Submitted 20 November 2017Accepted 24 May 2018Published 13 June 2018

Corresponding authorsXiaolin Li kerrylee_twsinacomXiaoping Zhangzhangxiaopingphd126com

Academic editorHauke Smidt

Additional Information andDeclarations can be found onpage 16

DOI 107717peerj4975

Copyright2018 Zhang et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

Dynamic succession of substrate-associated bacterial composition andfunction during Ganoderma lucidumgrowthBo Zhang12 Lijuan Yan3 Qiang Li45 Jie Zou1 Hao Tan2 Wei Tan2Weihong Peng2 Xiaolin Li2 and Xiaoping Zhang1

1Department of Microbiology College of Resources Sichuan Agricultural University Chengdu China2 Soil and Fertilizer Institute Sichuan Academy of Agricultural Sciences Chengdu China3Chair for Aquatic Geomicrobiology Institute of Biodiversity Friedrich Schiller University Jena JenaGermany

4Biotechnology and Nuclear Technology Research Institute Sichuan Academy of Agricultural SciencesChengdu China

5College of Life Sciences Sichuan University Chengdu ChinaThese authors contributed equally to this work

ABSTRACTBackground Ganoderma lucidum a valuable medicinal fungus is widely distributedin China It grows alongside with a complex microbial ecosystem in the substrate Assequencing technology advances it is possible to reveal the composition and functionsof substrate-associated bacterial communitiesMethods We analyzed the bacterial community dynamics in the substrate during thefour typical growth stages of G lucidum using next-generation sequencingResults The physicochemical properties of the substrate (eg acidity moisture totalnitrogen total phosphorus and total potassium) changed between different growthstages A total of 598771 sequences from 12 samples were obtained and assigned to22 bacterial phyla Proteobacteria and Firmicutes were the dominant phyla Bacterialcommunity composition and diversity significantly differed between the elongationstage and the other three growth stages LEfSe analysis revealed a large number ofbacterial taxa (eg Bacteroidetes Acidobacteria and Nitrospirae) with significantlyhigher abundance at the elongation stage Functional pathway prediction uncoveredsignificant abundance changes of a number of bacterial functional pathways betweenthe elongation stage and other growth stages At the elongation stage the abundanceof the environmental information processing pathway (mainly membrane transport)decreased whereas that of the metabolism-related pathways increasedDiscussion The changes in bacterial community composition diversity and predictedfunctions were most likely related to the changes in the moisture and nutrientconditions in the substrate with the growth ofG lucidum particularly at the elongationstage Our findings shed light on theG lucidum-bacteria-substrate relationships whichshould facilitate the industrial cultivation of G lucidum

Subjects Agricultural Science Biodiversity Biotechnology MicrobiologyKeywords Ganoderma lucidum Dynamic change NGS G lucidumndashbacteria-substrate Func-tional pathways Bacterial composition

How to cite this article Zhang et al (2018) Dynamic succession of substrate-associated bacterial composition and function during Gano-derma lucidum growth PeerJ 6e4975 DOI 107717peerj4975

INTRODUCTIONGanoderma lucidum belongs to the phylum Basidiomycota and its growth mainly dependson lignin as a carbon source (Mizuno et al 1995) The fruiting bodies and spores ofG lucidum are highly appreciated as health products in China for their richness inpolysaccharides and triterpenoids which are demonstrated to strengthen the immunesystem and inhibit tumor formation (Wang et al 2002 Sakamoto et al 2016) Becauseof its high medicinal value the planting area of G lucidum is expanding G lucidum hasbecome the main economic pillar in some places due to the advantages of its cost-effectiveproduction management eg requiring a small investment having a short life cycle andyielding benefits fast (Boh et al 2007) Like other edible fungi the growth of G lucidumdepends on many environmental factors (eg temperature enzyme activity and microbialcommunity) which likely induce changes in the content of nutrients such as polysaccharidesand microelements in its fruiting bodies (Stajic et al 2002 Tanaka et al 2016) Li et al(2014) demonstrated that the nutrient content in different tissues of G lucidum changedwith its growth and that it attributed to the extracellular enzyme activities Significantgrowth-related differences were present in crude polysaccharides and triterpenes in thefruiting bodies of most of the G lucidum strains that were tested (Fu et al 2008) A greaternumber of trace elements and heavy metals were found in the mycelia than in the fruitingbodies or spores of G lucidum (Xing et al 2001) Studies have characterized the subtlechanges of various substances in the body of G lucidum during its growth howeverchanges in microbial community in the surrounding cultivating environment or in thesubstrate have seldom been studied

As previously reported bacteria in the surrounding soil or the culture media werelikely to change into endophytic bacteria and play an important role in the growth ofedible fungi (Gagne et al 1987 Elvira amp Van 2000) The endophytic bacteria assist theirhost with nitrogen fixation growth promotion and disease resistance (Mano amp Morisaki2008 Zhang 2010 Wang Chen amp Peng 2016) Qiu et al (2011) revealed that a varietyof microorganisms existed in the mushroom substrate and significantly affected the hostdevelopment In particular tiny changes inmicrobial communities in the culture substratesmay impact the growth and development of edible fungi (Cho et al 2003) Ma Chen ampChen (2016) characterized the significant allelopathic effects of the dominant microbes(mainly molds and bacteria) on the growth of G lucidum in a continuous croppingsoil using culture-based methods and demonstrated that bacteria such as ClostridiumAlkaligenes and Bacillus had stronger allelopathic effects on G lucidum In additionpollution rates were associated with changes in microbial communities in the industrialproduction of Pleurotus eryngii (Lin et al 2010)

