The phylogeny of marine and freshwater species of the genus Chloromyxum Mingazzini, 1890 (Myxosporea: Bivalvulida) based on small subunit ribosomal RNA gene sequences

The small subunit ribosomal RNA gene (SSU rDNA) of two freshwater and one marine species of the genus Chloromyxum Mingazzini, 1890 were sequenced. The SSU rDNA trees obtained show the phylogenetic position of the marine species Chloromyxum leydigi Mingazzini, 1890 to be at the base of the freshwater clade, being well supported by a high bootstrap value. Chloromyxum cyprini Fujita, 1927 is closely related to Chloromyxum truttae Léger, 1906 and they represent a sister branch to raabeia sp., Myxidium sp. and Myxidium truttae Léger, 1930. Chloromyxum legeri Tourraine, 1931 is in a position ancestral to Myxidium lieberkuehni Bútschli, 1882 and Sphaerospora oncorhynchi Kent, Whitaker et Margolis, 1993. Three newly sequenced species of the genus Chloromyxum represent three separate lineages within the myxosporean tree and do not support the monophyly of this genus.     

Sphaerospora elwhaiensis sp.n. (Myxosporea: Sphaerosporidae) from landlocked sockeye salmon Oncorhynchus nerka (Salmoniformes: Salmonidae) in Washington State, USA

A new species of sphaerosporid myxosporean, Sphaerospora elwhaiensis sp. n., is described from kidney of non-anadromous sockeye salmon (kokanee) Oncorhynchus nerka (Walbaum) from Lake Sutherland in the northern Olympic Peninsula, Washington, USA. Infection with the parasite was detected in 45% of 177 kokanee examined over 5 years. While conforming to the morphological criteria by which members of the genus are defined, the parasite is distinguished from congeners in salmonids of western North America by a unique combination of valvular sculpting of the myxospore, the relatively large size of the myxospore and monosporous development within the pseudoplasmodium. In addition, nucleotide sequences of the parasite's small and large subunit ribosomal RNA gene are unique. Phylogenetic analyses of these sequences suggested that the parasite is most closely related to freshwater Myxidium spp. and Zschokkella spp. The molecular data have provided further evidence for a polyphyletic association previously recognized among members of the genus and emphasize the need for a taxonomic revision of Sphaerospora Thélohan, 1892 and related genera.     

Development of a PCR-based diagnostic test for Spongospora subterranea f.sp. nasturtii, the causal agent of crook root of watercress (Rorippa nasturtium-aquaticum)

The polymerase chain reaction (PCR) technique was utilized to obtain internal transcribed spacer ribosomal DNA (ITS rDNA) and small-subunit (18S) rDNA sequences from UK isolates of Spongospora subterranea f.sp. nasturtii , a plasmodiophorid pathogen of watercress ( Rorippa nasturtium-aquaticum ). ITS sequence data obtained from S. subterranea isolated from a range of UK sites were found to be identical. PCR primers were designed using these sequences and were shown to be capable of specific amplification of S. subterranea f.sp. nasturtii DNA from plant tissue and from water samples containing zoospores of the pathogen. As little as 5 ng total genomic DNA from infected plant material, or 1000 zoospores, was required for consistently successful amplification of DNA. A filtration-based method for obtaining pathogen DNA for PCR from watercress-bed water was developed.     

