Systematics of Hanseniaspora Zikes and Kloeckera Janke

The physiological and morphological characteristics of eighty-two strains of Hanseniaspora and Kloeckera, representing twenty-nine described species, were examined. These results along with DNA base composition and DNA/DNA reassociation experiments revealed that the genus Hanseniaspora comprises six distinct species, viz. H. valbyensis, H. uvarum, H. guilliermondii, H. occidentalis, H. osmophila and H. vineae, with K. japonica, K. apiculata, K. apis, K. javanica, K. corticis and K. africana, respectively, as their imperfect states.     

Saccharomyces cerevisiae CCMI 885 secretes peptides that inhibit the growth of some non-Saccharomyces wine-related strains

The nature of the toxic compounds produced by Saccharomyces cerevisiae CCMI 885 that induce the early death of Hanseniaspora guilliermondii during mixed fermentations, as well as their ability to inhibit the growth of other non-Saccharomyces wine-related strains, was investigated. The killing effect of mixed supernatants towards H. guilliermondii was inactivated by protease treatments, thus revealing the proteinaceous nature of the toxic compounds. Analysis of the protein pattern of mixed supernatants on Tricine SDS-PAGE showed that this S. cerevisiae strain secretes peptides (<10 kDa), which were detected only when death of H. guilliermondii was already established. Death-inducing supernatants were ultrafiltrated by 10 and 2 kDa membranes, respectively, and the inhibitory effect of those permeates were tested in H. guilliermondii cultures. Results indicated that the (2–10) kDa protein fraction of those supernatants seemed to contain antimicrobial peptides active against H. guilliermondii. Thus, the (2–10) kDa protein fraction was concentrated and its inhibitory effect tested against strains of Kluyveromyces marxianus, Kluyveromyces thermotolerans, Torulaspora delbrueckii and H. guilliermondii. Under the growth conditions used for these tests, the (2–10) kDa protein fraction of S. cerevisiae CCMI 885 supernatants exhibited a fungistatic effect against all the strains and a fungicidal effect against K. marxianus.     

Deoxyribonucleic acid homologies among staphylococci: Coagulase-positive reference strains

DNA hybridization results confirm the proposed separation of coagulase-positive staphylococci into two distinct species. Strains ofStaphylococcus aureus representing the various biotypes and different phage typing groups of the human biotype gave high values of reassociation with DNA fromS. aureus reference strain RN 450, at both optimal and restrictive reassociation temperatures. Similar results were obtained between strains ofS. intermedius and its reference strain K 3. Interspecific reassociation between the two coagulase-positive species was low, and each reference strain showed low DNA sequence homology with 10 coagulase-negative species.S. staphylolyticus, strain PS 73, and putative pleiotropic mutants ofS. aureus were shown to be unrelated toS. aureus.     

Structure of rat liver nuclei after depletion and reassociation of histone H1

Chromatin in isolated rat liver nuclei was compared with chromatin in (i) nuclei depleted of H1 by acid extraction; (ii) nuclei treated at pH 3.2 (without removal of H1), and (iii) depleted nuclei following reassociation of H1. Electron microscopy and digestion by DNase I, micrococcal nuclease and endogenous Ca/Mg endonuclease were used for this comparative examination. Electron micrographs of H1-depleted nuclei showed a dispersed and finely granular appearance. The rate of DNA cleavage by micrococcal nuclease or DNase I was increased several-fold after H1 removal. Discretely sized intermediate particles produced by Ca/Mg endonuclease in native nuclei were not observed in digests of depleted nuclei. Digestion by micrococcal nuclease to chromatin particles soluble in 60 mM NaCl buffer appeared not to be affected in depleted nuclei. When nuclei were treated at pH 3.2, neither the appearance of chromatin in electron micrographs nor the mode or rate of nuclease digestion changed appreciably. Following reassociation of H1 to depleted nuclei, electron micrographs demonstrated the reformation of compacted chromatin, but the lower rate of DNA cleavage in native nuclei was not restored. Further, H1 reassociation produced a significant decrease in the solubility of nuclear chromatin cleaved by micrococcal nuclease or Ca/Mg endonuclease. In order to evaluate critically the reconstitution of native chromatin from H1-depleted chromatin we propose the use of digestion by a variety of nucleases in addition to an electron microscopic examination.     

