Progress 08/04/15 to 06/30/20
Outputs
Target Audience:North American Mushroom Industry Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training and professional development for Kim Paley, Research Technician Training for an average of 50 growers per year How have the results been disseminated to communities of interest?Annual Mushroom Short Courses 2015-2019 - 150 participants/year County Pesticide Credit meetings (two/yr.) - Approximately 50 total participants, per meeting On-farm visits - tenone-day visits per year; average of eightcontacts per visit What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1- Evaluate effects of Mushroom Compost (MC) as an ingredient in Phase II and or Phase III substrate on yield and mushroom quality (Beyer) Several cropping tests were done adding MC to Phase II substrate at spawning and Phase III substrate at casing. Previously cropped MC supplementing Phase II or Phase III compost doesn't improve yield even with additional supplements and chelating additives. Evaluate epidemiology and etiology of Syzygites mold, a new pathogen of A. bisporus (Beyer and Pecchia) The thermal death point of S. megalocarpus sporangiospores was determined to be between 36.6C and 37.2C thus, mushroom composting and post-crop steaming are more than adequate to kill the pathogen. No significant differences were found in susceptibility to S. megalocarpus among different commercial A. bisporus strains or inoculation method, though hybrid brown strain stumps appeared more susceptible to spore suspension infection. All mushroom strains had similar time to infection, except for unwounded white strain, which took, on average, twice as long until signs of infection were observed. Objective 2- Improved nutrient uptake based on increased enzyme production of A. bisporus (Pecchia) In an attempt to increase the bioefficiency of A. bisporus we studied lignin peroxidase and Arabinofuranosidase to efficiently breakdown substrates. A. bisporus was transformed with one or both enzymes. Modified A. bisporus colonized the compost faster than commercially available strains, though none of the strains outperformed commercial strains for yield in cropping experiments. Faster vegetative growth in compost may not be a good indicator of yield potential. Investigate the role that indigenous microbial populations (Bacillus spp.) play in suppressing green mold caused by Trichoderma aggressivum (Pecchia) A quick, new plate challenge technique is being used to screen Phase II compost for susceptibility to green mold development. Suppressive Phase II composts generate a zone of inhibition of T. aggressivum when co-inoculated whereas susceptible composts do not and are overgrown with T. aggressivum. Suppressive compost samples were frozen for future study to identify unifying biological or chemical characteristics. Early testing in the lab has demonstrated that both the living cells as well as the cell-free solution suppressed T. aggressivum; however, small-scale control experiments utilizing compost have not been as successful. We investigated the interaction of fourdifferent Trichoderma spp. on mushroom yield and substrate microbiology and microbial analyses of the substrate is ongoing. Likewise, work investigating B. amyloliquefaciens for suppression of T. aggressivum and investigations into fungal ecology are ongoing. Objective 3- Investigate the role of that compost and casing characteristics have on Trichoderma disease (Pecchia) Volatile compounds (VCs) emitted by T. aggressivum and A. bisporus were evaluated for their potential role in fungal interactions. Differentiation in VCs were detected as early as spawn run day six. Microbiome studies were initiated to determine what microbes are present under different cropping conditions and pathogen presence. We determined and published the effects of Phase II composting pasteurization temperatures on microbial populations and subsequent compost conditioning. Indigenous Bacillus spp. were screened in vitro for inhibition of T. aggressivum. Several Bacillus spp. were identified that show excellent activity. Study morel mushroom cultivation indoors (Pecchia) Utilizing fourdifferent species (Morchella importuna, Morchella rufobrunnea, Morchella Americana and Morchella exuberans) from the Penn State Spawn collection we attempted indoor cultivation of morels. M importuna and M. rufobrunnea produced the highest number of mushroom pins in a cropping trial at the Mushroom Research Center (MRC). We studied the effects of pH, light and temperature on mycelial growth and pseudosclerotia formation for the four selected Morchella isolates. PH and light had a significant effect on mycelial growth for three of the Morchella isolates, with no affect observed for M. importuna, while pH had no effect. With the exception of M. exuberans, which had the most mycelial growth at 30C, mycelial growth and pseudosclerotia formation were maximized at 20C. M. americana and M. exuberansdid not produce any pseudosclerotium under any of the tested conditions. Objective 4- Evaluate potential bio-pesticides for control of fungal pathogens (Beyer and Pecchia). Most bio-fungicides tested were not effective against either T. aggressivum and L. fungicola, causing green mold and dry bubble, respectively. However, a commercial Streptomyces griseoviridis fungicide was as effective as the registered chemical treatment in reducing disease development of dry bubble. Additionally, a Gliocladium catenulatum biofungicide was effective in reducing the incidence and severity of green mold disease. A commercially available Bacillus spp. biocontrol product not labeled for use on mushrooms was at least as effective as the currently available labeled control products. Although only minor differences in yield results were noted, the incidence of dry bubble was significantly lower on the inoculated Metrafenone treatments when compared to the other inoculated treatments and equivalent to the un-inoculated control. Additionally, results