Delimiting cryptic pathogen species causing apple Valsa canker with multilocus data

Fungal diseases are posing tremendous threats to global economy and food safety. Among them, Valsa canker, caused by fungi of Valsa and their Cytospora anamorphs, has been a serious threat to fruit and forest trees and is one of the most destructive diseases of apple in East Asia, particularly. Accurate and robust delimitation of pathogen species is not only essential for the development of effective disease control programs, but also will advance our understanding of the emergence of plant diseases. However, species delimitation is especially difficult in Valsa because of the high variability of morphological traits and in many cases the lack of the teleomorph. In this study, we delimitated species boundary for pathogens causing apple Valsa canker with a multifaceted approach. Based on three independent loci, the internal transcribed spacer (ITS), β‐tubulin (Btu), and translation elongation factor‐1 alpha (EF1α), we inferred gene trees with both maximum likelihood and Bayesian methods, estimated species tree with Bayesian multispecies coalescent approaches, and validated species tree with Bayesian species delimitation. Through divergence time estimation and ancestral host reconstruction, we tested the possible underlying mechanisms for fungal speciation and host‐range change. Our results proved that two varieties of the former morphological species V. mali represented two distinct species, V. mali and V. pyri, which diverged about 5 million years ago, much later than the divergence of their preferred hosts, excluding a scenario of fungi–host co‐speciation. The marked different thermal preferences and contrasting pathogenicity in cross‐inoculation suggest ecological divergences between the two species. Apple was the most likely ancestral host for both V. mali and V. pyri. Host‐range expansion led to the occurrence of V. pyri on both pear and apple. Our results also represent an example in which ITS data might underestimate species diversity.     

引起蓝莓茎溃疡病的葡萄座腔菌属Botryosphaeria二新种

在我国华南地区发现了两个引起蓝莓茎溃疡病的Bofryosphaeria新种,即B.dolichospermatii和B.fujianensis,本研究对这两个物种进行了详细的形态学描述,并制作了图版.基于/TS、LSU、tef1-α 和TUB等4个基因片段的综合系统学分析,B.dolichospermatii和B.fu...     

PCR-based strategy to detect and identify species of Phaeoacremonium causing grapevine diseases

Species of Phaeoacremonium (especially Phaeoacremonium aleophilum) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants. Phaeoacremonium species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of Phaeoacremonium that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect Phaeoacremonium directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of Phaeoacremonium species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for Phaeoacremonium viticola and Phaeoacremonium angustius, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial beta-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate P. angustius from P. viticola, respectively. An easy and fast protocol was developed to detect and identify species of Phaeoacremonium in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of Phaeoacremonium spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.     

Browsed twig environmental DNA: diagnostic PCR to identify ungulate species

Ungulate browsing can have a strong effect on ecological processes by affecting plant community structure and composition, with cascading effects on nutrient cycling and animal communities. However, in the absence of direct observations of foraging, species‐specific foraging behaviours are difficult to quantify. We therefore know relatively little about foraging competition and species‐specific browsing patterns in systems with several browsers. However, during browsing, a small amount of saliva containing buccal cells is deposited at the bite site, providing a source of environmental DNA (eDNA) that can be used for species identification. Here, we describe extraction and PCR protocols for a browser species diagnostic kit. Species‐specific primers for mitochondrial DNA were optimized and validated using twigs browsed by captive animals. A time series showed that about 50% of the samples will amplify up to 12 weeks after the browsing event and that some samples amplify up to 24 weeks after browsing (12.5%). Applied to samples of natural browsing from an area where moose (Alces alces), roe deer (Capreolus capreolus), fallow deer (Cervus dama) and red deer (Cervus elaphus) are sympatric, amplification success reached 75%. This method promises to greatly improve our understanding of multispecies browsing systems without the need for direct observations.     

