Molecular and biochemical characterization of Trichoderma isolates inhibiting a phytopathogenic fungi Aspergillus niger Van Tieghem

Molecular and biochemical characterization of Trichoderma isolates inhibiting a phytopathogenic fungi Aspergillus niger Van Tieghem

Physiological and Molecular Plant Pathology 74 (2010) 274e282 Contents lists available at ScienceDirect Physiological and Molecular Plant Pathology ...

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Physiological and Molecular Plant Pathology 74 (2010) 274e282

Contents lists available at ScienceDirect

Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp

Molecular and biochemical characterization of Trichoderma isolates inhibiting a phytopathogenic fungi Aspergillus niger Van Tieghem H.P. Gajera*, D.N. Vakharia Department of Biotechnology, College of Agriculture, Junagadh Agricultural University, Junagadh-362 001, Gujarat, India

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 7 April 2010

Random Amplified Polymorphic DNA (RAPD) was used to examine the genetic variability among twelve isolates of Trichoderma representing three species and their ability to antagonize Aspergillus niger Van Tieghem causing collar rot in peanut using dual culture assay for correlation among RAPD products and their hardness to A. niger. One hundred and three of the 108 bands, using random decamer fungal primers, were polymorphic with an average frequency of 11.4 bands. The calculated Polymorphism Information Content (PIC) values for RAPD markers ranged from 0.172 to 0.401 and RAPD primer index (RPI) ranged from 0.99 to 6.01. RPI showed that RFu C-5 gave best results of polymorphism among the primer used in the experiment. RAPD analysis showed 10 marker loci for diagnosis of Trichoderma viride 60 and/or Trichoderma harzianum 2J, first two highest inhibitory acting antagonists. A UPGMA dendrogram constructed on the basis of Jaccard’s similarity coefficient using NTSYS 2.2 program which illustrated two distinct clusters of 12 isolates of Trichoderma and A. niger pathogen, and shared only 19% similarity. However, the in vitro highest A. niger growth inhibitory Trichoderma isolates e T. viride 60 (86.2%) and T. harzianum 2J (80.4%) were in same out group and shared 63% similarity. Relationship was found between the polymorphism showed by the Trichoderma isolates and their hardness to A. niger, in terms of in vitro production of cell wall degrading enzymes- chitinase, b-1,3 glucanase and protease, during antagonism. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Trichoderma isolates Aspergillus niger Antagonism Cell wall degrading enzymes Molecular markers

1. Introduction Aspergillus niger Van Tieghem causes a disease called black mold on certain fruits and vegetables such as grapes, onions, and peanut, and is a common contaminant of food. Some strains of A. niger have been reported to produce potentmycotoxins called ochratoxins [1]. The A. niger causing collar rot disease on peanut seedlings was first reported by Jochem [2]. A. niger may cause an average 5 per cent loss in yield but in some parts it may cause as high as 40 per cent losses in Peanut. Collar rot is more serious problem in sandy soil [3,4]. The measures preconized for its control, such as rotation of crops, use of resistant varieties and treatment of seeds and/or soil with fungicides, a lot of times become unsuitable or not effective, mainly due to the genetic variability presented by the pathogen to the hosts range, capacity to survive in the soil and in the seeds, and physiologic flexibility to infect different hosts [5]. The use of antagonistic microorganisms to A. niger has been investigated as an alternative control method, species of Trichoderma, applied as treatment of seeds or soil, have been * Corresponding author. E-mail address: [email protected] (H.P. Gajera). 0885-5765/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2010.04.005

demonstrated to control the pathogen in a variety of cultures in the greenhouse and field studies [6e9]. Knowledge concerning the behavior of these fungi as antagonists is essential for their effective use since they can act against target organisms in several ways. Species of Trichoderma can produce extracellular enzymes and antifungal antibiotics, but they may also be competitors to fungal pathogens, promote plant growth, and induce resistance in plants [8]. The commercial use of Trichoderma must be preceded by precise identification, adequate formulation, and studies about the synergistic effects of their mechanism of bio-control [10]. The ability of Trichoderma to control A. niger varies considerably, and it is possible to improve its biological control efficiency by the selection of isolates with high antagonistic potential and adapted to certain ecological or geographical areas [11]. Fujimori and Okuda [12] examined 74 strains of Trichoderma by RAPD profiles and the results were consistent with the morphological, physiological and ecological data of these strains, what suggests that the technique can aid to eliminate strains duplicated in a program for microbial selection. Using RAPD, Schlick et al. [13] analyzed strains of Trichoderma harzianum and mutants induced by gamma radiation originated from one wild isolate, verifying that with RAPD it was possible to differentiate all the mutants strains for

