Botryosphaeriaceae as potential pathogens of prunus species in South Africa, with descriptions of Diplodia africana and Lasiodiplodia plurivora sp. nov

Botryosphaeriaceae are common dieback and canker pathogens of woody host plants, including stone fruit trees. In the present study the diversity of members of the Botryosphaeriaceae isolated from symptomatic wood of Prunus species (plum, peach, nectarine and apricot) was determined in stone fruit-growing areas in South Africa. Morphological and cultural characteristics as well as DNA sequence data (5.8S rDNA, ITS-1, ITS-2 and EF-1a) were used to identify known members and describe novel members of Botryosphaeriaceae. From the total number of wood samples collected (258) 67 isolates of Botryosphaeriaceae were obtained, from which eight species were identified. All species were associated with wood necrosis. Diplodia seriata (= "Botryosphaeria" obtusa) was dominant, and present on all four Prunus species sampled, followed by Neofusicoccum vitifusiforme and N. australe. First reports from Prunus spp. include N. vitifusiforme, Dothiorella viticola and Diplodia pinea. This is also the first report of D. mutila from South Africa. Two species are newly described, namely Lasiodiplodia plurivora sp. nov. from P. salicina and Diplodia africana sp. nov. from P. persica. All species, except Dothiorella viticola, caused lesions on green nectarine and/or plum shoots in a detached shoot pathogenicity assay.     

Response of Scots pine (Pinus sylvestris) seedlings subjected to artificial infection with the fungus Sphaeropsis sapinea

Plant Molecular Biology Reporter - The ascomycete Sphaeropsis sapinea (syn. Diplodia pinea), the causal agent of Diplodia blight of pine, is also known to be an endophyte with a latent pathogenic...     

High‐resolution melting analysis: a genotyping tool for population studies on Daphnia

Determining genetic variation at the DNA level within and between natural populations is important for understanding the role of natural selection on phenotypic traits, but many techniques of screening for genetic variation are either cost intensive, not sensitive enough or too labour‐ and time‐consuming. Here, we demonstrate high‐resolution melting analysis (HRMA) as a cost‐effective and powerful tool for screening variable target genes in natural populations. HRMA is based on monitoring the melting of PCR amplicons. Owing to saturating concentrations of a dye that binds at high concentrations to double‐stranded DNA, it is possible to genotype high numbers of samples rapidly and accurately. We analysed digestive trypsins of two Daphnia magna populations as an application example for HRMA. One population originated from a pond containing toxic cyanobacteria that possibly produce protease inhibitors and the other from a pond without such cyanobacteria. The hypothesis was that D. magna clones from ponds with cyanobacteria have undergone selection by these inhibitors, which has led to different trypsin alleles. We first sequenced pooled genomic PCR products of trypsins from both populations to identify variable DNA sequences of active trypsins. Second, we screened variable DNA sequences of each D. magna clone from both populations for single nucleotide polymorphisms via HRMA. The HRMA results revealed that both populations exhibited phenotypic differences in the analysed trypsins. Our results indicate that HRMA is a powerful genotyping tool for studying the variation of target genes in response to selection within and between natural Daphnia populations.     

Early detection of Biscogniauxia nummularia in symptomless European beech (Fagus sylvatica L.) by TaqMan quantitative real-time PCR

AIMS: To develop a quantitative real-time PCR (Rt PCR) assay for the early detection of Biscogniauxia nummularia, a xylariaceous fungus that causes strip-canker and wood decay on European beech (Fagus sylvatica L.). METHODS AND RESULTS: The molecular assay was based on TaqMan chemistry using species-specific primers and a fluorogenic probe designed on the ITS1 sequence of rRNA gene clusters. The specificity of the oligonucleotides and the probe were tested using the DNA of B. nummularia isolates from different geographic areas, of phylogenetically related species, and of some fungi commonly colonizing European beech bark and wood. A total of 31 symptomless and symptomatic shoots of European beech were collected from three forest sites in the Apennine Mountains of Italy. The percentage of positive detections of B. nummularia with the TaqMan assay was 78.6%, compared with only 14.3% of positive isolations on growth media for two sites. CONCLUSIONS: In shoots, the quantitative Rt PCR assay detected down to 8.0-fg fungal DNA per microgram of total DNA extracted. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed in quantitative Rt PCR, by using TaqMan chemistry, revealed a rapid and sensitive method useful for the early detection of B. nummularia in symptomless European beech twigs.     

