Source: N Y AGRICULTURAL EXPT STATION submitted to NRP
COMBATTING STEMPHYLIUM LEAF BLIGHT OF ONION: A CRITICAL THREAT TO THE SUSTAINABILITY AND PROFITABILITY OF ONION PRODUCTION IN NEW YORK
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1025149
Grant No.
2021-68008-34101
Cumulative Award Amt.
$299,575.00
Proposal No.
2020-04951
Multistate No.
(N/A)
Project Start Date
Jan 15, 2021
Project End Date
Jan 14, 2024
Grant Year
2021
Program Code
[A1701]- Critical Agricultural Research and Extension: CARE
Recipient Organization
N Y AGRICULTURAL EXPT STATION
(N/A)
GENEVA,NY 14456
Performing Department
Plant Path./Plant Microbe Biol
Non Technical Summary
New York is the fifth largest producer of onions in the United States, producing 142,246 t from 2,832 ha (USDA NASS 2017). The majority of onions are grown in localized regions on muck (high organic matter) soils, where foliar disease management is a major determinant of onion productivity and profitability. Over the last tenyears Stemphylium leaf blight caused by the fungus Stemphylium vesicarium has become ubiquitous in fields throughout New York, causing premature death of foliage and preventing onion bulbs from attaining optimum size.Where the disease is not adequately controlled, yield losses of over 30-40% have been recorded. The fungus has rapidly developed widespread resistance to many of the commonly used fungicides, and options for controlling this important disease are becoming limited. This project will provide growers with an integrated management strategy through gaining a better understanding of pathogen diversity, undertanding the influence of agronomic practices on the severity of disease epidemics, and testing and fine-tuning a disease forecaster based on weather variables as a predictor of disease outbreaks as a means of achievingbetter timing of control strategies.
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121451117050%
2121451116050%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1451 - Onion, garlic, leek, shallot;

Field Of Science
1160 - Pathology; 1170 - Epidemiology;
Goals / Objectives
This project aims to reducecrop loss for the New York onion industrythrough development of an integrated management strategy forthe dominant disease, Stemphylium leaf blight, caused by the fungus Stemphylium vesicarium. Stemphylium leaf blight is a re-emerging disease problem for growers in northern USA, and is now ubiquitous in commercial fields in New York. The disease causes premature death of foliage, preventing onion bulbsfrom achieving optimum size, leading tolosses in yield of30-40%.Control of SLB has relied heavily on the use of fungicides. However, the pathogen has developed widespread resistance to several fungicides, and fungicide options for control are now limited. This project will develop anintegrated management strategy guided through new knowledge on the pathogen population structure, the lifecycle of the pathogen, the influence of agronomic factors,and disease forecasting based on environmental variables, to advise growers of optimum timing of fungicide application.
Project Methods
Genotyping marker development. The laboratories of PD Hay and co-PD Pethybridge has produced de novo genome assemblies for two S. vesicarium isolates (Sharma et al. 2020). These will be used as a scaffold for the identification of microsatellite motifs using the Tandem Repeat Finder version 408 software, identifying sequences with tri- and hexa-nucleotide repeat motifs, a minimum score of 50, matching point of two, and copy number of at least five. Fifty loci will be initially selected for primer design. Only one microsatellite per contig will be selected for primer design to minimize the probability of physical linkage of loci. The genome within 5 kb of each of the microsatellite motifs will be searched using the Basic Local Alignment Search Tool within the NCBI nuclear ribosomal DNA database in GenBank to ensure the loci are not linked to genic regions. Primers for each microsatellite will be designed using the program, Primer 3, with like melting temperatures (58 to 60°C) and product sizes between 90 and 450 bp to enable variation for multiplexing purposes. The primer pairs will be initially tested individually on DNA extracted from 30 S. vesicarium isolates collection from onion fields across New York from each of three years (2015 to 2017) and 15 promising primer sets will be selected based on amplification on single bands of expected sizes for all isolates visualized on an agarose gel. Promising microsatellite markers will be labelled with selected fluorophores (e.g. 6-FAM, NED, PET, VIC) at the 5' end of the forward primer for multiplexing. PCR assays testing the ability to multiplex will be conducted on the same 30 S. vesicarium isolates. Fragments of the fluorescently labelled PCR products will be analyzed with a GeneScan-600 LIZ size standardand alleles will be scored using a Microsatellite Plugin within the software, Geneious. PCR assays and fragment analyses will be repeated on this initial sub-set of isolates to evaluate reproducibility and estimate errors rates for all loci, and PCR products sequenced using Illumina sequencing to ensure