South African Isolates Research Articles

Genome Analysis and Genetic Stability of the Cryptophlebia leucotreta Granulovirus (CrleGV-SA) after 15 Years of Commercial Use as a Biopesticide.

Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) is an indigenous pest in southern Africa which attacks citrus fruits and other crops. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme incorporating the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV-SA) as a biopesticide has been implemented. This study investigated the genetic stability of a commercially produced CrleGV-SA product that has been applied in the field since 2000. Seven representative full-genome sequences of the CrleGV-SA isolate spanning a 15-year period were generated and compared with one another. Several open reading frames (ORFs) were identified to have acquired single nucleotide polymorphisms (SNPs) during the 15-year period, with three patterns observed and referred to as “stable”, “reversion”, and “unstable switching”. Three insertion events were also identified, two of which occurred within ORFs. Pairwise multiple alignments of these sequences showed an identity ranging from 99.98% to 99.99%. Concentration-response bioassays comparing samples of CrleGV-SA from 2000 and 2015 showed an increase in virulence toward neonate T. leucotreta larvae. The CrleGV-SA genome sequence generated from the 2015 sample was compared to the Cape Verde reference genome, CrleGV-CV3. Several fusion events were identified between ORFs within these genomes. These sequences shared 96.7% pairwise identity, confirming that CrleGV-SA is a genetically distinct isolate. The results of this study indicate that the genome of CrleGV-SA has remained stable over many years, with implications for its continued use as a biopesticide in the field. Furthermore, the study describes the first complete baculovirus genome to be sequenced with the MinION (Oxford Nanopore, Oxford, UK) platform and the first complete genome sequence of the South African CrleGV isolate.

Comparison of a South African and Canadian Isolate of the Nucleopolyhedrosis Virus Infecting the InsectTrichoplusia ni

The nucleotide and amino acid sequence of the ecdysteroid UDP-glucosyltransferase (egt), polyhedrin (pol), inhibitor of apoptosis (iap2 and iap3), late expression factor (lef) 9 and vp1054 genes of a South African isolate of Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) was compared to a Canadian isolate of TnSNPV. Nucleotide and amino acid sequence identity for the compared genes ranged from 89%to 98%and 95%to 99%, respectively. The deduced amino acid sequences of the TnSNPV egt, pol, iap3, lef-9 and vp1054 genes were used to infer phylogenetic trees and these were compared to the tree inferred from combined data sets consisting of the amino acid sequences of polyhedrin/granulin, lef-8 and lef-9 genes of 48 completely sequenced baculoviruses. The topologies of trees for the baculovirus core genes pol, lef-9 and vp1054 were better resolved than that of the auxiliary genes iap2, iap3 and egt when compared to the tree inferred from complete genome sequences. Sequence and phylogenetic analysis confirm...

Virulence Diversity of Phakopsora pachyrhizi Isolates From East Africa Compared to a Geographically Diverse Collection.

Soybean rust, caused by the biotrophic pathogen Phakopsora pachyrhizi, is a highly destructive disease causing substantial yield losses in many soybean producing regions throughout the world. Knowledge about P. pachyrhizi virulence is needed to guide development and deployment of soybean germplasm with durable resistance against all pathogen populations. To assess the virulence diversity of P. pachyrhizi, 25 isolates from eight countries, including 17 isolates from Africa, were characterized on 11 soybean genotypes serving as differentials. All the isolates induced tan lesions with abundant sporulation on genotypes without any known resistance genes and on soybean genotypes with resistance genes Rpp4 and Rpp5b. The most durable gene was Rpp2, where 96% of the isolates induced reddish brown lesions with little or no sporulation. Of the African isolates tested, the South African isolate was the most virulent, whereas those from Kenya, Malawi, and some of the isolates from Tanzania had the lowest virulence. An Argentinian isolate was virulent on most host differentials, including two cultivars carrying multiple resistance genes. Ten distinct pathotypes were identified, four of which comprised the African isolates representing considerable P. pachyrhizi virulence. Soybean genotypes carrying Rpp1b, Rpp2, Rpp3, and Rpp5 resistance genes and cultivars Hyuuga and UG5 were observed to be resistant against most of the African isolates and therefore may be useful for soybean-breeding programs in Africa or elsewhere.

