h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Environmental
Microbiology
Trichoderma
harzianum
MTCC
5179
impacts
the
population
and
functional
dynamics
of
microbial
community
in
the
rhizosphere
of
black
pepper
(Piper
nigrum
L.)
Palaniyandi
Umadevi
a,b,
Muthuswamy
Anandaraj
a,∗,
Vivek
Srivastav
a,
Sailas
Benjamin
baICAR-IndianInstituteofSpicesResearch,Kerala,India
bUniversityofCalicut,DepartmentofBotan,BiotechnologyDivision,Kerala,India
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received27September2016
Accepted16May2017
Availableonline29November2017
AssociateEditor:JerriZilli
Keywords:
Rhizosphere
Populationabundance
Functionalabundance
a
b
s
t
r
a
c
t
Employing IlluminaHiseq wholegenome metagenomesequencing approach, we
stud-ied the impact ofTrichoderma harzianum on altering the microbial community andits
functionaldynamicsintherhizhospheresoilofblackpepper(PipernigrumL.).The
metage-nomicdatasetsfromtherhizospherewith(treatment)andwithout(control)T.harzianum
inoculationwereannotatedusingdualapproach,i.e.,standaloneandMG-RAST.The
pro-bioticapplicationofT.harzianumintherhizhospheresoilofblackpepperimpactedthe
populationdynamicsofrhizospherebacteria,archae,eukaryoteasreflectedthroughthe
selectiverecruitmentofbacteria[Acidobacteriaceaebacterium(p=1.24e−12),Candidatus
korib-acterversatilis(p=2.66e−10)]andfungi[(Fusariumoxysporum(p=0.013),Talaromycesstipitatus
(p=0.219)andPestalotiopsisfici(p=0.443)]intermsofabundanceinpopulationandbacterial
chemotaxis(p=0.012),ironmetabolism(p=2.97e−5)withthereductioninabundancefor
pathogenicityislands(p=7.30e−3),phagesandprophages(p=7.30e−3)withregardto
func-tionalabundance.Interestingly,itwasfoundthattheenrichedfunctionalmetagenomic
signaturesonphytoremediationsuchasbenzoatetransportanddegradation(p=2.34e−4),
anddegradationofheterocyclicaromaticcompounds(p=3.59e−13)inthetreatment
influ-encedtherhizospheremicroecosystemfavoringgrowthandhealthofpepperplant.The
populationdynamicsandfunctionalrichnessofrhizosphereecosysteminblackpepper
influenced bythetreatment withT.harzianumprovidestheecological importanceofT.
harzianuminthecultivationofblackpepper.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis
anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mail:[email protected](M.Anandaraj).
https://doi.org/10.1016/j.bjm.2017.05.011
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC
Introduction
Plantscontributetotheestablishmentofspecificecological
niches of microbes inthe rhizosphere by playing key role
asecosystem engineers.1 Themicrobial community atthe
rhizospherereflectsits functional specificityatthelevel of
plant–microbe interactions.It suggests that
taxonomically-contrastedplantgrowthpromotingstrainsmaycoexistinsoil
and colonizethe same rhizosphere. Theprobiotic
commu-nityenrichmentbytheplantisthemajorelementinplant
responsetovariousbioticandabioticstresses,coupledwith
theapplicationofplantgrowthpromotingmicrobes.2Inthe
plantrhizosphere,theplantgrowth-promotingmicrobesplay
mainrolessuchasmodifyingtherootfunctioning,improving
plantnutritionand itsintake, and influencingthe
physiol-ogyofentire plant.Secondary metabolites secreted bythe
soilmicrobeshasroleincontrollingbioticinteractions.3The
chemicalecologyresearchfieldthatfocusonthe
understand-ingthespecificinteractionmediatedbytheproducerorganism
withthe targetmicrobeandwiththemicrobialcommunity
isof immense importancein rhizosphere microniche. The
experimentalapproaches ontherole ofsecondary
metabo-litessuggeststhattheycanacttoslowdownthegermination
of spores in order to bring less competitive environment
forthegrowth,actasagentsofsymbiosisand competitive
weaponsagainstother competing organisms.4 Hence,
inte-grating functional and ecological knowledge on microbial
populationsinsoilwillbeaprerequisiteindevelopingnovel
managementstrategiesforsustainableagricultureforwhich
thepopulationabundanceofsoilmicrobiomeisanimportant
component.
