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Dynamics of ascocarp development, ascospore maturation, and dispersal in Erysiphe necator were studied over a 4-year period, from the time of ascocarp formation to the end of the ascosporic season at the end of June in the following spring. Naturally dispersed chasmothecia were collected from mid-August to late November (when leaf fall wits complete); the different collections were used to form three to five cohorts of chasmothecia per year, with each cohort containing ascocarps formed in different periods. Chasmothecia were exposed to natural conditions in a vineyard and periodically sampled. Ascocarps were categorized as containing mature or immature ascospores, or as empty; mature ascospores inside chasmothecia were enumerated starting from late February. Ascospore discharge was determined using silicone-coated slides that were placed 3 to 4 cm from sections of the vine trunk holding the chasmothecia. Before complete leaf fall, 34% of the chasmothecia had mature ascospores, 48% had immature ascospores, and 18% were empty; in the same period, the trapped ascospores represented 56% of the total ascospores trapped in an ascosporic season (i.e., from late summer until the next spring or early summer). The number of viable chasmothecia diminished over time: 11 and 5% of chasmothecia had mature ascospores between complete leaf fall and bud break and after bud break, respectively. These ascocarps discharged ≈2 and 42% of the total ascospores, respectively. All the ascocarp cohorts released ascospores in autumn, survived the winter, and discharged viable ascospores in spring; neither ascospore numbers nor their pattern of temporal release was influenced by the time when chasmothecia were collected and exposed in the vineyard. Abundance of mature ascospores in chasmothecia wits expressed as a function of degree-days (DD) (base 10°C) accumulated before and after bud break through a Gompertz equation (R² = 0.92). Based on this equation, 90% of the ascospores were mature whet) 153 DD (confidence interval, 100 to 210 DD) had accumulated after bud break. Most ascospores were trapped in periods with >2 mm of rain; however, a few ascospores were airborne with <2 mm of rain and, occasionally, in wet periods of ≥3.5 h not initiated by rain. [ABSTRACT FROM AUTHOR]

Pierce's disease (PD), caused by Xylella fastidiosa, represents one of the most damaging diseases of cultivated grape. Management of PD in the vineyard often relies on the removal of infected individuals, which otherwise serve as a source of inoculum for nearby healthy vines. Effective implementation of such control measures requires early diagnosis, which is complicated by the fact that infected vines often harbor high titers of the pathogen in advance of visual symptom development. Here, we report a biomarker system that simultaneously monitors Xylella-induced plant transcripts as well as Xylella ribosomal (r)RNA. Plant biomarker genes were derived from a combination of in silico analysis of grape expressed sequence tags and validation by means of reverse-transcriptase polymerase chain reaction (RT-PCR). Four genes upregulated upon PD infection were individually multiplexed with an X. fastidiosa marker rRNA and scored using either real-time RT-PCR or gel-based conventional RT-PCR techniques. The system was sufficiently sensitive to detect both host gene transcript and pathogen rRNA in asymptomatic infected plants. Moreover, these plant biomarker genes were not induced by water deficit, which is a component of PD development. Such biomarker genes could have utility for disease control by aiding early detection and as a screening tool in breeding programs. [ABSTRACT FROM AUTHOR]

Dual epidemics are defined as epidemics developing on two or several plant organs in the course of a cropping season. Agricultural pathosystems where such epidemics develop are often very important, because the harvestable part is one of the organs affected. These epidemics also are often difficult to manage, because the linkage between epidemiological components occurring on different organs is poorly understood, and because prediction of the risk toward the harvestable organs is difficult. In the case of downy mildew (DM) and powdery mildew (PM) of grapevine, nonlinear modeling and logistic regression indicated nonlinearity in the foliage-cluster relationships. Nonlinear modeling enabled the parameterization of a transmission coefficient that numerically links the two components, leaves and clusters, in DM and PM epidemics. Logistic regression analysis yielded a series of probabilistic models that enabled predicting preset levels of cluster infection risks based on DM and PM severities on the foliage at successive crop stages. The usefulness of this framework for tactical decision-making for disease control is discussed. [ABSTRACT FROM AUTHOR]

In northern Italy, Erysiphe necator overwinters almost exclusively as chasmothecia. From 2004 to 2008, we investigated the occurrence of natural parasitism of grapevine powdery mildew chasmothecia by Ampelomyces quisqualis in the Trentino-Alto Adige region, in northern Italy. The survey was conducted in 18 vineyards in autumn 2004 and 2005 and in 45 vineyards in autumn 2006 and 2007. The incidence of powdery mildew signs (white powdery mycelia and conidia), the number of chasmothecia and their development pattern, and the incidence of parasitism by A. quisqualis were assessed. The production of 13. necator chasmothecia on leaves is related to the incidence and severity of the disease on leaves at the end of the season and is not correlated with the elevation of the vineyard, which is inversely related to the temperature. A limited amount of natural parasitism of E, necator by Ampelomyces spp. (0.17 to 3.51%) was observed in all Of the years of the survey. Pycnidia and conidia of Ampelomyces spp. parasitizing E. necator chasmothecia were found in conventional, organically grown, and untreated vineyards. Some of the isolated Ampelomyces strains have conidia that are shaped differently than those of the commercial A. quisqualis strain (AQ10) and are phylogenetically different from AQ10. [ABSTRACT FROM AUTHOR]

