Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Jul 1, 2020
Project End Date
Jun 30, 2025
Grant Year
Project Director
Pecchia, JO, A.
Recipient Organization
Performing Department
Plant Pathology & Environmental Microbiology
Non Technical Summary
There are 2 overlying goals of our research.1) To discover mechanisms of disease control, both chemical and biological, to reduce crop losses associated with fungal pathogens. We hope to increase our understanding of the role that Bacillus spp., as well as other indigenous populations found in the raw materials and substrate, play in affecting varying levels of substrate (crop) disease susceptibility.2) To improve on production and yield consistency of morel cultivation so that diversified and specialty growers can expand their production opportunities. Results of this long-term project will help both large and small mushroom growers survive and be profitable.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
These following objectives will be investigated simultaneously and each will continue for the duration of the project:Objective 1 - Survey Bacillus populations in raw materials and different commercially produced Phase II compost to test for efficacy of different isolates against fungal pathogens, focusing on Trichoderma aggressivum.Objective 2 - Investigate the role that compost and casing indigenous microbial communities play on Bacillus spp. survival and growth.Objective 3 - Study the epidemiology and etiology of the pathogen Syzygites megalocarpus.Objective 4 - To evaluate OMRI certified biopesticide formulations and macrobials that are commercially available in vitro and in vivo against indigenous fungal isolates.Objective 5 - Evaluate the yield potential of several Morchella spp. under varying growing conditions in both outside and indoor production systems.
Project Methods
Objective 1 - Survey Bacillus populations in raw materials and different commercially produced Phase II compost to test for efficacy of different isolates against fungal pathogens, focusing on Trichoderma aggressivum(Pecchia)Samples of compost raw materials and Phase II compost will be collected from commercial sites and from the MRC. Samples will be analyzed focusing on isolating naturally occurring Bacillus spp. that shows antagonistic effects against T. aggressivumin. Identification of native Bacillus isolates that shows inhibitory effects against Trichoderma will be carried out by sequencing the full 16S rRNA gene and by multi-locus sequence typing (MLST). These sequences will be compared with available sequences of Bacillus spp. at NCBI ( and Silva ( databases.The efficacy of commercial Bacillus products to control T. aggressivum will be evaluated for the commercial and experimental compost samples. T. aggressivum spore suspensions will be applied in different concentrations (104 - 106 per experimental unit) and inoculation methods (direct to the compost at spawning day and/or mixed with the spawn prior to deliveryto the compost). Commercial Bacillus products will also be applied in different concentrations and utilizing different application methods.Objective 2 - Investigate the role that compost and casing indigenous microbial communities play on Bacillus spp. survival and growth (Pecchia)Microbial communities will be screened from raw materials and compost during the cropping process by using three high-throughput sequencing. First, short amplicon sequencing (i.e., metataxonomic analysis) will be used to map the most abundant bacteria and fungi at the genus level. Second, selected samples will be submitted to long amplicon sequencing which relies on PacBio technologies and can generate higher taxonomic resolution for bacterial communities at the species/strains level. Third, another batch of samples will be used for metagenomic analysis with the hopes of mapping the bacterial communities' functionality. Basically, metagenomics analyses include the sequencing of bacterial communities' genomes by Illumina or PacBio technologies, assembly of these genomes by bioinformatics packages and characterization of genes present in these communities.Objective 3 - Study the epidemiology and etiology of the pathogen Syzgites megalocarpus (Beyer)We propose to use established procedures for determining the influence temperature, relative humidity, volatiles, pH, disinfectants and bio-pesticides and other physical and environmental conditions have on spore germination and disease development. Syzygites can be grown in the laboratory on relatively nutrient-rich media but there has been some controversy regarding the environmental conditions that promote thick-walled zygospore formation in culture (Kaplan and Boog, 1992). Davis (1967) induced zygospore production by growing Syzygites at 10oC in continuous dark (Davis, 1967). It was reported that zygospores were produced in cultures grown at 19oC on GYP medium with 0.2 M KCl regardless of the amount of light (Kaplan and Goos, 1992). We propose to test these conditions to produce zygospores to test their thermal death point to determine if post-crop steaming is adequate to eradicate zygospores. Several commercially available disinfectants will be tested in vitro and in vivo for their efficacy on Syzygites.We also will determine the influence of spore concentration on the timing of symptom development.The infestation by Syzygites spores most likely occurs after first break, when dead or dying mushroom tissue is present. We will attempt to determine if the concentration of spores or the timing of infection influences which symptom (pathogen covering fruit body or spotting of mushroom) will develop.Objective 4 - To evaluate as OMRI certified biopesticide formulations that are commercially available in vitro and in vivo against indigenous fungal isolates (Beyer)The fungal pathogens T. aggressivum, L. fungicola, Cladobotryum sp. S. megalocarpus and a commercial hybrid strain of A. bisporus will be grown in triplicate in petri plates containing potato dextrose yeast agar (PDYA) medium amended with available bio-fungicides. The mean diameter of the cultures will be determined after 3-10 days and 2-3 weeks of growth at 24oC for the fungal pathogens and A. bisporus, respectively. Fungicides inhibiting the radial growth of the pathogens without significant toxicity to the mushroom fungus will be advanced to the next phase, cropping trials. Fungal pathogens will be introduced, and compounds applied as follows: L. fungicola will be inoculated with a spore suspension sprayed on the surface of the casing. An un-inoculated treatment will be included as a control. Test compounds will be applied at rates determined from the in vitro trials and in a volume that will provide complete coverage of the casing layer. Non-treated fungicide controls will be included in each trial. The incidence and severity of disease development will be measured, and the data statistically analyzed. We will screen products like MilStop®, Mildew Cure®, Actinovate® and Nu-Cop®.It has been reported that the degree to which T. aggressivum develops on mushroom grain spawn following inoculation with T. aggressivum is positively correlated with disease severity in actual mushroom production trials.Therefore, the extent to which a fungicide protects the spawn from green mold attack would be predictive of the level of disease control achieved in cropping trials. Commercial A. bisporus spawn will be treated with an available bio-pesticide at concentrations based on the findings from the in vitro trials. For each concentration of compound, three treated spawn grains will be plated on a lawn of non-germinated T. aggressivum spores overlaid on PDYA contained in a 15-cm dia. petri plate. The average diameter of the zone of inhibition, which is the area surrounding the spawn grains where T. aggressivum failed to grow, will be determined after 5-7 days of incubation at 24oC. Compounds not significantly inhibiting mycelial growth of A. bisporus but producing a zone of inhibition will progress to the cropping trials. We will screen products like MilStop®, Mildew Cure®, Actinovate® and Nu-Cop®.Objective 5 - Evaluate the yield potential of several Morchella spp. under varying growing conditions in both outside and indoor production systems (Pecchia)We propose to study both outdoor and indoor cultivation during this project. The indoor experiments will be conducted at Penn State University's Mushroom Research Center which has climate controlled growing rooms with precise temperature, lighting, humidity, air flow and carbon dioxide control. The growing media will consist of a soil mixture already tested by our group. Initial experiments will be set up to test 2 morel species (currently in Penn State's culture collection) to determine which species fruit best under prescribed environmental conditions. Replicated trials will be conducted to test optimum environmental parameters and to select the best cultivars. Replicated plots will test nutrient supplementation and soil organic amendments. Morels will be spawned in the fall with data collection and yield assessment occurring in spring. Indoor cropping experiments can be conducted on a continual basis while outdoor cropping will take place annually.

