David M. Gadoury

Physiological and environmental aspects of ascospore discharge in Gibberella zeae (anamorph Fusarium graminearum)

We investigated ascospore discharge in the perithecial fungus, Gibberella zeae. In a wind tunnel study that simulated constant rain and varying day and night lengths, the rate of ascospore release was approximately 8–30% greater under light than in complete darkness. Under constant light, ascospore discharge occurred at maximal rates at relative humidity levels greater than 92%. When perithecia were placed under conditions of high external osmolarity, ascospore discharge was significantly reduced. Ascospores were discharged from asci along with droplets of fluid, the epiplasm, from within the ascus. Analysis of discharged epiplasmic fluid by GC-MASS Spectrometry revealed that mannitol was the major simple sugar component of the fluid. Activity of mannitol dehydrogenase, which catalyzes the conversion of fructose to mannitol, was higher in protein extracts from mature perithecia than in extracts from vegetative tissue. Several inhibitors of K+ and Ca++ ion channels inhibited ascospore discharge, which suggested that ascospore discharge resulted from the buildup of turgor pressure generated by ion fluxes and mannitol accumulation.

Partial control of grape powdery mildew by the mycoparasite Ampelomyces quisqualis.

Ampelomyces quisqualis normally infects senescent colonies of Uncinula necator in late summer. Our objective was to introduce the mycoparasite at the start of an epidemic, and thereby reduce the rate of disease increase. Prior to establishing field trials, isolates of A. quisqualis were evaluated for pathogenicity, virulence, and host range in greenhouse and laboratory assays. Infection of powdery mildew colonies only occurred when plants were kept wet and resulted in sporulation of A. quisqualis within 10 days. Two isolates of A. quisqualis (G5 and G273) were evaluated for pathogenicity and virulence against 18 monoconidial isolates of U. necator on grape seedlings and showed little evidence of pathogenic specialization. Three isolates (G273, SF419, and SF423) were equally pathogenic to Sphaerotheca fuliginea on cucurbit, S. macularis on strawberry, and U. necator on grape seedlings. All A. quisqualis isolates appear to have a broad host range and cause significant damage to powdery mildew colonies. Pycnidia of A. quisqualis G273 were produced on cotton wicks saturated with malt extract agar or wheat bran malt extract agar. Wicks were suspended above grapevines of Vitis vinifera «Riesling» and Vitis interspecific hybrid Aurore. Conidia were dispersed during rain to infect powdery mildew colonies while leaf surfaces were wet. Conidia were released for 3 months in 1990 from a single deployment of wicks. Higher numbers of conidia were released during the entire growing season in 1992 and 1993 due to replenishment of colonized wicks at monthly intervals. Wicks released conidia for 1 to 2 months in 1992 and 1993 before becoming depleted. Powdery mildew development was reduced on Riesling vines in 1990 following deployment of colonized wicks at 15 cm of shoot growth. Disease severity but not incidence was reduced on Aurore vines in 1992 under A. quisqualis-colonized wicks. High rainfall in 1992 provided ample opportunities for dispersal of inoculum of the mycoparasite and the wet conditions were conducive to parasitism. Disease development was late and much reduced in 1993, which was a drier season than 1992. Consequently, no differences were observed in A. quisqualis-treated and untreated plots in the same vineyard that year

Ontogenic Resistance and Plant Disease Management: A Case Study of Grape Powdery Mildew

