Variation in two components of pathogenicity and its relationship with genetic diversity in Plasmopara halstedii (sunflower downy mildew)

The specificity of the two components of pathogenicity: virulence and aggressiveness and its relationship with genetic variability were analysed in a local Plasmopara halstedii (sunflower downy mildew) population. Pathogenic and molecular analyses were carried out on seven isolates including five progeny isolates of five races arising from two parental races 100 and 710. P. halstedii isolates showed significant differences for all aggressiveness criteria and important genetic variations. Three cases of relationship between virulence and aggressiveness for progeny isolates as compared with parental ones were found as positive, negative or uncorrelated. For solving the specificity of these cases, relationship between the two components of pathogenicity among the isolates of three different races localised in the same genetic clade was positive. The hypothesis explaining these cases is discussed.     

Evolution of pathogenicity in Plasmopara halstedii (sunflower downy mildew)

Comprehension of the processes of co-evolution between the pathogen and its host plant is very important, particularly in the case of obligate pathogen as Plasmopara halstedii which cannot develop only on sunflower. The influence of selection pressure exercised by qualitative resistance in sunflower plants on evolution of pathogenicity was analysed in pathogenic populations of P. halstedii. This selection pressure led a new virulence to appear in P. halstedii isolates carrying several levels of aggressiveness. It seems that the qualitative resistance selection pressure plays an important role in the evolution of this pathogen, and these changes on the level of pathogenicity may help to a better adaptation of P. halstedii in the presence of intensive use of qualitative resistance.     

Relationship between aggressiveness and zoosporangia viability in Plasmopara halstedii (sunflower downy mildew)

Relationship between aggressiveness and zoosporangia viability was studied in seven Plasmopara halstedii (sunflower downy mildew) isolates of races 100, 300, 304, 314, 710, 704 and 714. Aggressiveness criteria including latent period and sporulation density were analysed on sunflower inbred line showing a high level of quantitative resistance. There were significant differences between pathogen isolates for the two aggressiveness criteria. Viability analyses were performed on oval and spheric zoosporangia. The number of zoospores released from oval zoosporangia was significantly higher than those released from spheric ones. The oval zoosporangia for more aggressive isolates of races 100 and 3xx produced more zoospores than the oval ones for less aggressive isolates of races 7xx. There was a significant correlation between aggressiveness criteria and the number of zoospores released from oval zoosporangia and vice versa for zoospores released from spheric ones. It is concluded that the relationship between aggressiveness and oval zoosporangia viability may be established in P. halstedii.     

Virulence cost in Plasmopara halstedii (sunflower downy mildew)

Virulence cost (trade-off between virulence and aggressiveness) was studied in seven Plasmopara halstedii (sunflower downy mildew) isolates of races 100, 300, 304, 314, 710, 704 and 714. The seven isolates were divided, according to their virulence and aggressiveness, into two main groups as more aggressive isolates of the 100 and 3xx races that do not overcome the sunflower differential host D3, and less aggressive isolates of 7xx races that can overcome D3. Consequently, the 100 and 3xx avirulent races had a virulence cost measured by differences in aggressiveness (from 58.3 to 78.2%) compared to 7xx virulent races carrying unnecessary virulence gene.     

Aggressiveness variation in Plasmopara halstedii and its alternation with non-race-specific resistance in sunflower

Aggressiveness variation and its alternation with non-race specific resistance in sunflower were studied in 19 Plasmopara halstedii isolates belonging to several races. Regarding aggressiveness criteria, percentage infection, latent period, sporulation density and dwarfing, on two sunflower inbred lines showing different levels of non-race specific resistance resistance FU and BT, there were significant differences in aggressiveness for P. halstedii isolates. The index of aggressiveness varied between 9.4 and 31.4. The inbred line BT, rather susceptible in the field, showed a higher percentage infection, a higher sporulation density, a shorter latent period and less reduced hypocotyl length than inbred line FU, which showed a greater resistance in the field. Percentage infection on FU was 1.4% less than BT, latent period on BT was 12.4% less than FU, sporulation density on FU was 22.3% less than BT and reduced hypocotyl length on BT was 15.3% less than FU. Consequently, it seems that the criteria as latent period, sporulation density and reduction of hypocotyl length could be used to measure non-race specific resistance in sunflower to P. halstedii under controlled conditions.     

