Mycologia, 101(5), 2009, pp. 583–591. DOI: 10.3852/07-194 # 2009 by The Mycological Society of America, Lawrence, KS 66044-8897 Issued 26 August 2009 Volatile C8 compounds and pseudomonads influence primordium formation of Agaricus bisporus Ralph Noble1 Andreja Dobrovin-Pennington Warwick HRI, University of Warwick, Wellesbourne, Warwick, CV35 9EF, United Kingdom INTRODUCTION Philip J. Hobbs Institute of Grassland and Environmental Research, Okehampton, Devon, EX20 2SB, United Kingdom Jemma Pederby Alison Rodger Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom Abstract: Primordium formation of Agaricus bisporus depends on the presence of a casing layer containing stimulatory bacteria and on sufficient air exchange. The influence of specific pseudomonad populations and volatile organic compounds (VOC) on primordium formation of A. bisporus was studied in microcosm cultures. VOC produced by A. bisporus mycelium were predominantly C8 compounds, some of which could inhibit primordium formation, with 1octen-3-ol being most inhibitory. A VOC produced by the rye grain substrate, 2-ethyl-1-hexanol, on which A. bisporus was grown also inhibited primordium formation. 2-Ethyl-1-hexanol and 1-octen-3-ol were metabolized by pseudomonad populations and adsorbed by activated charcoal, with both modes of removal enabling primordium formation in the casing. Removal of VOC by ventilation also enabled primordium formation to occur under axenic conditions. The presence of 2-ethyl-1-hexanol and 1-octen-3-ol in the microcosms resulted in higher total bacterial and pseudomonad populations in the casing. The stimulatory effects of the casing and its microbiota and air exchange on primordium formation of A. bisporus at least partly are due to the removal of inhibitory C8 compounds produced by the mycelium and its substrate. Monitoring and controlling the levels of these inhibitory VOC in mushroom culture should enable primordium formation of A. bisporus to be more efficiently and precisely controlled. Key words: casing, mushroom, 1-octen-3-ol, Pseudomonas putida, 2-ethyl-1-hexanol Accepted for publication 7 April 2009. 1 Corresponding author. E-mail:
[email protected] Fructification in the button mushroom (Agaricus bisporus) begins with the aggregation of mycelium into thick strands, followed by the formation of primordia, some of which develop further into sporophores. This is a highly inefficient process with generally fewer than 7% initiated primordia developing further into sporophores (Flegg 1979, Noble et al 2003). Primordium formation is stimulated by a reduction in temperature and an increase in ventilation, which is considered to have an effect through reduced CO2 concentration (Tschierpe and Sinden 1964, Flegg and Wood 1985). A. bisporus also requires a casing layer (casing), which has imprecisely defined chemical and microbiological properties that stimulate primordium formation (Wood 1976, Flegg 1979). The presence of stimulatory bacteria in the casing, of which Pseudomonas putida is regarded the most significant (Hayes et al 1969, Rainey et al 1990), is usually necessary for primordium formation to occur. Activated charcoal and other adsorbent materials can obviate the requirement for stimulatory pseudomonads (Eger 1961, Long and Jacobs 1974, Noble et al 2003), possibly by adsorbing compounds that inhibit primordium formation. Attempts to isolate putative inhibitors to primordium formation from activated charcoal casing have been confounded by the large number of compounds adsorbed and identified (Grove 1981). However several volatile C8 compounds were identified that were characteristic of A. bisporus and of sufficiently low molecular weight to be readily adsorbed by activated charcoal. Of these 1octen-3-ol was found to inhibit primordium formation in vitro on malt agar without inhibiting mycelial growth (Wood and Blight 