快速检测大豆疫莓菌的酶联免疫吸附技术研究

利用已经制备的大豆疫霉菌的抗血清，对大豆疫霉菌的纯培养和大豆病组织进行检测。结果表明，间接法（Ｉ－ＥＬＩＳＡ）和夹心法（ＤＡＳ－ＥＬＳＩＡ）能较好地检测纯培养的大豆疫霉菌，但在检测病组织时，夹心ＥＩＳＡ的特异性较强，能够可靠地检测到大豆病组织内的病原菌。     

三种ELISA方法检测苹果褪绿叶斑病毒和苹果茎沟病毒的比较

苹果褪绿叶斑病毒（ＡＣＬＳＶ）和苹果茎沟病毒（ＡＳＧＶ）是感染苹果和其它一些果树的重要病毒。作者应用ＰＡＳ－ＥＬＩＳＡ法成功地检测了苹果组培苗中的这两种病毒。为了简化操作步骤,试验了ＤＡＳ－ＥＬＩＳＡ和改良ＤＡＳ－ＥＬＩＳＡ法,并与ＰＡＳ－ＥＬＩＳＡ的检测结果比较。试验结果表明,ＤＡＳ－ＥＬＩＳＡ能检测出ＡＳＧＶ,却不能检测出ＡＣＬＳＶ,与此同时,这两种病毒均可用改良ＤＡＳ－ＥＬＩＳＡ检测。ＤＡＳ     

应用单克隆抗体检测香蕉束顶病

应用香蕉束顶病毒（ＢＢＴＶ）的单克隆抗体（ＭｃＡｂ）建立了检测香蕉束顶病的间接ＥＬＩＳＡ方法，其检测香蕉病组织汁液的最大稀释度为１：１２８０，健康香蕉组织汁液无非特异性反应。用间接ＥＬＩＳＡ方法检测了采自广东、广西９个市（县）的香蕉产区的８０个大田香蕉束顶病样本和２５个荫棚香蕉试管苗样本，其中大田样本ＢＢＴＶ的带毒率为７６．５％，试管苗样本的带毒率为８．０％。香蕉束顶病株不同部位ＢＢＴＶ含量检测表     

酶联放大系统提高常规酶联检测灵敏度的研究

以莴苣花叶病毒（Ｌｅｔｕｃｅｍｏｓａｉｃｖｉｒｕｓ）及其单克隆和多克隆抗体为试材，以直接ＥＬＩＳＡ、生物素抗生物素ＥＬＩＳＡ和异种动物双抗体夹心ＥＬＩＳＡ为方法，分别用酶联放大系统进行比较，发现酶联放大系统比常规的方法灵敏度高３～１０倍，其中以生物素抗生物素酶联放大系统效果最好，灵敏度达１ｎｇ     

用单克隆抗体结合酶联免疫吸附试验检测棉铃虫幼虫体内的核多角…

在常规ＥＬＩＳＡ间接法的基础上，应用单克隆抗体检测感染棉铃虫核型多角体病毒的棉铃虫幼虫体内病毒粒子。３龄幼虫饲喂表层涂有ＨａＮＰＶ人工饲料后９小时，即可在幼虫抽提液中检出病毒辣粒子抗原，而典型病虫显症需５－６天后才出现。因此本法是一种灵敏、快速、特异性强的检测昆虫杆状病毒辣的方法。利用单克隆抗体结合ＥＬＩＳＡ试验分别检测来自江苏和山东不同地区棉田自然死亡的棉铃虫幼虫，表明在江苏和山东不同棉区可检测     

