Source: UNIVERSITY OF GEORGIA submitted to NRP
CONTROL ALT DELETE: ENHANCING RESILIENCY OF BROCCOLI PRODUCTION BY MITIGATING ALTERNARIA LEAF BLIGHT AND HEAD ROT IN THE EASTERN UNITED STATES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1023417
Grant No.
2020-51181-32062
Cumulative Award Amt.
$2,731,129.00
Proposal No.
2020-02604
Multistate No.
(N/A)
Project Start Date
Sep 1, 2020
Project End Date
Aug 31, 2025
Grant Year
2020
Program Code
[SCRI]- Specialty Crop Research Initiative
Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
(N/A)
Non Technical Summary
Alternaria blight and head rot (ABHR) in broccoli can severely impact yields and increase production costs in the absence of effective control options and is a threat to the long-term viability and profitability of the industry. A coordinated, multi-state effort is required to determine pathogen biology, population structure and fungicide resistance profile of Alternaria sp. causing ABHR in broccoli. Assessing the effect of specific production practices, environmental conditions, and inoculum sources on disease outbreaks would be used to design practical, economically viable, and environmentally-sound strategies to limit losses to ABHR. Our research and extension team will characterize the pathogen(s) (utilizing comparative genomics, population biology and fungicide resistance profiles to identify population structure and develop diagnostic tools for identifying Alternaria sp. causing ABHR in broccoli), the host (screening of commercial varieties), and environment (evaluating production practices, and inoculum sources), which collectively constitute the disease triangle. We will integrate economic assessments and extension efforts into each objective that will further strengthen our project. This project meets the legislatively mandated SCRI focus areas of "efforts to identify and address threats from pests and diseases" and "efforts to improve production efficiency, handling and processing, productivity, and profitability.? This project aims to reduce economic losses in broccoli production caused by Alternaria sp. by providing growers the tools for making informed management decisions that increase profitability. Our outreach plan (Outreach Plan, page 32) will demonstrate how pathogen population genetics, pathogen diagnostics, fungicide sensitivity screening, varietal screening and evaluation of cultural practices can be employed in farm operations to control ABHR of broccoli. We aim to conduct our outreach activities through a synchronized effort of team members and will communicate progress of the project to stakeholders through multiple avenues (face-to-face interactions and multi-media). Impact of our extension programs will be evaluated by determining the rate of adoption of improved recommendations, and use of improved varieties, cultural practices and effective fungicides.ABHR has substantially increased the risk of growing brassica crops including broccoli in the eastern U.S. Efforts to limit losses in fields, and storage, have not been successful, likely due to recent shifts in pathogen population and potential resistance development to 'Quinone outside inhibitor (QoI)' fungicides. Potential economic benefits: The molecular diagnostic tools for detecting QoI resistance in the pathogen, population structure of pathogen, and management strategies resulting from the project will reduce losses to ABHR in broccoli across the production chain, maximizing productivity and profitability. Environmental benefits: Optimized production practices will reduce fungicide applications and minimize the over-use of QoI fungicides, decrease losses to ABHR, and increase the efficient use of land, water, pesticides and other resources for broccoli production. Social benefits: Broccoli growers along with researchers, extension and private industry professionals, particularly from the eastern U.S., are working together to manage a pressing issue and will collaborate and reinforce the eastern brassica industry as a team. This project will facilitate training of future agricultural scientists such as graduate students and post-doctorates. Improved sustainability and resiliency of broccoli production in the eastern U.S. will benefit rural agricultural communities, where brassica crops are important aspect of their local economy.
Animal Health Component
55%
Research Effort Categories
Basic
45%
Applied
55%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21214401160100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1440 - Cole crops;

Field Of Science
1160 - Pathology;
Goals / Objectives
Statement of goals: A coordinated, multi-state effort is required to determine pathogen biology, population structure and fungicide resistance profile of Alternaria sp. causing ABHR in broccoli. Assessing the effect of specific production practices, environmental conditions, and inoculum on disease outbreaks can be used for practical, economically viable, and environmentally-sound strategies to limit losses to ABHR.Objective 1: Utilize genomics, population genetics, and fungicide resistance profiling to characterize population structure and develop diagnostic tool(s) for Alternaria sp. causing ABHR in broccoli (45%).Objective 2: Examine if different sources of inoculum and production practices exist that contribute to ABHR outbreaks and develop management programs that are environmentally and economically sustainable (55%).
Project Methods
Objective 1: Utilize genomics, population genetics, and fungicide resistance profiling to characterize population structure and develop diagnostic tool(s) for Alternaria sp. causing ABHR in broccoli. Differences in the pathogen population, whether due to being different species, having different modes of reproduction, and/or differences in fungicide sensitivity are essential to the development of effective mitigation strategies that are sustainable. Economic surveys through stakeholder engagement will be conducted to estimate the economic impact of ABHR on broccoli growers. Hypotheses: a) Knowledge of the large-scale, regional population structure of the pathogen can be used to gain insight into the modes and mechanisms of introduction of the pathogen into broccoli production systems and whether there is more than one distinct species; b) Fine-scale within-field population genetic structure analyses will provide insight into the biology of Alternaria sp. and mechanisms of spread within the field, enabling accurate disease management recommendations; c) Complementary fungicide resistance profiling will be used to determine the prevalence QoI resistance in Alternaria sp. from broccoli production systems across the eastern US; and d) Field-ready, robust detection assays to identify different species (if present) and/or QoI resistance in Alternaria sp. will allow for real-time fungicide use recommendations that are effective and mitigate disease.Using standardized survey protocols, brassica crops and cruciferous weeds (around the broccoli fields) will be surveyed for ABHR symptoms in three states representing two primary eastern U.S. regions of broccoli production (8-10 fields/state surveyed 4-5 times/season). Isolates will form a basis for the population biology and comparative genomics study. We will study pathogen population diversity using genetic tools such as genotyping by sequencing. Population structure of isolates will be compared with isolates of Alternaria sp. collected previously from NY, VA and GA. This will enable us to determine how the pathogen population has changed over time. Understanding the diversity and prevalence of Alternaria sp. as well as the QoI fungicide resistance profile in these regions will aid in development of practical methods, not only to detect Alternaria sp. but also to screen for QoI resistance within different populations. Economic surveys through stakeholder engagement will be conducted to estimate the economic impact of ABHR on broccoli growers.Objective 2: Examine if different sources of inoculum and production practices exist that contribute to ABHR outbreaks and develop management programs that are environmentally and economically sustainable. Hypotheses: a) The role and presence of different sources of inoculum, especially seeds and crucifereous weeds, will provide new disease-mitigation strategies; b) Field studies on how different production practices affect ABHR outbreaks will identify disease management options; c) Recommendations resulting from this objective will be adopted by broccoli producers that will enable them to reduce losses due to ABHR; 4) Economic and stakeholder assessment of ABHR management strategies stemming from this project will improve resiliency and sustainability of broccoli production in eastern US.We will coordinate with three different states (GA, NY and VA) and evaluate how following production practices impact ABHR outbreaks: irrigation (type and frequency); fertility practices; and fungicide programs. We will also co-ordinate with the Eastern Broccoli Project (SCRI PROPOSAL NO: 2016-04937) group. This group has identified cultivars that are suitable for the eastern US for productivity and profitability; however, they did not screen for ABHR tolerance. This project will provide an opportunity for screening these cultivars along with other commercial cultivars in GA, NY and VA and the results obtained will benefit both projects. Disease severity for ABHR symptoms, yield, quality and marketability will be evaluated in replicated trial plots in GA, NY and VA. We will assess how seeds and weeds play role in ABHR infection. Field and greenhouse trials with naturally and artificially infested seeds will be used to assess seed-to-seedling and seedling-to-seedling transmission. Further chemical and biological seed treatments will be utilized to reduce seed infection and seedling transmission in broccoli. We will conduct economic and stakeholder advisory panel assessments from regional trials each year that will enable us to determine the cost-benefit trade off of recommended management strategies. Standard extension platforms that will integrate feedback from the stakeholders will be used to disseminate outcomes of project, which will ensure highest likelihood of adoption.