China is the center of origin of kiwifruit (Actinidia spp.) and the largest producing country in both terms of yield and acreage (Ferguson 2015). Kiwifruit virus diseases usually cause leaf yellowing, mosaic spots, fruit malformation, resulting in reduced yield and quality (Blouin et al. 2013). In August 2019, virus-like symptoms including chlorotic spots, crinkle, yellowing, and deformation were observed on leaves of kiwifruit grown in a 2,000-m2 commercial orchard in Chongqing, China, with disease incidence reaching approximately 20%. Twenty-eight leaf samples (CQ1-28) were randomly collected from symptomatic plants and mixed in equal amounts. Extraction of total RNA from above pooled sample, removal of ribosomal RNA and subsequent high-throughput sequencing (HTS) were conducted as previously reported (Wen et al. 2020). After filtering low-quality reads (Q30>94.16%), a total of 82,730,664 clean reads with an average length of 150 nucleotides (nt) were obtained and de novo assembled into contigs using CLC Genomics Workbench 11.0 (Qiagen) with k-mer values ranging from 31 to 121. BLASTx against the NCBI GenBank database showed that 10 contigs, 344 to 2,905 nt in length, were annotated as apple latent spherical virus (ALSV) (AB030940.1 and AB030941.1), which belongs to the Cheravirus genus and was originally identified in apple (Li et al. 2000). The nt identity between these contigs and the reference genome ranged from 75.2 to 85.7%. Primers CP-F/CP-R (5′-RTAGGHCARGGKGCTTGYTTGAGTA-3′/5′-CCAATAGGCTGTAGTRCTGGCC ATA-3′) targeting a 750 bp fragment CP gene of ALSV were designed based on the obtained contigs and the AB030941.1 sequence to verify the sequencing results in the individual samples. Total RNA was individually extracted from leaf samples CQ1-28 using a cetyltrimethylammonium bromide (CTAB) based method (Li et al., 2008) and subjected to reverse transcription polymerase chain reaction (RT-PCR). Target products were obtained from four samples (CQ2, CQ6, CQ8 and CQ15) and Sanger sequenced. Alignment of the four sequences showed that they shared 90.4 to 100% nt identity with each other and 75.7 to 77.3% nt identity with the corresponding AB030941.1 sequence. Furthermore, 49 symptomatic leaf samples were collected from Hubei (6), Jiangxi (6), Henan (31), Shanxi (4) provinces and Shanghai (2), China, and were tested for the presence of ALSV by RT-PCR with primers CP-F/CP-R. Three additional samples (Z2, ZZ19 and ZZ21) from Henan province were found ALSV positive. The detection rate of ALSV in Chongqing city and Henan province was 14.3% and 9.7%, respectively. To analyze the phylogenetic relationship of the ALSV isolates identified in this study, the seven CP sequences (GenBank accession nos. PV931796.1 to PV931802.1) of ALSV-positive kiwifruit samples together with the available sequences of ALSV from apple and Angelica sinensis (OP038546.1) (Jin et al. 2023) were aligned and a phylogenetic tree was constructed. The ALSV isolates obtained from kiwifruit clustered in the same branch with those infecting apple and A. sinensis. Since the mixed infections of ALSV with one or more of Actinidia virus 1, citrus leaf blotch virus, Actinidia virus A, Actinidia virus B and Actinidia seed borne latent virus, ALSV pathogenicity in kiwifruit remains challenging to confirm. To our knowledge, this is the first report of ALSV naturally infecting kiwifruit, expanding the host range of this virus and providing a crucial basis for viral disease monitoring.

