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

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this reason the previously commercially applied grapefruit cross-protecting source GFMS (grapefruit mildstrain) 12 has been replaced by GFMS 35.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

On the chromatic roots of generalized theta graphs

The generalized theta graph \Theta_{s_1,...,s_k} consists of a pair of endvertices joined by k internally disjoint paths of lengths s_1,...,s_k \ge 1. We prove that the roots of the chromatic polynomial $pi(\Theta_{s_1,...,s_k},z) of a k-ary generalized theta graph all lie in the disc |z-1| \le [1 + o(1)] k/\log k, uniformly in the path lengths s_i. Moreover, we prove that \Theta_{2,...,2} \simeq K_{2,k} indeed has a chromatic root of modulus [1 + o(1)] k/\log k. Finally, for k \le 8 we prove that the generalized theta graph with a chromatic root that maximizes |z-1| is the one with all path lengths equal to 2; we conjecture that this holds for all k.Comment: LaTex2e, 25 pages including 2 figure

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

Truthmakers and modality

This paper attempts to locate, within an actualist ontology, truthmakers for modal truths: truths of the form or . In section 1 I motivate the demand for substantial truthmakers for modal truths. In section 2 I criticise Armstrong’s account of truthmakers for modal truths. In section 3 I examine essentialism and defend an account of what makes essentialist attributions true, but I argue that this does not solve the problem of modal truth in general. In section 4 I discuss, and dismiss, a theistic account of the source of modal truth proposed by Alexander Pruss. In section 5 I offer a means of (dis)solving the problem

Fungal viruses unveiled : a comprehensive review of my coviruses

DATA AVAILABILITY: No new data was created or analyzed in this study. Data sharing is not applicable to this article.Mycoviruses (viruses of fungi) are ubiquitous throughout the fungal kingdom and are currently classified into 23 viral families and the genus botybirnavirus by the International Committee on the Taxonomy of Viruses (ICTV). The primary focus of mycoviral research has been on mycoviruses that infect plant pathogenic fungi, due to the ability of some to reduce the virulence of their host and thus act as potential biocontrol against these fungi. However, mycoviruses lack extracellular transmission mechanisms and rely on intercellular transmission through the hyphal anastomosis, which impedes successful transmission between different fungal strains. This review provides a comprehensive overview of mycoviruses, including their origins, host range, taxonomic classification into families, effects on their fungal counterparts, and the techniques employed in their discovery. The application of mycoviruses as biocontrol agents of plant pathogenic fungi is also discussed.National Research Foundation of South Africahttps://www.mdpi.com/journal/virusesBiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Diversity of viroids infecting grapevines in the South African Vitis germplasm collection

DATA AVAILABILITY : All sequence data generated or analysed during this study have been Submitted to Genbank under Accession numbers provided in the supplementary information files (Table 6).Seven viroid species and one putative viroid species have been reported to infect grapevine namely, hop stunt viroid (HSVd), grapevine yellow speckle viroid 1 (GYSVd-1), grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd), Japanese grapevine viroid (JGVd), grapevine latent viroid (GLVd), and citrus exocortis viroid (CEVd), as well as a grapevine hammerhead viroid-like RNA (GHVd), so far. In this study, RNA sequence (RNA-Seq) data, from 229 Vitis accessions from the field-maintained vineyard of the South African Vitis germplasm collection, were analysed to determine the diversity of the viroids present. Five of the seven known grapevine-infecting viroids and one putative grapevine-infecting viroid species were very commonly found, with 214 of the 229 samples containing at least one viroid species. HSVd, GYSVd-1, GYSVd-2, AGVd, and JGVd, as well as GHVd, were identified in the RNA-Seq data of the samples and confirmed using RT-PCR and Sanger sequencing. The HSVd sequences indicated the presence of two variants, with one showing multiple nucleotide insertions. AGVd and GYSVd-2 did not display significant sequence diversity, confirming past international studies. GYSVd-1 occurs as four major variants worldwide and representatives of all four variants were identified in this vineyard. This is the first report on the diversity of viroids infecting grapevine in South Africa and the first report of JGVd outside of Japan and GHVd in South Africa. Further studies are needed to fully assess the population and to identify potentially new viroid species.http://link.springer.com/journal/11262hj2024Forestry and Agricultural Biotechnology Institute (FABI)SDG-02:Zero Hunge