suggest there is no Metrafenone phytotoxicity for A. bisporus at the labeled and higher dosages. These results strongly suggested that Metrafenone was effective in controlling dry bubble. We tested the efficacy of sorghum flavonoids, which have been shown to be toxic to plant pathogenic fungi, against T. aggressivum and L. fungicola. Sorghum flavonoids are genotype specific and in preliminary experiments sorghum genotypes that are a viable source of antifungal compounds which may be effective against these pathogens were identified. Objective 5- Evaluate the use of wood-based, lignin-rich, or non-composted substrate combinations for growth and production of A. bisporus and other specialty mushrooms (Beyer and Pecchia) Corn stover formulations (50 or 100%) yielded as high as traditional straw-based compost formulations when composted properly. Corn stover may be used in place of straw to produce A. bisporus compost in regions with limited straw supplies. A single experiment comparing Cottonseed Gin Waste (CSW) to Cottonseed Hulls (CSH) suggests that the CGW will perform as well as the CSH in wheat-straw bedded manure and a partial synthetic hay-based substrate. Though changes in raw materials should be incorporated in a judicious manner. Objective 6- Developing bilingual educational materials and short IPM videos for organic mushroom producers and employees for management of mushroom flies and diseases (Beyer) "Steam-Off-Post-Crop Pasteurization to Maintain Low Pest Population" poster was developed. We are currently developing bilingual factsheets for organic mushroom producers and employees for management of mushroom flies and diseases. Once finalized we will develop the short videos on the following topics: A) Basic Integrated Pest Management (IPM) practices for organic mushroom farms; B) Biology and Control of the Mushroom and other Nuisance Flies; C) Biology and Control of Mushroom Diseases. Develop bilingual IPM educational materials for organic mushroom production (Beyer) We have conducted IPM related trainings for organic mushroom production during pesticide meetings and at the 2018 Mushroom Short Course. We are planning on making additional presentations on these topics during future pesticide meetings and Mushroom Short Courses.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Beyer, B. M. Research on Alternative Materials for Compost and Casing - Part I. Mushroom News. (67) (8), (pp. 9). American Mushroom Institute.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Beyer, D. M. Interpreting Compost and Casing Lab Results. Mushroom News. (67)(8), (pp. 5). American Mushroom Institute.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Beyer, D. M. USDA-NIFA Organic Research and Extension Initiative Project, 2019 Update. Mushroom News. (67)(5), (pp. 3). American Mushroom Institute.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Beyer, D. M. The Science Behind Spawn Growth and Nutrient Uptake 61st Mushroom Industry Conference, Chester County. Kennett Square, PA.
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:North American Mushroom Industry Changes/Problems:Objective 1 was changed to look at the cultural factors for controlling Syzygites pathogen. Objective 2 will be the bioassay of biocontrol of Trichoderma green mold. Objective 3 is new and will cover Morel cultivation research. Objective 5 and 6 are new and will include bilingual extension activities. What opportunities for training and professional development has the project provided?Marilyn Bonham - Masters student Xiangrong (Ryan) Guo - Masters student Mariella Butler-Martinez, Zamuel Ziegler, Corey Bullock, Charles Stinson - Undergraduate Students How have the results been disseminated to communities of interest?Annual Mushroom Short Course - 140 participants Four County Pesticide Credit meetings - Approximately 120 total participants On-farm visits - 15 one day visits; average of 5 contacts per visit. Pesticide Meetings - Fall 2018 and Spring 2019 PSU; Spring 2019 - Lambert pesticide talk Shiitake Workshops - October 2018 - Montgomery County ~25 participants May 2019 - Lehigh County ~ 25 participants October 2019 - Allegheny County ~ 15 participants What do you plan to do during the next reporting period to accomplish the goals?Objective 4: Evaluate potential bio-pesticides for control of fungal pathogens.We plan to evaluate commercially available essential oils and biopesticidesin vitroagainst indigenous isolates collected at farms around the country. The fungal pathogensTa2,V. fungicolaand a commercial hybrid strain ofA. bisporuswill be grown in triplicate in 5-cm dia. petri plates containing potato dextrose yeast agar (PDYA) medium (Difco) amended with 5 to 50 μL/L active ingredient. The mean diameter of the cultures will be determined after 3-10 days and 2-3 weeks of growth at 24?C for the fungal pathogens andA. bisporus, respectively. Compounds inhibiting the radial growth of the pathogens without significant toxicity to the mushroom fungus will be advanced to cropping trials. Cropping experiments and a small scale growth chamber bio-assay will be used to evaluate the successfulin vitrocompounds for phytotoxicity and efficacy of control of the fungal pathogens. ForTa2cropping trials,spawn will be dusted with a mixture of calcium sulphate and dry compounds at a concentration determined to be effective duringin vitrobioassay trials described above. For theVerticilliumcropping trials the fungal pathogen will be inoculated with a 104spore suspension sprayed on the surface of the casing. Inoculation will take place several days after casing. An un-inoculated treatment will be included as a control. Just prior to pin set, compounds will be applied at rates determined from thein vitrotrials and at a volume that will provide complete coverage of the casing layer. The incidence and severity of disease development will be measured and the data statistically analyzed.
Impacts
What was accomplished under these goals?
