شناسایی مولکولی و تعیین خصوصیات محرک رشدی سویه‌های باکتری جدا شده از ریزوسفر گندم

نوع مقاله : مقالات پژوهشی

نویسندگان

1 دانشگاه آزاد اسلامی واحد کرج

2 موسسه تحقیقات خاک و آب

چکیده

باکتری‌های محرک رشد گیاه‌ از طریق مکانیسم های مختلف مانند حل‌کنندگی فسفات، تولید اکسین و سیدروفور باعث افزایش عملکرد گیاهان زراعی می‌شوند. در این تحقیق جداسازی و شناسایی باکتری‌های ریزوسفر گندم انجام شد و برخی از خصوصیات محرک رشدی از قبیل تولید اکسین، سیدروفور و حل‌کنندگی فسفات معدنی مورد اندازه گیری قرار گرفت. برای غربالگری ژنتیکی 20 جدایه‌ مورد بررسی از روش آنالیز محدودگر دی‌ان‌ای ریبوزمی تکثیری یا ARDRA استفاده شد. بدین منظور پس از انجام PCR با استفاده از آغازگرهای عمومی 27F و 1492R برای تکثیر ناحیه ژنی 16S rDNA، از آنزیم‌های برشی HpaIIو RsaI برای هضم ناحیه ژنی 16S rDNA استفاده شد. نتایج غربالگری ژنتیکی نشان داد پس از هضم ناحیه تکثیر شده با آنزیم‌های برشی ‌تنوع قابل ملاحظه ای مشتمل بر هفت الگوی برشی قابل مشاهده است. گونه‌های Chryseobacterium ginsenosidimutans،C. lathyri ، C. piperi، C. taiwanense، Novosphingobium aromaticivorans، Pedobacter duraquae و Sphingomonas koreensis شناسایی شدند. نتایج نشان داد که تمامی جدایه‌ها قادر به تولید اکسین بودند که بیشترین اکسین تولید شده (93/25 میلی‌گرم در لیتر) مربوط به جدایه F1 بود. همچنین تنها سه جدایه F3، F45 و F46 توانایی تولید سیدروفور در محیط CAS آگار را دارا بودند. در مورد توان حل‌کنندگی فسفات معدنی، نتایج نشان داد که تنها دو جدایه توانایی حل‌کنندگی فسفات را داشتند که در این میان جدایه F6 (16/4 قطر هاله به کلنی) بیشترین توان حل‌کنندگی را داشت.
نتایج نشان داد که تمامی جدایه ها قادر به تولید اکسین بودند که بیشترین اکسین تولید شده (93/25 میلی گرم در لیتر) مربوط به جدایه F1 بود. همچنین تنها سه جدایه F3، F45 و F46 توانایی تولید سیدروفور در محیط CAS آگار را دارا بودند. در مورد توان حل کنندگی فسفات معدنی، نتایج نشان داد که تنها دو جدایه توانایی حل کنندگی فسفات را داشتند که در این میان جدایه F6 (16/4 قطر هاله به کلنی) بیشترین توان حل کنندگی را داشت.

کلیدواژه‌ها


عنوان مقاله [English]

Identification and Evaluation of PGP Traits in Some Bacterial Strains Isolated from Wheat Rhizosphere

نویسندگان [English]

  • Vida Hemmati 1
  • Hadi Asadi Rahmani 2
  • Shokofeh Rezaee 1
1 Islamic Azad University
2 Soil and Water Research Institute
چکیده [English]

