ABSTRACT Mycosphaerella brassicicola causes ringspot on Brussels sprouts, which can result in sub... more ABSTRACT Mycosphaerella brassicicola causes ringspot on Brussels sprouts, which can result in substantial yield loss in commercial production. Brussels sprout buttons are downgraded if this pathogen occurs on them. In this study, the effect of temperature and wetness duration was investigated on infection of Brussels sprouts using controlled environments (CE). The effect of temperature and wetness duration on inoculum production and ascospore discharge was also investigated. Infection by M. brassicicola was described using a mathematical model and was compared to estimates of ascospore availability obtained via a volumetric air sampler and immunofluorescence (IF). Infection of M. brassicicola was correlated (r = 0.92) with temperature during leaf wetness periods. The relationship between temperature and time to discharge of 5 and 50% of the cumulative total number of ascospores from ringspot lesions was r = 0.99 and 0.98, respectively (P < 0.001). In field experiments, an optimal wind run (the product of the average wind speed and the period over which that average speed was measured) of 250 to 500 km day(-1) was required for the dissemination of ringspot inoculum to field bait plants. Quantification of M. brassicicola inoculum in collected field aerosols was possible using a monoclonal antibody in a plate-trapped antigen enzyme-linked immunosorbent assay. Precoating of the air sampler wells with sodium azide prevented trapped spores from germinating. Ringspot inoculum could be detected and quantified in air samples from commercial crops of Brussels sprouts in the United Kingdom. Low levels of ringspot inoculum measured within crops did not lead to disease development.
A monoclonal antibody that recognises components of the wall of sporangia of Peronospora destruct... more A monoclonal antibody that recognises components of the wall of sporangia of Peronospora destructor was raised. Tests using spores of higher fungi and other species of mildew demonstrated the specificity of the monoclonal. The antibody was used to develop lateral flow devices for sporangia of P. destructor. A competitive lateral flow format was developed which could detect onion downy mildew sporangia. Five-microliter gold anti-mouse IgM solution pre-mixed with 10 μl of P. destructor monoclonal antibody (EMA 242) proved the optimal concentration for detection of sporangia of P. destructor when applied to sample pads of lateral flow devices. Limits of approximately 500 sporangia of P. destructor could be detected by the absence of a test line on the lateral flow device within test samples. Using a scanning densitometer improved the sensitivity of detection. Further development and validation of the test is required if it is to be used for risk assessments of onion downy mildew in the field.
A membrane filtration test has been developed for the detection of viable zoospores of Pythium sp... more A membrane filtration test has been developed for the detection of viable zoospores of Pythium species. Zoospore suspensions were filtered through 5 (m nitrocellulose membranes and the membranes incubated overnight in 0.07 m glucose, rifamycin (30 mg litre-1) and pimaricin (100 mg litre-1). Zoospore germlings were detected using a polyclonal antiserum, raised to mycelial surface washings of five Pythium spp., and visualised with Sigma fast red. The assay gave positive results for all Pythium spp. tested and also to zoospores of Phytophthora cryptogea. Of 10 fungal species isolated from commercial irrigation water, two were detected by the polyclonal antiserum in ELISA tests but only one produced detectable zoospore germlings. The latter isolate was later identified as a Pythium sp. Irrigation water samples collected from commercial UK nurseries yielded zoospores of both Pythium and Phytophthora spp. which, using the assay, were positively identified. Results indicated greater sensitivity than was seen with conventional plating methods. This is a test which could be adapted for on-site use in commercial nurseries.
ABSTRACT Two immunodiagnostic detection assay procedures were compared with two conventional assa... more ABSTRACT Two immunodiagnostic detection assay procedures were compared with two conventional assays for their sensitivity in detecting propagules of Pythium ultimum var. sporangiiferum, Pythium Group F, Phytophthora cactorum and P. cryptogea in dilution series in sterile distilled water. The most sensitive assay for all four species was the zoospore trapping immunoassay (ZTI). Conventional membrane filtration-dilution plating gave similar results to ZTI with the two Phytophthora spp., but was less sensitive in Pythium detection. Immunodiagnostic dipstick assays and conventional bait tests showed similar sensitivities in the dilution series, and were generally about two orders of magnitude less sensitive than ZTI. The four techniques were also compared for their detection efficacy with water samples collected from horticultural nurseries and in in situ tests of infected root zones of Chamaecyparis, tomato and Chrysanthemum. In these comparisons, ZTI was again the most sensitive test for water samples, although membrane filtration-dilution plating proved to be a more consistent test. Dipstick and baiting assays were the best techniques for in situ testing, and dipsticks provided epidemiologically valuable, quantitative data on pathogen propagule numbers.
