Different Trichoderma formulations and their application methods

INTRODUCTION

 

Trichoderma:

 

Trichoderma species are widespread, free-living filamentous fungi found in root and soil environments. In ecosystems like roots, soil, and leaves, they are quite interactive. They release a wide range of chemicals that cause plants to develop either localized or systemic defense mechanisms. Trichoderma strains have been used for a long time to control a variety of soil-borne plant pathogens, including Phytophthora, Rhizoctonia, Sclerotium, Pythium, and Fusarium. They are also important cellulase producers for biotechnology applications and increase their ability to increase root growth and development, crop productivity, resistance to biotic stresses, and the uptake and use of nutrients. Entrepreneurs are currently mass-growing its three primary species, T. harzianum, T. viride, and T. koningii.

 

 

Figure 1: Macroscopic and Microscopic view of Trichoderma spp.

 

TAXONOMIC CLASSIFICATION:

 

Position	Asexual stage	Sexual stage
Kingdom	Fungi	Fungi
Phylum	Ascomycota	Ascomycota
Sub-division	Deuteromycotina	Ascomycotina
Class	Hyphomycetes	Pyrenomycetes
Order	Monilliales	Sphariales
Family	Monilliaceae	Hypocreaceae
Genus	Trichoderma	Hypocrea

 

 

MASS PRODUCTION OF TRICHODERMA:

 

There are two major methods of inoculum production

1. Solid-state fermentation

 

2. Liquid state fermentation

 

1. Solid-state fermentation:

 

In laboratory research, mass synthesis of Trichoderma spp. is frequently accomplished using solid fermentation. In Petri plates, the fungus is grown, and spores and other propagules are then collected and prepared. Another method of solid fermentation is the bulk synthesis of bio-control fungus using a variety of inexpensive agricultural wastes and byproducts. As substrates, a variety of low-cost cereal grains, including ragi, millets, and sorghum, are employed.

 

The grains are soaked in water overnight and water is decanted in the morning.  The soaked grains are taken in heat-resistant bags (Polypropylene bags) and sterilized for 30 minutes in an autoclave or pressure cooker. The sterilized grains are inoculated with Trichoderma spore suspension and incubated for 10-15 days. 

 

Trichoderma produces a dark green spore coating on the grains. These grains can be powdered finely and used as a seed treatment or the grains can be used as it is for enriching FYM for soil application. This type of fermentation results in a product that is generally used as is rather than being formulated further.  These products are likely to obstruct agricultural machinery and thus are probably not feasible for commercial use (Cumagun, 2014).

 

Press mud-based formulation:

 

As a byproduct of the sugar refinery, press mud is available and can be employed as a substrate for mass multiplication. 

The procedure involves evenly blending a 9-day-old Trichoderma spp. culture cultured in potato dextrose broth with 120 kg of press mud. To keep it moist, water was intermittently poured. Gunny bags were used to cover this in order to allow airflow and trap moisture in the shadow. The mother culture for subsequent multiplication is ready in 25 days. The same was blended completely, added to 8 tonnes of press mud, and then incubated for 8 days in the shade before being used in the field.

 

Coffee husk-based formulation: 

 

In Karnataka, coffee husks are waste from the coffee industry. This product was very effective in managing Phytophthora foot rot in black pepper and is widely used in Karnataka and Kerala (Das et al., 2021).

 

Banana waste-based formulations: 

 

Balasubramanian proposed the bulk multiplication method of Trichoderma sp. in banana trash (Manoharachary et al., 2020). Urea, rock phosphate, Bacillus polymixa culture, P. sajor caju, and T. viride were used for the banana trash. Sheath, pseudo stem, core, and other banana trash are all sliced into lengths between 5 and 8 cm five layers of various components are arranged in a prepared pit.

 

One tonne of banana waste, 5 kg of urea, 125 kg of rock phosphate, and 1L of a broth culture of B. polymixa, P. sajor caju, and T. viride are all present in each layer.
The banana is produced in five different layers, each of which is thoroughly combined.
Within 45 days, banana waste decomposes, and the enriched culture is widely accessible for use in the field.

 

Talc-based formulation:

 

The talc-based formulation is used in TNAU Coimbatore for seed treatment of pulse crops and rice. Trichoderma grown in the liquid medium is mixed with talc powder in a ratio of 1:2 and dried to 8% moisture under shade.  Shelf life of 3 to 4 months. This type of formulation is popular in India to manage soil-borne diseases of various crops through seed treatment at 4 to 5 g/kg seed.

