Selection, isolation, and identification of fungi for bioherbicide production

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

Environmental

Microbiology

Selection,

isolation,

and

identiﬁcation

of

fungi

for

bioherbicide

production

Angélica

Rossana

Castro

de

Souza

a

_,

_Daiana

_Bortoluzzi

_Baldoni

b

_,

_Jessica

_Lima

a

_,

Vitória

Porto

a

_,

_Camila

_Marcuz

a

_,

_Carolina

_Machado

a

_,

_Rafael

_Camargo

_Ferraz

d

_,

Raquel

C. Kuhn

a

_,

_Rodrigo

_J.S.

_Jacques

b

_,

_Jerson

_V.C.

_Guedes

c

_,

_Marcio

_A.

_Mazutti

a,∗ a_Universidade_Federal_de_Santa_Maria_(UFSM),_Departamento_de_engenharia_química,_Santa_Maria,_RS,_Brazil

b_Universidade_Federal_de_Santa_Maria_(UFSM),_Departamento_em_ciência_do_solo,_Santa_Maria,_RS,_Brazil c_Universidade_Federal_de_Santa_Maria_(UFSM),_Departamento_de_protec¸ão_de_plantas,_Santa_Maria,_RS,_Brazil d_Universidade_Federal_do_Pampa,_Sant’Ana_do_Livramento,_RS,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19August2015

Accepted2May2016

Availableonline4October2016

AssociateEditor:FernandoDini

Andreote Keywords: Bioprospecting Diaporthe Bioproduct Liquidfermentation

a

b

s

t

r

a

c

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Production ofa bioherbicide forbiological controlof weedsrequires a series ofsteps,

fromselectionofasuitablemicrobialstraintoﬁnalformulation.Thus,thisstudyaimed

toselectfungiforproductionofsecondarymetaboliteswithherbicidalactivityusing

bio-logicalresourcesoftheBrazilianPampabiome.Phytopathogenicfungiwereisolatedfrom

infectedtissuesofweedsinthePampabiome.Aliquidsyntheticculturemediumwasused

forproductionofmetabolites.Thephytotoxicityoffungalmetaboliteswasassessedvia

bio-logicaltestsusingtheplantCucumissativusL.,andthemostpromisingstrainwasidentiﬁed

bymolecularanalysis.Thirty-ninefungiwereisolated,and28presentedsomephytotoxic

symptomsagainstthetargetplant.FungusVP51belongingtothegenusDiaportheshowed

themostpronouncedherbicidalactivity.TheBrazilianPampabiomeisapotentialresource

forthedevelopmentofnewandsustainablechemicalcompoundsformodernagriculture.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Brazilhostsapproximately20%ofthewholeworld’s

biolog-icaldiversity, whichcanbeemployedasaresourceforthe

developmentofnewandsustainableecosystemmanagement

toolsand opportunitiesforbioprospecting.1 _This_large

bio-diversityisdistributedwithinsixbiomes.Amongthose,the

Pampabiome,whichisrestrictedtopartofRioGrandedoSul

∗ _{Corresponding}_author.

E-mail:mazutti@ufsm.br(M.A.Mazutti).

State,presentsdistinctcharacteristicsofvegetation,climate,

andsoiltypes,makingitauniqueecosystemontheplanet,

capableofmaintainingahighplantandanimaldiversity.

How-ever,PampaistheleastknownBrazilianbiomeintermsofits

biodiversity.2

Despiteitsimportance,Brazilianmicrobialdiversityisstill

considered largely unknown. Discovery of microorganisms

for use as a source of commercially exploitable products

maysupportprogramsfocusedontheapplicationof

biosyn-http://dx.doi.org/10.1016/j.bjm.2016.09.004

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theticorbiodegradationprocesses.1,3_Fungi_represent_a_part

ofthemicroﬂoraofnaturalecosystemsandmaybepromising

sourcesfortheproductionofvariouscompounds.Itis

esti-matedthatthereareabout150,300–263,900fungalspeciesin

Brazil.4_Evidence_showing_that_biological_sources_can_provide

naturalproductswithphytotoxic activity opensanew

per-spectiveforthepreservationofmicrobialspeciesinthePampa

biome.

