Yeast two-hybrid and pull-down assays propose an interaction between P50 of apple chlorotic leaf spot virus and PR-10 of Malus sylvestris cv. R12740-7A.

Apple chlorotic leaf spot virus (ACLSV) movement protein (P50) is involved in cell-to-cell transport and influences the long-distance spread of silencing activity. Previously, we obtained 69 P50-interacting proteins from Malus sylvestris cv. R12740-7A and using bioinformatics analyzed their biological functions. In this study, we used the GAL4-based two-hybrid yeast system and His pull-down assays to confirm an interaction between PR-10 of M. sylvestris cv. R12740-7A and ACLSV P50. Our results provide a theoretical basis for further research on the biological function of PR-10 in ACLSV infection and the interacting mechanism between host and virus.

Viruses of Apple Are Seedborne but Likely Not Vertically Transmitted.

Many viruses occur in apple (Malus domestica (Borkh.)), but no information is available on their seed transmissibility. Here, we report that six viruses infecting apple trees, namely, apple chlorotic leaf spot virus (ACLSV), apple green crinkle-associated virus (AGCaV), apple rubbery wood virus 2 (ARWV2), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), and citrus concave gum-associated virus (CCGaV) occur in seeds extracted from apple fruits produced by infected maternal trees. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR) assays revealed the presence of these six viruses in untreated apple seeds with incidence rates ranging from 20% to 96%. Furthermore, ASPV was detected by RT-PCR in the flesh and peel of fruits produced by infected maternal trees, as well as from seeds extracted from apple fruits sold for fresh consumption. Finally, a large-scale seedling grow-out experiment failed to detect ACLSV, ASGV, or ASPV in over 1000 progeny derived from sodium hypochlorite surface sterilized seeds extracted from fruits produced by infected maternal trees, suggesting no detectable transmission via embryonic tissue. This is the first report on the seedborne nature of apple-infecting viruses.

Seed Transmission of Three Viruses in Two Pear Rootstock Species Pyrus betulifolia and P. calleryana.

Viral seed transmission causes the spread of many plant viral diseases. Pyrusbetulifolia and P. calleryana are important rootstock germplasms for pear production in China. This study revealed the widespread infection of apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and apple stem pitting virus (ASPV) in maternal trees of P. betulifolia and P. calleryana by nested multiplex reverse transcription-polymerase chain reaction (nmRT-PCR) assays. Seeds from eight P. betulifolia and two P. calleryana trees had positive rates of 15.9-73.9%, 0-21.2%, and 40.4% for ASGV, ASPV, and ACLSV, respectively. At the cotyledon and 6-8 true leaf stages, seedlings grown from seeds of infected trees gave positive rates of 5.4% and 9.3% for ASGV, 6.7% and 15.6% for ACLSV, and 0% and 2.7% for ASPV, respectively. Incidence in nursery P. betulifolia seedlings of 10.1%, 5.3%, and 3.5% were determined for ASGV, ACLSV, and ASPV, respectively. The nucleotide sequences of coat protein (CP) and movement protein coding genes of both ASGV and ASPV, and CP gene of ACLSV from maternal trees, seeds, and seedlings were analyzed. Sequence identities and phylogenetic comparison with corresponding sequences from GenBank demonstrated that molecular variation occurred within ASGV, ACLSV, and ASPV isolates, with most sequences determined here had close relationships with reported isolates infecting pear or formed independent clades. This is the first report on the seed transmission and the molecular characteristics of these viruses infecting two rootstock species. These findings provided important evidence in management effort for pear viral diseases.

Apple Russet Ring and Apple Green Crinkle Diseases: Fulfillment of Koch's Postulates by Virome Analysis, Amplification of Full-Length cDNA of Viral Genomes, in vitro Transcription of Infectious Viral RNAs, and Reproduction of Symptoms on Fruits of Apple Trees Inoculated With Viral RNAs.