they contain the target repeat motif of interest.The selected polymorphic markers will be multiplexed into two or three PCR sets containing four to six markers per set. Reproducibility of this assay will also be evaluated by conducting repeat PCR assays and fragment analysis on the same isolates used for marker development. The number of markers required to quantify genetic variability of the entire S. vesicarium population will be evaluated by inspection of a genotype accumulation curve using poppr (Kamvar et al. 2015).S. vesicarium populations. S. vesicarium isolates previously collected from New York onion fields in 2016 and 2018, and to be collected in 2021, will be subjected to genotyping. To assess genotypic diversity and differentiation in S. vesicarium populations between fields, regions and seasons, at least 20 isolates of S. vesicarium from each of four fields, in four counties (Genesee, Wayne, Oswego and Orange), representing the main onion growing regions in New York will be genotyped from the 2016, 2018 and 2021 collections. An additional 30 S. vesicarium isolates collected from low input farms in similar regions will be included in the study for comparative purposes. To assess within-field variation, 50 to 100 isolates collected along linear transects in each of three fields (in 2018) and to be collected in 2021, will be genotyped. The spatial position of each isolate along each transect will be recorded, and this metadata will be preserved in the analyses.In August 2021, at least 10 isolates of S. vesicarium will be collected from diseased onion leaves collected from four fields in each of the four counties. Leaves will be incubated under high humidity to encourage sporulation, conidia removed with a sterile needle and placed on V8 juice agar under a 12 h photoperiod to encourage mycelial growth and sporulation. Single conidia will be removed, sub-cultured and grown on V8 juice agar, and isolates stored (as above). For the within field study, two 150 m transects will be established in each of three commercial fields in August 2021. Diseased leaves (n = 5) will be collected at 3 m intervals along each of two transects, with a total of 50 sample points per transect. Leaves will be maintained under high humidity and single-spored isolates will be obtained and stored (as above), aiming to collect at least one isolate per sample point (100 isolates per field).For genotyping, all isolates will be removed from storage, cultured in clarified V8 juice broth amended with 100 mg/ml ampicillin and incubated at room temperature on an orbital shaker at 80 rpm for 7 days before harvest of the fungal mycelia. Genomic DNA will be extracted from all isolates with the Wizard Extraction Kit (Promega Corporation, Madison, WI). The concentration of the resultant DNA will be quantified with a Nanodrop spectrophotometer ND-1000, diluted to 25 ng/ml, and stored in sterile distilled water at -20°C.Populations will be described with statistics to quantify genetic diversity in clone-corrected datasets as appropriate. These statistics have been well described in our recent publication (Koenick et al. 2019). In brief, these will include measures of allelic diversity, allelic richness, number of multilocus genotypes, genotypic diversity, evenness, and clonal fraction. Analysis of molecular variance will be used to determine genetic structure among S. vesicarium populations. Complementary statistics to quantify population differentiation will be selected.

Progress 01/15/21 to 01/14/24

Outputs
Target Audience:Onions are a valuable vegetable crop in NY, with production ofover 250 million pounds of onions from 7200 acres, and an annual value of $43 M. NY onion production ispredominately onmuck histosols, and accountsfor 97% of the production in Northeast USA . Regular updates of this project were given to growers from 16 farms who participate in Hoepting's onion scouting program which involves weekly meetings during the growing season to share scouting data, spray records, and management recommendations. This interaction accounted for some 75% of muck onion acreage in NY. Each year of the project several growers properties were surveyed to assess the extent of SLB in volunteer onion and transplant onion. Results were communicated to participating growers on a one-to-one basis and to the wider grower community at grower events. Changes/Problems:The main changes occurred in objective 3. This objective assessed the forecasting model BSPcast, which was developed in Europe to predict risk of brown spot of pear caused by Stemphylium vesicarium for its ability to predict risk of SLB in onion. BSPcast requires inputs of the duration of periods of leaf wetness and air temperature during periods of leaf wetness to calculate a daily risk value 'R'. Calibration of the model required determining the threshold value of R, above which infection occurs, and below which infection does not occur. We attempted to emulate a range of environmental conditions in plots of onions by comparing ambient conditions with plots treated with periods of supplemental misting to exacerbate disease. However, in the 2021 season there were frequent weekly rain