Evaluation of genetic variation of the causal agent of leaf spot of citrus (Alternaria sp.) isolates based on RFLP-IGS
Subject Areas : MycologyAlireza Niazmand 1 , Maryam Rahmani 2 , Gilda Najafipoor 3
1 - Assistant Professor, Department of Plant Pathology, Jahrom Branch, Islamic Azad University, Jahrom Iran.
2 - MS.c., Department of Plant Pathology, Jahrom Branch, Islamic Azad University, Jahrom Iran
3 - Assistant Professor, Department of Plant Pathology, Jahrom Branch, Islamic Azad University, Jahrom Iran.
Keywords: lime, ITS1, Alternaria alternata, Orange, Sweet lemon,
Abstract :
Background and Objectives: Nowadays RFLP test from IGS region is a used for determination of interspecies diversities. The aim of this study was to determine pathogenic species of Alternaria from leaf spots of citrus in Mamasani region based on morphological and ITS1 sequencing and to determine genetic variation of isolates based on RFLP-IGS. Materials and Methods: This descriptive study was performed on 70 leaf spotted samples collected from different citruses species and cultivars of Mamasany orchards including orange (Valencia, Navel and leaf spoon Cv.), lime and sweet lemon (Wikova and local Cv.) during In autumn 2011. The isolates were cultured on PDA medium and purified by using single spore method and their species were identified based on morphological characters. The pathogenicity of the isolates was evaluated by based on Koch’s rules through exposure of isolates to leaf. DNA was extracted from mycelia by CTAB and the ITS1 and IGS regions were amplified by using specific primers. The ITS1 region was sequenced and the IGS region was digested by HinfI, BsnI, RsaI, BssmI and AluI restricted enzymes. Results: All isolates were identified as Alternaria alternata based on morphological characters and sequencing of ITS1 region. Constructed dendrogram based on RFLP patterns showed existence of three divergent groups in 51% similarity. Orange, lime and sweet lemon isolates grouped in divergent groups. Also Wikova isolates grouped in different clade. Conclusion: The obtained results clearly showed that the enzyme patterns of IGS can reveal the classification defects of Alternaria alternata form citrus hosts.
1. Fao: Food and Agriculture Organization of the United Nations, 2012. FAO statistical yearbook.
2. Annamous: Statics of Agriculture. In., vol. 2. Tehran: Minestry of agriculture; 2000: 275. [In Persian]
3. Timmer LW, Solel Z, Gottwald TR, Ibanez AM, Zitko SE. Environmental factors affecting production, release, and field populations of conidia of Alternaria alternata, the cause of brown spot of citrus. Phytopathology 1998, 88(11):1218-1223.
4. Gol Mohammadi M, Rahimian H, Bani HS, Taheri H. Occurrence of Brown Spot Disease of Page Mandarin in West of Mazandaran. J Agr Sci Nat. Resour. 2005. [In Persian]
5. Peever TL, Ibanez A, Akimitsu K, Timmer LW: Worldwide phylogeography of the citrus brown spot pathogen, Alternaria alternata. Phytopathology 2002, 92(7):794-802.
6. Takamatsu S, Hirata T, Sato Y. Phylogenetic analysis and predicted secondary structures of the rDNA internal transcribed spacers of the powdery mildew fungi (Erysiphaceae). Mycoscience 1998, 39(4):441-453.
7. Hillis DM, Dixon MT. Ribosomal DNA: molecular evolution and phylogenetic inference. Q Rev Biol 1991:411-453.
8. Anderson JB, Stasovski E. Molecular phylogeny of northern hemisphere species of Armillaria. Mycologia 1992:505-516.
9. Henrion B, Le Tacon F, Martin F. Rapid identification of genetic variation of ectomycorrhizal fungi by amplification of ribosomal RNA genes. New Phytol 1992:289-298.
10. Edel V, Steinberg C, Avelange I, Laguerre G, Alabouvette C. Comparison of three molecular methods for the characterization of Fusarium oxysporum strains. Phytopathology 1995, 85(5):579-585.
11. Banik MT, Volk TJ, Burdsall Jr HH. Armillaria species of the Olympic Peninsula of Washington state, including confirmation of North American biological species XI. Mycologia 1996:492-496.
12. Guidot A, Lumini E, Debaud JC, Marmeisse R. The nuclear ribosomal DNA intergenic spaces as a target sequence to study intraspecific diversity of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum directly on Pinus roof systems. Appl Environ Microb 1999(65):903-909.
13. Molina FI, Jong S-C, Huffman JL. PCR amplification of the 3' external transcribed and intergenic spacers of the ribosomal DNA repeat unit in three species of Saccharomyces. FEMS Microbiol lett 1993, 108(3):259-263.