Evidence for asexual genetic recombination in sunflower downy mildew,Plasmopara halstedii

Field isolates and single sporangium lines of the biotrophic Oomycete Plasmopara halstedii, differing in host preference and fungicide sensitivity, were used simultaneously for infection of sunflower. Dual infections led to asexually formed zoosporangia which gave rise to a new phenotype combining the characteristics of the parental strains. The new phenotype showed the metalaxyl-tolerance of one parent and virulence behaviour characteristic of the other, thus being able to infect a specific and fungicide treated sunflower line that neither of the parental strains could infect alone. These characteristics were inherited over many generations and did not occur spontaneously when parental strains were propagated separately. DNA fingerprints with minisatellite and simple sequence repeat primers showed characteristic differences between the patterns of the parental strains and the new phenotype. PCR experiments with mixed parental DNA resulted in additive patterns, but did not show the amplification product specific for the new phenotype. Since sexual reproduction was excluded under the experimental conditions used, the results provide evidence for genetic recombination through parasexual events in dual infections of sunflower downy mildew.     

Relationship between virulence and aggressiveness in Plasmopara halstedii (sunflower downy mildew)

Relationship between virulence and aggressiveness was studied in seven Plasmopara halstedii (sunflower downy mildew) pathotypes including five progeny pathotypes of races 300, 304, 314, 704 and 714 arising from two parental pathotypes of races 100 and 710. Aggressiveness criteria including percentage infection, latent period, sporulation density and reduction of hypocotyl length were analysed in one sunflower inbred line showing a high level of quantitative resistance. There were significant differences between P. halstedii pathotypes for all aggressiveness criteria. Pathogenicity of progeny pathotypes as compared with parental ones (relationship between virulence and aggressiveness) seems to be positive, negative or uncorrelated. Hypothesis explaining these cases are discussed.     

Variation in form and size of Plasmopara halstedii (sunflower downy mildew) zoosporangia

Zoosporangia form and size were studied on a collection of 94 strains of Plasmopara halstedii (sunflower downy mildew). Both oval and round forms were present in all strains analysed. The proportion of two forms varied significantly according to strain and plant age but more especially to host plant genotype. Whatever the strain or host genotype, oval zoosporangia were larger than round ones, but there was no relation between the proportion of the oval form and mean zoosporangia size. There was no relation between zoosporangia form or size and race virulence profiles or aggressiveness criteria, with the possible exception of zoosporangia size and sporulation density. It is concluded that, for this obligate parasite, although form and size of zoosporangia depend on pathogen strain, these characters also vary according to growth conditions of Plasmopara halstedii, in particular to the genotype of the plant host.     

Acclimation of Plasmopara halstedii (sunflower downy mildew) in relationship with trade-off between virulence and aggressiveness in a local pathogen population

The acclimation in relationship with virulence cost was analysed for seven Plasmopara halstedii (sunflower downy mildew) isolates including five progeny isolates of several races descending from two parental isolates of races 100 and 710. Aggressiveness criteria were analysed in one sunflower inbred line showing a high level of quantitative resistance. Isolates of races 100 and 3xx were characterised with shorter latent period and higher sporulation density than isolates of races 7xx. All isolates showed high percentage infection values and caused a large reduction in seedling size except for one isolate involved in dwarfing. The seven isolates were divided, according to their virulence and aggressiveness, into two main groups as more aggressive isolates of the 100 and 3xx races which do not overcome the sunflower differential host D3, and less aggressive isolates of 7xx races which can overcome D3. Consequently, the 100 and 3xx avirulent races had a virulence cost measured by differences in aggressiveness (from 45.5 to 76.3%) compared to 7xx virulent races carrying unnecessary virulence gene.     

Glasshouse evaluation of fungicides for the control of sunflower downy mildew (Plasmopara halstedii)

A precise, reproducible and easy-to-handle glasshouse test is described for the evaluation of the systemic activity of chemicals for the control of Plasmopara halstedii, the downy mildew pathogen of sunflower. Four-day-old sunflower germlings were inoculated by immersing them in a zoosporangium suspension. Seedlings were then immersed in appropriate concentrations of the chemicals to be tested. Plants were grown in a glasshouse and assessed on three occasions to determine successively antisporulant, curative (systemic fungistatic), and eradicant effects. Sporulation in general was inhibited by lower concentrations than those required to exert an eradicant effect. There was a highly significant correlation between the ED₅₀ values for visually recognised disease symptoms (stunting, dampingsff and leaf chlorosis) and for both curative and eradicant effects. Among 13 compounds tested, metalaxyl, RE 26745, furalaxyl, LAB 149202F and cymoxanil showed sufficient eradicant activity, to justify field evaluation for eradication of seed infections.     

