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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code HH2309SMU

2. Project title

Mushroom Virus X

3. Contractororganisation(s)

Warwick HRIUniversity of WarwickWellesbourneWarwickCV35 9EF                    

54. Total Defra project costs £ 913525(agreed fixed price)

5. Project: start date................ 01 July 2003

end date................. 31 March 2007

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.Mushroom virus X (MVX) is a disease of the commercial mushroom, Agaricus bisporus. When first recorded in 1996 it was associated with localised areas of pin suppression. It is now thought to be responsible for a range of symptoms. It seriously affected the UK mushroom industry in 2000/01 and has now been reported in Ireland, Holland and a number of other mushroom growing countries. The seriousness and complexity of the disease led Defra and HDC to put in place a major research programme at Warwick HRI.  Analysis of diseased mushrooms has identified double stranded RNA (dsRNA) molecules associated with MVX symptoms. dsRNA molecules indicate the presence of viruses in the mushroom. Evidence collected to date suggests that MVX is a complex of viruses, rather than a single virus. Hitherto 27 dsRNA elements have so far been identified as separate bands on agarose gels. Invariably MVX-infected mushrooms contain some, but not all of these dsRNA bands. Three dsRNAs routinely occur in asymptomatic mushrooms and up to seven other dsRNA elements are also considered asymptomatic.

Experiments carried out at Warwick HRI have focused on understanding the complex epidemiology of MVX, characterising some of the viral entities and establishing diagnostic technologies.

MVX DiagnosticsExtraction and visualisation of dsRNA bands associated with mushroom virus X (MVX) has been achieved routinely from mushroom caps and stipes. Confirmation of the presence of three dsRNA bands (bands 3 [14.4kb], 15 [3.6kb] and 19 [1.8kb]) has also been possible from caps, stipes and Agaricus mycelium grown on agar medium using RT-PCR. Increased sensitivity has been achieved through the use of nested PCR. Additionally, a real-time RT-PCR assay was developed using SYBR Green I for relative quantification of MVX dsRNAs. Detailed statistical analysis of MVX dsRNAs found in more than 1000 industry samples suggests that visualization of dsRNA molecules by gel electrophoresis remains the method of choice for reliable detection of MVX due to variability in the presence of individual dsRNA Forty commercial spawns and 74 cultures from the Agaricus Resource Programme (the world collection of wild Agaricus bisporus) were tested using RT-PCR for the presence of MVX dsRNA. None of the commercial spawn tested at Warwick HRI proved positive for the presence of MVX. One wild culture (ARP 250) tested positive for the presence of dsRNA element MVX 14.4. This is the only evidence to date that MVX elements occur in wild Agaricus strains.

Sequence analysisSequence analysis, homology searches, phylogenetic analysis, and genomic organization, all support the conclusion that the dsRNA element MVX14.4 is a new species of virus in the genus Endornavirus. It is proposed that appropriate nomenclature for this virus is Agaricus bisporus endornavirus 1 (AbEV1). This is the first molecular characterization of an endornavirus that infects a homobasidiomycete and AbEV1 is the first dsRNA element from the MVX complex to be fully sequenced and characterized.Transmission of MVXExperiments have demonstrated that MVX elements can be efficiently transferred by horizontal transmission between strains following anastomosis in dual culture tests. Vertical transmission (through fungal spores) was demonstrated through the production of single spore isolates (SSI) from an infected

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mushroom. Detection of MVX14.4 dsRNA in SSI cultures using RT-PCR demonstrates the potential for vertical transmission of these elements. The inability to detect the MVX14.4 element in all single spores suggested that partitioning of the dsRNAs may take place during basidiospore development. Demonstration of very effective horizontal transmission and that vertical transmission can occur underline the importance of crop hygiene for growers to prevent spread of the disease.

Potential sources of resistance to MVXExperiments were designed to further evaluate the potential of natural tolerance in (i) commercial and wild varieties of A. bisporus and (ii) alternative species from the genus Agaricus. Of ten species tested, three appeared resistant to the transmission of MVX. These included A. arvensis and 2 cultures of A. subrufescens. Of fourteen commercial Agaricus varieties screened, two (C54 carb9 control and BELLA) tested negative for the presence of MVX following challenge. C54 carb9 had been challenged with 1283 during previous studies and in both cases MVX dsRNAs were not transmitted. Fifty-seven wild Agaricus were tested and 7 appear resistant to the transmission of the MVX dsRNAs present in isolate 1283. These results suggest that further effort should be made to exploit possible resistance to MVX.

Transfer MVX diagnostic capability to CSL and production of a commodity PRAA detailed list of diagnostic protocols have been produced and supplied to CSL and training in their use provided for CSL staff at Warwick HRI. In addition, a detailed list of pathogens, pests and weed moulds affecting mushroom production have been used to produce a commodity Pest Risk Analysis for mushrooms in conjunction with CSL staff.

Virus Management StrategyThe results generated from this project have provided information on molecular characterisation and identification of a complex of viruses infecting the cultivated mushroom. Diagnostic techniques developed have enable detailed epidemiological experiments to be undertaken, providing data on routes of transmission, speed of spread and also a possible source of infection. Results of this work have been presented to UK mushroom growers and guidance provided on control through improved hygiene.At the end of this project in 2007, the incidence of MVX in the Uk industry is currently at its lowest level since 2001.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Background

Hollings (1962) reported the presence of virus particles of different sizes in diseased mushrooms. A number of viruses have been isolated from mushrooms including a bacilliform particle measuring 50 x 19nm, spherical particles of 24 and 29 nm and a 50 nm particle . A serious cropping problem known as ‘La France’ disease was later shown to be caused by a 34nm virus particle containing a set of nine dsRNA molecules. The UK mushroom industry suffered severe losses associated with this virus during the 1960/70s until the hygiene measures put in place enabled growers to control the problem. It is now increasingly rare to find either 34nm particles or the dsRNAs associated with them in Britain

In recent years outbreaks of a virus-like disease have resulted in severe cropping problems for UK growers. In 2000-2001 more than 700 samples were examined for presence of dsRNA by the Horticulture Research International Mushroom clinic. None were found to contain La France virus whereas more than 80% contained a complex of other viruses described as ‘mushroom virus X’ (MVX).

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Different symptoms have been associated with the presence of the novel dsRNA banding patterns; pinning suppression resulting in crop delays of 2-5 days through to bare areas with no mushrooms premature-opening of mushrooms; ‘brown’ mushrooms in an otherwise white crop; loss of quality and distortions, a reduction in crop yield.

UK industry sources suggested that in 2000, approximately 85% of UK production was affected by this problem and that the yield losses were in the region of 15%.

Further evidence from around the country suggests that bulk phase III production has been the most severely affected by MVX, with an approximate 50% reduction in the quantity being produced, although non-bulk farms and systems continue to be affected. The intransigence of MVX on some bulk phase III sites has had a devastating effect on the confidence in bulk phase III technology and it was thought that it would be a major setback if the advantages of this technology could not be exploited in Britain as it is by Britain's main competitors, Ireland and the Netherlands. The British mushroom industry cannot afford to lose the advantages in quality and productivity that bulk phase III production allows. It was thought at the outset of this work that the threat to bulk phase III from MVX should be fully understood so that adequate control measures could be put in place. The objectives of the HDC funded work (M39c) during this period were (1) to determine the importance of mushroom spores in transmitting MVX to healthy compost; (2) to confirm the relationship between very low levels of MVX contamination (0.0001%) of freshly spawned compost and MVX transmission and (3) to further examine the interactions between different MVX strains and composts in terms of symptom expression.