Detection and enumeration of air-borne spores of fungal plant pathogens has generally been achiev... more Detection and enumeration of air-borne spores of fungal plant pathogens has generally been achieved by microscopic examination of surfaces on which spores have been impacted and conventional agar-plating techniques. However, this requires considerable amounts of time and expertise. Such methods are inaccurate for obligate pathogens and unrealistic where there is no selective medium or where there are spore types of similar morphology (such as those produced by ascosporic fungi). In field disease-transmission studies, the use of a Burkard 7-day volumetric spore trap combined with an immunofluorescence test has, however, enabled the detection and quantification of field-trapped ascosporic inoculum of Mycosphaerella brassicicola (the ringspot pathogen of brassicas). This test has also been found useful in the validation of more rapid user-friendly immunoassay-based trapping procedures. A microtitre immunospore trapping device, which uses a suction system to trap air particulates directly by impaction into microtitre wells, has been used successfully for the rapid detection and quantification of ascosporic inoculum of M. brassicicola. The system shows potential for field detection of air-borne ascosporic inoculum of the ringspot pathogen.
Pythium oligandrum was recovered, identified and quantified from air‐dried soil plated on 1.5% wa... more Pythium oligandrum was recovered, identified and quantified from air‐dried soil plated on 1.5% water agar containing 0.1% glucose. Isolations of P. oligandrum over 2 years from soils treated with single applications of metalaxyl plus mancozeb were consistently lower than those from untreated soil from the same fields. In three fields in the first year P. oligandrum was reduced from a
Pythium violae Chesters & Hickman was isolated for the first time from cavity spot lesions on car... more Pythium violae Chesters & Hickman was isolated for the first time from cavity spot lesions on carrots in Israel and the resulting isolates were compared with isolates from the UK, France and Spain. The isolates from all locations responded similarly to temperature and had similar colony morphology, oogonial diameters and soluble protein patterns.
Polyclonal antisera were raised to whole and sonicated fractions of resting spores of Plasmodioph... more Polyclonal antisera were raised to whole and sonicated fractions of resting spores of Plasmodiophora brassicae, the causal agent of clubroot disease. Cross reactivity of antisera was assessed against a wide range of soil-borne pathogens. Antiserum raised to whole resting spores was used to probe artificially infested soils by indirect immunofluorescence. Resting spores could be detected at a concentration as low as to 1 × 102 spores per g in artificially infested soil, and were readily identified in naturally infested soils, with negligible fluorescence in soil thought to be clubroot-free. Antiserum raised to sonicated fractions of the resting spore was used in an indirect ELISA assay of soil. When bound antiserum was labelled with anti-rabbit IgG conjugated to alkaline phosphatase, there was a negative correlation between absorbance levels and resting spore numbers in artificially infested soil. In contrast a close to linear response, with a correlation coefficient of 0.971, was recorded when infested soils were assayed with bound antiserum labelled with protein-A peroxidase.
ABSTRACT Mycosphaerella brassicicola causes ringspot on Brussels sprouts, which can result in sub... more ABSTRACT Mycosphaerella brassicicola causes ringspot on Brussels sprouts, which can result in substantial yield loss in commercial production. Brussels sprout buttons are downgraded if this pathogen occurs on them. In this study, the effect of temperature and wetness duration was investigated on infection of Brussels sprouts using controlled environments (CE). The effect of temperature and wetness duration on inoculum production and ascospore discharge was also investigated. Infection by M. brassicicola was described using a mathematical model and was compared to estimates of ascospore availability obtained via a volumetric air sampler and immunofluorescence (IF). Infection of M. brassicicola was correlated (r = 0.92) with temperature during leaf wetness periods. The relationship between temperature and time to discharge of 5 and 50% of the cumulative total number of ascospores from ringspot lesions was r = 0.99 and 0.98, respectively (P < 0.001). In field experiments, an optimal wind run (the product of the average wind speed and the period over which that average speed was measured) of 250 to 500 km day(-1) was required for the dissemination of ringspot inoculum to field bait plants. Quantification of M. brassicicola inoculum in collected field aerosols was possible using a monoclonal antibody in a plate-trapped antigen enzyme-linked immunosorbent assay. Precoating of the air sampler wells with sodium azide prevented trapped spores from germinating. Ringspot inoculum could be detected and quantified in air samples from commercial crops of Brussels sprouts in the United Kingdom. Low levels of ringspot inoculum measured within crops did not lead to disease development.
A monoclonal antibody that recognises components of the wall of sporangia of Peronospora destruct... more A monoclonal antibody that recognises components of the wall of sporangia of Peronospora destructor was raised. Tests using spores of higher fungi and other species of mildew demonstrated the specificity of the monoclonal. The antibody was used to develop lateral flow devices for sporangia of P. destructor. A competitive lateral flow format was developed which could detect onion downy mildew sporangia. Five-microliter gold anti-mouse IgM solution pre-mixed with 10 μl of P. destructor monoclonal antibody (EMA 242) proved the optimal concentration for detection of sporangia of P. destructor when applied to sample pads of lateral flow devices. Limits of approximately 500 sporangia of P. destructor could be detected by the absence of a test line on the lateral flow device within test samples. Using a scanning densitometer improved the sensitivity of detection. Further development and validation of the test is required if it is to be used for risk assessments of onion downy mildew in the field.
