HH1759SFV_4240_FRP

CSG 15 (9/01) 1

DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS CSG 15 Research and Development

Final Project Report (Not to be used for LINK projects)

Two hard copies of this form should be returned to: Research Policy and International Division, Final Reports Unit DEFRA, Area 301 Cromwell House, Dean Stanley Street, London, SW1P 3JH. An electronic version should be e-mailed to [email protected]

Project title Studies on the transmission of foliar fungal pathogens of vegetable brassicas for the prediction of disease risk

DEFRA project code HH1759SFV

Contractor organisation and location

Horticulture Research International, Wellesbourne, Warwickshire CV35 9EF

Total DEFRA project costs 288,654

Project start date 01/04/99 Project end date 31/03/02

Executive summary (maximum 2 sides A4)

Background Many air-borne fungal diseases affecting vegetable brassicas have become common in UK production. Ringspot (Mycosphaerella brassicicola, and dark leaf spot (Alternaria brassicae) are the most serious problems with the most widespread geographic distribution. In vegetable brassica production areas 6 - 8 fungicide applications have been used to control those diseases and maintain the high quality of produce demanded by the market. These levels of fungicide usage often result from the need to prevent disease establishment within the crop. All vegetable brassicas crops in the UK are transplanted and at the time of transplanting are free of disease. This means that in the field disease must enter the crop from some other source (usually a crop in the locality owned by another grower) if it is to become a problem in the freshly transplanted crop. Infection conditions within vegetable brassica crops are not limiting which also means that if inoculum is available from some other source it will infect the freshly transplanted clean crop. The availability of inoculum is not known because it depends on the number of infected crops and their location in the area. The grower has thus a problem in that he can not ascertain if inoculum is available or not as he has not the capability to continuously wander around the locality inspecting crops. To avoid his crop becoming infected would require blanket applications of fungicide which may still prove ineffective. However by measuring the availability of inculum in the air the grower can avoid the need to do this and improve his control of disease by stopping its entry into the crop thus using minimal pesticide applications. Many air-borne pathogens (white blister; light leaf spot and ringspot) of vegetable brassicas have long latent periods (time between infection and appearance of disease). This means that success or failure of control is only apparent in some cases weeks after fungicide applications. Often this leads to diseases becoming well established in crops before the disease is really visible. Additionally many of these diseases are difficult to diagnose correctly, as they first appear as small black spots. A major problem exists in differentiating ringspot from dark leaf spot in its early stages of development. Within this project new and rapid methods of detecting and quantifying pathogenic inoculum have been developed by using air samples. By developing methods to detect the inoculum of air-borne pathogens the subsequent counts obtained for different pathogens can be used to improve forecasting systems for any target pathogen. When used in this way the measured inoculum will indicate (when used in conjunction with disease forecasting systems summarising the effect of

Project title

Studies on the transmission of foliar fungal pathogens of vegetable brassicas for the prediction of disease risk