 

Advantages of solid formulation

 

i. Process is simple 

ii. Cost effective 

iii. Less effluent release, reduce pollution 

iv. High titers (high production yield) 

v. Aeration process is easy 

vi. Resembles the natural habitat of some fungi and bacteria

vii. Easier downstream processing

viii. Sterilization of media is not required

 

Disadvantages of solid formulation:

 

i. Only those microbes that can survive in low moisture conditions can be used 

ii. Precise monitoring and accurate regulation of the fermentation process are not possible 

iii. The growth rate of microbes on solid substratum is relatively slow 

iv. Scale-up of the fermentation process is difficult 

v. Fermentation usually produces excessive heat in the medium 

vi. Bacterial contamination is sometimes problematic

 

2. Liquid fermentation

 

Under Lab Condition: Preparation methodology: Trichoderma is grown in liquid media generally PDA broth for obtaining biomass (1lt capacity).  After Trichoderma, culture is fully grown, the entire biomass is mixed with talc powder in a ratio of 1:2 and dried to 8% moisture under shade.  Carboxymethyl cellulose (CMC)/ Arabic gum powder should be added during mixing.  After drying and breaking the clots, the formulation is packed in milky white polythene bags (Herrera et al., 2020).

 

For large-scale production: In any liquid medium under stationary, shaker, or fermenter culture conditions, Trichoderma can be cultivated. Full spore production in stationary growth settings will take between 10 and 15 days; in shaker circumstances, it may take 7 days; and in fermenter conditions, peak production happens after 3 days.

Mother culture: By extracting a fungal disc from a culture that is 10 days old and inoculating it in broth, which serves as mother culture, T. viridae culture is introduced into the medium. (Broth: molasses yeast medium: 30 g of molasses, 5 g of yeast, and 1 liter of distilled water.) In a fermenter, the broth is made and sterilized. 50 liters of medium and 1.5 liters of mother culture are introduced to the fermenter and incubated for 10 days. 

Medium and carrier materials are combined in a 1:2 ratio, primarily talc powder. Carrier substrate is sterilized at 20 psi for 30 minutes on two separate days after having its pH adjusted to 7 by adding CaCo₃ at a rate of 150 g/kg. For product adherence, the mixture is air dried and combined with CMC at a rate of 5g/kg. used to treat the seeds and packaged in plastic bags.

 

Vermiculite-wheat bran-based formulation:

 

Multiplied in the molasses-yeast medium for 10 days. 100 g vermiculite and 33 g wheat bran are sterilized in an oven at 70°C for 3 days. The entire biomass with HCl is added, mixed well, and dried in shade.

 

Advantages of liquid fermentation:

 

i. Utilizes free-flowing liquid substrates, such as molasses and broths.

ii. Substrates are utilized quite rapidly, and need to be replaced constantly.

iii. Best suited for bacteria that require high moisture content.

iv. Inoculum ratio is usually small

 

Disadvantages of liquid fermentation:

 

i. High cost of media

ii. Large reactors needed

iii. Risk of contamination

iv. Behavior of organisms cannot be predicted at times

 

Table: Commercial formulations of Trichoderma available in India

 

Product	Bio-agent(s)	Developing agency
Antagon-TV	T.viride	Green Tech Agro Products, Coimbatore
Bioderma	T.viride + T.harzianum	Biotech International Limited, New Delhi
Bioguard	T.viride	Krishi Rasayan Export Pvt. Ltd., Solan (H.P.)
Ecoderma	T.viride + T.harzianum	Margo Biocontrol Pvt Ltd., Bangalore
Ecofit	Trichoderma viride	Hoechst and Schering AgrEvo Ltd., Mumbai
Funginil	T.viride	Crop Health Bioproduct Research Centre, Gaziabad
Pant Biocontrol Agent-1	T.harzianum	G.B.Pant University of Agriculture Technology, Pantnagar
Trichoguard	T.viride	Anu Biotechk International Ltd.,k Faridabad
Tricho-X	T.viride	Excel Industries Limited, Mumbai

 

 

METHODS OF APPLICATION

 

1. Seed treatment: Seed priming, in which seeds are mixed with an organic carrier and then moisture content is brought to a level just below that required for seed treatment has been used to deliver T. harzianum to control Pythium-induced damping-off on cucumber.  In another process of seed treatment, an industrial film-coating process that was developed for the application of chemicals and biological crop protection agents is being utilized for the application of Trichoderma spp.  Trichoderma spp. applied on radish and cucumber seeds through a film coating were shown to be effective against damping-off (Mukherjee et al., 2019

 

2. Soil amendment: Numerous attempts have been made to control several soil-borne pathogens by incorporating natural substrates colonized by antagonists of pathogens into the soil.

 

3. Soil drench: Seed priming has been used to administer T. harzianum to reduce Pythium-induced damping-off on cucumbers. In this method, seeds are combined with an organic carrier, and then the moisture content is brought to a level just below that required for seed treatment. In a different method of seed treatment, Trichoderma spp. is being applied using an industrial film-coating process that was created for the administration of chemical and biological crop protection agents. It has been demonstrated that Trichoderma spp. used as a film coating on cucumber and radish seeds can effectively prevent damping-off.

