Isolation and Identification of Bacterial Soft Rot Disease Agent Pectobacterium Isolates on Potato Tubers Delivered for Consumption in Yozgat and Kırşehir Provinces

Yıl 2022, , 332 - 342, 31.05.2022

Murat Ozturk , Soner Soylu

https://doi.org/10.33462/jotaf.974350

Cited By: 3

Öz

Pectobacterium spp. are ubiquitous bacterial plant pathogens with a wide host range which cause soft rot disease on the invaded plant tissue. This study aimed to isolate the bacterial soft rot disease agents belonging to Pectobacterium spp. on potato tubers delivered for consumption in the local bazaar, grocery and markets in the Center district of Yozgat province and Kaman district of Kırşehir province of Turkey between 2018 and 2019. Local bazaar, market and grocery stores were surveyed and 168 potato tubers were collected. The humidity cell test was performed to investigate the presence of pectinolytic bacteria in asymptomatic and soft rotted potato tubers. Bacterial isolation from 54 different soft rotted tuber slices was performed on Crystal Violet Pectate (CVP) medium. A total of 42 bacterial isolates, that formed pits on CVP medium, were obtained following isolation. Among these, 27 isolates were observed to have shiny, transparent, irregular, crater-shaped colony morphology on Nutrient Agar (NA) plates. All isolates were gram and oxidase negative, catalase-positive, facultative anaerobe, non-fluorescent morphological characteristics and caused soft rot on potato tuber slices. Following PCR analysis, 19 of 54 symptomatic tuber samples were determined as infected by P. carotovorum subsp. carotovorum (9 isolates), P. atrosepticum (6 isolates) and Pectobacterium spp. (4 isolates). In 8 of 114 asymptomatic tuber samples, P. carotovorum subsp. carotovorum (5 isolates), P. atrosepticum (2 isolates) and P. carotovorum subsp. brasiliense (1 isolate) were determined as responsible agents of latent infections. The presence of Pectobacterium isolates in the stored potato tubers brought for consumption suggests the pathogen can be transferred to different regions. Potato tubers offered for sale for consumption are thought to be an important inoculum source for the transfer of Pectobacterium isolates to disease-free areas.

Anahtar Kelimeler

Pectobacterium spp., Soft Rot, Latently Infected Tuber, Potato, PCR

Proje Numarası

6602c-ZF/18-237

Yozgat ve Kırşehir İllerinde Tüketime Sunulmuş Patates Yumrularında Bakteriyel Yumuşak Çürüklük Hastalığı Etmeni Pectobacterium İzolatlarının İzolasyonu ve Tanılanması

