These results demonstrated that the LY2835219 in vivo nematicidal ingredients were could not be evaporated and possessed a molecular weight of <1000 Da. After the supernatants were extracted using three organic solvents, the aqueous solution of the respective extracts and products in the aqueous phases were tested in the presence of nematodes, respectively. The aqueous solution corresponding to each of the three organic extracts had no nematicidal activity but the substances in each of the three aqueous phases resulted in 100% mortality rates of M. javanica juveniles at 12 h. These results demonstrated that the active nematicidal substances present in the supernatants were strongly polar and could

not be extracted using organic solvents such as ethyl acetate, chloroform or butanol. To test the stability of the nematicidal properties, extracts were subjected to Pifithrin-�� cold or heat. Regardless of the ‘inactivation’ strategy, extracts retained 100% efficacy following a 12-h incubation with M. javanica juveniles, suggesting that the nematicidal active ingredients were highly stable. OKB105 strain mutants were constructed using the pMarA plasmid to identify nematicidal-associated genes. Approximately

2000 kanamycin-resistant mutants were isolated and screened for nematicidal activity. One nematicidal-defective strain was identified and designated M1 (Table 4). Southern blot analyses were used to verify whether the TnYLB-1 transposon was inserted

into the M1 genome. A 0.14-kb probe was generated by PCR by amplifying an internal fragment of the TnYLB-1 kanamycin-resistance gene using primers P11/P12. Because there were no EcoRI restriction sites in TnYLB-1, the M1 chromosomal DNA was digested with EcoRI; hybridization identified a single band in the M1 mutant (Fig. 1), indicating that a single transposon was inserted into the M1 genome. To determine which M1 gene was disrupted, inverse PCR was performed using primers P13/P14. Amplified DNA was sequenced using the P15 primer and sequences aligned against the 168 sequences constituting the B. subtilis genome. The results demonstrated that the Dimethyl sulfoxide purL gene of the M1 mutant had been disrupted by the TnYLB-1 transposon, which located at 1314 bp downstream of the ATG start codon of the purL gene. The purL gene encodes a 5′-phosphoribosylformylglycinamidine synthase II (FGAM synthase II, EC 6.3.5.3) (Saxild & Nygaard, 1988); in B. subtilis it is positioned between the purQ and purF genes of the purine biosynthetic operon. The B. subtilis pur operon is organized into three groups of overlapping genes, followed by the last gene: purE-K-B; purC-S-Q-L-F; purM-N-H; and purD (Saxild & Nygaard, 2000). The FGAM synthase catalyzes the conversion of 5′-phosphoribosyl-N-formylglycinamide (FGAR) into 5′-phosphoribosyl-N-formyl-glycinamidine (FGAM) in the de novo purine nucleotide biosynthetic pathway.