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EXPERIMENTAL INVESTIGATIONS

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4. EXPERIMENTAL INVESTIGATIONS:

The present investigations on “Molecular characterization of Trichoderma

harzianum and Bio-management of disease complex in Gerbera” were carried

out at Nematology laboratory, Division of Entomology and Nematology, Indian

Institute of Horticulture Research (IIHR), Hessaraghatta Lake P.O., Bangalore –

560 089.

For all experimental purpose, Borosil glassware and Tarsons plasticware,

were used. Unless and otherwise mentioned, all the chemicals and reagents

were obtained from Hi-media (India). Composition of reagents, preparation

methods and common laboratory techniques like sterilization, isolation of

nematodes, inoculation, serial dilution, mass production of bio-agents,

enrichment of organic substrates etc., were given in the appendices.

The bio-control fungus - Trichoderma harzianum, bacteria - Pseudomonas

fluorescens and plant pathogens – Meloidogyne incognita and Phytophthora

cryptogea. used in this research work were isolated at Nematology laboratory,

Division of Entomology and Nematology, Indian Institute of Horticultural

Research (IIHR), Hessaraghatta Lake P.O., Bangalore – 560 089.

The tissue culture gerbera seedlings (cv. Debora) were procured from

SPIC biotech Pvt. Ltd., Coimbatore for poly-house and screen house

experiments. For open field evaluation gerbera seedlings (cv. Arka Krishika)

were obtained from the breeder, IIHR, Bangalore.

Experiments related to the bio-management of nematode induced disease

complex were carried out using the combination of formulations of

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T. harzianum with P. fluorescens developed and mass produced according to

the standard protocols developed in Nematology Laboratory (These bio-agent

strains were also identified by Indian Type Culture Collection (ITCC) of IARI,

New Delhi, We deposited these strains in ITCC and the field evaluation was

carried in experimental plots of Division of Entomology and Nematology, IIHR,

Bangalore.

4.1 MOLECULAR CHARACTERIZATION OF SELECTED STRAINS OF T. HARZIANUM :

4.1.1 Isolation of bio-agents:

4.1.1.1 Survey for bio-agent strains and isolation of fungus Trichoderma

spp.:

With the objective of isolation of indigenous strains of Trichoderma spp.

extensive surveys were undertaken to collect root and rhizospheric soil samples

from different agro climatic regions, foot-knot nematode affected plants and

also from undisturbed forest ecosystems of Southern India. Representative soil

and root samples were labeled and brought to the laboratory for analysis.

Collected the details of type of soil, previous crop, existing crop age, fertilizers

and chemicals used.

Isolated strains of Trichoderma spp. from the soil samples by dilution

plate techniques on Trichoderma Selective Medium (TSM) (Elad and Chet,

1983).

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4.1.1.2 Isolation of Trichoderma spp. from nematode infected root

Samples:

One gram of root sample was washed with sterile distilled water and

ground with the help of sterile pestle and mortar in 9 ml of 0.05% of agar

solution to remove cortex tissue. Dilution series were made to get 10-1 to 10-7.

From each dilution, 0.2 ml was pipetted on to the Trichoderma Selective

Medium (TSM) under aseptic conditions and incubated at 25 +1oC for two

weeks. Five replications were maintained for each dilution and dark conditions

were provided (to increase the growth) during incubation. Observations for

colonies of Trichoderma spp. were made after two weeks.

4.1.1.3 Isolation from soil samples:

One gram of soil sample was dissolved in sterile distilled water and made

dilutions up to 10-6. One ml of aliquot of appropriate dilutions (10-4, 10-5 & 10-6)

was poured in a Petri Dish and 15 ml of molten autoclaved TSM was poured in

each plate and incubated at 27 ± 2oc.

4.1.2 Morphological characterization:

The cultures grown in the TSM was purified by point inoculation method. The

resulting colonies of Trichoderma were observed on the medium after three

days of incubation and were transferred aseptically to PDA slants for future

use. They were identified on the basis of the morphological characters (Rifai

1969; Bissett 1991). The purified and identified cultures of Trichoderma spp.

were maintained on Potato Dextrose Agar (PDA) medium and stored at 4oC for

future use. Morphological characteristics of these newly isolated strains of

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Trichoderma were observed on various media viz., Potato dextrose Agar (PDA),

Carrot Agar (CA), Corn meal agar (CMA), Trichoderma selective media (TSM),

and potato dextrose broth (PDB) incubated at room temperature for up to 72

hrs. and observations were recorded for every 24 hrs.

In order to get fresh active cultures for the experimentation each strain of

Trichoderma spp. was grown on potato dextrose agar (PDA) for a week at room

temperature (27oc ± 2oc).

Observations on growth rates and colony characteristics were made from

the cultures grown at 25, 30, and 35°C for about one week in these media. The

radial growth of the fungus was measured by measuring the diameter of the

colony. The sporulation was estimated based on the number of conidia

observed per microscopic field. Growth patterns were recorded as per the

presence or absence of aerial mycelium or subdued growth. Pigmentation of

varying shades was recorded visually. All the stages were observed under -

OLYMPUS MO81 inverted microscope.

4.1.3 Phenotypic characterization:

10 ml of agar medium was prepared by mixing streptomycin sulphate

and chloramphenicol each at 10 ppm. About 0.5 ml of molten agar medium

amended with antibiotics- streptomycin sulphate, chloramphenicol was poured

into each of the cavities of a 3 cavity glass slide and allowed to solidify. Point

inoculation method was followed to inoculate in the cavities of the glass slide

for each strain. The glass slides were kept in sterile Petri dishes of 15 cm. The

inner portion of the top and bottom of the Petri dish was placed with a blotting

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paper cut to the diameter of the plate, and moistened to maintain the moisture

inside the Petri dish. They are maintained at 27 ± 2oC in BOD and allowed to

grow for 3 days.

Observations were recorded on the structure of the conidia and

ornamentation of conidiophores in fungal hyphae of each strain.

