A novel subspecies of 'Candidatus Liberibacter africanus' found on native Teclea gerrardii (Family : Rutaceae) from South Africa

The phloem limited bacterium „Candidatus Liberibacter africanus‟ is associated with citrus greening disease in South Africa. This bacterium has been identified solely from commercial citrus in Africa and the Mascarene islands, and its origin may lie within an indigenous rutaceous host from Africa. Recently, in determining whether alternative hosts of Laf exist amongst the indigenous rutaceous hosts of its triozid vector, Trioza erytreae, three novel subspecies of Laf were identified i.e. „Candidatus Liberibacter africanus subsp. clausenae‟, „Candidatus Liberibacter africanus subsp. vepridis‟ and „Candidatus Liberibacter africanus subsp. zanthoxyli‟ in addition to the formerly identified „Candidatus Liberibacter africanus subsp. capensis‟. The current study expands upon the range of indigenous rutaceous tree species tested for liberibacters closely related to Laf and its subspecies. A collection of 121 samples of Teclea and Oricia species were sampled from Oribi Gorge and Umtamvunu nature reserves in KwaZulu Natal. Total DNA was extracted and the presence of liberibacters from these samples determined using a generic liberibacter TaqMan real-time PCR assay. Liberibacters from positive samples were further characterised through amplification and sequencing of the 16S rRNA, outer-membrane protein (omp) and 50S ribosomal protein L10 (rplJ) genes. A single Teclea gerrardii specimen tested positive for a liberibacter and, through phylogenetic analyses of the three genes sequenced, was shown to be unique, albeit closely related to „Ca. L. africanus‟ and „Ca. L. africanus subsp. zanthoxyli‟. We propose that this newly identified liberibacter be named „Candidatus Liberibacter africanus subsp. tecleae‟.Citrus Research International (CRI), the National Research Foundation-THRIP program (NRF-THRIP) and the Department of Science and Technology (DST)/NRF Centre of Excellence in Tree Health Biotechnology (CTHB).http://link.springer.com/journal/104822018-03-31hb2017Microbiology and Plant Patholog

First report of the detection of Bean yellow mosaic virus (BYMV) on Tropaeolum majus ; Hippeastrum spp. and Liatris spp. in South Africa

The potyvirus, Bean yellow mosaic virus (BYMV) is an economically important plant virus which infects many leguminous crops (family Fabaceae) as well as members of the Liliaceae. BYMV has been detected in South Africa on Freesia spp., Gladiolus hortulanus, Lathyrus odoratus, Lupinus albus, Viola odoratus (Gorter, 1977) and Pisum sativum (Jooste et al., 2001), but few further studies have been conducted on this virus locally. During the current study, a RT-PCR capable of generic detection of potyviruses (Zheng et al., 2010) was utilised to detect these viruses from plant samples submitted by growers and previously shown to contain potyvirus-like flexuous rod-shaped particles by electron microscopy.http://apsjournals.apsnet.org/loi/pdishb2017Microbiology and Plant Patholog

Genetic diversity of ‘Candidatus Liberibacter africanus’ in South Africa based on microsatellite markers

Citrus Greening disease (CG) in South Africa (SA) is associated with the fastidious bacterium ‘Candidatus Liberibacter 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.Citrus Research International and the European Union’s Horizon 2020 research and innovation programme.https://link.springer.com/journal/10658hj2021BiochemistryGeneticsMicrobiology and Plant Patholog

Three novel lineages of 'Candidatus Liberibacter africanus' associated with native rutaceous hosts of Trioza erytreae in South Africa

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.Citrus Research International (CRI), the National Research Foundation (NRF) and the Department of Science and Technology (DST)/NRF Centre of Excellence in Tree Health Biotechnology (CTHB).http://ijs.sgmjournals.orghb2016Forestry and Agricultural Biotechnology Institute (FABI)Microbiology and Plant Patholog

Genomic characterization of two novel viruses infecting Barleria cristata L. from the genera Orthotospovirus and Polerovirus