A greater understanding of the development of powdery mildew epidemics on vines would improve disease management by making assessments of the risk of invasion more accurate. We characterized the spatiotemporal spread of epidemics in the vineyard, quantified their variability, and identified the factors responsible for it. We described changes in the probability of infection of a leaf in a plot over time and as a function of distance from a source of disease. Logistic models were fitted to field data from artificially inoculated plots. The velocity of spread decreased along the row and increased in the direction of the prevailing winds. The rate of progression over time was plot dependent, and the velocity was dependent on the vigor of the vine (0.1 to 0.27 m day-1 in areas of moderate vigor and 1.1 m day-1 in areas of high vigor). When applied to a larger plot with natural primary foci, the spatiotemporal logistic model showed that the velocity and the slope of the gradient in space depended on the foci; however, the velocity remained in the same range. During the period of highest susceptibility for grape, the probability of a leaf becoming infected increased from 2.5 to 13%. Our logistic model was able to predict changes in disease over time of its extension within the plot; however, the crop heterogeneity prevented prediction of variability of disease at the vine scale. [ABSTRACT FROM AUTHOR]

Botrytis cinerea (anamorph of Botryotinia fuckeliana) causes gray mold on a high number of crop plants including grapes. In this study, we investigated the genetic properties of a grape pathogenic population of B. cinerea in the area of Eger, Hungary. A total of 109 isolates from 12 areas were sampled. Based on the sequence of the β-tubulin (tub1) locus, they all belong to group II, a phylogenetic species within B. cinerea. Seventy-four isolates were classified as transposa, with both the Flipper and Boty transposons, and 10 were classified as vacuma, lacking both transposons. The remaining isolates contained either only Flipper (13) or Boty (12). Multilocus analysis of sequences from tub1 and two other loci (elongation factor 1-alpha, tef1, and a minisatellite from the intron of an ATPase, MSB1) led to poor phylogenetic resolution of strains in individual clades. Analysis of five microsatellites (Bc2, Bc3, Bc5, Bc6, and Bc10) resulted in 55 microsatellite haplotypes within the 109 strains. No correlation was detected among individual haplotypes and the presence/absence of Flipper and/or Boty, the geographic origin, or the year of isolation. Application of the index of association, the chi-square test, and the phi test consistently indicated that the population of Hungarian isolates of B. cinerea undergoes sexual reproduction. However, the index of association test suggested the presence of some clonality, and the fixation index showed a low or occasionally moderate level of fixation in the Flipper populations. We conclude that the B. cinerea populations in Hungary consist of a strongly recombining group II phylogenetic species. [ABSTRACT FROM AUTHOR]

Eutypa lata is a vascular pathogen of woody plants. In the present study we (i) determined which component(s) of the cell wall polymers were degraded in naturally infected grapevines and in artificially inoculated grape wood blocks; (ii) compared the pattern of wood decay in the tolerant grape cv. Merlot versus the susceptible cv. Cabernet Sauvignon; and (iii) identified secondary metabolites and hydrolytic enzymes expressed by E. lata during wood degradation. Biochemical analyses and a cytochemical study indicated that glucose-rich polymers were primary targets of E. lata. Structural glucose and xylose of the hemicellulose fraction of the plant cell wall and starch were depleted in infected woods identically in both cultivars. Moreover, the more tolerant cv. Merlot always had more lignin in the wood than the susceptible cv. Cabernet Sauvignon, indicating that this polymer may play a role in disease resistance. In vitro assays demonstrated the production by E. lata of oxidases, glycosidases and starch degrading enzymes. Phytotoxic secondary metabolites were also produced but our data suggest that they may bind to the wood. Finally, we demonstrated that free glucose in liquid cultures repressed primary but not secondary metabolism. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The role of fimbrial and afimbrial adhesins of Xylella fastidiosa in biofilm formation was assessed by visualization of cell aggregates of mutant strains after incubation on glass surfaces. FimA- or FimF- fimbrial mutants adhered as solitary cells at a slightly lesser frequency to glass surfaces than the parental strain; however, cell aggregates were not formed, unlike the wild-type strain. Conversely, whereas the XadA- and HxfB- nonfimbrial mutants also exhibited a much lower frequency of adherence to glass surfaces than the wild-type strain, most of the cells retained on the surfaces were in cell aggregates of different sizes, much like that of the parental strain. Neither fimbrial or afimbrial mutants formed a mature biofilm on the sides of flasks of broth cultures, unlike the dense biofilm formed by the wild-type strain. Although FimA- and FimF- mutants did not form cell aggregates on glass surfaces when incubated as individual strains, aggregates of a FimA- or FimF- mutant were observed when co-incubated with either a XadA- mutant or HxfB- mutant, respectively. These results are consistent with a model in which the fimbrial adhesins FimA and FimF are involved preferentially in cell-to-cell aggregate formation whereas the afimbrial adhesions XadA and HxfB preferentially contribute to initial cell binding to surfaces, whereupon further cell aggregation can occur. In each of five separate experiments, FimA, FimF, XadA, and HxfB mutants of X. fastidiosa all were less virulent to grape than the corresponding wild-type strain. Fimbrial and afimbrial mutants might produce a reduced biofilm within vessels of grape and, hence, be deficient in various cell-density-dependent traits required for movement through the plant and, thus, virulence. [ABSTRACT FROM AUTHOR]

