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Citrus virus A (CiVA), a novel negative-sense single-stranded RNA virus assigned to the species Coguvirus eburi in the genus Coguvirus, was detected in South Africa with the use of high-throughput sequencing after its initial discovery in Italy. CiVA is closely related to citrus concave gum-associated virus (CCGaV), recently assigned to the species Citrus coguvirus. Disease association with CiVA is, however, incomplete. CiVA was detected in grapefruit (C. paradisi Macf.), sweet orange [C. sinensis (L.) Osb.], and clementine (C. reticulata Blanco) in South Africa, and a survey to determine the distribution, symptom association, and genetic diversity was conducted in three provinces and seven citrus production regions. The virus was detected in ‘Delta’ Valencia trees in six citrus production regions, and a fruit rind symptom was often observed on CiVA-positive trees. Additionally, grapefruit showing symptoms of citrus impietratura disease were positive for CiVA. This virus was primarily detected in older orchards that were established prior to the application of shoot tip grafting for virus elimination in the South African Citrus Improvement Scheme. The three viral-encoded genes of CiVA isolates from each cultivar and region were sequenced to investigate sequence diversity. Genetic differences were detected between the Delta Valencia, grapefruit, and clementine samples, with greater sequence variation observed with the nucleocapsid protein (NP) compared with the RNA-dependent RNA polymerase (RdRp) and the movement protein (MP). A real-time detection assay, targeting the RdRp, was developed to simultaneously detect citrus-infecting coguviruses, CiVA and CCGaV, using a dual priming reverse primer to improve PCR specificity.

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.

Certain species of Brevipalpus (Trombidiformes: Tenuipalpidae) are able to transmit viruses of the citrus leprosis complex, a serious non-systemic disease of citrus. This disease is endemic to Central and South America, and is characterised by necrotic lesions with chlorotic halos on twigs, leaves and fruit. Citrus leprosis is caused by two types of RNA viruses: cytoplasmic (CL-C) and nuclear (CL-N). In South Africa, CL-N symptoms were detected in 2018 in citrus orchards in the Sundays River Valley (SRV) of the Eastern Cape (Fig 1), and found to be associated with trees testing positive for orchid fleck virus (OFV) (Cook et al. 2019). Mites from this site identified as B. californicus s.l. tested positive for OFV. It is assumed that the entry pathway of OFV virus into South Africa was via the trade of orchid pot plants, and that it was then transmitted to citrus by viruliferous B. californicus. This is the first CL virus-vector association and putative transmission from orchids to citrus identified in South Africa.

BackgroundHigh-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount.MethodsPlant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis.ResultsThe study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols.ConclusionsThis study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

Determination of virus genomes and differentiation of strains and strain variants facilitate the linkage of biological expression to specific genetic units. For effective management of stem pitting disease of citrus tristeza virus (CTV) by cross-protection, an understanding of these links is necessary. The deliberate field application of a biological agent such as a virus first requires a thorough assessment of the long-term impact before it can be applied commercially. Three CTV sources were genetically characterized as different variants of the T68 strain, and their long-term effects on stem pitting and production were investigated. The different CTV sources were inoculated to ‘Star Ruby’ grapefruit trees and evaluated for a number of biological parameters in a field trial in the Limpopo Province of South Africa over a 10-year period. Significant differences were observed in stem pitting severity, impact on tree growth, yield, and the percentage of small fruit produced. These T68 variants were also associated with different stem pitting phenotypes. The variants differed in only 44 nucleotide positions across their genomes, and these minor genetic differences can therefore be used to identify possible genome regions affecting stem pitting.

Two isolates of the T68 genotype of citrus tristeza virus (CTV) were derived from a common source, GFMS12, by single aphid transmission. These isolates, named GFMS12-8 and GFMS12-1.3, induced stem pitting with differing severity in ‘Duncan’ grapefruit (Citrus × paradisi [Macfad.]). Full-genome sequencing of these isolates showed only minor nucleotide sequence differences totaling 45 polymorphisms. Numerous nucleotide changes, in relatively close proximity, were detected in the p33 open reading frame (ORF) and the leader protease domains of ORF1a. This is the first report of full-genome characterization of CTV isolates of a single genotype, derived from the same source, but showing differences in pathogenicity. The results demonstrate the development of intragenotype heterogeneity known to occur with single-stranded RNA viruses. Identification of genetic variability between isolates showing different pathogenicity will enable interrogation of specific genome regions for potential stem pitting determinants.