广东桉树黄化(丛枝)病植原体分子鉴定与检测

从表现黄化(丛枝)症状的桉树上采集病叶,抽提主脉总DNA,采用植原体通用引物与巢式引物进行PCR和巢式PCR扩增,对扩增产物进行克隆和序列测定,获得了植原体的近全长16S rRNA基因及部分16～23S rRNA基因间隔区序列.序列分析揭示,所获得的序列与已知植原体基因组相应区段的序列高度同源,与柳叶菜变叶植原体(epilobium phyllody)和白腊树丛枝植原体(ash witches'-broom)相应序列(GenBank登录号:AY101386和AY566302)同源率为99.9%,与白腊树黄化植原体(aster yellows BD2)相应序列和番茄巨芽植原体(tomato big bud)相应序列同源率分别为99.6%和99.3%.该序列构建的系统进化树表明,引起我国广州地区桉树黄化(丛枝)病的植原体属于16SrI组(即翠菊黄化组),将其暂命名为桉树黄化(丛枝)植原体广东株系(Eucalyp-tus yellowing and witches'-broom phytoplasma strain Guangdong,EYWB-Gd).建立了桉树植原体巢式PCR检测方法,对疑似病样及桉树组培苗进行了检测,多数疑似病样检测结果为阳性,供试的10株组培苗未发现阳性样品.     

New data on aetiology of nodular gill disease in rainbow trout, Oncorhynchus mykiss

We studied amoebae associated with nodular gill disease (NGD) outbreaks in rainbow trout Oncorhynchus mykiss (Walbaum) in fish farms in South-Western Germany. Gills of 12 diseased rainbow trout were examined in fresh, by isolation attempts, histologically and using in situ hybridisation (ISH). A total of nine amoeba strains of the genera Acanthamoeba (1), Hartmannella (2), Naegleria (1), Protacanthamoeba (1) and Vannella (4) were isolated and determined using light microscopical, ultrastructural and molecular methods. Specific molecular probes designed from the SSU rDNA sequences of individual amoeba strains were used for non-radioactive ISH in histological sections. Association of Naegleria sp. with NGD and a direct ISH proof of Naegleria trophozoites attached to hyperplastic gill epithelium are novel findings, expanding the number of possible agents of NGD and supporting the hypothesis on multicausal aetiology of this disease.     

<Emphasis Type="Italic">Bradyrhizobium</Emphasis> isolated from huanglongbing (HLB) affected citrus trees reacts positively with primers for <Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus

Bradyrhizobium sp., a slow-growing nitrogen-fixing symbiotic bacterium of legumes and common root endophyte of other plants, is closely related to Candidatus Liberibacter asiaticus (Las), the uncultured putative pathogen associated with citrus huanglongbing (HLB). In attempts to isolate Las on a low-nutrient medium that had been used for the isolation of several uncultured bacteria of the alpha subclass of proteobacteria, slow-growing Bradyrhizobium spp. were isolated and identified by sequencing of 16S rDNA. The individual isolates tested weakly positive (Ct = 31.2–36.0) with the USDA primers commonly used in qPCR assays for Las in foliar tissues. Direct DNA extracts from roots of HLB symptomatic trees that contained sequences of Bradyrhizobium sp. had Ct values ranging from 31.2 to 36.5; sequences of Las were not present in those samples. Potential cross-reaction between DNA of members of the Rhizobiales and sequences amplified by the Las primers were tested in silico with the Primer-BLAST tool in NCBI. Similar to Las, Bradyrhizobium generated predicted 16S rDNA amplicon sizes of 78–79 bp with the qPCR primers and of 1167-1172 bp with the conventional PCR primers. Bradyrhizobium sequences of 16S rDNA had 1–7 mismatches and only 1 mismatch at the 3′ end of qPCR and conventional PCR primers confirming potential cross-reactivity. As Bradyrhizobium is usually not found in foliage, the USDA qPCR primers can be safely used to check leaves for the presence of Las, but a threshold value of 31.0 is recommended for Las detection in roots. Other primers should be tested for potential cross-reaction with members of the Rhizobiales.     

