Identifcation and Characterization of Pectolytic Bacteria which is Causing Soft Rot in some Fruits and Vegetables

Yıl 2017, Cilt: 7 Sayı: 1, 155 - 161, 31.03.2017

Fatih Dadaşoğlu , Recep Kotan

Öz

In this study, it is aimed to be determined make a defnitive diagnosis of Pectobacterium caratovorum
subsp. atrosepticum and Erwinia chrysanthemi, which are the agent of Soft Rot for some fruits and vegetables. For
this purpose, initially microbial diagnostic systems (MIS) are defned using, causing soft rot strains in different
fruits and vegetables, and 7 different strains of P. carotovorum subsp. atrosepticum and 5 different strains of E.
chrysanthemi were obtained. Also identifed using the system of BIOLOG and it was observed that the results
are different from results obtained in the MIS system. According to hypersensitive reaction (HR), pathogenicity
and pectolytic activity tests results it was determined that all of the pathogenic strains. Several biochemical and
molecular tests (16S rDNA PCR and spesifc PCR) for the diagnosis and characterization of certain strains were
made identifed as pathogen. The diagnosis of bacterial species conducted using the 16S rDNA PCR and all of the
strains given band of approximately 1500 bp in length was determined. P. carotovorum subsp. atrosepticum strains
of identifed as pathogen in strawberry, mulberry, parsley, cabbage, potatoes, peppers and eggplant and the strains
of E. chrysanthemi; 2 of them in onion, 2 of them in pepper and one of them also in parsley, it has been found to
cause soft rot disease.

Anahtar Kelimeler

MIS, Molecular identifcation, Pectobacterium, Soft rot

Bazı Sebze ve Meyvelerde Yumuşak Çürüklük Oluşturan Pektolitik Bakterilerin Tanı ve Karakterizasyonu

Öz

Bu çalışmada meyve ve sebzelerde yumuşak çürüklük hastalığına sebep olan Pectobacterium caratovorum
subsp. atrosepticum ve Erwinia chrysanthemi türlerinin klasik ve moleküler yöntemler kullanılarak kesin tanısının
yapılması hedeflenmiştir. Bu amaçla farklı meyve ve sebzelerde yumuşak çürüklük etmeni olduğu tespit edilen
izolatlar ilk olarak mikrobiyal tanı sistemi (MIS) kullanılarak tanılanmış ve 7 farklı P. c. subsp. atrosepticum
izolatı ile 5 farklı E. chrysanthemi izolatı elde edilmiştir. Ayrıca, strainler yine BIOLOG sistemi kullanılarak da
tanılanmış olup elde edilen sonuçların MIS sisteminde alınan sonuçlardan farklı olduğu gözlemlenmiştir. Yapılan
hypersensitive reaction (HR), patojenite ve pektolitik aktivite testleri sonucu bu strainlerin tamamının patojen
olduğu belirlenmiştir. Patojen olduğu belirlenen strainlerin kesin tanı ve karakterizasyonu için çeşitli biyokimyasal
ve moleküler testler (16S rDNA PCR ve spesifk PCR) yapılmıştır. 16S rDNA PCR testleri sonucunda strainlerin
cins düzeyinde tanısı yapılmış olup, izolatların tamamının yaklaşık 1500 bp uzunluğunda bant verdiği tespit
edilmiştir. Patojen olduğu belirlenen ve tanılanan P. carotovorum subsp. atrosepticum izolatlarının çilek, dut,
maydanoz, lahana, patates, biber ve patlıcanda, E. chrysanthemi izolatlarının ise; 2 tanesinin soğanda, 2 tanesinin
biberde ve 1 tanesinin de maydanozda yumuşak çürüklük hastalığına neden olduğu belirlenmiştir.