Phytopathogenicfungiproducetoxinsthatmayplayarole

inthedevelopmentofplantdiseases.Weedsareasigniﬁcant

problemincropproduction,andtheirmanagementiscrucial

formodernagriculturetoavoidyieldlossesandtoensurefood

safety.Traditionalchemicalcontroloptionsarelimiteddueto

ecodegradation,healthhazards,andthedevelopmentof

her-bicideresistanceinweeds.5Herbicide-resistantweedsarethe

mainprobleminweedcontrolduetothenumberofweed

bio-typesresistanttoherbicidesthatconstantlyincreasesbythe

continuoususeofthe sameproductsforyears.6_In_the_last

20years,nochemicalhasbeensynthesizedwithadifferent

modeofactionthanthosediscoveredsofar.7_Such_compounds

couldpresentaconsiderablepotentialasmodelsfor

develop-ingherbicideswithnewmodesofaction.8,9

Basedontheseaspects,themainobjectiveofthisworkwas

toisolatefungifromthePampabiomefortheproductionof

bioactivemoleculeswithherbicidalactivity.Thirty-nine

phy-topathogenicfungiwereisolated fromplantsofthe Pampa

biome.Theproductionofmetaboliteswasperformedinliquid

syntheticculturemedia.Thephytotoxicityofthefungiwas

assessedviabiologicaltests,andthemostpromisingstrain

wasidentiﬁedbymolecularanalysis.

Materials

and

methods

Isolationandselectionofmicroorganisms

Phytopathogenicfungiwereisolatedfrominfectedtissuesof

weedsofirrigatedriceandrangelandsatthreelocationsofthe

BrazilianPampabiome.Table1presentsthelocationsofthe

collectionsitesaswellastheweedscollectedateachlocation.

Thestrategyusedforcollectionwasbasedonselectingweeds

withsomesymptomsofinfection.Collectionwascarriedout

from December 2012toApril 2013indifferentareas ofthe

Pampabiome.Thesampleswerestoredpackedinplasticbags

andmaintainedat4◦CduringtransportationtotheLaboratory

ofBioprocesseswheretheisolationoffungiwascarriedout.

EachinfectedtissuewastransferredtoaPetridishcontaining

potatodextroseagar(PDA)andincubated at28◦Cforseven

daysinthedark.Afterthis,eachsamplewassubculturedthree

timestoobtainapureculture,whichwastransferredtoaPDA

slantinatesttubeandstoredat4◦C.

Liquidfermentation

Thegrowthofallphytopathogenicfungiisolatedinthe

pre-viousstepwascarriedoutinaliquidmedium,aimingatthe

productionofbioactivemoleculeswithherbicidalaction.For

thepre-inoculumproduction,myceliumfromeachtesttube

containingonefunguswasinoculatedonPDAinaPetridish

andincubatedforeightdaysat28◦C,whichwassufﬁcientfor

thefungalgrowthtocovertheentiresurfaceoftheagar.

After-wards,theagarsurfaceinthePetridishwaswashedwith5mL

ofautoclavedwater,andthesuspensionwastransferredfor

fermentation.

Thefermentationswerecarriedoutin250-mLErlenmeyer

ﬂasks containing125mLoffermentation mediumat28◦C,

120rpm for seven days (Innova 44R, New Brunswick). The

mediumwascomposedof(gL−1):glucose,10.0;yeastextract,

7.5;peptone,10.0;(NH4)2SO4,2.0;FeSO4·7H2O,1.0;MnSO4·H2O,

1.0;andMgSO4,0.5,andtheinitialpHwasadjustedto6.0.10

Afterthefermentation,cellswereseparatedby

centrifu-gationat4000rpmfor10min(Eppendorf,model5804R),and

thesupernatantwasﬁlteredthrougha0.45-␮mpolyvinylidene

ﬂuoride(PVDF)membrane.Theﬁlteredsamplewasusedto

evaluateitsbioherbicidalactivityinthebioassay.Eachofthe

39fungiwasconsideredadifferentbioherbicide.

