Molecular fingerprinting of Botrytis cinerea population structure from different hosts

Abstract

Botrytis cinerea (teleomorph: Botryotinia fuckeliana) causes gray mold disease on vegetable crops in greenhouses. Profound knowledge on pathogen diversity is necessary for efficiently disease management. In this study, forty-two B. cinerea isolates collected from 36 different greenhouses in Antalya province of Turkey were investigated. Twelve SRAP (sequence-related amplified polymorphism) and 18 ISSR (inter simple sequence repeat) primers producing high polymorphic fragments were used to genetic diversity of B. cinerea isolates infecting dill, basil, lettuce, bean, cucumber, tomato, pepper and eggplant. The unweighted pair-group method with arithmetic average analysis (UPGMA) was used to evaluate of combined ISSR and SRAP data showing a similarity range 0.15-0.90 among the isolates. Cophenetic correlation of the tree was high level (r=0.93). Interestingly, cluster analysis showed a divergent group consisting of lettuce isolates which were genetically different from the other isolates. On the other hand, transposable elements (Flipper and Boty) were detected among isolates from all the hosts. Isolates containing only the Fliper element were detected. The results showed that genetically characterized B. cinerea populations by a high level of genetic diversity were associated with genotype flow and the evolutionary potential of B. cinerea. In further studies, the newly tested molecular markers are useful and can be suggested for analyzing of genetic diversity and population structure of this pathogen on different hosts.

Keywords

• ISSR,SRAP,ISSR,SRAP,Gray mold,Genetic diversity,Host differentiation,ISSR,SRAP,Transposable elements

Farklı konukçulardan elde edilen Botrytis cinerea populasyon yapısının moleküler tanılanması

Abstract

Botrytis cinerea (teleomorph: Botryotinia fuckeliana) örtüaltı sebze yetiştiriciliğinde kurşuni küf hastalığı etmenidir. Patojende oluşan farklılıkların bilinmesi hastalıkla mücadelenin etkinliğini arttırmaktadır. Çalışmada, Türkiye’nin Antalya ilinde yer alan 36 farklı seradan 42 adet izolat kullanılmıştır. On iki SRAP (sequence-related amplified polymorphism) primer kombinasyonu ve 18 ISSR (inter simple sequence repeat) primeri dereotu, fesleğen, marul, fasulye, hıyar, domates, biber ve patlıcandan elde edilen B. cinerea izolatlarının genetik farklılıklarının belirlenmesinde oldukça yüksek polimorfizm sağlamışlardır. ISSR ve SRAP markırlardan elde edilen sonuçlar UPGMA (The unweighted pair-group method with arithmetic average analysis) analizine göre izolatlar arasında 0.15-0.90 oranında değişen benzerlik elde edilmiştir. Ayrıca, cophenetic correlation değeri r=0.93 ile oldukça yüksek bulunmuştur. Cluster analizi sonuçları değerlendirildiğinde marul izolatları diğer izolatlara göre oldukça uzak kümelenmiştir. Ayrıca, tüm izolatlar için tranpozabl elementler (Flipper ve Boty) araştırılmış ve sadece Flipper element tespit edilmiştir. Elde edilen genetik karakterizasyon sonuçlarına göre, B. cinerea populasyonunda oldukça yüksek seviyede genetik farklılıklar bulunmuştur. Bu durum, B. cinerea’nın evrimselleşme potensiyeli ve gen akışlarından kaynaklanabilir. Farklı konukçulardan elde edilen bu patojenin genetik farklılıklarının belirlenmesinde kullanılan moleküler markırlar, ileride yapılacak çalışmalara da ışık tutmaktadır.

Keywords

• ISSR,SRAP,ISSR,SRAP,Kurşuni küf,Genetik farklılık,Konukçu farklılığı,ISSR,SRAP,Transpoazabl elementler

