The extracellular subtilisin-like protease activity of several
fungal pathogens of nematodes, insects and plants was recorded after
batch cultivation. Nematophagous and entomophagous isolates of Verticillium,
Paecilomyces, Beauveria and Metarhizium produced
Suc-(Ala)2-Pro-Phe-pNA hydrolysing enzymes that differed in
serological properties based on Western blot analysis with polyclonal
antibodies. The antibodies used were raised against the main subtilisin-like
proteases from one species from each of the four genera.
From each strain of V. chlamydosporium tested, between one and
four isoforms with different pI values were separated by isoelectric
focusing and visualized by enzymoblots. The occurrence of multiple isoforms
with unique N-terminal sequences in single strains
might suggest the presence of subtilisin gene families. Using as a probe
a fragment of the Pr1 gene from M. anisopliae, RFLPs
were
observed that confirmed qualitative differences between subtilisin-like
genes within and between species. In contrast to these
pathogens of invertebrates, plant-pathogenic species of Verticillium
did not produce subtilisin-like proteases, nor were homologous
genes detected. Such activity, however, and the corresponding genes, were
detected in species weakly pathogenic to plants, or
saprotrophs.