Molecular evolution and phylogenetic analysis of biocontrol genes acquired from SCoT polymorphism of mycoparasitic Trichoderma koningii inhibiting phytopathogen Rhizoctonia solani Kuhn

Infection, Genetics and Evolution 45 (2016) 383–392

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Research paper

Molecular evolution and phylogenetic analysis of biocontrol genes acquired from SCoT polymorphism of mycoparasitic Trichoderma koningii inhibiting phytopathogen Rhizoctonia solani Kuhn H.P. Gajera ⁎, Darshna G. Hirpara, Zinkal A. Katakpara, S.V. Patel, B.A. Golakiya Department of Biotechnology, College of Agriculture, Junagadh Agricultural University, Junagadh-362 001, India

a r t i c l e

i n f o

Article history: Received 24 December 2015 Received in revised form 12 September 2016 Accepted 30 September 2016 Available online 05 October 2016 Keywords: Rhizoctonia solani Trichoderma Biocontrol mechanism SCoT polymorphism Biocontrol genes Functional annotations

a b s t r a c t The biocontrol agent Trichoderma (T. harzianum, T. viride, T. virens, T. hamantum, T. koningii, T. pseudokoningii and Trichoderma species) inhibited variably (15.32 - 88.12%) the in vitro growth of Rhizoctonia solani causing root rot in cotton. The T. koningii MTCC 796 evidenced highest (88.12%) growth inhibition of test pathogen followed by T. viride NBAII Tv23 (85.34%). Scanning electron microscopic study confirmed mycoparasitism for MTCC 796 and Tv23 which were capable of completely overgrowing on R. solani by degrading mycelia, coiling around the hyphae with hook-like structures. The antagonists T. harzianum NBAII Th1 and, T. virens NBAII Tvs12 exhibited strong antibiosis and formed 2-4 mm zone of inhibition for 70.28% and 46.62%, respectively growth inhibition of test pathogen. Mycoparasitism is a strong mode of action for biocontrol activity compared with antibiosis. The antagonists Trichoderma strains were performed for start codon targeted (SCoT) polymorphism to acquire biocontrol genes from potent antagonist. The six unique SCoT fragments amplified by genomic DNA of best mycoparasitic antagonist MTCC 796 strain are subjected to DNA sequencing resulted to confirm two functional sequences for activity related to biocontrol genes. The phylogenetic and molecular evolution of functional 824 bp of SCoT-3(920) and 776 bp of SCoT-6(806) fragments signify sequence homology with biocontrol genes endochitinase (partial cds of 203 amino acids) and novel hmgR genes (partial cds of 239 amino acids), respectively and the same were annotated and deposited in NCBI GenBank database. The hmgR gene is liable to be express hmg - CoA reductase which is a key enzyme for regulation of terpene biosynthesis and mycoparasitic strains produced triterpenes during antagonism to inhibit growth of fungal pathogen as evidenced with GC-MS profile. The biocontrol genes are found in best antagonist T. koningii MTCC 796 for mycoparasitic activity to restrain the growth of test pathogen R. solani. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Cotton (Gossypium spp.) is the leading fibre crop worldwide and is grown commercially in the temperate and tropical regions between 40° north and 30° south of the equator. The seed and soil borne fungi like Rhizoctonia spp, Pythium spp., Phoma exigua (Ascochyta), and Fusarium spp. are the vital fungi to cause seedling diseases viz., seed rot, root rot, pre-emergence damping off, and post-emergence damping off which encompassed huge losses in cotton yield. The root rot caused by Rhizoctonia solani Kuhn or Rhizoctonia bataticola (Taub) Butler is one of the most seroius diseases of cotton where it grows under warm climate and irregular rainfed conditions (Monga and Raj, 1994). The hirsutum and arboreum cotton species are affected with root rot disease. The disease is caused by seed borne pathogen and survive in soil for a long periods with host debris. There is a hard to control the disease

⁎ Corresponding author. E-mail address: [email protected] (H.P. Gajera).

http://dx.doi.org/10.1016/j.meegid.2016.09.026 1567-1348/© 2016 Elsevier B.V. All rights reserved.

by chemical means. Biologic control of root rot caused by Rhizoctonia is eco friendly and viable aspects to diminish the disease incidence. Trichoderma is one of the most important filamentous fungi common in soil and root ecosystems and used as an effective biocontrol agents for soil borne fungal plant pathogens. Trichoderma have ability to provide systemic resistance to plants by inducing defense related enzymes against phytopathogens (Gajera et al., 2015; Harman et al., 2004). The biocontrol exercised by Trichoderma can occur by means of several antagonistic mechanisms such as antibiosis, nutrient competition, suppression, mycoparasitism, induced resistance, hypovirulence and predation (Ruiz-Herrera, 1992; Schirmbock et al., 1994). Mycoparasitism is a strong antagonistic mechanism in which biocontrol agent Trichoderma first recognized the host pathogen followed by coiling surround host hyphae and then degraded the cell wall of pathogen by producing extracellular cell wall degrading enzymes (Kubicek et al., 2001; Viterbo et al., 2002). Major advances were made in chitinase and β -1,3-glucanase expression by various Trichoderma spp. but the nature of the inducers and repressors which affect the biocontrol enzymes activities are yet remain to distinguish (Kubicek et al., 2001;

