Viral Diversity in Mixed Tree Fruit Production Systems Determined through Bee-Mediated Pollen Collection.

Commercially cultivated Prunus species are commonly grown in adjacent or mixed orchards and can be infected with unique or commonly shared viruses. Apple (Malus domestica), another member of the Rosacea and distantly related to Prunus, can share the same growing regions and common pathogens. Pollen can be a major route for virus transmission, and analysis of the pollen virome in tree fruit orchards can provide insights into these virus pathogen complexes from mixed production sites. Commercial honey bee (Apis mellifera) pollination is essential for improved fruit sets and yields in tree fruit production systems. To better understand the pollen-associated virome in tree fruits, metagenomics-based detection of plant viruses was employed on bee and pollen samples collected at four time points during the peak bloom period of apricot, cherry, peach, and apple trees at one orchard site. Twenty-one unique viruses were detected in samples collected during tree fruit blooms, including prune dwarf virus (PDV) and prunus necrotic ringspot virus (PNRSV) (Genus Ilarvirus, family Bromoviridae), Secoviridae family members tomato ringspot virus (genus Nepovirus), tobacco ringspot virus (genus Nepovirus), prunus virus F (genus Fabavirus), and Betaflexiviridae family member cherry virus A (CVA; genus Capillovirus). Viruses were also identified in composite leaf and flower samples to compare the pollen virome with the virome associated with vegetative tissues. At all four time points, a greater diversity of viruses was detected in the bee and pollen samples. Finally, the nucleotide sequence diversity of the coat protein regions of CVA, PDV, and PNRSV was profiled from this site, demonstrating a wide range of sequence diversity in pollen samples from this site. These results demonstrate the benefits of area-wide monitoring through bee pollination activities and provide new insights into the diversity of viruses in tree fruit pollination ecosystems.

Complete genome sequence of a novel mitovirus isolated from the phytopathogenic fungus Alternaria alternata causing apple leaf blotch.

In this study, a novel mitovirus, tentatively designated as "Alternaria alternata mitovirus 2" (AaMV2), was isolated from the fungus Alternaria alternata f. sp. mali causing apple leaf blotch disease. The complete genome of AaMV2 is 3,157 nucleotides in length, with an A+U content of 68.10%. The genome has a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) protein with a molecular mass of 98.10 kDa. BLAST analysis revealed that AaMV2 has the highest sequence identity to Leptosphaeria biglobosa mitovirus 6, with 79.76% and 82.86% identity at the amino acid and nucleotide level, respectively. Phylogenetic analysis suggested that AaMV2 is a new member of the genus Duamitovirus within the family Mitoviridae. This is the first report of the complete genome sequence analysis of a mitovirus in A. alternata.

A viroid-derived small interfering RNA targets bHLH transcription factor MdPIF1 to regulate anthocyanin biosynthesis in Malus domestica.

Fruit colour is a critical determinant for the appearance quality and commercial value of apple fruits. Viroid-induced dapple symptom severely affects the fruit coloration, however, the underlying mechanism remains unknown. In this study, we identified an apple dimple fruit viroid (ADFVd)-derived small interfering RNA, named vsiR693, which targeted the mRNA coding for a bHLH transcription factor MdPIF1 (PHYTOCHROME-INTERACTING FACTOR 1) to regulate anthocyanin biosynthesis in apple. 5' RLM-RACE and artificial microRNA transient expression system proved that vsiR693 directly targeted the mRNA of MdPIF1 for cleavage. MdPIF1 positively regulated anthocyanin biosynthesis in both apple calli and fruits, and it directly bound to G-box element in the promoter of MdPAL and MdF3H, two anthocyanin biosynthetic genes, to promote their transcription. Expression of vsiR693 negatively regulated anthocyanin biosynthesis in both apple calli and fruits. Furthermore, co-expression of vsiR693 and MdPIF1 suppressed MdPIF1-promoted anthocyanin biosynthesis in apple fruits. Infiltration of ADFVd infectious clone suppressed coloration surrounding the injection sites in apple fruits, while a mutated version of ADFVd, in which the vsiR693 producing region was mutated, failed to repress fruit coloration around the injection sites. These data provide evidence that a viroid-derived small interfering RNA targets host transcription factor to regulate anthocyanin biosynthesis in apple.

