In this study, we investigated the nematicidal effects of Bacillus spp. against plant-parasitic nematodes and identified potential genes associated INCB024360 molecular weight with nematicidal activity. The bacterial strains

and plasmids used in this study are described in Table 1. Escherichia coli strain DH5α was used as the host for all plasmids. The pMarA shuttle plasmid carries the TnYLB-1 transposon, the mariner-Himar1 transposase and a Gram-positive thermosensitive replicon (Breton et al., 2006). New plasmids and strains constructed in this study are described in the text. Luria broth (LB) was used for growing E. coli, Bacillus spp. and the OKB105 mutants. Landy culture medium was used to ferment all Bacillus isolates and mutants (Landy et al., 1948). When required, Landy medium was supplemented with adenine (12 μg mL−1) and thiamine (0.8 μg mL−1), with 5-aminoimidazole-4-carboxamide riboside (AICA-riboside, an intermediate in the purine biosynthesis pathway, 4 mM), with O-diazoacetyl-l-serine (azaserine, 550 μM) or with sulfamethoxazole (250 μM), separately. Bacterial cultures were grown in 250-mL flasks with shaking (200 r.p.m.) at 37 °C for 48 h. When required, antibiotics were added at the following final concentrations: ampicillin (Ap), 100 μg mL−1; chloramphenicol (Cm), 5 μg mL−1; kanamycin (Km), 5 or 50 μg mL−1. Nematodes

used in this study are described in Supporting Information, Table S1 and maintained as described (Iwahori Loperamide et al., 1998; Wang et al., 2007, 2009). The four cultured Selumetinib nematodes were separated using the Baerman funnel technique (Gray, 1984), surface sterilized with 3% H2O2 or 1% NaClO for 5 min and then rinsed three times with sterile distilled water before use. Fermented cultures (50 mL) were centrifuged at 17000 g for 15 min at 4 °C and supernatants then collected and tested for nematicidal activity. Fifty sterilized nematodes were transferred to a 60-mm-diameter watch

crystal with 1.5 mL of the respective supernatants. The watch crystal was placed in Petri dishes and incubated at 25 °C. The method for determining nematicidal activity was based on previously described procedures (Huang et al., 2005) and nematode viability was assessed by observation under a dissecting microscope. The respective supernatants were tested in triplicate and each experiment was repeated five times. Cold resistance of the bacterial metabolites was tested by separately incubating culture filtrates at 4 and −25 °C for 5, 10, 15, 20, 25, 30, 35 or 40 days and subsequently testing for nematicidal activity as described above. Heat resistance was tested by supernatants incubating in a 100 °C water bath for 25 min or sterilizing in an autoclave at 121 °C for 20 min before testing for nematicidal activity. Assessment of nematicidal activity of the culture filtrate components was carried out in one of two ways: 1 100-mL culture filtrates were dried with a vacuum rotary evaporator at 60 °C.