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Chapter 2: Isolation, Molecular Characterization and Predatory

Activity of an Indian Isolates of Nematode-Trapping Fungi

2.1 INTRODUCTION

Food is fundamental need of any organism. The increasing population of world

has lead to high demands of food. In 1996 the World Food Summit (WFS) set the

target of ''eradicating hunger in all countries up to 2015". Subsequently, in 2012 UN

announced to ―create a world where no one is hungry‖ (http://www.fao.org/).

However, on the other hand, an infectious disease appears to cause great economic

looses in agricultural production. Several pathogenic bacteria, fungi, viruses and pests

attack important crops worldwide and ultimately the global food production.

Nematodes are small roundworm and there are thousands of species of this pest is

living on the earth. These nematodes rely on different sources for nutrition including

plants, bacteria, fungi, algae, insects etc. and they are one of the components of the

food chain. On the other hand, there are several species which can infect various

plants and animals. Plant-parasitic nematodes feed on plant tissues and cause diseases.

They interfere in agriculture crop production across the globe (Oka et al., 2000). The

root-knot nematodes, Meloidogyne spp. infects a variety of crop plants and it has been

considered the most damaging plant parasitic nematodes (Regaieg et al., 2010). They

damage the root system and thus reduced the growth of crops. Several strategies have

been employed to control these parasitic nematodes (Mukhtar et al., 2014). Among

these, conventional methods included soil sanitation, soil management, organic

amendment, chemical nematicides, use of resistant variety, biological control and

others (Collange et al., 2011). Chemical nematicides are widely used to control the

nematode population due to their instantaneous result although, the excess uses of

nematicides have resulted in the development of resistance in nematode population

and moreover, these chemical are toxic to plant, soil and environment (Anand et al.,

2010). These incidents have directed the attention of agricultural scientists to develop

bioproducts to control the parasitic nematodes. In fact use of biological products to

protect plant form infectious diseases has got momentum in the field of agriculture as

it is an eco friendly and economically feasible approach.

The activities of soil microorganisms shape the soil characteristics as well as

plant health and productivity (Carvalhais et al., 2012). Soil harbours a diverse range of

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fungi and many of them are rivals of nematodes (Singh et al., 2006). Nematode-

trapping fungi form a peculiar trapping structures by which it trap the nematodes and

kill them. These fungi are possible candidate to be used as a biocontrol agent.

Nematophagous fungi nowadays become a research of interest for the scientists all

over the globe to control parasitic nematodes. Many researchers have isolated and

studied their antagonist potential. Species of Arthrobotrys have been found to show

the predatory activity against both plant and animal parasitic nematodes (Carvalho et

al., 2011; Wang et al., 2013). On the other hand, Duddingtonia flagrans has been

extensively studied against animal parasitic nematodes (Nagee et al., 2001; Braga et

al., 2009; Santurio et al., 2011) and it been also reported to produce chlamydospores

on large scale which can undergo gut passage in small ruminants (Ferreira et al., 2011;

Tavela et al., 2013).

As discussed above, increasing population of world has end up in the

chemicalization of agriculture due of high demand of food. Now a day’s pesticides are

extensively used in agriculture to protect crop plants from pathogen. However,

majority of theses pesticides does not seem to be specifically targeting the specific

organism but conjointly have an effect on non target organisms (Johnsen et al., 2001;

Eisenhauer et al., 2009; Cloyd, 2012; Shi et al., 2013). So it may happen that when

one apply nematophagous fungi together with such pesticides, pesticides may interfere

with their performance. Thus, for it to be successfully integrated into field,

nematophagous fungi have to be assess their compatibility with the variety of

fungicides, insecticides and fungicides which are currently widely employed in

agriculture to protect agriculture crop form infectious diseases. Fungi which may

resist such pesticides are often successfully implemented in the field for biocontrol

purpose. In the present work, effect of different fungicides, herbicides and insecticides

on growth of two nematode-trapping fungi i.e. A. conoides and D. flagrans was also

checked under laboratory conditions. The pesticides that we have selected in the

present work are well known and extensively used in agriculture crop protection.

Many researches from India have reported various biological agents to control

root-knot nematodes (Shankar et al., 2011; Sowmya et al., 2012). However, in India

less attention has been paid on biocontrol of plant parasitic nematodes using

nematophagous fungi. In India till date Arthrobotrys oligospora, Dactylaria

brochopaga and Monacrosporium eudermatum have been reported for the control

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Meloidogyne spp. (Singh et al., 2012; Usman and Siddiqui, 2012; Singh et al., 2013a;

Singh et al., 2013b). In the present work, attention has been paid on the control of

Meloidogyne spp. of root-knot nematodes which cause major crop loss. In the present

study, two nematode trapping fungi form agriculture soil of Anand district of Gujarat

was isolated and characterized based on morphology and 18S rDNA gene sequences.

Additionally, their predatory activity against Meloidogyne spp. was investigated.