Progress 07/01/20 to 09/30/20

Target Audience:The target audience consists of US commercial mushroom producers and their suppliers, as well as small, diversified mushroom and vegetable growers. Changes/Problems:The biggest problem faced so far has been limitations with personnel (new graduate student and additional undergraduate students) working on the project due to COVID 19. What opportunities for training and professional development has the project provided?A post doc participated in Microbiome seminars to improve data analyses skills and share knowledge with other faculty, staff and students. Graduate students trained in traditional microbial and molecular techniques, in addition to, trainingin lab techniques and morel mushroom cultivation procedures. Dr. Beyer discussed findings to date with mushroom professionals. How have the results been disseminated to communities of interest?Verbally discussed findings with growers What do you plan to do during the next reporting period to accomplish the goals?Objective 1:Additional sampling of compost from Phase II, spawn run and during cropping is scheduled for October and November 2020 from each of the four farms. Further work analyzing the substrate microbiome and isolation and identification of Bacillus spp. will be done with samples that demonstrate suppressiveness to green mold caused by Trichoderma aggressivum. We plan to publish results. Objective 2:The newly collected compost samples obtained for use in objective one will be also be screened for suppressiveness and susceptibility to green mold. We plan to present information at International Congress and publish results. Objective 3:Additional efficacy testing of fungicides and disinfectants against S. megalocarpus will be completed at the MRC. We plan to publish results. Objective 4:Completion of the secondtrial along with a statistical analyses of yield and disease development will be completed. An additional biologically based fungicide will also be tested in an upcoming cropping trial. Objective 5:Work will continue to determine the mating types of the M. rufobrunnea isolates that we currently have in the lab. The high tunnel cropping site at Rock Springs will be reinoculated with spawn in fall 2020 and irrigated to test production potential of the twochosen species. Both mating types (and a combination of both) of M. importuna will be used as spawn to determine if mating type effects outdoor yields in cropping trials conducted in Maine. Indoor cropping trials will be conducted using mating types of both species to determine how mating types affect fructification and yield under controlled climate conditions at the MRC. We plan to present results at International Congress and publish results.

What was accomplished under these goals? Objective 1 (Pecchia):Raw material and Phase I mushroom compost sampling from fourcommercial button mushroom farms was initiated in September, 2020. One farm is located in Berks County, PAand threeof the farms are located in Chester County, PA. Bacterial isolation and testing of Bacillus isolates is currently underway in the lab. No data has been collected yet since this work was initiated in September, 2020. Objective 2 (Pecchia): Our lab has continued screening Phase II compost, from each Mushroom Research Center (MRC) crop, for susceptibility to green mold development;however, a delay in sample collection took place during the summer of 2020 due to COVID 19 ramping down of research activities at the MRC. Objective 3 (Beyer and Pecchia):No additional work with S. megalocarpus has been completed since this new project was initiated. Objective 4 (Beyer and Pecchia):A cropping trial testing the efficacy of three fungicides (Milstop, Mildew Cure and Nu-cop) against Lecanicillium fungicola, the causal agent of dry bubble, was recently completed at the MRC. A repeat of this experiment is also currently underway at the MRC. Data collection and analyses from these trials has not yet been completed. Objective 5 (Beyer):We have obtained 15 isolates of two Morchella species: M. importuna and M. rufobrunnea (species selected based on previous MRC cropping experiments). Using primers described in the literature we amplified the mating type genes for each isolate to determine if we have both mating types of both species. Preliminary results indicate that we have both mating types for M. importuna and we are still working on analyzing M. rufobrunnea. We are currently producing M. importuna spawn of each mating type to be used in an outdoor field cropping study in Maine. We are also currently producing M. importuna and M. rufobrunnea spawn (using the same isolates that were previously used for cropping studies) to reinoculate an outdoor field site at the PSU Plant Pathology farm at Rock Springs.