Characterization of a novel citrus tristeza virus genotype within three cross-protecting source GFMS12 sub-isolates in South Africa by means of Illumina sequencing

Tristeza disease (caused by citrus tristeza virus, CTV) is currently controlled in South Africa by means of cross-protection. In this study, we characterized the CTV populations of three grapefruit mild strain 12 (GFMS12) single-aphid-transmission-derived sub-isolates at the whole-genome level using Illumina sequencing technol­ogy. A novel South African isolate (CT-ZA3, of the T68 genotype) was shown to be the dominant genotype in all GFMS12 sub-isolates tested, along with reads unique to various other genotypes occurring as minor components. Uncertainty remains as to the significance of these minor components.Citrus Research International (CRI)http://link.springer.com/journal/705hb201

Development of a strand-specific RT-PCR to detect the positive sense replicative strand of Soybean blotchy mosaic virus

Soybean blotchy mosaic virus (SbBMV), a plant virus of the genus Cytorhabdovirus is an economically important virus of soybean reported only from the warmer, lower-lying soybean production areas in South Africa. The virus consistently appears in soybean crops annually in spite of the absence of soybean plants in winter. One possible reason for this may be that the virus replicates and hence persists in the SbBMV vector, a leafhopper, Peragallia caboverdensis. RNA viruses with antisense genomes as inferred for SbBMV produce positive sense RNAs as intermediate replicative forms during replication in their hosts, and detection of the positive strand in the plant host or vector is evidence of virus replication. In this study, a positive-strand specific RT-PCR (pss-RT-PCR) was developed to detect the positive RNA strand of SbBMV and validated on nine SbBMV isolates from soybean. The effect of tagged reverse transcription (RT) primers for cDNA synthesis, coupled with PCR using a tag-specific primer, as well as removal of unincorporated RT primers following cDNA synthesis was assessed. The positive RNA strand of SbBMV in infected plants was successfully detected following this protocol. Reverse transcription with forward and unmodified reverse primers confirmed that the assay was not able to detect the genomic sense RNA or self-primed cDNAs, lacking the non-viral tag, respectively. However, Exonuclease I (ExoI) treatment of cDNA was required to eliminate false-positive results during PCR amplification.The Association of African Universities (AAU) and through the National Research Foundation Incentive Grant for Rated Scientists.http://www.elsevier.com/locate/jviromet2019-09-01hj2018Forestry and Agricultural Biotechnology Institute (FABI)Microbiology and Plant Patholog

PCR bias associated with conserved primer binding sites, used to determine genotype diversity within Citrus tristeza virus populations

Citrus tristeza virus (CTV) is present in almost all of the major citrus production areas where it continues to reduce the profitability of citriculture. The accurate characterisation of CTV populations, which are usually made up of a number of disparate strains, requires the use of robust PCR protocols. Mismatches between primers and their corresponding binding sites may introduce primer-associated bias during amplification. The primer-associated bias of four sets of CTV specific primers, targeting the A and F regions and the p33 and p23 genes, were evaluated. This was done through the amplification of defined templates followed by their characterisation using the sequencing of multiple clones, as well as Illumina next generation sequencing. High levels of bias were found to be associated with the primer pairs targeting the A and F regions. The p33 gene primers were found to be biased against two genotypes and suggestions for preventing this apparent bias are discussed. The primer pair targeting the conserved p23 gene was found to have very little associated bias. Primers should undergo rigorous screening before being used to characterize virus populations that are known to exhibit high levels of variation, especially within primer binding sites.Citrus Research International (CRI) and the NRF THRIP.http://www.elsevier.com/locate/jviromet2017-11-30hb2017Microbiology and Plant Patholog

Comparative phylogenomics and multi-gene cluster analyses of the Citrus Huanglongbing (HLB)-associated bacterium Candidatus Liberibacter

<p>Abstract</p> <p>Background</p> <p>Huanglongbing (HLB, previously known as citrus greening), is associated with <it>Candidatus </it>Liberibacter species and is a serious threat to citrus production world-wide. The pathogen is a Gram negative, unculturable, phloem-limited bacterium with limited known genomic information. Expanding the genetic knowledge of this organism may provide better understanding of the pathogen and possibly develop effective strategies for control and management of HLB.</p> <p>Results</p> <p>Here, we report cloning and characterization of an additional 14.7 Kb of new genomic sequences from three different genomic regions of the <it>Candidatus </it>Liberibacter asiaticus (Las). Sequence variation analyses among the available <it>Ca</it>. Liberibacter species sequences as well as the newly cloned 1.5 Kb of <it>rpo</it>B gene from different <it>Ca</it>. Liberibacter strains have identified INDELs and SNPs. Phylogenetic analysis of the deduced protein sequences from the cloned regions characterizes the HLB-associated <it>Candidatus </it>Liberibacter as a new clade in the sub-division of the α-proteobacteria.</p> <p>Conclusion</p> <p>Comparative analyses of the cloned gene regions of <it>Candidatus </it>Liberibacter with members of the order Rhizobiales suggest overall gene structure and order conservation, albeit with minor variations including gene decay due to the identified pseudogenes. The newly cloned gene regions contribute to our understanding of the molecular aspects of genomic evolution of <it>Ca</it>. Liberibacter.</p