2.2 MATERIALS AND METHODS

2.2.1 Isolation of nematophagous fungi and its morphological characterization

A total 23 soil samples were collected from the vicinity of tomato roots from various

agricultural fields infected with nematodes in Anand district of Gujarat during

Decembers and January 2011.

2.2.1.1 Harvesting of root-knot nematodes.

Root-knot nematodes used in the present study were collected from infected

roots of tomato plants. Infected roots were rinsed with tap water to remove soil

particles followed by three times washing with sterile water amended with tetracycline

(50µL/mL). After this, roots were chopped into small pieces and dipped into sterile

distilled water containing tetracycline (30μg/mL) for 30 min. Nematodes that were

coming out into the water were collected by centrifugation at 2000rpm for 3 min.

Again nematodes were washed three times with sterilized water containing

tetracycline and collected each time as mentioned above. Throughout the study

whenever nematodes were needed, they were harvested as described above.

2.2.2.2 Isolation of nematode-tapping fungi.

Nematode-trapping fungi were isolated on water agar (Hi-Media, India), pH

7.0 as per the method described by Nagee et al (2001). Briefly, 1g of each soil

samples were sprinkled on 2% water agar plates amended with tetracycline (50μg/mL)

to avoid bacterial contamination. Plates were incubated at 25+1ºC in dark. After 10

days, as different fungi stared growing, 1mL of nematode suspension (~2000-3000

live nematodes) were added to each plate and further incubated. Afterwards plates

were regularly monitored under a light microscope to visualize trapping of nematodes.

Fungi which showed trapping of nematodes were subsequently purified by sub-

culturing on Czapek Dox Agar (CDA) and Corn Meal Agar (CMA). Purified cultures

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were maintained and preserved on 1.7% CMA by inoculating and incubating at

28+1ºC for 6 days. Pattern of trapping structures and morphology of conidiospores

were used to identify the fungi on the basis of morphology according to (Drechsler,

1937; Cooke and Godfrey, 1964). Microscopy and micrometry of fungi were made in

light microscope under magnification 10X and 40X attached with camera (Leica,

MD2500). For further morphological based identification, conidial size was measured

after growing fungi on nine different media described by (Singh et al., 2005). Length

of 10 conidia form each medium was measured and average length was calculated.

Nematode-trapping fungi were partially identified based on their morphology.

2.2.2 Evaluation of predatory activity against living Meloidogyne spp. of

nematodes

Nematode trapping efficiency of isolated fungi was performed in 90 mm

diameter Petri plates containing half strength CMA (0.85%). 8mm diameter agar disk

having mycelial growth form one week old CMA plate previously grow at 28+1ºC

was inoculated upside down on half strength CMA and allowed to grow in dark for 5

days. On 6th

day 200 μL nematode suspension containing ~1000-1100 nematodes

(adult and larvae) were added to each plates and further incubated in dark at 28+1ºC

for 24hr. Plates devoid of fungi were served as control. There were three replicates for

each fungal treatment and control. Over a period of 24hr incubation, plates were

observed in light microscope attached with camera (Leica, MD2500) under 10X and

40X magnifications. After 24 hours, plates were further observed under the light

microscope to evaluate the morphological variation in the fungi in the presence of

living nematodes. After this, plates were flooded with 2 mL normal saline solution to

recover untrapped nematodes. 100 μL of this suspension was deposited on glass slide

6 times in 10mm diameter quarter previously marked and the number of nematodes

was counted under light microscope under 4X magnification. The mean number of

nematodes in these aliquots was used to find the total number of nematodes trapped by

fungi. The results were statistically analyzed by one way ANOVA to compare

parasitism of isolates against a control group.

2.2.3 Optimization of growth conditions

As a fact, environmental factors viz., temperature, nutrient source, pH of soil

as well as other parameters affect the performance of biocontrol agent under the field.

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Optimum growth conditions i.e. suitable medium, pH and temperature was evaluated

for the isolates.

2.2.3.1Effect of media

Effect of nine different media described by (Singh et al., 2005) and other was

assessed to know which medium promotes luxurious growth of our isolates. For all the

media, agar plates were prepared and inoculated centrally with 8mm diameter disc

with mycelial growth from previously grown fungi. Plates were incubated at 28+1ºC

for 5 days. After 5 days, radial diameter of growth was measured and mean from three

plates were calculated and compared.

2.2.3.2 Effect of pH

To investigate which pH supports healthy growth of the isolated nematode-

trapping fungi, isolates were subjected to grow on corn meal agar having different pH

i.e. 4, 5, 6, 7, 8 and 9. The desired pH of medium was adjusted by adding 0.1N NaOH

or 0.1N HCL. Plates were incubated at 28+1ºC for 5 days and effect of pH was

assessed by measuring the radial diameter of growth.

2.2.3.3 Effect of temperature

Both the isolates were further subjected to different temperature conditions for

determination of optimum temperature for growth. Corn Meal Agar (CMA) (pH7) was

prepared, inoculated centrally and incubated at variable temperatures viz., 4+1, 15+1,

25+1, 28+1, 37+1 and 42+1ºC and incubated in the dark for 5 days. Effect of

temperature was evaluated as described above. All the above experiments i.e. effect of

media, pH and temperature were performed in triplicate.

