Biocontrol potential of saline- or alkaline-tolerant Trichoderma asperellum mutants against three pathogenic fungi under saline or alkaline stress conditions

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Bacterial,

Fungal

and

Virus

Molecular

Biology

Biocontrol

potential

of

saline-

or

alkaline-tolerant

Trichoderma

asperellum

mutants

against

three

pathogenic

fungi

under

saline

or

alkaline

stress

conditions

Ruiting

Guo,

Zhiying

Wang,

Ying

Huang,

Haijuan

Fan,

Zhihua

Liu

NortheastForestryUniversity,SchoolofForestry,Harbin,China

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24September2017 Accepted14February2018 Availableonline13April2018 AssociateEditor:VâniaMelo

Keywords:

Biocontrol

Trichodermaasperellum

Mutantlibrary Salinityandalkalinity Fungaldisease

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b

s

t

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t

Salinityandalkalinityare majorabioticstressesthatlimitgrowthanddevelopmentof poplar.Weinvestigatedbiocontrolpotentialofsaline-andalkaline-tolerant mutantsof

TrichodermaasperellumtomediatetheeffectsofsalinityoralkalinitystressesonPopulus

david-iana×P.albavar.pyramidalis(PdPappoplar)seedlings.AT-DNAinsertionmutantlibraryof

T.asperellumwasconstructedusinganAgrobacteriumtumefaciensmediatedtransformation

system;thisprocessyieldedsixtyfivepositivetransformants(T1–T65).Thesalinitytolerant mutant,T59,grewinPotatoDextroseAgar(PDA)containingupto10%(1709.40mM)NaCl. UnderNaCl-richconditions,T59wasmosteffectiveininhibitingAlternariaalternata(52.00%). Thealkalinitytolerantmutants,T3andT5,grewinPDAcontainingupto0.4%(47.62mM) NaHCO3.TheabilityoftheT3andT5mutantstoinhibitFusariumoxysporumdeclinedas

NaHCO3concentrationsincreased.NaHCO3toleranceofthePdPapseedlingsimproved

fol-lowingtreatmentwiththesporesoftheWT,T3,andT5strains.Thesalinitytolerantmutant (T59)andtwoalkalinitytolerantmutants(T3andT5)generatedinthisstudycanbeapplied todecreasetheincidenceofpathogenicfungiinfectionundersalineoralkalinestress.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

Trichoderma sp. is an important biocontrol agent against

pathogenicfungi.1 However,growthand physiologicalstate

ofTrichodermasp.isaffectedbybothbioticandabioticstress

factorsinthenaturalenvironment.Forexample,salinityand alkalinityare majorabioticstressesthatlimitinggrowth of

∗ _{Correspondingauthor.}

E-mail:[email protected](Z.Liu).

Trichoderma.2,3_{Thus,weconstructedaTrichodermasp.mutant}

librarytoobtainsaline-oralkaline-tolerantmutantsandthen tested their effectiveness as antifungal agents under both salineandalkalineconditions.

Most existing Trichoderma mutant libraries have been focused on cellulases in efforts to generate commercially applicable cellulolyticstrains. For example,Trichoderma ree-sei mutantsMCG77(ATCC56764)andQM9414(ATCC26921) produce more cellulase than the parent strain.4 _{T. reesei} mutant NG-14 hasincreasedfilter paper-degrading activity (five times higher) and specific activity (two times higher)

https://doi.org/10.1016/j.bjm.2018.02.008

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

comparedwithT. reeseimutantQM9414.5 _{Trichoderma viride} mutantstrainQM9123secretestwiceasmuchcellulaseasthe parentstrain.6_{T.viridemutantT100-14hasbetterconversion} ofcellobiosetoglucosethan theparentstrain.7 _Trichoderma

atroviridemutantTUBF-1505andT.citrinoviridemutantEB-104

bothproduce higherlevels of cellulases than their respec-tiveparent strains.8,9 _{There are a few previous studies on} biocontrol ofTrichoderma inregards to isonitrile antibiotic, cyanidehydratase,Cdresistance,andsalinitytolerance.10–13 OneexampleisthataT.harzianummutantcanexcrete isoni-trileantibiotic.11 _{TrichodermakoningiimutantT21hashigher} cyanidehydrataseactivitythanthewildtype.13_Tenof200T.

koningiimutantshavehigherCdtoleranceand canbeused

toremediateCd-contaminatedsoils.14_{FourTrichoderma} iso-lateshave been identified asstablemutants. Two ofthese are salt-tolerant mutants (Th50M6 and Th50M11), both of whicharesignificantlymoreeffectivethanthewildtypestrain atinhibitingFusariumoxysporumandimprovingtheyieldof tomatoplantsundersalineconditions.3_{Seedlingsraisedfrom} salinitytolerantTrichodermaisolateshadhigherlevelsoftotal phenols and superoxide dismutaseactivity. Saline tolerant

Trichodermamutantscaneffectivelyreducethephysiological

damagecausedbysaltstress10,15_{;however,therearefew} stud-iesregardingT.asperellumoralkanitytolerantmutants.

