基于细胞色素氧化酶基因和激发素基因的大豆疫霉菌特异性PCR引物

引起大豆疫霉根腐病的大豆疫霉菌(Phytophthora sojae)是危害大豆的破坏性病原菌之一,也是我国重要的检疫性植物病原菌。简单、快速、准确的鉴定和检测技术是阻止大豆疫霉菌传入和病害早期诊断的有效工具。本研究从大豆疫霉菌细胞色素氧化酶基因Ⅱ(coxⅡ)序列和两个激发素(elicitin)家族基因EST序列中开发了3对大豆疫霉菌特异引物:Cox3-F/Cox3-R、PSEL1-F/PSEL1-R和PSEL2-F/PSEL2-R。这3对引物在大豆疫霉菌中分别扩增出450、289bp和370bp的特异性片段,其检测大豆疫霉菌基因组DNA的灵敏度分别为20、2pg/μL和2pg/μL。3对引物能够有效检测大豆疫霉菌侵染的大豆病株,可以用于病害诊断和鉴别。     

疫霉菌对霜脲氰抗性遗传及对霜脲氰和甲霜灵的交互抗性

就疫霉菌对霜脲氰抗性的产生和遗传,以及疫霉菌对霜脲氰和甲霜灵的交互抗性进行了研究。结果表明,苎麻疫霉容易对霜脲氰产生抗药性突变,但抗性在连续３代单游动孢子分离过程中逐渐丧失。大雄疫霉对霜脲氰不易产生抗性突变,恶疫霉、大雄疫霉和苎麻疫霉对甲霜灵和霜脲氰不存在交互抗性     

两株对辣椒疫霉菌有拮抗作用的拮抗菌分泌蛋白的研究

用平板对峙生长法筛选到两株对辣椒疫霉菌具有较强拮抗作用的细菌,经鉴定为芽孢杆菌属。测定了无菌滤液的抑菌活性及两菌产生拮抗物的热稳定性。用硫酸铵沉淀法提取到对辣椒疫霉菌有拮抗作用的抗菌蛋白。发酵条件的研究表明,用Ｇ培养基、ｐＨ７、培养３２小时,ＸＡ１、ＸＦ１能产生具有较强拮抗作用的拮抗物。     

Detection of phytopathogens of the genus Dickeya using a PCR primer prediction pipeline for draft bacterial genome sequences

This study used a novel computational pipeline to exploit draft bacterial genome sequences in order to predict, automatically and rapidly, PCR primer sets for Dickeya spp. that were unbiased in terms of diagnostic gene choice. This pipeline was applied to 16 draft and four complete Dickeya genome sequences to generate >700 primer sets predicted to discriminate between Dickeya at the species level. Predicted diagnostic primer sets for both D. dianthicola (DIA‐A and DIA‐B) and ‘D. solani’ (SOL‐C and SOL‐D) were validated against a panel of 70 Dickeya reference strains, representative of the known diversity of this genus, to confirm primer specificity. The classification of the four previously sequenced strains was re‐examined and evidence of possible misclassification of three of these strains is presented.     

The quarantine root-knot nematode Meloidogyne enterolobii – a potential threat to Portugal and Europe

In 2017, during a survey on subsistence farms and gardens in Coimbra region, Portugal, 40 infected root samples were collected and 47 root-knot nematode (RKN) isolates identified, based on esterase phenotype. The phenotypes A2, H1, Hi2/Hi4, I1/I2/I3 and J3 associated to five Meloidogyne species (M. arenaria, M. hapla, M. hispanica, M. incognita and M. javanica) were found in 43 RKN isolates. The esterase phenotype En2/En4/En5, corresponding to M. enterolobii (=M. mayaguensis), was detected in four RKN isolates from Cereus hildmannianus (Cactaceae), Lampranthus sp. (Aizoaceae), Physalis peruviana (Solanaceae) and Callistemon sp. (Myrtaceae) infected roots. In order to validate the biochemical identification of the M. enterolobii isolates, molecular studies performed with species-specific primers yielded the expected fragment of c.520 bp, and the amplification of cytochrome oxidase subunits I and II regions of 800 bp. The DNA sequences of one of the isolates were compared with available Meloidogyne species sequences in databases. The Portuguese isolate grouped with 99–100% bootstrap support with all M. enterolobii sequences included for comparison, confirming the presence of this RKN species in Portugal. In the EPPO region, M. enterolobii has been reported in France and Switzerland and intercepted in the Netherlands, Germany and the UK associated with plant material from Asia, South America and Africa. Taking into account the pathogen aggressiveness and its distribution, there is a high probability of its spread not only in the Mediterranean region but also in Europe, and of it becoming a threat to the agricultural economy, where there are no effective strategies for its control.     

Genotyping Cephalosporium gramineum and development of a marker for molecular diagnosis

This study investigated the genetic variation of 40 isolates of Cephalosporium gramineum, the causal agent of cephalosporium stripe disease of wheat, based on variations in internal transcribed spacers (ITS) and intergenic spacers (IGS) of rDNA. Of the isolates, 29 were from Japan and the rest from the USA and Europe. The ITS region was about 600 bp and almost identical among these isolates. In the IGS region (～5 kbp), restriction fragment length polymorphism analysis detected four genotypes among the 40 isolates. One representative isolate was selected from each of the four genotypes, and the IGS region was sequenced. Attempts to design a genotype‐specific marker based on the size of PCR products amplified with selected primers failed to differentiate among the four genotypes. Alternatively, a species‐specific primer set (CGIGS1 and CGIGS2) was developed that annealed within the conserved region, producing a DNA fragment of about 1·8 kbp. Tests of this primer set on a wide range of other fungi from 11 genera confirmed that it was specific to C. gramineum. This primer set could serve as an effective tool in the molecular diagnosis of C. gramineum and has the potential to assist in a better understanding of the host–pathogen interaction.     

