Evaluation of antisera raised against Phytophthora fragariae for detecting the red core disease of strawberries by enzyme-linked immunosorbent assay (ELISA)

Antiserum was raised against pooled mycelial suspensions from five isolates (designated Pf 1, Pf 2, Pf 3, Pf 10 and Pf 11) representing five physiologic races of Phytophthora fragariae. In enzyme-linked immunosorbent assay (ELISA), this antiserum detected homologous soluble antigens at protein concentrations as low as 2 ng/ml.
Fungal antigens could also be detected in extracts of strawberry plants infected with P. fragariae. Root extracts prepared from the alpine strawberry Fragaria vesca and F. ananassa cv. Cambridge Favourite infected with any of the five isolates studied produced strong reactions in ELISA. In F. vesca , ELISA-positive material was detectable 6-8 days after inoculation before macroscopic symptoms appeared. The cultivar Red Gauntlet, which is resistant to Pf 1, 2 and 3 but susceptible to Pf 10 and 11, reflected this differential response in ELISA; the absorbance produced by extracts of plants infected with virulent isolates was significantly higher than that obtained with the corresponding extracts of plants inoculated with a virulent isolates. The recently introduced cultivars Hapil, Ostara and Providence were found to be susceptible to all isolates in this study: the corresponding root extracts were also positive in ELISA.
The antiserum also detected P cactorum infections. Nevertheless, the ELISA test described should prove valuable in screening certified strawberry stocks.     

Observations of apothecia of Tapesia yallundae and the cultural phenotypes of their progeny

Apothecia of Tapesia yallundae, the perfect state of Pseudocercosporella herpotrichoides, were found in March 1989 on wheat and barley stubble at nine of 37 naturally infested sites and also at one inoculated site in Germany. Apothecia were found on about 1% of stems collected from the naturally infested sites and 5% of stems from the inoculated site, and were first found in the second week of March, coinciding with the onset of an early spring. Apothecia were not found on either green plants or second-year stubble. In total, 143 single-spore isolates were obtained from the apothecia and described according to a range of morphological characteristics. The majority were ascribed to P. herpotrichoides var. herpotrichoides. This is the first isolation of this variety from T. yallundae apothecia in Germany. Even single-conidial isolates from one apothecium showed much variation, some being like var. herpotrichoides, some like var. acuformis and some intermediate.     

The identification of a pathotype-specific DNA probe for the R-type of Pseudocercosporella herpotrichoides

A 6·7-kb DNA fragment has been isolated from an R-type isolate of Pseudocercosporella herpotrichoides that exhibits specific hybridization to R-type isolates and not to W, C or S pathotypes or to P. anguioides. R-type isolates are polymorphic with respect to this fragment and three sizes were present (11·6, 6·7 and 4·5 kb) in Eco RI-digested DNA from isolates of a world-wide collection. Infection of rye seedlings was demonstrated by hybridization of this probe to DNA extracted from infected plants.     

Detection and recovery of Rhizoctonia solani in naturally infested glasshouse soils using a combined baiting, double monoclonal antibody ELISA

A combined baiting, double monoclonal antibody immunoassay was developed that allows specific and sensitive detection of the economically important soil-borne plant pathogen Rhizoctonia solani in naturally infested soils. The assay is quick, taking only three days to complete from receipt of soil samples and the immunoassay format allows recovery of Rhizoctonia isolates from colonized baits for determination of anastomosis group (AG) affiliation and pathogenicity. The assay was tested on naturally infested soils from commercial glasshouses used to grow lettuce. Using the immunoassay, conventional anastomosis tests against known AG isolates, and pathogenicity tests, it was shown that R. solani isolates recovered from soil samples were pathogenic towards lettuce and belonged to AG4. Furthermore, those isolates that exhibited strong pathogenicity towards lettuce were recovered from sites that had experienced severe Rhizoctonia damage in previous lettuce crops. The possibility of developing a preplanting test to predict damage to specific crop plants due to the presence of particular AGs in the soil is discussed.     

Pseudocercosporella anguioides, a weakly pathogenic fungus associated with eyespot in winter wheat at a site in England

Pseudocercosporella anguioides is reported for the first time from Britain. Isolates were made from the leaf sheaths of four plants of a sample of 2716 with suspected eyespot lesions taken in April 1986 from a winter wheat crop. The eyespot pathogen, P. herpotrichoides , was isolated from 724 plants in the same sample. P. anguioides was not found in a sample of eyespot-infected straws taken from the same crop in July 1986. In infection tests, the P. anguioides isolates produced no obvious lesions on the leaf sheaths of wheat seedlings grown in pots, but were sometimes reisolated from symptomless leaf sheaths. Although P. anguioides occurred infrequently, care is needed to distinguish it from P. herpotrichoides when monitoring the eyespot pathogen.     