Genotypic diversity of citrus tristeza virus within red grapefruit, in a field trial site in South Africa

Grapefruit cultivars (Citrus paradisi Macfad.) are extremely sensitive to Citrus tristeza virus (CTV) infections and are pre-immunized with mild-strain crossprotecting sources not containing components that elicit symptoms such as stempitting and decline, to ensure longer periods of productivity. However, preimmunizing sources often lose their efficiency and for this reason the previously commercially applied grapefruit cross-protecting source GFMS (grapefruit mildstrain) 12 has been replaced by GFMS 35. This study was undertaken to determine the diversity of CTV genotypes within trees that were inoculated with either GFMS 12 or GFMS 35. Samples were collected from a number of different trees of two red grapefruit cultivars (cv. Star Ruby and cv. Flame), planted 10 years prior to sampling in the Malelane production area of South Africa. Reverse transcription-polymerase chain reaction amplification of a 5‘ variable region (A-region) and a 3‘ conserved region (p23 gene) was followed by cloning, sequencing of multiple clones and phylogenetic analyses. The genotypic identities of clones were determined based on their relatedness to reference CTV strains. Sequence types within the VT genotypic group dominated in all of the samples, with T30-like sequence types being a minor component in some populations of the field collected samples. The original preimmunising populations of GFMS 12 and GFMS 35 were characterised on greenhouse maintained plants and compared with the populations exposed to field infections by aphids. While the methodology employed only allows a coarse representation of the genotype composition of the CTV population, this study provides insight into which genotypes of CTV must be incorporated within a mildstrain cross-protecting source within the South African Citrus Improvement Scheme (SACIS).Citrus Research International, Agricultural Research Council-Plant Protection Research Institute and the National Research Foundation-THRIP program.http://link.springer.com/journal/106582016-07-30hb201

Rapid detection of Grapevine leafroll-associated virus type 3 using a reverse transcription loop-mediated amplification method

Grapevine leafroll disease (GLD) is the most important disease of Grapevines in South Africa. Grapevineleafroll-associated virus type 3 (GLRaV-3) has a close association with the disease and is prevalent in SouthAfrican vineyards. GLD can be controlled using a combination of virus-free planting material, systemicinsecticides to control vector populations and removal of infected vines by roguing. Infected vines areidentified each autumn using either symptom display (in red cultivars) or ELISA (in white cultivars). WhileELISA is a simple, reliable means of testing for GLRaV-3, it is time consuming, laborious and insensitiveand a quicker, more sensitive method of detecting GLRaV-3 in the field is needed. A single-tube one-stepreverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay combined with a simpleRNA extraction protocol was developed for the rapid and easy detection of GLRaV-3. Hydroxy naptholblue was included as an indicator and under isothermal conditions at 60◦C the target viral gene couldbe amplified in under 2 h and positive results could be easily seen by examining the colour change fromviolet to sky blue. Using this method, 50 samples could be also pooled together with a single positivesample still being detected. A direct comparison of ELISA, nested PCR and RT-LAMP showed that RT-LAMPis as sensitive as nested PCR and could be performed in a much shorter time with less equipment. Thisassay is may be a possible alternative to ELISA for the detection of GLRaV-3 in the field.http://www.elsevier.com/locate/jviromethb201

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

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

Widespread occurrence of "Candidatus liberibacter africanus subspecies capensis" in Calodendrum capense in South Africa

Recent studies in citrus orchards confirmed that Citrus Greening, a heat sensitive citrus disease, similar to Huanglongbing (HLB), is associated with the presence of “Candidatus Liberibacter africanus” (Laf) in South Africa. Neither “Candidatus Liberibacter asiaticus” (Las), associated with HLB, “Candidatus Liberibacter americanus”, nor “Candidatus Liberibacter africanus ssp. capensis” (LafC), previously detected in the Western Cape, South Africa on an indigenous Rutaceous species, Calodendrum capense (L. f.) Thunb. (Cape Chestnut), were detected in citrus. The current study aims to determine the potential role of C. capense in the epidemiology of Citrus Greening in South Africa and whether LafC poses a risk to citriculture. A total of 278C. capense samples were collected throughout South Africa and tested for Liberibacters using realtime PCR. While LafC was found in 100 samples, distributed from all areas where collected, no evidence of Laf infection in any sample was found . The identity of the LafC present was confirmed by sequencing the amplicon derived from conventional PCR of the ßoperon of the ribosomal protein gene region of the first 17 infected trees found and of a representative sample from each district. The Liberibacter status of 44C. capense and 272 citrus (Midnight Valencia) trees growing in close proximity to each other for over 15 years was determined. Out of 44C. capense specimens, 43 were infected with LafC, but none of the citrus trees were infected with LafC. Based on the results of this it appears that natural spread of LafC to citrus does not occur.Citrus Research International (CRI), Nelspruithttp://www.springerlink.com /content/100265