The DNA-based community-fingerprinting methods such as DGGE and T-RFLP(Szeacutekely et al 2009) are cost-effective ways to explore the changes in microbial communitystructure in the environment Nevertheless these methods lack a clear description of themicrobial taxonomy and tend to underestimate microbial diversity with a relatively lowresolution Currently next-generation sequencing technology has overcome these issuesand is widely utilized to explore the distribution of microorganisms in diverse ecological

Zhang et al (2018) PeerJ DOI 107717peerj4975 223

conditions including freshwater lakes marine water agriculture soil forest soil thermalvents and even in some valuable herbs (Patel amp Jain 2015 Gigliotti et al 2015 Xia et al2016Meier 2016 Aacutevila et al 2016 Su et al 2017)

Previous studies onG lucidumwere mainly related to the cultivation technology and theactive components in fruiting bodies There have been few reports on the dynamic changesin microbial communities in the substrate at different growth stages of G lucidum In thisstudy next-generation sequencing of the V3ndashV4 region of bacterial 16S rRNA gene wasused to determine the composition and diversity of bacterial communities and to predictthe potential functions of the dominant microorganisms in the substrate during the fourgrowth stages of G lucidum (hyphal stage budding stage elongation stage and maturestage)

MATERIALS AND METHODSCultivation of Ganoderma lucidumThe Ganoderma lucidum cultivar Chuan Yuanzhi No 1 provided by the Soil and FertilizerInstitute at the Sichuan Academy of Agricultural Sciences has been deposited in the ChinaGeneral Microbiological Culture Collection Center (CGMCC) with the strain number ofCGMCC 13174 on October 21st 2016 The substrate was composed of cottonseed hull(90) wheat bran (5) corn flour (4) and gypsum (1) (Fig S1) all of which werefresh dry and unspoiled The substrate was put into polypropylene cultivation bags (size17 cmtimes 33 cmtimes 0005 cm) One side of the polypropylene bag was covered with breathablepaper for air entry during the cultivation of G lucidum The cultivation bags were thenautoclaved at 121 C for 2 h The purpose of autoclaving was to create a relatively asepticenvironment for mycelium germination The existence of microbes in the substrate wouldlikely acidify the substrate affecting the germination and growth of G lucidum Thereforeit was a good cultivation practice to sterilize the substrate before inoculation of the fungusG lucidum After sterilization the bags were cooled to room temperature and placed ina laminar flow cabinet for inoculation of G lucidum (Fig S2) A small piece of colonisedgrain (approximately 80 g) that was filled with mycelium of G lucidum with great vitalitywas inoculated into each cultivation bag After inoculation the cultivation bags wereplaced in a greenhouse in the cultivation site at Zhaojia Jintang China (N 3048prime1645primeprimeE 10435prime4879primeprime) The space of the cultivation site had been previously ventilated cleanedand simply disinfected with lime before the experiment was carried out The cultivationbags were arranged in parallel in two rows and two layers with seven to eight bags in eachon the ground in the greenhouse and the edge of each group was reinforced with woodenpiles (Fig S3)

The sampling of G lucidum was done at the four growth stages hyphal stage buddingstage elongation stage and mature stage After inoculation the mycelia of G lucidumbegan to germinate The first sampling was done at the hyphal stage (approximately 35days after the inoculation) when the mycelia of G lucidum spread and subsequently filledthe whole culture medium The mycelia twisted together The second sampling was doneat the budding stage (approximately 46 days after the inoculation) when the primordia

Zhang et al (2018) PeerJ DOI 107717peerj4975 323

formed and started to differentiate The third sampling was done at the elongation stage(approximately 56 days after inoculation) when the promordia grew longer and the stipewas formed Cap differentiation began after elongation The last sampling was done atthe mature stage (approximately 66 days after the inoculation) when the spores appearedon the pileus surface and gradually covered the yellow edges Disposable disinfectedgloves sterilized tweezers and knives were used for sampling At each growth stage threecultivation bags were brought to the lab and the substrate materials taken from differentparts of each cultivation bag were pooled together and homogenized (Fig S4) Finally atotal of twelve samples were collected in the four growth stages The fresh samples werestored at minus20 C in 2 mL Eppendorf tubes prior to DNA extraction

Chemical analysis of substrate materialsSubstratematerials at different growing stages ofG lucidumwere collected and the chemicalproperties were determined including pH value moisture total nitrogen total phosphorusand total potassium The samples were first digested with sulfuric acid hydrogen peroxideThen the treatment solution of each sample was analyzed with the conventional methodaccording to Thomas Sheard amp Moyer (1967)

DNA extraction PCR amplification and MiSeq sequencingNo less than 500 mg of substrate materials per sample were collected for DNA extractionThree biological replicates of samples taken at each growth stage were treated independentlyto ensure the methodological reproducibility The EZNA Rcopy Soil DNA kit (OMEGABio-Tek Norcross GA USA) was used to isolate DNA from the substrate following themanufacturerrsquos protocol DNA concentration wasmeasured using a UV spectrophotometer(Bio Photometer Eppendorf Hamburg Germany) The quality and size of the extractedDNA was checked by 08 agarose gel electrophoresis

The PCR amplification was performed by Shanghai Personal Biotechnology Co Ltd(Shanghai China) Both the primer information and the PCR protocol have been describedin detail in Srinivasan et al (2012) PCR amplification was performed using the bacterial16S rRNA gene-specific primers 338F (5prime-ACTCCTACGGGAGGCAGCA-3prime) and 806R(5prime-GGACTACHVGGGTWTCTAAT-3prime) with the following conditions 98 C for 2 min(initial denaturation) 25 cycles of 98 C for 15 s (denaturation) 55 C for 30 s (annealing)and 72 C for 30 s (extension) and 72 C for 5 min (final extension) (Langenheder ampSzeacutekely 2011) The PCR products were purified with Agencourt AMPure Beads (BeckmanCoulter Indianapolis IN) and quantified using a Quant-iT Pico Green dsDNA AssayKit with a microplate reader (FLx800 Bio-Tek Norcross GA USA) and were mixedbased on the concentration of each sample Amplicon sequencing was performed onIlluminarsquos MiSeq platform (Personalbio Shanghai China) The samples were barcodedbefore pooling The barcodes and adapters were trimmed with FASTX Toolkit All raw datawere submitted to the Sequence Read Archive (SRA) database with the accession numbersSRR5801759ndashSRR5801768 and SRR5801783ndashSRR5801784