events throughout the season leading to high weekly R values and all plots exhibiting weekly disease increase, with insufficient dry events for comparison. In the 2022 season we utilized low tunnels with open ends for reduced humidity oversome plots to maintain dry conditions. This led to a more robust data set with both dry and wet events. However,statistical analysis determined a very poor relationshipbetween R values from BSPcast and weekly changes in disease incidence. In 2023, we further amended the trial to include a higher intensity of leaf sampling. However, again BSPcast showed very poor predictive power. Due to the lack of accurate prediction by BSPcast, the model could not be recommended to growers as a means of scheduling fungicides. What opportunities for training and professional development has the project provided?One graduate student and one early-career researcher were involved in the project and learned laboratory techniques to characterize fungal populations (microsatellites) and sequencing of fungal genes associated with fungicide resistance. This resulted in authorship/co-authorship of one publication in a scientific journal, two poster presentations at conferences, and two oral presentations at grower events. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Use a population biology approach to elucidate the dominant sources of inoculum for SLB epidemics in onion crops in New York. This study successfullydeveloped a set of polymorphic and reproducible microsatellite (SSR) markers for population biology analyses of Stemphylium vesicarium. Of 26 non-genic loci originally selected, afinal set of 11 polymorphic and reproducible SSR markers were found suitable for population genetic studies. The eleven SSR markers were divided in two multiplex reactions. The PCR products were resolved by fragment analysis in an ABI 3730cl DNA Analyzer at the Cornell University Institute of Biotechnology. In total, 141 isolates of S. vesicarium collected from different fields, counties, states, and years were genotyped with the markers . In addition, SSR's were used to determine the importance of volunteer onion and transplant onion as sources of inoculum. A scientific paper on the development of microsatellites for S.vesicarium has been publishedin Plant Disease. Objective 2. Determine the role of agronomic practices of inter-seeding with barley on SLB epidemics in onion fields in New York. ?Barley windbreaks in onion crops.Barley is often seeded between rows of direct seeded onion to protect the slower emerging onion from wind damage. Barley is reportedly a host of S. vesicarium, and S. vesicarium can colonize and sporulate on dying grass species, so barley wind barriers were tested as sources ofS. vesicarium onto onion. Barley leaves (n=30) were collected from inter-row barley from each of eight commercial fields in May 2021. Leaves were incubated in plastic bags in high humidity for 7days and examined under a microscope for the presence of conidia of S. vesicarium.Stemphylium vesicarium was detected in barley from two fields at low incidence (3.3% or less), suggesting that inter-row barley did not constitute a significant source of S. vesicarium for the emerging onion crop. Volunteer onion. In April/May 2023, volunteer onion at the 3-4 leaf stage was collected from 10 fields in Elba, NY which had just been planted with onion or were due to be planted. Ten volunteer onion plants from each field were incubated under high humidity and examined as above. Stemphylium vesicarium occurredin volunteer onion from all 10 fields, at 20 to 90% incidence, indicating that volunteer onion was a significant source of overwintered S. vesicarium inoculum into the following onion crop, or crops grown in close proximity. Transplant onion. Some 75% of onion crops in NY are direct seeded with the rest planted from bareroot transplants imported from other States. In April/May 2022 and 2023, bare-rooted transplant onion of 8 and 7 varieties respectively were collected soon after delivery to growers. Stemphylium vesicarium was detected in allvarieties in 2022 and 2023, with the incidence of S. vesicarium ranging from 11.2--100% and 60.3-100% respectively. SLB is not usually severe on transplant onion as itmatures rapidly and is harvested earlier than direct seeded crops. However,transplant onion couldbe a significant source of inoculum into direct seeded fields planted in close proximity. Seed. Stemphyliumvesicarium is seedborne in onion (Aveling 1993). A total of 120 seed from 27 seedlots brought into NY in 2022 were plated onto water agar, incubated and examined under the microscope. S. vesicarium was not detected in any seedlot indicating that seed can currently be considered a minor source of inoculum in NY. Objective 3. Evaluate the potential of forecasting systems based on weather-based risk to support decisions surrounding fungicide application for SLB control. The forecasting model BSPcast, which was developed in Europe to predict risk of brown spot of pear caused by S.vesicarium was assessed for its ability to predictSLB in onion. BSPcast requires inputs of the duration of periods of leaf wetness and air temperature during