Hypothesis of durable resistance in sunflower plants against Plasmopara halstedii

Sunflower downy mildew is considered as one of the most serious diseases. Therefore, vertical resistance has been used intensively, but with the appearance of many races since 2000, research on more durable resistance has been undertaken. In this review, we present new results concerning the evolution of pathogenicity under artificial conditions in order to underline a mixture model assuming durable resistance against Plasmopara halstedii. Examples of host–parasite interactions including the influence of plant mixture against pathogens and durable resistance are presented to integrate in our couple P. halstedii/Helianthus annuus.     

Interaction between sunflower plants and five isolates of Plasmopara halstedii on the level of pathogenicity

The interaction between sunflower plants showing a high level of quantitative resistance and five Plasmopara halstedii (the causal agent of downy mildew) isolates of several races were studied using five single zoosporangium isolates per pathogen isolate. Aggressiveness criteria were analyzed for 25 P. halstedii single zoosporangium isolates. Based on the reaction for the P. halstedii isolates to four sunflower hybrids H1–H4 varying only in their downy mildew resistance genes, there were differences in virulence spectrum in pathogen isolates. Analysis of five single zoosporangium isolates for P. halstedii isolates showed significant variability within pathogen isolate for all aggressiveness criteria but not for all pathogen isolates. The hypothesis explaining the interaction between P. halstedii and its host plant was discussed on the level of pathogenicity.     

The genetics of resistance to five races of downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.)

 These studies were undertaken to determine whether downy mildew resistance genes in sunflower were independent as first reported, or linked as suggested by more recent hypotheses. The segregations for downy mildew reaction of 111 F₃ progenies from a cross between a susceptible line and a line with Pl2 were used to locate this gene on the sunflower consensus RFLP linkage map. It was shown that Pl2 was linked to the same RFLP markers on linkage group 1 as Pl1 and Pl6, mapped earlier, and at a very similar distance. The F₃ progenies showed exactly the same segregation patterns when tested with race 1 and race D. One hundred and fifty four progenies from a cross between a susceptible line and HA335, containing Pl6 (considered as giving resistance to all Plasmopara halstedii races), were tested with the five French downy mildew races, 1, A, B, C and D. Two progenies were observed to show segregation for races 1 and D, while appearing homozygous-resistant to races A , B and C. Tests on F₄ progenies confirmed this separation of resistances with fixation of susceptibility to races 1 and D and resistance to races A, B and C. It is concluded that the Pl6 gene is not a “strong” gene, giving resistance to all downy mildew races, but rather a cluster of genes, each providing resistance to one, or a few, downy mildew races. The genes giving resistance to races 1 and D, on one hand, and to races A, B and C, on the other hand, must be very closely linked, with about 0.6 cM between the two groups. Received: 23 December 1996 / Accepted: 18 April 1997     

Identification of a second linkage group carrying genes controlling resistance to downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.)

A sunflower line, XRQ, carrying the gene Pl5, which gives resistance to all French downy mildew races shows cotyledon-limited sporulation in seedling immersion tests; consequently, segregations in crosses with other downy mildew resistance sources were tested both by this method and by a secondary infection on leaves. Pl5 was found to segregate independently of Pl7 (HA338) but to be closely linked, or allelic, with Pl8 (RHA340). F₃ and F₄ progenies from a cross with a line containing Pl2 showed that Pl5 carries resistance to race 100 which segregates independently of Pl2. The Pl5 gene was mapped on linkage group 6 of the Cartisol RFLP map, linked to two RFLP markers, ten AFLP markers and the restorer gene Rf1. Tests with downy mildew race 330 distinguished Pl5 and Pl8, the first being susceptible, the second resistant, whereas both these genes were active against race 304 to which Pl6 (HA335) and Pl7 gave susceptibility. It is concluded that Pl5 and Pl8 are closely linked on linkage group 6 and form a separate resistance gene group from Pl6/Pl7 on linkage group 1. The origins of these groups of downy mildew resistance genes and their use in breeding are discussed. Received: 10 November 2000 / Accepted: 8 February 2001     

