Effector loss drives adaptation of Pseudomonas syringae pv. actinidiae biovar 3 to Actinidia arguta.

A pandemic isolate of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars of Actinidia chinensis. In contrast, A. arguta (kiwiberry) is not a host of Psa3. Resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infected A. arguta leaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease in A. arguta have been isolated and analyzed, revealing a 51 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent in A. arguta plantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growth in planta for knockouts of four effectors-AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a -suggesting a resistance response in A. arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes in Actinidia for breeding durable resistance into future kiwifruit cultivars.

A New TaqMan Real-Time PCR Assay for Detecting the Blueberry Pathogen Monilinia vaccinii-corymbosi.

Monilinia vaccinii-corymbosi (Mvc) is an important fungal pathogen of blueberry, causing mummy berry disease. While the symptoms of the advanced stages of the disease can be obvious, diagnosing its early stages can be challenging. To facilitate fast and sensitive screening of asymptomatic or latently infected plant material for Mvc, we developed a specific TaqMan real-time PCR assay targeting the internal transcribed spacer (ITS) region. The assay was shown to be specific to Mvc and did not cross react with any of the other tested Monilinia species or other blueberry pathogens. Using the multicopy ITS region ensured high analytical sensitivity, enabling very low concentrations of Mvc DNA (0.1 pg) to be detected both in water and host DNA matrix. Comparable results were obtained in interlaboratory testing, showing that the assay is robust, and can be effectively used in other laboratories. Assay sensitivity was also confirmed on infected plant tissue, showing that it is effective in detecting the pathogen in infected asymptomatic stem tissue, as well as infected tissue that was mixed with healthy tissue at a ratio of 1:10 by weight. The assay was duplexed with a plant internal control (cytochrome oxidase gene) for simultaneous amplification of the pathogen and plant internal control in a single reaction. This new diagnostic tool can be used for sensitive and rapid screening of blueberry plants for the presence of Mvc in many different settings, e.g., for breeding programs, research, or biosecurity diagnostics.

Real-Time PCR and Droplet Digital PCR Are Accurate and Reliable Methods To Quantify Pseudomonas syringae pv. actinidiae Biovar 3 in Kiwifruit Infected Plantlets.

Pseudomonas syringae pv. actinidiae is the etiological agent of kiwifruit canker disease, causing severe economic losses in kiwifruit production areas around the world. Rapid diagnosis, understanding of bacterial virulence, and rate of infection in kiwifruit cultivars are important in applying effective measures of disease control. P. syringae pv. actinidiae load in kiwifruit is currently determined by a labor-intense colony counting method with no high-throughput and specific quantification method being validated. In this work, we used three alternative P. syringae pv. actinidiae quantification methods in two infected kiwifruit cultivars: start of growth time, quantitative PCR (qPCR), and droplet digital PCR (ddPCR). Method performance in each case was compared with the colony counting method. Methods were validated using calibration curves obtained with serial dilutions of P. syringae pv. actinidiae biovar 3 (Psa3) inoculum and standard growth curves obtained from kiwifruit samples infected with Psa3 inoculum. All three alternative methods showed high correlation (r > 0.85) with the colony counting method. qPCR and ddPCR were very specific, sensitive (5 × 102 CFU/cm2), highly correlated to each other (r = 0.955), and flexible, allowing for sample storage. The inclusion of a kiwifruit biomass marker increased the methods' accuracy. The qPCR method was efficient and allowed for high-throughput processing, and the ddPCR method showed highly accurate results but was more expensive and time consuming. While not ideal for high-throughput processing, ddPCR was useful in developing accurate standard curves for the qPCR method. The combination of the two methods is high-throughput, specific for Psa3 quantification, and useful for research studies (e.g., disease phenotyping and host-pathogen interactions).

Evaluating Biodiversity Metric Response to Forecasted Land Use Change in the Northern Rio Grande Basin.