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:The primary target for dissemination of this research includes the stakeholders and growers producing broccoli in the eastern US, and the broader scientific community. Results of this research have been disseminated to targets including industry, academia, postdocs, graduate students, undergraduate students, and members of government organizations, such as the United States Department of Agriculture, at APS Northeastern and Southern Division and Plant Health. Targets, including administration, professors, postdoctoral scientists, graduate students, undergraduates, stakeholders, and the general public at UConn events, such as the Vegetable and Small Fruit Growers Conference, and One Health Conference. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The postdoc on the project has attended several workshops for professional development purposes. She also has co-presented with Chris Smart at grower meetings and for a webinar. Ag-clientele have been informed / educated about the current resistance situations in CT, MA, NY, VA, and GA and resistance management options through presentations at annual conferences. Provided ongoing mentoring of three graduate students, fourpostdocs, and five undergraduate students. The graduate students and postdoc presented their research at various scientific meetings and two students are on track for graduate degree completion in 2025. Three undergraduate students from UConn involved in this project received training in microbiology and molecular genetic techniques. Also involved were postdocs, Navjot Kaur, Hirut Betaw, Mychelle Batista, and Roy Davis II, who mentored graduate students and undergraduates on these projects. How have the results been disseminated to communities of interest?The results were disseminated through various platforms including peer-reviewed journal articles, newsletters, extension bulletins, webinar, national, regional, and state conferences, and vegetable grower meetings. Thefindings from this project resulted in the identification of issues related to circulation of Alternaria-contaminated broccoli seeds in domestic market for which no seed health testing are currently conducted. PD Dutta has communicated results with the major seed industries and apprised them about the current issue. In addition we also informed them on the circulation of fungicide resistant Alternaria brassicicola isolates in contaminated broccoli seeds. Further, based on the findings of age-related susceptibility of broccoli, we recommended modification of fungicide programs to better manage ABHR, which has been disseminated across the broccoli growers through above-mentioned outlets. We will continue to extend our results to our stakehodlers beyond the project period. What do you plan to do during the next reporting period to accomplish the goals?We completed all our proposed objectives and disseminated our findings through various outlets. We will continue to publish our findings in journal articles, present conference papers and dessiminate information to stakeholders.

Impacts
What was accomplished under these goals? We collected more than 500 single conidia isolates in CT, MA, GA, NY, and VA and identified most isolates as A. brassicicola. Using microplate assay based on optical density (OD) measurements to estimate inhibition of spore germination indirectly, we screened 350 A. brassicicola isolates against azoxystrobin. We found that A. brassicicola was sensitive to azoxystrobin with low EC50 values ranging from 0.005 ppm to 0.521 ppm and a median EC50 value of 0.0284 ppm. In addition to A. brassicicola, we found A. alternata in NY in 2021, CT in 2022 and VA in 2022 and 2023. We evaluated fungicide sensitivity using the microplate assay and screened for mutations that confer resistance to QoIs. We found that multiple A. alternata (n = 45) isolates had reduced sensitivity to azoxystrobin. EC50 values ranged from 0.012 to 83.9 ppm, and a median EC50 value of 4.35 ppm. Among 21 A. alternata sequenced at the cytb gene, 19 isolates showed the mutation G143A that confers complete resistance to QoIs. Studies are still ongoing to screen all A. alternata isolates. This study may explain why QoIs have reportedly failed and enable us to make more informed disease management recommendations.The Co-PI Smart conducted Boscalid sensitivity test for 76 isolates collected in 2021 and 2023. A group of 48 isolates were found to have reduced sensitivity to boscalid while the remaining 28 were sensitive. This is critical information as growers in NY use the fungicide Endura (active ingredient - boscalid) to control ABHR. The Everhart Lab has developed, multiplexed, and tested primers for amplicon sequencing of single nucleotide polymorphisms (SNPs) identified in A. brassicicola genomes. One 96-well plate has been run using these primers as an initial pilot trial. Additional plates will be run shortly to identify SNPs in isolates. Fragment analysis has also been completed on 142 A. brassicicola isolates from four states (one in CT, one in MA, and two in GA) collected in 2022. In two regions, the fields were less than 10 miles apart, and this study was designed to determine if population structure was more similar among closer fields. The 11 novel microsatellite primers designed in the Everhart Lab were used to describe the population structure. Using these primers, the population structure of A. brassicicola was found to be highly genetically diverse in all fields. Despite the distance between the two field regions, there were very few shared multilocus genotypes between any fields. We hypothesize that there may be multiple introductions of A. brassicicola that caused this population structure and that additional fine-scale sampling will be required to identify clones. Furthermore, while the organic CT and MA fields seem to have more clones present, the two conventional fields in GA are more likely to have sexual recombination occurring, despite only one mating type being present in the fields. Fungicide programs were evaluated in GA, VA and NY. The fungicide programs include Program 1 - Bravo WS, Miravis Prime, Quadris Top; Program 2 - Bravo WS, Priaxor, Inspire Super; and Program 3 - Bravo WS, Quadris top, Bravo WS. Some of the common findings include: significant differences among fungicide programs were observed only on susceptible cultivar 'Emerald crown' both in leaf and head disease reduction, fungicide program 2 (Bravo WS, Priaxor, Inspire Super) was efficacious in reducing leaf and head disease severity and yield loss in Emerald crown. Programs 1 and 2 significantly reduced leaf disease compared to program 3, whereas programs 2 and 3 were efficient in reducing head disease and yield loss as expressed by % marketable weight per plant. Field trials to evaluate irrigation, N-fterlity and varietal suscepetibility were concluded in year 3. The results were disseminated through various outlets (peer-reviewed journal articles, conference and extension presentations, webinar). Significant differences were observed among varieties for marketable head number and head weight per plant. Emerald crown had the lowest marketable head number but the highest average head weight per plant. The highest yield as expressed by average head weight is related to the cultivar's yield potential. There was no difference between programs for both average head weight and marketable head number. Multiple years of yield and efficacy data from cultivar, fungicide, irrigation, and nitrogen field trials in GA, NY and VA were synthesized with market price data as part of an economic analysis evaluating the balance between cost, efficacy, and revenue for growers considering potential adoption. Previous analysis indicated that most growers perceive themselves as only somewhat to moderately equipped, in terms of chemicals, cultivars, and knowledge, to effectively manage ABHR, highlighting the need for greater scientific