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Research Project: Resilient, Sustainable Production Strategies for Low-Input Environments

Location: Crops Pathology and Genetics Research

2023 Annual Report

Objectives
Objective 1: Develop crop production strategies that integrate water and nutrient input management and the environment for healthy, sustainable vineyards. [NP 305, Component 1, Problem Statement 1B] • Subobjective 1.A. Characterize varied responses of grapevine genotypes to drought in order to improve detection and interpretation of water stress signals for local and remote proximal sensors and to develop precision irrigation techniques tailored to genotype- specific root responses. • Subobjective 1.B. Determine the molecular basis associated with the differential responses to drought stress among grapevine genotypes. • Subobjective 1.C. Identifying threshholds for organoleptic volatile phenols and their glycosidically-bound derivatives in wine grape varieties exposed to smoke taint across different growing regions. Expected benefits include standardized chemical analyses of smoke taint compounds in exposed and unexposed vineyards for the wine varietals growing in CA, OR, and WA with the goal of identifying and quantifying genotype-specific environmental threshold levels. Objective 2: Analyze the interaction of soil health and vineyard floor management for the enhancement of vine and fruit quality. [NP 305, Component 1, Problem Statement 1B] • Subobjective 2.A. Determine relationships among soil and grape must microbiomes and their structure in the wine grape production system. Objective 3: Develop improved strategies for controlling grapevine disease using preventative and post-infection management strategies. [NP 305, Component 1, Problem Statement 1B] • Subobjective 3.A. Characterize the role of wood-decay fungi in trunk diseases, to develop post-infection practices that return vines to productivity. • Subobjective 3.B. Identify when trunk pathogens sporulate and the infection courts by which they infect, to develop preventative practices that protect susceptible host tissues.

Approach
The approaches for each objective range from experimentation under controlled conditions in the greenhouse to experimentation under natural field conditions, with commercial vineyards making up the majority of field study sites. Prior to hypothesis testing, some level of methods development (e.g., imaging water flowing through the vessels of living plants, pathogen detection from environmental samples of microscopic spores) is required for each objective, in part because grape is not a model study system. For objective 1, parallel sets of physiological experiments are focused on measuring anatomical, physiological, and transcriptional responses of leaves and fine roots, under normal levels of irrigation versus under drought stress. Whole plants of Vitis vinifera wine-grape varieties (Cabernet-Sauvignon, Chardonnay) and rootstocks with differential drought tolerance will be examined by X-ray microCT, followed by sections of leaves and roots examined by transmittance electron microscopy and Laser Capture Microdissection. RNA-seq techniques will then be used to seek out transcriptional differences at a molecular scale. For Sub-Objective 1.C.-The approach will combine field experimentation in the vineyard, winemaking and distilling processes in the experimental winery, and laboratory analyses of smoke-related compounds using, for e.g., gas chromatography/mass spectrometry (GC/MS). Compositional changes in the fruit of different cultivars, with exposure to smoke, will be characterized and quantified. Smoke-related compounds in wines made from the smoke-exposed fruit will also be characterized and quantified. Grape and wine quality analytical methods will be developed to detect key smoke-related compounds in the fruit and the wine, and acceptable limits will be established. Further, endproduct processing methods will be developed to help mitigate such compounds. For objective 2, the interaction of host genotype by environment (soil and climate, specifically) by management is examined. High-throughput amplicon sequencing of soil fungi and bacterial communities will be used to compare those of vine rows under different floor-management practices. Samples from the must will evaluate whether vineyard floor management practices impact the microbiome during fermentation. Diffuse reflectance Fourier transformed mid-infrared spectroscopy (DRIFTS) will be used to characterize changes in SOM chemical composition in particulate organic matter and other soil C fractions. For objective 3, inoculations of potted plants in the greenhouse will be used to test hypotheses at the plant scale about which combinations of pathogens and sequences of infection cause disease symptoms, and also about how differential tissue susceptibility affects whether an infection spreads throughout an individual plant. At the vineyard scale, spore trapping in diseased vineyards and evaluations of pruning-wound susceptibility will be used to determine when grapevines are at greatest risk of infection.