Poplarisamodelorganismfortheapplicationofgenetic engineering to forestry. Furthermore, poplar species are widelyusedforwoodproductioninshort-rotationforestry,16 andarecultivatedworldwide.17 _{Popularisalsousedto} elu-cidatethephysiologicalandmolecularmechanismsofstress toleranceinTrees.18_{Therearemanystressors,suchassalinity} andalkalinity,thatcancausedamagetopoplarsanddecrease theirproductivity.19_{Trichodermatreatmentshavebeenshown} toalleviateNaCl stress(70, 150and240mMNaCl) and sig-nificantlyincreasethelengthandfreshweightoftheshoot androot, numberofleaves,leafarea,and chlorophyll con-tentcomparedwiththecontrol.20_{TrichodermaasperellumQ1} canenhancecucumbergrowthbyinducingphysiological pro-tectionundersalinitystress.21_{Itispossiblethat salinityor} alkalinitytolerantT.asperellummutantscanimprovethe resis-tanceofpoplarseedlingstosalineoralkalinestressors.

In this study, a T-DNA insertional mutant library of

T. asperellum was constructed by Agrobacterium tumefaciens

mediated transformation system (ATMT). Some salinity or alkalinity tolerant mutants of T. asperellum were obtained. Then,the biocontrolpotentialofthe mutantstrainsunder NaClorNaHCO3stresswastested.And,thefunctionof alle-viatingsaline or alkalinestress to the poplar seedlings of themutantstrainsunderNaClorNaHCO3stresswastested. Ourresultsprovidetheoreticalsupportandapractical refer-enceforthedevelopmentofTrichodermastrainsforalleviating saline or alkaline stress and managing fungal diseases in salineoralkalinesoils.

Materials

and

methods

Strains,plantmaterialsandprimers

T.asperellumACCC30536wasobtainedfrom theAgricultural

Culture Collection of China. Alternaria alternata CFCC82114

(poplarleafwither),RhizoctoniasolaniCFCC86328,andF. oxyspo-rumCFCC86068wereobtainedfromtheChinaForestryCulture Collection Center.A.tumefaciens AGL-1,which contains the binary vector pBHt22 _{and a hygromycin B-resistance gene} (hph),wasprovidedbyProf.FucongZheng(SouthChina Uni-versityofTropicalAgriculture,China).

Populus davidiana×P. alba var. pyramidalis Louche (PdPap

poplar) seedlings were cultured aseptically for 15din Liq-uid RootingMedium(MS mediumwithNAA0.25mg/L and Sucrose20g/L)orDifferentialMedium(MSmediumwithNAA 0.05mg/L,6-BA 0.5mg/L, Sucrose20g/L, and Agar8g/L) at 25◦C23,24_{;theyallhadsimilarheights(about5cm)anda} sim-ilarnumberofleaves(6–8leaves).24

TheprimersHph-L(5-GAAACCGACGCCCCAGCACT-3)and Hph-R (5-AGTGCTGGGGCGTCGGTTTC-3) were designed to detect the hygromycin B-resistance gene (hph) using the Primer6.0software(PREMIERBiosoft,USA)andsynthesized bySangonBiotech(Shanghai,China)Co.,Ltd.

ConstructionoftheT-DNAinsertionalmutantlibraryfor T.asperellumandanalysisofthemutantsgeneticstability

T. asperellum was transformed using the ATMT method.24

200␮Mof35-Dimethoxy-4-HydroxyAcetophenone(AS)was addedtotheinducedandco-cultivated(pH5.0)media,and theconcentrationofT.asperellumwas106_spores/mL.25,26

Todeterminemitoticstability,80putative transformants were selectedrandomly,culturedfiveconsecutive timeson PotatoDextroseAgar(PDA)withouthygromycinB,and trans-ferredtoPDAcontaining300␮g/mLhygromycinB.24