Objective 1 - This objective was not addressed during the 2018-19 reporting year. Objective 2 - This objective was not addressed during the 2018-19 reporting year. Objective 3- Our lab has been screening Phase II compost, from each MRC crop, for susceptibility to green mold development using a quick, new plate challenge technique. The method involves placing Phase II compost directly onto ½ of a PDA plate and placing a plug ofT. aggressivumon the other half. Suppressive composts generate a zone of inhibition at the interface whereas susceptible composts do not and are quickly overrun withT. aggressivumon the plate. In conjunction with the plating challenge, we are freezing compost samples from each test so that we can go back and further analyze composts that naturally suppressive green mold development to determine if any consistent biological or chemical characteristics can be identified. Early testing in the lab has demonstrated that both the living cells as well as the cell-free solution suppressedTrichoderma, however, small-scale control experiments utilizing compost have not been as successful. A crop was also completed investigating the interaction of 4 differentTrichoderma spp. on mushroom yield and substrate microbiology. The microbial analyses of the substrate is still ongoing. Two crops at the MRC were initiated (one completed and one ongoing) testingB. amyloliquefaciensagainstT. aggressivum. The fungal ecology of the substrate was evaluated using who genome sequencing to determine what role fungal populations play in mushroom nutrition and disease development. The results are complete, and a draft manuscript is completed and will be submitted for publication in 2019. Objective 4 -Several bio-fungicides continue to be screened for efficacy against a fungal pathogen. Cueva and MilStop showed no control of Lecanicillium dry bubble disease. Objective 5 -This objective was not addressed during the 2018-19 reporting year.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2019
Citation:
Samuel, M., Trexler, R., Vieira, F., Pecchia, J., Kandel, P., Hockett, K, Bell, T, and Bull, C. 2019. Comparing approaches for capturing bacterial assemblages associated with symptomatic (bacterial blotch) and asymptomatic mushroom (Agaricus bisporus) caps. Phytobiomes Journal.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2019
Citation:
Osdaghi, E., Martins, S. J., Sepulveda, L., Vieira, F. R., Pecchia, J. A., Beyer, D. M., Bell, T. H., Yang, Y., Hockett, K. L., Bull, C. T. 2019. 100 Years since Tolaas: Bacterial Blotch of Mushrooms in the 21st Century. Plant Dis.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Mazin, M., Harvey, R., Andreadis, S., Pecchia, J., Cloonan, K., & Rajotte, E. G. 2019. The mushroom sciarid fly, Lycoriella ingenua (Diptera:Sciaridae) adults and larvae vector Mushroom Green Mold (Trichoderma aggressivum f. aggressivum) spores. Applied Entomology and Zoology. http://https://link.springer.com/journal/13355. [In Press]. ISBN/ISSN #/Case #/DOI #: Online ISSN: 1347-605X
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Vieira, F., Pecchia, J., Segato, F., & Polikarpov, I. 2019 "Exploring oyster mushroom (Pleurotus ostreatus) substrate preparation by varying Phase I composting: changes in bacterial communities and physicochemical composition of biomass impacting mushroom yields." Applied Microbiology and Biotechnology. 126(3):931-944.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:North American Mushroom Industry Changes/Problems:Objective 1 will be discontinued based on the negative results of previous years testing. Objective 2 will be discontinued as the funding from the Swayne Endowment will not be used to support the technician doing this research. Objective 3 will be changed to look at the cultural factors for controlling Syzygites pathogen. Objective 5 will be changed as the funding from the Swayne Endowment will not be used to support the technician doing original objective, so we will look to evaluate alternative casing and compost ingredients and novel cultural practices and tools. What opportunities for training and professional development has the project provided?Graduate students: John De Soto, Jr. Post doc: Fabricio Vieira How have the results been disseminated to communities of interest?Annual Mushroom Short Course - 150 participants Four County Pesticide Credit meetings - Approximately 160 total participants On-farm visits - 28-day visits; average of 8 contacts per visit. Impact - growers have increased their knowledge of cultivation, their yields and profits continue to increase. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 - no further work, based on the poor results to date. Objective 2 - Discontinued, lack of funding. Objective 3 - Changed to look at several commercial disinfectants to be tested for efficacy against Syzygites mold, a pathogen of A. bisporus. Spore suspensions and agar plugs with Syzygites mycelium will be used to for testing commercially available strains of A. bisporus to provide information on the relative susceptibility of the different strains as well as the pathogenicity and virulence of sporangiospores versus its hyphae. Documentation of strain susceptibility could help growers limit disease on their farms by choosing less susceptible strains. Determination of susceptibility to infection caused by mycelia versus spores may help in the development of future control practices. Another objective of this project is to design a reliable biocontrol approach that uses a cell-free solution instead of living bacterial cells, against Trichoderma species associated with mushroom production. Objective 1: Test the efficacy of cell-free biocontrol agents against green mold by direct application to spawn, supplement, compost and casing in a cropping system. Objective 2: Search for low-cost ingredients to develop media culture to optimize production of bioactive agents in a large scale, cell-free solution. Compare the growth ability on compost/casing of the major Trichoderma species (T. aggressivum f. aggressivum, T. viride, T. harzianum and T. lixii) associated with the A. bisporus production and determine their sensitivity to the Bacillus spp. cell-free solution. Build a genetic database (genomes) of the Trichoderma species associated with domestic mushroom production to improve on the current PCR based diagnostic test. Objective 4 - Identifying different bio-pesticides to be tested for efficacy against mushroom fungal pathogens. Based on this year's results we have shown that several bio-fungicides are not effective, so we need to continue screening new or different products. Objective 5 - Funding eliminated so we are changing it to look at alternative casing and compost ingredients. Biochar, produced from mushroom compost, will be tested as an alternative to peat moss as a mushroom casing material. It will also be tested as a compost ingredient and spawning supplement. Mobil app technology will be looked at for use in pest and disease control. Mechanization to reduce labor will be evaluated.
