Three-Dimensional Ultrastructure of Arabidopsis Cotyledons Infected with Colletotrichum higginsianum.

We used serial block-face scanning electron microscopy (SBF-SEM) to study the host-pathogen interface between Arabidopsis cotyledons and the hemibiotrophic fungus Colletotrichum higginsianum. By combining high-pressure freezing and freeze-substitution with SBF-SEM, followed by segmentation and reconstruction of the imaging volume using the freely accessible software IMOD, we created 3D models of the series of cytological events that occur during the Colletotrichum-Arabidopsis susceptible interaction. We found that the host cell membranes underwent massive expansion to accommodate the rapidly growing intracellular hypha. As the fungal infection proceeded from the biotrophic to the necrotrophic stage, the host cell membranes went through increasing levels of disintegration culminating in host cell death. Intriguingly, we documented autophagosomes in proximity to biotrophic hyphae using transmission electron microscopy (TEM) and a concurrent increase in autophagic flux between early to mid/late biotrophic phase of the infection process. Occasionally, we observed osmiophilic bodies in the vicinity of biotrophic hyphae using TEM only and near necrotrophic hyphae under both TEM and SBF-SEM. Overall, we established a method for obtaining serial SBF-SEM images, each with a lateral (x-y) pixel resolution of 10 nm and an axial (z) resolution of 40 nm, that can be reconstructed into interactive 3D models using the IMOD. Application of this method to the Colletotrichum-Arabidopsis pathosystem allowed us to more fully understand the spatial arrangement and morphological architecture of the fungal hyphae after they penetrate epidermal cells of Arabidopsis cotyledons and the cytological changes the host cell undergoes as the infection progresses toward necrotrophy. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Antifungal Activity of Alpha-Mangostin against Colletotrichum gloeosporioides In Vitro and In Vivo.

We investigated alpha-mangostin (α-mangostin, α-MG), a xanthone natural product extracted from the pericarp of mangosteen (Garcinia mangostana), for its antifungal activities and possible mechanism against Colletotrichum gloeosporioides, which causes mango anthracnose. The results demonstrated that α-MG had a relatively high in vitro inhibitory activity against C. gloeosporioides among 20 plant pathogenic fungi. The median effective concentration (EC50) values of α-MG against mycelial growth were nearly 10 times higher than those of spore germination inhibition for both strains of C. gloeosporioides, the carbendazim-sensitive (CBD-s) and carbendazim-resistant (CBD-r). The results suggested that α-MG exhibited a better inhibitory effect on spore germination than on the mycelial growth of C. gloeosporioides. Further investigation indicated that the protective effect could be superior to the therapeutic effect for mango leaves for scab development. The morphological observations of mycelium showed that α-MG caused the accumulation of dense bodies. Ultrastructural observation further revealed that α-MG caused a decrease in the quantity and shape of the swelling of mitochondria in the mycelium cells of C. gloeosporioides. In addition, bioassays disclosed that the inhibitory activity of α-MG on spore germination was reduced by adding exogenous adenosine triphosphate (ATP). These results suggested that the mode of action of α-MG could be involved in the destruction of mitochondrial energy metabolism. The current study supports α-MG as a natural antifungal agent in crop protection.

Chitosan-citric acid edible coating to control Colletotrichum gloeosporioides and maintain quality parameters of fresh-cut guava.

This study aimed to verify the action of edible chitosan-citric acid (CHI-CA) coating to control Colletotrichum gloeosporioides and maintain quality parameters of fresh-cut guava. Chitosan was obtained from Litopenaeus vannamei shells using high temperature and short exposure times. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of CHI-CA against C. gloeosporioides were determined by macrodilutions at 28 °C/120 h in the absence/presence of CHI-CA (0-10 mg/mL). Scanning electron microscopy was used to evaluate morphological changes in the fungus. Guava slices were coated with CHI-CA (MIC) or 5 mg/mL glycerol (control). Rot incidence and physicochemical, physical, and microbiological factors were determined at 0, 3, 7, and 14 days at 24 °C and 4 °C. Chitosan presented typical structural characterization, 64% deacetylation, and a molecular weight of 1.6 × 104 g/mol. CHI-CA exhibited MIC and MFC values of 5 mg/mL and 10 mg/mL, respectively, and promoted changes in the morphology and cell surface of fungal spores. The fresh-cut guava coated with CHI-CA maintained quality parameters during storage and preserved their sensorial characteristics. Therefore, the use of CHI-CA as a coating is a promising strategy for improving postharvest quality of fresh-cut fruits.

