Draft Genome Sequence of Xanthomonas arboricola pv. pruni Strain Xap33, Causal Agent of Bacterial Spot Disease on Almond.

We report the annotated genome sequence of Xanthomonas arboricola pv. pruni strain Xap33, isolated from almond leaves showing bacterial spot disease symptoms in Spain. The availability of this genome sequence will aid our understanding of the infection mechanism of this bacterium as well as its relationship to other species of the same genus.

First Report on Almond in Europe of Bacterial Spot Disease of Stone Fruits Caused by Xanthomonas arboricola pv. pruni.

Symptoms characteristic of bacterial spot disease of Prunus spp. (4) were observed on almond trees (Prunus dulcis (Mill.) Webb) in 14 localities of Comunidad Valenciana (eastern Spain) and Aragón (northeastern Spain) between 2006 and 2009. Symptoms were first noted in the spring and were observed until leaf fall. Initial infections began on leaves as small, angular, water-soaked spots, which mainly developed toward the tip and along the leaf margins. These water-soaking lesions were surrounded by chlorotic tissue, although chlorosis did not extend more than a few millimeters. Subsequently, the lesions turned light brown, necrotic, and sometimes the necrotic spots fell out. When the lesions coalesced, they produced large necrotic areas. Sometimes premature leaf drop of infected leaves was observed in severely affected trees. Infected fruits initially displayed sunken, corky lesions that oozed gum, which later became raised when the mesocarp dehydrated. Infected fruits either dropped prematurely or remained on trees after harvest. Cankers typical of bacterial spot disease of stone fruit trees were observed on branches and shoots. Isolations from diseased leaves and fruits yielded Xanthomonas-like colonies on YPGA medium (yeast extract, peptone, and glucose agar), which were subsequently purified and characterized. All strains were gram-negative rods, oxidase negative, and strictly aerobic and showed typical biochemical characteristics of the Xanthomonas genus (3). A collection of 70 strains were further identified by PCR with primers Y17CoF/Y17CoR (1) as Xanthomonas arboricola pv. pruni by comparison with reference strains ISPaVe B4 and ISPaVe B6 isolated from Prunus salicina in Italy. A selection of 46 strains were also analyzed by immunofluorescence (IF) and ELISA using commercial polyclonal antibodies from NEOGEN Europe Ltd. (Ayrshire, Scotland, UK) and SEDIAG S.A.S. (Longvic, France), respectively), although ELISA antibodies proved to be not specific for X. arboricola pv. pruni. Pathogenicity was confirmed by inoculation of 70 almond strains and the reference strains on leaves of potted almond trees and/or on detached leaves (2) with bacterial suspensions (107 CFU per ml). One leaf was inoculated at 8 to 10 sites per strain. Characteristic bacterial spot disease symptoms (4) appeared on all inoculated leaves after 1 week of incubation at 25°C and high humidity, but not on the negative controls infiltrated with sterile distilled water. The original pathogen was reisolated from lesions of inoculated leaves and confirmed by biochemical tests, IF and PCR. As observed in Spain, the disease produces serious damage on the most susceptible almond cultivars like Antoñeta, Guara, Marta, Mas Bovera, and Vayro and can be very harmful, with severity of infection depending upon the relative cultivar susceptibility and environmental conditions. Appropriate eradication measures were taken after the causal agent was confirmed as X. arboricola pv. pruni. This pathogen was previously reported on almond in Japan and New Zealand (4). To our knowledge, this is not only the first report on almond in Spain but also in Europe. References: (1) M. C. Pagani. Ph. D. thesis, North Carolina State University, Raleigh, 2004. (2) P. S. Randhawa and E. L. Civerolo. Phytopathology 75:1060, 1985. (3) L. Vauterin et al. Int. J. Syst. Bacteriol. 45:472, 1995. (4) J. M. Young. N. Z. J. Agric. Res. 20:105, 1977.

Characterization of citrus HSVd isolates.

Seven citrus isolates of Hop stunt viroid (HSVd) were subjected to retrotranscription and DNA amplification (RT-PCR), cloning and sequencing. Single stranded polymorphism (SSCP) analysis demonstrated the existence of variability among and within cachexia inducing sources of HSVd. The electrophoretic profiles of SSCP appeared to be able to discriminate between non-cachexia and cachexia sources of HSVd. Sequence analysis demonstrated that the variable (V) domain was very conserved among the cachexia variants. Five nucleotide differences, affecting both the upper (3 nucleotides) and the lower (2 nucleotides) strands of the V domain, were identified as a motif discriminating cachexia and non-cachexia sequences. These five nucleotides affect the organization of a short helical region and two flanking loops of the V domain probably modifying the three-dimensional geometry of the molecule. The stability of the minimum free energy rod-like conformation of the cachexia sequences is lower than the non-cachexia. Information regarding the host effect on the evolution and variability of viroid quasispecies is also provided.

Borage (Borago officinalis) is a New Host of Pseudomonas cichorii in the Ebro Valley of Spain.

Borage (Borago officinalis L.) is an important vegetable crop for consumption in the Ebro Valley of northeastern Spain. During the autumn and spring of the last 10 years, black necrotic lesions were observed in leaf petioles of white-flowered borage plants in greenhouses and seedbeds in Zaragoza, Spain. These lesions developed from the soil line and spread upward to the central vein of leaves. Severely infected leaves of mature and occasionally young plants become rotted. Longitudinal sections through the crown revealed severely necrotic cortical, vascular, and pith tissues. Isolations from infected roots, petioles, and leaves onto King's B medium yielded a gram-negative, rod-shaped bacterium with colonies that were fluorescent under UV light. Bacterial colonies were purified and characterized. The isolates were strictly aerobic, negative for levan production, soft rot of potato, and arginine dihydrolase activity, oxidase positive, and induced hypersensitive reaction in tobacco leaves (2). The bacteria were further identified as Pseudomonas cichorii by comparison of the 49 carbohydrate utilization profiles, API 50 CH (bioMérieux, Marcy l'Etoile, France), with the reference strain ICPPB 2827. Ten lettuce plants used as indicators and borage plants were inoculated by root and petiole injections of bacterial suspensions (108 CFU/ml) of the borage strains and the P. cichorii reference strain ICPPB 2827. Inoculated plants and controls were maintained in a growth chamber at 20 to 25°C with nearly 100% relative humidity. Symptoms of varnish spot, described in lettuce (1), and the black lesions initially observed in borage roots, petioles, and leaves were evident on all inoculated plants at 7 and 5 days after inoculation, respectively. No symptoms developed on control plants. A bacterium with identical characteristics to those described above was reisolated from the black lesions on inoculated plants. To our knowledge, this is the first report of P. cichorii as a pathogen of B. officinalis. Successful infection of borage plants was dependent on high humidity conditions, which is present because of the greater density of mature crops. References: (1) R. G. Grogan et al. Phytopathology 67:957, 1977. (2) R. A. Lelliot et al. J. Appl. Bacteriol. 29:470, 1966.