پديد آورندگان :
كشاورز، طيبه سازمان تحقيقات آموزش و ترويج كشاورزي - موسسه تحقيقات گياه پزشكي كشور، تهران , حاج نجاري، حسن سازمان تحقيقات آموزش و ترويج كشاورزي - موسسه تحقيقات علوم باغباني - پژوهشكده ميوه هاي معتدله و سردسيري،
كليدواژه :
اليزا , كلكسيون ارقام سيب , واكنش زنجيره‌اي پلي‌مراز , ويروس لكه سبزرد سيب
چكيده فارسي :
كلكسيون ملي ارقام تجاري بومي و وارداتي سيب مستقر در ايستگاه تحقيقات باغباني كمال‌شهر از اهميت خاصي براي برنامه‌هاي سالم‌سازي با هدف تأمين هسته‌هاي اوليه و پيش‌تكثير جهت توليد نهال سالم برخوردار است. تاكنون مطالعه متمركزي در زمينه غربالگري درختان سيب موجود در اين كلكسيون از نظر آلودگي به بيماري‌هاي ويروسي صورت نگرفته است. ويروس لكه سبزرد سيب (Apple chlorotic leaf spot virus, ACLSV) ويروس مهمي است كه باعث ايجاد آلودگي درختان ميوه در سراسر جهان مي‌شود. جهت ارزيابي وقوع و ميزان شيوع اين ويروس در كلكسيون سيب كمال‌شهر از مجموع 50 رقم سيب جديد، اميدبخش، بومي پر‌محصول و ارقام وارداتي سازگار بر روي پايه‌هاي بذري، نمونه‌برداري انجام شد و به روش الايزاي مستقيم با آنتي‌بادي اختصاصي ACLSV ارزيابي گرديد و جهت تأييد نتايج اليزا، تعدادي از اين ارقام با استفاده از روش واكنش زنجيره‌اي پلي مراز به روش نسخه‌برداري معكوس (RT-PCR) و جفت آغازگر تكثير‌كننده بخشي از پروتئين پوششي ACLSV مورد بررسي قرار گرفتند. نتايج اليزا نشان داد كه تعداد 39 رقم از 50 رقم مورد بررسي، به اين ويروس آلوده بوده و در آزمون RT-PCR نيز از 18 رقم مورد بررسي تنها پنج رقم، آلوده به ويروس تشخيص داده شد. تعدادي از ارقام شامل مكينتاش، ردرم بيوتي، استاركينگ، اردبيل 1، نايان ارنگه، خورسيجان، رداسپور كوپر، يلو ترنسپارنت 1، IR6-1 و اردبيل 2، در هر دو آزمون اليزا و RT-PCR عاري از ويروس تشخيص داده شدند. بنابراين اين ارقام پس از بررسي از نظر آلودگي به سه ويروس مهم ديگر شامل ويروس ساقه گودي سيب (Apple stem pitting virus, ASPV)، ويروس ساقه شياري سيب (Apple stem grooving virus, ASGV) و ويروس لكه حلقوي گوجه فرنگي (Tomato ring spot virus, ToRSV) مي‌توانند در برنامه توليد مواد تكثيري سالم و گواهي‌شده مورد استفاده قرار گيرند.
چكيده لاتين :
Introduction: Pome fruits are affected by many viruses that cause diseases with adverse effects in orchards
worldwide. Apple chlorotic leaf spot virus (ACLSV) is one of the most widespread and economically important
latent viruses that naturally affect many Prunus species, apples (Malus domestica Borkh.), pears (Pyrus
communis L.) and the other rosaceous species. ACLSV infection rates of up to 80–100% in many commercial
apple cultivars with yield losses of the order of 30–40% have been reported. In most commercial apple cultivars,
the infection generally is latent, but insensitive cultivars, such as apple trees grown on Marubakaido (Malus
prunifolia cv. Ringo) rootstocks, malformation and reduction in leaf size and chlorotic rings or line patterns are
common. The severity of symptoms induced by ACLSV depends largely on the plant species and virus strains.
