Molecular Characterization of Botrytis cinerea Isolates from Different Host Plants
Botrytis cinerea Isolation
DOI:
https://doi.org/10.63939/4y4dq916Keywords:
Strawberry gray mold, Botrytis cinerea, fungicides, ITS-rDNA, Molecular identification, Plant extracts, Biological controlAbstract
Background: Botrytis cinerea gray mold is one of the most significant diseases that limits strawberry productivity under field and postharvest conditions. It is essential to accurately identify the pathogen and examine potential control options to develop effective disease-management strategies. Objective: This study aimed to isolate and identify Botrytis cinerea associated with gray mold of strawberry, validate representative isolates through ITS-rDNA sequencing, determine their pathogenicity, and perform an in vitro assessment of the efficacy of selected fungicides, antagonistic microorganisms (AMO) and botanical extracts. Methodology: From Dec. 2024 to Mar. 2025, symptomatic strawberry samples were collected from various growing areas of northern Iraq. Isolation of the fungal isolates was performed on potato dextrose agar and subsequently purified with the hyphal-tip technique. Identification was performed first through colony and microscopic characteristics, then confirmed with pathogenicity tests and amplifying the internal transcribed spacer region of ribosomal DNA (ITS-rDNA) using primers ITS1 and ITS4. PCR products were sequenced, compared with reference sequences in the NCBI database using BLAST and analyzed phylogenetically using MEGA. In vitro evaluations were conducted to assess the inhibitory effects of selected fungicides, microbial antagonists, and plant extracts. Data were analyzed by analysis of variance and means compared using Tukey's test at p < 0.05 when appropriate. Results: B. cinerea was confirmed in infected strawberry tissues by morphology determination and pathogenicity test. ITS-rDNA sequencing of representative isolates revealed significant homology with reference B. cinerea sequences found in GenBank, and phylogenetic analysis confirmed that the tested isolates fell within the B. cinerea group. Isolates evaluated differed in virulence. In vitro, all tested fungicides decreased fungal growth, compared to the control with dicarboximide having the lowest EC50 followed by benzimidazole and then other fungicides. Biological Control Trichoderma spp. showed the highest antagonistic activity. The botanical extracts tested also inhibited the growth of fungi, and all separated these 12 strains into three antifungal bands; Allium sativum had the maximum antifungal effect, followed by Mentha spicata and Tagetes patula, Asystasia gangetica & Syzygium cumini & Rosmarinus officinalis & Achillea millefolium. Conclusion: Based on the results obtained, the current study concluded the presence of abnormally shaped colonies through a multi-faceted approach that included pathogenesis tests and ITS-rDNA sequencing to provide accurate identification of gray mold in strawberries (Botrytis cinerea). The study demonstrated significant variability among the isolates, highlighting the importance of accurate pathogen identification before implementing control measures. The results indicate that dicarboxymides, Trichoderma species, and garlic extract (Allium sativum) were the most effective chemical, biological, and phytochemical treatments, respectively. These findings support the feasibility of integrated management approaches that combine biological, phytochemical, and conventional fungicides for controlling gray mold in strawberries.
Downloads
References
1. Prasannath, K.; Galea, V.J.; Akinsanmi, O.A. (2023). Diversity and pathogenicity of species of Botrytis, Cladosporium, Neopestalotiopsis and Pestalotiopsis causing flower diseases of macadamia in Australia. Plant Pathol. 2023, 72, 881–899.
2. Brauna-Morževska, E.; Stoddard, F.L.; Bankina, B.; Kaņeps, J.; Bimšteine, G.; Petrova, I.; Fridmanis, D. (2023). Evaluation of pathogenicity of Botrytis species isolated from different legumes. Front. Plant Sci. 2023, 14, 1069126.
3. Moparthi, S.; Parikh, L.P.; Gunnink Troth, E.E.; Burrows, M.E. (2023). Identification and prevalence of seedborne Botrytis spp. in dry pea, lentil, and chickpea in Montana. Plant Dis. 2023, 107, 382392.
4. Nielsen, K.A.; Skårn, M.N.; Strømeng, G.M.; Brurberg, M.B.; Stensvand, A. (2022). Pervasive fungicide resistance in Botrytis from strawberry in Norway: Identification of the grey mould pathogen and mutations. Plant Pathol. 2022, 71, 1392–1403.
5. Maia, J.N.; Beger, G.; Pereira, W.V.; De Mio, L.L.M.; Duarte, H.D.S.S. (2021). Gray mold in strawberries in the Paraná state of Brazil is caused by Botrytis cinerea and its isolates exhibit multiple-fungicide resistance. Crop Prot. 2021, 140, 105415.
