Research Article Detection of Bacterial Wilt Pathogen and...

Research ArticleDetection of Bacterial Wilt Pathogen and Isolation of ItsBacteriophage from Banana in Lumajang Area Indonesia

Hardian Susilo Addy12 Norita Fatatik Azizi1 and Paniman Ashna Mihardjo1

1Department of Plant Protection Faculty of Agriculture University of Jember Jember 68121 Indonesia2Center for Development of Advanced Sciences and Technology University of Jember Jember 68121 Indonesia

Correspondence should be addressed to Hardian Susilo Addy hsaddyfapertaunejacid

Received 14 October 2015 Accepted 6 March 2016

Academic Editor Othmane Merah

Copyright copy 2016 Hardian Susilo Addy et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Bacterial wilt disease on banana is an important disease in Lumajang District and causes severe yield loss Utilizing bacteriophageas natural enemy of pathogenic bacteria has been widely known as one of the control strategies This research was aimed atdetermining the causing agent of bacterial wilt on banana isolated from Lumajang area to obtain wide-host range bacteriophagesagainst bacterial wilt pathogen and to know the basic characteristic of bacteriophages particularly its nucleic acid type Causativeagent of bacterial wilt was isolated from symptomatic banana trees from seven districts in Lumajang area on determinative CPGplates followed by rapid detection by PCR technique using specific pair-primer Bacteriophages were also isolated from soil ofinfected banana crop in Sukodono District Morphological observation showed that all bacterial isolates have similar characteristicas common bacterial wilt pathogen Ralstonia solanacearum In addition detection of FliC region in all isolates confirmed thatall isolates were R solanacearum according to the presence of 400 bp of FliC DNA fragment Moreover two bacteriophages wereobtained from this experiment (120601RSSKD1 and 120601RSSKD2) which were able to infect all nine R solanacearum isolates Nucleic acidanalysis showed that the nucleic acid of bacteriophages was DNA (deoxyribonucleic acid)

1 Introduction

The most popular crop cultivated in East Java Indonesia isbanana tree due to existence of about 33 of banana germplasmin this area [1] The production of banana increases annuallyand is affected by environmental factors such as plant disease[2] Bacterial wilt disease caused by Ralstonia solanacearumis one of the most important pathogens on banana plan-tation [3] Nasir et al [4] described that bacterial wilt oninfected banana caused by R solanacearum showed uniquesymptoms as yellowing on affected leaf appearance of bac-terial ooze and rotting on infected-plant parts In additionbacterial colonies on cassaminoacid peptone glucose (CPG)agar medium are fluidal-white but become pink spot onwhite colony while growing onCPG agarmedium containingtriphenyle tetrazolium chloride (TZC) [5] Elphinstone [6]reported that this bacterium causes yield loss about 80 to100 in diseased banana crop On the other hand another

bacterial wilt on banana has also been identified as bananaXanthomonas wilt disease caused by Xanthomonas vasicolapv musacearum with similar morphological symptom asshown in bacterial wilt disease caused byR solanacearum [7]

R solanacearum is soil borne pathogen very diverse in itsspecies and belongs to group of Ralstonia-species complexDue to its diversity in species and strain suitable diagnosticprocedure should be done to identify this pathogen accuratelyincluding molecular diagnostic such as polymerase chainreaction using specific primer [8] In the other hand diag-nosis of pathogen is also possible by using bacteriophage dueto its specificity to infect very narrow bacterial host withinfamily genus or species [9]

Several management techniques have been tried to con-trol bacterial wilt with less effectiveness such as the use ofchemical pesticides resistant plant and culture techniquemethods [10] In addition Addy et al [11] suggested a newmethod to control this pathogen by using bacteriophage

Hindawi Publishing CorporationInternational Journal of AgronomyVolume 2016 Article ID 5164846 7 pageshttpdxdoiorg10115520165164846

