Final Report Summary - MUSHTV (EU Title of project: Solutions for the mushroom industry to emerging disease threats from Trichoderma and Virus)
Executive Summary:
The MushTV Consortium is a network of 17 mushroom compost producers, grower associations, businesses and research organisations from across Ireland, United Kingdom, the Netherlands, Belgium and Poland working together to tackle real industry concerns. One of the key challenges facing the mushroom industry is the regulatory obligation to adopt an ‘Integrated Pest Management’ (IPM) approach to pest and disease control, as stipulated in the Sustainable Use of pesticides Directive 2009/128/EC (SUD), in order to safeguard our environment and the consumer.
Two compost-related diseases concerned the MushTV network at the start of this project; ‘compost green mould’ caused by Trichoderma aggressivum and ‘Brown Cap Mushroom Disease’ which causes mushrooms to turn brown and loose quality. Both can infect at an early stage in the cycle and severely reduce production. In addition, the industry has few chemical products available to control disease outbreaks so it relies heavily on understanding the biology of the problem organisms and excellent hygiene standards. Another area of concern was finding IPM-compatible products for the control of other diseases such as Cobweb, Dry Bubble and Wet Bubble for when they get out of control as there are few products available for use. Research outputs from the MushTV project have resulted in major advances being made in our knowledge and understanding of all these diseases: how they spread and how they can be controlled, and this has enabled us to provide practical IPM solutions, technologies and advice to the sector.
Diagnostic Tools. New diagnostic methods for the detection of both Trichoderma and Brown Cap Mushroom Disease in compost have been developed within MushTV providing the industry with the means to monitor their presence. Such monitoring provides an early warning system to growers and composters and can alert them to the need to review procedures in their hygiene systems that may have allowed unwanted organisms to slip through unnoticed.
Knowledge Transfer. MushTV scientists have worked with industry to identify weaknesses in current systems and to find solutions to address them. A series of technical factsheets, which have IPM-compatible strategies at their core, have been produced and disseminated to MushTV growers and composters, providing them with new knowledge and advice on (a) effective use of disinfectants in mushroom production; (b) prevention of Brown Cap Mushroom Disease, (c) control of fungal diseases of mushrooms and (d) understanding Trichoderma aggressivum in bulk Phase 3 compost.
Advancing the frontiers of science. Research by MushTV scientists has advanced the scientific knowledge base in several areas. They have characterised and identified 16 fungal viruses new to science, including two that are associated with the Brown Cap Mushroom Disease. These have been named Agaricus bisporus Virus 16 (AbV-16) and Agaricus bisporus Virus 6 (AbV-6). All the genome sequences of the novel viruses will be published in 2015 and will be available to the scientific community. A new qPCR based method to detect the viruses has been developed as well as a new method for the isolation of RNA from humic-rich mushroom compost. The way in which T. aggressivum is dispersed within large batches of Phase 3 compost has been elucidated and described. Surveys of grower and compost facilities have identified weaknesses in current hygiene systems, highlighting where action is needed to minimise contamination of facilities. Research has also identified a volatile-based diagnostic method for T. aggressivum in Phase 3 compost. In experimental phase 3 tunnels, a group of biochemically-related volatile compounds were repeatedly detected in artificially-infected compost during the Phase 3 process. The pattern of the emitted group of compounds was also different between infected and non-infected compost so a detection technique may be suitable at commercial scale, although further development is needed before commercialisation can take place. These advances in methodologies and knowledge will be published in peer review publications over the coming year.
Collaboration. By working together to resolve common problems, MushTV has fostered a sense of collaboration and friendship between people who are often competitors in the mushroom market place. This, above all has been the outstanding achievement of the MushTV project. The group look forward to working together in the future to address other challenges of mutual concern.
Project Context and Objectives:
At the outset of the project, one of the key challenges facing the European mushroom industry was the regulatory obligation to adopt an ‘Integrated Pest Management’ (IPM) approach to pest and disease control. The EU legal requirements were outlined in the Sustainable Use of pesticides Directive 2009/128/EC (SUD) and individual countries had to then enforce what are known as the “Sustainable Use of pesticides Regulations”. The underlying principles of the SUD regulations are to safeguard human health and the environment, but it does require a change in attitude by composters and growers towards pest and disease management. The objectives of MushTV were to provide information and solutions that were compatible with an IPM ethos.
Integrated Pest Management is described as ‘a program of prevention, monitoring, and control which offers the opportunity to eliminate or reduce the use of pesticides, and to minimise the risk to human health and the environment’. Managers and technical staff must have a greater understanding of the life cycle and biology of the pests and diseases they are trying to control. They must monitor levels of pests and diseases, know how they spread around the farm or facility, know the best hygiene measures to control them, ensure that hygiene measures are employed correctly, use biological and non-chemical control measures where possible, keep updated with advances in IPM control measures, and, only as a last resort, use the least toxic chemicals available when other avenues have been exhausted. The mushroom industry is already well placed to be IPM compatible, but it can still encounter serious pest and disease issues which may be challenging to control.
At the same time, within the mushroom sector, there has been a shift towards increased automation, mechanisation and expansion of production facilities. While mechanisation and expansion have distinct advantages in terms of efficiency and quality, one disadvantage is that should a disease become established, it can be much harder to control, incurring large scale losses and disruption to production.
MushTV is a 39 month project that aims to provide research-based solutions for the mushroom industry, primarily to deal with two relatively new major diseases affecting mushroom production: - Trichoderma aggressivum, which causes compost green mould and Mushroom Virus X (MVX) disease, which causes mushroom browning, crop delay and less-productive crops. Both diseases can have quite severe effects if they occur on bulk phase 3 compost facilities. The SME Associations in the project consortium have identified a need for (a) scientific knowledge of how these pathogens exploit mushroom production systems and (b) technological solutions to controlling them. The SME Associations also want more information on the effectiveness of disinfectants at killing these two pathogens in their various forms. There is an imminent need to identify an effective replacement-disinfectant for Formaldehyde, which is no longer approved for use in mushroom production facilities in most European countries. In addition, there is an imminent need to find effective pesticides or biocontrol agents to control other mushroom diseases such as dry bubble, wet bubble and cobweb, as there is generally only one approved fungicide for mushroom control (prochloraz) in most European countries. Growers and composters increasingly need to have a scientific-based understanding of how pathogens proliferate and spread on their facilities, and how their spread can be controlled. Such knowledge means they will be less reliant on disinfectants and pesticides and they can be more targeted with their use.
The SME Associations and Other mushroom businesses recognise that they do not have the research capacity or resources to conduct this type of research for the sector and they have selected research performers with the necessary expertise and track record to solve the problems on their behalf. The FP7 mechanism ‘Research for the Benefit of SME Associations’ provides an opportunity for collaboration between the appropriate SME-AGs, SMEs, Other businesses and RTD performers. The MushTV Consortium comprises 17 members across five countries. This is the first time that so many European mushroom-related businesses have come together to work towards solutions to common problems despite the fact that individuals within the consortium are direct competitors.
The objectives of MushTV are covered by seven research work packages which are summarised as follows:
Project Objectives:
(1) To identify alternative disinfectant products and methods of disinfection that perform well in large air volumes, and on large pieces of machinery contaminated with high levels of organic matter
(2) To identify the viral entities that make up MVX and use the obtained genetic sequence information, in conjunction with existing data, to improve and further develop sensitive diagnostic tests for use on compost and mushrooms
(3) To develop a T. aggressivum diagnostic method based on detecting unique volatiles from infected compost in the re-circulating air in compost incubation tunnels
(4) To identify where reservoirs of T. aggressivum and MVX inoculum occur at compost and grower facilities so they can be targeted with improved hygiene methods as part of an integrated pest management solution
(5) To evaluate the efficacy of a promising biopesticide based on Bacillus subtilis for the control of Dry Bubble, Wet Bubble, Cobweb as well as T. aggressivum and make recommendations for its use
(6) To describe the pattern of growth of T. aggressivum in a bulk compost-incubation tunnel and determine how it is distributed in the compost when the compost is removed, bulk handled and transported to the grower.
(7) To track the incidence and spread of MVX inoculum on MVX-prone compost and grower facilities before, during and after an outbreak.
There is also a Dissemination and Training work package to disseminate results to all grower-members and technical staff represented by the Consortium across Europe.
Project Results:
WP1: Identification of alternative disinfectants and methods.
WP1 was conducted by researchers at PRI (Partner 6) in the Netherlands and Inagro (Partner 7), in Belgium. It was responsible for identifying alternative disinfectant products to those previously used within the European mushroom industry, which are either being withdrawn from use due to EU regulations, or which are no longer being used due to national regulations or commercial decisions. Effective disinfectants (or biocides) are an essential component of the mushroom industry to ensure all surfaces and equipment are free of undesirable organisms such as bacteria, fungi and viruses, especially those that can cause damage to the mushroom crop. The members of the MushTV consortium have provided us with a list of biocides they wanted to be included in a survey on efficacy against Agaricus bisporus (a model system for MVX-infected A. bisporus) and Trichoderma aggressivum, two serious diseases of mushroom crops. Based on the information delivered to us by the members of the MushTV consortium, the different products were grouped according to the active principle of the biocides. A total of 12 groups could be discriminated; biocides based on 1) mixtures of aldehydes / quaternary ammonium compounds, 2) peracetic acid/hydrogen peroxide, 3) hypochlorite/chlorine based, 4) quaternary ammonium compounds only, 5) radicals and hypochlorite, generated in water by electricity (electrochemically activated water), 6) beneficial organisms, 7) phenolics, 8) purified proteins from plant and mineral sources, 9) ozone, 10) benzoic acid, 11) mixture of amphoteric compounds/quaternary ammonium, and 12) peroxomonosulfate based. From most groups a single product was chosen to serve as a model for the whole group. Some biocides were excluded from further testing based on previous research showing low efficacy.
In laboratory tests, efficacy of the selected biocides was tested both at the recommended dose and time (mostly 15 minutes) and at a double dose and prolonged time (mostly 1 hour), and results were compared with the effectiveness of formalin (at recommended doses). Formalin is currently the industry standard disinfectant in the Netherlands but it is not approved in many European countries. Disinfectants were tested against basidiospores or mycelium of A. bisporus and compost particles that were colonised by A. bisporus mycelium.
• all disinfectants were able to kill the basidiospores of A. bisporus at the concentrations and disinfection times tested except for Eco Des (based on quaternary ammonium only), gaseous ozone and Tersan (amphoterics and quaternary ammonium based),
• Mycelium of A. bisporus could be killed by all disinfectants, with the exception of ozone and electrochemically activated water.
• When present in compost particles, A. bisporus could only be killed effectively with formalin, although at the concentrations and disinfection times tested Eco Des (quaternary ammonium based), Prophyl (phenolics based) and Menno Clean (benzoic acid based) were also quite effective.
Disinfectants were also tested on T. aggressivum.
• Conidiospores of T. aggressivum were killed by all disinfectants, except gaseous ozone.
• At the concentrations and disinfection times tested, mycelium of T. aggressivum could be killed by formalin, Virocid (based on a combination of aldehyde and quaternary ammonium) and Menno Clean. Neither gaseous ozone nor electrochemically activated water had any effect on mycelium.
• None of the disinfectants was able to kill T. aggressivum in infected compost particles at the recommended dose and disinfection time – including Formalin
• Considerable research effort was put into finding a dosage and disinfection time needed for killing T. aggressivum in infected compost particles. Only high concentrations of formalin (6% or 8% for a minimum of 30 minutes) or Menno Clean (8% for a minimum of 2 hours) were able to kill T. aggressivum under laboratory conditions.
Practical relevance of these findings has been discussed with the members of the MushTV consortium. Next to this the disinfectants were compared with each other with respect to EU registration status, possible application methods (spraying, fogging), risks to the user and the environment, possible residues and corrosivity.
Next, attempts were made to extrapolate the laboratory results to the practical conditions in commercial mushroom cultivation. For such extrapolations, various types of surface that may occur in the mushroom industry (rubber mats, stainless steel, mats from growing rooms and tunnels, aluminium, concrete, insulation panel and coated steel) were contaminated with either basidiospores of Agaricus bisporus or conidiospores of Trichoderma aggressivum. Based on efficacy and the lack of leaving a residue, three disinfectants were chosen for testing under “semi-commercial” conditions. The products chosen were ECAS (electrochemically activated water), a product based on hypochlorite and a product based in a combination of peracetic acid and hydrogen peroxide. Results showed that ECAS was not very effective under these semi-commercial conditions. The products based on hypochlorite or on peracetic acid/hydrogen peroxide however, showed good efficacy. Both these products however are corrosive.
One of the compost companies was using a peracetic acid/hydrogen peroxide based product for disinfection of the spawning hall. Their experiences with the product were good, but they have been taking care of protecting the construction of their facilities with a special coating. Within the project the compost company has started to use swabs for monitoring the presence of yeasts and moulds on their facilities. Own research by the compost company has shown to them that in their facilities, the peracetic acid/hydrogen peroxide based product has good efficacy in killing the yeasts and moulds.
As disinfection needs to be preceded by thorough cleaning, a number of Dutch and Belgian growers have started to experiment with the use of industrial foam based detergents. For these foam based detergents to work properly, they need to be applied with specialised equipment and high pressure supply of water and air. As this equipment is quite expensive, the farmers have experimented with cheaper solutions with varying degrees of success.
