Pathogenicity analysis and seed transmission of watermelon virus A in bottle gourd.

Seed transmission is among the primary strategies utilized by plant viruses for long-distance dissemination, leading to the widespread occurrence of viral diseases globally. Watermelon virus A (WVA) is a novel wamavirus first found in watermelon. However, the pathogenicity and transmission mode of WVA are still unclear. Our previous work found that the incidence of WVA in bottle gourd is very high. Based on that, the pathogenicity and seed transmission mode of WVA in bottle gourd were studied. Compared with healthy plant, bottle gourd infected by WVA showed no visible disease symptom. Moreover, in the seeds of 20 bottle gourd cultivars, the occurrence of WVA varies from 0 to 90%, and one cultivar even reaches 100%. We also found that the transmission rate from seeds to the resulting seedlings was 100%. Furthermore, WVA was present in both the seed coat and embryo, and seed disinfection cannot eliminate WVA. Besides the seed and leaf, WVA can also be detected in stem, flower, and fruit, but not in the root. To our surprise, the level of transmission from WVA-infected plants to seeds was more than 85%. In addition, the viral accumulations of both WVA and CGMMV were increased in plants with co-infection of WVA and CGMMV. Taken together, these findings reveal that WVA is a seed-transmitted virus which causes no disease symptom in bottle gourd, and there may be synergism between WVA and CGMMV.

Comparative genomics and host range analysis of four Ralstonia pseudosolanacearum strains isolated from sunflower reveals genomic and phenotypic differences.

BACKGROUND: Bacterial wilt caused by Ralstonia solanacearum species complex (RSSC) is one of the devastating diseases in crop production, seriously reducing the yield of crops. R. pseudosolanacearum, is known for its broad infrasubspecific diversity and comprises 36 sequevars that are currently known. Previous studies found that R. pseudosolanacearum contained four sequevars (13, 14, 17 and 54) isolated from sunflowers sown in the same field. RESULTS: Here, we provided the complete genomes and the results of genome comparison of the four sequevars strains (RS639, RS642, RS647, and RS650). Four strains showed different pathogenicities to the same cultivars and different host ranges. Their genome sizes were about 5.84 ~ 5.94 Mb, encoding 5002 ~ 5079 genes and the average G + C content of 66.85% ~ 67%. Among the coding genes, 146 ~ 159 specific gene families (contained 150 ~ 160 genes) were found in the chromosomes and 34 ~ 77 specific gene families (contained 34 ~ 78 genes) in the megaplasmids from four strains. The average nucleotide identify (ANI) values between any two strains ranged from 99.05% ~ 99.71%, and the proportion of the total base length of collinear blocks accounts for the total gene length of corresponding genome was all more than 93.82%. Then, we performed a search for genomic islands, prophage sequences, the gene clusters macromolecular secretion systems, type III secreted effectors and other virulence factors in these strains, which provided detailed comparison results of their presence and distinctive features compared to the reference strain GMI1000. Among them, the number and types of T2SS gene clusters were different in the four strains, among which RS650 included all five types. T4SS gene cluster of RS639 and RS647 were missed. In the T6SS gene cluster, several genes were inserted in the RS639, RS647, and RS650, and gene deletion was also detected in the RS642. A total of 78 kinds of type III secreted effectors were found, which included 52 core and 9 specific effectors in four strains. CONCLUSION: This study not only provided the complete genomes of multiple R. pseudosolanacearum strains isolated from a new host, but also revealed the differences in their genomic levels through comparative genomics. Furthermore, these findings expand human knowledge about the range of hosts that Ralstonia can infect, and potentially contribute to exploring rules and factors of the genetic evolution and analyzing its pathogenic mechanism.

Transcriptome and Metabolome Analyses Reveal That Jasmonic Acids May Facilitate the Infection of Cucumber Green Mottle Mosaic Virus in Bottle Gourd.

