Butterfly pea (Clitoria ternatea), also known as Asian pigeonwings is a perennial herbaceous plant belongs to the Fabaceae family has a great source of natural food colorants and antioxidants (Suarna et al., 2021). It is a multipurpose forage that produces bioactive compounds, acts as a cover crop and improves soil fertility through nitrogen fixation (Gomez et al, 2003). Rhynchosia Yellow Mosaic Virus (RhYMV) which is transmitted by the whitefly (Bemisia tabaci) was first reported from Pakistan in leguminous weed least snoutbean, Rhynchosia minima (Illias et al., 2009). There is no record on the infection of RhYMV in butterfly pea in India or anywhere in the world. In 2022 and 2023, 250 whole butterfly pea plants were collected from few home stead gardens from ten districts (Alipurduar, Darjeeling, Jalpaiguri, Cooch Behar, Malda, Nadia, Birbhum, Hoogly, Purulia and East Midnapore) within the West Bengal province in India, showed extensive yellow mosaic symptoms and distorted flower with bleaching of blue color of petals (Supplementary file 1). The detection of the virus was carried out in the Advanced Plant Virus Diagnostic Centre, Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India. DNA extraction was conducted by using plant genomic DNA isolation kit (Sigma-Aldrich, St Louis, USA). The concentration and purity of DNA samples were determined by DeNovix-DS11 spectrophotometer. The presence of the virus was confirmed through polymerase chain reaction (PCR) using begomovirus-specific degenerated primer (Li et al., 2004) followed by RhYMV specific primer (Ilyas et al., 2009). Out of total 250 test samples, 241 were positive for begomovirus specific degenerated primer (96.40%) and out of 241 positive samples, 178 plants were positive to RhYMV specific primer which confirmed 73.85% samples infected to RhYMV. After confirmation of the virus whole genome obtained through Rolling Circle Amplification (RCA) (TempliPhi 100 Amplification Kit, Cytiva) followed by sequencing. After assembled and analyzing the data in CLC Genomics version Workbench 21.0.5 (Matvienko, 2015) whole genome of DNA-A sequence was depositd in the NCBI GenBank database (accession number- PP735226). The BLASTn analyses of the sequence indicated that the isolate from West Bengal, had the 95.44% identity (2616 bp out of 2741 bp) with a RhYMV isolate from snoutbean (Rhyncosia minima) from Pakistan (FM208847). For pathogenecity test (Supplementary file 2), whiteflies reared under polyhouse conditions, acquired the virus from infected plants over 24 hrs (AAP) then allowed viruliferous whiteflies to inoculate the healthy plants for 24 hrs (IAP) and symptoms were monitored under an insect-proof cage. Around two weeks of post- inoculation, all Clitoria plants exhibited mild yellow mosaic symptoms. At 28 dpi, bright yellow to green mosaic appearance, downward curling, and wrinkling of the leaves accompanied with thin tendril with severely distorted flowers were seen which was similar to the samples collected from the different districts. For further confirmation, the infected samples tested positive for RhYMV specific primer were subjected to RCA and whole genome sequencing and found same lineage of RhYMV (accession number- PQ360900). To the best of our knowledge, this is the first report on the incidence of RhYMV in butter fly pea which is to be considered as a new host of RhYMV in India could also act as a reservoir of the virus for further spread to other leguminous plants.