Trichoderma (telemorph Hypocrea) is an asexual fungal
genusinhabiting the soilofall climatic zones;many ofits
speciesareusedaseffectivebiofertilizerandbiocontrolagents
forplantsgrowningreenhouseaswellasfields.5–7The
mech-anismmediatedbyTrichodermaspp. includesthe antibiotic
activity,8 mycoparasitism,9 cell wall-lytic enzyme action,10
competitionfornutrients,11theinductionofsystemic
resis-tancetopathogensinplants5;andnutrientsupplythrough
thedegradationofbiomass.6,7
Blackpepper(PipernigrumL.)–anativetoIndiaand
pop-ularlyknown as theking of spices– isanexport oriented
importantspicecropgrownintropicalcountries.Thefootrot
diseasecausedbyPhytophthoracapsici,anoomycetepathogen
contributes to the major crop loss as it infects the vine
bothinnurseryand fields.12 Theelegantstudieson
Tricho-dermaharzianum(MTCC5179)towarditsgrowthpromotion13,14
anddiseasesuppression15–17 activitiesmadethisfungusan
importantcomponentintheintegrateddiseasemanagement
moduleofthecultivationstrategyofblackpepperinIndia.
Thus,wehypothesizedthattheprobioticapplicationof
Tricho-dermawouldalterthecommunitycompositionordynamics
ofothersoilfungiandbacteria atthe rhizosphereofblack
pepper;and that mightcontributetothe planthealthin a
betterwaythan therhizospherecommunitywithout
Tricho-derma.Inthelightofthishypothesis,thisstudyisdesigned
withthreeobjectives:(a)toinoculatetherhizosphereofblack
pepperwithT.harzianum(MTCC5179)forassessingitsimpact
onmicrobialcommunitydynamicsintherhizosphere,(b)to
subjecttherhizhospheresoiltowholegenomemetagenomics
analysis, and(c)tobringoutthe taxonomicand functional
abundanceforunderstandingthecommunitydynamics.
Materials
and
methods
Raisingofexplant
SinglenodecuttingsfromSreekaravarietyofblackpepperwere
washed withTween20for15min,followed byrunningtap
water.Thecuttingsweresubsequentlysurfacesterilizedwith
copperoxychloride(0.2%)for15min,andwashedtwicewith
steriledoubledistilledwater(ddH2O).Thecuttingswereagain
surfacesterilizedwithmercuricchloride(0.1%)for5min,
fol-lowedbywashwithddH2Otwice.Thecutendsofthecuttings
were quick dippedinindole-3-butyricacid (8000ppm), and
planted in protray on sterileperlite medium fortifiedwith
sterileHoagland’ssolution.18
Theprotrays with the preparationasabove were
main-tainedingreenhousewithtopportionsealedwithaluminum
foil.ThecuttingsweresprayedwithHoaglandsolutiononce
in a day. After 2months ofgrowth (whenplants attained
24–26cm height with 4–5 leaves), the rhizosphere (perlite)
samples from the plants were collected and analyzed for
the presence or absence of Trichoderma spp. by plating
(spread/pourplate method).Subsequently,saplingswithno
association ofTrichoderma spp.weretransferredtothe pots
filledwithtopsoil(composition:197Ca;173K,71Mg;18S;
11.38Fe;5.56Mn;3.24Zn;1.64P;0.92Cu;0.16B(allinppm);
and1.6%organiccarbon,pH:4.35).Twosetsofexperiments
[inoculatedwithT.harzianum(MTCC5179),thetreatmentand
withoutinoculationofT.harzianum,thecontrol]with4
repli-cateshaving3plantsperreplicaweredesignedforthestudy.
TalcformulationofT.harzianum(MTCC5179)(3.5g/3kgsoil)
was usedfor inoculating the soil. Growth parameters viz.,
heightoftheplant,stemgirth(1cmabovefromthesoilregion)
andtheleafareaindex(LAI)wererecorded.TheLAIwas
calcu-latedusingtheformula:length(cm)×width(cm)×0.6.After
120days,plantswereuprooted,therhizospheresoil(adhered
totherootsofpepperplants)samplewerecollectedfrom3
biologicalreplicatesofbothtreatmentandcontrol,andstored
at−80◦C.Theweightsofshootandroot(freshanddry)were
alsorecorded.