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Sequence and statistical analysisReads containing ambiguous lsquoNrsquo or with length lt120 nt or gt140 nt were discardedHigh-quality sequences with 97 or greater similarity were clustered into OTUs usingUCLUST (Edgar 2010) a sequence alignment tool using QIIME pipeline version 170(Caporaso et al 2010) All non-bacterial sequences were removed after classificationThe most abundant sequence of each OTU was selected as the representative sequenceof this OTU The relative abundances of the OTUs were calculated The OTUs withrelative abundance lower than 0001 of the total sequences across all samples wereremoved (Bokulich amp Mills 2013) The multivariate statistical analyses were done using theOTU relative abundance data in R environment (R Core Team 2016) An unconstrainedordination (non-metric multidimensional scaling NMDS) was used to visualize the broadpattern of the distribution of bacterial communities PERMANOVA was used to test thesignificance of the difference in bacterial communities between different growth stagesof G lucidum based on 999 permutations Both NMDS and PERMANOVA analysis wereperformed based on weighted UniFrac distance using the R vegan package (McArdle ampAnderson 2001 Oksanen et al 2008) The pairwise PERMANOVA was used to test thedifference in the bacterial community composition between two growth stages at each timewhen the growth stage was found significant to affect the bacterial community compositionin the overall term PERMANOVA test The numbers of shared OTUs were presented in aVenn diagram using the R VennDiagram package (Chen amp Boutros 2011) Bacterial alphadiversity indices including observed OTUs Chao1 ACE Shannon and Simpson wererarefied and calculated based on the smallest library size of the samples LEfSe analysis(Segata et al 2011) was used to reveal the bacterial taxa that showed differential abundancebetween different growth stages of G lucidum at all taxonomic levels PICRUSt software(Langille et al 2013) was used to predict the metabolic functions of bacterial communitiesbased on the microbial metabolic function categories in the KEGG database All significantdifferences were concluded at P lt 005

RESULTSChemical analysis of the substrate materialsThe physicochemical properties of the substrate materials (eg acidity moisture contenttotal nitrogen content total phosphorus content and total potassium content) changedbetween the four growth stages ofG lucidum (Table 1) The substrate was acidic throughoutthe growth ofG lucidum Substrate pHwas lowest at the hyphal stage (434) After reachinga peak at the budding stage (529) the substrate pH decreased at the later growth stagesSubstrate moisture content declined along with growth of G lucidum Particularly themoisture content dropped sharply at the mature stage We observed a lower total nitrogencontent in the substrate at the budding and the elongation stages than at the hyphal andmature growth stages of G lucidum Total phosphorus and potassium in the substratedisplayed the identical fluctuation pattern with the growth of G lucidum Both of themshowed the highest content at the budding stage and the lowest content at the elongationstage

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Table 1 Chemical properties of substrateMeanplusmn standard deviation Statistical analysis was carried out by ANOVA using SPSS 190 softwareThe Post Hoc tests were done with LSD method Different lower-case letters showed significant difference (P lt 005) in the substrate chemical prop-erties between the different growth stages of G lucidum

Growth stage pH Moisture () Total nitrogen (gkg) Total phosphorus (gkg) Total potassium (gkg)

Hyphal stage 434plusmn 011 c 6460plusmn 108 a 1353plusmn 030 a 274plusmn 013 b 1331plusmn 034 bBudding stage 529plusmn 015 a 5801plusmn 122 b 1075plusmn 029 b 336plusmn 008 a 1545plusmn 013 aElongation stage 505plusmn 001 b 5448plusmn 202 c 1125plusmn 059 b 164plusmn 007 d 880plusmn 019 dMature stage 489plusmn 006 b 3856plusmn 154 d 1260plusmn 004 a 243plusmn 003 c 1147plusmn 068 c

Taxonomy-based analysis of bacterial communityIn total 598771 sequences from the 12 samples were clustered into 1200 OTUs at 97similarity A total of 157 of OTUs were unclassified at the phylum level The high qualityreads ranged from 25996 to 69618 OTUs between samples (Table S1) A total of 295shared bacterial OTUs were found between the four growth stages of G lucidum in thesubstrate (Fig 1) A total of 22 phyla were detected in the substrate at the four growth stagesof G lucidum As shown in Table 2 the most abundant phylum was Proteobacteria whichaccounted for 4147ndash7286 (average 5723) of all the bacterial sequences followedby Firmicutes (2250ndash4033 average 3412) Together these two phyla represented8012ndash9593 of the bacterial species The less dominant phyla (average abundancegt1) included Bacteroidetes (330) Acidobacteria (275) and Actinobacteria (203)A total of 195 genera were identified in the samples Most of the identified genera belongedto the phyla of Proteobacteria Firmicutes Bacteroidetes and Actinobacteria (Table S2) Ofthe 24 major bacterial families (relative abundance gt1) showed in Fig 2 20 belongedto Proteobacteria and Firmicutes Among these major families two families of Firmicutes(eg Streptococcaceae and Bacillaceae) showed high relative abundance across all thegrowth stages The relative abundance of proteobacterial families such as RhizobiaceaeBradyrhizobiaceae and Enterobacteriaceae changed greatly between the different growthstages

