DEVELOPING CULTURAL AND BIOLOGICAL
MANAGEMENT STRATEGIES FOR CROWN GALL DISEASE

T. J. Burr, E. A. Momol and C. L. Reid
Department of Plant Pathology
New York State Agricultural Experiment Station, Cornell University, Geneva, NY

One objective of our research is to improve the sensitivity and specificity of methods for indexing grapevine cuttings for A. vitis. This has involved using a highly sensitive technique, the polymerase chain reaction (PCR). PCR amplifies unique DNA sequences to a detectable level, thus verifying the presence of the organism of interest. We have produced the DNA primers (short, specific DNA sequences) that are necessary to drive PCR for the detection of A. vitis. One primer amplifies a chromosomal gene rfbD that is unique to all A. vitis (pathogenic and nonpathogenic strains). Two other primers identify the presence of the Ti (tumor-inducing) plasmid. One amplifies the virA gene that is present in two pathogenic groups of A. vitis and the other amplifies virE2 that is present in the other group. By using these three primers together in multiplex PCR it is possible to identify pathogenic and nonpathogenic A. vitis. When only the rfbD product is detected, the strain is nonpathogenic but if virA or virE2 is also detected the strain is pathogenic. We are currently evaluating this multiplex PCR scheme directly on grape cuttings that are contaminated with A. vitis.

A second research objective is to determine if strains of A. vitis from wild grape can acquire a Ti-plasmid from pathogenic strains and thus become pathogenic. Pathogenic and nonpathogenic wild grape strains were mated on the surface of grape crown galls and the resulting bacteria were analyzed to see if the Ti plasmid had been transferred (which would make the wild grape strains able to cause crown gall). From 72 matings that were done, 338 potential transconjugants (bacteria that may have received the Ti plasmid) were isolated. Over 150 have been analyzed thus far and in no case has stable transfer of the Ti plasmid to a wild grape strain been verified. The remainder of the strains will be analyzed in 1997. From these experiments and from other work in our lab we find that A. vitis from wild grape has a genetically diverse chromosome that differs significantly from that of pathogenic strains. These genetic differences may in part be responsible for the apparent inability of wild grape strains to harbor a Ti plasmid and become pathogenic.

The third objective of our work involves testing biological controls for crown gall in the field. Two new field experiments were initiated in 1996. One is at the Long Island Horticultural Research Laboratory and the other is at Swedish Hill Winery in Romulus, NY. These will provide valuable information on how well the biological controls work in the field. In related research, we plan to measure the survival and spread of the biological control bacterium, F2/5, in these field experiments. Because it may take 3 - 5 years for crown gall to be expressed in the field plots (or longer if trunks are not injured by cold temperatures), we will continue to initiate more field experiments even before the current ones can be evaluated.

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GENETIC ENGINEERING OF GRAPE
FOR DISEASE RESISTANCE

Ming-Mei Chang
Department of Biology
SUNY-Geneseo, Geneseo, NY

As described in the previous reports, we established tissue culture techniques for in vitro propagation of three grape cultivars: Cabernet Sauvignon (Vitis vinifera), Concord (Vitis x labruscana) and Niagara (Vitis x labruscana). We were able to regenerate plants from all three cultivars through organogenesis (shoot or bud formation). Also, we determined the optimal concentration of kanamycin (50 mM/l) for eliminating the bacteria after Agrobacterium co-cultivation.

Among these three cultivars, we were able to induce embryo production from the leaf explants of Cabernet Sauvignon. The best embryogenesis inducing media tested so far is the Nitsh & Nitsh's (NN) agar medium containing 10 mM KT-30 (N-(2-chloro-4-pyridyl)-N'-phenylurea) and 5 or 10 mM 2,4,5- T (2,4,5 trichlorophenoxy acetic acid). Callus proliferation was observed within a month. These callus containing leaf explants were transferred to the same NN medium containing 10.7 mM NAA (a- naphthalene acetic acid) and 0.9 mM BAP (6- benzylamino purine) for another 2 months. Finally, they were transferred to a hormone-free medium. White, opaque embryos were visible within a week. All of the embryos were placed on a woody plant (WP) medium supplemented with 1 mM BAP to enhance shoot development. After a week, root formation was observed. We are in the process of developing techniques for embryo induction of the other two cultivars, Niagara and Concord.