ExtractionofrhizospheresoilDNAandsequencing
TherhizospheresoilDNAfromthetreatedandcontrolplants
were extracted from 100mg of soil using MoBio kit (MO
BIOLaboratories, Inc. USA),according tothe instructionof
themanufacturer.DNAfromthreebiologicalreplicateswas
pooledforthedownstreamanalysis.TheintegrityoftheDNA
was assessedbynanodrop spectrophotometer (2000/2000C,
Thermo Scientific, USA), and 2L of each sample was
subjectedtoelectrophoresison1%agarosegelusing1×
tris-borate-EDTAbuffer.Gelswerestainedwithethidiumbromide
andviewedusingGelimagingSystem(SyngeneTechnologies
Inc,USA).DNAlibrarywaspreparedusingNEBNextultraDNA
library prepkit for Illumina.Sequencing ofthe paired end
Readqualityassessment
The paired end reads generated were examined for read
length,totalnumberofreads,percentageofGCcontentand
meanbasequalitydistributionusingFastQCtoolkit.Allreads
werequalityfilteredwithanaveragePhredqualityof20,and
cutadapt(version1.8.3)wasusedforadapterremovalfromthe
sequences.
Denovoassemblyandannotation
Assemblywasperformedwithdefaultk-merlength(31-size)
usingde-bruijngraphmethod.InhousePERLandPythoncode
wereusedtoparsethefastqfilesforthedownstreamanalysis.
ThesequenceswereassembledwithRayMeta19usingak-mer
sizeof31.Thecontigswithmorethan150bpwere filtered
andtakenaspre-processedreadsfordownstreamanalysis.
Glimmer-MGv0.3.220 was usedtopredictthe protein
cod-ingregionsinthecontigs.Eachsamplereadswascompletely
assembledinabout 5days.Thisruntimeincluded denovo
contigandscaffoldassemblyprocess.
Taxonomy/functionalanalysis
Thetaxonomytreewasgeneratedbasedonneighbor-joining
methodusingMEGANsoftware.Thehierarchyofcomparative
taxonomicabundanceinall thesampleswasbasedonthe
contigabundancewiththenumberofreadsassignedtothe
taxonomy.Functionalannotationwasperformedusing
DIA-MONDversion0.7.921forpredictedgenesagainsttheprotein
databaseusingtheBLASTversion2.2.29+22 withane-value
of1e−5. The functional analyses ofall hits were analyzed
usingthe KEGG and SEEDoptions providedin theMEGAN
software.23
AnalysisbyMG-RAST
Theresults from the standalone workflow were compared
withMetaGenomeRapidAnnotationusingSubsystem
Tech-nology(MG-RAST).24Taxonomicclassificationwasperformed
to view the taxonomic level in the samples against the
M5NRpublicdatabaseusingbestfitclassificationwith1e−5
asmaximume-valuecutoff,and 60%asminimum identity
cutoff. Functional analysis for the distribution of
func-tional categories using subsystems was carried out using
the hierarchical classification with 1e−5 as maximum
e-value cutoff, and 60% as minimum identity cutoff. Alpha
diversitypresentinthetreatmentandcontrolsampleswere
estimated. Statistics
Forthegrowthparameters,theexperimentaldesignadopted
wascompletelyrandomizeddesign,andthedatawere
ana-lyzed byt-test. Analyses ofdifferential/relative abundance
features(ofmetagenomedata)weredoneusingSTAMP
soft-ware package.25 The differential abundance between the
sampleswascalculatedusingG-test(w/Yates’)+Fisher’stest
fortwosampleanalysisinSTAMPtool.
Results
Growthparameters
ThepHofT.harzianumtreatedsoilwas5.2,after120daysof
inoculation;whilethatofcontrolwas4.6.Growthparameters,
viz.,thefreshroot,freshshoot,dryroot,dryshoot,LAI(Leaf
AreaIndex),heightoftheplantweresignificantlyincreasedin
thetreatment(Table1).
Metagenomics:sequencingandassembly
Paired End (251bp × 2) sequencing yielded 2,121,934 and
2,123,836 reads fortreatmentand control samples,
respec-tively.Majorityofthesamplereadshad40–70%GCcontent.
