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An updated real-time multiplex quantitative polymerase chain reaction (qPCR) assay was designed and validated for the simultaneous detection of three ‘ Candidatus Liberibacter species’ (CLsp), ‘ Ca. Liberibacter asiaticus’ (CLas), ‘africanus’ (CLaf), and ‘americanus’ (CLam), associated with the huanglongbing disease of citrus. The multiplex assay was designed based on the qPCR assay published in 2006 by Li et al., considering all available CLsp 16S rRNA gene sequences in GenBank and the MIQE guidelines and workflow for qPCR optimization, which became available after 2006. When using the updated multiplex CLsp qPCR assay compared with singleplex qPCR, no significant increase in quantitative cycle (Cq) values was detected. The specificity and sensitivity of the updated qPCR assay was optimal, and measuring the intra- and interassay variations confirmed the reproducibility and repeatability of the assay. The assay was also successfully used with a large number of diverse samples at independent laboratories in four countries, thus demonstrating its transferability, applicability, practicability, and robustness as different qPCR reaction conditions or instruments had a minor effect on Cq values. This updated multiplex CLsp qPCR assay can be used in a variety of citrus surveys, germplasm, or nursery stock programs that require different pathogen detection tools for their successful operation. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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.

During the 2019 winter wheat season, symptoms of severe chlorosis and stunting were observed on wheat in the irrigation production areas of South Africa. RNAtag-seq data were generated for seven samples from KwaZulu-Natal province and one from Limpopo. Analysis of assembled contigs indicated the presence of a putatively novel member of the genus Tenuivirus, tentatively named "wheat yellows virus" (WhYV). The genome is made up of four segments, which are 8952, 3451, 2338, and 2045 nucleotides in length and code for a total of seven ORFs. Phylogenies of each segment (nucleotide) and the polymerase gene (amino acid), as well as amino acid sequence comparisons of each gene product, showed that WhYV is most closely related to rice stripe virus.

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.

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 '

RNAtag-seq of maize samples collected in Tanzania revealed the presence of a previously undescribed nucleorhabdovirus, tentatively named "Morogoro maize-associated virus" (MMaV), in three samples. The MMaV genome is 12,185-12,187 nucleotides long and shares a 69-70% nucleotide sequence identity with taro vein chlorosis virus. Annotation of the genomes showed a typical nucleorhabdovirus gene organization. PCR was unable to detect the same virus in the remaining 35 samples collected in the region.

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...

Nineteen samples from members of the plant genera Agapanthus, Clivia, Hippeastrum, and Scadoxus were collected from gardens in the Gauteng and Western Cape provinces of South Africa. The plants displayed highly variable symptoms of viral disease, including chlorosis, necrosis, streaking, and ringspot. RNAtag-seq was used to characterize the associated viral populations. Plants of the genus Agapanthus were found to be associated with three novel viruses from the families Caulimoviridae, Closteroviridae, and Betaflexiviridae; plants of the genus Clivia were associated with novel members of the families Potyviridae and Betaflexiviridae; and plants of the genus Scadoxus were associated with a novel member of the family Tospoviridae. Nerine latent virus was associated with plants of the genera Agapanthus, Clivia, and Hippeastrum, while hippeastrum mosaic virus was associated exclusively with a Hippeastrum cultivar.

Barleria cristata L. has become naturalized in South Africa, where it is commonly used as an ornamental. In 2019, plants of B. cristata showing putative viral symptoms were collected from two locations in Gauteng, South Africa. RNAtag-seq libraries were prepared and sequenced using an Illumina HiSeq 2500 platform. De novo assembly of the resulting data revealed the presence of a novel member of the family Tospoviridae associated with the plants from both locations, and this virus was given the tentative name "barleria chlorosis-associated virus". Segments L, M, and S have lengths of 8752, 4760, and 2906 nt, respectively. Additionally, one of the samples was associated with a novel polerovirus, provisionally named "barleria polerovirus 1", with a complete genome length of 6096 nt. This is the first study to show the association of viruses with a member of the genus Barleria.

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.