棉花枯萎病菌的血清学研究

本研究以棉花枯萎病菌公安菌株为材料，用Ｄ．Ｉａｎｎｅｌｉ法提取菌丝孢子蛋白抗原，按改进的Ｊ．Ｅ．Ｄｅｖａｙ免疫方案制备抗血清。琼脂双扩散测得效价达１／６４，用琼脂双扩散、（比较）对流免疫电泳、间接ＥＬＩＳＡ及ＳＰＡ－ＥＬＩＳＡ测定抗血清的特异性和灵敏度。结果表明４种检测方法基本上均能鉴别到尖孢镰刀菌种的水平，但不能鉴别到种以下的分类单位，４种方法检测所达的灵敏度依次为４．８６２５×１０－２，６．０７８×１０－３，３．７９９×１０－４，３．７９９×１０－４ｍｇ×ｍｌ－１。除此之外，本研究对琼脂双扩散法在真菌血清学上的应用上作了摸索     

梨中AVERMECTIN B1残留的酶联免疫吸附测定

本文建立了检测梨中Ａｖｅｒｍｅｃｔｉｎ Ｂ１的间接竞争酶联免疫吸附测定法（ＥＬＩＳＡ）。以结合物４″－Ｏ－（单）琥珀酰Ａｖｅｒｍｅｃｔｉｎ Ｂ１－ＢＳＡ免疫家兔，制备特异性针对Ａｖｅｒｍｅｃｔｉｎ Ｂ１的多克隆抗体。梨样本经甲醇提取后用ＥＬＩＳＡ检测。在６．０￣６０ｍｇ／ｋｇ添加浓度范围内，Ａｖｅｒｍｅｃｔｉｎ Ｂ１的回收率为８６％￣１０５％，变异系数（ＣＶ）为４％￣８％。样本检测限为０．１ｍｇ／     

苹果褪绿叶斑病毒PBM1（李假痘）毒株的提纯、抗血清制备及ELISA检测

从木本植物分离的苹果褪绿叶斑病毒（ａｐｐｌｅｃｈｌｏｒｏｔｉｃｌｅａｆｓｐｏｔｖｉｒｕｓ，Ｔｒｉｃｈｏｖｉｒｕｓ组）的ＰＢＭ１毒株能引起李品种“Ｈａｕｓｚｗｅｔｓｈｅ”的假痘病，将其繁殖在昆诺藜（Ｃｈｅｎｏｐｏｄｉｕｍｑｕｉｎｏａ）上。感染ＰＢＭ１毒株的昆诺藜叶组织用两次蔗糖梯度离心提纯。每１００ｇ叶组织的病毒产量平均为１．６８ｍｇ。在电镜下可观察到弯曲状病毒颗粒上的螺纹结构。通过ＳＤＳ－聚丙烯酰胺凝胶电泳分析，病毒外壳蛋白的分子量为２１．５ｋＤａ。用提纯的病毒免疫家兔制备的抗血清和ＥＬＩＳＡ法，对感染ＰＢＭ１的昆诺藜叶片和桃花进行了灵敏的检测。     

福建西番莲病毒病的发生及其病原黄瓜花叶病毒亚组鉴定

１９８８－１９９５年对福建西番莲病毒的发生为害进行调查,发现在西番莲栽培区病毒病普遍发生,发病率通常在３０－４０％,严重的达９０％以上,田间症状主要表现为叶片环斑、皱缩、花叶、环斑花叶、死面和果实木质化等。从田间病株采集４０份样本,经电镜观察和采用Ａ蛋白夹心ＥＬＩＳＡ（ＰＡＳ＿ＥＬＩＳＡ）,用西番莲化木质病毒ＰＷＶ）,西番莲黄花叶病毒ＰＦＹＭＶ）、紫果西番莲花叶病毒（ＧＭＶ）、烟草花叶病毒（ＴＭＶ     

几种危险性病毒抗血清的制备及检测方法的建立

番茄环斑病毒、烟草环斑病毒和南芥菜花叶病毒用改进的方法提纯后免疫兔子，制备出高质量的抗血清。琼脂扩散效价为１／３２０，用ＳＰＡ－ＥＬＩＳＡ方法检测病汁液可达１０－３～１０－４。３种血清之间无交叉反应，与健康寄主和常见的自然寄主蛋白也无反应。应用３种血清分别对２３种植物１００多个采集的样品和进口种子进行了ＳＰＡ－ＥＬＩＳＡ检测，未发现有上述３种病毒存在     

