the effect of B cereus SC-1 on sclerotial germination was 178

conducted according to Kamal et al (2015b) SEM analysis of sclerotia was performed 179

according to Benhamou amp Chet (1996) with minor modification Sclerotia were vapour-fixed 180

in 2 (wv) aqueous solution of osmium tetroxides (ProSciTech) for 40 hours at room 181

temperature The fixed samples were sputter-coated with gold-palladium using a K550 182

sputter coater (Emitech) Micrographs were taken to study the surface morphology of 183

sclerotia using a Hitachi 4300 SEN Field Emission-Scanning Electron Microscope (Hitachi 184

High Technologies) at an accelerator potential of 3thinspkV The SEM studies were conducted on 185

10 sclerotia and repeated twice 186

187

Transmission electron microscopy studies 188

Samples of mycelia from a dual culture assay were taken from the edge of challenged and 189

control mycelium which were infiltrated and embedded with LR White resin (Electron 190

Microscopy Sciences) in gelatine capsules and polymerised at 55degC for 48 h after fixing 191

post-fixing and dehydration Based on the observations of semi-thin sections using a light 192

microscope a diamond knife (DiATOME) was used to cut ultrathin sections of the samples 193

and collected onto 200-mesh copper grids (Sigma-Aldrich) After contrasting with uranyl 194

acetate (Polysciences) and lead citrate (ProSciTech) the sections were visualized with a 195

Hitachi HA7100 transmission electron microscope (Hitachi High Technologies) 196

197

Sclerotia of S sclerotiorum collected from bacterial challenged assays and controls were 198

fixed immediately in 3 (volvol) glutaraldehyde in 01M sodium cacodylate buffer (pH 72) 199

for 2 hours at room temperature Post fixation was performed in the same buffer with 1 200

80

(wv) osmium tetroxide at 4oC for 48 hours Upon dehydration in a graded series of ethanol201

samples were embedded in LR white resin (Electron Microscopy Sciences) From LR white 202

block 07microm thin sections were cut with glass knives and stained with 1 aqueous toluidine 203

blue prior to visualization with a Zeiss Axioplan 2 (Carl Zeiss) light microscope Ultrathin 204

sections of 80 nm were collected on Formvar (Electron Microscopy Sciences) coated 100 205

mesh copper grids stained with 2 uranyl acetate and lead citrate then immediately 206

examined under a Hitachi HA7100 transmission electron microscope (Hitachi High 207

Technologies) operating at 100kV From each sample 10 ultrathin sections were visualized 208

209

Amplification of genes corresponding to lipopeptide antibiotics and mycolytic enzymes 210

A single bacterial bead containing B cereus SC-1 preserved in Microbank was introduced 211

into an Erlenmeyer flask containing medium optimum for lipopeptide production (MOLP) 212

(Gu et al 2005) and incubated on a shaker at 150 rpm at 28oC for 7 days To detect mycolytic213

enzyme producing genes B cereus SC-1 was grown in minimal media (MM) (Solanki et al 214

2012) supplemented with homogenized mycelium of S sclerotiorum (1 wv) then incubated 215

at 28oC for 7 days For DNA extraction 250 microl of the cell suspension from each culture was216

individually transferred into a 05 ml microfuge tube centrifuged at 13000g for 5 minutes 217

and the supernatant discarded To lyse the cells 50 microl of 1X PCR-buffer (Promega) and 1microl 218

Proteinase K (10 mgml) (Sigma-Aldrich) was added to the cells which were then frozen at -219

80degC for 1 h The cells were digested for 1 h at 60degC and subsequently boiled for 15 minutes 220

at 95degC The genomic DNA was stored at -20degC for further investigation The corresponding 221

fragments involved in the production of lipopeptides and enzymes were amplified by PCR 222

using gene specific primers (Table 2) PCR amplification was carried out in a 25 microl reaction 223

mixture consist of 125 microl of 2X PCR master mix (Promega) 025 microl (18 mML) of each 224

primer 2 microl (20 ng) of template DNA and 10 microl of nuclease free water Amplified PCR 225

81

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

reactions were separated on 15 agarose stained with gel red (Bioline) The PCR

products were purified using a PCR Clean-up kit (Axygen Biosciences)

according to the manufacturerrsquos instructions and sequenced by the Australian Genome

Research Facility (AGRF Brisbane Queensland) All sequences were aligned using

MEGA v 62 (Tamura et al 2013) and compared to sequences available in GenBank

using BLAST (Altschul et al 1997)

Data Analysis

ANOVA was conducted using GENSTAT (16th edition VSN International) As there was

no significant difference observed in any repeated trials the data were combined to test

the differences between treatments using Fisherrsquos least significant differences (LSD) at P =

005

RESULTS

Antifungal spectrum of B cereus SC-1 cell free culture filtrates

The cell free culture filtrates of B cereus SC-1 were evaluated against hyphal growth and

sclerotial germination on PDA amended with 50 (vv) culture filtrate The in vitro

inhibition of hyphal growth by culture filtrates was significantly different to controls

(Ple005) (Table 1) The results demonstrated that radial mycelial growth was 481 mm in the

treated plates whereas mycelium was completely inhibited in Petri plates at 4 days post

inoculation Sclerotial germination was inhibited when sclerotia were placed on PDA

amended with culture filtrate whereas all sclerotia were germinated in control plates After

submerging sclerotia in culture filtrates for 10 days degradation of melanin pores on the

sclerotial surface and failure to germinate on PDA was observed The melanin layer remained

intact in sclerotia submerged in distilled water (Fig 1A B) Application of culture filtrates on

10-day-old canola cotyledon significantly suppressed (Ple005) lesions of S sclerotiorumThe

250

82

average lesion diameter of culture filtrate treated and control cotyledons were 238 mm and 251

1172 mm respectively The inhibition of lesion development was not significant (Ple005) 252

when the filtrate was applied 1 day post pathogen inoculation with mycelia and compared to 253

controls 254

255

Microscopic observations of B cereus SC-1 on S sclerotiorum 256

Mycelial samples collected from the zone of interaction between B cereus SC-1 and S 257

sclerotiorum were visualised with a SEM The hyphal tips of S sclerotiorum growing 258

towards the bacterial colonies were deformed and elongation was inhibited (Fig 2A) The 259

hyphae were observed to be vacuolated and loss of cytoplasm was evident when samples 260

were examined at higher magnification (Fig 2B) Control non-parasitized sclerotia of S 261

sclerotiorum appeared as spherical structures characterized by their intact globular rind layer 262

(Fig 2C) B cereus SC-1 treated sclerotia demonstrated pitted collapsed fractured and 263

ruptured outmost rind cells of the sclerotia Bacterial cells were abundantly present inside the 264

ruptured rind cells of parasitized sclerotia (Fig 2D) Cell to cell connection of bacteria 265

through thin mucilage was observed in most samples studied (Fig 2E) The rind layer cells 266

were completely destroyed and the broken walls of rind cells were discernible (Fig 2F) 267

Abundant presence of bacterial cells as well as disintegration of sclerotial rind cells was 268

frequently visible 269

270

Hyphal ultrastructure was micrographed using a TEM (Fig 3AB) The cell wall of control 271

hyphae was comparatively thinner and fibrillar structure was clearer than that of the sclerotial 272

cell wall The thickness of a single layered hyphal cell wall varied from 01 microm to 02 microm In 273

the hyphal tips the cytoplasmic membrane was significantly folded In most sections small 274

vesicles or tubules were observed between the cytoplasmic membrane and cell wall which 275

83

was not observed in sclerotia cells The presence of dark bodies were observed in hyphal cells 276

indicating polysaccharides (Bracker 1967) Generally ribosomes and mitochondria were 277

more abundant in hyphal cells than in sclerotia indicating that metabolic activity may be 278

lower in sclerotial cells 279

280

Additional information on the structural organization of untreated and treated sclerotia was 281

obtained from ultrathin sections using TEM The histological appearance of normal and 282

abnormal sclerotia was similar to that described by Huang (1982) Ultrastructural analysis of 283

sclerotia in the control treatment demonstrated that the rind layer was approximately three to 284

four cells in thickness The rind cells were surrounded by thick and electron dense cell walls 285

which contained dense cytoplasm The inner layer known as the medulla consisted of 286

loosely organised pleomorphic cells which were surrounded by thick electron lucent cell 287

walls Small polymorphic vacuoles and electron-opaque inclusions were visible in the 288

cytoplasm A number of electron lucent vacuoles were visible in the outer cells underneath 289

the rind cells which were surrounded by electron dense inclusions and comparatively less 290

electron dense cell walls Fine granular ground matrix was observed inside vacuoles A 291

comparatively reduced electron density of cell walls and reduced cytoplasmic density were 292

noticed in inner rind cells but the number of vacuoles and inclusions remained similar The 293

presence of an electron-opaque middle lamella was observed between the junctions of 294

contiguous cells The loosely arranged medullary cells were overlaid by a fibrillar matrix 295

which acted as a bridge between medullary cells that contained electron dense inclusions 296

(Fig 3C) 297

298

The effect of B cereus SC-1 on the rind cells of sclerotia was observed as collapsed and 299

empty cells and a few contained only cytoplasmic remnants The normal properties of control 300

84

sclerotia as described earlier were not apparent and delineation of the cell wall indicated that 301

all cells belonging to the rind and medulla were completely disintegrated by the action of the 302

bacterial metabolites Cells had lost all cytoplasmic organisation and aggregated as well as 303

non-aggregated cytoplasmic remnants were visible Both electron dense and electron lucent 304

cell walls were observed with clear signs of collapse indicating that metabolites of B cereus 305

SC-1 penetrated the cell wall prior to affecting the cytoplasmic organelles Finally 306

observations of various sections from bacterial challenged sclerotia revealed that the cell wall 307

of the rind and medulla were extensively altered and disintegrated Bacterial invasion caused 308

complete breakdown of adjacent cell walls which resulted in transgress of cytoplasmic 309

remnants among cells Massive alteration disorganization and aggregation of cytoplasm as 310

well as degradation of the fibrillar matrix were observed in medullary cells (Fig 3D) 311

312

PCR amplification of non-ribosomal lipopeptide and hydrolytic enzyme synthetase 313

genes 314

The gene clusters corresponding to bacillomycin D iturin A fengycin surfactin chitinase 315

and β-1-3 glucanase biosynthesis were successfully amplified from the B cereus SC-1 The 316

bacillomycin D biosynthetic cluster specific primers yielded a ~875 bp PCR product (Fig 317

4A) with 98 homology to the bamC gene of bacillomycin D operon of type strain B318

subtilis ATTCAU195 A ~647 bp PCR amplicon was amplified with the gene specific primer 319

pair iturin A operon (Fig 4B) The purified amplicon showed 97 homology to the ituD 320

gene of iturin A operon of B subtilis RB14 in Genbank PCR amplification with fenD gene 321

specific primer pairs yielded a ~220 bp PCR product (Fig 4C with 99 homology to the 322

fenD gene of B subtilis QST713 in Genbank) Amplification of genomic DNA with surfactin 323

specific primers yielded a ~441 bp product (Fig 4D) which showed 98 homology with the 324

srfDB3 gene of the surfactin operon of B subtilis B3 strain in Genbank The A ~270 bp band 325

85

corresponding to the chitinase (chiA) gene demonstrated 99 sequence similarity to a 326

Aeromonas hydrophila chitinase A (chiA) gene (Fig 4E) Finally PCR amplification of the 327

β-glucanase gene resulted in a ~415 bp size product which showed 100 sequence homology 328

to the β-glucanase gene of B subtilis 168 (Fig 4F) 329

330

DISCUSSION 331

This study examined the biocontrol mechanism of a potential bacterial antagonist B cereus 332

SC-1 associated with the suppression of S sclerotiorum The experimental results 333

demonstrated that antibiosis may be the most dominant mode of action attributed to the 334

bacterium B cereus SC-1 was used in this study for its biocontrol properties described in 335

previous investigations where it exhibited strong antifungal activity against Sclerotinia 336

diseases of canola and lettuce (Kamal et al 2015a Kamal et al 2015b) Cell-free 337

concentrated culture filtrates of B cereus SC-1 were able to inhibit the mycelial growth of S 338

sclerotiorum on PDA suggesting that this is most likely due to diffusible metabolites 339

produced by the bacterium A significant suppression of mycelial growth and complete 340

inhibition of sclerotial germination was observed on PDA containing 50 culture filtrate of 341

B cereus SC-1 The filtrates caused alteration of hyphal growth lysis of cells degradation of342

hyphal tips and bleaching of sclerotia due to the scarification of melanin The sclerotia 343

displayed reduced firmness and were completely degraded Similar studies demonstrated that 344

the cell free supernatant of B subtilis MB14 and B amyloliquefaciens MB101 were able to 345

significantly inhibit the growth of R solani when 10 and 20 culture filtrates were 346

incorporated into an artificial growth medium (Solanki et al 2013) The culture filtrate from 347

B amyloliquefaciens strain NJZJSB3 exhibited profound antifungal activity against the in348

vitro mycelial growth and sclerotial germination of S sclerotiorum (Wu et al 2014) When 349

the culture filtrate was diluted 60-fold the mycelia and sclerotial germination of S 350

86

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

sclerotiorum was inhibited by 804 and 100 respectively Zucchi et al (2010) also

reported that the actinobacterium Propionicimonas sp is able to release secondary

metabolites in culture filtrates causing severe degradation of the sclerotia of S sclerotiorum

Bioassays of culture filtrates on 10-day-old canola cotyledons one day prior to inoculation

with S sclerotiorum reduced lesion development by the pathogen However when the culture

filtrates were applied one day after inoculation with S sclerotiorum no significant difference

in lesion development compared to the controls was observed This may be attributed to the

preventive nature of the metabolites within the culture filtrate Similar results were obtained

by Ramarathnam et al (2011) who demonstrated that culture extracts of P chlororaphis

DF190 and PA23 B cereus DFE4 and B amyloliquefaciens DFE16 were able to

significantly reduce blackleg lesion development on canola cotyledon when inoculated 24 h

and 48 h prior to inoculation of the pathogen Yoshida et al (2001) also reported that

antifungal compounds in culture filtrates of B amyloliquefaciens RC-2 were able to

significantly reduce Colletotrichum dematium on mulberry leaves when applied prior to

inoculation of the pathogen

Electron microscopy studies have provided valuable insight into the mechanism of bacterial

antagonism at a physiological and cellular level Scanning and transmission electron

microscopy studies revealed that B cereus SC-1 caused alterations in the cell walls and loss

of cytoplasmic material in the hyphae of S sclerotiorum and this may be attributed to the

bioactive antifungal metabolites produced by the bacterium The deleterious effect of B

cereus SC-1 on sclerotia was observed from the outmost rind cells into the inner medullary

cells The loss of cytoplasmic content and cellular lysis could be attributed to the extensive

degradation of cell walls and cytoplasmic membrane Our results revealed that B cereus SC-375

87

1 successfully invaded and colonized the sclerotia of S sclerotiorum causing significant 376

cytological alterations SEM observations of hyphal tips growing towards the bacterial 377

colonies in dual culture showed that mycelial growth was inhibited in the moiety of bacterial 378

metabolites with the eventual complete loss of hyphal content Bacterial invasion of sclerotia 379

revealed that the outmost rind cells were ruptured and heavily colonized Ultrathin sections 380

from parasitized sclerotia demonstrated that the bacteria ingress towards the inner medullary 381

cells by causing retraction of the plasmalemma cytoplasmic disorganization and finally 382

complete loss of protoplasm All melanised and non-melanised cells and their contents were 383

disintegrated most likely due to the highly bioactive nature of the bacterial metabolites 384

Our results concur with the observations of Zucchi et al (2010) who also showed that the 385

direct action of bioactive metabolites released from actinobacterium Propionicimonas sp 386

strain ENT 18 induced pronounced cytoplasmic damage Observations regarding cytoplasmic 387

degradation of medullary cells in the presence of bacterial contact suggested that strong 388

diffusible compounds may be responsible for such results Studies conducted by Benhamou 389

and Chet (1996) investigated the interaction of Trichoderma harzianum and sclerotia of 390

Sclerotium rolfsii at a cellular level Their ultrastructural observations demonstrated that 391

Trichoderma invasion caused massive alteration of sclerotial cells including retraction and 392

aggregation of cytoplasm and breakdown of vacuoles To our knowledge this is the first time 393

that the antagonism of a biocontrol bacterium on mycelium and sclerotia of S sclerotiorum 394

has been rigorously studied by SEM and TEM 395

396

The present study attempted to amplify markers associated with the genes responsible for 397

production of antifungal antibiotics Amplification of PCR products using previously 398

published antibiotic gene specific primers revealed the presence of bacillomycin D iturin A 399

surfactin and fengycin lipopeptides operons in B cereus SC-1 The presence of particular 400

88

antibiotic bio-synthetic operon in a bacterial strain indicates its ability to produce antibiotics 401

(McSpadden Gardener et al 2001) The cyclic lipopeptide bacillomycin D is an important 402

member of the iturin family which undergo non-ribosomal synthesis from Bacillus spp and 403

has strong antifungal activity (Besson and Michel 1992 Koumoutsi et al 2004) The 404

bioactive lipopeptide iturin A released from Bacillus spp penetrates the lipid bilayer of the 405

cytoplasmic membranes and causes pore formation and cellular leakage (Maget-Dana and 406