1982). Because the function of the casing and mechanism of primordium formation are not fully understood fructification in button mushroom culture has relied on the empirical selection of casing materials (Noble et al 2003, Beyer 2004) and by reducing temperature and CO2 concentration at an appropriate stage (Flegg and Wood 1985). Attempts to reduce the stimulation of excess numbers of primordia and improve the efficiency of fructification, by manipulation of temperature or ventilation, or selection of less conducive casing materials, have resulted in excessive vegetative mycelial growth in the casing (‘‘stroma’’) and/or reduced sporophore production (Flegg 1979, Flegg and Wood 1985). 583 resuspended in sterile distilled water to 109 cfu mL21. Loughborough. The taxonomy of these isolates was determined by comparison of the 16S rRNA gene sequences with sequences that have been obtained from related Pseudomonas isolates in the EMBL database with the program FASTA (Genetics Computer Group. P. poae (n12. The bacteria were harvested by centrifugation.).0 98. UK) and pseudomonas isolation agar (PIA) (Difco Laboratories.584 MYCOLOGIA TABLE I. consisting of a mixture of black peat and CaCO3 (4 : 1 v/v) or activated charcoal (granular.5% v/v followed by 0. P. This substrate was covered with 60 g (about 17 mm layer) of casing.08–0. bisporus mycelium in the casing and in the headspace of axenic and nonaxenic microcosms.25 mm. bisporus strain A15 (Sylvan Spawn.— Seven isolates of P. The experiment was repeated four times. Paw8 and Paw340) were obtained from the Warwick HRI culture collection. UK). Bacterial suspensions (1 mL) were inoculated onto axenic peat + CaCO3 casing in A.— Two methods involving gas chromatography-mass spectrometry (GC-MS) were used to analyze the VOC produced by A. veronii (MAR2 and MAR12). The resulting samples (1 mL) were injected onto a GC column (SPB1. Casing (5 g) was put in the porous thimble of a Soxhalet apparatus and VOC were extracted in 45 mL of diethyl ether for 1 h. CO2 concentrations (. bisporus microcosm cultures.e. putida and related Pseudomonas species defined by 16S rRNA sequences (Elomari et al 1996. University of Wisconsin) (TABLE I). Temperatures (25 C for 7 d followed by 16 C). 30 3 0.6 This work aimed to improve the understanding of the mechanism of primordium formation of A. P. MATERIALS AND METHODS Axenic microcosm tests. P. P.25 mm thick film.5 100. Darmstadt. Microcosms were arranged in an incubator in a randomized block design with each incubator shelf containing a complete replicate block of each set of treatments.0 97. poae PsI PsI putida putida putida putida putida veronii veronii DSM14936T AF105387 AF105387 D85994 D85994 D85994 KT2440 KT2440 CFML 92-134 CFML 92-104 Sequence length 1529 1407 1407 1259 1259 1259 302614 302614 1430 1430 Similarity percent 100. There were three replicate microcosms of each Pseudomonas isolate and axenic or nonaxenic culture treatment. except where stated. Bacterial isolates and populations in casing materials. 1 mm diam) were recorded 28 d after the microcosms were filled (i. P. Similarity of the 16S rRNA gene sequence of Pseudomonas isolates to the best matches in the EMBL database with the program FASTA Pseudomonas isolate n12 NSC4 NSC6 4Alux T1/4 T2/6 Paw8 Paw340 MAR2 MAR12 Species name with greatest homology and EMBL accession No.0 99. Pennsylvania) of a Shimadzu QP 5000 GC-MS system (Shimadzu UK. and an unnamed species. Poole. Axenic and nonaxenic casing were used as controls. P. UK). Bacterial numbers in the casing were recorded before and after mushroom culture as described below. Sigma Aldrich. (ii) measuring the effect of the casing microbiota on the concentration of specific VOC produced by mushroom mycelium and its substrate and (iii) determining the effect of these VOC on primordium formation and on the populations of bacteria in the