ThehphgenewasassayedusingPCRintheputative trans-formantswhichhadmitoticstability.Thesenseprimerwas Hph-L (5-GAAACCGACGCCCCAGCACT-3)and theantisense primerwasHph-R(5-AGTGCTGGGGCGTCGGTTTC-3).25 GrowthconditionsoftheWTandmutantstrainsinPDA withdifferingconcentrationsofNaClorNaHCO3

TheWTandmutantstrainswereculturedat28◦Cfor5din PDA.Myceliumdiscs(5mmdiameter)wereobtainedfromthe edgesofthePDAplateswithaholepunch,inoculatedintonew PDA,andculturedat28◦Cfor5d.Finally,myceliumdiscsof 5mmdiameterwereobtainedfromtheedgesofPDAplates, inoculatedintothePDAwithdifferentconcentrationsofNaCl or NaHCO3, and culturedat28◦Cfor3–5d. The concentra-tionsofNaClwere2,4,6,8,and10%,andtheconcentrations ofNaHCO3were0.1,0.2,0.3,and0.4%.Thecolonydiameters wererecordedeverydayduringculturing.Theexperimentwas completelyrandomizedwiththreereplicates.

ThebiocontrolpotentialoftheWTandmutantstrainsin PDAwithdifferentconcentrationsofNaClorNaHCO3

TheWTand mutantstrainswerechallengedwiththe phy-topathogensA.alternata,R.solani,andF.oxysporum.PDAwith differentconcentrationsofNaClorNaHCO3wereusedinthe challenge experiments, and PDA was used ascontrol. The concentrationsofNaClwere2,4,6,8,and10%,andthe concen-trationsofNaHCO3were0.1,0.2,0.3,and0.4%.Symmetrical positionsweremarkedontheplates.Myceliumdiscs(5mm

diameter)wereobtainedfromthesamepartofthePDAplates, inoculatedontonewPDAonthesymmetricalpositions,and culturedat28◦Cfor5–10d.Theexperimentwascompletely randomizedwiththreereplicates.IR=(DC−DE)/(DC)×100%, where:“IR”istheabbreviationofinhibitionrate;“DC”is abbre-viation of the diameter ofthe control group, and; “DE” is abbreviationofthediameteroftheexperimentgroup.27 TheNaClorNaHCO3toleranceofthePdPapseedlings

co-culturedwiththeWT,T3,andT5strains

ThePdPapseedlingswereco-culturedwithsporesoftheWT, T3,andT5strainsunderNaCl(1and2%)andNaHCO3(0.05,0.1 and0.2%)stress.Thecellconcentrationswere106_spores/mL. LeavesfromthePdPapseedlingswereassayedforSOD,CAT, and POD afterco-culturing for0, 1, 3, 5, 7,and 15dusing commerciallyavailablekits(NanjingJiancheng Bioengineer-ingInstitute).

Results

TheT-DNAinsertionalmutantlibraryofT.asperellum andthegeneticstabilityoftheputativetransformants ThetransformationefficiencyofATMTisabout60mutantsper 106_{spores.Toinvestigatethemitoticstabilityoftheintegrated} T-DNA,eightyputativetransformantswiththevectorpBHt1 weretransferredrepeatedlytoPDA.Afterfiveroundsofgrowth onPDA,myceliumdiscsweretransferredtoselectivePDAwith 300␮g/mLhygromycinB.Alltransformantsthatgrewunder theseconditionshadhighmitoticstability.

Sixtyfivepositivetransformantswereobtained.PCR detec-tion of the target gene was performed on the 80 selected putative transformants. Presence of the hph gene in the hygromycinBresistantcolonieswasconfirmedwiththe Hph-LandHph-Rprimers.The1005bpPCRproductwasfoundinall 65putativetransformants;thissuggeststhatthehphgenewas successfullyintegratedintotheT.asperellumgenome(Fig.1). GrowthconditionsoftheWTandmutantstrains,aswell astheirbiocontrolpotential,underdifferentNaCl concentrations

GrowthoftheWTstraincanbepromoted by2%NaCl dur-ingtheearlystagesofgrowth.However,inthelaterstagesof growth,thiseffectgraduallydecreasesandthendisappears. Thegrowth oftheWTstrainwascompletelyinhibitedata NaClconcentrationof10%(Fig.2).

ThemutantstrainswerescreenedforNaCltoleranceusing PDA with 10% NaCl. Of the 65 mutant strains, only one (T59)grewonPDAwith10%NaCl(Fig.2).AlthoughtheT59 mutantgrewquicklyunderdifferentNaClconcentrations,the myceliumgrowthconditiondidnotchange;however,its abil-ity toproducespores wasinhibitedunderNaCl stress.The NaCltoleranceoftheT59strainwashigherthanthatofthe WTstrain.

