During the winter of 2003-2004, dieback symptoms were observed on Pinus radiata and P. pinaster in pine nurseries in Asturias (northern Spain). Small groups of affected seedlings appeared randomly distributed throughout the nurseries. The seedlings died rapidly, showing basal needle dieback, stem lesions, resin exudations, and wilting. Isolations from infected material onto potato dextrose agar (PDA) supplemented with 0.5 mg/ml of streptomycin sulfate and Komada's medium consistently yielded Fusarium sp. cultures. The isolates were transferred to PDA and Spezieller Nährstoffarmer agar and incubated at 25°C for 10 days with a 12-h photoperiod. The cultures were identified as Fusarium circinatum Nirenberg & O'Donnell (= Fusarium subglutinans Wollenweb. & Reinking), causal agent of pitch canker disease, on basis of the presence of polyphialides and characteristic sterile, coiled, hyphae (2). To further confirm their identity, a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) based on histone H3 gene sequences (4) and a test based on the F. circinatum-specific primers, CIRC1A-CIRC4A, which amplifies a 360-bp DNA fragment of the intergenic spacer region of the nuclear ribosomal operon (3), were used. Results obtained with both techniques confirmed the morphological identification of the cultures. A representative culture has been placed in the Centraalbureau voor Schimmelcultures (CBS 117843). The pathogen was isolated only from seedlings of P. radiata and P. pinaster. Other species such as P. nigra, P. sylvestris, and Pseudotsuga menziesii, which were also grown in these nurseries, did not show symptoms. Pathogenicity was confirmed by inoculating 6- to 9-month-old P. radiata and P. pinaster seedlings. Small strips of bark (10 × 1 mm) were cut from the stems and similar sized pieces of PDA colonized by F. circinatum were placed in contact with the open wounds and covered with parafilm. Basal needle dieback was observed 10 days after inoculation that resulted in wilting of the seedlings. F. circinatum was reisolated from the affected stems fulfilling Koch's postulates. Later in the year, symptoms of pitch canker were also observed on 20-year-old P. radiata in one forest plantation in Cantabria (northern Spain). Infected branches and shoots of the trees exudated abundant resin, resulting in resinous cankers. The needles, distal to branch tip infections, wilt, fade to yellow then red, and fall from the tree. Affected trees showed noticeable crown dieback. The isolations from the cankers also yielded F. circinatum cultures that were identified as described above. Although a nonrefereed report appeared in 1998 (1), to our knowledge, this is the first report of F. circinatum on P. radiata and P. pinaster in Spain and in Europe. References: (1) L. D. Dwinell et al. Int. Congr. Plant Pathol. 7th. 3:9, 1998. (2) H. I. Nirenberg and K. O'Donnell. Mycologia 90:434, 1998. (3) W. Schweigkofler et al. Appl. Environ. Microbiol. 70:3512, 2004. (4) E. T. Steenkamp et al. Appl. Environ. Microbiol. 65:3401, 1999.
Ribotyping of Salmonella serotype Typhimurium strains was optimised as a tool for epidemiological and phylogenetic purposes. Of five restriction endonucleases evaluated on a series of 84 isolates, HincII, San and PvuII were the most useful, generating 13, 9 and 9 ribotypes with 17, 11 and 18 polymorphic restriction sites, and attaining a discrimination index (DI) of 0.81, 0.53 and 0.59, respectively. The combination of results from tests with the three enzymes provided further discrimination (19 ribotypes, DI = 0.84). It proved useful for clonal analysis, defining 19 clonal lines with a remarkable degree of genetic heterogeneity, that were grouped into two major clusters (including 12 and 7 lines, respectively) at a significance level of 0.65. When the attributes of this system were compared with those of phage typing, it was found that ribotyping showed higher typability and sensitivity, supporting its use as an appropriate molecular method. In tracing the molecular epidemiology of Typhimurium strains in Asturias, six lines were found that could be considered endemic and were represented by organisms implicated in salmonellosis throughout the period of study; another four lines included organisms isolated from meat, water or both.
The capacity to differentiate Salmonella serotype Enteritidis strains by PCR ribotyping; RAPD typing with three arbitrary primers and ribotyping with a mixture of PstI and SphI or 'PS ribotyping', was evaluated on a series of 65 strains associated with human infections and 11 reference strains. The series had been analysed previously by phage typing and ribotyping performed with PstI and Sph I, separately. All methods typed all the strains; however, only ribotyping showed good reproducibility and sensitivity. Twenty-two PS ribotypes (discrimination index = 0.74) were identified, differentiating strains ascribed to seven phage types (PTs 1, 4, 6, 6a, 7, 8 and RDNC) as well as phage untyped strains. Conversely, some strains of PTs, 1, 4, 5a, 6, 6a, 7, 34 and RDNC showed the most frequent PS ribotype. By PCR ribotyping a single profile was found; while by RAPD typing, one, two or three RAPD types were identified with the primers MK22, OPB6 and OPB17, respectively. All Spanish strains were assigned to a single combined RAPD type, except PTl1 strains which showed a different and specific RAPD type with OPB17. The banding patterns defining the PS ribotypes were interpreted more easily and the patterns could be compared more accurately than the banding patterns defining RAPD types. A similarity dendrogram generated from the 22 PS ribotypes was traced and compared with RAPD types and phage types. Data from this work indicated that 'PS ribotyping' was the most useful genetic procedure to differentiate Enteritidis strains, and, therefore, it can be used as a complementary or alternative typing method to phage typing within this serotype.
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