Bacterial selection for biological control of plant disease: criterion determination and validation

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

Environmental

Microbiology

Bacterial

selection

for

biological

control

of

plant

disease:

criterion

determination

and

validation

Monalize

Salete

Mota

_,

_Cesar

_Bauer

_Gomes

_,

_Ismail

_Teodoro

_Souza

_Júnior

_,

Andréa

Bittencourt

Moura

a_{UniversidadeFederaldePelotas,DepartamentodeFitopatologia,Pelotas,RS,Brazil} b_{EmbrapaClimaTemperado,Pelotas,RS,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received17July2015

Accepted20May2016

Availableonline4October2016

AssociateEditor:JerriÉdsonZilli

Keywords: Rhizobacteria Antibiosis Mesocriconemaxenoplax Obligateparasites Perennialplants

a

b

s

t

r

a

c

t

Thisstudyaimedtoevaluatethebiocontrolpotentialofbacteriaisolatedfromdifferent

plantspeciesandsoils.Theproductionofcompoundsrelatedtophytopathogen

biocon-troland/orpromotionofplantgrowthinbacterialisolateswasevaluatedbymeasuringthe

productionofantimicrobialcompounds(ammoniaandantibiosis)andhydrolyticenzymes

(amylases,lipases,proteases,andchitinases)andphosphatesolubilization.Ofthe1219

bac-terialisolates,92%producedoneormoreoftheeightcompoundsevaluated,butonly1%of

theisolatesproducedallthecompounds.Proteolyticactivitywasmostfrequentlyobserved

amongthebacterialisolates.Amongthecompoundswhichoftendeterminethesuccess

ofbiocontrol,43%producedcompoundswhichinhibitmycelialgrowthofMonilinia

fructi-cola,butonly11%hydrolyzedchitin.Bacteriafromdifferentplantspecies(rhizosphereor

phylloplane)exhibiteddifferencesintheabilitytoproducethecompoundsevaluated.Most

bacterialisolateswithbiocontrolpotentialwereisolatedfromrhizosphericsoil.Themost

efficientbacteria(producingatleastfivecompoundsrelatedtophytopathogenbiocontrol

and/orplantgrowth),86intotal,wereevaluatedfortheirbiocontrolpotentialbyobserving

theirabilitytokilljuvenileMesocriconemaxenoplax.Thus,weclearlyobservedthatbacteria

thatproducedmorecompoundsrelatedtophytopathogenbiocontroland/orplantgrowth

hadahigherefficacyfornematodebiocontrol,whichvalidatedtheselectionstrategyused.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis

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

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

Introduction

Concernsregardingfoodsafetyandtheenvironmenthaveled

toreduceduseofagrochemicalsandthedevelopmentof

sus-tainable agriculture.In thiscontext, thefocus ofbiological

controlstudiesreflectsthedesireofseveralsectorstodevelop

∗ _{Correspondingauthor.}

E-mail:monalizem@gmail.com(M.S.Mota).

sustainablemethodsforplantdiseasecontrol.1_However,

effi-cient antagonistsmustbeobtainedforbiologicalcontrolto

becomeareality.

Soilmicroorganismscoexistinassociationwithplantroots

andinterferewithplantperformanceandmicrobial

commu-nitystructure.Bacteriaareestimatedtooccupybetween7%

http://dx.doi.org/10.1016/j.bjm.2016.09.003

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC

and15%ofthetotalrootsurfacearea.2_Ofthese,some

bacte-riapositivelyaffectplantsandhavebeendesignatedasplant

growth-promotingrhizobacteria(PGPR).3

Rhizobacteriacanindirectlyor directlypromotepositive

effectsonplants.Indirectly,theysuppresspathogens

medi-ated by competition and the production of antimicrobial

compoundsandlyticenzymes.Directly,theysolubilize

min-eralsandcauseawiderangeofchangesintherhizosphere,

whichpromoteshigherefficiencyintheabsorptionofwater

andmacro-andmicronutrientsbyplantsandchangesin

phy-tohormoneconcentrations,nitrogenfixation,andsiderophore

production.4–6

Inadditiontotherhizosphere,thephylloplaneisasource

ofantagonists;however,bacteriafromthisareaarestill

under-used as biological control agents, especially compared to

rhizobacteria.7 _{Kishore et al.,}8 _{found that both rhizoplane}

and phylloplane bacteria promote peanut seedling growth.

Inaddition,bacteria thatcolonizethe shootshaveabetter

chanceofsurvivingandmultiplyinginanutrition-rich

envi-ronmentsuchasthesoil,whereasinthephylloplane,theyare

exposedtohightemperatures,moisturecontentfluctuations,

andlimitednutrientavailability.

