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The First Report of Panaeolus Olivaceus and Panaeolus Guttulatus from Iran

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The First Report of Panaeolus Olivaceus and Panaeolus Guttulatus from Iran Archive of SID 2 Taxonomy and Biosystematics, 11th Year, No. 39, Summer 2019 The First Report of Panaeolus Olivaceus and Panaeolus Guttulatus from Iran Elham Seidmohammadi 1, Saeed Abbasi 2*, Mohammad Reza Asef 3 1 MSc Graduate of Plant Protection, Faculty of Agriculture, Razi University, Kermanshah, Iran. 2 Associate Professor, Department of Plant Protection, Faculty of Agriculture, Razi University, Kermanshah, Iran 3 Assistant Professor, Department of Botany, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran Abstract During macrofungal surveys continually undertaken from 2014 to 2017 at different locations of Eslamabad-e Gharb and Gilan-e Gharb, Kermanshah Province, a large number of agaric fungi including two specimens belonging to the genus Panaeolus were collected. At the collection site, while recording geographical coordinates, habitat characteristics, growth patterns, and macroscopic morphological features were recorded and photographed. Microscopic features including basidiospores, basidia, and cystidia were observed, measured, and illustrated using a light microscope, OLYMPUS BX51. Also, the DNA sequence of the rDNA-ITS region was amplified and sequenced using ITS1 and ITS4 primers. The phylogenetic analysis and the determination of species relationships were performed by the Maximum Likelihood method with 1000 repetitions of 1000 bootstrap replicates using the MEGA6 software. Based on the results obtained from morphological examinations along with data obtained from ITS- rDNA sequences, two species including Panaeolus olivaceus and Panaeolus guttulatus were identified. According to the literature, both species were new records for mycobiota of Iran. In the present paper, the morphological description of the mentioned species is presented. Key words: Eslamabad-e Gharb, Agaric, Macrofungi, Kermanshah. * [email protected] Copyright©2019, University of Isfahan. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/BY-NC-ND/4.0), which permits others to download this work and share it with others as long as they credit it, but they cannot change it in any way or use it commercially. www.SID.ir Archive of SID تاکسونومی و بیوسیستماتیک، سال یازدهم، شماره سی و نهم، تابستان 1398، صفحه 30-23 دریافت مقاله: 23/04/1398 پذیرش نهایی: 1399/04/18 اولین گزارش از Panaeolus olivaceus و Panaeolus guttulatus در ایران 3 2 1 الهام صید محمدی ، سعید عباسی *، محمدرضا آصف 1 دانش آموخته کارشناسی ارشد گیاهپزشکی، دانشکدۀ کشاورزی، دانشگاه رازی، کرمانشاه، ایران 2 دانشیار گروه گیاهپزشکی، دانشکدۀ کشاورزی، دانشگاه رازی، کرمانشاه، ایران 3 استادیار بخش تحقیقات رستنیها، مؤسسۀ تحقیقات گیاهپزشکی کشور، سازمان تحقیقات آموزش و ترویج کشاورزی، تهران، ایران چکیده طی نمونه برداری های مستمر انجامشده از سال 1393 تا 1396 در مناطق مختلفف شهرسفتان هفای اسف م آبفاد رفرو و گی ن ررو واقع در استان کرمانشفاه، تعفداد زیفادی از قفار هفای ک هفک دار ازجملفه دو نمونفه متعلفق بفه جفن Panaeolus جمع آوری شد. در محل نمونه برداری، ضمن ثبت مختصات جغرافیایی، ویژگفی هفای زیسفتگاه و نحفوۀ رویش، ویژگیهای ریختشناختی ماکروسکوپی ثبت و از آنهفا عکف بفرداری شفد. ویژگفی هفای ریخفت شفناختی میکروسکوپی