Hans-Börje Jansson

Effect of chitosan on hyphal growth and spore germination of plant pathogenic and biocontrol fungi

Aims:u2002 To investigate the toxic effect of chitosan on important root pathogenic and biocontrol fungi (nematophagous, entomopathogenic and mycoparasitic).

Fungal root endophytes from natural vegetation in Mediterranean environments with special reference to Fusarium spp

Surveys (in 2002 and 2003) were performed for fungal endophytes in roots of 24 plant species growing at 12 sites (coastal and inland soils, both sandy soils and salt marshes) under either water or salt stress in the Alicante province (Southeast Spain). All plant species examined were colonized by endophytic fungi. A total of 1830 fungal isolates were obtained and identified by morphological and molecular [internal transcribed spacer (ITS) and translation elongation factor-1alpha gene region (TEF-1alpha) sequencing] techniques. One hundred and forty-two fungal species were identified, belonging to 57 genera. Sterile mycelia were assigned to 177 morphospecies. Fusarium and Phoma species were the most frequent genera, followed by Aspergillus, Alternaria and Acremonium. Fungal root endophytic communities were influenced by the soil type where their respective host plants grew, but not by location (coastal or inland sites). Fusarium oxysporum, Aspergillus fumigatus and Alternaria chlamydospora contributed most to the differences found between endophytic communities from sandy and saline soils. Host preference was found for three Fusarium species studied. Fusarium oxysporum and Fusarium solani were especially isolated from plants of the family Leguminosae, while Fusarium equiseti showed a preference for Lygeum spartum (Gramineae). In some cases, specificity could be related to intra-specific variability as shown by sequencing of the TEF-1alpha in the genus Fusarium.

Chitosan permeabilizes the plasma membrane and kills cells of Neurospora crassa in an energy dependent manner

Chitosan has been reported to inhibit spore germination and mycelial growth in plant pathogens, but its mode of antifungal action is poorly understood. Following chitosan treatment, we characterized plasma membrane permeabilization, and cell death and lysis in the experimental model, Neurospora crassa. Rhodamine-labeled chitosan was used to show that chitosan is internalized by fungal cells. Cell viability stains and the calcium reporter, aequorin, were used to monitor plasma membrane permeabilization and cell death. Chitosan permeabilization of the fungal plasma membrane and its uptake into fungal cells was found to be energy dependent but not to involve endocytosis. Different cell types (conidia, germ tubes and vegetative hyphae) exhibited differential sensitivity to chitosan with ungerminated conidia being the most sensitive.

Interactions Between Nematophagous Fungi and Plant-Parasitic Nematodes: Attraction, Induction of Trap Formation and Capture

The behaviour of five plant-parasitic nematodes was compared with that of a bacteria-feeding nematode. The attraction of the plant-parasitic nematodes to thirteen nematophagous fungi was tested. The two plant-parasitic nematodes also known to be mycophagous were attracted to all fungi. In the other nematodes there was no distinct attraction pattern. The ability of different fungi to attract tended to increase with increasing dependence of the fungi on nematodes for nutrients. The rapidity with which the nematodes induced trap formation depended on their motility and on the concentration of nutrients in the culture medium. The ability of a nematode to induce trap formation was not correlated with feeding behaviour (bacteria-feeding, mycophagous or plant-parasitic). All nematodes were rapidly captured when traps were present.

Membrane fluidity determines sensitivity of filamentous fungi to chitosan

The antifungal mode of action of chitosan has been studied for the last 30 years, but is still little understood. We have found that the plasma membrane forms a barrier to chitosan in chitosan‐resistant but not chitosan‐sensitive fungi. The plasma membranes of chitosan‐sensitive fungi were shown to have more polyunsaturated fatty acids than chitosan‐resistant fungi, suggesting that their permeabilization by chitosan may be dependent on membrane fluidity. A fatty acid desaturase mutant of Neurospora crassa with reduced plasma membrane fluidity exhibited increased resistance to chitosan. Steady‐state fluorescence anisotropy measurements on artificial membranes showed that chitosan binds to negatively charged phospholipids that alter plasma membrane fluidity and induces membrane permeabilization, which was greatest in membranes containing more polyunsaturated lipids. Phylogenetic analysis of fungi with known sensitivity to chitosan suggests that chitosan resistance may have evolved in nematophagous and entomopathogenic fungi, which naturally encounter chitosan during infection of arthropods and nematodes. Our findings provide a method to predict the sensitivity of a fungus to chitosan based on its plasma membrane composition, and suggests a new strategy for antifungal therapy, which involves treatments that increase plasma membrane fluidity to make fungi more sensitive to fungicides such as chitosan.