The activity of the bioherbicides obtained from the

phytopathogenic fungi was demonstrated using

cucum-ber (Cucumis sativus L., variety Wisconsin), a target plant

frequently used in bioassays of herbicides. A completely

Table1–Geographicalcoordinatesofcollectionpointsaswellastheinfectedweedscollectedforisolationoffungi.

Local Coordinates Infectedweed

Latitude Longitude

DonaFrancisca(DF) −29.634086 −53.353015 Commelinaerecta(Commelinaceae)

Solanumpaniculatum(Solanaceae)

Sagittariamontevidensis(Alismataceae)

Solanumerianthum(Solanaceae)

RestingaSeca(RS) −29.845675 −53.402069 Ipomoeatriloba(Convolvulaceae)

Sorghumhalepense(Poaceae)

VilaParaíso(VP) −29.325004 −54.958734 Conocliniummacrocephalum(Asteraceae)

Passiﬂoraedulis(Passiﬂoraceae)

Solanumstipulaceum(Solanaceae)

Solanumamericanum(Solanaceae)

Baccharisdracuntifolia(Asteraceae)

Eryngiumhorridum(Apiaceae)

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randomizeddesign,composedof39treatments(eachselected

strainwas considered onetreatment) and four repetitions,

whereeachrepetitionrepresentedatraycontaining20

pro-pylene vessels with a volume of 200mL of a commercial

substrate(MacPlant®)withoutanytreatment,wasemployed.

Threeseedsweresownineachvessel, andafterthe

emer-gence, only one plant was maintained in each vessel and

transferredattheseedlingstagetoagreenhouselocatedat

theFederalUniversityofSantaMaria.Theseedsusedinthe

experimentwereobtainedfromalocalmarket anddidnot

undergoanytreatmentbeforeseeding.

Avolumeof30mLoffermentedbrothwasappliedatthe

sametimetoeachbioassayusingagardensprayer.Control

assayswereperformedusingtheculturemediuminsteadof

thefermentedbroth.After21days,plantinjurywasvisually

estimatedasapercentageofgrowthreductionincomparison

withtheuntreatedcontrols, where100%represented

com-pleteplantdeathand0%representednoeffect.11_In_addition,

other parameters were also investigated, including (i) the

heightoftheplant;(ii)thelengthoftheroot;(iii)freshweight

oftheaerialandrootparts;and(iv)dryweightoftheaerial

androotparts,whichwereevaluatedaftertheapplicationof

abioherbicide.

All treatments were normalized by dividing the value

obtainedinthetreatmentbythevalueobtainedinthecontrol

test. Basedontheplantdevelopment,the followingresults

wereobtained:“−”forinhibitionbetween0and0.95,“N”fora

zeroornon-signiﬁcanteffect(0.95–1.05),and“+”foragrowth

effect(higherthan1.05).Alldataweresubjectedtotheanalysis

ofvariance(ANOVA)andtoTukey’stest(p<0.05)tocompare

themeans.

Fungalidentiﬁcation

Themostpromisingfungusfortheproductionofa

bioherbi-cidewasidentiﬁed.FungalDNAwasextractedfromaliquots

Table2–Inhibitoryeffectofthefermentedbrothobtainedafterfermentationofeachisolatedfungusonthetargetplant (C.sativus).

