Progress 09/01/12 to 08/31/17

Outputs
Target Audience:The target audience is the 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?Workshops Conferences On-farm consultations What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Cropping experiments were used to evaluate bio-pesticides for efficacy in controlling Trichoderma aggressivum aggressivum, (Ta2) (Green Mold) and L. fungicola (Dry Bubble) two fungal pathogens of Agaricus bisporus, white and brown button mushroom. 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. Only the Thyme lowered the incidence of dry bubble disease by 60-75% with no effect of mushroom yield. 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. Results also suggest that a bio-fungicide Gliocladium catenulatum may be effective in reducing the incidence and severity of Ta2 green mold disease. Ta2 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. 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). Differentiation between A. bisporus and Ta2 VCs detected as early as spawn run day 6. Isolation of potential candidates for biocontrol for Ta2 was tested by isolating bacteria from samples were taken during the composting phase I and II. A strain of Ta2 was co-inoculated to verify the inhibition pressures with bacterial colonies. Those that showed a potential inhibition against T. aggressivum were re-cultured and purified. The Rep-PCR method showed that the species were members of Bacillus genus (Bacillus subtilis and Bacillus amyloliquefaciens). Several in vitro experiments showed that all isolates have inhibition affect against T. aggressivum. Co-culturing of B. subtilis or B. amyloliquefaciens and T. aggressivum on plates showed a higher suppression effect. On compost lab scale experiments, the inhibition effect was less efficient than in plates. In addition, filtered cultures, i.e., cell-free solution, showed similar results than co-culturing on plates, which drove us to look into the compounds released in the liquid media. The spawn run in "race tubes" showed that A. bisporus is not affected by cell-free supernatant. The culture cells filtered by a 50 KDa membrane showed inhibitory activity against both pathogens, T. aggressivum and S. megalocapus. Subsequently, ammonia sulfate precipitation revealed higher inhibitory activity in precipitates between 30 % to 60 % of ammonia sulfate precipitations. On the other hand, lower inhibitory activity was detected using precipitates between 0 % to 30 % ammonia sulfate precipitations. Lower or no activity was observed with precipitates between 60 % - 90 %. The cell-free solutions exposed to different temperatures showed high stability and effective inhibition activity against T. aggressivum in vitro. 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. We have made progress in identifying the sex pheromone of Megaselia halterata, which differs from the structure identified in 1983 by Ray Baker and colleagues that was never shown to be behaviorally active. In anticipating the use of the sex pheromone that we are nearing the identification of for Lycoriella ingenua, we examined whether delayed mating of females caused by mating disruption of this species could reduce fecundity and fertility. We performed extensive experiments delaying the mating of females by from one to seven days. We found that there was no negative effect on female fecundity or fertility when mating was delayed by up to five days. We have successfully extracted pheromone from wild-caught M. halterata females and shown using coupled gas chromatograph/electroantennogram recordings (GC/EAG) that there are several EAG-active peaks using male antennae but not from female antennae. We have performed coupled GC-Mass Spectrometer (GC/MS) analyses of these peaks and found two peaks that do not conform to the previously identified sex pheromone of this species by Ray Baker and colleagues (1983). We are pursuing obtaining synthetic samples of this prior-identified compound plus chemically characterizing these other two EAG-active peaks. 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. However, when used as a premise spray, Botanigard is effective at killing adults of both species, when flies are exposed to the sprayed surface soon after application. However, while the speed of kill was found to be significantly different to the control population for both fly species, it was not sufficiently fast (8 days and 11 days to 100% mortality for sciarid and phorid flies respectively), to recommend its use to mushroom growers. Since the lack of efficacy of B. bassiana had been established by the end of 2016, a range of alternative biopesticide products, which are registered for use on other crops, were obtained and evaluated for efficacy. Products evaluated using direct spray application to egg, larval and pupal stages of Sciraird and phorid flies: Entrust®, Dow AgroSciences, PFR-97, Certis, Requiem, Bayer Crop Sciences, Grandevo and Venerate, Marrone Bio Innovations, BioCeres, BioSafe Systems and AzaGuard, BioSafe Systems. Of these, Entrust, PFR-97 and Grandevo, showed promising results and were progressed to a larger bioassay system in compost cups. While B. bassiana did not prove to be a promising control agent for either fly species, subsequent screening of alternative registered biopesticide products did turn up three promising products that could provide growers with novel tools for fly management. New funding support from IR-4 will enable further development of use strategies and optimization of dose rates for these products. If these trials are successful, label extensions for use of these products in mushroom crops could be obtained.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Cloonan, K., Andreadis, A., Jenkins, N., and Baker, T.C. (2017) Attraction, oviposition and larval survival of the fungus gnat, Lycoriella ingenua, on fungal species isolated from adults, larve and mushroom compost. Mushroom News, In Press.