A needle full of culture of each strain from PDA was transferred on to the glass

slide and was stained by using Phenol cotton blue according to the standard

protocol (Chakrabarti, Chandrashekar et al. 2009). Observations were made on

the structures of conidia, conidiophore ornamentation and the distance

between mycelia/ hyphal septae, under inverted microscope.

4.1.4 Biochemical characterization of Trichoderma spp.:

Commercially available biochemical kits – HiCarbohydrateTM kits were

used for understanding the metabolic carbohydrate requirement for the growth

of Trichoderma spp. It contains 3 sets (A, B & C) with a standardized

colorimetric identification system based on the principle of pH change and

substrate utilization. It contains test system for 35 various carbohydrates.

Point inoculation method is followed to inoculate these kits for all the newly

isolated strains of Trichoderma spp. and incubated at 35 ± 2oC for 24 hours.

Results were recorded.

4.1.5 Molecular identification of Trichoderma spp. by ITS regions:

The strains identified as T. harzianum were taken for molecular level

species identification for the confirmation of the results obtained. The universal

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primers ITS1 and ITS4 were used for PCR analysis for species level

identification of newly isolated strains of Trichoderma spp. ITS RNA regions of

these strains were amplified by PCR by using the ITS1 and ITS 4 universal

primers. The strains identified as T. harzianum by molecular methods. Thus

the results from morphological and biochemical tests were confirmed by

molecular detection methods of T. harzianum.

4.1.5.1 Confirmation of the presence of the nematicidal genes:

Based on the literature survey 2 fungal genes which was reported to be

responsible for nematicidal activity was selected. The two genes were β-tubulin

and PRA1 (Belen Suarez, et al., 2004; Li, et al., 2010). These genes proved to

be effective against nematodes which were supposed to be present in the

strains with nematicidal activity.

4.1.5.2 Primer Designing:

1. The required nematicidal compound producing PRA1 gene in

T. harzianum was identified by literature search and the FASTA format of

the gene was collected.

2. The collected nucleotide sequence of the gene was confirmed for its

identity in the same species.

3. Then the sequence was uploaded in the Primer 3 online version and the

required parameters for the type of primers required were mentioned.

4. The list of required primer sequences with the maximum similarity with

the gene was produced by automated program.

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5. The appropriate primers with maximum % of amplification regions were

selected and sent for preparation of the primers to Bioserve. Pvt. Ltd.

4.1.5.3 DNA isolation of T. harzianum:

A protocol with a slight modification in the method of DNA isolation

(Raeder and Broda, 1985) was followed. Five day old T. harzianum cultures

inoculated in a 250 ml conical flask containing 100 ml PDB and incubated at

27 ± 2oc were used for the DNA isolation.

5 day old Mycelium was harvested and dried using sterile filter papers. It

was grounded into fine powder in a sterile mortar and pestle using liquid

nitrogen. 50 mg of mycelium powder was transferred into a 1.5 ml eppendorf

tube. Extraction buffer of 500 µl was added and homogenized thoroughly by

vortexing. Phenol: Chloroform: Isoamyl alcohol (25:24:1) of 500 µl was added

and vortexed. It was incubated at 60oC for 20 minutes and centrifuged at 4oC,

10,000 RPM for 60 minutes. After centrifugation 3 layers were formed. Using a

pipette the upper aqeous layer was transferred carefully to another 1.5 ml

eppendorf tube. Ribonuclease A (10 mg/ml conc) of 4 µl was added and

incubated in a hot air oven at 37oC for 10 minutes. Chloroform: Isoamyl

alcohol (24:1) of 500 µl was added and centrifuged at 4oc at 10,000 RPM for 20

minutes. After centrifugation 2 layers were formed. Using a pipette the upper

aqueous layer was transferred carefully to another 1.5 ml eppendorf tube. To

this a 3/4th volume of ice-cold iso-proponal was added to precipitate the DNA

and centrifuged for 2 minutes at 4oC at 10,000 RPM and the liquid portion was

removed leaving the pellet formed behind. To the pellet 100 µl 70% ethanol

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was added, tapped at the bottom and centrifuged for 50 seconds. The liquid

portion is removed leaving the pellet. It was kept for drying over night. Later 40

– 100 µl TE buffer was added and DNA concentration was checked by Nano

Drop method. The aliquots of 20 ng DNA concentration were made and stored

at -20oC for the experimental purposes.

4.1.5.4 PCR analysis:

A 25µl PCR mixture was prepared with the following protocol. All the chemicals

and enzymes of molecular grade used were procured from GeneiTM

TABLE No. 4.1 RECIPIE FOR 25µl PCR MIXTURE

Sl.

No.

Chemical / enzyme Quantity(µl)

1 Millicure PCR grade water 19.7

2 10x PCR buffer 2.5

3 10mM DNTP mix 0.5

4 Forward primer 0.5

5 Reverse primer 0.5

6 Template DNA 1.0

7 Taq polymerase 0.3

For each reaction 25µl PCR mixture as mentioned above were used.

Specific Forward and reverse primers designed for the target gene were

obtained from BioserveTM company, Hyderabad. DNA isolated from different

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strains of new isolates of T. harzianum was used as the template DNA in each

reaction mixture.

A program for amplification was standardized as follows:

1. Initial denaturation of template DNA at 94oc - 5 minutes.

2. Secondary denaturation at 94oc - 45 seconds.

3. Annealing temperature at 43oc - 35 seconds.

4. Initial extension at 72oc - 40 seconds.

5. Final extension at 72oc - 10 minutes.

Steps from 2-4 were repeated for 35 cycles.

4.1.5.5 Screening of PCR products by Gel electrophoretic method:

The resulting PCR products were screened for the presence or the

absence of the target nematicidal gene. Agarose gel of 2% with well size of 10 µl

was prepared. Five µl of PCR product was mixed with 3 µl loading dye –

bromophenol blue were loaded in the wells. 1kb ladder (GeneRulerTM-

Fermentas) was used as a reference. It was run in 0.5x TBE buffer at 90 volts.

The resulting amplified gene gel picture was captured.

