Cross-reactivity of antiserum raised against Phytophthora fragariae with other Phytophthora species and its evaluation as a genus-detecting antiserum

Antiserum (anti-PfM) raised against mycelial suspensions of Phytophthora fragariae isolates reacted strongly with antigens from several Phytophthora species. Some cross-reactions with antigens from Pythium species were decreased by fractionating on an affinity column of Sepharose 4B bound to extracts of Fragaria vesca roots infected with P. fragariae. The affinity-purified anti-PfM retained its high cross-reactivity with the various Phytophthora species tested. It also detected infection of raspberry and strawberry roots by some Phytophthora species. This antiserum could, therefore, prove useful as a broad-spectrum Phytophthora-detecting antiserum.
Anti-PfM could not be made specific for P. fragariae because it was raised against components shown to be antigenically similar in all Phytophthora species tested. However, immunoblotting with the affinity-purified anti-PfM produced distinct patterns for P. fragariae, P. erythroseptica and P. cactorum: three serotypes were identified for the latter species. This antiserum might therefore prove useful in classifying Phytophthora species.     

Crown rot caused by Phytophthora cactorum in Norwegian strawberry production

Crown rot of strawberry, caused by Phytophthora cactorum , was detected for the first time in Norway in 1992. This paper reports on surveys for P. cactorum in Norwegian certified strawberry plant production and on the distribution of the pathogen in regular strawberry production. In 1996 and 1997, samples of plant material from all certified strawberry plant growers in the country were investigated by isolation on artificial growth medium and using an enzyme-linked immunosorbent assay (ELISA). P. cactorum was not detected in any of the samples. A total of 171 isolations from plants with symptoms resembling crown rot were made from plants in a survey of the distribution of Phytophthora fragariae var. fragariae and from other samples. P. cactorum was detected at 35 different strawberry-producing farms in 11 of the 19 counties of Norway. The fungus was most frequently isolated from cv. Korona (at 18 locations), followed by cv. Inga (at 10 locations).     

"Candidatus Phlomobacter fragariae" Is the Prevalent Agent of Marginal Chlorosis of Strawberry in French Production Fields and Is Transmitted by the Planthopper Cixius wagneri (China)

ABSTRACT Marginal chlorosis has affected strawberry production in France for about 15 years. A phloem-restricted uncultured bacterium, "Candidatus Phlomobacter fragariae," is associated with the disease. A large-scale survey for marginal chlorosis in French strawberry production fields and nurseries by polymerase chain reaction amplification of "Ca. P. fragariae" 16S rDNA revealed that symptoms of marginal chlorosis were not always induced by "Ca. P. fragariae" and that the stolbur phytoplasma could induce identical symptoms. "Ca. P. fragariae" was found to be predominant in strawberry production fields, whereas the stolbur phytoplasma was predominantly detected in nurseries. Two transmission periods of the disease, one in spring and the other from late summer to early fall, were evident. Cixius wagneri planthoppers captured on infected strawberry plants were demonstrated to be efficient vectors of "Ca. P. fragariae." The involvement in natural disease spread of the whitefly Trialeurodes vaporariorum, previously shown to acquire and multiply "Ca. P. fragariae" under greenhouse conditions, remains uncertain.     

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.     

Sensitive real-time PCR detection of Xanthomonas fragariae in strawberry plants

Xanthomonas fragariae , the causal agent of angular leaf spot on strawberry, is a quarantine organism in strawberry propagation material in the European Union. For the reliable screening of planting material for latent infections, a real-time PCR assay based on Taqman® chemistry for the detection of X. fragariae was developed. Primers and probe sequences were based on a DNA fragment amplified by a previously reported X. fragariae -specific technique. The sequence of this genomic fragment had no significant similarity with any published GenBank sequence. Specificity of the designed assay was tested with an extended range of X. fragariae collection strains and isolates, with other Xanthomonas spp. and with unidentified bacterial isolates from strawberry plants. A nested PCR, which until now was the reference method for sensitive detection in planta , cross-reacted with the reference strain of Xanthomonas campestris pv. campestris . In combination with an elaborated DNA extraction procedure, the Taqman® PCR enabled reliable detection down to 300 colony forming units in a 100 mg strawberry leaf sample. The assay offers a new tool for epidemiological research and for sanitary control of plant material with low level or latent infections of X. fragariae .     