Diverse sources of infection and cryptic recombination revealed in South African Diplodia pinea populations

This study considers the population diversity and structure of Diplodia pinea in South Africa at different spatial scales from single trees to plantations, as well as comparing infections on healthy and diseased trees. A total of 236 isolates were characterized using 13 microsatellite markers. Analysis of these markers confirmed previous results that D. pinea has a high level of gene and genotypic diversity in South Africa, with the latter values ranging from 6% to 68% for the different plantations. The data also reflect a fungus with randomly associated alleles in populations at local plantation scales and for the population as a whole. These results suggest that recombination is occurring in D. pinea and that it most likely has a cryptic sexual state. The study also reveals the sources of endophytic infection and stress related disease out-breaks as diverse infections that have occurred over a long time period. In contrast, wound-associated die-back appears to be caused by clones of the pathogen occurring in narrow time frames.     

Scanning of mutations in short amplicons: Optimization of DNA melting method

The high resolution melting analysis (HRMA) is a new highly efficient method for genotyping and mutation scanning. HRMA is conducted immediately after PCR in closed-tube format, which enables the high throughput of the method. However, the closed-tube format makes HRMA dependent on the conditions of PCR and, thus, limits its capabilities. The open-tube format, which we have already developed (postamplification shortening of amplicons and optimized composition of ion medium), is applicable to the scanning of mutations of the K-RAS oncogene in tumor tissue and formalin-fixed paraffin-embedded samples. It is found that the open-tube format of DNA melting significantly increases the sensitivity of finding mutant alleles when using instruments both with and without an HRMA module. The higher sensitivity of the DNA melting compared to “Sanger” sequencing allows one to decrease the number of false-negative results of the mutation test, which is highly important for some forms of cancer.     

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA). This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts. Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.     

DNA-Based Identification of <Emphasis Type="Italic">Valeriana officinalis</Emphasis> s.l.: a Multiplexed Amplification Refractory Mutation System (MARMS) and High Resolution Melting Curve Analysis (HRMA)

The wide distribution of Valeriana officinalis as a herbal remedy as well as the considerably higher concentration of putative mutagenic valepotriate metabolites in other drug-delivering valerian species like Valeriana procera Kunth and Valeriana jatamansi Jones ex Roxb. illustrate the necessity of secure authentication of roots of Valeriana officinalis s.l., especially as the morphologically similar roots of the acutely toxic Veratrum album can be mistaken for those of Valeriana officinalis. We developed two DNA-based systems, a multiplex amplification refractory mutation system (MARMS), and a high-resolution melting curve analysis (HRMA) assay, both based on a sequence mutation within the atpB-rbcL region. With both methods, identification of Valeriana officinalis s.l. was possible. With the HRMA, the characteristic melting curve of 33 samples of Valeriana officinalis s.l. and of two commercial samples of Valerianae radix was distinct from the melting curves of all other Valeriana species (60 accessions), and from the closely related genera Centranthus and Valerianella. Since adulteration of Valeriana with toxic Veratrum species was reported previously, Veratrum primers were included in a multiplex PCR-HRM analysis. This system allowed the detection of a Veratrum admixture down to the level of 0.01 %. Although the advantages, in terms of sensitivity, specificity and practicality of the HRM for analysis of degraded plant material were superior to the MARMS assay, both methods are suitable for routine analysis. The results demonstrated the general ability of HRMA to detect specific (toxic) adulterations in drugs in a semiquantitative way.     

DNA phylogeny, morphology and pathogenicity of Botryosphaeria species on grapevines

Several species of Botr yosphaeria are known to occur on grapevines, causing a wide range of disorders including bud mortality, dieback, brown wood streaking and bunch rot. In this study the 11 Botryosphaeria spp. associated with grapevines growing in various parts of the world, but primarily in South Africa, are distinguished based on morphology, DNA sequences (ITS-1, 5.8S, ITS-2 and EF1-α) and pathological data. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina and a Diplodia sp. are confirmed from grapevines in South Africa, while Diplodia porosum, Fusicoccum viticlavatum and F. vitifusiforme are described as new. Although isolates of B. dothidea and B. stevensii are confirmed from grapevines in Portugal, neither of these species occurred in South Africa, nor were any isolates of B. ribis confirmed from grapevines. All grapevine isolates from Portugal, formerly presumed to be B. ribis, are identified as B. parva based on their EF1-α equence data. From artificial inoculations on grapevine shoots, we conclude that B. australis, B. parva, B. ribis and B. stevensii are more virulent than the other species studied. The Diplodia sp. collected from grapevine canes is morphologically similar but phylogenetically distinct from D. sarmentorum. Diplodia sarmentorum is confirmed as anamorph of Otthia spiraeae, the type species of the genus Otthia (Botryosphaeriaceae). A culture identified as O. spiraeae clustered within Botryosphaeria and thus is regarded as probable synonym. These findings confirm earlier suggestions that the generic concept of Botryosphaeria should be expanded to include genera with septate ascospores and Diplodia anamorphs.     

