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For the first time, the specific activities of chitinases, esterases, lipases and a serine protease (VCP1) produced by different isolates of the nematophagous fungus Pochonia chlamydosporia were quantified and compared. The isolates were grown for different time periods in a minimal liquid medium or media supplemented with 1 % chitin, 0.2 % gelatin or 2 % olive oil. Enzyme-specific activities were quantified in filtered culture supernatants using chromogenic p-nitrophenyl substrates (for chitinases, lipases and esterases) and a p-nitroanilide substrate (to measure the activity of the proteinase VCP1). Additionally, information on parasitic growth (nematode egg parasitism) and saprotrophic growth (plant rhizosphere colonisation) was collected. Results showed that the production of extracellular enzymes was influenced by the type of medium (p < 0.05) in which P. chlamydosporia was grown. Enzyme activity differed with time (p < 0.05), and significant differences were found between isolates (p < 0.001) and the amounts of enzymes produced (p < 0.001). However, no significant relationships were found between enzyme activities and parasitic or saprotrophic growth using Kendall's coefficient of concordance or Spearman rank correlation coefficient. The results provided new information about enzyme production in P. chlamydosporia and suggested that the mechanisms which regulate the trophic switch in this fungus are complex and dependent on several factors.

PCR-based methods to detect Verticillium chlamydosporium on infected plant roots were developed. Arbitrary ERIC primers and those based on rRNA genes, to identify fungi grown in pure culture, were unsuitable for DNA extracted from nematode-infested roots, because of interference by plant and nematode DNA. A novel method utilizing specific primers designed from an amplified and cloned fragment of the V. chlamydosporium β-tubulin gene was developed. Although it could not discriminate between different isolates of V. chlamydosporium, one primer set could identify the fungus on tomato roots infested with root-knot nematodes. The V. chlamydosporium β-tubulin sequence data showed close homology to sequences from plant endophytic Acremonium and Epichloe species and the saprotrophic Trichoderma viride.&nbsp

The adhesion of conidia of Drechmeria coniospora to the nematode Panagrellus redivivus was reduced after treatment of the conidia with Pronase E, or the detergents sodium dodecyl sulphate (SDS) and dodecyltrimethylammonium bromide (DTAB) suggesting involvement of proteinaceous compounds in the adhesion process. In the TEM the thick extracellular pad covering the adhesive bud of the conidia was completely removed after treatment with Pronase E. After treatment with SDS or DTAB the proteinaceous compounds appeared to be dissolved leaving mainly carbohydrates in the pad as observed on OsO4 without and OsO4 with Ruthenium red-stained material, respectively. The detergent extracts after SDS and DTAB treatments contained nine and seven peptides, respectively, with molecular masses in the range from 6 to 80 kDa on SDS-PAGE gels, and five biotinylated peptides were found in each extract, after blotting to nitrocellulose membranes, indicating that these were surface proteins. None of the detergent extracts was able to reduce adhesion of the conidia after treatment of the nematodes. The detergent extracts contained protease- and phosphatase activity. The protease inhibitor, chymostatin, inhibited infection of nematodes and growth of the conidia, suggesting the involvement of chymotrypsin-like proteases in the infection process. On gelatin-containing substrate gel electrophoresis two proteases were clearly chymostatin sensitive.

The infection process of the nematophagous fungus Verticillium chlamydosporium (isolate Vc1O) was studied in two nematode species that differed in susceptibility according to standardized in vitro assays. After 14 d, V. chlamydosporium infected 9 and 80% of Globodera rostochiensis and Meloidogyne incognita eggs, respectively. The purified proteinase VCPl, from V. chlamydosporium, removed the protein coat from eggs of M. incognita but not of G. rostochiensis. VCPl was more effective at hydrolysing proteins from M. incognita eggs than the related enzymes Prl and proteinase K, whereas the latter two were slightly more active against G. rostochiensis eggs than VCPl. Pretreatment of eggs with VCPl increased susceptibility to V. chlamydosporium from 80–100% in M. incognita eggs and from 9 to 29% in G. rostochiensis eggs. These observations suggest that VCPl is a host specificity-determining factor and also confirm it to be a pathogenicity determinant which works best in concert with other enzymes secreted by the actively growing fungus. Fungal growth on the surface of eggs of both species was commensurate with the infection frequencies observed; growth was limited and fewer appressoria were observed on eggs of G. rostochiensis compared with those of M. incognita. Eggs of M. incognita denatured by heat or killed with sodium azide, like eggs treated with VCPl, were more susceptible than untreated, healthy eggs. In contrast heat and azide treatment of G. rostochiensis eggs did not increase the number of infections. Appressoria were observed on the eggs of both nematode species in all treatments, but the numbers and types varied greatly. On M. incognita, the dominant type was swollen and branched laterally from hyphae, while on G. rostochiensis lateral inconspicuous appressoria prevailed. Differentiation of appressoria on live and dead nematode eggs showed that their production was not limited to the parasitic state of the fungus.

Morphological, pathogenic and genetic variability among 25 isolates of the nematophagous fungus Hirsutella rhossiliensis was quantified and compared. Genetic variability was assessed with 36 RAPD markers that were generated with eight random decamer primers. Cluster analysis of the PCR products indicated substantial genetic variability among isolates of this species. Out of 300 possible isolate-isolate combinations, 193 had less than 50% similarity (shared polymorphisms). In contrast, most of the isolates were similar morphologically and pathogenically, regardless of host or geographical origin. We suggest that, in spite of substantial molecular divergence in isolated populations of this fungus, the environment selects for similar characteristics associated with pathogenicity. Isolates from nematodes in the Hoplolaimidae (Rotylenchus robustus and Hoplolaimus galeatus), however, grew slower on agar, produced larger conidia, and were less pathogenic towards nematodes than were isolates from other hosts.

