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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.

Common hollyhock (Alcea rosea) is a ubiquitous ornamental in temperate climates but is highly adaptable and can be found growing in the tropics and subtropics. In 2019, an A. rosea plant showing symptoms of irregular chlorotic flecking on the basal leaves, with symptoms becoming gradually less severe toward the apex, was sampled in Pretoria, Gauteng province, South Africa. Total RNA was used to prepare an RNAtag-seq library, which was sequenced using an Illumina HiSeq 2500 instrument. Subsequent analysis of the data revealed the presence of two bipartite RNA viruses, namely orchid fleck virus (OFV) (segment 1: MW073772; segment 2: MW073773) and sweet potato chlorotic stunt virus (SPCSV) (segment 1: MW073774; segment 2: MW073775). OFV from this study was closely related to a strain from South Africa, associated with citrus leprosis disease, while SPCSV represented a novel strain. RT-PCR and bidirectional Sanger sequencing were used to confirm the presence of both viruses. Further samples were collected in 2020, which showed severe interveinal chlorosis, and were tested with RT-PCR; however only SPCSV was associated with these plants. This is the first time that both viruses have been associated with A. rosea, which should be considered a potential reservoir host of these agriculturally important viruses.

Tomato matilda virus (TMaV) is an iflavirus-like virus that was first reported by Saqib et al. (2015) in symptomless tomato plants in Australia. Those authors demonstrated using phylogenetic analys...

This is the first report of the presence of alstroemeria mosaic virus (AlMV) in South Africa, and describes the second complete genome to date. AlMV was identified from a sample of Alstroemeria pulchella via RNAtag sequencing and RT-PCR. The diversity and distribution of AlMV, as well as the potential threat to the cutflower industry, still needs to be determined.

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.

‘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 Greening disease (CG) in South Africa (SA) is associated with the fastidious bacterium ‘CandidatusLiberibacter africanus’ (Laf). It has been observed that Laf isolates obtained from different geographic localities in SA differed in the rate of transmission during grafting experiments leading to the hypothesis that genetic variation of Laf may exist in this country. To determine this, 167 Laf isolates obtained from Limpopo, North West, Mpumalanga and the Western Cape were subjected to microsatellite analyses, using four polymorphic markers. From UPGMA and STRUCTURE analysis, it was shown that most sources belong to one of two major genetic groups of Laf and these comprise 25 distinct haplotypes. Four samples included within this study did not group with these two major groups, suggesting a potential third and fourth genetic group of Laf being present, which can be validated by further sampling. Results further indicate that Laf populations in SA are formed by geographic locality. The high genetic diversity observed for Laf within this study is consistent with the hypothesis that Laf originated on the African continent, warranting further genetic analysis of Laf populations from Africa. This is the first study to unveil the genetic diversity of Laf.