Bacterial alpha diversityBacterial alpha diversity indices significantly differed between the four growth stages ofG lucidum (Table 3) The richness indices (eg observed OTUs Chao1 and ACE) weresignificantly higher at the late growth stages (eg elongation and mature stages) thanat the early growth stages (eg hyphal and budding stages) The Shannon diversity wassignificantly higher at the elongation stage than at the hyphal stage There was no differencein the Simpson index between the different growth stages of G lucidum

Bacterial beta diversityPERMANOVA test was used to test the effect of the growth stage of G lucidum onthe bacterial community composition in the substrate According to the pairwisePERMANOVA test (Table 4) bacterial community composition significantly differedbetween the elongation stage and the other three growth stages NMDS an unconstrainedordination was used to visualize the patterns of bacterial community distribution Theseparation of the bacterial community samples at the elongation stage from the other three

Zhang et al (2018) PeerJ DOI 107717peerj4975 623

118

182

63

26

66

295

58

12

17

1125 39

20

17

62

Hyphal stage Budding stage

Elongation stage Mature stage

Figure 1 Venn diagram showing the number of shared OTUs between different growth stages ofG lu-cidum

Full-size DOI 107717peerj4975fig-1

Table 2 The average relative abundance of different bacterial phyla in the substrate during the fourgrowth stages ofG lucidum Others are all unclassified phyla

Phylum Abu () athyphal stage

Abu () atbudding stage

Abu () atelongation stage

Abu () atmature stage

[Thermi] 001 001 002 001Acidobacteria 308 211 564 004Actinobacteria 111 087 337 258Bacteroidetes 165 100 866 148Chloroflexi 000 000 003 001Cyanobacteria 009 002 154 003Elusimicrobia 001 000 002 001Firmicutes 4037 3496 4006 2070Fusobacteria 004 003 007 004Gemmatimonadetes 001 001 004 000Nitrospirae 000 000 003 000Planctomycetes 001 000 003 000Proteobacteria 536 6099 4043 7493Tenericutes 000 000 000 002Verrucomicrobia 000 000 001 001Others 001 000 006 014

Zhang et al (2018) PeerJ DOI 107717peerj4975 723

0

20

40

60

80

100

Hyphal stage Budding stage Elongation stage Mature stage

Rel

ativ

e ab

un

dan

ce (

)

Others

An unclassified family of Cyanobacteria

Lactobacillaceae

Rhodospirillaceae

An unclassified family of Proteobacteria

Sphingomonadaceae

Brucellaceae

Clostridiaceae

Nocardiaceae

Xanthomonadaceae

Comamonadaceae

An unclassified family of Acidobacteria

Moraxellaceae

Chitinophagaceae

Oxalobacteraceae

Staphylococcaceae

Pseudomonadaceae

Caulobacteraceae

Enterococcaceae

Enterobacteriaceae

Bradyrhizobiaceae

Burkholderiaceae

Bacillaceae

Streptococcaceae

Rhizobiaceae

Figure 2 OTU average relative abundances of the major bacterial families in the substrate ofG lu-cidum during all growth stages Others the families with relative abundances lower than 1 at eachgrowth stage of G lucidum

Full-size DOI 107717peerj4975fig-2

Table 3 Bacterial alpha diversity indicesMeanplusmn standard deviation Statistical analysis was carried out by ANOVA using SPSS 190 softwareThe Post Hoc tests were done with LSD method The index of the observed OTUs was used to evaluate the observed OTU richness whereas theChao1 and ACE were used to estimate the total (observed and unobserved) OTU richness of the bacterial community The indices of Shannon andSimpson were used to access the richness and evenness of bacterial community respectively Different lower-case letters showed significant differ-ence (P lt 005) in the diversity indices between the different growth stages of G lucidum

Sample Observed OTUs Chao1 ACE Simpson Shannon

Hyphal stage 363plusmn 21 b 19700plusmn 2166 b 26022plusmn 4072 b 085plusmn 010 a 402plusmn 062 bBudding stage 357plusmn 13 b 18900plusmn 2458 b 27017plusmn 3529 b 088plusmn 000 a 408plusmn 009 abElongation stage 505plusmn 50 a 37200plusmn 7055 a 47966plusmn 9977 a 090plusmn 006 a 477plusmn 033 aMature stage 491plusmn 33 a 33667plusmn 5346 a 42242plusmn 5642 a 087plusmn 003 a 421plusmn 013 ab

stages were clearly displayed in the NMDS ordination (Fig 3) The bacterial communitysamples taken at the elongation stage were separated from the hyphal and budding stageson the first axis whereas they were separated from the mature stage on the second axis inthe NMDS ordination plot

Biomarker discoveryLEfSe analysis was used to reveal the bacterial taxa that showed differential abundancesbetween the four different growth stages of G lucidum at all taxonomic levels (Fig 4)

Zhang et al (2018) PeerJ DOI 107717peerj4975 823

Figure 3 Nonmetric Multidimensional Scaling ordination of bacterial communities based onweighted UniFrac distance

Full-size DOI 107717peerj4975fig-3

Table 4 PEMANOVA analysis of bacterial community between different growth stages based onweighted UniFrac distance Significance of the differences in bacterial communities between the differentgrowth stages were tested using 999 permutations

Significance

Hyphal stage Budding stage 05014Elongation stage 00014Mature stage 01000

Budding stage Elongation stage 00014Mature stage 01000

Elongation stage Mature stage 00014

Hyphal stage Budding stage Elongation stage Mature stage 00014

LEfSe analysis revealed a significantly higher abundance of the order Rhizobiales (eggenus Rhodoplanes) and the genus Sphingobium at the budding stage than at the otherthree growth stages of G lucidum The family Frankiaceae and the genera of Alkaliphilusand Erwinia were significantly enriched in the substrate at the hyphal stage with regardto other growth stages A large number of bacterial taxa exhibited a significantly higherabundance at the elongation stage Those taxa included the phyla of Bacteroidetes (eg

