Detection of Pythium violae and Pythium sulcatum in carrots with cavity spot using competition ELISA

Conventional methods indicated that Pythium violae was most commonly isolated from carrot cavity spot samples from 14 UK sites. For one site the most frequently isolated species was Pythium sulcatum. Results of similar isolation work were compared with the assay of cavity spot lesions using polyclonal antibodies, raised to P. violae or P. sulcatum, in competition ELISA. Where lesions were artificially induced the test confirmed which pathogen was causal. With cavities developed on the field-grown carrots P. violae again predominated and the ELISA confirmed this. In one sample P. sulcatum was also isolated from a small number of lesions and was not detected in ELISA. The competition ELISA did not indicate presence of either Pythium in a range of non-cavity spot lesions from which attempts at isolation were negative.     

Monoclonal antibodies to cell surface components of zoospores and cysts of the fungusPythium aphanidermatum reveal species-specific antigens

A panel of monoclonal antibodies (MAbs) designated PA1 to PA8 has been raised against cell surface components of zoospores and cysts of the pathogenic fungusPythium aphanidermatum. The antibodies were selected on the basis of binding assays using indirect immunofluorescence. Four binding patterns were observed: PA1 labeled the entire zoospore surface including both flagella, PA2 binding was restricted to the anterior flagellum, PA3–PA6 bound to the adhesive cell coat secreted by zoospores during encystment, and PA7 and PA8 labeled zoospores and the cyst cell wall. Electron microscopic immunogold labeling of zoospores showed that PA2 bound to the mastigonemes on the anterior flagellum. The MAbs were tested for binding to zoospores and cysts of several isolates ofP. aphanidermatum, and to zoospores and cysts of several species ofPythium, Phystophthora, Aphanomyces, andSaprolegnia. The results showed that the antigens recognized by MAbs PA1–PA6 were restricted toP. aphanidermatum, whereas those recognized by PA7 and PA8 occurred on all species tested.     

Cross-Reactivity and Neutralizing Ability of Monoclonal Antibodies against Microcystins

Monoclonal antibodies (MAbs) against the microcystin-leucine-arginine variant (MCYST-LR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. The specificity of the MAbs and their ability to neutralize the toxin were investigated by an indirect enzyme-linked immunosorbent assay (ELISA) and by a neutralizing test in mice, respectively. All MAbs reacted with MCYST-LR and also with the microcystin-arginine-arginine variant (MCYST-RR), 3, 7-didesmethylmicrocystin (MCYST-3, 7-dDMLR) and 7-desmethylmicrocystin (MCYST-7-DMLR). Furthermore, the antibodies reacted with cell-extracts of toxic and non-toxic M. aeruginosa strains. The MAbs can apparently recognize the common configuration, but not the variant-specific structure, in the microcystin molecules. The non-toxic strains apparently contain some substance(s) related antigenically to microcystin. The in vivo toxin-neutralizing ability of MAbs was minimal.     

Antifungal activity of aluminium‐containing salts against the development of carrot cavity spot and potato dry rot

As an alternative to the use of synthetic chemical fungicides to control plant disease, aluminium‐containing salts were evaluated for their effects on the mycelial growth of various fungal or fungus‐like pathogens and their ability to control carrot cavity spot (Pythium sulcatum) and potato dry rot (Fusarium sambucinum). Results showed that various aluminium‐containing salts provided strong inhibition of all the tested pathogens (Alternaria solani, Botrytis cinerea, F. sambucinum, P. sulcatum and Rhizopus stolonifer) with minimal inhibitory concentration of 1–10 mM. Aluminium chloride and aluminium sulphate were generally the most effective, inhibiting mycelial growth of pathogens by as much as 47% and 100%, respectively, at a salt concentration of 1 mM. Applied at 5 mM, aluminium sulphate also provided 28% and 100% inhibition of dry rot and cavity spot, respectively. Aluminium chloride (5 mM) reduced dry rot by 25% whereas aluminium lactate (5 mM) decreased cavity spot lesions by 86%. These results indicate that various aluminium‐containing salts may provide an alternative to the use of synthetic fungicides to control these pathogens.     

