Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521
Performing Department
Microbiology & Plant Pathology
Non Technical Summary
Xylella fastidiosa, a bacterial pathogen, causes Pierce's disease (PD) of grapevine and poses a serious threat to the viticulture industry. My research shows that treating grapevines with several different and diverse plant-associated bacteria triggers vines to become resistant to PD. I hypothesize that pre-treatment of vines with these bacteria stimulates the grape immune system to impart protection against encounters with the X. fastidiosa pathogen, somewhat analogous to how a vaccine would work in humans. This vaccination protection results in significantly less PD in vines. The purpose of this project is to test how long these bacteria can live in grapevines after being applied as treatments and to determine the timing of application that provides the best protection against PD. I will also conduct experiments to determine exactly how these bacteria lead to a vine's resistance to PD, and such information on the mode of action will facilitate registration and approval considerations by the Environmental Protection Agency.
Animal Health Component
20%
Research Effort Categories
Basic
75%
Applied
20%
Developmental
5%
Goals / Objectives
On a broad scale, this project focuses on the molecular basis of host immunity to xylem-dwelling bacterial plant pathogens. Specifically, I am focusing on the bacterium, Xylella fastidiosa. X. fastidiosa is the causal agent of several serious diseases that affect commodities central to California's agricultural economy, such as grape, almond, citrus and oleander. The project described here will drive the discovery and advance the knowledge of how X. fastidiosa circumvents the plant host immune system and colonizes and thrives in the xylem environment, particularly in the initial stages of infection. Unlocking the mechanisms of pathogenicity/virulence during the early phase of infection is fundamental to developing effective control measures against diseases caused by this significant plant pathogen. In order to test these control strategies and disseminate this information to the growers and to the public, my laboratory is in close communication with UCR Cooperative Extension faculty that directly communicate with farm advisors and growers. This avenue serves as a direct line to effectively distribute knowledge gained from this project, particularly involving Pierce' s disease of grapevine, caused by X. fastidiosa, currently a major problem in California.The Objectives of this proposal are : Objective 1. Develop sustainable biocontrol using California biocontrol isolates Objective 2. Field testing for efficacy and persistence of biocontrol applicationsObjective 3. Characterize the mode of action of biocontrol agentsThe short term goal of this project is to functionally explore critical virulence factors and host specificity determinants for X. fastidiosa. Importantly, this includes what sectors of the grapevine immune system are important in the interface between the bacterium and the plant host. The long term goal is to identify the tools necessary for X. fastidiosa to successfully invade their plant host, thereby determining the pertinent steps of infection that can be targeted for disease control.
Project Methods
Objective 1. Develop sustainable biocontrol using California biocontrol isolates. These experiments are designed to test the California isolates, P. viridiflava and A. xylosoxidans as singlet microbial applications and as part of a mixed two member consortia (both microbes) application that contains P. viridiflava and A. xylosoxidans. We will use a 7-day time frame as a scaffold to structure the experiments in this objective where we will include both pre- and post-pathogen treatments. We will also incorporate a foliar spray inoculation method (Baccari et al, 2019).1a. Greenhouse testing of pre-X. fastidiosa biological control treatment applications: For the needle inoculation experiments, we will test four different time points of biocontrol applications that will include four pre-treatments of the biocontrol. The pre-X. fastidiosa treatment time points will consist of inoculating the biocontrols 8 hours, 7 days, 14 days or 21 days prior to challenge with X. fastidiosa. These time points were chosen to encompass a time frame that mimics early exposure to X. fastidiosa. We will inoculate 1-year old greenhouse grown vines (15 per treatment) initially with 40µl of a 108cfu/ml suspension of live P. viridiflava, A. xylosoxidans cells or a mixed consortium of a 1:1 ratio of P. viridiflava and A. xylosoxidans in 1X phosphate buffered saline (1X PBS buffer). A replicate set of plants (15 per treatment) will be inoculated in the same manner with a mock biocontrol treatment of 40µl of 1X PBS buffer. All plants (the biocontrol or 1X PBS inoculated plants) will then be inoculated with 40µl of a 108cfu/ml suspension of live X. fastidiosa (Hill and Purcell, 1995) cells at either 8 hours, 7 days, 14 days or 21 days post-biocontrol application. For the foliar spray applications, microbial applications will be formulated as described in Baccari et al. (2019). The resulting microbial applications will be applied to all the leaves of the grapevines using a hand-held sprayer that delivers a fine mist until runoff. A replicate set of plants (15 per treatment) will be sprayed in the same manner with a mock biocontrol treatment of 1X PBS buffer containing 0.2% Breakthru. These will serve as the controls for