Isolation and phenotypic and morphological ...

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Isolation and phenotypic and morphological characterization of the first Podoviridae lytic bacteriophages ϕA38 and ϕA41 infecting Pectobacterium parmentieri (former Pectobacterium wasabiae) A. Smolarska & L. Rabalski & M. Narajczyk & R. Czajkowski Accepted: 6 July 2017 /Published online: 12 July 2017 # The Author(s) 2017. This article is an open access publication Abstract Two bacteriophages, ϕA38 and ϕA41, in- fecting Pectobacterium parmentieri strain SCC 3193 (former Pectobacterium wasabiae strain SCC 3193) were isolated from arable soil samples collected in different regions of Poland. ϕA38 and ϕA41 have a typical morphology of the members of the family Podoviride and order Caudovirales, with a head diameter of ca. 60 nm and tail length of ca. 20 nm. Phages ϕA38 and ϕA41 exhibited a similar RFLP pattern with Csp6I restriction endonuclease. They were stable in a range of pHs, temperatures and osmolarities but were rapidly inactivated by UV light. During the first 20 min., 74 and 69% of ϕ A38 and ϕ A41 phages, respectively, were adsorbed to SCC 3193 cells. In one-step growth experiments, ϕA38 and ϕA41 showed latent period of ca. 2030 min and burst size of 102 and 141 phages, respectively. The optimal multiplicity of infection (MOI) was calculated to be 0.01 for both bacteriophages. In the host range experiments, both phages were able to infect six from 21 of the tested P. parmentieri isolates but the phages were unable to infect other members of the Pectobacterium spp. or Dickeya spp. In the proof-of-concept experiments, ϕA38 and ϕA41 were able to inhibit the growth of P. parmentieri strain SCC 3193 and to protect potato tuber tissue maceration caused by the bacterium. The potential use of ϕA38 and ϕA41 bacterio- phages for the biocontrol of P. parmentieri in potato is discussed. Keywords Biological control . Erwinia carotovora subsp. wasabiae . Survival . Stability . Application Introduction Potato blackleg and tuber soft rot caused by pectinolytic bacteria Pectobacterium and Dickeya species (also called soft rot Enterobacteriaceae SRE) may result in important losses in (seed) potato production worldwide (Toth et al. 2011; Pérombelon 2002). To date, five SRE species are reported to cause potato blackleg in Europe viz. P. atrosepticum (Pba), D. dianthicola, D. solani, P. carotovorum subsp. brasiliense (Pcb) and P. parmentieri (former P. wasabiae) (Ppa, former Pwa) (de Haan et al. 2008; Toth et al. 2011; Waleron et al. 2013). In 2010, P. wasabiae was described for the first time as a new pathogen of potato in New Zealand, responsible for high blackleg levels in field crops Eur J Plant Pathol (2018) 150:413425 DOI 10.1007/s10658-017-1289-3 A. Smolarska : R. Czajkowski (*) Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Abrahama 58, 80-307 Gdansk, Poland e-mail: [email protected] L. Rabalski Department of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Abrahama 58, 80-307 Gdansk, Poland M. Narajczyk Laboratory of Electron Microscopy, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland

Transcript of Isolation and phenotypic and morphological ...

Page 1: Isolation and phenotypic and morphological ...

Isolation and phenotypic and morphological characterizationof the first Podoviridae lytic bacteriophages ϕA38 and ϕA41infecting Pectobacterium parmentieri (former Pectobacteriumwasabiae)

A. Smolarska & L. Rabalski & M. Narajczyk &

R. Czajkowski

Accepted: 6 July 2017 /Published online: 12 July 2017# The Author(s) 2017. This article is an open access publication

Abstract Two bacteriophages, ϕA38 and ϕA41, in-fecting Pectobacterium parmentieri strain SCC 3193(former Pectobacterium wasabiae strain SCC 3193)were isolated from arable soil samples collected indifferent regions of Poland. ϕA38 and ϕA41 have atypical morphology of the members of the familyPodoviride and order Caudovirales, with a headdiameter of ca. 60 nm and tail length of ca. 20 nm.Phages ϕA38 and ϕA41 exhibited a similar RFLPpattern with Csp6I restriction endonuclease. Theywere stable in a range of pHs, temperatures andosmolarities but were rapidly inactivated by UVlight. During the first 20 min., 74 and 69% ofϕA38 and ϕA41 phages, respectively, wereadsorbed to SCC 3193 cells. In one-step growthexperiments, ϕA38 and ϕA41 showed latent periodof ca. 20–30 min and burst size of 102 and 141phages, respectively. The optimal multiplicity ofinfection (MOI) was calculated to be 0.01 for both

bacteriophages. In the host range experiments, bothphages were able to infect six from 21 of the testedP. parmentieri isolates but the phages were unable toinfect other members of the Pectobacterium spp. orDickeya spp. In the proof-of-concept experiments,ϕA38 and ϕA41 were able to inhibit the growth ofP. parmentieri strain SCC 3193 and to protect potatotuber tissue maceration caused by the bacterium.The potential use of ϕA38 and ϕA41 bacterio-phages for the biocontrol of P. parmentieri in potatois discussed.