Peypoux 1994) Fengycin also shows strong antifungal activity and its production in B 407

cereus has been documented (Ramarathnam et al 2007) These studies indicate that B cereus 408

SC-1 harbours genes for four different lipopeptide antibiotics which may contribute to the 409

biocontrol of S sclerotiorum 410

411

The fungal cell wall is mostly constituted of chitin β-13-glucan and protein (Inglis and 412

Kawchuk 2002 Sahai and Manocha 1993) The presence of chitinase and β-glucanase 413

encoding genes in the genome of B cereus SC-1 indicates the production of mycolytic 414

enzymes which may be responsible for the destruction of fungal cell walls along with 415

lipopeptide antibiotics Avocado roots inhabited by four B subtilis strains were reported to 416

control Fusarium oxysporum fsp radicis-lycopersici and Rosellinia necatrix by producing 417

hydrolytic enzymes (glucanases or proteases) and antibiotic lipopeptides (surfactin fengycin 418

andor iturin) (Cazorla et al 2007) Solanki et al (2012) reported that four strains of Bacillus 419

harbour chitinase and β-glucanase producing genes and attributed their role against R solani 420

to the action of mycolytic enzymes Our results agree with the aforementioned studies which 421

suggest that amplified genes from B cereus SC-1 corresponding to production of chitinase 422

and β-glucanase might also play a role in the biological control of S sclerotiorum through 423

destruction of mycelia and sclerotia 424

425

89

A comparatively low percentage of environmental bacteria have the ability to produce several 426

antibiotics and mycolytic enzymes simultaneously B cereus SC-1 contains the genes for the 427

production of lipopeptide antibiotics and hydrolytic enzymes that may deploy a joint 428

antagonistic action towards the growth of S sclerotiorum Understanding the mode of action 429

of B cereus SC-1 through multiple approaches would provide the opportunity to improve the 430

consistency of controlling S sclerotiorum either by improving the mechanism through 431

genetic engineering or by creating conducive conditions where activity is predicted to be 432

more successful Whole genome sequencing would pave the way to further understand the 433

biocontrol mechanisms exhibited by B cereus SC-1 and would assist in designing gene 434

specific primers which could be used for more efficient selection of potential antagonistic 435

bacteria for biological control of plant diseases 436

437

ACKNOWLEDGEMENTS 438

This study was funded by a Faculty of Science (Compact) Scholarship Charles Sturt 439

University Australia We thank Jiwon Lee at the Centre for Advanced Microscopy (CAM) 440

Australian National University and the Australian Microscopy amp Microanalysis Research 441

Facility (AMMRF) for access to Hitachi 4300 SEN Field Emission-Scanning Electron 442

Microscope and Hitachi HA7100 Transmission Electron Microscope 443

444

REFERENCES 445

Altschul SF Madden TL Schaumlffer AA et al (1997) Gapped BLAST and PSI-BLAST a new 446

generation of protein database search programs Nucleic Acids Res 199725 3389-447

402 448

449

90

Baysal O Ccedilalişkan M Yeşilova O (2008) An inhibitory effect of a new Bacillus subtilis 450

strain (EU07) against Fusarium oxysporum f sp radicis-lycopersici Physol Mol 451

Plant P 73 pp 25-32 452

Benhamou N Chet I (1996) Parasitism of sclerotia of Sclerotium rolfsii by Trichoderma 453

harzianum Ultrastructural and cytochemical aspects of the interaction 454

Phytopathology 86 pp 405-416 455

Besson F Michel G (1992) Biosynthesis of bacillomycin D by Bacillus subtilis Evidence for 456

amino acid-activating enzymes by the use of affinity chromatography FEBS Lett 308 457

pp 18-21 458

Bolton MD Thomma BP Nelson BD (2006) Sclerotinia sclerotiorum (Lib) de Bary biology 459

and molecular traits of a cosmopolitan pathogen Mol Plant Pathol 7 pp 1-16 460

Bracker CE (1967) Ultrastructure of Fungi Annu Rev Phytopathol 5 pp 343-372 461

Cazorla F Romero D Perez-Garcia A Lugtenberg B de Vicente A Bloemberg G (2007) 462

Isolation and characterization of antagonistic Bacillus subtilis strains from the 463

avocado rhizoplane displaying biocontrol activity J Appl Microbiol 103 pp 1950-464

1959 465

Emmert EAB Klimowicz AK Thomas MG Handelsman J (2004) Genetics of Zwittermicin 466

A Production by Bacillus cereus Appl Environ Microbiol 70 pp 104-113 467

Fernando WGD Nakkeeran S Zhang Y (2004) Ecofriendly methods in combating 468

Sclerotinia sclerotiorum (Lib) de Bary In Recent Research in Developmental and 469


Fuller P Coyne D Steadman J (1984) Inheritance of resistance to white mold disease in a 471

diallel cross of dry beans Crop Sci 24 pp 929-933 472

91

Gao X Han Q Chen Y Qin H Huang L Kang Z (2014) Biological control of oilseed rape 473

Sclerotinia stem rot by Bacillus subtilis strain Em7 Biocontrol Sci Techn 24 pp 39-474

52 475

Gu X Zheng Z Yu HQ Wang J Liang F Liu R (2005) Optimization of medium constituents 476

for a novel lipopeptide production by Bacillus subtilis MO-01 by a response surface 477

method Process Biochem 40 pp 3196-3201 478

Hind-Lanoiselet T (2006) Forecasting Sclerotinia stem rot in Australia Charles sturt 479

University 480

Huang H Bremer E Hynes R Erickson R (2000) Foliar application of fungal biocontrol 481

agents for the control of white mold of dry bean caused by Sclerotinia sclerotiorum 482

Biol Control 18 pp 270-276 483

Huang HC (1982) Morphologically abnormal sclerotia of Sclerotinia sclerotiorum Can J 484

Microbiol 28 pp 87-91 485

Inglis G Kawchuk L (2002) Comparative degradation of oomycete ascomycete and 486

basidiomycete cell walls by mycoparasitic and biocontrol fungi Can J Microbiol 48 487

pp 60-70 488

Kamal MM Lindbeck K Savocchia S Ash G (2015a) Bacterial biocontrol of sclerotinia 489

diseases in Australia Acta Hort p (Accepted) 490


stem rot of canola using antagonistic bacteria Plant Pathol doi101111ppa12369 492

Koumoutsi A et al (2004) Structural and functional characterization of gene clusters 493

directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus 494

amyloliquefaciens strain FZB42 J Bacteriol 186 p 1084 495

Maget-Dana R Peypoux F (1994) Iturins a special class of pore-forming lipopeptides 496

Biological and physicochemical properties Toxicology 87 pp 151-174 497

92

McSpadden Gardener BB Mavrodi DV Thomashow LS Weller DM (2001) A rapid 498

polymerase chain reaction-based assay characterizing rhizosphere populations of 24-499

diacetylphloroglucinol-producing bacteria Phytopathology 91 pp 44-54 500

Moyne AL Cleveland TE Tuzun S (2004) Molecular characterization and analysis of the 501

operon encoding the antifungal lipopeptide bacillomycin D FEMS Microbiol Lett 502

234 pp 43-49 503

Mueller DS et al (2002) Efficacy of fungicides on Sclerotinia sclerotiorum and their 504

potential for control of Sclerotinia stem rot on soybean Plant Dis 86 pp 26-31 505

Murray GM Brennan JP (2012) The Current and Potential Costs from Diseases of Oilseed 506

Crops in Australia Grains Research and Development Corporation Australia 507

Nishikori T Naganawa H Muraoka Y Aoyagi T Umezawa H (1986) Plispastins new 508

inhibitors of phospholopase 42 produced by Bacillus cereus BMG302-fF67 III 509

Structural elucidation of plispastins J Antibiot 39 pp 755-761 510

Ordentlich A Elad Y Chet I (1988) The role of chitinase of Serratia marcescens in 511

biocontrol of Sclerotium rolfsii Phytopathology 78 p 84 512

Pal KK Gardener BMS (2006) Biological control of plant pathogens Plant healt instruct 2 513

pp 1117-1142 514

Palumbo JD Yuen GY Jochum CC Tatum K Kobayashi DY (2005) Mutagenesis of beta-515

13-glucanase genes in Lysobacter enzymogenes strain C3 results in reduced 516

biological control activity toward Bipolaris leaf spot of tall fescue and Pythium 517

damping-off of sugar beet Phytopathology 95 pp 701-707 518

Ramaiah N Hill R Chun J Ravel J Matte M Straube W Colwell R (2000) Use of a chiA 519

probe for detection of chitinase genes in bacteria from the Chesapeake Bay FEMS 520

Microbiol Ecol 34 pp 63-71 521

93

Ramarathnam R (2007) Mechanism of phyllosphere biological control of Leptosphaeria 522

maculans the black leg pathogen of canola using antagonistic bacteria University of 523

Manitoba 524

Ramarathnam R Fernando WD de Kievit T (2011) The role of antibiosis and induced 525

systemic resistance mediated by strains of Pseudomonas chlororaphis Bacillus 526

cereus and B amyloliquefaciens in controlling blackleg disease of canola BioControl 527

56 pp 225-235 528

Ramarathnam R Sen B Chen Y Fernando WGD Gao XW de Kievit T (2007) Molecular 529

and biochemical detection of fengycin and bacillomycin D producing Bacillus spp 530

antagonistic to fungal pathogens of canola and wheat Can J Microbiol 53 pp 901-531

911 532

Roongsawang N et al (2002) Isolation and characterization of a halotolerant Bacillus subtilis 533

BBK-1 which produces three kinds of lipopeptides bacillomycin L plipastatin and 534

surfactin Extremophiles 6 pp 499-506 535

Sahai AS Manocha MS (1993) Chitinases of fungi and plants their involvement in 536

morphogenesis and host-parasite interaction FEMS Microbiol Rev 11 pp 317-338 537

Saharan GS Mehta N (2008) Sclerotinia diseases of crop plants Biology ecology and 538

disease management Springer Science Busines Media BV The Netherlands 539

Smith E Boland G (1989) A Reliable Method for the Production and Maintenance of 540

Germinated Sclerotia of Sclerotinia sclerotiorum Can J Plant Pathol 11 pp 45-48 541

Solanki MK Robert AS Singh RK Kumar S Srivastava AK Arora DK Pandey AK (2012) 542

Characterization of mycolytic enzymes of Bacillus strains and their bio-protection 543

role against Rhizoctonia solani in tomato Curr Microbiol 65 pp 330-336 544

94

Solanki MK Singh RK Srivastava S Kumar S Kashyup PL Srivastava AK (2013) 545

Characterization of antagonistic potential of two Bacillus strains and their biocontrol 546

activity against Rhizoctonia solani in tomato J Basic Microb 55 pp 82-90 547

Tamura K Peterson D Peterson N et al (2013) MEGA5 molecular evolutionary genetics 548

analysis using maximum likelihood evolutionary distance and maximum parsimony 549

methods Mol Biol Evol 28 pp2731-2739 550

Vollenbroich D Ozel M Vater J Kamp RM Pauli G (1997) Mechanism of inactivation of 551

enveloped viruses by the biosurfactant surfactin from Bacillus subtilis Biologicals 25 552

pp 289-297 553


of Bacillus amyloliquefaciens strain NJZJSB3 against Sclerotinia sclerotiorum a 555

causal agent of canola stem rot J Microbiol Biotechn 24 pp 1327ndash1336 556

Yoshida S Hiradate S Tsukamoto T Hatakeda K Shirata A (2001) Antimicrobial activity of 557

culture filtrate of Bacillus amyloliquefaciens RC-2 isolated from mulberry leaves 558


Zhang X Sun X Zhang G (2003) Preliminary report on the monitoring of the resistance of 560

Sclerotinia libertinia to carbendazim and its internal management Chinese J Pestic 561

Sci Admin (In Chinese) 24 pp 18-22 562

Zucchi TD Almeida LG Dossi FCA Cocircnsoli FL (2010) Secondary metabolites produced by 563

Propionicimonas sp (ENT-18) induce histological abnormalities in the sclerotia of 564

Sclerotinia sclerotiorum BioControl 55 pp 811-819 565

566

567

568

569

95

Tables 570

Table 1 In vivo and in vitro effects of culture filtrates of Bacillus cereus SC-1 on Sclerotinia 571

sclerotiorum 572

Treatment

Mycelial diameter

(mm) in vitro

Lesion diameter (mm) on cotyledonsa

One day before

pathogen inoculation

One day after


B cereus SC1

culture filtrate 481plusmn05 a 238plusmn02 a 1036plusmn07 a

Control 850plusmn0 b 1172plusmn08 b 1147plusmn09 a

aLesion diameter measured 4 days after inoculation with S sclerotiorum 573

Values in columns followed by the same letters were not significantly different according to 574

Fisherrsquos protected LSD test (P=005) 575

96

Table 2 Gene specific primers used in this study 576

Antibiotic

Enzyme

Primer Primer sequence

(5´-3´)

PCR conditions References

Iturin A

F GATGCGATCTCCTTGGATGT

Initial denaturation 94oC for 3 min 35

cycles of at 94oC for 1min annealing at

60oC for 30 s extension at 72

oC for 1min

45s final extension 72oC for 6 min

(Ramarathnam

2007 Ramarathnam

et al 2007)

R ATCGTCATGTGCTGCTTGAG

Bacillomycin D

F GAAGGACACGGCAGAGAGTC

R CGCTGATGACTGTTCATGCT

Surfactin

F ACAGTATGGAGGCATGGTC

R TTCCGCCACTTTTTCAGTTT

Fengycin

F CCTGCAGAAGGAGGAGAAGTG

AAG


cycles of at 94oC for 35 s annealing at


oC for 45 s

final extension 72oC for 5 min

(Solanki et al

2013)

R TGCTCATCGTCTTCCGTTTC

Chitinase

F GATATCGACTGGGAGTTCCC


cycles of at 92oC for 1 min annealing at

58oC for 1 min extension at 72

oC for 1

min final extension 72oC for 7 min

(Ramaiah et al

2000)

R CATAGAAGTCGTAGGTCATC

β-glucanase

F AATGGCGGTGTATTCCTTGA CC




oC for 1


(Baysal et al 2008)

R GCGCGTAGTCACAGTCAAAGTT

577

97

Figures 578

579

580

Fig 1 Effect of culture filtrates of Bacillus cereus SC-1 against sclerotia of Sclerotinia 581

sclerotiorum A Culture filtrate treated sclerotia showing removal of melanin on the surface 582

after 10 days of submergence B Control sclerotia submerged into water showing intactness 583

of melanin 584

98

585

Fig 2 Scanning electron micrographs of mycelium and sclerotia of Sclerotinia sclerotiorum 586

A Mycelia excised at the edges towards the bacterial colonies showing inhibition of mycelial 587

growth in the moity of bacterial metabolites B Cross section of bacteria treated hyphae 588

demonstrating the vacuolated structure and loss of hyphal components C Outer rind layer of 589

control sclerotia D Bacteria treated sclerotia showing perforation of outer rind layer and 590

eruption of cell contents E Massive colonization and cell to cell communication (arrow 591

indicated) was observed inside of ruptured cell F Bacteria treated sclerotia showing complete 592

disintegration of outer rind layer and heavy bacterial colonization 593

99

594

Fig 3 Transmission electron micrographs of ultrathin section of vegetative hyphae and 595

sclerotia of Sclerotinia sclerotiorum A Control hyphae showing intactness of cellular 596

contents B Bacteria treated sclerotia showing disintegration of cellular contents C Control 597

sclerotia showing intactness of electron dense cell walls and cellular contents D Bacteria 598

treated sclerotia showing massive disintegration of cell wall and transgression of cellular 599

contents 600

601

602

603

604

605

100

606

Fig 4 PCR amplification of genes corresponding to lipopeptide antibiotics and mycolytic 607

enzymes in Bacillus cereus SC-1 A bamC gene (~875 bp) of the bacillomycin D operon B 608

ituD gene (~647 bp) of the iturin A operon C fenD gene (~220 bp) of the fengycin D srDB3 609

gene (~441bp) of the surfactin E chiA gene (~270 bp) of Chitinase and F β-glucanase gene 610

(~415 bp) synthetase biosynthetic cluster A 100-bp DNA size standard (Promega) was used 611

to measure the size of the amplified products The visualization of bands in amplification 612

products indicates that the target genes were present in the isolate 613

101


MM Kamal S Savocchia KD Lindbeck and GJ Ash (2015) Rapid marker-assisted

selection of antifungal Bacillus species from the canola rhizosphere FEMS Microbiology

Ecology (Formatted)

Chapter 7 describes a novel marker-assisted approach using multiplex PCR to screen

antagonistic Bacillus spp with potential as biocontrol agents against Sclerotinia sclerotiorum

Upon identification of marker selected strain CS-42 as Bacillus cereus using 16S rRNA gene

sequencing the strain was subsequently evaluated to control the growth of S sclerotiorum in

vitro and in planta Scanning and transmission electron microscopy studies of the zone of

interaction in dual culture and of treated sclerotia have indicated the mode of action of the B

cereus CS-42 strain against S sclerotiorum The findings in this chapter have shown the

opportunity for rapid and efficient selection of pathogen-suppressing Bacillus strains instead

of time consuming direct dual culture methodologies and could accelerate microbial

biopesticide development This chapter has been formatted according to ldquoFEMS

Microbiology Ecologyrdquo journal

102

Rapid marker-assisted selection of antifungal Bacillus species from the canola 1

rhizosphere 2


1 Kurt D Lindbeck

2 and Gavin J Ash

13




2NSW-Department of Primary Industries Wagga Wagga Agricultural Institute Pine Gully 7