Invivobiocontrolagentselectionisnotasimpletaskdue

tothediversityofagentsandinteractionswiththehostplant,

andtherefore,efficientsearchmethodsarerequired.Thus,it

isnecessarytodevelopefficientselectionstrategiestoreduce

costsandincreasethepossibilityofselectingorganismsthat

canbeproducedinalargescaleatlowcostandthatmaintain

theirviabilityandefficiencyforlongperiods.In1997,Schisler

andSlininger9_{dividedtheselectionprocessintothree}

cate-gories:(i)choosingtheappropriatepathosystem,(ii)choosing

the adequate method, and (iii) characterizing the isolates

and evaluatingefficiency. Inrecent years,this concept has

evolved,andothergroupsofresearchershaveproposedinitial

selectioncriteriabasedonevaluationsintheabsenceofthe

host.10,11

Inthissense,invitrotestsareappropriateduringthe

ini-tialselectionstepsduetothelargenumberofmicroorganisms

thatcanbeevaluatedand,especially,theirlowcost.Thus,due

totheneedforalternativemanagementstrategiesfor

difficult-to-controlpathogens,andconsideringthattheinitialstepsfor

biocontrolagentselectionshouldbeperformedintheabsence

ofthehost,thepresentstudyaimedto(i)characterize

bacte-riatodeterminetheirinvitropotentialfortheproductionof

compoundsrelatedtophytopathogenbiocontroland/or

pro-motionofplantgrowth(CRBPGs);(ii)selectbacterialisolates

withthehighestnumberofCRBPGs;and(iii)validatethe

selec-tionprocessbystudyingtheeffectofthebacteriaselectedon

theringedpeachnematode.

Material

and

methods

Originofthebacteria

A total of1219 bacteria that belonged tothe collection of

thePlantBacteriologyLaboratory(LaboratóriodeBacteriologia

Vegetal–LBV)attheUniversidadeFederaldePelotas,Brazil,

wereused.Thebacteriawereobtainedfromdifferentniches

(phylloplane,rhizosphere,andsoil)andgroupedaccordingto

theirisolationsource:figtree(FicuscaricaL.–55),Gramineae

(72),Leguminoseae(151),Liliaceae(219),peachtree(Prunus

per-sicaL.–297),Tagetessp.(51),non-rhizosphericsoil(309),and

others(65–tomatoplant(SolanumlycopersicumL.),Brassicae,

andculturemediumcontaminants).

EvaluationofCRBPGproduction

Theabilities toproduceantimicrobial compounds

(antibio-sis and ammonia production) and hydrolytic enzymes

(amylases, lipases, proteases, and chitinases) and to

solu-bilize phosphates were evaluated. Bacteria that previously

exhibitedordidnotexhibittheabilitytoproduceeach

com-poundstudiedwereusedaspositiveandnegativecontrols,

respectively.

Theabilityofbacteria toproduce antibioticcompounds

againstMonilinia fructicola (Winter)Honey,the causal agent

of peach brown rot was evaluated. Four bacteria arranged

equidistant at the edges were streaked on each Petri dish

containing523medium12_{andinthecenter,amycelialdisk}

containingthefunguspreviouslygrowninapotatodextrose

agar(PDA).Aftersevendaysat22±2◦C,scoreswereassigned

accordingtotheinhibitionzoneofthefungalgrowthas

fol-lows:0–noinhibition;1–≤10mm;2–≥11and≤20mm;and

3–≥21mm).

Ammonia production was observed by the presence of

a yellow-orangeprecipitate afterfivedays ofincubationat

28◦C.13

Starchhydrolysiswasevaluatedaccordingtothemethod

ofSchaad(1988).Afterfourdaysofincubationat28◦C,the

additionofLugol’sIodineallowedthevisualization ofclear

zonesaroundthebacterialcolony,indicativeofstarch

hydrol-ysis,andthebacteriawereclassifiedusingthescaledescribed

forantibioticcompounds.

Lipidhydrolysiswasevaluatedinmediumcontaining1%

Tween80,accordingtothemethodusedbyFahyandPersley,14

afterfourandsevendaysofincubationat28◦Candverified

bythepresenceofamilkywhiteprecipitatesurroundingthe

colonies.

Two substrates were used toevaluate the abilityof the

bacteriatohydrolyzeproteins,Litmus® _{milk(Difco)and5%}

gelatinmedium,asdescribedbySchaad.15_{Afterfourandten}

daysofincubationat28◦C,themediumchangedfrommilky

totranslucent(Litmus)orbecameliquefiedafterbeing

refrig-eratedat4◦Cfor1h(gelatin).Chitinhydrolysiswasevaluated

using0.5%chitinmediumasthesolecarbonsource16_and

cal-ciumphosphatesolubilizationwasevaluatedinNBRIPculture

medium,17_{bothactivitieswereobservedatseven,14,and21}

daysofincubationat28◦Ctoverifythedegradationzoneof

eachcompound.

Thehydrolysisofchitin,lipidandproteins(Litmus®_milk

and gelatin) was determined in a semiquantitative way,

using different incubation timesfor evaluating.The

inten-sity of production was low for positive reaction after 10

(lipidsandproteins)or21days(chitin)ofincubation;middle

if the positive reaction happened after 14 days of

incuba-tion (chitin); andhigh when thepositive reactionoccurred

afterfour (lipids andproteins) or sevendaysofincubation

Table1–Percentageofbacteriaproducingcompoundsrelatedtobiocontroland/orpromotinggrowthaccordingto numberofproducedcompoundsandtotalisolatedbygroupingastheoriginofthebacterialisolates(Ficuscarica, Gramineae,Leguminoseae,Liliaceae,Prunuspersica,soil,Tagetesandothers–total=1219isolates).