The effects of future land use change on arid and semi-arid watersheds in the American Southwest have important management implications. Seamless, national-scale land-use-change scenarios for developed land were acquired from the US Environmental Protection Agency Integrated Climate and Land Use Scenarios (lCLUS) project and extracted to fit the Northern Rio Grande River Basin, New Mexico relative to projections of housing density for the period from 2000 through 2100. Habitat models developed from the Southwest Regional Gap Analysis Project were invoked to examine changes in wildlife habitat and biodiversity metrics using five ICLUS scenarios. The scenarios represent a US Census base-case and four modifications that were consistent with the different assumptions underlying the A1, A2, B1, and B2 Intergovernmental Panel on Climate Change global greenhouse gas emission storylines. Habitat models for terrestrial vertebrate species were used to derive metrics reflecting ecosystem services or biodiversity aspects valued by humans that could be quantified and mapped. Example metrics included total terrestrial vertebrate species richness, bird species richness, threatened and endangered species, and harvestable species (e.g., waterfowl, big game). Overall, the defined scenarios indicated that the housing density and extent of developed lands will increase throughout the century with a resultant decrease in area for all species richness categories. The A2 Scenario, in general, showed greatest effect on area by species richness category. The integration of the land use scenarios with biodiversity metrics derived from deductive habitat models may prove to be an important tool for decision makers involved in impact assessments and adaptive planning processes.

Genomic analysis of the Kiwifruit pathogen Pseudomonas syringae pv. actinidiae provides insight into the origins of an emergent plant disease.

The origins of crop diseases are linked to domestication of plants. Most crops were domesticated centuries--even millennia--ago, thus limiting opportunity to understand the concomitant emergence of disease. Kiwifruit (Actinidia spp.) is an exception: domestication began in the 1930s with outbreaks of canker disease caused by P. syringae pv. actinidiae (Psa) first recorded in the 1980s. Based on SNP analyses of two circularized and 34 draft genomes, we show that Psa is comprised of distinct clades exhibiting negligible within-clade diversity, consistent with disease arising by independent samplings from a source population. Three clades correspond to their geographical source of isolation; a fourth, encompassing the Psa-V lineage responsible for the 2008 outbreak, is now globally distributed. Psa has an overall clonal population structure, however, genomes carry a marked signature of within-pathovar recombination. SNP analysis of Psa-V reveals hundreds of polymorphisms; however, most reside within PPHGI-1-like conjugative elements whose evolution is unlinked to the core genome. Removal of SNPs due to recombination yields an uninformative (star-like) phylogeny consistent with diversification of Psa-V from a single clone within the last ten years. Growth assays provide evidence of cultivar specificity, with rapid systemic movement of Psa-V in Actinidia chinensis. Genomic comparisons show a dynamic genome with evidence of positive selection on type III effectors and other candidate virulence genes. Each clade has highly varied complements of accessory genes encoding effectors and toxins with evidence of gain and loss via multiple genetic routes. Genes with orthologs in vascular pathogens were found exclusively within Psa-V. Our analyses capture a pathogen in the early stages of emergence from a predicted source population associated with wild Actinidia species. In addition to candidate genes as targets for resistance breeding programs, our findings highlight the importance of the source population as a reservoir of new disease.

Things we still haven't learned (so far).

Null hypothesis significance testing (NHST) is like an immortal horse that some researchers have been trying to beat to death for over 50 years but without any success. In this article we discuss the flaws in NHST, the historical background in relation to both Fisher's and Neyman and Pearson's statistical ideas, the common misunderstandings of what p < .05 actually means, and the 2010 APA publication manual's clear, but most often ignored, instructions to report effect sizes and to interpret what they all mean in the real world. In addition, we discuss how Bayesian statistics can be used to overcome some of the problems with NHST. We then analyze quantitative articles published over the past three years (2012-2014) in two top-rated sport and exercise psychology journals to determine whether we have learned what we should have learned decades ago about our use and meaningful interpretations of statistics.

Comparison of the complete genome sequence of two closely related isolates of 'Candidatus Phytoplasma australiense' reveals genome plasticity.