Öz

Pectobacterium spp. geniş bir konukçu yelpazesine sahip, her ortamda bulunan bakteriyel bitki patojenlerden biri olup, istila ettiği bitki dokusunda Bakteriyel Yumuşak Çürüklük olarak bilinen hastalığa neden olur. Bu çalışmada 2018-2019 yılları arasında Yozgat Merkez ve Kırşehir Kaman ilçesindeki yerel pazar, manav ve zincir marketlerde satışa sunulan patates yumrularında bakteriyel yumuşak çürüklük hastalığına neden olan Pectobacterium spp. izolasyonu, biyokimyasal, patojenisite ve moleküler yöntemlerle tanılanması amaçlanmıştır. Survey yapılan market ve manavlardan 168 yumru örneği toplanmıştır. Tipik yumuşak çürüklük belirtisi gösteren ve göstermeyen şüpheli yumrularda pektolitik izolatların varlığının araştırılması amacıyla nemli hücre testi yapılmıştır. Çürüme belirtileri gösteren 54 farklı yumru kesitlerinden pektolitik izolatların izolasyonu Kristal Violet Pektat (CVP) besiyeri üzerinde yapılmıştır. CVP besi yerinde çukur oluşturan pektolitik koloniler NA besi yerine saflaştırılması ile toplam 42 adet bakteri izolatı elde edilmiştir. Elde edilenler bakteri izolatları arasında 27 farklı izolatın Nutrient Agar (NA) besi yerinde şeffaf, parlak, düzensiz, krater formda koloni oluşturduğu belirlenmiştir İzolatların tamamı gram ve oksidaz negatif, katalaz pozitif, fakültatif anaerob, floresen olmayan koloni morfolojisine sahip özelliklerde sergilemiş ve patates dilimi üzerinde yumuşak çürüklüğe neden olmuştur. Elde edilen izolatlar ile yapılan PCR çalışmasında, 54 simptomlu yumru örneğinin 19 adedinde P. carotovorum subsp. carotovorum (9 izolat), P. atrosepticum (6 izolat) ve Pectobacterium spp. (4 izolat) enfeksiyona neden olan etmenler olarak tanılanmıştır. Tipik hastalık belirtisi göstermeyen 114 yumru örneğinin 8 adedinde P. carotovorum subsp. carotovorum (5 izolat), P. atrosepticum (2 izolat) ve P. carotovorum subsp. brasiliense (1 izolat) etmenleri latent enfeksiyon sorumlusu olarak belirlenmiştir. Bu çalışma ile tüketime yönelik olarak satışa sunulmuş patates yumrularında Pectobacterium izolatlarının varlığı belirlenerek patojenin farklı bölgelere transfer edilebileceği belirlenmiştir. Tüketim için satışa sunulan patates yumrularının hastalıktan ari bölgelere Pectobacterium izolatlarının nakledilmesinde önemli bir inokulum kaynağı olduğu düşünülmektedir.

Anahtar Kelimeler

Pectobacterium spp., Yumuşak Çürüklük, Latent Enfekteli Yumru, Patates, PCR

Destekleyen Kurum

Yozgat Bozok Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi

Proje Numarası

6602c-ZF/18-237

Teşekkür

Bu çalışma, Yozgat Bozok Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi 6602c-ZF/18-237 nolu proje bütçesinden desteklenmiştir.