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Benitez et al., 2004). Despite many studies, the induction of chitinolytic and glucanolytic genes as biocontrol mechanism of Trichoderma and their regulation is still a subject of speculation. The genes for biocontrol activity activated synergistically during mycoparasitism. Thus, understanding the induction process from these genes is necessary in order to select the most efficient Trichoderma biocontroller. The numerous fungal bio-control agents are studied as substitute to chemical pesticides and molecular markers proposed important tools for monitoring the genetic and environmental fate of bio-control agents including Trichoderma. Molecular characterization of antagonists Trichoderma distinguished genetic identity, relatedness, diversity of potent antagonist and thereby selection of proper candidates for biological control. The random amplified polymorphic DNA (RAPD) technique has been applied to Trichoderma strains in previous study for phylogenetic variations among Trichoderma variably inhibits the growth Aspergillus niger causing collar rot in groundnut and also proven to be useful for strain specification identification of best antagonists (Abbasi et al., 1999; Gajera and Vakharia, 2010). The start codon targeted (SCoT) polymorphism is a simple and novel DNA marker technique reported by Collard and Mackill (2009). In this technique, primers are designed exclusive of needing the genomic sequence information (Joshi et al., 1997; Sawant et al., 1999). The 18mer single primers are designed on the basis of conserved region surrounding the translation initiation codon, ATG and used for polymerase chain reaction (PCR) against genomic template DNA. The PCR products are resolve using standard agarose gel electrophoresis or capillary electrophoresis genetic analyzers. The SCoT markers amplified gene targeted fragments through which new information corresponds to biological traits can be generated while random DNA markers like RAPD, AFLP and ISSR could not be distinguished the same. The SCoT polymorphism was confirmed with rice (Collard and Mackill, 2009), peanut (Xiong et al., 2009), mango (Gajera et al., 2014; Luo et al., 2010, 2011, 2012) and Jatropha (Mulpuri et al., 2013) for molecular diversity analysis, understanding the genetic relationships for germplasm management and cultivar identification. Present study reported exceptional first time for trait specific functional molecular characterization of biocontrol gene from mycoparasitic Trichoderma using SCoT polymorphism. All Trichoderma strains could not work equally against specific soil borne disease as Trichoderma antagonists have different mechanisms of pathogen recognition. In the backdrop of this scenario, the aim of the present study was (1) to evaluate the antagonistic potentials of Trichoderma isolates as a biocontrol agent against pathogen R. solani causing root rot in cotton, (2) to observe mechanism of antagonist for growth inhibition of R. solani using scanning electron micrographs, and (3) to appraise functional molecular analysis of antagonist Trichoderma using SCoT markers and the unique bands found with best mycoparasitic antagonist were eluted, cloned, sequenced and utilized for functional annotation; and phylogenetic and molecular evolution of biocontrol genes for specific activity. 2. Materials and methods 2.1. Sources of Trichoderma isolates Trichoderma isolates viz., T1. T. harzianum NBAII Th1; T2. T. harzianum NRRL 13879; T3. T. harzianum NRRL 20565; T4. T.harzianum Local; T5. T. viride NBAII Tv23; T6. T. viride NRRL 6418; T7. T. virens NBAII Tvs12; T8. T. hamantum NBAII Tha 1; T9. T. koningii MTCC 796; T10. T. pseudokoningii MTCC 2048; T11. T. species NRRL 5242 were procured either from Indian Type Culture Collection (ITCC, indicating accession number with NBAII), New Delhi; Microbial Type Culture Collection (MTCC), Chandigarh; or Agricultural Research Service Culture Collection (NRRL), Illinois, USA (Table S1). The Trichoderma strains are available at their respective culture collection centre for future uses. One local isolate of T. harzianum was collected from culture collection of Department of Plant Pathology, Junagadh Agricultural University, Junagadh.

2.2. Isolation and characterization of phytopathogenic fungi The cotton plants showing typical symptoms of root rot were collected from field of Cotton Research Station of Junagadh Agricultural University. The infected seedlings may reveal dark lesions on the stem and root. Often the taproot is destroyed, and only shallow-growing lateral roots remain to support the plant. The sore shin phase of seedling disease is characterized by reddish brown, sunken lesions at or below ground level. These lesions enlarge, girdle the stem and cause it to shrivel. The fresh collected seedling diseased plants of cotton showing stem blight and shredded bark on the roots were thoroughly washed under running tap water to remove surface debrish and contaminants. The cotton root tissues infected with pathogen were cut out from the leading edge of lesion, and subjected to dip in 1% sodium hypochlorite for five minutes followed by washing with sterile distilled water and dried on sterile filter paper. The pathogen was isolated by hyphal tip method (Sinclair and Dhingra, 1985) from dry small pieces of root plated onto PDA media and incubated at 28°C. The cultures were transferred periodically on other PDA plate and repeated until pure culture obtained; and then maintained on PDA by storing it under refrigeration (4°C). The pathogenic fungal isolates was identified by Lactophenol cotton blue staining and plating on sabouraud dextrose agar media. Morphological observations of isolated pathogen were found to be dark brown mycelia / Sclerotia with irregular in shape and size and have a dimension range of 3.80 to 1.58 μM (Fig. S1). The pathogen was identified as Rhizoctonia solani Kuhn (JAU-Cotton) based on micro and macro-morphological characteristics and compared with features of other closely related species like R. bataticola (black mycelia with irregular in shape and a dimension range of 1.05 to 2.37 μM, pycnidia stage to cause disease known as Macrophomina phaseolina, sclerotia usually occur at a depth of 15-30 cm, thermal death point at 68°C) (Monga and Raj, 1994). This mono hypha-tip isolate was also confirmed for pathogenicity (virulence) test on cotton causing root rot and the pathogen selected as R. solani was used for antagonism study. 2.3. In vitro antagonism of Trichoderma against R. solani The antagonism between antagonistic fungi and pathogenic fungi were examined by dual culture method as (Dennis and Webster, 1971). A 5 mm diameter mycelial disc from the margin of the Trichoderma one week-old culture and the pathogen R. solani were placed on the opposite of the plate at equal distance from the periphery. The completely randomized block design with three petri dishes for each antagonist was used an experimental design. The sole pathogen R. solani inoculated disc without antagonists Trichoderma is used as a control plates. The plates were placed into incubator at 28 ± 2°C and observed after 10 days for growth of test fungus. The per cent growth inhibition of R. solani as index of antagonism was determined by following the method of Watanabe (1984). Growth inhibition of test pathogen by Trichoderma antagonist were carried out in three independent replications and data were interpreted statistically using completely randomized block design (Fisher and Yates, 1948). 2.4. Scanning electron micrographs of best antagonists inhibiting R. solani The best antagonists showing mycoparasitism (T9: T. koningii MTCC 796, T5: T. viride NBAII Tv23) or forming an inhibition zone with test pathogen (T1: T. harzianum NBAII Th1 and T7: T. virens NBAII Tvs12) were targeted for scanning electron micrographs. Mycelial samples from the interaction region (Trichoderma–pathogen hyphae) in dualculture tests were removed and fixed in 3% glutaraldehyde (Sigma, USA) in 0.1M phosphtae buffer (pH 7.0) (Elad et al., 1983). The slides were kept for refrigeration (12h) followed by dehydration with acetone. The dehydrated samples were coated with gold palladium using Polaron E500 sputter coater (Polaron Equipment, England) and observed

