Unravelling the Transcriptional Response of Agaricus bisporus under Lecanicillium fungicola Infection.

Mushrooms are a nutritionally rich and sustainably-produced food with a growing global market. Agaricus bisporus accounts for 11% of the total world mushroom production and it is the dominant species cultivated in Europe. It faces threats from pathogens that cause important production losses, including the mycoparasite Lecanicillium fungicola, the causative agent of dry bubble disease. Through quantitative real-time polymerase chain reaction (qRT-PCR), we determine the impact of L. fungicola infection on the transcription patterns of A. bisporus genes involved in key cellular processes. Notably, genes related to cell division, fruiting body development, and apoptosis exhibit dynamic transcriptional changes in response to infection. Furthermore, A. bisporus infected with L. fungicola were found to accumulate increased levels of reactive oxygen species (ROS). Interestingly, the transcription levels of genes involved in the production and scavenging mechanisms of ROS were also increased, suggesting the involvement of changes to ROS homeostasis in response to L. fungicola infection. These findings identify potential links between enhanced cell proliferation, impaired fruiting body development, and ROS-mediated defence strategies during the A. bisporus (host)-L. fungicola (pathogen) interaction, and offer avenues for innovative disease control strategies and improved understanding of fungal pathogenesis.

Radon Flux Measurement System.

ABSTRACT: Disposal of naturally occurring radioactive material (NORM) and technologically enhanced naturally occurring radioactive material (TENORM) waste in the State of Oregon is prohibited unless it can be demonstrated that the material is nonradioactive as defined by its radionuclide content and potential for emission into the environment. It was determined that a radon flux on the surface of the waste no greater than 0.37 Bq m -2 s -1 would meet this requirement. This article provides a method to estimate the radon flux through indirect measurement of the radon mass exhalation rate. It describes a device that consists of a radon accumulation chamber coupled with a continuous radon monitor and software to process the results and calculate the radon mass exhalation rate and radon flux for an unknown sample of approximately 500 g. The chamber system was tested with a uranium ore sample.

Comparison of Doses from Disposals of Technologically Enhanced Naturally Occurring Radioactive Materials in Kentucky and Oregon.

ABSTRACT: Oil and natural gas fracking waste contains technologically enhanced naturally occurring radioactive material (TENORM) and has increasingly been disposed of in unpermitted landfills, causing concern among regulators and the public about potential exposures. There are numerous issues with TENORM waste, including the lack of Federal regulations on its disposal and the lack of permitted landfills capable of accepting these waste streams. This paper examines two situations in which TENORM was placed in unpermitted landfills, one in Kentucky and one in Oregon. The same modeling and dose calculation methods were used in both cases, allowing for a comparison between the two sites. Site-specific differences, source terms, and doses from the disposals and potential remediation options are discussed and compared. Predicted groundwater concentrations are shown and compared against the relevant regulations for each site. Despite the differences in site and TENORM waste characteristics, it was more protective of the community and the environment to leave the waste in place at both sites. Radiation doses to landfill workers on site and to members of the public were low, both during the original disposal and for the remediation alternatives evaluated. Removal of the TENORM material in either case presents significant non-radiological risks that outweigh any benefit from the long-term dose reduction.

Potential Airborne Releases and Deposition of Radionuclides from the Santa Susana Field Laboratory during the Woolsey Fire.

