The CfKOB1 gene related to cell apoptosis is required for pathogenicity and involved in mycovirus-induced hypovirulence in Colletotrichum fructicola.

Colletotrichum fructicola is a globally significant phytopathogenic fungus. Mycovirus-induced hypovirulence has great potential for biological control and study of fungal pathogenic mechanisms. We previously reported that the mycovirus Colletotrichum alienum partitivirus 1 (CaPV1) is associated with the hypovirulence of C. fructicola, and the present study aimed to further investigate a host factor and its roles in mycovirus-induced hypovirulence. A gene named CfKOB1, which encodes putative protein homologous to the ß-subunit of voltage-gated potassium channels and aldo-keto reductase, is downregulated upon CaPV1 infection and significantly upregulated during the early infection phase of Nicotiana benthamiana by C. fructicola. Deleting the CfKOB1 gene resulted in diminished vegetative growth, decreased production of asexual spores, hindered appressorium formation, reduced virulence, and altered tolerance to abiotic stresses. Transcriptome analysis revealed that CfKOB1 regulates many metabolic pathways as well as the cell cycle and apoptosis. Furthermore, enhanced apoptosis was observed in the ΔCfKOB1 mutants. Viral RNA accumulation was significantly increased in the CfKOB1 deletion mutant. Additionally, our findings demonstrated that CaPV1 infection in the WT strain also induced cell apoptosis. Collectively, these results highlight the diverse biological roles of the CfKOB1 gene in the fungus C. fructicola, while it also participates in mycovirus-induced hypovirulence by regulating apoptosis.

A novel partitivirus conferring hypovirulence by affecting vesicle transport in the fungus Colletotrichum.

Colletotrichum spp. are economically important phytopathogenic fungi that cause anthracnose in a variety of plant species worldwide. Hypovirulence-associated mycoviruses provide new options for the biological control of plant fungal diseases. Here, we found a novel partitivirus from Colletotrichum alienum and named it Colletotrichum alienum partitivirus 1 (CaPV1). CaPV1 contained two dsRNA segments encoding an RNA-dependent RNA polymerase and a capsid protein and was classified under the genus Gammapartitivirus of the family Partitiviridae. CaPV1 significantly decreased host virulence, mycelial growth, appressorial development, and appressorium turgor but increased conidial production with abnormal morphology. In addition, CaPV1 could be successfully transfected into other Colletotrichum species, including C. fructicola, C. spaethianum, and C. gloeosporioides, and caused hypovirulence, indicating the broad application potential of this virus. CaPV1 caused significant transcriptional rewiring of the host fungus C. alienum. Notably, some genes related to vesicle transport in the CaPV1-infected strain were downregulated, consistent with the impaired endocytosis pathway in this fungus. When the Rab gene CaRab7, which is associated with endocytosis in vesicle transport, was knocked out, the virulence of the mutants was reduced. Overall, our findings demonstrated that CaPV1 has the potential to control anthracnose caused by Colletotrichum, and the mechanism by which Colletotrichum induces hypovirulence is caused by affecting vesicle transport.IMPORTANCEColletotrichum is a kind of economically important phytopathogenic fungi that cause anthracnose disease in a variety of plant species worldwide. We found a novel mycovirus of the Gammapartitivirus genus and Partitiviridae family from the phytopathogenic fungus Colletotrichum alienum and named it CaPV1. This study revealed that CaPV1 infection significantly decreased host virulence and fitness by affecting mycelial growth, appressorial development, and appressorium turgor. In addition, CaPV1 could also infect other Colletotrichum species, including C. fructicola, C. spaethianum, and C. gloeosporioides, by viral particle transfection and resulting in hypovirulence of these Colletotrichum species. Transcriptomic analysis showed that CaPV1 caused significant transcriptional rewiring of the host fungus C. alienum, especially the genes involved in vesicle transport. Moreover, endocytosis and gene knockout assays demonstrated that the mechanism underlying CaPV1-induced hypovirulence is, at least in part, caused by affecting the vesicle transport of the host fungus. This study provided insights into the mechanisms underlying the pathogenesis of Colletotrichum species and mycovirus-fungus interactions, linking the role of mycovirus and fungus vesicle transport systems in shaping fungal pathogenicity.

Characterization and biocontrol mechanism of Streptomyces olivoreticuli as a potential biocontrol agent against Rhizoctonia solani.

Rhizoctonia solani is a widespread and devastating plant pathogenic fungus that infects many important crops. This pathogen causes tobacco target spot, a disease that is widespread in many tobacco-growing countries and is destructive to tobacco. To identify antagonistic microorganisms with biocontrol potential against this disease, we isolated Streptomyces strains from forest inter-root soil and screened a promising biocontrol strain, ZZ-21. Based on in vitro antagonism assays, ZZ-21 showed a significant inhibitory effect on R. solani and various other phytopathogens. ZZ-21 was identified as Streptomyces olivoreticuli by its phenotypic, genetic, physiological and biochemical properties. Complete genome sequencing revealed that ZZ-21 harbored numerous antimicrobial biosynthesis gene clusters. ZZ-21 significantly reduced the lesion length in detached inoculated leaf assays and reduced the disease index under greenhouse and field conditions. Based on an in vitro antagonistic assay of ZZ-21 culture, the strain exhibited an antifungal activity against R. solani in a dose-dependent manner. The culture filtrate could impair membrane integrity, possibly through membrane lipid peroxidation. ZZ-21 could secrete multiple extracellular enzymes and siderophores. According to a series of antifungal assays, the extracellular metabolites of ZZ-21 contained antimicrobial bioactive compounds composed of proteins/peptides extracted using ammonium sulfate precipitation, which were stable under stress caused by high temperature and protease K. The EC50 value for ammonium sulfate precipitation was determined to be 21.11 µg/mL in this study. Moreover, the proteins/peptides also exhibited biocontrol ability and were observed to alter the plasma membrane integrity of R. solani which were evaluated by biocontrol efficacy assays on detached tobacco leaves and PI staining. Overall, strain ZZ-21 shows the potential to be developed into a biopesticide against tobacco target spot disease.