Citrus tatter leaf virus (CTLV), a strain of apple stem grooving virus, is a virus of citrus that is of commercial importance for trifoliate orange (Poncirus trifoliata) and trifoliate hybrid rootstocks. Bud-union crease is a CTLV-induced incompatibly symptom observed with these rootstocks and citrus scions which results in stunted tree growth. CTLV is a regulated pathogen for most citrus budwood certification programs worldwide. Due to the requisite virus testing, commercially supplied scion budwood would therefore seldom be a point of contamination. However, citrus rootstocks are primarily propagated by seed and the potential for seed transmission of CTLV in citrus is unclear and remains a potential source of infection. A single, unconfirmed report of seed transmission of CTLV in ‘Eureka’ lemon (Citrus × limon) exists, and although this report was not of a rootstock cultivar, it does point to the risk of seed transmission in citrus and close relatives in the family Rutaceae. CTLV-positive ‘Meyer’ lemon (C. × limon) trees in an experimental plot in the Eastern Cape Province of South Africa presented an opportunity to again test seed transmission in citrus. Seed was obtained from the infected ‘Meyer’ lemon trees, and 1164 seedlings were grown in an insect-secure greenhouse and screened for CTLV in pooled batches by conventional and real-time RT-PCR. No positive samples were detected and results indicated a lack of virus seed transmission in ‘Meyer’ lemon. Findings of this study are therefore not supportive of CTLV seed transmission in citrus.

Citrus black spot (CBS), caused by

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 took50 years to identify the causative agent as '

Author(s): Aldrich, Dirk Jacobus; Bester, Rachelle; Cook, Glynnis; du Plessis, Anton; Burger, Johan Theodorus; Maree, Hans Jacob | Abstract: Citrus tristeza virus (CTV) is the most important viral pathogen of citrus. CTV-induced stem pitting negatively impacts grapefruit and sweet orange production. The mechanisms of stem pitting development in CTV-infected citrus remain unclear. This study evaluated the utility of high-resolution CT scanning as a tool to study stem pitting in live citrus material. CT scans were used to easily identify pits based on differences in tissue density. Stem pits were also mapped and modelled three-dimensionally along the length of the stem. Nano-CT scanning proved to be a potentially valuable, non-destructive method for stem pitting characterization in citrus.