References

1. Aka-Kacar, Y., Demirel, A., Tuzcu, O., Yesiloglu, T., Ulas, M., & Yildirim, B. (2005). Preliminary results on fingerprinting lemon genotypes tolerant to Mal Secco (Phoma tracheiphila Kanc. et Ghik) disease by RAPD markers. Biologia Bratislava, 60(3):295-300.
2. Angelini, R.M., Rotolo, C., Masiello, M., Pollastro, S., Ishii, H., & Faretra, F. (2012). Genetic analysis and molecular characterisation of laboratory and field mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to QoI fungicides. Pest Management Science, 68(9):1231-1240.
3. Asadollahi, M., Fekete, E., Karaffa, L., Flipphi, M., Árnyasi, M., Esmaeili, M., Váczy, K.Z., & Sándor, E. (2013). Comparison of Botrytis cinerea populations isolated from two open-field cultivated host plants. Microbiological Research, 168(6):379-388.
4. Baysa,l Ö., Karaaslan, Ç., Siragusa, M., Allesandro, R., Carimi, F., De Pasquale, F., & Teixeira Da Silva, J.A. (2013). Molecular markers reflect differentiation of Fusarium oxysporum forma specials on tomato and forma on eggplant. Biochemical Systematics and Ecology, 47:139-147.
5. Baysal, Ö., Mercati, F., Ikten, H., Çetinkaya Yıldız, R., Carimi, F., Aysan, Y., & Teixeira da Silva, J.A. (2011). Clavibacter michiganensis subsp. michiganesis: Tracking strains using their genetic differentiations by ISSR markers in Southern Turkey. Physiological and Molecular Plant Pathology, 75(3):13-119.
6. Baysal, Ö., Siragusa, M., İkten, H., Polat, İ., Gümrükcü, E., Yiğit, F., Carimi, F., & Teixeira da Silva, J.A. (2009). Fusarium oxysporum f.sp. lycopersici races and their genetic discrimination by molecular markers in West Mediterranean region of Turkey. Physiological and Molecular Plant Pathology, 74(1):68-75.
7. Beever, R.E., & Weeds, P.L. (2004). Taxonomy and genetic variation of Botrytis and Botryotinia. pp. 29-52 In: Elad Y, Williamson B, Tudzynski P, Delen N, (eds). Botrytis, Biology, Pathology and Controls. Kluwer Academic Publisher. Netherland.
8. Brent, K.J., & Hollomon, D.W. (1998). Fungicide resistance: the assessment of risk. FRAC Monograph No. 2, Brussels.

