jotaf Tekirdağ Ziraat Fakültesi Dergisi 1302-7050 2146-5894 Tekirdağ Namık Kemal Üniversitesi 10.33462/jotaf.1259380 Phytopathology Fitopatoloji Ayçiçeğinde Sclerotinia sclerotiorum (Lib.) De Bary’ye Karşı Bazı Bakteriyel Antagonistlerin In vitro ve In vivo Etkinliklerinin Belirlenmesi Determination of In Vitro and In Vivo Efficacy of Some Bacterial Antagonists Against Sclerotinia sclerotiorum (Lib.) De Bary in Sunflowers https://orcid.org/0000-0002-8221-0452 Koçak Raziye SELCUK UNIVERSITY, ÇUMRA VOCATIONAL SCHOOL https://orcid.org/0000-0001-6822-9360 Boyraz Nuh SELÇUK ÜNİVERSİTESİ, ZİRAAT FAKÜLTESİ 03 13 2024 21 2 362 374 03 10 2023 06 22 2023 Copyright © 2005, Tekirdağ Ziraat Fakültesi Dergisi 2005 Tekirdağ Ziraat Fakültesi Dergisi

Bu çalışma Konya ve Aksaray illeri ayçiçek ekim alanlarında kök ve kök boğazı çürüklüğüne neden olan Sclerotinia sclerotiorum’a karşı bazı bakteriyel biyoajanların in vitro ve in vivo etkinliklerini belirlemek amacıyla 2017-2018 yıllarında yürütülmüştür. Konya ve Aksaray illeri ayçiçek ekim alanlarından toplanan hastalıklı bitkilerden izole edilip, tanılaması yapıldıktan sonra patojenisite testleri sonucu seçilen en virulent olan iki S. sclerotiorum (Hırkatol ve Eskil) izolatına karşı yine aynı alanlardan sağlıklı ayçiçek bitkilerinin rizosfer bölgesindeki topraktan izole edilen 16 bakteri izolatının antifungal etkileri değerlendirilmiştir. Öncelikli olarak ikili kültür testleri ile en etkili bakteriyel biyoajanlar belirlenmiştir. In vitro testler sonucunda en geniş zon çapı oluşturan toplam 5 bakteri izolatının 16S rRNA’ya göre moleküler olarak tanılaması yapılmış ve saksı denemelerinde kullanılmıştır. Bakteriler Bacillus cereus, Bacillus simplex, Brevibacterium frigoritolerans, Bacillus toyonensis (2 izolat) olarak teşhis edilmiş ve GenBank veritabanının (NCBI) BLAST programı kullanılarak kodlanmıştır. In vitro da S. sclerotiorum’un her iki izolatında da en yüksek etki %49.19-57.95 engelleme oranıyla Bacillus cereus ve Bacillus simplex’ de gözlenmiştir. In vivo da test edilen bakteri türlerinden Bacillus toyonensis (B1) hariç bütün bakteriler kontrole göre lezyon gelişimini azaltmış veya durdurmuştur. Uygulama sonucunda biyolojik mücadele ajanı bakterilerden Bacillus cereus ve Bacillus simplex in vivo koşullarda S. sclerotiorum’un her iki izolatının da gelişmesine tamamen (%100) engel olmuştur. Yapılan etkinlik çalışmalarında bakteriyel izolatların hem ayçiçeği bitkisinin sağlıklı gelişmesine neden olduğu hem de uygulama yapılmış bitkilerde hastalık oluşumunu kontrollerdeki bitkilerle karşılaştırıldığında önemli düzeyde engellediğini göstermiştir. Bu sonuçlar bu tür çalışmaların bölgemizde kolaylıkla uygulanabilecek sürdürülebilir tarım uygulamalarının geliştirilmesi için bir araç olarak önemini vurgulamakta aynı zamanda ayçiçeğinde S.sclerotiorum’a karşı B.cereus ve B.simplex’in biyolojik mücadelede kullanılabilecek potansiyel bakteriyel biyoajanlar olabileceklerini göstermektedir. Ayrıca çalışmada etkin bulunan bakteri izolatlarının ticari preparatlarının geliştirilmesine yönelik çalışmaların yapılması yararlı olacaktır.