Huanglongbing (HLB, Asian Citrus Greening), the most devastating disease of citrus has not been detected in southern Africa (Gottwald, 2010). HLB is associated with 'Candidatus Liberibacter asiaticus' (CLas), a phloem-limited bacterium vectored by Diaphorina citri Kuwayama (Hemiptera: Liviidae), the Asian Citrus Psyllid (ACP). African Citrus Greening, associated with 'Candidatus Liberibacter africanus' (CLaf) and its vector the African Citrus Triozid, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae), are endemic to Africa, although not previously reported from Angola. African Greening is less severe than HLB, largely due to heat sensitivity of CLaf and its vector. Introduction of HLB into southern Africa would be devastating to citrus production in commercial and informal sectors. Concern was raised that CLas or ACP might hae inadvertently been introduced into Angola. In July 2019, a survey was conducted in two citrus nurseries in Luanda and Caxito and in different orchards on 7 farms surrounding Calulo and Quibala. Yellow sticky traps for insects were placed at the various localities and collected after c. 3 weeks. Breeding signs of T. erytreae (pit galls) were observed on citrus in some locations, but no insect vectors were detected on traps. Trees were inspected for signs and symptoms of citrus pests and diseases, particularly those that resemble HLB (foliar blotchy mottle, shoot chlorosis, vein yellowing and corking, lopsided fruit with aborted seeds and colour inversion) and its vectors (pit galls on leaves or waxy exudates). Leaves and shoots with suspect symptoms were sampled for laboratory analysis (43 samples). DNA was extracted from petiole and midrib tissue of leaves using a modified CTAB extraction protocol of Doyle and Doyle (1990). Real-time PCR was done using universal Liberibacter primers of Roberts et al. (2015), CLaf specific primers of Li et al. (2006) and CLas specific primers of Bao et al. (2019). All real-time PCR protocols indicated the presence of CLaf in 6 samples (Tab. S1). CLas or other citrus Liberibacter species were not detected. The presence of CLaf in sample 37 was confirmed by constructing a library (NEXTFLEX® DNA Sequencing Kit, PerkinElmer) with extracted DNA and performing high-throughput sequencing on an Ion Torrent™ S5™ platform (Central Analytical Facility, Stellenbosch University). To improve the quality of the reads, all 233,617,700 obtained reads were trimmed from the 3' end to a maximum length of 240 nt using Trimmomatic (Bolger et al. 2014). The high quality reads were mapped to the Citrus sinensis reference genome (NC_023046.1) using Bowtie 2.3.4 (Langmead and Salzberg 2012) to subtract all the reads that had high identity to the host plant (number of mismatches allowed in the seed was set to 1). The 14,691,369 unmapped reads (6.2% of original data) were mapped to the CLaf reference genome NZ_CP004021.1 using CLC Genomics Workbench 10.1.1 (Qiagen) (Length fraction = 0.8; Similarity fraction = 0.9). A CLaf consensus genome was generated that spanned 99.7% of the reference genome and the 163001 mapped reads had a 22.9 mean read coverage. The consensus sequence was 99.7% identical to NZ_CP004021.1 and was submitted to Genbank as accession: CP054879. The positive CLaf detections were from trees with typical HLB or African Citrus Greening symptoms, viz. lopsided fruit with green stylar ends, aborted seed and stained columella at base of fruit button; yellow shoots with leaves showing symptoms of blotchy mottle and vein yellowing and corking (Fig. S1) in a commercial citrus farm outside Calulo and included 2 'Ponkan' mandarin (C. reticulata), 2 Valencia and 1 'Navelina' tree (C. sinensis), and a citrus nursery in Luanda (1 lime tree; C. aurantifolia) (Tab. S1). This first report of CLaf in Angola highlights the need to prevent spread by removing infected trees and managing the insect vector, as well as the need for further surveys to determine the occurrence of African Greening and its vectors in other provinces and to confirm the absence of exotic citrus pests and diseases. References Bao, M. et al. 2020. Plant Dis. 104:527 Bolger, A. M. et al. 2014. Bioinformatics. 30:2114-2120. Doyle, J.J. and Doyle, J.L. 1990. Focus 12:13 Gottwald, T.R. 2010. Annu. Rev. Phytopathol. 48:119 Langmead, B. and Salzberg, S. 2012. Nature Methods. 9:357-359. Li, W. et al. 2006. Jnl. Microbiol. Methods 66:104 Roberts, R. et al. 2015. Int. J. Syst. Evol. Micr. 65:723.

Citrus (Citrus spp.) production continues to decline in East Africa, particularly in Kenya and Tanzania, the two major producers in the region. This decline is attributed to pests and diseases including infestation by the African citrus triozid, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae). Besides direct feeding damage by adults and immature stages, T. erytreae is the main vector of 'Candidatus Liberibacter africanus', the causative agent of Greening disease in Africa, closely related to Huanglongbing. This study aimed to generate a novel barcode reference library for T. erytreae in order to use DNA barcoding as a rapid tool for accurate identification of the pest to aid phytosanitary measures. Triozid samples were collected from citrus orchards in Kenya, Tanzania, and South Africa and from alternative host plants. Sequences generated from populations in the study showed very low variability within acceptable ranges of species. All samples analyzed were linked to T. erytreae of GenBank accession number KU517195. Phylogeny of samples in this study and other Trioza reference species was inferred using the Maximum Likelihood method. The phylogenetic tree was paraphyletic with two distinct branches. The first branch had two clusters: 1) cluster of all populations analyzed with GenBank accession of T. erytreae and 2) cluster of all the other GenBank accession of Trioza species analyzed except T. incrustata Percy, 2016 (KT588307.1), T. eugeniae Froggatt (KY294637.1), and T. grallata Percy, 2016 (KT588308.1) that occupied the second branch as outgroups forming sister clade relationships. These results were further substantiated with genetic distance values and principal component analyses.