Progress Report
This report documents progress for project 2032-21220-008-000D "Resilient, Sustainable Production Strategies for Low-Input Environments", which started in March 2020 and continues research from 2032-21220-007-000D, "Sustainable Vineyard Production Systems". The project objectives focus on grapes and, to a limited extent, two other woody perennial crops in California (walnut, almond). The deliverables include labor-saving practices for preventing grapevine trunk diseases and technology to give growers precise measurements of water-use in real-time. Scientific advances are also an important outcome of the project, with development of novel study tools for researchers to examine the physiological bases of drought tolerance in grape rootstocks and the diversity of microbes originating in the vineyard that contribute to the fermentation of wine. Some of the work also addresses the complex impacts of global climate change on California agriculture, including wildfires, drought, changing rainfall patterns, less irrigation water, lower quality water, and heatwaves. In support of Sub-objective 1A, ARS researchers in Davis, California, in collaboration colleagues from other ARS locations, universities, and industry partners, continued efforts to validate and implement crop water-use and stress estimates, with remote-sensing tools. These long-term projects are known as ‘Grape Remote-sensing Atmospheric Profile and Evapotranspiration eXperiment (GRAPEX)’ and ‘Tree crop Remote Sensing of Evapotranspiration eXperiment (T-REX)’. Towers and sensor arrays were built in commercial vineyards and orchards, to measure water and carbon dioxide (CO2) exchange. These ground-based data, along with physiological, micrometeorological, and biophysical data, were analyzed and compared with controlled environment measurements to identify indicators specific to water and heat stress for each crop. New study sites were added to the projects to compare traditional irrigation versus regenerative practices. In support of Sub-objective 1B, ARS researchers in Davis, California, successfully developed a method for genomic DNA and total Ribonucleic acid (RNA) extraction from root tissues of six grapevine rootstocks, including three drought-sensitive, (namely Riparia Gloire, 101-14 Mgt Millardet, 420 A Mgt), and three drought-resistant genotypes (110R, 140 Ruggeri, 1103 Paulsen). Extracted DNA and RNAs have been subject to for gene-expression analysis on drought stress resistance by different rootstock genotypes. Experiments confirmed that the drought-resistant genotypes may have deep root system architecture, compared to the drought-sensitive rootstocks under drought conditions. Genes related to the regulation of root system architecture and possible enhancement of drought resistance have been isolated from grapevine rootstocks. In addition, some regulatory genes such as transcription factors (master switcher of gene expression) have been isolated from extremely drought resistant resurrection plant Myrothamnus flabellifolia and functionally characterized in a model plant Arabidopsis. Orthologous genes of these transcription factors have been identified from grapevine rootstocks and could serve as molecular markers for screening drought stress resistance in different grape genotypes. Toward this goal, a newly developed miniature CRISPR-associated protein has been modified and constructed for improving the efficiency of functional gene analysis in grapevines. In support of Sub-objective 1C, ARS researchers in Davis, California, investigated the efficacy of utilizing nanoparticle technology to detect volatile phenols in grapes. In collaboration with university researchers, cerium oxide nanoparticles (nanoceria) have been identified to successfully react with all the volatile phenols currently associated with the negative sensory characteristic termed as ‘smoke taint’ in wines. The nanoceria undergo a redox reaction specifically with phenolic compounds, generating a unique optical signal, the color and intensity of which represents the composition and concentration of the phenolic compound. The goal of this research is to provide an inexpensive, quick, and simple tool for grape growers/wine makers to measure volatile phenols in grapes before and after a smoke event. By comparing results to current analytical methodologies, this detection tool will promote the development of threshold values of smoke-derived compounds in grapes as well as baseline values across genotypic and environmental factors. Lastly, ARS researchers have successfully constructed a smoke chamber that can reproducibly expose grapevines to smoke. Smoke will be generated from various fuel types across California, Oregon, and Washington to determine how fuel type will impact the uptake and fate of important organoleptic compounds. In support of Sub-objective 2A, ARS researchers in Davis, California, examined the spatial distribution of soil microbial communities in 15 Vitis vinifera ‘Pinot noir’ vineyards, spanning southern Oregon to southern California. A goal of this research is to identify a soil microbial community that characterizes different aspects of both the soil and the quality of the wine from grapes at each vineyard. Microbial samples were collected from soil in the vine rows (in the irrigated ‘drip zone’ and non-irrigated dry zone) and from the alleys in between the rows. The roles of management histories and climatic conditions help us evaluate the interactive effects of climate, vineyard management and soil microbes on the chemical, biological and physical attributes of the soil. Ninety-six soil microbe metagenomes were sequenced. Analysis is underway to reveal how desirable soil microbe functions are associated with vineyard management practices. The process of creating assemblies of the metagenomes and additional data analysis for effects of climate, vineyard management and soil attributes on microbial function were initiated. For Objective 3, ARS researchers in Davis, California, identified different hyperspectral reflectance patterns among leaves of plants with grapevine trunk diseases Botryosphaeria dieback (Neofusicoccum parvum) and Esca (Phaeomoniella chlamydospora and Tropicoporus texanus). Trunk diseases go undetected because the causal fungi have long incubation periods in the permanent, woody structure of the vine, during which time leaves appear healthy. As a new approach for disease detection, we used a hyperspectral camera to take images of leaves (at wavelengths of 400 to 1,000 nanometers (nm)), on potted plants in the greenhouse, the woody stems of which we inoculated with each of the three fungi. We tested the hypothesis that the biochemical, anatomical, and/or transcriptomic changes in the leaves, which are indirect responses to the infection in the stem, are associated with changes in hyperspectral reflectance. Different hyperspectral responses among all inoculated plants were detected at two months post-inoculation, compared to non-inoculated plants. Reflectance patterns that were unique to the different fungi may be due to the different types of damage they cause to woody cells (enzymatic degradation, production of phytotoxic chemicals, physical blockage of the xylem by the host).

