Pure culture of root-knot nematode inoculum
M. incognita was the tested species of root knot nematode, identified from nematode adult female on the basis of the morphological characteristics of the female perineal pattern (Taylor and Sasser 1978). Pure culture of M. incognita was reared on eggplant cv. Ice in a screen house of Nematology Lab., Plant Pathology Department, National Research Centre at 30 ± 5oC by using a single egg mass of this nematode. Newly hatched second stage juveniles (J2s) and eggs were used as inocula.
Fungus culture
Isolates of B. bassiana, M. anisopliae, and P. lilacinus were obtained from Assiut University, Mycological Center, Faculty of Science. The isolates were cultured on Sabouraud dextrose yeast agar (SDYA) medium (Sabouraud 1892) which contained 40 g glucose, 20 g peptone, 20 g agar, and 2 g yeast extract in 1000 ml of distilled water in flasks which were autoclaved at 21 °C for 15–20 min.
Preparation of spore suspension
Fungal cultures grown on Sabouraud dextrose yeast agar (SDYA) medium were incubated at 25 ± 2 °C in darkness for 14 days. Conidial medium suspensions were prepared by scraping cultures with a sterile objective glass and transferred to 10 ml of sterile water containing 0.05% Tween 80 in a laminar flow chamber. The conidia were harvested by scraping the surface of the culture with inoculation needle. The mixture (spores+ hyphae) was stirred for 10 min and the hyphae were removed by filtering the mixture through fine mesh sieve. The conidial concentration of final suspension was determined (1 × 108 viable conidia) by direct count using hemocytometer. Serial dilutions were prepared in distilled water containing 0.1% Tween 80 and preserved at 5 °C until used. In vitro nematode tests were applied to evaluate efficacy of fungal spores against root knot nematode, M. incognita eggs. A volume of the adjustable concentrations (1 × 106, 1 × 107, and 1 × 108) viable conidia were directly applied to the eggs.
Preparation of supernatant
The filtrates of B. bassiana, M. anisopliae, and P. lilacinus were produced on broth semi-synthetic Sabouraud dextrose yeast. The medium was prepared and adjusted at PH (5.5–6.6). After sterilization, flasks were inoculated with the fungal species and incubated for 2 weeks at 25 °C and 50–60% Rh. At the end of the incubation period, the supernatant was separated from the mats by filtration through Whatman filter paper No.1 under aseptic conditions and the supernatant at different dilutions [S (Standard), S/2 and S/] were used for bioassay against nematodes (Barker 1985).
Laboratory tests
In vitro test was carried out to determine the effect of culture filtrates of the studied fungi, B. bassiana, M. anisopliae, and P. lilacinus, at dilutions, S, S/2, and S/4 M. incognita egg hatching from infected tomato roots. Eggs were extracted by Clorox (NaOCl 1.0%.), then the suspension was poured onto a 500 mesh sieve and washed by excess tap water to remove NaOCl (Hussey and Barker 1973). Then, extracted eggs were transferred to into clean beaker with sterilized water. One milliliter of distilled water containing 300 nematode eggs was put in plastic capsule with 9 ml of each fungal filtrate dilution. Control treatment was made by adding 1 ml of distilled water containing 300 nematode eggs to 9-ml distilled water as comparison. There were 5 replicates for each treatment.
Also, in vitro test was applied to evaluate efficacy of three conidial spore concentrations from B. bassiana, M. aniisopliae, and P. lilacinus against root knot nematode, M. incognita eggs. Concentrations of 1 × 106, 1 × 107, and 1 × 108 viable conidia were directly applied to eggs by adding 1 ml distilled water containing 300 eggs in plastic capsule with 9 ml of each fungal spore’s suspension concentration. Equal number of eggs was also transferred to separate plastic capsule containing 9-ml distilled water to serve as control.
Observations on the number of non-hatched eggs by light microscope were made 24, 48, 72, and 96 h after treatment. Data on non-hatched eggs were converted to the percentages of egg inhibition at each period and dilution according to Abbott’s formula (Abbott 1925) as follows:
$$ \mathrm{Egg}\kern0.28em \mathrm{inhibition}\ \left(\%\right)=\left(m-n\right)/\left(100-n\right)\times 100 $$
where m and n stand for the percentages of non-hatched eggs in the treatment and control, respectively. Net percentage egg inhibition was calculated by subtracting percentage of recovery (hatched eggs in distilled water) from the percentage inhibition after 96 h.
