Non Technical Summary
A wide range of specialty crops including fruit crops (specifically blueberry), ornamentals, vegetable transplants, and row crops such as sugar beet are negatively affected by the fungal pathogen Botrytis cinerea. This pathogen is the most prevalent pathogen infecting and causing significant economical losses to the ornamental and vegetable transplant industry in California, and the blueberry and sugar beet production in Michigan. Disease management of this pathogen mainly relies on fungicide applications, presuming the development of fungicide resistance. The development of fungicide resistance in the aforementioned crops have not been addressed. This information is critical for an effective disease management program that eradicates the pathogen and reduces economical losses due to this disease. In this project we propose to characterize fungicide resistance of Botrytis populations associated to selected crops, so stake holders are aware of which chemistries are no longer effective. Furthermore, to detect fungicide resistant populations timely, we will validate and develop a rapid molecular tool that can detect these populations. To achieve this, we will sample at least five of the selected crops in California and Michigan to build a Botrytis collection. In the laboratory, we will conduct experiments to determine the fungicide resistance profile testing five different fungicides regularly used by producers and validate and develop molecular tools to yield faster results. With our project outcomes, we will create updated disease management strategies for growers, develop protocols and trainings for public and private diagnostics laboratories. Our overarching goal is to contribute to reduce fungicide application of ineffective chemistries, protecting agriculture workers and the environment. Promote the use of less hazardous chemistries, combined with cultural practices to improve disease management and secure sustainable food production in the U.S.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
216	2410	1160	100%

Knowledge Area
216 - Integrated Pest Management Systems;

Subject Of Investigation
2410 - Cross-commodity research--multiple crops;

Field Of Science
1160 - Pathology;

Goals / Objectives
B. cinerea is a fungal pathogen with broad host range, that causes negative economic impacts across the U.S. from specialty crops such as blueberry, ornamentals, vegetable transplants and row crops such as sugar beet. Botrytis blight is the most prevalent pathogen infecting ornamental and vegetable transplant crops in California. In Michigan, several Botrytis species infect blueberry blossoms and sugar beet storage piles. Disease management mainly relies on fungicide applications, presuming development of fungicide resistance. The development of fungicide resistance in the aforementioned crops has not been addressed. This information is critical for disease management. Providing stakeholders with information on effective modes of action to control this pathogen will help to mitigate disease and improve management. We propose to:1) Characterize fungicide resistance profiles of understudiedBotrytispopulations associated with select crops, 2) Validate transferability of molecular diagnostics tools into less knownBotrytispopulations,3) Improve and augment diagnostics toolsfor fungicide resistance inBotrytisand4) Create disease management extension materials, protocols and trainings to share with growers, the National Plant Diagnostics Network (NPDN) and private laboratories.In line with CPPM priorities, our goal is to "ensure sustainable food production and respond effectively to societal pest management challenges with comprehensive IPM approaches: economically viable, ecologically prudent, and safe for human health".Outcome: Contribute to reduce fungicide applications of ineffective chemistries, protecting agriculture workers and the environment. Promote use of less hazardous chemistries, combined with cultural practices to improve disease management. Project would provide rapid detection tools for fungicide resistance testing of Botrytis isolates infecting a wide range of crop in the U.S.

Project Methods
Characterize fungicide resistance profiles of understudied Botrytis populations associated to specialty crops.Sampling. We will sample in the first year of the project plants exhibiting Botrytis blight symptoms. In California, from October to April we will sample at five ornamental and vegetable transplant nurseries. In Michigan, , we will sample at five blueberry orchards and five sugar beet storage piles. In the laboratory, plants will be incubated in humid chambers and surface disinfected to isolate and single spore Botrytis isolates.Fungicide phenotyping. We will assess fungicide resistance profiles from Botrytis culture collections, implementing a high-throughput amended agar method, based on measuring (with a microscope) Botrytis isolates germ tube (Alzohairy et al., 2021; Naegele et al. 2022). Isolates will be determined fungicide resistance if germ tube length was >50%, and fungicide-sensitive if < 50% relative to the control growth (Weber & Hann, 2011). Fungicide concentrations tested will be based on discriminatory doses already reported in the literature: 1 and 50 mg/ liter for boscalid, fenhexamid, and fluopyram, 10 mg/liter for azoxystrobin and 0.1 and 10 mg/ l for pyraclostrobin. Controls will consist of non-amended agar. Experiments will be arranged as completely randomized block design and be replicated three times. Results will determine the percentage of isolates resistant to five active ingredients currently used by growers. To evaluate if there were differences in resistance to the five fungicides, we will implement a generalized linear mixed model, in which fungicide sensitivity will be set as a binomial response (0 sensitive, 1 resistant), fungicide treatment will be a fixed factor. To determine if host, year, and location of collection have an effect on fungicide resistance status (resistant, susceptible), generalized linear mixed model analyses will be carried, where isolates, host, year, and location will be considered fixed variables. The poison agar method will be used on a subset of isolates to confirm that fungicide resistant profiles match what is predicted through molecular assays.Evaluating Botrytis genetic diversity. DNA from the Botrytis culture collection will be extracted, and each isolate will be evaluated using a panel of genetic markers previously used to describe genetic diversity of Botrytis from small fruits in CA (DeLong et al., 2020; Naegele et al., 2022) as well as species-specific markers to confirm what Botrytis species are present on the crops evaluated (Fan et al., 2015). These data will provide an overview of the level of diversity, species variability, population structure related to host, and to check for associations between fungicide resistance and genetic diversity.Validate transferability of molecular diagnostics tools into less known Botrytis populations.The Miles lab has developed molecular diagnostics tools to detect fenhexamid (Alzohairy et al., 2021) and SDHI (Alzohairy et al., 2023) B. cinerea resistant populations associated with small fruits. This objective will evaluate if the markers already developed are effective at detecting B. cinerea resistant populations from California vegetable transplant and ornamental crops and to determine if these markers can detect resistant isolates from other Botrytis species associated to blueberry blossoms and sugar beet piles in Michigan.B. cinerea marker transferability based on host. Based on the fungicide resistant profiles identified in Objective 1, isolates will be grouped as resistant to: 1. Succinate dehydrogenase inhibitors-SDHIs (boscalid and fluopyram), 2. Hydroxyanilides (fenhexamid), and 3. Quinone outside inhibitors -QoI (azoxystrobin and pyraclostrobin). To confirm that resistant genotypes from California can be detected with the molecular probes already developed for B. cinerea from fruits, we will conduct qPCR assays on DNA from B. cinerea isolates grouped in each of the resistant groups identified in objective 1. For group 1 we will use the probes sdhB, sdhC, sdhD and for group 2 erg27. Isolates from group 3 will be address in objective 3 (development of a new detection tool). qPCRs for each isolate will be run in triplicates.B. cinerea transferability to other Botrytis species. As preliminary work indicates that there is more than one Botrytis species associated to blueberry blossoms and sugar beet piles, we intend to evaluate if the probes specifically developed to detect B. cinerea resistant isolates are equally effective to detect other Botrytis species. To do so, we will conduct qPCR assays on DNA from Botrytis sp. isolates grouped in each of the resistant groups identified in objective 1, as explained above.Improve and augment diagnostics tools for fungicide resistance in BotrytisThere are no molecular tools that detect Botrytis populations resistant to quinone outside inhibitors (QoI) (FRAC 11). For powdery mildew, the Miles' lab developed a molecular assay to detect a SNP associated with QoI resistance in Erysiphe necator (Miles et al., 2021). QoI resistance in many plant pathogens, such as E. necator, is due to the G143A mutation in the cytochrome b gene (cytb). However, QoI resistance in B. cinerea is due to G143A mutation as well as the removal of an intron between the 143rd and 144th codons in cytb (Banno et al., 2009). In this objective we will develop new molecular assays to detect QoI resistance in Botrytis populations. To do so, we will evaluate if the molecular assay developed to detect a SNP in E. necator can be transferable to Botrytis species, and develop a molecular assay able to detect any changes between the 143rd and 144th codons in cytb gene.Stakeholders and scientific community (diagnosticians) outreachStakeholders. During the project, we will communicate progress reports to participating producers every six months. Results will be communicated in virtual meetings with producers. We will participate in extension meetings targeting the specialty crops involved. In California, we will participate in the Vegetable Transplant Network Association (VTNA) meetings held in the fall of each year, as well as in the California Nursery Crops meeting geared to ornamental producers, and in County based extension meetings held in the Counties we will be sampling. In Michigan, we will present results in the extension meeting Great Lakes Expo, as well as in the Blueberry Commission annual meeting. To evaluate if the information provided is useful and can be implemented by producers, we will conduct surveys at the end of each extension meeting. Outreach materials presented at extension meetings and fact sheets will be shared online in the Del Castillo and Miles laboratories. We will produce two newsletter articles a year for publication in the University of California Nursery and Floriculture Alliance (UCNFA), Greenhouse Management Magazine and Michigan State University (MSU) Extension News.Scientific outreach. Based on objectives 2 and 3, we will develop protocols to use molecular markers to detect resistance in B. cinerea and other Botrytis species associated to a wide host range of crops including ornamentals, vegetable transplants (tomato, broccoli, cabbage, lettuce), blueberry plants and fruits, and sugar beet. These protocols will be shared and validated with diagnostics laboratories from the National Plant Diagnostics Network (NPDN) including the MSU Plant & Pest Diagnostics, Del Castillo Lab (Greenhouse and Nursery Pathology Lab) and the NPDN's Protocol and Validation Committee. In this project we will organize a workshop at Michigan State University, and another one at UC Davis, where more NPDN and private laboratories will be invited to participate. Detection protocols will be also shared to a wider scientific community by publishing them in the Phytofrontiers Journal. Results will also be presented at the annual American Phytopathological Society meeting.