Farklı Konukçu Bitkilerden İzole Edilen Agrobacterium tumefaciens İzolatlarının Fenotipik ve Genotipik Karakterizasyonu
Öz
Bu çalışmada, 2008-2011 yılları boyunca 4 farklı ilde 10 farklı konukçu bitkiden tipik ur belirtisi gösteren enfekteli bitki örnekleri toplanmıştır. Toplanan enfekteli bitki dokularından toplam 174 bakteri izolatı elde edilmiştir. Elde edilen izolatların patojenisite testlemeleri havuç (Daucus carota L.) dilimi, kalonşe (Kalanchoe daigremontiana), domates (Solanum lycopersicum L.) ve ayçiçeği (Helianthus annus L.) fideleri üzerinde yapılmıştır. Patojenisite testleri sonucunda 414 re-izolat elde edilmiş olup, bu izolatlar arasından 138 re-izolat detaylı çalışmalarda kullanılmak üzere seçilmiştir. Seçilmiş olan tüm izolatlar klasik ve moleküler yöntemlerin sonuçlarına göre Agrobacterium tumefaciens olarak teşhis edilmiştir. Test edilen 138 izolat ve 6 referans izolatın tamamı kalonşe bitkisi ve havuç dilimi üzerinde tipik ur belirtilerine neden olurken, bu izolatlar arasından 67 izolat domates bitkisinde, 61 izolat ise ayçiçeği bitkisi üzerinde tipik ur belirtisi oluşturamamıştır. Bu tez kapsamında 3-ketolaktoz üretimi, % 2’lik NaCl’de büyüme, 35ºC’de gelişme, litmus milk’de reaksiyon, eritritol ve melezitozdan asit oluşturma, malonik asit, L-tartarik asit ve mucic asitten alkali oluşturma, demir amonyum sitrat kullanımı, sitrat kullanımı, PDA+CaCO3 besiyerinde asit temizleme, potasyum hidroksit testiyle gram reaksiyon ve oksidaz testi kullanılmıştır Yapılan morfolojik, fizyolojik ve biyokimyasal testlere göre, test edilen izolatlardan 3 tanesi dışında, izolatlar arasında önemli farklılık gözlenmemiştir. Bu izolardaki farklılıkta sözkonusu138 izolat arasından 48 izolat detaylı çalışmalar için seçilmiş, genetik profilleri REP PCR, ERIC PCR, BOX PCR ve RFLP teknikleri kullanılarak analiz edilmiştir. Bildiğimiz kadarıyla, şeftali, erik, kiraz, armut, trabzon hurması, meşe ve kalonşe bitkilerinden bu etmenin ülkemizdeki varlığı ilk kez bu çalışmayla ortaya konmuştur. Elde edilen sonuçlar farklı konukçulardan elde edilen Agrobacterium. tumefaciens izolatlarının benzerlik ve farklılıklarını ortaya koymuştur. Sonuç olarak, 5 ana grupta, 8 farklı küme ve 26 özgün profile sahip 34 genotip belirlenmiştir. İzolatlar ile konukçu bitki ve coğrafik bölge arasında ilişki kurulamamıştır.
Anahtar Kelimeler
Agrobacterium tumefaciens Spesifik PCR Genetik profilleme Patojenite testleri RFLP
Destekleyen Kurum
Çukurova Üniversitesi BAP
Proje Numarası
ZF2009D22
Teşekkür
Bu çalışma doktora tezinden hazırlanmış olup Çukurova Üniv. Bap tarafından ZF2009D22 numaralı proje ile desteklenmiştir.
Kaynakça
- Anonım (2012). TUIK verileri,www.tuik.gov.tr, (Erişim tarihi: 25 Aralık 2013)
- Anonymus, (2011). Fao statistics, www.fao.org, (Erişim tarihi: 23 Aralık 2013)
- Aysan, Y. & Sahın, F., Mırık, M., Donmez, M.F. and Tekman, H. (2003). First report of crown gall of apricot (Prunus armeniaca) caused by Agrobacterium tumefaciens in Turkey. Plant Pathology, 52 (6): 793-795.
- Aysan, Y. & Sahin, F. ( 2003). An outbreak of crown gall disease on rose caused by Agrobacterium tumefaciens in Turkey. Plant Pathology, 52 (6):780-783.
- Aysan, Y. & Sahin, F., 2003. An outbreak of crown gall disease on rose caused by Agrobacterium tumefaciens in Turkey. Plant Pathology, 52 (6):780-783.
