DEVELOPING A SUSTAINABLE MANAGEMENT PLAN FOR STEMPHYLIUM LEAF BLIGHT IN ONION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1023820
• Project Start Date: Oct 1, 2020
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
N Y AGRICULTURAL EXPT STATION
(N/A)
GENEVA,NY 14456

Performing Department
Geneva - Plant Pathology/Plant Microbe Biology

Non Technical Summary
Onion production is an important component of agriculture in New York. Thestate is ranked fifth in national production of onion, producing 142,246 t from 2,832 ha with a value of $45.7 M in 2017 (U.S. Dept. Ag. 2017). Stemphylium leaf blight (SLB) caused byS. vesicarium has emerged in recent years to become the most severe foliar diseases of onion in NY. Concern among growers of the destructive nature of SLB has led to overuse of fungicides, and development of multiple fungicide resistance. SLB iswidespread in NY, and can cause yield lossesof 30-40% or more. Widespread resistance to fungicides in Fungicide Resistance Action Committee (FRAC) groups 11 andsome group 7 has developed, with developing resistance to FRAC 2 and 9. This leaves onion growers with FRAC 3 and some FRAC 7 products, complicating the adoption of Cornell guidelines for resistance management, i.e. limiting applications of each group per season, and rotation between groups. There is an urgent need to develop strategies to slow the development of resistance to the few remaining fungicides available to growers. PCR techniques will be developed to more effectively monitor resistance to other FRAC groups. In addition, field trials will be conducted to identify additional fungicide FRAC groups for SLB control, to expand options for fungicide rotation. Existing environmental models developed for Stemphylium in pear will be tested for reliability in predicting SLB in onion, leading to more judicious use of fungicides and reduced selection pressure for resistance through improved timing of application. The work is of importance for maintaining thesustainability and profitability of onion production in NY, especially importantfor communities on muck soils, such as Elba and Orange Co., where onionproduction is a major component of local agriculture.

Animal Health Component

75%

Research Effort Categories

Basic

20%

Applied

75%

Developmental

5%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1451	1160	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

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

Field Of Science
1160 - Pathology;

Keywords

plant disease forecasting

fungicide resistance monitoring

onion disease management

Goals / Objectives
Goal: To develop a sustainable management plan for Stemphylium leaf blight (SLB) of onion.Obj. 1: Fungicide evaluation and grower adoption.Annually monitor for developing field resistance to fungicidescurrently used for the control of SLB, and screen for efficacious fungicides in FRAC groups not currently used by the NY onion industry through on-farm small-plot fungicide evaluations and field scouting. Results will be conveyed to growers through weekly discussion groups during the onion-growing season at which time growers will be supplied with fungicide recommendations based on field assessments and outcomes of research objectives.Obj. 2. Disease prediction.Field-test disease predictive models based on environmental conditions as a tool for sustainedfungicide use for SLB.Obj. 3: Development of PCR techniques to detect fungicideresistance.Develop PCR techniques to detect mutations in the hexokinase(HK), cyp51 and succinate dehydrogenase genes in which mutations have been associated with resistance in other fungi to FRAC 2, 3 and 7 fungicides respectively , amplify DNA from isolates with known fungicide sensitivity phenotype and determine sequence differences associated with fungicideinsensitivity.Obj. 4: Monitoring of fungicide resistance.Quantify the incidence of mutations associated with fungicideinsensitivity in S. vesicarium collected from the four main onionproducing regions, to determine the extent of insensitivity, and shifts in sensitivity over time.

