Analysis of population structure of Erysiphe necator using AFLP markers

An analysis of the population structure of Erysiphe necator from Spain was carried out to determine if population structure of grapevine powdery mildew is partitioned by origin of primary inoculum, i.e. overwintering ascomata on grapevine bark and mycelia in dormant buds. Ten of the 31 isolates collected were from flag shoots, i.e. shoots covered with sporulating mycelia that arise from dormant infected buds. Genetic variation was assessed using 61 AFLP markers. Cluster analysis revealed two distinct groups with 63% genetic similarity. Principal coordinates analysis ( pccorda ) produced a similar distribution of isolates in two groups. Isolates were not well identified within their groups by the origin of primary inoculum. Analysis of molecular variance ( amova ) determined that the origin of primary inoculum was not a significant component in the genetic variation in the population of E. necator . It was concluded that these two subpopulations are not separated by the type of primary source of inoculum.     

No indication of strict host associations in a widespread mycoparasite: grapevine powdery mildew (Erysiphe necator) is attacked by phylogenetically distant Ampelomyces strains in the field

Pycnidial fungi belonging to the genus Ampelomyces are common intracellular mycoparasites of powdery mildews worldwide. Some strains have already been developed as commercial biocontrol agents (BCAs) of Erysiphe necator and other powdery mildew species infecting important crops. One of the basic, and still debated, questions concerning the tritrophic relationships between host plants, powdery mildew fungi, and Ampelomyces mycoparasites is whether Ampelomyces strains isolated from certain species of the Erysiphales are narrowly specialized to their original mycohosts or are generalist mycoparasites of many powdery mildew fungi. This is also important for the use of Ampelomyces strains as BCAs. To understand this relationship, the nuclear ribosomal DNA internal transcribed spacer (ITS) and partial actin gene (act1) sequences of 55 Ampelomyces strains from E. necator were analyzed together with those of 47 strains isolated from other powdery mildew species. These phylogenetic analyses distinguished five major clades and strains from E. necator that were present in all but one clade. This work was supplemented with the selection of nine inter-simple sequence repeat (ISSR) markers for strain-specific identification of Ampelomyces mycoparasites to monitor the environmental fate of strains applied as BCAs. The genetic distances among strains calculated based on ISSR patterns have also highlighted the genetic diversity of Ampelomyces mycoparasites naturally occurring in grapevine powdery mildew. Overall, this work showed that Ampelomyces strains isolated from E. necator are genetically diverse and there is no indication of strict mycohost associations in these strains. However, these results cannot rule out a certain degree of quantitative association between at least some of the Ampelomyces lineages identified in this work and their original mycohosts.     

Spatial and Genetic Analysis of a Flag Shoot Subpopulation of Erysiphe necator in Italy

ABSTRACT Erysiphe necator overwinters as ascospores in cleistothecia and mycelium in dormant buds of grapevines. Shoots developing from infected buds early in the growing season are covered with dense mycelium and are known as "flag shoots". Combining epidemiological and genetic analyses, the objective of this study was to analyze the spatial and genetic structure of a flag shoot subpopulation of E. necator as a way to assess the contribution of flag shoots as primary inoculum, and to determine if flag shoot subpopulations are clonal with only one mating type. One vineyard in Tuscany, Italy was surveyed intensively for flag shoots for 8 years; isolations of E. necator were made from flag shoots for 5 years. We observed distinct disease foci developing around flag shoots early in epidemics, demonstrating a steep dispersal gradient of conidia and the importance of flag shoots as primary inoculum sources. Flag shoots were spatially aggregated within and between years, most likely as a result of short-distance dispersal of conidia from flags early in the season when dormant buds for the next year's shoots are formed and are susceptible to infection. The two mating types were found in 1:1 ratios in this flag shoot subpopulation. Genotypic diversity, based on inter-simple sequence repeat markers, was high in all years with only two haplotypes occurring twice, and subpopulations were genetically differentiated between years. Similarities between haplotypes were not spatially autocorrelated. One multilocus analysis of population structure is consistent with the hypothesis of random mating but another is not. These results are not consistent with expectations for a strictly clonal or strictly randomly mating flag shoot subpopulation. Instead, the hypothesis that the flag shoot subpopulation of E. necator may reproduce clonally and sexually needs further testing.     

