CHAPTER - III. Material and methods

Transcription

1 CHAPTER - III Material and methods

2 CHAPTER-III Materials and Methods MATERIALS AND METHODS 3.1 GLASSWARE Glassware made of Corning or Borosil were used throughout the present investigation. The glassware was first washed with a detergent followed by thorough cleaning with tap water before placing them in cleaning solution for 24 hrs and finally rinsed with distilled water for 3-4 times and were air dried before use. The composition of cleaning solution: Potassium dichromate : 60 g Conc.Sulphuric acid : 60 ml Distilled water : 1000 ml 3.2 CHEMICALS Chemicals of analytical reagent (AR) and guaranteed reagent (GR) grades of standard make were used. The ph of the media was adjusted to 7.0 using either 0.1 N Hcl or 0.1 N NaOH EQUIPMENT Compound microscope (10X, 40X magnification) was used for observing the fungi. Hot air oven and autoclave were used for sterilization of glassware, media and soil respectively. The incubator was used for incubating test materials at different temperatures. Fungal cultures were sub cultured onto PDA slants and stored in refrigerator. Weightments were done on a single pan electronic balance with a sensitivity of g for weightments of Root, Shoot fresh and dry weight. Laminar Air Flow was used for isolated pathogens and other microflora. Millipore filters (0.44 um) and whatman filter papers ware used for mycelium and spore free culture collection. PCR machine, Gel DOC, Electrophoresis, DNA extraction kit, DNA purification kit for molecular characterization of Trichoderma Spp. Other 52

3 material which were used in the present investigation for various purposes include camel hair brush, inoculation needles, earthen pots etc., 3. 4 CULTURE MEDIA USED Trichoderma specific medium (TSM, Elad and Chet, 1983; modified by Saha and Pan( 1997) used for selective isolation of native isolates of Trichoderma spp. Components Quantity (g l -1 ) MgSO4. 7 H2O 0.2 K2HPO4 0.9 KCl 0.15 NH4NO3 1.0 D Glucose 3.0 Agar Agar 20.0 Distill water 1.0 lit ph After autoclaving following components were added Components Quantity (g l -1 ) Chloramphenicol 250 Methyl orange 300 Rose Bengal Captan 50 WP Potato dextrose agar (PDA, Riker and Riker, 1936) Used for routine culture growth and maintenance of both pathogens and antagonists and for assessing antagonistic potential of Trichoderma spp. Against some soil borne plant pathogens. Components Quantity (g l -1 ) Peeled potato Dextrose 20.0 Agar agar 20.0 Distilled water 1.0 L 53

4 3.4.3 Potato dextrose broth (PDB) Materials and Methods PDB was prepared by using same components as in PDA except agar, whenever required. This is a general purpose medium used for various experiments on antagonism and maintenance of culture. Potato Dextrose Broth (PDB) was prepared using the compositions without agar and was used for the production of antibiotics and biomass produced by different isolates of antagonists Oat Meal Agar (OMA, Johnson and Curl, 1977) Used for cultural and morphological characterization of Trichoderma spp Components Quantity (g l -1 ) Oat meal 17 Agar 20 Distilled water to 1 lit Malt Extract Agar (MEA) Components Quantity (g l -1 ) Malt extract 15 K 2 HPO 4 4 Ammonium chloride 1 Agar 20 Distil water 1 lit Czapek Dox Agar (CZA) Used for cultural characterization of Trichoderma spp Components Quantity (g l -1 ) Sucrose 30 Sodium nitrate 2 Dipotassium phosphate 1 Magnesium sulphate 0.5 Potassium chloride 0.5 Ferrous sulphate 0.01 Agar 15 Final ph Distil water 1 lit 54