Road Wagga Wagga NSW 2650 8

Corresponding Author Mohd Mostofa Kamal Email mkamalcsueduau9

Keywords Biocontrol Marker Antagonists Bacillus Canola Rhizosphere 10

Running title Rapid detection of antagonistic Bacillus spp 11

ABSTRACT 12

A marker-assisted approach was adopted to search for Bacillus spp with potential as 13

biocontrol agents against stem rot disease of canola caused by Sclerotinia sclerotiorum 14

Bacterial strains were isolated from the rhizosphere of canola and screened using multiplex 15

PCR for the presence of surfactin iturin A and bacillomycin D peptide synthetase 16

biosynthetic genes from eight groups of Bacillus isolates consisting of twelve pooled 17

isolates Among the 96 isolates screened only CS-42 was positively identified as containing 18

all three markers It was subsequently identified as Bacillus cereus using 16S rRNA gene 19

sequencing This strain was found to be effective in significantly inhibiting the growth of S 20

sclerotiorum in vitro and in planta Scanning electron microscopy studies at the dual culture 21

interaction region revealed that mycelial growth was curtailed in the vicinity of bacterial 22

metabolites Complete destruction of the outmost melanised rind layer of sclerotia was 23

observed when treated with the bacterium Transmission electron microscopy of ultrathin 24

sections challenged with CS-42 showed partially vacuolated hyphae as well as degradation of 25

organelles in the sclerotial cells These findings suggest that genetic marker-assisted selection 26

103

may provide opportunities for rapid and efficient selection of pathogen-suppressing Bacillus 27

strains and the development of microbial biopesticides 28

29

INTRODUCTION 30

The rhizosphere has been considered as a ldquoBlack Boxrdquo of microorganisms that provides a 31

front line defence and protection of plant roots against pathogen invasion (Bais et al 2006 32

233-66 Weller 1988 379-407) A number of plant and soil associated beneficial bacteria are33

able to suppress plant pathogens through multiple modes of action including antagonism (Pal 34

and Gardener 2006 1117-42) The genus Bacillus occurs ubiquitously in soil and many 35

strains are able to produce versatile metabolites involved in the control of several plant 36

pathogens (Ongena and Jacques 2008 115-25) The persistence and sporulation under harsh 37

environmental conditions as well as the production of a broad spectrum of antifungal 38

metabolites make the organism a suitable candidate for biological control (Jacobsen et al 39

2004 1272-5) Members of the Bacillus genus possess the ability to produce heat and 40

desiccation resistant spores which make them potential candidates for developing efficient 41

biopesticide products These spores are often considered as factories of biologically active 42

metabolites which in turn have been shown to suppress a wide range of plant pathogens 43

Bacillus spp are known to produce cyclic lipopeptide antibiotics such as surfactin (Kluge et 44

al 1988 107-10) iturin A (Yu et al 2002 955-63) and bacillomycin D (Moyne 2001 622-45

9) and exhibit strong antifungal activity against a number of plant pathogens The selection46

and evaluation of these bacteria from natural environments using direct dual culture 47

techniques is a time consuming task The discovery of lipopeptide synthetases genes 48

associated with novel biocontrol and the development of gene specific markers has provided 49

the opportunity to detect antagonistic Bacillus species from large numbers of environmental 50

samples (Joshi and McSpadden Gardener 2006 145-54) 51

104

A number of PCR-based methods have been developed for screening antagonistic bacteria 52

(Gardener et al 2001 44-54 Giacomodonato et al 2001 51-5 Park et al 2013 637-42 53

Raaijmakers et al 1997 881) Recently a high-throughput assay was developed to screen 54

for antagonistic bacterial isolates for the management of Fusarium verticillioides in maize 55

(Figueroa-Loacutepez et al 2014 S125-S33) There is no doubt of the contribution of these 56

studies to facilitate the selection of potential biocontrol organisms However a more efficient 57

and affordable molecular based approach is crucial for the detection of bacterial antagonists 58

that can inhibit fungal growth disintegrate cellular contents and protect plants from pathogen 59

invasion Therefore the objective of this investigation was to develop a rapid and reliable 60

screening method to identify Bacillus isolates from the canola rhizosphere with antagonistic 61

properties towards S sclerotiorum The antagonism of marker selected bacteria was further 62

evaluated against S sclerotiorum in vitro and in glasshouse grown canola plants In addition 63

scanning electron microscopy (SEM) and transmission electron microscopy (TEM) studies 64

were conducted to explore the inhibitory effect of bacteria on cell organelles in mycelium and 65

sclerotia 66

67

METHODS 68

69

Rhizosphere sampling 70

71

The root system of whole canola plants from Wagga Wagga (35deg07008PrimeS 147deg22008PrimeE) 72

were collected to a depth of 15 cm using a shovel and immediately placed in plastic bags for 73

transport to the laboratory Samples were either immediately processed or placed in a cold 74

room (8degC) within 3 h of sampling and stored for a maximum of 3 days prior to processing 75

Soil samples from 10 individual canola rhizopheres were processed by removing the intact 76

root system and adhering soil from each plant using a clean scalpel Each rhizosphere sample 77

of 3 g was placed in a falcon tube containing 30 mL of sterile distilled water To dislodge the 78

105

bacteria and adhering soil from the roots each sample was vortexed four times for 15s 79

followed by a 1 minute sonication Aliquots of 1 mL of the suspensions were stored in 80

individually labelled microfuge tubes for further investigation 81

82

Bacillus culture and maintenance 83

Microfuge tubes containing 1 mL of each soil suspension were incubated in a water bath at 84

80oC for 15 minutes and then allowed to cool to 40

oC Using a sterile glass rod 50 microl of the85

soil suspension was then spread onto tryptic soy agar (TSA) and incubated for 24-48 h at 86

room temperature Following incubation individual colonies were inoculated into 96 well 87

microtiter plates (Costar) containing 200 microl of tryptic soy broth (TSB) and incubated at 40oC88

for 24 h A 10 microl aliquot of each liquid culture was transferred to fresh TSB in a new 89

microtiter plate and incubated at room temperature overnight A Genesys 20 90

spectrophotometer (Thermo Scientific) was used to assess the optical density of bacterial 91

growth at 600 nm with a reading of ge005 being scored as positive Replicate plates were 92

prepared by mixing individual cultures with 35 sterile glycerol (vv) which were stored at 93

-80degC94

95

Preparation of combined isolates and lysis of bacterial cell 96

The bacterial isolates from 12 wells of the TSB cultures were combined by transferring 10 97

microl of each into a single well of a 96 well PCR plate (BioRad) This resulted in 8 groups from 98

a total of 96 isolates DNA was isolated from each combined sample using a freeze thaw 99

extraction protocol Briefly 90 microl of 15x PCR buffer (Promega) was placed into each well of 100

a PCR plate and 10 microl of combined liquid culture was added and mixed thoroughly The PCR 101

plates were then placed into a -80oC freezer for 8 minutes before being transferred to a FTS102

960 thermocyler (Corbert Research) at 94oC for 2 minutes The incubation procedure was103

repeated thrice and the lysed cells stored at -20oC104

106

Development of PCR assays 105

The target specific primers used in this study were as described by Ramarathnam et al 106

(2007 901-11) The combined template DNA (representing 12 isolates) was amplified using 107

gene specific primers corresponding to iturin A (~647 bp) bacillomycin D (~875 bp) and the 108

surfactin lipopeptide antibiotic biosynthetase operon (~441 bp) (Table 1) DNA of individual 109

isolates from combinations resulting in positive amplification were then re-amplified with the 110

same primers to detect target isolates The PCR reaction volume was 25 microl which comprised 111

of 125 microl of 2X PCR master mix (Promega) 05 microl mixture of primers (18 mmoleL) 2 microl 112

(20 ng) of mix template DNA and 10 microl of nuclease free water The targets were amplified 113

using the cycling conditions as initial denaturation 94oC for 3 min 35 cycles of at 94

oC for 114

1min annealing at 60oC for 30s extension at 72

oC for 1 min 45s and final extension 72

oC for 115

6 min The re-amplification of the positive strain group was conducted with individual 116

template DNA using the same conditions Amplified PCR reactions were separated on 15 117

agarose gels stained with gel red (Bio-Rad) in Trisacetate-EDTA buffer and gel images were 118

visualized with a Gel Doc XR+ system (Bio-Rad) A 100-bp DNA size standard (Promega) 119

was used to measure the size of the amplified products 120

121

Identification of genes and bacteria 122

Each amplicon gel was excised and purified using the Wizardreg SV Gel and PCR Clean-Up123

System (Promega) according to the manufacturerrsquos instructions The purified DNA fragments 124

were sequenced by the Australian Genome Research Facility (AGRF Brisbane Queensland) 125

All sequences were aligned using MEGA v 62 (Tamura et al 2013a 2731-9) then 126

corresponding genes were identified through a comparative similarity search of all publicly 127

available GenBank entries using BLAST (Altschul et al 1997 3389-402) Bacterial DNA 128

extracted from isolates that positively amplified with all three target primers were subjected 129

to 16S rRNA gene sequencing following partial amplification using universal primers 8F and 130

107

1492R (Turner et al 1999 327-38) The amplified DNA was purified and sequenced as 131

described earlier The sequence data was analyzed by BLASTn software and compared with 132

available gene sequences within the NCBI nucleotide database Strains were identified to 133

species level when the similarity to a type reference strain in GenBank was above 99 134

135

Bio-assay of bacterial strains in vitro 136

The inhibition spectrum of marker selected Bacillus strains against mycelial growth and 137

sclerotial germination were conducted according to the method described by Kamal et al 138

(2015a) S sclerotiorum used in this study was collected from a severely diseased canola 139

plant as described in Kamal et al (2015a) The isolate was subcultured onto potato dextrose 140

agar (PDA) and incubated at 25oC for 3 days Agar plugs (5 mm diameter) colonized with141

fungal mycelium were transferred to fresh PDA and incubated at room temperature in the 142

dark The marker-selected bacterial strain CS-42 was cultured in TSB at 28oC After 24 h143

fresh juvenile cultures of bacterial isolates were measured using a Genesys 20 144

spectrophotometer (Thermo Scientific) and diluted to 108 CFU mL

-1 which was subsequently145

confirmed by dilution plating A 10 microl aliquot of two different bacterial strains were dropped 146

at two equidistant positions along the perimeter of the assay plate and incubated at room 147

temperature in dark for 7 days The inhibition of sclerotial germination by selected bacterial 148

isolates was also investigated Sclerotia grown on baked bean agar (Hind-Lanoiselet 2006) 149

were dipped into the bacterial suspensions and placed onto PDA and incubated at 28oC150

Sclerotia dipped into sterile broth were considered as controls After 7 days incubation the 151

germination of sclerotia was observed using a Nikon SMZ745T zoom stereomicroscope 152

(Nikon Instruments) All challenged sclerotia were transferred onto fresh PDA without the 153

presence of bacteria to observe germination capability Both experiments were conducted in a 154

randomized block design with five replications and assayed three times The inhibition index 155

for each replicate was calculated as follows 156

108

Inhibition () R1 = Radial mycelial colony growth of S sclerotiorum in 157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

control plate R2 = Radial mycelial colony growth of S sclerotiorum in bacteria treated plate

Disease inhibition bioassay in planta

Antagonism of the potential biocontrol strain was evaluated against S sclerotiorum on 50-

day-old canola plants (cv AV Garnet) in the glasshouse A suspension of the antagonistic

bacterial strain CS-42 was adjusted to 108 CFU mL

-1 amended with 005 Tween 20 and

sprayed until runoff with a hand-held sprayer (Hills) at a rate of 5 mL per plant The plants

were covered with transparent moistened polythene bags and incubated for 24 h A

homogenized mixture of 3-day-old mycelial fragments (104

fragments mL-1

) of S

sclerotiorum was sprayed at a rate of 5 mL per plant as described by Kamal et al (2015a)

The plants were covered again and kept for 24 h within a polythene bag to retain moisture

The plant growth chamber was maintained with at a temperature of 25oC and 100 relative

humidity Disease incidence was assessed at 10 day post inoculation (dpi) of the pathogen

The experiment was conducted in a randomized complete block design with 10 replicates and

repeated twice Percentage disease incidence was calculated by observing disease symptoms

and disease severity was recorded using a 0 to 9 scale where 0 = no lesion 1 = lt5 stem

area covered by lesion 3 = 6-15 stem area covered by lesion 5= 16-30 stem area covered

by lesion 7 = 31-50 stem area covered by lesion and 9 = gt50 stem area covered by lesion

(Yang et al 2009 61-5) Disease incidence disease index and control efficacy was

calculated as described below

Disease incidence = (Mean number of diseased stems Mean number of all investigated

stem) times 100

Disease index = [sum (Number of diseased stems in each index times Disease severity index)

(Total number of stem investigated times Highest disease index)] times 100 181

R1-R2 R1

109

Control efficacy = [(Disease index of control - Disease index of treated group) Disease 182

index of control)] times100 183

184

Scanning electron microscope studies 185

From the dual culture assay mycelia were excised at the edges closest to the bacterial 186

colonies and processed as described by Kamal et al (2015b) The samples of mycelia were 187

fixed in 4 (vv) glutaraldehyde (Electron Microscopy Sciences) at 4degC for 48 h then rinsed 188

with 01 M phosphate buffer (pH 68) and dehydrated in a graded series of ethanol The 189

dehydrated samples were subjected to critical-point drying (Balzers CPD 030) mounted on 190

stubs and sputter-coated with gold-palladium The hyphal surface morphology was 191

micrographed using a Hitachi 4300 SEN Field Emission-Scanning Electron Microscope 192

(Hitachi High Technologies) at an accelerator potential of 3thinspkV The SEM studies was 193

conducted with 10 excised pieces of mycelium and repeated two times From the co-culture 194

assay SEM analysis of sclerotia was performed according to Kamal et al (2015b) Sclerotia 195

were vapour-fixed in 2 (wv) aqueous solution of osmium tetroxides (ProSciTech 196

Australia) for 40 h at room temperature then sputter-coated with gold-palladium 197

Micrographs were taken to study the sclerotial surface morphology using a Hitachi 4300 198

SEN Field Emission-Scanning Electron Microscope (Hitachi High Technologies) at an 199

accelerator potential of 3thinspkV The SEM studies were conducted with 10 sclerotia and repeated 200

two times 201

202

Transmission electron microscope studies 203

Samples of mycelia from the dual culture assay were taken from the edge of challenged 204

and control mycelium were infiltrated embedded with LR White resin (Electron Microscopy 205

Sciences USA) in gelatine capsules and polymerized at 55degC for 48 h after fixing post-206

110

fixing and dehydration Based on the observations of semi-thin sections on the light 207

microscope ultrathin sections were cut and collected on 200-mesh copper grids (Sigma-208

Aldrich) After contrasting with uranyl acetate (Polysciences) and lead citrate (ProSciTech) 209

the sections were visualized with a Hitachi HA7100 transmission electron microscope 210

(Hitachi High Technologies) Sclerotia of S sclerotiorum collected from the bacterial 211

challenged and control test were fixed immediately in 3 (vv) glutaraldehyde in 01 M 212

sodium cacodylate buffer (PH 72) for 2 h at room temperature Post fixation was performed 213

in the same buffer with 1 (wtvol) osmium tetroxides at 4oC for 48 h Upon dehydration in a214

graded series of ethanol samples were embedded in LR White resin (Electron Microscopy 215

Sciences) From LR white block 07microm thin sections were cut and stained with 1 aqueous 216

toluidine blue prior to visualize with Zeiss Axioplan 2 (Carl Zeiss) light microscope 217

Ultrathin sections of 80nm were collected on Formvar (Electron Microscopy Sciences) 218

coated 100 mesh copper grids stained with 2 uranyl acetate and lead citrate then 219

immediately examined under a Hitachi HA7100 transmission electron microscope (Hitachi 220

High Technologies) operating at 100kV From each sample 10 ultrathin sections were 221

examined 222

223

RESULTS 224

225

Detection and identification of antibiotic producing bacteria 226

Ninety six Bacillus strains were isolated from the rhizosphere of field grown canola plants 227

and screened for three lipopeptide antibiotics (surfactin iturin A and bacillomycin D) that are 228

antagonistic to S sclerotiorum DNA extracted from each group of 12 combined Bacillus 229

strains were used for multiplex PCR amplification through a combination of three gene 230

specific primers (Table 1) Only one group of bacterial isolates resulted in positive 231

amplification for all three genes corresponding to surfactin iturin A and bacillomycin D 232

111

synthetase biosynthetic cluster (Figure 1A) The positively amplified single group of isolates 233

was individually screened with the same multiplex primers and only strain CS-42 resulted in 234

all three amplicons being amplified (Figure 1B) The multiplex gene specific primers yielded 235

a PCR product of approximately 875 bp with 98 homology to the bamC gene of 236

bacillomycin D operon of type strain B subtilis ATTCAU195 A PCR product of 237

approximately 647 bp was amplified with the gene specific primers with 99 homology to 238

the ituD gene of iturin A operon of B subtilis RB14 in Genbank Amplification of genomic 239

DNA with surfactin specific primers yielded a PCR product of approximately 441 bp with 240

98 homology to the srfDB3 gene of the surfactin operon of B subtilis B3 strain in Genbank 241

