بررسی خصوصیات و تنوع ژنتیکی Xanthomonas translucens عامل بیماری باکتریایی نواری گندم در استان کرمانشاه

نوع مقاله : علمی پژوهشی-فارسی

نویسندگان

1 دانشجوی کارشناسی‌ارشد، گروه گیاه‌پزشکی، دانشکده کشاورزی، پردیس کشاورزی و منابع طبیعی، دانشگاه رازی، کرمانشاه، ایران

2 دانشیار، گروه گیاه‌پزشکی، دانشکده کشاورزی، پردیس کشاورزی و منابع طبیعی، دانشگاه رازی، کرمانشاه، ایران

چکیده

بیماری باکتریایی نواری یکی از مهم‌ترین بیماری‌های گندم است که در صورت مهیا بودن شرایط محیطی خسارت قابل‌توجهی به میزبان خود در مناطق مختلف دنیا وارد می‌نماید. این بیماری در سال­های اخیر در استان کرمانشاه شیوع پیداکرده و موجب خسارت قابل‌توجه روی گندم شده است. به‌منظور بررسی خصوصیات عامل بیماری، طی سال‌های 1398 و 1399 از بذور و برگ‌های دارای علائم بیماری در مناطق عمده گندم‌کاری استان نمونه‌برداری انجام شد. جداسازی باکتری‌ها از بافت‌های گیاهی آلوده به روش معمول تهیه سوسپانسیون از برگ‌ها و بذور خردشده در آب مقطر سترون و کشت روی محیط کشت آگار غذایی (NA) به‌صورت خطی انجام شد. پس از رشد، 140 پرگنه‌ زردرنگ شفاف، براق و گرد خالص‌سازی شدند. برای تعیین خصوصیات فنوتیپی و بیوشیمیایی جدایه‌ها آزمون‌های باکتری‌شناسی مرسوم انجام شد. برای اثبات بیماری­‌زا بودن جدایه‌ها و نیز تعیین پاتووار عامل بیماری، آزمون بیماری‌زایی در گلخانه روی چهار میزبان اصلی شامل گندم، جو، چاودار و تریتیکاله انجام شد. برای شناسایی دقیق‌تر عامل بیماری، ناحیه حفاظت‌شده 16S rRNA در جدایه‌های منتخب، با آزمون PCR و استفاده از آغازگرهای 27F و 1492R تکثیر و تعیین توالی گردید. تنوع ژنتیکی بین جدایه‌ها با استفاده از روش rep-PCR و با آغازگرهای BOX و ERIC بررسی شد. طبق نتایج آزمون‌های فنوتیپی و بیوشیمیایی، جدایه‌ها به گونه X. translucens تعلق داشتند. بر اساس نتایج آزمون بیماری‌زایی، جدایه‌ها در دو گروه قرار گرفتند. اعضای گروه اول فقط روی گندم بیماری‌زا بودند و علائم خفیفی در برگ‌های تریتیکاله ایجاد کردند اما اعضای گروه دوم در هر چهار میزبان ایجاد بیماری کردند، به‌این‌ترتیب اعضای گروه اول به پاتووار undulosa و اعضای گروه دوم به پاتووار cerealis تعلق داشتند. نتایج تعیین توالی 16S rRNA، شباهت نوکلئوتیدی 100% جدایه‌های منتخب را با گونه‌ی X. translucens (CP043500 - MK356430) موجود در پایگاه ژن‌بانک نشان داد. بررسی تنوع ژنتیکی نشان داد بین جدایه‌های مناطق مختلف استان تنوع زیادی وجود دارد، اما ارتباطی بین این تنوع و مناطق نمونه‌برداری دیده نشد. ضمن اینکه rep-PCR نتوانست پاتووارهای شناسایی‌شده را از یکدیگر تفکیک نماید.