5 3.4.7 Other media used Materials and Methods Sorghum bran (20%), Farm Yard Manure (FYM) and talc were used for mass multiplication of Trichoderma isolates and pathogens, respectively in vivo studies. The sclerotia of S. rolfsii produced by growing it in PDA medium for 15 days at 28 ± 1 C Sterilization of glassware, media and soil Liquid and solid were sterilized at C at 15 psi for 15 min for two consecutive days. The glassware and other metallic apparatus were sterilized in hot air oven at 180 C for 2 hr after wrapping with brown paper. Flame sterilization was used for inoculation needle, forceps, etc. during transfer of culture. For isolation of pathogen, surface sterilization was done by dipping the soft tissues in 1% sodium hypochlorite solution for 2-3 min and hard tissue for 4-5 min. The inoculation chamber (Laminar air flow chamber) was sterilized by switching on the UV lamps for at least min and hands were sterilized with dehydrated alcohol. 3.5 SURVEY A roving Survey was conducted between December and March in the four major chickpea growing areas of Andhra Pradesh. The Mandal in each region were selected randomly. A total of 100 fields covering 14 Mandals were surveyed. The number of fields visited per district ranged from 15 to 20 and a distance of 2-3 km was allowed between sites, but the distance was greater where chickpea fields were far apart, resulting in a smaller number of sites visited in such Mandals. 3.6 ISOLATION AND IDENTIFICATION OF PATHOGENS Isolation of pathogens The three soil borne plant pathogens viz., Sclerotium rolfsii Sacc. (Athelia rolfsii Curzi.), (Collar rot of Chickpea), Rhizoctonia bataticola(taub.) Butler (dry root rot) and Fusarium oxysporum f. sp. ciceri (Padwick) Matuo and K. Sato) wilt were isolated from infected plant parts of chickpea seedlings by following tissue segment method (Rangaswami, 1958). The sample were washed thoroughly with tap water, cut into small bits (1-2 cm), again washed with sterile 55

6 distilled water 2-3 times, dipped into 1% Sodium hypochlorite solution, rinsed twice in sterile water, decanted excess water in the plant parts and plated on potato dextrose agar (Masago et al. 1977). Culture plates were incubated at 25 C for 7 days in the dark and was purified by repeated hyphal tip transfers. Identification of pathogen was done based on cultural, morphological characteristics observed under microscope. The cultures were maintained in PDA slants and stored at 4 C for further use Identification of pathogen The pathogens were identified as Sclerotium rolfsii, Rhizoctonia bataticola and Fusarium oxysporum f. sp. ciceri based on description given by Barnett and Hunter, ISOLATION AND IDENTIFICATION OF MYCOFLORA Serial dilution technique was used to isolate antagonistic mycoflora from rhizosphere soil of chickpea, Among that there were, 20 Trichoderma isolates, Fusarium sps, Aspergillus niger, Aspergillus flavus, Rhizopus stolonifer were isolated based on morphological characters Isolation of Trichoderma from Rhizosphere soil sample The soil samples were collected from the different locations of Kurnool, Kadapa and Anantapur districts of Andhra Pradesh at a depth ranging 5-6 cm, by removing top 2 cm surface soil. The soil collected from rhizosphere soil of healthy chickpea plants. All collected soil samples were mixed thoroughly to make a composite sample. A working samples 1 gram (dry weight basis) soil was taken from composite sample. 1 gram soil was mixed into 10 ml of sterile Distilled water then 1 ml of suspension was taken into another tube containing 9 ml of sterile distilled water. This serial dilution technique was continued up to10-2 to From the 10-6 dilution, 0.1 ml suspension was transferred to each of the three Petri dishes, 20 ml of melted Trichoderma Specific Medium (TSM) (Elad and Chet, 1983) was poured in each plate and mixed with the suspension by giving a gentle whirling motion to the plate and allowed them to incubate in room temperature (Islam et al, 2001; Bhagat, 2008, Bhagat and Pan, 2010). The probable colonies were observed 56