PCR-RFLP based diagnostic tests for Moxostoma Species in Ontario

Six Moxostoma species are found in Ontario, including two federally listed species. Knowledge of their distribution and status has been limited by incorrect field identifications, collections that report Moxosotoma or sucker species, and new site records without voucher specimens. To develop species diagnostic genetic markers, we amplified and sequenced two mitochondrial genes (ATPase subunits 6 and 8). Species-specific sequence differences were easily identified (of 830 analyzed bases, 132 substitutions were phylogenetically informative). Based on sequence differences, four restriction enzymes were identified and tested for their reliability as species diagnostic tools. Restriction digests of previously sequenced samples matched predicted species-specific patterns. Overall, 90% of samples digested matched. Disagreement between 20% of samples provided by various government agencies and species-specific digest patterns indicate a need for greater care during field identifications.     

Discrimination between four Simocephalus species from Slovakia using a PCR-RFLP technique

Planktonic crustaceans are traditionally identified based on morphological and morphometric characters. However, such characters may be hardly distinguishable and often overlap between species. A probability of misidentification is thus relatively high. Molecular techniques may increase the accuracy of identification if appropriate markers are used. Aim of our work was to develop a simple molecular procedure enabling discrimination between four species of Simocephalus occurring in Europe. PCR-RFLP technique proved to be suitable for such discrimination. Within the 709 bp fragment of mitochondrial cytochrome c oxidase subunit 1 gene we found unique combinations of restriction sites of the BbsI and SacI enzymes for Simocephalus vetulus, S. exspinosus, S. serrulatus and S. congener. PCR products of samples from several locations in Slovakia were digested with the two enzymes and electrophoresed on an agarose gel. The restriction patterns were clearly visible and easily distinguishable. This method is applicable for identifying the four species in any life-stage. Considering its simplicity and cost-effectiveness it can be widely used as a diagnostic tool for discriminating between Simocephalus species with overlapping morphologic characters.     

A saltwater flotation technique to identify unincubated eggs

ABSTRACT.   Field studies on nesting birds sometimes involve questions related to nest initiation dates, length of the incubation period, or changes in parental incubation behavior during various stages of incubation. Some of this information can be best assessed when a nest is discovered before the eggs have undergone any incubation, and this has traditionally been assessed by floating eggs in freshwater. However, because the freshwater method is not particularly accurate in identifying unincubated eggs, we developed a more reliable saltwater flotation method. The saltwater method involves diluting a saturated saltwater solution with freshwater until a salt concentration is reached where unincubated eggs sink to the bottom and incubated eggs float to the surface. For Laughing Gulls ( Leucophaeus atricilla ), floating eggs in freshwater failed to identify 39.0% ( N = 251) of eggs that were subsequently found by candling to have undergone incubation prior to collection. By contrast, in a separate collection of gull eggs, no eggs that passed the saltwater test ( N = 225) were found by a later candling to have been incubated prior to collection. For Double-crested Cormorants ( Phalacrocorax auritus ), floating eggs in freshwater failed to identify 15.6% ( N = 250) of eggs that had undergone incubation prior to collection, whereas in a separate collection, none of the eggs that passed the saltwater test ( N = 85) were found by a later candling to have been incubated prior to collection. Immersion of eggs in saltwater did not affect embryo survival. Although use of the saltwater method is likely limited to colonial species and requires calibrating a saltwater solution, it is a faster and more accurate method of identifying unincubated eggs than the traditional method of floating eggs in freshwater.     

SeqAnt: A web service to rapidly identify and annotate DNA sequence variations

Background  

The enormous throughput and low cost of second-generation sequencing platforms now allow research and clinical geneticists to routinely perform single experiments that identify tens of thousands to millions of variant sites. Existing methods to annotate variant sites using information from publicly available databases via web browsers are too slow to be useful for the large sequencing datasets being routinely generated by geneticists. Because sequence annotation of variant sites is required before functional characterization can proceed, the lack of a high-throughput pipeline to efficiently annotate variant sites can act as a significant bottleneck in genetics research.     

