Improved Precision of Powdery Mildew Management through Early Detection of Erysiphe (Unicinula) Necator in the Air of Washington Vineyards

Current risk-assessment models designed to facilitate the management of grapevine powdery mildew use vine phenology and prevailing weather conditions to assess disease risk. Inoculum availability and concentration are not model components at this time. The purpose of this study is to devise an reliable, inexpensive, and rapid means of detecting propagules of E. necator in the vineyard air prior to disease onset and, in doing so, add an ?inoculum availability? component to current models. One of our primary challenges was to overcome difficulties inherent in the positive and rapid identification of E. necator propagules using conventional techniques of light microscopy. We have developed primers that when used in PCR reactions can differentiate E. necator from 46 other powdery mildews common in the Pacific Northwest. In our extensive 2006 laboratory studies, we found the primer pairs sensitive enough to amplify the DNA from as few as 100-500 spores placed on glass air sampling rods. Regression analysis revealed a significant (F = 47.3; P= 0.005) relationship [y=1.6*exp(-exp(-(x-74.1)/75.4))] between the numbers of conidia placed on glass sampling rods and successful PCR amplifications with a coefficient of determination (r2) of 0.97. The primers were also used to identify E. necatorin vineyard air samples prior to disease onset. In 2006 E. necator was detected in the vineyard air using Rotorod air samplers situated within the vineyard prior to disease onset. Vineyard air samples were devoid of the pathogen prior to bud burst and prior to the initial ascospore release. The earliest indication of the presence of E. necator in the air occurred during a rain event of 9.9 mm that occurred during the prebloom stage. The presence of airborne ascospores during this rain event was confirmed using a Burkard volumetric air sampler. Subsequent negative sampling results intimated that the vineyard was apparently devoid of E. necatorpropagules, or the concentration of airborne propagules was below the detection thresholds of the sampling method, during incubation and latent periods. The detection of E. necator resumed several days prior to the visual appearance of powdery mildew signs and continued during subsequent disease development. When the stationary Rotorod method indicated the presence of E. necator in the vineyard air for the first time after bud burst, the information was used to initiate a fungicide spray program. In vineyards under high disease pressure, there was good correlation between predicted ascospore release and initial detection in all appellations where air-sampling studies were conducted. In a vineyard under high disease pressure in 2006, the use of sampling-driven mildew fungicide programs resulted in a 1-2 fewer applications, depending upon treatment regime. In a second vineyard trial use of the sampling-driven approach reduced fungicide usage by 86{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}. We are currently working with a local analytical company interested in providing PCR service to the Washington grape industry.

Map-Based Cloning of a Powdery Mildew Resistance Locus in Vitis

We analyzed powdery mildew resistance in 272 vines from the cross between Horizon (susceptible) and Ill. 547-1 (resistant). Segregation for resistance was normally distributed. Our work shows that the actual distance between two DNA markers for powdery mildew resistance identified earlier is actually 12 cM, rather than the 1.8 cM calculated earlier from a small population.

As a result of bulked segregant analysis using AFLP markers, we found 68 candidate markers for mapping to the powdery mildew resistance locus. Efforts have begun to saturate the genetic map in the region of this resistance gene locus. To date, 57 of the 68 markers were examined, yet of these just seven were tentatively placed on the map in the same region as the powdery mildew resistance locus.

New technologies are being developed in other laboratories that may allow a more thorough and detailed analysis of the many genes involved in a plant’s defense again disease. Our project to date has been focused on just a single gene (or a chromosomal region with several genes). Given the relative difficulty to clone even a single gene based on map position, I have decided to pause this project pending examination of the best techniques currently available to reach our goal of cloning grapevine genes for disease resistance and understanding more about the grapevine disease resistance responses. Undoubtedly, the material we now possess is unique, and our present knowledge of the map location of this gene for powdery mildew resistance will be of value as new approaches are examined to reach our original goals.

