Genomic characterization of novel viruses associated with Olea europaea L. in South Africa.

South Africa has a small but growing olive industry. Until now, no virological research has been carried out on this crop locally. Seventeen samples were collected from various olive cultivars from a single producer in the Stellenbosch growing area of South Africa. RNAseq was performed on total RNA, and the compositions of the metaviromes were determined. Olive leaf yellowing-associated virus was detected for the first time in South Africa, as well as four novel viruses from the family Closteroviridae and one each from the families Tymoviridae and Solemoviridae.

Genomic characterization of a novel potyvirus infecting Barleria repens in South Africa.

The complete RNA genome sequence of a novel member of the genus Potyvirus infecting Barleria repens has been determined. A plant showing symptoms of virus-like leaf mosaic was sampled in the Western Cape province of South Africa, and the associated virus has been tentatively named "Barleria repens mottle virus" (BaRMoV). The genome of BaRMoV consists of 9,561 nucleotides and encodes a typical potyvirus polyprotein that is 3,105 amino acids long. Pairwise comparisons showed that the BaRMoV genome shared an average of 70% nucleotide sequence identity with that of yam mosaic virus (YMV), and the corresponding polyprotein shared an average of 76.6% amino acid sequence identity with that of YMV. Phylogenetic analysis of the BaRMoV polyprotein amino acid sequence and those of other extant potyviruses confirmed the relationship between BarMoV and YMV. This is the first time that a member of the family Potyviridae has been identified in a member of the species Barleria.

Genomic characterization of soybean blotchy mosaic virus, a cytorhabdovirus from South Africa.

Samples showing blotchy mottle symptoms were collected from soybeans in North-West province, South Africa. The assembly of high-throughput sequencing data from three samples yielded contigs of 13,426 to 13,435nt, which represent the first complete genome sequences of soybean blotchy mosaic virus (SbBMV). SbBMV shows a typical cytorhabdovirus gene organization (3'-N-P-P3-M-G-L-5'), with each putative gene product being most similar, but with only 49.1-71.1% sequence identity, to those of cucurbit cytorhabdovirus 1. Given the species demarcation thresholds for rhabdoviruses, SbBMV is thus a distinct member of the genus Cytorhabdovirus.

Characterisation of the Alarm Pheromone of Bathycoelia Distincta (Pentatomidae).

Bathycoelia distincta (Pentatomidae) is the dominant pest in South African macadamia orchards, where adults are responsible for causing severe yield losses. Similar to other hemipterans, B. distincta release volatile compounds from scent glands that can deter natural enemies and act as an alarm signal among conspecifics. The overall aim of this study was to characterise the alarm pheromone of B. distincta. We: (i) analysed the scent gland contents of individual adult B. distincta by gas chromatography-mass spectrometry (GC-MS), (ii) quantified volatiles released from live stink bugs after stress, and (iii) evaluated the electrophysiological and behavioural activity of alarm pheromone compounds with dose-response experiments. A blend of fourteen compounds was identified in the scent gland extracts of adult stink bugs. Of these, six compounds were detected in the effluvia of live stressed stink bugs [(E)-2-hexenal, (E)-2-decenal, tridecane, dodecane, (E)-4-oxohex-2-enal and (E)-2-decenyl acetate]. No qualitative or quantitative differences were observed between sexes. Tridecane was the most abundant compound, comprising ∼50% of total secretions. Only (E)-2-hexenal, (E)-2-decenal, and (E)-4-oxohex-2-enal elicited an antennal response in both sexes. Finally, exposure to a mixture of (E)-2-hexenal, (E)-2-decenal, and (E)-4-oxohex-2-enal resulted in an increase in the speed and distance travelled by walking bugs and a decrease in time spent resting compared to unexposed bugs. Our results show that the blend of (E)-2-hexenal, (E)-2-decenal, and (E)-4-oxohex-2-enal can induce an alarm response in B. distincta.

Increased abundance of secreted hydrolytic enzymes and secondary metabolite gene clusters define the genomes of latent plant pathogens in the Botryosphaeriaceae.

