EVALUATION OF SELECTED ECO-FRIENDLY

COMPONENTS AGAINST PHOMOPSIS FRUIT ROT

(Phomopsis vexans) OF EGGPLANT

SUMAIYA KHANAM

DEPARTMENT OF PLANT PATHOLOGY

SHER-E-BANGLA AGRICULTURAL UNIVERSITY

DHAKA-1207

JUNE, 2015

EVALUATION OF SELECTED ECO-FRIENDLY

COMPONENTS AGAINST PHOMOPSIS FRUIT ROT

(Phomopsis vexans) OF EGGPLANT

SUMAIYA KHANAM

REGISTRATION NO. 09-03292

A Thesis

Submitted to the Faculty of Agriculture, Dept. of Plant Pathology

Sher-e-Bangla Agricultural University, Dhaka,

in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

IN

PLANT PATHOLOGY

SEMESTER: JANUARY - JUNE, 2015

Approved by:

Department of Plant Pathology

Supervisor

__________________________________

Dr. Md. Belal Hossain

Associate Professor

Chairman

Department of Plant Pathology

Sher-e-Bangla Agricultural University

Dr. Md. Rafiqul Islam

Professor

Supervisor

Dr. Nazneen Sultana

Professor

Department of Plant Pathology

Co-supervisor

Dedicated to My

Beloved Parents

CERTIFICATE

This is to certify that the thesis entitled, “EVALUATION OF

SELECTED ECO-FRIENDLY COMPONENTS AGAINST

PHOMOPSIS FRUIT ROT (Phomopsis vexans) OF

EGGPLANT” submitted to the Department of Plant Pathology, Faculty of

Agriculture, Sher-e-Bangla Agricultural University, Dhaka, in the partial

fulfillment of the requirements for the degree of MASTER OF SCIENCE

(M.S.) IN PLANT PATHOLOGY, embodies the result of a piece of bona

fide research work carried out by SUMAIYA KHANAM bearing

Registration No. 09-03292 under my supervision and guidance. No part of

the thesis has been submitted for any other degree or diploma.

I further certify that such help or source of information, as has been availed

of during the course of this investigation has duly been acknowledged.

Dated: 26 May, 2016 Prof. Dr. Md. Rafiqul Islam

Place: Dhaka, Bangladesh Department of Plant Pathology

Sher-e-Bangla Agricultural University

Supervisor

Department of Plant Pathology Fax: +88029112649

Sher - e - Bangla Agricultural Universit y Web site: www.sau.edu.bd

Dhaka - 1207 , Bangladesh

ACKNOWLEDGEMENTS

All admiration and praises are solely to “Almighty Allah” Whose mercy

absolutely enabled the author to pursue the higher education in Agriculture

discipline and successful completion of M.S. courses and research work for

the degree of M.S. in Plant Pathology.

The author expresses her immense respect and deepest sense of gratitude and

heartfelt thanks to her most reverend teacher and research Supervisor,

Professor Dr. Md. Rafiqul Islam, Department of Plant Pathology, Sher-e-

Bangla Agricultural University, Dhaka for his inestimable help, dynamic

and cordial guidance, planning, ingenious and valuable suggestion,

continuous encouragement, constructive criticism and scholastic supervision

throughout the research work and preparation of the thesis.

The author also expresses her gratitude, profound respect and immense

indebtedness to her honorable teacher and research Co-supervisor, Professor

Dr. Nazneen Sultana, Department of Plant Pathology, Sher-e-Bangla

Agricultural University, Dhaka for her sincere guidance, keen interest,

valuable advices, helpful suggestions and sympathetic co-operation regarding

this research work.

The author also wishes to pay her deep respect to Dr. Md. Belal Hossain,

Chairman, Department of Plant Pathology, Sher-e-Bangla Agricultural

University, Dhaka for his valuable advice and continuous effort throughout

the study and research period.

The author also wishes to express her sincere gratitude to all other respected

teachers of the Department of Plant Pathology, Sher-e-Bangla Agricultural

University, Dhaka for their valuable suggestion, kind cooperation and help

during the entire study and research period.

Cordial appreciation and thanks are extended to the staff of the laboratory

of Department of Plant Pathology and Horticulture Farm, Sher-e-Bangla

Agricultural University, Dhaka for their responsible help during the

experimental work.

Finally, the author expresses her immense gratefulness to her beloved

parents, husband, little daughter and well wishers whose inspiration,

sacrifice, advice, continuous encouragement and moral support opened the

gate and paved the way to higher studies.

The Author

EVALUATION OF SELECTED ECO-FRIENDLY COMPONENTS AGAINST

PHOMOPSIS FRUIT ROT (Phomopsis vexans) OF EGGPLANT

ABSTRACT

An eco-friendly bioagent Trichoderma harzianum, four plant extracts viz. garlic clove

extract, allamonda leaf extract, neem leaf extract and lemon grass extract, a fungicide

Bavistin 50WP and two soil amendment tools saw dust and poultry waste were evaluated

against Phomopsis vexans for the management of phomopsis fruit rot disease of eggplant.

The experiment was carried out in the horticultural farm of Sher-e-Bangla Agricultural

University during Rabi season (October, 2014 to March, 2015). Trichoderma harzianum

and Bavistin 50WP showed promising performance in controlling the disease. The lowest

disease incidence was 9.54, 14.77 and 16.52, respectively at 105, 114 and 123 DAT and

the disease severity was 3.73, 6.09 and 4.83, respectively at 105, 114 and 123 DAT in

Trichoderma harzianum treated plot. The fungicide Bavistin 50WP treated plot showed

disease incidence 10.77, 16.53 and 18.43, respectively at 105, 114 and 123 DAT. The

disease severity was also found 5.09, 9.64 and 10.01 in Bavistin 50WP treated plot at 105,

114 and 123 DAT, respectively. The plant extracts also showed significant effects in

controlling the disease reducing the disease incidence and disease severity. Among the

plant extracts, allamonda leaf (1:2) and neem leaf (1:2) extract were found very effective

against Phomopsis vexans. Allamonda leaf extract showed disease incidence (14.16, 17.96

and 18.60) and disease severity (7.50, 10.01 and 12.73) at 105, 114 and 123 DAT,

respectively. Disease incidence recorded in neem leaf extract treated plot was 16.66, 19.65

and 24.16 at 105, 114 and 123 DAT, respectively and the disease severity was 9.98, 10.46

and 15.14 at 105, 114 and 123 DAT, respectively. The effect of other plant extracts also

found moderate in reducing the disease incidence and severity. Saw dust and poultry

waste showed poor performances against the disease.

