Source: PENNSYLVANIA STATE UNIVERSITY submitted to
FOCUSING ON NOVEL PEST AND DISEASE MANAGEMENT STRATEGIES FOR US MUSHROOM FARMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1031448
Grant No.
2023-51181-41162
Cumulative Award Amt.
$3,470,035.00
Proposal No.
2023-05649
Multistate No.
(N/A)
Project Start Date
Sep 1, 2023
Project End Date
Aug 31, 2025
Grant Year
2023
Program Code
[SCRI]- Specialty Crop Research Initiative
Project Director
Beyer, D. M.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
(N/A)
Non Technical Summary
Our long-term goal is to transition the management of mushroom pathogens and flies, beyond short-term reliance on pesticides that are rapidly and repeatedly lost due to resistance and regulatory processes. Integrate disease management tools, techniques, and diagnostics, with pathogen biology. Increase awareness and implementation of novel and effective IPM solutions by mushroom growers and improve community relations. A previous suite of applications was developed, called Cropsmarts, used for web and mobile crop operations monitoring and data collection/analysis. With this proposed extension to that work, we will continue the cycle of design-development-test working with commercial mushroom farms. Develop and facilitate the adoption of effective IPM strategies to manage mushroom flies on farms. Develop bilingual educational materials for mushroom producers and employees for the management of mushroom flies and diseases. Evaluate natural and mushroom-derived compounds to inhibit growth or disrupt virulence mechanisms of blotch and fungal pathogens and flies. Confirm the thermal death points of several mushroom pathogens in culture and then using an extensive array of remote thermometers, we will monitor post-crop steaming procedures on mushroom farms to confirm the efficiency of their process. Re-investigate the virus symptoms on new hybrid strains that develop under different spore loads and times of infection. Validation of virus testing protocols for diagnostics and improvements to differentiate and detect various virus diseases.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2161470116060%
2111470113010%
2121470110110%
6011470301010%
4021470303010%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 601 - Economics of Agricultural Production and Farm Management; 211 - Insects, Mites, and Other Arthropods Affecting Plants; 216 - Integrated Pest Management Systems; 402 - Engineering Systems and Equipment;

Subject Of Investigation
1470 - Mushrooms and other edible fungi;

Field Of Science
1130 - Entomology and acarology; 1101 - Virology; 1160 - Pathology; 3010 - Economics; 3030 - Information and communication;
Goals / Objectives
The investigators will pursue seven related research and extension objectives focused on mushrooms, integrated pest management, and education/ outreach:?Objective 1: Detect and manage virus diseases in mushrooms.Objective 2: Design and facilitate the adoption of IPM tools to control mushroom fly populations on mushroom farms.Objective 3: Bacterial blotch management and impact on cap and substrate microbiomes.Objective 4: Determine precise thermal death points for fungal and viral pathogens during the post-crop steaming process.Objective 5: Novel broad-spectrum flavonoids from Sorghum bicolor to control fungal pathogens and insect pests of mushrooms.Objective 6: Continued design, development, integration, and usability testing of the Cropsmarts web and mobile apps for monitoring environmental variables, pests, and diseases on mushroom farms.Objective 7: Increase awareness, implementation, and economic impact of novel IPM solutions for mushroom growers.
