Determining role of bacteria associated with bacterial spot on pumpkin and squash fruit in the North Central Region of the United States

Bacterial spot, caused by Xanthomonas cucurbitae, is an important disease on pumpkin and squash in the United States. To assess the occurrence of bacterial spot in pumpkin and winter squash fields in the North Central Region (NCR), field surveys were conducted within three weeks of harvest in 2012 and 2013. During the surveys, 82 pumpkin and 51 squash fields in 2012, and 98 pumpkin and 28 squash fields in 2013 were visited in nine states. Symptomatic fruit were observed in 70 of 82 pumpkin and 47 of 51 squash fields in 2012, and 89 of 98 pumpkin and 24 of 28 squash fields in 2013. The average incidence of fruit with bacterial spot in all pumpkin and squash fields surveyed was 27.20 and 18.70% in 2012, and 22.72 and 18.21% in 2013, respectively. The highest incidence of fruit with bacterial spot was 96.67 and 93.33% in pumpkin fields in 2012 and 2013, respectively. Infected fruit had developed lesions, measuring 1-3 mm in diameter. Lesions measuring up to 10 mm in diameter were observed on some pumpkin and squash cultivars. X. cucurbitae was isolated from symptomatic fruit and identified based on the colony morphology, polymerase chain reaction (PCR) test using RST2/RST3 primers, and pathogenicity test on pumpkin ‘Howden’ in a greenhouse. Both multilocus sequence typing (MLST) and restriction associated DNA sequence analysis (RADseq) were used to study genetic variation among X. cucurbitae isolates. Twenty X. cucurbitae isolates from Illinois, Indiana, Kansas, Ohio, and Wisconsin were subjected to MLST analysis with three housekeeping genes (fusA, gltA, and gyrB) sequenced for each isolate. Results of MLST analysis showed no genetic variation among 20 X. cucurbitae isolates. A phylogenetic tree was generated for various Xanthomonas species using concatenated sequences, which shows a closer phylogenetic relationship between X. cucurbitae and X. campestris. In RADseq analysis, a total of 156,985 single nucleotide polymorphisms (SNPs) were detected across 81 X. cucurbitae isolates, a X. cucurbitae strain from American Type Culture Collection, and an Escherichia coli strain. Seven clusters were distinguished based on genetic distance matrix (Clusters I, II, III, IV, V, VI, and VII), among which Illinois isolates exhibited more genetic diversity. To study non-X. cucurbitae bacteria associated with bacterial spot on pumpkin and squash fruit, a total of 925 and 425 non-X. cucurbitae isolates were collected from symptomatic fruit in 2012 and 2013, respectively. To identify non-X. cucurbitae bacteria at the genus level, 208 isolates from 2012 and 104 isolates from 2013 were randomly selected and restriction fragment length polymorphism (RFLP) was used. Based on the RFLP results, the selected isolates were first separated into groups and then 10 genera were identified, which included Arthrobacter, Enterobacter, Enterococcus, Erwinia, Exiguobacterium, Microbacterium, Pantoea, Peanibacillus, Pectobacterium, and Pseudomonas. In-vitro studies of interactions between non-X. cucurbitae and X. cucurbitae showed that Pantoea isolates had antibacterial activity against X. cucurbitae. None of the tested non-X. cucurbitae isolates was pathogenic in pumpkin ‘Howden’ plants in the greenhouse tests