Progress 09/01/15 to 08/31/16

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?Workshops Conferences On-farm visits Publications What do you plan to do during the next reporting period to accomplish the goals?Our future research will attempt to optimize a dedicated VC monitoring system, a Portable-MS, to identify T. aggressivum in a phase III tunnel system and to monitor phase II activity to identify and attempt to correlate VCs to inadequate pasteurization and poor conditioning. Additional questions: Are volatiles produced by T. aggressivum inhibitory to A. bisporus? What is the impact of repurposing phase III T. aggressivum infested compost for use in phase I and phase II? We are working on separating the proteins found mushroom compost in the extract through filtration to see if by chance we can find a certain size protein (group of proteins) that demonstrate inhibition of fungal pathogens on the plate. Continue a thorough screening of identified bio-control fungicides and some commercial trials are planned. Over the past 12 months, we have developed a successful rearing program for phorid flies and are currently running productive colonies of both sciarid and phorid flies for use in experiments and product screening. Utilizing our supply of phorid flies, we will continue to conduct series of replicated bioassays to determine the potential efficacy of other bio-insecticides for control of phorids. We are currently attempting to characterize the precise stereochemical structure of this very difficult and first-ever discovered form of germacradienol, and have formed collaborative research efforts with top-notch sesquiterpene chemists from Germany, England, the U. S., and Costa Rica. Additional manuscripts are being prepared on a) the use of Botanigard for phorid control and b) methods for optimization and maintenance of phorid colonies. We are continuing to evaluate the likely impact of Botanigard® for control of sciarid and phorid adults at the population level before recommending this technology for adoption by the mushroom industry. In view of the very limited options currently available to the industry for phorid control, we have commenced screening of five alternative biopesticide products for evaluation of efficacy against larval and adult stages. These experiments are ongoing and we will report on efficacy of these products along with recommendations for possible label extensions in the next annual report.

Impacts
What was accomplished under these goals? Two prominent fungal pathogens Trichoderma aggressivum and Lecanicillium fungicola currently cause the most widespread diseases, Green Mold and Dry Bubble respectively, of the cultivated Agaricus bisporus mushroom production worldwide. With the decrease in number of registered and effective chemical fungicides available to growers, the industry needs to look for more environmentally friendly alternatives. Cropping experiments were conducted at the Pennsylvania State University Mushroom Research Center to determine if several novel bio-fungicides and essential oils were effective in controlling these fungal pathogens. 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 would potentially be predictive of the level of disease control achieved in cropping trials. Commercial spawn of an off-white hybrid mushroom strain was treated with calcium sulphate as the bio-fungicide carrier (control) and a mixture of calcium sulphate and different bio-fungicides or essential oils were compared to commercially available chemical or biological fungicides. 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. Horse bedding based, finished phase II compost, acquired from industry, was spawned with A. bisporus inoculum acquired from a North American spawn supplier. Spores, from Penn State culture collection Ta2 strain #342, were used as an inoculum to infest the substrate at spawning time. 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. We received a favorable review and have re-submitted the edited manuscript for publication.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Andreadis, S. S., Cloonan, K. R., Myrick, A. J., Chen, H. and Baker, T. C. (2015) Isolation of a female-emitted sex pheromone component of the fungus gnat, Lycoriella ingenua, attractive to males. J. Chem. Ecol. 41: 1127-1136.
• 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.
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Cloonan, K. R., Andreadis, S. S., Chen, H, Jenkins, N. E. and Baker, T. C. Attraction, oviposition and larval survival of fungus gnats, Lycoriella ingenua, on fungal species isolated from adults, larvae, and mushroom compost. Entomol. Exp. Appl.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Beyer, D. M., Pecchia, Paley, K. (2016) Evaluation of bio-fungicides for the control of fungal diseases on Agaricus bisporus in Proceedings for the International Society of Mushroom Science. www.ISMS.com
• Type: Other Status: Published Year Published: 2016 Citation: Andreadis, S. S., Cloonan, K. R., and Baker, T. C. (2016) progress in identifying the sex pheromone of the fungus gnat, Lycoriella ingenua, & other attractive substances. Mushroom News. 64: 4  13.

