Phage PPPL-1, A New Biological Agent to Control Bacterial Canker Caused by Pseudomonas syringae pv. actinidiae in Kiwifruit

Abstract

Pseudomonas syringae pv. actinidiae (Psa) is a Gram-negative bacterium that causes bacterial canker disease in kiwifruit. Copper or antibiotics have been used in orchards to control this disease, but the recent emergence of antibiotic-resistant Psa has called for the development of a new control agent. We previously reported that the bacteriophage (or phage) PPPL-1 showed antibacterial activity for both biovar 2 and 3 of Psa. To investigate the possibility of PPPL-1 to control bacterial canker in kiwifruit, we further tested the efficacy of PPPL-1 and its phage cocktail with two other phages on suppressing disease development under greenhouse conditions using 6 weeks old kiwifruit plants. Our results showed that the disease control efficacy of PPPL-1 treatment was statistically similar to those of phage cocktail treatment or AgrimycinTM, which contains streptomycin and oxytetracycline antibiotics as active ingredients. Moreover, PPPL-1 could successfully kill streptomycin-resistant Psa isolates, of which the treatment of BuramycinTM carrying only streptomycin as an active ingredient had no effect in vitro. The phage PPPL-1 was further characterized, and stability assays showed that the phage was stable in the field soil and at low temperature of 0 ± 2 °C. In addition, the phage could be scaled up quickly up to 1010 pfu/mL at 12 h later from initial multiplicity of infection of 0.000005. Our results indicate that PPPL-1 phage is a useful candidate as a biocontrol agent and could be a tool to control the bacterial canker in kiwifruit by Psa infection in the field conditions.

Full text





Antibiotics2021,10,554.https://doi.org/10.3390/antibiotics10050554www.mdpi.com/journal/antibiotics
Article
PhagePPPL‐1,aNewBiologicalAgenttoControlBacterial
CankerCausedbyPseudomonassyringaepv.actinidiae
inKiwifruit
Yu‐RimSong
1
,NguyenTrungVu
1
,JungkumPark
1
,InSunHwang
1
,Hyeon‐JuJeong
2
,Youn‐SupCho
2

andChang‐SikOh
1,3,
*
1
DepartmentofHorticulturalBiotechnology,CollegeofLifeScience,KyungHeeUniversity,
Yongin17104,Korea;yulimy@khu.ac.kr(Y.‐R.S.);nguyen12sh@gmail.com(N.T.V.);
jungkuum@naver.com(J.P.);hongkong10@hanmail.net(I.S.H.)
2
FruitResearchInstitute,JeollanamdoAgriculturalResearchandExtensionServices,
Haenam‐gun59021,Korea;pob1256@korea.kr(H.‐J.J.);aktis@korea.kr(Y.‐S.C.)
3
GraduateSchoolofBiotechnology,KyungHeeUniversity,Yongin17104,Korea
*Correspondence:co35@khu.ac.kr;Tel.:+82‐31‐201‐2678
Abstract:Pseudomonassyringaepv.actinidiae(Psa)isaGram‐negativebacteriumthatcausesbacterial
cankerdiseaseinkiwifruit.Copperorantibioticshavebeenusedinorchardstocontrolthisdisease,
buttherecentemergenceofantibiotic‐resistantPsahascalledforthedevelopmentofanewcontrol
agent.Wepreviouslyreportedthatthebacteriophage(orphage)PPPL‐1showedantibacterialac‐
tivityforbothbiovar2and3ofPsa.ToinvestigatethepossibilityofPPPL‐1tocontrolbacterial
cankerinkiwifruit,wefurthertestedtheefficacyofPPPL‐1anditsphagecocktailwithtwoother
phagesonsuppressingdiseasedevelopmentundergreenhouseconditionsusing6weeksoldki‐
wifruitplants.OurresultsshowedthatthediseasecontrolefficacyofPPPL‐1treatmentwasstatis‐
ticallysimilartothoseofphagecocktailtreatmentorAgrimycin
TM
,whichcontainsstreptomycin
andoxytetracyclineantibioticsasactiveingredients.Moreover,PPPL‐1couldsuccessfullykillstrep‐
tomycin‐resistantPsaisolates,ofwhichthetreatmentofBuramycin
TM
carryingonlystreptomycin
asanactiveingredienthadnoeffectinvitro.ThephagePPPL‐1wasfurthercharacterized,and
stabilityassaysshowedthatthephagewasstableinthefieldsoilandatlowtemperatureof0±2
°C.Inaddition,thephagecouldbescaledupquicklyupto10
10
pfu/mLat12hlaterfrominitial
multiplicityofinfectionof0.000005.OurresultsindicatethatPPPL‐1phageisausefulcandidateas
abiocontrolagentandcouldbeatooltocontrolthebacterialcankerinkiwifruitbyPsainfectionin
thefieldconditions.
Keywords:bacterialcanker;diseasecontrol;kiwifruit;phage;Pseudomonassyringaepv.actinidiae

1.Introduction
BacterialcankercausedbyPsahasbeenconsideredasthemostdevastatingdisease
inbothActinidiadeliciosa(greenkiwifruit)andActinidiachinensis(yellowkiwifruit)[1–3].
Thediseasesymptomscanoccuronthevariousorgansofkiwifruitplantssuchasred
oozeoncaneandtrunk,darkbrownspotswiththeyellowishhalosonleaves,wilting
vines,andnecrosisinflowers[4,5].Amongthem,thewidedeathofvinesleadstothe
mosteconomicloss[6].TheseriousdamagebyPsainfectiononthekiwifruitindustrywas
reportedinthemajorkiwifruitgrowingcountriessuchasChina,Italy,andNewZealand
[7–10].
PsawasfirstisolatedinJapanin1984[11],andsporadicoutbreakswerereportedin
Korea[4,12],Portugal[13],Spain[14],France[15],Turkey[16],Slovenia[17],Greece[18],
andGeorgia[19].Basedongeographical,genetic,andbiologicalcharacteristics,Psa
Citation:Song,Y.‐R.;Vu,N.T.;Park,
J
.;Hwang,I.S.;Jeong,H.‐J.;Cho,
Y.‐S.;Oh,C.‐S.PhagePPPL‐1,aNew
BiologicalAgenttoControlBacterial
CankerCausedbyPseudomonas
syringaepv.actinidiaeinKiwifruit.
A
ntibiotics2021,10,554.https://
doi.org/10.3390/antibiotics10050554
AcademicEditor:CarlaPereira
Received:13April2021
Accepted:7May2021
Published:10May2021
Publisher’sNote:MDPIstaysneu‐
tralwithregardtojurisdictional
claimsinpublishedmapsandinstitu‐
tionalaffiliations.

Copyright:©2021bytheauthors.Li‐
censeeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsandcon‐
ditionsoftheCreativeCommonsAt‐
tribution(CCBY)license(http://crea‐
tivecommons.org/licenses/by/4.0/).