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at least one primer and concluding that the method was valuable for identification and fast differentiation of strains. Molecular markers are important to develop tools that monitor the genetic and environmental fate of bio-control agents-Trichoderma as more fungal bio-control agents are registered as alternatives to chemical pesticides. Molecular characterization provides an immense source of data that can assist to the scientists in the study of identity, relatedness, diversity and selection of proper candidates for biological control. Our goal was to document the genetic variability of twelve isolates of three Trichoderma species (Trichoderma virens, Trichoderma viride, T. harzianum) using the RAPD technique and their antagonistic potential against A. niger. Our observations provide information about the relationship between antagonistic capacity of Trichoderma isolates, production of cell wall degrading enzymes during antagonism and RAPD markers. 2. Materials and methods 2.1. Collection and isolation of the fungus Twelve isolates of Trichoderma (six of T. harzianum, five of T. viride, one of T. virens) were isolated by serial dilution technique from different soil rhizosphere of Saurashtra region (Gujarat, India) and sent for species identification at IARI, New Delhi. Peanut seedlings which showed typical symptoms of collar rot were cut into small bits with the help of sterilized blade and the pure culture of pathogen (A. niger) was made by hyphal tip isolation method [14] on the solidified PDA medium in petri plates. All microbes were maintained throughout the study by periodical transfers on PDA medium under aseptic condition to keep the culture fresh and viable. 2.2. Fungal growth condition and DNA extraction Cultures were maintained on PDA at 28  C  2  C were grown in potato dextrose broth (PDB) for 48 h. Hyphae were collected on filter paper in a Buchner funnel, washed with distilled water, frozen, and were used for DNA extraction. Total genomic DNA was extracted from the acetone dried mycelium by a modified sodium dodisylsulphate (SDS) method [15]. The genomic DNA was stored in 50 ml TE buffer at 20  C for further use. 2.3. Random amplified polymorphic DNA (RAPD) The Polymerase chain reactions (PCR) were carried out in a 200 ml thin walled PCR tubes. The PCR for RAPD were carried out in a 25 ml of reaction mixture as described by Abbasi et al. [16]. A set of 10 RAPD fungal primers was tested across twelve Trichoderma isolates and pathogen A. niger. RAPD primers and PCR related chemicals were obtained from Bangalore genei, India. Amplification reactions were carried out in a total volume of 25 ml containing, 10 PCR buffer with 1.5 mM MgCl2 and 10 mM Tris, 2.25 U Taq polymerase, 0.20 mM each dNTPs, 1 mM primer and 50 ng template DNA. Amplification was carried out on Bio-Rad Thermal-Mycycler with the following programme as described by Shalini et al. [17]. The samples were initially heated to 94  C for 5 min and then subjected to 45 cycles of denaturation at 94  C for 1 min, annealing at 37  C for 1 min and 30 s, and extension at 72  C for 2 min followed by a final extension for 10 min. After the process, samples of 15 ml of the amplification products were assayed by electrophoresis in 1.5% agarose gel containing ethidium bromide, running with TBE buffer. The electrophoresis was carried out at 80 V (constant) for about 100 min using Bio-Rad submerge gel electrophoresis system. The gel was viewed under UV trans-illuminator for visualizing

