F.A. Mohiddin, Imran Bashir, Shahid A. Padder and Mushtaq A. Bhat

Division of Plant Pathology, SKUAST-K, Shalimar, Shalimar, Srinagar J&K- 190 025, India.

Abstract

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 μg 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.

Key words: Trichoderma, Biochemical Characterization, Morphological Characterization.

Introduction

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 biochemicals 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.

MATERIALS AND METHODS

Sample Collection

Rhizospheric soil samples were collected from various commercially grown chilli fields and kitchen gardens of district Anantnag (Bangidar, Bagi-Wanpoh, 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.

Biochemical Characterization

Trichoderma species isolated from soil samples were characterized for carbohydrate metabolization (Bakker and Schipper, 1987), phosphate solubilization (Pikovskaya, 1948), ammonia (Bakker and Schipper, 1987), chitinase (Okay et al., 2008), HCN (Bakker and Schipper, 1987) and IAA production (Brick et al., 1991).

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).

 

RESULTS AND DISCUSSION

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).

Screening of Trichoderma isolates for ammonia production, chitinase activity, HCN production and phosphate solubilisation

During ammonia production test thirteen isolates (AT1, AT2, AT3, AT4, AT5, AT6, AT7, BT1, BT7, BT8, BT10, BT11 and BT12) were found to be positive and seven isolates (BT2, BT3, BT4, BT5, BT6, BT9 and BT13) were found negative for ammonia production (Table 2). These findings are in agreement with the earlier reports (Aarti and Meenu, 2015). Similar findings were reported by Chadha et al. (2015) in Mucor hiemalis, Aspergillusnigerand Fusarium moniliforme. Ammonia production by T. harzianum, KT6, SE6, KT28 and BRT11 has widely been documented (Rawat and Tewari, 2011) as means to offer nitrogen to plant and culminate the pathogens in the vicinity as a result of its toxicity. The production of lytic enzymes by Trichoderma species is known as one of the major mechanisms for biocontrol activity against phytopathogenic fungi (Lorito et al., 1994). Chitinase activity is one of the important beneficial character exhibited by Trichoderma species as chitinases are known to contribute to the biocontrol properties of Trichoderma atroviride (Limon et al., 1999; Woo et al., 1999; Viterbo et al., 2001). This might be due to the reason that chitinases attack directly on the fungal structural components (Sela-Buurlage et al., 1993), while checking chitinase activity twelve isolates (AT2, AT3, AT7, BT1, BT3, BT4, BT7, BT8, BT10, BT11, BT12 and BT13) were found to be positive and eight isolates (AT1, AT4, AT5, AT6, BT2, BT5, BT6 and BT9) were found negative (Table 2). This is in agreement with earlier reports (Sharaf et al., 2012). Lytic enzymes like chitinases and β-1,3-glucanases, proteases and cellulases are potential mechanism associated with the ability of Trichoderma to control phytopathogens (Harighi et al., 2007).

 

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

District Location Isolate name Source
Anantnag Danter AT1 Kitchen garden
Bangidar AT2 Commercial field
Bangidar AT3 Commercial field
Harnag AT4 Commercial field
Harnag AT5 Kitchen garden
Bagi-Wanpoh AT6 Commercial field
Bagi-Wanpoh AT7 Commercial field
Baramulla Delina BT1 Kitchen garden
Delina BT2 Kitchen garden
Delina BT3 Kitchen garden
Arampora BT4 Commercial field
Arampora BT5 Commercial field
Arampora BT6 Commercial field
Arampora BT7 Commercial field
Palhalan BT8 Commercial field
Palhalan BT9 Commercial field
Palhalan BT10 Commercial field
Johama BT11 Kitchen garden
Johama BT12 Kitchen garden
Johama BT13 Kitchen garden

 

 

 

 

 

 

 

 

 

 

