Antagonistic Potential Improvement of Edible Mushroom, Genus Pleurotus and Its Protease for Controlling Root-Knot Nematode (Meloidogyne incognita)

Abstract

Edible mushroom Genus Pleurotus have been reported as the antagonistic fungi against plant-parasitic nematodes. One of the main rules of their nematicidal ability is the production of protease, that is important to degrade cuticle and eggshell of nematodes, and affect the development of nematode juvenile. Therefore, the objectives of this study were (1) to select Genus Pleurotus for controlling root-knot nematode (Meloidogyne incognita) in laboratory and control root knot disease in tomato under greenhouse condition, (2) to find out the nematicidal efficiency of crude protease from Pleurotus, and (3) to find out the optimal factors for protease production under the laboratory condition. From eight isolates of tested Pleurotus, P. pulmonarius No.3 and P. ostruatus showed the ability to inhibit egg hatching and increase the mortality of infective juveniles. These two Pleurotus could produce protease when primary detected by agar diffusion method. Specific activity of protease under protein-enriched media was 0.27 ± 0.04 U/mg protein from P. pulmonarius No.3., that higher than protease produced from P. ostruatus (0.06 ± 0.00 U/mg protein). Therefore, P. pulmonarius No.3 was selected to find out the optimal condition affecting protease production under solid state cultivation in the laboratory. It was found that the highest protease activity was 0.41 ± 0.00 U/ml when cultured on solid state media contained 6 grams of coarse oats, 6-8 ml of 1.5-2.5 % (w/v) KH2PO4 pH 5.0, incubated at 28 oC for 7 days, and 50 mM acetate buffer pH 5.0 was used for protease extraction. The activity of crude protease stabilized when incubated at 40-50 oC for 4 hr and the activity was decreased when incubated at 60-70 oC for 1 hr. At -20 oC was the optimal temperature for long term storage of crude protease for over 6 months. Under laboratory condition, the optimized Pleurotus (P. pulmonarius No. 3), called “OP”, completely killed infective juvenile (J2) of root-knot nematode (percentage of mortality as 100) at 4 day post inoculation and OP in combination with crude protease inhibited nematode egg hatching 100 %, as well. Regarding under greenhouse condition, the number of root gall 35.67 ± 5.24, egg masses 15.00 ± 0.89 and reproduction factor (Rf) was 0.68 ± 0.02 when tomato plants were treated by the OP in concentration of 14 g per pot (OP2). There was no significant difference when compared to OP2 in combination with crude protease, the number of galls, egg masses and Rf were 37.67 ± 2.88, 17.33 ± 1.37 and 0.61 ± 0.02, respectively. However, the treatments of individual OP2 and OP2 in combination with crude protease significantly inhibited root gall disease in tomato (p<0.05) when compared to untreated control. After staining with acid fuchsin, slowly developing of J2 to 3rd stage juvenile (J3) or 4th stage juvenile (J4) and to adult female was presented in tomato roots with OP treatment.