275

separated bands, and photographed. The low range ruler (100 bp to 3 kb) was used as molecular weight size markers. Amplifications were repeated once more for each RAPD primer and only consistent bands were considered for scoring. The NTSYS. PC (Numerical Taxonomy System Applied Biostatistics, Setauket, New York) system version 2.2 by Exeter Software was used for data analysis [18]. The data (band presence or absence) were introduced in the form of a binary matrix and a pair wise similarity matrix was constructed using the Jaccard’s coefficient. The SIMQUALK programme was used to calculate Jaccard’s similarity coefficient and a graphical phenogram (dendrogram) of the genetic relatedness among the different isolates was produced by means of the unweighted pair group method with arithmetic average (UPGMA) analysis [19]. Size of specific bands of DNA was determined using software Alphaimager 2200 manufactured by Alpha Ease FC, USA. A polymorphic information index (PIC) for RAPD profile was calculated as PIC ¼ 1  p2  q2, where, p is band frequency and q is no band frequency [20]. PIC values were than used to calculate a RAPD primer index (RPI), which was generated by adding up the PIC values of all the markers amplified by the same primer. 2.4. Antagonistic activity The dual culture technique described by Dennis and Webster [21] was used to test the antagonistic ability of Trichoderma against A. niger. The test fungus and twelve Trichoderma isolates were grown on PDA for a week at 28  2  C in incubator. Disk of 5 mm of the target fungus (A. niger) cut from the periphery were transferred to the petri plate with PDA. Trichoderma was also transferred aseptically in the same plate. Each plate received two disks, one with Trichoderma mycelium and another with A. niger, placed at a distance of 7 cm away from each other. The experiment was conducted in five replications for each antagonist. The plates were incubated at 28  2  C temperatures and observed after six days for growth of antagonist and test fungus. Index of antagonism as per cent growth inhibition of A. niger was determined by following the method of Watanabe [22]. 2.5. Extraction of cell wall degrading enzymes from antagonistic petri plates Cell wall degrading enzymes was extracted from petri plates containing both fungal pathogen and Trichoderma along with the test fungus alone as a control (C) at 6 days after inoculation (DAI). For that, 25 ml of 100 mM phosphate buffer (pH-5.5) containing 50 mM sodium chloride was added to each petri plate and transferred mycelia mat to conical flask. For enzyme induction, 1% of either carboxymethyl cellulose (CMC), sodium polypectate, chitin, laminarin or casein was added into culture medium of conical flask and pH adjusted to 5.5 [23,24]. Cultures were then shaken well in orbital shaker at 120 rpm at 28  C for about 6 h [25]. After that, it was transferred to 50 ml centrifuge tubes and centrifuged at 14 000 rpm for 10 min. Supernatant was collected and stored at 20  C until it used for assay of enzymes activities (cellulase, poly galacturonase (PG), chitinase, b-1, 3 glucanase, protease). The method of FolineLowry [26] was used to estimate the protein content in culture supernatant and used for calculating specific activity of the enzyme. 2.6. Enzymes assays Cellulase activity (EC 3.2.1.21) was determined by measuring the amount glucose formed from CMC [27]. Reactions were conducted for 15 min at 55  C [28]. Poly galacturonase (PG) activity (EC 3.2.1.15) was measured at 37  C using sodium polypectate as the substrate

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[28]. The galacturonic acid released was measured by the DNSA method [27]. Chitinase activity (EC 3.2.1.14) was assayed at 50  C [29] monitoring the amount of sugar N-acetylglucosamine from acetylated chitin, measured by DMAB method [30]. b-1, 3 glucanase activity (EC 3.2.1.39) was assayed on the release of glucose from laminarin by DNSA method [27] Reactions were carried out at 37  C for 10 min [31]. Specific activity of cellulase, PG, chitinase and b-1,3 glucanase were expressed as Unit mg1 protein. However, Unit activity was defined as the amount of enzyme necessary to produce one mM of corresponding reducing sugar in 1 min of one ml culture supernatants. Non enzymatic controls were also performed using boiled enzymes and were subtracted from the enzymatic values. Protease activity (EC 3.4.21.4) was measured using casein as substrate as described by Malik and Singh [32]. Blank was treated as zero time incubation. The amount of released total free amino acids was estimated by ninhydrin method [33]. Specific activity of protease was expressed as Unit mg1protein and one unit of protease activity was defined as the amount of protein necessary to produce mg free amino acids in 1 min of one ml culture supernatant. 3. Results 3.1. Primer selection Amplified products were observed when the genomic DNA of microbes (Trichoderma isolates and A. niger) was subjected to RAPD analysis using 10 random decamer primers. These 10 random fungal primers were obtained from Bangalore Genei, India. Initially, 10 primers were examined and of these, one primer (RFu C-8) failed to give any amplified products of DNA. Possibly this may be due to absence of complementary sequence in the genomic DNA. Thus, 9 out of 10 fungal primers were selected for evaluating molecular differences existing in 12 isolates of Trichoderma which belongs to 3 species for inhibiting the growth of fungal pathogen A. niger. However, molecular characterization of pathogen A. niger also carried out to identify genetic variation and diversity, and compared it with various isolates of Trichoderma. The accession number of each primer is given in Table 1. 3.2. DNA polymorphism analysis of the Trichoderma isolates and A. niger A total 108 bands were produced by 9 RAPD primers with an average frequency of 12 bands per primer (Figs. 1 and 2). Total 103 polymorphic bands were evident out of which 66 were polymorphic and shared between at least two individuals, and 37 bands