2.2.4 Molecular characterization of fungi

Fungi were identified based on 18S rDNA gene sequencing. For DNA

isolation, 70 mL of 2.4 % potato dextrose medium (PDB) (Hi-media, India) was

prepared in 250 mL Erlenmeyer flask. Medium was inoculated with spore suspension

and allowed to grow on rotary shaker 125rpm at 28+1ºC for 7 days. Mycelia were

harvested by filtering the medium using sterilized Whatman filet paper No.1. After

filtering, approximately 100-150mg of mycelia were crushed to fine powder in

mortar-pestle using liquid nitrogen followed by grinding with glass beads (Sigma) in

lysis buffer containing 0.1M Tris (pH 7.1), 0.3M EDTA (pH 8.0) and 1% SDS. To

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this 70µL of 0.1 % β mercaptoethanol was added per 1 mL of lysis buffer.

Homogenized mycelia were incubated at 70ºC for 1hr. After this the mixture was

centrifuged at 10,000rpm for 10minutes. Supernatant was transferred to new 2mL

microfuge tube and protein was precipitated using equal volume of Tris saturated

phenol (pH 8) and centrifuged at 10,000rpm for 10 minutes. This was followed by

mixing of aqueous phase with an equal volume of phenol-chloroform and again

centrifuged at 10, 000rpm for 10 minutes. Finally DNA was precipitated form the

aqueous phase using double volume of chilled ethanol (75%) and incubated at -20 ºC

for 1hr. Precipitated DNA was palleted by centrifuging at 12,000rpm for 10minutes.

DNA was washed with an absolute alcohol and again collected by centrifuging as

mentioned above. Plates were allowed to air dry and resuspended into 25µL nuclease

free water. Quality of DNA was assessed using gel electrophoresis and quantified

using NanoDrop 1000 spectrophotometer (Thermo scientific, USA). The partial

sequence of 18S rDNA encoding gene was amplified by using the forward 5'

AGGGTTCGATTCCGGAGA 3' and reverse 5' TTGGCAAATGCTTTCGC 3'.

Primers were synthesized from Sigma, India. 25 µL PCR reaction mixture was

prepared as follow.

Table 2.1: PCR reaction mixture used to amplify the 18S rDNA gene sequence.

Reagent Quantity

(µL)

Genomic DNA (50-70ng/µL) 1.0

10X TaqA assay buffer 2.5

Forward primer (10 µM) 1.0

Reverse primer (10 µM) 1.0

dNTPs mix (2.5 mM each) 2.5

Taq DNA polymerase (2.0U/ µL) 0.5

MiliQ water 16.5

Total 25.0

Taq DNA polymerase, 10X TaqA assay buffer and dNTPs were purchased from

Genei, Bangalore, India. PCR conditions was optimized and amplification of the

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target sequence was carried out in thermocycler (Corbett, Korea) with cycling profile

describe below.

Table 2.2: PCR cycling conditions applied to amplify the 18S rDNA gene

sequence.

PCR products were electrophoreses at 100V in submarine system in 1%

agarose gel amended with ethidium bromide dye along with 100 bp DNA marker and

visualized on UV transiluminator. Amplified products were sent to Eurofins

Genomics India Pvt. Ltd., Bangalore for sequencing from both the sides using prime

pair which was used to amplify the target sequence. Sequences were analyzed using

CodonCode aligner v9 (http://www.codoncode.com/aligner/) to make single contig.

Sequences were identified by BLASTn search against NCBI non redundant databse

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) Phylogenetic relationship with 13 different

species of nematophagous fungi having different mechanism to infect/trap nematodes

was conducted by MEGA5.1 (Tamura et al., 2011) using Neighbor-Joining (N-J)

method (Saitou and Nei, 1987). Related nucleotide sequences (Table 2.3) were

downloaded from NCBI nucleotide collection and comparisation was made.

Nucleotide sequences encoding for partial 18S ribosomal small subunit were

deposited in GenBank using Bankit tool

(http://www.ncbi.nlm.nih.gov/WebSub/?tool=genbank).

Temperature °C Time (Minutes) Cycles

94 5 1

94 1

35 58 1

72 1

72 7 1

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Table 2.3: 18S rDNA gene sequences downloaded from NCBI nucleotides

collection for phylogenetic analysis.

Name of fungi Mode of killing

nematodes

Accession No. Reference

Arthrobotrys oligospora

NBAII AO1

Adhesive network JX403728 Direct submission

Orbilia auricolor Adhesive network DQ471001 (Spatafora et al., 2006)

A. musiformis Adhesive network AJ001985 Direct submission

A. robusta Adhesive network AJ001988

Monacrosporium

doedycoides

Adhesive network AJ001994 Direct submission

A. superba Adhesive network EU977561 Direct submission

Dactylella oxyspora Adhesive knob AY902793 (Li et al., 2006)

Gamsyllela gephyropaga Adhesive columns EF445990 (Smith and Jaffee,

2009)

Hirsutella minnesotensis Endoparasitic DQ078757 Direct submission

Pochonia chlamydosporia Egg parasitic EU266591 Direct submission

Verticillium

chlamydosporium

Egg parasitic AJ291806 (Morton et al., 2003)

Paecilomyces lilacinus Egg parasitic FR775537 Direct submission

Nematoctonus concurrens Endoparasitic EF546658 Direct submission

2.2.5 Effect of various pesticides on growth of the isolates

Chemicals

All the experiments were performed in Corn Meal Agar (CMA) 1.7% (Hi-

media, India) pH 7.0. Commercial formulations of different pesticides that are

available in market were used for experiment, only benomyl was purched from Sigma

Aldrich, India.

