Genetic diversity in Monilinia laxa populations in stone fruit species in Hungary

The objectives of this study were firstly, to determine the genetic diversity of Monilinia laxa isolates from Hungary, using the PCR-based inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) technique; secondly, to prepare genetic diversity groups based on the dendrograms; and finally, to select some relevant isolates to study their fungicide sensitivity. 55 and 77 random amplified polymorphic ISSR and RAPD markers, of which 23 and 18 were polymorphic and 32 and 59 monomorphic, respectively, were used to assess the genetic diversity and to study the structure of M. laxa populations in Hungary. 27 isolates out of 57 ones were confirmed as M. laxa from several orchards (subpopulations) in three geographical regions, in various inoculum sources and in various hosts, were used. 10 fungicides and 12 isolates selected from genetic diversity groups based on the ISSR dendrograms were used to determine the fungicide sensitivity of the selected isolates. The analysis of population structure revealed that genetic diversity within locations, inoculum sources and host (H _S ) accounted for 99 % of the total genetic diversity (H _T ), while genetic diversity among locations, inoculum sources and host represented only 1 %. The relative magnitude of gene differentiation between subpopulations (G _ST ) and the estimate of the number of migrants per generation (Nm) averaged 0.005–0.009 and 53.9–99.2, respectively, for both ISSR and RAPD data set. The results obtained in dendrograms were in accordance with the gene diversity analysis. Grouping of isolates in the dendrograms was irrespective of whether they came from the same or different geographical locations. There was no relationship between clustering among isolates from inoculum sources and hosts. In the fungicide sensitivity tests, five isolates out of 12 were partly insensitive to boscalid+piraclostrobin, cyprodinil, fenhexamid or prochloraz. Obtained results in genetic diversity of M. laxa populations are discussed together with implications for the management of brown rot.     

Circulating tumor cell isolation,culture, and downstream molecular analysis

Circulating tumor cells (CTCs) are a major contributor of cancer metastases and hold a promising prognostic significance in cancer detection. Performing functional and molecular characterization of CTCs provides an in-depth knowledge about this lethal disease. Researchers are making efforts to design devices and develop assays for enumeration of CTCs with a high capture and detection efficiency from whole blood of cancer patients. The existing and on-going research on CTC isolation methods has revealed cell characteristics which are helpful in cancer monitoring and designing of targeted cancer treatments. In this review paper, a brief summary of existing CTC isolation methods is presented. We also discuss methods of detaching CTC from functionalized surfaces (functional assays/devices) and their further use for ex-vivo culturing that aid in studies regarding molecular properties that encourage metastatic seeding. In the clinical applications section, we discuss a number of cases that CTCs can play a key role for monitoring metastases, drug treatment response, and heterogeneity profiling regarding biomarkers and gene expression studies that bring treatment design further towards personalized medicine.     

Monilinia species causing brown rot of peach in China

In this study, 145 peaches and nectarines displaying typical brown rot symptoms were collected from multiple provinces in China. A subsample of 26 single-spore isolates were characterized phylogenetically and morphologically to ascertain species. Phylogenetic analysis of internal transcribed spacer (ITS) regions 1 and 2, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), β-tubulin (TUB2) revealed the presence of three distinct Monilinia species. These species included Monilinia fructicola, Monilia mumecola, and a previously undescribed species designated Monilia yunnanensis sp. nov. While M. fructicola is a well-documented pathogen of Prunus persica in China, M. mumecola had primarily only been isolated from mume fruit in Japan. Koch's postulates for M. mumecola and M. yunnanensis were fulfilled confirming pathogenicity of the two species on peach. Phylogenetic analysis of ITS, G3PDH, and TUB2 sequences indicated that M. yunnanensis is most closely related to M. fructigena, a species widely prevalent in Europe. Interestingly, there were considerable differences in the exon/intron structure of the cytochrome b (Cyt b) gene between the two species. Morphological characteristics, including spore size, colony morphology, lesion growth rate, and sporulation, support the phylogenetic evidence suggesting the designation of M. yunnanensis as a new species. A new multiplex PCR method was developed to facilitate the detection of M. yunnanensis and differentiation of Monilinia spp. causing brown rot of peach in China.     