Induction of a defense response in strawberry mediated by an avirulent strain of <Emphasis Type="Italic">Colletotrichum</Emphasis>

In the strawberry crop area of Tucumán (north-west Argentina) the three species of Colletotrichum causing anthracnose disease (C. acutatum, C. fragariae and C. gloeosporioides) were detected. Among all isolates characterized, one of them identified as C. acutatum (M11) and another as C. fragariae (F7) were selected due to their conspicuous interaction with the strawberry cultivar Pájaro. Whereas isolate M11 produced a strong compatible interaction in cv. Pájaro with clear disease symptoms (DSR = 5.0), the isolate F7 brought about a typical incompatible interaction (DSR = 1.0). When plants of cv. Pájaro were inoculated with F7 prior to the inoculation with M11, the former avirulent strain prevented the growth of the latter virulent pathogen. Experimental evidence indicated that the time elapsed between the first inoculation with the avirulent pathogen and the second inoculation with the virulent one was crucial to inhibit the growth of the latter. The growth of F7 on the plant without provoking damage and the fact that there was no in vitro antagonistic effect between the pathogens, suggests that the avirulent strain triggers a plant defensive response against M11. The defense response was further confirmed by the detection of an early oxidative burst occurring within 4 h after the first inoculation and by the observation of anatomical changes associated with defense mechanisms that lasted 50 days after the inoculation with F7. Results obtained support the hypothesis that the plant resistance against the virulent strain M11 is elicited by one or more diffusible(s) compound(s) produced by the avirulent strain F7.     

Detection of a mycoplasma-like organism in peanut plants with witches' broom using indirect enzyme-linked immunosorbent assay (ELISA)

The mycoplasma-like organism (MLO) associated with peanut (groundnut) witches' broom (PWB) from India was partially purified and an antiserum produced against it. Using a protein A indirect enzyme-linked immunosorbent assay (ELISA) procedure, PWB MLO was detected in crude extracts of leaves. stems and pegs of infected peanut plants, although stems were a better source than leaves and pegs. Extracts of infected tissues of three diseases of assumed MLO etiology in India, little leaf of brinjal (eggplant). I med rosed witches "broom, and Daturd sp. witches' broom, failed to react with the PWB MLO antiserum.     

Development and optimization of a real-time detection assay for Xanthomonas fragariae in strawberry crown tissue with receiver operating characteristic curve analysis

Angular leaf spot of strawberry is caused by the bacterium Xanthomonas fragariae. The disease is transmitted primarily through systemically infected nursery stock. This creates problems for nurseries wishing to export plants to Europe because of quarantine restrictions. Currently, field inspections for symptoms are used to certify plants free of X. fragariae, but visual inspection is not useful for detecting plants infected systemically. To detect systemic infections, polymerase chain reaction (PCR) is the desired tool because of its sensitivity, specificity, and ease of use. In this study, we developed three sets of real-time PCR primers and probes and determined optimal reaction conditions for use of these primers for the detection of the bacterium X. fragariae in strawberry crown tissue. Real time detection proved to be both more sensitive and specific than standard PCR. Moreover, the detection of X. fragariae in crown tissue extract was possible with real-time PCR but not with standard PCR which is a significant improvement over standard PCR. The information on sensitivity and specificity of the primer sets was used to evaluate the performance of these primers with receiver operating characteristic (ROC) curve analysis under different tolerances. The results of this analysis can be used to provide guidance on threshold selection to manage disease below unacceptable levels. The results of this research may be useful to regulators and inspectors who must certify that plants meet European and Mediterranean Plant Protection Organization standards.     

