The citrus industry is threatened by a devastating bacterial disease known as Huanglongbing (HLB) or citrus greening. Current control measures largely rely on chemical suppression of insect vectors, primarily Diaphorina citri and Trioza erytreae. A promising alternative lies in the biotechnological use of viruses that specifically infect these vectors. Although much progress has been made in elucidating the virome of D. citri, there is no data on the viral population that Trioza erytreae can harbor and/or infect it. In this study, we analyzed the virome of T. erytreae from the Iberian Peninsula, Madeira, the Canary Islands, São Tomé and Principe, the Reunion Islands, and South Africa. We identified seven new insect-specific viruses (ISV) belonging to the families Narnaviridae, Totiviridae, Solemoviridae, Phenuiviridae, Flaviviridae, and Tombusviridae, the last three detected only in South African populations, four mycoviruses of the Ambiguiviridae, Botourmiaviridae, and Partitiviridae families, and three viruses known to infect citrus. Furthermore, comparison with the DNA sequence database has allowed us to identify the presence of 22 actively expressed endogenous viral elements (EVEs; virus-derived genetic material integrated into the genome of a nonviral host). Our findings provide new insights into the virome of this important insect vector and new perspectives to its potential for biocontrol strategies against HLB.

Citrus production in South Africa is affected by seven mealybug (Hemiptera: Coccomorpha: Pseudococcidae) species, with Planococcus citri (Risso) being the most prevalent and the pest status of the other six species being generally minor or even negligible. Infestations can reduce fruit production and quality due to sooty mould development. Accurate species-level identification of mealybugs is valuable for effective agricultural pest management and in some cases may be of phytosanitary relevance. Traditional morphological identification is challenging. Slide mounted adult female specimens are usually required for accurate morphological identification, a process that is time consuming and requires specialist skills. Molecular diagnostics targeting mitochondrial genes, particularly the cytochrome c oxidase subunit I ( COI ), have become valuable rapid tools for reliable species discrimination in most insect groups. The use of molecular techniques in combination with morphological assessments provides stronger support for accurate species identification. This study reports the first validated mitochondrial genome of P. citri , an important citrus pest, alongside partial mitochondrial sequences for the six other mealybug species previously reported on citrus and two other species that are not associated with citrus in South Africa. Using high-throughput and Sanger sequencing combined with manual curation, a high-confidence P. citri mitogenome was assembled while draft mitogenomes were generated for the other species. Two complementary molecular assays were developed and validated. These consisted of a set of multiplex PCRs and restriction enzyme digest assays that enable rapid, same-day differentiation of the nine mealybug species. The multiplex PCR assays provide sensitive and specific detection, while the restriction digest assays act as a confirmatory tool and fail-safe mechanism, allowing early identification of discrepancies. In one of the species, Nipaecoccus viridis , greater genetic diversity was uncovered, suggesting cryptic speciation. This study underscores the complexities of unarmoured scale insect mitogenomics, including repetitive regions, low gene conservation and scarce reference sequences while providing a robust and efficient molecular framework that enhances accurate mealybug identification.

Background ONT sequencing has been previously evaluated for its ability to detect plant viruses and viroids. Its advantages, such as longer read lengths and real-time analysis, compete with extensively validated Illumina platforms for possible incorporation into routine pathogen detection. The continuous development and improvement of ONT sequencing along with the discontinuation of older equipment and reagents, necessitate a renewed comparison of these sequencing platforms, specifically in citrus where only limited research is available. Results This study compared the ability of Oxford Nanopore Technologies (ONT) sequencing, using the MinION flow cell, with Illumina sequencing, on the NovaSeqX platform, to accurately detect three viruses and three viroids in citrus. Both technologies were able to identify all pathogens using both reference-dependent and independent methods. While Illumina sequencing re-established the high sensitivity, coverage and accuracy seen previously, ONT compensated for fewer pathogen reads and lower depth with longer reads that enabled reasonable genome coverage and sequencing identities comparable to that of Illumina. Moreover, the pooling of data from different ONT barcode datasets from a single sample, improved comparability to Illumina results as small variations in library preparation, sample loading and flow cells can lead to a significant decrease in sequencing data. Reference gene expression profiles were also investigated to evaluate internal controls and check outlier samples. The ONT platform also had a shorter turnaround compared to Illumina sequencing. Conclusion The use of ONT sequencing may offer advantages for small-scale routine pathogen detection. It has the potential to accurately detect pathogens and discover novel viral agents. This comparison between Illumina and ONT platforms highlights the strengths associated with each approach and offers new insights into the possible application of high-throughput sequencing (HTS) within plant health surveillance and biosecurity programs.