Distribution and Expression of Elicitin‐like Protein Genes of the Biocontrol Agent Pythium oligandrum

Pythium oligandrum has the ability to induce plant defence reactions, and four elicitin‐like proteins (POD‐1, POD‐2, POS‐1 and oligandrin) that are produced by this oomycete have been identified as elicitor proteins. The first three are cell wall protein elicitors (CWPs), and the latter is an extracellular protein. Pythium oligandrum isolates have been previously divided into two groups based on the CWPs: the D‐type isolate containing POD‐1 and POD‐2, and the S‐type isolate containing POS‐1. We identified the genes encoding these elicitin‐like proteins and analyzed the distribution of these genes among 10 P. oligandrum isolates. A genomic fosmid library of the D‐type isolate MMR2 was constructed and genomic regions containing the elicitin‐like protein genes were identified. Southern blot analyses with probes derived from pod‐1 and an oligandrin gene indicated that the 10 P. oligandrum isolates could be divided into the same groups as those based on the CWPs. The D‐type isolates carried pod‐1, pod‐2 and two oligandrin genes, termed oli‐d1 and oli‐d2, while the S‐type isolates carried pos‐1 and one oligandrin gene termed oli‐s1. Phylogenetic analysis of POD‐1, POD‐2, POS‐1, Oli‐D1, Oli‐D2 and Oli‐S1 with the previously defined elicitins and elicitin‐like proteins of Phytophthora and Pythium species showed the specific clade. These genes occurred as single copies and were present in the P. oligandrum genomes but not in the other nine Pythium species (Pythium iwayamai, Pythium volutum, Pythium vanterpoolii, Pythium spinosum, Pythium torulosum, Pythium irregulare, Pythium ultimum, Pythium aphanidermutum and Pythium butleri). Furthermore, RT‐PCR analysis demonstrated that all of these genes were expressed during the colonization of tomato roots by P. oligandrum, supporting the idea that they encode potential elicitor proteins. To investigate the genetic relationships between the D‐type and the S‐type isolates, physical maps of the flanking regions around pod‐1, pod‐2, pos‐1 and the oligandrin genes were constructed. The maps suggest that the D‐type isolates may be derived from the S‐type isolates due to gene duplication and deletion events.     

Production and properties of monoclonal antibodies to Solanum nodiflorum mottle sobemovirus

Black raspberry necrosis virus (BRNV) reaches only very low concentrations in herbaceous plants and is difficult to maintain in culture. However, in a mixed culture with an unrelated virus, Solanum nodiflorum mottle (SNMV), in the genus Sobemovirus, the concentration of BRNV particles increases about 1000‐fold. In attempts to produce monoclonal antibodies (MAbs) to BRNV for diagnostic use, purified virus particles from the mixed virus culture were used as immunogen and the resultant antibodies screened against cultures of SNMV alone, BRNV+SNMV and healthy plant extracts. None of the virus‐specific MAbs obtained in this way was specific to BRNV but six were specific to SNMV. Although the original objective was not achieved, the SNMV MAbs were characterised and used to study serological properties of SNMV and other Sobemoviruses. Characterisation of the six SNMV MAbs showed that four were IgG3, one IgG1 and the other IgG2b. SNMV was detected by all six MAbs in ELISA, by five in Western blotting, by three in agarose gel double diffusion tests, but only one was suitable for trapping virus particles in immuno‐electron microscopy (IEM). In Western blotting using virus in sap extracts of Nicotiana clevelandii, each of the five MAbs detected a single major band of Mc. 31 000 in sap containing SNMV, and additional bands of lower mass attributed to degradation of coat protein. In various serological tests, no cross‐reactions were detected between SNMV and seven other viruses from the genus Sobemovirus. However, in IEM but not in Western blotting, significant cross‐reactions were observed between SNMV and Velvet tobacco mottle virus, another species from the genus Sobemovirus. The significance of these different findings is discussed.     