Host-Specific Toxin Production by Rhizoctonia solani, the Rice Sheath Blight Pathogen

ABSTRACT Rhizoctonia solani, the rice sheath blight pathogen, produces a toxin that reproduces all symptoms of the disease. The toxin has been partially purified and it was found to be a carbohydrate containing glucose, mannose, N-acetylgalactosamine, and N-acetylglucosamine. The toxin was also detected in infected leaves. Highly virulent isolates produced more toxin than less virulent isolates. Several R. solani isolates from rice and one each from cotton and tomato produced a similar toxin. All rice cultivars tested were susceptible to the pathogen and sensitive to the toxin. Host specificity of the toxin has been demonstrated using hosts and nonhosts of the pathogen.     

Effect of temperature and host genotype on the production of inoculum by Phytophthora fragariae var. fragariae from the roots of infected strawberry plants

A bioassay was used to monitor the release of inoculum in drainage water from strawberry plants inoculated with zoospores of Phytophthora fragariae var. fragariae. The fungus was detected in drainage water from plants that had been held at temperatures between 2 and 20 C. but not from plants held at 26°C. The lag phase before secondary inoculum was first released, the maximum and total amounts of inoculum released, and the length of time over which inoculum was released were all greater at the lower temperature regimes, especially those below 10 C. The results were consistent with observations on the effect of temperature on zoospore production from agar discs and on zoospore motility: more zoospores were produced at lower temperatures and they remained motile for longer. From this it is concluded that the inoculum detected consists mainly of motile zoospores. In most experiments with standardized suspensions c. 10-15 were sufficient to initiate infection of the plants in the bioassay. In general, more inoculum was produced by host genotype/fungal isolate combinations in which there were marked root rot symptoms than in combinations in which the host was resistant.     

The location of virus-like particles in the male gametophyte of birch, walnut and cherry naturally infected with cherry leaf roll virus and its relevance to vertical transmission of the virus

Virus-like particles (VLPs) in close-packed paracrystalline arrays were observed in cells forming the anther walls and in the sperm cell cytoplasm of immature pollen grains developing within cherry leaf roll virus (CLRV)-infected birch ( Betula pendula Roth.). VLPs within tubules, that were in some instances multiple and membrane bound, were also observed in anther cells and in pollen grains of CLRV-infected walnut ( Jugians regia L.). VLPs rarely coated the outer surfaces of developing grains. Washings from intact freshly collected pollen did not contain infective agents but pollen triturates were infectious after 12 months storage at-70°C. Purified CLRV (concentration 6·4 ng/ml) was readily detected by enzyme-linked immunosorbent assay (ELISA). CLRV-specific antigens (detected by ELISA) and VLPs (detected on grids coated with an antiserum prepared against CLRV) were readily removed by washing intact pollen grains from infected birch, walnut and cherry ( Prunus avium L. cv. F12/1). The antigen was less tenaciously held to the surfaces of anemophilous (birch and walnut) than entomophilous (cherry) pollen. Treatment of grains before ELISA testing with CLRV-specific γ globulin virtually eliminated the antigenicity of pollen washings whereas γ globulin from a pre-immune serum had no such effect. When anti-CLRV γ globulin-treated pollen grains were disrupted, CLRV-specific antigens were liberated. VLPs trapped on CLRV-antiserum coated grids to which pollen washings or extracts from disrupted grains had been applied were identified by decoration; a halo of antibody molecules enveloped VLPs treated with CLRV-antiserum but not those treated with antiserum prepared against poplar mosaic virus.     

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.     