The Phred score distribution (≥Q30) of the paired-end
metagenomereadsfortreatmentwas79.22%,while80.82%
wasforthecontrol.Theassemblyofreadsformed1,827,461
and1,879,703contigsandN50of210and212,respectivelyin
treatmentandcontrol.
AnalysisbyMG-RAST
Outof4,121,006(97.1%)sequencesthatpassedqualitycontrol,
93.5% sequencesproduced 3,389,349 predictedprotein
cod-ing regionsofthe metagenomeinthe treatment.Ofthese,
33.7% sequences were assigned with annotation by M5NR
database;76.0%ofannotated featuresfrom M5NRdatabase
wereassignedwithfunctionalcategories.Fromcontrol
sam-ple,outof4,162,647sequencespassedqualitycontrol(98%),
94.5%produced3,558,779predictedasproteincodingregion.
Of these, 33.9% were assigned with annotation by M5NR
database, and 74.7% of annotated features were assigned
tofunctional categories.Themean sequence length,mean
Table1–Tableshowingthegrowthparametersofblackpepper:with(treatment)andwithout(control)inoculationofT.
harzianum.Thegrowthparametersat120daysareshowninthetable(n=12).
S.No Parametersobserved T1mean(withTrichoderma) T2mean(withoutTrichoderma) Pr>(t)
1 Shootweight(fresh) 7.7 3.0 <0.0001
2 Rootweight(fresh) 44.5 26.6 0.0050
3 Leafareaindex(LAI) 802.5 430.4 0.0028
4 Stemgirth 0.1225 0.1400 0.3896
5 Heightoftheplant 78.5 44.4 0.0023
6 Rootweight(dry) 1.7 0.7950 0.0018
GCcontentfortreatedandcontrolwere248±13bp,63±7%
and 249±12bp, 62±8%, respectively. Thedouble approach
we used (stand alone and MG-RAST) for the analysis of
metagenomeyieldedcoherentresultsinbothtaxonomyand
functional categories. The comparative analysis on these
metagenomesusingMG-RASTisdiscussedfurther.
Populationdynamics
The alpha diversity (Shanon diversity index) of the
metagenome of both treatment and control samples were
489,569and455,862species,respectively.Fromtheanalysisof
relativeabundance(percentageproportion)fortop10
bacte-rialspecies,viz.,AcidobacteriaceaebacteriumKBS96,Candidatus
koribacterversatilis,Ktedonobacterracemifer,Candidatus solibac-terusitatus,Pedosphaeraparvula,Sphingomonassp.,URHD0057,
Gemmatimonadates bacterium, Pyrinomonasmethylalipathogens, Chthonomonascalidiroseaandunculturedbacteria[ofwhichA. bacterium(p=1.24e−12)andC.koribacterversatilis(p=2.66e−10)
showedstatisticalsignificance]werefoundabundantinthe
treatment, while uncultured bacteria found were more in
control sample (p=0.024) (Fig. 1). The abundance of these
bacteria suggests thatprobiotic application ofT. harzianum
in black pepper imparted the rhizosphere competence for
the bacteria to colonize the roots as the presence of A.
bacteriumandC.koribacterversatilishasprovenasthemajor
rhizospherecompetentbacteriainvolvingunique metabolic
pathwayatthe rhizosphere. Analysisof the relative
abun-dance oftop10 fungi, viz.,Rhizophagusirregularis,Fusarium
oxysporum, Oidiodendran maius, Pseudogymnoasus pannorum, Talaromyces stipitatus, Pestalotiopsis fici, Mortierella verticillata
andT.harzianumshowedthatF.oxysporum(p=0.013),T. stip-itatus(p=0.219)and P.fici(p=0.443)werehighintreatment,
whilethecontrolshowedhigherabundanceofR.irregularis
(p=0.034), Pseudogymnoasuspannarum (ahumanpathogenic
fungus,p=0.488)andOidiodendran(p=0.484).TheTrichoderma
readswere recorded onlyintreatment sample.Thehigher
abundanceofF.oxysporum,T.stipitatusandP.ficiintreatment
suggeststhatT.harzianumselectivelyenrichesthebiocontrol
fungiintherhizosphere.Thereductionofpathogenicfungi,
inturn,providesstrongevidencethatT.harzianumisableto
reducethehumanpathogeniceffectoftheamendedsoil,in
comparisontothecontrol.
