Genetic Diversity of a Natural Population of Apple stem pitting virus Isolated from Apple in Korea.
ABSTRACT: Apple stem pitting virus (ASPV), of the Foveavirus genus in the family Betaflexiviridae, is one of the most common viruses of apple and pear trees. To examine variability of the coat protein (CP) gene from ASPV, eight isolates originating from 251 apple trees, which were collected from 22 apple orchards located in intensive apple growing areas of the North Gyeongsang and North Jeolla Provinces in Korea, were sequenced and compared. The nucleotide sequence identity of the CP gene of eight ASPV isolates ranged from 77.0 to 97.0%, while the amino acid sequence identity ranged from 87.7 to 98.5%. The N-terminal region of the viral CP gene was highly variable, whereas the C-terminal region was conserved. Genetic algorithm recombination detection (GARD) and single breakpoint recombination (SBP) analyses identified base substitutions between eight ASPV isolates at positions 54 and 57 and position 771, respectively. GABranch analysis was used to determine whether the eight isolates evolved due to positive selection. All values in the GABranch analysis showed a ratio of substitution rates at non-synonymous and synonymous sites (dNS/dS) below 1, suggestive of strong negative selection forces during ASPV CP history. Although negative selection dominated CP evolution in the eight ASPV isolates, SLAC and FEL tests identified four possible positive selection sites at codons 10, 22, 102, and 158. This is the first study of the ASPV genome in Korea.
Project description:Apple stem pitting foveavirus (ASPV) is one of the most important and widespread virus infecting apples in the world. Of late, the virus has been found to be invariably associated with most of the apple plantations of Shimla district of Himachal Pradesh based on DAS-ELISA results. Bioassay of viruses in vegetatively propagated crops including apple is considered to be an essential component in indexing programmes for the production of virus free propagating material. Woody indicator Malus pumila 'Spy 227' was used for the detection of ASPV through double grafting method. Graft incompatibility and epinasty symptoms were observed on Malus pumila Spy 227 indicator plants. Further, molecular identification of the virus isolate was done by cloning and sequencing of the test isolate. Partial sequence analysis of the coat protein gene showed 89 % nucleotide identity in BLASTN analysis with ASPV isolate from China (Accession No. JF895517). This is the first record of ASPV producing Graft incompatibility on Spy 227 indicator plants.
Project description:BACKGROUND:Although the canonical function of viral coat protein (CP) is to encapsidate the viral genome, they have come to be recognized as multifunctional proteins, involved in almost every stage of the viral infection cycle. However, CP functions of Apple stem pitting virus (ASPV) has not been comprehensively documented. This study aimed to characterize the functions of ASPV CP and any functional diversification caused by sequence diversity of six ASPV CP variants and studied their biological, serological, pathogenic and viral suppressor of RNA silencing (VSR) functions. METHODS:Six ASPV CP variants that have previously been shown to belong to different subgroups were selected here to study their diversity functions. Agrobacterium mediated infiltration (Agroinfiltration) was used to express YFP-ASPV-CPs in Nicotiana. benthamiana and infect Nicotiana. occidental with PVX-ASPV-CPs in. Confocal microscopy was used to detect YFP-ASPV-CPs florescence. CPs expressed in Escherichia coli BL21 (DE3) were induced by IPTG. RESULTS:In this study, we showed that recombinant CPs expressed in Escherichia coli BL21 (DE3) had different levels of serological reactivity to three anti-ASPV antibodies used to detect ASPV. Furthermore, fusion CPs with YFP (YFP-CPs) expressed in N. benthamiana cells differed in their ability to form aggregates. We also showed that ASPV isolates that harbour these CPs induced different biological symptoms on its herbaceous host N. occidentalis. At the same time, we found that all six CPs when expressed in PVX vector showed similar VSR activity and produced similar symptoms in N. occidentalis, despite their differences in amino acids. CONCLUSIONS:Different ASPV isolates induced different symptoms in N. occidentalis, however, ASPV CP variants expressed in PVX vector showed the same symptoms in N. occidentalis plants. Also, we showed that ASPV CP variants has the same level of VSR activity, but they have different abilities to aggregate in N. benthamiana.