Comparison of isoenzyme and DNA markers for differentiating W-, R- and C-pathotypes of Pseudocercosporella herpotrichoides

Biochemical differences between the wheat, rye and couch-grass pathotypes of the eyespot pathogen, Pseudocercosporella herpotrichoides, have been identified by comparing isoenzyme profiles and DNA markers from several isolates. The wheat (W) and rye (R) pathotypes were clearly differentiated by both techniques, with isoenzyme profiling also separating the couch-grass isolates from the W- and R-type isolates. The isoenzyme profiles separated the P. herpotrichoides pathotypes from the two related species, P. anguioides and P. aestiva. The isoenzyme profiles of P. anguioides were closer to those of the R-type isolates of P. herpotrichoides than the W-type isolates, whereas the banding pattern of P. aestiva was very different. The isoenzyme profiles of the two German isolates, originally obtained from Nirenberg's laboratory, P. herpotrichoides var. herpotrichoides and var. acuformis, were similar to those of the wheat and rye pathotypes, respectively, isolated from UK-infected material.     

<Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> groups IA and II are represented among isolates from naturally infected lilies

Four cucumber mosaic virus isolates (named Cas, CB, P26 and Simp2) found in naturally infected lily plants were characterized on the basis of their serological properties and the results of analysis of RNA3 sequence fragments containing coat protein and movement protein genes. The properties of lily isolates were compared with those of eight other virus isolates originating from dahlia, delphinium, impatiens, honeysuckle, cucumber and redcurrant plants. On the basis of the reaction with group-specific monoclonal antibodies and RNA3 sequence analysis, two lily isolates (Cas and CB) were classified to group I of CMV, similarly to all previously reported virus isolates found in lily plants. Surprisingly, sequences of coat protein and movement protein genes of two other lily isolates (P26 and Simp2) showed more than 98% similarity to CMV group II isolates, and only 77% similarity to group I isolates. Results of ELISA with CMV group II-specific monoclonal antibodies confirmed the classification of isolate Simp2 as a member of group II. Isolate P26 reacted neither with CMV group I nor with group II-specific monoclonal antibodies. In order to explain the lack of reaction of P26 isolate with monoclonal antibodies, comparative analysis of predicted amino acid sequence of the coat protein was done. This revealed a mutation—change from alanine at position 138 to threonine, probably responsible for particular serological properties of isolate P26.     

Screening for potential antagonists of Pseudocercosporella herpotrichoides, the causal agent of eyespot disease of cereals

Bacteria isolated from wheat seedlings, plants or straw from several field sites were screened for antagonism towards the cereal eyespot pathogen Pseudocercosporella herpotrichoides on several media of differing nutrient status. Thirteen out of 348 isolates inhibited pathogen growth on low-nutrient media and several also prevented spore germination or reduced germ tube extension. These were selected for further tests on wheat seedlings inoculated with the eyespot fungus. Twelve known bacterial antagonists of other fungal plant pathogens were tested in vitro using the same methods, and the majority showed some activity towards P. herpotrichoides. Selected isolates were equally inhibitory to both W and R pathotypes of the fungus. Effects of potential antagonists on disease development were assessed by scoring lesions or by counting the number of infection plaques formed by the fungus on leaf sheaths. Two isolates of Pseudomonas fluorescens , along with a commercial strain of Streptomyces griseoviridis , showed activity both in vitro and in subsequent infection trials with plants and may therefore be of potential value as antagonists of P. herpotrichoides.     

