h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Environmental
Microbiology
Antagonistic
endophytic
bacteria
associated
with
nodules
of
soybean
(Glycine
max
L.)
and
plant
growth-promoting
properties
LongFei
Zhao
a,∗,
YaJun
Xu
a,
XinHe
Lai
baShangqiuNormalUniversity,CollegeofLifeSciences,KeyLaboratoryofPlant-MicrobeInteractionsofHenan,Shangqiu,Henan,PR
China
bTheFirstAffiliatedHospitalofWenzhouMedicalUniversity,InstituteofInflammation&Diseases,Wenzhou,China
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received5January2017 Accepted19June2017
Availableonline13October2017 AssociateEditor:FernandoAndreote
Keywords:
Endophytes Soybean Antagonisis
Phytophthorasojae
Plantgrowth-promotingpotential
a
b
s
t
r
a
c
t
Atotalof276endophyticbacteriawereisolatedfromtherootnodulesofsoybean(Glycine
maxL.)grownin14sitesinHenanProvince,China.Theinhibitoryactivityofthese bacte-riaagainstpathogenicfungusPhytophthorasojae01wasscreenedinvitro.Sixstrainswith morethan63%inhibitoryactivitieswerefurthercharacterizedthroughoptical epifluore-scencemicroscopicobservation,sequencing,andphylogeneticanalysisof16SrRNAgene, potentialplantgrowth-promotingpropertiesanalysis,andplantinoculationassay.Onthe basisofthephylogenyof16SrRNAgenes,thesixendophyticantagonistswereidentified asbelongingtofivegenera:Enterobacter,Acinetobacter,Pseudomonas,Ochrobactrum,and Bacil-lus.ThestrainAcinetobactercalcoaceticusDD161hadthestrongestinhibitoryactivity(71.14%) againsttheP.sojae01,whichcausedmorphologicalabnormalchangesoffungalmycelia; suchchangesincludefracture,lysis,formationofaprotoplastballattheendofhyphae,and splitends.ExceptforOchrobactrumhaematophilumDD234,otherantagonisticstrainsshowed thecapacitytoproducesiderophore,indoleaceticacid,andnitrogenfixationactivity. Regres-sionanalysissuggestedasignificantpositivecorrelationbetweensiderophoreproduction andinhibitionratioagainstP.sojae01.Thisstudydemonstratedthatnoduleendophytic bacteriaareimportantresourcesforsearchingforinhibitorsspecifictothefungiandfor promotingeffectsforsoybeanseedlings.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
∗ Correspondingauthorat:CollegeofLifeSciences,ShangqiuNormalUniversity,298WenhuaMiddleRoad,Shangqiu,Henan476000,PR
China.
E-mail:hnzhaolongfei@163.com(L.Zhao).
https://doi.org/10.1016/j.bjm.2017.06.007
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
The root nodules of legume plants are symbiotic organs induced by soil bacteria known as rhizobia. As part of the root system, root nodules harbor symbiotic bacte-ria and many endophytes, including Agrobacterium tumefacien,1 A.rhizogenes,2 Phyllobacterium, Stenotrophomonas,
Enterobacteriaceae,3 Bacillus species,4 Bacillus, Bordetella,
Cur-tobacterium, and Pantoea.5 Aside from their diversity,which has been studied extensively, the effect of nodule endo-phytesonhostlegumeswasrevealed.Thenoduleendophytic
Agrobacterium strains specifically inhibit the nodulation of
Rhizobiumgallicum on the common bean(Phaseolus vulgaris
L.)6 or facilitate the unspecific nodulation of Sinorhizobium
meliloti on woody legumes.7 Somenodule endophytesthat belong toBacillus,Bordetella, Curtobacterium,or A. rhizogenes
couldpromotethegrowthornodulationofredclover.5These phenomenaaresimilartothatofendophytesisolatedfrom other partsofplantsand couldbenefithostplantsby pro-ducingphytohormones,1-aminocyclopropane-1-carboxylase (ACC) deaminase, and antibioticcompounds, aswell as by fixingnitrogen,solubilizingphosphate, orsuppressing phy-topathogens through the competence ofinvasion sites.8–11 Owingtothe abovementionedadvantages,endophytesare considerednovelresourcesinthebiocontrolofplantdiseases andinthepromotionofplantgrowth.12–14
Asamajorlegumecrop,soybean(GlycinemaxL.)playsan importantroleinsustainableagricultureandintheeconomy ofmanycountries.Soybeanhasagreatnitrogen-fixing abil-itydue toits symbiosis withrhizobiainrootnodules. The presenceofBradyrhizobiumjaponicum,B.liaoningense,B. yuan-mingense,B.elkanii,15,16 B.huanghuaihaiense,17B.daqingense,18
B.pachyrhizi,B.iriomotense,B.canariense,19Sinorhizobiumfredii, and S. sojae20 has been reported in China, which is the centeroforiginofsoybean.21,22Similartootherplants, endo-phytic bacteria have been isolated from different parts of soybean,19,23–26 andsomeofthesepartsshowed antagonis-ticandgrowth-promotingpotential.27–29Diverseendophytic bacteria, including Pantoea, Serratia, Acinetobacter, Bacillus, Agrobacterium,andBurkholderia,havealsobeenisolatedfrom soybeannodules.30However,antagonisticendophytic bacte-riawithinnodulesofsoybeanforP.sojaeinHenanProvince havenotbeensufficientlystudied.
