Biodiversity and Molecular Characterization of Trichoderma spp. and Exploring its Synergistic Action for the Management of Cucumber Damping Off Incited by Pythium aphanidermatum.
MATERIALS AND METHODS
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 [+ or -] 2[degrees]C for 4 to 7 days. Visible fungal colonies were transferred to Potato dextrose agar (PDA) plates and incubated at 28 [+ or -] 2[degrees]C for 5 days and maintained on PDA medium for subsequent
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 [+ or -] 2[degrees]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 50[micro]l 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 1/4l mixture containing 50 mM KCl, 20 mM Tris HCl (pH 8.4), 2.0 mM MgCl2, 200 1/4M of each of the four deoxynucleotide triphosphates (dNTPs), 0.2 %M of each primer, 40 g/ %l of template and 2.5 U of Taq polymerase. The cycle parameters included an initial denaturation of 1 min at 95[degrees]C, followed by 35 cycles of 1 min at 95[degrees]C, 30 s at 60[degrees]C and 1.5 min at 72[degrees]C, with a final extension of 10 min at 72[degrees]C. The PCR products were resolved in 1% agarose gel, purified PCR product was sequenced in SciGenome Labs Pvt Ltd, Kerala.
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[degrees]C [+ or -] 2[degrees]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 [+ or -] 2[degrees]C), and transferred into 250 ml conical flasks containing 150 ml potato dextrose broth (PDB). It was incubated at 28 [+ or -] 2[degrees]C for 5 days. After complete colonization of the medium, the mycelium was harvested by filtration through sterile filter paper and stored at -80 [degrees]C until used for DNA extraction. DNA was extracted from the harvested 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[degrees]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 1/4l mixture containing 50 mM KCl, 20 mM Tris HCl (pH 8.4), 2.0 mM MgCl2, 200 1/4M of each of the four deoxynucleotide triphosphates (dNTPs), 0.2 1/4M of each primer, 40 g/1/4l of template and 2.5 U of Taq polymerase. Amplification was conducted with a total reaction volume of 50[micro]l in Eppendorf Master Cycler, German. The PCR settings used were as follows: a hold of 2 min at 95[degrees]C, 30 cycles of 1min at 94[degrees]C, 30 sec at 54[degrees]C and 1 min at 72[degrees]C and a final extension of 10min at 72[degrees]C. The PCR products were resolved on 1% agarose gel at 50 V, stained with ethidium bromide (0.5[micro]g/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 [+ or -] 2[degrees]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 [+ or -] 2[degrees]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.
Testing the efficacy of Trichoderma spp. against P aphanidermatum in green house
The efficacy of Trichoderma spp. against damping off pathogen was evaluated with the four effective Trichoderma isolates viz., T. virens (TRI 37), T. harzianum isolates (TRI 36, TRI 35) and T. asperellum isolate (TRI 9) in pot culture. Treatment details include T1- Biopriming (BP) with TRI 37 @ 10g/kg of seeds, T2- BP with TRI 36 @ 10g/kg of seeds, T3- BP with TRI 35 @ 10g/kg of seeds, T4-BP with TRI 9 @ 10g/kg of seeds,T5- BP with (TRI 37+TRI 36+TRI 35+TRI 9) @ 10g/kg of seeds, T6-Soil application (SA) with TRI 37 @ 2.5kg/ha at 15 and 30th days after seeding, T7-SA with TRI 36 @ 2.5kg/ha at 15 and 30th days after seeding, T8-SA with TRI 35 @ 2.5kg/ha at 15 and 30th days after seeding, T9-SA with TRI 9 @ 2.5kg/ha at 15 and 30th days after seeding, T10-SA with (TRI 37+TRI 36+TRI 35+TRI 9) @ 2.5kg/ha at 15 and 30th days after seeding, T11-BP+SA with TRI 37 @ 10g/kg of seeds+@ 2.5kg/ha at 15 and 30th days after seeding,T12- BP+SA with TRI 36 @ 10g/kg of seeds+@ 2.5kg/ha at 15 and 30th days after seeding, T13-BP+SA with TRI 35 @ 10g/kg of seeds+@ 2.5kg/ha at 15 and 30th days after seeding, T14- BP+SA with TRI 9 @ 10g/kg of seeds+@ 2.5kg/ha at 15 and 30th days after seeding, T15-BP+SA with (TRI 37+TRI 36+TRI 35+TRI 9) @ 10g/kg of seeds+@ 2.5kg/ha at 15 and 30th days after seeding, T16-BP+SA with Metalaxyl 2g/kg of seeds + 0.1% @ 15 and 30th days after seeding, T17- Un treated control. The treatments were replicated thrice and pathogen inoculated control was maintained. Five cucumber seeds were planted in each pot containing sterile potting medium (red soil: sand: FYM at 1:1:1 w /w/w). The pathogen was multiplied in sand maize medium and incorporated @ 10g per pot up to the depth of 10cm @[10.sup.5]cfu/g. Trichoderma was delivered through bio priming of seed and soil application with different combinations. Seeds were bioprimed with talc based bio formulation @ 10 g/ kg followed by two soil applications on 30 and 45 days after sowing @ 2.5 kg/ha. Plants inoculated with the pathogen alone served as control. Healthy controls were also maintained. Disease incidence was recorded after 20 days of sowing and per cent disease incidence was calculated as follows.