شامل بازیدیوسپور، بازیدیوم و سیستیدیوم بفا اسفتفاده از میکروسفکوو نفوری مفدل BX51 مشفاهده، اندازهگیری و عک برداری شد؛ همچنین توالی DNA مربوط به ناحیۀ ITS-rDNA با استفاده از آرازگرهفای ITS1 و ITS4 تکثیففر و تعیففین تففوالی شففد. تجزیففه و تحلیففل تبففارشففناختی و تعیففین روابففن گونففههففا بففه روش Maximum Likelihood با 1000 تکرار بوت استراو و با نرمافزار MEGA6 انجام شد. براساس نتفایج ماالعفات ریخفت شفناختی، در کنار دادههای حاصل از توالیهفای ناحیفۀ ITS-rDNA، دو گونفۀ P. olivaceus و P. guttulatus شناسفایی شفد. طبق منابع موجود، هر دو گونه برای بیوتای قارچی ایران، جدید هستند. در پژوهش حاضر، توصیف ریخفت شفناختی این گونهها ارائه شده است. واژههای کلیدی: اس مآباد ررو، آگاریک، قار های ماکروسکوپی، کرمانشاه. مقدمه بلففوآ آنهففا ریریکنواخففت اسففت؛ بففه همففین دلیففل در Panaeolus (Fr.) Quél جفففن دربرگیرنفففدۀ بخش هایی از تیغه که اسپور ها بالغ شده انفد، رنف تیغفه گونففههففای آگاریففک سففاپروترون بففا انففدام بففار دهی نسبت به قسمت هفایی کفه اسفپورها هنفوز نابفالغ هسفتند، کوچک تا متوسن و اسپورهای تیفره اسفت. ایفن جفن تیره تر است و تیغه هفا افاهری خفال دار دارنفد. برخفی از Kirk, 2019 شامل حدود 77 گونه میشود) (. گونههفای گونه های این جفن دارای ترکیف هفای سیلوسفایبین و Panaeolus جن بیشتر کو ددوست هسفتند و تعفدادی درنتیجه توهمزا هستند)Webster and Weber, 2007(. از آنها در مراتع رشد می کننفد. اسفپور هفا، سفیاه رنف و گونه هفای جفن Panaeolus کفه در گذشفته در زمفرۀ * [email protected] www.SID.ir Archive of SID 24 تاکسونومی و بیوسیستماتیک، سال یازدهم، شماره سی و نهم، تابستان 1398 اعضففای تیففرۀ Coprinaceae طبقففهبنففدی مففی شففدند در خانوادهای جدید و در موقعیتی بین Bolbitiaceae و )Singer, 1986(، با تغییراتفی کفه در طبقفه بنفدی انجفام Psathyrellaceae قرار گیرند)Tóth et al., 2013(. شد، در خانوادۀ Psathyrellaceae قفرار گرفتنفد؛ حفال تاکنون هشت گونه از ایفن جفن از ایفران گفزارش آنکففه برخففی قففار شناسففان ایففن جففن را در خففانوادۀ شففده اسففت )جففدول 1(. در ایففن ماالعففه، ضففمن ارائففۀ Bolbitiaceae طبقهبندی کفرده انفد ) ,.Matheny et al ارزیابی هفای تبارشفناختی، توصفیف ریخفت شفناختی دو 2006(؛ بفا ایفن حفال موقعیف ت سیسفتمات یک ایفن جفن گونۀ جدیدی ارائه شده است که پفیش از ایفن از ایفران هنفوز معلفوم نیسفت )Kirk, 2019(. براسفاس دادههفا ی گزارش نشدهاند. موجود، Panaeolus و آرایه های وابستۀ آن بهتفر اسفت جدول 1- گونههای گزارششدۀ جن Panaeolus از ایران Speices Locality References Panaeolus fimicola Arasbaran (Asef, 2007) P. sphinctrinus Ahar (Watling and Sweeney, 1974) P. papilionaceus Tonekabon, (Saber, 1995) P. phalaenarum Chalus (Watling and Sweeney, 1974) P. rickenii Mashhad (Zokaei, 2001) P. serotinus - (Saber, 1994) P. stipticus - (Saber, 1994) P.subbalteatus Golestan (Arefipour et al., 2006) P. subfirmus - (Saber, 1994) P. teutonicus Amol (Watling and Gregory, 1977) مواد و روشها مختصات جغرافیایی، از دستگاه مکان یاو GPS استفاده نمونهبرداری و شناسایی ریختشناختی شد. در محل نمونه بفردار ی، ضفمن ثبفت ویژگفی هفا، از جمعآوری نمونههای قارچی طی سال های 1393 تفا تمامی نمونه هفا از زوایفای مختلفف، بفه کمفک دوربف ین 1396 با نمونه برداری هفای مسفتمر و در فصفول مختلفف دیجیتال Samsung S6 عک برداری شفد. نمونفه هفا در سال بهویژه فصل بهار و پاییز انجام شفد؛ بفه ایفن منرفور پاکففتهففای کارففذی بففه آزمایشففگاه منتقففل شففدند. در مناطق مختلف ارتفاعات ق جه و دشتهای اطفران، در آزمایشگاه از نمونه ها برش های میکروسکوپی تهیه شفد. حد فاصل شهرهای اس مآباد ررو و گی ن ررو واقفع برش های میکروسکوپی در آو مقار و معرن ملفزر بفا در اسففتان کرمانشففاه