Genetic Diversity of Alternaria alternata Causal Agent of Early Blight of Tomato in Khuzestan Province Using SSRs Marker

Document Type : Research Article

Authors

• Khoshnood Nourollahi
• mohsen hasani

Ilam University

10.22067/jpp.v30i4.42644

Abstract

Introduction: The early blight of tomato caused by Alternaria alternata is one of the most important and destructive diseases in Khuzestan province. Study genetic structure of A. alternata populations provides different levels of information in the management of early blight disease in tomato farms. Short sequence repeat (SSR) markers were used to determine the genetic structure and estimate genetic diversity in A. alternata isolates in Khuzestan province.
Materials and Methods: In this study to evaluate the genetic diversity and genetic populations of A. alternata pathogen, sampling was randomly carried out on aerial parts of tomato plants with leaf brown lesions in farms and glasshouses from five different regions in Khuzestan province such as: Shoshtar, Omidiyeh, Dashte azadagan, Ahvaz, and Dezful. Each sample was cut into 2–5-mm long pieces, were surface-sterilized with 1% sodium hypochlorite for 3 min and rinsed three times with sterile distilled water and air-dried with sterile filter paper. The sterilized samples were placed onto a general medium potato dextrose agar (PDA). A total of 64 A. alternata isolates were obtained from infected samples. Pathogenicity test was carried out on local susceptible cultivar under an artificial condition in the greenhouse. For obtaining the mycelia mass, liquid cultures were initiated by adding 2–4 mm 2 pieces of filter paper to 250-mL Erlenmeyer flasks containing 100 mL PDB medium (potato dextrose broth). Mycelium was collected by filtration through sterile filter paper with a vacuum funnel. Mycelia were harvested, frozen and stored at -20°C. DNA was extracted using a modified hexadecyl trimethyl-ammonium bromide (CTAB) procedure. A set of five paired sequence repeat primers (SSR) were used to determine the genetic diversity of A. alternata isolates. PCR amplification was performed in a 25 μl reaction volume. The bands generated by SSR primers that were repeatable and clearly visible with a high intensity were scored manually for the presence (1) or absence (0) of bands in each isolate. Data analyses for evaluating of genetic diversity of isolates were calculations using molecular software such as: NTysis, Gene Alex, and POP GENE.
Results and Discussion: A total of 21 alleles were produced by SSR primers with an average of 4.2 alleles in all populations. The highest and lowest amounts of alleles were related to locus AEM13 with eight alleles and loci of AEM6 and AEM9 with two alleles respectively. The average of allelic variability per locus was the highest in Shoshtar population and the lowest in Dezful population. Observed allele number and effective numbers of alleles were higher in Shoshtar in comparison of other populations. A Comparison of genetic diversity parameters in five population showed that Shoshtar population has the highest genetic diversity but lower values were estimated for Dashte azadagan. The highest and lowest genetic distance was detected between Ahvaz-Dezfol (0.066) and Shoshtar-Omidieh (0.005), respectively. Based on dendrogram of populations revealed two distinct groups, one group contained Dezful and the other Shoshtar, Omidiyeh, Dashte azadagan and Ahvaz. Analysis of molecular variance showed that 85 percent of the genetic diversity of all the isolates and 14% is allocated to different geographical areas. There was the high genetic similarity between isolates from different regions. High genetic similarity can be attributed to the migration of genes or genotypes of different factors. With according to of Cluster analysis based on UPGMA and Dice similarity coefficient at 62% level, eight groups were revealed. On the basis of microsatellite data indicated high genetic diversity within the isolates; this number of alleles could not lead in separation, on the basis of geographical locations between samples. In this study, the relationship detected between isolates within the six populations were probably due to exchange of tomato seeds between sampled regions and geographical closeness as well.
Conclusion: This study have been carried out for the first time in Iran, and in comparison of international populations, a different level of diversity was detected within and between populations of worldwide A. alternata isolates. In this study, the high genetic diversity of A. alternata detected in five populations exposed a potential risk to tomato farms. Genetic diversity of A. alternata in Khuzestan province as an air born pathogen is a warning for a breeder to apply the successful use of resistance genes in local disease management. This gene diversity helps breeders for screening potential resistant cultivars according to gene diversity of A. alternata population in order to develop of durable resistant. Quarantine regulations will need to prevent the introduction of more diverse isolates into these populations and prevent transmission any isolates from this area to other regions of the country. Understanding the genetic structure of pathogen populations in the present study may provide insights into the epidemiology and evolutionary potential of pathogens and could lead to improved strategies for managing the disease. The obtained results indicating the high genetic diversity due to mutation, recombinant and a sexual mating ability of the pathogen in the Khuzestan province. Results in this study will be useful in breeding for tomato early blight resistant cultivars and developing necessary control measures.

Keywords

• Early blight
• Genetic similarity
• Molecular variance
• SSR

20.1001.1.20084749.1395.30.4.20.6

---

References

1. Aradhya M.K., Chan H.M., and Parfitt D.E. 2001. Genetic variability in the pistachio late blight fungus, Alternaria alternate. Mycological Research, 105: 300–306.