Impacts
What was accomplished under these goals?
Objective 1 - Results to date have shown that Mushroom Compost (MC) cannot be used to supplement Phase II or Phase III compost. No further testing is planned as wehad hopedto show substituting Phase II compost with MC (material from a previous mushroom crop) would reduce the amount of raw ingredients used to produce mushrooms. The results showed that MC added to replace Phase II compost did not yield as well as the control treatments, that was just Phase II compost with no MC. Therefore, trying to replace Phase II substrate with MC would not be economical for the growers. Objective 2 - We continued a project to increase the bioefficiency of Agaricus bisporus yields on currently available composted substrate. Last year we targeted the lignin degrading capacity of A. bisporus by introducing a lignin degrading enzyme (lignin peroxidase) from other white rot fungi. Unfortunately, we did not observe an increase in production through the use of transformed mushrooms with an increased ability to produce lignin degrading enzymes. This year we changed the target and focused on an enzyme needed to completely break down complex carbon molecules during xylan and cellulose degradation (Arabinofuranosidase). Recent work out of the Netherlands suggests that A. bisporus is unable to completely degrade certain carbon molecules thereby limiting the production potential on composted substrates. Our next step was to transform A. bisporus to produce both lignin peroxidase as well as arabinofuranosidase to see if a double pronged approach would work better. After successfully completing the transformations, initial grow tube experiments showed that the modified lines colonized the compost faster than commercially available strains. However, these strains were not yet tested in a cropping experiment. Though many of the transformed strains appeared to grow faster than commercial strains in grow tubes, none of the strains have outperformed commercial strains in cropping experiments. One explanation for this discrepancy may be that faster vegetative growth in compost may not be a good indicator of yield potential. Objective 3 - Microbiome studies were initiated to investigate the role that the casing and compost microbes play in nutrient utilization, fruit body formation and disease development of A. bisporus mushrooms. The initial step in these studies is to determine what microbes are present under different cropping conditions with subsequent objectives focusing on determining the functionality of those mircrobes. We determined the effects that Phase II composting pasteurization temperatures had on microbial populations and how that subsequently effected conditioning of the compost.Bacterial diversity, based on 16S ribosomal RNA obtained by high-throughput sequencing and classified in operational taxonomic units (OTUs), was greater than previously reported using culture-dependent methods. Alpha diversity estimators showed a lower diversity of OTUs under a high-temperature pasteurization condition. Bacillales order showed a relatively higher OTU abundance under a high-pasteurization temperature, which was alsorelated to higherammonia emission measurements during Phase II composting.Agaricus bisporus mycelium growth during a standard spawn run period was slower in the compost pasteurized at high temperature. Our lab has also been screening a series of indigenous Bacillus spp. (found in the compost) against green mold. All of the preliminary testing has been conducted in the lab, to screen for the best candidates. Several Bacillus spp. have been identified that show excellent efficacy in the lab (both cell and cell-free solutions of the culture). Objective 4 -In preliminary experiments, we tested the efficacy of sorghum flavonoids against these pathogens, Green Mold and Dry Bubble caused by Trichoderma aggressivum and Lecanicillium fungicola, respectively. Sorghum produces a rare suite of flavonoid compounds and several derivatives have been identified. The relative abundance of these compounds is dependent upon the sorghum genotype. Related flavonoid compounds have been shown to be toxic to pathogenic fungi such as Cochliobolus heterostrophus and Colletotrichum graminicola but safe for human consumption. Although these compounds are induced in response to biotic and abiotic stresses, the plant also produces copious amounts of these as it senesces. Therefore, we have identified sorghum genotypes that are viable source of these antifungal compounds. The first experimental crop was testing Metrafenone phytotoxicity on A. bisporus, the commercial white and brown mushrooms, when applied after casing at three rates. The results of this trial suggested that there is no significant difference in fresh mushroom yield when Metrafenone was applied at the recommended label rate (1x), twice that rate (2x) and four times the labeled rate (4x). These results suggest there is no Metrafenone phytotoxicity for A. bisporus at the labeled and higher dosages. The second cropping experiment was testing the influence of Metrafenone on the incidence of Lecanicillium dry bubble disease at the end of cropping. Two rates of Metrafenone on un-inoculated treatments were compared to an un-inoculated control and two rates of Metrafenone on Lecanicillium inoculated treatments were compared treatments of an inoculated control, a Mertect (Thiabendazole) application and a Bravo (Chlorothalonil) application. Fresh mushroom yield of the un-inoculated treatments showed no significant difference between the Metrafenone and untreated control, suggesting there was no phytotoxicity. The yield results for third break did correlate with the end of crop incidence of disease. The incidence of Lecanicillium was not significantly different between the inoculated Metrafenone treatments and the un-inoculated control, but was significantly lower than the other inoculated treatments, control and chemical application. The third and fourth cropping experiments were a repeat of the second trial. Basically both crops confirmed the results of the first cropping experiment. Although some minor differences in yield results were noted, the incidence of disease was significantly lower on the inoculated Metrafenone treatments when compared to the other inoculated treatments and significant the equivalent as the un-inoculated control. These results strongly suggested that Metrafenone was effective in controlling dry bubble. We