Functionalleveldynamics
Functionalabundance(Fig.2)betweenthetreatmentand
con-trolsamplesusinghierarchicalclassificationwithsubsystem
annotationsourcesshowedthatrhizosphereinthetreatment
waswithabundantreadsforvirulence,diseaseanddefense
(54,857), motility and chemotaxis (11,992), and ion
acquisi-tionandmetabolism(8151);whilethecontrolrecorded51,271
readsforvirulence,diseaseanddefense,11,564formotility
andchemotaxis,and7276forionacquisitionandmetabolism.
The relative abundance (percentage proportion) for the
specificfeatures(ironacquisitionand bacterialchemotaxis)
from stamp toolanalysis isgiveninFig. 3. Theheme and
heminuptakeandutilizationsystemsinGramnegative
bacte-ria (p=0.036) and iron acquisition in red pigmented Vibrio
(p=2.97e−5)wereabundantintreatmentmetagenomethan
incontrol.ThisindicatesthattheprobioticapplicationofT.
harzianumincreasedthemicrobialactionforthemetabolism
and absorptionof iron bythe plant. The bacterial
chemo-taxis washigherintreatedsample(p=0.012),whichshows
theactive/increasedinteractionofrhizospheremicrobeson
theblackpepperrootsbytheapplicationofT.harzianum.The
treatedsamplerecordedreducedabundanceonpathogenicity
islands,phagesandprophages(p=7.30e−3)(Fig.3).
Thereductionofpathogenicityislandandphagesin
treat-ment,whencomparedtocontrol,providesstrongevidencefor
theselectivecommunityrecruitedbytheT.harzianumtoward
the beneficial use in the cultivation system of black
pep-per.Though metagenomeofcontrolsampleshowedhigher
abundance (reads) globally for other functional category
(Fig.2),specificfeatureswereobservedatthehighest
func-tionaldistributionclassificationintreatment,whichincludes
metabolismofaromaticcompounds,viz.,benzoatetransport
anddegradation(p=2.34e−4)anddegradationofheterocyclic
aromaticcompounds(p=3.59e−13).Theincreasedabundance
forthesemetabolismofaromaticcompoundsbringsoutthat
the probiotic applicationofT. harzianuminblackpepperis
capableofcreatingtheunique communityforthe
phytore-mediation. 95% confidence intervals Ktedonobacter racemifer Sphingomonas sp. URHD0057 Gemmatimonadetes bacterium KBS708 Pyrinomonas methylaliphatogenes Chthonomonas calidirosea Acidobacteriaceae bacterium KBS 96
Candidatus koribacter versatilis Uncultured bacterium Candidatus solibacter usitatus Pedosphaera parvula
0.0 50.9 -15 -10 -5 0 5
Difference between proportions (%) Proportions (%) < 1e-15 < 1e-15 < 1e-15 < 1e-15 < 1e-15 < 1e-15 < 1e-15 1.24e-12 2.66e-10 0.024 P-v alue (corrected)
Fig.1–Specieslevelextendederrorbarchartprofilefortop10bacteriafromSTAMPtool.T.harzianumtreatmentisdenoted bybluebarandcontrolbyorangebar.ThedifferentialabundancebetweenthesampleswerecalculatedwithG-test
Clustering-based subsystems Carbohydrates Amino acids and derivatives Miscellaneous Confactors, Vitamins, Prosthetic Groups, Pigments Respiration RNA metabolism DNA metabolism Fatty acids, Lipids, and Isoprenoids Membrane transport Cell wall and capsule Virulence, Disease and defense Nucleosides and nucleotides Stress response Metabolism of aromatic compounds Phages, Prophages, Transposable elements, Plasmids Regulation and cell signaling Cell division and cell cycle Sulfur metabolism Nitrogen metabolism Phosphorus metabolism Motility and chemotaxis Potassium metabolism Iron acquisition and metabolism Secondary metabolism Dormancy and sporulation Photosynthesis
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Protein metabolism
Fig.2–ClassificationbasedonfunctionalabundancebyMG-RAST.Blueline:T.harzianumtreatmentisdenotedbybluebar andcontrolbygreenbar.Motilityandchemotaxis,ironacquisition,andvirulenceanddiseasefunctionsarewithhigh abundanceintreatment.
