Kızıl Kaliforniya solucanı sölom sıvısının Alternaria solani’ye karşı in vitro biyokontrol potansiyeli

Öz

Bu çalışmada, Kızıl Kaliforniya solucanından (Eisenia fetida) elektro-stimülasyon yöntemiyle elde edilen sölom sıvısının, domateste erken yanıklık hastalığına neden olan önemli fitopatojen Alternaria solani üzerindeki in vitro antagonist ve biyokontrol potansiyeli araştırılmıştır. Sölom sıvısının biyoaktivitesi üzerindeki metodolojik etkileri belirlemek amacıyla Tek Santrifüjlü (TS) ve Çift Santrifüjlü (ÇS) formülasyonlar ile Ham ve Steril Filtreli (0.22 µm) uygulamalar ikili kültür testlerinde karşılaştırılmıştır. Elde edilen bulgular, filtresiz ham sölom sıvısı ortamında matrisin doğasından kaynaklanan endojen Bacillus sp. ve sekonder mikrobiyota gelişiminin gözlendiğini; doğrudan sölomolitik hümoral etkinin net olarak değerlendirilebilmesi için steril filtrasyonun zorunlu olduğunu göstermiştir. Steril filtre edilmiş sölom sıvısı testlerinde, inkübasyonun 18. gününde kontrol grubunda inhibisyon bölgesi tamamen kapanırken (0.50 mm), Tek Santrifüjlü (TS) steril sölom sıvısı 24.34 mm'lik güçlü ve kararlı bir inhibisyon zonu oluşturarak fungal misel gelişimini yüksek oranda baskılamıştır. Buna karşın Çift Santrifüjlü (ÇS) grupta inhibisyon zonu 6.81 mm'ye gerilemiştir. Sonuç olarak, sterilize edilmiş Tek Santrifüjlü (TS) E. fetida sölom sıvısının, A. solani kontrolünde çevreci ve sürdürülebilir bir biyopestisit bileşeni olarak güçlü bir potansiyel taşıdığı ortaya konmuştur.

Anahtar Kelimeler

• Antifungal aktivite
• Eisenia fetida
• inhibisyon zonu
• toprak solucanı

In vitro biocontrol potential of coelomic fluid from the red Californian earthworm against Alternaria solani

Öz

In this study, the in vitro antagonist and biocontrol potential of coelomic fluid extracted from the Red Californian earthworm (Eisenia fetida) via electro-stimulation was investigated against Alternaria solani, an important phytopathogen causing early blight in tomatoes. To determine the methodological effects on the bioactivity of the coelomic fluid, Single-Centrifuged (TS) and Double-Centrifuged (CS) formulations, along with Crude and Sterile-Filtered (0.22 μm) applications, were compared using dual-culture assays. The findings revealed that endogenous Bacillus sp. and secondary microbiota naturally present in the crude coelomic matrix colonized the unfiltered culture media, demonstrating that sterile filtration is essential to accurately evaluate the direct coelomolytic humoral effect. In sterile-filtered coelomic fluid assays, while the inhibition zone in the control group was almost completely closed (0.50 mm) on the 18th day of incubation, the Single-Centrifuged (TS) sterile coelomic fluid created a strong and stable inhibition zone of 24.34 mm, highly suppressing fungal mycelial growth. Conversely, in the Double-Centrifuged (CS) group, the inhibition zone dropped to 6.81 mm. In conclusion, sterilized Single-Centrifuged (TS) E. fetida coelomic fluid shows strong potential as an eco-friendly and sustainable biopesticide component for controlling A. solani.