Disease incidence (%) = Number of plants affected/Total number of plant x 100
The experimental design was completely randomized with three replicates (pots) for each treatment and repeated twice.
RESULTS AND DISCUSSION
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 (ITS 1 -5'TCTGTAGGTGAACCTGCG 3') and ITS 4-5'TCCTCCGCTTATTGATATGC 3') of ITS region yielded the genomic product of 600 bp (Fig 1) in the reactions performed with 34 isolates of Trichoderma species. Absence of size variation among the isolates collected suggest that, majority of the isolates belong to Trichoderma.
The size of the amplicon containing the ITS 1, ITS2 and 5.8S r RNA was around 600 bp. In order to ascertain the Trichoderma orgin of sequence, the sequences were initially analyzed in BLAST. In the BLAST analysis isolates TRI 1 to TRI 16, TRI 19 to TRI 21, TRI 23- TRI 29, 38, 50, 60 and 70 had highest identity with T. asperellum. The query coverage was between 93-100% and identity was between 96-100%. In the case of other next three isolates TRI 35, 36 and TRI 37, isolate TRI 35 and 36 exhibited maximum identities with T. harzianum isolate TRI 37 with T. virens. On the basis of identity search in BLAST the isolates collected in the present study could be clearly categorized into three groups. 1. Major group comprising isolates were identified as T. asperellum and another small group of two isolates belonging T. harzianum and one belonging to T. virens(table) To overcome this problem the International Commission of Taxonomy of Fungi has recommended the use of DNA barcode tools for correct identification of Hypocrea and Trichoderma species. Therefore the ITS neucleotide sequence of the 34 isolates of the present study were analysed in TrichOKEY programme (www.isth.info) contrasting to results obtained in BLAST search in TrichOKEY analysis. The 34 isolates could be differentiated into 4 groups, one major group comprise isolate TRI 2-13, 16, 19, 20, 24, 26, 27, 28, 38, 50, 60 and 70 belonging to T. koningiopsis pertaining to the Rufa clade. The second group consists of isolates TRI 35 and TRI 36 which were confirmed as T. harzianum under catopteron clade, third group comprised isolate TRI 37 which was identified as T. virens belonging to virens clade, fourth group comprised of isolate TRI 15 which was identified as T. asperellum belonging to pachybasium A clade. Comparision of the results of TrichOKEY with BLAST indicate that the isolates TRI 2-13, 16, 19, 20, 24, 26, 27, 28, 38, 50, 60 and 70 under BLAST search were identified as T. asperellum. The other two group of isolates were identified as T. harzianum and T. virens by BLAST and appeared to be similar to TrichOKEY analysis. The six isolates (TRI 1, 14, 21, 23, 25 and 29) identified as T. asperellum in BLAST were found to have only genus specific hall mark sequences in TrichOKEY. However further species identification was not possible in TrichOKEY since species specific hall mark were not detectable, the isolates are therefore considered as unidentified species under the genus Trichoderma.
Diversity study of Trichoderma spp.
