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

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

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زیست شناسی خاک
دوره 7، شماره 2
آذر 1398
صفحه 135-148

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