An Outstandingly Rare Occurrence of Mycoviruses in Soil Strains of the Plant-Beneficial Fungi from the Genus Trichoderma and a Novel Polymycoviridae Isolate.

In fungi, viral infections frequently remain cryptic causing little or no phenotypic changes. It can indicate either a long history of coevolution or a strong immune system of the host. Some fungi are outstandingly ubiquitous and can be recovered from a great diversity of habitats. However, the role of viral infection in the emergence of environmental opportunistic species is not known. The genus of filamentous and mycoparasitic fungi Trichoderma (Hypocreales, Ascomycota) consists of more than 400 species, which mainly occur on dead wood, other fungi, or as endo- and epiphytes. However, some species are environmental opportunists because they are cosmopolitan, can establish in a diversity of habitats, and can also become pests on mushroom farms and infect immunocompromised humans. In this study, we investigated the library of 163 Trichoderma strains isolated from grassland soils in Inner Mongolia, China, and found only four strains with signs of the mycoviral nucleic acids, including a strain of T. barbatum infected with a novel strain of the Polymycoviridae and named and characterized here as Trichoderma barbatum polymycovirus 1 (TbPMV1). Phylogenetic analysis suggested that TbPMV1 was evolutionarily distinct from the Polymycoviridae isolated either from Eurotialean fungi or from the order Magnaportales. Although the Polymycoviridae viruses were also known from Hypocrealean Beauveria bassiana, the phylogeny of TbPMV1 did not reflect the phylogeny of the host. Our analysis lays the groundwork for further in-depth characterization of TbPMV1 and the role of mycoviruses in the emergence of environmental opportunism in Trichoderma. IMPORTANCE Although viruses infect all organisms, our knowledge of some groups of eukaryotes remains limited. For instance, the diversity of viruses infecting fungi-mycoviruses-is largely unknown. However, the knowledge of viruses associated with industrially relevant and plant-beneficial fungi, such as Trichoderma spp. (Hypocreales, Ascomycota), may shed light on the stability of their phenotypes and the expression of beneficial traits. In this study, we screened the library of soilborne Trichoderma strains because these isolates may be developed into bioeffectors for plant protection and sustainable agriculture. Notably, the diversity of endophytic viruses in soil Trichoderma was outstandingly low. Only 2% of 163 strains contained traces of dsRNA viruses, including the new Trichoderma barbatum polymycovirus 1 (TbPMV1) characterized in this study. TbPMV1 is the first mycovirus found in Trichoderma. Our results indicate that the limited data prevent the in-depth study of the evolutionary relationship between soilborne fungi and is worth further investigation.

Identification of CNGCs in Glycine max and Screening of Related Resistance Genes after Fusarium solani Infection.

Cyclic nucleotide-gated channels (CNGCs), non-selective cation channels localised on the plasmalemma, are involved in growth, development, and regulatory mechanisms in plants during adverse stress. To date, CNGC gene families in multiple crops have been identified and analysed. However, there have been no systematic studies on the evolution and development of CNGC gene families in legumes. Therefore, in the present study, via transcriptome analysis, we identified 143 CNGC genes in legumes, and thereafter, classified and named them according to the grouping method used for Arabidopsis thaliana. Functional verification for disease stress showed that four GmCNGCs were specifically expressed in the plasmalemma during the stress process. Further, functional enrichment analysis showed that their mode of participation and coordination included inorganic ion concentration regulation inside and outside the membrane via the transmembrane ion channel and participation in stress regulation via signal transduction. The CNGC family genes in G. max involved in disease stress were also identified and physiological stress response and omics analyses were also performed. Our preliminary results revealed the basic laws governing the involvement of CNGCs in disease resistance in G. max, providing important gene resources and a theoretical reference for the breeding of resistant soybean.

Occurrence, Distribution, and Transmission of Alfalfa Viruses in China.

