First Report of Bacterial Leaf Spot of Crown-of-Thorns (Euphorbia milii) Caused by Xanthomonas axonopodis pv. poinsettiicola in Taiwan

Abstract

Crown-of-thorns (Euphorbia milii; Euphorbiaceae) is a popular plant widely grown as a potted flower or hedge in many countries (Marys and Romano 2011). In 2017, leaf spot symptoms were observed on potted E. milii in a plant market located in Nantun District, Taichung, Taiwan. Over 90% of the plants onsite were infected. Leaf lesions were round, had necrotic centers, and had margins that appeared yellow when viewed from the upper leaf surface and water-soaked when observed from underneath. Symptomatic tissues were cut and examined under a light microscope. Samples with bacterial streaming were streaked onto nutrient agar, and yellow, round colonies were recovered. Strains Eu1, Eu2, and Eu3 were obtained from three different plants. They produced yellow mucoid colonies on yeast dextrose carbonate (YDC) agar, grew at 35°C or on SX medium (a semiselective medium), and could degrade starch, esculin, casein, and lipid, similar to many known xanthomonads (Schaad et al. 2001). The isolates also induced hypersensitive reaction on tobacco (cv. Taiyen 5) 48 h postinfiltration. Identification of Eu1 to Eu3 using the Biolog microbial identification system (Biolog, Hayward, CA) showed that they are Xanthomonas campestris pv. poinsettiicola (X. axonopodis pv. poinsettiicola) with probabilities of 84 to 98%. Multilocus sequence analysis targeting partial sequences of the lacF, lepA, fusA, gapA, gltA, and gyrB genes (Almeida et al. 2010) were conducted, and Eu1 to Eu3 had identical sequences for every fragment (accession nos. MH068853 to MH068858). Comparing their concatenated sequences (2,744 bp) with those from reference/type strains of other xanthomonads able to produce symptoms on spurges (Rockey et al. 2015) revealed that Eu1 to Eu3 share 98.65% sequence identity with X. axonopodis pv. poinsettiicola LMG849ᴾᵀ and have lower sequence similarity with X. hortorum pv. pelargonii LMG7314ᴾᵀ (94.06%), X. codiaei LMG8678ᵀ (93.55%), and X. campestris pv. campestris LMG568ᴾᵀ (93.51%). Koch’s postulates were fulfilled by spraying bacterial suspensions (OD₆₀₀ = 0.3; on average 7 × 10⁸ CFU/ml; in 0.02% Silwet L-77) onto all aboveground parts of cutting-propagated E. milii (approximately 10 cm tall; var. Olympus). The test was conducted with every isolate, each inoculated onto three plants. As controls, three plants were sprayed with bacteria-free solutions (0.02% Silwet L-77). Inoculated E. milii were placed separately in plastic bags for 48 h to maintain humidity. Within 1 month, all inoculated plants developed leaf spots similar to those found in the plant market, and the controls did not. Bacteria reisolated from inoculation-infected plants had the same colony morphologies as Eu1 to Eu3 on YDC agar. Colony polymerase chain reaction and sequencing of gapA of the reisolates showed that their sequences were identical to those of Eu1 to Eu3. A representative strain, Eu1, was spray-inoculated onto four poinsettia plants (approximately 8 cm in height) using the same inoculum density as mentioned above. Leaf spots appeared on all of the treated plants but not on four plants sprayed with bacteria-free solution as mentioned above. Natural infection of poinsettia and E. milii by X. axonopodis pv. poinsettiicola has been reported in the United States (Rockey et al. 2015). In Taiwan, however, only bacterial leaf spot of poinsettia has been recorded (Lee et al. 2006). This study is the first report of X. axonopodis pv. poinsettiicola naturally infecting E. milii in Taiwan. Because many nurseries and plant markets in Taiwan often grow E. milii and other spurges in close proximity and at high density, care should be taken to prevent the spread of the disease within and across host species.