4.1.5.5.1 PCR product purification and gene sequencing:

The PCR products having required gene amplification of required no. of

base pairs were purified to remove the traces of salts by following Bioserve PCR

Purification kit method. The resulting product was sent for sequencing to

Bioserve, Hyderabad.

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4.1.5.5.2 Confirmation of the nematicidal traits of T. harzianum by

using BLAST program:

The results of gene sequencing were obtained in Chromas format from

Bioserve. It was converted to FASTA format. Using NCBI mega blast it was

uploaded for confirming its species identity and for the nematicidal activity for

ITS and PRA1 respectively.

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4.2 INVESTIGATING THE MODE OF ACTION OF T. HARZIANUM OF

M. INCOGNITA:

Two experiments were designed under this objective to investigate the

mode of action of T. harzianum on M. incognita by evaluating the effect of its

culture filtrate on egg mass hatching and on mortality of juveniles (J2) of

M. incognita as follows.

4.2.1 Investigation on the effect of cell free culture filtrates of

T. harzianum on M. incognita egg mass hatching:

Trichoderma harzianum was inoculated into Potato Dextrose Agar

solidified dishes to obtain the fresh culture and allowed to grow for 7-days.

A 5 mm fresh culture disc of T. harzianum was inoculated in to the

250ml flask containing 100ml sterilized potato dextrose broth prepared as per

(Johnston and and Booth 1983). The flasks were incubated at 27° ± 1ºC for 8

days. 50 ml broth containing of 2.8 x 106 spores (CFU/ml) was centrifuged at

13000 RPM at 4o C for 20 minutes (Eppendorf refrigerated centrifuge 5415).

The pellet was discarded, and supernatant was collected and passed through

syringe filter 0.45 µm (Millipore PVDF Durapore13mm diameter). The culture

filtrate thus obtained was tested for the absence of any fungal spores by

placing it on Potato Dextrose Agar medium.

The culture filtrate was made into two concentrations of 50 and 100% by

adding sterile distilled water. Three ml of culture filtrate of 100% or 50%

concentration was transferred in to sterile Petri-dishes (Borosil) of 5 cm

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diameter. Freshly collected egg masses of M. incognita were surface sterilized

using 0.1% streptomycin sulphate for 45 minutes (Shawney and Webster 1975)

followed by rinsing several times with sterile distilled water. These egg masses

(five nos.) were placed in the Petri dishes containing culture filtrate of 100 or

50%. One set of control with sterile distilled water and another set of control

with PDB were maintained. Each treatment was replicated 5 times for all the

strains of T. harzianum (Th1 – Th5).

Observations on the hatching and mortality of juveniles were recorded

after 24, 48, 72 and 96 hours. After 96 hours of incubation, the cumulative

percentages of hatched juveniles were calculated. Then the egg-masses were

transferred to new petridishes with sterile distilled water. The no of nematodes

hatched for up to 24 hrs was observed. To determine the inhibitory effects and

viability of eggs treated with culture filtrates after 5 days, egg masses were

washed in sterile distilled water followed by 1% Sodium hypochlorite for 1

minute, counted and were transferred to new Petri dishes with 3 ml fresh

sterile distilled water and observed for the percentage egg hatch after another

48 hours. Sodium hypochlorite treatment separates all the un-hatched eggs

from egg-masses when they are shifted to distilled water. This helps in

counting the no. of un-hatched eggs after 48 hours and for computing the

percentages of egg hatch.

4.2.2 Investigation on the effect of cell free culture filtrates of

T. harzianum on juveniles of M. incognita.

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Trichoderma harzianum was inoculated into Potato Dextrose Agar

solidified dishes to obtain the fresh culture and allowed it to grow for 7-days.

A 5 mm fresh culture disc of T. harzianum was inoculated in to the

250ml flask containing 100ml sterilized potato dextrose broth prepared as per

Johnston and Booth 1983. The flasks were incubated at 27° ± 1ºC for 8 days.

50 ml broth containing 2.8 x 106 CFU was centrifuged (Eppendorf refrigerated

centrifuge 5415) at 13000 RPM at 4o C for 20 minutes. The pellet was

discarded, collected supernatant and passed through syringe filter 0.45 µm

(Millipore PVDF Durapore13mm diameter). The culture filtrate thus obtained

was tested for the absence of any fungal spores by placing it on Potato Dextrose

Agar medium.

The culture filtrate was made into two concentrations 50 and 100%

concentration by adding sterile distilled water. 3 ml of culture filtrate of 100 or

50% concentration, was transferred in to sterile Petri-dishes (Borosil) of 5 cm

diameter. Freshly hatched juveniles of M. incognita were surface sterilized using

0.1% streptomycin sulphate for 45 minutes (Shawney and Webster, 1975)

followed by rinsing several times with sterile distilled water. Juveniles (200

nos.) of M. incognita were added to the Petri dishes containing culture filtrate of

100% and 50% respectively. One set of control with sterile distilled water and

another set of control with PDB were maintained. Each treatment was

replicated 5 times for all the strains of T. harzianum (Th1 – Th5). Observations

on the mortality of juveniles were recorded after 24, 48, 72, 96 and 120 hours.

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4.3 TO EVALUATE THE BIO-EFFICACY OF T. HARZIANUM and

P. FLUORESCENS AGAINST M. INCOGNITA AND P. CRYPTOGEA IN VITRO

AND IN SCREEN HOUSE CONDITIONS.

4.3.1 Evaluation of compatibility of T. harzianum and P. fluorescens in

vitro

A fresh culture of P. fluorescens was prepared in 100 ml of Kings’B broth.

A loopful of P. fluorescens culture was inoculated, 24 hrs before the start of the

experiment. It was incubated at room temperature and used for the following

experiments.

Five mm culture disc of T. harzianum was inoculated onto Potato

Dextrose Agar solidified plates and incubated at 27 ± 2oc for 7-days to obtain

the fresh cultures. 5 mm diameter circular culture discs were punched and

used for all the inoculation purposes.