Presence of <Emphasis Type="Italic">Ascosphaera apis</Emphasis>, the causative agent of chalkbrood disease,in honeybees <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera: Apidae) in Japan

The presence of Ascosphaera apis, a fungus that is the causative agent of chalkbrood disease, was surveyed in Japan using a diagnostic polymerase chain reaction (PCR). A total of 336 individual European honeybees Apis mellifera were taken from 25 different apiaries in various regions of Japan. Of the 112 colonies surveyed, A. apis was detected in 27 colonies (24.1%). Positive results by PCR were obtained from 49 out of 336 surveyed individuals (14.6%). Based on these results, the distribution of A. apis in A. mellifera is widespread across Japan and does not exhibit significant differences between geographic areas. DNA sequences of the ITS and 5.8S rRNA region from all 17 isolates of A. apis were identical, even though they were from geographically distinct areas in Japan. It is suggested that no intra-species variation may be due to a recent bottleneck effect probably caused by humans before geographical expansion of the fungus.     

Growth promotion effect of Pichia guilliermondii in chilli and biocontrol potential of Hanseniaspora uvarum against Colletotrichum capsici causing fruit rot

In the present investigation, we have attempted to identify the potential two epiphytic yeast strains for growth promotion and management of chilli fruit rot. Seed treatment with Pichia guilliermondii showed increased seedling vigour index (55%), fresh weight (96%) and dry weight (45%) over untreated control. Furthermore, P. guilliermondii showed higher root colonisation ability, indole-3-acetic acid (IAA) production and phosphate solubilisation ability. On the other hand, seedling dip with Hanseniaspora uvarum induced higher levels of defence-related compounds in chilli seedlings challenge-inoculated with Colletotrichum capsici under glasshouse conditions. Among the different media tested, higher biomass of P. guilliermondii and H. uvarum was obtained in pine juice broth and sugarcane juice broth, respectively. Glycerol buffer formulation showed viability (>70%) of P. guilliermondii up to 4 months and H. uvarum up to 9 months when stored at ambient conditions. Seedling dip and foliar sprays with H. uvarum showed 37– 40% reduction in chilli fruit rot incidence under field conditions. It also showed higher (cumulative) accumulation of defence-related compounds in chilli leaves and ripe fruits under field conditions. The results of current investigation indicated a clear difference among the two epiphytic yeast strains. P. guilliermondii was identified as growth promoter of chilli and H. uvarum as antagonist of chilli fruit rot pathogen, C. capsici.     

Differentiation of metaxylem cell line in the root ofAllium cepa

Summary The first step of differentiation in the root segments ofAllium cepa containing metaxylem cells in different stages of differentiation were studied by DNA reassociation curves and compared to meristem cell extracted DNA. Upon sonication of DNA samples to about 400 base pairs, the reassociation profiles of the heat denatured DNA, were spectrophotometrically followed at two different concentrations. The kinetic complexities,i.e., the number of base pairs per haploid genome of a given sequence and its redundancy were calculated. Differences were found at the level of highly and medium repetitive sequences, thus demonstrating that some DNA reassociation classes may undergo amplification during root development.     

Phylogenetic analysis of Methanobrevibacter isolated from feces of humans and other animals

Comparative 16S rRNA gene sequence and genomic DNA reassociation analyses were used to assess the phylogenetic relationships of Methanobrevibacter fecal isolates. The 16S rRNA gene sequences of Methanobrevibacter smithii strain PS and the human fecal isolates B181 and ALI were essentially identical, and their genomic DNA reassociated at values greater than 94%. The analysis of 16S rRNA sequences of the horse, pig, cow, rat, and goose fecal isolates confirm that they are members of the genus Methanobrevibacter. They had a high degree of sequence similarity (97–98%) with the 16S rRNA gene of M. smithii, indicating that they share a common line of descent. The 16S rRNA genes of the horse and pig isolates had 99.3% sequence similarity. Sequence analysis of the 16S rRNA gene of the sheep fecal isolate showed that it formed a separate line of descent in the genus Methanobrevibacter. Genomic DNA reassociation studies indicate that the horse, pig, cow, and goose fecal isolates represent at least three new species. The horse and pig isolates were the only animal isolates that had > 70% genomic DNA reassociation and represent strains of a single species. The cow, goose, and sheep isolates had little or no genomic DNA reassociation with M. smithii or with each other. The relationship of the rat isolate to the other animal isolates was not determined. An evaluation of the relationship of 16S rRNA gene sequence similarity and genomic DNA reassociation of Methanobrevibacter and other methanogenic archaea indicated that genomic DNA reassociation studies are necessary to establish that two methanogenic organisms belong to the same species. Received: 17 November 1997 / Accepted: 16 January 1998     

