DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS CSG 15 Research and Development Final Project Report (Not to be used for LINK projects)

Two hard copies of this form should be returned to: Research Policy and International Division, Final Reports Unit DEFRA, Area 301 Cromwell House, Dean Stanley Street, London, SW1P 3JH. An electronic version should be e-mailed to [email protected]

Project title Determining the Physiological and Genetic Mechanisms of Initiation to Control Mushroom Population Densities

DEFRA project code HH1334SMU

Contractor organisation Warwick HRI and location Wellesbourne Warwick CV35 9EF

Total DEFRA project costs £ 236,595

Project start date 01/04/01 Project end date 31/03/04

Executive summary (maximum 2 sides A4)

Purpose of the Project Achieving the correct number of mushroom primordia on the casing layer is critical in obtaining the maximum yield of quality mushrooms. Sparse initiation results in depressed yields, whereas too many primordia result in dense populations of poor quality mushrooms which are difficult to pick 1,2,3. However, the mechanism and controlling factors of initiation have remained largely unknown. A tentative hypothesis was that initiation is controlled by the production of self-inhibitory compounds by the Agaricus mycelium. Lowering the level of this (these) compound(s) to below a threshold level is necessary for initiation to occur. The specific objective of this project was to test the above hypothesis of the mechanism of initiation. The long-term aim of this work was to control the amount and timing of mushroom initiation to improve crop uniformity, quality and ease of picking.

Main Findings of the Project • By using gas chromatography - mass spectrometry (GC-MS), mushroom mycelium was found to produce mainly 8-carbon compounds during growth in the casing. The compounds included 2- ethyl hexanol, 3-octanone, 3-octene, 1-octen-3-ol, 1-octanol. • One of these compounds, 2-ethyl hexanol was found to completely suppress the formation of mushroom primordia in peat-based casing, although it did not appear to affect mycelial growth • Several casing materials (charcoal, anthracite coal, zeolite) were able to adsorb the 8-carbon inhibitors and enable primordia formation and mushroom fruiting • Repeated use of an adsorbent casing (charcoal) resulted in saturation with 8-carbon compounds and an inability to promote initiation

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• The presence of 2-ethyl hexanol resulted in an increase in the Pseudomonas bacteria population in the casing, which may therefore be able to metabolise the inhibitor. • Strains of Pseudomonas putida and closely related Pseudomonas species were found to differ in terms of stimulating mushroom initiation, with some strains stimulating few or no primordia. • Analysis of 16 S rRNA genes showed that the Pseudomonas strains used were in the P. taetrolens / P. poae, P. tolaasii / P. veronii and P. putida groups. • Strains in the P. taetrolens / P. poae group stimulated more proimordia than the P. tolaasii / P. veronii group. Strains in the P. putida group were intermediate in stimulatory behaviour. • The initiating ability of Pseudomonas strains does not appear to be correlated with a common plasmid. • The numbers of primordia produced by different Agaricus bisporus strains was related to the quantities of 8-carbon compounds produced by the mycelium. Strains that produced more (inhibitory) 8-carbon compounds tended to produce fewer primordia. • Inoculation of non-sterile casing with Pseudomonas strains resulted in significant changes in the numbers of primordia and subsequent mushroom sporophores compared with uninoculated casing.

Implications of the Findings for Policy and Future Research The results of this work support the original hypothesis that mushroom initiation is controlled by the production of self-inhibitory compounds by the Agaricus mycelium, and their subsequent removal or metabolism in the casing. The results show that it may be possible to manipulate the level of mushroom primordia formation and subsequent mushroom population densities by • the application 8-carbon compounds (2-ethyl hexanol) to the casing or in the growing environment • by the inclusion of adsorbant materials in the casing • by the inoculation of the casing with strains of Pseudomonas spp. that are able to control the number of primordia formed. The work corresponded with DEFRA policy objectives in HH13 'Environments for protected cropping'. In particular, the project will improve the ability of growers to optimise efficiency of production in the use of picking labour and substrates, to manipulate cropping patterns, and to improve crop quality and uniformity.

Action to Follow the Research Technology Transfer The effects of the following factors on mushroom initiation and subsequent mushroom populations should be investigated further in large-scale experiments and commercial scale tests: • application of 8-carbon compounds (2-ethyl hexanol) in the growing room, as a possible method of controlling the amount and timing of initiation • application of Pseudomonas strains to casing to control the amount of primordia and developing sporophores • use of adsorbent materials in the casing. Science Objectives The genetic mechanism of initiation of Agaricus bisporus should be further examined and manipulated: • Identify, sequence and quantify expression analysis of key initiation genes of Agaricus bisporus. • Generate transgenic strains of A. bisporus with modified expression of key genes. • Culture transgenic strains in microcosm, aerated flask and non-sterile tray systems and determine the effects of gene manipulation on initiation behaviour, sporophore development, gene expression and responses to agronomic variables. The metabolism of 2-ethyl hexanol by Pseudomonas species as a possible mechanism for the stimulatory behaviour should be examined.