An apparatus for collecting total conidia of Blumeria graminis f.sp. hordei from leaf colonies using electrostatic attraction

Conidia from living conidiophores of barley powdery mildew ( Blumeria graminis f.sp. hordei ) on host leaves were collected consecutively using an electrostatic spore collector. The collector consisted of an electrical conductor plate linked to an electrostatic voltage generator and insulator plates placed abreast on a timed conveyer. The conductor plate was negatively charged by the potential supplied from the voltage generator. The negatively charged conductor plate caused dielectric polarization of the insulator plate, and the surface charge on the insulator plate attracted mature conidia abstricted from conidiophores on colonies growing on leaves placed 2 cm from the insulator plate. The surface charge on the insulator plate was proportional to the voltage applied to the conductor plate. Under optimized conditions, abstricted conidia were attracted to the electrostatically activated insulator plates without any detriment to their survival. During a colony's life span of c . 460 h, conidia were released throughout the day and c . 12 × 10⁴ conidia were collected during the lifetime of the colony. This is the first report on the direct quantification of progeny conidia produced by powdery mildew infecting host leaves.     

A single nucleotide polymorphism in the β-tubulin gene distinguishing two genotypes of Erysiphe necator expressing different symptoms on grapevine

The biotrophic fungus Erysiphe necator (formerly Uncinula necator ), for which two genetic groups have been described in European vineyards, is the causal agent of grapevine powdery mildew. By analysing the pathogen population with respect to polymorphism in the sequence of the β-tubulin gene, which distinguishes two groups of isolates, a new tool was developed for epidemiological and population studies and tested in the vineyard. As in many ascomycetes, the β-tubulin gene of E. necator ( Entub ) includes six introns and seven exons and encodes a 447-amino-acid protein. A single nucleotide polymorphism (SNP) in the intron-3 region of the Entub gene distinguished two genetic groups (A and B). This method was used to examine differences in the ratio of the two groups from a total of 289 grape powdery mildew samples collected at the beginning of the growing season from either flag shoots or leaves with sparse-spot symptoms in four different vineyards. The SNP in the intron-3 region of the β-tubulin gene, similar to SNPs in the CYP51 gene, was associated with genotypes A and B of E. necator and confirmed the existence of two sympatric populations of the pathogen in the French vineyards. Differences in the relative proportions of each group varied with the presence or absence of flag-shoot symptoms and with the region in which isolates had been collected.     

Formation of overwintering structures of Uncinula necator and colonization of grapevine under field conditions

Developing shoots of grapevine ( Vitis vinifera cv. Kerner) were inoculated with conidia of the powdery mildew, Uncinula necator , at well-defined phenological stages of the host to provoke the development of flag shoots in the field and to investigate these shoots as early as possible in the following growing season for the presence of the pathogen. The disease progress was monitored and fungal growth and development on samples from a field trial were analysed by means of low-temperature scanning electron microscopy (LT–SEM). Mycelium was detected on the surface and in the interior parts of buds. The suitability of the field plot to analyse flag shoots was proven by the occurrence of such shoots in the following spring. Furthermore, early stages of cleistothecia development on berries were described for the first time. Establishment of U. necator in dormant buds of grapevine, giving rise to flag shoots in the following spring, is considered to play a significant role in overwintering of the pathogen in the vineyards of southern Germany.     

