Biodiversity and Molecular Characterization of Trichoderma spp. and Exploring its Synergistic Action for the Management of Cucumber Damping Off Incited by Pythium aphanidermatum

Bioversity of fungal antagonist, Trichoderma from different locations of Nilgiri district of Tamil Nadu, India, were characterized through molecular methods. Thirty four isolates were tentatively identified as Trichoderma and confirmed upto species level through molecular tools. PCR amplification of the 18s-28s rRNA gene region revealed that all thirty four isolates produced amplicon size of 600bp and were further confirmed through sequencing. The sequences of various Trichoderma spp. were compared with both NCBI and TrichOKEY database to validate their molecular identity. Among 34 isolates, 31 isolates were identified as T. asperellum (KT462693, KU361372, KX533978 to KX533999, KX523262 to KX523264, KX555650, KX147092 to KX147094, KX5334000), 2 isolates as T. harzianum (KX533989, KX533990) and 1 isolate as T. virens (KU666466) through NCBI data base. However, all the T. asperellum isolates identified through NCBI database were identified as T. koningiopsis using TricHOKEY data base. Identity of T. harzianum isolates (TRI 35 and TRI 36) and T. virens isolate (TRI 37) were same in both NCBI and TricHOKEY database. Antagonistic assay with diverse species of Trichoderma revealed that T. virens (TRI 37) was effective in inhibiting the radial growth of Pythium aphanidermatum (87.78%) followed by T. harzianum (TRI 35), (TRI 36) and T. asperellum (TRI 9) in vitro. The effective isolates T. virens (TRI 37), T. harzianum (TRI 35, TRI 36) and T. asperellum (TRI 9) were compatible with each other. Biopriming of cucumber seeds with talc based formulation of the consortia comprising of T. virens isolate ( TRI 37), T. harzianum isolates (TRI 35 and TRI 36) and T. asperellum TRI 9 @ 108 cfu/g and soil application suppressed damping off to an extent of 76.82% over untreated control.

rRNA genes are universally conserved, while the ITS region and intergenic spacer (IGS) are highly variable 19 .The ITS and IGS region are the fastest evolving regions and varies among the species within a genus.Thus, the sequences of these regions were used for identification of closely related species 34 .The diversity of Trichoderma has been used for the management of soil-borne diseases.Among the soil-borne diseases, damping off caused by the genus Pythium is a common problem in fields and greenhouse grown crops which kills the seedlings.This disease complex usually involves other pathogens such as Fusarium, Phytophthora and Rhizoctonia.Pre -and post-emergence damping-off caused by Pythium spp. in vegetable crops are economically important worldwide 33 .Rapid germination of sporangia of Pythium in 1.5-2.5 h after exposure to exudates or volatiles from seeds or roots 22 followed by immediate infection makes management of the pathogen very difficult 33 .Pythium spp.tends to be generalistic and non-specific in their host range, which causes extensive and devastating root rot is often very difficult to prevent or control 17 .With this background the present study was undertaken for characterization of bio geographical diversity of Trichoderma by morphological and molecular means to explore the antagonistic potential against damping off pathogen in cucumber under protected cultivation.

Sampling and Isolation of Trichoderma
Rhizospheric soil samples were collected from different crop fields of Nilgiri district, Tamil Nadu, India.Trichoderma were isolated from the rhizospheric soil samples on Trichoderma selective medium 10 using serial dilution technique 26 .The plates were incubated at 28±2°C for 4 to 7 days.Visible fungal colonies were transferred to Potato dextrose agar (PDA) plates and incubated at 28±2°C for 5 days and maintained on PDA medium for subsequent studies.

Molecular characterization of Trichoderma spp. Genomic DNA extraction from Trichoderma isolates
Extraction of genomic DNA of all the isolates of Trichoderma spp.were extracted by harvesting the mycelium grown in potato dextrose broth for 3-4 days at 28±2°C.Mycelial mat was collected on filter paper, washed with distilled water for 2-3 times , frozen and used for DNA extraction.Genomic DNA was extracted as per the protocol described by Raeder and Broda 24 .DNA was suspended in 50µl of TE buffer and quantified with ethidium bromide fluorescence.

PCR amplification and sequencing
Primers ITS1 (5'-TCCGTAGGTGAACCT GCGG-3') and ITS4 (5'-TCCTCCGCTTAT TGATATGC-3') described by White et al 34 were used to amplify a fragment of rDNA including ITS1 and ITS2 and the 5.8S rDNA gene.The PCR amplification reactions were performed in a 50 ¼l mixture containing 50 mM KCl, 20 mM Tris HCl (pH 8.4), 2.0 mM MgCl2, 200 ¼M of each of the four deoxynucleotide triphosphates (dNTPs), 0.2 ¼M of each primer, 40 •g/ ¼l of template and 2.5 U of Taq polymerase.The cycle parameters included an initial denaturation of 1 min at 95°C, followed by 35 cycles of 1 min at 95°C, 30 s at 60°C and 1.5 min at 72°C, with a final extension of 10 min at 72°C.The PCR products were resolved in 1% agarose gel, purified PCR product was sequenced in SciGenome Labs Pvt Ltd , Kerala.

