Apple Chlorotic Leaf Spot Virus Isolates Research Articles

Molecular variability analyses of Apple chlorotic leaf spot virus capsid protein

The complete sequences of the coat protein (CP) gene of 26 isolates of Apple chlorotic leaf spot virus (ACLSV) from India were determined. The isolates were obtained from various pome (apple, pear and quince) and stone (plum, peach, apricot, almond and wild Himalayan cherry) fruit trees. Other previously characterized ACLSV isolates and Trichoviruses were used for comparative analysis. Indian ACLSV isolates among themselves and with isolates from elsewhere in the world shared 91-100% and 70-98% sequence identities at the amino acid and nucleotide levels, respectively. The highest degree of variability was observed in the middle portion with 9 amino acid substitutions in contrast to the N-terminal and C-terminal ends, which were maximally conserved with only 4 amino acid substitutions. In phylogenetic analysis no reasonable correlation between host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other Trichoviruses revealed the TaTao ACLSV peach isolate to be phylogenetically closest to Peach mosaic virus, Apricot pseudo chlorotic leaf spot virus and Cherry mottle leaf virus. Recombination analysis (RDP3 ver.2.6) done for all the available ACLSV complete CP sequences of the world and Indian isolates indicate no significant evidence of recombination. However, one recombination event among Indian ACLSV-CP isolates was detected. To the best of our knowledge, this is the first report of complete CP sequence variability study from India and also the first evidence of homologous recombination in ACLSV.

Biological and Molecular Characterization of Apple chlorotic leaf spot virus Causing Chlorotic Leaf Spot on Pear (Pyrus pyrifolia) in Taiwan

Pear plants (Pyrus pyrifolia var. Hengshen) showing symptoms of chlorotic spots on leaves were observed in orchards in central Taiwan in 2004. The sap of diseased leaves reacted positively to Apple chlorotic leaf spot virus (ACLSV) antiserum. A purified virus isolate (LTS1) from pear was characterized by host range, electron microscopy, phylogenetic analyses, serological property, and back-inoculation experiments to pear. Fifteen of 28 species of tested plants were susceptible to this virus after mechanical inoculation. Pathogenicity of ACLSV isolate LTS1 was verified by back-inoculating to pear seedlings. Filamentous virions of ≈12 × 750 nm were observed in the preparations of purified virus. Virus particles accumulated in the cytoplasm were observed in the ultrathin sections of LTS1-infected pear leaf tissue. Sequence analyses of the coat protein (CP) gene of LTS1 and the CP gene of LTS2, which originated from a distinct symptomatic pear sample, shared 81.4% to 92.6% nucleotide and 87.6% to 98.4% amino acid identities with those of the CP of 35 ACLSV isolates available in GenBank. ACLSV isolates were grouped into four clusters, i.e., Asia I, II, III, and Europe, and isolates LTS1 and LTS2 were classified as members of cluster Asia II and Asia I, respectively, based on phylogenetic data. Moreover, the variability of amino acid sequences of the CP gene of 37 ACLSV isolates showed geographically associated clustering in the phylogenetic tree. To our knowledge, this is the first study on the characterization of ACLSV causing the leaf chlorotic spot disease of pear in Taiwan. This study also provides the phylogenetic relationships among ACLSV populations based on amino acid sequences of CPs, which are correlated with their geographic origins.

EVALUATION OF RESISTANCE TO APPLE CHLOROTIC LEAF SPOT VIRUS (ACLSV) IN CONTROLLED GREENHOUSE CONDITIONS IN THE APRICOT BREEDING PROGRAMME OF CEBAS-CSIC IN MURCIA (SPAIN)

Apple chlorotic leaf spot virus (ACLSV) seems to be the causal agent of apricot viruela disease. This disease has become an important problem for apricot production in Spain and, for this reason, in the CEBAS-CSIC of Murcia we are setting up a method to evaluate ACLSV resistance. In this method, seedling rootstocks were inoculated by grafting an infected bark and later they were grafted with the apricot genotype to be evaluated. The different steps of the process were: selection of ACLSV isolate (APR60, APR61 or APR62), rootstock selection ('GF305' peach or 'Real Fino' apricot seedlings) and resistance evaluation (observation of symptoms on leaves and ELISA). Results showed an asymptomatic behaviour of the three ACLSV isolates assayed in apricot and peach leaves. ELISA was a suitable method to evaluate the presence (susceptibility) or absence (resistance) of virus in the leaves of the rootstocks and cultivars. APR62 showed the highest optical densities (OD) and so it was selected for our evaluation assays. In addition, 'GF305' had higher OD than 'Real Fino' apricot and it is better for evaluation. The first results on the evaluation of apricot resistance to ACLSV in controlled greenhouse conditions are presented.

Incidence and molecular variability of Apple stem pitting and Apple chlorotic leaf spot viruses in apple and pear orchards in Greece.

