Performing Department
(N/A)
Non Technical Summary
In light of expected increases in pest and pathogen pressure and decreases in pollinator availability under climate change, we must harness plants' natural abilities to manipulate biotic interactions to increase sustainability and maintain high yields in our crop systems. Across land plants, plants use volatile organic compounds (VOCs) as specialized multifunctional metabolites which act as direct signals of infection and damage, relaying complex cues across species boundaries, providing information to neighboring plants concerning pest and pathogen outbreaks. From VOC cues, neighboring plants enter a 'primed' state in which they can elicit a faster and more potent response when inevitably challenged with a pest or pathogen (e.g., systemic acquired resistance(SAR)). In developing plants with greater consistent production of defensive abilities, there are high long-term metabolic costs that lead to a reduction in yield. Efforts concerning SAR and induced defenses have inherent metabolic costs that are high in the short term and low over the plant's lifespan, decreasing negative effects on yield. VOC production may further reduce these yield diminishes as several studies indicate no significant reduction in yield from high VOC production. In addition to inducing SAR, VOCs can act directly by inhibiting pathogen growth or indirectly by altering the pathogen's transmission and infection success on new tissues. Thus, VOCs may be harnessed as an attractant or deterrent mechanism at a low metabolic cost, with further potential to induce SAR in neighboring plants, providing a holistic plant-derived biocide.While initiatives based on VOC use in agricultural practices are a promising avenue for sustainable agricultural research, few studies have assessed variation in VOC efficacy as a biocide against genetically diverse pathogen isolates on various host crops and further how to leverage VOC-induced SAR across diverse host crops. In this work, we leverage a genetically diverse association mapping population of Botrytis cinerea to explore the relationship between isolate-specific infection and the expression of VOC biosynthetic enzymes in several crop hosts. B. cinerea is a fungal pathogen that can infect leaves, flowers, and fruits, severely affecting yield across hundreds of food, cash, plantation, and horticulture crops globally at every step of production, storage, and distribution. B. cinerea's infection potential leads to a global control cost of over a billion USD annually. To develop novel ways to control B. cinerea, we will evaluate how general and species-specific VOCs affect B.cinerea virulence and transmission potential and describe the genetic associations of VOC resistance and susceptibility in B. cinerea. Finally, using the effective VOC dose necessary to alter B. cinerea virulence and transmission, we will evaluate the effectiveness of VOC fumigation in reducing B. cinerea infections and how well individual VOCs induce SAR in comparison to blends of VOC cues from infected plants of the same species. The results of this work will test the potential of VOCs as novel mediators of plant-pathogen interactions and develop fumigation guidance using plant-derived VOCs to control an important plant pathogenacross a significant proportion of crop species diversity.
Animal Health Component
0%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
This project addresses the AFRI area of 'Plant Health and Production and Plant Products' as fungal infections, specifically by the generalist pathogen B. cinerea, pose a significant challenge. B. cinerea can infect leaves, flowers, and fruits, severely affecting yield across hundreds of food, cash, plantation, and horticulture crops globally at every step of production, storage, and distribution. According to a 2012 study, B. cinerea was the second most important plant fungal pathogen with a global control cost of over $1,000,000,000 annually.Mycelial growth and spore viability are the primary targets of reducing disease severity and transmission in fungal infections, and because of the lack of large effect resistance genes in the host, this predominantly revolves around the identification of fungicides.However, B. cinerea has standing genetic variation enabling populations to adapt to fungicides rapidly. As such, there is a need to extend to find new sources of resistance. The projectaims to harness VOCs to develop novel and innovative technology for sustainable control of major fungal pathogens using existing omics data to identify candidate VOCs. Growing evidence shows VOCs can inhibit B. cinerea mycelial growth, spore viability and induce SAR. The half-life of many VOCs in the atmosphere ranges from a few hours to a day, decreasing the risk of off-target effects due to environmental persistence.The project's application of VOCs as natural plant-derived compounds with limited side effects has the potential to transform fumigation and plant breeding applications. This work has short-term goals of providing fungicides and SAR-inducing VOCs, which will furtherlong-term goals of breeding plants for specific VOCs, which confer resistance at limited to no negative effects on yield and developing complex VOC mixtures based on pathogen populations. By studying a wide range of eudicots, we will identify a set of VOCs that may enable the formation of blends that could counter the tendency for pathogens to develop resistance. In addition to using multiple eudicots to identify different VOCs, it will be necessary to conduct dose-response and standing genetic variation assays on these compounds. The proposed work will allow the identification of VOCs effective at both low doses and have ubiquitous effects across all the genotypes of a pathogen. The extensive collection of specific B. cinerea isolates maintained within the lab provides a unique opportunity to assay both of these parameters to optimize any downstream application.