First Report of Stemphylium lycopersici Causing Leaf spot on Sedum plumbizincicola in Hunan Province of China.

Sedum plumbizincicola is a perennial succulent herb that can hyperaccumulate high concentrations of cadmium and zinc (Liu et al. 2017). In October 2021, a leaf spot disease occurred on S. plumbizincicola seedlings in a nursery in Changsha (28°13' N; 112°56'E), the Hunan Province of China. Almost 30% of the nearly 1 million seedlings were infected. Symptoms initially appeared as small brown spots on the leaf surface or edges, gradually enlarged, becoming oval, and bearing chlorotic lesions with dark brown borders. Eventually, the center of the lesions became sunken and then fell off. Eight symptomatic plant samples were collected by five-point sampling method (Zheng et al. 2018). Small pieces of 5×5 mm were excised from the lesion margins, sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 40 s, rinsed with sterile distilled water three times, and then cultured on potato dextrose agar (PDA) at 26 °C for 5 days in the dark. Fungal colonies showing similar morphology were observed from all the isolated samples and, in total, eight fungal strains were obtained. On PDA, fungal colonies were initially white, and later become light gray. After cultured on V8 juice agar (V8A, each litre of medium contains 200 mL of V8 juice, 3 g of CaCO3 and 15 g of agarose) for 14 days (Hyowon et al. 2016), conidia of a representative isolate SY-1 were produced, which were oblong, muriform, with blunt ends and conical apex, pale to light brown, and constricted at the 1 to 3 major transverse septa, 38.34-46.68 µm×11.67-18.34 µm (n=50). These morphological characteristics were consistent with that of Stemphylium lycopersici (Nasehi et al. 2016). The internal transcribed spacer (ITS) region of rDNA and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of representative isolates SY-1 to SY-3 were amplified and sequenced using the primer pairs ITS4/ITS5 and gpd1/gpd2 as described previously (Woudenberg et al. 2017). BLASTn analysis showed that ITS sequences of isolates SY-1, SY-2 and SY-3 (accession nos. OP317641, OQ852042 and OQ852043) had more than 99% identity with Stemphylium sp, while GAPDH sequences (OP331223, OQ858620 and OQ858621) had 100% identity with S. lycopersici KR911813 (Sun et al. 2016). A concatenated ITS-GAPDH phylogenetic tree grouped our isolates within the S. lycopersici clade. For the pathogenicity test, one-month-old potted S. plumbizincicola seedlings were inoculated with conidia suspension (105 conidia/ml), which was induced on V8A. Four sites of each leaf of the potted S. plumbizincicola plants were dropped with a conidia suspension of strain SY-1, with 10 µL per site. Leaves treated with sterile water were served as controls. All of the inoculated seedlings were placed in a growth chamber at 26°C with a photoperiod of 12 h. The pathogenicity tests were repeated twice, with each had three replicative plants. After 7 days, all the inoculated leaves developed brown spots resembling those observed in the nursery, whereas the control plants remained symptomless. Stemphylium lycopersici was specifically re-isolated and identified by morphological and molecular methods (accession nos. OQ852045 for ITS and OQ858622 for GAPDH, respectively), thus fulfilling Koch's postulates. To our knowledge, this is the first report of S. lycopersici causing leaf spot on S. plumbizincicola in China. Since S. plumbizincicola played an important role and widely planted for heavy metal pollution treatment (Jiang et al. 2010), and this disease might seriously influence the S. plumbizincicola seedling breeding, identification of the pathogen might provide a foundation for the diagnosis and control of the disease.

First Report of Colletotrichum siamense Causing Anthracnose on Dioscorea alata in China.

Dioscorea alata is an annual or perennial dicotyledonous plant which is a vegetatively propagated tuberous food crop (Mondo et al. 2021). In 2021, symptoms of leaf anthracnose occurred on D. alata plants at a plantation in Changsha, the Hunan Province of China (28°18' N; 113°08'E). Symptoms initially showed as small, brown water-soaked spots on the leaf surface or margins, and enlarged to irregular, dark brown or black, necrotic lesions, with a lighter center and darker edge. At latter, lesions extended to most of the leaf surface causing leaf scorch or wilting. Almost 40% of the plants surveyed were infected. Symptomatic leaf samples were collected, and small pieces were taken at their disease-healthy junction, sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 40 s, rinsed three times with sterile distilled water, and then placed on potato dextrose agar (PDA) for incubation at 26 °C for 5 days in the dark. Fungal colonies with similar morphology were observed and, in total, 10 isolates were obtained from 10 plants. On PDA, colonies were initially white with fluffy hyphae, and later became light to dark gray, showing faint concentric rings. Conidia were hyaline, aseptate, cylindrical, and rounded at both ends, measuring 11.36 to 17.67 × 3.45 to 5.9 µm (n = 50). Appressoria were dark brown, ovate, globose, measuring 6.37 to 7.55 × 10.11 to 12.3 µm. These morphological characteristics were typical of Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, the internal transcribed spacer (ITS) region of rDNA, and partial sequences of actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes of a representative isolate Cs-8-5-1 were amplified and sequenced using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF/GDR as described previously (Weir et al. 2012). These sequences were deposited in GenBank (accession nos. OM439575 for ITS, OM459820 for ACT, OM459821 for CHS-1, and OM459822 for GAPDH). BLASTn analysis showed 99.59 to 100 % identity to the corresponding sequences of C. siamense strains. A Maximum likelihood phylogenetic tree based on the concatenated ITS, ACT, CHS-1 and GAPDH sequences was generated by MEGA 6. It revealed that the Cs-8-5-1 was clustered with the C. siamense strain CBS 132456 with 98% bootstrap support. For pathogenicity test, conidia suspension (105 spores/ml) was prepared by harvesting conidia from 7-day-old cultures growing on PDA, and 10 uL was dropped onto leaves of potted D. alata plants (8 droplets per leaf). Leaves treated with sterile water were served as controls. All the inoculated plants were placed in humid chambers (with 90% humidity) at 26°C with a photoperiod of 12 h. The pathogenicity tests were performed twice, with each had three replicated plants. Seven days after inoculation, the inoculated leaves showed symptoms of brown necrosis resembling that observed in fields, however, the control leaves remained symptomless. The fungus was specifically re-isolated and identified by morphological and molecular methods, thus fulfilling Koch's postulates. To our knowledge, this is the first report of C. siamense causing anthracnose on D. alata in China. Since this disease might seriously affect the photosynthesis of the plants, which can influence the yield, prevention and management strategies should be adopted to control this new disease. Identification of this pathogen will provide a foundation for the diagnosis and control of this disease.