Progress 09/01/14 to 08/31/15

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?Results have been presented at workshops and annual mushroom conferences and as well as at Plant Pathology and Entomology conference. What do you plan to do during the next reporting period to accomplish the goals?Continue as planned, we have added a new graduate student to work on one of the objectives to assist us in acomplishing it.

Impacts
What was accomplished under these goals? The bio-fungicides applications were either applied according the label's Active Ingredient rate or at a 2x rate. The timing of the applications were as follows: at casing, 7-9 days after casing, before first break harvest and between first and second break. The casing was sprayed with the Verticillium spore suspension 9 days after casing. First break harvest began 10 days after the fresh air flush, 17 days after casing. Mushrooms were harvested for 2 or 3 growing cycles, or breaks, depending on disease severity. The number of symptomatic mushrooms (spotting, bubble or split stems) were counted daily and totaled for each break (7 day total) and reported as percentage of mushrooms infected. Results suggest that Myco-stop demonstrated significantly better control of the disease. The other products showed little control over the disease.In another cropping experiment three commercial Phase II composts and the standard Mushroom Research Center compost were inoculated with Trichoderma aggressivum the day after spawning. Two of the commercial compost showed more disease development by first break than the other commercial and research compost. One of the un-inoculated commercial substrate developed green mold by 1st break, suggesting that the substrate was infected at the commercial operation. By the end of the crop no significant difference in green mold growth was noted between all substrates.In another cropping experiment to 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 green mold 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. We have identified substances from mushroom cultivation that are attractive to female sciarid mushroom flies that may explain how infestations get started in mushroom houses. We have discovered that certain fungi such as the green mold Trichoderma aggressivum, are attractive to female mushroom flies. We have also isolated an active female-emitted sciarid mushroom fly sex pheromone component that is highly active in attracting males.Behavioral bioassays using this olfactometer showed that mushroom compost with A. bisporus mycelia growing in it was not more attractive than compost lacking growing mycelia. We also found that female flies were more attracted to compost lacking A. bisporus mycelia than to the actual commodity, the white button mushroom fruiting bodies. Flies were not, however, attracted to sterilized compost, suggesting the attraction is due to volatiles produced by microbial metabolism in the compost.We also found that female L. ingenua flies were attracted to the mycoparasitic green mold Trichoderma aggressivum. Flies preferred mushroom compost that had T. aggressivum growing in it over compost lacking T. aggressivum, providing an experimental outcome consistent with the anecdotal belief that L. ingenua flies are vectors of T. aggressivum spores that can infest mushroom growing houses.Among the importance of our findings during this progress reporting period is that this is the first evidence, to our knowledge, of attraction of sciarid flies to T. aggressivum actually growing in mushroom compost. Our results indicate that future experiments should build on this relationship to examine the spore- or mycelial-carrying vectorial capacity of L. ingenua adults that might visit this compost and be exposed to T. aggressivum life stages.Our current findings during this progress-reporting period showing the superior attractiveness of mature phase II compost over sterilized compost, may guide mushroom growers in their efforts to exclude L. ingenua flies.During this progress-reporting period, we made great progress in isolating and identifying the female-emitted sex pheromone of the mushroom fly, L. ingenua. Our results definitely show that a previous report showing that the sex pheromone is comprised of n-C15-n-C18 saturated straight-chain hydrocarbons was erroneous. Our investigations using coupled gas chromatography electro-antennographic detection, plus a rarely used gas chromatography-coupled behavioral bioassay, resulted in a behaviorally active pheromone component being isolated and partially characterized via gas chromatography-mass spectrometry (GC/MS). This component was found definitively to not be n-heptadecane or any of the other n-C15-n-C19 hydrocarbons previously erroneously identified, but rather appears to be one of the isomers of a sesquiterpene alcohol.The sciarid fly colony continues to thrive and serves as a resource for all SCRI related studies using sciarids. A phorid colony has now also been established and is maintained in a separate room to the sciarid colony to prevent cross contamination. Two replicated, large-scale sciarid experiments in collaboration with the MRC to evaluate the efficacy and effect on mushroom yield of the commercial Beauveria bassisana product (Botanigard) and two different formulations of B. bassiana from our Penn State laboratory. The results indicated that a single application of B. bassiana (regardless of formulation) at time of spawning, does not affect mushroom yield, but is insufficient to prevent the successful development of sciarid larvae in compost. In response to this negative result we conducted a series of maximum challenge bioassays in the laboratory, in order to better understand which life stages were susceptible to fungal attack.Spore suspensions prepared from the commercial Botanigard product, and were sprayed directly onto sciarid eggs, larvae and pupae in Petri dishes. Daily observations were made to determine mortality or progression to the next life stage in comparison to controls sprayed only with water. We also conducted evaluations on the potential efficacy of premise sprays of Botanigard and the Penn State Beauveria based product Aprehend to control adult sciraid flies. The results of bioassays conducted on sciarid eggs, larvae and pupae based on percentage emergence either to the next life stage (eggs to larvae) or from larvae or pupae to adults following direct spray exposure to Botanigard at label application rates.The results indicate that Botanigard is not effective at killing sciarid eggs or larvae and has only a minimal effect on pupae, even when sprayed directly onto the insects (maximum challenge). The effect of exposing newly emerged adult sciarids to a surface previously sprayed with Aprehend (the newly developed biopesiticde from Penn State). Adults exposed to Aprehend treated surfaces died within 6 days, whereas the two control populations (exposed to surfaces sprayed with either blank oil formulation or left unsprayed) died naturally within 11 to 13 days. The comparative efficacy of Aprehend and Botanigard as premise sprays. While exposure to surfaces sprayed with Botanigard resulted in increased mortality in comparison to the control, adult sciarids exposed to Aprehend sprayed surfaces died significantly faster (5 days in comparison to 8 days for Botanigard).While these results suggest that Beauveria, and particularly the Aprehend-premise spray formulation, may be potentially interesting as a control measure for adult sciraids. We still need to evaluate the likely impact at the population level before recommending this technology for adoption by the mushroom industry.We will also conduct the same set of experiments as described above using phorid flies to evaluated the control potential of Beauveria products for phorid control. The phorid colony at Penn State is finally beginning to grow and stabilize and we anticipate completing these experiments within the next 12 months.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Cloonan, K., Andreadis, S., and T. C. Baker. Attraction of female fungus gnats Lycoriella ingenua to mushroom-growing substrates and the green mold Trichoderma aggressivum. Submitted to Entomologia Experimentalis et Applicata.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Andreadis, S., Cloonan, K., Myrick, A., Chen, H., and T. C. Baker. Isolation of a behaviorally active sex pheromone component of the mushroom fly Lycoriella ingenua, using GC/EAD and an unusual, coupled GC/Behavior Assay. Submitted to Journal of Chemical Ecology