casing. colonized with the A. Germany).12% v/v) and casing matric potential (21 to 22 kPa) in the microcosms were maintained as described in Noble et al (2003). P. Three further isolates of P.0 98. P. UK). Oxoid. Michigan) and incubated at 25 C for 48 h to determine the total bacterial populations as colony-forming units per g casing (cfu g21) and to estimate the proportion that were pseudomonads. Casing material was autoclaved at 121 C for 2 h for axenic treatments. P. Dorset.7 99. P. The flow of the . A solvent extraction method involved washing of casing in diethyl ether followed by low temperature analysis of the solvent.6 97. 12–16 d after the first primordia had reached this size). Serial dilutions of the suspension were plated on nutrient agar (CM309. Anzai et al 2000). P. The Pseudomonas isolates were grown in nutrient broth (LB broth [Miller] Merck. Bellefonte. Analysis of volatile organic compounds in microcosms. Milton Keynes). Bacterial populations in the casing in microcosms were determined by preparing suspensions of 1 g casing in 9 mL sterile Ringers solution (Fisher Scientific.6 97. which served as a nutritional substrate. The numbers of primordia (. 0. 0. Basingstoke. 4–8. bisporus in vivo by (i) determining the stimulatory effect of isolates of P.— A 500 mL glass microcosm culture system (Noble et al 2003) was used to examine growth in axenic casing materials. putida or closely related species were obtained from casing materials and other growing media (Fermor et al 2000): P. The microcosms were filled with a 10 mm layer (30 g) of sterile rye grain spawn. putida (T2/6 and T1/4). P. Peterborough. PsI (NSC4 and NSC6). putida (4Alux. Supelco.7 96. Detroit. 34 mm film with a 1 m. due to higher resolution than the solvent extraction method. Samples of adsorbent or casing were analyzed respectively with the solvent extraction and thermal desorption methods before use for A. at the time of transfer from 25 C to 16 C. bisporus inoculum.2 mm and a 0.2 s to give responses in the ng range. 2-Ethyl-1-hexanol also was used at 1 mL per beaker. 2-ethyl-1hexanol. bisporus inoculum were prepared to test for background VOC in the culture. Effect of C8 compounds on primordium formation and casing bacteria. deactivated fused silica guard column (internal diameter 0.05 or if stated P . Hammond and Nichols 1976). was 0. The jars were incubated under sterile conditions 14 d at 25 C. helium. The concentrated VOC were desorbed thermally from 220 mg samples of the adsorbents into a GC-MS system for identification and quantification. Eight replicate microcosms PSEUDOMONADS 585 were prepared for each A. RESULTS Effect of Pseudomonas isolates on primordium formation. octane. After autoclaving (axenic jars only) the substrate was inoculated at 2% w/w with rye grain spawn of the A.75 mL min21.NOBLE ET AL: VOLATILES. The flow of the eluting gas. Chromatographic retention time and mass spectral matching were used to confirm VOC identity. These VOC (10 mL) were placed in 20 mL glass beakers in microcosms. bisporus strains. total bacteria and pseudomonads in the casing were recorded 20 d after the casing was applied as previously described. Gaithersburg. UK) with five jars per flask. Microcosms without A. bisporus strains A15 and 5776 (Le Lion. GC oven conditions were an initial temperature of 40 C. Sterile water to a depth of 30 mm in the base of the flasks maintained a relative humidity . Crucibles containing the Carbotrap or Tenax adsorbents were placed in each microcosm after 7 d. An optic temperature programmable injector (Ai Cambridge.11% v/v. Primordium formation and production of VOC by A. The numbers of primordia. helium. a thermal desorption method was used to analyze VOC in the headspace of microcosms. Pampisford. Varrains. Axenic and nonaxenic cultures aerated without VOC were used as controls. These concentrations were subjected to a logarithmic transformation before analysis. The level of VOC was maintained by replenishment at 4–5 d intervals and by placing the 300 mL flasks in ice to reduce volatilization. eluting gas. octane. Bacterial and primordial populations showed evidence of a mean-variance relationship. VOC levels showed evidence of a mean-variance relationship.001. Maryland). 