effects of nutrient loading on sediment bacterial and

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Effects of nutrient loading on sediment bacterial and pathogen communities within seagrass meadows

Citation Liu Songlin Jiang Zhijian Deng Yiqin Wu Yunchao Zhang Jingping Zhao Chunyu Huang Delian Huang Xiaoping and Trevathan-Tackett Stacey M 2018 Effects of nutrient loading on sediment bacterial and pathogen communities within seagrass meadows MicrobiologyOpen vol 7 no 5 e00600 pp 1-11

DOI httpsdoiorg101002mbo3600

copy2018 The Authors

Reproduced by Deakin University under the terms of the Creative Commons Attribution Licence

Downloaded from DRO httphdlhandlenet10536DRODU30106856

MicrobiologyOpen 20187e600 emsp|emsp1 of 11httpsdoiorg101002mbo3600

wwwMicrobiologyOpencom

1emsp |emspINTRODUCTION

Seagrass meadows are incredibly productive ecosystems (Hemminga amp Duarte 2000) and support a high diversity of microorganisms in their sediments (Bourque Vega- Thurber amp Fourqurean 2015 Garciacutea- Martiacutenez Loacutepez- Loacutepez Calleja Marbagrave amp Duarte 2009) due to the

release of root exudates (amino acids sugars) and oxygen into the rhi-zosphere (Christiaen McDonald Cebrian amp Ortmann 2013 Ingemann Jensen Kuumlhl Glud Joslashrgensen amp Priemeacute 2005 Jensen Kuumlhl amp Priemeacute 2007) Microbes play a fundamental role in several biogeochemical cycling processes including the oxidation of organic carbon nitrogen fixation nitrification denitrification iron cycling and sulfate reduction

Received14November2017emsp |emsp Revised12January2018emsp |emsp Accepted12January2018DOI 101002mbo3600

O R I G I N A L R E S E A R C H


Songlin Liu12 emsp|emspZhijian Jiang1emsp|emspYiqin Deng3emsp|emspYunchao Wu12emsp|emspJingping Zhang1emsp|emsp Chunyu Zhao12emsp|emspDelian Huang12emsp|emspXiaoping Huang12emsp|emspStacey M Trevathan-Tackett4

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicensewhichpermitsusedistributionandreproductioninanymediumprovided the original work is properly citedcopy2018TheAuthorsMicrobiologyOpen published by John Wiley amp Sons Ltd

Joint first author

1Key Laboratory of Tropical Marine Bio-resources and Ecology South China Sea InstituteofOceanologyChineseAcademyofSciences Guangzhou China2UniversityofChineseAcademyofSciencesBeijing China3Key Laboratory of South China Sea Fishery Resources Exploitation amp Utilization Ministry ofAgricultureSouthChinaSeaFisheriesResearchInstituteChineseAcademyofFishery Sciences Guangzhou China4School of Life and Environmental Sciences Centre for Integrative EcologyDeakinUniversityVicAustralia

CorrespondenceXiaoping Huang South China Sea Institute of OceanologyChineseAcademyofSciencesGuangzhou ChinaEmail xphuangscsioaccn

Funding informationNational Basic Research Program of China GrantAwardNumber2015CB452902National Basic Research Program of China GrantAwardNumber2015CB452905National Natural Science Foundation of ChinaGrantAwardNumbernos41730529National Specialized Project of Science andTechnologyGrantAwardNumber2015FY110600

AbstractEutrophication can play a significant role in seagrass decline and habitat loss Microorganisms in seagrass sediments are essential to many important ecosystem pro-cesses including nutrient cycling and seagrass ecosystem health However current knowledge of the bacterial communities both beneficial and detrimental within sea-grass meadows in response to nutrient loading is limited We studied the response of sediment bacterial and pathogen communities to nutrient enrichment on a tropical sea-grass meadow in Xincun Bay South China Sea The bacterial taxonomic groups across all sites were dominated by the Gammaproteobacteria and Firmicutes Sites nearest to the nutrient source and with the highest NH4

+ and PO43minus content had approximately

double the relativeabundanceofputativedenitrifiersVibrionalesAlteromonadalesand Pseudomonadales Additionally the relative abundance of potential pathogengroups especially Vibrio spp and Pseudoalteromonas spp was approximately 2- fold greater at the sites with the highest nutrient loads compared to sites further from the source These results suggest that proximity to sources of nutrient pollution increases the occurrence of potential bacterial pathogens that could affect fishes invertebrates and humans This study shows that nutrient enrichment does elicit shifts in bacterial community diversity and likely their function in local biogeochemical cycling and as a potential source of infectious diseases within seagrass meadows

K E Y W O R D S

bacteria denitrification eutrophication putative pathogens seagrass meadows

2 of 11emsp |emsp emspensp LIU et aL

within the rhizosphere and surrounding sediments (Christiaen et al 2013 Hemminga amp Duarte 2000 Marbagrave Holmer Gacia amp Barron 2007) Most of these processes are primarily driven by bacterial com-munities (Joslashrgensen 1982) For example nitrogen fixation by cyano-bacteria and sulfate- reducing bacteria can significantly contribute to seagrass nutrient requirements (Hansen Udy Perry Dennison amp Lomstein 2000 Welsh 2000) In addition the presence of bacterial pathogens in coastal waters and sediments are important indicators of environmental health (Bally amp Garrabou 2007 Luna et al 2010) In fact seagrass meadows have been shown to be involved in reducing the abundance of bacterial pathogens linked to infections and diseases in humans and marine organisms (Lamb et al 2017)

Unfortunately seagrass beds have been severely degraded by anthropogenic disturbances and climate change with rapid rate of decline worldwide (~7 per year Waycott et al 2009) In particular seagrass beds have been adversely affected by eutrophication and this pressure is predicted to increase over the coming decades due to increased coastal aquaculture coastal development and concur-rent runoff (Burkholder Tomasko amp Touchette 2007 Ralph Tomasko Moore Seddon amp Macinnis- Ng 2006) In addition to directly affect-ing seagrass health through light reduction ammonium toxicity and water- column nitrate inhibition (Burkholder et al 2007) eutrophi-cation has been shown to affect sediment bacterial metabolism and function (Howard Perez Lopes amp Fourqurean 2016 Loacutepez Duarte Vallespinoacutes Romero ampAlcoverro 1998) Loacutepez etal (1998) foundthat the addition of inorganic nitrogen to Posidonia oceanica sediments significantly increased ammonification rates and bacterial exoenzy-matic activities which resulted in enhanced bacterial decomposition ofseagrass-derivedcarbonAdditionally thebacterialpathogenanddisease occurrence have been consistently correlated with high nu-trient loads in near- shore environments (National Research Council 2000 Vega Thurber et al 2014 Zaneveld et al 2016) However

studies that investigate the effect of nutrient loading on sediment bac-terial community structure within seagrass meadows are otherwise rare (Guevara Ikenaga Dean Pisani amp Boyer 2014) Filling this gap could help us understand the factors driving nutrient cycling and the potential for disease outbreaks resulting from nutrient loading within seagrass meadows

In this study we used Next- generation sequencing to answer the question To what extent does nutrient enrichment affect the sediment bacterial community structure including putative pathogens in a sea-grass meadow We sampled sediments from mixed seagrass commu-nities with increasing distance from nutrient loads coming from fish cages in Xincun Bay South China Sea (Liu et al 2016 Zhang Huang amp Jiang 2014) Our previous research has indicated that high nutrient levels in Xincun Bay stimulate the seagrass macroalgal and epiphytic biomass production resulting in higher organic carbon availability for bacteria or contribution to sediment carbon stocks (Liu et al 2016 2017) We hypothesized that higher nutrient loads from the fish cages will alter the bacterial community structure and bacterial pathogens in seagrass sediments The results of this study on how microbial com-munity composition changes under eutrophication scenarios will help clarify the reciprocal relationships of microbes and the biogeochemical environment in degraded seagrass meadows and increase our under-standing of the microbial ecology in these ecologically and socioeco-nomically important ecosystems