For Agrobacterium transformation, we were able to transfer either glucanase or chitinase genes into grape cells that then developed into calli (undifferentiated cells). Kanamycin resistant calli were obtained from leaf or petiole explants of all three cultivars after Agrobacterium transformation. Polymerase chain reaction (PCR) was used to detect gene insertions in these grape calli. The positive DNA bands (673 bp or 373 bp) were observed in the PCR-amplified DNAs derived from these Kmr calli, indicating insertions of the glucanase or chitinase gene in these cells. We are in the process of inducing embryo formation from these kanamycin resistant calli.

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INCREASING THE EFFECTIVENESS OF NATIVE EGG PARASITES
AND BIORATIONAL INSECTICIDES FOR GRAPE BERRY MOTH
AND EASTERN GRAPE LEAFHOPPER CONTROL

G. English-Loeb and T.E. Martinson¹, M. Saunders and S. Katti, A. Muza²
Department of Entomology
New York State Agricultural Experiment Station, Cornell University, Geneva, NY¹
Lake Erie Regional Grape Research and Extension Center
The Pennsylvania State University, North East, PA²

The overall goal of this project is to develop pest management practices incorporating both use of selective insecticides and biological control of grape berry moth (GBM) and eastern grape leafhopper by two egg parasites, Trichogramma minutum and Anagrus epos. The general strategy is to: 1) reduce detrimental impacts of insecticide applications on native parasite populations by modifying timing or insecticides used; and 2) increase the numbers of parasites through cultural practices (e.g. ground covers in row middles) or direct releases of mass-reared parasites. Progress on each of the four objectives follows:

Objective 1. Conduct small scale field trials to test the efficacy of releases of Trichogramma against Grape Berry Moth.

Seven field releases of Trichogramma minutum were made in a replicated trial at North East, PA, comparing 'Release' and 'No Release' blocks. In 'release' blocks, parasitization of eggs placed in Concord canopies at each release was significantly higher than in the 'no release' blocks. GBM infestations at harvest were lower in 3 of 4 replicates, but not statistically significant.

Objective 2. Complete testing of the toxicity of currently-used insecticides, fungicides, and insecticides to Trichogramma, in laboratory bioassays.

Toxicity of major insecticides (carbaryl, guthion, imidan, and penncap-M) and fungicides (Abound, Cu+lime, Stylet oil, Nova, Rubigan, Sulfur, and Ziram) to Trichogramma was tested in laboratory and field bioassays. Carbaryl and Penncap-M were most toxic to adults, while carbaryl and JMS Stylet Oil reduced emergence of adults from parasitized eggs. None of the fungicides were toxic to Trichogramma adults or parasitized eggs.

Objective 3. Evaluate timing and efficacy of alternative insecticides for GBM and leafhoppers that are non-toxic to egg parasites.

Seven insecticides were tested against GBM in a spray trial. Dipel and MVPII, mycrobial Bacillus thuringiensis insecticides (BT's), performed well against the first GBM brood, and are viable, registered, options for controlling GBM while avoiding disruption of biological control agents for leafhopper and GBM. Confirm, a selective, unregistered insect growth regulator insecticide, gave excellent control of both first and second brood GBM, and is also reported to be 'soft' on predatory and parasitic insects. With leafhoppers, an early (prebloom) application of low rates of carbaryl or penncap-m gave equivalent performance to a 10-day postbloom application of carbaryl. JMS Stylet oil provided partial control, while Neemix (a botanical insecticide) had no effect.

Objective 4. Evaluate the use of flowering ground covers in row middles to enhance and retain populations of both egg parasites in vineyards.

Plots with buckwheat, clover or sod growing in row middles were evaluated for their effect on numbers and distribution of native Anagrus epos (leafhopper parasite) and mass-released Trichogramma minutum (GBM parasite) at two field sites, one in a vinifera vineyard in the Finger Lakes, and the other in a Concord vineyard at North East, PA. In the Finger Lakes, parasitization of leafhopper eggs by Anagrus was higher in the buckwheat treatment (when flowering) than in the non-flowering sod and clover treatments. Parasitization of eggs by mass released Trichogramma also was higher in the buckwheat during one release. These results suggest that incorporating flowering plants (that supply nectar to the parasites) into vineyards may increase the effectiveness of biological control in vineyards. Leafhopper and A. epos populations at the Lake Erie site were very low in 1996 and it was not possible to assess the impact of buckwheat.