BACKGROUND: The Botryosphaeriaceae are important plant pathogens, but also have the ability to establish asymptomatic infections that persist for extended periods in a latent state. In this study, we used comparative genome analyses to shed light on the genetic basis of the interactions of these fungi with their plant hosts. For this purpose, we characterised secreted hydrolytic enzymes, secondary metabolite biosynthetic gene clusters and general trends in genomic architecture using all available Botryosphaeriaceae genomes, and selected Dothideomycetes genomes. RESULTS: The Botryosphaeriaceae genomes were rich in carbohydrate-active enzymes (CAZymes), proteases, lipases and secondary metabolic biosynthetic gene clusters (BGCs) compared to other Dothideomycete genomes. The genomes of Botryosphaeria, Macrophomina, Lasiodiplodia and Neofusicoccum, in particular, had gene expansions of the major constituents of the secretome, notably CAZymes involved in plant cell wall degradation. The Botryosphaeriaceae genomes were shown to have moderate to high GC contents and most had low levels of repetitive DNA. The genomes were not compartmentalized based on gene and repeat densities, but genes of secreted enzymes were slightly more abundant in gene-sparse regions. CONCLUSION: The abundance of secreted hydrolytic enzymes and secondary metabolite BGCs in the genomes of Botryosphaeria, Macrophomina, Lasiodiplodia, and Neofusicoccum were similar to those in necrotrophic plant pathogens and some endophytes of woody plants. The results provide a foundation for comparative genomic analyses and hypotheses to explore the mechanisms underlying Botryosphaeriaceae host-plant interactions.

Insect egg-induced physiological changes and transcriptional reprogramming leading to gall formation.

Gall-inducing insects and their hosts present some of the most intricate plant-herbivore interactions. Oviposition on the host is often the first cue of future herbivory and events at this early time point can affect later life stages. Many gallers are devastating plant pests, yet little information regarding the plant-insect molecular interplay exists, particularly following egg deposition. We studied the physiological and transcriptional responses of Eucalyptus following oviposition by the gall-inducing wasp, Leptocybe invasa, to explore potential mechanisms governing defence responses and gall development. RNA sequencing and microscopy were used to explore a susceptible Eucalyptus-L. invasa interaction. Infested and control material was compared over time (1-3, 7 and 90 days post oviposition) to examine the transcriptional and morphological changes. Oviposition induces accumulation of reactive oxygen species and phenolics which is reflected in the transcriptome analysis. Gene expression supports phytohormones and 10 transcription factor subfamilies as key regulators. The egg and oviposition fluid stimulate cell division resulting in gall development. Eucalyptus responses to oviposition are apparent within 24 hr. Putative defences include the oxidative burst and barrier reinforcement. However, egg and oviposition fluid stimuli may redirect these responses towards gall development.

The Sex Pheromone of the Pine Brown-Tail Moth, Euproctis terminalis (Lepidoptera: Erebidae).

The pine brown tail moth, Euproctis terminalis (Walker 1855), is a periodic pest in pine plantations in South Africa. The larvae feed on pine needles and can cause severe defoliation when population densities are high. Population densities fluctuate temporally and spatially, complicating the prediction of potential growth loss and tree mortality. The aim of this study was to identify the sex pheromone of the pine brown tail moth to provide stakeholders with a tool for monitoring it. Gas chromatography-electroantennogram detection and gas chromatography/mass spectrometry analyses of female pheromone gland extracts identified the major component as (Z,Z,Z,Z)-7,13,16,19-docosatetraen-1-ol isobutyrate. Traps baited with (Z,Z,Z,Z)-7,13,16,19-docosatetraen-1-ol isobutyrate caught more males than unbaited traps. A delta trap was shown to be a superior design compared to a bucket funnel trap. This pheromone can now be used for monitoring E. terminalis in pine plantations.

A Genome-Wide Association Study for Resistance to the Insect Pest Leptocybe invasa in Eucalyptus grandis Reveals Genomic Regions and Positional Candidate Defense Genes.