Cucumber green mottle mosaic virus (CGMMV) is a typical seed-borne tobamovirus that mainly infects cucurbit crops. Due to the rapid growth of international trade, CGMMV has spread worldwide and become a significant threat to cucurbit industry. Despite various studies focusing on the interaction between CGMMV and host plants, the molecular mechanism of CGMMV infection is still unclear. In this study, we utilized transcriptome and metabolome analyses to investigate the antiviral response of bottle gourd (Lagenaria siceraria) under CGMMV stress. The transcriptome analysis revealed that in comparison to mock-inoculated bottle gourd, 1929 differently expressed genes (DEGs) were identified in CGMMV-inoculated bottle gourd. Among them, 1397 genes were upregulated while 532 genes were downregulated. KEGG pathway enrichment indicated that the DEGs were mainly involved in pathways including the metabolic pathway, the biosynthesis of secondary metabolites, plant hormone signal transduction, plant-pathogen interaction, and starch and sucrose metabolism. The metabolome result showed that there were 76 differentially accumulated metabolites (DAMs), of which 69 metabolites were up-accumulated, and 7 metabolites were down-accumulated. These DAMs were clustered into several pathways, including biosynthesis of secondary metabolites, tyrosine metabolism, flavonoid biosynthesis, carbon metabolism, and plant hormone signal transduction. Combining the transcriptome and metabolome results, the genes and metabolites involved in the jasmonic acid and its derivatives (JAs) synthesis pathway were significantly induced upon CGMMV infection. The silencing of the allene oxide synthase (AOS) gene, which is the key gene involved in JAs synthesis, reduced CGMMV accumulation. These findings suggest that JAs may facilitate CGMMV infection in bottle gourd.

Cotton leaf curl Multan virus differentially regulates innate antiviral immunity of whitefly (Bemisia tabaci) vector to promote cryptic species-dependent virus acquisition.

Begomoviruses represent the largest group of economically important, highly pathogenic, DNA plant viruses that contribute a substantial amount of global crop disease burden. The exclusive transmission of begomoviruses by whiteflies (Bemisia tabaci) requires them to interact and efficiently manipulate host responses at physiological, biological and molecular scales. However, the molecular mechanisms underlying complex begomovirus-whitefly interactions that consequently substantiate efficient virus transmission largely remain unknown. Previously, we found that whitefly Asia II 7 cryptic species can efficiently transmit cotton leaf curl Multan virus (CLCuMuV) while MEAM1 cryptic species is a poor carrier and incompetent vector of CLCuMuV. To investigate the potential mechanism/s that facilitate the higher acquisition of CLCuMuV by its whitefly vector (Asia II 7) and to identify novel whitefly proteins that putatively interact with CLCuMuV-AV1 (coat protein), we employed yeast two-hybrid system, bioinformatics, bimolecular fluorescence complementation, RNA interference, RT-qPCR and bioassays. We identified a total of 21 Asia II 7 proteins putatively interacting with CLCuMuV-AV1. Further analyses by molecular docking, Y2H and BiFC experiments validated the interaction between a whitefly innate immunity-related protein (BTB/POZ) and viral AV1 (coat protein). Gene transcription analysis showed that the viral infection significantly suppressed the transcription of BTB/POZ and enhanced the accumulation of CLCuMuV in Asia II 7, but not in MEAM1 cryptic species. In contrast to MEAM1, the targeted knock-down of BTB/POZ substantially reduced the ability of Asia II 7 to acquire and accumulate CLCuMuV. Additionally, antiviral immune signaling pathways (Toll, Imd, Jnk and Jak/STAT) were significantly suppressed following viral infection of Asia II 7 whiteflies. Taken together, the begomovirus CLCuMuV potentiates efficient virus accumulation in its vector B. tabaci Asia II 7 by targeting and suppressing the transcription of an innate immunity-related BTB/POZ gene and other antiviral immune responses in a cryptic species-specific manner.

First report of tomato yellow mottle-associated virus infecting Solanum nigrum in China.