2emsp |emspMATERIALS AND METHODS

21emsp|emspStudy area and sampling sites

The study was performed at Xincun Bay (18deg24prime34primeprimeNndash18deg24prime42primeprimeN109deg57prime42primeprimeEndash109deg57prime58primeprimeE) located in southeastern Hainan IslandSouth China Sea (Figure 1) In recent years cage aquaculture located

F IGURE 1emspSampling sites in Xincun Bay at Hainan Island in the South China Sea which were divided by distance from the cageaquaculture(transectsABandC)and distance from the shore (stations 1 2 and 3)

emspensp emsp | emsp3 of 11LIU et aL

near the entrance of the bay has developed rapidly and currently includes more than 450 floating cage units (Zhang etal 2014)At the south-eastern part of the fish farm shallow- water seagrass meadows dominated by Thalassia hemprichii(EhrenbexSolms)AschandEnhalus acoroides (Lf) Royle occupy an area of approximately 200 ha (Huang et al 2006) Three transects(ABC)wereselectedaccordingtothedistancetofishfarmin the seagrass bed and represented a nutrient load gradient (Liu et al 2016) The first transect was 500 m from the fish farm with the other transects ~800 m apart from each other For each transect independent samples were taken at sampling stations at 50 400 and 750 m from the shore (stations 1 2 and 3 respectively) E acoroides was not found at sta-tions B1 C1 and C2 and T hemprichiiwasnotfoundatA2StationsA1A3B2B3andC3includedbothseagrassspecies(Figure1)Eachsamplewas annotated according to transect station and seagrass species for ex-ample T hemprichiiinA1canberepresentedasA1T

22emsp|emspSample collection

During low tide (~ 10 cm depth) 1 L of seawater above the sediment was sampled for nutrient analysis Additionally a surface sedimentsample (0ndash3cm)wascollected inbothT hemprichii and E acoroides meadows at each site using a sterile 10 cm diameter sampling core Each sediment sample was divided into two subsamples one subsam-plewasfrozenatminus20degCforsedimentorganiccarbon(SOC)andsedi-ment total nitrogen (TN) analysis The other subsample was preserved atminus80degCuntilextractionofgenomicDNA

23emsp|emspSample preparation and analysis

231emsp|emspSeawater nutrients SOC and sediment TN analysis

The seawater was filtered onto precombusted GFF filters (Whatman 450degC 3 hr) The filtered seawater was analyzed for NO3

minusndashNNO2

minusndashN NH4+ndashN and PO4

3minus using an AQ-2 Automated DiscreteAnalyzer (Seal Analytical Inc) The sum of NO3

minusndashN NO2mdashN and

NH4+ndashNrepresentsthetotalconcentrationofdissolvedinorganicni-

trogen(DIN)Thefrozen(minus20degC)sedimentsampleswerefreeze-driedand composited by plot in order to make the samples homogeneous The composite samples were sieved through a 500- μm screen to re-move coarse sediment and detrital materials The samples were then ground and homogenized with a mortar and pestle Sediment was acidified with 1 molL HCl overnight at room temperature to remove carbonateAcidifiedsedimentswerewashedwithdistilledwateranddried at 40degC in an oven SOC (acidified) and sediment TN (unacidi-fied) was determined using an elemental analyzer (Vario EL Elemental AnalysersystemeGmbHGermany)

232emsp|emspDNA extraction PCR amplification and Illumina sequencing

DNAwas extracted from 05g of sediment (wet weight) using anEZNAreg Soil DNA Kit (Omega Bio-Tek Inc Norcross GA USA)

according to the manufacturerrsquos instructions The quality and quantity of the extractedDNAwere verified bymeasuring theOD260280(asymp18)withaNanodrop2000spectrophotometerandagarosegelelec-trophoresis respectively Primer set 515F806R was used to amplify theV4regionof16SrRNAgene (Mckirdyetal2010)Sequencingwas performed on an Illumina MiSeq platform at Novogene Genomics Technology Co Ltd Beijing China

233emsp|emspSequence analyses OTU clustering and bioinformatics analysis

Sequences were analyzed using the QIIME version 191 pipeline(Caporaso et al 2010b) Raw sequences were demultiplexed and quality filtered using the default parameters in QIIME Sequenceswere then clustered into operational taxonomic units (OTUs) which was defined as gt97 16S rRNA gene sequence similarity usingUCLUST (Edgar 2010) with the open reference clustering proto-col The resulting representative sequences set were aligned using PyNAST(Caporasoetal2010a)andgivenataxonomicclassificationusing RDP (Wang Garrity Tiedje amp Cole 2007) retrained with the Greengenes version 135 (McDonald et al 2012) The resulting OTU table was used to determine the Chao 1 Shannon and rarefaction di-versityindicesusingQIIMEMETAGENassistwasusedforWeightedUnifracanalysis(Arndtetal2012)Inadditionwereviewedpaperscontaining information pertinent to the putative pathogens (genus level) that can cause fish and invertebrate disease or death or cause humanillnessthroughtheconsumptionofseafoodorskincontactAlist of putative pathogen OTUs (total abundance of all samples gt01) was compiled for further statistical analysis

24emsp|emspStatistical analysis

The raw data were log- or exponent- transformed in order to fulfill the assumptions of homogeneity and normality in cases where these assumptions were not met Since the effect of nutrient loading was the treatment we were primarily interested in testing we ran a pre-liminaryone-wayanalysisofvariance(ANOVA)toexaminetheeffectof distance to shore on seawater nutrients (DIN NO3

minusndashNNO2minusndashN

NH4+ndashNandPO4

3minus) SOC sediment TN and sediment CN Distance from the shore was not a significant treatment affecting the response variables (pgt05)andthuswerepooledforfurtherstatisticaltestsAone-wayANOVAtestedtheeffectoftransectonseawaternutrientswhile a two-wayANOVAanalyzed the effects of transectand sea-grass species on the SOC sediment TN and sediment CN Whenever significantdifferenceswereobservedfromanANOVAaTukeyrsquosposthoc test was run to identify the significantly different components of dependent variables

The weighted UNIFRAC resemblance matrix (Table S1) wasused for the bacterial community analyses while the filtered puta-tive pathogen counts were used for pathogen community analyses For both datasets a preliminary one- way permutational multivar-iate ANOVA (PERMANOVA) showed that distance to shore wasnot a significant treatment (Pseudo- F = 08236 P-perm = 0569)


A two-way PERMANOVAwas subsequently performed to deter-mine the statistically significant differences among the transects andseagrassspeciesAllmainandinteractioneffectswithα lt 005 were considered statistically significant A similarity percentageanalysis (SIMPER) was used to identify the bacterial and patho-genic taxa driving the differences in treatments The relationships between environmental parameters (seawater nutrients SOC and sediment TN) and the entire bacterial community relative abun-dance data were evaluated by distance- based redundancy analyses (db-RDA)(LegendreampAnderson1999)Theabove-mentionedsta-tisticalanalyseswereperformedwithPRIMER6ampPERMANOVA+(Clarke amp Gorley 2006) and IBM SPSS Statistics 190 software respectively

3emsp |emspRESULTS

31emsp|emspVariations of seawater nutrient and sediment parameters

The DIN and PO43minus concentrations of the seawater ranged from 457

to 1529 μmolmiddotLminus1 and 032 to 097 μmolmiddotLminus1 respectively (Table 1) The DIN species was dominated by NH4