PDF: Map-Based Cloning of a Powdery Mildew Resistance Locus in Vitis

Development of a “Soft” Powdery Mildew Control Program for Sustainable and Organic Growers in the North Coast

A spray trial for powdery mildew control on winegrapes was conducted at Roederer Estate US in Philo in a block of conventionally farmed “Chardonnay” grapes. Nine treatments were used, including Rallye, MKP and Elexa, Elexa only, Erase and Rallye, Erase only, MKP only, AQ-10, and a water control. A randomized complete block design consisting of 4 replications of 3 vines was used. Materials were applied at 7-10 day intervals depending on powdery mildew pressure as indicated by an Adcon Weather Monitorinig Station. Experimental lots of wine were made for taste evaluation (six lots).

Unfortunately, powdery mildew failed to develop in the plot due to overall low pressure, and an effective prebloom micronized wettable sulfur spray program. In the coming year, the plot will be relocated to a higher pressure area, and prebloom sulfur sprays will not be applied to the test plot area.

PDF: Development of a “Soft” Powdery Mildew Control Program for Sustainable and Organic Growers in the North Coast

Development of a “Soft” Powdery Mildew Control Program for Sustainable and

A spray trial for powdery mildew control on winegrapes was conducted at Roederer Estate US in Philo in a block of conventionally farmed ‘Chardonnay’ grapes. Nine treatments were used, including Rallye, MKP and Elexa, Elexa only, Erase and Rallye, Erase only, MKP only, AQ-10, and a water control. A randomized complete block design consisting of 4 replications of 3 vines was used. Materials were applied at 7-10 day intervals depending on powdery mildew pressure as indicated by an Adcon Weather Monitorinig Station. Experimental lots of wine were made for taste evaluation (six lots). Unfortunately, powdery mildew failed to develop in the plot due to overall low pressure, and an effective prebloom micronized wettable sulfur spray program. In the coming year, the plot will be relocated to a higher pressure area, and prebloom sulfur sprays will not be applied to the test plot area.

Development of Powdery Mildew Resistant Table and Wine Grape Cultivars and

Genetic variability is suspected among different isolates of Uncinula necator. Therefore, different isolates must be screened to ensure the validity of the research. Isolates of U. necator have been collected from Kern, Santa Barbara, Sacramento, and Napa Counties. Single spore colonies have been produced from each isolate. These isolates have been maintained on Carignane seedlings in a growth chamber maintained at 75 F. Isolates are now being transferred to additional seedlings to increase inoculum to conduct resistance studies on 23 varieties of Vitis selected for this project. The Vitis varieties were produced at ARS in Fresno, transported to Davis and are being maintained in greenhouses at high temperature to ensure that they are initially free of the pathogen when testing is done. Sulfur pots are also being utilized to ensure clean plants. The vines are being fertilized to induce production of a large amounts of new tissue to be used in testing their resistance to U. necator. Resistance is being assessed using an artificial inoculation method in the greenhouse and the leaf disk bioassay method and will be compared to total plant resistance in greehouse and field tests. For the artificial inoculation method, plants are sealed in four separate growth frames, one for each isolate, and are placed randomly throughout each frame. These plants were inoculated with a spore suspension, from one of the four U. necator isolates. Plants would then be individually rated to determine the degree of their resistance to the differing U. necator isolates. To this date, cuttings have been rooted from each of the 23 varieties in order to produce enough plants to conduct this portion of the experiment. In this process, cuttings were taken from the parent plant and soaked in de-ionized water. They were then dipped in rooting hormone and placed in a rooting sponge (Grow-Tech Inc.). Leaves on the cuttings were trimmed to reduce water loss due to transpiration. Cuttings were placed in a styrofoam tray and then set on heating mats under a misting system. In the leaf disk bioassay, a number of six day old leaves were collected from each Vitis variety. Leaf disks were cut from leaves using a one-centimeter cork borer. Three petri dishes containing eight leaf disks each were prepared for each variety. These petri dishes were placed in the vacuum-powered spore-settling tower and inoculated with one of the four U. necator isolates. The spore-settling tower was used to ensure uniform inoculation of leaf disks. Blank coverslips were placed in the tower with the leaf disks to determine the inoculum density which was determined after 24 hours by placing the cover slips on a hemocytometer and counting the number of spores per square centimeter. Leaf disks were rated ten days after inoculation using a dissecting microscope. The percentage of leaf disk coverage by powdery mildew was estimated to the nearest ten percent, providing an accurate means of selection for powdery mildew resistance.