Pear ring rot disease (Botryosphaeria dothidea) is a significant threat to the healthy development of the pear industry. Recent research has identified the functional role of long non-coding RNAs (lncRNAs) in various biological processes of plants. The role of lncRNAs in the pear defense response remains unknown. In this study, transcriptome sequencing was used to analyze lncRNAs in pear stem infected with B. dothidea. It identified 3555 lncRNAs, of which 286 were significantly differentially expressed. GO and KEGG analyses showed that cis- and trans-regulated target genes were enriched in multiple disease resistance-related pathways. More specifically, MSTRG.32189, predicted as an endogenous target mimic (eTM), was significantly down-regulated in response to B. dothidea infection, and was confirmed to inhibit the cleavage effect of PcmiR399b on PcUBC24. OE-MSTRG.32189 transgenic Arabidopsis exhibited lower Pi content and weaker disease resistance to Botrytis cinerea compared with wild type. In pear callus, overexpression of MSTRG.32189 negatively regulated PcmiR399b, which decreased Pi content and reduced disease resistance. Overexpressing PcmiR399b in pear callus exhibited the opposite effects compared with OE-MSTRG.32189. Overexpression and knockout of PcUBC24 further clarified that PcUBC24 negatively regulates Pi content and disease resistance to B. dothidea infection. Furthermore, the ROS levels and expressions of disease resistance pathway-related genes were regulated by the MSTRG.32189-PcmiR399b-PcUBC24 module in transgenic pear callus, which contributed to disease resistance. Overall, our results demonstrated the role of lncRNAs in the pear defense response, revealing that the MSTRG.32189-PcmiR399b-PcUBC24 module regulates phosphate accumulation and disease resistance to B. dothidea infection in pear.

Pear ring rot disease, the pathogen of Botryosphaeria dothidea causes significant threat to the healthy development of the pear industry, therefore the exploration of disease-resistant gene resources is crucial for disease prevention and control. Members of the R2R3-MYB subfamily play important roles in regulating pathogen resistance in plants, however the gene function in regulating host resistance in pear remains unclear. In this study, the role of PcMYB44 were investigated in regulating host resistance disease in pear calli using both forward and reverse genetic approaches. Overexpression of PcMYB44 positively regulates the disease resistance, whereas knockout of PcMYB44 results in a phenotype with decreased resistance. Our results further demonstrated that PcMYB44 could directly affect lignin content and resistance to fungal diseases by regulating the PcmiR397-PcLACs module and lignin biosynthesis gene expression levels. Additionally, overexpressing PcMYB44 also elevated expression levels of key genes of JA/SA/ET pathway. The obtained results revealed that PcMYB44 regulated host resistance to ring rot disease through synergistic regulation the lignification and activating disease-resistance gene expression of JA/SA/ET defense pathways as a underlying secondary mechanism, which provide valuable genetic resources for molecular breeding for disease resistance.

The full text of this preprint has been withdrawn by the authors due to author disagreement with the posting of the preprint. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author.

China is a major producer of pears in the world and anthracnose is the most important disease, which may include fruit rot and early defoliation, and further brings enormous economic losses. In August of 2023, a sudden outbreak of anthracnose disease, ranging from 70% to 90% disease incidence, occurred on fruits of Pyrus pyrifolia (Burm.f.) Nakai in 200 acres of three pear orchards at Baiyi town, Guiyang city, Guizhou Province of China. Thirty diseased fruits were randomly collected three pear orchards, i.e., 10 fruits from each, and transported to the laboratory for analysis. The symptoms showed dark brown lesions that extended rapidly causing sunken fruit rot lesions harboring orange conidial clumps. To identify the pathogen, three from the 10 diseased fruits per orchard were used for isolation. Small tissue pieces of diseased fruits were disinfected and rinsed twice with 75% alcohol and sterile water, placed on a potato dextrose agar (PDA), and cultured at 25℃ in a dark (Fang, 1998). Ten isolates with same colony morphology were obtained. Two isolates, GZ-3 and GZ-5, were randomly selected for further analysis. The GZ-3 and GZ-5 isolates grew on PDA plates at an average daily growth rate of 0.8~1.0 cm/d in the darkness at 25℃. The colonies were white to gray on the front side and greyish-green to orange on the reverse side. The conidia were hyaline, smooth, nonseptate, and fusiform, measuring 9.0-19.2×2.5-6.1 μm, with the average mean of 13.9±2.2×4.4±0.9 μm (n=50) for GZ-3, and the size of 9.5-15.4×2.3-4.8 μm, with average size of 13.1±1.5×3.7±0.5 μm (n=50) for GZ-5, respectively. The morphological characteristics of the GZ-3 and GZ-5 colonies were consistent with those of Colletotrichum acutatum complex (Damm et al. 2012). To confirm the pathogenicity, the 6 × 106 mL-1 spore suspension of GZ-3 and GZ-5 were inoculated on healthy P. pyrifolia (Burm.f.) Nakai fruits using the non-invasive spray method, while sterile water was used to inoculate control fruits. The fruits inoculated with the two isolates exhibited dark brown spots in the early stage of the disease, then the lesions expanded to rot and produced orange conidia. GZ-3 and GZ-5 were reisolated from the fruits, fulfilling Koch's postulates. The internal transcribed spacer (ITS) rDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase 1 (CHS-1) and β-tubulin (TUB2) were partially amplified using the primers designed previously (Weir et al. 2012) and sequenced. The above five gene sequences from GZ-3 showed 99.63% to 100% identity with those of GZ-5, respectively. The GZ-3 sequences were deposited in GenBank (ACT: PP825836; TUB2: PP825837; CHS-1: PP825838; GAPDH: PP825839; ITS: PP821112). The sequence alignment was performed based on the concatenated sequence of ACT-TUB2-CHS-1-GAPDH-ITS. A phylogenetic tree, constructed using the Neighbor-Joining method with bootstrap replication 2200 bootstrap alignments in MEGA11.0, showed GZ-3 and GZ-5 clustering with reference C. nymphaeae isolates. Based on the obtained morphological characterization and phylogenetic analysis results, GZ-3 and GZ-5 were identified as C. nymphaeae. Currently, 12 Colletotrichum species causing pear anthracnose have been reported in China (Fu et al. 2019). To our knowledge, this is the first report of C. nymphaeae causing pear anthracnose in the country. The findings are significant for a quick monitoring and control of pear anthracnose.

