First Report of Canker Disease Caused by Neofusicoccum australe on Eucalyptus and Pistachio in Spain.

In 2005 and 2006, dieback and branch cankers were observed in 12-year-old Eucalyptus globulus Labill. plantations in Gijón (northern Spain) and a 20-year-old pistachio (Pistacia vera L.) plantation in Constantí (northeastern Spain). Isolations were made from symptomatic branches. Small pieces of necrotic tissues were surface sterilized for 1 min in 1.5% NaOCl and plated onto malt extract agar amended with 0.5 g L-1 streptomycin sulfate. Plates were incubated at 25°C in the dark and all growing colonies were transferred to potato dextrose agar (PDA). A Neofusicoccum sp. was consistently isolated from necrotic tissues of both host species. On PDA at 25°C, isolates developed a moderately dense mycelium, initially with a pale yellow pigment diffusing into the medium but becoming olivaceous gray after 5 to 6 days. Pycnidia were produced on sterile eucalyptus and pistachio twigs placed on the surface of water agar after 1 month. Conidia were hyaline, fusiform, aseptate, with granular contents. Conidia from eucalyptus isolates measured (22.5-) 25.4 (-28.1) × (5-) 6.2 (-7.5) µm, (n = 40) and (20.0-) 23.6 (-28.0) × (6.5-) 7.1 (-8.0) µm, (n = 40) from pistachio isolates. Isolates were identified as Neofusicoccum australe (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips (1,2). DNA sequences of the rDNA internal transcribed spacer region (ITS), part of the beta-tubulin (BT2), and part of the translation elongation factor 1-alpha (EF1-α) genes from isolates CBS 122027 (pistachio) and CBS 122026 and CBS 122025 (eucalyptus) were used to confirm the identifications through BLAST searches in GenBank. Representative sequences of all studied regions were deposited in GenBank (ITS: EU375516 and EU375517; BT2: EU375520; EF1-α: EU375518 and EU375519). Pathogenicity tests were conducted on 8-month-old eucalyptus seedlings and 2-year-old pistachio plants with the three N. australe strains mentioned above. A mycelial plug taken from the margin of an actively growing colony of each isolate was put in a shallow wound (0.4 cm2) made with a scalpel on the stem of each plant. Inoculation wounds were wrapped with Parafilm. Controls were inoculated with sterile PDA plugs. Ten replicates for each isolate and plant species were used, with an equal number of control plants. Plants were maintained in a greenhouse at 25°C. After 3 weeks, all eucalyptus seedlings showed leaf wilting, stem canker, and pycnidia formation around the inoculation site. No foliar symptoms were observed in pistachio plants after 3 months, but depressed cankers variable in size and pycnidia formation developed around the inoculation site. Vascular necroses that developed on the inoculated plants were 10.2 ± 1.2 cm long in eucalyptus and 6.4 ± 1.6 cm long in pistachio, significantly greater than their respective controls (P < 0.01). There were no significant differences in necrosis lengths among the three N. australe isolate inoculations, irrespective of the inoculated host. These results point to a high susceptibility of eucalyptus to N. australe. No symptoms were visible in the control seedlings and no fungus was isolated from them. The pathogen was reisolated from all inoculated plants. To our knowledge, this is the first report of N. australe causing canker disease on eucalyptus and pistachio trees in Spain. References: (1) P. Crous et al. Stud. Mycol. 55:235, 2006. (2) B. Slippers et al. Mycologia 96:1030, 2004.

First Report of Leaf Spot and Twig Blight of Rhododendron spp. Caused by Phytophthora hibernalis in Spain.

During the early spring of 2004, an estimated 20% of containerized nursery stocks of Rhododendron spp. in Asturias (northern Spain) were affected by a foliar disease that has reoccurred annually. Leaf spots were dark brown to almost black, generally oval to round, visible from both sides of the leaf, and expanded to affect the entire leaf including the petiole. Affected leaves abscised from the plant. A Phytophthora sp. was consistently isolated from symptomatic leaf tissues on PARBH medium (3) and hyphal tips were transferred onto potato dextrose agar (PDA). Colonies grown on PDA at 20°C were submerged, had a growth rate of 2.2 mm/day, and had lobes of compact mycelium. Sporangia were semipapillate and caducous with a pedicel (20.0-) 37.7 (-52.5) µm long. Sporangia were asymmetrical in shape with the broadest point near the apex: 25.2 to 40.4 µm long × 10.2 to 15.8 µm wide (average 33.1 × 12.6 µm), and length/width ratio was 2.8:1. Chlamydospores were not observed. Isolates were homothallic and oogonia ranged from 26.5 to 27.5 µm in diameter. Antheridia were mostly amphigynous but occasionally paragynous. Oospores were plerotic and 23.1 to 25.5 µm in diameter. These characteristics conformed to those of Phytophthora hibernalis Carne (2). Sequences of the internal transcribed spacer regions on the isolates and comparison with other sequences in GenBank showed that they were identical to P. hibernalis (Accession No. AY827556.1 from Citrus sp.). For pathogenicity tests, four isolates of P. hibernalis were used to inoculate detached leaves of Rhododendron hybrid Brigitte. The underside of five detached leaves was inoculated with a drop of 40 µL of a suspension of 104 zoospores/ml. Controls were inoculated with a 40-µL drop of sterile distilled water. Leaves were incubated in a moist chamber at 20°C in the dark. A quantification of the lesion area was made 8 days after inoculation using the software Assess-APS. All inoculated leaves developed necrotic lesions that ranged from 0.246 to 1.512 cm2. P. hibernalis was reisolated from infected tissue. Symptoms were not detected on the controls. The test was repeated twice and similar results were obtained each time. P. hibernalis has been described previously as causing brown rot on citrus in Spain (4) and was isolated from rhododendron plants in California and Oregon (1). To our knowledge, this is the first record of P. hibernalis causing foliar blight on Rhododendron species in Spain as well as in Europe. References: (1) C. Blomquist et al. Online publication. doi:10.1094/PHP-2005-0728- 01-HN. Plant Health Progress, 2005. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul MN. 1996. (3) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986. (4) J. J. Tuset. An. Inst. Nac. Investig. Agrar. Ser. Prot. Veg. N.7, 1977.