Ultrastructure of Hanseniaspora ascospores

A comparative study of the ultrastructure in sections of the ascospores of six Hanseniaspora species showed three types of spores: (1) hat-shaped in H. valbyensis and H. guilliermondii, (2) spherical with an equatorial or subequatorial ledge, smooth or rough in H. occidentalis and H. uvarum, (3) spherical with warts in H. osmophila and H. vineae. Development and germination of the spores of Hanseniaspora guilliermondii is described in more detail.This investigation was supported by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) I     

蜜蜂球囊菌的microRNA鉴定及其调控网络分析

【目的】本研究利用small RNA-seq技术对球囊菌的纯培养进行测序,对球囊菌的micro RNAs miRNAs)进行预测、鉴定和分析,进而构建miRNAs-mRNAs的调控网络。【方法】利用Illumina Hiseq Xten平台对球囊菌菌丝与孢子进行测序,通过相关生物信息学软件对球囊菌的miRNAs进行预测和分析,通过茎环(Stem-loop)PCR对部分miRNAs进行鉴定,利用Cytoskype软件构建miRNAs-mRNAs的调控网络。【结果】本研究共获得48268696条clean reads,预测出118个球囊菌的miRNAs,它们的长度分布介于18–25 nt之间,不同长度的mi RNA的首位碱基偏好性差异明显。Stem-loop PCR验证结果显示共有10个miRNAs能够扩增出符合预期的目的片段,说明多数miRNAs可能真实存在。共预测出6529个球囊菌miRNAs的靶基因,其中5725个能够注释到Nr、Swissprot、KOG、GO和KEGG数据库。进一步分析结果显示有24个靶基因注释在MAPK信号通路。Cytoskype软件分析结果显示球囊菌的miRNAs与mRNAs之间存在复杂的调控网络,绝大多数的miRNAs处于调控网络的内部且同时结合多个mRNAs。【结论】本研究率先对球囊菌的miRNAs及miRNAs-mRNAs调控网络进行全面分析,研究结果丰富了对球囊菌miRNAs的认识,为其基础生物学信息提供了有益补充,也为阐明球囊菌致病的分子机理打下了一定基础。     

侵染中华蜜蜂6日龄幼虫的蜜蜂球囊菌的微小RNA差异表达谱及调控网络

【目的】蜜蜂球囊菌(Ascosphaeraapis,简称球囊菌)是专性侵染蜜蜂幼虫的致死性真菌病原。MicroRNA(miRNA)作为一类重要的基因表达调控因子,能够广泛参与真菌及其宿主的相互作用过程。本研究通过比较分析球囊菌孢子(AaCK)和侵染中华蜜蜂(Apis cerana cerana,简称中蜂) 6日龄幼虫肠道内的球囊菌(AaT)的smallRNA(sRNA)组学数据对球囊菌的差异表达miRNA(differentiallyexpressed miRNA,DEmiRNA)、靶mRNA及二者间的调控网络进行全面解析,旨在揭示miRNA介导的球囊菌对中蜂幼虫的侵染机制。【方法】对于球囊菌侵染的中蜂6日龄幼虫肠道的small RNA-seq (sRNA-seq)数据,利用BLAST工具连续比对东方蜜蜂(Apiscerana)和球囊菌的参考基因组筛滤得到AaT的sRNA组学数据。分别将AaCK和AaT的sRNA组学数据比对miRBase数据库,对球囊菌侵染宿主前后miRNA的数量和结构特征进行分析。联用RNAhybrid+svm_light、Miranda和Tar...     