Northern species of earth tongue genus Thuemenidium revisited, considering morphology, ecology and molecular phylogeny

Thuemenidium is a small earth tongue genus with three recognized white-spored species. Within Thuemenidium, T. atropurpureum and T. arenarium have been reported only from the northern hemisphere while T. berteroi is known solely in the southern hemisphere. We reviewed the ecology, examined the morphology and inferred the systematic positions of northern species of Thuemenidium from LSU-rDNA gene phylogeny of 48 taxa in Pezizomycotina including recent collections. Our results suggest that Thuemenidium in its current sense is polyphyletic and that T. atropurpureum, closely related to Microglossum and Leotia species, is a member of Leotiaceae (Helotiales, Leotiomycetes). Our phylogeny placed the other northern species, T. arenarium, in Geoglossaceae (Geoglossales, Geoglossomycetes), retaining genus Thuemenidium, pending further investigation.     

Sex determination of three raptor species using morphology and molecular techniques

ABSTRACT.   Accurate identification of sex is important for many raptor studies, but may be difficult to determine in the field for some species. Because of size differences between males and females, morphological measurements have often been used to sex raptors. However, few investigators have examined the accuracy of using measurements of individuals made at one location to sex individuals of the same species at another location. Our objective was to develop more accurate region-specific methods for determining the sex of Red-tailed Hawks ( Buteo jamaicensis ), Red-shouldered Hawks ( B. lineatus ), and Cooper's Hawks ( Accipiter cooperii ) migrating through and wintering in California. We determined sex using a polymerase chain reaction (PCR)-based genetic test and grouped individuals based on sex, age, and geographic area. We did not combine groups due to differences in measurements between age classes and geographic areas. We then compared a suite of morphological measurements between males and females of each combination, and developed both a discriminant function and a flowchart to determine the sex of Red-tailed and Red-shouldered hawks in the field. The flowcharts were more accurate than the functions for both species. We also confirmed the accuracy of the current flowchart used to determine the sex of Cooper's Hawks migrating along the California coast. These region-specific methods for Red-tailed and Cooper's hawks were generally more accurate than published methods, possibly indicating different populations of these species and highlighting the importance of validating sexing methods when using them in different locations.     

New record for Galba neotropica (d'Orbigny, 1835) in Argentina,with a detailed analysis of its morphology and molecular characteristics

We report for the first time the presence of Galba neotropica in Santa Fe province, Pampean Argentina. Until the present work, the identity of the intermediate host of Fasciola hepatica in this region, the most important livestock production area of Argentina, was unknown. This report extends the geographic distribution of G. neotropica and is the first to provide molecular and morphological information on this species in Argentina. During summer 2013, snails were collected on private livestock farms with high prevalence of fasciolosis in cattle. Specimens were identified by using the nuclear sequences of the internal transcribed spacers ITS-1 and ITS-2, the shell and the shape and size of the male reproductive organs. Molecularly, ITS-1 and ITS-2 nuclear sequences exhibit 100% nucleotide identity with G. neotropica from Lima, Peru. Morphologically, G. neotropica from Santa Fe province was indistinguishable from Galba viatrix and Galba cubensis, the other two cryptic species within this South American group of lymnaeids. The combination of molecular and morphological analyses is strongly recommended to identify lymnaeids at species level. The identity of the intermediate host and the subsequent knowledge of its susceptibility, behaviour, distribution, ecology and biology are important components in developing effective measures to control fasciolosis.     

Propagule morphology and river characteristics shape secondary water dispersal in tree species

Plant migration is a multi-stage process often driven by multiple dispersal vector systems. Water-mediated dispersal (hydrochory) is known to move propagules of nonaquatic species over long distances, but whether propagule morphology affects floating processes is an open question. We used a multi-species approach to assess the role of propagule morphology in the dispersal of primarily wind-dispersed tree species in different urban rivers; the impact of hydraulic structures (locks, spillways) on floating was also considered. We released tagged propagules of eight tree species (Acer platanoides, Acer negundo, Acer saccharinum, Ailanthus altissima, Fraxinus excelsior, Robinia pseudoacacia, Tilia platyphyllos, Ulmus glabra) in the main lowland Spree River and in the small tributary Panke River (Berlin, Germany) and directly observed the fate of the floating propagules over river sections of 1,200 m. Our results demonstrate the following: (1) Water is an effective dispersal agent for wind-dispersed tree species, extending typical wind-related transport distances by several times. (2) Interspecific differences in transport distances reflect propagule characteristics (dry weight, maximum wing width) and river system. (3) Propagule morphology also affects deposition patterns as it was generally the large propagules that were trapped along semi-natural embankments in slow flow areas. (4) Hydraulic structures hampered but did not entirely stop water-mediated dispersal and diminished the effects of propagule morphology on floating processes. These results provide novel insights into the functioning of hydrochory as an important dispersal vector of tree species in river systems and as a driver of plant invasions.     