Association of Apple Scar Skin Viroid (ASSVd) Infection with an Emerging Disease in 'Saiwaihong' Apples.

A novel disease affecting small immature fruits has surfaced in 'Saiwaihong' apples (Malus pumila), a recently developed variety extensively cultivated across more than 20,000 ha in China. In an effort to pinpoint the causal agent(s) responsible for this ailment, RNA sequencing analysis was conducted on four symptomatic and four asymptomatic apple samples. The results revealed a diverse range of viruses and viroids, indicating mixed viral infection in diseased samples. However, a more focused examination involving 152 symptomatic and 122 asymptomatic fruit samples, using RT-PCR and dot-blotting hybridization techniques, highlighted a close association between the disease and the presence of apple scar skin viroid (ASSVd). Among the ASSVd variants obtained from diseased 'Saiwaihong' apples, 20 were identified, and they were either identical or closely related to isolates from various apple varieties cultivated in different regions and countries. This suggests that ASSVd isolates in 'Saiwaihong' might have been introduced from other apple varieties. Furthermore, the analysis indicates the possibility of two separate introductions, as the ASSVd 'Saiwaihong' isolates exhibited two distinct phylogenetic groups. These insights provide valuable guidance for disease control strategies and emphasize the significance of ongoing monitoring for ASSVd, both in its familiar forms and potential new variants.

Genomic insights into novel Erwinia bacteriophages: unveiling their Henunavirus membership and host infection strategies.

Erwinia amylovora, the primary causative agent of blight disease in rosaceous plants, poses a significant threat to agricultural yield worldwide, with limited effective countermeasures. The emergence of sustainable alternative agents such as bacteriophages is a promising solution for fire blight that specifically targets Erwinia. In this study, we isolated pEp_SNUABM_01 and pEa_SNUABM_55 from a South Korean apple orchard soil, analyzed their genomic DNA sequences, and performed a comprehensive comparative analysis of Hena1 in four distinct sections. This study aimed to unveil distinctive features of these phages, with a focus on host recognition, which will provide valuable insights into the evolution and characteristics of Henunavirus bacteriophages that infect plant pathogenic Erwinia spp. By elucidating the distinct genomic features of these phages, particularly in terms of host recognition, this study lays a foundation for their potential application in mitigating the risks associated with fire blight in Rosaceae plants on a global scale.

Comparing Apples and Oranges: Advances in Disease Resistance Breeding of Woody Perennial Fruit Crops.

Apple and citrus are perennial tree fruit crops that are vital for nutritional security and agricultural economy and to achieve the Sustainable Development Goals of the United Nations. Apple scab and fire blight, along with Huanglongbing, canker, and tristeza virus, stand out as their most notorious diseases and annually destabilize fruit supply. An environmentally sound approach to managing these diseases is improving tree resistance through breeding and biotechnology. Perennial fruit tree germplasm collections are distributed globally and offer untapped potential as sources of resistance. However, long juvenility, specific pollination and flowering habits, and extensive outcrossing hinder apple and citrus breeding. Advances in breeding approaches include trans- and cis-genesis, genome editing, and rapid-cycle breeding, which, in addition to conventional crossbreeding, can all facilitate accelerated integration of resistance into elite germplasm. In addition, the global pool of available sources of resistance can be characterized by the existing genetic mapping and gene expression studies for accurate discovery of associated loci, genes, and markers to efficiently include these sources in breeding efforts. We discuss and propose a multitude of approaches to overcome the challenges of breeding for resistance in woody perennials and outline a technical path to reduce the time required for the ultimate deployment of disease-resistant cultivars.