ABSTRACT: The Santa Susana Field Laboratory (SSFL), located in southern California, is a former research facility, and past activities have resulted in residual radioactive contamination in Area IV of the Site. The Woolsey Fire burned across the site, including some of the contaminated areas, on 8-11 November 2018. Atmospheric transport modeling was performed to determine where the smoke plume went while the fire burned across the SSFL and the deposition footprint of particulates in downwind communities. Any radionuclides on vegetation and in surface soil released by the fire were assumed to follow particulate matter transport path and deposition. The predicted deposition footprint was used to guide confirmatory soil sampling at 16 locations including background. Highest offsite deposition was determined to be northeast of the Oak Park community, which is located about 6 km southwest of SSFL. Depth-profile sampling was used to evaluate whether radionuclides of SSFL origin were potentially emitted and deposited during the Woolsey Fire. If radionuclides had been deposited from the Woolsey Fire at sufficient concentrations, then they would be detected in the surface layer and would be expected to be higher within the plume footprint than outside it. An upper bound estimate of the hypothetical effective dose to a person in Oak Park based on measured radionuclide concentrations in soil and vegetation on the SSFL was less than 0.0002 mSv. The occurrence of naturally occurring radionuclides at concentrations above the established background for the SSFL was attributed to natural variability in geologic formations and not SSFL. No anthropogenic radionuclides were measured at levels above those expected from global fallout. The soil sampling confirmed that no detectable levels of SSFL-derived radionuclides migrated from SSFL at the locations sampled because of the Woolsey Fire or from past operations of the SSFL.

Methods of improving brain dose estimates for internally deposited radionuclides.

The US National Council on Radiation Protection and Measurements (NCRP) convened Scientific Committee 6-12 (SC 6-12) to examine methods for improving dose estimates for brain tissue for internally deposited radionuclides, with emphasis on alpha emitters. This Memorandum summarises the main findings of SC 6-12 described in the recently published NCRP Commentary No. 31, 'Development of Kinetic and Anatomical Models for Brain Dosimetry for Internally Deposited Radionuclides'. The Commentary examines the extent to which dose estimates for the brain could be improved through increased realism in the biokinetic and dosimetric models currently used in radiation protection and epidemiology. A limitation of most of the current element-specific systemic biokinetic models is the absence of brain as an explicitly identified source region with its unique rate(s) of exchange of the element with blood. The brain is usually included in a large source region calledOtherthat contains all tissues not considered major repositories for the element. In effect, all tissues inOtherare assigned a common set of exchange rates with blood. A limitation of current dosimetric models for internal emitters is that activity in the brain is treated as a well-mixed pool, although more sophisticated models allowing consideration of different activity concentrations in different regions of the brain have been proposed. Case studies for 18 internal emitters indicate that brain dose estimates using current dosimetric models may change substantially (by a factor of 5 or more), or may change only modestly, by addition of a sub-model of the brain in the biokinetic model, with transfer rates based on results of published biokinetic studies and autopsy data for the element of interest. As a starting place for improving brain dose estimates, development of biokinetic models with explicit sub-models of the brain (when sufficient biokinetic data are available) is underway for radionuclides frequently encountered in radiation epidemiology. A longer-term goal is development of coordinated biokinetic and dosimetric models that address the distribution of major radioelements among radiosensitive brain tissues.

Characterising the proteomic response of mushroom pathogen Lecanicillium fungicola to Bacillus velezensis QST 713 and Kos biocontrol agents.

The fungal pathogen Lecanicillium fungicola causes dry bubble disease in Agaricus bisporus cultivation and affected mushrooms significantly reduce the yield and revenue for mushroom growers. Biocontrol agents may represent an alternative and more environmentally friendly treatment option to help control dry bubble on mushroom farms. Serenade ® is a commercially available biocontrol product used for disease treatment in plant crops. In this work, the in vitro response of L. fungicola to the bacterial strain active in Serenade, Bacillus velezensis (QST 713) and a newly isolated B. velezensis strain (Kos) was assessed. B. velezensis (QST713 and Kos) both produced zones of inhibition on plate cultures of L. fungicola, reduced the mycelium growth in liquid cultures and damaged the morphology and structure of L. fungicola hyphae. The proteomic response of the pathogen against these biocontrol strains was also investigated. Proteins involved in growth and translation such as 60S ribosomal protein L21-A (-32-fold) and 40S ribosomal protein S30 (-17-fold) were reduced in abundance in B. velezensis QST 713 treated samples, while proteins involved in a stress response were increased (norsolorinic acid reductase B (47-fold), isocitrate lyase (11-fold) and isovaleryl-CoA dehydrogenase (8-fold). L. fungicola was found to have a similar proteomic response when exposed to B. velezensis (Kos). This work provides information on the response of L. fungicola to B. velezensis (QST 713) and indicates the potential of B. velezensis Kos as a novel biocontrol agent. Supplementary Information: The online version contains supplementary material available at 10.1007/s10658-022-02482-1.

