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Fruit of Siraitia grosvenorii (family Cucurbitaceae) are used as a sweetener in beverage and traditional herbal medicine production in China. Some viruses have been known to infect S. grosvenorii, such as papaya ringspot virus (PRSV), watermelon mosaic virus (WMV), and zucchini yellow mosaic virus (ZYMV) (Cai et al. 2001; Liao et al. 2005; Qin et al. 2005). In August 2017, six S. grosvenorii samples (nos. LHG-1 to LHG-6) that showed mosaic, chlorosis, and mottling of leaves were collected from Yongfu, Guangxi province, China. All symptomatic samples were tested by enzyme-linked immunosorbent assay (ELISA) in double antibody sandwich format for 10 known cucurbit-infecting viruses, including cucumber green mottle mosaic virus (CGMMV), cucumber mosaic virus, kyuri green mottle mosaic virus, PRSV, squash mosaic virus, tobacco mosaic virus (TMV), watermelon silver mottle virus, WMV, zucchini green mottle mosaic virus, and ZYMV. The sample LHG-1 with chlorosis and mottle symptoms reacted positively against the antisera of CGMMV and TMV (Adgen, U.K.) but negatively against antisera of the other viruses. The other samples were negative in ELISA for all viruses tested. Total RNA was extracted from the LHG-1 sample using TRIzol reagent (Takara Dalian, China) following the manufacturer’s instructions. Reverse transcription polymerase chain reaction (RT-PCR) assays using CGMMV-specific primer pair CGM1/CGM2 (Huang et al. 2007) and TMV-specific primers (Li et al. 2000) showed a positive RT-PCR product of the expected size (650 bp) for CGMMV but not for TMV. The amplicon was further cloned into pMD-18-T vector (Takara Dalian) and sequenced (GenBank accession no. MK858182). Sequence analysis revealed that the RT-PCR product showed 99% nucleotide identity to the sequences of CGMMV isolates available in GenBank (accession nos. HM008919, KY753928, KX883833, KP868654, KM873786, and MH271443). The virus in LHG-1 was therefore identified as an isolate of CGMMV and named as CGMMV-LHG. Additionally, to confirm the infectivity of CGMMV-LHG, leaf sap extract in phosphate buffer (0.1 M, pH 7.0) of LHG-1 was mechanically inoculated on leaves of S. grosvenorii and Lagenaria siceraria seedlings. Ten days after inoculation, S. grosvenorii produced symptoms similar to the diseased plants in the field, and systemic mosaic and vein-banding were observed on L. siceraria. The control seedlings inoculated with phosphate buffer remained symptomless. Presence of CGMMV in inoculated plants was confirmed by RT-PCR. To the best of our knowledge, this is the first report of CGMMV infection on S. grosvenorii under natural conditions. The virus can be transmitted by seeds, infects various cucurbit species, and causes yield losses all over the world (Liu et al. 2014; Wu et al. 2011). Use of virus-free seeds and seedlings will be helpful to control CGMMV in S. grosvenorii production areas.

Whitefly-transmitted geminiviruses belong to the Geminiviridae family, which includes a group of plant DNA viruses that occurs in tropical and subtropical regions worldwide and caused severe leaf curl diseases on many crops. In July 2011, symptoms of mild yellowing and curling of leaves were observed on Zinnia elegans in Hue City, Vietnam. To identify possible begomoviruses, two samples with typical symptoms (Z1 and Z2) were collected and total DNA was extracted using PlantGen DNA Kit (CoWin Biotech Co., China). Using begomovirus-specific degenerate primer pair AV494 and AC1048 to amply geminivirus capsid protein gene (3), a 586-bp DNA fragment was amplified from both collected samples. The PCR products from Z1 and Z2 were cloned and sequenced. The partial sequences from two isolates had 96.8% similarity to each other and most closely related to Ageratum yellow vein China virus (AYVCNV) (98% identity to GenBank Accession Nos. AM940137.1 and AJ849916.1). Using two degenerate primer pairs bego1 (5′-ACMGGCCYATGWAYMGGAAG-3′)/bego6 (5′-ACNGGDAARACRATGTGGGC-3′) and bego2 (5′-CTTDATRTTBTCRTCCATCC-3′)/bego3 (5′-CCWGGATTGCANARSAMGAT-3′) to amplify full-length viral genome from Z1 (1). The complete DNA-A sequence from Z1 were determined to be 2,741 nucleotides (JX911332), showed high levels of sequence identity (89% [JN809812] to 98% [AJ849916.1 and AJ558120]) with AYVCNV and shared 98% nucleotide sequence identity with AYVCNV-G68 (AJ849916.1) and AYVCNV-G13 (AJ558120). No DNA-B component nor DNA-β could be detected using primers described previously (4). Zinnia leaf curl virus, Ageratum enation virus, Tobacco leaf curl virus, and Alternanthera yellow vein virus have been reported to be associated with Zinnia leaf curl disease (2). To our knowledge, this is the first report of AYVCNV infecting Z. elegans. AYVCNV was first reported in A. conyzoides on Hainan Island, China, and could infect L. esculentum (Solanaceae), A. conyzoides (Asteraceae), or tobacco (Solanaceae). Our results further corroborate that AYVCNV can infect Asteraceae plants. Therefore, this virus may pose a serious threat to many plants. References: (1) L. Lin et al. Plant Prot. 37:44, 2011. (2) P. Naveen et al. J. Biotechnol. Bioinform. 2:6, 2012. (3) S. D. Wyatt et al. Phytopathology 86:1288, 1996. (4) X. P. Zhou et al. J. Gen. Virol. 84:237, 2003.

Cotton leaf curl disease (CLCuD) is a major constraint to cotton production, causing great economic losses in Pakistan and India. In China, CLCuD has been discovered in the field of Nanning, GuangXi. To better understand this disease, we sequenced the virus-associated small DNA molecules.We purified total DNA from cotton and okra plants exhibiting leaf curl symptoms; PCR amplified and sequenced CLCuMV satellite DNA (DNAbeta) -related small DNA molecules.We identified 2 novel recombinant DNA molecules with 1384 nucleotides in cotton and 754 nucleotides in okra. The 1384 nt molecule contains partial DNA-A and DNAbeta of CLCuMV GX1. It includes the intergenic region, adjacent AV2 and AC1 coding sequences, and reverse complementary AC3 of DNA-A and A-rich region of DNAbeta. Common nucleotides were found around the junction points of DNA-A and DNAbeta sequences, suggesting they were the sites of recombination. Comparison with previous reported CLCuMV recombinants produced in lab showed that the intergenic region of DNA-A and A-rich region of DNAbeta were conserved on the recombination process. The 754 nt molecule was produced by deletion of CLCuMV DNAbeta in the C1 open reading frame and A-rich region.We identified a novel recombinant molecule originated from CLCuMV DNA-A and DNAbeta and a small defective molecule of DNAbeta. This is the first report of sequence recombination and deletion of CLCuMV in China, which may be helpful to understand the CLCuMV evolution and host adaptation.