Some characteristics of monoclonal antibodies to a British M A V-like isolate of barley yellow dwarf virus

Rat monoclonal antibodies (MAbs) specific for a British F (MAV-like) isolate of barley yellow dwarf virus (BYDV) were produced and studied. In indirect ELISA using an antiserum to BYDV-F to trap virus from infected sap, the MAbs were shown to be specific for MAV-like isolates of BYDV from Britain, USA and Sweden but, in this test, they did not detect PAV-, RPV-, SGV- or RMV- like isolates of BYDV. In similar tests using homologous antisera to trap the viruses, the MAbs did not detect BYDV-PAV or -RPV or two other luteoviruses (potato leafroll and beet western yellows). One of the MAbs (MAFF 2) was partially purified from ascitic fluid, and used successfully in ELISA as a coating antibody and when conjugated to the enzyme alkaline phosphatase. Also, MAFF 2 successfully trapped BYDV-F particles when used to coat electron microscope grids. In indirect ELISA using three MAbs (MAFF 2, MAC 91 and MAC 92) it was possible to type the three major strain groups of BYDV, viz. MAV, PAV and RPV-like strains from Britain, USA and Europe.     

Detection of three whitefly-transmitted geminiviruses occurring in Europe by tests with heterologous monoclonal antibodies

Selected monoclonal antibodies (MAbs), prepared to particles of African cassava mosaic or Indian cassava mosaic geminiviruses, detected three geminiviruses that occur in Europe: abutilon mosaic virus in Abutilon pictum ‘Thompsonii’, tobacco leaf curl virus in Lonicera japonica var. aureo-reticulata and tomato yellow leaf curl virus in Lycopersicon esculentum. All three viruses were detected in indirect ELISA by MAbs SCR 17 and SCR 20 but they were differentiated by their reactions with SCR 18 and SCR 23. Tobacco leaf curl virus was detected only when reducing agents were included in the leaf extraction medium. Inclusion of sodium sulphite slightly improved detection of tomato yellow leaf curl virus but reducing agents were not needed for detection of abutilon mosaic virus.     

Production and Use of Monoclonal Antibodies to Pseudomonas andropogonis

Pseudomonas andropogonis is an important pathogen of worldwide distribution in ornamental and other plant species from 15 families. This paper reports the production and characterization of monoclonal antibodies (MAbs) to P. andropogonis and evaluation of their use in the detection of the pathogen in carnation cuttings. Ten stable hybridoma cell lines were produced. Results of indirect ELISA and indirect immuno-fluorescence showed that MAb 6B3 was specific for P. andropogonis; MAb 3D5W1 reacted with both P. andropogonis and P. caryophylli; six other MAbs reacted with all strains of seven species of rRNA group II pseudomonads tested except P. solanacearum and P. pickettii. Eight of the ten MAbs failed to cross-react with other non-fluorescent or fluorescent pseudomonads, xanthomonads and other bacteria tested. P. andropogonis was similar in protein profile to other rRNA group II pseudomonads tested except P. solanacearum and P. pickettii. Epitopes were clearly located within the cell by immunogold labelling. Of four MAbs that were isotyped, two possessed IgGl and two the IgM heavy chain. P. andropogonis was readily detected by combining immunofluorescence and detached carnation leaf assay using an initial inoculum of 4 × 10° colony forming units (cfu) ml^?1 after enrichment at room temperature for 4 days.     

Cavity spot of carrot (Daucus carota)

Cavity spot disease of carrot (Daucus carota) has been one of the intractable problems for both growers and scientists. Carrots are rejected at grading with one or two visible lesions, and when disease incidence passes a relatively low threshold it becomes uneconomic to harvest crops. For the scientist, there has been considerable pressure to produce both information on the cause of the disease and a cure. Many putative causes have been advanced over the years, but these were almost always contradicted by subsequent work. The first solid indication of involvement of a pathogen was when three different fungicides with activity against Oomycete fungi all reduced disease. Very quickly the causal agents Pythium violae and Pythium sulcatum were isolated from cavity spot lesions and Koch's postulates satisfied. The species are not typical of the more common pythia, having slow growth at normal temperatures, which means that in the context of isolation work, plates may be overgrown by other species before they are seen. Metalaxyl fungicide was identified as the most effective in controlling cavity spot caused by P. violae, but P. sulcatum is naturally tolerant of the fungicide. Recently, metalaxyl has been shown to be subject to enhanced microbial degradation. This phenomenon has been associated with failure to control cavity spot. No other fungicide has been shown to be consistently effective in the field, and none has been registered for disease control. For the future, this means that control of cavity spot can not be based solely on fungicidal control. Other, complementary strategies are necessary for reducing disease. Calcium carbonate is known to have significant effects on cavity spot, probably by inducing a soil microflora inhibitory to filamentous fungi. Management of agronomic aspects such as irrigation, soil cultivation and the length of time for which crops are grown may all be used, while carrot cultivars with some field resistance may be beneficial. However, one of the most significant factors is disease avoidance by not selecting fields with high inoculum levels. One serology‐based risk assessment test has been produced and commercialised, and molecular probes which could be the basis of more sensitive tests are available for both pathogens. The potential for disease reduction via a management strategy combining several key components is discussed.     

