906 Eur J Plant Pathol (2012)133:899-910 1.2 18000 1 16000 14000 1200 0.6 CKG-12G-24G-36G-48R-12R-24R-36R-48 Ro-P Rs.CG-12R-12G24R-24G-36R-36 of R callus 56 day and Rs :R.12.R.24 .R-36:Numb 2h336h :G-12G-24 Rs- R-34 and R-48: b in ner odes soakee 61 Rs-C and Rs-P were two populations ep inoculatin inoculated untreated Rs-C wer sRNA being Rs- (P nd Rs- and Rs- Af with b Th respective atode and did vith in 36 gth of. ime.Ner any t gro from Rs-C had the 0.05 rP=0.05 the RNAi effect of Rs eng-16 o on pa 4 h city of r similis wa also studied na odes The nd plant weight an b dsRNA for 12 h and 24 h th、 :li h d 36 h the diffa Rs-P were significantly lower (P=0.05)than for non (P=0.05)bc nd th noculated with untreated rs-c had lower root weight and above-ground plant weight compared to plants weight w significant (P=005)bet the inoculated with untreated Rs-P.The numbers of nem nd the r atodes in the rhizosphere and the disease severity level Ib dsRNA for 12 h and 24 h,and gip dsRNA for 12 Springerreproduction was studied by respectively inoculating treated and untreated nematodes on carrot callus. The results showed that after being soaked in Rs-eng-1b dsRNA and inoculated onto a carrot callus for 56 days (about two lifecycles of R. similis), Rs-C nematodes treated with Rs-eng-1b dsRNA for 12 h, 24 h, and 36 h had significantly lower (P00.05) reproduction than untreated Rs-C and Rs-P nematodes, and Rs-C nemat￾odes treated with gfp dsRNA, respectively (Fig. 6). The reproduction of nematodes treated with gfp dsRNA was significantly lower (P00.05) than untreat￾ed nematodes and did not decrease with increasing length of exposure time. Nematodes without any treat￾ment from Rs-C had the greatest reproduction. So, with exposure to Rs-eng-1b RNAi treatment increased, nematode reproduction decreased. In addition, the RNAi effect of Rs-eng-1b on path￾ogenicity of R. similis was also studied by respectively inoculating treated and untreated nematodes to anthur￾ium for 56 days. The results showed that above￾ground plant weight and root weight of anthurium inoculated with either untreated R. similis Rs-C or Rs-P were significantly lower (P00.05) than for non￾nematode treated anthurium (Fig. 7a–b). Anthurium inoculated with untreated Rs-C had lower root weight and above-ground plant weight compared to plants inoculated with untreated Rs-P. The numbers of nem￾atodes in the rhizosphere and the disease severity level of anthurium inoculated with untreated Rs-C were significantly higher (P00.05) than for anthurium in￾oculated with untreated Rs-P (Fig. 7c–d). Anthurium plants were inoculated with Rs-C nem￾atodes that had been treated with either Rs-eng-1b dsRNA or gfp dsRNA, or with untreated Rs-C and Rs-P nematodes. After infection for 56 days, fresh anthurium above-ground plants and roots were weighed and compared. The results (Fig. 7a–b) showed that anthurium plants inoculated with the Rs￾C nematodes treated with Rs-eng-1b dsRNA for 36 h had the heaviest above-ground plant and root weights. Its above-ground weight was significantly greater than those in other treatments (P00.05), and its root weight was remarkably heavier (P00.05) than those with gfp dsRNA treatments for 24 h and 36 h, as well as the untreated Rs-C and Rs-P nematodes. For above￾ground plant weight of anthurium inoculated with Rs-C nematodes untreated and treated with Rs-eng- 1b dsRNA for 12 h and 24 h, or with gfp dsRNA for 12 h, 24 h and 36 h, the difference in above-ground plant weight was only significant (P00.05) between the untreated nematodes and the nematodes treated with Rs-eng-1b dsRNA for 24 h. Differences in root weight were significant (P00.05) between the untreat￾ed nematodes and the nematodes treated with Rs-eng- 1b dsRNA for 12 h and 24 h, and gfp dsRNA for 12 h, Fig. 5 Expression of the endoglucanase Rs-eng-1b in Radopho￾lus similis population Rs-C treated with Rs-eng-1b double￾stranded (ds) RNA. CK: expression of Rs-eng-1b in untreated nematodes; G-12, G-24, G-36, and G-48: expression of Rs-eng- 1b in control nematodes soaked by non-endogenous gfp dsRNA solution for 12 h, 24 h, 36 h, and 48 h, respectively; R-12, R-24, R-36, and R-48: expression of Rs-eng-1b in nematodes soaked by Rs-eng-1b dsRNA for 12 h, 24 h, 36 h, and 48 h, respective￾ly. Bars indicate standard errors of mean data (n03) and differ￾ent letters indicate significant differences (P00.05) between treatments Fig. 6 Number of Radopholus similis on carrot callus 56 days after inoculation of 30 females, respectively. Rs-P and Rs-C: untreated population Rs-P and Rs-C; R-12, R-24, R-36: Number of R. similis after inoculating 30 females Rs-C treated by Rs-eng- 1b dsRNA for 12 h, 24 h, and 36 h, respectively; G-12, G-24, and G-36: Number of R. similis after inoculating 30 females of Rs-C treated by non-endogenous gfp dsRNA solution for 12 h, 24 h and 36 h, respectively. Rs-C and Rs-P were two populations of R. similis collected from roots of ornamental plants Calathea makoyana and Philodendron cv Green Emerald, respectively. Bars indicate standard errors of mean data (n05) and different letters indicate significant differences (P00.05) among treatments 906 Eur J Plant Pathol (2012) 133:899–910