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Scientific report (maximum 20 sides A4)

Introduction A casing layer, colonised by stimulatory bacteria (Pseudomonas putida) is normally necessary for mushroom initiation to occur. Activated charcoal can replace the function of stimulatory bacteria in casing4, possibly by adsorbing compounds which were inhibitory to initiation. A number of compounds have been isolated from activated charcoal casing, of which 1-octen-3-ol inhibited initial formation in pure cultures of A. bisporus5. Several key findings have been made in MAFF project HH1325SMU6, which have both improved the understanding of the initiation process and enabled several new scientific opportunities3. 1. Ranges of adsorbent materials which either promote or inhibit initiation under axenic conditions were identified. 2. Identification of significant differences between P. putida isolates in their ability to promote initiation. 3. Observation of a much wider range in initiation behaviour of wild Agaricus bisporus isolates than is found in commercial mushroom strains. 4. The successful fruiting of an A. bisporus isolate B430 which is capable of axenic initiation, which should enable the genetic and physiological mechanisms of initiation to be identified. 5. A particular population of mushrooms results from only 1 - 2 % of all initials, most of which are produced before the first flush. The above discoveries support the tentative hypothesis that initiation is controlled by the production of self-inhibitory compounds by the Agaricus mycelium. Lowering the level of this (these) compound(s) to below a threshold level, specific to each Agaricus isolate, is probably necessary for initiation to occur. The threshold level may be achieved through the metabolism of these compounds by the casing microflora (P. putida isolates), or by adsorption on specific casing materials.

Schematic of tentative hypothesis of mushroom initiation

Agaricus mycelium metabolism by ↓ adsorption by P. putida isolates ← self-inhibitory compound(s) → casing material ↓ ↓ threshold level(s) of compound(s) for initiation

temperature, CO2……………→↓

initiation

The discovery in the previous MAFF project of a wider range of materials capable of stimulating axenic fruiting (different carbonised materials and specific types of zeolite) may enable selective adsorption of compounds of particular molecular weight from mushroom mycelium. This may facilitate the isolation of specific inhibitory compounds, which could then be used to control initiation in mushroom culture. One of these materials, a carbonised by-product from the coal industry has been patented7 and about 5,000 m3 are now used in the UK mushroom industry. A previous MAFF project (HH1312SMU)8 showed that it may be possible to manipulate the casing microflora, since a genetically marked P. putida isolate could survive and multiply in a sterile casing throughout the cropping period. The isolate could also displace a naturally occurring P. putida population in non-sterile casing. It may therefore be possible to inoculate the casing layer with less or more stimulatory P. putida isolates, and thereby control the metabolism

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of inhibitory compounds and level of initiation more precisely than relying on the naturally occurring P. putida population. A mutant A. bisporus strain (B430) has been isolated which is capable of axenic fruiting in culture on defined media9. Work has shown that this strain is capable of fruiting on axenic casing, and of producing mature sporophores. The availability of A. bisporus isolates which are capable of axenic fruiting or mass and minimal initiation should help to determine the genetic and physiological mechanisms of initiation, in particular, the production of self-inhibitory compounds. Such isolates could be used to produce mushroom strains which have more desirable or controllable initiation patterns than current commercial strains. The aim of this project was to establish whether the tentative hypothesis of self-inhibitory compounds is a correct interpretation of the mechanism of initiation, and to identify several pathways by which the population of initials on the casing layer can be manipulated.

Materials and Methods

Axenic microcosm tests. A microcosm (500 mL, Kilner jar, Ravenhead Glass Ltd, St Helens, Lancs., UK) culture system, modified from Rainey10 was used to examine growth in axenic casing materials (Fig. 1). The microcosms were filled with a 10 mm layer (30 g) of sterile rye grain 'spawn', which served as a nutritional substrate, colonised with the appropriate A. bisporus strain. The strain Sylvan A15 was used, except where stated. This substrate was covered with 60 g (about 17 mm layer, surface area 64 cm2) of casing material, consisting of a mixture of black peat and CaCO3 (4:1 v/v) or other materials. Casing material was autoclaved for 2 h for sterile treatments. The microcosms were incubated at 25 C until mycelium had just started to colonise the surface of the casing layer, normally 3–5 d after the jars were filled. To promote conditions in the microcosms for initiation to occur, the CO2 concentration was reduced from about 0.4% v/v to 0.08–0.12% v/v by placing two sterile plastic caps, each containing 5 g self-indicating, 1–2.5 mm mesh soda lime granules, in each microcosm. The microcosms were then transferred to a 16 C cabinet; the soda lime was replaced when necessary and the casing layer irrigated with up to 15 mL sterile water after about 14 d to maintain a matric potential of -1 to -2 kPa. The matric potential, pH, and electrical conductivity of casing materials were determined according to Noble et al11. The numbers of primordia > 1 mm diameter were recorded 21 d after transfer to 16 C; axenic or non-axenic conditions were maintained in the microcosms for a further 21 d to determine if primordia developed into mature sporophores, i.e. stage 5 (Hammond and Nichols12) pileus with broken veil and gills exposed.

Large-scale non-axenic culture. A. bisporus strains were cultured using polypropylene trays (60 x 40 x 18 [deep] cm) in a controlled environment room, with cultural conditions as described in Noble et al11. Each tray contained 9 kg of the above composted substrate colonised with A. bisporus strain Sylvan A15 following inoculation with 1% w/w rye grain spawn as mentioned above. The substrate was covered with a 28 mm depth casing layer which consisted of a 4:1 v/v 11 mixture of black peat and CaCO3 (Noble et al ). The number of primordia (live and dead) were recorded at 3-day intervals, and the number of mushrooms at stage 5 were recorded and removed (Fig. 2).

Measurement of bacterial numbers in casing materials. In all the experiments, bacteria were isolated from casing materials on nutrient and Pseudomonas isolation agars (PIA) (Difco Laboratories, Detroit, Michigan) to determine the total bacterial populations as colony forming units per g dry weight casing material (cfu g-1), and to estimate the proportion which were Pseudomonas spp. An estimate of the proportion of Pseudomonas spp. which were P. putida isolates was obtained using the following tests (Stanier et al 1966, Lelliott and Stead 1987) on 30

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cfus from the PIA isolation: (i) fluorescence under UV light on Pseudomonas Agar F (PAF) (Merck Ltd, Poole, Dorset, UK); (ii) inability to hydrolyze gelatin; (iii) arginine dihydrolase test positive (Figs. 3 and 4). The strains were also tested with the 'white line test' for identifying pathogenic isolates of P. tolaasii13.