evidence on potential management solutions. Field experiments in VA exploring the susceptibility of different varieties to ABHR found significant differences in both efficacy and marketable yield. Economic analysis revealed that high-performing varieties in the Virginia region could yield substantially larger economic returns compared to low-performing varieties, which experienced significant ABHR rates during trials. For instance, the best-performing cultivar compared to the worst-performing cultivars in the trials had an approximately $4,000 difference in revenue per acre due to the substantially lower marketable yield for cultivars highly susceptible to ABHR. In GA and NY, economic analysis of fungicides showed that, relative to untreated control, various fungicide products were effective in reducing the percentage of unmarketable broccoli heads due to ABHR. Among the effective products, there was a clear positive economic return for growers applying the fungicides to their crop, particularly in GA, where disease pressures were markedly higher compared to NY. In such conditions, appropriate fungicide application is essential to avoid significant economic losses. Previous survey data from growers involved in this project revealed that most broccoli producers were uncertain if, or to what degree, excessive nitrogen fertilization might impact loss rates from ABHR. Experimental evidence from NY trials, which considered nitrogen rates 50% below and 50% above the grower standard, showed some yield loss at very low or very high nitrogen application rates. When yields were converted into revenue streams under alternative broccoli prices and considering a range of nitrogen prices from the historical average to the peak nitrogen prices of the past five years, the results indicated that, even when facing record-high nitrogen prices, optimizing the nitrogen rate yielded the greatest revenue-adjusted profit for growers. A fourth production practice considered was the timing of overhead irrigation. Data from Georgia, considering morning, noon, and evening irrigation, revealed a significant difference in ABHR rates and the percentage of unmarketable broccoli heads. This finding is particularly important economically, as optimizing the timing of irrigation to reduce losses is potentially a win-win for growers, increasing revenue at no additional cost, provided there is sufficient irrigation capacity to water the crop within the optimal irrigation windows. Overall, the economic analysis underscores the importance of optimizing production systems, including cultivars, fertilization, fungicides, and irrigation. Given the high value of broccoli, measures that effectively reduce ABHR were generally found to increase per-acre profit as the increased revenue generated through reduced losses due to unmarketable broccoli heads outweighs the increased cost of inputs.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Betaw, H, Day, CTC, Hoepting, C and Smart CD (2024) Aggressiveness and fungicide sensitivity of Alternaria brassicicola isolates collected in New York. Plant Disease.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Betaw, H, Day, CTC, Hoepting, C and Smart, CD (2024) Control strategies for Alternaria brassicicola on broccoli. Plant Health Progress
  • Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Davis II, R. L., Cerritos-Garcia, D. G., Martin, A. G., Fenton Jr, M. F., Nieto-Lopez, E. H., Bach, R. K., Dutta, B., and Everhart, S. E. 2023. First report of Alternaria japonica, causal agent of Alternaria blight and head rot on broccoli in Georgia, U.S.A.
  • Type: Journal Articles Status: Accepted Year Published: 2024 Citation: Cassity-Duffey K., B. Dutta, T. McAvoy, and T. Coolong. 2024. Assessing broccoli cultivar susceptibility to Alternaria brassicicola in Georgia. Plant Health Progress.
  • Type: Other Status: Published Year Published: 2024 Citation: Dutta, B, T. Coolong, T. McAvoy, and A. Sparks. 2024 Fresh market broccoli production in Georgia. UGA Coop. Ext. Bull 1460.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Kaur, N., and Dutta, B. 2024. Aggressive Alternaria brassicicola with reduced fungicide sensitivity can be associated with naturally infested broccoli seeds. Plant Disease. 108:2154-2161.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Kaur, N., and Dutta, B. 2024. Characterization of seed-to-seedling transmission of Alternaria brassicicola in broccoli. Plant Disease. 108:2046?2052.
  • Type: Other Status: Other Year Published: 2024 Citation: Webinar- Control Alt Delete: Enhancing resiliency of broccoli production by mitigating Alternaria leaf blight and head rot in the eastern United States
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2024 Citation: Kaur N and Dutta B. 2024. Characterization of Alternaria brassicicola from naturally infested broccoli seeds. Southern Division APS meeting (2024), Columbia, South Carolina. Poster Presentation.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2024 Citation: Gangurde, S. S. Kaur, N., and Dutta. B. 2024. Leaf cuticular wax- and hormone-mediated resistance to Alternaria brassicicola in broccoli. APS National Meeting (Plant health 2024), Memphis, Tennessee. Poster Presentation.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2024 Citation: Everhart, S. E., Davis II, R. L., Cerritos-Garcia, D. G., Martin, A. G., Nieto-Lopez, E. H., and Koch-Bach, R. 2024. Epidemiology and etiology of Alternaria blight and head rot of broccoli in the Eastern United States. 13th International Epidemiology Workshop. Foz do Iguazo, Brazil. Invited oral presentation.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2024 Citation: Cerritos-Garcia, D. G., Davis II, R. L., Martin, A. G., Fenton Jr., M. F. and S.E. Everhart. 2024. Why QoI fungicides are failing to control Alternaria leaf blight and head rot of broccoli in the Eastern US. UConn CAHNR Graduate Student Research Forum. Storrs, CT. Poster Presentation.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Martin, A. G., Davis II, R. L., Cerritos-Garcia D. G., Kodati, S., and Everhart, S. E. 2024. Temperature and leaf wetness (pilot) trial and errors. UConn CAHNR Graduate Student Research Forum. Storrs, CT. Poster Presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Davis II, R. L., Cerritos-Garcia, D. G., Martin, A. G., Fenton Jr., M. F., Legault, C. L., Everhart, S. E. 2024. Characterization of the pathogenicity and composition of Alternaria spp. in the Alternaria blight and head rot disease complex in broccoli. One Health Conference. Storrs, CT. Poster Presentation
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Cerritos-Garcia, D. G., Davis II, R. L., Martin, A. G., Fenton Jr, M. F., and Everhart, S. E. Understanding why QoI fungicides are failing to control Alternaria leaf blight and head rot of broccoli in the Eastern US. One Health Conference. Storrs, CT. Poster Presentation
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Martin, A. G., Davis II, R. L., Cerritos-Garcia D. G., Kodati, S., and Everhart, S. E. 2024. Temperature and Leaf Wetness (Pilot) Trials and Errors. One Health Conference. Storrs, CT. Poster Presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Davis II, R. L., Cerritos-Garcia, D. G., Martin, A. G., and Everhart, S. E. 2024. Kochs Postulates to confirm pathogenicity of Alternaria alternata and Alternaria japonica on broccoli. APS  Northeastern Division. Ithaca, NY. Oral Presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Cerritos-Garcia, D. G., Davis II, R. L., Martin, A. G., Fenton Jr., M. F., Betaw, H., Smart, C. D., Hoepting, C., Langston, D., Rideout, S., Patel, K., Dutta, B., and Everhart, S. E. 2024. Fungicide sensitivity of Alternaria spp. to azoxystrobin reveals presence of resistance to QoIs in some broccoli producing states in the East Coast. APS Northeastern Division Meeting, Ithaca, NY. Oral Presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Martin, A. G., Davis II, R. L., Cerritos-Garcia, D. G., Langston, D., Rideout, S., Dutta, B., Everhart, S. E. 2024. Comparative analysis on the optimal temperature and leaf wetness for disease development of three species of Alternaria. APS Northeast Division. Ithaca, NY. Oral Presentation.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Davis II, R. L., Cerritos-Garcia, D. G., Martin, A. G., Fenton Jr., M. F., Legault, C. L., Everhart, S. E. 2024. Alternaria in Connecticut brassica production. UConn Extension Vegetable and Small Fruit Growers Conference. Storrs, CT
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2024 Citation: Saint-Preux, C., D. Langston, and S. Rideout. 2024. Assessment of Alternaria spp. Distribution in Brassica Crops and Weeds across Virginia: Implications for Disease Management. Plant Health, Memphis, TN, July 2024. Poster Presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Saint-Preux, C., D. Langston, and S. Rideout. 2024. Leveraging Multispectral Imaging for Effective Detection and Severity Assessment of Alternaria Blight and Head Rot in Broccoli Fields. APS Potomac Division Meeting, Martinsburg, WV. Oral Presentation.


