A novel narnavirus isolated from Colletotrichum curcumae strain 780-2T.

The complete genome of a novel mycovirus, Colletotrichum curcumae narnavirus 1 (CcNV1), derived from the phytopathogenic fungus Colletotrichum curcumae strain 780-2T, was sequenced and analyzed. The full sequence of CcNV1 is 3,374 nucleotides in length and contains a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) of 1,087 amino acids with a molecular mass of 124.2 kDa that shares the closest similarity with that of Monilinia narnavirus H (53.02% identity). RdRp phylogeny analysis showed that CcNV1 is a new member of the proposed genus "Betanarnavirus" within the family Narnaviridae. This is the first report of a novel narnavirus infecting the phytopathogenic fungus C. curcumae, the causal agent of leaf blight of Curcuma wenyujin.

Mycoviruses infecting Colletotrichum spp: a comprehensive review / Micoviroses em Colletotrichum spp: uma revisão abrangente

Colletotrichum is one of the most economically important fungal genera, which affects a wide range of hosts, specifically tropical and subtropical crops. Thus far, there have been several records of mycovirus infection in Colletotrichum spp., primarily by viruses of the Partitiviridae family. There have also been records of infections by mycoviruses of the Chrysoviridae family. Mycoviruses are (+)ssRNA and dsRNA genome viruses, which may or may not be enveloped. To date, no mycovirus with a DNA genome has been isolated from Colletotrichum spp. Typically, mycoviruses cause latent infections, although hypo- and hypervirulence have also been reported in Colletotrichum spp. In addition to its effects on pathogenic behavior, mycovirus infection can lead to important physiological changes, such as altered morphological characteristics, reduced vegetative growth, and suppressed conidia production. Therefore, research on mycoviruses infecting phytopathogenic fungi can help develop alternative methods to chemical control, which can cause irreversible damage to humans and the environment. From an agricultural perspective, mycoviruses can contribute to sustainable agriculture as biological control agents via changes in fungal physiology, ultimately resulting in the total loss of or reduction in the virulence of these pathogens.

Colletotrichum é um dos gêneros fúngicos mais importantes economicamente, afetando uma ampla gama de hospedeiros, especialmente em cultivos tropicais e subtropicais. Atualmente já existem diversos registros de infecção por micovírus em Colletotrichum spp., sendo a maioria dos já identificados classificados na família Partitiviridae. Ocorrem registros também de micovírus pertencentes à família Chrysoviridae. Compreendem vírus de genoma de (+)ssRNA e dsRNA que podem ser ou não envelopados. Ainda não foram identificados micovírus com genoma de DNA isolados de Colletotrichum. A infecção por micovírus pode ocorrer de forma latente, mas já foi observado em Colletotrichum spp. o fenômeno de hipo e hipervirulência. Além de influenciar no comportamento patogênico, a infecção pode causar mudanças fisiológicas importantes como alterações das características morfológicas, redução do crescimento vegetativo e redução na produção de conídios. O estudo com micovírus em fungos fitopatogênicos traz uma alternativa ao controle químico que é um método capaz de causar danos irreversíveis ao homem e o meio ambiente. Sob a perspectiva agrícola, os micovírus podem contribuir para agricultura sustentável como agentes de controle biológico. Isso porque obsevam-se mudanças importantes na fisiologia fúngica resultando na perda total ou redução da virulência desses patógenos.

A Novel Heptasegmented Positive-Sense Single-Stranded RNA Virus from the Phytopathogenic Fungus Colletotrichum fructicola.