Fungicides: Tebuconzole (Folicur, 25.9% EC), Mancozeb (85% w/w) and

Carbendazim (Bavistin, 50% WP).

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Herbicides: Glyphosate (Roundup, 41% S.L IPA salt), Methsulfuron methyl (Algrip,

20% WG) and Paraquate trichloride (Grammoxone, 43.8%).

Insecticides: Profenofos (Rocket, 50% EC) and Ethion (Fosmite, 50% EC).

Stock solutions of each pesticide were prepared in sterile distilled water except

benomyl which was dissolved in dimethyl sulfoxide (DMSO) and appropriate volume

was added under aseptic conditions in sterilized CMA so as to achieve the required

final concentrations of active moiety. Fungicides were tested at 10-50 µg/ml

concentrations while herbicides and insecticides were tested at 50, 100, 500 and 1000

µg/ml concentrations. All the plates were inoculated centrally with 8 mm mycelial

disk from CMA plates previously grown at 28+1ºC for 7 days. Control plates contain

only CMA except for benomyl for which, the control pale was amended with DMSO.

Plates were incubated at 28+1ºC for 6 days, and radial growth diameter of fungus

colony in centimeter was measured and % inhibition was calculated by using

following formula

% of growth inhibition = (C-T)/C×100

Where: C= Radial diameter of the fungus growth in control plate.

T= Radial diameter of the fungus growth in treated plate (Johnsen et al., 2001)

.

All the studies were preformed in triplicate and mean values of radial growth diameter

for each concentration were used for calculation.

2.3 RESULTS AND DISCUSSION

2.3.1 Isolation and morphological characterization of nematophagous fungi

Isolation of nematophagous fungi from soil is a little bit complicated due to its

less abundance and over growth of other soil fungi. Water agar is nutritionally poor

medium which permits sluggish growth of organisms so it can be use for isolation of

scarce microorganisms. Two different nematode-trapping fungi were isolated from the

soil samples collected from different fields of Anand district of Gujarat. After 20-25

days of incubation, two different fungi showed trapping of nematodes (Figure 2.1).

Both the fungi were subsequently purified by sub-culturing on CMA/CZP agar plates.

Isolates were designated as RPAN10 and RPAN12. Morphology of conidia and type

of trapping structure forms the basis of identification of nematophagous fungi. Isolate

RPAN12 trap nematodes by means of three dimensional net made up of 3-7 rings

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(Figure 2.2). Initially the trapping device looks like a ring (Figure 2.2a) afterward it

turns in to fully developed net like structure. It produced a mono-septet elongated

conidiospores and the length of conidia range form 19.11-31.61µm with an average

length of 24.13±2.1µm (Table 2.1 and figure 2.4) on erect conidiophores (Figure

2.3c). Conidiophores are unbranched and have up to 18 conidia. These characteristics

are found similar to Arthrobotrys conoides described by (Drechsler, 1937) except for

size of conidia which is reported 30-46 µm and number of conidia on conidiophore

upto 30. Size of conidia is also varying from A. conoides isolate CED (GenBank

accession JN191309) reported from Brazil (Falbo et al., 2013).

Figure 2.1: Nematode trapped on water agar plates by the isolates after

incubation with living nematodes in dark for 20-25 days. (a) & (b) nematodes

trapped into three dimensional networks produced by isolate RPAN12 under 4X

magnification and (c) nematode trapped by ring like structure of isolate RPAN10

under 10X magnification.

Another isolate, RPAN-10 forms ring like trapping structure but not typical

networks likes that of A. conoides (Figure 2.5 a&b). Conidia are simple, single septet

on erect conidiophore (Figure 2.5c) measuring in length range 21.38-53.37µm with an

average length of 33.1±5.71µm (Table 2.1 and figure 2.6). The conidiophore produces

consecutive conidia slightly below and to one side of elder conidia (Figure 2.5d).

These characteristics are analogous to Duddingtonia flagrans reported by (Cooke,

1969) exception in size of conidia. Isolate RPAN10 also produced thick walled

chlamydospores (Figure 2.5c). Based upon these morphological characteristics, isolate

RPANT10 may be D. flagrans.

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Figure 2.2: Morphological variation found in trapping structures of A. conoides

(RPAN12) on corn meal agar under 40X objective. (a) developing trapping

structure and (b), (c), (d), (e) & (f) fully developed adhesive networks.

