Chilli pepper (Capsicum annuum L.) is one of the most important crops in Yunnan Province, China. An orthotospovirus isolate 14YV855 was isolated from a diseased chilli pepper plant exhibiting yellow ringspots and necrosis on leaves in Shiping County, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province in 2014. The complete genome sequence of 14YV855 was determined. The small, medium, and large RNAs are 3,428, 4,781, and 8,917 nucleotides long, respectively. The complete nucleocapsid (N) protein of 14YV855 shares a high amino acid identity of 84.8 to 89.9% to that of Capsicum chlorosis virus (CaCV), Groundnut bud necrosis virus (GBNV), Watermelon bud necrosis virus (WBNV), and Watermelon silver mottle virus (WSMoV), which is slightly less than the 90% identity threshold for the demarcation of new Orthotospovirus sp. Phylogenetic analyses revealed that the N protein and RNA-dependent RNA polymerase of 14YV855 are the most related to WSMoV, while the NSs, NSm, and Gn/Gc proteins are similar to those of GBNV. As expected, 14YV855 is serologically related to CaCV, GBNV, WBNV, and WSMoV when the monoclonal antibody against the N protein of WSMoV was used; however, 14YV855 can be distinguished from other orthotospoviruses by reverse-transcription PCR using the specific primers. Our results indicate that 14YV855 is a new Orthotospovirus sp. belonging to the WSMoV serogroup and is provisionally named Chilli yellow ringspot virus.

Sanqi (Panax notoginseng (Burk.) F. H. Chen) is a traditional Chinese medicinal plant with a long planting cycle of 2-3 years that makes it vulnerable to root diseases caused by several pathogens, including Fusarium solani, Alternaria panax, Phytophthoracactorum, and Pseudomonas sp. In April 2019, root soft rot samples of Sanqi were collected from a plantation site in Songming, southwest of China. Typical symptoms included root softening and necrosis, yellow leaf, and stem wilting. Ten diseased roots samples were collected and sterilized with 0.1% HgCl2 for 1 min, 75% ethanol for 2min, and then rinsed thrice with sterile water. Sterilized roots were cut into small pieces of 5 × 5 mm and cultured on the nutrient agar (NA) medium for 48 h at 28°C. From the root cultures, a total of thirteen bacterial strains were obtained. Three strains, SM 2-5, SM 2-13, and SM 2-14 were selected for further study. These three strains were gram-negative, short rod-shaped (1～2×0.5～1μm), non-spore-forming and had polar tufted flagella as observed under a transmission electron microscope (TEM). Also, the strains were positive for oxidase, beta-galactosidase, arginine dihydrolase, and lysine decarboxylase while negative for amylase and urease tested by biochemical methods (Wang 2017). To further determine the pathogenic species, genomic DNA of these three strains was extracted using a Genomic DNA Kit (Tsing Ke, Beijing, China), to PCR amplify 16S rDNA using universal primers 27F/1492R (Wang et al. 2017). Also, S. maltophilia 23S rDNA specific primers SM1/SM4 (Whitby et al. 2000) were used for PCR amplification to confirm the species. 16S rDNA sequence analysis showed that SM 2-5 (GenBank Accession No. MW555227), SM 2-13 (GenBank Accession No. MW555228), and SM 2-14 (GenBank Accession No. MW555229) shared the highest identity (>99.9%) with the S. maltophilia strains (GenBank Accession No. MT323142, MH669295, MN826555). Furthermore, 23S rDNA sequence analysis of SM 2-5 (GenBank Accession No. MZ707732), SM 2-13 (GenBank Accession No. MZ645941) and SM 2-14 (GenBank Accession No. MZ707733) revealed their high identity (>99.8%) with the S. maltophilia species. 16S and 23S rDNA phylogenetic analysis (Mega6.06) using the neighbor-joining (NJ) method with 1,000 bootstrap replicates revealed the three strains clustering with the other S. maltophilia strains. Therefore, based on morphology, metabolic profile, and sequence analysis, the three strains were identified as Stenotrophomonas maltophilia. To test pathogenicity, the strains were grown in the nutrient broth (NB) medium for 48h at 28°C until bacterial suspension reached to OD600≈1.0 (2.0×109CFU/mL). Then, healthy roots of one-year-old Sanqi plants, pre-washed with sterilized water and -poked with a sterilized needle, were soaked in bacterial suspension (2.0×109CFU/mL) of the three strains separately for inoculation 10min. Sterilized water treatment was used as a control. Subsequently, bacteria-inoculated plants were planted in sterile soil pots and cultured in a greenhouse at 28°C with shading rate of 70%. Each treatment group included 3 plants with 3 replicates. Ten days post inoculation, symptoms similar to the ones in natural conditions were observed in the bacteria-inoculated plants. Based on the disease index (Li et al. 2020), we found that among the three strains, SM 2-13 displayed the highest virulence, while no symptoms were observed in the control plants. The same bacterial strains were re-isolated from these inoculated roots and identified by the methods described above. Previous studies showed that some Stenotrophomonas species cause plant diseases such as rice white stripe (Singh et al. 2001), strawberry leaf black spot (Wang et al. 2017), Cyclobalanopsis patelliformis leaf spot (Bian et al. 2020), and Jatropha curcas L. seed borne and stem necrosis (Wang et al. 2018). To our knowledge, this is the first report confirming Stenotrophomonas maltophilia causing root soft rot of Panax notoginseng in China.

