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The development of durable resistance to broomrape (Orobanche cumana Wallr.) in sunflower (Helianthus annuus L.) requires detailed characterization of the genetic and physiological bases of resistance. The objective of the present study was to map the resistance gene accurately, and to characterize the mechanism of resistance to broomrape observed in a sunflower inbred line (PHSC1102). PHSC1102, which was consistently resistant against race F and race G populations of broomrape, was crossed with PHSC1201, which was susceptible to races F and G. A mapping population of 150 F2 genotypes was phenotyped by evaluating F2:3 families for resistance to broomrape races FGV and GTK. The use of single nucleotide polymorphism (SNP) markers mapped the OrSII gene to Linkage Group 4 (LG4) of the sunflower genome. Mini‐rhizotron and histological studies of the resistant line revealed that the attachment of broomrape to host roots was similar in both the resistant and susceptible lines and that the resistance was observed at a late stage (i.e., after tubercle development). Interestingly, the resistance of the PHSC1102 line was associated with the production of phenolic compounds, which were hypothesized to restrict the parasite's growth. This research provides novel and valuable information about the host–parasite interactions between sunflower and broomrape.

Downy mildew of sunflower (Helianthus annuus L.), caused by the oomycete Plasmopara halstedii, is a major constraint to production worldwide. The inbred line RHA 340 has been extensively used to produce resistant hybrids. It harbors the Pl8 gene derived from H. argophyllus Torrey and Gray (Miller and Gulya 1988), providing resistance to all pathotypes of P. halstedii in Europe described to date (Markell et al. 2016). In spring 2017, sunflowers with downy mildew were observed in one commercial field in Cortona (Italy), where a hybrid allegedly carrying Pl8 was grown. In June 2018, plants showing stunting and chlorotic leaves were collected in commercial fields of sunflower hybrids carrying Pl8 in Gers county in France (three fields) and in Cortona (Arezzo county) in Italy (two fields). Disease incidence ranged between 40 and 70%. On the underside of chlorotic lesions, P. halstedii-like sporulation was observed particularly in young leaves. One isolate was recovered from a single plant in each field, and the morphology of sporangiophores and sporangia led to the identification of P. halstedii Farl. Berl. & de Toni (Hall 1989). The identity of the pathogen was molecularly confirmed by amplification of the nuclear DNA coding for the large ribosomal unit (28S rDNA) using LR0R and LR6-O primers (Riethmüller et al. 2002). Sequences between 1,236 and 1,048 bp and 99% identical to P. halstedii (EF553469.1) in the NCBI database were deposited in GenBank (accessions MK249853, MK249854, MK249862, MK249876, and MK294561). The pathotype of the five isolates was determined by inoculation of nine differential sunflower inbred lines according to previously used methodology (Molinero-Ruiz et al. 2008). Additionally, RHA 340, which is used as the donor of resistance in our program, and the two commercial hybrids affected in the prospected fields were included. For each differential and isolate, two replications of 10 seedlings were used. Seedlings with 2- to 5-mm-long radicles were immersed in suspensions of 40 × 103 sporangia/ml for 4 h at 18°C, transplanted in perlite, and grown in a greenhouse (15 to 25°C, 14-h photoperiod) until expansion of the first pair of true leaves. Pathogen sporulation was enhanced by plant incubation for 24 h at 20°C and 100% relative humidity in darkness. Genotype resistance was recorded by the absence of sporulation on the first pair of true leaves, and susceptibility was considered when intense sporulation on cotyledons and leaves occurred. The five isolates were characterized as pathotype 714 and pathogenic to the genotypes carrying Pl8 (sporulation in cotyledons and leaves). Commercial hybrids with Pl8 have been grown throughout Europe and remained asymptomatic since 2012. Pathotype 714 has been reported in the Czech Republic, France, Hungary, and the United States (Viranyi et al. 2015) and is now identified also in Italy. However, the most important outcome of this research is the finding in Europe of this new pathotype, provisionally termed as 714-Pl8, which overcomes Pl8. This work demonstrates the need for updating the system for the identification of P. halstedii pathotypes by including RHA 340 as a publicly available line carrying Pl8. Breeding programs focused in European markets may be compromised from the emergence of these 714-Pl8 populations. Until sources of resistance are identified, monitoring should be implemented to prevent spread.