Progress 09/01/13 to 08/31/14

Outputs
Target Audience: The target audience is the 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? Three Extension and Pesticide Credit meetings 2013-2014 Mushroom Short Course - annual conferences FY 2013 and 2014 What do you plan to do during the next reporting period to accomplish the goals? Investigate the role of anaerobic compost and other compost characteristics have on Trichoderma disease development for Phase II and Phase II composts and to determine the influence of inoculant carriers, casing materials, formulas and or environmental factors on the growth and development of fungal pathogens. To continue to evaluate as many essential oils and bio-fungicides that are commercially available in vitro and in vivo against indigenous fungal isolates collected at farms around the country. To evaluate the role of volatiles in disease development. Evaluate disease resistance to registered commercial fungicides from isolates recently collected in the field and compared to isolates collected previous years. Evaluation of efficacy of B. bassiana against adult sciraid flies is ongoing and is investigating both lethal and sub lethal effects. We will look more closely at the insect pathogen interactions for each life stage to determine if targeting the most susceptible life stages and careful timing and placement of biopesticide applications can increase efficacy of B. bassiana. Over the coming months, all the experiments described above will be repeated using phorids, to evaluate the efficacy of B. bassiana for phorid fly control. With the GC/EAD system, we have made progress in starting to determine the activity of female L. mali extract and which elution times (GC retention times) contain EAG-active compounds. The quadroprobe EAG system will also been useful in determining the types of compounds that the male, vs. the female, L. mali antennae are responsive to.