Protein extract from Cereus jamacaru (DC.) inhibits Colletotrichum gloeosporioides growth by stimulating ROS generation and promoting severe cell membrane damage.

Mandacaru (Cereus jamacaru DC.), is a cactaceous symbol of caatinga vegetation at Brazilian Northeast region, however, there are no much studies about biochemical properties of this species. Here, the pioneering study brings very relevant data to highlight the importance of research with endemic plants of the caatinga. Afterward, the presence of enzymes such as peroxidase, protease, chitinase, ß-1,3-glucanase, and serine (trypsin) and cysteine (papain) protease inhibitors were evaluated. The peroxidase activity was higher in roots than other tissues. The ß-1,3-glucanase and proteolytic activity were prominent in stem and roots. The chitinase activity and protease inhibitor for both classes analyzed were detected in the stem and fruit peel. Antifungal activity against Colletotrichum gloeosporioides showed the root extract has a promising inhibitory activity on this economical important phytopathogenic fungus. After the contact of the hyphae with root extract increase in membrane permeability, based on Propidium Iodide (PI) uptake, and production of reactive oxygen species (ROS) were detected, compared to negative control. In addition, Scanning Electron Microscopy (SEM) analysis showed morphological damage on hyphae structure indicating that the treatment debilitates either cell membrane or cell wall leading to the cell death C. gloeosporioides.

ZnO-based nanofungicides: Synthesis, characterization and their effect on the coffee fungi Mycena citricolor and Colletotrichum sp.

In this work we compare the antifungal capacity of zinc oxide nanoparticles (ZnO-NPs) synthesized by a chemical route and a ZnO-based nanobiohybrid obtained by green synthesis in an extract of garlic (Allium sativum). To find out the characteristics of the materials synthesized, X-ray diffraction (XRD), IR spectroscopy and absorption in UV-Vis were used, as well as both scanning (SEM) and transmission (TEM) electron microscopy. The results showed that the samples obtained were of nanometric size (<100 nm), with a predominance of the wurtzite crystal phase of ZnO and little crystallization of the nanobiohybrids. Their antifungal capacity on two pathogenic fungi of coffee, Mycena citricolor (Berk and Curt) and Colletotrichum sp. was also evaluated. Both nanomaterials showed an efficient antifungal capacity, particularly the nanobiohybrids, with ~97% inhibition in growth of M. citricolor, and ~93% for Colletotrichum sp. The microstructural study with high resolution optical (HROM) and ultra-structural microscopy (using TEM) carried out on the fungi treated with the synthesized nanomaterials showed a strong nanofungicidal effect on the vegetative and reproductive structures and fungal cell wall, respectively. The inhibition of the growth of the fungi and micro and ultra-structural affectations were explained considering that the size of the nanomaterials allows them to pass easily through the cell membranes. This indicates that they can be absorbed easily by the fungi tested here, causing cellular dysfunction. Nanofungicide effects are also attributable to the unique properties of nanomaterials, such as the high surface-to-bulk ratio of atoms and their surface physicochemical characteristics that could directly or indirectly produce reactive oxygen species (ROS), which affect the proteins of the cell wall.

The expression of the genes involved in redox metabolism and hydrogen peroxide balance is associated with the resistance of cowpea [Vigna unguiculata (L.) Walp.] to the hemibiotrophic fungus Colletotrichum gloeosporioides.