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.SUPPLEMENTARY INFORMATION : Supplementary Figure 1: Foliar symptoms associated with Barleria cristata plants that were sampled in this study. Large, diffuse chlorotic spots were associated with the single infection of barleria severe mosaic virus (BSMoV) (19-3031), while a more defined mosaic was associated with the mixed involving both BSMoV and barleria polerovirus 1 (19-3037).Supplementary Figure 2: Maximum likelihood phylogeny based on the amino acid sequences of the N-protein of barleria chlorosis-associated virus (indicated by solid circle markers) and selected members of the Tospoviridae family. The phylogeny represents the tree with the highest log likelihood and was generated in MEGA X using the best-fit (Le Gascuel) model with gamma distribution (n=4). Bootstrapping was applied (1000 replicates) and the percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap percentages lower than 50 are not shown. The cognate amino acid sequence of Guaroa virus was used as an outgroup.Supplementary Figure 3: Maximum likelihood phylogeny based on the amino acid sequences of the RNA-dependant RNA polymerase of barleria polero virus 1 (indicated by solid circle markers) and selected members of the Luteoviridae family. The phylogeny represents the tree with the highest log likelihood and was generated in MEGA X using the best-fit (Jones-Taylor-Thornton) model. Bootstrapping was applied (1000 replicates) and the percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap percentages lower than 50 are not shown. The cognate amino acid sequences of two enamoviruses were used as outgroups.http://link.springer.com/journal/7052022-07-01hj2022Forestry and Agricultural Biotechnology Institute (FABI

Genetic and Biological diversity of 'candadatus liberibacters' from South Africa

Citrus greening disease (CG) in South Africa is associated with the phloem-limited bacterium, ‘Candidatus Liberibacter africanus’ (Laf). This disease has been known to cause yellowing leaf symptoms as well as the formation of unprofitable fruit for nearly a century in this country. In addition to Laf, Liberibacters have been described from indigenous trees belonging to the Rutaceae family in this country. The first of these to be described was ‘Ca. L. africanus subsp. capensis’ and was followed by the description of ‘Ca. L. africanus subsp. clausenae’ (LafCl), ‘Ca. L. africanus subsp. vepridis’ (LafV) and ‘Ca. L. africanus subsp. zanthoxyli’ (LafZ). It has been speculated that either one of these Laf-subspecies may have given rise to Laf senso stricto associated with commercial citrus in South Africa. This dissertation aimed to expand on the indigenous rutaceous host species evaluated for the presence of Laf either as alternative hosts, or harbouring close relatives of Laf. From Oricia, Teclea and Agathosma spp sampled, a novel Liberibacter was only described from a Teclea gerrardii collected in Southern KwaZulu-Natal. This Liberibacter was named ‘Ca. L. africanus subsp. tecleae (LafT)’. It was further assessed whether any of these Laf-subspecies infect commercial citrus species in South Africa. Previous epidemiological studies on CG in South Africa found that Laf was the sole agent associated with this disease, with the current study supporting these findings. Finally, the genetic diversity of Laf was assessed across four provinces from different citrus types using microsatellite markers. This analysis indicated that the genetic diversity of Laf in South Africa is comparatively high, and that the genetic populations observed were mainly influenced by geographic distribution rather than citrus type. This study gave support to the hypothesis that Laf originated on the African continent from an indigenous species present in the country. The study further supports that Laf is well adapted to its commercial citrus host as genetically distinct populations are formed based on geographical populations.Thesis (PhD)--University of Pretoria, 2019.Microbiology and Plant PathologyPhDUnrestricte

Orchid fleck virus and a novel strain of sweet potato chlorotic stunt virus associated with an ornamental cultivar of Alcea rosea L. in South Africa

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.Supplementary figure 1: Images of leaves collected from Alcea rosea plants expressing symptoms suspected of being of viral infection. B - Basal leaves; M - Leaves collected from the midpoint of the main stem; A – Leaves collected from the apex of each plant.Supplementary figure 2: Agarose gel image showing the bands of PCR confirmation products for orchid fleck virus (OFV) and sweet potato chlorotic stunt virus (SPCSV).The National Research Foundation of South Africahttps://link.springer.com/journal/10658hj2022Forestry and Agricultural Biotechnology Institute (FABI

Novel viruses associated with plants of the family Amaryllidaceae in South Africa