Microbial community profiles in intercellular fluid of rice

Plants harbor microorganisms that are thought to stimulate plant defense systems or promote plant growth. Individual species in these intercellular microbial communities are often not sufficiently abundant to be easily described, although some endophytic microorganisms amenable to culture have been characterized. To better understand the microbial population of plants, we collected intercellular fluid (IF) from leaf blades and sheaths of rice and subsequently isolated DNA from the IF. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S and 18S rDNA fragments amplified from IF DNA by PCR indicated that these band patterns were distinguishable from those of a leaf surface-wash fluid (SF). Analysis of a set of rDNA fragments amplified from IF DNA of rice with different genotypes, paddies or growth stages for the primary survey of overall microbial community in the IF suggested that this approach is suitable for analyzing microbial diversity in the IF from various plant samples. Actually, comparative analysis of amplified rDNA fragments of rice and other five plant species indicated that the microbial diversity in IF is likely to vary substantially among plant species. We can also use sequence analysis of 16S rDNA fragments amplified from rice IF DNA to identify species including unculturable bacteria and proteobacteria and Xanthomonas and 18S rDNA fragments to identify Tilletiaria anomala, Tilletia iowensis, Ustilago maydis and unculturable eukaryotes. Thus, IF DNA analysis seems to be a good tool to further study the microbial ecology of plants.     

樱桃花变绿病植原体的分子鉴定

 植原体(phytoplasma)是一类没有细胞壁,不能人工培养,存在于植物筛管细胞中的类似植物病原细菌的原核生物。迄今为止,世界各地报道的1 000余种植物病害与植原体有关,引起的症状主要包括丛枝、黄化、花变绿、花变叶、花器退化等。     

两种沙漠微藻的形态学和分子生物学鉴定

文中从古尔班通古特沙漠采集的沙样中,运用96孔板有限稀释法分离出两种形态各异的微藻物种GTD8A1和GTD9C2,形态学初步鉴定分别为绿藻门小球藻目和环藻目。利用GenBank中绿藻门不同科属种类序列的18S核糖体RNA基因保守区和28S核糖体RNA基因保守区分别设计18S rDNA特异性引物和5.8S rDNA-ITS区特异性引物。以自行设计的18S rDNA特异性引物与5.8S rDNA-ITS区特异性引物分别对以上两个物种进行PCR和测序后,经blastn比对,系统发育树及遗传距离分析,结果表明GTD8A1可能与Chlo-rella sp.MBIC10595为同一个物种,GTD9C2可能为Desmodesmus multivariabilis种内的一个变种。     

瘿蚊科3种昆虫核糖体DNA的克隆及序列分析

瘿蚊科是双翅目昆虫中的重要类群?本研究对瘿蚊科3种昆虫—麦红吸浆虫?菊花瘿蚊和食蚜瘿蚊的核糖体DNA进行PCR扩增?克隆?测序及序列分析, 并对ITS-1在麦红吸浆虫4个地理种群中的遗传变异情况进行分析?结果表明, 从3种昆虫中获得的核糖体DNA序列包括:部分的18S rDNA(44 bp)?28S rDNA(37 bp), 全部的ITS-1(487~535 bp), 5.8S rDNA(121 bp)及ITS-2(336~352 bp)序列?3种昆虫ITS序列的碱基差异百分比在17.21%~29.59%之间, 共含有206个变异位点?ITS-1序列在麦红吸浆虫4个地理种群中比较保守, 只有4个变异位点, 单倍型多样性为0.311 7~0.796 5, 核苷酸多样性为0.000 6~0.002 2?本研究为今后瘿蚊科昆虫的分类鉴定?系统发育和遗传进化等相关研究提供了基础?     

First record of metacestodes of Mesocestoides sp. in the common starling (Sturnus vulgaris) in Europe, with an 18S rDNA characterisation of the isolate

Metacestodes of Mesocestoides sp. were recorded from Sturnus vulgaris (Passeriformes: Stumidae) in the Czech Republic in April 2002. They were found in a cutaneous cyst and in the thoracic region of the body cavity of the bird. This is the first record of metacestodes of Mesocestoides sp. in this host species in Europe as well as the first finding of the formation of a cutaneous cyst provoked by this parasite. Additional specimens from Apodemus agrarius (Mammalia: Rodentia) from Bulgaria and Lacerta agilis (Reptilia: Squamata) from the Czech Republic were compared with that from S. vulgaris. Sequence data from the V4 variable region (18S rDNA) were used to compare genetic variability among these and previously characterized isolates of Mesocestoides spp. A number of distinct clades were recognized, with metacestodes from L. agilis showing the highest degree of relative divergence.     

