Comparison of immunofluorescence colony-staining in media,selective isolation on pectate medium,ELISA and immunofluorescence cell staining for detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in cattle manure slurry

Various isolation and serological techniques were compared for the detection ofErwinia carotovora subsp.atroseptica (Eca) andE. chrysanthemi (Ech) in cattle manure slurry containing c. 10⁸ colony forming units (cfu) per ml. The slurry samples could be preserved at –80°C for 8 months without reduction in the number of bacteria but not at –20°C. Samples stored at –80°C were inoculated with concentrations of the target bacterium ranging from 10² to 10⁸ per ml. Only immunofluorescence colony-staining (IFC) in combination with selective media was able to detect the target organism at a concentration of 100 cells per ml. No IFC-positive colonies were found in pour plates of the non-inoculated cattle slurry. The recovery of the target bacterium from slurry inoculated with 10² cfu of Ech per ml was 64% in PT medium (containing polygalacturonic acid) and 19% in crystal violet pectate medium (CVP). Recoveries of Eca were 32% and 82%, respectively. Ech and Eca could be detected at levels of 10³ cfu per ml of slurry by isolation on CVP. Crude filtration procedures were necessary for analysis of slurry samples with immunosorbent immunofluorescence (ISIF) cell staining. The detection level of ISIF for Ech was 10⁵ cells per ml of slurry. IF-positive cells were incidentally observed in the non-inoculated slurry. Detection of Ech and Eca with ELISA was only possible in slurry inoculated with 10⁸ cells of the target bacterium per ml.Samenvatting Verschillende isolatie- en serologische methoden werden vergeleken om de doelbacteriënErwinia carotovora subsp.atroseptica (Eca) enE. chrysanthemi (Ech) aan te tonen in runderdrijfmest met een natuurlijke bacterieflora van ca 10⁸ kolonievormende eenheden(cfu) per ml. De mestmonsters konden gedurende de onderzoekperiode tenminste 8 maanden bij –80°C worden bewaard zonder dat er een afname van het aantal levende mestbacteriën werd geconstateerd, terwijl bij bewaring bij –20°C wel een afname werd gevonden. De ontdooide mestmonsters werden geïnoculeerd met de doelbacterie in concentraties tussen 10² en 10⁸ per ml. De laagste concentratie van de doelbacterie, 10² cfu per ml, kon alleen worden aangetoond met de immunofluorescentie-kleuring van bacteriekolonies (IFC) in een selectief medium. Met deze techniek was het percentage herisolatie vanuit drijfmest geïnoculeerd met 10² Ech cfu per ml respectievelijk 64% in PT-medium (bevat polygalacturonzuur) en 19% in kristalviolet pectine medium (CVP). Voor Eca bedroegen deze percentages respectievelijk 82% en 32%. In de niet-geïnoculeerde mestmonsters werden geen IFC-positieve kolonies gevonden. Via isolatie op CVP konden 10³ of meer cfu van Eca en Ech worden aangetoond. Ruwe filtratie van de mestmonsters was nodig voor het aantonen van Eca- en Ech-cellen met immunoadsorptie immunofluorescentie microscopie. De detectiedrempel lag voor deze techniek op 10⁵ bacteriecellen per ml mestmonster. In niet-geïnoculeerde mest werden incidenteel IF-positieve bacteriën gevonden. Het aantonen van Ech en Eca met ELISA was slechts mogelijk in mest geïnoculeerd met 10⁸ of meer cellen van de doelbacterie per ml.     

Immunofluorescence Colony-staining (IFC) for Detection and Quantification of Ralstonia (Pseudomonas) solanacearum Biovar 2 (Race 3) in Soil and Verification of Positive Results by PCR and Dilution Plating