The possibility of factors other than phytoalexin accumulation preventing fungal growth in the incompatible interactions of soybean with Phytophthora megasperma f. sp. glycinea

Soybean ( Glycine max ) cv. Harosoy 63 is resistant to race 1 and susceptible to race 9 of Phytophthora megasperma f. sp. glycinea (Pmg). In detached primary leaves inoculated with zoospores, growth of race 1 was completely suppressed 16 h after inoculation, while race 9 was unaffected. The amount of the phytoalexin glyceollin that accumulated, however, was not significantly different in either the incompatible or compatible interaction 16 h after inoculation. At the circumference of the inoculated area, a slight accumulation of phytoalexin was observed only in the incompatible interaction 20 h or more after inoculation. Tolerance of race 9 to the phytoalexin was significantly higher than that of race 1 when the phytoalexin was added to agar. Moreover, race 9 degraded glyceollin faster than race 1. On leaves inoculated at separate points with either race, the lesion associated with race 9 never colonized areas inoculated with race 1. These results suggest that factor(s) other than the accumulation of phytoalexin in soybean tissue might cause cessation of growth of Pmg.     

苜蓿花叶病毒ELISA检测方法的建立及其在大豆病毒调查和抗性资源鉴定中的应用

苜蓿花叶病毒(alfalfa mosaic virus, AMV)是一种世界性分布、宿主范围广、具有严重危害性的植物病毒,能引起大豆的严重病害。本研究利用原核表达的AMV CP蛋白制备的抗血清,建立了高效、准确的AMV间接ELISA检测方法,并应用于病害调查和抗性鉴定,结果表明制备的3份抗血清对重组蛋白和AMV感染的大豆植物粗提液的效价均达到256 000倍,血清特异性分析结果显示3份抗血清仅识别感染AMV的大豆叶片,不识别感染大豆花叶病毒(soybean mosaic virus, SMV)的大豆叶片。通过建立的AMV间接ELISA与常规RT-PCR同时对采集的50份疑似感染AMV的大豆样品进行检测,有46份样品检测结果一致,符合率达92%。利用建立的AMV ELISA方法和课题组已建立的SMV ELISA方法对吉林省大豆主产区的大豆样品进行病毒检测的结果表明,病毒检出率为38.30%,SMV的检出率达30.85%,AMV的检出率达17.06%,复合侵染率为9.61%。对接种AMV的40个大豆品种进行抗性鉴定,结果显示40份大豆全部感染AMV,但是病毒载量存在差异,部分品种表现出AM...     

大豆疫霉菌对甲霜灵(Metalaxyl)的敏感性及变异性

大豆疫霉菌(Phytophthora megasperma f.sp.glycinea)对甲霜灵的敏感性及变异性研究结果表明,17个中国大豆疫霉菌侏菌丝生长对甲霜灵较敏感,Ec50值平均为0.0098ppm,Ec95值平均为2.6295ppm。但不同菌株对甲霜灵的敏感性不同,Ec50值范围在0.0042～0.0297ppm之间,相差7倍左右。Ec95值范围在0.7688～6.1682ppm之间,相差8倍左右。3个美国菌株菌丝体生长对甲霜灵有一定耐药性,Ec50值范围在0.3725～0.4813ppm之间,Ec95值范同在125.97～280.59ppm之间,分别比中国大豆疫霉菌株Ec50值和Ec95值平均高43倍和77倍。单孢分离物菌丝体生长对甲霜灵敏感性变异较大,B1菌株单孢分离物Ec50值范围在0.0058～0.2537ppm之间,S367菌株的单孢分离物的Ec50值范围在0.0069～0.1735ppm之间,分别相差43倍和25倍。有的单孢分离物对甲霜灵的耐药性增高。各菌株孢子囊和卵孢子产生对甲霜灵敏感性有一定差异,但1.0ppm的甲霜灵对其抑制率均为100％。活体测定结果表明,1.0ppm的甲霜灵对该病的防效达94～100％。     