Correlations between the transcriptional responses of genes that encode superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxiredoxin (Prx) enzymes and Colletotrichum gloeosporioides development in cowpea leaves were assessed. Each of these genes is involved in the redox metabolism and hydrogen peroxide balance. Although electron microscopy revealed that conidia adhered to and germinated on the leaf cuticle, the inoculated cowpea leaves did not show any characteristic anthracnose symptoms. The adhered and germinated conidia showed irregular surfaces and did not develop further. This was apparently due to increased leaf H2O2 levels in response to inoculation with C. gloeosporioides. During the early stages post inoculation, cowpea leaves elevated the H2O2 content and modulated the defense gene expression, as well as associated pathways. During the later stages, the increased expression of the CuZnSODI and CuZnSODII genes suggested an active superoxide dismutation to further elevate H2O2 levels, which indicated that higher H2O2 content may function as a toxic agent that kills the fungus. The second increase in H2O2 production above the threshold level was correlated with the expression of the APXI, CATI, CATII, PrxIIBCD, and PrxIIE genes, which resulted in a coordinated pattern to establish an appropriate balance between H2O2 generation and scavenging. Therefore, appropriate H2O2 content in cowpea leaves inhibited C. gloeosporioides development and maintained intracellular redox homeostasis to avoid uncontrolled programmed cell death and necrosis in cowpea leaves.

Genomics-guided discovery and structure identification of cyclic lipopeptides from the Bacillus siamensis JFL15.

In this research, a strain with broad-spectrum antimicrobial activities was isolated from the gastrointestinal tract of hairtail (Trichiurus haumela) and identified as Bacillus siamensis JFL15 through morphological, 16S rRNA, and average nucleotide identity analyses. The genome of B. siamensis JFL15 was sequenced, and three gene clusters involved in the biosynthesis of surfactin (srf), bacillibactin (dhb), and fengycin (fen) were predicted through antiSMASH analysis. The combined genomics-metabolics profiling of the strain revealed 20 active compounds, which belong to four main types of cyclic lipopeptides produced by Bacillus species: bacillibactin, iturin, fengycin, and surfactin. Among these lipopeptides, two high-purity antifungal components, namely, components b and c, were successfully identified as iturin A and bacillomycin F. The minimum inhibitory concentrations (MICs) of iturin A for Magnapothe grisea, Rhizoctorzia solani, and Colletotrichum gloeosporioides were 125.00, 62.50, and 125.00 µg/ml, respectively, whereas the MICs of bacillomycin F for these three organisms were 62.50, 31.25, and 62.50 µg/ml, respectively. The mechanism of bacillomycin F and iturin A against M. grisea was also investigated. Scanning electron microscopy (SEM) indicated that the surface of the hypha treated with iturin A or bacillomycin F became sunk, lumpy, and wrinkled. The diversity of the identified and predicted compounds from B. siamensis JFL15 suggested that this strain might be a promising biocontrol agent for an effective and environmentally friendly control of pathogenic microorganisms. To the best of our knowledge, this study is the first to describe cyclic lipopeptides purified and identified from B. siamensis.

A Fungus-Inducible Pepper Carboxylesterase Exhibits Antifungal Activity by Decomposing the Outer Layer of Fungal Cell Walls.

Colletotrichum species are major fungal pathogens that cause devastating anthracnose diseases in many economically important crops. In this study, we observed the hydrolyzing activity of a fungus-inducible pepper carboxylesterase (PepEST) on cell walls of C. gloeosporioides, causing growth retardation of the fungus by blocking appressorium formation. To determine the cellular basis for the growth inhibition, we observed the localization of PepEST on the fungus and found the attachment of the protein on surfaces of conidia and germination tubes. Moreover, we examined the decomposition of cell-wall materials from the fungal surface after reaction with PepEST, which led to the identification of 1,2-dithiane-4,5-diol (DTD) by gas chromatography mass spectrometry analysis. Exogenous DTD treatment did not elicit expression of defense-related genes in the host plant but did trigger the necrosis of C. gloeosporioides. Furthermore, the DTD compound displayed protective effects on pepper fruits and plants against C. gloeosporioides and C. coccodes, respectively. In addition, DTD was also effective in preventing other diseases, such as rice blast, tomato late blight, and wheat leaf rust. Therefore, our results provide evidence that PepEST is involved in hydrolysis of the outmost layer of the fungal cell walls and that DTD has antifungal activity, suggesting an alternative strategy to control agronomically important phytopathogens.