Detection and identification of the phytoplasma associated with pear decline in Taiwan

Pear decline (PD) is an important phytoplasmal disease that occurs mainly in Europe and North America. In 1994, pear trees exhibiting symptoms typical of PD disease were observed in orchards of central Taiwan. The sequence of 16S rDNA and 16S–23S rDNA intergenic spacer region (ISR) of the causative agent of pear decline in Taiwan (PDTW) were amplified with polymerase chain reaction (PCR) using a DNA template prepared from the diseased leaves. Sequence analysis of 16S rDNA revealed that the PDTW agent was closely related to the phytoplasmas of the apple proliferation group that cause diseases in stone fruits, pear and apple. Consistent with the result of 16S rDNA sequence analysis, sequence analysis of the 16S–23S rDNA ISR and putative restriction site analyses of 16S rDNA and 16S–23S rDNA ISR sequences provided further support for the view that the PDTW phytoplasma causing pear decline in Taiwan may represent a new subgroup of the apple proliferation group. According to the rDNA sequence of PDTW phytoplasma, two specific PCR primer pairs, APf2/L1n and fPD1/rPDS1, were designed in this study for the detection of the etiological agent in pear trees and insect vectors. Based on the sequence analyses of the PCR-amplified fragments, two species of pear psyllas, Cacopsylla qianli and Cacopsylla chinensis, were found to carry PDTW phytoplasma.     

Characterization of a gamma-3 Proteobacteria Responsible for the Syndrome "Basses Richesses" of Sugar Beet Transmitted by Pentastiridius sp. (Hemiptera, Cixiidae)

ABSTRACT The disease syndrome "basses richesses" (SBR) has affected sugar beet crops in Burgundy (France) since 1991. It mainly is associated with an uncultivable phloem-restricted bacterium-like organism (BLO) called SBR BLO. Transmission tests showed that field-collected Pentastiridius sp. (Hemiptera, Cixiidae) were able to transmit the SBR BLO to sugar beet. In the present work, sequences of a 1,507-bp 16S ribosomal (r)DNA fragment of SBR BLO were amplified from DNA extracts of SBR-affected field sugar beet plants, of field-collected Pentastiridius sp. plant-hoppers, and of Pentastiridiussp.-exposed sugar beet seedlings that expressed SBR symptoms. The sequences showed total identity, confirming the role of SBR BLO in the etiology of SBR and the vector role of Pentastiridius sp. Our surveys on SBR-affected sugar beet plants and Pentastiridius sp. planthoppers collected in different fields and different years suggest that a unique BLO was involved in SBR. Furthermore, comparison of 16S rDNA sequences permitted the identification of the SBR BLO as a new plant-pathogenic gamma-3 proteobacteria different from 'Candidatus Phlomobacter fragariae,' another BLO responsible for marginal chlorosis disease of strawberry in France. Phylogenetic analysis revealed a close relationship between the SBR bacterium and several bacteria described as endosymbionts of hemipteran insects.     

云南花生丛枝植原体16S rRNA和核糖体蛋白基因序列分析

 利用植原体16S rRNA基因及核糖体蛋白基因(ribosomal protein, rp)通用引物对发生在云南元谋的花生丛枝病病株DNA进行PCR扩增,并对扩增片段进行序列测定。扩增获得的云南元谋花生丛枝植原体(PnWB-YNym)16S rDNA、16S-23S rDNA和23S DNA片段总长1 806 bp,rp基因扩增片段长1 171 bp。云南株系与来源于台湾和海南的花生丛枝植原体均有较高同源性。比较16S rDNA片段,发现云南株系在5个位点上与来自台湾或海南的株系存在碱基差异,其中有1个位点的差异是云南元谋株系特异的;再分别比较核糖体蛋白rplV-rpsC 2个基因所编码的氨基酸序列,发现云南株系rpsC编码的第194位氨基酸与台湾和海南的株系存在差异。经16S rDNA片段系统进化及iPhyClassifier在线分析,表明PnWB-YNym在分类上属于16SrII-A亚组成员,与候选种‘Candidatus Phytoplasma australasiae’相关;基于rp基因构建的系统进化树表明,PnWB-YNym与16SrII-A亚组各成员聚为同一亚进化支(iii)。     