Preparation and Characterization of a Monoclonal Antibody to Prunus necrotic ringspot virus

A cell line named PVRSV1D11 secreting monoclonal antibody (McAb) against the prokaryotically expressed coat protein (CP) of Prunus necrotic ringspot virus (PNRSV) was developed using hybridoma technology including animal immunization, cell fusion, cell line culture and enzyme‐linked immunosorbent assay (ELISA)‐based for screening. The specificity, titre and detection sensitivity of the McAb were determined by indirect ELISA to establish optimal conditions. The antibody reacted strongly with PNRSV and showed no cross‐reactions with the proteins of Plum pox virus, Prunus dwarf virus, Apple stem pitting virus, Apple stem grooving virus, Apple mosaic virus or Apple chlorotic leafspot virus. The ascites developed with PNRSV1D11 cell line showed high absorbance until it was diluted to over 6.6 × 10⁷ fold. The McAb belonged to IgG_2a isotype and was diluted by 1.28 × 10⁵ folds as an optimal detection concentration. The detection sensitivity of the monoclonal antibody was 11.7 ng/ml protein of PNRSV. The results indicated that the McAb against the CP of PNRSV is suitable for PNRSV detection in the plants and for monitoring the dynamics of the virus by using indirect ELISA.     

Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato mop-top virus

The coat protein (CP) coding regions of two Czech Potato mop‐top virus (PMTV) isolates were sequenced and shown to be identical. One, the Korneta isolate CP gene, was cloned in several expression vectors. The recombinant PMTV‐CP was expressed in Escherichia coli and the purified recombinant protein was used to produce PMTV‐specific polyclonal antibodies. The antiserum had a titre of 1 : 2000 in an indirect enzyme‐linked immunosorbent assay (ELISA) and reacted specifically in immunoblotting and IPTA‐ ELISA (indirect plate‐trapped antigen (PTA)‐ELISA).     

Development and Application of Pathovar-Specific Monoclonal Antibodies That Recognize the Lipopolysaccharide O Antigen and the Type IV Fimbriae of Xanthomonas hyacinthi

The objective of this study was to develop a specific immunological diagnostic assay for yellow disease in hyacinths, using monoclonal antibodies (MAbs). Mice were immunized with a crude cell wall preparation (shear fraction) from Xanthomonas hyacinthi and with purified type IV fimbriae. Hybridomas were screened for a positive reaction with X. hyacinthi cells or fimbriae and for a negative reaction with X. translucens pv. graminis or Erwinia carotovora subsp. carotovora. Nine MAbs recognized fimbrial epitopes, as shown by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy; however, three of these MAbs had weak cross-reactions with two X. translucens pathovars in immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides and yielded a low-mobility ladder pattern on immunoblots. Subsequent analysis of MAb 2E5 showed that it specifically recognized an epitope on the O antigen, which was found to consist of rhamnose and fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all X. hyacinthi strains tested showed that this O antigen is highly conserved within this species. MAb 1B10 also reacted with lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and immunoblotting experiments with cells and extracts from other pathogens. No cross-reaction was found with 27 other Xanthomonas pathovars tested or with 14 other bacterial species from other genera, such as Erwinia and Pseudomonas, indicating the high specificity of these antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the detection of X. hyacinthi in infected hyacinths.     