Sourcegroups/(no.isolates) Numberofthecompoundsproduced

0 1 2 3 4 5 6 7 8 Ficuscarica 10.9* _{18.2 23.6 14.5 9.1 10.9 9.1 3.6 0.0} Gramineae 8.3 11.1 16.7 15.3 11.1 6.9 20.8 8.3 1.4 Leguminoseae 7.9 13.9 17.9 7.9 12.6 15.9 17.9 6.0 0.0 Liliaceae 2.3 6.4 8.2 16.9 13.7 19.2 17.8 15.5 0.0 Prunuspersica 10.8 16.8 18.2 20.5 12.5 12.1 6.1 2.7 0.3 Soil 11.0 12.0 11.0 17.5 12.9 14.6 13.9 5.5 1.6 Tagetes 0.0 2.0 23.5 21.6 19.6 15.7 13.7 3.9 0.0 Others** _{6.2 10.8 18.5 20.0 12.3 10.8 13.8 7.7 0.0}

∗ _{Percentagevaluesfor1219bacterialisolatestested.}

∗∗ _{Others=Solanumlycopersicum,Brassicae,andculturemediumcontaminants.}

Validationofbiocontrolbacterialselection–evaluationof nematicideactivity

Atthisstep,thebacteriathatproducedatleastfiveoftheeight

CRBPGsanalyzedwere used,aswellasDFs1985(whichdid

notproduceanyofthecompoundsevaluated)andDFs2180

(which produced one compound), forcontrast effects. The

ringednematode(Mesocriconemaxenoplax(Raski)Loofandde

Grisse)speciesassociatedwithPeachTreeShortLifedisease

waschosenasthetargetspeciestoevaluatetheefficiencyof

bacterialbiocontrol.

Mobileforms(juvenileandadult)ofthenematode(25per

plot)were treatedwithabacterialsuspension(A540=0.5)or

withsalinesolution(control)ata1:1ratio(v/v).Themicrotiter

plates containing the treatments were incubated at 25◦C

for 24h and arranged in a completely randomized design

(86 bacterialtreatments infour replicates). Thenumber of

deadnematodeswasevaluatedbyadding10␮Lof1NNaOH,

according toChenand Dickson.18 _{Thevalueswere sinacsc}

√

x/100transformed, subjected toanalysisofvariance, and

groupedbythemethodofScottandKnott.19

Bacterialidentification

The bacteria with best control were identificated by their

16SrRNAsequences.TotalDNAwasextractedwithWizard

genomic DNA purification Kit(Promega Corporation,USA).

Partial amplicom of 16S rRNA of samples were produced

byPCRwithprimers27Fand1492R.20 _{Theampliconswere}

cleansedwithWizardSVGelandPCRClean-upkit(Promega)

andsentforsequencingbyLudwigSequenciamento.

Results

Ofthe1219bacterialisolatesstudied,92%wereabletoproduce

oneormoreoftheeightcompoundsevaluated.However,only

1%ofthe isolatesproduced allthe compounds.Theability

ofthebacteriatoproducecompoundswascalled‘production

ofcompoundsrelatedtobiocontroland/orpromotinggrowth

(CRBPG)’, and most isolates (17%) produced three CRBPGs

(Table1).

Uponanalysisofthesourcegroupsandthenumberof

com-poundsproduced(Table1),threegroupsexhibitedbacterial

isolatesthatproducedallthecompoundsevaluated(soils–

1.6%,grasses–1.4%,andpeachtree–0.3%).Additionally,a

largenumberofisolatesfromLiliaceae(15.5%)wereableto

produceatleastsevenCRBPGs.TheisolatesfromTagetes

com-prisedtheonlygroupinwhich100%oftheisolatesexhibited

productionofatleastoneofthecompounds.Incontrast,the

groupofisolatesfromsoilsexhibitedthehighestpercentage

ofisolates,withoutproducinganyofthecompounds,followed

byisolatesfromthefigtreeandpeachtree.

Ingeneral,thegroupsexhibiteddifferentpercentage

dis-tributionsamongthenumbersofcompoundsproduced.The

highestpercentageforbacteria isolatedfrom figtree,

legu-minousplantsandTageteswasobservedfortwocompounds

andforthebacteriaisolatedfrompeachtree,soilandothers,

tookplaceinthreecompounds.Theisolatesobtainedfrom

thesoilhadamorehomogeneousdistribution,rangingfrom

11%to17.5%forzerotosixcompoundsproduced,incontrast

toTagetes,whichconcentratedthedistributionbetweentwo

andsixcompoundsproduced(85.9%).

Whenobservingeachoftheevaluatedcompounds(Fig.1A),

itisnoteworthythatproteolyticactivitywasmostfrequently

observedforbothsubstrates,andchitinhydrolysiswasleast

frequentlyobserved.Intheothertests,thequantityofisolates

that producedcompounds variedbetween 36%(amylolytic)

and50%(lipolytic).However,whencomparingtheeightgroups

originating from the bacterial isolates (Fig. 1B), it is

pos-sible todistinguish which isolates have higher production

potentialforeachofthecompoundsevaluated. TheTagetes

group exhibitedthe highestpercentage ofphosphate

solu-bilizers,protease-producersingelatin,ammonia-producers,

andamylase-producers.ThebacterialisolatesfromLiliaceae

were notable as protease-producers and for their

antibio-sisactivity againstM. fructicola.Thebacterialisolates from

grassesprovedtobethebestlipase-producers,andthosefrom

legumeswerethebestchitinase-producers.