Zhang et al (2018) PeerJ DOI 107717peerj4975 923

Figure 4 A cladogram showing the differentially abundant bacterial taxa at each of the four growing stages ofG lucidum based on LEfSe analy-sis (P lt 005 LDA scoregt 2)

Full-size DOI 107717peerj4975fig-4

genera Flavobacterium and Sediminibacterium) Acidobacteria (eg genus CandidatusKoribacter) Nitrospirae (eg genus Nitrospira) Cyanobacteria (eg order Streptophyta)and other taxa such as the orders of Xanthomonadales and Rhodospirillales and the familyof Comamonadaceae The phylum of TM7 and the orders of Clostridiales (eg familiesPeptostreptococcaceae and Lachnospiraceae) andPseudomonadales (eg generaAcinetobacterand Pseudomonas) were significantly more abundant at the mature stage than at othergrowth stages

Functional prediction of substrate bacteria communityA total of 39 KEGG pathways were identified in the study (Fig S5) Of these KEGGpathways 4893 were related to metabolism 1724 to environmental informationprocessing and 1423 to genetic information processing The tenmost prevalent pathwayswere related to four function types metabolism environmental information processinggenetic information processing and unclassifiedMembrane transport was the predominantKEGG pathway predicted by PICRUSt accounting for an average of 1593 during thegrowth of G lucidum Carbohydrate metabolism (on average 1003) and amino acid

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Table 5 PICRUSt predicted KEGG pathways of bacterial community in the substrate (relative abundance gt 1 in at least one growth stage)at different growth stages ofG lucidum Meanplusmn standard deviation Statistical analysis was carried out by ANOVA using SPSS 190 software ThePost Hoc tests were done with LSD method Different lower-case letters showed significant difference (P lt 005) in the relative abundance of thepathways between the different growth stages of G lucidum

Function type KEGG pathway Relative abundance ()

Hyphalstage

Buddingstage

Elongationstage

Maturestage

Cellular processes Cell motility 277plusmn 005 a 297plusmn 032 a 275plusmn 032 a 295plusmn 032 aMembrane transport 1740plusmn 196 a 1631plusmn 180 a 1136plusmn 081 b 1408plusmn 072 abEnvironmental information

processing Signal transduction 214plusmn 005 a 220plusmn 009 a 227plusmn 005 a 235plusmn 016 aFolding sorting and degradation 177plusmn 010 c 179plusmn 004 bc 218plusmn 004 a 198plusmn 011 abReplication and repair 606plusmn 027 b 599plusmn 014 b 691plusmn 015 a 606plusmn 012 bTranscription 244plusmn 010 ab 241plusmn 002 ab 254plusmn 009 a 226plusmn 005 b

Genetic informationprocessing

Translation 346plusmn 023 b 342plusmn 012 b 411plusmn 016 a 358plusmn 007 abAmino acid metabolism 981plusmn 016 a 1011plusmn 046 a 1038plusmn 012 a 1007plusmn 027 aBiosynthesis of other secondarymetabolites

093plusmn 002 ab 092plusmn 004 ab 103plusmn 011 a 080plusmn 004 b

Carbohydrate metabolism 1007plusmn 026 b 1009plusmn 011 b 1068plusmn 005 a 994plusmn 028 bEnergy metabolism 485plusmn 007 c 480plusmn 004 c 542plusmn 005 a 505plusmn 001 bEnzyme families 180plusmn 009 b 177plusmn 002 b 201plusmn 005 a 167plusmn 006 bGlycan biosynthesis andmetabolism

176plusmn 009 b 173plusmn 004 b 218plusmn 025 a 174plusmn 015 b

Lipid metabolism 375plusmn 001 a 386plusmn 019 a 389plusmn 010 a 393plusmn 010 aMetabolism of cofactors andvitamins

358plusmn 001 b 358plusmn 003 b 394plusmn 014 a 374plusmn 011 ab

Metabolism of other amino acids 189plusmn 004 b 202plusmn 015 ab 194plusmn 005 ab 213plusmn 007 aMetabolism of terpenoids andpolyketides

198plusmn 003 b 206plusmn 010 b 204plusmn 004 ab 222plusmn 007 a

Nucleotide metabolism 297plusmn 009 a 290plusmn 014 a 316plusmn 010 a 298plusmn 005 a

Metabolism

Xenobiotics biodegradation andmetabolism

437plusmn 020 ab 461plusmn 028 a 373plusmn 023 b 486plusmn 042 a

Cellular processes and signaling 371plusmn 013 a 373plusmn 003 a 399plusmn 009 a 389plusmn 034 aGenetic information processing 198plusmn 016 b 205plusmn 008 b 245plusmn 006 a 248plusmn 018 ametabolism 272plusmn 008 a 276plusmn 004 a 272plusmn 016 a 296plusmn 012 a

Unclassified

Poorly characterized 490plusmn 012 a 491plusmn 003 a 529plusmn 007 a 513plusmn 027 a

metabolism (on average 981) were the second and the third most abundant pathwaysduring the growth of G lucidum

The predicted pathways that had relative abundances over 1 at different growthstages of G lucidum were presented in Table 5 A number of predicted bacterial functionalpathways in the substrate were different between the elongation stage and the other threegrowth stages of G lucidum At the elongation stage the relative abundance of membranetransport and xenobiotics biodegradation andmetabolism were decreased other predictedpathways such as folding sorting and degradation replication and repair translationcarbohydrate metabolism energy metabolism enzyme families glycan biosynthesis andmetabolism and metabolism of cofactors and vitamins were enriched in the substrate