First report of tomato yellow mottle-associated virus infecting Solanum nigrum in China Zhenggang Li, Yafei Tang, Xiaoman She, Guobing Lan, Lin Yu, and Zifu He† Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, P. R. China. Tomato yellow mottle-associated virus (TYMaV) is a newly found cytorhabdovirus associated with epinasty of leaflet blades, yellow spots, puckering, and mottling symptoms in tomato plants in China (Xu et al., 2017). In May 2020, Solanum nigrum plants exhibiting leaf crinkling and mosaic symptoms (eXtra S1) were found in Shantou city, Guangdong, China. To identify the causal pathogens, the leaves of three symptomatic plants were collected and subjected to total RNA extraction with TRIzol Reagent (Takara, Kusatsu, Japan). About 100 µg RNA mixture, which consisted of an equal amount of total RNA extracted from the three samples, was subjected to small RNA deep sequencing and assembly (sRSA) (Kreuze et al., 2009). Small RNA cDNA library was constructed with the method described previously (Mi et al., 2008). Small RNA deep sequencing was performed with Illumina HiSeq X Ten platform. VirusDetect (Zheng et al., 2017) was used to analyze the sequence data. The result showed that the sequence data includes about 11 million reads and generated 194 unique contigs after removal of host-derived contigs. Subsequently, the unique contigs were screened using BLASTn search against the virus database. One hundred and five unique contigs were mapped to TYMaV genome (reference sequence, KY075646), 21 unique contigs were mapped to RNA1 segment of tomato chlorosis virus (ToCV) genome (reference sequence, KY618796), 67 unique contigs were mapped to RNA2 segment of ToCV genome (reference sequence, KY618797), and one unique contig was mapped to pepper veinal mottle virus (PVMV) genome (reference sequence, FJ617225) (eXtra S1). To verify the sRSA result for TYMaV detection, RT-PCR was performed with two primer pairs TYMaV-F1/R1 (5'-TCATTAGACTCAGGCCTAATCCTCA AAGT-3'/5'-GATATGGAGACGTCCAAGTTCAAAGGGATGGA-3'), and TYMaV-F2/R2 (5'-TATGCGGCAGCTTTCATGTCTATAGACCCT-3'/5'-ATGACCTAGCTTCAATAACAGTCGCG-3'), which are designed according to the sRSA result. All the symptomatic samples tested positive for TYMaV (eXtra S2). Western blot with TYMaV N protein-specific antibody further verified the result (eXtra S2). To obtain the nearly full-length sequence of TYMaV identified in Shantou, 13 primer pairs were designed to amplify the viral fragments. The amplified PCR products were then introduced into pMD19T (Takara, Kusatsu, Japan) and sequenced by Sangon Biotech Co. (Shanghai, China). The nearly full-length sequence of TYMaV Shantou isolate (TYMaV-ST) was assembled from the 13 overlapping sequences (reference sequence, MW527091). TYMaV-ST genome comprises of 13401 nt and shares 84.93% nucleotide sequence identity with the reference genome (KY075646). In addition, 37 S. nigrum samples and 20 tomato samples nearby with viral disease symptoms were collected from different sites of Guangdong province, China. Six S. nigrum samples and five tomato plant samples tested positive for TYMaV by RT-PCR, suggesting a wide spread of the virus in the surveyed region. These results together with those of the sRSA assay also suggest that the disease symptoms shown in the original S. nigrum plants may not necessarily be caused by TYMaV or by TYMaV alone. To our knowledge, this is the first report of TYMaV infecting S. nigrum in China. S. nigrum is a common weed which belongs to the family Solanaceae and may serve as a reservoir for TYMaV in the fields. Further research is needed to verify whether this is indeed the case, and to understand the characteristics of this virus including its transmission, pathogenicity, and economic significance. The authors declare no conflict of interest. Funding This work was supported by the Key Research and Development Program of Guangdong Province (2018B020202006), the Agricultural Competitive Industry Discipline Team Building Project of Guangdong Academy of Agricultural Sciences (202103TD and 202105TD), the Science and Technology Program of Guangzhou (202102020504), and Special Fund for Scientific Innovation Strategy-Construction of High-Level Academy of Agriculture Science (R2019PY-QF003). References: Kreuze, J. F., et al. 2009. Virology. 388: 1. Mi, S., et al. 2008. Cell. 133: 116. Xu, C., et al. 2017. J Virol. 91: 11. Zheng, Y., et al. 2017. Virology. 500: 130.