Project description:Deep sequencing has generated 52 contigs derived from five viruses; Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple green crinkle associated virus (AGCaV), and Apricot latent virus (ApLV) were identified from eight apple samples showing small leaves and/or growth retardation. Nucleotide (nt) sequence identity of the assembled contigs was from 68% to 99% compared to the reference sequences of the five respective viral genomes. Sequences of ASPV and ASGV were the most abundantly represented by the 52 contigs assembled. The presence of the five viruses in the samples was confirmed by RT-PCR using specific primers based on the sequences of each assembled contig. All five viruses were detected in three of the samples, whereas all samples had mixed infections with at least two viruses. The most frequently detected virus was ASPV, followed by ASGV, ApLV, ACLSV, and AGCaV which were withal found in mixed infections in the tested samples. AGCaV was identified in assembled contigs ID 1012480 and 93549, which showed 82% and 78% nt sequence identity with ORF1 of AGCaV isolate Aurora-1. ApLV was identified in three assembled contigs, ID 65587, 1802365, and 116777, which showed 77%, 78%, and 76% nt sequence identity respectively with ORF1 of ApLV isolate LA2. Deep sequencing assay was shown to be a valuable and powerful tool for detection and identification of known and unknown virome in infected apple trees, here identifying ApLV and AGCaV in commercial orchards in Korea for the first time.
Project description:Apple stem pitting virus (ASPV) mainly infects apple, pear and their closely related species in the world. ASPV causes some symptoms like leaf etiolation and stony pit in cultivated pear, but produces few symptoms in cultivated apple. We inoculated tobacco with ASPV, which originates from Nanking cherry (Prunus tomentosa), and we obtained tobacco RNA-sequencing data through high-throughput sequencing. In total, 17,401,736 clean reads were obtained after sequencing and removing adaptor sequences, contamination and low-quality reads. An RNA-seq data has been uploaded to Sequence Read Archive (SRA), which enables researchers to access the RNA-sequencing data of tobacco inoculated with ASPV.
Project description:Apple is the major commercial horticulture crop in Himachal Pradesh and other hill states of Jammu & Kashmir, Uttarakhand and some parts of Northeastern states of India. In order to gather data on health status and incidence of virus and virus-like pathogens in apple orchards, survey was conducted in the month of June and September, 2010 in Hatkoti, Rohru, Kuthara, Jubbal and Khadapathar areas of major apple producing Shimla district of Himachal Pradesh. A total of 250 samples were collected and analyzed by DAS-ELISA, NASH and RT-PCR. NASH results indicated that a total of 117 samples were infected with Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple scar skin viroid (ASSVd). Results showed that ASSVd is predominant in these areas with highest infection rate of 27.6% followed by ASPV (17.2%), ACLSV (16.8%), ApMV (15.2%) and ASGV (12%). Mixed infection of these viruses and viroid was frequently detected in apple trees in Himachal Pradesh. The trees, which were positive for viruses and viroids, showed a variety of fruit deformation and rusting symptoms besides leaf deformation, mosaic and chlorosis.
Project description:Apple mosaic virus (ApMV), an Ilarvirus is one of the most common pathogens of apple worldwide. During field surveys in commercial plantations of Himachal Pradesh and Jammu & Kashmir, observations of bright chlorotic mosaic like symptoms on apple trees indicated probable infection by the virus, which was later detected by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA). An incidence of 24 and 28% (based on ELISA) was obtained as 6/25 and 15/53 samples from HP and J&K were positive, respectively. An amplification of approximately 700 and 850 bp was obtained for coat and movement protein genes (CP and MP), respectively. The CP was 223 amino acids in length and showed 87-99% identity when compared to 21 ApMV isolates. Whereas, MP (286 amino acids) showed 91-95% identity with other isolates. However, the gene sequences were quite conserved among Indian isolates and grouped together phylogenetically. CP of the Indian isolates showed maximum identity of 95% with Korean isolate (AY 125977) in apple and in other host these showed a maximum identity of 98% to Czech Republic pear isolate. MP showed maximum identity with Chinese isolate i.e., 95%. The diversity study will also help in analyzing variability among the isolates and also to formulate diagnostic and resistance strategies.
