Evolutionary history of the cytochrome P450s from Colletotrichum species and prediction of their putative functional roles during host-pathogen interactions.

The genomes of species belonging to the genus Colletotrichum harbor a substantial number of cytochrome P450 monooxygenases (CYPs) encoded by a broad diversity of gene families. However, the biological role of their CYP complement (CYPome) has not been elucidated. Here, we investigated the putative evolutionary scenarios that occurred during the evolution of the CYPome belonging to the Colletotrichum Graminicola species complex (s.c.) and their biological implications. The study revealed that most of the CYPome gene families belonging to the Graminicola s.c. experienced gene contractions. The reductive evolution resulted in species restricted CYPs are predominant in each CYPome of members from the Graminicola s.c., whereas only 18 families are absolutely conserved among these species. However, members of CYP families displayed a notably different phylogenetic relationship at the tertiary structure level, suggesting a putative convergent evolution scenario. Most of the CYP enzymes of the Graminicola s.c. share redundant functions in secondary metabolite biosynthesis and xenobiotic metabolism. Hence, this current work suggests that the presence of a broad CYPome in the genus Colletotrichum plays a critical role in the optimization of the colonization capability and virulence.

First Report of Colletotrichum graminicola Causing Maize Anthracnose in Galicia, Northwestern Spain.

Maize (Zea mays) is one of the most important crops worldwide, and fungal diseases are responsible for major losses in food production. Anthracnose caused by Colletotrichum graminicola can infect all maize tissues, although stalk rot and seedling blight cause more significant economic damage (Munkvold and White, 2016). Anthracnose stalk rot is characterized by a distinctive external blackening of the lower stalks resulting in large black streaks, while the pith turns dark brown and has a shredded appearance. Like most stalk rots, the most obvious symptom is a sudden death of plants before grain maturity, and plant lodging. Symptoms commonly appear late in the season, suspicious maize stems of cultivar Tuy exhibiting symptoms of anthracnose stalk rot were collected from a field in Pontevedra, Galicia, Spain (Geographical coordinates: 42°23'27.1" N - 8°30'46.3" W) between June and December of 2022. Stem samples, approximately 50 mm2, were dissected and surface-disinfected for 90 seconds in 20% sodium hypochlorite (v/v) and rinsed three times in sterile distilled water. The samples were transferred to one half-strength acidified potato dextrose agar (PDA) supplemented with ampicillin (100 µg/mL) and lactic acid 90% (1.5 mL/L) and incubated for 5 days at 25 ºC (Sukno et al. 2008). Single spores were transferred to fresh PDA plates to obtain pure culture isolates. A total of six isolates were obtained, and among them, two were selected for further characterization (SP-36820-1 and SP-36820-3). Colonies grown on PDA have dark gray aerial mycelium with orange-colored spore masses. Conidia are falcate, slightly curved, tapered toward the tips, and are produced in acervuli with setae, measuring 37.65 to 24.84 x 8.02 to 4.67 µm, respectively (n = 100). These morphological characteristics are in agreement with C. graminicola previously described by Bergstrom and Nicholson (1999). Isolates were grown in potato dextrose broth (PDB) for 3 days at 25 ºC and total genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA). The internal transcribed spacer region of rDNA and the manganese-type superoxide dismutase gene (SOD2) were amplified using primers ITS4/ITS5 (White et al. 1990) and SOD625/SOD507 (Fang et al. 2002) and consequently sequenced. GenBank BLAST analysis revealed that the sequences were 100% identical to strains of C. graminicola. All sequences were deposited in GenBank (see e-Xtra 1 for accession numbers). To confirm Koch's postulates, plants of a derivative of maize inbred line Mo940 (developmental stage V3) were placed horizontally in a tray for inoculation and 20 droplets (7.5 µL) of a suspension of 3 x 105 conidia per milliliter were placed on the surface of the third leaf. The trays were closed to retain moisture and incubated overnight at 23ºC. The next day, the plants were returned to a vertical position and incubated in a growth chamber at 25ºC with 80% humidity and a light cycle of 16 h of light and 8 h of dark (Vargas et al. 2012). After four days inoculated leaves presented brown elongated lesions with necrotic centers consistent with C. graminicola infection, whereas control plants remained asymptomatic. The strains reisolated from infected leaves were morphologically identical to the original isolates. To our knowledge, this is the first report of Colletotrichum graminicola causing maize anthracnose in Spain. Recently, maize anthracnose was also reported in Bosnia and Herzegovina and China (Duan et al. 2019; Cuevas-Fernández et al. 2019), suggesting the pathogen's geographic range is increasing, which may be a threat to maize cultivation in locations with optimal humid conditions for disease development.

