Effective Application of DAS-ELISA for Detection of Grapevine Leafroll Associated Closterovirus-3 Using a Polyclonal Antiserum Developed from Recombinant Coat Protein

A polyclonal antiserum (As163) specific to grapevine leafroll associated closterovirus-3 (GLRaV-3) was developed using a recombinant coat protein expressed in E. coli from a cDNA clone identified after immunoscreening of a cDNA library. Specificity of the antiserum to GLRaV-3 was shown by Western blot and immunosorbent electron microscopy. With this antiserum, an effective double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for GLRaV-3 detection. To evaluate the sensitivity of the antiserum in DAS-ELISA for virus detection, different combinations of antibodies were compared. Although best results were obtained when As163 was used for coating and a monoclonal antibody (MabNY1.1) was used as an enzyme conjugate, good results were also obtained when As163 was used both for coating and as an enzyme conjugate. Using this As163–Mab system in DAS-ELISA, we confirmed the presence of GLRaV-3 in a diverse collection of leafroll infected vines.     

核盘菌转录因子SsMCM1原核表达与亚细胞定位

 核盘菌是一类寄主范围广泛的植物病原真菌。MADS-box蛋白家族基因广泛存在于生物体中,参与调控细胞识别、新陈代谢、细胞周期等。核盘菌转录因子SsMCM1调控其子实体形成与致病性。为进一步揭示SsMCM1的作用机制,本研究以核盘菌野生型菌株uf-70的cDNA为模板,扩增得到SsMCM1基因,并与原核表达载体pET-28a(+)连接,构建成重组质粒转化到大肠杆菌BL21(pLysS)表达菌株中,通过IPTG的诱导和亲和层析,纯化得到SsMCM1蛋白,并以此为抗原获得抗血清,完成效价测定。利用特异性抗血清,与核盘菌不同组织蛋白进行特异性的免疫结合,发现其与核盘菌总蛋白、核盘菌细胞质总蛋白均特异性结合,而核盘菌细胞壁蛋白未发现特异性反应,表明SsMCM1不定位于核盘菌细胞壁上。此外,构建其亚细胞定位载体pCG-1301::SsMCM1,并将重组质粒转化到农杆菌中,通过农杆菌的侵染作用进行瞬时表达,发现SsMCM1定位于细胞核中,作为转录因子调控核盘菌的子实体发育与致病性。     

Monitoring of plant and airborne inoculum of Sclerotinia sclerotiorum in spring oilseed rape using real‐time PCR

Sclerotinia stem rot of spring oilseed rape (Brassica napus) is caused by Sclerotinia sclerotiorum. In Sweden, the disease leads to severe crop damage that varies from year to year. A real‐time PCR assay was developed and used to determine the incidence of S. sclerotiorum DNA on petals and leaves of spring oilseed rape as well as in air samples, with the aim of finding tools to improve precision in disease risk assessment. Five field experiments were conducted from 2008 to 2010 to detect and study pathogen development. Assessments of stem rot showed significant differences between experimental sites. The real‐time PCR assay proved fast and sensitive and the relationship between percentage of infected petals determined using a conventional agar test and the PCR assay was linear (R² > 0·76). There were significant differences in S. sclerotiorum incidence at different stages of flowering. The incidence of S. sclerotiorum DNA on the leaves varied (0–100%), with significantly higher incidence on leaves at lower levels. In one field experiment, S. sclerotiorum DNA was not detected on petals during flowering, whereas the pathogen was detected on leaves, with a corresponding stem rot incidence of 7%. The amount of S. sclerotiorum DNA in sampled air revealed that spore release did not coincide with flowering on that experimental site. Thus, using a real‐time PCR assay to determine the incidence of S. sclerotiorum on oilseed rape leaves, rather than on petals, could potentially improve disease risk assessment.     

Development of monoclonal antibody-based immunological assays for the detection of live propagules of Rhizoctonia solani in soil

Mouse monoclonal antibodies (mAbs) and rabbit (polyclonal) antiserum were used to develop DIAGNOSTIC-ELISA, double-antibody-sandwich-ELISA (DAS-ELISA), DIP-STICK and immuno-fluorescence colony staining immunoassays for the specific detection of Rhizoctonia solani in soil. mAbs were raised against an anastomosis group 4 isolate of R. solani. Mice were immunized using either phosphate-buffered saline (PBS) suspensions of lyophilized mycelium plus Quil A adjuvant, or with a solubilized acetone precipitate prepared from cell-free surface washings from solid slant cultures. Polyclonal antisera were raised in rabbits using PBS suspensions of lyophilized mycelium and Quil A adjuvant. Hybridoma supernatants and rabbit antisera were screened by ELISA. Four of the cell lines raised produced mAbs that were species-specific. They recognized antigens from R. solani by ELISA and immunofluorescence, but not other related or unrelated species of soil-borne fungi. The remaining cell line produced mAbs that cross-reacted slightly, by ELISA, with antigens from R. cerealis. These mAbs did not recognize R. cerealis by immunofluorescence, or other related or unrelated soil-borne fungi, by ELISA.     