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Primary audience of the project is the brassica producers in eastern US. This includes broccoli producers, packers, shippers, agronomists, crop consultants, farm managers, field workers, seed companies, and dealers; and broccoli storage and shipping/transport personnel and companies. Changes/Problems:An inadvertent change in potting mix containing high amounts of fertilizer resulted on one planting of brassicas for use in pathogenicity assays to be unusable. Also, the water sources at our labs were switched to accommodate some buildings being switched to the public water system. We have found that a new water source we had been using to irrigate brassicas used for pathogenicity assays had a pH of ca. 5.0 which resulted in at least 3 plantings being unusable. To offset these setbacks a new incubator was purchased which will double the number of pathogenicity bioassays we conduct. What opportunities for training and professional development has the project provided?Technicians, graduate students and postdocs have interacted with colleagues at other universities during quarterly meetings that are part of the grant, expanding their network and skillset. In NY a high school student and an undergraduate from a local community college are currently working on the project over the summer of 2023 as part of an internship program. This has provided them with the experience of working on a plant pathology project that has an impact for broccoli growers. Hirut Betaw, Roy Davis and Navjot Kaur the postdocs on the project, had the opportunity to present their work at the annual meeting of the American Phytopathological Society (APS) in August 2022 and 2023. PD Dutta, Co-PD Langston, Co-PIs Smart, Everhart, Hoepting and Coolong also presented GA, VA and NY. Post-docs also mentored seven undergraduates, across four states. How have the results been disseminated to communities of interest?Results were dessiminated through journal articles, extension reports and bulletins, presentations at national, regional, state and county meetings, extenstion trainings and podcast. What do you plan to do during the next reporting period to accomplish the goals?We would like to finish following activities during the next reporting period. 1. Activities related to population genomics and fungicide sensitivity. 2. Activities related to charcaterizing weed isolates and determine optimized seed treatments to reduce Alternaria ionculum from seeds. 3. Activities related to optimizing fungicides and irrigation timing. 4. Economic asessments and analysis of Year 3 field trials. 5. Publish peer-reviewed journal artciles, extension reports and bulletins. 6. Extension activities at the national, regional, state and county level.

Impacts
What was accomplished under these goals? Objective 1:Survey of broccoli crops for Alternaria spp.A total of 638 isolates were collected from commercial broccoli fields in NY, VAand GA and identified as Alternaria brassicicola or A. japonicabased on PCR amplification using species-specific primers. Nearly 85% of the isolates were A. brassicicola and only 15% were identified as A. japonica. Considerable variation in aggressiveness was observed with isolates from both species on broccoli, cabbage, kale and collard.Population genetics:Approximately 300 A. brassicicola isolates that were collected between 2021 and 2022 in CT, MA, NY, VA, and GA were screened for sensitivity to azoxystrobin. Most isolates that were screened are sensitive to azoxystrobin with EC50s lower than one ppm, with the exception of about 25 isolates collected from two fields in VA. The cytb gene has been sequenced for 19 of these isolates, however no known mutations were identified. An improved single spore isolation method was used to expedite the collection of A. brassicicola isolates from the leaf lesions collected during the 2022 surveys. To identify SNP and SSR loci, the six A. brassicicola genomes (A18, A48, A63, A71, F6A2, F6A9) that were previously assembled as a part of this project in were used. A detection pipeline was developed to use sequence A18 as a reference against which the other five were mapped. Following the detection of variants, flanking sequences were extracted from the data and primers were developed surrounding the SNP loci. Microsatellite (SSR) primers were either designed using WebSAT or taken from previously published sources. Primers were divided into two groups for SNPs (190 pairs) and SSRs (161 pairs) and pooled using oPools from IDT DNA for amplicon sequencing (AmpSeq). A 96-well plate with representative samples from each state was prepared for an initial AmpSeq trial to determine the attrition rate of the pooled primers. Objective 2:Fungicide programs: NY tested 12 products in a conventional fungicide efficacy trial and 9 products (one of which was the conventional product Bravo) in an organic fungicide efficacy trial. All conventional fungicides, with the exception of a new copper zinc (CuZn) formulation, significantly reduced the disease severity compared with the non-treated controls (NTC). GA tested 11 products in a conventional fungicide efficacy trial and 9 products (one of which was the conventional product Bravo) in an organic fungicide efficacy trial. Among the conventaional fungicide products, Miravis Prime, Topguard EQ, Luna Sensation, Priaxor, Inspire Super, Quadris Top, Quadris and Switch had significantly lower head rot (%) compared with other products (Fontelis, Endura and Bravo). Head rot (%) ratings for the plots treated with OSO at both rates (13 fl oz and 6.5 fl oz), and Microthiol Dispress sulfur had significantly lower incidence compared to other treatmentsand the NTC. VA conducted a biological fungicide evaluation trial on broccoli in Blacksburg, VA in Fall 2022. Foliar disease severity was significantly lower in the plots treated with OSO 5SC, Guarda 3F, and LifeGard 40WGcompared to the NTC plots. Evaluation of irrigation practices:NY evaluated following treatments in irrigation-timing trial 1) 15 minutes of overhead irrigation at 7 AM; 2) 15 minutes of overhead irrigation at noon; 3) 15 minutes of overhead irrigation at 6:00 PM; 4) no additional overhead irrigation. No significant difference was observed among the treatments. GA conducted similar trial in Athens using Emerald Crown as the variety. Overall, there was significantly less disease in the no misting and 6 AM misting treatments compared to treatments that received misting during the day and this was reflected in the marketable heads from each plot in both study years. VA conducted one trial evaluating drip vs. overhead irrigation and the effect of leaf wetness period on ABHR.No disease was detected therefore no disease data is available. Broccoli cultivar trials for ABHR tolerance:NY evaluated the response of 28 commercial broccoli cultivars to ABHR disease. Based on the assessments, cultivars (Burney, Belstar, Millenium, Iron man, Diplimat, Imperial, Burgandy) had significantly lower area under diseae progress curve (AUDPC) on head compared with Eastern