Apple Chlorotic Leaf Spot Virus Research Articles

PATHOGENICITY OF TEN ISOLATES OF APPLE CHLOROTIC LEAF SPOT VIRUS TO PRUNUS SPECIES

Apple chlorotic leaf spot virus (ACLSV; genus Trichovirus; family Flexiviridae), one of the main viruses affecting fruit trees, can cause heavy losses to the yield of plum, prune, and apricot. For instance, virulent ACLSV strains are known to elicit severe symptoms on the leaves and fruits of apricot (butteratura or viruela). To assess the pathogenicity of this virus on a wider range of stone fruits, ten viral isolates from different geographical origins (Italy, Spain, Lebanon and Hungary) that had previously been characterized serologically and molecularly (Alrwahnih et al., 2004) were further evaluated via assays in the field. In September 2003, all isolates were inoculated by chip-budding on two cultivars each of the following Prunus species: apricot ('Cafona' and 'Bulida'), cherry ('Ferrovia' and 'Giorgia'), plum ('Black Amber', 'Florenzia', 'Stanley'), and peach ('Dixired' and 'Cardinal') with six replicates for each isolate-cultivar combination. Negative controls were included in the trial. During the four growing seasons that followed, all trees were assayed by ELISA, and foliar and fruit symptoms were recorded (in the case of fruits, controls were limited to two years). High variability was observed among the ten virus isolates, ranging from latency to induction of severe symptoms. In particular, isolates PE154, from Hungary, and PL112, from Italy, were the most severe as they induced, in both apricot cultivars, symptoms of marked vein clearing and yellow mottling on the leaves, and butteratura on the fruits. The isolate PE151 and PL110 caused vein clearing on leaves of the peach cultivar Cardinal, while the isolate APR61 caused yellow spotting on leaves of cvs. Cardinal and Dixired. The PL45 isolate caused heavy vein clearing and deformation on Dixired cultivar. Up to now, no symptoms have been observed on cherry cultivars. Most of the isolates induced discoloration and light deformation on the leaves of both plum cultivars.

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.

Putative Recombination Events and Evolutionary History of Five Economically Important Viruses of Fruit Trees Based on Coat Protein-Encoding Gene Sequence Analysis

To enhance the knowledge of recombination as an evolutionary process, 267 accessions retrieved from GenBank were investigated, all belonging to five economically important viruses infecting fruit crops (Plum pox, Apple chlorotic leaf spot, Apple mosaic, Prune dwarf, and Prunus necrotic ringspot viruses). Putative recombinational events were detected in the coat protein (CP)-encoding gene using RECCO and RDP version 3.31beta algorithms. Based on RECCO results, all five viruses were shown to contain potential recombination signals in the CP gene. Reconstructed trees with modified topologies were proposed. Furthermore, RECCO performed better than the RDP package in detecting recombination events and exhibiting their evolution rate along the sequences of the five viruses. RDP, however, provided the possible major and minor parents of the recombinants. Thus, the two methods should be considered complementary.

First Report of Cherry virus A in Sweet Cherry Trees in China.