2 International Journal of Agronomy

Fujiwara et al [12] successfully utilized bacteriophage tocontrol bacterial wilt on tomato In citrus Ahmad et al[13] also demonstrated that utilization of XacF1 suppressesthe development of citrus canker disease caused by X citriMoreover utilization of bacteriophage to control pathogenic-bacteria has been suggested as a tool in disease managementstrategy called phage therapy [14] besides its utilization asdetection tool for specific bacteria [15]

According to bacterial status determination of pathogenshould be accurately done efficiently and effectively More-over according to benefit of utilization of bacteriophageisolation of bacteriophage should be the first step in allprocedures prior to exploring and use of bacteriophage [11]Therefore the present study was conducted to determinebacterial wilt pathogen isolates of banana in LumajangIndonesia by PCR technique and to isolate virulent bacte-riophage that is able to infect all isolates of bacterial wiltpathogen

2 Method

21 Plant and Soil Sampling Sampling was done in sevendistricts in Lumajang Indonesia such as Sukodono RanduAgung Kedung Jajang Tempeh Tekung Yosowilangun andPasru Jambe (Figure 1) For isolation of bacteriophage soilsample was taken from Sukodono District A hundred gramsof soil sample was collected from 10 to 30 cm deep using pipe(3 cm in diameter) and was put into plastic bag Sample wasstored in 4∘C until further use

22 Isolation Bacterial Wilt Pathogen of Banana Stem piecesof wilted banana plants were disinfected using 70 alcoholsolution and were placed in Petri dish under moist conditionto spur the formation of bacterial ooze on the stem piecessurface Bacterial ooze which appeared on stempiece surfacewas then streaked on the CPG medium containing 001 ofTZC After 48 hours of incubation at 28∘C a fluidal colonywith pink spot on white colony on the plate was selectedas a putative R solanacearum The colony was soluble in3 of KOH solution and was infiltrated into tobacco leaf ashypersensitive response test The single colony of each isolatewas regrown on CPG broth for 24 hours and subjected toDNA extraction and purification and PCR detection followedprotocol of Sambrook et al [16]

23 Detection by Polymerase Chain Reaction To determineand confirm isolates as R solanacearum molecular methodlike PCR-based identification was done by using a FliC pair-primerwhich is specific to amplify the flagellin gene sequencein R solanacearum [17] The primer sequences for FliC-R and FliC-L were (51015840-GAACGCCAACGGTGCGAACT-31015840) and (51015840-GGCGGCCTTCAGGGAGGTC-31015840) respectivelyand the expected PCR product size was 400 bp Briefly PCRcondition was predenaturation at 94∘C for 3 minutes 35cycles of denaturation at 94∘C for 30 seconds annealingat 63∘C for 2min and elongation at 72∘C for 1min Finalextension was done at 72∘C for 3min PCR product wasthen visualized using UVGel Documentation System (Major

Sciences) on 1 of agarose gel electrophoresis stained usingGelGreen (Biotium)

24 Isolation Bacteriophage andNucleic Acid Bacteriophageswere isolated from soil samples collected from Sukodonodistrict according to the method described by Susianto et al[18] Isolated pure bacteriophages were stored in SM buffer at4∘C conditions until future use [11] Phages nucleic acid wasisolated and purified following Susianto et al [18] and wasused for characterization of nucleic acid by nuclease enzymessuch as DNAse and RNAse as described below

25 Host Range Assay of Bacteriophage Host range assaywas done in vitro to determine the lysing ability of eachbacteriophage against R solanacearum The assay was doneas spot testmethod as described by Susianto et al [18] Briefly400 120583L suspensions of R solanacearum isolates (at OD

600

of 1 with 24-hour culture) were mixed with soft agar (03agarose) and poured onto CPG agar plates Once soft agarsolidified a 2 120583L phage suspension (using amicropipette) wasspot-inoculated on the surface of agar and allowed to dry atroom temperature prior to incubation at 28∘C Lysed areas onthe spot inoculations were observed until 48 hours