Anahtar Kelimeler

MIS, Moleküler tanı, Pectobacterium, Yumuşak çürüklük

Kaynakça

• Arda M, 1995. Biyoteknoloji (Bazı Temel İlkeler), Kükem Derneği Bilimsel Yayınları, Ankara, 432s.
• Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, 1994. Current protocols in molecular biology. John Wiley and Sons, New York. 2.0.1–2.14.8
• Aysan Y, Karatas A, Cinar O, 2003. Biological control of bacterial stem rot caused by Erwinia chrysanthemi on tomato. Crop Protection, 22 :6, 807-811.
• Boyraz N, Bastas KK, Maden S, Yasar A, 2006. Bacterial leaf and peduncle soft rot caused by Pectobacterium carotovorum on Tulips in Konya, Turkey. Phytoparasitica, 34:3, 272-280.
• Brenner JD, Kieg NR, Garrity MG, 2007. Bergey's Manual of Systematic Bacteriology. 2nd ed p, 721-733.
• Choi O, Kim J, 2013. Pectobacterium carotovorum subsp. brasiliense Causing Soft Rot on Paprika in Korea. Journal of Phytopathology, 161:2, 125-132.
• Dadaşoğlu F, 2007. Isolation and identification of bacterial strains with insecticidal activities against greenhouse and field pest. Atatürk University, Graduate School of Natural and Applied Science Institute, Department of Plant Protection, Master Thesis, 93 p.
• Dickey RS, Kelman A, 1988. Erwinia: "Carotovora" or soft rot group. In: Laboratory Guide for Identification of plant pathogenic bacteria.p. Schaad, N.W. 2 ed. St. Paul: APS. pp, 44-59.
• Gallelli A, Galli M, De Simone D, Zaccardelli M, Loreti S, 2009. Phenotypic and Genetic Varıability of Pectobacterium carotovorum Isolated from Artichoke in the Sele Valley. Journal of Plant Pathology, 91:3, 757-761.
• Hugh R, Leifson E, 1953. The taxonomic significance of fermentative vs. Oxidative metabolism of carbohydrates by various Gram negative bacteria. J. Bact. 66: 24.
• Kang HW, Kwon SW, Go SJ, 2003. PCR-based specific and sensitive detection of Pectobacterium carotovorum ssp. carotovorum by primers generated from a URP-PCR fingerprinting-derived polymorphic band. Plant pathol 52: 127-133.
• Khoodoo MHR, Juafeerally-Fakim Y. 2004. RAPD-PCR fingerprinting and Southern analysis of Xanthomonas axonopodis pv. dieffenbachiae strains isolated from different aroid hosts and locations, Plant Dis. 88: 980–988.
• Kotan R, 2002. Doğu Anadolu Bölgesi’nde yetiştirilen yumuşak çekirdekli meyve ağaçlarından izole edilen patojen ve saprofitik bakteriyel organizmaların klasik ve moleküler metodlar ile tanısı ve biyolojik mücadele imkânlarının araştırılması. Atatürk Üniversitesi, Fen Bilimleri Enstitüsü, Doktora Tezi, 217s.
• Lazo GR, Gabriel DW, 1987. Conservation of plasmid DNA sequences and pathovar identification of strains of Xanthomonas campestris. Phytopathology 77: 448-453.
• Lelliott RA, Dickey RS, 1984. Genus VII. Erwinia. In: Bergey's manual of systematic bacteriology, Williams and Wilkins, Baltimore, USA. pp. 469-476.
• Maisuria VB, Nerurkar AS, 2012. Characterisation and differantiation of soft rot causing Pectobacterium carotovorum of Indian origin. Eur. J. Plant Pathol. 136:1, 87-102.
• Mikicinski A, Sobiczewski P, Sulikowska M, Pulawska J, Treder J, 2010. Pectolytic bacteria associated with soft rot of calla lily (Zantedeschia spp.) tubers. Journal of Phytopathology 158: 201-209.
• Miller LT, 1982. Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids. J. Clin. Microbiol, 16: 584-586
• Miller I, Berger T, 1985. Bacteria identification by gas chromatography of whole cell fatty acids. Hewlett Packard Gas Chromatography Application Note, Hewlett Packard Co., Alto, CA, 228-238.
• Ngadze E, Brady CL, Coutinho TA, Van Der Waals JE, 2012. Pectinolytic bacteria associated with potato soft rot and blackleg in South Africa and Zimbabwe. European Journal of Plant Pathology, 134:533–549.
• Palacio-Bielsa J, Cambra MA, Lopez MM, 2006. Characterisation of potato isolates of Dickeya chrysanthemi in Spain by a microtitre system for biovar determination. Annals of Applied Biology. 148: 157-164.
• Perombelon MCM, Kelman A, 1980. Ecology of the Soft Rot Erwinias. Ann. Rev. Phytopathol, 18: 361-387.
• Pitman AR, Harrow SA, Visnovsky SB, 2010. Genetic characterisation of Pectobacterium wasabiae causing soft rot disease of potato in New Zealand. Eur J Plant Pathol. 126: 423–435.
• Rahman MM, Eaqub Ali M, Khan AA, Hashim U, Akanda AM, Hakim MA, 2012. Characterization and identification of soft rot bacterial pathogens in Bangladeshi potatoes. African Journal of Microbiology Research, 6:7, 1437-1445.
• Rattanasuk S, Ketudat-Cairns M, 2009. Chryseobacterium indologenes, novel mannanase producing bacteria, Songklanakarin. J. of Sci and Tech. 31:4, 395-399
• Santana MA, Rodrıguez M, Matehus J, Faks J, Bocsanczy A, Gerstl A, Romay G, Montılla J, Fernande CE, Moreno Zambrano N, Marval D, 2012. A New Bacterial Disease of Cassava in Venezuela Caused by Enterobacter cloacae. International Journal of Agriculture Biology, 14:2, 183-189.
• Sarris PF, Trantas E, Pagoulatou M, Stavrou D, Ververidis F, Goumas DE, 2011. First report of potato blackleg caused by biovar 3 Dickeya sp. (Pectobacterium chrysanthemi) in Greece. New Disease Reports, 24, 21.
• Saygılı H, Aysan Y, Sahin F, Mirik M, Üstün N, 2005. Phenotypic characterization of tomato pith necrosis pathogens in Turkey. APS Annual Meeting, July 30-Agust 3, Austin, Texas, USA.
• Schochetman G, Ou CY, Jones WK, 1988. Polymerase chain reaction. J Infect Dis;158: 1154-7.
• Suparyono Pataky, JK, 1989. Influence of host resistance and growth stage at the time of inoculation on Stewart's wilt and Goss' wilt development and sweetcorn hybrid yield. Plant Disease, 73: 339-345.
• Tavasoli E, Marafat AR, Hassazadah N, 2011. Identity and genetic diversity of pectobacterium spp., causal agents of potato soft rot in Zanjan,Iran. Africa journal of plant science, 5:6, 329-336.
• Van Der Merwe JJ, Coutinho TA, Korsten L, Van Der Waals JE, 2010. Pectobacterium carotovorum subsp. brasiliensis causing blackleg on potatoes in South Africa. Eur J Plant Pathol 126: 175– 185.
• Van Vaerenbergh J, Baeyen S, De Vos P, Maes M, 2012. Sequence Diversity in the Dickeya fliC Gene: Phylogeny of the Dickeya Genus and TaqMan® PCR for 'D. solani', New Biovar 3 Variant on Potato in Europe. PLoS ONE 7:5, e 35738.