Crown (a commercial standard). GA evaluated 26 commercial broccoli cultivars at two locations; Athens and Tifton, GA in Fall 2022 where cultivar 'Belstar' and 'Marathon' performed consistently better at both locations. VA conducted two cultivar evaluation trials in Blacksburg, VA: in Fall 2022 and Spring 2023. In both assessments lowest foliar disease severity was observed with Vallejo, Avenger, and Emerald Jewel. The effect of nitrogen fertilizer rate: NY compared five following treatments: 50, 75, 100, 125 and 150% of locally recommended N treatment. Half of the recommended fertilizer was applied at planting for the entire plot of the trial. No significant differences were found for all the parameters taken. GA evaluated five levels of nitrogen fertilizer ranging from 50% to 150% of recommended rates for broccoli in GA. While there were significant differences between fertilizer rate and disease incidence, there were no clear trends evident, with the exception of the lowest N application rate, which had a lower incidence of disease compared to other rates. Evaluation of inoculum sources (seed and weed):Seed as a inoculum source: Natural seed infestation of A. brassicicola in commercial seedlots was investigated. Two hundred commercial seedlots from two broccoli varieties; Emerald Crown (n=100) and Eastern Crown (n=100) were evaluated under in-vitro conditions using seedling grow-out assay (SGO). Seed infestation was detected in 35% (Emerald Crown) and 30% (Eastern Crown) of the commercial seedlots.Our results suggest that natural seed infestation with pathogenic A. brassicicola can occur in commercial broccoli seedlots, which could potentially play a role in seasonal outbreaks of ABHR in production fields.Weed as a inoculum source: We collected 26 isolates collected from non-brassica crops and weeds in and around broccoli production fields.These results imply that weed species may be an important source of inoculum leading to ABHR outbreaks. We are currently collecting isolates from more weed species and we plan on conducting studies into possible seed infestation of weed species. Economic analysis:Yield and efficacy data from cultivar, fungicide, and nitrogen field trials in Georgia, New York, and Virginia were synthesized with market price data as part of an economic analysis to evaluate potential returns to adoption for growers. Year one trials in Georgia found a 40% greater revenue for Eastern Crown compared to Imperial despite a greater percentage of unmarketable heads. Cultivar trials in Virginia found a significantly higher level of revenue from Expo compared to other varieties due to the greater marketable yield observed. Fungicide trials in Georgia found significantly greater revenue from broccoli grown treated with eight different commercial fungicides compared to nontreated control due to extremely high losses without fungicide treatment. Although not as extreme, similar findings were found in New York trials but not Virginia trials due to low disease pressure. Economic analysis of New York nitrogen trials (50%, 75%, 100%, 125%, and 150% N) found similar cost-adjusted revenues per acre for 75%, 100%, and 125% N, and significantly lower cost-adjusted revenue for 50% and 150% N. The finding is robust to broccoli price sensitivity analysis. Results indicate that, even when growers are faced with record nitrogen prices, the loss of yield resulting from under application will reduce grower profitability.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Kaur, N and Dutta, B. 2023. Natural seed infestation of broccoli with Alternaria brassicicola. Annual Meeting of American Phytopathological Society (APS), Denver, Colorado.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Petkar, A. and Dutta, B. 2023. Host range, aggressiveness and fungicide sensitivity of Alternaria brassicicola affecting broccoli in Georgia, USA. Indian Phytopathological Society, Mysore, India
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Petkar, A., and Dutta, B. 2023. AN OVERVIEW OF ALTERNARIA BLIGHT IN BROCCOLI IN GEORGIA, USA. International Congress of Plant Pathology, Lyon, France
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Dutta, B. 2023. Fungicide update for vegetable crops in Georgia. Southeast Fruit and Vegetable Conference, Savannah, GA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Dutta, B. 2023. Management of Alternaria leaf blight and head rot in broccoli in Georgia. Southeast Fruit and Vegetable Conference, Savannah, GA.
  • Type: Book Chapters Status: Published Year Published: 2022 Citation: Dutta, B. 2022. Recommendation for broccoli diseases. Georgia Pest Management Handbook. Ed. E. Cabrera. University of Georgia Cooperative Extension Service Special Bulletin 29.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Dutta, B., and Foster, M.J. 2023. Evaluation of organic fungicides for the management of Alternaria leaf blight and head rot in broccoli in Tift County, Georgia, 2022. PDMR 17:V134
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Dutta, B., and Foster, M.J. 2023. Evaluation of conventional fungicides for the management of Alternaria leaf blight and head rot in broccoli in Tift County, Georgia, 2022. PDMR 17:V135
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Cerritos-Garcia D, Nieto-Lopez EH, Koch-Bach R, Petkar R, Smart CD, Hoepting CA, Langston DB, Rideout SL, Dutta B, Everhart SE. 2023 Species-dependent sensitivity to azoxystrobin observed among causal agents of Alternaria leaf blight and head rot in brassica crops in the eastern US. Annual Meeting of American Phytopathological Society (APS), Denver, Colorado.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Davis II RL, Cerritos-Garcia D, Martin AG, Fenton Jr. MF, Patel KD, Betaw H, Hoepting Ca, Rideout SL, Langston DB, Smart CD, Dutta B, and Everhart SE. 2023. Collection and isolation of Alternaria brassicicola samples to understand population structure along the East Coast, United States. APS  Northeastern
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Cerritos-Garcia D, Davis II, RL, Betaw H, Smart C, Hoepting C, Langston D, da Silva MB, Rideout S, Patel K, Dutta B, and Everhart SE. High throughput fungicide sensitivity assay for Alternaria brassicicola isolated from brassica crops fields in the Eastern US. 2023. Annual Meeting of American Phytopathological Society (APS), Denver, Colorado.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Davis II RL (presenting author), Cerritos-Garcia D, Martin AG, Fenton Jr. MF, Patel KD, Betaw H, Hoepting Ca, Rideout SL, Langston DB, Smart CD, Dutta B, and Everhart SE. Characterizing the population structure of Alternaria blight and head rot of broccoli on the East Coast of the United States. Annual Meeting of American Phytopathological Society (APS), Denver, Colorado.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Martin AG, Davis RL, Cerritos-Garcia D, and Everhart, SE. Identification of Alternaria japonica in Connecticut, Minnesota, Virginia, and Georgia. Annual Meeting of American Phytopathological Society (APS), Denver, Colorado.
