Denis Tourvieille de Labrouhe

Single nucleotide polymorphisms reveal multiple introductions into France of Plasmopara halstedii, the plant pathogen causing sunflower downy mildew.

Plasmopara halstedii, the causal agent of sunflower downy mildew, displays a gene-for-gene interaction with its host plant, Helianthus annuus and other species of the genus. Monitoring of the evolution of virulent races in France over a 19-year period led to the identification of 14 different races (or pathotypes). Twelve expressed sequence tag (EST)-derived markers displaying SNPs and insertion-deletion variations have recently been identified in P. halstedii. We used these markers to study the genetic structure and the evolution of sunflower downy mildew races. Bayesian assignment analysis identified three genetically differentiated groups of isolates organized around the first three races described in France. Strong genetic substructuring according to geographic origin of races was observed, confirming that these three groups corresponded to three separate introductions into France of isolates with different genetic and phenotypic backgrounds. Our results suggest that multiple introductions of P. halstedii isolates may have provided the raw material for more complex processes in the evolution of races, such as recombination between races or clonal evolution through mitotic instability.

Resistance to metalaxyl in isolates of the sunflower pathogen Plasmopara halstedii.

Plasmopara halstedii isolates showing an atypical reaction to metalaxyl were collected in France, in 1995 and 1996, and tested in the laboratory for their level of sensitivity to this fungicide. Primary and secondary infections caused by one of these isolates were not controlled by the metalaxyl concentration registered for seed treatment. The EC50 of this isolate was 12 800 mg a.i. kg-1 compared with 22 mg a.i. kg-1 for sensitive isolate, indicating a 582-fold decrease in sensitivity to the compound. There was no reduction in the agressiveness of the resistant isolate. Using other anti-oomycete fungicides, it appeared that propamocarb, contact fungicides (fluazinam, folpet, mancozeb) and the mixed formulations dimethomorph + mancozeb, cymoxanil + mancozeb and ofurace + folpet were effective against primary infections made with metalaxyl resistant and sensitive isolates, but not against secondary infections. Metalaxyl mixed with fluazinam, folpet or mancozeb was more effective against primary infections with the resistant isolate than metalaxyl alone. The EC50 of five other isolates ranged from 5 800 to 32 900 mg a.i. kg-1, indicating a variability in metalaxyl sensitivity of resistant sunflower downy mildew isolates. This is the first report of physiological resistance to metalaxyl in Plasmopara halstedii.

Colocation of downy mildew (Plasmopara halstedii) resistance genes in sunflower (Helianthus annuus L.)

SummaryThe Pl6 locus in the inbred sunflower (Helianthus annuus L.) line HA335 giving resistance to French races of downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni. was localized by molecular techniques. A bulked segregant analysis was made on the F2 progeny from a cross between this line and H52, a downy mildew susceptible line. The resistance gene in HA335 was found to have the same linked RFLP marker loci as those determined for Pl1 (resistance to race 1 in the line RHA266) on linkage group 1 of the consensus RFLP map of the cultivated sunflower. Pl1 and Pl6 thus appear either to be allelic or closely linked. The implications for sunflower breeding are discussed.

Resistance of sunflower inbred lines to various forms of attack by Sclerotinia sclerotiorum and relations with some morphological characters

SummaryTwenty sunflower inbred lines were studied for their reactions to 7 Sclerotinia sclerotiorum tests on different plant parts. A principal component analysis (P.C.A.) and t-tests on the means of resistant and susceptible groups indicated that reactions of inbred lines to infection of capitula by ascospores are independent from those to ascospore infections of terminal buds. They are even more contrasted with the results of any test measuring mycelial extension. However, for the last, there is a close association between the reactions of roots, leaves and capitula. Of the inbred lines, some showed good levels of resistance to most forms of attack, others were generally susceptible and there were some with good resistance to one particular form of attack. A P.C.A. of 18 morphological and field characters showed no general association between these characters and Sclerotinia sclerotiorum test results, although it was found that lines resistant to mycelial extension on capitula were generally earlier than those that were more susceptible. It is proposed that breeding programmes for general resistance to Sclerotinia sclerotiorum should include a combination of two or three tests.

Accumulation of defense related transcripts in sunflower hypocotyls (Helianthus annuus L.) infected with Plasmopara halstedii

A cDNA clone encoding a sunflower chitinase was obtained using degenerated primers in PCR amplifications and RACE procedures. This clone, a phenylalanine ammonia-lyase (PAL) clone and ubiquitin clone were used to analyse the resistance of sunflower (Helianthus annuus) to downy mildew. The differential regulation of amounts of PAL (involved in the general pathway of phenylpropanoid synthesis), chitinase (a pathogenesis-related protein) and ubiquitin (involved in proteolytic pathways) mRNA was studied in hypocotyls during the early stages after an aerial infection of sunflower inbred line RHA274 with zoospores from either race 1 (incompatible, host resistant) or race B (compatible, host susceptible) of Plasmopara halstedii. Northern analyses showed that transcript levels of PAL, chitinase and ubiquitin were rapidly and strongly increased after infection in incompatible interactions but not in the compatible ones, suggesting that regulation of these mRNAs is an important component of the resistance mechanisms in sunflower.

Emerging virulence arising from hybridisation facilitated by multiple introductions of the sunflower downy mildew pathogen Plasmopara halstedii.

