Improving Orchard Productivity Using Novel Disease Management Practices for Control of Apple Powdery Mildew in the Northeast USA

Progress report for GNE20-240

Project Type: Graduate Student
Funds awarded in 2020: $14,434.00
Projected End Date: 03/31/2023
Grant Recipient: Cornell University
Region: Northeast
State: New York
Graduate Student:
Faculty Advisor:
Kerik Cox
Cornell University
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Project Information

Summary:

Apple powdery mildew is an endemic foliar disease of apple in the northeastern United States. The pathogen, Podosphaera leucotricha, is largely managed by programs for apple scab, the primary fungal pathogen of concern to apple growers in the northeast. However, global climate change projections forecast that the northeast US region will experience greater periods of heat and increased variability in precipitation in the coming decades; environmental conditions that are conducive to the development of powdery mildew. Fungicides are the primary means of disease management, and application schedules are routine and prophylactic, resulting in unnecessary applications that leads to increased production costs and off-target environmental consequences. This project will develop new methods with which to manage apple powdery mildew using biopesticides and reduced-risk single-site fungicide products, improved management timing based on disease forecasting, and a robust molecular assay to screen for fungicide resistance should it develop. Outreach activities will include providing on-farm fungicide resistance risk consultations based on our molecular assays, presentation of results at regional grower schools and field days, and the development of extension deliverables.

Project Objectives:

The goal of this multi-year project is to leverage novel technologies and ideas to better manage the disease apple powdery mildew in the northeast USA, and proactively develop methods to assess resistance to highly effective single-site fungicides so that these tools are not lost in later years. Specific objectives include:

(1) Evaluate fungicide management programs that integrate commercially available biopesticide and reduced-risk single-site fungicide products for effective management of apple powdery mildew.

(2) Innovate timing of management practices using weather metrics as a form of disease forecasting to improve control and reduce fungicide inputs.

(3) Continued support for development of a robust PCR assay to identify mutations in target genes involved in single-site fungicide resistance in Podosphaera leucotricha.

Introduction:

The purpose of this project is to leverage contemporary disease management technologies and novel research for improved control of apple powdery mildew in the northeast United States. Powdery mildew may be of mounting importance in northeastern commercial apple production due to climate change’s influence on precipitation patterns causing unexpected periods of dry weather during susceptible developmental stages, which increases disease risk. Climate assessments report that since the 1990s the northeastern United States has experienced more pronounced year-to-year precipitation variability (more in the winter, less in the summer) and an average temperature increase regionwide (Hayhoe et al., 2008). These trends are predicted to continue and raise concern that apple powdery mildew may become a greater threat as the region’s general climate becomes more conducive for the disease in the next decade. Increasingly earlier springs will also favor early budbreak in apples (Wolfe et al., 2005), providing the pathogen additional time for mildew epidemics to develop and spread. Concern also exists that the geographical distribution of both the host and pathogen will also be shifted further due to fluctuating environmental shifts at the continental scale (Coakley et al., 1999), placing production regions in the northeast into warmer climates favoring pathogen development.

Research

Materials and methods:

Objective 1: Evaluate fungicide management programs that integrate commercially available biopesticide and reduced-risk single-site fungicide products for effective management of apple powdery mildew and apple scab.

Field studies will be conducted at Cornell AgriTech in a research orchard to evaluate fungicide management programs that integrate commercially available biopesticide and reduced-risk single-site fungicide products for effective management of apple powdery mildew. The orchard is planted to semi-dwarfing 22-year-old ‘Jonagold’ trees on M.111 rootstocks in a randomized block design with four replications. The block has history of severe powdery mildew epidemics and the cultivar ‘Jonagold’ is highly susceptible to powdery mildew. Fungicide programs being evaluated are listed in Table 1, and all applications will be made in 7-10-day intervals from tight cluster to second cover. Biopesticide products were chosen based on promising efficacy shown in preliminary trials, and single site fungicide products were chosen based on their low-risk for resistance development. Powdery mildew disease incidence will be assessed in late summer, defined as the percentage of leaves with sporulating lesions for eight fully expanded leaves from the terminal end of ten shoots. The incidence of apple scab will be similarly assessed to determine the impacts of these programs on a disease that co-occurs with powdery mildew. Disease incidence data will be subjected to analysis of variance (ANOVA) for a randomized block design using accepted statistical procedures and software (i.e. Generalized Linear Mixed Models (GLIMMIX)) procedure of SAS (version 9.4; SAS Institute Inc., Cary, NC). All percentage data will be subjected to arcsine square root transformation prior to analysis.

