Diversity of Babesia and Theileria species in symptomatic and asymptomatic dogs in Croatia

Babesiosis, the disease caused by tick-borne hematozoan parasites of the genus Babesia, is particularly common in dogs, and is caused by several “large” species of Babesia, as well as by an increasing number of “small” species of Babesia, some of which appear to be more closely related to members of the genus Theileria. In this work, blood samples were collected from 848 randomly selected, asymptomatic dogs and from 81 symptomatic dogs, microscopically positive for Babesia, and characterised by PCR and sequence analysis of a fragment of the ssrRNA gene. A prevalence of 3.42% (29 of 848) was found in asymptomatic dogs and sequence analysis revealed the presence of Babesia canis canis in 20 dogs (69%), Babesia gibsoni in six dogs (21%), Babesia canis vogeli in two dogs (7%) and Theileria annae in one dog (3%). In the group of symptomatic dogs, which were all positive by PCR, B. canis canis was the predominant species (78 dogs, or 96%), followed by single infections with B. canis vogeli, Babesia caballi and Theileria equi. Our study has confirmed that dogs are infected with a wide range of both large and small piroplasm species and subspecies, including B. caballi and T. equi, two parasites usually found in horses. The detection of the pathogenic species B. canis canis and B. gibsoni in asymptomatic dogs indicates that the relationship between parasite species/subspecies and clinical signs of infection in dogs deserves further investigation. Finally, the identities of the tick vectors transmitting T. annae and B. caballi remain to be elucidated.     

Cucumis sativus L-type lectin receptor kinase (CsLecRK) gene family response to Phytophthora melonis, Phytophthora capsici and water immersion in disease resistant and susceptible cucumber cultivars

L-type lectin receptor kinase (LecRK) proteins are an important family involved in diverse biological processes such as pollen development, senescence, wounding, salinity and especially in innate immunity in model plants such as Arabidopsis and tobacco. Till date, LecRK proteins or genes of cucumber have not been reported. In this study, a total of 25 LecRK genes were identified in the cucumber genome, unequally distributed across its seven chromosomes. According to similarity comparison of their encoded proteins, the Cucumis sativus LecRK (CsLecRK) genes were classified into six major clades (from Clade I to CladeVI). Expression of CsLecRK genes were tested using QRT-PCR method and the results showed that 25 CsLecRK genes exhibited different responses to abiotic (water immersion) and biotic (Phytophthora melonis and Phytophthora capsici inoculation) stresses, as well as that between disease resistant cultivar (JSH) and disease susceptible cultivar (B80). Among the 25 CsLecRK genes, we found CsLecRK6.1 was especially induced by P. melonis and P. capsici in JSH plants. All these results suggested that CsLecRK genes may play important roles in biotic and abiotic stresses.     

AFLPs detect low genetic diversity for Phytophthora nemorosa and P. pseudosyringae in the US and Europe

In California and Oregon, two recently described oomycete forest pathogens, Phytophthora nemorosa and P. pseudosyringae, overlap in their host and geographic ranges with the virulent P. ramorum, causal agent of “sudden oak death.” Epidemiological observations, namely broader geographic distribution and lack of landscape-level mortality, led to the hypothesis they are native to this region, whereas multiple lines of evidence indicate P. ramorum is exotic to North America. We used AFLP analysis to measure genetic variability in the homothallic P. nemorosa and P. pseudosyringae and to evaluate the hypothesis of endemism. We analysed 39 P. nemorosa and 48 P. pseudosyringae isolates (29 American and 19 European) from throughout their geographic and host ranges. In the US, both P. nemorosa and P. pseudosyringae have a dominant AFLP clone with several closely related variants. There is no evidence that genetic diversity is partitioned by host or location in P. nemorosa, but the US P. pseudosyringae clonal lineage is largely nested within a more genetically variable European group. Though the absence of highly variable sampled source populations does not allow us to determine whether each species is native or introduced in the western US with certainty, the results are most consistent with the hypothesis that both are introduced — P. pseudosyringae perhaps from Europe. Invasive Phytophthora species are increasingly being implicated in emergent forest diseases, highlighting the need to identify and characterize both native and previously unknown introduced forest Phytophthoras.     

