Background High-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. Methods Plant 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. Results The 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. Conclusions This 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.

‘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.

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.

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