Zhang et al (2018) PeerJ DOI 107717peerj4975 1123

bacterial communityOther pathwayswere not significantly different in abundance betweendifferent growth stages (eg cell motility signal transduction amino acid metabolism andcellular processes and signaling)

DISCUSSIONIn this study we observed significant changes in the physicochemical properties of thesubstrate materials during the growth of G lucidum The substrate materials togetherwith the aeration and temperature used in our study provided an optimum conditionfor the cultivation of G lucidum For cultivation of edible andor medicinal mushroomsthe substrate rich in essential bio-available nutrients with optimum pH moisture andair content is preferred (Chang amp Wasser 2018) Cottonseed hulls provide fine waterholding capacity and nitrogen to ensure the water and nutrient supply for the cultivationof edible mushrooms such as Pleurotus ostreatus (Lu amp Qu 1984) and aerobic bacteria(Slifkin amp Pouchet 1975) The mixture of cottonseed hulls with other materials such aswheat bran corn flour and gypsum was used for industrial production of G lucidum inChina The growth of G lucidum slightly increased substrate pH especially at the buddingstage However the substrate still remained acidic with pH values ranging between 4and 55 at different growth stages The moisture content continued declining with thevegetative growth of G lucidum The observed sharp decline of moisture content waslikely associated with the development of the fruiting bodies at the mature stage whenthe water demand was the largest The lowered total nitrogen content in the substratelikely indicated the higher uptake of nitrogen of G lucidum at the budding and elongationstages than at the hyphal and mature stages This phenomenon might be related tothe rapid mycelia growth and protein synthesis processes of G lucidum at the latergrowth stages Nitrogen content is one limiting factor that should be seriously takeninto consideration during mushroom cultivation Nitrogen supplementation improves thenutritional content in substrates (Lelley amp Janssen 1993) contributing to a higher yieldof mushrooms (Yang Chienyan amp Chen 2003) Dependent on the fungus physiology andthe medium composition nitrogen source was demonstrated to affect enzyme synthesis(Couto et al 2004 Kapich et al 2004) In addition the C N ratio can greatly impactthe mycelia growth and fruiting body formation of G lucidum (Hsieh amp Yang 2004) Ithas been previously stated that more enzymes are involved in cell wall synthesis duringfruiting body formation than at the mycelium and primordial stages However enzymesrelated to cell wall component degradation were higher expressed at the earlier stagesof mushroom growth (Rahmad et al 2014) Total phosphorus and potassium contentin the substrate was lowest at the elongation stage likely attributable to a high nutrientdemand of G lucidum at this stage G lucidum grows most prosperously at the elongationstage a critical period for biomass and nutrient production Minerals such as phosphorusand potassium were strongly correlated with the yield and biological efficiency of someedible fungi (eg Ganoderma lucidum and Lentinula edodes) (Ozcelik amp Peksen 2007Peksen amp Yakupoglu 2009)

Proteobacteria and Firmicutes were the dominating phyla observed in the bacterialcommunity in the substrate of G lucidum with variations in relative abundance between

Zhang et al (2018) PeerJ DOI 107717peerj4975 1223

different growth stages of G lucidum Proteobacteria widely exist in high abundance insoils with diverse morphologies physiologies and metabolisms and is considered to beadvantageous in global carbon nitrogen and sulfur cycling (Janssen 2006 Kersters et al2006 Spain Krumholz amp Elshahed 2009) A recent study suggested that the membersof the phylum Firmicutes play important roles in the conversion of wheat straw intobio-utilizable sugars throughout the Agaricus bisporus mushroom cropping process(Mcgee et al 2017) Durrer et al (2016) characterized the role of biological connectionsin agricultural soils and revealed that competition among various bacteria may cause adecline in the abundance of some taxa The relative abundance of Firmicutes decreasedwith time in our study likely due to the reduced bioavailability of the wheat straw and theresource competition between other organisms in the substrate at the late growth stageof G lucidum Besides Firmicutes other lignocellulose degrading bacteria belonging toActinobacteria Proteobacteria and Bacteroidetes (Ntougias et al 2004 Zhang et al 2014Liang et al 2015) were also present with relatively high abundance in the mushroomsubstrate ensuring an effective simultaneous degradation system of lignocellulose incollaboration with the mushroom G lucidum

In our study bacterial alpha diversity in the substrate increased significantly duringthe growth of G lucidum Bacterial alpha diversity indices (eg observed OTUs Chao1ACE and Shannon) peaked at the elongation stage Vajna et al (2010) attributed the higherdiversity of bacterial community around the mushrooms to the growth of the mushroomsNevertheless the evenness of bacterial community (Simpson index) remained unchangedwith time Bacterial community composition in the substrate changed significantly duringthe growth of G lucidum Particularly bacterial community composition significantlydiffered between the elongation stage and the other three growth stages of G lucidumwhich might be linked to the changes in the substrate environment and the growth ofG lucidum