Detection and identification of the maize bushy stunt phytoplasma in corn plants in Brazil using PCR and RFLP

Plants of corn (Zea mays L.) exhibiting symptoms of stunting and leaf reddening were assayed for the presence of phytoplasma gene sequences through the use of phytoplasma rRNA and ribosomal protein gene and maize bushy stunt (MBS) phytoplasma-specific oligonucleotide primers in polymerase chain reactions (PCR). Polymorphisms in 16S rDNA amplified from diseased plants were those characteristic of phytoplasmas classified in the16S rRNA gene group 16SrI, subgroup IB, of which MBS phytoplasma is a member. Amplification of ribosomal protein (rp) gene sequences in PCR primed by phytoplasma-specific primers confirmed presence of a phytoplasma in the diseased plants. Restriction fragment length polymorphism (RFLP) patterns of the amplified phytoplasma rp gene sequences were similar or identical to those observed for a known strain of MBS phytoplasma. In separate PCR, an MBS-specific oligonucleotide pair primed amplification of a MBS-characteristic DNA from templates derived from the diseased corn. Our data provide the first firm evidence for the presence of maize bushy stunt phytoplasma in corn in Brazil.     

Impact of a new biopesticide produced by Paenibacillus sp. strain B2 on the genetic structure and density of soil bacterial communities

The effect of paenimyxin, a new biopesticide produced by Paenibacillus sp. strain B2, on the density of soil bacterial communities was assessed by colony counting and by 16S rDNA and nirK quantitative polymerase chain reaction (PCR). Paenimyxin had a negative effect on the bacterial colony-forming unit (CFU) number, which was significantly reduced 2 and 4 days after treatment. The effect of paenimyxin on cultivatable bacteria was negligible 7 days after treatment. Approximately 10(7) 16S rDNA sequences per gram of soil (dry weight) were detected by quantitative PCR in all samples. Paenimyxin did not affect the quantification of 16S rDNA or of the denitrifying bacterial community. In addition, RISA fingerprinting showed that the genetic structure of the bacterial communities was significantly modified 2 days after paenimyxin application at 50 microM and 4 days after treatment at lower concentrations (0.5 and 5 microM). The impact of paenimyxin treatment on the genetic structure of soil bacterial communities was transient, as no effect could be observed after 7, 14 and 28 days when compared with the untreated control.     

Development of PCR-based Detection Methods for the Quarantine Phytopathogen <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis>, Causal Agent of Potato Wart Disease

PCR-based methods were developed for the detection and quantification of the potato pathogen Synchytrium endobioticum in soil extracts and in planta. PCR primers, based on the internal transcribed spacer region of the multi-copy gene rDNA were tested for specificity, sensitivity and reproducibility in conventional and real-time PCR assays. Soil extraction procedures compared included the Hendrickx centrifugation (HC) procedure, nested wet sieving (NWS) and a method used by the Plant Protection Service (PPS). The primers amplified a 472 bp product from S. endobioticum DNA, but did not amplify DNA from other potato pathogens, other plant pathogens, and related species. Standard cell disruption and DNA extraction and purification methods were optimized for amplification of S. endobioticum DNA from resting sporangia. DNA was successfully amplified from a single sporangium and equivalent DNA preparations from soil extracts. Low levels of target DNA in water did not amplify, possibly due to DNA loss during final purification steps. A real-time PCR assay, developed for soil-based extracts using primers and probe based on the rDNA gene sequences, involved co-amplification of target DNA along with an internal DNA fragment. Both conventional and real-time PCR methods performed well with HC- and NWS-extracts having a threshold sensitivity of 10 sporangia per PCR assay. Of the three soil extraction methods, only with the HC method could 100 g soil samples be efficiently processed in one single PCR assay. Such a high capacity assay could be useful for routine soil analysis in respect to disease risk assessments and to secure de-scheduling according to EPPO guidelines.     

