جداسازی و شناسایی باکتری‌های آزوسپیریلوم از ریزوسفر گیاهان گندم و ذرت و تعیین برخی خصوصیات محرک رشدی آن‌ها

نوع مقاله: مقاله پژوهشی

نویسندگان

1 دانشجوی دکتری رشته‌ی بیماری‌شناسی گیاهی، دانشکده کشاورزی، دانشگاه زابل

2 دانشیار بیماری‌شناسی گیاهی، دانشکده کشاورزی، دانشگاه زابل

3 دانشیار موسسه تحقیقات خاک و آب کشور

10.22092/sbj.2019.124371.152

چکیده

باکتری Azospirillum یکی از مهمترین ریزوباکتریایی محرک رشد گیاهان (PGPR) است. آنها در ریزوسفر بسیاری از گیاهان زراعی وجود دارند. در این تحقیق از مزارع گندم و ذرت استان اصفهان نمونه­برداری انجام شد و نمونه­های خاک و ریشه در محیط کشت NFB (Nitrogen-Free Bromothymol blue) کشت گردید. پس از رشد جدایه­ها، نمونه­های دارای هاله­ی شفاف به محیط کشت­های اختصاصی و نیمه اختصاصی نظیر NFB و RC (Rojo Congo=Red Congo) جامد منتقل و جدایه­ها براساس مرفولوژی کلنی­ها بر روی آن­ها انتخاب و خالص­سازی گردید. پس از انجام آزمون­های گرم، کاتالاز، اکسیداز و هوازی-بی­هوازی، تأیید و تشخیص جدایه­های Azospirillum توسط آزمایش­های مولکولی با استفاده از آزمون واکنش زنجیر­ه­ای پلیمراز (PCR) با آغازگرهای اختصاصی Az16S-A و Azo494-F/Azo756-R و توالی­یابی ژن rRNA S16 انجام شد. خصوصیات محرک رشد گیاهان (PGP) در جدایه­های باکتری Azospirillum، با بررسی حضور ژن­های NifH و NifD از آنزیم نیتروژناز توسط آغازگرهای PolF/PolR و FdB260/FdB261 و خصوصیات محلول­سازی فسفات، تولید کیتیناز، تولید HCN، تولید سیدروفور و تولید ایندول استیک اسید بوسیله­ی آزمون­های اختصاصی تعیین گردید. تکثیر قطعات 646، 263، 360 و 390 جفت بازی توسط آغازگرهای Az16S-A، Azo494-F/Azo756-R، PolF/PolR و FdB260/FdB261 به ترتیب وجود باکتری­های آزوسپیریلوم و حضور ژن­های NifH و NifD در آن­ها را تأیید نمود. آزمون­های اختصاصی همچنین توانایی تولید ایندول استیک اسید با میانگین 15 میلی­گرم در لیتر، انحلال فسفات نامحلول با میانگین 8/18 میلی­گرم در لیتر و تولید سیدروفور را دربین جدایه­ها آشکار نمود و نشان داد که بعضی از جدایه­ها دارای خصوصیات PGP متعدد بوده و برخی دیگر دارای یک خصوصیت یا فاقد آن­ها می­باشند. در­نهایت توالی­یابی ژن S rRNA16 تعدادی از جدایه­ها، وجود حداقل سه گونه­ی Azospirillum brasilense،A. lipoferum  و A. zeae را در ریزوسفر گیاهان گندم و ذرت مشخص نمود. این مطالعه برای استفاده از این باکتری­ها به­عنوان مایه­ی تلقیح در کشاورزی پایدار و ارگانیک می­تواند مفید باشد.

کلیدواژه‌ها


عنوان مقاله [English]

Isolation and identification of Azospirillum bacteria of wheat and maize rhizosphere and determination of some plant growth promoting characteristics

نویسندگان [English]

  • mahmood shahabi 1
  • Naser Panjekeh 2
  • H. A. Rahmani 3
  • M. Salari 2
1 ...Ph. D. Student, Department of Plant Protection, University of Zabol, Iran
2 Associate Professor, Department of Plant Protection, University of Zabol, Iran
3 Assistant professor, Soil and Water Research Institute of Iran
چکیده [English]