Table 1: Proposed Management Programs using Biopesticides integrated with Conventional Single-Site Fungicides

Treatment Number

Disease Management Program

Timing

1

Untreated Control (no fungicides)

-

2

3 lbs Koverall 75 (mancozeb) + 2.5lbs Captan 80 Alternated with Microthiol Disperss 15 lbs/A

TC – 2nd Cover

3

Rampart (phosphorous acid) 48 fl.oz/A alternated with Cevya (mefentrifluconazole) 5.0 fl.oz/A

TC – 2nd Cover

4

Rampart (phosphorous acid) 48 fl.oz/A alternated with Flint Extra (trifloxystronbin) 5.0 fl.oz/A

TC – 2nd Cover

5

Double Nickel LC 1qt/A alternated with Cevya (mefentrifluconazole) 5.0 fl.oz/A

TC – 2nd Cover

6

Double Nickel LC 1qt/A alternated with Flint Extra (trifloxystronbin) 5.0 fl.oz/A

TC – 2nd Cover

Objective 2: Innovative timing of management practices using weather metrics as a form of disease forecasting to improve control and reduce fungicide inputs.

A trial to evaluate weather-based protocols for timing fungicide applications for apple powdery mildew will be conducted at Cornell AgriTech. The trial will be conducted in three research orchards with four apple cultivars of differing susceptibility to powdery mildew. Orchard 1 has cultivars ‘GingerGold’ and ‘Cortland’, which were planted in 2008 on B.9 rootstocks. Orchard 2 has cultivar ‘Jonagold’ was planted in 1998 on M.111. Orchard 2 has a breeding selection, ‘HH1501’ (progeny of a ‘Golden Delicious’ cross) was planted in 2016 on G.935 rootstocks. Weather-based application protocols (treatments) will be arranged randomized complete block design with four single-tree replications with buffer trees present. Three weather-based protocols for timing fungicide applications will be evaluated (detailed below): the standard calendar-based application schedule (positive control), applications based on a forecasting model developed to manage barley powdery mildew, and applications based on the apple powdery mildew conidia release model. An untreated (negative) control will be included to assess effectiveness of the protocols. Irrespective of the protocol, a standard fungicide management program will be applied to protect trees from apple scab, which will include fungicides that have not impact on powdery mildew. This program will occur from tight cluster until terminal bud set. Each application protocol will implement a standard program for powdery mildew consisting of single-site fungicides (DMIs and QoIs) and sulfur, in rotation as necessary to adhere to label requirements. The application protocols will begin at tight cluster and end ay 2nd cover when powdery mildew risk is highest. Fungicides found to be successful against apple powdery mildew in research trials in Geneva will be used and include: sulfur (Microthiol Disperss, FRAC Code M02, UPI), Rhyme (flutriafol, DMI FRAC code 3, FMC Agricultural Solutions), Flint Extra (trifloxystrobin, QoI, FRAC Code: 11, Bayer CropScience), and Sercadis (fluxapyroxad, FRAC Code: 7. BASF) will be used in each a, which are detailed below. Whenever seven days have past, but weather conditions do not dictate a protocol-based application, Captan (Captec 4L Drexel), which has no effect on powdery mildew, but will be used to suppress apple scab.

 

  1. Calendar-based Management Program

Fungicides applications will be made every 7-10 days until terminal bud set (typically late July, early August in Geneva).

