23
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
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