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Plants release extracellular vesicles (EVs) for various reasons, such as unconventional protein secretion, RNA transport, and defence against pathogens. However, very little is known about plant EV biosynthesis and diverse and bioactive luminal contents, including how they alter recipient cell biology and function. To study EVs, researchers often isolate them from apoplastic wash or juice fluids using various methods. A method is needed to isolate EVs from non-model plants with tougher leaves such as species in the Pinus genus. Because of their spindly and sharp shape, pines have strong acicular-shaped leaves that are commonly called needles. Unlike all other taproots, pine seedlings grow into a primary root first, which gives rise to lateral roots later. There are also various dimensions and shapes of pine needles, but they are relatively strong compared to the leaves of other plants such as Arabidopsis thaliana, where most EV studies have been concentrated. Extracting EVs from a juice fluid is highly effective for plants with tougher leaves because it ensures that the cells have been broken down and the vesicles are present. Given these reasons, we describe our method for the isolation and purification of EVs from needles and roots of different pine species. A detailed protocol is provided that can guide the collection of juice fluid from pine needles and roots in a clean and efficient manner. A number of other plant species with similar morphologies can greatly benefit from the use of this protocol. Basic protocol 1: Isolation of extracellular vesicles from juice fluid of pine leaves and roots Basic protocol 2: Isolation of pine extracellular vesicles from juice fluid using size exclusion chromatography

Hybridization is recognized as a notable driver of evolution and adaptation, which closely related species may exploit in the form incomplete reproductive barriers. Three closely related species of Ceratocystis (i.e., C. fimbriata, C. manginecans and C. eucalypticola) previously have been shown to hybridize by mating naturally occurring self-sterile strains with an unusual laboratory-generated sterile isolate type, which could have impacted conclusions regarding the prevalence of hybridization and inheritance of mitochondria. In the current study we investigated whether interspecific crosses between fertile isolates of these three species are possible, and if so how mitochondria are inherited by the progeny. For this purpose, a PCR-RFLP method and a mitochondrial DNA-specific PCR technique were custom-made and applied in a novel approach of typing complete ascospore drops collected from the fruiting bodies in each cross to distinguish between self-fertilizations and potential hybridization. These markers showed hybridization between C. fimbriata and C. eucalypticola and between C. fimbriata and C. manginecans, while no hybridization was detected in the crosses involving C. manginecans and C. eucalypticola. In both sets of hybrid progeny, we detected biparental inheritance of mitochondria. This study was the first to successfully produce hybrids from a cross involving self-fertile isolates of Ceratocystis, and also provided the first direct evidence of biparental mitochondrial inheritance in the Ceratocystidaceae. This work lays the foundation for further research focused on investigating the role of hybridization in the speciation of Ceratocystis species, and if mitochondrial conflict could have influenced the process.

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.

The pathogenFusarium circinatum, causal agent of pitch canker disease, is currently one of the biggest fungal threats to pine health worldwide. Symptomatic infections are associated with a high mortality rate, particularly for nursery and established plants. Pitch canker disease results in reduced growth and significant annual losses in the South African forestry industry. Pines respond to insect damage and fungal infection by forming traumatic resin ducts, as well as upregulating the production of defence compounds. These phytochemicals include terpenes, the main chemical constituents of pine resin, and phenolics, produced in specialized cells of the secondary phloem. Many of these compounds belonging to these two phytochemical groups are known to have inhibitory or lethal effects on pine pests and pathogens. Although mostPinusspecies are susceptible toF. circinatum, there is variation in susceptibility to this pathogen among the different species and their hybrids. Resistance of a pine species to the pitch canker fungus is a major determining factor in the species' value to the pine industry; however, the underlying mechanisms of resistance are poorly understood. To explore defense responses between resistant and susceptible pines, we used GCMS and LCMS to characterize the phytochemical changes in youngP. patulaXP. tecunumaniihybrid clones in response toF. circinatuminfection. A significant increase was observed in the concentration of terpenes and phenolics between five- and 14-days post-inoculation. More resistant hybrids plants showed moderate disease symptoms as well as lower concentrations of defensive phytochemicals, both constitutively and in response toF. circinatum infection. These findings suggest that increased concentrations of terpenoid oleoresin and phenolics are not part of the defence strategy of pine against infection byF. circinatum. UnderstandingF. circinatum’s ability not only to overcome, but seemingly benefit from a more severe phytochemical defense response, will help explain the enduring pervasiveness of this global pine pathogen.

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.

Escherichia coli comprises diverse strains with a large accessory genome, indicating functional diversity and the ability to adapt to a range of niches. Specific strains would display greatest fitness in niches matching their combination of phenotypic traits. Given this hypothesis, we sought to determine whether E. coli in a peri-urban pond and associated cattle pasture display niche preference. Samples were collected from water, sediment, aquatic plants, water snails associated with the pond, as well as bovine feces from cattle in an adjacent pasture. Isolates (120) were obtained after plating on Membrane Lactose Glucuronide Agar (MLGA). We used the uidA and mutS sequences for all isolates to determine phylogeny by maximum likelihood, and population structure through gene flow analysis. PCR was used to allocate isolates to phylogroups and to determine the presence of pathogenicity/virulence genes (stxI, stxII, eaeA, hlyA, ST, and LT). Antimicrobial resistance was determined using a disk diffusion assay for Tetracycline, Gentamicin, Ciprofloxacin, Meropenem, Ceftriaxone, and Azithromycin. Our results showed that isolates from water, sediment, and water plants were similar by phylogroup distribution, virulence gene distribution, and antibiotic resistance while both snail and feces populations were significantly different. Few of the feces isolates were significantly similar to aquatic ones, and most of the snail isolates were also different. Population structure analysis indicated three genetic backgrounds associated with bovine, snail, and aquatic environments. Collectively these data support niche preference of E. coli isolates occurring in this ecosystem.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the