Dosimetry for the study of medical radiation workers with a focus on the mean absorbed dose to the lung, brain and other organs.

BACKGROUND: The reconstruction of lifetime radiation doses for medical workers presents special challenges not commonly encountered for the other worker cohorts comprising the Million Worker Study. METHODS: The selection of approximately 175,000 medical radiation workers relies on using estimates of lifetime and annual personal monitoring results collected since 1977. Approaches have been created to adjust the monitoring results so that mean organ absorbed doses can be estimated. RESULTS: Changes in medical technology and practices have altered the radiation exposure environments to which a worker may have been exposed during their career. Other temporal factors include shifts in regulatory requirements that influenced the conduct of radiation monitoring and the changes in the measured dose quantities. CONCLUSIONS: The use of leaded aprons during exposure to lower energy X rays encountered in fluoroscopically based radiology adds complexity to account for the shielding of the organs located in the torso when dosimeters were worn over leaded aprons. Estimating doses to unshielded tissues such as the brain and lens of the eye become less challenging when dosimeters are worn at the collar above the apron. The absence of leaded aprons in the higher energy photon settings lead to a more straightforward process of relating dosimeter results to mean organ doses.

Dosimetry associated with veterans who participated in nuclear weapons testing.

BACKGROUND: This article summarizes the methodology, results, and challenges of the reconstruction of red bone marrow and male breast doses for a 1982-person sub-cohort of ∼114,270 U.S. military veterans who participated in eight atmospheric nuclear weapons tests between 1945 and 1962. These doses are being used in an epidemiological investigation of leukemia and male breast cancer as part of a study of one million U.S. persons to investigate risk from chronic low-dose radiation exposure. METHODS: Previous doses to these veterans had been estimated for compensation and tended to be biased high but newly available documentation made calculating individual doses and uncertainties using detailed exposure scenarios for each veteran possible. The techniques outlined in this report detail the methodology for developing individual scenarios and accounting for bias and uncertainty in dose based on the assumptions made about exposure. RESULTS: Doses to the atomic veterans in this sub-cohort were relatively low, with about two-thirds receiving red bone marrow doses <5 mGy and only four individuals receiving a red bone marrow dose >50 mGy. The average red bone marrow dose for members of the sub-cohort was 5.9 mGy. Doses to male breast were approximately 20% higher than red bone marrow doses. DISCUSSION AND CHALLENGES: Relatively low uncertainty was achieved as a result of our methodology for reconstructing exposures based on knowledge of the individual veterans' locations and activities from military records. Challenges did arise from use of military records to determine probability of participation in specific activities but accounted for in estimates of uncertainty.

Asbestos exposure and mesothelioma mortality among atomic veterans.