iv

CONTENTS

CHAPTER TITLE PAGE

ACKNOWLEDGEMENTS i

ABSTRACT iii

CONTENTS iv

LIST OF TABLES vii

LIST OF FIGURES viii

LIST OF APPENDICES ix

1 INTRODUCTION 1-3

2 REVIEW OF LITERATURE 4-12

2.1 Symptom of phomopsis blight and fruit rot of eggplant 4

2.2 Incidence and severity of phomopsis blight and fruit rot

of eggplant 6

2.3 Morphology of Phomopsis vexans, the causal agent of

phomopsis fruit rot of eggplant 7

2.4 Seed infection and its impact 8

2.5 Management of phomopsis fruit rot through plant

extracts and biological control agent 10

3 MATERIALS AND METHODS 13-28

3.1 In vitro Experiment 13

3.1.1 Experimental site 13

3.1.2 Experimental period 13

3.1.3 Preparation of culture media 13

v

3.1.4 Isolation and maintenance of culture of

Phomopsis vexans 14

3.1.5 Preparation of spore suspension of Phomopsis

vexans 14

3.2 Field Experiment 15

3.2.1 Experimental site 15

3.2.2 Experimental period 15

3.2.3 Experimental design and layout 15

3.2.4 Test material (cultivar) 15

3.2.5 Raising of seedlings 15

3.2.6 Land preparation and fertilizer application 18

3.2.7 Transplanting of of seedlings to the main field 18

3.2.8 Intercultural operation 21

3.2.9 Plant extracts used in the experiment 21

3.2.10 Preparation of plant extracts 21

3.2.11 Spraying of plant extracts 23

3.2.12 Spraying of bioagent as spore suspension 23

3.2.13 Spraying of fungicide 25

3.2.14 Inoculation of eggplant by Phomopsis vexans 25

3.2.15 Data collection 27

3.2.16 Seed extraction 27

3.2.17 Analysis of data 30

vi

4 RESULTS 31-47

4.1 Laboratory experiment 31

4.1.1 Isolation and identification of causal agent 31

4.2 FIELD EXPERIMENT 33

4.2.1

Development of symptoms in brinjal plants following inoculation

with Phomopsis vexans 33

4.2.2 Evaluation of different treatments against disease incidence of

eggplant by Phomopsis vexans 35

4.2.3 Evaluation of different treatments against disease severity of

eggplant by Phomopsis vexans 38

4.2.4 Effect of different treatments on plant growth characteristics 42

4.2.4.1 Effect of different treatments on height of plant 42

4.2.4.2 Effect of different treatments on number of branch of eggplant 42

4.2.4.3

Effect of different treatments on number of leaf per branch of

eggplant 43

4.2.5 Effect of different treatments on fruit yield and seed yield of

eggplant 45

5 DISCUSSION 48-52

6 SUMMARY AND CONCLUSION 53-54

7 REFERENCES 55-64

8 APPENDICES 65-67

vii

LIST OF TABLES

Table Title Page

1. Effect of different treatments on disease incidence of fruit rot of

Eggplant at different days after transplanting (DAT) 37

2. Effect of different treatments on disease severity of fruit rot of

Eggplant at different days after transplanting (DAT) 41

3. Effect of different treatments on growth characteristics of eggplant 44

4. Effect of different treatments on fruit and seed yield of eggplant 47

viii

LIST OF PLATES

Plate Title Page

1 Eggplant seedlings (Variety-BARI Begun 8) raised in plastic tray 17

2 Different growth stages of eggplant showing in the experimental

field 19

3 A view of experimental field in Horticultural Farm of SAU 20

4 Preparation of different plant extracts. 22

5 Pure culture of Trichoderma harzianum 24

6 Trichoderma harzianum under compound microscope 24

7 Inoculation of eggplant by Phomopsis vexans 26

8 Harvesting of fruit for seed production 28

9 Extraction of seed from fruit 29

10 Pure culture of Phomopsis vexans 32

11 Alpha conidia of Phomopsis vexans under compound microscope 32

12 Infected plant showing phomopsis fruit rot symptom 34

ix

LIST OF APPENDICES

APPENDIX TITLE PAGE

NO.

I Map showing the experimental site under study 65

II Physiochemical properties of soil of the experimental

site 66

III

Monthly average relative humidity, maximum and

minimum temperature, rainfall and sunshine hour of

the experimental period (October 2014 to March 2015)

67

1

Introduction

The eggplant (Solanum melongena) belonging to the family Solanaceae originates

from India and is now generally grown as a vegetable throughout the tropical, sub-

tropical and warm temperate areas of the world. It is a popular, nutritious and widely

grown vegetable in Bangladesh as well as in the world and has got multifarious use as

a dish item (Rashid, 1976; Bose and Som, 1986).About 8 million farm families are

involved in eggplant cultivation. Its position in terms of acreage production is second

in vegetable crops in Bangladesh (BBS, 2003).The total cultivable area of eggplant is

44,377 acres with total annual production of 3,68,000 metric tons (BBS, 2013). A

large number of cultivars are grown in Bangladesh, which is showing a wide range of

variation in yield performance. This gives small, marginal and landless farmers a

continuous source of income and provides employment facilities for the rural people.

For most of the time, except peak production period, market price of eggplant

compared to other vegetables remains high which is in favor of the farmer’s solvency.

Therefore, it plays a vital role to boost up our national economy. It is grown round the

year especially during the lean period when the seasonal vegetables are in a scarcity

in the market. Eggplant, thus regarded as a cash crop to the farmers which provides

them continuous harvesting and financial assistance.

Such a potential crop is known to suffer from 12 diseases and among them phomopsis

fruit rot caused by Phomopsis vexans Harter has been treated as one of the major

constraints in eggplant cultivation in the country (Khan, et al., 2002; Das, 1998). The

disease was first reported from Gujrat in 1914 and since then from many parts of

India. Fakir (1983) and Ahmad (1987) have reported occurrence of this disease in

Bangladesh. The infected fruit develops pale and sunken spots that progress covering

the entire surface and cause dropping. Affected portion is mummified producing huge

2

pycnidia on the surface and becomes non-edible (Singh, 1992; Ashrafuzzaman,

1986).

Phomopsis vexans is viable for about 14 months in soil debries and in the seed from

infected fruits. The pathogen is reported both externally and internally seed borne

(Islam, 2005). It has become a major constraint for intensive cultivation of eggplant.

The disease affects the crops from seedling to maturity (Singh, 1992). Crop losses

due to this disease are evident, loss ranges from 15-20% in general but 30-50% in

severe case (Das, 1998).

Efforts for controlling the disease through different measures have been made by

many researchers (Khan, 1999; Hawlader, 2003; Nazimuddin, 2004 and Rummana,

2004). Plant extracts might be a substantial alternative of chemical pesticides in

controlling phomopsis blight and fruit rot of eggplant. Bavistin is reported to be an

effective fungicide against P. vexans (Khan et al., 2002; Meah et al., 1998; Mohanty

et al., 1994). Chemicals, though seriously discouraged in present plant protection

system especially for horticultural crops by Montreal Protocol, it might be

incorporated in the disease management program as the last option in absence of any

alternative.

Seed is the infection source of P. vexans and may serve as a substrate for pathogen

survival (Pan et al., 1995). The pathogens remain on the seed coat and the cotyledons

of eggplant seeds causing various degrees of seed discoloration and minute black

pycnidial bodies which is distinctly observed on the surface of the dry seed (Karuna

et al., 1994). Therefore, by the management of phomopsis fruit rot we can control

seed infection caused by P. vexans and thus seed yield is higher. Many plants

extracts, bioagents and hot water are reported to be antimicrobials against plant

pathogenic fungi (Bowers et al., 2000; Lawson et al., 1998; Hossain, 2004; Hassan,

3

2000; Cook, 1993; Harman, 1989). Those components may address against P. vexans.

Besides, seed dressing fungicide Bavistin may be assayed against the pathogen

Phomopsis vexans.

Under such scenario, developing a biologically based and environmentally safe eco-

friendly approach for management of phomopsis fruit rot is essential. To screen out

the effective components for management of phomopsis blight and fruit rot of

eggplant, the present experiment was undertaken with the following objectives:

1. To isolate and identify the causal organism of phomopsis fruit rot of eggplant.

2. To evaluate eco-friendly components for the management of phomopsis fruit rot of

eggplant for seed production.

4

REVIEW OF LITERATURE

Eggplant (Solanum melongena L.) is a popular Solanaceous vegetable crop of

Bangladesh. It is cultivated throughout the year and used by people of all strata of the

country (Rashid, 1993). Brinjal suffers from many diseases caused by fungi, bacteria,

virus, nematode and mycoplasma. Of them, phomopsis blight and fruit rot of eggplant

caused by the fungus Phomopsis vexans (Sacc and Syd) is a serious disease which

attacks all above ground parts of plant. It is mentionably damaging to the crop and is

a threat particularly in kharif season and late crop in winter season.

Phoma solani Hals. was first described as the organism of fruit rot of eggplant by

Halsted in 1982 in the United States of America (Harter, 1914). Since the name had

been used for another fungus it was changed to Phoma vexans by Saccardo and

Sydow in 1899 (Harter, 1914). Harter (1914) and Punithalingam and Holliday (1972)

have reported that brinjal (Solanum melongena L.) is the only economic host of

Phomopsis vexans. Evidences of research work regarding phomopsis blight and fruit

rot of eggplant is very dearth. However, it is known from literatures that emphasis

have been given to control the disease through biological control agents and plant

extracts. Literatures on the symptom, incidence and severity of phomopsis fruit rot,

its seed borne nature and control by eco-friendly components are given below.

2.1 Symptom of phomopsis blight and fruit rot of eggplant

Phomopsis blight occurs on fruit and occasionally on leaves or stems. Infection of

foliage and stem is less important than fruit infection.

5

Harter (1914) reported that on the fruits the symptoms appear first as minute sunken

greyish spots with a brownish halo, which later enlarge and coalesce, producing

concentric rings of yellow and brown zones. These spots increase in size and form

large rotten areas on which conidiomata often develop concentrically, covering most

of the rotten fruit surface. Pycnidia on fruit are larger than those on stems and leaves.