Plants in the genus Prunus of the family Rosaceae are important fruit and ornamental trees in China. In June of 2007, sweet cherry (Prunus avium) trees with mottling and mosaic symptoms were observed in a private garden near Kunming, Yunnan Province. Twenty-four samples, six each from sweet cherry, sour cherry (P. cerasus), flowering cherry (P. serrulata), and peach (P. persica) were collected from trees in private and community gardens in the area. The peach and sour and flowering cherry trees did not show any symptoms. Total nucleic acids were extracted using a cetyltrimethylammoniumbromide (CTAB) extraction method, and the extracts were tested for the following eight viruses by reverse transcription (RT)-PCR: American plum line pattern virus, Apple chlorotic leaf spot virus, Cherry green ring mottle virus, Cherry necrotic rusty mottle virus, Cherry virus A (CVA), Little cherry virus 1, Prune dwarf virus, and Prunus necrotic ringspot virus. Only CVA was detected in two symptomatic sweet cherry trees by RT-PCR with forward (5'-GTGGCATTCAACTAGCACCTAT-3') and reverse (5'-TCAGCTGCCTCAGCTTGGC-3') primers specific to an 873-bp fragment of the CVA replicase gene (2). The CVA infection of the two trees was confirmed by RT-PCR using primers CVA-7097U and CVA-7383L that amplified a 287-bp fragment from the 3'-untranslated region (UTR) of the virus (1). Amplicons from both amplifications were cloned and sequenced. Analysis of the predicted amino acid sequences of the 873-bp fragments (GenBank Accession Nos. EU862278 and EU862279) showed that they were 98% identical with each other and 97 to 98% with the type isolate of CVA from Germany (GenBank Accession No. NC_003689). The 286-bp sequences of the 3'-UTR (GenBank Accession Nos. FJ608982 and FJ608983) were 93% identical with each other and 93 to 98% with the type isolate. The sequence indicated that the three isolates were very similar and should be considered to be the same strain. CVA is a member of the genus Capillovirus in the family Flexiviridae and has been previously reported in Europe, North America, and Japan. The contribution of CVA to the symptoms observed and its distribution in China remain to be evaluated. To our knowledge, this is the first report of CVA in sweet cherry in China.

Molecular evidence for the presence of Apple chlorotic leaf spot virus in infected peach trees in India

During surveys of Himachal Pradesh (HP) and Punjab states in northern India, Apple chlorotic ring spot virus (ACLSV) was detected in infected peach trees using Double Antibody Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA). Identity of the virus was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) giving an amplification of ∼800 bp product using primer pair to amplify the complete coat protein (CP) gene. Multiple alignment of sequences of the CP from samples collected from Kullu and Solan in HP with sequences of other isolates of ACLSV originating from peach trees showed identity of 69–84% and 69–93% at the nucleotide and protein level, respectively. This variation is similar to that observed with other isolates of ACLSV. This is the first report of molecular characterization of ACLSV infecting peach trees in India.

Inhibition of long-distance movement of RNA silencing signals in Nicotiana benthamiana by Apple chlorotic leaf spot virus 50 kDa movement protein

Apple chlorotic leaf spot virus 50 kDa movement protein (P50) acts as a suppressor of systemic silencing in Nicotiana benthamiana. Here, we investigate the mode of action of P50 suppressor. An agroinfiltration assay in GFP-expressing N. benthamiana line16c (GFP-plant) showed that P50 could not prevent the short-distance spread of silencing. In grafting experiments, the systemic silencing was inhibited in GFP-plants (scion) grafted on P50-expressing N. benthamiana (P50-plant; rootstock) when GFP silencing was induced in rootstock. In double-grafted plants, GFP-plant (scion)/P50-plant (interstock)/GFP-plant (rootstock), the systemic silencing in scion was inhibited when GFP silencing was induced in rootstock. Analysis of P50 deletion mutants indicated that the N-terminal region (amino acids 1–284) is important for its suppressor activity. In gel mobility shift assay, P50 lacks binding ability with siRNAs. These results indicated that P50 has a unique suppressor activity that specifically inhibits the long-distance movement of silencing signals.

Viruses and viroids of stone fruits in Algeria.

Field surveys were carried out in the major stone fruit growing areas of eastern Algeria to assess the sanitary status of stone fruits. A total of 454 samples from peach, apricot, almond, sweet and sour cherry, plum and myrobalan were tested by ELISA or RT-PCR for the presence of Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Apple mosaic virus (ApMV), Apple chlorotic leaf spot virus (ACLSV) Apricot latent virus (ApLV), Cherry virus A (CVA), Cherry green ring mottle virus (CGRMV), and Plum bark necrosis stem pitting-associated virus (PBNSPaV). The overall average infection level was 10.4%. The most frequent virus was PNRSV (56.8%), followed by PDV (27.2%) and ApMV (22.7%). The most infected species was cherry (21.9%) and the less almond (4.4%). ACLSV, ApLV, CVA, CGRMV and PBNSPaV were not detected. Of 531 samples tested for the presence of viroids by tissueprint hybridization, 28 (5.2%) were infected. Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) were detected in 15 and 13 samples, respectively. This is the first large-scale study on viruses and viroids of stone fruits in Algeria and reports for the first time the presence of HSVd in the country.