The sunflower downy mildew pathogen Plasmopara halstedii is an invasive plant pathogen in Europe of American origin. Despite efforts to produce resistant host varieties, nationwide monitoring in France has revealed the rapid emergence of new virulent races increasing the number from one founder identified in 1966 to as many as 14 today. We have genotyped 146 samples (including all 14 races) using 13 nuclear and one mtDNA marker. Samples of the same race were found to share alleles/mtDNA haplotype and the two most common races had individuals with multiple matching genotypes. Cluster analyses confirmed that the samples form three groups to which races strongly adhere. Clusters were highly differentiated (F(ST) 0.65) and characterised by high inbreeding coefficients. Despite this, samples of recently emergent races, including six that are unique to France had mixed ancestry between the groups suggesting they have arisen in situ due to hybridisation. Five such samples also had conflicting mtDNA and nuclear DNA profiles. This demonstrates that multiple introductions have aided the establishment of this pathogen in France, and suggests recombination facilitated by these introductions is driving the emergence of new and endemic races in response to host resistance.

Transgenic tomatoes showing higher glutathione peroxydase antioxidant activity are more resistant to an abiotic stress but more susceptible to biotic stresses

The function of selenium independent glutathione peroxidase (GPx) in response to biotic and abiotic stresses was investigated in transgenic tomato plants overexpressing an exogenous GPx and exhibiting a 50% increase in total GPx activity. GPx-overexpressing and control plants were challenged either by a mechanical stress or by infection with the biotrophic parasite Oidium neolycopersici or the necrotrophic parasite Botrytis cinerea. In mechanically stressed plants, internode growth was significantly less modified in GPx-overexpressing plants compared to controls. This stress resistant phenotype was not accompanied with any change in the global antioxidant response of the plants other than their increased GPx activity. Following infection by O. neolycopersici or by B. cinerea, lesion extension was increased in GPx-overexpressing plants compared with controls. These results showed that GPx overexpression provoked opposite effects in situations of biotic and abiotic challenges, suggesting a key role for this scavenger enzyme in controlling biotic and abiotic stress responses.

Molecular Variability Within Diaporthe/Phomopsis helianthi from France.

ABSTRACT Diaporthe/Phomopsis helianthi causes brown stem canker of sunflower (Helianthus annuus) and is responsible for considerable yield loss. This species shows considerable variation for morphological characters, growth, and pathogenicity. Molecular variability of two sample groups was assessed with amplified fragment length polymorphism (AFLP) markers. Isolates of the first sample were collected from infected sunflower tissues from the main regions in France where the crop is grown, whereas isolates from the second sample came from stems within a single field of sunflower. A soybean strain was taken as an outgroup for AFLP analyses. Within sample one, the greatest genetic distance among isolates was 0.97, whereas it was 0.44 within sample two isolates. For the whole of France, the average genetic distance was 0.68, whereas in the one field it was 0.12. Neis genetic diversity indices were 0.20 and 0.06 for France and for one field, respectively. The greatest genetic distance was found between isolates from the most northern crops. The greatest genetic distance between D. helianthi isolates and the strain isolated from soybean was similar to that observed for D. helianthi isolates from different geographical areas. The problems in defining the genus Phomopsis are discussed. It is shown that internal transcribed spacer sequencing could be a useful criteria for Diaporthe/Phomopsis species determination. The considerable genetic variability of the pathogen could lead to the occurrence of new strains that could be more aggressive or more resistant to chemical control.

Interactions Between French Isolates of Phomopsis/Diaporthe Helianthi Munt.-Cvet. et al. and Sunflower (Helianthus annuus L.) Genotypes

Three artificial infection tests measuring the rate of mycelial growth of 7 Phomopsis/Diaporthe helianthi isolates were used on leaves, stems and capitula of 6 sunflower hybrids. Isolates and hybrids were chosen to cover the range of variability and resistance levels known at the present time. Significant genotype and isolate effects and isolate×genotype interactions were shown in all the tests, with some changes in order of hybrids according to the isolate used for infection. Consequences of interactions in breeding for stable resistance to P./D. helianthi are discussed.

An Optimized Duplex Real-Time PCR Tool for Sensitive Detection of the Quarantine Oomycete Plasmopara halstedii in Sunflower Seeds

Plasmopara halstedii, the causal agent of downy mildew of sunflower, is an oomycete listed as a quarantine pathogen. This obligate parasite resides in a quiescent state in seeds of sunflower and can be spread from seed production areas to areas of crop production by international seed trade. To prevent the spread or the introduction of potentially new genotypes or fungicide-tolerant strains, an efficient method to detect P. halstedii in sunflower seed is required. This work reports the optimization of a real-time detection tool that targets the pathogen within sunflower seeds, and provides statistically validated data for that tool. The tool proved to be specific and inclusive, based on computer simulation and in vitro assessments, and could detect as few as 45 copies of target DNA. A fully optimized DNA extraction protocol was also developed starting from a sample of 1,000 sunflower seeds, and enabled the detection of <1 infected seed/1,000 seeds. To ensure reliability of the results, a set of controls was used systematically during the assays, including a plant-specific probe used in a duplex quantitative polymerase chain reaction that enabled the assessment of the quality of each DNA extract.