The thrips-borne tospoviruses Calla lily chlorotic spot virus (CCSV), Tomato zonate spot virus (TZSV) and a new species provisionally named Tomato necrotic spot associated virus (TNSaV) infect similar crops in southwestern China. The symptoms exhibiting on virus-infected crops are similar, which is difficult for distinguishing virus species by symptomatology. The sequences of nucleocapsid proteins (NPs) of CCSV, TNSaV and TZSV share high degrees of amino acid identity with each other, and their serological relationship was currently demonstrated from the responses of the previously reported monoclonal antibodies (MAbs) against the NP of CCSV (MAb-CCSV-NP) and the nonstructural NSs protein of Watermelon silver mottle virus (WSMoV) (MAb-WNSs). Therefore, the production of virus-specific antibodies for identification of CCSV, TNSaV and TZSV is demanded to improve field surveys. The NP of TZSV-13YV639 isolated from Crinum asiaticum in Yunnan Province, China was bacterially expressed and purified for producing MAbs. Indirect enzyme-linked immunosorbent assay (ELISA) and immunoblotting were conducted to test the serological response of MAbs to 18 tospovirus species. Additionally, the virus-specific primers were designed to verify the identity of CCSV, TNSaV and TZSV in one-step reverse transcription-polymerase chain reaction (RT-PCR). Two MAbs, denoted MAb-TZSV-NP(S15) and MAb-TZSV-NP(S18), were screened for test. MAb-TZSV-NP(S15) reacted with CCSV and TZSV while MAb-TZSV-NP(S18) reacted specifically to TZSV in both indirect ELISA and immunoblotting. Both MAbs can be used to detect TZSV in field-collected plant samples. The epitope of MAb-TZSV-NP(S18) was further identified consisting of amino acids 78–86 (HKIVASGAD) of the TZSV-13YV639 NP that is a highly conserved region among known TZSV isolates but is distinct from TNSaV and TZSV. In this study, two MAbs targeting to different portions of the TZSV NP were obtained. Unlike MAb-CCSV-NP reacted with TNSaV as well as CCSV and TZSV, both TZSV MAbs can be used to differentiate CCSV, TNSaV and TZSV. The identity of CCSV, TNSaV and TZSV was proven by individual virus-specific primer pairs to indicate the correctness of serological responses. We also proposed an serological detection platform using MAb-CCSV-NP, MAb-TZSV-NP(S15) and MAb-TZSV-NP(S18) to allow researchers and quarantine staff to efficiently diagnose the infections of CCSV, TNSaV and TZSV in China and other countries.

In July 2016, more than 60% hydroponic commercially grown spinach (Spinacia oleracea) suddenly wilted in commercial greenhouse production in Songming (25°N, 103°E), Yunnan province. The infected pl...

Viral diseases are becoming an important problem in Amorphophallus production due to the propagation of seed corms and their trade across regions. In this study, combined-High-Throughput Sequencing, RT-PCR, electron microscopy, and mechanical inoculation were used to analyze virus-like infected Amorphophallus samples in Yunnan province to investigate the distribution, molecular characterization, and diversity and evolution of Amorphophallus-infecting viruses including three isolates of dasheen mosaic virus and three orthotospoviruses: mulberry vein banding associated virus (MVBaV), tomato zonate spot virus (TZSV) and impatiens necrotic spot virus (INSV). The results showed that DsMV is the dominant virus infecting Amorphophallus, mixed infections with DsMV and MVBaV to Amorphophallus were quite common in Yunnan province, China. This is the first report on infection of Amorphophallus with MVBaV, TZSV, and impatiens necrotic spot virus (INSV) in China. This work will help to develop an effective integrated management strategy to control the spread of Amorphophallus viral diseases.