Occurrence and characterization of mcr-1-harbouring Escherichia coli isolated from pigs in Great Britain from 2013 to 2015.

To determine the occurrence of mcr-1 -harbouring Escherichia coli in archived pig material originating in Great Britain (GB) from 2013 to 2015 and characterize mcr-1 plasmids. Enrichment and selective culture of 387 archived porcine caecal contents and recovery from archive of 1109 E. coli isolates to identify colistin-resistant bacteria by testing for the presence of mcr-1 by PCR and RT-PCR. mcr-1 -harbouring E. coli were characterized by WGS and compared with other available mcr-1 WGS. Using selective isolation following enrichment, the occurrence of mcr-1 E. coli in caeca from healthy pigs at slaughter from unique farms in GB was 0.6% (95% CI 0%-1.5%) in 2015. mcr-1 E. coli were also detected in isolates from two porcine veterinary diagnostic submissions in 2015. All isolates prior to 2015 were negative. WGS analysis of the four mcr-1 -positive E. coli indicated no other antimicrobial resistance (AMR) genes were linked to mcr-1 -plasmid-bearing contigs, despite all harbouring multiple AMR genes. The sequence similarity between mcr-1 -plasmid-bearing contigs identified and those found in GB, Chinese and South African human isolates and Danish, French and Estonian livestock-associated isolates was 90%-99%. mcr-1- harbouring plasmids were diverse, implying transposable elements are involved in mcr-1 transmission in GB. The low number of mcr-1 -positive E. coli isolates identified suggested mcr-1 is currently uncommon in E. coli from pigs within GB. The high sequence similarity between mcr-1 plasmid draft genomes identified in pig E. coli and plasmids found in human and livestock-associated isolates globally requires further investigation to understand the full implications.

Genetic characterization of canine parvovirus type 2 subtypes in Maputo, Mozambique.

Canine parvovirus type 2 (CPV-2) comprises three antigenic subtypes (2a, 2b and 2c) that have been reported in many countries. These subtypes cause serious disease in dogs with characteristic gastroenteritis signs. Little information has been documented in Africa about the genetic characterization of CPV-2. The aim of this study was to detect and to characterize the CPV-2 subtypes circulating in dogs admitted to Veterinary Clinics from two cities of Mozambique, Maputo and Matola, in 2010. A total of 40 field fecal samples were collected and tested for CPV-2 by polymerase chain reaction assay. The partial length VP2 gene of the positive samples were sequenced and genetically analyzed. Twenty-six (65%) fecal samples were positive for CPV-2. The restriction fragment length polymorphism analysis was also performed from positive samples and did not reveal the presence of CPV-2c subtype. The results of the sequencing revealed the presence of CPV-2a (n=9) and CPV-2b (n=17). No CPV-2 and CPV-2c were detected. Sequence analysis comparison showed nucleotide identities of 99.6-100% among our CPV-2 isolates. Amino acid analysis showed predicted amino acid changes. Phylogenetically, all of the CPV-2a strains isolated formed a cluster together with South African and Nigerian isolates. Most of Mozambican CPV-2b isolates also tended to cluster together with South African isolates; however, four were more closely related to French strain and one isolates to the American strain. The present study was the first to characterize the CPV-2 circulating in the Mozambican dog population.

Pepo aphid-borne yellows virus: a new species in the genus Polerovirus.

Pepo aphid-borne yellows virus (PABYV) has been proposed as a putative representative of a new species in the genus Polerovirus in the family Luteoviridae. The genomes of two South African (SA) isolates of cucurbit-infecting PABYV were described in this record. Total RNA, extracted from a pattypan (Cucurbita pepo L.) and a baby marrow (C. pepo L.) leaf samples, was subjected to next-generation sequencing (NGS) on the HiSeq Illumina platform. Sanger sequencing was subsequently used to authenticate the integrity of PABYV's genome generated from de novo assembly of the NGS data. PABYV genome of SA isolates consists of 5813 nucleotides and displays an organisation typical of poleroviruses. Genome sequence comparisons of the SA PABYV isolates to other poleroviruses support the classification of PABYV as a new species in the genus Polerovirus. Recombination analyses showed that PABYV and Cucurbit aphid-borne yellows virus (CABYV) shared the same ancestor for the genome part situated between breaking points. Phylogenetic analyses of the RNA-dependent RNA polymerase and the coat protein genes showed that SA PABYV isolates shared distant relationship with CABYV and Suakwa aphid-borne yellows virus. Based on our results, we propose that PABYV is a distinct species in the genus Polerovirus.