This WP produced a technical factsheet entitled ‘Use of chemical disinfectants in mushroom production’ which was produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP2: MVX characterisation, diagnostics and biology
WP2 was conducted by researchers at EMR, England (Partner 8) in collaboration with researchers at Teagasc, Ireland (Partner 1). In the 1990s, a set of disease symptoms emerged in the European mushroom industry which caused severe economic damage, for example in the UK, the disease caused £50 M of damage including farm closures (Adie el al, 2004). The symptoms varied from loss of yield, cap distortions and brown discolouration of caps. The disease was found to be associated with the presence of double-stranded RNA molecules (presumed viral) in the mushrooms and so it was named Mushroom Virus X (MVX) disease (Grogan et al, 2003) but it was not known if the viruses, or which virus(es) were responsible for the disease. After extensive cleaning and hygiene precautions in the industry one MVX symptom persisted, the cap browning. This occurrence of the brown cap disease is both sporadic but economically serious as the affected mushrooms are downgraded or discarded due to the associations of the brown discolouration with poor quality and old mushrooms.
The MushTV project was aimed at understanding mushroom diseases and their control and prevention, in particular MVX and Green Mould Disease. Work package 2 (WP2) had two objectives designed to increase our understanding of the nature of the viruses which infect and cause MVX disease in mushrooms, and to develop techniques to detect each virus to enable disease prevention: (1) to fully sequence and characterise the viral dsRNAs associated with MVX brown mushroom symptoms and (2) to identify and optimise the most effective diagnostic method to detect MVX viral presence in mushrooms and compost.
(1) Sequence and characterise the viral RNAs. The biotechnological technique known as Next Generation Sequencing was applied to sequence all the RNA viruses from nine diseased samples of mushrooms and one sample of symptom-free mushrooms. Approximately 45 million short sequence reads (150 – 200bp in length) were produced which were then assembled into longer length ‘contigs’ using a series of bioinformatics tools. Thirty-one contigs were identified as virus-like RNA molecules. The sequences of these contigs were then examined to identify regions which code for viral proteins. The key protein in this search is the replication enzyme known as RNA-dependent-RNA-polymerase or RdRp. Nineteen RdRps were identified and therefore the 31 contigs comprise of 19 different viruses. Some of these viruses contain more than one RNA molecule (AbV6, AbV9 and AbV16 have 2, 3 and 5 RNAs respectively). In addition, 5 viral molecules were identified which were not associated with any particular virus. Only 3 of the 19 viruses had been sequenced previously and so this research has identified 16 new viruses to science. The viruses were classified based on previously described fungal virus taxonomy based on the similarity of their protein sequences (particularly RdRp) as follows: 8 from the Timovirales class, 3 Hypoviruses, one Endornavirus, two Benyviruses, one Barnavirus, one Mitovirus, two Narnaviruses and a new classification of viruses for AbV16. The concept of multiple viral infections is a new and emerging area of science and leads to the conclusion that many of these are not pathogenic but possibly symbiotic. Symptom-free mushrooms contain at least 11 different viruses at significant levels.
The newly sequenced viruses have been given names following current scientific nomenclature. The naming uses the letters AbV (after Agaricus bisporus Virus) followed by a number. As two previously described viruses have been given the names AbV1 and AbV4, the numbering of the newly described viruses starts with ‘2’, i.e. AbV2 (Agaricus bisporus Virus 2) and continues as: AbV2, AbV3, AbV5, AbV6 AbV7, 8, 9,.......AbV16.
Further experimentation in association with WP7, has led to the hypothesis that AbV16 is the virus which causes the brown cap symptoms. The evidence for this hypothesis is that the degree of brown discolouration on diseased mushrooms correlates (with statistical significance) with the levels of the individual RNA components of AbV16 (which comprises of 5 different RNA molecules). In addition two other viruses, AbV6-2 and MBV, may also have some involvement in the disease occurrence.
(2) Identify and optimise the most effective diagnostic method to detect MVX viral presence in mushrooms and compost. After the association was first made between RNAs and MVX symptoms, the presence of RNA was detected by a technique involving electrophoretic separation and observation of dye-stained RNA (Grogan et al, 2003). This technique was able to push on the study of MVX but it was not sufficiently sensitive to detect RNAs at very low levels which is necessary to identify the early stages of the disease. Ideally if the viruses can be detected in the mushroom mycelium growing in compost then the detection technique would provide advanced warning before mushrooms are grown. A project prior to MushTV, the HDC Project M51, developed a proof of principle for a more sensitive test based on a technique called Quantitative-PCR for two of the viruses in the MVX complex (Burton et al, 2010). Work package 2 of the MushTV project aimed at further improving these preliminary tests. There were two components of these investigations: improvements in the extraction techniques of RNA from compost and the use of the newly identified RNA sequences to design ‘PCR’ primers’ for all 31 RNA molecules.
Extraction of RNA from mushroom compost to a high purity is a technically difficult procedure because of the high levels of contaminating chemicals found in mushroom compost for instance, humic substances and phenolics. The experiments conducted compared (a) different extraction buffers, (b) a variety of ‘clean-up’ methodologies, and (c) mechanical treatments to break up the compost into sufficiently small particles to allow for maximum extraction efficiency. A method was developed for RNA extraction from mushroom compost involving milling the compost using metal ball bearings, extraction of RNA with Trizol buffer followed by a clean-up step using silica. This technique was robust, repeatable and economically viable.
PCR primers were designed for each of the 31 virus molecules. The Quantitative-PCR tests were conducted using these primers and the improved RNA extraction technique on a large number of compost samples and all of the 31 virus RNAs were detected, thereby validating that the tests can detect and quantify all of the viruses in compost.
Exploitation and Commercialisation of Virus Tests and IP issues. The newly developed tests to detect and quantify the levels of each virus from compost (and mushrooms) have great use-value for the European mushroom industry and potentially commercial value for technical disease testing laboratories. For the mushroom industry, the tests could be used for early warning disease detection so that sources of infection can be identified, and for routine monitoring of compost producing and mushroom production facilities.
There are 2 potential IP outputs concerning the tests: 1) Methodology to extract RNA from mushroom compost, and 2) The nucleotide sequences of the identified RNA viruses. After discussion with the Exploitation Committee of Mush TV it was concluded that the IP should not be protected because (i) there is insufficient novelty for any formal IP protection (i.e. patent) for the RNA extraction methodology, and (ii) while there is a novelty in the sequences there a need to discuss this result freely with other researchers to formulate a clear understanding of the biology of this fungus/multiple virus interactions and (iii) the market for the testing is unlikely to be sufficiently large in financial terms to pay for the costs of patenting. The delay and constraints of patenting may discourage laboratories from offering these tests. The aim of the MushTV consortium is to provide help to the mushroom industry. Therefore the IP will be made available to laboratories by the publication of two scientific papers.
East Malling Research (Partner 8) investigated the Route to Exploitation in collaboration with its sister company East Malling Services, an independent limited company. These discussions led to a test-marketing to the MushTV consortium of two products of virus detection tests: The cheaper test which measures the levels of the viruses associated with the browning symptom (the five AbV16 RNAs, AbV6-2 and MBV) and a more expensive test whereby the levels of all 31 viral RNAs are measured. The MushTV consortium provided positive feedback on this offering but concluded that before a commercial service was offered or was of benefit to the industry, additional knowledge was required on (a) what are the background levels of the viruses at facilities using the new test; and (b) how should the results be interpreted i.e. knowledge is needed of the threshold levels of each virus that signal a potential threat (early warning) that would recommend that increased hygiene measures should be taken.
References
Adie B, Choi I, Soares A, Holcroft S, Eastwood D, Mead A, Grogan H, Kerrigan R, Challen M and Mills P (2004) MVX disease and dsRNA elements in Agaricus bisporus. Mushroom Science 16, 411-420. In: Science and Cultivation of Edible and Medicinal Fungi, Eds: Romaine, Keil, Rinker and Royse.
Burton KS, Baker A, Eastwood DC and Grogan H. (2010) Developing an accurate, quantitative and predictive test for Mushroom Virus X, Report to Horticulture Development Company, Stoneleigh, Warwickshire, UK. http://www.hdc.org.uk/sites/default/files/research_papers/M51%20Final%20Report.pdf
Grogan HM, Adie BAT, Gaze RH, Challen MP and Mills PR (2003). Double-stranded RNA elements associated with the MVX disease of Agaricus bisporus. Mycological Research 107: 147–154.
Scientific data from this WP will be published in 2015. Some of the information was included in a technical factsheet entitled ‘Brown cap
Mushroom Virus Prevention’ which was produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP3. A volatile-based diagnostic method for Trichoderma aggressivum detection in Phase 3 compost.
WP3 was conducted by researchers at PRI (Partner 6) in the Netherlands and AFBI (Partner 9), in Northern Ireland and worked closely with the compost producers within the Consortium. In recent years green mould (Trichoderma aggressivum) has presented big problems to the European mushroom industry. Trichoderma aggressivum infects compost at a very early stage. Infection most likely takes place at the compost facility during spawn-run (Phase 3 process). In Western Europe the spawn run process is frequently done in large tunnels, in bulk, at the compost yards and the end product is referred to as bulk phase 3 compost. During this process, spawned compost is incubated in tunnels and ventilated with large volumes of air to control compost temperature. During this process the compost is inaccessible for sampling, e.g. for PCR based techniques.
Many organisms, including plants and fungi, produce volatile organic compounds (VOCs) during their growth phase. When interacting with other organisms either in a mutualistic or antagonistic way, the emitted VOC blends can change because the metabolism is affected, e.g. it changes from growth phase to a more defensive stage (Müller et al. 2013; Mumm and Dicke 2010; Unsicker et al. 2009). Mumpuni et al. (1998) have shown that Agaricus bisporus and T. aggressivum use VOCs to affect each other’s growth rate in vitro. In WP3 we aimed to develop a non-invasive technique based on fungal VOCs in the process air to detect an infection by T. aggressivum in compost during spawn-run without the necessity to enter the tunnel. Using this as a starting point for MushTV this work package has six main objectives (tasks):
3.1. Scale up from small scale to medium scale compost quantities
3.2. Study variability of VOC patterns during commercial phase 3 production
3.3. Study specificity of VOC patterns in small to medium scale phase 3 composting systems
3.4. Improvement and simplification of the data processing of the detection method
3.5. Translate method into an application
3.6. Demonstrate proof of principle under commercial conditions
The VOC emission was studied by dynamic headspace trapping on adsorbent cartridges and thermodesorption gas-chromatography mass spectrometry (TDGC-MS). Raw data were processed using a combined untargeted metabolomics and data mining approach in order to identify candidate VOCs. First experiments on a small scale using glass vessels with 300g of Phase 2 compost showed a clear difference in VOC profiles between T. aggressivum and non-infected compost.
In Objective 3.1 we carried out experiments with volumes of Phase 2 compost ranging from medium scale ~50kg (referred to as Mini-tunnels; at PRI) to semi-industrial scale (~1800kg; at Partner 9, AFBI) to study different partial infection rates of T. aggressivum. Several experiments with Mini-tunnels showed that VOC profiles of infected compost significantly differed from non-infected compost at day 14 and day 18 of spawn-run. The majority of the VOCs that were significantly more abundant in T. aggressivum-infected and having a good predictive capacity belong to a group of biochemically related natural compounds that are widely present in plants and microorganisms. When comparing VOCs being significantly more abundant in the T. aggressivum infected compost in the small scale trials with data from the medium scale trials, it was difficult to assign one or a small set of VOCs reliably present in the majority of trials. Alternatively, we found that the temporal pattern of the total signal of the emitted candidate compounds (as determined by the joint mass spectral signal of the molecular weight) was different between infected and non-infected compost during spawn-run. In healthy Phase 3 compost, emission of the sum of candidate compounds peaked at around day 11 and then steadily decreased until despatch of the compost but the emission from infected compost peaked around day 14. This consistent difference in the pattern of emissions of these compounds for healthy Phase 3 compost compared to T. aggressivum infected Phase 3 was detected on small (50kg) scale and semi-industrial scale (1800kg) across several independent trials.
In Objective 3.2. we carried out several trials to determine whether VOC emission including the candidate compounds can be reliably monitored during commercial phase 3 production. Early experiments using the small scale Mini-tunnels filled with ~50kg Phase 2 compost from different compost producers showed clear differences in the VOC pattern between the different sampling days, and were detected in compost of all three compost producers. The VOC profiles of non-infected compost of the three compost producers behaved similarly during spawn-run. Later experiments worked with commercial companies. VOCs from the process air from commercial Phase 3 tunnels in the Netherlands and Ireland were collected at different days of spawn-run. Dutch and Irish compost differ in the composition of ingredients. Irish compost uses mainly straw and poultry manure while Dutch compost frequently has horse manure mixed with the straw. A large number of tunnels were sampled at each facility with a number of different spawn strains. The results from these commercial scale trials demonstrated that VOC emission patterns during the Phase 3 process obtained under commercial conditions resemble those detected on lab scale (50-1800kg compost). This indicates that the detection method of monitoring the VOC emission can also be applied on commercial scale.
In Objective 3.3. we tested whether the VOC patterns emitted from compost infected with different mould species (T. aggressivum, T. harzianum, T. atroviride, Aspergillus fumigatus, or Penicillium citreonigrum) were similar to or different to the volatile pattern from clean compost or compost infected with T. aggressivum. Experiments were carried out on small scale using Mini-Tunnels. With the exception of P. citreonigrum all tested mould species including T. aggressivum induced significant changes in the VOC profiles of phase 3 compost. No VOCs were uniquely induced by only one mould species but by combining the pattern of different induced VOCs, single mould species may be discriminated.
