A New Report of Xanthomonas cucurbitae Causing Bacterial Leaf Spot of Watermelon in Georgia, USA

Abstract

In June 2012, watermelon leaves (Citrullus lanatus (Thunb.) Matsum. & Nakai) were observed with angular, necrotic spots with chlorotic halos in a field in Telfair County, GA. The field exhibited 20 to 25% disease incidence with no observable symptoms on fruits. Isolations were made from foliar lesions of 30 leaves onto yeast extract-dextrose–CaCO3 (YDC) agar medium (3). Yellow-pigmented, Xanthomonas-like colonies were observed after 48-h incubation at 28°C from 100% of the samples. Bacteria harvested were gram-negative, oxidase-negative, indole-negative, hydrolyzed starch and esculin, and formed pits on crystal violet pectate and carboxymethyl cellulose media. The bacterial isolates did not produce nitrites from nitrates but produced hypersensitive reactions on tobacco upon inoculation with 1 × 108 colony-forming units (CFU)/ml. These characteristics are typical of members of the Xanthomonas campestris group. The genus Xanthomonas was confirmed using conventional PCR with genus-specific primers RST2 (5′AGGCCCTGGAAGGTGCCCTGGA3′) and RST3 (5′ATCGCACTGCGTACCGCGCGCGA3′), which produced an 840-bp band. Universal primers fD1 and rD1 (1) were used to amplify the 16S rRNA gene from four isolates and amplified products were sequenced and BLAST searched in GenBank. The nucleotide sequences of the isolates showed 97 to 98% similarity to X. cucurbitae (Accessions AB680438.1 and Y10760), X. campestris (HQ256868.1), X. arboricola (JF835910.1), X. oryzae pv. oryzicola (CP003057.1) and X. campestris pv. raphani (CP002789.1). PCR amplification and sequencing of the atpD gene (ATP synthase, 720 bp) showed 99% similarity with X. cucurbitae when BLAST searched in GenBank (HM568911.1). X. cucurbitae was not present in the database of BIOLOG (Biolog, Hayward, CA); therefore, substrate utilization tests of three isolates were compared with substrate utilization patterns of Xanthomonas groups reported by Vauterin et al. (4). The watermelon isolates displayed 93.7, 89.5, and 89.5% similarity with the reported BIOLOG metabolic profiles of X. campestris, X. cucurbitae, and X. hortorum, respectively, of Xanthomonas groups 15, 8, and 2. However, none of the isolates were amplified using a conventional PCR assay with X. campestris pv. campestris and X. campestris pv. raphani-specific primers (2), indicating a closer relationship with X. cucurbitae. When 2-week old watermelon seedlings cv. Crimson sweet (n = 4/isolate/experiment) were inoculated by spraying with a suspension of 1 × 108 CFU/ml, 100% of the seedlings developed symptoms (water soaked angular lesions that developed into necrotic spots) 14 days after planting under greenhouse conditions (~30°C and ~70% RH). Ten control plants inoculated with sterile water remained asymptomatic. Bacterial colonies were reisolated from symptomatic seedlings that showed similar characteristics to those described above. The identity of isolated colonies was confirmed by amplifying and sequencing the 16S rRNA gene, which showed 97 to 98% similarity to X cucurbitae accessions in GenBank. To our knowledge, this is the first report of X. cucurbitae on watermelon in Georgia since the 1950s. References: (1) Y. Besancon et al. Biotechnol. Appl. Biochem. 20:131, 1994. (2) Leu et al. Plant Pathol. Bull. 19:137, 2010. (3) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. APS Press. St. Paul, MN, 2001. (4) Vauterin et al. Int. J. Syst. Bacteriol. 45:472, 1995.

First report of bacterial spot caused by Xanthomonas cucurbitae on pumpkin in Italy

“…The geographical origin of the pathogen is unknown, but its introduction might be connected with the international trade of pumpkin seed. Our observation of X. cucurbitae leaf spot on watermelon supports the report of Dutta et al (2013). Further research is needed to assess the host range of the Italian isolates of X. cucurbitae amongst cucurbits.…”

Development of Bacterial Spot, Incited by Xanthomonas cucurbitae, in Pumpkin Fields

“…Field location (county) Dutta et al, 2013;Williams and Zitter, 1996) and other pumpkin-growing areas of the World (Lamichhane et al, 2010;Pruvost et al, 2008;Williams and Zitter, 1996). Bacterial spot also occurs widely on gourds and winter squash in Illinois (unpublished data).…”

“…RST2/3 primers have been widely used to identify Xanthomons spp. from other bacteria (Dutta et al, 2013;Pruvost et al, 2008;Schaad et al, 2001). These primers produce 1400 bp amplicon with X. cucurbitae, while the amplicons produced by other Xanthomonas spp.…”

“…This disease was first reported as bacterial leaf spot on 'Hubbard' squash in New York in 1926(Bryan, 1930. Subsequently, the disease was identified in other cucurbit-growing areas in Asia, Australia, Europe, and North America on cucumber, pumpkin, squash, and watermelon (Bineeta et al, 1999;Dutta et al, 2013;Lamichhane et al, 2010;Pruvost et al, 2008;Pruvost et al, 2009;Williams and Zitter, 1996).…”

First report of bacterial spot (Xanthomonas cucurbitae) of pumpkin in Ontario, Canada

“…Substrate utilisation profiles (Biolog, Hayward, CA) of Ontario isolates CT12PT1A and CT12PT3 were compared with those described for Xanthomonas DNA homology groups (Vauterin et al 1995). Utilisation patterns of the 95 substrates in the BIOLOG assay for CT12PT3 and CT12PT1A had 96.8% and 94.7% identity with members of Group 8 (X. cucurbitae), and both had 95.8% identity with at least one of the three X. cucurbitae isolates reported by Dutta et al (2013) (Dutta, pers. communication).…”