The galling insect, Leptocybe invasa, causes significant losses in plantations of various Eucalyptus species and hybrids, threatening its economic viability. We applied a genome-wide association study (GWAS) to identify single-nucleotide polymorphism (SNP) markers associated with resistance to L. invasa. A total of 563 insect-challenged Eucalyptus grandis trees, from 61 half-sib families, were genotyped using the EUChip60K SNP chip, and we identified 15,445 informative SNP markers in the test population. Multi-locus mixed-model (MLMM) analysis identified 35 SNP markers putatively associated with resistance to L. invasa based on four discreet classes of insect damage scores: (0) not infested, (1) infested showing evidence of oviposition but no gall development, (2) infested with galls on leaves, midribs or petioles and (3) stunting and lethal gall formation. MLMM analysis identified three associated genomic regions on chromosomes 3, 7 and 8 jointly explaining 17.6% of the total phenotypic variation. SNP analysis of a validation population of 494 E. grandis trees confirmed seven SNP markers that were also detected in the initial association analysis. Based on transcriptome profiles of resistant and susceptible genotypes from an independent experiment, we identified several putative candidate genes in associated genomic loci including Nucleotide-binding ARC- domain (NB-ARC) and toll-interleukin-1-receptor-Nucleotide binding signal- Leucine rich repeat (TIR-NBS-LRR) genes. Our results suggest that Leptocybe resistance in E. grandis may be influenced by a few large-effect loci in combination with minor effect loci segregating in our test and validation populations.

The Draft Genome of Deladenus siricidicola.

The nematode Deladenus siricidicola is used as biological control agent against the invasive woodwasp Sirex noctilio, a serious invasive pest of Pinus plantations globally. The draft genome of this ecologically and economically important entomoparasitic nematode was determined.

EuGI: a novel resource for studying genomic islands to facilitate horizontal gene transfer detection in eukaryotes.

BACKGROUND: Genomic islands (GIs) are inserts of foreign DNA that have potentially arisen through horizontal gene transfer (HGT). There are evidences that GIs can contribute significantly to the evolution of prokaryotes. The acquisition of GIs through HGT in eukaryotes has, however, been largely unexplored. In this study, the previously developed GI prediction tool, SeqWord Gene Island Sniffer (SWGIS), is modified to predict GIs in eukaryotic chromosomes. Artificial simulations are used to estimate ratios of predicting false positive and false negative GIs by inserting GIs into different test chromosomes and performing the SWGIS v2.0 algorithm. Using SWGIS v2.0, GIs are then identified in 36 fungal, 22 protozoan and 8 invertebrate genomes. RESULTS: SWGIS v2.0 predicts GIs in large eukaryotic chromosomes based on the atypical nucleotide composition of these regions. Averages for predicting false negative and false positive GIs were 20.1% and 11.01% respectively. A total of 10,550 GIs were identified in 66 eukaryotic species with 5299 of these GIs coding for at least one functional protein. The EuGI web-resource, freely accessible at http://eugi.bi.up.ac.za , was developed that allows browsing the database created from identified GIs and genes within GIs through an interactive and visual interface. CONCLUSIONS: SWGIS v2.0 along with the EuGI database, which houses GIs identified in 66 different eukaryotic species, and the EuGI web-resource, provide the first comprehensive resource for studying HGT in eukaryotes.

Evolution of the mating types and mating strategies in prominent genera in the Botryosphaeriaceae.

Little is known regarding mating strategies in the Botryosphaeriaceae. To understand sexual reproduction in this fungal family, the mating type genes of Botryosphaeria dothidea and Macrophomina phaseolina, as well as several species of Diplodia, Lasiodiplodia and Neofusicoccum were characterized from whole genome assemblies. Comparisons between the mating type loci of these fungi showed that the mating type genes are highly variable, but in most cases the organization of these genes is conserved. Of the species considered, nine were homothallic and seven were heterothallic. Mating type gene fragments were discovered flanking the mating type regions, which indicates both ongoing and ancestral recombination occurring within the mating type region. Ancestral reconstruction analysis further indicated that heterothallism is the ancestral state in the Botryosphaeriaceae and this is supported by the presence of mating type gene fragments in homothallic species. The results also show that at least five transitions from heterothallism to homothallism have taken place in the Botryosphaeriaceae. The study provides a foundation for comparison of mating type evolution between Botryosphaeriaceae and other fungi and also provides valuable markers for population biology studies in this family.