Keywords Biological control .Erwinia carotovorasubsp. wasabiae . Survival . Stability . Application

Introduction

Potato blackleg and tuber soft rot caused bypectinolytic bacteria Pectobacterium and Dickeyaspecies (also called soft rot Enterobacteriaceae –SRE) may result in important losses in (seed) potatoproduction worldwide (Toth et al. 2011; Pérombelon2002). To date, five SRE species are reported to causepotato blackleg in Europe viz. P. atrosepticum (Pba),D. dianthicola, D. solani, P. carotovorum subsp.brasiliense (Pcb) and P. parmentieri (formerP. wasabiae) (Ppa, former Pwa) (de Haan et al.2008; Toth et al. 2011; Waleron et al. 2013).

In 2010, P. wasabiae was described for the firsttime as a new pathogen of potato in New Zealand,responsible for high blackleg levels in field crops

Eur J Plant Pathol (2018) 150:413–425DOI 10.1007/s10658-017-1289-3

A. Smolarska : R. Czajkowski (*)Department of Biotechnology, Intercollegiate Faculty ofBiotechnology, University of Gdansk and Medical University ofGdansk, Abrahama 58, 80-307 Gdansk, Polande-mail: [email protected]

L. RabalskiDepartment of Recombinant Vaccines, Intercollegiate Faculty ofBiotechnology, University of Gdansk and Medical University ofGdansk, Abrahama 58, 80-307 Gdansk, Poland

M. NarajczykLaboratory of Electron Microscopy, Faculty of Biology,University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland

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(Pitman et al. 2010). In the following years,P. wasabiae was isolated from symptomatic potatoplants in different countries worldwide includingCanada, New Zeeland, Iran, South Africa, Zimbabwe,Finland, France, Germany, Poland, the Netherlands,Serbia, Scotland and USA (Khayi et al. 2016). Like-wise, a number of isolates classified previously asP. carotovorum were re-classified as P. wasabiaedue to the advances in development of genome-based taxonomical methods. The results of thesephylogenomic studies indicated that P. wasabiaewas present in association with potato in Europe al-ready for a long time and it is not an invasivePectobacterium spp. recently introduced to Europefrom outside (Khayi et al. 2016). Lately, it has beenproposed to phylogenetically separate P. wasabiaestrains isolated from potato and other hosts and col-lected in USA, Europe and Canada from strains in-fecting horseradish in Asia (Yuan et al. 2014;Pritchard et al. 2016). What is more, last year Khayiand co-workers (Khayi et al. 2016) reclassifiedpotato-associated P. wasabiae strains to a new taxonnamed P. parmentieri (Khayi et al. 2016) - a distinctcluster from the one grouping P. wasabiae strainsisolated from horseradish plants. In the last six years,potato-associated Pwa (now P. parmentieri) has con-tinued to be an important causative agent of potatoblackleg and soft rot in Europe leading toeconomically-important and increasing losses (vander Wolf et al. 2017).

Like in case of other soft rot Enterobacteriace-ae, efficient control of Ppa with the use of tradi-tional chemical and physical agents has not beenachieved yet (Czajkowski et al. 2011). Similarly,there are no soft rot Enterobacteriaceae-immunepotato cultivars available (Lapwood and Read1986; Lapwood and Harris 1982) and it is doubt-ful whether such cultivars would appear on themarket in the nearest future (Davidsson et al.2013). Consequently, only hygienic practices, in-cluding but not limited to the use of certified seedmaterial, avoidance of pathogen introduction inclean seed tubers and soil drainage to decreaseoxygen depletion and water film on tubers plantedin soil, can minimize the possibility of infectionand spread of the pathogen in potato. These mea-sures are, however, insufficient to eradicate black-leg and soft rot in potato entirely (van der Wolfand de Boer 2007; Czajkowski et al. 2011).

Bacteriophages (phages) are viruses able to in-fect and kill bacterial hosts (Duckworth and Gulig2002). They are ubiquitous in all environmentscontaining bacteria and have been found in soil,sewage, in and on animals and on plants (Abedon2008; Gill and Abedon 2003). Bacteriophages havebeen proposed as potential biological controlagents against plant pathogenic bacteria and con-sequently, they have been evaluated against differ-ent pathogens including Erwinia amylovora,Xanthomonas pruni, Pseudomonas tolaasii, Strep-tomyces scabies and Ralstonia solanacearum ondifferent host plants (Jones et al. 2008, 2012).S imi la r ly, phages were tes ted to cont ro lPectobacterium spp. and Dickeya spp. but onlylimited attempts have been made to characterizethese lytic bacteriophages in detail (Czajkowskiet al. 2014, 2015; Adriaenssens et al. 2012). Forexample, no phages acting against Ppa have beenreported in literature so far (Czajkowski 2016).The purpose of this study was to isolate andcharacterize novel lytic bacteriophages specific toP. parmentieri with the main idea to evaluate theirinteraction with bacterial host as well as phagepotential as biological control agents.

Materials and methods

Bacterial strains and media

All bacterial strains used in this study are listed inTable 1. For routine tests and maintenance, bacte-ria were grown at 28 °C for 24–48 h on tryptonesoya agar (TSA, Oxoid) prior to use. For liquidpreparations, bacterial cultures were grown intryptone soya broth (TSB, Oxoid) with agitation200 rpm at 28 °C. For long-term preservation,bacterial cultures were kept in sterile 40% (v/v)glycerol at −80 °C.

Isolation of bacteriophages from environmental samples

Bulk soil and rhizosphere soil samples were collectedbetween April and September 2013 in different arableregions in Poland. To isolate bacteriophages from theenvironmental samples we used protocol described pre-viously (Czajkowski et al. 2014).