بازدیففد شففد. بففرای ثبففت ارتفففا و اسففففتفاده از میکروسففففکوو Olympus مففففدل BX51 www.SID.ir Archive of SID اولین گزارش از Panaeolus olivaceus و Panaeolus guttulatus در ایران 25 بررسی شدند. بهمنرور ثبت ویژگی هفای میکروسفکوپی توالی های به دستآمفده بفرا ی هفر یفک از نمونفه هفا، بفا و سایر ساختارهای میکروسکوپی، از حفداکثر 20 مفورد اسففتفاده از نسففخۀ هفففت نففرمافففزار BioEdit، ویففرایش با عدسی چشفمی Dino eye lens 30 mm و نفرم اففزار شدند. توالیهای نوکلئوتیدی ویرایششده در مجموعف ۀ Dino capture عک برداری و ابعاد اسفپور بفه کمفک دادههففففای ژنففففی http://www.ncbi.nlm. NCBI) همففین نففرمافففزار انففدازهگیففری شففد. بففهمنرففور شناسففایی (nih.gov/genbank ثبفففت و شفففمار ۀ دسترسفففی آنهفففا آرایففههففا از کلیففدهای شناسففایی معتبففر ) ,Gerhardt دریافت شد. Hansen and Knudsen, 1992; 2008( و مقالف ههفای تخصصی مرتبن استفاده شد. ارزیابیهای تبارشناختی توالی های نوکلئوتیدی با استفاده از نرمافزار استخراج DNA و تکثیر ناحیۀ ITS-rDNA جستجوی BLAST استخراج DNA نمونهها با استفاده از کیت استخراج (http://www.blast.ncbi.nlm.nih.gov/blast.cgi) با DNA شرکت دنازیست )Denazist Asia( انجفام شفد . دیگر توالی های موجود در مجموعۀ دادههای ژنی توالی نوکلئوتیدی DNA ناحیۀ ترانویس یشفوند ۀ داخلف ی )NCBI, USA( مقایسه و نزد یکترین توالی ها ریبوزومی (ITS1-5.8S-ITS2) با آرازگرهفا ی ITS1 و شناسایی شد. رد یفچینی چندگانه با نرمافزار Clustal ITS4 تکثیر شد. واکنش زنجیفر ه ای پلیمفراز بفا دسفتگاه W انجام شد. تجزیه و تحلیل تبارشناختی و تعیین روابن ترموسففففففففایکلر (مففففففففدل Biometra، شففففففففرکت گونهها به روش Maximum Likelihood با تکرار Analytic Jena، آلمان( انجام شد. مخلوط واکفنش در 1000 بوتاستراو با استفاده از نرمافزار MEGA6 حجفم نهفایی 50 میکرولیتفر، شفامل 25 میکرولیتفر 2X انجام شد)Tamura et al., 2013(. PCR Master Mix )شفففرکت سفففیناکلون(، یفففک میکرومول از هرکدام از پرایمرهفا و 50 نفانوگرم DNA نتایج و بحث ژنومی بود. واکنش زنجیره ای پلیمراز براساس برنامفه ای بین گونه های شناسایی شده در این ماالعه، دو نمونفه P.olivaceus Panaeolus زمففانی شففامل واسرشففتگی ابتففدایی در دمففای 90 درجففۀ از جففففففن شففففففامل P.guttulatus (MH592651) (MH593015) سانتیگراد به مدت دو دقیقفه و بفه دنبفال آن 35 چرخفه و برپایۀ شففامل واسرشففتگی در دمففای 95 درجففۀ سففانتیگففراد بففه ویژگی های ریختشناختی شناسایی شدند. دربارۀ گونفۀ DNA P.olivaceus مدت 40 ثانیه، همجوشی در دمای 57 درجۀ سانتیگراد ، درصففد تشففابه تففوالی بففا دیگففر بفففه مفففدت 40 ثانیفففه، گسفففترش در دمفففای 72 درجفففۀ توالی های همفین گونفه در بانفک دادههفای ژنف ی، تشفابه سانتیگراد به مدت 50 ثانیه و گسترش نهفایی در دمفای بیش از 99 درصدی نشفان داد؛ امفا براسفاس جسفتجوی P.guttulatus 72 درجۀ سانتیگراد به مدت ده دقیقه، انجام شد. ب سفففت دربفففارۀ ، بفففا وجفففود انابفففا محصوﻻت واکنش زنجیفره ای پلیمفراز بفرای تعیفین ویژگی های ریختشناختی نمونۀ موجفود بفا ایفن گونفه، DNA ترادن به شرکت ماکروژن کفر ۀ جنفوب ی ارسفال شفدند. نمونۀ حاضر ازنرر تفوالی دارای قرابفت کفافی بفا www.SID.ir Archive of SID 26 تاکسونومی و بیوسیستماتیک، سال یازدهم، شماره سی و نهم، تابستان 1398 توالیهای موجود P.guttulatus در بانک دادههای ژنی اسففت. ابعففاد اسففپور 8/12-6/10 × 1/9-2/7 میکرومتففر نیست؛ از این رو به نرر می رسد که گونۀ P.guttulatus است )شفکل c-1(. بازیفدیوم هفا، گفرزی شفکل و دارای گونه ای کمپلک است؛ با این حفال تفکیفک گونفه هفا، چهار استریگمای بزرگ است که بفا فاصفله از یکفدیگر نیازمند دسترسی به تفوالی هفای چنفد ژن بفرای تجزیفه و قففرار دارنففد )شففکلb-1(.
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