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A fluorimetric method for the determination of endochitinolytic activity using N-fluorescein-labeled chitin (FITC-Chitin) is proposed, and a procedure for FITC-Chitin preparation with a degree of FITC content of 2.2 mol% (one FITC molecule per 45 glucosamine residues) is described. FITC-Chitin is capable to distinguish endochitinase and exochitinase (beta-N-acetylglucosaminidase) activities.

•  The ability of the nematode-trapping fungus Arthrobotrys oligospora and the nematode egg parasite Verticillium chlamydosporium to colonize barley (Hordeum vulgare) and tomato (Lycopersicum esculentum) roots was examined, together with capability of the fungi to induce cell wall modifications in root cells. •  Chemotropism was studied using an agar plate technique. Root colonization was investigated with light microscopy and scanning electron microscopy, while compounds involved in fungus-plant interactions were studied histochemically. •  Only A. oligospora responded chemotropically to roots. Colonization of barley and tomato by both fungi involved appressoria to facilitate epidermis penetration. V. chlamydosporium colonized tomato root epidermis and produced chlamydospores. Papillae, appositions and lignitubers ensheathing hyphae on tomato were also found. Phenolics (including lignin), protein deposits and callose were present in papillae in both hosts. Both fungi were still present in epidermal cells 3 months after inoculation. •  Nematophagous fungi colonized endophytically monocotyledon and dicotyledon plant roots. Arthrobotrys oligospora seemed to be more aggressive than V. chlamydosporium on barley roots. Both fungi induced cell wall modifications, but these did not prevent growth. The response of root cells to colonization by nematophagous fungi may have profound implications in the performance of these organisms as biocontrol agents of plant parasitic nematodes.

Culture filtrates of the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium growing on colloidal chitin showed increasing chitinolytic activity and production of two (32- and 43-kDa) main proteins. Maximum activity was found 18-20 days after inoculation, but V. suchlasporium always displayed higher activity. Zymography of such filtrates on carboxymethyl-chitin-Remazol brilliant violet 5R/acrylamide gels showed five bands of substrate degradation for V. suchlasporium and three for V. chlamydosporium. Filtrates with maximum activity were chromatographed on macroporous cross-linked chitin affinity matrix, showing a peak of main (50-60%) activity, which only contained a 43-kDa protein for both fungi. Zymography and colloidal chitin degradation showed that it was a single endochitinase (CHI43) with optimum pH range of 5.2-5.7. The main isoforms had pIs of 7.6 for V. suchlasporium and 7.9 for V. chlamydosporium. Eggs of the nematode Globodera pallida treated with CHI43 and the serine protease P32 from V. suchlasporium alone or in combination showed surface damage in comparison with controls when examined by scanning electron microscopy.

Barley roots were readily colonised by the nematophagous fungus Verticillium chlamydosporium. Light microscopy (LM) but also low temperature scanning electron microscopy (LTSEM) revealed details of the colonisation process. Hyphae were found on the rhizoplane often with dictyochlamydospores. Hyphae of V. chlamydosporium penetrated epidermal cells, often by means of appressoria. A hyphal network was formed in epidermal and cortical cells. Likewise, hyphal coils were found within root cells next to transverse cell walls. Cortical cells were the limits of fungal colonisation, since no hyphae were seen in the vascular cylinder. Modifications of root cell contents (phenolic droplets and callose appositions) were common three weeks after inoculation with V. chlamydosporium. These features may indicate induction of plant defence reactions in late stages of root colonisation by the fungus. Both LTSEM and LM have proved extremely useful to describe root colonisation by the fungus. The results found may have implications in the mode action of nematophagous fungi against plant parasitic nematodes.

We have characterized biologically and physiologically eight Verticillium lecanii strains from several origins including insect pests. Of all the temperatures tested, 25 degrees C was the best for growth and at 40 degrees C none of the strains could grow. At 4 and 7 degrees C, growth was reduced in comparison to warmer temperatures. The strains had better development at pH close to 7 (F = 27.64, P < 0,01) than at pH 3. Self-inhibition of germination of strain 50 was found when more than 0.78 conidia/cm(2) were plated on corn meal agar (CMA). Germination of conidia was close to 100% for all strains except one, three days after inoculation. Among extracellular enzymatic activities studied the fungal strains showed strongest proteolytic activities followed by lipolytic and chitinolytic activities. Some strains showed significant differences (P < 0.05) in conidia production. Most of the fungicides tested (especially benomyl) inhibited radial growth of strain 50 on CMA. Pathogenicity (as median lethal time, LT50) of V. lecanii strains on larvae of Galleria mellonella varied from 2.66 -/+ 0.33 to 4.27 -/+ 0.25 days. We conclude that in vitro tests per se are not sufficient to select the best biocontrol strains of entomopathogenic fungi. Pathogenicity is a complex process in which the presence, timing and regulation of many factors including those covered in this paper, as well as their interactions, are probably involved.

Almond mesocarp (AM) has been evaluated for production of Verticillium lecanii. Microbial flora of AM has been studied. After ground AM dilution plating, 5.3 x 10(5) +/- 2.6 x 10(4) fungal CFU.g-1 of dry AM were found. Common fungal saprophytes (Rhizopus sp. and Alternaria spp.) were found in more than 80% of the samples. Aspergillus sp. and yeasts were found less commonly. Rhizopus sp., Alternaria spp., and Aspergillus sp. inhibited growth of several V. lecanii; therefore, AM was treated with sterilization agents to eliminate endogenous mycoflora. Small samples (10 g) of AM saturated in distilled water treated with steam (120 degrees C and 100 kPa) were completely sterilized after 15 min. Ground AM incorporated on agar increased the biomass of V. lecanii compared with controls. This suggested AM as suitable substrate for the production of the fungus. In petri dishes, 9.7 x 10(7) +/- 2.9 x 10(7) conidia.g-1 of dry AM were produced after inoculating 10 conidia.g-1 of AM and incubating for 2 weeks. Viability of conidia produced was more than 90%. These conidia (5 x 10(4) conidia per larvae) caused Galleria mellonella mortality, calculated as median lethal time (LT50 5.3 +/- 1.6 days), that was significantly higher (F = 10.93; P < 0.05) than untreated controls (LT50 11.3 +/- 1.1 days). Larger scale tests have to be optimized before mass production.