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Tab

le1

Pectobacteriumspp.andDickeya

spp.strainsused

inthisstudyandthehostrangeof

bacteriophages

ϕA38

andϕA41

isolated

initiallyagainstP

ectobacteriumparm

entieristrain

SCC3193

No

Strain

Species

Relevantcharacteristics

ϕA38

ϕA41

Reference

1SCRI140/

IFB5310

P.parm

entieri

Solanumtuberosum,A

rizona,U

SA

++

Waleron

etal.2013

2SCC3193/IFB

5395

P.parm

entieri

Solanumtuberosum,F

inland,1987,reference

strain

++

Pirhonen

etal.1988

3IFB5417

P.parm

entieri

soil,

Poland,2013

++

Collectionof

IntercollegiateFaculty

ofBiotechnology

UGandMUG(IFB)

4IFB5434

P.parm

entieri

Solanumtuberosum,P

oland,2013

++

Collectionof

IntercollegiateFaculty

ofBiotechnology

UGandMUG(IFB)

5IFB5501

P.parm

entieri

Solanumtuberosum,P

oland,2013

++

Wiken

Deesetal.2017

6IFB5563

P.parm

entieri

Solanumtuberosum,P

oland,1996

++

Collectionof

IntercollegiateFaculty

ofBiotechnology

UGandMUG(IFB)

7IFB0310

P.parm

entieri

Solanumtuberosum,P

oland,2011

−−

Collectionof

IntercollegiateFaculty

ofBiotechnology

UGandMUG(IFB)

8CFB

P3304/IFB

5302

P.parm

entieri

Japanese

horseradish,Japan,1985

−−

GotoandMatsumoto1987

9SCRI103/

IFB5309

P.parm

entieri

Solanumtuberosum,S

cotland,U

K,1977

−−

Collectionof

IntercollegiateFaculty

ofBiotechnology

UGandMUG(IFB)

10IPO1955/IFB5396

P.parm

entieri

Solanumtuberosum,the

Netherlands,2001

−−

deHaanetal.2008

11IPO1949/IFB5397

P.parm

entieri

Solanumtuberosum,the

Netherlands,2002

−−

deHaanetal.2008

12IFB5408

P.parm

entieri

Solanumtuberosum,P

oland,2013

−−

Collectionof

IntercollegiateFaculty

ofBiotechnology

UGandMUG(IFB)

13IFB5427

P.parm

entieri

(unknown)

weedcollected

atthepotato

field,

Poland,2013

−−

Collectionof

IntercollegiateFaculty

ofBiotechnology

UGandMUG

14IFB5441

P.parm

entieri

Solanumtuberosum,P

oland,2013

−−

Collectionof

IntercollegiateFaculty

ofBiotechnology

UGandMUG(IFB)

15IFB5485

P.parm

entieri

Solanumtuberosum,B

elgium

,2012

−−

Collectionof

IntercollegiateFaculty

ofBiotechnology

UGandMUG(IFB)

16IFB5486

P .parm

entieri

Solanumtuberosum,B

elgium

,2012

−−

Collectionof

IntercollegiateFaculty

ofBiotechnology

UGandMUG(IFB)

17IPO1957/IFB

5495

P.parm

entieri

Solanumtuberosum,the

Netherlands,2001

−−

deHaanetal.2008

18IFB5498

P.parm

entieri

Solanumtuberosum,P

oland,2013

−−

Collectionof

IntercollegiateFaculty

ofBiotechnology

UGandMUG(IFB)

19IFB5553

P.parm

entieri

Solanumtuberosum,P

oland,1996

−−

Collectionof

IntercollegiateFaculty

ofBiotechnology

UGandMUG(IFB)

20Ecc

71P.carotovorumsubsp.carotovorum

Solanumtuberosum,1987,referencestrain

−−

Willisetal.1987

21SC

RI1043

P.atrosepticum

−−

Hintonetal.1989

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Enrichment of bacteriophages in their host bacteriaand purification of individual phage particles

Pectobacterium parmentieri strain SCC 3193 (Pirhonenet al. 1988; Khayi et al. 2016) was used to enrich lyticbacteriophages from soil as previously described (Twestand Kropinski 2009). For this 1 ml of filter-sterilizedsoil extract was added to 9 ml of SCC 3193 bacterialculture grown in TSB, containing ca. 108 colonyforming units (cfu) ml−1 and incubated at 28 °C for24 h with shaking (200 rpm). After incubation, bacteriawere removed by centrifugation (8000×g, 5 min) andsupernatants were filter-sterilized with 0.22 μm filters(sterile active cellulose syringe filters, VWR). Purifica-tion of individual phage particles was done using a softtop agar method as previously described (Bertani 1951).

Bacteriophage morphology under transmission electronmicroscopy

Microscopic analyses were performed in the Laboratoryof Electron Microscopy, Faculty of Biology, Universityof Gdansk, Poland, using negative staining with uranylacetate as described previously (Gasic et al. 2011;Czajkowski et al. 2014).

Determination of bacteriophages’ host range

A host specificity assay was performed using 19 bacte-rial isolates of P. parmentieri collected from symptom-atic potato plants and other crops in different years andcountries, and on five reference/type strains of other softrot Enterobacteriaceae viz. P. carotovorum subsp.carotovorum strain Ecc71, P. atrosepticum strain SCRI1043, P. carotovorum subsp. brasiliense strain JJ56,Dickeya dadantii strain 3937 and D. solani strainIPO2222 as previously described (Czajkowski et al.2014) (Table 1).

