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this reason the previously commercially applied grapefruit cross-protecting source GFMS (grapefruit mildstrain) 12 has been replaced by GFMS 35.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

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

Survey of Citrus tristeza virus (CTV) diversity in pigmented Citrus x paradisi (Macfad.) (Grapefruit) trees in north-western Argentina

Citrus tristeza virus (CTV) is the most severe viral pathogen of citrus and elicits a wide range of devastating disease symptoms. Grapefruit cultivars (Citrus x paradisi) are the most sensitive among citrus to the effects of CTV infections. Grapefruit is an important crop within the north-western Argentine citrus industry; however, production has been affected by CTV stem-pitting. In general, CTV diversity within South America is poorly studied, with data on grapefruit CTV populations being particularly limited. In this study, 50 samples were collected from Star Ruby, Henninger’s Ruby and Ruben Pink cultivars, within the provinces of Tucumán, Salta and Jujuy in north-western Argentina. The CTV p33 gene was PCR amplified and the resulting amplicons sequenced with Sanger sequencing. A subset of these amplicons was sequenced with Illumina MiSeq sequencing. AT-1-like sequences were dominant within the majority of populations, as determined by Sanger sequencing, followed by sequences clustering within the unresolved Kpg3/SP/T3 and RB clades. Sequencing by Illumina MiSeq confirmed this, as well as detecting minor sequence types within the HA 16–5, VT, B165 and A18 clades.The National Research Foundation (South Africa) and Ministerio de Ciencia, Tecnologia e Innovación Productiva (Argentina) via the South Africa-Argentina Bilateral agreement programme.http://link.springer.com/journal/106582019-06-01hj2017Microbiology and Plant Patholog