Genetic diversity in European Pisum germplasm collections.

The distinctness of, and overlap between, pea genotypes held in several Pisum germplasm collections has been used to determine their relatedness and to test previous ideas about the genetic diversity of Pisum. Our characterisation of genetic diversity among 4,538 Pisum accessions held in 7 European Genebanks has identified sources of novel genetic variation, and both reinforces and refines previous interpretations of the overall structure of genetic diversity in Pisum. Molecular marker analysis was based upon the presence/absence of polymorphism of retrotransposon insertions scored by a high-throughput microarray and SSAP approaches. We conclude that the diversity of Pisum constitutes a broad continuum, with graded differentiation into sub-populations which display various degrees of distinctness. The most distinct genetic groups correspond to the named taxa while the cultivars and landraces of Pisum sativum can be divided into two broad types, one of which is strongly enriched for modern cultivars. The addition of germplasm sets from six European Genebanks, chosen to represent high diversity, to a single collection previously studied with these markers resulted in modest additions to the overall diversity observed, suggesting that the great majority of the total genetic diversity collected for the Pisum genus has now been described. Two interesting sources of novel genetic variation have been identified. Finally, we have proposed reference sets of core accessions with a range of sample sizes to represent Pisum diversity for the future study and exploitation by researchers and breeders.

First Report of Bacterial Blight Caused by Pseudomonas viridiflava on Pea in Spain.

Because production of dry peas (Pisum sativum L.) is increasing in Spain, disease surveys were carried out from 2004 to 2006 in Castilla y Leon, the largest pea-producing region. In May of 2004, a leaf and stem blight caused an estimated 25% loss in yield in pea (cv. Messire) fields in El Cerrato (Palencia). Bacteria were isolated on King's B medium from 10 symptomatic plants from different fields (3). Thirty gram-negative isolates produced fluorescent, yellowish mucoid colonies. All isolates showed oxidative glucose metabolism on Hugh-Leifson medium and were levan and oxidase negative, potato soft rot positive, arginine dihydrolase negative, and tobacco hypersensitive positive. They also hydrolyzed esculine and gelatine. These results were different than those expected by Pseudomonas syringae pv. pisi and P. syringae pv. syringae (3). API 50 CH tests (bioMerieux, Marcy l'Etoile, France) revealed that all the isolates used the following carbon sources: glycerol, erythritol, l-arabinose, ribose, d-xylose, galactose, d-glucose, d-fructose, d-manose, inositol, manitol, sorbitol, d-raffinose, d-fucose, and d-arabitol. This nutritional profile is identical with that of P. viridiflava strain CFBP 6730, originally from pea plants in France. Therefore, these isolates were tentatively identified as P. viridiflava (2). Since a preliminary test demonstrated that 9 of the 30 isolates were pathogenic on pea plants, pathogenic isolates P44, P45, and P46 were selected arbitrarily for further tests. These three isolates plus strains HRI-W 1704 (P. syringae pv. pisi type race 6) and CFBP 1769 (P. syringae pv. syringae) were inoculated onto 10 pea seedlings (cv. Messire) each in two identical trials, following a described protocol (1). Seedlings inoculated with sterile distilled water were used as controls. After 10 days of incubation in a growth chamber at 22°C and 80% relative humidity, severe rotting and collapse similar to symptoms observed in fields appeared on pea seedlings inoculated with isolates P44, P45, and P46, while water-soaked leaf spots and necrotic symptoms were caused by P. syringae pv. pisi and P. pv. syringae. No symptoms were observed on plants inoculated with sterile water. Isolates recovered from symptomatic stems showed the same morphological and biochemical features of the original isolates. Sequences of 1,399 bp long from the three isolates (GenBank Accession Nos. GQ398128, GQ398129, and GQ398130) were 100% identical to P. viridiflava 16S rDNA database reference sequences. To our knowledge, this is the first report of P. viridiflava causing a disease of pea in Spain. The disease has been reported in New Zealand (4) and France (2). References: (1) E. M. Elvira-Recuenco et al. Eur. J. Plant Pathol. 109:555, 2003. (2) C. Grondeau et al. Plant Pathol. 41:495, 1992 (3) N. W. Schaad et al., eds. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001. (4) J. D. Taylor et al. N. Z. J. Agric. Res. 5:432, 1972.

