شناسايي مخمر Cryptococcus magnus به عنوان عامل شانكر درختان ميوه هسته‌دار در برخي از استان‌هاي مركزي ايران

Cryptococcus magnus as the causative agent of stem and branch canker of stone fruit trees in some central provinces of Iran

شانكر تنه و شاخه يكي از مهم‌ترين بيماري‌هاي درختان ميوه هسته‌دار است كه بارزترين علايم آن ايجاد زخم‌هاي فرورفته روي شاخه‌ها همراه با تراوش صمغ مي‌باشد. دو باكتري Xanthomonas arboricola pv. pruni و Pseudomonas syringae pv. syringae به عنوان عوامل اصلي اين بيماري قبلا از مناطقي از ايران گزارش شده‌اند. به منظور شناسايي و تعيين پراكنش عوامل مولد شانكر از نواحي مركزي ايران، نمونه‌هاي داراي علايم شانكر از باغ‌هاي اين مناطق جمع آوري گرديد. پس از كشت نمونه‌ها روي محيط آگار غذايي حاوي سوكرز (NAS) كلني‌هاي برجسته، كرم متمايل به صورتي و خميري مانند جدا شد. بيماري‌زايي نماينده‌هايي از جدايه‌ها روي نهال‌هاي هلو (Prunus persica) به اثبات رسيد. بررسي‌هاي ميكروسكوپي، فنوتيپي و مولكولي براي شناسايي جدايه‌ها انجام شد. با استفاده از آغازگرهاي ITS4 و ITS5 اين قطعه در واكنش‌هاي زنجيره‌اي پلي‌مراز (PCR) در جدايه‌هاي نماينده تكثير و توالي يابي شد. توالي‌هاي به دست آمده با استفاده از نرم‌افزار بلاست در ژن بانك (NCBI) با توالي‌هاي موجود در اين پايگاه مقايسه و ميزان شباهت تعيين گرديد. بخشي از توالي ژن‌هاي 26S، RPB1 و RPB2 نيز براي تاييد شناسايي استفاده شد. در بررسي ميكروسكوپي جدايه‌ها سلول‌هايي كروي شكل كه برخي از آن‌ها در حال جوانه زدن بودند مشاهده شد. در مقايسه توالي قطعه ITS در ژن بانك، جدايه‌ها بيشترين شباهت را به گونه Cryptococcus magnus نشان دادند. توالي ژن‌هاي 26S، RPB1 و RPB2 نيز اين شباهت را تاييد كردند.

Canker is one of the most damaging diseases of stone fruit trees which incite depressed brown to black lesions, often accompanied by exudation of gum, on twigs, branches and trunk of affected trees. The most common causal agents of bacterial canker in the world as well as in Iran are Pseudomonas syringae pv. syringae and Xanthomonas arboricola pv. pruni. To determine the predominant causative microorganism in some major fruit growing areas of the central provinces of Iran, samples were taken from the canker-affected stone fruit trees in these regions and macerates of the affected bark tissues were cultured on sucrose nutrient agar (NAS). The predominant colonies appearing on NAS were whitish and yeast-like in appearance. Pathogenicity of representative isolates on peach (Prunus persica) seedlings was confirmed. Microscopic, phenotypic and genotypic characteristics of pure cultures of the isolates were determined. DNAs of representative isolates were extracted and the rDNA ITS regions were amplified and sequenced. The sequences were compared with those deposited in GenBank. Partial sequences of the 26S rRNA, RPB1 and RPB2 genes were determined and compared with those existing in GenBank. Budding globose cells were predominant in cell suspensions studied under the microscope. The isolates showed the highest identity with Cryptococcus magnus based on the sequences of the ITS regions. The identity of the isolates as C. magnus was further verified by comparison of the nucleotide sequences of their 26S, RPB1 and RPB2 genes with the homologous regions in Cryptococcus species. It is concluded that canker of stone fruit trees in the central provinces of Iran is predominantly caused by the yeast species C. magnus.