Melanins are dark-brown to black-colored biomacromolecules which have been thoroughly studied in animals and microorganisms. However, the biochemical and molecular basis of plant melanins are poorly understood. We first characterized melanin from the black radish (Raphanus sativus var. niger) 'HLB' through spectroscopic techniques. p-Coumaric acid was identified as the main precursor of radish melanin. Moreover, a joint analysis of transcriptome and coexpression network was performed for the two radish accessions with black and white cortexes, 'HLB' and '55'. A set of R2R3-type RsMYBs and enzyme-coding genes exhibited a coexpression pattern, and were strongly correlated with melanin formation in radish. Transient overexpression of two phenol oxidases RsLAC7 (laccase 7) or RsPOD22-1 (peroxidase 22-1) resulted in a deeper brown color around the infiltration sites and a significant increase in the total phenol content. Furthermore, co-injection of the transcriptional activator RsMYB48/RsMYB97 with RsLAC7 and/or RsPOD22-1, markedly increased the yield of black extracts. Spectroscopic analyses revealed that these extracts are similar to the melanin found in 'HLB'. Our findings advance the understanding of structural information and the transcriptional regulatory mechanism underlying melanin formation in radish.

Pear is popular among people, which is an important pillar industry in China. In March of 2023, dark brown necrotic lesions were discovered on the trunks of Pyrus pyrofolia cv. Osmanthus pear in orchard, Liuzhou City, Guangxi Zhuang Autonomous Region. In August, field investigation and sample collection were conducted in orchard. Forty pear trees were selected for symptomatic observation, which of 21 had lesions ranging from 10 to 24 per tree, and 19 with 1 to 8 lesions, respectively. To isolate the pathogen, small tissue pieces of 3 diseased pear trunk samples were disinfected with 75% ethanol for 1 minute, rinsed with sterile water, and dried with filter paper. The tissue pieces were placed on potato dextrose agar (PDA) plates and cultured in a dark incubator at 25℃. Six isolates with the similar morphology were obtained. One of the six isolates was randomly selected as the representative strain and named as GX-3. Mycelium grows with an average rate of 4.26 cm/d. The hypha is highly aerial, and is initially white and then turns black. Subsequently, pycnidia formed and secreted black mucus on the PDA medium after 28 days. The immature conidia were ellipsoid, colorless, hyaline, and unicellular, mostly becoming brown bicellular with longitudinal stripes at maturity. The conidial size was 22.5 to 32.6×12.1 to 19.7μm, and the average size was 28.4±2.3×16.7±2.0 μm (n=50), respectively. GX-3 colony morphology was consistent with that of Lasiodiplodia pseudotheobromae (Alves et al.2008). For molecular identification, the internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (TEF1-α), and β-tubulin regions were amplified using the primers ITS1/4, EF1-728F/986R, and Bt2a/Bt2b, respectively (White et al.1990; Carbone and Kohn 1999; Glass and Donaldson 1995). The obtained sequences of GX-3 were deposited in NCBI with Accession numbers OR655421, OR661231, and OR661230, respectively. The sequences of ITS, TEF1-α, and β-tubulin from GX-3 are 99.44%、99.67% and 99.78% identities with those of L. pseudotheobromae CBS 447.62, respectively. The phylogenetic analysis was performed by maximum likelihood method, revealing that GX-3 is closely clustered with the isolates of L. pseudotheobromae. Therefore, the GX-3 strain was identified as L. pseudotheobromae. GX-3 was further analyzed for its pathogenicity on pear. Firstly, the GX-3 mycelium plugs and spraying spore suspension with the concentration of 1×107 conidia/ml were applied on the stems of 4-month-old healthy birch-leaf pear (Pyrus betulifolia Bunge) potted seedlings by acupuncture needle method, meanwhile PDA and sterile water were used as controls. After 3 days of inoculation, stem surface of the birch-leaf pear exhibited dark brown lesions with slight surface depression, obvious dryness, and canker symptoms, while the control treatment showed no symptoms. The GX-3 was also inoculated on in vitro branches of 'Hosui', 'Hongxiangsu', 'Bodoqing' and 'Xuehua', showing dark brown canker lesions. The same pathogen can be successfully isolated from diseased stems and branches but not from the controls, which accomplishes Koch's postulates. L. pseudotheobromae has been widely reported that it can cause rot and canker on apple, walnut, hackberry, and so on (Xue et al. 2019; Wang et al. 2023; Liang et al. 2020). This is the first report of necrosis and canker disease caused by L. pseudotheobromae on pear in China, which is a potential threat to pear industry.