were polymorphic and unique while 5 bands were monomorphic (Table 1). Four (RFu C-1, RFu C-3, RFu C-4, RFu C-6) of the 9 RAPD primers produced monomorphic profiles. Primer RFu C-6 generated the maximum 19 bands, whereas RFu C-9 generated the lowest with 5 bands. The per cent polymorphism furnished by each primer ranged between 84.6 and 100 (Table 1). The calculated PIC values for RAPD markers were ranged from 0.172 to 0.401 and RAPD primer index (RPI) ranged from 0.99 to 6.01 (Table 1). The lowest PIC and RPI values obtained by RFu C-2 and highest was with RFu C-5. Thus, RPI showed that RFu C-5 (Fig. 1) gave best results among the primer used here. Dendrogram was constructed using the similarity matrix. The similarity coefficient ranged from 0.19 to 0.80 (Fig. 4). The dendrogram obtained indicates that there was a major cluster consisting of 12 isolates of Trichoderma of total 13 microbes, whereas one fungal pathogen (A. niger) were found to be different from the rest of the microbes e Trichoderma. The major cluster A and cluster B consisted of 12 isolates of Trichoderma and 1 pathogen A. niger and shared 19% similarity (Fig. 4). Total 5 out of 9 primers generated 8 unique marker loci for A. niger. Primer RFu C-2 amplified DNA of pathogen A. niger only and produced two highly dense unique markers (1100, 786 bp) which may be useful to identify pathogen from Trichoderma isolates. However, primer RFu C-1 generated 1675 and 1381 bp RAPD markers from DNA of A. niger. Similarly, RFu C-10 associated with 1023 and 565 bp marker for A. niger. RAPD markers linked to antagonism of 12 isolates of Trichoderma with pathogen A. niger was tabulated in Table 2. 3.3. In vitro growth inhibition of A. niger by Trichoderma isolates Per cent growth inhibition of pathogen (A. niger) was significantly higher in T6 (86.2%) antagonist followed by T8 (80.4%), T3 (74.3%), T2 (71.9%), T1 (60.9%) and T12 (50.6%) at 6 DAI (Table 3). However, other antagonists were recorded below 30% growth inhibition of fungal pathogen. Thus, it was observed that T6 antagonist (i.e., interaction between T. viride 60 and pathogen A. niger) have a better growth of inhibition of test fungus A. niger compared to other bio-control agents (Fig. 3). 3.4. Production of cell wall degrading enzymes during antagonism Twelve Trichoderma isolates, tested for antagonism with fungal pathogen, produced and secreted on induction substantial amounts of various cell wall degrading enzymes in comparison to control T13 (A. niger alone). Maximal specific activity (2.64 U mg1 protein) of cellulase was produced by control T13 (A. niger alone). This activity was 2.43 fold and 1.36 fold higher than those determined for T6 (T. viride 60 X A. niger) and T8 (T. harzianum 2J X A. niger) during 6

Table 1 Polymorphism obtained with different RAPD primers generated from twelve isolates of Trichoderma and pathogen A. niger. Sr. No.

1 2 3 4 5 6 7 8 9 10 Total

Name of primer

Accession no.

RFu C-1 RFu C-2 RFu C-3 RFu C-4 RFu C-5 RFu C-6 RFu C-7 RFu C-8 RFu C-9 RFu C-10

AM AM AM AM AM AM AM AM AM AM

911695 911696 911697 773320 911698 765822 911699 773321 773779 765832

Polymorphic bands S

U

T

9 0 7 5 15 10 6 e 4 10 66

4 7 4 4 0 8 3 e 1 6 37

13 7 11 09 15 18 9 e 05 16 103

Mono morph. bands

Total bands

Polymor. (%)

PIC

RPI value

1 0 2 1 0 1 0 e 0 0 5

14 7 13 10 15 19 9 e 5 16 108

92.8 100.0 84.6 90.0 100.0 94.7 100.0 e 100.0 100.0 95.4

0.189 0.172 0.198 0.251 0.401 0.260 0.260 e 0.265 0.281

2.65 0.99 2.58 2.51 6.01 4.80 2.34 e 1.33 4.50

S ¼ Shared; U ¼ Unique; T ¼ Total Polymorphic Bands; PIC ¼ Polymorphism Information Content; RPI ¼ RAPD Primer Index.