The Role of Plant Hormones in the Interaction of Colletotrichum Species with Their Host Plants.

Colletotrichum is a plant pathogenic fungus which is able to infect virtually every economically important plant species. Up to now no common infection mechanism has been identified comparing different plant and Colletotrichum species. Plant hormones play a crucial role in plant-pathogen interactions regardless whether they are symbiotic or pathogenic. In this review we analyze the role of ethylene, abscisic acid, jasmonic acid, auxin and salicylic acid during Colletotrichum infections. Different Colletotrichum strains are capable of auxin production and this might contribute to virulence. In this review the role of different plant hormones in plant-Colletotrichum interactions will be discussed and thereby auxin biosynthetic pathways in Colletotrichum spp. will be proposed.

Genome Sequence Resources of Colletotrichum truncatum, C. plurivorum, C. musicola, and C. sojae: Four Species Pathogenic to Soybean (Glycine max).

Colletotrichum is a large genus of plant pathogenic fungi comprising more than 200 species. In this work, we present the genome sequences of four Colletotrichum species pathogenic to soybean: C. truncatum, C. plurivorum, C. musicola, and C. sojae. While C. truncatum is globally considered the most important pathogen, the other three species have been described and associated with soybean only recently. The genome sequences will provide insights into factors that contribute to pathogenicity toward soybean and will be useful for further research into the evolution of Colletotrichum.

Whole-Genome Sequence of the Orchid Anthracnose Pathogen Colletotrichum orchidophilum.

Colletotrichum orchidophilum is a plant-pathogenic fungus infecting a wide range of plant species belonging to the family Orchidaceae. In addition to its economic impact, C. orchidophilum has been used in recent years in evolutionary studies because it represents the closest related species to the C. acutatum species complex. Here, we present the first-draft whole-genome sequence of C. orchidophilum IMI 309357, providing a resource for future research on anthracnose of Orchidaceae and other hosts.

Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters.

BACKGROUND: The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. RESULTS: Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications. CONCLUSION: The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.

A Fungal Effector With Host Nuclear Localization and DNA-Binding Properties Is Required for Maize Anthracnose Development.

Plant pathogens have the capacity to manipulate the host immune system through the secretion of effectors. We identified 27 putative effector proteins encoded in the genome of the maize anthracnose pathogen Colletotrichum graminicola that are likely to target the host's nucleus, as they simultaneously contain sequence signatures for secretion and nuclear localization. We functionally characterized one protein, identified as CgEP1. This protein is synthesized during the early stages of disease development and is necessary for anthracnose development in maize leaves, stems, and roots. Genetic, molecular, and biochemical studies confirmed that this effector targets the host's nucleus and defines a novel class of double-stranded DNA-binding protein. We show that CgEP1 arose from a gene duplication in an ancestor of a lineage of monocot-infecting Colletotrichum spp. and has undergone an intense evolution process, with evidence for episodes of positive selection. We detected CgEP1 homologs in several species of a grass-infecting lineage of Colletotrichum spp., suggesting that its function may be conserved across a large number of anthracnose pathogens. Our results demonstrate that effectors targeted to the host nucleus may be key elements for disease development and aid in the understanding of the genetic basis of anthracnose development in maize plants.

Gene family expansions and contractions are associated with host range in plant pathogens of the genus Colletotrichum.

