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.

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.

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

AIMS: To investigate the toxic effect of chitosan on important root pathogenic and biocontrol fungi (nematophagous, entomopathogenic and mycoparasitic). METHODS AND RESULTS: We have used standard bioassays to investigate the effect of chitosan on colony growth and developed bioassays to test spore germination. The results showed that the root pathogenic and mycoparasitic fungi tested were more sensitive to chitosan than nematophagous and entomopathogenic fungi. Chitosanases (and perhaps related enzymes) are involved in the resistance to chitosan. Two fungi, one sensitive to chitosan, Fusarium oxysporum f. sp. radicis-lycopersici, and one less sensitive, Pochonia chlamydosporia, were selected for ultrastructural investigations. Transmission electron microscopy revealed differences in the ultrastructural alterations caused by chitosan in the spores of the plant pathogenic fungus and in those of the nematophagous fungus. Confocal laser microscopy showed that Rhodamine-labelled chitosan enters rapidly into conidia of both fungi, in an energy-dependent process. CONCLUSIONS: Nematophagous and entomopathogenic fungi are rather resistant to the toxic effect of chitosan. Resistance of nematophagous and entomopathogenic fungi to chitosan could be associated with their high extracellular chitosanolytic activity. Furthermore, ultrastructural damage is much more severe in the chitosan sensitive fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this paper suggest that biocontrol fungi tested could be combined with chitosan for biological control of plant pathogens and pests.

Endophytic colonization of date palm (Phoenix dactylifera L.) leaves by entomopathogenic fungi.

Light and scanning electron microscopy together with fungal isolation techniques were used to detect entomopathogenic fungi within young and adult date palm (Phoenix dactylifera) petioles and to assess fungal survival in leaf tissues. The entomopathogenic fungi Beauveria bassiana, Lecanicillium dimorphum and Lecanicillium c.f. psalliotae survived inside leaf tissues at least 30 days after inoculation. Entomopathogenic fungi colonized inoculated petioles endophytically and were recovered up to 3cm from the inoculation site. Fungi were detected inside the parenchyma and sparsely within vascular tissue using microscopy techniques. Our results show that the entomopathogenic fungi used in this study survived and colonized date palm tissues in bioassays both under laboratory and field experimental conditions with no evidence of significant damage.

Colonization of plant roots by egg-parasitic and nematode-trapping fungi.

• 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.

Rhizosphere Colonization and Control of Meloidogyne spp. by Nematode-trapping Fungi.

The ability of nematode-trapping fungi to colonize the rhizosphere of crop plants has been suggested to be an important factor in biological control of root-infecting nematodes. In this study, rhizosphere colonization was evaluated for 38 isolates of nematode-trapping fungi representing 11 species. In an initial screen, Arthrobotrys dactyloides, A. superba, and Monacrosporium ellipsosporum were most frequently detected in the tomato rhizosphere. In subsequent pot experiments these fungi and the non-root colonizing M. geophyropagum were introduced to soil in a sodium alginate matrix, and further tested both for establishment in the tomato rhizosphere and suppression of root-knot nematodes. The knob-forming M. ellipsosporum showed a high capacity to colonize the rhizosphere both in the initial screen and the pot experiments, with more than twice as many fungal propagules in the rhizosphere as in the root-free soil. However, neither this fungus nor the other nematode-trapping fungi tested reduced nematode damage to tomato plants.

Adhesion of Conidia of Drechmeria coniospora to Caenorhabditis elegans Wild Type and Mutants.

Adhesion of conidia of the endoparasitic fungus Drechmeria coniospora to the cuticles of the wild type and four different head defective mutants of Caenorhabditis elegans, and subsequent infection, was studied. The conidia adhered around the sensory structures in the head region, vulva, and occasionally to other parts of the cuticle in both mutant and wild type hosts. Infection took place after adhesion to the head region by penetration through the cuticle, and, following adhesion around the vulva, through the natural orifice. Infection was not observed after adhesion to other parts of the cuticle. Adhesion was reduced after treatment of the nematodes with Pronase E. Adhesion returned towards normal again within 2 hours, indicating that the proteinaceous material emanating from the sensory structures was rapidly replaced.

Panagrellus redivivus: failure to find evidence for the occurrence and biosynthesis of sialic acids.