Fungal attachment to nematodes

The adhesion mechanisms in three nematophagous fungi are reviewed. In all these fungi the infection and subsequent digestion of nematodes is initiated by the firm adhesion of the fungus to the nematode surface. In Arthrobotrys oligospora the adhesive phase is restricted to special three-dimensional structures. Drechmeria coniospora conidia attach to the nematode cuticle by an adhesive bud while Catenaria anguillulae uses an adhesive phase of zoospore development for this purpose. In A. oligospora , the adhesion of nematodes to the traps is mediated by a layer of extracellular fibrillar polymers. The ultrastructure of this layer changes during adhesion, the fibrils become more dense and oriented in one direction. The surface layer consists mainly of carbohydrate-containing fibrils and a low-molecular-weight protein. Previous and present studies show that the low-molecular-weight protein of A. oligospora is a lectin. In contrast, the adhesive layer of D. coniospora does not seem to change during the adhesion process. The adhesin of C. anguillulae appears to consist mainly of protein. The studies suggest that adhesion of nematodes to A. oligospora involves a recognition event using a lectin-carbohydrate interaction which might trigger the reorganization of the surface polymer layer and release of enzymes, leading to the firm binding of the nematode. A similar process may take place also in other nematophagous fungi.

Attraction of Nematodes to Living Mycelium of Nematophagous Fungi

Methods were designed to detect attraction and repulsion of nematodes by fungi and determine the attraction intensity of different fungi. Of 23 fungi tested, 15 attracted the bacteria-feeding nematode Panagrellus redivivus. Of the 14 nematophagous fungi tested, ten attracted and one repelled nematodes, whereas three were neutral. Among nine non-nematophagous fungi, five attracted nematodes. In general, the attraction intensity increased with increasing dependence of the fungi on nematodes for nutrients.

Real-time PCR quantification and live-cell imaging of endophytic colonization of barley (Hordeum vulgare) roots by Fusarium equiseti and Pochonia chlamydosporia

*New tools were developed for the study of the endophytic development of the fungal species Fusarium equiseti and Pochonia chlamydosporia in barley (Hordeum vulgare) roots. These were applied to monitor the host colonization patterns of these potential candidates for biocontrol of root pathogens. * Molecular beacons specific for either F. equiseti or P. chlamydosporia were designed and used in real-time polymerase chain reaction (PCR) quantification of fungal populations in roots. Genetic transformation of isolates with the green fluorescent protein (GFP) gene was carried out using an Agrobacterium tumefaciens-mediated transformation protocol, and spatial patterns of root colonization were investigated by laser confocal microscopy. * Quantification of endophytes by real-time PCR in roots of barley gave similar results for all fungi, and was more accurate than culturing methods. Conversely, monitoring of root colonization by GFP-expressing transformants showed differences in the endophytic behaviours of the two species, and provided evidence of a plant response against endophyte colonization. * Both F. equiseti and P. chlamydosporia colonized barley roots endophytically, escaping attempts by the host to prevent fungal growth within root tissues. This strongly supports a balanced antagonism between the virulence of the colonizing endophyte and the plant defence response. Development of real-time PCR techniques and GFP transformants of these fungal species will facilitate future work to determine their biocontrol capacity.

Pre-penetration events in fungal parasitism of nematode eggs

The present investigation deals with the main factors involved in early infection of nematode eggs by fungal parasites. We studied the effect of hydrophobicity on appressorium formation by germlings of Pochonia rubescens (syn. Verticillium suchlasporium), P. chlamydosporia (syn. V. chlamydosporium) and Lecanicillium lecanii (syn. V. lecanii). Appressoria were frequently formed on hydrophobic surfaces such as polyvinyl chloride or polystyrene and were infrequently formed on hydrophilic materials such as glass or aluminium. Infected eggs probed with the FITC-labelled lectin Concanavalin-A showed intense labelling corresponding to appressoria formed by fungal parasites on the eggshell surface. Proteolytic activity was found in extracts from conidia and germlings of fungal parasites (especially P. chlamydosporia) in the absence of nematode eggs. Addition of the serine proteinase inhibitors phenylmetylsulphonyl fluoride (PMSF) or diisopropyl fluorophosphonate (DFP) to the extracts reduced their proteolytic activity. PMSF was the most effective inhibitor. Zymography also revealed proteolytic activity in extracts from the three fungi tested. This activity mostly corresponded to bands of Rfs of substrate degradation similar to that of purified main protease (P32) from P. rubescens. Other bands with molecular weight higher than P32 (low Rf) were found especially for P. chlamydosporia extracts. For L. lecanii only bands of low Rf were found. Serum anti-P32 partially inhibited proteolytic activity of extracts from conidia and germlings. Application of PMSF and DFP to the inoculum, reduced egg penetration for the three species studied. PMSF caused the highest reduction in eggs infected by L. lecanii, while DFP significantly reduced egg infection by both P. chlamydosporia and L. lecanii. Our results therefore show hydrophobicity, appresorium formation and protease production as factors involved in early parasitism of nematode eggs.

Attraction of nematodes to endoparasitic nematophagous fungi

The endoparasites, Meria coniospora and Cephalosporium balanoides , have adhesive conidia, which attract nematodes. The non-adhesive conidia of Harposporium anguillulae and those of the nematode-trapping Arthrobotrys oligospora did not attract nematodes. The mycelia of all fungi tested attracted the bacterial feeding nematode Panagrellus redivivus , with M. coniospora and H. anguillulae attracting the most nematodes. The attraction of nematodes to conidia was confined to the living state and seems to be connected with the adhesiveness of the conidia.