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Funded Project

Funding Program: Enhancement Grants - Special Projects

Project Title: Evaluating Zinc Supplementation for Management of Pierce's Disease

Project Directors (PDs): Jeff A Brady [1] Travis R Faske [2] McGahan G Donald [3]

Lead State: TX Lead Organization: Texas AgriLife Research

Undesignated Funding: $25,000

Start Date: May-01-2009 End Date: Apr-30-2010

No-Cost Extension Date: Feb-28-2011

Pests Involved: Xylella fastidiosa

Site/Commodity: Grape

Summary: Pierce's disease of grape is caused by Xylella fastidiosa, a xylem-limited, insect-vectored bacterium that blocks water transport in the plant. Once infected, grapes develop typical disease symptoms such as leaf scorch, abnormal leaf abscission, uneven periderm formation on developing canes, and reduced yield. For many European winegrape cultivars, the disease is lethal in one to five years. The incidence of Pierce's disease is particularly severe in the southern U.S., where environmental conditions favor successful overwintering of the insect vectors and the bacterium. Disease management has focused on insecticidal control of the vectors and removal of potential sources of inocula from in and around the vineyard. Recent testing indicates that zinc supplementation may mitigate both disease severity and bacterial colonization of grape tissue. This project will quantitatively and qualitatively document the effects of increased zinc supplementation on bacterial numbers and disease symptomology in grape. Results from the project will be used to develop zinc supplementation as an additional tactic to manage Pierce's disease. Objectives: 1. Evaluate zinc supplementation as a means to chemically manage X. fastidiosa proliferation and PD symptomology in grape. 2. Quantify zinc toxicity levels and measure effects of zinc toxicity in grape. 3. Determine if zinc is the only nutrient responsible for bacterial suppression in grape. 4. Begin to assess the environmental and enological impacts of an increased zinc supplementation regime.

Final Report:

Outputs Winegrape extension in Texas is conducted primarily by 4 viticulture extension specialists who interact one on one with both prospective and established vineyard owners. One of the extension specialists, Fran Pontasch, is located at the Stephenville Research and Extension Center where this work was conducted, and she was heavily involved in this project. Ms. Pontasch has disseminated information generated in this project to individual growers, as well as the other viticulture extension specialists so that they may inform growers. Some growers had been overapplying zinc due to rumors that it may prevent or mitigate development of PD in their vines. We have been instrumental in spreading word through the viticultural extension specialists that there is no direct evidence that heavy applications of zinc mitigate development of PD, and that zinc supplementation should follow established recommendations for general plant health and grape production. The PI has additionally interacted directly on multiple occasions with growers attending the Texas PD Research and Education Program symposia. The Texas PD program growers' advisory board also meets every year to accept research proposals, and these meetings have been an opportunity for discussions of micronutrients as a PD management tactic as well. A poster presentation at the national APS meeting (listed below) led to an ongoing collaboration and numerous grant submissions with Dr. Leonardo De La Fuente of Auburn University, who is studying in vitro zinc toxicity mechanisms in X. fastidiosa. In addition to informal discussions about the ongoing micronutrient work, this project directly or indirectly led to the following formal presentations at national, regional, and local scientific meetings: Ator, R., Faske, J., Rathburn, H., Mitchell, F., and Brady, J. DNA probe-melt genotyping of Xylella fastidiosa. Second Place Undergraduate, Second Place Life Sciences, 2010 Texas A&M Pathways Student Research Symposium, West Texas A&M University, Canyon, TX. Brady, J.A., Faske, J., Faske, T., and McGahan, D. Evaluating the impact of nutritional treatments on Xylella fastidiosa in grapevine. National Meeting of the American Phytopathological Society, Charlotte, NC. August 7-11, 2010. Castaneda-Gill JM, King JL, Laney R, Rathburn HB, Mitchell FL, Brady J. Multilocus Melt Typing of Xylella fastidiosa subspecies using real-time PCR. First Place Graduate Oral Presentation, 2008 Tarleton State University Student Research Symposium, Stephenville, TX. Faske, J, Castaneda-Gill, J, King, J, Laney, R, Rathburn, H, Mitchell, F, Brady, J. Multiple fluorescent markers for Xylella fastidiosa subspecies. National Meeting of the American Phytopathological Society, August 2009. Myers, B.A., Brady, J.A., Rathburn, H.B., and Mitchell, F.L. Genotyping Xylella fastidiosa using multiplex PCR. National Meeting of the American Phytopathological Society, Charlotte, NC August 7-11, 2010. Ruiz, J., McGahan, D. G. 2011 - 48th Annual Soil Survey and Land Resources Workshop and Symposia, USDA-NRCS; AgriLife Research Texas A&M System; AgriLife Extension Texas A&M, Texas A&M University, College Station, TX, "Assessing Micronutrient Availability in a North Central Texas Vineyard". (February 3, 2011). Ruiz, J., McGahan, D. G., 9th Annual Pathways Student Research Symposium, Texas A&M University System, West Texas A&M University; Canyon, TX., "Assessing Micronutrient Availability by Landform in a North Central Texas Vineyard". (October 22, 2010). Ruiz, J. (Presenter & Author), McGahan, D. G., 9th Annual Tarleton Student Research Symposium, Tarleton State University, Stephenville TX, "Assessing Micronutrient Availability by Landform in a North Central Texas Vineyard". (October 16, 2010). Shrum, S.L., Faske, J.B., Mitchell, F.L., and Brady, J.A. Multi-locus DNA melt analysis of Xylella fastidiosa strains. 2011 Texas A&M Pathways Student Research Symposium, Texas A&M University, College Station, TX. Additionally, this project is producing a number of peer-reviewed publications. In order to efficiently screen a large number of grapevine tissue DNA extractions for X. fastidiosa, a more efficient DNA extraction protocol was developed and published. The necessity of marking X. fastidiosa to be used as experimental inocula led to the creation of a fluorescent strain that will be described in an upcoming publication and has already been described in a presentation at a national meeting (above). Contamination of plant tissue with non-grape strain X. fastidiosa bacteria and the problem of distinguishing different X. fastidiosa strains culminated in the publication of a methods paper for a new bacterial genotyping method that can quickly discriminate bacterial strains from plant or insect DNA extractions or from culture. Development of the genotyping method provided a master's thesis project for two graduate students and involved numerous student presentations. This project directly or indirectly led to the following peer-reviewed publications and master's theses, and several additional publications are in preparation: Brady, J. A., J. B. Faske, et al. (2012). "Probe-based real-time PCR method for multilocus melt typing of Xylella fastidiosa strains." J. Microbiol. Meth. 89: 12-17. Brady, J. A., J. B. Faske, et al. (2011). "High-throughput DNA isolation method for detection of Xylella fastidiosa in plant and insect samples." J. Microbiol. Meth. 86(3): 310-312. Castañeda-Gill, J. M. (2009). Multilocus melt typing of Xylella fastidiosa subspecies using real-time PCR. Department of Biological Sciences. Stephenville, Texas, Tarleton State University. Master's thesis: 114. Hassell, A., J. Brady, et al. (2012). "Xylella fastidiosa leaf disk feeding assay." Environmental Entomology: In preparation. Myers, B. A. (2011). Development of a high throughput assay for genotyping Xylella fastidiosa subspecies using multiplex PCR. Biological Sciences. Stephenville, Tarleton State University. Master's thesis: 88.