Main airborne Ascomycota spores: characterization by culture, spore morphology, ribosomal DNA sequences and enzymatic analysis

The aim of this work was to identify the main allergy-related Ascomycetes fungal spores present in the atmosphere of Porto, using different and complementary techniques. The atmospheric sampling, performed in the atmosphere of Porto (Portugal) from August 2006 to July 2008, indicated Cladosporium, Penicillium, Aspergillus and Alternaria as the main fungal spore taxa. Alternaria and Cladosporium peaks were registered during summer. Aspergillus and Penicillium highest values were registered from late winter to early spring. Additionally, the Andersen sampler allowed the culture and isolation of the collected viable spores subsequently used for different identification approaches. The internal-transcribed spacer region of the nuclear ribosomal repeat unit sequences of airborne Ascomycetes fungi isolates revealed 11 taxonomically related fungal species. Among the identified taxa, Penicillum and Aspergillus presented the highest diversity, while only one species of Cladosporium and Alternaria, respectively, were identified. All selected fungal spore taxa possessed phosphatase, esterase, leucine arylamidase and β-glucosidase enzymatic activity, while none had lipase, cystine arylamidase, trypsin or β-glucuronidase activity. The association between the spore cell wall morphology, DNA-based techniques and enzymatic activity approaches allowed a more reliable identification procedure of the airborne Ascomycota fungal spores.     

Postharvest biocontrol of Monilinia laxa,Monilinia fructicola and Monilinia fructigena on stone fruit by two Aureobasidium pullulans strains

The antagonistic effects of yeasts, L1 and L8, isolated from carposphere of ‘Redhaven’ peaches were tested for the first time in the same experiment against three Monilinia species (Monilinia laxa, Monilinia fructicola and Monilinia fructigena) in in vitro and in vivo trials. The two antagonists were selected after preliminary assays for their ability to reduce brown rot in peaches and nectarines, and both were identified by molecular and morphological tools as Aureobasidium pullulans. In in vivo trials, neither the autoclaved cells, nor the sterile culture filtrates of either antagonist showed any significant reduction of rot incidence produced by inocula of the three Monilinia species, while the washed cells of L1 and L8 completely inhibited M. laxa and M. fructicola rots and reduced M. fructigena infections by 70% and 90%, respectively. In other trials, nectarines treated with antagonist cells and inoculated with the pathogens were stored at 0 °C for 21 days, plus 7 days at 20 °C. The low temperature reduced brown rot development, since all fruit were free from disease symptoms on removal from cold storage. However after 7 d at 20 °C, untreated fruit were rotted over 45% depending on the Monilinia species but the antagonists completely inhibited M. laxa and M. fructicola, while M. fructigena infections were reduced by 89.8% and 91.2% by L1 and L8, respectively. For both strains, 10⁸ CFU ml^?1 was the most active concentration, although L1 showed good activity at a concentration of 10⁷ CFU ml^?1. Isolate L8 at the concentration of 10⁷ CFU ml^?1 was ineffective against M. fructicola and M. fructigena, showing no difference between treated fruit and the control, excepting the case of nectarines inoculated with M. laxa, where L8 at the concentration of 10⁷ CFU ml^?1 reduced the brown rot infections with respect to the control. The increase in population density of A. pullulans strains L1 and L8 in the wounds of nectarines stored at 0° or 20 °C was low but sufficient to control brown rot. In conclusion, the present preliminary study identified two antagonistic strains of A. pullulans as active ingredients for the development of biofungicides for postharvest application against three Monilinia species that are responsible for high economic losses in stone fruit crops.     

Diospyros species in Thailand: Their distribution, fruit morphology and uses

Diospyros species distributed widely in Thailand were classified into four ecotypes, according to their habitat; constantly humid area, alternately dry and wet area, mountainous cool area and all area. Some of them inhabit near dwelling areas or in the paddy field in the village. The young fruit is covered with dense pubescence in most species. The size, shape, and color of mature fruit greatly vary greatly with the species. In most species, the mature fruit has a soft pulp and hard skin. The fruit of six species has been used for dying. Four species produce edible fruits, with color and flavor favorable for breeding of edibleDiospyros species. The fruit of some species contains some chemicals useful as fish poisoning or of medicines, although the active components have not yet been identified. The edible fruit contained many tannin cells, but the fruit used as fish poisoning and medicines had only a few.     