核盘菌对油菜、向日葵和大豆的侵染及其致病性分化研究

 通过对从陕西大荔采集的油菜及其后茬向日葵上的核盘菌和从新疆阿勒泰的向日葵上采集的核盘菌样品进行单菌核分离、培养和纯化,比较其菌丝生长速度、菌丝干重、菌落形态、致病力强弱及菌株的草酸累积等,将两地的核盘菌分成A、B、C三种类型,其中A类来源于陕西大荔的油菜和向日葵,B、C类来源于新疆阿勒泰的向日葵。不同类型核盘菌对于不同寄主的致病力存在着分化现象,A、B类菌株生长正常、菌落均匀旺盛,B类对油菜、向日葵和大豆的致病力较强,草酸产量较高;而A类仅对油菜和向日葵的致病力较强对大豆的致病力很弱,但草酸产量最高。C类菌株生长异常,菌落稀疏不均匀,对3种作物的致病力均弱,草酸产量较低。2001年田问调查亦表明:A类菌株可导致油菜、向日葵菌核病的发生,但未见其使大豆致病,由此提出油菜茬后,不宜种植向日葵,二者应与小麦、玉米等实行较长周期的轮作。本文也同时对各菌株进行了RAPD分析和菌丝体亲和性研究,结果表明,菌株间的遗传多样性表现丰富但未发现与其致病性分化相关。     

雷帕霉素对4种植物病原真菌的抑菌活性

采用菌丝生长速率法,测定了雷帕霉素对番茄灰霉病菌、油菜菌核病菌、水稻纹枯病菌和棉花枯萎病菌的抑菌活性,比较了嘧菌酯、丙烷脒及雷帕霉素对番茄灰霉病菌的抑菌效果,并通过电子显微镜观察了雷帕霉素对番茄灰霉病菌菌丝生长的影响。结果表明:雷帕霉素对供试4种植物病原真菌菌丝均表现出了极强的抑制活性,其中对油菜菌核病菌的抑制作用最强,EC50值为2.23×10-4μg/mL,对番茄灰霉病菌、水稻纹枯病菌和棉花枯萎病菌的EC50值分别为1.32×10-3、4.05×10-3及3.82×10-3μg/mL;雷帕霉素对番茄灰霉病菌菌丝的抑制活性显著高于嘧菌酯(EC50值为3.24μg/mL)和丙烷脒(EC50值为3.81μg/mL)。电镜观察发现,经雷帕霉素处理后,番茄灰霉病菌菌丝表现出提前衰老等症状。研究结果可为深入探讨雷帕霉素对植物病原真菌的作用机制奠定基础。     

Early stages of infection of rapeseed petals and leaves by Sclerotinia sclerotiorum revealed by scanning electron microscopy

The primary ascospore inoculum of Sclerotinia sclerotiorum initially infects rapeseed (Brassica napus var oleifera) via petals. Infected petals fall onto leaf surfaces, resulting in infection of those organs. A scanning electron microscopy (SEM) study of this process was undertaken to elucidate the host-parasite relationship and to determine the best plant organ for detection by serology of early field infection as an aid to disease forecasting and cost-effective disease control. The behaviour of ascospores deposited on young petals and on leaves was compared. Ascospores were deposited by inverting a mature apothecium above either a leaf disc, a young petal or young petal placed on a leaf surface. Spore germination, host penetration and colonization were examined by SEM. On young petals, the following steps in pathogenesis were observed: ascospore adhesion and germination, penetration of the host from short germ tubes and collapse of epidermal cells. Petals were then covered with extensive mycelium. From these sites, the mycelium invaded leaf tissues and infection proceeded. In contrast, ascospores landing directly on leaf surfaces failed to germinate. The role of petals as sites of pre-election in the aetiology of the disease is discussed in relation to the published literature.     