Botryosphaeriaceae associated with the die-back of ornamental trees in the Western Balkans.

Extensive die-back and mortality of various ornamental trees and shrubs has been observed in parts of the Western Balkans region during the past decade. The disease symptoms have been typical of those caused by pathogens residing in the Botryosphaeriaceae. The aims of this study were to isolate and characterize Botryosphaeriaceae species associated with diseased ornamental trees in Serbia, Montenegro, Bosnia and Herzegovina. Isolates were initially characterized based on the DNA sequence data for the internal transcribed spacer rDNA and six major clades were identified. Representative isolates from each clade were further characterized using DNA sequence data for the translation elongation factor 1-alpha, ß-tubulin-2 and large subunit rRNA gene regions, as well as the morphology of the asexual morphs. Ten species of the Botryosphaeriaceae were identified of which eight, i.e., Dothiorella sarmentorum, Neofusicoccum parvum, Botryosphaeria dothidea, Phaeobotryon cupressi, Sphaeropsis visci, Diplodia seriata, D. sapinea and D. mutila were known taxa. The remaining two species could be identified only as Dothiorella spp. Dichomera syn-asexual morphs of D. sapinea, Dothiorella sp. 2 and B. dothidea, as well as unique morphological characters for a number of the known species are described. Based on host plants and geographic distribution, the majority of Botryosphaeriaceae species found represent new records. The results of this study contribute to our knowledge of the distribution, host associations and impacts of these fungi on trees in urban environments.

Insect Gallers and Their Plant Hosts: From Omics Data to Systems Biology.

Gall-inducing insects are capable of exerting a high level of control over their hosts' cellular machinery to the extent that the plant's development, metabolism, chemistry, and physiology are all altered in favour of the insect. Many gallers are devastating pests in global agriculture and the limited understanding of their relationship with their hosts prevents the development of robust management strategies. Omics technologies are proving to be important tools in elucidating the mechanisms involved in the interaction as they facilitate analysis of plant hosts and insect effectors for which little or no prior knowledge exists. In this review, we examine the mechanisms behind insect gall development using evidence from omics-level approaches. The secretion of effector proteins and induced phytohormonal imbalances are highlighted as likely mechanisms involved in gall development. However, understanding how these components function within the system is far from complete and a number of questions need to be answered before this information can be used in the development of strategies to engineer or breed plants with enhanced resistance.

Sirex woodwasp: a model for evolving management paradigms of invasive forest pests.

The Sirex woodwasp, Sirex noctilio, and its fungal mutualist, Amylostereum areolatum, together constitute one of the most damaging invasive pests of pine. Despite a century of research and well-established management programs, control remains unpredictable and spread continues to new areas. Variable success in managing this pest has been influenced by complex invasion patterns, the multilayered nature of biological interactions, the varying local ecologies, and microevolutionary population processes in both the biocontrol organisms and in the wasps. Recent research findings are challenging the historical perspectives on methods to manage the Sirex woodwasp, calling for management programs to incorporate the variable local dynamics affecting this pest complex. In this regard, the Sirex woodwasp provides a superb model to illustrate the need for a different approach to develop efficient and sustainable management tools to deal with the growing and global nature of pest invasions in forests and plantations.

The Transcriptome and Terpene Profile of Eucalyptus grandis Reveals Mechanisms of Defense Against the Insect Pest, Leptocybe invasa.