HDC funded epidemiology studies had been unable to reproduce all of the symptoms associated with MVX disease. One of the objectives in HDC project M39b was to examine the relationship between symptom expression and dsRNA profiles (as present in infected mushrooms), as a function of time and concentration of inoculation. Symptom expression was thought to be the result of a combination of factors and the dual approaches of HDC and DEFRA work should help to determine the factors which are important in symptom expression.

Further HDC funded work (M39c) built on this work and examined the interactions between contrasting MVX strains as a function of time of inoculation and compost source.

Since MVX became commonplace in GB, La France virus has not been detected, even though it was present on three farms prior to them developing MVX. La France virus appears to have been easier to control than MVX on virus-vulnerable bulk phase III sites. Evidence suggests that MVX is more infectious than La France and has now displaced it in GB. Proof of this would be useful in understanding the epidemiology and evolution of mushroom viruses in general.

Warwick HRI (WHRI) provided a general mushroom diagnostic clinic and a specific MVX diagnostic service for mushroom growers for many years. Since the closure of this general clinic in 2005, Defra Plant Health Division have expressed concerns about mushroom pest diagnostic capabilities within the UK with regard to future quarantine-related needs; PHD are responsible for quarantine-related mushrooms issues under the Plant Health Order for England and Wales. It was therefore proposed that WHRI transfer existing pathogen diagnostic expertise to Defra CSL in order to support future surveillance activities and to ensure a UK-based diagnostic capability. In this respect, such a transfer would go some way to addressing the National Audit Office report’s comments regarding MVX in the context of surveillance and reporting of future new mushroom pests and pathogen (NAO Report HC 1186, 29 October 2003 - Protecting England and Wales from plant pests and diseases). In addition, WHRI would provide technical input into a commodity-based pest risk analysis which would identity future risks and pathways and inform future policy in the advent of an introduction of any new exotic mushroom pests or pathogens.

This project aimed to provide essential underpinning research for HDC funded work, to determine the relationship between dsRNA bands, to identify and characterise the number of viruses in MVX, to evaluate the Agaricus culture collection for virus tolerance, and to develop strategies for on farm control of the disease.

Scientific Objectives

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1. To determine the relationship between all major dsRNA bands present in the MVX complex and to sequence characterise major components.

2. To develop an RT-PCR diagnostic test for separate viruses within the MVX complex and test commercial spawns and ARP cultures for virus presence.

3. Screen antisera for ability to detect A.bisporus on farm using MTIST 4. Characterise transmission of dsRNA elements between strains, assess effects of cross infection

and determine significance of dsRNA partitioning during transmission through spores.5. Identify potential sources of resistance/tolerance to MVX in Agaricus spp. 6. Determine the rate of spread of MVX into healthy compost from a point source7. Design virus management strategy based on output of experimental programme and present to,

and discuss with growers. 8. To localise and quantify MVX dsRNA elements within infected tissues9. Complete optimisation of diagnostic tests for MVX for transfer to CSL10. Transfer MVX diagnostic capability and general pathogen diagnostic capability to CSL11. Contribute relevant information to CSL to create a commodity-based PRA

Our scientific report is presented as sections relating to each of the eleven Scientific Objectives (above). All objectives have been met or exceeded with the exception of Objective 8 which was curtailed following discussions with the Project Officer as not being cost effective. Detailed data tables, figures and references are all presented in the Appendix.

OBJECTIVE 1; To determine the relationship between all major dsRNA bands present in the MVX complex and to sequence characterise major components.Typical MVX dsRNA profiles and seizes are shown in Appendix 1.

Fifteen MVX dsRNA elements were purified, end-labelled, partially digested using T1 ribonuclease and gel electrophoresis optimised. Preliminary results showed that it was possible to identify differences between MVX dsRNA segments with the aid of this technique. In further experiments, five distinct terminal fingerprinting analytical gels were obtained. Legitimate comparisons between dsRNA elements could only be made within a single experiment (bands post-fixed with *, exhibited different fingerprints). Experiment I compared MVX bands 3*, H2, 9, 18, 19, 15* and H3*; Experiment II compared bands H3*, 19*, 22*, 23*, 2+H1* dsRNA mix and a single La France* sample; Experiment III compared 2+H2* mix with H3*, 22*, 23*; Experiment IV compared MVX bands H2, 9, 18, 19, and a La France* dsRNA element; Experiment V compared H1, H3, 9*. Many other dsRNA combinations were fingerprinted and fractionated but did not yield clear fingerprints. It was anticipated that the terminal fingerprinting would reveal substantial similarities between MVX dsRNA elements, indicate probable defective interfering particles, and provide insight into the number of discrete viruses in the MVX complex. Excluding a single group (H2, 9, 18, 19), minor fingerprint polymorphisms were detected in all MVX dsRNA elements tested.

Sequence characterisation of a novel Endornavirus from Agaricus bisporus

Endornaviruses have been mainly reported in plants, such as barley, bell pepper, broad bean, kidney bean, melon, bottle gourd, malabar, rice, spinach, seagrass (Pfeiffer, 1998; Gibbs et al., 2000; Coutts, 2005; Fukuhara et al., 2006; Valverde & Gutierrez, 2005). However, some non-plant endornaviruses have been recently reported in protists such Phytophtora spp. isolate P441 (Hacker et al., 2005) and fungi such as Helicobasidium mompa and Gremmeniella abietina type B (Osaki et al., 2006; Tuomivirta & Hantula, 2006).

Methods

dsRNA isolationA single-spore isolate (SSI 61), which harboured only MVX14.4 was used as the source of the MVXdsRNA element. MVX14.4 was extracted from SSI 61 mushrooms as follows;Mushroom sporophores were ground to a fine powder in liquid nitrogen, using a mortar and pestel. Powdered samples were transferred to 250 ml pots containing 16 ml of 1x STE, 2 ml of 10% w/v SDS, 1 ml of 2% w/v bentonite aqueous solution and 18 ml of acid phenol (pH 4.3; Sigma, cat. No. P-4682). The mixture was emulsified by shaking for 30 min at 4 C and then centrifuged for 15 min at 9,000 xg (Europa 24 M- MSE). Supernatants were transferred to 50 ml tubes for further centrifugation (10 min,

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9,000 xg, Hermle Z382K). Absolute ethanol was added to the purified aqueous phase to a final concentration of 15% v/v. For each sample two columns were prepared, comprising a sterile 20 ml plastic syringe plugged with glass wool, equilibrated with 25 ml of 1x STE/ 15% v/v ethanol and 1.5 g of CF-11 cellulose (Whatman, cat. No. 4021050). Samples were added to each column and washed with 40 ml of 1x STE/ 15% v/v ethanol. The dsRNA from the column was eluted by adding 10 ml of 1xSTE and collected in sterile tubes. Absolute ethanol was again added to each tube to a final concentration of 15% v/v and the wash process repeated, apart from a final elution with 6 ml of 1xSTE. Nucleic acids were precipitated at -20C for 2h using 20 ml of absolute ethanol and 1 ml of 3M sodium acetate (pH 5.2). Samples were centrifuged at 9,000 xg for 25 min (Hermle Z382K) and the supernatants discarded. The pellets were resuspended in 100 l DMPC water and further purified using Qiaquick PCR purification kit (Qiagen, cat. No. 28106), following the manufacturer’s protocol. Samples were finally eluted in 30 l DMPC water.

MVXdsRNA was further digested with DNase and S1 nuclease to remove any remaining traces of DNA and ssRNA, and used as template for downstream RT-PCR fill in protocol.

cDNA Cloning, Sequencing and Sequence AnalysisAmplified RT-PCR products were gel purified and ligated into pGEM®-T Easy vector. To confirm the presence of the clonal inserts, pGEM®-T Easy clones were restriction enzyme digested using Not I endonuclease (Roche, cat. No. 1014706), according to the manufacturer’s protocol. Sequencing reactions were performed to generate sequences from both ends of clonal inserts using ABI Prism® BigDye™ Terminator cycle Sequencing Ready Reaction Kit with Amplitaq® DNA Polymerase, Fs (Perkin-Elmer Applied Biosystems, cat. No. 403044).