Treatment Height(cm) Freshweight(g) Dryweight(g) Phytotoxiceffect

Aerial Root Aerial Root Aerial Root

DF11 1.23 (+) 0.98 N 1.30 (+) 1.20 (+) 0.81 (−) 1.00 N 0 DF12 0.73 (−) 0.96 N 0.69 (−) 1.63 (+) 0.78 (−) 0.78 (−) 40 DF13 0.78 (−) 0.78 (−) 0.63 (−) 1.40 (+) 0.73 (−) 0.85 (−) 30 DF21 0.60 (−) 0.94 (−) 0.65 (−) 0.93 (−) 0.69 (−) 0.83 (−) 40 DF23 0.74 (−) 1.51 (+) 0.73 (−) 1.19 (+) 0.71 (−) 0.81 (−) 10 DF24 0.73 (−) 1.08 (+) 0.59 (−) 1.52 (+) 0.70 (−) 0.65 (−) 30 DF25 1.09 (+) 0.91 (−) 1.10 (+) 1.82 (+) 1.27 (+) 1.03 N 20 DF3 1.05 N 0.98 N 0.94 (−) 1.07 (+) 0.89 (−) 0.84 (−) 10 DF41 1.05 (+) 0.91 (−) 0.96 N 1.07 (+) 1.05 N 1.23 (+) 0 DF42 1.13 (+) 1.21 (+) 1.22 (+) 1.62 (+) 1.02 N 1.35 (+) 0 RS11 0.77 (−) 0.89 (−) 0.80 (−) 1.20 (+) 0.85 (−) 0.83 (−) 10 RS12 1.01 N 0.94 (−) 0.84 (−) 1.06 (+) 0.77 (−) 1.17 (+) 0 RS13 0.71 (−) 1.02 N 0.73 (−) 1.64 (+) 0.77 (−) 0.83 (−) 30 RS22 1.24 (+) 1.01 N 1.05 (+) 1.15 (+) 1.23 (+) 1.28 (+) 0 RS24 1.21 (+) 0.95 (−) 1.19 (+) 1.33 (+) 1.12 (+) 1.05 (+) 0 RS25 0.69 (−) 1.00 N 0.83 (−) 0.88 (−) 1.04 N 1.24 (+) 30 RS26 0.64 (−) 0.90 (−) 0.68 (−) 1.43 (+) 0.66 (−) 0.78 (−) 20 VP11 1.11 (+) 0.94 (−) 1.22 (+) 1.54 (+) 1.20 (+) 1.62 (+) 0 VP14 1.19 (+) 1.10 (+) 1.15 (+) 1.62 (+) 1.12 (+) 1.32 (+) 10 VP21 1.05 N 0.95 (−) 1.12 (+) 1.40 (+) 1.02 N 1.17 (+) 0 VP22 1.19 (+) 1.10 (+) 1.26 (+) 1.24 (+) 1.20 (+) 0.99 N 0 VP23 1.08 (+) 0.94 (−) 1.39 (+) 2.45 (+) 1.23 (+) 1.03 N 0 VP41 0.96 N 0.54 (−) 1.05 N 0.98 N 0.79 (−) 0.91 (−) 20 VP43 0.88 (−) 0.50 (−) 0.91 (−) 0.99 N 0.68 (−) 0.85 (−) 20 VP44 0.98 N 0.45 (−) 1.00 N 1.13 (+) 0.76 (−) 1.04 N 10 VP45 0.84 (−) 0.57 (−) 0.92 (−) 0.98 N 0.75 (−) 0.70 (−) 40 VP51 0.65 (−) 0.82 (−) 0.89 (−) 0.80 (−) 0.64 (−) 0.78 (−) 60 VP52 0.92 (−) 0.83 (−) 0.92 (−) 0.70 (−) 0.75 (−) 0.73 (−) 20 VP53 0.74 (−) 1.50 (+) 1.05 N 1.57 (+) 1.04 N 1.04 N 20 VP55 0.77 (−) 0.79 (−) 0.74 (−) 0.74 (−) 0.75 (−) 0.89 (−) 30 VP56 1.22 (+) 0.98 N 1.11 (+) 1.28 (+) 1.12 (+) 1.68 (+) 40 VP62 0.83 (−) 1.32 (+) 1.02 N 1.51 (+) 0.95 N 2.08 (+) 10 VP63 0.89 (−) 0.58 (−) 0.94 (−) 1.29 (+) 0.85 (−) 1.19 (+) 20 VP68 0.89 (−) 1.50 (+) 1.20 (+) 1.78 (+) 1.11 (+) 1.11 (+) 20 VP72 0.91 (−) 1.26 (+) 1.06 (+) 1.62 (+) 0.81 (−) 1.44 (+) 10 VP73 0.89 (−) 1.10 (+) 0.93 (−) 1.10 (+) 0.90 (−) 0.96 N 30 VP76 1.47 (+) 0.91 (−) 1.39 (+) 1.48 (+) 1.44 (+) 1.32 (+) 0 VP81 0.94 (−) 1.21 (+) 0.98 N 1.13 (+) 0.73 (−) 0.73 (−) 10 VP88 1.07 (+) 1.12 (+) 0.90 (−) 0.60 (−) 0.83 (−) 0.83 (−) 10

