Time and Location of Study Colonisation Test

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III. MATERIALS AND METHODS

3.1 Time and Location of Study

This research was conducted in Nematology Laboratory Department of Plant Protection, Faculty of Agriculture and in greenhouse at Cikabayan, Bogor Agricultural University, Bogor West Java Province, Indonesia from February to August 2009. 3.2 Exploration of Endophytic Fungi 3.2.1 Isolation and identification of endophytic fungi Healthy and nematode infected tomato plant samples were collected for isolation of endophytic fungi from tomato plant roots in highland and lowland areas. The method used for isolation of endophytic fungi was that proposed by Rodrigues 1994 that has already been modified. Tomato plant roots were washed thoroughly using flowing water to remove all the soil particles from the roots. Sterilization of the root surface was done by dipping the roots in 70 ethanol for one minute, and then to 1 NaOCl for three minutes after which the roots were rinsed three times with sterile water then dried on dry sterile blotting paper. The roots were then cut into small pieces and placed on petri dishes already filled with PDA under laminar airflow, in each petri dish three root pieces were placed and replicated three times then incubated at room temperature and observed for a period of one week, after which the fungal colonies that developed from the cut root tissues were purified on new PDA media and the isolates that developed were tested for their pathogenicity potential then identified based on colony colour and morphology as well as observation of microscopic features using identification keys according to Watanabe 2002.

3.2.2 Selection of endophytic fungi based on pathogenicity test

To ensure that the isolated endophytic fungi were not pathogenic and produce disease symptoms to the host plants later after inoculation then there was high need to test for the pathogenicity potential of the isolates to be used, this was 24 done by growing tomato seeds on the petri dish containing pure colonies of the isolated fungi. The fungal colonies where tomato seeds grew were proved to be non pathogenic while colonies where there were no growth at all or growth was inhibited were proved to be potential pathogenic isolates, selection of endophytic and pathogenic isolates was based on the pathogenicity test, the seeds were also grown on a control petri dish filled only with PDA. At this stage 12 out of the 20 isolates were proved to be potential isolates of endophytic fungi and were used for further inoculation. The resulting isolates of endophytic fungi included both sporulating and non-sporulating fungi. These isolates were further used for in planta test to assess their effect against root-knot nematodes as well as growth promotion in tomato plants, and in vitro test to estabilish their antagonistic mechanism against the RKN.

3.2.3 Inoculation of seeds with suspension spore

The potential endophytic fungi selected that were preserved in test-tube agar slopes in the refrigerator were re-cultured on 60 mm diameter petri dishes filled with PDA enriched with 50 mg streptomycine-sulphate per litre to prevent bacterial contamination all these were incubated under laboratory conditions with natural photoperiod of 12 hours daylight and darkness for 7 days. Spore suspension for inoculating tomato seeds were produced in 500-ml erlenmeyer flasks containing 200 ml of half strength potato dextrose broth PDB. Half strength PDB was prepared by dissolving 12 g of PDB per litre of sterile distilled water the flasks containing PDB were sterilized by autoclaving at 121°C for 15 minutes then allowed to cool down. Mycelia blocks of each endophytic fungi isolate were cut from 1 week old culture on PDA and aseptically transferred to PDB under laminar airflow. Two replicate flasks were used for each endophytic fungi isolate. Two flasks containing non-inoculated PDB were used as controls. Flasks were incubated under laboratory conditions using rotary shaker for two week to allow for fungal sporulation and to disperse spores throughout the PDB medium. The fungal spores were harvested by filtering the suspension through a sterile cheese cloth to remove mycelial fragments. The spore density of sporulating fungi was then estimated using the heamocytometer and the 25 suspension was standardized to provide a final spore concentration of 1.5 × 10 6 sporesml. Tomato seeds were soaked in the already made spore suspension culture of each endophytic fungi for 12 hours after which the seeds were planted on the sterilized soil media that was already prepared on planting trays. The seeds were left for germination and routine cultural practices were carried out.

3.2.4 Re-inoculation of tomato plants with suspension spores

At transplanting stage tomato seedlings were selected based on their uniformity in size and growth vigour, the selected seedlings were transplanted followed by immediate reinoculation whereby suspension spore of each particular endophytic fungi isolate were watered at the holes made around the roots of the tomato plants. The flasks containing the suspension spores were shaken occasionally to resuspend the spores in the solution. The plants were then maintained in the greenhouse conditions with frequent cultural practices carried out on daily basis.

3.3 Colonisation Test

To detect endophytic fungi colonization of plants, several methods for in-situ detection of endophytic fungi in plant tissues have been developed, microscopic examination of differentially stained samples of endophyte infected plants Saha et al. 1988, other methods for in-situ detection of endophytes include the use of monoclonal antibodies Hiat et al. 1997; Hiat et al. 1999, tissue printing immunoblotting Gwinn et al. 1991, tissue print immunoassay Hahn et al. 2003, electron microscopy Sardi et al. 1992 and autoradiography You et al. 1995. In this research re-isolation was done to determine the ability of the endophytic fungi to colonise tomato plant tissues. Re-isolation was done from the roots three weeks after re-inoculation of endophytic fungi to tomato plants at transplanting stage, where by the roots of seven tomato plants to be used for re-isolation per endophytic fungi treatment were selected at random from each treatment, surface sterilised by dipping the roots in 70 ethanol for one minute, then to 1 NaOCl for three minutes to eliminate root epiphytic microorganisms 26 after which the roots were rinsed three times with sterile distilled water then dried on dry sterile blotting paper. The sterilized roots were cut into small pieces under laminar airflow and plated on PDA medium in 60 mm diameter petri dishes, from each tomato plant root, three root segments were plated per petri dish and the petri dishes incubated for seven days under laboratory conditions. Frequency of colonization was determined by observing the total number of roots samples per treatment where colonies of endophytic fungi emerged.

3.4 Meloidogyne incognita Egg Mass Inoculation