SequencingThe sequencing of inserts cloned in pGEM®-T Easy vector was performed in 10 µl volume reactions comprising: 2 µl ABI Prism® BigDye™ terminator cycle Sequencing Ready Reaction Kit with Amplitaq® DNA Polymerase, 1 µl of 5xBuffer, 0.2 µl primer T7 or SP6 (10 pmol/µl), 800-1000 ng purified plasmid. Sequencing primers T7 (5- TAATACGACTCACTATAGGG-3’) and SP6 (5’-ATTTAGGTGACACTATAGAA-3’) annealed flanking regions of the pGEM®-T Easy cloning site. BigDye® terminator cycle sequence reactions were performed using GeneAmp 9600 Thermal Cycler according to the following parameters: 25 cycles of rapid thermal ramp (1o C/s) to 96o C, 96o C for 10s, rapid thermal ramp to 50o C, 50o C for 5 s, rapid thermal ramp to 60o C, 60o C for 4 min. Reaction products were sequenced in the Warwick-HRI Genomic Centre using an automated DNA capillary system (3130xl Genetic Analyzer, Applied Biosystems).

RT-PCR fill inRT-PCR fill in using sequence-specific primers was used to expand the MVX14.4 sequence and bridge the gaps between non-overlapping clones. Given that the orientation and relative position of contigs were unknown, ca 80 primers targeting various contigs were designed and used as a pool (maximum 5 primers-pool) in RT-PCR fill-in. Primers were designed in such a way to span at least 100 bp of the known sequence.

cDNA synthesis reactions were prepared as follows: 0.5 µl of each 10 µM PA primer (designed for a target contig) were mixed with 0.5 µl of each 10 µM PB primer (designed for a different contig), 300 ng of purified RNA template and DMPC water up to 10 µl total volume. Reactions were boiled for 5 min and then placed on ice. The second step-reaction was performed in 20 µl volume and comprised the following: 1 µl Superscript™ II Reverse Transcriptase (200 U/µl, Invitrogen cat. No.18064-022), 4 µl of 5x cDNA synthesis buffer, 2 µl dNTPs mix (10 mM), 1 µl DTT (0.1M), 1 µl Rnase OUT ™ (40 U/µl, Invitrogen cat. No.11146-024), and 10 µl template (from the previous step). cDNA synthesis was progressed using a HYBAID MBS 0.5 G Thermal Cycler with the following parameters: 50 C for 45 min (cDNA synthesis), 85 C for 5 min (termination). To remove any remaining RNA template, each cDNA reaction was treated with 1 l E. coli RNase H (2 U/µl, Invitrogen) at 37 C for 20 min.

PCR amplification of cDNA products were performed in 50 µl volume reactions as follows: 0.5 µl Expand Long Template enzyme mix (Roche, cat. No. 1681834), 5 µl of 10x buffer I, 2 µl dNTPs (10 mM mix), 0.5 µl of 10 µM PA primer (already used for cDNA synthesis), 0.5 µl of 10 µM PB primer (already used for cDNA synthesis), 3 µl cDNA. PCR reactions were progressed using a HYBAID MBS 0.2 G Thermal Cycler according to the following parameters: initial denaturation at 94o C for 2 min, followed

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by 10 cycles of 94o C for 15 s, 50o C for 30 s, 68o C for 8 s, subsequently 20 cycles of 94o C for 15 s, 50o

C for 30 s, 68o C for 8s with 5 s increment each cycle, and a final extension at 72o C for 5 min.

Single Primer Amplification Technique (SPAT)To determine the MVX14.4 dsRNA termini, an adaptation of the SPAT technique published by Shapiro et al. (2005) and modified by A. Soares (pers. comm.) was performed. Extracted dsRNA (500 ng,) was ligated to a single stranded anchor-primer, 5’-GACCTCTGAGGATTCTAAAC/iSp9/TCCAGTTTAGAATCC-3’ (iSp9 is a carbon chain spacer) using 10U T4 RNA ligase in a 10 µl reaction (New England Biolabs, cat. No M0204S). The ligation reaction was incubated at 10o C for 12 h and the product was precipitated at room temperature using Pellet Paint® NT Co-Precipitant (Novagen, cat. No. 70748) in 100 µl total volume as follows: 2 µl of Pellet Paint® were added to the ligation reaction, followed by 0.1 volume of 3M sodium acetate and 2 volumes of 100% ethanol. After gently mixing, the reaction was centrifuged at 12,000 xg for 5 min. The pellet was rinsed with 2 volumes of 75% v/v ethanol, centrifuged again and air-dried prior to resuspension in 8 µl of water to the pellet.

Results

The completed consensus sequence of MVX14.4 dsRNA, assembled from more than 130 overlapping RT-PCR fill in sequences was 12750 bp (Appendix 1.2). All regions were sequenced from more than two independently synthesized clones. The MVX14.4 dsRNA termini were determined from at least three cDNA clones recovered from independent dsRNA extractions. For the 5’ end, 7 sequences were generated, whereas 12 sequences were recovered for the 3’end. Integrity of the assembled consensus sequence was confirmed by RT-PCR screening using primers that span different regions of the entire sequence. A single open reading frame (ORF) was found in the plus strand, starting at nt 29 and ending at nt 12679, which encodes a putative protein of 4216 aa (369.13 kDa) (Appendix 1.3). This would imply a 5’-untranslated region (UTR) of 28 nt. The first methionine codon nt 29-31 is in a favourable context for translation initiation, with purine (A) residues at the -3 and +1 positions, according to Kozak’s rules for ribosomal scanning (Kozak, 1986; Lütcke et al., 1987).

The 3’-untranslated region (UTR) was shown to be 71 bp long, including a run of seven G residues at the 3’ terminus. Other significant ORFs were not detected in alternative reading frames of the plus and minus strands of MVX14.4 dsRNA. A BLASTX search using the complete sequence of the MVX14.4 dsRNA showed significant similarity to putative polyprotein sequences encoded by members of the novel virus genus Endornavirus, recently accepted by the ICTV (data not shown). The best sequence similarity identified was with Helicobasidium mompa endornavirus 1-670 (HmEv1-670, composite E value = 11e-132), followed by Vicia faba endornavirus (VFV, composite E value = 5e-118), Phytophthora endornavirus 1 (PEV1, composite E value = 15e-115), Oryza sativa endornavirus (OSV, composite E value= 8e-111). Other than the Endornavirus genus, the next most similar alignments were with regions of RdRp of several viruses belonging to the Closteroviridae family, such as Mint vein banding virus (E value = 4e-11) and Strawberry chlorotic fleck associated virus (E value = 3e-10). The size of MVX14.4 (12751 bp) was most similar to PEV1 (13883 bp) (Appendix 1.4). The amino acid sequence of MVX14.4 showed a wide range of similarity with non-plant and plant endornaviruses (Table 1.1).