First Report of the Isolation of the Symbiotic Bacterium Photorhabdus luminescens subsp. laumondii Associated with Heterorhabditis safricana from South Africa.

Photorhabdus luminescens subsp. laumondii is closely associated with the entomopathogenic nematode Heterorhabditis bacteriophora and has, to date, not been isolated from other nematode species. This study is the first report of P. luminescens subsp. laumondii from two South African isolates of entomopathogenic nematodes, Heterorhabditis safricana SF281 and H. bacteriophora SF351. Both symbiotic bacterial strains are phenotypically closely related to P. luminescens subsp. laumondii previously isolated and described from H. bacteriophora. The genetic relatedness between P. luminescens subsp. laumondii strains SF281B and SF351B was confirmed by comparing 16S rDNA, recA, gyrB and gltX sequences with sequences of P. luminescens subsp. laumondii, including the type strain (TT01T) and strain E21.

HSV-2 ΔgD elicits FcγR-effector antibodies that protect against clinical isolates.

A single-cycle herpes simplex virus (HSV) deleted in glycoprotein D (ΔgD-2) elicited high titer HSV-specific antibodies (Abs) that (i) were rapidly transported into the vaginal mucosa; (ii) elicited antibody-dependent cell-mediated cytotoxicity but little neutralization; (iii) provided complete protection against lethal intravaginal challenge; and (iv) prevented establishment of latency in mice. However, clinical isolates may differ antigenically and impact vaccine efficacy. To determine the breadth and further define mechanisms of protection of this vaccine candidate, we tested ΔgD-2 against a panel of clinical isolates in a murine skin challenge model. The isolates were genetically diverse, as evidenced by genomic sequencing and in vivo virulence. Prime and boost immunization (s.c.) with live but not heat- or UV-inactivated ΔgD-2 completely protected mice from challenge with the most virulent HSV-1 and HSV-2 isolates. Furthermore, mice were completely protected against 100 times the lethal dose that typically kills 90% of animals (LD90) of a South African isolate (SD90), and no latent virus was detected in dorsal root ganglia. Immunization was associated with rapid recruitment of HSV-specific FcγRIII- and FcγRIV-activating IgG2 Abs into the skin, resolution of local cytokine and cellular inflammatory responses, and viral clearance by day 5 after challenge. Rapid clearance and the absence of latent virus suggest that ΔgD-2 elicits sterilizing immunity.

Genome sequence analysis of two South African isolates of Moroccan watermelon mosaic virus infecting cucurbits.

Moroccan watermelon mosaic virus (MWMV) has been prevalent in cucurbits in the Republic of South Africa (RSA) since it was first reported in 1987. However, full genome studies of the South African isolates have never been conducted previously. The full genome of two MWMV isolates infecting cucurbits (Cucurbita pepo L.) in the province of KwaZulu-Natal, RSA, was compared with the genome of the Tunisian isolate in this communication. The genome sequences of the RSA MWMV isolates were elucidated using next-generation sequencing and Sanger sequencing. The analyses performed included nucleotide and amino acid sequence comparison, determination of the genetic distances, detection of potential recombination, and phylogeny. The genome sequences of the RSA MWMV isolates were found to be 9719 nucleotides long, excluding the poly(A) tail. Sequence homology, genetic distances, and phylogenetic analyses indicated close relationships between the RSA isolates. This record will contribute to building up the MWMV isolate sequences from the different countries where the virus occurs, a useful step toward understanding MWMV evolution.