Project Methods
A postdoctoral researcher with experience in the development of Taqman-based qPCR protocols will work closely with the Pecchia and Kantor Labs and Dr. Mark Wach's research group at Sylvan Inc., (the leading authority of LFIV virus in the western hemisphere) to develop target sequences for the new protocol. Standard curves will be developed from samples with known virus titres and will be used as positive controls in all experiments. After validation of the method, qPCR and standard PCR protocols will be used to determine the incidence and titre of symptomatic and asymptomatic samples (n=10 per farm) taken from commercial A. bisporus farms in CA, FL, and PA. The type of symptoms that develop under varying spore loads and the timing of infection will be documented by measuring yield, mushroom morphology, and mycelial growth in the casing and substrate. Mushrooms showing symptoms will be confirmed as virus-infected mushrooms using the standard PCR or qPCR (when available) analysis described in the previous objective. The timing of infection influence on symptoms and severity experiment will use pure culture derived from virus-infected mushrooms or mycelium inoculated onto sterile grains and then used to infest the substrate at spawning and fully colonized substrate prior to casing following established inoculation protocols at Penn State's MRC. We will generate a large set of manually annotated images of phorid and sciarid flies captured on Pennsylvania Fly Monitor sticky traps using LabelMe software to train our CNN. Preliminary data suggests that thousands of annotated flies from < 20 iPhone and Google Pixel-captured images can produce 87%-92% accuracy in fly detections. The effectiveness of nematodes and mites to control mushroom phorid and sciarid larvae will first be assessed in laboratory studies. Standard colony-rearing cups (12 oz plastic containers filled with colonized compost) will be used in biocontrol efficacy studies. Application rates of the biocontrol agents and timing of applications (with respect to larval development) will be assessed. We have developed a novel and effective screening technique for evaluating the efficacy of pesticide formulations on adult flies. Twenty adult flies are transferred into cone-shaped exposure arenas 24h after pesticides are applied to exposure arenas at their maximum label rate. Flies are then transferred from exposure arenas to release cages after 1 minute, and adult mortality is recorded at 0-hours, 1-h, and 24-h post-exposure. Insecticides will be compared for efficacy against both fly species using analysis of variance (or non-parametric ANOVA) to determine differences in mortality between treatments. We will work across the fields of natural products chemistry, comparative genomics, bacterial taxonomy, and microbiome analysis to identify or develop novel materials for the management of the wide array of bacterial blotch pathogens in the US. We will investigate natural products for inhibition of the full diversity of pathogens and disease control to prevent the emergence of unmanaged blotch species. We will use a published bioassay-guided HPLC fractionation approach to isolate and characterize antimicrobial compounds from fruiting bodies of commercially cultivated or wild edible mushroom species (referred to as medicinal mushrooms). Three fruiting bodies of a minimum of 10 medicinal mushroom types will be inoculated with members of the pathogen diversity panel. we will determine the thermal death times of spores from fungal pathogens as well as A. bisporus spores harboring LFIV. Spores will be collected using a moistened sterile cotton-tipped applicator and placed in 50 ml of sterile water. The spore suspension is filtered through cheesecloth to remove larger pieces of hyphae and quantified using a Bright-LineTM Hemocytometer following the manufacturer's instructions. Nine 100 µl samples containing 3-5 x 104 spores will be prepared for each treatment along with a 100 µl sterile water uninoculated control. Eight of the nine samples containing sporangiospores will be placed into a gradient thermocycler for 10, 15, 30, and 60 minutes, exposing the samples to different temperatures between 34-60 °C. The remaining spore sample (positive control) will be kept at room temperature along with the negative control. We will use an extensive array of remote thermometers to monitor post-crop steaming procedures on mushroom farms to confirm the efficiency of their process. We also propose to set up a lab-based replicate of wood used on farms and the time/temperature needed to penetrate the wood to the thermal death points of Agaricus and pathogen mycelium. Suitable sensors must also include Bluetooth or other reliable communications capabilities to interface with the Cropsmarts mobile data capture platform. We will construct a test-bed apparatus consisting of simulated wooden growing beds at Penn State's MRC. Once a set of candidate temperature sensors have been identified, we will conduct an iterative series of development 'sprints' (design-build-test) to integrate these into the Cropsmarts mobile and web application suite. To accumulate flavonoids compounds, at flowering when 3-DAs accumulation is at its peak in leaves, plants will be harvested/chopped with a silage harvester. Forced air dried biomass will be used and multiple batches will be processed. Flavonoids will be isolated by boiling dry biomass in 2N HCl in a 10L glass vessels followed by isoamyl alcohol extraction and fungal growth assays will be performed. Fungal minimal media in a microtiter plate (200 μl/well) will be supplemented with the 3-DAs at varying concentrations of 0.50, 1.0, 2.0, and 5.0, 10.0, 15 μg/ml and inoculated with the fungal spores. Controls will consist of media with acidified methanol, spores, water only, and uninoculated media. Cultures will be incubated at 25C in the dark. Fungal growth will be quantified by measuring the optical density (λ = 590 nm) over five days as well as the area under the growth curve. We will also test 3-DAs against bacterial blotch. Two methods will be employed for sciarid and phorid fly assays. Twenty-five insects will be loaded per replicate (1-2 days post eclosion) into arenas made of electrostatic screens (powder assay) or plastic food bowls (liquid assay). In the liquid assay, the plastic arenas are evenly coated with 3-DAs, dried for 24 h and flies will be allowed to contact for 1 minute. At the end of treatment, flies will be removed from the arenas, transported to mesh cages (no 3-DAs) and observed for response/mortality for 24 h. We will extend the Cropsmarts application to support image capture and persistence for all major crop management records including crop inputs, crop measures, and crop outputs. This will provide growers with a visual record of all important crop events. Images will be captured using the Cropsmarts mobile app with smartphone camera. When a user records a crop input, crop measure, or crop output, they can elect to link an image to this record using either the phone camera or by accessing the image gallery. These images will be uploaded to the Cropsmarts web application and permanently linked to the associated record. A bilingual IPM manual will be developed and distributed to all participants at in-person training sessions. It will be used as supporting material during training and used for reference during on-farm implementation. A quick-reference laminated poster for fungal, bacterial, and on-farm use of fly IPM techniques with step-by-step instructions on how to properly implement and maintain the techniques will be developed and distributed. Finally, we will develop updated bilingual fact sheets on mushroom pests and diseases using data from this project.