Transmission studies of the newly described apple chlorotic fruit spot viroid using a combined RT-qPCR and droplet digital PCR approach.

The transmission of the apscaviroid tentatively named apple chlorotic fruit spot viroid (ACFSVd) was investigated using a one-step reverse-transcription (RT) droplet digital PCR assay for absolute quantification of the viroid, followed by quantification of relative standard curves by RT-qPCR. Our results indicate that ACFSVd is effectively transmitted by grafting, budding and seeds. No transmission has yet been observed to the viroid-inoculated pome fruit species Pyrus sp. and Cydonia sp. ACFSVd was detected in viruliferous aphids (Myzus persicae, Dysaphis plantaginea) and in codling moths (Cydia pomonella). The viroid was also detected systemically in the infected hemiparasitic plant Viscum album subsp. album (mistletoe).

ALSV-Based Virus-Induced Gene Silencing in Apple Tree (Malus × domestica L.).

Virus-induced gene silencing (VIGS) is a fast and efficient tool to investigate gene function in plant as an alternative to knock down/out transgenic lines, especially in plant species difficult to transform and challenging to regenerate such as perennial woody plants. In apple tree, a VIGS vector has been previously developed based on the Apple latent spherical virus (ALSV) and an efficient inoculation method has been optimized using biolistics. This report described detailed step-by-step procedure to design and silence a gene of interest (GOI) in apple tree tissues using the ALSV-based vector.

Determination of Protein Interactions among Replication Components of Apple Necrotic Mosaic Virus.

Apple mosaic disease is one of the most widely distributed and destructive diseases in apple cultivation worldwide, especially in China, whose apple yields account for more than 50% of the global total. Apple necrotic mosaic virus (ApNMV) is a newly identified ilarvirus that is closely associated with apple mosaic disease in China; however, basic viral protein interactions that play key roles in virus replication and the viral life cycle have not been determined in ApNMV. Here, we first identify an ApNMV-Lw isolate that belongs to subgroup 3 in the genus Ilarvirus. ApNMV-Lw was used to investigate interactions among viral components. ApNMV 1a and 2apol, encoded by RNA1 and RNA2, respectively, were co-localized in plant cell cytoplasm. ApNMV 1a interacted with itself at both the inter- and intramolecular levels, and its N-terminal portion played a key role in these interactions. 1a also interacted with 2apol, and 1a's C-terminal, together with 2apol's N-terminal, was required for this interaction. Moreover, the first 115 amino acids of 2apol were sufficient for permitting the 1a-2apol interaction. This study provides insight into the protein interactions among viral replication components of ApNMV, facilitating future investigations on its pathogenicity, as well as the development of strategies to control the virus and disease.

Complete genome sequence of a mite-associated virus obtained by high-throughput sequencing analysis of an apple leaf sample.

We provide the complete sequence of a virus tentatively named "Tetranychus urticae-associated picorna-like virus 1PK13" (TuaPV1-PK13) obtained from the high-throughput sequencing of a symptomless apple leaf sample. Although the virus sequence was originally derived from apple leaves, the data suggest that the virus is associated with the two-spotted mite Tetranychus urticae.

Development of RT-qPCR assays for the detection of three latent viruses of pome.

Latent fruit tree viruses present economic threat to the industry and nurseries as diseases they cause not only reduce fruit quality and production yield, but can also be spread inadvertently through propagation due to the lack of viral symptoms on an infected mother plant. As a result, these viruses require appropriate detection tools for effective management. In this study we developed RT-qPCR assays for the detection of three latent viruses of pome, apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), and apple mosaic virus (ApMV), using the alignment of representative sequences from the NCBI database. The optimized assays were shown to be specific by successfully amplifying the target from positive controls without showing any detectable amplification in negative and non-target controls, and revealed high sensitivity by reliably detecting as low as 101 copies per reaction. The results also demonstrated that both the choice of extraction method and the reagents used for RT-qPCRcould play a critical role in virus detection outcome. These assays were both reliable and robust compared to the extant RT-PCR methods, and they could be a viable tool for making informed management decisions.