Factors that Influence Disease Onset from Overwintering Powdery Mildew

Isolates of Uncinula necator have been collected from Sacramento, Napa, Santa Barbara and Kern counties and were being maintained on Carignane seedlings in individual grow tubes housed in a 75F chamber. Seedling development was found to be hampered by this unnatural environment and poor infection rate resulted, requiring a change in system. The isolate inoculum source seedlings are now kept in separate growth chambers, where they receive adequate light and air for proper growth. Sufficient inoculum is being built up in these chambers for mass inoculation of the test seedlings. Each isolate will be tested at 3 different temperatures and 3 different humidities twice. 30 Carignane seedlings will be inoculated by means of a spore settling tower and transferred by sealed boxes to growth chambers set at either 15, 25 or 33 C. Slip covers were inoculated along with the seedlings to determine the density of the inoculation. One growth chamber has been obtained for each isolate and equipped with 30 humidity subchambers comprised of an airtight plastic contained filled with a salt solution. 10 subchambers will maintain 23{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} RH, 10 will maintain 60{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} RH and 10 will maintain 95{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} RH. Through experimentation with different compounds, Lithium chloride, Manganese chloride and sterile water, were found to best establish the determined humidities (respectively). In February, separate inoculations of 3 isolates were performed on 30 4-leaf seedlings each and the youngest leaf from each inoculated plant was placed in the humidity subchamber to incubate. After 10 days of incubation, the mildew on the leaves was too thick because of an excess of spore inoculum, making individual colony measurement impossible. Furthermore, the weather stripping that seals around the petioles had become saturated from multiple mixings of the solutions and was causing some phytotoxicity on the tissues that were touching it. It was determined through trials that a pure silicon sealant would work better at sealing off the container without imbibing any of the salt solution. Some follow up trials were done with the new sealant and 6-leaf seedlings whose youngest leaves have a thicker epidermis to resist phytotoxicity. The seedlings showed none of the earlier problems. 3 more inoculations were performed on various days in the second week of March. Inoculum concentration was reduced from 6 fully infected leaves to 1 fully infected leaf. The first chamber inoculated is ready to be measured and shows numerous distinct colonies on each leaf. No phytotoxicity is present in any of the chambers with the new silicon sealant and the larger seedlings. Several lesions from each leaf will be measured at 10 day intervals for rate of growth in mm/time and some will be destructively sampled for spore concentration/lesion. Slides of spores produced in each subchamber will be incubated at their respective humidities and the percentage of germination will be counted. The fourth chamber is in the process of being fitted with the silicon sealant and will be filled with the fourth isolate inoculation this week.

Factors that Influence Disease Onset from Overwintering Powdery Mildew Mycelium in Grapevine Buds and Establishment of Protocols for Risk Assessment and Control

Powdery mildew, caused by Uncinula necator, can overwinter in the dormant buds of grapevines and become a source of primary inoculum in the spring. Grapevine dormant buds develop during the previous season to become the current seasons fruiting canes. Developing dormant buds were collected periodically following the onset of shoot growth in the spring of 1998. The buds were sectioned and examined microscopically for the presence hyphae and infection. In 1997 and 1998, a field site in Madera County, California was observed for infected buds resulting from perennated U. necator. Early season treatments were tested for the reduction of bud infections over time and the number of infections were counted and mapped. Leaf haustoria of U. necator were observed to be ovoid in shape, ranging in size from 3.75 x 7.5 u.m to 7.5 x 12.5 urn with a mean size of 6.86 x 8.56 iim. These sizes are consistent with those previously measured. Mycelia and haustoria were found in the buds from the earliest collection date, approximately three weeks after shoot growth. The size and shape of haustoria were consistent with the size range and shape of haustoria in the inoculated leaves, and the hyphae averaged 3.76|um, which was consistent with previous observations. Specific tissues observed to carry infections in the buds were trichomes and prophylls. Infected buds on each vine in each 1/3 acre block were mapped. The early control procedures yielded a reduction in the incidence of bud infections over time in the treatment blocks. A repeated measures ANOVA using the year as the within subject, showed a significant difference in the treatment blocks from 1997 to 1998. The number of infections per vine and the total number of infections per block significantly decreased. The control blocks, however, increased slightly overall but there was no significant difference between years.

