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.     

Leaf Disk Inoculation — a Useful Tool for Selecting Infections of Sunflower Downy Mildew at Low Inoculum Concentration, but Inappropriate to Pathotype Characterization

A modified technique for leaf disk inoculation of sunflower with zoosporangia of the downy mildew pathogen Plasmopara halstedii was developed. Infection with low concentrations of inoculum was obtained down to the level of single sporangia. No significant difference in the infection rate was seen between disks from cotyledons and true leaves. This makes leaf disk inoculation particularly suitable for infections at low sporangium density and for the investigation on plant tissue which cannot be infected with whole seedling inoculation. The technique seems, however, to be inappropriate for pathotype characterization using differential host lines.     

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.     

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.     

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.     

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.     

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.     

Transition of secondary to systemic infection of sunflower with Plasmopara halstedii – An underestimated factor in the epidemiology of the pathogen

Plasmopara halstedii, the downy mildew pathogen of sunflower causes significant economic loss world-wide, mostly through soilborne systemic infection of seedlings. Natural infection of sunflower with P. halstedii was monitored in a sunflower field cultivated for ornamental purpose in soil where no sunflower had been grown before. Local and systemic infections were observed in plants of different developmental stages which were sown in five consecutive field plots between Apr. and Jul. The airborne origin of the infection by zoosporangia was concluded from field history, pathogenic symptoms, time course of infection and microscopic investigation of mycelium distribution in stems. A high potential for transition from local to systemic infection was found, at least in ornamental sunflower cultivation under the typical weather conditions in Central Europe. This questions the paradigm that economically and epidemiologically relevant sunflower downy mildew incidences are only derived from subterranean infections. Airborne secondary infections, as they may occur in all developmental stages and on all organs of the host plant, are responsible for late systemic infection and can play a key role in the production of contaminated seeds carrying the pathogen into the next season.     

Specificity in the symbiotic association of Lotus corniculatus and Rhizobium loti from natural populations

To test whether Rhizobium loti are coadapted to nodulate local plant genotypes, we competed R. loti strains in a common environment with clonally propagated Lotus corniculatus. Both the plants and bacterial strains were originally collected from natural populations in three localities and the R. loti strains used were distinguishable by enzyme electrophoretic markers and differed in geographical origin relative to host plant origin. The proportions of nodules occupied by symbiont strains varied widely and depended on both host plant and symbiont genotype. Nonrandom nodulation patterns resulted primarily from preferential nodulation of host genotypes by the symbiont strain that had been associated with the host in the natural environment. Symbionts nodulating their original hosts were preferentially found in nodules on adventitious tap roots as opposed to the younger, lateral roots (for one host-symbiont pair) or in large nodules, independent of location on the root system (for a second host-symbiont pair). The proportion of nodules occupied by a symbiont on novel host genotypes varied, ranging from nearly random expectation to a significant reduction in the proportion of nodules occupied. The analysis of the bacteria recovered from 994 nodules by multilocus enzyme electrophoresis revealed that 952 (95.8%) nodules were occupied by one of the four inoculant strains and 11 (1.1%) were co-occupied by two inoculant strains. A total of 31 (3.1%) nodules were occupied by strains that did not match the electrophoretic profiles of the original inoculant strains. Based on the comparison of multilocus profiles for 23 enzyme loci, we concluded that these bacteria were foreign strains and not recombinants of the original inoculant strains. Our findings indicate a strong host genotype by strain interaction underlying the outcome of rhizobial competition for nodulation sites and suggest there are distinct mechanisms leading to differential recognition of compatible host and symbiont genotypes.     

