اولين گزارش از گروه بيماري‌زايي 3 و4 Leptosphaeria maculans عامل شانكر ساقه كلزا از شمال ايران

First report of pathogenicity group 3 and 4 Leptosphaeria maculans, the causal agent of blackleg disease of oilseed rape in Northern Iran

كلزا به عنوان يكي از مهمترين محصولات توليد روغن در سراسر دنيا بوده است و بيماري ساق سياه با عامل بيمارگر Leptosphaeria maculans ازبيماري‌هاي مهم آن در ايران است. از بين گروه‌هاي بيماري‌زايي، گروه بيماري‌زايي1 PG- براي عامل بيمارگر L. biglobosa و PG-2و PG-T براي L. maculans از شمال ايران گزارش شده است (Fernando et al. 2007, Mirabadi et al. 2010). نمونه‌هاي آلوده كلزا از قسمت‌هاي گوناگون گياه با علايم تغيير شكل، رنگ پريدگي و حضور پكنيديوم در سال 95-1392 از مزارع شمال ايران جمع‌آوري شدند. صد و دو جدايه بدست آمده از بافت‌هاي آلوده ابتدا بر اساس مورفولوژي جدايه‌هاو تشگيل رنگدانه (Boerema et al. 2004) و سپس بر اساس مولكولي با استفاده از واكنش زنجيره‌ايي پليمراز با جفت آغازگراختصاصي به عنوانL. maculans شناسايي شدند (Liu et al. 2006 ). پيكنيدهاي قارچ عامل بيماري سياه رنگ، با سلول پايه متورم، قسمت هايي از پيكنيديوم همراه با قطرات صورتي كم رنگ در انتهاي منفذ برآمده ديده شدند. سلولهاي آسك قلوه اي شكل و كشيده باريك داراي هشت اسپور بودند. علاوه بر آن همه جدايه‌ها در گروه عامل بيمارگر پر آزار با استفاده از جفت آغازگر اختصاصي تعيين بيماري‌زايي، شدند. بيماريزايي جدايه‌هاي قارچ عامل بيماري با تعيين غلظت سوسپانسيون سلولي كنيدي (2×107 )در هر ميلي ليتر و سپس محلول پاشي اسپورها در مرحله برگچه هاي اوليه روي ارقام افتراقي وستار، كوينتا و گلاسير انجام شد. همه گياهان تلقيح شده در شرايط اتاقك رشد با دماي Cº22 و رطوبت نسبي 80% با فاصله زماني 16 ساعت روشنايي و تاريكي نگهداري شدند. گروه بيماري‌زايي 32 جدايه بر روي سه رقم افتراقي گلاسير، كوينتا و وستار از نظر حضور لكه‌هاي نكروزه يا هاله كلروتيك و تشكيل پيكنيد مطابق روش كن و فرناندو (Chen and Fernando 2006) مشخص شدند (شكل 1). شانزده جدايه در گروه بيماري‌زايي PG-4 قرارگرفتند و توسط توانايي آنها در توليد اسپور و واكنش حساسيت روي سه رقم افتراقي مشخص شدند. سه جدايه‌PG-3 روي ارقام وستار و گلاسير اسپور مي‌دهند و ايجاد زخم‌هاي قهوه‌ايي و بدون اسپور روي رقم كوينتا مي‌كنند. نتايج نشان داده است، كه از بررسي تغييرجمعيت قارچ بيمارگر در توسعه روش موفقيت آميز در مديريت بيماري مي‌توان استفاده نمود

The rapeseed (Brassica napus) is an important oilseed crop throughout the world for oil production. Blackleg disease which caused by Leptosphaeriamaculans is one of the most important disease of rapeseed in Iran. Among pathogenicity groups PG-1 (Leptosphaeria biglobosa) and PG-2, PGT (L. maculans) has been reported on rapeseed from Mazandaran and Golestan provinces of northern Iran (Fernando et al. 2007, Mirabadi et al. 2010). The samples (leaves, stem, flowers and seed) with symptoms of diseases such as discoloration, deformation, wrinkled and presence of pycnidia were collected during 2013-2016 from canola crops in North Iran. In addition, some of the stem debris with stem canker symptoms was examined for the presence and absence of pathogen sexual (perfect) state. Diseased tissues (leaves, stems, flowers and seed) were surface sterilized to obtain pathogen isolates following the procedure described by West et al. (2002) and Chen et al. (2010). Single ascosporic cultures of the fungus were isolated from psedothecia using the method developed by Li et al. (2004). In all 102 isolates when the causal pathogen was isolated from infected tissues, it was identified as L. maculans, by colony morphology/ pigment production on potato dextrose agar (PDA) and potato dextrose broth (PDB) respectively (Boerema et al. 2004), and species specific polymerase chain reaction (PCR) (Liu et al. 2006). The pycnidia of the fungus were black, globose to subglobose in shape, parts of pycnidia have pale pink droplet overflowed from the ostiole and Asci are clavate to cylindrical, bitunicate and 8 spores. Moreover, all isolates were classified as highly virulent using PCR with virulence type specific primers (Kuusk et al. 2002). Thirty-two isolates of L. maculans were used for determining the pathogenicity test using a cotyledon inoculation method. Pathogenicity of the isolates was confirmed by spraying a conidial suspension of the fungus (2×107 conidia mL-1) on cotyledons of Westar, Quinta and Glacier cultivars. All plants were maintained in a growth chamber at 22°C with 16 h light period and relative humidity of 80%. The first review of the plants was performed after 5 days and changes during experimenting plants have been observed after 12 days using the scale of 0–9 developed by Chen & Fernando (2006) which 0 to 3 reaction was considered as resistant (R), 4 to 6 as intermediate (I) and 7 to 9 as susceptible (S) (Fig. 1). Sixteen of isolates were classified as belonging to pathogenicity group PG-4. They were characterized by their ability to sporulate on the three differential hosts (S=7-9). Three isolates were PG-3, they sporulated on Westar and Glacier, and caused brown, non sporulating lesion on Quinta (I=4-6). The remaining isolates belonged to PG-2 and PGT. Moreover, our findings provided evidence that L. maculans (PG-4) is present on oilseed rape seeds in the North Iran. Our results showed that an understanding of possible shift in fungus populations will be of value in developing strategies for successful management of blackleg disease such as longer rotation between canola crops, new resistant cultivars against all PG isolates and strict regulations concerning crop seed trade. In addition, the current study is the first report on the identification of L. maculans (PG-3, PG-4) from oilseed in Northern Iran.