انگشت نگاری ژنومی گونه های مختلف آزوسپیریلوم جدا شده از مزارع گندم و ذرت با استفاده از روش Rep-PCR
محورهای موضوعی : میکروب شناسی گیاهیمحمود شهابی 1 , ناصر پنجه که 2 , هادی اسدی رحمانی 3 , محمد سالاری 4
1 - دانشجوی دکتری، رشته بیماری شناسی گیاهی، دانشکده کشاورزی، دانشگاه زابل
2 - دانشیار، بیماری شناسی گیاهی، دانشکده کشاورزی، دانشگاه زابل
3 - دانشیار، موسسه تحقیقات خاک و آب کشور
4 - دانشیار، بیماری شناسی گیاهی، دانشکده کشاورزی، دانشگاه زابل
کلید واژه: روش, توالی یابی, آزوسپیریلوم, rep-PCRتنوع ژنتیکی,
چکیده مقاله :
سابقه و هدف: بیماری فیتوپلاسمایی تورم جوانه گوجه فرنگی در سال های اخیر در مناطق مختلف کشور شیوع پیدا کرده است. این پژوهش با هدف تعیین غلظت و الگوی گسترش کاندیداتوس فیتوپلاسما استرالیزیا در گوجه فرنگی، به منظور ردیابی سریع تر بیمارگر به ویژه در دوره کمون بیماری و مدیریت بهتر آن انجام شد.مواد و روش ها: در یک آزمایش فاکتوریل در قالب طرح کاملاً تصادفی، سه گیاه گوجه فرنگی به وسیله پیوند به کاندیداتوس فیتوپلاسما استرالیزیا آلوده شدند. در فواصل زمانی 10، 20، 40 و 70 روز پس از مایه زنی، نمونه برداری از برگ های انتهایی گیاهان تیمار، برگ ها از ساقه های بالا و پایین محل پیوند و نیز ریشه های فرعی انجام شد. سپس با روش واکنش زنجیره ای پلی مراز در زمان واقعی، غلظت و الگوی گسترش فیتوپلاسما در گیاهان یاد شده تعیین شد. همچنین، روش های واکنش زنجیره ای پلی مراز مستقیم و آشیانه ای در ردیابی فیتوپلاسمای همراه با بیماری استفاده و مقایسه شدند.یافته ها: نتایج نشان دادند که کاندیداتوس فیتوپلاسما استرالیزیا پس از ورود به گیاه، به سمت بالا و پایین حرکت کرده و به همین دلیل در برگ های بالایی و ریشه های گیاهان آلوده، غلظت آن بیشتر از برگ های میانی و پایینی بود. براساس یافته های این پژوهش، میانگین دوره کمون بیماری حدود 40 روز تخمین زده شد. واکنش زنجیره ای پلی مراز آشیانه ای حساس تر از واکنش زنجیره ای پلی مراز مستقیم در ردیابی این فیتوپلاسما به ویژه در دوره کمون بیماری بود.نتیجه گیری: برای ردیابی و تشخیص بیماری تورم جوانه گوجه فرنگی ناشی از کاندیداتوس فیتوپلاسما استرالیزیا، بهتر است از برگ های انتهایی و ریشه ها نمونه برداری و از روش واکنش زنجیره ای پلی مراز آشیانه ای استفاده شود.