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Fig. 1. RAPD profiles of Trichoderma isolates and A. niger obtained with RFu C-1, RFu C-3, RFu C-4 and RFu C-5 primers. (M ¼ 0.1e3.0 Kbp ruler, 1 ¼ T. viren BAN; 2 ¼ T. viride BAN; 3 ¼ T. viride JND; 4 ¼ T. harzianum BAN; 5 ¼ T. viride 54; 6 ¼ T. viride 60; 7 ¼ T. viride 62; 8 ¼ T. harzianum 2J; 9 ¼ T. harzianum 4J; 9 ¼ T. harzianum 4J; 10 ¼ T. harzianum 5J; 11 ¼ T. harzianum 6J; 12 ¼ T. harzianum JND; 13 ¼ A. niger).

days of antagonism. The highest PG activity was also produced by control T13 (6.73 U mg1 protein). The activities produced by T6 and T8 antagonists were 1.14 fold and 2.34 fold smaller respectively (Table 3). The chitinolytic activity induced in some antagonists’ plates comprises growth of isolates. This activity was 11 fold and 8.72 fold higher in T6 (T. viride 60 X A. niger) and T8 (T. harzianum 2J X A. niger) antagonists respectively than the activity produced by control T13 petri plate (Table 3). The b-1,3 glucanase activity produced by T12, T11, T10 and T13 antagonists were smaller and non-significantly differed. However, the same activity was higher about 3.46 fold and 2.85 fold in T6 and T8 antagonists. It is also expected that antagonists’ fungi synthesize proteases which may act on host cell wall. The antagonists T6 and T8 secreted about 9 fold higher proteolytic activity as recorded on 6 DAI. This activity was found slightly higher in T8 compared to T6 but a difference was not significant. The specific activities of chitinase, b-1,3 glucanase and protease increased in antagonism plates containing T. viride 60 and T. harzianum 2J isolates at 6 DAI which correlated to the higher 86.2% and 80.4% growth inhibition of test fungus A. niger, respectively. Petri plate containing control A. niger (T13) had maximum specific activities of cellulase and PG and lower activities of chitinase and protease. However, b-1,3 glucanase activity was found lowest in T12 (T. harzianum JND  AN) interaction which inhibited

the test fungus by 50.6%. Thus, Chitinase activities played important role in inhibiting the test fungus followed by protease. However, b-1,3 glucanase inhibited the growth of pathogens in synergistic cooperation with chitinase during antagonism. 3.5. RAPD profiles and the antagonism of Trichoderma isolates against A. niger The intraspecific and interspecific genetic variation among the isolates was clear (Fig. 4). Although belonging to the same strain, the isolates presented low similarity, except T. harzianum 5J and T. harzianum 6J. The T. viride 60 and T. harzianum 2J presented stronger antagonistic action than other isolates, in agreement with the in vitro antagonism study (Table 3). These isolates were in the same group in the dendrogram, although they were of different species and shared 63% similarity. The similarity degree of among the isolates that presented better antagonistic acting was around 60%. However, isolates of T. viride and T. harzianum were mixed up in cluster based on species but formed a cluster based on antagonistic action against pathogen A. niger. Thus, dendrogram showed mostly grouping of the Trichoderma isolates by the level of antagonism. The technique of RAPD was efficient in demonstrating the DNA polymorphism of these isolates of Trichoderma showing highest intraspecific genetic variability.

Fig. 2. RAPD profiles of Trichoderma isolates and A. niger obtained with RFu C-6, RFu C-7, RFu C-9 and RFu C-10 primers. (M ¼ 0.1e3.0 Kbp ruler, 1 ¼ T. viren BAN; 2 ¼ T. viride BAN; 3 ¼ T. viride JND; 4 ¼ T. harzianum BAN; 5 ¼ T. viride 54; 6 ¼ T. viride 60; 7 ¼ T. viride 62; 8 ¼ T. harzianum 2J; 9 ¼ T. harzianum 4J; 9 ¼ T. harzianum 4J; 10 ¼ T. harzianum 5J; 11 ¼ T. harzianum 6J; 12 ¼ T. harzianum JND; 13 ¼ A. niger).

Table 2 RAPD markers associated with characterization of twelve isolates of three Trichoderma species and pathogen A. niger. Sr. No.

Name of Primer

Molecular markers (bp) Trichoderma isolates

AN

1

2

3

4

5

6

7

8

9

10

11

12

13

1.

RFu C-1

e

e

e

e

e

712 580

e

712 580

1381 484

e

e

e

2.

RFu C-2

e

e

e

e

e

e

e

e

e

e

e

e

3.

RFu C-3

e

670

305

e

e

e

e

305

e

RFu C-4

e

e

e

e

e

1015 703 305 e

e

4.

e

296

296

e

1340

5. 6.

RFu C-5 RFu C-6

1899 1032

114 e

1899 e

e e

e e

151

1833 1032

151 1032

e 1032

e e

688 1032

688 e

7.

RFu C-7

639

929

e

735

e

1015 703 305 1528 1100 916 e 2002 1789 681 951

1675 1381 484 1100 786 747

e

e

e

185

e

e

8. 9.

