Progress 09/01/24 to 08/31/25
Outputs
Target Audience:An ongoing quarterly survey conducted by the American Mushroom Institute (AMI) and Penn State University (PSU) reported that both the phorid fly (Megaselia halterata) and sciarid (Lycoriella ingenua) were widespread, with phorids being the predominant pest in Chester County, PA. The survey also reported that Trichoderma Green Mold (Trichoderma aggressivum), Dry Bubble (Lecanicillium fungicola), Hairy Mold (Syzygites megalocarpus), and Cobweb (Cladobotryum sp.) were the major fungal pathogens. The Trichoderma Green Mold crop loss estimates for 40 respondents approached $3M per year. Likewise, Bacterial Blotch disease of mushrooms is one of the most economically important production and postharvest problems faced by mushroom producers worldwide; it is estimated that 10% of mushroom crops worldwide are lost due to bacterial diseases, and 100% losses occur on a healthy crop in only a few days. A recent 2022-23 informal polling of growers suggested that average yield and quality losses from pests and diseases are approximately. 10-15%. These diseases and flies remain significant problems for mushroom growers in North America, and management options are limited. Conventional and organic growers have an urgent need for new technology, basic science, outreach, and management strategies for mushroom flies, La France Isometric Virus (LFIV) disease, bacterial blotch, and fungal pathogens. In response, we have formed a team of experts in plant pathology, environmental microbiology, entomology, computer science and technology, economics, and extension to collaborate on this project because mushroom cultivation involves complex interactions and engineered systems. 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?The Extension programming coordinated a diverse range of educational meetings and strategic planning sessions. These activities supported integrated pest management (IPM), disease prevention, and long-term industry sustainability, while promoting engagement with both growers and institutional partners. In-person and hybrid pesticide meetings were held in Chester and Berks Counties and featured presentations in both English and Spanish. Topics included a new species of beneficial nematode for controlling mushroom phorid flies; a detailed presentation on the life cycles of mushroom pathogens and how they relate to symptom development. In April 2025, a Mushroom Industry Strategic Planning Meeting was held in Kennett Square, PA. What do you plan to do during the next reporting period to accomplish the goals?Continue to determine the influence of spore concentrations and timing of infection on symptom development, using spores from symptomatic mushrooms. Currently, we are conducting experiments using varying levels of viral load at inoculation and monitoring infection dynamics at different developmental stages of the mushroom. Completion and statistical analysis of all experimental datasets to determine the impact of LFIV quantification and symptom expression. Optimization of the qPCR protocol for higher throughput and potential field deployment. Explore novel strategies to optimize the existing qPCR protocol for potential use in field conditions. This future direction has emerged from repeated discussions with farmers and industry partners who expressed a strong need for a practical, field-deployable LFIV detection tool. During on-farm sampling, yield and disease incidence will be assessed to quantify virus impact to determine statistical correlations between infectivity, titres, and yield reduction. Repeat S. carpocapsae optimization field studies on more farms to increase the sample size. Perform comprehensive field studies to compare S. scimitus application timings and rates on mushroom farms. Develop our hybrid deep learning model that includes the characteristics of both CNN and vision transformers. We also plan to re-train the system with annotations that include groups of insects close to each other, since most of the false positives we observed existed when this happened. Incremental Learning/Active Learning-based approaches: Use the current model to generate predictions for new samples; Use the ambiguous predictions to annotate new samples (Active or human intervention); Create a combined dataset merging the old and new samples; Re-train a new model. In addition, we plan to test the functionality of our web-based deep-learning system when connected to the Cropsmarts mobile application on a cooperating mushroom farm, and work with the Cropsmarts development team to improve user experience based on user feedback. Present at the Mushroom Short Course, Pesticide Credit Meetings, Phorid Fly Action Council meetings, and individually with growers on their farms. Complete a bilingual fact sheet about S. carpocapsae nematodes and applications. Begin writing a fact sheet on S. scimitus mites and applications. We will complete the identification of pathogens from TN and OK and compare these to pathogens from PA and CA. Findings from metagenomes indicate that non-Pseudomonas blotch pathogens may be present on mushroom cap samples from Pennsylvania. We will use semi-selective media for these pathogens to try to isolate them. Inhibition of the blotch pathogen by GRAS compounds will be initiated. The activity (killing vs. inhibition) will be further described using the panels of pathogens identified in the surveys. Alternative bioassays will overcome potential issues with the oxidation of resazurin by some mushroom extracts that interfere with the analysis. 