**5. Viability in the storage and field**

One of the critical problems in the commercialization of bio-agents is the loss of viability of the propagules over time. The shelf life of the bio-control product is dependent on the storage temperature and carriers as used in the formulation of bio control agents. The shelf life of bio- control agent plays a significant role in successful marketing. *Trichoderma* spp. are multiplied on bio-degradable substrates for long shelf-life and is also beneficial for field application. Bio-control agents are a biomass product, maintaining their viability at the end of the course [104]. Talc based *Trichoderma virens* conidia keep 82% viability at 5°C in refrigerator after 6 months, while at room temperature was observed for 3 months [105]. The viable propagule of *Trichoderma* in talc formulation was reduced by 50% after 120 days of storage [77]. Increasing shelf life of talc formulations of *Trichoderma* using various ingredients (chitin and glycerol) in production medium fermentation was carried out the shelf of talc formulation of *Trichoderma* up to 1 year [106, 107]. *Trichoderma* on coffee husk has a shelf life of more than 18 months. Talc, peat, lignite and kaolin based formulations of *Trichoderma*, have a shelf life of 3–4 months. In the storage polypropylene

bags use of various colors, *Trichoderma viride* showed maximum shelf life in milky white bags of 100-micron thickness. The *Trichoderma* fungus in the storage temperatures is less than 4°C.

### **6. Mode of action**

Bio-control agents are playing an important role in controlling of plant pathogens, especially soil borne fungal pathogens. Biological control agents reduce the disease of the target crop usually by one or more of the modes of action manly antibiosis, competition, mycoparasitism, cell wall degrading enzymes and induced resistance. The indirect interaction with pathogens is competition for nutrients and space and directly with the pathogen by hyperparasitism or antibiosis [108]. Bio-control agents might directly interact with the pathogens by hyperparasitism [109], and antibiosis [110]. Bio-agents induce resistance enhanced in plants against pathogens, competitions for nutrients and spaces [111]. Various chemical compounds such as lectins during the initial contact, recognition and cell wall-degrading enzymes such as β-1,3-glucanases, chitinases, proteinases, and lipases, during the penetration [112]. In hyper-parasitism growth of bio control agent towards the target organism, coiling, final attack and dissolution of target pathogens cell wall by the activity of enzymes [86].

Mycoparasitism is one of the most important direct antagonism mechanisms that attack one fungus on another [113]. and causes complete death of fungal propagules or destruction and lysis [114]. Mycoparasitism is a complex process which involves chemotrophic growth, recognition and coiling, the interaction of hyphae and secretion of specific lytic enzymes [113]. *Trichoderma* hyphae, initial recognition and wind around the pathogen's hyphae by forming a hook, the appressorium permeates into the pathogen cell, and chitin is broken down by enzymes such as chitinase and glucanase [109]. The fungal cell walls contain chitin and glucan are the major constituents of many fungal cells [115]. *Trichoderma* strains have antagonistic potential and are mainly characterized by their ability to secrete enzymes such as chitinases, glucanases, and proteases that hydrolyze the cell walls of pathogens [116]. Chemotrophic response fungus induces the released the cell wall degrading enzymes from *Trichoderma viz.* β-1, 3 glucanase, proteases, lipases and chitinases [117]. The role of proteases in biocontrol of *Botrytis cinerea* by *T. harzianum* [57]. In mycoparasitism has been attributed to the role of chitinases [118]. The proteases reduced the activities of the pathogen enzymes exo- and endo polygalacturonase, pectin methylesterase, pectate lyase, chitinase, cutinase, and β-1,3-glucanase that are essential during host infection. *Trichoderma* hyphae contact and start coiling around the attachment of hyphae [119]. The hyphae grow along the host hyphae and secrete different lytic enzymes such as glucanase, chitinase and pectinase that are involved in mycoparasitism and ultimately degeneration of the target fungus [119]. *Trichoderma* produces low molecular weight compounds that have antifungal and antibacterial properties, these substances inhibit cell wall synthesis [118]. *Trichoderma* hyphae release antibiotic compounds which penetrate the pathogen's hyphae and inhibition of host cell wall synthesis [59, 120]. The mycoparasitism of *Trichoderma* spp. towards *Pythium ultimum* and *Sclerotium rolfsii* [18]. The parasitism of *Rhizoctonia solani* hyphae by the *Trichoderma virens* in controlling citrus seedling disease [121, 122]. *Trichoderma* species such as *T. atroviride*, *T. virens,* and *T. reesei* have ability of mycoparasitism [7, 123]. *Trichoderma harzianum* is excellent mycoparasitic activity against *Rhizoctonia solsni* [11] and also involve chitinase and β-1, 3 glucanase [1]. *Trichoderma* spp. is known to produce antimicrobial metabolites that act via hyperparasitism [10].