DEFRA project code

HH1759SFV

CSG 15 (9/01) 2

environment on each pathogen) the type and actual amount of disease which will occur. With this more precision approach there will be reductions in the amounts of fungicide required to control disease by eliminating unnecessary fungicide applications which are based on weather information alone. For all plant diseases inoculum must be present if the disease is to develop. Disease occurring on plants which exists in a form which does produce inoculum will not develop but die out within the crop due to the continuous production and shedding of leaves in vegetable crops. For this reason systems based on the detection of air-borne diseases such as downy mildews using systems which detect disease in the plants will be inferior to those sytems which can detect inoculum in air (the pathogens form of spread and development). By detecting inoculum predictions can be made about what diseases will develop as the inoculum will be detected before it infects the plant or crop. An additional advantage is that only relatively small amounts of inoculum in air samples can be detected. However to detect low levels of diseases in crops using leaf tissues will require huge amounts of leaf samples with the need for many tests which renders the procedure uneconomic both from a time and material viewpoint. These tests are also prone to huge error depending on the leaf sampling regime used. It is therefore likely that using the human detection system " the eye " will be able to detect low levels of disease in crops before systems based on using leaf material can detect the disease. Using leaf material should only be used to differentiate different diseases or spots where there is similar symptoms. Fortunately detecting inoculum in the air is not affected by these difficulties although proper spatial sampling regimes will still be required. However it was not an objective of this project to ascertain spatial sampling regimes for air-borne diseases. New methods for the quantification of inoculum would require new trapping technology and new specific tests as the conventional methods of measuring inoculum (visual counts) were too slow and prone to inaccuracy. Objective one New specific tests for different types of airborne pathogens affecting vegetable brassicas were successfully developed within objective one using specific antibody based probes. A co-immunogen approach was used for raising a monoclonal antiserum to M. brassicicola, Alternaria brassicicola and A. brassicae. Using this method a highly specific MAb to M. brassicicola was produced (EMA 187). Cross-reactivity studies using MAb EMA 187 have shown that there was no fungal asexual spores. Only ascospores of Pyrenopeziza brassicae reacted with the antiserum. By using Mab EMA 187 there was a good correlation between the number of ascospores of M. brassicicola trapped by the MTIST spore trapping device and the absorbance values generated by ELISA. The amount of ringspot disease, which appeared on exposed trap plants placed inside the controlled environment cabinet, and the number of MTIST device-trapped ascospores of M. brassicicola also demonstrate a clear relationship between PTA ELISA values and lesion numbers on plants. Mixed spore population studies suggested that there is likely to be little interaction between large and small propagules trapped. There was also a very good correlation between existing antibody based trapping systems (the B-7 Day immunofluorescence test) and the PTA ELISA indicating a useful bench mark for assessing the efficiency of other trapping systems. However trapping technology was required to successfully "catch" the spores in the air and to which new methods could be applied (objective two see below). Objective two Objective two was very successfully completed by employing new novel trapping systems (MTIST spore trap) and the spore type and number differentiated which reacted with the antibodies developed under objective one. The collection efficiency of the MTIST trap was improved for use under field conditions by adding wind directional features. However the collection efficiency of the system was unaffected by speed of airflow although the system was more efficient at collecting small propagules (Penicillium spores). Potential for optimisation of the system was demonstrated by employing surface coatings on the microtiter wells which the spores were collected into (see objective seven). Objective seven A range of microtiter well (the vessel into which spores were "caught") coatings were tested for their ability to retain the trapped spores. Microtiter well coatings containing 2% BSA and 2 % Casein were significantly poorer at collecting and retaining smaller spores (Penicillium). Well coatings containing paraffin wax and hexane were significantly better at collecting and retaining larger spores used in this study (Lycopodium, Erysiphe and Botrytis). The well coating used would be of great importance because of the numerous washing steps involved in ELISA procedure. This means that unless spores are properly attached to the microtiter well surface they will be lost during the stage where they are quantified. The objective was successfully completed in that in was ascertained that well coating containing paraffin wax could be used without affecting the quantification step. However for some immunological tests germination of the spore may or may not be necessary. Microtiter well coatings may be necessary which halt any further biological activity and reduce cross-reactivity. This could be observed where the system was used under field conditions. By pre-coating microtiter wells with 0.5mg ml-1 sodium azide prior to exposure to in the field there was inhibition of spore germination so reducing this source of error. Pre-coating microtiter wells with a higher concentration of sodium azide inhibited the ELISA assay reducing the sensitivity of M. brassicicola ascospore detection. Sodium azide inhibits the action of horseradish peroxidase used in this assay format. For these reasons it is very important that spore trapping assays should be optimised to give the greatest accuracy. The new system required optimisation against conditions found in the field which were investigated under objective three.