According to Walker (1952) the first phase of phomopsis blight is a blight of young

seedling. The stem is girdled slightly above the soil line. The seedlings topple over

and die. The stem lesion is dark brown, becoming gray in the center as the pycnidia

develop. Round or oval brown spots develop on the leaf, becoming irregular in shape

as they enlarge upto 3 cm in diameter. The centers become gray and contain pycnidia.

The fruit is attacked while on the plant. The decay being first soft and watery and

later the fruits become black and mummified as the pycnidia develop abundantly over

the surface.

Pawar and Patel(1957) reported that the symptoms of phomopsis blight range from

poor germination and seedling blight to fruit rot. If the infection enters the fruits

through the calyx, the whole fruit may become mummified due to dry rot. Post-

emergence damping-off of seedlings results from infection of the stem just above the

soil surface. The symptoms on leaves are more prominent during the early stages of

plant growth. The lesions first are small, more or less circular, and buff to olive, later

becoming cinnamon buff, with an irregular blackish margin. Irregular spots result

from coalescence. After transplanting, leaves coming in contact with the soil may

become infected directly or develop leaf spot due to infection by conidia. Lesions on

the petiole or the lower part of the midrib can result in death of the entire leaf.

6

Affected leaves may drop prematurely, and the blighted areas become covered with

numerous black pycnidia.

Ashrafuzzaman (1986) stated that due to this disease damping off takes place at

seedling stage. Leaf may be attacked at any time. Generally the first symptom appears

on the lower leaves. Spots are clear, circular and grayish. Numerous pycnidia are

formed on aged spots. Infected leaves become yellowish and eventually may die.

Cankers observed at the base of the stem, bark is cracked and woody portion opened.

Light coloured, sunken spots are observed on fruit. Black pycnidia are formed in

spots and the fruits become mummified.

Kumar et al. (1986) described that Phomopsis vexans caused fruit rot of eggplant

which appeared as minute, circular, water soaked, sunken, grayish spots with

brownish halo and have a light coloured center which later enlarged to produce

concentric rings and brownish zones. Spots increased in size and formed larger rotten

areas on which pycnidia developed causing blackening of the affected area.

Ken et al (2009) described that on older plants, round or oval spots develop on the

leaf and stem, which enlarge and become more irregular. Fruits are infected while on

the plant and exhibit spots which appear as pale sunken areas which may cover the

entire fruit if not treated. The small black pycnidia are present in abundance in the

fruit spots.

If conditions become dry, infected fruit become shrivelled, dry, and form black

mummies (Howard et al, 2009).

7

2.2 Incidence and severity of phomopsis blight and fruit rot of eggplant

Meah et al., (2002) recorded up to 100% disease incidence and 9.8% disease severity

in cultivar Dohazari. Meah et al., (2002) also reported a loss equivalent to Taka 808

million (US$ 134 million) per annum due to phomopsis blight and fruit rot of

eggplant in Bangladesh. Khan (1999) reported that phomopsis blight and fruit rot of

eggplant causes about 21% fruit rot and 7% seed rot in eggplant.

Meah (2003) did an epidemiological survey on the incidence and severity of

phomopsis blight and fruit rot of eggplant in 18 major growing areas of Bangladesh

and reported that sandy loam soil, soil moisture in the range of 50-60%, moderate air

temperature (20- C) and high atmospheric moisture (65-78 % RH) significantly

influenced the prevalence of the disease. The prevalence was higher in the southern

part of Bangladesh with moderate weather than the middle and northern part of

Bangladesh with severe weather.

2.3 Morphology of Phomopsis vexans, the causal agent of phomopsis fruit rot of

eggplant

According to Edgerton and Moreland, 1921, pycnidia of Phomopsis vexans is

subepidermal, erumpent, dark, thick-walled and flattened to globose shaped, varying

in size, often 100-300 µm in diameter, with or without a beak. Phialides are hyaline,

simple or branched, sometimes septate, 10-16 µm long and arising from the

innermost layer of cells lining the cavity. Alpha conidia are hyaline, aseptate, sub-

cylindrical and 5-8 × 2-3 µm in size. Beta conidia are filiform, curved, hyaline,

septate, 18-32 x 0.5-2.0 µm in size and non- germinating. Hyphae are hyaline, septate

and 2.5-4 µm diameter.

8

Gratz (1942) stated that, perithecia in culture of Phomopsis vexans are usually in

clusters, 130-350 µm diameter and beaked. Beaks are sinuous, carbonaceous,

irregular and 80-500 µm long. Asci clavate, sessile, 24-44 × 5-12 µm, eight-spored.

Ascospores are biseriate, hyaline, narrowly ellipsoid to bluntly fusoid, one-septate,

constricted at the septum and 9-12 × 3-4.5 µm in size.

Punithalingum and Holliday (1972) reported that, pycnidia of Phomopsis vexans on

stems, leaves and fruits are solitary or gregarious, initially immersed, becoming

erumpent, black, globose to irregular, up to 350µm wide having opening by 20-50

wide ostioles. Conidiophores (phialides) are hyaline, simple or branched, sometimes

septate,10-16µm long, arising from the innermost layers of cells lining the cavity.

Conidia are fusoid to ellipsoidal, hyaline, filiform, curved, rarely erect and 20-30 ×

0.5-1µm in size.

2.4 Seed infection and its impact

The fungus P. vexans is seed borne at significant levels (Edgerton and Moreland,

1921; Toole et al., 1941)

Infection in seed adversely affects the seed quality, causing seed discoloration,

reduced seed weight and density, poor germinability and reduced viability (Toole et

al., 1941; Porter, 1943)

Phomopsis vexans is an important fungus isolated from surface sterilized eggplant

seeds and stated to be seed borne by Walker (1952) in USA. Panwar (1957) reported

that the causal organism of the disease remained viable for about 14 months in soil

debris and in the seeds from infected fruits, which were poor in germination.

9

Infected seeds contain profuse branched septate mycelium aggregated in the seed

coat, between the seed coat and endosperm and in the embryo region of the seeds.

Pycnidia are produced in the seed coat, between the seed coat and endosperm, and in

the endosperm tissue (Vishunavat and Kumar, 1994).

Karuna et al. (1994) studied on seed borne inocula of P. vexans and the effect of

infection on seed quality in eggplant and found that seed borne infection of varieties

PBR -7, PBR-5, MHB-1 and Pant rituraj were 29%, 36%, 35% and 56% respectively.

Seed infection caused various degrees of seed discoloration. On the surface of dry

seed, the fungus was observed as black pycnidial bodies. Pre-treatment of seeds with

0.1% HgCl2 solution reduced seed infection. P. vexans infection adversely affected

seed quality.

Karuna et al. (1994) also studied locations of infection of P. vexans in brinjal seeds

and observed that component plating of seed of the arburgine cultivar PBR-5 yielded

22% and 12% infection in seed coats and cotyledons, respectively. Pycnidia and

mycelium were seen on seed coats and in the embryo.

Pan et al. (1995) studied the seed borne nature of P. vexans and reported that P.

vexans was present on seed coat and on the cotyledons of arburgine seeds collected

from disease fruit in West Bengal, India. It was suggested that the seeds were an

infection source and might serve as a substrate for pathogen survival.

Gangadharaswamy et al. (1997) studied the impact of P. vexans on seed quality of

brinjal and reported that 13% seed infection was found in variety Erenagere collected

from farmers and no seed infection was found in variety Erenagere collected from

farmers and no seed infection was found in variety “Pusa purple” collected from

10

National Seed Corporation. P. vexans even at low incidence caused failure of

emergence of root, ultimately rotting of seeds.

Khan et al. (2002) analyzed 22 seed samples collected from farmers of different

eggplant growing areas of Bangladesh and recorded 0.5-7.0% infection of P. vexans.

2.5 Management of phomopsis fruit rot through plant extracts and biological

control agent

Mohanty et al. (1995) investigated the allelopathic control of Phomopsis vexans,

causal agent of phomopsis fruit rot of brinjal by aqueous leaf extracts of five plants.

Fungal growth was inhibited to a maximum by leaf extracts of Allamonda cathertica

(93.75%) followed by Aegle mermelos (85.38%). Leaf extracts of Catheranus roseus,

Polyalthia longifolia and Azadirachta indica were equally effective, but that of

Octimum sanctum was the least effective causing 52.23% growth inhibition.

Khaleduzzaman (1996) reported that out of four plant extracts, garlic bulb extract was

found best in reducing seed-borne prevalence and increasing germination percentage

of seed followed by ginger, neem and bishkatali extracts. The medicinal plant extracts

reduced seed borne prevalence of all the fungi. The reduction of different fungi

differed significantly from the control. However, among the 6 extracts, ginger extract

performed better in reducing seed-borne prevalence of all the major fungi and

increasing germination followed by followed by garlic, Bishkatali and ghagra extract.