Background & Objectives: Azospirillum comprises rhizobacteria that live freely in the soil where they change gaseous nitrogen to nitrite and nitrate and as a result promote growth and yield of some economically important plants. This study aimed to isolate and identify different native species of Azospirillum and to investigate their genetic diversity as plant growth-promoting factors. Materials & Methods: In this research, some soil and root samples from wheat and maize fields of Isfahan province were collected and cultured in the tubes containing nitrogen-free bromothymol blue (NFB) semisolid media. The Azospirillum isolates were chosen based on morphological properties on NFB and RC (Rojo Congo) media and then were purified. The detection and identification of Azospirillum isolates was done by molecular tests using PCR (Polymerase Chain Reaction), 16S rRNA gene sequencing, and genomic fingerprinting with rep-PCR (repetitive extragenic palindromic elements-polymerase chain reaction). Results: The replication of 646 and 263 bp fragments by specific primers, confirmed the presence of Azospirilum bacteria. Azospirillum species were also identified as A. brasilense, A. lipoferum, and A. zeae using replication and sequencing of the 16S rRNA gene. The genomic profiling of different Azospirillum isolates produced unique band patterns through application of repetitive element BOX (BOX-AIR), ERIC (Enterobacterial Repetitive Intergenic Consensus), and REP and showed high genetic diversity between the isolates. Conclusion: According to this study, the Azospirillum isolates of wheat and maize fields of Isfahan province have different species and genetic diversity and band patterns amplified using rep-PCR can be useful for diagnosis and classification of Azospirillum species.
growth promoting Rhizobacteria isolated from the rhizosphere of wheat (Triticum durum Desf.) in
Algeria. Afr J Microbiol Res. 2013; 7(23): 2893-2904.
2. Bashan Y, De-Bashan LE. How the plant growth-promoting bacterium Azospirillum promotes plant
growth—a critical assessment. In Adv Agron. 2010; 108: 77-136. Academic Press.
3. Eleni V, Anastasia V, Maria D, Iordanis C, Anastasia T, Katinakis P. Phylogenetic analysis of
Azospirillum species isolated from the rhizosphere of field‐grown wheat based on genetic and
phenotypic features. Mol Microbial Ecol Rhizosphere. 2013; 1: 203- 210.
4. Saikia SP, Bora D, Goswami A, Mudoi KD, Gogoi A. A review on the role of Azospirillum in the
yield improvement of non leguminous crops. Afr J Microbiol Res. 2012; 6(6): 1085-1102.
5. Lin SY, Shen FT, Young CC. Rapid detection and identification of the free-living nitrogen fixing
genus Azospirillum by 16S rRNA-gene-targeted genus-specific primers. Antonie Leeuwenhoek. 2011;
99(4): 837-844.
6. Lin SY, Liu YC, Hameed A, Hsu YH, Lai WA, Shen FT, Young CC. Azospirillum fermentarium sp.
nov., a nitrogen-fixing species isolated from a fermenter. Int J Syst Evol Microbiol. 2013; 63(10):
3762-3768.
7. Zhou S, Han L, Wang Y, Yang G, Zhuang L, Hu P. Azospirillum humicireducens sp. nov., a nitrogenfixing bacterium isolated from a microbial fuel cell. Int J Syst Evol Microbiol. 2013; 63(7):
2618-2624.
8. Wisniewski-Dye F, Borziak K, Khalsa-Moyers G, Alexandre G, Sukharnikov LO, Wuichet K, Hurst
GB, McDonald WH, Robertson JS, Barbe V, Calteau A. Azospirillum genomes reveal transition of
bacteria from aquatic to terrestrial environments. PLoS Genet. 2011; 7(12): e1002430.
9. Kaneko T, Minamisawa K, Isawa T, Nakatsukasa H, Mitsui H, Kawaharada Y, Nakamura Y,
Watanabe A, Kawashima K, Ono A, Shimizu Y. Complete genomic structure of the cultivated rice
endophyte Azospirillum sp. B510. DNA Res. 2010; 17(1): 37-50.
10. Sant'Anna FH, Almeida LG, Cecagno R, Reolon LA, Siqueira FM, Machado MR, Vasconcelos AT,
Schrank IS. Genomic insights into the versatility of the plant growth-promoting bacterium
Azospirillum amazonense. BMC genomics. 2011; 12(1): 409.
11. dos Anjos Borges LG, Dalla Vechia V, Corção G. Characterisation and genetic diversity via
REP-PCR of Escherichia coli isolates from polluted waters in southern Brazil. FEMS Microbiol
Ecol. 2003; 45(2): 173-180.
12. Najafi Pour G, Taghavi SM. Comparison of P. syringae pv. syringae from different hosts based on
pathogenicity and BOX-PCR in Iran. J Agr Ssi Tech-Iran. 2011; 13(3): 431-442.