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RE-EVALUATION OF THE IMPACT OF ROOT-FEEDING PHYLLOXERA
ON CONCORD GRAPES

M. C. Saunders¹, A. J. Muza and S. N. Katti², G .M. English-Loeb and T. E. Martinson³
Department of Entomology
The Pennsylvania State University, University Park, PA¹
Lake Erie Regional Grape Research and Extension Center, North East, PA²
Department of Entomology
New York State Agricultural Experiment Station, Cornell University, Geneva, NY³

Objective: Investigate the potential benefits of treating established Concord vineyards infested with phylloxera with a new systemic insecticide.

A new systemic insecticide Proguard (cinnamic aldehyde) was evaluated for efficacy against root-feeding phylloxera.The experiment was conducted in a mature Concord vineyard trained to the single curtain (no tie) system at the Lake Erie Regional Grape Research and Extension Center in North East, PA.The experiment consisted of 5 replications involving three treatments. Treatments included: 1) Control (Untreated); 2) Proguard 1% (1 gal cinnamic aldehyde/100 gal spray solution); and 3) Proguard 3% (3 gal cinnamic aldehyde/100 gal spray solution). Each treatment plot consisted of 9 vines with a six vine buffer between plots. Applications were made on: July 2, July 11 and August 6 using a Friend small plot sprayer equipped with a hooded boom. Treatments were applied at 100 gal/A with pressure adjusted to 100 psi for the first spray and increased to 200 psi for the last two sprays.

Phylloxera populations were monitored by excavating roots (total of 15 root systems/5 replicates) and checking for the presence of phylloxera and nodosities (root galls caused by injection of saliva during feeding). Samples of rootlets were removed from each vine before washing and root tissue with nodosities was separated from healthy tissue. Nodosities were examined and the number of live and dead phylloxera were recorded. From these data we computed the number of live phylloxera/gram of root and total number of phylloxera/gram of root. Post-washed root samples were also examined for galls. Nodosities and healthy tissue from both pre-washed and post-washed samples were placed in a drying oven at 158OF for 16 hours then weighed. The % of root tissue (by weight) with nodosities was calculated for each sample by dividing the dry weight of nodosities/total dry root weight x 100.

The data was subjected to square root transformation (x+ 0.5) 1/2 and analyzed using Waller-Duncan's k-ratio t-test (k-ratio=100). The control treatment was not statistically different from the Proguard 1% or 3% treatments for either the % of root tissue (by weight) with nodosities, live phylloxera/gram of root, or total phylloxera/gram of root.

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DEVELOPMENT OF PRACTICAL MODELS FOR USE
IN THE MANAGEMENT OF GRAPE POWDERY MILDEW

D. M. Gadoury, R. C. Seem and W. F. Wilcox
Department of Plant Pathology
New York State Agricultural Experiment Station, Cornell University, Geneva, NY

Objective: The overall goal of the project has been to develop simple, weather-driven, models to serve as an early-warning system for the development of powdery mildew.

After four years, we have demonstrated that the stage is set for success or failure of disease management programs during the first 6-8 weeks after bud break. Fungicide use as early as 1 inch of shoot growth on susceptible cultivars greatly reduces early establishment of powdery mildew. Because the subsequent rate of spread is insensitive to fungicide use, early spraying has far-reaching consequences with respect to final disease levels on mildew-susceptible cultivars. An analysis of disease development and weather showed that years with 5 or more infection periods (>0.1 inch of rain and temperature >50 F) are typified by severe disease pressure throughout the region. Conversely, dry years with 2 or less infection periods between prebloom and fruit set are typified by very mild disease.