Outcomes Greenhouse and field trials testing the impact of micronutrient supplementation on Xylella fastidiosa (Xf) proliferation were conducted starting in 2009 and were recently completed. A brief description of nutrient/micronutrient evaluations follows: Materials and Methods Vines for greenhouse testing were treated with a hot water dip method before propagation to reduce potential Xylella fastidiosa (Xf) contamination of experimental vines. Vines were inoculated on 3 occasions by the pin-prick method with Xylella fastidiosa subsp. fastidiosa (Xff) grape isolate STX1, a PD-causing Texas strain isolated from the research plots of the Texas AgriLife Research and Extension Center in Stephenville, TX. The strain used in greenhouse experiments was labeled with a genomic insert of the sea anemone fluorescent protein AmCyan that served as a DNA target for qPCR amplification to measure proliferation of the inoculated bacterium in the vines. The same Xf strain was used in field trial inoculations, but it did not contain the fluorescent transgene. Greenhouse vines were planted in 1 gallon pots in MetroMix400 potting soil, watered weekly with 500 ml of 18 M Ohm reagent grade water and fertilized weekly with 500 ml of Hoagland's solution #2, which consisted of 6.0 x 10-3 M KNO3, 4.0 x 10-3 M Ca(NO3)2, 1.0 x 10-3 M NH4H2PO4, 2.0 x 10-3 M MgSO4, 4.6 x 10-5 M H3BO3, 9.0 x 10-6 M MnCl2*4H2O, 7.65 x 10-7 M ZnSO4*7H2O, 3.0 x 10-7 M CuSO4*5H2O, 1.0 x 10-7 M NaMoO4*2H2O, and 5.0 x 10-5 g/L of Sprint 330 iron. Plants subjected to nutrient deficiencies had one or more micronutrients withheld from the Hoagland's solution, while plants subjected to excess micronutrient supplementation had an individual micronutrient supplied in excess. A 2009 greenhouse trial was initiated on susceptible cabernet sauvignon vines. Each treatment consisted of 8 experimental reps. Tests included the following nutrient regimes applied as soil drenches: 1) Cabernet sauvignon, Non-inoculated, Complete Hoagland's 2) Cabernet sauvignon, Complete Hoagland's, inoculated 3) Cabernet sauvignon, Complete Hoaglands, Bacillus thuringiensis inoculated 4) Cabernet sauvignon, Non-pathogenic Xf inoculated, Complete Hoagland's 5) Cabernet sauvignon, Hoagland's minus zinc starting at inoculation 6) Cabernet sauvignon, Hoagland's minus Mn starting at inoculation 7) Cabernet sauvignon, Water only after inoculation 8) Cabernet sauvignon, Hoagland's minus micronutrients starting at inoculation 9) Cabernet sauvignon, Hoagland's plus 1.75 mM Zn starting at inoculation 10) Cabernet sauvignon, Hoagland's plus 1.75 mM Zn starting 2 weeks prior to inoculation 11) Cabernet sauvignon, Only 1.75 mM Zn starting at inoculation 12) Cabernet sauvignon, Hoagland's plus 1.8 mM Mn starting at inoculation 13) Cabernet sauvignon, Only 1.8 mM Mn starting at inoculation 14) Cabernet sauvignon, Only S starting at inoculation 15) Cabernet sauvignon, Only 60 uM Cu starting at inoculation 16) Cabernet sauvignon, Hoagland's plus 1.75 mM Zn, 1.8 mM Mn, 60 uM Cu, and S supplementation starting at inoculation Petioles were collected from the plants and Xf were quantified in petiole tissue by qPCR on three separate occasions by two different qPCR assays. A 2010 greenhouse trial was initiated on highly susceptible chardonnay vines. Tests included the most promising micronutrient treatments from the 2009 greenhouse trial, and a number of additional plant defense compounds, insecticides, and a bio-control agent as well. The top 5 treatments were watered with reagent grade water and Hoagland's solution supplementation, while the rest were watered with the greenhouse garden hoses (well water). 1) Chardonnay, Hoagland's non-inoculated control 2) Chardonnay, Hoagland's minus zinc starting at inoculation 3) Chardonnay, Hoagland's plus 1.75 mM Zn starting at inoculation 4) Chardonnay, Hoagland's plus 1.8 mM Mn starting at inoculation 5) Chardonnay, Hoagland's plus sulfur starting at inoculation 6) Chardonnay, BTN+ 7) Chardonnay, imidacloprid (widow) prophylactic soil drench 8) Chardonnay, venom 70S, prophylactic soil drench 9) Chardonnay, venom 70S foliar, prophylactic 10) Chardonnay, Centric 40G, prophylactic soil drench 11) Chardonnay, employ harpin spray, prophylactic soil drench 12) Chardonnay, myke, prophylactic soil drench 13) Chardonnay, Actigard, prophylactic soil drench 14) Chardonnay, salicylic acid, prophylactic, 1 mM soil drench 15) Chardonnay, 1-aminocyclopropane carboxylic acid, prophylactic, 0.5 mM 16) Chardonnay, 2,6 dichloropyridine-4 carboxylic acid, prophylactic, 100 mM foliar 17) Chardonnay, abscisic acid, 100 uM soil