STANDARD OPERATING

PROCEDURE
FOR ANALYSIS OF
Trichoderma viride
&
Trichoderma harzianum

1
 SPECIFICATION & METHOD OF ANALYSIS OF Trichoderma spp.

1. Form and appearance

2. pH: 6.0-8.0
3. CFU/g of the product : 2 x 106 /gm or ml min.
4. Percent content of the Biocontrol organism in the formulation & nature of biomass.
5. Percentage of carrier/filler, wetting/ dispending agent, stabilizers/ emulsifiers,
contaminants/ impurities etc.
6. Moisture content: 8% max
7. CFU counts: Trichoderma 2x106 CFU/ml or gm. (Stability at 30oC and 65% RH).
8. Contaminants:
 Biological Contaminants:
 Pathogenic Contaminants: such as gram negative bacteria Salmonella, Shigella, Vibrio etc.:
absent
 Other contaminants should not exceed 1x104/ml or g
 Chemical/ botanical pesticides contaminants: absent.

METHOD OF ANALYSIS:

CFU counts by serial dilution and examination under regular compound research
microscope with bright field optics.

Estimation of Colony Forming Units count:-

Take 1 g. of product and mix it in 9 ml of sterilized distilled water in a clean and

sterilized test tube to make 10-1 dilution (1:10). Shake well and take 1 ml. of the
suspension to 9 ml. of sterile water in a tube to make 10-2 dilution (1:100). Make four
more serial dilutions in the same way to get 10-6 dilution. Transfer 1 ml. of this
suspension to sterile Petri Plates and add 15-20 ml. of sterilized, melted and cooled
Trichoderma spp. selective media. Rotate the plates gently and allow it to solidify.
Incubate the Perti plates in BOD incubator under the fluorescent illumination at 25 oC ±
2 oC and R.H. at 65% ± 5 % for five to seven days. Observe the development of typical
Trichoderma spp. colony and calculate the number of colony unit per gram of the sample
as the following formula.

CFU/g.= Average number of colonies/dilution factor

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PRECAUTIONS:-

The following precaution may be taken during the analysis of Trichoderma spp. samples:-
1. Chemicals & Glasswares:- Analytical Grade (AR) quality chemicals and Schott
Duran or Borosil glassware should be used.
2. Cleaning and Sterilization: - The glasswares used for testing should be cleaned with
detergent like Lavoline and Sterilized in Hot air Oven at 180 oC for 3 hours.
3. Sterilization: - Media, distilled water and micro tips should be autoclaved at 1.05
Kg/cm2 (15 psi) pressure for 20 minutes.
4. Inoculation: - Inoculation of test material into sterile media should be carried out
under aseptic conditions using Laminar Air Flow Cabinet.
5. Incubation: - After inoculation all the Perti plates should be incubated in a BOD
Incubator under florescent illumination at 25 oC ± 2 oC and at 65% ± 5 % R.H. for five
to seven days.

SELECTIVE MEDIA FOR TRICHODERMA (ELAD AND CHET, 1983)

S. No. Components Quantity

1. Magnesium sulphate 0.2 g.
2. Dipotassium hydrogen phosphate 0.9 g.
3. Ammonium nitrate 1.0 g
4. Potassium chloride 0.15 g.
5. Glucose 3.0 g.
6. Metalaxyl 0.3 g.
7. Rose Bengal 0.15 g.
8. Chloramphenical 0.25 g.
9. Agar 15.0 g.
10. Distilled Water 1 litre
Calculation:-

Number of colony forming unit /plates

Dilution No. of colony forming units/ plate Average
factor
Y R1 R2 R3 R4 X

CFU/g = average no. of colonies (X)/dilution factor (Y)

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ESTIMATION OF pH

pH (acidity or alkalinity test) of bio-pesticides is conducted as per the IS: 6940- 1982.
Take 10g of sample in 50 ml of glass beaker & mixed with 25 ml of distilled water. pH of
the suspension is observed by using the meter.
R1 R2 R3 R4 Average
X
pH of sample = X

MOISTURE CONTENT:-

Moisture content to be determined as per the method describes in IS; 6940-1982.