Figure 2.3: Morphology of conidiospores of A. conoides (RPAN 12) after growing

fungi on CMA at 28ºC for 7 days. (a) & (b) monosepted conidia of A. conoides

under 10X & 40X objective respectively and (c) conidia on conidiophore under

10X objective.

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Figure 2.4: Variation in size of conidia of A. conoides on different media after

incubation at 28°C for 7 days. Upper three from left to right on CDA, CMA &

MM plates and lower three from left to right RM, SDA & YEPSSM.

It is well documented that chlamydospores of D. flagrans can survive gut

passage of rumen and hence can be used as biocontrol agent against animal parasitic

nematodes (Waller et al., 2001; Gomez-Rincon et al., 2006; Assis et al., 2012; Tavela

Ade et al., 2013).

Previously A. oligospora, A. oviformis, A musiformis and D. flagrans have

been isolated from the Gujarat and their predatory activity against animal parasitic

nematodes Haemonchus contortus is reported by (Nagee et al., 2001; Chauhan et al.,

2002b; Sanyal and Mukhopadhyaya, 2003). Here we have first time isolated A.

conoides form the Gujarat region. Moreover, previous studies carried out form the

Gujarat were particularly on parasitism against animal parasitic nematodes, using D.

flagrans and other fungi however, in this study attention have been made on root-knot

using A. conoides and D. flagrans.

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Figure 2.5: Morphological characterization of D. flagrans (RPAN10) after

growing on CZP agar at 28ºC for 7 days. (a) & (b) ring like trapping structure (c)

conidia and chlamydospores under 40X magnification.

Figure 2.6: Variation in size of conidia of D. flagrans on different media after

incubation at 28°C for 7 days. Upper three from left to right on CDA, CMA &

MM plates and lower three from left to right RM, NA & YEPSSM.

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Table 2.4: Variation in size of conidia of A. conoides and D. flagrans on different

media after incubation at 28°C for 6 days.

2.3.2 Predatory activity against living Meloidogyne spp. of root-knot nematodes.

There is an imperative need to develop new technologies for crop protection

against the parasitic nematodes as these tiny creatures are getting resistant against

chemical pesticides and cause great economic loose. Use of biocontrol agents is one of

the prominence methods which could be useful in this prospect. In the present study,

in vitro predatory activity of both the isolates of nematode-trapping fungi was

evaluated against the root-knot nematodes. Predatory activity of both the isolates was

performed against living root-knot nematodes in order to estimate which isolate is

Medium A. Conoides D. flagrans

Length

range (μm)

Average size

conidia (μm)

Length

range (μm)

Average size

conidia (μm)

Corn meal agar 22.27–27.68 25.64±1.24 21.65-47.5 36.1±8.22

Czapek’s agar 21.74–25.32 23.9±1.25 31.17-53.37 40.65±6.53

Sabouraud’s

dextrose agar

21.61–28.25 24.99±2.3 28.88-38.92 29.97±5.37

Potato dextrose agar 20.08–30.8 23.7±3.07 23.26-40.46 33.44±4.77

Richard’s agar 19.11–26.28 23.13±2.04 21.38-38.92 29.97±5.37

Jensen’s agar 23.45–25.31 23.01±1.89 23.57-42.09 31.61±5.64

Yeast extract

peptone soluble

starch agar

20.84–28.46 24.63±2.61 22.28-40.25 33.75±6.39

Martin’s agar 21.78–31.61 25.12±2.66 21.61-30.01 26.16±2.92

Nutrient agar 20.89–27.46 23.01±1.89 28.19-44.8 36.26±6.19

Average 24.13±2.1 33.1±5.71

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better to manage plant parasitic nematodes. After 5 days, ~1000-1100 nematodes were

added to growing fungal plates and predatory efficacy was assessed after 24 hours.

Results showed that very few trapping structures were produced by both the isolates

on half strength CMA after 5 days. The numbers of trapping structures were

drastically increased when nematodes were added to growing fungi. This shows that

in the presence of nematodes, both the isolates got induced. Previously this response

was thought to be because of peptides secreted by nematodes (Pramer and Stoll, 1959;

Pramer and Kuyama, 1963; NORDBRING‐HERTZ, 1973; Nordbring-Hertz, 1977).

However, recently pheromones identified as a ascarocides have been reported by

(Hsueh et al., 2013) which might trigger some signaling pathways in A. oligospora

and ultimately lead to aggressive behavior of this fungus. The response of A. conoides

(RPAN12) was superior compared to D. flagrans (RPAN10). A. conoides started

trapping of nematodes after 5-6 hours of incubation and traps almost 92-95%

nematodes within in 24 hours. Compared to A. conoides, D. flagrans showed very low

trapping efficiency and only 26-29% nematodes were trapped within 24 hours. A

significant difference (P<0.001) in trapping efficiency was observed among control,

A. conoides and D. flagrans (Figure 2.7).

Figure 2.7: Trapping efficacy of isolated nematode-trapping fungi. Bar line

indicate standard devotion of three replicates.