+ Significantly higher DIN (F = 7571 p = 000) NH4

+ (F = 13572 p = 000) and PO43minus (F = 1473

p=005) concentrationswere found in transectA compared to theother transects (Tukeyrsquos post hoc test) The SOC sediment TN and sediment CN ranged from 009 to 073 0015 to 0053 and 740 to 1607 respectively with the highest SOC TN and CN found at site A1T (Table1) Additionally the sediment CN ratios were not observed to be significantly different among transects (F = 208 p = 188) however there was significantly higher SOC (F = 8936 p = 009) and sediment TN (F = 1653 p = 001) content observed in transectAcomparedto theother transects (Tukeyrsquosposthoc test)SOC sediment TN and sediment CN were not affected by the sea-grass species and the interactions of transect and seagrass species (p gt 05)

32emsp|emspTotal bacterial community structure

Sequencing of the sediment microbial communities using the 16S rRNAgene resulted inmore than388k sequencesand25kOTUsfrom all the sampling sites On average each sample had ~ 28000 se-quences and ~1850 OTUs (Table S2) The α- diversity indices (Chao1 Shannon and rarefaction) indicated that there was lower diversity in transectAcomparedtotheothertransects(TableS2)

Though there was no significant effect on bacterial community structure associated with seagrass species (Pseudo- F = 21021 P-perm = 0115) or the transect x species interaction (Pseudo- F = 1988 P-perm = 0129) the effect of transect location on the bac-terial communities was marginally significant (Pseudo- F = 23613 P-perm = 0079) SIMPER analysis showed that the 10 most abun-dant orders accounted for more than 60 of the dissimilarity be-tween transects and was dominated by the Gammaproteobacteria andFirmicutes(Figure2andFigureS1TableS3)IntransectAtheaverage relative abundance of Vibrionales Alteromonadales andPseudomonadales belonging to denitrifying bacteria groups were up to twofold higher compared to the other two transects while the Bacillales and Clostridiales were higher in transects B and C (Figure 2) Furthermore the relative abundance of Clostridiales BacillalesandExiguobacterales increasedfromtransectAtotran-sect C while that of Pseudomonadales showed the opposite trend (Figure 2)

Thedb-RDAanalysesofthe16SrRNAgenedataexplained481of thevariation in the first two axes (Figure3)The db-RDA analy-ses confirmed the clear separation of transects according to above- mentioned environmental factors although much of this difference lookstobedrivenbyonesample(A1T)Axis2(db-RDA2)separatedthe sites furthest from fish farms sites (including transect B and C) fromthenearersites(transectA)inXincunBayThetransectprofilesreflected an apparent shift in SOC and nutrient concentrations be-tweenAandBC(Figure3)TherewerestrongnegativecorrelationsbetweenSOCanddb-RDA1(r=minus755Table2)andstrongpositive

TABLE 1emspSeawater nutrients and sediment elemental content among all the sampling stations

Parameters

Stations

A1 A2 A3 B1 B2 B3 C1 C2 C3

DIN (μmolmiddotLminus1) 1286 1529 1271 562 516 641 487 551 457

NH4+ (μmolmiddotLminus1) 1068 1246 1138 407 375 488 356 386 309

NO3minus (μmolmiddotLminus1) 194 267 112 135 122 148 114 148 129


PO43minus (μmolmiddotLminus1) 097 068 073 042 047 032 045 040 035

Thalassia hemprichii SOC () 073 033 016 014 017 013 009 014

Sediment TN () 0053 0039 0025 0021 0018 0015 001 0013

Sediment CN 1607 987 747 778 1102 1011 105 1256

Enhalus acoroides SOC () 026 022 032 01 016 015

Sediment TN () 0041 0029 0035 0015 0025 0021

Sediment CN 740 885 1067 778 747 833


correlations between NH4+ (r = 532) PO4

3minus (r=52) and db-RDA2respectively (Table 2)

33emsp|emspPutative pathogens

There were 18 potentially opportunistic putative pathogens found in the seagrass sediments in this study including those associated with human fish invertebrate and mammal diseases (Table 3) The puta-tive pathogens (at genus level) accounted for about 2425 of total bacterial community among all the transects In general these taxa presentedinhigherrelativeabundancesintransectAparticularlyatthe T hemprichii stations in transectA thanotherallotherstations

(Figure4andFigureS2)AlthoughthePERMANOVA indicatedthatthe overall bacterial community composition was not significantly dif-ferent among the three transects total pathogenic relative abundance intransectAwas18timesthatoftheothertwotransectsTheaver-agedissimilaritiesoftransectAversustransectBandtransectAver-sus transect C were twice as that of transect B versus transect C (Table S4)AbundancedifferencesinVibrio spp and Pseudoalteromonas spp contributed to more than 60 of the dissimilarities between transect A and the other transects (Table S4)Moreover the relative abun-dances of Vibrio spp and Pseudoalteromonas spp were both more thantwofoldhigherintransectAcomparedtotransectsBandC

4emsp |emspDISCUSSION

The objective of this work was to study the effects of aquaculture- associated nutrient loading on the bacterial community structure in seagrass sediments including the presence of putative pathogens Overall the nutrient concentrations of this study were generally higher than other nutrient-impacted seagrass beds (Apostolaki HolmerMarbagrave amp Karakassis 2010 Guevara et al 2014) which can mainly

F IGURE 2emspMicrobial community composition of the top 10 most abundant orders averaged over each transect Values show means and 1 standard error (n=4ndash5)Relative abundances at each site are provided in Figure S1

F IGURE 3emspDistance-basedRedundancyAnalysis(db-RDA)ordinationofmicrobialcommunitydata(WeightedUNIFRACresemblance matrix calculated from relative abundance data) fitted toenvironmentalvariablesTheplotrepresentsadb-RDAordinationbasedupontheBrayndashCurtisdistanceofallthesamplingsitesCorrelations can be found in Table 2

TABLE 2emspMultiple partial correlations between Distance- based RedundancyAnalysis(db-RDA)coordinateaxesandenvironmentalvariables

Variable db- RDA1 db- RDA2

SOC () minus0755 0334

Sediment TN () minus0342 minus0263

DIN (μmolL) minus0264 0418

NH4+ (μmolL) 0022 0532

NO3minus (μmolL) 0112 0148

NO2minus (μmolL) minus0398 minus0262

PO43minus (μmolL) 0269 052


Taxon Infectious organisms References

Arcobacter spp Human Collado Inza Guarro and Figueras (2008)

Bacillus spp Fishes and invertebrates Webster(2007)andAustinAustinAustinandAustin(2012)

Chryseobacterium spp Fishes Austinetal(2012)

Clostridium spp Human Gorbach and Thadepalli (1975)

Corynebacterium spp Human Roux et al (2004)

Edwardsiella spp Human and Fishes Bullock and Herman (1985) and ObasohanAgbonlahorandObano(2010)

Flavobacterium spp Fishes Farkas (1985)

Francisella spp Fishes Mauel Soto Moralis and Hawke (2007)

Halomonas spp Human Stevens Hamilton Johnson Kim and Lee (2009)

Mycobacterium spp Human and Fishes Primm Lucero and Falkinham (2004) and Watral and Kent (2007)

Mycoplasma spp Human and invertebrates Paillard Le Roux and Borrego (2004) and Waites Katz and Schelonka (2005)

Pseudoalteromonas spp Invertebrates Chistoserdov Gubbala Smolowitz Mirasol and Hsu (2005)

Pseudomonas spp Human and invertebrates Gilardi (1972) and Webster (2007)

Psychrobacter spp Human Bowman (2006)

Shewanella spp Human and invertebrates Li et al (2010) and Janda (2014)

Streptococcus spp Fishes Baeck Kim Gomez and Park (2006)

Tenacibaculum spp Fishes Austinetal(2012)