Milestone 01/01 Devise a method for chemically analysing self-inhibitory compounds in casing materials.

GC-MS Methods Two methods involving gas chromatography - mass spectrometry (GC-MS) have been used for analysing the compounds produced by mushroom mycelium in casing materials in sterile and non-sterile microcosms: washing of casing material in a solvent (diethyl ether) followed by low temperature analysis of the solvent and analysis of casing materials using thermal desorption of casing volatiles on to adsorption 'traps' (Carbotrap, Tenax and silica gel) followed by thermal desorption of the traps. Mushroom cultures were grown in the above microcosm system and volatiles were either analysed in the casing material using the solvent extraction method or adsorbent traps using the thermal desorption method. Microcosms without mushroom inoculum (blanks) were also prepared to test for background compounds in the culture system.

Solvent Extraction Method. We prepared samples for analysis using a solvent extraction method as illustrated in Fig. 5. In each case, 5 g of the charcoal was put into a thimble and 45 mL of the solvent (diethyl ether) added. The extraction was left to run for approximately one hour. The resulting samples were then injected on to the column (column: Supelco SPB1, 30 x 0.25 mm, 0.25 µ M film thickness). The GC method was adapted to bring the first peaks off the column quickly. The temperature ramp was set to: rate: temperature (C): time (s) (0:50:5, 20:150:0 and 4:250:5).

Thermal desorption method. Volatile compounds were pre-concentrated by adsorption on to Tenax (TA 60/80, Supelco Inc., Supelco Park, Bellefonte, PA, USA) and Carbotrap (Supelco 20/40 mesh) adsorbents. The concentrated volatiles were then thermally desorbed from the adsorbents into the GC-MS system for identification and quantification. Chromatographic retention time and mass spectral matching were used to confirm volatile identity. A Hewlett Packard (hp) (hp Ltd, Heathside Park Road, Cheadle Heath, Stockport, Cheshire, UK) GC-MS system consisting of a 5890 II Series gas chromatograph and a 5972A mass selective detector (MSD II) was used for analysis. A 25 m fused silica (cross linked methyl siloxane) hp-1 column with an internal diameter of 0.2 mm and a 0.34 µm film with a 1 m, ‘Q plot’, deactivated fused silica guard column (internal diameter 0.53 mm), containing a porous polymer of divinylbenzene (Supelco Inc.) was used. The flow rate of the eluting gas, helium, was 0.75 ml min-1. An Optic temperature programmable injector (Ai Cambridge Ltd, Pampisford, Cambridge, UK) was used to desorb headspace samples from the adsorbents and is initially at 30oC and heated at 16oC s-1 to 250 oC. An electronic pressure controller was used to offset peak pressure broadening with increasing GC column temperature. The GC oven conditions were an initial temperature of 40oC, then increased to 220oC at 15oC min-1 and remaining at 220oC for 1 min. The GC-MS interface was at 280oC. The mass spectrometer scanned from 35 to 250 mass units every 0.2 s to give responses in the ng range. Volatile organic compounds detected by the mass spectrometer were identified using a probability based matching algorithm and a NIST mass spectral library (National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD 20899-0001, USA). Compounds were declared unknown if their matching probability was less than 80 (100 being a perfect match).

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Milestone 02/01 Agree optimum experimental designs and levels of replication for experiments with HRI Biometrics department and inform MAFF (DEFRA) Based on previous experimental data using the sterile microcosms (MAFF Report HH1325SMU6), six replicates were needed to detect a difference of 40% of the mean (12 initials per microcosm using peat casing), which was considered to be adequate. A blocking structure with layers in the incubator was found to minimise residual variation. A method for analysing the number of initials produced on casing materials was developed by integrating the number of initials produced with time. Bacterial and primordial populations showed evidence of a mean-variance relationship. These populations were respectively subjected to a logarithmic or square root transformation prior to analysis. All differences in the results section were significant at P < 0.05, or if stated, P < 0.01 or 0.001.

Milestone 02/02 Determine differences inhibitory compounds between casing materials with different adsorption characteristics The following casing materials were used: peat, activated charcoal, anthracite coal, zeolite, coir, vermiculite, bentonite, pumice, perlag, quartz and silica. Sterile and non-sterile casing materials of each were used in microcosms. Mushroom volatiles were determined using Tenax and Carbotrap adsorbents and thermal desorption GC-MS as previously described. Mushroom primordia and bacterial numbers in the casing were recorded at the end of the experiment.

Milestone 02/03 Threshold levels of inhibitory or stimulatory compounds will be determined by the repetitive use of an adsorbent casing. An experiment was conducted whereby a sterile activated charcoal casing was repetitively used in the sterile microcosm culture system. On each of four occasions, the number of mushroom primordia produced was determined and a sample of casing analysed for the presence of volatile compounds using the thermal desorption GC-MS. Volatile compounds produced by the substrates in the microcosms were identified and subtracted by using blank, uninoculated controls.

Milestone 02/04 Determine the effect of adding identified inhibitory compounds on initiation, and the interactions with P. putida isolates The following compounds (10 ml) were placed in 20 ml glass beakers in the microcosms, after 7 and 17 days: 1-octen-3-ol, 2-ethyl hexanol, 3-octanone, trans-3-octene. Beakers with water were used as a control. Casing was prepared from sterile activated charcoal and non-sterile peat and lime. The numbers of primordia and the bacterial population of the casing were recorded in the microcosms at the end of the experiment.

