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.