مقایسه تنوع جمعیت باکتری‌های همراه بلوط ایرانی در دو روش شناسایی مبتنی بر کشت و مستقل از کشت (متاژنومیکس)

نویسندگان

1 دانشجوی دکتری بیوتکنولوژی جنگل، گروه جنگلشناسی و اکولوژی جنگل دانشگاه علوم کشاورزی و منابع طبیعی گرگان.

2 *نویسنده مسئول: دانشیار، گروه جنگلشناسی و اکولوژی جنگل دانشکده علوم جنگل دانشگاه علوم کشاورزی و منابع طبیعی گرگان، گرگان، ایران

3 کرج پژوهشکده بیوتکنولوژی کشاورزی ایران

4 استاد، گروه بیوتکنولوژی میکروبی پژوهشگاه بیوتکنولوژی کشاورزی ایران (ABRII)، کرج، ایران

5 دانشیار، دانشکده علوم و زیست فنآوری، دانشگاه شهید بهشتی، تهران، ایران.

چکیده

هدف: در طی یک دهه گذشته حدود 30-20 درصد جنگلهای زاگرس با معضلی به نام زوال بلوط مواجه شده­اند. تحقیق حاضر با هدف بررسی تنوع باکتریایی مناطق جنگلی سالم و زوال یافته از طریق مقایسه نتایج دو روش مبتنی بر کشت و مستقل از کشت طراحی و انجام شد.
مواد و روش‌ها: از بافت­های مختلف درختان بلوط و خاک اطراف آنها در جنگل­های مناطق مختلف استان ایلام نمونه برداری انجام شد. در روش مبتنی بر کشت با استفاده از روش­های میکروبیولوژی و مولکولی، باکتری­های موجود در نمونه­ها جداسازی و شناسایی تا سطح جنس و گونه شناسایی شدند. به منظور مطالعه متاژنومیکس در روش مستقل از کشت، DNA میکروبی به طور مستقیم از نمونه­ها استخراج و با آماده سازی کتابخانه متاژنومی با استفاده از آغازگرهای عمومی و نشانگر، تنوع باکتریایی با استفاده از پلتفرم Miseq کمپانی Illumina مورد بررسی قرار گرفت و در ادامه نتایج دو روش مورد مقایسه قرار گرفت.
نتایج: بر اساس روش مبتنی بر کشت، تنها 3 فیلوم شناسایی شد، در صورتیکه در روش متاژنومیکسی، تعداد 9416 OTU بدست آمد که به 12 فیلوم تقسیم بندی شدند. در هر دو روش 3 خانواده Bacillaceae، Enterobacteriacea و Xanthomonadaceae به عنوان خانواده­های غالب مشاهده شدند. تنوع گونه­ایی در یک نمونه (تنوع آلفا) در نمونه رایزوسفر و در مناطق ایوان و گله جار نسبت به سایر نمونه­ها بیشتر بود که در روش مبتنی بر کشت نیز این دو منطقه از تنوع گونه­ای بیشتر برخوردار بودند. تنوع گونه­ایی در بین نمونه­ها (تنوع بتا) در مناطق ایوان، گله­جار، چوار، تنگ­دالاب و ارغوان از نظر ترکیب گونه­ای مشابه بودند. اما جامعه باکتریایی ساقه و برگ نسبت به سایر نمونه­ها شباهت بیشتری داشتند و بالک و رایزوسفر هم از نظر ترکیب گونه­ای شباهت بیشتری با یکدیگر داشتند.
نتیجه‌گیری: در روش متاژنومیکس جنس­های با تنوع خیلی کم هم شناسایی می­شود در صورتیکه در روش مبتنی بر کشت احتمال این موضوع ضعیف است. ضمنا در روش مستقل از کشت می­توان تنوع آلفا و بتا را به شکل جامع­تری بررسی کرد. در روش مبتنی بر کشت می­توان جمعیت باکتریایی را تا سطح گونه شناسایی کرد در صورتیکه در روش مستقل از کشت معمولا تا سطح جنس شناسایی می­شود. لذا پیشنهاد می­شود در مطالعات بررسی جوامع میکروبی ترکیب دو روش مبتنی و مستقل از کشت همزمان استفاده شود.

کلیدواژه‌ها

عنوان مقاله [English]

A comparative study of culture dependent and independent techniques (metagenomics) of bacterial communities associated with Persian oak tree

نویسندگان [English]

  • Elaheh Ahmadi 1
  • Davoud Azadfar 2
  • Mozhgan Kowsari 3
  • Gholamreza Salehi Jouzani 4
  • Masood Tohidfar 5
1 PhD Candidate, Department of Silviculture and forest ecology, Gorgan university of Agricultural Science and Natural Resources
2 Corresponding author. Associate Professor, Department of Silviculture and forest ecology, Gorgan university of Agricultural Science and Natural Resources, Gorgan, Iran
3 Assistant Professor, Microbial Biotechnology Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Iran
4 Professor, Microbial Biotechnology Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Iran
5 Science and biotechnology faculty, Shaid beheshti. Tel.: 02129903244. Fax: 021 29903244
چکیده [English]

Objective
During the last decade, about 20-30% of the Zagros forests have faced a problem called oak decline. The aim of present study was to investigate the bacterial diversity in the healthy and declined forest areas by comparing two methods culture-dependent and culture-independent methods.
Materials and methods
Sampling was done from different tissue and soil of oak trees in the forests of Ilam province. In the culture-based method, using microbiological and molecular methods the bacteria in the samples were isolated and identified to the genus and species level. In order to study metagenomics in culture-independent method, total microbial DNA was extracted directly from the samples and prepared metagenomic library using conventional and index primer, the diversity of bacterial was examined using Illumina's Miseq platform and then the results of the two methods were compared.
Results
Based on the culture-based method, only 3 phylum were identified, while in the metagenomics method, 9416 OTUs were obtained, that were divided into 12 phylum. In both methods, 3 families of Bacillaceae, Enterobacteriacea and Xanthomonadaceae were observed as the dominant families. The species diversity within a sample (Alpha diversity) was higher in the rhizosphere sample and in the Eyvan and Gale jar areas than other samples. The species diversity between samples (Beta diversity) was in Eyvan, Gale jar, Chavar, Tangedalab and Arghavan appear more similar to each other than the sites samples. Bacterial community of stems and leaves were more similar and also bulk and rhizosphere were more similar in bacterial species composition.
Conclusions
In the metagenomics method, very few species are identified, while in the culture method, the probability of this is low. Also, in the culture-independent method, alpha and beta diversity can be studied more comprehensively. In the culture-based method, the bacterial population can be identified up to the species level, while in the culture-independent method, it is usually identified up to the genus level. Therefore, it is suggested to use a combination of two methods simultaneously in studies of microbial communities.

کلیدواژه‌ها [English]

  • Bacterium
  • Diversity
  • Oak Decline
  • Phylogeny
  • Metagenome

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مجله بیوتکنولوژی کشاورزی
دوره 14، شماره 1
اردیبهشت 1401
صفحه 21-46

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