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

Outputs
Target Audience:North American Mushroom Industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?North American Mushroom Conference - several posters and presentations Chester Co. Pesticide Credit Meeting - 2 presentations Berks Co. Pesticide Credit Meeting - 2 presentations 65 on-farm visits 2 webinars 255 phone calls 443 emails What do you plan to do during the next reporting period to accomplish the goals?Research and extension activities will continue as planned.

Impacts
What was accomplished under these goals? A postdoc was hired to work on the project's La France Isometric Virus (LFIV) objective. Vaskar Thapa, Ph.D., is a fungal virologist with extensive virus experience. During this initial phase of the project, we conducted a comprehensive review of the La France Isometric Virus (LFIV) literature, screened commercial mushroom farms in Chester County, PA, for suspected infections, tested our virus detection protocol, and experimented with methods to establish virus-infected mushroom cultures in the lab. We have recently initiated quantitative polymerase chain reaction (qPCR) experiments to detect and quantify multiple viral genome segments. Additionally, we are exploring strategies to sequence previously un-sequenced viral segments. We utilized a dsRNA enrichment protocol to screen for viral infections in mushroom tissue samples. This protocol detects large dsRNA molecules, which serve as a proxy for the virus since most fungal viruses possess dsRNA genomes or use dsRNA as an intermediate in their life cycle. LFIV infection shows a characteristic dsRNA profile, including 7-9 distinct bands in gel electrophoresis. RT-PCR further confirmed this finding using specific primers for multiple viral segments with available sequence information. We are now employing this protocol regularly in the lab for initial virus screening. We pursued two methods to establish LFIV-infected cultures in the lab. In the first approach, we inoculated healthy mushroom spawn with LFIV-infected spore suspension at a rate of approximately 50,000 spores per 20 grams of spawn. After allowing the infection to spread for two weeks, we screened for the virus using the dsRNA protocol. In the second approach, we inoculated a 2-week-old healthy compost culture of the commercial strain, starting with 1 gram of spawn per 100 grams of compost, with a suspension of 10,000 LFIV-infected spores. The resulting mushrooms were then screened for the virus. The first approach proved more successful than the second. These experiments utilized spores collected in 2006 and 2012, and we plan to repeat them using currently circulating LFIV strains. Preliminary qPCR experiments have been initiated to detect LFIV in freshly infected mushroom samples. We designed primers specific to five genomic segments with available sequence information and ran RT-PCR to validate and optimize these primers. Once PCR conditions were optimized, we performed qPCR tests using SYBR Green dye in the BioRad CFX 96 system. The experiment with cDNA templates produced amplification curves for all segments. We are conducting additional qPCR experiments to validate these results and will run experiments with appropriate standard curve fitting for quantification. We are also investigating reliable strategies to sequence LFIV segments for which sequence information is currently unavailable. Our working hypothesis is that LFIV disease severity correlates with the titer of viral genomic segments. Comprehensive sequence information will be crucial to test this hypothesis and determine if any segments have a major role in disease development. Two LaFrance Virus (LFIV) infected Agaricus bisporus strains, an "off-white hybrid" and "Brown," were procured from the Penn State Mushroom Spawn Culture Lab. From the cultures, LFIV-infected grain spawn was prepared. This grain spawn was used to spawn compost in an attempt to produce LFIV mushrooms to collect LFIV-infected spores. Two tubs were made using 200g LFV infected off-white spawn. In a third tub, 200g of uninfected Agaricus spawn was mixed throughout before 15g of LFIV-infected off-white variety rye grain spawn was placed in the center, buried about 1 inch deep, and marked with a sterile toothpick. Another tub was prepared with 200g uninfected Agaricus spawn mixed throughout for a control treatment. The substrate was fully colonized at the time of casing. Unfortunately, very little to no growth was observed in the LFIV-infected treatments. This test will be repeated using less LFIV-infected spawn so the healthy spawn may dominate the substrate and have fewer LFIV particles in the system. For the entomology objective, an effective dosage for nematode applications was determined. It was also determined