ABSTRACT A fundamental principle of integrated pest management is that actions taken to manage disease should be commensurate with the risk of infection and loss. One of the less-studied factors that determines this risk is ontogenic, or age-related resistance of the host. Ontogenic resistance may operate at the whole plant level or in specific organs or tissues. Until recently, grape berries were thought to remain susceptible to powdery mildew (Uncinula necator) until late in their development. However, the development of ontogenic resistance is actually quite rapid in berries, and fruit become nearly immune to infection within 4 weeks after fruit set. Our objective was to determine how and at what stage the pathogen was halted in the infection process on ontogenically resistant berries. Adhesion of conidia, germination, and appressorium formation were not impeded on older berries. However, once berries were approximately 3 weeks old and older, few germlings were able to form secondary hyphae. Ontogenically resistant berries responded rapidly to infection by synthesis of a germin-like protein that had been previously shown to play a role in host defense against barley powdery mildew. On susceptible berries, cell discoloration around penetration sites indicated the oxidation of phenolic compounds; a process that was followed by localized cell death. However, the pathogen was still able to infect such cells prior to their death, continue secondary growth, and thereby colonize young berries. Formation of papillae was not involved in the differential resistance mechanism of older berries. In susceptible berries, papillae formed frequently at infection sites but did not always contain the pathogen, whereas in resistant berries, the pathogen was always halted prior to the formation of papillae. The host defense, which conditions ontogenic resistance, operates in the earliest stages of the infection process, in the absence of gross anatomical barriers, prior to the formation of a functional haustorium and prior to the development of a conspicuous penetration pore. We also found that diffuse powdery mildew colonies that were not visible in the field predisposed berries to bunch rot by Botrytis cinerea, increased the levels of infestation by spoilage microorganisms, and substantially degraded wine quality. Our improved understanding of the nature, causes, and stability of ontogenic resistance in the grapevine/ powdery mildew system has supported substantial changes in how fungicides are used to control the disease. Present applications are more focused on the period of maximum fruit susceptibility instead of following a calendar-based schedule. This has improved control, reduced losses, and in many cases reduced the number of fungicide applications required to suppress the disease. Particularly where fungicides are deployed in a programmatic fashion and ontogenic resistance is dynamic, there may be equivalent improvements to be made in other hostpathogen systems through studies of how host susceptibility changes through time.

Seasonal Development of Ontogenic Resistance to Downy Mildew in Grape Berries and Rachises

ABSTRACT Clusters of Vitis vinifera and V. labrusca are reported to become resistant to Plasmopara viticola at stages of development ranging from 1 to 6 weeks postbloom. It has been suggested that resistance is associated with loss of the infection court as stomata are converted to lenticels, but the time of onset, cultivar variation, and seasonal variation in ontogenic resistance has remained uncertain, as has the comparative susceptibility of stem tissue within the fruit cluster. In New York, we inoculated clusters of V. vinifera cvs. Chardonnay and Riesling and V. labrusca cvs. Concord and Niagara at stages from prebloom until 5 to 6 weeks postbloom. Berries were infected and supported profuse sporulation until 2 weeks postbloom, and pedicel tissue remained susceptible until 4 weeks postbloom. Although berries on later-inoculated clusters failed to support sporulation, discoloration and necrosis of berry tissues was often noted, and necrosis of the pedicel within such clusters often led to further discoloration, shriveling, reduced size, or loss of berries. When the epidermis of discolored berries that initially failed to support sporulation was cut, the pathogen emerged and sporulated through incisions, indicating that lack of sporulation on older symptomatic berries was due to infection at an early stage of berry development followed by conversion of functional stomata to lenticels during latency. We repeated the study on Chardonnay and Riesling vines in South Australia and found that the period of berry and rachis susceptibility was greatly increased. The protracted susceptibility of the host was related to the increased duration and phenological heterogeneity of bloom and berry development in the warmer climate of South Australia. The time of onset and subsequent expression of ontogenic resistance to P. viticola may thus be modified by climate and should be weighed in transposing results from one climatic area to another. Our results can be used to refine forecast models for grapevine downy mildew to account for changes in berry and rachis susceptibility, and to focus fungicide application schedules upon the most critical periods for protection of fruit.

Cleistothecia of Uncinula necator : An additional source of inoculum in Italian vineyards

Density and viability of populations of cleistothecia of Uncinula necator from bark, leaves, and soil were determined in three vineyards in the Florence and Siena provinces of Tuscany for 3 years. A higher density of cleistothecia was found on fallen leaves than on bark. However, the percentage of viable cleistothecia was higher on bark. No viable cleistothecia were recovered from soil. U. necator overwintered as mycelium in dormant infected buds, which gave rise to flag shoots, only in Santa Cristina, where 20 and 92 flag shoots per hectare were detected before bloom in 1994 and 1995, respectively. Disease incidence and severity increased similarly at Corti, Fornace, and at Santa Cristina, although powdery mildew epidemics started from ascospores only in Corti and Fornace, whereas flag shoots were present at Santa Cristina. Cleistothecia were formed in autumn in both 1994 and 1995, and their dispersal started in late September to mid-October, with the maximum number of cleistothecia trapped in funnels during the second half of October. Cleistothecia appear to function as the sole source of primary inoculum for grape powdery mildew in some Italian vineyards and serve as additional sources of inoculum where the pathogen also overwinters in infected buds. In Australia but not in New York, the pathogen also overwinters as cleistothecia on fallen leaves.

