Molecular identification and genetic variation of Alternaria species isolated from tomatoes using ITS1 sequencing and inter simple sequence repeat methods

Authors

Department of Biology, Faculty of Science, Lorestan University, Khorramabad, Iran

Abstract

Background and Purpose: Alternaria is one of the most abundant fungi that exists in numerous places around the world. This saprophytic fungus causes diseases in plants and accounts for the spoilage of cereals in warehouses. The aim of this study was to identify Alternaria isolates based on their morphological characteristics and internal transcribed spacer ribosomal RNA (ITS rRNA) sequencing method. To this end, genetic diversity in the isolates was also examined using inter simple sequence repeat (ISSR) markers.

Materials and Methods: To conduct this research, a total of 60 tomato samples with black spots were collected from supermarkets in Khorramabad City, Iran, in the winter of 2017. The specimens were cultured on a potato dextrose agar medium. After the purification of the fungus by the single-spore method, the identification of the species was carried out using morphological characteristics and ITS rRNA sequencing by polymerase chain reaction. The genetic diversity of the identified species with four primers was evaluated using the ISSR marker.

Results: Based on the sequencing of the ITS1 region, all the isolates were identified as A. alternata. Cluster diagrams for the ISSR marker were classified into six distinct groups. The mean polymorphism information content was obtained as 0.35, indicating the effectiveness of the primers in the separation of the isolates.

Conclusion: The sequencing of ITS1 led to the identification of Alternaria species that are morphologically similar. The production of various mycotoxins by A. Alternata species leads to the contamination of livestock and human food. Regarding this, the investigation of the genetic diversity of A. alternata species using the ISSR marker would facilitate the identification of suitable and effective strategies for controlling the fungal and mycotoxin contamination of human nutrition.