Development of PCR markers for the<Emphasis Type="Italic"> Pl5/Pl8</Emphasis> locus for resistance to<Emphasis Type="Italic"> Plasmopara halstedii</Emphasis> in sunflower,<Emphasis Type="Italic"> Helianthus annuus</Emphasis> L. from complete CC-NBS-LRR sequences

Sunflower downy mildew, caused by Plasmopara halstedii, is one of the major diseases of this crop. Development of elite sunflower lines resistant to different races of this oomycete seems to be the most efficient method to limit downy mildew damage. At least two different gene clusters conferring resistance to different races of P. halstedii have been described. In this work we report the cloning and mapping of two full-length resistance gene analogs (RGA) belonging to the CC-NBC-LRR class of plant resistance genes. The two sequences were then used to develop 14 sequence tagged sites (STS) within the Pl5/Pl8 locus conferring resistance to a wide range of P. halstedii races. These STSs will be useful in marker-assisted selection programs.Communicated by C. Möllers     

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.     

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     

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.     

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

Summary The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH₄NO₃ (50 to 250 g N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N.Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.     

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.     

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.     

Induction of resistance against downy mildew on sunflower by rhizobacteria

Abstract

Induction of resistance to downy mildew caused by Plasmopara halstedii in sunflower was studied after treatment with PGPR (plant growth promoting rhizobacteria) strain INR7 (Bacillus spp). Treatment of sunflower seeds with 1×10⁸cfu/ml of PGPR strain INR7 resulted in decreased disease severity and offered 51 and 54% protection under green house and field conditions, respectively. The induction of resistance to P. halstedii by PGPR strain INR7 was accompanied by the accumulation of various host defense-related enzymes in susceptible sunflower seedlings. Enhanced activation of catalase (CAT), phenylalanine ammonia-lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO) and chitinase (CHI) was evident at 6, 9, 12, 12 and 12h post inoculation, respectively, in sunflower seedlings raised from seeds treated with PGPR strain INR7. This enhanced and early activation of defense-related responses in the susceptible cultivar after treatment with PGPR strain INR7 was comparable to that in the resistant cultivar. The results indicate that PGPR strain INR7 induced resistance against P. halstedii in sunflower is mediated through enhanced expression of defense mechanism.     

Characterization of a Plasmopara species on Ambrosia artemisiifolia, and notes on P. halstedii, based on morphology and multiple gene phylogenies

Common ragweed (Ambrosia artemisiifolia) is an invasive and highly allergenic plant species, on which two species, Plasmopara halstedii and Plasmopara angustiterminalis, have been recognized to cause downy mildew disease. In this study, morphological and molecular patterns of seven Plasmopara specimens collected from A. artemisiifolia in Canada, Hungary, and USA were compared with those of P. halstedii and P. angustiterminalis from Helianthus and Xanthium, respectively. Analyses of partial sequences of three genes, namely those for the large subunit (28S) of rDNA, cytochrome c oxidase subunit II (COX2), and NADH dehydrogenase subunit I (ND1) of mtDNA, were carried out to examine the phylogenetic relationships among these specimens using both Bayesian and maximum parsimony methods. All the phylogenetic analyses revealed that the downy mildew pathogens infecting A. artemisiifolia in Hungary and North America clearly represent a lineage distinct from other Plasmopara taxa investigated. The shape of sporangia and the width of trunks and branches also allowed the separation of the specimens parasitic to A. artemisiifolia from P. halstedii on Helianthus annuus and P. angustiterminalis on Xanthium strumarium. Surprisingly, the Hungarian and the Canadian specimens were more closely related to each other than to those from the USA based on COX2 and ND1 mtDNA data, although the D1/D2/D3 sequences of 28S rDNA were identical in all these Plasmopara specimens. The regional distribution of the mtDNA haplotypes seen in this study suggests a transatlantic migration has occurred and would be interesting to follow up with a more detailed sampling. To investigate the diversity within P. halstedii sensu lato, infecting different host plant species, specimens from six asteraceous genera, Ambrosia, Flaveria, Helianthus, Siegesbeckia, Solidago, and Xanthium, were also included in molecular analyses. These represented six distinct lineages according to the host plant genera. These findings might serve as a basis for a taxonomical reassessment of the P. halstedii complex and also for the delimitation of several well-defined species within this complex.     

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.