Cucurbit Leaf Crumple Virus (CuLCrV) is Seed Transmitted in Yellow Squash (Cucurbita pepo L.).

The traditional understanding of begomovirus transmission exclusively through the whitefly Bemisia tabaci (Gennadius) has shifted with findings of seed transmission in some begomoviruses over the last decade. We investigated the seed transmissibility of cucurbit leaf crumple virus (CuLCrV), a bipartite begomovirus that has recently emerged as a severe constraint for yellow squash (Cucurbita pepo L.) production in the southeastern United States. We found high concentration of CuLCrV in male and female flower tissues of infected squash, including pollen and ovules. The virus infiltrated the fruit tissues including the endocarp and funiculus, which are anatomically positioned adjacent to the seeds. In seeds, CuLCrV was detected in the endosperm and embryo where there are no vascular connections, in addition to the seed coat. The virus was detected in the radicle, plumule, cotyledonary leaves, and true leaves of seedlings grown from seeds collected from infected fruits. In the grow-out test conducted, CuLCrV infections ranged from 17-56% of the progeny plants. To ensure that partial viral genome fragments were not being mistaken for replicative forms of the virus, we performed RCA ̶ PCR and amplified complete DNA-A and DNA-B of CuLCrV from seed tissues, seedlings, progeny plants of CuLCrV infected squash. Near complete DNA-A and DNA-B sequences of CuLCrV were recovered from a progeny plant, further validating our findings. Our results demonstrate that CuLCrV can translocate from vegetative to reproductive tissues of yellow squash, persist within the seeds, and subsequently induce infection in progeny plants, confirming its capacity for seed transmission.

First Report of Watermelon Crinkle Leaf-Associated Virus-2 (WCLaV-2) in Watermelon (Citrullus lanatus) in Georgia, USA.

Watermelon (Citrullus lanatus) is a key horticultural crop in Georgia with farmgate value of 142 million USD (2022 Farm Gate Value Report), yet faces challenges from whitefly-transmitted viruses, especially during periods of elevated whitefly populations in the fall. Foliar symptoms on watermelon plants including yellow mottling and chlorosis, wrinkling, bunching, and upward curling, were observed in experimental fields at UGA Tifton and commercial fields in Colquitt County in the fall of 2023. These were similar to those described for watermelon crinkle leaf-associated viruses (WCLaV-1 and WCLaV-2) from Florida (Hendrick et al, 2021) and Texas (Hernandez et al., 2021). The disease incidence reached 100% in both locations. WCLaV-1 was previously identified in Georgia (Adeleke et al., 2022a); however, WCLaV-2 remained undetected in further surveys (Adeleke et al., 2022b). Total nucleic acid was extracted from symptomatic leaf tissues with the MagMAXTM 96 Viral RNA isolation kit (ThermoFisher Scientific, USA), following the manufacturer's guidelines, with the omission of DNAse treatment. The presence of WCLaV-1 was identified through reverse transcription-polymerase chain reaction (RT-PCR) (Hernandez et al., 2021) in 17 out of 24 samples in Tift County, and 12 out of 15 samples from Colquitt County. Within the same set of samples, WCLaV-2 was identified in three samples from Tift County and four samples from Colquitt County by RT-PCR directed at the RdRp gene (Hernandez et al., 2021). WCLaV-1 was not detected in the three samples from Tift County that had WCLaV-2, while mixed infections of WCLaV-2 and WCLaV-1 were observed in the samples form Colquitt County. Two whitefly transmitted viruses, previously reported in Georgia were also identified as mixed infection in these samples (Table S1). The presence of WCLaV-2 was further confirmed by amplifying the movement protein (MP) gene of WCLaV-2 by RT-PCR assays (Hernandez et al., 2021). The amplicons, with expected sizes of 968bp for the RdRp gene and 562bp for the MP gene of WCLaV-2, located on RNA 1 and RNA 2 segments respectively, were directly sequenced from both directions (Genewiz, USA) from a sample collected in Tift County. The resulting data were analyzed via BLASTn search. The MP gene fragment (PP178543) shared 100% identity with isolates from Brazil (LC636074.1), Texas (MW559086.1), and Florida (MZ325858.1). RdRp gene (PP178542) shared >99.7% identity with isolates from Brazil (LC636073.1), Texas (MW559083.1) and Florida (MZ325855.1). WCLaV-1 and WCLaV-2, initially discovered in Asia (Xin et al., 2017), have been assigned to the genus Coguvirus, in the family Phenuiviridae (Walker et al., 2022). Subsequent reports from the USA (Hendrick et al., 2021; Hernandez et al., 2021), Australia (Mulholland et al., 2023), and Brazil (Maeda et al., 2022) indicate the global spread of these viruses. Watermelon is the primary host of WCLaV-2. Despite these findings, biological information, including vector relations, for both viruses and other members of the genus Coguvirus remains elusive. The impact of these viruses on watermelon production and yield in the identified regions remains largely unknown, underscoring the need for further investigations.