Milestone 03/01 Test range of Pseudomonas putida isolates for ability to stimulate initiation under axenic conditions. The Pseudomonas isolates used in this work were obtained from peat or peat-based casing (Table 1). The taxonomy of these strains was identified using both the enzymatic and isolation plate tests previously described and molecular (DNA-based) techniques. The DNA encoding of the 16 S rRNA gene of the Pseudomonas isolates was amplified by polymerase chain reaction (PCR) and sequenced 14. The 16 S rRNA gene sequences were compared with sequences that have been obtained from related strains in this cluster by both distance and parsimony methods14. The Pseudomonas isolates were grown in nutrient broth. The bacteria were harvested by centrifugation, re-suspended in sterile distilled water to 109 cfu ml-1. Bacterial suspensions (1 ml) were then inoculated on to sterile casing in microcosms. Sterile and non-sterile casing were used as controls. Bacterial numbers in the casing and mushroom primordia were recorded at the end of the experiment as previously described.

Milestone 03/02 Determine differences between Pseudomonas putida isolates with respect to metabolism of inhibitory compounds.

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An experiment was conducted to compare the metabolism of 8-C compounds identified in 02/03 by P. putida isolates and in sterile and non-sterile casing. Isolates were selected that were stimulatory (n12) or non-stimulatory (MAR2) to initiation. A further experiment was conducted to determine the metabolism of the mushroom initiation inhibitor, 1-octen-3-ol, by P. putida strains. Two strains were selected, n12 (promotes initiation) and MAR2 (does not promote initiation). The isolates were grown on minimal media with a range in concentrations of 1-octen-3-ol. Milestone 03/03 Determine the effect of additions of P.putida inocula to the casing on initiation in a large-scale culture system Mushroom cultures were prepared using the large-scale non-axenic culture system previously described (Fig. 2). The following Pseudomonas isolates were added to the casing after 7 days: Control (distilled water), n12, NSC4, T2/6, MAR2. Bacterial suspensions or water (50 ml) containing 109 cfu ml-1 were added to the casing. The numbers of initials were recorded at 3-daily intervals and the number of mushrooms reaching stage 5 of development were harvested and recorded over a 3-week picking period. The experiment was repeated twice.

Milestone 04/01 Devise a method for plasmid profiling of Pseudomonas putida isolates. A method for plasmid profiling of bacteria was developed which was based on the QIAGEN plasmid purification procedure. Bacterial cultures were grown on Luria-Bertani (LB) medium. Plasmid DNA was eluted in a high-salt buffer, and then concentrated and desalted by isopropanol precipitation. Purified plasmids were analysed on an agarose gel.

Milestone 04/02 Plasmid profiling of stimulatory and non-stimulatory P. putida isolates The method from Milestone 04/01 was used for plasmid profiling of the following Pseudomonas isolates: non-stimulatory - PAW340 (no plasmids), PAW8 (plasmids inserted), MAR2, MAR12; stimulatory - n12, NSC4, NSC6, T1/4, T2/6. Strains PAW340 and PAW8 were obtained from the HRI culture collection and known to be P. putida and were genetically modified so that plasmids were removed or known plasmids inserted.

Milestone 05/01 Determine the production of self inhibitory compounds from Agaricus bisporus strains showing different initiation behaviour An experiment was conducted to examine the production of volatile compounds from six A. bisporus isolates with different initiation behaviours. These were 3 commercial strains (Sylvan A15, Le Lion X22, Italspawn F58) and 3 experimental strains (B430, 5776, 8369). The isolates were cultured in sterile and non-sterile microcosms. Volatile compounds were trapped using two different adsorbents (Carbotrap and Tenax) and determined using thermal desorption GC-MS. The following Agaricus bisporus isolates were also tested for initiation behaviour under sterile conditions using peat -based casing: commercial strains Le Lion X22 & X25, Italspawn F56 & F58 and experimental strains ARP174.

Results

Milestone 01/01 Devise a method for chemically analysing self-inhibitory compounds in casing materials. Although some volatiles from mushroom mycelium were detected in the casing using the solvent extraction method for GC-MS analysis, the sensitivity of the method was low compared with adsorbent trapping and thermal desorption method of GC-MS analysis. An example of an extraction from peat casing using the solvent extraction method is shown in Fig. 6. The best material for adsorbing mushroom volatiles in the microcosms was fond to be 'Tenax', although volatiles were also adsorbed in Carbotrap. Silica was found to be a poor adsorbent trap for

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mushroom volatiles. Over 30 compounds were identified in chromatograms of the Tenax traps. However, some of these compounds e.g. naphthalene and benzoic acid were also present in the microcosms without mushroom inoculum (Fig. 7). These compounds were therefore omitted from the analysis. Compounds that were present in the inoculated microcosms were predominantly 8- carbon compounds (Table 4).

Milestone 02/02 Determine differences inhibitory compounds between casing materials with different adsorption characteristics Primordia were produced on sterile activated charcoal, coal and zeolite, but not on peat, coir, vermiculite, bentonite or silica (Fig. 8). Direct analysis of the casing materials with thermal desorption GC-MS was not possible due to the high moisture content. However, the use of Tenax traps showed that the putative initiation inhibitors (8-carbon compounds) could only be detected in the microcosms containing casing in which initiation did not occur (Table 5). There was no putative inhibitor in microcosms containing activated charcoal, coal or zeolite casing materials (in which initiation occurred), indicating that these materials had adsorbed the inhibitor.

