Kök Kanserine Karşı Biyolojik Mücadele Ajanı Bakterilerin Belirlenmesi

Öz

Bu çalışma kök kanseri hastalığının [Rhizobium radiobacter (Agrobacterium tumefaciens)] in vitro ve yarı in vivo testlerle biyolojik mücadelesine yönelik yeni biyoajan bakterilerinin belirlenmesi amacıyla yapılmıştır. Farklı cinslere ait toplam 2012 adet potansiyel biyoajan bakteri straini patojene karşı in vitro koşullarda test edilmiştir. In vitro test sonucu etkili bulunan biyoajan bakteri strainlerinin mikrobiyal identifikasyon sistem tanıları (MIS) bazı konvansiyonel testler ile desteklenmiştir. Daha sonra etkili bulunan izolatların yarı in vivo biyokontrol aktiviteleri havuç dilimi ve kabak meyvesinde test edilmiştir. Havuç dilimlerinde yüzey kaplama yüzdesi kullanılırken, kabak meyvesinde ise ur sayı ve büyüklüğüne göre veriler elde edilmiş ve istatistiki analizi yapılmıştır. Daha sonra en etkili bulunan biyoajan ve patojen bakteri strainlerinin tanıları moleküler olarak belirlenmiştir. Elde edilen bulgulara göre; R. radiobacter’e karşı in vitro koşullarda 106 adet biyoajan bakteri straini (66 adedi antibiyozis etki; 40 adedi hiperparazitik etki) etkili bulunmuştur. Biyoajan bakterilerin konvansiyonel tanı test sonuçları ile mikrobiyal identifikasyon tanı sonuçlarının birbirini destekler nitelikte olduğu belirlenmiştir. Yarı in vivo biyokontrol aktivite test sonucu 106 adet biyoajan bakteri straininden 8 adetinin pektolitik aktivite oluşturmadığı belirlenmiş ve yarı in vivo test sonucu 8 adet biyoajan bakteri straini değerlendirilebilmiştir. Havuç dilimi ve kabak meyvesinde patojenin gelişimini baskılayan en etkili strainin RK 1986 (havuç dilimi 1.78±0.47; kabak meyvesi 0.26±0.04) olduğu, onu RK 570A (havuç dilimi 2.89±0.82; kabak meyvesi 0.35±0.03) ve RK 1074 (havuç dilimi 3.44±0.99; kabak meyvesi 0.46±0.05) strainlerinin takip ettiği tespit edilmiştir. Moleküler tanı sonuçlarına göre en etkili olan biyoajan bakteri straini (RK 1986) Bacillus mojavensis, patojen bakteri strainin ise (1B) Rhizobium radiobacter olduğu kaydedilmiştir.

Anahtar Kelimeler

• Agrobacterium tumefaciens
• Bacillus mojavensis
• Biyolojik mücadele
• Kök kanseri
• Rhizobium radiobacter.

Etik Beyan

Tekiner Aydın N. and Kotan R., declare that they have no competing interests. This study includes a part of Nasibe TEKİNER AYDIN’s doctoral thesis accepted by Atatürk University Institute of Science.

Teşekkür

I would like to thank Supersol Biotechnology Company and Kotan Biotechnology R&D for their material and equipment support throughout my studies.

Determination of Biological Control Agent Bacteria Against Crown Gall

Öz

This study was carried out to determine new bioagent bacteria for in vitro and semi-in vivo biological control of crown gall disease [Rhizobium radiobacter (Agrobacterium tumefaciens)]. A total of 2012 potential bioagent bacterial strains belonging to different genera were tested in vitro against the pathogen. Microbial identification systems (MIS) diagnoses of bioagent bacterial strains found to be effective as a result of in vitro tests were supported by some conventional tests. Then, the strains' semi-in vivo biocontrol activities found to be effective were tested in carrot slices and squash fruits. Statistical analysis of the data was made according to the percentage of surface coverage in carrot slices and the number and size of tumors in squash fruits. Then, the most effective bioagent and pathogenic bacterial strain diagnoses were determined molecularly. According to the results; 106 bioagent bacterial strains (66 antibiosis; 40 hyperparasitic effects) were found to be effective against R. radiobacter in vitro conditions. It was determined that conventional test results of bioagent bacteria and MIS results supported each other. As a result of semi-in vivo biocontrol activity, it was determined that 8 bioagent bacterial strains out of 106 bioagent bacterial strains did not produce pectolytic activity, and 8 bioagent bacterial strains could be evaluated as a result of semi-in vivo test. The most effective strain suppressing the development of the pathogen in carrot slices and squash fruits was RK 1986 (carrot slices 1.78±0.47; squash fruits 0.26±0.04), followed by RK 570A (carrot slices 2.89±0.82; squash fruits 0.35+0.03) and RK 1074 (carrot slices 3.44±0.99; squash fruits 0.46±0.05) strains were followed. According to the results of molecular identification, the most effective bioagent bacterial strain (RK 1986) was Bacillus mojavensis, and the pathogenic bacteria strain (1B) was R. radiobacter.

