Carrasco et al 2019165:611-624 INTRODUCTION ental samples and to dete production rdwide 2)The mmercial sultivation of Agam ricus bisporus (Lange) The formulation of the casing material has been postulated casing material to inty to ases in mercial material most commonly employed by the muh room industr y were to stage on as essential for mushroom crop ing because of their bene the 50 50 on sing composition an e cas rop discases 8),ame the most dama ewaatngdieasedeuopmeantndaaiosgtfcpp with either L.fungicola or M.pe nd Gams,and the thet the unin ted treatment, wet WBD) sis to analyse the composition and dynamics of the casing oms of DBDand WBDare detected ditionally,the micro over the casing layer,when the host e omes was alsc e(e METHODS tion and Casing characterization and crop design ons on acteriz mercially available casing materials were acquired the phases of the B.V .CI:b epreviously e compos ex KF,Valimex microbiome 115 161:the evaluatio of enzymatic activity n B.V and val KEIng materi- n onls I 19. Compost and casing physice chemicl characterization was ring pH conduct 22);and the quantification and water holding capacity at the be ginning of the trials and al ong the crop cycle in trial 1 (Ta ble available in e)2 Next-generation seq ncing (NGs)offers a ne of possibilities to characterize the microbe-rich environ ooms pro vided with climate control(auto nati ental niche in w s and develops System)that available at Centro Tecnc mrnchmcntoroliot and so CHAMP. Autol,Spai 4 hor aeoR23Cme,oARntnnoemetOn8rytar6eR0erdoX,HKCetronTeoo8aee5cen ogrono.Spai o@plants.ox.ac.uk 99r next-nr seque rin I fatt Us,perational R (ORA) this in ct D PRJNA 612INTRODUCTION Mushrooms provide a healthy contribution to the human diet, and the production of cultivated mushrooms is a dynamic and fast-growing industry worldwide [1, 2]. The commercial cultivation of Agaricus bisporus (Lange) Imbach involves the production of a selective compost, which must be covered with a layer of casing material to achieve a profitable crop [3]. This casing material possesses physical and chemical characteristics that facilitate the shift from the vegetative tissue (hyphae) to the reproductive stage (basidiomes) during mushroom fructification [4]. Some of the natural casing inhabitants have been described as essential for mushroom cropping because of their bene￾ficial impact on the development of the mycelium and fructification [5–7]. Casing composition and the casing microbiota may also affect the development of mushroom crop diseases [8], among the most damaging of which are dry bubble disease (DBD) caused by Lecanicillium fungi￾cola (Preuss) Zare and Gams, and wet bubble disease (WBD) caused by Mycogone perniciosa (Magnus) Dela￾croix [8, 9]. The symptoms of DBD and WBD are detected over the casing layer, when the host parasitized by the harmful fungus develops undifferentiated masses of tissue called bubbles (Fig. 1) [10, 11]. Despite the potential importance of the casing microbiota for mushroom fructification and disease control, most recent publications have focused on the characterization of the micro-organisms present in mushroom compost along the different phases of the composting process [12–14]. Techniques that have previously been used to characterize the composition and dynamics of the compost and casing microbiomes include the characterization of the culturable microbiome [15, 16]; the evaluation of enzymatic activity in compost [17, 18]; the phospholipid fatty acid (PLFA) profile [15, 19, 20]; 16S ribosomal deoxyribonucleic acid (rDNA)-based denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length poly￾morphism (T-RFLP) [7, 19, 21, 22]; and the quantification of chitin and laccase activity [20]. Next-generation sequencing (NGS) offers a new spectrum of possibilities to characterize the microbe-rich environ￾mental niche in which A. bisporus grows and develops. Metagenomics conducted through NGS does not rely on enrichment or isolation, and so it is possible to work with crude environmental samples and to detect and quantify non-culturable micro-organisms. Compared to classical techniques such as DGGE and T-RFLP, NGS metagenomics provides a more holistic approach with a more powerful workflow [23]. The formulation of the casing material has been postulated to be a factor conditioning susceptibility to fungal diseases in mushroom crops [24]. In the present work two of the com￾mercial materials most commonly employed by the mush￾room industry were evaluated to compare their natural suppressive effect on DBD and WBD. Black peat (mostly employed for the local industry to produce mushrooms for the fresh market), blonde peat (mostly used to produce for the canning industry) and a 50 : 50 mixture of black and blonde peat were employed as casing layers in a crop trial evaluating disease development and yield losses when crops were artificially inoculated with either L. fungicola or M. per￾niciosa. During the trial, the uninoculated treatment, cased with the 50 : 50 mixture, was subjected to metagenomic anal￾ysis to analyse the composition and dynamics of the casing microbiome along the crop cycle. Additionally, the micro￾biome of compost samples and basidiomes was also sequenced. METHODS Casing characterization and crop design Commercially available casing materials were acquired from local providers to run the experiment. C1: black peat, based on peat moss (Euroveen B.V., BVB Substrates, Grub￾benvorst, Limburg, The Netherlands); C3: blonde peat, based on Spaghnum peat moss (Valimex KF, Valimex SL, Valencia, Spain); and C2: a mixture of both casing materi￾als (50 % each) (Euroveen B.V. and Valimex KF). Compost and casing physico-chemical characterization was carried out by measuring pH, electrical conductivity (EC), moisture content, ash, organic matter, nitrogen, C/N ratio and water holding capacity at the beginning of the trials and along the crop cycle in trial 1 (Table S1, available in the online version of this article) [25]. The trial (September 2017) was conducted in three separate rectangular growing rooms provided with climate control (automatic Fancom System) that were available at Centro Tecnológico de Investigación del Champiñón de La Rioja (CTICH) (ASO￾CHAMP, Autol, Spain). Ninety blocks of phase III Received 14 October 2018; Accepted 14 March 2019; Published 17 April 2019 Author affiliations: 1 Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1, UK; 2 Centro Tecnológico de Investigación del Champiñón de La Rioja (CTICH), Autol, Spain; 3 Plataforma de Genómica y Bioinformatica, Centro de Investigación Biom  edica de La Rioja (CIBIR), Logroño, Spain. *Correspondence: Jaime Carrasco, jaime.carrasco@plants.ox.ac.uk Keywords: Agaricus bisporus; casing material; next generation sequencing; metagenomics; microbiome; mycoparasites. Abbreviations: DBD, dry bubble disease; DGGE, denaturing gradient gel electrophoresis; NGS, next-generation sequencing; OTUs, operational transcriptomic units; PLFA, phospholipid fatty acid; T-RFLP, terminal restriction fragment length polymorphism; WBD, wet bubble disease. Data associated with this project have been uploaded in Oxford Research Archives (ORA) and can be downloaded from this link: https://ora.ox.ac.uk/ objects/uuid:603f6945-84f5-41d7-b76c-64ebecd86815. Sequence data for this study are also deposited in NCBI Bioproject collection under BioProject ID PRJNA477500 and will be available upon publication. Six supplementary tables and four supplementary figures are available with the online version of this article. Carrasco et al., Microbiology 2019;165:611–624 612