The semiquantitative component of the evaluations,

throughtheassignmentofgradesorevaluationsduring

dis-tinctincubationperiods(Fig.2),allowedustosummarizethe

resultsofcompoundproductionintensityintonegative,low

(type I zone or slowest isolates), medium (type II zone or

28 41 41 38 79

61

17 47

Leguminoseae

28 39 43 46 66 ₆₃ 12 51 Others 43 49 63 48 84 70 11 71 Liliaceae 36 ₃₄ 38 37 47 27 4 25 Prunus persica 31 46 36 41 65 57 16 54

AMI AMO ANT FOS GEL LIT QUI TWN

Soil 55 59 37 57 96 31 10 35

AMI AMO ANT FOS GEL LIT QUI TWN

Tagetes 38 35 ₃₁ 36 56 47 2 35 Ficus carica 32 42 38 40 74 49 10 81 Gramineae

B

A

AMI = 36% AMO = 44% ANT = 43% FOS = 43 % GEL = 69% LIT = 52% QUI = 11% TWN = 50%

Fig.1–Percentbacteriaproducingcompoundsrelatedtobiocontroland/orpromotinggrowthdependingonthetotal isolatesnumber.(A)AMI,amylase;AMO,ammonia;ANT,antibiotics(halostypeI–≤10mm;II–≥11and≤20mm;andIII– ≥21mm);FOS,phosphatesolubilization;GEL,proteasesmediumgelatin;LIT,proteinasemediumLitmus®_{;CHI,chitinase;} TWN,lipasesmediumTween80.(B)Groupsformedastheisolationorigin(Ficuscarica,Gramineae,Leguminoseae,others, Liliaceae,Prunuspersica,soil,Tagetes).

time),andhigh(typeIIIzoneorfastisolates)categories.Thus,

protease-producingisolates(bothsubstrates)predominated

uponthenegativeones.Amongthepositiveresults,thehigh

intensitywasmorefrequenttoisolatesthathydrolyzedlipids,

gelatinandmilkorsolubilizedphosphate(Fig.2).

Incontrast, consideringonlythe isolatesthat produced

eachcompoundandtheirisolationniches(Fig.3),rhizospheric

isolatespredominatedforallthecompounds(rangingfrom

53to38%),exceptforchitinase(Fig.3i)andlipaseproduction

(Fig.3ii),wherethethreenichesexhibitedpracticallythesame

percentage.

Thus,upondeterminationoftheisolatesabilitytoproduce

CRBPGs,it waspossibletoselect86 bacteriathatproduced

fromfivetoeightcompoundsevaluated(threebacteriathat

producedeight,32thatproducedseven,23thatproducedsix,

and28thatproducedfivecompounds).

Evaluationofringednematodemortalityuponexposureto

abacterialsuspensionfor24hallowedustogrouptheisolates

into10 groupswithdifferentefficiencylevels (a–j,Table2).

Eightofthesegroupspositivelydifferedfromthecontrol,i.e.,

comprisedbacteriathatcausedmortalityinjuvenileM.

xeno-plax(groupsa–h);onewasequaltothecontrol(groupi);and

anotherreducedtheirmortality(groupj).Ofthese,89.4%of

bacteriawereeffective,andmostbacteria(42.5%)resultedin

atleast96.7%mortality(groupa).

In contrast, when considering the bacteria grouped by

numberofCRBPGsproduced,weclearlyobservedthatbacteria

thatproducedmoreCRBPGshadahighernematicide

percent-age(Table2).Thus,allisolatesevaluatedthatproducedeight

CRBPGsresulted in100% nematodemortality;producersof

sevenCRBPGshad43.7%oftheirisolatesinthesuperiorgroup;

producersofsixCRBPGshad34.8%oftheirisolatesingroup

a; andamongthosethatproduced fiveCRBPGs,only21.4%

exhibitedhighefficiency.

Thepartial16S rRNAgenesequencing(Table3)resulted

85–100%identityand76%isolatesidentifiedbelongingtothe

genusBacillus.

Discussion

In general, all the plant species and soils used to isolate

bacteriawerehostcandidatesforpathogenbiocontrol.Each

57% 16% 17% 10% 64% 18% 13% 5% 56% 44% 31% 12% 57% 48% 10% 42% 50% 29% 2% Negative 3% 6% 89%

i

ii

iii

iv

v

vi

_vii

_viii

Low Middle High

Fig.2–Percentageofisolates(total=1219)groupedasreactionintensity:(i)chitinolytic;(ii)lipolytic;(iii)proteolyticon gelatinmedium;(iv)proteolyticLitmus®_{milkmedium;(v)ammonia-production;(vi)amylase-production;(vii)antibiotic} againstMoniliniafructicola;(viii)phosphate-solubilization.

ofCRBPGsproduced.Theisolatesthatoriginatedfromgrasses,

legumes,andLiliaceaeexhibitedhigherpercentagesfor

pro-ductionofsixorsevenCRBPGs,althoughthetwofirstgroups

comprisedapproximately20%oftheisolatesthatdidnot

pro-duceanyoroneofthesecompounds.

However, the group of isolates obtained from Tagetes

is noteworthybecause different species from these plants

are considered antagonists to species of Pratylenchus and

Meloidogyne.21 _{TherearenoreportsofantagonismofTagetes}

bacterialisolatesagainstspeciesofMesocriconema.

Addition-ally,studiesinthe1990sshowedthatusingisolatesfromroots

ofantagonisticplantsincreasedthechancesoffindinggood

candidatesforbiocontrolagentsbyuptosixtimesininvivo

tests.22,23_{Thus,thehighpercentagesofCRBPGproducers(98%}

between2and7CRBPGs)isolatedfromtheTagetesrhizosphere

observedinthepresentstudyreflectthispremise.