drench 18) Chardonnay, jasmonic acid, 100 uM soil drench 19) Cabernet sauvignon non-inoculated control A 2011 greenhouse trial was initiated on highly susceptible chardonnay vines. Tests included 28 Xf strains inoculated for potential bio-control against the PD strain. The treatments were watered with the greenhouse garden hoses (well water). A 2010 field trial was initiated on moderately resistant blanc du bois vines and susceptible cabernet sauvignon vines. Field plots contained 3 treatments: inoculated controls, zinc soil treatment (Zinc-Gro Maxi Granular ZnSO4 monohydrate, Tetra Micronutrients, Fairbury, NE) consisting of a 250 ml volume of fertilizer applied 1 ft from the base of each vine on both sides of the vine and buried in a 4 inch deep pit directly under an irrigation emitter, zinc soil plus foliar treatment, consisting of the soil treatment described above, plus weekly foliar application during the growing season of a 1.75 mM solution of ZnSO4*7H2O. All plants received weekly irrigation during the growing season. Control, zinc soil, and zinc soil plus foliar treatments were conducted in 5 randomized blocks, with each block containing 4 plants from each of the 3 treatments. All field plants were inoculated by the pin prick method at two stem locations on 3 separate dates spanning a 3 week period in May 2010. A 2011 field trial was conducted on moderately resistant blanc du bois vines and susceptible cabernet sauvignon vines to follow up on 2010 field trial results. Because zinc concentrations in petiole tissue were below 100 ppm, the foliar zinc treatment was increased 10X compared to 2010 levels, to a 10.75 mM solution. Field plots contained 3 treatments: inoculated controls, zinc soil plus foliar treatment (Zinc-Gro Maxi Granular ZnSO4 monohydrate, Tetra Micronutrients, Fairbury, NE) consisting of a 250 ml volume of fertilizer applied 1 ft from the base of each vine on both sides of the vine and buried in a 4 inch deep pit directly under an irrigation emitter plus weekly foliar application during the growing season of a 10.75 mM solution of ZnSO4*7H2O, and a third treatment consisting of foliar application of 25 ml/L elemental sulfur spray (Red Ball EM-53 liquid Sulphur, Georgia Gulf Sulfur Corp.). All plants received weekly irrigation during the growing season. Control, foliar sulfur, and zinc soil plus foliar treatments were conducted in 5 randomized blocks, with each block containing 4 plants from each of the 3 treatments. All field plants were inoculated by the pin prick method at two stem locations on 3 separate dates spanning a 3 week period in May 2011. For both greenhouse and field trials, three samples from petioles nearest to the inoculation points were taken for each experimental rep at multiple timepoints throughout a period of 4 months. DNA was extracted from the petioles by a novel high-throughput method (listed above) and the samples were subjected to qPCR to quantify Xf proliferation during micronutrient treatments. The oligos used for AmCyan qPCR detection in greenhouse samples were forward primer AmCyanF 5'- CGATGCTTTACTGCGTATCCTACC-3', reverse primer AmCyanR 5'- TTTCTCAAAAGATGGGTCCCAACC-3', and the 5' hydrolysis probe AmCyanP 5'- TCTTCGCCATCACAGGTCCATCAGCAGG-3'. In addition to the AmCyan qPCR reactions, samples were also subjected to a qPCR assay using the method of Schaad et al., 2001, to detect the ITS region of Xf. Field samples were non-transgenic and were only subjected to qPCR of the ITS region. PCR reactions contained 500 nM forward and reverse primer and 500 nM probe. Each PCR reaction contained 2.5 ul of 2X PCR master mix [80 mM Tris-Cl, pH 9.0, 50 mM KCl, 10 mM MgCl2, 1.2 uM dUTP (Roche Applied Science, Indianapolis, IN), 0.4 uM dATP, 0.4 uM dCTP, 0.4 uM dGTP (Promega, Madison, WI), 0.5 U platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), 0.5 U LightCycler uracil-DNA glycosylase (Roche Applied Science, Indianapolis, IN), and 1 mM Na2EDTA, pH 8.0]. PCR reactions contained 1 ul of template from a DNA extraction in an overall reaction volume of 5 ml. The thermocycling protocol consisted of 95 °C for 10 minutes, followed by 45 amplification cycles of 95 °C for 10 seconds, 60 °C for 10 seconds, and 72 °C for 10 seconds. Crossing points were calculated using the fit points method with LightCycler® analysis software. Each qPCR run contained 30 negative control reactions and 5 positive control reactions. DNA copy number in each sample was calculated with LightCycler® analysis software by comparison to an external standard curve created with a serial dilution (1 x 10^1 to 1 x 10^8 DNA templates/PCR