Onthebasisoftheabovementionedbackground knowl-edgeandconsideringthenoduleendophytesasanewbacteria resourcewithpotentialinbiotechnology,weconductedthis study(1)toscreenantagonisticendophyticbacteriafrom soy-bean nodules against P. sojae; (2) to explore the potential plant-beneficialtraitsofendophyticbacteria;and(3)toassay theseedlinggrowthresponseofsoybeantotheinoculationof endophyticbacteria.
Materials
and
methods
Collectionofrootnodules,soilsamples,phytopathogenic fungus,andsoybeanseeds
NodulesfromcultivatedsoybeanwerecollectedfromJulyto August2012,whentheplantswereblooming.Sampleswere
obtainedfromfieldsof14 sitessubordinateto9districtsof HenanProvince,China(mapavailableasSupplementaryFig. 1).21,22 Three healthy rootnodules with similar sizes were excisedfromthelateralrootsofeachplant.Soildebriswas brushedawayfromthenodules,andthenoduleswerestored in sterileplastic bags at4◦C untilthey wereprocessedfor isolationwithin24h.
Ineachsite,soilcoresweresampledatfivelocationswith adepthof15–20cmand5cmawayfromthetaproots,which werebulkedandthoroughlymixedtoformcomposite sam-ples.Soilsampleswerestoredinlooselytiedplasticbagsat 4◦C.Soiltexturewasdefinedaccordingtotheinternational institutiontrianglecoordinategraph,andsoilpHwas deter-minedasdescribedinZhaoetal.31
Aphytopathogenicfungus,P.sojae01,wasprovidedbythe CollegeofLifeSciencesofNorthwestA&FUniversityinChina andwasincubatedonpotatodextroseagarplate(PDA:extract of200g potato,20g ofglucose,18gofagar, 1L ofdistilled water)at30◦Cfor3daysandmaintainedat4◦Cfortemporary storage.
Theseedsofsoybean(G.maxL.)cultivarZhonghuang13, whichistheprinciplecultivarusedinthesampling region, were bred bytheInstitute ofCrop Sciencesofthe Chinese AcademyofAgriculturalSciences.
Isolationandpurificationofsoybeannoduleendophytes Bacteria were isolated from root nodules according to a standardmethodasdescribedbyMaetal.32andMilleretal.33 Asinglecolonyoftheisolatewasrepeatedlystreakedonthe same mediumand examined witha microscope.Pure cul-tureswerepreservedonplatesat4◦Cfortemporarystorage orinsterilevialswith30%(v/v)glycerolforlong-termstorage at−80◦C.Toconfirmifthesurfacesterilizationprocesswas successful,severalsurface-sterilizednoduleswererolledover nutrientagar(NA)platesandaliquotsofwaterfromfinalrinse solutionsandthenplatedontoNAplates.34Plateswithoutany contaminantswereconsideredeffectivelysurface-sterilized, andthecorresponding plateswereusedfortheisolationof endophytes.