Milestone 02/03 Threshold levels of inhibitory or stimulatory compounds will be determined by the repetitive use of an adsorbent casing. Volatile compounds which were in the highest concentrations were 8-C compounds: 2-ethyl hexanol, 3-octanone, 3-octene, 1-octen-3-ol, 1-octanol. The number of primordia declined with each repetitive use of the casing, so that no initials were produced on the casing material used for the fourth time (Table 2). This corresponded to an increase in the concentration of 8-C carbon compounds.

Milestone 02/04 Determine the effect of adding identified inhibitory compounds on initiation, and the interactions with P. putida isolates The numbers of initials produced on sterile activated charcoal and non-sterile peat/lime casing in the presence of different 8-carbon compounds is shown in Fig. 8. The sterile charcoal casing resulted in significantly more primordia than the non-sterile peat casing. The presence of 2-ethyl hexanol completely suppressed the formation of primordia in the microcosms containing peat casing. The compounds did not appear to affect mycelial growth. There was no effect of the compounds in the charcoal casing (Fig. 9 ). The presence of the 8-carbon compounds also affected the bacterial population of the casing. Bacterial and Pseudomonas spp. numbers were higher in the presence of 1-octen-3-ol and 2- ethyl hexanol than in the control jars and in the jars with 3-octanone or trans 3- octene (Fig. 10).

Milestone 03/01 Test range of Pseudomonas putida isolates for ability to stimulate initiation under axenic conditions The results of the enzymatic tests for identifying Pseudomonas species are shown in Table 1 and Figs. 3 and 4. Fluorescence under UV light distinguished the fluorescent Pseudomonads from the other Pseudomonads and E. coli (Table 1). However, the gelatin hydrolysis and arginine dihydrolase tests were not reliable in distinguishing P. putida from the other Pseudomonads. None of the strains were identified as being pathogenic P. tolaasii using the P. reactans white line test. Reliable information on the taxonomy of the Pseudomonads strains used in this work was obtained from the 16 S rRNA gene sequences. The parsimony analysis of 16 S rRNA genes of the Pseudomonas strains used in this project and selected Pseudomonas species is shown in Fig. 12. This showed that the isolates used here fell into three main taxonomic groups: P.

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tolaassii / P. veronii group (MAR2 and MAR12), P. taetrolens / P. poae group (n12, NSC4 and NSC6), and P. putida group (A4lux, T2/6 and T1/4). Strains of Pseudomonas putida and closely related Pseudomonas species were found to differ in terms of stimulating mushroom initiation, with some strains stimulating few or no primordia. None of the strains added to sterile casing stimulated as many primordia as non-sterile casing (Fig.11). Significant differences in stimulatory behaviour were found between Pseudomoinas isolates, with isolates n12 and NSC4 (P. taetrolens group) stimulating the production of more primordia than other isolates (Fig. 11). P. putida PAW340 (plasmids removed) and Pseudomonas isolate MAR2 did not stimulate a significant production of primordia in Agaricus bisporus (Fig. 11). Bacterial numbers at the end of the experiment were similar in all the treatments (Fig. 14). None of the Pseudomonas strains resulted in cap spotting associated with bacterial blotch caused by P. tolaasii or P. gingerii13.

Milestone 03/02 Determine differences between Pseudomonas putida isolates with respect to metabolism of inhibitory compounds The concentration of 8-C compounds was lowest in non-sterile casing, which also produced the most primordia (Table 4). The isolate n12 metabolised slightly more 8-C compounds than the non-stimulatory isolate Mar2 (Table 3). Levels of 8-C compounds were highest in sterile casing, which produced no primordia. No differences in growth between the P. putida strains or between the 1-octen-3-ol concentrations were observed. This showed that the mushroom initiation inhibitor does not provide a nutritional carbon source for P. putida, although it may be degraded into non-inhibitory products.

Milestone 03/03 Determine the effect of additions of P.putida inocula to the casing on initiation in a large-scale culture system The number of primordia and mushrooms following inoculation of the casing with different bacterial strains is shown in Table 6. Pseudomonas strain n12 produced significantly fewer small and total initials than the control (uninoculated treatment). Pseudomonas strain NSC4 produced significantly more initials and mushrooms than the control treatment. Total weight of mushrooms from the NSC4 treatment (2.52 kg/tray) was also slightly higher than that from the control treatment (2.36 kg/tray).

Milestone 04/01 Devise a method for plasmid profiling of Pseudomonas putida isolates. Figure 13 shows the DNA profiles from several bacterial strains. These strains included E. coli and P. putida isolates with known plasmid profiles, and known mushroom initiation stimulators. The results show the method is capable of analysing and distinguishing plasmid profiles of P. putida isolates.

Milestone 04/02 Plasmid profiling of at least six stimulatory and six non-stimulatory P. putida isolates Plasmid isolation from control Pseudomonas putida strains (PAW340+ and PAW8-) enabled plasmid containing Pseudomonas strains to be easily detected. Plasmids were detected in strains that did and did not readily initiate Agaricus bisporus (eg. MAR2) (Fig. 13). Some initiating Pseudomonas putida strains had plasmids of a common size, but not all strains. The initiating ability of Pseudomonas strains does not appear to be correlated with a common plasmid.

