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 tomato zonate spot virus (TZSV) poses a significant threat to agriculture. Therefore, the elucidation of the functional roles and interactions of its encoded proteins is crucial for the development of effective control strategies. The aim of this study was to investigate the interaction network between the TZSV nucleocapsid (N), the non-structural M-segment (NSm) and the non-structural S-segment (NSs) proteins, with a focus on the functional characterization of the NSm protein. Yeast two-hybrid (Y2H) analysis indicated that both the N protein (N-N) and the NSm protein (NSm-NSm) exhibit self-interaction in vitro, with successful expression of all fusion proteins confirmed by Western blotting. Subsequently, we used bimolecular fluorescence complementation (BiFC) and luciferase complementation imaging (LCI) assays in epidermal cells of Nicotiana benthamiana to confirm that N and NSm proteins self-interact. In addition, heterologous interactions between NSs-N, N-NSm and NSs-NSm were also detected. BiFC and co-localization experiments with fusion proteins elucidated the interaction place of the cell: N-N and NSm-N interactions occurred in both the cytoplasm and nucleus, with NSm-NSm interaction occurring in the nucleus, whereas NSs-N and NSs-NSm interactions only occurred in the cytoplasm. Subcellular localization studies showed that the N protein is distributed in both the cytoplasm and the nucleus, whereas the NSm and NSs proteins are predominantly localized in the cytoplasm. In particular, NSm was found to specifically target plasmodesmata (PD) and co-localize with the known PD marker protein PDLP8. Interestingly, TZSV NSm was demonstrated to mediate the cell-to-cell movement of a cucumber mosaic virus mutant (ΔCMV-GFP) lacking its native movement protein (3a). This was evidenced by the spread of approximately 50 fluorescent foci to neighboring cells observed at 6 dpi. This study comprehensively describes the intricate interaction network between the N, NSm and NSs proteins of TZSV and clarifies their subcellular localizations within plant cells. Crucially, we provide conclusive evidence that the NSm protein of TZSV is a functional movement protein essential for facilitating viral intercellular transport which promotes viral spread within the host during systemic infection. These findings offer important insights into the infection mechanism of TZSV and provide potential targets for the control of TZSV.