Novel and diverse mycoviruses co-infecting a single strain of the phytopathogenic fungus Alternaria dianthicola.

Alternaria dianthicola is a pathogenic fungus that causes serious leaf or flower blight on some medicinal plants worldwide. In this study, multiple dsRNA bands in the range of 1.2-10 kbp were found in a Alternaria dianthus strain HNSZ-1, and eleven full-length cDNA sequences of these dsRNA were obtained by high-throughput sequencing, RT-PCR detection and conventional Sanger sequencing. Homology search and phylogenetic analyses indicated that the strain HNSZ-1 was infected by at least nine mycoviruses. Among the nine, five viruses were confirmed to represent novel viruses in the families Hypoviridae, Totiviridae, Mymonaviridae and a provisional family Ambiguiviridae. Virus elimination and horizontal transmission indicated that the (-) ssRNA virus, AdNSRV1, might be associated with the slow growth and irregular colony phenotype of the host fungus. As far as we know, this is the first report for virome characterization of A. dianthus, which might provide important insights for screening of mycovirus for biological control and for studying of the interactions between viruses or viruses and their host.

Identification and Characterization of Colletotrichum fructicola and Colletotrichum siamense Causing Anthracnose on Luffa Sponge Gourd in China.

Luffa sponge gourd (Luffa cylindrica) is an important cucurbitaceous vegetable and is known as the source of loofah. From 2020 to 2021, a leaf disease occurred on luffa leaves in the Hunan Province of China. Symptoms were displayed as oval to irregular chlorotic lesions surrounded by yellow halos. The pathogens were isolated from the affected leaves. According to morphological characterization and molecular identification using multi-locus phylogenetic analysis of the internal transcribed spacer (ITS), actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB2), and partial mating type (Mat1-2) gene (ApMAT) regions, the pathogens were identified as two Colletotrichum species: Colletotrichum fructicola and C. siamense. Koch's postulates were identified by a pathogenicity test and re-confirmation. To the best of our knowledge, C. fructicola and C. siamense are two new species associated with luffa sponge gourd anthracnose.

First Report of Southern Blight on Aloe vera Caused by Athelia rolfsii in China.

Aloe vera (L.) Burm f., which belongs to the family Aloaceae, is a perennial succulent plant and cultivated for its medicinal, cosmetic, vegetable and ornamental uses. In summer of 2021, about 15% (60 infected among 400 surveyed plants) of A. vera (A. barbadensis) plants in two gardens in Lishui, Zhejiang Province of China showed symptoms of southern blight disease. Symptomatic plants primarily exhibited slightly sunken water-soaked, dark brown lesions on taproot and basal part of the stems. As the disease progressed, leaves in the basal part of stems and subsequently the whole plant rotted and withered, with white mycelial mats occurring on infected stems and leaves. Numerous brown, spherical sclerotia were observed on the colonized tissues and soil surfaces around the infected plants. Mycelial fragments and sclerotia from symptomatic leaves were plated directly to potato dextrose agar (PDA) amended with 100 µg/ml streptomycin and incubated at 26°C in the dark. By hyphal-tip method, a total of five pure isolates were obtained from five diseased leaf samples. When cultured on PDA at 26°C for three days, colonies showed white and thick aerial mycelium, with a radial growth rate of 23.7 mm/day. Typical clamp connection structures were observed microscopically after three days and numerous globoid, rapeseed shape sclerotia, measuring 1 to 2 mm in diameter (n=50) formed after six days. These sclerotia were initially white and gradually turned dark brown with age. On the basis of morphological characteristics, the fungal isolates were identified as Athelia rolfsii (Curzi) C.C. Tu & Kimbr (anamorph Sclerotium rolfsii Sacc) (Mordue 1974). The internal transcribed spacer (ITS) and translation elongation factor 1-α gene (TEF1) regions of a representative isolate LHBJ2-4 were amplified and sequenced using the primers ITS4/ITS5 (White et al. 1990) and EF1/EF2, respectively (accession no. MZ956758 and OL365370). BLASTn search showed that the amplified ITS and TEF1 sequences had 99.71% (680/682 bp) and 99.80% (498/499 bp) identity with the A. rolfsii isolates CBS 115.22 (MH854711.1) and Sr_286 (JF267815), respectively. Neighbor-joining phylogenetic tree based on the ITS sequences revealed that LHBJ2-4 clustered with A. rolfsii isolates. For pathogenicity test, three potted A. vera plants (~30 cm tall) were inoculated by placing a 0.5 cm mycelial plug of isolate LHBJ2-4 (three-day old) at the base of each A. vera plant. Three A. vera plants inoculated with sterile PDA plugs served as controls. All the inoculated plants were placed in a growth chamber at 27°C under a 12/12 h light/dark cycle. The inoculation assays were carried out twice. After 5 to 7 days, stem bases of the inoculated plants showed brown lesions that were similar to those observed in the field. However, control plants remained symptomless. Athelia rolfsii was re-isolated from all the inoculated plants and identified using morphological and molecular method described above, thus confirming Koch's postulates. Although A. rolfsii has been reported to cause disease on A. vera in India (Dubey and Pandey 2009), to the best of our knowledge, this is the first report of A. rolfsii causing southern blight on A. vera in China. Because A. rolfsii has a wide host range and is difficult to control (Punja 1985), occurrence of southern blight in China might be a serious threat for A. vera production and appropriate management strategies should be developed to control this disease.