A survey was conducted in the major pome fruit growing districts of Greece to assess the incidence of Apple stem pitting virus (ASPV) and Apple chlorotic leaf spot virus (ACLSV) using RT-PCR assays amplifying part of the RNA-dependent RNA polymerase (RdRp) and the coat protein genes of the viruses, respectively. ASPV was predominant in apple (91.8%) and pear (51.3%) and was detected in almost all the cultivars and rootstocks tested. It was also found for the first time in the Japanese pear (Pyrus serotina). The incidence of ACLSV in apples was also high (65.7%) and mixed infections with both viruses were frequently encountered. In pear, ACLSV incidence was lower (20.7%). This is the first extensive survey conducted in Greece for monitoring ACLSV and ASPV using molecular assays. Nucleotide sequencing of different apple and pear ASPV and ACLSV isolates and comparative analysis with already published homologous genomic regions revealed a significant level of variation up to 29% and 22% for the RdRp and the CP genes, respectively. However, high identity rates were found among isolates of both viruses from different geographical origins. Our results show the high impact of the use of infected plant material in the dissemination of ASPV and ACLSV and illustrate the need to implement programs for the production of certified pome fruit propagating material in Greece, in order to prevent further spread of the viruses.

Indexação biológica múltipla e RT-PCR para detecção de vírus latentes em macieiras

O objetivo deste trabalho foi avaliar a viabilidade da indexacao biologica multipla de virus latentes de macieiras em indicadoras lenhosas. O metodo consistiu na enxertia de cinco especies de indicadoras e duas borbulhas da cultivar a ser indexada em um unico porta-enxerto. Concluiu-se que a eficacia e a sensibilidade do metodo biologico multiplo e adequada para a deteccao confiavel de Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) e Apple chlorotic leaf spot virus (ACLSV) em programas de limpeza clonal e certificacao de matrizes de fruteiras. A RT-PCR confirmou o diagnostico biologico de ASPV e ASGV. Entretanto, ACLSV diagnosticado pela indicadora LL-S5 nao foi detectado por RT-PCR em tres dos nove acessos analisados. O gene de proteina capsidica de ACLSV de um dos acessos estudados foi clonado e sequenciado; a regiao de pareamento dos iniciadores de PCR foi comparada com dados do Genbank como subsidio a interpretacao da nao amplificacaode certos isolados de ACLSV.

Himalayan wild cherry (Prunus cerasoides D. Don): a new host of Apple chlorotic leaf spot virus

SummaryHimalayan wild cherry (Prunus cerasoides), widely distributed in the Himalayas, was found to exhibit mild virus‐like symptoms. Leaf samples from affected plants were subjected to enzyme‐linked immunosorbent assay indexing for known pome and stone fruit viruses, viz., Apple mosaic virus, Apple chlorotic leaf spot virus (ACLSV), Prunus necrotic ring spot virus and Apple stem grooving virus. Results showed the presence of ACLSV. For further characterization, the coat protein gene was amplified by reverse transcription‐polymerase chain reaction (RT‐PCR). Sequencing of the amplicon confirmed the presence of ACLSV. The isolate shared 98% amino acid sequence identity with P1R9D9 (Bulgarian) and C‐2 (Hungarian) isolates of ACLSV. This study reports the presence of ACLSV in Himalayan wild cherry.

Characterisation ofApple chlorotic leaf spot virusinfecting almonds in India

Restricted field surveys were conducted to determine the presence and incidence of virus infections in almonds. Representative asymptomatic samples from six locations were tested by enzyme linked immunosorbent assay and reverse transcription and polymerase chain reaction for known stone fruit viruses. Only Apple chlorotic leaf spot virus (ACLSV) infection was detected in one of the six almond samples from the state of Himachal Pradesh. Comparison of sequenced amplicons with two available sequences of ACLSV-almond isolates revealed 92% and 94% amino acid identity. However, the Indian ACLSV-almond isolate shared maximum (98%) homology at the amino acid level to ACLSV-peach (AM498050) and wild apricot (AM498048) isolates from India. The C-terminus is highly conserved within the Indian ACLSV isolates with the exception of a few residues which in the case of the Indian almond isolates are more typical of peach and wild apricot isolate sequences.

Molecular Typing of Turkish Apple Chlorotic Leaf Spot Virus Isolates Based on Partial Coat Protein Gene

Apple chlorotic leaf spot virus (ACLSV) isolates from various hosts and geographic locations in Turkey were molecularly characterized by RFLP, nucleotide sequence analysis and the construction of a phylogenetic tree including ACLSV isolates from GenBank. Based on nucleotide sequence alignment and the phylogenetic tree, we proposed a classification of ACLSV isolates in which isolates were divided into three major groups. The first group contained mainly Far-Eastern isolates, the second group the Hungarian (eastern-European) ACLSV isolates, and the third group, which contained isolates of variable characteristics, was again divided into two subgroups, subgroup I containing mixed European isolates, and subgroup II containing central European isolates. Three representative Turkish ACLSV isolates belonged to the third group; of these, one was from the mixed European cluster (subgroup I) and two from the central European cluster (subgroup II). The nucleotide sequence divergence and geographic origin of the ACLSV isolates were correlated, which indicated the possible extraction of the Turkish isolates.

