Luis Vicente Lopez-Llorca

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).

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.

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.

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.

Sequencing and functional analysis of the genome of a nematode egg-parasitic fungus, Pochonia chlamydosporia

Pochonia chlamydosporia is a worldwide-distributed soil fungus with a great capacity to infect and destroy the eggs and kill females of plant-parasitic nematodes. Additionally, it has the ability to colonize endophytically roots of economically-important crop plants, thereby promoting their growth and eliciting plant defenses. This multitrophic behavior makes P. chlamydosporia a potentially useful tool for sustainable agriculture approaches. We sequenced and assembled ∼41 Mb of P. chlamydosporia genomic DNA and predicted 12,122 gene models, of which many were homologous to genes of fungal pathogens of invertebrates and fungal plant pathogens. Predicted genes (65%) were functionally annotated according to Gene Ontology, and 16% of them found to share homology with genes in the Pathogen Host Interactions (PHI) database. The genome of this fungus is highly enriched in genes encoding hydrolytic enzymes, such as proteases, glycoside hydrolases and carbohydrate esterases. We used RNA-Seq technology in order to identify the genes expressed during endophytic behavior of P. chlamydosporia when colonizing barley roots. Functional annotation of these genes showed that hydrolytic enzymes and transporters are expressed during endophytism. This structural and functional analysis of the P. chlamydosporia genome provides a starting point for understanding the molecular mechanisms involved in the multitrophic lifestyle of this fungus. The genomic information provided here should also prove useful for enhancing the capabilities of this fungus as a biocontrol agent of plant-parasitic nematodes and as a plant growth-promoting organism.

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.

Immunocytochemical localization of a 32-kDa protease from the nematophagous fungusVerticillium suchlasporium in infected nematode eggs

Abstract Fluorescein-labeled anti-rabbit antiserum showed that a serine protease designated P32, produced by the nematophagous fungus Verticillium suchlasporium , is secreted during infection of nematode eggs. Increased fluorescence in appressoria of the fungus on eggshells of the plant parasitic nematode Heterodera schachtii indicated the presence of P32 in these fungal structures. Appressoria are involved in host penetration and these results support a role for P32 in the pathogenicity of the fungus to nematode eggs. These findings agree with similar results of entomogenous fungi penetrating the insect cuticle.

Mode of Action and Interactions of Nematophagous Fungi

Nematophagous fungi are potential candidates for biological control of plant-parasitic nematodes, and an important constituent in integrated pest management programs. In this chapter we describe various aspects on the biology of these fungi. Nematophagous species can be found in most fungal taxa, indicating that the nematophagous habit evolved independently in the different groups of nematophagous fungi. Regarding their mode of action we discuss recognition phenomena (e.g. chemotaxis and adhesion), signaling and differentiation, and penetration of the nematode cuticle/eggshell using mechanical, as well as enzymatic (protease and chitinase) means. The activities of nematophagous fungi in soil and rhizosphere is also discussed.

Infection of the Red Palm Weevil (Rhynchophorus ferrugineus) by the entomopathogenic fungus Beauveria bassiana: A SEM study

The Red Palm Weevil (Rhynchophorus ferrugineus) is a devastating pest of palms in the Mediterranean, Middle East, and Eastern countries. No effective control measures are available. R. ferrugineus has been found naturally infected by the entomopathogenic fungus Beauveria bassiana, but its infection process in this host is unknown. We have studied the infection of R. ferrugineus larvae and adults by B. bassiana using dry conidia and conidia suspensions using scanning electron microscopy (SEM). In early stages, SEM revealed acquisition of B. bassiana conidia by cuticle ornamentation in legs, antennae, and elytra of R. ferrugineus adults. Subsequently, conidia germinated and frequent episodes of hyphal/conidial fusion were found. Appressoria, signs of adhesion and cuticle degradation led to penetration (even direct) and colonization of R. ferrugineus hosts by the fungus. B. bassiana conidiophores were found in a R. ferrugineus cuticle, which indicate the completion of the life cycle of the fungus in the insect host. SEM has proven that dry conidia of B. bassiana is an adequate inoculum for R. ferrugineus infection. SEM revealed that conidia of B. bassiana attached to the cuticle of R. ferrugineus can germinate and differentiate appressoria. Microsc. Res. Tech., 2010.