Phylogenetic analysis
The rDNA homology searches were performed using ITS gene sequences by BLAST program (http://www.ncbi.nlm.nih.gov).Sequeces were compared with Trichoderma spp.isolates retrieved from the Genbank database.Newly obtained sequences were submitted in Genbank database (NCBI).Sequences were analyzed in pairwise and multiple sequence alignment and the identity was scored with the Bio-Edit V 7.0.5 11 .Phylogenetic tree was constructed by the neighbor joining method and tree topologies were evaluated by performing bootstrap analysis of 1000 data sets performed with MEGA 6 (Molecular Evolutionary Genetic Analysis) software 31 .The rDNA homology searches were performed using ITS gene sequences by TrichOKEY program (http://www.isth.com).Sequences were compared with Trichoderma spp.isolates retrieved from the TrichOKEY database.

Isolation of Pythium from infected cucumber plants
The pathogen Pythium was isolated from damping off affected cucumber plants collected from major cucumber growing areas of Coimbatore, Erode and Madurai districts of Tamil Nadu.The infected plant tissue was washed with sterile water and cut into small pieces from the leading edges of lesions.Then surface sterilized with 0.1% mercuric chloride, washed with sterile distilled water thrice and shade dried on sterile filter paper.The dried pieces were plated on PDA and incubated at 28°C ± 2°C for 5 days.Molecular Characterization of Pythium isolate Isolation of genomic DNA of Pythium sp.
Genomic DNA was extracted from the suspension cultures of Pythium by the Cetyl Trimethyl Ammonium Bromide (CTAB) method as described by Lee and Taylor 18 .The isolate of Pythium was grown at room temperature (28 ± 2°C), and transferred into 250 ml conical flasks containing 150 ml potato dextrose broth (PDB).It was incubated at 28 ± 2°C for 5 days.After complete colonization of the medium, the mycelium was harvested by filtration through sterile filter paper and stored at -80°C until used for DNA extraction.DNA was extracted from the harvested VASUMATHI et al.: STUDY OF Trichoderma spp.mycelia according to the procedure described by Mahuku 20 .Mycelia were ground to a fine powder in liquid nitrogen and suspended in CTAB buffer.
The mixture was incubated at 65°C for 30 min.DNA was precipitated using ice-cold isopropanol and the pellet was washed with 70% ethanol, dried and dissolved in TE buffer.Identification of Pythium sp.
To identify the species of Pythium isolates of 16S rDNA intervening sequence specific Pa1-(5'TCCACGTGAACCGTTGAAATC3');ITS2-(5'GCTGCGTTCTTCATCGATGC-3') primers were used to get an amplicon of 210 bp size 13 .PCR amplification reactions were performed in a 50 ¼l mixture containing 50 mM KCl, 20 mM Tris HCl (pH 8.4), 2.0 mM MgCl2, 200 ¼M of each of the four deoxynucleotide triphosphates (dNTPs), 0.2 ¼M of each primer, 40 •g/¼l of template and 2.5 U of Taq polymerase.Amplification was conducted with a total reaction volume of 50µl in Eppendorf Master Cycler, German.The PCR settings used were as follows: a hold of 2 min at 95°C, 30 cycles of 1min at 94°C, 30 sec at 54°C and 1min at 72°C and a final extension of 10min at 72°C.The PCR products were resolved on 1% agarose gel at 50 V, stained with ethidium bromide (0.5µg/ml) and analyzed using gel documentation system.

Screening of Trichoderma spp against P. aphanidermatum
The antifungal activity of Trichoderma spp. was tested by dual culture technique 7 .The pathogen and Trichoderma were grown on PDA for a week at room temperature (28 ± 2°C), about nine mm diameter mycelial disc of the pathogen (Pythium aphanidermatum) was cut from the periphery and transferred to the Petri plate with PDA and nine mm diameter mycelial disc of Trichoderma was placed simultaneously at opposite sides of same Petriplate aseptically and incubated at room temperature 28 ± 2°C with alternate light and darkness for 7 days and observed periodically.The experiment was replicated thrice and per cent growth inhibition was calculated by the formula of I = (C-T)/C x 100, where C is mycelial growth in control plate, T is mycelial growth of test organisms in inoculated plate and I is inhibition of mycelial growth.Hyperparasitism was calculated by measuring the overgrowth of Trichoderma isolates on the pathogen from the zone of interaction of Trichoderma with pathogen in centimeter.

Number of plants affected Disease incidence (%) =
x 100 Total number of plant The experimental design was completely randomized with three replicates (pots) for each treatment and repeated twice.

Isolation of Trichoderma spp.
A total of 34 isolates of Trichoderma were isolated from different rhizosphere soil samples of different crop plants.Isolate code, species identification, location, NCBI accession numbers, TrichOKEY identification and isolation details of Trichoderma strains are furnished in Table 1.