These results demonstrated that only strain CS-42 harboured surfactin iturin A and 242

bacillomycin D biosynthesis genes in its genome The strain CS-42 was subjected to 16S 243

rRNA gene sequencing and showed greater than 99 similarity to B cereus UW 85 in 244

Genbank 245

246

Inhibition of S sclerotiorum by Bacillus strains in vitro and in planta 247

The marker selected B cereus CS-42 strain was evaluated against S sclerotiorum on PDA 248

using a dual culture assay to correlate the positive amplification signal with antagonistic 249

activity The dual culture assay demonstrated that strain CS-42 significantly inhibited 250

(802) mycelial growth of S sclerotiorum compared to the control (Figure 2A) and no 251

significant difference in inhibition (819) was observed in comparison to our type 252

biocontrol strain Bacillus cereus SC-1 (Table 2) In contrast one group of isolates that 253

positively amplified both surfactin and iturin A but negative with bacillomycin D 254

demonstrated limited antifungal activity (Figure 2B) There was no mycelial inhibition 255

observed by the strains which were amplified only with surfactin on PDA (Figure 2C) The 256

antifungal spectrum of marker selected strains on sclerotial germination was evaluated on 257

112

PDA After dipping sclerotia into 108 CFU mL

-1 bacterial suspension the results258

demonstrated that the three gene harbouring strain CS-42 completely restricted sclerotial 259

germination after 5 days (Figure 2E) Whereas sclerotia dipped in sterile broth germinated 260

and completely occupied the whole plate (Figure 2F) To confirm the in vitro results strain 261

CS-42 was used in an in planta bioassay in the glasshouse Inoculation of bacteria one day 262

before pathogen inoculation revealed that the disease incidence (394) and disease index 263

(276) was significantly (P=005) reduced in comparison to the control Control plants 264

showed typical symptoms of infection (Figure 2H) with S sclerotiorum by 24 hpi with a 265

dramatic rise in disease incidence with further incubation reaching 100 by 10 dpi The 266

challenge strain CS-42 provided a control efficacy of 698 which was not significantly 267

different (736) to that shown by type strain B cereus SC-1 268

269

SEM and TEM analysis 270

The region of interaction in dual culture of the bacterium and fungus was observed using 271

SEM In this region the hyphal tips of S sclerotiorum displayed extreme morphological 272

alteration characterized by swollen and disintegrated hyphal cells TEM of the hyphal tips 273

from the interaction region revealed extensive vacuolization and disorganization of cell 274

contents In most cases a degrading thin cell wall and folded cytoplasmic membrane was 275

observed Granulated cytoplasm and scattered vacuoles were observed inside the cell 276

indicating abnormal cell structure 277

278

The sclerotia from the co-culture bioassay were also subjected to SEM analysis The 279

bacteria treated sclerotia of S sclerotiorum displayed deformed spherical structures 280

characterized by their severely ruptured collapsed fractured and pitted outmost rind layer 281

Abundant multiplication of bacterial cells inside the ruptured rind cells of parasitized 282

sclerotia were frequently observed Ultrastructural analysis of bacteria treated sclerotia was 283

113

obtained from ultrathin sections through TEM The micrograph demonstrated that most of 284

cells were collapsed and empty whereas a few contained only cytoplasmic remnants The 285

normal properties of the control sclerotia were lost and delimitation of the cell wall indicated 286

that all cells belonging to rind and medulla were completely disintegrated by bacterial 287

metabolites The complete loss of cytoplasmic organization in cells and aggregated 288

cytoplasmic remnants were frequently visible Appearance of electron dense and an electron 289

lucent cell wall demonstrated clear signs of collapse which indicated that damage of cell wall 290

occurred before damage to cytoplasm organelles The rind and medullary cells were 291

extensively altered and disintegrated and cytoplasmic remnants moved among the cells 292

293

DISCUSSION 294

Several members of the Bacillus genus are inhibitory to plant pathogens and could be used 295

as biocontrol agents (Emmert and Handelsman 1999 1-9) The spore forming ability and 296

high level of resistance to heat and desiccation makes these bacteria suitable candidates in 297

stable biopesticide formulations (Ongena and Jacques 2008 115-25) The genus Bacillus 298

exhibit diversity in the production of a wide range of broad spectrum ribosomally or non-299

ribosomally synthesized antibiotics (Stein 2005 845-57) The antimicrobial activity of non-300

ribosomally synthesized lipopeptide antibiotics including surfactins iturins and fengycins 301

have been well documented (Ongena and Jacques 2008 115-25) The in vitro dual culture 302

method of bioassay has been widely used to detect potential antagonistic bacteria from the 303

natural environment However screening of large populations of bacteria using in vitro dual 304

culture assays is laborious and time consuming (De Souza and Raaijmakers 2003 21-34) 305

Molecular techniques have accelerated the detection of some antibiotic producing bacterial 306

strains from huge populations of bacterial isolates (Park et al 2013 637-42) Here we 307

demonstrate a marker-assisted selection approach where desired strains can be selected using 308

primers to target specific antibiotic producing genes Our previous study revealed that gene 309

114

specific primers could amplify surfactin iturin A and bacillomycin D lipopeptide antibiotic 310

corresponding antagonistic biosynthetic operon from antagonistic strain B cereus SC-1 using 311

the same PCR conditions (Kamal et al 2015a) This has led to the design of a multiplex PCR 312

approach for detection of target Bacillus species from mixed populations This genus has 313

been targeted for screening as they have previously been demonstrated to confer 314

antimicrobial activity (Maget-Dana and Peypoux 1994 151-74 Maget-Dana et al 1992 315

1047-51 Moyne et al 2001 622-9 Ongena et al 2007 1084-90 Peypoux et al 1991 101-316

6) 317

318

A rapid marker-based method was introduced to detect novel environmental Bacillus 319

isolates as putative biocontrol agents of a major pathogen of canola S sclerotiorum DNA 320

extracted from combined Bacillus isolates was used as a template for multiplex PCR 321

amplification with a mixture of three sets of previously described primers corresponding to 322

surfactin iturin A and bacillomycin D biosynthetic genes (Ramarathnam et al 2007 901-323

11) From the eight groups of isolates one group of combined isolates was positive for the 324

all three genes The individual re-amplification of this group demonstrated that all three genes 325

together are only contained in one strain The production of amphiphilic lipopeptides has 326

been reported by endospore-forming bacteria including B subtilis (Peypoux et al 1999 553-327

63) Surfactin is a bacterial cyclic lipopeptide which is commonly used as an antibiotic and 328

largely prominent for its exceptional surfactant activity (Mor 2000 440-7) Surfactin was 329

observed to exhibit antagonism against bacteria viruses fungi and mycoplasmas (Ongena et 330

al 2007 1084-90 Singh and Cameotra 2004 142-6) Although surfactins are not directly 331

responsible for antagonism to fungal pathogens they are associated with developmental 332

functions and catalyze synergistic effects that accelerate the defence activity of iturin A 333

(Hofemeister et al 2004 363-78 Maget-Dana et al 1992 1047-51) In addition they can 334

115

interrupt phospholipid functions and are able to produce ionic pores in lipid bilayers of 335

cytoplasmic membranes (Sheppard et al 1991 13-23) 336

337

The cyclic lipopeptide iturins such as iturin A iturin C iturin D iturin E bacillomycin D 338

bacillomycin F bacillomycin L bacillomycin Lc and mycosubtilin have been classified into 339

nine groups on the basis of variation of amino acids in their peptide profile (Pecci et al 340

2010 772-8) Among all the iturins iturin A has been shown to be secreted by most Bacillus 341

strains and found to exhibit a broad spectrum of antifungal activity against pathogenic yeast 342

and fungi (Klich et al 1994 123-7) It interacts with the cytoplasmic membranes of the 343

target cell forming ion conducting pores Its mode of action could be attributed to its 344

interaction with sterols and phospholipids In the current study only one group was positive 345

for iturin A Iturin A confers strong antimycotic activity against several phytopathogens and 346

is commonly produced by Bacillus spp (Maget-Dana et al 1992 1047-51 Ramarathnam et 347

al 2007 901-11) Bacillomycin D produced by Bacillus spp also exhibits strong antifungal 348

activity against a wide range of fungal plant pathogen (Moyne et al 2001 622-9) The 349

limited detection of isolates containing the gene conferring bacillomycin D production among 350

the groups of isolates tested indicates their low frequency in the natural environment This 351

result is supported by Ramarathnam et al (2007 901-11) who showed that the percentage of 352

bacteria producing bacillomycin D are comparatively low in the environment and their 353

capability to produce the antibiotic may be variable 354

355

We observed the presence of either surfactin or iturin A producing genes in another group 356

of isolates From each group individual isolates were subjected to further PCR screening and 357

yielded two positive for surfactin and one for iturin A It is interesting to note that in vitro 358

dual culture assays with iturin A positive isolates demonstrated limited inhibition and the 359

surfactin positive isolate showed zero inhibition of mycelial growth and sclerotial 360

116

germination of S sclerotiorum This finding indicates that a combined synergistic effect of 361

these antibiotics maybe necessary to suppress the pathogen The production of several 362

lipopeptide antibiotics is often a very important factor in mycolytic activity against S 363

sclerotiorum which was also evident in this study Challis and Hopwood (2003 14555-61) 364

described that the production of multiple antibiotics by sessile actinomycetes has a 365

synergistic action in the competition for food and space with other microorganisms Another 366

study revealed that B subtilis M4 is a multiple antibiotic producer however only fengycin 367

was recovered from protected apple tissue upon bacterial inoculation against Botrytis cinerea 368

(Ongena et al 2005 29-38) Studies to better understand these antibiotic producing bacteria 369

their synthesis and activity against Sclerotinia spp are required 370

371

The antagonistic strain CS-42 was identified as B cereus through 16S rRNA gene 372

sequencing In vitro dual culture assay with CS-42 revealed that the strain was able to 373

suppress mycelial growth significantly and completely restricted sclerotial germination 374

Electron microscopy of the interaction region in the dual culture and parasitized sclerotia 375

demonstrated that the mode of action of this bacterium may be via antibiosis The inhibition 376

of fungal radial mycelial growth by agar-diffusible metabolites produced by bacteria is an 377

indication of antibiosis (Burkhead et al 1995 1611-6) The scanning electron micrograph 378

demonstrated that mycelial growth towards the bacteria was restricted in the vicinity of 379

bacterial metabolites and the hyphae became vacuolated The transmission electron 380

micrograph of hyphal tips from the interaction region revealed damage to hyphal cell walls as 381

well as disintegrated cell contents SEM analysis of parasitized sclerotia revealed that the 382

outmost rind layer was ruptured and heavy colonization of bacteria was observed The TEM 383

study indicated the complete destruction of medullary cells and transgresses of the cytoplasm 384

was evident among cells along with other cell contents This result was in agreement with our 385

117

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

previous study where B cereus SC-1 destroyed mycelium and sclerotia similarly observed by

SEM and TEM (Kamal et al 2015) The histological abnormalities of sclerotia have also

been reported to be caused by secondary metabolites produced by Propionicimonas sp

(ENT-18) (Zucchi et al 2010 811-9) Moreover in planta evaluation of strain CS-42 against

S sclerotiorum demonstrated significant disease suppression Our previous study revealed

that B cereus SC-1 demonstrated profound antifungal activity against mycelial growth and

sclerotial germination of S sclerotiorum This strain has provided a significant reduction of

disease incidence in canola both in glasshouse and field conditions (Kamal et al 2015a)

Another similar study showed that foliar application of lipopeptide producing B

amyloliquefaciens strains ARP23 and MEP218 conferred significant disease reduction of

Sclerotinia stem rot in soybean (Alvarez et al 2012 159-74)

Antagonistic Bacillus strains can be rapidly detected by gene specific primers from

enormous natural rhizosphere bacterial communities of particular ecosystems The direct

search for antagonistic bacteria from combined samples via genetic markers may accelerate

the selection of biocontrol agents against specific plant pathogens The canola rhizosphere

associated bacteria isolated in this study has demonstrated lt1 representation of potential

antagonism which might be surprising that such a very low abundance of soil bacteria have

significant functional impact on pathogens such as S sclerotiorum However similar research

has previously demonstrated that only 1 of 24-diacetylphloroglucinol producing soil

inhabiting pseudomonads demonstrated significant antagonism towards soil borne pathogens

(Beniacutetez and Gardener 2009 915-24 McSpadden Gardener et al 2005 715-24) Finally we

propose that this approach may assist in rapid selection of bacterial antagonists and improve

selection accuracy The present marker-assisted selection protocol could be adapted towards

various microbes having existing markers and iterative application of such selection methods 410

118

may lead to the development of more effective pathogen-suppressing bacteria against various 411

diseases including Sclerotinia stem rot of canola 412

413


We are thankful to Professor Brian McSpadden Gardener of Ohio State University USA for 415

providing the training necessary to conduct the experiments We also thank Jiwon Lee at the 416

Centre for Advanced Microscopy (CAM) Australian National University and the Australian 417

Microscopy amp Microanalysis Research Facility (AMMRF) for access to the Hitachi 4300 418

SEN Field Emission-Scanning Electron Microscope and Hitachi HA7100 Transmission 419

Electron Microscope 420

421

FUNDINGS 422

The study was funded by a Faculty of Science (Compact) scholarship Charles Sturt 423

University Australia 424

425

Conflict of interest None declared 426

427

REFERENCES 428

Altschul SF Madden TL Schaumlffer AA et al Gapped BLAST and PSI-BLAST a new 429


402 431

Alvarez F Castro M Priacutencipe A et al The plant associated Bacillus amyloliquefaciens strains 432

MEP218 and ARP23 capable of producing the cyclic lipopeptides iturin or surfactin 433

and fengycin are effective in biocontrol of sclerotinia stem rot disease J Appl 434

Microbiol 2012112 159-74 435

119

Bais HP Weir TL Perry LG et al The role of root exudates in rhizosphere interactions with 436

plants and other organisms Annu Rev Plant Biol 200657 233-66 437

Beniacutetez MS Gardener BBM Linking sequence to function in soil bacteria sequence-directed 438

isolation of novel bacteria contributing to soilborne plant disease suppression Appl 439

Environ Microbiol 200975 915-24 440

Burkhead KD Schisler DA Slininger PJ Bioautography shows antibiotic production by soil 441

bacterial isolates antagonistic to fungal dry rot of potatoes Soil Biol Biochem 442

199527 1611-6 443

Challis GL Hopwood DA Synergy and contingency as driving forces for the evolution of 444

multiple secondary metabolite production by Streptomyces species Proc Natl Acad 445

Sci U S A 2003100 14555-61 446

De Souza JT Raaijmakers JM Polymorphisms within the prnD and pltC genes from 447

pyrrolnitrin and pyoluteorin-producing Pseudomonas and Burkholderia spp FEMS 448

Microbiol Ecol 200343 21-34 449

Emmert EAB Handelsman J Biocontrol of plant disease a (Gram-) positive perspective 450

FEMS Microbiol Lett 1999171 1-9 451

Figueroa-Loacutepez AM Cordero-Ramiacuterez JD Quiroz-Figueroa FR et al A high-throughput 452

screening assay to identify bacterial antagonists against Fusarium verticillioides J 453

Basic Microbiol 201454 S125-S33 454

Gardener BBM Mavrodi DV Thomashow LS et al A rapid polymerase chain reaction-based 455

assay characterizing rhizosphere populations of 24-diacetylphloroglucinol-producing 456

bacteria Phytopathology 200191 44-54 457

Giacomodonato MN Pettinari MJ Souto GI et al A PCR-based method for the screening of 458

bacterial strains with antifungal activity in suppressive soybean rhizosphere World J 459

Microbiol Biotechnol 200117 51-5 460

120

Hind-Lanoiselet T Forecasting Sclerotinia stem rot in Australia School of Agricultural and 461

Wine Sciences volume PhD Wagga Wagga NSW Australia Charles sturt 462

University 2006 463

Hofemeister J Conrad B Adler B et al Genetic analysis of the biosynthesis of non-464

ribosomal peptide and polyketide-like antibiotics iron uptake and biofilm formation 465

by Bacillus subtilis A13 Mol Genet Genomics 2004272 363-78 466

Jacobsen B Zidack N Larson B The role of Bacillus-based biological control agents in 467

integrated pest management systems Plant diseases Phytopathology 200494 1272-468

5 469

Joshi R McSpadden Gardener BB Identification and characterization of novel genetic 470

markers associated with biological control activities in Bacillus subtilis 471

Phytopathology 200696 145-54 472

Kamal MM Lindbeck KD Savocchia S et al Biological control of sclerotinia stem rot of 473

canola using antagonistic bacteria Plant Pathol 2015adoi101111ppa12369 474

Kamal MM Savocchia S Lindbeck K et al Mode of action of the potential biocontrol 475

bacterium Bacillus cereus SC-1 against Sclerotinia sclerotiorum BioControl 476

2015b(Unpublished work) 477

Klich MA Arthur KS Lax AR et al Iturin A a potential new fungicide for stored grains 478

Mycopathologia 1994127 123-7 479

Kluge B Vater J Salnikow J et al Studies on the biosynthesis of surfactin a lipopeptide 480

antibiotic from Bacillus subtilis ATCC 21332 FEBS Lett 1988231 107-10 481

Maget-Dana R Peypoux F Iturins a special class of pore-forming lipopeptides Biological 482

and physicochemical properties Toxicology 199487 151-74 483

121

Maget-Dana R Thimon L Peypoux F et al Surfactiniturin A interactions may explain the 484

synergistic effect of surfactin on the biological properties of iturin A Biochimie 485