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 T. koningiopsis in TrichOKEY. However identification of the other isolates as T. virens, T. harzianum tally both in BLAST and TrichOKEY search.
The relationship between the Trichoderma isolates of the present study and the established species in the TrichOKEY programme were assessed further. The type sequences of the identified species, T. koningiopsis, T. asperellum, T. virens and T. harzianum belonging to clade Rufa, Pachybasium A, Virens and Catopteron (harzianum clade) were retrived from database and analyzed in multiple alignment in CLUSTAL-W programme. A phylogenetic tree was constructed based on alignment clearly revealed the species identity of the isolates under study. The TRI isolates 2-13, 15, 16, 19, 20, 22, 24, 26-28, 38, 50, 60 and 70 occupied the same position as the T. koningiopsis the member of rufa clade, it is intresting to note here that T. asperellum which belong to pachybasium clade also occupy the same branch as that of rufa clade which suggest that on the basis of nucleotide identity these two species are not easily distinguishable. The isolate TRI 37 was grouped with virens clade, TRI 35 and TRI 36 was aligned with catopteron clade. Very clearly these three rufa, virens and catopteron clade branch off distinctly. Interesting results are observed with TRI isolates TRI 23, 25, 21, 29 and 14 which could not be identified at species level in TrichOKEY. They have origin along with rufa and pacybacium clade but branch off distinctly. They may represent a new species group which need to be validated by taking more phylogenetic marker genes.
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 4th and 5th introns of translation elongation factor 1 alpha (tef1-EF-1 [+ or -]) and the coding region of endochitinase-42(ech 42) aid in resolving the species
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 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 al (36), 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.
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 30th 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 30th 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 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.
I would like to duly acknowledge UGC grant and DST-FIST for providing the facilities of instrumentation in Department of Plant Pathology, Tamil Nadu Agricultural University.
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S. Vasumathi, K. Eraivan Arutkani Aiyanathan and S. Nakkeeran *
Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore - 641 003, India.Y
(Received: 08 October 2016; accepted: 03 December 2016)
* To whom all correspondence should be addressed.
Caption: Fig. 1. Molecular confirmation of Trichodema isolates with ITS primers
Caption: Fig. 2. Phylogenetic tree of the Trichoderma isolates. The numbers given over branches indicate bootstrap coefficient
Caption: Fig. 3. Antagonistic efficacy of Trichoderma spp. against cucumber damping off pathogen under in vitro condition