Molecular characterization of Trichoderma spp.
PCR amplification with the conserved primer The result of the phylogenetic analysis based on the 18S-28S-rRNA gene sequences of different species of Trichoderma isolates were analyzed and results revealed that three different clusters were formed in phylogenetic tree (Fig 2).The evolutionary history was inferred using the Neighbor-Joining method Saitou and Nei 25 .The optimal tree with the sum of branch length = 1.69400390.The difficulty in identification of species using NCBI similarity search tool, BLAST (http:// blast.ncbi.nlm.nih.gov).has been expressed by several workers 8 .The lacunae in identifying species on the basis of similarity search in BLAST are absence of quality control of species authentification, sequences deposited under the original names and not under the names after verification.Kredics et al 16 suggested that more than 40% of Hypocreae and Trichoderma sequences available in Genbank database are unidentified or misidentified at the species level .In the present study the isolate which had maximum hit with T. asperellum were identified as The shortcoming in identification of species by using only ITS marker has been reported by several workers 30,17 .Druchian and kubich 9 evaluated along 11 gene loci and formed that the 4 th and 5 th introns of translation elongation factor 1 alpha (tef1-EF-1±) and the coding region of endochitinase -42(ech 42) aid in resolving the species

Antagonistic activity
Dual culture assay revealed that all the isolates of Trichoderma spp inhibited the mycelial growth of P.aphanidermatum more than 50% over control (Table 2, Fig 3).However, the maximum inhibition of 87.78% of the mycelia growth of P.aphanidermatum was observed with the T. virens isolate TRI 37. It was followed by the T. harzianum isolates TRI 35 and TRI 36, which inhibited the mycelial growth to an extent of 85.5% over control.The next best isolates TRI 7, TRI 9, TRI 26 and 38 which inhibited the growth of pathogen to an extent of 81.5, 81.3, 80.0 and 80.0 per cent over the control were T. asperellum respectively.Similarly Anita et al 2 reported that, interaction between Trichoderma and isolated Pythium species in dual culture technique, range of inhibition was observed ranging from 56.92 -86.67%.The significant inhibition was observed in case of T. viride against P. viniferum 86.67% .Studies on hyperparasitism indicated that the T.virens isolate (TRI 37) overgrew on P. aphanidermatum up to 2.16

Bioefficacy of Trichoderma formulation on the management of cucumber damping-off
The effective isolate s of T. virens, T. harzianum isolates (TRI 35 and 36) and T. asperellum isolate (TRI9) were evaluated for the management of cucumber damping off under pot culture in green house through biopriming of seeds and soil application either as individual isolate or as consortia.Results of the investigation emphasized ingeneral that bio-priming, soil application and bio priming coupled with soil application with consortia of Trichoderma isolates comprising of T. virens (TRI 37), T. harzianum isolates (TRI 35 and 36) and T. asperellum (TRI 9) were effective in the suppression of damping off rather than the application of individual isolates of Trichoderma compared to untreated control.However, bio priming and soil application with the consortia comprising of T.virens (TRI37), T.harzianum isolates (TRI 35 and 36) and T.asperellum (TRI 9) suppressed damping off to an extent of 76.82% over untrated control and was followed by the soil application of consortia comprising of (TRI 37+TRI 36+TRI 35+TRI 9),which was applied on 15 and 30 th days after seeding(74.08% reduction over control).
Comparison of Trichoderma consortia, delivered through biopriming and soil application with T. virens isolate (TRI 37) and T. harzianum isolates (TRI 35 and 36) was only next to seed treatment with metalaxyl coupled with soil application of metalaxyl 0.1% on 15 and 30 th days after seeding, which reduced damping off upto 85.02% over control(Table 3).Similar results were also reported by Abd-El-Khair et al 1 and Singh et al 28 that confirms our findings.They reported that the incidence of damping-off was found maximum VASUMATHI et al.: STUDY OF Trichoderma spp. in the pathogen inoculated control (54.67%) and lowest in the plants treated with the consortium of Trichoderma isolates BHU51+BHU105 (22.00%) rather than the individual application of Trichoderma isolate BHU51 and BHU105 on to seeds.Singh and Singh 27 also reported that the use of mixture of Trichoderma, increase the level of defence releted enzymes in the plant that protect the plant from the infection caused by Macrophomina.

Fig. 3 .
Fig. 3. Antagonistic efficacy of Trichoderma spp.against cucumber damping off pathogen under in vitro condition

Table 1 .
Identification,NCBI Genebank accession number and isolation details of different isolates of Trichoderma

Table 2 .
36 vitro efficacy of Trichoderma spp.againstPythium aphanidermatum by dual culture method Means followed by a common letter are not significantly different at the 5% level by DMRT; Figures in parentheses are square root transformed values cm from the zone of interaction, indicating the hyperparasite nature of the T.virens isolate TRI 37. Hyperparasitism by the T.virens indicate the capability of these isolate produce hydrolytic enzyme followed by lysis of pathogen.Similarly hyperparasitism nature reported by Yang et al36, In co-culture in vitro, isolates of Trichoderma spp., including Tri01003, Tri01090 and Tri01091, displayed the ability to steadily colonize and aggressively attack the mycelia of P. ultimum, and finally produce conidia on the Pythium colony.

Table 3 .
Effect of bioformulations of Trichoderma spp. on the incidence of cucumber damping off under glasshouse conditions