Nematodes used for the experiment were reared on tomato seedlings of cv Early Urbana inoculated with 5000 second-stage juveniles (J2) as explained in Ebadi et al. (2009). At harvest, plant roots were washed, cut into pieces, placed in a jar of 0.5% commercial NaOCl and shaken for 4 min. The suspension was washed with tap water through 75- and 20-μm sieves and their numbers were counted with a counting slide under a light microscope (Hussey and Barker 1973).
The four strains of P. chlamydosporia var. chlamydosporia (Pcc isolated with the accession numbers of Pcc10, Pcc20, Pcc30 and Pcc60) used in this study had been maintained on corn meal agar at 5 °C in the Nematology Department Collection, Iranian Research Institute of Plant Protection, Tehran.
The fungal inoculum preparations were made according to the procedure of De Leij et al. (1993). Conical flasks were filled with a mixture of sand + milled barley (1:1 v/v) and 30 ml distilled water was added for each 100 g of mixture and autoclaved at 121 °C for 20 min on two consecutive days. Flasks were inoculated with plugs of isolates, kept at 25 °C and occasionally shaken for even growth. After 3 weeks, 5 g of the sand/barley substrate, mixed well with 100 ml distilled water, was transferred to a blender (Waring) and blended for 2 min. The contents were washed onto a 45-μm aperture sieve, and the chlamydospores were collected on a nested 10-μm sieve (De Leij et al. 1993). The chlamydospores were counted by a hemocytometer.
A suspension of washed chlamydospores was mixed with 50 g sterilized sand, added to 1 kg natural soil collected from a pistachio orchard, and the resulting mixture was used to fill a 14-cm diameter plastic pot, to give a final count of 1 × 104 chlamydospores/g soil. This spore density was used for all four fungal isolates and two further sets of chlamydospore densities were prepared to give 1 × 103 and 5 × 103 chlamydospores/g soil for isolate Pcc60 only and designated Pcc60A and Pcc60B respectively and Pcc60C (1 × 104 chlamydospores/g soil).
The nematode genera present in soil were mostly non-parasitic nematodes with very few Tylenchidae, and there were no root-knot nematodes present.
Pistachio cv Kaleghochi was chosen for the experiment. Prior to planting, seeds were disinfested for 4 min in 1% NaOCl, rinsed with sterile distilled water, immersed in 1% pentachloronitrobenzen, followed by soaking overnight in sterilized water and then pre-germinated in the dark on moist filter paper in Petri dishes.
One seedling was planted in each pot and allowed to establish for a month, when each relevant pot was inoculated with a suspension of nearly 3000 eggs of M. javanica added to three holes around each plant. Treatments included: nematode + isolate of Pcc10, Pcc20, Pcc30 and Pcc60C (10,000 cfu/g soil); nematode + isolate Pcc60B (5000 cfu/g soil), nematode + isolate Pcc60A (1000 cfu/g soil), nematodes alone and pistachio alone. Each treatment was replicated five times, and the pots were arranged in a completely randomized design on a glasshouse bench, with average temperature of 27.5 °C, and irrigated as required.
Plants were harvested after 4 months. Roots were washed in water, blotted gently dry and the fresh weights of the pistachio shoots and roots were taken. The numbers of galls or egg masses on roots were rated based on the 0–5 scale of Hartman and Sasser (1985).
To determine nematode multiplication rates, eggs were extracted from the roots by the NaOCl, using the same procedure as described above (Hussey and Barker 1973), treated roots were further processed in a blender to extract any possibly hidden eggs within the roots. The extracts were filtered through 75- and 20-μm sieves, and the numbers of eggs were estimated from the contents of the latter.
The populations of J2 were measured in 200 g soil from each pot of each treatment combination by means of modified Whitehead trays (Whitehead and Hemming 1965). The final total population density of healthy nematodes was calculated by combining the total numbers of J2 and healthy eggs. For estimation of the numbers of healthy eggs, the total numbers of infected eggs were subtracted from the total numbers of eggs. A reproduction factor was estimated by dividing the final nematode population density by the initial population density (Pf/Pi) (Ebadi et al. 2009).
To verify the percentage of egg infection, 10 egg masses/replicate (there were few egg masses at the time) were incubated in 0.05% NaOCl between a glass slide and coverslip and observed under × 400 magnification. To re-isolate and confirm identification of these infected eggs, a 0.2-ml sub-sample of a suspension made from these eggs was taken, spread over the surface of 0.8% water agar, containing 50 ppm each of tetracycline, chloramphenicol and streptomycin, and the grown hyphae were sub-cultured on PDA for further examination.
Viability and abundance of the fungi in soil and on the roots at the end of the experiment were checked, using a SSM (semi-selective medium) (De Leij and Kerry 1991). A sub-sample of 1 g soil from each replicate was added to 9 ml of 0.05% sterile water agar and used to prepare dilution series of 10− 1 to 10− 4. An aliquot of 0.2 ml of each dilution was transferred onto a 9-cm Petri dish containing SSM, with three replicates per dilution. Dishes were kept in an incubator at 25 °C for 1 or 2 weeks and the final numbers of cfu were counted.
Semi-selective medium contained the following: 37.5 mg carbendazim, 37.5 mg thiabendazole, 75 mg rose bengal, 17.5 mg NaCl, 50 mg each of streptomycin sulphate, chlortetracycline hydrochloride and chloramphenicol, 3 ml Triton X-100, and 17 g corn meal agar (Difco) in a liter of distilled water.
Roots of each replicate were cut into small pieces and then a 1 g sub-sample was rinsed with sterile distilled water, crushed with a sterilized pestle and mortar and added to 9 ml of 0.05% sterile water agar solution. As in the soil test, a dilution series was prepared and the suspensions were transferred onto the SSM. Colonies were counted after 1 week (Bourne et al. 1996).
All data were subjected to analysis of variance (ANOVA), using SPSS version 16. Data were checked for homogeneity of variance before being pooled. Data for percent egg infection, the reproduction factor and number of healthy eggs and J2/pot were transformed to arcsin (x), sqrt (x + 0.5) and log (x + 1), respectively, before ANOVA. Means were separated using the least significant difference (LSD) test (P ≤ 0.05).