13. Vicente JG, Roberts SJ. Discrimination of Pseudomonas syringae isolates from sweet and wild
cherry using rep-PCR. Eur J Plant Pathol. 2007; 117(4): 383-392.
14. Adiguzel G, Atasever M. Phenotypic and genotypic characterization of lactic acid bacteria isolated
from Turkish dry fermented sausage. Rom Biotechnol Lett. 2009; 14(1): 4130-4138.
15. Versalovic J, Koeuth T, Lupski R. Distribution of repetitive DNA sequences in eubacteria and application to finerpriting of bacterial enomes. Nucleic Acids Res. 1991; 19(24): 6823-6831.
16. Rebecca LH, Zothansanga Singh BP, Gurusubramanian G, Senthil NK. DNA finger printing of
Bacillus thuringiensis based on repetitive DNA sequences using ERIC-PCR. Sci Vis. 2011; 11(3):
147-154.
17. Louws FJ, Rademaker JL, De Bruijn FJ. The three Ds of PCR-based genomic analysis of
phytobacteria: diversity, detection, and disease diagnosis. Annu Rev Phytopathol. 1999; 37(1):
81-125.
18. Ishii S, Sadowsky MJ. Applications of the rep‐PCR DNA fingerprinting technique to study
microbial diversity, ecology and evolution. Environ Microbiol. 2009; 11(4): 733-740.
19. Yadegar A, Alebouyeh M, Lawson AJ, Mirzaei T, Mojarad EN, Zali MR. Differentiation of
non-pylori Helicobacter species based on PCR–restriction fragment length polymorphism of the 23S
rRNA gene. World J Microbiol Biotechnol. 2014; 30(6): 1909-1917.
20. Patton TG, Katz S, Sobieski RJ, Crupper SS. Genotyping of clinical Serratia marcescens isolates: a
comparison of PCR-based methods. FEMS Microbiol Lett. 2001; 194(1): 19-25.
21. Sabat AJ, Budimir A, Nashev D, Sá-Leão R, Van Dijl JM, Laurent F, Grundmann H, Friedrich AW,
ESCMID Study Group of Epidemiological Markers (ESGEM. Overview of molecular typing
methods for outbreak detection and epidemiological surveillance. Euro Surveill. 2013; 18(4): 20380.
22. Kanimozhi K, Panneerselvam A. Studies on isolation and nitrogen fixation ability of Azospirillum
spp. isolated from Thanjavur district. Der Chemica Sinica. 2010; 1(3): 138-145.
23. Radif HM, Hassan SS. Detection of hydrolytic enzymes produced by Azospirillum brasiliense
isolated from root soil. World J Exp Biosci. 2014; 2: 36-40.
24. Garibyan L, Avashia N. Research techniques made simple: polymerase chain reaction (PCR). J
Invest Dermatol. 2013; 133(3): e6.
25. Mao DP, Zhou Q, Chen CY, Quan ZX. Coverage evaluation of universal bacterial primers using the
metagenomic datasets. BMC Microbiol. 2012; 12(1): 66.
26. Shime–Hattori A, Kobayashi S, Ikeda S, Asano R, Shime H, Shinano T. A rapid and simple PCR
method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria.
J Appl Microbiol. 2011; 111(4): 915-924.
27. Senthilraj R, Prasad GS, Janakiraman K. Sequence-based identification of microbial contaminants
in non-parenteral products. Braz J Pharm Sci. 2016; 52(2): 329-336.
28. Mazinani Z, Asgharzadeh A. Genetic diversity of Azotobacter strains isolated from soils by
amplified ribosomal DNA restriction analysis. Cytol Genet. 2014; 48(5): 293-301.
29. Lu G, Moriyama EN. Vector NTI, a balanced all-in-one sequence analysis suite. Brief Bioinform.
2004; 5(4): 378-388.
30. Mamaril JC, Trinidad LC. Use of rep-PCR to fingerprint the genome of Azospirillum spp. In
Molecular Microbial Ecology of the Soil. Springer, Dordrecht. 1998; pp. 155-160.