14. Selosse MA, Costa G, Battista CD, Tacon FL, Marfin F. Meiotic segregation and recombination of the intergenic spacer of the the ribosomal DNA of the oomycetous fungus Pythium ultimum. Curr Genet 1996(17):125-127.
15. Vilgalys R, Hester M. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 1990, 172(8):4238-4246.
16. Chen W. Restriction fragment length polymorphism in enzymatically amplified ribosomal DNAs of three heterothallic Pythium species. Phytopathology 1992, 82(12):1467-1472.
17. Edel V, Steinberg C, Gautheron N, Alabouvette C. Evaluation of restriction analysis of polymerase chain reaction (PCR)-amplified ribosomal DNA for the identification of Fusarium species. Mycol Res 1997, 101(2):179-187.
18. Simmons EG. Alternaria: an identification manual: Am Soc Microbiol, 2007.
19. Stewart Jr CN, Via LE. A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Biotechniques 1993, 14(5):748-750.
20. Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR protocols: a guide to methods and applications: Academic press; 1990.
21. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications. Edited by Innis MA, Gelfand DH, Sninsky JJ, White TJ. San Diego, CA: Academic Press; 1990: 315-322.
22. Hong SG, Cramer RA, Lawrence CB, Pryor BM. Alt a 1 allergen homologs from Alternaria and related taxa: analysis of phylogenetic content and secondary structure. Fungal Genet Biol 2005, 42(2):119-129.
23. Ershad D. Fungi of Iran. Publications Department of botany 1995(10):277. [In Persian]
24. Rouhibakhsh A, Ershad D. An investigation on mycoflora of citrus necrotic spots in Western part of Mazandaran. Iranian J Plant Pathol 1997(33):94 - 110. [In Persian]
25. Banihashemian S, Zad J, Hedjaroud GA, Okhovvat SM, Mosahebi GH. Albinism in citrus seedlings. Iranian J Agric Sci 2001, 32(2):345-354. [In Persian]
36. Kohmoto K, Akimitsu K, Otani H. Correlation of resistance and susceptibility of citrus to Alternaria alternata with sensitivity to host-specific toxins. Phytopathology 1991, 81(7):719-722.
27. Kohmoto K, Scheffer RP, Whiteside JO. Host-selective toxins from Alternaria citri [from lemon and tangerine trees in Florida]. Phytopathology 1979, 69.
28. Palm ME, Civerolo EL. Isolation, pathogenicity, and partial host range of Alternaria limicola, causal agent of mancha foliar de los citricos in Mexico. Plant Dis 1994, 78.
29. Peever TL, Olsen L, Ibanez A, Timmer LW. Genetic differentiation and host specificity among populations of Alternaria spp. causing brown spot of grapefruit and tangerine and grapefruit hybrids in Florida. Phytopathology 2000, 90(4):407-414.
30. Timmer LW, Peever TL, Solel Z, Akimitsu K. Alternaria diseases of citrus-novel pathosystems. Phytopathol Mediterr 2003, 42(2):99-112.
31. Golmohammadi M, Andrew M, Peever TL, Peres NA, Timmer LW. Brown spot of tangerine hybrid cultivars Minneola, Page and Fortune caused by Alternaria alternata in Iran. Plant Pathology 2006, 55(4): 578-587.
32. Peay KG, Kennedy PG, Bruns TD. Fungal community ecology: a hybrid beast with a molecular master. Bioscience 2008, 58(9):799-810.
33. Pryor BM, Michailides TJ. Morphological, pathogenic, and molecular characterization of Alternaria isolates associated with Alternaria late blight of pistachio. Phytopathology 2002, 92(4):406-416.
34. Appel DJ, Gordon TR. Relationships among pathogenic and nonpathogenic isolates of Fusarium oxysporum based on the partial sequence of the intergenic spacer region of the ribosomal DNA. Mol. Plant Microbe In1996, 9(2):125-138.
35. Pecchia S, Mercatelli E, Vannacci G. PCR amplification and characterization of the intergenic spacer region of the ribosomal DNA in Pyrenophora graminea. FEMS Microbiol lett 1998, 166(1):21-27.
36. Slippers B, Wingfield BD, Coutinho TA, Wingfield MJ. DNA sequence and RFLP data reflect geographical spread and relationships of Amylostereum areolatum and its insect vectors. Mol Ecol 2002, 11(9):1845-185.
37. Pantou MP, Mavridou A, Typas MA. IGS sequence variation, group-I introns and the complete nuclear ribosomal DNA of the entomopathogenic fungus Metarhizium: excellent tools for isolate detection and phylogenetic analysis. Fungal Genet Biol 2003, 38 (2): 159-174.
38. Hong SG, Liu D, Pryor BM. Restriction mapping of the IGS region in Alternaria spp. reveals variable and conserved domains. Mycol Res 2005, 109(1):87-95