Keywords

References

1. MacDonald S, Prickett T, Wildey K, Chan D. Survey of ochratoxin A and deoxynivalenol in stored grains from the 1999 harvest in the UK. Food Addit Contam. 2004; 21(2):172-81.
2. Pitt JI, Hocking AD. Fungi and food spoilage. New South Wales, Australia: Blackie Academic & Professional; 1997.
3. Tutelyan VA. Deoxynivalenol in cereals in Russia. Toxicol Lett. 2004; 153(1):173-9.
4. Thomma BP. Alternaria spp.: from general saprophyte to specific parasite. Mol Plant Pathol. 2003; 4(4):225-36.
5. Samson RA, Houbraken J, Thrane U, Frisvad JC, Andersen B. Food and indoor fungi. Netherlands: CBS-KNAW Fungal Biodiversity Centre; 2010.
6. Pastor FJ, Guarro J. Alternaria infections: laboratory diagnosis and relevant clinical features. Clin Microbiol Infect. 2008; 14(8):734-46.
7. Escrivá L, Oueslati S, Font G, Manyes L. Alternaria Mycotoxins in food and feed: an overview. J Food Qual. 2017; 2017:1569748.
8. Gabriel MF, Postigo I, Tomaz CT, Martínez J. Alternaria alternata allergens: markers of exposure, phylogeny and risk of fungi-induced respiratory allergy. Environ Int. 2016; 89:71-80.
9. Mirhendi H, Fatemi MJ, Bateni H, Hajabdolbaghi M, Geramishoar M, Ahmadi B, et al. First case of disseminated phaeohyphomycosis in an immunocompetent individual due to Alternaria malorum. Med Mycol. 2013; 51(2):196-202.
10. Barkai-Golan R, Paster N. Mycotoxins in fruits and vegetables. New York: Elsevier; 2011.
11. Schrader TJ, Cherry W, Soper K, Langlois I, Vijay HM. Examination of Alternaria alternata mutagenicity and effects of nitrosylation using the Ames Salmonella test. Teratog Carcinog Mutagen. 2001; 21(4):261-74.
12. Faostat F. Disponível. Available at: URL: http://faostat.fao.org. Acessoem; 2012.
13. Samuel O, Orji MU. Fungi associated with the spoilage of post-harvest tomato fruits sold in major markets in Awka, Nigeria. Univ J Microbiol Res. 2015; 3(2):11-6.
14. Polívková Z, Šmerák P, Demová H, Houška M. Antimutagenic effects of lycopene and tomato purée. J Med Food. 2010; 13(6):1443-50.
15. Borguini RG, Ferraz da Silva Torres EA. Tomatoes and tomato products as dietary sources of antioxidants. Food Rev Int. 2009; 25(4):313-25.
16. Zhang CX, Ho SC, Chen YM, Fu JH, Cheng SZ, Lin FY. Greater vegetable and fruit intake is associated with a lower risk of breast cancer among Chinese women. Int J Cancer. 2009;
125(1):181-8.
17. Andersen B, Frisvad JC. Natural occurrence of fungi and fungal metabolites in moldy tomatoes. J Agric Food Chem. 2004; 52(25):7507-13.
18. Barkai-Golan R, Paster N. Mouldy fruits and vegetables as a source of mycotoxins: part 1. World Myc J. 2008; 1(2):147-59.
19. Arzanlou M, Abeln EC, Kema GH, Waalwijk C, Carlier J, Vries Id, et al. Molecular diagnostics for the Sigatoka disease complex of banana. Phytopathology. 2007; 97(9):1112-8.
20. Loganathan M, Venkataravanappa V, Saha S, Rai AB, Tripathi S, Rai RK, et al. Morphological, pathogenic and molecular characterizations of Alternaria species causing early blight of tomato in Northern India. Proc Natl Acad Sci India B Biol Sci. 2016; 86(2):325-30.
21. Leiminger JH, Auinger HJ, Wenig M, Bahnweg G, Hausladen H. Genetic variability among Alternaria solani isolates from potatoes in Southern Germany based on RAPD-profiles. J Plant Dis Protect. 2013; 120(4):164-72.
22. Reddy MP, Sarla N, Siddiq EA. Inter simple sequence repeat (ISSR) polymorphism and its application in plant breeding. Euphytica. 2002; 128(1):9-17.
23. Wolfe AD. ISSR techniques for evolutionary biology. Methods in enzymology. Massachusetts: Academic Press; 2005. P. 134-44.
24. Simmons EG. Alternaria: an identification manual. Am Soc Microbiol. 2007; 51:687.
25. Mohammadi A, Shams‐Ghahfarokhi M, Nazarian‐Firouzabadi F, Kachuei R, Gholami‐Shabani M, Razzaghi‐Abyaneh M. Giberella fujikuroi species complex isolated from maize and wheat in Iran: distribution, molecular identification and fumonisin B1 in vitro biosynthesis. J Sci Food Agric. 2016; 96(4):1333-40.
26. Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR protocols: a guide to methods and applications. Massachusetts: Academic Press; 1990.
27. Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, et al. The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol Breed. 1996; 2(3):225-38.
28. Temitope FP, Oluchi UE. Studies on the antifungal activity of Lactobacillus plantarum and Lactobacillus fermentum on spoilage fungi of tomato fruit. J Microbiol Res. 2015; 5(3):95-100.