the experiment. All plants (the biocontrol or 1X PBS- BreakThru sprayed plants) will then be needle-inoculated with 40µl of a 108cfu/ml suspension of live X. fastidiosa (Hill and Purcell, 1995) cells at either 8 hours, 7 days, 14 days or 21 days post-biocontrol foliar application.1b. Greenhouse testing of post-X. fastidiosa biological control treatment applications: Here we will explore if post-applications of P. viridiflava and A. xylosoxidans can provide effective biological control of PD after vines have already been exposed to X. fastidiosa. For the needle inoculation experiments, we will inoculate vines (15 per treatment) with 40µl of a 108cfu/ml suspension of live X. fastidiosa cells. A replicate set of plants (15 per treatment) will be inoculated with a mock pathogen treatment of 40µl of 1X PBS buffer only to serve as controls for the experiment. All plants (the X. fastidiosa or 1X PBS inoculated plants) will then be inoculated with 40µl of a 108cfu/ml suspension (prepared as described above for the needle inoculation pre-treatments) of a post-pathogen application of P. viridiflava, A. xylosoxidans cells or a mixed consortium of a 1:1 ratio of P. viridiflava and A. xylosoxidans in 1X PBS buffer 1 week, 4 or 8 weeks after initial inoculation with X. fastidiosa. For the foliar sprays, we will inoculate vines (15 per treatment) with 40µl of a 108cfu/ml suspension of live Xf cells. A replicate set of plants (15 per treatment) will be inoculated with a mock pathogen treatment of 40µl of 1X PBS buffer only to serve as controls for the experiment. All plants (the Xf or 1X PBS inoculated plants) will then receive a foliar spray until runoff with a post-pathogen application of P. viridiflava, A. xylosoxidans cells or a mixed consortium of a 1:1 ratio of P. viridiflava and A. xylosoxidans prepared as described above for the pre-treatment foliar sprays 1 week, 4 or 8 weeks after initial inoculation with X. fasitidiosa.Pierce's Disease severity assessments: All plants for both the pre- and post-pathogen biological control experiments (Objs. 1a and 1b) will be visually examined for PD symptom development over a period of twelve weeks on an arbitrary disease rating scale as previously described (Guilhabert and Kirkpatrick, 2005, Ingel et al., 2019, Deyett et al. 2019b). We will quantify X. fastidiosa populations in both local and systemic petioles when the positive control grapevines (inoculated with X. fastidiosa only and a 1X PBS mock biocontrol pre- or post- X. fastidiosa applications in Objs. 1a and 1b) as previously described (Deyett et al., 2019b).Objective 1c. Monitor the persistence of singlet and mixed biocontrols in grapevines. The purpose of this objective is to monitor persistence and population growth dynamics of the biological control bacteria in grapevines over time. For the needle inoculation and foliar treatments, we will monitor populations of the biological controls both locally (at the point of application) and systemically (10th node above the point of application) using the subset of time points from the pre- and post- X. fastidiosa exposure treatments that we ascertained were the most effective at controlling PD from Objs. 1a,b. We will design species specific quantitative (qPCR) primers using whole genome sequence analysis that will be obtained as described below. These species-specific primers, will allow us to quantify the absolute abundance of each of the three biocontrols in comparison to absolute abundance levels of X. fastidiosa using qPCR.Objective 2. Field testing for efficacy and persistence of biocontrol applications. We will establish the vineyard in year 1 of the project and plant vines in the spring of year 2. The experimental design will consist of a total of 100 vines in a replicated block design with 5 blocks of 20 plants and 5 vines per treatment within each block. The 4 treatments include a 1) 1X PBS control (mock inoculation), 2) P. viridiflava, 3) A. xylosoxidans, 4) and a mixture of the 2 biocontrols. In the late spring of 2021, we will inoculate all the shoots of all grapevines with each biological control treatment or mock treatment each year at bloom, when they reach 20-30cm in length using the needle inoculation approach (Hill and Purcell 1995; Clifford et al 2013). Vines will be subjected to the natural disease pressure occurring at AgOps due to the presence of GWSS in nearby citrus orchards and adjacent to urban citrus. We will rate the development of PD symptoms over the course of a growing season. We will rate individual vines using our PD severity scale (1= no to mild symptoms; 2 = intermediate symptoms; 3 = severe symptoms) designed for field assessments of PD as previously described (Deyett et al., 2019a). We will also quantify the absolute abundance of each biological control organism as well as X. fastidiosa using the qPCR techniques described in Obj. 1c. Statistical analyses will be conducted using R and as described in Deyett et al. (2019b).Objective 3: Characterize the mode of action of the biological control agents. These experiments will focus on transcriptional analysis of grapevines that receive the biocontrol applications that showed the greatest efficacy and persistence from Objs. 1 and 2. The pre- and post-treatments will be applied to grapevines as described in Obj. 1a, b. In all cases, one petiole and cognate stem tissue will be harvested from the point of inoculation (POI) tissue (local) and 10 nodes above the POI (systemic) for the RNA-Seq studies as previously described (Rapicavoli et al., 2018).