A procedure was developed for specific and sensitive quantitative detection of Ralstonia (Pseudomonas) solanacearum biovar 2 (race 3) in soil. It is based on immunofluorescence colony-staining (IFC) followed by confirmation of the identity of fluorescent colonies by PCR-amplification or dilution plating on a semi-selective medium, SMSA. Addition of sucrose and the antibiotics cycloheximide and crystal violet to the non-selective trypticase soy broth agar resulted in increased colony size and staining intensity of R. solanacearum in IFC. Verification of IFC-results by picking cells from IFC-positive colonies followed by dilution plating of the suspended cells on SMSA was highly efficient. The success rate was 92% and 96% with spiked and naturally contaminated soils respectively. Several other bacterial species which cross-reacted with polyclonal antibodies in IFC also grew on SMSA and were difficult to distinguish from R. solanacearum, thereby necessitating confirmation of the results. Rapid verification of IFC-positive results directly by PCR-amplification with primers D2/B specific to division 2 of R. solanacearum had a success rate of 86% and 96% with spiked and naturally contaminated soil samples, respectively. Primers D2/B reacted with all R. solanacearum division 2 strains, and strains of R. syzygii and the banana blood disease bacterium, but not with saprophytic bacteria cross-reacting in IFC with R. solanacearum antibodies. In comparative tests, IFC was able to detect consistently ca. 100 cfu g^–1 of soil, a detection level similar to that found with direct plating on SMSA, but less laboriously, whereas detection level with a bioassay on tomato plants was only 10⁴–10⁵ cfu g^–1 of soil.     

Development of a semi-selective medium for isolation of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv<Emphasis Type="Italic">. musacearum</Emphasis> from banana plants

Banana Xanthomonas wilt, caused by Xanthomonas campestris pv. musacearum, is a new threat to banana cultivation in eastern Africa. The causal bacterium grows slowly in culture and is easily overgrown by contaminants. A selective culture medium for isolation of X. c. pv. musacearum will facilitate disease study. A medium that suppressed saprophytic growth and possessed diagnostic characters for the pathogen was developed. Various carbon sources were tested with two isolates of X. c. pv. musacearum, and sucrose was selected as main carbon source. The susceptibility of X. c. pv. musacearum and other bacterial strains was tested with 29 different antibiotics. Cephalexin and cycloheximide had no effect on X. c. pv. musacearum but cephalexin inhibited most of the saprophytes and cycloheximide inhibited the fungal contaminants. Based on these studies, we have developed a semi-selective medium YTSA-CC containing yeast extract (1%), tryptone (1%), sucrose (1%), agar (1.5%), cephalexin (50 mg l⁻¹) and cycloheximide (150 mg l⁻¹), pH 7.0. The pathogen X. c. pv. musacearum was easily identified as yellowish, mucoid and circular colonies on YTSA-CC medium. This simple semi-selective medium was effective for isolation of X. c. pv. musacearum from infected banana tissues and soil, and it should be a valuable tool in ecological and epidemiological studies.     

New approach in detecting phytopathogenic bacteria by combined immunoisolation and immunoidentification assays1

Antibodies coated onto a suitable solid phase, e. g. polystyrene beads, rods for inoculating agar plates, or petri dishes, enable selective trapping of homologous bacteria and of immunologically related bacteria by immunoaffinity. After washing away unbound organisms, the bound organisms are desorbed and plated on a suitable medium. Selective immunoisolation has been demonstrated for various phytopathogenic bacteria including Corynebacterium michiganense ssp. michiganense, Erwinia chrysanthemi, E. carotovora ssp. atroseptica, Xanthomonas campestris pv. begoniae and Pseudomonas syringae pv. phaseolicola. Three serological methods are described for additional rapid immunoidentification of colonies directly on agar plates and for screening for cross-reacting microorganisms: (a) direct immunodiffusion on dilution plates, (b) agar mixed-antibody assay, (c) fluorescent antibody colony staining. Schemes are presented for increasing the reliability and sensitivity of sample screening for quality indexing of plant material, and for efficient screening and isolation of possible cross-reacting microorganisms to enable production of more specific antisera.     

Suppression of potato cyst nematode root penetration by the endoparasitic nematophagous fungusHirsutella rhossiliensis