Project Methods
Obj. 1: A small-plot field trial will be conducted in one commercial onion fieldannually, similar to Hoepting (2018 a,b,). Trials will be arranged in aRandomized Complete Block design with four replications and at least sixtreatments annually. FRAC 2, 3, 7 and 9 fungicides including premixes, pipeline products and biological fungicides (e.g. FRAC 44, MB02) will be tested for their efficacy on Stemphylium leaf blight (SLB). Fungicide treatments and a 'maintained' control will also receive pesticides tocontrol downy mildew and thrips. In addition a 'no pesticides' control willreceive no pesticide applications for comparison. Fungicides will be applied at least five times during the season with a CO2-pressurized backpack sprayer at 40gpa and 31-35 psi using three TeeJet 8005 VS flat fan nozzles spaced 19 in. apart. Treatments will be initiated in July when onion are approximately 8-9 leaf/0.5-1 in. bulb stage, and continued every 7 to 10 days until 40-70% lodging in August. Disease assessments will be conducted at least three times during the season on 10 plants/plot including total number of Stemphylium leaf blight (SLB) target lesions per plant, and visual estimation of percent leaf dieback/plant and percentSLB colonization of dieback tissue/plant. Data will be analyzed by general analysis of variance (ANOVA) and Fisher's Protected Least Significant Differencetest (Statistix 10).Commercial onion fields (20) will be scouted every week during the growingseason, and growers provided with weekly SLB incidence and severity data, coupled with research-based fungicide recommendations.Obj. 2: In a current NIFA CPPM project, Hay will conduct preliminary work to adapt the BSPcast predictive model developed for Stemphylium brown spot in pear to SLB in onion. This FCF project, will allow on-farm evaluation and ground-truthing of this model in an additional ten onion fields. Dataloggers(Spectrum Technologies) measuring air temperature and leaf wetness will be deployed within a non fungicide treated plot (20 ft x 20 ft) within each field and downloaded at weekly intervals and input into an Excel template which willcompute daily disease severity according to BSPcast as lesions per leaf (S) using inputs of daily wetness duration (W) and mean air temperature during wetness periods (T) according to: Log10(S) = -1.70962 + 0.0289T + 0.04943W + 0.00868TW - 0.002362W2 -0.000238T2W.Daily periods will be considered from 8:00 am of the previous day to 8:00 am of the current day. The maximum daily disease severity (S) predicted by the model is 3.79, therefore a relative daily infection risk (R) is calculated as R = S/3.79, to allow R to range from 0 to 1. A cumulative daily infection risk (CR) is obtained by summing R values for the previous 3 days, with R and CR calculated daily. In pear orchards in Europe fungicide applications in orchards are triggered by CR values of 0.4-0.5 or 0.6 for orchards with moderate or low disease pressure respectively. The accuracy of prediction will be determined by comparing CR values with changes in disease incidence and severity at weekly intervals. For disease severity, 30 leaves will be collected from individual plants in a nontreated plot (20 ft x 20 ft) within each commercial onion field and assessed visually for percentage leaf dieback. For disease incidence, leaves will be visually inspected under a microscope (50 x) for the presence of Stemphylium conidia or pseudothecia upon collection. Any leaves without sporulation will be incubated in humid bags and observed again after 7 days, to capture any latent infections at the time of sampling. The final incidence will be recorded weekly. For days on which disease assessments are conducted, receiver operatingcharacteristic (ROC) curve analysis will be used to assess the relationshipbetween weekly changes in disease incidence (i.e. disease incidence on day (n)minus incidence on day (n-7)) and various CR values on days n-7 to n-14, torepresent a range of potential incubation periods. Data will be divided intogroups of 'cases' (intervals in which disease incidence increases between day nand day n-7 or 'controls' (intervals in which disease incidence does not increase). ROC analysis will be performed to evaluate the ability of a particular CR valueat various times prior to day n (i.e. days n-7 to n-14) to predict infection (i.e. increase in disease incidence/severity between sample times). The true positiveproportion (TPP) will be calculated by dividing the number of true positives (i.e.intervals where SLB increased) by the total number of cases. The false positiveproportion (FPP) will be calculated as 1-TPP. ROC curves will be constructedusing the ROCR package in R (Sing et. al. 2005), with TPP plotted on the verticalaxis and FPP on the x axis. The area under the ROC curve (AUROC) will becalculated to evaluate the accuracy of prediction, with values of 0.5 to 0.7, 0.7 to 0.9 and >0.9 indicating low, moderate and high predictive power respectively (Tegg et al. 2015). Youden's index (J) will be calculated (Youden 1950) to furtherassess predictive power. The CR value associated with the highest level of J will be considered optimal (Bewick et al. 2004; Hay et al. 2016). ROC analysis will beconducted in a similar manner to determine the best relationship between CR values at preceding times and changes in the incidence of leaf infection between weekly sample times. Model predictions will be included in the weekly onion scouting reports to growers (Obj. 1) and used to fine-tune fungicide recommendations.Should the model prove to have utility in predicting SLB in onion in NY,discussions will be held with NYS IPM to develop the model for inclusion in the Network for Environment and Weather Applications (NEWA) pest and diseaseforecasting platform.Obj. 3: Analysis of our recently assembled genome of S. vesicarium (Sharma et al.2020) has identified the HK, cyp51 and SDH genes in which mutations have beenassociated with resistance in other fungi to FRAC 2, 3 and 7 fungicides respectively. Potential primers to amplify these regions in S. vesicarium will be developed using Primer 3, and assessed for specificity by a BLAST search of theGenBank database. DNA will be extracted using the Wizard Extraction Kit, from mycelium grown in V8 broth. PCR conditions will be optimized in a series of experiments and used to amplify DNA from isolates collected in 2018/2019 with known fungicide sensitivity phenotype. PCR products will be quantified,prepared for sequencing with ExoSAP-IT and sequenced in both directions atthe Cornell, Institute of Biotechnology, BRC. Forward and reverse sequences for each PCR product will be aligned using Geneious to obtain a consensus sequence and compared visually between isolates of S. vesicarium with either sensitive or insensitive phenotype, for single nucleotide polymorphisms (SNP's) associated with resistance, and compared with SNP's associated with resistance in other species.Obj. 4: Ten isolates of S. vesicarium previously collected from each of three fieldsin the four main onion growing regions in 2018/2019 collected in 2021 and 2022 (as part of this project) will be grown in V8 broth, DNA extracted and subjectedto PCR developed in Obj. 3. PCR amplicons will be sequenced and examinedusing Geneious, for SNP's associated with fungicide resistance. Data will beanalyzed to determine any regional differences in types and incidence of SNP'sassociated with fungicide resistance, and to determine changes in the frequencyof fungicide resistance within regions over time.

