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

      Dygico, Lionel Kenneth; Gahan, Cormac G.M.; Grogan, Helen; Burgess, Catherine; Department of Agriculture, Food and the Marine; 14F881 (Elsevier, 2020-03-16)
      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.
    • Effectiveness of current hygiene practices on minimization of Listeria monocytogenes in different mushroom production‐related environments

      Pennone, Vincenzo; Dygico, Kenneth Lyonel; Coffey, Aidan; Gahan, Cormac G.M.; Grogan, Helen; McAuliffe, Olivia; Burgess, Catherine M.; Jordan, Kieran; Department of Agriculture, Food and the Marine; 14/F/881 (Wiley, 2020-05-20)
      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.
    • Exposure of Agaricus bisporus to Trichoderma aggressivum f. europaeum leads to growth inhibition and induction of an oxidative stress response

      Kosanovic, Dejana; Grogan, Helen; Kavanagh, Kevin; Irish Research Council; Science Foundation Ireland; GOIPD/2018/115; 12/RI/2346 (Elsevier BV, 2020-09)
      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.
    • Exposure of Agaricus bisporus to Trichoderma aggressivum f. europaeum leads to growth inhibition and induction of an oxidative stress response

      Kosanovic, Dejana; Grogan, Helen; Kavanagh, Kevin; Science Foundation Ireland; Irish Research Council; 12/RI/2346.; GOIPD/2018/115 (Elsevier, 2020-07-23)
      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.
    • First Report of Shot Hole Disease on Cherry Laurel (Prunus laurocerasus) Caused by Micrococcus aloeverae in Ireland

      Smith, L.; Gibriel, H. A. Y.; Brennan, C.; del Pino de Elias, M.; Twamley, A.; Doohan, F.; Grogan, Helen; Feechan, A.; Department of Agriculture, Food and the Marine; 15/S/759 (Scientific Societies, 2020-08-13)
      First Report of Shot Hole Disease on Cherry Laurel (Prunus laurocerasus) Caused by Micrococcus aloeverae in Ireland
    • First Report of Shot Hole Disease on Cherry Laurel (Prunus laurocerasus) Caused by Micrococcus aloeverae in Ireland

      Smith, L.; Gibriel, H. A. Y.; Brennan, C.; del Pino de Elias, M.; Twamley, A.; Doohan, F.; Grogan, Helen; Feechan, A.; Department of Agriculture, Food and the Marine; 15/S/759 (American Phytopathological Society, 2020-08-13)
      First Report of Shot Hole disease on Cherry Laurel (Prunus laurocerasus) Caused by Micrococcus aloeverae in Ireland
    • Genetic regulation of compost and plant degradation mechanisms in Agaricus bisporus

      Dunne, Keith; O' Donoghue, Martin-Timothy; Grogan, Helen; Heneghan, Mary; IT Sligo; Department of Agriculture, Food and the Marine (2021-06-16)
      Agaricus bisporus (common button mushroom) is an economically significant mushroom with an annual global value in excess of $4.7 billion (Eastwood et al, 2015). When commercially grown, A. bisporus mushrooms are mostly picked from the first and second flush. This is due to the third flush resulting in reduced yields (Royse and Sanchez, 2008), which are also often more prone to disease. This occurs despite significant nutrients and nitrogen being available in the compost for A. bisporus to utilise. To further understand why this is occurring, microarray analysis was carried out on compost samples throughout a full commercial growth cycle, with the aim of identifying genes that may be responsible for this reduction in yield.
    • Hydrogen Sulfide Gas Emissions during Disturbance and Removal of Stored Spent Mushroom Compost

      Velusami, Balasubramanian; Curran, Thomas P; Grogan, Helen; Teagasc Walsh Fellowship Programme; UCD School of Biosystems Engineering; Department of Agriculture, Food and the Marine, Ireland (American Society of Agricultural and Biological Engineers, 2013-12)
      Spent mushroom compost (SMC) is a by-product of the mushroom industry that is used as an agricultural fertilizer. In Europe, SMC storage and use are governed by EU Nitrates Directive 91/676/EEC to protect waterways against pollution by nitrates. A health and safety risk was identified during the removal of stored SMC for land application, as the stored SMC released high levels of toxic H2S gas into the atmosphere when disturbed. In this study, emissions of H2S were monitored at two outdoor and two indoor locations where stored SMC was being removed for land application. A repeating peaktrough pattern of H2S emissions was detected at all sites, with peaks corresponding to periods of active disturbance of SMC. The highest H2S concentrations (10 s average) detected at the SMC face were, respectively, 680 and 2083 ppm at outdoor sites 1 and 2, and 687 and 89 ppm at indoor sites 3 and 4. Higher concentrations of H2S were released from older SMC compared to newer material. Indoor-stored SMC had lower moisture content (53% to 65%) compared to outdoor-stored material (66% to 72%), while the temperature of indoor-stored SMC was higher (33°C to 51°C) compared to outdoorstored material (24°C to 36°C). The current short-term exposure limit (STEL) of 10 ppm was exceeded at all sites except site 4, which was smaller than the other sites, indicating a significant health and safety risk associated with working in the vicinity of stored SMC when it is being actively disturbed. Results suggest that SMC stored under cover in small heaps (600 m3) emits less H2S during disturbance and removal compared to SMC stored outdoors in large heaps (>1500 m3). This should be taken into consideration in the design, construction, and management of SMC storage facilities. Health and safety protocols should be in place at SMC storage facilities to cover the risks of exposure to toxic H2S gas during disturbance of stored SMC.
    • Hydrogen Sulfide Gas Emissions in the Human-Occupied Zone during Disturbance and Removal of Stored Spent Mushroom Compost