Host Barriers and Responses to Uncinula necator in Developing Grape Berries

ABSTRACT Grape berries are highly susceptible to powdery mildew 1 week after bloom but acquire ontogenic resistance 2 to 3 weeks later. We recently demonstrated that germinating conidia of the grape powdery mildew pathogen (Uncinula necator) cease development before penetration of the cuticle on older resistant berries. The mechanism that halts U. necator at that particular stage was not known. Several previous studies investigated potential host barriers or cell responses to powdery mildew in berries and leaves, but none included observation of the direct effect of these factors on pathogen development. We found that cuticle thickness increased with berry age, but that ingress by the pathogen halted before formation of a visible penetration pore. Cell wall thickness remained unchanged over the first 4 weeks after bloom, the time during which berries progressed from highly susceptible to nearly immune. Autofluorescent polyphenolic compounds accumulated at a higher frequency beneath appressoria on highly susceptible berries than on highly resistant berries; and oxidation of the above phenolics, indicated by cell discoloration, developed at a significantly higher frequency on susceptible berries. Beneath the first-formed appressoria of all germinated conidia, papillae occurred at a significantly higher frequency on 2- to 5-day-old berries than on 30- to 31-day-old fruit. The relatively few papillae observed on older berries were, in most cases (82.8 to 97.3%), found beneath appressoria of conidia that had failed to produce secondary hyphae. This contrasted with the more abundantly produced papillae on younger berries, where only 35.4 to 41.0% were located beneath appressoria of conidia that had failed to produce secondary hyphae. A pathogenesis-related gene (VvPR-1) was much more highly induced in susceptible berries than in resistant berries after inoculation with U. necator. In contrast, a germin-like protein (VvGLP3) was expressed within 16 h of inoculation in resistant, but not in susceptible berries. Our results suggest that several putative barriers to infection, i.e., cuticle and cell wall thickness, antimicrobial phenolics, and two previously described pathogenesis-related proteins, are not principal causes in halting pathogen ingress on ontogenically resistant berries, but rather that infection is halted by one or more of the following: (i) a preformed physical or biochemical barrier near the cuticle surface, or (ii) the rapid synthesis of an antifungal compound in older berries during the first few hours of the infection process.     

Effects of sunlight exposure on grapevine powdery mildew development

Natural and artificially induced shade increased grapevine powdery mildew (Erysiphe necator) severity in the vineyard, with foliar disease severity 49 to 75% higher relative to leaves in full sun, depending on the level of natural shading experienced and the individual experiment. Cluster disease severities increased by 20 to 40% relative to those on check vines when ultraviolet (UV) radiation was filtered from sunlight reaching vines in artificial shading experiments. Surface temperatures of leaves in full sunlight averaged 5 to 8°C higher than those in natural shade, and in one experiment, filtering 80% of all wavelengths of solar radiation, including longer wavelengths responsible for heating irradiated tissues, increased disease more than filtering UV alone. In controlled environment experiments, UV-B radiation reduced germination of E. necator conidia and inhibited both colony establishment (hyphal formation and elongation) and maturity (latent period). Inhibitory effects of UV-B radiation were significantly greater at 30°C than at 20 or 25°C. Thus, sunlight appears to inhibit powdery mildew development through at least two mechanisms, i.e., (i) UV radiation's damaging effects on exposed conidia and thalli of the pathogen; and (ii) elevating temperatures of irradiated tissues to a level supraoptimal or inhibitory for pathogen development. Furthermore, these effects are synergistic at temperatures near the upper threshold for disease development.     

Development of a grower‐conducted inoculum detection assay for management of grape powdery mildew

Management of grape powdery mildew (Erysiphe necator) and other polycyclic diseases often relies on calendar‐based pesticide application schedules that assume the presence of inoculum. An inexpensive, loop‐mediated isothermal amplification (LAMP) assay was designed to quickly detect airborne inoculum of E. necator to determine when to initiate a fungicide application programme. Field efficacy was tested in 2010 and 2011 in several commercial and research vineyards in the Willamette Valley of Oregon from pre‐bud break to véraison. In each vineyard, three impaction spore traps were placed adjacent to the trunk. One trap was maintained and used by the grower to conduct the LAMP assay (G‐LAMP) on‐site and the other two traps were used for laboratory‐conducted LAMP (L‐LAMP) and quantitative PCR assay (qPCR). Using the qPCR as a gold standard, L‐LAMP was comparable with qPCR in both years, and G‐LAMP was comparable to qPCR in 2011. Latent class analysis indicated that qPCR had a true positive proportion of 98% in 2010 and 89% in 2011 and true negative proportion of 96% in 2010 and 64% in 2011. An average of 3·3 fewer fungicide applications were used when they were initiated based on spore detection relative to the grower standard practice. There were no significant differences in berry or leaf incidence between plots with fungicides initiated at detection or grower standard practice plots, suggesting that growers using LAMP to initiate fungicide applications can use fewer fungicide applications to manage powdery mildew compared to standard practices.     