199274 1047-51 486

McSpadden Gardener BB Gutierrez LJ Joshi R et al Distribution and biocontrol potential of 487

phlD+ pseudomonads in corn and soybean fields Phytopathology 200595 715-24 488

Mor A Peptide‐based antibiotics A potential answer to raging antimicrobial resistance Drug 489

Dev Res 200050 440-7 490

Moyne AL Shelby R Cleveland T et al Bacillomycin D an iturin with antifungal activity 491

against Aspergillus flavus J Appl Microbiol 200190 622-9 492

Ongena M Jacques P Bacillus lipopeptides versatile weapons for plant disease biocontrol 493

Trends Microbiol 200816 115-25 494

Ongena M Jacques P Toureacute Y et al Involvement of fengycin-type lipopeptides in the 495

multifaceted biocontrol potential of Bacillus subtilis Appl Microbiol Biotechnol 496

200569 29-38 497

Ongena M Jourdan E Adam A et al Surfactin and fengycin lipopeptides of Bacillus subtilis 498

as elicitors of induced systemic resistance in plants Environ Microbiol 20079 1084-499

90 500

Pal KK Gardener BMS Biological control of plant pathogens Plant healt instruct 20062 501

1117-42 502

Park JK Lee SH Han S et al Marker‐assisted selection of novel bacteria contributing to 503

soil‐borne plant disease suppression Molecular Microbial Ecology of the 504

Rhizosphere Volume 1 amp 2 2013 637-42 505

Pecci Y Rivardo F Martinotti MG et al LCESI‐MSMS characterisation of lipopeptide 506

biosurfactants produced by the Bacillus licheniformis V9T14 strain J Mass Spectrom 507

201045 772-8 508

122

Peypoux F Bonmatin J Wallach J Recent trends in the biochemistry of surfactin Appl 509


Peypoux F Bonmatin JM Labbe H et al Isolation and characterization of a new variant of 511

surfactin the [val7] surfactin Eur J Biochem 1991202 101-6 512

Raaijmakers JM Weller DM Thomashow LS Frequency of antibiotic-producing 513

Pseudomonas spp in natural environments Appl Environ Microbiol 199763 881 514

Ramarathnam R Mechanism of phyllosphere biological control of Leptosphaeria maculans 515

the black leg pathogen of canola using antagonistic bacteria Department of Plant 516

Science volume PhD Winnipeg Manitoba Canada University of Manitoba 2007 517

Ramarathnam R Sen B Chen Y et al Molecular and biochemical detection of fengycin and 518

bacillomycin D producing Bacillus spp antagonistic to fungal pathogens of canola 519

and wheat Can J Microbiol 200753 901-11 520

Sheppard J Jumarie C Cooper D et al Ionic channels induced by surfactin in planar lipid 521

bilayer membranes Biochim Biophys Acta 19911064 13-23 522

Singh P Cameotra SS Potential applications of microbial surfactants in biomedical sciences 523

Trends Biotechnol 200422 142-6 524

Stein T Bacillus subtilis antibiotics structures syntheses and specific functions Mol 525

Microbiol 200556 845-57 526

Tamura K Peterson D Peterson N et al MEGA5 molecular evolutionary genetics analysis 527

using maximum likelihood evolutionary distance and maximum parsimony methods 528

Mol Biol Evol a201328 2731-9 529

Turner S Pryer KM Miao VPW et al Investigating deep phylogenetic relationships among 530

cyanobacteria and plastids by small subunit rRNA sequence analysis J Eukaryot 531

Microbiol 199946 327-38 532

123

Weller DM Biological control of soilborne plant pathogens in the rhizosphere with bacteria 533

Ann Rev Phytopathol 198826 379-407 534

Yang D Wang B Wang J et al Activity and efficacy of Bacillus subtilis strain NJ-18 against 535

rice sheath blight and Sclerotinia stem rot of rape Biol Control 200951 61-5 536

Yu GY Sinclair JB Hartman GL et al Production of iturin A by Bacillus amyloliquefaciens 537

suppressing Rhizoctonia solani Soil Biol Biochem 200234 955-63 538

Zucchi TD Almeida LG Dossi FCA et al Secondary metabolites produced by 539


Sclerotinia sclerotiorum BioControl 201055 811-9 541

542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

124

Tables 559


Antibiotic Primer Primer sequence Reference

Surfactin SUR3F ACAGTATGGAGGCATGGTC

(Ramarathnam 2007

Ramarathnam et al

2007)

SUR3R TTCCGCCACTTTTTCAGTTT

Iturin A ITUD1F GATGCGATCTCCTTGGATGT

ITUD1R ATCGTCATGTGCTGCTTGAG

Bacillomycin

D

BAC1F GAAGGACACGGCAGAGAGTC

BAC1R CGCTGATGACTGTTCATGCT

561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

125

585

Table 2 Effect of Bacillus cereus CS-42 (108 CFU mL

-1) against Sclerotinia sclerotiorum 586

and comparison with the type strain Bacillus cereus SC-1 in vitro and in the glasshouse 587

Strain

In vitro In planta

Mycelial colony

diameter (mm)

Inhibition

()

Disease

incidence ()

Disease

index ()

Control

efficacy ()

CS-42 181 b 802 a 394 b 276 b 698 a

SC-1 154 b 819 a 376 b 242 b 736 a

Control 850 a - 100 a 916 a -



590

591

592

593

594

595

596

597

598

599

600

126

Figures 601

602 603

Fig 1 Detection of antibiotic target sequences in bacteria from canola rhizosphere samples 604

(A) Eight groups of Bacillus (12 in each) consisting of 96 isolates were amplified605

simultaneously and showed the presence of three genes (surfactin iturin A and bacillomycin 606

D) in the 4th

group (B) Multiplex PCR amplification of the 4th

group and comprised of 12607

individual isolates produced three amplicons for isolate CS-42 and a single amplicon for 608

isolates CS-38 and CS-45 A 100-bp DNA size standard (M) (Promega) was used to measure 609

the size of the amplified products The visualization of bands in amplification product 610

indicates that target genes were present in the isolate 611

612

613

127

614

615

Fig 2 Antagonism assessment of Bacillus strains against Sclerotinia sclerotiorum In vitro 616

dual culture inhibition of mycelial growth by (A) CS-42 (B) CS-38 (C) CS-45 and (D) 617

Control at 7 dpi The clear zone between bacteria and fungi indicates bacterial antagonism 618

(E) Inhibition of sclerotial germination by dipping the sclerotia into 108 CFU mL

-1 of CS-42619

strain at 5 dpi showing inhibition of germination (F) Control sclerotia germinate and occupy 620

the entire plate Evaluation of bacterial antagonism on an entire canola plant at 10 dpi (G) 621

CS-42 treated plant showing disease-free appearance (H) Control plants showing a gradual 622

increase in typical stem rot symptoms 623

624

625

626

627

628

629

630

631

632

128

633

Fig 3 Scanning electron micrographs of mycelium and sclerotia of S sclerotiorum (A) 634

Mycelia excised at the edges towards the bacterial colonies showing inhibition and 635

destruction of mycelial growth in the vicinity of bacterial metabolites (B) Control hyphae 636

demonstrated aggressive growth (C) Bacteria treated sclerotia showing disintegration of 637

outer rind layer and heavy colonization (B) Control sclerotia showing intact globular outer 638

rind layer 639

640

641

642

643

644

645

646

129

647


sclerotia of S sclerotiorum (A) Bacteria treated sclerotia appearing hollow with a lack of 649

cellular contents (B) Control sclerotia showing intact cellular organelles (C) Bacteria treated 650

sclerotia showing massive disintegration of cell wall and transgression of cellular contents 651

(D) Control sclerotia showing intact electron dense cell walls and cellular contents652

653

654

Chapter 8 General Discussion

This thesis reports on the development of a promising bacterial biocontrol agent with

multiple modes of action for sustainable management of sclerotinia stem rot in canola The

disease is an important threat to canola and other oilseed Brassica production in Australia and

around the world This research was prompted by surveys conducted in 1998 1999 and 2000

in southeastern Australia where the incidence of stem rot was found to exceed 30 and

resulted in yield losses of 15-30 (Hind-Lanoiselet et al 2003) More recently yield loss

was reported as 039-154 tha in southern NSW Australia (Kirkegaard et al 2006) Murray

and Brennan (2012) estimated the annual losses in Australia due to stem rot of canola to be

AUD 399 million and the cost of fungicide to control stem rot was reported to be

approximately AUD 35 per hectare In 2012 and 2013 an increase in inoculum pressure was

observed in high-rainfall zones of NSW and an outbreak of stem rot in canola was also

reported across the wheat belt region of Western Australia (Khangura et al 2014)

The potential threat of sclerotinia to canola production in Australia raised the question as to

whether the pathogen could have the potential to cause epidemics in other countries such as

Bangladesh S sclerotiorum has been reported on mustard (Hossain et al 2008) chilli

aubergine cabbage (Dey et al 2008) and jackfruit (Rahman et al 2015) Being a

comparatively new pathogen in Bangladesh research regarding distribution of the pathogen

and incidence of sclerotinia stem rot in an intense mustard growing area was explored An

intensive survey was conducted in eight northern districts of Bangladesh in 2013-14

Sclerotinia stem rot was observed in all districts with petal and stem infection ranging from

237 to 487 and 23 to 74 respectively It is noteworthy that S sclerotiorum was

isolated from both symptomatic petals and stems whereas S minor was isolated for the first

130

time only from symptomatic petals The survey demonstrated that sclerotinia stem rot poses a

similar degree of threat to oilseed industries in Australia and Bangladesh

Sustainable management of S sclerotiorum still remains a challenging issue globally

Sclerotia are black melanised compact and hard resting structure that inhabit soil and remain

viable up to 5 years (Fernando et al 2004) Apart from myceliogenic germination of

sclerotia millions of ascospores are released through carpogenic germination The senescing

petals of canola serve as a nutrient source for ascospores which assist in proliferation and the

establishment of pathogenicity (Saharan amp Mehta 2008) Therefore management of sclerotia

resting in the soil as well as in petals and other aerial plant parts need to be protected from the

ingress of ascospores Several methods ranging from cultural control to host resistance have

been used to control S sclerotiorum There is no doubt of the contribution of cultural

methods in reducing sclerotinia disease incidence though seed treatment altering row wider

plant spacing using erect and non-lodging varieties organic amendment incorporation soil

solarisation and crop rotation but their efficacy is not satisfactory to develop a reliable

strategy for managing sclerotinia stem rot in canola and mustard crops (Fernando et al

2004) The wide host range limited host resistance and inappropriate timing of fungicide

application at the correct ascospore release stage have reduced the efficacy of techniques

which can be adopted for management of S sclerotiorum (Sharma et al 2015)

Scientists are now facing one of the greatest ecological challenges the development of eco-

friendly alternatives to chemical pesticides against crop diseases The potential use of

antagonistic micro-organisms formulated as bio-pesticides has attracted attention worldwide

as a promising rational and safe disease management option (Fravel 2005) The terms

131

ldquobiological controlrdquo and its abbreviated synonym ldquobiocontrolrdquo was defined as lsquothe reduction

of inoculum density or disease producing activities of a pathogen or parasite in its active or

dominant state by one or more organisms accomplished naturally or through manipulation

of the environment host or antagonist or by mass introduction of one or more antagonistsrsquo

(Baker amp Cook 1974) Recently it was redefined as lsquothe purposeful utilisation of introduced

or resident living organisms other than disease resistant host plants to suppress the activities

and populations of one or more plant pathogensrsquo (Pal amp Gardener 2006) The first recorded

attempt at biological control was the control of damping off of pine seedlings using

antagonistic fungi (Hartley 1921) The success of disease reduction through the use of

antagonists between 1920 to 1940 led to researchers conducting more experiments with

diverse organisms (Millard amp Taylor 1927) Successful management of take-all of wheat

through natural biocontrol agents opened a new era of relevant research between 1960 to

1965 (Cook 1967) They found that suppression of take-all was achieved through the

presence of another natural microorganism This understanding raised the question of

whether wheat could grow better by reducing take-all through the introduction of natural

biocontrol agents (Gerlagh 1968) Today scientists are explaining biological control as the

exploitation of a natural phenomenon The key player is one or more antagonists that have the

potential to interfere in the life cycles of plant pathogens such as fungi bacteria virus

nematodes protozoa viroids and trap plants (Cook amp Baker 1983) A potential biocontrol

agent should have properties including an ability to compete persist colonise proliferate be

inexpensive be produced in large quantities maintain viability and be non-pathogenic to the

host plant and the environment Production must result in biomass with an extended shelf life

and the delivery and application must permit full expression of the agent (Wilson amp

Wisniewski 1994)

132

Amid these scientific developments a number of fungal based biocontrol agents have

emerged as promising tools for the management of S sclerotiorum The fungal mycoparasite

Coniothyrium minitans has been formulated and commercialised as Contans WG for the

management of sclerotinia diseases in susceptible crops However Contans WG has often

displayed inconsistent activity under field conditions (Fernando et al 2004) Several studies

have been conducted on the exploration of fungal biocontrol agents but research on bacterial

antagonist for the management of S sclerotiorum is scarce Very recently our laboratory has

conducted pioneering work in Australia by using bacterial antagonists for the management of

S sclerotiorum in canola and lettuce Our study identified Bacillus cereus SC-1 B cereus P-

1 and Bacillus subtilis W-67 antagonists from 514 naturally occurring bacterial isolates

which mediated biocontrol of stem rot in the phyllosphere of canola (Kamal et al 2015) The

rationale for the collection of bacterial isolates from canola petals and sclerotia was inspired

by the study conducted by Upper (1991) who described that in general a unique variety of

bacterial species are present in the phyllosphere and are able to compete as natural enemies

against pests and pathogens

Inglis and Boland (1990) demonstrated that application of ascospores on bean petals prior to

surface sterilisation significantly reduced disease incidence of S sclerotiorum which

indicated that the microorganisms of a particular habitat play an important role in suppressing

pathogens It is interesting to note that the most promising antagonistic strain B cereus SC-1

B cereus P-1 and B subtilis W-67 were isolated from sclerotia and canola petal which

confirmed the presence of natural antagonists in the surrounding environment of the invading

pathogen Among the antagonistic bacteria Bacillus spp are known to produce antifungal

antibiotics (Edwards et al 1994) heat and desiccation resistant endospores (Sadoff 1972)

and have high efficacy against pathogens as aerial leaf sprays (Ramarathnam 2007) The

133

bacterial biocontrol agents developed in this study belong to Bacillus spp and showed

significant antagonism in vitro through the production of non-volatile and volatile

metabolites These antagonistic strains significantly reduced the viability of sclerotia Spray

treatments of bacterial strains not only reduced disease incidence in the glasshouse but also

showed similar control efficiency of stem rot of canola as the recommended commercially

available fungicide Prosaro 420SC (Bayer CropScience Australia) under field conditions

Timing of bacterial application to the crop is crucial to control sclerotinia infection This

study established that disease incidence and control efficiency was significantly reduced

when the bacteria were applied in the field at 10 flowering stage of canola The bacterial

colonisation at early blossom stage may have prevented the establishment of ascospores This

investigation could provide the opportunity to utilise a natural biocontrol agent for

sustainable management of sclerotinia stem rot of canola in Australia

Application of chemical fungicides for the management of lettuce drop is a serious health

concern as lettuce is often freshly consumed (Subbarao 1998) This investigation

demonstrated that soil drenching with B cereus SC-1 restricted the sclerotial activity

reduced sclerotial viability below 2 and provided complete protection of the seedlings from

pathogen invasion The antagonistic bacterium B cereus SC-1 might have the potential to kill

or disintegrate overwintering sclerotia through parasitism Incorporation of bacteria into the

soil might break the lifecycle of the pathogen by killing overwintering sclerotia Apart from

disease protection B cereus SC-1 also promoted lettuce growth both in vitro and under

glasshouse conditions The production of volatile organic compounds by B cereus SC-1

appears to not only suppress pathogen growth by activating induced systemic resistance but

also induced growth promotion Plant growth promotion by volatile organic compounds

emitted from several rhizobacterial species have been previously documented (Farag et al

134

2006 Ryu et al 2003) B cereus strain SC-1 might directly regulate plant physiology by

mimicking synthesis of plant hormones which promotes the growth of lettuce Identification

of bacterial volatiles that trigger growth promotion would pave the way for better

understanding of this mechanism The growth promotion of lettuce in the glasshouse study

might be attributed to a combined production of metabolites by this bacterium that trigger

certain metabolic activities which enhance plant growth The ability to colonise and

prolonged survival are important properties of potential biocontrol agents B cereus strain

SC-1 showed optimum rhizosphere competency as well as sclerotial colonisation ability by

reaching noteworthy population levels and survivability Detailed investigations of strain SC-

1 in natural field conditions could provide the necessary information for successful biocontrol

of sclerotinia lettuce drop

This study demonstrated that strong antifungal action resulting from cell free culture filtrates

was probably due to direct interference of bioactive compounds released from B cereus

strain SC-1 In addition we observed that bioactive molecules released from B cereus SC-1

inhibited S sclerotiorum and showed greater efficacy than B subtilis strains against