Anahtar Kelimeler

• Antifungal activity
• earthworm
• Eisenia fetida
• inhibition zone

Kaynakça

1. Anonim, 2024. Domates, patlıcan ve patateste erken yanıklık hastalığı (Alternaria solani). Kayseri İl Tarım ve Orman Müdürlüğü, Kayseri.
2. Anonim, 2025. Zirai Mücadele Teknik Talimatları, Cilt 3: Domates, Patlıcan ve Patateste Erken Yanıklık Hastalığı (Alternaria solani Ell. & Mart.). T.C. Tarım ve Orman Bakanlığı, Tarımsal Araştırmalar ve Politikalar Genel Müdürlüğü (TAGEM), Ankara.
3. Atherton J.G. & J. Rudich, 1986. The tomato crop: A scientific basis for improvement. Chapman and Hall, London. Aydilek S., 2012. Toprak solucanlarından elde edilen sölom sıvılarının çeşitli hücreler üzerine etkisi. Yüksek Lisans Tezi, İstanbul Üniversitesi Fen Bilimleri Enstitüsü, İstanbul.
4. Batista D.C., M.A. Lima, F. Haddad, L.A. Maffia & E.S.G. Mizubuti, 2006. Validation of decision support systems for tomato early blight and potato late blight under Brazilian conditions. Crop Protection, 25(7): 664-670.
5. Beschin A., M. Bilej, F. Hanssens, J. Raymakers, E. Van Dijck, H. Revets, ... & P. De Baetselier, 1998. Identification and cloning of a 42-kDa coelomic fluid protein, CCF-1, a novel pattern recognition protein of Eisenia foetida reacting with β-1,3-glucan and lipopolysaccharide. Journal of Biological Chemistry, 273(38): 24944-24948.
6. Bilej M., L. Brys, A. Beschin, R. Lucas, E. Vercauteren, R. Hanušová & P. De Baetselier, 1995. Identification of a cytolytic protein in the coelomic fluid of Eisenia foetida earthworms. Immunology Letters, 45(1-2): 123-128.
7. Bilej M., De Baetselier P. & A. Beschin, 2000. Antimicrobial defense of the earthworm Eisenia foetida. Folia Microbiologica, 45(4): 283-300.
8. Bilej M., P. Procházková, M. Šilerová & R. Josková, 2010. Earthworm immunity. Advances in Experimental Medicine and Biology, 708: 66-79.