Kaynakça

• Adeolu, M., Alnajar, S., Naushad, S., & Gupta, R. S. (2016). Genome-based phylogeny and taxonomy of the ‘Enterobacteriales’: proposal for Enterobacterales ord. nov. divided into the families Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov. International Journal of Systematic and Evolutionary Microbiology, 66(12): 5575-5599.
• Agrios, GN. (2005). Plant diseases caused by prokaryotes. Bacteria and Mollicutes. Plant Pathology. 5th ed.
• Aksoy HM, Kaya Y, Ozturk M, Secgin Z, Onder H, Okumus A 2017. Pseudomonas putida–Induced response in phenolic profile of tomato seedlings (Solanum lycopersicum L.) infected by Clavibacter michiganensis subsp. michiganensis. Biological Control, 105: 6-12.
• Anonim, (2020). Tarımsal ekonomi ve politika geliştirme enstitüsü (TEPGE), ürün raporu, patates, 2020. https://arastirma.tarimorman.gov.tr/tepge/Belgeler/PDF%20%C3%9Cr%C3%BCn%20Raporlar%C4%B1/2020%20%C3%9Cr%C3%BCn%20Raporlar%C4%B1/Patates%20%C3%9Cr%C3%BCn%20Raporu%202020-326%20TEPGE.pdf.
• Arıoğlu, H., Çalışkan, M.E., Onaran H. 2010. Türkiye’de Patates Üretimi, Sorunları ve Çözüm Önerileri. IV. Ulusal Patates Kongresi, Niğde. https://link.springer.com/content/pdf/10.1007%2F978-3-030-28683-5.pdf
• Aysan, Y., Mirik, M., Saygili, H., Sahin, F. (2004). New symptoms of tomato soft rot diseases in Turkey. In: I International Symposium on Tomato Diseases 695 (pp. 291-294).
• Basım, H., Basım, E., Baki, D., Turgut, A. (2019). Wet rot disease of banana (Musa sp.) caused by Pectobacterium carotovorum subsp. carotovorum in Turkey. Canadian Journal of Plant Pathology, 41(2): 174-187.
• Baştaş, K.K., Hekimhan, H., Maden, S., Tör, M. (2009). First report of bacterial stalk and head rot disease caused by Pectobacterium atrosepticum on sunflower in Turkey. Plant Disease 93(12): 1352-1352.
• Benlioğlu K., 1991. Bolu, Nevşehir ve Niğde illerinde patates üretim alanlarında Erwinia spp.’nin yaygınlık oranları, tanılanması ve inokulum kaynakları üzerinde araştırmalar. Ege Üniversitesi, Fen Bilimleri Enstitüsü, Doktora tezi, İzmir
• Benlioğlu, K,, Öktem Y.E., Özakman M., 1991. Bacterial diseases of potatoes in the major potato‐growing areas in Turkey. EPPO Bulletin 21(1): 67-72.
• Boyraz, N., Bastas K.K., 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.
• Bozkurt, İ.A., Soylu, S. (2019). Elma kök uru hastalığı etmeni Rhizobium radiobacter’e karşı epifit ve endofit bakteri izolatlarının antagonistik potansiyellerinin belirlenmesi. Tekirdağ Ziraat Fakültesi Dergisi, 16:348-361.
• Cetinkaya-Yildiz R, Mirik M, Aysan Y, Kusek M, Sahin F. (2004). An outbreak of bacterial stem rot of Dieffenbachia amoena caused by Erwinia carotovora subsp. carotovora in the Eastern Mediterranean Region of Turkey. Plant Disease 88(3): 310-310.
• Cigna, J., Dewaegeneire, P., Beury, A., Gobert, V., Faure, D. (2017). A gapA PCR-sequencing assay for identifying the Dickeya and Pectobacterium potato pathogens. Plant disease, 101(7): 1278-1282.
• Charkowski, A., Sharma, K., Parker, M. L., Secor, G. A., Elphinstone, J. (2020). Bacterial diseases of potato. The potato crop, 351-388.
• Czajkowski, R., Perombelon, M. C., van Veen, J. A., van der Wolf, J. M. (2011). Control of blackleg and tuber soft rot of potato caused by Pectobacterium and Dickeya species: a review. Plant pathology, 60(6): 999-1013.
• Czajkowski, R., De Boer, W. J., Van der Zouwen, P. S., Kastelein, P., Jafra, S., De Haan, E. G., Van der Wolf, J. M. (2013). Virulence of ‘Dickeya solani’and Dickeya dianthicola biovar‐1 and‐7 strains on potato (Solanum tuberosum). Plant Pathology, 62(3): 597-610.
• Czajkowski, R., Pérombelon, M. C. M., Jafra, S., Lojkowska, E., Potrykus, M., Van Der Wolf, J. M., Sledz, W. (2015). Detection, identification and differentiation of Pectobacterium and Dickeya species causing potato blackleg and tuber soft rot: a review. Annals of Applied Biology, 166(1): 18-38.
