Characterization of Biocontrol Agents Isolated From Temperate Region of India

Trichoderma species are potential fungal bio-control agents used against a wide range of soil borne plant pathogens. In the present study a total of 20 Trichoderma isolates viz., AT1, AT2, AT3, AT4, AT5, AT6, AT7, BT1, BT2, BT3, BT4, BT5, BT6, BT7, BT8, BT9, BT10, BT11, BT12, BT13 were isolated from vegetable fields of Kashmir valley and their efficacy was tested by using various biochemical tests. Thirteen isolates of Trichoderma viz., AT1, AT2, AT3, AT4, AT5, AT6, AT7, BT1, BT7, BT8, BT10, BT11 and BT12 were found to be positive for ammonia production. Similarly twelve isolates viz., AT2, AT3, AT7, BT1, BT3, BT4, BT7, BT8, BT10, BT11, BT12 and BT13 were found to be positive as far as the chitinase activity is concerned. In the IAA production assay maximum IAA was produced by isolate BT9 (6.605 μg mL-1) followed by BT6 (5.278 μg mL-1), while minimum IAA was produced by isolate AT1 (1.538 ìg mL-1). Only five isolates viz., AT1, AT2, AT3, AT4 and AT5 metabolized lactose and sucrose while seven isolates viz., AT1, AT2, AT3, AT4, AT5, AT6 and AT7 were found to metabolize maltose. Hydrocyanic acid (HCN) was observed to be produced by only three isolates of Trichoderma viz., AT3, AT5 and AT7. Trichoderma isolate AT3 qualifying most of the biochemical tests were morphological characterized as Trichoderma harzianum.

The practices of integrated agricultural management, where chemicals are either replaced or minimized by by-products, are the most suitable option (Cavalcante et al., 2008).Biological control agents (BCAs) are by-products based on microorganisms that cause harmful alterations to plant pathogens by chemical or physical processes (Vinale et al., 2008).BCA differ from chemical agents in that to be effective, they need to grow and successfully colonize and therefore they need to be applied in high and frequent quantities (Lo et al., 1998).Fungi from the genus Trichoderma spp.have a long history of successful control of plant diseases (Benitez et al., 2004).Trichoderma play an important role not only in controlling the plant diseases but also in increasing growth and yield of the plants (Chadha et al., 2015), so different biochemical attributes leading to its effective use against various pathogens are of paramount importance.There is a vast number of biochemical being released by Trichoderma species to bring about suppression of a huge number of phytopathogens and these biochemicals may be released constitutively or may be induced by the pathogen presence (Aarti and Meenu, 2015;Rawat and Tewari, 2011) .
What we observe and define as biocontrol may be the culmination of a number of different mechanisms working synergistically to achieve disease control.Our knowledge of the complexity of these systems is currently limited by our ability to perceive them, and a great deal of research will have to be undertaken in order to fathom exactly what is taking place during the biocontrol process.As with so many other aspects of science, basic knowledge about the mechanisms involved in the biocontrol process will be of immense value to that intent on developing new methods for utilizing biocontrol agents.With this aim present study was undertaken to study the various biochemicals involved in the process of plant growth promotion by Trichoderma species.

Sample Collection
Rhizospheric soil samples were collected from various commercially grown chilli fields and kitchen gardens of district Anantnag (Bangidar, BagiWanpoh, Danter, Harnag) and Baramulla (Delina, Arampora, Palhalan and Johama) of Kashmir valley.Twenty (20) samples from each district were randomly collected, out of which 10 were taken from commercially grown chilli fields and 10 from local kitchen gardens.

Morphological Characterization
Distinct cultural and morphological characteristics were observed for identification, and the plates were stored at 4 °C.Cultural and morphological characteristics which were studied include colony growth rate, colony colour, reverse colour, colony edge, mycelial form, conidiophore branching, conidial colour and presence or absence of chlamydospores (Shahid et al., 2013).

Isolation of Trichoderma species from vegetable fields
A total of 20 (7 from Anantnag and 13 from Baramulla) Trichoderma isolates (plate 1) were isolated from 40 randomly collected rhizospheric soil samples from various commercially grown chilli fields and kitchen gardens of district Anantnag and Baramulla of Kashmir valley by using Trichoderma specific medium.Nomenclature given to these Trichoderma isolates, the collection sites and source of soil samples is detailed in Table 1.Similarly Trichoderma species were isolated from chilli rhizosphere by Wani et al. (2014) and the technique used for isolation of Trichoderma species is in agreement with chaudhari et al. (2011) and Khandelwal et al. (2012).
HCN production is also an important trait found in various soil micro-organisms as it indirectly promotes plant growth by controlling

Production of IAA by various isolates of Trichoderma specie
Microbial synthesis of the phytohormone auxin (indole-3-acetic acid/indole acetic acid/IAA) has been known for a long time.It is reported that 80% of microorganisms isolated from the rhizosphere of various crops possess the ability to synthesize and release auxins as secondary metabolites (Patten and Glick, 1996).During IAA production test all the isolates were found to produce IAAhowever their production amount varied considerably.Maximum IAA was produced by isolate BT9 (6.605 ìg mL -1 ) followed by BT6 (5.278 ìg mL -1 ), BT11 (3.408 ìg mL -1 ) and AT3 (3.317 ìg mL - 1 ) while minimum IAA was produced by isolate AT1 (1.538 ìg mL -1 ) (Fig. 1

Carbohydrate metabolization by various isolates of Trichoderma species
During these tests, it was found that all the isolates metabolized glucose.Similarly all the isolates metabolized Fructose except isolates BT5 and BT6.It was found that only five isolates (AT1, AT2, AT3, AT4 and AT5) metabolized lactose and sucrose while seven isolates (AT1, AT2, AT3, AT4, AT5, AT6 and AT7) were found to metabolize maltose (Table 3).Kubicek et al. (2003) also detected the species-specific metabolic properties of Trichoderma.The carbon sources supported best growth in all species detected were: dmannitol, N-acetyl-d-glucosamine, l-erythritol, glycerol, fructose, fucose, l-arabinose, d-galactose, and xylitol and thus authenticatingour findings further even though there were some isolates lacking the ability to metabolize lactose and sucrose may be as a result of certain enzymatic complications.Similar findings were observed by Monga (2001) and Mehta et al. (2012).

Cultural and morphological characterization of Trichoderma isolate (AT3)
Although cultural and morphological characteristics of all the isolates were studied, our main focus was on the potential Trichoderma isolate (AT3).The characteristics which were studied include colony growth rate, colony colour, reverse colour, colony edge, mycelial form, conidiophore branching, conidial colour and presence or absence of chlamydospores (Table 4).After studying these characteristics Trichoderma isolate (AT3) was found to resemble Trichoderma harzianum.
). Resende et al. (2014) also reported the production of IAA by various isolates of Trichoderma and the amount of IAA produced varied from as low as 1.21ìg mL -1 and as high as 2.18 ìg mL -1 and thus supporting our findings.Similar findings were recorded by Aarti and Meenu (2015), Gravel et al. (2007) and Badawi et al. (2011) in Trichoderma species.

Table 1 .
Details of Trichoderma isolates isolated from rhizospheric soils of chilli fields

Table 2 .
Screening of various isolates of Trichoderma species for ammonia production, chitinase activity, HCN production and Phosphate solubilisation

Table 3 .
Cultural and morphological characteristics of Trichoderma isolates

Table 4 .
Carbohydrate Fig. 1.Production of IAA by various isolates of Trichoderma species