Petri dishes (90 mm) containing Potato Dextrose Agar were used in the

experiments conducted for evaluation of compatibility of T. harzianum with

P. fluorescens in vitro. All the plates with P. fluorescens were inoculated 6 hrs

earlier to the inoculation of T. harzianum. Culture discs of T. harzianum were

inoculated 20 mm from the periphery of the plate.

Four different set of experiments were conducted to evaluate the

compatibility of different strains/ isolates of T. harzianum (Th1 – Th5) with

P. fluorescens (Pf1 and Pf2) in vitro. Five replications of each were maintained

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and all the experimental plates were incubated at 27° ± 1ºC. Observations were

recorded on the compatibility interactions in vitro for about a week.

4.3.1.1 Set – 1

Half a portion of a Petri dish was streaked with fresh culture of P. fluorescens.

After 6 hours a 5mm fresh culture disk of T. harzianum was placed in the other

half plate (20 mm from the periphery) on the same day. It was repeated for two

different strains of P. fluorescens. Each strain of P. fluorescens was evaluated

for compatibility with all strains of T. harzianum (strains Th1 – Th5) separately.

Petri dishes inoculated only with T. harzianum (strains Th1 – Th5) separately

without inoculation of P. fluorescens were treated as control.

4.3.1.2 Set - 2

Fresh P. fluorescens broth 500 µl was spread in a Petri dish by using a

disposable L- spreader. After 6 hours a 5mm fresh culture disk of T. harzianum

was placed at the centre of the plate. It was repeated for two different strains of

P. fluorescens. Each strain of P. fluorescens was evaluated for compatibility

with all strains of T. harzianum (strains Th1 – Th5) separately.



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4.3.1.3 Set - 3

A loopful of fresh culture of P. fluorescens was used to make 2 straight streaks

on the plate dividing it into 4 quadrants. After 6 hours, 5mm fresh culture

disks (2 nos.) of T. harzianum were placed 20 mm from the periphery in 2

quadrants facing each other. It was repeated for two different strains of

P. fluorescens. Each strain of P. fluorescens was evaluated for compatibility

with all strains of T. harzianum (strains Th1 – Th5) separately.



4.3.1.4 Set - 4

A loopful of fresh culture of P. fluorescens was taken and a zigzag streak was

made in one side of plate. After 6 hours, a 5mm fresh culture disk of

T. harzianum was placed 20 mm from the periphery on the other side of the

plate. It is repeated for two different strains of P. fluorescens. Each strain of

P. fluorescens was evaluated for compatibility with all strains of T. harzianum

(strains Th1 – Th5) separately.



Percent compatibility was calculated by using the following formula:

% compatibility = 100 - % ‘I’ (Where I is % Inhibition or % Increase)

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The per cent increase in the colony diameter was calculated using the

following formula :

% increase =

Colony diameter in the treated - Colony diameter in control

× 100

Colony diameter in control

The per cent inhibition in the colony diameter was calculated using the

formula

% inhibition =

Colony diameter in control - Colony diameter in treated

× 100

Colony diameter in control


vitro by mycelia dry weight method:

This experiment has been carried out to confirm the results obtained

from the dual culture methods. The fresh culture of P. fluorescens (Pf1) was

prepared in 100ml of Kings’B broth by inoculating a loopful of P. fluorescens

culture on just previous day of the start of the experiment.

500µl of P. fluorescens of each strain was inoculated into 500ml screw

capped bottle with 200 ml freshly prepared Potato Dextrose broth. A 5mm fresh

culture disc of T. harzianum was added to the same broth. Each strain of

P. fluorescens was evaluated for compatibility with all strains of T. harzianum

(strains Th1 – Th5) separately. Culture bottles inoculated only with

T. harzianum (strains Th1 – Th5) separately without inoculation of

P. fluorescens were maintained as controls. Three replicates were maintained

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for each. The culture bottles were incubated at 27° ± 1ºC. Observations were

recorded on the compatibility interactions in the plates for about a week.

The flasks were incubated at 27° ± 1ºC for 10 days. After 10 days the

observations were recorded on mycelial dry weight. The culture bottle was

thoroughly shaken and 1 ml sample was taken to check CFU by dilution

plating method on PDA and Kings B agar media. The whole media with culture

was made to pass through a Whatman Filter paper #1, placed in a funnel. The

residue collected on the filter paper was dried in hot air oven at 37oC for 4 hrs.

The dry weight of the culture mat was recorded.

Percent compatibility was calculated by using the following formula

% compatibility= 100 - % ‘I’ (where ‘I’ is Inhibition or Increase)

The per cent increase in the colony diameter was calculated using the

following formula :

% increase =

Growth weight in dual culture - Growth weight in control

× 100

Growth weight in control

The per cent inhibition in the colony diameter was calculated using the formula

% inhibition =

Growth weight in control - Growth weight in dual culture

× 100


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vivo

Tomato was taken as target crop for evaluation of compatibility of

T. harzianum and P. fluorescens as it is possible to get precise data on this

important aspect on tomato.

The T. harzianum strains (Th1 – Th5) which proved effective against the

pathogens in vitro were mass produced. The mass produced T. harzianum

strains (Th1 – Th5) were evaluated for their compatibility with P. fluorescens

(Pf1 and Pf2) on tomato (cv. Arka vikas) individually and in combination by

means of seed treatment and substrate application. Seeds were treated with

talc based formulation of different strains of T. harzianum and P. fluorescens in

combinations. 1 kg substrate (coco peat) mixed with 2 grams of T. harzianum +

2 grams P. fluorescens was filled in each seedling tray (with 98 wells). The

treatments were:

T1- Seed and substrate (coco peat) were treated with T. harzianum – Th1

formulation.

T2 - Seed and substrate (coco peat) were treated with T. harzianum – Th2

formulation.

T3 – Seed and substrate (coco peat) were treated with T. harzianum – Th3

formulation.

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formulation.


formulation.

T6 – Seed and substrate (coco peat) were treated with P. fluorescens – Pf1

formulation

T7 - Seed and substrate (coco peat) were treated with P. fluorescens – Pf2

formulation

T8 - Seed and substrate (coco peat) were treated with a combination of

formulation of T. harzianum (Th1) and P. fluorescens (Pf1)


formulation of T. harzianum (Th2) and P. fluorescens (Pf1).