 

  1. Barley Powdery Mildew Forecasting Model (Polley and King 1973)

Applications of fungicides will be made whenever a “high risk period” is forecast until terminal bud set. High risk weather is defined at point which three of the four following weather conditions listed below are met:

            Daily maximum temperature > 60.08°F (15.6°C)

            Dew point deficit temperature > 41°F (5°C)

            Daily rainfall < 0.0393 in (1 mm)

            Day run of wind > 153 mi (246 km)

 

  1. Apple Powdery Mildew Conidia Release Forecasting Model (Sutton and Jones 1979)

Applications of fungicides will be made whenever a “high risk period” is forecast until terminal bud set. High risk weather is defined at point which three of the four following weather conditions listed below are met:

            Daily maximum temperature > 68°F (14.4°C)

            Average wind speed > 5 mph (8.04 kmph)

            Average relative humidity > 60%

            Average solar radiation > 224.7 Lang/hr

 

To determine the impact of each management program on disease development, disease severity will be assessed for each of the programs every two weeks until terminal bud set in August. Powdery mildew severity is defined as the percentage of leaf surface area covered with sporulating lesions for eight fully expanded leaves from the terminal end of ten shoots per replicate plot. Disease progress curves based on severity will then be used to calculate Area Under the Disease Progress Curves (AUPDC) to assess the effectiveness of each forecasting system in a quantitative manner. Mean AUDPC values for each replicated plot will be subject to ANOVA for a randomized block design using the (GLIMMIX) procedure of SAS (version 9.4; SAS Institute Inc., Cary, NC.).

 

Objective 3: Continued support for development of a robust PCR assay to identify mutations in target genes involved in single-site fungicide resistance in Podosphaera leucotricha.

A robust PCR assay is currently in development to screen fungal isolates of Podosphaera leucotricha for single nucleotide polymorphisms (SNPs) that are known to confer fungicide resistance to most used classes of single-site fungicides (DMIs, QoIs, and SDHIs) in the northeast USA. Some of the funds from this grant would be used in the continued support of this long-term project. To develop and evaluate the assay in this objective, a series of steps are required:

 

Genome and transcriptome assembly of P. leucotricha:

During the early summer months, conidia of P. leucotricha will be isolated from visibly sporulating colonies on the leaves of susceptible cultivars. To reduce the chance of collecting conidia from multiple sources, sporulating colonies will only be selected if no other colonies were present on a leaf, and only one colony will be selected from an infected shoot. Genomic DNA will be extracted with a modified CTAB protocol (Doyle and Doyle, 1990). Total RNA will be extracted using an optimized E.Z.N.A. Plant RNA Kit (Omega Bio-Tek) then delivered to the Cornell Biotechnology Resource Center (BRC) for quality assurance and a reverse transcription procedure to generate cDNA. Genomic DNA and transcriptomic cDNA sequencing will be performed by the Cornell BRC with an Illumina Mi-Seq 250bp paired-end run. A de novo genome assembly will be constructed from the genome sequence data using SPAdes (v3.13.2, St. Petersburg State University). A de novo transcriptome assembly will be constructed from the cDNA sequence data using Trinity (v2.8.6, Broad Institute).

 

Characterization of target genes and PCR assay development:

Using the draft genome/transcriptome assemblies of P. leucotricha and previously published gene sequences of interest from related fungal species, areas of high homology will be identified, and primers designed to target the P. leucotricha CYP51, cytb, and SdhB genes. Gene sequences will be amplified and submitted to the Cornell BRC for Sanger sequencing, then analyzed and annotated in CLC Main Workbench (v.8.1.2, Qiagen, Redwood City) to confirm the PCR assay successfully and reliably amplifies the regions of interest in fungicide resistance development.

 

Screening northeastern US P. leucotricha isolates for SNPs:

Powdery mildew isolates will be collected from research and commercial orchards from around the northeast US region, and genomic DNA and extracted as described previously. The PCR assay targeting the regions of interest in each gene where known SNPs conferring fungicide-resistance reside will validate whether fungicide resistance to DMIs, QoIs, or SDHIs is present in populations of P. leucotricha regionwide.

Research results and discussion:

Objective 1: Evaluate fungicide management programs that integrate commercially available biopesticide and reduced-risk single-site fungicide products for effective management of apple powdery mildew and apple scab.