Screeningofantagonisticendophyticbacteria
Theantifungalactivityofendophytesagainstpathogenic fun-gus P. sojae01 wasdetectedbyusingthe pointinoculation method.35SporesoffungalcultureswereinoculatedonPDA plates,andasmallblockofagarwithfungalmyceliacutwith asterilepuncher(Ø=4mm)wasplacedinthecenterofafresh plate.TestedstrainswerespotinoculatedontheedgeofPDA plates approximately 25mmfrom thecenter. After incuba-tion at28◦C for7days,the inhibitionzonewasmeasured. Fungalmyceliathatwerecultivatedwithoutinoculationwere includedascontrol.36Experimentswereperformedin tripli-cateforeachbacterialisolate.
Secondaryscreeningofantifungalactivitywasperformed similartotheprimaryscreeningmethod,but bacteriawere spot inoculatedasbacterialsuspension(OD600≈1). Antago-nisticactivitieswereevaluatedbymeasuringinhibitionzones betweenpathogensandtestedbacteria.
Microscopicobservationofphytopathogenicfungimycelia Todeterminetheeffectofendophyticbacteriaonpathogenic fungus, treated and untreated pathogenic fungi were cul-turedfor2daysonPDAmedium.Themorphologicalchanges ofpathogenicfunguscausedbyendophyteswereexamined underanopticalepifluorescencemicroscope(BX50Olympus) at200-foldmagnificationandcomparedwiththestructures of the control groups. The mycelium of each pathogenic fungusonthe growthPDA mediumwasdirectly examined andphotographedfromtheplatesbyusingadigitalcamera (Olympus).
Sequencingandphylogeneticanalysis
Thetotal genomic DNA was extracted from the culture of nodule isolates by using the previous method.37 The 16S rRNAgenewasamplifiedfromthegenomicDNAbyPCRwith theuniversal forwardprimer P1(5-CGGGATCCAGAGTTT GATCCTGGCTCAGAACGAACGCT-3)andreverseprimer P6(5-CGGGAT CCT ACGGCT ACC TTG TTA CGA CTT CAC CCC-3),respectively,whichcorrespondedtothepositionsof 8–37bpand1479–1506bpinEscherichiacoli16SrRNAgene.38 AnaliquotofPCRproductofisolateswasdirectlysequenced by Sangon Biotech (Shanghai) Co., Ltd. using the same primersmentioned above. Acquired andrelated sequences were matched with ClustalX1.81 software, imported into Bioedit4.8.4,andmanuallycorrected.Aphylogenetictreewas constructedusingtheJukes–Cantormodelandthe neighbor-joiningmethod39 inTREECONpackage(version1.3b).40 The similarityofeachtested strainwascomputedbyusingthe DNAMANapplication(version6.0.3.40,LynnonCorporation). Theacquired 16SrRNA genesequences were submitted to NCBIGenBank(http://www.ncbi.nlm.nih.gov/).TheGenBank accessionnumbersofthesequencesobtainedinthisstudyare KF843714–KF843719.
Siderophoreproduction
Bacteria were cultured inLuria–Bertani (LB) broth at 30◦C withshakingat130rpmuntiltheexponentialgrowthphase (OD600≈1) was achieved. The production of siderophores by the bacteria was determined according to the chrome azurol-S (CAS) analytical method.41 The supernatant was obtainedby centrifugationat9000×g for10min and then mixed with 1mL of CAS assay solution.42 A medium mixed with the CAS assay solution at a 1:1 ratio was included as blank control, and the difference of OD630 betweenthe treatmentand blankwasestimatedas values ofsiderophoreproduction.43Experimentswereperformedin triplicate.
NitrogenfixationandnifHgeneamplification
Thefixationofatmosphericnitrogenbythebacteriumwas testedqualitativelyusingAshby’sN-freemedium(NFM:10g ofmannitol,0.2gofKH2PO4,0.2gofMgSO4·7H2O,0.2gofNaCl, 0.1gofCaSO4·2H2O,5gofCaCO3,pH7.0–7.5,1.8gagarinlLof distilledwater).23Plateswereincubatedat28◦Cfor3days,and strainsthatgrewnormallyinNFMmediaweredefinedas
can-didatesofnitrogenfixers.30,44 Experimentswere performed intriplicate. Theforward primernifH40F (5-GGNATCGGC AAG TCS ACS AC-3), reverse primernifH817R (5-TCR AMC AGC ATGTCC TCSAGCTC-3),and theproceduredescribed byVinuesa et al.45 were usedfor nifHgene specific ampli-ficationbyPCR.PCRproductswereseparated byhorizontal electrophoresisin1%(w/v)agarosegels, andpatternswere visualized.