Impacts
What was accomplished under these goals? Cropping experiments using Verticillium fungicola as a pathogen were conducted to evaluate the efficacy of several bio-fungicides for control of disease development and influence on mushroom yield. A conidial suspension (1 × 105 conidia ml-1) of V. fungicola was prepared just prior to the experimentation. Mertect 340 F (thiabendazole) and or Bravo (chlorothalonil) was used as commercial fungicides, while paraffin oil, natamycin, and Biosol (fungal mass fertilizer/bio-fungicide) were used as alternative fungicides. The bio-fungicides applications were either applied according the label’s Active Ingredient rate or at a 2x rate. The timing of the applications was as follows: at casing, 7-9 days after casing, before first break harvest and between first and second break. The casing was sprayed with the Verticillium spore suspension 9 days after casing. First break harvest began about 17 days after casing. Mushrooms were harvested for 2 or 3 growing cycles, or breaks, depending on disease severity. Yield data was statistically analyzed for a completely randomized design using the Waller Duncan K-ratio t-test at a significance level of 0.05 to separate the means. The number of symptomatic mushrooms (spotting, bubble or split stems) were counted daily and totaled for each break (7 day total) and reported as percentage of mushrooms infected. Results suggest that Bio-sol had little control of the disease and a slight negative influence on yield. The paraffin oil and natamycin showed some control over the disease for 1st break but less disease control on 2nd break. The un-inoculated treatments had a slight negative effect on yield, but most were not significantly different than the un-inoculated control. To investigate the role of Phase I and Phase II anaerobic compost characteristics on Trichoderma disease development, a cropping experiment was designed to have 4 treatments: anaerobic conditions during Phase I composting, during Phase II composting and combinations of each. In the first experiment, Phase I oxygen levels were not low enough to be considered anaerobic, however, Phase II oxygen levels were in the 3-5% range, and considered to be anaerobic. Additionally, anaerobic Phase II compost appeared off-colored and demonstrated a typical sour, anaerobic smell. A 105 spore suspension of T. aggressivum was used to inoculate a single point in the center of the tub the day after spawning. The percentage of Trichoderma growth on the compost surface was assessed during the 17 day spawn run. Results suggest that more disease development was observed on substrate that was anaerobic during Phase II when compared to an aerobic Phase II substrate. No differences were observed between the two Phase I composts, which was not surprising due to the difficulties faced making anaerobic Phase I compost for this experiment. The sciarid fly colony continues to thrive and serves as a resource for all SCRI related studies using sciarids. A phorid colony has now also been established and is maintained in a separate room to the sciarid colony to prevent cross contamination. Both colonies are maintained at 20 oC and 90 % humidity in small cages containing mushroom compost from the Penn State mushroom research facility. We have conducted two replicated, large-scale sciarid experiments in collaboration with the MRC to evaluate the efficacy and effect on mushroom yield of the commercial Beauveria bassisana product (Botanigard) and two different formulations of B. bassiana from our Penn State laboratory. Efficacy of sciarid control was evaluated by artificially infesting each replicate with 100 sciarid eggs and counting the daily emergence of adult sciarid flies from each replicate. Results indicate that a single application of B. bassiana (regardless of formulation) at time of spawning, does not affect mushroom yield, but is insufficient to prevent the successful development of sciarid larvae in compost. Further investigations into the ability of B. bassiana to infect and kill the various life stages of sciarids were conducted