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.     

Can percentage infection and dwarfing be used to differentiate aggressiveness in Plasmopara halstedii?

Aggressiveness was studied in seven Plasmopara halstedii (sunflower downy mildew) parental isolates of races 100, 300, 304, 314, 704, 710 and 714 using five single zoosporangium isolates per parental isolate. Aggressiveness criteria, including percentage infection and dwarfing (reduction of hypocotyl length), were analysed in one sunflower inbred line showing a high level of quantitative resistance. Analysis of five single zoosporangium isolates of each parental isolate showed variability within parental isolate for the two aggressiveness criteria, but not for all parental isolates for percentage infection and vice versa for the reduction of hypocotyl length. Percentage infection showed high values irrespective of the parental isolate used. However, all the parental isolates caused a large reduction in seedling size except for the isolate of race 314. Although percentage infection and reduction of hypocotyl length could be used to differentiate aggressiveness in P. halstedii, it seems that these criteria played a limited role to define P. halstedii isolates according to their aggressiveness.     

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 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.     

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.     

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.     

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.     

Different pathotypes of the sunflower downy mildew pathogen Plasmopara halstedii all contain isometric virions†

Eight pathotypes of Plasmopara halstedii were screened to investigate the occurrence of virions and the potential viral influence on the pathogenicity of the sunflower downy mildew pathogen. In 23 of 26 P. halstedii isolates derived from eight countries in Europe, North America and South America, virions were detected by transmission electron microscopy. By contrast, there were no ultrastructural indications of virus‐like particles in eight other related Oomycetes. The virions of representative P. halstedii isolates were morphologically and biochemically characterized and compared among each other. Regardless of their host's pathotypes, the geographical origin of the isolate and the sensitivity towards the fungicide metalaxyl, the viral characters obtained were uniform. The virions were isometric and measured approximately 37 nm in diameter. One polypeptide of c. 36 kDa and two segments of single‐stranded RNA (3.0 and 1.6 kb) were detected. Both viral RNA segments were detected by capillary electrophoresis in the three remaining P. halstedii isolates where virions were undetectable by transmission electron microscopy. Virus‐specific primers for the 1.6 kb‐segment were synthesized and used to determine and compare a partial sequence of the viral coat protein among virions of different P. halstedii pathotypes. In all tested isolates, fragments of 0.7 kb were amplified which were directly sequenced. Sequence variation was insignificant. As both less aggressive and more aggressive P. halstedii isolates contained virions, the presence or absence of virions could not explain the diverse aggressiveness of the downy mildew pathogen towards sunflower. Moreover, the results indicated that pathogenicity of P. halstedii was not related to variation in morphological or biochemical characters of the virions.     

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.     

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.     

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.     

Isozyme Analysis and Soluble Mycelial Protein Pattern in Iranian Isolates of Several formae speciales of Fusarium oxysporum

A total of 13 representative isolates of Fusarium oxysporum f. sp. melonis (FOM) from Iran, USA and France, eight isolates of seven formae speciales from Iran and one isolate of F. oxysporum f. sp. niveum from the USA were compared based on isozyme analysis and soluble mycelial protein pattern. Isozyme analyses of alkaline phosphatase (ALP), catalase (CAT), esterase (EST), malate dehydrogenase (MDH), superoxide dismutase (SOD) and xanthine dehydrogenase (XDH) revealed polymorphism among the F. oxysporum isolates in which 22 electrophoretic phenotypes (EP) were determined. At least 10 putative loci for these six enzymes were detected and they were all polymorphic. Maximum genetic diversity was observed in CAT, EST and XDH loci. Using UPGMA, the 22 isolates were separated into three main groups with one of the groups divided into two subgroups. Group I included isolates belonging to five formae speciales from Iran, whereas group II that included FOM isolates from both Iran and the USA was divided into two subgroups each containing the vast majority of the respective isolates from either country. Group III constituted FOM isolates from France and one pathogenic isolate on pepper from Iran. FOM isolates representing five different geographical regions from Iran belonged to two different races of 1 and 1,2Y and one vegetative compatibility group (VCG)0134 and thus were genetically homologous. Isozyme polymorphism in these isolates was highly correlated with VCG and geographical origins and to a lesser extent with races. Variations in soluble protein profile in FOM isolates were correlated with genetic distances determined in isozyme analysis. This study suggests that isozyme analysis could be a useful tool for identifying genetic diversity not only in FOM but also several formae speciales of F. oxysporum.     