Natural occurrence of tomato chlorosis virus on tomatillo (Physalis philadelphica) in the United States.

Tomatillo (Physalis philadelphica L.) is an annual plant native to Mexico and Guatemala, and cultivated in other tropical and subtropical regions. In October 2023, tomatillo plants with interveinal yellowing of leaves, marginal chlorosis, leaf thickening, and leaf rolling symptoms (Figure 1) were observed at Colquitt and Tift County, Georgia, US. The disease incidence ranged from 80-100 % which reduced fruit quality and marketability. Twenty tomatillo leaves exhibiting severe symptoms were collected, and, sub-sampled of the leaves were pooled into microcentrifuge tubes. Further, MagMAX 96 viral RNA isolation kit (Thermo Fisher Scientific, US), was used for the extraction of (n=4) total nucleic acid (TNA) (Kavalappara et al. 2021). Symptomatic leaves were tested for the presence of insect-transmitted viruses such as begomovirus (tomato yellow leaf curl virus, TYLCV), potyvirus (turnip mosaic virus, TuMV), crinivirus (tomato infectious chlorosis virus, TICV; tomato chlorosis virus, ToCV), and tospovirus (orthotospovirus tomatomaculae, TSWV). Polymerase chain reaction (PCR) was performed for detecting TYLCV, using gene-specific primers (Kumar et al., 2023). However, for ToCV, TuMV and TICV detection, cDNA was prepared using 100 ng of TNA as a template, followed by the PCR ( Liu et al., 2012). Moreover, the detection of TSWV was conducted using immuno-strips (Adgia, US) following the manufacturer's instructions. ToCV was detected from all the tested samples, while TuMV, TICV, TYLCV and TSWV were not detected in any symptomatic tissues. In addition, RT-PCR was performed using gene-specific primers targeting the RNA-dependent RNA polymerase (RdRP) gene and the heat-shock protein 70 (Hsp70) gene of ToCV. The PCR amplicon of 439 bp encoding Hsp70 and 643 bp corresponding to RdRP was gel-purified and Sanger sequenced (Azenta Life Sciences, US). BLASTn analysis shows RdRP gene from ToCV-tomatillo (OR905600) has 100 % identity with ToCV of RNA1 segment (RdRP, GenBank accession no. AY903447, Florida, US), while Hsp70 gene (OR900219) has 100 % identity with ToCV of RNA2 segment (Hsp70, GenBank accession no. LC778246, Cairo, Egypt). In addition, the symptomatic tomatillo leaves were studied for transmission assay using tomato, employing non-viruliferous whiteflies (Bemisia tabaci) with 48 h of acquisition access period. Further, two weeks post-infection, the presence of ToCV was detected from the test plants while other whitefly-transmitted viruses remins undetected. In 2023, ToCV is widespread in tomato-growing counties, infecting commercially grown tomato cultivars with intermediate resistance against TYLCV-IL (Israel strain). However, tomatillo plants infected with TuMV in California (Liu et al., 2012), TSWV in Georgia, (Díaz-Pérez and Pappu 2000) and TYLCV in Mexico (Gámez-Jiménez et al. 2009) were reported. This study suggests that tomatillo could be a permissive host for ToCV while restrictive to other prevalent viruses in the region. A recent investigation speculates a potential synergistic interaction between ToCV and TYLCV-IL, exacerbating the breakdown of host resistance in tomato (Fiallo-Olivé et al. 2019, Kumar et al. 2023). To the best of our knowledge, this is the first report for the natural incidence of ToCV on tomatillo within the US. The findings will contribute to developing more effective management strategies against emerging viral threats.