Results Moisture Content % by mass = 100V
M
OR
Moisture content % by mass:-
Weigh 5 g sample of product and pour into beaker of 50 g. capacity. Beaker containing
weighed sample is kept in hot air oven at 65 OC for 30 minutes. After 30 minutes beaker
is taken out from oven and again weigh it for determination of mass content.

Moisture content per cent = (M-m) x 100

M
Where,
M= Initial weight of product
M= mass of product found after hot air
oven treatment
Moisture content % bymass : X w/w Mean
R1 R2 R3 X

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PATHOGENIC CONTAMINANTS

5
6
OTHER MICROBIAL CONTAMINANTS

ANTAGONISTIC CAPABILITY

7
Appendix-I
Bioassay for plant disease antagonists based on disease severity and root colonization.

The target pathogen to be tested against has to be grown in Sand maize medium. The Sand-maize
medium is prepared by adding sand 90g, maize 10g. and water 10ml in a saline or any glass bottle of
300ml capacity and then autoclaved twice. Then 5 mycelial discs of the test pathogen are transferred into
the bottle and left for incubation for 15 days. Once the culture has grown well, the sand maize medium is
mixed along with the fungal growth and 1g from this preparation is used as the inoculum after adjusting
the cfu to 1 x10/g by addition of sand. The plastic cups (5-6 cm diameter) filled with soil and FYM
(3:1) have to be used. In each cup the filling should be done upto ¾th level. The pathogen inoculum is
mixed with sand has to be applied upto 2cm depth in the plastic cups.

The bioefficacy of the bioagent shall be tested by both seed treatment and soil application. For
seed treatment, the recommended dose of the formulation has to be used (5 to 10g.). For soil application,
the bioagent is added at the rate of 1g of formulation (minimum cfu should be the 2x106). The
germination percentage, disease intensity and seedling vigour are to be recorded.

Another set of plastic cups filled with sterile soil and sterile FYM has to be used to confirm
whether the bioefficacy was due to the isolate of the bioagent tested or due to the native isolates of the
bioagent present in the soil.

The keys for grading the efficacy mentioned below shall be used (Srivastava et al., 2002).
However, for the registration purpose, the bioagents that are Highly Efficient, Efficient or Moderately
Efficient in the plastic cup test under glass house condition (in the presence of pathogen) can be allowed
(i.e.) germination percentage of 70% or above, disease incidence of 30% or less can be considered for
registration.
Disease Grading Key
Disease Description Rating of
incidence bioefficacy of
(%) bioagents
0 Germination>90%, no seed rotting, seedling healthy, root and Highly Efficient
shoot portions well developed (HE)
1-15 Germination 80-90%, infection on main as well as lateral roots, Efficient (E)
seedlings are well developed
16-30 Germination 70-80%, development of roots restricted and Moderately
growth is less compared to Score 1. Infection occurred on roots. Efficient (ME)
Shoot portions developed but growth retarded compared to
Score 1.

31-45 Germination 60-70%, length of roots and shoots short compared Moderately
to Score 1. Germination of seeds inhibited. 50% of root area Inefficient (MI)
infected. Shoot portions also showed infection
46-60 Seed germination 50-60%. Development of roots and shoots Efficient (I)
greatly retarded. Shoot portions also showed infection.
Above 60 Less than 50% germination and seed rotting Highly
Inefficient (HI)

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 For the root colonization assay, the rhizosphere region of the plants tested above have to be
collected and the soil adhering to the root surface has to be removed by gently tapping the roots. The root
bits have to be cut into 1 cm bits and randomly 25 bits should be selected for each treatment. They have to
be plated on (TSM) and the percentage of root bits colonized has to be recorded. This has to be
performed in the sterile soil and non sterile soil. One control treatment without the Biocontrol agent,
being tested, should be kept for both the sterile and non-sterile soil to rule out of the possibility of
interference of native micro flora in the bioefficacy assay.