大豆根腐病原镰孢菌种群多样性DGGE分析及其致病性研究

 应用变性梯度凝胶电泳(DGGE)技术以及主成分分析方法,结合植物病原菌传统分离、鉴定及致病力测定,检测位于黑龙江省海伦市大豆连作定位试验区根腐病原镰孢菌的种群构成及其致病力。采用真菌传统分离和鉴定方法共确定镰孢菌6个种,包括燕麦镰孢Fusarium avenaceum、木贼镰孢F. equiseti、禾谷镰孢F. graminearum、腐皮镰孢F. solani、尖镰孢F. oxysporum和拟轮枝镰孢F. verticillioides。其中,尖镰孢菌的分离频率最高,为56.7%。结合DGGE条带克隆测序及系统发育分析鉴定出8种镰孢菌,较传统方法增加了黄色镰孢F. culmorum和一个尖镰孢近似种。同时,DGGE图谱中尖镰孢所占百分比下降为37.4%。采用主成分分析方法将大豆生育指标相关的多种变量进行降维分析,得到第一主成分贡献值为91.2%。按照贡献值的正负结果,将30株镰孢菌分为致病和非致病类群。第二主成分贡献值为5.8%,根据其正负值可以矫正被错误估计的致病能力。综合所有研究结果,该定位试验区大豆根腐病主要病原镰孢菌为尖镰孢、禾谷镰孢和燕麦镰孢,且以尖镰孢菌为优势病原菌。     

Identification of Phytophthora species on fruit crops in Turkey by polyacrylamide gel electrophoresis1

To identify 11 Phytophthora isolates obtained from lemon, peach and apple trees, and from strawberry plants, in the Mediterranean region of Turkey, protein, acetylesterase, peroxidase and catalose band patterns of these isolates and the same bands of 10 identified isolates of Phytophthora citrophthora, P. cinnamami and P. cactarum were compared. These 11 regional isolates were identified on the basis of the similarity of their protein and enzyme band patterns to those of identified Phytophthora isolates. It was concluded that protein band patterns could be used for identification of Phytophthora species. With enzyme band patterns, however, it may be possible to identify at species level but it is more practical to use this method only for identification of subspecies.     

Distribution analyses of Meloidogyne spp. and Pratylenchus coffeae sensu lato in coffee plots in Costa Rica and Guatemala

The distribution of the plant-parasitic nematodes Pratylenchus coffeae sensu lato and Meloidogyne spp. were studied in two plots, one in Guatemala ( P. coffeae and M. paranaensis ) and the other in Costa Rica ( P. coffeae and M. exigua ). The quantity of nematodes per g fresh weight root were counted for each coffee tree sampled. The distributions were aggregated, and generally fitted well to negative binomial distributions. Population aggregation was greater when a smaller number of nematodes were involved, suggesting that initial colonization develops in foci. Analyses of the relationships between population levels of the species suggested that there was competition between Pratylenchus coffeae and Meloidogyne spp. This competition was expressed differently depending on the relative population density of the different species.     

多重RT-PCR同时检测6种马铃薯病毒及1种类病毒

病毒病是限制我国马铃薯生产的重要因子之一。本研究通过引物设计和体系优化建立了一套多重RT-PCR检测方法,可以在一个反应体系中同时检测6种马铃薯病毒和1种马铃薯类病毒以及1个马铃薯内参基因,包括马铃薯Y病毒(potato virus Y, PVY)、马铃薯M病毒(potato virus M, PVM)、马铃薯S病毒(potato virus S, PVS)、马铃薯X病毒(potato virus X, PVX)、马铃薯A病毒(potato virus A, PVA)、马铃薯卷叶病毒(potato leaf curl virus, PLRV)、马铃薯纺锤块茎类病毒(potato spindle tuber viroid, PSTVd)和细胞色素c氧化酶亚基Ⅰ(cytochrome c oxidase subunitⅠ,CoxⅠ)基因的特异片段,产物大小依次为181、226、275、565、630、681、359、500 bp,符合理论预期。采用建立的方法以相应病毒和类病毒的质粒作模板进行检测,其检测灵敏度在1.6×10^-3～1.8×10^-1 ...     

