نخستين گزارش از Alternaria alternata به عنوان عامل لكه برگي Sambucus ebulus در ايران

First report of Alternaria alternata causing leaf spot on Sambucus ebulus in Iran

گياهان دارويي از ديرباز ارتباط نزديكي با زندگي انساني داشته و براي درمان برخي بيماري‌هاي انساني استفاده شده اند. گياه آقطي با نام علمي Sambucus ebulus (Caprifoliaceae) از گونه هاي ارزشمند و دارويي از زمان قديم است و بومي مناطق جنوبي و مركزي اروپا، شمال غربي آفريقا و جنوب غربي آسيا (به ويژه شمال ايران) مي‌باشد (Westwood 1985; Jabbari et al. 2017). اين گونه نقش مهمي در طب سنتي بسياري از كشورها در مناطق مختلف دنيا دارد (Jabbari et al. 2017). در تابستان سال 1393 بيماري لكه‌برگي روي گياهان آقطي در استان‌هاي گيلان و مازندران مشاهده شد. علائم بيماري بر روي برگ‌هاي آلوده به صورت لكه‌هاي نكروزه، قهوه اي رنگ با هاله‌ قهوه‌اي تيره و به شكل دايره‌اي تا نامنظم مشاهده گرديد. در تابستان سال 1393 و 1394 از گياهان داراي علائم بيماري در استان‌هاي گيلان و مازندران نمونه برداري صورت پذيرفت و جداسازي قارچ‌ها در محيط كشت‌هاي آب آگار 2% (WA 2%) و سيب زميني دكستروز آگار (PDA) انجام گرفت. خالص سازي جدايه‌هاي قارچي به روش برداشتن نوك ريسه در محيط كشت PDA انجام شد. به منظور بررسي ريخت شناختي، جدايه‌ها روي محيط سيب زميني-هويج-آگار (PCA) كشت و در شرايط نور سفيد فلورسنت با دوره روشنايي/ تاريكي 16/8 ساعته و دماي 25 درجه سلسيوس به مدت 7 روز قرار گرفتند (Simmons 2007). پرگنه روي محيط PCA به رنگ سبز زيتوني تيره تا قهوه‌اي و قطر رشد آن پس از 7 روز برابر با 70-65 ميلي‌متر اندازه گيري شد. كنيديوفورهاي اوليه اغلب به صورت منفرد و به طول 70 ميكرومتر مي‌باشند و زنجيره‌ه اي كنيديومي بلند و فاقد انشعاب از كنيديوم‌هاي را توليد مي‌كنند. كنيديوم‌ها اغلب به صورت تخم مرغي يا گرزي شكل حاوي 5-3 ديواري عرضي و 1-2 ديواره طولي بوده و به ابعاد 54-20×12-8 ميكرومتر مي‌باشند (شكل 1، A-D). بر اساس خصوصيات ريخت شناسي قارچ‌هاي جدا شده به عنوان گونه A. tenuissima شناسايي شدند (Simmons 2007). در مطالعات انجام شده توسط ودنبرگ و همكاران (2015) اين گونه در حال حاضر تحت نام A. alternata نامگذاري شده است

Medicinal plants have undoubtedly been considered by human beings since ancient times. Sambucus ebulus (Caprifoliaceae) is one of the best known medicinal herbs since ancient times and is native to Southern and Central Europe, Northwest Africa, and Southwest Asia (especially Northern Iran) (Westwood 1985; Jabbari et al. 2017). It plays an important role in traditional medicine in many countries from different regions of the world (Jabbari et al. 2017). During the summer of 2014, leaf spots have been observed on S. ebulus plants in Gilan and Mazandaran provinces, Iran. Symptoms on infected leaves appeared as brown necrotic spots that surrounded by distinct dark brown haloes and were circular to irregularly in shape. During the summer of 2014 and 2015, sampling was done in Gilan and Mazandaran provinces from symptomatic leaves of S. ebulus and the fungus was isolated on 2% water agar (WA 2%) and potato dextrose agar (PDA). Pure fungal cultures were obtained by picking up a single spore on PDA. For morphological identification, the isolates were cultured on Potato Carrot Agar medium (PCA) and incubated at 25 °C under cool-white fluorescent with 16 h dark, 8 h light photoperiod for 14 days (Simmons 2007). The seven-days-old colonies on PCA were dark olive to brown in color and 70 mm in diameter. Primary conidiophores were often simple, reached to 70 µm in length and produced numerous conidia in a simple long chain. The conidia were 8–12 × 20–45 µm in size, egg or club shaped and contained 3-5 transverse and 1-2 longitudinal septa (Fig. 1a-c).According to macro- and micro morphological characters, all recovered isolated were identified as Alternaria tenuissima which was later synonymized under the name of A. alternata by Woudenberg et al. (2015). PCR amplification of the nuclear ITS-rDNA and RNA polymerase II second largest subunit regions was performed using primers ITS1/ITS4 and RPB2–5F2/fRPB2–7cR respectively (White et al. 1990, Sung et al. 2007; Liu et al. 1999). The sequence of NG1 isolate was deposited into the GenBank with the accession numbers MK212914 for ITS-rDNA and MK262741 for rpb2 region and living cultures of this strain were deposited in the Agriculture Biotechnology Research Institute of Iran culture collection (ABRII 10119). Phylogenetic estimates were evaluated using the Maximum Parsimony Analyses in MEGA 6.0 (Tamura et al. 2013). The results of phylogenetic study showed, NG1 isolate was clustered in a well-supported clade (100%) including sec. Alternaria and related to Alternaria alternate isolates (Fig. 2). Pathogenicity test was performed in greenhouse condition on the detached leaves taken from healthy S. ebulus. Ten healthy leaves per isolates were cleaned with sterile water and were inoculated with conidial suspension containing 1 × 106 conidia/mL using the spray method. The control leaves were inoculated with distilled water. Leaf spots similar to the original symptoms were observed on all inoculated leaves after 5 days since inoculation and the fungus re-isolated from leaf lesions (Fig. 1e-g). Also, no symptoms were seen in controls.