BACKGROUND: The United States (U.S.) conducted 230 above-ground atmospheric nuclear weapons tests between 1945 and 1962 involving over 250,000 military personnel. This is the first quantitative assessment of asbestos-related mesothelioma, including cancers of the pleura and peritoneum, among military personnel who participated in above-ground nuclear weapons testing. METHODS: Approximately 114,000 atomic veterans were selected for an epidemiological study because they were in one of eight series of weapons tests that were associated with somewhat higher personnel exposures than the other tests and because they have been previously studied. We were able to categorize specific jobs into potential for asbestos exposure based on a detailed database of the military activities of the atomic veterans. Standardized mortality ratios (SMR) were calculated by service, rank (officer/enlisted) and ratings (occupation code and work location aboard ship) after 65 years of follow-up. RESULTS: Mesothelioma deaths were significantly increased overall (SMR 1.56; 95% CI 1.32-1.82; n = 153). This increase was seen only among those serving in the PPG (SMR 1.97; 95% CI 1.65-2.34; n = 134), enlisted men (SMR 1.81; 95% CI 1.53-2.13; n = 145), and the 70,309 navy personnel (SMR 2.15; 95% CI 1.80-2.56; n = 130). No increased mortality rates were seen among the other services: army (SMR 0.45), air force (SMR 0.85), or marines (SMR 0.75). Job categories with the highest potential for asbestos exposure (machinist's mates, boiler technicians, water tender, pipe fitters, and fireman) had an of SMR 6.47. Job categories with lower potential (SMR =1.35) or no potential (SMR =1.28) for asbestos exposure had non-significantly elevated mesothelioma mortality. CONCLUSIONS: The large excess of mesothelioma deaths seen among atomic veterans was explained by asbestos exposure among enlisted naval personnel. The sources of exposure were determined to be on navy ships in areas (or with materials) with known asbestos content. No excess of mesothelioma was observed in other services or among naval personnel with minimal exposure to asbestos in this low-dose radiation exposed cohort.

Analysis of the effect of Bacillus velezensis culture filtrate on the growth and proteome of Cladobotryum mycophilum.

Cladobotryum mycophilum, the causative agent of cobweb disease on Agaricus bisporus results in significant crop losses for mushroom growers worldwide. Cobweb disease is treated through strict hygiene control methods and the application of chemical fungicides but an increase in fungicide resistant Cladobotryum strains has resulted in a need to develop alternative biocontrol treatment methods. The aim of the work presented here was to evaluate the response of C. mycophilum to a Bacillus velezensis isolate to assess its potential as a novel biocontrol agent. Exposure of 48 hr C. mycophilum cultures to 25% v/v 96 hr B. velezensis culture filtrate resulted in a 57% reduction in biomass (P < 0.0002), a disruption in hyphal structure and morphology, and the appearance of aurofusarin, a secondary metabolite which is a known indicator of oxidative stress, in culture medium. Proteomic analysis of B. velezensis culture filtrate revealed the presence of peptidase 8 (subtilisin), peptide deformylase and probable cytosol aminopeptidase which are known to induce catalytic activity. Characterisation of the proteomic response of C. mycophilum following exposure to B. velezensis culture filtrate revealed an increase in the abundance of a variety of proteins associated with stress response (ISWI chromatin-remodelling complex ATPase ISW2 (+24 fold), carboxypeptidase Y precursor (+3 fold) and calmodulin (+2 fold). There was also a decrease in the abundance of proteins associated with transcription (40 S ribosomal protein S30 (-26 fold), 40 S ribosomal protein S21 (-3 fold) and carbohydrate metabolism (l-xylulose reductase (-10 fold). The results presented here indicate that B. velezensis culture filtrate is capable of inhibiting the growth of C. mycophilum and inducing a stress response, thus indicating its potential to control this important pathogen of mushrooms.

Transmission of mushroom virus X and the impact of virus infection on the transcriptomes and proteomes of different strains of Agaricus bisporus.

Cultivation of Agaricus bisporus is a large horticultural industry for many countries worldwide, where a single variety is almost grown exclusively. Mushroom virus X (MVX), a complex of multiple positive-sense single stranded RNA (ss(+)RNA) viruses, is a major pathogen of typical A. bisporus crops. MVX can manifest a variety of symptoms in crops and is highly infective and difficult to eradicate once established in host mycelium. Currently our knowledge regarding the molecular response of A. bisporus fruit bodies to MVX infection is limited. In order to study the response of different A. bisporus strains with different susceptibilities to MVX, we designed a model system to evaluate the in-vitro transmission of viruses in A. bisporus hyphae over a time-course, at two crucial phases in the crop cycle. The symptom expression of MVX in these varieties and the transcriptomic and proteomic response of fruit bodies to MVX-infection were examined. Transmission studies revealed the high potential of MVX to spread to uninfected mycelium yet not into the fruit bodies of certain strains in a crop. MVX affected colour and quality of multiple fruit bodies. Gene expression is significantly altered in all strains and between times of inoculation in the crop. Genes related to stress responses displayed differential expression. Proteomic responses revealed restriction of cellular signalling and vesicle transport in infected fruit bodies. This in-depth analysis examining many factors relevant to MVX infection in different A. bisporus strains, will provide key insights into host responses for this commercially important food crop.