Susceptibility of grapevine buds to infection by powdery mildew Erysiphe necator

Bud colonization and perennation of powdery mildew ( Erysiphe necator ) was studied by inoculating shoots of grapevine ( Vitis vinifera cv. Carignane) at different phenological stages. Disease incidence and severity assessments indicated that buds were most susceptible at the three- to six-unfolded-leaf stage. Incidence of powdery mildew colonies on the surface of buds collected from these shoots 7 weeks postinoculation was highest at these stages (68 and 62%, respectively), which indicates that colonization of the bud interior via the infected bud surface is likely to occur within this period. Histological analyses of buds revealed hyphae with haustoria, conidiophores and conidia on all parts of the bud interior except for the meristems. In particular, trichomes were frequently parasitized by haustoria. In total, 13·2% of all buds analysed, and 32·3% of all buds originating from shoots inoculated at the three-unfolded-leaf stage, were infected by E. necator . In the spring of the following year, buds from inoculated shoots yielded 18 flag shoots (1·6% of all emerging shoots). These primary infections caused an epidemic 28 days after the appearance of the first flag shoot. A linear regression analysis on the frequency of infections of the bud exterior, bud interior and flag shoots revealed that incidence of external bud infection in the first season is strongly correlated with flag shoot incidence in the following season ( R ² = 0·94). Hence predictions of flag shoot incidence may be reliably based on the incidence of infection on the outer bud scales in the preceding season.     

New tools for studying epidemiology and resistance of grape powdery mildew to DMI fungicides

Using a Random Amplified Polymorphic DNA (RAPD) assay, we investigated the genetic polymorphism existing among 62 European isolates of the grape powdery mildew fungus (Uncinula necator [Schw.] Burr.). Isolates overwintering as mycelium in buds were genetically distinct from isolates overwintering as ascospores, suggesting the existence of two genetically isolated powdery mildew populations, and consequently of two independent sources of inoculum in the vineyard. Isolates resistant to fungicides inhibiting sterol 14α-demethylation (DMIs) were found in both populations, suggesting that resistance to DMIs may arise independently in the two powdery mildew populations. A PCR assay targeting the gene encoding U. necator 14α-demethylase has been developed which will permit an early, specific detection of U. necator infections, and may be useful for spraying programmes. ©1997 SCI     

宁夏温室瓜菜白粉病菌鉴定及病害流行预测模型构建

为明确宁夏回族自治区温室瓜菜白粉病菌的分类地位,对采自该地区温室的南瓜、黄瓜和甜瓜上的白粉病菌基于ITS序列分析进行分子鉴定;利用孢子捕捉器对温室中甜瓜白粉病菌的孢子量进行监测,分析环境因子、孢子量和病情指数之间的关系,并采用逐步回归分析法构建温室甜瓜白粉病的流行预测模型。结果表明,基于ITS序列的分子鉴定结果,3种瓜菜白粉病的病原菌均为单囊壳白粉菌Podosphaera xanthii。发病期间,每日温室中甜瓜白粉病菌的孢子量在12:00—16:00时段最多,占24 h内总孢子量的34%~81%,20:00—08:00时段最少;白粉病菌孢子的释放与光照强度有关,相关系数为0.602。第t天的病情指数与标准累积温度、标准累积湿度、t-4 d前08:00—12:00时段的累积孢子量、第t-4天16:00—20:00时段的孢子量均具有显著的相关性,相关系数分别为0.935、0.938、0.956和0.921。以标准累积湿度和第t-4天16:00—20:00时段的孢子量为预测变量构建了温室甜瓜白粉病流行预测模型,决定系数为0.962,表明该模型具有较好的实际应用价值。     

Real-time PCR quantification of latent infection of wheat powdery mildew in the field