This results make clear that A. conoides is more proficient in trapping of root-

knot nematodes then D. flagrans. The difference in efficiency may be attributed to the

difference in trapping weapons that fungi use to trap nematodes. A. conoides make use

of adhesive network made up of 3-7 rings (Figure 2.2) while D. flagrans produces ring

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like structure but did not form network characteristic like that of A. conoides (Figure

2.5 a&b). Moreover, nematophagous fungi are host specific to some extent. As

previously stated, D. flagrans has been extensively studied against animal parasitic

nematodes rather than plant parasitic nematodes (Vilela et al., 2012; Assis et al., 2013)

whereas A. conoides have been found to be a superior in controlling Meloidogyne

infection. For the first time (Al-Hazmi et al., 1982) reported reduction of Meloidogyne

incognita under microplote and greenhouse conditions. Afterward (Campos and

Campos, 1997; Kalele et al., 2010; Falbo et al., 2013) also said that that A. conoides

can be used for the control of root-knot nematodes. A. conoides is also successful

against animal parasitic nematodes. However, very few researchers studied the effect

of A. conoides on animal parasitic nematodes. Say for example (Maciel et al., 2009)

reported reduction of Ancylostoma spp. by A. conoides. Recently (Paula et al., 2013)

have studied successful trapping of Angiostrongylus cantonensis which causes

eosinophilic meningoencephalitis in humans. The present study reveals the trapping

efficiency of local isolates in plate assay, so further research need to be carried out for

suitable formulation and application methodology for A. conoides so it can serve a

more robust prospective mean to control Meloidogyne spp. infection under the field

applications.

2.3.3 Optimization of the growth conditions

For field application, large scale production of fungi is required. Further,

different abiotic factors especially temperature and soil pH affect the efficiency of

biocontrol agents under field conditions (Hasanzadeh et al., 2012). Here, different

cultural conditions viz., pH, temperature and media for favorable growth of the

isolated nematode-trapping fungi were optimized. Of the 9 different media tested,

Corn Meal Agar (CMA) showed luxurious growth of both the fungi followed by

Martinson’s Medium (MM), Jenson’s Medium (JM), Yeast Extract Peptone Soluble

Starch Medium (EEPSTM) and Remington’s Medium (RM). D. flagrans also grow

well on Czapek Dox Agar (CZP) whereas A. conoides showed moderate growth on

RM as compared to D. flagrans. Both isolates showed slow growth on Sabouraud

Dextrose Agar (SDA), Potato Dextrose Agar (PDA) and Nutrient Agar (NA) (Figure

2.8).

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Figure 2.8: Effect of different media on growth of the isolates. AC-Arhtrobotrys

conoides, DF-Duddingtonia flagrans.

While comparing growth of both isolates, A. conoides was found to be slow

growing compared to D. flagrans. All the media contain different pure as well as

crude carbon and nitrogen sources; still both the fungi were able to grow on all the

media tested. This highlighted the saprophytic nature of nematophagous fungi because

in soil, different carbon and other nutrients are available at varying proportions and

fungi have to manage with this. In case of temperature, both the isolates grew

optimally at 25 and 28ºC. These results are comparable with optimum temperature

reported for A. oligospora (Duponnois et al., 1995; Morgan et al., 1997).

Figure 2.9: Efeect of temperature on Figure 2.10: Effect of pH on growth of

the growth the isolates. of the isolates.

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Stunted growth of both fungi was observed at 42ºC while at 4ºC, isolates were failed

to grow this outcome clearly explained that very high and low temperatures is

inhibitory for both the fungi. A. conoides found to be little bit cold loving than D.

flagrans as it showed enhanced growth at 15 ºC and very poor growth at 37 ºC while

this was reverse in case of D. flagrans (Fig. 2.9). In case of pH, healthy growth of the

isolates was observed between pH 6 to 8. These results are comparable with the

optimum pH reported for A. oligospora (Nagesh et al., 2005) and Pochonia

chlamydosporia (Nagesh et al., 2007). At pH 5 and 9 moderate growth was observed

and no growth was observed at pH 4 (Fig. 2.10). A. conoides showed relatively fair

growth at pH 5 and pH9 than D. flagrans. This result showed that very acidic and

alkaline conditions are not conducive for the growth of both the fungi. These results

are analogous with results of (Hasanzadeh et al., 2012). Thus temperature 25-28 ºC

and pH 6-9 was found to suitable for both the isolates. These parameters should be

kept in mind together with other abiotic and biotic factors at the time of field

application otherwise it may be lead to unanticipated results.

2.3.4 Molecular characterization of the isolates

Nematode-trapping fungi produced different types of trapping devices to catch

and kill nematodes. They make three fundamental types of trapping devices adhesive

knobs, constricting rings and adhesive networks (Rubner, 1996). Previously

nematophagous fungi were identified and classified solely based on the type of

trapping devices and morphology of conidia. However, only morphological base

identification does not lead to concluding results. Knowledge of DNA sequence has

become very essential in any biology study. For the very first time, Ahren and their

colleagues make use of 18S rDNA sequences to study the phylogeny of nematode-

trapping fungi (Ahren et al., 1998). They have concluded that 18S rDNA based

phylogeny is supported with the type of trapping structure produced by fungi.