Anahtar Kelimeler

• Agrobacterium tumefaciens
• Bacillus mojavensis
• Biological control
• Crown gall
• Rhizobium radiobacter.

Etik Beyan

There is no need to obtain permission from the ethics committee for this study.

Teşekkür

I would like to thank Supersol Biotechnology Company and Kotan Biotechnology R&D for their material and equipment support throughout my studies.

Kaynakça

1. Abdallah, B. D., Tounsi, S., Gharsallah, H., Hammami, A. and Gargouri, F. O. (2018). Lipopeptides from Bacillus amyloliquefaciens strain 32a as promising biocontrol compounds against the plant pathogen Agrobacterium tumefaciens. Environmental Science and Pollution Research, 25: 36518-36529.
2. Agrios, G. N. (1997). Plant Pathology. Academic Press, U.S.A.
3. Aktan, Z.C. (2018). Determination of in vitro effects of antagonist and plant growth promoting bacteria against soilborne fungal disease agents on almond trees. (MsC. Thesis) Hatay Mustafa Kemal University The Institute of Science, Hatay, Turkey.
4. Araujo, F. F., Henning, A. A. and Hungria, M. (2005). Phytohormones and antibiotics produced by Bacillus subtilis and their effects on seed pathogenic fungi and on soybean root development. World Journal of Microbiology and Biotechnology, 21(8-9):1639-1645.
5. Banerjee, G., Gorthi, S. and Chattopadhyay, P. (2018). Beneficial effects of bio-controlling agent Bacillus cereus IB311 on the agricultural crop production and its biomass optimization through response surface methodology. Annals of the Brazilian Academy of Sciences, 90(2): 2149-2159.
6. Bozkurt, I. A. and Soylu, S. (2019). Determination of antagonistic potential of epiphytic and endophytic bacterial strains against apple crown gal disease agent Rhizobium radiobacter. Journal of Tekirdag Agricultural Faculty, 16(3): 349-361.
7. Cazelles, O., Epard, S. and Sımon, J. L. (1991). The effect of disinfection with oxyquinoline sulfate of the Berl. X Rip. 5C rootstock on the expression of crown gall in grape propagation. Revue Suisse de Viticulture d’Arboricet d’Horticulture, 23: 285-288.
8. Commare, R. R., Nandakumar, R., Kandan, A., Suresh, S., Bharthi, M., Raguchanger, T. and Samiyappan, R. (2002). Pseudomonas fluorescens based formulation for the management of sheath blight disease and leaf folder insect in rice. Crop Protection, 21: 671-677.