Paradoxical Hypersusceptibility of Drug-resistant Mycobacteriumtuberculosis to β-lactam Antibiotics

Mycobacterium tuberculosis (M. tuberculosis) is considered innately resistant to β-lactam antibiotics. However, there is evidence that susceptibility to β-lactam antibiotics in combination with β–lactamase inhibitors is variable among clinical isolates, and these may present therapeutic options for drug-resistant cases. Here we report our investigation of susceptibility to β-lactam/β–lactamase inhibitor combinations among clinical isolates of M. tuberculosis, and the use of comparative genomics to understand the observed heterogeneity in susceptibility. Eighty-nine South African clinical isolates of varying first and second-line drug susceptibility patterns and two reference strains of M. tuberculosis underwent minimum inhibitory concentration (MIC) determination to two β-lactams: amoxicillin and meropenem, both alone and in combination with clavulanate, a β–lactamase inhibitor. 41/91 (45%) of tested isolates were found to be hypersusceptible to amoxicillin/clavulanate relative to reference strains, including 14/24 (58%) of multiple drug-resistant (MDR) and 22/38 (58%) of extensively drug-resistant (XDR) isolates. Genome-wide polymorphisms identified using whole-genome sequencing were used in a phylogenetically-aware linear mixed model to identify polymorphisms associated with amoxicillin/clavulanate susceptibility. Susceptibility to amoxicillin/clavulanate was over-represented among isolates within a specific clade (LAM4), in particular among XDR strains. Twelve sets of polymorphisms were identified as putative markers of amoxicillin/clavulanate susceptibility, five of which were confined solely to LAM4. Within the LAM4 clade, ‘paradoxical hypersusceptibility’ to amoxicillin/clavulanate has evolved in parallel to first and second-line drug resistance. Given the high prevalence of LAM4 among XDR TB in South Africa, our data support an expanded role for β-lactam/β-lactamase inhibitor combinations for treatment of drug-resistant M. tuberculosis.

Prevalence of virulence and antimicrobial resistance genes in Salmonella spp. isolated from commercial chickens and human clinical isolates from South Africa and Brazil.

Salmonellosis is a significant public health concern around the world. The injudicious use of antimicrobial agents in poultry production for treatment, growth promotion and prophylaxis has resulted in the emergence of drug-resistant strains of Salmonella. The current study was conducted to investigate the prevalence of virulence and antimicrobial resistance genes from Salmonella isolated from South African and Brazilian broiler chickens as well as human clinical isolates. Out of a total of 200 chicken samples that were collected from South Africa 102 (51%) tested positive for Salmonella using the InvA gene. Of the overall 146 Salmonella-positive samples that were screened for the iroB gene, most of them were confirmed to be Salmonella enterica with high prevalence rates. All the Salmonella isolates obtained were subjected to antimicrobial susceptibility testing with ten antibiotics. Salmonella isolates from South African chickens exhibited resistance to almost all antimicrobial agents used. All the samples were further subjected to the Polymerase Chain Reaction in order to screen some common antimicrobial and virulence genes of interest, namely spiC, pipD, misL, orfL, pse-1, tet A, tet B, ant (3”)-la, sul 1 and sul. All the Salmonella-positive isolates exhibited resistance to at least one antimicrobial agent; however, antimicrobial resistance patterns demonstrated that multiple drug resistance was prevalent. The findings provide evidence that broiler chickens are colonised by pathogenic Salmonella harbouring antimicrobial resistance genes. Therefore, it is evident that there is a need for prudent use of antimicrobial agents in poultry production systems in order to mitigate the proliferation of multiple drug resistance across species.

Microsatellite characterisation of South African Puccinia striiformis races

Since the first appearance of wheat stripe rust in 1996 in South Africa, four Puccinia striiformis races have been described. The first detected race, 6E16A−, was proposed to be a foreign introduction from Central or Western Asia that subsequently gained additional virulence through step-wise mutations. Simple sequence repeat markers were used to determine the genetic relationships between the four races, as well as a P. striiformis isolate collected from wild rye and two Kenyan isolates. A single isolate of P. striiformoides collected from Dactylis glomerata was included as an out-group. While the genetic similarity between P. striiformoides and the two Kenyan P. striiformis isolates was low, both shared only 48% genetic similarity with the South African isolates. In contrast, the South African P. striiformis isolates shared at least 74% genetic similarity with each other, with 6E16A−, 6E22A− and 7E22A− being the most similar. A minimum spanning network analysis confirmed this close relationship with several hypothetical intermediates occurring between individual races. This suggests that the South African P. striiformis population is clonal. However, the fact that P. striiformis was introduced into the country suggests that further incursions of more aggressive isolates could occur, which could threaten wheat production in the country.