Frequency of virulence phenotypes of Phytophthora fragariae in the field

The virulences of 102 single-zoospore cultures of Phytophthora fragariae from one field site were determined on a range of strawberry differentials, and used to assign the cultures to four clusters (I, II A, IIB, IIC) using cluster analysis. On cv. Favourite, which is susceptible to all known races of the pathogen, isolates in cluster I were recovered most frequently and had the narrowest spectrum of virulence. On cv. Saladin isolates in cluster IIB were more common and were pathogenic to this cultivar. However, c. 30% of the single zoospores from field isolates from Saladin were avirulent on this cultivar and belonged to cluster I. Hyphal-tip and single-zoospore cultures from selected field isolates in cluster IIB did not always have the same virulence phenotype as the parent isolates. One hyphal-tip culture from a field isolate in cluster IIB had a virulence phenotype (IID) which had not been recorded before in Europe, and it attacked some cultivars not previously affected by red core. When cultivars such as Saladin were inoculated with mixtures of zoospores from two isolates from different clusters, with and without the corresponding virulence factors, the isolate with the corresponding virulence factor was selected. However, on the universally susceptible cv. Favourite the results depended on the relative competitiveness of the isolates and not on virulence factors.     

Antibody production and identity of MLOs associated with sugar-cane whiteleaf disease and bermuda-grass whiteleaf disease from Thailand

Cuttings from sugar-cane plants with symptoms of whiteleaf disease and bermuda-grass plants also showing whiteleaf symptoms were collected in Thailand and transferred to a glasshouse at East Mailing. Using these plants as source material, procedures were devised for the partial purification of MLO-associated immunogens. Antisera were raised separately to the sugar-cane whiteleaf and bermuda-grass whiteleaf immunogens. These antisera exhibited specificity for their homologous MLO antigens in F(ab')₂ indirect enzyme immunosorbent assays (ELISA). Detection of MLO infection was possible in crude tissue extracts diluted several hundredfold. No cross-reactions were observed in reciprocal tests between these two MLO sources and their antisera, nor were there reactions between either antiserum and extracts of other graminaceous hosts showing symptoms of natural whiteleaf infection. No cross-reactions were obtained in reciprocal tests with Spiroplasma citri or aster yellows MLO and their homologous antibodies.     

Indian peanut clump virus (IPCV) infection on wheat and barley: symptoms, yield loss and transmission through seed

Wheat and barley crops were shown to be susceptible to Indian peanut clump virus (IPCV) under field conditions. In wheat, the Hyderabad isolate of IPCV (IPCV-H) induced symptoms resembling the rosette caused by soil-borne wheat mosaic virus, and these were apparent only three weeks after emergence. Early-infected plants were severely stunted and dark green, with chlorotic streaks on the youngest leaves, which turned necrotic as the plants aged; most of these plants died. Late-infected plants were also stunted and were conspicuous in the field because of their dark green appearance as a result of delayed maturity. The virus was detected by ELISA and nucleic acid hybridization in all plants with symptoms. These plants usually produced fewer tillers than healthy ones. Spikes were malformed, often did not emerge from the flag leaf, and they contained few, shrivelled seeds. Grain yield was decreased, on average, by 58%. In barley, IPCV-H caused severe stunting and general leaf chlorosis. As the plants aged, the leaves became necrotic and the few infected plants that reached maturity produced small spikes. IPCV-H antigens were detected by ELISA in every wheat seed from infected plants and the virus was transmitted through wheat seed at a frequency of 0.5–1.3%. Storage at 4°C for more than a year did not affect seed transmission frequency. The virus was detected in leaves and roots of seed-transmitted plants. Seed transmission was not detected in barley. The Durgapura isolate (IPCV-D) was detected in wheat crops (cv. RR-21) at 3 different locations in Rajasthan State, India. Infected plants showed reduced growth without any overt symptoms.     

Virulence and aggressiveness of single-zoospore isolates of Phytophthora fragariae

A system for scoring the virulence of isolates of Phytophthora fragariae based on a scale of root rot from 0 ( no symptoms ) to 5 (76-100% roots roiled) on a series of strawberry cultivars is described. Thirty-two single-zoospore isolates from one field site were compared by subjecting their root rot scores to cluster analysis and this grouped them into two major clusters equivalent to physiologic races B66–3 and B66-11, Different sub-clusters of isolates of race B66-11 produced different degrees of rotting on the same hosts. Apart from differences in virulence between the sub-clusters there was some evidence for differences in aggressiveness between isolates within sub-clusters.
Increasing inoculum concentration by over 300-fold increased rotting by c . 25% but did not alter the rankings of different isolate/host combinations. Repeated passage of isolates through cultivars of differing susceptibilities did not affect their pathogenicity.     