Soluble expression and purification of Brucella cell surface protein (BCSP31) of Brucella melitensis and preparation of anti-BCSP31 monoclonal antibodies

Brucella cell surface protein (BCSP31) is potentially useful for diagnosing brucellosis. We aimed to establish a monoclonal antibody (MAb) against Brucella melitensis BCSP31 and to investigate its distribution in diagnosis. Soluble recombinant BCSP31 was successfully expressed and purified. Two MAbs (1F1 and 1E5) against B. melitensis BCSP31, effective in detecting both recombinant and cellular proteins, were obtained and characterized. The MAbs did not react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus aeruginosus, but strongly reacted with BCSP31 and B. melitensis by ELISA and Western blot analysis. We also tested different Brucella species and brucellosis using the prepared anti-BCSP31 MAbs. BCSP31 and anti-BCSP31 MAbs may play important roles in future research in diagnosing brucellosis.     

Specific identification of pathogenic Yersinia enterocolitica by monoclonal antibodies generated against recombinant attachment invasion locus (rAil) protein

Classical pathogenic strains of Yersinia enterocolitica produce a 17 kDa outer membrane protein, Ail (attachment-invasion locus), which mediates bacterial attachment to some cultures epithelial cell lines and invasion of others. In the present study, hybridomas were developed for the production of monoclonal antibodies (MAbs) against Ail protein of Y. enterocolitica. A set of five stabilized hybridoma cell lines were generated, of which, two MAbs, YEA 302 and YEA 303, exhibited specific reaction to the native Ail protein (17 kDa) present in whole cell lysate of Y. enterocolitica strains beside having reaction with rAil. The other three MAbs, YEA 5, 17 and 32, had some cross reactions with proteins other than Ail. Two out of five MAbs were IgG1, two were IgG2b and one in IgM in nature. MAbs (YEA 302 and YEA 303) did not show any cross-reaction with whole cell lysate of Brucella abortus, Vibrio cholerae, Salmonella typhimurium and Escherichia coli and other species of Enterobacteriaceae including Y. pestis in ELISA and Western blot analysis. The presence of Ail protein among the strains recovered from pork and milk samples was evaluated by these sets of MAbs and the results were compared with the duplex PCR. Collectively, the data suggest that these MAbs may have the potential for their use in the detection of pathogenic Y. enterocolitica reliably, rapidly and at a relatively low cost.     

Generation and characterization of a lipopolysaccharide-specific murine monoclonal antibody to <Emphasis Type="Italic">Proteus vulgaris</Emphasis> OX19

The three strains of non-pathogenic Proteus species namely, Proteus vulgaris OX2, P. vulgaris OX19 and Proteus mirabilis OXK used in the Weil–Felix test are the group-specific cross-reactive antigens for Rickettsia and Orientia species. Earlier studies have revealed that the group specific and cross-reactive antigens responsible for the Weil–Felix test lie mostly in the lipopolysaccharide (LPS) moiety of the bacterial cell wall [Amano et al. (1993a) Infect Immun 61:4350–4355, (1993b) Microbiol Immunol 37:927–933, (1998) Infect Immun 66:923–926]. The three Proteus strains (OX2, OX19 and OXK) were used to raise murine monoclonal antibodies (MAbs) by hybridoma technology. Several MAb-producing hybridomas could be stabilized following limiting dilution. Affinity and specificity of these MAbs were checked by indirect ELISA using a battery of homologous and heterologous antigens including LPS. Amongst these, one MAb was found to be specific for P. vulgaris OX19 LPS. Since the Weil–Felix reaction is based on the cross-reactivity between the LPS based epitopes, this MAb could be of potential use in mapping of epitopes on the cross-reactive LPS and may also be useful as a potential diagnostic reagent.     

