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

Keywords: Cucurbita pepo, gyrase subunit B, lesion, soft rot, cucurbits, bacterial disease

In August 2012, leaves of pumpkin (Cucurbita pepo cvs. Gladiator, Aladdin, Apollo, and Super Hero) in Kent County, Ontario, Canada were observed with 1-4 mm irregular-shaped light brown to tan lesions, often with a chlorotic halo. Mature fruit had 2-4 mm light brown to tan sunken lesions with dark borders (Fig. 1), and later developed severe soft rot. Approximately 35 ha were affected with more than 50% of foliage and 60% of fruit damaged. Isolations were made from fruit using the method described by Cuppels et al. (1990) but with tryptic soy agar. More than 95% of colonies isolated were opaque, light to bright yellow, glistening, circular, and flat. Isolates were gram-negative. 

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). The 16S DNA sequences of CT12PT3 and CT12PT1A were obtained with primers designed by the Pest Diagnostic Clinic, University of Guelph (5’GCYTAACACATGCAAGTCGA-3’ and 5’-GTGTGTACAAGNCCCGGGAA-3’). A BLAST search of the GenBank database revealed that both sequences (GenBank Accession Nos. KJ817203 and KJ817204) had 100% nt identity to the 16S DNA sequences of Xanthomonas dyei (NR_104949), Xanthomonas pisi (AB680442), Xanthononas vesicatoria (AY288080 and AF123088) and Xanthononas cucurbitae (AB680438). 

In addition, 928 bp of the gyrase subunit B gene (gyrB) was sequenced with primers gyrB-F and gyrB-R (Hamza et al., 2012). Both Ontario isolates (KJ817205 and KJ817206) had 100% nt identity with gyrB of X. cucurbitae (HM569161.1 and HM569162.1). The next closest matches were Xanthomonas axonopodis pv. citrumelo (CP002914.1) and Xanthomonas campestris pv. raphani (CP002789.1), with 94% nt identity. A neighbour-joining tree from a ClustalW multiple sequence alignment of the overlapping regions of these gyrB sequences and 14 other Xanthomonas species showed that sequences from both Ontario isolates clustered only with HM569161 and HM569162 with high bootstrapping values (Fig. 2). These results strongly suggest the isolates are X. cucurbitae.

To confirm pathogenicity of the isolates, a bacterial suspension (1 x 10⁷ cfu/ml) of each isolate was applied to the leaves of four pumpkin plants (cv. Howden) using a hand-held mist sprayer. Plants were covered for 24h under a translucent plastic box and maintained under artificial light with 16h day length at 24-28°C. Small lesions with slight chlorotic haloes were observed on all inoculated plants 10 days post inoculation (Fig. 3). No symptoms were observed in the water control. Bacteria were isolated from lesions as previously described. The gyrB sequences of these isolates had 100% nt identity to the original isolates, and the BIOLOG substrate utilisation tests showed 100% and 98.9% similarity to CT12PT3 and CT12PT1A respectively. The presence of bacterial spot caused by X. cucurbitae poses a new threat to pumpkin and squash production in Ontario.