A Tale of Genome Compartmentalization: The Evolution of Virulence Clusters in Smut Fungi.

Smut fungi are plant pathogens mostly parasitizing wild species of grasses as well as domesticated cereal crops. Genome analysis of several smut fungi including Ustilago maydis revealed a singular clustered organization of genes encoding secreted effectors. In U. maydis, many of these clusters have a role in virulence. Reconstructing the evolutionary history of clusters of effector genes is difficult because of their intrinsically fast evolution, which erodes the phylogenetic signal and homology relationships. Here, we describe the use of comparative evolutionary analyses of quality draft assemblies of genomes to study the mechanisms of this evolution. We report the genome sequence of a South African isolate of Sporisorium scitamineum, a smut fungus parasitizing sugar cane with a phylogenetic position intermediate to the two previously sequenced species U. maydis and Sporisorium reilianum. We show that the genome of S. scitamineum contains more and larger gene clusters encoding secreted effectors than any previously described species in this group. We trace back the origin of the clusters and find that their evolution is mainly driven by tandem gene duplication. In addition, transposable elements play a major role in the evolution of the clustered genes. Transposable elements are significantly associated with clusters of genes encoding fast evolving secreted effectors. This suggests that such clusters represent a case of genome compartmentalization that restrains the activity of transposable elements on genes under diversifying selection for which this activity is potentially beneficial, while protecting the rest of the genome from its deleterious effect.

Genetic characterization of human herpesvirus type 1: Full-length genome sequence of strain obtained from an encephalitis case from India

Background & objectives:Human herpes simplex virus 1 (HSV-1) is the most common cause of sporadic encephalitis in humans that contributes to >10 per cent of the encephalitis cases occurring worldwide. Availability of limited full genome sequences from a small number of isolates resulted in poor understanding of host and viral factors responsible for variable clinical outcome. In this study genetic relationship, extent and source of recombination using full-length genome sequence derived from a newly isolated HSV-1 isolate was studied in comparison with those sampled from patients with varied clinical outcome.Methods:Full genome sequence of HSV-1 isolated from cerebrospinal fluid (CSF) of a patient with acute encephalitis syndrome (AES) by inoculation in baby hamster kidney-21 (BHK-21) cells was determined using next-generation sequencing (NGS) technology. Phylogenetic analysis of the newly generated sequence in comparison with 33 additional full-length genomes defined genetic relationship with worldwide distributed strains. The bootscan and similarity plot analysis defined recombination crossovers and similarities between newly isolated Indian HSV-1 with six Asian and a total of 34 worldwide isolated strains.Results:Mapping of 376,332 reads amplified from HSV-1 DNA by NGS generated full-length genome of 151,024 bp from newly isolated Indian HSV-1. Phylogenetic analysis classified worldwide distributed strains into three major evolutionary lineages correlating to their geographic distribution. Lineage 1 containing strains were isolated from America and Europe; lineage 2 contained all the strains from Asian countries along with the North American KOS and RE strains whereas the South African isolates were distributed into two groups under lineage 3. Recombination analysis confirmed events of recombination in Indian HSV-1 genome resulting from mixing of different strains evolved in Asian countries.Interpretation & conclusions:Our results showed that the full-length genome sequence generated from an Indian HSV-1 isolate shared close genetic relationship with the American KOS and Chinese CR38 strains which belonged to the Asian genetic lineage. Recombination analysis of Indian isolate demonstrated multiple recombination crossover points throughout the genome. This full-length genome sequence amplified from the Indian isolate would be helpful to study HSV evolution, genetic basis of differential pathogenesis, host-virus interactions and viral factors contributing towards differential clinical outcome in human infections.