periods of leaf wetness to calculate a daily risk value 'R', with R values >0.2 signifying days conducive for infection. In 2021, 15 plots of onion were established on the Cornell AgriTech research farm with transplant onion tested to be free of S. vesicarium at planting. All plots received sub-surface drip irrigation, while an additional 5 plots received eithera low or high level of supplemental mist irrigationto encourage disease. Buffer rows of onion were inoculated with conidia of S. vesicarium to act as an inoculum source. Leaves from 25plants per plot were retrieved at weekly intervals, incubated under high humidity and observed for conidia of S. vesicarium to determine weekly change indisease incidence. Air temperature and leaf wetness were monitored in all plotsand weather data was input into the model BSPcast to identify periods conducive to spore germination and infection. The 2021 season had frequent weekly rainfall events which contributed to infection periods in all treatments and disease increase in all plots in each assessment week. The trial was repeated in 2022 with five plots covered with low tunnels to ensure dry conditions insome plots. Methods were as for 2021 with some plots receiving additional overhead misting to ensure infection events.However, ROC analysis of the dataset showed a poor predictive power between the R values calculated by BSPcast and weekly changes in the incidence of S. vesicarium. The trial was modifiedin 2023, with four plots each treated with 1) no treatment (ambient only), 2) low mist ,3) high mist,and 4) plots covered with tunnels. Alarger number of leaves (n=40/plot) were sampled in 2023to estimate incidence. Despite these improvements, ROC analysis of the dataset showed a poor predictive power between the output of BSPcast and changes in incidence of S. vesicarium. Results determined that BSPcast was not sufficiently accuratefor growers to useas a means to schedulingfungicide application for SLB in onion. Objective 4. Develop an integrated disease management strategy for SLB in onion and support the dissemination of findings through on-farm demonstrations, newsletter articles, research bulletins, and presentations at extension meetings. Two articles were published in the VegEdge publication. Hoepting, C.A., and Hay, F. 2021. Part I: Onion fungicide research updates and new recommendations for control of Botrytis and Stemphylium leaf blights, 2021. VegEdge 17(12):8-10. Hoepting, C.A., andHay, F. 2021. Part II: Onion fungicide research updates and new recommendations for control of Botrytis and Stemphylium leaf blights, 2021. VegEdge 17(13), 8-9. Six presentations were made at vegetable grower events. Pineros-Guerrero, N. 2024. Stemphylium leaf blight of onion in New York - research update. Empire State Producers Expo, Oncenter Syracuse, January 22, 2024. Hay, F.S., Hoepting, C., Heck, D., Klein, A., Pethybridge, S.J. 2024. Stemphylium leaf blight - a continually evolving problem for onion growers in New York. Invited speaker Onions, N.Z. Pukekohe Research Station, Pukekohe, New Zealand. Jan 17, 2024. Pethybridge, S.; Hay, F.S. et al., 2023. Fight the blight. Stemphylium leaf blight an emerging threat to US onion production. Poster presented at National Onion Association/National Allium Research Committee meeting San Antonio, November 2023. Heck, D. 2023. Update on Stemphylium leaf blight of onion and development of fungicide resistance. Empire State Producers Expo, Oncenter, Syracuse, February 8, 2023. Heck, D., Hay, F., Hoepting, C.A., Pethybridge, S.J. 2021. 'Stemphylium leaf blight in onion - a continually evolving problem for growers in New York'. 69thAnnual Muck Vegetable Growers Conference 2021. Virtual meeting. April 1, 2021. Hay, F., Heck, D., and Pethybridge S.J. 2021. 'Stemphylium leaf blight. W3008 Multistate Project Annual Meeting. February 3, 2021.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Heck, D.W., Hay, F., Pethybridge, S.J. 2023. Enabling population biology studies of Stemphylium vesicarium from onion with microsatellites. Plant Disease https://apsjournals.apsnet.org/doi/10.1094/PDIS-04-23-0706-RE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Pineros-Guerrero, N., Hay, F. S., Heck, D. W., Klein, A., Hoepting, C., and Pethybridge, S. J. 2024. Determining the contribution of onion volunteers to the population genetics of Stemphylium vesicarium in New York, USA, using microsatellite markers. Proc. APS-North East Division Meeting, Ithaca, NY. Pp. 10. 6 March 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Pineros-Guerrero, N., Hay, F. S., Heck, D. W., Klein, A., Hoepting, C. A., and Pethybridge, S. J. 2023. Determining the contribution of onion transplants to the population genetics of Stemphylium vesicarium in New York, USA using microsatellite markers. Proc. International Congress of Plant Pathology, Lyon, France. 20-25 August 2023.


Progress 01/15/22 to 01/14/23

Outputs