BACKGROUND: Many species belonging to the genus Colletotrichum cause anthracnose disease on a wide range of plant species. In addition to their economic impact, the genus Colletotrichum is a useful model for the study of the evolution of host specificity, speciation and reproductive behaviors. Genome projects of Colletotrichum species have already opened a new era for studying the evolution of pathogenesis in fungi. RESULTS: We sequenced and annotated the genomes of four strains in the Colletotrichum acutatum species complex (CAsc), a clade of broad host range pathogens within the genus. The four CAsc proteomes and secretomes along with those representing an additional 13 species (six Colletotrichum spp. and seven other Sordariomycetes) were classified into protein families using a variety of tools. Hierarchical clustering of gene family and functional domain assignments, and phylogenetic analyses revealed lineage specific losses of carbohydrate-active enzymes (CAZymes) and proteases encoding genes in Colletotrichum species that have narrow host range as well as duplications of these families in the CAsc. We also found a lineage specific expansion of necrosis and ethylene-inducing peptide 1 (Nep1)-like protein (NLPs) families within the CAsc. CONCLUSIONS: This study illustrates the plasticity of Colletotrichum genomes, and shows that major changes in host range are associated with relatively recent changes in gene content.

Identification of horizontally transferred genes in the genus Colletotrichum reveals a steady tempo of bacterial to fungal gene transfer.

BACKGROUND: Horizontal gene transfer (HGT) is the stable transmission of genetic material between organisms by means other than vertical inheritance. HGT has an important role in the evolution of prokaryotes but is relatively rare in eukaryotes. HGT has been shown to contribute to virulence in eukaryotic pathogens. We studied the importance of HGT in plant pathogenic fungi by identifying horizontally transferred genes in the genomes of three members of the genus Colletotrichum. RESULTS: We identified eleven HGT events from bacteria into members of the genus Colletotrichum or their ancestors. The HGT events include genes involved in amino acid, lipid and sugar metabolism as well as lytic enzymes. Additionally, the putative minimal dates of transference were calculated using a time calibrated phylogenetic tree. This analysis reveals a constant flux of genes from bacteria to fungi throughout the evolution of subphylum Pezizomycotina. CONCLUSIONS: Genes that are typically transferred by HGT are those that are constantly subject to gene duplication and gene loss. The functions of some of these genes suggest roles in niche adaptation and virulence. We found no evidence of a burst of HGT events coinciding with major geological events. In contrast, HGT appears to be a constant, albeit rare phenomenon in the Pezizomycotina, occurring at a steady rate during their evolution.

Compositions of fungal secretomes indicate a greater impact of phylogenetic history than lifestyle adaptation.

BACKGROUND: Since the first fungal genome sequences became available, investigators have been employing comparative genomics to understand how fungi have evolved to occupy diverse ecological niches. The secretome, i.e. the entirety of all proteins secreted by an organism, is of particular importance, as by these proteins fungi acquire nutrients and communicate with their surroundings. RESULTS: It is generally assumed that fungi with similar nutritional lifestyles have similar secretome compositions. In this study, we test this hypothesis by annotating and comparing the soluble secretomes, defined as the sets of proteins containing classical signal peptides but lacking transmembrane domains of fungi representing a broad diversity of nutritional lifestyles. Secretome size correlates with phylogeny and to a lesser extent with lifestyle. Plant pathogens and saprophytes have larger secretomes than animal pathogens. Small secreted cysteine-rich proteins (SSCPs), which may comprise many effectors important for the interaction of plant pathogens with their hosts, are defined here to have a mature length of ≤ 300 aa residues, at least four cysteines, and a total cysteine content of ≥5%. SSCPs are found enriched in the secretomes of the Pezizomycotina and Basidiomycota in comparison to Saccharomycotina. Relative SSCP content is noticeably higher in plant pathogens than in animal pathogens, while saprophytes were in between and closer to plant pathogens. Expansions and contractions of gene families and in the number of occurrences of functional domains are largely lineage specific, e.g. contraction of glycoside hydrolases in Saccharomycotina, and are only weakly correlated with lifestyle. However, within a given lifestyle a few general trends exist, such as the expansion of secreted family M14 metallopeptidases and chitin-binding proteins in plant pathogenic Pezizomycotina. CONCLUSIONS: While the secretomes of fungi with similar lifestyles share certain characteristics, the expansion and contraction of gene families is largely lineage specific, and not shared among all fungi of a given lifestyle.

Genome evolution and transcriptome plasticity is associated with adaptation to monocot and dicot plants in Colletotrichum fungi.