Vibrio spp Human fishes and invertebrates

Colwell and Grimes (1984) Janda PowersBryantandAbbott(1988)and Vaseeharan and Ramasamy (2003)

TABLE 3emspAlistofputativepathogensofhuman fishes and invertebrates identified in this study

F IGURE 4emspPresence of putative pathogens at the genus level averaged over the three transects Values show means and 1 standard error (n=4ndash5)Relativeabundances at each site are provided in Figure S2


be attributed to the large amount of floating fish cage units in a nearly closed- system Xincun Bay On a local scale the nutrient concentra-tions in the seawater were highest in the areas closest to the fish farm-ing area which was similar to that observed in previous studies carried out in Xincun Bay (Zhang et al 2014) Liu et al (2016) revealed that the SOC source in seagrass meadows of Xincun Bay was of marine autochthonous origin with more algal organic carbon contribution in the high nutrient areas This indicated that the aquaculture- induced nutrient loading enhanced more labile organic carbon inputs and the import of aquaculture- sourced particulate organic matter to seagrass meadows was negligible Therefore the results of this study provided evidence that eutrophic inputs including labile organic carbon inputs could be shifting the sediment bacterial community toward functional groups that can utilize the excess nutrients The elevated denitrifying communities and putative pathogens in the high nutrient- loaded area (transectA)coupledwiththereducedseawaternutrientloadandshiftin microbial communities in transects B and C also suggest that the seagrass plants themselves and their associated microbial communi-ties could be playing a role in minimizing the influence and spread of nutrients and pathogens to nearby areas potentially through meta-bolic and biogeochemical cycling and filtration

41emsp|emspCharacteristics of bacterial and pathogen community structure

In this study high bacterial community diversity (Shannon index of this study372ndash872otherstudies371ndash400IkenagaGuevaraDeanPisani amp Boyer 2010 Guevara et al 2014) could contribute to the maintenance function and stability of the environment and thus im-prove the resilience of ecosystems suffering from human disturbance (Parnell Crowl Weimer amp Pfrender 2009) Consistent with a previ-ous study in Xincun Bay (Jiang et al 2015) the Gammaproteobacteria and Firmicutes were the dominant bacterial taxonomic groups in the surface sediments Fish farming typically generates a large amount of feces that are abundant in Firmicutes (Wu et al 2010) which explains the relative high abundance of Firmicutes in this study However this study showed some dissimilar results compared with other regional seagrass meadowsrsquo studies that showed sediments pri-marily dominated by Deltaproteobacteria and Bacteroidetes (Garciacutea- Martiacutenez et al 2009 Guevara et al 2014 Ikenaga et al 2010) Most Deltaproteobacteria are sulfate reducers and occur in anaerobic con-ditions (de Moraes Franco Pellizari amp Sumida 2014 Loacutepez- Garciacutea et al 2003) Bacteroidetes were associated with substrates rich in or-ganic carbon (Fierer Bradford amp Jackson 2007) The sandy substrate and the low sediment organic matter in the seagrass beds of Xincun Bay (Liu et al 2016 2017) likely promote deeper oxygen penetra-tion which could reduce the microbially mediated sulfate reduction metabolic pathways in sediments (Bourque et al 2015) This possibly explains the relatively low abundances of the Deltaproteobacteria and Bacteroidetes in this study

Alongsimilartrendsthepathogenicgroupsweremainlyassignedto Gammaproteobacteria and Firmicutes Our sequences aligned with 18 of 42 potentially pathogenic genera described in Lamb et al (2017)

Lamb et al (2017) reported that the relative abundance of potentially pathogenic genera was less than 1 in seawater in seagrass meadows atSpermondeArchipelago IndonesiaThis concentrationwasmuchlower than the sediment pathogenic relative abundance (24) in this study It has been previously shown that pathogen abundance can be up to 100- fold greater in sediment when compared with the water column (Ghaderpour et al 2014 Perkins et al 2014) and this may indicate that the sediment in seagrass meadows may be a sink for pathogens in the water column that move across a seagrass canopy However more work needs to be done to link the high relative abun-dances of putative pathogenic genera to the actual pathogenic species or strain as well as to actual cell counts in order to fully understand the risks of pathogenic accumulation to human and ecosystem health

42emsp|emspResponses of bacterial and pathogenic community structure to nutrient load

We found preliminary evidence that sediment bacterial commu-nity structure could be influenced by the proximity to point source nutrients loads The db-RDA2 axis indicated high nutrient load sampling stations were separated from the other sampling stations although the PERMANOVA results indicated no statistical differ-ence These disparate results were likely due to the variability in seagrass meadow diversity as well as low replication sampling within a transectHoweverdespite the lowstatisticalpower thedb-RDAanalysis indicated that NH4

+ and PO43minus contents were important driv-

ers of the microbial community structure between transectA andtransects B and C that is proximity to nutrient source It was pre-viously shown that microbial communities of salt marsh sediments can also be stabilized in response to nutrient loading (Bowen et al 2011 Kearns et al 2016) In this study it is possible that NH4

+ and PO4

3minus typically the limiting factors for heterotrophic bacteria produc-tion (Kirchman 1994 Thingstad Zweifel amp Rassoulzadegan 1998 Wheeler amp Kirchman 1986) are directly synthesized into bacterial biomass and promoting bacterial growth Furthermore higher eu-trophic conditions can also lead to higher labile organic carbon inputs whichwasapparentintransectA(Liuetal2016)Elevatednutrientload in combination with labile organic carbon availability favors the fast- growing r- strategist microbes (Fernandes Kirchman Michotey Bonin amp LokaBharathi 2014 Pinhassi amp Berman 2003 Trevathan- Tackett et al 2017) and is likely linked to the higher relative abun-dances ofGammaproteobacteria likeVibrionales AlteromonadalesandPseudomonadalesintransectA

In general the Gammaproteobacteria represent abundant denitri-fying communities in marine sediments (Bhatt Zhao Monteil- Rivera amp Hawari 2005) Here the results indicate that the carbon- limited conditions of this study area indicated by low sediment CN ratios and SOC content (this study average value was 022 global data average value was 18 Kennedy et al 2010) and high proportion of microbial biomass carbon in SOC (previous study in Xincun Bay average value was 1574 Liu et al 2016) would favor denitrifi-cation processes (Burgin amp Hamilton 2007) The process of nitrate being converted to N2 would be enhanced in high nutrient areas due


to the potential high denitrification activity This may be benefiting nearby areas by reducing nitrate loads in areas closest to the fish farming area Conversely at the relative low nutrient concentration areas there were increases in Firmicutes including members of the Bacillales Clostridiales and Exiguobacterales Liu et al (2016 2017) have found that bacterial biomass and labile organic carbon decreased with decreasing nutrient concentrations in Xincun Bay The Firmicutes is relative stable group of bacteria under various substrate availability conditions (Kampmann et al 2012) therefore the increase in their rel-ative abundance at the lower nutrient transects might be due to the relative shifts in other bacterial groups