T12 – Seed and substrate (coco peat) were treated with a combination of


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T18 - Control with no treatment

Each treatment was replicated 5 times in a completely randomized block

design.

4.3.3.1 Estimation of plant growth parameters

Thirty days after sowing, five tomato seedlings were randomly uprooted

from each seedling tray (replicate). Observations were made on length, weight

of shoot and root according to standard protocol (Bridge and Page, 1980).

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4.3.3.2 Estimation of the extent of root colonization:

One gram of root sample was washed, grounded and the CFU was

checked by the serial dilution followed by pour plate method.

Serial dilutions up to 10-6 concentration were prepared. 1ml from each of

10-4, 10-5 and 10-6 dilutions were pipetted into the Petri dishes and spread

completely in the plate. Freshly prepared PDA or Kings B agar media was

poured into each plate and made to spread evenly by pour plate method and

allowed for solidification. Three replicates for each dilution were maintained

with controls and CFU was recorded.

4.3.4 Evaluation of bio-efficacy of combination of T. harzianum and

P. fluorescens against P. cryptogea f.sp gerbera spp. in vitro.

A fresh culture of P. fluorescens was prepared in 100 ml of Kings’B broth.

A loopful of P. fluorescens culture was inoculated, 24 hrs before the start of the

experiment. It was incubated at room temperature and used for the following

experiments.

5 mm culture disc of T. harzianum was inoculated onto PDA solidified

plates and P. cryptogea (method of isolation mentioned in appendices) was

inoculated onto carrot agar solidified plates. They were incubated at 27 ± 2oc

for 7-days to obtain the fresh cultures. 5 mm diameter circular culture discs

were punched and used for all the inoculation purposes.

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Petri dishes (90 mm) containing carrot agar, pre- inoculated with 500 µl

of P. fluorescens (24 hrs old broth) spread completely by streaking thrice in all

directions (using a sterile swab) were used in the experiments conducted for

evaluation of bio-efficacy of T. harzianum and P. fluorescens against

P. cryptogea in vitro.

4.3.4.1 Set - 1

T1 - Culture discs of T. harzianum and P. cryptogea were placed each 20 mm

from the periphery, opposite to each other simultaneously.

T2 - Culture disc of P. cryptogea was placed (20 mm from the periphery) and

incubated at 27 ± 2oc for 48 hours. Later a culture disc of T. harzianum

was placed (20 mm from the periphery) opposite to P. cryptogea growth.

T3 - Culture disc of T. harzianum was placed (20 mm from the periphery) and

incubated at 27 ± 2oc for 48 hours. Later a culture disc of P. cryptogea

was placed (20 mm from the periphery) opposite to T. harzianum growth.

T4 - Culture disc of T. harzianum was placed 20 mm from the periphery.

T5 - Culture disc of P. cryptogea was placed 20 mm from the periphery.

T6 - In a Petri dish without P. fluorescens inoculation, a disc of P. cryptogea

was placed and incubated at 27 ± 2oc for 48 hours. Later a disc of

T. harzianum was placed. These discs were placed 20 mm from the

periphery opposite to each other.

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T7 - In a Petri dish without P. fluorescens inoculation, a disc of T. harzianum

was placed and incubated at 27 ± 2oc for 48 hours. Later a disc of

P. cryptogea was placed. These discs were placed 20 mm from the

periphery opposite to each other.

T8 - Control plates containing carrot agar, inoculated only with T. harzianum.

T9 - Control plates containing carrot agar, inoculated only with P. cryptogea

4.3.4.2 Set – 2

Petri dishes plated with solidified carrot agar were used in this experiment.

T1 - A loopful of P. fluorescens was streaked (zigzag) onto one periphery. A

culture disc of T. harzianum was placed on other side of the plate (20 mm

from the periphery) another culture disc of P. cryptogea was placed at

centre of the plate.

T2 - A loopful of P. fluorescens was streaked (zigzag) onto one periphery.

Culture disc of P. cryptogea was placed on other side of the plate (20 mm

from the periphery).

T3 - Culture disc of T. harzianum was placed onto one periphery. Culture

disc of P. cryptogea was placed on the other side of plate (20 mm from

the periphery).

T4 - Control plates containing carrot agar, inoculated only with T. harzianum.

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T5 - Control plates containing carrot agar, inoculated only with P. cryptogea

All the plates were incubated at 27° ± 1ºC. Observations on the inhibition

pattern were recorded daily up to 7 days.

The per cent inhibition in the colony diameter was calculated using the formula

4.3.4.3 Poison baiting test:

Petri dish (90 mm) containing carrot agar was inoculated with a culture disc of

P. cryptogea at centre and allowed to grow completely. Later, Whatman filter

paper discs (3 nos.) of 5 mm diameter were soaked for 10 minutes in

P. fluorescens broth culture (24 hr old). They were placed on P. cryptogea

growth at equal distance to each other. It was incubated at room temperature

for 5 days. Observations on zone of inhibition were recorded.

4.3.5 Evaluation of bio-efficacy of various strains of T. harzianum in vivo

against nematodes:

Tomato was taken as target crop for evaluation of bioefficacy of

T. harzianum on nematodes, as it is possible to get precise data on this

important aspect on tomato.

% inhibition =

Growth weight in control - Growth weight in dual culture

× 100


56

The T. harzianum strains (Th1 – Th5) which are proved to be effective against

the pathogens in vitro were mass produced using standard protocols. The mass

produced T. harzianum strains (Th1 – Th5) were evaluated for their effect on

tomato (cv. Arka vikas) individually and in combination by means of seed

treatment and substrate application. Seeds were treated with talc based

formulation of different strains of T. harzianum. 1 kg substrate (coco peat) was

treated with 10 grams of each strain of this bio-agent. Coco peat was filled in

each seedling tray (with 98 wells) and seeds were sown. The treatments were:

T1 - Seed treatment with T. harzianum – Th1 formulation.




