Farklı Konukçu Bitkilerden İzole Edilen Agrobacterium tumefaciens İzolatlarının Fenotipik ve Genotipik Karakterizasyonu

Year 2021, Volume: 18 Issue: 2, 247 - 259, 01.05.2021

Eda Geylani Yüzbaşıoğlu , Yeşim Aysan

https://doi.org/10.33462/jotaf.750544

Cited By: 2

Abstract

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.

Keywords

Agrobacterium tumefaciens, Spesifik PCR, Genetik profilleme, Patojenite testleri, RFLP

Supporting Institution

Çukurova Üniversitesi BAP

Project Number

ZF2009D22

Thanks

Bu çalışma doktora tezinden hazırlanmış olup Çukurova Üniv. Bap tarafından ZF2009D22 numaralı proje ile desteklenmiştir.

Isolation and Identification of Agrobacterium tumefaciens Strains From Different Hosts In Turkey

Abstract

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.

Keywords

Agrobacterium tumefaciens, Pathogenicity tests, Specific PCR, Genetic profiling, RFLP

Project Number

ZF2009D22

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