Anthracnose: A New Leaf Disease on Radermachera sinica (China Doll) in China.

Radermachera sinica (China doll) is a popular evergreen horticultural crop worldwide. However, little information has been provided to describe the anthracnose disease of R. sinica. In 2018, symptoms suspected of leaf anthracnose were observed on R. sinica in gardens and commercial greenhouses in Guangzhou, China. Lesions on diseased leaves showed thinned and grayish white centers, dark-brown to black borders, and raised black spots. Twenty-seven single-conidia isolates were obtained from symptomatic leaf lesions. Based on morphological characteristics and multilocus phylogenetic analysis, 19 isolates were identified as Colletotrichum siamense and six and two isolates were identified as C. fructicola and C. karstii, respectively. An in vivo pathogenicity test was conducted on leaves of R. sinica plants, and it was discovered that C. siamense was more aggressive under wounded conditions than under unwounded conditions, and caused symptomatic necrotic lesions on the leaf. Afterward, the same pathogen was reisolated from lesions of inoculated leaves to fulfill Koch's postulates. However, neither C. fructicola nor C. karstii caused visible lesions on leaves of R. sinica under wounded or unwounded conditions, indicating that they may be asymptomatic endophytes or opportunistic pathogens on R. sinica. To our knowledge, this study is the first report of Colletotrichum spp. associated with anthracnose disease on R. sinica in China.

Complete genome sequence of a previously undescribed monopartite begomovirus and betasatellite infecting Malvastrum coromandelianum in Cambodia.

A previously undescribed monopartite begomovirus was identified in Kampot province, Cambodia, in Malvastrum coromandelianum plants exhibiting yellow vein symptoms characteristic of begomovirus infections. The apparently full-length viral component was cloned and sequenced following enrichment of circular DNA by rolling-circle amplification and restriction enzyme digestion. The genome of the virus was 2737 nucleotides in length (KP188831) and exhibited an organization like that of other monopartite begomoviruses, sharing the highest nucleotide sequence similarity (87.7% identity) with ageratum yellow vein virus (AM940137). A satellite molecule was amplified from total DNA by PCR amplification, using the betasatellite-specific primer pair ß01/ß02. The satellite molecule (1346 nt, KP188832) had structural characteristics like those of other betasatellites associated with begomoviruses and shared the highest nucleotide sequence similarity (84.8% identity) with malvastrum yellow vein betasatellite (MN205547). According to the criteria established for species demarcation for classification of begomoviruses (family Geminiviridae) and betasatellites (family Tolecusatellitidae), respectively, the virus isolate from M. coromandelianum in Cambodia is a previously undescribed novel monopartite begomovirus, for which the name "malvastrum yellow vein Cambodia virus" (MaYVCV) is proposed, and the betasatellite is a previously undescribed novel betasatellite, for which the name "malvastrum yellow vein Cambodia betasatellite" (MaYVKHB) is proposed.

C4, the Pathogenic Determinant of Tomato Leaf Curl Guangdong Virus, May Suppress Post-transcriptional Gene Silencing by Interacting With BAM1 Protein.

Tomato leaf curl Guangdong virus (ToLCGdV) is a begomovirus associated with a Tomato yellow leaf curl disease (TYLCD) epidemic in Guangdong province, China. Being the least conserved protein among geminivirus proteins, the function of C4 during ToLCGdV infection has not been elucidated. In this study, the infectious clones of ToLCGdV and a ToLCGdV mutant (ToLCGdVmC4) with disrupted C4 ORF were constructed. Although ToLCGdV and ToLCGdVmC4 could infect Nicotiana benthamiana and tomato plants, ToLCGdVmC4 elicited much milder symptoms compared with ToLCGdV. To further verify the role of C4 in viral pathogenesis, C4 was expressed in N. benthamiana from Potato virus X (PVX) vector. The results showed that ToLCGdV C4 enhanced the pathogenicity of PVX and induced more severe developmental abnormalities in plants compared with PVX alone or PVX-mC4. In addition, ToLCGdV C4 suppresses systemic gene silencing in the transgenic N. benthamiana line 16c, but not local gene silencing induced by sense GFP in wild-type N. benthamiana plants. Moreover, C4 suppresses transcriptional gene silencing (TGS) by reducing the DNA methylation level of 35S promoter in 16c-TGS N. benthamiana plants. Furthermore, C4 could also interact with the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1), suggesting that C4 may suppress gene silencing by interfering with the function of BAM1 in the cell-to-cell spread of RNAi. All these results suggest that C4 is a pathogenic determinant of ToLCGdV, and C4 may suppress post-transcriptional gene silencing (PTGS) by interacting with BAM1.