Target Audience:During 2022, fourpresentations were made to growers and twotours of field trialswere held. In addition Hoepting and team hosted weekly grower meetings to discuss scouting data and pesticide recommendations(June 7, 14, 21, 28, July 5, 12, 19, 26, August 2, 9, 16, 23 and 30, 13 weeks). Hoepting and team also conducted the CCE CVP Onion Scouting Program in commercial onion fields in Elba muck (10 fields), Wayne Co. (5 fields) and Oswego Co. (6 fields) which involved weekly scouting from June 6 to August 29, and September 12 and 27 (15 weeks), included 14 weekly scouting reports provided to participating growers. Three newsletter articles on onion disease controlwere published in Veg Edge. Changes/Problems:For objective 1, one of the fungicide field trials was oversprayed with fungicide by the co-operating grower which compromised late season and yield data. However, early assessments of this trial, and other trials conducted as part of this objective, have provided sufficient information to compensate for this. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Presentations: Hoepting, C.A. 2022. Research highlights from 2021 onion fungicide trials and new fungicide recommendations for 2022 growing season. CCE Cornell Vegetable Program Muck Donut Hour. Elba, NY: June 21, 2022 (10 participants). Hoepting, C.A. 2022. Research highlights from 2021 and new onion fungicide recommendations for 2022. Annual Muck Onion Twilight Meeting. Butler, NY: June 16, 2022 (74 attendees). Hoepting, C.A. 2022. Fungicide programs in the face of fungicide resistance: Updates from the field. Annual Orange County Onion School. Ottisville, NY: March 10, 2022 (51 attendees). Hay, F.S. 2022. Update on fungicide sensitivity testing. Cornell Co-operative Orange County Onion School (by Zoom) March 10, 2022. (51 attendees) Tours: C.A. Hoepting. 2022. Tour of on-farm onion fungicide trial for Botrytis leaf blight and Stemphylium leaf blight. Elba, NY: September 9, 2022 (6 attendees). Caldwell, S.K. and C.A. Hoepting. 2022. Tour of on-farm onion fungicide trial for Botrytis leaf blight and Stemphylium leaf blight. Fulton, NY: September 8, 2022 (5 attendees). Weekly grower meetings Hoepting, C.A. 2022. Elba Muck Donut Hour.Weekly grower meetingsto discuss scouting data pesticide recommendations. Met on June 7, 14, 21, 28, July 5, 12, 19, 26, August 2, 9, 16, 23 and 30 (13 weeks): 110 contacts. Hoepting, C.A., S.K. Caldwell and N Gropp. 2022. CCE CVPOnion Scouting Programin Elba muck (10 fields), Wayne Co. (5 fields) and Oswego Co. (6 fields). Weekly scouting from June 6 to August 29, and September 12 and 27 (15 weeks), included14 weekly scouting reports. Newsletter articles: Hoepting, C.A. and F.S. Hay. 2022. 2021 fungicide research highlights for Stemphylium leaf blight in onion - The fall of the FRAC 3s and keeping onions green despite poor SLB control. Veg Edge, 18(13): 3-5. Hoepting, C.A. 2022. Slim pickings for fungicides to control Stemphylium leaf blight of onion in 2022. Veg Edge, 18(12): 4-5. Hoepting, C.A. 2022.Best fungicide options for control of Botrytis leaf blight in onion: It depends on what kind of spot you got. Veg Edge, 18(11): 4-6. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Fungicide evaluation and grower adoption. Data from three on-farm trials to screen fungicide efficacy will be analyzed and results shared with growers. Further field trials will be established in 2023. Objective 2. Disease prediction. A third field trial will be established in 2023 to determine the capacity of disease forecasters to predict Stemphylium leaf blight disease. Objective 3. PCR techniques to identify fungicide resistance Further investigations will be conducted to identify gene mutations associated with FRAC 2 and 3 resistance in Stemphylium vesicarium. Objective 4. Monitoring SLB for fungicide resistance. Further screening of fungicide sensitivity will be undertaken for S. vesicarium isolates collected in 2022 and 2023.

Impacts
What was accomplished under these goals? Objective 1 - Fungicide evaluation and grower adoption: (Milestones 1.1 and 2.1) Year 1 trials to screen fungicides for efficacy against SLB were successfully completed. Data was analyzed in Winter 2022 and shared with growers through several medium in 2022. Highlights from 2021 on-farm fungicide trials: Keeping onions green with FRAC 3 + 3 and P07 Several combinations of fungicides were identified which significantly improved onion health. For example, FRAC 3 + 3 + P07 treatments resulted in the greenest foliage in the trial (33-37%) with the fewest plants that died standing up (2-3%). Keeping onions green with FRAC 7 premixes/tankmixes Several FRAC 7 premixes and combinations with FRAC 2 significantly improved onion health. For example, Luna Tranquility (7 + 9), Luna Experience (7 + 3c) and Miravis Prime (7 + 12) had significantly 2-, 3- and 4-times more green foliage than FRAC 7 alone (Endura), which was not significantly different than the untreated. Compensating for poor SLB control with improved plant health In this trial, several FRAC 2, 3 and 7 active ingredients alone were not effective at controlling SLB spores or target spots or preventing leaf dieback, but some combinations improved plant health and resulted in acceptable yield and bulb quality. (Milestone 4.1) Harvest and analysis of data for year 2 small plot fungicide trial. In 2022, several fungicide treatments were evaluated for control of SLB and leaf dieback in three on-farm small-plot trials in Oswego (1) and Elba (2). All trials were arranged as randomized complete block designs with 4 replications. Individual plots were 4 onions rows (= 5 ft) wide and 15 ft long with 2 ft between tiers. Data was collected from the inner 2 rows per plot. On 8-10 randomly selected plants per plot disease assessments were taken approx. 