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Pseudomonas viridiflava Causing Necrotic Leaf Spots and Defoliation on Hebe spp. in Northern Spain

Hebe spp. is gaining interest as an ornamental crop in Spain. In October of 2003, plants of Hebe spp. (cv. Pink Paradise, Recurva, and Topiaria) grown in a nursery located in a northern region of Spain (Principality of Asturias) developed dark reddish spots in leaves. The spots (favored by high-humidity conditions) appeared initially in internal and lower leaves, but eventually progressed to the rest of the plant causing extensive defoliation. Samples from symptomatic leaves were processed for microbiological analysis. From the samples, Stemphylium sp. and fluorescent Pseudomonas sp., identified by conventional microbiological methods (1,3), were consistently recovered. Two isolates of the fungi and two of the bacterium (LPPA 365 and LPPA 366) were used in artificial inoculations of healthy plants (cv. New Zeeland) and all were performed in duplicate. A Stemphylium sp. was inoculated by irrigation of four seedlings with a suspension obtained by homogenization of the mycelium collected from two plates of potato dextrose agar in 100 ml of sterile distilled water. An equal number of seedlings were sprayed with a bacterial suspension (10⁸ CFU/ml) in yeast extract peptone glucose broth. The same methods were used to inoculate all four combinations of fungi and bacteria. The seedlings were maintained at 22°C during 1 month with a 16/8-h photo-period, but in inoculations with bacteria they were initially kept under transparent plastic bags for 48 h to facilitate entry. Seedlings treated with sterile distilled water were included as controls. Although a powdery film of sooty mold was observed in some of the leaves irrigated with one of the two Stemphylium sp. strains, rusty lesions and defoliation did not occur on plants inoculated with the fungal isolates alone. In contrast, symptoms comparable with those observed in the field developed in plants inoculated with the bacteria, either alone or together with the fungus. Reisolation of fluorescent Pseudomonas sp. from symptomatic leaves taken from inoculated plants confirmed pathogenicity on Hebe spp. The pathogenic isolates were tentatively identified as atypical Pseudomonas viridiflava on the basis of biochemical tests (2,3), and their identity was then confirmed by sequencing of the gene encoding the 16S rRNA (3; GenBank Accession Nos. AM182933 and AM182934 for LPPA 365 and 366, respectively). To our knowledge, this is the first report of P. viridiflava as the cause of necrotic leaf spots and defoliation in Hebe spp.

References: (1) H. L. Barnett and B. B. Hunter. Illustrated Genera of Imperfect Fungi. 3rd ed. Burgess Publishing Co, Minneapolis, MN, 1972. (2) A. J. González et al. Appl. Environ. Microbiol. 69:2936, 2003. (3) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470. 1966.