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ofgrowthintheliquidmediumusingtheZRFungal/Bacterial

DNAMiniPrepkit(Zymo Research).Afterextractionoftotal

DNA,theinternaltranscribedspacer1(ITS1)-5.8SrDNA-ITS2

regionofnuclearribosomalDNAwasampliﬁedwithprimers

ITS1 and ITS4. The reaction of ampliﬁcation was carried

outaccordingtoBaldonietal.12_{Ampliﬁcation}_of_the_correct

fragmentwas veriﬁedbyelectrophoresis ina1.5%agarose

gelwith1×Tris–borate–ethylenediaminetetraaceticacid(TBE)

buffer.TheDNAfragmentswerestainedwithBlueGreen

Load-ing Dye I® _(LGC _{Biotecnologia,} _Cotia, _Brazil) _and _analyzed

inultravioletlight.Theproductsofpolymerasechain

reac-tion (PCR) were puriﬁed using the GenElute PCR Clean-Up

Kit®_(Sigma,_St._Louis,_MO,_USA)_following_the_{manufacturer’s}

instructions.Sequencingofthesampleswascarriedoutusing

theABI PRISM3100GeneticAnalyzer(Applied Biosystems).

The sequenced fragments were analyzed by the program

StadenPackage2.0.0btoobtainaconsensussequence.13_The

consensussequence was deposited to GenBank (accession

number KU523580), and a comparative search of GenBank

sequenceswascarriedoutusingtheBLASTntool.The

addi-tionalsequencesretrievedfrom GenBankincludedthoseof

Brazilianspeciesdescribedforthisgenus.14_For_the

identiﬁca-tionofthefungus,allthesequenceswerealignedusingthe

programBioEditv.7.2.5withtheClustalWalgorithm.15

Thephylogenywasreconstructedbymaximumlikelihood

basedontheanalysisoftheITSregionusingMEGA5.0.16_A

total of1000bootstrap replicateswere usedforthe

recon-struction.TheKimuratwo-parameternucleotide-substitution

modelwasusedwithModelTestrunwithuniformratesand

partial deletion (95%) parameters.17 _The_sequences _of

Dia-portheambigua(KC343015)andDiaporthellacorylina(KC343004)

wereusedastheoutgroups.18,19

Results

and

discussion

In this work,39 phytopathogenic fungi were isolated from

weeds of the Pampa biome. Table 2 presents the results

obtainedinthebioassays.Twenty-eightfungishowed

phyto-toxiceffectsagainstthetargetplant,andthemostpronounced

effect was shown byfungusVP51 withan activity of60%.

Otherfungi(DF12,DF21,VP45,andVP56)alsoshowed

activ-ityatthelevelof40%.Thesesamefungialsoproducedgood

results in the growth inhibition ofaerial parts of the

tar-get,withareductionoftheheightandfreshweightaround

35–40%relativetothecontrol.Thegrowth inhibitionofthe

rootpartwaslesspronouncedincomparisonwiththeaerial

part.Generally,theinhibitionwasaround20%,anditdidnot

C

D

B

A

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Table3–ComparisonofmeanamongtheeffectsoftreatmentsontheaerialandrootpartsofC.sativus.