31. Rohlf JF. NTSYSpc, Numerical taxonomy and multivariate analysis system, version 2.1 (© 2002 by
Applied Biostatistics, Inc). Exeter Software. New York: Setauket. 2000.
دنیای میکروبها، سال دوازدهم شماره چهارم زمستان .1389انگشت نگاری ژنومی گونه های مختلف آزوسپیریلوم جدا شده از مزارع گندم و ذرت با روش ..Rep-PCRشهابی و همکاران
375
32. Ayyaz K, Zaheer A, Rasul G, Mirza MS. Isolation and identification by 16S rRNA sequence analysis
of plant growth-promoting azospirilla from the rhizosphere of wheat. Braz J Microbiol. 2016; 47(3):
542-550.
33. Bashan Y, Holguin G, De-Bashan LE. Azospirillum-plant relationships: physiological, molecular,
agricultural, and environmental advances (1997-2003). Can J Microbiol. 2004; 50(8): 521-577.
34. Ilyas N, Bano A. Azospirillum strains isolated from roots and rhizosphere soil of wheat (Triticum
aestivum L.) grown under different soil moisture conditions. Biol Fertil Soils. 2010; 46(4): 393-406.
35. Tarrand JJ, Krieg NR, Döbereiner J. A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum
(Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Can J Microbiol. 1978; 24(8): 967-980.
36. Mehnaz S, Weselowski B, Lazarovits G. Azospirillum zeae sp. nov., a diazotrophic bacterium
isolated from rhizosphere soil of Zea mays. Int J Syst Evol Microbiol. 2007; 57(12): 2805-2809.
37. Doleans-Jordheim A, Cournoyer B, Bergeron E, Croize J, Salord H, Andre J, Mazoyer MA, Renaud
FN, Freney J. Reliability of Pseudomonas aeruginosa semi-automated rep-PCR genotyping in
various epidemiological situations. Eur J Clin Microbiol Infect Dis. 2009; 28(9): 1105-1111.
38. Ishii S, Ohno H, Tsuboi M, Otsuka S, Senoo K. Identification and isolation of active N2O reducers
in rice paddy soil. ISME J. 2011; 5(12): 1936.
39. Tejera NL, Lluch C, Martinez-Toledo MV, Gonzalez-Lopez J. Isolation and characterization of Azotobacter and Azospirillum strains from the sugarcane rhizosphere. Plant Soil. 2005; 270(1): 223-232.
_||_
growth promoting Rhizobacteria isolated from the rhizosphere of wheat (Triticum durum Desf.) in
Algeria. Afr J Microbiol Res. 2013; 7(23): 2893-2904.
2. Bashan Y, De-Bashan LE. How the plant growth-promoting bacterium Azospirillum promotes plant
growth—a critical assessment. In Adv Agron. 2010; 108: 77-136. Academic Press.
3. Eleni V, Anastasia V, Maria D, Iordanis C, Anastasia T, Katinakis P. Phylogenetic analysis of
Azospirillum species isolated from the rhizosphere of field‐grown wheat based on genetic and
phenotypic features. Mol Microbial Ecol Rhizosphere. 2013; 1: 203- 210.
4. Saikia SP, Bora D, Goswami A, Mudoi KD, Gogoi A. A review on the role of Azospirillum in the
yield improvement of non leguminous crops. Afr J Microbiol Res. 2012; 6(6): 1085-1102.
5. Lin SY, Shen FT, Young CC. Rapid detection and identification of the free-living nitrogen fixing
genus Azospirillum by 16S rRNA-gene-targeted genus-specific primers. Antonie Leeuwenhoek. 2011;
99(4): 837-844.
6. Lin SY, Liu YC, Hameed A, Hsu YH, Lai WA, Shen FT, Young CC. Azospirillum fermentarium sp.
nov., a nitrogen-fixing species isolated from a fermenter. Int J Syst Evol Microbiol. 2013; 63(10):
3762-3768.
7. Zhou S, Han L, Wang Y, Yang G, Zhuang L, Hu P. Azospirillum humicireducens sp. nov., a nitrogenfixing bacterium isolated from a microbial fuel cell. Int J Syst Evol Microbiol. 2013; 63(7):
2618-2624.