7 closely and picked up from Petri plates and transferred to PDA slants and finally pure culture was obtained by repeated subculture. They were identified on the basis of their morphological characters (Rifai, 1969; Bhagat and Pan, 2010). The details of Trichoderma spp. given in Table Cultural characteristics of Trichoderma spp. The cultural characteristics of 20 isolates of Trichoderma were studied in three media, viz., potato dextrose agar (PDA), oat meal agar (OMA), Cazpex Dox Agar (CZA) and malt extract agar (MEA). Mycelial discs (6 mm) of young growing culture of respective isolates of Trichoderma was inoculated edge of the Petriplates containing either solidified PDA or OMA or MEA or CZA medium and incubated at 28 ± 1 C for one week. The growth pattern of isolates of Trichoderma was observed daily and all the distinguishing characters were recorded. The experiment was replicated three times. The characters recorded were colony, growth rate, presence or absence of pigments, hyphae and presence of any distinguishing odour. Table 3.1 The details of Trichoderma isolates Sl. Isolation Village Mandal Crop No. code 1 KJ-12 Dannavalapadu Jammulamadugu Chickpea Rhizospere 2 KNP-1 Marduru Panyam Chickpea Rhizospere 3 ATPU-4 Raketla Urvakonda Chickpea Rhizospere 4 KNP-3 Mittnala Panyam Chickpea Rhizospere 5 KT6 Maduru Thonduru Chickpea Rhizospere 6 KP 10 Rampathadu Pendlimari Chickpea Rhizospere 7 APTU-6 Raketla Urvakonda Chickpea Rhizospere 8 KR-4 Korrapadu Rajupalem Chickpea Rhizospere 9 KNN-2 Bollavaravarum Nandikotkur Chickpea Rhizospere 10 KT-7 Thonduru Thonduru Chickpea Rhizospere 11 KNO-2 Guttapadu Orvakallu Chickpea Rhizospere 12 KNK-1 Dornipadu Kovelakuntla Chickpea Rhizospere 13 KNK-9 Dornipadu Kovelakuntla Chickpea Rhizospere 14 ATPPE-6 Chimmalavagu Pedappapuru Chickpea Rhizospere 15 KT-13 Kasunuru Thonduru Chickpea Rhizospere 16 KNO-9 Nannur Orvakallu Chickpea Rhizospere 17 KNN-4 Nandikotkur Nandikotkur Chickpea Rhizospere 18 ATPP-6 Senagagudurur putluru Chickpea Rhizospere 19 KNPG-3 Pathakota Pagidyala Chickpea Rhizospere 20 ATPU-1 Raketla Uravakonda Chickpea Rhizospere 57

8 3.7.3 Morphological characterization of Trichoderma isolates Morphological/ Anamorphic characterization of Trichoderma isolates were done by growing them in potato dextrose agar medium in order to unequivocally verify taxa and species. Morphological characteristics viz., conidiophores length, width and branching pattern, phialides width at tip, middle and base, phialides length and shape, phialospores and chlamydospores shape, length and width including colouration. For morphological characterization, slide culture technique was used and incubated at 25 C with alternating 12 hr dark and 12 hr cool white fluorescent light. All conidiophores, phialides conidia and chlamydospores were measured following the methods of Lieckfeidt et al. (2001). At least 50 observations were randomly recorded while measuring the morphological characteristics of Trichoderma spp Molecular characterization of native Trichoderma isolates Genomic DNA extraction from Trichoderma isolates Isolation of fungal genomic DNA was done by growing the fungi for 3-4 days. The mycelia were incubated with lysis buffer containing 250 mmtris-hcl (ph 8.0), 50 mmedta (ph8.0), 100 mmnacl and 2% SDS, for 1 hr at 60 C followed by centrifugation at 12,000 rpm for 15 min. The supernatant was then extracted with equal volume of water saturated phenol and further centrifuged at 12,000 rpm for 10 min; the aqueous phase was further extracted with equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) and centrifuge at 12,000 rpm for15 min; the aqueous phase was then transferred in a fresh tube and the DNA was precipitated with chilled ehanol (100%). DNA was pelleted by centrifuging at rpm for 15 min and washed in 70% ethanol by centrifugation. The pellets were air dried and suspended in TE buffer (Tris EDTA buffer) (ph 8.0) Qualitative and quantitative estimation of DNA The extraction of total genomic DNA from the Trichodrma isolates as per the above procedure was followed by RNAase treatment. Genomic DNA was re suspended in 100 μl 1 X TE buffer and incubated at 37 C for 30 min with RNAse (60 μg). After incubation the sample was re extracted with PCI (Phenol: 58