Treatment Aerial Root

Height(cm) Freshweight(g) Dryweight(g) Height(cm) Freshweight(g) Dryweight(g)

DF21 0.604b _0.648b _0.688a _0.938a _0.933a _0.835a

VP51 0.650b _0.885ab _0.638a _0.824a _0.803a _0.776a

VP52 0.923a _0.922a _0.746a _0.827a _0.703a _0.733a

VP55 0.771ab _0.742ab _0.755a _0.791a _0.738a _0.889a

Differentletters(a,b)inthecolumnrepresentasigniﬁcantdifferenceat95%(p<0.05–Tukeytest).

resultintheplantdeath.Thelowherbicidalactivitymightbe relatedtothefactthattheactivemetabolitewassigniﬁcantly dilutedinthecrudeextract.However,itisimportantto con-siderthatabioherbicidemaynotnecessarilycausethesame effectonplantsasachemicalherbicide.Bioherbicideshave thepotential toprovidea competitive advantagefor grow-ingseedlingsthroughtheinfectionanddelayofthegrowth ofweedseedlings.20

Fig.1showssomeeffectsonaerialparts,suchasyellowing,

leafspots,andblight.Themostpronouncedeffectsonaerial

partswereobtainedwithfungiVP76,DF24,andVP51,which

arepresentedinFig. 1(A–C),respectively, whereitis

possi-bletocomparetheireffectswiththatofthecontrol(Fig.1D).

TheblastingsymptomseeninFig.1AandCwasalsonoticed

byChungetal.21_when_evaluating_the_potential_of_a

bioher-bicidefrom Plectosporiumtabacinumforgrowth inhibitionof

Sagittariatrifolia.Berneretal.22_reported_that_one_of_the

phy-totoxicsymptomscausedbyfungiofthegenusCercosporella

wassmallbrownspotsonleaves.

Leafspots(Fig.1B)wereobservedintheﬁrst72hafterthe

applicationoftheDF24bioherbicide.Theseplantspresented

mild, irregularlydistributedlesions,havingadarkgreento

darkbrowncolor,whichwerelimitedtothesprayedleaves.

Theyellowingaroundthespotsbecamewidespreadintheleaf

blade,formingnecrosisfromthetipsandedgesofthesheet.

Theleavesthatemergedaftertheinoculationwerefreeofthe

disease.Theleafspoteffects,followedbyyellowing,werealso

describedbyYandocetal.23_who_analyzed_the_effects_of_the

fungiBipolarissacchariandDrechsleragiganteaonthecontrol

plantImperatacylindrical.

Similarsymptomswere reportedbyother authorswhen

a fermented broth was used for weed control. Inhibition

ofgrowth was alsoaphytotoxic effect ofbacterialisolates

on weeds, as observed by Weissmann et al.24 _Gronwald

etal.25 _obtained_a_reduction_of_31%_in_plant_height,

report-ing that the inhibition of growth was an important factor

in determining the action of a bioherbicide. Walker and

Tilley26_also_observed_a_reduction_in_the_dry_weight_of_Senna

Table4–Specimensincludedinthisstudy.AccessionGenbanknumbersinboldreferredtotheITSsequencesobtained fromDiaporthesp.inPampabioma,SouthernBrazil.