A fungus capturing nematodes in 3-celled constricting rings was isolated from a sample of rabbit dung collected from oak woods in Wellington Country Park in Hampshire. The fungus was initially found predating an adult nematode (Plectus) which was present in the dung sample. The trapped nematode had a constricted ring around the anterior of its body. The ring was disconnected from any mycelium and the nematode was still alive and swimming. This ring was used to propagate the fungus. A comparative review of the conidia of constricting ring fungi was made; and TEM was used to make an initial examination of the constricting rings and the ring/stalk connection of this fungus.

Infection of nematodes by conidia of Drechmeria coniospora was studied by optical- and electron microscopy. After penetration, trophic hyphae invaded solely via the pseudocoel; penetration of internal organs was never observed. The invading hyphae had a characteristic wave-like appearance, this pattern possibly having the advantage of preventing rupture of hyphae due to host movements. After death of the host 30–48 h after infection, trophic hyphae contained numerous lipid droplets often associated with microbodies (peroxisomes) which were characterized by the presence of catalase and the β-oxidation enzyme thiolase. Conidiophores developed from tips of trophic hyphae; the process of outgrowth being similar to that for initial penetration in that it occurred via enzymic action and mechanical force. Intimate association between fungal cell wall and nematode cuticle suggested that leakage of nematode contents was prevented after outgrowth. Conidiophores possessed an electron dark layer at the outside of their wall which was not continuous with developing conidia. Numerous spores were successively formed from each individual peg present on the conidiophore. Pegs were ordered at regular distances, located in close proximity to septa and on the apical end of the conidiophore. Invariably immature spores are formed; development of the adhesive knob occurred after release from the peg. Approximately 5000–10 000 spores were formed at the expense of a single nematode.

The nematophagous fungus, Duddingtonia flagrans is a potential biocontrol agent against nematode parasites of ruminants. Improved methods for the rapid and accurate detection of D. flagrans would aid the evaluation of this fungus as a biocontrol agent and its suitability for environmental release. To date, detection and identification of D. flagrans is reliant on morphological methods, which can be laborious, time-consuming, and error prone. In this study, a PCR assay using species-specific primers located in the ITS regions was developed for the rapid and accurate identification of D. flagrans. The PCR assay was specific to five different isolates of D. flagrans and was capable of detecting a minimum concentration of 100 chlamydospores per gram of soil. In contrast to cultured-based detection and identification methods, this assay is amenable to high throughput screening of environmental samples. The assay detected D. flagrans in faecal, leaf litter, and soil samples collected from 80 % of the Irish farms tested indicating that the fungus is abundant in Ireland.

The nematophagous fungus Pochonia chlamydosporia is a potential biocontrol agent against root knot and cyst nematodes. Genetic transformation of the fungus to introduce visual marker genes, novel traits, or changes in expression levels of endogenous genes, would greatly enhance understanding of its behaviour on nematode-infested roots and of its interactions with other soil and rhizosphere microorganisms. A transformation system for the introduction of novel genes into P. chlamydosporia has been developed. Methods to generate protoplasts, introduce DNA and regenerate transformed viable fungal mycelium have been optimised, using plasmids carrying the green fluorescent protein marker gene gfp and the hygromycin resistance gene hph. Cultures of P. chlamydosporia were resistant to high levels of a range of fungal inhibitors, including hygromycin, that are commonly used with dominant selectable marker genes in the transformation of other fungi. However, regenerating protoplasts transformed with hph could be selected by their ability to grow through an agar overlay containing 1 mg ml(-1) hygromycin. Green fluorescence was observed in protoplasts and regenerating mycelium after transformation with gfp, but the GFP phenotype was lost on subculture. Maintenance of introduced genes was not stable, and during subculture, PCR assays indicated that the transformants lost both hph and gfp. When these genes were introduced on the same plasmid, segregation of hph and gfp was observed prior to their loss. It was unclear whether the introduced plasmids were able to replicate autonomously in P. chlamydosporia, or if they integrated transiently into the fungal genome. Possible reasons for the instability of the transformants are discussed.

The mitosporic fungus Pochonia chlamydosporia is a potential biocontrol agent for cyst (Heterodera spp. and Globodera spp.) and root knot (Meloidogyne spp.) nematodes, which are important agricultural plant pests. 54 isolates from diverse geographical regions and several nematode hosts were used in this study. Genetic variation was examined using enterobacterial repetitive intergenic consensus (ERIC) primed PCR and sequences from the internal transcribed spacer (ITS) rRNA region. ERIC PCR yielded 35 scorable binary characters from all the fungi tested and cluster analysis of the data showed that isolates from cyst nematodes were more genetically variable than those from root knot nematodes. The ITS regions were highly conserved, the only significant difference being an extra thymidine in isolates from Meloidogyne spp. Assays with nematode eggs indicated that isolates differ in their ability to infect different nematode genera. The results indicate host related variation in P. chlamydosporia. This finding has significant implications for the application of P. chlamydosporia as a biocontrol agent.

A new nematophagous-predacious hyphomycete, Candelabrella shahriari sp. nov., which captures nematodes by means of adhesive networks, is described and illustrated.