Identification and First Report of Fusarium andiyazi Causing Sheath Rot of Zizania latifolia in China.

Zizania latifolia is a perennial plant native to East Asia. The swollen culm of Z. latifolia is a popular vegetable and traditional herbal medicine consumed in China and some other Asian countries. From 2019 to 2021, a sheath rot disease was found in Zhejiang Province of China. Symptoms mainly occurred in the leaf sheath showing as brown necrotic lesions surrounded by yellow halos. The pathogen fungal isolates were isolated from the affected sheaths. Ten representative isolates were selected for morphological and molecular identification by phylogenetic analyses of the translation elongation factor 1-α (TEF1) and the RNA polymerase II subunit beta (RPB2) gene regions. Based on the combined datasets, the fungal isolates were identified as Fusarium andiyazi. Koch's postulates were confirmed by pathogenicity test, re-isolation and re-identification of the fungal isolates. To the best of our knowledge, this is the first report of sheath rot caused by F. andiyazi in Z. latifolia in China.

First Report of Colletotrichum cliviicola Causing Leaf Spot on Tobacco (Nicotiana tabacum) in Hunan Province of China.

Tobacco (Nicotiana tabacum L.) is an annual, leafy, herb of the genus Nicotiana in the family Solanaceae. It is an important commercial crop in China. In 2020, a leaf spot disease was observed on tobacco leaves in commercial fields in the Hunan Province of China. Symptoms appeared as water-soaked, yellow-green spots, then turned dark brown, and coalesced into larger necrotic lesions, often leading to leaf wilt. Approximately 20% of the plants in a 50-ha area were infected, exhibiting symptomatic spots on 60% of these leaves. Symptomatic leaf samples were collected and cut into small pieces, sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 40s, rinsed with sterile distilled water for three times, plated on potato dextrose agar (PDA) and incubated at 26°C in the dark. Isolates with similar morphology were developed from ten samples. Fungal isolates produced densely, white to dark green, aerial mycelium. Conidia were straight, hyaline, aseptate, cylindrical, contained oil globules, and 15 to 25 µm × 3.0 to 4.0 µm (n=50). Appressoria were dark brown, irregularly shaped, 5.5 to 10.0 µm × 4.5 to 6.5 µm (n=50). These morphological characteristics were typical of Colletotrichum cliviicola (Yang et al. 2009). For molecular identification, the internal transcribed spacer (ITS) region of rDNA, actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and chitin synthase (CHS-1) genes of a representative isolate CS16-2 were amplified and sequenced using the primer pairs as described previously (Weir et al. 2012). These sequences were deposited in GenBank (GenBank Accession Nos. MW649137 for ITS, MW656181 for ACT, MW656182 for GAPDH and MW656183 for CHS-1). BLAST analysis showed that they had 99.46% to 100% identity to the corresponding sequences of C. cliviicola strains. A concatenated phylogenetic tree was generated, using the ACT, GAPDH and CHS-1 sequences of the isolate CS16-2 and other closely matching Colletotrichum species obtained from the GenBank. We found that the CS16-2 was grouped with the C. cliviicola clade with 97% bootstrap support, including the C. cliviicola strain AH1B6 (Wang et al. 2016). Pathogenicity was tested spraying 2-month-old potted tobacco plants until runoff with a conidial suspension (105 spores/ml). Leaves were mock inoculated with sterilized water. The pathogenicity tests were performed twice, with three replicate plants each. Plants were kept in humid chambers at 26°C with a 12-h photoperiod. Five days post-inoculation, the inoculated plants developed symptoms of consisting of the yellow-brown necrotic lesion resembling the symptoms that were observed in fields, while the control plants remained symptomless. C. cliviicola was re-isolated and identified by morphological and molecular methods as described above. Currently, C. cliviicola has been reported to be the causal agent of anthracnose in some plants, such as soybean (Zhou et al. 2017) and Zamioculcas zamiifolia (Barbieri et al. 2017). However, to our knowledge, this is the first report of C. cliviicola causing leaf spot on tobacco in China and even in the word. Given that the may greatly affect the yield and quality of tobacco production, growers should be prepared to manage this new disease. This work might provide further insight for disease diagnosis on tobacco as some other Colletotrichum species, such as C. fructicola (Wang et al. 2016) and C. karsti (Zhao et al. 2020), have also been responsible for anthracnose.

A Novel Partitivirus That Confer Hypovirulence to the Plant Pathogenic Fungus Colletotrichum liriopes.

Here, we report a novel double-stranded RNA virus designated Colletotrichum liriopes partitivirus 1 (ClPV1) from the plant pathogenic fungus C. liriopes. ClPV1 genome has two double stranded RNAs (dsRNAs), named as dsRNA 1 and dsRNA 2, which in the lengths of 1,807 and 1,706 bp, respectively. The dsRNA 1 and dsRNA 2 encoded proteins showing significant amino acid (aa) sequence identity to the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) of partitiviruses, respectively. Phylogenetic analysis using the aa sequences of RdRp and CP indicated that ClPV1 was grouped to members of the putative Epsilonpartitivirus genus in the Partitiviridae family. Spherical viral particles in approximately 35 nm in diameter and packaging the ClPV1 genome were isolated. Virus elimination and virus transfection with purified viral particles, and biological comparison revealed that ClPV1 could reduce the virulence and conidia production of C. liriopes. To the best of our knowledge, this is the first report of mycovirus in C. liriopes fungus.

First Report of Leaf Spot Caused by Botrytis cinerea on Cardamine hupingshanensis in China.