The occurrence of sialic acids in the free-living nematode Panagrellus redivivus was studied by periodate oxidation/[3H]sodium borohydride reduction of about 10(7) nematodes. In parallel, the capability of sialic acid biosynthesis was examined by metabolic labeling of the same number of nematodes with N-[3H]acetylmannosamine. In both experiments, radioactivity was incorporated into the nematodes. Mild acid hydrolysis, however, did not release radioactively labeled sialic acids or derivatives as tested by radio thin-layer chromatography, suggesting that P. redivivus does not contain or synthesize sialic acids.

Lectin studies of surface carbohydrates and induction of gland secretion in the free-living stages ofSchistosoma mansoni.

Secretion from glands was observed when cercariae ofSchistosoma mansoni were exposed to certain lectins. Lectins fromMaclura pomifera, Pisum sativum, andTriticum vulgaris, were effective at 7.5 µ/ml. The effects of cercarial gland secretion caused byT. vulgaris agglutinin andArachis hypogaea agglutinin were blocked by pretreatment with the inhibiting glycan. Discharge of glands was not visualized after exposure of miracidia to lectins. The distribution of five labeled lectins was determined on live miracidia and cercariae. OnlyT. vulgaris agglutinin generally labeled the cercarial and miracidial bodies. Specific labeling occurred with the other lectins on the anterior, in glands or on their secretions, in flame cells in both stages, and on an unidentified ring of cells in miracidia. The possible mechanisms involved in changes caused by the lectins are discussed.

Control of Root-knot Nematodes on Tomato by Lectins.

Significant control of tomato root knot was achieved by applications of the lectins Concanavalin A (Con A) and Limax flavus agglutinin in greenhouse, growth chamber, and microplot trials. Four consecutive weekly applications at lower concentrations of Con A yielded better control than single applications at a higher total concentration. The present state of knowledge on binding of Con A to soil nematodes and the in vitro effect of this lectin in chemotactic behavior are discussed. The mode of action of Con A on root-knot control is unknown.

Quantification of predatory and endoparasitic nematophagous fungi in soil.

Methods were developed to quantify predatory and endoparasitic fungi in soil. The methods were based on previously developed detection techniques and combined with a most probable number estimation. The methods were applied to an agricultural soil fertilized with farmyard manure. Large amounts of farmyard manure resulted in increased amounts of organic matter, numbers of propagules of predatory and endoparasitic fungi, and numbers of bacteria and nematodes.

Caenorhabditis elegans: comparisons of chemotactic behavior from monoxenic and axenic culture.

Significant differences in chemotactic response of Caenorhabditis elegans were demonstrated for nematodes from monoxenic culture as compared to nematodes from axenic culture. These results support those of a previous study in which large differences in growth, development, behavior, and longevity were shown for C. elegans in comparative assays of the monoxenic and axenic regimes.

Susceptibility of Neoaplectana spp. and Heterorhabditis heliothidis to the Endoparasitic Fungus Drechmeria coniospora.

Adhesive conidia of the nematophagous fungus, Drechmeria coniospora (Drechsler) W. Gams and Jansson (Moniliales: Deuteromycetes), would occasionally attach but never penetrate the infective stages of insect parasitic Neoaplectana carpocapsae, N. glaseri, N. bibionis, N. intermedia, and Heterorhabditis helfothidis (Rhabditida). However, adult and pre-infective stages of Neoaplectana spp. became infected by the fungus.

Fluorescent and Ferritin Labelling of Cuticle Surface Carbohydrates of Caenorhabditis elegans and Panagrellus redivivus.

Caenorhabditis elegans and Panagrellus redivivus were investigated for surface carbohydrates using fluorescent-labelled and ferritin-labelled lectins. Rhodamine-labelled Concanavalin A was specifically located in the cephalic region of both species. Rhodamine-labelled wheat germ agglutinin was located over the entire cuticle of P. redivivus but was absent on C. elegans. Rhodamine-labelled peanut agglutinin and Limax flavus agglutinin did not label nematodes of either species. Galactose and sialic acid were not detected on either species, whereas mannose-glucose residues were specifically localized in the head areas of both species. No detectable N-acetylglucosamine occurred on C. elegans, but it was evenly distributed over the cuticle surface of P. redivivus.