3-octanone. VOC detected by the mass spectrometers were identified with a probability-based matching algorithm and a NIST mass spectral library (National Institute of Standards and Technology. Although unsuitable for analysis of high moisture content casing.—Pseudomonas isolates added to axenic casing . Two replicate multi-adapter flasks aerated with each VOC and four replicate axenic and nonaxenic controls were prepared. A 25 m fused silica (cross-linked methyl siloxane) hp-1 column with an internal diameter 0. 1-octanol. VOC from the microcosm headspace first were first preconcentrated onto adsorbents (Carbotrap 20/40 mesh and Tenax TA 60/ 80. The colonized substrate was covered with 80 g of the above axenic or nonaxenic peat + CaCO3 casing and the jars placed in 10 L Quickfit multi-adapter flasks (Fisher Scientific. at the same time of recording numbers of primordia and casing bacteria. An electronic pressure controller was used to offset peak pressure broadening with increasing GC column temperature. which was moistened with 10 mL sterile water on each jar. 3-octanone. The temperature ramp of the GC oven was set to rate: temperature (C): time (s) (0 : 50 : 5. The flasks were transferred to a room at 18 C and connected to a humidified. Q plot. increased to 220 C at 15 C min21 and remaining at 220 C for 1 min. The mass spectrometer scanned 35–250 mass units every 0. sporophores (developmental stage 4. Compounds were declared unknown if their matching probability was less than 80 (100 being a perfect match).01 or 0. The flasks were sealed with bacterial air vents and incubated at 25 C about 8 d until mycelium became visible at the surface of the casing. Effect of VOC on primordium formation and casing bacteria in ventilated flasks. The adsorbents were analyzed by the thermal desorption method after a further 21 d. 1-octen-3-ol or cis-3-octen-1-ol. France) were selected on the basis of known difference in primordium formation in culture. Microcosms were prepared with axenic activated charcoal and nonaxenic peat + CaCO3 casing as previously described. The GC-MS interface was 280 C.3 g) contained within 6 mL porcelain crucibles placed on the casing surface.— The effect of introducing specific VOC identified from the above experiment into the atmosphere of microcosms was examined. A Hewlett Packard (HP. UK) GC-MS system consisting of a 5890 II series gas chromatograph and a 5972A mass selective detector (MSD II) was used for analysis. bisporus strain Sylvan A15.7–0. Supelco. giving a total of 96 microcosms. Cambridge. All differences in RESULTS were significant at P . giving a total of 48 microcosms. containing a porous polymer of divinylbenzene (Supelco) was used. The number of primordia and sporophores was recorded after a further 10 d. The A. bisporus culture to test for background VOC. filtered air flow of 15 L h21 to maintain a CO2 concentration of 0. Loughborough. These populations were subjected respectively to a logarithmic or square root transformation before analysis. Cheshire. trans3-octene and 1-octen-3-ol. after 7 and 17 d: 2-ethyl-1-hexanol.1 mL min21. Beakers containing water were used as control. 0. 90%.53 mm). UK) was used to desorb headspace samples from the adsorbents and initially was set to 30 C and heated at 16 C s21 to 250 C. Three replicate microcosms of each VOC and casing material treatment were prepared. was 1. 0.— Composted substrate (Noble et al 1998) (100 g) was filled into 350 mL glass jars. Stockport.