9. Byzov B.A., N.V. Khomyakov, S.A. Kharin & A.V. Kurakov, 2009. Fate of soil bacteria and fungi in the gut of earthworms. European Journal of Soil Biology, 45(3): 209-215.
10. Chaerani R. & R.E. Voorrips, 2006. Tomato early blight (Alternaria solani): The pathogen, genetics, and breeding for resistance. Journal of General Plant Pathology, 72(6): 335-347.
11. Cooper E.L., E. Kauschke & A. Cossarizza, 2002. Digging for innate immunity since Darwin in Lumbricus terrestris. BioEssays, 24(4): 319-333.
12. Coperchini F., P. Engelmann, B. Opper, C. Bodó & P. Nemeth, 2015. Effect of temperature and pH variation on the cytotoxic and hemolytic activity of Eisenia fetida coelomic fluid. Developmental & Comparative Immunology, 50(2): 115-121.
13. Danafar N. & M. Shokoohi, 2025. In vitro investigations of coelomic fluid of Eisenia fetida: Protein analysis, antioxidant activities and antibacterial effects on diabetic wounds' bacteria. Iranian Journal of Microbiology, 17(1): 163-170.
14. Dennis C. & J. Webster, 1971. Antagonistic properties of species-groups of Trichoderma: I. Production of non-volatile antibiotics. Transactions of the British Mycological Society, 57(1): 25-39.
15. Domínguez J. & C.A. Edwards, 2011. Biology and ecology of earthworm species used for vermicomposting. In: Edwards C.A., N.Q. Arancon & R.L. Sherman (Eds.), Vermiculture Technology: Earthworms, Organic Wastes, and Environmental Management. CRC Press, Boca Raton, pp. 25-37.
16. Dura S. & Ö. Murat, 2024. 2023 ve 2024 yıllarına ait Türkiye domates üretiminin değerlendirilmesi. Tarım Gündemi Dergisi.
17. Ečimović S., K. Vrandečić, M. Kujavec, M. Žulj, J. Ćosić & M. Velki, 2021. Antifungal activity of earthworm coelomic fluid obtained from Eisenia andrei, Dendrobaena veneta and Allolobophora chlorotica on six species of phytopathogenic fungi. Environments, 8(10): 102.
18. Fuller-Espie S.L., D.R. Harris, J.H. Daly & J.M. Jakeman, 2015. Optimization of cell culture conditions for the earthworm Eisenia hortensis: A study investigating the effects of media, carbon dioxide, temperature, serum, and anti-fungal agents. Journal of the Pennsylvania Academy of Science, 89(2): 57-68.
19. Gudeta K., A. Bhagat, J.M. Julka, R. Sinha, R. Verma, A. Kumar, S. Kumari, F. Ameen, S. A. Bhat, R. Amarowicz & M. Sharma, 2022. Vermicompost and its derivatives against phytopathogenic fungi in the soil: A review. Horticulturae, 8(4): 311.
20. Hua Z., Y.H. Wang, H.W. Cao, L.J. Pu & Y.D. Cui, 2011. Purification of a protein from coelomic fluid of the earthworm Eisenia foetida and evaluation of its hemolytic, antibacterial, and antitumor activities. Pharmaceutical Biology, 49(3): 269-275.
21. Kauschke E., W. Mohrig & E.L. Cooper, 1997. Coelomic fluid properties of earthworms. Invertebrate Survival Journal, 1(1):1-12.
22. Knogge W., 1996. Fungal infection of plants. The Plant Cell, 8(10): 1711-1722.
23. Kobayashi H., M. Ohtomi, Y. Sekizawa & N. Ohta, 2001. Toxicity of coelomic fluid of the earthworm Eisenia foetida to vertebrates but not invertebrates: Probable role of sphingomyelin. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 128(3): 401-411.
24. Kocakurt H., 2022. Kırmızı toprak solucanı Eisenia fetida'dan elde edilen sölom sıvısının özelliklerinin araştırılması. Yüksek Lisans Tezi, Bursa Uludağ Üniversitesi Fen Bilimleri Enstitüsü, Bursa.
25. Kumari T. & V. Shukla, 2022. Comparative impact of fungicides on biomolecules of Eisenia fetida. Bulletin of Pure and Applied Sciences-Zoology (Animal Science), 41(1): 1-13.
26. Lassegues M., A. Milochau, F. Doignon, L. Du Pasquier & P. Valembois, 1997. Sequence and homology of an antibacterial protein, fetidin, from the earthworm Eisenia fetida andrei. Biochemical and Biophysical Research Communications, 238(3): 680-684.
27. Milochau A., M. Lassegues & P. Valembois, 1997. Purification, characterization and activities of two hemolytic proteins from Eisenia fetida andrei. Biochimica et Biophysica Acta (BBA)-General Subjects, 1337(1): 123-132.
28. Mutschler M., T. Zitter & C. Born, 2006. Tomato lines for the northeast combining early blight and late blight resistance. 22nd Annual Tomato Disease Workshop, Vegetable Program, New York.
29. Plavšin I., M. Velki, S. Ečimović, K. Vrandečić & J. Ćosić, 2017. Inhibitory effect of earthworm coelomic fluid on growth of the plant parasitic fungus Fusarium oxysporum. European Journal of Soil Biology, 78: 1-6.
30. Popović M., M. Milutinović & M. Stojanović, 2005. Enzymatic activities in coelomic fluid of earthworms and their potential role in phytopathogen inhibition. Soil Biology and Biochemistry, 37(8): 1540-1547.
31. Prakash M., N. Karmegam & T. Daniel, 2018. Extracellular enzymatic profiles of coelomic fluid extracted from Eisenia fetida. Journal of Advanced Zoology, 39(1): 45-52.
32. Procházková P., M. Šilerová, J. Felsberg, R. Josková, A. Beschin, P. De Baetselier & M. Bilej, 2006. Relationship between coelomic fluid proteins and defense mechanisms in Eisenia fetida. Developmental & Comparative Immunology, 30(11): 981-988.
33. Rotem J., 1994. The genus Alternaria: Biology, epidemiology and pathogenicity. APS Press, St. Paul.
34. Sanaiirad S., M. Seifi, N. Hemmati, A. Khosravi & D. Nikaein, 2025. In vitro study of antifungal effects of earthworm coelomic fluid obtained from Eisenia fetida on three opportunistic fungal pathogens. Current Medical Mycology, 11:e1637.
35. Sekizawa Y., T. Kubo, H. Kobayashi, T. Nakajima & S. Natori, 1997. Molecular cloning of cDNA for lysenin, a novel protein in the coelomic fluid of earthworm Eisenia foetida, inducing contraction of rat vascular smooth muscle. Gene, 191(1): 97-102.
36. Toyota K. & M. Kimura, 2000. Microbial community in the earthworm cast and gut. Biology and Fertility of Soils, 31(5): 365-371.
37. Tutar U. & İ. Karaman, 2017. Investigation of antibacterial properties of mucus and coelomic fluid obtained from Eisenia fetida. Cumhuriyet Science Journal, 38(3): 427-434.
38. Tutar U., 2013. Toprak solucanlarından elde edilen vermikompostun bazı bitki patojenleri üzerindeki antimikrobiyal aktivitelerinin araştırılması. Yüksek Lisans Tezi, Cumhuriyet Üniversitesi Fen Bilimleri Enstitüsü, Sivas.
39. Van der Waals J.E., L. Korsten & B. Slippers, 2004. Genetic diversity among Alternaria solani isolates from potatoes in South Africa. Plant Disease, 88(9): 959-964.
40. Yanqin L., S. Yan, S. Zhenjun, L. Shijie, W. Chong, L. Yan & G. Yuhong, 2007. Coelomic fluid of the earthworm Eisenia fetida induces apoptosis of HeLa cells in vitro. European Journal of Soil Biology, 43: 143-148.
41. Yıldız M., O. Gürkan, C. Turgut, Ü. Kaya & G. Ünal, 2005. Tarımsal savaşımda kullanılan pestisitlerin yol açtığı çevre sorunları. VI. Türkiye Ziraat Mühendisliği Teknik Kongresi, TMMOB Ziraat Mühendisleri Odası, 3-7 Ocak 2005, Ankara, s. 22.