9 Chloroform: Isoamylalcohol (25:24:1) solution and RNA free DNA was precipitated with chilled ethanol as described earlier. The quality and quantity of DNA was analyzed.all isolates of Trichoderma were taken up for ITS-PCR in 0.8% agarose gel. The DNA from all isolates produced clear sharp bands, indicating good quality of DNA PCR amplification of its region of Trichoderma isolates Amplification Genomic DNA was amplified by mixing the template DNA (50 ng), with the polymerase reaction buffer, dntp mix, primers and Taq polymerase. Polymerase Chain Reaction was performed in a total volume of 100 μl, containing 78 μl deionized water, 10 μl 10 X Taq pol buffer, 1 μl of 1 U Taq polymerase enzyme, 6 μl 2 mmdntps, 1.5 μl of 100 mm reverse and forward primers and 1 μl of 50 ng template DNA. PCR was programmed with an initial denaturing at 94 C for 5 min. followed by 30 cycles of denaturation at 94 C for 30 sec, annealing at 59 C for 30 sec and extension at 70 C for 2 min and the final extension at 72 C for 7 min in a Primus 96 advanced gradient Thermocycler. PCR product (20 μl) was mixed with loading buffer (8 μ1) containing 0.25% bromophenol blue, 40% w/v sucrose in water and then loaded in 2% Agarose gel with 0.1% ethidium bromide for examination with horizontal electrophoresis Sequence Alignment For direct sequencing of the PCR products, a total of 20 isolates of Trichoderma PCR products produced sequences that could be aligned and showed satisfactory homology from the NCBI Gene bank data base. The priming sites of the ITS1and ITS4 primers were determined in order to confirm that the sequences obtained corresponding to the actual ITS 1 region (Table 2). A multiple sequence alignment was carried out that included the ITS 1 region, including gaps and the complete sequences align. There were quite a number of gaps that were introduced in the multiple sequence alignment within the ITS-1 region that were closely related and similar sequence indicated. 20 Trichoderma isolates were used in the multiple sequence alignment. From the sequence alignment, variations were observed among species of Trichoderma isolates 59

10 Sequence Analysis of 5.8S-ITS Region Materials and Methods The nucleotide sequences of 5.8S-ITS region were aligned using Molecular Evolutionary Genetics Analysis (MEGA) version 6.0. The forward and reverse sequences were checked and edited manually when needed. Then, a consensus sequence was generated from each alignment made by using Clustal X algorithm. The sequences were then compared with the sequences deposited in Gen Bank database using Basic Local Alignment Search Tool (BLAST), where a nucleotide blast program was chosen. Table 3.2: The nucleotide sequence used for ITS Region Primer Sequence Reference ITS 1-5.8s-ITS2 region of r DNA ITS TCCGTAGGTGAACCTGCGG- ITS TCCTCCGCTTATTGATATGC- 3 1 Cheverri et al 2004 ITS1 ITS1 ITS3 ITS2 18S 5.8S 28S ITS2 ITS4 Fig.3. 1: Schematic representation of rdna showing the internal transcribed spacer (ITS)-1 region and 5.8S gene with primers (primers: ITS1 and ITS2) amplifying different parts of the spacer. Primer sequences are given in the text above. 3.8 EVALUATION OF ANTAGONISTIC POTENTIAL OF TRICHODERMA ISOLATES Rapid Screening Rapid screening of Trichoderma isolates was done against 3 soils borne plant pathogens following the methods of Roy and Pan (2002). Five isolates of Trichoderma harzianum, five isolates of T. viride and five isolates of T. asperellum and five isolates T.longibrachiatum were screened for their antagonistic potential 60

11 against three soil borne fungal plant pathogens through multiple culture followed by dual culture in potato dextrose agar on the basis of their relative growth rate measured as a function of incubation period. Six mm dia mycelial discs picked up from the margin of young growing 3-4 days old culture of Trichoderma and pathogens, were inoculated at the peripheral region at equal distance from centrally inoculated test pathogens and incubated for 5-6 days at 28 ± 1ºC. The relative growth rate of test antagonist was observed and recorded. The most efficient isolate was sorted out with each pathogen. The Trichoderma isolates were rated on the basis of their ability to suppress the mycelial growth of pathogen following the methods of modified Bell s scale (Bell et al., 1982 modified by Saha and Pan, 1997). S1: The antagonist completely overgrew the pathogen (100% over growth) S2: The antagonist overgrew at least 2/3rd growth of the pathogen (75% Overgrowth) S3: The antagonist colonized half of the growth of pathogen (50% Overgrowth) S4: The pathogen and antagonist (locked at the point of contact) S5: The pathogen overgrew the antagonist Dual Culture Method: (Antagonistic Activity) The Trichoderma isolates were evaluated against three soil borne pathogenic fungi by dual culture technique as described by Morton and Strouble (1955). A 6 mm diameter mycelial disc from the margin of the 7 days-old culture of Trichoderma isolates and the soil borne plant pathogens (Sclerotium rolfsii, Rhizoctonia bataticola and Fusarium oxysporum f. sp. ciceri) were inoculated at peripheri region opposite to each other at equal distance. The experimental design used was a completely randomized with three Petri dishes for each isolates. In control plates (without Trichoderma), a sterile agar disc was placed at opposite side of the soil borne inoculated plates. Inoculated plates were incubated at 28 ± 1ºC for 7 days. 2-3 days after the incubation period, radial growth of pathogen isolates was measured and per cent inhibition of average radial growth was calculated in relation to mycelial growth of the controls as follows (Vincent 1947). 61