Plasmopara viticola populations collected from three islands in the Ionian Sea—an arm of the Mediterranean Sea to the west of Greece—were analyzed with microsatellite molecular markers in order to investigate the pathogen population structure. Downy mildew populations from mainland regions previously studied were found to have high genotypic diversity and limited clonality; however, populations under Mediterranean island conditions mostly showed limited variation and the epidemics basically were driven by the multiple clonal infections of one or a few genotypes. Populations from different islands were differentiated from each other, whereas genetic divergence also was found among subpopulations of the same plot. Polyploid individuals and individuals that over-wintered in asexual form were observed in some cases. The findings obtained by this population genetics study improve our understanding of the biology of the pathogen and lead to potential alternative control measures for the disease. [ABSTRACT FROM AUTHOR]

Plasmopara viticola is a strictly biotrophic oomycete that causes downy mildew, which is one of the most important grapevine diseases. Control of the disease is most often achieved by fungicide applications, which may have severe environmental consequences. Therefore, alternative control strategies based on biocontrol agents (BCAs) are currently in development. Thousands of potential BCAs have to be screened for their antagonist efficacy against Plasmopara viticolo. Evaluation of their effect on the pathogen can be achieved by detecting the amount of R viticola DNA in leaves treated with potential antagonists and infected with the pathogen. In this study, a rapid high throughput method was developed for relative quantification of P viticola DNA directly from Vitis vinifera leaves by means of multiplex real time quantitative polymerase chain reaction (PCR) with TaqMan chemistry. This method allows simultaneous amplification, but independent detection, of pathogen and host DNA by using species specific primers and TaqMan probes that are labeled with different fluorescent dyes. Including detection of E vinifera DNA in the tests is fundamental because it provides an endogenous reference and allows normalization for variations caused by sample to-sample differences in DNA extraction, PCR efficiencies, and pipetting volumes. The developed method allows highly sensitive and specific detection of R viticola DNA (minimal detectable quantity of 0.1 pg). Moreover, high precision and reproducibility of TaqMan assays were observed over a linear range of four orders of magnitude, confirming the reliability of the developed PCR assay. Potential applications range from screening for BCA efficiency to evaluation of fungicide efficacy, or assessment of host resistance. [ABSTRACT FROM AUTHOR]

Tests with a real-time reverse transcription-polymerase chain reaction (RT-PCR) were performed on specimens of Xiphinema index collected from the rhizosphere of Grapevine fanleaf virus (GFLV)-infected grapevines at Palagiano, Italy. A 1,157-bp fragment of the GFLV RNA-2 coat protein (CP) gene was amplified and sequenced. A fluorescent Scorpion probe was designed to detect a highly conserved CP region. A second region with isolate-specific multiple nucleotide polymorphisms was used to detect GFLV isolates using molecular beacons (MB). The Scorpion probe allowed quantitative estimation of GFLV RNA-2 in single nematodes, using a dilution series of a 692-nucleotide transcript of the CP gene. The assay allowed detection of GFLV RNA-2 in individual X. index, with a minimum template threshold of 800 fg or 2.8 × 106 RNA-2 molecules per nematode. The CP fragment used for GFLV detection with the Scorpion probe appeared highly conserved among isolates. The probes were tested against other GFLV isolates, which were recognized by the species-specific Scorpion probe and by the corresponding MB specific to the particular isolate. Both tests appeared useful as diagnostic tools or for studies on GFLV in acquisition, retention, and transmission experiments. [ABSTRACT FROM AUTHOR]