On day threeof culture,the T59 strain was exposed to thepathogenicfungi.Onthefifthofculture,theT59mutant totallycoveredallthreefungalphytopathogenfungi(Fig.2). TheT59strainhadthehighestinhibitionrate(52.00%)against

Alternariaalternataunder10%NaClconditions.Meanwhile,the

WTstraingrewslowlyunderNaClstresses.TheT59strainhad abetterbiocontrolpotentialthantheWTunderNaClstress.

GrowthconditionsoftheWTandmutantstrains,aswell astheirbiocontrolpotential,underdifferent

concentrationsofNaHCO3

In later stages of growth, the inhibition of the WT strain wasweakerunder0.05,0.1,and0.2%NaHCO3concentrations; thus, under theseconditions NaHCO3 does notinhibit the growth ofthe WTstrain.ThegrowthoftheWTstrainwas completelyinhibitedunder0.4%NaHCO3.

After3dculturingtheT3andT5mutantsgrewonthePDA with0.4%NaHCO3.ThegrowthoftheT5mutantwasfaster thanthatoftheT3mutant(Fig.3).TheNaHCO3tolerancesof theT3andT5mutantsarehigherthanthatoftheWTstrain. TheT5mutantwasexposedtoR.solani,andafter4dtheT5 mutantinhibitedthegrowthofR.solaniunder0.1%NaHCO3 conditions.TheT5mutantissensitivetohigherNaHCO3 lev-els;however,R.solaniexhibitedsimilargrowthunder0.2,0.3, and0.4%NaHCO3,whichwashigherthanunder0.1%NaHCO3. TheT5mutantwasexposedtoR.solaniafter6dofexposure toboth0.2%and0.3%NaHCO3,andexposedtoR.solaniafter 7dofexposureto0.4%NaHCO3.However,theT5mutantdid notcompletelyinhibitR.solaniat0.4%NaHCO3after10dof exposure(Fig.4AandD).

TheT5mutantinhibited A.alternataafter5dofgrowth; however,after10dofgrowththemutantdidnotcompletely inhibit A. alternata at 0.1% NaHCO3. At0.2% NaHCO3, the growthofA.alternatawasslowerthanatotherNaHCO3 con-centrations.TheT5mutantinhibitedA.alternataafter6dof growth;however,after10dofgrowththeT5mutantdidnot coveredinhibitA.alternata.At0.3%NaHCO3,theT5mutant inhibitedA.alternataafter5dofgrowth;however,after10dof growththeT5mutantdidnotcompletelyinhibitA.alternata.

1005bp 100bp 250bp 750bp 1000bp 2000bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 500bp 20

Fig.1–PCRdetectionofthehphgeneinassumedtransformants.TheHphgenewasamplifiedwiththeHph-LandHph-R primers.Partialagargelelectrophoresis.Lanes1–18:18putativetransformants;lane19:wild-typeT.asperellum;lane20: DL2000DNAmarker(TaKaRaCo.).

6% NaCl

8% NaCl

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4% NaCl

10% NaCl

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b

c

d

Fig.2–BiocontrolpotentialoftheT59mutantunderNaClstressafter6d:thegrowthoftheWTandT59strainsinPDA(A andC);thegrowthoftheWTandT59strainsinPDAwith10%NaCl(BandD),and;confrontationoftheT59mutantwith

Alternariaalternata,Fusariumoxysporum,orRhizoctoniasolaniunderdifferentNaClconcentrations(E–G).

a

d

g

b

c

f

e

Fig.3–GrowthoftheWTandmutantstrainsunderNaHCO3stressafter4d:theWT,T3,andT5strainsinPDA(A–C);the

WT,T3,andT5strainsinPDAwith0.4%NaHCO3(D–F),and;challengeoftheT3strainwiththeT5straininPDAwith0.4%

NaHCO3(G).

d

a

b

c

e

f

Fig.4–ConfrontationoftheT5mutantwithR.solani,F.oxysporum,orA.alternataafter10d:challengeoftheT5mutant withR.solani,F.oxysporum,orA.alternataunderdifferentNaHCO3stresses(A–C,thereverseside);challengeoftheT5

At0.4%NaHCO3,theT5mutantdidnotcompletelyinhibitA.

alternataafter7dor10dofgrowth(Fig.4BandE).

