Development of specific PCR primers for identification and detection of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> Leon

A PCR-based method was developed for the identification and detection of Phytophthora capsici in pepper plants. Three PCR primers (CAPFW, CAPRV1 and CAPRV2) specific for P. capsiciwere designed based on the sequence of its internal transcribed spacer regions. CAPFW/CAPRV1 amplify a 452 bp product from P. capsici DNA whereas CAPFW/CAPRV2 a 595 bp fragment; neither set amplifies DNA from pepper or several fungi pathogenic to pepper. In conventional (single-round) PCR, the limit of detection was 5 pg DNA for both primer sets, whereas in nested PCR the detection limit for both was of 0.5 fg. However, when the dilution series of target DNA were spiked with plant DNA, amplification declined two-fold in both conventional and nested PCR. The CAPFW/CAPRV2 set in conventional PCR was used to detect P. capsici DNA in inoculated plants. Detection occurred as soon as 8h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils.     

Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR

A diagnostic procedure, based on a polymerase chain reaction method (PCR) was developed to detect infection of crayfish with the Oomycete Aphanomyces astaci. A set of oligonucleotide primers was designed to specifically amplify A. astaci DNA in the ITS region surrounding the 5.8S rDNA gene. The PCR amplifies a 115 bp amplicon. The specificity of the primers was demonstrated by testing on 27 A. astaci strains and against 20 non-A. astaci Oomycetes and 5 fungal species. Most of the non-A. astaci Oomycete or fungal species included in the study are either known parasites of freshwater crayfish cuticle or can be found in their natural environment. Specificity was also tested against crayfish tissue and some known parasites and bacteria infecting crayfish.

A protocol for the extraction of A. astaci DNA from infected crayfish tissue was developed. The optimised method allows the detection of two genome equivalents of purified A. astaci genomic DNA.

The method was tested on noble crayfish (Astacus astacus), artificially infected with A. astaci. Detection of A. astaci was possible at the very first time of sampling, which was 2 days after the beginning of spore exposure.     

斯氏艾美耳球虫ITS1-5.8S rRNA-ITS2序列的克隆及PCR检测方法的建立

通过对多种鸡球虫和松鼠球虫18SrRNA和28SrRNA进行序列比对分析,在18SrRNA 3′端和28SrRNA 5′端保守区设计艾美耳属通用引物,以斯氏艾美耳球虫洛阳分离株LY卵囊基因组DNA为模板首次成功克隆到斯氏艾美耳球虫完整的ITS1-5.8SrRNA-ITS2序列,其大小为1 178bp,其中ITS1序列长度为423bp,5.8SrRNA为155bp,ITS2为600bp,斯氏艾美耳球虫LY株ITS1/2序列高度变异,与鸡球虫、啮齿动物球虫的序列相似性低于60%。然后在斯氏艾美耳球虫ITS1/2序列超变区设计种特异引物,建立了灵敏、特异的PCR检测方法。本研究结果将为兔球虫强致病种的临床诊断和揭示兔球虫种群遗传特征提供有效的分子工具。     

利用ITS序列和trnL-F序列探讨赖草属6个物种的系统发育关系

本研究克隆了赖草属6个物种Leymus karelinii,L.angustus,L.racemosus,L.arenarius,L.triticoides和L.ambiguus的ITS序列和trnL-F序列,并选用3个新麦属物种的ITS序列和27个小麦族二倍体物种的trnL-F序列,均以Bromus catharticus为外类群,用最大简约法构建系统发育树。结果发现:不同组的赖草属物种分别聚在一起;赖草属植物与新麦草属植物亲缘关系较近;新麦草属植物是这6个赖草属物种的母本来源。     

华南虎蛔虫ITS及5.8S rDNA序列扩增及分析

以从我国广州动物园华南虎肠道中采集的2条蛔虫作为研究对象,利用核糖体DNA(rDNA)中转录间隔区序列(ITS)的遗传标记特点,用保守引物NC5和NC2对提取的DNA进行PCR扩增,经胶回收纯化后,连接到pGEM-TEasy载体上,然后转化并鉴定出阳性菌落。对菌落进行PCR扩增并测序,将测序结果与GenBank公布的相关蛔虫ITS序列进行比对分析。结果显示,来自广州动物园的2条蛔虫ITS及5.8S rDNA序列总长均为858 bp,序列相似性为100%,与狮弓蛔虫(Toxascaris leonina)、猫弓首蛔虫(Toxocara cati)、犬弓蛔虫(T.canis)的ITS-1序列相似性分别为97%、81%、82%;5.8S序列的相似性分别为100%、98%、99%;ITS-2序列与GenBank上公布的狮弓蛔虫序列相似性94.6%,与猫弓首蛔虫、犬弓首蛔虫、马来西亚弓首蛔虫(T.malaysiensis)序列相似性均小于60%。研究结果证明此次分离的华南虎蛔虫属于狮弓蛔虫。     

两株水牛伊氏锥虫内转录间隔区基因序列的测定与系统进化关系

为研究从水牛分离到的伊氏锥虫广西株和湖北株的分类学地位,对2株伊氏锥虫的核糖体基因内转录间隔区(ITS1-5.8S-ITS2)基因进行了克隆和测序分析.用CLUSTALXl.83软件多重比对分析了序列差异,应用MEGA4.0软件绘制系统进化树.结果表明,这2株水牛伊氏锥虫的ITS1-5.8S-ITS2 rRNA基因扩增产物分别为1 102 bp和1 095 bp,序列存在多态性,其中广西株的ITS-1序列长为340 bp,ITS-2序列长为582 bp,湖北株的ITS-1序列长为339bp,ITS-2序列长为587 bp.系统进化分析显示,这2株伊氏锥虫分布在同一大枝的不同亚群中.     