Table 1. Identification, JSICBI Genebank accession number and isolation details of different isolates of Trichoderma s. Isolate GPS Location Source of No. code Area Longitude Latitude culture 1 TRI 1 Coonoor 11.3530 fN 76.7959 fE Lillium 2 TRI 2 Coonoor 11.3530 fN 76.7959 fE Lillium 3 TRI 3 Gudalur 11.5029 fN 76.4917 fE Tea 4 TRI 4 Gudalur 11.5029 fN 76.4917 fE Tea 5 TRI 5 Baraliayur 11.3429 fN 76.8500 fE Tea 6 TRI 6 Chinnaka- 11.3347 fN 76.7705 fE Silver oak rumpalam 7 TRI 7 Chinnaka- 11.3347 fN 76.7705 fE Tea rumpalam 8 TRI 8 Baraliayur 11.3429 fN 76.8500 fE Chrysanthemum 9 TRI 9 Kaikatti 11.3128 fN 76.6909 fE Tea 10 TRI 10 Edavanalli 12.5207 fN 77.9855 fE Lillium 11 TRI 11 Edavanalli 12.5207 fN 77.9855 fE Lillium 12 TRI 12 Kallar 11.3390 fN 76.8652 fE Tea 13 TRI 13 Kallar 11.3390 fN 76.8652 fE carnation 14 TRI 14 Kasolai 11.3151 fN 76.6992 fE Carnation 15 TRI 15 Kasolai 11.3151 fN 76.6992 fE Gerbera 16 TRI 16 Kasolai 11.3151 fN 76.6992 fE Tea 17 TRI 19 Katteri 11.3341 fN 76.7879 fE Chrysanthemum 18 TRI 20 Kunnakombai 11.3149 fN 76.7111 fE Tea 19 TRI 21 Kunnakombai 11.3149 fN 76.7111 fE Silver oak 20 TRI 22 Kothagiri 11.4086 fN 76.8720 fE Rose 21 TRI 23 Kothagiri 11.4086 fN 76.8720 fE Lillium 22 TRI 24 Kodanad 11.5015 fN 76.9063 fE Lillium 23 TRI 25 Kodanad 11.5015 fN 76.9063 fE Chrysanthemum 24 TRI 26 Kodanad 11.5015 fN 76.9063 fE Carnation 25 TRI 27 Denad 11.44906916 76.9420 fE Gerbera 26 TRI 28 Masiangudi 11.5634 fN 76.6338 fE Tea 27 TRI 29 Masiangudi 11.5634 fN 76.6338 fE Chrysanthemum 28 TRI 35 Devasola 11.3204 fN 76.6868 fE Lillium 29 TRI 36 Devasola 11.3204 fN 76.6868 fE Silver oak 30 TRI 37 Devasola 11.3204 fN 76.6868 fE Tea 31 TRI 38 Chinnaka- 11.3347 fN 76.7705 fE Tea rumpalayam 32 TRI 50 Kallar 11.3390 fN 76.8652 fE Chrysanthemum 33 TRI 60 Maniyapuram 11.3157 fN 76.7080 fE Carnation 34 TRI 70 Maniyapuram 11.3157 fN 76.7080 fE Gerbera s. Isolate Genebank Identity of Species No. code accession number NCBI TricHOKEY 1 TRI 1 KX533988 T. asperellum T.koningiopsis 2 TRI 2 KX533979 T. asperellum T.koningiopsis 3 TRI 3 KX533983 T. asperellum T.koningiopsis 4 TRI 4 KX533985 T. asperellum T.koningiopsis 5 TRI 5 KX533991 T. asperellum T.koningiopsis 6 TRI 6 KT462693 T. asperellum T.koningiopsis 7 TRI 7 KX533980 T. asperellum T.koningiopsis 8 TRI 8 KX533992 T. asperellum T.koningiopsis 9 TRI 9 KX533993 T. asperellum T.koningiopsis 10 TRI 10 KX533994 T. asperellum T.koningiopsis 11 TRI 11 KX533986 T. asperellum T.koningiopsis 12 TRI 12 KU361372 T. asperellum T.koningiopsis 13 TRI 13 KX533978 T. asperellum T.koningiopsis 14 TRI 14 KX533987 T. asperellum Unidentified 15 TRI 15 KX533984 T. asperellum T. asperellum 16 TRI 16 KX533995 T. asperellum T.koningiopsis 17 TRI 19 KX533996 T. asperellum T.koningiopsis 18 TRI 20 KX523262 T. asperellum T.koningiopsis 19 TRI 21 KX533997 T. asperellum Unidentified 20 TRI 22 KX523263 T. asperellum T.koningiopsis 21 TRI 23 KX147092 T. asperellum T.koningiopsis 22 TRI 24 KX084067 T. asperellum T.koningiopsis 23 TRI 25 KX533998 T. asperellum Unidentified 