Alfalfa (Medicago sativa L.) is one of the most important quality forages worldwide and is cultivated throughout China. Alfalfa is susceptible to a variety of viral diseases during its growth, which has caused huge amounts of commercial losses. However, the profile of the alfalfa virus in China remains ambiguous and the viruses transmitted by Odontothrips loti (Haliday), dominant insect pests in alfalfa, are also poorly understood. In the present study, virus diversity was investigated in the primary alfalfa-growing areas in China. A total of 18 alfalfa viruses were identified through RNA-sequencing (RNA-seq) and reverse transcription-polymerase chain reaction (RT-PCR). Two new plant viruses, Medicago sativa virus 1 (MsV1) and Medicago sativa luteovirus 1 (MsLV1), were detected for the first time. Another four viruses, including the Alfalfa ringspot-associated virus (ARaV), Alfalfa virus F (AVF), Alfalfa enamovirus 1 (AEV1), and Alfalfa deltaparitivirus (ADPV), were reported in China for the first time as well. Both Alfalfa mosaic virus (AMV) and Medicago sativa alphapartitivirus 2 (MsAPV2) are the dominant pathogens, with an infection incidence of 91.7-100%, and 74.4-97.2%, respectively. Additionally, O. loti with first- and second-instar nymphs were shown to acquire the AMV within 0.25 h of feeding on a virus-infected alfalfa. Transmission by thrips to healthy alfalfa plants was also demonstrated. Additionally, we clarified the dynamic changes in the AMV in pre-adult stages of O. loti, which indicated that the AMV is propagated in the nymph stage of O. loti. These findings provide valuable information for understanding the alfalfa virome, confirm the role thrips O. loti plays in alfalfa virus transmission, and improve our fundamental knowledge and management of diseases in China.

The Newly Identified Trichoderma harzianum Partitivirus (ThPV2) Does Not Diminish Spore Production and Biocontrol Activity of Its Host.

A new partititvirus isolated from a Trichoderma harzianum strain (T673), collected in China, was characterized and annotated as Trichoderma harzianum partitivirus 2 (ThPV2). The genome of ThPV2 consists of a 1693 bp dsRNA1 encoding a putative RNA-dependent RNA polymerase (RdRp) and a 1458 bp dsRNA2 encoding a hypothetical protein. In comparative studies employing the ThPV2-infected strain (T673) and a strain cured by ribavirin treatment (virus-free strain T673-F), we investigated biological effects of ThPV2 infection. While the growth rate of the virus-infected fungus differed little from that of the cured variant, higher mycelial density, conidiospore, and chlamydospore production were observed in the virus-infected strain T673. Furthermore, both the ThPV2-infected and the cured strain showed growth- and development-promoting activities in cucumber plants. In vitro confrontation tests showed that strains T673 and T673-F inhibited several important fungal pathogens and an oomycete pathogen in a comparable manner. Interestingly, in experiments with cucumber seeds inoculated with Fusarium oxysporum f. sp. cucumerinum, the ThPV2-infected strain T673 showed moderately but statistically significantly improved biocontrol activity when compared with strain T673-F. Our data broaden the spectrum of known mycoviruses and provide relevant information for the development of mycoviruses for agronomic applications.

Molecular characterization of strawberry vein banding virus from China and the development of loop­mediated isothermal amplification assays for their detection.

Strawberry vein banding virus (SVBV) is one of the serious viral pathogens infecting strawberry worldwide. To understand the molecular characterization of SVBV from China, complete genome sequences of sixteen SVBV isolates were cloned and sequenced. Sequence comparison showed they shared high nucleotide sequence identity (93.6-99.5%) with isolates from China and Japan (96.6-98.4%), while relatively low identity with the isolates from Canada (91.9-93.7%) and USA (85.5-85.9%). Phylogenetic analyses based on the complete genome sequence or coat protein (CP) gene showed the SVBV isolates clustered into three clades correlated with geographic distribution. Recombination analyses identified 13 recombinants and 21 recombinant events, indicating frequent and multiple recombinations in SVBV evolution. Furthermore, a sensitive loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of SVBV isolates, which could be especially suitable for seedling propagation, virus-free culture and routine diagnostics in field investigation. This study offers new understanding of the molecular evolution and may help to improve the management of SVBV.

The first complete genome sequence of iris severe mosaic virus.

The first complete genome sequence of ISMV was determined by deep sequencing of a small RNA library constructed from ISMV-infected samples and rapid amplification of cDNA ends (RACE) PCR. The ISMV genome consists of 10,403 nucleotides excluding the poly(A) tail and contains a large open reading frame encoding a polyprotein of 3316 amino acids. Putative proteolytic cleavage sites were identified by BLAST analysis. The ISMV polyprotein showed highest amino acid sequence identity to that encoded by onion yellow dwarf virus. Phylogenetic analysis of the polyprotein amino acid sequence confirmed that ISMV forms a cluster with shallot yellow stripe virus, Cyrtanthus elatus virus A, narcissus degeneration virus and onion yellow dwarf virus. These results confirm that ISMV is a distinct member of the genus Potyvirus.