Dose Assessment for Technologically Enhanced Naturally Occurring Radioactive Materials Disposal in Landfills.

ABSTRACT: Technologically enhanced naturally occurring radioactive material (TENORM) is gaining notoriety in the public sector, as the oil and gas industry looks for disposal locations for its slightly radioactive waste streams. Due in part to both the lack of federal regulations on the disposal of TENORM and the lack of permitted landfills that are designated for TENORM waste, occasionally it ends up being unknowingly placed in municipal landfills. It was alleged that a municipal landfill in Kentucky accepted 1.05 × 106 kg of TENORM over approximately 8 mo starting in July 2015. This matter is still in litigation, and many facts, including whether the material in question actually constituted TENORM, are still in dispute. The authors had no means available to independently verify the actual composition of the material. Therefore, for purposes of this article only, we assume that the material in question did constitute TENORM. This qualification allows us to evaluate potential doses while respecting the litigation process. Doses from the disposals and for two remediation alternatives, (1) closure-in-place and monitoring and (2) excavation and redisposition of waste, were evaluated, taking into consideration the landfill construction, local geology and hydrology, meteorology, background radiation, population distribution, and current and future land uses. This study outlines appropriate methods for calculating doses to potential receptors for a variety of exposure pathways that are broadly applicable to municipal or chemical/hazardous waste landfills. As this study demonstrates, doses to landfill workers and members of the public are low, both during the disposal and for the remediation alternatives evaluated, and well below regulatory limits. Removal of the materials does not reduce present day doses, and it presents other risks that outweigh any benefit from the long-term dose reduction.

Analysis of Environmental Data to Support Quantification of Historical Releases from a Former Uranium Processing Facility in Apollo, Pennsylvania.

ABSTRACT: This paper describes how environmental measurement data were used to help quantify the spatial impact and behavior of uranium released to the environment from a uranium manufacturing facility in Apollo, PA. The Apollo facility released enriched uranium to the environment while it operated between 1957 and 1983. Historical monitoring data generated by the site, along with other independent data sources, provided a long-term record documenting the presence and behavior of uranium in the local environment. This record of evidence, together with reconstructed estimates of facility releases, has been used to estimate environmental concentrations during facility operations and potential exposures to members of the public. Historical environmental measurement data were also used to confirm predictions of deposition and concentrations in air. The data are used here to derive atmospheric deposition velocities for the uranium emissions. Based on the spatial pattern of measurements and calculated deposition velocities around the facility, the released material contained larger particles that deposited close to the facility, and the released material remains largely in the surface layers of the soil, indicating limited downward mobility. Evidence of measurable impacts was determined to extend a relatively short distance (<500 m) from the facility. The soil data collected around Apollo are also compared to findings related to uranium mobility at another facility where uranium was released to the environment, and similar behavior was observed at both sites.

Viral Interactions and Pathogenesis during Multiple Viral Infections in Agaricus bisporus.

