Trichoderma is a member of the Division Deuteromycotina of fungi that is present in almost all types of soils and other diverse habitats. It occurs frequently in soils having acidic pH. It can easily be isolated from soil on potato dextrose agar medium.
1. Culture Characteristics:
Most species of Trichoderma are photosensitive, sporulating on many natural and artificial substrates in a form of alternating concentric rings. The rings develop due to its growth in alternate light and darkness period.
Conidia (asexually produced exogenous spores) are produced during the light period of growth. The white areas do not contain spores, while the green areas are covered with dense masses of conidia.
Under microscope hyaline (colourless) conidiophores bearing several branches can be observed. These branches are called phialides that produce spherical exogenous conidia at its end. The organism grows and ramifies as typical fungal hyphae having 5-10 µm diameters.
Asexual sporulation occurs as single-celled, usually green conidia (typically 3 to 5 µm in diameter) that are released in large numbers. Intercalary resting chlamydospores are also formed. Clamydospores are also single celled, although two or more chlamydospores may be fused together.
2. Species of Trichoderma:
There are many species of Trichoderma, namely T. harzianum, T. koninghii, T. pseudokoninghii, T. viride, etc. In addition there are several variations within a species; these are called strains. Most strains are highly adapted to an asexual life. In the absence of meiosis, chromosome plasticity is the norm, and different strains have different numbers and sizes of chromosomes.
Most cells have many nuclear. Sometimes some vegetative cells possess more than 100 nuclei. Various asexual genetic factors, such as parasexual recombination, mutation and other processes contribute to variation between nuclei. In this way Trichoderma spp. are highly adaptable and evolve rapidly.
T. harzianum Rifai strain T-22 is a naturally occurring fungus. It is used to protect crops and seeds from various fungi that cause plant diseases. It is used primarily in greenhouses and nurseries, as well as by consumers. The active ingredient is not expected to cause adverse effects to humans, pets or the environment. There are certain crops where it is not approved for use.
3. Why Trichoderma spp. as Biopesticide (or Bio-control Agent)?
Species of Trichoderma grow rapidly in the close vicinity of root region called rhizosphere to which they colonize readily. Some strains are highly rhizosphere competent i.e., able to colonize and grow on roots as they develop. The most strongly rhizosphere competent strains can be added to soil or seeds by any method. Depending on the strain when they come into contact with roots, they colonize the root surface or cortex.
Thus, if added as a seed treatment, the best strains will colonize root surfaces even when roots a meter or more below the soil surface and they can persist at useful numbers up to 18 months after application. However, most strains lack this ability.
With or without legal registration, these are used for the control of plant diseases such as damping off, root rots, wilts, etc. There are several reputable companies that manufacture the products.
4. Mode of Action against Plant Pathogens:
In addition to colonizing roots, Trichoderma spp. attack, parasitize and otherwise gain nutrition from other fungi also. Since Trichoderma spp. grow and proliferate best when the plant roots are healthy, they have evolved numerous mechanisms for both attack of other fungi and for enhancing plant and root growth.
Several new general methods for both biocontrol and for causing enhancement of plant growth have recently been demonstrated. It is now clear that there must be hundreds of separate genes and gene products involved in these processes. Some of the mechanisms are as below:
(i) Mycoparasitism (act of parasitizing the hyphae of fungal pathogens, and coiling and penetration of hyphal cells),
(ii) Antibiosis (production of substrate specific extracellular enzymes),
(iii) Competition for nutrients or space,
(iv) Tolerance to stress through enhanced root and plant development,
(v) Solubilization and sequestration of inorganic nutrients,
(vi) Induced resistance, and
Inactivation of the pathogen’s enzymes involved in causing diseases through production of extracellular enzymes and toxins.
Rhizoctonia solani is a plant pathogen that resides in soil and causes diseases called damping off, root rots, etc., fragmentation and lysis of hyphae of Macrophomina phaseolina on culture medium due to secretion of metabolites by Trichoderma viride.
This type of mechanism of action is called antibiosis. M. phaseolina is a fungal pathogen and causes charcoal rot, dry rot or blight symptoms on about 500 plant species.
It shows a scanning electron micrograph of the surface of a hypha of Pythium ultimum after removal of T. harzianum hypha. Where the Trichoderma hypha penetrated P. ultimum through cell wall degrading enzymes, several tiny holes were formed in the hyphae of P. ultimum. Here T. harzianum is a parasite over a fungal pathogen P. ultimum-, therefore, this mode of action is called mycoparasitism.
5. Plant Growth Promotion:
For many years, the ability of these fungi to increase the rate of plant growth and development, including, their ability to cause the production of more robust roots has been known. The mechanisms for these abilities are only just now becoming known.