Antibiotics2021,10,5542of13

strainscanbegroupedintobiovars1,2,3,5,and6[20].Biovar4wasreclassifiedasP.
syringaepathovaractinidifoliorum[21].InKorea,severalPsastrainsbelongingtobiovar2
wereisolatedfromgreenkiwifruitcv.‘Hayward’(e.g.,JYS5)andyellowkiwifruitcv.
‘Hort16A’(e.g.,KBE9)[4].Biovar3strains,whichwasfirstisolatedinItalyin2008[1],
alsoappearedinKorea(e.g.,SYS1)[22].Recently,biovar3strainshavebeenfoundin
Europe,NewZealand,Chile,andChina,resultinginseveredamageintheinternational
kiwifruitindustry[23–25].
Currently,onlyafeweffectivetherapeuticdefinitivemethodsaredemonstratedfor
cankerdiseaseinkiwifruit.Copperorstreptomycinproductshavebeentraditionallyused
[26],butPsastrainsresistanttocopperandstreptomycinhavebeenreportedinseveral
countriesincludingKorea[27–29].Moreover,continuoususageorenvironmentalcontam‐
inationwiththesechemicalssignificantlycontributestogenerationofresistantbacteria.
Hence,alternativesmethodstomanagebacterialcankerinkiwifruitareneeded.
Aphageisavirusthatinfectsandkillshostbacterialcells,anditismostlyspecies
specific.Itisnotharmfultohumanandenvironment,thusithasbeenconsideredasan
eco‐friendlyagenttocontrolplantpathogenicbacteria,inparticular,antibiotic‐resistant
pathogenicbacteria[30,31].Phageshavetheself‐replicatingpropertyandhighhostspec‐
ificity.Thesecharacteristicsmakethempromisingasanalternativemethodtoantibiotics
forcontrolofbacterialcanker[32].Tocontrolphytopathogenicbacteria,severalphage‐
basedproductssuchasAgriPhage™(SaltLakeCity,UT,USA)havealreadybeendevel‐
opedandcommercialized,andmanystudiesshowingsignificantbiocontrolefficacyof
phagesforthemanagementoftheseveralimportantphytopathogenicbacteriainrecent
yearshavebeenreported[33].
AlthoughthelargenumberofphagestargetingtoPsawereisolatedandcharacter‐
ized[34–37],onlyafewofthemsuchasPseudomonasphageφ6weredemonstratedto
showtheircontrolefficacyexvivoonbacterialcanker[38].Wepreviouslyreportedviru‐
lentphages,PPPL‐1,KHUφ34,andKHUφ38,whichcouldkillPsastrainsbelongingto
bothbiovar2and3andotherP.syringaepathovars[37,39].PPPL‐1andKHUφ38belong
toPodoviridaefamily,whileKHUφ34isamemberofMyoviridaefamily.Additionally,
PPPL‐1genomeanalysisillustratedthatitisavirulentphagewithgenesencodingaclass
IIholinandRz‐likelysisprotein,butnogenesrelatingtolysogeniccycle[39].Further‐
more,thisphagewasstableunderdiverseenvironmentalconditionswherekiwifruittrees
aregrowing.Thus,weaimedtofurtherexaminethepotentialofPPPL‐1phageincontrol‐
lingbacterialcankerdiseaseinkiwifruitaswellasinmanagingantibiotic‐resistantPsa
isolates.Furthermore,thecombinationofPPPL‐1withKHUφ34andKHUφ38incontrol‐
lingbacterialcankerdiseaseinkiwifruitwasalsoevaluated.
2.Results
2.1.ControlEfficacyofPPPL‐1PhagetoPreventBacterialCankerinPlanta
TotestthecontrolefficacyofPPPL‐1phageagainstbacterialcankerinkiwifruit,108
pfu/mLofphages(equivalenttoMOI1.0)werefirsttreatedonbothsidesof12kiwifruit
leavesof5plantsundergreenhouseconditions,followedbyapplicationofPsaKBE9
(biovar2)andSYS1(biovar3)mixture2hafterphagetreatment.ThePPPL‐1phageap‐
plicationsignificantlyprotectedthetreatedleaveswithPsa,basedonthereductionin
visiblesymptomaticspotscomparedtotheuntreatedone(Figure1a).Indeed,thenumber
ofvisiblesymptomaticspotsinthePPPL‐1‐pretreatedleavesmaintainedatalowerrate
around16.7±4.63(mean±standarderror)spotsafter14daysafterinoculation(dai),while
itcontinuouslyincreasedandreachedtoanaverageof512.9±144.35spotsat14daiinthe
untreatedleaves(Figure1b).Furthermore,thediseaseincidenceinthePPPL‐1‐pretreated
leaveswasnotstatisticallydifferentfromthatinleavestreatedwithcommercialantibiotic
product‐Agrimycin™(approximately41.7spots±16.83)(Figure1b).Theseassayswere
repeatedthreetimeswiththesimilarresults.Theseresultsindicatethatthepretreatment

Antibiotics2021,10,5543of13

ofPPPL‐1phagecanefficientlycontrolbacterialcankerinkiwifruitasmuchasthetreat‐
mentoftheantibioticsproduct.

Figure1.ControlefficacyofPPPL‐1phagetreatmentagainstbacterialcankercausedbyPseudomo‐
nassyringaepv.actinidiae(Psa)KBE9andSYS1mixtureinplanta.(a)Thekiwifruitleaves10days
afterpathogeninoculation(dai)withphagesorantibioticpesticide.Bottomfiguresareenlarged
photographsoftopleaves.Theyellowarrowsindicatesymptomaticspots.Thesterilizedwater
wasusedfordilutionofphagesandbacteria.Water,PPPL‐1(10
8
pfu/mL),orAgrimycin™(0.4
g/L)wasapplied2hbeforeinfectionwithPsa(10
8
cfu/mL).(b)Themeanofnumbersofsympto‐
maticspotsonthetreatedleaves(n=12).Theerrorbarsindicatethestandarderror,andthediffer‐
entalphabetsinthenextoflinesindicatethedifferentgroupsbasedonsignificantdifferencesatp
<0.05byDuncan’smultiplerangetestat14dai.
Previously,wereportedotherPsaphages,KHUφ34andKHUφ38,whichbelongto
MyoviridaeandPodoviridae,respectively,andtheirlyticactivityagainstPsaSYS1(biovar
3)waslessthanthatagainstPsaKBE9(biovar2)invitro[37].Therefore,wetriedtocom‐
paretheircontrolefficacyagainstPsaKBE9andSYS1mixtureinplanta.Consistentlyto
thepreviousresult,inthepresenceofPsaKBE9infection,theindividualtreatmentof
KHUφ34andKHUφ38phagesshowedlesseffectscomparedtoPPPL‐1inplanta,butthe
phagecocktailwiththreephagesshowedthesimilarefficacytoPPPL‐1phagealone(Fig‐
ure2).WhiletheleavestreatedwithPsaonlyshowedanaverageof512.9±144.35symp‐
tomaticspotsat14dai,theleavespretreatedwithKHUφ34andKHUφ38showed234.17
±98.46and428.33±112.15spots,comparedwithonly16.67±4.63and27.08±10.16spots

Antibiotics2021,10,5544of13

bypretreatmentwithPPPL‐1andthephagecocktail,respectively(Figure2b).Theseas‐
sayswererepeatedtwicewiththesimilarresults.Overall,theseresultsindicatethatthe
controlefficacyofPPPL‐1phageissignificantlysimilartophagecocktailtreatmentand
betterthanthoseoftwootherphages,consistentwithinvitrolyticactivity.