A bushel of viruses: Identification of seventeen novel putative viruses by RNA-seq in six apple trees.

Apple decline in Washington state has been increasing in incidence, particularly on Honeycrisp trees grown on G.935 rootstock. In this disease the trees exhibit dieback with necrosis at the graft union and in the rootstock. The cause of this disease remains unknown. To identify viral candidates, RNA-seq was performed on six trees: four trees exhibiting decline and two healthy trees. Across the samples, eight known viruses and Apple hammerhead viroid were detected, however none appear to be specifically associated with the disease. A BLASTx analysis of the RNA-seq data was performed to identify novel viruses that might be associated with apple decline. Seventeen novel putative viruses were detected, including an ilarvirus, two tombus-like viruses, a barna-like virus, a picorna-like virus, three ourmia-like viruses, three partiti-like viruses, and two narna-like viruses. Four additional viruses could not be classified. Three of the viruses appeared to be missing key genes, suggesting they may be dependent upon helper viruses for their function. Others showed a specific tropism, being detected only in the roots or only in the leaves. While, like the known apple viruses, none were consistently associated with diseased trees, it is possible these viruses may have a synergistic effect when co-infecting that could contribute to disease. Or the presence of these viruses may weaken the trees for some other factor that ultimately causes decline. Additional research will be needed to determine how these novel viruses contribute to apple decline.

Construction of full-length infectious cDNA clones of Apple stem grooving virus using Gibson Assembly method.

Apple stem grooving virus (ASGV) belongs to the genus Capillovirus within the family Betaflexiviridae. In this work, we described the construction of full-length infectious cDNA clones of ASGV isolate jilin-shaguo (JL-SG) using the Gibson Assembly approach (New England BioLabs). The isolate was previously detected in a Chinese pear-leaf crab apple (Malus asiatica Nakai.) in Baicheng, Jilin province, China. Two full-length cDNA clones of ASGV JL-SG were obtained, and they are identical to each other in sequence. The full-length cDNA clone was infectious on Chenopodium quinoa, Nicotiana glutinosa, and N. occidentalis 37B via agroinfiltration. Through sap inoculation, the infection was additionally spread to C. amaranticolor. N. benthamiana could not be infected, neither through agroinfiltration nor sap inoculation. In infected herbaceous plants, typical ASGV particles with morphology of flexuous filaments were observed by transmission electron microscope (TEM). Moreover, seeds of infected N. glutinosa and N. occidentalis 37B were collected and germinated, the seedlings were ASGV-free in RT-PCR test, suggesting ASGV JL-SG is not seed-transmissible in the tested Nicotiana species. In addition, the cDNA clone was agroinfiltrated into seedlings of Malus pumila cv. Fuji. The infection was symptomless, and can be spread to C. quinoa via sap inoculation, causing typical symptoms. ASGV JL-SG was also detected by RT-PCR in the infected Fuji plants, however, no virion was observed by TEM.

Genomic characterization of Malus domestica virus A (MdoVA), a novel velarivirus infecting apple.

Screening of apple samples using a high-throughput sequencing (HTS) approach led to the discovery of a novel virus, tentatively named "Malus domestica virus A" (MdoVA). Its genomic organisation and phylogenetic relationship showed relatedness to viruses of the genus Velarivirus in the family Closteroviridae. It is not clear whether MdoVA has any impact on its host, as the analysed apple tree contained other viruses and a viroid.

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.

Apple chlorotic fruit spot viroid: a putative new pathogenic viroid on apple characterized by next-generation sequencing.