桧木醇衍生物的合成及抑菌活性

桧木醇是具有?酚酮骨架的单萜类天然化合物, 设计并合成了17个新型桧木醇衍生物, 其结构经核磁共振波谱及高分辨质谱分析确证。抑菌活性测定结果表明,目标化合物在50 μg/mL下对水稻纹枯病菌Rhizoctonia solani、番茄灰霉病菌Botrytis cinerea、油菜菌核病菌Sclerotinia sclerotiorum、苹果树腐烂病菌Valsa mali和黄瓜炭疽病菌Colletotrichum orbiculare均表现出较好的抑菌活性,其中化合物 3a 对水稻纹枯病菌、 3j 对番茄灰霉病菌、 3m 对油菜菌核病菌的EC₅₀值分别为1.84、2.47和1.05 μg/mL,表现出比桧木醇 (2.00、11.3和5.40 μg/mL) 更优的活性。     

核盘菌致病性分化研究

为了解核盘菌的致病性分化,本研究用活体定位穿刺接种法,以油菜为供试寄主,对采自四川省10个地区23个县、9种寄主的108个菌株进行了致病性测定,结果表明,所有菌株对供试油菜品种均能致病,但各菌株所致病斑长度差异很大(2.7～82.0 mm),说明核盘菌种群内存在明显的致病性分化,这种分化与地理来源和寄主来源没有明显的关系。     

A Laboratory Simulation for Vectoring of Trichosporon pullulans by Conidia of Botrytis cinerea

ABSTRACT A mechanism that could contribute to the suppression of Botrytis cinerea during pathogen sporulation was examined in this study. Yeasts capable of binding to B. cinerea were formulated with a cellulose carrier and applied to sporulating colonies of the pathogen. The particles from this yeast/cellulose product attached to B. cinerea conidia in the sporulating colony. Inoculum from treated colonies was harvested and applied to tomato stem tissue to test for subsequent pathogenicity. Disease development from inoculum obtained from cultures that had been treated with Trichosporon pullulans was significantly retarded (P = 0.0001) compared with cellulose-only controls. However, between 5 and 11% of conidia applied were attached to yeast cells. The removal of conidia not attached to yeast resulted in inoculum composed of >90% of conidia attached to yeast, and from this inoculum, disease development was significantly retarded (P < 0.05). When inoculum from treated B. cinerea colonies was applied to nutrient limiting agar and then incubated, the B. cinerea conidia germinated, and yeast cells infested the new hyphal growth. Constraints of the formulation of the yeast used in this study, and the implications of this vectoring approach for the suppression of B. cinerea during pathogen sporulation are discussed.     

木霉T97菌株对几种植物病原真菌的拮抗作用机制和温室防治试验

从土壤中分离了一木霉Trichoderma sp.菌株T97.竞争及对峙培养结果表明,木霉T97对豌豆根腐病菌Fusarium solani f.sp.pisi、番茄灰霉病菌Botrytis cinerea、茄子黄萎病菌Verticillium dahliae、黄瓜枯萎病菌Fusarium oxysporum f.sp. cucumerinum、小麦全蚀病菌Gaeumannomyces graminis var. tritici、小麦根腐病菌Bipolaris sorokiniana和立枯丝核病菌Rhizoctonia solani等7种病原菌有较强的生长竞争优势.光学显微观察表明,木霉T97通过缠绕、附着和穿透的方式寄生立枯丝核菌、番茄灰霉病菌和小麦全蚀病菌.受T97作用后,茄子菌核病菌的菌丝尖端肿大、变粗,豌豆根腐病菌和黄瓜枯萎病菌的菌丝出现断裂等溶菌现象.用T97培养物(0.6%(w/w))处理土壤,对茄子黄萎病和菌核病、黄瓜枯萎病和菌核病以及豌豆根腐病的苗期病害防治效果达66%～81%.用T97孢子悬浮液108cfu/mL在花期喷雾保护黄瓜、辣椒和番茄叶面,对灰霉病的防治效果相当于50%速克灵WP 3 000倍液.     

利用SRAP分析油菜品种对核盘菌遗传分化的影响

 本文采用茎秆牙签接种法将单一核盘菌菌株接种至不同油菜品种茎秆,再从46个油菜品种茎秆内收集接种后形成的菌核进行分离纯化和培养,并采用SRAP技术对46株核盘菌菌株进行了遗传分化分析。从12对检测引物中共获得357个位点,其中多态性位点273个,占76.47%;UPGMA聚类分析显示,在相似系数为0.77时,46株核盘菌菌株能够分为7组。当以寄主的抗(耐)病程度、寄主品种类型和品种选育地来源为标准将菌株分为不同群体时,AMOVA(analysis of molecular variance)结果表明核盘菌菌株在各群体内变异率分别为98.50%、105.16%和95.36%,均达到极显著水平(P<0.001),而寄主品种选育地群体间的遗传变异达到极显著,变异率为4.64%。结果表明:核盘菌菌株接种不同油菜品种后,菌株间存在明显的遗传分化,这种分化与油菜品种的选育地来源有密切关系。     