Figure2.ControlefficacyofPPPL‐1phagecomparedtoKHUφ34,KHUφ38,orphagecocktail
withallthreephagesagainstbacterialcankercausedbyPseudomonassyringaepv.actinidiae(Psa)
KBE9andSYS1mixtureinplanta.(a)Thekiwifruitleaves10daysafterPsainoculation(dai)with
phagesorantibioticpesticide.Bottomfiguresofeachtreatmentareenlargedphotographsoftop
leaves.Theyellowarrowsindicatesymptomaticspots.Thesterilizedwaterwasusedfordilution
ofphagesandbacteria.Buffer,eachphage(10
8
pfu/mL),orAgrimycin™(0.4g/L)wastreated2h
beforetreatmentwithbacterialsuspension(10
8
cfu/mL).Mockandbufferaresterilizedwater.(b)
Themeanofnumberofsymptomaticspotsonthetreatedleaves(n=12).Theerrorbarsindicate
thestandarderror,andthedifferentlettersontopofeachbarindicatethedifferentgroupsbased
onsignificantdifferencesatp<0.05byDuncan’smultiplerangetestat10dai.

Antibiotics2021,10,5545of13

2.2.ConcentrationandTreatmentTimingofPPPL‐1PhageforEfficientControlofBacterial
CankerinPlanta
Toexamineifphageconcentrationcouldbereducedfordiseasecontrol,thecontrol
efficacyofMOI0.1wascomparedwiththatofMOI1.0in20leavesof5plantsunder
greenhouseconditions.WhileleavestreatedwithPsaKBE9onlyshowed62.7±20.7spots
5weeksaftertreatment,about12.6±4.29and27.1±7.29spotsfromMOI1.0andMOI0.1,
respectively,wereobserved(Figure3a).Theseassayswererepeatedtwicewiththesimilar
results.Theseresultsindicatethat,althoughbothMOItreatmentssignificantlyreduced
thediseaseseveritybyPsainkiwifruitleaves,MOI1.0wasmoreefficient,anditwas
statisticallysimilartoAgrimycin
TM
treatment.

Figure3.Effectsofphageconcentrationandtimingofphagetreatmentoncontrolefficacyof
PPPL‐1phagetreatmentagainstbacterialcankercausedbyPseudomonassyringaepv.actinidiae
(Psa)KBE9inkiwifruitleaves.(a)Leavesweretreatedwithphagesat10
7
and10
8
pfu/mLby
brushingonbothsidesofleaves,thenPsawasappliedonthesamesides2hlater.(b)Leaveswere
inoculatedwithPsaonbothsidesofleavesbybrushing,thenphagesat10
7
and10
8
pfu/mLwere
appliedonthesamesidesbybrushing.Thefronttreatmentofeachtreatmentlabelwasfirstap‐
plied,andthebackonewastreated2hlater.Theerrorbarsindicatethestandarderror(n=20),
andthedifferentalphabetsinthenextoflinesindicatethedifferentgroupsbasedonsignificant
differencesatp<0.05byDuncan’smultiplerangetestat5weeksor7daysaftertreatment.
InadditiontotheprophylacticefficacyofPPPL‐1phage,itstherapeuticefficacywith
MOI1.0wasalsoexamined.TheplantswereinoculatedwithPsabacteriaandthenphages
wereapplied2hlater.Asaresult,thecontrolefficacyofphagetreatmentonkiwifruit

Antibiotics2021,10,5546of13

leavesafterpathogentreatmentexhibitedstatisticallynodifferencefromthatofnophage
treatment(Figure3b).TheseresultsindicatenotherapeuticefficacyofPPPL‐1phagefor
applicationinplanta.
2.3.AntibacterialEffectsofPPPL‐1PhageonStreptomycin‐ResistantPsaIsolatesInVitro
Streptomycin‐basedproductsaremainlyusedforcontrolofPsa[26,28].However,
manystudieshavereportedtheemergenceofstreptomycin‐resistantPsaisolates
[27,29,40].Fourstreptomycin‐resistantPsastrainsisolatedfromSouthKorea(YCS3,JYS5,
KACC10584,andKACC10595)wereusedtoexaminetheantibacterialeffectsofPPPL‐1
againsttheminvitro.Forthisassay,Buramycin
TM
containingastreptomycinasanactive
compoundandAgrimycin™containingbothstreptomycinandoxytetracyclineasactive
compoundswereusedascontrols.Buramycin
TM
didnotsuppressbacterialgrowthof
streptomycin‐resistantPsaisolatesatall,whileitcausedtheformationofclearzone
againstonlyPsastrainKBE9,astreptomycin‐sensitiveisolate(Figure4a,b).Incontrast,
bothAgrimycin™andPPPL‐1phageformedclearzonesagainstallPsastrains,andtheir
antibacterialactivitieswerestatisticallyverysimilaratp<0.05,althoughthesizesofclear
zonesslightlyvariedamongPsaisolates.TheseresultsindicatethatPPPL‐1phagecould
beusedtocontrolstreptomycin‐resistantPsaisolateslikeantibioticsproducts.

Figure4.AntibacterialeffectsofPPPL‐1onstreptomycin‐resistantPsaisolates.(a)Theimagesof
platesshowingclearzones.Themosttopleftfigureshowedthetreatmentofotherfigures.(b)
Lengthofclearzonecausedbyeachtreatment.Theerrorbarsindicatethestandarddeviation(n=
3),andthealphabetsontopofeachbarindicatethedifferentgroupsbasedonsignificantdiffer‐
encesatp<0.05byDuncan’smultiplerangetestineachstrain.sH
2
O,sterilizedwater;Buramy‐
cin™(1.25g/L);Agrimycin™(0.4g/L);PPPL‐1(10
8
pfu/mL).