Indoleaceticacid(IAA)production
IAAproductionwasestimatedbyinoculatingabacterial sus-pension (1×108cfumL−1)in10mLLB broththatcontained l-tryptophan(100gmL−1)andshakenat30◦Cfor72hinthe dark.Fivemillilitersofeachculturewerecentrifuged(20min, 6000×g),andIAAproductionwasmeasuredasindolic com-poundsin2mLofsupernatantmixedwith2mLofSalkowski reagent,andtheabsorbancewasreadat535nmafter30min incubationinthedark.46Astandardcurvewasusedfor cali-brationtoquantify.Threereplicateswereperformedforeach IAAsynthesismeasurement.
Plantinoculationstudieswithendophyticbacteria
Antifungal endophytic bacteria were cultured in TY agar medium at 30◦C to the mid-log phase.47 Cells were pel-leted by centrifugation (3440×g, 10min at 4◦C), washed twicewithasterilesalinesolution,andpreparedfor bacte-rialsuspensions(approximately108cfumL−1).Thetreatment ofsoybeanseedswas thesame asthesurface sterilization ofnodules.31 Ineach sterilePetridish,30surface-sterilized seeds were placed separately on moist filter paper for germination at 28◦C. Germinated seeds were immersed in bacterial suspension for 8h. Experiments were con-ducted intriplicate.Thecontrolwasimmersedwithsterile water.
Inoculated seedlingswere sowninpots filledwith190g sterilized vermiculite and then incubated in the green-house withaphotoperiod of16hdaylightat22◦C,anight temperature of 20◦C, and 65% relative humidity. After the first main leaf appeared, each seedling was inoculated with 108cfu of the tested strain every week, and steril-ized water was poured every 3 days to maintain relative humidity.47Seedlingswithout inoculationwereincludedas blank control. Plants were harvested after 6 weeks, and root length, fresh weight, and chlorophyll content were determined.
Statisticalanalysis
Datacollectedfromgrowthpromotionandendophytic inocu-lationexperimentswereexaminedwithANOVAusingtheIBM SPSS17.0package(bytheDataTheoryScalingSystemGroup, FacultyofSocialandBehavioralSciences,LeidenUniversity, TheNetherlands).Theeffectsofsixendophyticbacteriaon shootlength,rootlength,freshweight,andchlorophyll con-tentofsoybeanseedlingswereanalyzedwithGraphPadPrism 5.01software.
2 3 4 5 10 13 15 17 16 14 12 11 9 8 7 6 1
G
F
E
D
C
B
A
Fig.1–Morphologicalchangesofthemyceliaofplantpathogenicfungiuponinteractionwithendophytesisolatedfrom soybeannodules.(A)NormalmyceliaofPhytophthorasojae01(CK);(B)myceliabecamewrappedwithbiofilmformedby endophyticbacteriaDD222(1);(C)myceliabecamefractured(2),lysis(3)undereffectbyendophyticbacteriaDD161;(D) hyphaeendbecameprotoplastconcentrationandformedaball(4,5,6,and7)formyceliaunwrappedbybiofilmunderthe actionofDD201;(E)someaerialhyphaeshowedsarciniform(8)wrappedaroundeachother(8)andtwisted(9,10)underthe actionofDD198.(F)Aerialhyphaebecamethin,transparent,andbent,andformedtransparentliquiddroplets(11,12,13, and14)undertheactionofendophyticbacteriaDD234;(G)hyphaeendbecamesplitends(15,17)andprotoplast
concentrationappearedspherical(16)undertheactionofendophyticbacteriaDD176.
Results
Isolationandscreeningofantagonisticbacteria
Atotalof276bacterialisolateswereobtained,ofwhich31 showedsignificantinhibition(inhibitionratio>42%)against
P. sojae 01in the initialand secondaryscreenings (Supple-mentaryTableS1).Sixisolatesthatshowedmorethan63% inhibitionformycelialgrowthofP.sojae01onPDAplatewere selectedforfurtherstudy.TheseisolateswereDD161(71.14% inhibition),DD176(70.30%),DD198(68.43%),DD222(64.32%), DD201(63.40%),andDD234(63.16%).