The Glycosylphosphatidylinositol Anchor Biosynthesis Genes GPI12, GAA1, and GPI8 Are Essential for Cell-Wall Integrity and Pathogenicity of the Maize Anthracnose Fungus Colletotrichum graminicola.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is one of the most common posttranslational modifications of proteins in eukaryotic cells and is important for associating proteins with the cell surface. In fungi, GPI-anchored proteins play essential roles in cross-linking of ß-glucan cell-wall polymers and cell-wall rigidity. GPI-anchor synthesis is successively performed at the cytoplasmic and the luminal face of the ER membrane and involves approximately 25 proteins. While mutagenesis of auxiliary genes of this pathway suggested roles of GPI-anchored proteins in hyphal growth and virulence, essential genes of this pathway have not been characterized. Taking advantage of RNA interference (RNAi) we analyzed the function of the three essential genes GPI12, GAA1 and GPI8, encoding a cytoplasmic N-acetylglucosaminylphosphatidylinositol deacetylase, a metallo-peptide-synthetase and a cystein protease, the latter two representing catalytic components of the GPI transamidase complex. RNAi strains showed drastic cell-wall defects, resulting in exploding infection cells on the plant surface and severe distortion of in planta-differentiated infection hyphae, including formation of intrahyphal hyphae. Reduction of transcript abundance of the genes analyzed resulted in nonpathogenicity. We show here for the first time that the GPI synthesis genes GPI12, GAA1, and GPI8 are indispensable for vegetative development and pathogenicity of the causal agent of maize anthracnose, Colletotrichum graminicola.

Anthracnose disease of centipedegrass turf caused by Colletotrichum eremochloae, a new fungal species closely related to Colletotrichum sublineola.

Colletotrichum is a cosmopolitan, anamorphic fungal genus responsible for anthracnose disease in hundreds of plant species worldwide, including members of the Poaceae. Anthracnose disease of the widely planted, non-native, warm-season lawn grass, Eremochloae ophiuroides (centipedegrass), is commonly encountered in the southern United States, but the causal agent has never been identified. We use DNA sequence data from modern cultures and archival fungarium specimens in this study to determine the identity of the fungus responsible for centipedegrass anthracnose disease and provide experimental confirmation of pathogenicity. C. eremochloae sp. nov., a pathogen of centipedegrass, is proposed based on phylogenetic evidence from four sequence markers (Apn2, Apn2/ Mat1, Sod2, ITS). C. eremochloae isolates from centipedegrass shared common morphology and phenotype with C. sublineola, a destructive pathogen of cultivated sorghum and Johnsongrass weeds (Sorghum halepense, S. vulgaris). Molecular phylogenetic analysis identified C. eremochloae and C. sublineola as closely related sister taxa, but genealogical concordance supported their distinction as unique phylogenetic species. Fixed nucleotide differences between C. eremochloae and C. sublineola were observed from collections of these fungi spanning 105 y, including the 1904 lectotype specimen of C. sublineola. C. eremochloae was identified from a fungarium specimen of centipedegrass intercepted at a USA port from a 1923 Chinese shipment; the multilocus sequence from this specimen was identical to modern samples of the fungus. Thus, it appears that the fungus might have migrated to the USA around the same time that centipedegrass first was introduced to the USA in 1916 from China, where the grass is indigenous. The new species C. eremochloae is described and illustrated, along with a description and discussion of C. sublineola based on the lectotype and newly designated epitype.

Sequential delivery of host-induced virulence effectors by appressoria and intracellular hyphae of the phytopathogen Colletotrichum higginsianum.

Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death.

Cell death of Nicotiana benthamiana is induced by secreted protein NIS1 of Colletotrichum orbiculare and is suppressed by a homologue of CgDN3.

Colletotrichum orbiculare, the causal agent of cucumber anthracnose, infects Nicotiana benthamiana. Functional screening of C. orbiculare cDNAs in a virus vector-based plant expression system identified a novel secreted protein gene, NIS1, whose product induces cell death in N. benthamiana. Putative homologues of NIS1 are present in selected members of fungi belonging to class Sordariomycetes, Dothideomycetes, or Orbiliomycetes. Green fluorescent protein-based expression studies suggested that NIS1 is preferentially expressed in biotrophic invasive hyphae. NIS1 lacking signal peptide did not induce NIS1-triggered cell death (NCD), suggesting apoplastic recognition of NIS1. NCD was prevented by virus-induced gene silencing of SGT1 and HSP90, indicating the dependency of NCD on SGT1 and HSP90. Deletion of NIS1 had little effect on the virulence of C. orbiculare against N. benthamiana, suggesting possible suppression of NCD by C. orbiculare at the postinvasive stage. The CgDN3 gene of C. gloeosporioides was previously identified as a secreted protein gene involved in suppression of hypersensitive-like response in Stylosanthes guianensis. Notably, we found that NCD was suppressed by the expression of a CgDN3 homologue of C. orbiculare. Our findings indicate that C. orbiculare expresses NIS1 at the postinvasive stage and suggest that NCD could be repressed via other effectors, including the CgDN3 homologue.