- Aysan, Y. & Sahin, F., Mirik, M., Donmez, M.F. and Tekman, H., 2003. First report of crown gall of apricot (Prunus armeniaca) caused by Agrobacterium tumefaciens in Turkey. Plant Pathology, 52 (6): 793-795. Bozkurt, I. A. & Soylu, S., 2011. ‘Hatay İli Elma Bahçelerinde Agrobacterium tumefaciens’in Neden Olduğu Kök Boğazı uru Hastalığının Belirlenmesi’. Türkiye 4. Bitki Koruma Kongresi, 28-30 Haziran 2011, Kahramanmaraş, Sayfa, 315 sayfa.
- De Boer, S. H. & Ward, L.J. (1995). PCR detection of Erwinia carotovora subsp. atroseptica associated with patoto tissue. Phytopathology, 85: 854-858. De Cleene, M. & De Ley, J. (1976). The host range of crown gall. Bot. Rev, 42: 389-466.
- Dong, L. C., Sun, C. W., Thıes, K. L., Luthe, D. S. & Graves, C. H. (1991). Use of polymerase chain reaction to detect pathojenic strains of. Agrobacterium. Phytopathology, 82: 434-439.
- Eastwell, K. C., Wıllıs, L. G. & Cavıleer, T. D. (1995). A rapid and sensitive method to detect Agrobacterium vitis in grapewine cuttingns using the Polimerase chain reaction. Plant Disease, 79: 822-827.
- Karaca, I. (1977). Fitobakteriyoloji ve Bakteriyel Hastalıklar. Ege Üniversitesi, Ziraat Fakültesi Yayınları, No: 294, 270s.
- Lipincott, J. A., Lipincott, B. B. & Starr, M. P. (1980). The genus Agrobacterium. (In: Star MP edıtor) Phytopathogenic Bacteria, 842-855. springer- Verlag, New York.
- Lipincott,, J. A. & Lipincott,, B. B. (1975). The genus Agrobacterium and plant tumorigenesis. Annual Rev. Microbiol. 29: 377-405. Minnemeyer, S., L., Lighrfoot & Matthysse, A., G. (1991). A semiguantitatue bioassay for relative virulence of Agrobacterium tumefaciens strains on Bryophyllum daigoromontiana. Jour. Bacteriology, 173: 7723-7724.
- Mirik, M., Aysan, Y. & Sahin, F. (2011). Characterization of Pseudomonas savastanoi pv. Savastanoi strains isolated from several host plants in Turkey and report of Fontanesia as a new host.Journal of plant Pathology, 93 (2), 263-270.
- Moore, L. W., Bouzar, H. & Burr, T. (2001). Agrobacterium. P: 17-35 In Laboratory Guidek for Identification of Plant Pathogenic Bacteria, 3rd.Ed., (Schaad, N.W., Jones, J.B., Chun, W. editors), American Phytopathological Press, St. Parl, Minnesota. MOORE, L., W, 1988. Use of Agrobacterium radiobacter in agricultural ecosystem. Microbiological Sciences, 5: 92-95.
- Nester, E. W., Gordon, R. M., Amasıno, R. M. & Yanofssky, M. F. (1984). Crown gall: Molecular and physiological analysis. Annual Review of Plant Physiology, 35: 387-413.
- Oden,S., 1991. İzmir ve İstanbul İllerinde Önemli Süs Bitkilerinde Görülen Bakteriyel Hatalıklar ve Etmenlerin Saptanması Üzerine Araştırmalar. Ege Üniversitesi, Fen bilimleri Entstitüsü, Bitki Koruma anabilim dalı, Doktara Tezi, 84 sayfa
- Oger, P., Desaux, Y., Petıt, A., Gardon, L., Mancaeau, C., Chomel, C. & Nesme, X. (1998). Validity sensitivity and resolution limit of thePCR-RFLP analysis of the rrs (16S rRNA gene) as a tool to identfy soil-borne and plant –associated bacterial populations. Genet. Sel. Evol., 30 suppl. 1: 311-321 Peluso, R., Raıo, A., Morra, F. & Zoina, A. (2003). Physiolgical, biochemical and molecular analyses of Italian collection of Agrobacterium tumefaciens strain Eurepean Journal of Plant Pathology, 109: 291-300.
- Pıonnat, S., Keller, H., Herıcher, D., Bettachini, A.,Dessaux, Y., Nesme, X. & Poncet, C. (1999). Ti plazmids from Agrobacterium characterize rookstock clones that initiated a spread of crown gall disease in mediterranean countries. Apllied and Enviromental Microbiology, Vol, 65, No. 9: 4197-4206.