Table 1.1. Regions of significant similarity between MVX14.4 and PEV1, HmEV1-670, OSV, and VFV amino acid sequences identified by pairwise comparison using NCBI database

AMINO ACID RESIDUES IDENTITY (%) SIMILARITY (%)MVX14.4 PEV1

543-1580 543-1673 21% 38%

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2737-2802 3106-3171 31% 53%3716-4214 4117-4612 35% 53%

MVX14.4 HmEV1- 670488-2796 933-3577 20% 36%2800-3700 - No identity No similarity3723-4198 4883-5357 34% 54%

MVX14.4 GaEV1-3700 - No identity No similarity

3727-4158 2913-3343 26% 45%

MVX14.4 OSV495-1614 558-1785 21% 38%1615-3600 - No identity No similarity3604-4208 3984-4566 33% 51%

MVX14.4 VFV294-818 1593-2226 22% 40%

820-3700 - No identity No similarity3722-4184 5239-5736 34% 52%

The region of MVX14.4 dsRNA polyprotein with the highest sequence similarity to Endornavirus was located near the C-terminus. Inspection of this sequence showed that conserved motifs characteristic of RNA-dependent RNA polymerase (RdRp) were present in MVX14.4 dsRNA. Motifs found (between ca aa 3700 and aa 4150) represented the signature of ssRNA virus superfamily III (Koonin & Dolja, 1993). MVX14.4 RdRp motifs showed high conservation with those of other endornaviruses. Neighbour-joining phylogenetic analysis of RdRp (motifs III-VI) of various endornaviruses and ssRNA viruses showed that MVX14.4 clustered within an Endornavirus clade (79% bootstrap support, Appendix 1.5). Preliminary phylogenetic analyses, using more diverse RdRps confirmed the highest similarity between endornaviruses and ssRNA viruses belonging to the alpha-like superfamily (data not shown). Pairwise identity analysis indicated a plant endornavirus (OSV) RdRp as the most similar to the MVX14.4 RdRp (59% similarity; Appendix 1.6).

Another region of MVX14.4 polyprotein with high similarity to endornaviruses was located near the N-terminal part of the protein. Further inspection of this region (between ca aa 1300 and aa 1580) showed conserved motifs characteristic of RNA helicases of superfamily I (Koonin & Dolja, 1993). When MVX14.4 sequence identified as helicase motif I-VI was used for BLASTP searching and pairwise identity analysis, the best match was with PEV1 (Appendix 1.6), which has previously been reported to contain helicase-like domains (Hacker et al., 2005). The next most similar alignment was with helicase-like regions of Phaseolus vulgaris dsRNA element (E value = 3e-14) and VFV (E value = 2e-13).

Sequence analysis, homology searches, phylogenetic analysis, and genomic organization, all support the conclusion that MVX14.4 is a new species of the genus Endornavirus. Agaricus bisporus endornavirus 1 (AbEV1) is proposed as an appropriate nomenclature of this virus. This is the first molecular characterization of an endornavirus that infects a homobasidiomycete and AbEV1 is the first dsRNA element from the MVX complex to be fully sequenced and characterized.

Members of the genus Endornavirus exhibit no significant homology with dsRNA viruses outside their genus, but rather their RdRp and helicase domains are most closely related to ssRNA viruses of alpha-like superfamily.Although endornaviruses have been reported in various plants, their presence in other organisms has not been extensively studied. Few reports have described endornaviruses in fungi. Results reported here support the hypothesis that MVX14.4 is the first ever endornavirus to be characterized in edible fungi.

Further sequence analysis was conducted on other dsRNA elements, but not presented here as none was sequenced to completion.

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OBJECTIVE 2; To develop an RT-PCR diagnostic test for separate viruses within the MVX complex and test commercial spawns and ARP cultures for virus presence.

RT-PCRAn RT-PCR protocol was developed for detection of MVX dsRNA components (Appendix 2.1). Further development of this method is presented under Objective 9 of this report.A real-time RT-PCR assay using SYBR Green I for relative quantification of MVX dsRNAs was developed. In this method, data are normalized against the 18S rRNA (Appendix 2.2)

Commercial spawns and ARP cultures. Forty commercial spawns and 74 cultures from the Agaricus Resource Programme (ARP; the world collection of wild Agaricus bisporus) were tested using RT-PCR for the presence of MVX dsRNA elements 14.4, 9.4a, 3.6, 1.8. None of the cultures tested at Warwick HRI proved positive for the presence of these dsRNA elements. A wild culture kindly supplied by Dr Rick Kerrigan (ARP partner), ARP 250 tested positive for the presence of dsRNA element 14.4. (Appendix 2.3)

OBJECTIVE 3; Screen antisera for ability to detect A.bisporus on farm using MTIST

Airborne transmission of Agaricus bisporus was monitored within a commercial spawning hall at Blue Prince Mushroom Farm at Horley. Three standard Burkard glass slide suction samplers and two Burkard Microtitre immunospore traps (MTIST) were operated continuously over the 8 day period. Transmissible inoculum of A. bisporus (spores), although at a low level during spawning, was recorded at each of the activities of Phase III production. An increase in the level of A. bisporus transmission (spores and mycelial fragments) was observed during ‘pulling’ of Phase III production. The use of a formaldehyde fog proved effective in removing the greater part of this inoculum from the airborne environment. Nevertheless the potential exists for airborne transmission of mushroom Virus X via Agaricus inoculum at each stage of the process.

Comparative studies employing the Burkard standard suction sampler and the new MTIST spore trap proved highly correlative (r2= 0.805, linear) in the collection of trapped A. bisporus spores. The use of the MTIST spore trap, together with developed antisera, enabled the rapid quantification of the trapped target inoculum by PTA ELISA (plate-trapped antigen ELISA).

OBJECTIVE 4; Characterise transmission of dsRNA elements between strains, assess effects of cross infection and determine significance of dsRNA partitioning during transmission through spores

Although spread (transfer) of the MVX disease from infected to non-infected plots had been produced in cropping trials, definitive transmission of dsRNA elements from one strain (donor) to another (recipient) had not previously been demonstrated. This experiments were designed to assess whether the MVX dsRNAs could transmit equally well into different varietal types.

StrainsDual culture transmission experiments were conducted using three different donor strains (1283, 2735 and 1994) and three different recipient strains (C43 carb9, C54 carb8 and C63 x 422) (Appendix 4; Table 2). The donor strains, previously isolated from MVX infected tissue, and harboured a range of specific MVX dsRNAs. Recipient strains were free of MVX dsRNA (as assessed using PCR) and were genetically marked carboxin resistance mutants (Challen & Elliott 1987) in order to monitor/confirm that MVX had moved between strains. Three different recipient varieties were used (smooth white, off-white and hybrid spawn varieties).

TransmissionAcceptor and Donor strains were grown on CE-CYM medium at 25°C for three weeks. Two plugs were taken from the growing edge of two replicate plates and donor and acceptor plugs were positioned

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10mm apart on replicate plates of CE-CYM and allowed to anastomose. Sample isolations (sub-cultures) were made from the periphery on each side (donor/acceptor) at 4 time points (3, 6 12 and 35 days) post anastomosis. Sample isolations were plated of CE-CYM and incubated at 25°C for three weeks.

RT-PCR Subcultures from 3, 6, 12 and 35 days post-anastomosis were screened using primers for 18S (RNA positive control), and MVX dsRNAs MVX 14.4, 9.4a, 3.6, 1.8. 18S primers were tested individually, but MVX specific primers were combined in a multiplex.

Transmission RT-PCRInitial RT-PCR screening of the acceptor and donor strains recovered expected results for the presence of MVX dsRNAs. All the nucleic acid templates yielded 18S rtPCR products (control) and all donor samples proved positive for the appropriate MVX dsRNAs. MVX dsRNAs were first detected in acceptor strains after 6-12 day pairings (Appendix 4; Table 3). Not all dsRNA transferred equally to the acceptor strains. The hybrid acceptor strain (C63-carb) most readily accepted dsRNAs and virtually all the test dsRNAs were detected in pairings using this strain (Appendix 4; Table 3). With the smooth white (C43-carb) acceptor only 1 dsRNA (MVX1.8) appeared transmitted. In pairings with the off-white (C54-carb) acceptor, no MVX transmission could be detected (Appendix 4; Table 3). Post-anastomosis provenance of all the acceptor strains was confirmed by growth on carboxin modified agar (Challen & Elliott 1987).