In Objective 3.4. the aim was to improve and simplify the data processing of the VOC detection method. The developed method focusses on the sampling and analysis of the emission pattern of a specific group of VOCs which can be done in a very targeted manner, e.g. by using mass spectrometry techniques either as stand-alone technique or hyphenated with a separation technique such as gas chromatography. By using a targeted approach the detection sensitivity can be highly improved and processing of the data is more straightforward compared to the untargeted data mining approach that was used at the beginning of the project.
To tackle Objective 3.5 we showed as a proof of concept that VOC emissions can successfully be monitored using an online mass spectrometry technique (Proton Transfer Reaction- Mass Spectrometer (PTR-MS)) during the commercial Phase 3 process. Patterns were similar to what had been shown for small and semi-industrial scale trials. The proof of principle of this method under commercial conditions (as stated under Objective 3.6.) was not achieved because we did not get notice of any green mould incidents during the life of the project.
References.
Müller, A., Faubert, P., Hagen, M., zu Castell, W., Polle, A., Schnitzler, J.-P. and Rosenkranz, M. 2013. Volatile profiles of fungi – Chemotyping of species and ecological functions. Fungal Genet Biol 54(0): 25-33.
Mumm, R., and Dicke, M. 2010. Variation in natural plant products and the attraction of bodyguards involved in indirect plant defense. Can J Zool 88(7): 628-667.
Mumpuni, A., Sharma, H.S.S. and Brown, A.E. 1998. Effect of metabolites produced by Trichoderma harzianum biotypes and Agaricus bisporus on their respective growth radii in culture. Appl Environ Microbiol 64(12): 5053-5056.
Unsicker, S.B. Kunert, G., and Gershenzon, J. 2009. Protective perfumes: the role of vegetative volatiles in plant defense against herbivores. Curr Opin Plant Biol 12(4): 479-485.
Scientific data from this WP will be published in 2015. Some of the information was also included in the technical factsheets on ‘Understanding Trichoderma aggressivum in Bulk Phase 3 compost’ which were produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP4: Investigations to locate reservoirs of Trichoderma and MVX inoculum mushroom compost and mushroom grower facilities.
WP4 was conducted by researchers at Teagasc, Ireland (Partner 1) in collaboration with AFBI (Partner 9), in Northern Ireland. This work package worked in confidence with composters and growers and all individual results are confidential, as detailed in Table 3.2b in Annex 1. WP4 had two objectives. Objective 4.1 looked to identify the most likely locations on mushroom facilities to detect Trichoderma aggressivum and MVX if it is present. Pilot studies were carried out at compost and grower facilities to identify a list of critical sample points which would be used in the wider investigations of industry facilities in Objective 4.2.
The pilot studies for MVX and Trichoderma on compost facilities were carried out together because both pathogens are likely to be spread in a similar fashion (spores, infected compost and compost-debris). Visits were conducted and samples were taken from various locations. Samples were processed in the laboratory and tested for presence of MVX and T. aggressivum. For MVX testing, pure cultures of Agaricus bisporus were obtained from compost and compost-debris samples and tested for presence of MVX dsRNAs using MVX-specific PCR primers. For T. aggressivum testing, all samples were plated onto Malt Agar medium (with antibiotics) and any moulds that grew were identified. Any Trichoderma species present were isolated into pure culture and tested using T. aggressivum-specific PCR primers. The results from the pilot study were evaluated and sample locations that gave consistent and useful results were then selected for inclusion on the “Critical Control Point” list which would be used for a wider survey of industry compost facilities in Objective 4.2.
The Pilot studies of the grower facilities for MVX and Trichoderma were carried out on two separate facilities. Sampling for presence of Trichoderma on the grower facility involved taking compost samples as well as taking swabs of equipment, shelving and surfaces which might harbour T. aggressivum spores as well as infected compost debris. Sampling for presence of MVX on mushroom growing units focused on taking samples of compost and mushrooms as these are the best types of samples for MVX. A grower facility with on-going MVX issues was targeted and a separate grower facility with Trichoderma problems was also targeted. Various samples were taken from a range of locations, the samples were processed by both molecular and microbiological techniques and the results evaluated. Two separate lists of Critical Control Points for grower facilities were compiled, one for MVX and one Trichoderma. The Critical Control Points were discussed and agreed with the Consortium. Once the three lists of Critical Control Points for sampling of industry facilities were complete, the survey of industry facilities across Europe began.
Objective 4.2 looked to gather information on how widespread Trichoderma and MVX were on mushroom compost and growing facilities across Europe. All compost facilities within the Consortium were surveyed using the Critical Control Points identified in the pilot studies, but occasionally additional samples were also taken. The results of sample testing were communicated to each composter confidentially. A number of growing facilities across Europe were also surveyed, using the Critical Control Points identified in the pilot studies. Results were communicated to the management of each facility confidentially. As part of this objective, a case study on Trichoderma spread and transmission was carried out at the experimental research facilities of Partner 9, AFBI, in Northern Ireland, where studies on the growth of Trichoderma in Phase 3 compost were being conducted. Sampling of Partner 9 facilities took place on six occasions from the end of one T. aggressivum growth trial to the same point at the end of a second T. aggressivum growth trial.
Results of the case study show that Trichoderma is very easily transmitted once it is present on a facility, even when an apparently very rigid hygiene protocol is in place. The number of Trichoderma- positives being detected increased over the duration of the second trial as T. aggressivum became established in an experimental crop. Most of the positives were within the growing room (house 3) containing the trial. However by the end of the crop, T. aggressivum had spread outside the growing room into the corridor, onto the exterior door surface, the control panel for the room as well as into offices, canteen and transport vehicles, increasing the likelihood of further spread and contamination of new crops. A critical conclusion of the case study was that an improved and more intensive cleaning and disinfection programme was necessary to eliminate all instances of T. aggressivum cross contamination on the facility. Infected crops must be thoroughly cooked-out (65OC for a minimum of 8 hours with compost in the growing room followed by a second cook-out of 65OC for a minimum of 8 hours once the compost has been removed). In addition, all structures, equipment and surfaces must be thoroughly cleaned and disinfected, with all parts of the facility being targeted.
Some of the information from the WP was included in the technical factsheets on ‘Brown cap Mushroom Virus Prevention’ and ‘Understanding Trichoderma aggressivum in Bulk Phase 3 compost’ which were produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP5 – Evaluation of the biopesticide Bacillus subtilis for the control of mushroom pathogens
WP5 was conducted by researchers at Inagro, in Belgium (Partner 7) and Teagasc, Ireland (Partner 1). The objective of this WP was to evaluate the efficacy of a promising biopesticide based on Bacillus subtilis for the control of the diseases known as Dry Bubble, Wet Bubble, Cobweb as well as T. aggressivum, and, if successful results were obtained, to make recommendations for its use. A commercially available product containing B. subtilis was used. Cultures of the four pathogens Trichoderma aggressivum, Verticillium (Lecanicillium) fungicola var. fungicola (Dry Bubble), Mycogone perniciosa (Wet Bubble) and Cladobotryum mycophilum (Cobweb) were obtained from research partners PRI, in the Netherlands, and Teagasc.
Initial work consisted of laboratory scale tests. In vitro antagonism of B. subtilis was examined by testing different concentrations against fungal mycelium plugs. In vitro dual culture studies were done following standard microbiological techniques using a range of different culture media (MEA, PDA and Sabouraud). Plates were incubated for several days at 25°C and inhibition of fungal growth monitored daily by recording the diameter of the inhibition zone. Each dual confrontation was performed in duplicate. Cladobotryum was not inhibited by Bacillus and overgrew the Bacillus colonies completely within 3 to 7 days. Trichoderma growth was not inhibited by Bacillus on Sab agar plates, but some inhibition occurred on PDA and MEA plates. Fungal growth of both Verticillium and Mycogone was strongly inhibited by Bacillus. Agaricus growth was somewhat reduced on PDA plates but inhibition was less obvious on MEA plates. As results from laboratory tests are not always a predictor of how products will work under cropping conditions, a series of small cropping experiments in 0.2 m² containers were carried out according to standard practices on mushroom farms.
Evaluation of B. subtilis against T. aggressivum – compost green mould. A small scale cropping trial was set up to test the efficacy of Bacillus subtilis against T. aggressivum under bulk phase 3 conditions. In this trial 1N and 2N rates were applied and three applications were made at different points in the crop cycle: (1) applied to mushroom spawn prior to its incorporation into compost; (2) sprayed onto fully colonised compost at the end of spawn-run after it has been broken up (bulk handled) and emptied from the incubation container and (3) applied as a drench to the casing in the last watering before the crop was vented (Day 7 approx.). Trichoderma aggressivum inoculum consisted of mushroom compost that was pre-colonised with T. aggressivum as this is the most likely source of infection at bulk Phase 3 facilities. Three applications of Bacillus failed to prevent or reduce the development of green mould in the crop.
Evaluation of B. subtilis against Mycogone – wet bubble. To evaluate the efficacy of B. subtilis, against Mycogone, a small cropping experiment was set up. Bacillus was applied following a three application treatment schedule (at casing, at last watering before aeration and after first flush). Infected control plots were treated with the industry standard prochloraz-Mn (46%; Sporgon, BASF) at the manufacturer’s recommended rates. Observations confirmed the efficacy of Sporgon. However, plots treated with Bacillus showed high levels of wet bubble disease symptoms, despite its antifungal activity against Mycogone on agar plate tests. A second trial was conducted and results confirmed that Bacillus did not show any control against wet bubble, compared to prochloraz.
Evaluation B. subtilis against Verticillium – dry bubble – and Cladobotryum – cobweb. Similar trials to those described for Mycogone were also conducted for Verticillium and Cladobotryum. Results of duplicate trials for each pathogen also confirmed that Bacillus did not show any control against dry bubble or cobweb, compared to prochloraz.
Evaluation of selected alternative products against Verticillium and Cladobotryum.
When the biopesticide B. subtilis was shown to be ineffective against three mushroom pathogens, the consortium agreed to evaluate two extra candidate products against dry bubble and cobweb. The first was a commercially available biopesticide product (Product C) with no approval for use in Europe. The second was a commercially available chemical fungicide (Product B) with limited approval for use in Europe. In duplicate small scale cropping trials Product C did not give any control against either Verticillium or Cladobotryum, compared to prochloraz. Duplicate small scale cropping trials with Product B however confirmed that it was effective at controlling both Verticillium. or Cladobotryum, compared to prochloraz. There is interest in getting the product registered for the European mushroom Industry. However, in the absence of an effective biopesticide, non-chemical disease control still relies heavily on effective hygiene methods.
Phytotoxicity and residue tests. All three products were assessed in large scale cropping trials for any potential adverse effects on the growth and yield of mushrooms. All products were tested at single and double or x1.5 doses. There was no significant effect of any product on total yield but there were some minor changes to the flushing pattern. Mushrooms from the standard and double rate treatments for Product B were tested for residues of the active ingredient. Residues were detected but they were below the MRL.
SIGNIFICANT RESULTS
• Bacillus subtilis gave no control against T. aggressivum green mould in bulk Phase 3 compost.
• Bacillus subtilis gave no control against wet bubble, dry bubble or cobweb disease, compared to prochloraz.
• A second biopesticide (Product C) gave no control against dry bubble or cobweb disease, compared to prochloraz.
• A chemical fungicide (Product B) gave very good control against dry bubble and cobweb disease, compared to prochloraz
Some of the information from this WP was included in the technical factsheets on Fungal Diseases of Mushrooms and their Control (English, Dutch and Polish) which was produced in three languages and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP6 Trichoderma Growth in Phase 3 Compost.
WP6 was conducted by researchers at AFBI (Partner 9) in Northern Ireland in collaboration with Teagasc, Ireland (Partner 1). The work focussed on characterising the growth, dispersal and impact on yield of T. aggressivum in bulk-incubation tunnels. In these compost systems, large volumes of re-circulated air are forced through an unsegregated compost mass so theoretically the potential to infect all the compost within the tunnel could be high. Initial trials were undertaken to specifically investigate how T. aggressivum grew in compost during a typical 17 day spawn run performed in bulk. Experimental mini composting tunnels (ca 1800 kgs) were subdivided along four vertical and three horizontal planes and a spore suspension inoculum positioned in the back, lower quadrant. Results indicated that distances travelled by T. aggressivum within the bulk spawn-run (compost temperature ca 25±1OC) were generally limited to just ca 0.5 -1.0 m. These growth rates are in broad agreement with previous in vitro and in vivo studies. T. aggressivum was not generally visible at the end of bulk spawn-run so composters would be oblivious to any compost infection. The compost was removed from the experimental mini tunnels in an artificial process, removing 12 blocks of unmixed compost from each of the four vertical and three horizontal planes. This contrasts sharply with commercial practices where the bulk handling operations will usually involve several distinct mixing processes. Results indicated that the severity of the subsequent mushroom crop production yield losses was highly correlated with the position of the compost in the tunnel relative to the growth of the Trichoderma. Whilst crop yield was largely unaffected in compost from the front two or three compost slices, average yields from the back slice closest to the inoculation point were reduced by 47% (range 2 - 100%) and 56% (range 8 - 98%) respectively in two trials. These initial compost trials have been essential in increasing our understanding of how far and in what direction Trichoderma can grow during the 17 day bulk spawn-run period.