ANALYSIS OF AUTOCHTHONOUS PLUM GENOTYPES (PRUNUS DOMESTICA L.) IN CROATIA FOR THE PRESENCE OF PLUM POX VIRUS

SUMMARY Plum pox virus (PPV) is increasingly endangering plum production in Croatia. This study aimed at determining the presence of this virus in autochthonous plum genotypes and at identifying plum trees of particular genotypes, not infected by it. To sort out the broad array of synonyms used in plum variety naming, the samples were submitted to genetic analysis, for defining their affiliation to a particular genotype (variety). In addition, the presence and frequency was assessed of Apple chlorotic leaf spot virus (ACLSV) and Prunus necrotic ringspot virus (PNRSV) infections in autochthonous plum genotypes. In 2005, leaf and fruit samples from 54 trees were collected in seven Croatian counties, Osjecko-baranjska, Vukovarsko-srijemska, Pozesko-slavonska, Brodsko-posavska, Viroviticko-podravska, Sisacko-moslavacka and Lickosenjska. The variety Bistrica is cultivated in all seven areas while the other cultivars (Bela staroverska, Bijela rana, Bjelica jajara, Brdaklija, Cericanka, Crnica, Dabinka, Debeljara, Glodara, Kalaca, Kamenjara, Komocanka, Mandalenka, Miholjcanka, Pasjara, Pintara, Plavica, Ruzica, Sitna bijelica, Torgulja, Ilinjaca, Trnovaca and Valpovka) were found in particular locations of specific counties. Serological tests were used for establishing the presence of the three viruses. PPV was detected in four plum genotypes, three of which (cv. Glodara from Dolci, cv. Pasjara from Viljevo and cv. Bistrica from Podvrsko) contained the virus in both leaves and fruits, while cv. Glodara from Crkvari contained PPV only in the leaves. All the other plum genotypes tested were found to be PPV-free. PNRSV was detected in two genotypes. It was found both in the leaves and fruits of cv. Komocanka from Caglin, while only the leaves of cv. Debeljara from Batina were infected by this virus. None of the tested plum accessions was infected by ACLSV. Likewise, none of the three viruses was detected in 48 of 54 trees investigated.

PLUM POX VIRUS SILENCING OF C5 TRANSGENIC PLUMS IS STABLE UNDER CHALLENGE INOCULATION WITH HETEROLOGOUS VIRUSES

Transgenic C5 cv. Honey Sweet is a clone of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP). This transgenic plum displays post-transcriptional gene silencing (PTGS) which makes it highly resistant to PPV infection. To test the effect of heterologous viruses on the efficacy and stability of PTGS against PPV, transgenic C5 trees were graft-inoculated with different combinations of Prunus necrotic ringspot virus (PNRSV), Apple chlorotic leaf spot virus (ACLSV), Prune dwarf virus (PDV) and PPV-D strain. The potential for suppression of the silencing mechanism mediated by these viruses was evaluated. Challenge experiments were performed under greenhouse, nursery and field conditions in Romania and Spain, comprising two different environments, continental and Mediterranean, respectively. Virus infections were evaluated by visual monitoring of symptom and by serological and molecular assays. The engineered resistance to PPV in C5 transgenic plums was stable and was not suppressed by the presence of the challenging heterologous viruses over a three-year experimental period across all trials.

The complete nucleotide sequence of an isolate of apple chlorotic leaf spot virus from peach (Prunus persica (L.) Batch)

The complete nucleotide sequence of an isolate of apple chlorotic leaf spot virus from peach (Prunus persica (L.) Batch)

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.

