Pathogenic groups identified among Plasmopara halstedii isolates belonging to several races

Pathogenic groups among 50 Plasmopara halstedii (sunflower downy mildew) isolates belonging to races 100, 300, 304, 314, 710, 704 and 714 were identified. Based on the reaction for the P. halstedii isolates to sunflower differential line D3, these isolates were divided into two groups as more virulent isolates of the 7xx races and the less virulent isolates of 100 and 3xx races. Index of aggressiveness (sporulation density/latent period) was calculated for each isolate and the presence of significant differences between isolates of 100 and 3xx races (more aggressive) and isolates of 7xx races (less aggressive) was revealed. Consequently, it seems that P. halstedii isolates may be divided into two pathogenic groups as more virulent and less aggressive isolates of 7xx races and less virulent and more aggressive isolates of the 100 and 3xx races.     

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.     

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.     

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.     

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.     

Pathogenicity and its alternation with both morphological and genetic variation in Plasmopara halstedii (sunflower downy mildew)

Morphological, pathogenic and genetic variation was studied in seven Plasmopara halstedii (sunflower downy mildew) isolates of several races using five singlezoosporangium isolates per pathogen isolate. Aggressiveness criteria were analysed in one sunflower inbred line showing a high level of quantitative resistance. Genetic relationships were detected between the single zoosporangium isolates using 12 expressed sequence tags (EST)-derived markers. Analysis of the five single zoosporangium isolates for P. halstedii isolates showed variability within pathogen isolates for all aggressiveness criteria, but not for all pathogen isolates. Isolates of races 100 and 3xx were characterised with shorter latent period and higher sporulation density than the isolate of races 7xx. All pathogen isolates showed high percentage infection values and caused a large reduction in seedling size except for one isolate involved in dwarfing. There was no relation between zoosporangia form or size and race virulence profiles or aggressiveness criteria. There was no intra-genetic variation for all pathogen isolates, but it was observed an important genetic variation between single zoosporangium isolates of all races. No correlation was detected between pathogenicity traits and EST genotypes.     

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.     

A Method to Measure Aggressiveness of Plasmopara halstedii (Sunflower Downy Mildew)

For the first time, a method was used to measure aggressiveness of two Plasmopara halstedii races (100 and 710), the parasite causing sunflower downy mildew. Two sunflower lines showing different levels of quantitative resistance were used to measure two aggressiveness criteria: latent period and sporulation density. A strain of race 100 had a shorter latent period and greater sporulation density than a strain of race 710. The sunflower inbred line BT, rather susceptible in the field, presented a greater sporulation density and a shorter latent period than another inbred line FU, which shows greater resistance in the field. These results indicated that race 100 was more aggressive than race 710. The behaviour in the field of the two inbred lines was confirmed in the laboratory observations.     

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.     

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.     

Isolation of Oospores of Sunflower Downy Mildew, Plasmopara halstedii, and Microscopical Studies on Oospore Germination

A method was elaborated to isolate oospores of Plasmopara halstedii from tissue of its host, Helianthus annuus . Isolated oospores were studied microscopically and germination was documented with respect to the time course and the mode of germination. Formation of primary sporangia was similarly observed in oospores, harvested from 4- to 6-week-old systemically infected sunflower plants, grown under constant conditions at 16°C, as well as from field plants, harvested late in the season. Pretreatment of oospores with cold temperatures, previously assumed to stimulate the rate and to accelerate the speed of oospore germination, did not result in such effects. Germination usually occurred within 10–30 days of incubation at a highly variable rate of about 1 to 17% (average 6.7%) in deionized water.     

Allozyme variation in domesticated annual sunflower and its wild relatives

 The annual sunflower (Helianthus annuus L.) is a morphologically and genetically variable species composed of wild, weedy, and domesticated forms that are used for ornament, oilseed, and edible seeds. In this study, we evaluated genetic variation in 146 germplasm accessions of wild and domesticated sunflowers using allozyme analysis. Results from this survey showed that wild sunflower exhibits geographically structured genetic variation, as samples from the Great Plains region of the central United States were genetically divergent from accessions from California and the southwestern United States. Sunflower populations from the Great Plains harbored greater allelic diversity than did wild sunflower from the western United States. Comparison of genetic variability in wild and domesticated sunflower by principal coordinate analysis showed these groups to be genetically divergent, in large part due to differences in the frequency of common alleles. Neighbor-Joining analyses of domesticated H. annuus, wild H. annuus and two closely related wild species (H. argophyllus T. & G. and H. petiolaris Nutt.) showed that domesticated sunflowers form a genetically coherent group and that wild sunflowers from the Great Plains may include the most likely progenitor of domesticated sunflowers. Received: 2 December 1996/Accepted: 4 April 1997     