柑橘内生细菌YS45的鉴定、抗菌物质分析及其对油菜菌核病的防治作用

 从柑橘枝条中分离到26株对油菜菌核病菌(Sclerotinia sclerotiorum)具有拮抗作用的内生细菌,其中YS45菌株的拮抗活性最强。16S rRNA基因序列分析及形态学和生理生化鉴定结果表明YS45菌株为枯草芽孢杆菌(Bacillus subtilis)。高效液相色谱及质谱分析结果显示其主要的抑菌活性物质为一组fengycin同系物,包括fengycins A、fengycins B和一种稀少fengycin类型化合物。油菜离体叶片接种试验中,YS45菌株发酵液对油菜菌核病的防效在70%以上,与五氯硝基苯相当;田间小区接种试验表明,YS45菌株发酵液对油菜菌核病的防效也在50%以上。     

Development of a monoclonal antibody immunoassay for the eyespot pathogen Pseudocercosporella herpotrichoides

A double-antibody-sandwich ELISA test has been developed for the detection of Pseudocercosporella herpotrichoides using a highly specific monoclonal antibody PH-10 as the capture antibody and genus-specific rabbit polyclonal antiserum as the detector antibody. The assay recognizes extracts from plants both artificially and naturally infected with P. herpotrichoides giving at least three-fold higher absorbance values with extracts from Pseudocercosporella-infected tissue than with extracts from healthy tissues or from tissues naturally infected with Microdochium nivale, Rhizoctonia cerealis or material artificially inoculated with P. anguioides. The assay tested positively against all isolates of P. herpotrichoides , including both W-type and R-type isolates. In this assay system, extraction of the antigen from the stem bases of infected plants is a one-step process not requiring any dilution procedures. The assay can be used to detect the pathogen in presymptomatic infected seedlings. The immunogen used to generate the specific monoclonal antibody and the rabbit antiserum was a mycelial extract from which the high-molecular-weight proteins and glycoproteins had been removed by ammonium sulphate precipitation. The high-molecular-weight fraction was shown to contain cross-reactive antigens; it induced antiserum in mice that cross-reacted with the other stem-base fungi even at high dilutions. The monoclonal antibody PH-10 is an IgM antibody. Heat and periodate treatment of the antigen indicate that it is a glycoprotein and that the epitope recognized by the antibody is a protein.     

Occurrence of fungal pathogens of carrots on wooden boxes used for storage

Infested wooden boxes, previously used for carrot storage, were sampled in four commercial carrot production farms in Bradford Marsh, Ontario, and screened for fungal occurrence. At least 128 and 465 fungal isolates were recovered from these boxes in 2001 and 2002, respectively, and were classified into 10 taxonomic groups, including Alternaria spp., Aspergillus spp., Botrytis cinerea , Fusarium spp., Mucor spp., Penicillium spp., Rhizoctonia carotae , Rhizopus spp., Sclerotinia sclerotiorum and Trichoderma spp. A subsample of 27 putative pathogenic isolates was further tested for the ability to cause disease on carrots and to colonize wood surfaces under growth room and cold storage conditions. Approximately 60% of the taxa growing on wood caused lesions upon contact with intact carrots in cold storage. Isolates of S. sclerotiorum , B. cinerea and R. carotae caused the most severe diseases, developed most extensively on wooden surfaces in cold storage, and represented 12% of the recovered fungi. Isolates of Alternaria spp., Aspergillus spp., Fusarium spp., Mucor spp., Penicillium spp., Rhizopus spp. and Trichoderma spp. caused negligible or no disease on carrots and represented 88% of recovered fungi. Several of these fungi, however, showed potential to colonize wooden surfaces and cause disease on sliced carrots. This study suggests that pathogenic inocula occurring on used wooden boxes can initiate disease upon contact with healthy carrots and reusing infested boxes can affect carrots in storage.     