reaction) of Xf genomic DNA. Each sample was subjected to 3 qPCR replicate amplification reactions. DNA copy numbers were exported into SAS v9.2 (SAS Institute, Cary, NC), log transformed and submitted to analysis of variance to identify copy number differences using the GLM procedure. Student Newman-Keuls (SNK) post-hoc tests determined differences between means in the model. Characterization of the effects of zinc application to the soils at the Stephenville AgriLife Research plots is being conducted by a Tarleton State University student as a part of a master's thesis project. Soil pits have been excavated in the vineyard to characterize soil horizons, and numerous samples have been collected to characterize the concentration of zinc and other nutrients in close proximity to the test plots and throughout the vineyard. DTPA extractions have been performed and nutrient and micronutrient analyses are currently being conducted. Expected completion date for the thesis is August 2012, and publication of the results in a peer-reviewed journal will occur sometime thereafter. Enological impacts of additional zinc supplementation were determined by comparing wine produced from plants that were part of the experimental control with wine produced from the soil zinc and the soil plus foliar zinc field treatments. Fractions were saved throughout the winemaking process and sent to the Texas A&M Soil Testing Laboratory for nutrient analysis. Similarly, nutrient levels in grapevine petioles following nutrient treatments were determined by harvesting petioles, washing them in 0.1 M HCl, drying them to the touch, and sending them to the Texas A&M Soils Testing Laboratory for analysis. Results/Discussion 2009 Greenhouse Trial Evaluation of bacterial numbers per gram of cabernet sauvignon plant tissue revealed statistically significant (p<0.05) differences in each test, but post-hoc tests revealed that no treatment consistently suppressed bacterial proliferation in planta throughout the course of the experiment. Several of the treatments were repeated on chardonnay plants in the 2010 greenhouse trial. 2010 Greenhouse Trial ANOVA performed on the qPCR results of 160 chardonnay plants in 20 greenhouse test groups in 2010 revealed no significant differences between the means of test groups, therefore no multiple comparison test was performed. Large variation in bacterial counts within treatments impacted the 2010 greenhouse trial. 2011 Greenhouse Trial Although none of the potential bio-control Xf strains produced a statistically significant difference in Xf proliferation, one strain came close (p<0.063) when t-tested against inoculated control plants. 2010 and 2011 Field Trials In both 2010 and 2011, qPCR measuring the mean bacterial numbers in petiole tissues was not significantly different between treated and untreated vines when ANOVA was conducted on 120 vines from a tolerant variety (Blanc du Bois) and a susceptible grape variety (Cabernet Sauvignon), although the average number of bacteria per gram of petiole tissue was lower in the tolerant variety. Zinc levels in petioles of field treated vines were highly significantly different from one another (p<0.001) when compared to control vines that received no additional zinc supplementation. The two grape varieties were also highly significantly different from one another in petiole zinc concentrations, with average cabernet sauvignon petiole zinc level being almost twice as high as in blanc du bois (215 ppm vs. 118 ppm in the soil + foliar application). Soil only applications of zinc resulted in more modest petiole concentrations, only about 15 ppm above untreated control vines, which had baseline petiole zinc concentrations of 75 ppm for cabernet sauvignon and 49 ppm for blanc du bois. The level of zinc in wines made from zinc soil + foliar treated vines was significantly different (p<0.05) than untreated control vines. Although some of the zinc ended up in precipitates discarded during the winemaking process, the finished wines from zinc soil + foliar treated vines averaged about 5 ppm zinc compared to control wine at about 1 ppm zinc. Greenhouse plant biomass was highly significantly different (p<0.001) between zinc treated cabernet sauvignon plants and control plants supplemented with Hoagland's solution in the 2010 greenhouse trial. It should be noted that the level of soil drench zinc supplementation used in the greenhouse trial is toxic to cabernet sauvignon grapevines over the course of one year, and surviving vines were very stunted and displayed red discoloration in surviving leaf tissue.

