First Report of Xanthomonas hortorum pv. hederae Causing Bacterial Leaf Spot of Hedera helix in Taiwan

Abstract

Hedera helix (ivy) is a popular evergreen commonly grown worldwide as outdoor and indoor plants. In 2018, symptoms similar to bacterial leaf spot were observed on potted ivies in two nurseries, one located in Tianwei Township, Changhua County (observed in January) and the other in Nantun District, Taichung City, Taiwan (observed in May). In both locations, leaf spot symptoms were seen on over 90% of the plants. The lesions were irregularly shaped and were necrotic or water-soaked. A total of seven infected plants were sampled and brought to the laboratory, one collected in Changhua and six sampled in Taichung. Diseased tissues were cut, and bacterial streaming was observed under a light microscope. The samples were streaked onto nutrient agar (NA) and onto NA with 0.2% yeast extract and then were incubated at 25°C. Round yellow colonies were recovered from all seven plants, and seven independent bacterial strains (Hed1 to Hed7) were isolated (each from a different plant). Hed1 was obtained from the plant collected in Changhua, whereas the others were isolated from plants collected in Taichung. All seven strains produced yellow mucoid colonies on yeast dextrose calcium carbonate agar, similar to most known xanthomonads (Schaad et al. 2001). They tested positive for esculin degradation, weak positive for casein hydrolysis, and negative for the abilities to degrade Tween 80 and starch (Schaad et al. 2001). Infiltration of these strains’ suspensions into tomato leaves (cv. Known-you 301) induced hypersensitive responses. Identification of Hed1 to Hed7 was conducted via multilocus sequence analysis targeting the fusA, gyrB, gapA, gltA, lacF, and lepA genes (Almeida et al. 2010). Gene fragments were sequenced for all seven strains. For every fragment tested, Hed1 to Hed7 all had the same sequences (GenBank accession nos. MK124762 to MK124767). Comparing the concatenated 2,745-bp sequences of Hed1 to Hed7 among each other and against sequences included in the Plant-Associated Microbes Database (http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl) (Almeida et al. 2010) revealed that all seven strains shared identical sequences with the type strain of Xanthomonas hortorum pv. hederae (LMG733) and had lower than 98% identity with the other Xanthomonas species and pathovars. To fulfill Koch’s postulates, three strains (Hed1, Hed2, and Hed6) were randomly selected and spray inoculated onto potted ivies at a concentration of approximately 1 × 10⁸ CFU/ml (in 0.02% Silwet L-77). Each strain was inoculated onto three plants. Three additional plants sprayed with 0.02% Silwet L-77 solution without bacteria served as controls. The plants were then bagged in plastic bags for 3 days to maintain high humidity. Within 3 weeks, necrotic leaf spots developed on all bacteria-inoculated plants but not on the control plants. Bacterial strains were reisolated from the plants inoculated with Hed1, Hed2, and Hed6, and all reisolates shared the same gapA sequence with the original strains. Bacterial leaf spot on ivy has been reported in Greece, Japan, and other parts of the world (Pirc et al. 2012; Suzuki et al. 2002; Trantas et al. 2016). The present study is, to our knowledge, the first report of the occurrence of this disease in Taiwan. Ivies are often planted in shaded/indoor areas at high densities and are watered frequently in nurseries. These conditions could favor the development of bacterial leaf spot and the transmission of its pathogen.