- Pulawska, J. and Sobiczewskı, P., 2005. Devolepment of a semi-nested PCR based method for sensitive detection of tumorigenic Agrobacterium in soil. J. Appl. Microbiology, 98 (3): 710-721.
- Rademaker, J. L. W. & BRUJIN, F. J. (1997). Characterization and classification of microbes by REP-PCR genomic finger printing and computer asisted pattern analysis. Journal Applied Bacteriology, 68: 728-738.
- Raıo, A., Peluso, R., Nesme, X. & Asfolfo, Z. (2004). Chromosomal and plazmid diversity of Agrobacteriumstrains isolated from Ficus benjamina tumors. European Journal of Plant Pathology, 110: 163-174.
- Rhouma, A., Boubaker, A., Nesme, X. & Desaux, Y., (2006). Plasmid and chromosomal diversity of a Tunisian collection of Agrobacterium tumefaciens strains. Tunisian Journal of Plant Protection 1: 73-84.
- Sawada, H., Jekı, H. & Matsuda, I. (1995). PCR Detection Ti and Ri Plazmids from Phytopathogenic Agrobacterium strains. Applied and Environmental Microbiology, February: 828-831.
- Slater, S.C., Barry, S. G., Goodner, B., Setubal, J. C., Farrand, S.K., Nester, E. W., Burr, J., Banta, L., Dıckerman, A.W., Paulsen, I., Otten, L., Suen, G., Welch, R., Almeıda, E., Godsy &E, Heısel, S. (2009). Genome sequences of three Agrobacterium biovars help elucidate the evoltion of multicchromosome genomes in bacteria. Journal of Bacteriology, Vol. 191, No. 8, p: 2501-2511. T. C. Gıda, Tarım ve Hayvancılık Bakanlığı. Zirai Karantina Yönetmeliği (2009).
- Tan, B. S, Yabukı, J., Matsumoto, S., Kageyama, K. & Fukuı, H. (2003). PCR primers for identification of opine types of Agrobacterium tumefaciens in Japan. Journal of General Plant Pathology, 69 (4): 258-266.
- Tenover, F. C. Arbeıt, R. D. & Goorıng. R.U. (1997). How to select and interpret molcular strain typing methods for epidemiological studies of bacterial infections: a review for health care epidemiologists. Infect Control Hosp. Epidemiology, 18: 426-439
- Versalovıc, J., Schneıder, M, De brujn, F. J. & Lupskı, J. R. (1994). Genomic fingerprintig of bacteria using repetetive sequence-based polimerase chain reaction. Methods in Molecular and Cellular Biology, 5: 25-40.
- Zeanen, I., Van Larebeke, N., Tenchy, H., Van montagu, M. & Schell, J. (1974). Supercoiled circular DNA in crown gall inducing Agrobacterium strains. J. Mol. Biol. 86: 109-127.
Öz
During 2008-2011 growing season, 148 infected plant samples, showing tumor (gall) symptom, were collected from ten different host plants in 4 different province of Turkey. Total of 174 bacterial isolates were obtained from infected samples. Pathogenicity tests were conducted on carrot (Daucus carota L.) slice, kalonche (Kalanchoe daigremontiana), tomato (Solanum lycopersicum L.) and sunflower (Helianthus annus L.) seedlings. From pathogenicity tests, 414 isolates were re-isolated and total of 138 bacterial re-isolates were selected and used in further studies. All these selected isolates were identified as Agrobacterium tumefaciens according to classical and molecular identification tests. Although all 138 isolates caused typical galls on kalonche and carrot slices, 67 isolates on tomato and 61 isolates on sunflower seedlings were failed to cause typical gall symptoms. In the study, 3-ketolactose production, growth in 2% NaCl, development at 35ºC, reaction in litmus milk, acid formation from erythritol and hybridis, alkali formation from malonic acid, L-tartaric acid and mucic acid, use of iron ammonium citrate, use of citrate, acid clearing in PDA+CaCO3 medium, gram reaction with potassium hydroxide test and oxidase test were used. Apart from three isolates, no major differences were observed among the bacterial isolates tested according to morphological, physiological and biochemical tests. Among these138 isolates, 48 bacterial isolates were selected and their genetic profiles were analyzed by using REP PCR, ERIC PCR, BOX PCR and RFLP techniques. As far as we know, peach, plum, cherry, pear, persimmon, oak and kalonchoe from the plants in our country, the presence of these factors has been demonstrated in this study for the first time. This study clearly showed the differences and similarities of the bacterial isolates of Agrobacterium tumefaciens obtained from different host plants. As a result, 34 genotypes in 5 main group and 8 different clusters, 26 unique profiles were determined. With isolates of different host plants and geographic areas can be concluded that the relationship between the genotyped.