A mutagenesis-based screen to rapidly identify phosphorylation sites in mitogen-activated protein kinase substrates

Electroporation is an important approach for genetic engineering experiments allowing for introduction of foreign DNA in a selected host. Here, we describe for the first time the use of glycine betaine as an osmoprotectant for electroporation of gram-positive bacteria Bacillus subtilis. High electroporation efficiency (up to 5×10(5) cfu/μg) was obtained using 7.5% glycine betaine. The new method improved the transformation efficiency of B. subtilis with linear integrative DNA nearly 700-fold compared with existing Bacillus transformation techniques.     

A metabolomics approach to characterise and identify various Mycobacterium species

We investigated the potential use of gas chromatography mass spectrometry (GC-MS), in combination with multivariate statistical data processing, to build a model for the classification of various tuberculosis (TB) causing, and non-TB Mycobacterium species, on the basis of their characteristic metabolite profiles. A modified Bligh-Dyer extraction procedure was used to extract lipid components from Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium kansasii cultures. Principle component analyses (PCA) of the GC-MS generated data showed a clear differentiation between all the Mycobacterium species tested. Subsequently, the 12 compounds best describing the variation between the sample groups were identified as potential metabolite markers, using PCA and partial least-squares discriminant analysis (PLS-DA). These metabolite markers were then used to build a discriminant classification model based on Bayes' theorem, in conjunction with multivariate kernel density estimation. This model subsequently correctly classified 2 "unknown" samples for each of the Mycobacterium species analysed, with probabilities ranging from 72 to 100%. Furthermore, Mycobacterium species classification could be achieved in less than 16 h, and the detection limit for this approach was 1×10(3)bacteriamL(-1). This study proves the capacity of a GC-MS, metabolomics pattern recognition approach for its possible use in TB diagnostics and disease characterisation.     

A hybridisation technique to identify anthelmintic resistance genes in Haemonchus

The identification of genes associated with anthelmintic resistance can be facilitated in Haemonchus contortus by the ability of this species to hybridise with Haemonchus placei. Although the hybrid males are sterile, the lines can be rescued by backcrossing the females to either parental species. Resistance genes can be retained in Haemonchus hybrids, while the unwanted contortus background is removed through backcrossing to H. placei and anthelmintic selection of the progeny. Under this selection, genes involved in resistance would retain the H. contortus nucleotide sequence, while those that are not would either be H. placei or a random mixture of both, depending on the amount of backcrossing that had occurred. The first candidate gene to be tested in this system was a Haemonchus P-glycoprotein, hcpgp-1. hcpgp-1 was amplified, cloned and sequenced from H. contortus and H. placei. Two restriction sites were then identified in the sequenced product; one specific to H. contortus hcpgp-1 and the other found only in the H. placei gene. These genes were identified from macrocyclic lactone selected and non-selected worms by restricting PCR products from individual worms. Fitted occurrence of the H. contortus allele was 49% of unselected worms and 69% of macrocyclic lactone selected worms. The probability of this percentage occurring by chance was P=0.006. Thus macrocyclic lactone selection was acting to increase the percentage of hcpgp-1 from macrocyclic-lactone-resistant CAVRS.     

Somatic plant regeneration from selected common cypress (Cupressus sempervirens L.) clones resistant to the bark canker disease

Regeneration of nodular cultures involves the production of new individuals from organogenic nodules. Despite the existence of well-established protocols for bromeliads species, many gaps in understanding the mechanisms of this route still remain, particularly the regeneration of new shoots, a stage little explored. Therefore, this work aimed to monitor the morpho and histodifferentiation processes of shoot regeneration from nodular cultures of Billbergia zebrina. To accomplish this, nodular cultures previously obtained from nodal segments of B. zebrina were transferred to regeneration induction medium consisting of semi-solid MS medium supplemented with 1.0 µM of naphthalene acetic acid and 2.0 µM of benzylaminopurine, respectively. Cultures were maintained in a growth chamber over 8 weeks, and samples were collected weekly for analysis under light, transmission electron, and scanning electron microscopy. Cells in the shoot formation region presented characteristics of mitotically active cells, such as the presence of numerous plasmodesmata and conspicuous microtubules. New shoots maintained vascular connection to the node. Cells binding the nodule vascular bundle toward the shoot showed degeneration of organelles, which, in turn, originated vascular neoelements. The dome presented a depressed or prominent surface, depending on shoot development. Rounded stomata were conspicuous in the leaf primordia and generally higher in number as compared to the epidermal surface. This study elucidated the nodular culture in vitro system in aspects associated to the regeneration of shoots and it is anticipated that such data will lead to the optimization of present nodular cultures based protocols with the aim of conserving endangered bromeliad species.     

