et al 2019-165:611-624 Proteobacteria,Firmicutes,Actinobacteria and Bacterioidetes study all displayed good agronomical characteristics and were the most abundant phyla n mpost in com peat the casing(49.5%)than with the phase III compost(11.8%) (Fig.5b),an so it scems ely that th suspens mat ous trial conducted in the summer of 2017 with the same madura and Ther cross-com Table of the of i microbiome in compos hat the three casing materials tste In the case of the fungal communities in the samples of 山2n8e diseases ) persion of bubble priate to place empha is on because the predominance of OTUs corresponding to A. ected the bisporus in b ced the even healthy basidiomes in the control treatment of the trial of the and belo ted in tion of the fungal microbiome betw een the casing,where omaed in this study c in compost has becn The presence oi abundant sp authors found a similar relative abundance of Lfungicola in Detection of the causative agent of bul ble ease by NGS clc-butn the exponentially by the end (Table S6),which is consistent he tnal (no macnsitop器enabled us to c were served in mushroom y of ager applied in the cas 84 e that the州 L.fung in the casing AF342784.described as cau casing ir Iosence of disdse syelaed to the natural suppressive effect hreofgincG casing layer by the en heless,we cannot ignore the possibility that,at low inocu- of the crop cycle oms are a h two of the samples and three of Gs and ence of endomicrobes 42,43).which could include the condary metabolites such as volatile organic inhibi ple of G7 and one of G8. ciosa required to generate macro scopic mptoms of bubble d DISCUSSION isms that limit dis have been venue for future work. Our sequencing results on the e II cropsProteobacteria, Firmicutes, Actinobacteria and Bacterioidetes were the most abundant phyla in compost phase III, casing and basidiomes (Fig. 2a). However, the basidiomes showed a higher number of bacterial components in common with the casing (49.5 %) than with the phase III compost (11.8 %) (Fig. 5b), and so it seems likely that the bacterial diversity in A. bisporus basidiomes is configured more by the casing than by the compost. This similarity was also apparent with the abundance of genera observed, since 6 of the 10 domi￾nant genera in casing samples were detected among the most abundant in basidiomes, while Acinetobacter, Actino￾madura and Thermobifida were only detected as major components of the bacterial microbiome in compost (Table S5). In the case of the fungal communities in the samples of phase III compost, casing and basidiomes, it seems inappro￾priate to place much emphasis on differences between the fungal communities present in compost and the basidiomes because the predominance of OTUs corresponding to A. bisporus in basidiome samples (Fig. 2) reduced the relative abundance of the remaining OTUs to 10 % and below, although significant differences were noted in the composi￾tion of the fungal microbiome between the casing, where Ascomycota was dominant, and the rest of the samples (Figs 3c, d, 4b). Detection of the causative agent of bubble disease by NGS Interestingly, although the blocks sampled for the character￾ization of the microbiome remained asymptomatic during the trial (no macroscopic symptoms of bubble disease were detected), the sensitivity of the NGS analysis enabled us to detect the presence of the causative agents of bubble disease in asymptomatic crops. Lecanicillium fungicola (GenBank accession no. FJ810136), which shows >98 % base identity with the species L. fungi￾cola var. aleophilum EF641885 or L. fungicola var. fungicola AF342784, described as causative agents for dry bubble dis￾ease in mushroom crops [40]), was detected in the casing in three samples of G2, two of G4, two of G5, two of G6 and three of G7, increasing in relative abundance along the crop cycle to be the fourth most abundant fungal species in the casing layer by the end of the crop cycle (Table S6). In addi￾tion, the presence of L. fungicola was also detected in the compost, in two of the samples of G1 and three of G8, and even in one of the basidiomes tested that showed none of the macroscopic symptoms of disease. The presence of M. perniciosa was also detected, but only residually in one sam￾ple of G7 and one of G8. DISCUSSION Although bubble diseases have been reported as a major cause of yield losses due to biotic agents [9], relatively little is known about the natural suppressive effect of the casing material on the expression of these fungal diseases. The three commercially relevant casing materials used in this study all displayed good agronomical characteristics and supported comparable yield to commercial production under control conditions, with black peat and a 50 : 50 mixture of black and blonde peat supporting the highest production. However, none of the three casing materials evaluated significantly reduced the severity of bubble dis￾eases when conidial suspensions of the causative agents were inoculated on the casing material. Notably, in a previ￾ous trial conducted in the summer of 2017 with the same experimental design and the same facilities, we observed cross-contamination with L. fungicola in the control room, associated with a high pressure of flies during cropping (data not shown). In this earlier trial, we also observed that the three casing materials tested were equally ineffi￾cient in preventing natural infection by bubble disease car￾ried by flies, the main vectors for the dispersion of bubble diseases [9]. However, interestingly, metagenomics analysis detected the ubiquitous presence of L. fungicola in compost, casing and even healthy basidiomes in the control treatment of the trial reported in this study, even though crops remained asymp￾tomatic. The presence of L. fungicola in compost has been reported previously through MiSeq sequencing as the fourth most abundant species [13]. However, in this study the authors found a similar relative abundance of L. fungicola in compost along the crop cycle. In our experiment the relative abundance of this parasite in compost was very low but detectable at the beginning of the crop cycle, but it increased exponentially by the end (Table S6), which is consistent with the higher impact of DBD observed in mushroom crops when the crop ages [9, 11]. The severity of disease symptoms resulting from DBD has been already directly related to the amount of causative agent applied in the cas￾ing [41], therefore we hypothesize that the relative abun￾dance of L. fungicola detected in the casing by NGS sequencing was not sufficient to generate detectable out￾breaks of the disease. Also, as reported previously, the absence of disease symptoms in the presence of the causa￾tive agent could be related to the natural suppressive effect that casing material possesses against mycoparasites, which is associated with the native casing microbiome [8]. None￾theless, we cannot ignore the possibility that, at low inocu￾lum densities, cultivated mushrooms are also protected by defence mechanisms that may be innate, or linked to the presence of endomicrobes [42, 43], which could include the production of secondary metabolites such as volatile organic compounds that inhibit the germination of L. fungicola [44]. The minimum inoculum densities of L. fungicola and M. perniciosa required to generate macroscopic symptoms of bubble diseases in mushroom crops are unknown, and uncovering the mechanisms that limit bubble disease devel￾opment at low inoculum densities would be an interesting avenue for future work. Our sequencing results indicate that L. fungicola popula￾tions can be present and increase in abundance in both phase III compost and casing, even in asymptomatic crops. Carrasco et al., Microbiology 2019;165:611–624 618