菊欧文氏菌分子检测技术的研究

 蝴蝶兰细菌性软腐病对蝴蝶兰的生长危害严重, Erwinia chrysanthemi(菊欧文氏菌)、Erwinia carotovora subsp. carotovora(胡萝卜软腐欧文氏菌胡萝卜软腐亚种)是引起蝴蝶兰软腐病的主要病原细菌, 其中E.chrysanthemi被列入我国三类检疫性有害生物。本文对菊欧文氏菌分子检测技术进行了研究, 设计出针对该病原细菌的特异性引物, 应用实时荧光PCR方法检测样品中存在的菊欧文氏菌, 检测灵敏度达到10²cfu/mL。     

盆栽条件下水分对植物组织建成的影响

盆载条件下以三种梯度控水处理两个多月的６种植物，解剖观察其组织建成情况。发现水分状况对细胞组织建成有影响。且不同组织所受影响的程度不同。其中，轻度干旱（７０％供水）与充分供水（１００％）条件下所观察的多数组织发育良好，而中度干旱（４０％供水）则组织发育不佳。证明即使对旱生植物，幼苗期足够的供水也是必须的。有关植物旱性结构以及不同水分状况下植物内部结构差异的报道，近些年来日渐增多（１－５）。但不同水     

Phytophthora lateralis discovered in an old growth Chamaecyparis forest in Taiwan

The geographic origins of the invasive Phytophthora species, P. lateralis and P. ramorum are unknown. In 2008 soil samples were collected in an old growth yellow cedar (Chamaecyparis obtusa var. formosana) stand in the Ma‐kau Ecological Park in north eastern Taiwan and subjected to Phytophthora baiting procedures at 18°C. Cedar needle baits yielded isolates of a slow growing Phytophthora culture from one soil sample, together with P. cinnamomi. Phytophthora bisheria sp. nov. was obtained from another sample. The slow growing isolates conformed closely to P. lateralis in the morphology of their sporangia and chlamydospores, growth–temperature relationships, absence of gametangia and their ITS and cox II sequences. The isolates’ sporangia were partially caducous, with short pre‐formed pedicels of ca. 3–5 μm, a highly unusual feature in a non‐papillate Phytophthora. The isolates also produced multicellular stromata on cedar decoction agar. Small morphological and molecular differences were observed between the Taiwan‐isolates and Oregon‐control isolates. Taiwan may lie within the geographic centre of origin of P. lateralis. By analogy Japan may also lie within the natural range of P. lateralis; and Japan, along with Taiwan and Yunnan, could be an origin for the closely related P. ramorum.     

香蕉穿孔线虫(Radopholus similis)特异性分子检测技术研究

 香蕉穿孔线虫(Radopholus similis)是一种国际公认的极具毁灭性的有害线虫,也是我国重要的进境检疫性有害生物。利用线虫通用引物对香蕉穿孔线虫7个不同群体的ITS进行PCR扩增、克隆及测序,获得ITS片段序列长度为706bp。经与国外香蕉穿孔线虫种群及近缘种的ITS序列进行比对及同源性分析,构建设计了1对香蕉穿孔线虫的特异性引物RsF1/RsR1,特异性扩增片段长度为271 bp;同时引入D2A/D3B作内标,研究出特异性检测香蕉穿孔线虫的一步双重PCR分子技术和方法,该项检测技术特异性好,耗时较短、操作简便、可靠。     

马铃薯腐烂茎线虫特异性分子检测技术研究

 本研究利用通用引物(rDNA1/rDNA2)研究了21个国内甘薯茎线虫(Ditylenchus destructor)群体和1个韩国马铃薯茎线虫(D.destructor)群体的rDNA-ITS序列,从21个国内群体中扩增出2个大小不同的ITS片段,分别约为940bp和1100bp;经克隆、序列测定和分析比对发现其ITS区存在特异性差异,分别命名为A型和B型,其中18个群体DdTH、DdCL、DdJN、DdMY1、DdYX1、DdZZ、DdLN,DdDX1、DdFN,DdYX2、DDSX1、DdDX2、DdXY,DdLL、DdSX2、DdLY,DdMY2和DdPY的ITS扩增产物约为940bp,称之为A型马铃薯腐烂茎线虫(940bp),3个群体DdSH,DdTS,DdYS为B型马铃薯腐烂茎线虫(1100bp)。设计构建并筛选出A型和B型马铃薯腐烂茎线虫2对特异性引物DdS1/DdS2和DdL1/DdL2,分别扩增出A型马铃薯腐烂茎线虫、B型马铃薯腐烂茎线虫群体的特异片段252bp和485bp;引入D3A/D3B作为内标,设计出一步双重PCR检测技术;同时优化了检测体系和PCR反应程序。该技术具有较高的特异性和灵敏性,能快速、准确地检测出不同型的马铃薯腐烂茎线虫群体。     

盆栽条件下不同供水处理对六个树种蒸腾速率的影响

在三种供水条件下，研究了六个树种的生境土壤含水量，蒸腾速率、气孔阻抗和叶温与气温差。土壤含水量和蒸腾速率随供水量的减少而下降，气孔阻抗和叶温与气温差则增加，测定分析表明，六个树种的最优水分条件都应是充分供水，旱生树种并非喜旱，仅是耐旱而已。对土壤水分的消耗，旱生灌木低于乔木，其中梭梭属最低。     

菟丝子属杂草普查及防治试验简报

本文报道江西省在普查中新发现的菟丝子属杂草的寄主植物3l科75种以及4种除草剂对荒地菟丝子属杂草的防治效果，提出菟丝子属杂草的综合防治方法。