Pear black spot (Alternaria spp.) disease widespread in most pear production regions in China, causes early defoliation, which leads to the decline of fruit quality and heavily restricts the healthy development of the pear industry. Presently, no efficient way was found to biocontrol pear black spot disease. The mycovirus-mediated hypovirulent strain is regarded as an important measure of biocontrol of the fungal disease. Therefore, it is particularly urgent to excavate mycoviral resources with hypovirulence in Alternaria spp. strains. This study obtained partial or complete genomes of 14 mycoviruses from 83 Alternaria spp. strains from Wuhan city of China by high throughput sequencing, including 3 double-stranded (ds) RNA and 11 positive single-stranded (+ss) types, which were grouped into 8 distinct lineages, belonging to Chrysoviridae (isolate of AaCV1), Curvulaviridae (one virus), Partitiviridae (one virus), Mitoviridae (three viruses), Botourmiaviridae (five viruses), Deltaflexiviridae (one virus), Fusariviridae (one virus) and Togaviridae (one virus). The multiple mycoviral coinfections were common and prevalent, accounting for 78 % (65/83), with each strain harboring different numbers of mycoviruses, ranging from two to seven in Alternaria spp. Furthermore, the effects of the mycoviruses on the host were analyzed to screen the viruses-inducing hypovirulent strains. The derivative isolates from hypovirulent HB-145 with different mycoviruses were obtained by vertical transmission. It revealed that the mycoviruses have different effects on the virulence of derivative isolates from HB-145. In conclusion, these findings provide important molecular information for the classification, origin, and genetic variety of mycoviruses, and candidate materials for the exploitation of mycoviruses as biocontrol agents.

Circular single-stranded DNA (ssDNA) viruses have been rarely found in fungi, and the evolutionary and ecological relationships among ssDNA viruses infecting fungi and other organisms remain unclear. In this study, a novel circular ssDNA virus, tentatively named Diaporthe sojae circular DNA virus 1 (DsCDV1), was identified in the phytopathogenic fungus Diaporthe sojae isolated from pear trees. DsCDV1 has a monopartite genome (3185 nt in size) encapsidated in isometric virions (21-26 nm in diameter). The genome comprises seven putative open reading frames encoding a discrete replicase (Rep) split by an intergenic region, a putative capsid protein (CP), several proteins of unknown function (P1-P4), and a long intergenic region. Notably, the two split parts of DsCDV1 Rep share high identities with the Reps of Geminiviridae and Genomoviridae, respectively, indicating an evolutionary linkage with both families. Phylogenetic analysis based on Rep or CP sequences placed DsCDV1 in a unique cluster, supporting the establishment of a new family, tentatively named Gegemycoviridae, intermediate to both families. DsCDV1 significantly attenuates fungal growth and nearly erases fungal virulence when transfected into the host fungus. Remarkably, DsCDV1 can systematically infect tobacco and pear seedlings, providing broad-spectrum resistance to fungal diseases. Subcellular localization analysis revealed that DsCDV1 P3 is systematically localized in the plasmodesmata, while its expression in trans-complementation experiments could restore systematic infection of a movement-deficient plant virus, suggesting that P3 is a movement protein. DsCDV1 exhibits unique molecular and biological traits not observed in other ssDNA viruses, serving as a link between fungal and plant ssDNA viruses and presenting an evolutionary connection between ssDNA viruses and fungi. These findings contribute to expanding our understanding of ssDNA virus diversity and evolution, offering potential biocontrol applications for managing crucial plant diseases.