In this study, a novel positive-sense single-stranded RNA (+ssRNA) mycovirus, tentatively named Colletotrichum fructicola RNA virus 1 (CfRV1), was identified in the phytopathogenic fungus Colletotrichum fructicola. CfRV1 has seven genomic components, encoding seven proteins from open reading frames (ORFs) flanked by highly conserved untranslated regions (UTRs). Proteins encoded by ORFs 1, 2, 3, 5, and 6 are more similar to the putative RNA-dependent RNA polymerase (RdRp), hypothetical protein (P2), methyltransferase, and two hypothetical proteins of Hadaka virus 1 (HadV1), a capsidless 10- or 11-segmented +ssRNA virus, while proteins encoded by ORFs 4 and 7 showed no detectable similarity to any known proteins. Notably, proteins encoded by ORFs 1 to 3 also share considerably high similarity with the corresponding proteins of polymycoviruses. Phylogenetic analysis conducted based on the amino acid sequence of CfRV1 RdRp and related viruses placed CfRV1 and HadV1 together in the same clade, close to polymycoviruses and astroviruses. CfRV1-infected C. fructicola strains demonstrate a moderately attenuated growth rate and virulence compared to uninfected isolates. CfRV1 is capsidless and potentially encapsulated in vesicles inside fungal cells, as revealed by transmission electron microscopy. CfRV1 and HadV1 are +ssRNA mycoviruses closely related to polymycoviruses and astroviruses, represent a new linkage between +ssRNA viruses and the intermediate double-stranded RNA (dsRNA) polymycoviruses, and expand our understanding of virus diversity, taxonomy, evolution, and biological traits. IMPORTANCE A scenario proposing that dsRNA viruses evolved from +ssRNA viruses is still considered controversial due to intergroup knowledge gaps in virus diversity. Recently, polymycoviruses and hadakaviruses were found as intermediate dsRNA and +ssRNA stages, respectively, between +ssRNA and dsRNA viruses. Here, we identified a novel +ssRNA mycovirus, Colletotrichum fructicola RNA virus 1 (CfRV1), isolated from Colletotrichum fructicola in China. CfRV1 is phylogenetically related to the 10- or 11-segmented Hadaka virus 1 (HadV1) but consists of only seven genomic segments encoding two novel proteins. CfRV1 is naked and may be encapsulated in vesicles inside fungal cells, representing a potential novel lifestyle for multisegmented RNA viruses. CfRV1 and HadV1 are intermediate +ssRNA mycoviruses in the linkage between +ssRNA viruses and the intermediate dsRNA polymycoviruses and expand our understanding of virus diversity, taxonomy, and evolution.

Molecular characterization of a novel single-stranded RNA virus, ChRV1, isolated from the plant-pathogenic fungus Colletotrichum higginsianum.

In this study, a novel single-stranded RNA virus was isolated from the plant-pathogenic fungus Colletotrichum higginsianum strain HTC-5, and the virus was named "Colletotrichum higginsianum ssRNA virus 1" (ChRV1). The complete genome of ChRV1 is 3850 nucleotides in length with a GC content of 52% and contains two in-frame open reading frames (ORFs): ORF1 (smaller) and ORF2 (larger). ORF1 encodes a protein with the highest sequence similarity to proteins encoded by Phoma matteucciicola RNA virus 1 (PmRV1, 47.99% identity) and Periconia macrospinosa ambiguivirus 1 (PmAV1, 50.73% identity). ORF2 encodes a protein with a conserved RNA-dependent RNA polymerase (RdRp) domain with similarity to the RdRps of PmRV1 (61.41% identity) and PmAV1 (60.61% identity), which are recently reported unclassified (+) ssRNA mycoviruses. Phylogenetic analysis of the RdRp domain showed that ChRV1 grouped together with PmRV1, PmAV1, and other unclassified (+) ssRNA mycoviruses and had a distant relationship to invertebrate viruses and plant viruses of the family Tombusviridae. This is the first report of a novel (+) ssRNA virus infecting the phytopathogenic fungus C. higginsianum.

The complete genome sequence of a new mitovirus from the phytopathogenic fungus Colletotrichum higginsianum.

In this study, a novel mitovirus designed "Colletotrichum higginsianum mitovirus 1" (ChMV1) was isolated from the phytopathogenic fungus Colletotrichum higginsianum. The genome of this mitovirus is 2,893 nt in length with an A + U content of 61% and contains a large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). A BLASTp analysis revealed that the RdRp domain of ChMV1 had 30.25% to 61.72% sequence identity to those of members of the genus Mitovirus and showed the highest degree of similarity (61.72% identity) to Botrytis cinerea mitovirus 3 (BcMV3). Phylogenetic analysis further indicated that ChMV1 is a member in the genus Mitovirus of the family Mitoviridae. To the best of our knowledge, this is the first report of a mitovirus in C. higginsianum.

The dsRNA mycovirus ChNRV1 causes mild hypervirulence in the fungal phytopathogen Colletotrichum higginsianum.

The genus Colletotrichum comprises a large number of filamentous fungi responsible for anthracnose diseases in many tropical and subtropical fruits and vegetables. In particular, Colletotrichum higginsianum infects Brassicaceae species, including Arabidopsis. The C. higginsianum strain IMI349063A is naturally infected with a dsRNA virus, named Colletorichum higginsianum non-segmented virus (ChNRV1). Here, we investigated the biological effect of ChNRV1 in C. higginsianum by comparing strains with and without the virus. ChNRV1 does not have an effect on C. higginsianum growth under salt and cell-wall stress conditions. However, thermal stress reduced C. higginsianum growth rate, this effect being more evident in the wild-type C. higginsianum strain containing the virus. Although ChNRV1 had no effect in conidiation, conidia were narrower when the virus is present. More importantly, ChNRV1 causes a mild increase in C. higginsianum virulence (hypervirulence) when infecting Arabidopsis plants. These findings indicated that, whereas the ChNRV1 mycovirus does not impair growth and conidiation of C. higginsianum, it confers hypervirulence to the fungal host. These findings will help in future research on the effect of mycoviral infection on pathogenic fungi in plant species of agronomical relevance.