These experiments demonstrate for the first time, transmission of MVX dsRNAs into novel acceptor strains. The MVX donor strains were isolated from commercial samples and are thus all of hybrid varieties. Restricted transmission of dsRNA elements to the C43/C54 strains was most likely to be the result of limited anastomosis (vegetative incompatibility) of quondam varieties with hybrid donors. The observation that not all donor strains were equally infected with MVX dsRNAs is interesting and suggests there may be potential for using different strains as ‘virus breakers’ to reduce the commercial impact of MVX.

Cropping of selected crosses on the Mushroom Unit supported the results obtained from initial plate tests. The full dsRNA extraction performed on the cropping material showed further exchange of MVX related dsRNAs to the acceptor strains. Crosses of 1944 and C43 carb9 also produced MVX associated symptoms on some of the trial material. We believe this is the first time that these symptoms have been duplicated in a trial situation. It therefore supports the conclusion that these bands are related with the symptomatic browning of white mushroom varieties.

dsRNA partitioning during transmission through sporesMethodsA single MVX-infected donor (isolate 1283) mushroom was sterilised using absolute ethanol and placed on a mushroom stand. The mushroom was allowed to open and spores were collected over a 24 hour period. Single spore cultures were made using sterile distilled water at x 50 and x 100 dilution. The cultures were spread on plates of CE-CYM. Single spores were microscopically identified and spores transferred individually to new media. 162 single spores were grown, but 60 were selected for further study. Sixty single spore isolate (SSI) cultures from 1283 were tested using RT-PCR with 18sRNA (positive control) and primers for three MVX elements.

MVX infected spawnSix agar plugs of freshly grown SSI cultures were used to inoculate screw-capped glass jars containing 150g of sterilised, pre-cooked, rye grain. Jars were incubated at 25°C for 2-4 weeks and shaken weekly to ensure good growth. When the rye grain was well colonised, the jars of infected spawn were stored at 4°C.

CroppingWarwick HRI Phase II compost was used to fill plastic bags (3-3.5kg). Spawn was mixed by hand and the spawn run compost was then transferred to 125mm (10 inch) flower pots. Three replicates were grown of each SSI and pots were arranged using a random block design. Positive (1283) and negative (A15) control strains were included. Compost was spawn run and the casing applied 14 days later. Two flushes were harvested from each SSI for dsRNA profiling by full dsRNA extraction and agarose gel

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electrophoresis Yield data was produced for mushroom crops and compared with the 1283 and non-infected acceptor strains.

ResultsAll the nucleic acid templates yielded 18S products and 47 yielded the MVX14.4 amplicon (Figure 1.). Thirteen of the single spore isolates had no detectable product (Figure 1.). All 60 SSI cultures tested negative for the presence of bands 9.4a and 3.6.

Figure 1. RT-PCR screening of single spore progeny from MVX strain 1283. Primer B3198 amplifies a 315bp

product from MVX 14.4. Gel image shows results for all 60 SSI cultures.

Full dsRNA extractions performed on SSI material grown on the unit showed that the 5 isolates which tested negative for MVX dsRNAs using RT-PCR, contained no MVX related dsRNAs (9, 27, 84, 127, 134). Four out of five of the isolates which tested positive for the presence of band 3 using RT-PCR were shown to contain band 3 in the full extraction (61, 121, 140, 160). One of these isolates (SSI 140) also contained MVX band 15b which is present in the parent strain. One isolate, SSI1 proved to be non-self sterile and therefore produced no mushrooms.

Yields for crops that were negative for MVX band 3 produced similar yield patterns to the A15 control. However, yields for the crops positive for MVX band 3 were highly variable which would suggest a lack of correlation between the presence of band 3, and poor performance. The detection of MVX14.4 dsRNA in SSI cultures using RT-PCR demonstrates the potential for vertical transmission of these elements. The inability to detect the MVX14.4 element in all single spores suggested that partitioning of the dsRNAs may take place during basidiospore development. Partitioning of dsRNAs might be expected if more that one virus was present and not all spores received all viruses equally. Segragation of dsRNA elements has been observed in other fungi. It is however, unusual that only 2 of the 6 MVX dsRNAs present in the parent were detected in the single spore progeny. Also, mushrooms grown from the SSIs did not contain any of the asymtomatic bands (H2 and H3) present in the MVX parent strain and often in commercial mushrooms.

OBJECTIVE 5; Identify potential sources of resistance/tolerance to MVX in Agaricus spp.

Experiments were designed to further evaluate the potential of natural tolerance in (i) commercial and wild varieties of A. bisporus and (ii) alternative species from the genus Agaricus. It is anticipated that tolerance could be exploited directly using a strain-rotation approach (virus-breaking) or, if only present

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in wild species, by the exploitation of resistance genes via transgenic and/or selective breeding strategies.

MethodsSeventy-seven strains were included in this study to screen for the presence of MVX dsRNAs and their resistance to MVX through dual plate studies. Fifty-three ARP cultures (wild A. bisporus) were obtained from the Agaricus Resource Programme. Fourteen commercial strains and a further 10 strains belonging to the genus Agaricus were also chosen. Alternative species were selected on the basis of their relationships with A. bisporus. (Appendix 5.1).

Transmission experiment Fifty-three ARP cultures (wild A. bisporus), 14 commercial strains and a further 10 strains from the genus Agaricus were ‘challenged’ by anastomosis with an MVX donor strain (1283-2). Control strains, included carboxin marked mutants C43 carb9, C54 carb8, C63 422 and A15.

Acceptor and donor strains were grown on CE-CYM medium at 25°C for three weeks. Plugs of donor and acceptor mycelium were positioned 10mm apart on replicate plates of CE-CYM and allowed to anastomose. Sample isolations (sub-cultures) were made from the periphery on each side (donor/acceptor) at 3 time points (13, 35 and 62 days) post anastomosis. Sample isolations were plated of CE-CYM and incubated at 25°C for three weeks.

RT-PCR of transmission crossesSub cultures were screened using primers for 18S (RNA positive control), and MVX dsRNA’s MVX 14.4, 9.4a and 3.6 (Appendix 5.2). Initially, sub-samples taken at day 35 (point 4) and day 62 (point 6) from both A and B plates were combined and tested together. If the result was negative for the presence of MVX dsRNAs, samples were re-tested individually.

Purified extraction was boiled for 5 minutes with Random hexamer primers (Invitrogen Life technologies, Paisely). Tubes were immediately placed on ice for 5 minutes, centrifuged to consolidate and the following added in order; sterile ultra pure water (1µl), 5 x 1st strand buffer (4µl), 0.1M DTT (2µl), 10mM dNTP (1µl)(Invitrogen Life Technologies, Paisely) and SuperScript II RT (1µl). The reaction was performed in a hybaid thermocycler in the following way: 10 min at 25°C, 60 min at 42°C, 15 min at 70°C with a 4°C hold. 1µl of RNase H (Invitrogen Life Technologies, Paisely) and tubes incubates at 37°C for 20 minutes. Amplification of the template was performed in a reaction volume of 50µl containing: sterile ultra pure water (33µl),10x PCR buffer (5µl), 75mm MgCl2 (1.5µl), 20mm dNTPs (1µl), 20pmoles each (forward and reverse) primer (Table 1), 2.5 Units platinum Taq DNA polymerase (Life Technologies) and 5µl of cDNA from the RT reaction. PCR conditions were as follows: 2 min at 96°C (1 cycle), 30 sec at 94°C, 30 sec at 55°C, 3 min at 72°C (30 cycles) and a final extension of 10 min at 72°C (1 cycle).

