Erratum to Variation in aggressiveness of Plasmopara halstedii (sunflower downy mildew).

Journal of Plant Diseases and Protection -     

Inter-isolate variation for virulence in Plasmopara halstedii (sunflower downy mildew) from Hungary

A total of 82 isolates of Plasmopara halstedii , collected from all production areas of Hungary between 1976 and 1993, were assessed for their virulence pattern on a standard set of sunflower differentials under glasshouse conditions. The isolates were classified into six pathogenic races each representing a particular virulence phenotype. From 1976 until 1988 all the isolates were found to be virulent only on sunflowers possessing no known resistance genes, thus classified as race 1. There was an apparent shift in the virulence of the P. halstedii population collected after 1988. Six races (1, 2, 3, 4, 8 and 9) were identified among the 45 samples collected between 1989 and 1993, with races 1 and 3 predominant, at a frequency of 35% each. While the increase in race virulence is undoubtedly due to selection imposed by resistant hybrids, the origin of the new races is unknown. Whether new races have arisen from the indigenous P. halstedii population, or whether they have been imported from abroad, can only be reliably determined by DNA techniques, such as fingerprinting.     

向日葵霜霉病——我国向日葵的一种新病害

向日葵霜霉病在我国迄今未见报导。1960—1962年,在黑龙江省牡丹江和合江农垦区进行病害稠查时,发现这一病害,在上述地区普遍发生,个别地区为害较重。1962年黑龙江八一农垦     

Development of a PCR test to detect the downy mildew causal agent Plasmopara halstedii in sunflower seeds

Plasmopara halstedii , the causal agent of downy mildew of sunflower, is an obligate parasite but viable sporangia and oospores of the pathogen may be found in a quiescent state in seeds of sunflower and therefore may be transported with sunflower seeds in international commercial exchanges. In order to prevent the spread of this pathogen, especially the introduction of potentially new races, an efficient method to analyse sunflower seed samples is required. In this study, a P. halstedii -specific polymerase chain reaction (PCR) test was developed based on the ribosomal large sub unit (LSU) DNA. The forward (PHAL-F) and reverse (PHAL-R) PCR primers were designed from two polymorphic regions of LSU. After screening 22 isolates of P. halstedii corresponding to different races and countries and 32 other oomycete, deuteromycete and ascomycete isolates, the PHAL-F/R primers amplified only a single PCR band of c. 310 bp from P. halstedii . The PHAL-F/R PCR test could detect as little as 3 pg of P. halstedii genomic DNA per 20  µ L reaction volume and enabled the direct detection of P. halstedii in 35 g sunflower seed samples without the need for any prior biological baiting step. An internal amplification control (IAC) was developed to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. The test was successfully used on samples of naturally contaminated seeds. These new molecular tools should be of great interest for quarantine seed testing purposes.     

Microscopical studies of the effect of metalaxyl on the interaction between sunflower,Helianthus annuus L. and downy mildew,Plasmopara halstedii

The mode of action of the fungicide metalaxyl againstPlasmopara halstedii, the causal agent of sunflower downy mildew, was studied following its application before, during and after artificial contamination of seedlings. The development of the fungus within the treated seedlings was examined microscopically and compared to that occuring in untreated genetically susceptible or resistant genotypes. Hypersensitive-like reactions and necrotic zones leading to the inhibition of fungus growth within the hypocotyl were observed for the three modes of application. This suggests that sunflower defence mechanisms activated by genetical resistance are also involved in the control of downy mildew by metalaxyl.     

Advances in sunflower downy mildew research

This review summarises the progress in research on sunflower downy mildew as reported in publications of the past 10 to 15 years, the period since the last comprehensive review on Plasmopara halstedii. Particular attention is paid to subjects that showed much progress and may be of particular interest to sunflower pathologists, mycologists or molecular biologists. Accordingly, single sections are devoted to the problems of taxonomic and phylogenetic aspects, host specificity, the host—pathogen interaction including resistance phenomena, as well as epidemiology and disease management. Reflecting the progress achieved in some fields and illuminating the deficits in others should stimulate the reader's interest in this very significant pathosystem.     

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.     

Pathogenic variability of Plasmopara halstedii infecting sunflower in the Czech Republic

Plasmopara halstedii was isolated from diseased sunflowers collected from eight locations in the Czech Republic from 2007 to 2014. Races of the pathogen were determined based on 84 isolates collected during the study. In total, eight races of P. halstedii were detected using a set of nine sunflower differential lines. Races 700, 704, 705, 710, 714 and 715 were proven by soil drench inoculation, and two additional races (730 and 770) proposed by the previously applied leaf disc inoculation method. Race 700 was the most dominant in the Czech P. halstedii populations, with race 710 being the second most frequent. Races 704 and 714 were found over three seasons, while other races were recorded only in one growing season (race 730 in 2010, and the new races 705 and 715 in 2014). A comprehensive study was further conducted for isolates collected in 2013–14 using an extended differential set consisting of 15 sunflower lines. According to the latter methodology which marks races with five‐digit virulence codes, races 70060, 70471, 70571, 71060, 71461 and 71571 were recorded. The growing complexity of P. halstedii pathogenicity exhibited by the ability to infect higher numbers of differential genotypes and resulting in determination of the new pathogen races (virulence profiles) 70571, 71461 and 71571 is alarming. Although the limited number of isolates studied cannot characterize the entire pathogen diversity in the Czech Republic, the trend towards more diverse virulence in P. halstedii populations is clearly demonstrated by the new records of races 704, 705, 714 and 715, all capable of overcoming the resistance gene Pl₆.     