Crude extracts and dilution extracts (1:1v/v) of the selected plant species were used

for treating the seeds. Neem bark, garlic clove, bishkatali gave better results. Further

investigation in controlling seed borne fungi in wheat seed using alcoholic extracts of

neem bark and garlic clove also gave best results.

11

Panda et al. (1996) tested the efficacy of leaf extracts from Polyalthia longifolia,

Aegle mermelos, Azadirachta indica, Catheramus roseus, Octimum sanctum and

Allamonda cathertica for control of phomopsis blight (caused by P. vexans). Leaf

extracts of Allamanda cathertica had excellent potential as a fungicide.

Khan (1999) studied the effect of plant extracts (Allamanda, Bael, and Neem) for the

management of phomopsis blight and fruit rot of eggplant in field condition. Among

three plant extracts, allamanda spray was found most effective. He also found that the

incidence and severity of phomopsis fruit rot varied from one plant extract to another

when aqueous leaf extract of neem, allamanda and bael applied at flowering, fruiting

and peak fruiting stages in different doses (S, S/10 and S/100).

Meah (2003) reported that garlic bulbs extract (1:1) and allamonda leaves extract

efficiently controlled P. vexans in the laboratory, nursery house and in the field

reducing severity of leaf blight and fruit rot by 71-75%.

Hawlader (2003) reported that seed treatment with allamanda leaf extracts (1:1)

effectively increased germination of eggplant seeds and considerably decreased

nursery diseases.

Islam (2004) found garlic bulbs and allamonda leaves extract cause 76-100%

inhibition of mycelial growth of Phomopsis vexans. Diethyl ether, Dichloromethane

and water acted as effective solvents. TCL studies showed the presence of a number

of compounds having very low polarity in garlic bulbs and allamanda leaf extracts

acting against the pathogen.

12

Antagonistic Pseudomonas fluorescens and Trichoderma harzianum seed treatment

and spray treatment were found to be effective against P. vexans (Srinivas et al.,

2005)

Alam (2005) tested 11 botanicals to control phomopsis blight and fruit rot of

eggplant. Out of 11 botanicals, garlic (Allium sativum L.) bulb and allamanda

(Allamanda cathertica L.) leaf extracts were found promising arresting mycelial

growth and inhibiting spore germination of Phomopsis vexans in in vitro condition

and controlled phomopsis blight and fruit rot of eggplant in the field significantly. He

also found that combinations of apparently healthy seed, seed treated with garlic bulb

extract and soil treated with Trichoderma harzianum completely controlled damping

off, tip over and seedling blight in the nursery bed in the net house and increased

germination by 48.83% over control.

13

Materials and methods

3.1 In vitro EXPERIMENT

3.1.1 Experimental site

The In vitro experiments were conducted at M.S. Laboratory of the Department of

Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka.

3.1.2 Experimental period

The experiments were conducted in August-September, 2014.

3.1.3 Preparation of culture media

Composition of Potato Dextrose Agar (PDA) media are as follows:

Potato (Peeled and sliced) 200g

Dextrose 20g

Agar 20g

Water 1000ml

Peeled and sliced potato was boiled to collect the extract by sieving with a fine piece

of cloth. Afterwards, the other ingredients were mixed up and then heated gently for

few minutes. After preparation, the contents were poured into 250ml Erlenmeyer

flasks, plugged with cotton and were sterilized in autoclave at C under 15 PSI

for 20 minutes. This medium was acidified with 30 drops of 50% lactic acid per 250

ml medium to inhibit the growth of bacteria. These 20 ml of the medium was poured

in to each petri-plate (9cm dia.) and allowed to solidify.

14

3.1.4 Isolation and maintenance of culture of Phomopsis vexans

The pathogen, Phomopsis vexans was isolated by tissue planting method from

naturally infected brinjal fruit collected from the Horticulture Garden of Sher-e-

Bangla Agricultural University, Dhaka (Islam et al.). The sample showed typical

phomopsis fruit rot symptom having pale, sunken spots on the mummified fruit (CMI

Description no.338). The infected fruit was repeatedly washed in fresh water and

surface sterilized with 10% Clorox for 1 minute followed by three times washing in

distilled water to eliminate traces of mercury. Infected tissues were cut and placed in

PDA media and incubated at 22±2 for 10 days. After incubation, it was observed that

white mycelia and pycnidia were formed. Several slides were prepared and examined

under compound microscope. The fungus was identified according to CMI

description (No. 338). The pathogen was purified and multiplied subsequently

through block culture on PDA medium and culture was stocked in PDA slant for

future use.

3.1.5 Preparation of spore suspension of Phomopsis vexans

Phomopsis vexans was grown on PDA medium in petridish at C temperature.

After formation of pycnidia (in about 15-20 days), 5ml/plate sterile water was added

and the spore masses was scraped away with sterile needle/scalpel. The conidial

suspension was made with additional 45ml water and blended in Moulinex blender

for 2 minutes in medium speed and filtered through sterile cheesecloth. The

suspension was adjusted to 105conidia/ml solution and stored at C for future use.

15

3.2 FIELD EXPERIMENT

3.2.1 Experimental site

The experiments were conducted in the experimental field of Department of

Horticulture, Sher-e-Bangla Agricultural University, Dhaka.

3.2.2 Experimental period

The experiments were conducted during the winter season (October, 2014 to March,

2015).

3.2.3 Experimental design and layout

The experiment was laid out in a Randomized Block Design (RCBD) with three

replications where unit plot size was 3.5 sq.m. (3.5m x 1m). Plot to plot distance was

maintained 1m and row to row distance was1.5m. Plant to plant distance 75cm. Soil

in between two plots were taken over to make it raised, thereby facilitating free

movement for nursing and free drainage of excess water during rain. There were three

replications in the experiment. Four plant extracts, one biological control agent, one

fungicide, manuring with saw dust, poultry waste constituted the treatments.

3.2.4 Test material (Cultivar)

Eggplant cultivar “BARI Begun 8” was used for the experiment. The seeds were

collected from BARI (Bangladesh Agricultural Research Institute), Gazipur.

16

3.2.5 Raising of seedlings

Seedlings were raised in plastic trays with proper care and management. Trays were

prepared by mixing soil, sand and well decomposed cowdung in proportion of 2:1:1.

The mixture were sterilized in autoclave. Then the trays were filled with sterilized

soil and the seeds were sown. Shade was provided to save the young and delicate

seedlings from heavy showering and scorching sunlight. Seedlings were observed

regularly and watering was done as per necessary up to transplanting in the main

field.

17

Plate 1. Eggplant seedlings (Variety-BARI Begun 8) raised in plastic tray

18

3.2.6 Land preparation and fertilizer application

A piece of medium high land with well drainage system was selected and prepared by

ploughing, followed by laddering. The soil was well pulverized for good tilth

condition. Weeds and stubbles were removed. During field preparation, application of

fertilizers and manuring was done with recommended doses (Anonymous, 1997). The

experimental plot was partitioned into the unit plots in accordance with the

experimental design.

Fertilizers and manures used in experimental field

Fertilizer Rate (Kg/ha)

Urea 130

TSP 125

MP 100

Cowdung 10000

Oilcake 500

Whole amount of well decomposed cowdung, TSP and half amount of MP were

applied during land preparation. Urea and remaining half of MP were applied in three

installments as top dressing.

3.2.7 Transplanting of seedlings to the main field

After proper growth of the seedlings they were transplanted in the field followed by

watering. Five seedlings were planted in each subplot maintaining plant-to-plant

distance 75 cm and line-to-line distance 0.5m.

19

a) 20 DAT b) 30 DAT

c) 45 DAT d) 60 DAT

e) 80 DAT f) 90 DAT

Plate 2(a-f).Different growth stages of eggplant showing in the experimental field

20

Plate 3. A view of experimental field in Horticultural farm of SAU

21

3.2.8 Intercultural operation

Various intercultural operations were accomplished for better growth and

development of the seedlings. Weeding, manuring and irrigation were done time to

time according to plants’ need. The plants were also kept free from insect pest attack

by spraying insecticides as required intervals.