The endoparasitic nematophagous fungusHirsutella rhossiliensis was tested for its ability to suppress root penetration and cyst formation by the potato cyst nematode speciesGlobodera pallida. Isolates ofH. rhossiliensis were obtained from infected potato cyst nematode juveniles from different starch potato fields in The Netherlands. The isolates showed no difference in spore adhesion to juveniles on agar plates (adhesion rate: ±90%). The most rapid growing isolate, CBS 108.94, was used for experiments. Vegetative mycelial colonies ofH. rhossiliensis CBS 108.94, grown in potato dextrose broth, were used as soil inoculum. During submerged cultivation the mycelial colonies produced phialides (spore-bearing cells) but no spores. Exposed to the air, however, spores were rapidly formed. The effect of different soil inoculum densities of mycelial colonies on root penetration byGlobodera pallida was examined in an experiment in 250-ml pots. Up to a mycelial colony concentration representing a potential spore density of 10⁴ g^–1 soil no suppression occurred. At approximated densities of 2.5×10⁴ and 10⁵ spores g^–1 soil the numbers of juveniles which penetrated roots were reduced by 30% and 34%, respectively. The distribution of the inoculum could be improved by fragmentation of the mycelial colonies before soil inoculation. Using mycelial fragments, again no suppression of root penetration was observed up to a potential spore density of 10⁴ g^–1 soil, but at densities of 10⁵ and 10⁶ g^–1 a suppression of 54% and 88%, respectively, was measured. In a greenhouse experiment, soil inoculation with mycelial colonies with a potential spore production of 2.5×10⁵ g^–1 soil resulted in a suppression of root penetration of 37% and 51% after 5 and 6 weeks, respectively, but the number of newly formed cysts after 18 weeks in soil was not different for control and inoculated pots. It is concluded thatH. rhossiliensis may be useful for the reduction of root damage caused by juveniles of potato cyst nematodes, but the usefulness for population control is doubtful.     

Detection of Xanthomonas sp., the Causal Agent of Onion Bacterial Blight, in Onion Seeds Using a Newly Developed Semi-selective Isolation Medium

Onion bacterial blight, caused by Xanthomonas sp., is a potentially severe disease in several tropical and subtropical areas. Although little research has been undertaken on this pathosystem, seed transmission of the pathogen has been hypothesized. Because of an important bacterial microflora naturally associated with onion seeds, detection of the pathogen is difficult using non-selective agar media. A new semi-selective medium, whose selectivity was obtained by a combination of four antibiotics, was developed. The new NCTM1 medium contained (per liter) yeast extract 7g, peptone 7g, glucose 7g, agar 15g, neomycin 10mg, cephalexin 30mg, trimethoprime 3mg, pivmecillinam 100mg and propiconazole 20mg. Plating efficiencies, using 16 pure cultures of the pathogen, ranged from 79% to 142%, with an average of 110% compared to the basal medium. All onion Xanthomonas sp. strains from several countries grew on NCTM1 medium. The pathogen was repeatedly isolated using this medium from seed samples containing approximately 10⁶ saprophytic bacteria per gram, as well as from symptomless plant material. Xanthomonas sp. was detected only in seeds originating from one infected seed production site. This is the first report of selective isolation of Xanthomonas sp. from onion seeds. NCTM1 medium should be a valuable tool to study the ecology and epidemiology of Xanthomonas sp. causing onion bacterial blight.     

A diagnostic medium for the semi-selective isolation and enumeration of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis>pv. <Emphasis Type="Italic">vignicola</Emphasis>

A semi-selective medium for isolation of Xanthomonas axonopodis pv. vignicola from cowpea (Vigna unguiculata) plant and soil samples was developed. Twelve carbon and five nitrogen sources were tested with four strains of X. axonopodispv.vignicola, and 25 antibiotics were screened against saprophytes. -cellobiose (10g) was selected as the optimal carbon source. Among the antibiotics, cefazoline inhibited growth of most of the saprophytes with little effect on strains of the pathogen. ,-methionine enhanced growth of X. axonopodispv.vignicola. Boric acid along with ammonium chloride suppressed growth of Pseudomonas fluorescens. The semi-selective medium designated as cefazoline-cellobiose-methionine (CCM) medium contained K₂HPO₄ 1.34g, KH₂PO₄ 0.4g, MgSO₄ 0.3g, H₃BO₃ 0.2g, NH₄Cl 1.0g, -cellobiose 10g, cycloheximide 0.2g, ,-methionine 1.0g, cefazoline 10mg and agar 14g per l of water (pH 7.2). Colonies of X. axonopodispv.vignicola on CCM medium were whitish, round, raised and 0.2–1.8mm in diameter 96h after incubation. CCM medium generally inhibited growth of Pantoea agglomerans, Bacillus subtilis and saprophytes isolated from cowpea leaves. Colonies of Pseudomonas fluorescens and a saprophytic bacterium, which were not completely suppressed by CCM, could be differentiated from X. axonopodispv.vignicola by their smaller size and different color. The CCM medium proved useful for isolation of X. axonopodispv.vignicola from cowpea plant and soil samples. This is the first report of a semi-selective medium developed for detection of X. axonopodispv.vignicola.     

