HOT report

1. UNIVERSITY OF AGRICULTURAL
SCIENCES, BENGALURU
COLLEGE OF SERICULTURE, CHINTAMANI
HOT REPORT
2018-2019
COURSE:
TOPIC:
SUMITTED TO:
Dr. Mahesh, M.
Dept. of Pathology,
College of Sericulture,
Chintamani.
SUBMITTED BY:
Madhu .J (ALC7032),
Malleshnaik H. (ALC7034),
Manmatha .G (ALC7037),
Naveen .N. (ALC7043),
Shreedevi. R (ALC7060),

2. Isolationof Biocontrolagentsviz.Trichodermasp.andPseudomonassp.from
the rhizosphere of Plantation and Fruit crops
Introduction
Soil biodiversity plays a key role in the sustainability of agriculture systems
and indicates the level of health of soil, especially when considering the richness of
microorganisms that are involved in biological control of soil borne diseases.
Rhizosphere soil samples were taken from an assay with different plantation crops like
Cashew, Coconut, Coffee and fruit crops like Mango, Guava and Sapota. This
experiment was conducted at College of Sericulture, Chintamani in Hands On
Training (HOT) courses 2020-2021
Soil sample should be collected from a field where the pathogen is known to be
present but disease occurrence is low. Areas where a pathogen was introduced but not
established, and areas of monoculture of crops where disease intensity has decreased
over the years on a susceptible crop provides excellent chances of finding a suitable
biocontrol agent.
Collection of soil sample from the Rhizosphere zone
 Soil samples were collected separately from fruits and plantation crops at the depth
of 30cm in the rhizosphere zone.
 The collected sample were brought to the laboratory and debris from the soil were
removed, clods were crushed and kept for shade drying.
 Powdered soil was taken and sieved using 20mm sieve.
 The powdered soil sample of plantation and fruit crops were weighed to 10g
separately and packed in a zip cover.

3. Sterilization of the equipments
The equipments required were Petri plates, conical flask, tests tubes,beakerand
glass rods.
Procedure
 Petri plates were rolled in newspaper.
 Each conical flask was cotton plugged and about 9ml of sterilized water was
measured and poured to each test tubes blanks and were cotton plugged.
 The above equipments were kept in autoclave for sterilization.
 Autoclave was set with a temperature of 121.6 0C and 15psi of pressure and kept
for 20 minutes.

4. Preparation of the culture media
General media for fungi (PDA) and bacteria (NA) were prepared. To isolate
specific organism, specific media for Trichoderma sp.is (TSM)and for
Pseudomonas fluorescensisKing’sBmediawere prepared and autoclaved.
Materials required for media preparation(for 1Litre)
Ingredients For PDA
Potato(peeled) 200gm
Dextrose 20gm
Agar agar 20gm
Distilled water 1000ml
Preparationfor Autoclaving

5. NAME OF
THE MEDIA
QUANTITY
OF THE
MEDIA
(g)
QUANTITY
OF WATER
ADDED
(ml)
QUANTITY
OF AGAR
ADDED
(g)
SPECIFIC
TO
ORGANISM
Trichoderma
specific media
(TSM)
23.30 1000 20 Trichoderma
sp.
Potato
dextrose agar
(PDA)
20 1000 20 Fungi
King’s B 42.23 1000 20 Pseudomonas
sp.
Nutrient agar
(NA)
28 1000 20 Bacteria

6. After the preparation of media and autoclaving, serial dilution of collectedsoil sample
as to be done.
 Switch on UV light in Laminar air flow for 1-2 minutes before staring of serial
dilution.
 10 g of soil was mixed in 100 ml of distilled water and stirred well for 15 min
using the glass rod.
 Using micropipette 1 ml of the soil suspension taken and prepared serial dilutions
from 10-1 to 10-9
 One ml of each dilution viz., 10-3& 10-4was poured on to Trichoderma Specific
Medium (TSM) and potato dextrose agar (PDA)and media should be treated with
streptomycin.
 Like that One ml of each dilution viz., 10-7& 10-8was poured on to king’s B media
and nutrient agar media (NA).
 Respective Petri plate ware labelled with date, media used, dilution factor,
method of media pouring (pour plate method & spread plate method).
 After that Petri plates were subjected to incubated in incubation chamber about 5-
7 days for the growth of fungi and bacteria for 24-48 hrs.
 Temperature maintained for bacteria was 30 oC& Fungi 28oC