Determining the Factors that Influence Disease Onset from Overwintering Powdery

Objectives:

  1. Determine how temperatures and chilling affect disease onset from grape buds harboring mycelium of Uncinula necator.
  2. Establish effective control strategies for bud perennation.
  3. Using the data from the first and second objectives, construct a model and subsequent risk assessment program for the control of the infected buds as primary inoculum sources.

Objective 1: From the first collection in December 1996, no bud infections were observed.

These buds were kept at 4°C for 8 months prior to planting in growth chambers due to lack of growth chamber space at the time. It is postulated that the extended length of constant cold temps may have had an affect on the buds. Surplus wood collected from the UC Davis Carignane vineyard is now being stored in a cold chamber maintained at 2-4° C. Buds will be randomly selected and pushed at 20°C to assess what the incidence of infection with different rates of chilling. The second collection was made in January 1998. Buds were observed daily for infection, sporulation, bud-swell, bud-break, flat leaf stage, and infection. Each shoot was measured and any symptoms of stunting were noted. Infected buds were observed in all temperatures except 30°C. The incidence of infected buds was highest at 15 and 20° C (Table 1). Preliminary data show a positive correlation between degree days and infection as well as between days to infection and temperature. Rates of sporulation of Uncinula necator do change with temperature but it is doubtful that is the only reason for the decrease in infected shoots at the higher temperatures. In fact, 25°C is close to optimum temperature for the growth of the pathogen. Further research will be needed to understand the dynamic association between accumulated chilling units and the response of the pathogen to the host growth rate. Material from the March collection is currently being assessed.

Objective 2: In year one of the study (1997), infected buds on each vine in each 1/3 acre replication were mapped (Fig. 1).

In April 1998 we will re-visit these blocks and assess disease incidence. We hypothesize that the vines in the treatment blocks will have a lower incidence of infected shoots in 1998 relative to those in the control blocks simply because early season infection in treated blocks did not occur on adjacent shoots. Therefore, bud infection on adjacent shoots should be reduced. After recording this information we will repeat the protocol as in 1997 and follow the treatment and control blocks into a third season (1999). Prior to treatment application in 1997, there were 734 infected buds in the blocks designated to be treated with Rally 4 oz/A and 762 infected buds in the non-treated control blocks.

Objective 3. Nothing as of yet has been constructed for a model.

Upon completion of mis years second growth chamber study and second field study, data will be analyzed. Model construction will be done in 1999.

Identification of Powdery Mildew Races or Biotypes from Grapes and Their Control by Minimum Required Fungicide Doses

Powdery mildew samples were collected during late summer 1992 and stored under -80° for laboratory evaluation. Buds and scale samples were collected during early spring (1993) and stored under laboratory conditions. The stored samples were rubbed onto the early shoots to observed mildew incidence and severity. The study is still underway. The mildew is now showing up and appropriate fungal sprays are being made. The results will be collected in July 1997.

Epidemiology and Control of Grapevine Powdery Mildew in California

Powdery mildew can overwinter in cleistothecia lodged in bark. Cleistothecia become lodged in the bark when they wash off of leaves during fall rains. If cleistothecia fall to the soil instead of the bark, they do not usually remain viable. In the spring, cleistothecia in the bark release ascospores during mild prolonged wetness periods. These same temperatures (15 to 25 C) favor germination and infection of grape leaves. Cleistothecia do not remain viable for prolonged periods of 30 C. Canopies which do not have the majority of the leaves growing over the bark are less likely to have as many cleistothecia lodged in the bark. Most infections occur within 10 cm of the bark. Cleistothecia were less viable if they originated from the Central Valley or Southern California than from Coastal regions. This may be why most early infections in the southern areas are the result of infected dormant buds. However, prolonged temperatures over 30 C kill the fungus at any point in its life cycle. Cleistothecia can form as early as July and produce viable ascospores as early as August. Therefore, diligent disease control is important, including post harvest. Spring applications of wettable sulfur (5 lb / 100 gal / acre) were most effective in controlling early season mildew. Spraying should start when shoots are at the 0 to 2 inch stage. Applications should be repeated at ten day intervals when rains, soaking fogs and dews keep grape leaves wet for 12 hours or more.