The pathogenic population was about twofold greater in high nutri-ent load areas than that of the relatively lower nutrient areas This was consistent with previous theoretical and empirical studies that nutrient enrichment often enhances pathogen abundance due to high resource availability including abundant labile carbon substrates (Johnson et al 2010 Lafferty amp Holt 2003 Liu et al 2016 McKenzie amp Townsend 2007) Previous work on the causative agent of cholera (Vibrio cholerae) provided a good example of how a pathogen can be affected by nutri-ent load and related marine plankton bloom (McKenzie amp Townsend 2007 Vezzulli Pruzzo Huq amp Colwell 2010) Secondly high nutrient loads have led to a decline in seagrass biomass and cover in this study area (Liu et al 2016) which could reduce their lsquopathogen filteringrsquo abilities within the seagrass meadows (Lamb et al 2017) Thirdly the elevated nutrient load could also increase pathogen fitness and vir-ulence or change host susceptibility (Johnson et al 2010 McKenzie amp Townsend 2007) In this study Vibrio spp and Pseudoalteromonas spp were mainly responsible for the differences in pathogen abun-dances under different nutrient conditions The nutrient load induced by fish farms could heighten the prevalence risk of infectious diseases in natural fishery resources as well as aquaculture organisms in Xincun Bay(IwamotoAyersMahonampSwerdlow2010Johnsonetal2010Oetamaetal2016)Additionallytherearemorethan12speciesofVibrio known as human pathogens (Blazer 1988) resulting in disease as a consequence of toxin intake (Martinez- Urtaza et al 2012 Oetama et al 2016) Furthermore the sediment pathogens can be grazed as-sociated with sediment organic matter at the bottom by meiofauna worms prawns cockles and demersal fishes sequentially may move up the food chain and reach humans (Ghaderpour et al 2014)

5emsp |emspCONCLUSION

We found that higher nutrient load into a seagrass meadow elevated the relative abundance of denitrifying communities as well as poten-tial pathogen groups Changes of sediment bacteria and pathogen relative abundance not only should be attributed to nutrient enrich-ment but also associated labile organic carbon On one hand these findings imply that high nutrient enrichment enhanced the potential denitrification activity yet present a risk of infectious diseases in na-ture What is unknown however is how the bacterial and pathogenic activitieschangeinresponsetonutrientloadingAsamatterforfu-ture research we recommend meta- transcriptomic and metabolomics

analyses of seagrass sediment microbial communities to evaluate functional gene expression associated with nutrient cycling and viru-lence products among different nutrient load levels

ACKNOWLEDGMENTS

This research was supported by the National Basic Research Program of China (2015CB452905 2015CB452902) the National Natural Science Foundation of China (nos 41730529) and the National Specialized Project of Science and Technology (2015FY110600)

CONFLICT OF INTEREST

None declared

ORCID

Songlin Liu httporcidorg0000-0002-6478-4938

REFERENCES

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ArndtDXiaJLiuYZhouYGuoACCruzJASinelnikovIBudwillKNesboslashCLWishartDS(2012)METAGENassistAcomprehen-sive web server for comparative metagenomics Nucleic Acids Research 40W88ndashW99httpsdoiorg101093nargks497

Austin B Austin DA Austin B ampAustin DA (2012) Bacterial fishpathogens Heidelberg Germany Springer

Baeck G W Kim J H Gomez D K amp Park S C (2006) Isolation and characterization of Streptococcus sp from diseased flounder (Paralichthys olivaceus) in Jeju Island Journal of Veterinary Science 7 53ndash58httpsdoiorg104142jvs20067153

Bally M amp Garrabou J (2007) Thermodependent bacterial pathogens andmassmortalities in temperatebenthiccommunitiesAnewcaseof emerging disease linked to climate change Global Change Biology 132078ndash2088httpsdoiorg101111j1365-2486200701423x

Bhatt M Zhao J-S Monteil-Rivera F amp Hawari J (2005) Biodegradation of cyclic nitramines by tropical marine sediment bacteria The Journal of Industrial Microbiology and Biotechnology 32261ndash267httpsdoiorg101007s10295-005-0239-9

Blazer V S (1988) Bacterial fish pathogens Environmental Biology of Fishes 2177ndash79httpsdoiorg101007BF02984445

BourqueASVega-ThurberRampFourqureanJW(2015)Microbialcom-munity structure and dynamics in restored subtropical seagrass sedi-ments Aquatic Microbial Ecology 7443ndash57httpsdoiorg103354ame01725

Bowen J L Ward B B Morrison H G Hobbie J E Valiela I Deegan LAampSoginML(2011)Microbialcommunitycompositioninsed-iments resists perturbation by nutrient enrichment ISME Journal 5 1540ndash1548httpsdoiorg101038ismej201122

Bowman J P (2006) The genus Psychrobacter In A Balows H GTruumlper M Dworkin W Harder amp K H Schleifer (Eds) The pro-karyotes (pp 920ndash930) New York NY USA Springer httpsdoiorg1010070-387-30746-X

Bullock G amp Herman R L (1985) Edwardsiella infections of fishes US Fish and Wildlife Service Fish disease leaflet 71 Kearneysville West Virginia


BurginAJampHamiltonSK(2007)Haveweoveremphasizedtheroleofdenitrification inaquaticecosystemsA reviewofnitrate removalpathways Frontiers in Ecology and the Environment 589ndash96httpsdoiorg1018901540-9295(2007)5[89HWOTRO]20CO2

BurkholderJMTomaskoDAampTouchetteBW (2007)Seagrassesand eutrophication Journal of Experimental Marine Biology and Ecology 35046ndash72httpsdoiorg101016jjembe200706024

CaporasoJGBittingerKBushmanFDDeSantisTZAndersenGLampKnightR(2010a)PyNASTAflexibletoolforaligningsequencesto a template alignment Bioinformatics 26 266ndash267 httpsdoiorg101093bioinformaticsbtp636

Caporaso J G Kuczynski J Stombaugh J Bittinger K Bushman F DCostello EK FiererN PentildeaAGGoodrichJKGordonJIHuttleyGA (2010b)QIIME allows analysis of high-throughputcommunity sequencing data Nature Methods 7335ndash336httpsdoiorg101038nmethf303

ChistoserdovAY Gubbala S L Smolowitz RMirasol F amp HsuA(2005)AmicrobiologicalassessmentofepizooticshelldiseaseintheAmericanlobsterindicatesitsstrictlydermaletiologyLobster shell dis-ease workshop(pp12ndash20)BostonMAUniversityofMassachusetts

ChristiaenBMcDonaldACebrianJampOrtmannAC(2013)Responseof the microbial community to environmental change during seagrass transplantation Aquatic Botany 10931ndash38httpsdoiorg101016jaquabot201303008

Clarke K amp Gorley R (2006) User manualtutorial Plymouth PRIMER-E Ltd 93

Collado L Inza I Guarro J amp Figueras M J (2008) Presence of Arcobacter spp in environmental waters correlates with high levels of fecal pollution Environmental Microbiology 101635ndash1640httpsdoiorg101111j1462-2920200701555x

Colwell R amp Grimes D (1984) Vibrio diseases of marine fish populations Helgolaumlnder Meeresunters 37 265ndash287 httpsdoiorg101007BF01989311

de Moraes P C Franco D C Pellizari V H amp Sumida P Y G (2014) Effect of plankton- derived organic matter on the microbial community of coastal marine sediments Journal of Experimental Marine Biology and Ecology 461257ndash266httpsdoiorg101016jjembe201408017

Edgar R C (2010) Search and clustering orders of magnitude faster thanBLASTBioinformatics 262460ndash2461httpsdoiorg101093bioinformaticsbtq461

Farkas J (1985) Filamentous Flavobacterium sp isolated from fish with gill diseases in cold water Aquaculture 44 1ndash10 httpsdoiorg1010160044-8486(85)90037-7

Fernandes S O Kirchman D L Michotey V D Bonin P C amp LokaBharathi P (2014) Bacterial diversity in relatively pristine and anthropogenically- influenced mangrove ecosystems (Goa India) The Brazilian Journal of Microbiology 45 1161ndash1171 httpsdoiorg101590S1517-83822014000400006

FiererNBradfordMAampJacksonRB (2007)Towardanecologicalclassification of soil bacteria Ecology 88 1354ndash1364 httpsdoiorg10189005-1839

Garciacutea-MartiacutenezMLoacutepez-LoacutepezACallejaMLMarbagraveNampDuarteCM (2009) Bacterial community dynamics in a seagrass (Posidonia oce-anica) meadow sediment Estuaries and Coasts 32276ndash286httpsdoiorg101007s12237-008-9115-y