Wheat powdery mildew, caused by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a destructive wheat disease worldwide. The key issue for the disease forecast is to timely and accurately estimate and quantify the latent infection levels in volunteer seedlings where the pathogen over-winters or over-summers to serve as sources of initial inoculum of epidemics. To improve the conventional method, a real-time PCR assay had been established in this study to quantify latent infection level of wheat leaves. Artificially and naturally infected leaves in wheat fields at different geographical locations in China were collected and processed to determine the latent infection levels. Linear relationships between the molecular disease index (MDX) and the observed disease index (DX) were obtained from artificial inoculation experiments. Field experiments showed that the spatial distribution patterns of MDX matched well with those of DX in the most cases. This study demonstrated that the real-time PCR assay was a useful tool to rapidly and accurately quantify the latent infection levels of wheat powdery mildew and to efficiently estimate the initial inoculum potentials of epidemics in the fields.     

Persistence and spatial autocorrelation of clones of Erysiphe necator overwintering as mycelium in dormant buds in an isolated vineyard in northern Italy

The population structure of the grape powdery mildew fungus, Erysiphe necator (formerly Uncinula necator), has been hypothesized to vary from being clonal to highly diverse and recombining. We report here on the structure of an E. necator population sampled during a 4-year period from an isolated vineyard in northern Italy (Voghera, Pavia Province). We obtained 54 isolates of E. necator that overwintered asexually as mycelium in grapevine buds and caused severe symptoms on the emerging shoots, known as flag shoots. All isolates were genotyped for mating type, four multilocus polymerase chain reaction (PCR)-based markers (a total of 64 loci were scored), and two single-copy loci designed to identify genetic subgroups in E. necator. All isolates had the same mating type and single-locus alleles that correlate to isolates from flag shoots in other areas. Only 2 of the 64 loci scored from multilocus markers were polymorphic; 46 of the 54 isolates had the same multilocus haplotype. Seven isolates had a second haplotype that was recovered over 3 years, and only a single isolate was found with a third haplotype. Both variant haplotypes differed from the main clonal haplotype by single loci. Spatial autocorrelation analyses showed that vines with flag shoots were not aggregated within years, but they were aggregated between consecutive years. These results demonstrate that this subpopulation of E. necator on flag shoots is composed of a single clonal lineage that has persisted for at least 4 years. We speculate that the lack of diversity in the flag shoot subpopulation in this vineyard is the result of restricted immigration from surrounding areas and genetic drift operating through founder effects and periodic bottlenecks. We propose a model that integrates epidemiology and population genetics to explain the variation observed in genetic structure of E. necator flag shoot subpopulations from different vineyards or viticultural regions.     

Effects of Diffuse Colonization of Grape Berries by Uncinula necator on Bunch Rots, Berry Microflora, and Juice and Wine Quality

ABSTRACT Production of grape (principally cultivars of Vitis vinifera) for high-quality wines requires a high level of suppression of powdery mildew (Uncinula necator syn. Erysiphe necator). Severe infection of either fruit or foliage has well-documented and deleterious effects upon crop and wine quality. We found that berries nearly immune to infection by U. necator due to the development of ontogenic resistance may still support diffuse and inconspicuous mildew colonies when inoculated approximately 3 weeks post-bloom. Fruit with diffuse mildew colonies appear to be healthy and free of powdery mildew in late-season vineyard assessments with the naked eye. Nonetheless, presence of these colonies on berries was associated with (i) elevated populations of spoilage microorganisms; (ii) increased evolution of volatile ethyl acetate, acetic acid, and ethanol; (iii) increased infestation by insects known to be attracted to the aforementioned volatiles; (iv) increased rotting by Botrytis cinerea; and (v) increased frequency of perceived defects in wines prepared from fruit supporting diffuse powdery mildew colonies. Prevention of diffuse infection requires extending fungicidal protection until fruit are fully resistant to infection. Despite a perceived lack of improvement in disease control due to the insidious nature of diffuse powdery mildew, potential deleterious effects upon crop and wine quality thereby would be avoided.     