Pectinolytic enzyme production by Colletotrichum truncatum, causal agent of soybean anthracnose

Antecedentes. Colletotrichum truncatum es el hongo patógeno más comúnmente asociado con la antracnosis de la soja, enfermedad de alta prevalencia en Argentina. Las enzimas pectinolíticas se relacionan con la patogenicidad de un amplio rango de hongos fitopatógenos. Objetivos. Investigar la producción de enzimas pectinolíticas por cepas aisladas de plantas de soja enfermas de diferentes regiones de nuestro país, y con ello contribuir a la caracterización fisiológica de dichos aislamientos como paso preliminar para esclarecer el aún desconocido rol biológico de las enzimas pectinolíticas en la interacción Colletotrichum spp-soja. Métodos. Se investigó la producción in vitro de enzimas pectinolíticas, en un medio sintético con pectina como fuente de carbono, de diez aislamientos de C. truncatum. Resultados. Todas las cepas crecieron en dicho medio, detectándose actividades polimetilgalacturonasa (PMG), poligalacturonasa (PG) y pectin liasa (PL). En general, el pico de galacturonasas precedió al día de máximo crecimiento, en cambio el de PL se registró posteriormente. La cepa BAFC 3097 (originaria de la Provincia de Santa Fe) produjo altos títulos de las tres enzimas tras 7–10 días: 1,08U/ml PG, 1,05U/ml PMG, 156U/ml PL. C. truncatum, cultivado en un medio con glucosa como fuente de carbono, produjo PG y PMG (pero no PL), aunque su síntesis disminuyó marcadamente sugiriendo que estas enzimas son constitutivas. Conclusión. La disparidad registrada en la producción enzimática entre cepas no puede atribuirse al crecimiento fúngico; tampoco se corresponde con su distribución geográfica; pero podría relacionarse con diferencias en su virulencia, que aún no se han investigado(AU)

Background. Colletotrichum truncatum is the most common pathogenic fungus associated with soybean anthracnose, a prevalent disease in Argentina. Pectinolytic enzymes are involved in the pathogenicity of a wide range of plant pathogenic fungi. Objectives. To explore pectinolytic enzyme production in Argentinian Colletotrichum strains isolated from diseased soybean plants from different geographic locations, as a preliminary step to establish the biological role of the pectinolytic enzymes in the Colletotrichum spp.–soybean system, yet unknown. Methods. Ten strains were screened for in vitro pectinolytic enzyme production on a defined medium based on pectin as carbon source. Results. All isolates were able to grow in this medium and polymethylgalacturonase (PMG), polygalacturonase (PG) and pectin lyase (PL) activities were detected. On the whole, the peak of polygalacturonases activities preceded the day of maximum growth, while PL activity reached its highest level afterwards. Strain BAFC 3097 (from Santa Fe province) yielded high titles of the three enzymes (1.08U/ml PG, 1.05U/ml PMG, 156U/ml PL), after a short incubation period (7–10 days). Low synthesis of polygalacturonases in cultures containing glucose as unique carbon source suggests that these enzymes are constitutive in contrast with PL, which was not detected. Conclusions. The disparity observed in enzyme production among strains cannot be related to fungal growth, since no major differences in mycelial yield were found; it was not connected with their geographic origin, but might be associated with differences in virulence among strains not yet evaluated(AU)

Occurrence and characterization of Colletotrichum dematium (Fr.) grove.