7 days after the 3rd, 6th and 8th sprays in the first Elba trial, after the 5th and 8th sprays in the second Elba trial, and after the 5th and 7th sprays in the Oswego trial. Green foliage (%) per plot was visually estimated 17 and 12 days after the 8th (last) sprays in the first and second two Elba trials, respectively, and 11 and 19 days after the 9th (final) spray in the Oswego trial. The Oswego trial was harvested and graded in the field on Aug 31, and the second Elba trial was harvested on Oct 3-4, and graded on Oct 14, 17-12, 26-27. The grower cooperator sprayed over top of the trial area with fungicides for the last 3 spray weeks of the first Elba trial, which caused SLB for the last disease ratings to be very low. Since there were no meaningful differences in green foliage/plant health due to very low disease pressure, yield data was not collected. Data entry, analysis and summary are currently underway. Objective 2 - Disease prediction (Milestone 4.2) A replicated field trial was conducted to examine the accuracy of the disease forecaster BSPcast in predicting SLB in onion. Greenhouse raised onion (var. Expression) was transplanted into May 31. Each plot was 20 ft. long and consisted of 2 rows of onions transplanted through black plastic at a spacing of 6 inches (80 plants/plot). Plots were separated by 20 ft. buffers. To achieve different levels of foliar wetness, 4 plots were covered with plastic rain shelters with sides and ends exposed to reduce humidity, 4 plots received rainfall only, 4 plots received high levels of leaf wetness with misting in addition to rainfall, 4 plots received a low level of additional misting in addition to rainfall. Leaves (n = 25) were collected from individual plants in each plot on Jun 27, Jul 12, 19 and 26, Aug 2, 9, 16 and 23 and visually assessed for percent necrosis. Leaves were incubated in high humidity for 10 d, and incidence of S. vesicarium (conidia or pseudothecia) recorded. Air temperature and leaf wetness within each plot was measured at 15 min intervals with Spectrum Technologies 1650 dataloggers. In addition, an Easylog UL-USB-2 datalogger (Lascar Electronics) in a Stevenson screen was placed in each plot to measure air temperature and humidity at 15 min. intervals. Statistical analysis will be conducted to determine the association between BSPcast prediction in a plot in each week and the change in SLB incidence for that week. Objective 3 - Development of PCR techniques to identify fungicide resistance (Milestone 4.3): Mutations in the succinate dehydrogenase genes (Sdh) are known to be associated with resistance in fungi to FRAC 7 fungicides. In 2021, several mutations associated with FRAC 7 resistance in Stemphylium vesicarium were identified on the SdhB (P230H), SdhC (G79R, H134R, C135R) and SdhD (Q12K, D126E, S120P and D126N) genes. In 2022, isolates with particular mutations were tested for sensitivity to FRAC 7 active ingredients fluopyram and fluxapyroxad in agar plates amended with 0, 0.2, 1, 5, 10 and 25 µg a.i./ml. For each isolate a plug of mycelium was placed in the center of agar plates with three replicate plates per concentration. The diameter of colonies was recorded at 6 days and the concentration of fungicide necessary to reduce colony diameter by 50% in comparison to the control (EC50) was calculated by probit analysis. Different mutations conferred different levels of sensitivity to FRAC 7 a.i.'s. However, the mutations of most common occurrence in field isolates of S. vesicarium (H134R, C135R and G79R) were all associated with high levels of fungicide insensitivity for FRAC 7 a.i.'s. Resistance to FRAC 2 fungicides in fungi has been associated with mutations in the Hexokinase (HK) genes, and such mutations have been reported in S. vesicarium in Europe (Alberoni et al., 2010). The HK region of three isolates of S. vesicarium which were sensitive, slightly insensitive and moderately insensitive to iprodione were sequenced, with a mutation detected only in the HK of the slightly insensitive isolate. New PCR primers have been developed to amplify the HK and will be tested against a greater number of isolates. Objective 4 - Monitoring SLB for fungicide resistance (Milestone 4.4): Isolates of S. vesicarium collected from diseased onion fields in Elba, Wayne Co., Oswego Co., and Orange Co., NY in 2020 (n=114) and 2021 (n=81) were tested for fungicide sensitivity to three active ingredients (a.i.) in the FRAC 3 group (difenoconazole, propiconazole and tebuconazole). Results suggest the rate of development of insensitivity to difenoconazole is slower than tebuconazole, with propiconazole intermediate between these. Some regional differences are also apparent. For example, for difenoconazole, the percentage of isolates in the highly sensitive fungicide category (EC50 < 1.0 µg/ml) was 51.5, 47.8, 64.3 and 0% from Elba, Oswego, Wayne and Orange respectively. For tebuconazole the percentage of isolates in the highly insensitive fungicide category (EC50 > 10 µg/ml) was 33.3, 56.5, 33.3 and 100 % from Elba, Oswego, Wayne and Orange respectively. This suggests a faster rate of resistance development in Orange Co., compared to other regions. For propiconazole there was little evidence of regional differences with 12.1, 17.4, 20.0 and 27.3% of isolates in the highly insensitive fungicide category (EC50 > 10 µg/ml) from Elba, Oswego, Wayne and Orange respectively. Results will be used to inform changes in fungicide programs necessary to maintain disease control and slow the development of fungicide resistance. Isolates ofStemphylium vesicarium(n=498)were collected from Orange, Oswego and Wayne Counties, and from the Elba region during 2022, for future analysis of fungicide sensitivity.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: C. A. Hoepting, E. R. van der Heide, and S. K. Caldwell. 2022. Efficacy of fungicide tank mixes for control of Botrytis leaf blight and Stemphylium leaf blight in onion, Elba, 2021. Plant Disease Management Reports 16:V138 http://www.plantmanagementnetwork.org/pub/trial/pdmr/reports/2022/V138.pdf
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: C. A. Hoepting, E. R. van der Heide, and S. K. Caldwell. 2022. Efficacy of FRAC 3 and 7 mixes for control of Botrytis leaf blight and Stemphylium leaf blight in onion, 2021. Plant Disease Management Reports 16:V139 http://www.plantmanagementnetwork.org/pub/trial/pdmr/reports/2022/V139.pdf
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: C. A. Hoepting, E. R. van der Heide, and S. K. Caldwell. 2022. Efficacy of fungicide products for control of Botrytis leaf blight and Stemphylium leaf blight in onion in Oswego, 2021. Plant Disease Management Reports 16:V140. http://www.plantmanagementnetwork.org/pub/trial/pdmr/volume16/abstracts/v140.asp
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Pineros-Guerrero, N., F.S. Hay, D.W. Heck, A. Klein, C.A. Hoepting and S.J. Pethybridge. 2022. Sensitivity of Stemphylium vesicarium isolates to demethylation inhibitors (Poster). In: Proceedings of the American Phytopathological Society Annual Meeting, Pittsburg, PA, USA: August 6-10, 2022.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Heck, D.W., A. Klein, N. Pineros-Guerrero, F.S. Hay, C.A. Hoepting, S.J. Pethybridge. 2022. Lack of sensitivity in Stemphylium vesicarium isolates to succinate dehydrogenase inhibitors from onion in New York (Poster). In: Proceedings of the American Phytopathological Society Annual Meeting, Pittsburg, PA, USA: August 6-10, 2022.
  • Type: Other Status: Published Year Published: 2022 Citation: Hoepting, C.A. 2022. Cornell onion (dry bulb) fungicide Cheat Sheet for control of leaf diseases in New York, 2022. Cheat Sheet: Cornell Cooperative Extension  Cornell Vegetable Program Website. Posted: July 5, 2022 (2 pages). Online: chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/ https://rvpadmin.cce.cornell.edu/uploads/doc_1076.pdf. Updated annually: 2019, 2020, 2021, 2022.
  • Type: Other Status: Published Year Published: 2022 Citation: Hoepting, C.A. 2022. 2022 Relative performance of fungicides including tank mixes for control of Botrytis leaf blight (Table 1) and Stemphylium leaf blight (Table 2) in onion. Cheat Sheet: Cornell Cooperative Extension  Cornell Vegetable Program Website. Posted: July 5, 2022 (2 pages). Online: chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://rvpadmin.cce.cornell.edu/uploads/doc_1077.pdf
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Hay, F. S., Heck, D. W., Sharma, S., Klein, A., Hoepting, C., and Pethybridge, S. J. 2022. Stemphylium leaf blight of onion. Plant Disease Lesson. The Plant Health Instructor 22: 10.1094/PHI-P-2022-01-0001


Progress 01/15/21 to 01/14/22

Outputs
Target Audience: Nothing Reported Changes/Problems:Limited data was obtained from the field trial due to atypical weather patterns in 2021, which resulted in rainfall periods each week. This resulted in insufficient differences in leaf wetness between 'no', 'low' and 'high' leaf wetness treatments. In 2022 we will utilize low tunnels over the 'no' leaf wetness treatments to prevent rainfall from affecting results. In addition, some weather data was lost due to equipment malfunctions despite equipment being purchased new and tested prior to the field trial. Following discussions with the manufacturer, some faulty leaf wetness sensors have been replaced. In 2022 wewill also utilize humidity sensors in each plot to act as a surrogate for leaf wetness, should further malfunctions occur. What opportunities for training and professional development has the project provided?A Post-doctorate has gained skills in population biology and microsatellite marker development. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The population biology component of the project willbe analyzed to determine any differences in genotype of S. vesicarium between regions. A secondfield trial will be established to assessforecasting models for predicting infection periods.