Antibiotics2021,10,5547of13

2.4.StabilityofPPPL‐1PhageintheFieldSoilandLowTemperature
Inthepreviousreport,weshowedthatthelyticactivityofPPPL‐1phageisstable
underthefieldtemperature(averagetemperatureinkiwifruit‐growingregionsinKorea
overtheyear:0to26°C)andpH4–11[39].However,here,wecheckedhowlongthelytic
activityofPPPL‐1phagecanbesustainedinthefieldsoilat26°C.First,tocheckthevia‐
bilityofPPPL‐1phageinsidethefieldsoil,10
8
pfu/mLofPPPL‐1phagewasinoculatedin
thefieldsoilcollectedfromthekiwifruitorchardinKorea.ThePPPL‐1phagekeptitslytic
activityagainstPsaformorethan240hinthefieldsoilat26°C(Figure5a).Forlong‐term
storage,whetherthelyticactivityofPPPL‐1phagecanbekeptstableatlow‐temperature
(0±2°C)ornotiscritical.Therefore,about2×10
10
pfu/mLofPPPL‐1phagesolutionas
theinitialtiterwaskeptatthistemperature.ThedatashowsthatPPPL‐1phagewasvery
stableformorethan7daysatlowtemperature(Figure5b).Theseresultsindicatethat
PPPL‐1phagecanbeusedinthefieldwherekiwifruittreesaregrowingandcanbekept
atlowtemperatureforstorage.

Figure5.StabilityofPPPL‐1phageinthefieldsoilat26°C(a)andlowtemperature(0–2°C)(b)
andalsominimumMOIforscaled‐upproduction(c).Thephagesof10
8
pfu/mLfor(a)and10
10

pfu/mLfor(b)wereusedforassays.Thephagewasincubatedwithhostbacteria(OD
600
=0.5)at
threedifferentMOIs,andthephageconcentrationwasmeasuredattheindicatedtimepoints(c).
Errorbarsindicatestandarddeviationsofthreereplicates(n=3),andthealphabetsonthetopof
errorbars(a)orinthenextlines(b)indicatethedifferentgroupsbasedonsignificantdifferences
atp<0.05byDuncan’smultiplerangetest.
2.5.OptimalConditionsforScaled‐upProductionofPPPL‐1Phage
Theoptimalconditionsforscaled‐upproductionofPPPL‐1phageneedtobedeter‐
mined,andwetesteddifferentparameterstooptimizephagetiters.Twoofcriticalcondi‐
tionsforefficientmassiveproductionofthephagearetheoptimalstageorconcentration
ofhostbacteriaandtheminimumMOI.First,wecheckedtheoptimalbacterialstagefor

Antibiotics2021,10,5548of13

efficientphageproductionwithMOI0.1.Thedatahighlightedthat0.5ofOD600(approxi‐
mately5×108cfu/mL)wasmoreefficientthan0.2or1.0ofOD600(datanowshown).Next,
wecheckedtheminimumMOIwith0.5ofOD600ofhostbacteria.Forthis,wesetupthree
differentMOIs,MOI0.0005,0.00005,and0.000005,andcheckedphagetitersatseveral
timepoints.ThephagetiterinallthreeMOIsincreasedduringthefirst6hafterinocula‐
tionofPPPL‐1phage(Figure5c).However,onlythephagetiterinMOI0.000005increased
moreuntil12hafterinoculation,anditreachedtothehighestamount(Figure5c).These
resultsindicatethatMOI0.000005withOD6000.5ofhostbacteriainliquidmediumisop‐
timalforscalingupproductionofPPPL‐1phage.
3.Discussion
BacterialcankercausedbyPsaisadestructivediseaseofkiwifruitandfurthercauses
seriouseconomiclossofkiwifruitproductionworldwide[41].Recently,aseriesofpapers
aboutthepracticalbacterialdiseasecontrolwithPseudomonasphagehavebeenpub‐
lished.However,thesestudiesfocusondemonstratingphageactivityinvitro,whileafew
studiestodatehavepreviouslyinvestigatedphageactivitybothinvitroandexvivo
[38,42].Oneofthechallengesinbiocontrolwithphagesistheincompatibilityofphage
efficacyunderinvitroandinvivoconditions[33].However,inthisstudy,wedemonstrate
theefficacyofPPPL‐1phageinsuppressingcankerdiseaseinvivo.
TheresultsofPPPL‐1applicationinkiwifruitplantsbeforePsainfectionunder
greenhouseconditionsdemonstrateddiseasesuppressionaseffectiveasAgrimycin™—a
commercialantibiotic‐basedproduct(Figures1and2).Thus,thisphagemightbeaprom‐
isingbiologicalagentforcontrolofbacterialcankerdisease.However,whenthephage
wasappliedafterPsainfection,itwasunabletosuppressdiseasedevelopment(Figure
3b).Unlikeourresults,theapplicationofaphagecocktailwithfourphages,CHF1,CHF7,
CHF9,andCHF21,in2‐year‐oldkiwifruitplants(cv.‘Hayward’)1hpostinfectionwith
Psasignificantlysuppressedbothbacterialgrowthanddiseasedevelopmentafter30days
[42].TheMOIusedinthisstudywas10,whichwas10‐or100‐foldmorethanthoseinour
study.Moreover,Psatiterwas100‐foldlessthanthatofourstudy.Theusageofhigher
concentrationofphagesandlesstiterofPsaintheinvivoexperimentmightresultin
significantcontrolefficacy.Therefore,wesuggestapplyingthisphageearlyspringbefore
thediseaseisoccurredandshouldnotbeusedafterpathogeninfectskiwifruittreesinthe
fieldorchards.Thetimingtoapplychemicalproductsforcontrolofbacterialcankerin
fieldisverycritical[43].
Theapplicationofphagesinfieldfaceswithdifferentchallengessuchastoleranceto
environmentalconditions[33].PPPL‐1phageisstablebythewayitistreatedorunder
theenvironmentinkiwifruitcultivationregions[39].Thispreviousstudyshowedthat
PPPL‐1phagewasdemonstratedtobestableupto40°C,atpHrangeof3–11,andunder
UV‐A.Furthermore,inthisstudy,weshoweditslongevityintheorchardsoilat26°Cand
itsstabilityatlowtemperature(Figure5).Ourresultstogetherwiththepreviousstudies
supportthepotentialofPPPL‐1phageforbacterialcankercontrolinfieldwherekiwifruit
treesaregrowing.Thestabilityofphagesatnaturalenvironmentincrop‐growingregions
isoneofcriticalfactorsforgoodcontrolefficacyagainstbacterialdiseases.Therearemany
caseswherephagesarestableinthecrop‐growingconditionsinfield[42,44,45].
Currently,phagecocktailhasemergedasasolutiontoovercomethelimitationof
singlephagetreatment[33].Wangetal.[46]explainedthreedifferentwaysthatthephage
cocktaildecreasedthebacterialwiltincidenceintomatoincluding(1)individuallyinfect‐
ingandkillingtargetbacteria,thusreducingthepathogendensity,(2)theslowdevelop‐
mentofpathogenstrainsviaenforcingphage‐resistantdevelopment,or(3)encouraging
thedevelopmentofantagonisticbacterialspecies.Furthermore,tousephagecocktailmay
slowdowntheappearanceofresistantpathogensifphagesinphagecocktailaregenet‐
icallyandmorphologicallydifferent.Ourphagecocktail,KHUφ34,KHUφ38,andPPPL‐
1,couldsuppressdiseasebythemixedPsainfectionandshowednostatisticaldifference
withPPPL‐1treatmentaloneinsuppressingdisease(Figure3).Therefore,itwasfailedto