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology

We describe the isolation and the ultrastructural characteristics of adult bovine articular chondrocytes in vitro. Slices of bovine articular cartilage undergo sequential digestions with pronase and collagenase in order to release cells. Chondrocytes are plated at high density (1 x 10(5) cells/cm2) in culture dishes or roller bottles with Ham's F-12 medium, supplemented with 10% fetal bovine serum. Before culture, chondrocytes are freed of surrounding territorial matrix. Within the first few days of culture they re-establish a territorial matrix. As time progresses, chondrocytes synthesize both territorial and extraterritorial matrices. The matrices are rich in collagen fibrils and ruthenium red-positive proteoglycans. These features are most apparent in mass roller cultures in which aggregates of cells and matrix appear as long streaks and nodules. This morphology reveals an organization of chondrocytes and their matrices that is similar to that of the parent articular cartilage in vivo.     

Trees within trees: genes and species, molecules and morphology

The construction and interpretation of gene trees is fundamental in molecular systematics. If the gene is defined in a historical (coalescent) sense, there can be multiple gene trees within the single contiguous set of nucleotides, and attempts to construct a single tree for such a sequence must deal with homoplasy created by conflict among divergent histories. On a larger scale, incongruence is expected among gene tree topologies at different loci of individuals within sexually reproducing species, and it has been suggested that this discordance can be used to delimit species. A practical concern for such topological methods is that polymorphisms may be maintained through numerous cladogenic events; this polymorphism problem is less of a concern for nontopological approaches to species delimitation using molecular data. Although a central theoretical concern in molecular systematics is discordance between a given gene tree and the true "species tree," the primary empirical problem faced in reconstructing taxic phylogeny is incongruence among the trees inferred from different sequences. Linkage relationships limit character independence and thus have important implications for handling multiple data sets in phylogenetic analysis, particularly at the species level, where incongruence among different historically associated loci is expected. Gene trees can also be reconstructed for loci that influence phenotypic characters, but there is at best a tenuous relationship between phenotypic homoplasy and homoplasy in such gene trees. Nevertheless, expression patterns and orthology relationships of genes involved in the expression of phenotypes can in theory provide criteria for homology assessment of morphological characters.     

Community analysis of Belarusian lakes: relationship of species diversity to morphology, hydrology and land use

In the glacial lake district of northern Belarus, limnologistscollected extensive biological, chemical and physical data on550 lakes (51% of all Belarusian glacial lakes). This largedata set provided a unique opportunity to examine subtle relationshipswith great statistical power. Our purpose was to use multivariateand correlation analyses to explore relationships of speciesrichness with morphological and hydrological parameters. A multivariateanalysis of the environmental data suggests that the Belarusianlakes can be separated along gradients of size, hydraulic residencetime and watershed development (land use). In most instances,species richness for major planktonic and benthic groups wascorrelated significantly with lake size and land use. Speciesrichness values were correlated less with watershed size orhydraulic residence time. In each community, there was a groupof species characterized by higher correlations with principalcomponent analysis (PCA) axes. These groups are as follows:for phytoplankton—diatoms, for zooplankton—rotifers,and for zoobenthos—molluscs. For lakes both in pristineand developed watersheds, we found small but significant negativespecies–area correlations for littoral crustaceans. Asurvey of the relevant literature shows scale dependence forthe correlation between species richness and lake size. Forpelagic crustaceans, the species–area correlation wassignificant (and positive) for lakes with developed watershedsbut not for pristine watersheds.     