Afterward (Li et al., 2006) proved the same theory. Yet identification of the

nematophagous fungi using small and large ribosome coding gene sequences is

considered to be more accurate than exclusively morphology based (Yang and Liu,

2006).

A good quality and quantity of genomic DNA (Figure 2.11) was isolated from

both the fungi as described in the methodology.

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Figure 2.11: Genomic DNA isolated form AC- A. conoides and DF- D. flagrans on

1.0 % agarose gel.

Figure 2.12: Amplified 18s rDNA encoding gene partial sequence with 100bp

ladder. AC- A. conoides and DF- D. flagrans.

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Figure 2.13: A Neighbor joining tree showing relationship of our isolates with

other nematophagous fungi.

A 552 and 559bp 18S rDNA ITS gene from both the fungi was amplified

(Figure 2.12) and sequenced. Sequences obtained were further subjected to BLASTn

search against NCBI non-redundant databse. Blast results confirmed that isolates

RPAN-12 is Arthrobotrys conoides and RPAN-10 is Duddingtonia flagrans which

also matches with morphology based data. Sequences were submitted to GenBank

under the accession No. JX979095 and JX979094 for A. conoides and D. flagrans

respectively. The phylogenetic relationship of nematophagous fungi based on 18S

rRNA gene, nuclear loci and elongation factor 1-alpha (EF1-a) sequences is been

reported previously (Ahren et al., 2003; Mayer et al., 2005; Li et al., 2006). Form

these data it can be concluded that, trapping device plays a very important role in

classifying nematophagous fungi. Nematophagous fungi have been classified into

three main group i.e. nematode trapping, endoparasitic and egg/cyst parasitic fungi

based upon the mechanisms they use to trap the nematodes. All these groups form

separate clades when phylogeny of nematophagous fungi is studied on the basis of

18S rDNA gene sequences. Figure 2.13 shows N-J tree, representing the association

of our isolate A. conoides and D. flagrans. Here three different clades with bootstrap

values 98, 51 and 74 is clearly seen, in that nematode-trapping fungi are disconnected

from other nematophagous fungi. A. conoides and D. flagrans both are nematode-

trapping fungi falls in the cluster of nematode-trapping fungi with bootstrap value 98

Chapter 2 2014

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while other fungi which forms either adhesive knob (D. oxyspora) adhesive columns

(G. gephyropaga), endoparasitic or egg parasitic fungi (H. minnesotensis, P.

chlamydosporia, V. chlamydosporium, P. lilacinus and N. concurrens) forms separate

clade (Fig.8). These results are analogous with the previous studies described.

2.3.5 Effect of various pesticides on the growth of isolates

In order to develop a successful biocontrol agent against plants parasitic

nematodes, the antagonist or the predator must sustain the abiotic stresses as well as

should be resistant to diverse pesticides that are readily used by framers to control

other plant pathogens. Further, combination of biological and chemical control agent

also called Integrated Pest Management (IPM) is more effective means to control pest

for sustainable agriculture (Talebi et al., 2008; Unlu et al., 2012). Here in this study,

we also checked compatibility of our isolates with different pesticides that are rapidly

used in agricultural practices.

2.3.6.1 Effect of fungicides

A. conoides and D. flagrans are found to sensitive to all the fungicides tested at

40-50 µg/ml, except D. flagrans which showed a very stunted growth i.e. 92.5%

growth inhibition at 40 µg/mL of mancozeb (Figure 2.14). Among all the fungicides,

carbendazim (Bavistin) found to be more toxic than the others. Carbendazim causes

100% growth inhibition even at 10µg/mL of both the isolates. Isolates were resistant

to benomyl compared to other fungicide tested at 10µg/mL concentration as it causes

only 20 and 18.8% growth inhibition of A. conoides and D. flagrans respectively

(Figure 2.15). Moreover, isolates were partially resistant to tebuconzole at 10 µg/mL

which causes only 32.3 and 26.2% growth inhibition of A. conoides and D. flagrans

respectively (Figure 2.16). On comparing both the fungi, D. flagrans was found to

more resistant to fungicides than A. conoides.

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Figure 2.14: Effect of mancozeb on radial Figure 2.15: Effect of benomyl on

growth of the isolates. radial growth of the isolates.

Figure 2.16: Effect of tebuconazole on radial growth of the isolates.

Various findings have evaluated the adverse effect of fungicides on

economically important fungi. Kubilay and Gokce have reported that, fungicides

(captan, dichlofluanid, iprodione, benomyl, and carbendazim) interfere the

performances of Paecilomyces fumosoroseus (Er and Gökçe, 2004). Khalil et al have

also reported incompatibility of Verticillium lecannii an entomopathogenic fungus

with certain pesticides including mancozeb and benomyl (Khalil et al., 1985). There

are several reports on the adverse effect of fungicides on the growth of various

nematophagous fungi. Grewal and Sohi reported that commonly used pesticides

inhibit the growth of A. conoides and A. oligospora (Grewal and Sohi, 1988).