Mitochondrial DNA polymorphisms in Phytophthora infestans: New haplotypes are identified and re-defined by PCR

Polymorphisms of mitochondrial DNA (mt-DNA) are particularly useful for monitoring specific pathogen populations like Phytophthora infestans. Basically type I and II of P. infestans mt-DNA were categorized by means of polymorphism lengths caused by an ~ 2 kb insertion, which can be detected via restriction enzyme digestion. In addition genome sequencing of haplotype Ib has been used as a simple Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR–RFLP) method to indirectly identify type I and II alterations through EcoR I restriction enzyme DNA fragment patterns of the genomic P4 area. However, with the common method, wrong mt-DNA typing occurs due to an EcoR I recognition site mutation in the P4 genomic area. Genome sequencing of the four haplotypes (Ia, Ib, IIa, and IIb) allowed us to thoroughly examine mt-DNA polymorphisms and we indentified two hypervariable regions (HVRs) named HVRi and HVRii. The HVRi length polymorphism caused by a 2 kb insertion/deletion was utilized to identify mt-DNA types I and II, while another length polymorphism in the HVRii region is caused by a variable number of tandem repeats (n = 1, 2, or 3) of a 36 bp sized DNA stretch and was further used to determine mt-DNA sub-types, which were described as R_{n=1, 2, or 3}. Finally, the P. infestans mt-DNA haplotypes were re-defined as IR₁ or IIR₂ according to PCR derived HVRi and HVRii length polymorphisms. Twenty-three isolates were chosen to verify the feasibility of our new approach for identifying mt-DNA haplotypes and a total of five haplotypes (IR₁, IR₂, IR₃, IIR₂ and IIR₃) were identified. Additionally, we found that six isolates determined as type I by our method were mistakenly identified as type II by the PCR–RFLP technique. In conclusion, we propose a simple and rapid PCR method for identification of mt-DNA haplotypes based on sequence analyses of the mitochondrial P. infestans genome.     

Two novel species representing a new clade and cluster of Phytophthora

Phytophthora stricta sp. nov. and Phytophthora macilentosa sp. nov. are described based on morphological, physiological and molecular characters in this study. Phytophthora stricta represents a previously unknown clade in the rRNA internal transcribed spacer (ITS)-based phylogeny. Phytophthora macilentosa, along with nine other species, consistently forms a high temperature-tolerant cluster within ITS clade 9. These observations are supported by the sequence analysis of the mitochondrial cytochrome c oxidase 1 gene. Both species are heterothallic and all examined isolates are A1 mating type. Phytophthora stricta produces nonpapillate and slightly caducous sporangia. This species is named after its characteristic constrictions on sporangiophores. Phytophthora macilentosa produces nonpapillate and noncaducous sporangia, which are mostly elongated obpyriform with a high length to breadth ratio. Both species were recovered from irrigation water of an ornamental plant nursery in Mississippi, USA and P. stricta was also recovered from stream water in Virginia, USA.     

变叶海棠及其伴生植物峨眉小檗的水分利用策略

变叶海棠(Malus toringoides)是分布在我国四川省甘孜州炉霍县亚高山地区的野生植物种,其嫩叶可制茶,为一种纯正的天然保健珍品。它根系发达、抗逆性强,也是川西亚高山干旱地区退化植被生态恢复的良好树种。运用氢稳定同位素示踪技术,比较分析了变叶海棠和伴生植物峨眉小檗(Berberis aemulans)茎水与其潜在水源(降水、土壤水和河水)的δD值,结果表明:变叶海棠与峨眉小檗植物水主要来源于降水和深层土壤水。生长在河边的变叶海棠并不利用河水。在干季,降水10-20 mm后,变叶海棠对降水的利用率为33.50%-70.06%,而峨眉小檗为26.17%-45.17%;在雨季,降水10-25 mm后,变叶海棠对降水的利用率为40.64%-69.01%,而峨眉小檗为28.44%-71.41%;无论干季还是雨季,两种植物在雨后对降水利用的格局相似,但变叶海棠对降水的利用率皆显著高于峨眉小檗(P<0.01)。两种植物水分利用策略与其根系分布相一致。为川西甘孜州亚高山干旱地区退化植被生态恢复的树种选择以及变叶海棠的扩繁与利用提供科学理论依据。     

Prenylated flavanones and flavanonols as chemical markers in Glycosmis species (Rutaceae)

Fifteen prenylated or geranylated flavanones and flavanonols were isolated from the leaf extracts of different Glycosmis species collected in Thailand and Malaysia. All structures were elucidated by spectroscopic methods, especially 1D and 2D NMR. Six compounds were described for the first time and two were only known so far as synthetic products. The chemotaxonomic significance of flavanoid accumulation within the genus Glycosmis is highlighted.     