Bacterial blight caused by Pseudomonas syringae pv. pisi is an important disease of pea (Pisum sativum L.). Knowledge about the genes or mechanisms acting against this pathogen is scarce. A genetic mapping study of the resistance was performed using a recombinant inbred line mapping population generated by crossing the susceptible cultivar Cheyenne to the resistant line ZP0104. Resistance to race 2 was a dominant monogenic trait mapped at 109.4 cM on LGVII between AB114 and AB122 simple sequence repeat markers. It is assumed to be the previously Ppi2 based on its location. Resistance to races 4 and 8 behaved also as dominant monogenic traits and presumably due to a single gene conferring resistance to both races. It was mapped on LGIII at 11.2 cM of the AD57 microsatellite marker. This gene has been named Ppi8 and it is proposed as a new resistance gene. Ppi8 and Ppi2 are associated to the pea consensus map for the first time. To identify genes related to resistance and susceptibility, a DeepSuperSAGE genome-wide transcription profiling approach was conducted producing 45,261 different pea unitags. A set of 651 unitags were differently represented in the susceptible versus the resistant response with a significance of P 4. These putative differentially expressed set includes genes previously related to responses to biotic stresses (pathogenesis-related or related to disease resistance responses), abiotic stresses or of unknown function, among other.

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.

Verticillium wilt and leaf mottle of sunflower, caused by the fungus Verticillium dahliae (Vd) has become a major constraint to sunflower oil production in temperate European countries. Information about Vd from sunflower is very scarce despite genetics, molecular traits and pathogenic abilities of fungal strains affecting many other crops being widely known. Understanding and characterizing the diversity of Vd populations in those countries where sunflowers are frequent and severely affected by the fungus are essential for efficient breeding for resistance. In this study, we have analyzed genetic, molecular and pathogenic traits of Vd isolates affecting sunflower in European countries. When their genetics was investigated, almost all the isolates from France, Italy, Spain, Argentina, and Ukraine were assigned to vegetative compatibility group (VCG) 2B. In Bulgaria, Turkey, Romania, and Ukraine, some isolates were assigned to VCG6, but some others could not be assigned to any VCG. Genotyping markers used for Vd affecting crops other than sunflower showed that all the isolates were molecularly identified as race 2 and that markers of defoliating (D) and non-defoliating (ND) pathotypes distinguished two well-differentiated clusters, one (E) grouping those isolates from Eastern Europe and the other (W) all those from the Western Europe and Argentina. All the isolates in cluster W were VCG2B, while the isolates in cluster E belonged to an unknown VCG or to VCG6. When the host range was investigated in the greenhouse, the fungus was highly pathogenic to artichoke, showing the importance of farming alternatives in the management of Verticillium attacks. Sunflower genotypes were inoculated with a selection of isolates in two experiments. Two groups were identified, one including the isolates from Western Europe, Argentina, and Ukraine, and the other including isolates from Bulgaria, Romania, and Turkey. Three pathogenic races were differentiated: V1, V2-EE (Eastern Europe) and V2-WE (Western Europe). Similarly, three differentials are proposed for race identification: HA 458 (universal susceptible), HA 89 (resistant to V2-EE, susceptible to V2-WE) and INRA2603 (susceptible to V2-EE, resistant to V2-WE). The diversity found in Vd affecting sunflower must be taken into account in the search for resistance to the pathogen for European environments of sunflower production.

The diploid annual legume species, Lathyrus sativus L., also known as grass pea, and L. cicera L., also known as chickling pea, are neglected species although they are widely grown as pulse crops for human food and also as grain feed or as forage in many of the harshest agro-environments of the world. The surface devoted to these two crops is increasing in Spain, where they were traditionally grown. The presence of bacterial blight among these crops was detected in fields located in the North part of the Central Spanish Meseta during the 2008–9 and 2009–10 seasons. Metabolic-biochemical tests, inoculation experiments in different hosts and molecular markers identified the causal agent as Pseudomonas syringae pv. syringae. This work represents the first description of the occurrence of P. syringae pv. syringae in Lathyrus spp. Bacteria isolated from these two plant species were also pathogenic and very aggressive in other cool season legume species. Rep-PCR patterns showed a close similarity among the P. syringae pv. syringae samples collected within each Lathyrus species, and that the bacterial isolates from Lathyrus spp. were very similar to strains of this pathogen isolated from common and hairy vetch, and from pea. Multilocus sequencing typing confirmed the similarity of one of these isolates with database P. syringae pv. syringae sequences. Most of the L. cicera landraces evaluated for resistance to P. syringae pv. syringae were susceptible or highly susceptible, but some promising resistance sources to this pathogen were found under controlled conditions.