Fruit protection against infection by Phyllosticta citricarpa, the causal agent of citrus black spot (CBS), relies heavily on fungicides. Quinone-outside inhibitors (QoIs), which have a site-specific mode of action, have been in use since the early 2000s in South African citrus orchards. This study investigated the occurrence of the F129L, G137R or G143A mutations in codons 129, 137 and 143, respectively, in cytochrome b (cytb) of P. citricarpa. Molecular analysis detected the F129L mutation in cytb of 73 of 191 isolates tested, but the G143A and G137R mutations were not detected. A non-synonymous nucleotide substitution from TTC to TTA was predominantly detected in 69 isolates, while a codon change from TTC to CTC was detected in only four isolates. Both mutations result in an amino acid substitution from phenylalanine to leucine. To investigate the practical impact of this mutation on the field efficacy of QoI fungicides, trials were performed on a lemon orchard with a history of high CBS pressure and 100% detection rate of the F129L mutation. Results revealed a strong association between the occurrence of the F129L mutation and reduced efficacy in controlling CBS by pyraclostrobin and azoxystrobin, when applied outside the requirements of registration, i.e. not in a mixture with a fungicide from another FRAC group. This study is the first report of the F129L mutation and a shift in sensitivity to QoI fungicides in P. citricarpa populations.

BACKGROUND:Certification and disease management practices are reliant on accurate pathogen detection assays. Effective nucleic acid extraction is the cornerstone of various molecular detection techniques, and many virus and viroid RNA extraction protocols have been developed, each with accompanying advantages and limitations. The extraction method influences the type, quantity and quality of RNA and, therefore, the overall sensitivity of the assay. METHODS:Plants from two citrus types were infected with multiple viruses and viroids. Ten plants from each were selected. RNA was extracted from bark and leaf samples using four protocols: CL (CTAB/LiCl), CE (CTAB/ethanol), TL (acid phenol buffer/LiCl and isopropanol) and SI (SDS/isopropanol). Extracts were subjected to RT-qPCR for pathogen quantitation. Results were confirmed with Illumina high-throughput sequencing (HTS). Leaf material from a citrus type was resampled, RNA extracted with CE, SI and CP (CTAB/isopropanol) and pathogen quantities determined with RT-qPCR. An additional five plants were also selected and subjected to RNA extraction with CP, SI and a modified CE. RT-qPCRs and HTS were applied to all of these extracts. Protocols were evaluated based on pathogen quantities and various bioinformatic approaches. Reference genes and CTV genotypes were also assessed with HTS. RESULTS:The extraction protocol had a remarkable influence on RT-qPCR and HTS pathogen detection ability. The CL method performed best for CTV while SI proved more sensitive for the other viruses. Viroid detection had increased variability with CE extracts containing more viroid RNA although CL extracts showed comparable amounts of CDVd. The CE protocol indicated potential debilitating effects on virus detection. The additional sampled plants subjected to the modified CE protocol, showed increased virus and decreased viroid amounts. The extraction protocol did, however, not significantly influence CTV genotype composition and the investigation of reference genes provided insight into appropriate genes to be used as internal controls for cross sample comparisons. CONCLUSIONS:Despite the differential pathogen extraction amongst protocols, all target pathogens could be detected with appropriate RT-qPCR replicates and combined bioinformatic approaches. This study emphasises that although multiple protocols can be effective, further selection and optimisation could enhance the performance. Present research serves as a foundation for the refinement of diagnostic pipelines in citrus pathology.