Genetically depauperate but widespread: the case of an emblematic Mediterranean pine

Genetic variation is generally considered a prerequisite for adaptation to new environmental conditions. Thus the discovery of genetically depauperate but geographically widespread species is unexpected. We used 12 paternally inherited chloroplast microsatellites to estimate population genetic variation across the full range of an emblematic circum-Mediterranean conifer, stone pine (Pinus pinea L.). The same chloroplast DNA haplotype is fixed in nearly all of the 34 investigated populations. Such a low level of variation is consistent with a previous report of very low levels of diversity at nuclear loci in this species. Stone pine appears to have passed through a severe and prolonged demographic bottleneck, followed by subsequent natural- and human-mediated dispersal across the Mediterranean Basin. No other abundant and widespread plant species has as little genetic diversity as P. pinea at both chloroplast and nuclear markers. However, the species harbors a nonnegligible amount of variation at adaptive traits. Thus a causal relationship between genetic diversity, as measured by marker loci, and the evolutionary precariousness of a species, cannot be taken for granted.     

Systemic induction of phloem secondary metabolism and its relationship to resistance to a canker pathogen in Austrian pine

The mechanisms and conditions affecting expression of systemic induced resistance (SIR) in pine are not clearly understood. Two hypotheses were tested here: that SIR against a pathogen induced by either a pathogen or an insect involves coordinated shifts in phloem secondary metabolism; and that fertility affects the production of these compounds. To test these hypotheses, a tripartite system was used comprising Austrian pine (Pinus nigra) grown under three different fertility regimes, the fungal pathogen Diplodia pinea, and the defoliator Neodiprion sertifer. Fungal induction led to systemic accumulation of lignin, phenolic glycosides and stilbenes, whereas insect defoliation led to an increase in germacrene D concentration in branch phloem. Fertility affected the concentrations of only the phenolic glycosides. Multivariate analyses showed coregulation of compounds within at least three consistent groupings: phenolic glycosides, stilbenes and monoterpenes. As groups and as individual compounds, accumulation of phenolic glycosides and stilbenes was negatively correlated with disease susceptibility. The experimental manipulation of the phenolics and terpenoids metabolic networks achieved in this study by biotic induction and changes in nutrient availability suggests that lignin, phenolic glycosides and stilbenes are important biochemical factors in the expression of SIR against the pathogen in this system.     

Differentiation of Staphylococcus spp. by high-resolution melting analysis

High-resolution melting analysis (HRMA) is a fast (post-PCR) high-throughput method to scan for sequence variations in a target gene. The aim of this study was to test the potential of HRMA to distinguish particular bacterial species of the Staphylococcus genus even when using a broad-range PCR within the 16S rRNA gene where sequence differences are minimal. Genomic DNA samples isolated from 12 reference staphylococcal strains (Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri, and Staphylococcus xylosus) were subjected to a real-time PCR amplification of the 16S rRNA gene in the presence of fluorescent dye EvaGreen?, followed by HRMA. Melting profiles were used as molecular fingerprints for bacterial species differentiation. HRMA of S. saprophyticus and S. xylosus resulted in undistinguishable profiles because of their identical sequences in the analyzed 16S rRNA region. The remaining reference strains were fully differentiated either directly or via high-resolution plots obtained by heteroduplex formation between coamplified PCR products of the tested staphylococcal strain and phylogenetically unrelated strain.     

Chloroplast DNA Diversity among Trees, Populations and Species in the California Closed-Cone Pines (Pinus Radiata, Pinus Muricata and Pinus Attenuata)

The amount, distribution and mutational nature of chloroplast DNA polymorphisms were studied via analysis of restriction fragment length polymorphisms in three closely related species of conifers, the California closed-cone pines-knobcone pine: Pinus attenuata Lemm.; bishop pine: Pinus muricata D. Don; and Monterey pine: Pinus radiata D. Don. Genomic DNA from 384 trees representing 19 populations were digested with 9-20 restriction enzymes and probed with cloned cpDNA fragments from Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] that comprise 82% of the chloroplast genome. Up to 313 restriction sites were surveyed, and 25 of these were observed to be polymorphic among or within species. Differences among species accounted for the majority of genetic (haplotypic) diversity observed [G(st) = 84(+/-13)%]; nucleotide diversity among species was estimated to be 0.3(+/-0.1)%. Knobcone pine and Monterey pine displayed almost no genetic variation within or among populations. Bishop pine also showed little variability within populations, but did display strong population differences [G(st) = 87(+/-8)%] that were a result of three distinct geographic groups. Mean nucleotide diversity within populations was 0.003(+/-0.002)%; intrapopulation polymorphisms were found in only five populations. This pattern of genetic variation contrasts strongly with findings from study of nuclear genes (allozymes) in the group, where most genetic diversity resides within populations rather than among populations or species. Regions of the genome subject to frequent length mutations were identified; estimates of subdivision based on length variant frequencies in one region differed strikingly from those based on site mutations or allozymes. Two trees were identified with a major chloroplast DNA inversion that closely resembled one documented between Pinus and Pseudotsuga.     