  • Type: Other Status: Other Year Published: 2023 Citation: Roy Davis and Daniel Cerritos Garcia. Alternaria survey results and fungicide sensitivity evaluation. UConn Vegetable IPM Field Workshop. Storrs, CT. 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Saint-Preux, C., Vahidi, M., Shafian, S., Langston, D., and Rideout, S. 2023. Enhancing Alternaria Blight and Head Rot Management in Broccoli: Integrating Biological Fungicides and Drone-based Aerial Imaging for Effective Disease Control. Abstract for Oral presentation at the 2023 Tri society: ASA  CSSA  SSSA International Annual Meeting, St. Louis Missouri, October 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Saint-Preux, C., Langston, D., and Rideout, S. 2023. Integrating drone technology and disease control strategies to manage Alternaria blight and head rot of brassicas. Poster presentation at the 2023 American Phytopathological Society annual meeting, Denver, Colorado, August 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Saint-Preux, C., Langston, D., and Rideout, S. 2023. Assessment of Management strategies for Alternaria blight and head rot of brassicas. Oral presentation at the 2023 APS Potomac division annual meeting, Fairfax, Virginia, March 2023.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2023 Citation: Saint-Preux, C., Langston, D., and Rideout, S. 2023. Effect of biological fungicides at reducing Alternaria blight and head rot (ABHR) on broccoli, 2022. Plant Disease Management Reports.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2023 Citation: Saint-Preux, C., Langston, D., and Rideout, S. 2023. Evaluation of broccoli cultivars susceptibility to Alternaria blight and head rot (ABHR), 2022. Plant Disease Management Reports.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Coolong, T., T. McAovy, B. Dutta, and K. Cassity-Duffey. Role of nitrogen rate and irrigation timing on Alternaria brassicicola infection in broccoli (Brassica oleracea var. italica). SRASHS. Feb. 5, 2023, Oklahoma City, OK.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Cassity-Duffey, K, T. Coolong, and B. Dutta. 2022. Cultivar trials for Alternaria leaf blight and head rot of broccoli. HortScience 57:S35.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Betaw, H, Day, CTC, Herrmann, T and Smart CD. 2023. Aggressiveness of Alternaria brassicicola isolates and control strategies. Presentation at Plant Health 2023 Denver, Colorado August 12-16, 2023


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Primary audience of the project is the brassica producers in eastern US. This includes broccoli producers, packers, shippers, agronomists, crop consultants, farm managers, field workers, seed companies, and dealers; and broccoli storage and shipping/transport personnel and companies.Other targets for dissemination include the broader scientific community concerned with the evolution of fungicide resistance and population genomics of plant pathogens.Targets also include postdocs, graduate students, and undergraduate students who receive training in microbiology, molecular genetics, genomics, and scientific communication. Changes/Problems:Co-Everhart has moved from University of Nebraska to University of Connecticut. Her move to a new institution did not cause much delay on her objectives. Co-PI Rideout has moved from Easternshore, VT to Blacksburg, VA. His move has delayed some of the trials in VA. What opportunities for training and professional development has the project provided?In GA and VA, two technicians, three post doctoral associates and three graduate students were trained under this project. In CT, one technician, two graduate students and one post-doctoral associate were trained. In NY, technicians and postdoc have interacted with colleagues at other universities during quarterly meetings that are part of the grant, expanding their network and skillset. Three high school students worked on the project over the summer as part of an internship program. This has provided them with the experience of working on a plant pathology project that has an impact for broccoli growers. They really enjoyed seeing all the different broccoli cultivars. Hirut Betaw, the postdoc on the project, had the opportunity to present her work at the annual meeting of the American Phytopathological Society in August 2022. Co-PIs Smart and Hoepting also present the work at grower meetings across NY. This project has thus far provided training for two PhD students, one of whom presented his research at a scientific meeting this summer. Another PhD student submitted an article to Plant Disease Journal as a short communication and it was accepted for publication with minor revisions and will be presenting a poster at American Phytopathological Society annual conference 2022. Also involved is a postdoc, Rachel Koch, who has mentored a graduate student on this work thus far. How have the results been disseminated to communities of interest?All together 25 county extension meetings across three states were conducted where outcomes of the year 1 and year 2 trials were disseminated. Apart from this PD Dutta and Co-PI Smart participated in a webinar (round-table discussion) for broccoli growers in the US. Information were also disseminated in regional and national extension and research conferences by the PD, and Co-PIs. Results were also disseminated through oral and poster presentations, plant disease management reports, artcles in trade journals and peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?We will accomplish goals outlined for year 3 of the project across all objectives.

Impacts
What was accomplished under these goals? Objective 1: In GA, a total of 163 isolates were collected from ten commercial broccoli fields. In NY, over a dozen broccoli fields were surveyed, and four fields were identified that had Alternaria outbreaks. Alternaria isolates (n=119) were collected from four fields in four counties across NY including Niagara, Orleans, Ontario, and Broome Counties. In VA, over 160 Alternaria isolates were collected over 2021-2022 from different brassica species and weeds. Our results suggest that thus far, only A. brassicicola and A. japonica are responsible for causing ABHR in the target region. Surprisingly, A. japonica was only identified from the isolates that were collected in Georgia. In all three states, more than 90% of the isolates were identified as A. brassicicola. Additionally, we identified single nucleotide polymorphisms (SNPs) in several of our phylogenetic loci, including the major allergen (Alt a1) and ATPase, in the subset of our isolates, suggesting there is geographic structuring of A. brassicicola.Alternaria isolates from GA were highly aggressive on broccoli (65%), cabbage (46%) and collard (44%) than on kale (8%). In NY, all 119 isolates were highly aggressive onbroccoli and collards while cabbage and cauliflower were least susceptible to all isolates. In VA, Alternaria isolates are being tested for their pathogenicity and aggressiveness on broccoli, cabbage, collard and kale.In GA, conidial germination assay for a sub-set of isolates on azoxystrobin-amended agar medium indicated 100% of the isolates (n=145) were sensitive at both concentrations (10 and 100 µg/ml). Fungicide sensitivity assay for A. japonica is underway. In addition, Co-PI Everhart have screened 15 A. brassicicola (five each from NY, VA and GA) and seven A. japonica (all from Georgia) isolates for sensitivity to azoxystrobin. These isolates represent historical collections (isolated prior to the use of QoI fungicides) and new collections. Thus far, we have detected a range in EC50 values, suggesting that some of the isolates are intermediately sensitive to azoxystrobin, but none are resistant. None of the three known point mutations that confer QoI resistance are present in the 15 A. brassicicola isolates that we used in the phylogenetic analysis and fungicide sensitivity screening. We are currently in the process of optimizing primers that target the cytb gene in A. japonica so that we can screen those isolates for QoI resistance.Population genetics: Currently, wesequenced six A. brassicicola genomes, and assembled and annotated them. We are currently in the process of mapping the raw reads back to a JGI-generated reference to call SNPs and create a pipeline for understanding the genetic diversity.Development of diagnostic tools: To this end, we are continuing to screen Alternaria isolates for the point mutations in the cytB gene. Additionally, we are also generating QoI-resistant A. brassicicola mutants in the lab to verify that we can expect to find the known mutations in the cytB genes in resistant isolates. Objective 2:Fungicide programs: In GA, six Organic Material Review Institute (OMRI)-listed fungicides were evaluated and based on our results significantly lower area under disease progress curve (AUDPC) value was observed with Double Nickel compared to other treatments and the non-treated check. Furthermore, we also evaluated 11 conventional fungicides.Disease severity was significantly higher for Switch, Quadris and non-treated check compared to Fontelis, Miravis Prime, Topguard EQ, Bravo Weather Stik, Luna sensation and Priaxor. In NY, during the 2021 season, 12 conventional fungicides were evaluated.All conventional fungicides significantly reduced the disease severity compared with the non-treated control. In NY, OMRI-listed fungicides were also screened in a separate experiment. A total of 9 treatments including 6 OMRI-listed fungicides, 2 conventional fungicides and one non-sprayed control were compared for Alternaria leaf blight and head rot. Among the OMRI-listed fungicides, broccoli plants treated with OSO 5%SC had the lowest disease severity.Evaluation of irrigation practices:In GA, an irrigation timing trial was implemented with treatments: no overhead, overhead irrigation at 6 AM, 12 Noon, and 6 PM. No significant differences were determined in total yield between misting treatments, but significant differences were determined in the percent disease incidence, number of heads harvested per plot, and marketable heads from each plot. In NY, irrigation timing treatments included 1) 15 minutes of overhead irrigation at 7 AM; 2) 15 minutes of overhead irrigation at Noon; 3) 15 minutes of overhead irrigation at 6:30 PM; 4) no additional overhead irrigation. No significant difference was observed among the treatments. In VA, we conducted a trial evaluating drip vs. overhead irrigation and the effect of leaf wetness period on disease incidence and severity. We did not observe differences between treatments in this trial.Broccoli cultivar trials for ABHR tolerance:In GA, varieties (n=26) were screened for resistance to A. brassicicola in Athens and Tifton, GA in Fall 2021. Varieties with low disease in each location included: Marathon, Belstar, Vallejo, and Emerald Pride. Varieties with low resistance and high disease included: Eastern Crown, Mellinium and Asteroid. In NY, a field experiment was conducted to determine response of 27 commercial broccoli cultivars to disease. Significant difference was observed in susceptibility among the broccoli cultivars with 'BC 1611', 'Montebello', and 'Astroid' being the least susceptible and 'Green Magic', 'Emerald Star' and 'Lieutenant' the most susceptible. In VA, Co-PD Langston and Co-PI Rideout are currently assessing and harvesting a 26 cultivar broccoli trial that has moderate/severe disease pressure.The effect of nitrogen fertilizer rate:In GA, five levels of nitrogen fertilizer were applied ranging from 50% to 150% of recommended rates for each location (Athens and Tifton).No significant differences between fertilizer rate and disease were found at both locations. However, higher yields were obtained in the Athens location. In NY, five treatments (50, 75, 100, 125 and 150% of locally recommended N treatment) were compared. Plots fertilized with 100% N had significantly lower AUDPC value compared to 50% N treated plots. In VA, no significant differences among fertility treatments (50, 75, 100, 125 and 150% of locally recommended N treatment) were observed, but the data did trend towards lower rates of nitrogen having higher disease levels as observed in NY.Evaluation of inoculum sources:We determined the ability of seed-to-seedling transmission of A. brassicicola in broccoli using artificial infestation of seeds under in-vitro conditions. Based on our results, seed-to-seedling transmission of A. brassicicola in broccoli can occur as 100% of the inoculated seedlot transmitted the pathogen indicating seeds could be potential source of inoculum. We also evaluated commercial seedlots for natural infestation of broccoli seeds with Alternaria. We recovered 73 isolates from 100 commercial broccoli seedlots, which was further confirmed as Alternaria spp. In terms of weeds as a source of inoculum, we have collected and isolated Alternaria spp. from cutleaf evening primrose, nutsedge and wild brassica. An isolate found in nutsedge tested positive for A. brassicicola. Currently, more studies are being conducted to verify the other isolates pathogenicity on brassicas species.Economic analysis.Co-PI Colson is currently conducting economic analysis on field trials conducted in GA, NY and VA for 2021 and 2022 seasons.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Cassity-Duffey, K., T. Coolong, and B. Dutta. Cultivar trials for Alternaria leaf blight and head rot in broccoli. ASHS. July 31, 2022. Chicago, Ill. (Poster).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Kaur, N., and Dutta, B. 2022. Seed-to-seedling transmission of Alternaria brassicicola in broccoli. Poster: American Phytopathological Society Annual Meeting 2022 at Pittsburgh, PA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Petkar, A., Doss, T., Patel, K., Torrance, T., and Dutta, B. 2022. Host range, aggressiveness and fungicide sensitivity of Alternaria brassicicola affecting broccoli in Georgia. Poster: American Phytopathological Society Annual Meeting 2022 at Pittsburgh, PA.
  • Type: Other Status: Published Year Published: 2021 Citation: Dutta, B. Update on the national Alternaria project in broccoli. Great Lakes vegetable producers network weekly roundtable, Sep 1, 2021. (Webinar; invited)
  • Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Nieto-Lopez EH, Cerritos Garcia D, Koch Bach RA, Petkar A, Smart C, Hoepting C, Langston D, Rideout S, Dutta B, Everhart SE. 2022. Species identification and fungicide sensitivity of fungi causing Alternaria Leaf Blight and Head Rot in cole crops across the Eastern U.S. Plant Disease
  • Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Cassity-Duffey, K., Coolong, T., and Dutta, B. 2022. Evaluation of the effect of timing of simulated leaf wetness on disease severity in Alternaria leaf blight and head rot in broccoli, Oconee County, GA 2021. PDMR 16:V153
  • Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Kaur, N., and Dutta, B. 2022. Evaluation of fungicide programs for the management of Alternaria leaf blight and head rot in broccoli in Tift County, Georgia, 2021. PDMR 16:V169.
  • Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Dutta, B., and Kaur, N. 2022. Evaluation of organic fungicides for the management of Alternaria leaf blight and head rot in broccoli in Tift County, Georgia, 2021. PDMR 16:V130.
  • Type: Other Status: Published Year Published: 2021 Citation: Dutta, B. and Petkar, A. 2021. Management of Alternaria blight and head rot in brassica. Vegetable and Specialty Crop News, June 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Hirut B, Day, CTC, Smart CD. (2022). Improving control strategies for Alternaria blight and head rot. Presentation at Plant Health 2022 Pittsburgh, Pennsylvania, August 6-10, 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Nieto-Lopez EH (presenting author), Cerritos-Garcia D, Koch RA, Doss T, Dutta B, Petkar A, Langston D, Rideout S, Smart CD, Everhart S. 2021. Investigation into the loss of fungicide efficacy for Alternaria leaf blight and head rot of broccoli and cruciferous crops in the Eastern USA. Poster: American Phytopathological Society North Central Division Meeting, virtual.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Species-dependent sensitivity to azoxystrobin observed among causal agents of Alternaria leaf blight and head rot in brassica crops in the eastern US. Poster: American Phytopathological Society Annual Meeting 2022 at Pittsburgh, PA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Petkar, A., Doss, T., Patel, K., Torrance, T., and Dutta, B. 2022. Fungicide sensitivity of Alternaria brassicicola affecting broccoli in Georgia. Poster: Annual Meeting of National Institute of Antimicrobial Resistance Research and Education. May 16, Lincoln, Nebraska.


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Primary audience of the project is the brassica producers in eastern US. This includes broccoli producers, packers, shippers, agronomists, crop consultants, farm managers, field workers, seed companies, and dealers; and broccoli storage and shipping/transport personnel and companies.Other targets for dissemination include the broader scientific community concerned with the evolution of fungicide resistance and population genomics of plant pathogens.Targets also include postdocs, graduate students, and undergraduate students who receive training in microbiology, molecular genetics, genomics, and scientific communication. Changes/Problems:Prior to trial establishment, Co-PIs conducting the irrigation experiment felt that changing the irrigation timing from a weekly schedule to a daily schedule to better measure the effect of daily leaf wetness period based on the timing of overhead misting. Timing treatments were decided to be: 1) once early in the morning; 2) once mid-day; and 3) once before dark. The hypothesis is that the effect of leaf wetness provided by overhead irrigation would be greater if leaf wetness occurred at different times of the day as opposed to frequency during the week. What opportunities for training and professional development has the project provided?This project has thus far provided training for four PhD students, four post-doctoral associates, extension agents and consultants. How have the results been disseminated to communities of interest?The results have been disseminated through various national, regional and state or local meetings. Two of the abstracts and posters that stemmed from this project were presented at the annual Americal Phytopathological Society Meeting (Aug 2021). PD Dutta, Co-PD Langston, Co-PIs Smart, Rideout, and Coolong have presented the outline of the project and the preliminary findings at various regional (SE Fruit and Vegetable Conference), state and local county extension meetings in GA, VA, and NY. What do you plan to do during the next reporting period to accomplish the goals?We will continue our statewide survey in three states for Alternaria spp. in commercial broccoli fields. We will finish characterizing (species identity, pathoegnicity and aggressiveness on brassica crops, fungicide sensitivity) isolates from year 1 and 2 in all three states. We will select represenative Alternria isolates from each species from three different states for genome sequencing and will population genetics study. We will continue our effort to find the source of Alternaria isolates (seed and weed). We will continue our efforts on evaluating cultural practices (N-fertility, irrigation), cultivars, and fungicide programs for ABHR managment in eastern US. We will continue our extension efforts to disseminate information via. peer-reviewed journal articles, extension articles, plant disease management reports, extension presentations (regiona, state and local).

