Expression Of Substrate-Pecificity Extracellular Protease Associated With Zoophthora Radicans (Entomophthorales: Entomophthoraceae) Virulence Induced By Plutella Xylostella (Lepidoptera: Yponomeutidae)

Extracellular protease (ECPase) is an important virulence factor during insect infection by entomopathogenic fungi, playing a critical role at many aspects including degradation of cuticle structural protein and activation of phenoloxidase system etc. While various extracellular proteases have different substrate-specificity and exhibit different pathogenicity against different hosts. In this study, based on bioassay of various passages of Zoophthora radicans against Plutella xylostella, detection of types and activities of proteases produced in various liquid media and analysis of their correlation to virulence, the function of induction of extracellular proteases associated with virulence by host substrate was identified. The results also proved up partial mechanism of virulence variation during adaption to non-original host by the isolate and the importance of substrate-specificity extracellular proteases in improving its virulece.Passages through Plutella xylostella and variation of virulence Inoculation by both Zoophthora radicans isolates F99101 and ARSEF1342 respectively with their optimum dose resulted in great increase in the mortalities of Plutella xylostella. F99101 could not be reisolated successfully in that the cadavers killed by F99101 discharged few conidia. In addition, large numbers of resting spores were found in a few cadavers indicating the substrates of Plutella xylostella are unsuitable for the growth and propagation of F99101. Whereas the cadavers killed by the original strain of ARSEF1342 were densely covered with conidiophores and large numbers of conidia were discharged. Thus, the passages R1~R4 may be re-isolated in turn and the virulence of isolate against Plutella xylostella showed a distinct rising tendency along with passages reisolates. After adaption with four passages, the cumulative mortality in 8d of Plutella xylostella after inoculation (250 conidia/mm2) increased from 14.9% to 27.3% and the peak of Plutella xylostella death appeared on Day 2 instead of Day 5, which demonstrates the rapidity of non-original host infectionby the isolate had been improved.Induced expression of ECPases and correlation to their virulence DifferentZoophthora radicans isolates produced different extracellular proteases during liquidculture. A conservative protease band with molecular weight of 162kD was found for ARSEF1100, ARSEF1136 and ARSEF2699. Furthermore, two substrate-specificity protease bands of 153kD and 148kD were found for ARSEF1100 and only 153kD or 148kD band was found for ARSEF1136 and ARSEF2699 respectively. Whereas F99101 produced two protease bands of 218kD and 207kD. The overall proteases activity of isolates was correlated to their virulence against Plutella xylostella in a certain extent. Like the virulence of ARSEF1342 which had extremely lowest proteases production was also much lower than that of other isolates and there was a parallelism between maximal proteases activity and the death peak of the challenged hosts. But the correlation does not reach significant level (r=0.76). After passage through Plutella xylostella , ARSEF1342, the original strain (Ro), only produced two protease bands corresponding to molecular masses of 153kD and 162kD during liquid culture, whereas the band of 148kD was lost in the Z. radicans reisolates (R1 ~R4) , but the 153kD band gradually became obvious along with passages of reisolates. The same effect was obtained during Plutella xylostella powder liquid culture. While the band of 148kD may be reproduced to a high level by induction of Pier is rapae powder indicating that the proteases zymogram of the strain may be selectively expressed according to its demand by induction of different host substrates. The correlations between various proteases and virulences against Plutella xylostella were widely divergent. The correlation coefficient was 0.784 during liquid culture and extremly markedly correlation was identified during Plutella xylostella powder liquid culture but no correlation was found during Pieris rapae powder liquid cultu...