Azospirillum bacteria are one of the most important plant growth promoting rhizobacteria which exist in the rhizosphere of many crop plants. In this research, some soil and plant roots were collected from wheat and maize rhizosphere of the fields in Isfahan and were cultured in the tubes containing Nitrogen Free Bromothymol (NFB) medium. In order to isolate Azospirillum strains, the grown bacteria pellicles were streaked on NFB and Rojo Congo (RC) specific and semi-specific media. The bacteria were isolated and purified using morphological characteristics on the medium. After gram staining, catalase, oxidase and oxidative-fermentative tests, the detection and identification of Azospirillum isolates was done by molecular tests using PCR test with Az16S-A and Azo494-F/Azo756-R specific primers and 16S rRNA gene sequencing. Plant growth promoting characteristics (PGP) of Azospirillum isolates were determined by investigating the presence of NifH and NifD genes of nitrogenase enzyme by PolF/PolR and FdB260/FdB261 specific primers respectively. The properties of phosphate solubilization and production of chitinase, HCN, siderophores and indole acetic acid of isolates were performed by using specific tests. The replication of 646, 263, 360, and 390 bp fragments by Az16S-A, Azo494-F/Azo756-R, PolF/PolR and FdB260/FdB261 primers respectively, confirmed the presence of Azospirilum bacteria and the presence of NifH and NifD genes. Specific tests also revealed the ability of indole acetic acid production, insoluble phosphate solubilizationwith average value of 15 and 18.8 mg/l respectively and siderophore production in isolates. The results showed that some of the isolates possessed several PGP properties, while some of them had one or no characteristic. Finally, the sequencing of the 16S rRNA gene in some isolates determined and three species of A. brasilense, A. lipoferum and A. zeae identified in rhizosphere of wheat and maize plants.

کلیدواژه‌ها [English]