Effects of yeast antagonist in combination with UV-C treatment on postharvest diseases of pear fruit

The yeast antagonist Candida guilliermondii and ultraviolet-C (UV-C) treatment were investigated for controlling infection following artificial inoculation with Penicillium expansum or Botrytis cinerea, or natural infection in pear fruit stored at 20°C. Applied separately, both C. guilliermondii and UV-C (5 kJ m⁻²) effectively inhibited decay caused by P. expansum or B. cinerea, and natural infection. The combination of C. guilliermondii and UV-C showed better control efficacy. Application of UV-C did not affect the growth of C. guilliermondii in pear fruit wounds, while UV-C induced a significant increase in the activities of chitinase, β-1,3-glucanase, catalase and peroxidase in pear fruit. The mechanism by which UV-C enhanced the biocontrol efficacy of C. guilliermondii may be related to the elicitation of defense responses in pear fruit. The combination of C. guilliermondii and UV-C radiation could be a promising method for the control of P. expansum and B. cinerea in pear fruit.     

球囊菌胁迫中华蜜蜂幼虫肠道过程中病原的转录组学研究

【目的】本研究利用RNA-seq技术对球囊菌胁迫的中华蜜蜂(中蜂)幼虫肠道进行深度测序,经趋势分析得到差异表达基因(DEGs)的显著表达模式,进而对胁迫过程中的球囊菌进行转录组学分析。【方法】利用Illumina HiSeq 2500平台对球囊菌胁迫的中蜂幼虫肠道进行深度测序,并利用相关软件进行了深入分析。最后,通过RT-qPCR对RNA-seq数据进行了验证。【结果】本研究共得到球囊菌的41133932条高质量clean reads。22865个DEGs共聚类为8个基因表达模式,其中,16769个DEGs聚类为2个显著上调趋势与2个显著下调趋势。GO富集分析结果显示,显著上调与显著下调趋势中的DEGs分别富集于40与37个GO term,基因富集数最多的为细胞进程(2486 unigenes)。KEGG代谢通路(pathway)富集分析结果显示,显著上调与显著下调趋势中的DEGs分别富集于119和112个pathway,基因富集数最多的分别是氨基酸生物合成(127 unigenes)与核糖体(98 unigenes)。进一步分析表明球囊菌在胁迫中蜂幼虫肠道的过程中通过提高物质合成促进其增殖,而宿主通过抑制球囊菌的蛋白合成抵御病原入侵。富集在MAPK信号通路的11个DEGs的表达水平随着胁迫时间的延长而逐渐下降,推测中蜂幼虫通过抑制该通路而阻遏球囊菌增殖。【结论】本研究不仅为揭示白垩病过程中的球囊菌-中蜂幼虫互作提供了重要信息,也为阐明不同抗性蜂种的球囊菌抗性差异奠定了基础。     

DNA reassociation kinetics in relation to genome size in four amphibian species

DNA reassociation kinetics were studied, by means of the hydroxyapatite chromatography method, for four species of Amphibians with different nuclear DNA content: Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) of the Anura subclass and Trituras cristatus (23 pg) and Necturus maculosus (52 pg) of the Urodela subclass.Within each subclass the two species studied were found to have about the same absolute amount of unique DNA. The differences of total nuclear DNA can be accounted for by quantitative variations of the repetitive sequence classes, at least in part due to changes in the number of copies of the various sequences. On the contrary the great difference in nuclear DNA between the two subclasses, Anura and Urodela, involves all sequence classes in parallel; the slowly reassociating fraction appears to be unique in spite of a tenfold difference in absolute amount.The dependence of reassociation kinetics on DNA fragment length for the four species indicates for all of them an interspersed organization of the various sequence classes.     

Association of viruses with two protozoal pathogens of the honey bee

Undisturbed honey bee colonies near Rothamsted were commonly infected with a filamentous DNA virus (FV) which was usually associated with Nosema apis in infected adult individuals and occurred most frequently about June. No symptoms were caused by FV, which appeared less harmful than either black queen-ceil virus (BQCV) or bee virus Y (BVY), two isometric RNA viruses also associated with N. apis. Bee virus X, another isometric RNA virus distantly related to BVY, was not associated with N. apis, but prevailed in winter and was then associated significantly with Malpighamoeba mellificae in dead individuals, although it frequently multiplied alone. Results of laboratory experiments supported conclusions made from the field observations about these relationships and their pathology.     