Species Strain Locality GenBankcode

DiaportheactinidiaeN.F.Sommer&Beraha ICMP:13683 NewZealand KC145886

Diaportheactinidiae JL2 China KT163360

DiaportheambiguaNitschke CBS187.87 Italy KC343015

DiaporthecitriF.A.Wolf CBS199.39 Italy KC343051

Diaporthecitri CBS199.39 Italy KC343051

DiaportheendophyticaR.R.Gomes,C.Glienke&Crous LGMF935 Brazil KC343070

Diaportheendophytica LGMF928 Brazil KC343068

DiaporthekongiiR.G.Shivas,S.M.Thompson&A.J.Young 042 KR024725

Diaporthekongii 021 KR024720

DiaporthemelonisBeraha&M.J.O’Brien CBS507.78 USA KC343142

Diaporthemelonis CBS435.87 Indonesia KC343141

DiaporthemiriciaeR.G.Shivas,S.M.Thompson&Y.P.Tan BRIP55662c Australia KJ197283

Diaporthemiriciae BRIP54736j Australia KJ197282

Diaporthephaseolorum(Cooke&Ellis)Sacc. M69 Brazil JQ936148

Diaporthephaseolorum 8.1.1 Ecuador KP133195

DiaportheschiniR.R.Gomes,C.Glienke&Crous CBS133181 Brazil NR111861

Diaportheschini B125 Brazil KR812222

Diaporthesp. VP51 Brazil KU523580

DiaporthetecomaeSacc.&P.Syd. CBS100547 Brazil KC343215

DiaportheterebinthifoliiR.R.Gomes,C.Glienke&Crous CBS133180 Brazil NR111862

Diaportheterebinthifolii B135 Brazil KR812223

Diaporthevexans(Sacc.&P.Syd.)Gratz CBS127.14 USA KC343229

DiaporthellacorylinaLar.N.Vassiljeva CBS121124 China KC343004

PhomopsisdiacheniiSacc. PH10-1 Lithuania KR870866

Phomopsisdiachenii PH1 CzechRepublic KR870844

PhomopsisfoeniculiDuManoir&Vegh PH03 Germany KR870843

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obtusifoliatreatedwithabrothfermentedbyMyrothecium ver-rucaria.

Todetermine themost potentialtreatment forthe

pro-ductionofbioherbicides,somefungi(DF21,VP51,VP52,and

VP55)showingthehighestherbicidalactivityandthehighest

inhibitionoftheheightandweightofplantswerescreened.

Todetermine ifthere were differences amongthesefungi,

thedatafromTable2wereanalyzedbyANOVA,followedby

Tukey’s test (p<0.05). Theresults are compiled in Table3.

For the aerialpart, fungiDF21 and VP51showed the most

pronounced inhibitory effects on the plant height, which

werestatisticallydifferentfromthoseshownbytheothers.

Regardingfreshweight,theeffectshownbyfungusDF21was

statisticallydifferentfromthoseshownbytheothers,whereas

therewerenoveriﬁedsigniﬁcantdifferencesintheeffectson

dryweight,aswellasrootparts.Basedontheseresults,itcan

beinferredthatthefungishowedeffectsmainlyontheaerial

partsofthetarget,andthemostprominentwasVP51.This

fungusdemonstratedconsiderablephytotoxicityandaffected

morphologyofthetarget.Forthisreason,itwasselectedfor

molecularidentiﬁcation.

ThemolecularanalysisoftheITS1-5.8S-ITS2regionofVP51

showeditshighsimilaritytothespeciesDiaportheschini(100%),

D.terebinthifolii(99%),andD.phaseolorum(99%)(Table4),among

thenucleotidesequencesavailableintheNationalCenterfor

BiotechnologyInformation(NCBI)database.However,no

sig-niﬁcantdivergencesamongthesespecieswerefoundinthis

regiontoallowidentiﬁcationofVP51atthespecieslevel.The

phylogeneticcladeformeddidnotsupportthespecies

sepa-ration(lowbootstrapvalues)(Fig.2).

Therefore,thisresultisnotsufﬁcienttoprovidefull

identi-ﬁcation, andatthis time,itisonlypossibletosay thatthe

fungusbelongs tothe genus Diaporthe.Gomes et al.14

sug-gestedredeﬁningtheclassiﬁcationofthespecieswithinthe

genus Diaporthebased onmorphological and cultural

char-acteristics,thematingtype,andDNAsequencestoobtaina

satisfactorydelineationofthespecieswithinthegenus

Dia-porthe.