200 pathogens from the surveys will be sequenced using Nanopore technology to further describe potential virulence factors. Full TnSeq library generation in progress for 4 strains. These strains will be used to evaluate genes involved in the fitness of these pathogens under various conditions. The impact of applications of virus, bioactive mushroom extracts, and GRAS compounds will be evaluated on mushroom cap microbiomes. Confirm the Thermal Death Time (TDT) of mushroom pathogens. The TDT is the time necessary to kill a given number of organisms and is established by maintaining a constant temperature while time is determined. Because microorganisms are more heat sensitive during the active growing phase than during the stationary spore stage phase of the life cycle, we will determine the TDT of spores from Trichoderma. Lecanicillium, Syzygites and Cladobotryum spp., as well as A. bisporus spores harboring LFIV. We will examine typical fuel requirements (fuel oil and natural gas) needed to raise to the temperatures determined necessary for pasteurization and maintain that temperature in growing rooms in BTU equivalents. A sensitivity analysis model will be created to compare various energy requirements and total costs of pasteurization based on assorted energy prices for oil and natural gas. We are testing casing layers in combination with different bioactive compounds for their effectiveness against mushroom fungi and flies. We are profiling flavonoid bioactives from 400 sorghum accessions and identifying high biomass sorghum accessions to harvest large amounts of bioactives to test against fungi and insect larvae. Identify the top 10 high-biomass sorghum accessions with high bioactivity of flavonoids and test these high-biomass accessions for large-scale flavonoid extractions. We will be fractionating the mixture of bioactive flavonoids to identify flavonoid species with high levels of bioactivity. Continue design, development, and testing of functionality in the Cropsmarts mobile application to capture images associated with recorded crop inputs, crop measures, and crop outputs. 2) Continue design, development, and testing of an architecture to send images captured through the capability described above to an external URL for processing. This is intended to support the machine learning algorithms being developed by the University of Delaware team 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. Continue exploring opportunities to leverage crop performance data through data analytics, data visualizations, and integration of artificial intelligence (AI). Continue usability walkthroughs with mushroom growers and other stakeholders in the commercial mushroom agriculture domain. Previous work in this area has led to many usability enhancements to the application, especially in the area of speeding manual data capture across the mushroom crop cycle. Revise and refine the Cropsmarts web app deployment process to lower AWS costs and streamline system administration tasks. We will develop a non-destructive methodology for early disease detection and fly presence utilizing a combination of multi-spectral remote sensing images and machine learning algorithms. We will generate a large set of spectral images to train the network to develop an algorithm to detect early symptom development. We will continue to explore commercialization opportunities. 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 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. We will develop updated bilingual fact sheets on mushroom pests and diseases using data from this project. We will also demonstrate the cost-effectiveness of IPM by calculating NPVs for each technique. Demonstrate and provide detailed instructions on how to use the Cropsmarts mobile app. Conduct on-farm meetings and training events for growers on post-crop steaming, IPM techniques to control flies and diseases, and for mobile app testing. Stakeholder feedback and strategic planning meetings. Publications will be written and translated for other stakeholders.
Impacts
What was accomplished under these goals?
A strain of Agaricus bisporus white button mushrooms, which is currently being used in industry, was chosen as a source of spores for inoculation studies. Sterile grain flasks were inoculated with mycelium from a symptomless mushroom that had been confirmed to have La France Isometric Virus (LFIV). The LFIV infection was confirmed by performing a quantitative polymerase chain reaction (qPCR) procedure. Mushrooms were grown and collected to make spore prints to prepare several concentrations of virus spore solutions used to inoculate compost and casing at different times during mushroom development. Regardless of time or concentration of LFIV spore application, there were no symptoms consistent with LFIV infection on any of the tubs. Further testing with a symptomatic mushroom will be done. During this period, we successfully established qPCR protocols targeting five genomic segments of the LFIV. This achievement fulfills one of the project's primary objectives, aimed at developing a sensitive and robust method for LFIV detection. qPCR was utilized to quantify viral load, incorporating a statistically significant number of samples to ensure data robustness. This multi-segment approach significantly improves detection efficiency compared to traditional single-segment PCR. We hypothesized that asymptomatic-infected mushrooms harbor lower viral titers than symptomatic ones. To test this, we quantified the L1 segment that encodes for the RNA-dependent RNA polymerase (RdRp) gene, using qPCR. Samples were collected from virus-infected beds, comprising 6 symptomatic and 9 asymptomatic mushrooms of the same strain. Our observation shows that double-stranded RNA extracted from asymptomatic-infected mushrooms fails to display the L1 segment in gel-electrophoresis; however, L1 is detected in qPCR, and the L1 copy number in the symptomatic-infected samples is over 240-fold higher than in the asymptomatic-infected samples, which explains why the L1 segment from the asymptomatic-infected samples does not appear in gel-electrophoresis. Protocols were optimized for S. carpocapsae nematode applications on mushroom farms. Optimized nematode applications showed reduced mushroom fly adult emergence by 41% on mushroom farms in field tests. Laboratory studies on S. carpocapsae revealed that chlorinated water (like on farms) does not kill the nematodes, but it does affect their ability to infest and