Plum pox virus detection in dormant plum trees by PCR and ELISA

An immunocapture polymerase chain reaction (IC-PCR) protocol and ELISA were compared for their effectiveness in detecting plum pox virus (PPV) in dormant plum material. Although the IC-PCR was about one thousand times more sensitive than ELISA, PPV was detected by ELISA in 71–80% of bark samples collected in December, January and March 1996/97 from pot-grown rootstock trees inoculated with PPV the previous March, compared with 85–86% detection in the same samples by IC-PCR. In similar samples from one-year-old shoots taken from infected branches of orchard trees, 66–81% were positive by ELISA compared with 81–87% by IC-PCR. With bulked samples taken from the fibrous roots of the pot-grown trees, PPV was detected in 92–100% of samples by IC-PCR in winter compared with only 38–65% by ELISA. These results were confirmed in samples from the roots and shoots of the same trees in 1997/98. Three samples per shoot would have been sufficient to detect PPV by ELISA in 87 of the 88 infected shoots tested during the two winters. However, infected shoots are irregularly distributed in diseased trees and PCR assays of root samples offer the potential for improving the reliability of identifying trees infected with PPV.     

Molecular detection of Fusarium solani f. sp. glycines in soybean roots and soil

A polymerase chain reaction (PCR)-based method was developed to detect DNA of Fusarium solani f. sp. glycines , the cause of soybean sudden death syndrome. Two pairs of primers, Fsg1/Fsg2 designed from the mitochondrial small subunit ribosomal RNA gene, and FsgEF1/FsgEF2 designed from the translation elongation factor 1-α gene, produced PCR products of 438 and 237 bp, respectively. Primer specificity was tested with DNA from 82 F. solani f. sp. glycines , 55 F. solani non-SDS isolates, 43 isolates of 17 soybean fungal pathogens and the oomycete Phytophthora sojae , and soybean. The sensitivity of primer Fsg1/Fsg2 was 10 pg while that of FsgEF1/FsgEF2 was 1 ng when using F. solani f. sp. glycines total genomic DNA or down to 10³ macroconidia g⁻¹ soil. Nested PCR increased the sensitivity of the PCR assay 1000-fold to 10 fg using primers Fsg1/Fsg2, and 1 pg using primers FsgEF1/FsgEF2. F. solani f. sp. glycines DNA was detected in field-grown soybean roots and soil by PCR using either single pairs of primers or the combination of two pairs of primers. The occurrence of F. solani f. sp. glycines was determined using nested PCR for 47 soil samples collected from soybean fields in 20 counties of Illinois in 1999. F. solani f. sp. glycines was detected in soil samples from all five Illinois Agricultural Statistic Districts including 100, 89, 50, 92 and 50% of the samples from East, Central, North-east and West Districts, respectively.     

枯草芽孢杆菌B006对黄瓜枯萎病菌和辣椒疫霉病菌的抑制作用及其抗菌组分分析

枯草芽孢杆菌菌株B006发酵液的甲醇提取物对黄瓜枯萎病菌Fusarium oxysporum f. sp. cucumerinum菌丝的抑制作用大于对辣椒疫霉病菌Phytophthora capsici菌丝的抑制作用;但对P. capsici游动孢子萌发的抑制作用明显大于对黄瓜枯萎病菌孢子萌发的抑制作用,提取物原液在5 min内可使辣椒疫霉的游动孢子全部崩解。提取物经60、90和120℃加热处理10 min后仍保持抑菌活性,其盐酸水解液在TLC层析板显现橙红色斑点,说明提取物具有环肽结构。HPLC-ESI-MS分析可得到m/z为995、1009、1023、1037、1051和1436、1450、1478和1492的质谱峰,与抗菌物质surfactin和fengycin的分子量大小一致。以终浓度为10^6 cfu·g^-1的接种量将B006菌剂施入无菌育苗基质中,在黄瓜和辣椒4～6片叶期时用固相萃取法提取根际的抗菌物质,并进行HPLC-ESI-MS分析,可从根际检测到surfactin和fengycin,但未从对照处理根上检测到脂肽类抗生素。本研究对理解枯草芽孢杆菌在田间状况下的防病机理有一定意义。     

