Ralph Dean

We seek to understand the genetic basis of non-race-specific resistance to fusiform rust disease caused by Cronartium quercuum f. sp. fusiforme (Cqf) in Pinus taeda, an economically critical pine species. In previous research, our group mapped two major resistance QTL with high genetic resolution in the genome of a P. taeda resistance donor. In a parallel bulked-segregant RNAseq experiment, we identified candidate resistance genes with SNP highly associated with resistance to Cqf. These genes were part of the nucleotide-binding leucine-rich repeat. Here, we will leverage our newly gained knowledge of the genetics of host resistance to generate a pine population segregating for the same two resistance QTL. To understand the genetics of avirulence in the pathogen, the pine population will then be challenged with a diverse basidiospore mixture of Cqf in an artificial inoculation experiment. Following symptom development, fungal strains capable of growing on each of four host resistance genotypes will be sampled directly from diseased tissue and sequenced. Following SNP discovery, the fungal genome will be scanned for the presence of selective sweeps that would indicate proximity to genes selected for virulence against one or the other QTL, such as effectors.

The main goal of this proposal is to improve the resilience and sustainability of U.S. tomato farms through the identification and utilization of novel genetic determinants that confer resistance to Verticillium Wilt incited by non-race 1 isolates of Verticillium dahlia (Vdn). The pathogen can live for 20 years or more in the soil and rotation is not feasible for control of the disease because other crops are also hosts. We propose that the critical solution to mitigate VW damage may be host resistance and the most feasible and economic control is the use of verticillium-tolerant tomato cultivars. There are numerous resistant cultivars effective against race 1; however, no source of resistance to non-race 1 isolates is commercially available. Multiple on-farm trials in Vdn infested grower fields enabled us to successfully identify novel sources of Vdn resistant tomato germplasm. The following objectives express a two-tiered approach to achieve and deliver the proposed outcomes to the stakeholders. a) identify and fine-map Vdn resistant locus (loci) in three NCSU tomato breeding lines. b) Utilize Vdn resistance to develop new tomato hybrids stacked with additional disease resistances The addition of Vdn resistance will broaden the disease-resistant spectrum of the elite NCSU tomato cultivars minimizing economic risk for growers in the U.S. and worldwide. Improved cultivars will be selected in a farmers' participatory selection process and released for commercial use. Findings from this research will be published in relevant journals and may also provide a genetic tool to combat VW in other economically important crops.

Planned Activity, Objectives, and Methods Most plant pathogens produce effectors, proteins that are introduced into the plant cell to facilitate the pathogenesis process. Plants carry nucleotide-binding leucine rich repeat (NLR) proteins which, upon recognition of specific effectors, trigger a defense response called effector-triggered immunity, usually including a hypersensitive response (HR), a rapid cell death at the point of infection. The maize Rp1-D gene encodes an NLR resistance protein that confers resistance to common rust disease conferred by the fungus Puccinia sorghi. We previously used genetics and molecular biological approaches to identify several host proteins responsible for controlling the activity of Rp1-D21, an auto-active derivative of Rp1- D. In this project, we will use complementary approaches, including bioinformatics, functional genomics, cell biology, and spectroscopy techniques, to identify and analyze the molecular components of the interaction deriving from P. sorghi as well as other important host-derived components. We will identify effectors associated with the control of host cell death and suppression of the host defense response. We will define how these effectors influence important physiological changes in host cells, such as changes in pH, reactive oxygen species production, and calcium flux, and will characterize their subcellular localizations. We will also examine the maize HR with respect to these same physiological changes and the organelle dynamics in the cell. We will examine in particular the formation of stromules, narrow stromafilled tubes that extend from plastids, often connected to other subcellular compartments, including the nucleus, that are believed to facilitate the exchange of signaling components between the plastids and nucleus during HR. Finally, we will characterize the physical interactions of all the host- and pathogenderived components that interact with Rp1-D and are likely to constitute components of the Rp1-D signaling complex, the ???????????????resistosome??????????????????. The proposal explicitly addresses the focus of the PBI program to ????????????????support ??????????????? fundamental ??????????????? research on the mechanisms and principles that mediate the interaction of plants with their biotic partners???????????????. Intellectual merit Despite significant progress, there remains much to learn about NLR-mediated resistance. This is particularly true in monocots. This project employs state-of-the-art biochemical and cell biology techniques to augment and extend our understanding of the control of the defense response mediated by Rp1-D focusing on pathogen derived components. This will result in an understanding of the control of the NLRmediated response that is unique in maize and among the most detailed in any plant species. Broader impacts The broader impacts of this proposal are twofold. The proposed research will elucidate a pivotal defense mechanism in maize which is both a model species for plant quantitative genetics and the number one crop in the U.S. Our results will be of direct relevance to efforts to genetically improve this important crop. Since the HR is a general defense response found in all multicellular plants, our findings will be relevant to improving other important crop species, particularly other grasses. The second impact is through the planned outreach activities with the NCSU Science House. All outreach activities will educate the public on genetics, plant breeding, biotechnology, and associated societal implications. They build on existing successful programs that have developed several instructional modules for teachers and students.