Plants have evolved complex defenses that allow them to protect themselves against pests and pathogens. However, there is relatively little information regarding the Eucalyptus defensome. Leptocybe invasa is one of the most damaging pests in global Eucalyptus forestry, and essentially nothing is known regarding the molecular mechanisms governing the interaction between the pest and host. The aim of the study was to investigate changes in the transcriptional landscape and terpene profile of a resistant and susceptible Eucalyptus genotype in an effort to improve our understanding of this interaction. We used RNA-seqencing to investigate transcriptional changes following L. invasa oviposition. Expression levels were validated using real-time quantitative PCR. Terpene profiles were investigated using gas chromatography coupled to mass spectometry on uninfested and oviposited leaves. We found 698 and 1,115 significantly differentially expressed genes from the resistant and susceptible interactions, respectively. Gene Ontology enrichment and Mapman analyses identified putative defense mechanisms including cell wall reinforcement, protease inhibitors, cell cycle suppression and regulatory hormone signaling pathways. There were significant differences in the mono- and sesquiterpene profiles between genotypes and between control and infested material. A model of the interaction between Eucalyptus and L. invasa was proposed from the transcriptomic and chemical data.

Fungi associated with black mould on baobab trees in southern Africa.

There have been numerous reports in the scientific and popular literature suggesting that African baobab (Adansonia digitata) trees are dying, with symptoms including a black mould on their bark. The aim of this study was to determine the identity of the fungi causing this black mould and to consider whether they might be affecting the health of trees. The fungi were identified by sequencing directly from mycelium on the infected tissue as well as from cultures on agar. Sequence data for the ITS region of the rDNA resulted in the identification of four fungi including Aureobasidium pullulans, Toxicocladosporium irritans and a new species of Rachicladosporium described here as Rachicladosporium africanum. A single isolate of an unknown Cladosporium sp. was also found. These fungi, referred to here as black mould, are not true sooty mould fungi and they were shown to penetrate below the bark of infected tissue, causing a distinct host reaction. Although infections can lead to dieback of small twigs on severely infected branches, the mould was not found to kill trees.

Multiple Phytophthora species associated with a single riparian ecosystem in South Africa.

The diversity of Phytophthora spp. in rivers and riparian ecosystems has received considerable international attention, although little such research has been conducted in South Africa. This study determined the diversity of Phytophthora spp. within a single river in Gauteng province of South Africa. Samples were collected over 1 y including biweekly river baiting with Rhododendron indicum leaves. Phytophthora isolates were identified with phylogenetic analyses of sequences for the internal transcribed spacer (ITS) region of the ribosomal DNA and the mitochondrial cytochrome oxidase c subunit I (coxI) gene. Eight Phytophthora spp. were identified, including a new taxon, P. taxon Sisulu-river, and two hybrid species from Cooke's ITS clade 6. Of these, species from Clade 6 were the most abundant, including P. chlamydospora and P. lacustris. Species residing in Clade 2 also were encountered, including P. multivora, P. plurivora and P. citrophthora. The detection of eight species in this investigation of Phytophthora diversity in a single riparian river ecosystem in northern South Africa adds to the known diversity of this genus in South Africa and globally.

MAT gene idiomorphs suggest a heterothallic sexual cycle in a predominantly asexual and important pine pathogen.

Diplodia pinea (=Sphaeropsis sapinea) is a well-known and economically important latent pathogen of Pinus spp. in many parts of the world. Despite intensive scrutiny, its sexual state has never been observed and the fungus has thus been considered exclusively asexual. It was, therefore, surprising that a recent population genetics study showed high genotypic diversity and random association of alleles in a number of populations, suggesting that the pathogen has a cryptic sexual stage. Using the genome sequence of two individual D. pinea isolates, we interrogated the structure of the MAT locus in this fungus. The results suggested that D. pinea is heterothallic (self-sterile) with complete and apparently functional copies of the MAT genes containing the α-1 and HMG domains present in different isolates. In addition to the MAT1-2-1 and MAT1-1-1 genes, we found a MAT1-1-4 gene in the MAT1-1 idiomorph and a novel MAT1-2-5 gene in the MAT1-2 idiomorph. Importantly, the frequencies of occurrence of both idiomorphs in populations examined were not significantly different from a 1:1 ratio, which would be expected in sexually reproducing populations. Although the sexual state has never been observed, the results strongly suggest that D. pinea has a cryptic, heterothallic sexual cycle.