Viral interactions during multiple viral infections were examined in Agaricus bisporus cultures harboring 9 viruses (comprising 18 distinct viral RNAs) by statistically analyzing their relative abundance in fruitbodies. Four clusters of viral RNA were identified that suggested synergism and coreplication. Pairwise correlations revealed negative and positive correlations between clusters, indicating further synergisms and an antagonism involving a group containing a putative hypovirus and four nonhost ORFan RNAs (RNAs with no similarity to known sequences) possibly acting as defective interfering RNAs. The disease phenotype was observed in 10 to 15% of the fruitbodies apparently randomly located among asymptomatic fruitbodies. The degree of symptom expression consistently correlated with the levels of the multipartite virus AbV16. Diseased fruitbodies contained very high levels of AbV16 and AbV6 RNA2; these levels were orders of magnitude higher than those in asymptomatic tissues and were shown statistically to be discretely higher populations of abundance, indicating an exponential shift in the replicative capacity of the virus. High levels of AbV16 replication were specific to the fruitbody and not found in the underlying mycelium. There appeared to be a stochastic element occurring in these viral interactions, as observed in the distribution of diseased symptoms across a culture, differences in variance between experiments, and a number of additional viruses undergoing the step-jump in levels between experiments. Possible mechanisms for these multiple and simultaneous viral interactions in single culture are discussed in relation to known virus-host regulatory mechanisms for viral replication and whether additional factors could be considered to account for the 1,000-fold increase in AbV16 and AbV6 RNA2 levels.IMPORTANCE How viruses interact in a multiple-virus infection was examined by quantifying the levels of 18 viral RNAs in fruiting cultures of the agriculturally cultivated fungus Agaricus bisporus and statistically analyzing and modeling their abundance. Synergistic, antagonistic, and neutral interactions occurred simultaneously in cultures. The viral RNAs were grouped into four clusters, each displaying similar relative abundance, and between clusters, further interactions were found with positive, negative, or no correlations. Mushroom fruitbodies showing disease symptoms were distributed apparently randomly across the culture. These symptoms were associated with the presence of viral RNAs from two different clusters at very high levels, 1,000-fold higher than asymptomatic fruitbodies. The role of viral interaction together with stochastic factors and the regulation of host antiviral defenses in pathogenesis are discussed.

Reconstruction of Enriched Uranium Released to Air from the Former Apollo Facility, Apollo, Pennsylvania.

ABSTRACT: The former Apollo facility converted enriched uranium hexafluoride into uranium oxide for shipment to nuclear fuel fabrication plants from 1957 to 1983. This paper describes quantification of the source term from the Apollo facility in terms of quantities of uranium released, particle size, and solubility characteristics. Releases occurred through stacks, rooftop vents, and an incinerator that operated from 1964 to 1969. Incidental and accidental releases are addressed as part of this analysis. Atmospheric releases of uranium from plant operations were estimated from stack sampling and production records. Roof vents, both filtered and unfiltered, were the major emission points from the plant. The total estimated release of uranium activity (including 234U, 235U, and 238U) to the air was 28 GBq. Measurements by others found that the releases were primarily associated with large particles and that their solubility was variable but generally low (Class Y). The release estimates presented here and those findings were incorporated into a sophisticated atmospheric transport model to estimate atmospheric concentrations and soil contamination levels due to the releases and to reconstruct historical doses to individuals that lived in the vicinity of the former Apollo facility.

Exposure of Agaricus bisporus to Trichoderma aggressivum f. europaeum leads to growth inhibition and induction of an oxidative stress response.

Green mould disease of mushroom, Agaricus bisporus,is caused by Trichodermaspecies and can result in substantial crop losses.Label free proteomic analysis of changes in the abundance of A. bisporusproteins following exposure to T. aggressivumsupernatantin vitroindicated increased abundance of proteins associated with an oxidative stress response (zinc ion binding (+6.6 fold); peroxidase activity (5.3-fold); carboxylic ester hydrolase (+2.4 fold); dipeptidase (+3.2 fold); [2Fe-2S] cluster assembly (+3.3 fold)). Proteins that decreased in relative abundance were associated with growth: structural constituent of ribosome, translation (-12 fold), deadenylation-dependent decapping of nuclear-transcribed mRNA (-3.4 fold), and small GTPase mediated signal transduction (-2.6 fold). In vivoanalysis revealed that 10-4 T. aggressivuminoculum decreased the mushroom yield by 29% to 56% and 10-3 T. aggressivuminoculum decreased the mushroom yield by 68% to 100%. Proteins that increased in abundance in A. bisporusin vivofollowing exposure to T. aggressivumindicated an oxidative stress response and included proteins with pyruvate kinase activity (+2.6 fold) and hydrolase activity (+2.1 fold)). The results indicate that exposure of A. bisporusmycelium to T. aggressivum in vitroand in vivoresulted in an oxidative stress response and reduction in growth.