6. Kulkarni, S.J. (2022). An insight into research and investigations of gray mold focused on Botrytis cinerea. In Driving Factors for Venture Creation and Success in Agricultural Entrepreneurship; Arafat, M.Y., Saleem, I., Ali, J., Khan, A., Balhareth, H.H., Eds.; IGI Global: Hershey, PA, USA, 2022; pp. 273–289.
7. Backmann, L., Schmidtmann, K., Wegmann-Herr, P., Jürgens, A. and Scharfenberger-Schmeer, M. (2024). Molecular biological methods to assess different Botrytis cinerea strains on grapes. Microbiology Research, 15(2), 567-581. https://doi.org/10.3390/micr obiolres150 20037.
8. Gaber, M.A., Wagih, E.E., Shehata, M.R., Fahmy, M.M., and Abdel Wahab, H.A. (2020). Detection and characterization of Botrytis cinerea isolates from vegetable crops in Egypt. International Journal of Phytopathology, 8(3), 77-85. https://journals.esciencepress.net/i ndex. php/phytopath/article/view/2945.
9. Tuite, J. (1969) Plant Pathological Methods: Fungi and Bacteria. Burgess Publishing Company, 239 p.
10. Park, D., Shafer, O. T., Shepherd, S. P., Suh, H., Trigg, J. S., Taghert, P. H. (2008): The Drosophila basic helix - loop - helix protein DIMMED directly activates PHM, a gene encoding a neuropeptide - amidating enzyme. Molecular and Cellular Biology 28 (1): 410-421.
11. Shih, M., Ambady, N., Richeson, J. A., Fujita, K., & Gray,
H. M. (2002). Stereotype performance boosts: The impact of
self-relevance and the manner of stereotype activation.
Journal of Personality and Social Psychology, 83, 638–647
12. Möller, E., Bahnweg , G., Sandermann, H. Geiger, H. (1992): A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. - Nucleic Acids Research 20 (22): 6115.
13. Sanger, F., Air, G. M., Barrell, B. G., Brown, N. L., Coulson, A.R., Fiddes, J. C., Hutchison, C. A., Slocombe, P. M. & Smith, M. (1977). Nature 265,687-695
14. Kumar, S., Stecher, G., Tamura, K. (2016): MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. - Molecular Biology and Evolution 33(7): 1870 1874.
15. Fillinger, S. and Elad, Y. (Eds.). (2016). Botrytis-the fungus, the pathogen and its management in agricultural systems (Vol. 486). Cham, Switzerland: Springer International Publishing. https://link.springer.com/content/pdf/10.1007/978-3-319-23371 -0.pdf.
16. Perez, C., Pauli, M. and Bazerque, P. (1990). An Antibiotic Assay by Agar Well Diffusion Method. Acta Biologiae et Medicinae Experimentalis, 15, 113-115.
17. Steel, R.G.D., Torrie, J.H. and Dickey, D.A. (1997) Principles and Procedures of Statistics: A Biometrical Approach. McGraw Hill Book Company, New York.
18. Parducci, A., & Haugen, R. (1967). The frequency principle for com- parative judgments. Perception & Psychophysics, 2, 81-82
19. Petrasch, S. Knapp. S. J. Van Kan, J. A., Blanco - Ulate, B. (2019): Grey mould of strawberry, a devastating disease caused by the ubiquitous necrotrophic fungal pathogen
20. Hassan, I., Mehmood, N., Riaz, A., Naz, F. (2019): Geographical distribution and morpho - molecular characterization of pre - harvest gray mold of strawberry in Punjab, Pakistan. - Mycopath 15 (2).
21. Card, S. D. (2005). Biological control of Botrytis cinerea in lettuce & strawberry crops. - A thesis submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy at Lincoln University, Canterbury, New Zealand
22. Fernández - Ortuño, D., Li, X., Wang. F., Schnabel, G. (2012): First report of gray mold of strawberry caused by Botrytis caroliniana in North Carolina. - Plant Disease 96 (6): 914
23. Maia, J. N., Beger, G., Pereira, W. V., De Mio, L. L. M., Duarte, H. d. S. S. (2021): Gray mold in strawberries in the Paraná state of Brazil is caused by Botrytis cinerea and its isolates exhibit multiple - fungicide resistance. - Crop Protection 140: 105415.
24. Chen, X., Wang, Y., Gao, Y., Gao, T., Zhang, D. (2019): Inhibitory abilities of Bacillus isolates and their culture filtrates against the gray mold caused by Botrytis cinerea on postharvest fruit. - The Plant Pathology Journal 35 (5): 425.
25. Chen, X., Wang, Y., Gao, Y., Gao, T., Zhang, D. (2019): Inhibitory abilities of Bacillus isolates and their culture filtrates against the gray mold caused by Botrytis cinerea on postharvest fruit. - The Plant Pathology Journal 35 (5): 425.
