Status of the Apple chlorotic leaf spot virus Infection in Native and Imported Apple Tree Cultivars in the National Collection of Kamalshahr Horticulture Research Station

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.

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.

The ELISA results showed that 39 out of 50 cultivars were infected by ACLSV. RT-PCR 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.