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.SUPPLEMENTARY INFORMATION : Supplementary Fig. 1: Foliar symptoms associated with each plant sampled in this study. The unique sample accession number is shown in the bottom right-hand corner of each image. Images A to L show the Agapanthus cultivars sampled, M to Q the Clivia cultivars, R is a cultivar of Hippeastrum and S shows the single plant of Scadoxus puniceus that was sampled.Supplementary Fig. 2: Alignment of the minus-strand priming site of Agapanthus tungro virus (AgTV) with whole genome sequences of Rice tungro bacilliform virus (RTBV).Supplementary Figure 3.1: Maximum likelihood phylogeny based on the complete nucleotide sequences of Agapanthus tungro virus variants (sources from this study indicated by a solid circle marker) and selected members of the Caulimoviridae family. The phylogeny represents the tree with the highest log likelihood and was generated in MEGA X using the General Time Reversible model with gamma distribution (n=5). Bootstrapping was applied (1000 replicates) and the percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap percentages lower than 50 are not shown. The viral genus corresponding to each reference is shown in brackets, while the sample number is indicated for samples related to this study. Supplementary Figure 3.2: Maximum likelihood phylogeny based on the amino acid sequences of the heat shock protein homolog of Agapanthus velarivirus variants (sources from this study indicated by solid circle markers) and selected members of the Closteroviridae family. The phylogeny represents the tree with the highest log likelihood and was generated in MEGA X using the Le Gascuel model with gamma distribution (n=5). Bootstrapping was applied (1000 replicates) and the percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap percentages lower than 50 are not shown. The viral genus corresponding to each reference is shown in brackets, while the sample number is indicated for samples related to this study. A heat shock protein 70 amino acid sequence of Arabidopsis thaliana was used as an outgroup. Supplementary Figure 3.3: Maximum likelihood phylogeny based on the amino acid sequences of the replicase gene of members of the Betaflexiviridae family found in this study (sources from this study indicated by solid circle markers) and selected members of the same family. These include the nerine latent virus, well as novel viruses, clivia carlavirus A and agapanthus virus A, as well as. The phylogeny represents the tree with the highest log likelihood and was generated in MEGA X using the Le Gascuel model with gamma distribution (n=5). All positions containing gaps and missing data were eliminated (complete deletion option). Bootstrapping was applied (1000 replicates) and the percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap percentages lower than 50 are not shown. The viral genus corresponding to each reference is shown in brackets, while the sample number is indicated for samples related to this study. Supplementary figure 3.4: Maximum likelihood phylogeny based on the amino acid sequences of the polyprotein of Hippeastrum mosaic virus and the novel clivia yellow stripe virus strains (sources from this study indicated by solid circle markers) and selected members of the Potyvirus genus. The phylogeny represents the tree with the highest log likelihood and was generated in MEGA X using the Le Gascuel model. All positions containing gaps and missing data were eliminated (complete deletion option). Bootstrapping was applied (1000 replicates) and the percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap percentages lower than 50 are not shown. Representatives from other genera of the Potyviridae family form the outgroup, with the genus name corresponding to each genus shown in brackets. Supplementary figure 3.5: Maximum likelihood phylogeny based on the amino acid sequences of the produ ct of the N gene of scadoxus chlorotic ringspot virus (sources from this study indicated by a solid circle marker) and selected members of the Orthotospovirus genus. The phylogeny represents the tree with the highest log likelihood and was generated in MEGA X using the Le Gascuel model with gamma distribution (n=5). All positions were used in the analysis. Bootstrapping was applied (1000 replicates) and the percentage of trees in which the associated taxa clustered together is shown next to the branches. Bootstrap percentages lower than 50 are not shown.Supplementary table 1: The pre- and post-assembly read numbers associated with each sample. The number of contigs exceeding 1000bp, for both the CLC Genomics Workbench and metaSPAdes assemblies, are listed. The datasets are linked to sample specific NCBI Biosample accession numbers.Supplementary Table 2: Primers used in all PCR, RT-PCR and RACE assays. The genomic target region, melting temperature (Tm) and product size (RT-PCR) are listed. Key: CP – Coat protein; Pol – Polyprotein; AgTV – Agapanthus tungro virus; AgVV – Agapanthus velarivirus; AgVA – Agapanthus virus A; AgCVB – Agapanthus carlavirus B; ClCVA – Clivia carlavirus A; CYSV – Clivia yellow stripe virus, HiMV – Hippeastrum mosaic virus; NeLV – Nerine latent virus; ScSRV – Scadoxus chlorotic ringspot virus; GSP – Gene specific primer; -F – Forward; -R – Reverse.Supplementary Table 3: List of viruses from this study with their associated sample and NCBI GenBank accession numbers. Genome length, assembling software used for the specific genome’s assembly, the number of reads involved in the assembly, as well as the average coverage and the percentage of total reads, are also presented. AgTV – Agapanthus tungro virus; AgVV – Agapanthus velarivirus, AgVA – Agapanthus virus A, ClCVA – Clivia carlavirus A, NeLV – Nerine latent virus, HiMV – Hippeastrum mosaic virus, ScCRV – Scadoxus chlorotic ringspot virus.http://link.springer.com/journal/7052022-07-19hj2022Forestry and Agricultural Biotechnology Institute (FABI

Diversity and distribution of Maize-associated totivirus strains from Tanzania.

Research Article published by SpringerTypically 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

Molecular characterization of Morogoro maize-associated virus, a nucleorhabdovirus detected in maize (Zea mays) in Tanzania.

Research Article published by Springer ViennaRNAtag-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