26. Williamson, B., Tudzynski, B., Tudzynski, P. and Van Kan, J.A.L. 2007. Botrytis cinerea: The cause of grey mould disease. Molecular Plant Pathology, 8(5), 561–580. https://doi.org/10.11 11 /j.1364-3703.2007.00417. x.
27. Staats, M., van Baarlen, P. and van Kan, J.A.L. 2005. Molecular phylogeny of the plant pathogenic genus Botrytis and the evolution of host specificity. Molecular Biology and Evolution, 22(2), 333–346. https://doi.org/10.1093/molbev/ msi020.
28. Elad, Y., Williamson, B., Tudzynski, P. and Delen, N. (Eds.). 2007. Botrytis: biology, pathology and control (pp. 1-8). Sprin. https://link.springer.com/content/pdf/10.1007/978-1-4020-2626-3.pdf.
29. Basu, A., Nguyen, A., Betts, N. M., Lyons, T. J. (2014): Strawberry as a functional food: an evidence - based review. - Critical Reviews in Food Science and Nutrition 54 (6): 790 – 806.
30. Bertetti, D., Garibaldi, A., Gullino, M. (2008): Resistance of Botrytis cinerea to fungicides in Italian vineyards. - Communications in Agricultural and Applied Biological Sciences 73 (2): 273-282.
31. A R Joseph. Improved Management Strategies for Botrytis Fruit Rot, Macrophomina Root Rot, and Verticillium Wilt in California Strawberry Production. DigitalCommons - CalPoly (California State Polytechnic University), https://digitalcommons.calpoly.edu/theses/3144 (2025, accessed February 2026).
32. Jameel SM, Rehman A, Rajput N, et al. MOLECULAR CHARACTERIZATION OF THE PATHOGEN CAUSING GREY MOLD IN STRAWBERRY AND ITS MANAGEMENT. Applied Ecology and Environmental Research 2022; 20: 465–478.
33. Naegele RP, Abdelsamad N, DeLong J, et al. Fungicide Resistance and Host Influence on Population Structure in Botrytis spp. from Specialty Crops in California. Phytopathology 2022; 112: 2549–2559.
34. Radi M, Lahlali R. Management Strategies of Grey Mould Disease in Berries. Plant Health Cases. Epub ahead of print 24 March 2025. DOI: 10.1079/planthealthcases.2025.0010.
35. Abbey J, Alzohairy SA, Neugebauer KA, et al. Fungicide resistance in Botrytis cinerea and identification of Botrytis species associated with blueberry in Michigan. Frontiers in Microbiology 2024; 15: 1425392–1425392.
36. El‐Beltagi HS, Abbas E, Almutairi HH, et al. Controlling Botrytis gray mold in strawberry fruit by bioactive protein isolated from kidney bean. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 2024; 52: 13783–13783.
37. Singh R, Caseys C, Kliebenstein DJ. Genetic and molecular landscapes of the generalist phytopathogen Botrytis cinerea. Molecular Plant Pathology; 25. Epub ahead of print 1 December 2023. DOI: 10.1111/mpp.13404.
38. Iqbal M, Flöhr A, Andréasson E, et al. Breeding for Integrated Pest Management (B-IPM): a new concept simultaneously optimising plant resistance and biocontrol. Frontiers in Plant Science 2025; 16: 1659069–1659069.
39. Rehman A, Ali F, Hameed A, et al. Management Strategies for Botrytis Rot in Strawberry. In: IntechOpen eBooks. IntechOpen. Epub ahead of print 8 November 2023. DOI: 10.5772/intechopen.1002225.
40. Capriotti L, Molesini B, Pandolfini T, et al. RNA interference-based strategies to control Botrytis cinerea infection in cultivated strawberry. Plant Cell Reports 2024; 43: 201–201.
41. Šavli K, ŽUNIČ JZ, Celar F. Non-chemical control options against grey mould (Botrytis cinerea) on strawberry (Fragaria x anannasa). Journal of Central European Agriculture 2025; 26: 1015–1027.
42. Kim G, Son D, Choi S-G, et al. Monitoring of Benzimidazole Resistance in Botrytis cinerea Isolates from Strawberry in Korea and Development of Detection Method for Benzimidazole Resistance. The Plant Pathology Journal 2023; 39: 614–624.
43. Neugebauer KA, Mattupalli C, Hu M, et al. Managing fruit rot diseases of Vaccinium corymbosum. Frontiers in Plant Science 2024; 15: 1428769–1428769.
44. Keldibekova M, Knyazev SD. Genes and Loci Controlling the Resistance of Strawberry (F. ananassa Duch.) to Pathogens. Indian Journal of Agricultural Research. Epub ahead of print 17 November 2023. DOI: 10.18805/ijare.af-808.
Downloads
Published
Issue
Section
License
Copyright (c) 2026 International Journal of Environmental and Biological Sciences
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