Cardamine hupingshanensis is a selenium (Se) and cadmium (Cd) hyperaccumulator plant distributed in wetlands along the Wuling Mountains of China (Zhou et al. 2018). In March of 2020, a disease with symptoms similar to gray mold was observed on leaves of C. hupingshanensis in a nursery located in Changsha, Hunan Province, China. Almost 40% of the C. hupingshanensis (200 plants) were infected. Initially, small spots were scattered across the leaf surface or margin. As disease progressed, small spots enlarged to dark brown lesions, with green-gray, conidia containing mold layer under humid conditions. Small leaf pieces were cut from the lesion margins and were sterilized with 70% ethanol for 10 s, 2% NaOCl for 2 min, rinsed with sterilized distilled water for three times, and then placed on potato dextrose agar (PDA) medium at 22°C in the dark. Seven similar colonies were consistently isolated from seven samples and further purified by single-spore isolation. Strains cultured on PDA were initially white, forming gray-white aerial mycelia, then turned gray and produced sclerotia after incubation for 2 weeks, which were brown to blackish, irregular, 0.8 to 3.0 × 1.2 to 3.5 mm (n=50). Conidia were unicellular, globose or oval, colourless, 7.5 to 12.0 × 5.5 to 8.3 µm (n=50). Conidiophores arose singly or in group, straight or flexuous, septate, brownish to light brown, with enlarged basal cells, 12.5 to 22.1 × 120.7 to 310.3 µm. Based on their morphological characteristics in culture, the isolates were putatively identified as Botrytis cinerea (Ellis 1971). Genomic DNA of four representative isolates, HNSMJ-1 to HNSMJ-4, were extracted by CTAB method. The internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate dehydrogenase gene (G3PDH), heat-shock protein 60 gene (HSP60), ATP-dependent RNA helicaseDBP7 gene (MS547) and DNA-dependent RNA polymerase subunit II gene (RPB2) were amplified and sequenced using the primers described previously (Aktaruzzaman et al. 2018) (MW820311, MW831620, MW831628, MW831623 and MW831629 for HNSMJ-1; MW314722, MW316616, MW316617, MW316618 and MW316619 for HNSMJ-2; MW820519, MW831621, MW831627, MW831624 and MW831631 for HNSMJ-3; MW820601, MW831622, MW831626, MW831625 and MW831630 for HNSMJ-4). BLAST searches showed 99.43 to 99.90% identity to the corresponding sequences of B. cinerea strains, such as HJ-5 (MF426032.1, MN448500.1, MK791187.1, MH727700.1 and KX867998.1). A combined phylogenetic tree using the ITS, G3PDH, HSP60 and RPB2 sequences was constructed by neighbor-joining method in MEGA 6. It revealed that HNSMJ-1 to HNSMJ-4 clustered in the B. cinerea clade. Pathogenicity tests were performed on healthy pot-grown C. hupingshanensis plants. Leaves were surface-sterilized and sprayed with conidial suspension (106 conidia/ mL), with sterile water served as controls. All plants were kept in growth chamber with 85% humidity at 25℃ following a 16 h day-8 h night cycle. The experiment was repeated twice, with each three replications. After 4 to 7 days, symptoms similar to those observed in the field developed on the inoculated leaves, whereas controls remained healthy. The pathogen was reisolated from symptomatic tissues and identified using molecular methods, confirming Koch's postulates. B. cinerea has already been reported from China on C. lyrate (Zhang 2006), a different species of C. hupingshanensis. To the best of our knowledge, this is the first report of B. cinerea causing gray mold on C. hupingshanensis in China and worldwide. Based on the widespread damage in the nursery, appropriate control strategies should be adopted. This study provides a basis for studying the epidemic and management of the disease.

First report of Epicoccum sorghinum causing leaf spot on Hemerocallis citrina in China.

Hemerocallis citrina Baroni, also called yellow flower vegetable (huang hua cai in Chinese), is belonging to the family Xanthorrhoeaceae and is widely planted in China, the Korea Peninsula and Japan for ornamental purposes and vegetable value. In addition, they could also be used as a traditional Chinese medicinal and modern medicinal plant (Du et al. 2014). In August 2019, a leaf spot disease was observed on H. citrina plants in Zhejiang Province of China, with approximately 85% incidence in almost 700 ha. Symptoms were firstly displayed as small, water-soaked, pale chlorotic spots, with yellow halos enlarged into large fusiform spots with brown edge and gray centers. Later, infected leaves were badly damaged and became wilted. Small pieces of infected tissue were excised from the margin of necrotic lesions, surface disinfected with 70% ethanol for 8s, 0.1% HgCl2 for 1 min, rinsed with sterile distilled water for three times, and incubated on potato dextrose agar (PDA, amended with 100 mg/L streptomycin sulfate) at 26°C in the dark. Fungal colonies with similar cultural morphology were consistently obtained from repeated isolations. When cultured on PDA, colonies were villose, regular, grayish-green, and turned gray-brown, with the reverse side became reddish-brown. Chlamydospores were gray, unicellular or multicellular, nearly spherical, 11 to 27 × 10 to 23 µm. Pycnidia and conidia were produced on PDA when the fungal colonies were exposed to ultraviolet light for 12 h with a distance of 40 cm to the late source. Pycnidia were brown, mostly spheroid, and measured 90 to 138 × 120 to 210 µm. Conidia were hyaline, ellipsoidal, unicellular, aseptate, 4.3 to 5.5 × 1.8 to 2.4 µm. These morphological characteristics agreed with the descriptions of Epicoccum sorghinum (Zhou et al. 2018). The DNA of a representative strain HHC6-2 was extracted using CTAB method and the rDNA internal transcribed spacer (ITS), actin (ACT) and ß-tubulin (TUB) genes were amplified and sequenced, using the primers ITS4/ITS5 (White et al. 1990), ACT512F/ACT783R (Carbone and Kohn 1999) and Bt-1/Bt-2 (Glass and Donaldson 1995), respectively. BLASTn searches of the resulting ITS, ACT and TUB sequences (accession nos. MW073403, MW080522, MW080521) revealed 98.58 to 100% identity to the E. sorghinum sequences (MT125854, MN956831 and MF987525). The pathogenicity test was carried out by inoculation of potted H. citrina plants using conidial suspensions. H. citrina seedlings were planted in pots with sterilized soil. Before inoculation, leaves were surface-disinfected with 70% ethanol and sterile distilled water. Leaves were inoculated by placing small droplets of conidial suspensions (105 conidia/ml) on one side of the midvein, and 3 to 5 drops were used per leaf. Sterile water was used as control. All the inoculated plants were placed in humid chambers at 25°C for 48h, and then maintained in a greenhouse at 25°C with a 16 h day-8 h night cycle. The pathogenicity assays were performed twice with three replications. Four days after inoculation, yellow to brown spots resembling those observed in the fields developed on the inoculated leaves. However, no symptoms were observed on the controls. E. sorghinum was re-isolated and identified based on morphological and molecular techniques as described above. To our knowledge, this is the first report of E. sorghinum causing leaf spot on H. citrina. It seems to be a threat for H. citrina planting in China and should be considered in order to reduce losses caused by this disease. This study might provide the basis for diagnosis and control of the disease.