Improved method to induce sporulation of <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis>, causal fungus of grape ripe rot

Colletotrichum gloeosporioides and C. acutatum are causal agents of grape ripe rot, but with available methods, sporulation of C. gloeosporioides on plate media has been unstable and inferior to that of C. acutatum. To facilitate studies on C. gloeosporioides, I developed an improved method to induce conidiation of this fungus. Isolates of C. gloeosporioides were pre-cultured in potato dextrose broth for 1 week, then pulverized in whole broth. The homogenate was then spread on diluted oatmeal agar (15–20% commercial oatmeal agar medium, 1.5% agar) plates. After the plates were cultured at 25°C under continuous light for another week, the C. gloeosporioides isolates sporulated stably on the plate medium.     

Biotrophic mycoparasitism byVerticillium biguttatum onRhizoctonia solani

Verticillium biguttatum cannot utilise cellulose or nitrate-nitrogen and it requires biotin for growth, yet it grew and sporulated abundantly onRhizoctonia solani on cellulose, obtaining at least organic carbon, nitrogen and biotin fromR. solani. Videomicroscopy of inter-hyphal interactions on films of water agar showed thatV. biguttatum behaved as a biotrophic mycoparasite. From germinating spores, it penetrated the hyphae ofR. solani and formed haustorium-like branches without killing the host cells, and the haustoria supported an external mycelial network of the mycoparasite. Later the mycoparasite sporulated, and the infected host cells died. On cellulosic substrataV. biguttatum did not reduce the growth ofR. solani, and often enhanced the rate of cellulose degradation. However,V. biguttatum drastically reduced the production of sclerotia byR. solani, often completely suppressing sclerotium production when the mycoparasite infected only a localized region of the host colony. This is ascribed to the creation of a nutrient sink by the parasite, consistent with biotrophy. On plates of cellulose agar the suppression of sclerotia was not confined to parasitized colonies but extended to adjacent colonies ofR. solani that had successfully anastomosed with the parasitized colony. There was no effect on adjacent vegetatively incompatible colonies, where attempted anastomoses caused cytoplasmic death. In comparable experiments the necrotrophic mycoparasiteGliocladium roseum had no long-distance effect on sclerotium production byR. solani.Suppression of sclerotium production may explain the reported success ofV. biguttatum in biocontrol of black scurf of potato in experimental field conditions.     

Verification of immunofluorescence colony-staining of Erwinia carotovora subsp. atroseptica by reisolation and immunodiffusion or PCR

In this study, the reliability and efficiency of three procedures for verification of IFC-positive colonies of Erwinia carotovora subsp. atroseptica were compared: (1) PCR amplification, (2) reisolation on a non-selective medium (trypticase soy agar) followed by direct immunodiffusion (TSA-DID), developed for isolation of target and cross-reacting bacteria, and (3) reisolation on a selective medium (crystal-violet pectate) and characterization of selected isolates with Ouchterlony double diffusion (DLCVP-ODD), developed for isolation of pectinolytic Erwinia spp. The reliability of a PCR amplification procedure for characterization of E.c. atroseptica was evaluated. Specific amplification products could be produced from DNA of all 187 European strains of the bacterium, while no amplification products were obtained from DNA of four distinctive serological groups of bacteria cross-reacting with antibodies against E.c. atroseptica , nor from DNA of randomly selected saprophytic bacteria isolated from potato peel extracts. All 60 immunofluorescent-positive target colonies from a potato peel extract with added E.c. atroseptica tested were positive by PCR compared with 68 and 72% successful determinations by TSA-DID and DLCVP-ODD, respectively. PCR enabled verification of fluorescent colonies from IFC preparations of naturally infected seed lots with an efficiency of 93%, compared with 48 and 71% successful determinations by TSA-DID and DLCVP-ODD, respectively. It is concluded that PCR is useful for routine confirmation of the identity of fluorescent colonies in IFC.     