Phomopsis phaseoli as reported by Etchegaray et al (2008) In planta bioassays of culture

filtrates on 10-dayold canola cotyledons reduced lesion development when applied one day

prior to pathogen inoculation However it is noteworthy that when the culture filtrates were

applied one day after pathogen inoculation no significant difference was observed in lesion

development compared to the controls This may be attributed to the bioactive metabolites

within the culture filtrate that display a preventive rather than curative nature against the

pathogen Electron microscopic observation revealed that B cereus strain SC-1 caused

unusual swellings alteration of cell wall structure damage to the cytoplasmic membrane of

both mycelium and sclerotia and caused emptying of cytoplasmic content from cells The loss

135

of cytoplasmic material may be attributed to the increase in porosity of both the cell wall and

cytoplasmic membrane by metabolites Incubation of sclerotia in culture filtrates of B cereus

strain SC-1 not only removed the melanin from the outmost rind layer but also damaged the

medullary cells of the sclerotia The action of lipopeptide antibiotics andor hydrolytic

enzymes might have contributed to cell damage A comparatively low percentage of

environmental bacteria have the ability to produce several antibiotics and hydrolytic enzymes

simultaneously B cereus strain SC-1 was shown to have genes for the production of

bacillomycin D iturin A fengycin and surfactin as well as chitinase and β-glucanase which

may release lipopeptide antibiotics and hydrolytic enzymes that deploy a joint antagonistic

action towards the growth of S sclerotiorum

For sustainable management of sclerotinia diseases emphasis should be given to the

management of sclerotia which are the primary source of inoculum in the environment

(Bardin amp Huang 2001) Several investigations on the control of Sclerotinia spp have

focused mainly on the restriction of ascospores and mycelium however information

regarding the mode of action of bacterial metabolites on sclerotia is comparatively scarce

(Zucchi et al 2010) Further confirmation of the presence of these lipopeptides and enzymes

in broth culture using spectrometric analysis followed by a rigorous biosafety investigation

may assist in providing further evidence for the positive use of B cereus strain SC-1 andor

the derived compounds for commercial application against S sclerotiorum

The rapid and efficient selection of potential biocontrol agents is important to the

development of microbial pesticides The use of direct dual culture evaluation to screen

biocontrol potential bacteria is a time consuming process Marker assisted selection is a

process whereby a molecular marker is used for indirect selection of a genetic determinant or

136

determinants of a trait of interest which is mainly used in plant breeding to develop certain

desired varieties (Collard amp Mackill 2008) DNA markers would provide the opportunity to

accelerate the selection process for antagonists and improve selection accuracy It is also

noteworthy that after rigorous screening of thousands of strains a single potential strain may

still not be present among those isolates The current study revealed a unique marker-assisted

approach to screen Bacillus spp with biocontrol potential to combat sclerotinia stem rot of

canola Canola rhizosphere inhabiting strains of bacteria were screened using multiplex PCR

by combining three primers for three corresponding antibiotic genes Positive amplification

of the three genes was observed in B cereus strain CS-42 This strain was found to be

effective in significantly inhibiting the growth of S sclerotiorum in vitro and in planta

Interestingly Bacillus harbouring a single antibiotic gene showed very little or no inhibition

This might be attributed to the synergistic effect of these lipopeptide antibiotics against the

pathogen

Scanning electron microscopy studies of the dual culture interaction region revealed that

mycelial growth was inhibited in the vicinity of bacterial metabolites Complete destruction

of the outmost melanised rind layer was observed when sclerotia were treated with B cereus

Sc-1 or CS-42 Transmission electron microscopy of ultrathin sections of hyphae and

sclerotia of S sclerotiorum challenged with B cereus strain CS-42 showed vacuolated

hyphae as well as degradation of organelles in the sclerotial cells This study has shown that

the presence of antibiotic biosynthesis genes in certain Bacillus strains can be detected by

multiplex PCR in the natural canola rhizosphere bacterial community The use of direct PCR

instead of direct dual culture could accelerate the search for potential biocontrol strains in

specific crop pathogen interactions which are also better adapted to soil conditions than

foreign strains In view of the results obtained from this study we propose a rapid and

137

efficient method through amplification of combined rhizosphere bacterial DNA to detect

useful Bacillus strains with antifungal activity which should lead to the more rapid

development of promising microbial biopesticides

Though the research presented in this thesis on biological control of S sclerotiorum with

Bacillus spp could pave the way for better disease management strategies the following

research needs to be conducted for the further development of a potential biocontrol agent

1 Development of an economically viable large-scale production approach for inoculum of

B cereus SC-1 and standardisation of biocontrol formulations Evaluation of the performance

of formulations should be conducted over a wide range of field conditions for the suppression

of S sclerotiorum infection of canola

2 Bio-efficacy improvement of B cereus SC-1 using gene technology and evaluation of

performance in different ecological niches The population stability of B cereus SC-1 should

be monitored in relation to pathogen population and their ecological parameters to ensure

biological balance and to regulate the augmentation of biocontrol inoculum

3 Research on compatibility with agrochemicals is essential to use the biocontrol agent as a

tool in Integrated Disease Management (IDM) The IDM strategy including cultural

chemical biological with B cereus SC-1 and host resistance should be refined retested and

revalidated under natural field conditions in regional trials

4 Intensive marker assisted screening should be conducted to detect and develop a widely

adaptable consortium of native biocontrol agents (BCA) in comparison to B cereus SC-1 and

CS-42 that display high competitive saprophytic ability enhanced rhizosphere competence

while providing higher magnitude of biological suppressive activity against S sclerotiorum

and other plant pathogens

138

References

Agrios GN 2005 Plant Pathology New York Elsevier Academic

Anonymous 2014 Canola Crop report (June) In Australian Oilseed Federation 1-3

Bailey KL 1996 Diseases under conservation tillage systems Canadian Journal of Plant

Science 76 635-9

Baker K Cook RJ 1974 Biological control of plant pathogens WH Freeman and Company

Barbetti MJ Banga SK Fu TD et al 2014 Comparative genotype reactions to Sclerotinia

sclerotiorum within breeding populations of Brassica napus and B juncea from India and

China Euphytica 197 47-59

Bardin S Huang H 2001 Research on biology and control of Sclerotinia diseases in Canada

Canadian Journal of Plant Pathology 23 88-98

Boland GJ Hall R 1994 Index of plant hosts of Sclerotinia sclerotiorum Canadian Journal of

Plant Pathology 16 93-108

Collard BCY Mackill DJ 2008 Marker-assisted selection an approach for precision plant

breeding in the twenty-first century Philosophical Transactions of the Royal Society B

Biological Sciences 363 557-72

Colton B Potter T Canola in Australia the first thirty years In Salisbury PA Potter TD

Mcdonald G Green AG eds Proceedings of the 10th International Rapeseed Congress

1999 Canberra Australia Australian Oilseed Federation 1-4

Cook R 1967 Gibberella avenacea sp n perfect stage of Fusarium roseum f sp cerealis

avenaceum Phytopathology 57 732-6

Cook RJ Baker KF 1983 The nature and practice of biological control of plant pathogens

American Phytopathological Society

de Souza JT Raaijmakers JM 2003 Polymorphisms within the prnD and pltC genes from

pyrrolnitrin and pyoluteorin-producing Pseudomonas and Burkholderia spp FEMS

Microbiology Ecology 43 21-34

Dey TK Hossain MS Walliwallah H Islam K Islam S Hoque E 2008 Sclerotinia

sclerotiorum An emerging threat for crop production In BSPC BARI Bangladesh 12

Edwards S Mckay T Seddon B 1994 Interaction of Bacillus species with phytopathogenic

fungi Methods of analysis and manipulation for biocontrol purposes In Blakeman JP

Williamson B eds Ecology of plant pathogens Wallingford CAB International 101-18

Etchegaray A De Castro Bueno C De Melo IS et al 2008 Effect of a highly concentrated

lipopeptide extract of Bacillus subtilis on fungal and bacterial cells Archives of Microbiology

190 611-22

139

140

Farag MA Ryu CM Sumner LW Pareacute PW 2006 GC-MS SPME profiling of rhizobacterial

volatiles reveals prospective inducers of growth promotion and induced systemic resistance

in plants Phytochemistry 67 2262-8

Fernando W Ramarathnam R Krishnamoorthy AS Savchuk SC 2005 Identification and use of

potential bacterial organic antifungal volatiles in biocontrol Soil Biology and Biochemistry

37 955-64

Fernando WGD Nakkeeran S Zhang Y 2004 Ecofriendly methods in combating Sclerotinia

sclerotiorum (Lib) de Bary In Recent Research in Developmental and Environmental

Biology Kerala India Research Signpost 329-47

Fravel D 2005 Commercialization and implementation of biocontrol Annual Review of

Phytopathology 43 337-59

Gardener BBM Mavrodi DV Thomashow LS Weller DM 2001 A rapid polymerase chain

reaction-based assay characterizing rhizosphere populations of 24-diacetylphloroglucinol-

producing bacteria Phytopathology 91 44-54

Gerlagh M 1968 Introduction of Ophiobolus graminis into new polders and its decline

European Journal of Plant Pathology 74 1-97

Giacomodonato MN Pettinari MJ Souto GI Mendez BS Lopez NI 2001 A PCR-based

method for the screening of bacterial strains with antifungal activity in suppressive soybean

rhizosphere World Journal of Microbiology amp Biotechnology 17 51-5

Hartley CP 1921 Damping-off in forest nurseries US Dept of Agriculture

Hind-Lanoiselet T Ash GJ Murray GM 2003 Prevalence of sclerotinia stem rot of canola in

New South Wales Animal Production Science 43 163-8

Hossain MD Rahman MME Islam MM Rahman MZ 2008 White rot a new disease of

mustard in Bangladesh Bangladesh Journal of Plant Pathology 24 81-2

Inglis G Boland G 1990 The microflora of bean and rapeseed petals and the influence of the

microflora of bean petals on white mold Canadian Journal of Plant Pathology 12 129-34

Kamal MM Lindbeck KD Savocchia S Ash GJ 2015 Biological control of sclerotinia stem

rot of canola using antagonistic bacteria Plant Pathology doi101111ppa12369

Khangura R Burge AV Salam M Aberra M Macleod WJ 2014 Why Sclerotinia was so bad

in 2013 In Understanding the disease and management options Department of

Agriculture and Food Western Australia

Kimber DS McGregor DI eds 1995 Brassica oilseeds-production and utilization Wallingford

UK CAB International

Kirkegaard JA Robertson MJ Hamblin P Sprague SJ 2006 Effect of blackleg and sclerotinia

stem rot on canola yield in the high rainfall zone of southern New South Wales Australia

Australian Journal of Agricultural Research 57 201-12

141

Millard W Taylor C 1927 Antagonism of micro-organisms as the controlling factor in the

inhibition of scab by green manuring Annals of Applied Biology 14 202-16

Murray GM Brennan JP 2012 The Current and Potential Costs from Diseases of Oilseed Crops

in Australia In Grains Research and Development Corporation Australia 91

Paas E Pierce G 2002 An Introduction to Functional Foods Nutraceuticals and Natural Health

Products In Winnipeg Manitoba Canada National Centre for Agri-Food Research in

Medicine

Pal KK Gardener BMS 2006 Biological control of plant pathogens Plant health instructor 2

1117-42

Park JK Lee SH Han S Kim JC Cheol Y Gardener BM 2013 Marker‐Assisted Selection of

Novel Bacteria Contributing to Soil‐Borne Plant Disease Suppression In De Bruijn FJ ed

Molecular Microbial Ecology of the Rhizosphere Hoboken NJ USA John Wiley amp Sons

Inc 637-42

Purdy LH 1979 Sclerotinia sclerotiorum history diseases and symptomatology host range

geographic distribution and impact Phytopathology 69 875-80

Raaijmakers JM Weller DM Thomashow LS 1997 Frequency of Antibiotic-Producing

Pseudomonas spp in Natural Environments Applied and Environmental Microbiology 63

881-7

Rahman M Dey T Hossain D Nonaka M Harada N 2015 First report of white mould caused

by Sclerotinia sclerotiorum on jackfruit Australasian Plant Disease Notes 10 1-3

Ramarathnam R 2007 Mechanism of phyllosphere biological control of Leptosphaeria

maculans the black leg pathogen of canola using antagonistic bacteria Winnipeg

Manitoba Canada University of Manitoba PhD

Ryu C-M Farag MA Hu C-H et al 2003 Bacterial volatiles promote growth in Arabidopsis

Proceedings of the National Academy of Sciences 100 4927-32

Sadoff HL 1972 The antibiotics of Bacillus species their possible roles in sporulation

Progress in Industrial Microbiology 11 1-27

Saharan GS Mehta N 2008 Sclerotinia diseases of crop plants Biology ecology and disease

management The Netherlands Springer Science Busines Media BV

Sharma P Meena PD Verma PR et al 2015 Sclerotinia sclerotiorum (Lib) de Bary causing

sclerotinia rot in oilseed brassicas A review Journal of oilsees brassica 6 (Special) 1-44

Singh J Faull JL 1988 Antagonism and biological control In Mukerji KG Garg KL eds

Biocontrol of Plant Diseases Boca Raton Florida USA CRC Press 167-77

Skidmore AM 1976 Interactions in relation to bological control of plant pathogens In

Dickinson CH Preece TF eds Microbiology of Aerial Plant Surfaces London UK

Academic Press Inc (London) Ltd 507-28

142

Solanki MK Robert AS Singh RK et al 2012 Characterization of mycolytic enzymes of

Bacillus strains and their bio-protection role against Rhizoctonia solani in tomato Current

Microbiology 65 330-6

Subbarao KV 1998 Progress toward integrated management of lettuce drop Plant Disease 82

1068-78

Upper C 1991 Manipulation of Microbial Communities in the Phyllosphere In Andrews J

Hirano S eds Microbial Ecology of Leaves Springer New York 451-63

Villalta O Pung H 2010 Managing Sclerotinia Diseases in Vegetables (New management

strategies for lettuce drop and white mould of beans) Department of Primary Industries

Victoria 1 Spring Street Melbourne 3000

Wilson CL Wisniewski ME 1994 Biological control of postharvest diseases theory and

practice CRC press

Zucchi TD Almeida LG Dossi FCA Cocircnsoli FL 2010 Secondary metabolites produced by

Propionicimonas sp (ENT-18) induce histological abnormalities in the sclerotia of

Sclerotinia sclerotiorum BioControl 55 811-9

143

Appendices

(Conference abstracts and newsletter articles)

144

Concurrent Session 3-Biological Control of Plant Diseases 39

Sclerotinia sclerotiorum (Lib) de Bary is a polyphagous

necrotropic fungal pathogen that infects more than 400

plant species belonging to almost 60 families and causes

significant economic losses in various crops including

canola in Australia Five hundred bacterial isolates from

sclerotia of S Sclerotorium and the rhizosphere and

endosphere of wheat and canola were evaluated

Burkholderia cepacia (KM-4) Bacillus cereus (SC-1)

and Bacillus amyloliquefaciens (W-67) demonstrated

strong antagonistic activity against the canola stem rot

pathogen Sclerotinia sclerotiorum in vitro A phylogenetic

study revealed that the Burkholderia strain (KM-4)

does not belong to the potential clinical or plant pathogenic

group of B cepacia These bacteria apparently

produced antifungal metabolites that diffuse through the

agar and inhibit fungal growth by causing abnormal

hyphal swelling They also demonstrated antifungal

activity through production of inhibitory volatile compounds

In addition sclerotial germination was restricted

upon pre-inoculation with every strain These results

demonstrate that the aforementioned promising strains

could pave the way for sustainable management of S

sclerotiorum in Australia

P03017 Microbial biocontrol of Sclerotinia stem rot of canola

MM Kamal GJ Ash K Lindbeck and S Savocchia

EH Graham Centre for Agricultural Innovation (an

alliance between Charles Sturt University and NSW DPI)

School of Agricultural and Wine Sciences Charles Sturt

University Boorooma Street Locked Bag 588 Wagga

Wagga NSW- 2678 Australia

Email mkamalcsueduau

145

146

DISEASE MANAGEMENT

POSTER BOARD 64

Cotyledon inoculation A rapid technique for the evaluation of bacterial antagonists against Sclerotinia sclerotiorum in canola under control conditions

Mohd Mostofa Kamal Charles Sturt University mkamalcsueduau

MM Kamal K Lindbeck S Savocchia GJ Ash

Graham Centre for Agricultural Innovation (an alliance between Charles Sturt University and NSW DPI) School of Agricultural and Wine Sciences Charles Sturt University Boorooma Street Locked Bag 588 Wagga Wagga NSW- 2678 Australia

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum (Ss) is a devastating disease of canola in Australia Prior to field testing a rapid antagonistic bacterial evaluation involving a cotyledon screening technique was adapted against SSR in canola Isolates of Bacillus (W-67 W-7-R SC-1 P-1 and E-2-1) previously shown to be antagonistic to Ss invitro were

used for further evaluation Ten days old canola cotyledons were detached surface sterilized and inoculated with 10microL of

a suspension of the Bacillus strains (1x108 CFU mL

-1) on moist

blotting paper in sterile Petri dishes for colonization After 24 hours macerated mycelium (1x10