AM真菌和镰刀菌对西瓜根系膜脂过氧化作用和膜透性的影响

 在温室盆栽条件下研究了丛枝菌根(arbuscular mycorrhiza,AM)真菌Glomus versiforme (Karsten) Berch和西瓜枯萎镰刀菌(Fusarium oxysporum f. sp. niveum)对西瓜根系中膜脂过氧化产物丙二醛(MDA)含量和细胞膜透性的影响。结果表明,接种AM真菌的西瓜根系中MDA含量、组织自动氧化速率和膜透性均低于对照,先接种G. versiforme,后接种F. oxysporum f. sp. niveum处理的MDA含量、组织自动氧化速率和细胞膜透性均低于只接种F. oxysporum f. sp. niveum的处理。接种G. versiforme感枯萎病西瓜品种"郑杂5号"MDA含量、组织自动氧化速率和膜透性降低幅度大于抗病品种"京欣1号"的接种处理,说明G. versiforme可降低感病西瓜品种的膜透性和MDA的产生,从而有效地保护细胞膜系统,减轻F. oxysporum f. sp. niveum对西瓜的为害程度。     

The Role of Ethylene Production in Virulence of Pseudomonas syringae pvs. glycinea and phaseolicola

ABSTRACT The importance of ethylene production for virulence of Pseudomonas syringae pvs. glycinea and phaseolicola was assayed by comparing bacterial multiplication and symptom development in bean and soybean plants inoculated with ethylene-negative (efe) mutants and wild-type strains. The efe mutants of Pseudomonas syringae pv. glycinea were significantly reduced in their ability to grow in planta. However, the degree of reduction was strain-dependent. Population sizes of efe mutant 16/83-E1 that did not produce the phototoxin coronatine were 10- and 15-fold lower than those of the wild-type strain on soybean and on bean, and 16/83-E1 produced very weak symptoms compared with the wild-type strain. The coronatine-producing efe mutant 7a/90-E1 reached fourfold and twofold lower population sizes compared with the wild-type strain on soybean and bean, respectively, and caused disease symptoms typical of the wild-type strain. Experiments with ethylene-insensitive soybeans confirmed these results. The virulence of the wild-type strains was reduced to the same extent in ethylene-insensitive soybean plants as the virulence of the efe mutants in ethylene-susceptible soybeans. In contrast, the virulence of Pseudomonas syringae pv. phaseolicola was not affected by disruption of the efe gene.     

Development of a recombinant antibody ELISA test for the detection of Polymyxa betae and its use in resistance screening

An ELISA test was developed for the quantitative detection of the obligate parasite Polymyxa betae , the vector of Beet necrotic yellow vein virus (BNYVV), in infected sugarbeet roots. The test used monoclonal and polyclonal antibodies raised to a recombinantly expressed glutathione-S-transferase (GST) from P. betae . A close correlation was found between the number of P. betae zoospores in serially diluted suspensions and absorbance values in the ELISA test. Time-course studies of plants grown in naturally infested soils in controlled environment tests demonstrated the value of the ELISA test in screening for P. betae resistance. In preliminary tests, P. betae -resistant accessions of the wild sea beet ( Beta vulgaris ssp. maritima ), which might be used to restrict the transmission of BNYVV, were identified.     

利用小种标记分析云南省两个县的小麦条锈菌群体

 小麦条锈病是由小麦条锈菌（Puccinia striiformis f.sp. tritici）引起的气流传播病害,在世界各主要麦区均有发生,也是我国小麦生产上危害最严重的病害之一。小麦条锈菌毒性变异频繁,易产生新毒性小种,导致小麦品种的抗病基因失效,引起小麦条锈病周期性流行。因此,了解小麦种植区的条锈菌群体结构组成及变异,对于制定更为有效的条锈病控制方法具有重要的意义。