26 Characterization of Bacteriophage Nucleic Acid Phagesnucleic acid was characterized using DNAse and RNAseby incubating 1 120583g120583L of nucleic acid in 20120583L of reactioncontaining 1 unit ofDNAse I orRNAseAll reactions (mixtureof ddH

2O nucleic acid enzyme buffer and enzyme) were

incubated at 37∘C for 2 hours prior to visualization on agarosegel electrophoresis

3 Results

About 40 of districts in Lumajang have been chosen aslocation for sampling related to the occurrence of banana wiltsymptom such as districts of Yosowilangun Randu AgungTempeh Pasrujambe Tekung Sukodono andKedung JajangAll of the banana trees showed similar symptoms such aswilt-ing (Figure 2(a))with yellowing on fruits (Figure 2(b)) brownand red spots were observed on symptomatic fruits (internalsymptom) when sliced vertically and horizontally (Figures2(c) and 2(d)) In addition the bananarsquos stem also showedsimilar brown and red lines along the stem (Figure 2(e))Interestingly brown-red liquid mass appeared after 48 hourswhile stem section (Figure 2(f)) was incubated under humidcondition (Figure 2(g)) To confirm the causative pathogenof this disease we isolated the agent using determinationmedium (CPG) containing 001 of triphenyl tetrazoliumchloride (TZC) The result showed that white colonies whitewith red spot were grown on the media as bacterial colonies(Figures 3(a) and 3(b))

About nine isolates were collected from 7 locationsand different banana hosts such as variety of Raja (Musatextilia) and variety of Kepok (Musa acuminata balbisiana)All isolates were Gram-negative bacteria according to theKOH solubility test and showed similar reaction while thebacterial suspension was infiltrated onto the tobacco leaves

International Journal of Agronomy 3

Table 1 Bacterial isolates used in this study

No Origin Bananarsquos varietya Isolate code KOH solubility testHypersensitive

response induced intobacco leaves

1 Tempeh Kepok TP1 + +TP2 + +

2 Randu Agung Kepok RA1 + +RA2 + +

3 Pasru Jambe Raja PJ1 + +4 Kedung Jajang Raja KJ2 + +5 Yosowilangun Kepok Yoso + +6 Tekung Raja TKG + +7 Sukodono Kepok SKD + +aVariety of Kepok refers toMusa acuminata variety of Raja refers toMusa textilia

6

lowast1

lowast2

lowast5

lowast43

lowast

lowast

lowast

7

Figure 1 Location of bananarsquos sampling in Lumajang East Java Indonesia Asterisk (lowast) refers to sampling spot area (modified from GoogleMaps) such as Kedung Jajang (1) Pasru Jambe (2) Sukodono (3) Randu Agung (4) Tekung (5) Tempeh (6) and Yosowilangun (7)

(Table 1) Interestingly all isolates have 400 bp fragments afterPCR amplification using specific primer (FliC primer) for Rsolanacearum (Figure 3(c))

Isolation of bacteriophage that is able to infect bacterialwilt pathogen was done by exploring soil sample fromSukodono District and it was found that about two kinds ofphages namely 120601RSSKD1 and 120601RSSKD2 were able to infect

all bacterial isolates However phage-plaques morphologywere different which were turbid and clear plaques (Table 2Figure 4) Furthermore we also analyzed phages nucleic acidtype by treating the purified phages genome with DNAse andRNAse The result showed that all bacteriophages had DNAas their nucleic acid due to it sensitivity to DNAse but not toRNAse (Figure 4)


(a) (b)

(c) (d)

(e) (f)

(g)

Figure 2 Wilt symptom on banana trees (a) bunches (b) fruits that are horizontally sliced (c) fruits that are vertically sliced (d) stems thatare vertically sliced (e) stems that are horizontally sliced (f) and bacterial massooze on the stem slice surface (g)