Chitinase and peroxidase activities in sunflower hypocotyls: Effects of BTH and inoculation with <Emphasis Type="Italic">Plasmopara halstedii</Emphasis>

Systemic acquired resistance (SAR) can be induced in plants by incompatible pathogens, pathogen derived extracts, or certain chemicals as benzothiadiazole (BTH). The aim of this work was to compare changes in peroxidase and chitinase activities, enzymes considered as PR-proteins, caused by BTH and the pathogen Plasmopara halstedii. Hypocotyls from susceptible and resistant BTH-treated sunflower seedlings showed increased peroxidase and chitinase activities. Inoculation with P. halstedii increased chitinase and peroxidase activities in inoculated hypocotyls from susceptible but not from resistant sunflower seedlings.     

RFLP and RAPD mapping of the sunflower Pl1 locus for resistance to Plasmopara halstedii race 1

The Pl1 locus in sunflower, Helianthus annuus L., conferring resistance to downy mildew, Plasmopara halstedii, race 1 has been located in linkage group 1 of the consensus RFLP map of the cultivated sunflower. Bulked segregant analyses were used on 135 plants of an F₂ progeny from a cross between a downy mildew susceptible line, GH, and RHA266, a line carrying Pl1. Two RFLP markers and one RAPD marker linked to the Pl1 locus have been identified. The RFLP markers are located at 5.6 cM and 7.1 cM on either side of Pl1. The RAPD marker is situated at 43.7 cM from Pl1. The significance and applications of these markers in sunflower breeding are discussed.     

Leaf expansion as related to plant water availability in a wild and a cultivated sunflower

This study aimed to determine if two species of sunflower, Helianthus annus L. cv. Hysun 31 (cultivated, single-stemmed genotype) and Helianthus petiolaris Nuttall ssp. fallax (wild, many-hranched genotype) differed in the response of leaf growth to water deficits. Earlier published studies, concerned only with H. annuus, failed to reveal differences in the response of sunflowers to water stress. Plants of the two species were paired in large containers of soil and grown under high radiation in a glasshouse. One batch of plants was irrigated and the other allowed to dry so that predawn leaf water potentials declined at an average of 0.072 MPa day^?1. The dry batch was rewatered when predawn leaf water potentials reached ?0.85 MPa. The stress imposed was sufficient to curtail leaf growth so that plants in the dry treatment had only 60% of the leaf area of irrigated plants at the onset of rewatering. Both species were affected by stress to the same relative extent, though their leaf areas at this stage differed 7-fold. Both genotypes also recovered to the same degree in the long term, finally having leaf areas and gross dry matter distribution patterns which were indistinguishable from plants which were irrigated throughout. However, water stress resulted in different distribution patterns of leaf area: H. annuus produced larger leaves at the top of its single stem which compensated for the reduced area in lower leaves, whereas H. petiolaris compensated in the leaves on its branches. Leaves which emerged after the time of stress were most able to compensate in area subsequently. For example, those leaves of H. annuus which emerged one week after stress-relief were more than three times larger than comparable leaves on plants irrigated continuously. Leaf expansion rates were affected earlier in the stress cycle than leaf conductance in H. annuus, but not in H. petiolaris. But as with other plant responses to water stress, the differences between the two species were small.     

Marker-assisted selection for two rust resistance genes in sunflower

In this study we report on the identification of molecular markers, OX20₆₀₀ and OO04₉₅₀, linked to the geneR _Adv in the proprietary inbred line P2. This gene confers resistance to most of the pathotypes of Puccinia helianthi identified in Australia. Analysis indicates these RAPD markers are linked to the resistance locus at 0.0 cM and 11 cM respectively. SCAR markers SCX20₆₀₀ and SCO04₉₅₀ derived from these two RAPD markers, and SCT06₉₅₀ derived from a previously reported RAPD marker linked at 4.5 cM from the R ₁ rust resistance gene were developed. SCX20₆₀₀ and SCO04₉₅₀ were linked at similar distances from their resistance locus as the RAPD markers. SCTO6₉₅₀ co-segregated completely with rust resistance. The robustness of the R ₁ SCAR marker was demonstrated through the amplification of the marker in a diverse range of sunflower germplasm considered to possess the R ₁ gene. The SCAR markers forR _Adv were not amplified in the sunflower rust differential set thereby supporting the contention that this is a novel resistance gene. They did amplify in a number of proprietary lines closely related to the line P2. This locus is under further investigation as it will be useful in our attempts to use molecular-assisted breeding to produce durable resistance in sunflower to P. helianthi.