in the lab, using maximum challenge tests. Spore suspensions were sprayed directly onto sciarid eggs, larvae and pupae in Petri dishes. Daily observations were made to determine mortality or progression to the next life stage in comparison to controls. Results indicate that the pupal stage is the most susceptible to infection by B. bassiana, whereas eggs and larvae appear to be mostly unaffected, even under maximum challenge conditions. One goal of this research project is to identify semiochemicals that attract or repel adult fungus gnats, L. mali. Proactive control measures, as opposed to reactive control measures, may reduce overall fly pressure in a growing house such proactive control measures depend on the identification of insect semiochemicals, or "message-carrying” chemicals including female-produced sex pheromones that attract males. The blend of pheromone components might also be able to be applied in conjunction with small amounts of adulticides for an "attract and kill" control strategy. Our initial efforts at finding attractants for L. mali have centered around developing discriminating behavioral assays to assess the attractiveness of various materials, including compost, spawn, Trichoderma and the mushroom-crop itself. In order to investigate possible volatile attractants we developed a two-choice, static-flow olfactometer constructed out of small plastic cups and a Petri plate. This assay system is powerful because it can dependably detect and register the first choice of an egg-laying female that is attracted to the volatile emissions of whatever is in the test chamber and compare it with the attractiveness of material that is in the second chamber. We have used it so far to determine convincingly that mushroom compost containing A. bisporus mycelia is highly attractive to egg-laying L. mali females. Qualitative observations revealed that female flies are able to enter the choice container through these straws from the arena floor, but have difficulty passing the opposite direction from the choice container back to the release arena. We performed preliminary experiments examining the effects of different substrates on the development and emergence of adult L. mali flies. For these experiments we added 250 g of each substrate to rearing containers placed them individually into rearing cages and aspirated 200 gravid females into these rearing cages to allow them to lay eggs into the substrate. These cages were then left in colony conditions for 21 days until adults began to emerge. Each day after adults began to emerge the cups were placed into a clean rearing cage and the number of adults were counted and recorded until no more adults emerged from the substrate. The simple two-choice open chamber olfactometer revealed that female flies were more likely to be found on Trichoderma spores as compared to blank filter paper controls. These experiments also suggest that female flies may be more attracted to spawned mushroom compost infected with Trichoderma vs. uninfected spawned mushroom compost.

Publications

• Type: Other Status: Published Year Published: 2013 Citation: Cloonan, K., George, J., and Baker, T.C. 2014. Efforts to Identify Attractants to Help Control the Fungus Gnat, Lycoriella mali. Mushroom News (7): 2014, pp. 4-9.
• Type: Other Status: Published Year Published: 2013 Citation: Pecchia J.A., and D.M. Beyer, D.M., 2013. The Status of Trichoderma Green Mold in the United States. Mushroom News 61 (7): 16-17
• Type: Other Status: Published Year Published: 2013 Citation: Pecchia , J.A. and Beyer, D.M. 2013. Pest Management on US Commercial Mushroom Farms. Outlooks on Pest Management 24(1):28-29.
• Type: Other Status: Published Year Published: 2013 Citation: Beyer, D., Pecchia, J., and Elias, R. 2013. Investigations into Cultural Factors Influencing the Development of Trichoderma Green Mold. Mushroom News 61 (7): 4-11.
• Type: Other Status: Published Year Published: 2013 Citation: Beyer, D. M., K. O'Donnell, K. Paley, and M. P. Wach. 2013. First Report of Syzygites megalocarpus Web Mold. Mushroom News 61(4): 16-17