TheT5mutantinhibitedF.oxysporumafter5dofgrowth; however,itdidnotcompletelyinhibitF.oxysporumafter10dof growthat0.1%NaHCO3.ThegrowthofF.oxysporumincreased withNaHCO3concentrationsinthePDA.Itturnsoutthatthe growthofF. oxysporumispromoted byNaHCO3 andthe T5 mutantwasunabletoinhibitF.oxysporumat0.2,0.3,or0.4% NaHCO3(Fig.4CandF).

TheT3mutanthad similar abilitiestoinhibit the three pathogenic fungiunder different NaHCO3 levels. However, sincethegrowth oftheT3mutantwasslower thanthatof theT5mutantunderhigherNaHCO3conditions,theabilityof theT3mutanttoinhibitA.alternataat0.3%and0.4%NaHCO3 waslowerthanthatoftheT5mutant(Fig.5).

GrowthofPdPapseedlingsunderNaClorNaHCO3stress

ThePdPapseedlingswitheredinMSwith1or2%NaCl (espe-cially2%NaCl),andtheseedlingsdiedafter4d.Inthelater stagesofculture,theinhibitionofthepathogenicfungiwas weakerunderNaClstress.

At0.05%NaHCO3,theleaves,stems,androotsofthePdPap seedlingsdidnotchangeafter15dofculture.At0.1%NaHCO3, therootsbegantobrownafter15dculture.At0.2%NaHCO3, therootswerebrownafter3dofculture,buttheleavesand stemswerenormal.At0.3%NaHCO3,therootswerebrown after2dofculture,andtheleavesandthestemswere with-ered.

NaClandNaHCO3toleranceofPdPapseedlingstreated

withtheWT,T59,T3,orT5spores

After2dofco-culture,thesporesoftheWTandT59strains germinatedunderNaClstressandgatheredaroundtheroots of the PdPap seedlings. The NaCl tolerance of the PdPap seedlingsdidnotchange,butthePdPapseedlingswithered. Thus,the NaCltoleranceofthe PdPapseedlings cannotbe improvedbytreatmentwithWTorT59spores.

Intheearlystageofculture,thePdPapseedlingswere seri-ouslydamagedat0.3%NaHCO3;however,followingtreatment withWT,T3,orT5spores,theroots,leaves,andstemswere normalafter2d.After5dofculture,theleavesandthestems inthegroupstreatedwithsporeswere inbetterconditions thanthoseinthegroupsnottreatedwithspores.Thus,the toleranceofthePdPapseedlingstoNaHCO3canbeenhanced byWT,T3,orT5spores.

EnzymaticactivityofthePdPapseedlingstreatedwith WT,T3,orT5sporesunderNaHCO3conditions

At0.05% NaHCO3, the SOD,CAT,and POD activitiesin the control group increased, decreased, and slightly increased, respectively,comparedtothecontrol.TheSODandCAT activi-tiesofthePdPapseedlingstreatedwithWT,T3,andT5spores wasmaintainedduring3–7doftreatment.TheSODactivity wasincreasedaftertreatment,andthenremainedconstant (Fig.6C).TheWT,T3,andT5sporesallhadsimilarimpacts onSOD,CAT,andPODactivitiesinthePdPapseedlings(no significantdifferencesatp≤0.05).Therewerenodifferences intheconditionsoftheleaves,stems,androotsofthePdPap seedlingsfollowingthefourdifferenttreatments(Fig.6D).

At0.1%NaHCO3,theSODandPODactivitieswasincreased, decreased, and then increased compared with the control group.TheSODactivityofthePdPapseedlingstreatedwith WT,T3,andT5sporesremainedconstantduring1–15dof cul-ture(Fig.7C).ThePODactivityofthePdPapseedlingstreated withWT,T3,andT5sporesremainedconstantduring3–5dof cultureandthendecreased(Fig.7A).TheCATactivitiesofthe PdPapseedlingstreatedtheWT,T3,andT5sporesremained constantfrom3–7dofculture,thendecreasedrapidlyonthe 15thdayofculture(Fig.7A).TheWT,T3,andT5sporeshad similar impactsontheSOD, CAT,and PODactivitiesofthe PdPap seedlings, and there were no significant differences (p≤0.05).ThebiomassofthePdPapseedlingstreatedwiththe WTsporeswerehigherthanthosetreatedwithsporesofthe otherstrains(Fig.7D).