Epidemiological differentiation of pathogenic strains of Salmonella enteritidis by ribotyping.

The usefulness of two-way ribotyping, performed with SphI and PstI, as a genetic marker for a series of pathogenic Salmonella enteritidis strains is reported. Eighteen combined ribotypes were differentiated, a discrimination index of 0.77 was reached, a genetic relationship dendrogram was traced, and the results were applied in an epidemiological study.

Discriminatory power and application of ribotyping of Yersinia enterocolitica O:3 in an epidemiological study.

Ribotyping performed with five restriction endonucleases was used in an attempt to subtype Yersinia enterocolitica serotype O:3 and also as a tool for clonal analysis. DNA from organisms under study (48 isolates from diarrheic human feces, 24 from food, and 5 reference strains) was tested by Southern hybridization using a DNA probe carrying an rRNA operon from Escherichia coli. Strains were grouped into seven ribotypes by the HindIII restriction endonuclease, into five ribotypes by Ncil, Bg/l, and Sa/l; and into two ribotypes by EcoRI, resulting in a discrimination index (DI) of 0.37, 0.17, 0.43, 0.13, and 0.03 for the five endonucleases. By combining the results obtained with two or more restriction endonucleases, a further discrimination was registered, the most efficient combination (in terms of discriminatory power vs. cost in work, time, and money) for routine typing being HindIII-Bg/l (9 types, DI=0.58). In the clonal analysis, results obtained with the five restriction endonucleases allowed us to define 11 groupings or clonal lines, which showed a remarkable degree of genetic heterogeneity (genetic distance coefficients between 0.03 and 0.73) and were grouped into two major clusters. One cluster included 93% of the strains and eight lines. At least two of the most frequent lines can be considered endemic in Asturias, Spain, because organisms belonging to these lines have been circulating and causing human yersiniosis in recent years and have also been isolated from commercial raw meat products. Two Ncil ribotypes from the series under study (92.2% of strains included in the prevalent cluster) were similar but not identical to ribotypes of 0.3 organisms from other geographic areas described in the literature, indicating that the genetic structure of prevalent human pathogens of this serotype is basically clonal.

Yersinia enterocolitica O:3. Antimicrobial resistance patterns, virulence profiles and plasmids.

Antimicrobial resistance patterns (ARP), virulence profiles and plasmids in clinical isolates of Yersinia enterocolitica serotype O:3 were studied. The ARP was tested using the disk method. All strains were susceptible to amoxicillin/clavulanic acid, cefoxitin, fosfomycin, gentamicin, kanamycin, neomycin, tetracycline, nalidixic acid, norfloxacin, ciprofloxacin and trimethoprim. All of them presented resistance to ampicillin, all with the exception of one to cephalotin, differing in resistance or susceptibility to chloramphenicol, streptomycin (Sm), sulfadiazine (Sd) and cotrimoxazole. Due to these differences they were grouped into 8 ARP. Twenty-nine strains carried plasmids and were grouped into 5 plasmid profiles. All strains carrying 42 MDa plasmids were positive for virulence tests (calcium dependence, crystal violet binding, Congo red binding and autoagglutination). No correlation between ARP and plasmid profile was found, although small plasmids of 6.5 and 4.1 MDa mediated resistance to Sm-Sd, as was shown by transformation to Escherichia coli strains. DNAs from plasmids were analyzed by restriction enzymes. All 42 MDa plasmids showed identical EcoRI and HindIII profiles. The two 6.5 MDa plasmids showed identical BglII, AvaI and SalI restriction profiles and the four 4.1 Mda plasmids also yielded restriction profiles similar to each other, but different from the 6.5 ones.