3.2.9 Plant extracts used in the experiment

Local name Sceintific name Plant parts used Concentration (w/v)

Garlic Allium sativum Clove 1:2

Allamanda Allamanda cathertica Leaf 1:2

Neem Azadirachta indica A. Juss. Leaf 1:2

Lemon grass Cymbopogon flexuosus Leaf 1:2

3.2.10 Preparation of plant extract

For preparation of plant extracts, required amount of respective parts of each plant

was taken, washed in tap water, crushed in a mortar and pestle. The crushed materials

were blended in an electric blender adding equal amount of sterile water for 1:1

solution. The blend was filtered through sterile cheesecloth. The supernatant was

diluted in equal amount of sterile water for 1:2 solutions.

22

Plate 4. Preparation of different plant extracts. A. Blender B. Garlic clove

C. Garlic clove extract. D. Allamonda leaf E. Allamonda leaf extract F. Neem

leaf G. Neem leaf extract H. Lemon grass I. Lemon grass extract

A

B C

D E

F G

H I H

23

3.2.11 Spraying of plant extracts

The extracts were sprayed at fruiting stage as pre-inoculation spray (before 24 hr. of

inoculation) and post inoculation spray (after 24 hr. of inoculation). The spraying was

done by a hand sprayer to cover whole surface of plant leaf, flower and fruits. An

amount of 50ml solution was sprayed in one plant. Precautions were taken to avoid

drifting of spray materials to neighbouring plants with polythene barrier.

3.2.12 Spraying of bioagent as spore suspension

Spraying of eggplants with spore suspension (5 x 106 spore /ml) of Trichoderma sp.

was done at fruiting stages of the crop through spraying conidial suspension onto all

leaves and fruits of a plant @ 100ml/plant.

24

Plate 5. Pure culture of Trichoderma harzianum

Plate 6. Trichoderma harzianum under compound microscope

25

3.2.13 Spraying of fungicide

The fungicide (Bavistin 50WP) was sprayed at fruiting stage of the crop by a hand

sprayer. Fungicide solution was prepared by taking requisite amount of fungicide

(2gm/L) for optimum concentration.

3.2.14 Inoculation of eggplant by Phomopsis vexans

All leaves and fruits of an eggplant were inoculated with Phomopsis vexans before

and after 24 hr. of treatment with plant extracts. Spore suspension of Phomopsis

vexans (107spores/ml) was sprayed with an atomizer @ 70 ml/plant. The inoculated

plants were kept covered with moist polythene sheet for 24 hr. (Plate. 5)

26

Plate 7. Inoculation of eggplant by Phomopsis vexans

27

3.2.15 Data collection

After inoculation and spraying of fungicide and plant extracts, data were taken at

every 10 days intervals for the following parameters:

1. % Fruit infection

2. % FAD (Fruit area diseased)

Percent FAD (Fruit Area Diseased) was measured by eye estimation. Area of single

fruit was considered as 100%. Deducting the healthy area, the diseased area was

estimated. Average of %FAD was then calculated by dividing the total diseased areas

by the total number of investigated fruits.

Total diseased areas

% FAD = ----------------------------------------------

Total number of investigated fruits

Seed weight of individual plot was also measured after ripening of the fruit.

3.2.16 Seed extraction

The harvested fruits were stored for three to four days until they become soft. This

allows the seed to mature fully. Seeds were extracted by cutting, crushing and

macerating with a mechanical extractor. After extraction, seeds were washed and

cleaned with extra water in a container. Seed drying was done by spreading the wet

seeds in the sun. Seeds were stired with hands at least 2-3 times a day, turning them

over to dry uniformly. The seed should be completely dry to about 8% moisture

content. Amount of seed production was counted in case of each plot.

28

Plate 8. Harvesting of fruit for seed production

29

Plate 9. Extraction of seed from fruit

30

3.2.17 Analysis of data

All collected data were tabulated and analyzed following statistical computer program

(MSTAT-C). Treatment means were compared with Duncan’s Multiple Range Test

(DMRT).

31

RESULTS

4.1 LABORATORY EXPERIMENT

4.1.1 Isolation and identification of causal agent

The causal fungus was isolated from infected fruits and studied in the laboratory. The

fungus was purified and identified as Phomopsis vexans (Fig. 8.). In PDA, the fungus

grew with whitish mycelia which later developed dark gray colony. Conidia of

Phomopsis vexans were hyaline and single celled. There are two types of conidia

(Alpha and Beta) of Phomopsis vexans. Alpha conidia was globose and fusoid and

Beta conidia was hyaline and filiform shaped.

32

Plate 10. Pure culture of Phomopsis vexans

`

Plate 11. Alpha conidia of Phomopsis vexans under compound microscope

33

4.2 FIELD EXPERIMENT

4.2.1 Development of symptoms in brinjal plants following inoculation with

Phomopsis vexans

Seven days after inoculation of the plants by inoculum, leaves showed clearly

defined, circular gray to brown spots with rough coloured centre. The spots coalesced

and affected leaves turned yellow and eventually died. The old spots showed

numerous black pycnidia.

Most of the flowers dropped after 5 days of inoculation. Fruit rot appeared as minute,

globose, water soaked spots with brownish colour, which later enlarged to produce

concentric rings. The outer most ring got separated from the healthy fruit surface.

Spots increased in size and formed large rotten area which produced blackish

pycnidia distributed throughout the rotten fruit. Fruits were mummified. Some

affected fruits dropped off.

Biological control agent Trichoderma harzianum, plant extracts viz. garlic clove

extract, allamonda leaf extract, neem leaf extract, lemon grass extract and fungicide

Bavistin 50WP were applied on cultivated high yielding variety of BARI Begun 8 at

fruiting stage after inoculation of Phomopsis vexans. Saw dust and poultry waste

were also applied in the soil before seedling transplanting to evaluate their

effectiveness against Phomopsis vexans.

34

Plate 12. Infected plant showing phomopsis fruit rot symptom

35

4.2.2 Evaluation ofdifferent treatments against disease incidence of eggplant by

Phomopsis vexans

The effect of Biological control agent Trichoderma harzianum, plant extracts,

Bavistin 50WP, saw dust and poultry waste on disease incidence (percent fruit

infection) at 105 DAT was summarized in table 3. Trichoderma harzianum and

Bavistin 50WP showed significant effect in respect of percent fruit infection in

comparison to control. The highest percent fruit infection was observed in control

plot (26.86) and the lowest percent fruit infection was observed in Trichoderma

harzianum treated plot (9.54). Bavistin 50WP treated plot showed 10.77% fruit

infection. The plant extracts (garlic clove extract, allamonda leaf extract, neem leaf

extract and lemon grass extract) also showed significant effect in reducing percent

fruit infection in comparison to control. However, the effects were statistically similar

among themselves. Among the plant extracts, the lowest fruit infection was observed

in allamonda leaf extract treated plot (14.16%) followed by neem leaf extract

(16.66%), garlic clove extract (20.78%) and lemon grass extract (21.51%).Saw dust

and poultry waste treated plot showed 14.79% and 15.54% fruit infection

respectively.

The effect of biological control agent Trichoderma harzianum, plant extracts,

Bavistin 50WP, saw dust and poultry waste on disease incidence at 114 DAT was

summarized in table 3.Application of fungicide (Bavistin 50WP) and Trichoderma

harzianum significantly reduced percent fruit infection. The lowest percent fruit

infection was observed in Trichoderma harzianum treated plot (14.77) followed by

Bavistin 50WP treated plot (16.53). Among the plant extracts, the lowest fruit

infection was observed in Allamonda leaf extract treated plot (17.96%) followed by

Garlic clove extract (18.29%), Neem leaf extract (19.65%) and Lemon grass extract

36

(24.82%).Saw dust and poultry waste also showed significant effect in reducing

percent fruit infection where20.91% fruit infection was recorded in saw dust treated

plot and 21.46% fruit infection was found in poultry waste treated plot. The control

plot showed 23.64% fruit infection.

The biological control agent Trichoderma harzianum, plant extracts, Bavistin 50WP,

saw dust and poultry waste showed significant effect in terms of disease incidence

(i.e.; percent fruit infection) at 123 DAT in comparison to control. The lowest percent

fruit infection (%FI) was observed in Trichoderma harzianum treated plot (16.52).

Bavistin 50WP treated plot showed 18.43% fruit infection. The plant extracts (garlic

clove extract, allamonda leaf extract, neem leaf extract and lemon grass extract)

showed significant effect in reducing percent fruit infection compared to control. But

effects were statistically similar among themselves. Among the plant extracts, the

lowest fruit infection was found in allamonda leaf extract treated plot (18.60%)

followed by lemon grass extract (20.24%), neem leaf extract (24.16%) and garlic

clove extract (25.31%). Saw dust and poultry waste also showed significant effect in

reducing percent fruit infection (%FI) and percent fruit area diseased (%FAD).