9. Dandurishvili, N., Toklikishvili, N., Ovadis, M., Eliashvili, P., Giorgobiani, N., Keshelava, R., Tediashvili, M., Vainstein, A., Khmel, I., Szegedi, E. and Chernin, L. (2010). Broad-range antagonistic rhizobacteria Pseudomonas fluorescens and Serratia plymuthica suppress Agrobacterium crown gall tumors on tomato plants. Journal of Applied Microbiology, 110: 341-352.
10. De Freitas, J. R., Banerjee, M. R. and Germida, J. J. (1997). Phosphate solubilizing rhizobacteria enhance the growth and yield but not phosphorus uptake of canola (Brassica napus L). Biology and Fertility of Soils, 24: 358-364.
11. Eastwell, K. C., Sholberg, P. L. and Sayler, R. J. (2006). Characterizing bacterial biocontrol agents for supression of Rhizobium vitis, a casual agent crown gall disease grapevines. Crop Protection, 25: 1191-1200.
12. EPPO (2019). European and Mediterranean Plant Protection Organization (EPPO). www.eppo.org (Accessed Date: 13.05.2019).
13. Farrand, S. K. and Wang, C. (1992). Do We Really Understand Crown Gall Control by Agrobacterium radiobacter strain K84? In: Tjamos ES et al (eds.) Biological Control of Plant Diseases. Plenum Press, New York, U.S.A.
14. Frandberg, E. and Schnurer, J. (1994). Chitinolytic properties of Bacillus pabuli KI. Journal of Applied Bacteriology, 76(4): 361-367.
15. Frikha‐Gargouri, O., Ben Abdallah, D., Ghorbel, I., Charfeddine, I., Jlaiel, L., Triki, M. A. and Tounsi, S. (2017). Lipopeptides from a novel Bacillus methylotrophicus 39b strain suppress Agrobacterium crown gall tumours on tomato plants. Pest Management Science, 73(3): 568-574.
16. Gloyer, W. O. (1934). Crown gall and hairy root of apples in nursery and orchard. Bulletin of the New York State Agricultural Experiment Station, 638: 1-30.
17. Gokce, A. Y. and Kotan, R. (2016). Investigation of biological control possibilities of wheat root rot disease caused by Bipolaris sorokiniana (Sacc.) using PGPR and bio-control bacteria in controlled condition. Plant Protection Bulletein, 56(1): 49-75.
18. Goodman, R. N., Grimm, R. and Frank, M. (1993). The influence of grape rootstocks on the crown gall infection process and on tumor development. American Journal of Enology and Viticulture, 44: 22-26.
19. Guerinot, M.L. and Colwell, R.R. (1985). Enumeration, isolation, and characterization of N2-fixing bacteria from seawater. Applied and Environmental Microbiology, 50(2): 350-355.
20. Gupta, A. K. and Kamal, B. (2006). Pre-planting application of soil sterilants and herbicides for management of crown gall disease on ‘colt’ cherry rootstock. Indian Journal Agricultural Science, 76: 426-429.
21. Gupta, A. K., Khosla, K. and Bhardwaj, S. S. (2007). Biological control of crown gall of stone fruits. Indian Phytopathology, 61: 385.
22. Gupta, A. K., Khosla, K., Bhardwaj, S. S., Thakur, A., Devi, S., Jarial, R. S., Sharma, C., Singh, K. P., Srivastava, D. K. and La, R. (2010). Biological control of crown gall on peach and cherry rootstock Colt by native Agrobacterium radiobacter strains. The Open Horticulture Journal, 3: 1-10.
23. Hammami, I., Rhouma, A., Jaouadi, B., Rebai, A. and Nesme, X. (2008). Optimization and biochemical characterization of a bacteriocin from a newly straind Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains. Letters in Applied Microbiology, 48:253-260.
24. Hardoim, P. R., Van Overbeek, L. S. and Van Elsas, J. D. (2008). Properties bacterial endophytes and their proposed role in plant growth. Trends Microbiology, 16: 463-471.
25. Inam-ul-Haq, M., Javed, M., Ahmad, R. and Rehman, A. (2003). Evaluation of different strains of Pseudomonas fluorescens for the biocontrol of Fusarium wilt of chickpea. Pakistan Journal of Plant Pathology, 2: 65-74.
26. Jiang, Y. M., Zhu, X. R. and Li, Y. B. (2001). Postharvest control of litchi fruit rot by Bacillus subtilis. Lebensmittel-Wissenschaft Und-Technologie-Food Science and Technology, 34(7): 430-436.
27. Khmel, I. A., Sorokina, T. A., Lemanova, N. B., Lipasova, V. A., Metlitski, O. Z., Burdeinaya, T. V. and Chernin, L. S. (1998). Biological control of crown gall in grapevine and raspberry by two Pseudomonas spp. with a wide spectrum of antagonistic activity. Biocontrol Science and Technology, 8(1): 45-57.
28. Kim, H. S., Park, J., Choi, S. W., Choi, K. H., Lee, G. P., Ban, S. J., Lee, H. L. and Kim, C. S. (2003). Isolation and characterization of Bacillus strains for biological control. Journal of Microbiology, 41: 196-201.
29. Klement, Z., Mavridis, A., Rudolph, K., Vidaver, A., Perombelon, M. C. and Moore, L. W. (1990). Inoculation of Plant Tissues Methods in Phytobacteriology, Akademiai Kiado, Budapest, Hungary.
30. Kotan, R. and Tozlu, E. (2021). Determination of bactericidal effects of some pesticides on useful and pathogenic bacteria. Journal of Tekirdag Agricultural Faculty, 18(2): 197-212 (In Turkish).