The effect of volatiles from cv. Isabella (Vitis labrusca) on the growth of Botrytis cinerea was tested in vitro and in situ, in the latter case on 'Roditis' grapes (V vinifera), at various temperatures. The goal of the research was to determine whether the volatiles emitted by Isabella grapes could be effective biocontrol agents of Botrytis cinerea. The closed Mariotte system was used as a bioassay method to analyze quantitatively the biological action of these volatiles on fungal growth and disease development. The in vitro experiments revealed the inhibitory action of the Isabella volatiles on the sporulation and sclerotia formation of the fungus, as well as the stimulating action of the Roditis volatiles on the sporulation of the fungus. The in situ study confirmed the antifungal action of the Isabella volatiles as they reduced the inoculum and pathogenicity of B. cinerea. The antibiotic action was more pronounced at 21°C. The study indicates that Isabella volatiles act as biocontrol agents of B. cinerea. [ABSTRACT FROM AUTHOR]

The epidemiology and control of black rot (Guignardia bidwellii) was studied from 1995 to 1999 in vineyards in Dresden and Naples, NY, where disease pressure was moderate and extreme, respectively. The efficacy of serial applications of myclobutanil, provided at 2-week intervals and varied with respect to their number and time of initiation, was examined within the context of host phenology, inoculum availability, and sanitation. At Dresden, sprays applied over 4 weeks through the immediate prebloom stage provided only 13 to 91% control of diseased clusters, despite the release of 95% of the season's ascosporic inoculum during the period of fungicidal protection. However, applications immediately prior to bloom plus 2 and 4 weeks later, which afforded protection while fruit are highly susceptible to infection, provided virtually complete control. At Naples, where mummified berries were retained in the canopy after mechanical pruning, this same regime provided only approximately 80% disease control, but applying a fourth spray 2 weeks prebloom generally improved control. Hand-pruning mummies to the ground in selected plots significantly (P &2le; 0.05) improved control in some spray regimes. Although this sanitation practice did not affect inoculum dynamics through bloom, very few spores were recovered thereafter from mummies collected from the ground, whereas abundant ascospores and conidia were recovered from mummies in the trellis for an additional 6 to 8 weeks. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Auto fluorescent 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 paplillae 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. [ABSTRACT FROM AUTHOR]

Little information is available concerning the expression of pathogenesis-related (PR) proteins in grapevine (Vitis vinifera) and their effect properties on the major fungal pathogens of grape. A systematic study was performed on the effect of total or individual grape proteins on mycelial growth, spore germination, and germ tube growth of Uncinula necator, Phomopsis viticola, and Botrytis cinerea. Two proteins, identified as PR proteins by immunological methods and by N-terminal sequencing as osmotin and thaumatin-like protein, exhibited strong antifungal activities in vitro, blocking the growth of Phomopsis viticola and Botrytis cinerea mycelia. In addition, they inhibited spore germination and germ tube growth of U. necator, Phomopsis viticola, and Botrytis cinerea. The presence of both proteins displayed a synergistic effect. The expression of osmotin and thaumatin-like protein was induced in grapevine leaves and berries infected with U. necator and Phomopsis viticola. Thaumatin previously was thought to occur exclusively in berries. Immunoblot analyses revealed the accumulation of the two PR proteins in infected leaves and berries, supporting a role in vivo in increasing the resistance of grapevine to fungal attack. [ABSTRACT FROM AUTHOR]

The effects of humidity on powdery mildew development on grape seedlings and the germination of Uncinula necator conidia in vitro were examined. Studies were conducted at an optimum temperature of 25 ± 2°C. Disease on foliage was markedly affected by humidity levels in the test range of 39 to 98% relative humidity (RH), corresponding to vapor pressure deficits (VPD) of 1,914 to 61 Pa. Incidence and severity increased with increasing humidity to an optimum near 85% RH, and then appeared to plateau or decrease marginally at higher values. Conidial density and chain length also were proportional to humidity, but were influenced less strongly. There was a strong, positive linear relationship between humidity level and frequency of conidium germination with RH treatments of ≤84%. However, germination frequency fell sharply at RH levels above a mean of 87%. All measures of humidity were equally accurate in predicting germination responses; however, VPD was slightly more effective than RH in accounting for effects on disease development and pathogen spomlation, and both were more effective than absolute humidity. Humidity appears to play a significant role in grapevine powdery mildew epidemiology, confirming the benefits of management practices to avoid and mitigate high humidity in the vineyard canopy. [ABSTRACT FROM AUTHOR]

Presents abstracts on plant pathology in historical perspective submitted for presentation at the American Phytopathological Society 2004 Meeting in Anaheim, California, on July 31-August 4, 2004. "Bacteriology and plant pathology in late 19th century America: A short-lived (and forgotten) marriage"; "The living soil: Soil bacteria, ecology and social cooperation"; "The great dying on the vine: Scientific responses to the phylloxera grapevine disaster, France 1867-1900."