Identification and Genetic Characterization of Ralstonia solanacearum Species Complex Isolates from Cucurbita maxima in China.

Ralstonia solanacearum species complex is a devastating phytopathogen with an unusually wide host range, and new host plants are continuously being discovered. In June 2016, a new bacterial wilt on Cucurbita maxima was observed in Guangdong province, China. Initially, in the adult plant stage, several leaves of each plant withered suddenly and drooped; the plant then wilted completely, and the color of their vasculature changed to dark brown, ultimately causing the entire plant to die. Creamy-whitish bacterial masses were observed to ooze from crosscut stems of these diseased plants. To develop control strategies for C. maxima bacterial wilt, the causative pathogenic isolates were identified and characterized. Twenty-four bacterial isolates were obtained from diseased C. maxima plants, and 16S rRNA gene sequencing and pathogenicity analysis results indicated that the pathogen of C. maxima bacterial wilt was Ralstonia solanacearum. The results from DNA-based analysis, host range determination and bacteriological identification confirmed that the 24 isolates belonged to R. solanacearum phylotype I, race 1, and eight of these isolates belonged to biovar 3, while 16 belonged to biovar 4. Based on the results of partial egl gene sequence analysis, the 24 isolates clustered into three egl- sequence type groups, sequevars 17, 45, and 56. Sequevar 56 is a new sequevar which is described for the first time in this paper. An assessment of the resistance of 21 pumpkin cultivars revealed that C. moschata cv. Xiangyu1 is resistant to strain RS378, C. moschata cv. Xiangmi is moderately resistant to strain RS378, and 19 other pumpkin cultivars, including four C. maxima cultivars and 15 C. moschata cultivars, are susceptible to strain RS378. To the best of our knowledge, this is the first report of C. maxima bacterial wilt caused by R. solanacearum race 1 in the world. Our results provide valuable information for the further development of control strategies for C. maxima wilt disease.

Genome Sequencing of Ralstonia solanacearum Race 4, Biovar 4, and Phylotype I, Strain YC45, Isolated from Rhizoma kaempferiae in Southern China.

Ralstonia solanacearum is an important phytopathogen that attacks over 400 plant species, including Zingiberaceae plants. Here, we report the complete genome sequence of strain YC45, which was isolated from Rhizoma kaempferiae in southern China.

Identification and molecular characterization of a single-stranded circular DNA virus with similarities to Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1.

A putative circular single-stranded DNA (ssDNA) virus was recovered from Hypericum japonicum collected in Vietnam. The viral isolate was tentatively named Hypericum japonicum-associated circular DNA virus (HJasCV). HJasCV shares 58.7-65.4% nucleotide sequence identity with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) and SsHADV-1-like viruses. Like this group of viruses, the genome of HJasCV (2 200 nt) has two large ORFs, one in the virion-sense and the other in the complementary-sense DNA. The proteins encoded in the virion-sense and complementary-sense ORFs share 39-46 % and 45-67 % amino acid sequence identity with the putative capsid and replication-associated proteins (Reps), respectively, of SsHADV-1 and SsHADV-1-like viruses. The putative Rep of HJasCV contains all of the motifs related to rolling-circle replication. Its 111-bp intergenic region (IR) contains a hairpin structure with a geminivirus-like nonanucleotide sequence, TAATGTTAT, at the apex of the loop. Phylogenetic analysis revealed that HJasCV forms a monophyletic clade with SsHADV-1 and SsHADV-1-like viruses.