31. Kotan, R., Dikbas, N. and Bostan, H. (2009). Biological control of post harvest disease caused by Aspergillus flavus on stored lemon fruits. African Journal of Biotechnology, 8(2): 209-214.
32. Kovacs, N. (1956). Identication of Pseudomonas pyocyanea by the oxidase reaction. Nature, 170-173.
33. Lazo, G. R., Roffey, R. and Gabriel, D. W. (1987). Pathovars of Xanthomonas campestris are distinguishable by restriction fragment length polymorphism. International Journal of Systematic and Evolutionary Microbiology, 37: 214-217.
34. Lelliot, R. A. and Stead, D. E. (1987). Methods for the Diagnosis of Bacterial Diseases of Plants. Black Well Scientific Puplication, U.S.A.
35. Limanska, N., Korotaeva, N., Biscola, V., Ivanytsia, T. and Merlich, A. (2015). Study of the potential application of lactic acid bacteria in the control of ınfection caused by Agrobacterium tumefaciens. Journal of Plant Pathology and Microbiology, 6(8): 292.
36. Mohammadi, P. (2018). Evaluation of biological control of (Clavibacter michiganensis subsp. michiganensis (Smith) Davis et al.) bacterial wilt and canker of tomato by antagonistic bacteria. (Ph.D. Thesis) Atatürk University, The Institute of Science, Erzurum, Türkiye.
37. Moore, L. W. and Canfield, M. (1996). Biology of Agrobacterium and management of crown gall disease. In: Hall R (ed) Principles and practice of managing soilborne plant pathogens, APS Press, Minnesota, U.S.A.
38. Moore, L. W., Bouzar, H. and Burr, T. J. (2001). Agrobacterium. In: Schaad NW, Jones JB, Chun W (eds.) Laboratory Guide for Identification of Plant Pathogenic Bacteria. American Phytopathological Society Press, Minnesota, U.S.A.
39. Moore, L. W. (1988). Use of Agrobacterium radiobacter in agricultural ecosystems. Microbiological Science, 5: 92-95.
40. Nautiyal, C. S. (1999). An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiology Letters, 170: 65-270.
41. Parke, J. L. and Gurian-Sherman, D. (2001). Diversity of the Burkholderia cepacia complex and implications for risk assessment of biological control strains. Annual Reviews Phytopathology, 39: 225-258.
42. Peluso, R., Raio, A., Morra, F. and Zoina, A. (2003). Physiolgical, biochemical and molecular analyses of Italian collection of Agrobacterium tumefaciens strain. Eurepean Journal of Plant Pathology, 109: 291-300.
43. Rhouma, A., Ferchichi, H., Hofsa, M. and Boubaker, A. (2004). Efficacy of the non pathogenic Agrobacterium strains K84 and K1026 against crown gall in Tunisia. Phytopathologia Mediterranea, 43(2): 167-176.
44. Schallmey, M., Singh, A. and Ward, O. P. (2004). Developments in the use of Bacillus species for industrial production. Canadian Journal of Microbiology, 50: 1-17.
45. Tekiner Aydın, N. and Kotan, R. (2022). Pathogenicity of different Rhizobium radiobacter (Agrobacterium tumefaciens) isolates and their identification with conventional methods. KSU Journal of Agriculture and Nature, 25(Suppl 1): 149-157.
46. Tekiner, N., Kotan, R., Tozlu, E. and Dadasoglu, F. (2019). Determination of some biological control agents against Alternaria fruit rot in quince. Alınteri Journal of Agricultural Sciences, 34(1): 25-31.
47. Tekiner, N., Tozlu, E., Kotan, R. and Dadasoglu, F. (2020). Biological control of Botrytis cinerea and Alternaria alternata with bioagent bacteria and fungi under in vitro conditions. Fresenius Environmental Bulletein, 29(1): 640-649.
48. Tiwari, K. and Thakur, H. K. (2014). Diversity and molecular characterization of dominant Bacillus amyloliquefaciens (JNU-001) endophytic bacterial strains straind from native neem varieties of Sanganer region of Rajasthan. Journal of Biodiversity, Bioprospecting and Development, 1: 115.
49. Tolba, I. H. and Soliman, M. A. (2013). Efficacy of native antagonistic bacterial strains in biological control of crown gall disease in Egypt. Annals of Agricultural Science, 58(19): 43-49.
50. Tozlu, E., Tekiner, N., Kotan, R. and Ortucu, S. (2018). Investigation on the biological control of Alternaria alternata. Indian Journal of Agricultural Sciences, 88(8): 1241-1247.
51. Vrain, T. C. and Copeman, R. J. (1987). Interactions between Agrobacterium tumefaciens and Pratylenchus penetrans in the roots of two red raspberry cultivars. Canadian Journal of Plant Pathology, 9: 236-240.
52. Zhang, J. and Dou, H. (2002). Evaluation of Bacillus subtilis as potential biocontrol agent for postharvest green mold control on Valencia orange. Proceedings of the Florida State Horticultural Society, 115: 60-64.
53. Zhang, C. M., Lu, S. H., Wei, C. M. and Zhao, J. H. (1991). Bacterium strain 1-1-4 Pseudomonas fluorescens for biocontrol of peach crown gall disease. Acta Agriculturae Shanghai, 7(4): 65-69.