Milestone 05/01 Determine the production of self inhibitory compounds from Agaricus bisporus strains showing different initiation behaviour

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The volatile compounds which were in the highest concentrations were 8-C compounds: 2-ethyl hexanol, 3-octanone, octane, 3-octene, 1-octen-3-ol, 1-octanol, 3,7-dimethyl-1,6-octadienol (Table 4). All the strains produced 2-ethyl hexanol in similar quantities. There was a negative relationship between the concentration of the other 8-C compounds and the number of primordia produced by the different A. bisporus isolates under non-sterile conditions (Fig. 15). No primordia were produced in the sterile microcosms, but the concentrations of trapped 8- Carbon compounds were higher than in the non-sterile microcosms. The volatile concentrations were also higher from the sparse fruiting strain 5776 than from the normal hybrid strain. Primordia formation in Agaricus bisporus strains A15 and 5776 in non-sterile casing is shown in Fig. 16. None of the A. bisporus strains tested produced primordia under sterile conditions, although they all produced primordia and sporophores under normal, non-sterile culture (Fig. 17).

Discussion and Conclusions

This work has confirmed earlier research 5,14 that the volatiles produced by mushroom mycelium are predominantly 8-carbon compounds. The results have shown that one of these compounds, 2-ethyl hexanol, can inhibit the production of primordia by mushroom mycelium on peat-based casing. Adsorption of 8-carbon compounds by certain casing materials (activated charcoal, coal, zeolite) enabled primordia formation. Repetitive use of an adsorbent casing material (charcoal) resulted in an increase in concentration of 8-carbon compound to the point where initiation was no longer able to occur. 2-ethyl hexanol did not inhibit primordia formation on charcoal casing. This was probably due to the greater adsorption capacity of charcoal compared with peat. The presence of 2-ethyl hexanol and the other 8-carbon compounds did not appear to affect the mycelial growth of the mushroom in the casing. Wood5 noted that 1-octen-3-ol inhibited fruitbody formation but not mycelial growth. Here, no effect of 1-octen-3-ol was observed. The presence of 2-ethyl hexanol resulted in a higher Pseudomonas spp. population in the casing. These bacteria may therefore be able to metabolise this inhibitor. Analysis of 16S rRNA genes of Pseudomonas strains showed that strains in the P. taetrolens / P. poae group stimulated more proimordia than the P. tolaasii / P. veronii group. Strains in the P. putida group were intermediate in stimulatory behaviour. Previous work has identified P. putida as being stimulatory to mushroom initiation 3,10,15. However, work here has shown that fluorescence and enzymatic tests are not able to distinguish P. putida from closely related species. The initiating ability of Pseudomonas strains does not appear to be correlated with a common plasmid. None of the Pseudomonas strains were able stimulate primordia formation in axenic casing to the same extent as a naturally occurring microbiota in non-sterile casing. This confirms earlier work by Eger 16. Inoculation of non-sterile casing with Pseudomonas strains resulted in significant changes in the numbers of primordia and subsequent mushroom sporophores compared with uninoculated casing. The technique of adding Pseudomonas strains to 'normal' non-sterile casing was not been previously investigated. The results of this work support the original hypothesis that mushroom initiation is controlled by the production of self-inhibitory compounds by the Agaricus mycelium, and their subsequent removal or metabolism in the casing. Although 1-octen-3-ol was not metabolised by Pseudomonas spp., it is possible that 2-ethyl hexanol could be metabolised. This mode of action for stimulatory behaviour of Pseudomonas strains should be further investigated.

Future Work

Technology Transfer The effects of the following factors on mushroom initiation and subsequent mushroom populations should be investigated further in large-scale experiments:

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• application of 8-carbon compounds (2-ethyl hexanol) in the growing room, as a possible method of controlling the amount and timing of initiation • application of Pseudomonas strains to casing to control the amount of primordia and developing sporophores • use of adsorbent materials in the casing. If successful, the above technologies should then be investigated in commercial scale tests.

Science Objectives The genetic mechanism of initiation of Agaricus bisporus should be further examined and manipulated: • Conduct suppression Subtractive Hybridisation (SSH) to determine differentially expressed genes between vegetative mycelium, undifferentiated aggregates, differentiated primordia and mushroom pins (early stage sporophores). • Produce micro-arrays and their use in determining gene expression profiles during initiation under standard cropping conditions and comparative gene expression in response to different initiation stimuli. • Identify, sequence and quantify expression analysis of key initiation genes. • Generate transgenic strains with modified expression of key genes. • Culture transgenic strains in microcosm, aerated flask and non-sterile tray systems and determine the effects of gene manipulation on initiation behaviour, sporophore development, gene expression and responses to agronomic variables. The metabolism of 2-ethyl hexanol by Pseudomonas species as possible mechanism for the stimulatory behaviour should be examined.

References

1. Noble R, Reed JN, Miles S, Jackson AF & Butler J (1997) Influence of mushroom strains and population density on the performance of a robotic harvester. J. Agric. Eng. Res. 68: 215 - 222. 2. Flegg P.B. & Wood D.A. (1985) Growth and fruiting. In: The Biology and Technology of the Cultivated Mushroom. (eds P.B. Flegg, D.M. Spencer & D.A. Wood) pp 141-178. John Wiley & Son, Chichester, UK. 3. Fermor T.R., Lincoln S., Noble R., Dobrovin-Pennington A., (2000) Microbiological properties of casing. Science and Cultivation of Edible Fungi (ed. LJLD van Griensven), Balkema, Rotterdam, Netherlands, 447-454. 4. Long P.E., & Jacobs L. (1974) Aseptic fruiting of the cultivated mushroom Agaricus bisporus. Tans. Br. Myc. Soc. 63: 99-107. 5. Wood DA (1982) Sporophore initiation in axenic culture. Rep. Glasshouse Crops Res. Inst. 1981, 140. 6. MAFF Project Final Report HH1325SMU 2001. Control of mushroom initiation by manipulation of the casing microflora and microenvironment. 7. Noble R and Bareham L . 2002. Casing material and its use in crop cultivation. Patent: PCT/GB02/01172. 2003. Horticulture Research International, Warwick, UK. 8. MAFF Project Report HH1312SMU (1998) Physiological influences of the properties of the casing layer on the growth, productivity and quality of the mushrooms. 9. Elliott TJ & Wood DA (1978) A developmental variant of Agaricus bisporus. Trans. Br Myc. Soc. 70: 373-381.