Resistance of Phytophthora capsici to metalaxyl in plastic‐house capsicum crops in southern Italy*

In Calabria (southern Italy), control of crown and root rot of capsicum caused by Phytophthora capsici has relied primarily on soil drenches of metalaxyl. However, severe outbreaks occur every year in glasshouse crops, in which the practice of using plastic mulch and furrow irrigation favours the disease. Single‐hypha isolates of P. capsici collected in Calabria in 1992/1998 were tested in vitro for their level of sensitivity to metalaxyl. Isolates of other species of Phytophthora were used as reference. Fungicide sensitivity was determined by plating mycelial plugs onto potato dextrose agar amended with metalaxyl, at final concentrations ranging from 0.1 to 1000μg mL^?1 a.s. Inhibition of radial growth (%) was determined when colonies on unamended medium had covered approximately two‐thirds of the plate. The ED₅₀ values for inhibition of mycelial growth of P. capsici isolates ranged from 1.41 to44.6μg mL^?1 a.s. More than 80% of the P. capsici isolates from commercial plastic‐house crops in Calabria showed a moderate level of resistance as they were inhibited less than 60% at 5 μg mL^?1 but more than 60% at 100μg mL^?1     

Horizontal and vertical distribution of airborne conidia of Botrytis cinerea in a gerbera crop grown under glass

The horizontal and vertical distribution of airborne conidia ofBotrytis cinerea in a gerbera crop in two glasshouses (100 m² and 350 m²) was studied during 18 months in 1988 and 1989. Conidia ofB. cinerea were caught in simple spore traps consisting of agar in Petri dishes placed in a regular pattern at three different heights in the glasshouse and counted as colonies, after incubation. Lesions due to conidial infection were counted on gerbera petals. The horizontal and vertical distribution of conidia ofB. cinerea in a gerbera crop grown under glass was fairly uniform in both distinct glass-houses. Conidia ofB. cinerea trapped in a glasshouse can originate from sources inside and outside the glasshouse. No significant interaction was found between location and time for the colony counts and for the log transformed (ln(N+1)) lesion counts. The results of this study suggest that spore trapping at one height and at a limited number of locations and dates is sufficient for efficient monitoring ofB. cinerea in a glasshouse.     

Detection of soft rot Erwinia spp. on seed potatoes: conductimetry in comparison with dilution plating, PCR and serological assays

Automated conductance measurements in polypectate medium were used for the detection of pathogenic soft rot Erwinia spp. in potato peel extracts. The detection threshold for Erwinia carotovora subsp. atroseptica (Eca) in inoculated peel extracts was ca. 10⁴ colony forming units (cfu) ml^-1 when samples were considered positive on the basis of a response within 48 h at 20 °C. Detection of E. chrysanthemi (Ech) was less sensitive, only 10⁵ cfu ml^-1 peel extract were detected within 36 h at 25 °C. The linear correlation between detection times in conductimetry and inoculum levels of Eca and Ech in peel extracts was used for a quantitative estimation of Eca and Ech in naturally contaminated peel extracts. Samples giving a positive conductimetric response had to be confirmed with an enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) for the presence of Eca and Ech, because E. carotovora subsp. carotovora (Ecc) also generated a conductance response. Conductimetry was sensitive and efficient for detection of contamination levels of Eca higher than 10⁴ cfu ml^-1 peel extract. For Ech, conductimetric detection was less sensitive and inefficient due to low contamination levels of Ech and the presence of high numbers of Ecc in many samples after enrichment, which interfered with the test. Immunofluorescence cell staining (IF) combined with enrichment and immunofluorescence colony staining (IFC) were suited to detect and quantify low numbers of Eca and Ech at less than 10⁴ cfu ml^-1 in peel extracts. However, since false positive and negative reactions in serology were observed, the use of PCR after enrichment, or in combination with IFC to confirm positive results, was required for accurate detection.     

Conditional expression of harpinPsscauses yeast cell death that shares features of cell death pathway with harpinPss-mediated plant hypersensitive response (HR)