In this study, the prevalence and genome diversity of apple stem pitting virus (ASPV) in commercial pear orchards in South Africa were investigated. Leaves were collected from trees in two geographically distinct regions, representing six different pear cultivars covering 12 orchards. Total RNA was extracted and assayed for ASPV using RT-PCR. A subset of 18 samples was subjected to high-throughput sequencing (HTS) for a more detailed analysis. Bioinformatic analyses of the HTS data, led to the identification and sequencing of a divergent ASPV variant with 85.10

Grapevine leafroll disease is the most damaging viral disease afflicting global grape and wine production. Of the five viruses likely to be associated with the disease, grapevine leafroll-associated virus 3 (GLRaV3) is believed to be the predominant agent, albeit its role as the causal agent has remained uncertain. GLRaV3 (species Ampelovirus trivitis, genus Ampelovirus, family Closteroviridae) has the third largest single-stranded, positive-sense RNA genome among plant viruses at ~18.5 kb, only surpassed by two other members of the family Closteroviridae, citrus tristeza virus and GLRaV1. GLRaV3 is unique among plant viruses in several ways, including the size of its genome, the long non-coding regions, and its association with the outer mitochondrial membrane for viral replication. Unfortunately, our understanding of the molecular mechanisms governing GLRaV3 genome replication, gene expression, and virus-host interactions is poor due to many factors. These include the unavailability of infectious cDNA clones and, until recently, an effective experimental system to initiate grapevine infection with viral clones. In this review, we capture recent advances in GLRaV3 research towards the establishment of infectious clones, grapevine inoculation systems, as well as approaches to elucidating the function of GLRaV3-encoded proteins. We also present a working model to explain GLRaV3 pathogenesis.

Huanglongbing, a devastating citrus disease, is associated with ‘ Candidatus Liberibacter asiaticus’, ‘ Ca. L. africanus’ or ‘ Ca L. americanus’, bacteria transmitted by the psylloids Diaphorina citri and Trioza erytreae . Using a DNA-Seq and metabarcode sequencing integrated approach, the first catalogue of endosymbionts associated with T. erytreae from the Iberian Peninsula, South Africa and African Islands, was generated. The almost complete genome of two new bacteria, one facultative and one obligate, tentatively named Asaia-like endosymbiont of T. erytreae and Sodalis-like endosymbiont of T. erytreae, respectively, was assembled and annotated. The complete mitochondrial genomes of T. erytreae from the geographical areas studied were also assembled and phylogenetic analyses were performed, suggesting that T. erytreae populations currently present in the Iberian Peninsula and specimens analyzed from South Africa may have originated from a common ancestor. Similar results were obtained when the genetic distances between Sodalis-like endosymbiont of T. erytreae were taken into consideration, thus supporting the symbiont–host codivergence which suggests that this bacterium is approaching to an obligate status. Finally, a new genetic marker of T. erytreae, an insertion in the mitochondrial tRNA-Ser gene, was identified only in some European samples, showing for the first time the existence of two mixed subpopulations of T. erytreae . The integrated DNA-Seq and metabarcode sequencing approach used in this study, besides generating a catalogue of T. erytreae endosymbionts, provided novel data on the sequence variability of bacterial and insect mitochondrial genomes from different geographic areas, highlighting the possible original sources of currently spreading T. erytreae populations may be more complex than previously reported.

The complete genome sequence of fig leaf mottle-associated virus 2 (FLMaV2), a positive-sense single-stranded RNA virus with a genome length of 16,925 nucleotides, was determined via RT-PCR and rapid amplification of cDNA ends (RACE). Its genome organization resembles those of little cherry virus 2 (LChV2) and yam asymptomatic virus 1 (YaV1), both of which are currently classified as members of the genus Ampelovirus but differ from typical ampeloviruses. Phylogenetic analysis based on amino acid sequences of the RNA-dependent RNA polymerase and coat protein of members of the family Closteroviridae showed that FLMaV2 clustered with LChV2 and YaV1. Because of their unusual genome organization, FLMaV2, LChV2, and YaV1 might represent a distinct genus within the family Closteroviridae.