2020 Annual Report Update: Objective 1 has not yet been undertaken as the grant period began after the 2020 growing season in which the trial could've been accomplished. The trial detailed in the Objective 1's methods section below will be undertaken during the summers of 2021 and 2022, and the results provided in those years' respective annual reports.

 

Objective 2: Innovative timing of management practices using weather metrics as a form of disease forecasting to improve control and reduce fungicide inputs.

2020 Annual Report Update: Objective 2 has not yet been undertaken as the grant period began after the 2020 growing season in which the trial could've been accomplished. The trial detailed in the Objective 2's methods section below will be undertaken during the summers of 2021 and 2022, and the results provided in those years' respective annual reports.

 

Objective 3: Continued support for development of a robust PCR assay to identify mutations in target genes involved in single-site fungicide resistance in Podosphaera leucotricha.

2020 Annual Report Update: The work detailed in Objective 3 is well underway. Short-read sequencing technology (Mi-Seq by Illumina) has been used to generate both de novo genome and transcriptome assemblies for Podosphaera leucotricha (causal agent of apple powdery mildew), as described in the Methods section above. However, an obstacle to this work has appeared: these short-read assemblies proved unable to resolve the entire length of the three genes of interest for this study (CYP51, cytb, and SdhB). Although powdery mildew genomes are poorly characterized and we cannot be certain, we believe this may be due to large amounts of repeatable genetic elements in the P. leucotricha genome that may render short-read sequencing technology inefficient to the task. To overcome this obstacle, in 2021-2022 we will explore the use of novel long-read sequencing technology (Sequel II by PacBio) that has recently become commercially available to generate a new de novo genome assembly that allows us to resolve the entire lengths of the genes of interest. In December 2020, I attended a virtual PacBio Sequencing workshop hosted by the Cornell Bioinformatics facility to learn more about this technology.

 

Meanwhile, should the de novo genome efforts prove futile, efforts have been made to screen partial lengths of the three genes of interest (CYP51, cytb, and SdhB) using primers designed from previously published CYP51, cytb, and SdhB gene sequences from related fungal species that show of high homology (similarity). In short, this will allow me to screen for the known single nucleotide polymorphisms (SNPs) that confer fungicide resistance in other  fungal organisms which may do the same in P. leucotricha. However, this method provides information only to the partial length of the genes of interest and cannot resolve novel SNPs that may be conferring fungicide resistance; hence the goal to resolve the whole gene lengths with de novo sequencing as described above. Ten (10) P. leucotricha isolates collected from the research orchards at Cornell AgriTech have been screened for these historically known SNPs in 2020. No SNPs were identified that correspond to fungicide resistance conferment in the causal pathogen. In 2021-2022, more fungal isolates will be collected from the AgriTech research orchards and (pending Cornell's easement of Covid-19 travel restrictions) commercial orchards around the Northeast to better characterize the P. leucotricha population.

 

Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary:

Education/outreach description:

Results will be communicated via well-established channels relied upon by apple growers statewide for orchard recommendations, as follows. Presentations will be given at regional industry and extension meetings, such as the Cumberland-Shenandoah Fruit Workers Conference held annually in November, The Empire State Producers Expo and the ENY Fruit & Vegetable Conference and Lake Ontario Fruit Schools held annually in February. Findings will be reported as articles in industry publications, such as in NY Fruit Quarterly. I presently write a yearly update on apple powdery mildew for the seasonally relevant extension publication Scaffolds, and will include the research outcomes of this proposal in that yearly update and weekly forecast predictions. Information will also be conveyed through direct conversations between extension educators, as well as on-farm consultations with farmers whose orchards are screened for fungicide-resistant populations of apple powdery mildew.

 

2020 Annual Report Update:

Due to the ongoing Covid-19 pandemic and Cornell University's travel restrictions, on-farm consultations to collect powdery mildew samples were not undertaken this year. In 2021 I will be able to complete this task should Cornell's travel restrictions ease. With respect to presentations, many grower conferences were cancelled or delayed due to the pandemic. I anticipate being able to virtually attend some events in early 2021 to present this work.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.