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.

First Report of Pepper as a Natural Host for Pelargonium zonate spot virus in Spain.

Pelargonium zonate spot virus (PZSV) was first reported on Pelargonium zonale (L.) L'Hér. ex Aiton and later on tomato in Italy, Spain, France (1), and the United States (2). In Spain, PZSV was first detected in 1996 in tomato plants of cv. Royesta from greenhouses in Zaragoza Province (3) and subsequently in tomato in the Catalonia and Navarra areas. In April 2006, symptoms of PZSV were found at high incidence on tomato in a greenhouse in Huesca, Aragón (northeastern Spain). Randomly distributed pepper plants (Capsicum annuum L.) of cv. Estilo F1 growing in the same greenhouse showed severe foliar chlorotic ringspots and line patterns similar to those observed in tomato. Samples from symptomatic peppers and tomatoes and one asymptomatic weed of Rubia tinctorum L. tested positive by double-antibody sandwich (DAS)-ELISA using polyclonal antibodies against PZSV (Agdia Inc., Elkhart, IN and DSMZ, Braunschweig, Germany) as did a Spanish PZSV isolate used as a positive control (3). Sap extracts from two tomatoes, three peppers, and the single R. tinctorum plant were mechanically inoculated to 22 indicator species, including pepper and tomato. On 17 of 22 species inoculated, sap from symptomatic tomatoes and peppers elicited local or systemic symptoms similar to those reported earlier for PZSV isolates (3). Systemic symptoms were mainly mosaic, chlorotic, and necrotic line patterns and ringspots on leaves of most indicator species, closely resembling those observed on the greenhouse pepper and tomato plants. Symptoms on inoculated tomatoes also included stem necrosis and death. Reactions of indicator species did not indicate the presence of any other pepper- or tomato-infecting viruses. Both field infected and mechanically inoculated plants of pepper cvs. Yolo Wonder and Doux des Landes were maintained in the greenhouse until the development of fruit symptoms. Only fruits of cv. Yolo Wonder showed dark green and slightly depressed circles on their surface. Local and systemic infection by PZSV was confirmed by DAS-ELISA in most inoculated plants. Total RNA from leaves of field or inoculated plants was used as template for amplification by reverse transcription (RT)-PCR with primers R3-F and R3-R that are specific for the PZSV 3a gene (2), and amplicons were sequenced directly. The sequences of 697 nt from pepper and tomato isolates from the same greenhouse were identical (GenBank Accession Nos. CQ178217 and CQ178216, respectively) and had 96.1% identity to nucleotides 384 to 1,080 in PZSV RNA-3 (NC_003651). Our results confirm the natural infection of pepper plants in Huesca by PZSV. To our knowledge, this is the first report of pepper as a natural host for PZSV, a significant finding considering the potential risks of PZSV dispersion whenever tomato and pepper coexist, particularly in greenhouses and nurseries. References: (1) M. Finetti-Sialer and D. Gallitelli. J. Gen. Virol. 84:3143, 2003. (2) H. Y. Liu and J. L. Sears. Plant Dis. 91:633, 2007. (3) M. Luis-Arteaga and M. A. Cambra. Plant Dis. 84:807, 2000.

First Report of Natural Infection of Greenhouse-Grown Tomato and Weed Species by Pelargonium zonate spot virus in Spain.