First Report of Diaporthe hongkongensis Causing Fruit rot on Peach (Prunus persica) in China.

Peach [Prunus persica (L.) Batsch] is an important deciduous fruit tree in the family Rosaceae and is a widely grown fruit in China (Verde et al., 2013). In July and August 2018, a fruit rot disease was observed in a few peach orchards in Zhuzhou city, the Hunan Province of China. Approximately 30% of the fruit in more than 400 trees was affected. Symptoms displayed were brown necrotic spots that expanded, coalesced, and lead to fruit being rotten. Symptomatic tissues excised from the margins of lesions were surface sterilized in 70% ethanol for 10 s, 0.1% HgCl2 for 2 min, rinsed with sterile distilled water three times, and incubated on potato dextrose agar (PDA) at 26°C in the dark. Fungal colonies with similar morphology developed, and eight fungal colonies were isolated for further identification. Colonies grown on PDA were grayish-white with white aerial mycelium. After an incubation period of approximately 3 weeks, pycnidia developed and produced α-conidia and ß-conidia. The α-conidia were one-celled, hyaline, fusiform, and ranged in size from 6.0 to 8.4 × 2.1 to 3.1 µm, whereas the ß-conidia were filiform, hamate, and 15.0 to 27.0 × 0.8 to 1.6 µm. For molecular identification, total genomic DNA was extracted from the mycelium of a representative isolate HT-1 and the internal transcribed spacer region (ITS), ß-tubulin gene (TUB), translation elongation factor 1-α gene (TEF1), calmodulin (CAL), and histone H3 gene (HIS) were amplified and sequenced (Meng et al. 2018). The ITS, TUB, TEF1, CAL and HIS sequences (GenBank accession nos. MT740484, MT749776, MT749778, MT749777, and MT749779, respectively) were obtained and in analysis by BLAST against sequences in NCBI GenBank, showed 99.37 to 100% identity with D. hongkongensis or D. lithocarpus (the synonym of D. hongkongensis) (Gao et al., 2016) (GenBank accession nos. MG832540.1 for ITS, LT601561.1 for TUB, KJ490551.1 for HIS, KY433566.1 for TEF1, and MK442962.1 for CAL). Pathogenicity tests were performed on peach fruits by inoculation of mycelial plugs and conidial suspensions. In one set, 0.5 mm diameter mycelial discs, which were obtained from an actively growing representative isolate of the fungus on PDA, were placed individually on the surface of each fruit. Sterile agar plugs were used as controls. In another set, each of the fruits was inoculated by application of 1 ml conidial suspension (105 conidia/ml) by a spray bottle. Control assays were carried out with sterile distilled water. All treatments were maintained in humid chambers at 26°C with a 12-h photoperiod. The inoculation tests were conducted twice, with each one having three fruits as replications. Six days post-inoculation, symptoms of fruit rot were observed on inoculated fruits, whereas no symptoms developed on fruits treated with agar plugs and sterile water. The fungus was re-isolated and identified to be D. hongkongensis by morphological and molecular methods, thus fulfilling Koch's Postulates. This fungus has been reported to cause fruit rot on kiwifruit (Li et al. 2016) and is also known to cause peach tree dieback in China (Dissanayake et al. 2017). However, to our knowledge, this is the first report of D. hongkongensis causing peach fruit rot disease in China. The identification of the pathogen will provide important information for growers to manage this disease.

First Report of Fusarium commune causing Stem Rot of Tobacco (Nicotiana tabacum) in Hunan Province, China.

Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves. It is one of the most important cash crops in China. In April of 2020, tobacco stems in commercial tobacco fields developed a brown to dark brown rot, in the Hunan Province of China. Almost 20% of the plants were infected. Symptoms appeared as round water-soaked spots, then turned dark black and developed into brown necrotic lesions leading to the stem becoming girdled and rotted. Diseased stem tissue was cut and sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 2 min, rinsed with sterile distilled water three times, and then plated on potato dextrose agar (PDA) and incubated at 26°C in the dark. Six isolates with similar morphology were obtained. Colonies cultured on PDA have morphological characteristics of Fusarium spp. producing white to orange-white, densely aerial mycelium with magenta to dark violet pigmentation. Macroconidia were produced on carnation leaf agar plates (Xi et al. 2019), which were slightly curved, with apical and basal cells curved, and usually contained three or five septa, 25.50 to 41.50×3.55 to 5.80 µm (n=50). Microconidia were cylindrical, ovate-oblong, straight to slightly curved, aseptate and 5.80 to 13.75 × 3.10 to 4.10 µm (n=50). For molecular identification, the translation elongation factor 1-alpha (EF1-α), the largest subunit of RNA polymerase II gene sequences (RPB2) and the mitochondrial small subunit rDNA (mtSSU) of a representative isolate CZ3-5-6 were amplified using the primer pairs ef1/ef2 (O'Donnell et al. 1998), 5F2/7Cr (O'Donnell et al. 2010) and NMS1/ NMS2 (Li et al. 1994). The obtained EF1-α, RPB2 and mtSSU sequences (GenBank accession nos. MT708482, MT708483 and MW260121, respectively) were 99.70 %, 100% and 100% identical to strains of F. commune (HM057338.1 for EF1-α, KU171700.1 for RPB2 and MG846025 for mtSSU). Moreover, Fusarium-ID database searches revealed that the EF1-α and RPB2 were 100% identical to F. commune strains (FD_01140_EF-1a and FD_02411_RPB2). Based on the morphological and molecular characteristics of the representative isolate, the fungal species was identified as F. commune. Pathogenicity testing of a representative isolate was performed by inoculating tobacco plants, which were grown for 2.5 months in a sterile pot with autoclaved soil. Each tobacco stem was injected with 20 µl of conidial suspension (105 spores/ml). Plants inoculated with sterilized water served as control. The pathogenicity tests were performed twice using three replicate plants, and all plants were kept in humid chambers (80 × 50 × 80 cm) at 26°C with a 12-h photoperiod. After 10 days, dark brown necrotic symptoms around the inoculated site, similar to those observed in natural field, were developed in all inoculated plants, whereas no symptoms were observed on the control plants. The pathogenic fungus was re-isolated from symptomatic tissue and identified as F. commune but was not recovered from the control plants. Fusarium commune has been reported to cause root rot or stalk and stem rot on some plants, such as sugarcane (Wang et al. 2018), Gentiana scabra (Guan et al. 2016) and maize (Xi et al. 2019). However, to our knowledge, this is the first report of F. commune causing stem rot on tobacco in China. Identification of F. commune as a stem rot causing pathogen might provide important insights for disease diagnosis on tobacco caused by different Fusarium species. Overall, this disease might bring a threat to tobacco production, and appropriate control measures should be adopted to reduce losses in tobacco fields.

First Report of Damping-off on Sedum plumbizincicola Caused by Rhizoctonia solani AG 2-1 in China.

Sedum plumbizincicola X.H. Guo et S.B. Zhou sp. nov. is a plant species of the family Crassulaceae that has the ability to hyperaccumulate cadmium and zinc in high concentrations (Liu et al. 2017). In April of 2018 and 2019, a disease of damping-off was observed on S. plumbizincicola seedlings in a nursery in Changsha (28°13'N; 112°56'E), the Hunan Province of China, in which nearly 1 million seedlings were planted. Approximately 40% of the surveyed plants were infected. The affected plants displayed water-soaking on the shoots and stems, and chlorosis on the leaves. As the disease spread upward, leaf stalks or the whole plants became wilted and collapsed. Five diseased stem and leaf samples were collected. Symptomatic tissues were excised and surface sterilized with 70% ethanol for 10 s, and 0.1% HgCl2 for 2 min, washed with sterile distilled water for three times, and then cultured on potato dextrose agar (PDA) at 26°C in darkness. Fungal colonies were similar in morphology: white, light gray to brown, with hyphae branched at nearly right angles, septa near the branching point and constrictions at the base of hyphal branches. After 10 days, white-gray to brown sclerotia were produced. The morphological characteristics were consistent with those of Rhizoctonia solani J.G. Kühn (Sneh et al. 1991). Genomic DNA of a representative isolate was extracted using the cetyltrimethylammonium bromide method. The internal transcribed spacer (ITS) region of rDNA was amplified and sequenced with the primer pairs ITS4/ITS5 (White et al. 1990). When analyzed by the BLASTn program, the ITS sequence (GenBank Accession No. MN961664) had 100% identity to the corresponding gene sequence of R. solani anastomosis group (AG) 2-1(Accession Nos: LC202869.1 and MH862641.1). In addition, primers Rhsp1/ITS4B and Rhsp2/ITS1F specific for R. solani, and AG21sp/ITS4B specific for R. solani AG 2-1 were also used (Salazar et al. 2000). Results revealed that our isolate was R. solani AG 2-1. Pathogenicity was confirmed via in vivo inoculation of one-month-old S. plumbizincicola seedlings in sterilized nursery soil with four representative isolates. For each pot, five 5-mm-diameter mycelial plugs from 7-days old colonies on PDA were placed in the soil near the base of the stems. Plants inoculated with agar plugs without mycelium served as controls. The inoculated plants were kept in a growth chamber at 25°C with a 12/12 h light/dark cycle. Pathogenicity tests were performed twice, with three replicative potted plants for each isolate in each test. Approximately 25 days after inoculation, the damping-off symptoms resembling those observed in the field were displayed on the inoculated plants, while no obvious symptoms were observed on the control plants. R. solani was re-isolated from all infected plants and molecularly characterized, thus confirming Koch's postulates. R. solani has been previously reported as the pathogen of damping-off disease in many plants, such as canola (Paulitz et al. 2006) and oat (Zhang et al. 2016). However, to the best of our knowledge, this is the first report of R. solani causing damping-off of S. plumbizincicola in China. S. plumbizincicola is widely planted for heavy metal pollution treatment in China. The occurrence of this disease could seriously affect the production of the seedlings, and management strategies should be developed.

First Report of Fusarium xylarioides Causing Root and Stem Rot on Aloe vera in China.