Antibiosisis is the condition of antagonistic to the suppression of pathogenic microorganisms due to toxic compounds (antibiotics). Antibiotic is a secondary metabolite with a low molecular weight that is harmful to the other microorganisms at a low concentration [124]. The antibiotic is produced by bio-control agents and is the main contributing mechanism under soil conditions [125]. Soil-borne microorganisms have different strains of *Trichoderma* species [5]. Secondary metabolites secreted in situ and effects against pathogens at low amounts [110].

Competition is the **f**orm of microbial in soils and living plant surfaces for nutrient limited environments [126]. Bio- control agents and pathogens compete with one another for the nutrients and space in the environment. The competition is considered to be an indirect interaction between the pathogen and bio control agent [127]. The competition for nutrients of bio-control agents fights for the essential micronutrients such as iron and manganese in soils. The bio control agents have more efficient for utilizing micro-nutrient uptake for the substances than the pathogens [128]. Iron competition is a limiting factor in alkaline soils for microbial growth and development [129]. Siderophore is low-molecular weight chelators with is a very high and specific affinity for Fe called siderophores [130]. *Trichoderma* spp. produces highly efficient low molecular weight ferric iron chelators termed siderophores that stop the growth of other fungi [131]. Siderophore is a chealate of the Fe ions that bind and take up the Siderophore-Fe-complex and making iron unavailable to the pathogen [132]. *Trichoderma* species as bio-control antagonists release siderophores that chelate iron (Fe3+) prevent the growth and development of fungal pathogens [90]. *Trichoderma asperellum* producing iron-binding siderophores controls *Fusarium* wilt [133].

Induce resistance is indirect mechanism in host physio-biochemical pathways that trigger defense cascades inside the plants and lead to suppression of disease development. Induced defense mechanisms involve the production of reactive oxygen species, phytoalexins, phenolic compounds, pathogenesis-related proteins, physical barriers [134]. The role of *Trichoderma* in plants defense as involved in induced immunity. Concept of induces resistance on cucumber seedling disease with *T. harzianum* [43]. The roots are recognized fungal-derived molecules that changes occur locally and systemically in gene expression, increasing salicylic acid, jasmonic acid, and phytoalexin levels in plants. The induced resistance is enhanced against infections by a pathogen in the plant without direct antagonistic interaction with the pathogen [135, 136]. *Trichoderma* application cause induces resistance against the diseases in plants and provides long-term protection [137]. *Trichoderma* in the rhizosphere can protect plants against aerial pathogen infections, through the induction of resistance via a hypersensitive response (HR), systemic acquired resistance (SAR) and induced systemic resistance (ISR) in plants [10]. Induced resistance was demonstrated through the induction of *Trichoderma* against foliage disease of beans caused by *Colletotrichum lindemuthianum* and *Botrytis cinerea* [138]. *Trichoderma* produces several metabolites that act as elicitors of plant results in the synthesis of phytoalexins, PR proteins that increase in resistance against several plant pathogens [139] and in abiotic conditions [10].

#### **7. Diseases management**

*Trichoderma* spp. and *Gliocladium* spp. were the first bio-control agents that effectively manage plant pathogens such as *Sclerotium rolfsii*, *Rhizoctonia solani*, and *Fusarium solani,* cause diseases on groundnut, bean, and apple, respectively [94, 138, 140]. *Trichoderma viride, Trichoderma harzianum* and *Trichoderma virens* are being successfully used for the control of diseases such as foot rots, root rots, damping off, collar rots and *Fusarium* wilts of horticultural crops. *Trchoderma* are effective against foliar and soil borne plant pathogens [141]. The talc based formulations of *Trichoderma* manage several soil-borne diseases of various crops by seed treatment at 4 g–5 g/kg seed. Soil borne plant pathogens are successfully manage through seed coating, furrow application and root dip of seedlings. Successfully managed *S. rolfsii* and *Pythium* spp. on radish and pea by seed coating of *T. hamatum* as reported [142]. *Trichoderma harzianum* application in the field with wheat bran colonized rapidly in the soil and inhibits the *Rhizoctonia solani* and *S. rolfsii* in beans [26, 143]. *Trichoderma* spp. has potential in controlling wilt and damping-off diseases caused by *Fusarium* sp. and *Rhizoctonia solani* [28, 121]. *Pythium,* and *Phytophthora, Rhizoctonia, Fusarium and Sclerotium*, spp. are soil borne plant pathogens causing diseases in several crops, the diseases manage through *Trichoderma* spp. as reported [22]. *Trchoderma* has the potential to manage fungal and nematode diseases as well as host defense inducing ability in plants [64]. Nano-particles (Ag and Au) of *Trichoderma asperellum* showed antifungal activity at different concentrations, the maximum radial growth inhibition was observed at 200 ppm against *Rhizoctonia solani* as compared with the chemical [23].
