908 Eur J Plant Pathol (2012)133:899-910 Discussion and juvenile of R.similis may secrete abundantly EGases for infection.In addition to infection,the During feeding and migration in the plant,parasitic female is also responsible for breeding,and therefore nematodes inject enzymes through the stylet into plant requires additional nutrients (Gowen et al.2005).So, tissue to degrade the cell wall.The most extensively females may secrete more EGases than juveniles due studied nematode cell wall-degrading enzyme is B-1, to their higher activity in gaining nutrents.In contrast, 4-endoglucanase (EGase)(Haegeman et al.2009).Rs males of R.similis have a degraded stylet and pharyn eng-/b is known as EGases which functions in geal glands are non-parasitical (Williams and Siddiqi degrading cellulose(Goellner et al.2001),a key com 1973;Luc 1987;Gowen et al.2005).Eggs need the ponent in forming cell walls.In this study,Rs-eng-1b least nutntion due to their immobility.Smant et al was isolated and obtained according to our constructed (1998)reported that the EGases gene was only SSH library built from mixed-stage nematodes of Rs expressd in the mobile stages of G C and Rs-P populations with different pathogenic abil namely in pre-parasitic and parasitic J2 and in adul ity on Musa paradisiaca.The sequence of Rs-eng-1b males.but not in sedentary females.Our present find in here was 1,427 bp that had a 99%similarity with ings and the results reported by Smant ct al.(1998 rst repor ,OS-g-1b(E414859)( nd nay on of Rs-eng-/b n out hig nif 11g1 a DNA T and in th vell de glands (willia and Siddiai 1973 t al 2005)EGas tto be 公 de cell walls of hosts 16hto361 hel odes to gh the tissue and absorb nutrition from there e.So the fen (2009 ile SpringerDiscussion During feeding and migration in the plant, parasitic nematodes inject enzymes through the stylet into plant tissue to degrade the cell wall. The most extensively studied nematode cell wall-degrading enzyme is β-1, 4-endoglucanase (EGase) (Haegeman et al. 2009). Rs￾eng-1b is known as EGases which functions in degrading cellulose (Goellner et al. 2001), a key com￾ponent in forming cell walls. In this study, Rs-eng-1b was isolated and obtained according to our constructed SSH library built from mixed-stage nematodes of Rs￾C and Rs-P populations with different pathogenic abil￾ity on Musa paradisiaca. The sequence of Rs-eng-1b in here was 1,427 bp that had a 99 % similarity with the first report of Rs-eng-1b (EU414839) (Haegeman et al. 2008). The expression of Rs-eng-1b in Rs-C was about 2.7 times higher than the expression in Rs-P, and the pathogenicity of Rs-C was significantly greater than that of Rs-P. Consequently, the expression of Rs-eng-1b was positively correlated with the pathoge￾nicity of R. similis. The expression of Rs-eng-1b in the nematodes treated by Rs-eng-1b dsRNA for more than 12 hr decreased significantly, and the Rs-eng-1b dsRNA treatment caused the nematodes to have a lower reproduction than gfp dsRNA treated and un￾treated Rs-C. Furthermore, the growth of anthurium inoculated by the nematodes treated by Rs-eng-1b dsRNA was better than that of untreated nematodes and nematodes treated with gfp dsRNA, and the effect of Rs-eng-1b RNAi treatment was enhanced with extending time of Rs-eng-1b RNAi treatment. In view of these results, it is assumed that a higher expression of Rs-eng-1b results in more degradation of cellulose, and therefore contributes to nematode invasion of the hosts resulting in more serious damage to the plant. Analysis of the expression of Rs-eng-1b performed at different nematode stages of Rs-C showed that Rs￾eng-1b expression was highest in females and lowest in eggs. Interestingly, the expression difference of Rs￾eng-1b in females, males, juveniles, and eggs, con￾forms to their individual developmental features. The female and juvenile of R. similis are the infective forms which have a powerful stylet and well devel￾oped pharyngeal glands (Williams and Siddiqi 1973; Gowen et al. 2005). EGases is thought to be secreted in order to degrade cell walls of hosts during infection, and may help nematodes to migrate through the host tissue and absorb nutrition from there. So the female and juvenile of R. similis may secrete abundantly EGases for infection. In addition to infection, the female is also responsible for breeding, and therefore requires additional nutrients (Gowen et al. 2005). So, females may secrete more EGases than juveniles due to their higher activity in gaining nutrients. In contrast, males of R. similis have a degraded stylet and pharyn￾geal glands are non-parasitical (Williams and Siddiqi 1973; Luc 1987; Gowen et al. 2005). Eggs need the least nutrition due to their immobility. Smant et al. (1998) reported that the EGases gene was only expressd in the mobile stages of G. rostochiensis, namely in pre-parasitic and parasitic J2 and in adult males, but not in sedentary females. Our present find￾ings and the results reported by Smant et al. (1998) indicate that EGases may be related to parasitism and nematode infection. Haegeman et al. (2008) reported the expression of EGases in different life stages of R. similis by a semi-quantitative RT-PCR. In their study Rs-eng-1b was expressed at a very low level in eggs, and higher levels in males and females, but not in juveniles. They concluded that other EGases not iden￾tified in juveniles might complement the role of Rs￾eng-1b. Employing RNAi to study gene function of nem￾atodes, there are in principle three approaches to input dsRNA: microinjection, feeding approaches using bacteria with expression of dsRNA, and soak￾ing nematodes in dsRNA (Urwin et al. 2002). However, the first two approaches are unrealistic: due to the small size of nematodes it is difficult to microinject dsRNA, nor do PPNs feed upon bacte￾ria. Therefore RNAi input on PPNs is generally performed by soaking the nematodes for more than 4 h for effective gene silencing (Rosso et al. 2009). Chen et al. (2005) soaked G. rostochiensis in dsRNA of Gr-eng-1 for 24 h, Bakhetia et al. (2007) soaked H. glycines in dsRNA of Hg-eng-1 for 16 h and Cheng et al. (2010) soaked Bursaphelenchus xylophilus in dsRNA of Bx-eng-1 for 24 h for the best RNAi effect. However as shown in this study, overlong periods can also cause decreased expression. In this study, R. similis soaked in dsRNA of Rs-eng-1b for 36 h, expression of Rs-eng-1b of R. similis decreased significantly. All these results indicate that RNAi treatment for eng-1 in different nematodes requires 16 h to 36 h exposure to display an effective silencing. Haegeman et al. (2009) silenced the putative 908 Eur J Plant Pathol (2012) 133:899–910