‘Candidatus Liberibacter africanus’ is associated with citrus greening (huanglongbing [HLB]) in South Africa. Various unpublished reports have suggested that the related bacterium ‘Ca. L. asiaticus’ associated with HLB in citrus might be seed transmissible based on real-time PCR results. Seed transmission poses a risk of long distance disease spread, especially with the dissemination of rootstock seed. Therefore, it was essential to determine whether ‘Ca. L. africanus’ is seed transmitted in citrus. Fruit from 26 ‘Ca. L. africanus’-infected branches of six citrus cultivars showing greening symptoms were collected and the seed was harvested. Cultivars included were Minneola tangelo (Citrus reticulata × C. paradisi); sweet oranges (C. sinensis) Premier midseason, Clanor midseason, and Olinda Valencia; Eureka lemon (C. limon) and Troyer citrange (Poncirus trifoliata × C. sinensis) rootstock variety. Branches bearing each fruit were collected and confirmed to contain ‘Ca. L. africanus’ by real-time PCR testing using Taqman probe HLBp and HLBaf and HLBr primers as described by Li et al. (3). The seed of each sample was sorted into five categories ranging from healthy looking to totally aborted based on their appearance before planting. Germination was done in seed trays under vector-free conditions at 24 to 28°C. Thereafter the seedlings were planted in small, plastic bags and monitored for greening-like symptoms or other abnormalities for up to 2 years. A slow-release, balanced fertilizer was applied and supplemented with micro-nutrient sprays for plant maintenance. Plants showing abnormal symptoms were potted into larger pots and closely monitored. These samples and a number of other seedlings showing growth abnormalities were tested for ‘Ca. L. africanus' by real-time PCR as described above. In total, 1,570 seedlings were obtained. Some abnormal symptoms such as small chlorotic leaves, interveinal chlorosis, yellow veins, and stunting were seen in some seedlings. Most symptoms resembled deficiencies, and no blotchy mottle typical of ‘Ca. L. africanus’ infection was noted on any of the seedlings. Abnormal seedlings arose from normal and abortive seed. One hundred and eighteen of these seedlings (8 Minneola tangelo; 24 Premier midseason, 42 Clanor midseason, 33 Olinda Valencia, and 11 Troyer citrange seedlings) were individually tested using real-time PCR for ‘Ca. L. africanus’ detection. These seedlings had germinated from essentially healthy-looking seed (category 1) to seeds with severe abnormalities (category 5) and 33, 24, 23, 30, and 8 seedlings, respectively, were tested from each of the five seed categories. No samples tested positive with real-time PCR based on a positive/negative threshold Cq value of 35. Buds of some seedlings that yielded the lowest Cq values above 35 were grafted onto healthy ‘Madam vinous’ sweet orange (C. sinensis) seedlings and monitored for symptom development for 3 months. No symptoms developed and all these indicators also tested negative for ‘Ca. L. africanus’, indicating the absence of a transmissible agent. Just as other researchers (1,2) have recently indicated a lack of evidence for seed transmission of ‘Ca. L. asiaticus’, no seed transmission of ‘Ca. L. africanus’ could be demonstrated in this experiment either. References: (1) U. Albrecht and K. D. Bowman. HortScience 44:1967, 2009. (2) J. S. Hartung et al. Plant Dis. 94:1200, 2010. (3) W. Li et al. J. Microbiol. Methods 66:104, 2006.

