TRAINING-WORKSHOP ON SOIL AND NUTRIENT

MANAGEMENT AND ASSESSING SOIL HEALTH

LARGE-SCALE PRODUCTION
OF
Trichoderma harzianum
FOR REGIONAL AND PROVINCIAL LABORATORIES OR THOSE WITH EXISTING LABORATORY

Aurora M. Manalang
Senior Agriculturist
OIC Head, Soil Microbiology Section

August 30, 2022

Via Zoom and Facebook Live
What is Trichoderma harzianum?

Trichoderma is a fungus, also known

as Compost Fungus Activator (CFA)
used for rapid composting of
agricultural waste in just 3-4 weeks.

It is also used as biological control

agent for the suppression of plant
pathogenic microorganisms.
Basic Requirements for a Large Scale Production of CFA

THE ROOM
❖ The room should be at least 4m x 4m or
bigger with open shelves, big working
table, 2-3 stools, sink and faucet.
❖ The walls and floors should be solid.
❖ Proper ventilation, electrical outlets and
water supply should be present.
❖ The doors and windows can be locked
from inside.
Basic Requirements for a Large Scale Production of CFA

THE EQUIPMENT

Weighing Balance pH Meter Blender

Basic Requirements for a Large Scale Production of CFA

THE EQUIPMENT

Autoclave/ Pressure Cooker Refrigerator or Bioref

Basic Requirements for a Large Scale Production of CFA

Storage Shelves for Inoculated Bottles

Basic Requirements for a Large Scale Production of CFA

Open shelves for bottles/ incubating newly inoculated Tricho cultures

Basic Requirements for a Large Scale Production of CFA

Plastic trays for incubation of Tricho

Basic Requirements for a Large Scale Production of CFA

Inoculating Chamber with UV Light

Basic Requirements for a Large Scale Production of CFA

MATERIALS AND SUPPLIES

MEDIA PREPARATION

MICROBIOLOGY KIT FOR INOCULATION

DISINFECTION AND WASHING

Materials and Supplies

MEDIA PREPARATION

✓ White gulaman powder/gulaman bar cut

into very small pieces)
✓ Instant oat
✓ Distilled or deionized water
✓ Empty ice cream plastic container (1
gal) or plastic pitcher (1.5mL)
✓ Beaker or plastic scoop (30mL
capacity)
Materials and Supplies

MEDIA PREPARATION

✓ Polyprophylene bags (6 x 12in and 9 x

18in) or lapad bottles (300mL)
✓ Weighing boat
✓ Spatula
✓ Aluminum foil
✓ Cut pp bags (as cover for lapad bottles)
✓ Heavy duty rubber bands
Materials and Supplies
MEDIA PREPARATION

✓ Plastic ladle
✓ Cotton plugs
✓ Plastic funnels
✓ Pipette tips or glass pipettes (1mL)
✓ Pipettor (1mL) or aspirator
✓ Filter paper
✓ Plastic cups
✓ Wash bottles
✓ Plastic spoon
✓ Sulfuric acid or Datu Puti Vinegar
Materials and Supplies
MICROBIOLOGY KIT FOR INOCULATION
✓ Alcohol lamp
✓ Inoculating loop or spatula
✓ Ethyl alcohol (95%)
✓ Safety matches
✓ Permanent markers
✓ Spray bottles
✓ Erlenmeyer flask (100mL, 250mL and 500mL)
✓ Rags
✓ Plastic trays
✓ Face mask
✓ Laboratory gown and lab shoes
✓ Petri dish
Materials and Supplies
DISINFECTION AND WASHING
✓ Disinfecting spray ✓ Brush for bottles
✓ Microbiological liquid ✓ Hand gloves
soap ✓ Heat resistant gloves
✓ Detergents ✓ Sprayer
✓ Scrubbing pads ✓ Hand towels
✓ Rags

OTHERS
✓ Scissors ✓ Pen or pencil
✓ Trash can with cover ✓ Sando bags
✓ Record book ✓ Brown paper
Preparation of Gulaman-Oat Media

1. Weigh 100 grams instant oat.

2. Weigh 25 to 30 grams white gulaman bars, cut

into small pcs or 2 packs of white powdered
gulaman.
Preparation of Gulaman-Oat Media
Preparation offor
Media Preparation Gulaman-Oat Media
Trichoderma Production

3. Measure 850 to 900 ml deionized or

distilled water using graduated
cylinder and pour in a blender.
Preparation offor
Media Preparation Gulaman-Oat Media
Trichoderma Production

4. Add the gulaman bar and

blend very quickly.

5. Pour the blended gulaman

bars into an empty container.
Preparation offor
Media Preparation Gulaman-Oat Media
Trichoderma Production

6. Add the oats and mix well.

Preparation offor
Media Preparation Gulaman-Oat Media
Trichoderma Production

7. Set pH buffer at 7.0, 4.0, 7.0.

Get pH reading of the prepared
media.
Preparation offor
Media Preparation Gulaman-Oat Media
Trichoderma Production

The ideal pH of media is between 4.2 to 4.5.