Fungi are important parasitic microorganism of plants, which mainly include saprophytes and pathogens species. However, differences of mycotoxins production by different fungi still remain challenging. To address this bottleneck, saprophytes and pathogens Alternaria spp. are used for inoculation and metabolites production analysis are process via liquid chromatography- high resolution mass spectrometry (LC-HRMS). Characteristic neutral loss is present for modified mycotoxins screening and feature fragments which match chemical similarity are applied for annotation. The results revealed that Saprophytic Alternaria spp. are the dominant secondar metabolites producing fungi with extremely differences. Glucose conjugations, ALU-G, AME-G, and TeA-G, are identified as modified mycotoxins. In addition, molecular networking result reveals that metabolic pathway of Alternaria mycotoxins mainly include acetylation, dehydration, didehydration, dimethylamination, hydrogenation, isomerization, methylation, combination of methylation and hydroxylation, and combination of demethylation and dihydroxylation.

Positive-sense RNA viruses remodel cellular cytoplasmic membranes as the membranous sources for the formation of viral replication organelles (VROs) for viral genome replication. In plants, they traffic through plasmodesmata (PD), plasma membrane-lined pores enabling cytoplasmic connections between cells for intercellular movement and systemic infection. In this study, we employed turnip mosaic virus (TuMV), a plant RNA virus to investigate the involvement of RTNLB3 and RTNLB6, two ER (endoplasmic reticulum) membrane-bending, PD-located reticulon-like (RTNL) non-metazoan group B proteins (RTNLBs) in viral infection. We show that RTNLB3 interacts with TuMV 6K2 integral membrane protein and RTNLB6 binds to TuMV coat protein (CP). Knockdown of RTNLB3 promoted viral infection, whereas downregulation of RTNLB6 restricted viral infection, suggesting that these two RTNLs play contrasting roles in TuMV infection. We further demonstrate that RTNLB3 targets the α-helix motif 42LRKSM46 of 6K2 to interrupt 6K2 self-interactions and compromise 6K2-induced VRO formation. Moreover, overexpression of AtRTNLB3 apparently promoted the selective degradation of the ER and ER-associated protein calnexin, but not 6K2. Intriguingly, mutation of the α-helix motif of 6K2 that is required for induction of VROs severely affected 6K2 stability and abolished TuMV infection. Thus, RTNLB3 attenuates TuMV replication, probably through the suppression of 6K2 function. We also show that RTNLB6 promotes viral intercellular movement but does not affect viral replication. Therefore, the proviral role of RTNLB6 is probably by enhancing viral cell-to-cell trafficking. Taken together, our data demonstrate that RTNL family proteins may play diverse complex, even opposite, roles in viral infection in plants.

Pear ring rot disease is an important branch disease, caused by Botryosphaeria dothidea. With the discovery of fungal viruses, the use of their attenuated properties for biological control provides a new strategy for the biological control of fungal disease. RNA silencing is a major antiviral defense mechanism in plants, insects, and fungi. Viruses encode and utilize RNA silencing suppressors to suppress host defenses. Previous studies revealed that Botryosphaeria dothidea chrysovirus 1 (BdCV1) exhibited weak pathogenicity and could activate host gene silencing by infecting B. dothidea. The aim of our study was to investigate whether BdCV1 can encode a silencing suppressor and what effect it has on the host. In this study, the capability of silencing inhibitory activity of four BdCV1-encoded proteins was analyzed, and the P3 protein was identified as a BdCV1 RNA silencing suppressor in the exotic host Nicotiana benthamiana line 16c. In addition, we demonstrated that P3 could inhibit local silencing, block systemic RNA silencing, and induce the necrosis reaction of tobacco leaves. Furthermore, overexpression of P3 could slow down the growth rate and reduce the pathogenicity of B. dothidea, and to some extent affect the expression level of RNA silencing components and virus-derived siRNAs (vsiRNAs). Combined with transcriptomic analysis, P3 had an effect on the gene expression and biological process of B. dothidea. The obtained results provide new theoretical information for further study of interaction between BdCV1 P3 as a potential silencing suppressor and B. dothidea.