The demand for organic tomatoes in the Southeast is high, but production is limited due to lack of regionally adapted high yielding varieties. Organic growers have requested research disease management practices including improved varieties with superior fruit quality so that they can take advantage of the ever increasing market demand. Our long-term goal is to develop sustainable approach of disease management for organic production by integrating organic disease management system and resistance breeding well-adapted to organically growing conditions in the Southeast. The proposed project will benefit farmers in the U.S. in general, and in the Southeast in particular, who need high-value crops that can be grown on small acreages. This proposal was developed through direct interaction with the organic growers. The objectives of this proposal are: 1: Determine genotypic differences for foliar and soil-borne fungal disease resistance, and fruit quality in heirloom tomato varieties grown under organic conditions 2: Production system: grafting for the management of major diseases and enhancing fruit quality under organic transition conditions 3: Identification of suitable tomato varieties for organic production through participatory variety selection for high yield, disease resistance, and fruit quality 4: Disseminate knowledge gained on tomato varieties and production systems grown in organic conditions to farmers, extension agents, industry, and the general public The proposed research is relevant to the Organic Transition program and will facilitate the development of organic agriculture production, biodiversity, and soil health by minimizing the pressure of soil-borne pathogens, and integrating novel technology into organic system.

The phasing out of methyl-bromide has left many agricultural systems vulnerable to soil-borne plant pests. Bacterial wilt and Verticillium wilt are existential threats to many crop systems. Addressing these threats requires a multifaceted approach. Understanding how to restore tired soils will involve researching how plant genetics, microbiomes, and grower inputs are interconnected. NC State has a large number of proprietary sequenced tomato lines and access to commercial lines with diverse genetic backgrounds known to improve soil health. To better understand how to utilize this rich source of genetics, we propose to study the impacts of these tomato lines on soil health in disease infested fields. Several locations with bacterial wilt and Verticillium wilt infested soils are available to us. In cooperation with growers, we propose to study the impact of this diverse tomato collection on soil health under various conditions [fumigated, non-fumigated, and anaerobic soil disinfestation (ASD)]. The utilization of these tomato lines will also be studied in grafted and non-grafted systems to allow growers the usage of cultivars with their preferred characteristics. Soil health will be assessed by measuring plant-available nutrient content, microbiome diversity, disease severity, and subsequent increases in yields.

Plants recognize pathogens either through detecting molecules that are generally associated with microbes--called Pathogen-associated molecular patterns or PAMPs--or by recognizing events that lead to multi-faceted defense responses. We are interested in dissecting the machinery controlling these responses. In particular we are interested in understanding how the plant suppresses the effector triggered response so that it does not cause undue harm to the plant. We have identified a process mediated by protein degradation that appears to be a general mechanism for degrading the proteins that recognize effectors and trigger the defense response. We are also interested in identifying the receptors that recognize PAMPs in maize.

Botrytis cinerea, the causal agent of grey mold, is one of the most important pathogens of strawberry and many other crops. It is the target of fungicide applications to limit pre- and post-harvest losses. Indeed, in the SE USA, strawberries are typically sprayed weekly during the growing season. However, the pathogen adapts under high fungicide use patterns and through mutations, acquires resistance to an array of fungicides and multi-fungicide resistant isolates have emerged. The objective of this work is to assess the efficacy of novel RNAi technologies to suppress Botrytis, with emphasis on Botrytis strains that have resistance to one or more fungicides. Through microplate assays with liquid media, we will determine the efficacy of RNAi to limit growth and development of Botrytis strains with no or to single chemical resistance in the presence or absence of the most important fungicides commercially used to control the pathogen. We will also test effectiveness against its close relative, Sclerotinia sclerotiorum, the causal agent of white mold.

It is well known that during times of stress, possibly due to herbivory or pathogen infection, plants emit a wide range of volatile organic compounds (VOC). These VOCs serve to activate the plant defenses, attract beneficial insects, and warn adjacent plants of an impending attack. There are no sensors available today that can detect the volatomic ???????????????fingerprint?????????????????? in the field or storage conditions with sufficient sensitivity and selectivity to help with early diagnosis of plant disease. The innovation proposed in this project is 1) identify the volatomic fingerprints of specific plant diseases for model crops through lab analysis of collected samples from plants in simulated field conditions; 2) develop the low-power, field-deployable, connected electronic nose (e-nose) systems with high sensitivity and selectivity, optimized to detect the volatomic fingerprints identified through lab analysis.

Tomato growers in the southeastern USA (SEUS) continue to face many challenges and opportunities to optimize and advance integrated management of soilborne diseases and weeds without methyl bromide as a pre-plant soil fumigant. Through a long-term inter-disciplinary, multi-state and stakeholder-driven program, all our growers have transitioned to alternative fumigants or farming systems. Through this participatory model, our long-term goal is to foster a vegetable industry that is competitive, sustainable, and conducive to SEUS farm viability. In practical terms, our goal is to develop an arsenal of integrated approaches to manage soilborne pests for a diverse range of vegetable farmers who farm a few to hundreds of acres. To that extent, we seek to optimize current farming methods for short to mid-term benefits and we also seek to advance novel approaches that have mid- to long-term outcomes. We propose to 1: Optimize weed and soilborne disease management systems with current and emerging tools. 2: Develop and advance alternative and novel chemistry to manage weeds and diseases; 3: Perform economic analysis of alternative farming practices and systems; and 4: Translate research outcomes and grower experiences through extension and outreach efforts into information and products useful to growers and other stakeholders. Outcomes of this proposal will advance the science and practice of weed science, plant pathology, horticultural science, farming systems research and agricultural economics and enable more growers to successfully manage soilborne pests and improve crop productivity using fumigant- and biologically-based systems.

Through the collaboration of Engineers and Plant Pathologists at North Carolina State University, a new sensor technology based on capacitive micromachined ultrasonic transducers (CMUT) has been designed, developed, and fabricated for the detection of plant volatile organic compounds. These sensors will be used to facilitate early detection of fungal infection of plants for more targeted intervention of crop management strategies. Plants emit a unique volatile bouquet under different stresses, that acts like a fingerprint. Such volatiles can be identified and then used to modify the CMUT sensor to detect distinctive plant stressors, such as fungal infection. For this study, the first project objective will be to detect, and discriminate VOC profiles produced by economically significant crop plants infected with different fungal pathogens. that are responsible for causing several major crop diseases. These fungi will be used to infect plants for volatile collection and sensor testing. Plants will be infected with each specific fungal pathogen and placed into a PETG volatile chamber. Over a period of several days, the headspace of the infected plants will be pumped out of the chamber and periodically run over the sensor along with clean air to determine if the sensor can detect the volatiles from the infected plants. Up to 8 different polymer coated gravimetric sensors will be developed and tested. After the physical sensor systems are fabricated, combinations of different functionalization layers will be tested, and data will be analyzed to find the set of polymers for maximizing sensitivity and selectivity to target analytes. This data will be used to form the fingerprint volatile emission for each plant stress, specifically for each fungal pathogen or general fungal infection. We expect fungal infected plants to produced detectable volatiles and will be unique to each pathogen. So far in our studies, we have been able to detect and differentiate plant volatiles from several crops and pathogens. The second project objective of this study is to identify and characterize volatiles produced during infection and could be likely candidates for triggering the sensor using GC-MS analysis. To achieve this objective headspace from fungal pathogen infected and water treated control plants will be collected from a variety of timepoints and compared. Volatiles present in only the infected plant headspace will be considered candidates. The most likely candidates include volatiles from the terpenoid and lipoxygenase pathway. The third project objective of this study is to test volatile compounds identified by the GC-MS in their pure and singular form against the CMUT sensor to verify which volatile triggers a response from the sensor. The desirable outcome for this objective would be that the specific volatiles identified in the second objective produced the same pattern as seen in the first objective. Once a pattern has been established for each fungal pathogen, the sensor can be further developed for testing in field settings.