Discussion
Theprimeobjectives ofthis study wasto assess the
com-munitychangesattherhizosphereofblackpepperpursuant
to the inoculation of T. harzianum, and also to unveil the
significant effects ofT. harzianum on the selective
recruit-mentofspecificmicrobes,andtheirfunctionalassignments
inrhizosphere ofblackpepper.Theresults clearlyshowed
that T. harzianum significantly influenced in the selective
abundanceofbeneficialbacteriaandfungi,andsubsequent
growthpromotioninblackpepper;andtheimpactat
func-tionallevelwasidentifiedasincreasedbacterialchemotaxis,
virulence, disease and defense, ion metabolism. From the
results,increase inthe growth parameters, viz.,freshroot,
freshshoot,dryroot,dryshoot,leafareaindex,heightofthe
plantrevealsthegrowth promotionactivity ofT. harzianum
inblackpepper,asindicatedbyotherauthorstoo.Anandaraj
and Sarma14 reported that the application ofT. harzianum
(MTCC 5179) resulted inenhanced growth in black pepper
withincreasednumberofnodes,andconsequentlythe
num-berofcuttings.Sibi13alsoshowedthepositiveinfluenceofT.
harzianum(MTCC5179) ontheimprovementsinthe
forma-tionoffreshrootandshoot,followedbyincreaseinthedry
weightofrootandshootinblackpepper.TreatmentwithT.
harzianum(MTCC 5179)individuallyimpartedbettergrowth
promotionanddiseasesuppressionthanthatofaconsortia
ofplantgrowth-promotingrhizobacteriaaloneorin
combina-tionwithT.harzianum(MTCC5179).13Thesestudiesindicated
growthpromotionandtheorganismwasrecommendedasa
componentofintegrateddiseasemanagementandwithouta
clearunderstandingofothermechanisms.Thepresentstudy
unravelstheunderlyingmicrobialdynamicsandmajor
func-tionalprocesses.
Thoughthepopulationabundanceofbacteria,archeaand
eukaryote werea littleless intreatment thanincontrol, it
showedselectiveabundance(morepercentageproportion)of
bacteria, viz.,A.bacteriumand C.koribacterversatili– outof
top10bacterialspecies;thesebacteriabelongtothephylum
Acidobacteriaceae,theavidcolonizeroftherhizospherewith
potentrhizospherecompetence.26A.bacteriumiscapableof
growingondiversecollectionofcomplexorganiccompounds
includingxylan,cellulose,methylcellulose,syringate,pectin
andferulate.27Candidatussp.containsabundanceof
carboxy-laseactiveenzymes(CAZyme)familyandareinvolvedinthe
breakdown,utilizationandbiosynthesisofdiversestructural
and storage polysaccharides and resistance to fluctuating
temperatureandnutrientdeficientconditions.28This
selec-tiveabundantrecruitmentofthesebeneficialbacteriainthe
treatment might be the major impact for the growth
pro-motionactivitybytheactivebreakdownofcomplexorganic
compounds by theseorganisms, thereby creating
microcli-mates for the colonization of microbes in the roots and
subsequentinteraction withthe communitiesatthe
rhizo-sphere.Further,the analysisofblackpepperrootexudates
and actionofthesebacteriaonthe rootmetaboliteswould
givethespecificroleofthesebacteriaattherhizosphereof
blackpepper.
Unlikeincontrol,themetagenomeoftreatmentshowed
abundant reads of the beneficial fungi, viz. F. oxysporum,
Talaromycessp.Pestalotiopsissp.and T.harzianum;apositive
correlationwithexpectedbeneficialactivitiesaspointedout
bydifferentauthors:EparvierandAlabouvette29showedthat
increasedpopulationofF.oxysporumwasbetterforthe
biocon-trol anddisease suppressionactivityinFlax;manyisolates
ofTalaromycesspp.were showntopromoteplantgrowth.30
Elegant studies have demonstrated that T. flavus
95% confidence intervals 95% confidence intervals 95% confidence intervals 1.97e-6 2.97e-5 0.012 0.036 Heme hemin uptake and utilization systems in GramP...
Iron acquisition in red pigmented vibrio
Bacterial Chemotaxis
Heme hemin uptake and utilization systems in GramN...