Characterisation of Meloidogyne species from China using Isozyme Phenotypes and Amplified Mitochondrial DNA Restriction Fragment Length Polymorphism

Forty-six Meloidogyne populations from 14 provinces of China were characterised in terms of malate dehydrogenase, esterase phenotypes and HinfI restriction profiles of amplification products from the mitochondrial DNA (mtDNA) region between the COII and lrRNA genes. Isozyme phenotyping revealed that 29 of the populations were M. incognita, six were M. javanica, six were M. arenaria, three were M. hapla and two were M. enterolobii. HinfI restriction patterns of the COII-lrRNA region correlated with nematode isozyme phenotypes and enabled reliable differentiation and identification of the five root-knot nematodes occurring in China. The size and sequence of the mtDNA amplification product were determined for the first time for M. enteroloii, a potentially economically important crop pathogen. Sequence comparison showed that the sequence of the intergenic region between the COII and lrRNA genes for M. enterolobii was identical to that reported for M. mayaguensis. Together with published observations on morphology, host range and esterase phenotype of the two nominal species, the mtDNA sequence evidence suggests that M. mayaguensis could be conspecific with M. enterolobii.     

PCR-based sensitive and specific detection of Pectobacterium atrosepticum using primers based on Rhs family gene sequences

A sensitive and specific assay was developed to detect potato blackleg caused by Pectobacterium atrosepticum in potato. Primers PEAF and PEAR from the Rhs family gene homologous to RhsA (cell envelope biogenesis – outer membrane) were used to amplify a 904 bp DNA fragment. PCR was used to detect the pathogen in artificially inoculated potato seed tubers. The PCR product was only produced from 12 isolates of P. atrosepticum from various countries among 36 isolates of other species of Pectobacterium , Pseudomonas , Xanthomonas , as well as Escherichia coli and the soilborne fungus Fusarium oxysporum f.sp. dianthi .     

Starch gel electrophoresis of lactate dehydrogenase and glucose phosphate isomerase of poultry coccidia using the LKB multiphor

A modification of thin-layer starch gel horizontal electrophoresis is described. The original method of Wraxall and Culliford (1986) is improved so that the preparation of starch gel is as simple as preparing the agarose gel. Thus the commercially supplied kits and instruments for the agarose gel electrophoresis can be also used for the starch gel electrophoresis. Furthermore, a method of preparing the permanent dry enzymograms from the starch gels with visualized enzymes is presented. The described procedure was used in the LDH and GPI analyses of poultry coccidia.     

我国甜菜褐斑病菌的PCR快速检测

由甜菜尾孢菌Cercospora beticola引起的甜菜褐斑病(Cercospora leaf spot, CLS)是甜菜生产中危害较重的叶部真菌病害,导致甜菜产量和含糖量下降。由于CLS早期症状不易识别,传统病斑形态目测诊断法易错过最佳防治时期,因此分子检测方法对于CLS的准确监测十分重要。迄今为止,国外已报道的分子检测法主要有基于甜菜尾孢菌 rRNA-genes的actin gene编码区和ITS区结合限制性酶切的分子检测法、基于 rRNA-genes的ITS区结合测序的检测方法和基于大量尾孢属真菌RAPD多态性分析而设计的特异引物Cebe2检测法。目前,我国对甜菜尾孢菌的分子检测工作尚未见报道,为此,本试验对甜菜尾孢菌上述3种主要分子检测方法进行了比较,并进一步验证了特异引物Cebe2的可靠性,初步确立了我国甜菜尾孢菌的快速分子检测技术体系,为我国CLS的早期准确检测和有效控制提供技术支持。     

Detection and quantification of Pratylenchus thornei in DNA extracted from soil using real-time PCR

