Characterization of bacterial knot disease caused by Pseudomonas savastanoi pv. savastanoi on pomegranate (Punica granatum L.) trees: a new host of the pathogen.

UNLABELLED: This study aimed to isolate and identify the causal organism causing hyperplastic outgrowths (knots) on stems and branches of pomegranate trees in the Eastern Mediterranean region of Turkey. Bacterial colonies were isolated from young knots on plates containing selective nutrient media. Biochemical tests, fatty acid analysis and PCR were performed to identify possible causal disease agent. Representative isolates were identified as Pseudomonas.pv.savastanoi (Psv) using biochemical tests, fatty acid profiling and PCR. Following inoculation of pomegranate plants (cv. hicaz) with bacterial suspensions, 25 of 54 bacterial isolates caused typical knots at the site of inoculation. PCR analysis, using specific primer for Psv, generated a single amplicon from all isolates. The similarity of the sequence of Turkish pomegranate isolate was 99% similar to the corresponding gene sequences of Psv in the databases. Based on symptoms, biochemical, molecular, pathogenicity tests and sequence analyses, the disease agent of knots observed on the pomegranate trees is Psv. To the best of our knowledge, this research has revealed pomegranate as a natural host of Psv, which extends the list of host plant species affected by the pathogen in the world and Turkey. SIGNIFICANCE AND IMPACT OF THE STUDY: Pomegranate trees were affected by the disease with outgrowths (galls or knot) disease. Currently, there is no published study on disease agent(s) causing the galls or knots on pomegranate trees in worldwide. Bacterial colonies were isolated from young knots. The causal agent of the knot Pseudomonas savastanoi pv.savastanoi (Psv) was identified based on symptoms, biochemical, molecular methods, pathogenicity tests and sequence analysis. To the best of our knowledge, this is the first report of Psv on pomegranate as a natural host, which extends the growing list of plant species affected by this bacterium in the world and Turkey.

First Report of Bacterial Blight of Pomegranate Caused by Xanthomonas axonopodis pv. punicae in Turkey.

Pomegranate (Punica granatum L.) is an increasingly important fruit crop that is widely cultivated in Turkey. Typical bacterial blight symptoms were observed since spring of 2011 in pomegranate orchards located in Antalya Province. Symptoms were characterized by dark brown, angular to irregularly shaped spots on leaves and fruit; cankers on stems, branches, and trunks; and split trunks. The pathogen was isolated from leaf spots on naturally infected plants showing typical symptoms onto yeast dextrose chalk agar. Bright yellow bacterial colonies were consistently isolated. Bacterial strains were characterized as gram negative, oxidase negative, catalase positive, tobacco hypersensitivity positive, and able to produce acid from L-arabinose, D-galactose, D-glucose, and D-mannitol but not from D-xylose. Pathogenicity of the representative bacterial strain Serik-4 was performed on 2-year-old pomegranate plants cv. Hicaz. Leaves were sprayed until runoff with bacterial cell suspensions containing 107 CFU/ml. Inoculated plants were covered with transparent plastic bags to maintain moisture for 48 h. Negative control plants were inoculated with sterile distilled water. Plants were then incubated in a greenhouse at 30°C for 14 days. Symptoms on leaves included dark brown, angular to irregularly shaped water soaked lesions along the veins of the inoculated plants 10 days after inoculation. No lesions developed on the control plants. The symptoms on inoculated plants were similar to those on naturally infected plants. Yellow bacterial colonies were re-isolated from the inoculated plants and identified as the same as the original strain by conventional tests and FAME analysis, thus fulfilling Koch's postulates. Fatty acid methyl ester profiling of the representative strain Serik-4 using GC-MIDI (Microbial Identification Inc, Newark, DE) identified the genus of the bacterium as Xanthomonas. The identity of Serik-4 was further confirmed by amplifying the 16S rRNA gene with the universal primers 27F and 1492R (3) and sequence analysis (GenBank Accession No. KM007073). The 16S rRNA gene sequences of Serik-4 was 99% identical to the corresponding gene sequences of the Xanthomonas axonopodis pv. punicae strain present in the NCBI database (JQ067629.1). High incidence of bacterial blight caused by X. axonopodis pv. punicae on pomegranate has been previously reported in India (2), Pakistan (1), and South Africa (4). To our knowledge, this is the first report of bacterial blight on pomegranate caused by X. axonopodis pv. punicae in Turkey. References: (1) M. A. Akhtar and M. H. R. Bhatti. Pakistan J. Agric. Res. 13:95, 1992. (2) R. Chand and R. Kishun. Indian Phytopathol. 44:370, 1991. (3) D. J. Lane. Page 115 in: Nucleic Acid Techniques in Bacterial Systematics, 1991. (4) Y. Petersen et al. Australas. Plant Pathol. 39:544, 2010.