Wenxing Xu

Hypovirulence of the Phytopathogenic Fungus Botryosphaeria dothidea: Association with a Coinfecting Chrysovirus and a Partitivirus

ABSTRACT Botryosphaeria dothidea is an important pathogenic fungus causing fruit rot, leaf and stem ring spots and dieback, stem canker, stem death or stool mortality, and decline of pear trees. Seven double-stranded RNAs (dsRNAs; dsRNAs 1 to 7 with sizes of 3,654, 2,773, 2,597, 2,574, 1,823, 1,623, and 511 bp, respectively) were identified in an isolate of B. dothidea exhibiting attenuated growth and virulence and a sectoring phenotype. Characterization of the dsRNAs revealed that they belong to two dsRNA mycoviruses. The four largest dsRNAs (dsRNAs 1 to 4) are the genomic components of a novel member of the family Chrysoviridae (tentatively designated Botryosphaeria dothidea chrysovirus 1 [BdCV1]), a view supported by the morphology of the virions and phylogenetic analysis of the putative RNA-dependent RNA polymerases (RdRps). Two other dsRNAs (dsRNAs 5 and 6) are the genomic components of a novel member of the family Partitiviridae (tentatively designated Botryosphaeria dothidea partitivirus 1 [BdPV1]), which is placed in a clade distinct from other established partitivirus genera on the basis of the phylogenetic analysis of its RdRp. The smallest dsRNA, dsRNA7, seems to be a noncoding satellite RNA of BdPV1 on the basis of the conservation of its terminal sequences in BdPV1 genomic segments and its cosegregation with BdPV1 after horizontal transmission. This is the first report of a chrysovirus and a partitivirus infecting B. dothidea and of a chrysovirus associated with the hypovirulence of a phytopathogenic fungus. IMPORTANCE Our studies identified and characterized two novel mycoviruses, Botryosphaeria dothidea chrysovirus 1 (BdCV1) and Botryosphaeria dothidea partitivirus 1 (BdPV1), associated with the hypovirulence of an important fungus pathogenic to fruit trees. This is the first report of a chrysovirus and a partitivirus infecting B. dothidea and of a chrysovirus associated with the hypovirulence of a phytopathogenic fungus. BdCV1 appears to be a good candidate for the biological control of the serious disease induced by B. dothidea. Additionally, BdPV1 is placed in a clade distinct from the established genera. The BdCV1 capsid has two major structural proteins, and the capsid is distinct from that made up by a single polypeptide of the typical chrysoviruses. BdPV1 is the second partitivirus in which the putative capsid protein shares no significant identity with any mycovirus protein. A small accompanying dsRNA that is presumed to be a noncoding satellite RNA of BdPV1 is the first of its kind reported for a partitivirus.

Actinidia chlorotic ringspot‐associated virus: a novel emaravirus infecting kiwifruit plants

By integrating next-generation sequencing (NGS), bioinformatics, electron microscopy and conventional molecular biology tools, a new virus infecting kiwifruit vines has been identified and characterized. Being associated with double-membrane-bound bodies in infected tissues and having a genome composed of RNA segments, each one containing a single open reading frame in negative polarity, this virus shows the typical features of members of the genus Emaravirus. Five genomic RNA segments were identified. Additional molecular signatures in the viral RNAs and in the proteins they encode, together with data from phylogenetic analyses, support the proposal of creating a new species in the genus Emaravirus to classify the novel virus, which is tentatively named Actinidia chlorotic ringspot-associated virus (AcCRaV). Bioassays showed that AcCRaV is mechanically transmissible to Nicotiana benthamiana plants which, in turn, may develop chlorotic spots and ringspots. Field surveys disclosed the presence of AcCRaV in four different species of kiwifruit vines in five different provinces of central and western China, and support the association of the novel virus with symptoms of leaf chlorotic ringspots in Actinidia. Data on the molecular features of small RNAs of 21-24 nucleotides, derived from AcCRaV RNAs targeted by host RNA silencing mechanisms, are also reported, and possible molecular pathways involved in their biogenesis are discussed.