Colletotrichum siamense, a Mycovirus-Carrying Endophyte, as a Biological Control Strategy for Anthracnose in Guarana Plants

Abstract Guarana, the fruit of Paullinia cupana, is known for its stimulating and medicinal properties by the Amazonian indigenous population and communities. However, it presents serious phytopathological problems, such as anthracnose disease caused by Colletotrichum spp. The objective of this study was to verify if C. siamense, a mycovirus-carrying endophytic fungus, could protect guarana seedlings, by reducing or by eliminating characteristic disease symptoms. Other physiological changes in the plant caused by the presence of this endophyte were also evaluated. The cuttings of the Cereçaporanga cultivar were dipped in a biological control suspension and planted in a specific substrate. After four months in the greenhouse, the seedlings were sprayed with a suspension of phytopathogen conidia, and a portion of these seedlings received the fungicide indicated for the crop to be compared with the control seedlings. After 28 days, the number of lesions, morphophysiological and macro characteristics, and leaf micronutrients were evaluated. The seedlings treated with C. siamense showed a lower percentage of lesions and an increased aerial part and root system compared to the other treatments. There were no significant differences between treatments regarding the percentage of macronutrients and micronutrients.

Molecular characterization of a novel mycovirus from the plant pathogenic fungus Colletotrichum gloeosporioides.

In this study, we report the molecular characterization of a novel mycovirus in a phytopathogenic fungus of the species Colletotrichum gloeosporioides, which we named "Colletotrichum gloeosporioides RNA virus 1" (CgRV1). The virus has a dsRNA genome of 2,975 bp and possesses two non-overlapping open reading frames (ORFs 1 and 2). The smaller ORF1 encodes a protein of unknown function, and the larger ORF2 encodes the RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis based on the RdRp sequence showed that CgRV1 clustered with and is closely related to unclassified mycoviruses that are distinct from members of the family Partitiviridae.

A novel ourmia-like mycovirus isolated from the plant pathogenic fungus Colletotrichum gloeosporioides.

In this study, a novel mycovirus designed Colletotrichum gloeosporioides ourmia-like virus 1 (CgOLV1) was isolated from a filamentous phytopathogenic fungus, Colletotrichum gloeosporioides. The virus has a genome of 2,516 nucleotides and contains a large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis indicated that CgOLV1 is located in the ourmia-like mycovirus clade, whose members are related to plant ourmiaviruses. To the best of our knowledge, this is the first report of an ourmia-like mycovirus in C. gloeosporioides.

Complete genome sequence of a novel mitovirus from the ectomycorrhizal fungus Geopora sumneriana.

A double-stranded RNA (dsRNA) segment was extracted from the ectomycorrhizal fungus Geopora sumneriana (Cooke) M. Torre, and its full-length cDNA sequence, comprising 3146 nucleotides, was determined. Sequence analysis revealed the presence of a large open reading frame (ORF) on the positive strand of this dsRNA segment when the mold mitochondrial genetic code was applied. The ORF encodes a putative RNA-dependent RNA polymerase (RdRp), which shares the highest degree of similarity with Tuber excavatum mitovirus, with 37.52% identity. This dsRNA segment represents the genome replication intermediate of a novel mitovirus that was tentatively designated as "Geopora sumneriana mitovirus 1" (GsMV1). Phylogenetic analysis further suggested that GsMV1 is a member of the family Narnaviridae. This is the first study reporting on a mitovirus genome sequence in the ectomycorrhizal fungus G. sumneriana.

A dsRNA virus with filamentous viral particles.

Viruses with double-stranded RNA genomes form isometric particles or are capsidless. Here we report a double-stranded RNA virus, Colletotrichum camelliae filamentous virus 1 (CcFV-1) isolated from a fungal pathogen, that forms filamentous particles. CcFV-1 has eight genomic double-stranded RNAs, ranging from 990 to 2444 bp, encoding 10 putative open reading frames, of which open reading frame 1 encodes an RNA-dependent RNA polymerase and open reading frame 4 a capsid protein. When inoculated, the naked CcFV-1 double-stranded RNAs are infectious and induce the accumulation of the filamentous particles in vivo. CcFV-1 is phylogenetically related to Aspergillus fumigatus tetramycovirus-1 and Beauveria bassiana polymycovirus-1, but differs in morphology and in the number of genomic components. CcFV-1 might be an intermediate virus related to truly capsidated viruses, or might represent a distinct encapsidating strategy. In terms of genome and particle architecture, our findings are a significant addition to the knowledge of the virosphere diversity.Viruses with double-stranded RNA (dsRNA) genomes form typically isometric particles or are capsid-less. Here, the authors identify a mycovirus with an eight-segmented dsRNA genome that forms exceptionally long filamentous particles and could represent an evolutionary link between ssRNA and dsRNA viruses.