4 Discussion

Banana in Lumajang area that shows wilting yellowingand drying symptoms was particular symptom of bacterialwilt pathogen infection caused by R solanacearum The

symptom becomes more contrasting while showing rottingin the center area of sliced fruit This observation was similarto Nasir et alrsquos [4] during their observation on bananawilt disease in West Sumatra Province Indonesia Moreconfirmation was also done by observing the morphological


(a) (b)M 1 2 3 4 5 6 7 8 9

925

400

Base pair

(c)

Figure 3Morphological colonies of bacterial wilt pathogen grew around infected stem (a) and after purification of colony onCPG containing001TZC (b)whichwas detected by PCRusing FliC primer (c) LaneM120582StyI Lanes 2ndash9were based on isolate fromSukodono PasrujambeTempeh 1 Kedung Jajang Tekung Yosowilangun Tempeh 2 Randu Agung 1 and Randu Agung 2 respectively

Table 2 Host range of bacteriophage against R solanacearum andits plaque morphology

Isolate codes Sensitivitya Plaque morphology by phages120601RSSKD1 120601RSSKD2

TP1 + Turbid TurbidTP2 + Turbid TurbidRA1 + Clear ClearRA2 + Clear ClearPJ1 + Clear ClearKJ2 + Clear ClearYoso + Clear ClearTKG + Clear ClearSKD + Clear ClearaResults from five independent replications

colonies of the causative agent and showed that all isolateshad similar morphological colonies as described by Feganand Prior [19] Moreover Liestiany et al [20] described thatthe colony of R solanacearum is fluidal and viscous and hasirregular colony edge Using PCR technique all isolates canbe determined using specific pair-primer such as FliC whichwas designated to detect the presence of specific flagellin genefragment on R solanacearum species [17] The FliC primerwas widely used by some researchers to detect and identifyR solanacearum because it is highly specific and sensitive onPCR-based detection system as done by Huang et al [21] who

detectedR solanacearum from soil andKubota et al [22] whodetected R solanacearum isolated from banana

On the other hand we also isolated two bacteriophages120601RSSKD1 and 120601RSSKD2 from Sukodono soil Both phageshave similar ability to infect all bacterial isolates but differ insize of plaque There were two types of bacterial host isolatesrelated to plaque morphology two isolates were given turbidplaques while the remained seven isolates were given clearplaques Interestingly we obtained wide-host bacteriophagesthat are able to infect all isolated bacterial hosts Fujiwara etal [12] demonstrated that all different phages 120601RSA1 120601RSB1and 120601RSL1 were able to infect R solanacearum but withdifferent host range Askora et al [23] also found two typesof phages that have different host Not all of these phagesare able to infect all bacterial strains We suggested that allisolates are the same strain even though it was isolated fromdifferent districts and banana cultivars This suggestion issimilar to Clokie et alrsquos [24] who demonstrated that plaquesappearances may be affected by phages life cycle phages hostand phage type Both phage types have the same nucleicacid type that is DNA due to the sensititivity to DNAseCounis and Torriglia [25] suggested that nucleic acid typewill be DNA if sensitive to DNAse but not to RNAse Thuswe conclude that all isolates of bacterial wilt pathogen are Rsolanacearum and both phages 120601RSSKD1 and 120601RSSKD2 arephages with DNA as their nucleic acid

Identification and determination of the causative agentof the disease is the critical step in the effort to determineappropriate control strategies [26] There are many tactics


(a) (b)

MUndigested

Treatment

DNAse RNAse

1933

774622425347

(kbp)

(c)