A second series of trials studied the effect of three mixing processes (action of the compost tunnel winch, horizontal layered filling of transportation vehicles and vertical lorry emptying for shelf growing at grower holdings) on dispersal and spread of T. aggressivum during 3 differentiated handling operations (compost emptied unmixed, compost emptied mixed within each of 4 individual tunnel slices, compost emptied mixed and layered across all 4 tunnel slices). The latter most closely simulating bulk handling operations carried out at commercial premises. Consistently, no dispersal from the original point of inoculation was evident in compost from unmixed slices; the infection was dispersed through the inoculated slice only in compost from individually mixed slices, and compost fully mixed across all 4 tunnel slices dispersed the initial localised T. aggressivum infection (previously contained close to the point of inoculation in the back, bottom quadrant of the compost tunnel) throughout all compost in that tunnel. The severity of the yield losses was highly correlated with the degree of mixing – essentially 0% yield loss from unmixed compost except in the inoculated quadrant where it was 100%; ca 100% yield loss in the inoculated mixed slice but close to 0% yield loss from the remaining 3 slices in the tunnel and ca 100% yield loss across all compost from the tunnel in the fully mixed treatments. These trials further indicated that T. aggressivum can readily infect and colonise otherwise healthy, productive mushroom compost as evidenced by typical yields of 25 - 34 kgm2-1 in control plots and uninfected compost.
In additional trials, the use of equipment that had handled T. aggressivum infected compost was also shown to infect clean compost from adjacent newly opened tunnels, spreading the infection further. In addition, where the infection occurs in a tunnel is critical; if it is near the front, all subsequent compost will come in contact with now infected machinery; if it is near the back then a large proportion of compost from the front of the tunnel should yield normally. This new knowledge could explain previous observations where several growers getting compost from the same tunnel report completely different levels of infection ranging from none to total crop wipe-out.
Crucially, these studies have now confirmed that fully colonised phase 3 compost is susceptible to T. aggressivum infection. Results clearly indicate that the bulk spawn-run process potentially increases the risk of a small compost infection becoming a serious disease outbreak, however not as a result of significant growth within the compost during the 17 day spawn-run but as a direct consequence of the bulk handling and mixing operations that disperse and spread small infections through much larger compost masses. Cross contamination was also shown to occur on the growing unit from an infected crop to a newly filling crop and handling Trichoderma infected Phase 3 readily dispersed the disease around the growing unit. This was clearly identified at the critical sampling locations tested as part of WP4. Further, the results from WP1, which highlighted that disinfectants were ineffective in killing T. aggressivum in colonised compost, have important ramifications for best practice guidelines at growing units. Stringent hygiene procedures and cleaning up of infected materials/facilities following crop termination were effective at containing the disease. Post crop steam sterilisation (65-70˚C in the compost for a minimum of 8 hours applied both with compost in the growing room and again when it had been removed) was effective as independently verified by Partner 1 (Teagasc) under WP4.
A second objective of this work-package aimed to quantify the concentration of T. aggressivum in Phase 3 mushroom compost at various production stages. A number of Trichoderma detection methods - Compost Fragment Plate (CFP), Compost Plate, Most Probable Number, PCR and real-time PCR have been tested. Trichoderma was detected on air fallout plates positioned close to the mini-tunnel door during emptying of the back infected tunnel slice in almost all trials but less consistently on plates positioned at a far wall in the spawning hall. This is consistent with investigations that have shown Trichoderma spores, though not readily airborne, are easily transmitted on dust particles and compost fragments. Air fallout plates can therefore provide a very simple but useful monitoring tool for compost manufacturers and several of the consortium partners have already incorporated this monitoring procedure in their routine hygiene programmes. Accurate diagnostic testing that can be applied directly to large compost masses is notoriously difficult not least because sample size is prohibitively small to be classed as representative. In the mini tunnel trials, 10 or 100g samples represent just 0.0025% – 0.025% of the compost; in commercial operations processing much larger compost volumes this problem is compounded. In the initial trials, in the absence of a compost mixing process there was an acknowledged limitation in the sampling protocol that meant the assays used showed varying abilities to detect the inoculated T. aggressivum. In trials that incorporating compost three mixing procedures as standard, all assays efficacy improved significantly although the representative sampling size limitation stands.
PCR and real-time PCR methods generally proved effective with the strict proviso that compost must be thoroughly mixed before sampling to ensure assay efficacy. However, using CFP detection methods with two samples, both Partners 9 and 1 identified T. aggressivum at low levels that was undetected by real-time PCR (Ct = 40). The latter method currently records Ct values up to 40 cycles, with a Ct of 40 = zero, but there may be a limit of detection as this was clearly not zero as further evidenced by crop production yield losses and T. aggressivum assessments at crop termination. Undoubtedly, as a quantitative tool, real time PCR provides valuable additional information to composters on the extent of the infection and likely impact at the mushroom production stage. In addition, the quicker sample turn-around time of real-time PCR would make it the preferred detection method for composters, however, it is still an “end of process” assay and reliant on representative samples from prohibitively large volumes of well mixed compost. Volatile organic compound (VOCs) samples in adsorbent cartridges taken from both within the compost and from the head space above the compost during these WP6 trials were provided to Partner 6 for the volatile diagnostic work and are reported separately in WP3. This alternative “in process” or “online” VOC assay (WP3) to distinguish Trichoderma infected compost on experimental scale facilities may also be applicable under commercial conditions. This would offer significant advantages in the decision making process on how to manage compost infected with Trichoderma to limit further exposure of both compost and growing production facilities.
Some of the information from this WP was included in the technical factsheet on Understanding Trichoderma aggressivum in Bulk Phase 3 compost (English, Dutch and Polish) which was produced in three languages and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP7: Investigations to track the incidence and spread of MVX inoculum on compost and grower facilities.
WP7 was conducted by researchers at Teagasc, Ireland (Partner 1) in collaboration with researchers at EMR, England (Partner 8). It had one objective, OBJECTIVE 4.1 –to gather information on the incidence and spread of MVX (before during and after an outbreak) on compost and grower facilities, which was subdivided into three tasks.
TASK 7.1 set out to validate the improved MVX diagnostic test and extraction method developed in WP2 under the controlled conditions of an experimental cropping trial. A 4m trough of A15 spawned Phase 2 compost was inoculated at one end, at spawning time, then incubated as normal. At the end of the spawn run period sections were removed from the trough at 1m intervals, then cased and cropped individually. Samples of compost were also taken at the end of spawn run along the length of the trough for the detection of two MVX dsRNAs “AbV16” and “AbV6” using the existing RT-PCR method and the new qPCR method. Following cropping, mushroom samples and end of crop compost samples were also taken for analysis by RT-PCR and qPCR. The newly developed methodology for extraction of RNA from compost and qPCR diagnostics worked effectively to quantify the levels of viruses in the compost samples and the results matched with the corresponding RT-PCR results for both mushrooms and mycelia grown off compost. Therefore the improved diagnostics proved to be effective for their intended purpose for use with spawn-run compost.
TASK 7.2 studied MVX Epidemiology as it relates to compost facilities. Background monitoring of MVX on compost facilities was carried out at a number of compost facilities and results were reported back to each company. Monitoring was done in two ways: (i) a full round of Phase 3 compost samples from each tunnel on the facility over an emptying cycle and (ii) monthly samples on one day per month from each of the facilities for five months, this monthly sampling included two debris type samples and a sample of the Phase 3 compost from any tunnels being emptied that day. The Phase 3 compost samples were tested by RT-PCR for the MVX dsRNAs and the debris type samples were used in bioassay cropping experiments to determine if MVX dsRNAs would be transmitted to healthy compost and if brown mushroom symptoms would be observed. Brown mushroom symptoms were measured in the cropping experiments trials using a chromameter. Following an outbreak of MVX in autumn 2013, a period of intense sampling took place over the course of 5 months. MVX dsRNSAs (MVX band 19 - now known as AbV16-1, and MVX band 15 - now known as AbV6-2) were detected in a small number of Phase 3 compost samples. The compost debris samples used in the bioassay experiments did not result in symptoms being observed, however MVX dsRNAs were detected in mushrooms from a treatment inoculated with one of the debris samples. The conclusion from this task was that MVX dsRNAs are present in some samples of Phase 3 compost at tunnel emptying and in some compost debris on compost facilities, and infection of healthy phase 3 compost with such material can transmit MVX viruses (AbV-16 and AbV-6) into the healthy compost.
TASK 7.3 studied MVX Epidemiology as it relates to mushroom growing facilities. Background sampling was carried out at several grower facilities which had recently experienced MVX to identify locations where MVX was present. An additional grower facility, experiencing significant MVX issues, was targeted for intensive sampling in conjunction with the compost facility supplying the Phase 3 compost. These samples were tested for MVX dsRNAs and also used in bioassay experiments. Samples of debris were also obtained from all grower facilities for use in bioassay experiments.
The initial background sampling detected MVX only in samples of mushrooms or compost from crops with symptoms and not in samples from other locations on the facilities. However, the samples of compost from a grower facility experiencing significant problems had MVX dsRNAs in all samples tested: Phase 3 compost from the compost facility at emptying, compost from the truck at delivery to the grower facility, compost from the freshly filled shelf and compost from under symptomatic mushrooms. In bioassay experiments, where small amounts of all these “virus-positive” compost samples were added into healthy compost as inoculum and then cropped, only the treatment inoculated with compost from under the symptomatic mushrooms re-produced symptomatic mushrooms upon cropping. Mushrooms from all other treatments however tested positive for the MVX dsRNAs, consistent with those of the inoculum, but did not exhibit symptoms.
The debris samples from all grower facilities were also used in a separate bioassay experiment. Mushrooms from all treatments inoculated with the debris exhibited no symptoms and all tested negative by RT-PCR, however one ‘end of crop’ compost sample from one of the treatments at the end of the bioassay tested positive for MVX dsRNA band 19 (AbV16-1).
SIGNIFICANT RESULTS
• The new RNA extraction method developed in WP2 was an effective and efficient method of extracting RNA from mushroom compost
• The qPCR tests developed in WP2 for the mushroom viruses AbV16-1 and AbV6-2 were effective and sensitive at detecting their presence in compost samples, compared with existing detection methods for mushrooms and mycelium by RT-PCR.
• In bioassay experiments MVX dsRNAs were transmitted from infected Phase 3 compost into healthy Phase 3 compost but generally the mushrooms showed no symptoms. Only when infected 'end of crop compost' from a symptomatic crop was used to infect the healthy Phase 3 compost, were brown mushroom symptoms observed in the resulting crop. Thus, low levels of MVX viruses can be present in crops but with no symptoms.
• Debris samples from compost and grower facilities were found to be a means of transmitting viruses to healthy compost.
Some of the information from this WP was included in the technical factsheet on ‘Brown cap Mushroom Virus Prevention’ (English, Dutch and Polish) which was produced in three languages and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
Potential Impact:
MushTV has produced a range of new technical knowledge and advice for the mushroom industry, which has been distributed in the form of factsheets, workshops, seminars and popular articles. MushTV has developed a new diagnostic method to detect MVX viruses in mushroom compost and work is in progress to make it available as a commercial service. MushTV has developed methodology to underpin a novel volatile-based diagnostic method for detecting Trichoderma aggressivum in Phase 3 compost. Dissemination events in face to face settings have reached in the region of 200 industry personnel from across all SME AGs and the various MushTV partners and their customers. The MushTV Industry partners have indicated how they have benefited significantly through their participation in MushTV. Many partners have altered their own practices in response to knowledge and information gained from the project, particularly in the areas of disinfectant use and control of Trichoderma green mould and Mushroom Virus X. This new information will make the mushroom industry as a whole more knowledgeable and more efficient at dealing with disease threats thereby reducing their reliance on, and use of pesticides. This will have important economic benefits for the industry. Reduced pesticide use also has a positive social and environmental impact in the context of high quality food and protection of the environment. Specific results are outlined below.
WP1 identified that it is very difficult to kill T. aggressivum with disinfectants when it is present in compost particles so that it is essential to ensure all compost fragments are cleaned off equipment and surfaces prior to disinfection. A factsheet entitled ‘Use of chemical disinfectants in mushroom production’ was produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers. .
WP2 has characterised and named the viruses that make up MVX and developed improved highly sensitive tests for their detection. The MVX virus that causes the brown cap mushroom symptoms has been sequenced and named as Agaricus bisporus Virus 16 (AbV16).
WP3 has the potential to deliver a volatile-based diagnostic method to detect T. aggressivum in Phase 3 compost however it requires further development and is far from commercial implementation at this point in time.
WP4 has gathered data from growers and composters about the incidence of T. aggressivum and MVX on facilities during the project period. This information has helped individual SME’s to be more informed of disease risks on their facilities.
WP5 has evaluated three products (two biopesticides and one chemical) for the control of mushroom diseases. Only the chemical product has shown promise as a potential new product for the industry. As yet, non-chemical disease control still relies on effective hygiene methods. In addition, a technical factsheet entitled ‘Fungal diseases of mushrooms and their control’ was produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP6 generated new knowledge on the growth and spread of T. aggressivum within the bulk Phase 3 system thereby increasing our understanding of how easily this pathogen can have a severe impact on mushroom production. This information, along with information from WPs 3 and 4, above, was used to compile a technical factsheet entitled ‘Understanding Trichoderma aggressivum in Bulk Phase 3 compost’. It was produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP7 generated new knowledge on how MVX spreads within mushroom compost. It identified that the AbV16 virus that causes Brown Cap Mushroom disease can be present in both compost and mushrooms in the absence of symptoms, leading to a false sense of security that the virus is absent. This information, along with information from WPs 2 and 4, above, were used to compile a technical factsheet entitled ‘Brown Cap Mushroom Virus prevention’. It was produced in three languages (English, Dutch and Polish) and provided in electronic and hard copy format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
The MushTV SMEAgs and all Consortium members agreed to make all the factsheets available to the wider mushroom industry on the MushTV and other websites .