BACKGROUND: Colletotrichum fungi infect a wide diversity of monocot and dicot hosts, causing diseases on almost all economically important plants worldwide. Colletotrichum is also a suitable model for studying gene family evolution on a fine scale to uncover events in the genome associated with biological changes. RESULTS: Here we present the genome sequences of 30 Colletotrichum species covering the diversity within the genus. Evolutionary analyses revealed that the Colletotrichum ancestor diverged in the late Cretaceous in parallel with the diversification of flowering plants. We provide evidence of independent host jumps from dicots to monocots during the evolution of Colletotrichum, coinciding with a progressive shrinking of the plant cell wall degradative arsenal and expansions in lineage-specific gene families. Comparative transcriptomics of 4 species adapted to different hosts revealed similarity in gene content but high diversity in the modulation of their transcription profiles on different plant substrates. Combining genomics and transcriptomics, we identified a set of core genes such as specific transcription factors, putatively involved in plant cell wall degradation. CONCLUSIONS: These results indicate that the ancestral Colletotrichum were associated with dicot plants and certain branches progressively adapted to different monocot hosts, reshaping the gene content and its regulation.

Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052.

Production of butanol by solventogenic clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome cell sensitivity to solvents, stress-directed evolution methodology was used three decades ago on Clostridium beijerinckii NCIMB 8052 that spawned the SA-1 strain. Here, we evaluated SA-1 solventogenic capabilities when growing on a previously validated medium containing, as carbon- and energy-limiting substrates, sucrose and the products of its hydrolysis d-glucose and d-fructose and only d-fructose. Comparative small-scale batch fermentations with controlled pH (pH 6.5) showed that SA-1 is a solvent hyper-producing strain capable of generating up to 16.1 g l(-1) of butanol and 26.3 g l(-1) of total solvents, 62.3 % and 63 % more than NCIMB 8052, respectively. This corresponds to butanol and solvent yields of 0.3 and 0.49 g g(-1), respectively (63 % and 65 % increase compared with NCIMB 8052). SA-1 showed a deficiency in d-fructose transport as suggested by its 7 h generation time compared with 1 h for NCIMB 8052. To potentially correlate physiological behaviour with genetic mutations, the whole genome of SA-1 was sequenced using the Illumina GA IIx platform. PCR and Sanger sequencing were performed to analyse the putative variations. As a result, four errors were confirmed and validated in the reference genome of NCIMB 8052 and a total of 10 genetic polymorphisms in SA-1. The genetic polymorphisms included eight single nucleotide variants, one small deletion and one large insertion that it is an additional copy of the insertion sequence ISCb1. Two of the genetic polymorphisms, the serine threonine phosphatase cbs_4400 and the solute binding protein cbs_0769, may possibly explain some of the observed physiological behaviour, such as rerouting of the metabolic carbon flow, deregulation of the d-fructose phosphotransferase transport system and delayed sporulation.

Plant defense mechanisms are activated during biotrophic and necrotrophic development of Colletotricum graminicola in maize.

Hemibiotrophic plant pathogens first establish a biotrophic interaction with the host plant and later switch to a destructive necrotrophic lifestyle. Studies of biotrophic pathogens have shown that they actively suppress plant defenses after an initial microbe-associated molecular pattern-triggered activation. In contrast, studies of the hemibiotrophs suggest that they do not suppress plant defenses during the biotrophic phase, indicating that while there are similarities between the biotrophic phase of hemibiotrophs and biotrophic pathogens, the two lifestyles are not analogous. We performed transcriptomic, histological, and biochemical studies of the early events during the infection of maize (Zea mays) with Colletotrichum graminicola, a model pathosystem for the study of hemibiotrophy. Time-course experiments revealed that mRNAs of several defense-related genes, reactive oxygen species, and antimicrobial compounds all begin to accumulate early in the infection process and continue to accumulate during the biotrophic stage. We also discovered the production of maize-derived vesicular bodies containing hydrogen peroxide targeting the fungal hyphae. We describe the fungal respiratory burst during host infection, paralleled by superoxide ion production in specific fungal cells during the transition from biotrophy to a necrotrophic lifestyle. We also identified several novel putative fungal effectors and studied their expression during anthracnose development in maize. Our results demonstrate a strong induction of defense mechanisms occurring in maize cells during C. graminicola infection, even during the biotrophic development of the pathogen. We hypothesize that the switch to necrotrophic growth enables the fungus to evade the effects of the plant immune system and allows for full fungal pathogenicity.