LEfSe analysis revealed that a large number of bacterial taxawere enriched in the substrateat the elongation stage than at other growth stages of G lucidum including Bacteroidetes(eg genera Flavobacterium and Sediminibacterium) Acidobacteria (eg genus CandidatusKoribacter) Nitrospirae (eg genus Nitrospira) Cyanobacteria (eg order Streptophyta)and other taxa such as the orders of Xanthomonadales and Rhodospirillales and the family ofComamonadaceae The increased relative abundance of these taxa were likely attributableto the colonization of these bacteria and the changes in the physicochemical condition ofthe substrate at the elongation stage of G lucidum These bacteria were characterized bytheir capacity in the biogeochemical cycling of major biogenic elements such as carbonnitrogen phosphorus and micronutrients So the nitrogen phosphorus and potassiumand other nutrients in the substrate were likely more demanded by these bacteria at theelongation stage than at other stages The reduction in substrate total nitrogen phosphorusand potassium at the elongation stage highly supported this assumption The activities ofthe bacterial community in the cultivation substrate significantly affect growth and qualityof the edible mushrooms (Miller et al 1990 Stoumllzer amp Grabbe 1991) Nitrogen fixationgenes were detected in the genus of Burkholderia (Minerdi et al 2001) Nitrospirae playsimportant roles in nitrification during themushroom cropping process (Mcgee et al 2017)

Zhang et al (2018) PeerJ DOI 107717peerj4975 1323

Other bacteria may provide essential growth factors such as thiamine (Henningsson 1967)The enrichment of these bacteria and the increased bacterial diversity at the elongationstage might be important to trigger the development of the fruiting bodies of G lucidum

G lucidum produces various enzymes to degrade lignin and synthesize substancesto accomplish its growth as other white rot fungi (Coelho et al 2010) Accordinglythe changes in the physicochemical properties of the substrate materials by G lucidumduring its growth might be an important driver of the successional changes in bacterialcommunity composition in the substrate As the main ingredient of the substrate materialcottonseed hull can affect the growth of G lucidum to a great extent Previous studiesreported compositional changes in a substrate with cottonseed hull which includedcellulose hemicelluloses and lignin during the growth period of mushrooms (Li Pang ampZhang 2001 Ni et al 2002) The abundances of most bacterial groups were dependent onorganic C and total N in the habitat (Zhang 2010) In our study the moisture contentof substrate continued decreasing with the growth of G lucidum likely to be anotherkey factor to affect the bacterial community composition and diversity Therefore thequalitative and quantitative change in the substrate physicochemical properties might havedriven the dynamic shifts in the composition and functions of bacterial community duringthe growth of G lucidum In addition bacterial communities can be directly affected byorganic substances that fungi secrete (Frey-Klett Pierrat amp Garbaye 1997) Metabolitesof mushrooms such as carbohydrates amino acids thiols and all kinds of enzymeswere secreted to change the physicochemical conditions in the substrate (Rangel-CastroDanell amp Pfeffer 2002 Zhu et al 2013) likely creating a suitable environment for bacterialcolonization and growth at the later stage In this study differentially abundant bacterialtaxa were uncovered in the substrate at different growth stages most likely associated withthe differed metabolic functions in the community

The reconstruction of metagenome based on marker genes allows inference of thepotential functional profile of the bacterial community in the substrate ofG lucidum Of theidentified KEGG pathways 1724 were related to environmental information processingin whichmembrane transport dominatedMembrane transport of the bacterial communitywas predicted more abundant at the budding and hyphal stages than at the elongationstage in the substrate The changes in the predicted functional pathways in the bacterialcommunities were likely attributable to the changes in the substrate physicochemicalconditions The pathway of membrane transport showed high abundance when themoisture content was high at the early stage The extracellular enzymes and other organicsubstances that microbes produce are transported by liquid phase diffusion (Yang et al2011) High moisture content facilitates the transport of nutrients and mobility of bacteriaincreasing the availability of nutrients to bacteria and intercommunication betweenmicroorganisms in the substrate For bacteria outer membrane proteins play essentialroles in bacterial adaptation (Lin Huang amp Zhang 2002) The membrane compositionsdiffer from bacterial species (Sohlenkamp amp Geiger 2016) The outer membranes areimportant for bacterial adaption as they are associated with the uptake of water energyand nutrient microbial interaction regulations and stress-resistance (Lin Huang amp Zhang2002 Schwechheimer amp Kuehn 2015) Outer-membrane vesicles make bacteria interact

Zhang et al (2018) PeerJ DOI 107717peerj4975 1423

with the environment and survive under stress conditions (Schwechheimer amp Kuehn2015) Therefore in addition to the effect of G lucidum the high abundance of predictedenvironmental information processing pathway (particularly membrane transport) in thebacterial community at the early stage ofG lucidumwas also likely related to the adaptationand colonization mechanisms of the bacterial settlers that entered into the substrate fromair

At the elongation stage the abundance of the predicted membrane transport andxenobiotics biodegradation and metabolism pathways reduced whereas the predictedgenetic information processing pathways (eg replication and repair and translation)metabolism pathways (eg carbohydratemetabolism energymetabolism enzyme familiesglycan biosynthesis and metabolism and metabolism of cofactors and vitamins) increasedsuggesting a functional change in the bacterial community from environmental adaptationand survival strategies to self-development with the growth of G lucidum The increase ofpredicted translation pathway at the elongation stage might be a result of rapid proteinsynthesis in bacterial cells in response to the rapid growth of G lucidum At this stagebacterial richness and diversity increased supported by the evidence of the reduction ofthe nutrients (eg total nitrogen phosphorus and potassium) and moisture content inthe substrate Thus the sufficient provision of nutrients and water to the substrate at theelongation stage might be important to sustain the growth and metabolic activities of thefungus and the substrate -associated microbiome