Antibiotics2021,10,5549of13

seetheadvantageofphagecocktailfordiseasecontrolinthisstudy,probablybecausethe
treatmentwithasinglephage,PPPL‐1,wassoefficient.However,atleast,therewasno
negativeeffectsofthreephagecocktailinkiwifruitleaves.Becausebiovar2and3ofPsa
arepresentinKoreaandalsothereispossibilityofappearanceofresistantpathogenvar‐
iantsagainstPPPL‐1phage,thephagecocktailmixtureshouldbeconsideredforlong‐
termtreatment.
Traditionally,antibioticshavebeenusedtocontrolpathogenicbacteriainnotonly
agriculturebutalsofoodprocessingandhumantherapy.However,theriskofappearance
ofantibiotic‐resistantpathogenswaswarned.Inthecaseofbacterialcankerdisease,strep‐
tomycin‐basedpesticideshavebeenusedtocontrolPsa,andstreptomycin‐resistant
strainswerealsoreported[29].PPPL‐1phagesuccessfullyinhibitedthegrowthofseveral
streptomycin‐resistantPsastrainsisolatedinSouthKorea,comparedtotwostreptomy‐
cin‐basedpesticides‐Buramycin™orAgrimycin™(Figure3).Thus,PPPL‐1phagemight
beusedforcontrollingstreptomycin‐resistantPsastrains.Furthermore,thecombination
ofPPPL‐1withantibiotics‐basedpesticidesmightalsoenhancethecontrolefficacyofthe
currentantibioticapplicationonlyandalsoreducetheusageofantibiotics‐basedpesti‐
cides.However,furtherexperimentsareneededtoconfirmthesepossibilities.
Althoughsomanypapersdemonstratedtheefficacyofphagesinplantdiseasecon‐
trol,thereareonlyafewcommercialphage‐basedproductssuchasAgriPhageTM,Erwiph‐
age,andBiolysesavailableworldwide[33].Todeterminethebestconditionforprocessing
ofphageproducts,theconcentrationofhostbacteriaandtheminimumMOIshouldbe
consideredandselected.Inthisstudy,0.5ofOD600ofhostbacteriaandMOIof0.000005
wereshowntobethesuitableconditionforscaled‐upproduction.Therelativehighcost
ofphageapplicationcomparedtotheconventionalmethodsisgenerallyoriginatedfrom
thescaled‐upproductionstep,andthisleadstothedifficultyinphagetherapyinfield
conditions[47].However,therapidincreaseinPPPL‐1concentrationuptoapproximately
1010pfu/mLwithin12hmightbesuitableforscaled‐upproductionwithinshort‐termpe‐
riod,thuscontributingtothepricereduction.ThesefeaturesofPPPL‐1phagewillbevery
usefultomakeaphageproductforcommercialization.
4.MaterialsandMethods
4.1.GrowthConditionsofBacterialStrains
SixPsastrainsincludingKBE9,SYS1,KACC10584,andKACC10595,isolatedfrom
A.chinensiscultivar(cv.);‘Hort16A’inSouthKorea;andJYS5andYCS3isolatedfromA.
deliciosacv.‘Hayward’inSouthKorea[4]wereusedinthisstudy.Allstrainscorre‐
spondedtobiovar2exceptSYS1(biovar3).Forexperimentalpurpose,asinglecolonyof
eachstrainonTrypticSoybrothAgar(TSA;Difco,FranklinLakes,NJ,USA)platewas
usedforculturingitin5mLofliquidTrypticSoyBroth(TSB;Difco,FranklinLakes,NJ,
USA)inashakingincubatorat140rpmand26°Cfor18h.Thestreptomycin‐resistant
strains(JYS5,YCS3,KACC10584,andKACC10595;[29])wereculturedonmediawith50
µg/mLofstreptomycin(DuchefaBiochemie,RVHaarlem,TheNetherlands).
4.2.PhageLysatePreparation
ThephagesPPPL‐1,KHUφ34,andKHUφ38[37,39]werestoredinsodiumchloride‐
magnesiumsulfate(SM)buffer(50mMTris‐HCl,100mMNaCl,and10mMMgSO4∙7H2O)
at4°Cforroutineuseandat−80°Cbyglycerolstockforlong‐termstorage.Asingle
plaqueofeachphagewascollectedbyrecoveryfromglycerolstockusingthepreviously
describedmethodsofplaqueassays[48]andthenresuspendedinSMbuffer.Briefly,the
phagestocksolutionwasinoculatedtomelted5mLTSAcontaining0.4%agar(~42°C)
and100μLofthebacterialsuspensionandthenpouredonTSAsolidplateandincubated
at26°Covernightafterproperlysolidifying.
Forphagelysatepropagation,100μLofplaqueresuspension(~108pfu/mL)wasin‐
oculatedwith5mLofovernight(18h)liquidcultureofPsaKBE9(OD600=0.5–0.6)ina