Figure 4 Spot assay on CPG medium showing different plaque morphologies which were clear (a) and turbid (b) plaques Phage genomeprofiles which were treateddigested by two nucleases DNAse and RNAse (c)

to control the disease particularly plant disease causedby phytopathogenic bacteria such as the use of biologicalcontrol agents Utilization of bacteriophage as biocontrolagent against phytopathogenic bacteria has been studied andinvestigated both at laboratory scale and on a field scalewhichexposed the strategies in utilizing bacteriophage relatedto the environment condition Fujiwara et al [12] utilizedbacteriophage like 120601RSA1 to treat soil borne pathogensR solanacearum with high efficacy without using specialformulation to phage They directly poured or sprayed phagesuspension into soil whichwas infested by bacterial hostThisprocedure implies that environmental conditions in the soildo not extremely affect stability of bacteriophage comparedto the condition on soil surface or plant part above soilsurface In contrast Iriarte et al [27] demonstrated the use ofbacteriophages 120601XV3-16 and 120601Xacm 2004-16 in formulatedway to combat Xanthomonas that infects tomato leaves Thisindicates that the phage stability and infectivity is influencedby the location of pathogen associated with the affectedplant parts especially those directly exposed to extremeenvironmental factorsThe use of this formula aims to protectbacteriophage from damage due to extreme environmental

conditions such as UV and high temperature [28] resultingin loss of phage infectivity

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] P E Prahardini Yuniarti andA Krismawati ldquoCharacterizationof superior banana of mas kirana and agung semeru varieties inlumajang districtrdquo Buletin PlasmaNutfah vol 16 no 2 pp 126ndash133 2010

[2] J D Van Elsas P Kastelein P M De Vries and L S VanOverbeek ldquoEffects of ecological factors on the survival andphysiology of Ralstonia solanacearum bv 2 in irrigation waterrdquoCanadian Journal of Microbiology vol 47 no 9 pp 842ndash8542001

[3] M F C Ilagan M T M Perez and A K Raymundo ldquoDirectdetection of race 2 Ralstonia solanacearum (Smith) yabuuchi


in banana fruit stalk using polymerase chain reactionrdquo ThePhilippine Agricultural Scientist vol 93 no 3 pp 307ndash316 2010

[4] N Nasir Jumjunidang and Riska ldquoDistribution of Fusariumwilt and bacterial wilt on germplasm location and field schoolof integrated pest management in West Sumatrardquo Jurnal Horti-cultura vol 15 no 3 pp 215ndash222 2005

[5] A Kelman ldquoThe relationship of pathogenicity in Pseudomonassolanacearum to colony appearance on a tetrazolium mediumrdquoPhytopathology vol 44 pp 693ndash695 1954

[6] J G Elphinstone ldquoThe current bacterial wilt situation aglobal overviewrdquo in Bacterial Wilt Disease and the Ralstoniasolanacearum Species Complex C Allen P Prior and A CHayward Eds pp 9ndash28 American Phytopathological SocietyPress St Paul Minn USA 2005

[7] D Ahimelash S T Alemu T Addis F L Turyagyenda and GBlomme ldquoBanana Xanthomonas wilt in Ethiopia occurrenceand insect vector transmissionrdquo African Crop Science Journalvol 16 no 1 pp 75ndash78 2010

[8] S E Seal L A Jackson J PWYoung andM JDaniels ldquoDiffer-entiation of Pseudomonas solanacearum Pseudomonas syzygiiPseudomonas pickettii and the blood disease bacterium bypartial 16S rRNA sequencing-construction of oligonucleotideprimers for sensitive detection by polymerase chain reactionrdquoJournal of General Microbiology vol 139 no 7 pp 1587ndash15941993

[9] S Hagens and M J Loessner ldquoApplication of bacteriophagesfor detection and control of foodborne pathogensrdquo AppliedMicrobiology and Biotechnology vol 76 no 3 pp 513ndash519 2007

[10] Yuliar Y A Nion and K Toyota ldquoRecent trends in con-trol methods for bacterial wilt diseases caused by Ralstoniasolanacearumrdquo Microbes and Environments vol 30 no 1 pp1ndash11 2015

[11] H S Addy A Askora T Kawasaki M Fujie and T YamadaldquoUtilization of filamentous phage 120593RSM3 to control bacterialwilt caused by Ralstonia solanacearumrdquo Plant Disease vol 96no 8 pp 1204ndash1209 2012

