Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Alson H. Smith, Jr. Agri Res & Ext Ctr
Non Technical Summary
Crown gall of grape is a very common disease of grapevines around the world. Symptoms are the formation of galls on the trunk or other parts of grapevines. When the vines are infected, the vascular system can be compromised due to the gall formation, thus, the infected vine can be killed from the disease. The pathogen of grapevine crown gall is a bacterium Rhizobium vitis. This pathogen causes systemic infection, thus, once the vine is infected, the only true remedy is the removal of the infected vine.Unfortunately, we do not have goodmeans of managing grapevine crown gall. The best practice is a hilling of the graft union of the vine where tissues are susceptible to the infection; however, it is cost prohibitive since you have to protect the graft union with the soil in the fall, and then remove the soil in the spring (otherwise it will negatively affect the vine).I launched an international collaborative project with Dr. Kawaguchi in National Food and Agricultural Research Organization in Japan and Kumiai Chemical in Japan to address this economically important disease. The aim of this project is the validation of a biological control agent Rhizobium vitis strain ARK-1. The preliminary results indicate a very good efficacy of ARK-1 against several R. vitis isolates from Virginia.The objectives of this project are to 1)investigatea practical method to apply ARK-1 in the field, and 2) find a potential biological agent(s) that can be applied by itself or with ARK-1 to enhance the efficacy. We will conduct a series of lab, greenhouse, and field assays to achieve these objectives.
Animal Health Component
70%
Research Effort Categories
Basic
10%
Applied
70%
Developmental
20%
Goals / Objectives
Crown gall of grape, caused by Rhizobium (=Agrobacterium) vitisis a chronic and very common issue among Virginia vineyards. Galls are water and nutrient sinks that weaken plants and can eventually lead to vine death. Hypertrophic cell growth forms galls at wounds on the roots or trunk of the grapevine (Vitis spp.). Wounds can result from a variety of causes, but winter injury or mechanical damage, such as graftingare primary causes. Rhizobium vitis causes crown gall by transferring the T-DNA region of the tumor-inducing bacterial plasmid (Ti-plasmid) to the host cell, which subsequently integrates into the plant host genome.The pathogen causes a systemic infection; consequently, the only true remedy is the removal of infected vines.Currently, cultural management strategies, such as hilling of the graft union during the winter, are the predominant methods used for control of crown gall. However, many of these strategies are not feasible or sustainable due to their cost and labor requirement.I collaborate with Dr. Kawaguchiat Okayama Prefecture Agricultural Research Center in Okayama, Japan, and Kumiai Chemical in Tokyo Japan to validate a biological control agent which is anR. vitis isolateARK-1. Our preliminary results showed that ARK-1 inhibits the production of galls when both tumorigenic (= can cause galls) and ARK-1 (non-tumorigenic) are co-inoculated. ARK-1 might interfere with the pathogenic isolates by disturbing several of the tumor-causing genes.I will conduct a series of lab, greenhouse, and field studies to determine whether ARK-1 can be practically and effectively used to mitigate crown gall. We will also investigate other potential biological control agents, and develop better detection/diagnostics methods.
Project Methods
Objective 1a) Evaluate application methods, and timing of application of ARK-1 in the greenhouse and field using a pre-release, commercial product: ARK-1 biocontrol efficacy assays in tomato. Tomato seedlings will be grown from seed for two weeks in a greenhouse. Bacterial inocula will be prepared by inoculating 5 ml of YEM broth in a 15 ml culture tube with a R. vitis colony. After 48 hours on a shaker at 25C, optical densities of cell cultures will be measured at 600 nm (OD600) and adjusted to OD600 of 0.1 (~ 108 cell/ml).Four inoculation methods (Stab, Paint, Soil application, and root dip), summarized in Table 2, will be used to examine the efficacy of inoculation methods to control a mixture of four tumorigenic R. vitis isolates. If it is not noted, the cell concentration to be used in the experiments is 5 x 105 cell/mL. We will determine the best timing of ARK-1 application for each inoculation method, and then compare the efficacy of these methods.Gall formation will be recorded and galls will be measured at their widest diameter using a digital caliper at 42 days post-inoculation. For each experiment, experimental unit will be a tomato seedling. There will be five stab wounds per plant, three internal replications (= plants) and at least three experimental runs.In planta ARK-1 efficacy assays in grapevine. Similar experiments will be conducted with Vitis vinifera cv. 