32% 30% 44% 26% 37% 35% 28% Rhizosphere

i

ii

iii

iv

v

vi

vii

viii

Fig.3–Percentageofisolateswithpositivereactiongroupedasisolationniche(phylloplane,rhizosphereandsoil):(i) individuallychitinolytic(135);(ii)lipolytic(604);(iii)proteolyticongelatinmedium(839);(iv)proteolyticmediumLitmus® milk(630);(v)ammonia-producers(531);(vi)amylolytic(443);(vii)antibioticsproducing(529);(viii)phosphatesolubilizers (530).Valuesbetweenbracketsrepresentthenumberofisolateswithpositiveresults.

Table2–MortalitypercentofMesocriconemaxenoplaxduetheactionofbacteriaproducingatleastfivecompounds relatedtobiocontroland/orpromotiongrowth(CRBPGs)after24hincubationat25◦C.

Bacterialtreatment Mortality(%) Bacterialtreatment Mortality(%) Bacterialtreatment Mortality(%)

ProducerseightcompoundsCRBPGs

DFs1391 100.00a* _{DFs1486 100.00a DFs19866 100.0a}

ProducerssevencompoundsCRBPGs

DFs0066 100.00a DFs2247 100.00a DFs0081 55.67f DFs0390 100.00a DFs2251 100.00a DFs1021 49.08g DFs0695 100.00a DFs0439 97.95a DFs0582 46.87g DFs0886 100.00a DFs1887 93.15b DFs1985 31.56h DFs1256 100.00a DFs1039 86.87b DFs1421 29.63h DFs1258 100.00a DFs0419 80.64c DFs0706 22.57i DFs1341 100.00a DFs0023 78.33c DFs2137 19.25i DFs1947 100.00a DFs1219 72.15d DFs1967 17.77i DFs2049 100.00a DFs0756 69.56e DFs1320 12.65j DFs2108 100.00a DFs0377 59.51f DFs1344 9.38j DFs2124 100.00a DFs2121 58.73f

ProducerssixcompoundsCRBPGs

DFs1983 100.00a DFs2191 90.23b DFs2172 50.00g DFs2007 100.00a DFs2122 85.42b DFs1934 28.37h DFs2126 100.00a DFs2175 82.89c DFs1923 22.55i DFs2156 100.00a DFs0032 81.68c DFs2162 22.33i DFs2227 100.00a DFs1691 71.94d DFs2048 16.11i DFs2229 100.00a DFs2265 67.09e DFs2062 13.29j DFs2236 100.00a DFs2052 65.06e DFs0422 7.09j DFs2110 96.74a DFs2246 62.75e

ProducersfivecompoundsCRBPGs

DFs2008 100.00a DFs2148 82.55c DFs2155 54.17f DFs2099 100.00a DFs2113 81.03c DFs1955 45.71g DFs2109 100.00a DFs0418 79.02c DFs1970 40.91g DFs2133 100.00a DFs2168 77.11d DFs1932 37.29h DFs2147 100.00a DFs1650 76.06d DFs2193 37.08h DFs1928 98.04a DFs2060 74.54d DFs1935 19.46i DFs1899 91.92b DFs1909 69.88e DFs1950 13.92i DFs2250 90.77b DFs1902 69.61e DFs2051 9.17j DFs2136 83.94c DFs2027 68.60e DFs1985** _31.56h DFs1958 83.90c DFs2089 64.12e DFs2180** _6.01j CV(%) 10.02 Control16.08i

∗ _{Originalvalueswereconvertedtoarcsen}√_{x/100whoseoriginalmeansfollowedbythesameletterinthecolumndonotdifferbyScott–Knott}

testat5%probability.

∗∗ _{Isolatedusedascontrast:DFs1985,notproducesevaluatedcompoundsandDFs2180,producingonecompound.}

In contrast,approximately 90% of the isolates obtained

frompeachtreesproducedsomeCRBPGs.Althoughthese

iso-latesare notamongthosewithhighestCRBPG production,

theyshouldreceivespecialattentionbecausepeachtreesare

affectedbytheringednematode,andbiocontrolagentsfrom

thehostsaredesirablebecausetheyhavebeenadaptedto

sur-viveinthishabitat.Thisaspectwassuccessfullyexploitedin

thecontroloftheringednematode.24–26_{Formostofthesource}

groups, the isolatesfrom the rhizosphereexhibited higher

productionofthe evaluated compounds, as well ashigher

intensities of CRBPG production. These results reflect the

richnessoftherhizosphericenvironment, whichis

respon-siblefortheoccurrenceandpreferenceofseveralorganisms,

includingthosethatexhibitantagonisticeffectsagainst

sev-eralpathogens,suchasnematodes.27

Ithasbeenpreviouslyestablishedthatenzymes,especially

lyticenzymes,are synthesizedbymicroorganismsand also

actasanantagonisticmechanism,which,inmanysituations,

maypartiallyexplainthebiologicalcontrolofplantdisease.28

Theisolatesstudiedhereexhibitedahigherfrequencyfor

pro-teinandlipidhydrolysis,whichisanimportantresult,asthese

compoundsare presentinthemembranessurrounding the

eggsandinthecuticleofmobileformsofdifferentnematode

species.26 _{Thus, these lytic activitieshave been associated}

withthebiologicalcontrolofnematodes.29

Chitinases are also important in controlling

phy-topathogens,aschitinisthemainconstituentofnematode

eggs26,30_{andthefungalwall.}31_{Thus,chitinolyticbacteriaare}

potential biocontrol agents for both pathogen groups.32–34

However, just over 10% of the isolates studied produced

chitinases,whichindicatesthatthisabilityismorecommon

in actinomycetes and less common in bacteria and fungi

presentinthesoil.35

Severalresearchersconsiderantimicrobialcompound

pro-ductiononeofthemostimportantantagonistmechanisms.