0 0 3839.0 -4 -3 -2 -2 -2 -1 -1 0 1 2 3 0 0 0 2629.0 -6 -4 4 6 8 10 2 2 1 3 4 5 Sequences Sequences 14124.0-3 Sequences Inoculated Inoculated Inoculated Uninoculated Uninoculated Uninoculated
Difference between proportions (%)
Difference between proportions (%)
Difference between proportions (%) Phages and prophages
Pathogenicity islands
Heterocyclic aromatic compounds degradation
Phenylpropanoid compound degradation
Benzoate transport & degradation
3.59e-13 1.25e-5 P-v alue (corrected) P-v alue (corrected) P-v alue (corrected) 2.34e-4 7.30e-3 7.30e-3
Fig.3–Functionallevelextendederrorbarchartprofileforironacquisitionandchemotaxis,phagesandprophages, pathogenecityislandsandheterocyclicaromaticcompoundsdegradationfromSTAMPtool.T.harzianumtreatmentis denotedbybluebarandcontrolbyorangebar.ThedifferentialabundancebetweenthesampleswerecalculatedwithG-test (w/Yate’s)+Fisher’stestfortwosampleanalysisinSTAMPtool.
abundanceofthespeciesofFusariumandTalaromycesin
treat-mentindicatestheecologicalsignificanceontheirpopulation
abundancedrivenbytheadditionofT.harzianumtowardthe
fitnessofblackpeppergrowthandsubsequentyield.
Rajanetal.15showedthebiocontrolanddisease
suppres-sionactivitiesofT. harzianum(MTTC 5179)in blackpepper
againstfootrotdiseaseatfieldconditions;whichwasfound
to be efficiently proliferating in the soil and remained in
the soil for long time, apart from imparting protection to
theroot systemagainstP. capsici.In thepresent study,the
metagenome analysiswas performedafter four monthsof
treatment,andprovedthatT.harzianum(MTCC5179)wasable
toremaininsoilforalongtime.Interestingly,theproportion
ofR.irregulariswashigherinthecontrolthanintreatment,
whichindicatestheinteractionofTrichodermawiththenative
VesicularArbuscularMycorrhiza(VAM)andmodulationofits
population.ThesporegerminationandhyphalgrowthofG.
mosseaewasstimulatedbyT.harzianumwiththeproduction
ofvolatilecompounds.33Inpresentstudy,thelessabundance
ofArbuscularmycorrhizalfungi(AMF)intreatedsoilmight
beduetothestimulatedgrowth ofAMFbythecommunity
theplant34ratherthantheirphysicalpresenceinthe
rhizo-sphereandviceversaincontrol.ApplicationofT.harzianum
improvedbettergrowthofblackpepper,whichwasatparwith
T.harzianumincombinationwithAMF.Thetreatmentswith
AMFaloneandincombinationwithPseudomonassp.failedto
enhancethegrowth.13 P. fici,anendophyteoftea produces
bioactivemetabolitesandnaturalproducts,35andthe
analy-sesofitsgenomeandtranscriptomeshowedthatitharbors
efficientgenesresponsible forthesynthesisofvarious
sec-ondarymetabolites.36Furtherfunctionalanalysisofthereads
onP.fici,fromthepresentmetagenomedatawouldgive
signif-icantinsightintoitsroleonblackpepperthroughinteraction
atrhizosphere.
The metagenome of the treatment in the present
study showed higher abundance for iron acquisition and
metabolisminredpigmentedVibrio,coupledwithhemeand
heminuptakeandutilizationsystemsinGramnegative
bacte-riathancontrol;whichevidencestheinfluenceofT.harzianum
inrhizosphere–microbeinteraction.Rhizospheremicrobiome
facilitatestheuptakeofspecifictraceelementssuchasiron.