Citrus tristeza virus (CTV) causes several disease syndromes in different citrus hosts; namely quick decline, seedling yellows, and stem pitting.CTV-induced stem pitting leads to substantial economic losses in sensitive citrus varieties, including grapefruit.The formation of stem pits has previously been linked to the ability of the virus to colonize xylem tissue outside of its typical phloem limitation, thereby disrupting normal xylem development.The nature of this compromised tissue has not been fully elucidated.In this study, stem pits were characterized at the molecular anatomical level using a combination of techniques to better understand the characteristics of the xylem and phloem tissues impacted by severe pitting.Biological staining was used to visualize CTV-induced stem pitting and was complemented with a novel technology that has not previously been used to study CTV-induced stem pitting, namely serial block-face scanning electron microscopy (SBF-SEM).This proofof-concept study yielded new insights into the morphology of stem pitting-affected tissue.The utility of SBF-SEM for stem pitting characterization was also demonstrated and an optimized protocol for its application on hard, woody material is presented.

This collaborative work by over 180 researchers from 40+ countries addresses the challenges posed by “phantom agents”—putative pathogenic agents named in literature without supporting data on their existence. Those agents remain on regulatory lists, creating barriers in trade and plant certification. Historically identified based solely on symptoms, these agents lack isolates or sequence data, making reliable detection or risk assessment impossible. After reviewing over 120 such agents across 10 key plant genera, we recommend their removal from regulatory lists and call for revised standards aligned with modern diagnostics. This effort seeks to streamline germplasm exchange, benefiting global agriculture by removing the constraints imposed by phantoms.

The management of plant diseases relies on the accurate identification of pathogens that requires a robust and validated tool in terms of specificity, sensitivity, repeatability, and reproducibility. High-throughput sequencing (HTS) has become the method of choice for virus detection when either a complete viral status of a plant is required in a single assay or if an unknown viral agent is expected. To ensure that the most accurate diagnosis is made from an HTS data analysis, a standardized protocol per pathosystem is required. This chapter presents a detailed protocol for the detection of viruses and viroids infecting citrus using HTS. The protocol describes all the steps from sample processing, nucleic acid extraction, and bioinformatic analyses validated to be an efficient method for detection in this pathosystem. The protocol also includes a section on citrus tristeza virus (CTV) genotype differentiation using HTS data.

Citrus tristeza virus (CTV) causes economically important stem pitting in sensitive citrus types however the exact mechanisms of stem pitting development in citrus remain unclear. In this study, CTV infectious clones were used to study stem pitting induction in ‘Duncan’ grapefruit and ‘Mexican’ lime. A panel of open reading frame (ORF) replacement clones was generated focusing on the CTV ORFs implicated in stem pitting development and pathogenicity, namely p33, p18, p13 and p23. ORF replacements from severe- and mild-pitting CTV isolates were introduced into a mild-pitting infectious clone (genotype T36) to determine if stem pitting could be induced. A broad range of stem pitting outcomes were observed with ORF p18 (from isolate T3-KB) and ORF p23 (from isolate GFMS12-1.3) associated with enhanced stem pitting development. Metabolomic trends underlying the different stem pitting outcomes were further assessed by untargeted metabolite profiling. In each citrus host, the metabolite profiling identified statistically significant compounds that differed between stem pitting groups. These compounds were mainly phenolic acids and phenolic glycosides and are known to function as antioxidant and stress-signaling molecules. These metabolites can serve as targets for future time-course observations to potentially use mass spectrometry profiling to inform CTV management practices.