29. Akinmusire OO. Fungal species associated with the spoilage of some edible fruits in Maiduguri Northern Eastern Nigeria. Adv Environ Biol. 2011; 157:62.
30. Ibrahim AD, Musa K, Sani AA, Aliero AA, Yusuf BS. Microorganisms associated with the production of volatile compounds in spoilt tomatoes. Res Biotechnol. 2011; 2(2):82-9.
31. Baker SE. Aspergillus niger genomics: past, present and into the future. Med Mycol. 2006; 44(Suppl 1):S17-21.
32. Kalyoncu F. Determination of fungi associated with tomatoes (Lycopersicum esculentum M.) and tomato pastes fatih kalyoncu, A. usame tamer and mustafa oskay. Plant Pathol J. 2005; 4(2):146-9.
33. Wogu MD, Ofuase O. Microorganisms responsible for the spoilage of tomato fruits, Lycopersicum esculentum, sold in markets in Benin City, Southern Nigeria. Sch Acad J Biosci. 2014; 2(7):459-66.
34. Ogodo AC, Nwaneri CB, Agwaranze DI, Inetianbor JE, Okoronkwo CU. Antimycotic and antibacterial activity of Aframomum melegueta seed extracts against bacteria and fungi species from food sources. Central Afr J Public Health. 2017; 3(4):44-50.
35. Laich FS, Alcoba-Flórez J, Pérez-Roth E, Bahaya Y, Luis Delgado J, Méndez-Álvarez S. Molecular characterization of Alternaria alternata causing ocular infection: detection of IGS-RFLP intraspecific polymorphism. Sabouraudia. 2008; 46(6):615-9.
36. Lee HB, Patriarca A, Magan N. Alternaria in food: ecophysiology, mycotoxin production and toxicology. Mycobiology. 2015; 43(2):93-106.
37. Logrieco A, Moretti A, Solfrizzo M. Alternaria toxins and plant diseases: an overview of origin, occurrence and risks. World Mycotoxin J. 2009; 2(2):129-40.
38. Perello A, Moreno M, Sisterna M. Alternaria infectoria species‐group associated with black point of wheat in Argentina. Plant Pathol. 2008; 57(2):379.
39. Zitter TA, Jones JB, Jones JP, Stall RE, Zitter TA. Tomato spotted wilt. Compendium of tomato diseases. St Paul: APS Press, The American Phytopathological Society; 1991.
40. Zitter TA, Drennan JL. Comparing fungicides for controlling the light phenotype Alternaria tomatophila in tomato, 2004. Fungic Nematic Tests. 2005; 60:V072.
41. Chaerani R, Voorrips RE. Tomato early blight (Alternaria solani): the pathogen, genetics, and breeding for resistance. J Genet Plant Pathol. 2006; 72(6):335-47.
42. Derbalah AS, El-Mahrouk MS, El-Sayed AB. Efficacy and safety of some plant extracts against tomato early blight disease caused by Alternaria solani. Plant Pathol J. 2011; 10(3):115-21.
43. Vijayan K. Inter simple sequence repeat (ISSR) polymorphism and its application in mulberry genome analysis. Int J Indust Entomol. 2005; 10(2):79-86.
44. Ng WL, Tan SG. Inter-simple sequence repeat (ISSR) markers: are we doing it right. ASM Sci J. 2015; 9(1):30-9.
45. Abadio AK, Lima SS, Santana MF, Salomão TM, Sartorato A, Mizubuti ES, et al. Genetic diversity analysis of isolates of the fungal bean pathogen Pseudocercospora griseola from central and southern Brazil. Genet Mol Res. 2012; 11(2):1272-9.
46. Röder MS, Korzun V, Wendehake K, Plaschke J, Tixier MH, Leroy P, et al. A microsatellite map of wheat. Genetics. 1998; 149(4):2007-23.
47. Kale S, Pardeshi V, Gurjar G, Gupta V, Gohokar R, Ghorpade P, et al. Inter-simple sequence repeat markers reveal high genetic diversity among Alternaria alternata isolates of Indian origin. J Mycol Plant Pathol. 2012; 42(2):194-200.
48. Bagherabadi S, Zafari D, Soleimani MJ. Genetic diversity of Alternaria alternata isolates causing potato brown leaf spot, using ISSR markers in Iran. J Plant Pathol Microbiol. 2015; 6(7):1-6.
49. Nielsen R, Slatkin M. An introduction to population genetics. Sunderland: Sinauer Associates, Inc; 2013.
50. Hartl DL, Clark AG, Clark AG. Principles of population genetics. Sunderland: Sinauer Associates; 1997.
51. Schmidt T, Schaechter M. Topics in ecological and environmental microbiology. New York: Elsevier; 2012.
52. Pozzi CR, Braghini R, Arcaro JR, Zorzete P, Israel ALM, Pozar IO, et al. Mycoflora and occurrence of alternariol and alternariol monomethyl ether in Brazilian sunflower from sowing to harvest. J Agr Food Chem. 2005; 53(14):5824-8.
Current Medical Mycology
Volume 5, Issue 2
June 2019
Pages 1-8

Files

History

  • Receive Date: 09 July 2019
  • Revise Date: 07 September 2020
  • Accept Date: 09 July 2019
  • First Publish Date: 09 July 2019

Share

How to cite

Statistics