In vitro sensitivity testing of Cladobotryum mycophilum to carbendazim and prochloraz manganese

Limited information of fungicide efficacy on cultivated mushrooms and resistance development potential is available. Minor crop industries in general have a smaller arsenal of protectants to rely on and the likelihood of resistance build-up is of greater concern. This study focused on Cladobotryum mycophilum's sensitivity to carbendazim and prochloraz manganese following recent reports on decreased efficacy of both fungicides. The median effective dose (ED50) values for carbendazim ranged between 0.02 mg/L and 4.31 mg/L with 60% of the South African isolates being moderately resistant. The highest resistance factor for carbendazim was 215. Prochloraz manganese ED50 values varied from 0.00001 mg/L to 0.55 mg/L. A significant difference in mean ED50 values for both fungicides tested was observed. Using cluster analysis, no discrimination of isolates previously exposed and unexposed to prochloraz manganese was observed. A wide range of differences in ED50 values indicated moderate resistance to carbendazim and high sensitivity to prochloraz manganese among isolates under investigation. Discriminant analysis indicated significant differences between clusters contributed by one or a few variables. This study provided evidence that prochloraz manganese remains highly fungitoxic to C. mycophilum. However, prochloraz manganese is to be used in a disease management strategy in combination with strict farm hygiene management strategies to retain product efficacy and ensure crop protection.

Evidence of genotypic diversity among Candida auris isolates by multilocus sequence typing, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and amplified fragment length polymorphism

Candida auris is a multidrug-resistant nosocomial bloodstream pathogen that has been reported from Asian countries and South Africa. Herein, we studied the population structure and genetic relatedness among 104 global C. auris isolates from India, South Africa and Brazil using multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). RPB1, RPB2 and internal transcribed spacer (ITS) and D1/D2 regions of the ribosomal DNA were sequenced for MLST. Further, genetic variation and proteomic assessment was carried out using AFLP and MALDI-TOF MS, respectively. Both MLST and AFLP typing clearly demarcated two major clusters comprising Indian and Brazilian isolates. However, the South African isolates were randomly distributed, suggesting different genotypes. MALDI-TOF MS spectral profiling also revealed evidence of geographical clustering but did not correlate fully with the genotyping methods. Notably, overall the population structure of C. auris showed evidence of geographical clustering by all the three techniques analysed. Antifungal susceptibility testing by the CLSI microbroth dilution method revealed that fluconazole had limited activity against 87% of isolates (MIC90, 64 mg/L). Also, MIC90 of AMB was 4 mg/L. Candida auris is emerging as an important yeast pathogen globally and requires reproducible laboratory methods for identification and typing. Evaluation of MALDI-TOF MS as a typing method for this yeast is warranted.

Streptomyces species associated with common scab lesions of potatoes in South Africa

Streptomyces scabiei is largely accepted as the causal organism of common scab on potato in South Africa, and other Streptomyces species associated with common scab are not often considered. This study therefore aims to determine the diversity and prevalence of Streptomycetes associated with common scab on potatoes in South Africa. Isolates from 11 of the 16 potato producing regions in South Africa were characterized morphologically, physiologically and genetically. Most isolates resembled S. scabiei based on morphology and physiology. Most pathogenic isolates were S. scabiei and S. stelliscabiei, and no S. acidiscabies or S. turgidiscabies isolates were found. All three pathogenicity/virulence genes (txtAB, nec1, tomA) were found in South African isolates. Pathogenicity could not be linked to the presence of a single one or any combination of two of the three genes. These results represent the most comprehensive published survey of Streptomycetes isolated from common scab lesions on potatoes in South Africa.

Burkholderia kirstenboschensis sp. nov. nodulates papilionoid legumes indigenous to South Africa

Despite the diversity of Burkholderia species known to nodulate legumes in introduced and native regions, relatively few taxa have been formally described. For example, the Cape Floristic Region of South Africa is thought to represent one of the major centres of diversity for the rhizobial members of Burkholderia, yet only five species have been described from legumes occurring in this region and numerous are still awaiting taxonomic treatment. Here, we investigated the taxonomic status of 12 South African root-nodulating Burkholderia isolates from native papilionoid legumes (Hypocalyptus coluteoides, H. oxalidifolius, H. sophoroides and Virgilia oroboides). Analysis of four gene regions (16S rRNA, recA, atpD and rpoB) revealed that the isolates represent a genealogically unique and exclusive assemblage within the genus. Its distinctness was supported by all other aspects of the polyphasic approach utilized, including the genome-based criteria DNA−DNA hybridization (≥70.9%) and average nucleotide identities (≥96%). We accordingly propose the name B. kirstenboschensis sp. nov. for this taxon with isolate Kb15T (=LMG 28727T; =SARC 695T) as its type strain. Our data showed that intraspecific genome size differences (≥0.81 Mb) and the occurrence of large DNA regions that are apparently unique to single individuals (16−23% of an isolate's genome) can significantly limit the value of data obtained from DNA−DNA hybridization experiments. Substitution of DNA−DNA hybridization with whole genome sequencing as a prerequisite for the description of Burkholderia species will undoubtedly speed up the pace at which their diversity are documented, especially in hyperdiverse regions such as the Cape Floristic Region.