kill phorid flies. Laboratory studies of Vestergaard netting revealed that the Deltamethrin + Piperonyl butoxide netting killed >90% of adult phorid flies after a 1-minute or 5-minute exposure. Laboratory studies of Vestergaard netting remained after initial exposure > 70% effective 7 months, and > 50% effective 12 months. Laboratory studies of Vestergaard netting revealed that the netting remained effective after being steamed, similar to field conditions. We developed a web-based deep-learning system to identify three classes of mushrooms and other insects. We have used fly trap images for training, extracting non-overlapping patches of 256 x 256 pixels. A web application is developed that receives the patches and generates the count and the insect class. We have completed the proposed surveys of mushroom farms for bacterial blotch pathogens and the initial microbiome surveys proposed. We isolated strains using the Prospector™ and our standard procedures from three mushroom houses in CA. We tested all strains for pathogenicity and fluorescence, and sequenced genes for identification. We are currently analyzing this data. Because no farms were identified in FL, two mushroom farms in TN and OK were identified and sampled for mushrooms with blotch. Putative pathogens have been isolated in storage. These bacteria will be evaluated for pathogenicity and genes sequenced for identification. This analysis indicates that non-Pseudomonas blotch pathogens may be present on mushroom cap samples from PA. We evaluated disease on 4 specialty mushrooms and White and Brown Button/Portobella mushrooms. We have made significant progress in extracting, partitioning, and evaluating the bioactive compounds in mushroom species. One partition from maitake was found to consistently inhibit blotch bacteria. Modern temperature sensors are used to measure core wood temperatures during the post-crop steaming process. Thermal probes that would collect and relay data to the newly created beta version of Cropsmarts were used. Two probes were inserted into two types of wooden bed boards from mushroom grow rooms. Based on the size and weight of each plank of wood, we calculated their density. Both the dense and less dense planks of wood reached pasteurization temperature, with the denser wood reaching target temperatures after the less dense wood. The next test was conducted to see if compost itself is working as an insulator. One sensor was placed in the center of a tub of compost another was placed within the wood that was buried in the compost. Both probes heated up slower than the room did, and slower than either the less dense or more dense wood exposed to the air in the room had heated. While both probes reached the same peak temperature, the probe in the wood with compost reached target temperatures about 1.5 hours before the buried probe. The sorghum specialized metabolite group's focus is on the identification of flavonoid bioactive compounds that are effective in increasing the mortality of mushroom flies as well as controlling fungal pathogens of mushrooms. This group has started screening sorghum germplasm accessions for the identification of diverse compounds. Four hundred sorghum accessions were grown at the PSU Agronomy Farm. These 400 accessions represent genetic and chemical diversity from grain, biomass, and sweet sorghum. Leaf samples were harvested and freeze-dried, and will be prepped for spectrophotometric analysis. We have also been testing selected metabolite extracts against mushroom flies in an initial attempt to find germplasm accessions that produce extracts performing better than the preliminary results. A suite of applications, called Cropsmarts, for web and mobile mushroom crop monitoring and data collection/analysis was developed. This application was extended to support image capture for all major crop management records, including crop inputs, crop measures, and crop outputs. This capability also supports other objectives in the project by providing the basic technology infrastructure for image processing. Images can be captured using a smartphone camera and this app. A user records a crop input, crop measure, or crop output, and they can elect to link an image to this record using either the phone camera or by accessing the image gallery. Images are uploaded to the Cropsmarts web application and permanently linked to the associated record. We have integrated thermocouple and other temperature sensors into the Cropsmarts application, and we have experimented with the use of remote sensor technology to capture environmental data from growing rooms, specifically, air temperature, humidity, and carbon dioxide. Subsequent work with commercial-off-the-shelf industrial sensor technologies has shown far more promise. The Extension programming coordinated a diverse range of educational meetings and strategic planning sessions. These activities supported integrated pest management (IPM), disease prevention, and long-term industry sustainability, while promoting engagement with both growers and institutional partners. In-person and hybrid pesticide meetings were held in Chester and Berks Counties and featured presentations in both English and Spanish. Topics included a new species of beneficial nematode for controlling mushroom phorid flies; a detailed presentation on the life cycles of mushroom pathogens and how they relate to symptom development. In April 2025, a Mushroom Industry Strategic Planning Meeting was held in Kennett Square, PA.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Inaugural Plants Insects and Microbes Symposium title Mushroom Microbiomes May 17, 2024 Auburn University, Auburn AL, C. T. Bull
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Best Management Practices for Phorid Fly Control Michael Wolfin. Chester County Pesticide Credit Meeting
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Fungqal Pathogens and Indicator Molds David Beyer, Chester County Pesticide Credit Meeting
|
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.)
|