A membrane filtration test has been developed for the detection of viable zoospores of Pythium sp... more A membrane filtration test has been developed for the detection of viable zoospores of Pythium species. Zoospore suspensions were filtered through 5 (m nitrocellulose membranes and the membranes incubated overnight in 0.07 m glucose, rifamycin (30 mg litre-1) and pimaricin (100 mg litre-1). Zoospore germlings were detected using a polyclonal antiserum, raised to mycelial surface washings of five Pythium spp., and visualised with Sigma fast red. The assay gave positive results for all Pythium spp. tested and also to zoospores of Phytophthora cryptogea. Of 10 fungal species isolated from commercial irrigation water, two were detected by the polyclonal antiserum in ELISA tests but only one produced detectable zoospore germlings. The latter isolate was later identified as a Pythium sp. Irrigation water samples collected from commercial UK nurseries yielded zoospores of both Pythium and Phytophthora spp. which, using the assay, were positively identified. Results indicated greater sensitivity than was seen with conventional plating methods. This is a test which could be adapted for on-site use in commercial nurseries.
ABSTRACT Two immunodiagnostic detection assay procedures were compared with two conventional assa... more ABSTRACT Two immunodiagnostic detection assay procedures were compared with two conventional assays for their sensitivity in detecting propagules of Pythium ultimum var. sporangiiferum, Pythium Group F, Phytophthora cactorum and P. cryptogea in dilution series in sterile distilled water. The most sensitive assay for all four species was the zoospore trapping immunoassay (ZTI). Conventional membrane filtration-dilution plating gave similar results to ZTI with the two Phytophthora spp., but was less sensitive in Pythium detection. Immunodiagnostic dipstick assays and conventional bait tests showed similar sensitivities in the dilution series, and were generally about two orders of magnitude less sensitive than ZTI. The four techniques were also compared for their detection efficacy with water samples collected from horticultural nurseries and in in situ tests of infected root zones of Chamaecyparis, tomato and Chrysanthemum. In these comparisons, ZTI was again the most sensitive test for water samples, although membrane filtration-dilution plating proved to be a more consistent test. Dipstick and baiting assays were the best techniques for in situ testing, and dipsticks provided epidemiologically valuable, quantitative data on pathogen propagule numbers.
Detection and enumeration of air-borne spores of fungal plant pathogens has generally been achiev... more Detection and enumeration of air-borne spores of fungal plant pathogens has generally been achieved by microscopic examination of surfaces on which spores have been impacted and conventional agar-plating techniques. However, this requires considerable amounts of time and expertise. Such methods are inaccurate for obligate pathogens and unrealistic where there is no selective medium or where there are spore types of similar morphology (such as those produced by ascosporic fungi). In field disease-transmission studies, the use of a Burkard 7-day volumetric spore trap combined with an immunofluorescence test has, however, enabled the detection and quantification of field-trapped ascosporic inoculum of Mycosphaerella brassicicola (the ringspot pathogen of brassicas). This test has also been found useful in the validation of more rapid user-friendly immunoassay-based trapping procedures. A microtitre immunospore trapping device, which uses a suction system to trap air particulates directly by impaction into microtitre wells, has been used successfully for the rapid detection and quantification of ascosporic inoculum of M. brassicicola. The system shows potential for field detection of air-borne ascosporic inoculum of the ringspot pathogen.
Pythium oligandrum was recovered, identified and quantified from air‐dried soil plated on 1.5% wa... more Pythium oligandrum was recovered, identified and quantified from air‐dried soil plated on 1.5% water agar containing 0.1% glucose. Isolations of P. oligandrum over 2 years from soils treated with single applications of metalaxyl plus mancozeb were consistently lower than those from untreated soil from the same fields. In three fields in the first year P. oligandrum was reduced from a
Pythium violae Chesters & Hickman was isolated for the first time from cavity spot lesions on car... more Pythium violae Chesters & Hickman was isolated for the first time from cavity spot lesions on carrots in Israel and the resulting isolates were compared with isolates from the UK, France and Spain. The isolates from all locations responded similarly to temperature and had similar colony morphology, oogonial diameters and soluble protein patterns.
Polyclonal antisera were raised to whole and sonicated fractions of resting spores of Plasmodioph... more Polyclonal antisera were raised to whole and sonicated fractions of resting spores of Plasmodiophora brassicae, the causal agent of clubroot disease. Cross reactivity of antisera was assessed against a wide range of soil-borne pathogens. Antiserum raised to whole resting spores was used to probe artificially infested soils by indirect immunofluorescence. Resting spores could be detected at a concentration as low as to 1 × 102 spores per g in artificially infested soil, and were readily identified in naturally infested soils, with negligible fluorescence in soil thought to be clubroot-free. Antiserum raised to sonicated fractions of the resting spore was used in an indirect ELISA assay of soil. When bound antiserum was labelled with anti-rabbit IgG conjugated to alkaline phosphatase, there was a negative correlation between absorbance levels and resting spore numbers in artificially infested soil. In contrast a close to linear response, with a correlation coefficient of 0.971, was recorded when infested soils were assayed with bound antiserum labelled with protein-A peroxidase.
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Papers by Alison J Wakeham