Project description:Apple mosaic disease has a great influence on apple production. In this study, an investigation into the incidence of apple mosaic disease in southwest China was performed, and the pathogen associated with the disease was detected. The results show that 2869 apple trees with mosaic disease were found in the Sichuan, Yunnan, and Guizhou Provinces, with an average incidence of 9.6%. Although apple mosaic virus (ApMV) is widespread in apples worldwide, the diseased samples were negative when tested for ApMV. However, a novel ilarvirus (apple necrotic mosaic virus, ApNMV) was identified in mosaic apple leaves which tested negative for ApMV. RT-PCR analysis indicated that ApNMV was detected in 322 out of 357 samples with mosaic symptoms. Phylogenetic analysis of coat protein (CP) sequences of ApNMV isolates suggested that, compared with ApMV, ApNMV was closer to prunus necrotic ringspot virus (PNRSV). The CP sequences of the isolates showed the diversity of ApNMV, which may enable the virus to adapt to the changeable environments. In addition, the pathology of mosaic disease was observed by microscope, and the result showed that the arrangement of the tissue and the shape of the cell, including the organelle, were seriously destroyed or drastically changed.
Project description:In the present study, an attempt was made to eliminate apple chlorotic leaf spot virus, apple mosaic virus, apple stem grooving virus and apple stem pitting virus from apple cultivar 'Oregon Spur-II'. Thermotherapy was carried out at 37-40 °C for 4 weeks followed by culturing of meristems of different sizes. During establishment of explants, highest survival percentage (62.35%) and proliferation (30.68%) was recorded during summer season. However, size of meristems and position of buds from where meristems were excised also influenced their survival. The meristems of size 0.6-0.7 mm were found to be the most appropriate for maximum establishment. Meristems excised from buds positioned on distil portions of actively growing shoots showed better results. MS medium supplemented with BA (1.0 mg/l), IBA (0.05 mg/l) and GA3 (0.1 mg/l) resulted in 56.62% establishment of explants, while maximum number of meristems proliferated with low BA (0.5 mg/l), IBA (0.08 mg/l) and same GA3 concentration. Two to fourfold multiplication was observed. Virus indexing of shoots raised from different sizes of meristems was carried out and found that 0.3-0.6 mm size was able to eliminate ACLSV, ApMV, ASGV and ASPV. However, some of 0.5-0.6 mm sized shoots were found infected with ACLSV. Larger meristems could not completely eliminate the viruses under study.
Project description:Marssonina coronaria associated with apple blotch disease causes severe premature defoliation, and is widely distributed in Korea. Thirteen isolates were collected from orchards located in Gyeongbuk Province from 2005~2007. All isolates displayed over 99.6% and 99.2% sequence similarity to each other in internal transcribed spacer regions and partial sequences of 28S rDNA, respectively. The isolates were phylogenetically closely related to Chinese isolates. Selected isolates did not differ in their pathogenicity. The optimum conditions for fungal growth were 20℃ and pH 6 on peptone potato dextrose agar (PPDA). Peptone and mannose were the best nitrogen and carbon source, respectively. Fungal growth was better on PPDA than on common potato dextrose agar. This study provides valuable information for integrated disease management program and facilitates the routine culturing of M. coronaria.
Project description:A rapid and sensitive two-step RT-PCR protocol for simultaneous detection of major apple viruses, namely Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd), was developed. Five specific primer pairs were tested and confirmed for these viruses and viroid together in a single tube, giving amplicons of ~198, ~330, ~370, ~547 and ~645 bp corresponding to ASGV, ASSVd, ASPV, ApMV and ACLSV, respectively. Using a guanidinium-based extraction buffer along with a commercial kit resulted in better quality RNA as compared to kit, suited for multiplex RT-PCR. A rapid CTAB method for RNA isolation from apple tissue was developed, which produce good yield and saves time. To the best of our knowledge, this is the first report on the simultaneous detection of five pathogens (four viruses and a viroid) from apple with NADH dehydrogenase subunit 5 (nad5) as an internal control.