Trichoderma : A scientific approach against soil borne pathogens

The members of the genus Trichoderma are widely used as bioagent for the control of phytopathogenic fungi in agriculture sectors. The members of this genus are reproducing asexually by the formation of cyanide and chlamydospore, in wild habitats they reproduce by formation of ascospores. Trichoderma species are well known for the production of cell wall degrading enzymes (CWDEs). These CWDEs, play a major role in biocontrol mechanism. We all know that all living organisms are made up of genes that code for a particular function. Similarly, in Trichoderma, some genes are responsible for the secretion of these CWDEs. These genes, which aid in the biocontrol action, are called biocontrol genes. These bacterial genes code for a particular enzyme and protein that degrade the pathogen cell wall. These biocontrol genes can be isolated and cloned for large scale commercial production. It has also been found that some of the genes of Trichoderma are also helpful in the abiotic and biotic stress. The mechanisms which are employed by Trichoderma for the phytopathogenic action are generally included atibiosis, mycoparasistism, competition for nutrients, etc.


INTRODUCTION
In recent times, excessive use of chemical pesticides has posed a threat on the environment.Trichoderma based biocontrol agents have better ability to promote plant defense response, promote plant growth and soil remediation.Trichoderma species has gained wide acceptance as effective biocontrol agents against several phytopathogens.Micropropagules of Trichoderma spp. in the form of conidia are preferred over chlamydospores and mycelia biomass because of its viability and stability in field application (Rosane et al., 2008;Chet, 1987).
The genus Trichoderma are commonly found in soils and on decaying wood and vegetable matter.Strains of Trichoderma are rarely associated with diseases of living plants, although an aggressive strain of Trichoderma causes a significant disease of the commercial mushroom and soil borne pathogens.Samuels (1996) provides a comprehensive review of Trichoderma spp.for enzyme production and biological control mechanisms.In Trichoderma spp., sexual reproduction not present are believed to be mitotic and clonal.The main *Corresponding author.E-mail: biocontrol.csa@gmail.comAuthor(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License problem with the nomenclature of Trichoderma is pleomorphisim present within the genus.In Trichoderma, there are two stages, the sexual stage is called Hypocrea (telomorphic) and the asexual stage is called Trichoderma (anamorphic).The genus is called Hypocrea/Trichoderma (Druzhinina et al., 2011).However, despite these significant advances in our knowledge about this genus, the full taxonomic history of Trichoderma is still not complete, and the detailed description of the taxonomic history of Trichoderma remains problematic.A refined classification and identification is necessary for predictive indications about ecology.
Trichoderma is widely used for the control of many soil borne plant pathogens.Trichoderma spp.are the efficient producer of cell wall degrading enzymes (Srivastava et al., 2014b); some of these enzymes are of commercial importance.Many research workers have proved that Trichoderma spp.possess some biocontrol genes that can be isolated and cloned for commercial large scale production (Massart and Jijakli, 2007).
The biococntrol ability of Trichoderma is of much importance as it does not acccumulate in food chain and thus do not harm plants, animals and humans.The genes involved in the biocontrol mechanisms of Trichoderma are of great importance.

TAXONOMY OF TRICHODERMA
Although the genus Trichoderma has been known since the 19th century.Its association with Hypocrea Fr. was discovered by the Tulasne brothers in 1865; its taxonomy has remained obscure until recent decades.Bisby (1939) thought that the morphological variation could be ascribed to a single species, Trichoderma viride.The first serious attempt to morphologically distinguish species, or rather "species aggregate", was made by Rifai (1969).Some new species subsequently described were keyed out by Domsch et al. (1980).Teleomorph connections were established by means of ascospore isolates by Dingley (1957) and Webster and Rifai (1968).In Japan, Doi (1969aDoi ( , 1972b) studied a number of telemorphs and described them with cultural and anamorph characters, but no cultures were preserved from that study.After this, no morphological differentiation was given.Bissett (1984Bissett ( , 1991b, c) , c) gave a detailed description of the morphological studies, who distinguished about 21 taxa.These studies have shown that the delimitation of biological species is extremely difficult in this genus on morphological grounds alone.
Apart from morphological studies, there are many other methods that are used in the taxonomy, such as study of secondary metabolites; this has shown a great diversity in this genus (Okuda et al., 1982).Physiological features that are detected by the microtiter plate assay are the useful tools that are used for the identification.Isoenzyme profiles are also used for taxonomic classification (Samuels et al., 1994;Leuchtmann et al., 1996).In modern era molecular techniques, such as sequences of its region of ribosomal DNA and fingerprinting techniques provide the finest resolution of taxonomic entities (Meyer et al., 1992;Fujimori and Okuda, 1994;Muthumeenakshi et al., 1994).