Microscopicobservationofphytopathogenicfungus
Thecoloniesofpathogenicfungusweremoreinhibitedafter 4daysofculturingwithendophyticbacteriaonPDAmedium comparedwiththecontrol.Microscopicobservationshowed that the fungal mycelia presented morphological changes inthetreatmentofendophyticbacteria.Thetreatedfungus becamefractured(Fig.1B)orlysed(Fig.1C)andwerewrapped
with biofilm formed bythe bacteria (Fig. 1Band C) unlike the control(Fig.1A).Inaddition,forthemyceliatreatedby endophyticbacteria,thehyphaeendsbecameprotoplastballs (Fig.1DandG) orsplit(Fig.1G)eventhoughtheywerenot wrappedbybiofilm.Someaerialhyphaeshowedsarciniform wrappedaroundeachother(Fig.1E)andtwisted(Fig.1E),as wellasafracturedandsphericalprotoplastend.Furthermore, someaerialhyphaebecamethin,transparent,andbent,and formedtransparentliquiddroplets(Fig.1F)undertheaction ofendophyticbacteria.
Phylogeneticanalysisofantagonisticendophyticbacteria Thephylogenyof16S rRNAgenes indicatedthat six endo-phyticantagonistsbelongedtofivegenera,asshowninFig.2
andTable1.DD198showed99.9%similaritywithEnterobacter
cloacaeXJU-PA-7(EU733519).DD161had100%sequence simi-laritieswithAcinetobactercalcoaceticus.DD201was100%similar toPseudomonasputida,DD234was100%similarto Ochrobac-trumhaematophilum,and DD222andDD176presented100% similaritieswithBacillusamyloliquefaciensandBacilluscereus,
0.1
Bacillus amyloliquefaciens NRRLB-14393T(EU138458) Erwinia psidii LMG 7034T(Z96085)
Acinetobacter lwoffii DSM 2403T(NR_026209)
Bacillus Bacillus limegachenifterium ormis IAM 13418DSM13T(D16273) T(NR_074923) Bacillus atrophaeus NRRLNRS-213T(EU138516) Acinetobacter genomo sp.3 LMG1035T(HQ180184)
Pseudomonas oryzihabitans IAM 1568T(AM262973) Enterobacter gergoviae JCM1234T(AB004748)
Pseudomonas putida ATCC 12633T(AF094736)
Enterobacter cloacae XJU-PA-7 (EU733519)
Pantoea stewartii LMG2632T(Y13251)
Pseudomonas oryzihabitans Cl-13(KC178587)
Bacillus subtilis 26A(KC295415) DD198 (KF843716)
Enterobacter pyrinus KCTC2520T(AJ010486)
Pantoea ananas LMG2665T(Z96081) Enterobacter dissolves LMG 2683T(Z96079)
Enterobacter cloacae ATCC13047T(AJ251469)
Enterobacter oryzae Ola 51T(EF488759)
Enterobacter radicincitans D5/23T(AY563134)
Acinetobacter baylyi CCM7195T(AM410709)
Acinetobacter soli B1T(EU290155)
Acinetobacter calcoaceticus MTCC127(AB859067) DD161 (KF843714)
Acinetobacter calcoaceticus DSM 30006T(AJ633632) Acinetobacter calcoaceticus 97424(HE651906)
Pseudomona sputida IHBB1369(GU186116) DD201(KF843717)
Ochrobactrum haematophilum JN54 (KF150363) DD234(KF843719)
Bacillus amyloliquefaciens CA81(KF040978)
DD222 (KF 843718)
Bacillus mojavensis BCRC17124T(EF433405) Bacillus subtilis NRRL NRS-744T(EU138520)
Bacillus mycoides ATCC6462T(AB021192)
Bacillus weihenstephanensis DSM11821T(AB021199)
Bacillus cereus SBTBC-008(KF601957)
DD176 (KF843715)
Bacillus thuringiensis IAM 12077T(D16281) 84 100 84 100 85 100 68 61 100 54 100 100 77 65 100 100 90 74 100 82 100 100 97 64 95 100 100 100 60 100 88 98 Enterobacter sp.CCBAU15492(DQ988939)
Bacillus cereus ATCC14579T(AF290547)
GroupI Enterobacter/ Pantoea/Erwinia GroupII Acinetobacter GroupIII Pseudomonas GroupIV Ochrobactrum GroupV Bacillus
Fig.2–Neighborjoiningtreebasedonalignmentofnucleotidesequencesofthe16SrRNAgenefromtestedstrains(shown inbold)andreferencestrains.GenBankaccessionnumberswereplacedinparentheses.Bootstrapvaluesgreaterthan50% wereindicated.Scalebarrepresentsthenumberofsubstitutionspersite.