A new approach to identify species in Xiphinema americanum sensu lato

Four putative species belonging to the X. americanum group are known to transmit American nepoviruses and these nematodes and viruses are listed in European quarantine legislation. Identification of species in this group is therefore of particular importance for phytosanitary purposes, but is problematic because of the similar morphology of the putative species. As part of the Synthesys project BE-TAF 1769, eight collaborative institutions contributed material to Fera for study. Video technology allowed the best records of type material and in conjunction with photographic images created a 'virtual' collection of images that relies less on the deteriorating quality of museum specimens. Revised definitions of lip region and tail shape are considered to be the most stable characters for differentiation. Position of the amphidial aperture, development of the odontostyle collar and some established morphometric characters are considered of limited use although they retain value for latter stages of identification.     

FiberID--a technique to identify fibrous protein subclasses

Fibrous proteins such as collagen, silk, and elastin play critical biological roles, yet they have been the subject of few projects that use computational techniques to predict either their class or their structure. In this article, we present FiberID, a simple yet effective method for identifying and distinguishing three fibrous protein subclasses from their primary sequences. Using a combination of amino acid composition and fast Fourier measurements, FiberID can classify fibrous proteins belonging to these subclasses with high accuracy by using two standard machine learning techniques (decision trees and Naïve Bayesian classifiers). After presenting our results, we present several fibrous sequences that are regularly misclassified by FiberID as sequences of potential interest for further study. Finally, we analyze the decision trees developed by FiberID for potential insights regarding the structure of these proteins. Proteins 2007. © 2006 Wiley‐Liss, Inc.     

Multiple nuclear gene sequences identify phylogenetic species boundaries in the rapidly radiating clade of Mexican ambystomatid salamanders

Delimiting the boundaries of species involved in radiations is critical to understanding the tempo and mode of lineage formation. Single locus gene trees may or may not reflect the underlying pattern of population divergence and lineage formation, yet they constitute the vast majority of the empirical data in species radiations. In this study we make use of an expressed sequence tag (EST) database to perform nuclear (nDNA) and mitochondrial (mtDNA) genealogical tests of species boundaries in Ambystoma ordinarium, a member of an adaptive radiation of metamorphic and paedomorphic salamanders (the Ambystoma tigrinum complex) that have diversified across terrestrial and aquatic environments. Gene tree comparisons demonstrate extensive nonmonophyly in the mtDNA genealogy of A. ordinarium, while seven of eight independent nuclear loci resolve the species as monophyletic or nearly so, and diagnose it as a well-resolved genealogical species. A differential introgression hypothesis is supported by the observation that western A. ordinarium localities contain mtDNA haplotypes that are identical or minimally diverged from haplotypes sampled from a nearby paedomorphic species, Ambystoma dumerilii, while most nDNA trees place these species in distant phylogenetic positions. These results provide a strong example of how historical introgression can lead to radical differences between gene trees and species histories, even among currently allopatric species with divergent life history adaptations and morphologies. They also demonstrate how EST-based nuclear resources can be used to more fully resolve the phylogenetic history of species radiations.     

A simple PCR-RFLP method for identification and differentiation of 11 Malassezia species

A PCR-RFLP method targeted toward 26S rDNA and with 2 restriction enzymes, CfoI and BstF51, was developed to identify 11 Malassezia species. Not only type and standard strains but also 13 clinical isolates were identified successfully in this study. The results of identifications were confirmed by DNA sequencing.