Performing Department
(N/A)
Non Technical Summary
The goalof this research is to elucidate the overwinter recovery phenomenon of an important plant pathogen. Xylella fastidiosa infects hundreds of plant species globally, including a number of important crops such as grapevines, almonds, olives, and citrus. Overwinter recovery is currently the only known "cure" of Pierce's disease of grapevines: infected plants recover from infections and are symptom- and pathogen-free after exposure to cold winter temperatures. Despite its importance to PD ecology and management, very little is understood about the climate conditions that induce recovery or the biological mechanism that kills offX. fastidiosacells. To clarify the cold exposure necessary to induce recovery, we have begun field trials that will be incorporated with past experimental results into a new recovery climate model. Then, we will determine the seasonal timing of recovery by comparing recovery across groups of infected stem segments excised at different stages of dormancy. With this timing information, we will be able to analyse the transcriptomes of grapevines from cultivars with known low and high recovery rates to determine gene expression patterns associated with recovery.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
This research project investigates the climate factors and biological changes associated with overwinter host recovery from an important plant pathogen,Xylella fastidiosa, supporting AFRI priority areaPHPPP.X. fastidiosacauses a number of crop diseases including Pierce's Disease (PD) of grapevine. The range of PD is limited by cold winter temperatures; exposure to cold winters eliminatesX. fastidiosafrom grapevines in a phenomenon known as winter recovery. Despite its importance to PD ecology and management, very little is understood about the climate conditions that induce recovery or the biological mechanism that kills offX. fastidiosacells. To clarify the cold exposure necessary to induce recovery, we propose field trials that will be incorporated with past experimental results into a new recovery climate model. Then, we will determine the seasonal timing of recovery by comparing recovery across groups of infected stem segments excised at different stages of dormancy. With this timing information, we will be able to sample recovering plants for RNAseq at the proper stage of dormancy: grapevines from cultivars with known low and high recovery rates will be sampled to determine gene expression patterns associated with recovery. Under direct mentorship from PM Almeida, PD Brown will complete the proposed work with the assistance of undergraduate research assistants, supporting the AFRI EWD goals of advancing science and creating research experiences and applied learning opportunities for undergraduates.
Project Methods
1.Overwinter recovery climate model (2022 - 2026): I will combine my own field-collected recovery data with published recovery experimental results to create a new overwinter recovery climate model. Recovery rates will be modeled in a generalized linear model as a function of cold exposure, cultivar, and other biological parameters. To generate more recovery data, I have organized field trials in three locations with differing winter temperatures: Berkeley, Hopland, and Blodgett. I inoculated 150 Cabernet Sauvignon grapevines in Spring 2022 withX. fastidiosaor saline buffer for negative controls and then tested for positivity by qPCR 12 weeks post-inoculation. Plants were moved to field sites in early October and returned to the UC Berkeley greenhouses in four cohorts six weeks apart, the weeks of Dec. 17th, Jan. 28th, Mar. 17th, and May 1st. Air and soil temperatures were monitored through the winter. Upon return, plants were grown in the greenhouse until symptoms appeared, or for at least 20 weeks, and then tested forX. fastidiosaby qPCR. DNA extraction and qPCR methods are standard protocols in our group (Kahnet al.2023, Sicardet al.2020). The same experiment is being repeated this winter, and the plants were again taken to each field site in early October 2023. In addition to my field data, I will include recovery data from published works and new recent unrelated experiments from our group to build a more robust model. Location and date information from publications will be used to extract past hourly temperature data from the NOAA's Integrated Surface Database.2.Timing of pathogendeath during recovery (2023-2026): Grapevines undergo substantial physiological changes moving into and out of dormancy, and recovery could be associated with a number of these dormancy or cold stress-induced changes. To determine when the elimination ofX. fastidiosacells occurs, individual canes from infected plants will be excised and rooted at different stages of dormancy and tested for recovery. Sixty Cabernet Sauvignon grapevines with four main canes will be needle-inoculated withX. fastidiosacells on each cane, with 5 plants inoculated with saline buffer only as negative controls. Plants will be taken to the field and kept in tents for the winter. Canes will be removed and rooted sequentially: one cane (and fall cuttings) rooted in mid-March, the second following spring sap bleeding but prior to bud break (early April), and the third after bud break (mid-April). Cane 4 will stay on the plant as an 'expected recovery' control. Rooted cuttings will be grown in the greenhouse for 20 weeks prior toX. fastidiosadetection by qPCR to determine recovery status. Recovery among cutting groups will be modeled as a function of removal date. Differences in recovery rates among the cutting groups will clarifywhich physiological change during dormancy is associated with recovery.3.Recovery transcriptome comparison across cultivars (2023 - 2027): To generate a list of candidate genes that may be involved in the recovery process, I will compare the transcriptomes of infected and recovered (R), still infected year 2 (not recovered) (I), and negative control (C) plants from cultivars with low and high recovery rates. Tinta Amarella, Mencia, and Albarino cultivars will be used for the low-recovery cultivars (16-26% recovery) and Tannat, Teroldego, and Tinta Francisca will be used as the high-recovery cultivars (60-80% recovery) (Almeida, unpublished data).Cane segments will be flash-frozen for RNAseq in the field at three time points during dormancy and three time points as grapevines leave dormancy and break bud. Samples will be stored and the samples used for sequencing will be determined based on the recovery timing results from Experiment 2.Biological replicates of 2 pooled cane samples will be formed after plants' recovery status is determined. A standard computational pipeline will be used to trim, map, and count reads and then test for differential gene expression (Castro et al 2023, Grimplet 2009). Then, DEG's between R, I, and C plants within each cultivar and between cultivars (within R, I and C) will be compared.