The genus Diaporthe (anamorph: Phomopsis) belongs to

the phylum Ascomycota,subphylum Pezizomycotina, class

Sordariomycetes,orderDiaporthalescharacterizedassexual

fungi. However, some fungi in this genus present asexual

Diaporthe citri KC343051 Diaporthe citri KC343051 Diaporthe tecomae KC343215 Diaporthe vexans KC343229 Phomopsis diachenii KR870866 Phomopsis diachenii KR870844 Phomopsis foeniculi KR870843 Phomopsis foeniculi KR870842 Diaporthe melonis KC343142 Diaporthe melonis KC343141 Diaporthe kongii KR024725 Diaporthe kongii KR024720 Diaporthe phaseolorum JQ936148 Diaporthe phaseolorum KP133195 Diaporthe terebinthifolii NR111862 Diaporthe terebinthifolii KR812223 Diaporthe schini NR111861 Diaporthe schini KR812222 Isolate VP51 Diaporthe endophytica KC343070 Diaporthe endophytica KC343068 Diaporthe actinidiae KC145886 Diaporthe actinidiae KT163360 Diaporthe miriciae KJ197283 Diaporthe miriciae KJ197282 Diaporthe ambigua KC343015 Diaporthella corylina KC343004 0.02 99 98 65 79 100 91 99 99 75 96

Fig.2–PhylogeneticreconstructionoftheDiaporthesp.obtainedfromITS1-5.8S-ITS2sequences.Bootstrapvalues(in%)are frommaximumlikelihood(ML)analysis(1000bootstraps).Onlybootstrapvaluesofatleast50%areshown.

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forms, leading to difﬁculty in identifying members of this

genusatthespecieslevel.Diaporthespp.areoftendescribed

asproducersofenzymes andsecondarymetabolites27 _with

the potential as antibiotics,28 _fungicides,29 _and _anticancer

agents,30 _as _well _as _that _for _preventing _herbivory _and

forbiologicalcontrolofweeds.31,32 _Ethyl

2,4-dihydroxy-5,6-dimethylbenzoate, phomopsilactone,33 _{phomopxanthone} _A

andB,34_taxol,30_{phomopsichalasin,}35_lactones,29_nonenolides,

phomonol,phomotone,36_and_phomophene_are_some_of_the

compoundsproducedbymembersofthegenus.

Someofthecompounds listed aboveshowed herbicidal

activity.PhomentrioloxinBcausedsmallnecroticspotsona

numberofplantspecies,whereasgulypyroneAcausedleaf

necrosis on Helianthus annuus plantlets.30 _Cimmino _et _al.8

testedseveralcompoundsproducedinliquidcultureby

Pho-mopsissp.(teleomorph:Diaporthegulyae)forthecontrolofthe

annualweedCarthamuslanatus.

Conclusions

Inthiswork,39 fungiwere isolatedfromthePampabiome

withthegoalofobtainingbiomoleculeswithherbicidal

activ-ityagainstweeds.Twenty-eightfungicausedsomephytotoxic

symptoms,but themostpronouncedeffectswere obtained

withfungiDF21,VP51,VP52,andVP55.Amongthose, VP51

showedthehighestherbicidalactivityandwassubjectedto

molecularidentiﬁcation.ThenucleotidesequenceofVP51was

comparedwithsequencesavailableintheNCBIdatabase,and

thefunguswasidentiﬁedasbelongingtothegenusDiaporthe,

membersofwhichhavealreadybeenreportedasproducers

ofbioherbicides.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

TheauthorsthankCAPES-CoordinationfortheImprovement

ofHigherEducationPersonnelandCNPQ-NationalCouncilfor

ScientiﬁcandTechnologicalDevelopmentforthescholarships

andthe StateDepartmentofDevelopment and Investment

Promotion(SDPI-RS)fortheﬁnancialsupportofthiswork.

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