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Smuts of Poaceous Plants in Markazi and Lorestan Provinces of Iran

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Smuts of Poaceous Plants in Markazi and Lorestan Provinces of Iran دانش بیماریشناسی گیاهی )شاپای ا:0926-9822، شاپای چ: 9982-2926( سال دهم، جلد 2، پاییز و زمستان 2322 Plant Pathology Science (eISSN:2588-6290, pISSN:2251-9270) Vol. 10(1), 2021 Research Article Smuts of poaceous plants in Markazi and Lorestan Provinces of Iran BAHRAM SHARIFNABI1, REZA RAGHEBI1, FARIBA GHADERI 2 1- Department of Plant Protection, Isfahan University of Technology, Isfahan, Iran 2- Department of Plant Protection, Yasouj University, Yasouj, Iran Received: 31.05.2021 Accepted: 04.07.2021 Sharifnabi B, Raghebi R, Ghaderi F (2021) Smuts of poaceous plants in Markazi and Lorestan Provinces of Iran. Plant Pathology Science 10(1):42-63. Doi: 10.2982/PPS.10.1.42. Abstract Introduction: Poaceous plants such as corn, wheat, barley, sorghum, oats and millet are an important part of agricultural ecosystems. Smuts are one of the most important fungal diseases of these plants, which often cause economic damage and the destruction of part or all of their yields. Materials and Methods: Poaceous smut infected plants in farms and pastures of Markazi and Lorestan provinces were sampled. Morphological characteristics of these fungi and their germination type of teliospores were studied with bright field and fluorescent microscopes and identified using valid keys. The phylogenetic relationship of these fungi with other smuts was also investigated based on ITS-rDNA region sequencing. Results: According to the type of host, morphological characteristics and mode of teliospores germination 12 species vs. U. maydis, U. hordei, U. turcomanica, U. avenae, U.nuda, U. bromivora, U. cynodontis, U. tritici, Tilletia laevis, Tilletia controversa Sporisorium reilianum and S. cruentum were diagnosed. The morphological characteristics and phylogenetic relationship of these fungi with other smuts are described. Conclusion: The smuts of poaceous plants in Markazi and Lorestan provinces include eight species of the genus Ustilago, two species of the genus Sporisorium and two species of the genus Tilletia. Keywords: Corn, Wheat, Ustilago, Tilletia, Sporisorium. [email protected] 42 سیاهکهای گیاهان تیره Poaceae در استانهای مرکزی و لرستان ایران شریفنبی و همکاران Plant Pathology Science Vol.10(1), 2021 مقاله پژوهشی سیاهکهای گیاهان تیره Poaceae در استانهای مرکزی و لرستان ایران بهرام شریفنبی 1، رضا راغبی 1، فریبا قادری 2 1- گروه گیاهپزشکی، دانشگاه صنعتی اصفهان، اصفهان 2- گروه گیاهپزشکی، دانشگاه یاسوج، یاسوج دریافت: 11/10/1011 پذیرش: 1011/10/10 شریفنبی ب، راغبی ر، قادری ف )1011( سیاهکهای گیاهان تیره Poaceae در استانهای مرکزی و لرستان ایران. دانش بیماریشناسی گیاهی Doi: 10.2982/PPS.10.1.42. .02- 30 :)1(11 چکیده مقدمه: گیاهان تیره Poaceae مانند ذرت، گندم، جو، سورگوم، انواع یوﻻف و ارزن بخش مهمی از زیستبومهای کشاورزی هستند. سیاهکها از مهمترین بیماریهای قارچی این گیاهان هستند که اغلب باعث خسارت اقتصادی و از بین رفتن بخشی یا تمام محصول آنها میشوند. مواد و روشها: نمونهبرداری از گیاهان تیره Poaceae آلوده به سیاهک در مزرعهها و مراتع استانهای مرکزی و لرستان انجام شد. ویژگیهای ریختشناسی