This study was carried out in 2017-2018 to determine the in vitro and in vivo activities of some bacterial bioagents against Sclerotinia sclerotiorum, which causes root and root-collar rot in sunflower cultivation areas of Konya and Aksaray provinces. Against the two most virulent S. sclerotiorum (Hırkatol and Eskil) isolates selected as a result of pathogenicity tests after being isolated and diagnosed from diseased plants which were collected from sunflower cultivation areas in Konya and Aksaray provinces, the antifungal effects of 16 bacterial isolates from the soil in the rhizosphere region of the healthy sunflower plants from the same areas were evaluated. Primarily, the most effective bacterial bioagents were determined by dual culture tests. As a result of the in vitro tests, a total of 5 bacterial isolates constituting the largest zone diameter were molecularly identified according to 16S rRNA and were used in pot experiments. The bacteria were identified as Bacillus cereus, Bacillus simplex, Brevibacterium frigoritolerans, Bacillus toyonensis (2 isolates) and were coded using the BLAST program of the GenBank database (NCBI). As per in vitro, the highest effect in both isolates of S. sclerotiorum was observed in Bacillus cereus and Bacillus simplex with an inhibition rate of 49.19-57.95%. Except for Bacillus toyonensis (B1), one of the bacterial species which were tested in vivo, all the bacteria reduced or stopped lesion development compared to the control. As a result of the application, the biological control agent completely prevented the growth of both the isolates of Bacillus cereus and Bacillus simplex S. sclerotiorum in in vivo conditions (100%). Efficacy studies have shown that bacterial isolates both cause healthy growth of sunflower plants and significantly prevent disease formation in treated plants when compared to control plants. These results emphasize the importance of such studies as a tool for the development of sustainable agricultural practices that can be easily applied in our region, and also show that B. cereus and B. simplex in sunflowers can be potential bacterial bioagents that can be used in biological control against S.sclerotiorum. In addition, it will be useful to carry out studies on the development of commercial preparations of the bacterial isolates found in the study.

 Sunflower White rot Biological control Sclerotinia sclerotiorum Ayçiçeği beyaz çürüklük biyolojik mücadele Sclerotinia sclerotiiorum Selçuk Üniversitesi Bilimsel Araştırma Projeleri Koordinatörlüğü 18101017 nolu proje Abdullah, M. T., Ali, N. Y. and Suleman, P. (2008). Biological control of Sclerotinia sclerotiorum (Lib.) de Bary with Trichoderma harzianum and Bacillus amyloliquefaciens. Crop Protection, 27(10): 1354-1359. Ajilogba, C. F., Babalola, O. O. and Ahmad, F. (2013). Antagonistic effects of Bacillus species in biocontrol of tomato Fusarium wilt. Studies on Ethno-Medicine, 7(3): 205-216. Arora, N. K., Khare, E. and Maheshwari, D.K. (2010). Plant growth promoting rhizobacteria: constraints in bioformulation, commercialization, and future strategies. In: Maheshwari, D.K. (Ed.), Plant Growth and Health Promoting Bacteria. Springer, Berlin, Heidelberg, 97–116. Ash, C., Farrow, J. A. E., Wallbanks, S. and Collins, M. D. (1991). Phylogenetic heterogeneity of the genus Bacillus revealed by comparative-analysis of small-subunit-ribosomal RNA sequences. Letters in applied microbiology, 13(4): 202–206. Bacon, C. W., Palencia, E. R. and Hinton, D. M. (2015). Abiotic and biotic plant stress-tolerant and beneficial secondary metabolites produced by endophytic Bacillus species. In: Arora, N.K. (Ed.), Plant Microbes Symbiosis: Applied Facets. Springer, India, pp. 163–177. Baniasadi, F., Bonjar, G. H. S., Baghizadeh, A., Nik, A. K., Jorjandi, M., Aghighi, S. and Farokhi, P. R. (2009). Biological control of Sclerotinia sclerotiorum, causal agent of sunflower head and stem rot disease, by use of soil borne Actinomycetes isolates. American Journal of Agricultural and Biological Sciences, 4(2): 146-151. Cawoy, H., Bettiol, W., Fickers, P. and Ongena, M. (2011). Bacillus based biological control of plant diseases. In: Stoytcheva, M. (Ed.), Pesticides in the Modern World-Pesticides Use and Management. InTech, Rijeka, Croatia, pp. 273–302. Connor, N., Sikorski, J., Rooney, A. P., Kopac, S., Koeppel, A. F., Burger, A., Cole, S. G., Perry, E. B., Krizanc, D., Field, N. C., Slaton, M. and Cohan, F. M. (2010). Ecology of speciation in the genus Bacillus. Applied and Environmental Microbiology, 76(5): 1349-1358. Davar, R., Darvishzadeh, A. M., Masouleh, A. K. and Ghosta, Y. (2012). The infection processes of Sclerotinia sclerotiorum in basall stem tissue of a susceptible genotype of Helianthus annuus L. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 40(1): 143–149. Duncan, R. W., Dilantha Fernandoa, W. G. and Rashidb, K. Y. (2006). Time and burial depth influencing the viability and bacterial colonization of Sclerotinia sclerotiorum. Soil Biology and Biochemistry, 38(2): 275-284. Earl, A. M., Losick, R. and Kolter, R. (2008). Ecology and genomics of Bacillus subtilis. Trends in microbiology, 16(6): 269–275. Erturk, Y., Ercisli, S., Haznedar, A. and Cakmakci, R. (2010). Effects of plant growth promoting rhizobacteria (PGPR) on rooting and root growth of kiwifruit (Actinidia deliciosa) stem cuttings. Biological Research, 43: 91–98. Fernando, W. G. D., Nakkeeran, S. and Zhang, Y. (2004). Ecofriendly methods in combating Sclerotinia sclerotiorum (Lib.) de Bary. Recent Research Developments in Environmental Biology, 1: 329–347. Fernando, W.G. D., Ramarathnam, R. and Krishnamoorthy, A. S. (2005). Identification and use of potential bacterial organic antifungal volatiles in biocontrol. Soil Biology and Biochemistry, 37(5): 955–964. Fira, D., Dimkić, I., Berić, T., Lozo, J. and Stanković, S. (2018). Biological control of plant pathogens by Bacillus species. Journal of Biotechnology, 285:44-55. Fravel, D. R. (2005). Commercialization and implementation of biocontrol. Annual Review of Phytopathology, 43: 337-359. Gao, X., Han, Q., Chen, Y., Qin, H. and Huang, L. (2014). Biological control of oilseed rape Sclerotinia stem rot by Bacillus subtilis strain Em7. Biocontrol Science and Technology, 24(1): 39-52. Georgakopoulos, D. G., Fiddaman, P., Leifert, C. and Malathrakis, N. E. (2001). Evaluation of antagonistic bacteria and fungi for biological control of sugar beet and cucumber damping-off caused by Pythium ultimum. Bulletin OILB/ SROP, 24 (3): 203-207. Ghildiyal, A. and Pandey, A. (2008). Isolation of cold tolerant strains of Trichoderma sp. from glacial sites of Indian Himalayan region. Research Journal of Microbiology, 3(8): 559–564. Guimaraes, L. M., Furlan, R. L., Garrido, L. M., Ventura, A., Padilla, G. and Facciotti, M. C. (2004). Effect of pH on the production of the antitumor antibiotic retamycin by Streptomyces olindensis. Biotechnology and applied biochemistry, 40(1): 107–111. Güldoğan, Ö., Aktepe, B. P., Aysan, Y. (2022). Use of different Bacillus species in the biological control of tomato bacterial speck disease. Journal of Tekirdag Agricultural Faculty, 19(4): 829-839. Gulya, T. J., Rashid, K. Y. and Masirevic, S. M. (1997). Sunflower diseases. In: Schneiter, A. A. (Ed.). Sunflower technology and production. Madison: American Society of Agronomy, 35: 263-379. Gutiérrez-Luna, F. M., López-Bucio, J., Altamirano-Hernández, J., Valencia-Cantero, E., de la Cruz, H. R. and Macías-Rodríguez, L. (2010). Plant growth-promoting rhizobacteria modulate root-system architecture in Arabidopsis thaliana through volatile organic compound emission. Symbiosis, 51: 75–83. Han, C. S., Xie, G., Challacombe, J. F., Altherr, M. R., Bhotika, S. S., Bruce, D, Campbell, C. S., Campbell, M. L., Chen, J. and Chertkov, O. (2006). Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis. Journal of Bacteriology,188(9): 3382-3390. Hassen, A. I. and Labuschagne, N. (2010). Root colonization and growth enhancement in wheat and tomato by rhizobacteria isolated from the rhizoplane of grasses. World Journal of Microbiology and Biotechnology, 26: 1837–1846. Ji, S. H. (2013). Biocontrol Activity of Bacillus amyloliquefaciens CNU114001 against fungal plant diseases. Mycobiology, 41: 234–242. Jiménez, G., Urdiain, M., Cifuentes,A., López-López, A., Blanch, A. R., Tamames, J., Kämpfer, P., Kolstø, A. B., Ramón, D., Martínez, J. F., Codoñer, F. M. and Rosselló-Mora, R. (2013). Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group, and pairwise genome comparisons of the species of the group by means of ANI calculations. Systematic and Applied Microbiology, 36(6):383–391. Jose, P. A., Santhi, V. S., Jebakumar, S. R. D. (2011). Phylogenetic- affiliation, antimicrobial potential and PKS gene sequence analysis of moderately halophilic Streptomyces sp. inhabiting an Indian saltpan. Journal of Basic Microbiology, 51(4): 348–356. Kamal, M. M., Lindbeck, K. D., Savocchia, S. and Ash, G. J. (2015). Biological control of Sclerotinia stem rot of canola using antagonistic bacteria. Plant Pathology, 64(6): 1375-1384. Kannahi, M. and Kowsalya, M. (2013). Efficiency of plant growth promoting rhizobacteria for the enhancement of Vigna mungo growth. Journal of Chemical and Pharmaceutical Research, 5(5): 46-52. Kloepper, J. W., Ryu, C. M. and Zhang, S. (2004). Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathology, 94(11): 1259-1266. Kotan, R. (2014). Faydalı bakterilerin tarımda kullanımı. Harman Time, 11: 44-48. Kotan, R. and Çelik, S. (2014). Mikrobiyal gübre ve biyopestisitlerin kullanımında dikkat edilmesi gereken hususlar. Harman Time, 14: 64-68. Küsek, M. (2007). Identification of grape crown gall disease caused by Agrobacterium vitis and research on possible control of the disease. (Ph.D. Thesis) Çukurova University, Institute of Natural and Applied Sciences, Plant Protection Department, Adana, Türkiye. Leslie, J. F. and Summerell, B. A. (2006). The Fusarium Laboratory Manual. Blackwell Publishing Professional, 2121 State Avenue, Ames, Iowa 50014, USA. pp. 388. Liu, S., Fu, L., Chen, J. et al. (2021). Baseline sensitivity of Sclerotinia sclerotiorum to metconazole and the analysis of cross-resistance with carbendazim, dimethachlone, boscalid, fluazinam, and fludioxonil. Phytoparasitica, 49(1): 123–130. Lucas, J. A. (1998). Plant Pathology and Plant Pathogens (3rd ed.). Blackwell Science, Oxford, pp. 274. Mansour, T., A., Nida, Y., A. and Patrice, S. (2008). Biological control of Sclerotinia sclerotiorum (Lib.) de Bary with Trichoderma harzianum and Bacillus amyloliquefaciens. Crop Protection, 27(10): 1354-1359. Mishra, J., Tewari, S., Singh, S. and Arora, N. K. (2015). Biopesticides: where We stand? In: Arora, N.K. (Ed.), Plant Microbes Symbiosis: Applied Facets,Springer, India, pp. 37–75. Moeinzadeh, A., Sharif-Zadeh, F., Ahmadzadeh, M. and Heidari Tajabadi, F. (2010). Biopriming