8. Wisniewski-Dye F, Borziak K, Khalsa-Moyers G, Alexandre G, Sukharnikov LO, Wuichet K, Hurst
GB, McDonald WH, Robertson JS, Barbe V, Calteau A. Azospirillum genomes reveal transition of
bacteria from aquatic to terrestrial environments. PLoS Genet. 2011; 7(12): e1002430.
9. Kaneko T, Minamisawa K, Isawa T, Nakatsukasa H, Mitsui H, Kawaharada Y, Nakamura Y,
Watanabe A, Kawashima K, Ono A, Shimizu Y. Complete genomic structure of the cultivated rice
endophyte Azospirillum sp. B510. DNA Res. 2010; 17(1): 37-50.
10. Sant'Anna FH, Almeida LG, Cecagno R, Reolon LA, Siqueira FM, Machado MR, Vasconcelos AT,
Schrank IS. Genomic insights into the versatility of the plant growth-promoting bacterium
Azospirillum amazonense. BMC genomics. 2011; 12(1): 409.
11. dos Anjos Borges LG, Dalla Vechia V, Corção G. Characterisation and genetic diversity via
REP-PCR of Escherichia coli isolates from polluted waters in southern Brazil. FEMS Microbiol
Ecol. 2003; 45(2): 173-180.
12. Najafi Pour G, Taghavi SM. Comparison of P. syringae pv. syringae from different hosts based on
pathogenicity and BOX-PCR in Iran. J Agr Ssi Tech-Iran. 2011; 13(3): 431-442.
13. Vicente JG, Roberts SJ. Discrimination of Pseudomonas syringae isolates from sweet and wild
cherry using rep-PCR. Eur J Plant Pathol. 2007; 117(4): 383-392.
14. Adiguzel G, Atasever M. Phenotypic and genotypic characterization of lactic acid bacteria isolated
from Turkish dry fermented sausage. Rom Biotechnol Lett. 2009; 14(1): 4130-4138.
15. Versalovic J, Koeuth T, Lupski R. Distribution of repetitive DNA sequences in eubacteria and application to finerpriting of bacterial enomes. Nucleic Acids Res. 1991; 19(24): 6823-6831.
16. Rebecca LH, Zothansanga Singh BP, Gurusubramanian G, Senthil NK. DNA finger printing of
Bacillus thuringiensis based on repetitive DNA sequences using ERIC-PCR. Sci Vis. 2011; 11(3):
147-154.
17. Louws FJ, Rademaker JL, De Bruijn FJ. The three Ds of PCR-based genomic analysis of
phytobacteria: diversity, detection, and disease diagnosis. Annu Rev Phytopathol. 1999; 37(1):
81-125.
18. Ishii S, Sadowsky MJ. Applications of the rep‐PCR DNA fingerprinting technique to study
microbial diversity, ecology and evolution. Environ Microbiol. 2009; 11(4): 733-740.
19. Yadegar A, Alebouyeh M, Lawson AJ, Mirzaei T, Mojarad EN, Zali MR. Differentiation of
non-pylori Helicobacter species based on PCR–restriction fragment length polymorphism of the 23S
rRNA gene. World J Microbiol Biotechnol. 2014; 30(6): 1909-1917.
20. Patton TG, Katz S, Sobieski RJ, Crupper SS. Genotyping of clinical Serratia marcescens isolates: a
comparison of PCR-based methods. FEMS Microbiol Lett. 2001; 194(1): 19-25.
21. Sabat AJ, Budimir A, Nashev D, Sá-Leão R, Van Dijl JM, Laurent F, Grundmann H, Friedrich AW,
ESCMID Study Group of Epidemiological Markers (ESGEM. Overview of molecular typing
methods for outbreak detection and epidemiological surveillance. Euro Surveill. 2013; 18(4): 20380.
22. Kanimozhi K, Panneerselvam A. Studies on isolation and nitrogen fixation ability of Azospirillum
spp. isolated from Thanjavur district. Der Chemica Sinica. 2010; 1(3): 138-145.