20.37% fruit infection was recorded in saw dust treated plot and 27.13% fruit

infection was found in poultry waste treated plot. The highest percent fruit infection

(%FI) was observed in control plot (29.73%) (Table 3)

37

Table 1. Effect of different treatments on disease incidence of fruit rot of

eggplant at different days after transplanting (DAT)

Treatment Disease Incidence

105 DAT 114 DAT 123 DAT

T1 9.54b 14.77 b 16.52 c

T2 20.78 ab 18.29 ab 25.31 abc

T3 14.16 b 17.96 ab 18.60 bc

T4 16.66 ab 19.65 ab 24.16 abc

T5 21.51 ab 24.82 a 20.24 bc

T6 10.77 b 16.53 ab 18.43 bc

T7 14.79 ab 20.91 ab 20.37 bc

T8 15.54 ab 21.46 ab 27.13 ab

T9 26.86 a 23.64 ab 29.73 a

CV (%) 27.90 30.66 21.28

Here,

T1= Trichoderma harzianum

T2= Garlic clove extract

T3= Allamonda leaf extract

T4= Neem leaf extract

T5= Lemon grass extract

T6= Bavistin 50WP

T7= Saw dust

T8= Poultry waste

T9=Control

38

4.2.3 Evaluation of different treatments against disease severity of eggplant by

Phomopsis vexans

The effect of Biological control agent Trichoderma harzianum, plant extracts,

Bavistin 50WP, saw dust and poultry waste on disease severity (percent fruit area

diseased) at 105 DAT was summarized in table 4. The biological control agent

Trichoderma harzianum and fungicide Bavistin 50WP showed significant effect in

terms of percent fruit area diseased in comparison to control. The highest percent fruit

area diseased (%FAD)was observed in control plot (14.69) and the lowest percent

fruit area diseased (%FAD) was observed in Trichoderma harzianum treated plot

(3.73). Bavistin 50WP treated plot showed 5.09% fruit area diseased (%FAD).The

plant extracts (garlic clove extract, allamonda leaf extract, neem leaf extract and

lemon grass extract) showed significant effect in reducing percent fruit area diseased

(%FAD) in comparison to control. Among the plant extracts, the lowest fruit area

diseased (%FAD) was recorded in allamonda leaf extract treated plot (7.50%)

followed by neem leaf extract (9.98%), garlic clove extract (10.77%) and lemon grass

extract (12.18%). Saw dust and poultry waste also showed significant effect in

reducing percent fruit area diseased in comparison to control. But the effects were

statistically similar between themselves. 8.03% fruit area diseased was observed in

poultry waste treated plot and 8.37% fruit area diseased was observed in saw dust

treated plot. The highest fruit area diseased 14.69% was recorded in control plot.

The effect of biological control agent Trichoderma harzianum, plant extracts,

Bavistin 50WP, saw dust and poultry waste on disease severity (percent fruit area

diseased) at 114 DAT was summarized in table 4. The biological control agent

Trichoderma harzianum and fungicide Bavistin 50WP showed significant effect in

terms of percent fruit area diseased in comparison to control. The lowest percent fruit

39

area diseased (%FAD) was observed in Trichoderma harzianum treated plot (6.09).

Bavistin 50WP treated plot showed 9.64% fruit area diseased (%FAD). Among the

plant extracts, the lowest fruit area diseased (%FAD) was recorded in allamonda leaf

extract treated plot (10.01%) followed by neem leaf extract (10.46%), garlic clove

extract (10.77%) and lemon grass extract (16.99%). Saw dust and poultry waste also

showed significant effect in reducing percent fruit area diseased in comparison to

control. But the effects were statistically similar between themselves. 12.28% fruit

area diseased was observed in poultry waste treated plot and 12.31% fruit area

diseased was observed in saw dust treated plot. 12.71% fruit area diseased was

recorded in control plot.

The effect of Biological control agent Trichoderma harzianum, plant extracts,

Bavistin 50WP, saw dust and poultry waste on disease severity (i.e.; percent fruit area

diseased) at 123 DAT was summarized in table 4. The biological control agent

Trichoderma harzianum and fungicide Bavistin 50WP showed significant effect in

terms of percent fruit area diseased in comparison to control. The highest percent fruit

area diseased (%FAD) was observed in control plot (18.95) and the lowest percent

fruit area diseased (%FAD) was observed in Trichoderma harzianum treated plot

(4.83). 10.01% fruit area diseased (%FAD) was recorded in Bavistin 50WP treated

plot. The plant extracts (garlic clove extract, allamonda leaf extract, neem leaf extract

and lemon grass extract) showed significant effect in reducing percent fruit area

diseased (%FAD) in comparison to control. Among the plant extracts, the lowest fruit

area diseased (%FAD) was recorded in lemon grass extract treated plot (10.48)

followed by allamonda leaf extract (12.73), neem leaf extract (15.14) and Garlic

clove extract (15.55). Saw dust and poultry waste also showed significant effect in

reducing percent fruit area diseased in comparison to control. However, the effects

40

were statistically similar between themselves. The disease severity (%FAD) were

13.94 and 17.45 in saw dust and poultry waste treated plot respectively.

41

Table 2. Effect of different treatments on disease severity of fruit rot of

eggplant at different days after transplanting (DAT)

Treatment Disease Severity (%FAD)

105 DAT 114 DAT 123 DAT

T1 3.737 c 6.090 c 4.830c

T2 10.77 abc 12.74 ab 15.55 ab

T3 7.507 bc 10.01 bc 12.73 ab

T4 9.983 abc 10.46 bc 15.14 ab

T5 12.18 ab 16.99 a 10.48 bc

T6 5.090 bc 9.647 bc 10.01 bc

T7 8.373 abc 12.31 ab 13.94 ab

T8 8.037 abc 12.28 ab 17.45 a

T9 14.69 a 12.71 ab 18.95 a

CV (%) 41.12 29.43 18.60

Here,

T1= Trichoderma harzianum

T2= Garlic clove extract

T3= Allamonda leaf extract

T4= Neem leaf extract

T5= Lemon grass extract

T6= Bavistin 50WP

T7= Saw dust

T8= Poultry waste

T9=Control

42

4.2.4 Effect of different treatments on plant growth characteristics

4.2.4.1 Effect of different treatments on height of plant

The effect of fungicide Bavistin 50WP and Biological control agent Trichoderma

harzianum on height of eggplant was summarized in table 1. Application of

fungicide (Bavistin 50WP) and Trichoderma harzianum significantly influenced

plant height. The highest (89.33cm) height was observed in Trichoderma harzianum

treated plot followed by Bavistin 50WP treated plot (84.33 cm). The effect of plant

extracts on plant height differed significantly in comparison to control. Among the

plant extracts, the highest height was observed in case of Garlic clove extract treated

plot (80 cm), preceded by neem leaf extract (78.67cm), lemon grass extract

(77.33cm) and allamonda leaf extract (76 cm). The effect of saw dust and poultry

waste in terms of plant height was statistically similar compared to control plot. The

lowest plant height was observed in control plot (69 cm).

4.2.4.2 Effect of different treatments on number of branch of eggplant

The effect of fungicide Bavistin 50WPand Biological control agent Trichoderma

harzianum on number of branch per plant was presented in table 1. The highest

number of branch per plant was observed in Trichoderma harzianum treated plot (9)

followed by Bavistin 50WP treated plot (8). The effect of plant extracts on number of

branch per plant were statistically identical among themselves but significantly

different in comparison to control. Among the plant extracts, the highest number of

branch per plant (7.33) was observed in garlic clove extract and neem leaf extract

treated plot preceded by lemon grass extract (6.66) and allamonda leaf extract (6.33).

The effect of saw dust and poultry waste in terms of number of branch per plant was

statistically similar to control. The number of branch per plant of poultry waste and

43

saw dust treated plots was consequently 5.66 and 5.33. The number of branch per

plant in control plot was 5.33.