Triticum mosaic virus (TriMV, genus Poacevirus, family Potyviridae) was first reported in 2006 (Seifers et al. 2008) to infect wheat, and since then, it has been established as a constraint for US wheat production (Byamukama et al. 2013). In the field, TriMV often exists as a coinfection with wheat streak mosaic virus (WSMV), and these two viruses interact synergistically to produce severe symptoms and greater yield loss (Byamukama et al. 2012; Tatineni et al. 2022). Both TriMV and WSMV are transmitted by wheat curl mites (Aceria tosichella Keifer) (McMechan et al. 2014). Wheat is the primary host reported for TriMV in the field, but Seifers et al. (2010) established oat, rye, barley, and several other cereals and grasses as hosts under controlled conditions. However, there are no documented cases of TriMV infecting oats in the field. Between 10-25 June, 2023, a total of 273 field oat plants showing foliar yellowing, yellow flecking, and streaking symptoms were collected from four different fields in Nebraska (Big Springs: 41.1029° N, 102.1451° W; Mead: 41.2292° N, 96.4938° W; Odell: 40.0459° N, 96.7984° W; Stumf: 40.5048° N, 101.4223° W). Total RNA was extracted using the MagMax Plant RNA Isolation kit (Thermo Fisher Scientific) and the KingFisher Flex Magnetic Particle Processor (Thermo Fisher Scientific) (Mondal et al. 2023). Sample RNA was assayed with a single-step multiplex reverse transcription polymerase chain reaction (RT-PCR) to determine presence of WSMV and TriMV. Out of 273 symptomatic oat plants, 254 (93.04%) tested positive for at least one virus. Out of total positive samples, 238 were positive for WSMV (93.70 %), 12 plants tested positive for both TriMV and WSMV (4.70%), and 4 plants were infected with TriMV alone (1.60%). As a secondary confirmation, amplified fragments from the TriMV single infection were gel purified using a gel extraction kit (QIAquick) and sequenced (Eurofins Genomics). The nucleotide sequences were analysed using the BlastN program, compiled, and edited in the BioEdit software (Hall 1999). Sequences were deposited in the NCBI GenBank database (accession number PP475806). Nucleotide BLAST searches of the target coat protein (CP) gene showed > 98% identity to the corresponding sequences in TriMV accession MK318274. For further validation, virus inoculum was prepared by grinding field-collected plant material from plants with only TriMV present in 20 mM sodium phosphate buffer, pH 7.0, and then mechanically inoculating two-week-old oats (cv. Shaw n=8) and wheat (cv. Sattler, n=8) plants. Three weeks post-inoculation, all the eight wheat plants exhibited mild yellowing and streaking symptoms, while oat plants did not show obvious foliar symptoms. All wheat and oat plants were further tested positive with DAC-ELISA (antibodies produced against TriMV CP at the USDA-ARS facility in Lincoln, NE) and with RT-PCR. The specific attribution of these symptoms to TriMV in oats is not possible as none produced prominent symptoms. Asymptomatic oat infection from symptomatic field-collected oat samples could be due to oat cultivar differences. Although the prevalence of TriMV in wheat has been established across the Great Plains of the United States, to our knowledge, this is the first report of TriMV infection in US oat fields. Our finding warrant further investigation into the incidence and impact of the virus in oat crop and its potential for serving as a asymptomatic virus reservoir.

Wheat streak mosaic disease (WSMD) is the most important viral disease affecting wheat production in the US Great Plains. WSMD is caused by a single or co-infection of wheat curl mite (WCM)-transmitted wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), and/or High Plains wheat mosaic virus (HPWMoV). Nebraska, one of the US Great Plains states, significantly contributes to the national small grain production and utilizes cereal crops as forage and cover crops. In this study, a state-wide sampling was conducted during the 2023-2025 growing seasons across Nebraska to examine the infection dynamics of the causal agents of WSMD in winter and spring cereal crops. A total of 1624 symptomatic leaf samples were assayed with multiplex RT-PCR. In 2024, co-infection of wheat with WSMV and TriMV was more common, whereas WSMV single infections dominated in 2023. In contrast, WSMV single infections were predominant in field oats compared to WSMV-TriMV coinfection. We found a high incidence of WSMV and TriMV as single and co-infections in other cereal and forage crops: rye (80%), triticale (93%), and barley (72%). HPWMoV was found only in a few wheat samples as co-infections with WSMV or WSMV+TriMV in 2023. The P1 and CP sequences of WSMV and TriMV from different cereal hosts showed minimal sequence diversity, indicating that these viruses circulate freely among diverse cereal hosts. Our data suggest that various commercial, forage, and cover cereal crops are infected with mite-transmitted viruses in field conditions and may serve as a green bridge for the continuum of WSMD in wheat.

Yellow dwarf viruses (YDV), comprising strains of barley yellow dwarf virus (BYDV) and cereal yellow dwarf virus (CYDV), cause economically important diseases in cereal crops in the United States. Although YDV are widespread in Nebraska, information on species distribution and their temporal variability is limited. A statewide survey was conducted during the 2023 and 2024 growing seasons to characterize YDV strain diversity and associated aphid vectors in cereal crops. Total RNA was extracted from symptomatic leaf samples collected from commercial and experimental fields, and viral detection was performed using a multiplex one-step reverse transcription polymerase chain reaction (RT-PCR). Aphid abundance was assessed separately using Berlese funnel from the field-collected plant materials, and species were determined morphologically. In 2023, 68.1% (644/946) of the tested samples were positive for at least one YDV strain, whereas in 2024, only 25.5% (144/564) of the samples tested positive. BYDV-SGV was the predominant strain in 2023, particularly in oats and wheat, whereas SGV+MAV and RPV+SGV strains were the most common coinfections. In contrast, CYDV-RPV and BYDV-MAV predominated in wheat and barley in 2024, whereas PAV was most frequently detected in oats. The bird cherry-oat aphid (Rhopalosiphum padi) was the most abundant aphid species across all crops and locations. These results demonstrate substantial year-to-year variation in the composition of YDV strains in Nebraska cereal crops and confirm the utility of multiplex RT-PCR for field diagnosis. Furthermore, the observed patterns of virus prevalence and aphid abundance emphasize the epidemiological relevance of these findings and support the role of aphid vectors in shaping strain distribution and composition.