Downloaded from www.microbiologyresearch.org by IP: 54.242.161.225 On: Thu, 03 Mar 2016 10:24:56 Microbiology (1995), 141, 775-784 Printed in Great Britain A basic serine protease from Paecilomyces lilacinus with biological activity against Meloidogyne hapla eggs Peter J. M. Bonants, Paul F. L. Fitters,t Hans Thijs,$ Eefje den Belder, Cees Waalwijk and Jan Willem D. M. Henflings Author for correspondence: Peter J. M. Bonants.Te1: +31 8370 76213. Fax: +31 8370 10113. e-mail: BONANTS@IPO.AGRO.NL DLO Research Institute for Plant Protection (IPO-DLO), PO Box 9060,6700 GW Wageningen, The Netherlands Scanning electron micrographs of the nematode-egg-parasitic fungus Paecilomyces lilacinus infecting eggs of the root-knot nematode Meloidogyne spp. suggested the involvement of lytic enzymes. When grown on a liquid mineral salts medium, supplemented with different substrates as the sole N- and C-source, the fungus produced an extracellular protease. Colloidal chitin, vitellin and intact eggs of the root-knot nematode Meloidogyne hapla induced proteolytic activity that was repressed by glucose. The protease was partially purified from the culture filtrate by affinity chromatography. It has a molecular mass of 33.5 kDa, a pH optimum of 103, a temperature optimum of 60 "C and an isoelectric point above pH 102. The enzyme was completely inhibited by PMSF. The amino acid sequence, as derived from the nucleotide sequence of a cDNA clone, had high homology with several subtilisin-like serine proteases. It was shown that the purified enzyme degrades vitellin. The protease quantitatively bound to nematode eggs, and eggs incubated with the purified protease eventually floated. Incubation of the purified protease with nematode eggs significantly influenced their development as demonstrated by time-lapse microscopy. Immature eggs were highly vulnerable to protease treatments, whereas those containing a juvenile were more resistant. In addition, hatched larvae were not visibly affected by the protease. It can be concluded that the serine protease might play a role in penetration of the fungus through the egg-shell of nematodes. Keywords : Paecilomyces lilacinus, Meloidogyne hapla, protease, nematode-egg-parasitic fungus, cDNA sequence INTRODUCTION Nematodes are serious pests of many crops. There is growing opposition to chemical control of these pests because of unwanted environmental side-effects. t Present address: St Patrick's College, Department of Biology, Maynooth, County Kildare, Ireland. $ Present address: Wageningen Agricultural University, Department of Genetics, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands. g Present address: NetherlandsInstitutefor FisheriesResearch RIVO-DLO, PO BOX 68, 1970 AB IJmuiden, The Netherlands. Abbreviations: BPB, bromophenol blue; MM, minimal medium; PVDF, polyvinylidene difluoride; SEM, scanning electron microscopy. The GenBank accession number for the nucleotide sequence of the protease cDNA clone pSP-3 reported in this paper is L29262. Alternatives for these control measures are being de- veloped, one of which may be biological control by parasitic fungi. Many fungi parasitize plant-parasitic nematodes, either by capturing nematodes or by parasitizing their eggs. Several fungi are capable of penetrating nematode eggs. The fungus Dact_ylellaoviparasitica grows rapidly through egg-masses of the root-knot nematode Meloidogwe spp. and hyphae of the fungus penetrate egg-shells (Stirling & Mankau, 1979). Fungal egg-parasites, isolated from eggs of the cyst nematode Heterodera avenae, were investigated with respect to their ability to infect cyst nematode eggs of H. scbachtii by Dackman et al. (1989). Of these isolates, Verticillitlm stlcblasporitlm appeared to be most effective. I/. cblamydosporitlm was evaluated in vitro for its ability to 0001-9500 0 1995 SGM 775