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 CSG 15 (Rev. 6/02) 1 Project Determining the Physiological and Genetic Mechanisms of DEFRA HH1334SMU title Initiation to project code Control Mushroom Population Densities • 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. CSG 15 (Rev. 6/02) 2 Project Determining the Physiological and Genetic Mechanisms of DEFRA HH1334SMU title Initiation to project code Control Mushroom Population Densities 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 CSG 15 (Rev. 6/02) 3 Project Determining the Physiological and Genetic Mechanisms of DEFRA HH1334SMU title Initiation to project code Control Mushroom Population Densities of inhibitory compounds and level of initiation