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under scanning electron microscope (SEM) (EVO-18 CARL ZEISS) for the presence of coiling structures of antagonists and wall disintegration of test pathogen. 2.5. DNA extractions and PCR-SCoT amplifications Total genomic DNA was extracted using CTAB method from growing mycelia (7 days) of Trichoderma isolates and pathogen R. solani inoculated with potato dextrose broth (Narayanasamy and Saravana, 2009). The purity of DNA was checked in 0.8% agarose gel electrophoresis and found single intact band. The quantity of genomic DNA isolated from Trichoderma strains and R. solani JAU-cotton was measured in piccodrop (Picodrop PET01) and 20 ng/μl DNA concentration was maintained for PCR-SCoT amplification. Primers for SCoT marker analysis were designed from the conserved region surrounding the translation initiation codon, ATG (Table 1) (Joshi et al., 1997; Sawant et al., 1999). It is a simple, reliable and novel gene targeted marker technique where amplification was derived from the translation initiation start codon. Total 80 SCoT primers were designed and synthesized from Merck Bioscience, India which were 18-mer and ranged in GC content between 50% and 72% (Collard and Mackill, 2009; Luo et al., 2010). Initially, PCR amplifications were carried out with two Trichoderma isolates (T1 and T9) using the 80 primers individually. The primers which gave apparent and polymorphic

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bands were used for further amplifications with all antagonists. As a result, 38 primers were selected for SCoT analysis of Trichoderma isolates and test pathogen (Table 1). The SCoT-PCR was carried out according to Luo et al. (2011) with some modifications. Each 20 μL amplification reaction consisted of 10 mM Tris-HCl (pH 8.0) containing 1.75 mM MgCl2, 2.5 mM dNTP, 0.25 mM primer, 1 unit of Taq DNA polymerase and about 25 ng template DNA. All chemicals were molecular grade and purchased from Merck Bioscience, India. PCR reaction was performed in a VeritiTm Thermal Cycler (Applied Biosystems) as follows: an initial denaturation step for 3 min at 94°C followed by forty cycles (94°C/45 s, 50°C/1 min, 72°C/2 min) and then a final extension cycle at 72°C for 5 min. The PCR amplifications were processed and amplified products were resolved on capillary electrophoresis genetic analyzers (30110 QIAxcel advanced, Qiagen) as per manufacturer protocol and guidelines. The unique bands were recorded using QIAxcel Screen Gel software (1.3.0) with resolution of five base pair differences. Amplifications were repeated twice and only consistent bands were considered for scoring. The unique DNA fragments having specific size was determined using software Alphaimager 2200, Alpha Ease FC, USA. The polymorphic information content (PIC) value of SCoT profile was calculated using equation PIC = 1 - p2 - q2, where, p is band frequency and q is no band frequency (Ghislain et al., 1999). The PIC values were used to calculate a primer index, which

Table 1 Polymorphism obtained with SCoT primers generated from Trichoderma isolates and pathogen R. solani

Name of Primers SCoT 1 SCoT 2 SCoT 3 SCoT 4 SCoT 5 SCoT 6 SCoT 7 SCoT13 SCoT14 SCoT15 SCoT21 SCoT22 SCoT23 SCoT24 SCoT26 SCoT27 SCoT28 SCoT29 SCoT35 SCoT36 SCoT37 SCoT38 SCoT39 SCoT40 SCoT41 SCoT44 SCoT45 SCoT47 SCoT51 SCoT52 SCoT53 SCoT55 SCoT56 SCoT63 SCoT65 SCoT70 SCoT72 SCoT73 Total Average

Sequences (5'....3')

Allele/ Band size (bp)

CAACAATGGCTACCACCA CAACAATGGCTACCACCC CAACAATGGCTACCACCG CAACAATGGCTACCACCT CAACAATGGCTACCACGA CAACAATGGCTACCACGC CAACAATGGCTACCACGG ACGACATGGCGACCATCG ACGACATGGCGACCACGC ACGACATGGCGACCGCGA ACGACATGGCGACCCACA AACCATGGCTACCACCAC CACCATGGCTACCACCAG CACCATGGCTACCACCAT ACCATGGCTACCACCGTC ACCATGGCTACCACCGTG CCATGGCTACCACCGCCA CCATGGCTACCACCGGCC CATGGCTACCACCGGCCC GCAACAATGGCTACCACC CAATGGCTACCACTAGCC CAATGGCTACCACTAACG CAATGGCTACCACTAGCG CAATGGCTACCACTACAG CAATGGCTACCACTGACA CAATGGCTACCATTAGCC ACAATGGCTACCACTGAC ACAATGGCTACCACTGCC ACAATGGCTACCACTGTC ACAATGGCTACCACTGCA ACAATGGCTACCACCGAC ACAATGGCTACCACTACC ACAATGGCTACCACTAGC ACCATGGCTACCACGGGC ACCATGGCTACCACGGCA ACCATGGCTACCAGCGCG CCATGGCTACCACCGCCC CCATGGCTACCACCGGCT

500-2000 248-3000 420-3200 450-2200 500-3000 175-3000 550-3000 100-2000 130-1500 210-1980 230-2000 250-2000 365-2000 260-2990 465-2000 500-3000 430-2500 220-2000 350-2000 700-2000 450-1992 350-3545 200-3000 470-2500 550-2500 1120 750-2000 410-3000 1000-3000 510-3000 260-2230 495-2000 480-2000 400-1900 230-2000 230-1995 240-2000 150-3330

Polymorphic bands S

U

Polymorphic (%)

5 7 9 5 7 7 8 7 15 17 9 11 10 9 12 6 12 11 8 4 3 4 9 4 3 0 3 5 2 4 5 7 6 8 9 14 14 10 289 7.61

4 1 3 3 2 4 1 6 3 6 3 0 1 6 1 4 0 1 0 1 1 2 2 7 2 1 1 5 4 3 4 1 3 2 3 1 2 4 98 2.58

100 100 100 100 100 100 100 100 100 100 100 84.6 100 100 100 100 100 100 100 100 80 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99.33

Total Bands 9 8 12 8 9 11 9 13 18 23 12 13 11 15 13 10 12 12 8 5 5 6 11 11 5 1 4 10 6 7 9 8 9 10 12 15 16 14 390 10.26

S = Shared; U = Unique; T = Total Polymorphic Bands; PIC = Polymorphism information content; SPI = (SCoT Primer Index) = Total number of bands X PIC

PIC

SPI

0.85 0.84 0.89 0.81 0.85 0.87 0.87 0.88 0.91 0.93 0.88 0.90 0.89 0.91 0.91 0.89 0.90 0.89 0.84 0.71 0.66 0.75 0.90 0.87 0.73 0.00 0.72 0.85 0.76 0.76 0.84 0.85 0.85 0.86 0.88 0.92 0.93 0.90 0.83

7.68 6.68 10.64 6.48 7.69 8.69 7.85 11.50 16.42 21.45 10.59 9.92 9.78 12.73 11.83 8.90 10.80 10.68 6.71 3.57 2.65 4.50 9.90 9.57 3.65 0.00 2.88 8.50 4.56 5.32 7.56 6.80 7.65 8.60 10.56 13.80 14.88 12.60 8.83