Impacts Multiple Texas growers have already begun applying zinc as a potential means of controlling PD, a decision based both on the threat that the disease poses and local claims based on anecdotal evidence that zinc mitigates or eliminates the effects of PD. Although in preliminary greenhouse tests a positive effect was seen, the results so far indicate initial claims that zinc can be used as a cure for PD may have been overstated, and interactions with genotype and/or environment greatly influence any effect that zinc may have on proliferation of Xf in plant tissue and on development of PD symptoms. Any disease suppression to date with zinc application has been episodic and temporary. Growers using zinc as a potential PD control have discussed applying zinc at levels that far exceed those being used in this study, even in areas on the Texas High Plains, with little or no natural PD pressure. The main impact of this study to date has been to temper some initial claims that zinc supplementation is the answer to the PD threat in the state, and perhaps this will reduce unnecessary input and excess application by some growers. This information could save producers the expense of unnecessary zinc applications and could prevent unnecessary introduction of zinc into the environment. Another concern at the outset of this study was that zinc supplementation would negatively affect the winemaking process or the product. Even with zinc supplementation exceeding recommended levels for grapevines, the zinc ending up in the final product was statistically significantly higher, but the amount (5 ppm) was not high when compared to other agricultural products. One question before this study was conducted concerned the impact of insecticides on the bacterial causative agent of PD. There had been suggestions that the insecticides might be affecting bacterial growth as a result of off-target affects impacting the bacterium. Results indicate that the systemic insecticides used to control the vectors do not directly affect bacterial proliferation in the vine and should only be applied for purposes indicated on the product label.