      Velusami, Balasubramanian; Curran, Thomas P; Grogan, Helen (American Society of Agricultural and Biological Engineers, 2013-12)
      Hydrogen sulfide (H2S) gas levels were monitored in the human-occupied zone at four spent mushroom compost (SMC) storage sites during removal of SMC for application on agricultural land. During SMC removal operations, H2S gas monitors were mounted on the outside of the tractor, positioned at the SMC periphery, and worn by individual tractor drivers. The highest H2S concentrations (10 s average) detected outside the tractor, at the SMC periphery, and for the tractor driver were, respectively, 454, 249, and 100 ppm for the outdoor sites and 214, 75, and 51 ppm for the indoor sites. The highest short-term exposure values (STEV, over a 15 min period) outside the tractor, at the SMC periphery, and for the tractor driver were 147, 55, and 86 ppm for the outdoor sites and 19, 9, and 10 ppm for the indoor sites. The values exceeded the current maximum permissible concentration limit of 10 ppm for all the sites except for the SMC periphery and tractor driver at the indoor sites. Results suggest that H2S levels detected at indoor storage sites during SMC removal are lower compared to outdoor storage sites. Results indicate that there is a substantial health and safety risk associated with working in the vicinity of stored SMC when it is being disturbed and removed for land application, and that the risk is great for the tractor driver. This article discusses possible control measures and lists recommendations to reduce the risks.
    • Identification and control of Cobweb disease on mushrooms

      Grogan, Helen; Gaze, Richard (Teagasc (Agriculture and Food Development Authority), IrelandHorticultural Development Council, 2008)
      This factsheet is a summary of the most important information currently available on Cobweb disease (Cladobotryum spp.). Its objective is to provide guidance on the recognition, prevention and control of the disease.
    • Identification and control of dry bubble disease of mushrooms

      Gaze, Richard; Grogan, Helen (Teagasc (Agriculture and Food Development Authority), IrelandHorticultural Development Council, 2008)
      This factsheet is a summary of the most important information currently available on Dry bubble disease of mushrooms (Verticillium fungicola). Its objective is to provide guidance to the recognition, prevention and control of the disease.
    • Lactococcus lactis subsp. lactis as a natural anti-listerial agent in the mushroom industry

      Dygico, Lionel K.; O'Connor, Paula M.; Hayes, Maria; Gahan, Cormac G M; Grogan, Helen; Burgess, Catherine; Department of Agriculture, Food & the Marine; 14F881 (Elsevier, 2019-01-28)
      Mushroom growth substrates from different commercial producers of mushrooms (Agaricus bisporus) were screened for the presence of bacteria with potential for use as biocontrol agents for controlling Listeria monocytogenes in the mushroom production environment. Eight anti-listerial strains were isolated from different sources and all were identified using 16s rRNA gene sequencing as Lactococcus lactis subsp. lactis. Whole-genome sequencing of the Lc. lactis isolates indicated that strains from different sites and substrate types were highly similar. Colony MALDI-TOF mass spectrometry found that these strains were Nisin Z producers but inhibitory activity was highly influenced by the incubation conditions and was strain dependant. The biofilm forming ability of these strains was tested using a crystal violet assay and all were found to be strong biofilm formers. Growth of Lc. lactis subsp. lactis using mixed-biofilm conditions with L. monocytogenes on stainless steel resulted in a 4-log reduction of L. monocytogenes cell numbers. Additional sampling of mushroom producers showed that these anti-listerial Lc. lactis strains are commonly present in the mushroom production environment. Lc. lactis has a generally regarded as safe (GRAS) status and therefore has potential for use as an environmentally benign solution to control L. monocytogenes in order to prevent product contamination and to enhance consumer confidence in the mushroom industry.
    • Multiple viral infections in Agaricus bisporus - Characterisation of 18 unique RNA viruses and 8 ORFans identified by deep sequencing

      Deakin, Gregory; Dobbs, Edward; Bennett, Julie M.; Jones, Ian M.; Grogan, Helen; Burton, Kerry S.; Teagasc Walsh Fellowship Programme; European Commission; 201043; 286836 (Nature Publishing Group, 26/05/2017)
      Thirty unique non-host RNAs were sequenced in the cultivated fungus, Agaricus bisporus, comprising 18 viruses each encoding an RdRp domain with an additional 8 ORFans (non-host RNAs with no similarity to known sequences). Two viruses were multipartite with component RNAs showing correlative abundances and common 3′ motifs. The viruses, all positive sense single-stranded, were classified into diverse orders/families. Multiple infections of Agaricus may represent a diverse, dynamic and interactive viral ecosystem with sequence variability ranging over 2 orders of magnitude and evidence of recombination, horizontal gene transfer and variable fragment numbers. Large numbers of viral RNAs were detected in multiple Agaricus samples; up to 24 in samples symptomatic for disease and 8–17 in asymptomatic samples, suggesting adaptive strategies for co-existence. The viral composition of growing cultures was dynamic, with evidence of gains and losses depending on the environment and included new hypothetical viruses when compared with the current transcriptome and EST databases. As the non-cellular transmission of mycoviruses is rare, the founding infections may be ancient, preserved in wild Agaricus populations, which act as reservoirs for subsequent cell-to-cell infection when host populations are expanded massively through fungiculture.
    • Mushroom Virus X (MVX) prevention

      Gaze, Richard; Grogan, Helen (Teagasc (Agriculture and Food Development Authority), IrelandHorticultural Development Council, 2007)
      This factsheet is a summary of the most important information currently available. Its objective is to provide guidance to the recognition, prevention and control of the disease.