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was generated by adding up the PIC values of all the markers amplified by the same primer. 2.6. PCR cloning and DNA sequencing of unique PCR-SCoT fragments from mycoparasitic antagonist T. koningii MTCC 796 The in vitro antagonism between antagonistic fungi and pathogenic fungi were examined by dual culture method and highest mycoparasitism was observed with antagonist T. koningii MTCC 796. The five SCoT primers (SCoT 3, 6, 21, 24, 51) amplified six unique fragments specific to mycoparasitic strain MTCC 796 were subjected to re-amplifications with PCR-SCoT procedures and resolved in 1.5% agarose gel. The six unique fragments were eluted, cloned, sequenced and blast for annotation and identification of biocontrol related genes. The unique fragments were eluted from the agarose gel with the GenElute Minus EtBr Spin Column kit (Sigma, USA) and ligated into the pCR®2.1-TOPO® vector from TOPO TA Cloning® Kit (Invitrogen, USA) according to the instructions of the manufacturer. The OneShot cells of Escherichia coli TOP10F were transformed with ligated DNA and plated on LB medium containing kanamycin (40 μg/ml), Xgal (50 μg/ml) and IPTG (isopropyl-b-D-thiogalactopyranoside) (125 mM). The mini-M Plasmid DNA extraction system was used to extract recombinant plasmid DNA and sequenced on both strands with M13 forward and reverse universal primers using automatic DNA sequencer (ABI 3130XL system). Removal of vector sequences was done online at national center for biotechnology information (NCBI). Homologies to known sequences were searched in the GenBank database using the basic alignment search tool (BLAST) available online from the NCBI (Altschul et al., 1990). The nucleotide and protein sequences of the fragments matching with biocontrol genes endochitnase and 3-hydroxy-3-methylglutaryl-coenzyme A reductaselike protein (hmgR) genes are deposited in the GenBank database as nucleotide accession No. KF723013.1; KF723014.1 and protein accession No. AHF57043.1; AHF57044.1, respectively. Dual and multiple gene specific alignments for homology search were performed using the ClustalW algorithm and further, phylogenetic tree and molecular evolutionary analysis for nucleotide and protein sequences was constructed using MEGA 6.0 software and maximum composite likely hood method with bootstrap values calculated from 1000 replicate runs in the MEGA software. 3. Results 3.1. Antagonist activity and SEM characterization of Trichoderma inhibiting R. solani The growth inhibition of R. solani during in vitro interaction with biocontrol agents Trichoderma at 10 DAI was found to be significant (Fig. 1). Percent growth inhibition of pathogen was evident highest in T. koningii MTCC 796 (T9) (88.12%) antagonist followed by, T. viride NBAII Tv23 (T5) (85.34%), T. harzianum NBAII Th1 (T1) (70.28%), T. virens NBAII Tvs12 (T7) (46.62%), and lowest examined with T. viride NRRL 6418 (T6) (15.18%) at 10 DAI. The antagonists T. viride (NRRL 6418) (T6), T. virens NBAII Tvs12 (T7) and T. pseudokoningi (MTCC 2048) (T10) were recorded below 25% growth inhibition of test pathogen. The interaction between T. koningii MTCC 796 and pathogen R. solani (T9) have a superior growth inhibition of test pathogen followed by T. viride NBAII Tv23 X R. solani (T5) compared to other Trichoderma antagonists. The interection effect between best antagonists and test pathogen were examined under microscope at 10 DAI (Fig. 2). The T. koningii MTCC 796 (T9) isolate overgrew completely on the pathogen with mycoparasitism as observed in SEM observations. The antagonists T. harzianum NBAII Th1 (T1) and, T. virens NBAII Tvs12 (T7) inhibited 70.28% and 46.62% growth of test pathogen, respectively with formation of about 2-4 mm zone of inhibition (antibiosis mode of action) after 10

Fig. 1. Percent growth inhibition of R. solani strain JAU-cotton during in vitro antagonism with Trichoderma strains at 10 DAI (Bars indicate standard error of mean between three replications) [T1 = T. harzianum (NBAII Th1) X R. solani; T2 = T. harzianum (NRRL 13879) X R. solani; T3 = T. harzianum (NRRL 20565) X R. solani; T = T.harzianum (Local) X R. solani; T5 = T. viride (NBAII Tv23) X R. solani; T6 = T. viride (NRRL 6418) X R. solani; T7 = T. virens (NBAII Tvs12) X R. solani; T8 = T. hamantum (NBAII Tha 1) X R. solani; T9 = T. koningi (MTCC 796) X R. solani; T10 = T. pseudokoningi (MTCC 2048) X R. solani; T11 = T. species (NRRL 5242) X R. solani; T12 = R. solani strain JAU-cotton (ITCC 8160.11) Control].

days in dual culture test. The SEM examinations confirmed the disintegration and degradation of pathogen mycelia without touch and coiling (Fig. 2). 3.2. Molecular characterization of Trichoderma by PCR-SCoT The SCoT polymorphism is a simple and novel DNA marker technique in which 18-mer single primers are designed on the basis of conserved region surrounding the translation initiation codon, ATG and used for PCR against genomic template DNA. The SCoT markers amplified gene targeted fragments through which new information corresponds to biological traits can be generated and hence SCoT is more selective than other random DNA markers like RAPD, AFLP and ISSR. Genomic DNA of eleven Trichoderma and one test pathogen R. solani were subjected to PCR-SCoT analysis. The polymorphic primers performed for PCR-SCoT across all Trichoderma strains and test pathogen. Total 38 SCoT primers amplified to generate 390 bands among eleven isolates Trichoderma and pathogen R. solani (Table 1). The SCoT15 primer produced maximum number of 23 bands of which 17 were shared polymorphic and six were unique polymoprphic (Fig. 3). Among the 387 polymorphic bands across eleven Trichoderma isolates, 289 bands were shared polymorphic within two or more Trichoderma strains, while 98 bands were unique-polymorphic. The average percent polymorphism obtained for SCoT primers were 99.33%. The PIC values for SCoT markers were ranged from 0.00 (SCoT44) to 0.93 (SCoT15, SCoT72) with an average value of 0.83 per primer and SCoT primer index (RPI) differed from 0.00 (SCoT44) to 21.45 (SCoT15) (Table 1). Based on PIC and RPI, the SCoT15 is most informative primer for polymorphism of genomic DNA of Trichoderma strains. Out of 38 SCoT, 35 primers amplified 98 unique bands for identification of eleven Trichoderma strains and test pathogen (Table 1). The genomic DNA of Trichoderma strains amplified maximum 7 unique bands by SCoT40 followed by 6 unique bands produced with SCoT13, SCoT15, SCoT24 markers to distinguish antagonists strains. The antagonists T. harzianum NBAII Th1 (T1) and T. virens NBAII Tvs12 (T7) demonstrated strong antibiosis and they were discriminated by two and seven unique fragments, respectively, generated by different SCoT primers (Table 2). However, best mycoparasitic antagonists - T. koningii MTCC 796 (T9) exhibited six unique fragments by five SCoT primers and T. viride NBAII Tv23 (T5) identified five unique fragments using four SCoT primers. The five SCoT primers (SCoT 3, 6, 21, 24, 51) amplified six unique fragments specific to T. koningii MTCC 796, which