'Cabernet Sauvignon' and 'Chardonnay' seedlings grown from seeds collected at AHS AREC. Unless tomato experiments result in very poor outcomes, all experiments described above will be repeated with grape seedlings. I also have been in contact with two nurseries to conduct experiments at the time of propagation.Objective 1b) Determine the effect of rate of ARK-1 to reduction of crown gall: A mixture of four isolates will be prepared as described, then a dilution series will be made to create cell suspensions of 1:1, 1:2, 1:4, 1:8, and 1:10 relative ration between ARK-1 (5 x 105 cell/mL) and the four-isolate mix. The effect of the relative ratio (= treatment) will be examined using tomato and grapes using the stab method. In addition, once we identified a preferred method(s) from the objectives 1a) and 1b), other inoculation methods will be tested using different doses of tumorigenic R. vitis mix.Statistical analysis: Effects of treatments on the probability of gall formation and the average gall diameter per treatment will be analyzed using the generalized linear mixed model (GLIMMIX) or finite mixture model (FMM) in SAS (ver. 9.4, SAS, Cary, NC). The logit (assuming either the binomial (in GLIMMIX) or the zero-inflated binomial distribution (in FMM)) and the identity link function will be used for the mean probability of gall formation, and gall diameter data, respectively. Treatment factors will be considered as fixed effects and experimental run factor will be considered as a random effect. Once the effect of a fixed factor is found to be significant (P < 0.05), Fisher's Least Significant Difference (LSD, a= 0.05) will be used as a multiple comparison method.Objective 2a) Screen/sample Virginia vineyards for indigenous, non-pathogenic R. vitis strains that might have potential use as biological agents for crown gall managementIn addition to current collection of isolates, more samples from Virginia and mid-Atlantic regions will be collected. Isolation of R. vitis will be conducted on a semi-selective Roy and Sasser (RS) medium (Roy and Sasser, 1983). A modified multiplex PCR procedure (Kawaguchi et al. 2005) will be used to identify these bacterial colonies to determine whether they are 1) R. vitis or not, and if it is, 2) whether R. vitis contains a piece of DNA that causes galls or not.For isolates that are R. vitis, but do not contain Ti-plasmid, the stab co-inoculation assay with tomato and grape seedlings will be conducted. If gall formation is not observed with co-inoculation with a mixture of four Virginia isolates with grape, DNA will be sequenced to examine the similarity to R. vitis ARK-1 and other known antagonistic strains. Using the final candidates, inoculation studies will be conducted to determine the efficacy of the new candidate(s), possibility of synergistic effect with ARK-1 co-inoculation. Experimental methods will be very similar to those of objective 1 stab method. Other research topics, such as tests for antibiosis, genome comparisons to ARK-1, and gene expression study can be considered in the future.Objective 2b) Determine population diversity of the US and Japanese R. vitis isolates. DNA fingerprinting of the collected R. vitis isolates in USA (Virginia and surrounding states) and Japan will be carried out using the repetitive sequence-based (rep)- PCR (using BOXA1R, REP, REP1R-I/REP2-I primer sets, Table 3) (Kawaguchi et al. 2017; Kawaguchi and Tanina 2014), and inter-simple sequence-repeat (ISSR)-PCR methods using primer sets GTGC4, GAGC4, and GTG5.Using gel electrophoresis, amplified PCR products will be separated, and then each DNA fragment with a distinct electrophoretic mobility will be assigned a position number and a score of either 1 or 0 for the presence or absence of the fragment, respectively. In order to improve accuracy, an isolate with known fragments will be used as a control, in addition to the gel ladder, and software such as Gel Analyzer (GelAnalyzer.com) will be used. The estimation of distance between a pair of tested genes will be calculated as [1 - Dice similarity coefficient]. For calculation of Dice similarity coefficient and distance estimation, R (ver. 3.5, R Development Core Team) package "proxy" will be used. Then a dendrogram will be created using arithmetic average (UPGMA) clustering. In addition to the cluster analysis, the multiple correspondence analysis (MCA)and nominal principal component analysis (PCA) will be conducted. For MCA, the country of origin will be also used as a supplemental variable. Both MCA and PCA will be conducted in JMP Pro (ver. 14, SAS Institute, Cary, NC, USA).Objective 3) Develop a mutiplex qPCR procedure to be able to detect relative quantity of ARK-1 in the grapevineWe will modify a singleplex qPCR protocol of R. vitisto develop a multiplex real-time PCR assay targeting cytochrome oxidase (COX), the R. vitis virD2 gene (for a tumorigenic R. vitis), and contig 19 (for ARK-1).?Once the protocol is established, selected isolates will be used to determine the efficacy of the qPCR protocol. Then, with the final protocol, there will be a series of studies to be conducted to determine interaction between a tumorigenic R. vitis and ARK-1. Treatments will be 1) ARK-1, 2) a tumorigenic R. vitis, and 3) simultaneous co-inoculation of ARK-1 and the tumorigenic R.vitis. At 0, 1, 5, 15, 24, 48, and 72 hours after inoculation, the surface of seedling will be sampled (destructively), and subjected to the multiplex-qPCR procedure. Based on the outcome, the time interval will be extended. The second experiment will examine the timing of ARK-1 application. ARK-1 will be inoculated -72, -48, -24, -15, -5, -1, 0, 1, 5, 15, 24, 48, and 72 hours before a tumorigenic isolate, and samples will be examined later to compare relative cell number between ARK-1 and the tumorigenic isolate. (Note: negative time point indicates the tumorigenic isolate will be inoculated first.) The experimental settings and analysis will be similar to those described in objective 1.