2. Baymani M. 2012. Determination of dominant causal agent of early blight of tomato and loss estimation and its chemical control in Khuzestan province. M.Sc thesis, Shahid Chamran University, Ahvaz, Iran.

3. Benichou S.A., Dongo D.E., Henni P., and Simoneau P. 2009. Isolation and characterization of microsatellite markers from the phytopathogenic fungus Alternaria dauci. Molecular Ecology Resources, 9: 390–392.

4. Bock C.H., Thrall P.H., Brubaker C.L., and Burdon J.J. 2002. Detection of genetic variation in Alternaria brassicicola using AFLP fingerprinting. Mycological Research, 106: 428-434.

5. Burdon J.J., and Silk J. 1997. Sources and patterns of diversity in plant-pathogenic fungi. Phytopathology, 87: 664-669.

6. Chowdhury A.K., Yonemoto Y., Kato H., and Macha M.M. 2005. Cultivar identification by morphometric descriptors and RAPD markers among some Acerola (Malpighia glabra Linn.) cultivars. Japanese Journal Of Tropical Agriculture, 49: 41-42.

7. Dini-Andreote F., Cristina Pietrobon V., Andreote F.D., Romao A.S, BellatoSposito M., and Araujo W.L. 2009. Genetic variability of Brazilian Isolates of Alternaria alternata detected by AFLP and RAPD techniques. Brazilian Journal of Microbiology, 40: 670-677.

8. Doyle J.J., and Doyle J.L. 1990. A rapid total DNA preparation procedure for fresh plant tissue. Focus, 12: 13-15.

9. Eatebarian H. 2006. Vegetable disease and their control. Third edition. Tehran University Press. (In Persian).

10. Esphandiari A. 1947. Field crop and fruit tree disease in north subtropical region of Iran. Issues No. 5, Institute Of Plant Pests And Diseases. (In Persian).

11. Geisler D.M., Pitt J.I., and Taylor J.W. 1998. Cryptic speciation and recombination in the aflatoxin-producing fungus Aspergillus flavus. Proceedings of National Academy of Sciences, USA, 95: 388-393.

12. Ghosta Y. 2004. A taxonomic study on the genus Alternaria from Iran, Ph.D Thesis, Tarbiat Modarres University, Tehran, Iran.

13. Hajianfar R., and Zarbakhsh A. 2006. Identification of causal organism of early blight and stem canker diseases on tomato in major production regions of country. Iranian Plant Protection Journal, 17: 182. (In Persian with English abstract)

14. Hajipour Jarchelo Z., Ghosta Y., and Rezaee S. 2012. Study of pathogenic species of Alternaria on tomato and potato in West Azerbaijan province. Journal of Plant Protection Science, 43: 155-163. (In Persian with English abstract).

15. Izadpanah K., Ashkan M., Banihashemi Z., Rahimian H., and Minasian V. 2011. Plant pathology. Volume II, Fifth Edition (translation), Tehran Aeezh publication. (Translated in Persian).

16. Jaafarnia S., and Homae M. 2008. Comprehensive Illustrated Guide greenhouse cucumber and tomato. Cultivaton, Sokhan Gostare Publishing, Mashhad. (in Persian with English abstract)

17. Jaliani N. 1992. Tomato early blight disease and its chemical control in Jiroft and Bam. 10th plant protection congress Kerman. Iran 118p (in Persian).

18. Kakvan N., Zamanizadeh H., Morid B., Taheri H., and Hajmansor S. 2012. Study on pathogenic and genetic diversity of Alternaria alternata isolated from citrus hybrids of Iran, based on RAPD-PCR technique. European Journal of Experimental Biology, 2 (3): 570-576.

19. Kale M.S., Pardeshi V.C., Gurjar G.S., Gupta V.S., Gohokar, R.S., Ghoropade P.B., and Kadoo N.Y. 2012. Inter simple sequence repeat markers reveal high genetic diversity among A. alternata isolates of Indian origin. Journal of Mycology and Plant Pathology, 42(2): 194-200.

20. Kolliker R., Jones E.S., Drayton M.C., Dupal M. P., and Forster J. W. 2001. Development and characterization of simple sequence repeat (SSR) marker for white clover (Trifolium repens L.). Theoretical and Applied Genetics, 102: 416-424.

21. Kumar M., Mishra G. P., Singh R., Kumar J., Naik P.K., and Singh S. B. 2009. Correspondence of ISSR and RAPD markers for comparative analysis of genetic diversity among different apricot genotypes from cold arid deserts of trans-Himalayas. Physiology and Molecular Biology of Plants, 15: 225-236.

22. Leiminger J., Bahnweg G., and Hausladen H. 2010. Population genetics consequences on early blight disease. Twelfth EuroBlight workshop.

23. Liang-Dong G., and Li X. 2004. Genetic variation of alternaria alternata, an endophytic fungus isolated from Pinus tabulaeformis as determined by random amplified microsatellites (RAMS). Fungal Diversity, 16: 53-65.