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10. Rainey PB, Cole ALJ, Fermor TR, Wood DA (1990) A model system for examining involvement of bacteria in basidiome initiation of Agaricus bisporus. Mycological Research 94: 191-194. 11. Noble R, Dobrovin-Pennington A, Evered CE, Mead A (1999) Properties of peat-based casing soils and their influence on the water relations and growth of the mushroom (Agaricus bisporus) Plant and Soil 207:1-13. 12. Hammond JBW, Nichols, R (1976) Carbohydrate metabolism in Agaricus bisporus (Lange) Sing.:changes in soluble carbohydrates during growth of mycelium and sporophore. J. Gen. Microbiol. 93:309-320. 13. Rainey PB, Brodey C, Johnstone K (1992) Biology of Pseudomonas tolaasii, cause of brown blotch disease of the cultivated mushroom. Advances in Plant Pathology 8: 95-117. 14. Grove JF (1981) Volatile compounds from the mycelium of the mushroom Agaricus bisporus. Phytochemistry 20: 2021-2022. 15. Hayes, WA, Randle PE, Last FT (1969) The nature of the microbial stimulus affecting sporophore formation in Agaricus bisporus (Lange) Sing. Annals of Applied Biology 64: 177- 187. 16. Eger G (1972) Experiments and comments on the action of bacteria on sporophore initiation in Agaricus bisporus. Mushroom Science VIII. 719-725.

Publications and other outputs

Publications R. Noble, T.R. Fermor, S. Lincoln, A. Dobrovin-Pennington, A. Mead, R. Li, C. Evered & T.J. Elliott (2003) Primordia initiation of mushroom (Agaricus bisporus) strains on axenic casing materials. Mycologia 95: 620-629. R. Noble & A. Dobrovin-Pennington (2001) Solving the initial problem (2001). HDC News 78: 21- 22. R. Noble & A. Dobrovin-Pennington (2002) Solving the initial problem. Mushroom Journal 629: 23-25. R. Noble (2002) Controlling mushroom initiation. HRI Annual Report for 2001/2002. p15. R Noble, A Dobrovin-Pennington (2004) . Substitution of peat in mushroom casing using fine particle tailings. Scientia Horticulturae (In Press)

Posters Noble, R., Dobrovin-Pennington, A. and Hobbs, P.J. Mushroom initiation. Mushroom Growers' Association Conference, Chester, November 2001. Noble, R., Dobrovin-Pennington, A. and Hobbs, P.J. Mushroom initiation. Mushroom Growers' Association Conference, Cambridge, September 2002. Noble, R., Dobrovin-Pennington, A. and Hobbs, P.J. Physiological and genetic mechanisms of initiation. XVI International Society for Mushroom Science Congress, Miami, USA, March 2004. Hobbs, P.J., R.Noble, R., Dobrovin-Pennington A., and Williams, J. Factors responsible for suppressing fruiting body formation in edible mushrooms. International Mass Spectrometry Conference, Edinburgh, 1st-5th Sept 2003.

Presentations R. Noble. Mushroom casing research. HRI Mushroom Subject Day, Wellesbourne, June 2002. A. Dobrovin-Pennington. Mushroom initiation. HRI Mushroom Subject Day, Wellesbourne, June 2003. R. Noble. Research at HRI on mushroom initiation. Pennsylvania State University, USA, January 2003.

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Table 1. Enzymatic diagnostic tests for distinguishing Pseudomonas species and strains Diagnostic test Bacterial species Fluorescent pigment Gelatin hydrolysis Arginine Dihydrolase E. coli - - - Pseudomonas gladioli + + + P. aeruginosa - - - P. syringae - - - P. putida PAW340 + + + n12 + + + NSC4 + + + NSC6 + - - MAR2 + - + MAR12 + - +

Table 2. Effect of repetitive use of sterile activated charcoal casing on mushroom initiation and concentration of 8-C compounds in the casing. Number of repetitions 1 2 3 4 LSD (P<0.05) Number of primordia per microcosm 42 14 4 0 5 Concentration of 8-C compounds in casing, ug/g casing 2.58 3.92 4.29 4.80 0.43

Table 3. Effect of Pseudomonas putida strains on the metabolism of 8-C compounds and primordia produced by the mushroom Agaricus bisporus. Casing treatment or P.putida strain sterile non- n12 Mar2 LSD (P<0.05) sterile Number of primordia per microcosm 0 51 8 0 6 Concentration of 8-C compounds in adsorbent traps, ug/g casing 1.32 0.16 0.73 0.98 0.21

Table 4. Analysis of mushroom volatiles and relationship with initiation sterility sterile non- sterile non-sterile sterile mushroom strain A15 A15 5776 5776

primordia / jar 0 49.4 0 4.0

Compound, µg g-1 3-octanone 23.21 0.08 32.91 8.71 1-octanol 0.03 0.05 0.05 0.03 1-octen-3-ol 8.38 0.16 8.93 3.65 octane 0.87 0.02 0.99 0.40 1,6-octadien-3-ol 7.21 0.06 6.24 0.30 1-bromo-octane 3.29 0.89 6.22 1.72 1-bromo-heptane 1.71 3.58 10.53 4.54 2-ethyl-1-hexanol 106.5 27.3 31.3 20.7

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Table 5. Analysis of mushroom volatiles and numbers of primordia produced in microcosms with different sterile casing materials, n.d. not detected

Material peat charcoal zeolite coal

primordia / jar 0 10 4 9

Compound, µg g-1 3-octanone 23.21 n.d. n.d. n.d. 1-octanol 0.03 n.d. n.d. n.d. 1-octen-3-ol 8.38 n.d. n.d. n.d. octane 0.87 n.d. n.d. n.d. 1,6-octadien-3-ol 7.21 n.d. n.d. n.d. 1-bromo-octane 3.29 n.d. n.d. n.d. 1-bromo-heptane 1.71 n.d. n.d. n.d. 2-ethyl-1-hexanol 106.5 n.d. n.d. n.d.