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.     

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.     

New Races of Sunflower Downy Mildew (Plasmopara Halstedii) in Germany

Plasmopara halstedii, the causal agent of downy mildew of cultivated sunflower (Helianthus annuus), was documented in Germany for the first time in commercial fields. The pathogen was first observed in the Württemberg area, where races 1 and 4 were identified using a set of differential lines. Later, commericial fields near Baden were found to be infected by race 5, which is the first occurrence of that race outside of North America. Withthe discovery of race 5, there are now eight races of the sunflower downy mildew fungus that have been found in Europe. The sunflower cultivars most frequently grown in Germany were investigated for resistance to race 1, 4 and 5; while all were resistant to race 1, none were resistant to either race, 4 or 5.     

New Races of Sunflower Downy Mildew (Plasmopara Halstedii) in Germany

Plasmopara halstedii, the causal agent of downy mildew of cultivated sunflower (Helianthus annuus), was documented in Germany for the first time in commercial fields. The pathogen was first observed in the Württemberg area, where races 1 and 4 were identified using a set of differential lines. Later, commercial fields near Baden were found to be infected by race 5, which is the first occurrence of that race outside of North America. With the discovery of race 5, there are now eight races of the sunflower downy mildew fungus that have been found in Europe. The sunflower cultivars most frequently grown in Germany were investigated for resistance to race 1, 4 and 5; while all were resistant to race 1, none were resistant to either race 4 or 5.     

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     

Reaction of Some Coffea arabica Genotypes to Strains of Colletotrichum kahawae, the Cause of Coffee Berry Disease

Pathogenicity tests were performed on 11 genotypes of Coffea arabica using single‐isolate suspensions of Colletotrichum Kahawae obtained from 90 monoconidial isolates. The objective of this study was to estimate the proportion of pathogenic variation corresponding 10 differences in aggressiveness and virulence (races). A large part of the variation in the pathogen population was due to aggressiveness. The differential effects were too small to suggest conclusively that races exist. This paper discusses the possible causes for the observed small differential interaction and suggests breeding strategies that not only prevent possible adaptation of the pathogen to resistant varieties but also limit variation for resistance due to differences in aggressiveness of the pathogen.     

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.     

Genome characterization of Pyrenophora tritici-repentis isolates reveals high plasticity and independent chromosomal location of ToxA and ToxB

The fungus Pyrenophora tritici - repentis (Died.) causes tan spot, an important leaf disease of wheat worldwide. Isolates of this pathogen have been collected and characterized into eight races on the basis of their ability to produce three different host-selective toxins. The karyotype of 47 isolates was determined by pulsed field gel electrophoresis. The collection originated from different parts of the world and included genotypes from all races. A single isolate was characterized for each of races 3, 4 and 6, whereas fourteen, five, nine, five and eleven isolates were karyotyped for races 1, 2, 5, 7 and 8, respectively. The survey showed that the chromosome number of P. tritici-repentis was highly variable, with some isolates having as few as eight chromosomes, but others having 11 or more. Similarly, the genome size ranged from 25.5 to 48.0 Mb, and individual chromosome sizes ranged from 1.3 to more than 5.7 Mb. Considerable variation was observed in karyotype patterns among the P. tritici-repentis isolates tested. A total of 29 different karyotypes was identified among the 47 isolates. These chromosome level variations were as variable for isolates within a race as for isolates across races. Southern blot analysis of the 47 isolates with ToxA and ToxB probes revealed that the toxin genes were always located on different chromosomes. Furthermore, with six chromosome-specific single-copy probes, the ToxA -carrying chromosome was shown to be homologous among the Ptr ToxA-producing isolates, with a related chromosome in the non-ToxA-producing isolates, suggesting that the chromosome on which ToxA generally resides is of an essential nature. Interestingly, a molecular rearrangement involving a translocation of ToxA to a different chromosome was identified in one isolate.