At0.2%NaHCO3,theSODandPODactivitiesfirstincreased, thendecreasedrapidly,andthenincreasedagaincompared

e

a

f

b

c

d

Fig.5–ConfrontationoftheT3mutantwithR.solani,F.oxysporum,orA.alternataafter10d:challengeoftheT3mutant withR.solani,F.oxysporum,orA.alternataunderdifferentNaHCO3stresses(A–C,thereverseside);challengeoftheT3

a

b

POD activity(U mg protein

-1)

SOD activity(U mg protein

-1) CA T activity(U mg protein -1) aaaa c c a POD 0.5%NaHCO3 0.05%NaHCO3 +the WT 0.05%NaHCO3 +the mutant T3

0.05%NaHCO3 +the mutant T5

b b b b b b a a a a _aaaa a a c aaaa b b b b b b b bc bc b b b a a a a a a a 30 35 40 45 50 25 20 15 10 5 0 CAT Induced time(d) Induced time(d) Induced time(d) SOD 50 60 40 30 20 10 0 0 1 3 5 7 15 aaaa a a b b c c a b a bba c a a b a a a a

d

c

Fig.6–EnzymaticactivitiesofthePdPapseedlinginteractedwiththeTrichodermasporesandunder0.05%NaHCO3stress:

SOD,CAT,andPODactivitiesofthePdPapseedlingstreatedwiththesporesoftheWT,T3,andT5strainsunder0.05% NaHCO3stress(A–C,respectively);PdPapseedlingstreatedwithsporesoftheWT,T3,andT5mutantsunder0.05%NaHCO3

stress(D).Valuesineachcolumnfollowedbythesameletterarenotsignificantlydifferent(p≤0.05).

tothecontrolgroup.TheSODandPODactivitiesofthePdPap seedlingstreatedwiththeWT,T3, andT5sporeswere the sameasthoseofthecontrolgroup.However,theSOD activ-ityofthePdPap seedlingstreatedwiththeWT,T3,and T5 sporeswashigherthanthatofthecontrolgroupduring3–7d ofculture,especiallyonthe5thdaywhentheSODandPOD activitieswere2.14and1.37 timesgreaterthaninthe con-trolgroup,respectively(Fig.8AandC).TheCATactivityinthe PdPapseedlingstreatedwiththeWT,T3,andT5sporeswas lowerthaninthecontrolgroup(Fig.8B).TheWT,T3,andT5 sporeshadsimilarimpactsontheactivitiesoftheSOD,CAT, andPODinthePdPapseedlings(nosignificantdifferencesat

p≤0.05).

Discussion

Soilsalinizationisaworldwideproblemcausedbythe long-term use of drip irrigation. It has become a major factor limitingagriculturalandforestryproduction.2_Wefoundthat the growth of the WT strain of T. asperellum can be pro-motedby 2%NaCl.However, asincubation time increased thepromotioneffectgraduallyweakenedandeventually dis-appeared.TheWT strainhas powerful NaCltolerance, but it cannot grow in PDA containing 10% (1709.40mM) NaCl. TheT59mutanthadbestNaCltoleranceofallthemutants, andcangrowinPDAcontaining10%(1709.40mM)NaCl.Two

T. harzianum salt-tolerant mutants (Th50M6 and Th50M11)

which cangrowinPDAcontainingup to69mMNaClwere obtained.3 _{Five of forty-five T. harzianum wild-type strains} (Th-13,Th-14, Th-19,Th-33,and Th-50)cangrowandform spores in growth containing up to 240mM NaCl.20 _{The T.}

asperellum wild-type strains (TaDORS3 and TaDOR693) can

growonPDAwith1000mMNaCl.27_{Meanwhile,theNaCl} tol-eranceoftheT59mutantis25timeshigherthantheTh50M6 and Th50M11mutants,3 _{seventimeshigherthan the} wild-type strains Th-13, Th-14, Th-19, Th-33, and Th-50,20 _and 1.7 times higher than the wild-type strains TaDORS3 and TaDOR693.27

TheTrichodermaWTstrainwasverysensitivetoNaHCO3;

meanwhile,theT3andT5mutantshadbestNaHCO3 toler-anceofall the mutants(T1-T65).Thereare afew previous studiesthat reporttheNaHCO3 toleranceofTrichodermasp. UnderNaClorNaHCO3cultureconditions,themutantsstill exhibited antagonistic abilities towards R. solani, F.

oxyspo-rum, or A. alternata. The mutantT59 grows quickly under

fourdifferentNaClconcentrations,andwasmoreprotective againstthethreekindsofpathogenicfungithantheWTstrain. TheTh50M6mutanthadbetterantagonisticactivitythanthe wild-typestrain,andhadamaximuminhibitionrateof60% againstF.oxysporuminsalinemedia.3_{Thesaline-tolerantT.}

harzianumstrainTh-14significantlyreducedtheincidenceof

F. oxysporum-inducedwiltdisease inchickpeaplants.20 _The

T5 mutantalso had antagonistic abilities towards R.solani

and A. alternata under NaHCO3 stress. TheT5 mutanthas

a

b

POD activity(U mg protein

-1)