A PCR detection system for South African basidiomycetous isolates from esca affected grapevine

Grapevine trunk diseases threaten the economic livelihood and lifespan of vineyards. One such disease, esca, involves the fungal colonisation and consequent blocking of vascular tissue, followed by tissue necrosis and eventual wood rot. Basidiomycetous fungi are the main causal agents of this wood rot and phylogenetic analysis group all South African isolates into ten taxa all belonging to the Hymenochaetales. Genera represented include Inocutis, Inonotus, Fomitiporella, Fomitiporia and Phellinus. To be able to identify large numbers of unidentified basidiomycetous field isolates, species-specific primers were developed to identify the ten taxa with a PCR assay based on the ribosomal ITS1 and ITS2 region. Furthermore isolates from fruiting bodies found on non-Vitis sp. woody hosts were identified as Fomitiporia capensis by using this newly developed assay.

Genome sequence and comparative analysis of a putative entomopathogenic Serratia isolated from Caenorhabditis briggsae.

BackgroundEntomopathogenic associations between nematodes in the genera Steinernema and Heterorhabdus with their cognate bacteria from the bacterial genera Xenorhabdus and Photorhabdus, respectively, are extensively studied for their potential as biological control agents against invasive insect species. These two highly coevolved associations were results of convergent evolution. Given the natural abundance of bacteria, nematodes and insects, it is surprising that only these two associations with no intermediate forms are widely studied in the entomopathogenic context. Discovering analogous systems involving novel bacterial and nematode species would shed light on the evolutionary processes involved in the transition from free living organisms to obligatory partners in entomopathogenicity.ResultsWe report the complete genome sequence of a new member of the enterobacterial genus Serratia that forms a putative entomopathogenic complex with Caenorhabditis briggsae. Analysis of the 5.04 MB chromosomal genome predicts 4599 protein coding genes, seven sets of ribosomal RNA genes, 84 tRNA genes and a 64.8 KB plasmid encoding 74 genes. Comparative genomic analysis with three of the previously sequenced Serratia species, S. marcescens DB11 and S. proteamaculans 568, and Serratia sp. AS12, revealed that these four representatives of the genus share a core set of ~3100 genes and extensive structural conservation. The newly identified species shares a more recent common ancestor with S. marcescens with 99 % sequence identity in rDNA sequence and orthology across 85.6 % of predicted genes. Of the 39 genes/operons implicated in the virulence, symbiosis, recolonization, immune evasion and bioconversion, 21 (53.8 %) were present in Serratia while 33 (84.6 %) and 35 (89 %) were present in Xenorhabdus and Photorhabdus EPN bacteria respectively.ConclusionThe majority of unique sequences in Serratia sp. SCBI (South African Caenorhabditis briggsae Isolate) are found in ~29 genomic islands of 5 to 65 genes and are enriched in putative functions that are biologically relevant to an entomopathogenic lifestyle, including non-ribosomal peptide synthetases, bacteriocins, fimbrial biogenesis, ushering proteins, toxins, secondary metabolite secretion and multiple drug resistance/efflux systems. By revealing the early stages of adaptation to this lifestyle, the Serratia sp. SCBI genome underscores the fact that in EPN formation the composite end result – killing, bioconversion, cadaver protection and recolonization- can be achieved by dissimilar mechanisms. This genome sequence will enable further study of the evolution of entomopathogenic nematode-bacteria complexes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1697-8) contains supplementary material, which is available to authorized users.