Characterization of Ribosomal DNA from Venturia inaequalis and Its Phylogenetic Relationship to rDNA from Other Tree-Fruit Venturia Species

ABSTRACT A portion of the 18S ribosomal DNA (rDNA) gene, the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA gene were polymerase chain reaction-amplified from strains and field populations of Venturia inaequalis and assessed for genetic variation. A previously reported optional group I intron in the 18S rDNA gene of V. inaequalis was detected in 75.0% of 92 strains collected worldwide and in 61.1 and 71.2% of 54 and 59 strains from two Michigan orchards, respectively. Sequence and restriction analysis of rDNA revealed four intron alleles, three of which were present both in worldwide strains and in each field population. Two ITS1 alleles were detected and found to be linked to specific intron alleles. The ITS1-5.8S-ITS2 sequences from V. asperata V. carpophila, V. cerasi, V. inaequalis, V. nashicola, V. pyrina, and Cladosporium caryigenum were compared using phylogenetic analysis. Strains of the Venturia species were placed in three distinct monophyletic groups in a phylogenetic tree. The first group comprised V. inaequalis; the second, V. pyrina and V. nashicola; and the third, V. cerasi, V. carpophila, and V. asperata. The described intron and ITS1 alleles in V. inaequalis provide genetic markers for subdividing populations of V. inaequalis, and the ITS1-5.8S-ITS2 sequences are valuable in determining the relationship of the species from tree-fruit crops with other Venturia species.     

西瓜蔓枯病分子诊断技术研究

 本文测定西瓜上的西瓜蔓枯病菌(Didymella bryoniae)、西瓜炭疽病菌(Colletotrichum orbiculare)及西瓜枯萎病菌(Fusarium oxysporum f. sp. niveum)的rDNA的ITS序列,比对近缘种及西瓜上不同病菌的ITS序列同源性,设计出特异性上游引物XM-2和下游引物XM-R2。经过对XM-2/XM-R2引物的PCR扩增条件的优化,可以扩增出一条344bp的西瓜蔓枯病菌特异性DNA条带。上述方法可以检测到pg以上的蔓枯病菌基因组DNA,并且可以准确扩增出西瓜蔓枯病自然病样中特异性的DNA片段。本文建立了一项西瓜蔓枯病分子检测技术,该方法准确、快速、可靠,可用于西瓜蔓枯病田间的快速诊断。     

Molecular characterization and diagnostics of stubby root and virus vector nematodes of the family Trichodoridae (Nematoda: Triplonchida) using ribosomal RNA genes

Plant parasitic nematodes of the family Trichodoridae cause substantial yield losses in many agricultural crops. Rapid and accurate identification of trichodorids to the species level is critical for selection of appropriate measures for control. This study analysed 99 sequences of the D2–D3 expansion segments of the 28S rRNA gene and 131 sequences of the 18S rRNA gene from the stubby nematodes belonging to the genera Nanidorus, Paratrichodorus and Trichodorus. Species delimiting was based on the integration of morphological identification, which is not provided in the present article, and molecular‐based phylogenetic inference and sequence analysis. Twenty‐two valid species and several species complexes were identified among nematodes included in the analysis. PCR‐RFLPs of the partial 18S rDNA and the D2–D3 expansion segments of the 28S rDNA were tested and proposed for identification of these nematodes. Gel PCR‐RFLP profiles and tables with restriction fragment lengths for several diagnostic enzymes are provided for identification. Some problems of taxonomy and phylogeny of nematodes of the family Trichodoridae are also discussed.