گیاه پزشکی

گیاه پزشکی

ارزیابی نرم افزاری و آزمایشگاهی PCR با آغازگرهای ITS1/ITS4 برای تفکیک مبتنی بر طول قطعه قابل تکثیر در Alternaria و Juniperus

نوع مقاله : علمی پژوهشی -انگلیسی

نویسندگان
1 گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران
2 گروه شیمی و بیوتکنولوژی، دانشکده علوم، محاسبات و فناوری‌های مهندسی، دانشگاه صنعتی سوینبرن، ملبورن، استرالیا
چکیده
جنس Alternaria شامل قارچ‌های اندوفیت و بیماری‌زای فرصت‌طلب است که اغلب با درختان ارس مرتبط هستند، استفاده از نشانگرهای مولکولی مبتنی بر منطقه ITS برای تمایز سریع و دقیق مستقل از روش های کشت و شناسائی کلاسیک بیماری شناسی گیاهی بسیار مورد توجه قرار گرفته است. نظر به حضور توالی ITS در فرمانروا گیاهان و قارچ ها، تمایز قابل اعتماد قطعات قابل ردیابی این منطقه در جدایه‌های قارچ و گیاه اهمیت بالائی دارد. در این مطالعه با ارزیابی درون‌رایانه‌ای توالی‌های مرجع ITS-rDNA معتبری برای گونه های Juniperus spp.(۴۲۳۱ جفت‌باز) و Alternaria alternata (۲۹۱۱ جفت‌باز) با استفاده از روش مونتاژ هدایت‌شده با توالی مرجع از داده‌های قابل دسترس عموم شامل ۱۹۳ رکورد از گونه‌های ارس ایران و ۸۰۵۰ رکورد از A. alternata ساخته شد. هم‌ترازی دو توالی مرجع اختلاف طول قابل‌توجهی را نشان داد به­طوری­که ناحیه کامل ITS (ITS1+5.8S+ITS2) در ارس حاوی حدود ۱۰۹۳ جفت‌باز و همین ناحیه در قارچAlternaria دارای حدود ۴۸۲ جفت‌باز است که اختلافی بیش از ۶۰۰ جفت‌باز را نشان می دهد. مکانیابی رایانه­ای از ۱۴ آغازگر عمومی قارچی بر روی هر دو توالی مرجع، جایگاه‌های اتصال آغازگرها را شناسایی و اندازه قطعات تکثیرشده را پیش‌بینی کرد. از میان جفت آغازگرها،ITS1/ITS4 برای تأیید آزمایشگاهی بر اساس محدوده اندازه مطلوب قطعه، جایگاه‌های اتصال کاملاً حفاظت‌شده و اختلاف اندازه واضح حدود ۶۰۰ جفت‌باز بین محصولات میزبان و قارچ انتخاب شد. در بخش آزمایشگاهی، واکنش زنجیره­ای پلیمراز با آغازگرهای ITS1/ITS4 بر روی DNA خالص Alternaria یک باند حدود ۵۷۰ جفت‌باز و بر روی DNA برگ ارس یک باند حدود ۲/۱ کیلوباز تولید کرد که نشان‌دهنده اختلاف اندازه قابل تشخیص برای تفکیک دو قطعه قابل تکثیر از محتوی ژنومی گیاه و قارچ در شرایط آزمایش‌شده است. این روش ساده، کم‌هزینه و بدون نیاز به کشت، یک ابزار غربالگری اولیه مبتنی بر اندازه برای تشخیص یک آمپلیکون قارچی در بافت‌های ارس فراهم می‌کند، اما تشخیص گونه جدایه‌ها قارچی و حتی تایید تعلق آن‌ها به جنس قارچی خاص نیازمند نشانگرهای تکمیلی است.
کلیدواژه‌ها
موضوعات

عنوان مقاله English

In silico and experimental evaluation of ITS1/ITS4 PCR for size-based discrimination between Alternaria and Juniperus amplicons

نویسندگان English

F. Shokouhifar 1
M. Mamarabadi 1
B. Zaferanloo 2
1 Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
2 Department of Chemistry & Biotechnology, School of Science, Computing and Engineering Technologies, Swinburne University of Technology, Melbourne, Australia
چکیده English

The genus Alternaria comprises endophytic and opportunistic pathogenic fungi frequently associated with Juniperus trees. The use of ITS-based molecular markers for rapid and accurate differentiation, independent of culture-based and classical plant pathology identification methods, has received considerable attention. Because ITS sequences occur in both plants and fungi, reliably distinguishing detectable fragments from fungal and plant isolates is critical. However, the standard ITS barcode has limited resolution for species-level identification. In this study, we conducted an integrated in silico and experimental evaluation of the ITS region for discriminating Alternaria from Juniperus host tissues. For the in silico component, we constructed validated ITS-rDNA reference sequences for Juniperus spp. (4,231 bp) and Alternaria alternata (2,911 bp) using reference-guided assembly of publicly available sequences, including 193 records from Iranian Juniperus species and 8,050 A. alternata records. Alignment of the two reference sequences revealed a marked length difference: the complete ITS region (ITS1+5.8S+ITS2) is approximately 1,093 bp in juniper but only approximately 482 bp in Alternaria, representing a difference of more than 600 bp. In silico mapping of 14 universal fungal primers onto both reference sequences identified primer-binding sites and predicted amplicon sizes. Among the primer pairs, ITS1/ITS4 was selected for experimental validation because of its optimal amplicon size range, fully conserved binding sites, and the clear ~600 bp size difference between the host and fungal products. For the experimental component, PCR with ITS1/ITS4 primers on pure Alternaria DNA produced a single band of approximately 570 bp. In contrast, juniper leaf DNA produced a single band of approximately 1.2 kb, indicating a detectable size difference between the plant- and fungal-derived amplifiable fragments under the tested conditions. This simple, low-cost, culture-free method provides a preliminary size-based screening tool for detecting a fungal amplicon in juniper tissues. However, species identification of fungal isolates, and even confirmation of their assignment to a specific fungal genus, requires additional markers.

کلیدواژه‌ها English

Agarose gel electrophoresis
amplicon size
in silico analysis
Juniperus excelsa
PCR-based detection
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