GhaderpourANasoriKNMChewLLChongVCThongKLampChai L C (2014) Detection of multiple potentially pathogenic bacteria in Matang mangrove estuaries Malaysia Marine Pollution Bulletin 83 324ndash330httpsdoiorg101016jmarpolbul201404029

Gilardi G (1972) Infrequently encountered Pseudomonas species causing infection in humans Annals of Internal Medicine 77211ndash215httpsdoiorg1073260003-4819-77-2-211

Gorbach S L amp Thadepalli H (1975) Isolation of Clostridium in human infections Evaluation of 114 cases Journal of Infectious Diseases 131 S81ndashS85httpsdoiorg101093infdis131SupplementS81

Guevara R Ikenaga M Dean A L Pisani C amp Boyer J N (2014)Changes in sediment bacterial community in response to long- term nu-trient enrichment in a subtropical seagrass- dominated estuary Microbial Ecology 68427ndash440httpsdoiorg101007s00248-014-0418-1

HansenJWUdyJWPerryCJDennisonWCampLomsteinBA(2000) Effect of the seagrass Zostera capricorni on sediment micro-bial processes Marine Ecology Progress Series 19983ndash96httpsdoiorg103354meps199083

HemmingaMAampDuarteCM (2000)SeagrassecologyCambridgeUK Cambridge University Press

HowardJLPerezALopesCCampFourqureanJW(2016)Fertilizationchanges seagrass community structure but not blue carbon storage Results from a 30- year field experiment Estuaries and Coasts 39 1422ndash1434httpsdoiorg101007s12237-016-0085-1

Huang X P Huang L M Li Y H Xu Z Z Fong C W Huang D J Han QHuangHTanYLiuS(2006)Mainseagrassbedsandthreatstotheir habitats in the coastal sea of South China Chinese Science Bulletin 51136ndash142httpsdoiorg101007s11434-006-9136-5

IkenagaMGuevaraRDeanALPisaniCampBoyerJN(2010)Changesin community structure of sediment bacteria along the Florida coastal everglades marshndashmangrovendashseagrass salinity gradient Microbial Ecology 59284ndash295httpsdoiorg101007s00248-009-9572-2

IngemannJensenSKuumlhlMGludRNJoslashrgensenLBampPriemeacuteA(2005) Oxic microzones and radial oxygen loss from roots of Zostera marina Marine Ecology Progress Series 293 49ndash58 httpsdoiorg103354meps293049

Iwamoto M Ayers T Mahon B E amp Swerdlow D L (2010)Epidemiology of seafood- associated infections in the United States Clinical Microbiology Reviews 23 399ndash411 httpsdoiorg101128CMR00059-09

Janda J M (2014) ShewanellaAmarinepathogenasanemergingcauseofhuman disease Clinical Microbiology Newsletter 3625ndash29httpsdoiorg101016jclinmicnews201401006

JandaJPowersCBryantRampAbbottS(1988)Currentperspectiveson the epidemiology and pathogenesis of clinically significant Vibrio spp Clinical Microbiology Reviews 1245ndash267httpsdoiorg101128CMR13245

Jensen S I KuumlhlM amp PriemeacuteA (2007) Different bacterial commu-nities associated with the roots and bulk sediment of the seagrass Zostera marina FEMS Microbiology Ecology 62 108ndash117 httpsdoiorg101111j1574-6941200700373x

Jiang Y-F Ling J Wang Y-S Chen B Zhang Y-Y amp Dong J-D (2015) Cultivation- dependent analysis of the microbial diver-sity associated with the seagrass meadows in Xincun Bay South China Sea Ecotoxicology 24 1540ndash1547 httpsdoiorg101007s10646-015-1519-4

JohnsonPTTownsendARClevelandCCGlibertPMHowarthR W McKenzie V J Rejmankova E Ward M H (2010) Linking environmental nutrient enrichment and disease emergence in hu-mans and wildlife Ecological Applications 20 16ndash29 httpsdoiorg10189008-06331

Joslashrgensen B B (1982) Mineralization of organic matter in the sea bedmdashthe role of sulphate reduction Nature 296 643ndash645 httpsdoiorg101038296643a0

Kampmann K Ratering S Kramer I Schmidt M Zerr W amp Schnell S (2012) Unexpected stability of Bacteroidetes and Firmicutes com-munities in laboratory biogas reactors fed with different defined substrates Applied and Environmental Microbiology 78 2106ndash2119httpsdoiorg101128AEM06394-11

KearnsPJAngellJHHowardEMDeeganLAStanleyRHampBowen J L (2016) Nutrient enrichment induces dormancy and de-creases diversity of active bacteria in salt marsh sediments Nature Communications httpsdoiorg101038ncomms12881

Kennedy H Beggins J Duarte C M Fourqurean J W Holmer M Marbagrave N amp Middelburg J J (2010) Seagrass sediments as a global


carbon sink Isotopic constraints Global Biogeochemical Cycles 241ndash8httpsdoiorg1010292010GB 003848

Kirchman D L (1994) The uptake of inorganic nutrients by heterotrophic bacteria Microbial Ecology 28 255ndash271 httpsdoiorg101007BF00166816

Lafferty K D amp Holt R D (2003) How should environmental stress af-fect the population dynamics of disease Ecology Letters 6654ndash664httpsdoiorg101046j1461-0248200300480x

Lamb J B van deWater J A Bourne D G Altier C Hein M YFiorenzaEAAbuNJompaJHarvellCD(2017)Seagrasseco-systems reduce exposure to bacterial pathogens of humans fishes and invertebrates Science 355 731ndash733 httpsdoiorg101126scienceaal1956

Legendre P amp Anderson M J (1999) Distance-based redundancyanalysis Testing multispecies responses in multifactorial ecolog-ical experiments Ecological Monographs 69 1ndash24 httpsdoiorg1018900012-9615(1999)069[0001DBRATM]20CO2

LiHQiaoGGuJ-QZhouWLiQWooS-HXuDHParkSI (2010) Phenotypic and genetic characterization of bacteria isolated from diseased cultured sea cucumber Apostichopus japonicus in north-eastern China Diseases of Aquatic Organisms 91223ndash235httpsdoiorg103354dao02254

LiuSJiangZWuYZhangJArbiIYeFHuangXMacreadiePI(2017) Effects of nutrient load on microbial activities within a seagrass- dominated ecosystem Implications of changes in seagrass blue carbon Marine Pollution Bulletin 117 214ndash221 httpsdoiorg101016jmarpolbul201701056

Liu S Jiang Z Zhang J Wu Y Lian Z amp Huang X (2016) Effect of nutrient enrichment on the source and composition of sediment organic carbon in tropical seagrass beds in the South China Sea Marine Pollution Bulletin 110 274ndash280 httpsdoiorg101016jmarpolbul201606054

Loacutepez N I Duarte C M Vallespinoacutes F Romero J amp Alcoverro T(1998) The effect of nutrient additions on bacterial activity in sea-grass (Posidonia oceanica) sediments Journal of Experimental Marine Biology and Ecology 224 155ndash166 httpsdoiorg101016S0022-0981(97)00189-5

Loacutepez-Garciacutea P Duperron S Philippot P Foriel J Susini J amp Moreira D (2003) Bacterial diversity in hydrothermal sediment and epsilon-proteobacterial dominance in experimental microcolonizers at the Mid-Atlantic Ridge Environmental Microbiology 5 961ndash976 httpsdoiorg101046j1462-2920200300495x

Luna G Vignaroli C Rinaldi C Pusceddu A Nicoletti L GabelliniM Danovaro R Biavasco F (2010) Extraintestinal Escherichia coli carrying virulence genes in coastal marine sediments Applied and Environmental Microbiology 765659ndash5668httpsdoiorg101128AEM03138-09