Citrus Viroid IV or Bark cracking viroid (CVd-IV) has been reported from various countries, mostly in the Near East, but was unknown in southern Africa. It can cause severe bark cracking of trifoliate orange (Poncirus trifoliata), but symptoms on the indicator, Arizona 861-S-1 Etrog (Citrus medica), are mild and transient, usually leaf epinasty (2–4). CVd-IV was detected for the first time in South Africa during the 2009 biennial indexing of mother trees maintained for the South African Citrus Improvement Scheme (CIS). Symptoms were observed on Etrog indicators budded with material from mother trees of DuRoi Valencia sweet orange (C. sinensis). Initially, leaf bend of a single leaf was noted on one plant, and later, petiole browning developed on all test plants. Reverse transcription (RT)-PCR testing confirmed CVd-IV infection by amplification of a 286-bp PCR product representing the full-length CVd genome using the primers CVd-IV-F3 5′-GGTGGATACAACTCTTGGG-3′ (1) and CVd-IV-SL3 5′-GGGTAGTTTCTATCTCAG-3′ (N. Duran-Vila personal communication). Samples of 78 field and 30 nursery trees of DuRoi Valencia supplied over a 7-year period from the CIS were budded on Etrog and tested 3 months after inoculation. Ninety positive samples were obtained from trees up to 7 years old. These trees were on either Swingle citrumelo (P. trifoliata × C. paradisi) or rough lemon (C. jambhiri) rootstocks and no symptoms of bark-cracking or other abnormalities were noted on these trees. Following the initial interception, a comprehensive screening of the CIS gene source, consisting of 450 cultivars, was initiated and CVd-IV was detected in five other accessions: SB navel (C. sinensis) collected in 1984 in Kwa-Zulu Natal Province of South Africa, Gillemberg navel (C. sinensis) collected in 1986 in Limpopo Province, Tarocco midseason (C. sinensis) imported from Sicily in 1987, Fortuna mandarin (C. reticulata) imported from Spain in 1987, and Westin midseason (C. sinensis) imported from Brazil in 1994. None of these accessions are planted on a commercial scale and field observations are therefore not possible. PCR amplicons of all positive samples were bidirectionally sequenced and sequences submitted to GenBank. Sequence comparisons showed those obtained from DuRoi Valencia (JN903763), Tarocco (JN903764), and SB Navel (JN903765), and GenBank reference sequences AB054633 (Japan) and HM042747 (China) to be identical. Likewise, the South African sequences from Westin (JN903766) and Gillemberg (JN903767) were identical to reference sequences X14638 and NC_003539 (Israel) and GQ260216 (Iran). The Fortuna sequence (JN903762) differed by two base pairs from the latter group. On the basis of the entry dates of the accessions, it is probable that CVd-IV has been in South Africa since at least 1984, but was not detected during indexing because of the erratic symptom expression of CVd-IV on Etrog. Existing CVd-IV-contaminated field material will be monitored for possible disease expression, but thus far the trifoliate hybrid and rough lemon rootstocks do not show the same susceptibility as reported for P. trifoliata (4). The detection of CVd-IV in field and gene-source material emphasizes the importance of intensive and specific indexing to ensure distribution of pathogen-free citrus in South Africa. References: (1) L. Bernad and N. Duran-Vila. Mol. Cell. Probes 20:105, 2006. (2) N. Duran Vila et al. J. Gen. Virol. 69:3069, 1988. (3) H. Putcha et al. Nucleic Acids Res. 19:6640, 1991. (4) C. Vernière et al. Plant. Dis. 88:1189, 2004.

Citrus tristeza virus (CTV) is endemic to southern Africa and the stem pitting syndrome that it causes was a limiting factor in grapefruit production prior to the introduction of cross-protection in the Citrus Improvement Scheme. This disease mitigation strategy, using various field-derived CTV sources, has significantly extended the productive lifespan of grapefruit orchards in South Africa. CTV commonly occurs as a population of various strains, masking the phenotypic effect of individual strains. Likewise, current South African CTV cross-protection sources are strain mixtures, obscuring an understanding of which strains are influencing cross-protection. The severity of various CTV strains has mostly been assessed on sensitive indicator hosts, but their effect on commercial varieties has seldom been investigated. Single-variant CTV isolates were used to investigate the phenotypic expression of CTV strains in commercial grapefruit varieties as well as CTV indicator hosts. They were biologically characterized for their ability to cause stem pitting and their rate of translocation and titer in the different hosts, monitored by enzyme-linked immunosorbent assay. Complete genome sequences for three CTV strain variants were generated. Isolates of CTV strains VT, T68, RB, and HA16-5 did not induce severe stem pitting in four grapefruit hosts in a glasshouse trial. Viral titers of the strains differed in the grapefruit hosts, but the RB isolate reached a higher titer in the grapefruit hosts compared with the VT, T68, and HA16-5 isolates. Additionally, horticultural assessment of two grapefruit varieties inoculated with the RB isolate in two field trials demonstrated that mild stem pitting did not negatively influence the horticultural performance of the grapefruit trees over an eight-year assessment period. ‘Star Ruby’ trees containing the CTV source GFMS35 showed less stem pitting than trees inoculated with the RB isolate, but had smaller canopy volumes and lower yields than trees containing the RB isolate. This suggests that the influence of CTV sources on tree performance is not limited to the effect of stem pitting.