— Microcosms were prepared with axenic and nonaxenic peat + CaCO3 casing as previously described. casing sterility and adsorbent treatments. 0. Samples of the casing materials were analyzed by the solvent extraction method. 20 : 150 : 0 and 4 : 250 :5 ). The supply of VOC to flasks was removed and the flasks aerated with fresh air only. The air flow to multi-adapter flasks was passed through sealed 300 mL flasks containing 3–5 mL ethanol. 001). A number of compounds with molecular weights exceeding 150 were detected in trace amounts (e.1 3 106 to 6. None of the Pseudomonas isolates added to axenic casing stimulated the formation of as many primordia as nonaxenic casing (FIG. 0. However the final population of pseudomonads in nonaxenic casing was significantly (P .2 3 104 cfu g21 without A. Hammond and Nichols 1976) in some of the microcosms. The total bacterial population of the casing declined from 1. 0. bisporus culture there were slight increases in the populations of pseudomonads. In the presence of A.01).5 3 105 to 8. 0. with the A. with 2-ethyl-1-hexanol. 3.0 3 105 to 7.6octadien-3-ol) (these are excluded from TABLES II and III).001). Final populations of total bacteria and pseudomonads in the casing of nonaxenic microcosms were unaffected by the A.001) smaller than the final populations in the axenic casing inoculated with Pseudomonas isolates. whereas P. The exception was 1-bromo-heptane. Numbers of primordia .001). veronii isolates MAR2 and MAR12 stimulated the formation of the fewest primordia although they had the highest pseudomonad populations in the casing at the end of the experiment (3. bisporus strain and adsorbent treatments.4 3 108–2.g. 1. 121 df). benzyl alcohol and benzone compounds being most abundant (TABLE III). 1Bromo-octane was detected only in microcosms inoculated with A.586 MYCOLOGIA FIG.5 3 105 cfu g21 in the presence and absence of A. bisporus inoculum.— Significant formation of primordia occurred only in nonaxenic microcosms.9 3 107 cfu g21 to 6. In nonaxenic casing during A. and in total bacteria. Isolates n12 and NSC4 stimulated the formation of more primordia than other isolates. 1). 0. which was greater in the microcosms containing A. which was most abundant in the axenic microcosms (P . bisporus metabolites of 1bromo-decane. By the time of primordia assessment one or two primordia had developed further into immature sporophores (stages 1 and 2. from 5. These halogenated VOC might be A. %) and on axenic casing inoculated with different Pseudomonas isolates (&). No VOC were detected in the adsorbents before use. Length of vertical bar indicates least significant difference between treatments (P 5 0. from 6. Several VOC were detected in both microcosms with and without A. 1) (P .7-dimethyl-1. No primordia formed on uninoculated sterile casing. bisporus inoculum respectively.7 3 108 cfu g21 respectively compared with 1.6 3 108 cfu g21 for other isolates). All VOC concentrations were highest in the axenic. VOC that were . Noble et al 2003). bisporus (P . Primordium formation and production of VOC by A. 1 mm diam (square root transformed values) that formed on nonaxenic peat + CaCO3 casing (control.5 3 108 and 3. 0. bisporus strain A15 forming more primordia than strain 5776 (TABLE II) (P . with concentrations of most VOC significantly higher in the Tenax than in the Carbotrap.0 3 106 cfu g21.6 3 105 cfu g21. bisporus inoculum. were found to differ in terms of growth in the casing and in stimulating primordium initiation (FIG.8 3 106 and 2. uninoculated microcosms and were reduced by the presence of the microbiota in the nonaxenic microcosms and the A.1 3 105 cfu g21 but declined to 1.05. bisporus strains. However the formation and development of primordia and sporophores was slower in the microcosms than in the larger scale flask culture due to the lower temperature (16 C compared with 18 C) (Flegg 1979. bisporus inoculum the pseudomonad population of nonaxenic casing increased from 7. bisporus (TABLE II). More than 30 VOC were identified in the chromatograms of the adsorbent traps from microcosms. bisporus inoculum. 