Introduction: Wheat is one of the most important food crops. In modern agriculture, due to the increase in human population and the detrimental effects of pesticides such as environmental pollution, concerns about human and animal health, adapting suitable alternatives which have none of these dangerous effects would be necessary. This is possible by increasing the production of bio-fertilizers. Plant growth-promoting rhizobacteria (PGPR) are the beneficial rhizosphere bacteria that can enhance plant growth directly or indirectly through a wide variety of mechanisms. PGPR can stimulate plant growth directly by supplying nutrients such as phosphorous and nitrogen or by the production of phytohormones such as auxins, cytokinins (CK), gibberellins (GAs) or ACC deaminase synthesis. They can also promote plant development indirectly by the suppression of pathogens by different mechanisms such as biosynthesis of antimicrobial molecules or antibiosis induced systemic resistance (ISR), rhizosphere competition, cell wall degrading enzymes like chitinase and HCN production. In this study, amplified ribosomal DNA restriction analysis was performed for screening the bacterial isolates. Then phosphate solubilization, siderophore and auxin release activities and effect of bacterial isolates on wheat seed germination traits were studied.
Materials and Methods: In order to isolate wheat rhizosphere bacteria, soil samples were taken from the wheat rhizosphere of Tehran, Qazvin, Zanjan, West and East Azerbaijan, Kurdistan and Hamadan provinces. Genomic DNA of each isolate was extracted by using a modified cetyl trimethylammonium bromide (CTAB) method. Amplified ribosomal DNA restriction analysis with HpaII and RsaI restriction enzymes was done for genetic screening. Growth stimulating factors were evaluated by auxin production, siderophore production, and inorganic phosphate solubilizing activity. Siderophore production was determined by measuring the diameters of the colony (mm) and of any orange halo (mm) formed from the blue medium surrounding bacterial growth on CAS Blue Agar medium. To examine Pi solubilization capability, 2µ bacteria suspension was placed on the plates containing Sperber’s medium. Cultures were incubated at 25 ± 2 °, when the diameters of the colony and of the halo zone surrounding it were measured and the mean ± SE of the ratios of halo (mm)/colony (mm) calculated. In order to evaluate the production of auxin, isolates were grown in 100ml flasks containing 25ml TSB medium for 48h on a rotary shaker. 1 ml supernatant was mixed with 2ml of Salkowsky reagent after centrifugation at 10000g for 15min. The absorbance of the complex was read at 535nm in a Spectrophotometer. To investigate the effect of bacterial isolates on germination traits, radicle and plumule fresh and dry weight, radicle and plumule length, germination percentage, germination rate, and germination average rate were measured. The data were analyzed with using SAS 9.1. Mean comparisons were performed by LSD and main effective interaction was found significant at P < 0.05.
Results and Discussion: 20 isolates of wheat rhizosphere bacteria were subjected to amplified ribosomal DNA restriction analysis. The 16S rDNA region was amplified by polymerase chain reaction and PCR products were digested by HpaII and RsaI restriction enzymes. From each pattern, one sample was sent to sequencing. Different species including; Chryseobacterium ginsenosidimutans, C. lathyri, C. piperi, C. taiwanense, Novosphingobium aromaticivorans, Pedobacter duraquae, and Sphingomonas koreensis were identified from the wheat rhizosphere. Bacteria were tested for their plant growth promoting qualities. All of the strains produced auxin from 1.90 to 25.93. Mean comparison of the data showed that the highest level of auxin was produced with F1 and the lowest amount was observed by F18. Phosphate solubilization measured as a halo zone on Sperber’s medium was observed with F6 and F56 isolates. The ratio of the diameter of the halo zone to the colony diameter was 2.86 with F6. The highest level of siderophore production by wheat rhizosphere bacteria, observed as halo formation around colonies on CAS Blue Agar medium, was obtained with F46, followed by F45 and F3. The ratio of the diameter of the orange halo surrounding bacterial growth to the colony diameter was 2.86 with F46. The result showed that the effect of wheat rhizosphere bacteria on germination traits such as radicle fresh and dry weight, plumule fresh weight, radicle and plumule length, germination percentage, germination rate, and germination average rate was significant at the one percent level and the effect of wheat rhizosphere bacteria on plumule fresh weight was significant at the five percent level.
Conclusion: Plant growth promoting bacteria enhance the growth and development of plants with different ways. These bacteria affect the growth and development of crops by phosphate solubilization, production of hydrogen cyanide, siderophore, and hormones such as auxin, gibberellic acid and cytokinins. According to the result, due to growth promoting characteristics such as siderophore and auxin production, phosphate solubilization, and the improvement of the seed germination traits, it can be possible to prepare bacterial inoculant for the field experiment in order to increase the availability of nutrients and improve the growth of plants.

کلیدواژه‌ها [English]

  • Auxin
  • PGPR
  • Siderophore
  • wheat
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