Chauhan and colleagues have also reported inhibitory effect of carbendazim on A.

musiformis (Chauhan et al., 2002a). Pullen et al reported adverse effect of benomyl

and other fungicides on hyphal growth of Hirsutella rhossoliensis at certain

Chapter 2 2014

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concentrations (Pullen et al., 1990). Furthermore, it has been said that tebuconazole

also affect non target soil microorganisms (Cycon et al.; Bending et al., 2007; Muñoz-

Leoz et al., 2011) and thus influence the soil fertility. In addition, our isolates showed

somewhat similar resistant profile towards mancozeb and carbendazim reported

against A. oligospora (Mohammadi Goltapeh et al., 2010). In this reverence, our

isolates were superior to some extent as isolates showed some degree of resistant to all

the fungicide tested up to 20µg/mL except tebuconazole. Results of the presrnt study

shows that our isolates of nematophagous fungi A. conoides and D. flagrans are

partially resistant to tested fungicides upto 10-20 µg/mL. Thus, once these fungi

applied in the field as a biocontrol agent, such fungicides should be used upto a

precise level otherwise it may interfere with the performance of both the fungi.

2.3.6.2 Effect of Herbicides

Herbicides are chemicals used to inhibit the growth of weeds that grow

unnecessarily with the crops. Both the isolates were resistant to glyphosate and

paraquote dichloride up to 100 µg/mL (Figure 2.17&2.18) whereas, metsulfuryl

methyl was found to toxic even at 50 µg/L (Figure 2.19). A. conoides was most

resistant to glyphosate up to 1000 µg/mL while growth of D. flagrans was inhibited at

500µg/mL concentration (Figure 2.17). Paraquote dichloride a contact herbicide and

Metsulfuryl methyl causes 100% inhibits growth of both the isolates at 500 and 1000

µg/ml respectively (Figure 2.18&2.19). A. conoides was partially resistant to

metsulfuryl methyl at 500 µg/mL showed 85.5% growth inhibition whereas, D.

flagrans failed to grow at this particular concentration (Figure 2.19). In contrast to

fungicides, A. conoides was found to more resistant to herbicides than D. flagrans.

Figure 2.17: Effect of glyphosate on radial Figure 2.18: Effect of paraquate

growth of the isolates. dichloride on radial growth of

the isolates.

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Figure 2.19: Effect of metsulfuryl methyl on radial growth of the isolates.

Poprawaski and Majchorwicz have studied the effect of herbicides on growth

of entomopathogenic fungi and concluded that herbicides interfere with the growth of

fungi (Poprawski and Majchrowicz, 1995). Similar results have been reported by

Paraquat and Abdel-Mallek for cellulose decomposing fungi (Paraquat and Abdel-

Mallek, 1987) and for Phymatotrichum omnivorum by Gunasekaran and Ahuja

(Gunasekaran and Ahuja, 1975). In contrast to these, our isolates of nematophagous

fungi were resistant to all the herbicides upto 100 µg/mL and for some herbicides upto

500 µg/mL. This indicates that these herbicides if applied in the field they may have

not much effect on growth of A. conoides (RPAN12) and D. flagrans (RPAN10).

2.3.6.3 Effect of Insecticides

Among the insecticides, profenofos was found to be more lethal for both the

isolates as 75% growth of both the isolates was diminished at 50 µg/mL concentration

(Figure 2.20). In contrast to this, isolates were resistant to ethion up to 100 µg/mL. A.

conoides was found to more resistant to ethion compare to D. flagrans as at A.

conoides is can survive 500 µg/mL concentration whereas D. flagrans failed to grow

(Figure 2.21). According to Vig et al application of insecticides that they have studied

have no any adverse impact on soil fungi (Vig et al., 2008). In contrast to this, (Asi et

al., 2010) reported significant inhibition of mycelial growth and conidial germination

by various insecticides. Das and Mukherjee have reported some beneficial and

harmful effect of insecticides on soil microorganisms. According to them some

insecticides are detrimental to some organisms whereas the some microorganisms

degrade it and utilize it for the growth (Das and Mukherjee, 2000). These result shows

Chapter 2 2014

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that insecticide ethion can be applied but profenophose can’t be applied together with

these isolates in the field.

Figure 2.20: Effect of profenophose on Figure 2.21: Effect of ethion on

radial growth radial growth of the isolates. growth of the isolates.

The overall results of in vitro study indicate that excluding fungicides and few

insecticides at higher concentration, isolated nematophagous fungi can be successfully

use for the control of nematode in the field as a sustainable biocontrol agent.

However, this in vitro study results do not escort to final conclusion because, these

effects may not necessarily reproduce once applied in the field. The reason is that, in

soil or in field a very complex phenomenon is going on. Further, as said by (Mensin et

al., 2013) different pesticides have different rate of effect on growth of various

nematophagous fungi and at present we have no knowledge regarding the mode of

action of these herbicides and insecticides on fungi.

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