Development of natural resistance in fruit of Concord, Chardonnay, and Riesling was studied. Concord fruit were nearly immune to powdery mildew within 2 weeks after fruit set. A single Nova spray applied to Concord at prebloom provided a high degree of control of fruit infection, highlighting the importance of the prebloom-fruit set period in fruit infection.Two applications of Nova at prebloom and 10 days later provided complete control of fruit infection. Two applications of Kocide, a material not highly regarded for powdery mildew control, on the above schedule provided good suppression of both powdery mildew and downy mildew on Concord. Control of powdery mildew on Concord fruit requires protection before the disease becomes conspicuous. Chardonnay and Riesling clusters inoculated at 10% bloom or 2-weeks postbloom became severely mildewed. Clusters inoculated at 4-weeks postbloom were covered with a diffuse, inconspicuous network of powdery mildew. The risk of infection dropped sharply between 2- and 4-weeks postbloom, but significant, albeit inconspicuous, damage to the crop occurred to clusters inoculated at 4-weeks postbloom. Both cultivars acquired substantial resistance to infection far earlier in development than was previously thought.

Previous reports stated that fruit remained susceptible to new infections until sugar levels reach 8%, and that infections continue to expand and produce spores until sugar levels reach 15%. However, we found that fruit became nearly immune to new infections more than one month before sugar levels reached 8%. Therefore, we believe that severe infection of Chardonnay and Riesling fruit is a consequence of events which occur early in fruit development, although the infections may not be conspicuous until later in summer. The period from prebloom to 2-weeks post bloom is undoubtedly the period during which most fruit infection occurs, although a few new infections may occur between 2 and 4 weeks postbloom. The high susceptibility of fruit during the critical period above, coupled with the closure of the canopy within 2 weeks after bloom, makes this the most important time to control powdery mildew. Earlier and later fungicide applications may be important to control foliar mildew. However, it is very doubtful that fruit infection can be controlled satisfactorily if spray coverage, material selection, rates, or the interval between applications is suboptimal during this period. Conversely, growers may have assurance of relatively clean fruit on Chardonnay and Riesling, based upon trace levels of powdery mildew observed during late-July.

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STRUCTURE AND DEVELOPMENT OF GRAPEVINES
IN THE NORTHEAST

Martin C. Goffinet
Department of Horticultural Sciences
New York State Agricultural Experiment Station, Cornell University, Geneva, NY

Growers' knowledge of the structure and development of grapevines is generally not sufficient to understand the many unique growth and management aspects of this crop. Efforts to develop a Cornell publication on the structure and development of grapevines in the Northeast have begun. Although the grapevine anatomy project at the New York State Agricultural Experiment Station in Geneva has accumulated much information over the years, there are many gaps in our ability to provide good examples of plant structure and development in several critical areas. A concerted effort was initiated in 1996 to secure enough information on how vines are constructed, grow and develop in our Northeastern region, so that a publication can be produced. The support of the New York Wine & Grape Foundation has allowed inroads into accumulating this information. Work in 1996 included collecting grapevine tissues not yet analyzed, making photographic records of their anatomy and development, and documenting how various organs and tissues relate to physiology and to the annual cycle of events important to vine growth and vine management. Specific inroads in 1996 included: structural details of minimal-pruned vines; leaf structure and function; root development; relation of buds to the flowering and fruiting cycle; cane and trunk cold injury and recovery mechanisms; use of reserve materials in bud break, shoot extension and the floral process; origin of the cluster and flower development; nature of abnormal flower formation; structure of the graft union. Charts, tables, graphs and diagrams have been made to show examples or to explain details of vine growth.

Continued work will proceed in 1997 to complete collection and analysis of vine tissues and to consolidate the information into a reference publication.

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NOVEL OPPORTUNITIES FOR BIOLOGICAL CONTROL
OF GRAPE DISEASES

W. Koeller
Department of Plant Pathology
New York State Agricultural Experiment Station, Cornell University, Geneva, NY

Spores of fungal plant pathogens secrete an enzyme called cutinase in order to penetrate the surface of plants they are attempting to infect. We have found recently that cutinases from pathogens not able to infect grapes can protect grape leaves from pathogens. Enzymes are of protein nature, are part of our nutrition and inherently non-toxic. They are, therefore, acknowledged by EPA as "biochemicals" and would be registered as a biocontrol agent. This finding opened the opportunity to exploit the enzyme in grape disease control. The development of an enzyme to be used as a spray material against plant diseases would represent an entirely new technology of disease control. It has become possible to isolate the genetic information (= gene) for producing cutinase from any organism, to transfer this gene into a different organism, and to let this genetically modified and more productive organism produce large amounts of the enzyme.