[12] A Fujiwara M Fujisawa R Hamasaki T Kawasaki M Fujieand T Yamada ldquoBiocontrol of Ralstonia solanacearum by treat-ment with lytic bacteriophagesrdquo Applied and EnvironmentalMicrobiology vol 77 no 12 pp 4155ndash4162 2011

[13] A A Ahmad A Askora T Kawasaki F Makoto and TYamada ldquoThe filamentous phage XacF1 causes loss of virulencein Xanthomonas axonopodis pv Citri the causative agent ofcitrus canker diseaserdquo Frontier Microbiology vol 5 no 321 pp1ndash14 2014

[14] B Balogh J B Jones F B Iriarte and M T Momol ldquoPhagetherapy for plant disease controlrdquo Current PharmaceuticalBiotechnology vol 11 no 1 pp 48ndash57 2010

[15] N R Dhany H S Addy and W S Wahyuni ldquoThe use of abacteriophage for detector kit of bacterial blight pathogen onsoybeanrdquo Jurnal Fitopatologi Indonesia vol 9 no 4 pp 116ndash1222013

[16] J Sambrook P Maccallum and D Russel Molecular CloningA Laboratory Manual Cold Springs Harbour Press New YorkNY USA 3rd edition 2001

[17] J Schonfeld H Heuer J D van Elsas and K Smalla ldquoSpecificand sensitive detection of Ralstonia solanacearum in soil onthe Basis of PCR amplification of fliC fragmentsrdquo Applied andEnvironmental Microbiology vol 69 no 12 pp 7248ndash72562003

[18] G Susianto M M Farid N R Dhany and H S Addy ldquoHostrange for bacteriophages that infect bacterial blight pathogen

on soybeanrdquo Procedia Environmental Sciences vol 20 pp 760ndash766 2014

[19] M Fegan and P Prior ldquoDiverse members of the Ralstoniasolanacearum species complex cause bacterial wilts of bananardquoAustralasian Plant Pathology vol 35 no 2 pp 93ndash101 2006

[20] E Liestiany E N Fikri and E Susilowati ldquoKemampuanPseudomonas kelompok fluorescens dari Kabupaten Tabalongmenekan pertumbuhanRalstonia solanacearum secara in vitrordquoAgripeat vol 13 no 1 pp 8ndash15 2012

[21] J Huang J Wu C Li C Xiao and G Wang ldquoSpecific andsensitive detection of Ralstonia solanacearum in soil with quan-titative real-time PCR assaysrdquo Journal of Applied Microbiologyvol 107 no 5 pp 1729ndash1739 2009

[22] R Kubota B G Vine AM Alvarez andDM Jenkins ldquoDetec-tion of Ralstonia solanacearum by loop-mediated isothermalamplificationrdquo Phytopathology vol 98 no 9 pp 1045ndash10512008

[23] A Askora T Kawasaki S Usami M Fujie and T YamadaldquoHost recognition and integration of filamentous phage 120601RSMin the phytopathogen Ralstonia solanacearumrdquo Virology vol384 no 1 pp 69ndash76 2009

[24] M R J Clokie A D Millard A V Letarov and S HeaphyldquoPhage in naturerdquo Bacteriophage vol 1 no 1 pp 31ndash45 2011

[25] M-F Counis and A Torriglia ldquoAcid DNases and their interestamong apoptotic endonucleasesrdquo Biochimie vol 88 no 12 pp1851ndash1858 2006

[26] R A Frampton A R Pitman and P C Fineran ldquoAdvances inbacteriophage-mediated control of plant pathogensrdquo Interna-tional Journal of Microbiology vol 2012 Article ID 326452 11pages 2012

[27] F B Iriarte B Balogh M T Momol L M Smith M Wilsonand J B Jones ldquoFactors affecting survival of bacteriophage ontomato leaf surfacesrdquo Applied and Environmental Microbiologyvol 73 no 6 pp 1704ndash1711 2007