4 mycelial fragments mL

-1)

of 3-day-old potato dextrose broth grown Ss was similarly drop inoculated onto canola cotyledons under controlled glass house

conditions (19plusmn1oC) Concurrently intact seedlings (ten days

old) were inoculated with using the same methods in a glasshouse Both experiments were repeated thrice with five replications Five days after inoculation no necrotic lesions were observed on seedlings inoculated with W-67 SC-1 P-1 a few lesions (le10) were formed on seedlings inoculated with W-7-R and E-2-1 strains while severe necrotic lesions covered more than 90 area in control cotyledons both invitro and in the glass house Further field investigation and correlation between field data and cotyledon inoculation assay may establish a relatively rapid technique to evaluate the performance of antagonistic bacteria against SSR of canola

147

148

8th Australasian Soil borne Disease Symposium 2014 Page 36

MARKER-ASSISTED SELECTION OF BACTERIAL BIO-

CONTROL AGENTS FOR THE CONTROL OF SCLEROTINIA

DISEASES

MM KamalA K D LindbeckA S SavocchiaA GJ AshA and BB McSpadden GardenerB

AGraham Centre for Agricultural Innovation (an alliance between Charles Sturt University


Boorooma Street Locked Bag 588 Wagga Wagga NSW- 2678 Australia BDepartment of

Plant Pathology The Ohio State University Ohio Agricultural Research and Development

Center Wooster 44691 USA


ABSTRACT The present investigation was conducted to search for potential bio-

control agents belong to Bacillus Acinetobacter and Klebsiella from crop

rhizospheres in Wooster OH Almost 1000 bacterial strains were isolated and

screened through DNA markers to identify isolates previously associated by

molecular community profiling studies with plant health promotion Upon

extraction of genomic DNA bacterial population of interest was targeted using

gene specific primers and positive strains were selectively isolated Restriction

Fragment Length Polymorphism analysis was performed with two enzymes MspI

and HhaI to identify the most genotypically distinct strains The marker selected

strains were then identified by 16S sequencing and phylogenetically characterized

Finally the selected strains were tested in vitro to evaluate their antagonism

against Sclerotinia sclerotiorum on Potato Dextrose Agar The results

demonstrated that all positive strains recovered have significant inhibitory effect

against S sclerotiorum Repeated application of this method in various systems

provides the opportunity to more efficiently select regionally-adapted pathogen-

suppressing bacteria for development as microbial biopesticides

149

150

151

152

153

154

Chapter 0

Chapter 1

Chapter 2

Chapter 3

Chapter 31

Chapter 32

Chapter 4

Chapter 5

Chapter 6

Chapter 7

Chapter 8

Chapter 9

Chapter 10

i

Table of Contents

Table of contentsi

Statement of authenticityiii

Acknowledgementsiv

Abstractv

Publications arising from this researchviii

Chapter 11

General Introduction

Chapter 26

Literature Review

Biology and biocontrol of Sclerotinia sclerotiorum (Lib) de Bary in oilseed Brassicas

Chapter 343

Prevalence of sclerotinia stem rot of mustard in northern Bangladesh

Chapter 4 51

Biological control of sclerotinia stem rot of canola using antagonistic bacteria

Chapter 5 62


Chapter 671

Elucidating the mechanism of biocontrol of Bacillus cereus SC-1 against Sclerotinia

sclerotiorum

Chapter 7101

Rapid marker-assisted selection of antifungal Bacillus species from the canola

rhizosphere

ii

Chapter 8130

General Discussion

References139

Appendices143













iv

Acknowledgements






















Mohd Mostofa Kamal

v

Abstract
























vi

























vii



















viii


Journal article









1375-1384 DOI 101111ppa12369









ix









Australasian


Zealand Pp138





Newsletter articles



Pp14-15



edition Pp11-12

x










1























2
























3























4













Bangladesh




in the glasshouse





sclerotiorum

5



















bibliographic style

6




(Accepted DOI 101007s13313-015-0391-2)








7



a Kurt D Lindbeck

b and Gavin J Ash

a2







9

Abstract 10













23


25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

Introduction

























8

51

9









S sclerotiorum60

















10







83

Plant infection 84


















11





Pathogenicity 106





















127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

















Dueck 1977)

Life cycle








12

151

13


















Epidemiology 169








14










Pearson 1982) 186


Host resistance 188













15






206


208


















226

16


























252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275



















Fungal antagonists







17

276

18


























19









310

















327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350


























20

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375













unpublished data)












2013)



21

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400





















cfu mL-1




(Zhang et al 2004)



22

401

23










1990b) 411
















24









Fernando 2004) 435















450

451

25

Mycoviruses 452

























26




Conclusion 480





















27




















References 520



28
















639 553




(1)47-59 557



29







18 (6)977-978 574






195 587






7867-76 598

30


















31

















32












26 (1)17-22 695









33




















34

















World Appl Sci J 12 (8)1192-1195 779


135 783

35



















36





















37




















38


(4)346-350 894













(3)169-172 921





39


















(4)759-764 964

965

966

40

Figures 967

968




972

973

974

975

976

977

978

979

980

981

982

983

41

984


986

987

988

989

990

991

42

992

993

994




43




19(2) 13-19








44






ABSTRACT



INTRODUCTION




45







46









47








48



CONCLUSION


ACKNOWLEDGEMENTS


REFERENCES



49




















50





51




DOI 101111ppa12369









journal



















Introduction







52





























































53






















































R1100

































54













R1100




Cotyledon bioassay





















following formulae




































55




























Data analysis






Results











100

100100

97

98

9769

72

43

0middot01










56




(a)

(b)

(c)

(d)

(e)

(f)







57








Discussion



glasshouse

Isolate



SC-1 132 a 376 a 116 a 242 a 879 b 736 b

W-67 184 b 424 b 173 b 298 b 819 a 675 a

P-1 218 b 472 b 202 b 338 b 789 a 633 a




Strain



SC-1 154 a 33 a 819 c 962 c 825 a 125 a

W-67 228 b 93 b 732 b 894 b 930 b 240 b

P-1 290 c 148 c 659 a 828 a 945 b 280 b





58








Treatment



SC-1 (91) 65 a 78 a 93 a 55 b 79 b 123 b 538 b 623 b 540 b


W-67 (91) 75 a 95 a 133 b 92 c 141 c 182 c 227 a 324 a 321 a

SC-1 + W-67 (91) 58 a 75 a 85 a 28 a 51 a 82 a 768 c 754 c 694 c






59






Acknowledgements


References















47ndash59













60
















































4577ndash84
























Management
























46 327ndash38



81 357ndash64















78 5942ndash4






18ndash22




1033ndash9





61













62

63







Abstract
























INTRODUCTION
















64













growth promotion
























-1 then 50 mL of









65





oC for 24






























CFU mL-1














66











and the CFU mL-1



with 10 replicates









-1



























67




























of soil


over seven time











al 2010) roots

CONCLUSION





68








agricultural crops

ACKNOWLEDGEMENTS



Literature Cited
















Mic 56 713-718



87 159-166




45 333-341






P Path (in press)






69


mic 63 541-556

















Bio Con 56 225-235



4932






1068-1078








Table


-1 of Bacillus



(cm)

Shoot length

(cm)

Head weight

(g)

Biomass

increase ()






70

Figures










standard error

BAA

A B

71















72


sclerotiorum 2


1 Kurt D Lindbeck

2 and Gavin J Ash

13







10

ABSTRACT 11













S sclerotiorum24

25


73

INTRODUCTION 27



















46






74















66











75












88














76



104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125







oC in





vitro










oC for 3 days


77

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150
























78

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174





fragments mL-1

) and placed






oC and 15

oC















79


twice 177










187










197




80









209

















81

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249







Data Analysis




005

RESULTS













250

82




controls 254

255















270






83





280

















(Fig 3C) 297

298



84












312


genes 314












85





330

DISCUSSION 331




















86

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374
























87





















396





88











411














425

89












437







444

REFERENCES 445



402 448

449

90



Plant P 73 pp 25-32 452






pp 18-21 458







1959 465








91



52 475





University 480








pp 60-70 488










92






234 pp 43-49 503











pp 1117-1142 514








93



Manitoba 524




56 pp 225-235 528




911 532













94









pp 289-297 553













566

567

568

569

95

Tables 570


sclerotiorum 572

Treatment

Mycelial diameter

(mm) in vitro


One day before


One day after


B cereus SC1






96


Antibiotic

Enzyme


(5´-3´)


Iturin A





oC for 1min


(Ramarathnam

2007 Ramarathnam

et al 2007)


Bacillomycin D



Surfactin



Fengycin


AAG




oC for 45 s


(Solanki et al

2013)


Chitinase





oC for 1


(Ramaiah et al

2000)


β-glucanase





oC for 1


(Baysal et al 2008)


577

97

Figures 578

579

580




of melanin 584

98

585









99

594






contents 600

601

602

603

604

605

100

606








101




Ecology (Formatted)












102


rhizosphere 2


1 Kurt D Lindbeck

2 and Gavin J Ash

13









ABSTRACT 12















103



29

INTRODUCTION 30






















104















sclerotia 66

67

METHODS 68

69


71








105




82













-80degC94

95










106











oC for 114



oC for 115






121










107





135























108


158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180










fragments mL-1

) of S













stem) times 100



R1-R2 R1

109



184

















two times 201

202





110
















examined 222

223

RESULTS 224

225








111













Genbank 245

246












112













269









278






113










293

DISCUSSION 294
















114








6) 317

318

















115



337


















355






116











371















117

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

























118



413








421

FUNDINGS 422



425


427

REFERENCES 428



402 431




Microbiol 2012112 159-74 435

119








199527 1611-6 443



Sci U S A 2003100 14555-61 446















120



University 2006 463






5 469















121



199274 1047-51 486




Dev Res 200050 440-7 490







200569 29-38 497



90 500


1117-42 502






201045 772-8 508

122


















Microbiol 200556 845-57 526



Mol Biol Evol a201328 2731-9 529



Microbiol 199946 327-38 532

123










542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

124

Tables 559




(Ramarathnam 2007

Ramarathnam et al

2007)




Bacillomycin

D



561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

125

585




Strain

In vitro In planta

Mycelial colony

diameter (mm)

Inhibition

()

Disease

incidence ()

Disease

index ()

Control

efficacy ()

CS-42 181 b 802 a 394 b 276 b 698 a

SC-1 154 b 819 a 376 b 242 b 736 a

Control 850 a - 100 a 916 a -



590

591

592

593

594

595

596

597

598

599

600

126

Figures 601

602 603




D) in the 4th







612

613

127

614

615





-1 of CS-42619





624

625

626

627

628

629

630

631

632

128

633






rind layer 639

640

641

642

643

644

645

646

129

647






653

654
























130























131
























Wisniewski 1994)

132

























133

























134

























135
























136













pathogen












137
























138

References




Science 76 635-9





























190 611-22

139

140






37 955-64































141







Medicine


1117-42




Inc 637-42





881-7



















142





1068-78







practice CRC press




143

Appendices


144

































145

146

DISEASE MANAGEMENT

POSTER BOARD 64








-1) on moist



-1)




147

148




DISEASES
























149

150

151

152

153

154

Chapter 0

Chapter 1

Chapter 2

Chapter 3

Chapter 31

Chapter 32

Chapter 4

Chapter 5

Chapter 6

Chapter 7

Chapter 8

Chapter 9

Chapter 10

ii

Chapter 8130

General Discussion

References139

Appendices143













iv

Acknowledgements






















Mohd Mostofa Kamal

v

Abstract
























vi

























vii



















viii


Journal article









1375-1384 DOI 101111ppa12369









ix









Australasian


Zealand Pp138





Newsletter articles



Pp14-15



edition Pp11-12

x










1























2
























3























4













Bangladesh




in the glasshouse





sclerotiorum

5



















bibliographic style

6




(Accepted DOI 101007s13313-015-0391-2)








7



a Kurt D Lindbeck

b and Gavin J Ash

a2







9

Abstract 10













23


25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

Introduction

























8

51

9









S sclerotiorum60

















10







83

Plant infection 84


















11





Pathogenicity 106





















127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

















Dueck 1977)

Life cycle








12

151

13


















Epidemiology 169








14










Pearson 1982) 186


Host resistance 188













15






206


208


















226

16


























252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275



















Fungal antagonists







17

276

18


























19









310

















327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350


























20

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375













unpublished data)












2013)



21

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400





















cfu mL-1




(Zhang et al 2004)



22

401

23










1990b) 411
















24









Fernando 2004) 435















450

451

25

Mycoviruses 452

























26




Conclusion 480





















27




















References 520



28
















639 553




(1)47-59 557



29







18 (6)977-978 574






195 587






7867-76 598

30


















31

















32












26 (1)17-22 695









33




















34

















World Appl Sci J 12 (8)1192-1195 779


135 783

35



















36





















37




















38


(4)346-350 894













(3)169-172 921





39


















(4)759-764 964

965

966

40

Figures 967

968




972

973

974

975

976

977

978

979

980

981

982

983

41

984


986

987

988

989

990

991

42

992

993

994




43




19(2) 13-19








44






ABSTRACT



INTRODUCTION




45







46









47








48



CONCLUSION


ACKNOWLEDGEMENTS


REFERENCES



49




















50





51




DOI 101111ppa12369









journal



















Introduction







52





























































53






















































R1100

































54













R1100




Cotyledon bioassay





















following formulae




































55




























Data analysis






Results











100

100100

97

98

9769

72

43

0middot01










56




(a)

(b)

(c)

(d)

(e)

(f)







57








Discussion



glasshouse

Isolate



SC-1 132 a 376 a 116 a 242 a 879 b 736 b

W-67 184 b 424 b 173 b 298 b 819 a 675 a

P-1 218 b 472 b 202 b 338 b 789 a 633 a




Strain



SC-1 154 a 33 a 819 c 962 c 825 a 125 a

W-67 228 b 93 b 732 b 894 b 930 b 240 b

P-1 290 c 148 c 659 a 828 a 945 b 280 b





58








Treatment



SC-1 (91) 65 a 78 a 93 a 55 b 79 b 123 b 538 b 623 b 540 b


W-67 (91) 75 a 95 a 133 b 92 c 141 c 182 c 227 a 324 a 321 a

SC-1 + W-67 (91) 58 a 75 a 85 a 28 a 51 a 82 a 768 c 754 c 694 c






59






Acknowledgements


References















47ndash59













60
















































4577ndash84
























Management
























46 327ndash38



81 357ndash64















78 5942ndash4






18ndash22




1033ndash9





61













62

63







Abstract
























INTRODUCTION
















64













growth promotion
























-1 then 50 mL of









65





oC for 24






























CFU mL-1














66











and the CFU mL-1



with 10 replicates









-1



























67




























of soil


over seven time











al 2010) roots

CONCLUSION





68








agricultural crops

ACKNOWLEDGEMENTS



Literature Cited
















Mic 56 713-718



87 159-166




45 333-341






P Path (in press)






69


mic 63 541-556

















Bio Con 56 225-235



4932






1068-1078








Table


-1 of Bacillus



(cm)

Shoot length

(cm)

Head weight

(g)

Biomass

increase ()






70

Figures










standard error

BAA

A B

71















72


sclerotiorum 2


1 Kurt D Lindbeck

2 and Gavin J Ash

13







10

ABSTRACT 11













S sclerotiorum24

25


73

INTRODUCTION 27



















46






74















66











75












88














76



104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125







oC in





vitro










oC for 3 days


77

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150
























78

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174





fragments mL-1

) and placed






oC and 15

oC















79


twice 177










187










197




80









209

















81

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249







Data Analysis




005

RESULTS













250

82




controls 254

255















270






83





280

















(Fig 3C) 297

298



84












312


genes 314












85





330

DISCUSSION 331




















86

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374
























87





















396





88











411














425

89












437







444

REFERENCES 445



402 448

449

90



Plant P 73 pp 25-32 452






pp 18-21 458







1959 465








91



52 475





University 480








pp 60-70 488










92






234 pp 43-49 503











pp 1117-1142 514








93



Manitoba 524




56 pp 225-235 528




911 532













94









pp 289-297 553













566

567

568

569

95

Tables 570


sclerotiorum 572

Treatment

Mycelial diameter

(mm) in vitro


One day before


One day after


B cereus SC1






96


Antibiotic

Enzyme


(5´-3´)


Iturin A





oC for 1min


(Ramarathnam

2007 Ramarathnam

et al 2007)


Bacillomycin D



Surfactin



Fengycin


AAG




oC for 45 s


(Solanki et al

2013)


Chitinase





oC for 1


(Ramaiah et al

2000)


β-glucanase





oC for 1


(Baysal et al 2008)


577

97

Figures 578

579

580




of melanin 584

98

585









99

594






contents 600

601

602

603

604

605

100

606








101




Ecology (Formatted)












102


rhizosphere 2


1 Kurt D Lindbeck

2 and Gavin J Ash

13









ABSTRACT 12















103



29

INTRODUCTION 30






















104















sclerotia 66

67

METHODS 68

69


71








105




82













-80degC94

95










106











oC for 114



oC for 115






121










107





135























108


158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180










fragments mL-1

) of S













stem) times 100



R1-R2 R1

109



184

















two times 201

202





110
















examined 222

223

RESULTS 224

225








111













Genbank 245

246












112













269









278






113










293

DISCUSSION 294
















114








6) 317

318

















115



337


















355






116











371















117

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

























118



413








421

FUNDINGS 422



425


427

REFERENCES 428



402 431




Microbiol 2012112 159-74 435

119








199527 1611-6 443



Sci U S A 2003100 14555-61 446















120



University 2006 463






5 469















121



199274 1047-51 486




Dev Res 200050 440-7 490







200569 29-38 497



90 500


1117-42 502






201045 772-8 508

122


















Microbiol 200556 845-57 526



Mol Biol Evol a201328 2731-9 529



Microbiol 199946 327-38 532

123










542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

124

Tables 559




(Ramarathnam 2007

Ramarathnam et al

2007)