Diseases caused by Alternaria alternata and Botryosphaeria dothidea diminish pear yield and quality, and restrict the pear agricultural industry. Lignification is a conserved mechanism for plant resistance against pathogen invasion. The regulatory mechanisms underlying defence-induced lignification in pear in response to fungal pathogen infection remain unknown. In this study, analysis of lignification level and lignin content in pear revealed that A. alternata and B. dothidea induced lignification, and transcriptomics showed that lignin biosynthesis was affected. To explore whether laccases (LACs) mediated by miR397 regulate lignification in pear, we investigated the role of PcmiR397 in repressing the expression of PcLACs using 5'-RNA ligase-mediated-RACE and co-transformation in tobacco. Opposite expression patterns for PcmiR397 and PcLAC target genes were observed in pear in response to pathogens. Transient transformation in pear demonstrated that silencing PcmiR397 and overexpressing a single PcLAC enhanced resistance to pathogens via lignin synthesis. To further reveal the mechanism underpinning the PcMIR397 response of pear to pathogens, the PcMIR397 promoter was analysed, and pMIR397-1039 was found to be inhibited by pathogen infection. The transcription factor PcMYB44 was up-regulated, and it bound to the PcMIR397 promoter and inhibited transcription following pathogen infection. The results demonstrate the role of PcmiR397-PcLACs in broad-spectrum resistance to fungal disease, and the potential role of PcMYB44 involved in the miR397-PcLAC module in regulating defence-induced lignification. The findings provide valuable candidate gene resources and guidance for molecular breeding to improve resistance to fungal disease in pear.

Diagnosis of fruit tree viruses has been challenging for a long time as viral titer is often low and unevenly distributed among different tissues and branches of fruit trees. It is necessary to develop effective and reliable detection systems to identify viral pathogens in fruit trees. In this chapter, I describe RT-PCR and its derivatives tube capture-based reverse-transcription PCR (TC-RT-PCR) and multiplex RT-PCR assays for detection and identification of latent viruses in apple and pear trees. Classical RT-PCR is composed of two steps including transcription of viral RNA using extracted total RNA and PCR amplification of viral cDNA. TC-RT-PCR includes a TC step to capture particles and nucleic acid mixtures from crude plant tissue extracts as template directly for the first single-strand DNA (cDNA) synthesis, followed by PCR to amplify the viral cDNA fragment for viral identification. The cDNA derived from total RNAs can also be used for a one-step multiplex PCR to simultaneously detect several viruses in a given sample. As perennial fruit trees are usually coinfected by several viruses in orchards, multiplex RT-PCR can save time and lower labor and material costs for viral detection. These nucleic acid-based methods are sensitive and may be adapted for detection and identification of diverse viruses from different tissue materials of fruit trees.

Pear chlorotic leaf spot associated virus (PCLSaV) belongs to the genus Emaravirus and possesses a genome composed of five negative-sense single-stranded RNA (-ssRNA) segments. This study developed a SYBR green-based reverse transcription quantitative PCR (RT-qPCR) assay for the detection of PCLSaV infecting pear trees. A set of two primers q5-F2/q5-R2 designed based on the viral RNA5 sequences showed high specificity and feasibility for PCLSaV detection. The standard curve was established. RT-qPCR assays showed that PCLSaV content was greatly higher in diseased branch and symptomatic leaf samples than that in un-diseased branch and asymptomatic leaf samples. The RT-qPCR was reliability in the detection of the virus in field and in-vitro cultured pear samples. This technique would be useful for the supervision of the viral disease and the certification of pear planting materials.