RFu C-9 RFu C-10

e e

159 980

e 899

e e

e e

427 879 254

e e

908 780 427 e

e e

e e

e e

e 1023 565

3

5

3

1

e

15

1

10

4

2

3

e 1698 1115 367 5

Total No.

e

10

1. T. virens BAN; 2. T. viride BAN; 3. T. viride JND; 4. T. Harzianum BAN; 5. T. viride 54; 6. T. viride 60; 7. T. viride 62; 8. T. harzianum 2J; 9. T. harzianum 4J; 10. T. harzianum 5J; 11. T. harzianum 6J; 12. T. harzianum JND; 13. A. niger (AN).

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Table 3 Specific activity of cell wall degrading enzymes during in-vitro antagonism of Trichoderma isolates with A. niger at 6 days after inoculation (DAI). No.

Antagonists

% Inhibition of A. niger

Cell wall degrading enzymes (U mg1protein) Cellulase a

1

2

3

4

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 S.Em. C.D. at 5% C.V. %

T. virens BAN X AN T. viride BAN X AN T. viride JND X AN T. harzianum BAN X AN T. viride 54 X AN T. viride 60 X AN T. viride 62 X AN T. harzianum 2J X AN T. harzianum 4J X AN T. harzianum 5J X AN T. harzianum 6J X AN T. harzianum JND X AN Control e A. Niger (AN)

60.9 71.9 74.3 26.1 42.4 86.3 23.7 81.6 32.1 40.2 9.81 50.6 e 0.90 2.60 3.35

1.59 1.47 1.50 2.26 1.96 0.93 1.90 1.35 2.71 2.33 2.64 2.03 3.19 0.05 0.16 4.74

Poly galacturonase 5

a

3.93 3.64 3.60 4.81 5.89 3.14 5.64 2.01 5.31 6.19 5.00 4.62 6.73 0.15 0.43 5.52

Chitinase 6

a

0.72 0.70 0.90 0.35 0.44 1.31 0.30 1.07 0.44 0.41 0.65 0.14 0.11 0.04 0.11 10.90

b-1,3 Glucanase 7

a

1.87 2.15 1.95 1.78 1.72 3.48 1.09 3.01 1.64 0.42 0.63 0.40 0.78 0.10 0.29 10.73

Protease 8a 3.21 3.68 3.43 2.63 2.75 5.24 1.57 5.63 3.04 2.50 4.27 3.56 0.55 0.11 0.33 6.07

a U mg1protein; 4. U ¼ mM glucose min1 ml1; 5. U ¼ mM galacturonic acid min1 ml1; 6 . U ¼ mM N acetylglucosamine min1 ml1; 7. U ¼ mM glucose min1 ml1; 8. U ¼ mg free amino acids min1 ml1.

The highest (86.2%) fungal growth inhibition (A. niger) was achieved by T. viride 60 during in vitro antagonism followed by T. harzianum 2J (80.4%). RAPD markers linked to this trait were noticed with some fungal primers. Primer RFu C-4 gave three amplified products (1528, 1100 and 916 bp) for T. viride 60 which were not yielded by any other Trichoderma Isolates. Similarly, RFu C-6 detected 2002, 1789 and 681 bp markers associated with T. viride 60. However, T. viride 60 and T. harzianum 2J (first two higher inhibitory acting antagonists) generated common RAPD markers (Table 2). These were 712 and 580 bp by primer RFu C-1, and 1015 and 703 bp by RFu C-3. Interestingly, these higher inhibitory acting antagonists also elevated production of cell wall degrading enzymes like chitinase, b-1,3 glucanase and protease in the culture medium of antagonism. So, the markers loci for T. viride 60 and T. harzianum 2J may have relationship with the production of cell wall degrading enzymes (chitinase, b-1,3 glucanase and protease). 4. Discussion Antagonism of 12 isolates of 3 Trichoderma species (T. virens, T. viride and T. harzianum) with A. niger Van Tieghem, causing collar rot in peanut, during in vitro interaction indicated maximum (86.2%) growth inhibition of test pathogen with T. viride 60 followed by T. harzianum 2J (80.4%) at 6 DAI (Table 3, Fig. 3). The antagonistic effect of Trichoderma spp. and Pseudomonas fluorescence against isolates of Fusarium oxysporum f. sp. carthami that causes wilt disease in sunflower was studied by Prameala et al. [34]. Among three antagonists tested, T. viride was found to be more effective than T. harzianum and P. fluorescens which confirm the present experimental results that T. viride was the best antagonist than T. harzianum and T. virens. Seventy Trichoderma isolates collected from different regions of Morocco were tested for their capacity to inhibit in vitro mycelial growth of Sclerotium rolfsii [35]. Four of these isolates (Nz, Kb2, Kb3 and Kf1) showed good antagonistic activity against S. rolfsii and were also highly competitive in natural soil. These isolates would therefore be candidates for development in biological control. However, Shalini and Kotasthane [36] screened seventeen Trichoderma strains against Rhizoctonia solani in vitro. All strains including T. harzianum, T. viride and Trichoderma aureoviride were more or less inhibited the growth of R. solani. It was observed that when T. viride 60 (T6) interact with A. niger and in vitro antagonism revealed the highest production of cell wall

