The Colletotrichum genus has been considered as one of the top 10 fungal pathogens in molecular plant pathology based on their scientific and agrobiological importance. Although the genus contains species with different lifestyles, most of the Colletotrichum sp. are known by their hemibiotrophic strategy of infection/invasion causing anthracnose disease in many economically important crops. Hemibiotrophy includes two sequential stages of infection, biotrophy and necrotrophy, in a series of steps that involve the participation of different virulence factors. In this review, we present the current status of the knowledge of such factors reported in this genus and a list of related genes identified in Colletotrichum sp. genomes.

BackgroundMicroorganisms produce cell-wall-degrading enzymes as part of their strategies for plant invasion/nutrition. Among these, pectin lyases (PNLs) catalyze the depolymerization of esterified pectin by a β-elimination mechanism. PNLs are grouped together with pectate lyases (PL) in Family 1 of the polysaccharide lyases, as they share a conserved structure in a parallel β-helix. The best-characterized fungal pectin lyases are obtained from saprophytic/opportunistic fungi in the genera Aspergillus and Penicillium and from some pathogens such as Colletotrichum gloeosporioides.The organism used in the present study, Colletotrichum lindemuthianum, is a phytopathogenic fungus that can be subdivided into different physiological races with different capacities to infect its host, Phaseolus vulgaris. These include the non-pathogenic and pathogenic strains known as races 0 and 1472, respectively.ResultsHere we report the isolation and sequence analysis of the Clpnl2 gene, which encodes the pectin lyase 2 of C. lindemuthianum, and its expression in pathogenic and non-pathogenic races of C. lindemuthianum grown on different carbon sources. In addition, we performed a phylogenetic analysis of the deduced amino acid sequence of Clpnl2 based on reported sequences of PNLs from other sources and compared the three-dimensional structure of Clpnl2, as predicted by homology modeling, with those of other organisms. Both analyses revealed an early separation of bacterial pectin lyases from those found in fungi and oomycetes. Furthermore, two groups could be distinguished among the enzymes from fungi and oomycetes: one comprising enzymes from mostly saprophytic/opportunistic fungi and the other formed mainly by enzymes from pathogenic fungi and oomycetes. Clpnl2 was found in the latter group and was grouped together with the pectin lyase from C. gloeosporioides.ConclusionsThe Clpnl2 gene of C. lindemuthianum shares the characteristic elements of genes coding for pectin lyases. A time-course analysis revealed significant differences between the two fungal races in terms of the expression of Clpnl2 encoding for pectin lyase 2. According to the results, pectin lyases from bacteria and fungi separated early during evolution. Likewise, the enzymes from fungi and oomycetes diverged in accordance with their differing lifestyles. It is possible that the diversity and nature of the assimilatory carbon substrates processed by these organisms played a determinant role in this phenomenon.

One of the most challenging environmental threats of the last two decades is the effects of emerging pollutants (EPs) such as pharmaceutical compounds or industrial additives. Diclofenac and bisphenol A have regularly been found in wastewater treatment plants, and in soils and water bodies because of their extensive usage and their recalcitrant nature. Due to the fact of this adversity, fungal communities play an important role in being able to safely degrade EPs. In this work, we obtained a sewage sludge sample to study both the culturable and non-culturable microorganisms through DNA extraction and massive sequencing using Illumina MiSeq techniques, with the goal of finding degraders adapted to polluted environments. Afterward, degradation experiments on diclofenac and bisphenol A were performed with the best fungal degraders. The analysis of bacterial diversity showed that Dethiosulfovibrionaceae, Comamonadaceae, and Isosphaeraceae were the most abundant families. A predominance of Ascomycota fungi in the culturable and non-culturable population was also detected. Species such as Talaromyces gossypii, Syncephalastrum monosporum, Aspergillus tabacinus, and Talaromyces verruculosus had remarkable degradation rates, up to 80% of diclofenac and bisphenol A was fully degraded. These results highlight the importance of characterizing autochthonous microorganisms and the possibility of selecting native fungal microorganisms to develop tailored biotransformation technologies for EPs.

Colletotrichum lindemuthianum is the etiological agent of anthracnose, one of the main diseases of bean (Phaseolus vulgaris). In this study, the complete cDNAs of two endo-β-1,4-xylanase genes (xyl1) from non-pathogenic (0) and pathogenic (1472) races of C. lindemuthianum were isolated and characterized. To get an insight into the role of endo-β-1,4-xylanases in their different lifestyles, xyl1 gene expression and enzyme activity in mycelia of both races grown in the presence of xylan or P. vulgaris cell walls were investigated. The xyl1 sequence analysis and Clustal alignment revealed the characteristic elements of genes coding for endo-β-1,4-xylanases of the GH11 family. The growth of the two races with glucose as the sole carbon source showed both basal transcription levels of xyl1 and endoxylanase activity. When glucose was substituted with xylan or plant cell walls, xyl1 transcription, and enzyme activity significantly increased in race 1472 as compared to race 0. The pathogenic race degraded xylan faster and grew better than the non-pathogenic counterpart. Seemingly, the regulation of xylanolytic gene expression, enzyme production and the nature of the assimilatory carbon substrates processed by these organisms play a determinant role in their lifestyle. Phylogenetic analyses of XYL1 and endo-β-1,4-xylanases from other fungi revealed a diversification process and separation of proteins from the same fungal species into different lineages.

Pectin lyases (PNLs) are important enzymes that are involved in plant cell wall degradation during the infection process. Colletotrichum is a diverse genus of fungi, which allows the study of the evolution of PNLs and their possible role in pathogen-host interactions and lifestyle adaptations. The phylogenetic reconstruction of PNLs from Colletotrichum and analysis of selection pressures showed the formation of protein lineages by groups of species with different selection pressures and specific patterns. The analysis of positive selection at individual sites using different methods allowed for the identification of three codons with evidence of positive selection in the oligosaccharide-binding region and two codons on the antiparallel sheet, which may influence the interaction with the substrate. Seven codons on the surface of the protein, mainly in the peripheral helices of the PNLs, could have an important function in evasion of plant defenses, as has been proposed in other enzymes. According to our results, it is possible that events of genetic duplication occurred in ancestral lines, followed by episodes of genetic diversification and gene loss, probably influenced by differences in the composition of the host cell wall. Additionally, different patterns of evolution in Colletotrichum appear to be molded by a strong purifying selection and positive selection episodes that forged the observed evolutionary patterns, possibly influenced by host interaction or substrate specificity. This work represents a starting point for the study of sites that may be important for evasion of plant defenses and biotechnological purposes.