The root-lesion nematode Pratylenchus thornei is one of the most important pests restricting productivity of wheat in the Pacific Northwest (PNW). It is laborious and difficult to use microscopy to count and identify the nematodes in soils. A SYBR Green I-based real-time polymerase chain reaction (PCR) assay was developed to detect and quantify this species from DNA extracts of soil. A primer set, designed from the internal transcribed spacer region (ITS1) of rDNA, was highly specific to P. thornei and did not amplify DNA from 27 isolates of other Pratylenchus spp., other nematodes, and six fungal species present in PNW wheat fields. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. The standard curve was supported by a high correlation between the numbers of P. thornei added to soil and the numbers quantified using real-time PCR. Examination of 15 PNW dryland field soils and 20 greenhouse samples revealed significant positive correlations between the numbers determined by real-time PCR and by the Whitehead tray and microscopic method. Real-time PCR is a rapid, sensitive alternative to time-consuming nematode extractions, microscopic identification, and counting of P. thornei from field and greenhouse soils.     

Microscopic detection of reactive oxygen species generation in the compatible and incompatible interactions of Alternaria alternata Japanese pear pathotype and host plants

 Reactive oxygen species (ROS) generation was examined in the interaction of Alternaria alternata Japanese pear pathotype and host plants using three methods: nitro blue tetrazolium (NBT) method for microscopic detection of O₂ ⁻, diaminobenzidine (DAB) methods for microscopic detection of H₂O₂, and cerium chloride methods for ultrastructural detection of H₂O₂. ROS generation was detected by NBT and DAB methods at appressoria on leaves of susceptible cultivars and heat-shocked leaves of resistant cultivars but not in leaves of resistant cultivars. Ultrastructural detection by the cerium chloride method identified ROS generation at cell walls of appressoria and penetration pegs in susceptible, resistant leaves and heat-shocked leaves. These differences in the ultrastructural and microscopic data in resistant areas were due to the restriction of ROS generation in limited areas, the side facing the plant surface, of appressoria and penetration pegs. Therefore, ROS generation was apparently induced regardless of the resistance or susceptibility of the cultivar with the difference being in the volumes generated. After evaluating the pathological role of ROS generation in fungal structures, such generation was found to be associated with early penetration of cell walls in pear plants. Additionally, ROS generation in plants was also found in degrading pectin layers near infected hyphae and in plasma membrane modification sites in susceptible leaves but not in resistant leaves. ROS generation in susceptible leaves might be accompanied with plasma membrane damage, although the role of ROS generation in the pectin layers is not clear. ROS generation in both fungal and plant cells during their interaction was likely associated with the expression of susceptibility. Received: June 3, 2002 / Accepted: July 31, 2002     

Simultaneous detection of eight viruses and two viroids affecting stone fruit trees by using a unique polyprobe

The use of riboprobes carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the simultaneous detection of up to six different pathogens using a non-radioactive molecular hybridization procedure. In the present report, we describe the development of a unique polyprobe (poly10) with the capacity to detect viruses and viroids commonly found infecting fruit trees. The poly10 covers eight viruses: Apple mosaic virus (ApMV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), American plum line pattern (APLPV), Plum pox virus (PPV), Apple chlorotic leaf spot virus (ACLSV), Apricot latent virus (ApLV), Plum bark necrosis and stem pitting-associated virus (PBNSPaV) and two viroids: Hop stunt viroid (HSVd) and Peach latent mosaic viroid (PLMVd). Poly10 is comparable to the individual riboprobes in terms of end-point dilution limit and specificity, allowing the detection of up to 2.5 picograms of viral or viroidal RNA. However, the polyprobe requires a hybridization temperature of 60°C instead of the standard 68°C. The validation of the new simultaneous detection strategy was confirmed by the analysis of 60 field samples, which came from seven different hosts. The use of the polyprobe as an alternative to other routinely used detection methods is discussed.     