Samples were analysed electrophytically using a 1% w/v agarose, ethidium bromide and TAE buffer (Sambrook, Fritsch & Maniatis 1989). Gel Images were captured using Grabber software v. 2.00 (Phoretix International, Newcastle-upon-tyne). Molecular weights were quantified using a 100bp ladder (Invitrogen, Paisely).

ResultsRT-PCR of acceptor strainsAll Seventy-seven strains including wild ARP, other Agaricus and commercial varieties tested positive for 18S RNA. None of the strains tested positive for the presence of MVX 14.4, 9.4a and 3.6.

RT-PCR of transmission crossesOf ten other species tested, three appear resistant to the transmission of donor MVX. These included A. arvensis and 2 cultures of A. subrufescens. Fourteen commercial Agaricus varieties were screened for donor MVX dsRNAs. Of the fourteen strains tested two (C54 carb9 control and BELLA) tested negative for the presence of MVX 14.4, 9.4a and 3.6. C54 carb9 had been challenged with 1283 during previous studies and in both cases MVX dsRNAs were not transmitted. The commercial variety BELLA is a brown strain, from Amycel. Fifty-seven wild Agaricus were tested and 7 appear resistant to the transmission of the MVX dsRNAs present in 1283.

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27/77 band H243/77 band 331/77 band15

12/77 no bands transmitted36/77 1 band transmitted23/77 2 bands transmitted6/77 3 bands transmitted

Full results are presented in Appendix 5.3.

OBJECTIVE 6; Determine the rate of spread of MVX into healthy compost from a point source

Experiments were conducted to quantify the rate of movement of dsRNAs through a crop and to determine whether there was a relationship between symptom expression and the presence and/or intensity of dsRNAs.

MethodsCompost and cropping containerSix troughs (2.75 m long, 23.5 cm wide, 11.5 cm deep) (Appendix 6) in which several holes have been drilled through to the bottom to allow for drainage were filled with 14 kg spawned (0,5%) Phase II compost

Inoculum and InoculationSterilised compost was colonised with infected Agaricus cultures containing strain 1283-2, strain 2735 or strain A15-1 as a control. 100 mg of infected compost was used to infect each trough (about 0.001%).Inoculum was positioned 10 cm in from one end of the trough at a depth of about 5 cm. Two replicate troughs were prepared per strain

Cropping conditionsThe inoculated troughs were cased to a depth of about 4 cm with standard commercial casing to which casing spawn was added Troughs were case-run and aired according to standard operating procedures on the mushroom unit

SamplingCompost samples were taken at 0.5 m intervals from the inoculation point (6 locations) on each sampling day (on days 12, 15, 28, 34, 42) for testing by RT-PCR. Casing samples were taken at 0.5 m interval once (day 15) when the casing was colonised to see if PCR tests can detect dsRNAsMushrooms samples were harvested and tested by RT-PCR and dsRNA extraction

SymptomsTroughs were monitored for the appearance of symptoms during the crop cycle (bare areas, crop delay, brown mushrooms, prematurely opening mushrooms)

ResultsResults indicated that MVX dsRNAs had spread from the point of inoculation to a distance of at least 2.5 m by the first flush with a reduction in dsRNA intensity with increasing distance from the inoculation point. MVX 14.4 was detected in the first flush of mushrooms 34 days after inoculation. This was estimated to be equivalent to at least c. 80mm per day.Crop delay symptoms were obvious up to 1.5 m from the inoculation point with MVX culture 1283 but not with culture 2735. No brown mushroom symptoms were recorded with culture 2735.

OBJECTIVE 7; Design virus management strategy based on output of experimental programme and present to, and discuss with growers.

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The results generated from this project have provided information on molecular characterisation and identification of a complex of viruses infecting the cultivated mushroom. Diagnostic techniques developed have enable detailed epidemiological experiments to be undertaken, providing data on routes of transmission, speed of spread and also a possible source of infection. Results of this work have been presented to UK mushroom growers at Warwick HRI/Mushroom Growers Association Subject days held at Wellesbourne, at regional meetings and through HDC News and the Mushroom Journal. Guidance has been provided on control through improved hygiene measures. Further details can be seen in the list of outputs form this project.At the end of this project in 2007, the incidence of MVX in the UK industry is currently at its lowest level since 2001.

OBJECTIVE 8; To localise and quantify MVX dsRNA elements within infected tissues

There has been little previous use of in situ hybridisation (ISH) with filamentous fungi. These experiments aimed to develop fluorescent (FISH) detection of MVX elements and exploit the technology for localisation and quantification. ISH experiments were performed using two different methods. Oligonucleotide probes were prepared for Ab18S RNA helices (control probes) and used with A15 mycelium to develop FISH protocols. Despite extensive experimentation and parameter variations FISH detection of the 18S RNAs proved unreliable. Oligonucleotide probes were also prepared for MVX band 3 and tested with 1283 mycelia. Although fluorescence was obtained using MVX probes against infected mycelia, penetration of the fungal cell wall proved difficult without tissue disruption. Following disruption it was not possible to ‘localise’ fluorescence signals. Further extensive method development would be required before FISH could be used to reliably detect MVX dsRNA elements. Discussions with the DEFRA project officer regarding milestone 02/03 (Fluorescent primers for quantitative detection of Band 3 optimised) concluded that further work would not be cost effective.

OBJECTIVE 9; Complete optimisation of diagnostic tests for MVX for transfer to CSL

Extraction and visualisation of dsRNA bands associated with mushroom virus X (MVX) has been achieved routinely from mushroom caps and stipes using a modification of a published protocol (Valverde, R.A.,1990). Confirmation of the presence of three dsRNA bands (bands 3 [14.4kb], 15 [3.6kb] and 19 [1.8kb]) has also been possible from caps, stipes and Agaricus mycelium grown on agar medium using RT-PCR following the extraction of dsRNA using a TRIREAGENTTM (Sigma-Aldrich) protocol. Neither of these procedures has been successfully applied to spawned compost due probably to the very low concentrations of MVX dsRNA present and also to the inhibitory influence in RT of humic compounds found in compost.

This objective sought to test alternative protocols to develop a reliable and sensitive process.

RNeasyTM RNA extraction kit (Qiagen)This commercial kit was used exactly according to the manufacturer’s instructions. The starting material was spawned compost heavily colonised with an Agaricus strain infected with MVX. A batch of 30g fresh weight was freeze dried to produce 9g dry weight and aliquots were used for all other protocols. Here 100mg was processed. An identical weight of freeze dried mushroom cap from the same MVX-infected Agaricus strain was used as a positive control. The final extracts were subjected to reverse transcription (RT) and then the cDNAs used in PCR with primer pairs for the 18S ribosomal RNA gene region (universal) and for the 3.6kb MVX dsRNA band (B15) described above. PCR products were only seen using the extracts from cap tissue. The presence of compounds inhibitory to RT in the spawned compost extract was confirmed by mixing the extracts from cap tissue and spawned compost together before RT and then PCR. No PCR products were seen.

PowerSoilTM DNA isolation kit (Mo Bio Laboratories)This commercial kit was used exactly according to the manufacturer’s instructions. The starting material was spawned compost heavily colonised with an Agaricus strain infected with MVX. An aliquot (200mg) of the freeze dried sample as described above was processed and an identical weight of freeze dried mushroom cap from the same MVX-infected Agaricus strain was used as a positive

SID 5 (Rev. 3/06) Page 15 of 19

control. Mixtures of these two samples (100mg of each) were also extracted to test for the removal of compounds inhibitory to RT. This protocol was effective at removing RT inhibitors from the mixed sample but did not allow amplification of an 18S or B15 product from the 200mg spawned compost. When 10mg of spawned compost was extracted no RT inhibition was observed but no B15 product resulted, possible due to insufficient virus dsRNA being present.