Marbagrave N Holmer M Gacia E amp Barron C (2007) Seagrass beds and coastalbiogeochemistryInAWDLarkumRJOrthampCMDuarte(Eds) Seagrasses Biology ecology and conservation (pp 135ndash157)Dordrecht Netherlands Springer

Martinez-Urtaza J Blanco-Abad V Rodriguez-Castro A Ansede-BermejoJMirandaAampRodriguez-AlvarezMX(2012)Ecologicaldeterminants of the occurrence and dynamics of Vibrio parahae-molyticus in offshore areas ISME Journal 6 994ndash1006 httpsdoiorg101038ismej2011156

MauelMSotoEMoralisJampHawkeJ(2007)Apiscirickettsiosis-likesyndrome in cultured Nile tilapiainLatinAmericawithFrancisella spp as the pathogenic agent Journal of Aquatic Animal Health 1927ndash34httpsdoiorg101577H06-0251

McDonald D Price M N Goodrich J Nawrocki E P DeSantis T Z ProbstAAndersenGLKnightRHugenholtzP (2012)An im-proved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea ISME Journal 6610ndash618 httpsdoiorg101038ismej2011139

McKenzieVJampTownsendAR(2007)Parasiticandinfectiousdiseaseresponses to changing global nutrient cycles EcoHealth 4384ndash396httpsdoiorg101007s10393-007-0131-3

Mckirdy D M Thorpe C S Haynes D E Grice K Krull E S Halverson G P amp Webster L J (2010) The biogeochemical evolution of the Coorongduringthemid-tolateHoloceneAnelementalisotopicandbiomarker perspective Organic Geochemistry 4196ndash110httpsdoiorg101016jorggeochem200907010

National Research Council (2000) Clean coastal watersmdashUnderstanding and reducing the problems from nutrient pollution Washington (p 450) DC NationalAcademyofSciencesPress

ObasohanEAgbonlahorDampObanoE(2010)WaterpollutionAreviewof microbial quality and health concerns of water sediment and fish in the aquatic ecosystem African Journal of Biotechnology 9423ndash427

OetamaVSPHennersdorfPAbdul-AzizMAMrotzekGHaryantiH amp Saluz H P (2016) Microbiome analysis and detection of pathogenic bacteria of Penaeus monodon from Jakarta Bay and Bali Marine Pollution Bulletin 110 718ndash725 httpsdoiorg101016jmarpolbul201603043

Paillard C Le Roux F amp Borrego J J (2004) Bacterial disease in marine bivalves a review of recent studies Trends and evolution Aquatic Living Resources 17477ndash498httpsdoiorg101051alr2004054

Parnell J J Crowl T A Weimer B C amp Pfrender M E (2009)Biodiversity in microbial communities System scale patterns and mechanisms Molecular Ecology 18 1455ndash1462 httpsdoiorg101111j1365-294X200904128x

Perkins T L Clements K Baas J H Jago C F Jones D L Malham S K amp McDonald J E (2014) Sediment composition influences spa-tial variation in the abundance of human pathogen indicator bacteria within an estuarine environment PLoS ONE 9e112951 httpsdoiorg101371journalpone0112951

Pinhassi J amp Berman T (2003) Differential growth response of colony- forming α- and γ- proteobacteria in dilution culture and nutrient addition experiments from Lake Kinneret (Israel) the Eastern Mediterranean Sea and the Gulf of Eilat Applied and Environmental Microbiology 69 199ndash211 httpsdoiorg101128AEM691199-2112003

PrimmT P LuceroCAamp FalkinhamJO (2004)Health impacts ofenvironmental mycobacteria Clinical Microbiology Reviews 1798ndash106httpsdoiorg101128CMR17198-1062004

Ralph P J Tomasko D Moore K Seddon S amp Macinnis-Ng C M (2006) Human impacts on seagrasses Eutrophication sedimentation andcontaminationInAWDLarkumRJOrthampCMDuarte(Eds)Seagrasses Biology ecology and conservation(pp567ndash593)DordrechtNetherlands Springer

Roux V Drancourt M Stein A Riegel P Raoult D amp La Scola B(2004) Corynebacterium species isolated from bone and joint in-fections identified by 16S rRNA gene sequence analysis Journal of Clinical Microbiology 42 2231ndash2233 httpsdoiorg101128JCM4252231-22332004

Stevens D A Hamilton J R Johnson N Kim K K amp Lee J-S(2009) Halomonas a newly recognized human pathogen causing infections and contamination in a dialysis center Three new spe-cies Medicine 88 244ndash249 httpsdoiorg101097MD0b013 e3181aede29

Thingstad T F Zweifel U L amp Rassoulzadegan F (1998) P limita-tion of heterotrophic bacteria and phytoplankton in the northwest Mediterranean Limnology and Oceanography 43 88ndash94 httpsdoiorg104319lo19984310088

Trevathan-Tackett S M Seymour J R Nielsen D A Macreadie PI JeffriesTC SandermanJ Baldock JHowesJM StevenAD Ralph P (2017) Sediment anoxia limits microbial- driven sea-grass carbon remineralization under warming conditions FEMS Microbiology Ecology 93 6 httpsdoiorg101093femsec fix033


Vaseeharan B amp Ramasamy P (2003) Control of pathogenic Vibrio spp by Bacillus subtilis BT23 a possible probiotic treatment for black tiger shrimp Penaeus monodon Letters in Applied Microbiology 36 83ndash87httpsdoiorg101046j1472-765X200301255x

VegaThurber R L Burkepile D E Fuchs C ShantzAAMcMindsR amp Zaneveld J R (2014) Chronic nutrient enrichment increases prevalence and severity of coral disease and bleaching Global Change Biology 20544ndash554httpsdoiorg101111gcb12450

Vezzulli LPruzzoCHuqAampColwellRR (2010)Environmentalreservoirs of Vibrio cholerae and their role in cholera Environmental Microbiology Reports 2 27ndash33 httpsdoiorg101111j1758-2229200900128x

Waites K B Katz B amp Schelonka R L (2005) Mycoplasmas and ureaplasmas as neonatal pathogens Clinical Microbiology Reviews 18 757ndash789httpsdoiorg101128CMR184757-7892005

WangQGarrityGMTiedjeJMampColeJR(2007)NaiveBayesianclassifierforrapidassignmentofrRNAsequencesintothenewbacte-rial taxonomy Applied and Environmental Microbiology 735261ndash5267httpsdoiorg101128AEM00062-07

Watral V amp Kent M L (2007) Pathogenesis of Mycobacterium spp in zebrafish (Danio rerio) from research facilities Comparative Biochemistry and Physiology Part C Toxicology amp Pharmacology 14555ndash60

Waycott M Duarte C M Carruthers T J Orth R J Dennison W C OlyarnikSCalladineAFourqureanJWHeckKLHughesARKendrickGA(2009)Acceleratinglossofseagrassesacrosstheglobethreatens coastal ecosystems Proceedings of the National Academy of Sciences of the United States of America 10612377ndash12381httpsdoiorg101073pnas0905620106

Webster N S (2007) Sponge disease A global threat Environmental Microbiology 9 1363ndash1375 httpsdoiorg101111j1462-2920 200701303x

Welsh D T (2000) Nitrogen fixation in seagrass meadows Regulation plantndashbacteria interactions and significance to

primary productivity Ecology Letters 3 58ndash71 httpsdoiorg101046j1461-0248200000111x

WheelerPAampKirchmanDL(1986)Utilizationofinorganicandorganicnitrogen by bacteria in marine systems Limnology and Oceanography 31998ndash1009httpsdoiorg104319lo19863150998