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Antagonisms

SEM images-interaction zone

Antagonisms

387

SEM images-interaction zone R

R

R

T

T1: T. harzianum NBAII Th1 x R. solani

T7: T. virens NBAII Tvs12 x R. solani

R T

T R T

T5: T. viride NBAII Tv23 x R. solani

T T

R

T9: T. koningii MTCC 796 x R. solani

Fig. 2. Antagonism of potentials Trichoderma isolate with test pathogen R. solani at 10 days after inoculations (DAI) and scanning electron micrographs during interactions (R = Hyphae of test pathogen R. solani; T = Hyphae of Trichoderma antagonist). T1 and T7 antagonists used antibiosis as biocontrol mechanism against R. solani and disintegrate and digests the hyphae of pathogen without touch and coiling. However, T5 and T9 antagonists exhibited mycoparasitism and interact around hyphae of R. solani and formed surround a coiling and hock like structure followed by disintigration and disruption of mycelia of pathogen.

elevated highest mycoparasitism and growth inhibition of test pathogen (Fig. 3). 3.3. Molecular evolution, functional annotation and phylogenetic analysis of biocontrol genes The six unique SCoT fragments amplified by genomic DNA of best mycoparasitic antagonist T. koningii MTCC 796 (T9) are subjected to DNA sequencing. The sequences of the six DNA fragments were distinct, with no sequence homology with each other. However, out of six sequences, two functional sequences 824 bp of SCoT-3(920) fragment and 776 bp of SCoT-6(806) fragment signify sequence homology with biocontrol genes and the same were annotated and deposited in NCBI GenBank database (Table S2). However, other four sequences did not match with any other gene sequences in the blast results and hence not utilized for further study. The 824 bp functional sequence from SCoT-3(920) showed sequence homology with endochitinase gene and the same were annotated and deposited in NCBI GenBank database for supporting their specificity with nucleotide accession No. KF723013.1 and protein accession No. AHF57043.1. The test sequence of 824 bp of T. koningii MTCC 796 and deduced amino acid sequence of 203 aa with partial cds join (b1..29,93..191,276..325,394..N824) is depicted in Fig. S2. Gene bank nucleotide searches revealed that amplified fragment had maximum homology with endochitinase gene of various Trichoderma strain. The endochitnase gene sequence based phylogenetic and molecular evolutionary tree showing the relationships between the test chitnolytic sequence of strain MTCC 796 and selected representatives of the chitinase gene (90% similarity) of genus Trichoderma or Hypocrea is given in Fig. 4. A similar amino acid homology study for the 824 bp sequenced fragments was carried out. The translated version of query sequence was compared with other proteins using BLASTx search. The deduced amino acid sequence evident high degree of identity and 96% similarity with other chitinases (Fig. 5). The 203 deduced amino acid sequence of endochitinase gene of mycoparasitic T. koningii MTCC 796 compared with other known sequences and accordingly phylogenetic and molecular evolutionary tree constructed which were found comparable with deduce amino acids sequence of other chitinase gene from Trichoderma isolates (Fig. 5).

The nucleotide sequence of SCoT-6(806) fragment from MTCC 796 strain showed 776 bp functional sequence and blast results exhibited sequence homology with hmgR gene. The same were annotated and deposited in NCBI GenBank database for supporting their specificity with nucleotide accession No. KF723014.1 and protein accession No. AHF57044.1 (Table S2). The test sequence of 776 bp of T. koningii MTCC 796 and deduced amino acid sequence of 239 aa with partial cds join (b 1..130,161..472,500..N776) are depicted in Fig. S3. Gene bank nucleotide searches revealed that amplified fragment had maximum homology with hmgR gene of various Trichoderma strain. The endochitnase gene sequence based phylogenetic and molecular evolutionary tree was constructed and it demonstrated the relationship between the test hmgR gene sequence of strain MTCC 796 and selected representatives of the hmgR gene (80% similarity) of genus Trichoderma or Hypocrea (Fig. 6). It is evident from phylogenetic analysis of hmgR gene that the isolate MTCC 796 represents a partial cds of hmgR. A similar amino acid homology study for the 776 bp sequenced fragments was carried out. The translated version of query sequence was compared with other proteins using BLASTx search. The deduced amino acid sequence evident high degree of identity and 80% similarity with other hmgR like protein (Fig. 7). Comparison of gene deduce amino acid sequence obtained from gene sequence and molecular evolutionary analysis of protein hmgR gene showed that mycoparasitic T. koningii MTCC 796 generated 239 deduced amino acids sequences and they were found comparable with deduce amino acids sequences of other hmgR gene from Trichoderma isolates (Fig. 7). The best antagonist T. koningii MTCC 796 exhibited highest growth inhibition of test pathogen R. solani with mycoparasitism and SCoT polymorphism of the same strain produced six unique fragments of which two are identified and characterized as biocontrol - endochitinse and hmgR genes. 4. Discussion Trichoderma are widely used in agriculture as biocontrol agents because of their ability to reduce the incidence of plant diseases caused by soil borne pathogens. The effect of Trichoderma on soil borne pathogens is higher as compared to chemicals and it persists in soil for longer period after application. The Trichoderma uses antibiosis, nutrient