also have been testing the efficacy of different biological control agents against Trichoderma aggressivum, the causal agent of green mold of mushrooms. A commercially available Bacillus spp. biocontrol product, not labelled for use on mushrooms, was been tested on 2 crops for efficacy against green mold. Initial results demonstrated that the product was at least as effective as the currently available labelled control products. A third replicate cropping trial is scheduled to confirm initial results. Objective 5 -Cottonseed hulls (CSH) are a common ingredient for mushroom substrate formulations, however the cost of the hulls is increasing, and they are becoming less available. Therefore, growers have a keen interest in finding a cost-effective replacement for the hulls. We compared substrate formulas of CGW to CSH. Using the mushroom cultural procedures and material employed in this experiment, these results suggest that the cotton gin waste will perform as well as the cottonseed hulls in a wheat-straw bedded manure formula. If used in a partial synthetic (hay based) formula it should do as well as CSH or other carbohydrates supplement such as corn cobs. It is concluded from this single test that cotton gin waste is acceptable for use in commercial mushroom composting. As with all changes on a mushroom farm, it is advisable that the material be incorporated into the standard practices in a cautious and judicious manner.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Vieira, F., & Pecchia, J. An exploration into the bacterial community under different pasteurization conditions during substrate preparation (composting - Phase II) for Agaricus bisporus cultivation. Microbial Ecology 75(7):318-330.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Vieira, F., Pecchia, J., Segato, F., & Polikarpov, I. "Exploring oyster mushroom (Pleurotus ostreatus) substrate preparation by varying Phase I composting: changes in bacterial communities and physicochemical composition of biomass impacting mushroom yields." Applied Microbiology and Biotechnology.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:North American Commercial Mushroom Industry Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate students: Garrett Morrison, Siyi Ge, Rob Harvey and John De Soto, Jr. Post doc: Fabricio Vieira How have the results been disseminated to communities of interest?Mushroom Review workshop, March 2017. 59th Mushroom Short Course, September 2017. Pesticide Credit Meetings, February and October 2017. Conference presentations, county extension meetings, on-farm visits, publications. What do you plan to do during the next reporting period to accomplish the goals?Continued screening of bio-pesticides for mushroom pest and diseases. Research will be conducted to determine the impact of a number of factors on the growth and sporulation of S. megalocarpus. This includes how environmental factors such as temperature, humidity, and pH affect its growth, survival, virulence, and spread in a mushroom industry setting and the possibility of a bacterial symbiont that might affect pathogenicity and virulence. This data will also assist other researchers in culturing S. megalocarpus for research purposes. We also propose to determine what chemicals effectively reduce the growth rate or kill this pathogen. Various compounds will be tested including the following: fungicides, disinfectants, and any available organically friendly alternatives such as biocontrol products or essential oils. This work will be carried out in vitro. In addition, we will determine what effect temperatures and humidity have on the growth of this pathogen and how growth conditions affect the disease cycle, mushroom tissue (both fresh and decaying), harvested from the Mushroom Research Center (MRC) at Penn State University will be inoculated with differing concentrations of sporangiospores (the asexual spores). This same procedure will be repeated using agar plugs with mycelia on them as inoculum. Updates and findings from the project will be presented quarterly to the advisory panel. During the quarterly meetings, members of the advisory panel will have an opportunity to hear, discuss and give their opinions on the direction of future research during the duration of the project. A new web page will be developed and dedicated to maintain communications of project progress. This web page will contain a link from Penn State University's mushroom web page as well as from the AMI industry resources webpage. Faculty, post-docs, and graduate and undergraduate students will have an opportunity to present their findings at extension meetings, Penn State's annual Mushroom Short Course as well as the North American Mushroom Conference. Annual progress reports will be given to the industry advisory panel as well as being submitted for publication in the Mushroom News, the industry trade journal. Scientific findings will be published in referred journals and presented at professional meetings.
Impacts
What was accomplished under these goals?
Cropping experiments were used to evaluate a naturally occurring bacterium, Streptomyces griseoviridis, a commercially available biological fungicide. The trials had combinations of un-inoculated and inoculated-untreated controls compared to a treatment of registered chemicals. It was found to be as effective at rates of 40 mg/sq. ft., when compared to the registered chemical treatment, combinations of Mertect and Bravo in reducing Lecanicillium fungicola (dry bubble disease) disease severity and incidence or disease. This past year we continue to work with different rates of application and timing of this bio-fungicides application. We found that lower rates of S. griseoviridis were not as effective in controlling L. fungicola when compared to previous crops at higher rates. Preliminary results also suggest that a bio-fungicide Gliocladium catenulatum was effective in reducing the incidence and severity of Trichoderma aggressivum f. aggressivum (Ta2) green mold disease. One goal of his research program was to identify volatile compounds emitted by the green mold pathogen and Agaricus mushrooms. Preliminary data has shown that different volatiles are formed when comparing Ta2 infested compost with Agaricus and compost with just A. bisporus mycelium. We identified key volatile organic compounds (VOCs) produced by (Ta2) and A. bisporus collected from cultures grown in Phase III compost. Differentiation between A. bisporus