Antibiotics2021,10,55410of13

shakingincubatorat140rpmfor6h.Then,thelysatewascollectedbycentrifugationfor
5minat8000×gtoremovetheremainedbacteriaaswellasitsdebris.Ifnecessary,the
supernatantwastreatedby1%chloroformfor30min,andthenthechloroformwasre‐
movedbycentrifugationfor15minat3000×g.Finally,thesupernatantwasfilteredwith
0.22μmporesizeofPVDFsyringefilter(Futecs,Deajeon,Korea)andstoredin4°C.To
determinethephagelysateconcentration,its10‐folddilutedserieswasdottedonthesoft
TSA(0.4%agar)plateinoculatedwith100μLofPsaKBE9(OD600=0.5–0.6).Itsconcentra‐
tionwascalculatedbycountingthenumberofplaquesformedinthedottedarea.
4.3.Scaled‐upProductionandPrecipitationofPhages
Todeterminetheoptimalstageofhostbacteriaforscaledproduction,theovernight
cultureofPsastrainKBE9wasdiluted1000‐foldin400mLliquidmedium,andtheywere
grownupto0.2(~2×108cfu/mL),0.5(~5×108cfu/mL),and1.0(~109cfu/mL)ofoptical
densityat600nm(OD600).Then,thePPPL‐1phageformultiplicityofinfection(MOI)0.1
wasaddedtothebacterialsuspension,anditstiterwaschecked12hlater.Next,todeter‐
minetheminimumMOIforscaled‐upproduction,400mLbacterialsuspensionofPsa
strainKBE9(OD600=0.5)wasinoculatedwith400μLof2.5×106,2.5×107,and2.5×108
pfu/mLofthephagelysatetoreachMOI0.0005,0.00005,and0.000005.After0,6,12,and
18hofshakingincubationat26°C,thephagelysatewascollectedbycentrifugationat
8000rpmfor10minandfiltrationusingfiltersystem(0.22μm;Corning,NY,USA)with
vacuumpumpsystem,andthephagetiterwascalculatedbytheplaqueassaymethod.
Togethigherphagetiter,thephagelysatewasprecipitatedusing10%polyethylene
glycol6000(DaejungChemical&metals,Siheung,Korea)supplementedwith1MNaCl
(LPSsolution,Daejeon,Korea)asfinalconcentrations.Afterovernightincubationat4°C,
thepelletswerecollectedbycentrifugationat4°Cand9000×gfor20minandthenresus‐
pendedwith2mLofSMbuffer.Finally,0.1MKClwasaddedtothesuspensionforwell
separationofphagesfromthepellets,andthesuspensionwascentrifugedat12,000×g
and4°Cfor10min.
4.4.ControlEfficacyTestofPPPL‐1PhageInVivo
ThegraftedkiwifruitplantswithA.chinensiscv.‘Haehyang’(originatedfromthe
highlysusceptiblecv.‘Hongyang’[3])asscionandA.deliciosacv.‘Hayward’asrootstock
wereplantedin6Lpotsinthegreenhouse(15–25°C)for6weeks.Forevaluatingthe
controlefficacyofPPPL‐1phageinvivo,12leaves(about15cmindiameter)of5plants
weretreatedwithphageresuspension(108pfu/mL)onbothsidesusingthesilicon
brusher.After2h,thebacteriasuspension(OD600=0.1,~108cfu/mL)ofPsaKBE9andSYS1
mixtureinsterilizedtapwater(10mMMgCl2bufferwasnotusedbecauseitcausedne‐
crosisinkiwifruitleaves)wastreatedusingthesamemethod.Forpositivecontrol,leaves
weretreatedwithAgrimycinTM(0.4g/L,SUNGBOChemicals,Seoul,Korea),andsterilized
tapwaterwasusedasmocktreatment.Moreover,thecontrolefficacyofPPPL‐1phage
wascomparedtotheindividualKHUφ34andKHUφ38phages[38]andalsotophage
cocktailwithallthreephages.Forphagecocktail,thesameamountofeachphagewas
mixedtoreachthefinalconcentrationofeachphageto108pfu/mL.Toenhancetheattach‐
mentoftreatedphageandbacterialcells,0.02%SilwetL‐77(Momentive,NY,USA)as
finalconcentrationwasaddedtoalltreatments.Thetreatedleaveswereobservedfor2
weeks,andthenumberofleafspotswerecountedtoevaluatethecontrolefficacy.
TooptimizetheamountofPPPL‐1phagefortreatment,twophageconcentrations
(107and108pfu/mL)wereexaminedin20leavesof5plantsat2hbeforeorafterinocula‐
tionofPsaKBE9.Theexperimentswereperformed,asdescribedabove.Thetreatedplants
wereobservedfor5weeksor7days.


Antibiotics2021,10,55411of13

4.5.AntibacterialEffectofPPPL‐1PhagebyFilterPaperDiscMethodInVitro
TheantibacterialactivityofPPPL‐1phagewastestedforcomparisonwithrecom‐
mendedpesticidesagainststreptomycin‐resistantPsausingdiscdiffusiontest.Briefly,the
sterilizedfilterpaperdiscs(Ø8mm;Advantech,Taipei,Taiwan)wereplacedonTSA
platesincubatedwith5mLofmeltingsoftagarand100μLofeitherPsaKBE9suspension
(OD600=0.5)orstreptomycin‐resistantPsastrainssuchasYCS3,JYS5,KACC10584,and
KACC10595.Aftercompletelysolidifying,40μLofBuramycin™(1.25g/L;FarmHan‐
nong,Seoul,Korea),Agrimycin™(0.4g/L)orPPPL‐1(108pfu/mL)wasdroppedontoeach
disc.Aftercompletedrying,eachplatewassealedandincubatedat26°C.Thediameter
(cm)ofclearzoneswasmeasuredastheindicationofantibacterialactivity.
4.6.StabilityTestofPPPL‐1PhageintheFieldSoilandLowTemperature
Forstabilitytestinsoil,108pfu/gofPPPL‐1phagewasinoculatedtothefieldsoil
collectedfromkiwifruitorchardandthenincubatedat26°C.Thelivephagetiterwas
determinedat0,1,2,4,and7daysafterinoculationusingplaqueassayagainstPsastrain
KBE9.Forstabilityatlowtemperature,1mLphagesuspension(~1010pfu/mL)waskept
at0–2°C,andthelivephagetiterwasmeasuredat7daysafterincubation.
4.7.StatisticalAnalysis
Tostatisticallyanalyzeallresults,Duncan’smultiplerangetest(p<0.05)wasper‐
formedwithSAS(version9.4forWindows;SASInstitute,Cary,NC,USA).Allexperi‐
mentswererepeatedmorethantwiceusingthreeormoreplantsineachassay.
AuthorContributions:Y.‐R.S.,N.T.V.,J.P.,I.S.H.,H.‐J.J.,Y.‐S.C.,andC.‐S.O.designedandper‐
formedexperiments.Y.‐R.S.,N.T.V.,andC.‐S.O.wroteapaper.Allauthorsreviewedandapproved
thefinalversionofthemanuscript.
Funding:ThisworkwascarriedoutwiththesupportofKoreaInstituteofPlanningandEvaluation
forTechnologyinFood,Agriculture,Forestry(IPET)throughAgri‐BioindustryTechnologyDevel‐
opmentProgram,fundedbyMinistryofAgriculture,FoodandRuralAffairs(MAFRA)(No.317012‐
4andNo.320041051HD020).
InstitutionalReviewBoardStatement:Notapplicable.
InformedConsentStatement:Notapplicable.
DataAvailabilityStatement:Alldataarecontainedwithinthearticle.
Acknowledgments:WethankYoungJinKohandKoreanAgriculturalCultureCollectionfor
providingPsastrains.
ConflictsofInterest:Theauthorsdeclarethattheyhavenoconflictofinterest.
References
1. Ferrante,P.;Scortichini,M.IdentificationofPseudomonassyringaepv.actinidiaeascausalagentofbacterialcankerofyellow
kiwifruit(ActinidiachinensisPlanchon)incentralItaly.J.Phytopathol.2009,157,768–770.
2. McCann,H.C.;Li,L.;Liu,Y.;Li,D.;Pan,H.;Zhong,C.;Rikkerink,E.H.;Templ e ton,M.D.;Straub,C.;Colombi,E.Originand
evolutionofthekiwifruitcankerpandemic.GenomeBiol.Evol.2017,9,932–944.
3. Wang,F.;Li,J.;Ye,K.;Liu,P.;Gong,H.;Jiang,Q.;Qi,B.;Mo,Q.AninvitroActinidiabioassaytoevaluatetheresistanceto
Pseudomonassyringaepv.actinidiae.PlantPathol.J.2019,35,372.
4. Koh,Y.;Kim,G.;Jung,J.;Lee,Y.;Hur,J.OutbreakofbacterialcankeronHort16A(ActinidiachinensisPlanchon)causedby
Pseudomonassyringaepv.actinidiaeinKorea.N.Z.J.CropHortic.Sci.2010,38,275–282.
5. Donati,I.;Buriani,G.;Cellini,A.;Mauri,S.;Costa,G.;Spinelli,F.Newinsightsonthebacterialcankerofkiwifruit(Pseudomonas
syringaepv.actinidiae).J.BerryRes.2014,4,53–67.
6. Vanneste,J.L.Thescientific,economic,andsocialimpactsoftheNewZealandoutbreakofbacterialcankerofkiwifruit(Pseu‐
domonassyringaepv.actinidiae).Annu.Rev.Phytopathol.2017,55,377–399.
7. Youn g,J.Pseudomonassyringaepv.actinidiaeinNewZealand.J.PlantPathol.2012,94,S1.5–S1.10.
8. Qin,H.;Gao,X.;Zhao,Z.;Zhu,S.;Li,J.;Huang,L.TheprevalencedynamicsandrulesofbacterialcankerofkiwifruitinShaanxi.
ActaPhytophylacicaSin.2013,40,225–230.