Effect of pH, temperature, chloroform, UV radiationand NaCl concentration on phage fitness under in vitroconditions

Phages were characterized for features which can beimportant factors in biological control applications,where phages are expected to be applied on tubersduring planting, in potato rhizosphere and/or haulms toprotect the growing plants against Ppa. Accordingly,they were tested for stability at a range of differentT

able1

(contin

ued)

No

Strain

Species

Relevantcharacteristics

ϕA38

ϕA41

Reference

Solanumtuberosum,S

cotland,U

K,1985,

referencestrain

22JJ56

P.carotovorumsubsp.brasiliensis

Solanumtuberosum,S

outh

Africa

−−

Czajkow

skietal.2015

233937

Dickeya

dadantii

Saintpaulia

sp.,France,1977,referencestrain

−−

Hugouvieux-Cotte-Pattatand

Robert-Baudouy

1994

24IPO2222

/IFB

0123

Dickeya

solani

TSolanumtuberosum,the

Netherlands,2007

−−

vanderWolfetal.2017

‘+’-lysisof

bacterialcells(plaqueform

ation),‘-‘–lack

ofbacterialcellslysis(noplaque

form

ation)

T–type

strain

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pHs (pH 2 – pH 12), temperature (−20, 4, 28, 37, 42 and85 °C) and osmolarities (different NaCl final concentra-tions: 0.05, 0.5, and 5.0 M NaCl) which may occur insoil environment (Czajkowski et al. 2012). Likewise,phages were tested for stability under UV light (PhilipsTUVG30 T8, UV dose 50 mJ cm2, 30 cm from the lightsource) to assess the possibility of leaf and stem appli-cations in the field (Jones et al. 2008) and for stability insolutions containing chloroform (20% final concentra-tion), which may be important for the isolation, purifi-cation and large scale commercial preparations of bac-teriophages as reported by others (Clokie and Kropinski2009). In each test, stability was assessed as the ratio ofthe bacteriophage particles which survived the experi-ment to the initial number of phages used. Each exper-iment was performed twice with the same setup and theresults from both experiments were averaged. Effects ofpH, temperature, chloroform, NaCl concentration andUV radiation were analyzed as previously described(Czajkowski et al. 2014).

Optimal multiplicity of infection (MOI)

Optimal multiplicity of infection was determined forphages ϕA38 and ϕA41. SCC 3193 culture in TSB(10 ml) was infected with phages at four different pfu/cfu ratios (MOI): 0.01, 0.1, 1.0 and 10.0 (1 ml). Afterovernight incubation at 28 °C with shaking (200 rpm),bacterial cultures were centrifuged (10,000×g, 10 min)and supernatants were assayed for phage presence asdescribed above. The MOI resulting in the highestphage titer (the highest pfu ml−1) was considered asoptimal. The experiment was independently repeatedthree times with the same setup and the results from allrepetitions were averaged.

Effect of temperature on the optimal MOI

In order to evaluate the effect of temperature on thebacteria-phage interaction, 100 μl of 105 cfu ml−1

P. parmentieri SCC 3193 suspensions were mixed withsuspensions containing individual bacteriophage (ϕA38or ϕA41) (103 pfu ml−1) (MOI 0.01) in Ringer’s buffer(Merck). Five ml of soft top agar (TSB supplementedwith 7 g l−1 agar) was added to each mixture, poured onthe surface of TSA plates and incubated at four temper-atures (10, 15, 22 and 28 °C) until plaques were formed.Phage titer (pfu ml−1) was calculated for each bacterio-phage and each treatment. Three independent repetitions

per temperature and per phage (ϕA38 or ϕA41) wereperformed and the results were averaged per phage. Theentire experiment was performed twice with the samesetup.

Phage adsorption

To determine the speed of phage adsorption to Ppa cells,1 ml of the log-phage SCC 3193 cells (ca. 108 cfu ml−1)was infected with phage suspension to reach MOI of 0.1(ca. 107 pfu ml−1) and incubated at 28 °C for 20 min.After 0 (control), 1, 2, 5, 10 and 20 min., two individualsamples per phage were collected and centrifuged at10000×g for 5 min to pellet the bacteria together withthe adsorbed phages. The resulting supernatants weresterilized with 0.22 μm syringe filter and assayed forfree, non-adsorbed phages. The experiment was repeat-ed independently three times with the same setup andthe results were averaged. Phage adsorption was calcu-lated as previously described (Czajkowski et al. 2014).

One-step growth

To determine the latent period and burst size for ϕA38and ϕA41, a one-step growth experiment was conduct-ed as previously described (Ellis and Delbrück 1939;Czajkowski et al. 2014) using P. parmentieri strain SCC3193 as a host for both bacteriophages.

Purification of bacteriophage genomic DNAfor restriction fragment length polymorphism (RFLP)

Purification of phage DNA was performed using theMaster Pure Complete DNA & RNA Purification Kit(Epicenter) following the manufacturer’s protocol forisolation of total DNA/RNA from cell samples. Afterpurification, phage genomic DNA was resuspended in20 μl of sterile demineralized water and stored at 4 °Cfor further use.