Table1–Phylogeneticsimilarityandplant-growthpromotingpropertiesofendophyticbacteria.
Strains Accession number
Themostclosestrelative(accessionnumber) 16SrRNA similarity (%) Siderophore production (gmL−1) IAA pro-duction (molmL−1)c Nitrogen fixation nifH gene
DD161 KF843714 AcinetobactercalcoaceticusMTCC127(AB859067) 100 54.33±0.093aab 2.24±0.11b + +
DD176 KF843715 BacilluscereusATCC14579T(KF601957) 100 48.32±0.067b 2.80±0.43b + +
DD198 KF843716 EnterobactercloacaeXJU-PA-7(EU733519) 99.9 20.70±0.418c 1.28±0.08bc + +
DD222 KF843718 BacillusamyloliquefaciensCA81(KF040978) 100 4.49±0.070d 1.17±0.11bc + +
DD201 KF843717 PseudomonasputidaIHBB1369(GU186116) 100 3.68±0.035e 16.50±1.82a + +
DD234 KF843719 OchrobactrumhaematophilumJN54(KF150363) 100 1.56±0.107f 0.10±0.01c − −
a Average(±,standarddeviation),thedataincolumnsisaveragevaluesofthreerepetitions.
b Thesamelettermeansnosignificantdifferencebetweentreatments(p=0.01).
c ControlforIAAassaywasLBbrothwithoutbacterialinoculationundersameincubationcondition.
−:negativeaction.
Characterizationofpotentialplant-beneficialtraitsof endophyticbacteria
Table1summarizestheresultsofPGPtraitevaluationinvitro.
ExceptforDD234,the other fivestrainsshowedthe capac-itytoproducesiderophoreandIAA,aswellasthecapacity tofixnitrogen.Differentbiosynthesesofsiderophoreswere foundamongthestrains.DD161,DD176,andDD198produced
54.3,48.3,and20.7gmL−1ofsiderophores,respectively,while DD222, DD201, andDD234 producedless than 5gmL−1 of siderophoresinthesameexperimentalconditions.Regression analysis showed a significant positive correlation between siderophoreproductionandinhibitionratioagainstP.sojae01 (R=0.9643, p=0.0019<0.05)(detailsavailablein Supplemen-taryFig.S2).IAAproductionofDD201wassignificantlyhigher (16.5gmL−1)thanthatoftheotherfive(<3gmL−1).
Con trol DD 234 DD2 01 DD 176 DD2 22 DD 161 DD1 98 Con trol DD2 34 DD 201 DD1 76 DD 222 DD1 61 DD 198 R oo t l e n g th (c m ) Co ntro l DD 234 DD 201 DD 176 DD2 22 DD 161 DD 198 8
Bacte rial strains
F re s h w e ig h t( g /f re sh p lan t) Contr ol DD 234 DD2 01 DD 176 DD2 22 DD 161 DD1 98 Bacterial strains Ch lo rop hy ll co n te n t( m g /g w t) 0 10 20 30 40
Bacte rial strains
Sh oo t l e n g th (c m ) 0 5 10 15
*
*
*
*
*
*
Bacterial strains 0 2 4 6*
*
*
*
0.0 0.5 1.0 1.5 2.0 2.5B
C
D
A
Fig.3–Effectofsixendophyticbacteriaonshootlength(A),rootlength(B),freshweight(C),andchlorophyllcontent(D)of soybeanseedlings.Eachvalueisthemeanof10replicates.Barsrepresentthestandarddeviationsofmean.Statistical significancewasdeterminedatp<0.05accordingtoTukey’stest.Asteriskrepresentssignificantdifference.
Seedlinggrowthresponsetotheinoculationofendophytic bacteria
Theresultsin Fig. 3showed thatinoculations withDD176 significantlyincreased(19.2%,p<0.05)theshootlengthof soy-beanseedlings.InoculationswithDD176,DD161,andDD198 resulted in a significant increase in root length (38.32%, 36.23%, and 29.82%, respectively) (p<0.05), fresh weight of plants(36.45%,20.47%,and17.00%,respectively)(p<0.05),and chlorophyllcontent(36.73%,17.09%,and13.75%,respectively). Overall,theseresultsshowedthatinoculationofthetested endophyticbacteriasignificantlyimprovedthegrowthof soy-beanseedlings.