degrading enzymes- chitinase and b-1,3-glucanase (Table 3), in addition to protease followed by T. harzianum 2J and other subsequent antagonists of various isolates of Trichoderma. However, levels of cellulase and PG were found to be the lowest in T6 antagonist and it was recorded highest in control e T1 (A. niger). Pectinase and cellulase had broken down pectin and cellulose, the two major polymers that maintain the firmness and structure of host cell walls. Production of cellulase and PG determined the pathogenicity of necrotrophic pathogen [37]. There are several mechanisms involved in Trichoderma antagonism namely antibiosis whereby the antagonic fungus shows production of antibiotics, competition for nutrients. In case of mycoparasitism, Trichoderma directly attacks the plant pathogen by excreting lytic enzymes such as chitinases, b-1,3 glucanases and proteases [38]. Because of the skeleton of pathogenic fungi cell walls contains chitin, glucan and proteins, enzymes that hydrolyze these components have to be present in a successful antagonist in order to play a significant role in cell wall lysis of the pathogen [39,40]. In present study, percent growth inhibition of test fungus and cell wall degrading enzymes e chitinases, b-1,3-glucanase and protease in the culture medium of antagonist treatment established a relationship to inhibit growth of fungal pathogen by increasing the levels of these lytic enzymes (Table 3, Fig. 3). Most of the bio-controlagents are known to produce chitinase and b-1,3-glucanases enzymes which could degrade the cell wall leading to the lysis of hyphae of the pathogen [41]. The pathogen cell wall and chitin induce nag1 gene, but it is only triggered when there is contact with the pathogen [40,42,43]. Chit36 inhibited the Botrytis cinerea spore germination and the growth of both S. rolfsii and F. oxysporum [44]. Other genes homologous to chit36 have been cloned from T. harzianum TM, Trichoderma atroviride P1 and Trichoderma asperellum Te203 [44]. Present findings showed higher specific activity of enzymeschitinases, b-1,3 glucanase and protease during antagonism of some T. isolates with fungal plant pathogen. Activity of these enzymes varied with various isolates of Trichoderma species. This may be due to the expression of certain gene in Trichoderma spp. during antagonism as Chit33 is expressed only during the contact phase and not before overgrowing R. solani [45]. However, chit36Y does not need the direct contact of the pathogen to be expressed. Chit33, chit42 and chit36 have been over expressed in Trichoderma

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Fig. 3. Antagonism between Trichoderma isolates and A. niger at 6 DAI (Antagonists petri dish (T1 to T12) have Trichoderma isolates at the top and pathogen A. niger at the bottom). (T1 ¼ T. viren BAN  AN; T2 ¼ T. viride BAN  AN; T3 ¼ T. viride JND  AN; T4 ¼ T. harzianum BAN  AN; T5 ¼ T. viride 54; T6 ¼ T. viride 60  AN; T7 ¼ T. viride 62  AN; T8 ¼ T. harzianum 2J  AN; T9 ¼ T. harzianum 4J  AN; T10 ¼ T. harzianum 5J  AN; T11 ¼ T. harzianum 6J  AN; T12 ¼ T. harzianum JND  AN; T13 ¼ Control-A. niger(AN)).

spp. in order to test the role of these chitinases in mycoparasitism, and the 42-kDa chitinase is believed to be a key enzyme [43]. Another enzymatic system that is involved in cell wall degradation by an antagonistic organism is b-glucan degrading enzymes. The production of b-1,3 glucanase was reported as an important enzymatic activity in bio-control microorganisms because b-1,3 glucan is a structural component of fungal cell walls. b-1,3 glucanase inhibited spore germination or the growth of pathogens in synergistic cooperation with chitinases [46,47]. Many b-1,3-glucanases have been isolated, but only a few genes have been cloned, e.g. bgn13.1 [46].