[28] J B Jones G E Vallad F B Iriarte et al ldquoConsiderations forusing bacteriophages for plant disease controlrdquo Bacteriophagevol 2 no 4 pp 208ndash214 2012

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Fujiwara et al [12] successfully utilized bacteriophage tocontrol bacterial wilt on tomato In citrus Ahmad et al[13] also demonstrated that utilization of XacF1 suppressesthe development of citrus canker disease caused by X citriMoreover utilization of bacteriophage to control pathogenic-bacteria has been suggested as a tool in disease managementstrategy called phage therapy [14] besides its utilization asdetection tool for specific bacteria [15]

According to bacterial status determination of pathogenshould be accurately done efficiently and effectively More-over according to benefit of utilization of bacteriophageisolation of bacteriophage should be the first step in allprocedures prior to exploring and use of bacteriophage [11]Therefore the present study was conducted to determinebacterial wilt pathogen isolates of banana in LumajangIndonesia by PCR technique and to isolate virulent bacte-riophage that is able to infect all isolates of bacterial wiltpathogen

2 Method

21 Plant and Soil Sampling Sampling was done in sevendistricts in Lumajang Indonesia such as Sukodono RanduAgung Kedung Jajang Tempeh Tekung Yosowilangun andPasru Jambe (Figure 1) For isolation of bacteriophage soilsample was taken from Sukodono District A hundred gramsof soil sample was collected from 10 to 30 cm deep using pipe(3 cm in diameter) and was put into plastic bag Sample wasstored in 4∘C until further use

22 Isolation Bacterial Wilt Pathogen of Banana Stem piecesof wilted banana plants were disinfected using 70 alcoholsolution and were placed in Petri dish under moist conditionto spur the formation of bacterial ooze on the stem piecessurface Bacterial ooze which appeared on stempiece surfacewas then streaked on the CPG medium containing 001 ofTZC After 48 hours of incubation at 28∘C a fluidal colonywith pink spot on white colony on the plate was selectedas a putative R solanacearum The colony was soluble in3 of KOH solution and was infiltrated into tobacco leaf ashypersensitive response test The single colony of each isolatewas regrown on CPG broth for 24 hours and subjected toDNA extraction and purification and PCR detection followedprotocol of Sambrook et al [16]

23 Detection by Polymerase Chain Reaction To determineand confirm isolates as R solanacearum molecular methodlike PCR-based identification was done by using a FliC pair-primerwhich is specific to amplify the flagellin gene sequencein R solanacearum [17] The primer sequences for FliC-R and FliC-L were (51015840-GAACGCCAACGGTGCGAACT-31015840) and (51015840-GGCGGCCTTCAGGGAGGTC-31015840) respectivelyand the expected PCR product size was 400 bp Briefly PCRcondition was predenaturation at 94∘C for 3 minutes 35cycles of denaturation at 94∘C for 30 seconds annealingat 63∘C for 2min and elongation at 72∘C for 1min Finalextension was done at 72∘C for 3min PCR product wasthen visualized using UVGel Documentation System (Major

Sciences) on 1 of agarose gel electrophoresis stained usingGelGreen (Biotium)

24 Isolation Bacteriophage andNucleic Acid Bacteriophageswere isolated from soil samples collected from Sukodonodistrict according to the method described by Susianto et al[18] Isolated pure bacteriophages were stored in SM buffer at4∘C conditions until future use [11] Phages nucleic acid wasisolated and purified following Susianto et al [18] and wasused for characterization of nucleic acid by nuclease enzymessuch as DNAse and RNAse as described below

25 Host Range Assay of Bacteriophage Host range assaywas done in vitro to determine the lysing ability of eachbacteriophage against R solanacearum The assay was doneas spot testmethod as described by Susianto et al [18] Briefly400 120583L suspensions of R solanacearum isolates (at OD