Conditional expression of harpin_Psscauses yeast cell death that shares features of cell death pathway with harpin_Pss-mediated plant hypersensitive response (HR).Pseudomonas syringae pv.syringae 61 hrp Z gene encodes harpin_Pss, a 34.7 kD extracellular protein that elicits a hypersensitive response (HR) in plants. Conditional expression of either full-length or truncated hrp Z sequences under the GAL1 promoter caused cell death in Saccharomyces cerevisiae Y187. Plating of pYEUT- hrp Z transformants on a medium containing galactose resulted in complete inhibition of colony formation, whereas their growth on a glucose-based medium was unaffected. Western blot analysis confirmed the expression of harpin_Pssin yeast cells transformed with pYEUT- hrp Z and grown in galactose-containing medium. A time-dependent decline in the percentage of trypan blue-excluding cells in cultures of pYEUT- hrp Z transformants was observed when cultured on galactose-containing medium. Similarly, the number of viable cells reduced to about 50% within 6 h. There were similarities in the harpin_Pss-mediated cell death in plants and yeast cell death (YCD). Galactose-induced cell death in pYEUT-hrp Z transformants of S. cerevisiae Y187 was suppressed by a protein kinase inhibitor K252a (10 μ M). The viability of pYEUT- hrp Z transformants was prolonged in the presence of 100 U ml⁻¹catalase suggesting a role for the oxidative burst in YCD that was further supported by the flow cytometric patterns of propidium iodide uptake by yeast cells. Overall, it appears that yeast provides a useful model system to understand the molecular mechanism of harpin_Pss-mediated cell death.     

A selective medium for improving quantitative isolation ofTrichoderma spp. from soil

ATrichoderma-selective agar medium (TSM) was developed for quantitative isolation ofTrichoderma spp. from soil. Selectivity was obtained by using chloramphenicol as a bacterial inhibitor, and pentachloronitrobenzene, p-dimethylaminobenzenediazo sodium sulfonate and rose-bengal as selective fungal inhibitors. The TSM also contains a low concentration of glucose which still allows relatively rapid growth and sporulation ofTrichoderma, enabling convenient and rapid identification ofTrichoderma colonies. All the 15Trichoderma isolates tested formed colonies and grew well on this medium. Recovery ofTrichoderma from artificially inoculated soils was high and was not affected by soil type or by other microorganisms. A positive correlation was observed betweenTrichoderma added to soil and counts ofTrichoderma colonies on TSM plates. When combined with a soil pellet sampler, the selective medium was also used successfully for recovery of the indigenousTrichoderma population of natural soils.     

A combined agar-absorption and BIO-PCR assay for rapid, sensitive detection of Xylella fastidiosa in grape and citrus

Application of the polymerase chain reaction (PCR) to disease diagnosis is limited in part by the presence of PCR inhibitors. Inhibition can be overcome and sensitivity increased by culturing bacteria on agar media prior to PCR (termed BIO-PCR). However, Xylella fastidiosa grows slowly, requiring 10–14 days for visible colonies to appear. In this study an agar-absorption BIO-PCR method for detecting X. fastidiosa in grape and citrus plants was developed. Optimum lengths of time for absorption of inhibitors by the agar medium or enrichment of bacteria on the medium were determined for Pierce's disease of grape and citrus variegated chlorosis. When petioles of grape and citrus leaves with symptoms were spotted onto agar media, the spots washed after various time intervals and assayed for X. fastidiosa by real-time PCR, 97% (31 out of 32) and 100% (six out of six) of spots were positive after 2 days and 4 h for grape and citrus, respectively. With direct PCR, only 12·5% (four out of 32) and 33% (two out of six) of spots were positive, respectively, and visible X. fastidiosa colonies were evident after 10 and 14 days, respectively. In a separate experiment with samples from a different vineyard, 46% (13 out of 28) of the grape samples (agar spots) were positive after 1 day and 93% (26 out of 28) after 5 days using agar-absorption PCR. In contrast, all samples were negative by direct PCR. Viable X. fastidiosa were recovered from all samples after 14 days. Further tests with eight randomly selected grape petioles from three Texas vineyards known to have Pierce's disease resulted in 50% being positive by a simple 24 h agar-absorption PCR assay, whereas none was positive by direct PCR. Overall, 10 out of 16 (63%) vines from five vineyards (two in California and three in Texas) were positive after the 24 h agar-absorption PCR assay. In contrast, only one vine was positive by direct PCR. This simple agar absorption-based PCR assay protocol should prove useful for the routine detection of X. fastidiosa and other slow-growing bacteria in the presence of PCR inhibitors.     