12 C T I = C Where, I = Per cent inhibition of growth Materials and Methods C = Mycelial growth in control plate T = Mycelial growth in test plate All strains were tested in three replications and were carried out twice for each isolate Mycoparasitism Hyphal interaction or mycoparasitism between potential Trichoderma isolates and the pathogen Rhizoctonia bataticola, Sclerotium rolfsii and F.oxysporum f.sp.ciceri were studied by dual culture method following Quimio and Cumagun (2001). A cellophane membrane was placed over solidified potato dextrose agar (PDA) medium into the Petri plates, the test pathogen and antagonist were inoculated at the peripheral region opposite to each other and incubated into BOD incubator at 28 ± 1 C for 3-4 days. The zone of point of contact between pathogen and antagonists were cut along with cellophane membrane using a new blade, placed on a cleaned slide, stained with 3% KOH and observed under a phase contrast microscope (Phase Contrast Microscope) Antibiosis Effect of non-volatile antibiotics The ability of Trichoderma iso9lates to produce the non-volatile substances was studied following methods described by Dennis and Webster (1971 a) and Quimio and Cumagun (2001). All test isolates of Trichoderma were grown in 100 ml sterilized potato dextrose broth (PDB) for 10 days in 250 ml Erlenmeyer flasks with periodical shaking. Culture filtrate of Trichoderma was harvested by filtering it through Whatmann filter no. 42 filter paper into sterilized flasks. The culture filtrate was centrifuged at 6000 rpm for 10 min, sterilized by passing it through Cellulose Millipore membrane filter paper (0.4 µm pore size). The required volume of culture 62

13 filtrate was added with known volume of melted PDA to obtain final concentration of 10%, 15% and 20% (v/v) culture filtrate. The amended media was poured into Petri dishes and inoculated with mycelial plug (6 mm dia) picked up from young culture of test pathogen and incubated at 28 ± 1 C for 4 days. The PDA medium without addition of culture filtrate of antagonist was served as control. The radial mycelial growth of test pathogen was measured and per cent inhibition of mycelial growth of pathogen was calculated according to (Vincent 1947) as mentioned earlier Effect of volatile antibiotics Productions of volatile metabolites by Trichoderma spp were assayed as described by Dennis and Webster 1971 and Goyal et al., 1994 with slight modifications. The Trichoderma isolates were centrally inoculated by placing 3mm disc taken from three days old cultures on the PDA plates and incubated at 28 ± 2 C for three days. The top of each petridish was replaced with bottom of PDA plate inoculated centrally with the pathogen. Petridish with PDA medium without Trichoderma spp at the lower lid and the upper lid with pathogens was maintained as control. The pair of each petridishes were sealed together with paraffin tape and incubated for 4-6 days. After incubation the inhibition of mycelia growth was calculated according to (Vincent 1947) Ecological fitness of Trichoderma isolates The ecological studies included the competitive parasitic ability and Rhizosphere colonization by isolates of Trichoderma. Two soil types, viz., natural and sterilized soil were used in both studies following the methods of Papavizas (1982) and Roy and Pan (2002) Preparation of mycelia and conidial form of inoculum For mycelial preparation of test isolates of Trichoderma, mycelial plug (6 mm dia) from young growing region of 4 days old culture of test isolates of Trichoderma was inoculated into Erlenmeyer flasks (250 ml) containing 100 ml potato dextrose broth medium (PDB). The inoculated flasks were incubated at 28 ± 1 C for 3-4 days into a BOD incubator. The mycelial mat was harvested by passing 63