Influence of the medium-solidifying agent, the nutrient, and the genotype on the production of gametangia by Phytophthora ramorum in vitro

The effect of different parameters, including the type of nutrients, the quality of the gelling agent, and the genotype of the strain, were evaluated in the production of gametangia by Phytophthora ramorum in vitro. By comparing different agar sources on a carrot-based medium, a delay or a failure in the production of oospores was observed in pairings carried out on media supplemented with technical agar. In contrast, oospores were produced on other agar types, the production on media supplemented with agarose being slightly higher. The formation of gametangia was also influenced by the genotype of the strains involved in the pairing. A European A1 strain producing very few chlamydospores was found to be a better mating partner than other A1 strains. Using a carrot-agarose medium and selected genotypes, all European isolates were characterized in terms of mating type. A macroscopic experiment highlighted a particular spatial distribution of P. ramorum oospores in vitro. A method using polycarbonate membrane was evaluated to assess the selfing ability of P. ramorum.     

异质性光照下匍匐茎草本狗牙根克隆整合的耗益

在一盆栽实验中,将匍匐茎草本植物狗牙根克隆分株对进行异质性光照处理,对远端分株(幼年)分别进行不同程度的遮荫,并对分株对之间的匍匐茎进行保持连接或是切断处理。结果显示:在中度遮荫时,切断匍匐茎连接显著降低了远端分株的生物量和净光合速率(Pn);在重度遮荫时,切断匍匐茎连接显著降低了远端分株的分株数、叶片数、生物量、净光合速率(Pn)、最大光量子产量(Fv/Fm)和实际光量子产量(ФPSⅡ),克隆整合缓解了遮荫胁迫对远端分株生长的影响。在中度遮荫时,保持匍匐茎连接显著提高了近端分株的净光合速率(Pn);在重度遮荫时,保持匍匐茎连接显著提高了近端分株的最大光量子产量(Fv/Fm)、实际光量子产量(ФPSⅡ)和净光合速率(Pn),克隆整合使得相连未受胁迫近端分株的光合效率表现出补偿性增加。相比于匍匐茎切断处理,当远端分株遭受重度遮荫胁迫时,克隆整合引起近端未受胁迫分株生物量显著降低,而整个克隆片段生物量没有显著变化。因此,当远端分株遭受重度遮荫胁迫时,匍匐茎草本狗牙根可能采取一种风险分摊策略以降低基株死亡风险,这样一种策略对于维持克隆植物在异质性生境中基株适合度具有重要意义。     

Distribution of littorine and other alkaloids in the roots of datura species

Littorine, various tropoyl and tigloyl esters and cuscohygrine are components of the roots of all nine Datura species studied. The genus exhibits a uniform alkaloid spectrum and its inclusion, with Solandra, in the tribe Datureae is supported on phytochemical grounds.     

Secondary metabolites from Eurotium species, Aspergillus calidoustus and A. insuetus common in Canadian homes with a review of their chemistry and biological activities

As part of studies of metabolites from fungi common in the built environment in Canadian homes, we investigated metabolites from strains of three Eurotium species, namely E. herbariorum, E. amstelodami, and E. rubrum as well as a number of isolates provisionally identified as Aspergillus ustus. The latter have been recently assigned as the new species A. insuetus and A. calidoustus. E. amstelodami produced neoechinulin A and neoechinulin B, epiheveadride, flavoglaucin, auroglaucin, and isotetrahydroauroglaucin as major metabolites. Minor metabolites included echinulin, preechinulin and neoechinulin E. E. rubrum produced all of these metabolites, but epiheveadride was detected as a minor metabolite. E. herbariorum produced cladosporin as a major metabolite, in addition to those found in E. amstelodami. This species also produced questin and neoechinulin E as minor metabolites. This is the first report of epiheveadride occurring as a natural product, and the first nonadride isolated from Eurotium species. Unlike strains from mainly infection-related samples, largely from Europe, neither ophiobolins G and H nor austins were detected in the Canadian strains of A. insuetus and A. calidoustus tested, all of which had been reported from the latter species. TMC-120 A, B, C and a sesquiterpene drimane are reported with certainty for the first time from indoor isolates, as well as two novel related methyl isoquinoline alkaloids.     