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

S. No.
Isolates
Ammonia production
Chitinase activity
HCN production
Phosphate solubilisation
1
AT1
+
2
AT2
+
+
3
AT3
+
+
+
4
AT4
+
5
AT5
+
+
6
AT6
+
7
AT7
+
+
+
8
BT1
+
+
9
BT2
10
BT3
+
11
BT4
+
12
BT5
13
BT6
14
BT7
+
+
15
BT8
+
+
16
BT9
17
BT10
+
+
18
BT11
+
+
19
BT12
+
+
20
BT13
+

 

 

 

 

 

 

Table 3: Cultural and morphological characteristics of Trichoderma isolates

Name of the isolate Colony color Colony edge Common characters among all isolates
AT1 Dark green Wavy Conidial color: Green

Conidiophore branching: Highly branched regular

Mycelial  form Floccose to Arachnoid

Reverse color : Colorless

Colony growth rate (cm/day) : 8-9 in 3 days

Mycelial color : Watery white

AT2 Dark green Wavy
AT3 Dark green Wavy
AT4 Dark green Wavy
AT5 Dark green Wavy
AT6 Dark green Wavy
AT7 Dark green Wavy
BT1 Light  green Wavy
BT2 Light  green Wavy
BT3 Light  green Smooth
BT4 Light  green Smooth
BT5 Light  green Smooth
BT6 Light  green Wavy
BT7 Light  green Wavy
BT8 Light  green Wavy
BT9 Light  green Smooth
BT10 Light  green Smooth
BT11 Light  green Smooth
BT12 Light  green Smooth
BT13 Light  green Smooth

 

 

 

 

 

 

 

 

 

 

Table 4:   Carbohydrate metabolization by various isolates of Trichoderma species

S. No.  

Isolates

Carbohydrate metabolisation
Glucose Fructose Lactose Sucrose Maltose
1 AT1 + + + + +
2 AT2 + + + + +
3 AT3 + + + + +
4 AT4 + + + + +
5 AT5 + + + + +
6 AT6 + + +
7 AT7 + + +
8 BT1 + +
9 BT2 + +
10 BT3 + +
11 BT4 + +
12 BT5 +
13 BT6 +
14 BT7 + +
15 BT8 + +
16 BT9 + +
17 BT10 + +
18 BT11 + +
19 BT12 + +
20 BT13 + +

HCN production is also an important trait found in various soil micro-organisms as it indirectly promotes plant growth by controlling some soil borne diseases (Kremer and Souissi, 2001; Siddiqui et al., 2006), while screening for HCN only three isolates (AT3, AT5 and AT7) were found positive and rest were found negative. This is in agreement with earlier reports (Aarti and Meenu., 2015). The metabolite HCN production by fungal isolates SE6, KT28 and BRT11 has widely been reported as a possible mechanism of disease control (Rawat and Tewari 2011). Similar results were obtained by Ngoma et al. (2013) in case of bacterial isolates. While in case of phosphate solubilisation test, none of the isolate was found to be positive. Trichoderma species are widely known for releasing phosphatases into soil and release the otherwise unavailable phosphate forms from tricalcium phosphate primirily (Saraf et al., 2013). Phosphate solubilizing efficiency of different isolates of T. harzianum was observed by Tallapragada and Gudimi (2011) on Sperber‘s medium with modifications and it was observed that many of the isolates of Trichoderma isolates do not solubilize phosphate.

Production of IAA by various isolates of Trichoderma species

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 IAA however their production amount varied considerably. Maximum IAA was produced by isolate BT9 (6.605 μg mL-1) followed by BT6 (5.278 μg mL-1), BT11 (3.408 μg mL-1) and AT3 (3.317 μg mL-1) while minimum IAA was produced by isolate AT1 (1.538 μg mL-1) (Fig. 1). 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 1.21μg mL-1 and as high as 2.18 μg mL-1and 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.

Fig. 1: Production of IAA by various isolates of Trichoderma species

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: d-mannitol, 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.

ACKNOWLEDGEMENTS

The financial assistance from the University Grants Commission, New Delhi through a research project (No. 43-15/2014 (SR) that supported this study is gratefully acknowledged.

 

 

 

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