of sunflower (Helianthus annuus L.) seed with Pseudomonas fluorescens for improvement of seed invigoration and seedling growth. Australian Journal of Crop Science, 4(7): 564-570. Montesinos, E., Bonaterra, A., Badosa, E, Francãç, J., Alemany, J., Llorente, I. and Moragrega, C. (2002). Plantmicrobe interactions and the new biotechnological methods of plant disease control. International Microbiology, 5(4): 169–175. Nelson, B., Duval, D. and Wu, H. (1988). An in vitro tecnique for large-scale production of sclerotia of Sclerotinia sclerotiorum. Phytopathology, 78: 1470-1472. Onaran, A. and Yanar, Y. (2011). Screening bacterial species for antagonistic activities against the Sclerotinia sclerotiorum (Lib.) De Bary causal agent of cucumber white mold disease. African Journal of Biotechnology,10(12): 2223-2229. Ouhaibi-Ben Abdeljalil, N., Vallance, J., Gerbore, J., Rey, P. and Daami-Remadi, M. (2016). Bio-suppression of Sclerotinia stem rot of tomato and biostimulation of plant growth using tomato-associated rhizobacteria. Journal of Plant Pathology and Microbiology,7(2): 1000331. Öztürk, Ö., Akınerdem, F., Bayraktar, N. and Ada, R. (2008). The ınvestigation of yield and ımportant agronomic characters of some hybrıd sunflower cultivars under Konya ırrigated conditions. Selçuk University The Agricultural Faculty Journal, 22(45): 11-20. Pal, K. K. and Gardener, B. (2006). Biological control of plant pathogens. The plant health instructor. https://doi.org/10.1094/PHI-A-2006-1117-02 (Accessed date: 13.09.2019). Rahman, M. M. E., Hossain, D. M., Suzuki, K., Shiiya, A., Suzuki, K. and Dey, T. K. (2016). Suppressive effects of Bacillus spp. on mycelia, apothecia and sclerotia formation of Sclerotinia sclerotiorum and potential as biological control of white mold on mustard. Australasian Plant Pathology, 45(1): 103-117. Rasheed, S., Dawar, S., Ghaffar, A. and Shaukat S. S. (2004). Seed borne mycoflora of groundnut. Pakistan Journal of Botany, 36(1): 199-202. Rocha, F. Y. O., Oliveira, C. M., Silva, P. R. A., Leona, Henrique Melo, V., Carmo, M. G. F. and Baldani, J. I. (2017). Taxonomical and functional characterization of Bacillus strains isolated from tomato plants and their biocontrol activity against races 1, 2 and 3 of Fusarium oxysporum f. sp. lycopersici, Applied Soil Ecology, 120: 8-19. Rosenberg, G., Steinberg, N., Oppenheimer-Shaanan, N., Olender, T., Doron, S., Ben-Ari, J., Sirota-Madi, A., Bloom-Ackermann, Z. and Kolodkin-Gal, I. (2016). Not so simple, not so subtle: The interspecies competition between Bacillus simplex and Bacillus subtilis and its impact on the evolution of biofilms. NPJ Biofilms Microbiomes, 2: 15027. Saharan, G. S. and Mehta N. (2008). Sclerotinia Diseases of Crop Plants: Biology, Ecology and Disease Management, Vol. LXII. Springer-Verlag GmbH, Heidelberg, Germany. Schwartz, A., Ortiz, I., Maymon, M., Herbold, C., Fujishige, N., Vijanderan, J., Villella, W., Hanamoto, K., Diener, A., Sanders, E., DeMason, D. and Hirsch, A. (2013). Bacillus simplex—a little known PGPB with anti-fungal activity—alters pea legume root architecture and nodule morphology when coinoculated with Rhizobium leguminosarum bv. viciae. Agronomy, 3(4):595–620. Sikorski, J. and Nevo, E. (2007). Patterns of thermal adaptation of Bacillus simplex to the microclimatically contrasting slopes of 'Evolution Canyons' I and II, Israel. Environ Microbiology, 9(3):716–726. Soylu, S., Soylu E. M., Kurt ġ. And Ekici Ö. K. (2005). Antagonistic potentials of rhizosphere-associated bacterial isolates against soil-borne diseases of tomato and pepper caused by Sclerotinia sclerotiorum and Rhizoctonia solani. Pakistan