Tomato (Lycopersicon esculentum Mill.) plants showing severe chlorotic and necrotic ringspots, line patterns on leaves, and concentric chlorotic ringspots on stems and fruits were observed in plastic greenhouse-grown tomato crops cv. Royesta during the spring of 1996 in Zaragoza province, Northeast Spain. Symptoms were similar to those associated with Pelargonium zonate spot virus (PZSV) infection on tomato in Italy (1,2). The causal agent was mechanically transmitted from leaf, fruit, and stem samples to several indicator species. The following host reactions were recorded: chlorotic local lesions on Chenopodium amaranticolor, C. quinoa, Cucumis sativus, and Cucurbita pepo, and systemic reactions, sometimes associated with localized reactions, on Capsicum annuum 'Doux des Landes' and 'Yolo Wonder', Datura stramonium, Gomphrena globosa, Nicotiana clevelandii, N. glutinosa, N. megalosiphon, N. rustica, N. sylvestris, N. tabacum 'Paraguay', 'Samsun', and 'Xanthi nc', Ocimum basilicum, Petunia hybrida, Physalis floridana, Solanum melongena, and Vigna unguiculata. Symptoms obtained in indicator species were erratic. During the spring of 1999, naturally occurring symptoms appeared again on tomato plants, cultivars Royesta and Bond, growing in greenhouses in the same area. Positive serological reactions with the enzyme-linked immunosorbent assay (ELISA) using a commercial PZSV antiserum (Agdia Inc.), developed against an Italian isolate of PZSV, were obtained with extracts from leaves, stems, and fruits of tomato plants naturally infected (1999) and from systemically infected indicator species mechanically inoculated with sap from tomato samples (1996 and 1999). Serological results were confirmed by molecular hybridization analysis using a PZSV-specific riboprobe (D. Gallitelli, personal communication). Some of the weeds growing around the greenhouses (Capsella bursa-pastoris, Diplotaxis erucoides, Picris echioides, and Sonchus oleraceus) also tested positive for PZSV (A405nm values greater than three times that of healthy plants). However, other weed species such as Anacyclus tomentosus, Beta maritima, Cardaria draba, Malva sylvestris, Medicago sp., Polygonum aviculare, Rumex sp., and Sisymbrium irio tested negative, while results from tests on Borago officinalis, Bromus rigidus, and Convolvulus arvensis were inconclusive. Symptoms like those of naturally infected tomato plants were reproduced by mechanically inoculating tomato seedlings with sap from PZSV-infected tobacco (Nicotiana glutinosa and N. tabacum 'Paraguay') or from Physalis floridana plants. References: (1) D. Gallitelli. Ann. Appl. Biol. 100:457, 1982. (2) C. Vovlas et al. Inform. Fitopatol. 2:39, 1986.

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.

Natural Infection of Field-Grown Borage (Borago officinalis) by Alfalfa mosaic virus in Spain.

Alfalfa mosaic virus (AMV) has a wide host range and is distributed throughout the world. It causes disease in several vegetable crops, including bean, celery, lettuce, pea, pepper, and tomato (1). In Spain, it has been found naturally infecting alfalfa, pepper, and tomato. During the autumn of 1999, in the area of Zaragoza (northeastern Spain), several plants expressing foliar yellow mosaic symptoms were observed in borage grown for human consumption in open field plots. The commercial value of the symptomatic plants was greatly reduced. The symptoms were similar to those previously obtained in greenhouse-grown borage plants mechanically inoculated with three tomato isolates of AMV (2). The following indicator species, including virus-free borage plants, were mechanically inoculated with sap from leaves of symptomatic borage plants, and reactions were recorded: chlorotic and necrotic local lesions on Tetragonia expansa and Vigna unguiculata; chlorotic local lesions and systemic mosaic on Chenopodium quinoa, C. amaranticolor, Cucumis sativus 'Marketmore', Gomphrena globosa, and Nicotiana glutinosa; systemic mosaic, sometimes associated with localized reactions, on Ocimum basilicum, Capsicum annuum 'Doux des Landes' and 'Yolo Wonder', N. benthamiana, N. clevelandii, N. rustica, N. sylvestris, N. tabacum 'Paraguay' and 'Xanthi nc', Petunia hybrida, Physalis floridana, and Solanum melongena 'Cerna krazavitska'. The reactions are in agreement with the indicator host reactions described for AMV (1). Symptoms on virus-free borage plants mechanically inoculated with sap from symptomatic borage and from inoculated C. amaranticolor and O. basilicum experimental hosts were similar to those observed in naturally infected borage. Positive serological reactions (A405 values more than three times greater than those of the negative controls) in double-antibody sandwich enzyme-linked immunosorbent assay using commercially prepared antiserum against AMV (Agdia, Inc., Elkhart, IN) were obtained with extracts of naturally infected borage leaves and with systemically infected indicator species. Alfalfa plots located in the vicinity of the symptomatic borage plants could be the source of virus for borage infections. To our knowledge, this is the first report of natural AMV infection in Borago spp. References: (1) E. M. J. Jaspars and L. Bos. CMI/AAB. No. 229, 1980. (2) M. Luis-Arteaga and J. M. Alvarez. Inf. Téc. Econ. Agr. 92:70, 1996.