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.

Distribution and retention of cleistothecia of Uncinula necator on bark of grapevines.

Clesitothecia of Uncinula necator were dispersed by late summer and early autumn rain to the bark of grapevines. Rain-dispersed ascocarps accumulated rapidly on bark during a 10-wk period and were then retained on bark through subsequent rain events between leaf abscission and budbreak the following spring. The density of populations on bark was significantly correlated (r = 0.72-0.88) with catches of cleistothecia in filter-paper funnels attached to the trunk of grapevines. The density of populations on bark during winter was not correlated with several measures of disease incidence and severity from the previous growing season. We concluded that while incidence and severity may determine the potential population available for dispersal, rain events determine the actual efficiency of transfer from infected organs to the bark of the vine. The percentage of ascocarps that reacted positively with the fluorescent vital stain fluorescein diacetate ranged from 50 to 62% and did not change significantly during overwintering until cleistothecia began to dehisce in spring. Therefore, the density and viability of populations of cleistothecia on bark at the time of budbreak appear to have been determined at the time of leaf fall the previous autumn and were not modified by subsequent environmental conditions. The densest aggregations of cleistothecia occurred on the cordons of cordon-trained vines, with successively lower densities occurring on the bark of the upper and lower trunks. The pruning and training system of vines of Vitis labrusca cv. Concord did not affect the density of populations of cleistothecia on bark

Delaying the onset of fungicide programs for control of apple scab in orchards with low potential ascospore dose of Venturia inaequalis

Conventional fungicide spray programs for the control of apple scab have long involved the application of fungicides prior to the pink stage of fruit-bud development. Our earlier studies, which showed a relationship between the relatively low levels of overwintering inoculum found in modern commercial orchards and the onset of scab epidemics, indicate that early-season fungicide sprays are not necessary in many commercial orchards in the northeastern United States. To test this hypothesis, experimental plots were established in several commercial orchards in New Hampshire and New York, and tests were run from 1985 to 1991 (.)

Primary Infection, Lesion Productivity, and Survival of Sporangia in the Grapevine Downy Mildew Pathogen Plasmopara viticola

ABSTRACT Several aspects of grapevine downy mildew epidemiology that are fundamental to model predictions were investigated. Simple rainfall-, temperature-, and phenology-based thresholds (rain > 2.5 mm; temperature > 11 degrees C; and phenology > Eichorn and Lorenz [E&L] growth stage 12) were evaluated to forecast primary (oosporic) infection by Plasmopara viticola. The threshold was consistent across 15 years of historical data on the highly susceptible cv. Chancellor at one site, and successfully predicted the initial outbreak of downy mildew for 2 of 3 years at three additional sites. Field inoculations demonstrated that shoot tissue was susceptible to infection as early as E&L stage 5, suggesting that initial germination of oospores, rather than acquisition of host susceptibility, was probably the limiting factor in the initiation of disease outbreaks. We also found that oospores may continue to germinate and cause infections throughout the growing season, in contrast to the widely-held assumption that the supply of oospores is depleted shortly after bloom. Lesion productivity (sporangia/lesion) did not decline with age of a lesion in the absence of suitable weather to induce sporulation. However, the productivity of all lesions declined rapidly through repeated cycles of sporulation. Extremely high temperatures (i.e., one day reaching 42.8 degrees C) had an eradicative effect under vineyard conditions, and permanently reduced sporulation from existing (but not incubating) lesions to trace levels, despite a later return to weather conducive to sporulation. In fair weather, most sporangia died sometime during the daylight period immediately following their production. However, over 50% of sporangia still released zoospores after 12 to 24 h of exposure to overcast conditions.

Integration of pesticide application schedules for disease and insect control in apple orchards of the northeastern United States

(…) To provide a framework to achieve this goal, we begin with the following objectives: reduce to a minimum the number of pesticide applications per season by optimizing the timing of applications to control the complex of economically important pests present at certain key times during the growing season; maintain or exceed current levels of insect and disease control; insofar as practical, use forecasting and monitoring systems to increase lead time for control decisions and actions. (…)