Progress 10/01/20 to 09/30/21

Outputs
Target Audience:NY onion growers participated in a number of extension events hosted by C. Hoepting. Hoepting, C.A. 2021. Tour of on-farm Oswego onion fungicide trial, Oswego, NY: August 27, 2021 (11 participants). Hoepting, C.A. 2021. Tour of on-farm Elba onion fungicide trials, Elba, NY: August 24, 2021 (9 participants). Hoepting, C.A. 2021. New onion fungicide recommendations for 2021. Annual Oswego County Onion Growers Twilight Meeting, Oswego, NY: June 24, 2021 (51 participants). Hoepting, C.A. 2021. New onion fungicide recommendations for 2021. Elba Muck Donut Hour, Elba, NY: June 22, 2021 (8 participants). In addition, a presentation was made by C. Hoepting to growers in the Republic of Serbia by Zoom.. Hoepting, C.A. 2021. Practical experiences in the fight against Stemphylium of onions. Belgrade, Republic of Serbia Grower Meeting, Serbia: January 18, 2021 (40 participants). In addition, a presentation was made to 20 onion researchers and extension educators as part of the W3008 Multistate project. Hay, F., Heck, D., Hoepting, C., Klein, A., Pethybridge S.J. 2021. Stemphylium leaf blight - current status of fungicide insensitivity. Update for W3008 Multistate Project Annual Meeting, February 3, 2021 (by Zoom). Changes/Problems:The initial methodology for the forecasting trials involved monitoring leaf wetness and air temperature in 10 commercial crops and collecting leaves at weekly intervals to assess disease incidence. Due to concerns with Covid-19 potentially affecting travel, it was decided to conduct this part of the project in 16 plotson the Cornellresearch farm. Differential overhead misting was used to simulate a range of environmental conditions. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Two field tours were held (Elba and Oswego) to show results of field trials to growers. Two presentations were made to grower groups. Presentations (by Zoom) were made to growers in Canada and the Republic of Serbia. What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will include a second on-farm trial to assess fungicides and a further field trial to collect more data to examine the accuracy of disease forecasating models.