08 5.10 22.53) (142.61 (0.42 0.00) 21.60) 1.76) (4.74) (45. the concentrations were higher in the axenic microcosms than in the nonaxenic microcosms.02 1.02) 1.19 1.5 3 105 cfu g21 to 1. 1 mL 2-ethyl-1-hexanol was sufficient to cause a suppressive effect.76) (7.94 1.05) 69 df 1.16 6.024) (8.00 22.32 22.45 0.16) Axenic 5776 0.83 1. Values are logarithmic transformations of mean VOC concentrations in Carbotrap and Tenax adsorbents and of A.72) (0.1 (4.67 (0.17 1.89 (0.74) (0. 2).25 (11.05 2.0) (36. The other C8 compounds did not significantly affect primordium formation in the microcosms. At the end of the experiment no C8 compounds were detected with the solvent method of extraction in any of the microcosms containing peat + CaCO3 casing or in the control microcosms containing axenic activated charcoal (FIG.61 2.18) Nonaxenic absent 24.41 22.08 0. No VOC were detected in the casing materials before use. both with and without Agaricus bisporus.08) (0.07) 1.04) (1.39 2. In the microcosms containing peat + CaCO3 casing. The numbers of primordia that formed on axenic activated charcoal and nonaxenic peat + CaCO3 casing in the presence of different C8 compounds is shown (FIG.70) (0.05) (32.49 2. bisporus were exclusively 8-carbon compounds (TABLE II).61 0.05) 69 df 0.65 present only in the microcosms inoculated with A.0) 1.72 3.71) (3.86 22.65) LSD (P 5 0.61 1.89) (0.39 21.90 21.01) (2. Values for primordia and VOC concentrations in adsorbents are respectively square root and logarithmic transformed means of microcosms containing Carbotrap and Tenax adsorbents.49 1.1) 1.21) (0.05) 2.1 3 106 cfu g21.68) LSD(P 5 0. with back-transformations shown in parentheses Sterility Agaricus bisporus VOC (mg g21) Benzoic acid Benzone compounds Benzyl alcohol 1-Bromo decane 1-Bromo heptane Diethyl ester 2-butanedioic acid 2-Ethyl-1-hexanol d-limonene Mequinol Naphthalene compounds 2-Pentyl furan Axenic absent 0.05) (0.06) (0. whereas the pseudomonad population increased from 7.22) (6.90 23.06 (49.87 1.38) Nonaxenic A15 7. Volatile organic compounds (VOC) detected in the highest concentrations in axenic and nonaxenic microcosms.62 (13.40 0.5) 3.30 2. The presence of 2-ethyl-1-hexanol in the microcosms suppressed primordium formation.63) (830.81 4.05) 2.22) (5.73 0.24) (0.88 1.19 3.39 (4.03) (8.21) (8.47) (305.16 (1.3 3 107 cfu g21 at the beginning and end respectively). bisporus strains A15 and 5776.19 (6. At the end of the experiment 2-ethyl-1-hexanol.71 0.60) 3.00) 21.87) (0. and numbers of primordia .29) (7.03) (1.91) (8. 0.17 1.17 20.98 20.40 21.13) (12.13 23.89 Axenic present Nonaxenic present 22. Final total bacterial TABLE III.62 0.96 23.99) (0.4) 20.5 3 107 and 2.73) .0) 0. 3octanone and trans-3-octene were detected in the activated charcoal casing at similar concentrations.30) (0.36 (0.81 3.NOBLE ET AL: VOLATILES.15 4.72 5. Volatile organic compounds (VOC) detected only in axenic and nonaxenic microcosms containing Agaricus bisporus inoculum.67 0.35 0.— The 8-carbon compounds in the microcosms did not affect mycelial growth in the casing. 1 mm diam.55 21. with back-transformations shown in parentheses Sterility A.83 0.44 (6.01).34) (4. Effect of 8-carbon compounds on primordium formation and casing bacteria.00) 1.49 0.93) Nonaxenic 5776 2.89 1. 2).72) (204.63 23.59) (0.10) (0.39 5.96 20.50 24. bisporus strain Primordia per microcosm VOC (mg g ) 1-bromo-octane 1.68 (0.39) (24.02) (0.15 2.99 (68.80 (0.05) (0. 