Controlling downy mildew (Plasmopara viticola) in field-grown grapevine with β-aminobutyric acid (BABA)

Two foliar sprays of BABA (DL-3-amino-n-butanoic acid, DL-β-aminobutyric acid), or a mixture of BABA and different fungicides at reduced rates, effectively controlled (>90%) downy mildew, caused byPlasmopara viticola, in the foliage of field-grown grapevines. In five field trials BABA sprays resulted in a significant reduction of infectious leaf area and fungal sporulation, and of necrosis of oilspots. The level of disease control for BABA in four trials was similar to that achieved by metalaxyl-Cu or ‘Acrobat Plus’ (dimethomorph + mancozeb). Two-way tank-mixtures of BABA + fosetyl-AI, BABA + folpet, or BABA + Bion (benzothiadiazole), each at half the recommended rate, provided an additive effect againstP. viticola, performing as well as the full rate of each fungicide alone. BABA was not phytotoxic and did not affect pH, total titratable acids, or °Brix of the juice, as determined by commercial fungicidal standards. The results indicate that foliar applications of BABA can efficiently be integrated into a downy mildew control program in vineyards.     

Involvement of phenolic compounds in the resistance of grapevine callus to downy mildew (Plasmopara viticola)

Callus tissue of different grapevines (Vitis spp.) was inoculated withPlasmopara viticola. Short, highly-branched hyphae with necrosis, and long hyphae with heavy sporulation were observed on resistant and susceptible callus respectively. Thin-layer chromatography and spectrophotometric analysis showed that resistant callus contained greater quantities of gallocatechin derivatives than susceptible callus. Regression analysis between the field disease rating of each variety and its gallocatechin derivatives content indicated 92.2% correlation. Histochemical studies showed that, after infection withP. viticola, flavonoids appeared in the superficial cell walls of the callus, to a lesser degree on susceptible callus than on resistant callus. At a late stage of infection, the superficial cells of resistant callus were suberized, which did not occur in susceptible callus. This study showed that the preformed gallocatechin derivatives, the induced flavonoids and suberized superficial cells might play a role in the resistance of grapevine callus tissue to this fungus.Abbreviations CallusI Callus ofV. riparia var. Gloire de Montpellier - CallusV Callus ofV. vinifera var. Grenache - TLC Thin Layer Chromatography - var. variety - GAD Gallic acid derivatives - GD gallocatechin derivatives - RC resistant callus - SC susceptible callus     

Importance of secondary inoculum of Plasmopara viticola to epidemics of grapevine downy mildew

To quantify the magnitude and the spatial spread of grapevine downy mildew secondary sporangia, 4685 Plasmopara viticola single lesion samples were collected from 18 plots spread across central Europe. Disease symptoms were collected on two to 22 sampling dates per plot between 2000 and 2002. Four multiallelic microsatellite markers were used for genotypic identification of pathogen samples. Genetic analysis showed more than 2300 site-specific P. viticola genotypes, indicating that populations are genetically rich demographic units. Approximately 70% of the genotypes were sampled once and 14% were sampled twice throughout the various epidemics. In the 18 populations only seven genotypes (0.3%) were identified more than 50 times. Three genotypes particularly successful in causing disease through secondary cycles showed mainly a clustered distribution. The distance of sporangial migration per secondary cycle was less than 20 m and their plot colonization rate was calculated at around 1–2 m² day⁻¹. Downy mildew epidemics of grapevine are therefore the result of the interaction of a multitude of genotypes, each causing limited (or a few) lesions, and of a dominant genotype able to spread stepwise at plot-scale. These findings contrast with current theories about grapevine downy mildew epidemiology, which postulate that there is massive vineyard colonization by one genotype and long-distance migration of sporangia.     

部分鲜食葡萄品种霜霉病抗性田间调查

以54个鲜食葡萄品种为试材,调查统计不同葡萄品种的霜霉病发病程度、发病速率、落叶率和新梢成熟度。结果表明,不同葡萄品种对霜霉病的抗性存在明显差异,欧美杂种品种群的葡萄对霜霉病的抗性显著高于欧亚品种群;病情指数与落叶率、新梢成熟度呈极显著的相关性,与发病速率之间关系不显著。     

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.