Colletotrichum dematium was isolated from caraway for the first time in Poland in 2005. Isolations of this fungus were repeated in 2006 and 2007. The cultures of fungus were obtained from superficially disinfected leaves, root necks, roots, stems and umbels. The isolates were identified on culture media: PDA and malt agar with addition of pieces of caraway stems and on the base of macro and microscopic structures. Studies on the biotic effect between C. dematium and other species of phyllosphere fungi of caraway showed that the majority of the studied species limited the growth and development of C. dematium, but the size of the limiting effect was different. The species from Trichoderma and Gliocladium genera were the most effective against C. dematium, causing degeneration and lysis of hyphae and precluded the formation of the pathogen's acervuli and conidia. C. dematium in dual culture with E. purpurascens, A. radicina, S. sclerotiorum, B. cinerea and R. solani produced an inhibition zone which indicated its capacity for antibiosis.

Peroxisome biogenesis factor PEX13 is required for appressorium-mediated plant infection by the anthracnose fungus Colletotrichum orbiculare.

Peroxisomes are ubiquitous organelles of eukaryotic cells that fulfill a variety of biochemical functions, including beta-oxidation of fatty acids. Here, we report that an ortholog of the Saccharomyces cerevisiae peroxisome biogenesis gene PEX13 is required for pathogenicity of Colletotrichum orbiculare. CoPEX13 was identified by screening random insertional mutants for deficiency in fatty acid utilization. Targeted knockout mutants of CoPEX13 were unable to utilize fatty acids as a carbon source. Expression analysis using green fluorescent protein fused to the peroxisomal targeting signals PTS1 and PTS2 revealed that the import machinery for peroxisomal matrix proteins was impaired in copex13 mutants. Appressoria formed by the copex13 mutants were defective in both melanization and penetration ability on host plants, had thin cell walls, and lacked peroxisomes. Moreover, the concentration of intracellular glycerol was lower in copex13 appressoria than those of the wild type. These findings indicate that fatty acid oxidation in peroxisomes is required not only for appressorium melanization but also for cell wall biogenesis and metabolic processes involved in turgor generation, all of which are essential for appressorium penetration ability.

Dual trypan-aniline blue fluorescence staining methods for studying fungus-plant interactions.

Understanding the infection biology of fungi is the key step in devising suitable control strategies for plant diseases. Recently, the Arabidopsis-Colletotrichum higginsianum (causal agent of anthracnose) system has emerged as a seminal paradigm for deciphering the infection biology underlying fungus-plant interactions. We describe here three staining methods coupled with confocal microscopy: trypan blue, aniline blue and dual trypan blue-aniline blue fluorescence staining. Trypan blue and aniline blue staining were employed to scan the infection structures of the hemibiotrophic fungus C. higginsianum and host response in A. thaliana leaf tissues. The two techniques then were combined to observe the contrast between in planta fungal infection structures, i.e., infection vesicles, primary hyphae and secondary hyphae, and the host plant defense responses, i.e., papilla formation and hypersensitive response. These staining techniques also were applied to the lentil-C. truncatum pathosystem to demonstrate their applicability for multiple pathosystems.

Systematic analysis of the falcate-spored graminicolous Colletotrichum and a description of six new species from warm-season grasses.

Species limits in the fungal genus Colletotrichum are traditionally distinguished by appressorial and/or conidial morphology or through host plant association, but both criteria are criticized for their inability to resolve distinct taxa. In previous research eight novel falcate-spored Colletotrichum species were identified from graminicolous hosts using multilocus molecular phylogenetic analysis. In the present work formal descriptions and illustrations are provided for six of the new taxa: C. hanaui sp. nov., C. nicholsonii sp. nov., C. paspali sp. nov., C. jacksonii sp. nov., C. miscanthi sp. nov. and C. axonopodi sp. nov.; and an emended description with epitypification is provided for C. eleusines. Comparison of hyphopodial appressoria and host association against phylogenetic species boundaries and evolutionary relationships in the graminicolous Colletotrichum group demonstrate that, while these characters can be useful in combination for the purpose of species diagnosis, erroneous identification is possible and species boundaries might be underestimated if these characters are used independently, as exemplified by the polyphyletic taxa C. falcatum. Appressoria have been subject to convergent evolution and were not predictive of phylogenetic relationships. Despite these limitations, the results of this work establish that in combination appressorial and host range characters could be used to generate informative dichotomous identification keys for Colletotrichum species groups when an underlying framework of evolutionary relationships, taxonomic criteria and nomenclature have been satisfactorily derived from molecular systematic treatments.