600

of 1 with 24-hour culture) were mixed with soft agar (03agarose) and poured onto CPG agar plates Once soft agarsolidified a 2 120583L phage suspension (using amicropipette) wasspot-inoculated on the surface of agar and allowed to dry atroom temperature prior to incubation at 28∘C Lysed areas onthe spot inoculations were observed until 48 hours

26 Characterization of Bacteriophage Nucleic Acid Phagesnucleic acid was characterized using DNAse and RNAseby incubating 1 120583g120583L of nucleic acid in 20120583L of reactioncontaining 1 unit ofDNAse I orRNAseAll reactions (mixtureof ddH

2O nucleic acid enzyme buffer and enzyme) were

incubated at 37∘C for 2 hours prior to visualization on agarosegel electrophoresis

3 Results

About 40 of districts in Lumajang have been chosen aslocation for sampling related to the occurrence of banana wiltsymptom such as districts of Yosowilangun Randu AgungTempeh Pasrujambe Tekung Sukodono andKedung JajangAll of the banana trees showed similar symptoms such aswilt-ing (Figure 2(a))with yellowing on fruits (Figure 2(b)) brownand red spots were observed on symptomatic fruits (internalsymptom) when sliced vertically and horizontally (Figures2(c) and 2(d)) In addition the bananarsquos stem also showedsimilar brown and red lines along the stem (Figure 2(e))Interestingly brown-red liquid mass appeared after 48 hourswhile stem section (Figure 2(f)) was incubated under humidcondition (Figure 2(g)) To confirm the causative pathogenof this disease we isolated the agent using determinationmedium (CPG) containing 001 of triphenyl tetrazoliumchloride (TZC) The result showed that white colonies whitewith red spot were grown on the media as bacterial colonies(Figures 3(a) and 3(b))

About nine isolates were collected from 7 locationsand different banana hosts such as variety of Raja (Musatextilia) and variety of Kepok (Musa acuminata balbisiana)All isolates were Gram-negative bacteria according to theKOH solubility test and showed similar reaction while thebacterial suspension was infiltrated onto the tobacco leaves








6

lowast1

lowast2

lowast5

lowast43

lowast

lowast

lowast

7






(a) (b)

(c) (d)

(e) (f)

(g)


4 Discussion




(a) (b)M 1 2 3 4 5 6 7 8 9

925

400

Base pair

(c)










(a) (b)

MUndigested

Treatment

DNAse RNAse

1933

774622425347

(kbp)

(c)




Competing Interests


References


































Journal of




Agronomy





Microbiology




Volume 2014































6

lowast1

lowast2

lowast5

lowast43

lowast

lowast

lowast

7






(a) (b)

(c) (d)

(e) (f)

(g)


4 Discussion




(a) (b)M 1 2 3 4 5 6 7 8 9

925

400

Base pair

(c)










(a) (b)

MUndigested

Treatment

DNAse RNAse

1933

774622425347

(kbp)

(c)




Competing Interests


References


































Journal of




Agronomy





Microbiology




Volume 2014

























(a) (b)

(c) (d)

(e) (f)

(g)


4 Discussion




(a) (b)M 1 2 3 4 5 6 7 8 9

925

400

Base pair

(c)










(a) (b)

MUndigested

Treatment

DNAse RNAse

1933

774622425347

(kbp)

(c)




Competing Interests


References


































Journal of




Agronomy





Microbiology




Volume 2014

























(a) (b)M 1 2 3 4 5 6 7 8 9

925

400

Base pair

(c)










(a) (b)

MUndigested

Treatment

DNAse RNAse

1933

774622425347

(kbp)

(c)




Competing Interests


References


































Journal of




Agronomy





Microbiology




Volume 2014

























(a) (b)

MUndigested

Treatment

DNAse RNAse

1933

774622425347

(kbp)

(c)




Competing Interests


References


































Journal of




Agronomy





Microbiology




Volume 2014






















































Journal of




Agronomy





Microbiology




Volume 2014
























Research Article Detection of Bacterial Wilt Pathogen and...

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