RFLP of phage genomes

Purified phage genomic DNA (ca. 150–400 ng μl−1)was subjected to single-enzyme restriction analysis withCsp6I, NcoI, NdeI, BamHI, HindIII, KpnI, SalI, AluI,XbaI, EcoRI, KspAI, AluI and RsaI (all from ThermoFisher Scientific) restriction endonucleases according tothe protocol provided by the manufacturer. Briefly,phage genomic DNA (ca. 200 ng per reaction) was

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digested for up to 24 h with 2.5 U of a restrictionendonuclease in a total volume of 10 μl. DigestedDNA was electrophoresed in 2% agarose gels in 0.5 xTBE. For estimation of the size of DNA fragments, λphage genomic DNA digested with HindIII and EcoRI(Thermo Fisher Scientific) was used. Agarose gels werestained with 5 mg ml−1 of GelRed (Biotum) for visual-ization of digested DNA as recommended by themanufacturer.

Host challenge assay and effect of bacteriophageson potato tuber tissue maceration causedby P. parmentieri SCC3193

Host challenge assay and the assay to verify the protec-tive effect of bacteriophages on potato tuber macerationwere performed as previously described (Czajkowskiet al. 2014) using P. parmentieri SCC 3193 as a hostfor both phages. Both experiments were repeated inde-pendently one timewith the same setup and the obtainedresults were averaged per assay.

Statistical analysis

Data were analyzed accordingly to the experimentaldesign used. To achieve approximate normality, countswere log-transformed adding a value 1 to avoid takinglogs of zero. Effects were considered to be significant atP = 0.05 and pair-wise differences were obtained usingt-test. All analyses were performed with the statisticalsoftware package Statistica v. 10 (www.statsoft.com).

Results

Isolation of P. parmentieri lytic bacteriophagesfrom environmental samples

Between April and September 2013, 164 environmentalsamples were collected from different regions in Polandand assayed for the presence of lytic bacteriophagesinfecting P. parmentieri strain SCC 3193. After enrich-ment of putative bacteriophages in SCC 3193 cultures,only two samples (both from arable soil collected inPomorskie and Lubelskie provinces, respectively,1.22% of all samples tested) yielded lytic bacterio-phages able to infec t and ki l l exc lus ive lyP. parmentieri SCC 3193 host. From each of the twopositive samples one distinct plaque was isolated and

further purified to obtain pure phage particles. Theobtained phages were named ϕA38 and ϕA41. Enrich-ment ofϕA38 andϕA41 in SCC 3193 resulted in phagesuspensions with high titer averaging 109–1010 pfu ml−1

after an overnight incubation at 28 °C. Both ϕA38 andϕA41 formed clear plaques, ca 1.1–1.4 mm in diameterwith sharp edges on lawns of P. parmentieri SCC 3193after 24 h incubation at 28 °C.

Transmission electron microscopy (TEM)and restriction fragment length polymorphism (RFLP)analyses of ϕA38 and ϕA41

Transmission electron microscopy analysis performedforϕA38 andϕA41 revealed that both phages belong tothe order Caudovirales and family Podoviridae basedon their morphology and presence of the non-envelopedicosahedral head (diameter ca. 60 nm) (n = 10 perphage) and no-contractile short tail (length ca. 20 nm)(n = 10 per phage) (Fig. 1). Genomic DNA of phagesϕA38 and ϕA41 was insensitive for digestion with allrestriction endonucleases tested, except Csp6I, forwhich genomic DNA obtained from phages ϕA38 andϕA41 expressed the same restriction-nuclease patternswith all common DNA fragments in both phages (datanot shown).

Host range of ϕA38 and ϕA41 phages

Both ϕA38 and ϕA41 exhibited the same host rangeand were able to infect 6 from 21 P. parmentieri isolatestested. The susceptible Ppa strains originated from po-tato samples collected in Poland, Finland and USA.Neither ϕA38 nor ϕA41 were able to infect isolatesbelonging to species other than P. parmentieri (Table 1).

Characterization of features involved in the stabilityand survival of ϕA38 or ϕA41 in the environment

Effect of pH

Stability of both phages followed the same trend.ϕA38and ϕA41 were stable in neutral pH (pH 6.8–7.0) buttheir numbers were reduced both in acidic and basic pHsafter incubation. A ca. 10-fold reduction of phage num-bers were recorded in pH 2 and pH 12 but both ϕA38and ϕA41 survived a 24 h incubation at each pH tested.

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Effect of chloroform

Phages ϕA38 and ϕA41 were sensitive to chloroformtreatment; 60 and 70% reduction of initial phage num-bers were recorded for phagesϕA38 andϕA41, respec-tively, after incubation in solution containing chloro-form for 1 h at room temperature.

Effect of temperature

Stability of ϕA38 and ϕA41 under six different tem-peratures (−20, 4, 28, 37, 42 and 85 °C) followed thesame trend. Both bacteriophages were more stable atlower (−20 and 4 °C) than at high temperatures (42 and85 °C). Both phages were able to survive at 85 °C for24 h, but incubation at this temperature resulted in atleast 100-fold decrease in phage numbers.

Effect of NaCl concentrations

ϕA38 or ϕA41 were incubated for 24 h in solutionscontaining 0.05, 0.5 and 5.0 M NaCl or in solutionwithout NaCl (control). No difference in phage survivalbetween control (phages incubated without NaCl) andtreatments containing NaCl was observed in bothexperiments.