9. Carriço, J.A., Pinto, F.R., Simmas, C., Nunes, S., Sousa, N.G., Frazao, N., Lencastre, H., & Almeida, J.S. (2005). Assesment of band-based similarity coefficient for automatic type and subtype classification of microbial isolates analyzed by pulse field gel electrophoresis. Journal of Clinical Microbiology, 43(11):5483-5490.
10. Choi, I.S., Kim, D.H., Lee, C.W., Kim, J.W., & Chung, Y.R. (1998). Analysis of genetic variation in B. cinerea isolates using Random Amplified Polymorphic DNA markers. Journal of Microbiology and Biotechnology, 8(5):490-496.
11. Cui, Z.J. (2013). Identification of sporulational group and analysis of genetic polymorphism of Botrytis cinerea. Master Thesis, China.
12. Datta, D., Gupta, S., Chaturvedi, S.K., & Nadarajan, N. (2011). Molecular markers in crop improvement. Indian Institute of Pulses Research, Kanpur - 208 024.
13. Dice, L.R. (1945). Measures of the amount of ecologic association between species. Ecology, 26:297-302.
14. Diolez, A., Marches, F., Fortini, D., & Brygoo, Y. (1995). Boty, a long-terminal-repeat retro-element in the phytopathogenic fungus Botrytis cinerea. Applied and Environmental Microbiology, 61(1):103-108.
15. Domsch, K.H., Gams, W., & Anderson, T. (1993). Compendium of Soil Fungi. New York, Academic Press.
16. Dorsaf Ben, A., & Hamada, W. (2005). Genetic diversity of some Tunisian Botrytis cinerea isolates using molecular markers. Phytopathologia Mediterranea, 44:300-306.
17. Doveri, S., Lee, D., Maheswaran, M., & Powell, W. (2008). Molecular markers: History, features and applications. In Principles and Practices of Plant Genomics, Volume 1, C.K.a.A.G. Abbott, ed. (Enfield, USA: Science Publishers), pp. 23-68.
18. Elad, Y., Williamson, B., Tudzynski, P., & Delen, N. (2007). Chapter 1: Botrytis spp. and diseases they cause in agricultural systems–an introduction. Y. Elad et al. (eds.), Botrytis: Biology, Pathology and Control, 1-8. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
19. FAO, (2014). Food and agriculture organisation of the United Nations. www.fao.org/faostat Accessed date:12 December 2016.
20. Finkers, R., van den Berg, P., van Berloo, R., ten Have, A., van Heusden, A.W., van Kan, J.A.L., & Lindhout, P. (2007). Three QTLs for Botrytis cinerea resistance in tomato. Theoretical and Applied Genetics, 114(4):585-593.
21. Fournier, E., Giraud, T., Loiseau, A., Vautri, D., Estoup, A., Solignac, M., Cornuet, J.M., & Brygoo, Y. (2002). Characterization of nine polymorphic microsatellite loci in the fungus Botrytis cinerea (Ascomycota). Molecular Ecology Notes, 2:253-255.
22. Hammer, R., Harper, D.A.T., & Ryan, P.D. (2001). PAST: Paleontological statistics software package for education and data analysis. Palaeontol, Electronica, 4(1):9.
23. Hennebert, G.L., (1973). Botrytis and botrytis-like genera. Persoonia, 7: 183-204.
24. Hildebrand, C.E., Torney, D.C., & Wagner, R.P. (1992). Mapping the genome. Informativeness of polymorphic DNA markers. Los Alamos Science, 20:100-102.
25. Ingram, D.M., & Meister, C.W. (2006). Managing Botrytis Gray Mold in Greenhouse Tomatoes Using Traditional and Bio-Fungicides. Plant Management Network.
26. Isenegger, D.A., Macleod, W.J., Ford, R., & Taylor, P.W.C. (2008). Genotypic diversity and migration of clonal lineages of Botrytis cinerea from chickpea fields of Bangladesh inferred by microsatellite markers. Plant Pathology, 57(5):967-973.
27. Jaccard, P. (1907). La distribution de la flore dans la zone alpine. Revue générale des sciences pures et appliqués, 18:961-967
28. Jarvis, J.W. (1980). The Biology of Botrytis. Edited by J.R. Coley-Smith, K. Verhoeff, W.R. Jarvis. Taxonomy. Academic Press. A Subsidiary of Harcourt Brace Jovanovich, Publishers. London New York Toronto Sydney San Francisco.
29. Jarvis, W.R. (1977). Botryotinia and Botrytis species: Taxonomy, Physiology and Pathogenicity, A guide to the Literature. Monograph No. 15, Canada Department of Agriculture, Ottawa, Canada.
30. Jensen, M.H. (2002). Controlled environment agriculture in deserts tropics and temperate regions – A world review. Acta Horticulturae, 578(1):19-25.
31. Kaur, G. (2015). Variability and management studies of Botrytis Cinerea causing grey mould in gladiolus. PhD Thesis. Dr Yashwant Singh Parmar University, India.
32. Kecskeméti, E., Brathuhn, A.,Kogel, K.H., Berkelmann-Löhnertz, B., & Reineke, A. (2014). Presence of transposons and mycoviruses in Botrytis cinerea ısolates collected from a German grapevine growing region. Journal of Phytopathology, 162(9):582-595.
33. Khazaeli, P., Zamanizadeh, H., Morid, B., & Bayat, H. (2010). Morphological and molecular identification of Botrytis cinerea causal agent of gray mold in rose greenhouses in central regions of Iran. International Journal of Agricultural Science and Research, 1(1):19-24.
34. Kumari, S., Tayal, P., Sharma, E., & Kapoor, R. (2014). Analyses of genetic and pathogenic variability among Botrytis cinerea isolates. Microbiological Research, 169(11): 862-872.
35. Levandowsky, M., & Winter, D. (1971). Distance between sets. Nature, 234(5):34-35.
36. Leyronas, C., Byrone, F., Duffaud, F., Troulet, C., & Nicot, P.C. (2015). Assessing host specialization of Botrytis cinerea on lettuce and tomato by genotypic and phenotypic characterization. Plant Pathology, 64(1):119-127.
