Secondly, the full length of the Cm gene and the partial sequence fragment of pMarA were amplified using primer pairs P7/P8 and P9/P10, respectively. The two sequences were joined together by overlap PCR with primers P7/P10, and the amplified fragments were purified and cloned into the SalI and SphI sites of the pUC18-L vector
to generate plasmid pUC18-purL. This suicide plasmid was transformed into strain M1 to create an integrated mutant (Fig. S1). To identify the double crossover mutants, one of transformants, which were screened on solid LB agar medium supplemented with 5 μg mL−1 chloramphenicol and 50 μg mL−1 kanamycin, was selected and designated M1-2. Primers P5/P6 were used to analyze the M1-2 purL regions. Sequence analysis of the PCR product confirmed that the integration of purL allele was successful. The purL gene of M1-2 was expressed under its own pur operon. The data were statistically analyzed using anova, followed Angiogenesis inhibitor by Fisher’s least-significant difference test (P=0.05) using spss software (SPSS Inc., Chicago, IL). Landy media filtrates collected from cultures of Bacillus strains OKB105, 69 and B3 demonstrated nematicidal activity against Aphelenchoides besseyi, Ditylenchus destructor, B. xylophilus and Meloidogyne javanica and Bacillus strain FZB42 had antagonistic activity against the same panel of nematodes with the exception of PF-02341066 manufacturer A. besseyi.
The 16 different bacteria/nematode combinations were analyzed at 12 h. At this time point, the significant (P=0.05) mortalities of M. javanica, A. besseyi, D. destructor,
B. xylophilus were 100% (OKB105), 10.6% (B3), 27.6% (OKB105) and 35.6% (69), respectively Thiamine-diphosphate kinase (Table 3). Mortality rates for all combinations, however, increased in a time-dependent manner. In addition, M. javanica juveniles were more sensitive to the effects of culture filtrates collected from the four Bacillus spp. than were nematodes of the three other species under similar conditions. Therefore, M. javanica was selected as the target for screening Bacillus spp. nematicidal properties. Culture filtrates of OKB105 significantly (P=0.05) caused 100% mortality of M. javanica juveniles compared with the culture filtrates 69 (84.8%), B3 (80.4%) and FZB42 (46%) at 12 h of incubation (Table 3d). Supernatants collected from four Bacillus spp. resulted in 100% mortality of M. javanica juveniles after 48 h with strain OKB105 possessing the highest level of activity. Controls had no effect on the four nematode species tested at 72 h. Therefore, M. javanica and B. subtilis strain OKB105 were chosen for subsequent studies. The root-knot nematodes M. javanica were incubated in the presence of various extracts derived from strain OKB105 filtrates. This analysis demonstrated M. javanica juvenile mortality of 0% and 100% at 12 h, depending on the fraction tested. That is, solutions B, C, F had no nematicidal activity, whereas incubation with solution A, D or E resulted in a 100%M.