24. Litt M., and Luty J.A. 1989. A hyper variable microsatellite revealed by in vitro amplification of a dinunleotide repeat within the cardiac muscle actin gene. American Journal of Human Genetics. 44: 397-401.

25. Lourenzo V., Rodriguez T., Campos A., Braganza C., Scheuermann K., Reis A., Brommonschenkel S., Maffia L., and Mizubuti S. 2011. Genetic structure of the population of Alternaria solani in Brazil. Journal of Phytopathology, 159: 233-240.

26. Martinezi S.P., Snowdon R., and Kuhnemann J.P. 2004. Variability of Cuban and international populations of Alternaria solani from different hosts and localities AFLP genetic analysis. European Journal of Plant Pathology 110: 399–409.

27. McDonald B.A., Zhan J., and Burdon J.J. 1999. Genetic structure of Rhynchosporium secalis in Australia. Phytopathology, 89: 639-645.

28. Mohammadi S.A., and Prasanna B.M. 2003. Analysis of genetic diversity in crop plant: Salien statistical tools and considerations. Crop Science, 43: 1248-1235.

29. Montazeri M., Greaves M.P., Pei M.H., and Ruiz C. 2005. An analysis of genetic diversity in hyphal tip isolates of promising mycoherbicide Alternaria alternata for control of Amaranthus retroflexus. Iranian Journal of Weed Science, 1: 51-65.

30. Morris P.F., Connolly M.S., and St-Clair D.A. 2000. Genetic diversity of Alternaria alternata isolated from tomato in California assessed using RAPDs. Mycological Research Journal, 104: 286-292.

31. Nasim G.H., Khan S., and Khokhar I. 2012. Molecular polymorphism and phylogenetic relationship of some Alternaria alternata isolates. Pakistan Journal of Botany, 44: 1267-1270.

32. Nourollahi K., Haghi Z., and Mehrabi Oladi A. 2014. Study of genetic diversity of Fusarium verticillioides isolates the causal agent of crown and root rot in rice in ilam province using SSR marker. Iranian Journal of plant protection science, 45: 29-37. (In Persian with English abstract)

33. Peakall R., and Smouse P.E. 2012. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics, 28: 2537-2539.

34. Perrier X., and Jacquemoud-Collet J.P. 2006. DARwin software, http://darwin.cirad.fr/darwin

35. Roder M.S., Korzun V., Wendehake K., Plaschke J., Tixier M.H., Leroy P., and Ganal M.W. 1998. A microsatellite map of wheat, Genetics, 149:20-23.

36. Rohlf F.J. 1998. NTSYS-pc. Numerical Taxonomy and Multivariate Analysis System, version 2.02, Exeter Software, Setauket, NY.

37. Rotem J. 1994. The genus Alternaria: biology, epidemiology, and pathogenicity. St Paul, Minnesota, USA: American Phytopathological Society, 326 pp.

38. Shahryari D., and Karimi Rozbahani A. 1998. Stem canker of tomato in Varamin. Journal of Plant Pests and Diseases, 65: 12-19.

39. Sharma T.R.T., and Tewari J.P. 1998. RAPD analysis of three Alternaria species pathogenic to crucifers. Mycological Research, 102: 807–814.

40. Slatkin M. 1987. Gene flow and the geographic structure of natural populations. Science, 236: 787-792.

41. Tautz D. 1989. Hyper variability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Research, 17: 6463-6470.

42. Timmer L.W., Peever T.L., Solel Z., and Akimitsu K. 2003. Alternaria diseases of citrus – novel pathosystems. Phytopathologia Mediterranea, 42: 99-112.

43. Tran-Dinh N., and Hocking A. 2006. Isolation and characterization of polymorphic microsatellite markers for Alternaria alternata. Molecular Ecology, 6: 405–407.

44. Vander Waals J. E., Korsten L., and Slippers B. 2004. Genetic diversity among Alternaria solani isolates from potatoes in South Africa. Plant Disease, 88: 959-964.

45. Vicent A., Armengol J., and Garcıa Jimenez J. 2007. Rain fastness and persistence of fungicides for control of Alternaria brown spot of citrus. Plant Disease, 91: 393–399.

46. Weber J. L., and May P.E. 1989. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. American Journal of Human Genetics, 44: 338-396.

47. Weir T.L., Huff D. R., Christ B.J., and Romaine C.P. 1998. RAPD-PCR analysis of genetic variation among isolates of Alternaria solani and A. alternata from potato and tomato. Mycologia, 90: 813-821.

48. Yeh F.C., Yang R.C., and Boyle T. 1999. Microsoft Window-based freeware for population genetic analysis (POPGENE), ver.1.31, ftp://ftp.microsoft.com/softlib/MSLFILES/HPGL.EXE.

49. Youssuf A.M., and Gherbawy H. 2005. Genetic variation among isolates of Alternaria spp. from select Egyptian crops. Phytopathology and Plant Protection, 38: 77- 89.

50. Zane L., Bargelloni L., and Patarnello T. 2002. Strategies for microsatellite isolation. A review, Molecular Ecology, 11:1-16.