BACKGROUND: 'Candidatus Phytoplasma australiense' is associated with at least nine diseases in Australia and New Zealand. The impact of this phytoplasma is considerable, both economically and environmentally. The genome of a NZ isolate was sequenced in an effort to understand its pathogenicity and ecology. Comparison with a closely related Australian isolate enabled us to examine mechanisms of genomic rearrangement. RESULTS: The complete genome sequence of a strawberry lethal yellows (SLY) isolate of 'Candidatus Phytoplasma australiense' was determined. It is a circular genome of 959,779 base pairs with 1126 predicted open reading frames. Despite being 80 kbp larger than another 'Ca. Phytoplasma australiense' isolate PAa, the variation between housekeeping genes was generally less than 1% at a nucleotide level. The difference in size between the two isolates was largely due to the number and size of potential mobile units (PMUs), which contributed to some changes in gene order. Comparison of the genomes of the two isolates revealed that the highly conserved 5' UTR of a putative DNA-directed RNA polymerase seems to be associated with insertion and rearrangement events. Two types of PMUs have been identified on the basis of the order of three to four conserved genes, with both PMUs appearing to have been present in the last common ancestor of 'Ca. Phytoplasma asteris' and 'Ca. Phytoplasma australiense'. Comparison with other phytoplasma genomes showed that modification methylases were, in general, species-specific. A putative methylase (xorIIM) found in 'Ca. Phytoplasma australiense' appeared to have no analogue in any other firmicute, and we believe has been introduced by way of lateral gene transfer. A putative retrostransposon (ltrA) analogous to that found in OY-M was present in both isolates, although all examples in PAa appear to be fragments. Comparative analysis identified highly conserved 5' and 3' UTR regions of ltrA, which may indicate how the gene is excised and inserted. CONCLUSIONS: Comparison of two assembled 'Ca. Phytoplasma australiense' genomes has shown they possess a high level of plasticity. This comparative analysis has yielded clues as to how rearrangements occur, and the identification of sets of genes that appear to be associated with these events.

Real-time quantification of microRNAs by stem-loop RT-PCR.

A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.

Psychosocial Factors and Sport Injuries: Meta-analyses for Prediction and Prevention.

BACKGROUND: Several studies have suggested that psychosocial variables can increase the risk of becoming injured during sport participation. OBJECTIVES: The main objectives of these meta-analyses were to examine (i) the effect sizes of relationships between the psychosocial variables (suggested as injury predictors in the model of stress and athletic injury) and injury rates, and (ii) the effects of psychological interventions aimed at reducing injury occurrence (prevention). METHODS: Electronic databases as well as specific sport and exercise psychology journals were searched. The literature review resulted in 48 published studies containing 161 effect sizes for injury prediction and seven effect sizes for injury prevention. RESULTS: The results showed that stress responses (r = 0.27, 80 % CI [0.20, 0.33]) and history of stressors (r = 0.13, 80 % CI [0.11, 0.15]) had the strongest associations with injury rates. Also, the results from the path analysis showed that the stress response mediated the relationship between history of stressors and injury rates. For injury prevention studies, all studies included (N = 7) showed decreased injury rates in the treatment groups compared to control groups. CONCLUSION: The results support the model's suggestion that psychosocial variables, as well as psychologically, based interventions, can influence injury risk among athletes.

Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a 'phenotype of interest' (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with 'Fuzzy-Spreader'-like morphologies were also identified through a visual screen. The second, a 'mutant of interest' (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either by DNA-binding proteins or by the architecture of the nucleoid.

Phylogenetic Analysis of "Candidatus Phytoplasma australiense" Reveals Distinct Populations in New Zealand.

ABSTRACT The phytoplasma "Candidatus Phytoplasma australiense" has been reported from New Zealand and Australia, where it has been associated with a range of host plants, especially since the 1970s. Partial tuf gene sequences of 36 New Zealand (NZ) isolates from four different host genera revealed nine different variants, which clustered into two distinct groups without any obvious correlation with host or geographic region. Phylogenetic analysis of these sequences, together with those available from Australian isolates, revealed three distinct clades: one found solely in Australia, one found solely in NZ, and a third with representatives from both countries. These divisions are consistent with differences observed in the 16-23S rRNA internal transcribed spacer region; therefore, we conclude that they represent three distinct subgroups: tuf 1, tuf 2, and tuf 3. We estimated a time of divergence for the three clades based on a synonymous substitution rate calculated by comparing the complete tuf gene sequence from the Loofah witches'-broom phytoplasma and "Candidatus Phytoplasma australiense". Using a calibration date of 110 million years, the estimated time to a common ancestor for all clades (6 to 9 million years ago) suggests divergence during the Miocene, well after the geological separation of NZ and Australia.

The SNPlex genotyping system: a flexible and scalable platform for SNP genotyping.