Wu C H Sercu B Van De Werfhorst L C Wong J DeSantis T Z Brodie E L Hazen T C Holden P A Andersen G L (2010)Characterization of coastal urban watershed bacterial communities leads to alternative community- based indicators PLoS ONE 5e11285 httpsdoiorg101371journalpone0011285

ZaneveldJRBurkepileDEShantzAAPritchardCEMcMindsRPayetJPWelshRCorreaAMLemoineNPRosalesSFuchsC (2016) Overfishing and nutrient pollution interact with temperature to disrupt coral reefs down to microbial scales Nature Communications 711833 httpsdoiorg101038ncomms11833

Zhang J P Huang X P amp Jiang Z J (2014) Physiological responses of the seagrass Thalassia hemprichii (Ehrenb) Aschers as indicators ofnutrient loading Marine Pollution Bulletin 83 508ndash515 httpsdoiorg101016jmarpolbul201312056

SUPPORTING INFORMATION

Additional Supporting Information may be found online in the supporting information tab for this article

How to cite this article Liu S Jiang Z Deng Y et al Effects of nutrient loading on sediment bacterial and pathogen communities within seagrass meadows MicrobiologyOpen 20187e600 httpsdoiorg101002mbo3600

coversheet

trevathan-tackett-effectsof-2018

MicrobiologyOpen 20187e600 emsp|emsp1 of 11httpsdoiorg101002mbo3600

wwwMicrobiologyOpencom

1emsp |emspINTRODUCTION

Seagrass meadows are incredibly productive ecosystems (Hemminga amp Duarte 2000) and support a high diversity of microorganisms in their sediments (Bourque Vega- Thurber amp Fourqurean 2015 Garciacutea- Martiacutenez Loacutepez- Loacutepez Calleja Marbagrave amp Duarte 2009) due to the

release of root exudates (amino acids sugars) and oxygen into the rhi-zosphere (Christiaen McDonald Cebrian amp Ortmann 2013 Ingemann Jensen Kuumlhl Glud Joslashrgensen amp Priemeacute 2005 Jensen Kuumlhl amp Priemeacute 2007) Microbes play a fundamental role in several biogeochemical cycling processes including the oxidation of organic carbon nitrogen fixation nitrification denitrification iron cycling and sulfate reduction

Received14November2017emsp |emsp Revised12January2018emsp |emsp Accepted12January2018DOI 101002mbo3600

O R I G I N A L R E S E A R C H


Songlin Liu12 emsp|emspZhijian Jiang1emsp|emspYiqin Deng3emsp|emspYunchao Wu12emsp|emspJingping Zhang1emsp|emsp Chunyu Zhao12emsp|emspDelian Huang12emsp|emspXiaoping Huang12emsp|emspStacey M Trevathan-Tackett4

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicensewhichpermitsusedistributionandreproductioninanymediumprovided the original work is properly citedcopy2018TheAuthorsMicrobiologyOpen published by John Wiley amp Sons Ltd

Joint first author

1Key Laboratory of Tropical Marine Bio-resources and Ecology South China Sea InstituteofOceanologyChineseAcademyofSciences Guangzhou China2UniversityofChineseAcademyofSciencesBeijing China3Key Laboratory of South China Sea Fishery Resources Exploitation amp Utilization Ministry ofAgricultureSouthChinaSeaFisheriesResearchInstituteChineseAcademyofFishery Sciences Guangzhou China4School of Life and Environmental Sciences Centre for Integrative EcologyDeakinUniversityVicAustralia

CorrespondenceXiaoping Huang South China Sea Institute of OceanologyChineseAcademyofSciencesGuangzhou ChinaEmail xphuangscsioaccn

Funding informationNational Basic Research Program of China GrantAwardNumber2015CB452902National Basic Research Program of China GrantAwardNumber2015CB452905National Natural Science Foundation of ChinaGrantAwardNumbernos41730529National Specialized Project of Science andTechnologyGrantAwardNumber2015FY110600

AbstractEutrophication can play a significant role in seagrass decline and habitat loss Microorganisms in seagrass sediments are essential to many important ecosystem pro-cesses including nutrient cycling and seagrass ecosystem health However current knowledge of the bacterial communities both beneficial and detrimental within sea-grass meadows in response to nutrient loading is limited We studied the response of sediment bacterial and pathogen communities to nutrient enrichment on a tropical sea-grass meadow in Xincun Bay South China Sea The bacterial taxonomic groups across all sites were dominated by the Gammaproteobacteria and Firmicutes Sites nearest to the nutrient source and with the highest NH4

+ and PO43minus content had approximately

double the relativeabundanceofputativedenitrifiersVibrionalesAlteromonadalesand Pseudomonadales Additionally the relative abundance of potential pathogengroups especially Vibrio spp and Pseudoalteromonas spp was approximately 2- fold greater at the sites with the highest nutrient loads compared to sites further from the source These results suggest that proximity to sources of nutrient pollution increases the occurrence of potential bacterial pathogens that could affect fishes invertebrates and humans This study shows that nutrient enrichment does elicit shifts in bacterial community diversity and likely their function in local biogeochemical cycling and as a potential source of infectious diseases within seagrass meadows

K E Y W O R D S

bacteria denitrification eutrophication putative pathogens seagrass meadows

















minusndashNNO2














NH4+ndashNandPO4





3emsp |emspRESULTS





+ (F = 13572 p = 000) and PO43minus (F = 1473







Parameters

Stations

A1 A2 A3 B1 B2 B3 C1 C2 C3







Sediment TN () 0053 0039 0025 0021 0018 0015 001 0013

Sediment CN 1607 987 747 778 1102 1011 105 1256


Sediment TN () 0041 0029 0035 0015 0025 0021

Sediment CN 740 885 1067 778 747 833













SOC () minus0755 0334



NH4+ (μmolL) 0022 0532





































+ and PO4









ACKNOWLEDGMENTS



None declared

ORCID


REFERENCES

































































































coversheet


















minusndashNNO2














NH4+ndashNandPO4





3emsp |emspRESULTS





+ (F = 13572 p = 000) and PO43minus (F = 1473







Parameters

Stations

A1 A2 A3 B1 B2 B3 C1 C2 C3







Sediment TN () 0053 0039 0025 0021 0018 0015 001 0013

Sediment CN 1607 987 747 778 1102 1011 105 1256


Sediment TN () 0041 0029 0035 0015 0025 0021

Sediment CN 740 885 1067 778 747 833













SOC () minus0755 0334



NH4+ (μmolL) 0022 0532





































+ and PO4









ACKNOWLEDGMENTS



None declared

ORCID


REFERENCES

































































































coversheet




3emsp |emspRESULTS





+ (F = 13572 p = 000) and PO43minus (F = 1473







Parameters

Stations

A1 A2 A3 B1 B2 B3 C1 C2 C3







Sediment TN () 0053 0039 0025 0021 0018 0015 001 0013

Sediment CN 1607 987 747 778 1102 1011 105 1256


Sediment TN () 0041 0029 0035 0015 0025 0021

Sediment CN 740 885 1067 778 747 833













SOC () minus0755 0334



NH4+ (μmolL) 0022 0532





































+ and PO4









ACKNOWLEDGMENTS



None declared

ORCID


REFERENCES

































































































coversheet














SOC () minus0755 0334



NH4+ (μmolL) 0022 0532





































+ and PO4









ACKNOWLEDGMENTS



None declared

ORCID


REFERENCES

































































































coversheet



































+ and PO4









ACKNOWLEDGMENTS



None declared

ORCID


REFERENCES

































































































coversheet








ACKNOWLEDGMENTS



None declared

ORCID


REFERENCES

































































































coversheet























































































coversheet


effects of nutrient loading on sediment bacterial and

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