Small RNA-Based Antiviral Defense in the Phytopathogenic Fungus Colletotrichum higginsianum.

Even though the fungal kingdom contains more than 3 million species, little is known about the biological roles of RNA silencing in fungi. The Colletotrichum genus comprises fungal species that are pathogenic for a wide range of crop species worldwide. To investigate the role of RNA silencing in the ascomycete fungus Colletotrichum higginsianum, knock-out mutants affecting genes for three RNA-dependent RNA polymerase (RDR), two Dicer-like (DCL), and two Argonaute (AGO) proteins were generated by targeted gene replacement. No effects were observed on vegetative growth for any mutant strain when grown on complex or minimal media. However, Δdcl1, Δdcl1Δdcl2 double mutant, and Δago1 strains showed severe defects in conidiation and conidia morphology. Total RNA transcripts and small RNA populations were analyzed in parental and mutant strains. The greatest effects on both RNA populations was observed in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains, in which a previously uncharacterized dsRNA mycovirus [termed Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1)] was derepressed. Phylogenetic analyses clearly showed a close relationship between ChNRV1 and members of the segmented Partitiviridae family, despite the non-segmented nature of the genome. Immunoprecipitation of small RNAs associated with AGO1 showed abundant loading of 5'U-containing viral siRNA. C. higginsianum parental and Δdcl1 mutant strains cured of ChNRV1 revealed that the conidiation and spore morphology defects were primarily caused by ChNRV1. Based on these results, RNA silencing involving ChDCL1 and ChAGO1 in C. higginsianum is proposed to function as an antiviral mechanism.

Investigation of mycoviruses in endophytic and phytopathogenic strains of Colletotrichum from different hosts.

Fungi belonging to the Colletotrichum genus can be categorized as endophytic or phytopathogenic. These fungi can be infected by viruses, termed mycoviruses, which are know to promote hypovirulence in infected fungi. However, there are few studies that have described mycoviral infections of endophytes. The production of secondary metabolites by endophytes with antimicrobial potential in inhibiting numerous pathogens has gained increasing attention. The aim of the current study was to investigate the presence of mycoviruses in endophytic and phytopathogenic fungi of the Colletotrichum genus, as well as to analyze the antimicrobial activity of crude extracts obtained from these samples. To detect the presence of mycoviruses in the samples, dsRNA was extracted, treated with enzymes, and analyzed following electrophoresis in agarose gel. Furthermore, isometric mycoviral particles were observed by transmission electron microscopy. Serial microdilution methodology was used to test crude extracts of Colletotrichum spp for antibacterial activity against Escherichia coli and Staphylococcus aureus, and antifungal activity against Fusarium solani. The results of the molecular and microscopic analyses indicated that a phytopathogenic strain presented infection by mycovirus. The antibacterial activity analysis revealed that the minimum inhibitory concentrations and minimum bactericidal concentrations were low for the fungal extracts of the two endophytes, indicating that these extracts were effective antibacterial agents. However, their antifungal activity against F. solani was not statistically different compared to that of the negative control.

Detection and characterization of a novel Gammapartitivirus in the phytopathogenic fungus Colletotrichum acutatum strain HNZJ001.

Spherical virus-like particles about 40nm in diameter were observed under transmission electron microscope (TEM) and two dsRNA bands (dsRNA-1 and dsRNA-2) were detected on agarose gel after extraction from the mycelial preparation of a Colletotrichum acutatum strain HNZJ001 that isolated from an anthracnose lesion on immature pepper fruit. The complete nucleotide sequences of the dsRNAs were determined. DsRNA-1 (1762 nt) and dsRNA-2 (1381 nt) each contained a single open reading frame and potentially encoded 62 kDa and 40 kDa proteins, respectively. The 62 kDa protein showed similarity to the RNA-dependent RNA polymerase (RdRp) of partitiviruses, while the 40 kDa product had no significant similarity to any published capsid protein throughout all databases, besides of low homology with the hypothetical "capsid" protein of a few partitiviruses in fungus Ustilaginoidea virens. Genome comparison and phylogenetic analysis indicated that the virus is closely related to the mycovirus in the family Partitiviridae. The results suggested a novel two-segment dsRNA virus be detected. We name it Colletotrichum acutatum partitivirus 1 (CaPV1). RT-PCR detection, using a primer pair based on the RdRp of the dsRNA-1 showed very high efficiency of CaPV1 transmission into the progenies of the fungus. Virus curing and fungal phenotype observation for evaluation of the impact of CaPV1 in host fungus were also carried out.

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)