  • Azospirillum
  • Siderophore
  • Chitinase
  • 16S rRNA
  1. Afzal, A. and Bano, A., 2008. Rhizobium and phosphate solubilizing bacteria improve the yield and phosphorus uptake in wheat (Triticum aestivum). International Journal of Agriculture and Biology 10(1):85-88.
  2. Ahmad, F., Ahmad, I. and Khan, M.S., 2008. Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiological Research 163(2):173-181.
  3. Ahmed, E.A., Hassan, E.A., El Tobgy, K.M.K. and Ramadan, E.M., 2014. Evaluation of rhizobacteria of some medicinal plants for plant growth promotion and biological control. Annals of Agricultural Sciences 59(2):273-280.
  4. Akbari, G.A., Arab, S.M., Alikhani, H.A., Allakdadi, I. and Arzanesh, M.H., 2007. Isolation and selection of indigenous Azospirillum spp. and the IAA of superior strains effects on wheat roots. World Journal of Agricultural Sciences 3(4):523-529.
  5. Ali, B., Sabri, A.N. and Hasnain, S., 2010. Rhizobacterial potential to alter auxin content and growth of Vigna radiata (L.). World Journal of Microbiology and Biotechnology 26(8):1379-1384.
  6. Ayyaz, K., Zaheer, A., Rasul, G. and Mirza, M.S., 2016. Isolation and identification by 16S rRNA sequence analysis of plant growth-promoting azospirilla from the rhizosphere of wheat. Brazilian Journal of Microbiology 47(3):542-550.
  7. Bashan, Y. and De-Bashan, L.E., 2010. How the plant growth-promoting bacterium Azospirillum promotes plant growth—a critical assessment. In Advances in agronomy (Vol. 108:77-136). Academic Press.
  8. Bashan, Y., Holguin, G. and De-Bashan, L.E., 2004. Azospirillum-plant relationships: physiological, molecular, agricultural, and environmental advances (1997-2003). Canadian Journal of Microbiology 50(8):521-577.
  9. Bhatt, P.V. and Vyas, B.R.M., 2014. Screening and characterization of plant growth and health promoting rhizobacteria. International Journal of Current Microbiology and Applied Sciences 3(6):139-155.
  10. Chen, Y.P., Rekha, P.D., Arun, A.B., Shen, F.T., Lai, W.A. and Young, C.C., 2006. Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Applied Soil Ecology 34(1):33-41.
  11. Dashti, A.A., Jadaon, M.M., Abdulsamad, A.M. and Dashti, H.M., 2009. Heat treatment of bacteria: A simple method of DNA extraction for molecular techniques. Kuwait Medical Journal 41(2):117-122.
  12. Dastager, S.G., Deepa, C.K. and Pandey, A., 2011. Potential plant growth-promoting activity of Serratia nematodiphila NII-0928 on black pepper (Piper nigrum L.). World Journal of Microbiology and Biotechnology 27(2):259-265.
  13. de-Bashan, L.E., Hernandez, J.P., Morey, T. and Bashan, Y., 2004. Microalgae growth-promoting bacteria as “helpers” for microalgae: a novel approach for removing ammonium and phosphorus from municipal wastewater. Water Research 38(2):466-474.
  14. Dey, R., Pal, K.K., Bhatt, D.M. and Chauhan, S.M., 2004. Growth promotion and yield enhancement of peanut (Arachis hypogaea L.) by application of plant growth-promoting rhizobacteria. Microbiological Research 159(4):371-394.
  15. El-Hadad, M.E., Mustafa, M.I., Selim, S.M., Mahgoob, A.E.A. and EI-Tayeb, T.S., 2010. In vitro evaluation of some bacterial isolates from Ethiopia and South Africa. Biological Control 45:72-84.
  16. Hayat, R., Ali, S., Amara, U., Khalid, R. and Ahmed, I., 2010. Soil beneficial bacteria and their role in plant growth promotion: a review. Annals of Microbiology 60(4):579-598.
  17. Herman, M.A.B., Nault, B.A. and Smart, C.D., 2008. Effects of plant growth-promoting rhizobacteria on bell pepper production and green peach aphid infestations in New York. Crop Protection 27(6):996-1002.
  18. Ilyas, N. and Bano, A., 2010. Azospirillum strains isolated from roots and rhizosphere soil of wheat (Triticum aestivum L.) grown under different soil moisture conditions. Biology and Fertility of Soils 46(4):393-406.
  19. Kanimozhi, K. and Panneerselvam, A., 2010. Studies on isolation and nitrogen fixation ability of Azospirillum spp. isolated from Thanjavur district. Der Chemica Sinica 1(3):138-145.
  20. Karlidag, H., Esitken, A., Turan, M. and Sahin, F., 2007. Effects of root inoculation of plant growth promoting rhizobacteria (PGPR) on yield, growth and nutrient element contents of leaves of apple. Scientia Horticulturae 114(1):16-20.
  21. Kloepper, J.W., Ryu, C.M. and Zhang, S., 2004. Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathology 94(11):1259-1266.
  22. Lin, S.Y., Shen, F.T. and Young, C.C., 2011. Rapid detection and identification of the free-living nitrogen fixing genus Azospirillum by 16S rRNA-gene-targeted genus-specific primers. Antonie van Leeuwenhoek 99(4):837-844.
  23. López-Reyes, L., Carcaño-Montiel, M.G., Lilia, T.L., Medina-de la Rosa, G. and Armando, T.H.R., 2017. Antifungal and growth-promoting activity of Azospirillum brasilense in Zea mays L. ssp. mexicana. Archives of Phytopathology and Plant Protection 50(13-14):727-743.
  24. Lu, G. and Moriyama, E.N., 2004. Vector NTI, a balanced all-in-one sequence analysis suite. Briefings in Bioinformatics 5(4):378-388.
  25. Mahbouba, B., Nadir, B., Nadia, Y. and Abdelhamid, D., 2013. Phenotypic and molecular characterization of plant growth promoting Rhizobacteria isolated from the rhizosphere of wheat (Triticum durum Desf.) in Algeria. African Journal of Microbiology Research 7(23):2893-2904.
  26. Manivannan, M. and Tholkappian, P., 2013. Prevalence of Azospirillum isolates in tomato rhizosphere soils of coastal areas of Cuddalore District, Tamil Nadu. International Journal of Recent Scientific Research 4:1610-1613.
  27. Mazinani, Z. and Asgharzadeh, A., 2014. Genetic diversity of Azotobacter strains isolated from soils by amplified ribosomal DNA restriction analysis. Cytology and Genetics 48(5):293-301.