In vitro activity of 15-azasterol (A25822B) against chalkbrood pathogen Ascosphaera apis in the honey bee

The antifungal agent 15-azasterol A25822B was examined for effects on the growth and development of Ascosphaera apis. The minimum inhibition concentration (MIC) of azasterol against A. apis was 1 m. Growth and development of A. apis was completely controlled at this concentration. At a concentration of 0.01 m growth of A. apis was retarded and although sporocysts were formed developing spores were not be able to reach maturation. A major effect of azasterol at this low concentration was the accumulation of lipid in the hyphae, sporocysts and immature spores. In addition it caused a conformational change in mitochondria and damage to the spore membrane structure. On the basis of these results, further investigations of azasterol for the treatment of chalkbrood disease in the honey bee are warranted.Work was performed during sabbatical leave at the University of California, Davis.     

DNA sequence organization in the genome of Nicotiana tabacum

The genome of Nicotiana tabacum was investigated by DNA/DNA reassociation for its spectrum of DNA repetition components and pattern of DNA sequence organization. The reassociation of 300 nucleotide DNA fragments analyzed by hydroxyapatite chromatography reveals the presence of three major classes of DNA differing in reiteration frequency. Each class of DNA was isolated and characterized with respect to kinetic homogeneity and thermal properties on melting. These measurements demonstrate that the genome of N. tabacum has a 1C DNA content of 1.65 pg and that DNA sequences are represented an average of 12,400, 252, and 1 times each. — The organization of the DNA sequences in the N. tabacum genome was determined from the reassociation kinetics of long DNA fragments as well as S1 nuclease resistance and hyperchromicity measurements on DNA fragments after annealing to C₀t values at which only repetitive DNA sequences will reassociate. At least 55% of the total DNA sequences are organized in a short period interspersion pattern consisting of an alternation of single copy sequences, averaging 1400 nucleotides, with short repetitive elements approximately 300 nucleotides in length. Another 25% of the genome contains long repetitive DNA sequences having a minimal genomic length of 1500 nucleotides. These repetitive DNA sequences are much less divergent than the short interspersed DNA sequence elements. These results indicate that the pattern of DNA sequence organization in the tobacco genome bears remarkable similarity to that found in the genomes of most animal species investigated to date.     

基于第三代长读段测序数据解析蜜蜂球囊菌基因的可变剪切与可变腺苷酸化

[目的]蜜蜂球囊菌(Ascosphaeraapis,简称球囊菌)是一种专性侵染蜜蜂幼虫的真菌病原,导致的白垩病是严重影响养蜂生产的顽疾,每年给养蜂业造成较大损失.本研究旨在基于已获得的第三代长读段测序数据对球囊菌菌丝(Aam)和孢子(Aas)中基因的可变剪切(alternative splicing,AS)和可变多聚腺...     

Satellite DNA properties of the germ line limited DNA and the organization of the somatic genomes in the nematodesAscaris suum andParascaris equorum

During the early cleavage divisions in some Ascarids, parts of the chromosomes are eliminated from the somatic blastomeres (chromatin diminution, Boveri, 1887) while the chromosomes in the germ line cells maintain their integrity. To characterize the germ line and soma genome, DNA was isolated from gametes and embryonic somatic cells of two Ascarid species,Parascaris equorum var. univalens andAscaris suum. It was shown that the germ line limited DNAs of these species have the same density and almost identical reassociation kinetics: in CsCl the predominant component of the germ line limited DNA ofP. equorum andA. suum has the buoyant density of 1.697g/cm³, while soma DNA of both species bands at 1.700 g/cm³. InP. equorum there is a small additional germ line limited satellite DNA component with the density of 1.690 g/cm³, identical to that of mitochondrial DNA of both organisms. Comparison of the reassociation kinetics of germ line and soma DNA demonstrates for both species that the eliminated DNA sequences are highly repetitive. In contrast to these similarities between the germ line limited DNAs ofP. equorum andA. suum the analysis of their base composition revealed differences (40% guanine plus cytosine inP. equorum and 36% inA. suum). The only very fast reassociating DNA sequences which we could isolate from soma DNA was demonstrated to be foldback DNA. The reassociation kinetics of totalA. suum soma DNA was investigated by hydroxylapatite chromatography. Least squares analysis of the data revealed about 10% of intermediate repetitive DNA sequences. Their interspersion between single copy DNA sequences was analyzed by comparing the reassociation kinetics of DNA fragments 0.35 and 7.2 kilobases long. Thus the DNA sequence arrangement ofAscaris does not follow the short period interspersion pattern observed in most organism.