‘Candidatus Liberibacter asiaticus’, the bacterium associated with citrus Huanglongbing (HLB), was reported from Uganda and tentatively from Tanzania, posing a threat to citriculture in Africa. Two surveys of citrus expressing typical HLB symptoms were conducted in Uganda, Kenya, and Tanzania to verify reports of ‘Ca. L. asiaticus’ and to assess the overall threat of HLB to eastern and southern African citrus production. Samples were analyzed for the presence of ‘Candidatus Liberibacter’ species by real-time PCR and partial sequencing of three housekeeping genes, 16S rDNA, rplJ, and omp. ‘Ca. L. africanus’, the bacterium historically associated with HLB symptoms in Africa, was detected in several samples. However, samples positive in real-time PCR for ‘Ca. L. asiaticus’ were shown not to contain ‘Ca. L. asiaticus’ by sequencing. Sequences obtained from these samples were analogous to ‘Ca. L. africanus subsp. clausenae’, identified from an indigenous Rutaceae species in South Africa, and not to ‘Ca. L. asiaticus’. Results indicate a nontarget amplification of the real-time assay and suggest that previous reports of ‘Ca. L. asiaticus’ from Uganda and Tanzania may be mis-identifications of ‘Ca. L. africanus subsp. clausenae’. This subspecies was additionally detected in individual Diaphorina citri and Trioza erytreae specimens recovered from collection sites. This is the first report of ‘Ca. L. africanus subsp. clausenae’ infecting citrus and being associated with HLB symptoms in this host.

Genotypic characterization of Citrus tristeza virus (CTV) strains has progressed significantly, but their phenotypic expression is poorly established as CTV naturally occurs as mixed-strain populations. A screening system for the analysis of mixed-strain populations is required for population studies and the correlation with symptom expression. In this study, a published CTV strain-specific detection assay was expanded and improved to facilitate detection of currently known CTV strains. Supplementary RT-PCR assays were developed for two variant groups of the RB strain and the HA16-5 strain, and assays for the T36 strain and generic CTV detection were improved. The value of the strain-specific assays was shown by the ability to identify the strain components of two CTV cross-protecting sources, GFMS35 and LMS6, used in the South African budwood certification scheme and to demonstrate the segregation of strains in budwood source trees.

Background Small RNA (sRNA) associated gene regulation has been shown to play a significant role during plant-pathogen interaction. In commercial citrus orchards co-infection of Citrus tristeza virus (CTV) and viroids occur naturally. Methods A next-generation sequencing-based approach was used to study the sRNA and transcriptional response in grapefruit to the co-infection of CTV and Citrus dwarfing viroid. Results The co-infection resulted in a difference in the expression of a number of sRNA species when comparing healthy and infected plants; the majority of these were derived from transcripts processed in a phased manner. Several RNA transcripts were also differentially expressed, including transcripts derived from two genes, predicted to be under the regulation of sRNAs. These genes are involved in plant hormone systems; one in the abscisic acid, and the other in the cytokinin regulatory pathway. Additional analysis of virus- and viroid-derived small-interfering RNAs (siRNAs) showed areas on the pathogen genomes associated with increased siRNA synthesis. Most interestingly, the starting position of the p23 silencing suppressor’s sub-genomic RNA generated a siRNA hotspot on the CTV genome. Conclusions This study showed the involvement of various genes, as well as endogenous and exogenous RNA-derived sRNA species in the plant-defence response. The results highlighted the role of sRNA-directed plant hormone regulation during biotic stress, as well as a counter-response of plants to virus suppressors of RNA-silencing. Electronic supplementary material The online version of this article (10.1186/s12985-017-0871-9) contains supplementary material, which is available to authorized users.