Apple russet ring and apple green crinkle are graft-transmitted diseases first reported more than 60 years ago, but at present, no association between a specific virus (variant) and the disease has been clearly demonstrated. In this study, we conducted the following series of experiments to identify the causal viruses (variants) of these apple diseases; (1) comprehensive analysis by next-generation sequencing of all viruses in each apple tree affected with russet ring or green crinkle disease, (2) amplification of full-length genomic cDNA of viruses using primers containing the T3 promoter and the in vitro transcription of infectious viral RNAs, (3) inoculation of viral RNA transcripts to both herbaceous and apple plants, (4) analysis of sequence variants of viruses present in infected plants, (5) back-inoculation of sequence variants of candidate viruses to apple seedlings combined with the virus-induced flowering technology using the apple latent spherical virus vector to reproduce the symptom on the fruit as soon as possible, and (6) reproduction of symptoms on the fruits of apple trees inoculated with sequence variants and the re-isolation of each virus variant from apples showing fruit symptoms. The results showed that one of the sequence variants of the apple chlorotic leaf spot virus causes a characteristic ring-shaped rust on the fruits of infected apple trees and that a sequence variant of the apple stem pitting virus probably causes green crinkle symptoms on an infected apple fruit. Thus, we were able to fulfill Koch's postulates to prove the viral etiology of both the apple russet ring and green crinkle diseases. We also propose an experimental system that can prove whether a virus found in diseased tissues is the pathogen responsible for the diseases when the etiology is undetermined.

A sensitive nested multiplex RT-PCR assay for the simultaneous detection of three common viruses infecting pear plants.

A highly sensitive nested multiplex reverse transcription-polymerase chain reaction (nmRT-PCR) assay was developed for the simultaneous detection of Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) infecting pear trees. In the assay, a set of three forward primers specific to each of the three viruses and a universal reverse primer was used as external primers in the first-round PCR, which was followed by a second-round PCR developed previously. The nmRT-PCR assay was 104 times more sensitive than conventional mRT-PCR assay in detecting the three viruses in in vitro pear plantlets. This assay was subsequently used to detect these viruses in leaf and bark samples of cultivated and wild pear trees from orchards and demonstrated to be highly sensitive and reliable. This is the first report describing a use of nmRT-PCR for the sensitive and simultaneous detection of the three viruses infecting pear plants. The assay would be useful for the certification of pear planting materials and surveillance of nursery stocks.

The detection of ACLSV and ASPV in pear plants by RT-LAMP assays.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV), two important viruses frequently occurring in pear trees. A set of four RT-LAMP primers designed based on the highly conserved region of each CP gene of the two viruses showed high specificity and feasibility for ACLSV and ASPV detections. The RT-LAMP assays for ACLSV and ASPV in pear samples were 104 and 103 times more sensitive than that of conventional RT-PCR assays. The RT-LAMP under optimal reaction condition was subsequently utilized in the detection of the two viruses in-vitro cultures of pear and field pear samples. This study provides a rapid and sensitive tool to determine the infection statues of the two viruses in pear certification program.

One-step reverse transcription loop-mediated isothermal amplification assay for detection of Apple chlorotic leaf spot virus.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of Apple chlorotic leaf spot virus (ACLSV). In this method, a set of four primers was designed based on the conserved regions in the coat protein gene of ACLSV, and the primers were synthesized for the RT-LAMP assay using total RNA extracted from ACLSV-infected leaf tissues. The optimal reaction temperature and assay time were determined to be 64°C and 75min, respectively. The sensitivity of RT-LAMP reactions was reliable up to a maximum dilution of 1:3125, which was more sensitive than the RT-PCR assay. The successful application of RT-LAMP to field-collected apple samples demonstrated its potential for broader applications in effectively diagnosing diseases and, consequently, its potential to control ACLSV from spreading further, particularly in many developing countries around the world. To our knowledge, this is the first application of RT-LAMP for the detection of ACLSV.