کلیدواژه‌ها

عنوان مقاله [English]

Characterization and genetic diversity of Xanthomonas translucens, the causal agent of bacterial stripe of wheat in Kermanshah province, Iran

نویسندگان [English]

  • S. Hosseini 1
  • A. Marefat 2
1 M.Sc. student, Department of Plant Protection, College of Agriculture, Campus of Agriculture and Natural Resources, Razi University, Kermanshah, Iran
2 Associate Professor, Department of Plant Protection, College of Agriculture, Campus of Agriculture and Natural Resources, Razi University, Kermanshah, Iran
چکیده [English]

Background and Objectives
Bacterial stripe caused by Xanthomonas translucens is one of the most important diseases of wheat worldwide. The disease was first reported on barley in America in 1917. In Iran, it was reported for the first time on wheat and barley in Kerman province in 1987. Based on the standards for naming pathovars, the currently accepted pathovars as pathogens infecting cereals include pv. cerealis, pv. translucens, pv. undulosa, and pv. secalis. Following climate changes, especially heavy rainfalls in recent years, and probably the transmission of infected seeds, the disease has shown significant outbreaks and damages on wheat in Kermanshah province, Iran. The present study was performed to (I) characterize the pathogen in Kermanshah province, (II) determine pathogen distribution, and (III) assess the genetic diversity of the strains.
Materials and Methods
Wheat seeds and leaves showing symptoms of the disease were collected from major wheat-growing areas in Kermanshah province during 2019 and 2020. In the laboratory, samples were rinsed with tap water and were macerated into a few drops of sterile distilled water. Two loops full of the resulting suspension were streaked on the nutrient agar medium. Plates were incubated at 25°C and pale yellowish colonies were purified. Physiological and biochemical tests were performed to identify and characterize 140 isolates. To determine the pathovar(s) of the pathogen, the pathogenicity of the strains was tested on wheat, barley, rye, and triticale in the greenhouse. Nearly complete 16S rRNA sequences were determined for two strains representatives of two pathovars distinguished by pathogenicity test using 27F and 1492R primers. Genetic diversity among the isolates was evaluated in rep-PCR utilizing BOX and ERIC primers.
Results
Based on physiological and biochemical tests, Xanthomonas strains from wheat in Kermanshah province were identified as X. translucens. Pathogenicity tests revealed two groups among the strains. Members of the first group induced disease symptoms on wheat and triticale exclusively and were identified as pv. undulosa, while the strains belonging to the second group infected wheat, barley, rye, and triticale, and were identified as pv. cerealis. The rep-PCR using ERIC and BOX primers demonstrated genetic variation among the strains. However, there was no significant difference between the results obtained by ERIC- and BOX-PCR. Nearly complete 16S rRNA sequences were determined as the representative strains of two pathovars distinguished by pathogenicity test and based on the comparison of sequences with the GenBank database. The two strains matched X. translucens with 100% similarity. The nucleotide sequences of the pathogen strains were deposited in the GenBank database under the accession numbers MW173461 and MW193070.
Discussion
According to the findings of this study, X. translucens strains collected from wheat in Kermanshah province belonged to pathovars undulosa and cerealis. Considering the wide host range of these pathovars, the disease could be found on other cereals and grasses belonging to the Poaceae in the province and this subject must be taken into consideration in future studies. Although rep-PCR showed significant diversity among the strains, there was no correlation between the rep-PCR groups and the pathovars identified in the pathogenicity test or the geographic origin of the strains. Biochemical and physiological tests, as well as rep-PCR and 16S rRNA sequencing, could not distinguish the two pathovars. This confirms that pathogenicity tests and host range are still the main methods for determining pathogen pathovar(s).

کلیدواژه‌ها [English]

  • Black chaff
  • Pathogenicity
  • Pathovar
  • rep-PCR

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  © 2022 by the authors. Licensee SCU, Ahvaz, Iran. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0 license) (http://creativecommons.org/licenses/by-nc/4.0/).
گیاه پزشکی
دوره 44، شماره 4
دی 1400
صفحه 89-105

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