In small scale greenhouse experiments, we could show that cutinase isolated from the apple scab fungus prevented the infection of grape leaves by the black rot fungus. The amounts of cutinase we can prepare from the apple scab fungus are extremely small and prohibit experimentation even on a greenhouse scale. The purpose of the request for funding was, therefore, to start preparing quantities of the cutinase sufficient for initial vineyard trials. With the funds received we were able to accomplish the preparation of a gene probe specific to the cutinase gene of Venturia inaequalis, the apple scab fungus. The strategy chosen was to determine internal amino acid sequences of the cutinase under question. The PCR technique allows to translate the amino acid sequences back into genetic information. The specific gene probe is 180 base pairs long and sufficient to be utilized in the cloning of the gene.

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SUPPLEMENTAL IRRIGATION, VINE PERFORMANCE
AND ECONOMIC FEASIBILITY FOR NORTHEAST VINEYARDS:
Interactions with Pruning in Mature Vineyards
and Improved Establishment of New Vineyards

A.N. Lakso¹, B. Shaffer² and R. Dunst³
Department of Horticultural Sciences¹
New York State Agricultural Experiment Station, Geneva, NY¹
Cornell Cooperative Extension² and Vineyard Laboratory, Fredonia, NY³

Although previous studies of irrigation in New York and Pennsylvania have shown little benefit on conventionally pruned vines, current pressures require heavy crops to be competitive. Generally, the heavier the crops the more sensitive the vines are to other stresses like drought. A long-term study was begun in 1990 to evaluate the long-term effects of supplemental irrigation on the performance of mature Concord vines with three different pruning regimes (Balance, 80 Node Constant, and Minimal). Secondly, the role of irrigation in the establishment of a newly planted Niagara vineyard and its continued productivity was also evaluated.

Over the experiment (3 wet, 2 relatively normal and 2 dry years), the results of the Concord study were (note that these were mature vines in deep gravel with no crop thinning, Roundup row-middle management, and excellent season-long under-trellis weed control): 1) There were no long-term benefits of irrigation on Balance-pruned and 80-Node pruned vines; however, occasional effects of dry years (reduced berry size, sugar accumulation, some reductions in pruning weight) were found; 2) Irrigation in these deep gravel soils has been beneficial only in the Minimally-pruned vines; 3) The primary effect of irrigation in Minimally-pruned vines was to greatly reduce variability among plots with different soil water-holding capacity within the vineyard. Minimal pruned vines in drier locations showed clearly poorer results without irrigation. Economic analysis indicated that the increased yields of irrigation in the average of the minimal-pruned plots would pay for the irrigation system, but not too much more.

The results of the Niagara experiment indicate that: 1) Niagaras have a great potential for high sustained growth and cropping if not stressed as the first 5 crops of irrigated vines averaged 12.4 tons/acre; 2) Very good water availability appears necessary for Niagaras to develop early growth and high early cropping and also to maintain good vine size and heavy cropping after establishment (40% increase with irrigation in total cropping was found [first 5 crops irrigated totaled 62 tons/acre versus 43 for non-irrigated]); 3) Irrigation appears to have great potential to improve yields and yield stability in Niagara plantings; and 4) Irrigation should be provided continuously, even after the vineyard is established. Economic analyses show strong increases in the value of irrigation in Niagaras.

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TESTING VARIETIES, CLONES, ROOTSTOCKS AND PRODUCTION METHODS
OF VITIS VINIFERA FOR COMMERCIAL PRODUCTION IN NEW YORK

R. Pool and S. Lerch
Department of Horticultural Sciences
New York State Agricultural Experiment Station, Cornell University, Geneva, NY

Reduced funding and new charges by Station administration greatly restricted activity of this research in 1996. Existing plots were maintained, and, where possible, data was collected. Data was taken for the first time on 7 varieties and 10 new clones of Chardonnay and Pinot noir. Rootstock effects were measured on Chardonnay; no relationship between vine size, hardiness, quality or yield was observed in this year of little winter injury. Research measuring bud cold hardiness was terminated after 8 years.