List of Websites:
www.MushTV.eu; Helen.grogan@teagasc.ie
The MushTV Consortium is a network of 17 mushroom compost producers, grower associations, businesses and research organisations from across Ireland, United Kingdom, the Netherlands, Belgium and Poland working together to tackle real industry concerns. One of the key challenges facing the mushroom industry is the regulatory obligation to adopt an ‘Integrated Pest Management’ (IPM) approach to pest and disease control, as stipulated in the Sustainable Use of pesticides Directive 2009/128/EC (SUD), in order to safeguard our environment and the consumer.
Two compost-related diseases concerned the MushTV network at the start of this project; ‘compost green mould’ caused by Trichoderma aggressivum and ‘Brown Cap Mushroom Disease’ which causes mushrooms to turn brown and loose quality. Both can infect at an early stage in the cycle and severely reduce production. In addition, the industry has few chemical products available to control disease outbreaks so it relies heavily on understanding the biology of the problem organisms and excellent hygiene standards. Another area of concern was finding IPM-compatible products for the control of other diseases such as Cobweb, Dry Bubble and Wet Bubble for when they get out of control as there are few products available for use. Research outputs from the MushTV project have resulted in major advances being made in our knowledge and understanding of all these diseases: how they spread and how they can be controlled, and this has enabled us to provide practical IPM solutions, technologies and advice to the sector.
Diagnostic Tools. New diagnostic methods for the detection of both Trichoderma and Brown Cap Mushroom Disease in compost have been developed within MushTV providing the industry with the means to monitor their presence. Such monitoring provides an early warning system to growers and composters and can alert them to the need to review procedures in their hygiene systems that may have allowed unwanted organisms to slip through unnoticed.
Knowledge Transfer. MushTV scientists have worked with industry to identify weaknesses in current systems and to find solutions to address them. A series of technical factsheets, which have IPM-compatible strategies at their core, have been produced and disseminated to MushTV growers and composters, providing them with new knowledge and advice on (a) effective use of disinfectants in mushroom production; (b) prevention of Brown Cap Mushroom Disease, (c) control of fungal diseases of mushrooms and (d) understanding Trichoderma aggressivum in bulk Phase 3 compost.
Advancing the frontiers of science. Research by MushTV scientists has advanced the scientific knowledge base in several areas. They have characterised and identified 16 fungal viruses new to science, including two that are associated with the Brown Cap Mushroom Disease. These have been named Agaricus bisporus Virus 16 (AbV-16) and Agaricus bisporus Virus 6 (AbV-6). All the genome sequences of the novel viruses will be published in 2015 and will be available to the scientific community. A new qPCR based method to detect the viruses has been developed as well as a new method for the isolation of RNA from humic-rich mushroom compost. The way in which T. aggressivum is dispersed within large batches of Phase 3 compost has been elucidated and described. Surveys of grower and compost facilities have identified weaknesses in current hygiene systems, highlighting where action is needed to minimise contamination of facilities. Research has also identified a volatile-based diagnostic method for T. aggressivum in Phase 3 compost. In experimental phase 3 tunnels, a group of biochemically-related volatile compounds were repeatedly detected in artificially-infected compost during the Phase 3 process. The pattern of the emitted group of compounds was also different between infected and non-infected compost so a detection technique may be suitable at commercial scale, although further development is needed before commercialisation can take place. These advances in methodologies and knowledge will be published in peer review publications over the coming year.
Collaboration. By working together to resolve common problems, MushTV has fostered a sense of collaboration and friendship between people who are often competitors in the mushroom market place. This, above all has been the outstanding achievement of the MushTV project. The group look forward to working together in the future to address other challenges of mutual concern.
Project Context and Objectives:
At the outset of the project, one of the key challenges facing the European mushroom industry was the regulatory obligation to adopt an ‘Integrated Pest Management’ (IPM) approach to pest and disease control. The EU legal requirements were outlined in the Sustainable Use of pesticides Directive 2009/128/EC (SUD) and individual countries had to then enforce what are known as the “Sustainable Use of pesticides Regulations”. The underlying principles of the SUD regulations are to safeguard human health and the environment, but it does require a change in attitude by composters and growers towards pest and disease management. The objectives of MushTV were to provide information and solutions that were compatible with an IPM ethos.
Integrated Pest Management is described as ‘a program of prevention, monitoring, and control which offers the opportunity to eliminate or reduce the use of pesticides, and to minimise the risk to human health and the environment’. Managers and technical staff must have a greater understanding of the life cycle and biology of the pests and diseases they are trying to control. They must monitor levels of pests and diseases, know how they spread around the farm or facility, know the best hygiene measures to control them, ensure that hygiene measures are employed correctly, use biological and non-chemical control measures where possible, keep updated with advances in IPM control measures, and, only as a last resort, use the least toxic chemicals available when other avenues have been exhausted. The mushroom industry is already well placed to be IPM compatible, but it can still encounter serious pest and disease issues which may be challenging to control.
At the same time, within the mushroom sector, there has been a shift towards increased automation, mechanisation and expansion of production facilities. While mechanisation and expansion have distinct advantages in terms of efficiency and quality, one disadvantage is that should a disease become established, it can be much harder to control, incurring large scale losses and disruption to production.
MushTV is a 39 month project that aims to provide research-based solutions for the mushroom industry, primarily to deal with two relatively new major diseases affecting mushroom production: - Trichoderma aggressivum, which causes compost green mould and Mushroom Virus X (MVX) disease, which causes mushroom browning, crop delay and less-productive crops. Both diseases can have quite severe effects if they occur on bulk phase 3 compost facilities. The SME Associations in the project consortium have identified a need for (a) scientific knowledge of how these pathogens exploit mushroom production systems and (b) technological solutions to controlling them. The SME Associations also want more information on the effectiveness of disinfectants at killing these two pathogens in their various forms. There is an imminent need to identify an effective replacement-disinfectant for Formaldehyde, which is no longer approved for use in mushroom production facilities in most European countries. In addition, there is an imminent need to find effective pesticides or biocontrol agents to control other mushroom diseases such as dry bubble, wet bubble and cobweb, as there is generally only one approved fungicide for mushroom control (prochloraz) in most European countries. Growers and composters increasingly need to have a scientific-based understanding of how pathogens proliferate and spread on their facilities, and how their spread can be controlled. Such knowledge means they will be less reliant on disinfectants and pesticides and they can be more targeted with their use.
The SME Associations and Other mushroom businesses recognise that they do not have the research capacity or resources to conduct this type of research for the sector and they have selected research performers with the necessary expertise and track record to solve the problems on their behalf. The FP7 mechanism ‘Research for the Benefit of SME Associations’ provides an opportunity for collaboration between the appropriate SME-AGs, SMEs, Other businesses and RTD performers. The MushTV Consortium comprises 17 members across five countries. This is the first time that so many European mushroom-related businesses have come together to work towards solutions to common problems despite the fact that individuals within the consortium are direct competitors.
The objectives of MushTV are covered by seven research work packages which are summarised as follows:
Project Objectives:
(1) To identify alternative disinfectant products and methods of disinfection that perform well in large air volumes, and on large pieces of machinery contaminated with high levels of organic matter
(2) To identify the viral entities that make up MVX and use the obtained genetic sequence information, in conjunction with existing data, to improve and further develop sensitive diagnostic tests for use on compost and mushrooms
(3) To develop a T. aggressivum diagnostic method based on detecting unique volatiles from infected compost in the re-circulating air in compost incubation tunnels
(4) To identify where reservoirs of T. aggressivum and MVX inoculum occur at compost and grower facilities so they can be targeted with improved hygiene methods as part of an integrated pest management solution
(5) To evaluate the efficacy of a promising biopesticide based on Bacillus subtilis for the control of Dry Bubble, Wet Bubble, Cobweb as well as T. aggressivum and make recommendations for its use
(6) To describe the pattern of growth of T. aggressivum in a bulk compost-incubation tunnel and determine how it is distributed in the compost when the compost is removed, bulk handled and transported to the grower.
(7) To track the incidence and spread of MVX inoculum on MVX-prone compost and grower facilities before, during and after an outbreak.
There is also a Dissemination and Training work package to disseminate results to all grower-members and technical staff represented by the Consortium across Europe.
Project Results:
WP1: Identification of alternative disinfectants and methods.
WP1 was conducted by researchers at PRI (Partner 6) in the Netherlands and Inagro (Partner 7), in Belgium. It was responsible for identifying alternative disinfectant products to those previously used within the European mushroom industry, which are either being withdrawn from use due to EU regulations, or which are no longer being used due to national regulations or commercial decisions. Effective disinfectants (or biocides) are an essential component of the mushroom industry to ensure all surfaces and equipment are free of undesirable organisms such as bacteria, fungi and viruses, especially those that can cause damage to the mushroom crop. The members of the MushTV consortium have provided us with a list of biocides they wanted to be included in a survey on efficacy against Agaricus bisporus (a model system for MVX-infected A. bisporus) and Trichoderma aggressivum, two serious diseases of mushroom crops. Based on the information delivered to us by the members of the MushTV consortium, the different products were grouped according to the active principle of the biocides. A total of 12 groups could be discriminated; biocides based on 1) mixtures of aldehydes / quaternary ammonium compounds, 2) peracetic acid/hydrogen peroxide, 3) hypochlorite/chlorine based, 4) quaternary ammonium compounds only, 5) radicals and hypochlorite, generated in water by electricity (electrochemically activated water), 6) beneficial organisms, 7) phenolics, 8) purified proteins from plant and mineral sources, 9) ozone, 10) benzoic acid, 11) mixture of amphoteric compounds/quaternary ammonium, and 12) peroxomonosulfate based. From most groups a single product was chosen to serve as a model for the whole group. Some biocides were excluded from further testing based on previous research showing low efficacy.
In laboratory tests, efficacy of the selected biocides was tested both at the recommended dose and time (mostly 15 minutes) and at a double dose and prolonged time (mostly 1 hour), and results were compared with the effectiveness of formalin (at recommended doses). Formalin is currently the industry standard disinfectant in the Netherlands but it is not approved in many European countries. Disinfectants were tested against basidiospores or mycelium of A. bisporus and compost particles that were colonised by A. bisporus mycelium.
• all disinfectants were able to kill the basidiospores of A. bisporus at the concentrations and disinfection times tested except for Eco Des (based on quaternary ammonium only), gaseous ozone and Tersan (amphoterics and quaternary ammonium based),
• Mycelium of A. bisporus could be killed by all disinfectants, with the exception of ozone and electrochemically activated water.
• When present in compost particles, A. bisporus could only be killed effectively with formalin, although at the concentrations and disinfection times tested Eco Des (quaternary ammonium based), Prophyl (phenolics based) and Menno Clean (benzoic acid based) were also quite effective.
Disinfectants were also tested on T. aggressivum.
• Conidiospores of T. aggressivum were killed by all disinfectants, except gaseous ozone.
• At the concentrations and disinfection times tested, mycelium of T. aggressivum could be killed by formalin, Virocid (based on a combination of aldehyde and quaternary ammonium) and Menno Clean. Neither gaseous ozone nor electrochemically activated water had any effect on mycelium.
• None of the disinfectants was able to kill T. aggressivum in infected compost particles at the recommended dose and disinfection time – including Formalin
• Considerable research effort was put into finding a dosage and disinfection time needed for killing T. aggressivum in infected compost particles. Only high concentrations of formalin (6% or 8% for a minimum of 30 minutes) or Menno Clean (8% for a minimum of 2 hours) were able to kill T. aggressivum under laboratory conditions.
Practical relevance of these findings has been discussed with the members of the MushTV consortium. Next to this the disinfectants were compared with each other with respect to EU registration status, possible application methods (spraying, fogging), risks to the user and the environment, possible residues and corrosivity.
Next, attempts were made to extrapolate the laboratory results to the practical conditions in commercial mushroom cultivation. For such extrapolations, various types of surface that may occur in the mushroom industry (rubber mats, stainless steel, mats from growing rooms and tunnels, aluminium, concrete, insulation panel and coated steel) were contaminated with either basidiospores of Agaricus bisporus or conidiospores of Trichoderma aggressivum. Based on efficacy and the lack of leaving a residue, three disinfectants were chosen for testing under “semi-commercial” conditions. The products chosen were ECAS (electrochemically activated water), a product based on hypochlorite and a product based in a combination of peracetic acid and hydrogen peroxide. Results showed that ECAS was not very effective under these semi-commercial conditions. The products based on hypochlorite or on peracetic acid/hydrogen peroxide however, showed good efficacy. Both these products however are corrosive.
One of the compost companies was using a peracetic acid/hydrogen peroxide based product for disinfection of the spawning hall. Their experiences with the product were good, but they have been taking care of protecting the construction of their facilities with a special coating. Within the project the compost company has started to use swabs for monitoring the presence of yeasts and moulds on their facilities. Own research by the compost company has shown to them that in their facilities, the peracetic acid/hydrogen peroxide based product has good efficacy in killing the yeasts and moulds.
As disinfection needs to be preceded by thorough cleaning, a number of Dutch and Belgian growers have started to experiment with the use of industrial foam based detergents. For these foam based detergents to work properly, they need to be applied with specialised equipment and high pressure supply of water and air. As this equipment is quite expensive, the farmers have experimented with cheaper solutions with varying degrees of success.
