First report of Potato Yellow Dwarf Nucleorhabdovirus infecting pepper (Capsicum spp.) in Oklahoma.

Pepper (Capsicum spp.) is an economically valuable crop used for spice and fresh vegetable (Tripodi, and Kumar. 2019). The total acreage of peppers in Oklahoma is low (NASS, 2020), but there are numerous farms that are growing peppers for commercial production and provide fresh produce to local farmer's markets and processing industries. During a field survey in 2021, pepper plants with leaf distortion, mottling of the leaf, apical yellowing, and vein banding were observed in commercial pepper fields (Supplementary Figure 1). Seventeen leaf samples were collected from symptomatic pepper plants in Caddo County of Oklahoma. Total RNA was extracted from each sample using the Spectrum Plant total RNA kit, and screened by RT-PCR for Pepper mild mottle virus (PMMoV) as described previously (Ali and Ali, 2015). Total RNA from two negative PCR samples (named Caddo57 and Caddo64 respectively) were analyzed by high throughput sequencing. A total read count of 45,780,855 (average length = 73.51bp) and 21,163,567 (average length = 73.86 bp) for Caddo57 and Caddo64 respectively, were assembled in de novo using CLC genomic workbench (QIAGEN) and used for BLASTn and BLASTx analysis. Three contigs: 4,259bp (average coverage 31776.33X), 4,378 (average coverage 21773.09X), and 4,206bp (average coverage 57419.46X) for Caddo57 isolate showed 90-92% nucleotide (nt) identities with partial sequences of several genes (M, G, L, P, N, X, and Y genes) of PYDV isolate (KY549567). Similarly, four contigs: 4,204bp (average coverage 57446.92X), 2,738bp (average coverage 16192X), 4,257bp (average coverage 31791X), and 1,510bp (average coverage 33051.35X) were obtained for Caddo64 isolate and showed 90-92% nt identities with the same genes of PYDV isolate (KY549567). To further confirm the presence of PYDV, total RNA from Caddo57 and Caddo64 samples were tested by RT-PCR assays using newly designed primers (Table 1) based on the contigs sequences obtained above. The expected PCR products from both isolates were directly sequenced. Using BLASTn, nucleotide sequences of both L gene (OP805375), and N gene (OP805377) for Caddo57 isolate showed 92.82% and 92.392% identities respectively with PYDV isolate (KY549567). Similarly, nucleotide sequences of L gene (OP805376) and N gene (OP805378) of Caddo64 isolate showed 92.27% and 92.08% identities respectively with PYDV isolate (KY549567). The species demarcation criteria for nucelorhabdoviruses is 50% in cognate genes (Walker et al. 2018). These results demonstrate that our isolates align with PYDV species, and do not constitute evidence for a divergent nucleorhabdovirus member. The remaining 15 samples were negative by RT-PCR assay to PYDV. Our results confirmed the presence of PYDV infecting pepper in Oklahoma. Currently, PYDV has been reported infecting potato, tobacco, marigold, pepper, tomato, and white clover. The geographical distribution of PYDV appears to be limited to the US with detection in Wisconsin, Minnesota, California, New York, and Maryland (Walker et al. 1939, Lockhart, 1989, Falk et al. 1981, Chiu et al. 1970, Hammond et al. 2017). The PYDV is naturally transmitted by aphids and leafhoppers (Ghosh et al. 2008). The recognition of PYDV in Oklahoma is of notable concern for local growers. Further studies are needed to expand upon the relationship of these PYDV isolates to the currently reported isolates in the USA. This is the first report of potato yellow dwarf nucleorhabdovirus (PYDV) infecting pepper in Oklahoma.