Collacyclumines A-D from the endophytic fungus Colletotrichum salsolae SCSIO 41021 isolated from the mangrove Kandelia candel.

Four undescribed alkaloids, namely collacyclumines A-D, along with a known analogue, agrocybenine, were isolated from the endophytic fungus Colletotrichum salsolae SCSIO 41021 derived from the mangrove plant Kandelia candel (L.) Druce. Collacyclumine A represents the first case of dimeric pyrrolidine alkaloid in nature. The structures of these compounds were elucidated by a combination of NMR spectra, HRESIMS data, and X-ray diffraction experiment. A proposed biosynthetic pathway of these isolated compounds were also discussed. None of compounds showed cytotoxic effects against ten cell lines.

Species boundaries in plant pathogenic fungi: a Colletotrichum case study.

BACKGROUND: Accurate delimitation of plant pathogenic fungi is critical for the establishment of quarantine regulations, screening for genetic resistance to plant pathogens, and the study of ecosystem function. Concatenation analysis of multi-locus DNA sequence data represents a powerful and commonly used approach to recognizing evolutionary independent lineages in fungi. It is however possible to mask the discordance between individual gene trees, thus the speciation events might be erroneously estimated if one simply recognizes well supported clades as distinct species without implementing a careful examination of species boundary. To investigate this phenomenon, we studied Colletotrichum siamense s. lat., which is a cosmopolitan pathogen causing serious diseases on many economically important plant hosts. Presently there are significant disagreements among mycologists as to what constitutes a species in C. siamense s. lat., with the number of accepted species ranging from one to seven. RESULTS: In this study, multiple approaches were used to test the null hypothesis "C. siamense is a species complex", using a global strain collection. Results of molecular analyses based on the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) and coalescent methods (e.g. Generalized Mixed Yule-coalescent and Poisson Tree Processes) do not support the recognition of any independent evolutionary lineages within C. siamense s. lat. as distinct species, thus rejecting the null hypothesis. This conclusion is reinforced by the recognition of genetic recombination, cross fertility, and the comparison of ecological and morphological characters. Our results indicate that reproductive isolation, geographic and host plant barriers to gene flow are absent in C. siamense s. lat. CONCLUSIONS: This discovery emphasized the importance of a polyphasic approach when describing novel species in morphologically conserved genera of plant pathogenic fungi.

Discovery of pathogenicity genes in the crucifer anthracnose fungus Colletotrichum higginsianum, using random insertional mutagenesis.

Agrobacterium tumefaciens-mediated transformation (ATMT) was used for random insertional mutagenesis to identify pathogenicity genes in the hemibiotrophic fungus Colletotrichum higginsianum. A high-throughput primary infection assay on Arabidopsis thaliana seedlings allowed the rapid screening of 8,850 transformants. Forty mutants showing reproducible pathogenicity defects on Arabidopsis and Brassica plants were obtained, and their infection phenotypes were characterized microscopically. Six mutants were impaired in appressorial melanization, fifteen had reduced penetration ability, 14 induced host papillae or hypersensitive cell death, and five were affected in the transition from biotrophy to necrotrophy. Southern blot analysis showed 58% of the transformants had single-site T-DNA integrations. Right-border flanking sequences were recovered from 12 mutants by inverse polymerase chain reaction (PCR) or thermal asymmetric interlaced PCR and were used to isolate the tagged genes from a genomic library. The putative pathogenicity genes encoded homologs of a major facilitator superfamily phosphate transporter, importin-beta2, ornithine decarboxylase, beta-1,3(4)-glucanase, ATP-binding endoribonuclease, carbamoyl-phosphate synthetase, and the polyprotein precursor of N-acetylglutamate kinase and N-acetylglutamyl-phosphate reductase. Six further loci were homologous to proteins of unknown function. None of these genes were previously implicated in the pathogenicity of any Colletotrichum species. The results demonstrate that ATMT is an effective tool for gene discovery in this model pathogen.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Proline suppresses apoptosis in the fungal pathogen Colletotrichum trifolii.

The role of reactive oxygen species (ROS) in cell communication, control of gene expression, and oxygen sensing is well established. Inappropriate regulation of ROS levels can damage cells, resulting in a diseased state. In Colletotrichum trifolii, a fungal pathogen of alfalfa, the mutationally activated oncogenic fungal Ras (DARas) elevates levels of ROS, causing abnormal fungal growth and development and eventual apoptotic-like cell death but only when grown under nutrient-limiting conditions. Remarkably, restoration to the wild-type phenotype requires only proline. Here, we describe a generally unrecognized function of proline: its ability to function as a potent antioxidant and inhibitor of programmed cell death. Addition of proline to DARas mutant cells effectively quenched ROS levels and prevented cell death. Treating cells with inhibitors of ROS production yielded similar results. In addition, proline protected wild-type C. trifolii cells against various lethal stresses, including UV light, salt, heat, and hydrogen peroxide. These observations appear to be general because proline also protected yeast cells from lethal levels of the ROS-generating herbicide methyl viologen (paraquat), suggesting a common protective role for proline in response to oxidative stress. The ability of proline to scavenge intracellular ROS and inhibit ROS-mediated apoptosis may be an important and broad-based function of this amino acid in responding to cellular stress, in addition to its well established role as an osmolyte.

Functional analysis of Ras in Colletotrichum trifolii.

Ras is a small monomeric GTP binding protein that transduces signals for growth and differentiation of eukaryotic organisms. Previously, a unique ras gene, designated Ct-ras, was cloned from the alfalfa fungal phytopathogen, Colletotrichum trifolii. Expression of Ct-Ras in mouse fibroblast cells (NIH3T3) demonstrated that Ct-ras is functionally similar to the mammalian ras genes since activating mutations of Ct-ras caused oncogenic phenotypes in nu/nu mice, including tumors. In C. trifolii, activated 'oncogenic' Ras (Val2) induced abnormal hyphal proliferation, defects in polarized growth and significantly reduced differentiation such as conidiation and appressorium formation in a nutrient dependent manner. Gene disruption of ct-ras was lethal. To further evaluate the function of Ct-Ras in C. trifolii, three different approaches were used: overexpression of cytosolic Ras by CAAX box deletion; expression of dominant negative Ct-RasT22N; and antisense ct-ras expression. Results showed that suppression of Ct-Ras activity significantly decreases fungal germination frequencies and hyphal growth rates. Taken together, these data suggest involvement of Ct-Ras in regulation of fungal cell growth and differentiation.

A Ras protein from a phytopathogenic fungus causes defects in hyphal growth polarity, and induces tumors in mice.

Ras is a low-molecular-weight guanine nucleotide (GDP/GTP)-binding protein that transduces signals for growth and differentiation in eukaryotes. In mammals, the importance of Ras in regulating growth is underscored by the observation that activating mutations in ras genes are found in many animal tumors. Colletorichum trifolii is a filamentous fungal pathogen of alfalfa which causes anthracnose disease. To investigate signaling pathways that regulate growth and development in this fungus, a gene encoding a Ras homolog (CT-Ras) was cloned from C. trifolii. CT-Ras exhibited extensive amino acid similarity to Ras proteins from higher and lower eukaryotes. A single amino acid change resulting in mutationally activated CT-Ras induced cellular transformation of mouse (NIH 3T3) fibroblasts and tumor formation in nu/nu mice. In Colletotrichum, mutationally activated CT-Ras induced abnormal hyphal proliferation and defects in polarized growth, and significantly reduced differentiation in a nutrient-dependent manner. These results show that C. trifolii Ras is a functional growth regulator in both mammals and fungi, and demonstrate that proper regulation of Ras is required for normal fungal growth and development.