This WP produced a technical factsheet entitled ‘Use of chemical disinfectants in mushroom production’ which was produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP2: MVX characterisation, diagnostics and biology
WP2 was conducted by researchers at EMR, England (Partner 8) in collaboration with researchers at Teagasc, Ireland (Partner 1). In the 1990s, a set of disease symptoms emerged in the European mushroom industry which caused severe economic damage, for example in the UK, the disease caused £50 M of damage including farm closures (Adie el al, 2004). The symptoms varied from loss of yield, cap distortions and brown discolouration of caps. The disease was found to be associated with the presence of double-stranded RNA molecules (presumed viral) in the mushrooms and so it was named Mushroom Virus X (MVX) disease (Grogan et al, 2003) but it was not known if the viruses, or which virus(es) were responsible for the disease. After extensive cleaning and hygiene precautions in the industry one MVX symptom persisted, the cap browning. This occurrence of the brown cap disease is both sporadic but economically serious as the affected mushrooms are downgraded or discarded due to the associations of the brown discolouration with poor quality and old mushrooms.
The MushTV project was aimed at understanding mushroom diseases and their control and prevention, in particular MVX and Green Mould Disease. Work package 2 (WP2) had two objectives designed to increase our understanding of the nature of the viruses which infect and cause MVX disease in mushrooms, and to develop techniques to detect each virus to enable disease prevention: (1) to fully sequence and characterise the viral dsRNAs associated with MVX brown mushroom symptoms and (2) to identify and optimise the most effective diagnostic method to detect MVX viral presence in mushrooms and compost.
(1) Sequence and characterise the viral RNAs. The biotechnological technique known as Next Generation Sequencing was applied to sequence all the RNA viruses from nine diseased samples of mushrooms and one sample of symptom-free mushrooms. Approximately 45 million short sequence reads (150 – 200bp in length) were produced which were then assembled into longer length ‘contigs’ using a series of bioinformatics tools. Thirty-one contigs were identified as virus-like RNA molecules. The sequences of these contigs were then examined to identify regions which code for viral proteins. The key protein in this search is the replication enzyme known as RNA-dependent-RNA-polymerase or RdRp. Nineteen RdRps were identified and therefore the 31 contigs comprise of 19 different viruses. Some of these viruses contain more than one RNA molecule (AbV6, AbV9 and AbV16 have 2, 3 and 5 RNAs respectively). In addition, 5 viral molecules were identified which were not associated with any particular virus. Only 3 of the 19 viruses had been sequenced previously and so this research has identified 16 new viruses to science. The viruses were classified based on previously described fungal virus taxonomy based on the similarity of their protein sequences (particularly RdRp) as follows: 8 from the Timovirales class, 3 Hypoviruses, one Endornavirus, two Benyviruses, one Barnavirus, one Mitovirus, two Narnaviruses and a new classification of viruses for AbV16. The concept of multiple viral infections is a new and emerging area of science and leads to the conclusion that many of these are not pathogenic but possibly symbiotic. Symptom-free mushrooms contain at least 11 different viruses at significant levels.
The newly sequenced viruses have been given names following current scientific nomenclature. The naming uses the letters AbV (after Agaricus bisporus Virus) followed by a number. As two previously described viruses have been given the names AbV1 and AbV4, the numbering of the newly described viruses starts with ‘2’, i.e. AbV2 (Agaricus bisporus Virus 2) and continues as: AbV2, AbV3, AbV5, AbV6 AbV7, 8, 9,.......AbV16.
Further experimentation in association with WP7, has led to the hypothesis that AbV16 is the virus which causes the brown cap symptoms. The evidence for this hypothesis is that the degree of brown discolouration on diseased mushrooms correlates (with statistical significance) with the levels of the individual RNA components of AbV16 (which comprises of 5 different RNA molecules). In addition two other viruses, AbV6-2 and MBV, may also have some involvement in the disease occurrence.
(2) Identify and optimise the most effective diagnostic method to detect MVX viral presence in mushrooms and compost. After the association was first made between RNAs and MVX symptoms, the presence of RNA was detected by a technique involving electrophoretic separation and observation of dye-stained RNA (Grogan et al, 2003). This technique was able to push on the study of MVX but it was not sufficiently sensitive to detect RNAs at very low levels which is necessary to identify the early stages of the disease. Ideally if the viruses can be detected in the mushroom mycelium growing in compost then the detection technique would provide advanced warning before mushrooms are grown. A project prior to MushTV, the HDC Project M51, developed a proof of principle for a more sensitive test based on a technique called Quantitative-PCR for two of the viruses in the MVX complex (Burton et al, 2010). Work package 2 of the MushTV project aimed at further improving these preliminary tests. There were two components of these investigations: improvements in the extraction techniques of RNA from compost and the use of the newly identified RNA sequences to design ‘PCR’ primers’ for all 31 RNA molecules.
Extraction of RNA from mushroom compost to a high purity is a technically difficult procedure because of the high levels of contaminating chemicals found in mushroom compost for instance, humic substances and phenolics. The experiments conducted compared (a) different extraction buffers, (b) a variety of ‘clean-up’ methodologies, and (c) mechanical treatments to break up the compost into sufficiently small particles to allow for maximum extraction efficiency. A method was developed for RNA extraction from mushroom compost involving milling the compost using metal ball bearings, extraction of RNA with Trizol buffer followed by a clean-up step using silica. This technique was robust, repeatable and economically viable.
PCR primers were designed for each of the 31 virus molecules. The Quantitative-PCR tests were conducted using these primers and the improved RNA extraction technique on a large number of compost samples and all of the 31 virus RNAs were detected, thereby validating that the tests can detect and quantify all of the viruses in compost.
Exploitation and Commercialisation of Virus Tests and IP issues. The newly developed tests to detect and quantify the levels of each virus from compost (and mushrooms) have great use-value for the European mushroom industry and potentially commercial value for technical disease testing laboratories. For the mushroom industry, the tests could be used for early warning disease detection so that sources of infection can be identified, and for routine monitoring of compost producing and mushroom production facilities.
There are 2 potential IP outputs concerning the tests: 1) Methodology to extract RNA from mushroom compost, and 2) The nucleotide sequences of the identified RNA viruses. After discussion with the Exploitation Committee of Mush TV it was concluded that the IP should not be protected because (i) there is insufficient novelty for any formal IP protection (i.e. patent) for the RNA extraction methodology, and (ii) while there is a novelty in the sequences there a need to discuss this result freely with other researchers to formulate a clear understanding of the biology of this fungus/multiple virus interactions and (iii) the market for the testing is unlikely to be sufficiently large in financial terms to pay for the costs of patenting. The delay and constraints of patenting may discourage laboratories from offering these tests. The aim of the MushTV consortium is to provide help to the mushroom industry. Therefore the IP will be made available to laboratories by the publication of two scientific papers.
East Malling Research (Partner 8) investigated the Route to Exploitation in collaboration with its sister company East Malling Services, an independent limited company. These discussions led to a test-marketing to the MushTV consortium of two products of virus detection tests: The cheaper test which measures the levels of the viruses associated with the browning symptom (the five AbV16 RNAs, AbV6-2 and MBV) and a more expensive test whereby the levels of all 31 viral RNAs are measured. The MushTV consortium provided positive feedback on this offering but concluded that before a commercial service was offered or was of benefit to the industry, additional knowledge was required on (a) what are the background levels of the viruses at facilities using the new test; and (b) how should the results be interpreted i.e. knowledge is needed of the threshold levels of each virus that signal a potential threat (early warning) that would recommend that increased hygiene measures should be taken.
References
Adie B, Choi I, Soares A, Holcroft S, Eastwood D, Mead A, Grogan H, Kerrigan R, Challen M and Mills P (2004) MVX disease and dsRNA elements in Agaricus bisporus. Mushroom Science 16, 411-420. In: Science and Cultivation of Edible and Medicinal Fungi, Eds: Romaine, Keil, Rinker and Royse.
Burton KS, Baker A, Eastwood DC and Grogan H. (2010) Developing an accurate, quantitative and predictive test for Mushroom Virus X, Report to Horticulture Development Company, Stoneleigh, Warwickshire, UK. http://www.hdc.org.uk/sites/default/files/research_papers/M51%20Final%20Report.pdf
Grogan HM, Adie BAT, Gaze RH, Challen MP and Mills PR (2003). Double-stranded RNA elements associated with the MVX disease of Agaricus bisporus. Mycological Research 107: 147–154.
Scientific data from this WP will be published in 2015. Some of the information was included in a technical factsheet entitled ‘Brown cap
Mushroom Virus Prevention’ which was produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP3. A volatile-based diagnostic method for Trichoderma aggressivum detection in Phase 3 compost.
WP3 was conducted by researchers at PRI (Partner 6) in the Netherlands and AFBI (Partner 9), in Northern Ireland and worked closely with the compost producers within the Consortium. In recent years green mould (Trichoderma aggressivum) has presented big problems to the European mushroom industry. Trichoderma aggressivum infects compost at a very early stage. Infection most likely takes place at the compost facility during spawn-run (Phase 3 process). In Western Europe the spawn run process is frequently done in large tunnels, in bulk, at the compost yards and the end product is referred to as bulk phase 3 compost. During this process, spawned compost is incubated in tunnels and ventilated with large volumes of air to control compost temperature. During this process the compost is inaccessible for sampling, e.g. for PCR based techniques.
Many organisms, including plants and fungi, produce volatile organic compounds (VOCs) during their growth phase. When interacting with other organisms either in a mutualistic or antagonistic way, the emitted VOC blends can change because the metabolism is affected, e.g. it changes from growth phase to a more defensive stage (Müller et al. 2013; Mumm and Dicke 2010; Unsicker et al. 2009). Mumpuni et al. (1998) have shown that Agaricus bisporus and T. aggressivum use VOCs to affect each other’s growth rate in vitro. In WP3 we aimed to develop a non-invasive technique based on fungal VOCs in the process air to detect an infection by T. aggressivum in compost during spawn-run without the necessity to enter the tunnel. Using this as a starting point for MushTV this work package has six main objectives (tasks):
3.1. Scale up from small scale to medium scale compost quantities
3.2. Study variability of VOC patterns during commercial phase 3 production
3.3. Study specificity of VOC patterns in small to medium scale phase 3 composting systems
3.4. Improvement and simplification of the data processing of the detection method
3.5. Translate method into an application
3.6. Demonstrate proof of principle under commercial conditions
The VOC emission was studied by dynamic headspace trapping on adsorbent cartridges and thermodesorption gas-chromatography mass spectrometry (TDGC-MS). Raw data were processed using a combined untargeted metabolomics and data mining approach in order to identify candidate VOCs. First experiments on a small scale using glass vessels with 300g of Phase 2 compost showed a clear difference in VOC profiles between T. aggressivum and non-infected compost.
In Objective 3.1 we carried out experiments with volumes of Phase 2 compost ranging from medium scale ~50kg (referred to as Mini-tunnels; at PRI) to semi-industrial scale (~1800kg; at Partner 9, AFBI) to study different partial infection rates of T. aggressivum. Several experiments with Mini-tunnels showed that VOC profiles of infected compost significantly differed from non-infected compost at day 14 and day 18 of spawn-run. The majority of the VOCs that were significantly more abundant in T. aggressivum-infected and having a good predictive capacity belong to a group of biochemically related natural compounds that are widely present in plants and microorganisms. When comparing VOCs being significantly more abundant in the T. aggressivum infected compost in the small scale trials with data from the medium scale trials, it was difficult to assign one or a small set of VOCs reliably present in the majority of trials. Alternatively, we found that the temporal pattern of the total signal of the emitted candidate compounds (as determined by the joint mass spectral signal of the molecular weight) was different between infected and non-infected compost during spawn-run. In healthy Phase 3 compost, emission of the sum of candidate compounds peaked at around day 11 and then steadily decreased until despatch of the compost but the emission from infected compost peaked around day 14. This consistent difference in the pattern of emissions of these compounds for healthy Phase 3 compost compared to T. aggressivum infected Phase 3 was detected on small (50kg) scale and semi-industrial scale (1800kg) across several independent trials.
In Objective 3.2. we carried out several trials to determine whether VOC emission including the candidate compounds can be reliably monitored during commercial phase 3 production. Early experiments using the small scale Mini-tunnels filled with ~50kg Phase 2 compost from different compost producers showed clear differences in the VOC pattern between the different sampling days, and were detected in compost of all three compost producers. The VOC profiles of non-infected compost of the three compost producers behaved similarly during spawn-run. Later experiments worked with commercial companies. VOCs from the process air from commercial Phase 3 tunnels in the Netherlands and Ireland were collected at different days of spawn-run. Dutch and Irish compost differ in the composition of ingredients. Irish compost uses mainly straw and poultry manure while Dutch compost frequently has horse manure mixed with the straw. A large number of tunnels were sampled at each facility with a number of different spawn strains. The results from these commercial scale trials demonstrated that VOC emission patterns during the Phase 3 process obtained under commercial conditions resemble those detected on lab scale (50-1800kg compost). This indicates that the detection method of monitoring the VOC emission can also be applied on commercial scale.
In Objective 3.3. we tested whether the VOC patterns emitted from compost infected with different mould species (T. aggressivum, T. harzianum, T. atroviride, Aspergillus fumigatus, or Penicillium citreonigrum) were similar to or different to the volatile pattern from clean compost or compost infected with T. aggressivum. Experiments were carried out on small scale using Mini-Tunnels. With the exception of P. citreonigrum all tested mould species including T. aggressivum induced significant changes in the VOC profiles of phase 3 compost. No VOCs were uniquely induced by only one mould species but by combining the pattern of different induced VOCs, single mould species may be discriminated.
In Objective 3.4. the aim was to improve and simplify the data processing of the VOC detection method. The developed method focusses on the sampling and analysis of the emission pattern of a specific group of VOCs which can be done in a very targeted manner, e.g. by using mass spectrometry techniques either as stand-alone technique or hyphenated with a separation technique such as gas chromatography. By using a targeted approach the detection sensitivity can be highly improved and processing of the data is more straightforward compared to the untargeted data mining approach that was used at the beginning of the project.
