First Report of Bacterial Stem and Pith Necrosis of Tomato Caused by Pseudomonas viridiflava in Spain

Stem necrosis caused by Pseudomonas viridiflava has been reported as an important disease of greenhouse-grown tomato (Aysan et al. 2004). In May 2014, wilting of branches was observed in tomato plants (Solanum lycopersicum) of a Roma-type variety (Eliseo) in a commercial greenhouse located in the Axarquía region (southern Spain). Lengthwise sections of symptomatic branches showed brown discoloration, necrosis, and often hollowing and collapse of the pith of the stem. Although wilting was severe and affected many plants, on apparently healthy branches, fruits developed and exhibited no symptoms. Surface-sterilized segments of symptomatic stems were placed on Luria Bertani plates, and bacterial growth was observed from tissue margins after 48 to 72 h at 24°C. When streaked on fresh plates, isolated colonies were yellowish in appearance. The initial determination of bacterial species was performed by molecular methods. A 877-bp DNA fragment of the 16S rDNA gene was amplified using primer F (5′-GTGCCAGCAGCCGCGGTAA-3′) and R (5′-AGGCCCGGGAACGTATTCAC-3′), and fully sequenced. The amplified fragment comprised the differential hypervariable (hv) region of Pseudomonas strains described by González et al. (2003) and BLAST analyses revealed a 100% match with Pseudomonas viridiflava, including the presence of a HinfI site and absence of a SacI site, regarded as a signature for this species. A second sampling performed one month later, when symptoms had extended to two other greenhouses in the same plot, confirmed identification of P. viridiflava. Molecular data were corroborated in two representative isolates with biochemical and physiological tests, including typical and atypical P. viridiflava isolates (González et al. 2003) and also P. syringae pv. tomato as a negative control. The bacteria isolated were aerobic and gram-negative. In King’s B medium, their fluorescence was similar to that of the atypical P. viridiflava isolate. In LOPAT tests, they behave mostly as typical P. viridiflava; they were oxidase- and arginine dehydrolase-negative, pectinolytic-positive, and induced hypersensitive reaction on tobacco leaves. However, they produced mucoid colonies on hypersucrose medium, as the atypical P. viridiflava. They produced acid from glucose, fructose, sorbitol, L(+)arabinose, D(-)mannitol, but not from D(-)arabinose or sucrose, and they hydrolyzed gelatin and did not grow at 37°C. P. viridiflava identification was fully confirmed with specific primers PVF/PVR, 7F/7R, and 20F/20R (Heydary et al. 2012). Pathogenicity assays were performed on tomato plants (cultivar Moneymaker) at the three-leaf stage by injecting ∼100 μl of a bacterial suspension (10⁸ CFU/ml) into the stems below the cotyledons. Control plants were mock inoculated in the same way with sterile water. All plants inoculated with our isolates and the typical P. viridiflava isolate developed symptoms—a characteristic thinning of the stems and leaf wilting—whereas control plants remained asymptomatic. Twelve plants were used per treatment and the assay was repeated twice. P. viridiflava was reisolated from symptomatic plants, fulfilling Koch´s postulates. To our knowledge, this is the first report of P. viridiflava as the cause of tomato pith necrosis in Spain.