Journal of Biological Sciences, 8(1): 43–48. Tara, N. and Saharan, B. S. (2017). Plant growth promoting traits shown by bacteria Brevibacterium frigrotolerans SMA23 Isolated from Aloe vera rhizosphere. Agricultural Science Digest, 37(3): 226-231. Tozlu, E. (2003). Pasinler ovası’nda ayçiçeğinde gövde çürüklüğü hastalığını oluşturan Sclerotinia sclerotiorum (lib.) de bary ve Sclerotinia minor jagger’ın yayılışı, tanılanması, patojeniteleri ve biyolojik kontrolü. (Doktora Tezi Basılmamış) Atatürk Üniversitesi Fen Bilimleri Enstitüsü, Erzurum, Türkiye. Tozlu, E., Mohammadi, P., Senol Kotan, M., Nadaroglu, H. and Kotan, R. (2016). Biological control of Sclerotinia sclerotiorum (Lib.) de Bary, the causal agent of white mould disease in red cabbage, by some bacteria. Plant Protection Science, 52: 188–198. Tsavkelova, E. A., Cherdyntseva, T. A., Botina, S. G. and Netrusov, A. I. (2007). Bacteria associated with orchid roots and microbial production of auxin. Microbiological Research, 162(1): 69–76. Vijayakumari, S. J., Sasidharannair, N. K., Nambisan, B. and Mohandas, C. (2013). Optimization of media and temperature for enhanced antimicrobial production by bacteria associated with Rhabditis sp. Iranian Journal of Microbiology, 5(2): 136–141. Vuong, T. D., Hoffman, D.D., Diers, B. W., Miller, J.F., Steadman, J. R. and Hartman, G.L. (2004). Evaluation of soybean, dry bean, and sunflower for resistance to Sclerotinia sclerotiorum. Crop Science, 44(3): 777–783. Wang, Y., Fang, X., An, F., Wang, G. and Zhang, X. (2011). Improvement of antibiotic activity of Xenorhabdus bovienii by medium optimization using response surface methodology. Microbial Cell Factories, 10: 98. Warcup, J. H. (1958). Distribution and Detection of Root- Disease Fungi. Plant Pathology Problems and Progress (Ed.) C. S: Hulton, G. W. Fulton, Helen Hert, SEA, Mc Callon The Ragents of the Universty of Wisconsen, 317–324. Weller, D. M. (1988). Biological control of soilborn plant pathogens in the rhizosphere with bacteria. Annual Review of Phytopathology, 26: 379–407. Williams, L. D., Burdock, G. A., Jiménez, G. and Castillo, M. (2009). Literature review on the safety of Toyocerin, a non-toxigenic and non-pathogenic Bacillus cereus var. toyoi preparation. Regulatory Toxicology and Pharmacology, 55(2): 236–246. Xiaoning, G., Qingmei, H., Yafei, C., Huqiang, Q., Lili, H. and Zhensheng, K. (2014). Biological control of oilseed rape Sclerotinia stem rot by Bacillus subtilis strain Em7. Biocontrol Science and Technology, 24: 39–52. Xu, D. and Côte, J. C. (2003). Phylogenetic relationships between Bacillus species and related genera inferred from comparison of 3′ end 16S rDNA and 5′ end 16S–23S ITS nucleotide sequences. International Journal of Systematic and Evolutionary Microbiology, 53(3): 695–704. Yörük, B. and Mirik, M. (2021). Determination of in vitro biocontrol potentials of antagonist bacterial isolates against walnut blight disease agent Xanthomonas arboricola pv. juglandis. Journal of Tekirdag Agricultural Faculty, 18(3): 569–577. Yörükçe, M. A., Aktaş, B., Geroğlu, Y., Poyrazoğlu Çoban, E. and Bıyık, H. H. (2017). Isolation and identification of bacteria from fruit garden soils in Aydın Province. International Journal of Secondary Metabolite, 4(2): 66–73. Zeng, W., Kirk, W. and Hao, J. (2012). Field management of Sclerotinia stem rot of soybean using biological control agents. Biological Control, 60(2): 141–147. Zhang J. X. and Xue A. G. (2010). Biocontrol of Sclerotinia stem rot (Sclerotinia sclerotiorum) of soybean using novel Bacillus subtilis strain SB24 under control conditions. Plant Pathology, 59(2): 382–391.