To tackle Objective 3.5 we showed as a proof of concept that VOC emissions can successfully be monitored using an online mass spectrometry technique (Proton Transfer Reaction- Mass Spectrometer (PTR-MS)) during the commercial Phase 3 process. Patterns were similar to what had been shown for small and semi-industrial scale trials. The proof of principle of this method under commercial conditions (as stated under Objective 3.6.) was not achieved because we did not get notice of any green mould incidents during the life of the project.
References.
Müller, A., Faubert, P., Hagen, M., zu Castell, W., Polle, A., Schnitzler, J.-P. and Rosenkranz, M. 2013. Volatile profiles of fungi – Chemotyping of species and ecological functions. Fungal Genet Biol 54(0): 25-33.
Mumm, R., and Dicke, M. 2010. Variation in natural plant products and the attraction of bodyguards involved in indirect plant defense. Can J Zool 88(7): 628-667.
Mumpuni, A., Sharma, H.S.S. and Brown, A.E. 1998. Effect of metabolites produced by Trichoderma harzianum biotypes and Agaricus bisporus on their respective growth radii in culture. Appl Environ Microbiol 64(12): 5053-5056.
Unsicker, S.B. Kunert, G., and Gershenzon, J. 2009. Protective perfumes: the role of vegetative volatiles in plant defense against herbivores. Curr Opin Plant Biol 12(4): 479-485.
Scientific data from this WP will be published in 2015. Some of the information was also included in the technical factsheets on ‘Understanding Trichoderma aggressivum in Bulk Phase 3 compost’ which were produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP4: Investigations to locate reservoirs of Trichoderma and MVX inoculum mushroom compost and mushroom grower facilities.
WP4 was conducted by researchers at Teagasc, Ireland (Partner 1) in collaboration with AFBI (Partner 9), in Northern Ireland. This work package worked in confidence with composters and growers and all individual results are confidential, as detailed in Table 3.2b in Annex 1. WP4 had two objectives. Objective 4.1 looked to identify the most likely locations on mushroom facilities to detect Trichoderma aggressivum and MVX if it is present. Pilot studies were carried out at compost and grower facilities to identify a list of critical sample points which would be used in the wider investigations of industry facilities in Objective 4.2.
The pilot studies for MVX and Trichoderma on compost facilities were carried out together because both pathogens are likely to be spread in a similar fashion (spores, infected compost and compost-debris). Visits were conducted and samples were taken from various locations. Samples were processed in the laboratory and tested for presence of MVX and T. aggressivum. For MVX testing, pure cultures of Agaricus bisporus were obtained from compost and compost-debris samples and tested for presence of MVX dsRNAs using MVX-specific PCR primers. For T. aggressivum testing, all samples were plated onto Malt Agar medium (with antibiotics) and any moulds that grew were identified. Any Trichoderma species present were isolated into pure culture and tested using T. aggressivum-specific PCR primers. The results from the pilot study were evaluated and sample locations that gave consistent and useful results were then selected for inclusion on the “Critical Control Point” list which would be used for a wider survey of industry compost facilities in Objective 4.2.
The Pilot studies of the grower facilities for MVX and Trichoderma were carried out on two separate facilities. Sampling for presence of Trichoderma on the grower facility involved taking compost samples as well as taking swabs of equipment, shelving and surfaces which might harbour T. aggressivum spores as well as infected compost debris. Sampling for presence of MVX on mushroom growing units focused on taking samples of compost and mushrooms as these are the best types of samples for MVX. A grower facility with on-going MVX issues was targeted and a separate grower facility with Trichoderma problems was also targeted. Various samples were taken from a range of locations, the samples were processed by both molecular and microbiological techniques and the results evaluated. Two separate lists of Critical Control Points for grower facilities were compiled, one for MVX and one Trichoderma. The Critical Control Points were discussed and agreed with the Consortium. Once the three lists of Critical Control Points for sampling of industry facilities were complete, the survey of industry facilities across Europe began.
Objective 4.2 looked to gather information on how widespread Trichoderma and MVX were on mushroom compost and growing facilities across Europe. All compost facilities within the Consortium were surveyed using the Critical Control Points identified in the pilot studies, but occasionally additional samples were also taken. The results of sample testing were communicated to each composter confidentially. A number of growing facilities across Europe were also surveyed, using the Critical Control Points identified in the pilot studies. Results were communicated to the management of each facility confidentially. As part of this objective, a case study on Trichoderma spread and transmission was carried out at the experimental research facilities of Partner 9, AFBI, in Northern Ireland, where studies on the growth of Trichoderma in Phase 3 compost were being conducted. Sampling of Partner 9 facilities took place on six occasions from the end of one T. aggressivum growth trial to the same point at the end of a second T. aggressivum growth trial.
Results of the case study show that Trichoderma is very easily transmitted once it is present on a facility, even when an apparently very rigid hygiene protocol is in place. The number of Trichoderma- positives being detected increased over the duration of the second trial as T. aggressivum became established in an experimental crop. Most of the positives were within the growing room (house 3) containing the trial. However by the end of the crop, T. aggressivum had spread outside the growing room into the corridor, onto the exterior door surface, the control panel for the room as well as into offices, canteen and transport vehicles, increasing the likelihood of further spread and contamination of new crops. A critical conclusion of the case study was that an improved and more intensive cleaning and disinfection programme was necessary to eliminate all instances of T. aggressivum cross contamination on the facility. Infected crops must be thoroughly cooked-out (65OC for a minimum of 8 hours with compost in the growing room followed by a second cook-out of 65OC for a minimum of 8 hours once the compost has been removed). In addition, all structures, equipment and surfaces must be thoroughly cleaned and disinfected, with all parts of the facility being targeted.
Some of the information from the WP was included in the technical factsheets on ‘Brown cap Mushroom Virus Prevention’ and ‘Understanding Trichoderma aggressivum in Bulk Phase 3 compost’ which were produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP5 – Evaluation of the biopesticide Bacillus subtilis for the control of mushroom pathogens
WP5 was conducted by researchers at Inagro, in Belgium (Partner 7) and Teagasc, Ireland (Partner 1). The objective of this WP was to evaluate the efficacy of a promising biopesticide based on Bacillus subtilis for the control of the diseases known as Dry Bubble, Wet Bubble, Cobweb as well as T. aggressivum, and, if successful results were obtained, to make recommendations for its use. A commercially available product containing B. subtilis was used. Cultures of the four pathogens Trichoderma aggressivum, Verticillium (Lecanicillium) fungicola var. fungicola (Dry Bubble), Mycogone perniciosa (Wet Bubble) and Cladobotryum mycophilum (Cobweb) were obtained from research partners PRI, in the Netherlands, and Teagasc.
Initial work consisted of laboratory scale tests. In vitro antagonism of B. subtilis was examined by testing different concentrations against fungal mycelium plugs. In vitro dual culture studies were done following standard microbiological techniques using a range of different culture media (MEA, PDA and Sabouraud). Plates were incubated for several days at 25°C and inhibition of fungal growth monitored daily by recording the diameter of the inhibition zone. Each dual confrontation was performed in duplicate. Cladobotryum was not inhibited by Bacillus and overgrew the Bacillus colonies completely within 3 to 7 days. Trichoderma growth was not inhibited by Bacillus on Sab agar plates, but some inhibition occurred on PDA and MEA plates. Fungal growth of both Verticillium and Mycogone was strongly inhibited by Bacillus. Agaricus growth was somewhat reduced on PDA plates but inhibition was less obvious on MEA plates. As results from laboratory tests are not always a predictor of how products will work under cropping conditions, a series of small cropping experiments in 0.2 m² containers were carried out according to standard practices on mushroom farms.
Evaluation of B. subtilis against T. aggressivum – compost green mould. A small scale cropping trial was set up to test the efficacy of Bacillus subtilis against T. aggressivum under bulk phase 3 conditions. In this trial 1N and 2N rates were applied and three applications were made at different points in the crop cycle: (1) applied to mushroom spawn prior to its incorporation into compost; (2) sprayed onto fully colonised compost at the end of spawn-run after it has been broken up (bulk handled) and emptied from the incubation container and (3) applied as a drench to the casing in the last watering before the crop was vented (Day 7 approx.). Trichoderma aggressivum inoculum consisted of mushroom compost that was pre-colonised with T. aggressivum as this is the most likely source of infection at bulk Phase 3 facilities. Three applications of Bacillus failed to prevent or reduce the development of green mould in the crop.
Evaluation of B. subtilis against Mycogone – wet bubble. To evaluate the efficacy of B. subtilis, against Mycogone, a small cropping experiment was set up. Bacillus was applied following a three application treatment schedule (at casing, at last watering before aeration and after first flush). Infected control plots were treated with the industry standard prochloraz-Mn (46%; Sporgon, BASF) at the manufacturer’s recommended rates. Observations confirmed the efficacy of Sporgon. However, plots treated with Bacillus showed high levels of wet bubble disease symptoms, despite its antifungal activity against Mycogone on agar plate tests. A second trial was conducted and results confirmed that Bacillus did not show any control against wet bubble, compared to prochloraz.
Evaluation B. subtilis against Verticillium – dry bubble – and Cladobotryum – cobweb. Similar trials to those described for Mycogone were also conducted for Verticillium and Cladobotryum. Results of duplicate trials for each pathogen also confirmed that Bacillus did not show any control against dry bubble or cobweb, compared to prochloraz.
Evaluation of selected alternative products against Verticillium and Cladobotryum.
When the biopesticide B. subtilis was shown to be ineffective against three mushroom pathogens, the consortium agreed to evaluate two extra candidate products against dry bubble and cobweb. The first was a commercially available biopesticide product (Product C) with no approval for use in Europe. The second was a commercially available chemical fungicide (Product B) with limited approval for use in Europe. In duplicate small scale cropping trials Product C did not give any control against either Verticillium or Cladobotryum, compared to prochloraz. Duplicate small scale cropping trials with Product B however confirmed that it was effective at controlling both Verticillium. or Cladobotryum, compared to prochloraz. There is interest in getting the product registered for the European mushroom Industry. However, in the absence of an effective biopesticide, non-chemical disease control still relies heavily on effective hygiene methods.
Phytotoxicity and residue tests. All three products were assessed in large scale cropping trials for any potential adverse effects on the growth and yield of mushrooms. All products were tested at single and double or x1.5 doses. There was no significant effect of any product on total yield but there were some minor changes to the flushing pattern. Mushrooms from the standard and double rate treatments for Product B were tested for residues of the active ingredient. Residues were detected but they were below the MRL.
SIGNIFICANT RESULTS
• Bacillus subtilis gave no control against T. aggressivum green mould in bulk Phase 3 compost.
• Bacillus subtilis gave no control against wet bubble, dry bubble or cobweb disease, compared to prochloraz.
• A second biopesticide (Product C) gave no control against dry bubble or cobweb disease, compared to prochloraz.
• A chemical fungicide (Product B) gave very good control against dry bubble and cobweb disease, compared to prochloraz
Some of the information from this WP was included in the technical factsheets on Fungal Diseases of Mushrooms and their Control (English, Dutch and Polish) which was produced in three languages and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP6 Trichoderma Growth in Phase 3 Compost.
WP6 was conducted by researchers at AFBI (Partner 9) in Northern Ireland in collaboration with Teagasc, Ireland (Partner 1). The work focussed on characterising the growth, dispersal and impact on yield of T. aggressivum in bulk-incubation tunnels. In these compost systems, large volumes of re-circulated air are forced through an unsegregated compost mass so theoretically the potential to infect all the compost within the tunnel could be high. Initial trials were undertaken to specifically investigate how T. aggressivum grew in compost during a typical 17 day spawn run performed in bulk. Experimental mini composting tunnels (ca 1800 kgs) were subdivided along four vertical and three horizontal planes and a spore suspension inoculum positioned in the back, lower quadrant. Results indicated that distances travelled by T. aggressivum within the bulk spawn-run (compost temperature ca 25±1OC) were generally limited to just ca 0.5 -1.0 m. These growth rates are in broad agreement with previous in vitro and in vivo studies. T. aggressivum was not generally visible at the end of bulk spawn-run so composters would be oblivious to any compost infection. The compost was removed from the experimental mini tunnels in an artificial process, removing 12 blocks of unmixed compost from each of the four vertical and three horizontal planes. This contrasts sharply with commercial practices where the bulk handling operations will usually involve several distinct mixing processes. Results indicated that the severity of the subsequent mushroom crop production yield losses was highly correlated with the position of the compost in the tunnel relative to the growth of the Trichoderma. Whilst crop yield was largely unaffected in compost from the front two or three compost slices, average yields from the back slice closest to the inoculation point were reduced by 47% (range 2 - 100%) and 56% (range 8 - 98%) respectively in two trials. These initial compost trials have been essential in increasing our understanding of how far and in what direction Trichoderma can grow during the 17 day bulk spawn-run period.
