First Report of Leaf Spot of Dieffenbachia picta and Aglaonema commutatum Caused by Burkholderia gladioli in Argentina.

Abstract

During May of 2008 (austral autumn), an uncharacterized disease was observed on Dieffenbachia picta (Lodd.) Schott and Aglaonema commutatum Schott in commercial greenhouses in Pontevedra (34°45'6″S, 58°42'42″W), Argentina. Affected plants showed irregular, brown lesions on leaves, approximately 15 to 20 mm in diameter, surrounded by water-soaked haloes that progressed inward from the margins. Water-soaked rotting symptoms were also observed in petioles. Disease incidence approached 80%. Abundant bacterial streaming was observed from lesions when examined at ×100. Bacteria consistently isolated from lesions formed cream-colored, glistening, convex colonies on sucrose peptone agar and produced a yellowish green, diffusible, nonfluorescent pigment on King's medium B. Four isolates from different symptomatic plants were selected for further study. All were aerobic, gram-negative rods that accumulated poly-β-hydroxybutyrate inclusions. In LOPAT tests, all induced a hypersensitive response in tobacco plants, caused soft rot of potato tubers, and were positive for levan, negative for arginine dihydrolase, and variable for oxidase. All isolates oxidized glucose, did not hydrolyze starch and were able to rot onion slices. Colonies developed at 41°C but not at 4°C. With the API 20NE test strips and database (bioMerieux, Buenos Aires, Argentina), all isolates matched (99% identity) Burkholderia cepacia, but their inability to metabolize cellobiose and sucrose further identified them as B. gladioli. For molecular identification, 23S rDNA was amplified by PCR using B. gladioli-specific primers LP1 and LP4, which yielded a 700-bp product (3), and PCR-restriction fragment length polymorphism of 16S rDNA using AluI (2). PCR products were identical to those from the type strain for B. gladioli, ICMP 3950, isolated from Gladiolus spp. that had been included in all tests for comparison. Pathogenicity was verified on D. picta and A. commutatum by spraying the plants with bacterial suspensions in sterile distilled water at 108 CFU/ml with and without wounding the leaves with a sterile needle and also by injection-infiltration of bacterial suspensions at 105 CFU/ml. In addition, another host plant, Gladiolus communis L., was inoculated in the same manner. Controls were sprayed or infiltrated with sterile distilled water. After 48 h in a humidity chamber, plants were kept at 25 ± 3°C in a greenhouse. In all hosts, symptoms were first detected 3 days after inoculation and lesions expanded to resemble natural infections within 4 to 7 days. All strains caused necrosis around the inoculation sites and lesions were identical to those induced by the ICMP reference strain. Bacteria were reisolated from each host tested and then the original and reisolated strains were compared by enterobacterial repetitive intergeneric consensus-PCR (1); DNA fingerprints of the reisolated strains were identical to those of the original strains, thereby fulfilling Koch's postulates. No lesions were observed on controls or on plants inoculated by spraying without wounding, suggesting that bacteria gain entry through wounds. On the basis of PCR and physiological tests the pathogen was identified as B. gladioli (2-4). To our knowledge, this is the first report of B. gladioli on Dieffenbachia and Aglaonema spp.