Valverde dsRNA extractionThe standard protocol was applied to 7g dry weight of the freeze dried sample of spawned compost as described above. Most (85%) of the dsRNA extracted was applied to an agarose gel (0.8%) and the separated nucleic acids visualised. No distinct dsRNA bands were observed. The remaining 15% was diluted tenfold and 15% of this (equivalent to 250mg of the original freeze dried sample) was used in reverse transcription. PCR was then done on the cDNAs generated and products were successfully amplified using all three MVX dsRNA primer pairs. This protocol is clearly reliable and can handle relatively large amounts of spawned compost; this is important if the level of mycelial colonisation or total quantity of MVX dsRNA present is low. It has the disadvantage of being a multi-step process that would only permit a single operator to process approximately 12 samples per day.

Modified TRIREAGENTTM extractionThe manufacturer’s protocol was followed exactly using 100mg aliquots of spawned compost as described above. The final step of dissolving the RNA pellet in 50μl water was replaced by dissolution in a salt/Tris/EDTA (STE) buffer containing 15% ethanol. This solution is used in the Valverde extraction protocol prior to binding the dsRNA to CF11 microfibrous cellulose (Whatman). The solubilised RNA was applied to a Micro Bio-Spin column (BioRad) filled with CF11 equilibrated in STE with 15% ethanol. The brown discoloration of the extract at the end of the TRIREAGENTTM extraction was removed by several washes of the CF11 spin column with STE + 15% ethanol. The bound dsRNA was eluted from the CF11 spin column with 50μl water and 8μl (equivalent to 16mg of the original freeze dried sample) was used in reverse transcription. PCR was then done on the cDNAs generated and products were successfully amplified using all three MVX dsRNA primer pairs. This modified protocol has been shown to detect MVX infected spawned compost at a very low level (at least one order of magnitude lower than that detected using the Valverde method). It also has the advantage of allowing a single operator to process up to 100 samples per day.

Increased sensitivity of detection of MVX dsRNA by nested PCRStandard PCR has the potential to amplify a single copy of DNA by approximately 1010 times if 35 cycles are carried out. If nested PCR is undertaken using 0.1μl of the product generated with the external primers the potential amplification is 1020. Three new primer pairs were designed from the sequences of bands 3, 15 and 19. The new primers amplified products of 266, 100 and 258bp (compared to 314, 162 and 322bp with the original primers). All three new primer pairs were shown to generate product yields that could be visualised on a gel (>10ng) from “invisible” yields using the external primers. This procedure could usefully be applied to samples that have given a negative result with the external primers to increase the confidence of the initial result.

OBJECTIVE 10; Transfer MVX diagnostic capability and general pathogen diagnostic capability to CSL

A detailed list of diagnostic protocols have been produced and supplied to CSL (see Appendix 10).Training was provided for CSL staff at Warwick HRI.

OBJECTIVE 11; Contribute relevant information to CSL to create a commodity-based PRA

A wide range of viral, fungal and bacterial pathogens [or pests in general] have the potential to cause serious damage of mushrooms. Warwick HRI (and its preceding organisations) has provided most of the UK input into research on a range of pathogens on mushrooms.

SID 5 (Rev. 3/06) Page 16 of 19

MethodologyWarwick HRI’s experience was used to inform development of a PRA and included a literature survey of all non-native mushroom pests worldwide, highlighting those that are known to be particularly damaging; a detailed breakdown of mushroom ("fruit" and spawn) import statistics (to help identify pathways) and any quality/phytosanitary (and mycosanitary) measures taken in the importing country and the UK, e.g. any measures taken to separate imports from home produced mushrooms; PRAs on the principal damaging non-native mushroom pests present in countries that are our key mushroom trading partners; a forecast for the future of mushroom trading, production and pest management (new trading partners, new species, pest control capabilities etc); descriptions/manuals/gross margin budgets etc for mushroom production and pest control practices in the UK; and a list of key references for the Info Centre to purchase.

ResultsThe analysis determined that 19 species of fungi, 11 species of bacteria and five species of viruses were directly pathogenic to mushrooms. Weed mould fungi, whilst not directly affecting the mushroom crop, can colonise compost and cause losses through competition. This study determined that there are at least 59 species of weed mould of mushroom composts. Of the invertebrate pests associated with mushroom production, 21 insect species, 22 Acarina (mites), 28 mycophagous nematodes and at least 18 saprophytic nematodes were listed.

The majority of mushroom pathogens, weed moulds and pests are present in the UK. However, one species of fungal pathogen, two species of bacterial pathogen and two species of weed mould competitor are not considered present in the UK. All known viral pathogens of mushroom are already known to occur in the UK. Of the invertebrate pests affecting mushrooms, one species of insect, eight mite species, 17 species of mycophagous nematodes and ten species of saprophytic nematodes are not present in the UK.

Mushroom growers are also advised to remain vigilant against species that are already present in the UK and have the potential to affect mushroom production, but have not yet been officially recorded in mushroom production environments. These species include several species of weed mould and nematode, and one species of fungal pathogen, Verticillium fungicola var. aleophilum. This pathogen may present a risk to UK mushrooms. It has been recorded affecting mushrooms in North America, but despite being present in other habitats in the UK, has not yet been found in UK mushroom crops.

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

Challen, M.P. & Elliott, T.J. (1987). Polypropylene straw ampoules for the storage of microorganisms in liquid nitrogen. Journal of Microbiological Methods 5, 11-23.

Coutts, R.H.A. (2005). First report of an Endornavirus in the Cucurbitaceae. Virus Genes 31, 361- 362.Fukuhara, T., Koga, R., Aoki, N., Yuki, C., Yamamoto, N., Oyama, N., Udagawa, T., Horiuchi, H.,

Miyazaki, S., Higashi, Y., Takeshita, M., Ikeda, K., Arakawa, M., Matsumoto, N. & Moriyama, N. (2006). The wide distribution of endornaviruses, large double-stranded RNA replicons with plasmid-like properties. Archives of Virology 151, 995-1002.

Gibbs, M.J., Koga, R., Moriyama, H., Pfeiffer, P. & Fukuhara, T. (2000). Phylogenetic analysis of some large double-stranded RNA replicons from plants suggests they evolved from a defective single-stranded RNA virus. Journal of General Virology 81, 227-233.

SID 5 (Rev. 3/06) Page 17 of 19

Hacker, C.V., Brasier, C.M. & Buck, K. W. (2005). A double-stranded RNA from Phytophthora species is related to the plant endornaviruses and contains a putative UDP glycosyltrnasferase gene. Journal of General Virology 86, 1561- 1570.

Hollings, M. (1962). Viruses associated with a die-back disease of cultivated mushroom. Nature 196, 962-965.

Koonin, E.V. & Dolja, V. (1993). Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Critical Reviews in Biochemistry and Molecular Biology 28, 375-430

Kozak, M. (1986). Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283- 292.

Lütcke, H.A., Chow, K.C., Mickel, F.S., Moss, K.A., Kern, H.F. & Scheele, G.A. (1987). Selection of AUG initiation codons differs in plants and animals. The EMBO Journal 6, 43- 48.

Osaki, H., Nakamura, H., Sasaki, A., Matsumoto, N. & Yoshida, K. (2006). An endornavirus from a hypovirulent strain of the violet root rot fungus, Helicobasidium mompa. Virus Research 118, 143-149.

Pfeiffer, P., Jung, J.L., Heitzler, J. & Keith, G. (1993). Unusual structure of double-stranded RNA associated with the "447" cytoplasmic male sterility in Vicia faba. Journal of General virology 74, 1167-1173.

Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989). Molecular Cloning: a laboratory manual. New York, USA: Cold Spring Harbor Laboratory press.

Shapiro, A., Green, T., Rao, S., White, S., Carner, G., Mertens, P.P.C. & Becnel, J.J. (2005). Morphological and molecular characterization of a cypovirus (Reoviridae) from the mosquito Uranotaenia sapphirina (diptera: Culicidae). Journal of Virology 79, 9430- 9438.

Tuomivirta, T.T. & Hantula, J. (2006). The first complete sequence of a fungal Endornavirus from Gremmeniella abietina type B. Direct submission (NC_007920).

Valverde, R.A. & Guttierez, D.L. (2005). First report of an endornavirus in bell pepper. Direct submission (ABB51642).

MVX Outputs 2002-2003

Poster sessions, conference contributions, grower events and abstractsGrogan, H.M. (2002). Mushroom Virus X. Invited speaker at IV International Conference on Mushroom Biology

and Mushroom Products, Mexico, February 2002. Grogan, H.M. (2002). How does Mushroom Virus X spread?. Abstract. HRI/HDC Mushroom Open Day, 20th

June 2002. Grogan, H.M. (2002). Mushroom Disease Update. Invited speaker to MGA Conference, Cambridge, Sep 19-21,

2002.Grogan, H.M. (2002). Mushroom Disease Update. Invited speaker to MGA Northern Area Meeting, Dec 5th,

2002.

Technical reportsGrogan, H.M., Tomprefa, N. (2002). Epidemiology of Virus X complex. Final Report for HDC Contract No. M

39a. 30 pp. Mills, P.R., Adie, B., Challen, M.P., Grogan, H.M., Wakeham, A.& Mead, A. (2002). Final Project Report on

Defra project HH2304SMU: Characterisation of dsRNA associated with mushroom Virus X. 23 pp.

Popular articles and other publicationsGrogan, H.M. (2003). HDC Research Review (21). Latest on Virus X. Mushroom Journal, No. 638, pp 22-25.Grogan, H.M. (2003). A disease update. Mushroom Journal, No. 637, pp 5-9.Mills, P.R., Adie, B., Challen, M.P., Grogan, H.M., Wakeham, A., Mead, A., Choi, I. & Clay, C. (2002).

Understanding Virus X. HDC News, 88, 24-26. Grogan, H.M. & Gaze, R.H.(2002). Latest on Virus X. HDC News, 86, 20-22. Grogan, H.M. (2002). IV International Conference on Mushroom Biology and Mushroom Products. The

Mushroom Journal, 631, 6-8.

MVX Outputs 2003-2004

Refereed publicationsGrogan, H.M., Adie, B.A.T., Gaze, R.H., Challen, M.P. & Mills, P.R. (2003). Double stranded RNA elements

associated with the MVX disease of Agaricus bisporus. Mycological Research, 107, 147-154.

SID 5 (Rev. 3/06) Page 18 of 19

Other Research PapersAdie, B., Choi, I., Soares, A., Holcroft, S., Eastwood, D., Mead, A., Grogan, H.M., Challen, M., Mills, P.R. (2004)

MVX disease and double-stranded RNA elements in Agaricus bisporus. Mushroom Science 16, 411-420.Grogan, H.M., Tomprefa, N., Mulcahy, J., Holcroft, S., Gaze, R.H. (2004) Transmission of Mushroom Virus X

disease in crops. Mushroom Science 16, 489-498.

Poster sessions, conference contributions, grower events and abstractsChallen, M.P. (2003) Virus X ‘Molecular Biology’ Update. Mushroom Day, HRI Wellesbourne, 17 July 2003Soares, A., Holcroft, S., Choi, I., Grogan, H., Challen, M. & Mills, P. (2003) Double-stranded RNA elements and

the MVX disease of Agaricus bisporus. Abstracts - Basidio2003, Horticulture Research International, Wellesbourne; 5 September 2003

Soares, A.X., Adie, B.A.T., Holcroft, S., Grogan, H.M., Challen, M. & Mills, P. (2003) Novel double-stranded RNA elements associated with the MVX disease of the button mushroom Agaricus bisporus. Abstracts - 8th International Symposium on double-stranded RNA viruses. Il Ciocco, Italy, 13-18 September 2003. Poster P7.27

Holcroft, S., Challen, M.P., Soares, A.X., Grogan, H.M. & Mills, P. (2003) Double-stranded RNAs and the MVX disease of Agaricus bisporus. Molecular Biology of Fungal Pathogens 14, Ambleside, 15-17 September 2003

Popular articlesGrogan, H.M. (2003). HDC Research Review (21). Latest on Virus X. Mushroom Journal, No. 638, pp 22-25.Grogan, H.M. (2003). A disease update. Mushroom Journal, No. 637, pp 5-9.Grogan, H.M. (2004). The effects of Virus X on cropping (DEFRA funded research). HDC News, 101, 29-30.

MVX Outputs 2004-2005

Edited conference proceedingsAdie, B., Choi, I., Soares, A., Holcroft, S., Eastwood, D., Mead, A., Grogan, H.M., Challen, M., Mills, P.R. (2004)

MVX disease and double-stranded RNA elements in Agaricus bisporus. Mushroom Science 16, 411-420.

Poster sessions, conference presentations, grower events and abstractsMaffettone, E., Soares, A.X., Holcroft, S., Grogan, H.M., Challen, M.P. & Mills, P. (2004) MVX disease and

dsRNA elements in the cultivated mushroom Agaricus bisporus. British Mycological Society Annual Scientific Meeting, University of Nottingham, 13-15 September 2004. Abstracts, pg 62. Poster presentation 4.7.

Mills P R, Challen M, Soares A, Grogan H, Maffettone E and Burrow S. Double stranded RNAs in the Mushroom virus X complex of Agaricus bisporus. Genetics and Cellular Biology of Basidiomyctes VI; Pamplona, Spain, June 2005.

Mills P R. Mushroom Pathology. BSPP Regional Meeting, University of Nottingham, June 2005.Maffettone E, Soares A, Holcroft S, Challen M and Mills P. MVX disease and dsRNA elements in the cultivated

mushroom Agaricus bisporus. Basidio 2005. University of Warwick April 2005.Maffettone E. et al. A new virus in the cultivated mushroom Agaricus bisporus. 2005 Mushroom industry

Conference and trade exhibitionPereira N. Mushroom virus X diagnostics. 2005 Mushroom industry Conference and trade exhibitionGoncharenko N et al. Movement and detection of MVX in compost. 2005 Mushroom industry Conference and

trade exhibitionGrogan H.M. Mushroom disease research; some key issues for successful control. 2005 Mushroom industry

Conference and trade exhibitionMills P R. Underpinning mushroom research (Defra) at Warwick HRI. 2004 Mushroom industry Conference and

trade exhibition Mushroom industry Conference and trade exhibitionMaffettone, E., Soares, A., Warner, P., Challen, M.P. & Mills, P.R. (2005) Molecular characterisation of MVX

disease dRNA elements from Agaricus bisporus. Abstracts - Exploitation of Fungi, Annual Scientific meeting, British Mycological Society, University of Manchester, 5-8 September 2005. Poster G.9

Maffettone, E., Soares, A., Challen, M.P. & Mills, P.R. (2005) The endornavirus AbEV1: a dsRNA element associated with MVX disease of Agaricus bisporus. Abstracts - Molecular Biology of Plant Pathogens XVI, Ambleside, 19-21 September 2005.

Popular articlesGrogan H M. Underpinning mushroom research (Defra) at Warwick HRI. Plant It! 2005Gaze R. Underpinning mushroom research (Defra) at Warwick HRI. The Mushroom Journal, Sept 2005

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