Discussion
Currently,endophyticmicroorganismsarebelievedtobean importantbioresourceformodernagriculture48becauseofthe beneficialeffectsofendophytesonplantgrowthpromotion, biocontrol,anddiseaseresistance.Asapartoftheroot sys-tem,rootnodulesarealsoahabitatforendophytes.However, this habitatisdifferentfrom other partsofplantsbecause theendophytesinthishabitathavetocompeteandco-exist
withsymbioticbacteriaandhelptheplantsthroughcertain mechanisms. In this study, asignificant inhibitory activity againstpathogenicfungusP.sojae01wasfoundamong11.2% (31/276)ofthenoduleendophyticbacteria(Table1and Supple-mentaryTableS1).Thehighproportionoffungalantagonistic bacteriaintherootnodulesrevealedinthisstudyandin pre-viousstudies49demonstratedthatantagonisticactivitymight beauniversalmechanismthroughwhichnoduleendophytic bacteriacanhelphostplants.
Inadditiontotheantagonismagainstpathogenicfungi,all sixstrainsproducedsiderophoresandIAA,whilefivestrains werecapableoffixingnitrogen.Theseresultsdemonstrated thatnoduleendophyticbacteriahavediversefunctionsinthe inhibitionofphytopathogensandinpromotinggrowth.
Currently,severalpossiblemechanismsaresuggestedfor theinhibitionofphytopathogensbyendophyticbacteria:(1) competitionwithpathogensfortheecologicalniche/substrate (siderophores) in the rhizosphere; (2) production of antibi-otics (cyclic lipopeptides,iturin, fengycin)50 and antifungal substances (2,4-diacetylphloroglucinol); (3) production of extracellularchitinaseandlaminarinasetolysefungalcells51 and degrade fusaric acid produced by fungal pathogens52; and(4)productionofvolatileorganiccompounds(suchas 2,3-butanediolandacetoin,whichactassignalingmoleculesto
mediateplant–microbeinteractions),53whichcouldstrongly inhibitpathogengrowthondifferenthosts54andelicitplant growthbyinducedsystemicresistance(ISR).50,55 Theresults obtainedinthecurrentstudymightindicateevidenceofthe firstandthethirdmechanisms.
Thehigh correlation (R2=0.93) of siderophores and the fungalinhibitionofnoduleendophyticbacteriainthisstudy (SupplementaryFig.S2)supportedtheideathatthestrong fer-rousabsorptionbyendophyticbacteriamightbeamechanism fortheinhibitionoffungalgrowth.Meanwhile,theformation ofbiofilmonthehyphae andthemorphologicalchangesof the myceliaofthe target fungus(Fig. 1) showed that anti-fungalsubstancesand fungalcell-lysingenzymes mightbe producedbytheendophyticbacteria,asreportedbyMauch etal.51 Theseresultsdemonstrated thatnoduleendophytic bacteriaareimportantresourcesforsearchingforinhibitors specifictothefungibutwithoutnegativeeffectsonsymbiotic bacteria.
Inaccordancewiththephylogenyof16SrRNAgenes(Fig.2), thesixmostefficient(>63%inhibition)antagonisticstrains, whichwerepreliminarilyidentifiedasbelongingtofive
gen-era(Table1),demonstratedagainthattherootnodulescould
beoccupiedbydiversebacteria.Thesefindingssupportedthe resultsofotherstudies.3,30,56,57Allthesefivegenera,namely,
Acinetobacter, Bacillus, Enterobacter, Ochrobactrum, and Pseu-domonas,havebeenreportedpreviouslyasnoduleendophytes ofdifferentlegumes,includingsoybean.3,58–60However,these endophytes were isolated from soybean nodules collected fromdifferentregionsofHenanProvince,therebysuggesting theantagonisticeffectofsoybeanendophytesonpathogenic fungusofdiversegeographicalsourcesandspecies.This diver-sitymaybeattributedtomultiplesymbioticrelationshipsin theparticularregion(theoriginalareaofsoybean).21,22During alongevolution period,soybean,rhizobia,and endophytes formedamultiplesymbioticrelationship,andsoybean pro-videsnutrientsandasuitableenvironmentforsymbioticand endophyticbacteria.Rhizobiaprovidednitrogennutritionfor plantsand endophytes,whileendophytesstrengthenedthe resistanceofplantsandsymbioticbacteriaagainstpathogens andbadenvironmentalfactors.