Impacts
What was accomplished under these goals? Objective 1 (Milestones 1.1 and 2.1) Year 1 small plot trials to screen fungicides for efficacy against SLB successfully completed. In 2021, a small-plot on-farm fungicide trials were conducted in Elba muck in Orleans Co. Ten FRAC groups including four sub-classes of FRAC 7 (sub-classes 1, 3, 4 & 6) and four active ingredients of FRAC 3 (a, b, c and d) were tested. Individual products included Quadris Top (FRAC 3b + 11), Tilt (FRAC 3a) Viathon (FRAC 3c + P07), Cevya (FRAC 3d), Luna Tranquility (FRAC 7(1) + 9a), Luna Experience (FRAC 7(1) + 3c), Folicur (FRAC 3c), Rampart (FRAC P07), Miravis Prime (FRAC 7(4) + 12), Endura (FRAC 7(3)), a.i. pyrazilfumid (FRAC 7(6)), Rovral (FRAC 2), Vivando (FRAC U13), Gatten (FRAC U08), Scala (FRAC 9a), Omega (FRAC 29), Gavel (FRAC 22 + M03) and Oso (FRAC 19). To determine effect of applying multiple FRAC 3 products on SLB control, Quadris Top + Tilt was compared to Quadris Top + Tilt + Viathon, and Cevya was compared to Cevya + Tilt. To determine the effect of P07 on SLB control, Rampart (FRAC 07) was compared to Viathon (FRAC 3c + P07), Folicur (FARC 3c), and Luna Experience (FARC 7(1) + 3c) + Rampart (P07), and Cevya (FRAC 3d) was compared to Cevya + Rampart. Co-application of other FRAC groups included Rovral (FRAC 2) co-applied with each of Luna Tranquility (FRAC 7(1) + 9a), Miravis Prime (FRAC 7(4) + 12) and Scala (FRAC 9a), Badge (FRAC M1) + Rampart, Omega (FRAC 29) + Prev-AM (a.i. sodium tetraborohydrate) and Gavel (FARC 22 + M03) + Oso (FRAC 19). To determine whether foliar application of micronutrients may alleviate SLB, Quadris Top + Tilt was also tested with each of Ele-Max Super Zinc, Yara Vita Stop It (Calcium), Zinc + Calcium and Green Stim. To determine importance of application timing of effective SLB treatments, Viathon + Tilt was applied bi-weekly (ACE) and started late (DEF) compared to weekly (A-F). There was also a program that included Miravis Prime (FRAC 7(4) + 12) A fb. Scala (FARC 9a) + Rovral (FRAC 2) fb. Viathon (FRAC 3c + P07) + Tilt (FRAC 3a) fb. Luna Tranquility (FRAC 7(1) + Rovral (FRAC 2) fb. Cevya (FRAC 3d) + Quadris Top (FRAC 3b + 11) fb. Viathon + Tilt. Each treatment was replicated four times. The first spray was initiated after first detection of SLB on Jul 12. Treatments were sprayed weekly for six weeks. Data was collected per plant on 10 plants/plot on % leaf tipburn/dieback, % SLB spore colonization of necrotic leaf tissue, SLB target lesion condition (presence of tan, black and purple target lesions on necrotic and green tissue, whether there were greater than 3 targets/plant, and if disease appeared to be primary or secondary in nature). Percent green foliage and % plants dying standing up was visually rated per plot. The trial was harvested, onions topped and graded and weighed by size. Data entry, analysis and summary is underway. Objective 2 (Milestones 1.2 and 2.2) Disease forecasting trial in 10 commercial fields was changed to accommodate monitoring in 16 plots in a replicated plot trial at Research South, Geneva. Due to concern regarding potential for periodic Covid-19 restrictions during 2021, it was decided to undertake a replicated trial at Research South, Geneva rather than conduct work on commercial farms. The replicated trial allowed a greater number of environmental scenarios than originally envisaged, i.e. 10 commercial fields. The trial involved 16 plots of onions with differential levels of leaf wetness duration applied by misters. Each plot was 10 feet long and consisted of 80 onion transplanted at the 4-leaf stage. Onion plants were arranged in 2 double rows at 6 in. spacing within rows. Onion was planted through black plastic with subsurface drip irrigation. Plots were bordered by 20 ft. buffers along rows. Subsamples of leaves (n=50) collected at transplanting indicated no infection by S. vesicarium. For inoculum, four isolates