Chromosome-level analysis of the Colletotrichum graminicola genome reveals the unique characteristics of core and minichromosomes.

The fungal pathogen Colletotrichum graminicola causes the anthracnose of maize (Zea mays) and is responsible for significant yield losses worldwide. The genome of C. graminicola was sequenced in 2012 using Sanger sequencing, 454 pyrosequencing, and an optical map to obtain an assembly of 13 pseudochromosomes. We re-sequenced the genome using a combination of short-read (Illumina) and long-read (PacBio) technologies to obtain a chromosome-level assembly. The new version of the genome sequence has 13 chromosomes with a total length of 57.43 Mb. We detected 66 (23.62 Mb) structural rearrangements in the new assembly with respect to the previous version, consisting of 61 (21.98 Mb) translocations, 1 (1.41 Mb) inversion, and 4 (221 Kb) duplications. We annotated the genome and obtained 15,118 predicted genes and 3,614 new gene models compared to the previous version of the assembly. We show that 25.88% of the new assembly is composed of repetitive DNA elements (13.68% more than the previous assembly version), which are mostly found in gene-sparse regions. We describe genomic compartmentalization consisting of repeat-rich and gene-poor regions vs. repeat-poor and gene-rich regions. A total of 1,140 secreted proteins were found mainly in repeat-rich regions. We also found that ~75% of the three smallest chromosomes (minichromosomes, between 730 and 551 Kb) are strongly affected by repeat-induced point mutation (RIP) compared with 28% of the larger chromosomes. The gene content of the minichromosomes (MCs) comprises 121 genes, of which 83.6% are hypothetical proteins with no predicted function, while the mean percentage of Chr1-Chr10 is 36.5%. No predicted secreted proteins are present in the MCs. Interestingly, only 2% of the genes in Chr11 have homologs in other strains of C. graminicola, while Chr12 and 13 have 58 and 57%, respectively, raising the question as to whether Chrs12 and 13 are dispensable. The core chromosomes (Chr1-Chr10) are very different with respect to the MCs (Chr11-Chr13) in terms of the content and sequence features. We hypothesize that the higher density of repetitive elements and RIPs in the MCs may be linked to the adaptation and/or host co-evolution of this pathogenic fungus.

Population Genomics Provide Insights into the Global Genetic Structure of Colletotrichum graminicola, the Causal Agent of Maize Anthracnose.

Understanding the genetic diversity and mechanisms underlying genetic variation in pathogen populations is crucial to the development of effective control strategies. We investigated the genetic diversity and reproductive biology of Colletotrichum graminicola isolates which infect maize by sequencing the genomes of 108 isolates collected from 14 countries using restriction site-associated DNA sequencing (RAD-seq) and whole-genome sequencing (WGS). Clustering analyses based on single-nucleotide polymorphisms revealed three genetic groups delimited by continental origin, compatible with short-dispersal of the pathogen and geographic subdivision. Intra- and intercontinental migration was observed between Europe and South America, likely associated with the movement of contaminated germplasm. Low clonality, evidence of genetic recombination, and high phenotypic diversity were detected. We show evidence that, although it is rare (possibly due to losses of sexual reproduction- and meiosis-associated genes) C. graminicola can undergo sexual recombination. Our results support the hypotheses that intra- and intercontinental pathogen migration and genetic recombination have great impacts on the C. graminicola population structure. IMPORTANCE Plant pathogens cause significant reductions in yield and crop quality and cause enormous economic losses worldwide. Reducing these losses provides an obvious strategy to increase food production without further degrading natural ecosystems; however, this requires knowledge of the biology and evolution of the pathogens in agroecosystems. We employed a population genomics approach to investigate the genetic diversity and reproductive biology of the maize anthracnose pathogen (Colletotrichum graminicola) in 14 countries. We found that the populations are correlated with their geographical origin and that migration between countries is ongoing, possibly caused by the movement of infected plant material. This result has direct implications for disease management because migration can cause the movement of more virulent and/or fungicide-resistant genotypes. We conclude that genetic recombination is frequent (in contrast to the traditional view of C. graminicola being mainly asexual), which strongly impacts control measures and breeding programs aimed at controlling this disease.