T5 – Seed treatment with T. harzianum – Th5 formulation.

T6 – Substrate (coco peat) treated with T. harzianum – Th1 formulation.

T7 – Substrate (coco peat) treated with T. harzianum – Th2 formulation.

T8 - Substrate (coco peat) treated with T. harzianum – Th3 formulation.



57

T11- Seed and substrate (coco peat) treated with T. harzianum – Th1

formulation.

T12 - Seed and substrate (coco peat) treated with T. harzianum – Th2

formulation.

T13 – Seed and substrate (coco peat) treated with T. harzianum – Th3

formulation.

T14 – Seed and substrate (coco peat) treated with T. harzianum – Th4

formulation.

T15 – Seed and substrate (coco peat) with T. harzianum – Th5 formulation.

T16 – Control with no treatment

Each treatment was replicated 10 times in a completely randomized block

design.

4.3.5.1 Evaluation of bio-efficacy against nematodes:

One month old tomato plants in the five replicates (seedling-trays (5

nos.) with 98 plants) of each treatment (T1-T16) were inoculated with freshly

hatched juveniles (J2) of M. incognita (20 J2/ plant). Five replicates of each

treatment without inoculation of nematodes were treated as controls as per

standard protocol (Nico, et al., 2002).

58

Thirty days after inoculation, 5 plants from each replicate were randomly

picked up with roots for the observations. Observations on plant growth

parameters (seedling length, weight of shoot and root), no. of galls present in

the roots per seedling and extent of root colonization of bio-agent was

estimated by means of CFU.

4.3.5.2 Estimation of the extent of root colonization:

Observations were also made on the extent of root colonization this bio-

agent. One gram of root sample was washed, grounded and the CFU was

checked by the serial dilution followed by pour plate method.

Serial dilutions up to 10-6 concentration were prepared. 1ml from each of

10-4, 10-5 and 10-6 dilutions were pipetted into the Petri dishes and spread

completely in the plate. Freshly prepared PDA media was poured into each

plate and made to spread evenly by pour plate method and allowed for

solidification. Three replicates for each dilution were maintained with controls

and CFU were recorded.

4.3.6 Evaluation of bio-efficacy of T. harzianum and P. fluorescens

against nematode induced disease complex in gerbera under screen

house conditions:

An experiment was conducted in the screen house (glass house) conditions

with the following treatments using the unsterilized soil mixed with FYM.

59

T1 – Seedlings of Gerbera (cv. Debora) were treated with P. fluorescens (CFU 2

x 108/ml) suspension and were transplanted in pots containing 2.5 kg of

soil. [PF-SD]

T2 - 25 g of P. fluorescens formulation was mixed with 2.5 kg of soil filled in

the pots and the untreated seedlings were transplanted. [PF-SB]

T3 - Seedlings treated with P. fluorescens (CFU 2 x 108/ml) sown in 2.5 kg of

soil treated with 25 g of the P. fluorescens. [PF SD+SB]

T4 - Seedlings treated with of T. harzianum (CFU 2 x 106 /ml) suspension

were sown in pots containing 2.5 kg of soil. [TH –SD]

T5 - 25 g of T. harzianum (CFU 2 x 106 /ml) formulation was mixed with 2.5

kg of soil filled in the pots and untreated seedlings were sown. [TH-SB]

T6 - Seedlings treated with T. harzianum (CFU 2 x 106 /ml) sown in 2.5 kg of

soil treated with 25 g of the T. harzianum. [TH SD+SB]

T7 - Seedlings were treated with combination of formulations of T. harzianum

and P. fluorescens at 1:1 ratio and were transplanted in pots containing

2.5 kg of soil. [PF+TH-SD]

T8 - 25 g of combination of formulations of T. harzianum and P. fluorescens

at 1:1 ratio was mixed with 2.5 kg of soil filled in the pots and the

untreated seedlings were sown. [PF+TH-SB]

60

T9 - Seedlings treated with T. harzianum and P. fluorescens combi-

formulation at 1:1 ratio were transplanted in 2.5 kg of soil treated with

25g combination of formulations of T. harzianum and P. fluorescens.

[PF+TH- (SD+SB)]

T10 - Control was maintained without any treatment.

Fifteen replicates of all the treatments were maintained, 5 replicates of

each treatment were infested with nematodes, 5 replicates with P. cryptogea

and 5 replicates with both the pathogens to produces disease complex.

61

4.4 DEVELOPING A STRATEGY OF BIO-MANAGEMENT USING

T. HARZIANUM AND P. FLUORESCENS OF M. INCOGNITA AND P. CRYPTOGEA ON GERBERA IN POLY-HOUSE CONDITIONS.

The present investigations on “Molecular characterization of Trichoderma

harzianum and Bio-management of disease complex in Gerbera” were carried

out at Indian Institute of Horticultural Research (IIHR), Hessaraghatta Lake

P.O., Bangalore – 560 089.

The tissue culture gerbera seedlings (cv. Debora) were procured from

SPIC biotech Pvt. Ltd., Coimbatore for poly-house and screen house

experiments. Gerbera seedlings (cv. Arka Krishika) were obtained from the

breeder Indian Institute of Horticulture Research, Bangalore for open field

evaluation.

Experiments related to the bio-management of nematode induced disease

complex were carried out using the combination formulation of Trichoderma

harzianum with Pseudomonas fluorescens developed in Nematology

Laboratory of Division of Entomology and Nematology, Indian Institute of

Horticulture Research, Bangalore.

4.4.1 Evaluation of effect of application of combinations of formulations of

T. harzianum and P. fluorescens in the management of disease complex

caused by M. incognita and P. cryptogea in gerbera (cv. Debora) in

protected (Poly-house) conditions.

4.4.1(A) An experiment was conducted using bio-pesticides enriched neem

cake in the poly-house in raised beds with plot size of 1 sq.m. Nine

62

seedlings were transplanted in each bed with a spacing of 35 x 15 cm.