23. Radif HM, Hassan SS. Detection of hydrolytic enzymes produced by Azospirillum brasiliense
isolated from root soil. World J Exp Biosci. 2014; 2: 36-40.
24. Garibyan L, Avashia N. Research techniques made simple: polymerase chain reaction (PCR). J
Invest Dermatol. 2013; 133(3): e6.
25. Mao DP, Zhou Q, Chen CY, Quan ZX. Coverage evaluation of universal bacterial primers using the
metagenomic datasets. BMC Microbiol. 2012; 12(1): 66.
26. Shime–Hattori A, Kobayashi S, Ikeda S, Asano R, Shime H, Shinano T. A rapid and simple PCR
method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria.
J Appl Microbiol. 2011; 111(4): 915-924.
27. Senthilraj R, Prasad GS, Janakiraman K. Sequence-based identification of microbial contaminants
in non-parenteral products. Braz J Pharm Sci. 2016; 52(2): 329-336.
28. Mazinani Z, Asgharzadeh A. Genetic diversity of Azotobacter strains isolated from soils by
amplified ribosomal DNA restriction analysis. Cytol Genet. 2014; 48(5): 293-301.
29. Lu G, Moriyama EN. Vector NTI, a balanced all-in-one sequence analysis suite. Brief Bioinform.
2004; 5(4): 378-388.
30. Mamaril JC, Trinidad LC. Use of rep-PCR to fingerprint the genome of Azospirillum spp. In
Molecular Microbial Ecology of the Soil. Springer, Dordrecht. 1998; pp. 155-160.
31. Rohlf JF. NTSYSpc, Numerical taxonomy and multivariate analysis system, version 2.1 (© 2002 by
Applied Biostatistics, Inc). Exeter Software. New York: Setauket. 2000.
دنیای میکروبها، سال دوازدهم شماره چهارم زمستان .1389انگشت نگاری ژنومی گونه های مختلف آزوسپیریلوم جدا شده از مزارع گندم و ذرت با روش ..Rep-PCRشهابی و همکاران
375
32. Ayyaz K, Zaheer A, Rasul G, Mirza MS. Isolation and identification by 16S rRNA sequence analysis
of plant growth-promoting azospirilla from the rhizosphere of wheat. Braz J Microbiol. 2016; 47(3):
542-550.
33. Bashan Y, Holguin G, De-Bashan LE. Azospirillum-plant relationships: physiological, molecular,
agricultural, and environmental advances (1997-2003). Can J Microbiol. 2004; 50(8): 521-577.
34. Ilyas N, Bano A. Azospirillum strains isolated from roots and rhizosphere soil of wheat (Triticum
aestivum L.) grown under different soil moisture conditions. Biol Fertil Soils. 2010; 46(4): 393-406.
35. Tarrand JJ, Krieg NR, Döbereiner J. A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum
(Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Can J Microbiol. 1978; 24(8): 967-980.
36. Mehnaz S, Weselowski B, Lazarovits G. Azospirillum zeae sp. nov., a diazotrophic bacterium
isolated from rhizosphere soil of Zea mays. Int J Syst Evol Microbiol. 2007; 57(12): 2805-2809.
37. Doleans-Jordheim A, Cournoyer B, Bergeron E, Croize J, Salord H, Andre J, Mazoyer MA, Renaud
FN, Freney J. Reliability of Pseudomonas aeruginosa semi-automated rep-PCR genotyping in
various epidemiological situations. Eur J Clin Microbiol Infect Dis. 2009; 28(9): 1105-1111.
38. Ishii S, Ohno H, Tsuboi M, Otsuka S, Senoo K. Identification and isolation of active N2O reducers
in rice paddy soil. ISME J. 2011; 5(12): 1936.
39. Tejera NL, Lluch C, Martinez-Toledo MV, Gonzalez-Lopez J. Isolation and characterization of Azotobacter and Azospirillum strains from the sugarcane rhizosphere. Plant Soil. 2005; 270(1): 223-232.