Antibiotics2021,10,55412of13

9. Donati,I.;Cellini,A.;Sangiorgio,D.;Vanneste,J.L.;Scortichini,M.;Balestra,G.M.;Spinelli,F.Pseudomonassyringaepv.actinidiae:
Ecology,infectiondynamicsanddiseaseepidemiology.Microb.Ecol.2020,80,81–102.
10. Guroo,I.;Wani,S.;Wani,S.;Ahmad,M.;Mir,S.;Masoodi,F.Areviewofproductionandprocessingofkiwifruit.Int.J.Food
Process.Techn ol.2017,8,699–705.
11. Tak i kawa,Y.;Serizawa,S.;Ichikawa,T.;Tsuyumu,S.;Goto,M.Pseudomonassyringaepv.actinidiaepv.nov.thecausalbacterium
ofcankerofkiwifruitinJapan.Jpn.J.Phytopathol.1989,55,437–444.
12. Koh,Y.;Park,S.;Lee,D.CharacteristicsofbacterialcankerofkiwifruitoccurringinKoreaanditscontrolbytrunkinjection.
KoreanJ.PlantPathol.1996,12,324–330.
13. Balestra,G.;Renzi,M.;Mazzaglia,A.FirstreportofbacterialcankerofActinidiadeliciosacausedbyPseudomonassyringaepv.
actinidiaeinPortugal.NewDis.Rep.2010,22,10.
14. Balestra,G.;Renzi,M.;Mazzaglia,A.FirstreportofPseudomonassyringaepv.actinidiaeonkiwifruitplantsinSpain.NewDis.
Rep.2011,24,10.
15. Vanneste,J.;Poliakoff,F.;Audusseau,C.;Cornish,D.;Paillard,S.;Rivoal,C.;Yu,J.FirstreportofPseudomonassyringaepv.
actinidiae,thecausalagentofbacterialcankerofkiwifruitinFrance.PlantDis.2011,95,1311.
16. Bastas,K.;Karakaya,A.FirstreportofbacterialcankerofkiwifruitcausedbyPseudomonassyringaepv.actinidiaeinTurkey.Plant
Dis.2012,96,452.
17. Dreo,T.;Pirc,M.;Ravnikar,M.;Žežlina,I.;Poliakoff,F.;Rivoal,C.;Nice,F.;Cunty,A.FirstreportofPseudomonassyringaepv.
actinidiae,thecausalagentofbacterialcankerofkiwifruitinSlovenia.PlantDis.2014,98,1578.
18. Holeva,M.;Glynos,P.;Karafla,C.FirstreportofbacterialcankerofkiwifruitcausedbyPseudomonassyringaepv.actinidiaein
Greece.PlantDis.2015,99,723.
19. Meparishvili,G.;Gorgiladze,L.;Sikharulidze,Z.;Muradashvili,M.;Koiava,L.;Dumbadze,R.;Jabnidze,N.Firstreportofbac‐
terialcankerofkiwifruitcausedbyPseudomonassyringaepv.actinidiaeinGeorgia.PlantDis.2016,100,517.
20. Sawada,H.;Kondo,K.;Nakaune,R.Novelbiovar(biovar6)ofPseudomonassyringaepv.actinidiaecausingbacterialcankerof
kiwifruit(Actinidiadeliciosa)inJapan.Jpn.J.Phytopathol.2016,82,101–115.
21. Cunty,A.;Poliakoff,F.;Rivoal,C.;Cesbron,S.;Fischer‐LeSaux,M.;Lemaire,C.;Jacques,M.;Manceau,C.;Vanneste,J.Charac‐
terisationofPseudomonassyringaepv.actinidiae(Psa)isolatedfromFranceandassignmentofstrainsPsabiovar4toadenovo
pathovar:Pseudomonassyringaepv.actinidifoliorumpv.nov.PlantPathol.2015,64,582–596.
22. Koh,Y.J.;Kim,G.H.;Koh,H.S.;Lee,Y.S.;Kim,S.‐C.;Jung,J.S.OccurrenceofanewtypeofPseudomonassyringaepv.actinidiae
strainofbacterialcankeronkiwifruitinKorea.PlantPathol.J.2012,28,423–427.
23. Mazzaglia,A.;Studholme,D.J.;Taratufolo,M.C.;Cai,R.;Almeida,N.F.;Goodman,T.;Guttman,D.S.;Vinatzer,B.A.;Balestra,
G.M.Pseudomonassyringaepv.actinidiae(PSA)isolatesfromrecentbacterialcankerofkiwifruitoutbreaksbelongtothesame
geneticlineage.PLoSONE2012,7,e36518.
24. Butler,M.I.;Stockwell,P.A.;Black,M.A.;Day,R.C.;Lamont,I.L.;Poulter,R.T.Pseudomonassyringaepv.actinidiaefromrecent
outbreaksofkiwifruitbacterialcankerbelongtodifferentclonesthatoriginatedinChina.PLoSONE2013,8,e57464.
25. McCann,H.C.;Rikkerink,E.H.;Bertels,F.;Fiers,M.;Lu,A.;Rees‐George,J.;Andersen,M.T.;Gleave,A.P.;Haubold,B.;Wohlers,
M.W.GenomicanalysisofthekiwifruitpathogenPseudomonassyringaepv.actinidiaeprovidesinsightintotheoriginsofan
emergentplantdisease.PLOSPathog.2013,9,e1003503.
26. Cameron,A.;Sarojini,V.Pseudomonassyringaepv.actinidiae:Chemicalcontrol,resistancemechanismsandpossiblealternatives.
PlantPathol.2014,63,1–11.
27. Han,H.S.;Koh,Y.J.;Hur,J.‐S.;Jung,J.S.OccurrenceofthestrA‐strBstreptomycinresistancegenesinPseudomonasspeciesiso‐
latedfromkiwifruitplants.J.Microbiol.2004,42,365–368.
28. Nakajima,M.;Masao,G.;Tadaaki,H.SimilaritybetweencopperresistancegenesfromPseudomonassyringaepv.actinidiaeand
P.syringaepv.tomato.J.Gen.PlantPathol.2002,68,68–74.
29. Lee,Y.S.;Kim,G.H.;Song,Y.‐R.;Oh,C.‐S.;Koh,Y.J.;Jung,J.S.StreptomycinResistantIsolatesofPseudomonassyringaepv.acti‐
nidiaeinKorea.Res.PlantDis.2020,26,44–47.
30. Jones,J.B.;Jackson,L.;Balogh,B.;Obradovic,A.;Iriarte,F.;Momol,M.Bacteriophagesforplantdiseasecontrol.Annu.Rev.
Phytopathol.2007,45,245–262.
31. Jassim,S.A.;Limoges,R.G.Naturalsolutiontoantibioticresistance:Bacteriophages‘TheLivingDrugs’.WorldJ.Microbiol.Bio‐
technol2014,30,2153–2170.
32. Brüssow,H.WhatisneededforphagetherapytobecomearealityinWesternmedicine?Virology2012,434,138–142.
33. Vu,N.T.;Oh,C.‐S.BacteriophageUsageforBacterialDiseaseManagementandDiagnosisinPlants.PlantPathol.J.2020,36,204–
217.
34. Yin,Y.;Ni,P.e.;Deng,B.;Wan g,S.;Xu,W.;Wang, D.IsolationandcharacterisationofphagesagainstPseudomonassyringaepv.
actinidiae.ActaAgric.Scand.BSoilPlantSci.2019,69,199–208.
35. DiLallo,G.;Evangelisti,M.;Mancuso,F.;Ferrante,P.;Marcelletti,S.;Tinari,A.;Superti,F.;Migliore,L.;D’Addabbo,P.;Frezza,
D.IsolationandpartialcharacterizationofbacteriophagesinfectingPseudomonassyringaepv.actinidiae,causalagentofkiwifruit
bacterialcanker.J.BasicMicrobiol.2014,54,1210–1221.
36. Frampton,R.A.;Tayl or,C.;Moreno,A.V.H.;Visnovsky,S.B.;Petty,N.K.;Pitman,A.R.;Fineran,P.C.Identificationofbacterio‐
phagesforbiocontrolofthekiwifruitcankerphytopathogenPseudomonassyringaepv.actinidiae.Appl.Environ.Microbiol.2014,
80,2216–2228.