Inthis study, twoofthe antifungal endophyticbacteria (DD176 and DD222) were identified as Bacillus sp. Bacil-lus is oneof the most abundant rhizosphere bacteria and noduleendophytes.34,58,60Thesebacteriacouldimprovethe yieldsofvariouscrops61–63bystimulatingplantgrowth(with hormones)andimprovingnutrientsupply(with phosphate-solubilizing siderophores)or by antagonism against phy-topathogens through protease or cellulose production.64–66 OurresultsshowedthatB.sp.DD176andB.sp.DD222produced siderophore and hormones (IAA) in addition to effectively inhibitingthepathogenicfungus(Table1).Theformationof biofilmandtheaccompanyingmorphologicalchangesinthe mycelia(Fig.1)supportedtheideathatBacillusspp.could pro-duceseveraltypesofenzymestodegradefungalcellwalls, whichresultedinaprotoplastballorsplitendsofthemycelia. Furthermore,B.amyloliquefaciensstrainsarecharacterizedby highrhizospherecompetenceandasignificantgenetic appa-ratusdevotedtothebiosynthesisofawiderangeofsubstances withantibiotic activity.55,67–71 All previous studies and our
study demonstrated thatnoduleendophyticBacillus strains arevaluablecandidatesforexploringbiofertilizers.
The isolate DD198 was identified as Enterobacter cloa-cae,whichisarhizopherebacterium34,58,60,72 thatproduces phytohormones, such as ethylene, auxins, cytokinins,65 siderophores,63,73 andfixesnitrogen.72 Inthepresentstudy, strain E. cloacae DD198 showed significant inhibitory activ-ityagainstP.sojae01invitroandpromotedeffectsforwheat seedlingswithinoculationtreatment.
Inourstudy,A.calcoaceticusDD161possessesthestrongest ability to produce siderophores and inhibit the growth of pathogenic fungus,aswell assynthesizeIAA.The compre-hensiveeffectindicatedthatthegrowthofsoybeanseedling inoculated with DD161 was significantly improved (Fig. 3). Interestingly,onlyafewreportsshowedthatA.calcoaceticus
strains indicatedbothnitrogenfixationactivity and inhibi-tion effect asidefrom their PGPR activity. Previousreports showed that A. calcoaceticus isolated from rhizosphere of wheat74,75 could synthesize IAAfrom tryptophan and pro-ducesiderophoresandphosphate-solubilizingorganicacids. Therefore,theA.calcoaceticusstrainmayimprovecropgrowth andyieldonthebasisofitsbiocontrolactivity,siderophore production,andnitrogenfixation.76
IsolateDD201wasidentifiedasPseudomonassp.,whichis anopportunisticbacteriumfoundinterrestrialandaquatic environments, and indicated biotechnological behaviors.77 In this context,P. sp. DD201 showed the highest IAA pro-duction (16.5molmL−1) but did not significantly improve thegrowthofsoybeanseedlingsininoculationtests(Fig.3). Previous studies confirmed that IAA could promote plant grow that low concentration and inhibit root growth at high concentration.78–80 Presumably, PGP bacteria use IAA as a part of their colonization strategy and as a signal molecule in bacteria–host communication.66 These func-tions might explain the reason for the production of IAA that was common among our six endophytic strains
(Table1).
Thisstudy provedthatfungal antagonistic strainDD234 was O.haematophilum(Table 1). Ochrobactrumisolates could assistplantnutrientuptakefromthesoilandpreventplant diseases.81 The plant growth-promoting characteristics of siderophoreproduction,IAAproduction,andphosphate sol-ubilization were found in some strains of this genus82 and might be the mechanism for increasing host plant growth.83
Conflicts
of
interest
Wedeclarethatwehavenoconflictofinteresttothiswork.
Acknowledgments
ThisworkwassupportedbyprojectsfromtheNational Sci-ence Foundation of China (U1204301), the Foundation for UniversityKeyTeacherbytheMinistryofEducationofHenan Province (2012GGJS166) and the University Key Scientific ResearchProjectofHenanProvince(17A180011).
Appendix
A.
Supplementary
data
Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2017.06.007.
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