Agapanthus praecox Willd. is an ornamental flowering plant that is indigenous to southern Africa and was reported to be a host of tomato spotted wilt orthotospovirus (TSWV) in Australia in 2000 (Wilson et al. 2000). Tomato spotted wilt orthotospovirus (TSWV) belonging to the genus Orthotospovirus of the family Tospoviridae is a single-stranded negative sense RNA virus known to cause disease symptoms in many crops and ornamental plant species. This virus is in the top 10 of most economically important plant viruses worldwide (Rybicki 2015; Scholthof et al. 2011). In May 2021, leaf material from three agapanthus (Agapanthus praecox) plants displaying chlorotic mottling, and yellow lesions (Supplementary material 1A) was collected in Mbombela, South Africa. One gram of symptomatic leaf material was used for total RNA extraction from each of the three samples using a CTAB extraction protocol (Ruiz-García et al. 2019). The three RNA extracts were pooled, and a sequencing library was constructed using the Ion Total RNA-Seq Kit v2.0 and RiboMinus™ Plant Kit for RNA-Seq (ThermoFisher Scientific) (Central Analytical Facility (CAF), Stellenbosch University). The RNA library was sequenced on an Ion Torrent Proton Instrument (CAF). A total of 34,392,939 single-end reads were obtained. Data was trimmed for quality with Trimmomatic (CROP:250, MINLEN:50). De novo assembly was performed on the remaining 32,281,645 trimmed reads (average readlength: 100 nt, range: 50-250 nt) using SPAdes 3.13.0 and resulted in 4,788 contigs. BLASTn analysis identified viral contigs longer than 1,000 nucleotides (nts) with high nucleotide (nt) identity to TSWV (6 contigs), as well as to the newly discovered viruses, agapanthus tungro virus (AgTV) (1 contig), and agapanthus velarivirus (AgVV) (4 contigs) (Read et al 2021). Read mapping was performed against the relevant reference sequence with the highest nt identity to the contigs. For TSWV, 4995, 21221 and 14574 reads mapped to segment L (KY250488), M (KY250489) and S (KY250490) of isolate LK-1, respectively resulting in 99.97%, 100.00% and 99.97% genome coverage of the reference accessions. The nt identity between the reference accessions and the consensus sequences generated (OP921761-OP921763) were 97.26%, 97.64% and 97.82% for segment L, M and S. The presence of TSWV was confirmed in the HTS sample using an RT-PCR assay (primers L1 and L2) targeting the L segment of TSWV (Mumford et al. 1994). In July 2022, additional leaf samples displaying symptoms of chlorotic mottling, streaking, and ringspots were collected from 31 symptomatic and 3 asymptomatic agapanthus plants in public gardens in Stellenbosch, South Africa. Using the above-mentioned RT-PCR assay, 13 of the symptomatic samples tested positive for TSWV. All six plants displaying ring spot symptoms (Supplementary material 1B) were infected with TSWV. However, plants that displayed yellow streaking (five samples) and chlorotic mottling (two samples) (Supplementary material 1C-D) were also positive for TSWV which could be due to the presence of other viruses, plant growth stage, infection time or just variable symptom expression in a single host species as reported previously (Sherwood et al. 2003). The 275 bp RT-PCR amplicons of the HTS sample and three additional positive samples were validated with bidirectional Sanger sequencing (CAF) and had 96% identity to accession KY250488. The pairwise nt identity between amplicons was 98.55-100%. This is the first report of TSWV infecting agapanthus in South Africa. This study contributes information towards the distribution and incidence of TSWV and highlights the need for nurseries to screen plant material before propagation.

Virus-like symptoms on fig tree leaves are a common occurrence worldwide and has mostly been attributed to fig mosaic disease (FMD). Even though only fig mosaic virus (FMV) has been shown to cause FMD, many other viruses have been identified in diseased fig trees. In 2021, total RNA was extracted from fig leaf samples displaying symptoms of mosaic and chlorotic mottling and was subjected to high-throughput sequencing (HTS) to construct the first virome profile of a South African fig tree. Bioinformatic analyses identified FMV, fig leaf mottle-associated virus 1 (FLMaV1), fig leaf mottle-associated virus 2 (FLMaV2), fig latent virus 1 (FLV1), fig badnavirus 1 (FBV1) and grapevine badnavirus 1 (GBV1) in the data. Reverse transcription polymerase chain reaction (RT-PCR) was conducted, for these viruses, on 24 additional fig leaf samples collected in the Western Cape. FBV1, GBV1, FMV, FLMaV1, FLV1, FLMaV2, and fig fleck-associated virus (FFkaV) were detected in 100%, 96%, 92%, 54%, 46%, 21%, and 12.5% of the samples, respectively. This is the first report on the presence of FMV, FLMaV2, FLV1, FFkaV, FBV1 and GBV1 in South Africa and offers a preliminary insight into the virus status of fig trees in the country.