  28. Mcgilloway, R.L., Weaver, R.W., Ming, D.W. and Pillai, S.D., 2002. A PCR-MPN based quantitative approach to enumerate nitrifying bacteria in zeoponic substrates. Journal of Rapid Methods & Automation in Microbiology, 10(1), pp.49-58.
  29. Mehanni, M.M. and El-Katatny, M.H., 2012. Evaluation of the antifungal activity of some Azospirillum strains for their possible role as biocontrol agents. Egypt. J. Bot. 2nd International conference, 29-30 April, Minia Univ 79 – 97.
  30. Mehnaz, S., Baig, D.N. and Lazarovits, G., 2010. Genetic and phenotypic diversity of plant growth promoting rhizobacteria isolated from sugarcane plants growing in Pakistan. Journal of Microbiology and Biotechnology 20(12):1614-1623.
  31. Mehta, S. and Nautiyal, C.S., 2001. An efficient method for qualitative screening of phosphate-solubilizing bacteria. Current Microbiology 43(1):51-56.
  32. Minorsky, P.V., 2008. On the inside. Plant Physiology 146(4):323-324.
  33. Murphy, J.A.M.E.S. and Riley, J.P., 1962. A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta 27:31-36.
  34. Naik, P.R., Raman, G., Narayanan, K.B. and Sakthivel, N., 2008. Assessment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil. BMC Microbiology 8(1):230.
  35. Nautiyal, C.S., Mehta, S. and Pushpangadan, P., Council of Scientific and Industrial Research (CSIR), 2003. Composition for qualitative screening of phosphate solubilizing microorganisms and a qualitative method for screening microorganisms. United States Patent 6:638,730.
  36. Pandey, A., Palni, L.M.S. and Hebbar, K.P., 2001. Suppression of damping-off in maize seedlings by Pseudomonas corrugata. Microbiological Research 156(2):191-194.
  37. Pashapour, S., Besharati, H., Rezazade, M., Alimadadi, A. and Ebrahimi, N., 2016. Activity screening of plant growth promoting rhizobacteria isolated from alfalfa rhizosphere. Biological Journal of Microorganism 4(16):65-76.
  38. Pedraza, R.O., 2015. Siderophores production by Azospirillum: biological importance, assessing methods and biocontrol activity. In Handbook for Azospirillum (251-262). Springer, Cham.
  39. Perrig, D., Boiero, M.L., Masciarelli, O.A., Penna, C., Ruiz, O.A., Cassán, F.D. and Luna, M.V., 2007. Plant-growth-promoting compounds produced by two agronomically important strains of Azospirillum brasilense, and implications for inoculant formulation. Applied Microbiology and Biotechnology 75(5):1143-1150.
  40. Pikovskaya, R.I., 1948. Mobilization of phosphorus in soil in connection with vital activity of some microbial species. Mikrobiologiya 17:362-370.
  41. Poly, F., Monrozier, L.J. and Bally, R., 2001. Improvement in the RFLP procedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil. Research in Microbiology 152(1):95-103.
  42. Radif, H.M. and Hassan, S.S., 2014. Detection of hydrolytic enzymes produced by Azospirillum brasiliense isolated from root soil. World Journal of Experimental Biosciences 2(2):36-40.
  43. Richardson, A.E., Hadobas, P.A., Hayes, J.E., O'hara, C.P. and Simpson, R.J., 2001. Utilization of phosphorus by pasture plants supplied with myo-inositol hexaphosphate is enhanced by the presence of soil micro-organisms. Plant and Soil 229(1):47-56.
  44. Saharan, B.S. and Nehra, V., 2011. Plant growth promoting rhizobacteria: a critical review. Life Sciences and Medical Research 21(1):30.
  45. Sahoo, R.K., Ansari, M.W., Pradhan, M., Dangar, T.K., Mohanty, S. and Tuteja, N., 2014. Phenotypic and molecular characterization of native Azospirillum strains from rice fields to improve crop productivity. Protoplasma 251(4):943-953.
  46. Sahu, P.K., Gupta, A., Sharma, L. and Bakade, R., 2017. Mechanisms of Azospirillum in Plant Growth Promotion. Scholars Journal of Agriculture and Veterinary Sciences 4(9):338-343.
  47. Saikia, S.P., Dutta, S.P., Goswami, A., Bhau, B.S. and Kanjilal, P.B., 2010. Role of Azospirillum in the Improvement of Legumes. In Microbes for Legume Improvement (389-408). Springer, Vienna.
  48. Serpil, S, 2012. An agricultural pollutant: chemical fertilizer. International Journal of Environmental Science and Development 3(1):77-80.
  49. Shime–Hattori, A., Kobayashi, S., Ikeda, S., Asano, R., Shime, H. and Shinano, T., 2011. A rapid and simple PCR method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria. Journal of Applied Microbiology 111(4):915-924.
  50. Sridevi, M. and Veera Mallaiah, K., 2007. Bioproduction of indole acetic acid by Rhizobium strains isolated from root nodules of green manure crop, Sesbania sesban (L.) Merr. Iranian Journal of Biotechnology 5(3):178-182.
  51. Stead, D.E., Elphinstone, J.G., Weller, S., Smith, N. and Hennessy, J., 2000. Modern methods for characterising, identifying and detecting bacteria associated with plants. Acta Horticulturae 530:45-59.
  52. Steenhoudt, O. and Vanderleyden, J., 2000. Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiology Reviews 24(4):487-506.
  53. Stoltzfus, J.R., So, R.M.P.P., Malarvithi, P.P., Ladha, J.K. and De Bruijn, F.J., 1997. Isolation of endophytic bacteria from rice and assessment of their potential for supplying rice with biologically fixed nitrogen. Plant and Soil, 194(1-2), pp.25-36.
  54. Tortora, M.L., Díaz-Ricci, J.C. and Pedraza, R.O., 2011. Azospirillum brasilense siderophores with antifungal activity against Colletotrichum acutatum. Archives of Microbiology 193(4):275-286.
  55. Vessey, J.K., 2003. Plant growth promoting rhizobacteria as biofertilizers. Plant and Soil 255(2):571-586.
  56. Yasmin, F., Othman, R., Saad, M.S., and Sijam, K. 2007. Screening for beneficial properties of Rhizobacteria isolated from sweet potato rhizosphere. Journal of Biotechnology 6:49-52.
  57. Zehr, J.P. and McREYNOLDS, L.A., 1989. Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii. Applied and Environmental Microbiology 55(10):2522-2526.