Table 6. Effect of bacterial strain on initiation and mushroom production on non-sterile casing. Mean of 2 experiments.

Bacterial Number of initials Mushrooms strain < 3 mm 4 mm dead total Stage 3 control 925 91 128 1144 191 n12 859 98 112 1068 187 NSC4 985 103 130 1219 211 T2/6 932 93 110 1134 190 MAR2 934 82 142 1158 207 LSD 87 29 31 66 15 (P<0.05)

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Fig.1 Kilner Jar microcosm for sterile culture experiments

Fig. 2 Non-sterile culture in plastic trays

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Fig. 3. Arginine dihydrolase test for identifying Pseudomonas species

Fig. 4 Gelatin hydrolysis test for identifying Pseudomonas species

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Fig. 5. Soxhlet apparatus for the extraction of solids

Fig. 6 GC-MS mass spectra for diethyl ether extracts from peat-based casing with and without mushroom mycelium

1-octanol

peak for octanol, as well as several other peaks, only observed with mushroom inoculum in upper spectra, lower spectra substrates only

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Fig.7 Thermal desorption GC-MS mass spectra of Tenax adsorbent traps from microcosms with non-sterile casing (upper) and sterile casing (lower)

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Fig. 8. Numbers of primordia produced in different sterile and non-sterile casing materials e 7 Sterile 6 LSD 5 P <0.05 Non-sterile 4

root) 3 2 1 0

Primordia per microcosm (squar Charc. Coal Coir Peat Silica Vermic. Zeolite (act.) Casing material

Fig.9. Effect of additon of 8 carbon compounds on initiation

35 NS peat 30 LSD S charcoal 25 P=0.05 20 15 10 5

Number of initials > 1 mm 0 Control 1-octene-3-ol 2 ethyl 3-octanone trans 3-octene hexanol Treatment

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Fig.10. Effect of additon of 8 carbon compounds on bacterial numbers

10 9 8

7 7 LSD 6 P= 0.05 5 4

cfu/g x 10 cfu/g x 3 2

Total bacteria in casing, Total bacteria 1 0 Non-sterile TT 1-octene-3-ol 2 ethyl hexanol 3-octanone trans 3-octene casing Treatments

Fig. 11 Pseudomonas putida isolates and mushroom initiation

10

8

6 LSD P=0.05 4

2 Primordia (sq root) 0

/6 4 6 8 r2 rile lux 2 C C r12 n12 aw te T1/4 T Ma sterile -s 4A NS NS aw340 P Ma P non Treatment

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Fig. 12. 16 S rRNA sequence analysis of Pseudomonas isolates used in the experiments

P. tolaasii-like

P. tolaasii af184918 P. tolaasii P.tolaasii af184918 P. tolaasii D86002 MAR2 MAR12 P. amygdali af105388 P. tolaasii P. amygdali ab021402 P. taetrolens-like P. amygdali P. amygdali P. amygdali P. amygdali NSC4 NSC6 P. taetrolens n12 P. agarici P. agarici P. taetrolens P. taetrolens P. chlraphis P. chlraphis P. putida-like P. chlraphis A4lux T2/6 T1/4 P. putida D85994 P. putida D85992 P. putida mt2 D83788 P. putida F1 P. aeruginosa P. aeruginosa P. aeruginosa P. mendocina P. olevorans P. mendocina P. stutzeri P. mendocina P. azotoformans P. azotoformans P. agarici D85995 P. syringae P. viridflavans

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Fig. 13. Plasmid profiles of Pseudomonas isolates used in the experiments. (explanatory footnote to figure) λ H, λ DNA cut with Hind III Paw +, Pseudomonas putida Paw 8 (pkt230) Paw -, Pseudomonas putida Paw 340 Plasmid isolation from control Pseudomonas putida strains (Paw+ and Paw-) and plasmid containing Pseudamonas strains n12, NSC4, NSC6 4Alux, Mar2 and Mar12 obtained from mushroom casing.

Fig. 14. Populations of different Pseudomonas putida isolates

10 9

-1 8 7 6 5 4 bacteria cfu g 3 10

log 2 1 0

e /4 6 ux 1 C6 40 teril n12 T T2/ S -s Sterile 4Al NSC4 N Paw8 Mar-2 Paw3 Mar-12 Non Treatment of P. putida isolate

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Fig.15. Relationship between the concentration of 8-C compounds and the number of primordia produced by different Agaricus bisporus isolates. Each point is the mean of 3 microcosms.

80 70 60 r = 0.88 50 40 30 20

primordia per microcosm 10 0 1 1.2 1.4 1.6 1.8 2 2.2 2.4 8-C compound in adsorbent traps, ug/g adsorbent

Fig. 16. Microcosms with sterile and non-sterile peat-based casing with Agaricus bisporus strains A15 and 5776 showing initials in non-sterile casing

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Fig. 17. Wild Agaricus bisporus ARP116 growing with non-sterile peat based casing

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