The conservation of plant biosecurity relies on the rapid identification of pathogenic organisms, including viruses. With next-generation sequencing (NGS), it is possible to identify multiple viruses within a metagenomic sample. In this study, we explored the use of electronic probes (e-probes) for the simultaneous detection of 11 recognized citrus viruses. E-probes were designed and screened against raw sequencing data to minimize the bioinformatic processing time required. The e-probes were able to accurately detect their cognate viruses in simulated datasets, without any false negatives or positives. The efficiency of the e-probe-based approach was validated with NGS datasets generated from different RNA preparations: double-stranded RNA (dsRNA) from ‘Mexican’ lime infected with different Citrus tristeza virus (CTV) genotypes, dsRNA from field samples, and small RNA and total RNA from grapefruit infected with the CTV T3 genotype. A set of probes was made available that is able to accurately detect CTV in sequence data regardless of the input dataset or the genotype that plants are infected with.

Biological process based network of genes differentially expressed across both grapefruit varieties. Figure S2. Molecular function based network of genes differentially expressed across both grapefruit varieties. Figure S3. Cellular component based network of genes differentially expressed across both grapefruit varieties. (PDF 2159 kb)

Trioza erytreae (Del Guercio) (Hemiptera: Triozidae), is a minor pest of citrus, but the significance of this triozid is attributed to its ability to vector the African citrus greening disease pathogen, ‘Candidatus’ Liberibacter africanus. The population fluctuation of T. erytreae is correlated with the flushing rhythm of the citrus host. However, seasonal fluctuation in the infection potential of populations carrying the greening pathogen has been difficult to monitor in the past due to limited detection methodologies. This study explored the fluctuation in infectivity in an orchard severely infected by African greening. Two seasons’ data are presented from a small Citrus aurantium (L.) (sour orange) orchard in the Nelspruit district in South Africa. Individual T. erytreae caught weekly on sticky traps placed in the orchard were tested using PCR. Additionally, a T. erytreae outbreak was observed in 2011 in another C. sinensis (L.) Osbeck (sweet orange) orchard which displayed a high incidence of greening also in the Nelspruit district. T. erytreae collected here were used in a challenge study and some transmission data are presented. T. erytreae population fluctuations followed the citrus growth flush cycles and were influenced by specific climatic factors. Fluctuations in the percentage infectivity of the T. erytreae populations were observed, with infectivity peaking with or just after the citrus flush seasons, but with peaks in infectivity differing to peaks in population of the vector. In a challenge study, the percentage pathogen transmission to citrus was much lower than the detected percentage infection of the triozids used for the

Author(s): Steyn, C; Cook, G; Burger, J T; Maree, H J | Abstract: Viroid species identified in citrus induce a range of symptoms in this host as well as in non-citrus hosts. Currently, 7 citrus viroid species are recognized including Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), Citrus bark cracking viroid (CBCVd), Citrus viroid V (CVd-V), Citrus viroid VI (CVd-VI) and Citrus exocortis viroid (CEVd). Cachexia-inducing variants of HSVd and CEVd, which causes exocortis, are considered severe pathogens of citrus, whereas other viroid species induce less severe symptoms such as stunting, either singly or in combination. Some viroid species, such as CDVd, have previously been used to deliberately induce stunting for high density planting of citrus, but studies on the effects of commercial orchards are limited. Research on the effect of viroid species requires the isolation of single viroid species. Biological isolation of single species from naturally infected citrus is challenging since viroids seldom occur as single infections and are often found in combination with various other pathogens. The production of single viroid species clones for in vitro transcription enables the generation of a single viroid species inoculum for research applications and circumvents the need for maintenance of sources in plants. Complete genomes of 7 viroid strains including CBLVd, 2 HSVd variants (CVd-IIa and CVd-IIb), CDVd, CBCVd, CVd-V and CEVd were cloned with a leading T7 promoter sequence to facilitate in vitro transcription. Circularized RNA transcripts were successfully used to transfect ‘Etrog’ citron (Citrus medica) by slash-inoculation that developed typical citrus viroid symptoms.