Impacts
What was accomplished under these goals? Objective 1. Use a population biology approach to elucidate the dominant sources of inoculum for SLB epidemics in onion crops in New York (Research). The goal of this study was to develop a set of polymorphic and reproducible microsatellite (SSR) markers for population biology analyses of Stemphylium vesicarium. The fungal isolates were isolated in 2016 and 2018 from symptomatic onions collected in different fields across New York state. The isolates were stored in a collection of the Epidemiology of Vegetable Diseases (EVADE) laboratory at Cornell AgriTech facilities in Geneva, NY. The genome of two isolates (On16-63 and On16-391; Sharma et al. 2019) were used for microsatellite library development. Twenty-six non-genic loci were selected based on the number of repeat motifs and physical distance in the genome. Primers were designed for all selected loci and were tested for amplification and reproducibility in seven isolates (On16-13, On16-20, On16-83, On18-4, On18-43, On18-247, and On18-478). Nineteen primers were selected, and the PCR products were sequenced to confirm the presence of microsatellite repeats. A final set of 11 polymorphic and reproducible SSR markers were used for population genetic studies of S. vesicarium. The 5' end of the forward primer was labeled with one of the four fluorophores (6-FAM, NED, PET, and VIC). The eleven SSR markers were divided in two multiplex reactions for optimization and amplification of 36 isolates. The multiplex genotyping was conducted in reactions of 12.5 µL volume with Multiplex 5x Master Mix (New England Biolabs) containing different concentrations of each primer pair. The PCR products were resolved by fragment analysis in an ABI 3730cl DNA Analyzer at the Cornell University Institute of Biotechnology, using a GeneScan-500 LIZ size standard. In total, 141 isolates of S. vesicarium collected from different hosts, fields, counties, states, and years were genotyped with the markers developed. Additionally, three S. beticola isolates collected from spinach seeds were used to inspect for cross-species applications. All the eleven SSR markers amplified for all 141 isolates of S. vesicarium. Meanwhile, only four markers amplified for S. beticola. The number of alleles ranged from three to 20 with an average of 8.36. The Nei's allelic diversity index ranged from 0.38 to 0.94 with an average of 0.73. Further analyses will be performed on this dataset to inspect additional genotypic diversity indices and genetic distance among populations, and indexes of association for random mating, population structure/admixture and linkage disequilibrium. Objective 2. Determine the role of agronomic practices of inter-seeding with barley on SLB epidemics in onion fields in New York (Research). Previous studies have indicated that barley may be a host of S. vesicarium,and that S. vesicarium can colonize and sporulate on dying grass species. Rows of barley are often seeded between rows of onion as a windbarrier, with the former emerging more quickly to act as a to protect the slower emerging onion. Barley leaves (n=30) were collected from inter-row barley from each of eight commercial fields on 25 May 2021. Barley in some fields had been killed with herbicide, and plants were either completely dead (1 field), with some green leaves (4 fields) or still green (3 fields). Leaves were incubated in plastic bags under high humidity for 7-10 days to encourage fungal growth,and examined under a microscope for the presence of conidia of S. vesicarium or pseudothecia of the sexual stage (Pleospora allii). Stemphylium vesicarium was detected in barley from two fields at low incidence (3.3% or less) (Table 1). Pseudothecia of the sexual stage (P. allii) were not detected. By contrast leaves from volunteer onion plants (n=11) collected from two fields had 81.8% incidence of S. vesicarium, with no evidence of P. allii. Results suggest that inter-row barley does not constitute a significant source of S. vesicarium for the emerging onion crop. However, volunteer onion emerging from bulbs remaining in the field from the previous year is likely to constitute an important source and growers should ensure to remove volunteer onion from fields. Objective 3. Evaluate the potential of forecasting systems based on weather-based risk to support decisions surrounding fungicide application for SLB control (Research/Extension). Fifteen onion plots were established with transplant onion (3-4 leaf stage) free of S. vesicarium at planting. All plots received sub-surface drip irrigation, while 5 plots received a low level of supplemental irrigation and 5 plots received a high level. Buffer rows of onion were inoculated with conidia of S. vesicarium to act as an inoculum source. Leaves from 50 plants per plot were retrieved at weekly intervals and incubated under high humidity and observed for conidia of S. vesicarium or pseudothecia of the sexual stage (P. allii) to determine weekly disease incidence. Air temperature and leaf wetness were monitored in all plots with Spectrum Technologies 1650 microstations and weather data was input into the model BSPcast to identify periods conducive to spore germination and infection. The trial was only partially successful and insufficient data was collected for statistical analysis. The 2021 season was unusual in having weekly rainfall events which contributed to infection periods in all treatments leading to no significant difference in disease incidence between treatments. Further, some weather data was lost due to malfunctions in loggers. Trials in 2022 will be modified to include plots covered with low tunnels to ensure dry conditions in some plots. Objective 4. Develop an integrated disease management strategy for SLB in onion and support the dissemination of findings through on-farm demonstrations, newsletter articles, research bulletins, and presentations at extension meetings (Extension). No activity on this objective for this reporting period.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Hay, F., Stricker, S., Gossen, B.D., McDonald, M.R., Heck, D., Hoepting, C., Sharma, S., and S.J. Pethybridge 2021. Stemphylium leaf blight of onion: a re-emerging threat to onion production in Eastern North America. Feature Article Plant Disease https://doi.org/10.1094/PDIS-05-21-0903-FE