We developed the SNPlex Genotyping System to address the need for accurate genotyping data, high sample throughput, study design flexibility, and cost efficiency. The system uses oligonucleotide ligation/polymerase chain reaction and capillary electrophoresis to analyze bi-allelic single nucleotide polymorphism genotypes. It is well suited for single nucleotide polymorphism genotyping efforts in which throughput and cost efficiency are essential. The SNPlex Genotyping System offers a high degree of flexibility and scalability, allowing the selection of custom-defined sets of SNPs for medium- to high-throughput genotyping projects. It is therefore suitable for a broad range of study designs. In this article we describe the principle and applications of the SNPlex Genotyping System, as well as a set of single nucleotide polymorphism selection tools and validated assay resources that accelerate the assay design process. We developed the control pool, an oligonucleotide ligation probe set for training and quality-control purposes, which interrogates 48 SNPs simultaneously. We present performance data from this control pool obtained by testing genomic DNA samples from 44 individuals. in addition, we present data from a study that analyzed 521 SNPs in 92 individuals. Combined, both studies show the SNPlex Genotyping system to have a 99.32% overall call rate, 99.95% precision, and 99.84% concordance with genotypes analyzed by TaqMan probe-based assays. The SNPlex Genotyping System is an efficient and reliable tool for a broad range of genotyping applications, supported by applications for study design, data analysis, and data management.

Complete DNA Sequence of Pseudomonas syringae pv. actinidiae, the Causal Agent of Kiwifruit Canker Disease.

Pseudomonas syringae pv. actinidiae is the causal agent of bacterial canker of kiwifruit, a disease that has rapidly spread worldwide. We have fully sequenced and assembled the chromosomal and plasmid DNA from P. syringae pv. actinidiae ICMP 18884 using the PacBio RS II platform.

Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data.

Changes in gene copy number are important in the setting of precision medicine. Recent studies have established that copy number alterations (CNAs) can be detected in sequencing libraries prepared by hybridization-capture, but there has been comparatively little attention given to CNA assessment in amplicon-based libraries prepared by PCR. In this study, we developed an algorithm for detecting CNAs in amplicon-based sequencing data. CNAs determined from the algorithm mirrored those from a hybridization-capture library. In addition, analysis of 14 pairs of matched normal and breast carcinoma tissues revealed that sequence data pooled from normal samples could be substituted for a matched normal tissue without affecting the detection of clinically relevant CNAs (>|2| copies). Comparison of CNAs identified by array comparative genomic hybridization and amplicon-based libraries across 10 breast carcinoma samples showed an excellent correlation. The CNA algorithm also compared favorably with fluorescence in situ hybridization, with agreement in 33 of 38 assessments across four different genes. Factors that influenced the detection of CNAs included the number of amplicons per gene, the average read depth, and, most important, the proportion of tumor within the sample. Our results show that CNAs can be identified in amplicon-based targeted sequencing data, and that their detection can be optimized by ensuring adequate tumor content and read coverage.

Deep sequencing of HPV16 genomes: A new high-throughput tool for exploring the carcinogenicity and natural history of HPV16 infection.

For unknown reasons, there is huge variability in risk conferred by different HPV types and, remarkably, strong differences even between closely related variant lineages within each type. HPV16 is a uniquely powerful carcinogenic type, causing approximately half of cervical cancer and most other HPV-related cancers. To permit the large-scale study of HPV genome variability and precancer/cancer, starting with HPV16 and cervical cancer, we developed a high-throughput next-generation sequencing (NGS) whole-genome method. We designed a custom HPV16 AmpliSeq™ panel that generated 47 overlapping amplicons covering 99% of the genome sequenced on the Ion Torrent Proton platform. After validating with Sanger, the current "gold standard" of sequencing, in 89 specimens with concordance of 99.9%, we used our NGS method and custom annotation pipeline to sequence 796 HPV16-positive exfoliated cervical cell specimens. The median completion rate per sample was 98.0%. Our method enabled us to discover novel SNPs, large contiguous deletions suggestive of viral integration (OR of 27.3, 95% CI 3.3-222, P=0.002), and the sensitive detection of variant lineage coinfections. This method represents an innovative high-throughput, ultra-deep coverage technique for HPV genomic sequencing, which, in turn, enables the investigation of the role of genetic variation in HPV epidemiology and carcinogenesis.

Polymorphisms in the DNA repair genes XPD, XRCC1, XRCC3, and APE/ref-1, and the risk of lung cancer among male smokers in Finland.