Detection of corn stunt spiroplasma in vivo by ELISA using antisera to extracts from infected corn plants (Zea mays)

Antisera were raised in rabbits to extracts from healthy corn plants and from plants infected with corn stunt spiroplasma (CSS); they were prepared by partially purifying sap or tissue homogenates from whole stems by differential centrifugation and filtration. The titres of CSS-specific antibody in the antisera to diseased plant extracts (DPE) were monitored by spiroplasma deformation tests. After cross-absorption against healthy plant extracts, the DPE antisera detected cultured CSS at concentrations exceeding 10⁶ cells/ml, compared with less than 10⁵ cells/ml for homologous antiserum, and reacted more strongly against extracts from diseased than from healthy plants. A comparison of techniques for extracting diseased plant tissues showed that the reaction of stems and midribs against cultured CSS antiserum was two to three times stronger if the samples were lyophilized before extraction. When extracts from lyophilized tissues were used as test antigens the DPE antisera discriminated between diseased and healthy corn stems, midribs and roots as effectively as antiserum to cultured CSS. Preliminary attempts to detect CSS in its vector, Dalbulus maidis , were complicated by non-specific reactions of insect tissue extracts but concentrations equivalent to at least one CSS-infected insect/ml of sample were detected by antiserum to cultured CSS.     

Estimation of amounts of Phytophthora infestans mycelium in leaf tissue by enzyme-linked immunosorbent assay

A polyclonal antiserum, prepared in a rabbit immunized with a mycelium extract of Phytophthora infestans , reacted in an enzyme-linked immunosorbent assay (ELISA) with mycelial extracts of two Phytophthora species but not with those of 10 other micro-organisms found on potato. P. infestans mycelium in leaf tissue was readily detected by ELISA using either the plate-trapped antigen or F(ab')₂ antibody-fragment techniques. The amount of mycelium in leaf extracts was estimated by comparing the values obtained in ELISA with those for known concentrations of P. infestans mycelium.     

Production of antiserum and immunodetection of Cucurbit chlorotic yellows virus,a novel whitefly-transmitted crinivirus

Cucurbit chlorotic yellows virus (CCYV), a whitefly-transmitted virus, is a new member of the Crinivirus genus, that causes major economic losses in cucumber, melon and watermelon crops. To develop immunodiagnostic methods for CCYV, we produced an antiserum by immunizing rabbits with bacterially expressed recombinant coat protein of CCYV and detected CCYV from CCYV-infected plants with western blotting and immunoelectron microscopy. CCYV in extracts from infected plants was also readily detected by double antibody sandwich enzyme-linked immunosorbent assay with high sensitivity and specificity. A tissue blot immunoassay indicated that CCYV localizes in the phloem tissues of the petiole and is distributed in tiny spots within the leaf lamina.     

Classification of a world-wide collection of isolates of Pseudocercosporella herpotrichoides by RFLP analysis of mitochondrial and ribosomal DNA and host range

Two profiles of mitochondrial DNA (mtDNA) were detected among 59 isolates of Pseudocercosporella herpotrichoides from diverse geographical sources. One profile (type II) was present only among isolates pathogenic on rye (R-type), and a second profile (type I) was present in W, C and S-pathotypes of the fungus. Mitochondrial DNA from P. herpotrichoides hybridized strongly to DNA of P. anguioides , but very weakly to DNA from P. aestiva , suggesting that the latter is not closely related to P. herpotrichoides. Fifteen profiles of ribosomal DNA (rDNA) were observed among isolates with type I mtDNA, and five of these were only found among seven isolates from Ireland. The other profiles present among type-I isolates appeared to be largely independent of geographic origin. Three rDNA profiles were observed among R-type (type II mtDNA) isolates.     

Rapid detection of Phytophthora cinnamomi using PCR with primers derived from the Lpv putative storage protein genes

Phytophthora cinnamomi is an ecologically and economically important pathogen. In this study, PCR assays were developed with primer pair LPV2 or LPV3 for rapid detection and identification of this organism. Both primer pairs were selected from putative storage protein genes. The specificity of these primer pairs was evaluated against 49 isolates of P. cinnamomi , 102 isolates from 30 other Phytophthora spp., 17 isolates from nine Pythium spp. and 43 isolates of other water moulds, bacteria and true fungi. PCR with both primer pairs amplified the DNA from all isolates of P. cinnamomi regardless of origin. The LPV3 primers showed adequate specificity among all other species tested. The LPV2 primers cross-reacted with some species of Pythium and true fungi, but not with any other Phytophthora species. PCR with the LPV3 primers detected the pathogen at levels of a single chlamydospore or 10 zoospores in repeated tests. The PCR assay was at least 10 times more sensitive than the plating method for detection of the pathogen from artificially infested soilless medium, and, to a lesser extent, from naturally infected plants. PCR with LPV3 primers can be a useful tool for detecting P. cinnamomi from soilless media and plant tissues at ornamental nurseries, whereas the LPV2 primers can be an effective alternative for identification of this species from pure culture. Applications of these assays for detection of P. cinnamomi in other environments were also discussed.     