Interactions of Plum pox virus strain Rec with Apple chlorotic leafspot virus and Prune dwarf viruses in field-grown transgenic plum Prunus domestica L., clone C5

Transgenic plums, <I>Prunus domestica</I> L. clone C5, were inoculated by bud grafting with <I>Plum pox virus</I> (PPV-Rec, recombinant strain originated from plum), PPV-Rec + <I>Apple chlorotic leafspot virus</I> (ACLSV), PPV-Rec + <I>Prune dwarf virus</I> (PDV), and PPV-Rec + ACLSV + PDV. Non-inoculated transgenic plums served as controls. Plants were grown in an open field for 5 years. They were evaluated by visible symptoms, by DAS-ELISA and RT-PCR. Mild PPV symptoms, diffuse spots or rings appeared two years after inoculation in some leaves of plants artificially inoculated with PPV-Rec, PPV-Rec + ACLSV, PPV-Rec + PDV, and PPV-Rec + ACLSV + PDV. Severe PPV symptoms appeared in leaves of shoots growing from infected buds used for inoculation. During the following three years, further weakening of PPV symptoms was observed in transgenic plants. In 2007, very mild PPV symptoms were found in only a few leaves, and over 60%, resp. 70% of the C5 trees showed no PPV symptoms. The presence of PPV was confirmed by ELISA, ISEM and RT-PCR. No difference in PPV symptoms was observed between PPV-Rec and combinations PPV-Rec + ACLSV, PPV-Rec + PDV, PPV-Rec + ACLSV + PDV. No symptoms of ACLSV appeared in combinations of ACLSV with PPV-Rec and PPV-Rec + PDV during 2004–2007, but the presence of ACLSV in leaves of transgenic plants clone C5 was proved by ELISA and RT-PCR. Neither synergistic nor antagonistic effects of ACLSV on PPV-Rec were observed. No symptoms of PDV appeared in combinations of viruses with PDV during 2004–2007. PDV was not detected by ELISA, and the presence of PDV was uncertain by RT-PCR in most of inoculated trees in 2006 and 2007. The results of RT-PCR will be further confirmed by sequence analysis and discussed. These results suggest a possible antagonistic interaction between PPV-Rec and PDV in plum clone C5.

Cydonia oblonga as reservoir of Apple chlorotic leaf spot virus in India

Cydonia oblonga as reservoir of Apple chlorotic leaf spot virus in India

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.

Results of in vitro thermotherapy of apple cultivars

Apple cultivars ‘Idared’ and ‘Sampion’ were selected for application of in vitro thermotherapy at 39 ± 0.5°C. The presence of viruses in selected initial trees was detected by enzyme-linked immuno-sorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) before the beginning of thermotherapy. Cultivars ‘Idared’ and ‘Sampion’ were both infected with Apple chlorotic leaf spot virus (ACLSV) and with Apple stem pitting virus (ASPV). Cultivar ‘Sampion’ was moreover infected with Apple stem grooving virus (ASGV). Both apple cultivars were successfully multiplied in in vitro cultures on Murashige and Skoog (MS) medium with 1.5 mg l−1 6-benzylaminopurine (BAP). After the end of thermotherapy and transfer to ex vitro conditions, three clones of apple cultivar ‘Idared’ were free of both viruses (ACLSV, ASPV) according to repeated RT-PCR and ELISA. On the contrary, the same viruses (ACLSV, ASGV and ASPV) as in the initial field-grown trees were detected by RT-PCR in all four clones of cultivar ‘Sampion’. Other sanitation and testing of apple cultivars will continue in the following years.

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.