و نحوه جوانهزنی تلیوسپور این قارچها با میکروسکوپهای نوری زمینهروشن و فلورسنت مطالعه و با استفاده از کلیدهای معتبر شناسایی شدند. رابطه فیلوژنتیکی این قارچها با سایر سیاهکها بر اساس توالییابی ناحیهITS-rDNA نیز مورد بررسی قرار گرفت. یافتهها: با توجه به نوع میزبان، خصوصیات ریختشناسی و نحوه جوانهزنی تلیوسپور 12 گونه U. bromivora، U.nuda، U. avenae، U. turcomanica، U. hordei ،U. maydis، Sporisorium reilianum ، Tilletia controversa،Tilletia laevis، U. tritici، U. cynodontis وSporisorium cruentum تشخیص داده شدند. مشخصات ریختشناسی و رابطه فیلوژنتیکی این قارچها با سایر سیاهکها شرح داده شده است. نتیجهگیری: سیاهکهای گیاهان تیره Poaceae در استانهای مرکزی و لرستان شامل هشت گونه از جنس Ustilago، دو گونه از جنس Sporisorium و دو گونه از جنس Tilletia هستند. واژگان کلیدی : ذرت، گندم، Sporisorium ،Tilletia ،Ustilago مقدمه Introduction سیاهکها از مهمترین بیماریهای قارچی گیاهان هستند که اغلب موجب خسارت اقتصادی به ویژه به گیاهان تیره Poaceae مانند ذرت، گندم، جو، سورگوم، انواع یوﻻف و ارزن میشوند. عوامل محیطی و شرایط جغرافیایی در پراکنش گونههای مختلف سیاهک نفش به سزایی دارند. آب و هوای [email protected] 43 دانش بیماریشناسی گیاهی سال دهم، جلد 1، پاییز و زمستان 1011 Plant Pathology Science Vol.10(1), 2021 معتدل، رطوبت نسبی زیاد، هوای مرطوب و رطوبت آزاد باﻻ برای توسعه گونهها مناسب است اگر چه بعضی در محدوده جغرافیایی نسبتاً کوچکی وجود دارند ولی بقیه هر جا که میزبان وجود داشته باشد آنها نیز یافت میشوند )Vanky 2005, Sert 2009 (. تقریباً 1301 گونه سیاهک حقیقی در دو زیر شاخه، چهار رده، هشت راسته، 20 تیره و 11 جنس طبقهبندی و گزارش شده است )Vanky 2008(. احتماﻻً اولین سیاهک از ایران توسط رابینهورست )1781( تحت عنوان .Tilletia sorghi-vulgaris L گزارش و معرفی شده است، ولی در حال حاضر 117 گونه از سیاهکها در ایران شناسایی شدهاند که مربوط به 13 جنس میباشند و میزبانهای اصلی آنها دو تیره Poaceae و Cyperaceae میباشند .)Ershad 2009( شناسایی ریختی هر سیاهک، براساس ویژگیهایی شامل نوع میزبان، نشانههای بیماری روی میزبان و خصوصیات ریختی هاگینهها )Sours( شامل شکل ، رنگ، اندازه، ساختار دیواره تلیوسپور، پیوستگی هاگها، تزیینات سطح خارجی دیواره، غﻻف اطراف هاگها، هاگهای نارس، سلولهای نازا و تندش هاگها انجام میشود )Ershad 2009(. هدف اصلی اجرای این تحقیق جمعآوری و شناسایی ریختی و مولکولی )بر اساس توالییابی ناحیه ITS-rDNA ( گونههای سیاهک روی گیاهان تیره Poaceae در دو استان مرکزی و لرستان بود. مواد و روشها Matherial and Methods نمونهبرداری سیاهکها نمونهبرداری از مزارع و مراتع مختلف در استانهای مرکزی )خنداب- ده سد، فرمهین- ایبک آباد، خنداب، خنداب – بابارئیس، شازند، آشتیان، ساروق، خنداب- مانیزان( و لرستان )ازنا- الیگودرز، الشتر، بدرآباد- خرم آباد، شورآب- خرم آباد، سراب چنگایی، ویسیان- پلدختر، کمالوند- خرمآباد، کوهدشت، دورود، بروجرد( انجام شد. نمونههای گیاهان آلوده به سیاهک همراه با نمونه گیاه سالم جمعآوری شده و به آزمایشگاه منتقل گردیدند. مطالعه خصوصیات ریختی سیاهکها ریختشناسی تلیوسپورها، در محلول شیار )Shear( )011 میلیلیتر محلول دو درصد استات پتاسیم،121 میلیلیتر گلیسرین، 171 میلیلیتر الکل اتیلیک 19 درصد( پس از تهیه اسﻻیدهای میکروسکوپی، برای اندازهگیری خصوصیات مختلف مورفومتریک تلیوسپورهای گونهها، 91 تلیوسپور طبیعی و بالغ بهطور تصادفی از هر گونه در این محلول توسط میکروسکوپ نوری معمولی با بزرگنمایی 111 بررسی شد. سورها نیز در زیر استریومیکروسکوپ مشاهده شدند و اجزای آنها مورد 44 سیاهکهای گیاهان تیره Poaceae در استانهای مرکزی و لرستان ایران