Antibioticsandtoxiccompoundsproducedbythebacteriacan

actonboththehatchinganddevelopmentofnematodes.26,36

Table3–Bacterialidentificationbyhomologyofpartialsequenceof16SrRNAusing27Fand1492Rprimers.

BacterialTreatment Description Identity Accession

DFs066 Bacillussubtilissubsp.subtilisstrainKISR-116SribosomalRNAgene,PSa _{98% KM068047.1}

DFs390 BacillusthuringiensisstrainBT-EM1416SribosomalRNAgene,PS 99% KT588443.1

DFs439 BacillussubtilisstrainCI116SribosomalRNAgene,PS 99% KU557409.1

DFs695 Bacillussp.AR493partial16SrRNAgene,strainAR493 100% LN829576.1

DFs886 BacillusthuringiensisBt40716SribosomalRNA,CSb _{98% NR102506.1}

DFs1256 Bacillussubtilissubsp.subtilisstrainDSM1016SribosomalRNA,PS 98% NR027552.1

DFs1258 Bacillussubtilisstrain1-316SribosomalRNAgene,PS 97% HQ693082.1

DFs1341 Bacillussubtilissubsp.subtilisstr.168strain16816SribosomalRNA,CS 99% NR102783.1

DFs1928 Paenobacilluspolymyxagene16SRNA,PS 99% AB689755.1

DFs1947 BacilluscereusstrainCF-S3016SribosomalRNAgene,PS 97% KJ781400.1

DFs1983 BacillusthuringiensisBt40716SribosomalRNA,CS 99% NR 102506.1

DFs2008 PseudomonaspoaeRE*1-1-14strainRE*1-1-1416SribosomalRNA,CS 97% NR102514

DFs2049 PseudochrobactrumsaccharolyticumstrainALK62616SribosomalRNAgene,PS 96% KC456591.1

DFs2099 BacilluscereusstrainYLB-P516SribosomalRNAgene,PS 99% KF376341.1

DFs2108 Pseudomonassp.VS-216SribosomalRNAgene,PS 98% JF699699.1

DFs2109 PseudomonasfluorescensstrainM516SribosomalRNAgene,PS 99% KT767962.1

DFs2110 BacilluscereusstrainNCIM210616SribosomalRNAgene,PS 100% KU218541.1

DFs2124 BacilluscereusATCC14579strainATCC1457916SribosomalRNA,CS 99% NR074540.1

DFs2126 Bacillussp. 85%

DFs2133 Bacillussp.SNG-316SribosomalRNAgene,PS 99% KP992135.1

DFs2147 PseudomonasputidastrainBB316SribosomalRNAgene,PS 98% KP314235.1

DFs2229 BacilluscereusATCC14579strainATCC1457916SribosomalRNA,CS 100% NR074540.1

DFs2236 BacilluscereusstrainFT9,CGc _{97% CP008712.1}

DFs2247 BacillusamyloliquefaciensFZB42strainFZB4216SribosomalRNA,CS 99% NR075005.1

DFs2251 BacilluscereusstrainKCd116SribosomalRNAgene,PS 99% FJ613630.1

a _{(PS)Partialsequence.}

b _{(CS)Completesequence.}

c _{(CG)Completegenome.}

M.fructicola,whichisusedasanindicatorofantibiotic

pro-ductionbecauseM.xenoplaxisanobligatepathogenandthus

cannotbeculturedinvitro.Therefore,itcannotbeaffirmed

thattheantibioticactivityisthesameforthenematode.

In addition, 44% of the isolates produced ammonia,

which isa compound that has previously been associated

with pathogen control26,37 _{and has the additional}

advan-tage of being volatile and the ability to expand through

soil pores, reaching a higher volume around the bacterial

coloniesproducingthecompound.Furthermore,ammoniais

acompoundthatactsduringorganicmatterdecomposition,

which improves soil structure and plant nutrition,

result-inginhigherproductivity bythecropand highertolerance

tophytoparasites.38 _{Thephosphate solubilization observed}

amongtheisolatesmayimproveplantnutrition.Thisaspect

isespeciallyimportant,asthedamagetopeachtreesbyM.

xenoplaxisaggravatedunderconditionsoflowsoilfertility.39

A high percentage (85%) of bacteria selected in vitro

exhibitedhighmortalityinmobileformsoftheringed

nema-tode.Thepotencyofthis abilitymayberelatedtodifferent

mechanismsofaction,whichisindicatedbythenumberof

bacteria-producedCRBPGs.Otherstudieshaveassociatedthe

individualproductionofsomecompoundswiththecontrol

ofdifferentnematodes.26,40,41_{However,noneofthese}

stud-iesexamined the diversityincompoundsproducedforthe

selectionofbiocontrolisolates.