24 TRI 26 KX533999 T. asperellum T.koningiopsis 25 TRI 27 KX533981 T. asperellum T.koningiopsis 26 TRI 28 KX533982 T. asperellum T.koningiopsis 27 TRI 29 KX5334000 T. asperellum Unidentified 28 TRI 35 KX533989 T.harzianum T.harzianum 29 TRI 36 KX533990 T.harzianum T.harzianum 30 TRI 37 KU666466 T. virens T. virens 31 TRI 38 KX523264 T. asperellum T.koningiopsis 32 TRI 50 KX555650 T. asperellum T.koningiopsis 33 TRI 60 KX 147094 T. asperellum T.koningiopsis 34 TRI 70 KX147093 T. asperellum T.koningiopsis Table 2. In vitro efficacy of Trichoderma spp. against Pythium aphanidermatum by dual culture method S. Isolates No. Mycelia Mycelia No. growth(cm) growth(cm) Pythium Trichoderma 1 TRI 1 (T.asperellum-KX533988) 2.67 6.3 2 TRI 2(T.asperellum-KX533979) 2.20 6.8 3 TRI 3(T.asperellum-KX533983) 2.37 6.6 4 TRI 4(T.asperellum-KX533985) 2.70 6.3 5 TRI 5(T.asperellum-KX533991) 2.63 6.4 6 TRI 6(T.asperellum-KT462693) 3.60 5.4 7 TRI 7(T.asperellum-KX533980) 1.40 7.6 8 TRI 8(T.asperellum-KX533992) 2.10 6.9 9 TRI 9(T.asperellum-KX533993) 1.37 7.6 10 TRI 10(T.asperellum-KX533994) 2.70 6.3 11 TRI 11(T.asperellum-KX533986) 2.50 6.5 12 TRI 12(T.asperellum-KU361372) 2.10 6.9 13 TRI 13(T.asperellum-KX533978) 3.57 5.4 14 TRI 14(T.asperellum-KX533987) 3.63 5.4 15 TRI 15(T.asperellum-KX533984) 2.37 6.6 16 TRI 16(T.asperellum-KX533995) 3.07 5.9 17 TRI 19(T.asperellum-KX533996) 2.37 6.6 18 TRI 20(T.asperellum-KX523262) 4.30 4.7 19 TRI 21(T.asperellum-KX533997) 2.13 6.9 20 TRI 22(T.asperellum-KX523263) 3.63 5.4 21 TRI 23(T.asperellum-KX147092) 2.40 6.6 22 TRI 24(T.asperellum-KX084067) 4.30 4.7 23 TRI 25(T.asperellum-KX533998) 2.67 6.3 24 TRI 26(T.asperellum-KX533999) 2.43 6.6 25 TRI 27(T.asperellum-KX533981) 4.20 4.8 26 TRI 28(T.asperellum-KX533982) 3.70 5.3 27 TRI 29(T.asperellum-KX5334000) 2.63 6.4 28 TRI 35(T.harzianum-KX533989) 1.33 7.7 29 TRI 36(T.harzianum-KX533990) 1.30 7.7 30 TRI 37(T.virens-KU666466) 1.10 7.8 31 TRI 38(T.asperellum-KX523264) 2.20 6.8 32 TRI 50(T.asperellum-KX555650) 2.13 6.9 33 TRI 60(T.asperellum-KX147094) 3.40 5.6 34 TRI 70(T.asperellum-KX147093) 3.57 5.4 35 Control 9.0 -- S. Isolates No. Hyperpar- Inhibition No. asitism(cm) over control (%) 1 TRI 1 (T.asperellum-KX533988) 1.8 70.33 (n) (56.99) 2 TRI 2(T.asperellum-KX533979) 1.7 75.56 (h) (60.37) 3 TRI 3(T.asperellum-KX533983) 1.8 73.67 (i) (59.78) 4 TRI 4(T.asperellum-KX533985) 1.9 70.00 (o) (56.78) 5 TRI 5(T.asperellum-KX533991) 1.62 70.78 (m) (57.27) 6 TRI 6(T.asperellum-KT462693) 1.60 60.00 (s) (50.76) 7 TRI 7(T.asperellum-KX533980) 1.98 84.44 (e) (66.76) 8 TRI 8(T.asperellum-KX533992) 1.8 76.67 (f) (61.12) 9 TRI 9(T.asperellum-KX533993) 1.08 84.81 (d) (67.06) 10 TRI 10(T.asperellum-KX533994) 1.08 70.00 (o) (56.78) 11 TRI 11(T.asperellum-KX533986) 1.98 72.22 (l) (58.19) 12 TRI 12(T.asperellum-KU361372) 1.44 76.67 (f) (61.11) 13 TRI 13(T.asperellum-KX533978) 0.72 60.37 (r) (50.98) 14 TRI 14(T.asperellum-KX533987) 1.8 59.63 (t) (50.55) 15 TRI 15(T.asperellum-KX533984) 1.44 73.70 (i) (59.14) 16 TRI 16(T.asperellum-KX533995) 1.98 65.93 (p) (54.28) 17 TRI 19(T.asperellum-KX533996) 1.44 73.70 (i) (59.14) 18 TRI 20(T.asperellum-KX523262) 0.36 52.22 (w) (46.27) 19 TRI 21(T.asperellum-KX533997) 1.8 76.30 (g) (60.86) 20 TRI 22(T.asperellum-KX523263) 1.08 59.63 (t) (50.55) 21 TRI 23(T.asperellum-KX147092) 1.98 73.33 (j) (58.90) 22 TRI 24(T.asperellum-KX084067) 0.36 52.22 (w) (46.27) 23 TRI 25(T.asperellum-KX533998) 1.98 70.37 (n) (57.02) 24 TRI 26(T.asperellum-KX533999) 1.98 72.96 (k) (58.66) 25 TRI 27(T.asperellum-KX533981) 1.08 53.33 (v) (49.90) 26 TRI 28(T.asperellum-KX533982) 1.98 58.89 (u) (50.11) 27 TRI 29(T.asperellum-KX5334000) 1.8 70.74 (m) (57.25) 28 TRI 35(T.harzianum-KX533989) 1.98 85.19 (c) (67.36) 29 TRI 36(T.harzianum-KX533990) 1.98 85.56 (b) (67.66) 30 TRI 37(T.virens-KU666466) 2.16 87.78 (a) (69.53) 31 TRI 38(T.asperellum-KX523264) 1.98 75.56 (h) (60.37) 32 TRI 50(T.asperellum-KX555650) 1.8 76.30 (g) (60.86) 33 TRI 60(T.asperellum-KX147094) 1.97 62.22 (q) (52.07) 34 TRI 70(T.asperellum-KX147093) 1.8 60.37 (r) (50.98) 35 Control -- -- Means followed by a common letter are not significantly different at the 5% level by DMRT; Figures in parentheses are square root transformed values Table 3. Effect of bioformulations of Trichoderma spp. on the incidence of cucumber damping off under glasshouse conditions S. Treatments Damping off Per cent No incidence (%) reduction over control T1 BP with TRI 37 @10g/kg of seed 38.00 (k) 53.44 (37.16) T2 BP with TRI 36 @10g/kg of seed 39.10(l) 53.15 (38.70) T3 BP with TRI 35 @10g/kg of seed 39.07 (l) 53.19 (38.68) T4 BP with TRI 9 @10g/kg of seed 39.20 (l) 53.03 (38.76) T5 BP with TRI37+TRI 36+TRI 37.17 (j) 55.46 35+TRI9 10g/kg of seed (37.56) T6 SA with TRI 37 @ 2.5 kg/ha at 15 29.66 (g) 64.46 and 30th days after seeding (32.99) T7 SA with TRI 36 @ 2.5 kg/ha at 15 32.71 (h) 60.81 and 30th days after seeding (34.88) T8 SA with TRI 35 @ 2.5 kg/ha at 15 32.69 (h) 60.83 and 30th days after seeding -34.87 T9 SA with TRI 9 @ 2.5 kg/ha at 15 35.27 (i) 57.74 and 30th days after seeding (36.43) T10 SA with TRI37+TRI 36+TRI 21.13 (c) 74.68 35+TRI 9 @ 2.5 kg/ha at (27.36) 15 and 30th days after seeding T11 BP +SA with TRI 37 @10g/kg of 23.04 (d) 72.39 seed+2.5 kg/ha at 15 and 30th days after seeding (28.68) T12 BP +SA with TRI 36 @10g/kg of 23.00 (d) 72.44 seed+2.5 kg/ha at 15 and (28.65) 30th days after seeding T13 BP +SA with TRI 35 @10g/kg of 26.45 (e) 68.31 (30.95) 30th days after seeding T14 BP +SA with TRI 9 @10g/kg of 27.24 (f) 67.36 seed+ 2.5 kg/ha at 15 and (31.46) 30th days after seeding T15 BP +SA with TRI37+TRI 36+TRI 19.34 (b) 76.82 35+TRI9 @10g/kg of seed+ (26.09) 2.5 kg/ha at 15 and 30th days after seeding T16 BP +SA with Metalaxyl 2g/kg 12.50 (a) 85.02 of seed+ 0.1% at 15 and (20.70) 30th days after seeding T17 Control 83.47 (m) -- (66.01)
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|Author:||Vasumathi, S.; Aiyanathan, K. Eraivan Arutkani; Nakkeeran, S.|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Mar 1, 2017|
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