Bacillomycin

D



561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

125

585




Strain

In vitro In planta

Mycelial colony

diameter (mm)

Inhibition

()

Disease

incidence ()

Disease

index ()

Control

efficacy ()

CS-42 181 b 802 a 394 b 276 b 698 a

SC-1 154 b 819 a 376 b 242 b 736 a

Control 850 a - 100 a 916 a -



590

591

592

593

594

595

596

597

598

599

600

126

Figures 601

602 603




D) in the 4th







612

613

127

614

615





-1 of CS-42619





624

625

626

627

628

629

630

631

632

128

633






rind layer 639

640

641

642

643

644

645

646

129

647






653

654
























130























131
























Wisniewski 1994)

132

























133

























134

























135
























136













pathogen












137
























138

References




Science 76 635-9





























190 611-22

139

140






37 955-64































141







Medicine


1117-42




Inc 637-42





881-7



















142





1068-78







practice CRC press




143

Appendices


144

































145

146

DISEASE MANAGEMENT

POSTER BOARD 64








-1) on moist



-1)




147

148

8th Australasian Soil borne Disease Symposium 2014 6



DISEASES
























149

150

151

152

153

154

Chapter 0

Chapter 1

Chapter 2

Chapter 3

Chapter 31

Chapter 32

Chapter 4

Chapter 5

Chapter 6

Chapter 7

Chapter 8

Chapter 9

Chapter 10













iv

Acknowledgements






















Mohd Mostofa Kamal

v

Abstract
























vi

























vii



















viii


Journal article









1375-1384 DOI 101111ppa12369









ix









Australasian


Zealand Pp138





Newsletter articles



Pp14-15



edition Pp11-12

x










1























2
























3























4













Bangladesh




in the glasshouse





sclerotiorum

5



















bibliographic style

6




(Accepted DOI 101007s13313-015-0391-2)








7



a Kurt D Lindbeck

b and Gavin J Ash

a2







9

Abstract 10













23


25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

Introduction

























8

51

9









S sclerotiorum60

















10







83

Plant infection 84


















11





Pathogenicity 106





















127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

















Dueck 1977)

Life cycle








12

151

13


















Epidemiology 169








14










Pearson 1982) 186


Host resistance 188













15






206


208


















226

16


























252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275



















Fungal antagonists







17

276

18


























19









310

















327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350


























20

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375













unpublished data)












2013)



21

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400





















cfu mL-1




(Zhang et al 2004)



22

401

23










1990b) 411
















24









Fernando 2004) 435















450

451

25

Mycoviruses 452

























26




Conclusion 480





















27




















References 520



28
















639 553




(1)47-59 557



29







18 (6)977-978 574






195 587






7867-76 598

30


















31

















32












26 (1)17-22 695









33




















34

















World Appl Sci J 12 (8)1192-1195 779


135 783

35



















36





















37




















38


(4)346-350 894













(3)169-172 921





39


















(4)759-764 964

965

966

40

Figures 967

968




972

973

974

975

976

977

978

979

980

981

982

983

41

984


986

987

988

989

990

991

42

992

993

994




43




19(2) 13-19








44






ABSTRACT



INTRODUCTION




45







46









47








48



CONCLUSION


ACKNOWLEDGEMENTS


REFERENCES



49




















50





51




DOI 101111ppa12369









journal



















Introduction







52





























































53






















































R1100

































54













R1100




Cotyledon bioassay





















following formulae




































55




























Data analysis






Results











100

100100

97

98

9769

72

43

0middot01










56




(a)

(b)

(c)

(d)

(e)

(f)







57








Discussion



glasshouse

Isolate



SC-1 132 a 376 a 116 a 242 a 879 b 736 b

W-67 184 b 424 b 173 b 298 b 819 a 675 a

P-1 218 b 472 b 202 b 338 b 789 a 633 a




Strain



SC-1 154 a 33 a 819 c 962 c 825 a 125 a

W-67 228 b 93 b 732 b 894 b 930 b 240 b

P-1 290 c 148 c 659 a 828 a 945 b 280 b





58








Treatment



SC-1 (91) 65 a 78 a 93 a 55 b 79 b 123 b 538 b 623 b 540 b


W-67 (91) 75 a 95 a 133 b 92 c 141 c 182 c 227 a 324 a 321 a

SC-1 + W-67 (91) 58 a 75 a 85 a 28 a 51 a 82 a 768 c 754 c 694 c






59






Acknowledgements


References















47ndash59













60
















































4577ndash84
























Management
























46 327ndash38



81 357ndash64















78 5942ndash4






18ndash22




1033ndash9





61













62

63







Abstract
























INTRODUCTION
















64













growth promotion
























-1 then 50 mL of









65





oC for 24






























CFU mL-1














66











and the CFU mL-1



with 10 replicates









-1



























67




























of soil


over seven time











al 2010) roots

CONCLUSION





68








agricultural crops

ACKNOWLEDGEMENTS



Literature Cited
















Mic 56 713-718



87 159-166




45 333-341






P Path (in press)






69


mic 63 541-556

















Bio Con 56 225-235



4932






1068-1078








Table


-1 of Bacillus



(cm)

Shoot length

(cm)

Head weight

(g)

Biomass

increase ()






70

Figures










standard error

BAA

A B

71















72


sclerotiorum 2


1 Kurt D Lindbeck

2 and Gavin J Ash

13







10

ABSTRACT 11













S sclerotiorum24

25


73

INTRODUCTION 27



















46






74















66











75












88














76



104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125







oC in





vitro










oC for 3 days


77

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150
























78

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174





fragments mL-1

) and placed






oC and 15

oC















79


twice 177










187










197




80









209

















81

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249







Data Analysis




005

RESULTS













250

82




controls 254

255















270






83





280

















(Fig 3C) 297

298



84












312


genes 314












85





330

DISCUSSION 331




















86

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374
























87





















396





88











411














425

89












437







444

REFERENCES 445



402 448

449

90



Plant P 73 pp 25-32 452






pp 18-21 458







1959 465








91



52 475





University 480








pp 60-70 488










92






234 pp 43-49 503











pp 1117-1142 514








93



Manitoba 524




56 pp 225-235 528




911 532













94









pp 289-297 553













566

567

568

569

95

Tables 570


sclerotiorum 572

Treatment

Mycelial diameter

(mm) in vitro


One day before


One day after


B cereus SC1






96


Antibiotic

Enzyme


(5´-3´)


Iturin A





oC for 1min


(Ramarathnam

2007 Ramarathnam

et al 2007)


Bacillomycin D



Surfactin



Fengycin


AAG




oC for 45 s


(Solanki et al

2013)


Chitinase





oC for 1


(Ramaiah et al

2000)


β-glucanase





oC for 1


(Baysal et al 2008)


577

97

Figures 578

579

580




of melanin 584

98

585









99

594






contents 600

601

602

603

604

605

100

606








101




Ecology (Formatted)












102


rhizosphere 2


1 Kurt D Lindbeck

2 and Gavin J Ash

13









ABSTRACT 12















103



29

INTRODUCTION 30






















104















sclerotia 66

67

METHODS 68

69


71








105




82













-80degC94

95










106











oC for 114



oC for 115






121










107





135























108


158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180










fragments mL-1

) of S













stem) times 100



R1-R2 R1

109



184

















two times 201

202





110
















examined 222

223

RESULTS 224

225








111













Genbank 245

246












112













269









278






113










293

DISCUSSION 294
















114








6) 317

318

















115



337


















355






116











371















117

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

























118



413








421

FUNDINGS 422



425


427

REFERENCES 428



402 431




Microbiol 2012112 159-74 435

119








199527 1611-6 443



Sci U S A 2003100 14555-61 446















120



University 2006 463






5 469















121



199274 1047-51 486




Dev Res 200050 440-7 490







200569 29-38 497



90 500


1117-42 502






201045 772-8 508

122


















Microbiol 200556 845-57 526



Mol Biol Evol a201328 2731-9 529



Microbiol 199946 327-38 532

123










542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

124

Tables 559




(Ramarathnam 2007

Ramarathnam et al

2007)




Bacillomycin

D



561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

125

585




Strain

In vitro In planta

Mycelial colony

diameter (mm)

Inhibition

()

Disease

incidence ()

Disease

index ()

Control

efficacy ()

CS-42 181 b 802 a 394 b 276 b 698 a

SC-1 154 b 819 a 376 b 242 b 736 a

Control 850 a - 100 a 916 a -



590

591

592

593

594

595

596

597

598

599

600

126

Figures 601

602 603




D) in the 4th







612

613

127

614

615





-1 of CS-42619





624

625

626

627

628

629

630

631

632

128

633






rind layer 639

640

641

642

643

644

645

646

129

647






653

654
























130























131
























Wisniewski 1994)

132

























133

























134

























135
























136













pathogen












137
























138

References




Science 76 635-9





























190 611-22

139

140






37 955-64































141







Medicine


1117-42




Inc 637-42





881-7



















142





1068-78







practice CRC press




143

Appendices


144

































145

146

DISEASE MANAGEMENT

POSTER BOARD 64








-1) on moist



-1)




147

148




DISEASES
























149

150

151

152

153

154

Chapter 0

Chapter 1

Chapter 2

Chapter 3

Chapter 31

Chapter 32

Chapter 4

Chapter 5

Chapter 6

Chapter 7

Chapter 8

Chapter 9

Chapter 10

v

Abstract
























vi

























vii



















viii


Journal article









1375-1384 DOI 101111ppa12369









ix









Australasian


Zealand Pp138





Newsletter articles



Pp14-15



edition Pp11-12

x










1























2
























3























4













Bangladesh




in the glasshouse





sclerotiorum

5



















bibliographic style

6




(Accepted DOI 101007s13313-015-0391-2)








7



a Kurt D Lindbeck

b and Gavin J Ash

a2







9

Abstract 10













23


25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

Introduction

























8

51

9









S sclerotiorum60

















10







83

Plant infection 84


















11





Pathogenicity 106





















127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

















Dueck 1977)

Life cycle








12

151

13


















Epidemiology 169








14










Pearson 1982) 186


Host resistance 188













15






206


208


















226

16


























252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275



















Fungal antagonists







17

276

18


























19









310

















327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350


























20

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375













unpublished data)












2013)



21

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400





















cfu mL-1




(Zhang et al 2004)



22

401

23










1990b) 411
















24









Fernando 2004) 435















450

451

25

Mycoviruses 452

























26




Conclusion 480





















27




















References 520



28
















639 553




(1)47-59 557



29







18 (6)977-978 574






195 587






7867-76 598

30


















31

















32












26 (1)17-22 695









33




















34

















World Appl Sci J 12 (8)1192-1195 779


135 783

35



















36





















37




















38


(4)346-350 894













(3)169-172 921





39


















(4)759-764 964

965

966

40

Figures 967

968




972

973

974

975

976

977

978

979

980

981

982

983

41

984


986

987

988

989

990

991

42

992

993

994




43




19(2) 13-19








44






ABSTRACT



INTRODUCTION




45







46









47








48



CONCLUSION


ACKNOWLEDGEMENTS


REFERENCES



49




















50





51




DOI 101111ppa12369









journal



















Introduction







52





























































53






















































R1100

































54













R1100




Cotyledon bioassay





















following formulae




































55




























Data analysis






Results











100

100100

97

98

9769

72

43

0middot01










56




(a)

(b)

(c)

(d)

(e)

(f)







57








Discussion



glasshouse

Isolate



SC-1 132 a 376 a 116 a 242 a 879 b 736 b

W-67 184 b 424 b 173 b 298 b 819 a 675 a

P-1 218 b 472 b 202 b 338 b 789 a 633 a




Strain



SC-1 154 a 33 a 819 c 962 c 825 a 125 a

W-67 228 b 93 b 732 b 894 b 930 b 240 b

P-1 290 c 148 c 659 a 828 a 945 b 280 b





58








Treatment



SC-1 (91) 65 a 78 a 93 a 55 b 79 b 123 b 538 b 623 b 540 b


W-67 (91) 75 a 95 a 133 b 92 c 141 c 182 c 227 a 324 a 321 a

SC-1 + W-67 (91) 58 a 75 a 85 a 28 a 51 a 82 a 768 c 754 c 694 c






59






Acknowledgements


References















47ndash59













60
















































4577ndash84
























Management
























46 327ndash38



81 357ndash64















78 5942ndash4






18ndash22




1033ndash9





61













62

63







Abstract
























INTRODUCTION
















64













growth promotion
























-1 then 50 mL of









65





oC for 24






























CFU mL-1














66











and the CFU mL-1



with 10 replicates









-1



























67




























of soil


over seven time











al 2010) roots

CONCLUSION





68








agricultural crops

ACKNOWLEDGEMENTS



Literature Cited
















Mic 56 713-718



87 159-166




45 333-341






P Path (in press)






69


mic 63 541-556

















Bio Con 56 225-235



4932






1068-1078








Table


-1 of Bacillus



(cm)

Shoot length

(cm)

Head weight

(g)

Biomass

increase ()






70

Figures










standard error

BAA

A B

71















72


sclerotiorum 2


1 Kurt D Lindbeck

2 and Gavin J Ash

13







10

ABSTRACT 11













S sclerotiorum24

25


73

INTRODUCTION 27



















46






74















66











75












88














76



104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125







oC in





vitro










oC for 3 days


77

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150
























78

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174





fragments mL-1

) and placed






oC and 15

oC















79


twice 177










187










197




80









209

















81

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249







Data Analysis




005

RESULTS













250

82




controls 254

255















270






83





280

















(Fig 3C) 297

298



84












312


genes 314












85





330

DISCUSSION 331




















86

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374
























87





















396





88











411














425

89












437







444

REFERENCES 445



402 448

449

90



Plant P 73 pp 25-32 452






pp 18-21 458







1959 465








91



52 475





University 480








pp 60-70 488










92






234 pp 43-49 503











pp 1117-1142 514








93



Manitoba 524




56 pp 225-235 528




911 532













94









pp 289-297 553













566

567

568

569

95

Tables 570


sclerotiorum 572

Treatment

Mycelial diameter

(mm) in vitro


One day before


One day after


B cereus SC1






96


Antibiotic

Enzyme


(5´-3´)


Iturin A





oC for 1min


(Ramarathnam

2007 Ramarathnam

et al 2007)


Bacillomycin D



Surfactin



Fengycin


AAG




oC for 45 s


(Solanki et al

2013)


Chitinase





oC for 1


(Ramaiah et al

2000)


β-glucanase





oC for 1


(Baysal et al 2008)


577

97

Figures 578

579

580




of melanin 584

98

585









99

594






contents 600

601

602

603

604

605

100

606








101




Ecology (Formatted)












102


rhizosphere 2


1 Kurt D Lindbeck

2 and Gavin J Ash

13









ABSTRACT 12















103



29

INTRODUCTION 30






















104















sclerotia 66

67

METHODS 68

69


71








105




82













-80degC94

95










106











oC for 114



oC for 115






121










107





135























108


158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180










fragments mL-1

) of S













stem) times 100



R1-R2 R1

109



184

















two times 201

202





110
















examined 222

223

RESULTS 224

225








111













Genbank 245

246












112













269









278






113










293

DISCUSSION 294
















114








6) 317

318

















115



337


















355






116











371















117

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

























118



413








421

FUNDINGS 422



425


427

REFERENCES 428



402 431




Microbiol 2012112 159-74 435

119








199527 1611-6 443



Sci U S A 2003100 14555-61 446















120



University 2006 463






5 469















121



199274 1047-51 486




Dev Res 200050 440-7 490







200569 29-38 497



90 500


1117-42 502






201045 772-8 508

122


















Microbiol 200556 845-57 526



Mol Biol Evol a201328 2731-9 529



Microbiol 199946 327-38 532

123










542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

124

Tables 559




(Ramarathnam 2007

Ramarathnam et al

2007)




Bacillomycin

D



561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

125

585




Strain

In vitro In planta

Mycelial colony

diameter (mm)

Inhibition

()

Disease

incidence ()

Disease

index ()

Control

efficacy ()

CS-42 181 b 802 a 394 b 276 b 698 a

SC-1 154 b 819 a 376 b 242 b 736 a

Control 850 a - 100 a 916 a -



590

591

592

593

594

595

596

597

598

599

600

126

Figures 601

602 603




D) in the 4th







612

613

127

614

615





-1 of CS-42619





624

625

626

627

628

629

630

631

632

128

633






rind layer 639

640

641

642

643

644

645

646

129

647






653

654
























130























131
























Wisniewski 1994)

132

























133

























134

























135
























136













pathogen












137
























138

References




Science 76 635-9





























190 611-22

139

140






37 955-64































141







Medicine


1117-42




Inc 637-42





881-7



















142





1068-78







practice CRC press




143

Appendices


144

































145

146

DISEASE MANAGEMENT

POSTER BOARD 64








-1) on moist



-1)




147

148




DISEASES
























149

150

151

152

153

154

Chapter 0

Chapter 1

Chapter 2

Chapter 3

Chapter 31

Chapter 32

Chapter 4

Chapter 5

Chapter 6

Chapter 7

Chapter 8

Chapter 9

Chapter 10

Microbial biocontrol of Sclerotinia stem rot of canola

Documents

Transcript of Microbial biocontrol of Sclerotinia stem rot of canola