Comparative transcriptomic provides novel insights into the soybean response to Colletotrichum truncatum infection.

Introduction: Soybean (Glycine max) is among the most important crops in the world, and its production can be threatened by biotic diseases, such as anthracnose. Soybean anthracnose is a seed-borne disease mainly caused by the hemibiotrophic fungus Colletotrichum truncatum. Typical symptoms are pre- and post-emergence damping off and necrotic lesions on cotyledons, petioles, leaves, and pods. Anthracnose symptoms can appear early in the field, causing major losses to soybean production. Material and Methods: In preliminary experiments, we observed that the same soybean cultivar can have a range of susceptibility towards different strains of C. truncatum, while the same C. truncatum strain can cause varying levels of disease severity in different soybean cultivars. To gain a better understanding of the molecular mechanisms regulating the early response of different soybean cultivars to different C. truncatum strains, we performed pathogenicity assays to select two soybean cultivars with significantly different susceptibility to two different C. truncatum strains and analyzed their transcriptome profiles at different time points of interaction (0, 12, 48, and 120 h post-inoculation, hpi). Results and Discussion: The pathogenicity assays showed that the soybean cultivar Gm1 is more resistant to C. truncatum strain 1080, and it is highly susceptible to strain 1059, while cultivar Gm2 shows the opposite behavior. However, if only trivial anthracnose symptoms appeared in the more resistant phenotype (MRP; Gm1-1080; Gm2-1059) upon 120 hpi, in the more susceptible phenotype (MSP; Gm-1059; Gm2- 1080) plants show mild symptoms already at 72 hpi, after which the disease evolved rapidly to severe necrosis and plant death. Interestingly, several genes related to different cellular responses of the plant immune system (pathogen recognition, signaling events, transcriptional reprogramming, and defense-related genes) were commonly modulated at the same time points only in both MRP. The list of differentially expressed genes (DEGs) specific to the more resistant combinations and related to different cellular responses of the plant immune system may shed light on the important host defense pathways against soybean anthracnose.

Identification and Comparison of Colletotrichum Secreted Effector Candidates Reveal Two Independent Lineages Pathogenic to Soybean.

Colletotrichum is one of the most important plant pathogenic genus of fungi due to its scientific and economic impact. A wide range of hosts can be infected by Colletotrichum spp., which causes losses in crops of major importance worldwide, such as soybean. Soybean anthracnose is mainly caused by C. truncatum, but other species have been identified at an increasing rate during the last decade, becoming one of the most important limiting factors to soybean production in several regions. To gain a better understanding of the evolutionary origin of soybean anthracnose, we compared the repertoire of effector candidates of four Colletotrichum species pathogenic to soybean and eight species not pathogenic. Our results show that the four species infecting soybean belong to two lineages and do not share any effector candidates. These results strongly suggest that two Colletotrichum lineages have acquired the capability to infect soybean independently. This study also provides, for each lineage, a set of candidate effectors encoding genes that may have important roles in pathogenicity towards soybean offering a new resource useful for further research on soybean anthracnose management.

Soybean anthracnose caused by Colletotrichum species: Current status and future prospects.