Effect of UV radiation

ϕA38 or ϕA41 were unable to survive a 5 and 10 minexposition to UV light in repeated experiments. For both

ϕA38 and ϕA41 bacteriophages, all phage particleswere readily inactivated with a 5 min UVexposition.

Phage adsorption, optimal MOI and one-step growth

Adsorp t ion of phages ϕA38 and ϕA41 toP. parmentieri strain SCC 3193 in TSB at 28 °C afterfirst 20 min was 74 and 69%, respectively (Fig. 2a).Optimal MOI was determined to be 0.01 for both bac-teriophages (data not shown). The latent period (notincluding the first 20 min adsorption step) was 20–25 min for both phages. The estimated average burstsize for ϕA38 and ϕA41 was 102 ± 7 and 141 ± 5‘progeny’ phage particles per infected host cell, respec-tively (Fig. 2b).

Effect of temperature on the optimal MOI

The rate of P. parmentieri SCC 3193 infection withphages ϕA38 and ϕA41 was tested at four differenttemperatures (10, 15, 22 and 28 °C) using bacterialcultures of 105 cfu ml−1 and viral suspensions of 103

pfu ml−1 (effective MOI 0.01). The highest ϕA38 andϕA41 infection incidence resulting in the highest num-ber of Bprogeny^ bacteriophages visualized as plaqueson the lawn of SCC 3193 was observed at 22 °C and28 °C, whereas at 10 °C, seven and two-fold lowernumber of phage plaques in comparison with 22 °Cand 28 °C was recorded for phages ϕA38 and ϕA41,respectively (data not shown).

Fig. 1 Transmission electron micrographs of P. parmentieri SCC3193 bacteriophagesϕA38 andϕA41 stained with uranyl acetate.Before microscopic analyses, the bacteriophages were purified bypassaging individual plaques four times using soft top agar methodand SCC 3193 as a host.ϕA38 and ϕA41 suspensions containing

ca. 1016 pfu ml−1 in Ringer’s buffer (Merck) were used for stain-ing. Each photograph represents a typical bacteriophage particle.At least 10 different photographs were taken for each sample andpreparation. Scale bar: 100 nm

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P. parmentieri challenge in vitro assay

Initial challenge trials with P. parmentieri SCC 3193and ϕA38 and ϕA41 bacteriophages were performed

to evaluate the potential of isolated bacteriophages toact as antimicrobials against P. parmentieri. Withinthe first 2 h, the bacterial counts (calculated on thebasis of OD600 measurement: for Ppa OD600 is equal

Fig. 2 Adsorption curves of bacteriophages ϕA38 and ϕA41 toP. parmentieri SCC 3193 (a) and one-step growth curves ofbacteriophages ϕA38 and ϕA41 (b). a For testing the speed ofphage adsorption to the bacterial host cells, 1 ml of log-phase SCC3193 cells, 108 colony-forming units ml−1, was infected with aphage suspension to reach multiplicity of infection (MOI) 0.1 (ca.107 plaque-forming units ml−1) and incubated at 28 °C. Afterincubation for 0 (control), 1, 2, 5, 10 and 20 min, two individualsamples per phage were collected and centrifuged at 10000 x g for5 min to pellet the bacteria together with the adsorbed bacterio-phages. The resulting supernatants were filter sterilized with a0.22 μm syringe filter and assayed for free, non-adsorbed phages.The experiment was repeated independently three times. b Two

milliliters of exponential-growth cultures of SCC 3193 were har-vested by centrifugation (10 min at 8000 x g) and resuspended infresh tryptone soya broth (TSB) to an OD600 of 0.5 (ca. 5 × 108

colony-forming units (cfu) ml−1. A 2ml aliquot of 5 × 108 cfu ml−1

was spiked with phage suspension (final concentration 107 plaque-forming units ml−1; multiplicity of infection, MOI = 0.1). Thephages were allowed to adsorb to bacterial cells for 20 min at28 °C. After this time, the suspension was diluted 10,000 times inTSB pre-warmed to 28 °C then incubated at 28 °C with shaking(ca. 200 rpm). Two samples of 100 μl were taken every 10 minover a period of 100 min for each phage. The number of viralparticles was determined by soft top agar method. Phage plaqueswere counted after 24 h incubation at 28 °C

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to 108 cfu ml−1) were similar in P. parmentieri cul-tures infected with bacteriophages and those unin-fected with ϕA38 or ϕA41 (control). After this time,the growth of bacterial cells was heavily inhibited inco-cultures containing ϕA38 or ϕA41 phages, wherethe number of bacteria did not increase above 107–108 cfu ml−1 during the first 12 h of incubation (Fig.3a). In the control experiment (uninfected with bac-teriophages P. parmentieri cultures), the bacterialcounts reached about 109–1010 cfu ml−1 after 12 hincubation (data not shown).

Suppression of soft rot development in potato slicesco-inoculated with SCC3193 and ϕA38 or ϕA41bacteriophages

ϕA38 and ϕA41 were tested for their ability to reducepotato tuber tissue maceration caused by P. parmentieriSCC3193. In the replicated experiments, ϕA38 andϕA41 bacteriophages were able to reduce potato tubertissuemaceration to at least 40–50% of that observed forthe control potato slices inoculated with SCC 3193 only(Fig. 3b).