环境因子对养殖条件下出蛰的东北林蛙存活和生长的影响

实验通过在设施环境下和控制条件下,研究出蛰温度、湿度及风速3种环境因素对林蛙生长和存活的影响,目的在于探索东北林蛙(Rana dybowskii)适宜的出蛰环境。结果表明:温度是影响东北林蛙出蛰存活和生长的重要因素,当出蛰温度低于16℃,东北林蛙有较高的成活率,而出蛰温度高于20℃,则会引起70%以上的东北林蛙死亡,出蛰期间温度缓慢提高有利于东北林蛙生长和存活。东北林蛙出蛰期间适宜的湿度在80%以上,湿度低于60%则对东北林蛙不利。出蛰环境以无风为好,有风会造成东北林蛙的死亡。在人工养殖过程中,可依据当地、当年的气候来进行出蛰。出蛰当日平均温度宜在15℃以下。     

Description of Brenneria roseae sp. nov. and two subspecies, Brenneria roseae subspecies roseae ssp. nov and Brenneria roseae subspecies americana ssp. nov. isolated from symptomatic oak

Gram-negative, facultatively anaerobic bacteria were isolated from symptomatic oak tissue in the UK and USA. Partial gyrB sequencing placed ten strains in the genus Brenneria, with B. goodwinii as the closest phylogenetic relative. The strains were investigated further using a polyphasic approach including MLSA (based on partial gyrB, rpoB, infB and atpD gene sequences), 16S rRNA gene sequencing, DNA–DNA relatedness studies and both phenotypic and chemotaxonomic assays. The MLSA and 16S rRNA gene analyses separated the strains into two groups based on origin, suggesting that they belong to Brenneria as two novel species. However, the DNA–DNA relatedness values revealed a closer relationship between the groups and indicated that they should belong to the same species. As the two groups of strains from the UK and USA can be differentiated from each other phenotypically and by ERIC PCR fingerprints, it is proposed to classify them as novel subspecies of a novel Brenneria species. The name Brenneria roseae sp. nov. (FRB 222^T = LMG 27714^T = NCPPB 4581^T) is proposed, with Brenneria roseae subsp. roseae ssp. nov. (FRB 222^T = LMG 27714^T = NCPPB 4581^T) for the strains from the UK and Brenneria roseae subsp. americana ssp. nov. (FRB 223^T = LMG 27715^T = NCPPB 4582^T) for the strains from the USA.     

Structure and development of the style base in Abildgaardia, Bulbostylis, and Fimbristylis (Cyperaceae,Cyperoideae, Abildgaardieae)

The Abildgaardieae tribe within the family Cyperaceae comprises six or seven genera, among which Abildgaardia, Bulbostylis and Fimbristylis pose a challenge regarding their morphological delimitation. Molecular phylogenetic analyses including species of Abildgaardieae are rare, but in most of those studies, Abildgaardia and Fimbristylis appear as more closely related to each other than to the Bulbostylis genus. Duration of the style base has been one of the most widely used characters for delimiting these three genera. The style base is a persistent structure in most species of Bulbostylis and deciduous in Abildgaardia and Fimbristylis. The reasons why the style base may persist or fall off have been scarcely discussed. The assumption that abscission layers are present in the style base of all three genera and the fact that tracheids have been observed in the style base of Bulbostylis suggest that this structure might have histological complexity. In view of this, a complete ontogenetic and anatomical study of the gynoecium has been carried out for all these three genera. It turned out that the style base is histologically simple in Abildgaardia, Bulbostylis and Fimbristylis and shows similar structure and development in all three genera. The fact that the style base has a shorter duration in Abildgaardia and Fimbristylis than in Bulbostylis might be related to the lower number of sclerotised cells that make up such structures in the mature fruit of the former two genera. Abscission of the style and style base may be the result of much simpler reasons than the differentiation of an abscission layer, resulting merely from mechanical shear force effects. Differences among genera have been observed in the shape of the style base and the development of the style. The histological simplicity of the style base is consistent with the homoplastic appearance of this structure in genera that are not closely related (e.g. Rhynchospora). Because of this, while the presence of the thickened style base seems to be a synapomorphy in species of Abildgaardieae, its persistence on or detachment from the fruit might have emerged repeatedly during this clade evolution and might not be a suitable character for genera delimitation.     