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SCoT 1

SCoT 3

SCoT 6

SCoT 15

SCoT 21

SCoT 24

SCoT 45

SCoT 51

Fig. 3. SCoT profile of antagonist Trichoderma strains and test pathogen using different start codon targeted primers [Alignment Marker= 15bp-10kb; M= 250bp-8kb size marker; 1= T. harzianum (NBAII Th1); 2= T. harzianum (NRRL 13879); 3= T. harzianum (NRRL 20565); 4= T. harzianum (Local); 5= T. viride (NBAII Tv23); 6= T. viride (NRRL 6418); 7= T. virens (NBAII Tvs12); 8= T. hamantum (NBAII Tha 1); 9= T. koningii (MTCC 796); 10= T. pseudokoningi (MTCC 2048); 11= T. species (NRRL 5242); 12 = R. solani (Control)]

competition, mycoparasitism as major biocontrol mechanism against phytopathogens. There are many genes which are responsible for biocontrol activity and these genes are called biocontrol genes (Carsolio et al., 1999; Dana et al., 2001; Howell, 2003). Present study demonstrates in vitro antagonism of eleven Trichoderma species with R. solani causing root rot in cotton. The results of dual culture revealed that antagonist T. koningii MTCC 796 (T9) evidenced highest growth inhibition of test pathogen followed by T. viride NBAII Tv23 (T5), T. harzianum NBAII Th1 (T1), T. virens NBAII Tvs12 (T7) at 10 DAI. The best two antagonists strains MTCC 796 and Tv23 overgrew on test pathogen with mycoparasitism as mode of action; and Th1 and Tvs12 evidenced strong antibiosis with formation of inhibition zone to inhibit growth of test

pathogen. SEM images inidcates that mycoparasitism is a potetial biocontrol mechanism than antibiosis for biocontrol activity against R. solani. These results were in confirmation with the finding of Melo and Faull (2000) who reported that the T. koningii and T. harzianum were found to be effective in reducing the radial growth of R. solani. The T. koningii strain produced toxic metabolites with strong activity against R. solani, inhibiting the mycelial growth. Ramezani (2001) also documented that T. harzianum significantly inhibited the growth of M. phaseolina. T. viride and T. harzianum had a greater inhibition on M. phaseolina than T. hamatum. Our study showed T. koningii MTCC 796 and T. viride NBAII Tv23 have a better growth inhibition of R. solani.

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Table 2 SCoT unique markers associated with best antagonists Trichoderma strains inhibiting pathogen R. solani during antagonism SCoT Primer

T. harzianum (NBAII Th1) (T1)

T. virens (NBAII Tvs12) (T7)

T. viride (NBAII Tv23) (T5)

T. koningii (MTCC 796) (T9)

Base pair (bp) SCoT1

1520

SCoT3

-

1985

-

1510 1150

SCoT6

-

-

480 198

748

SCoT15 SCoT21 SCoT24 SCoT38 SCoT41 SCoT44 SCoT45 SCoT47 SCoT51 SCoT65 SCoT73 TOTAL

305 -

222 3650 590 598 210 410 7

2

2000

Shalini and Kotasthane (2007) screened seventeen Trichoderma strains against R. solani in vitro. All strains including T. harzianum, T. viride and T. aureoviride were inhibited the growth of R. solani. The present study revealed the the best antagonists T. koningii MTCC 796 (T9) and T. viride NBAII Tv23 (T5) inhibited the growth of pathogen by using a mode of action mycoparasitism. The antagonists MTCC 796 and Tv23 grew over mycelia of test poathogen with surround coiling and hock like structure formation followed by disintigration and disruption of mycelia of pathogen as evidenced in SEM images. Formation of apressoria-like structures enabled the hyphae of Trichoderma to firmly attach to the surface of its host mycelium. This process occurred at different intensities depending on the Trichoderma spp., suggesting that various antagonists have different mechanisms of host recognition. Mycoparasitism involves morphological changes, such as coiling and formation of appressorium- like structures, which serve to penetrate the host (McIntyre et al., 2004). The first contact between Trichoderma spp. and pathogen R. solani occurred after 5 days of inoculations, followed by growth inhibition. Differential antagonistic activity has even been observed for various Trichoderma spp. which demonstrates a semi-specificity in the interaction of Trichoderma with its host (Schirmbock et al., 1994). Our results on SEM study revealed that T. koningi MTCC 796 and T. viride NBAII Tv23 showed effectively coiling on pathogen R. solani at 10 DAI, and may started the parasitism earlier during antagonism

1005 1995 -

420 498 1980 -

5

6

compared to other Trichoderma spp. Similar results have been reported for T. harzianum against Crinipellis perniciosa (Marco et al., 2000), Sclerotium rolfsii (El-Katany et al., 2001) and Rhizoctonia cerealis (Innocenti et al., 2003). SEM results explained that Trichoderma attached to the pathogen R. solani either by hyphal coils, hooks or appressoria followed by lysed sites and penetration holes were found in hyphae of the fungal pathogen subsequently removal of parasitic hyphae (Elad et al., 1983). Monteiro et al. (2010) reported that T. harzianum ALL42 were capable of overgrowing and degrading R. solani and M. phaseolina mycelia, coiling around the hyphae with formation of apressoria and hook-like structures. The interaction between fifteen isolates of T. harzianum and the soil borne plant pathogen, R. solani, was studied using light microscopy and transmission electron microscopy (TEM) which showed efficient coiling process followed by a substantial production of chtinolytic enzymes (Almeida et al., 2007). Howell (2003) examined Trichoderma attaches to the pathogen with cell-wall carbohydrates that bind to pathogen lectins. Once Trichoderma is attached, it coils around the pathogen and forms the appresoria. The following step consisted of the production of pathogenesis related enzymes and peptaibols. Gajera et al. (2012) reported pathogen specific mechanism of antagonists Trichoderma for biocontrol activity against M. phaseolina causing charcol rot in castor. T. koningii MTCC 796 was capable of overgrowing

Fig. 4. Phylogenetic and molecular evolutionary analysis of encoding endochitinase gene from antagonistic fungus T. koningii strain MTCC 796 (The numbers represent per cent bootstrap support of each node). N Seq1 gb| KF723013.1|: Trichoderma koningii strain MTCC 796 endochitinse gene, partial cds. (gi|572926915:b1..29,93..191,276..325,394..N824)

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Fig. 5. Phylogenetic and molecular evolutionary analysis of protein endochitinase from antagonistic fungus T. koningii strain MTCC 796 (The numbers represent per cent bootstrap support of each node). N Seq1 gb| AHF57043.1 |: endochitinse, partial [Trichoderma koningii] (gi|572926916:b1..29,93..191,276..325,394..N824)