Twelve polymorphic expressed sequence tags‐derived markers for Plasmopara halstedii,the causal agent of sunflower downy mildew

Twelve expressed sequence tags‐derived markers were isolated from Plasmopara halstedii (Oomycetes), the causal agent of sunflower downy mildew. A total of 25 single nucleotide polymorphisms and five indels were detected by single‐strand conformation polymorphism analysis and developed for high‐throughput genotyping of 32 isolates. There was a high level of genetic diversity (H_E = 0.484). Observed heterozygosity ranged from 0 to 0.143 indicating that P. halstedii is probably a selfing species. These markers were also useful in detecting significant genetic variations among French populations (F_ST = 0.193) and between French and Russian populations (F_ST = 0.23). Cross‐amplification tests on three closely related species indicated that no loci amplified in other Oomycete species.     

A QTL analysis of sunflower partial resistance to downy mildew ( Plasmopara halstedii) and black stem ( Phoma macdonaldii) by the use of recombinant inbred lines (RILs)

Partial resistance to downy mildew (Plasmopara halstedii) and to black stem (Phoma macdonaldii) in sunflower were investigated under natural field infection and a controlled growth chamber respectively. Genetic control for resistance to the diseases was determined in recombinant inbred lines (RILs) and their two parents, ’PAC-2’ and ’RHA-266.’ The experiments were undertaken in a randomized complete block design with two replications, in a field severely infected by downy mildew and in a controlled growth chamber with plants inoculated with an agressive French isolate of P. macdonaldii. Each replication consisted of three rows, 4.6-m long, giving 48 plants per RIL or parent in the field and 15 plants in the growth chamber. Genetic variability was observed among the RILs for resistance to both diseases. When 10% of the selected RILs were compared with the mean of the two parents genetic gain was significant for partial resistance to the diseases. Four putative QTLs for resistance to downy mildew on linkage groups 1, 9 and 17 were detected using composite interval mapping. The QTLs explained 54.9% of the total phenotypic variance. Major QTLs (dmr1–1 and dmr1–2) for resistance were found on linkage group 1 with up to 31% of the phenotypic variability explained by two peaks. QTL analysis of resistance to black stem showed seven QTLs on linkage groups 3, 6, 8, 9, 11, 15 and 17. The detected QTLs together explain 92% of the phenotypic variation of the trait. Crosses between RILs contrasted for their resistance to downy mildew and black stem, and exhibiting molecular polymorphism in detected QTLs, will be made in order to focus more-precisely on the genomic region of interest. Received: 28 February 2001 / Accepted: 14 June 2001     

Cytoskeletal responses during early development of the downy mildew of grapevine (Plasmopara viticola)

A host-free system was established to induce the early development of the obligate biotrophic pathogen Plasmopara viticola, the downy mildew of grapevine. This system was used to study cytoskeletal responses during encystation and germ tube formation. During these processes, both the actin and the tubulin cytoskeleton show a stage-specific pattern of distribution. Elimination of the cytoskeleton by the actin drug latrunculin B and the microtubule drug ethyl-N-phenyl-carbamate did not affect the release of mobile zoospores from the sporangia, nor the encystation process, but efficiently inhibited the formation of a germ tube. The data are discussed with respect to a role of both actin and microtubules for the establishment of the cell polarity guiding the emergence and the growth of the germ tube.     

Molecular analysis of a major locus for resistance to downy mildew in sunflower with specific PCR-based markers

Resistance of sunflower to the obligate parasite Plasmopara halstedii is conferred by specific dominant genes, denoted Pl. The Pl6 locus confers resistance to all races of P. halstedii except one, and must contain at least 11 tightly linked genes each giving resistance to different downy mildew races. Specific primers were designed and used to amplify 13 markers covering a genetic distance of about 3 cM centred on the Pl6 locus. Cloning and sequence analysis of these 13 markers indicate that Pl6 contains conserved genes belonging to the TIR-NBS-LRR class of plant resistance genes. Received: 9 April 2001 / Accepted: 10 August 2001