Molecular and serological diversity in Apple chlorotic leaf spot virus from sand pear (Pyrus pyrifolia) in China

Apple chlorotic leaf spot virus (ACLSV) isolates from sand pear (Pyrus pyrifolia) were characterized by analyzing the sequences of their coat protein (CP) genes and serological reactivity of recombinant coat proteins (rCPs). The sequences of CP genes from 22 sand pear isolates showed a high divergence, with 87.3–100% identities at the nucleotide (nt) level and 92.7–100% identities at the amino acid (aa) level. Phylogenetic analysis on the aa sequence of CP showed that the analyzed ACLSV isolates fell into different clusters and all isolates from sand pear were grouped into a large cluster (I) which was then divided into two sub-clusters (A and B). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot and enzyme-linked immunosorbent assay (ELISA) analyses demonstrated that rCPs of eight ACLSV isolates (PP13, PP15-2, PP24, PP43, PE, PP54, PP56 and ACLSV-C) from two sub-clusters had different mobility rates and serological reactivity. The rCPs of five isolates grouped into the sub-cluster A showed stronger reactivity with antibodies against rCPs of a sand pear isolate ACLSV-BD and virions of a Japanese apple isolate P-205 than that with the antibody against a Chinese apple isolate ACLSV-C. Three isolates grouped into the sub-cluster B showed stronger reactivity with the antibody against ACLSV-C. The antigenic determinants of CPs from these eight isolates and isolates ACLSV-BD and P-205 were predicted. These results contribute to a further understanding of molecular diversity of the virus and its implication in serological detection.

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.

Deep sequencing reveals the first fabavirus infecting peach

A disease causing smaller and cracked fruit affects peach [Prunus persica (L.) Batsch], resulting in significant decreases in yield and quality. In this study, peach tree leaves showing typical symptoms were subjected to deep sequencing of small RNAs for a complete survey of presumed causal viral pathogens. The results revealed two known viroids (Hop stunt viroid and Peach latent mosaic viroid), two known viruses (Apple chlorotic leaf spot trichovirus and Plum bark necrosis stem pitting-associated virus) and a novel virus provisionally named Peach leaf pitting-associated virus (PLPaV). Phylogenetic analysis based on RNA-dependent RNA polymerase placed PLPaV into a separate cluster under the genus Fabavirus in the family Secoviridae. The genome consists of two positive-sense single-stranded RNAs, i.e., RNA1 [6,357 nt, with a 48-nt poly(A) tail] and RNA2 [3,862 nt, with a 25-nt poly(A) containing two cytosines]. Biological tests of GF305 peach indicator seedlings indicated a leaf-pitting symptom rather than the smaller and cracked fruit symptoms related to virus and viroid infection. To our knowledge, this is the first report of a fabavirus infecting peach. PLPaV presents several new molecular and biological features that are absent in other fabaviruses, contributing to an overall better understanding of fabaviruses.

Virulence determination and molecular features of peach latent mosaic viroid isolates derived from phenotypically different peach leaves: a nucleotide polymorphism in L11 contributes to symptom alteration.

Symptoms of chlorosis along leaf edges (chlorosis-edge), along leaf veins (chlorosis-vein) and yellowing on peach leaves have been observed for a long history in the field, while the pathological factor(s) responsible for these symptoms remained unknown. Peach latent mosaic viroid (PLMVd) was detected in the leaves collected from three unique phenotypic peach trees showing above mentioned symptoms. The obtained PLMVd isolates were subjected to population structure analyses and biological assays to evaluate their pathogenicity on peach seedlings in an effort to elucidate the relationship between the PLMVd and the symptoms observed on peach trees in China. In addition, molecular features of PLMVd isolates were analyzed to obtain some insight into the structure-function relationships of this viroid. The results revealed that the symptoms of chlorosis-edge and yellowing were indeed incited by PLMVd, and a direct link between the nucleotide polymorphisms and the symptoms of yellowing and chlorosis-edge was established, i.e. residue U338 responsible for the yellowish symptom and C338 responsible for the chlorosis-edge symptom. This study provides an additional proof to endorse a previous proposal that PLMVd pathogenicity determinants reside in L11. The illustrative etiology of the disease, visualization of the symptoms progression and identification of the unique single nucleotide polymorphism possibly involved in the symptom induction will significantly increase understanding of the pathogenic mechanisms of PLMVd and will help in designing control strategies for the resulting disease.

Genetic variability and population structure of Grapevine virus A in China based on the analysis of its coat protein gene

Abstract In this study, reverse transcription PCR (RT–PCR) detection revealed a high infection rate of Grapevine virus A (GVA) in grapevine plants cultivated in China. Population structures of 14 GVA isolates from Chinese grapevines were studied by single-stranded conformation polymorphism (SSCP) of the coat protein (CP) gene. Results showed that while most isolates contained a population of sequence variants, with one being predominant, one isolate (AF) had three major variants, and two isolates (BSSL and BSBD) consisted of a number of sequence variants with almost the same detection frequencies. The estimation of genetic diversity intra isolates by sequence alignment analysis indicated a high variability in the CP gene and the occurrence of mixed infections in some grapevines by divergent sequence variants. RT–PCR analysis using variant-specific primers (H587/C995, GVA6591F/GVA6906R) and phylogenetic analyses of CP gene sequences suggested that all sequence variants from Chinese isolates were separated into two subgroups IA and IB in the group I.