Alternaria fungus can cause notable diseases in cereals, ornamental plants, vegetables, and fruits around the world. To date, an increasing number of mycoviruses have been accurately and successfully identified in this fungus. In this study, we discovered mycoviruses from 78 strains in 6 species of the genus Alternaria, which were collected from 10 pear production areas using high-throughput sequencing technology. Using the total RNA-seq, we detected the RNA-dependent RNA polymerase of 19 potential viruses and the coat protein of two potential viruses. We successfully confirmed these viruses using reverse transcription polymerase chain reaction with RNA as the template. We identified 12 mycoviruses that were positive-sense single-stranded RNA (+ssRNA) viruses, 5 double-strand RNA (dsRNA) viruses, and 4 negative single-stranded RNA (−ssRNA) viruses. In these viruses, five +ssRNA and four −ssRNA viruses were novel mycoviruses classified into diverse the families Botourmiaviridae, Deltaflexivirus, Mymonaviridea, and Discoviridae. We identified a novel −ssRNA mycovirus isolated from an A. tenuissima strain HB-15 as Alternaria tenuissima negative-stranded RNA virus 2 (AtNSRV2). Additionally, we characterized a novel +ssRNA mycovirus isolated from an A. tenuissima strain SC-8 as Alternaria tenuissima deltaflexivirus 1 (AtDFV1). According to phylogenetic and sequence analyses, we determined that AtNSRV2 was related to the viruses of the genus Sclerotimonavirus in the family Mymonaviridae. We also found that AtDFV1 was related to the virus family Deltaflexivirus. This study is the first to use total RNA sequencing to characterize viruses in Alternaria spp. These results expand the number of Alternaria viruses and demonstrate the diversity of these mycoviruses.

[目的]鉴定明确我国砂梨主产区白纹羽病的病原菌种类,为该病的有效防控提供理论依据.[方法]在我国砂梨主产区调查白纹羽病的发生危害特点并采集病根样品,通过组织分离法和单菌丝纯化获得分离株,对获得的菌株采用形态学和分子生物学相结合的方法进行种类鉴定和致病性验证.[结果]从湖北、福建、贵州、四川和山东等砂梨主产区采样到48份病根样品,共分离获得128个座坚壳(Rosellinia)菌株.所有菌株的菌落初期为白色,9 d后有黑色素沉淀;菌丝均无色透明,产生有分支,部分在隔膜处呈梨形膨大.36个代表菌株多基因(ITS、TUB、RPB2和LSU)系统发育分析的结果显示,所获得的座坚壳(Rosellinia)菌株均属褐座坚壳菌(R.necatrix),但不同来源菌株的基因序列有异,在系统进化树中聚集在3个不同的亚分支.从3个亚分支中选取其品种来源均为翠冠梨的3个菌株HB1-1-15、FJ1-2-2和GZ1-2-3,于小麦粒上培养14 d后拌土接种到杜梨生根试管苗后均可产生与田间相同的症状,但3个菌株的致病力存在显著差异.[结论]我国砂梨主产区白纹羽病的病原均为褐座坚壳菌(R.necatrix),不同来源菌株的基因序列之间存在明显差异.本研究是我国梨白纹羽病病原系统鉴定的首次报道.

A hypovirulent SZ-2-3y strain isolated from diseased Paris polyphylla was identified as Botrytis cinerea. Interestingly, SZ-2-3y was coinfected with a mitovirus, two botouliviruses, and a 3074 nt fusarivirus, designated Botrytis cinerea fusarivirus 8 (BcFV8); it shares an 87.2% sequence identity with the previously identified Botrytis cinerea fusarivirus 6 (BcFV6). The full-length 2945 nt genome sequence of the mitovirus, termed Botrytis cinerea mitovirus 10 (BcMV10), shares a 54% sequence identity with Fusarium boothii mitovirus 1 (FbMV1), and clusters with fungus mitoviruses, plant mitoviruses and plant mitochondria; hence BcMV10 is a new Mitoviridae member. The full-length 2759 nt and 2812 nt genome sequences of the other two botouliviruses, named Botrytis cinerea botoulivirus 18 and 19 (BcBoV18 and 19), share a 40% amino acid sequence identity with RNA-dependent RNA polymerase protein (RdRp), and these are new members of the Botoulivirus genus of Botourmiaviridae. Horizontal transmission analysis showed that BcBoV18, BcBoV19 and BcFV8 are not related to hypovirulence, suggesting that BcMV10 may induce hypovirulence. Intriguingly, a partial BcMV10 sequence was detected in cucumber plants inoculated with SZ-2-3y mycelium or pXT1/BcMV10 agrobacterium. In conclusion, we identified a hypovirulent SZ-2-3y fungal strain from P. polyphylla, coinfected with four novel mycoviruses that could serve as potential biocontrol agents. Our findings provide evidence of cross-kingdom mycoviral sequence transmission.