Specific detection of Phytophthora nicotianae using the polymerase chain reaction and primers based on the DNA sequence of its elictin gene ParA1

Six primers based on the sequence of the flanking and coding regions of the elicitin gene ParA1 of Phytophthora nicotianae were tested for specific detection of the fungus by the polymerase chain reaction (PCR). One combination, IL7/IL8, with IL7 in a flanking region and IL8 in a coding region of the gene, gave an intense 378 bp signal with a diverse collection of isolates of P. nicotianae, that included some from black shank disease of tobacco and others from a variety of hosts. The sequence of the amplification product obtained with an isolate that produces elicitin and one that does not, was homologous with the known sequence of the ParA1 gene. The same primer combination gave no signal with sixteen other Phytophthora species tested except for two isolates P. palmivora with which it gave a weak 800 bp signal. It gave no signal with DNA from healthy tobacco and tomato plants but P. nicotianae was detected in inoculated tobacco and tomato plants. Small numbers of zoospores (>100) trapped onto a nitrocellulose membrane after filtration from suspension were also detected after two successive rounds of PCR.     

山西省窖藏薯类块茎中2种短体线虫的种类鉴定

本文采用传统形态学与分子生物学相结合的方法,对采自山西省窖藏马铃薯和薯蓣块茎中的短体线虫进行了种类鉴定.结果 表明,从马铃薯块茎中分离出的短体线虫形态学特征与斯克里布纳短体线虫Pratylenchus scrib-neri一致,其SSU序列与P.scribneri美国群体相似性达99.8％.从薯蓣块茎中分离出的短体线虫...     

皖北高粱田杂草种类及群落特征分析

为明确皖北高粱Sorghum bicolor田杂草发生现状, 于2020年-2021年采用倒置“W”九点取样法对皖北6市52个乡镇田间杂草进行了调查?结果表明, 皖北高粱田共有杂草36种, 属17科, 其中禾本科?菊科?苋科种类最多?优势杂草为稗Echinochloa crus-galli?马唐Digitaria sanguinalis?牛筋草Eleusine indica?狗尾草Setaria viridis等4种, 区域性优势杂草4种, 常见杂草13种, 一般杂草15种?阜阳市杂草的优势度指数最高, Berger-Parker指数为4.89, Simpson指数?Shannon-Wiener指数?Pielou指数均高于其他5市, 优势杂草以稗?马唐?牛筋草?狗尾草?反枝苋Amaranthus retroflexus和苋Amaranthus tricolor为主; 亳州市杂草的优势度仅次于阜阳市, 以稗?马唐?牛筋草?狗尾草和铁苋菜Acalypha australis为主?淮北市杂草的丰富度指数最高, Margalef指数为3.16, 以稗?牛筋草?狗尾草?鸭跖草Commelina communis为主?此外, 青葙Celosia argentea?金色狗尾草Setaria pumila?马齿苋Portulaca oleracea?反枝苋Amaranthus retroflexus及碎米莎草Cyperus iria等5种杂草在部分区域内优势度较高?经聚类分析, 皖北杂草群落分为两组, 阜阳?亳州及宿州3市的杂草聚为第1组, 淮南?淮北市及蚌埠3市的杂草聚为第2组?造成各地市杂草群落差异较大的原因与当地农田管理水平?用药习惯和种植制度等因素关系密切?     

无锡地区麦田杂草种群的演变及防治

无锡地区麦田杂草第一次普查于190年，2000年进行了第二次调查。10a来随着种植业结构的调整，耕作制度的改变、化学除草剂的长期应用，麦田杂草优势种和优势种群所组成的群落结构发生了明显的变化。现把调查的结果与种群的变化综述如下。     

Occurrence and distribution of important weed species in German winter oilseed rape fields

Journal of Plant Diseases and Protection - Data on weed species currently found in winter oilseed rape, the extent of their occurrence and regional distinctions were collected in autumn 2005, 2006...     