Some of these abilities are likely to be quite profound. Recently, we have found that one strain increases the numbers of even deep roots (at as much as a meter below the soil surface).
These deep roots cause the crops, such as corn, and ornamental plants, such as turf grass, various national and international research papers. Since nitrogen fertilizer use is likely to be curtailed by Government to minimize damage of soil fertility, the use of this organism may provide a method for farmers to retain high agricultural productivity.
Besides, Trichoderma spp. possess innate resistance to most agricultural chemicals including fungicides. But the individual strains differ in their resistance. Some lines have been selected or modified to be resistant to specific agricultural chemicals.
Most manufacturers of Trichoderma strains for biological control have extensive lists of susceptibilities or resistance to a range of pesticides. In Integrated Pest Management (IPM) programme, use of Trichoderma with small dosages of pesticides, is also considered.
Biocontrol microbes must contain a large number of genes that encode products which permit the biocontrol to occur. Several genes have been cloned from Trichoderma spp. to produce crops resistant to plant diseases.
No such genes are yet commercially available, but many are in developmental stage. These genes, which are present in Trichoderma spp. and many other beneficial microbes, are the basis for natural organic crop protection and production.
6. Commercial Formulations:
Trichoderma spp. are active ingredients in a variety of commercial biofungicides used to control a range of economically important aerial and soil borne fungal pathogens. Many studies, primarily in vitro studies, have shed light on the molecular basis of the three-way relationship among the pathogen, the plant, and the biocontrol agent. However, complexities of these interactions have been poorly studied in situ.
For example, many of the factors involved in biocontrol are known, but the antifungal mechanisms, including mycoparasitism needs to be studied more. The fate of Trichoderma in soil and on the plant is not well understood.
Effective monitoring of biocontrol- related processes in vivo based on the use of vital markers provides a basis for development of new selection methods and improved applications.
7. Method of Application:
Trichoderma is applied in soil or compost farmyard manure (FYM) heap by using conventional application equipment. It must be ensured that the soil is moist and temperature is around 12°C. Biopesticides are applied through seed inoculation or soil inoculation method.
At the time of application, seeds are inoculated with Trichoderma-based product by preparing suspension. Sterilized seeds are soaked for 30 minutes in a suspension of Trichoderma-based product containing 1.1 x 103 propagules/ml. Then the seeds are dried and sown in field. Concentrations of both spores and propagules in the formulated products should be 2x 107 colony forming units (CFU) per gram of product.
8. Registered Microbial Pesticides:
Following are some of the registered microbial products which are sold in market:
Spores of Bacillus popilliae, B. thuringiensis subsp, Kurstaki, B. thuringiensis subsp. Israelensis, B. thuringiensis subsp. Aizawai GC-91, B. thuringiensis subsp. Isaelensis strain g2215, B. subtilis amyloliquefaciens strain fzb24, B. subtilis qst 713, B. subtilis GB03, Bacillus cereus strain BP01, Pseudomonas fluorescens A506, P. fluorescens 1629RS, P. syringae, P. aureofaciens st Tx-1, Streptomyces griseoviridis K61.
Trichoderma harzianum ATCC 20476, T. polysporum ATCC 20475, T. harzianum Rifai KRL-AG2, T. harzianum Strain T-39 Coniothyrium minitans St con/m/91 -08, killed fermentation solids and solubles of Myrothecium verrucaria, Paecilomyces fumorsoroseus Apopka strain 97.
Heliothis Nucleopolyhedrosis virus (NPV), Douglas fir tussock moth NPV, Gypsy moth NPV, Beet armyworm NPV, Cydia pomonella granulosis virus, Indian meal moth granulosis virus.
(iv) Non-viable Microbial Products (engineered):
Bt subsp. kurstaki delta-endotoxin in killed P. fluorescens, Bt subsp. san diego delta-endotoxin in killed P. fluorescens, Bt CrylA(c) & Cry 1(c) delta-endotoxin in killed P. fluorescens, Bt K CrylC in killed Pseudononus, Agrobacterium radiobacter Strain K1026.
(v) Genetically Engineered Plants:
Bt CrylllA potato PVSTBT02 (Monsanto), Bt CrylA(c) cotton PVCHBK04 (Monsanto), Bt corn CrylAb (Mon 810) PZ01502, Bt corn CrylAb, Potato Leafroll Virus (PLRV) replicase protein in Potato as produced in potato plants (Monsanto), Bt Cry IF com (PHI8999) Mycogen, Bt CrylA(b) in corn from PV CIB4431 (Norvatis/Mycog), Bt CrylA(b) delta-endotoxin and genetic material in corn (M) necessary for its production in corn (Monsanto).