and degrading M. phaseolina mycelia, coiling around the hyphae with apressoria and hook-like structures. Formation of apressoria-like structures enabled the hyphae of Trichoderma spp. to firmly attach to the surface of its host mycelium. However, some antagonists (T. viride NBAII Tv 23, T. hamatum NBAII Tha 1) used different mechanism against M. phaseolina just touched the hyphae without coiling. Whereas, T. pseudokoningii showed spore around pathogen not attached to hyphae. Present study agreed with the results suggesting all Trichoderma strains could not work equally against specific soil borne disease as various Trichoderma antagonists have different mechanisms of pathogen recognition. The amplification of functional gene and their corresponding traits by SCoT markers have become the markers of choice for crop plants due to their high polymorphism and reproducibility (Collard and Mackill, 2009; Luo et al., 2010). This may be due to SCoT detects polymorphisms in coding sequences, because the primers were designed to amplify from the short conserved region surrounding the ATG translation start codon (Collard and Mackill, 2009; Xiong et al., 2009). Therefore, functional genes and their corresponding traits related amplification fragments were generated by SCoT polymorphism. The other markers like RAPD detected polymorphism at random whereas ISSR and SSR markers perceived amplifications from microsatellite regions which nay not primarily represent functional genes. The SCoT polymorphisms was successfully utilized for genetic diversity analysis and regional specific molecular fingerprinting of crop varieties in rice, peanut and mango (Collard and Mackill, 2009; Xiong et al., 2009; Gajera et al., 2014; Luo et al., 2010, 2011, 2012). As a PCR-based gene target technique, SCoT analysis is low cost and effective to use. The present study reported first time SCoT polymorphism for characterization functional biocontrol genes in Trichoderma isolates inhabiting potentially phytopathogen R. solani. The results found with six unique molecular fingerprints to identify best mycoparasitic antagonist T. koningii MTCC 796 (T9). The unique fragments were eluted, cloned, sequenced and annotated for functional gene sequences. Out of six DNA sequences, two functional sequences 824 bp of SCoT-3(920) fragment and 776 bp of SCoT-6(806) fragment signify sequence homology with biocontrol genes endochitinase and hmgR

genes, respectively and the same were annotated and deposited in NCBI GenBank database. Present findings examined functional biocontrol gene endochitinase and hmgR gene in best mycoparasitic antagonist T. koningii MTCC 796 who inhibited maximum growth inhibition of test pathogen R. solani under mycoparasitism mode of action. The Chit33, chit42 and chit36 have been over expressed in Trichoderma spp. in order to test the role of chitinases in mycoparasitism, and the 42-kDa chitinase is believed to be a key enzyme (Howell, 2003). Present study confirms the characterization of endochitinase gene derived from 824 bp functional sequence of SCoT-3(920) fragment of best mycoparasitic antagonist MTCC 796 having deduced amino acid sequence of 203 aa with partial cds join b1..29,93..191,276..325,394..N824) (nucleotide accession No. KF723013.1 and protein accession No. AHF57043.1). It is evident from phylogenetic analysis of chitinase gene that the isolate MTCC 796 represents a partial cds of endochitinase gene. The activity of biocontrol genes from antagonists Trichoderma elevated when get contacted with pathogen cell wall. The pathogen cell-wall induces nag1 gene, but it is only triggered when there is contact with the pathogen (Carsolio et al., 1999; Harman et al., 2004; Howell, 2003). Activation of genes varied with various Trichoderma species. This may be due to the expression of certain gene in Trichoderma spp. during antagonism as Chit33 is articulated only during the contact phase and not before overgrowing R. solani (Dana et al., 2001). However, chit36Y is expressed without direct contact of the pathogen. The hmg - CoA reductase is involved in the synthesis of sterols, isoprenoids and other lipids. The enzyme is a key step in the mevalonate pathway and polytopic nature of protein (Brown and Goldstein, 1980). So far, the antifungal activity of hmg - CoA reductase expressed by hmgR gene in Trichoderma was elucidated by Cardoza et al. (2007). The cloning and characterization of T. harzianum CECT 2413 hmgR gene encoding a hmg - CoA reductase were explained for the biosynthesis of terpene compounds. Partial silencing of the hmgR gene gave rise to T. harzianum transformants with a higher level of sensitivity to lovastatin, a competitive inhibitor of the hmg - CoA reductase enzyme. The production of ergosterol was inhibited than the wild-type strain by hmgR

Fig. 6. Phylogenetic and molecular evolutionary analysis of encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase-like protein (hmgR) gene from antagonistic fungus T. koningii strain MTCC 796 (The numbers represent per cent bootstrap support of each node). N Seq1 gb| KF723014.1|: Trichoderma koningii strain MTCC 796 3-hydroxy-3-methylglutarylcoenzyme A reductase-like protein (hmgR)gene, partial cds. (gi|572926917:b1..130,161..472,500..N776)

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Fig. 7. Phylogenetic and molecular evolutionary analysis of 3-hydroxy-3-methylglutarylcoenzyme A reductase-like protein (hmgR) from antagonistic fungus T. koningii strain MTCC 796 (The numbers represent per cent bootstrap support of each node). N Seq1 gb |AHF57044.1 |: 3-hydroxy-3-methylglutaryl-coenzyme A reductase-like protein, partial [Trichoderma koningii]. (gi|572926918:b1..130,161..472,500..N776)

silenced transformants in a minimal medium containing lovastatin. The silenced transformants augmented the expression of erg7 gene and diminished hmgR gene expression. The results summarized that hmgR silenced transformants showed a reduction in their antifungal activity against phytopathogens R. solani and Fusarium oxysporum. Present study explained functional characterization of hmgR gene in best mycoparasitic antagonist T. koningii MTCC 796 inhibiting maximum growth of test pathogen R. solani. The study confirms characterization of hmgR gene derived from 776 bp functional sequence of SCoT-6(806) fragment of antagonist MTCC 796 having deduced amino acid sequence of 239 aa with partial cds joins (b1..130,161..472,500..N776) (nucleotide accession No. KF723014.1 and protein accession No. AHF57044.1). Further, trait specific sequence characterized amplified region (SCAR) markers may be designed from functional biocontrol genes sequences (endochinase and hmgR gene) and validated to indentify mycoparasitic antagonistic Trichoderma strains having potentials biocontrol activity. However, bioactive secondary metabolites of best mycoparasitic strains T. koningii MTCC 796 (T9) and T. viride NBAII Tv23 (T5); best antibiosis strains T. harzianum NBAII Th1 (T1), T. virens NBAII Tvs12 (T7) and least T. viride NRRL 6418 (T6) antagonists were evaluated using GC-MS profile (Supplementary information). The MTCC 796 strain under the influence of pathogen cell wall evidenced to produce undecane 3,8-dimethyl, ethyl iso-allocholate, 1,2-benzenedicarboxylic acid, triacontane, 1-bromo, octadecane, 3-ethyl-5- (2-ethylbutyl), hexadecane, 7,9-dimethyl, heptadecane, 8-methyl, hexatriacontane which were reported for antifungal, antioxidant and cytotoxic activities (Akpuaka et al., 2013; Muthulakshmi et al., 2012; Rajeswari et al., 2012). The phenol 3,5-bis(1,1-dimethylethyl), octadecane and eicosane were noticed in Tv23 strain with antifungal, antibacterial, antioxidant, cytotoxic effects (Akpuaka et al., 2013; Manorenjitha et al., 2013). Present study demonstrated that mycoparasitic strains amplified unique SCoT functional fragments which were eluted, cloned, sequenced and blast for characterization of biocontrol genes and one of the fragment confirmed as hmgR gene. This gene is responsible for expression of hmg CoA reductase which is a key enzyme for regulation of terpene biosynthesis and mycoparasitic strains produced triterpenes during antagonism to inhibit growth of fungal pathogen as evidenced with GC-MS profile. 5. Conclusions Trichoderma isolates inhibited the growth of R. solani pathogen by using two different mechanism viz., mycoparasitism and antibiosis. In mycoparatism, biocontrollers - T. koningi MTCC 796 and T. viride NBAII Tv23 continued to grow over pathogen without formation of inhibition zone during antagonism. However, T. harzianum NBAII Th1 and T. virens NBAII Tvs12 exhibited antibiosis and formed about 2-4 mm zone of inhibition after 10 DAI. The same were confirmed in dual culture test and SEM examinations. Mycoparasitism is a strong mode of action for biocontrol activity compared to antibiosis inhibited about 88.12% growth inhibition of test pathgen by MTCC 796 strain followed by Tv23 (85.34%). Mycoparasitic strains started the parasitism earlier and was capable of overgrowing, coiling around the hyphae with apressoria and hook-like structures followed by disintegration and disruption of