Associations between lung cancer risk and common polymorphisms in the DNA repair genes xeroderma pigmentosum complementation group D (XPD), X-ray repair cross-complementing group 1 (XRCC1), XRCC3 and apurinic/apyrimidinic endonuclease/redox factor 1 were examined within a randomized clinical trial designed to determine whether alpha-tocopherol, beta-carotene, or both would reduce cancer incidence among male smokers in Finland. We found no direct association between lung cancer risk and any of the DNA repair genotypes studied, however, the association between XPD codon 751 genotype and lung cancer was modified by alpha-tocopherol supplementation, and the association between XRCC1 codon 399 genotype and lung cancer was modified by the amount of smoking. Our results suggest that common alterations in single DNA repair genes are not major determinants of lung cancer susceptibility among smokers.

DIASPORE MORPHOLOGY AND SEED DISPERSAL IN SEVERAL WIND-DISPERSED ASTERACEAE.

I made measurements of morphology and settling velocity on seeds of 19 species of wind-dispersed Asteraceae. From the morphological measurements I calculated Reynolds numbers and approximate plume loadings for the species. Diaspore settling velocity increases linearly with the square root of plume loading. This relationship varies among species and among subfamilies, but not among life history types. Reynolds number is highly variable among subfamilies, less so within subfamilies. Diaspores with beaked achenes have significantly lower settling velocities than diaspores with unbeaked achenes, even though beaked and unbeaked achenes do not differ in plume loading or in Reynolds number. Reynolds numbers of all diaspores examined are well above the range in which Stokes' Law applies. I recommend that the use of formulae based on Stokes' Law be curtailed in studies of the relationship between plume loading and settling velocity. The results suggest that many seed characters may have evolved due to selection on dispersal ability. This is in spite of phyletic constraints on morphology reflected in the relative uniformity of Reynolds numbers within subfamilies.

AN ANALYSIS OF VARIABILITY IN SEED SETTLING VELOCITIES OF SEVERAL WIND-DISPERSED ASTERACEAE.

Dispersal is an important life history component. Seed settling velocity may be a useful surrogate for the measurement of dispersal ability in wind-dispersed plants, particularly those whose seeds have plumose dispersal structures. I measured settling velocities on seeds of eight species of Asteraceae, including annuals, biennials, and perennials, and including both native and introduced species. The species are Aster exilis, Picris echioides, Chrysopsis villosa, Heterotheca grandiflora, Conyza bonariensis, Sonchus oleraceous, Senecio vulgaris, and Taraxacum officinale. From these data I estimated components of total variation in seed settling velocities due to differences among species, among plants within species, and among inflorescences and seeds within plants. Significant amounts of variability were found at all levels. Contrasts among mean settling velocities showed that the five introduced species have lower settling velocities than the three native species; this result continues to be true when annuals are considered separately from biennials and perennials. Also, over all eight species, annuals have lower settling velocities than biennials and perennials. Variability among species apparently reflects different dispersal "strategies" employed by the species; these different strategies may be correlated with other life-history traits and with ecological characteristics. Variability within species also may have ecological consequences in that such variability may represent an example of risk-spreading.

Gene-environment interactions between the codon 194 polymorphism of XRCC1 and antioxidants influence lung cancer risk.

X-ray repair cross complementing group 1 (XRCC1) is a DNA repair gene whose polymorphisms appear to influence the risk of lung cancer. We explored the influence of antioxidants on the association between the codon 194 arganine to tryptophan substitution polymorphism of XRCC1 and lung cancer risk. In a case-control study nested within a cohort of tin miners the cases were those diagnosed with lung cancer over 6 years of follow-up (n = 108). Two controls, matched on age and sex, were selected for each case by incidence density sampling. Individuals with the variant Arg194Trp allele seemed to be at lower risk for lung cancer (odds ratio (OR): 0.7, 95% confidence interval (95%CL): 0.4-1.2). Furthermore, high serum alpha-tocopherol (OR: 0.4, 95%CL: 0.2-0.9) and retinol (OR: 0.4, 95%CL: 0.2-0.9) levels were associated with significantly reduced risk of lung cancer among individuals with the Arg194Trp variant allele, but not among individuals with the wild-type genotype. In addition, the Arg194Trp variant reduced the risk of lung cancer associated with increased serum carotenoids compared to those with the homozygous wild-type allele. Our results show that Arg194Trp XRCC1 variant modifies the association between serum antioxidants and lung cancer risk.