 

4. Aerial spraying / Wound dressing: To prevent illnesses that damage above-ground portions, Trichoderma species can be sprayed onto leaves. Foliar disease bio-control is not as advanced as soil-borne disease bio-control. The lack of examples of bio-control of foliar diseases may be due to the accessibility of affordable and efficient chemical fungicides, their simplicity in administration to foliage, and the fact that the outcomes of using bio-control agents were not as effective as those of using conventional fungicides. The application of Trichoderma to wounds on shrubs and trees during pruning, before decay fungi take hold, is a more effective example of the fungus being used to treat aerial plant part

 

5. Fluid Drill Technology: Bio-control agents are incorporated into fluid drill gels as part of this distribution strategy. In one study, before the seedlings were planted, vegetable or fruit tree seedlings were dipped into gels containing antagonists so that the root area was covered by a thin coating of gel. T. harzianum has been administered using fluid-drilling gels to control R. solani and S. rolfsii on apples. This novel method, which makes use of fluid drill technology's advantages, holds great potential for the creation and use of bio-control bacteria.

 

6. Multiple  delivery  systems:

Azadirachtin @ 3 mL/Lon 30 and 45 DAS foliar sprays followed by seed treatment and soil application of T. viride (2.5 kg/ha) were found to be significantly beneficial by recording the least disease incidence and maximum seed production in sesame.

 

Shelf life of Trichoderma formulations:

 

When preserving a formulation, a bio-control agent's shelf life is extremely important. Contrary to inert or inorganic food bases, antagonists amplified in an organic food base typically have a longer shelf life. Trichoderma in coffee husks has a shelf life of more than 18 months. Trichoderma formulation, which was based on talc, peat, lignite, and kaolin, had a four-month shelf life. The same product had a four-month shelf life in its gypsum-based version. According to research on the storage of T. viride formulation in polypropylene bags of different colours, milky white bags with a 100 gauge thickness had the highest T. viride population.

 

·       The formulated bio-control agent is stored at room temperature and a 1 g sample is drawn at monthly intervals both for Trichoderma.

·       One gram sample is diluted in 100ml of sterile water and serially diluted up to 10⁶ and plated on TSM for Trichoderma and record the number of colonies at different dilutions and intervals

 

Quality control:

 

Any bio-product used to control pests and diseases must maintain a certain level of stability throughout time and in various storage environments. The items are typically kept in a room-temperature environment. Although the temperature range is not extreme, over time the microorganisms' viability decreases. The talc- and lignite-based formulations are typically expected to endure for at least six months. A few producers have also asserted that it lasts for around a year. Checking the product's viability is therefore appropriate. Trichoderma is expected to meet the national criteria of 10⁶ cfu/g. Any product that doesn't meet these requirements at the time of testing won't be allowed for usage or sale (Ramanujam et al., 2010).          

 

Precautions:

 

—  Don’t use chemical fungicide after application of Trichoderma for 4-5 days.

—  Don’t use Trichoderma in dry soil, moisture is an essential factor for its growth and survivability.

—  Don’t put the treated seeds in direct sun rays.

—  Don’t keep the treated FYM for a longer duration.

 

Conclusion and future aspects:

 

For sustainable agriculture, the development of Trichoderma formulations has further improved the biological control of plant diseases. Future studies on Trichoderma biological control systems should investigate the effectiveness of emulation at controlling Plant Pathogens by its endophytic nature. To scale up production systems, including large-scale marketing of Trichoderma formulation in farmer’s fields, particularly in developing countries, it is necessary to improve research-industry partnerships.

 

References:

 

Cumagun, C. J. R. (2014). Advances in formulation of Trichoderma for biocontrol. In Biotechnology and biology of trichoderma (pp. 527-531). Elsevier.

 

Das, M. M., Aguilar, C. N., Haridas, M. and Sabu, A. (2021). Production of bio-fungicide, Trichoderma harzianum CH1 under solid-state fermentation using coffee husk. Bioresource Technology Reports, 15, 100708.

 

Herrera, W., Valbuena, O. and Pavone-Maniscalco, D. (2020). Formulation of Trichoderma asperellum TV190 for biological control of Rhizoctonia solani on corn seedlings. Egyptian Journal of Biological Pest Control, 30(1), 1-8.

 

Manoharachary, C., Singh, H. B. and Varma, A. (Eds.). (2020). Trichoderma: agricultural applications and beyond. Springer International Publishing. 

 

Mukherjee, P. K., Mehetre, S. T., Sherkhane, P. D., Muthukathan, G., Ghosh, A., Kotasthane, A. S. and Saxena, D. R. (2019). A novel seed-dressing formulation based on an improved mutant strain of Trichoderma virens, and its field evaluation. Frontiers in Microbiology, 10, 1910.

 

Ramanujam, B., Prasad, R. D., Sriram, S. and Rangeswaran, R. (2010). Mass production, formulation, quality control and delivery of Trichoderma for plant disease management. The journal of plant protection sciences, 2(2), 1-8.