• De Boer, S.H., Li, X., Ward, L.J. (2012). Pectobacterium spp. associated with bacterial stem rot syndrome of potato in Canada. Phytopathology, 102(10): 937-947.
• De Haan, E.G., Dekker-Nooren, T.C., van den Bovenkamp, G.W., Speksnijder, A.G., van der Zouwen, P.S., van der Wolf, J.M. (2008). Pectobacterium carotovorum subsp. carotovorum can cause potato blackleg in temperate climates. European Journal of Plant Pathology, 122(4): 561-569.
• Dees, M.W., Lebecka, R., Perminow, J. I. S., Czajkowski, R., Motyka, A., Zoledowska, S., Brurberg, M. B. (2017). Characterization of Dickeya and Pectobacterium strains obtained from diseased potato plants in different climatic conditions of Norway and Poland. European Journal of Plant Pathology, 148(4): 839-851.
• Duarte, V., De Boer, S. H., Ward, L. J., De Oliveira, A. M. R. (2004). Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil. Journal of applied microbiology, 96(3): 535-545.
• Du Raan, S., Coutinho, T. A., Van der Waals, J. E. (2016). Cardinal temperature differences, determined in vitro, between closely related species and subspecies of pectinolytic bacteria responsible for blackleg and soft rot on potatoes. European Journal of Plant Pathology, 144(2): 361-369.
• Frechon, D., Exbrayat, P., Helias, V., Hyman, L.J., Jouan B, Llop, P., Lopez, M.M., Payet N., Perombelon, M.C.M., Toth, I.K., van Beckhoven, J.R.C.M., van der Wolf JM, Bertheau Y 1998. Evaluation of a PCR kit for the detection of Erwinia carotovora subsp. atroseptica on potato tubers. Potato Research 41(2): 163-173.
• Gardan, L., Gouy, C., Christen, R., Samson, R. (2003). Elevation of three subspecies of Pectobacterium carotovorum to species level: Pectobacterium atrosepticum sp. nov., Pectobacterium betavasculorum sp. nov. and Pectobacterium wasabiae sp. nov. International Journal of Systematic and Evolutionary Microbiology, 53(2): 381-391.
• Kang, H.W., Kwon, S.W., Go, S.J. (2003). PCR‐based specific and sensitive detection of Pectobacterium carotovorum ssp. carotovorum by primers generated from a URP‐PCR fingerprinting‐derived polymorphic band. Plant pathology, 52(2): 127-133.
• Khayi, S., Cigna, J., Chong, T.M., Quêtu-Laurent, A., Chan, K.G., Hélias, V., Faure, D. (2016). Transfer of the potato plant isolates of Pectobacterium wasabiae to Pectobacterium parmentieri sp. nov. International Journal of Systematic and Evolutionary Microbiology, 66(12): 5379-5383.
• Kılıç M., Mirik, M, Aysan Y. (2012). Süs bitkilerinde yumuşak çürüklük etmeni Erwinia türleri ve alttürlerinin moleküler tanısı. Ç.Ü Fen ve Mühendislik Bilimleri Dergisi, 27:77-91.
• Klement, Z. (1990). Tobacco (HR) test for the quick demonstration of pathogenicity. Methods in Phytobacteriology. Klement, Z., Rudolph, K., and Sands, DC, eds. Akadémiai Kiadó, Budapest, 101-102.
• Lelliott, R.A., Stead, D.E. (1987). Methods for the diagnosis of bacterial diseases of plants. Blackwell Scientific Publications.
• Łojkowska, E., Kelman, A. (1994). Comparison of the effectiveness of different methods of screening for bacterial soft rot resistance of potato tubers. American Potato Journal, 71(2): 99-113.
• Ma, B., Hibbing, M. E., Kim, H. S., Reedy, R. M., Yedidia, I., Breuer, J., Charkowski, A. O. (2007). Host range and molecular phylogenies of the soft rot enterobacterial genera Pectobacterium and Dickeya. Phytopathology, 97(9): 1150-1163.
• Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, P., Foster, G.D. (2012). Top 10 plant pathogenic bacteria in molecular plant pathology. Molecular plant pathology, 13(6): 614-629.