Colutellin A, an immunosuppressive peptide from Colletotrichum dematium.

Colletotrichum dematium is an endophytic fungus recovered from a Pteromischum sp. growing in a tropical forest in Costa Rica. This fungus makes a novel peptide antimycotic, colutellin A, with a MIC of 3.6 microg ml(-1) (48 h) against Botrytis cinerea and Sclerotinia sclerotiorum. Collutellin A has a mass of 1127.7 Da and contains residues of Ile, Val, Ser, N-methyl-Val and beta-aminoisobutryic acid in nominal molar ratios of 3 : 2 : 1 : 1 : 1, respectively. Independent lines of evidence suggest that the peptide is cyclic and sequences of Val-Ile-Ser-Ile and Ile-Pro-Val have been deduced by MS/MS as well as Edman degradation methods. Colutellin A inhibited CD4(+) T-cell activation of interleukin 2 (IL-2) production with an IC(50) of 167.3+/-0.38 nM, whereas cyclosporin A in the same test yielded a value of 61.8 nM. Inhibition of IL-2 production by collutellin A at such a low concentration indicates the potential immunosuppressive activity of this compound. In repeated experiments, cyclosporin A at or above 8 microg ml(-1) exhibited high levels of cytotoxicity on human peripheral blood mononuclear cells, whereas collutellin A or DMSO (carrier) alone, after 24 and 48 h of culture, exhibited no toxicity. Because of these properties collutellin A has potential as a novel immunosuppressive drug.

Antifungal activity of strawberry fruit volatile compounds against Colletotrichum acutatum.

Eight volatile products characterizing strawberry aroma, which is generated from the oxidative degradation of linoleic and linolenic acids by a lipoxygenase (LOX) pathway, were examined because of their antifungal activity against Colletotrichum acutatum, one of the causal agents of strawberry anthracnose. In this study, the effects of aldehydes, alcohols, and esters on mycelial growth and conidia development were evaluated. (E)-Hex-2-enal was found to be the best inhibitor of mycelial growth [MID (minimum inhibitory doses)=33.65 microL L(-1)] and of spore germination (MID=6.76 microL L(-1)), while hexyl acetate was the least effective of all volatile compounds tested (MID=6441.89 microL L(-1) for mycelial growth and MID=1351.35 microL L(-1) for spore germination). Furthermore, the antifungal activity of (E)-hex-2-enal on susceptibility of strawberry fruits to C. acutatum was also confirmed. The presence of these molecules in jars containing strawberry fruits inoculated with a suspension of spores inhibited the fungus growth and prevented the appearance of symptoms. Moreover, a study of the effects of (E)-hex-2-enal on conidial cells of C. acutatum was evaluated by transmission electron microscopy. This volatile compound altered the structures of the cell wall and plasma membrane, causing disorganization and lysis of organelles and, eventually, cell death.

Saccharomyces cerevisiae SSD1 orthologues are essential for host infection by the ascomycete plant pathogens Colletotrichum lagenarium and Magnaporthe grisea.

Fungal plant pathogens have evolved diverse strategies to overcome the multilayered plant defence responses that confront them upon host invasion. Here we show that pathogenicity of the cucumber anthracnose fungus, Colletotrichum lagenarium, and the rice blast fungus, Magnaporthe grisea, requires a gene orthologous to Saccharomyces cerevisiae SSD1, a regulator of cell wall assembly. Screening for C. lagenarium insertional mutants deficient in pathogenicity led to the identification of ClaSSD1. Following targeted gene replacement, appressoria of classd1 mutants retained the potential for penetration but were unable to penetrate into host epidermal cells. Transmission electron microscopy suggested that appressorial penetration by classd1 mutants was restricted by plant cell wall-associated defence responses, which were observed less frequently with the wild-type strain. Interestingly, on non-host onion epidermis classd1 mutants induced papilla formation faster and more abundantly than the wild type. Similarly, colonization of rice leaves by M. grisea was severely reduced after deletion of the orthologous MgSSD1 gene and attempted infection by the mutants was accompanied by the accumulation of reactive oxygen species within the host cell. These results suggest that appropriate assembly of the fungal cell wall as regulated by SSD1 allows these pathogens to establish infection by avoiding the induction of host defence responses.