Cylindrocarpon olidum W1菌株代谢物中壳二孢氯素及其类似物的分离及抑菌活性

采用硅胶柱层析及高效液相色谱等技术从冬青卫矛内生真菌Cylindrocarpon olidum W1次生代谢物中分离鉴定出8个抑菌活性化合物W1-1~W1-8。经核磁共振氢谱和碳谱、高分辨质谱等技术,并结合相关文献确认其为壳二孢氯素及其类似物,分别为Ilicicolin A（W1-1）、5-Chlorocolletorin B（W1-2）、Ilicicolin B（W1-3）、Deacetylchloronectrin（W1-4）、Ilicicolin C（W1-5）、壳二孢氯素（W1-6）、Cylindrol A4（W1-7）和Cylindrol B（W1-8）。抑菌活性测定结果表明:壳二孢氯素及其类似物对烟草青枯病菌有较强的抑制作用,上述化合物的最小抑制浓度分别为6.25、3.13、>100、3.13、6.25、12.5、25和25 μg/mL;化合物W1-2、W1-4和W1-5对番茄灰霉病菌和油菜菌核病菌菌丝生长亦有较强的抑制作用,其中对番茄灰霉病菌的抑制中浓度（IC50）分别为36.45、21.60和26.69 μg/mL,对油菜菌核病菌的抑制中浓度分别为20.21、16.79和12.11 μg/mL。     

百合斑驳病毒CP与CI基因的融合表达、多抗制备及其应用

采用RT-PCR方法从甘肃地区引种的切花百合上克隆了百合斑驳病毒(Lily mottle virus,LMoV)的外壳蛋白(coatprotein,CP)基因与细胞质内含体(cytoplasmic inclusions,CI)基因的3′端600 bp片段,连接到原核表达载体pET-28a(+)上,构建了融合表达的重组质粒pET-28a-CP-CI200。重组质粒转化大肠杆菌BL21成功表达了融合蛋白CP-CI200。经镍柱亲和层析获得纯化的融合蛋白,进一步用其免疫家兔制备了多克隆抗体。Western Blot结果显示,该多抗与感染LMoV的百合叶片中的病毒CP与CI均发生特异性反应,而对健康百合叶片无反应。用该多抗建立双抗夹心酶联免疫吸附法(DAS-ELISA)检测百合叶片样品,相对于RT-PCR方法其灵敏度和特异性均达到了93.3%。研究结果为快速、准确检测百合斑驳病毒的试剂盒研制奠定了基础。     

竹红菌甲素Triton X-100微乳剂的制备、光热稳定性及抑菌活性

为解决竹红菌甲素(hypocrellin A,HA)易溶于有机溶剂却极难溶于水的问题,采用物理包埋法,以Triton X-100为载体,制备了对水稀释后稳定性良好的500 mg/L的竹红菌甲素Triton X-100微乳剂,并 测定了其光热稳定性及对植物病原菌的抑制活性。结果表明:在0~54℃、照度低于3 600 lx时, 该微乳剂的光热稳定性良好;在照度为7 200 lx时,50 mg/L的HA-TX-100微乳剂对番茄灰霉病菌Botrytis cinerea、松针褐斑病菌Lecanosticta acicola、油菜菌核病菌Sclerotinia sclerotiorum和小麦赤霉病菌Fusarium graminearum的抑制率均超过50%,其中对油菜菌核病菌的抑制率最高,达77.60%;1 000 mg/L的HA-TX-100微乳剂对活体番茄接种番茄灰霉病菌的保护作用与治疗作用抑制率分别为72.05%和64.73%,均优于相同条件下市售50%多菌灵可湿性粉剂的作用。研究结果表明,竹红菌甲素具有开发为光活化农药的潜力。     

Detection and quantification of airborne inoculum of Sclerotinia sclerotiorum using quantitative PCR

This paper reports the development of a new specific diagnostic technique to accurately quantify airborne inoculum of Sclerotinia sclerotiorum and discusses its potential use in disease-forecasting schemes, using examples of three contrasting epidemic seasons: 2007, when there was a severe epidemic of sclerotinia stem rot (SSR) in England and high numbers of airborne ascospores were trapped at Rothamsted, and, in contrast, 2003 and 2004, when the incidence of SSR in England was low and low numbers of airborne ascospores were trapped at Rothamsted. DNA was extracted from wax-coated plastic tapes, such as those used in Burkard (Hirst-type) spore traps and rotating-arm traps. A SYBR-green quantitative PCR (qPCR) method produced a linear relationship between ascospore numbers and S. sclerotiorum DNA (mean 0·35 pg DNA per spore) and was able to detect DNA representing as few as two ascospores. The technique was insensitive to DNA of the host plant, Brassica napus , and other plant pathogens, including Sclerotinia minor , S. trifoliorum and Botrytis cinerea , and common airborne fungal genera such as Cladosporium and Penicillium . There was no relationship between rainfall and numbers of airborne ascospores of S. sclerotiorum present at Rothamsted during the period of infection in the severe SSR season (2007).