A new spore precipitator with polarized dielectric insulators for physical control of tomato powdery mildew

ABSTRACT In an attempt to physically protect greenhouse tomato plants from the powdery mildew fungus Oidium neolycopersici, we developed a new electrostatic spore precipitator in which a copper wire conductor is linked to an electrostatic generator and covered with a transparent acrylic cylinder (insulator). The conductor was negatively charged by the generator, and the electrostatic field created by the conductor was used to dielectrically polarize the insulator cylinder. The dielectrically polarized cylinder also produced an electrostatic force without a spark discharge. This force was directly proportional to the potential applied to the conductor and was used to attract conidia of the pathogen. The efficacy of this spore precipitator in protecting hydroponically cultured tomato plants from powdery mildew was evaluated in the greenhouse. The hydroponic culture troughs were covered with a cubic frame installed with the spore precipitator, and the disease progress on precipitator-guarded and unguarded seedlings was traced after the conidia were disseminated mechanically from inoculum on tomato plants. Seedlings in the guarded troughs remained uninfected during the entire experiment, in spite of rapid spread of the disease to all leaves of the unguarded seedlings.     

Comparison of resistance-inducing abilities of virulent and avirulent races of Erysiphe graminis f.sp. hordei and a race of Erysiphe graminis f.sp. tritici in barley

Resistance to powdery mildew was induced in barley by preinoculation with virulent and avirulent races of barley powdery mildew ( Erysiphe graminis f.sp. hordei ), and with a race of wheat powdery mildew ( E. graminis f.sp. tritici ). Four inducer densities were tested in 13 different induction periods between 1 and 24 h. Generally, the resistance induced by barley powdery mildew increased up to 10-12 h of induction and was maintained in longer induction periods. The inducing abilities of virulent and avirulent races could not be distinguished up to 10-12 h of induction, after which the inducing ability of avirulent races increased significantly in relation to virulent races. Wheat powdery mildew was able to induce more resistance than barley powdery mildew in induction periods up to 8 h. In a single inoculation procedure the number of haustoria developing from virulent barley powdery mildew decreased as inoculum density increased. The effect was ascribed to induction of resistance. This reduction of infection efficiency in the compatible interaction was compared to induced resistance. However, the inoculum density needed for 50% resistance induction in the double inoculation procedure was approximately 40 times higher than the inoculum density needed for 50% reduction in infection efficiency in the single inoculation procedure.     

Identification of race-specific resistance in North American Vitis spp. limiting Erysiphe necator hyphal growth

Race-specific resistance against powdery mildews is well documented in small grains but, in other crops such as grapevine, controlled analysis of host-pathogen interactions on resistant plants is uncommon. In the current study, we attempted to confirm powdery mildew resistance phenotypes through vineyard, greenhouse, and in vitro inoculations for test cross-mapping populations for two resistance sources: (i) a complex hybrid breeding line, 'Bloodworth 81-107-11', of at least Vitis rotundifolia, V. vinifera, V. berlandieri, V. rupestris, V. labrusca, and V. aestivalis background; and (ii) Vitis hybrid 'Tamiami' of V. aestivalis and V. vinifera origin. Statistical analysis of vineyard resistance data suggested the segregation of two and three race-specific resistance genes from the two sources, respectively. However, in each population, some resistant progeny were susceptible in greenhouse or in vitro screens, which suggested the presence of Erysiphe necator isolates virulent on progeny segregating for one or more resistance genes. Controlled inoculation of resistant and susceptible progeny with a diverse set of E. necator isolates clearly demonstrated the presence of fungal races differentially interacting with race-specific resistance genes, providing proof of race specificity in the grape powdery mildew pathosystem. Consistent with known race-specific resistance mechanisms, both resistance sources were characterized by programmed cell death of host epidermal cells under appressoria, which arrested or slowed hyphal growth; this response was also accompanied by collapse of conidia, germ tubes, appressoria, and secondary hyphae. The observation of prevalent isolates virulent on progeny with multiple race-specific resistance genes before resistance gene deployment has implications for grape breeding strategies. We suggest that grape breeders should characterize the mechanisms of resistance and pyramid multiple resistance genes with different mechanisms for improved durability.