Simultaneous detection by multiplex PCR of the high-temperature-growing Pythium species: P. aphanidermatum, P. helicoides and P. myriotylum

The objective of this study was to develop a multiplex PCR detection method for the high-temperature-growing pathogens Pythium aphanidermatum, P. helicoides and P. myriotylum. Species-specific primer pairs were designed that targeted the rDNA ITS regions. The multiplex PCR was constructed with a universal primer pair for eukaryotes directed at the 18S rDNA as a positive control, in addition to the three species-specific primer pairs. When the multiplex PCR was applied to naturally infested soils, the expected species were reliably identified, suggesting that the method is suitable for the detection of the three Pythium pathogens in environmental samples.     

汉阴县麦田杂草优势种群及防除

陕西省汉阴县麦田杂草有13科23种,其中分布范围较广、密度较大的有看麦娘、日本看麦娘、菵草、长芒棒头草等单子叶杂草和繁缕、猪殃殃、大巢菜、藜、刺儿菜等双子叶杂草.水稻茬麦田杂草发生危害重于旱地麦田,水稻茬麦田的优势杂草主要有看麦娘、繁缕、日本看麦娘、菵草,旱地麦田优势杂草主要有繁缕、猪殃殃、大巢菜、刺儿菜.针对麦田杂草的发生特点,提出协调农业防除与化学防除的综合治理策略,化学防除要针对麦田的不同杂草优势种群,合理选用除草剂,以达到综合除草效果.     

免耕栽培模式下油菜菌核病的早期分子检测及预测模型建立

为建立免耕栽培模式下油菜菌核病的早期预测模型,通过巢式PCR法检测湖北省前茬分别为棉花和水稻的2种免耕油菜田花朵带菌率,结合田间调查分析茎秆菌核病发生率与病害主要流行影响因子之间的相关性,并采用主成分分析法建立免耕油菜田花期菌核病的预测模型。结果表明,2009—2012年棉花-油菜田花朵带菌率在同期比水稻-油菜田高,前者花朵带菌率为2.0%~58.2%,后者为0~41.0%。花朵带菌率、子囊盘密度和叶发病率对茎秆发病起主要作用,降雨量和温度作用次之;建立的棉花-油菜和水稻-油菜2种免耕类型田病害预测模型分别为:y=0.261x_1+4.89x_2+0.323x_3+0.32x_4+0.457x_5-9.438,y=0.361x_1+5.824x_2+0.323x_3+0.809x_4+0.333x_5-12.608;且预测值与实际值之间均具有较高的拟合度。表明在花期获得的花朵带菌率、子囊盘密度、叶发病率、降雨量及气温数据,经病害模拟方程可预测当年油菜菌核病发生情况。     

安徽省麦田杂草种类组成变化及群落特征分析

为明确近年来安徽省麦田杂草种类组成及群落结构变化情况,本研究采用倒置"W"取样法对安徽省麦田杂草进行了连续两年的跟踪调查,结果发现麦田杂草有19科72种,其中禾本科、菊科、蓼科种类最多。优势杂草有节节麦Aegilops tauschii、野燕麦Avena fatua、日本看麦娘Alopecurus japonicus、看麦娘A.aequalis、菵草Beckmannia syzigachne、猪殃殃Galium spurium等6种,区域性优势杂草有5种,常见杂草有8种,一般杂草有53种。此外,不同地区优势杂草存在差异,淮北草害区是以猪殃殃+节节麦+野燕麦为主的杂草群落;江淮丘陵草害区为日本看麦娘+看麦娘+猪殃殃+菵草;江南草害区为日本看麦娘+看麦娘+菵草。从杂草的分布来看,江淮丘陵草害区的杂草群落物种丰富度、多样性最高,杂草有56种,香农指数最高,为2.34;江南草害区杂草群落物种丰富度最低,有17种杂草,辛普森指数最高,为0.037,均匀度指数也较低,为0.29,优势杂草突出;从相似性指数来看,安徽省三大草害区之间的相似性指数都小于等于0.5,差异性较大。同时,安徽省麦田杂草入侵问题严重,节节麦甚至已经成为某些地区的优势杂草之一,而大穗看麦娘A.myosuroides在安徽省局部地区也多有发生。