Characterization of siRNAs derived from cucumber green mottle mosaic virus in infected cucumber plants.

Virus-derived small interfering RNAs (vsiRNAs) of cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, were characterised in cucumber plants by deep sequencing. CGMMV vsiRNAs of 21-22 nt in length predominated, suggesting that there might be a conserved mechanism of DCL2 and DCL4 involvement in the biogenesis of vsiRNAs, as well as a common RNA silencing pathway in CGMMV-infected cucumber plants. The 5'-terminal base of vsiRNAs was biased towards C/A/U, suggesting that CGMMV vsiRNAs might be loaded into diverse AGO-containing RISCs to disturb the gene expression of host plants. Possible targets for some of the vsiRNAs were also predicted.

Complete genomic sequence analysis reveals a novel fabavirus infecting cucurbits in China.

The complete genome sequence of a cucurbit-infecting fabavirus was determined. Sequence analysis revealed that it had a genomic organization typical of fabaviruses, with genome segment sizes of 5870 nt (RNA-1) and 3294 nt (RNA-2). It shared CP and Pro-Pol amino acid sequence identities of 52.0-58.9% with those of reported fabaviruses. ELISA and western blots gave no cross-reactions between this cucurbit virus and broad bean wilt viruses 1 and 2. Based on molecular and serological criteria for species demarcation in the genus Fabavirus, the virus represents a distinct species, for which the species name Cucurbit mild mosaic virus (CuMMV) is proposed.

Molecular characterization of two genotypes of a new polerovirus infecting brassicas in China.

The genomic RNA sequences of two genotypes of a brassica-infecting polerovirus from China were determined. Sequence analysis revealed that the virus was closely related to but significantly different from turnip yellows virus (TuYV). This virus and other poleroviruses, including TuYV, had less than 90% amino acid sequence identity in all gene products except the coat protein. Based on the molecular criterion (>10% amino acid sequence difference) for species demarcation in the genus Polerovirus, the virus represents a distinct species for which the name Brassica yellows virus (BrYV) is proposed. Interestingly, there were two genotypes of BrYV, which mainly differed in the 5'-terminal half of the genome.

Distribution and molecular diversity of three cucurbit-infecting poleroviruses in China.

Cucurbit aphid-borne yellows virus (CABYV) and Melon aphid-borne yellows virus (MABYV) have been found to be associated with cucurbit yellowing disease in China. Our report identifies for the first time a third distinct polerovirus, tentatively named Suakwa aphid-borne yellows virus (SABYV), infecting Suakwa vegetable sponge. To better understand the distribution and molecular diversity of these three poleroviruses infecting cucurbits, a total of 214 cucurbitaceous crop samples were collected from 25 provinces in China, and were investigated by RT-PCR and sequencing. Of these, 108 samples tested positive for CABYV, while 40 samples from five provinces were positive for MABYV, and SABYV was detected in only 4 samples which were collected in the southern part of China. Forty-one PCR-amplified fragments containing a portion of the RdRp gene, intergenic NCR and CP gene were cloned and sequenced. Sequence comparisons showed that CABYV isolates shared 78.0-79.2% nucleotide sequence identity with MABYV isolates, and 69.7-70.8% with SABYV. Sequence identity between MABYV and SABYV was 73.3-76.5%. In contrast, the nucleotide identities within each species were 93.2-98.7% (CABYV), 98.1-99.9% (MABYV), and 96.1-98.6% (SABYV). Phylogenetic analyses revealed that the polerovirus isolates fit into three distinct groups, corresponding to the three species. The CABYV group could be further divided into two subgroups: the Asia subgroup and the Mediterranean subgroup, based on CP gene and partial RdRp gene sequences. Recombination analysis suggested that MABYV may be a recombinant virus.

Complete sequence analysis reveals two distinct poleroviruses infecting cucurbits in China.

The complete RNA genomes of a Chinese isolate of cucurbit aphid-borne yellows virus (CABYV-CHN) and a new polerovirus tentatively referred to as melon aphid-borne yellows virus (MABYV) were determined. The entire genome of CABYV-CHN shared 89.0% nucleotide sequence identity with the French CABYV isolate. In contrast, nucleotide sequence identities between MABYV and CABYV and other poleroviruses were in the range of 50.7-74.2%, with amino acid sequence identities ranging from 24.8 to 82.9% for individual gene products. We propose that CABYV-CHN is a strain of CABYV and that MABYV is a member of a tentative distinct species within the genus Polerovirus.