of S. vesicarium from commercial farms were grown on V8 agarfor 14 d on 4 petri plates under fluorescent light (12 h: 12 h, light:dark) to encourage the formation of conidia. Plates were flooded with 10 ml of sterile water with surfactant (0.01% Tween 20) and the colony surface scraped lightly with a glass microscope slide to dislodge conidia. The suspension was diluted to 7300 conidia/ml and 100 ml applied to 10 plants in the middle of buffer rows with a Solo sprayer on July 7. Four replicate plots received either no additional overhead misting, a low level of mist or high level of mist. Misting was done arbitrarily based on prevailing weather conditions. From July 15, leaves (n = 25) were collected at weekly intervals from individual plants in each plot for 5 weeks. Leaves were visually assessed for percent leaf area with necrosis and then placed in individual plastic bags containing moistened tissue to provide high humidity. Leaves were incubated for 14 d and observed under the microscope for signs of S. vesicarium (conidia or pseudothecia). Air temperature and leaf wetness within each plot was measured at 15 min intervals using leaf wetness sensors and Spectrum Technologies 1650 dataloggers. Environmental data was processed and run through the BSPcast model to provide a daily R value, with R >0.2 indicating risk of infection. The trial was only partially successful. Some environmental data was lost due to periodic malfunction of leaf wetness sensors which reduced the number of data points collected. Some sensors were replaced by the manufacturer. In addition, the year 2020 was unseasonably wet with frequent rainfall events over the whole season. Of the 60 weekly data points collected, 55 had days with R > 0.2, and 54 recorded an increase in incidence of S. vesicarium. This resulted in insufficient data points in which in which R < 0.2 to allow ROC analysis of the accuracy of prediction. The trial is to be repeated in 2022. The 2021 data that was collected will be aggregated with data collected in 2022 to provide a larger data set. To improve success in the 2022 trial, plots which receive no supplemental misting will be covered with plastic tunnels. On days in which rainfall is expected, plastic will be rolled down to cover the plot at times during the season to ensure low levels of leaf wetness in some weeks, with daily R < 0.2. In addition, a logging humidity sensor will be placed in the canopy in each plot. Preliminary studies have determined a relationship between humidity and leaf wetness, suggesting that humidity could act as a surrogate for leaf wetness. Objective 3 (Milestones 1.3 and 2.3) Mutations in the succinate dehydrogenase genes (SdhA, SdhB, SDhC and SdhD) are known to be associated with resistance in fungi to FRAC 7 fungicides. PCR primers and protocols were successfully designed to amplify the Sdh genes of S. vesicarium. Sdh genes of 235 isolates of S. vesicarium were sequenced and mutations associated with FRAC 7 resistance were identified. Several mutations associated with FRAC 7 resistance were identified on the SdhB (P230H), SdhC (G79R, H134R, C135R) and SdhD (Q12K, D126E, S120P and D126N). The most frequently occurring mutations in fungicide resistant isolates occurred on the SdhC. Primers were designed to amplify the Cyp51 gene to examine mutations associated with FRAC 3 resistance. The Cyp51 gene of 8 isolates of S. vesicarium, including 4 isolates sensitive and 4 insensitive to FRAC 3 were sequenced. No evidence of mutation associated with fungicide insensitivity was detected, indicating the a mechanism other than a single nucleotide polymorphism confers resistance against FRAC 3. ?Objective 4. (Milestones 2.4) A collection of 550 isolates of S. vesicarium was obtained in 2020 from over 25 farms located in Elba, and Oswego, Wayne, and Orange Co. Isolates were single-spored and placed in storage. A subset will be tested for fungicide sensitivity.