Antibiotics2021,10,55413of13

37. Yu,J.‐G.;Lim,J.‐A.;Song,Y.‐R.;Heu,S.;Kim,G.H.;Koh,Y.J.;Oh,C.‐S.Isolationandcharacterizationofbacteriophagesagainst
Pseudomonassyringaepv.actinidiaecausingbacterialcankerdiseaseinkiwifruit.J.Microbiol.Biotechnol.2016,26,385–393.
38. Pinheiro,L.A.;Pereira,C.;Barreal,M.E.;Gallego,P.P.;Balcão,V.M.;Almeida,A.Useofphageϕ6toinactivatePseudomonas
syringaepv.actinidiaeinkiwifruitplants:Invitroandexvivoexperiments.Appl.Microbiol.Biotechnol.2020,104,1319–1330.
39. Park,J.;Lim,J.‐A.;Yu,J.‐G.;Oh,C.‐S.GenomicfeaturesandlyticactivityofthebacteriophagePPPL‐1effectiveagainstPseudo‐
monassyringaepv.actinidiae,acauseofbacterialcankerinkiwifruit.J.Microbiol.Biotechnol.2018,28,1542–1546.
40. Han,H.‐S.;Nam,H.‐Y.;Koh,Y.‐J.;Hur,J.‐S.;Jung,J.‐S.Molecularbasesofhigh‐levelstreptomycinresistanceinPseudomonas
marginalisandPseudomonassyringaepv.actinidiae.J.Microbiol.2003,41,16–21.
41. Scortichini,M.;Marcelletti,S.;Ferrante,P.;Petriccione,M.;Firrao,G.Pseudomonassyringaepv.actinidiae:Are‐emerging,multi‐
faceted,pandemicpathogen.Mol.PlantPathol.2012,13,631–640.
42. Flores,O.;Retamales,J.;Núñez,M.;León,M.;Salinas,P.;Besoain,X.;Yañez,C.;Bastías,R.CharacterizationofBacteriophages
againstPseudomonassyringaepv.actinidiaewithPotentialUseasNaturalAntimicrobialsinKiwifruitPlants.Microorganisms2020,
8,974.
43. Preti,M.;Scannavini,M.;Franceschelli,F.;Cavazza,F.;Antoniacci,L.;Rossi,R.;Bugiani,R.;Tommasini,M.;Collina,M.Field
efficacyofsomeproductsagainstthebacterialcankerofkiwifruitinEmilia‐RomagnaRegion,Italy.ActaHortic.2015,1243:39‐
48.
44. Iriarte,F.B.;Obradović,A.;Werns ing,M.H.;Jackson,L.E.;Balogh,B.;Hong,J.A.;Momol,M.T.;Jones,J.B.;Vall a d,G.E.Soil‐
basedsystemicdeliveryandphyllosphereinvivopropagationofbacteriophages:Twopossiblestrategiesforimprovingbacte‐
riophagepersistenceforplantdiseasecontrol.Bacteriophage2012,2,e23530.
45. Rombouts,S.;Volckaert,A.;Venneman,S.;Declercq,B.;Vandenheuvel,D.;Allonsius,C.N.;VanMalderghem,C.;Jang,H.B.;
Briers,Y.;Noben,J.P.CharacterizationofnovelbacteriophagesforbiocontrolofbacterialblightinleekcausedbyPseudomonas
syringaepv.porri.Front.Microbiol.2016,7,279.
46. Wang,X.;Wei,Z.;Yang, K.;Wan g,J.;Jousset,A.;Xu,Y.;Shen,Q.;Friman,V.‐P.Phagecombinationtherapiesforbacterialwilt
diseaseintomato.Nat.Biotechnol.2019,37,1513–1520.
47. Lang,J.M.;Gent,D.H.;Schwartz,H.F.ManagementofXanthomonasleafblightofonionwithbacteriophagesandaplantacti‐
vator.PlantDis.2007,91,871–878.
48. Kropinski,A.M.;Mazzocco,A.;Waddell,T.E.;Lingohr,E.;Johnson,R.P.Enumerationofbacteriophagesbydoubleagaroverlay
plaqueassay.InBacteriophages;Springer’sHumanaPress:Totowa,NJ,USA,2009;pp.69–76.