Soil in the poly-house was pre sterilized by 30% formalin fumigation

followed by solarisation. 100 kg of neem cake was enriched with 2.5 kg of

either of T. harzianum or P. fluorescens. The treatments were as follows :

T1 – Seedlings of gerbera (cv. Debora) were treated with of T. harzianum (CFU

2 x 106 /ml) suspension and were transplanted in the beds [TH –SD]

T2 - Seedlings were transplanted in the beds mixed with neem cake

(500g/sq.m) enriched with T. harzianum (CFU 2 x 106 /g) [TH+NC-SB]

T3 - Seedlings were treated with P. fluorescens (CFU 2 x 108/ml) suspension

and were transplanted in the beds [PF-SD]


(500g/sq.m) enriched with P. fluorescens (CFU 2 x 108/g) [PF+NC-SB]

T5 - Seedlings were treated with suspension of combination of T. harzianum

(CFU 2 x 106 /ml) and P. fluorescens (CFU 2 x 108/ml) and were

transplanted. [PF+TH-SD]


(500g/sq.m) enriched with combination of T. harzianum (CFU 2 x 106 /g)

and P. fluorescens (CFU 2 x 108/g). [PF+TH+NC-SB]

T7 - T1 + T2 [TH+NC- SD+SB]

T8 - T3 + T4 [PF+NC- SD+SB]

63

T9 - T5 + T6 [TH+PF+NC- SD+SB]


(500g/sq.m) [NC - SB]

T11 - Seedlings were transplanted in the beds treated with Ridomil (5g/ lit)

[RID - SB]

T12 - Seedlings were transplanted in the beds treated with Carbofuran (5g/ lit)

[CARB - SB]

T13- Control (without any treatment)

Fifteen replicates of each treatment were maintained in a randomized

block design (RBD). After 20 days of planting, 5 replicates in each treatment

were inoculated with only freshly hatched M. incognita (1 J2/g), 5 replicates in

each treatment with only P. cryptogea (CFU 2 x 10 6/g) and 5 replicates in each

treatment with both freshly hatched M. incognita (1 J2/g) and P. cryptogea

(CFU 2 x 10 6/g) in each treatment.

The conditions of the poly-house (lighting, temperature, moisture and

humidity) were maintained according to the standards (Sankar, et al., 2003).

The soil application treatments of bio-pesticides and neem cake were

repeated in respective experimental beds at 3 months interval.

Observations were made on no. of flowers harvested at the regular

intervals, vase life of the flowers and plant growth parameters. Observations

64

were also recorded on nematode, disease incidence after 12 months after

inoculation.

4.4.1(B) An experiment was conducted using bio-pesticides enriched

vermicompost in the poly-house in raised beds with plot size of 1 sq.mts.

Nine seedlings were transplanted in each bed with a spacing of 35 x 15

cm. Soil in the poly-house was pre sterilized by 30% formalin fumigation

followed by solarisation. 100 kg of vermicompost was enriched with 2.5

kg of either of T. harzianum or P. fluorescens. The treatments are as

follows :



T2 - Seedlings were transplanted in the beds mixed with vermicompost

(500g/sq.m) enriched with T. harzianum (CFU 2 x 106 /g) [TH+VER-SB]




(500g/sq.m) enriched with P. fluorescens (CFU 2 x 108/g) [PF+VER-SB]




65



and P. fluorescens (CFU 2 x 108/g). [PF+TH+VER-SB]

T7 - T1 + T2 [TH+VER- SD+SB]

T8 - T3 + T4 [PF+VER- SD+SB]

T9 - T5 + T6 [TH+PF+VER- SD+SB]


(500g/sq.m) [VER - SB]


[RID - SB]


[CARB - SB]








66



The soil application treatments of bio-pesticides and vermicompost were

repeated in respective experimental beds at 3 months interval.




inoculation.

4.4.1(C) An experiment was conducted using bio-pesticides enriched FYM

in the poly-house in raised beds with plot size of 1 sq.mts. Nine

seedlings were transplanted in each bed with a spacing of 35 x 15 cm.

Soil in the poly-house was pre sterilized by 30% formalin fumigation

followed by solarisation. 100 kg of FYM was enriched with 2.5 kg of

either of T. harzianum or P. fluorescens. The treatments are as follows :



T2 - Seedlings were transplanted in the beds mixed with FYM (500g/sq.m)

enriched with T. harzianum (CFU 2 x 106 /g) [TH+FYM-SB]



T4 - Seedlings were transplanted in the beds mixed with FYM (500g/sq.mt)

enriched with P. fluorescens (CFU 2 x 108/g) [PF+ FYM -SB]

67





enriched with combination of T. harzianum (CFU 2 x 106 /g) and

P. fluorescens (CFU 2 x 108/g). [PF+TH+ FYM -SB]

T7 - T1 + T2 [TH+ FYM - SD+SB]

T8 - T3 + T4 [PF+ FYM - SD+SB]

T9 - T5 + T6 [TH+PF+ FYM - SD+SB]


[FYM - SB]


[RID - SB]


[CARB - SB]





68






The soil application treatments of bio-pesticides and FYM were repeated

in respective experimental beds at 3 months interval.



were also recorded on nematode disease incidence after 12 months after

inoculation.

4.4.1(D) An experiment was conducted using bio-pesticides enriched

neemcake, vermicompost and FYM in the poly-house in raised beds with plot

size of 1 sq.mts. Nine seedlings were transplanted in each bed with a spacing of

35 x 15 cm. Soil in the poly-house was pre sterilized by 30% formalin

fumigation followed by solarisation. 100 kg of neemcake/ vermicompost/ FYM

was enriched with 2.5 kg of either of T. harzianum or P. fluorescens. The

treatments are as follows:

T1 – Seedlings were transplanted in the beds mixed with neem

cake(250g/sq.m) and vermicompost (250g/sq.m) enriched with

combination of T. harzianum (CFU 2 x 106 /g) and P. fluorescens (CFU 2

x 108/g). [PF+TH+NC+VER-SB]

69

T2 - Seedlings were transplanted in the beds mixed with neem

cake(250g/sq.m) and FYM (250g/sq.m) enriched with combination of

T. harzianum (CFU 2 x 106 /g) and P. fluorescens (CFU 2 x 108/g).

[PF+TH+NC+FYM-SB]


cake(250g/sq.m) and vermicompost (250g/sq.mt) [NC+VER - SB]


cake(250g/sq.m) and FYM (250g/sq.mt) [NC+FYM - SB]

T5 - Seedlings were transplanted in the beds treated with Carbofuran (2.5g/ lit)

+ Ridomil (2.5g/lit) [CARB+RID- SB]










70

The soil application treatments of bio-pesticides and neem cake/

vermicompost/ FYM were repeated in respective experimental beds at 3

months interval.

Observations were made no. of flowers harvested at the regular intervals,

vase life of the flowers and plant growth parameters. Observations were

recorded on nematode and disease incidence after 12 months after inoculation.

71

4.5 STANDARDIZING THE METHODS FOR BIO-MANAGEMENT OF DISEASE COMPLEX IN OPEN FIELD CONDITIONS.

4.5.1 Evaluation of effect of application of combinations of formulations

of T. harzianum and P. fluorescens in the management of disease

complex caused by M. incognita and P. cryptogea in Gerbera (cv.

Arka Krishika) in open field conditions.

4.5.1(A) An experiment was conducted in the open field conditions in a sick

plot infested with nematodes and other pathogens. Seedlings were

transplanted in raised beds with plot size of 1 sq.mts. Nine seedlings

were transplanted in each bed with a spacing of 35 x 15 cm. 100 kg of

neem cake was enriched with 2.5 kg of T. harzianum and P. fluorescens.




(300g/sq.m) enriched with T. harzianum (CFU 2 x 106 /g) [TH+NC-SB]




(300g/sq.mt) enriched with P. fluorescens (CFU 2 x 108/g) [PF+NC-SB]

72






and P. fluorescens (CFU 2 x 108/g). [PF+TH+NC-SB]

T7 - T1 + T2 [TH+NC- SD+SB]

T8 - T3 + T4 [PF+NC- SD+SB]

T9 - T5 + T6 [TH+PF+NC- SD+SB]


(300g/sq.m) [NEEM - SB]


[RID - SB]


[CARB - SB]



block design (RBD). The treatments were repeated by soil application of bio-

pesticides and neem cake in respective experimental beds at 3 months interval.

73



were also recorded on nematode, disease incidence after 12 months of

inoculation.

4.5.1(B) An experiment was conducted in the open field conditions in a sick

plot infested with M. incognita and other pathogens. Seedlings were

transplanted in raised beds with plot size of 1 sq.m. Nine seedlings were

transplanted in each bed with a spacing of 35 x 15 cm. 100 kg of

vermicompost was enriched with 2.5 kg of T. harzianum and P.

fluorescens.




(300g/sq.m) enriched with T. harzianum (CFU 2 x 106 /g) [TH+VER-SB]




(300g/sq.m) enriched with P. fluorescens (CFU 2 x 108/g) [PF+VER-SB]




74


(300g/sq.mt) enriched with combination of T. harzianum (CFU 2 x 106

/g) and P. fluorescens (CFU 2 x 108/g). [PF+TH+VER-SB]

T7 - T1 + T2 [TH+VER- SD+SB]

T8 - T3 + T4 [PF+VER- SD+SB]

T9 - T5 + T6 [TH+PF+VER- SD+SB]


(300g/sq.m) [VER - SB]


[RID - SB]


[CARB - SB]



block design. The treatments were repeated by soil application of bio-pesticides

and vermicompost were repeated in respective experimental beds at 3 months

interval.




inoculation.

4.5.1(C) An experiment was conducted in the open field conditions in a sick


75


transplanted in each bed with a spacing of 35 x 15 cm. 100 kg of FYM

was enriched with 2.5 kg of T. harzianum and P. fluorescens.




enriched with T. harzianum (CFU 2 x 106 /g) [TH+FYM-SB]




enriched with P. fluorescens (CFU 2 x 108/g) [PF+ FYM -SB]





enriched with combination of T. harzianum (CFU 2 x 106 /g) and P.

fluorescens(CFU 2 x 108/g). [PF+TH+ FYM -SB]

T7 - T1 + T2 [TH+ FYM - SD+SB]

T8 - T3 + T4 [PF+ FYM - SD+SB]

T9 - T5 + T6 [TH+PF+ FYM - SD+SB]

76


[FYM - SB]


[RID - SB]


[CARB - SB]


Fifteen replicates of each treatment were maintained in a completely

randomized block design. The treatments were repeated by soil application of

bio-pesticides and FYM were repeated in respective experimental beds at 3

months interval.




inoculation.

4.5.1(D) An experiment was conducted in the open field conditions in a sick



transplanted in each bed with a spacing of 35 x 15 cm. 100 kg of

neemcake/ vermicompost/ FYM was enriched with 2.5 kg of

T. harzianum and P. fluorescens. The treatments were as follows:

T1 – Seedlings were transplanted in the beds mixed with neem

cake(150g/sq.m) and vermicompost (150g/sq.mt) enriched with

77

combination of T. harzianum (CFU 2 x 106 /g) and P. fluorescens (CFU 2

x 108/g). [PF+TH+NC+VER-SB]


cake(150g/sq.m) and FYM (150g/sq.mt) enriched with combination of

T. harzianum (CFU 2 x 106 /g) and P. fluorescens (CFU 2 x 108/g).

[PF+TH+NC+FYM-SB]


cake(150g/sq.m) and vermicompost (150g/sq.mt) [NC+VER - SB]


cake(150g/sq.m) and FYM (150g/sq.mt) [NC+FYM - SB]

T5 - Seedlings were transplanted in the beds treated with Carbofuran (2.5g/

lit) + Ridomil (2.5g/lit) [CARB+RID- SB]



block design. The treatments were repeated by soil application of bio-pesticides

and neem cake/ vermicompost/ FYM were repeated in respective experimental

beds at 3 months interval.




inoculation.

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