SOD activity(U mg protein

-1) CA T activity(U mg protein -1) a a aa a b b b POD 0.1%NaHCO3 0.1%NaHCO3 +the WT

0.1%NaHCO3 +the mutant T3

0.1%NaHCO3 +the mutant T5

b b b b b b b a aa aa a a a a aaaa b b b b b b ba a a a a _a c a a a a a a 30 25 20 15 10 5 0 CAT Induced time(d) Induced time(d) Induced time(d) SOD 50 40 30 20 10 60 0 0 1 3 5 7 15 a a a a b a b b b b a b a a a a a a b a a b b a a b

d

c

Fig.7–EnzymaticactivitiesofthePdPapseedlinginteractedwiththeTrichodermasporesunder0.1%NaHCO3stress:SOD,

CAT,andPODactivitiesfromthePdPapseedlingstreatedwithsporesoftheWT,T3,andT5strainsunder0.1%NaHCO3

stress(A–C,respectively);PdPapseedlingstreatedwithsporesoftheWT,T3,andT5mutantsunder0.1%NaHCO3stress(D).

Valuesineachcolumnfollowedbythesameletterarenotsignificantlydifferent(p≤0.05).

0.2%(23.81mM)NaHCO3 conditions.Ragazzietal.reported thatdifferentFusariumspecies,includingF.oxysporum,were promoted under salt stress conditions.28 _{Our results agree} withthisreport,astheantagonisticabilitiesoftheT5mutant against F. oxysporum decreased as NaHCO3 concentrations increased.Theantagonistic abilitiesofthe T3mutantwere nearlythesameasthoseoftheT5mutant.The antagonis-ticabilitiesoftheT3mutanttowardsA.alternataunder0.3% (35.71mM) and0.4%(47.62mM)NaHCO3 cultureconditions wereweakerthantheseoftheT5mutant.

Saltrepressesplantgrowthandrootdevelopment.29_Ina previousstudy,tomato plantswere abletogrowwhile irri-gateddailywitha2800ppm(2.8mg/L)NaCl solution.3 _Grey poplar(Populustremula×alba,syn.Populuscanescens)cangrow inmoderatelevelsofNaCl(75mM).16_{TheshootgrowthofP.}

euphraticawasnotreducedeveninmediacontaining100mM

NaCl.However,theshootgrowthofChinesepoplars(Populus

tomentosaandP.albacv.pyramidalis)wasmarkedlyreducedin

10mMNaCl.30_{Photosynthesis,leafarea,height,andgrowth} diameter of poplars declined rapidly as salinity increased. Someplantmortalityhasbeenobservedafter14daysat1% (170.94mM)NaCl.31 _{Inthisstudy,theconcentrationofNaCl} intheMSmediawas1%(170.94mM)or2%(341.88mM).The PdPapseedlingsbecamewitheredunder1%(170.94mM)or2% (341.88mM)NaClconditions,andtheydiedafter4dofculture. TheNaCltoleranceofthePdPapseedlingswasnotimproved bytreatmentwithWTorT59spores.Thesporesgrewunder

NaClconditions.However,thePdPapseedlingswerecultured inMSsotherewasadifferencebetweentheexperimentaland naturalconditions.

Alkaline salts have been shown to havemuch stronger destructiveeffectsonplantsthanneutralsalts.Whenasaline soilcontainsHCO3−,plantsaresubjecttothedamagingeffects ofbothsaltandalkalistress.32_{Of12halophytes,mosthad} lower germinationpercentages inNaHCO3 than in NaCl.33 Saline-alkaline stress can cause an increase antioxidant enzyme activity.34 _{Superoxide dismutases (SODs) catalyze} the dismutation ofthe superoxide anionto hydrogen and molecular oxygen; this is the first step in active oxygen-scavenging systems. CATand POD are alsoveryimportant foroxygen-scavengingsystems.35_{Theyplayanimportantrole} in protecting cells against various environmental stresses, includingsalinityandalkalinitystress.36_{Theycanbeusedto} evaluatesalinityandalkalinityresistance.Inthisstudy,the impactoftheWT,T3,andT5strainsonSOD,CAT,andPOD activitiesinthePdPapseedlingsweresimilarunderNaHCO3 stress(nosignificantdifferencesatp≤0.05).Underthe0.05% NaHCO3condition,theSODactivityofPdPapseedlingstreated withthe WT,T3, andT5spores increasedsignificantly fol-lowing3–5dofco-culturing.Thisisconsistentwithprevious studies showingthatT.asperellumtreatmentincreasedSOD activityof‘XY335’and‘JY417’maizeseedlingsinsalinesoil and increased the activity of antioxidant enzymes under NaCl stress in cucumbers.10,37 _{In this study, CAT activity}

a

b

POD activity(U mg protein

-1)