An Apple Chlorotic Leaf Spot Virus Isolate from Ornamental Dwarf Flowering Almond (Prunus glandulosa 'Sinensis'): Detection and Characterization

An apple chlorotic leaf spot virus (ACLSV) isolate was detected by TAS-ELISA and RT-PCR in an ornamental dwarf flowering almond (Prunus glandulosa Thunb.). This plant, maintained at the Centre for Plant Health, Sidney, B.C., Canada, has been showing transient leaf symptoms during the spring seasons. A 390-bp fragment and a 1,350-bp product, in the RNA polymerase and the coat protein viral coding regions, respectively, were amplified by RT-PCR from the infected plant. A sequence comparison of the 390-bp fragment of this ACLSV isolate (designated as AL1292) with other published isolates, revealed a similarity of 81% to 84% at the nucleotide level and 88% to 100% at the amino acid level. In contrast to other ACLSV isolates, AL1292 has an exceptionally narrow range of experimental herbaceous and woody hosts, as determined by mechanical and graft inoculation assays. These standard bioassays may not be effective for the detection of the AL1292 isolate because of its limited host range. The results we report in this paper confirm P. glandulosa as a natural host of this virus. Currently it is not known how ACLSV is spread, other than by bud-grafting and possibly by root grafts. The use of virus-tested source plants for the preparation of planting material will minimize its spread.

Characterization of Apricot pseudo-chlorotic leaf spot virus, A Novel Trichovirus Isolated from Stone Fruit Trees

ABSTRACT A trichovirus closely related to Apple chlorotic leaf spot virus (ACLSV) was detected in symptomatic apricot and Japanese plum from Italy. The Sus2 isolate of this agent cross-reacted with anti-ACLSV polyclonal reagents but was not detected by broad-specificity anti- ACLSV monoclonal antibodies. It had particles with typical trichovirus morphology but, contrary to ACLSV, was unable to infect Chenopodium quinoa and C. amaranticolor. The sequence of its genome (7,494 nucleotides [nt], missing only approximately 30 to 40 nt of the 5' terminal sequence) and the partial sequence of another isolate were determined. The new virus has a genomic organization similar to that of ACLSV, with three open reading frames coding for a replication-associated protein (RNA-dependent RNA polymerase), a movement protein, and a capsid protein, respectively. However, it had only approximately 65 to 67% nucleotide identity with sequenced isolates of ACLSV. The differences in serology, host range, genome sequence, and phylogenetic reconstructions for all viral proteins support the idea that this agent should be considered a new virus, for which the name Apricot pseudo-chlorotic leaf spot virus (APCLSV) is proposed. APCLSV shows substantial sequence variability and has been recovered from various Prunus sources coming from seven countries, an indication that it is likely to have a wide geographical distribution.

Detection of Apple Chlorotic Leaf Spot and Apple Stem Grooving Viruses Using RT-PCR

Reverse transcriptase polymerase chain reaction (RT-PCR) procedures were used to amplify fragments specific for apple chlorotic leaf spot virus (ACLSV) and apple stem grooving virus (ASGV). Products visible in agarose gels were produced using templates consisting of as little as 5 pg of total nucleic acid extracted from infected apple leaves. Both viruses were also detected in fruit, bark, and root tissues of infected apples. These results suggest the viruses are distributed throughout actively growing trees. The systems were further validated by sequencing fragments amplified from leaves of a apple, cherry, and Asian pear trees growing in South Carolina. Comparisons with published sequences indicate that the fragments from the three ACLSV isolates have very high (≥97.6%) percent identity values to the corresponding region of a plum isolate and moderately high (≥81.4%) identities to an apple isolate from Japan. The fragments amplified from the ASGV isolates were found to be similar (≥88.5% identities) to the corresponding region of an apple ASGV isolate from Japan.

Use of monoclonal antibodies for the identification of different antigenic domains in apple chlorotic leaf spot virus

Thirteen monoclonal antibodies (Mabs) specific for apple chlorotic leaf spot virus (ACLSV), produced by the somatic cell hybridization technique, were used to investigate the antigenic structure of the virus. Epitope specificity studies showed that these Mabs defined in ACLSV particles seven independent antigenic domains, representing at least eight distinct epitopes. One of them was present only in virions and not in dissociated subunits. It appeared that the interaction between a Mab and the virus could, in some cases, induce conformational changes in the viral particles which enhanced the binding of others. Twenty nine virus isolates differing in geographical origin, primary hosts and symptomatology were tested with these monoclonal antibodies by ELISA. With the exception of two Mabs which did not react with three cherry isolates, and one Mab which did not react with one plum isolate, all of them recognized all ACLSV isolates tested.