Transformants overexpressing bgn13.1 have been reported to inhibit the growth of B. cinerea, R. solani and Phytophthora citrophthora. Our study also showed some molecular markers from Trichoderma isolates liked to higher antagonist activity against A. niger and production of cell wall degrading enzymes (chitinases, b-1,3 glucanase and protease) during antagonism. In addition to chitin and glucan, filamentous fungi cell wall contains proteins. Some antagonists like T. viride 60 and T. harzianum 2J secreted about 9 fold higher proteolytic activity during antagonism compared to control as recorded on 6 DAI. The production of

H.P. Gajera, D.N. Vakharia / Physiological and Molecular Plant Pathology 74 (2010) 274e282 T.virenB T.harziaB T.viride54 T.virideL J T.virideB T.harzia5J T.harzia6J

A

T.viride60 T.harzia2J T.harziaL J T.viride62 T.harzia4J A.niger 0.19

0.34

0.49 Jaccard's Coefficient

0.65

0.80

B

Fig. 4. UPGMA dendrogram depicting the genetic relationships among the twelve Trichoderma isolates and pathogen A. niger based on the RAPD data.

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and/or T. harzianum 2J viz., RFu C-4 (1528, 1100 and 916 bp), RFu C-6 (2002, 1789, 681 bp), RFu C-1 (712, 580 bp), RFu C-3 (1015, 703 bp). Primer RFu C-2 amplified DNA of pathogen A. niger only and produced 1100, 786 bp markers which may be useful to identify pathogen from Trichoderma isolates. Among the 12 isolates of Trichoderma, T. viride 60 was the best agent to inhibit the growth of fungal A. niger on PDA media. Further, T. viride 60 isolate was tested in pot culture study. Five varieties of peanut seeds (J-11, GG-2, GAUG-10, GG-13 and GG-20) treated with T. viride 60 (microbial load 1.83  106 cfu/g talc powder) and shown in A. niger infested potting mixture (microbial load 1.5  107 cfu/g soil) were resulted to reduce the collar rot incidence of peanut by about 58% under sick soil condition as recorded on 15 days after sowing. Thus, T. viride 60 might have a significant role in the control of collar rot disease by reducing the virulence of A. niger in the peanut rhizosphere. Appendix. Supplementary data

proteases may play a role in antagonism [48]. The role of proteases in mycoparasitism has been reinforced with the isolation of new protease-overproducing strains of T. harzianum [49]. Proteases involved in the degradation of heterologous proteins have been characterized by Delgado-Jarana et al. [50]. Marco et al. [24] demonstrated that antagonistic fungi Trichoderma synthesized proteases which may act on the host cell wall of pathogens in vitro. Identification of Trichoderma isolates based on morphological data, which used as a taxonomic tool have been confusing. Therefore, re-identification of isolates of Thichoderma using molecular tools (RAPD) was important [51]. In present study, the highest growth inhibition of pathogen A. niger was 86.2% and 80.4% by T. viride 60 and T. harzianum 2J respectively during in vitro study, which were also in same cluster and shared 63% similarity (Fig. 4). However, isolate specific markers for various Trichoderma were generated with specific primers (Table 2). The results showed that RAPD can be used for evaluating the molecular variation existing within a group of genotypes (intraspecific genetic variability). Several workers have used RAPD markers to detect genetic variations among the various Trichoderma isolates [16,52,53]. Similar to our study, Goes et al. [54] also used RAPD technique to examine the genetic variability among 14 isolates of Trichoderma and their ability to antagonize R. solani using a dual culture assay for correlation among RAPD products and their hardness to R. solani. They found that belonging to the same species or strains, the isolates presented low similarity around 55% which support present findings. Shalini et al. [17] carried out characterization of 17 bio-control strains identified as Trichoderma, and from R. solani using RAPD. They found 85 out of 102 bands polymorphic using 17 random primers. Fourteen strains of Trichoderma were studied by Santos [55], using molecular markers of RAPD among other techniques, and it was possible to verify the natural genetic variability and to divide the strains in similarity groups, as well as differentiating the original strains of different regions. Zimand et al. [56] used RAPD markers to distinguish strains of Trichoderma. Ten of the strains identified as T. harzianum exhibited similarities, and it was possible to distinguish the isolate T-39, used commercially as bio-control agent of B. cinerea. For efficient selection of species of Trichoderma with taxonomic finalities, this may support our results. The results reported in this work clearly indicated that the RAPD markers linked to antagonistic action are observed with some fungal primers. T. viride 60 and T. harzianum 2J (first two higher inhibitory acting antagonists), elevated production of cell wall degrading enzymes - chitinase, b-1,3 glucanase and protease in culture medium during in vitro antagonism with test fungus A. niger, generated RAPD markers. This showed 10 marker loci for diagnosis of T. viride 60

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