14 through the Whatman No. 42 filter paper and homogenized by a stirrer. The required concentration of mycelial inocula was prepared by adding sterilized distilled water and was used immediately or preserved at 4 C into a refrigerator for subsequent use. For conidial inocula, same procedures was followed upto the inoculation of antagonist in the medium (PDB) but incubated for 9 days instead of 3-4 days as in case of mycelial preparation. The conidia of Trichoderma isolates was separated from the mycelial mat by shaking the conical flasks clockwise and anticlockwise, the conidial suspension was collected into sterilized conical flask and centrifuged it at 6000 rpm for 10 min. The supernatant was decanted out from the centrifuge tube and pellets formed in the bottom of centrifuge tube were again resuspended in sterilized distilled water. The required concentration of conidial suspension was prepared by adding the sterilized distilled water and was used immediately Rhizosphere colonization The rhizosphere competence of test isolates of Trichoderma was conducted with Chickpea (Cicer arentium) using natural, sundried and sterilized soils. Two kg potting mixture of soil (natural, sundried and sterilized soil) and farm yard anure (2:1 v/v) was mixed thoroughly with 20 g of sorghum bran (20%) formulation of Trichoderma isolates (1 x 10 8 cfu/g of product), MHC of the potting mixture was adjusted to 50% and filled into the earthen pots. six seeds of chickpea were sown per pot. The experiment was replicated four times and seeds were allowed to germinate. The pots were supplied with irrigation water whenever required to maintain the MHC of potting mixture to 50%. The rhizosphere soil was collected by gently uprooting the chickpea plant and brushing the soil adhered to roots after 15 DAS. The observation was repeated at 15 days interval and the data were recorded upto 75 DAS. The rhizosphere soil collected from chickpea crops was mixed thoroughly in each case and the rhizosphere population of Trichoderma spp. was estimated by soil dilution technique (Dhingra and Sinclair, 1995). One Gram of soil samples of each isolate-crop combination was suspended in 10 ml sterilized distilled water. The suspension was serially diluted upto 10-8 and 1 ml each of these aliquot was plated on modified TSM with five replications. The inoculated Petri dishes were incubated at 28 ± 1 C for 7 days and the number of colony forming units per g of rhizosphere 64

15 soil for each isolate and test crop were counted. The total number of cfu/g of rhizosphere soil was the rhizosphere population of Trichoderma spp Competitive parasitic ability This experiment was conducted in two types of soils viz., natural and sterilized soils used to assess the ecological adaptability of Trichoderma spp. to a soil which is geographically located at different places. 200g air dried (natural and sterilized soil) was mixed thoroughly with mycelia and conidial inocula of test isolates of Trichoderma separately, moisture holding capacity of each soil type was adjusted to 50% and fitted into the earthen cups (100 ml). 25 sclerotia of S. rolfsii was buried cm deep, covered with perforated aluminum foil and incubated at 28 ± 1 C for 7 days. The sclerotia of S. rolfsii was harvested separately by floatation and sieving method (Rodriguez-Kabana et al., 1974) and plated on modified Trichoderma selective medium (TSM) after surface sterilization with 1.0% sodium hypochlorite solution for 2-3 min. and air dried. The Petridishes seeded with sclerotia of S. rolfsii, were incubated at 28 ± 1 C for 7 days and the sclerotia with colonization by Trichoderma spp. in each treatment were recorded. The per centage colonization of sclerotia of pathogens by Trichoderma spp. was calculated by dividing the number of sclerotia yielding Trichoderma by total number of sclerotia seeded in the Petridishes Production of sclerotia of S. rolfsii For production of sclerotia of S. rolfsii, mycelial plug (6 mm) of the fungus was inoculated into potato dextrose agar (PDA) medium and incubated at 28 ± 1 C for 25 days. The sclerotia were picked up by sterilized spatula. The sclerotia of S. rolfsii were collected, washed, air dried, surfaced sterilized with 1.0% sodium hypochlorite solution, again air dried and either used immediately or preserved at 4 C into the screw cap test tubes for subsequent use SEED PRIMING WITH BIOAGENTS The biopriming of seeds chickpea were done with 20 Trichoderma conidial forms of inoculum were prepared as described earlier. chickpea seeds were thoroughly washed with distilled water, air dried and finally dipped into the suspension of bioagents for few minutes, stirred thoroughly to ensure uniform 65

