Significant medium components for chitinase and gelatinase production by L. capsici YS1215 was optimized by statistical methods. Fractional factorial screening technique and central composite design were used to optimize the medium composition for the production of chitinase and gelatinase from L. capsici YS1215. The optimum conditions identi？ed were nutrient broth of 2.0 g/l, crab shell powder of 2.0 g/l and gelatin of 1.0 g/l. The statistical experimental approach was found effective in optimizing the medium components with 3.76, and 1.1 fold of the prior chitinase and gelatinase production, respectively. Crude enzymes obtained by precipitation with 80% ammonium sulfate were found in inhibition of pathogenic fungal mycelia against Colletotrichum gleosporioides, Phytophthora capsici, and Rhizoctonia solani.
The root-knot nematodes, Meloidogyne spp., are important pathogens causing serious diseases in various plants in agriculture. The bacterial strain, Lysobacter capsici YS1215, was isolated from the soil. In the present study, we investigated control of the root-knot nematode by the strain YS1215 in in vitro condition. One of the potential mechanisms against the Meloidogyne spp. may be the chitinolytic activity against the nematodes’ eggs. The chitinase secreted by L. capsici YS1215 was purified by protein precipitation with 80% ammonium sulfate, DEAE-Cellulose and Sephadex G-100. By chitinase active staining of the chitinase, a single band was obtained with an estimated molecular weight of 43.6 kDa. The optimal pH and temperature of the chitinase were 6.0 and 40oC, respectively. The chitinase degraded chitin layer of the shells of the nematodes’ eggs and reduced the egg hatch significantly. The activity of chitinase secreted by L. capsici YS1215 was not affected by ZnCl2, MnCl2, MgCl2, CuSO4 and CaCl2. The chitinase activity was enhanced by EDTA, while it was strongly inhibited by HgCl2 and AgNO3. The purified enzyme could also hydrolyze swollen chitin, glycol chitin, glycol chitosan, and chitin powder.
The effect of different concentrations of bacterial culture filtrate (BCF) of L. capsici YS1215 on the mortality of second-stage juveniles (J2) of Meloidogyne incognita was studied using 24-well plates. J2 mortality increased with increasing concentrations of BCF. To investigate role of gelatinases in the culture filtrate, the partial purification and characterization of gelatinolytic proteins was done. The partially purified proteins showed three clear bands with molecular weights estimated using zymography to be 255.7, 232.1, and 146.4 kDa. The optimal pH and temperature for the proteins were 8.0 and 40oC, respectively. EDTA, FeCl3, and 1, 10-phenanthroline inhibited the activity of the proteins, while it was activated by MnCl2. Moreover, the proteins could hydrolyze skimmed milk, collagen, gelatin, and bovine serum albumin as substrates, but not casein. The proteins could induce 75% J2 mortality in 5 days and degrade J2 bodies.
Lactic acid was isolated from culture filtrate of the YS1215, and its ovicidal activity was investigated. Purification and identification of lactic acid was performed by series of column chromatographies and identified by 1H and 13C-NMR spectrum and GC-MS analysis. Our results showed that bacterial culture filtrate containing lactic acid significantly inhibited egg hatch. The lowest egg hatch rate (5.9%) was found in high concentration (1,250 mM) of lactic acid at 5 days after incubation, followed by 1,000 (15.2%), 750 (23.7%), 500 (29.8%) and 250 (36.4%) mM while egg hatch in control (sterile distilled water) was 44.5%. Additionally, crude compounds extracted from bacterial culture filtrate with ethyl acetate also showed significant suppression of egg hatch.
Chitinase and gelatinase activities were induced by nematode eggs and juveniles supplemented in medium as substrates, and confirmed by SDS-PAGE showing active bands in chitinase active staining and gelatin zymography, respectively. The results of this study clearly indicated that applications of bacterial culture and crab shell powder significantly increased the chitinase and gelatinase activities as well as population of chitinolytic and gelatinolytic bacteria in the rhizosphere. Furthermore, bacterial culture of L. capsici YS1215 applied for tomato plants also promoted growths on shoot length due to decrease in the severity of diseases caused by the root-knot nematodes in comparison with control (fertilizer treatment). Hence, the results clearly demonstrated that L. capsici YS1215 and its lytic enzymes played important roles in suppression of M. incognita infection.