Identification of W-type and R-type isolates of Pseudocercosporella herpotrichoides

W-type isolates of Pseudocercosporella herpotrichoides grown on a maize-based agar and exposed to near- ultra-violet radiation at c . 13°C produced a greenish black colour, whilst R-type isolates produced a pink or pale brown colour in the agar medium. More colonies from directly plated lesions or from spore suspensions could be recognized as P. herpotrichoides and could be more easily differentiated as W-type or R-type or as mixtures of both by colour production on maize agar (MA) than by colony morphology on potato dextrose agar (PDA), despite the presence of other fungi. Isolates with intermediate morphology on PDA were positively identified as W-type or R-type on MA; their pathogenicities to wheat and rye seedlings were usually similar to those of W-type or R-type isolates with typical colony morphology, confirming their identification on MA. Drops of mixed suspensions of W-type and R-type spores on PDA formed fast-growing colonies with smooth margins which sometimes had slow-growing sectors with feathery margins. Drops of the same mixtures on MA formed greenish black colonies which sometimes had pink or pale brown sectors. However, when these mixtures were spread onto MA, W-type and R-type colonies could easily be differentiated by colour.     

Selective media for <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Selective media without pentachloronitrobenzene were developed for quantitative assays of Fusarium oxysporum in soils. Media Fo-G1 and Fo-G2 were effective for naturally infested soils, Fo-W1 and Fo-W2 for wild-type isolates in soils containing a nitrate-nonutilizing (nit) mutant, and Fo-N1 and Fo-N2 for nit mutants. Selective media were made using ammonium citrate dibasic, l-sorbose, econazole nitrate, 25% iminoctadine triacetate solution and 50% tolclofos-methyl wettable powder for soil dilutions of 100-fold or more (Fo-G1, FoW1 and Fo-N1) and 10-fold (Fo-G2, Fo-W2 and Fo-N2). Potassium chlorate was added to Fo-N1 and Fo-N2. The efficacy for selectively isolating F. oxysporum was confirmed using six soils naturally infested with one of six formae speciales of F. oxysporum and with soil dilutions containing conidia of wild-type strains or nit mutants from the six formae speciales. On Fo-G1 and Fo-G2, most colonies of F. oxysporum were compact and round with purplish or reddish pigment in the reverse. Cylindrocarpon sp. formed colonies as large as those of F. oxysporum but were distinguishable by their colony morphology. Other contaminants such as F. solani, F. moniliforme, and Trichoderma were suppressed by medium ingredients and colonies of F. oxysporum. On Fo-W1 and Fo-W2, colony morphology of F. oxysporum and contaminants corresponded to that on Fo-G1 and Fo-G2, although F. oxysporum failed to produce the pigment. On Fo-N1 and Fo-N2, nit mutants formed clear colonies from 100- and 10-fold soil dilutions, respectively, and contaminants seldom formed large colonies.     

Siderophore-mediated competition for iron and induced resistance in the suppression of fusarium wilt of carnation by fluorescent Pseudomonas spp

The mechanisms of suppression of fusarium wilt of carnation by two fluorescentPseudomonas strains were studied.Treatments of carnation roots withPseudomonas sp. WCS417r significantly reduced fusarium wilt caused byFusarium oxysporum f. sp.dianthi (Fod). Mutants of WCS417r defective in siderophore biosynthesis (sid^–) were less effective in disease suppression compared with their wild-type. Treatments of carnation roots withPseudomonas putida WCS358r tended to reduce fusarium wilt, whereas a sid^– mutant of WCS358 did not.Inhibition of conidial germination of Fod in vitro by purified siderophores (pseudobactins) of bothPseudomonas strains was based on competition for iron. The ferrated pseudobactins inhibited germination significantly less than the unferrated pseudobactins. Inhibition of mycelial growth of Fod by bothPseudomonas strains on agar plates was also based on competition for iron: with increasing iron content of the medium, inhibition of Fod by thePseudomonas strains decreased. The sid^– mutant of WCS358 did not inhibit Fod on agar plates, whereas the sid^– mutants of WCS417r still did. This suggests that inhibition of Fod by WCS358r in vitro was only based on siderophore-mediated competition for iron, whereas also a non-siderophore antifungal factor was involved in the inhibition of Fod by strain WCS417r.The ability of thePseudomonas strains to induce resistance against Fod in carnation grown in soil was studied by spatially separating the bacteria (on the roots) and the pathogen (in the stem). Both WCS417r and its sid^– mutant reduced disease incidence significantly in the moderately resistant carnation cultivar Pallas, WCS358r did not.It is concluded that the effective and consistent suppression of fusarium wilt of carnation by strain WCS417r involves multiple mechanisms: induced resistance, siderophore-mediated competition for iron and possibly antibiosis. The less effective suppression of fusarium wilt by WCS358r only depends on siderophore-mediated competition for iron.