Pseudomonas syringae pv. pisi is a seedborne pathogen distributed worldwide that causes pea bacterial blight. Previous characterization of this pathogen has been carried out with relatively small and/or geographically limited samples. Here, a collection of 91 strains are examined that include strains from recent outbreaks in Spain (53 strains) and from 14 other countries, and that represent all races and the new race 8, including the type race strains. This collection was characterized on the basis of 55 nutritional tests, genetic analysis (rep-PCR, amplification of AN3 and AN7 specific markers, and multilocus sequence typing (MLST)) and pathogenicity on the differential pea cultivars to identify races. Principal component analysis and distance dendrograms confirm the existence of two genetic lineages within this pathovar, which are clearly discriminated by the AN3/AN7 markers, rep-PCR and MLST. Strains from races 1 and 7 amplified the AN3 marker; those from races 2, 6 and 8 amplified AN7, while strains of races 3, 4 and 5 amplified either AN3 or AN7. Nevertheless, strains were not grouped by race type by any of the genetic or biochemical tests. Likewise, there was no significant association between metabolic and/or genetic profiling and the geographical origin of the strains. The Spanish collection diversity reflects the variability found in the worldwide collection, suggesting multiple introductions of the bacteria into Spain by contaminated seed lots.

A total of 242 Pisum accessions were screened for resistance to Pseudomonas syringae pv. pisi under controlled conditions. Resistance was found to all races, including race 6 and the recently described race 8. Fifty-eight accessions were further tested for resistance to P. syringae pv. syringae under controlled conditions, with some highly resistant accessions identified. Finally, a set of 41 accessions were evaluated for resistance to P. syringae pv. pisi and pv. syringae under spring- and winter-sowing field conditions. R2, R3 and R4 race-specific resistance genes to P. syringae pv. pisi protected pea plants in the field. Resistance sources to race 6 identified under controlled conditions were ineffective in the field. Frost effects were also evaluated in relation to disease response. Results strongly suggest that frost tolerance is effective in lowering the disease effects caused by P. syringae pv. pisi and pv. syringae under frost-stress conditions, even in the absence of disease resistance genes, although the highest degree of this protection is reached when frost tolerance and disease-resistance genes are combined in the same genetic background.

A total of 298 bacterial isolates were collected from pea cultivars, landraces and breeding lines in North-Central Spain over several years. On the basis of biochemical-physiological characteristics and molecular markers, 225 of the isolates were identified as Pseudomonas syringae, either pv. pisi (110 isolates) or pv. syringae (112), indicating that pv. syringae is as frequent as pv. pisi as causal agent of bacterial diseases in pea. Most strains (222) were pathogenic on pea. Further race analyses of P. syringae pv. pisi strains identified race 4 (59.1% of the isolates of this pathovar), race 2 (20.0%), race 6 (11.8%), race 5 (3.6%) and race 3 (0.9%). Five isolates (4.6%) showed a not-previously described response pattern on tester pea genotypes, which suggests that an additional race 8 could be present in P. syringae pv. pisi. All the isolates of P. syringae pv. syringae were highly pathogenic when inoculated in the tester pea genotypes, and no significant pathogenic differences were observed. Simultaneous infections with P. syringae pv. pisi and pv. syringae in the same fields were observed, suggesting the importance of resistance to both pathovars in future commercial cultivars. The search for resistance among pea genotypes suitable for production in this part of Spain or as breeding material identified the presence of resistance genes for all P. syringae pv. pisi races except for race 6. The pea cultivars Kelvendon Wonder, Cherokee, Isard, Iceberg, Messire and Attika were found suitable sources of resistance to P. syringae pv. syringae.

Russia devotes more than six million hectares to sunflower (Helianthus annuus L.) crop every year being, together with Ukraine, the most important producer countries worldwide. From 2014 an increase of wilt diseases is being observed in the country. Plants with symptoms of wilting and leaf yellowing and necrosis were observed in two commercial fields and one demonstrative field trial in Orenburg region in August 2016, and in one commercial field in Saratov region in August 2015. Disease incidence was estimated between 15-30% depending on the field and yield from infected plants was near to zero. Four symptomatic plants per location were collected for observation and pathogen isolation. From each sample, the basal part of the stem was surface sterilized for 5 min in 10% sodium hypochlorite solution, rinsed in water, cut longitudinally and sections of the cortex with brownish appearance indicative of possible fungal colonization were incubated in water-agar medium at 25oC in darkness. Fungal colonies were c...

Twelve different Ty1-copia and Ty3-gypsy group LTR retrotransposons were compared for their usefulness in SSAP marker development in two agriculturally important Vicia species. Three of the retrotransposons, PDR1, Tps19 and Tvf4, yielded useful SSAP marker systems in V. faba, and V. narbonensis. Another, Tvf1 was a good source of SSAP markers in V. narbonensis alone. The optimized SSAP marker systems were applied to the analysis of two diverse Vicia germplasm sets. Two hundred and two polymorphic Tvf1 SSAP markers were scored in 56 V. narbonensis samples and 196 polymorphic markers derived from the other three most useful retrotransposons were scored in a collection of 20 V. faba samples. The marker data were then used to construct phylogenetic trees. The trees for both species tend to show long-branch lengths, with rather little fine structure. Some V. narbonensis accessions cluster by geographical origin but many do not and a given geographical region is often represented by multiple diverse groups in the tree, suggesting a deep and ancient structure for the diversity of V. narbonensis that spans its current geographic range. The tree for the V. faba accessions also shows very limited clustering with geographical origin and no obvious correlation between diversity and morphology-based taxonomic groupings for the species.