Some virus isolates induce a severe disease in apricot and plum characterized by depressions and protuberances
that deform the fruit, often confused with the “sharka” disease due to Plum pox virus (PPV), and named for this
reason as “pseudopox.” ACLSV is a filamentous virus, 680–780 nm long and 12 nm in width as the type species
of the genus Trichovirus in the family Betaflexiviridae. ACLSV contains a single-stranded, positive-sense RNA
about 7.5–8 kb in size, with a polyadenylated 3" terminus and a cap at its5"-end, and also contain multiple copies
of a single coat protein (CP) of 21–24 kDa. The economic importance of ACLSV is largely due to its worldwide
distribution and its capacity to induce severe graft incompatibilities in some Prunus combinations, causing major
problems in nurseries. In apple trees, ACLSV frequently is detected in coinfection with Apple stem grooving
virus and Apple stem pitting virus. ACLSV is mainly transmitted by grafting. No natural virus vectors are known
for this virus and are not known to be seed or pollen transmitted. The older National Apple Collection of Native
and Imported apple cultivars in Kamalshahr Horticulture Research Station located in Karaj includes 85 cultivars
and promising genotypes on seed stocks benefit a high genetic variability. Though the cultivars were screened
for more biotic and abiotic factors, but no screening has been achieved yet for virus diseases, while this valuable
germplasm comprises a wide range of newly released cultivars or in releasing procedure, high yield natives and
those imported selected as adapt to Iranian climate, so a great need of healthy primary nucleus to establish
mother orchards for certified plant material. As this valuable germplasm is very important in providing healthy
primary nucleus to establish mother orchards for certified plant material so the aim of this research was an
evaluation of native and imported apple tree cultivars of this Collection to ACLSV infection.
Material and Methods: To assess the occurrence and the prevalence of this virus in the collection, a total of
fifty accessions were collected. The sample collection was carried out in summer (2016) and spring (2017). The
collecting method consisted of sampling leaves homogeneously distributed around the canopy of the plant. All
samples were screened for the presence of ACLSV by DAS-ELISA using the ACLSV specific polyclonal
antibody using commercial kits purchased from Bioreba Company, Switzerland. To confirm ELISA results some
cultivars were subjected to reverse transcription-polymerase chain reaction. Total RNA was isolated from some
apple cultivars using CTAB RNA extraction method and was used as a template for RT-PCR. Specific
oligonucleotide primers corresponding to a region of the ACLSV genome that encodes part of the CP, were used
in RT-PCR. The amplified PCR products were analyzed in 1% agarose gel stained by ethidium-bromide and
visualized under UV light after electrophoresis.
Result and Discussion: The ELISA results showed that 39 out of 50 cultivars were infected by ACLSV. RTPCR on total RNA from the ELISA positive samples resulted in the amplification of an expected 358 bp DNA
fragment. In RT-PCR out of 18 tested cultivars, five were infected. Some cultivars including Makintash, Red
Rome Beauty, Starking, Ardebil1, Nayan Arangeh, Khorsijan, Red spur cooper, Yellow transparent 1, IR6-1 and
Ardebil2 recognized ACLSV free in ELISA and RT-PCR, so could be used in the production and employment of
virus-free propagating material program after infection testing to the other three important viruses Apple stem
pitting virus, Apple stem grooving virus and Tomato ringspot virus. The results showed a high rate of ACLSV
infection of native and imported apple cultivars in the older National Apple Collection in Kamalshahr
Horticulture Research Station. Previous studies, in apple gardens and nurseries of Iran, have shown a high
percentage of ACLSV infection too. Similar results have also been obtained on the percentage of infection with
this virus in the Czech Republic, Romania, Albania, and Bosnia and Herzegovina. As germplasm exchanges are
the main source of transmission of new viruses to countries, one of the most important strategies to control
viruses in fruit trees is to prevent the introduction of virus-infected germplasm into the country.