16 coverage of seeds with suspension of bioagents. The treated seeds were spreaded on a cleaned blotter paper and allowed to shade dry. The treated seeds were seeded into Petri dishes lined with double layered moist blotter paper and covered with upper lid of Petri plate lined with moist blotter paper and incubated for one week at 28 ± 1 C. Treated seeds were sown in soil under greenhouse conditions. Seeds were sown in the earthen pots (30 diameter at 10 seeds/pot containing soil and manure in the ratio of 1:1. The germination of seeds was observed periodically and the root length, shoots length, root and shoot weight under wet and dry condition was measured. The vigour index I and II of respective crop seedlings were calculated on the basis of root and shoot length as follows: Vigour index I = [Root length (cm) + shoot length (cm)] x germination %) Vigour index II = [Dry weight of seedlings x germination %) 3.10 FIELD EFFICACY OF POTENTIAL TRICHODERMA SPP. AGAINST DRY ROOT OF CHICKPEA Field trial of some isolates of Trichoderma against dry root rot of Chickpea was conducted at RARS, Nandyala during , under sick plot condition by artificial inoculation of R.bataticola in the experimental plots. The cultivar JG-11 was sown at a spacing of 30 x 10 cm 2 in Randomized Block Design (RBD) with three replications. Pod yield and test weight measured at the time of harvest and per cent germination after 20 days. The detail treatment schedules followed is as follow: Treatments T.No Treatments T 1 Seed treatment with KNN 4 T 2 Soil application with KNN 4 T 3 T1+T2 T 4 Seed treatment with ATPU 1 T 5 Soil application with ATPU 1 T 6 T4+T5 T 7 Seed treatment with KNPG 3 T 8 Soil application with KNPG 3 T 9 T7+T8 T 10 Seed treatment with ATPP 6 T 11 Soil application with ATPP 6 T 12 T10+T11 Control T 13 66

17 Mass multiplication of R.bataticola Materials and Methods The pathogen R.bataticola was mass multiplied on sterilized sorghum grain. For this, 100 g of sorghum seeds were washed thoroughly in tap water and soaked in water for overnight in 250 ml conical flask with addition of 20 ml of 4% dextrose. The flasks were then autoclaved for 20 min at 15 psi. After cooling the flasks at room temperature, they were shaken well to separate the sterilized grains and were inoculated with 2-3 discs of 4 days old cultures of M. phaseolina and incubated at 28 ± 2 C for 7 days. After seven days, the inoculum was mixed with soil in 1kg/plot (Rajeswari et al., 1999) Mass production of Trichoderma isolates Preparation of powder formulation was carried by using stationary culture method. The biomass from 15 days culture of Trichoderma in flasks was used for preparation of formulation. The biomass along with medium in conical flasks was mixed with a carrier in the ration 1:2. The mixture is air dried for 3 to 4 days and blended to have a flour powder to which 5 g of Carboxy methyl cellulose was added (Jeyarajan et al., 1994). Sorghum-grain flour (50 g) was taken in each 250 ml conical flask and 10 ml tap water was added for adjusting moisture to 50 per cent (W/V).Mass multiplication of Trichoderma spp. with the substrate were inoculated with 2 to 3 discs of seven days old culture of potential Trichoderma spp. separately into conical flasks and incubated at room temperature for 28 ± 2 C Mass multiplication Trichoderma on FYM It is inexpensive and cheaply available material for multiplication of inoculum in large scale. It takes days for the colonization of the FYM substrates. Farm yard manure Sorghum grain multiplied Trichoderma : 10 kg : 200 gms Spread thick layer of FYM on ground under shade, and above it add a layer of Trichoderma multiplied sorghum grains and above it spread FYM. In the same 67

18 manner, make a heap with alternate layers of FYM and sorghum grains. Cover the heap with gunny bag and sprinkle water daily to maintain required moisture content (Kousalya and Jeyarajan, 1988) STATISTICAL ANALYSIS In the present investigation, all data were given appropriate statistical treatment before describing the results obtained. The results obtained invitro and invivo/field tests were subjected to CRD (Completely Randomized Design) and RBD (Randomized Block Design), respectively for their appropriate statistical treatment under one or more (interaction of two or more factors) factors. All the data were studied for the significance of results at 0.05% by using SAS software 9.3 and means were compared with Tukey s honestly significant difference (HSD) and the relevant data have also been indicated in each table. The antagonistic data were analyzed by one way ANOVA (analysis of variance). 68