Iron in soil, exists primarily in the insoluble ferric oxide
form,whichisnotavailableformicrobialgrowth.Basedon
thescarcityofavailableironsaswell asthetoxicityoffree
ironatelevatedconcentrationsintheenvironment,bacteria
employavarietyofmechanismstoregulatethe
intracellu-larironconcentrations.37Ontheotherhand,plantsalsoplay
crucialrole inincreasingthesolubility ofinorganic ironin
therhizosphere, whichmaybeduetothe interaction with
microbiome.38 Rhizobacteria are generally motile, and the
motilityiseitherrandomorchemotacticforinteractingwith
theplants.39Infact,thebacterialchemotaxiswasfoundas
abundantintreatmentthan incontrol,suggestingthatthe
probioticapplicationofT.harzianuminblackpepperwould
enableactiveinteraction oftherecruited bacterial
commu-nityintherootsystem.Anatomicaldatafromthetreatment
andcontrolalsoprovide ampleevidencesfortheaforesaid
inference.34Theabundanceofreadsonpathogenicityislands,
functionalityofphagesandprophageswerefoundtobeless
intreatmentthanincontrol.Thelessabundanceofhuman
pathogenic fungiasevidenced from the analysis of
taxon-omyabundanceishighlyrelatedtotheresultsoffunctional
analysis, which suggests the beneficial effect of probiotic
applicationofT.harzianum,especiallyinthecontextofhuman
health.
Rhizoremediationisaspecificformofphytoremediation
involvingplantsand theirassociatedrhizospheric
microor-ganisms (bacteria and fungi). Rhizoremediation can either
occurnaturallyorcouldbefacilitatedbyinoculatingsoilwith
microorganismscapableofdegradingenvironmental
contam-inants.Theplantassociatednon-pathogenicendophyticand
therhizosphericbacteriaarethemajorplayersinthe
degra-dation of toxic metabolites present in soil.40 Heterocyclic
aromaticcompoundsandbenzoatesaretoxiccompounds
per-sistforalongtimeinsoil,thatleadstoilleffectsinanimals
andhumans.Inthepresentstudy,metagenomeoftreatment
recorded higherabundanceofreads forthedegradation of
heterocyclicaromaticcompounds,benzoatetransportandits
degradation.Thisinformationwouldgivethepositiveimpact
ofT.harzianuminthecroppingsystemofblackpepper.Further,
thefunctionalmetagenomicswouldgivemoreinformationon
bacteriainvolvedintherhizoremediationthroughthe
rhizoe-cosysteminblackpepper.
In conclusion, the population dynamics and functional
richnessofrhizosphereecosysteminblackpepperinfluenced
bythe treatment withT. harzianumprovides the ecological
importanceofT.harzianuminthecultivationofblackpepper.
On thebasisofthe presentreportand previousstudies on
effectofT.harzianuminthefitnessofblackpepper;itcanbe
suggestedthatasmycorhizosphere,anothermicroecological
niche,viz.,‘trichorhizosphere’isalsocoexistsinalteringthe
communitydynamicsofbacteriaandsoilfungi;andthus,the
rhizospheremicroecosystemdevelopedbyT.harzianummight
contributeapivotalroleinimpartingplanthealth,whichis
unliketheloneeffectofT.harzianum.Themethodsemployed
inthisstudyshowasignificantsteptowardpossible
imple-mentation of metagenomics for the functional elucidation
ofT.harzianum–thevaluablebiocontrol,growthpromoting
fungusin the productionsystemof blackpepper.The
rhi-zosphere and the trichorhizospheremetagenomes ofblack
pepperelucidatedinthisstudywouldbecomeimportant
fac-torsindevelopinganyIDMmodulesintherootecosystemof
blackpepper.Further,targetedstudiesbasedonthepresent
metagenomicreadoneachorganismandateachcomponent
wouldgiveenormousinformationonthismicroclimate.
Conflicts
of
interest
Theauthorsdeclarethatthereexistsnoconflictofinterest.
Acknowledgements
This study was funded by Indian Council of Agricultural
Research, India, through outreachproject “PhytoFuRa
(Phy-tophthora, Fusarium and Ralstonia diseases of Horticultural
and Field Crops”. www.phytofura.net.in). The sequencing
servicewashiredfromScigenome,Kochi,Kerala.Theauthors
acknowledgeDrs.DevasahayamandSasikumar,ICAR–Indian
InstituteofSpicesResearch, Kozhikodeode,Keralafortheir
supportincarryingouttheexperiments.PUisgratefultoDrs.
V.Srinivasan,Hamza,SJEapen,K.KandiannanandD.Prasath,
ICAR–IndianInstituteofSpicesResearch,Kozhikode,Kerala
fortheirvaluablesuggestioninimplementingthiswork.
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