<i>Serratia ureilytica</i> causing cucurbit yellow vine disease in cucurbits in Indiana

Indiana is one of the major pumpkin-producing states in the U.S. with a production value reaching $40 million in 2024 (USDA-NASS 2024). In July 2025, several Cucurbita pepo L. (spaghetti winter squash and summer squash) and C. moschata Duchesne (winter squash) plants in Tippecanoe County, Indiana, exhibited yellowing, stunting, and scorching margins of leaves. Approximately 10% of the plants in a one-hectare field exhibited symptoms typical of cucurbit yellow vine disease (CYVD), caused by Serratia ureilytica (Mphande et al. 2025a), and vectored by the squash bug (Anasa tristis DeGeer). In the same year, samples of C. pepo (pumpkin), collected in a sentinel plot located in Knox County, Indiana, showed CYVD-like symptoms. To confirm the identity of the causal agent, a total of 37 samples across the two counties were collected and analyzed. Crown tissue (500 mg) was surface sterilized with 70% ethanol for 2 minutes and rinsed with sterile water. Tissue was macerated in 500 μL of water. The suspension was streaked on Luria Broth Media amended with tetracycline (20 μg/ml) and incubated at 28 °C for 24 hours (Mphande et al. 2025b). Five of the 37 samples tested positive for S. ureilytica. Pure colonies were transferred to new plates, and direct PCR was done on isolates 25236 (from C. pepo ‘Pinnacle’) and 25237 (from C. pepo ‘Yellow crookneck’) using Serratia-specific primers YV1-YV4, CYVD-specific primers A79F-A79R, and primers that amplify the gyrB gene (Zhang et al. 2005; Besler and Little 2016). Resulting amplicons were then Sanger-sequenced. Sequences for YV1-YV4 (acc. PX460593, PX460594), A79F-A79R (PX513359, PX513360), and gyrB (PZ019506, PZ019507) shared 99-100% nucleotide identity with S. ureilytica homologous sequences. To complete Koch’s postulate, plants were inoculated under greenhouse conditions using isolates 25236 and 25237, as well as both C. pepo cultivars mentioned above. For each type, four treatments were established: non-inoculated control, mock, isolates 25236, and 25237. Mocks were inoculated with PBS only. Each treatment consisted of five plants as biological replicates. Plants were inoculated using a syringe inoculation method (Mphande et al. 2025b). Briefly, 300 μL of a 1 OD600 bacterial suspension in 1X PBS buffer was used to pierce the plant stems three to four times at a 45-degree angle. Three weeks later, symptoms were seen in inoculated plants. In cv ‘Yellow crookneck’, 4 out of 5 plants inoculated with isolate 25236 showed symptoms, and 1 out of 5 plants inoculated with 25237 showed symptoms. In cv ‘Pinnacle,’ all plants inoculated with 25236 and 25237 showed symptoms. Isolations were made from the crowns of inoculated plants, and the isolates were subsequently amplified with the three sets of primers mentioned previously. The resulting sequences matched the original isolates. A maximum-likelihood phylogenetic tree was constructed using Multi-Locus Sequence Analysis of Serratia isolates 25236 and 25237. In the resulting tree, both isolates clustered with S. ureilytica sequences retrieved from GenBank. Mphande et al (2025a) utilized strains of S. ureilytica from multiple U.S. states, including Indiana, to perform phylogenomic analyses. However, to the best of our knowledge, this is the first report to formally confirm and document S. ureilytica as the causal agent of CYVD in Indiana. This finding provides critical information for growers, extension educators, and diagnosticians, to recognize and respond to this emerging disease.