Pseudomonas syringae pv. syringae causes extensive yield losses in the pea crop worldwide, although there is little information on its host specialization and its interactions with pea. A collection of 88 putative P. syringae pv. syringae strains (including 39 strains isolated from pea) was characterized by repetitive polymerase chain reaction (rep-PCR), multilocus sequence typing (MLST), and syrB amplification and evaluated for pathogenicity and virulence. rep-PCR data grouped the strains from pea into two groups (1B and 1C) together with strains from other hosts; a third group (1A) was formed exclusively with strains isolated from non-legume species. MLST data included all strains from pea in the genomospecies 1 of P. syringae pathovars defined in previous studies; they were distributed in the same three groups defined by rep-PCR. The inoculations performed in two pea cultivars showed that P. syringae pv. syringae strains from groups 1A and 1C were less virulent than strains from group 1B, suggesting a possible pathogenic specialization in this group. This study shows the existence of genetically and pathogenically distinct P. syringae pv. syringae strain groups from pea, which will be useful for the diagnostic and epidemiology of this pathogen and for disease resistance breeding.

Pseudomonas syringae is the main pathogen responsible for bacterial blight disease in pea and can cause yield losses of 70%. P. syringae pv. pisi is prevalent in most countries but the importance of P. syringae pv. syringae (Psy) is increasing. Several sources of resistance to Psy have been identified but genetics of the resistance is unknown. In this study the inheritance of resistance to Psy was studied in the pea recombinant inbred line population P665 × 'Messire'. Results suggest a polygenic control of the resistance and two quantitative trait loci (QTL) associated with resistance, Psy1 and Psy2, were identified. The QTL explained individually 22.2 and 8.6% of the phenotypic variation, respectively. In addition 21 SSR markers were included in the P665 × 'Messire' map, of which six had not been mapped on the pea genome in previous studies. © 2011 Springer Science+Business Media B.V., Financial support by AGL2008-01239, Ministerio de Ciencia e Innovación GEN2006-27798-C6-3-E/VEG and Junta de Castilla y León ITACyL 2004/845 projects is acknowledged.

A total of 122 accessions of different wild and cultivated Pisum sp. were analysed using retrotransposon-based insertion polymorphisms (RBIP) markers. The Pisum materials included wild and cultivated (landraces and cultivars) materials from the World core collection of the John Innes Centre (JI) representing all generally recognized Pisum taxa, landraces materials from the Spanish core collection, and commercial pea cultivars largely sown in Spain. The overall polymorphism detected by RBIP marker was high and all accessions, except two pairs, could be distinguished by their marker pattern. Principal component and phylogenetic analyses clearly discriminated P. fulvum and P. abyssinicum samples from both each other and P. sativum, while P. elatius and P. humile samples were scattered among the other taxa clusters, supporting the existence of three well defined taxa in the genus Pisum (P. abyssinicum, P. fulvum and P. sativum). These results also suggest that the Spanish pea core collection of landraces maintains a relatively high variability which is only partially represented in cultivars generally sown in Spain. Thus, Spanish landraces are still a source of genetic variability for breeding new pea cultivars. Se ha estudiado un total de 122 accesiones silvestres y cultivadas dePisum sp. usando marcadores basados en polimorfismos de inserción de retrotransposones (RBIP). Las accesiones de Pisum incluyen materiales silvestres y cultivados (cultivares y variedades locales) de la colección nuclear mundial del John Innes Centre (JI) representando a todos los taxones generalmente reconocidos de Pisum, variedades locales de la colección nuclear española, y por últimoalgunas variedades comerciales de guisante ampliamente cultivadas en España. Para el análisis genético se usaron 18 loci RBIP. El polimorfismo general detectado con los marcadores RBIP fue alto y todas las muestras, excepto dos pares, pudieron ser identificadas por un patrón particular de marcadores. Análisis de componentes principales y filogenéticos discriminaron claramente P. fulvum y P. abyssinicum entre ellas y de P. sativum, mientras que las muestras de P. humile y P. elatius se mezclaban con las de otros taxones en distintos grupos. Esto apoya la existencia de tres especies en el género Pisum (P. abyssinicum, P. fulvum y P. sativum). Los resultados indican que la colección nuclear española de guisante mantiene una variabilidad relativamente elevada que está sólo parcialmente representada en los cultivares generalmente sembrados en España. Por tanto, las variedades locales españolas representan aún una fuente de variabilidad genética para la mejora de nuevos cultivares.