Candida arabinofermentans, a new L-arabinose fermenting yeast

Candida arabinofermentans (type strain NRRL YB-2248, CBS 8468), a new yeast that ferments the pentose L-arabinose, is described. The three known strains of this new species were isolated from insect frass of pine and larch trees in the U.S. Phylogenetic analysis of nucleotide sequences from the D1/D2 domain of large subunit (26S) ribosomal DNA places C. arabinofermentans among the methanol-assimilating yeasts and most closely related to Candida ovalis. Strains of the new species produce 0.7-1.9 g/l ethanol from L-arabinose.     

The large pine weevil Hylobius abietis (L.) as a potential vector of the pathogenic fungus Diplodia sapinea (Fr.) Fuckel

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Rapid Identification of Medically Important Candida Isolates Using High Resolution Melting Analysis

An increasing trend in non albicans infections and various susceptibility patterns to antifungal agents implies a requirement for the quick and reliable identification of a number of medically important Candida species. Real-time PCR followed by high resolution melting analysis (HRMA) was developed, tested on 25 reference Candida collection strains and validated on an additional 143 clinical isolates in this study. All reference strains and clinical isolates inconclusive when using phenotypic methods and/or HRMA were analysed using ITS2 sequencing. Considering reference and clinical strains together, 23 out of 27 Candida species could be clearly distinguished by HRMA, while the remaining 4 species were grouped in 2 pairs, when applying the mean Tm ± 3 SD values, the shape of the derivative melting curve (dMelt curve) and, in some cases, the normalized and temperature—shifted difference plot against C. krusei. HRMA as a simple, rapid and inexpensive tool was shown to be useful in identifying a wide spectrum of clinically important Candida species. It may complement the current clinical diagnostic approach based on commercially available biochemical kits.     

California pignolia: Seeds of pinus sabiniana

The pignolia nut or seed of the Italian stone pine (Pinus pinea) is. well-known as a food item, especially in Mediterranean cuisine. By contrast, the seed of Pinus sabiniana, a species of pine found only in California and once much utilized by the aboriginal population, is little-known commercially. When examined in terms of nutritional value, shell-to-kernel ratio, and kernel weight, the seeds of these 2 species appear to be similar. It would seem that the difference between them for commercial purposes is the 2000-yr cultivation of P. pinea in Europe as opposed to the general neglect of the California species, at least since the Euro-american domination of the area.     

Efficient species identification of pine marten (<Emphasis Type="Italic">Martes martes</Emphasis>) and red fox (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>) scats using a 5′ nuclease real-time PCR assay

Monitoring wildlife species by DNA identification of samples collected non-invasively is an important tool in conservation management. DNA identification of species from faecal (scat) samples is problematic due to the small quantities and poor quality of the DNA isolated from such samples. This study demonstrates the use of real-time PCR technology in the identification of red fox (Vulpes vulpes) and pine marten (Martes martes). It is shown that real-time PCR can be used to identify fox and pine marten by either melting curve analysis (Tm determination) with SYBR Green 1 detection or by the use of species specific fluorogenic probes. The technique is shown to work efficiently with scat DNA.     

Accurate identification of closely related Dendrobium species with multiple species-specific gDNA probes

About 63 species of Dendrobium are identified in China, making the identification of the origin of a particular Dendrobium species on the consumer market very difficult. We report evaluation of multiple species-specific probes screened from genomic DNA for closely related Dendrobium species identification, based on DNA array hybridization. Fourteen species-specific probes were screened from five closely related Dendrobium species, D. aurantiacum Kerr, D. officinale Kimura et Migo, D. nobile Lindl., D. chrysotoxum Lindl. and D. fimbriatum Hook., based on the SSH-Array technology we developed. Various commercial Dendrobium samples and unrelated samples were definitely identified. The specificity and accuracy of the multiple species-specific probes for species identification was assessed by identifying various commercial Dendrobium samples (Herba Dendrobii). Hybridization patterns of these multiple probes on digested genomic DNAs of Dendrobium species indicated that there are distinct polymorphic sequence fragment in the higher eukaryotes. This is the first report on detection and utilization of multiple species-specific probes of Dendrobium in whole genomic DNA, and this could be useful tools not only for a new technical platform for the closely related species identification but also for epidemiological studies on higher eukaryotes.     

Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: Identification of novel mutations

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene.