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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.
Petri dishes inoculated only with T. harzianum (strains Th1 – Th5) separately
without inoculation of P. fluorescens were treated as control.
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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.
Petri dishes inoculated only with T. harzianum (strains Th1 – Th5) separately
without inoculation of P. fluorescens were treated as control.
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.
Petri dishes inoculated only with T. harzianum (strains Th1 – Th5) separately
without inoculation of P. fluorescens were treated as control.
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
4.3.2 Evaluation of compatibility of T. harzianum and P. fluorescens in
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
Growth weight in control
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4.3.3 Evaluation of compatibility of T. harzianum and P. fluorescens in
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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T4 – Seed and substrate (coco peat) were treated with T. harzianum – Th4
formulation.
T5 – Seed and substrate (coco peat) were treated with T. harzianum – Th5
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)
T9 - Seed and substrate (coco peat) were treated with a combination of
formulation of T. harzianum (Th2) and P. fluorescens (Pf1).
T10 - Seed and substrate (coco peat) were treated with a combination of
formulation of T. harzianum (Th3) and P. fluorescens (Pf1).
T11 - Seed and substrate (coco peat) were treated with a combination of
formulation of T. harzianum (Th4) and P. fluorescens (Pf1).
T12 – Seed and substrate (coco peat) were treated with a combination of
formulation of T. harzianum (Th1) and P. fluorescens (Pf1).
51
T13 – Seed and substrate (coco peat) were treated with a combination of
formulation of T. harzianum (Th1) and P. fluorescens (Pf2)
T14 – Seed and substrate (coco peat) were treated with a combination of
formulation of T. harzianum (Th2) and P. fluorescens (Pf2).
T15 - Seed and substrate (coco peat) were treated with a combination of
formulation of T. harzianum (Th3) and P. fluorescens (Pf2)
T16 - Seed and substrate (coco peat) were treated with a combination of
formulation of T. harzianum (Th4) and P. fluorescens (Pf2).
T17 - Seed and substrate (coco peat) were treated with a combination of
formulation of T. harzianum (Th5) and P. fluorescens (Pf2)
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.
53
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.
54
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.
55
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
Growth weight in control
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.
T2 - Seed treatment with T. harzianum – Th2 formulation.
T3 - Seed treatment with T. harzianum – Th3 formulation.
T4 - Seed treatment with T. harzianum – Th4 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.
T9 - Substrate (coco peat) treated with T. harzianum – Th4 formulation.
T10 - Substrate (coco peat) treated with T. harzianum – Th5 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]
T4 - Seedlings were transplanted in the beds mixed with neem cake
(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]
T6 - Seedlings were transplanted in the beds mixed with neem cake
(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]
T10 - Seedlings were transplanted in the beds mixed with neem cake
(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 :
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 vermicompost
(500g/sq.m) enriched with T. harzianum (CFU 2 x 106 /g) [TH+VER-SB]
T3 - Seedlings were treated with P. fluorescens (CFU 2 x 108/ml) suspension
and were transplanted in the beds [PF-SD]
T4 - Seedlings were transplanted in the beds mixed with vermicompost
(500g/sq.m) enriched with P. fluorescens (CFU 2 x 108/g) [PF+VER-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]
65
T6 - Seedlings were transplanted in the beds mixed with vermicompost
(500g/sq.m) 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]
T10 - Seedlings were transplanted in the beds mixed with vermicompost
(500g/sq.m) [VER - 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.
66
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 vermicompost 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
were also recorded on nematode, disease incidence after 12 months after
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 :
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 FYM (500g/sq.m)
enriched with T. harzianum (CFU 2 x 106 /g) [TH+FYM-SB]
T3 - Seedlings were treated with P. fluorescens (CFU 2 x 108/ml) suspension
and were transplanted in the beds [PF-SD]
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
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]
T6 - Seedlings were transplanted in the beds mixed with FYM (500g/sq.m)
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]
T10 - Seedlings were transplanted in the beds mixed with FYM (500g/sq.m)
[FYM - 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
68
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 FYM 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
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]
T3 - Seedlings were transplanted in the beds mixed with neem
cake(250g/sq.m) and vermicompost (250g/sq.mt) [NC+VER - SB]
T4 - Seedlings were transplanted in the beds mixed with neem
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]
T6- 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).
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.
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
(300g/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]
T4 - Seedlings were transplanted in the beds mixed with neem cake
(300g/sq.mt) enriched with P. fluorescens (CFU 2 x 108/g) [PF+NC-SB]
72
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]
T6 - Seedlings were transplanted in the beds mixed with neem cake
(300g/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]
T9 - T5 + T6 [TH+PF+NC- SD+SB]
T10 - Seedlings were transplanted in the beds mixed with neem cake
(300g/sq.m) [NEEM - 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). The treatments were repeated by soil application of bio-
pesticides and neem cake in respective experimental beds at 3 months interval.
73
Observations were made on no. of flowers harvested at the regular
intervals, vase life of the flowers and plant growth parameters. Observations
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.
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 vermicompost
(300g/sq.m) enriched with T. harzianum (CFU 2 x 106 /g) [TH+VER-SB]
T3 - Seedlings were treated with P. fluorescens (CFU 2 x 108/ml) suspension
and were transplanted in the beds [PF-SD]
T4 - Seedlings were transplanted in the beds mixed with vermicompost
(300g/sq.m) enriched with P. fluorescens (CFU 2 x 108/g) [PF+VER-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]
74
T6 - Seedlings were transplanted in the beds mixed with vermicompost
(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]
T10 - Seedlings were transplanted in the beds mixed with vermicompost
(300g/sq.m) [VER - 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. The treatments were repeated by soil application of bio-pesticides
and vermicompost 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
were also recorded on nematode, disease incidence after 12 months after
inoculation.
4.5.1(C) An experiment was conducted in the open field conditions in a sick
plot infested with M. incognita and other pathogens. Seedlings were
75
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 FYM
was enriched with 2.5 kg of T. harzianum and P. fluorescens.
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 FYM (300g/sq.mt)
enriched with T. harzianum (CFU 2 x 106 /g) [TH+FYM-SB]
T3 - Seedlings were treated with P. fluorescens (CFU 2 x 108/ml) suspension
and were transplanted in the beds [PF-SD]
T4 - Seedlings were transplanted in the beds mixed with FYM (500g/sq.m)
enriched with P. fluorescens (CFU 2 x 108/g) [PF+ FYM -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]
T6 - Seedlings were transplanted in the beds mixed with FYM (300g/sq.mt)
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
T10 - Seedlings were transplanted in the beds mixed with FYM (300g/sq.m)
[FYM - 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 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.
Observations were made on no. of flowers harvested at the regular
intervals, vase life of the flowers and plant growth parameters. Observations
were also recorded on nematode, disease incidence after 12 months after
inoculation.
4.5.1(D) 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
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]
T2 - Seedlings were transplanted in the beds mixed with neem
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]
T3 - Seedlings were transplanted in the beds mixed with neem
cake(150g/sq.m) and vermicompost (150g/sq.mt) [NC+VER - SB]
T4 - Seedlings were transplanted in the beds mixed with neem
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]
T6- Control (without any treatment)
Fifteen replicates of each treatment were maintained in a randomized
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.
Observations were made on no. of flowers harvested at the regular
intervals, vase life of the flowers and plant growth parameters. Observations
were also recorded on nematode, disease incidence after 12 months after
inoculation.