Potential Impacts Currently, there isn't a cure for PD, and infected vines die in 1 to 5 years. Transgenic vines with resistance mechanisms loom on the horizon, but their adoption is uncertain. Because the crop is perennial and the economic loss can continue for years, use of systemic insecticides to control the insect vectors of PD has enjoyed widespread adoption by growers in all grape producing areas of Texas with PD incidence. Systemic insecticide use has brought the disease under management in many areas of the state, but it can affect non-target species as well. Donald Hopkins (University of Florida) has identified a bio-control strain of Xf that can reduce disease symptoms and bacterial proliferation, although the mechanism of control is not clear. Several potential bio-control Xf strains were examined in association with this project, and one of the strains may be used in future tests as a potential PD management tactic. Neither transgenic plants nor bio-control strains will be effective for established vineyards with existing PD issues, and therapeutic measures are needed by many vineyards in the state, and throughout the southern U.S. This project evaluated a putative therapeutic for PD, and the results may dissuade growers from zinc oversupplementation as a potential means of controlling PD. Zinc was applied at a level high enough to display toxicity in the greenhouse vines, yet it did not offer statistically significant reduction of bacterial proliferation in greenhouse or field-grown vines.

Leveraged Funds This project was leveraged in the submission of 10 related grants for research on diseases caused by X. fastidiosa and on research studying micronutrient application, 5 of which were funded: McGahan, Donald G (PI). Vineyard soil and nutrient survey. Sponsored by Tarleton State University, $8,604.00. (November 1, 2010 - August 31, 2011). Funded. McGahan, Donald G (PI). Vineyard soil and nutrient survey," Sponsored by Tarleton State University, $6,992.00. (October 26, 2009 - August 1, 2010). Submitted Aug. 2009. Funded. Mitchell, Forrest (PI), Brady, Jeff (Co-I). Entomological research into Pierces disease in Texas. 2009-2010. $293,096.00. USDA-APHIS. Texas Pierces Disease Research and Education Program. Funded. Mitchell, Forrest (PI), Brady, Jeff (Co-I). Research into Pierces disease in Texas. 2011-2012. $159,102.00. USDA-APHIS. Texas Pierces Disease Research and Education Program. Funded. Mitchell, Forrest (PI), Brady, Jeff (Co-I). Neonicotinoid efficacy in Texas grape. 2010-2011. $99,000.00. USDA-APHIS. Texas Pierces Disease Research and Education Program. Funded. De La Fuente, Leonardo (PI), Brady, Jeff (Co-I). Genotypic and phenotypic diversity of Xylella fastidiosa based on virulence and environmentally-related traits. 2011-2013. $205,256.00. California Department of Food and Agriculture. Not funded. De La Fuente, Leonardo (PI), Brady, Jeff (Co-I). Phenotypic diversity and virulence of Xylella fastidiosa isolates infecting grape and blueberry. 2012-2016. $400,000.00. USDA. Not funded. Brady, Jeff (PI), Sanderlin, Randy (Co-I). Integrating plant-based treatments into management tactics for diseases of pecan and grape caused by the bacterium Xylella fastidiosa. 2010-2012. $153,367.00. USDA. Not funded. Brady, Jeff (PI), Mitchell, Forrest (Co-I). Plant nutrient effects on Xylella fastidiosa populations in grapevine. 2009-2010. $125,935.00. USDA-APHIS. Texas Pierces Disease Research and Education Program. Not funded. Mitchell, Forrest (PI), Brady, Jeff (Co-I). Management of Xylella fastidiosa in the Southern United States Grape Crops by Integration of Multiple Strategies. 2012-2017. $3,500,000. USDA-NIFA. Not funded. The Southern Region IPM seed grant funds for this project facilitated the acquisition of additional funding sources to study PD in Texas. In particular, the Texas PD Research and Education Program asked investigators to diversify their funding sources and augment Texas PD funds with outside sources. This seed grant aided the PI in acquiring additional funds as a co-investigator on other Texas PD grants. The seed grant funds provided partial support for a Tarleton State University masters student who graduated in May, 2011. TSU provided financial support to the student for teaching laboratory classes, enabling his contribution to the project. A second TSU masters student has received material support from this grant, and is expected to graduate in August 2012. TSU also provided financial support to this student, both in the form of a teaching stipend, and in the form of research grants. The grant also enabled use of greenhouse space at the Texas Pierces Disease Research and Education Program headquarters in Fredericksburg, TX, and allowed a third masters level student to participate in plant treatments and tissue processing, as he completed an unrelated research project. Four additional undergraduate students participated in this project to a smaller extent but received their funding from other granting sources.

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