Differential Adhesion and Infection of Nematodes by the Endoparasitic Fungus Meria coniospora (Deuteromycetes).

The conidia of the endoparasitic fungus Meria coniospora (Deuteromycetes) had different patterns of adhesion to the cuticles of the several nematode species tested; adhesion in some species was only to the head and tail regions, on others over the entire cuticle, whereas on others there was a complete lack of adhesion. After adhesion, the fungus usually infected the nematode. However, adhesion to third-stage larvae of five animal parasitic nematodes, all of which carry the cast cuticle from the previous molt, did not result in infection. M. coniospora infected animal parasitic nematodes when this protective sheath was removed. Seven preparations of sialic acid (N-acetylneuraminic acid) gave three types of response in adhesion-infection of nematodes: (i) a significant reduction in conidial adhesions; (ii) no interference with adhesion, but a 10-day delay in infection; and (iii) a delay in infection by 2 to 3 days. The current results support previous findings indicating involvement of sialic acids localized on nematode cuticles in recognition of prey by M. coniospora.

Caenorhabditis elegans: lectin-mediated modification of chemotaxis.

Binding of the lectins concanavalin A (Con A) and limulin to Caenorhabditis elegans wild type resulted in consistent, reproducible, partial inhibition of chemoattraction to sterile filtrates of Escherichia coli. Normal chemotaxis resumed within 8 hr following treatment with these lectins. Competitive displacement of Con A or limulin by flooding with the specific sugars resulted in rapid resumption of normal chemotactic behavior. The experimental protocol for Con A applied to three age groups (newly hatched larvae, young adults, and old adults) showed the same response for all groups tested. Two mutant C. elegans with morphological defects in the cephalic chemosensilla showed the same inhibition of chemotactic response after exposure to Con A, and rapidly resumed normal behavior after competitive displacement of the lectin. Limulin and Con A did not affect nematode growth, development, or longevity, demonstrating that the observed results were not attributable to toxic effects. These results and other experimental evidence support the premise that behavioral modification was caused by functional impairments caused by Con A and limulin to chemoreceptors located on sensory dendrites of the cephalic sensilla.

Control of Root-Knot Nematodes on Tomato by the Endoparasitic Fungus Meria coniospora.

The endoparasitic nematophagous fungus Meria coniospora reduced root-knot nematode galling on tomatoes in greenhouse pot trials. The fungus was introduced to pots by addition of conidia at several inoculum levels directly to the soil or addition of nematodes infected with M. coniospora to the soil; both methods reduced root galling by root-knot nematodes. These studies represent a part of a recently initiated effort to evaluate the potential of endoparasitic nematophagous fungi for biocontrol of nematodes.

Caenorhabditis elegans and Panagrellus redivivus: enzyme-mediated modification of chemotaxis.

Treatment with mannosidase or sialidase completely inhibited chemotactic responses of Caenorhabditis elegans wild type, C. elegans mutants CB1377 (daf-6)X and CB1379 (che-3)I, and Panagrellus redivivus to a source of attractants. Trypsin (EC3.4.21.4) caused a partial reduction in the level of chemoresponse. Normal chemotaxis was renewed within 20 hr following exposure to the enzymes. Other enzymes tested had no effect. Experimental and supporting evidence is presented that behavioral modification resulted from functional impairments to receptors located within chemosensory sensilla.

Life cycle of the endoparasitic nematophagous fungus Meria coniospora: a light and electron microscopic study.

The obligate endoparasitic fungus Meria coniospora lives its entire vegetative life within infected nematodes. Conidia of M. coniospora infect the nematode Panagrellus redivivus mainly in the mouth region. The infection, starting with adhesion of conidia to the nematode surface, growth of trophic hyphae, production of conidiophores and conidia, was followed using light, scanning and transmission electron microscopy.

Predacity by nematophagous fungi and its relation to the attraction of nematodes.

Predacity, the ability of nematophagous fungi to destroy nematodes, was investigated for eight species of fungi by a method using sterilized soil and the nematodePanagrellus redivivus. In addition, the ability of the fungi to attract nematodes was investigated using an agar plate technique. Predacity and attraction were highly correlated (r=0.98) in these tests. The presence of traps in cultures ofArthrobotrys oligospora increased the ability to attract nematodes by a factor of 2.