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mycelia of test pathogen R. solani. The Trichoderma strains subjected to SCoT analysis for functional molecular characterization and derived unique markers for best antagonists MTCC 796. The best antagonist T. koningii MTCC 796 identified with six unique SCoT fragments which were eluted, cloned and sequenced. The two sequences confirmed as a biocontrol gene after functional annotation and homologies to known sequences in the GenBank database using BLAST available online from the NCBI. The nucleotide and protein sequences of the biocontrol genes were annotated and submitted to NCBI as endochitinase gene (nucleotide accession No. KF723013.1 and protein accession No. AHF57043.1) and hmgR gene (nucleotide accession No. KF723014.1 and protein accession No. AHF57044.1). Acknowledgments Authors thank to Gujarat State Biotechnology Mission, Gandhinagar (India) for financial support to carry out some part of the research work. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2016.09.026. References Abbasi, P.A., Miller, S.A., Mealia, T., Hoitink, H.A.J., Kim, J.M., 1999. Precise detection and tracing of Trichoderma hamatum 382 in compost amended potting mixes by using molecular markers. Appl. Environ. Microbiol. 12, 5421–5426. Akpuaka, A., Ekwenchi, M.M., Dashak, D.A., Dildar, A., 2013. Biological activities of characterized isolates of n-hexane extract of Azadirachta indica (Neem) leaves. Nat. Sci. 11 (5), 141–147. Almeida, F.B.R., Cerqueira, F.M., Silva, R.N., Ulhoa, C.J., Lima, A.L., 2007. Mycoparasitism studies of Trichoderma harzianum strains against Rhizoctonia solani: evaluation of coiling and hydrolytic enzyme production. Biotechnol. Lett. 29, 1189–1193. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Benitez, T., Rincon, A.M., Limon, M.C., Codon, A.C., 2004. Biocontrol mechanisms of Trichoderma strains. Int. Microbiol. 7 (4), 249–260. Brown, M.S., Goldstein, J.L., 1980. Multivalent feedback regulation of HMG CoA reductase, a control mechanism coordinating isoprenoid synthesis and cell growth. J. Lipid Res. 21, 505–517. Cardoza, R.E., Hermosa, M.R., Vizcaíno, J.A., González, F., Llobell, A., Monte, E., Gutiérrez, S., 2007. Partial silencing of a hydroxy-methylglutaryl-CoA reductase-encoding gene in Trichoderma harzianum CECT 2413 results in a lower level of resistance to lovastatin and lower antifungal activity. Fungal Genet. Biol. 44 (4), 269–283. Carsolio, C., Benhamou, N., Haran, S., Cortés, C., Gutiérrez, A., Chet Herrera-Estrella, A., 1999. Role of the Trichoderma harzianum endochitinase gene, ech42, in mycoparasitism. Appl. Environ. Microbiol. 65, 929–935. Collard, B.C.Y., Mackill, D.J., 2009. Start codon targeted (SCoT) polymorphism: a simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Mol. Biol. Report. 27, 86–93. Dana, M.M., Limón, M.C., Mejías, R., Mach, R.L., Benítez, T., Pintor-Toro, J.A., 2001. Regulation of chitinase 33 (chit33) gene expression in Trichoderma harzianum. Curr. Genet. 38, 335–342. Dennis, C.J., Webster, J., 1971. Antagonism properties of species groups of Trichoderma, III Hyphal interaction. Trans. Br. Mycol. Soc. 57, 363–369. Elad, Y., Chet, I., Boyle, P., Henis, Y., 1983. Parasitism of Trichoderma spp. on Rhizoctonia solani and Sclerotium rolfsii - Scanning electron microscopy and fluorescence microscopy. Phytopathology 73, 85–88. El-Katany, M.H., Gudelj, M., Robra, K.H., Enaghy, M.A., Gubitz, G.M., 2001. Characterization of a chitinase and an endo-1,3-glucanase from Trichoderma harzianum Rifae T24 involved in control of the phytopathogen Sclerotium rolfsii. Appl. Microbiol. Biotechnol. 56, 137–143. Fisher, R., Yates, N., 1948. Statistical Methods for Research Workers. 12th ed. Oliver and Boyd, Edinburg, London, pp. 130–131 Biological Monograph and Manuals. Gajera, H.P., Vakharia, D.N., 2010. Molecular and biochemical characterization of Trichoderma isolates inhibiting a phytopathogenic fungi Aspergillus niger Van Tieghem. Physiol. Mol. Plant Pathol. 74, 274–282. Gajera, H.P., Bambharolia, R.P., Patel, S.V., Khatrani, T.J., Goalkiya, B.A., 2012. Antagonism of Trichoderma spp. against Macrophomina phaseolina: Evaluation of Coiling and Cell Wall Degrading Enzymatic Activities. J. Plant Pathol. Microbiol. 3, 149. Gajera, H.P., Bambharolia, R.P., Domadiya, R.K., Patel, S.V., Golakiya, B.A., 2014. Molecular characterization and genetic variability studies associated with fruit quality of indigenous mango (Mangifera indica L.) cultivars. Plant Syst. Evol. 300, 1011–1020. Gajera, H.P., Savaliya, D.D., Patel, S.V., Goalkiya, B.A., 2015. Trichoderma viride induces pathogenesis related defense response against rot pathogen infection in groundnut (Arachis hypogaea L.). Infect. Genet. Evol. 34, 314–325.

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