Soybean (Glycine max) is one of the most important cultivated plants worldwide as a source of protein-rich foods and animal feeds. Anthracnose, caused by different lineages of the hemibiotrophic fungus Colletotrichum, is one of the main limiting factors to soybean production. Losses due to anthracnose have been neglected, but their impact may threaten up to 50% of the grain production. TAXONOMY: While C. truncatum is considered the main species associated with soybean anthracnose, recently other species have been reported as pathogenic on this host. Until now, it has not been clear whether the association of new Colletotrichum species with the disease is related to emerging species or whether it is due to the undergoing changes in the taxonomy of the genus. DISEASE SYMPTOMS: Typical anthracnose symptoms are pre- and postemergence damping-off; dark, depressed, and irregular spots on cotyledons, stems, petioles, and pods; and necrotic laminar veins on leaves that can result in premature defoliation. Symptoms may evolve to pod rot, immature opening of pods, and premature germination of grains. CHALLENGES: As accurate species identification of the causal agent is decisive for disease control and prevention, in this work we review the taxonomic designation of Colletotrichum isolated from soybean to understand which lineages are pathogenic on this host. We also present a comprehensive literature review of soybean anthracnose, focusing on distribution, symptomatology, epidemiology, disease management, identification, and diagnosis. We consider the knowledge emerging from population studies and comparative genomics of Colletotrichum spp. associated with soybean providing future perspectives in the identification of molecular factors involved in the pathogenicity process. USEFUL WEBSITE: Updates on Colletotrichum can be found at http://www.colletotrichum.org/. All available Colletotrichum genomes on GenBank can be viewed at http://www.colletotrichum.org/genomics/.

A Major Effect Gene Controlling Development and Pathogenicity in Botrytis cinerea Identified Through Genetic Analysis of Natural Mycelial Non-pathogenic Isolates.

Botrytis cinerea is a necrotrophic plant pathogenic fungus with a wide host range. Its natural populations are phenotypically and genetically very diverse. A survey of B. cinerea isolates causing gray mold in the vineyards of Castilla y León, Spain, was carried out and as a result eight non-pathogenic natural variants were identified. Phenotypically these isolates belong to two groups. The first group consists of seven isolates displaying a characteristic mycelial morphotype, which do not sporulate and is unable to produce sclerotia. The second group includes one isolate, which sporulates profusely and does not produce sclerotia. All of them are unresponsive to light. Crosses between a representative mycelial non-pathogenic isolate and a highly aggressive field isolate revealed that the phenotypic differences regarding pathogenicity, sporulation and production of sclerotia cosegregated in the progeny and are determined by a single genetic locus. By applying a bulked segregant analysis strategy based on the comparison of the two parental genomes the locus was mapped to a 110 kb region in chromosome 4. Subcloning and transformation experiments revealed that the polymorphism is an SNP affecting gene Bcin04g03490 in the reference genome of B. cinerea. Genetic complementation analysis and sequencing of the Bcin04g03490 alleles demonstrated that the mutations in the mycelial isolates are allelic and informed about the nature of the alterations causing the phenotypes observed. Integration of the allele of the pathogenic isolate into the non-pathogenic isolate fully restored the ability to infect, to sporulate and to produce sclerotia. Therefore, it is concluded that a major effect gene controlling differentiation and developmental processes as well as pathogenicity has been identified in B. cinerea. It encodes a protein with a GAL4-like Zn(II)2Cys6 binuclear cluster DNA binding domain and an acetyltransferase domain, suggesting a role in regulation of gene expression through a mechanism involving acetylation of specific substrates.

PoGO: Prediction of Gene Ontology terms for fungal proteins.

BACKGROUND: Automated protein function prediction methods are the only practical approach for assigning functions to genes obtained from model organisms. Many of the previously reported function annotation methods are of limited utility for fungal protein annotation. They are often trained only to one species, are not available for high-volume data processing, or require the use of data derived by experiments such as microarray analysis. To meet the increasing need for high throughput, automated annotation of fungal genomes, we have developed a tool for annotating fungal protein sequences with terms from the Gene Ontology. RESULTS: We describe a classifier called PoGO (Prediction of Gene Ontology terms) that uses statistical pattern recognition methods to assign Gene Ontology (GO) terms to proteins from filamentous fungi. PoGO is organized as a meta-classifier in which each evidence source (sequence similarity, protein domains, protein structure and biochemical properties) is used to train independent base-level classifiers. The outputs of the base classifiers are used to train a meta-classifier, which provides the final assignment of GO terms. An independent classifier is trained for each GO term, making the system amenable to updating, without having to re-train the whole system. The resulting system is robust. It provides better accuracy and can assign GO terms to a higher percentage of unannotated protein sequences than other methods that we tested. CONCLUSIONS: Our annotation system overcomes many of the shortcomings that we found in other methods. We also provide a web server where users can submit protein sequences to be annotated.