and Ta2 VCs detected as early as spawn run day 6. Our lab worked on the thermal death point of asexual spores, the sporangiospores, of Syzygites megalocarpus a relatively new pathogen of A. bisporus. Multiple replications the thermal death point of S. megalocarpus sporangiospores was determined to fall in between 36.6 C and 37.2 C. We also determined that S. megalocarpus is not very susceptible to either fungicide, Bravo (Chlorothalonil) and Mertect (Thiabendazole), even at concentrations considerably higher than what is considered commercially acceptable. Isolation of potential candidates for biocontrol for Ta2 was tested by isolating bacteria from samples that were taken during Phase I and Phase II composting. A strain of Ta2 was co-inoculated to verify the inhibition pressures with bacterial colonies. Colonies that showed a potential inhibition against Ta2 were re-cultured and purified. The Rep-PCR method showed that the isolates were members of the Bacillus genus (Bacillus subtilis and Bacillus amyloliquefaciens). Several in vitro experiments demonstrated that all of the isolates inhibited Ta2 growth to some extent. On compost, lab-scale experiments, the inhibition of the Bacillus isolates was less efficient than on nutrient plates. In addition, filtered cultures, i.e., cell-free solution, showed similar results to unfiltered cultures on Ta2 inhibition on plates, which drove us to try to identify the compounds released into the liquid media. Spawn run in "race tubes" demonstrated that A. bisporus was not affected by the cell-free supernatant. Interestingly, A. bisporus growth was slightly faster in the presence of cell-free solution, which may be related to available nutrients left after the whole cultures filtration process. The culture cells filtered by a 50 KDa membrane showed inhibitory activity against both pathogens, Ta2and Syzygites megalocapus. Subsequently, ammonia sulfate precipitation revealed higher inhibitory activity in precipitates between 30 % to 60 % ammonia sulfate precipitations. On the other hand, lower inhibitory activity was detected using precipitates between 0 % to 30 % ammonia sulfate precipitations. Little or no activity was observed with precipitates between 60 % - 90 %. The cell-free solutions exposed to different temperatures also showed high stability and maintained the effective inhibition activity against Ta2 in vitro.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Beyer, D.M. and J. A. Pecchia. Efficacy of Bio-fungicides for Control of Mushroom Diseases. 59th Mushroom Short Course Kennett Square, PA September 2017.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Pecchia, J. A. and D. M. Beyer. Corn Stover for Mushroom Compost. 59th Mushroom Short Course Kennett Square, PA September 2017.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:North American Commercial 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?Conference presentation, county extension meeting, on-farm visits, publications. What do you plan to do during the next reporting period to accomplish the goals?Continued screening of bio-pesticides for mushroom pest and diseases. During production, the mushroom produces fruit bodies in a series of breaks or flushes that occur at 7-day intervals, with growers typically harvesting 3 breaks per crop. Fewer mushrooms are produced in each successive break, with a substantial reduction in yield between the second and the third. Although nutrients are still in the substrate it is more economical to replace the old crop and refill the house with a new crop. This project has several objectives aimed at improving growing efficiency and utilizing more sustainable raw materials in the production process. Several approaches are being taken to address the different hypotheses as to what factors may currently be limiting mushroom yields. Updates and findings from the project will be presented quarterly to the advisory panel. During the quarterly meetings, members of the advisory panel will have an opportunity to hear, discuss and give their opinions on the direction of future research during the duration of the project. A new web page will be developed and dedicated to maintain communications of project progress. This web page will contain a link from Penn State University's mushroom web page as well as from the AMI industry resources webpage. Faculty, post-docs, and graduate and undergraduate students will have an opportunity to present their findings at extension meetings, Penn State's annual Mushroom Short Course as well as the North American Mushroom Conference. Annual progress reports will be given to the industry advisory panel as well as being submitted for publication in the Mushroom News, the industry trade journal. Scientific findings will be published in referred journals and presented at professional meetings.
Impacts
What was accomplished under these goals?
Cropping experiments were used to evaluate combinations of Mertect, Bravo, JAZZ and Trilogy for efficacy against T. aggressivum and L. fungicola. L. fungicola was inoculated with a spore suspension several days after casing. The trials had combinations of un-inoculated and inoculated-untreated controls compared to a treatment of registered chemicals. Different rates of application and combinations of bio-fungicides were tested. The most efficient method and timing for these chemicals will be reported and efficacy was determined by fresh mushroom yields and quantification of diseased mushrooms, both bubbles and spotted mushrooms. Our results suggest that most bio-fungicides tested were not effective against either pathogen. However, a couple of essential oils were effective and a naturally occurring bacterium, Streptomyces griseoviridis, a commercially available biological fungicide, was as effective as the registered chemical treatment in reducing L. fungicola disease development. Preliminary results also suggest that a bio-fungicide Gliocladium catenulatum was effective in reducing the incidence and severity of T. aggressivum green mold disease. Trichoderma aggressivum aggressivum, (Ta2), the aggressive "green mold" of Agaricus, persists despite advanced composting technologies. Genetic approaches to detect Ta2 in bulk substrate are inadequate and tunnel systems limit regular sampling. Monitoring volatile compounds (VC) of phase III (P3) compost may provide an early detection method of Ta2. One goal of this research program is to identify volatile compounds emitted by the green