First Report of Bacterial Blight Caused by Pseudomonas syringae pv. syringae on Common Vetch in Spain.

Common vetch (Vicia sativa L.) is an important legume crop used for livestock feed in the Mediterranean Area. This crop has an important role for sustainable agriculture in dryland rotations in Spain, where the Castilla y León Region is the major production area. During the springs of 2007 and 2008, necrotic lesions on stems, leaves, and flowers were observed in five different common vetch plots around Medina de Rioseco (Castilla y León). Four of the plots were sown with cv. Buza. No information was available about the cultivar in the fifth plot. In many cases, lesions had expanded into the stems causing complete wilting. Disease incidence was estimated at approximately 20%. Two symptomatic plants per plot were sampled. One section per plant was individually surface disinfested in 0.5% NaOCl for 1 min, followed by three washes in sterile water. Macerates were plated in King's B medium (KB) agar (24°C for 48 h) (2). Colonies from all isolations on KB agar were pale yellow and blue-green fluorescent under UV light, as typical of fluorescent Pseudomonas spp. (2). Ten isolates (one per section) were characterized. These were identified as Pseudomonas syringae by the LOPAT scheme (2) and Hugh-Leifson reaction and all utilized erythritol, l-lactate, and dl-homoserine, but not l(+)-tartrate, as carbon sources. They were positive for aesculin and gelatine hydrolysis. The 10 isolates caused severe necrotic lesions when they were puncture inoculated (108 CFU/ml suspension, 50 µl per wound, and two replicates) on immature lemon fruits (Citrus × limon, cv. Primofioro), bean pods (Phaseolus vulgaris L., cv. Ancha Lisa), and pea pods (Pisum sativum L., cv. Ucero). PCR amplification of a 752-bp syrB fragment (3) was positive for all isolates. On the basis of these tests, the 10 isolates were identified as P. syringae pv. syringae (2). Subsequently, each isolate was inoculated into two sets of 10 plants of V. sativa cv. Buza by injecting 200 µl of a bacterial suspension (108 CFU/ml) into the stem (2); 10 plants were injected with sterile water as controls. Ten days after inoculation, necrotic symptoms were observed on all plants, and 1 week later, all plants were completely wilted and dead. These symptoms were similar to those observed in the field. Control plants remained symptomless. Isolations were made from two inoculated plants per each original isolate, and all reisolates were identical to the original isolates in the above biochemical tests and PCR of the syrB gene. P. syringae pv. syringae reference strains, CFBP1768 and CFBP1769 (Collection Française de Bactéries Phytopathogènes), gave the same results in all biochemical, pathogenicity, and PCR tests. To our knowledge, this is the first report of bacterial blight caused by this pathogen on vetch in Spain. This pathogen had been previously identified in this crop in France (4) and in V. villosa (a closely related species) in the United States (1). Therefore, to prevent the spread of this pathogen, research on efficient preventive and control measures is needed. References: (1) G. L. Ercolani et al. Phytopathology 64:1330, 1974. (2) N. W. Schaad et al., eds. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001. (3) K. N. Sorensen et al. Appl. Environ. Microbiol. 64:226, 1998. (4) C. Tourte and C. Manceau. Eur. J. Plant Pathol. 101:483, 1995.

Development of molecular markers using MFLP linked to a gene conferring resistance to Diaporthe toxica in narrow-leafed lupin ( Lupinus angustifolius L.).

Phomopsis stem blight (PSB) caused by Diaporthe toxica is a major disease in narrow-leafed lupin ( Lupinus angustifolius L.). The F(2) progeny and the parental plants from a cross between a breeding line 75A:258 (containing a single dominant resistance gene Phr1 against the disease) and a commercial cultivar Unicrop (susceptible to the disease) were used for development of molecular markers linked to the disease resistance gene. Two pairs of co-dominant DNA polymorphisms were detected using the microsatellite-anchored fragment length polymorphism (MFLP) technique. Both pairs of polymorphisms were isolated from the MFLP gels, re-amplified by PCR, sequenced, and converted into co-dominant, sequence-specific and PCR-based markers. Linkage analysis by MAPMAKER suggested that one marker (Ph258M2) was 5.7 centiMorgans (cM) from Phr1, and the other marker (Ph258M1) was 2.1 cM from Ph258M2 but further away from Phr1. These markers are suitable for marker-assisted selection (MAS) in lupin breeding.