Anahtar Kelimeler
Agrobacterium tumefaciens Pathogenicity tests Specific PCR Genetic profiling RFLP
Proje Numarası
ZF2009D22
Kaynakça
- Anonım (2012). TUIK verileri,www.tuik.gov.tr, (Erişim tarihi: 25 Aralık 2013)
- Anonymus, (2011). Fao statistics, www.fao.org, (Erişim tarihi: 23 Aralık 2013)
- Aysan, Y. & Sahın, F., Mırık, M., Donmez, M.F. and Tekman, H. (2003). First report of crown gall of apricot (Prunus armeniaca) caused by Agrobacterium tumefaciens in Turkey. Plant Pathology, 52 (6): 793-795.
- Aysan, Y. & Sahin, F. ( 2003). An outbreak of crown gall disease on rose caused by Agrobacterium tumefaciens in Turkey. Plant Pathology, 52 (6):780-783.
- Aysan, Y. & Sahin, F., 2003. An outbreak of crown gall disease on rose caused by Agrobacterium tumefaciens in Turkey. Plant Pathology, 52 (6):780-783.
- Aysan, Y. & Sahin, F., Mirik, M., Donmez, M.F. and Tekman, H., 2003. First report of crown gall of apricot (Prunus armeniaca) caused by Agrobacterium tumefaciens in Turkey. Plant Pathology, 52 (6): 793-795. Bozkurt, I. A. & Soylu, S., 2011. ‘Hatay İli Elma Bahçelerinde Agrobacterium tumefaciens’in Neden Olduğu Kök Boğazı uru Hastalığının Belirlenmesi’. Türkiye 4. Bitki Koruma Kongresi, 28-30 Haziran 2011, Kahramanmaraş, Sayfa, 315 sayfa.
- De Boer, S. H. & Ward, L.J. (1995). PCR detection of Erwinia carotovora subsp. atroseptica associated with patoto tissue. Phytopathology, 85: 854-858. De Cleene, M. & De Ley, J. (1976). The host range of crown gall. Bot. Rev, 42: 389-466.
- Dong, L. C., Sun, C. W., Thıes, K. L., Luthe, D. S. & Graves, C. H. (1991). Use of polymerase chain reaction to detect pathojenic strains of. Agrobacterium. Phytopathology, 82: 434-439.
- Eastwell, K. C., Wıllıs, L. G. & Cavıleer, T. D. (1995). A rapid and sensitive method to detect Agrobacterium vitis in grapewine cuttingns using the Polimerase chain reaction. Plant Disease, 79: 822-827.
- Karaca, I. (1977). Fitobakteriyoloji ve Bakteriyel Hastalıklar. Ege Üniversitesi, Ziraat Fakültesi Yayınları, No: 294, 270s.
- Lipincott, J. A., Lipincott, B. B. & Starr, M. P. (1980). The genus Agrobacterium. (In: Star MP edıtor) Phytopathogenic Bacteria, 842-855. springer- Verlag, New York.
- Lipincott,, J. A. & Lipincott,, B. B. (1975). The genus Agrobacterium and plant tumorigenesis. Annual Rev. Microbiol. 29: 377-405. Minnemeyer, S., L., Lighrfoot & Matthysse, A., G. (1991). A semiguantitatue bioassay for relative virulence of Agrobacterium tumefaciens strains on Bryophyllum daigoromontiana. Jour. Bacteriology, 173: 7723-7724.
- Mirik, M., Aysan, Y. & Sahin, F. (2011). Characterization of Pseudomonas savastanoi pv. Savastanoi strains isolated from several host plants in Turkey and report of Fontanesia as a new host.Journal of plant Pathology, 93 (2), 263-270.
- Moore, L. W., Bouzar, H. & Burr, T. (2001). Agrobacterium. P: 17-35 In Laboratory Guidek for Identification of Plant Pathogenic Bacteria, 3rd.Ed., (Schaad, N.W., Jones, J.B., Chun, W. editors), American Phytopathological Press, St. Parl, Minnesota. MOORE, L., W, 1988. Use of Agrobacterium radiobacter in agricultural ecosystem. Microbiological Sciences, 5: 92-95.
- Nester, E. W., Gordon, R. M., Amasıno, R. M. & Yanofssky, M. F. (1984). Crown gall: Molecular and physiological analysis. Annual Review of Plant Physiology, 35: 387-413.