Comparative pollen morphology of Brachylena, Tarchananthus and two species of Tubulifloridites (Asteraceae) from the Eocene, Knysna Lignite of South Africa

Two fossil taxa Tubulifloridites antipodica and T. viteauensis recovered from the Eocene Knysna Lignite of South Africa were examined with scanning (SEM) and transmission electron microscopy (TEM). The details of their sculpturing and wall structure are similar to the same species of fossil dispersed pollen taxa recovered from southwestern Africa and South America. Fifteen species of the woody South African taxa, Brachylaena (9 species) and Tarchonanthus (6 species) were investigated with SEM and TEM. All of the taxa are tricolporate, spherical to slightly prolate, microechinate to echinate and have a bilayered columellate infrastructure, except B.ilicifolia, which has a single columellate infrastructural level with the “granularization” of the outer portion of the infrastructural layer or the inner layer of the tectum. There is a similar distribution of plesiomorphic and derived pollen characters in a number of aster subfamilies and tribes suggesting a similar evolutionary progression of pollen, and pollen wall character evolution was occurring synchronously in a variety of aster subfamilies during the middle Tertiary and that these unique pollen features may be important to the evolution and diversification of the Asteraceae.     

Differences in chemical signals may explain species recognition between an island lizard, Podarcis atrata, and related mainland lizards, P. hispanica

Chemical signals can be the basis of interspecific recognition and speciation in many animals. The Columbretes Islands wall lizard, Podarcis atrata is very close genetically to the mainland Iberian wall lizard Podarcis hispanica. However, a previous study suggested that chemosensory interspecific recognition would avoid reproductive interactions and hybridization between these two species. These results suggested that chemicals used in intraspecific communication might differ in composition and/or proportions between these two species. In this paper, we used gas chromatography–mass spectrometry (GC–MS) to characterize the chemical composition of the lipophilic fraction from femoral gland secretions of male P. atrata and P. hispanica. The analysis showed that chemicals found in femoral secretions varied in composition and proportions between species and between populations. Seven steroids and two unidentified waxy esters, were exclusive of P. atrata lizards from the islands. In contrast, nine steroids and other six compounds were only found in mainland P. hispanica. There were also differences in proportions of shared compounds between species. Moreover, all these differences were higher between P. atrata and P. hispanica than between any population of P. hispanica. Chemical differences might be consequence of genetic differences, but they could also be explained by adaptation to different habitats with different climatic conditions or diet resources. Compounds that are specific of each species, or differences in the pattern of compounds, could explain species recognition. Therefore, these results of chemical composition and previous studies of chemosensory recognition reinforce the fact that the genetic differences between P. hispanica and P. atrata may result in an effective reproductive isolation between these two taxa.     

Species delimitation in the European species of Clavulina (Cantharellales, Basidiomycota) inferred from phylogenetic analyses of ITS region and morphological data

The identification of the conventionally accepted species of Clavulina (Cantharellales, Basidiomycota) in Europe (Clavulina amethystina, Clavulina cinerea, Clavulina cristata, and Clavulina rugosa) is often difficult and many specimens are not straightforwardly assignable to any of those four species, which is why some authors have questioned their identity. In order to assess the status of those species, a morphological examination was combined with the molecular analysis of the ITS region. The same six major clades were obtained in the Bayesian and parsimony phylogenetic analyses, and all six clades were well-supported at least by one of the analyses. Morphological characters, such as the overall branching pattern, the presence and intensity of grey colour, the cristation of the apices, and basidiospore size and shape were to various extents correlated with the phylogenetic signal obtained from the ITS region. The congruence between the molecular analyses and morphology, rather than geographical origin, suggests the existence of several species that can be delimited using a combined phylogenetic and morphological species recognition. The analyses revealed that C. cristata and C. rugosa are well-delimited species. In contrast, more than one taxa could be subsumed under the names C. amethystina and C. cinerea, the taxonomical complexity of which is discussed. The ITS region is proved to be adequate to separate phylogenetic species of Clavulina.     

New method for electroporation of Lactobacillus species grown in high salt

We here describe a new method for electroporation of Lactobacillus species, obligately homofermentative and facultatively heterofermentative, based on the cell-wall weakening resulting from growth in high-salt media. For L. casei, optimum transformation efficiency of up to 10⁵ transformants per microgram of plasmid DNA was achieved following growth in the presence of 0.9 M NaCl. Plasmids of different sizes and replication origins were also similarly transformed. These competent cells could be used either directly or stored frozen, up to 1 month, for future use, with similar efficiency. This protocol was assayed with different Lactobacillus species: L. delbrueckii subsp. lactis, L. paracasei, L. plantarum and L. acidophilus, and it was found that they were transformed with similar efficiency.