Typically associated with fungal species, members of the viral family Totiviridae have recently been shown to be associated with plants, including important crop species, such as Carica papaya (papaya) and Zea mays (maize). Maize-associated totivirus (MATV) was first described in China and more recently in Ecuador, where it has been found to co-occur with other viruses known to elicit maize lethal necrosis disease (MLND). In a survey for maize-associated viruses, 35 samples were selected for Illumina HiSeq sequencing, from the Tanzanian maize producing regions of Mara, Arusha, Manyara, Kilimanjaro, Morogoro and Pwani. Libraries were prepared using an RNA-tag-seq methodology. Taxonomic classification of the resulting datasets showed that 6 of the 35 samples from the regions of Arusha, Kilimanjaro, Morogoro and Mara, contained reads that were assigned to MATV reference sequences. This was confirmed with PCR and Sanger sequencing. Read assembly of the six MATV-associated datasets yielded partial MATV genomes, two of which were selected for further characterization, using RACE. This yielded two full-length MATV genomes, one of which is divergent from other available MATV genomes.

‘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.

During the past few years, an unknown disease, which resulted in various degrees of chlorosis, mosaic and streaking of wheat leaves was commonly found in the irrigation wheat growing areas of South Africa (SA). The yellowing symptoms resembled a viral infection but were not consistent with previously reported wheat viruses in SA. The observed symptoms suggested that this disease was likely caused by soil-borne virus, which may be transmitted by soil inhabiting microorganisms such as Polymyxa graminis. Therefore, to identify any virus associated with leaves and determine the presence of P. graminis in roots, wheat plants exhibiting characteristic symptoms of the disease were collected during 2018 from farmers’ fields and/or experimental plots across major wheat growing regions of SA. While the roots of the samples were tested for P. graminis using light microscopy, polymerase chain reaction (PCR) and Sanger sequence analysis, the leaves were tested for the presence of viruses using Next generation sequencing (NGS), with the results being confirmed with reverse transcription (RT) PCR. Combined, the microscopic and PCR results confirmed P. graminis in 96% of the symptomatic samples tested. NGS analysis performed on a subset of 22 symptomatic leaf samples resulted in the identification of wheat stripe mosaic virus (WhSMV) in all the samples tested. RT-PCR confirmed the presence of WhSMV in similar samples suggesting that WhSMV is the most likely cause of the observed yellowing of wheat leaves. Twenty-one of the 22 samples which tested positive for WhSMV were also positive for P. graminis indicating that WhSMV is most probably transmitted by P. graminis. WhSMV is a novel virus that was recently described on wheat in Brazil and Paraguay. To our knowledge, this is the first report of WhSMV in South Africa and the third report in the world. As WhSMV has been detected only recently, information on the epidemiology and control of this disease is not yet available.

Greening disease of citrus in South Africa is associated with ‘Candidatus Liberibacter africanus’ (Laf), a phloem-limited bacterium vectored by the sap-sucking insect Trioza erytreae (Triozidae). Despite the implementation of control strategies, this disease remains problematic, suggesting the existence of reservoir hosts to Laf. The current study aimed to identify such hosts. Samples from 234 trees of Clausena anisata, 289 trees of Vepris lanceolata and 231 trees of Zanthoxylum capense were collected throughout the natural distribution of these trees in South Africa. Total DNA was extracted from samples and tested for the presence of liberibacters by a generic Liberibacter TaqMan real-time PCR assay. Liberibacters present in positive samples were characterized by amplifying and sequencing rplJ, omp and 16S rRNA gene regions. The identity of tree host species from which liberibacter sequences were obtained was verified by sequencing host rbcL genes. Of the trees tested, 33 specimens of Clausena, 17 specimens of Vepris and 10 specimens of Zanthoxylum tested positive for liberibacter. None of the samples contained typical citrus-infecting Laf sequences. Phylogenetic analysis of 16S rRNA gene sequences indicated that the liberibacters obtained from Vepris and Clausena had 16S rRNA gene sequences identical to that of ‘Candidatus Liberibacter africanus subsp. capensis’ (LafC), whereas those from Zanthoxylum species grouped separately. Phylogenetic analysis of the rplJ and omp gene regions revealed unique clusters for liberibacters associated with each tree species. We propose the following names for these novel liberibacters: ‘Candidatus Liberibacter africanus subsp. clausenae’ (LafCl), ‘Candidatus Liberibacter africanus subsp. vepridis’ (LafV) and ‘Candidatus Liberibacter africanus subsp. zanthoxyli’ (LafZ). This study did not find any natural hosts of Laf associated with greening of citrus. While native citrus relatives were shown to be infected with Laf-related liberibacters, nucleotide sequence data suggest that these are not alternative sources of Laf to citrus orchards, per se.