Pathogenic variability in Ethiopian isolates of Fusarium oxysporum f. sp. ciceris and reaction of chickpea improved varieties to the isolates

Twenty-four isolates of Fusarium oxysporum f. sp. ciceris were isolated from wilted chickpea plants obtained from different districts and ‘wilt sickplots’ of central Ethiopia to assess variability in pathogenecity of the populations. Each isolate was tested on 10 different chickpea lines and eight improved chickpea varieties. Isolates showed highly significant variation in wilt severity on the differential lines and improved varieties. Based on the reaction types induced on differential lines, isolates were grouped into four corresponding races. Of the 24 isolates, F13, F20 and F22 were the most virulent. Isolates of race 3 were found in all of the districts and ‘wilt sickplots’ studied. Improved chickpea varieties also showed differential reactions to the isolates. All varieties were resistant to isolates of race 3, while varieties Arerti and DZ-10-4 were resistant to all isolates tested, showing the lowest mean wilt severity. Varieties DZ-10-11 and Maryie were susceptible to isolates F13, F20 and F22 and showed the highest mean wilt severity. Identification of races can be useful in breeding chickpea varieties resistant to wilt. The differential reactions of the improved varieties against different races might be important in managing chickpea wilt through gene deployment.     

草莓伪轻型黄边病毒的鉴定

 从6省13市约18品种草莓样本、分离纯化并研究鉴定,初步证实辽宁、哈尔滨及南京一些栽培田,有草莓伪轻型黄边病毒(SPMYEV)的发生分布。在春香、宝交早生、丹东鸡心,诺宾卡及80-3.1等品种上发病。病样先以小叶嫁接技术,接于草莓UC-4、EMC及"Alpine",约3~5周显症、下部叶片呈现黄色斑块,接UC-5、EMB不太敏感仅于老叶上现轻斑驳或褪绿斑,一种草莓中瘤钉毛蚜(Chaetosiphon sp.)及棉蚜(Aphis gossypii)作半持久性传播本病毒,桃蚜(Myzus persicae)不传播。UC-4对本病毒的繁殖、保存和嫁接鉴别最为适合,提纯病毒电镜观察其粒子线状,大小约630~680×12~13nm,县典型核蛋白吸收光谱,其最大吸收值位于2.63nm处;最小吸收值位于243nm处。A260/280=1.24。经SDS-聚丙烯酰胺凝胶电泳,测其外壳蛋白分子量约为37,000d。病叶制做超薄切片也观察到线形晶状病毒粒子,分散或聚集于寄主的薄壁组织细胞质中。本病毒系国内首报。     

Genetic polymorphism and virulence of Colletotrichum gloeosporioides isolated from strawberry (Fragaria × ananassa Duchesne)

To analyze genetic relationships among Colletotrichum gloeosporioides isolates; 34 isolates were collected from strawberries all over Japan, but primarily from Chiba Prefecture, and 20 were isolated from hosts other than strawberry. These isolates were assayed for virulence on strawberries and subjected to a fingerprint (FP) analysis by repetitive extragenic palindromic-PCR. Most of the isolates that were highly virulent to strawberries had relatively similar FPs even though they were isolated from various host plants (strawberry, cyclamen, nipplefruit and Japanese pear) in different regions. A cluster analysis revealed that the highly virulent and the weakly virulent isolates tended to form individual clusters, indicating that the highly virulent strains of C. gloeosporioides, which were genetically close to isolates with similar FPs and distinguishable from the weakly virulent strains, were responsible for strawberry anthracnose in Japan.