SOD activity(U mg protein

-1) CA T activity(U mg protein -1) a a aa a a a a POD 0.2%NaHCO3 0.2%NaHCO3 +the WT

0.2%NaHCO3 +the mutant T3 0.2%NaHCO3 +the mutant T5

b b b b b b a a a a a a a a a a aa a a b b b b b b b b b b c a a c c a a a a a 30 25 20 15 10 5 0 CAT Induced time(d) Induced time(d) Induced time(d) SOD 50 45 40 35 30 25 20 15 10 5 0 0 1 3 5 7 10 aaaa ab ba c a ba a aa b c a a b _b a aa

d

c

Fig.8–TheenzymaticactivitiesofthePdPapseedlingsinteractedwiththeTrichodermasporesunder0.2%NaHCO3stress:

SOD,CAT,andPODactivitiesfromthePdPapseedlingstreatedwithsporesoftheWT,T3,andT5strainsunder0.2% NaHCO3stress(A–C,respectively);PdPapseedlingstreatedwithsporesoftheWT,T3,andT5mutantsunder0.2%NaHCO3

stress(D).Valuesineachcolumnfollowedbythesameletterarenotsignificantlydifferent(p≤0.05).

increased;thesediffersfromapreviousreportshowingthat CAT activity was reduced in leaves and roots following T.

asperellumtreatment.37_{PODactivitywasdecreasedfollowing}

treatment.Previousstudieshaveshownthatsaline–alkaline stressincreasestheactivityofsome,butnotall,antioxidant enzymes.12,38,39 _{Under 0.1%NaHCO}

3 conditions, SOD, CAT, and PODactivitiesin thePdPap seedlingstreated withthe WT,T3,orT5sporeswerelowerthanfromplantsunderthe sameconditionsbutsubjectedto0.05%NaHCO3stress. How-ever, biomassof the PdPap seedlings treatedwith WT,T3, orT5sporeswerehigherthanthoseofthePdPapseedlings treatedwiththespores under0.05%NaHCO3 stress.Under 0.2%NaHCO3stress,theSODandPODactivitiesofthePdPap seedlings treated with WT, T3, and T5 spores was higher thaninthe controlgroup; however,CATactivity waslower than inthe controlgroup.HighCATactivity indicates that theplantsareunderoxidativestress.37 _{Ourresultssupport} thisconclusion.Efficientantioxidantactivitydoesnot neces-sarilyleadtotheup-regulationofallantioxidantenzymes.40 The demand of the oxygen-scavenging enzyme decreased under0.2%NaHCO3 stress.Itispossiblethat theactivities ofother antioxidant enzymes inthe PdPap seedlings were impactedby thespores ofthe WT,T3, or T5strains, lead-ingtoimprovementsintheNaHCO3toleranceofthePdPap seedlings.Thus,theNaHCO3toleranceofthePdPapseedlings canbeimproved bytreating withsporesfrom the WT,T3, and T5 strains grown under 0.05, 0.1, and 0.2% NaHCO3 stress.

Wegeneratedasaline-andtwoalkaline-tolerantT. asperel-lummutants(T59,andT3andT5,respectively);thesemutants had better biocontrol potential under NaCl or NaHCO3 stresses.WeintroduceanewapproachforthebiocontrolofT.

aspellumbygeneratingsaline-oralkaline-tolerantstrainsby

mutationselection.Thisapproachcouldbeusefulfor enhanc-ingsalt and/oralkaline-salttolerance,aswell astheability tobiocontrolforR.solani,A.alternata,andF.oxysporumunder salineoralkalinestressconditions.

Ethical

approval

Themanuscriptdoesnotcontainexperimentsusinganimal orhumanstudies.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Thiswork wassupported bythe National HighTechnology ResearchandDevelopmentProgram(the13thFive-YearPlan Program) [Grant Number 2016YFC0501505] and the Funda-mental Research Fundsfor the Central Universities, China [GrantNumber2572017AA03].

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