- Oden,S., 1991. İzmir ve İstanbul İllerinde Önemli Süs Bitkilerinde Görülen Bakteriyel Hatalıklar ve Etmenlerin Saptanması Üzerine Araştırmalar. Ege Üniversitesi, Fen bilimleri Entstitüsü, Bitki Koruma anabilim dalı, Doktara Tezi, 84 sayfa
- Oger, P., Desaux, Y., Petıt, A., Gardon, L., Mancaeau, C., Chomel, C. & Nesme, X. (1998). Validity sensitivity and resolution limit of thePCR-RFLP analysis of the rrs (16S rRNA gene) as a tool to identfy soil-borne and plant –associated bacterial populations. Genet. Sel. Evol., 30 suppl. 1: 311-321 Peluso, R., Raıo, A., Morra, F. & Zoina, A. (2003). Physiolgical, biochemical and molecular analyses of Italian collection of Agrobacterium tumefaciens strain Eurepean Journal of Plant Pathology, 109: 291-300.
- Pıonnat, S., Keller, H., Herıcher, D., Bettachini, A.,Dessaux, Y., Nesme, X. & Poncet, C. (1999). Ti plazmids from Agrobacterium characterize rookstock clones that initiated a spread of crown gall disease in mediterranean countries. Apllied and Enviromental Microbiology, Vol, 65, No. 9: 4197-4206.
- Pulawska, J. and Sobiczewskı, P., 2005. Devolepment of a semi-nested PCR based method for sensitive detection of tumorigenic Agrobacterium in soil. J. Appl. Microbiology, 98 (3): 710-721.
- Rademaker, J. L. W. & BRUJIN, F. J. (1997). Characterization and classification of microbes by REP-PCR genomic finger printing and computer asisted pattern analysis. Journal Applied Bacteriology, 68: 728-738.
- Raıo, A., Peluso, R., Nesme, X. & Asfolfo, Z. (2004). Chromosomal and plazmid diversity of Agrobacteriumstrains isolated from Ficus benjamina tumors. European Journal of Plant Pathology, 110: 163-174.
- Rhouma, A., Boubaker, A., Nesme, X. & Desaux, Y., (2006). Plasmid and chromosomal diversity of a Tunisian collection of Agrobacterium tumefaciens strains. Tunisian Journal of Plant Protection 1: 73-84.
- Sawada, H., Jekı, H. & Matsuda, I. (1995). PCR Detection Ti and Ri Plazmids from Phytopathogenic Agrobacterium strains. Applied and Environmental Microbiology, February: 828-831.
- Slater, S.C., Barry, S. G., Goodner, B., Setubal, J. C., Farrand, S.K., Nester, E. W., Burr, J., Banta, L., Dıckerman, A.W., Paulsen, I., Otten, L., Suen, G., Welch, R., Almeıda, E., Godsy &E, Heısel, S. (2009). Genome sequences of three Agrobacterium biovars help elucidate the evoltion of multicchromosome genomes in bacteria. Journal of Bacteriology, Vol. 191, No. 8, p: 2501-2511. T. C. Gıda, Tarım ve Hayvancılık Bakanlığı. Zirai Karantina Yönetmeliği (2009).
- Tan, B. S, Yabukı, J., Matsumoto, S., Kageyama, K. & Fukuı, H. (2003). PCR primers for identification of opine types of Agrobacterium tumefaciens in Japan. Journal of General Plant Pathology, 69 (4): 258-266.
- Tenover, F. C. Arbeıt, R. D. & Goorıng. R.U. (1997). How to select and interpret molcular strain typing methods for epidemiological studies of bacterial infections: a review for health care epidemiologists. Infect Control Hosp. Epidemiology, 18: 426-439
- Versalovıc, J., Schneıder, M, De brujn, F. J. & Lupskı, J. R. (1994). Genomic fingerprintig of bacteria using repetetive sequence-based polimerase chain reaction. Methods in Molecular and Cellular Biology, 5: 25-40.
- Zeanen, I., Van Larebeke, N., Tenchy, H., Van montagu, M. & Schell, J. (1974). Supercoiled circular DNA in crown gall inducing Agrobacterium strains. J. Mol. Biol. 86: 109-127.
Ayrıntılar
| Birincil Dil | Türkçe |
|---|---|
| Bölüm | Makaleler |
| Yazarlar | |
| Proje Numarası | ZF2009D22 |
| Yayımlanma Tarihi | 1 Mayıs 2021 |
| Gönderilme Tarihi | 10 Haziran 2020 |
| Kabul Tarihi | 2 Ocak 2021 |
| Yayımlandığı Sayı | Yıl 2021 Cilt: 18 Sayı: 2 |