Grapevine leafroll disease (GLD) is a globally important disease that affects the metabolic composition and biomass of grapes, leading to a reduction in grape yield and quality of wine produced. Grapevine leafroll-associated virus 3 (GLRaV-3) is the main causal agent for GLD. This study aimed to identify protein-protein interactions between GLRaV-3 and its host. A yeast two-hybrid (Y2H) library was constructed from Vitis vinifera mRNA and screened against GLRaV-3 open reading frames encoding structural proteins and those potentially involved in systemic spread and silencing of host defense mechanisms. Five interacting protein pairs were identified, three of which were demonstrated in planta. The minor coat protein of GLRaV-3 was shown to interact with 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 02, a protein involved in primary carbohydrate metabolism and the biosynthesis of aromatic amino acids. Interactions were also identified between GLRaV-3 p20A and an 18.1-kDa class I small heat shock protein, as well as MAP3K epsilon protein kinase 1. Both proteins are involved in the response of plants to various stressors, including pathogen infections. Two additional proteins, chlorophyll a-b binding protein CP26 and a SMAX1-LIKE 6 protein, were identified as interacting with p20A in yeast but these interactions could not be demonstrated in planta. The findings of this study advance our understanding of the functions of GLRaV-3-encoded proteins and how the interaction between these proteins and those of V. vinifera could lead to GLD.

The roles of proteins encoded by members of the genus Ampelovirus, family Closteroviridae are largely inferred by sequence homology or analogy to similarly located ORFs in related viruses. This study employed yeast two-hybrid and bimolecular fluorescence complementation assays to investigate interactions between proteins of grapevine leafroll-associated virus 3 (GLRaV-3). The p5 movement protein, HSP70 homolog, coat protein, and p20B of GLRaV-3 were all found to self-interact, however, the mechanism by which p5 interacts remains unknown due to the absence of a cysteine residue crucial for the dimerisation of the closterovirus homolog of this protein. Although HSP70h forms part of the virion head of closteroviruses, in GLRaV-3, it interacts with the coat protein that makes up the body of the virion. Silencing suppressor p20B has been shown to interact with HSP70h, as well as the major coat protein and the minor coat protein. The results of this study suggest that the virion assembly of a member of the genus Ampelovirus occurs in a similar but not identical manner to those of other genera in the family Closteroviridae. Identification of interactions of p20B with virus structural proteins provides an avenue for future research to explore the mechanisms behind the suppression of host silencing and suggests possible involvement in other aspects of the viral replication cycle.

Recent developments in high-throughput sequencing (HTS) technologies and bioinformatics have drastically changed research in virology, especially for virus discovery. Indeed, proper monitoring of the viral population requires information on the different isolates circulating in the studied area. For this purpose, HTS has greatly facilitated the sequencing of new genomes of detected viruses and their comparison. However, bioinformatics analyses allowing reconstruction of genome sequences and detection of single nucleotide polymorphisms (SNPs) can potentially create bias and has not been widely addressed so far. Therefore, more knowledge is required on the limitations of predicting SNPs based on HTS-generated sequence samples. To address this issue, we compared the ability of 14 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 21 variants of pepino mosaic virus (PepMV) in three samples through large-scale performance testing (PT) using three artificially designed datasets. To evaluate the impact of bioinformatics analyses, they were divided into three key steps: reads pre-processing, virus-isolate identification, and variant calling. Each step was evaluated independently through an original, PT design including discussion and validation between participants at each step. Overall, this work underlines key parameters influencing SNPs detection and proposes recommendations for reliable variant calling for plant viruses. The identification of the closest reference, mapping parameters and manual validation of the detection were recognized as the most impactful analysis steps for the success of the SNPs detections. Strategies to improve the prediction of SNPs are also discussed.