A combination of Lactobacillus buchneri and Pediococcus pentosaceus extended the aerobic stability of conventional and brown midrib mutants–corn hybrids ensiled at low dry matter concentrations by causing a major shift in their bacterial and fungal community

We evaluated the effects of applying a combination inoculant to four corn hybrids harvested at high moisture on their nutritive value and microbial populations. The treatment design was the factorial combination of corn hybrids ensiled with (INO) and without (CON) inoculant. The hybrids were TMF2R737 (MCN), F2F817 (MBR), P2089YHR (PCN), and PI144XR (PBR), ensiled at dry matter (DM) concentrations of 30.5%, 26.3%, 31.1%, and 31.5%, respectively; MBR and PBR were brown midrib mutants (BMR). The inoculant contained Lactobacillus buchneri and Pediococcus pentosaceus (4 × 10⁵ and 1 × 10⁵ cfu/g of fresh corn). The experiment had a complete randomized design with treatments replicated six times. Corn was treated or not with inoculant, packed into 7.6 L bucket silos, and stored for 100 d. At d 0, the relative abundance (RA, %) of Enterobacteriaceae was lower in PBR vs. the other hybrids [51.3 vs. x¯ = (average of) 58.4] and in the case of fungi, incertae sedis (i.s.) Tremellales and Mucoraceae were more and less abundant, respectively, in conventional hybrids vs. BMRs (x¯= 25.8 vs. x¯ = 13.9 and x¯ = 3.64 vs. x¯ = 7.52; P < 0.04). After ensiling, INO had higher LAB (9.3 vs. 7.1 log cfu/g of fresh corn) and acetic acid (3.44% vs. 1.32% of DM) and lower yeast (3.1 vs. 4.6) and molds (1.5 vs. 3.0), and also extended the aerobic stability (582 vs. 111 h) but decreased DM recovery (95.6% vs. 97.4%) vs. CON (P < 0.02). Inoculation reduced bacterial phylogenetic diversity (6.75 vs. 14.4) but increased fungal observed taxonomical units (46 vs. 20) vs. CON (P < 0.01). Also, a higher relative abundance (RA) for Lactobacillaceae (99.2% vs. 75.7%) and lower for Enterobacteriaceae (0.28 vs. 9.93) was observed due to inoculation (P < 0.001). For fungi, INO had a lower RA compared to CON for Monascaceae (12.6 vs. 44.7) and increased i.s. Tremellales (8.0 vs. 1.2) and i.s. Saccharomycetales (6.4% vs. 0.3%; P < 0.006). Inoculation changed the diverse bacterial community found in the phyllosphere across hybrids to a taxonomically uneven one dominated by Lactobacillaceae. In the case of fungi, INO application increased the fungal diversity at d 100 mainly by reducing the dominance of Monascaceae vs. CON. In conclusion, the INO treatment overwhelmed the disparate microbial populations found across BMR and conventional hybrids ensiled at low DM concentrations and ensured a significant concentration of acetic acid that modified fungal populations and in turn extended the aerobic stability of all hybrids.     

黑龙江与广州颚口线虫幼虫分离株的形态学观察及其分子鉴定

本研究从来自黑龙江及广州地区的泥鳅中分离到10条颚口线虫幼虫,采用光学显微镜观察幼虫的形态特征,并对虫体的ITS2与CO1基因进行扩增测序、系统发育分析以鉴定虫种。结果显示10条颚口线虫幼虫头球均有3环小钩,其形态学特征与日本颚口线虫(Gnathostoma nipponicum)第3期幼虫相符。序列分析结果显示与GenBank中登录的日本颚口线虫ITS2和CO1基因的同源性分别为100%和99%。邻位连接法构建的50%一致树也均与日本颚口线虫处于同一分支。表明从黑龙江及广州地区泥鳅中分离的颚口线虫幼虫为日本颚口线虫幼虫,首次证明了中国存在日本颚口线虫。     

Sequence and Phylogenetic Analysis of rDNA ITS of Three Eimeria Species in Rabbit

In order to study whether the internal transcribed spacers (ITS) sequence could be used as a molecular marker for the species identification of rabbit coccidian, the rDNA ITS of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were amplified by polymerase chain reaction (PCR), and were cloned into pGEM-T Easy vector subsequently. The positive recombinant plasmids were identified by PCR and then sequenced. By sequence comparison and comparative analysis with the relative sequences of rabbit Eimeria spp. available in GenBank, the results showed that the lengths of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were 1065, 1009 and 1047 bp, respectively, and the sequence homologies with the same species sequences were 99.2%, 99.0% and 94.5%, respectively, while were 55.3% to 82.1% compared with corresponding sequences of other different species sequences. The phylogenetic analysis using software Mega 5.0 showed that all rabbit coccidia clustered together in a clade, which was divided into two sister lineages, corresponding to the presence or absence of oocyst residuum. The result demonstrated ITS could be used as a molecular marker for the species identification of rabbit coccidia.     

甘肃甘南野生羊肚菌rDNA的ITS序列分析

根据形态学特征挑选了7株采自甘肃甘南境内的野生羊肚菌,应用rDNA-ITS序列进行了比对技术分析。结果表明,7株野生羊肚菌归属为4个种：粗柄羊肚菌Morchellav crassipes、羊肚菌M.esculenta、黑脉羊肚菌M.angusticeps和高羊肚菌M.elata。根据分子系统树发现,粗柄羊肚菌和羊肚菌属于黄羊肚菌类群,而黑脉羊肚菌和高羊肚菌属于黑羊肚菌类群。