Progress 09/01/12 to 08/31/13

Outputs
Target Audience: The target audience is the 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? Three Extension and Pesticide Credit meetings 2012-2013 Mushroom Short Course - annual conference What do you plan to do during the next reporting period to accomplish the goals? – Investigate the role of anaerobic compost and other compost characteristics have on Trichoderma disease development for Phase II and Phase II composts and to determine the influence of inoculant carriers, casing materials, formulas and or environmental factors on the growth and development of fungal pathogens. – To continue to evaluate as many essential oils and biorationals that are commercially available in vitro and in vivo against indigenous fungal isolates collected at farms around the country.

Impacts
What was accomplished under these goals? An isolate of Verticillium and a Benomyl and Thiophanate-methyl resistant strain of Trichoderma (Ta2)were obtained from the culture collection of the Department of Plant Pathology (Pennsylvania State University). The pathogens were cultured on potato dextrose agar. In this study, the inhibition of spore germination by the oils was evaluated. This experiment was done in test tubes containing 10 ml of the spore suspension supplemented with either TO or LGO essential oils to obtain a final concentration of 40, 80 and 200 μl ml-1. Four days after inoculation, the number of germinated spores was counted with a hemacytometer. Thyme oil at a minimum concentration of 160 μl l-1 was effective at controlling both pathogens. Although LGO demonstrated some control of the pathogens, it was found to be less effective than its counterpart, thyme oil. The spore germination trial confirmed the antifungal activities of the two oil, where TO was found to be the most effective at controlling both pathogens. The use of TO and LGO on a commercial spawning product was evaluated for the protection of the spawn against the two pathogens. For this experiment, 50 g of spawn was transferred into a Ziploc bag and 2.4 ml of the oil formulation [80, 160 or 200 μl l-1] was sprayed onto the spawn. The spawn was carefully mixed to ensure an overall contact of the spawn to the oil mixture. Five spawn-inoculated grains treated with the oil mixture were placed onto a Petri plate previously inoculated with a spore suspension of either Verticillium or Trichoderma. The application of TO or LGO on the spawn was able to protect the mycelia growing on the grain against the development of Verticillium and Ta2. Two cropping experiments were conducted using V. fungicola as a pathogen, to confirm the efficacy of these essential oils as a preventative application. A conidial suspension (1 × 105 conidia ml-1) of the pathogen was prepared just prior to the experimentation. 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 fungicide, while TO and LGO essential oils, were used as alternative fungicides. Tween-80 was used as surfactant in the second cropping experiment. These trials were done using environmentally controlled production rooms at the Mushroom Research Center at Penn State University. The Tom Baker and Nina Jenkins labs worked in early 2013 to successfully establish a colony of the target sciarid fly pest species, Lycorella mali, in a large walk-in growth chamber in their space on the Penn State campus. Attempts to establish a colony of the other pest species, the phorid fly Megasilia halterata, have thus far failed, although many colonies started to become established during 2013 before succumbing to high mite infestations and other problems. Following the establishment of the L. mali colony, the Baker lab, beginning in April 2013, began to develop several promising behavioral bioassay designs in order to be able to test the flies’ preferences for a variety of possible natural attractive substances. Baker’s lab is currently working on several other assay designs to potentially achieve better discrimination between treatments. Eventually, once olfactometer-type bioassays can be optimized, results from such assays will be further tested in our laboratory insect flight tunnel. Both types of behavioral assays will be complemented by neurophysiological experiments involving electroantennograms (EAGs) and single cell recordings from individual neurons on the antennae. Because we confirmed other reports that the L. mali flies have a propensity to deposit eggs on or near colonies of the green mold pest of mushrooms, Trichoderma aggressivum, we obtained colonies of T. aggressivum from the Nina Jenkins lab and tested this mold’s attractiveness to the flies in the choice