HomePlant DiseaseVol. 106, No. 5First Report of Apple rubodvirus 2 Infecting Pear (Pyrus communis) in South Africa Previous DISEASE NOTE OPENOpen Access licenseFirst Report of Apple rubodvirus 2 Infecting Pear (Pyrus communis) in South AfricaK. Bougard, H. J. Maree, G. Pietersen, J. Meitz-Hopkins, and R. BesterK. BougardDepartment of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaSearch for more papers by this author, H. J. Mareehttps://orcid.org/0000-0001-9639-4558Department of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaCitrus Research International, P.O. Box 2201, Matieland, 7602, South AfricaSearch for more papers by this author, G. PietersenDepartment of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaSearch for more papers by this author, J. Meitz-Hopkinshttps://orcid.org/0000-0002-8127-6978Department of Plant Pathology, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaSearch for more papers by this author, and R. Bester†Corresponding author: R. Bester; E-mail Address: [email protected]https://orcid.org/0000-0002-2226-0968Department of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaCitrus Research International, P.O. Box 2201, Matieland, 7602, South AfricaSearch for more papers by this authorAffiliationsAuthors and Affiliations K. Bougard1 H. J. Maree1 2 G. Pietersen1 J. Meitz-Hopkins3 R. Bester1 2 † 1Department of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa 2Citrus Research International, P.O. Box 2201, Matieland, 7602, South Africa 3Department of Plant Pathology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa Published Online:14 Mar 2022https://doi.org/10.1094/PDIS-08-21-1631-PDNAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Apple rubbery wood virus 2 (ARWV-2; Rott et al. 2018) belongs to the species Apple rubodvirus 2, a member of the genus Rubodvirus (family Phenuiviridae; Kuhn et al. 2020). ARWV-2 was first identified in apples and is associated with apple rubbery wood disease (ARWD), which is characterized by unusual flexibility of stems and branches, reduced growth, shortened internodes, and increased cold sensitivity (Jakovljevic et al. 2017; Rott et al. 2018). ARWD was first reported in 1935 in England on apple and has since been found on quince and pear (Jakovljevic et al. 2017; Rott et al. 2018). In January 2021, leaves were collected from a pear tree (Pyrus communis cv. ‘Forelle’, F514) in a commercial orchard near Villiersdorp, South Africa. The tree displayed no foliar or tree branch symptoms except for malformed fruits potentially due to insect feeding damage or pear stony pit disease previously associated with infection of apple stem pitting virus (ASPV) (Paunovic et al. 1999). Leaf petioles (1 g) were used for total RNA extraction, using a modified CTAB extraction protocol (Ruiz-García et al. 2019). A sequencing library was constructed (Illumina TruSeq Stranded Total RNA with plant Ribo-Zero) and sequenced on an Illumina HiseqX instrument (Macrogen, South Korea). A total of 30,709,182 paired-end reads (100 nt) were obtained and trimmed for quality with Trimmomatic (SLIDINGWINDOW:3:20, MINLEN:20) (Bolger et al. 2014). De novo assembly, using default parameters of CLC Genomics Workbench 11.0.1 (Qiagen), resulted in 97,294 contigs. BLASTn analysis identified 17 viral contigs, with 14 contigs having high nucleotide identity to ASPV and three to ARWV-2. The latter contigs included all three segments of ARWV-2. The L contig was 7,371 nt, M was 1,289 nt, and S was 1,463 nt in length, generated with 7,341, 626, and 9,161 reads for segments L, M, and S, respectively. Segment S had the highest read coverage (524.87×), followed by segments L (88.07×) and M (36.60×). The ARWV-2 GenBank accessions with the highest percentage identity to the contigs were MF062128.1 from the United States of America (98.2% to segment L), MN163134.1 from China (97.5% to segment M), and NC_055535.1 from Germany (93.5% to segment S). The contigs spanned 100, 80.92, and 100% of these accessions of segments L, M, and S, respectively, and were deposited in GenBank as accessions MZ593725 to MZ593727. Reverse transcription polymerase chain reaction was used to validate the presence of ARWV-2 in sample F514, using primers directed at segments L (con708_178F/con708_666R), M (ARWaV-2S1_38F/ARWaV-2S1_682R), and S (ARWaV-2M567F/ARWaV-2M1342R) (Rott et al. 2018). Amplicon sequences (510 bp [L], 645 bp [M], and 799 bp [S]) were confirmed with bidirectional Sanger sequencing. Fifty-nine additional pear samples were surveyed in 2021 for ARWV-2 using the M segment assay mentioned above. The survey included the Koue Bokkeveld and Elgin areas, and cultivars ‘Bosc’ (22 samples), ‘Abate’ (10 samples), ‘Rosemarie’ (3 samples), ‘Forelle’ (9 samples), ‘Packham’s Triumph’ (12 samples), and ‘Early Bon Chretien’ (3 samples). A total of 27 samples (11 samples from the Koue Bokkeveld region and 16 samples from the Elgin region) tested positive for ARWV-2, demonstrating the common presence of this virus in pears in South Africa. This is the first report of ARWV-2 infecting pear in South Africa. Although no association with disease symptoms were observed, this study expands the data on the incidence and distribution of this virus in South Africa.The author(s) declare no conflict of interest.References:Bolger, A. M., et al. 2014. Bioinformatics 30:2114. https://doi.org/10.1093/bioinformatics/btu170 Crossref, ISI, Google ScholarJakovljevic, V., et al. 2017. Eur. J. Plant Pathol. 148:637. https://doi.org/10.1007/s10658-016-1119-z Crossref, ISI, Google ScholarKuhn, J. H., et al. 2020. Arch. Virol. 165:3023. https://doi.org/10.1007/s00705-020-04731-2 Crossref, ISI, Google ScholarPaunovic, S., et al. 1999. J. Phytopathol. 147:695. https://doi.org/10.1046/j.1439-0434.1999.00449.x Crossref, ISI, Google ScholarRott, M. E., et al. 2018. Plant Dis. 102:1254. https://doi.org/10.1094/PDIS-06-17-0851-RE Link, ISI, Google ScholarRuiz-García, A. B., et al. 2019. Page 163 in: Citrus Tristeza Virus. Humana, New York, NY. Crossref, Google ScholarFunding: The project was funded by Hortgro (PO-20-USGEN-PM01).The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 106, No. 5 May 2022SubscribeISSN:0191-2917e-ISSN:1943-7692 Download Metrics Article History Issue Date: 28 Apr 2022Published: 14 Mar 2022First Look: 14 Oct 2021Accepted: 13 Oct 2021 Page: 1535 Information© 2022 The American Phytopathological SocietyFundingHortgroGrant/Award Number: PO-20-USGEN-PM01KeywordsApple rubbery wood virus 2 (ARWV-2)tree fruitsviruses and viroidsThe author(s) declare no conflict of interest.PDF downloadCited byOccurrence of apple rubbery wood virus 1 and apple rubbery wood virus 2 in pear and apple in Campania (southern Italy) and development of degenerate primers for the rapid detection of rubodviruses7 February 2023 | Journal of Plant PathologyApple rubbery wood virus 2CABI Compendium, Vol. CABI CompendiumIdentification and Characterization of Citrus Concave Gum-Associated Virus Infecting Citrus and Apple Trees by Serological, Molecular and High-Throughput Sequencing Approaches5 November 2021 | Plants, Vol. 10, No. 11

It has been nearly 100 years since citrus growers in two distinct regions in the northern provinces of South Africa noticed unusual symptoms in their citrus trees, causing significant crop losses. They had no idea that these symptoms would later become part of an almost global pandemic of a disease called greening or huanglongbing (HLB). The rapid spread of the disease indicated that it might be caused by a transmissible pathogen, but it took >50 years to identify the causative agent as 'Candidatus Liberibacter africanus'. Recently, the disease appeared in more African countries, spreading by both infected planting material and Trioza erytreae. To date, five 'Ca. L. africanus' subspecies have been identified in various rutaceous species, with 'Ca. L. africanus subsp. clausenae' the only subspecies for which a biovar was detected in citrus. Efforts to detect and differentiate HLB-causing Liberibacter species are ongoing, and recent developments are discussed here. This review focuses on aspects of the African form of HLB, including its specific bacterial species and subspecies, its main insect vector, its geographic distribution, and current management strategies.