4.2.4.3 Effect of different treatments on number of leaf per branch of eggplant

The effect of fungicide Bavistin 50WP and Trichoderma harzianum on number of

leaf per branch of eggplant was summarized in table 1. The highest number of leaf

per branch was observed in case of Trichoderma harzianum treated plot (27) and the

second highest (25) was observed in Bavistin 50WP treated plot. The effect of plant

extracts on number of leaf per branch differed significantly in comparison to control

but the effects between them was statistically similar. Among the plant extracts, the

highest number of leaf per branch was observed in garlic clove extract treated plot

(22) preceded by neem leaf extract (21.67), lemon grass extract (20.67) and

allamonda leaf extract (20). The effect of saw dust and poultry waste in terms of

number of leaf per branch differed significantly compared to control. The number of

leaf per branch of saw dust and poultry waste treated plots were respectively 17 and

16. The number of leaf per branch in control plot was 13.

44

Table 3. Effect of different treatments on growth characteristics of eggplant

Treatment Plant

height

(cm)

Number of branch

per plant

Number of leaf

per branch

T1 89.33 a 9.000 a 27.00 a

T2 80.00 c 7.333 abc 22.00 b

T3 76.00 d 6.333 bcd 20.00 b

T4 78.67 cd 7.333 bc 21.67 b

T5 77.33 cd 6.667 bcd 20.67 b

T6 84.33 b 8.000 ab 25.00 a

T7 71.33 e 5.333 d 17.00 c

T8 71.00 e 5.667 cd 16.00 c

T9 69.00 e 5.333 d 13.00 d

CV (%) 1.55 9.84 4.96

Here,

T1= Trichoderma harzianum

T2= Garlic clove extract

T3= Allamonda leaf extract

T4= Neem leaf extract

T5= Lemon grass extract

T6= Bavistin 50WP

T7= Saw dust

T8= Poultry waste

T9=Control

45

4.2.5 Effect of different treatments on fruit and seed yield of eggplant

The effect of fungicide Bavistin 50WP and biological control agent Trichoderma

harzianum on fruit yield was summarized in table 5. Application of fungicide

(Bavistin 50WP) and Trichoderma harzianum significantly increased fruit yield in

comparison to control. The highest (36.38 ton/ha) fruit yield was observed in

Trichoderma harzianum treated plot where the second highest (35.01 ton/ha) was

observed in Bavistin 50WP treated plot. The effect of plant extracts on fruit yield

differed significantly in comparison to control but the effects were statistically

similar. Among the plant extracts, the highest fruit yield was observed in lemon grass

extract treated plot (27.99 ton/ha) preceded by neem leaf extract (27.28 ton/ha), garlic

clove extract (27.11 ton/ha) and allamonda leaf extract (26.27 ton/ha).The effect of

saw dust and poultry waste in terms of fruit yield was statistically identical. The

plants of poultry waste and saw dust treated plots consequently produced 18.39 ton/ha

and 16.15 ton/ha fruit. 14.67 ton/ha fruit yield was found in control plot.

The effect of fungicide Bavistin 50WP and biological control agent Trichoderma

harzianum on seed yield was summarized in table 5. Application of fungicide

(Bavistin 50WP) and Trichoderma harzianum significantly increased seed yield. The

highest (35.67 gm/plot) seed yield was observed in Trichoderma harzianum treated

plot and the second highest (32.48 gm/plot) was observed in Bavistin 50WP treated

plot. The effect of plant extracts in terms of increasing seed yield differed

significantly in comparison to control. Among the plant extracts, the highest seed

yield was observed in garlic clove extract treated plot (28.51 gm/plot), preceded by

lemon grass extract (28.07 gm/plot), neem leaf extract (24.85 gm/plot) and allamonda

leaf extract (13.95 gm/plot). The effect of saw dust and poultry waste in terms of seed

yield was statistically similar compared to control plot. The plants of saw dust and

46

poultry waste treated plots consequently produced 9.88 gm/plot and 15.05 gm/plot

seed. Seed production found in control plot was 10.77 gm/plot.

47

Table 4. Effect of different treatments on fruit and seed yield of eggplant

Treatment Fruit yield (tonha-1

) Seed Yield (gm/plot)

T1 36.38a 35.67a

T2 27.11b 28.51bc

T3 26.27b 13.95d

T4 27.28b 24.85c

T5 27.99b 28.07bc

T6 35.01a 32.48ab

T7 16.15c 9.880d

T8 18.39c 15.05d

T9 14.67c 10.77d

CV (%) 9.32 9.48

Here,

T1= Trichoderma harzianum

T2= Garlic clove extract

T3= Allamonda leaf extract

T4= Neem leaf extract

T5= Lemon grass extract

T6= Bavistin 50WP

T7= Saw dust

T8= Poultry waste

T9=Control

48

DISCUSSION

The eggplant belongs to the family Solanaceae, is a popular, nutritious and widely

grown vegetable in Bangladesh as well as in the world. About 8 million farm families

are involved in eggplant cultivation in Bangladesh. Its position in terms of acreage

production is second in vegetable crops and the total acreage of eggplant is 44,377

acres with total annual production of 3,68,000 metric tons in the country (BBS,

2013). A large number of cultivars are grown in Bangladesh, which is showing a wide

range of variation in yield performance. It is a continuous source of income for the

rural farmers and provides employment facilities for the rural people. Therefore, it

plays a vital role to boost our national economy. It is grown round the year especially

during the lean period when the seasonal vegetables are in a scarcity in the market.

Eggplant, thus regarded as a cash crop to the farmers which provides them continuous

harvesting and financial assistance.

Such a potential crop is known to suffer from 12 diseases and among them phomopsis

fruit rot caused by Phomopsis vexans has been treated as one of the major constraints

in eggplant cultivation in the country (Khan, et al., 2002; Das, 1998).The infected

fruit develops pale and sunken spots that progress covering the entire surface and

cause dropping. Affected portion is mummified producing huge pycnidia on the

surface and becomes non-edible (Singh, 1992; Ashrafuzzaman, 1986).

The organism isolated from diseased fruits collected from the field and experimental

plot was resembled with those described by Punithalingum and Holliday (1972),

Islam (2005) and CMI description No. 338.

49

Effect of Trichoderma harzianum and plant extracts against phomopsis fruit rot

of eggplant caused by Phomopsis vexans

From the field study it was evident that the pathogen Phomopsis vexans was

effectively controlled by Trichoderma harzianum. Spraying of spore suspension of

Trichoderma harzianum reduced disease incidence to 9.54, 14.77 and 16.52 at 105,

114 and 123 DAT, respectively. Disease severity was reduced to 3.73, 6.09 and 4.83

at 105, 114 and 123 DAT, respectively. The findings of the present investigation are

well supported by the findings of Meah and Howlader (2003). Howlader (2003)

reported that spraying of spore suspension of T. harzianum CP yielded good result

against phomopsis blight and fruit rot of eggplant in the field.

Evidence of using Trichoderma spp. against plant pathogens especially against fungal

pathogens are available in the literature (Shamsuzzaman et al; 2003a; Bari et al; 2000;

Prassad and Rangeshwarn, 2000; Kaur and Mukhapaddhyay, 1992; Kumar and

Khare, 1990; Harman et al., 1989; Sivan, et al., 1984; Agrawal et al., 1977) however

research with Trichoderma spp. against P. vexans are few. Meah (2003) found that T.

harzianum CP significantly controlled the nursery diseases caused by Phomopsis

vexans and increased seed germination by 49% over control. He also reported that

spraying of spore suspension of T. harzianum CP was effective in controlling

phomopsis blight and fruit rot of eggplant in the field.

Different strains of T. harzianum act as strong competitors to other pathogenic

microorganisms in the rhizosphere colonizing the root zone. (Harman et al. 1989).

The account for disease reduction by Trichoderma strains was explained as the

competition for space and nutrients with the pathogenic fungi (Alabouvette et al.

1992) and the production of antibiotics by the antagonists (Larkin et al. 1996). Elad et

50

al., (1982) reported the lylic activity of extra cellular enzymes of T. harzianum that

aided to antagonize the pathogen.

The plant extracts also effectively controlled the disease reducing fruit infection

significantly. Garlic clove extract reduced disease incidence to 20.78, 18.29 and 25.31

at 105, 114 and 123 DAT respectively. In garlic clove extract treated plot disease

severity was reduced to 10.77, 12.74 and 15.55 at 105,114 and 123 DAT,

respectively. The efficacy of garlic clove extract in controlling P. vexans has been

reported by other workers. Inhibition of pathogenic fungi by garlic bulb extract might

be due to the presence of antimicrobial compounds in the extract. It has been reported

that the antibiotic substance present in garlic is the allyl compound of allyl

thiosulphate (Cavallito et al.1944). It is also reported that garlic contains an amino

acid alliin which on crush transferred into allicin by the action of allicinase enzymes

and this allicin is toxic to microorganism (http//:www.gourmetgarlicgardens.com).