Generation of an Infectious Clone of a New Korean Isolate of Apple chlorotic leaf spot virus Driven by Dual 35S and T7 Promoters in a Versatile Binary Vector.

The full-length sequence of a new isolate of Apple chlorotic leaf spot virus (ACLSV) from Korea was divergent, but most closely related to the Japanese isolate A4, at 84% nucleotide identity. The full-length cDNA of the Korean isolate of ACLSV was cloned into a binary vector downstream of the bacteriophage T7 RNA promoter and the Cauliflower mosaic virus 35S promoter. Chenopodium quinoa was successfully infected using in vitro transcripts synthesized using the T7 promoter, detected at 20 days post inoculation (dpi), but did not produce obvious symptoms. Nicotiana occidentalis and C. quinoa were inoculated through agroinfiltration. At 32 dpi the infection rate was evaluated; no C. quinoa plants were infected by agroinfiltration, but infection of N. occidentalis was obtained.

A multiple RT-PCR assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees.

Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) are three latent viruses frequently occurring in apple trees worldwide. In field orchards, these viruses are frequently found in a mixed infection with viroids in the genus Apscarviroid, including Apple scar skin viroid, and Apple dimple fruit viroid. Together these viruses and viroids could cause serious damage to apple fruit production worldwide. Rapid and efficient detection methods are pivotal to identify and select the virus-free propagation material for healthy apple orchard management. In this study a multiplex Reverse Transcription-PCR (RT-PCR) was developed and optimized for simultaneous detection and differentiation of the three latent viruses and apscarviroids. With newly designed specific primers for ACLSV, ASGV, APSV, and EF-1α (as an internal control), and a pair of degenerate primers for apscarviroids, optimized parameters for multiplex RT-PCR were determined. The resulting PCR products from each target virus and viroid could be easily identified because their product sizes differ by at least a 100bp. The multiplex RT-PCR method is expected to detect different variants of the viruses as the test results showed that a variety of isolates from different regions in China gave positive results. To the best of our knowledge, this multiplex RT-PCR assay is the first to simultaneously detect multiple viruses and viroids infecting apple trees in a single reaction tube. This assay, therefore, offers a useful tool for routine certification and quarantine programs.

Serological and Molecular Detection of Latent Viruses in the Apple Germplasm Bank of Santa Catarina

Abstract The Apple Germplasm Bank (AGB) of Santa Catarina Agricultural Research and Rural Extension Company - Epagri, AGB-Epagri, is the largest of the genus Malus in Brazil. Twenty-eight main accessions of this bank were virus screened through DAS-ELISA, RT-PCR and IC-RT-PCR during two consecutive reproductive cycles, and each accession showed latent mixed infection by at least two species, among ASGV, ASPV and ACLSV. The combined use of diagnostic methods helped overcome inconsistencies commonly found in apple virus detection and was shown essential for the AGB-Epagri can be safely used as a source of genetic variability and for the exchange of virus-free propagative material.

Simultaneous detection and differentiation of three viruses in pear plants by a multiplex RT-PCR.

A multiplex RT-PCR (mRT-PCR) assay was developed for detection and differentiation of the Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV), which are viruses frequently occurring in pear trees. Different combinations of mixed primer pairs were tested for their specificity and sensitivity for the simultaneous detection of the three viruses. Three primer pairs were used to amplify their fragments of 247bp, 358bp and 500bp, respectively. The primer pair for ASPV was designed in this work, while the primer pairs for ACLSV and ASGV were from previous reports. The sensitivity and specificity of the mRT-PCR assay for the three viruses were comparable to that of each uniplex RT-PCR. The mRT-PCR was applied successfully for the detection of three viruses in leaves of pear and apple plants, but was unreliable in the detection of ASGV in dormant barks. In conclusion, this mRT-PCR provides a useful tool for the routine and rapid detection and the differentiation of three pear viruses.