شریفنبی و همکاران Plant Pathology Science Vol.10(1), 2021 بررسی قرار گرفت و از تلیوسپورها عکسبرداری انجام شد. همچنین خصوصیات ریختشناسی هر یک از نمونهها توسط میکروسکوپ فلورسنت با بزرگنمایی 111 بررسی و عکسبرداری شد. بدین منظور، به نمونه مورد نظر روی اسﻻید، یک قطره هیدروکسید پتاسیم 19% )19 گرم هیدروکسید پتاسیم KOH، 21 میلیلیتر گلیسرول و 71 میلیلیتر آب مقطر( و یک قطره محلول کلکوفلور و 1111 میلی لیتر آب مقطر( اضافه و کامﻻً مخلوط گردید. نمونههای آماده شده در مدت زمان 21 دقیقه تا شش ساعت بسته به نوع جنس و گونه در شرایط تاریکی در داخل فویل آلومینیومی قرار گرفته شدند. سپس تحت شرایط نور UV بررسی و عکسبرداری شدند )Knaus et al. 2013(. جهت بررسی جوانهزنی تلیوسپورها از محیط کشت آب اگار استفاده شد که بعد از خنک شدن محیط کشت، 91 پی پیام از آنتی بیوتیک کلرامفنیکل به آن اضافه شد. هاگهای قارچ در آب مقطر سترون رقیق گردید و سپس 1-9/1 میلی لیتر از آن روی محیط کشت آب آگار ریخته شد و پخش گردید و در مدت زمانی 11 روزه در دمای c˚21 در شرایط تناوب نوری 12 ساعت )12 ساعت تاریکی، 12 ساعت روشنایی( قرار گرفتند، الگوی جوانهزنی آنها بررسی شد. برای شناسایی جنس و گونهها، ازکلیدهای گونه و جنس سیاهکها استفاده گردید )Vanky 2005, Vánky and Abbasi 2013 ). بررسی رابطه فیلوژنتیکی سیاهکها برای تأیید شناسایی ریختشناسی گونههای جنس Sporisorium ،Ustilago و Tilletia ابتدا استخراج DNA به روش موری و تامپسون انجام گرفت )Murray and Thompson 1980(. سپس از جفت آغازگر عمومیITS5 /ITS4 برای شناسایی گونههای مختلف سیاهکها استفاده گردید )White et al. 1990(. مخلوط واکنش PCR با حجم 21 میکرولیتر، 7 میکرولیتر آب دیونیزه استریل، 0 میکرولیتر 10X PCR Buffer، 9 میلیموﻻر MgCl2، 211 میلیموﻻر از هر نوکلئوتید، 21 پیکومول از هر آغازگر، یک واحد Taq DNA پلیمراز و سه میکرولیتر DNA با غلظت 11 نانوگرم تهیه شد. برنامه حرارتی در PCR شامل یک چرخه واسرشتسازی اولیه در دمای 13 درجه سانتیگراد به مدت پنج دقیقه، 09 چرخه شامل واسرشتسازی در دمای 13 درجه سانتیگراد به مدت 01 ثانیه، دمای اتصال آغازگرها به ناحیه هدف در دمایC° 93 به مدت 09 ثانیه و گسترش رشته در دمای 82 درجه سانتیگراد به مدت 31 ثانیه و یک چرخه گسترش نهایی رشته در دمای 82 درجه سانتیگراد به مدت هشت دقیقه انجام گرفت. بعد از تکثیر ناحیه مورد نظر، محصول PCR در روی ژل آگاروز یک درصد ارزیابی شد. جهت تعیین توالی، محصول حاصل از تکثیر نواحی ژن ITS-rDNA به شرکت .Macrogen Inc کره جنوبی فرستاده شد. برای اطمینان از صحت توالیهای به دست آمده از هر نمونه، هر یک از توالیها با استفاده از ابزار جستجوی Altschul et al. ( Blast 1997( با توالیهای موجود در بانک ژن NCBI مقایسه شد. برای مقایسه فیلوژنتیکی جدایههای 45 دانش بیماریشناسی گیاهی سال دهم، جلد 1، پاییز و زمستان 1011 Plant Pathology Science Vol.10(1), 2021 توالییابی شده، تعدادی توالی از گونههای مختلف جنس Sporisorium ،Ustilago و Tilletia از بانک ژن انتخاب گردید و Puccinia graminis نیز به عنوان آرایه خارج از گروه )جدول 1( انتخاب گردید. توالیها با استفاده از نرمافزار MAFFT v.7.043b همردیفسازی گردید ) Katoh and Standley 2013(. آنالیزهای فیلوژنتیکی با استفاده از روش فاصله )Distance method( انجام گردید. ماتریکس فاصله توالیهای مرتب شده با روش پارامتر دو کیمورا )Kimura,s two parameter( محاسبه جدول 2. مشخصات جدایههای گونههای Sporisorium ،Ustilago و Tilletia مورد استفاده در تجزیه و تحلیلهای فیلوژنتیکی در این مطالعه. Table1. Characteristics of Ustilago, Sporisorium and Tilletia species isolates used in phylogenetic analyses in this study.
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