1-octen-3-ol. PSEUDOMONADS 587 TABLE II.66) (0.33) (64.56 1.40) (359.24 (0.40 0.40) (0.13 (3.54 0.10) (0.6-octadien-3-ol octane 1-octanol 3-octanone 1-octen-3-ol 21 Axenic A15 0.0) (6.60 20.82 24.48 (4. in the microcosms containing axenic activated charcoal 10 mL were required.30 (1. The axenic charcoal casing resulted in more primordia than the nonaxenic peat + CaCO3 casing (P .54) (1.54 (34.29 3.59 1.06 0.49 (4.01) (0.06 1. The total bacterial population of the control treatment casing did not change significantly during the culture period (2. With the exception of 1-octanol.33 3.03) (23.28 (3.25) (8.14) 1.9) (0.62 20. 588 MYCOLOGIA FIG. 32 df). although the numbers of sporophores were greater in nonaxenic conditions (TABLE IV).— The presence of ethanol or C8 compounds into ventilated flask culture reduced mycelial growth in the casing. 0. which also was suppressed by 2-ethyl-1hexanol and cis-3-octen-1-ol. Length of vertical bar indicates least significant difference between treatments (P 5 0. 3. 32 df). Octane suppressed the FIG. 1 mm diam (square root transformed values) that formed on nonaxenic peat + CaCO3 casing (%) and axenic charcoal casing (&). Effect of C8 compounds (10 mL unless stated) in the atmosphere of microcosms on the numbers of primordia . Effect of C8 compounds (10 mL unless stated) in the atmosphere of microcosms on the populations of total bacteria (%) and pseudomonads (&) in nonaxenic peat + CaCO3 casing. 3). Length of vertical bar indicates least significant difference between treatments (P 5 0. Primordia and sporophores were produced in both axenic and nonaxenic ventilated flask culture. where measured (mg g21).05. Effect of VOC on primordium formation and casing bacteria in ventilated flasks. The presence of ethanol or 1octen-3-ol almost completely inhibited primordium formation. 3).05. Figures above bars are the concentrations of the respective C8 compound detected in the casing at the end of the experiment. 2. numbers in the peat + CaCO3 casing were higher in the presence of 1-octen-3-ol and 2-ethyl-1-hexanol than in the control microcosms and in the presence of the other C8 compounds (FIG.01) (FIG. . The pseudomonad population was increased by the presence of 2-ethyl-1hexanol in the microcosms (P . although this was not examined.5 14.NOBLE ET AL: VOLATILES.0 0. bisporus culture also was observed by Hayes and Nair (1974) and indicates the significance of pseudomonads in metabolizing VOC produced by A. bisporus is grown can inhibit primordial formation. Supplementation of the casing with ODA resulted in an increase in the number of sporophores produced. Inhibition was less with activated charcoal casing than with peat-based casing. The in vivo results from microcosm cultures support the hypothesis based on in vitro agar plate cultures (Wood and Blight 1982) that some of these C8 compounds can inhibit primordium formation with 1octen-3-ol being most inhibitory. 2-Ethyl-1-hexanol inhibited primordium formation at lower concentrations than C8 compounds produced by A.3 2. with both modes of removal enabling primordium formation.2) (1. Effect of volatile organic compounds on the numbers of mushroom primordia . 0.2 — — (22. 2-Ethyl-1-hexanol and 1-octen-3-ol increased the total bacterial population of the casing during the culture period (P . probably due to the greater adsorption capacity of activated charcoal compared with peat. Mau et al (1992) identified a compound 10-oxo-trans-8-decenoic acid (ODA) from A.1 1. DISCUSSION This work has confirmed research by Grove (1981) and Combet et al (2006) that VOC produced by A.4) (0. bisporus sporophores that stimulated mycelial growth and stipe elongation of A. primordium formation occurred within 7 d and at least one sporophore per jar was produced in all treatments. Wood and Blight (1982) noted that 1-octen-3-ol inhibited fruit-body formation but not mycelial growth on agar culture.3 0 13. bisporus and at concentrations below which a negative effect on mycelial growth was observed. The results also have shown that a VOC produced by the rye grain substrate.3 17.1 0.5 numbers of primordia but not of sporophores and 3octanone had no significant effect (TABLE IV). nonaxenic) Control (axenic) Control (nonaxenic) Ethanol 2-Ethyl-1-hexanol Octane 1-Octen-3-ol cis-3-Octen-1-ol 3-Octanone LSD (P 5 0.1 0. Primordia numbers are square root transformed values.6 Bacteria (ln cfu g21 casing) Total 0 16.7 5.9 13. PSEUDOMONADS 589 TABLE IV.0) Sporophores — — 0.4 3.9) (11. as suggested by Hayes et al (1969) and Eger (1972).7 18. bisporus mycelium are predominantly C8 compounds. 