Detection of three pear viruses by multiplex RT-PCR assays with co-amplification of an internal control

Apple stem pitting virus, Apple chlorotic leaf spot virus and Apple stem grooving virus are economically important and common pathogens of pear and apple cultivars. All are symptomless in commercial pear and apple cultivars. Two multiplex RT-PCR methods with co-amplification of an internal control from a plant mRNA sequence were used to detect these three viruses simultaneously. The approaches, including what materials were to be selected and how to extract nucleic acids, were designed to improve detection efficiency. Detection assays, using bark chips of 1- or 2-year-old pear branches, could be performed both in the growing season and in dormant trees. A specific internal RNA control, which could be amplified from total nucleic acids, was co-amplified with the target viruses. Therefore, purified RNA as an amplification template was not necessary. Isolation of nucleic acids from bark chips was more successful than isolation from leaves. Three virus-specific primer pairs were designed to allow two multiplex RT-PCR amplifications accompanied by one pair of primers of the mitochondrial nad5 gene. Four amplification products including three viruses and an internal control were sequenced to prove the specificity of all primer pairs. By using a nad5 gene in mitochondrial mRNA of the apple as an internal control of co-amplification, these methods reduced the risk of false negative results that may occur with routine RT-PCR assays.

VIRUSES AND VIROIDS OF STONE FRUITS IN EGYPT

SUMMARY Field surveys were carried out to assess the sanitary status of stone fruits in Egypt. A total of 716 samples was tested by ELISA for Prunus necrotic ring spot virus (PNRSV), Plum pox virus (PPV), Prune dwarf virus (PDV), Apple chlorotic leaf spot virus (ACLSV) and Apple mosaic virus (ApMV). The viruses most frequently detected were PNRSV (57%) and PPV (41%); PDV and ACLSV were found in 3% of samples and ApMV was not detected. Plum bark necrosis stem pitting-associated virus (PBNSPaV) and Apricot latent virus (ApLV) were detected for the first time in Egypt by using RTPCR. Of 693 samples tested by tissue-imprint hybridization (TIH), 101 (15%) were found to be infected by either Peach latent mosaic viroid (PLMVd) or Hop stunt viroid (HSVd).

Combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) in the coat protein of apple chlorotic leaf spot virus are crucial for infectivity

Amino acid sequences of apple chlorotic leaf spot virus (ACLSV) coat protein (CP) were compared between 12 isolates from apple, plum and cherry, and 109 cDNA clones that were amplified directly from infected apple tissues. Phylogenetic analysis based on the amino acid sequences of CP showed that the isolates and cDNA clones were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (Ala(40)-Val(59)-Phe(75)-Ser(130)-Met(184) or Ser(40)-Leu(59)-Tyr(75)-Thr(130)-Leu(184)) were highly conserved within each cluster. Site-directed mutagenesis using an infectious cDNA clone of ACLSV indicated that the combinations of two amino acids (Ala(40) and Phe(75) or Ser(40) and Tyr(75)) are necessary for infectivity to Chenopodium quinoa plants by mechanical inoculation. Moreover, an agroinoculation assay indicated that the substitution of a single amino acid (Ala(40) to Ser(40) or Phe(75) to Tyr(75)) resulted in extreme reduction in the accumulation of viral genomic RNA, double-stranded RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting that the combinations of the two amino acids at positions 40 and 75 are important for effective replication in host plant cells.

Occurrence and distribution of stone fruit viruses and viroids in commercial plantings of Prunus species in western Anatolia, Turkey.

Symptoms of virus and viroid infections have been observed during the last ten years in stone fruit crops, however, no relative incidences of virus and viroid diseases have been reported on stone fruit crops in western Anatolia, Turkey. Large-scale surveys were conducted from June to August between the years 2004 and 2006 in the main stone fruit growing orchards and mother blocks of western Anatolia to determine the seven most important virus and two important viroid affecting Prunus species. The results of serological (ELISA) and molecular (PCR and tissue-printing molecular hybridization) tests demonstrated the occurrence of Plum pox virus (PPV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Apricot latent virus (ApLV), Plum bark necrosis stem pitting-associated virus (PBNSPaV), Peach latent mosaic viroid (PLMVd), and Hop stunt viroid (HSVd) in tested 1732 specimens of stone fruits. The overall infection level with these graft-transmissible agents was 30%. The results showed that PDV is predominant in stone fruit crops. To our knowledge, PBNSPaV was reported for the first time in western Anatolia. The results obtained in this study illustrate a need for certification or clean stock program to prevent the occurrence and the spread of graft transmissible agents in western Anatolia.