Fig. 3 Host challenge assay with P. parmentieri SCC 3193 (a)and protective effect ofϕA38 andϕA41 bacteriophages on potatotuber tissue co-inoculated with bacteriophages and SCC 3193 (b).a ϕA38 and ϕA41 were analyzed for the ability to inhibit thegrowth of SCC 3193 in vitro in tryptone soya broth (TSB) at 28 °Cin vitro. At time 0, 109 colony-forming units (cfu) ml−1 overnightculture of P. parmentieri SCC3193 was diluted 1:50 in fresh TSBand spiked with bacteriophages to a final concentration of ca. 105

plaque forming units ml−1. Samples were collected each hour for12 h and used for measuring the OD600. Log (cfu + 1) ml−1 wascalculated at time 12 h from the OD600 measurements and plating.For the control, a 109 cfu ml−1 overnight culture of P. parmentieriSCC 3193, diluted 1:50 in fresh TSBmedium, incubated under the

same conditions and processed in the same manner, was used. Theexperiment was repeated independently twice and the results wereaveraged. No bacterial colonies grew after incubation with phagesfor 24 h, as visualized after plating on TSA. b The effect wasdetermined by measuring the diameter of rotting tissue (mm) after72 h incubation at 28 °C in a humid box. Wells in potato sliceswere filled with: water (negative control), 106 colony-formingunits (cfu) ml−1 P. parmentieri positive control, or co-inoculatedwith 104 plaque-forming units (pfu) ml−1 of phageϕA38 orϕA41and 106 cfu ml−1 of SCC 3193. Three potato slices with three wellseach, derived from three different potato tubers, were used pertreatment. The experiment was repeated independently and theresults were averaged. Vertical lines represent standard errors

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Discussion

This study was conducted to assess the presence of lyticbacteriophages infecting P. parmentieri in bulk andrhizosphere soil samples collected in different regionsof Poland with the major aim to isolate new bacterio-phages lytic exclusively to P. parmentieri. To the au-thors’ knowledge, no studies have previously docu-mented isolation and characterization of Podoviridaebacteriophages infecting Ppa.

Of all samples analyzed, only two contained lyticbacteriophages infecting specifically Ppa. This low fre-quency of phage isolation was comparable to that re-ported in previous studies in which bacteriophages wereisolated from ca. 1 to 20% of screened samples depend-ing on the season, location, environmental conditionsand presence of putative hosts (Gross et al. 1991; Marshand Wellington 1994; Miller 2001). Moreover, also inthe course of this study, enrichment of phages in hostbacterial cultures was always necessary prior to isolation(Czajkowski et al. 2014, 2015).

Phages ϕA38 and ϕA41 belong to Caudoviralesorder and Podoviridae family assessed on their mor-phology as determined by the transmission electronmicroscopy. Furthermore, they have icosahedral headsand short tail of diameters and sizes which would clas-sify them as C1 morphotype (Ackermann andEisenstark 1974) of the Podoviridae family. Althoughdifferent studies have shown that more than 90% of lyticbacteriophages isolated and characterized with transmis-sion electron microscopy so far belong to theCaudovirales order (Ackermann 2003; Ackermann1998), only ca. 14% of tailed phages belong to thePodoviridae family (Ackermann 2001). Up to date, onlyfour SRE-infecting bacteriophage isolates viz. PP1 andPPWS1 infecting P. carotovorum subsp. carotovorum(Lee et al. 2012; Hirata et al. 2016) and Peat1 and ϕM1infecting P. atrosepticum (Kalischuk et al. 2015; Bloweret al. 2017) were classified to Podoviridae family andneither of those infects specifically P. parmentieri. Thismay suggest that the SRE-infecting Podoviridae bacte-riophages are difficult to isolate using standard proce-dures and/or that the Podoviridae phages are rare in theenvironment (Ackermann 2011).

Phages ϕA38 and ϕA41 were indistinguishablefrom each other based on their morphology and RFLPprofiles; they were, however, isolated from samples

collected in distinct locations of Poland. There is nostraightforward explanation why phenotypically andmorphologically similar bacteriophages were isolatedexclusively in these two agricultural regions situatedca. 500 km from each other and not in samples collectedin different agricultural zones in the country. It is possi-ble that the phages were transferred on potato tubersoriginating from the same location and released to soilduring planting where they found alternative (soil-borne) hosts and therefore survived. However, it re-mains unidentified, whether the SRE-infecting bacterio-phages are more frequently isolated from environmentsinfested with the host bacteria than from the SRE-unpolluted locations (Czajkowski et al. 2015).

ϕA38 and ϕA41 were persistent under differenttemperatures, pH and osmolarities and when incubatedin the presence of chloroform, but were immediatelyinactivated by the UV radiation. This suggests, as ex-pected, that Ppa bacteriophages isolated in this studyshare the same pattern as other bacteriophages infectingplant pathogens (Gupta et al. 1995). Studies have dem-onstrated that bacteriophages are readily inactivated byexposure to sunlight and UV light, and that their popu-lations may diminish under high temperatures, underhigh and low pH and under high ionic concentrations(Jones et al. 2008; Czajkowski et al. 2015).