• Motyka-Pomagruk, A., Zoledowska, S., Sledz, W., Lojkowska, E. (2021). The occurrence of bacteria from different species of Pectobacteriaceae on seed potato plantations in Poland. European Journal of Plant Pathology, 159(2): 309-325.
• Nabhan, S., De Boer, S. H., Maiss, E., Wydra, K. (2012a). Taxonomic relatedness between Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum subsp. odoriferum and Pectobacterium carotovorum subsp. brasiliense subsp. nov. Journal of Applied Microbiology, 113(4): 904-913.
• Nabhan, S., Wydra, K., Linde, M., Debener, T. (2012b). The use of two complementary DNA assays, AFLP and MLSA, for epidemic and phylogenetic studies of pectolytic enterobacterial strains with focus on the heterogeneous species Pectobacterium carotovorum. Plant Pathology, 61(3): 498-508.
• Nabhan, S., De Boer, S. H., Maiss, E., Wydra, K. (2013). Pectobacterium aroidearum sp. nov., a soft rot pathogen with preference for monocotyledonous plants. International journal of systematic and evolutionary microbiology, 63(7): 2520-2525.
• Öner, E. K., & Aytaç, S. (2016). Bafra koşullarında turfanda patates (Solanum tuberosum L.)'te Dikim zamanları ve yumru ön uygulamalarının verim ve verim kriterlerine etkisi. Ordu Üniversitesi Bilim ve Teknoloji Dergisi, 6(2), 184-194.
• Ozturk, M., Aksoy, H.M., Ozturk, S., Potrykus, M., Lojkowska, E. (2016). First report of potato blackleg and soft rot caused by Pectobacterium wasabiae in Turkey. New Disease Reports 34(17): 2044-0588.
• Öztürk 2017. Orta Karadeniz bölgesinde patateste sorun olan Pectobacterium ve Dickeya spp. bakteriyel etmenleri üzerine araştırmalar. Ondokuz Mayıs Üniversitesi, Fen Bilimleri Enstitüsü, Doktora Tezi, Samsun.
• Ozturk, M., Aksoy, H. M., Potrykus, M., Lojkowska, E. (2018). Genotypic and phenotypic variability of Pectobacterium strains causing blackleg and soft rot on potato in Turkey. European Journal of Plant Pathology, 152(1): 143-155.
• Oztürk, M., Eroğlu, Z., Soylu S. (2019). First report of Pectobacterium betavasculorum associated with bacterial vascular necrosis and root rot disease of sugar beet in Turkey. New Disease Report, 39: 20
• Öztürk, M., Soylu, S. (2022). Yozgat ili beyaz baş lahana üretim alanlarında bakteriyel yumuşak çürüklük hastalığına neden olan Pectobacterium izolatlarının tanılanması. KSÜ Tarım ve Doğa Dergisi, 25 (3):495-503.
• Öztürk, M. (2022). First report of Pectobacterium polaris causing blackleg disease on potato plants in Turkey. Journal of Plant Pathology : (in press) https://doi.org/10.1007/s42161-022-01111-4.
• Panda, P., Fiers, M. A. W. J., Armstrong, K., & Pitman, A. R. (2012). First report of blackleg and soft rot of potato caused by Pectobacterium carotovorum subsp. brasiliensis in New Zealand. New Disase Report, 26(15): 2044-0588.
• Pasanen, M., Laurila, J., Brader, G., Palva, E. T., Ahola, V., Van Der Wolf, J., Pirhonen, M. (2013). Characterisation of Pectobacterium wasabiae and Pectobacterium carotovorum subsp. carotovorum isolates from diseased potato plants in Finland. Annals of applied biology, 163(3): 403-419.
• Pérombelon, M.C.M. (1992). Potato blackleg: epidemiology, host-pathogen interaction and control. Netherlands Journal of Plant Pathology, 98(2): 135-146.
• Pérombelon, M.C.M. (2002). Potato diseases caused by soft rot erwinias: an overview of pathogenesis. Plant pathology, 51(1): 1-12.
• Pitman, A. R., Wright, P. J., Galbraith, M. D., Harrow, S. A. (2008). Biochemical and genetic diversity of pectolytic enterobacteria causing soft rot disease of potatoes in New Zealand. Australasian Plant Pathology, 37(6): 559-568.
• Pitman, A. R., Harrow, S. A., & Visnovsky, S. B. (2010). Genetic characterisation of Pectobacterium wasabiae causing soft rot disease of potato in New Zealand. European Journal of Plant Pathology, 126(3): 423-435.