Microbes are ubiquitous and participate in the life activities of other organisms (Singh2015) It was noteworthy that those bacterial OTUs that were present throughout the wholelife cycle ofG lucidum accounted formore than 94of the bacterial community indicatingthat they were the first settlers and continued to be present in the substrate These bacterialOTUs might have great capacity to cope with the acidity of the substrate environment andthe resource competition withG lucidum and other organisms Some of these OTUs mightalso be beneficial to the growth of G lucidum Bacteria were involved in cell membranepermeability metabolic activity exudates absorption and nutrient bioavailability ofmycelia affecting the fruiting body growth (Fitter amp Garbaye 1994 Frey-Klett Pierrat ampGarbaye 1997) For instance bacterial metabolites were found related to fruiting bodyformation of Agaricus bisporus and help the mushroom resist pathogens (Urayama 1967Park amp Agnihotri 1969 Zhang 1999) Bacteria such as fluorescent pseudomonads wereparticularly beneficial to Pleurotus ostreatus budding which brings up a potential researchdirection for improving the yield and quality of Ganoderma lucidum by inoculatingbeneficial bacteria (Ouhdouch Barakate amp Finance 2001 Cho et al 2003) The phylaActinobacteria and Firmicutes were suggested to carry out the conversion of wheat strawinto utilisable sugars (Mcgee et al 2017) In our study a higher abundance of Bacilluswas observed at the early growth stages (eg hyphal stage and budding stage) of Glucidum Bacillus spp isolated from the cultivation substrate can optimize the growth ofPleurotus ostreatus by inhibiting Trichoderma harzianum growth through laccase induction(Velaacutezquez-Cedentildeo et al 2008) Bacillus provided relative protection mechanism againstother harmful fungi because mycelia of cultivated mushroom were easily contaminated

Zhang et al (2018) PeerJ DOI 107717peerj4975 1523

by other harmful fungi before primordia formation Hence the potential enhancement ofbacterial activities on the growth of G lucidum is worth further investigation

CONCLUSIONSThe study revealed a significant effect of G lucidum growth on the physicochemicalproperties and bacterial community composition of the substrate materials (eg pHmoisture and nutrient conditions) Changes in the substrate physicochemical propertiesassociate with the growth of G lucidum were likely the direct factor that drove the changesin bacterial community composition The diversity structure and the predicted functionalpathways of the substrate bacterial community greatly differed between the elongationstage and other growth stages The decrease of nutrient contents and moisture indicateda rigorous growth of G lucidum at the elongation stage We therefore suggest that thenutrient supply especially at the elongation stage is crucial to the growth of G lucidumthe diversity and the metabolic activities of the bacteria in the substrate Hence this studyincreased the understanding of the G lucidum-bacteria-substrate interactions to hopefullyfacilitate the industrial cultivation of G lucidum

ACKNOWLEDGEMENTSWe would like to thank American Journal Experts (httpwwwajecn) for providinglinguistic assistance during the preparation of this manuscript

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis work was supported by the Chinese Agricultural Research System (CARS-20) aswell as edible fungi breeding and cultivation project of the innovation team in Sichuanprovince The funders had no role in study design data collection and analysis decision topublish or preparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsChinese Agricultural Research System CARS-20Innovation team in Sichuan province

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Bo Zhang conceived and designed the experiments performed the experiments analyzedthe data prepared figures andor tablesbull Lijuan Yan performed the experiments analyzed the data contributed reagentsmateri-alsanalysis tools prepared figures andor tablesbull Qiang Li analyzed the data

Zhang et al (2018) PeerJ DOI 107717peerj4975 1623

bull Jie Zou performed the experimentsbull Hao Tan and Weihong Peng performed the experiments authored or reviewed drafts ofthe paperbull Wei Tan conceived and designed the experimentsbull Xiaolin Li conceived and designed the experiments analyzed the data prepared figuresandor tables authored or reviewed drafts of the paper approved the final draftbull Xiaoping Zhang conceived and designed the experiments authored or reviewed draftsof the paper approved the final draft

Data AvailabilityThe following information was supplied regarding data availability

NCBI accession numbers are SRR5801759ndashSRR5801768 and SRR5801783 SRR5801784in BioProject ID PRJNA392763

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj4975supplemental-information

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LiWT YuMYWeiW Xu XY Jiang N Zheng LY Yang ZR Luo X 2014 Variationof substance content during growth cycle of cultured Ganoderma lucidum andits mechanism Chinese Traditional amp Herbal Drugs 45552ndash557 [In Chinese]DOI 107501jissn0253-2670201404019

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TanakaM KnowlesW Brown R HondowN Arakaki A Baldwin S StanilandS Matsunaga T 2016 Biomagnetic recovery of selenium bioaccumulating ofselenium granules in magnetotactic bacteria Applied amp Environmental Microbiology823886ndash3891 DOI 101128AEM00508-16

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Vajna B Nagy A Sajben E Manczinger L Szijaacutertoacute N Kaacutedaacuter Z Bordaacutes D Maacuteri-aligeti K 2010Microbial community structure changes during oyster mush-room substrate preparation Applied Microbiology amp Biotechnology 86367ndash375DOI 101007s00253-009-2371-3

Velaacutezquez-CedentildeoM Farnet AMMata G Savoie JM 2008 Role of Bacillus sppn antagonism between Pleurotus ostreatus and Trichoderma harzianum in heat-treated wheat-straw substrates Bioresource technology 99(15)6966ndash6973DOI 101016jbiortech200801022

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Wang YY Khoo KH Chen ST Lin CCWong CH Lin CH 2002 Studies on theimmuno-modulating and antitumor activities of Ganoderma lucidum (Reishi)polysaccharides functional and proteomic analyses of a fucose-containing glyco-protein fraction responsible for the activities Bioorganic amp Medicinal Chemistry101057ndash1062 DOI 101016S0968-0896(01)00377-7

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Zhang ZZ 1999Microflora analysis ofMorchella rhizosphere and its root Edible fungi ofChina 18(2)25ndash26

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ZhuM Du F Zhang GWang H Ng T 2013 Purification a laccase exhibiting dyedecolorizing ability from an edible mushroom Russula virescens InternationalBiodeterioration amp Biodegradation 8233ndash39 DOI 101016jibiod201302010

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