Allamonda leaf extract also reduced disease incidence to 14.16%, 17.96% and 18.6%

at 105, 114 and 123 DAT, respectively and disease severity to 7.50%, 10.01% and

12.73% at 105, 114 and 123 DAT, respectively. The findings of the present

investigation are well supported by the findings of Khan et al.(2002), Kuprasvile

(1996), Panda et al.(1996) and Mohanty et al., (1995) who reported that allamonda

leaf extract inhibited the growth of P. vexans significantly. Tiwari et al., (2002)

reported that Allamonda cathertica acted as antidermatophitic agent against fungi.

Reports on the toxic compounds of allamonda leaf contains some substances toxic to

the micro-organisms (Hwang, 2001).Some unidentified compounds extracted from

Allamanda cathertica using organic solvents prevented growth of P. vexans in culture

at unspecified concentrations (Masuduzzaman et al., 2008). In the present

investigation, neem leaf extract also reduced disease incidence and disease severity

51

effectively. Disease incidence was reduced to 9.54, 14.77 and 16.52 at 105, 114 and

123 DAT, respectively and disease severity to 9.98, 10.46 and 15.14 at 105, 114 and

123 DAT, respectively. The present results of effectiveness of neem leaf extract

corroborate with the report of other workers. Neem and its products have been widely

reported to control plant fungal diseases (Vir and Sharma, 1985; Amadioha, 2000;

Dubey et al., 2009).The bio-efficacy of neem extract over pathogens can be attributed

to the fact that neem has active compounds such as azadirachtin, nimbin, nimbidin,

nimbinene and azadirone which are antifungal, antibactrial and anti-insecticidal in

nature (Bohra et al., 2006). From the field study it was evident that the pathogen

Phomopsis vexans was effectively controlled by lemon grass extract. Lemon grass

extract reduced upto 21.51, 24.82 and 20.24disease incidence at105, 114 and 123

DAT, respectively. It also reduced disease severity to 12.18, 16.99 and 10.48 at 105,

114 and 123 DAT, respectively. The findings of the present investigation are well

supported by the findings of Ogunlana et al. (1987) who described antimicrobial

effects of lemon grass, including activity against both gram-positive and gram-

negative bacterial pathogens and fungi.

Effect of Bavistin 50WP, saw dust and poultry waste against Phomopsis vexans

Bavistin 50WP was found promising against P. vexans in the field. Bavistin reduced

disease incidence to 10.77, 16.53 and 18.43 at 105,114 and 123 DAT respectively.

Disease severity was also reduced to 5.09, 9.64 and 10.01 at 105, 114 and 123 DAT,

respectively. The present findings are in agreement with the Meah (2003), Meah et al.

(1998), Islam and Sitansu (1992) and Islam et al.(1990). Meah (2003) reported that

Bavistin 50WP (0.1%) significantly controlled phomopsis blight and fruit rot of

eggplant in the nursery and as well as in the field condition. Meah et al. (1998) also

stated that Bavistin 50WP @ 0.2% applied both before and after inoculation of P.

52

vexans on the surface rough fruit showed effective performance in reducing percent

fruit infection and percent fruit area diseased producing the least sized lesion. Islam

and Sitansu (1992) reported that in a field trial against Phomopsis vexans, Bavistin

50WP (0.1%) provided nearly complete control of leaf blight and fruit rot of eegplant

with 3 sprays at 15 days interval. Islam et al. (1990) found in field trial following in

vitro screening of different fungicides, Bavistin 50WP @ 0.1% gave the best control

against phomopsis blight and fruit rot of eggplant. In the present experiment, saw dust

and poultry waste were also found effective against P. vexans. Saw dust showed

lower disease incidence than poultry waste. Saw dust treated plot showed 14.79,

20.91 and 20.37 disease incidence at105, 114 and 123 DAT, respectively. Poultry

waste treated plot showed 15.54, 21.46 and 27.13 disease incidence at 105, 114 and

123 DAT, respectively. In case of disease severity, it was found that saw dust treated

plot showed 8.37, 12.31 and 13.94 disease severity at 105,114 and123 DAT,

respectively. Poultry waste treated plot showed 8.03, 12.28 and 17.45 disease severity

at 105,114 and123 DAT, respectively. Saw dust and poultry waste are now being

considered as environment friendly approach that make the soil suppressive

improving the antagonistic activities of the soil microorganisms. The findings of the

present study corroborate with the findings of Bhuyan (2010) and Dataram (1988).

Bhuyan (2010) reported that poultry waste was found potential for controlling fungus

diseases like rhizome rot of ginger. Dataram (1988) reported that the incidence of

rhizome rot was low when Trichoderma viride was applied to soil along with wood

saw dust.

53

SUMMARY AND CONCLUSION

Phomopsis fruit rot is one of the most important biotic disease that significantly

reduce eggplant production. The fungus Phomopsis vexans is seed borne and can

survive in crop debris in the absence of the host. The fungus can survive for more

than a year in fields where a diseased crop was grown and is mainly favoured by

warm, wet weather. People often manage the disease with plant protection chemicals.

But it is environmentally hazardous and hampers our ecosystem. Thus, finding out the

alternatives of chemical fungicides with eco-friendly components the present

investigation has been undertaken to evaluate the effect of bio-agent, plant extracts

and soil amendments against the disease and find out the suitable management

components for controlling phomopsis fruit rot in our country. The present

experiments were conducted during Rabi season (October, 2014 to March, 2015) in

the Horticultural farm of Sher-e-Bangla Agricultural University. A biological control

agent (Trichoderma harzianum), four plant extracts viz. garlic clove extract,

allamonda leaf extract, neem leaf extract and lemon grass extract, a fungicide

(Bavistin 50WP), saw dust and poultry waste were evaluated against Phomopsis

vexans causing phomopsis fruit rot.

Trichoderma harzianum showed promising performance in reducing percent fruit

infection, percent fruit area diseased and seed infection. Fruit and seed yield was

found the highest in case of Trichoderma harzianum treated plot. Fruit yield and seed

yield was 36.38 ton/ha and 35.67 gm/plot in Trichoderma harzianum treated plot,

respectively. Fruit and seed yield were also found promising in Bavistin 50WP

treated plot where fruit yield and seed yield were 35.01 ton/ha and 32.48gm,

respectively.

54

Among the four plant extracts, allamonda leaf (1:2) extract and neem leaf (1:2)

extract found to be effective against Phomopsis vexans reducing percent fruit

infection and percent fruit area diseased. The fruit yield and seed yield were more or

less similar among the plant extracts treated plot and higher than the control plot. Saw

dust and poultry waste were also found effective in increasing fruit and seed yield.

Considering the performances of bio-agent, plant extracts, fungicide and soil

amendments evaluated in the experiment it is suggested that Trichoderma harzianum,

Bavistin 50WP, allamonda leaf extract and neem leaf extract at 1:2 concentration

could be used for successful management of phomopsis fruit rot of eggplant.

55

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65

APPENDICES

Appendix I. Map showing the experimental site under study

Source: https://www.google.com.bd/maps/place/horticulture-farm

66

Appendix II. Physiochemical properties of soilof the experimental site

Source: Soil Resources Development Institute (SRDI), Dhaka-1207. (Year-

2014)

Characteristics Value

Particle size analysis

% Sand

% Silt

% Clay

Textural class

pH

Organic carbon (%)

Organic matter (%)

Total N (%)

Phosphorus(µg/g soil)

Exchangeable K (me/100 g soil)

Sulphur (µg/g soil)

Boron (µg/g soil)

Zinc (µg/g soil)

25.68

53.85

20.47

Silty-loam

5.8-7.1

0.31

0.54

0.027

23.66

0.60

28.43

0.05

2.31

67

Appendix III. Monthly average relative humidity, maximum and minimum

temperature, rainfall and sunshine hour of the experimental period (October 2014

to March 2015)

Source: Bangladesh Meteorological Department (Climate division), Agargaon, Dhaka

-1207.

Month Average RH

(%)

Average Temperature (ºC) Total

Rainfall

(mm)

Average

Sunshine

hours Min. Max.

March 64 20.4 32.5 65.8 5.2

April 69 23.6 33.7 165.3 4.9

May 81 24.5 32.9 339.4 4.7

June 84 25.4 33.7 415.6 4.8

July 89 27.5 34.8 512.4 4.7