Biological and Molecular Characterization of Five Phomopsis Species Associated with Pear Shoot Canker in China

In recent years, a widespread canker disease that infects the branches of pear trees has been observed in many provinces in China; it kills the branches and results in high losses in fruit production. Symptomatic branches were collected for etiological isolation from 11 varieties of three pear species and from Malus pumila. Samples were collected from six provinces in China. In total, 143 Phomopsis isolates were obtained from 181 samples and these were identified as belonging to five species: Phomopsis fukushii (n = 69 isolates), Diaporthe eres (n = 31), P. amygdali (n = 22), P. longicolla (n = 13), and D. neotheicola (n = 8). Pathogenicity tests showed that only the first three species induced lesions on nonwounded branches of Pyrus pyrifolia var. Cuiguan. All the fungal species induced branch cankers following wound inoculations, and tests with additional pear varieties showed significantly higher virulence levels for the first three species than the latter two. A host range evaluation suggested that the five species could infect most fruit trees belonging to the Rosaceae family as well as some non-Rosaceous species. Virulence varied depending on the species of both host and pathogen. Isolates of Phomopsis amygdali had significantly higher virulence in all the tested Rosaceae plants. Correlations among the host, pathogen, and sampling regions were noted, and the morphology, growth rate, and sporulation of these species in varied media were also characterized. This study presents the first attempt to perform a broad survey and characterization of the Phomopsis spp. associated with the pear shoot cankers in China. This study shows that D. eres and P. amygdali are just as responsible for the pear shoot canker diseases as P. fukushii, and it expands the host and geographic ranges of the five species. This report provides useful information for understanding and improving management strategies for controlling this economically important disease.

First Report of Citrus exocortis viroid from Grapevine in China

Citrus exocortis viroid (CEVd) can induce bark scaling, dwarfing, leaf epinasty, and fruit yield loss in susceptible hosts. In citrus, CEVd is reported from around the world, but in grape, it is reported from fewer locations (Australia, Brazil, California, and Spain [1]). In 2009, leaves were collected from 40 grapevines (of several different cultivars and species) from Henan, Hubei, Shandong, and Liaoning provinces, China. Total RNA or double-stranded RNA was extracted from the leaves by a described method (3) and subjected to reverse transcription with a random primer (Takara, Dalian, China) and then PCR with primer CEV-AM3 and CEV-AP3 (2). Results showed that the target DNA fragments of 372 bp long were amplified only from the symptomless leaves collected from two grapevines of cv. White Rose grown for approximately 26 years within a small garden in Hubei Province. Amplified products were recovered and cloned into pMD18-T (Takara) and 10 positive clones of each isolate were sequenced and aligned. For both isolates, 20% of the clones represented the same variant (CEVd-hn-g-1; GenBank Accession No. GU592444). It showed a max identity of 94 to 99% with the variants (GenBank Accession Nos. Y00328.1 and DQ471996.1) from grape registered in NCBI, 91 to 100% (GenBank Accession Nos. DQ431993.1 and DQ831485.1) from citrus, 91 to 98% (GenBank Accession Nos. EF488068.1 and EF488050.1) from broad bean, and 89 to 94% (GenBank Accession Nos. AY671953.1 and S67446.1) from tomato. To our knowledge, this is the first report of CEVd from grape in China. References: (1) M. Eiras et al. Fitopatol. Bras. 31:440, 2006. (2) H. J. Gross et al. Eur. J. Biochem. 121:249, 1982. (3) W. X. Xu et al. J. Virol. Methods 135:276, 2006.

A rapid silica spin column-based method of RNA extraction from fruit trees for RT-PCR detection of viruses

Efficient recovery of high quality RNA is very important for successful RT-PCR detection of plant RNA viruses. High levels of polyphenols and polysaccharides in plant tissues can irreversibly bind to and/or co-precipitate with RNA, which influences RNA isolation. In this study, a silica spin column-based RNA isolation method was developed by using commercially available silica columns combined with the application of a tissue lysis solution, and binding and washing buffers with high concentration guanidinium thiocyanate (GuSCN, 50% w/v), which helps remove plant proteins, polysaccharides and polyphenolic compounds. The method was successfully used to extract high quality RNA from citrus (Citrus aurantifolia), grapevine (Vitis vinifera), peach (Prunus persica), pear (Pyrus spp.), taro (Colocosia esculenta) and tobacco (Nicotiana benthamiana) samples. The method was comparable to conventional CTAB method in RNA isolation efficiency, but it was more sample-adaptable and cost-effective than commercial kits. High quality RNA isolated using silica spin column-based method was successfully used for the RT-PCR and/or multiplex RT-PCR amplification of woody fruit tree viruses and a viroid. The study provided a useful tool for the detection and characterization of plant viruses.