BIOCONTROL GENES OF TRICHODERMA
Trichoderma is widely used for the control of many soil borne plant pathogens (Table 1).Trichoderma spp.Are efficient producers of cell wall degrading enzymes (Srivastava et al., 2014a), some of which are of commercial importance.Many research workers have proved that Trichoderma spp.possess some biocontrol genes that can be isolated and cloned for commercial large scale production (Massart and Jijakli, 2007).Kuc (2001) has proved that some genes, providing resistance to abiotic and biotic stress are present in Trichoderma (Table 2).Mycoparasistism (Figure 1), antibiosis and competition for the nutrients are the main strategies employed by Trichoderma for the phytopathogenic action (Janisiewicz and Korsten, 2002).Among the different species tested, Trichoderma harzianum was found to be the most promising strain (Gao et al., 2002).

GENOMICS OF TRICHODERMA
The genome size of filamentous fungi is very small as it is about 25 to 50 Mb.However, with the advancement of pulse field gel electrophoresis, karyotyping of filamentous fungi is possible.Karyotyping is helpful in the detection, translocations and variations in chromosome numbers.Through pulsed-field gel electrophoresis (PFGE), chromosomal DNA was separated from Trichoderma (Gilly and Sands, 1991;Mäntylä et al., 1992;Hayes et al., 1993;Herrera-Estrellaet et al., 1993).The expected genome size and chromosome number of Trichoderma ranges from 31 to 39 Mb and from 3 to 7, respectively.From the data obtained through DNA homology, it was found that T. harzianum and Trichoderma veins are closely related, and it was concluded that they may have the same phylogenetic origin (Herrera-Estrella et al., 1993).On the other hand, Mäntylä et al. (1992) determined molecular karyotypes of various strains of Trichoderma reesei that had undergone mutagenesis and screening for the hyper production of cellulase enzyme.The authors found that extensive alteration in the genome organization of these strains occurred.
The first member of the genus sequenced was T. reesei (Table 3).This fungus is the first choice, because the genome size of this organism is very small (33 Mb) and has only seven chromosomes.Fungal genomics laboratory of NCSU has expressed sequence tag (EST),

Tvsp1 Trichoderma virens
This gene encodes for serine protease.Rhizoctonia solani which affects the cotton seedlings has been controlled biologically by serine protease.

tri5 Trichoderma harzianum
This gene is responsible for the synthesis of trichothecene which inhibits the protein and DNA synthesis in the cells of the pathogens and inhibits their growth.

TgaA and TgaB Trichoderma virens
This gene exhibits antagonist activity against R. solani and Sclerotium rolfsii

ThPG1
Trichoderma harzianum This gene encodes for endopoly-galacturonase.This enzyme is involved in the cell wall degradation of the pathogens like R. solani and P. ultimum

Th-Chit Trichoderma harzianum
This gene is responsible for the antifungal activity in transgenic tobacco plant.