Preparation offor
Media Preparation Gulaman-Oat Media
Trichoderma Production

8. Fold the 6 x 12 polypropylene bags into

half (crosswise).

9. Pour 30ml. into a folded polypropylene

bag using plastic scoop.
Preparation offor
Media Preparation Gulaman-Oat Media
Trichoderma Production

10. After pouring the media, bring down

all the media at the lower portion of the
plastic, then fold the polypropylene bags
into half.

11. Fold the polypropylene bags again

into 4 and seal it with rubber band.
Preparation offor
Media Preparation Gulaman-Oat Media
Trichoderma Production

12. Combine 2 polypropylene bags

with media and seal it with rubber
band to keep media in upright
position during sterilization.

13. Arrange the paired media

bags in a clean tray.
Preparation of Gulaman-Oat Media

Recommended Way of Folding Polypropylene Bags

MEDIA STERILIZATION

1. Add distilled water in the

pressure cooker.
MEDIA STERILIZATION
2. Arrange the media in the wire baskets of the pressure cooker.
MEDIA STERILIZATION
3. Carefully put the wire baskets inside the pressure cooker.
MEDIA STERILIZATION
4. Set the pressure cooker at 121 ⁰C, 15psi and sterilize for 15mins.
MEDIA STERILIZATION

Actual photo of the sterilized media.

MEDIA PREPARATION

1. After sterilization, carefully bring down the

media at the bottom of the polypropylene
bag and mix well.
MEDIA PREPARATION
2. Spread the media up to the middle part of the polypropylene bags.
MEDIA PREPARATION

3. Allow the media to cool and harden.

MEDIA PREPARATION
4. Fold the pp bag into half and store in 9x18 polypropylene bag and seal with rubber band.
MEDIA PREPARATION
MEDIA PREPARATION
5. Put the prepared media in clean plastic tray. Store the media in a
refrigerator if inoculation will be performed the next day.
PRE-INOCULATION

1. Lift the upper part of the

polypropylene bag to separate the
media.

2. Fold the bottom corners of the

polypropylene bag to allow air to
enter the plastic.
INOCULATION SET-UP
INOCULATION PROCEDURE
1. Clean the entire inoculating chamber. Spray with
disinfecting spray and ethyl alcohol mixture.

2. Place all materials and supplies for inoculation including

the prepared media. Switch on the UV lamp of the
inoculating chamber for 30 minutes.

3. Under aseptic condition, heat the inoculating needle or

spatula and transfer a loopful of Trichoderma to the
surface of the sterile agar-oat medium.

4. Adhere the inoculated Trichoderma

into the media. Inflate the polypropylene
bags and seal with rubber bands.
INOCULATION PROCEDURE

5. After inoculation, seal the opening

of the polypropylene bag by folding it
twice, then gather the folded portion
carefully.
INOCULATION PROCEDURE

6. Fold the gathered portion away

from your direction twice and seal it
with rubber band.
INOCULATION PROCEDURE

7. Label the polypropylene bag by putting the

date of inoculation.

8. Incubate in clean air conditioned room

for at least a week.

Note:
Trichoderma grows best under diffused light condition.
Do not exposed Trichoderma under direct sunlight.

When the fungus starts to produce spores (green color

after 7 days), the inoculant is ready for use in
composting.
INOCULATION PROCEDURE

A view of freshly inoculated Trichoderma

TRICHODERMA PURE CULTURES
USING OF TRICHODERMA INOCULANT
USING OF TRICHODERMA INOCULANT

Procedure

1. Select a polypropylene bag/bottle

with fully grown inoculant and free
of contaminants.

2. Add tap water to the inoculant.

3. Shake, mix thoroughly the agar-oat

with fungus mycelia and spores.
USING OF TRICHODERMA INOCULANT
Procedure

4. Add the mixture to a 16-liter knapsack

sprayer containing clean water.

5. Shake the mixture vigorously to evenly

distribute the fungus mycelia and spores.

6. Spray closely to the agricultural wastes

(e.g. rice straw) previously soaked
overnight to hasten faster decomposition.
USING OF TRICHODERMA INOCULANT
Procedure

7. Visit the compost pile at least twice a week

and moisten the rice straw if necessary.

8. Start land preparation after 4 weeks.

Composted materials can now be
incorporated into the soil.
Rice straw 18 days after spraying Trichoderma
REFERENCES
Zin, N. and Badaluddin, N. (2020). Biological functions of Trichoderma
spp. for agriculture applications. Annals of Agricultural Sciences. 65
(2).168-178.
Technical Guide on Trichoderma Production
Technical Guide on Trichoderma Production
Thank you
and
God Bless
us all!