7. Observations:
The growth of bacteria and fungi in their respective media were observed and
recorded.
 Growth of fungal colonies was observed in both in TSM and PDA treated
media Specifically in TSM, growth of Trichoderma was observed.
 Growth of Pseudomonas sp.in King’s B media
Growth of Pseudomonas in King’s B
media
Growth ofTrichoderma on PDA

8. Subculture of bioagents
Materials required:
Cork borer, spirit lamp, sterilized Petri plates, cotton,inoculationloop, PDA
media, NA media, Streptomycin, ethanol.
Procedure:
Autoclaved Petri plates were taken,oven dried and subjected to UV light
treatment for about 15 minutes.
Subculturing of Trichoderma sp.
Switch on UV light in Laminar flow for 20 minutes before starting the experiment and
streptomycin as to be added to media to avoid bacterial growth.
 Well grown Pure culture of Trichoderma sp. was selected.
 Sterilisation of Cork borer with flame and alcohol as to be done.
 With the help of Cork borer and inoculation loop, small bit of Trichoderma from
pure culture transferred to Petri plates for sub culture.
 Petri plate were labelled and wrapped and kept for incubation.

9. Subculture of Pseudomonas sp.
 Pure culture of Pseudomonas sp. was selected.
 Using inoculation loop the inoculum of bacteria was taken and streaked on the
Petri plate containing NA media.
 The Petri plate was labelled and wrapped.
 It was then shifted to the incubation chamber.
Conformation test for Pseudomonas sp.
Pure culture of the bacteria was subjected to UV light, Green fluorescence was
observed and this was confirmed as Pseudomonas sp.
Subculture of bioagents in slants
Preparation of slants:
 Twelve slants were cotton plugged and autoclaved.
 Sterilized test tubes were selected.
 Under the laminar flow, 1/3rd of the PDA media was poured to each test tube
and kept for solidification.
 After solidification, pure culture of Trichoderma sp. was inoculated into the
slants.
 Each slantwas labelled and cotton plugged and were shifted to incubator for
the growth of organism.
 After two days white mycelial growth was observed.
Green colour fluorescence appearance when
exposed to UV light

10. Preparation of slants

11. Preparationof PDB (Potato Dextrose Broth)
 200 grams of potato were weighed and peeled off, for making the broth of 1lt.
 The weighed potatoes were cut into pieces.
 The cut pieces were cleaned and kept in a pressure cooker along with water for
the extraction of the potato broth.
 While boiling, the consistency of the broth was checked andconfirmed.
 Broth obtained was separated from the potato pieces,by filtering it with
agaugematerial.
 The filtered broth was poured into the flask.
 The dextrose was weighed (20 g per litre) and was dissolved in the distilled
waterwhichwas used to make up the volume.
 Then the distilled water was added to the broth to make up the volume of 1lt.
 After complete mixing,broth was measured and poured into each round bottom
flask (four litres per flask).
 After leaving the broth for one-day cooling, then it was inoculated with
Trichoderma sp. (inoculums in 3 round bottom flask) and Pseudomonas sp.(in
2 round bottom flask).
 Keep it for incubation for a period of 15 to 30 days.

12. We can see the Trichoderma mat as white colored mycelial growth initially, later on it turns
to green color as it sporulates.

13. Trichoderma sp. inoculum and Pseudomonas sp. inoculum was grinded in a mixer and
added to talc powder separately and mixed thoroughly.After mixing and drying it was
packed in a polythene cover which was labelled (1kg per pack).
Preparation of tricodermapowder

15. Distribution of trichoderma to farmers

16. .

25. NAME OF
THE
MEDIA
QUANTITY
OF THE
MEDIA
(in grams)
QUANTITY
OF
WATER
ADDED
(ml)
QUANTITY
OF AGAR
ADDED (g)
ORGANISM
Trichoderma
specific
media
(TSM)
23.30 1000 20 Trichoderma
spp
Potato
dextrose
agar (PDA)
20 1000 20 Fungi
Kings B 42.23 1000 20 Pseudomonas
spp
Nutrient
agar (NA)
28 1000 20 Bacteria