VCP1 PROTEASE DETECTION IN Pochonia chlamydosporia var. catenulata STRAIN IMI SD 187

Abstract

The mode of action of Pochonia as a biological control agent is the penetration by means of the hyphae into the target nematode eggs. The egg shell of nematodes has an outer vitelline membrane composed mainly by proteins. Thus, hyphal penetration is the result of a physic pressure and the specific hydrolytic activity of some enzymes such as proteases and chitinases. The most important protease studied in Pochonia chlamydosporia var. chlamydosporia is VCP1. The aim of this work was to detect and characterize the protease VCP1 in a strain of P. chlamydosporia var. catenulata, a potential biological control agent for root-knot nematodes. The specific activity of this protease was tested on different culture media supplemented with protein inductors using N-succinyl-Ala-Ala-Phe-p-nitroanilide as substrate. PCR and RFLP analyses with specific primers and probes were also done. Isolates from the chlamydosporia variety were used as positive controls. The medium supplemented with chitin was the only one capable of inducing some VCP1 activity but in a low level. The VCP1-encoding gene was not detected in strain IMI SD 187 by RFLP even when the stringency conditions were reduced to the minimum possible. The low level of enzymatic activity detected by biochemical techniques does not support the presence of protease VCP1 in this strain. The low enzymatic activity reached points out to the existence of a VCP1 isoform or another different protease. These hypotheses should be proved widening the range of catenulata variety strains and taking the chlamydosporia variety (strain 10) as a reference in a comparative study with conclusive results. The results of this enzymatic characterization related to the egg infection process of host nematodes are the first achieved in a catenulata variety strain >in the world.
(Key words: nematode parasites; RFLP; enzymes; protease)