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
• Type: Other Status: Published Year Published: 2021 Citation: Hoepting, C.A., Caldwell, S.K., and van der Heide, E.V. 2021. Efficacy of fungicide treatments for control of Botrytis Leaf Blight and Stemphylium leaf blight in onion, Elba, 2020. Plant Disease Management Reports 15: V162.
• Type: Other Status: Published Year Published: 2021 Citation: Hoepting, C.A., Caldwell, S.K., and van der Heide, E.V. 2021. Effect of fungicide application timing on control of Botrytis leaf blight and Stemphylium leaf blight in onion, 2020. Plant Disease Management Reports 15: V163
• Type: Other Status: Published Year Published: 2021 Citation: Hoepting, C.A., Caldwell, S.K., and van der Heide, E.V. 2021. Efficacy of fungicide treatments for control of Botrytis Leaf Blight and Stemphylium leaf blight in onion, Oswego, 2020. Plant Disease Management Reports 15: V123.
• Type: Other Status: Published Year Published: 2021 Citation: Hoepting, C.A., Caldwell, S.K., and van der Heide, E.V. 2021. Efficacy of fungicide treatments for control of Stemphylium leaf blight in onion, Elba, 2020. Plant Disease Management Reports 15: V155.
• Type: Other Status: Published Year Published: 2021 Citation: Hoepting, C.A., Caldwell, S.K., and van der Heide, E.V. 2021. Efficacy of fungicide combinations for control of Stemphylium leaf blight in onion, Elba, 2020. Plant Disease Management Reports 15: V156.
• Type: Other Status: Published Year Published: 2021 Citation: Hoepting, C.A. 2021. Running out of fungicide options for control of Stemphylium leaf blight in onion. Veg Edge, 17(18): 4-5.
• Type: Other Status: Published Year Published: 2021 Citation: Hoepting, C.A. 2021. Onion downy mildew prevention the crutch for limping SLB fungicides to manage DM-SLB complex. Veg Edge, 17(14): 8-9.
• Type: Other Status: Published Year Published: 2021 Citation: Hoepting, C.A. and Hay, F.S. 2021. Part II: Onion fungicide research updates and new recommendations for control of Botrytis and Stemphylium leaf blights, 2021. Veg Edge, 17(13): 8-9.
• Type: Other Status: Published Year Published: 2021 Citation: Hoepting, C.A. and Hay, F. S. 2021. Part I: Onion fungicide research updates and new recommendations for control of Botrytis and Stemphylium leaf blights, 2021. Veg Edge, 17(12): 8-10.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Heck, D., Hoepting, C., Klein, A., Pethybridge S.J., Hay, F. 2021. Stemphylium leaf blight management in onions. 69th Annual Muck Vegetable Growers Conference, Bradford, ON, Canada. April 1, 2021. (by Zoom).