that nematodes die within 4-5 days of application, and on-farm application protocols were revised to control mushroom flies. Field studies were begun to test revised nematode application methods in collaboration with Todd Watkins and Phil Coles to determine effective dosage for mite applications. It was determined that mites can survive >45 days in mushroom compost, and additional field studies were started to test predatory mite application methods. It was also determined that netting from Vestergaard with their "Roof" formulation effectively killed mushroom phorids for at least 4 months. Began studies to understand the effects of steaming on Vestergaard nets' efficacy. It was reported that all mesh sizes of Vestergaard netting are too big for exclusion, and the supplier is modifying the netting to improve exclusion. The organic bioinsecticide Organishield (sucrose octanoate ester) was found to be effective at killing phorids. Researchers and graduate students engaged in 86 Extension Activities, reaching almost 900 stakeholders and numerous phone calls, farm visits/meetings, and guest presentations. Another two graduate students and a research technologist were hired to work on the project. For the Bacterial Blotch objective, the UFL lab has started searching for alternative mushroom farms, as the facility in Florida listed in the grant was recently closed. Mushroom facilities in Tennessee, Texas, and Oklahoma have been identified, and mushroom sampling will be made this next year. In addition, the PSU has recruited an MS student and Postdoc who have begun to learn the mushroom production system.Preliminary extractions from medicinal mushrooms successfully resulted in some activity againstP. tolaasii. We received and prepared the materials needed for the random barcode TnSeq experiments. For the post-crop objectives of the grant, over the last year, we have continued to design, develop, and test the Cropsmarts applications to support several different objectives of this grant. Specific accomplishments include the following: We have incorporated an RTD temperature sensor to support the "thermal death" part of the study. This sensor was used over four trials to measure and compare internal wood, compost, and air temperatures during growing room steam-off. We have designed, developed, and tested functionality in the Cropsmarts mobile application to capture images associated with recorded crop inputs, measures, and outputs. We have designed, developed, and conducted initial testing of an architecture to send images captured through the capability described in #2 above to an external URL for processing. This is intended to support the machine learning algorithms the University of Delaware team developed to classify and count different species of flies in growing room fly traps. This architecture was designed to be general so that other future processing URLs could be easily added. We have conducted a series of usability walkthroughs with mushroom growers and other stakeholders in the commercial mushroom agriculture domain. These have led to many usability enhancements to the application, especially in speeding manual data capture across the mushroom crop cycle. In response to the trials in #4 above, we have developed a Spanish-language version of the Cropsmarts mobile application. This is still to be tested. Besides the above, we have continued regular internal usability testing of all the Cropsmarts functionality. Many application refinements have been made to enhance the application's ease of use and usefulness. We have also begun exploring commercialization opportunities for Cropsmarts, including participation in the regional NSF I-Corps program.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Delane, R. and Haynes, S.R., 2024. Enabling Smart Mushroom Agriculture. Poster. 2024 North American Mushroom Conference, February 2629, 2024, Las Vegas, NV.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Malachi M. Brought, M.M. 2024 Evaluating a new species of beneficial nematode to control mushroom phorid flies on mushroom farms. March 13  2024 Spring Mushroom Pesticide Meetings (Chester, Co.)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Beyer, D.M. 2024. Life Cycles of Mushroom Pathogens as Related to Symptom Development. March 13  2024 Spring Mushroom Pesticide Meetings (Chester, Co.)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Malachi M. Brought, M.M. 2024 Evaluating a new species of beneficial nematode to control mushroom phorid flies on mushroom farms. March 14  2024 Spring Mushroom Pesticide Meetings (Berks Co.)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Beyer, D.M. 2024. Life Cycles of Mushroom Pathogens as Related to Symptom Development. March 13  2024 Spring Mushroom Pesticide Meetings (Berks Co.)