应用改进的多克隆抗体Western blot技术研究柑桔速衰病毒蛋白

 本文利用多克隆抗体发展了一种无背景的Western blot技术并用于研究柑桔速衰病毒(CTV)的蛋白。结果表明,利用CTV兔多克隆抗体1212和1052发展的Western blot技术可以检测到感染CTV的墨西哥酸橙或Citrus excelsa植株内CTV的4种蛋白P1、P2、P3和P4。在健株或病株内杂交反应均无非特异性背景。从不同寄主上分离到的CTV不同分离物的蛋白条带是不同的。利用1212和1052抗体均可以检测到感染6个CTV分离物的墨西哥酸橙幼苗内的P1、P2和P3。利用1052抗体能检测到感染严重型分离物T36、T3和Mm2的墨西哥酸橙幼苗内微弱的P4,但感染轻型分离物T30、T26和T4的幼苗内则检测不到。利用1212抗体检测不到P4。在C. excelsa内,1212和1052抗体均能检测到感染所有分离物的病株内的P1。在感染T3、T26、T4或Mm2的病株内能检测到P2,但在感染T30和36分离物的病株内则检测不到。在感染T36、T3、T26、T4和Mm2的病株内可检测到P3,但在感染T30的病株内则检测不到。在大多数植物内,P1、P2、P3和P4的分子量分别约为25kDa,24kDa,21kDa和18kDa。在感染T36分离物的C. excelsa植株体内,P1和P3的分子量分别约为27kDa和22kDa,比感染其它分离物的C. excelsa和墨西哥酸橙内的P1和P3分子量略大。P1、P2和P3的分子量与利用单克隆抗体检测的CP,CP1和CP2的分子量相等。因此,P1、P2和P3可能是CTV的外壳蛋白CP,CP1和CP2。P4的特性不清楚。研究结果也表明,利用特异性的多克隆抗体进行的Western blot是研究CTV的一种有用的技术。应用该技术,病株内不同的CTV分离物或株系就可以通过特异性蛋白条带区分开来。     

利用常规PCR和实时荧光定量PCR检测杨梅凋萎病菌

凋萎病是近年来危害杨梅的主要病害。为了快速灵敏的检测杨梅凋萎病菌(Pestalotiopsis versicolor和P.microspora),本研究开发了常规PCR和SYBR Green实时荧光定量PCR技术各一套。利用P.versicolor(JN861773)和P.microspora(JN861776)的ITS1-5.8S rDNA-ITS2序列的相同部分设计引物对(Pvm1L/Pvm1R)。该引物对利用常规PCR技术能特异性扩增出杨梅凋萎病菌188 bp的目标产物而对照菌株则呈阴性。该常规PCR体系能够检测人工接种后21 d和田间自然发病的有病症杨梅组织中的凋萎病菌,检测下限是0.6×10~5拷贝数。利用Pvm1L/Pvm1R进行SYBR Green实时荧光定量PCR,检测灵敏度是常规PCR的100倍,检测下限是0.6×10~3拷贝数,能够检测出人工接种及田间已经感染但尚未表现症状的杨梅组织中的凋萎病菌。这两项技术简单、快速、灵敏特异性强,可以应用于杨梅凋萎病的诊断和苗木检疫。     

检测植物病毒ELISA异种动物抗体双夹心法的研究和应用

 制备南方菜豆叶花叶病毒、大豆花叶病毒、烟草花叶病毒、番茄不孕病毒、苜蓿花叶病毒的兔抗血清和鼠腹水抗体,组合成ELISA异种动物抗体双夹心试验。抗体球蛋白的适宜工作浓度为4微克/毫升;未经纯化的特异抗血清(抗体)的适宜工作浓度为10^-4(即1/10,000)稀释度。检测以上5种病毒的灵敏度:提纯抗原为10-0.64毫微克/毫升;病叶澄清液为1/10,000-1/100,000稀释度。本法灵敏度与ELISA双抗体夹心法相当或略高。一般8小时内可得出结果。适用于大量样品的检测。     

Conventional PCR and real time quantitative PCR detection of <Emphasis Type="Italic">Phytophthora cryptogea</Emphasis> on <Emphasis Type="Italic">Gerbera jamesonii</Emphasis>

A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 10³ P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.