• Potrykus, M., Sledz, W., Golanowska, M., Slawiak, M., Binek, A., Motyka, A., Lojkowska, E. (2014). Simultaneous detection of major blackleg and soft rot bacterial pathogens in potato by multiplex polymerase chain reaction. Annals of Applied Biology, 165(3): 474-487.
• Potrykus, M., Golanowska, M., Sledz, W., Zoledowska, S., Motyka, A., Kolodziejska, A., Lojkowska, E. (2016). Biodiversity of Dickeya spp. isolated from potato plants and water sources in temperate climate. Plant Disease, 100(2): 408-417.
• Samson, R., Legendre, J. B., & Christen, R. (2005). Fischer-Le Saux M., Achouak W., Gardan L. Transfer of Pectobacterium chrysanthemi (Burkholder et al., 1953) Brenner et al. 1973 and Brenneria paradisiaca to the genus Dickeya gen. nov. 60 as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species, Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp. nov. International J. of Systematic and Evolutionary Microbiology, 55: 1415.
• Schaad, N.W., Jones, J.B., Chun, W. (2001). Laboratory guide for the identification of plant pathogenic bacteria (No. Ed. 3). American Phytopathological Society (APS Press).
• Skelsey, P., Humphris, S.N., Campbell, E.J., Toth, I.K. (2018). Threat of establishment of non-indigenous potato blackleg and tuber soft rot pathogens in Great Britain under climate change. PloS one, 13(10): e0205711.
• Soylu, E.M., Soylu, S., Kara, M., Kurt, Ş. (2020). Sebzelerde sorun olan önemli bitki fungal hastalık etmenlerine karşı vermikomposttan izole edilen mikrobiyomların in vitro antagonistik etkilerinin belirlenmesi. KSU Tarım ve Doğa Dergisi, 23: 7-18.
• Soylu, S., Kara, M., Uysal, A., Kurt, Ş. Soylu, E.M., Üremiş, İ., Sertkaya, E., Bozkurt, İ.A., Öztürk, M. (2022). Amik ovası havuç ekim alanlarında sorun olan fungal ve bakteriyel hastalık etmenlerin belirlenmesi. KSU Tarım ve Doğa Dergisi, 25: (baskıda) https://doi.org/10.18016/ksutarimdoga.vi.1015936.
• Samancı, B., Özkaynak, E., Çetin, M. D. (2003). Antalya koşullarında turfanda patates (Solanum tuberosum L.) yetiştiriciliğinde bazı çeşitlerin verim ve verim ile ilgili özelliklerinin belirlenmesi. Akdeniz Üniversitesi Ziraat Fakültesi Dergisi, 16(1): 27-33.
• Toth, I. K., Van Der Wolf, J. M., Saddler, G., LOjkowska, E., Hélias, V., Pirhonen, M., Elphinstone, J.G. (2011). Dickeya species: an emerging problem for potato production in Europe. Plant pathology, 60(3): 385-399.
• Ustun, N., Arslan, N. (2016). Bacterial stem rot of globe artichoke (Cynara cardunculus var. scolymus) caused by Pectobacterium carotovorum subsp. carotovorum in Turkey. Journal of Plant Pathology 98: 91.
• Van der Wolf, J.M., De Haan, E.G., Kastelein, P., Krijger, M., De Haas, B. H., Velvis, H., Van Der Zouwen, P. S. (2017). Virulence of Pectobacterium carotovorum subsp. brasiliense on potato compared with that of other Pectobacterium and Dickeya species under climatic conditions prevailing in the Netherlands. Plant Pathology, 66(4): 571-583.
• Waleron M., Waleron K., Podhajska A.J., Łojkowska E. (2002) Genotyping of bacteria belonging to the former Erwinia genus by PCR-RFLP analysis of a recA gene treatment. Microbiology, 148: 583–595.
• Waleron, M., Waleron, K., Łojkowska, E. (2013). Occurrence of Pectobacterium wasabiae in potato field samples. European Journal of Plant Pathology, 137: 149-158.
• Yüzbaşıoğlu, E.G., Aysan, Y. (2021). Farklı konukçu bitkilerden izole edilen Agrobacterium tumefaciens izolatlarının fenotipik ve genotipik karakterizasyonu. Tekirdağ Ziraat Fakültesi Dergisi, 18 (2): 247-259.
• Zoledowska, S., Motyka, A., Zukowska, D., Sledz, W., Lojkowska, E. (2018). Population structure and biodiversity of Pectobacterium parmentieri isolated from potato fields in temperate climate. Plant disease, 102(1): 154-164.