Metaviromic characterization of betaflexivirus populations associated with a Vitis cultivar collection in South Africa

DATA AVAILABILITY STATEMENT : All RNAseq datasets are available at National Center for Biotechnology Information’s (NCBI) Sequence Read Archive (SRA), accession PRJNA626577. All assembled sequences have been submitted to NCBI GenBank, listed in Table S1.SUPPLEMENTARY MATERIALS : FIGURE S1: RNA-dependent RNA polymerase (RdRP) gene phylogeny of GRSPaV variants; FIGURE S2: pairwise ANI values shared between GRSPaV variants; FIGURE S3: RNA-dependent RNA polymerase (RdRP) gene phylogeny of GVA variants; FIGURE S4: pairwise ANI values shared between GVA variants; FIGURE S5: RNA-dependent RNA polymerase (RdRP) gene phylogeny of GVB variants; FIGURE S6: pairwise ANI values shared between GVB variants; FIGURE S7: RNA-dependent RNA polymerase (RdRP) gene phylogeny of GVE variants; FIGURE S8: pairwise ANI values shared between GVE variants; FIGURE S9: RNA-dependent RNA polymerase (RdRP) gene phylogeny of GVF variants; FIGURE S10: pairwise ANI values shared between GVF variants; FIGURE S11: RNA-dependent RNA polymerase (RdRP) gene phylogeny of GVH variants; FIGURE S12: pairwise ANI values shared between GVH variants; FIGURE S13: RNA-dependent RNA polymerase (RdRP) gene phylogeny of GVI variants; FIGURE S14: pairwise ANI values shared between GVI variants; FIGURE S15: RNA-dependent RNA polymerase (RdRP) gene phylogeny of GVM variants; FIGURE S16: pairwise ANI values shared between GVM variants; TABLE S1: sample information, number of reads, and BioSample and GenBank accession numbers for each accession; TABLE S2: mixed infections for cultivar accessions with more than one betaflexivirus present.South Africa is associated with a centuries-old viticultural industry, accompanied by a diverse range of wine and table grape cultivars and an extensive history of pervasive introductions of vine material and associated viruses. The Vitis D2 collection in Stellenbosch represents the most comprehensive collection of Vitis species, hybrids, and cultivars in South Africa. We collected leaf petiole material from 229 accessions from this collection. Our metaviromic analyses revealed a total of 406 complete/near complete genomes of various betaflexiviruses. Among these, we identified the presence of grapevine rupestris stem pitting-associated virus and grapevine viruses A, B, E, F, H (GVH), I (GVI), and M (GVM). Notably, this study marks the first report of GVH, GVI, and GVM in South Africa, which were confirmed via RT-PCR. This research significantly contributes to our understanding of viral diversity and introductions in South African viticulture and emphasizes the need for vigilant monitoring and management of viral infections. Our findings lay the groundwork for strategies that mitigate the impact of viruses on South Africa’s wine industry, which generates an annual revenue of approximately 500 million USD.National Research Foundation (NRF) of South Africa.https://www.mdpi.com/journal/virusesam2024BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologySDG-15:Life on lan

The perceived barriers to the inclusion of rainwater harvesting systems by UK house building companies

This work investigates the barriers that exist to deter the implementation of rainwater harvesting into new UK housing. A postal questionnaire was sent to a selection of large, medium and small house-builders distributed across the UK. Questions were asked concerning potential barriers to the inclusion of rainwater harvesting in homes separated into five sections; (1) institutional and regulatory gaps, (2) economic and financial constraints, (3) absence of incentives, (4) lack of information and technical knowledge, and (5) house-builder attitudes. The study concludes that although the knowledge of rainwater systems has increased these barriers are deterring house-builders from installing rainwater harvesting systems in new homes. It is further acknowledged that the implementation of rainwater harvesting will continue to be limited whilst these barriers remain and unless resolved, rainwater harvesting's potential to reduce the consumption of potable water in houses will continue to be limited