Accomplishments
1. Identifying ideal sampling times to track grapevine water stress with hyperspectral tools. Grape growers need new methods to accurately track vine-water stress, in order to optimize deficit-irrigation strategies that maximize fruit quality and yield. Current methods are labor intensive and typically assess only a few vines in a vineyard. Indices from hyperspectral reflectance measurements (HI) could inform management over larger acreage, while still providing resolution for individual vines, but require refinement to be a viable tool in commercial vineyards. ARS researchers in Davis, California, and other locations in collaboration with researchers from University of California, Davis, and industry, found that vine water-stress was best detected during morning and late afternoon. This suggests that satellite and drone overpasses, typically during midday, may miss the ideal times to detect water stress. Industry partners have altered measurement timings in response to our new results.

2. Transcription factors play pivotal roles for enhancement of drought stress resistance. To survive changing environmental conditions, plants have evolved sophisticated mechanisms to regulate their responses to drought stress. Gene expression master regulators, such as transcription factors, play pivotal roles in plant resistance to drought stress and other adverse conditions. An ARS researcher in Davis, California, in collaboration with researchers from Sichuan Agricultural University, Sichuan, China, found that over-expression of drought stress-inducible transcription factors, which they isolated from an extremely drought and salt resistant Myrothamnus flabellifoli. The validity of these drought tolerant transcriptome factors was verified using Arabidopsis as the model plant. These results will help identify similar genes from grapevine rootstocks, to help develop molecular markers to screen for drought resistance.

3. New fertilization, irrigation, and compost methods reduce greenhouse gas emissions and maintain crop yields. ARS researchers in Davis, California, and researchers at the University of California, Davis, completed a global structured review of the effects of cover crops on soil biological, physical, and chemical attributes, also known as ‘soil-health indicators’. Given the priority of both USDA and various state of California agencies to support Climate Smart Agriculture, we examined the benefits of cover crops for soil health, crop productivity, economic, and ecological factors. Cover crops were associated with higher levels of soil organic carbon– a prized soil-health indicator – and soil texture class influenced the magnitude/frequency of this and other soil-health indicators. This work demonstrates that, in nearly all soil texture classes, cover crops tend to be associated with the majority of soil health indicators, regardless of cover crop type, cropping system, and region of the world. However, reducing soil compaction was not a consistent effect of cover crops across all soil textures, suggesting other mitigating practices for soil compaction are needed in combination with cover crops. Our findings suggest that cover crops improve and/or sustain soil-health indicators, which may protect soils for crop production and promote Climate Smart Agriculture.

4. Confirmed efficacy of trunk renewal for post-infection treatment of the grapevine trunk disease Esca. ARS researchers in Davis, California, confirmed that a technique known as ‘trunk renewal’ was successful for treating ‘Sauvignon blanc’ vines with Esca, in that only one of 97 retrained vines developed symptoms five years later. The approach was to cut away the canopy of symptomatic vines (and the chronic wood infections caused by the Esca pathogens) and then retrain a new canopy from a shoot that grew from the base of the trunk, which was presumably healthy. Fruit from asymptomatic-retrained vines was not identical in chemical composition to that of healthy vines that were not retrained, likely because their canopies were 15 years younger. In contrast, symptomatic fruit from non-retrained vines, which is visibly covered in black spots (a host response to infection), was characterized by volatile compounds with green, grassy, or herbal aromas, and high concentrations of flavanol compounds, which suggest a delay in ripening or induction of a host-chemical response to infection. Because Esca infections are localized in the wood, symptomatic vines also have asymptomatic shoots with asymptomatic fruit, which was chemically similar to healthy fruit on healthy vines. This is good because it suggests that thinning the symptomatic fruit before harvest, but retaining the asymptomatic fruit, can contribute to consistent fruit quality throughout the vineyard.

5. Inexpensive, quick, and simple tool for detecting volatile phenols in grapes. Wildfire smoke impacts the chemical composition of grapes, which in turn impacts the sensory characteristics of wine. Methods to analyze free and bound volatile phenols in grapes, in order to make a decision about if or when to harvest a smoke-exposed vineyard, are time intensive and expensive. An ARS researcher in Davis, California, in collaboration with researchers from University of California, Davis, and Clarkson University, investigated the efficacy of cerium oxide nanoparticles (nanoceria) as an alternative method for measuring volatile phenols in grapes. Nanoceria react with phenolic compounds, generating a color change that represents the composition and concentration of specific phenolic compounds. Nanoceria have been shown to successfully react with all volatile phenols currently associated with smoke-exposed fruit. This research will offer an inexpensive and portable detection tool for grape growers and wine makers to analyze grape samples before and after a smoke event.

U.S. DEPARTMENT OF AGRICULTURE

Crops Pathology and Genetics Research: Davis, CA