PhagesϕA38 and ϕA41 were characterized for theirlatent periods, adsorption to SCC 3193 host cells andburst size. Both phages showed rapid adsorption (ca.70% particles adsorbed in the first 20 min) and largeburst size (more than 100 phages per infected cell),comparable with results obtained by others onCaudovirales bacteriophages (Weinbauer 2004). Burstsize is considered as one of evolutional traits, and lyticphages which have short latency and large size of theburst may spread in bacterial population faster andhence are evolutionary more successful (Chibani-Chennoufi et al. 2004; Wang 2006; Abedon et al.2001). The optimal multiplicity of infection (MOI) forboth ϕA38 and ϕA41 phages, resulting in the highestnumber of ‘progeny’ phage particles, was 0.01. Thisoptimal MOI was further influenced by temperaturewith the highest number of ‘progeny’ phages at relative-ly high temperature of 22 and 28 °C. Phage infection ofhost cells depends on the cell’s physiological state,which is modulated by external factors viz. temperature(Hadas et al. 1997). Whereas most in vitro research on

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phages is performed under optimal host growth temper-ature, in the natural environment the infection may takeplace under non-ideal conditions. According to du Raanet al. (2016) the optimal growth temperature for Ppa is29 °C with a range from 20 to 34 °C (du Raan et al.2016). This may suggest that ϕA38 and ϕA41 canefficiently infect Ppa in a variety of temperatures underwhich Ppa naturally persists in potato-associatedenvironment.

In our study, ϕA38 and ϕA41 exhibited similar hostrange, being able to infect only several of Ppa isolatesanalyzed and no other SRE bacteria. Specifically,ϕA38and ϕ41 were able to lyse six Ppa strains isolated frompotato in Poland, Finland and USA obtained in yearsfrom 1987 to 2013 and one strain of Ppa isolated fromsoil in Poland in 2013, they were unable however to killany other Ppa strains tested. Although, a number offactors determines resistance of a particular bacteriumto phage infection (Hyman and Abedon 2010), there isno direct explanation whyϕA38 andϕA41 have such anarrow ability to infect different Ppa strains. Likewise,no correlation was found between parameters such ascountry, plant host and year of isolation and Ppa sus-ceptibility to ϕA38 and ϕA41 infection. Both phageswere isolated initially on SCC 3193 which remains areference strain and the most studied isolate ofP. parmentieri (Nykyri et al. 2012) buy it may notnecessary be the prevalent Ppa strain in potato-associated environment. Only limited data is availableon the genetic diversity and ecology of Ppa (formerPwa) (Charkowski 2015; Pitman et al. 2010) and there-fore more work is needed to better understand Ppafitness under potato field conditions.

Finally, in vitro and in semi-in planta (potato sliceassay) proof-of-concept experiments, ϕA38 and ϕA41were able to inhibit the growth of Ppa and to protectpotato tuber tissue from maceration caused by SCC3193. Both phages were able to significantly reduce softrot of potato slices in comparison with those inoculatedwith Ppa only. Similar protection was obtained in ourprevious studies when lytic bacteriophages were ana-lyzed against D. solani (Czajkowski et al. 2014) andwhere broad-host bacteriophages were evaluated againstcombination of several SRE bacteria (Czajkowski et al.2015) using the same potato slice assay approach. Themajor concern of using bacteriophages to control plantpathogens is that the phages are restricted to certain

strains of target bacterium only (Jones et al. 2008,2012). As mentioned above, ϕA38 and ϕA41 exhibitvery narrow host range and therefore it is doubtfulwhether they could be used against Ppa in field applica-tions alone. This could be partially overcome by usingphage cocktails consisting of several phages with differ-ent host specificity against different SRE pathogens.Such approach has already been successfully introducedin veterinary (Chan et al. 2013) and food industry(García et al. 2008). In biological control of plant path-ogens, examples of using phage cocktails include con-trol of Erwinia amylovora (causing fire blight) (Gillet al. 2003), Xanthomonas campestris pv. vesicatoria(bacterial spot of tomato) (Balogh et al. 2003) andXanthomonas axonopodis pv. citri and X. citrumelo(citrus canker, citrus bacterial spot) (Balogh et al.2008), and Ralstonia solanacearum (bacterial wilt)(Fujiwara et al. 2011). Similarly, the number of charac-terized bacteriophages infecting soft rot Enterobacteri-aceae increases (Czajkowski 2016) and therefore itshould soon be possible to formulate a phage cocktailtargeting all major soft rot and blackleg potato patho-gens in one application.

In conclusion, these results indicate that ϕA38and ϕA41 may be considered as candidates forP. parmentieri antagonists in biological control ap-plications. Additional studies are however, requiredto assess the effectiveness and consistency of controlin the field, phage population dynamics, propertiming of application and long-term ecotoxicologicalrisks.

Acknowledgements The work was financially supported by theNational Center for Research and Development (Narodowe Cen-trum Badan i Rozwoju - NCBR), Poland via a LIDER programresearch grant (LIDER/450/L-6/14/NCBR/2015) to RobertCzajkowski. The authors would like to thank Sylwia Jafra andMagdalena Rajewska (University of Gdansk, Poland) for helpfuldiscussions and Jacquie van der Waals (University of Pretoria,South Africa) for providing the P. carotovorum subsp. brasiliensestrain JJ56 for the bacteriophages’ host specificity assay.

Compliance with ethical standards

Conflict of interest All authors are fully aware of this submis-sion and have declared that no competing interests exist.

Human and animal participants This study did not involveany human and/or animal participants.

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Open Access This article is distributed under the terms of theCreative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestrict-ed use, distribution, and reproduction in any medium, providedyou give appropriate credit to the original author(s) and the source,provide a link to the Creative Commons license, and indicate ifchanges were made.

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