Impacts
What was accomplished under these goals? PD Dutta surveyed eight commercial broccoli fields in fall 2020 and spring 2021. Overall, 108 Alternaria spp. were isolated, purified and single-spore cultures were stored at -80oC under further use. Another survey will be conducted in fall 2021. In VA, three broccoli farms operated by VA Department of Corrections were scouted for the presence of disease in the fall of 2020. Plants exhibiting symptoms of Alternaria blight and head rot (AHBR) were found in each of the three locations. Parker Farms in Oak Grove, VA was sampled both in the fall of 2020 and spring of 2021 for Alternaria sp. In addition, farms in Blacksburg and Wytheville, VA were sampled in the spring of 2021. Several Alternaria isolates were obtained and are being currently characterized. In NY, Co-PI Smart surveyed transplant production facilities and will continue collect isolates as soon as there are outbreaks in fall 2021. Co-PI Everhart utilized PCR amplification and multi-locus phylogenetic analysis on a subset of collected isolates from each region (NY, VA and GA). The results suggest that thus far, only A. brassicicola and A. japonica are responsible for causing ABHR in the target region. Additionally, Co-PI Everhart identified single nucleotide polymorphisms (SNPs) in several of our phylogenetic loci, including the major allergen (Alt a1) and ATPase, in the subset of our isolates, suggesting there is geographic structuring of A. brassicicola. Second, we verified the efficacy of a previously published set of Alternaria diagnostic primers for rapid identification of each of our isolates, obviating the need for sequencing. Utilizing A. brassicicola-specific primers, 108 Alternaria isolates from GA collected in fall 2020 and spring 2021 were assayed and confirmed as A. brassicicola.PD Dutta and collaborator Petkar has developed a detached leaf assay to assess aggressiveness of Alternaria spp. in brassicas. One hundred percent of the isolates collected were pathogenic on brassica hosts but aggressiveness varied considerably. Based on this assay, 83.7% of the Alternaria isolates were highly aggressive on broccoli whereas only 10.9 and 4.3% of the isolates were moderately and less-aggressive, respectively. Only, 1.1% of the isolates were non-pathogenic on broccoli. During this reporting period, Co-PI Everhart has developed and optimized the protocols that the entire SCRI team will use to measure fungicide sensitivity for all isolates collected over the three years of this project. Co-PI Everhart has screened 15 A. brassicicola (five each from NY, VA and GA) and five A. japonica (all from GA) isolates for sensitivity to azoxystrobin. Thus far, we have detected a range in EC50 values, suggesting that some of the isolates are intermediately sensitive to azoxystrobin, but none are resistant. To screen for fungicide resistance in A. brassicicola isolates, we used previously published primer sets that target the known point mutations in the cytb gene that are known to confer QoI resistance in ascomycete pathogens. None of the three known point mutations that confer QoI resistance are present in the 15 A. brassicicola isolates that we used in the phylogenetic analysis and fungicide sensitivity screening. We are currently in the process of optimizing primers that target the cytb gene in A. japonica so that we can screen those isolates for QoI resistance. An efficient and cost-effective DNA extraction procedure has been optimized for A. brassicicola and all collected isolates have extracted DNA thus far. In order to identify SNPs in our target Alternaria species, we aim to sequence three to four genomes for each species. Currently, we are in the process of deciding which A. brassicicola isolates to send for the preliminary genome sequencing. None of the three known point mutations that confer QoI resistance are present in the 15 A. brassicicola isolates that we used in the phylogenetic analysis and fungicide sensitivity screening. Before diagnostic tools can be developed to rapidly screen for fungicide resistance, we need to understand the genetic mechanism behind the resistance. To this end, we are continuing to screen Alternaria isolates for the point mutations in the cytB gene. Additionally, we are also generating QoI-resistant A. brassicicola mutants in the lab to verify that we can expect to find the known mutations in the cytB genes in resistant isolates. Co-PI Smart has completed the conventional and organic fungicide efficacy trials in NY, and are analyzing the data. The identical efficacy trails are being performed in GA and VA. There were 12 treatments in the conventional trial, and nine in the organic trial. Co-PI Smart (NY) has completed the irrigation practices experiment for 2021. To determine the role of timing of irrigation in disease severity, four overhead irrigation treatments were compared. The trials in GA and VA are currently planted. Cultivar screen trials have just been planted with 27 cultivars in GA, VA and NY. Co-PI Smart and collaborator Hoepting (NY) have completed the fertilizer rate experiment for 2021. The trials are underway in GA and VA, which will be completed by Nov 2021. PD Dutta and postdoctoral research associate Kaur will evaluate the host range of Alternaria spp. on wide range of Cruciferae, Solanaceous, Asteraceae and Cucurbitaceae weeds commonly found in the broccoli growing regions in GA. Co-PD Langston collected symptomatic weed samples from the broccoli fields and the Alternaria isolates from the samples are currently being characterized. Co-PI Colson (Economist) has developed two survey instruments (broccoli growers, project team/ stakeholder advisory panel) and administered to obtain estimates of the prevalence and severity of ABHR in broccoli, effectiveness of existing management strategies for ABHR and critical research and management needs of the broccoli industry. Both surveyed groups agree in this regard. Growers estimate that on average over the past five years 14% of the broccoli crop in their area has been lost due to ABHR. Nearly 25% of growers report experiencing catastrophic losses of more than 50% of the crop in their area during the past five years, as a direct result of ABHR. On average over the past five years, only 16% of growers report minor losses of less than 5%. Among an array of existing management strategies (e.g., different fungicides, planting practices, weed management, irrigation, etc.), all surveyed groups agree that there is no single very effective management strategy for reducing economic losses. The average broccoli grower can be characterized as being: concerned about ABHR; moderately familiar with and able to identify ABHR in broccoli; only moderately equipped in terms of management strategies to reduce economic losses; and uncertain about optimal farm management practices to address ABHR.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Nieto-Lopez EH (presenting author), Cerritos-Garcia D, Koch RA, Doss T, Dutta B, Petkar A, Langston D, Rideout S, Smart CD, Everhart S. 2021. Investigation into the loss of fungicide efficacy for Alternaria leaf blight and head rot of broccoli and cruciferous crops in the Eastern USA. Poster: American Phytopathological Society North Central Division Meeting, virtual.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Petkar, A (presenting author)., Doss, T., Koch, R.A., Ji, P., Everhart, S., and Dutta, B. 2021. Identification and host-range evaluation of Alternaria species complex affecting broccoli in GA. Poster: American Phytopathological Society North Central Division Meeting, virtual.
  • Type: Websites Status: Published Year Published: 2021 Citation: Web-page. https://alternariabroccoliproject.uga.edu/welcome/ A webpage was created to update the progress on different aspects of the project.