RESUMO
The pine wood nematode, Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, a quarantine organism, causes serious damage to pines worldwide. In Europe, it was first detected in Portugal in 1999 (3) and the pathogen was thought to be restricted to this area. However, in 2008, B. xylophilus was isolated from a single tree in the Cáceres Region (Extremadura) of Spain, bordering Portugal (2). The region of Galicia has approximately 383,632 ha of Pinus pinaster Aiton that constitutes more than 40% of the surface of Spain. Since 1999, we have analyzed 5,155 samples to monitor the presence of the pathogen. In 2008, a Spanish national contingency plan established three delimiting sampling areas, including a high risk area 20 km from the border with Portugal (2 × 2 km grid), a medium risk area 80 km wide (4 × 4), and another area covering the whole region (8 × 8). The plan required collecting samples from symptomatic trees. In 2010, in the high risk area, 307 sites were surveyed in coniferous forests. At each site, wood chip samples were collected from five pine trees. The collected wood chips were then incubated for 15 days in the lab and nematodes were extracted by Baermann's funnel method. B. xylophilus was detected from a decayed mass of P. pinaster from the As Neves Municipality (Pontevedra, Galicia). Affected specimens showed typical symptoms associated with pine wilt, including needle discoloration and death of branches. B. xylophilus was identified by morphological and molecular methods. Morphological characteristics included high lips, constricted heads, and short stylets with reduced basal knobs. Females had rounded tails, some with a short mucro, and flat vulva, while males had spicules curved with a cucullus. Measurements of these nematodes (10 females: body length = 720.99 ± 123.87 µm, a = 41.07 ± 5.83, b = 9.22 ± 3.44, c = 26.57 ± 4.13, V = 73.2, stylet length = 14.91 ± 1.65 µm; 10 males: body length = 576.4 ± 88.16 µm, a = 38.12 ± 5.36, b = 7.83 ± 0.39, c = 23.07 ± 2.59, stylet length = 14.63 ± 1.95 µm, spicules length = 22.5 ± 2.21 µm) were similar to the isolates found in Portugal described by Penas et al.(4) and smaller than described by Mota et al. (3). Molecular diagnosis was done following the protocols recommended by the EPPO (1): (i) Amplification of satellite DNA of B. xylophilus by PCR obtaining fragments of 160, 320, and 480 bp; (ii) PCR amplification of a region of 77 bp satellite DNA of B. xylophilus by Taqman Real Time; and (iii) PCR-restriction fragment length polymorphism of the internal transcribed spacer (ITS) region of Bursaphelenchus spp. nrDNA obtaining the restriction pattern for B. xylophilus. The ITS product amplified by PCR was also sequenced, showing a 99% homology with the sequences of B. xylophilus deposited in GenBank. A sequence of this nematode was submitted to GenBank database and assigned the number HQ646254. On the basis of these diagnostic characteristics, we have confirmed that B. xylophilus is now present in Galicia (northwestern Spain), which is one of the most productive and important region of Spain for forestry. References: (1) EPPO Bull. 39:344, 2009. (2) EPPO Rep. Serv. 3:2010/051, 2010. (3) M. M. Mota et al. Nematology 1:727, 1999. (4) A. C. Penas et al. J. Nematol. Morphol. Syst. 10:137, 2008.
RESUMO
Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae was first described in Japan and Korea and is currently an emerging disease that causes major losses in China, Italy, New Zealand, France, Portugal, and Chile. Gold kiwifruit (Actinidia chinensis), especially cvs. Jin Tao and Hort 16A, seem to be more susceptible than green kiwifruit (Actinidia deliciosa) cvs. Hayward and Summer. The bacterium affects male and female woody vines equally, with young vines being more susceptible. The most characteristic symptoms that appear in early spring are reddish orange or white exudates associated with cankers and wounds in branches and/or trunk, as well as brown leaf spots. Buds and fruits were also affected (1). In Spain, 1,132 ha of kiwifruit orchards yielded 25,285 t of fruit in 2009 (2). Most Spanish kiwifruit is cultivated in Galicia (northwest Spain), where the main cultivar is Hayward. In 2010, the first plantation of cv. Jin Tao and one plantation of cv. Summer were established in this area close to Hayward woody vine. In early spring 2011, 80% of the vines in one orchard had twigs with reddish exudates and branches and trunks as well as leaves with angular spots surrounded by yellow haloes. Isolations from both Actinidia spp. were conducted on nutrient agar with sucrose. One hundred and twelve isolates were obtained and seventy-seven were aerobic, gram negative and nonfluorescent on King's B medium. Biochemical tests performed were levan, oxidase, potato rot, arginine didhydrolase, hypersensitivity in tobacco, and utilization of 49 carbohydrates by the API 50 CH system (BioMérieux, Marcy l'Etoile, France). Three PCR protocols were used: two with pathovar-specific primers (PSAF1/PSAR2 and PSAF3/PSAR4) and one with nonspecific primers (PsITSF1/PsITSR2) (3). The results of all biochemical and molecular tests were in agreement with those expected for P. syringae pv. actinidiae. The 16S-23S region of strain EFA 37 isolated from A. deliciosa cv. Summer was sequenced (GenBank Accession No. JF815537) and had 100% sequence identity with P. syringae pv. actinidiae (GenBank Accession Nos. AY342165 and D86357). Pathogenicity tests were performed on 15 plants of A. deliciosa cv. Hayward (five plants per isolate) with the Spanish representative strain EFA 37 and compared with two reference strains isolated from both Actinidia species in Italy and five plants of an untreated control. Three buds per healthy vine were wounded with a sterile needle, inoculated with 30 to 50 µl of each bacterial suspension (108 CFU/ml), sealed, and then covered with plastic. Five leaves per healthy vine were also pierced with a sterile needle and then atomized with the same suspension. Symptoms began to appear after 5 days on inoculated vines, but not on untreated control vines. The bacterium, P. syringae pv. actinidiae, was reisolated from symptomatic plants. The kiwifruit orchard with affected plants was eradicated (25 ha). To our knowledge, this is the first report of P. syringae pv. actinidiae in Spain. References: (1) EPPO Alert List. Online publication. Retrieved from http://www.eppo.org/QUARATINE/Alert_List , June, 2011. (2) Ministerio de Medio Ambiente y Medio Rural y Marino (MARM). Anuario de Estadística, Online Publication. Retrieved from http://www.marm.es/estadistica/pags/anuario/2010 , June 2011. (3) J. Rees-George et al. Plant Pathol. 59:453, 2010.
RESUMO
Kale (Brassica oleracea L. acephala) is common in northwestern Spain where it is severely damaged by different insect pests. Damage could be reduced by using resistant varieties. The objectives of this work were to evaluate the resistance of kale populations to leaf damage by lepidopterous pests, to determine which traits are the best indicators of resistance, and finally to study the relationship between the glossy phenotype and resistance. Fifteen kale populations, sowed early and late, were evaluated at two locations in northwestern Spain. Significant differences among genotypes were found for all damage traits. Damage was not related to planting dates. Highest levels of damage were observed from July to November. Some populations with different performance under natural infestation in 1999 were again evaluated in 2000 under artificial infestation with Mamestra brassicae (L.) eggs. Two accessions, MBC-BRS0142 and MBG-BRS0170, showed resistance to attack by lepidopterous pests. Correlation coefficients among damage traits show that general appearance rating may be an useful indicator of resistance. Phenotype of kale with glossy leaves seems to be related to resistance although further research is needed.