Project Methods
?Objective 1:Assess variation in conserved and lineage-specific expression of VOC biosynthetic enzymes across a diverse set of eudicots infected with diverseB. cinereastrains.To survey relationships between potential VOC production during B. cinerea infection across the eudicots, the PD will explore associations between the expression of B. cinerea genes and plant VOC biosynthetic enzymes.From the resulting modeling efforts using a mixed linear model for genome-wide association, we expect, for each plant species, a list of VOC enzymes with expression patterns associated with the expression of specificB. cinereagenes, specificB. cinereaisolates, and/orB. cinereavirulence. We will compare results among plant species and select eight VOCs enzymes that have known or predicted products for use in Obj. 2. VOCs will consist of four with primarily positive coefficients ofB. cinereagene expression and four negative.Objective 2: Identify the genetic architecture of B. cinerea variation in susceptibility to general and lineage-specific VOCs.Certain fungi compensate for hazardous conditions by investing energy into higher spore production, potentially increasing the transmission potential at the cost of decreased mycelia growth. To assess post-treatment compensatory investment in spore production, we will determine spore count after five days. We will use sterile water to release spores from each plate. The resulting spore suspension will be filtered through glass wool to remove mycelial fragments and diluted counted with a hemocytometer. The same spores will be tested for viability using the protein stain sulforhodamine B (SRB) via a microplate reader.To test the potential for VOC fumigation to influence pathogen transmission by reducing germination success directly, we will measure the dose-response effects of VOCs on spores. Healthy spores will be isolated from all 96 B. cinerea isolates grown for five days in unadulterated Potato dextrose agar (PDA) in this experiment. We will add 100ul of different VOC concentrations to the spore suspensions based on the observed MIC of mycelia and reports from the literature to each spore suspension. Spore viability and germination rates will be measured as described above. The toxicology experiments serve as a significant labor-intensive portion of this proposed work. However, we have allocated sufficient funds to support undergraduate researchers (URs) to help. Obj. 2 will provide many direct, indirect, and function-valued phenotypes for a genome-wide association of direct and indirect effects of general and lineage-specific VOCs on B. cinerea pathogenicity and transmission potential.Objective 3: Validate the fumigation effects of select plant VOCs on reducing B. cinerea pathogenicity across eudicots.For 16 eudicot crop species, we will use the B. cinerea isolate with the highest virulence per species as a proxy for the most aggressive infection that would necessitate pesticide application to prevent yield loss. For each plant species x B. cinerea isolate combination, we will identify fumigation effects using the predetermined mycelial MIC of each plant compound from Obj. 2 in one preventative and reactionary treatment. A second reactionary treatment will be included based on the MIC of spore viability. Replication and controls for Obj. 3 will be the same as Obj. 2, and more isolates may be added depending on logistics. We will grow all 16 species for both experiments, collect leaves, and complete the experiment using a detached leaf assay. We will place detached leaves in individual Phyto-agar-lined polystyrene containers with holes covered in surgical tape to allow the gas exchange of O2 and CO2. In the preventative experiment, we will place a disc containing a VOC treatment in the chamber with the leaf for 24 hours, at which point the disc will be removed, and the leaf will be inoculated. The preventative treatment assesses the ability of the VOC to induce SAR and SAR's magnitude of resistance across species. In reactionary experiment A, the detached leaf will be inoculated with B. cinerea, and after 24 hours, the VOC treatment will be added for a subsequent 24 hours. Reactionary experiment A assesses the efficacy of VOC treatment on active lesions, as all species have been observed to produce visible lesions after 24 hours of inoculation. In reactionary experiment B, the detached leaf will be inoculated with B. cinerea and coincidently receive a VOC treatment for 24 hours based on the MIC of spore viability. Reactionary experiment B assesses the ability of VOC treatment to control initial spore dispersal onto new tissues. Lesions will be visualized every 12 hours for 72 hours post-inoculation and lesion development digitally measured as described above. Larger reductions in growth than expected based on observations from Obj. 2 and controls in Obj. 3 indicate synergistic effects and potentially the induction of SAR by specific VOCs. In contrast, antagonist effects are indicated by a more minor reduction in growth than expected, whereby the VOC increases the plant's susceptibility to B. cinerea.