mold pathogen and Agaricus mushrooms and to assess the interaction of volatiles between different fungi. Our Graduate Student has applied for an Endeavour Fellowship and the opportunity to work with students and faculty seeking to further understand the complex relationships among different microbial communities in mushroom production. Utilizing The University of Sydney's mushroom research facility, the Marsh Lawson Mushroom Research Unit, he will seek to identify microorganisms that are native to the mushroom substrate which may inhibit or accelerate the growth of Trichoderma species. The bulk of his research program seeks to identify and assess key volatile organic compounds (VOCs) produced by Trichoderma aggressivum f. aggressivum (Ta2) and Agaricus bisporus to determine their importance and relationships in the development of green mold outbreaks. Samples of VOCs will be collected from cultures grown in vitro and from compost bioreactors available at Penn State's Mushroom Research Facility (MRC) using solid-phase microextration (SPME) and analyzed with gas chromatography-mass spectrometry (GC-MS). Sample collection will be performed throughout the development of Ta2 and Agaricus bisporus to detect changes in VOCs produced throughout the fungi's life cycles, as it has been observed that Ta2 does not cause disease until the crop has reached a certain stage of growth. One such VOC produced by T. aggressivum, 6-Pentyl-2H-pyran-2-one, is known to be antimicrobial, fungicidal, and phytotoxic. Identified VOCs were selected and introduced into cultures to determine their impact on the growth of Agaricus bisporus. Phase III was completed in a small scale bioreactor system in which Airflow was maintained at rate equivalent to a phase III tunnel system and compost temperatures were maintained at 25-27°C. Headspace solid-phase microexactraction (SPME) fibers for VC sampling occurred at three-day intervals beginning day after spawning. Volatiles were analyzed using Agilent Technologies GC-MS method set to "scavenge" mode with a slow ramp speed over 36 minutes to detect identifier VCs. Our results suggest similarities between treatments require targeting of 150-300g/mol compounds using selective-ion monitoring (SIM). Eliminating manure, a 100% synthetic N based compost may reduce interference for higher precision on targeted compounds. Differentiation between A. bisporus and Ta2 VCs detected as early as spawn run day 6. We have identified a small set of fungal species present during mushroom cultivation that are attractive to female sciarid mushroom flies, i.e., the fungus gnat species, Lycoriella ingenua. Some of these were found on the bodies of gravid females and may explain how infestations get started in mushroom houses. Other fungal species having different degrees of attractiveness to females are those commonly found in mushroom compost. We found that some fungal species that were not attractive to females are very strong oviposition stimulants to females, and so we hypothesize that the volatiles from some fungi attract the females to parts of the compost, and other fungi then stimulate the females to lay their eggs there. We found that larvae can develop to adulthood by feeding on the mycelia of only three different fungal species, and for all the other species, the larvae die before making it past the first larval stage. We have also isolated an active female-emitted sciarid mushroom fly sex pheromone component, a sesquiterpene alcohol that is a type of germacradienol, that is highly active in attracting males. This work proves that a compound identified as a sex pheromone in 1980 by a different research group was an erroneous and misleading identification that has hampered research into finding a true and highly active sex pheromone such as the one we have isolated. Utilizing our supply of phorid flies, we have conducted a series of replicated bioassays to determine the potential efficacy of Beauveria (specifically Botanigard®) for control of phorids. Results obtained are similar to those previously reported for sciarid flies, and it appears that Botanigard® is not effective as a compost drench for phorid fly control. A paper titled 'Efficacy of Beauveria bassiana formulations against the fungus gnat Lycoriella ingenua", was submitted to Biological Control. Cropping experiments were conducted at the Pennsylvania State University Mushroom Research Center to determine if corn stover could be used at 50% and 100% of the bulk raw material in the Phase I compost formulation to produce a high yielding A. bisporus crop. Results showed that, when composted properly, mushroom yields grown on both 50 and 100% corn stover formulations yielded as high as traditional straw-based compost formulations. These results suggest that corn stover can be used in place of straw to produce A. bisporus compost in regions that have limited or no economically affordable straw supplies.
Publications
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2016
Citation:
Vieira, F., and Pecchia, J. 2016. A deep exploration into the microbial community in an experimental composting condition for Agaricus bisporus cultivation. Proceedings form the 19th International Society for Mushroom Science Congress. Amsterdam, The Netherlands www.ISMS.com.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2016
Citation:
Andreadis, ,Stefanos S, Cloonan, K. R., Bellicanta, G. S., Paley, K., Pecchia, J. A., Jenkins, N. E. (2016) Efficacy of Beauveria bassiana formulations against the fungus gnat Lycoriella ingenua Biological Control In Press.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Bishop, E., Pecchia, J., Istvan, A., Royse, D.J. 2016. Effects of Spent Mushroom Compost (SMC) as an Ingredient in Phase I Compost on Production of Agaricus bisporus. Compost Science & Utilization. 24(4):246-258.
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2016
Citation:
Beyer, D. M., Pecchia, Paley, K. (2016) Evaluation of bio-fungicides for the control of fungal diseases on Agaricus bisporus. Proceedings form the 19th International Society for Mushroom Science Congress. Amsterdam, The Netherlands www.ISMS.com.
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2016
Citation:
Pecchia, J, Beyer, D and Li, X. 2016. The use of corn stover to replace straw in compost formulations for the production of Agaricus bisporus. Proceedings form the 19th International Society for Mushroom Science Congress. Amsterdam, The Netherlands. www.ISMS.com.