Mortality of second stage juveniles (J2)
For determining the effect of fungal filtrates of B. bassiana, M. anisopliae, and P. lilacinus on second stage juvenile mortality (J2) of M. incognita, the number of J2 in the soil per pot was extracted using a sieving and decanting technique (Barker 1985) and counted. For extraction of second stage juveniles (J2) of M. incognita from roots, galled eggplant roots with egg masses per plant were washed thoroughly with tap water to avoid debris and cut into small pieces. Then, they were placed in plastic capsule containing sufficient water, covered to avoid loss of water by evaporation, and collected every 24 h (Young 1954). The same procedures were carried when 1 ml of distilled water containing 200 J2 was added to 9 ml of filtrate of each fungus. Control treatment was made by adding 9 ml of distilled water to 1 ml of nematode suspension containing the same number of nematodes.
Number of dead and alive juveniles per each treatment was determined under light microscope 24, 48, and 72 h after treatment. The J2 were considered dead when they did not move when probing with a fine needle. Data on nematode mortality were converted to the percentages of nematode mortality according to Abbott’s formula (Abbott 1925) as follows:
$$ \mathrm{Juvenile}\ \mathrm{mortality}\kern0.28em \left(\%\right)=\left(m-n\right)/\left(100-n\right)\times 100 $$
where m and n are for the percentages of dead juveniles in the treatment and control, respectively. Net percentage of mortality was calculated by subtracting percentage of nematode recovery in distilled water from the percentage of mortality after 72 h.
Screen house experiments
Pot experiment design
The experiment was carried out in pots in screen house of Plant Pathology Department, National Research Centre (NRC). Seeds of cowpea (Vigna unguiculata (L.) Walp.) cv. Baladi were sown in each pot in April 5, 2018 in pots (20-cm diameter) containing 2 kg of solarized sandy loamy soil. Each pot was inoculated with 2000 newly hatched juveniles (J2) + 1000 eggs of M. incognita in April 19, 2018. This inoculum was made in four holes made around the plant. At the same time of nematode inoculation, cowpea plants were treated with the tested three cultural filtrates of B. bassiana, M. anisopliae, and P. lilacinus. These fungi were tested at dilutions, S, S/2, and S/4 at the rate of 10 ml per pot from each dilution in four holes around the plant and nematode only with liquid medium (control) used as untreated check. Pots were arranged in a completely randomized design with 5 replicates for each treatment on a bench under screen house conditions maintained at 30 ± 5 °C. Then, the plants were irrigated as needed.
After 3 months of nematode inoculation (harvest stage of cowpea plant) in July 2018, plants of cowpea were carefully uprooted and roots were washed thoroughly with running tap water to avoid debris. Then, roots were cut into two halves. Numbers of J2 in soil and roots, egg masses, as well as number of galls per plant were counted in one half of roots. The number of J2 in the soil per pot was extracted using a sieving and decanting technique (Barker 1985) and counted. Then, the second half of roots was incubated in tap water by incubation method (Young 1954) to help hatching J2 from egg masses. All J2 numbers of nematodes were counted under a light microscope.
At the same time, plant growth criteria of cowpea including shoot length (cm), fresh and dry shoot weights (g), and fresh and dry weights of roots (g) were recorded. Also, number and weight of pods (g) were recorded.
Also, in the second experiment, the same procedures were applied except that three concentrations of 1 × 108, 1 × 107, and 1 × 106 spores of the tested fungi at the rate of 10 ml per each concentration were tested. Mean percentages of nematode reduction, plant growth, and yield increases were calculated by dividing sum percentages of all parameters of each treatment/number of these parameters. This measurement was used to compare among treatments within all groups.
Statistical analysis
This experiment has been carried out according to analysis of variance (ANOVA) procedures. Duncan’s multiple range test as reported by Snedecor and Cochran (1989) was used for comparing among treatments at 5% level of probability. This was done by Computer Statistical Package (COSTAT) User Manual Version 3.03, Barkley Co.