三种毛枝藻的培养研究及其异丝性特征

毛枝藻属Stigeoclonium植物是异丝性藻类, 由直立系统和匍匐系统共同组成; 直立系统的形态高度变化, 虽然匍匐系统的形态比直立系统的形态稳定, 但野外采来的标本匍匐系统不全, 影响鉴定的效果。将采自野外的10号毛枝藻属样的标本通过实验室培养, 观察到它们从孢子萌发至匍匐系统和直立系统发育全过程的细节, 确定其中3种毛枝藻的种名为夏毛枝藻S. aestivale (Haz.) Coll.、偏生毛枝藻S. subsecundum Kütz.和丰满毛枝藻S. farctum Berthold。夏毛枝藻和偏生毛枝藻孢子萌发大致为匍匐式, 夏毛枝藻匍匐系统是分枝的丝状体, 不产生假根, 细胞圆球形或短圆柱形, 内具叶绿体; 偏生毛枝藻的匍匐系统是假二叉分枝的假根, 假根由单列长圆柱形细胞组成, 最后叶绿体消失变为无色; 丰满毛枝藻的孢子萌发是严格的匍匐式, 匍匐系统由主枝和分枝构成假薄壁组织的盘状结构。这3种毛枝藻的匍匐系统都各具其独特而持久的形态特征。它们的直立系统都是分枝的丝状体, 夏毛枝藻和偏生毛枝藻分枝较多, 一侧生或互生, 无对生, 枝顶尖细或为毛状, 前者细胞宽7-9 μm, 长17-30 μm, 长为宽的3-4倍; 后者宽10-14 μm, 长18-36 μm, 长为宽的2-3倍, 细胞外胶质层厚。丰满毛枝藻分枝稀少或不发育, 细胞宽8-10 μm, 长9-32 μm, 长为宽的2-4倍。但此3种毛枝藻直立系统的形态特点是不稳定而可变的, 故毛枝藻的分类应主要参考它们的匍匐系统而定。首次报道了中国产毛枝藻的培养研究。     

Morphological and molecular characteristics of selected species of Minidiscus (Bacillariophyta,Thalassiosiraceae)

We report on taxonomically distinguishing morphological features of six diatom taxa from the marine, nanoplanktonic genus, Minidiscus, found in Eastern Canada and neighbouring waters. Four of the six were also investigated using genetic approaches. We found that the culture deposited in the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) as CCMP 496 was morphologically closest to the published type image of M. trioculatus, whereas other isolates were morphologically and/or genetically divergent. Consequently, we propose three new taxa. One monoclonal line isolated from the Bay of Fundy is proposed as a new variety, M. trioculatus var. monoculatus. The well-studied nanoplanktonic diatom strain, CCMP 495, currently considered to be ‘M. trioculatus’ was morphologically and genetically divergent from CCMP 496 and we propose specific status for it, M. variabilis. We isolated a new strain from the Gulf of St Lawrence that was nearly genetically identical in 18S and ITS regions to CCMP 495, but showed distinct morphological differences and we propose a new form for this strain. Culture-based data were supplemented by those from natural seawater whenever possible. Two species (M. comicus, M. chilensis) known from our region, but unavailable in culture, are presented for comparison.     

Drivers of macrofungal species composition in temperate forests,West Hungary: functional groups compared

The most influential environmental drivers of macrofungal species composition were studied in managed, even-aged, mixed forests of Őrség National Park, Hungary. Functional groups of macrofungi were analyzed separately by non-metric multidimensional scaling and redundancy analysis exploring their relations to tree species composition, stand structure, soil/litter conditions, microclimate, landscape, and management history. There was some evidence that macrofungi are related to drivers that are relatively easy to measure. Wood-inhabiting fungal species composition is driven primarily by the species composition of living trees, while substratum properties and microclimate play minor roles. The terricolous saprotrophic community was determined principally by a litter pH gradient involving tree species composition and soil/litter properties. Microclimate had no concordant effect. No obvious underlying gradients were detected on ectomycorrhizal fungal species composition; however, tree size and litter pH had significant effects. For each group, no clear responses to landscape or management history were detected.     

Phylogenetic relationships of Italian Bellevalia species (Asparagaceae), inferred from morphology,karyology and molecular systematics

The seven Bellevalia species and subspecies known from Italy, representing about 10% of the genus and three out of six sections, were studied. An integrated morphological, karyological and molecular approach was used to infer phylogenetic and systematic relationships among them. B. romana (the generitype) is the most distinctive species on karyotype asymmetry grounds. B. boissieri and B.dubia, usually considered as subspecies of one species (the latter endemic to Sicily), deserve specific status based on biparental nrDNA markers (internal transcribed spacer, ITS), since they do not form a single clade. The allotetraploid endemic B. pelagica, morphologically similar to B. romana, is sister to the latter under parsimony, both in morphological and ITS trees; it is also related with B. dubia, based on karyotype asymmetry and a uniparental cpDNA marker (trnL^(UAA)–trnF^(GAA) IGS (intergenic spacer)). A second allotetraploid endemic, B. webbiana, is closely related, on morphological, karyological and molecular grounds, with B. boissieri and B. ciliata, and also with B. trifoliata, three species that might all involved in its origin. B. sect. Conicae Feinbr. and sect. Nutantes Feinbr. are here typified, the former (type: B. ciliata) is most likely a synonym of the latter (type: B. trifoliata).