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Progress 08/04/15 to 09/30/15
Outputs
Target Audience:North American Commercial 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?57th Penn State Mushroom Short Course - 6 presentations were made reporting different aspects of this project. What do you plan to do during the next reporting period to accomplish the goals?One goal of this research program is to identify volatile compounds emitted by the green mold pathogen and Agaricus mushrooms and to assess the interaction of volatiles between different fungi. Our Graduate Student has applied for an Endeavour Fellowship and the opportunity to work with students and faculty seeking to further understand the complex relationships among different microbial communities in mushroom production. Utilizing The University of Sydney's mushroom research facility, the Marsh Lawson Mushroom Research Unit, he will seek to identify microorganisms that are native to the mushroom substrate which may inhibit or accelerate the growth of Trichoderma species. The bulk of his research program seeks to identify and assess key volatile organic compounds (VOCs) produced by Trichoderma aggressivum f. aggressivum (Ta2) and Agaricus bisporus to determine their importance and relationships in the development of green mold outbreaks. Samples of VOCs will be collected from cultures grown in vitro and from compost bioreactors available at Penn State's Mushroom Research Facility (MRC) using solid-phase microextration (SPME) and analyzed with gas chromatography-mass spectrometry (GC-MS). Sample collection will be performed throughout the development of Ta2 and Agaricus bisporus to detect changes in VOCs produced throughout the fungi's life cycles, as it has been observed that Ta2 does not cause disease until the crop has reached a certain stage of growth. One such VOC produced by T. aggressivum, 6-Pentyl-2H-pyran-2-one, is known to be antimicrobial, fungicidal, and phytotoxic. Identified VOCs will be selected and introduced into cultures to determine their impact on the growth of Agaricus bisporus.
Impacts
What was accomplished under these goals?
Cropping experiments using V. fungicola as a pathogen were conducted to evaluate the efficacy of several bio-fungicides for control of disease development and influence on mushroom yield. Mertect 340 F® and or Bravo Weatherstik® 720 (Syngenta Crop Protection Inc,), containing 54% active ingredient [0.72 g. of chlorothalonil per litre] was used as commercial fungicides, while Organocide, MBI 110, Contans WG, Prestop, Mycostop were used as alternative fungicides. Results suggest that Myco-stop demonstrated significantly better control of the disease compared to the other bio-pesticides tested. The other products showed little control over the disease. Further testing of rates and applications of MycoStop are being conducted. In another cropping experiment the influence of two bio-pesticides JAZZ and MBI 110 were used after casing on standard un-pasteurized casing and pasteurized casing. The commercially available JAZZ actually worked better than the MBI 110 and the inoculated control. When the casing was pasteurized the control of the JAZZ was negated and more Ta2 was noted on all inoculated pasteurized casing treatment. These results suggest that there may be some microbial interaction with the bio-pesticides used and the native microbial ecology to make the JAZZ more effective in controlling green mold. Construction of the composting reactor system was finally completed. The final steps involved the installation of the temperature, carbon dioxide and oxygen monitoring system. The data collection system is now completely operational and will allow us to monitor reactor temperatures as well as exhaust ammonia (via wet chemistry titration), carbon dioxide and oxygen levels. Each incubator can hold 3 bioreactors with individual reactor temperatures monitored continuously for the duration of an experiment. We can adjust the oxygen/carbon dioxide levels in the compost by reducing the air supply through individual flow restrictors based on exhaust oxygen and carbon dioxide readings. We are currently working with the reactor system to determine what air flow levels are required to maintain aerobic conditions within the compost and we are working on the ammonia titration protocol. Additionally, the exhaust purge times need to be determined under different air flows (to flush resonant exhaust streams between reactor sampling) for carbon dioxide and oxygen measurements, prior to running experiments in the reactors. Changes in bacteria populations in the casing layer were determined for a single crop using metagenomic analysis of extracted DNA. Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria were the most predominant phyla found in the casing. The Actinobacteria and Firmicutes decreased significantly after day 6 but increased on days 22 and 29. The Bacteroidetes increased from day 6 to day 13 to day 22 with a subsequent decline in numbers on day 29. The Proteobacteria, the most predominant phyla, increased from day 6 to day 13 but then decreased on days 22 and 29. This is just the first step in better understanding the biology and relationships of these microbes as they may relate to fruiting and disease development in a crop. We are looking at introducing manganese peroxidase and versatile oxidase genes from Pleurotus ostreatus into Agaricus bisporus as well as upregulating cellobiose dehydrogenase genes with hopes of increasing growing efficiency on current substrates. These enzymes are used to degrade lignin and complex carbohydrates in fungi. We would also like to allow for the use of novel substrates to grow A. bisporus on that are currently not utilized due to their high lignin content. If genes coding for enzymes from other fungi are able to be successfully introduced into A. bisporus, wood-based substrates may be an alternative raw material that could be efficiently used as a substrate for the production of the button mushroom. We evaluated the use of an entomopathogenic fungus, Beauvaria bassiana, to control phorid flies in a mushroom production system. Spawn run compost collected from commercial mushroom houses, infested with Phorid pupae, was evenly mixed with compost prepared at the MRC and filled into small pots. Each pot was then treated and placed in individual insect cage to prevent the flies from escaping. Due to the inability to rear phorid flies, infested compost was the best method of introducing flies into the system. However, by not knowing absolute starting numbers, there was considerable variability with fly numbers between replicates. Results from the first cropping showed that the commercially available B. bassiana product had no impact on adult fly emergence. Results from the second crop indicated that the application of the commercial product at spawning reduced adult fly emergence. B. bassiana appears to have some potential to control phorid (and possible sciarid) populations, however, application timing is critical in having a successful fly management program. Further work needs to be conducted to determine optimum B. bassiana formulation (the commercial oil-based product demonstrated mycotoxocity to the mushroom when applied after pin development) and application rates and timing.
Publications
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