Viroid-like symptoms were observed in 2016 on apple fruits of the cultivar "Ilzer Rose" in southern Burgenland, Austria. Preliminary molecular biological investigations indicated that the symptoms were caused by a new unknown viroid. Therefore, new primers were designed, and the whole genome sequence of the viroid (354 nt) was determined by next-generation amplicon sequencing using the Illumina MiSeq® platform (San Diego, California, USA). The viroid secondary structure has a rod-like conformation and contains conserved regions (the TCR, CCR upper strand, and CCR lower strand) that are characteristic of members of the genus Apscaviroid. Based on our results and the demarcation criteria for viroids, the tentatively named "apple chlorotic fruit spot viroid" should be considered a putative new member of the genus Apscaviroid.

The complete genome sequence of apple rootstock virus A, a novel nucleorhabdovirus identified in apple rootstocks.

We report the complete genome sequence of a novel nucleorhabdovirus, apple rootstock virus A (ApRVA), isolated from Malus spp. in South Korea. ApRVA has a 14,043-nt single-stranded negative-sense RNA genome. In the antigenome sense, it contains seven open reading frames, encoding the putative nucleocapsid protein, phosphoprotein, cell-to-cell movement protein, matrix protein, glycoprotein, RNA-dependent RNA polymerase, and an additional hypothetical protein, the gene for which is located between the genes for the matrix protein and glycoprotein. The complete genome sequence of ApRVA showed 47.45% nucleotide sequence identity to that of black currant-associated rhabdovirus 1. The genome organization, phylogenetic relationships, and sequence similarities to other nucleorhabdoviruses suggest that ApRVA is a new member of the genus Nucleorhabdovirus.

Evolving by deleting: patterns of molecular evolution of Apple stem pitting virus isolates from Poland.

In this study, 267 coat protein gene (CP) sequences from 48 Polish isolates of Apple stem pitting virus (ASPV) were determined. The genetic structure of the virus population was analysed and possible mechanisms of molecular evolution explored. We found evidence of recombination within the ASPV population and the presence of 17 ASPV molecular variants that differ in the length, number and arrangement of deletions in the CP. Population genetic analyses showed significant variation among isolates from pear and apple trees, between isolates from the same host species and, more interestingly, within isolates, supporting the existence of significant levels of variability within individual hosts, as expected by a quasispecies population structure. In addition, different tests support that selection might have been an important force driving diversification within isolates: positive selection was found acting upon certain amino acids. Phylogenetic analyses also showed that isolates did not classify according to the host species (pear or apple trees) but according to the pattern of deletions, suggesting a possible role for deletions during clade diversification.

Molecular variability of apple hammerhead viroid from Italian apple varieties supports the relevance in vivo of its branched conformation stabilized by a kissing loop interaction.

In the absence of protein-coding ability, viroid RNAs rely on direct interactions with host factors for their infectivity. RNA structural elements are likely involved in these interactions. Therefore, preservation of a structural element, despite the sequence variability existing between the variants of a viroid population, is considered a solid evidence of its relevant role in vivo. In this study, apple hammerhead viroid (AHVd) was first identified in the two apple cultivars 'Mela Rosa Guadagno' (MRG) and 'Agostinella' (AG), which are cultivated since long in Southern Italy, thus providing the first solid evidence of its presence in this country. Then, the natural variability of AHVd viroid populations infecting MRG and AG was studied. The sequence variants from the two Italian isolates shared only 82.1-87.7% sequence identity with those reported previously from other geographic areas, thus providing the possibility of exploring the impact of this sequence divergence on the proposed secondary structure. Interestingly, all the AHVd sequence variants considered in this study preserved a branched secondary structure stabilized by a kissing-loop interaction, resembling the conformation proposed previously for variants from other isolates. Indeed, most mutations did not modify the proposed conformation because they were co-variations, conversions of canonical into wobble base-pairs, or vice versa, as well as changes mapping at loops. Importantly, a cruciform structural element formed by four hairpins, one of which is implicated in the proposed kissing-loop interaction, was also preserved because several nucleotide changes actually resulted into two, three and up to five consecutive co-variations associated with other changes that did not affect the secondary structure. These data provide very strong evidence for the relevance in vivo of this cruciform structure which, together with kissing-loop interaction, likely contribute to further stabilizing the branched AHVd secondary structure.