A second series of trials studied the effect of three mixing processes (action of the compost tunnel winch, horizontal layered filling of transportation vehicles and vertical lorry emptying for shelf growing at grower holdings) on dispersal and spread of T. aggressivum during 3 differentiated handling operations (compost emptied unmixed, compost emptied mixed within each of 4 individual tunnel slices, compost emptied mixed and layered across all 4 tunnel slices). The latter most closely simulating bulk handling operations carried out at commercial premises. Consistently, no dispersal from the original point of inoculation was evident in compost from unmixed slices; the infection was dispersed through the inoculated slice only in compost from individually mixed slices, and compost fully mixed across all 4 tunnel slices dispersed the initial localised T. aggressivum infection (previously contained close to the point of inoculation in the back, bottom quadrant of the compost tunnel) throughout all compost in that tunnel. The severity of the yield losses was highly correlated with the degree of mixing – essentially 0% yield loss from unmixed compost except in the inoculated quadrant where it was 100%; ca 100% yield loss in the inoculated mixed slice but close to 0% yield loss from the remaining 3 slices in the tunnel and ca 100% yield loss across all compost from the tunnel in the fully mixed treatments. These trials further indicated that T. aggressivum can readily infect and colonise otherwise healthy, productive mushroom compost as evidenced by typical yields of 25 - 34 kgm2-1 in control plots and uninfected compost.
In additional trials, the use of equipment that had handled T. aggressivum infected compost was also shown to infect clean compost from adjacent newly opened tunnels, spreading the infection further. In addition, where the infection occurs in a tunnel is critical; if it is near the front, all subsequent compost will come in contact with now infected machinery; if it is near the back then a large proportion of compost from the front of the tunnel should yield normally. This new knowledge could explain previous observations where several growers getting compost from the same tunnel report completely different levels of infection ranging from none to total crop wipe-out.
Crucially, these studies have now confirmed that fully colonised phase 3 compost is susceptible to T. aggressivum infection. Results clearly indicate that the bulk spawn-run process potentially increases the risk of a small compost infection becoming a serious disease outbreak, however not as a result of significant growth within the compost during the 17 day spawn-run but as a direct consequence of the bulk handling and mixing operations that disperse and spread small infections through much larger compost masses. Cross contamination was also shown to occur on the growing unit from an infected crop to a newly filling crop and handling Trichoderma infected Phase 3 readily dispersed the disease around the growing unit. This was clearly identified at the critical sampling locations tested as part of WP4. Further, the results from WP1, which highlighted that disinfectants were ineffective in killing T. aggressivum in colonised compost, have important ramifications for best practice guidelines at growing units. Stringent hygiene procedures and cleaning up of infected materials/facilities following crop termination were effective at containing the disease. Post crop steam sterilisation (65-70˚C in the compost for a minimum of 8 hours applied both with compost in the growing room and again when it had been removed) was effective as independently verified by Partner 1 (Teagasc) under WP4.
A second objective of this work-package aimed to quantify the concentration of T. aggressivum in Phase 3 mushroom compost at various production stages. A number of Trichoderma detection methods - Compost Fragment Plate (CFP), Compost Plate, Most Probable Number, PCR and real-time PCR have been tested. Trichoderma was detected on air fallout plates positioned close to the mini-tunnel door during emptying of the back infected tunnel slice in almost all trials but less consistently on plates positioned at a far wall in the spawning hall. This is consistent with investigations that have shown Trichoderma spores, though not readily airborne, are easily transmitted on dust particles and compost fragments. Air fallout plates can therefore provide a very simple but useful monitoring tool for compost manufacturers and several of the consortium partners have already incorporated this monitoring procedure in their routine hygiene programmes. Accurate diagnostic testing that can be applied directly to large compost masses is notoriously difficult not least because sample size is prohibitively small to be classed as representative. In the mini tunnel trials, 10 or 100g samples represent just 0.0025% – 0.025% of the compost; in commercial operations processing much larger compost volumes this problem is compounded. In the initial trials, in the absence of a compost mixing process there was an acknowledged limitation in the sampling protocol that meant the assays used showed varying abilities to detect the inoculated T. aggressivum. In trials that incorporating compost three mixing procedures as standard, all assays efficacy improved significantly although the representative sampling size limitation stands.
PCR and real-time PCR methods generally proved effective with the strict proviso that compost must be thoroughly mixed before sampling to ensure assay efficacy. However, using CFP detection methods with two samples, both Partners 9 and 1 identified T. aggressivum at low levels that was undetected by real-time PCR (Ct = 40). The latter method currently records Ct values up to 40 cycles, with a Ct of 40 = zero, but there may be a limit of detection as this was clearly not zero as further evidenced by crop production yield losses and T. aggressivum assessments at crop termination. Undoubtedly, as a quantitative tool, real time PCR provides valuable additional information to composters on the extent of the infection and likely impact at the mushroom production stage. In addition, the quicker sample turn-around time of real-time PCR would make it the preferred detection method for composters, however, it is still an “end of process” assay and reliant on representative samples from prohibitively large volumes of well mixed compost. Volatile organic compound (VOCs) samples in adsorbent cartridges taken from both within the compost and from the head space above the compost during these WP6 trials were provided to Partner 6 for the volatile diagnostic work and are reported separately in WP3. This alternative “in process” or “online” VOC assay (WP3) to distinguish Trichoderma infected compost on experimental scale facilities may also be applicable under commercial conditions. This would offer significant advantages in the decision making process on how to manage compost infected with Trichoderma to limit further exposure of both compost and growing production facilities.
Some of the information from this WP was included in the technical factsheet on Understanding Trichoderma aggressivum in Bulk Phase 3 compost (English, Dutch and Polish) which was produced in three languages and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP7: Investigations to track the incidence and spread of MVX inoculum on compost and grower facilities.
WP7 was conducted by researchers at Teagasc, Ireland (Partner 1) in collaboration with researchers at EMR, England (Partner 8). It had one objective, OBJECTIVE 4.1 –to gather information on the incidence and spread of MVX (before during and after an outbreak) on compost and grower facilities, which was subdivided into three tasks.
TASK 7.1 set out to validate the improved MVX diagnostic test and extraction method developed in WP2 under the controlled conditions of an experimental cropping trial. A 4m trough of A15 spawned Phase 2 compost was inoculated at one end, at spawning time, then incubated as normal. At the end of the spawn run period sections were removed from the trough at 1m intervals, then cased and cropped individually. Samples of compost were also taken at the end of spawn run along the length of the trough for the detection of two MVX dsRNAs “AbV16” and “AbV6” using the existing RT-PCR method and the new qPCR method. Following cropping, mushroom samples and end of crop compost samples were also taken for analysis by RT-PCR and qPCR. The newly developed methodology for extraction of RNA from compost and qPCR diagnostics worked effectively to quantify the levels of viruses in the compost samples and the results matched with the corresponding RT-PCR results for both mushrooms and mycelia grown off compost. Therefore the improved diagnostics proved to be effective for their intended purpose for use with spawn-run compost.
TASK 7.2 studied MVX Epidemiology as it relates to compost facilities. Background monitoring of MVX on compost facilities was carried out at a number of compost facilities and results were reported back to each company. Monitoring was done in two ways: (i) a full round of Phase 3 compost samples from each tunnel on the facility over an emptying cycle and (ii) monthly samples on one day per month from each of the facilities for five months, this monthly sampling included two debris type samples and a sample of the Phase 3 compost from any tunnels being emptied that day. The Phase 3 compost samples were tested by RT-PCR for the MVX dsRNAs and the debris type samples were used in bioassay cropping experiments to determine if MVX dsRNAs would be transmitted to healthy compost and if brown mushroom symptoms would be observed. Brown mushroom symptoms were measured in the cropping experiments trials using a chromameter. Following an outbreak of MVX in autumn 2013, a period of intense sampling took place over the course of 5 months. MVX dsRNSAs (MVX band 19 - now known as AbV16-1, and MVX band 15 - now known as AbV6-2) were detected in a small number of Phase 3 compost samples. The compost debris samples used in the bioassay experiments did not result in symptoms being observed, however MVX dsRNAs were detected in mushrooms from a treatment inoculated with one of the debris samples. The conclusion from this task was that MVX dsRNAs are present in some samples of Phase 3 compost at tunnel emptying and in some compost debris on compost facilities, and infection of healthy phase 3 compost with such material can transmit MVX viruses (AbV-16 and AbV-6) into the healthy compost.
TASK 7.3 studied MVX Epidemiology as it relates to mushroom growing facilities. Background sampling was carried out at several grower facilities which had recently experienced MVX to identify locations where MVX was present. An additional grower facility, experiencing significant MVX issues, was targeted for intensive sampling in conjunction with the compost facility supplying the Phase 3 compost. These samples were tested for MVX dsRNAs and also used in bioassay experiments. Samples of debris were also obtained from all grower facilities for use in bioassay experiments.
The initial background sampling detected MVX only in samples of mushrooms or compost from crops with symptoms and not in samples from other locations on the facilities. However, the samples of compost from a grower facility experiencing significant problems had MVX dsRNAs in all samples tested: Phase 3 compost from the compost facility at emptying, compost from the truck at delivery to the grower facility, compost from the freshly filled shelf and compost from under symptomatic mushrooms. In bioassay experiments, where small amounts of all these “virus-positive” compost samples were added into healthy compost as inoculum and then cropped, only the treatment inoculated with compost from under the symptomatic mushrooms re-produced symptomatic mushrooms upon cropping. Mushrooms from all other treatments however tested positive for the MVX dsRNAs, consistent with those of the inoculum, but did not exhibit symptoms.
The debris samples from all grower facilities were also used in a separate bioassay experiment. Mushrooms from all treatments inoculated with the debris exhibited no symptoms and all tested negative by RT-PCR, however one ‘end of crop’ compost sample from one of the treatments at the end of the bioassay tested positive for MVX dsRNA band 19 (AbV16-1).
SIGNIFICANT RESULTS
• The new RNA extraction method developed in WP2 was an effective and efficient method of extracting RNA from mushroom compost
• The qPCR tests developed in WP2 for the mushroom viruses AbV16-1 and AbV6-2 were effective and sensitive at detecting their presence in compost samples, compared with existing detection methods for mushrooms and mycelium by RT-PCR.
• In bioassay experiments MVX dsRNAs were transmitted from infected Phase 3 compost into healthy Phase 3 compost but generally the mushrooms showed no symptoms. Only when infected 'end of crop compost' from a symptomatic crop was used to infect the healthy Phase 3 compost, were brown mushroom symptoms observed in the resulting crop. Thus, low levels of MVX viruses can be present in crops but with no symptoms.
• Debris samples from compost and grower facilities were found to be a means of transmitting viruses to healthy compost.
Some of the information from this WP was included in the technical factsheet on ‘Brown cap Mushroom Virus Prevention’ (English, Dutch and Polish) which was produced in three languages and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
Potential Impact:
MushTV has produced a range of new technical knowledge and advice for the mushroom industry, which has been distributed in the form of factsheets, workshops, seminars and popular articles. MushTV has developed a new diagnostic method to detect MVX viruses in mushroom compost and work is in progress to make it available as a commercial service. MushTV has developed methodology to underpin a novel volatile-based diagnostic method for detecting Trichoderma aggressivum in Phase 3 compost. Dissemination events in face to face settings have reached in the region of 200 industry personnel from across all SME AGs and the various MushTV partners and their customers. The MushTV Industry partners have indicated how they have benefited significantly through their participation in MushTV. Many partners have altered their own practices in response to knowledge and information gained from the project, particularly in the areas of disinfectant use and control of Trichoderma green mould and Mushroom Virus X. This new information will make the mushroom industry as a whole more knowledgeable and more efficient at dealing with disease threats thereby reducing their reliance on, and use of pesticides. This will have important economic benefits for the industry. Reduced pesticide use also has a positive social and environmental impact in the context of high quality food and protection of the environment. Specific results are outlined below.
WP1 identified that it is very difficult to kill T. aggressivum with disinfectants when it is present in compost particles so that it is essential to ensure all compost fragments are cleaned off equipment and surfaces prior to disinfection. A factsheet entitled ‘Use of chemical disinfectants in mushroom production’ was produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers. .
WP2 has characterised and named the viruses that make up MVX and developed improved highly sensitive tests for their detection. The MVX virus that causes the brown cap mushroom symptoms has been sequenced and named as Agaricus bisporus Virus 16 (AbV16).
WP3 has the potential to deliver a volatile-based diagnostic method to detect T. aggressivum in Phase 3 compost however it requires further development and is far from commercial implementation at this point in time.
WP4 has gathered data from growers and composters about the incidence of T. aggressivum and MVX on facilities during the project period. This information has helped individual SME’s to be more informed of disease risks on their facilities.
WP5 has evaluated three products (two biopesticides and one chemical) for the control of mushroom diseases. Only the chemical product has shown promise as a potential new product for the industry. As yet, non-chemical disease control still relies on effective hygiene methods. In addition, a technical factsheet entitled ‘Fungal diseases of mushrooms and their control’ was produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP6 generated new knowledge on the growth and spread of T. aggressivum within the bulk Phase 3 system thereby increasing our understanding of how easily this pathogen can have a severe impact on mushroom production. This information, along with information from WPs 3 and 4, above, was used to compile a technical factsheet entitled ‘Understanding Trichoderma aggressivum in Bulk Phase 3 compost’. It was produced in three languages (English, Dutch and Polish) and provided in electronic format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
WP7 generated new knowledge on how MVX spreads within mushroom compost. It identified that the AbV16 virus that causes Brown Cap Mushroom disease can be present in both compost and mushrooms in the absence of symptoms, leading to a false sense of security that the virus is absent. This information, along with information from WPs 2 and 4, above, were used to compile a technical factsheet entitled ‘Brown Cap Mushroom Virus prevention’. It was produced in three languages (English, Dutch and Polish) and provided in electronic and hard copy format to all SME AGs and MushTV partners to disseminate to their members, staff and customers.
The MushTV SMEAgs and all Consortium members agreed to make all the factsheets available to the wider mushroom industry on the MushTV and other websites .
List of Websites:
www.MushTV.eu; Helen.grogan@teagasc.ie