Effectiveness of current hygiene practices on minimization of Listeria monocytogenes in different mushroom production-related environments.

BACKGROUND: The commercial production of Agaricus bisporus is a three stage process: 1) production of compost, also called "substrate"; 2) production of casing soil; and 3) production of the mushrooms. Hygiene practices are undertaken at each stage: pasteurization of the substrate, hygiene practices applied during the production of casing soil, postharvest steam cookout, and disinfection at the mushroom production facilities. However, despite these measures, foodborne pathogens, including Listeria monocytogenes, are reported in the mushroom production environment. In this work, the presence of L. monocytogenes was evaluated before and after the application of hygiene practices at each stage of mushroom production with swabs, samples of substrate, casing, and spent mushroom growing substrates. RESULTS: L. monocytogenes was not detected in any casing or substrate sample by enumeration according to BS EN ISO 11290-2:1998. Analysis of the substrate showed that L. monocytogenes was absent in 10 Phase II samples following pasteurization, but was then present in 40% of 10 Phase III samples. At the casing production facility, 31% of 59 samples were positive. Hygiene improvements were applied, and after four sampling occasions, 22% of 37 samples were positive, but no statistically significant difference was observed (p > .05). At mushroom production facilities, the steam cookout process inactivated L. monocytogenes in the spent growth substrate, but 13% of 15 floor swabs at Company 1 and 19% of 16 floor swabs at Company 2, taken after disinfection, were positive. CONCLUSION: These results showed the possibility of L. monocytogenes recontamination of Phase III substrate, cross-contamination at the casing production stage and possible survival after postharvest hygiene practices at the mushroom growing facilities. This information will support the development of targeted measures to minimize L. monocytogenes in the mushroom industry.

Proteomic investigation of interhyphal interactions between strains of Agaricus bisporus.

Hyphae of filamentous fungi undergo polar extension, bifurcation and hyphal fusion to form reticulating networks of mycelia. Hyphal fusion or anastomosis, a ubiquitous process among filamentous fungi, is a vital strategy for how fungi expand over their substrate and interact with or recognise self- and non-self hyphae of neighbouring mycelia in their environment. Morphological and genetic characterisation of anastomosis has been studied in many model fungal species, but little is known of the direct proteomic response of two interacting fungal isolates. Agaricus bisporus, the most widely cultivated edible mushroom crop worldwide, was used as an in vitro model to profile the proteomes of interacting cultures. The globally cultivated strain (A15) was paired with two distinct strains; a commercial hybrid strain and a wild isolate strain. Each co-culture presented a different interaction ranging from complete vegetative compatibility (self), lack of interactions, and antagonistic interactions. These incompatible strains are the focus of research into disease-resistance in commercial crops as the spread of intracellular pathogens, namely mycoviruses, is limited by the lack of interhyphal anastomosis. Unique proteomic responses were detected between all co-cultures. An array of cell wall modifying enzymes, plus fungal growth and morphogenesis proteins were found in significantly (P < 0.05) altered abundances. Nitrogen metabolism dominated in the intracellular proteome, with evidence of nitrogen starvation between competing, non-compatible cultures. Changes in key enzymes of A. bisporus morphogenesis were observed, particularly via increased abundance of glucanosyltransferase in competing interactions and certain chitinases in vegetative compatible interactions only. Carbohydrate-active enzyme arsenals are expanded in antagonistic interactions in A. bisporus. Pathways involved in carbohydrate metabolism and genetic information processing were higher in interacting cultures, most notably during self-recognition. New insights into the differential response of interacting strains of A. bisporus will enhance our understanding of potential barriers to viral transmission through vegetative incompatibility. Our results suggest that a differential proteomic response occurs between A. bisporus at strain-level and findings from this work may guide future proteomic investigation of fungal anastomosis.