2-ethyl-1-hexanol.6 0.8 0.1 1. in spite of the inoculated casing having . In these experiments the population of pseudomonads in the casing tended to increase during A.7 1. 2-Ethyl-1-hexanol and 1-octen-3-ol were metabolized by the casing microbiota and adsorbed by activated charcoal casing.001) and all the VOC except 3-octanone increased the population of pseudomonads in the casing.3 4.0 14.9 17. The presence of 2-ethyl-1-hexanol and 1-octen-3-ol resulted in higher total bacterial and pseudomonad populations in the casing. whereas the total bacterial population remained stable or slightly declined. The increasing dominance of pseudomanads in the casing bacterial population during A.1 4. 2-Ethyl-1hexanol is a metabolite known to be produced by Acremonium obclavatum and Aspergillus versicolor (Enzeonu et al 1994. 1 mm diam and sporophores formed and on the casing bacterial populations in axenic and nonaxenic jar cultures contained within ventilated flasks. on which A. and they suggested that ODA might be involved in the initiation of fruiting.8 0. bisporus mycelium. However none of the Pseudomonas isolates tested stimulated primordia formation in axenic casing to the same extent as a naturally occurring microbiota in nonaxenic casing.4) (16. with back-transformations shown in parentheses Numbers per jar Volatile organic compound Control (start. It is well established that pseudomonads use C8 compounds as substrates (Chakrabarty et al 1973). These bacteria therefore were able to use and metabolize the inhibitory VOC.6 16.0) (20.0 1.1 14. bisporus culture. After the removal of the VOC from the air stream into the flasks.5 1.7 Pseudomonads 0 13. The pseudomonad population of the casing in the control treatment remained stable during the A. as suggested by Eger (1972) and Long and Jacobs (1974).05) 12 df Primordia — — 4.0) (4. bisporus.3 0. bisporus culture period although the total numbers of bacteria declined (TABLE IV).2 14.0) (31.9 0 15. axenic) Control (start.1 14.3 17. Pasanen et al 1997) although its source in the substrate in these experiments was not established.0 2.3 16. The biology and technology of the cultivated mushroom. LITERATURE CITED Anzai Y. Crow SL. Wakabayashi H. Chou G. tolaasii. This work has shown that the stimulatory effects of the casing and its microbiota and air exchange on primordium formation of A. Genetic regulation of octane dissimilation plasmid in Pseudomonas. Experiments and comments on the action of bacteria on sporophore initiation in Agaricus bisporus. Mushroom Sci 10:595–602. Eight carbon volatiles in mushrooms and fungi: properties. Flegg PB.590 MYCOLOGIA ACKNOWLEDGMENTS a significantly higher pseudomonad population. Science and cultivation of edible fungi. p 447–454. putida isolates were observed. including P. Simmons RB. Untersuchungen u ¨ ber die Function der Deckschicht bei der Fruchtko ¨ rperbildung des Kulturchampignons. veronii had little or no stimulatory effect on primordium formation. UK: John Wiley & Son. P. Chakrabarty AM. Mushroom Sci 8:719–725. The influence of different mushroom strains and substrates on the production of C8 compounds also should be investigated further. 1996. and two isolates of P. putida isolates. with several other Pseudomonas species. Combet E. Polishing up and managing your casing. Burton KS. 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