Aloe vera (L.) Burm f. is a perennial herb belonging to the family liliaceae. It is widely grown for medicinal, cosmetic and vegetable use. In 2018 and 2019, a root rot disease occurred on potted A. vera plants in a nursery in the Hunan Province of China. Symptoms of the disease include water soaking lesions, brown spots on taproot or basal part of the stem. The plants were easy to pull out when the taproot is rotten or necrotic. As the disease progressed upward, leaves in the basal part of stems became red-brown and gradually fell off. In severe cases, the whole plants became rotten and wilted. For isolation purposes, diseased tissues were excised from the lesion margins, surface disinfested with 70% ethanol for 10 s, 0.1% HgCl2 for 2 min, rinsed with sterile water thrice, and then placed on potato dextrose agar (PDA) and incubated at 26°C for 3 days in the dark. When cultured on PDA, fungal strains with similar morphology were consistently isolated and purified by single spore isolation. Colonies showed thick, pink aerial mycelium with a growth rate of 1.3 cm /day. The pigmentation was more intense in the colony center and became pale orange and white at the edge of colony. When cultured on SNA (Spezieller Nährstoffarmer agar), the fungus showed less pigmentation and thinner hyphae. Microconidia were abundantly produced, clavate and oval to kidney shaped, 7.1 to 15.2 µm × 2.5 to 5.1 µm, with 0 to 1 transverse septa. Macroconidia were sickle shaped, slender, slightly incurved in apical cell and foot-shaped in the basal cell, measured 27.9 to 53.2 µm × 2.5 to 3.5 µm, with 3 to 5 septa. These morphological characteristics were similar with those of Fusarium spp. (Booth 1971). For molecular identification, genomic DNA of the fungus was extracted by cetyl trimethyl ammonium bromide method. A portion of EF-1α (translation elongation factor 1-α) and RPB1 (the largest subunit of RNA polymerase) genes were amplified and directly sequenced using the EF-1/EF-2 and Fa/G2R primers (O'Donnell et al. 2010). The EF-1α and RPB1 were deposited in the GenBank with accession numbers MT755386 and MT755387. The EF-1α and RPB1 had 97.14% (ID FD_01334) and 99.62% identity (FD_03853), respectively, to F. xylarioides strains in the Fusarium-ID database (Geiser et al. 2004). In addition, the EF1-a showed 96.825% identity to the F. lateritium CBS 119871(AM295281) (a synonym of F. xylarioides), and the RPB1 showed 99.623% identity to the F. xylarioides NRRL 25486 (JX171517.1). Accordingly, the fungus was putatively identified to be F. xylarioides. For pathogenicity assay, A.vera seedlings were pot planted using sterilized nursery soil and inoculated with conidia suspension (1 × 105 conidia/ml), which were eluted from 7-day-old PDA cultures with sterilized water, according to the method described previously (Vakalounakis et al. 2015). The collar of each potted plant was poured with 20 ml of conidia suspensions. Plants mock inoculated with sterile water were used as control. All the inoculated plants were placed in a growth chamber at 25°C under 12/12 h light/dark cycle. The inoculation assays were carried out twice, with each one had three replicated plants. After 30 days, rot symptoms seen from the roots and basal part of stems were observed on the inoculated plants, but no visible symptoms were observed on control plants. The fungus was re-isolated from the inoculated plants and identified to be F. xylarioides by morphological and molecular characteristics, thus confirming Koch's postulates. As we know, many Fusarium species have been reported to cause root and stem rot disease in A.vera such as the F. oxysporum (Ji et al. 2007) and F. solani (Vakalounakis et al. 2015). However, to the best of our knowledge, this is the first report of F. xylarioides causing root and stem rot disease of A.vera in China. The identification of the pathogen fungus might provide a foundation for taking appropriate control strategies to this disease.

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.

Characterization of a Novel Ourmia-Like Mycovirus Infecting Magnaporthe oryzae and Implications for Viral Diversity and Evolution.

Here, the molecular characterization of a novel mycovirus that was isolated from a phytopathogenic fungus Magnaporthe oryzae and designed as Magnaporthe oryzae ourmia-like virus 4 (MOLV4) is reported. MOLV4 has a genome that is 2497 bp long and possesses a single open reading frame (ORF), which encodes the product RNA-dependent RNA polymerase (RdRp). Sequence similarities were found between the MOLV4 encoded RdRp and the counterparts of a few previously reported ourmia-like mycoviruses. Virus-curing and biological comparison indicate that the virus has no or mild effects on the morphology and mycelium growth rate of the host fungus. Phylogenetic analysis using the RdRp aa sequences was performed. The results show that MOLV4 is clustered with the ourmia-like mycoviruses, forming a clade closely related to ourmiaviruses but distinct from narnaviruses. In addition, database searches revealed that several MOLV4-related sequences are present in the transcriptome shotgun assembly (TSA) library, expressed sequence tag database (ESTdb), whole-genome shotgun (WGS) library, and genomic survey sequences (GSS) libraries of a few other species of eukaryote organisms. Our results show that MOLV4, together with other similar ourmia-like mycoviruses, might represent a virus clade that links the plant ourmiaviruses and fungal narnaviruses and has a wide range of hosts.

Diverse, Novel Mycoviruses From the Virome of a Hypovirulent Sclerotium rolfsii Strain.

Sclerotium rolfsii, which causes southern blight in a wide variety of crops, is a devastating plant pathogen worldwide. Mycoviruses that induce hypovirulence in phytopathogenic fungi are potential biological control resources against fungal plant diseases. However, in S. rolfsii, mycoviruses are rarely reported. In a previous study, we found a hypovirulent strain carrying a diverse pattern of dsRNAs. Here, we utilized the RNA_Seq technique to detect viral sequences. Deep sequencing, RT-PCR and Sanger sequencing validation analyses revealed that this strain harbors various new viral species that show affinity to the distinctly established and proposed families Benyviridae, Endornaviridae, Fusariviridae, Hypoviridae, and Fusagraviridae. Moreover, some viral sequences that could not be assigned to any of the existing families or orders were also identified and showed similarities to the Alphavirus, Ourmiavirus, phlegivirus-like and Curvularia thermal tolerance virus-like groups. In addition, we also conducted deep sequencing analysis of small RNAs in the virus-infecting fugal strain. The results indicated that the Dicer-mediated gene silencing mechanism was present in S. rolfsii. This is the first report of viral diversity in a single S. rolfsii fungal strain, and the results presented herein might provide insight into the taxonomy and evolution of mycoviruses and be useful for the exploration of mycoviruses as biocontrol agents.