FISHing in fungi: Visualisation of mushroom virus X in the mycelium of Agaricus bisporus by fluorescence in situ hybridisation.

Agaricus bisporus is a commercial mushroom crop susceptible to a disease caused by a complex of viruses known collectively as mushroom virus X (MVX). Symptoms of MVX include bare patches and mushroom cap discolouration (browning) in the fruiting bodies, phenotypes associated with the viruses AbV6 and AbV16, respectively. Limited understanding exists of the localisation and mobilisation of these viruses within the mycelium of A. bisporus. To this end, a non-destructive fluorescence in situ hybridisation (FISH) method was developed for in situ targeting of AbV6 and AbV16 in A. bisporus mycelium. An MVX strain associated with the bare patch disease phenotype revealed predominantly high signal towards the growing edges of cultures when probed for AbV6, with a 'halo-effect' of high signal intensity around putative vacuoles. An MVX strain associated with the browning disease phenotype showed high signal intensities within reticulating networks of hyphae in a highly compartmentalised manner when probed for AbV16. Localisation of the two viruses in MVX-infected cultures appears independent, as both viruses were found in completely discrete areas of the mycelium in differential patterns. FISH detected low level presence of the two viruses, AbV6 and AbV16 in a number of cultures which had tested negative for the viruses by RT-PCR. This suggests that FISH may be more sensitive at detecting viruses at low levels than molecular methods. This study demonstrates that FISH is a powerful tool in the field of mycovirology.

The ability of Listeria monocytogenes to form biofilm on surfaces relevant to the mushroom production environment.

Due to its ubiquitous nature, Listeria monocytogenes is a threat to all fresh fruits and vegetables, including mushrooms, which are Ireland's largest horticultural crop. Although fresh cultivated mushrooms (Agaricus bisporus) have not been previously linked with listeriosis outbreaks, the pathogen still poses a threat to the industry, particularly due to its ability to form biofilms. This threat is highlighted by the multiple recalls of mushroom products caused by L. monocytogenes contamination and by previous studies demonstrating that L. monocytogenes is present in the mushroom production environment. In this study, the biofilm formation potential of L. monocytogenes strains isolated from the mushroom production environment was investigated on materials and at temperatures relevant to mushroom production. A preliminary assessment of biofilm formation of 73 mushroom industry isolates was undertaken using a crystal violet assay on polystyrene microtitre plates. The biofilm formation of a subset (n = 7) of these strains was then assessed on twelve different materials, including materials that are representative of the materials commonly found in the mushroom production environments, using the CDC biofilm reactor. Vertical scanning interferometry was used to determine the surface roughness of the chosen materials. All the strains tested using the CDC biofilm reactor were able to form biofilms on the different surfaces tested but material type was found to be a key determining factor on the levels of biofilm formed. Stainless steel, aluminium, rubber, polypropylene and polycarbonate were all able to support biofilm levels in the range of 4-4.9 log10 CFU/cm2, for seven different L. monocytogenes strains. Mushroom industry-specific materials, including growing nets and tarpaulins, were found to support biofilms levels between 4.7 and 6.7 log10 CFU/cm2. Concrete was found to be of concern as it supported 7.7 log10 CFU/cm2 of biofilm for the same strains; however, sealing the concrete resulted in an approximately 2-log reduction in biofilm levels. The surface roughness of the materials varied greatly between the materials (0.7-3.5 log10 Ra) and was found to have a positive correlation with biofilm formation (rs = 0.573) although marginally significant (P = 0.051). The results of this study indicate that L. monocytogenes can readily form biofilms on mushroom industry relevant surfaces, and additionally identifies surfaces of specific concern, where rigorous cleaning and disinfection is required.