test bioassay chamber. Tested in two different sets of experiments with slightly different methods, the flies showed overall preferential movement to the side of the chamber that contained a petri dish with growing colonies of T. aggressivum compared to the side containing a petri dish with only auger. L. mali flies preferentially are attracted to the side of the bioassay chamber containing the T. aggressivum culture vs. the side containing a petri dish with clean auger. The flies were more active in this set of trials, and moved from the center of the chamber. Again working in collaboration with the Jenkins lab, we performed further choice assays. When the choice was between compost infected with T. aggressivum vs. compost with growing mycelia of the button mushroom Agaricus bisporus, the flies showed a slight, but not-significantly-different trend toward T. aggressivum. L. mali flies, when presented with a choice between compost infested with T. aggressivum vs. compost containing A. bisporus, they showed a slight, but not significant preference toward the T. aggressivum colonies. During 2013 we began working with these extremely tiny flies to try to develop ways to record from their antennae for neurophysiological assays. This is no small feat! We were able to get a suitable preparation developed to begin getting electroantennogram recordings. Once we determine which types of natural substrates are the most attractive to the flies in our behavioral bioassays, we will use solid phase microextraction (SPME) to collect volatiles in the headspaces of these substrates and inject them onto the GC to perform GC/EAD recordings to determine which compounds are most likely to be responsible for the attraction. Development of entomopathogen strain(s) and formulations for use as premise spray adulticides using production technology routinely used by mushroom spawn producers. (Jenkins) A stable colony of sciarid flies has been established at Penn State. Adult flies were collected from a Sylvan mushroom farm near Kennett Square. The colony is maintained in a dedicated control environment room (20 oC and 90 % humidity) in small cages containing mushroom compost from the Penn State mushroom research facility. A number of different rearing protocols were tested for optimal production of flies. We have now established a standard protocol, which enables us to use set numbers of freshly mated adult females for production of predicable numbers of viable eggs, which they lay in compost. We are also able to slow larval development time and hence produce freshly emerged adult populations on demand. Having developed this reliable and predictable method of production, we are able to use this system to evaluate the effect of biopesticide treatments when adult flies emerge into a treated environment. Figure 1 below illustrates the experimental set up for evaluating biopesticide spray applications to a single light source, which attracts the flies. Flies contacting the sprayed surface, acquire fungal spores and are monitored for mortality. Additionally, the fecundity of the treated population is evaluated by comparing emergence of the next generation with that of the control groups. Phorid flies were also collected from Kennett Square, but the colony was overtaken by fungus mites and we were obliged to freeze the whole colony to prevent spread of the mite infestation. We have been in regular contact with the mushroom growers since this incident, but phorid numbers have been naturally low all season and we have not had another opportunity to collect phorids for a starter colony. We are maintaining contract with the mushroom growers and will collect phorids as soon as an opportunity arises.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Beyer, D. M., K. O'Donnell, K. Paley, and M. P. Wach. 2012. First Report of Syzygites megalocarpus (Mucorales) Web Mold on the Commercial Portabella Button Mushroom Agaricus bisporus in North America. Plant Disease 97 (1):142-142.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Pecchia, J. A., and D. M. Beyer. 2012. The Status of Trichoderma aggressivum, the Causal Agent of Green Mold, in the United States as Determined in Response to 2010-2011 Quarterly Surveys Beijing, China. Pages 350-356.
• Type: Other Status: Published Year Published: 2012 Citation: Beyer, D. M., and J. Poppiti. 2012. The Safe and Proper Use of Disinfectants and Sanitizers on Mushroom Farms. Mushroom News 60 (7):8-15.
• Type: Other Status: Published Year Published: 2012 Citation: Pecchia, J. A., and D. M. Beyer. 2012. The Penn State Lines, Trichoderma Green Mold Survey. Mushroom News 60 (8):10-11.