Sequencingof16SrRNAallowstheaccurateidentification

ofbacterialgenerafromvariousplantspecies.Bacillusisan

importantbacteriagenusbecauseofthesynthesisofawide

rangeofmetaboliteswithdiverseproperties.42,43_However,for

the precisedefinitionofspeciesand subspeciesanalysisof

othergenesisneeded,forexamplegyrA.44

Itisveryimportanttodevelopfastandefficientmethodsto

selectbiocontrolmicroorganisms,especiallywhenevaluating

alargenumberofcandidates.Invitroevaluationsallowfor

areductioninthenumber ofisolates,whichmakesfurther

invivotestsviable.Thisaspectiscrucialwhenworkingwith

perennialplantsandlong-cycle,obligatepathogens,suchas

thepeachtree-M.xenoplaxpathosystem.

Thus,theresultsobtainedinthepresentstudyindicatethe

efficacyoftheselectionstrategyandvalidatetheuseofthe

CRBPGproductionprofileinreducingthenumberofisolates

forinvivoevaluation.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorswouldliketothanktheRioGrandedeSulState

Research Foundation (Fundac¸ão de Amparo a Pesquisa do

Estado do Rio Grande do Sul–FAPERGS) for financial

sup-port(10.10360),theBrazilianFederalAgencyfortheSupport

andEvaluationofGraduateEducation(ConselhoNacionalde

Desenvolvimento Científicoe Tecnológico– CAPES) for the

doctoralscholarshipgiventothefirstauthor,andtheBrazilian

National Council for Scientific and Technological

r

e

f

e

r

e

n

c

e

s

1.HyakumachiM.Researchonbiologicalcontrolofplant

diseases:presentstateandperspectives.JGenPlantPathol.

2013;79(6):435–440.

2.GrayEJ,SmithDL.IntracellularandextracellularPGPR:

commonalitiesanddistinctionsintheplant–bacterium

signalingprocesses.SoilBiolBiochem.2005;37:

395–412.

3.KloepperJW,SchrothMN.Plantgrowthpromoting

rhizobacteriaonradishes.In:Proceedingsofthe4th

InternationalConferenceonPlantPathogenicBacterial.Angers:

INRA;1978:879–882.

4.VanLoonLC.Plantresponsestoplantgrowthpromoting

bacteria.PlantPathol.2007;119:243–254.

5.LugtenbergB,KamilovaF.Plant-growth-promoting

rhizobacteria.AnnuRevMicrobiol.2009;63:541–556.

6.GlickBR.PlantGrowth-PromotingBacteria:Mechanismsand

Applications.HindawiPublishingCorporation,Scientifica; 2012:15.

7.LindowSE,LeveauJHJ.Phyllospheremicrobiology.CurrOpin

Biotechnol.2002;13:238–243.

8.KishoreK,PandeS,PodileAR.Phylloplanebacteriaincrease

seedlingemergence,growthandyieldoffield-grown

groundnut(ArachishypogaeaL.).ApplMicrobiol.

2005;40:260–268.

9.SchislerDA,SliningerPJ.Microbialselectionstrategiesthat

enhancethelikelihoodofdevelopingcommercialbiological

controlproducts.JIndMicrobiolBiotechnol.1997;19:

172–179.

10.ValidovS,KamilovaF,QIS,etal.Selectionofbacteriaableto

controlFusariumoxysporumf.sp.Radiceslycopersiciin

stonewoolsubstrate.JApplMicrobiol.2007;102:

461–471.

11.KöhlJ,PostmaJ,NicotP,RuoccoM,BlumB.Stepwise

screeningofmicroorganismsforcommercialusein

biologicalcontrolofplant-pathogenicfungiandbacteria.Bio

Control.2011;57:1–12.

12.KadoCI,HeskettMG.Selectivemediaforisolationof

Agrobacterium,Corynebacterium,Erwinia,Pseudomonasand

Xanthomonas.Phytopathology.1970;60:969–976.

13.CappuccinoJG,ShermanN.Microbiology–ALaboratoryGuide.

MenloPark:BenjaminCummingsSciencePublishing;

1998:477.

14.FahyPC,PersleGJ.PlantBacterialDiseases:ADiagnosticGuide.

Sydney:AcademicPress;1983:393.

15.SchaadNW.LaboratoryGuideforIdentificationofPlant

PathogenicBacteria.2nded.St.Paul:TheAmerican

PhytopathologySociety;1988:158.

16.RenwickA,CampbellR,COES.Assessmentofinvitro

screeningsystemsforpotentialbiocontrolagentsof

Gaeumannomycesgraminis.PlantPathol.1991;40: 524–532.

17.NautiyalCS.Anefficientmicrobiologicalgrowthmediumfor

screeningphosphatesolubilizingmicroorganisms.FEMS

MicrobiolLett.1999;170:265–270.

18.ChenSY,DicksonDW.Atechniquefordetermininglive

second-stagejuvenilesofHeteroderaglycines.JNematol.

2000;32(1):117–121.

19.ScottAJ,KnottM.Aclusteranalysismethodforgrouping

meansintheanalysisofvariance.Biometrics.

1974;30(2):507–512.

20.LaneDJ.16S/23SrRNAsequencing.In:StackebrandtE,

GoodfellowMN,eds.NucleicAcidTechniquesinBacterial

Systematics.Chichester:Wiley;1991:115–147.