37. Li, G., & Quiros, C.F., (2001). Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theoretical Applied Genetic, 103:455–461.
38. Ma, Z., & Michailides, T.J. (2005). Genetic structure of Botrytis cinerea populations from different host plants in California. Plant Disease, 89(10): 1083-1089.
39. Malvick, D., & Percich, J. (1998). Genotypic and pathogenic diversity among pea-infecting strains of Aphanomyces euteiches from the central and western United States. Phytopathology, 8889:915-921.
40. Milgroom, M.G. (1996). Recombination and the multilocus structure of fungal populations. Annual Review of Phytopathology, 34:457-77.
41. Mirzaei, S., Goltapeh, E.M., & Shams-Bakhsh, M. (2007). Taxonomical studies on the genus Botrytis in Iran. Journal of Agricultural Technology, 3(1):65-76.
42. Moyano, C., Alfonso, C., Gallego, J., Raposo, R., & Melgarejo, P. (2003). Comparison of RAPD and AFLP marker analysis as a means to study the genetic structure of Botrytis cinerea populations. European Journal of Plant Pathology, 109(5):515-522.
43. Paul, B. (2000). ITS1 region of rDNA of Pythium megacarpum sp. nov., its taxonomy and its comparison with related species. FEMS Microbiology Letters, 186(2):229-233.
44. Podani, J., & Miklós, I. (2002). On the horseshoe effect in ecological ordinations. 45th Symposium of the International Association for Vegetation Science, March 3-8, 2002, Porto Alegre, Brazil
45. Polat, İ., Baysal, Ö., Mercati, F., Kitner, M., Cohen, Y., Lebeda, A., & Carimi, F. (2014). Characterization of Pseudoperonospora cubensis isolates from Europe and Asia using ISSR and SRAP molecular markers. European Journal of Plant Pathology, 139(3):641-653.
46. Rigotti, S., Gindro, K., Richter, H., & Viret, O. (2002). Characterization of molecular markers for specific and sensitive detection of Botrytis cinerea Pers.: Fr. in strawberry (Fragaria X ananassa Duch.) using PCR. FEMS Microbiology Letters, 209(2):169-174.
47. Rosslenbroich, H.J., & Stuebler, D. (2000). Botrytis cinerea – history of chemical control and novel fungicides for its management. Crop Protection, 19(8-10):557-61.
48. Saitou, N., & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4):406-425.
49. Schlötterer, C. (2004). The evolution of molecular markers-just a matter of fashion? Nature Reviews Genetics, 5(1):63-69.
50. Shao, W., Ren, W., Zhang, Y., Hou, Y., Duan, Y., Wang, J., Zhou, M., & Chen, C. (2015). Baseline sensitivity of natural populations and characterization of resistant strains of Botrytis cinerea to fluazinam. Australasian Plant Pathology, 44(4):375-383.
51. Smith, J.S.C., Chin, E.C.L., Shu, H., Smith, O.S., Wall, S.J., Senior, M.L., Mitchel, S.E., Kresorich, S., & Tiegle, J. (1997). An evaluation of the utility of SSR loci as molecular markers in maize (Zea mays L.): comparisons with data from RFLPs and pedigree. Theoretical and Applied Genetics, 95(1-2):163-173.
52. Sun, H.Y., Wang, H.C., Chen, Y., Li, H.X., Chen, C.J., & Zhou, M.G. (2010). Multiple resistance of Botrytis cinerea from vegetable crops to carbendazim, diethofencarb, procymidone, and pyrimethanil in China. Plant Disease, 94(5):551-556.
53. Tanović, B., Delibašić, G., Milivojević, J., & Nikolić, M. (2009). Characterization of Botrytis cinerea isolates from small fruits and grapevine in Serbia. Archives of Biological Sciences, 61(3):419-429.
54. ten Have, A., van Berloo, R., Lindhout, P., & van Kan, J.A.L. (2007). Partial stem and leaf resistance against the fungal pathogen Botrytis cinerea in wild relatives of tomato. European Journal of Plant Pathology, 117(2):153-166.
55. Thompson, J.R., & Latorre, B.A. (1999). Characterization of Botrytis cinerea from Table Grapes in Chile Using RAPD-PCR. Plant Disease, 83(12):1090-1094.
56. TUIK, (2014). Turkish Statistical Institute. www.turkstat.gov.tr. Accessed date: 12 December 2016.
57. Tuzel, Y., & Leonardi, C. (2010). Protected cultivation in Mediterranean region: Trends and needs. Ege Üniversitesi Ziraat Fakültesi Dergisi, 46(3):215-223.
58. Ünlü, A., Baysal, Ö., Polat, İ., Sülü, S.M., İkten, H., Devran, Z., & Gümrükcü, E. (2017). Batı Akdeniz Bölgesi örtüaltı yetiştiriciliğinde sorun olan domateste Bakteriyel Benek (Pseudomonas syringae pv. tomato “Okabe” Y.D.&W) hastalık etmeni izolatlarının genetik farklılıklarının moleküler yöntemlerle tespiti. Derim, 34(2):122-130.
59. van Der Vlugt-Bergmans, C.J.B., Brandwagt, B.F., Vant Klooster, J.W., Wagemakers, C.A.M., & van Kan, J.A.L. (1993). Genetic variation and segregation of DNA polymorphisms in Botrytis cinerea. Mycological Research, 97(10):1193-1200.
60. Vercesi, A., Toffolatti, S.L., Venturini, G., Campia, P., & Scagnelli, S. (2014). Characterization of Botrytis cinerea populations associated with treated and untreated cv. Moscato vineyards. Phytopathologia Mediterranea, 53(1):108-123.
61. White, T.J., Bruns, T., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR protocols: Aguide to methods and applications (M.A. 9nnis, D.H. Gelfand, J.J.Sninsky and T.J. White, eds): 315-322. San Diego: Academic Press.
62. Williamson B., Tudzynski, B., Tudzynski, P., & van Kan, J.A.L. (2007). Botrytis cinerea: the cause of grey mould disease. Molecular Plant Pathology, 8(5):561-580.
63. Yohalem, D.S., Neilson, K., & Nicolaisen, M. (2003). Taxonomic and nomenclatural clarification of the onion neck rotting Botrytis species. Mycotaxon, 85:175-182.