Development of a diagnostic system for detection of specific antibodies and antigens against Middle East respiratory syndrome coronavirus

Middle East respiratory syndrome coronavirus (MERS‐CoV) is a single‐stranded RNA virus that causes severe respiratory disease in humans with a high fatality rate. Binding of the receptor binding domain (RBD) of the spike (S) glycoprotein to dipeptidyl peptidase 4 is the critical step in MERS‐CoV infection of a host cell. No vaccines or clinically applicable treatments are currently available for MERS‐CoV. Therefore, rapid diagnosis is important for improving patient outcomes through prompt treatment and protection against viral outbreaks. In this study, the aim was to establish two ELISA systems for detecting antigens and antibodies against MERS‐CoV. Using a recombinant full‐length S protein, an indirect ELISA was developed and found to detect MERS‐CoV‐specific antibodies in animal sera and sera of patient with MERS. Moreover, MAbs were induced with the recombinant S protein and RBD and used for sandwich ELISA to detect the MERS‐CoV S protein. Neither ELISA system exhibited significant intra‐assay or inter‐assay variation, indicating good reproducibility. Moreover, the inter‐day precision and sensitivity were adequate for use as a diagnostic kit. Thus, these ELISAs can be used clinically to diagnose MERS‐CoV.

     

Immunolocalization of the cell wall components inPinus densiflora pollen

Summary In order to compare cell wall formation in gymnosperm pollen with that in angiosperm pollen, the distribution of cell wall constituents in the pollen grain and pollen tube ofPinus densiflora was studied immunocytochemically with monoclonal antibodies JIM 5 (against non- or poorly esterified pectin), JIM 7 (against highly esterified pectin), JIM 13 (against arabinogalactan proteins, AGPs), and LM 2 (against AGPs containing glucuronic acid). In the pollen grain wall, only the outer layer of the intine was labeled with JIM 5 and weakly with JIM 7. The tube wall was scarcely labeled with JIM 5 and very weakly labeled with JIM 7. In contrast, the whole of both the intine and the tube wall was strongly labeled with JIM 13 and LM 2, and the generative-cell wall was also labeled only with LM 2. The hemicellulose B fraction, which is the main polysaccharide fraction from the pollen tube wall, reacted strongly with JIM 13 and especially LM 2, but not with antipectin antibodies. These results demonstrate that the wall constituents and their localization inP. densiflora pollen are considerably different from those reported in angiosperm pollen and suggest that the main components of the cell wall ofP. densiflora pollen are arabinogalactan and AGPs containing glucuronic acid.Abbreviations AGPs arabinogalactan proteins - ELISA enzymelinked immunosorbent assay - MAbs monoclonal antibodies     

Detection by using monoclonal antibodies of Yersinia enterocolitica in artificially-contaminated pork

Monoclonal antibodies against Yersinia enterocolitica were produced by fusion of NS‐1 mouse myeloma cells with spleen cells of ICR mice immunized with heat‐killed and heat‐killed plus SDS‐mercaptoethanol treated forms of Y. enterocolitica ATCC 27729 alone or mixed with Y. enterocolitica MU. The twenty‐five MAbs obtained from five fusions were divided into nine groups according to their specificities to different bacterial strains and species, as determined by dot blotting. The first five groups of MAbs were specific only to Y. enterocolitica, but did not recognize all of the isolates tested. MAbs in groups 6 and 7 reacted with all isolates of Y. enterocolitica tested but showed cross‐reaction with some Yersinia spp. and Edwardsiella tarda, especially in the case of group 7. MAbs in groups 8 and 9 reacted with all isolates of Y. enterocolitica and Yersinia spp., as well as other Gram‐negative bacteria that belong to the family Enterobacteriaceae. These MAbs recognized Y. enterocolitica antigens with apparent molecular weights ranging from 10 – 43 kDa by Western blotting, and could detect Y. enterocolitica from ～10³– 10⁵ colony forming units (CFUs) by dot blotting. The hybridoma clone YE38 was selected for detection of Y. enterocolitica in pork samples which had been artificially‐contaminated by inoculation with Y. enterocolitica ATCC 27729 at concentrations of ～10⁴– 10⁶ CFUs/g and incubation in peptone sorbitol bile broth at 4°C. Samples were collected and applied on a nitrocellulose membrane for dot blotting with trypticase soy and cefsulodin‐Irgasan‐novobiocin agars. After 48 hr of incubation, the detection limit was ～10²– 10³ CFU/g by dot blotting.