21.FabryCFS,FreitasLG,NevesWS,etal.Obtenc¸ãodebactérias

paraobiocontroledeMeloidogynejavanicapormeiode

aquecimentodesoloetratamentocomfiltradoderaízesde

plantasantagonistasafitonematoides.FitopatolBras.

2007;32:079–082.

22.KloepperJW,Rodriguez-KábanaR,McinroyJA,CollinsDJ.

Analysisofpopulationsandphysiologicalcharacterization

ofmicroorganisms.PlantSoil.1991;136:95–102.

23.KloepperJW,Rodriguez-KábanaR,McInroyJA,YoungRW.

Rhizospherebacteriaantagonistictosoybeancyst(Heterodera

glycines)androot-knot(Meloidyneincognita)nematodes:

identificationbyfattyacidanalysisandfrequencyof

biologicalcontrolactivity.PlantSoil.1992;139:

74–84.

24.McInnisT,KluepfelD,ZehrE.SuppressionofCriconemella

xenoplaxonpeachbyrhizospherebacteria.In:Second InternationalNematologyCongress,AbstractsofPapers106.

1990.

25.KluepfelDA,McInnisTM,ZehrEI.Involvementof

root-colonizingbacteriainpeachorchardsoilssuppressive

ofthenematodeCriconemellaxenoplax.Phytopathology.

1993;83(11):1240–1245.

26.WestcottSW,KluepfelD.InhibitionofCriconemellaxenoplax

egghatchbyPseudomonasaureofaciens.Phytopathology.

1993;83:1245–1249.

27.Zavaleta-MejiaE,VanGundySD.Effectsofrhizobacteriaon

Meloidogyneinfection.JNematol.1982;14(4):475–476.

28.DuffyB,SchoutenA,RaaijamakersJM.Pathogen

self-defense:mechanismstocounteractmicrobial

antagonism.AnnuRevPhytopathol.2003;41:501–538.

29.AaltenPM,VitourD,BlanvillainSR,GowenSR,SutraL.Effect

ofrhizospherefluorescentPseudomonasstrainsonplant

parasiticnematodesRadopholussimilesandMeloidogynespp.

LettApplMicrobiol.1998;27:357–361.

30.DunneC,Moenne-LoccozY,deBruijnFJ,O ´GaraF.

Overproductionofaninducibleextracellularserineprotease

improvesbiologicalcontrolofPythiumultimumby

StenotrophomonasmaltophiliastrainW81.Microbiology.

2000;146:2069–2078.

31.FeifilovaEP.Thefungalcellwall:modernconceptsofits

compositionandbiologicalfunction.Microbiology.

2010;79(6):711–750.

32.HallmannJ,Rodríguez-KábanaR,KloepperJW.

Chitin-mediatedchangesinbacterialcommunitiesofthe

soil,rhizosphereandwithinrootsofcottoninrelationto

nematodecontrol.SoilBiolBiochem.1999;31:551–560.

33.SlimeneIB,TabbeneO,GharbiD,etal.Isolationofa

chitinolyticBacilluslicheniformisS213strainexertinga

biologicalcontrolagainstPhomamedicaginisinfection.Appl

BiochemBiotechnol.2015;175(7):3494–3506.

34.BrzezinskaSM,JankiewiczU,BurkowskaA,WalczakM.

Chitinolyticmicroorganismsandtheirpossibleapplication

inenvironmentalprotection.CurrMicrobiol.2014;68:

71–81.

35.HsuSC,LockwoodJL.Powderedchitinagarasaselective

mediumtoenumerationofactinomycetesinwaterandsoil.

ApplMicrobiol.1975;29:422–426.

36.SiddiquiIA,HaasD,HeebS.Extracellularproteaseof

PseudomonasfluorescensCHA0,abiocontrolfactorwith

activityagainsttheroot-knotnematodeMeloidogyne

incognita.ApplEnvironMicrobiol.2005;71(9):5646–5649.

37.FravelDR.Roleofantibiosisinthebiocontrolofplant

diseases.AnnuRevphytopathol.1988;26:75–91.

38.McSorleyR.Alternativepracticesformanaging

plant-parasiticnematodes.AmJAlternAgric.1998;13:

98–104.

39.RitchieDF,ClaytonCN.Peachtreeshortlife:acomplexof

interactingfactors.PlantDis.1981;65(6):462–469.

40.AraújoFF,SilvaJFV,AraújoASF.InfluênciadeBacillussubtilis

naeclosão,orientac¸ãoeinfecc¸ãodeHeteroderaglycinesem

41.DawarSM,TariqM,ZakiMJ.ApplicationofBacillusspeciesin

controlofMeloidogynejavanica(Treub)Chitwoodoncowpea

andmashbean.PakistanJBot.2008;40(1):439–444.

42.JacobsenBJ,ZidackNK,LarsonBJ.TheroleofBacillus-based

biologicalcontrolagentsinintegratedpestmanagement

systems:plantdiseases.Phytopathology.2004;94:

1272–1275.

43.VelmuruganN,ChoiMS,HanSS,LeeYS.Evaluationof

antagonisticactivitiesofBacillussubtilisandBacillus

licheniformisagainstwood-stainingfungi:invitroandinvivo

experiments.JMicrobiol.2009;47:385–392.

44.ChunJ,BaeKS.PhylogeneticanalysisofBacillussubtilisand

relatedtaxabasedonpartialgyrAgenesequences.AntonVan