MASS MULTIPLICATION OF TRICHODERMA
Grains are cheap, easily available and act as best nutritive media for the mass multiplication of many microorganisms.Bajra (Pennisetum typhoides) grains should be completely soaked in 2% sucrose solution in water for 6 h.After draining out the excess water, the soaked 250 g seeds of bajra should be filled in autoclavable polypropylene (PP) bags of 30 × 20 cm 2 .The PP bags should be plugged with nonabsorbent cotton followed by autoclaving at 15 lbs pressure for 30 min.After autoclaving, the bags should be left for cooling overnight.The next day, the bags should be individually inoculated by using 5 ml stock solution (10 6 to 10 8 CFU/ml) of starter culture grown for 100 days, with syringe.Before inoculation, the place from where the inoculation is to be made should be marked out with a small circle with the help of marker pen.Punctured place of injection of the PP bag must be sealed with cellophane tape.The bags should be incubated at 25 ± 2°C for 15 days in a temperature controlled room.After 15 days of incubation, the contents of the bags should be taken out and kept in hot air oven for drying overnight at 35°C.During the 15 days of incubation visual check every day is essential to ensure detection and elimination of contaminated PP bag(s).Formulation thus prepared should be ground to fine powder, while ensuring that during the process temperature does not go beyond at 35°C.The powdered formulation thus obtained should be mixed with presterilized talc in 1:9 (Trichoderma spore:talc) ratio.Three samples should be taken from each, but lot during production and tested using a standardized method to determine the viability of the active ingredient expressed as colony forming units (CFU).The product thus prepared is ready for packaging at this stage.For storage, the finished product should be stored in vacuum filled plastic bags, covered by paper cartons of different sizes (250, 500 and 1000 g).These packets should then be kept in sealed cartons for transportation purpose.

ADVANTAGES OF TRICHODERMA
(1) Trichoderma spp.are very useful for fabric detergent, animal feed production, fuel production, alternative to conventional bleaching, effluent treatment, degradation of organochlorine pesticides and biocontrol of crop diseases.
(2) It is a potential bioagent for the management of fungal seed and soil borne pathogens.It is well known for its antagonistic activity against soil borne pathogens, such as Fusarium, Pythium, and Rhizoctonia.
(3) It is also known to suppress plant parasitic nematodes.(4) It does not lead to development of resistance in plant pathogens, no phytotoxic effects, do not create any pollution problems as it is eco-friendly, promote plant growth, induces resistance in host, solubilize phosphorus (5) It significantly minimizes losses due to crop diseases and reduces cost of production, increases yield, quality and profit.(6) Many Trichoderma spp.are of great economic importance producing hydrolytic enzymes, namely, cellulases, chitinases and xylanases, biochemicals and antibiotic products which have been applied to fields, such as food processing and pulp bleaching.In addition, some species produce heterologous proteins and others have been successfully used as biological control agents against a range of phytopathogens.

DISADVANTAGES OF TRICHODERMA
There are many advantages associated with the use of Trichoderma.However, in addition to their useful properties, there are some disadvantages associated with the use of Trichoderma.
(1) Some species of Trichoderma pose a threat to the horticultural industry.For example, reduction in mushroom yield by as much as 50% have been attributed to Trichoderma infection and hence it is considered as a harmful parasite of mushroom.
(2) It also affects the organ (liver) transplanted in human.
(3) The disease is the major constraint in economical production as it inflicts heavy crop losses.

CONCLUSION AND FUTURE RESEARCH
Chemical based control is very effective, but there are some disadvantages associated with the use of these chemicals.The most dangerous thing with the use of these chemicals is the toxicity which they impart to the soil.That is why today people avoid the use of chemical based fungicide.Biosynthetic design of fungicide has present a new era in the development of fungicide.The genes present in the fungi Trichoderma has the ability to enhance host plant's resistance against phytopathogenic fungi.
This gene helps in the production of Trichoderm in which shows antifungal activity against S. cerevisiae, Kluyveromyces marxianus, Candida albicans, C. glabrata, C. tropicalis and Aspergillus fumigates.erg1Trichoderma harzianum This gene encodes an enzyme named squalene peroxidase, which helps in the synthesis of ergosterol and silencing of this gene provides resistance to terbinafine, an antifungal compound.

Figure 1 .
Figure 1.Scanning electron micrograph on mycoparasitism of the F. oxysporum ciceri hyphae by the hyphae of T. harzianum with pincer shaped structure.

Fig. 1 :
Fig. 1:Scanning electron micrograph on mycoparasitism of the F. oxysporum ciceri hyphae by the hyphae of T. harzianum with pincer shaped structure Fig. 1:Scanning electron micrograph on mycoparasitism of the F. oxysporum ciceri hyphae by the hyphae of T. harzianum with pincer shaped structure

Table 1 .
Trichoderma species and its uses against different plant pathogens.
This gene has its role in mycoparasitic activity against R. solani and P. ultimumTrCCD1 Trichoderma reeseiThis gene is involved in carotenoid metabolism that helps in the development of conidiospores and hyphal growth in T. reesei

Table 3 .
Summary of some features of the sequenced genomes.