Project title


DEFRA project code

HH1759SFV

CSG 15 (9/01) 3

Objective three The collection efficiency of the modified MTIST spore trap was not affected by windspeed. This successful conclusion within objective three indicated that the MTIST trap could be used under field conditions where there was variable windspeed. However there was an indication that the conventional traps used for comparison with the new MTIST trap were affected by windspeed in their potential to trap larger spore types (Lycopodium). The conventional spore trap collected significantly higher numbers of Lycopodium than the MTIST spore trap however the MTIST trapped significantly higher numbers of Penicillium (a very small spore type) than Lycopodium, Erysiphe and Botrytis. It was successfully ascertain in experiments that inoculum of the ringspot pathogen of brassicas should be monitoring at heights of greater than one metre above the crop if the potential for movement of inoculum from infected crops to uninfected crops was to be ascertained. Further information on when spores become airborne within the crop was also needed and this was investigated within objective four. The rationale of catching spores only when conditions are right for them to become airborne is an important principal in measuring disease transmission. Objective four When used in the field the accuracy of the system was highest when optimised to trap inoculum only during periods when it is produced. This means that the if ascospores of M. brassicicola were to be trapped it was best to investigate the environmental conditions which give rise to their release and activate the trapping technology only when these conditions were fufilled. The results obtained in this way are very different to those obtained if trapping were to be carried out over time frames which were not consistant with spore release. This factor has contributed to much confusion in the past about the intrepretation of spore counts in air samples. It was observed that ascospores of M. brassicicola were produced only when the lesions were wet and when a light intensity exceeded 0.003kwm2. By using existing commercial or non commercial data loggers, trapping could be activated when a light intensity of >0.003kwm2 was observed together with a relative humidity (r.h.) of > 80 % (which mean't that the lesions were likely to be wet. These two conditions are required for release of ascospores of M. brassicicola. The system was thus ready to test as a means identifying when disease first enters vegetable crops. This was successfully carried out within objective five. Objective five Using the modified version of the MTIST spore trap in the field demonstrated that epidemiologically significant levels of M. brassicicola inoculum in the air can be detected both reliably and rapidly. Despite weather conditions when the results were less accurate (e.g. heavy rainfall) large numbers of ascospores were detected at the ringspot source and up to 1.6 kms away. The distance (and numbers) that ascospores will travel depends on the amount of disease at source. There was enough ascospores produced by an infected plot (of dimensions of 5x5m) to give high levels 1.6 kms from the source. The results successfully show that even a small patch of infected brassicas which are commonly grown in these types of dimensions on allotments have the ability to impact on larger areas of commercial production. The scales used in commercial production are much greater and the effect of large scale production on transmission of inoculum was successfully investigated in objective six. Objective six Within commercial crops there was a close relationship between the disease predictions using the Brassicaspot infection models and the onset of disease within a Brussels sprout crop at Skegness as detected by the presence of high levels of inoculum. Despite numerous infection periods there was only one critical infection period (7-8 July 2001) when there was sufficient ringspot inoculum present to initiate disease development. This successfully proves that inoculum is limiting and thesholds of inoculum are required to initiate epidemics. These results have great epidemiological significance for the control of ringspot and other diseases in the field. Overwintered unsprayed cauliflower plots with heavy levels of infection are common in field vegetable production areas. These can on average be approximately 10 to 20 hectares in size and must represent considerable sources of inoculum for plantings carried out during the new season (May onwards). It is likely that these crops have the potential to spread significant amounts of disease over entire production areas. However these risks can be measured by the system developed in this project. In addition it is also clear that summer-grown cauliflowers and broccoli which, are unsprayed, also act as considerable sources of disease for overwintered cauliflower and late season Brussels sprouts crops. The tests developed in this project can be optimised within lateral flow assays which means that the information on inoculum presence or absence and amount can be ascertained by the grower in the field. Overall Conclusions The information and the inoculum detection system developed in this project could be used within crop protection programmes to eliminate over spraying of fungicides. This would be particularly useful early in the season as a method of preventing disease transfer between over wintered crops and freshly transplanted crops. The results clearly show that by incorporating information on inoculum with information on disease risk (using predictive models) it will be possible to designate strategies, which can be used to reduce pesticide applications to one or two sprays per crop. The critical date for applying fungicide applications to the crop can be identified. However it is unclear how this information can be applied

Project title


DEFRA project code

HH1759SFV

CSG 15 (9/01) 4

to bigger cropping areas. One possible route might be to establish networks of traps (3-4 traps) which could be applied to larger cropping areas. With high sampling rates these traps if positioned to reflect prevailing wind patterns could be used to designate the onset of disease risk in different areas and pinpoint specific transmission events affecting different crops within the area. However further information is required on the source of error in the system resulting from variability in virulence of inoculum. There are many spots within crops which infect but fail to develop further or contribute to the epidemic indicating a potential variation in virulence. Information on inoculum will be required if the amount of pesticide used to control airborne diseases is to be reduced to low levels or if the efficiency of biological control agents is to be improved. As tests for pathogenic inoculum can be carried out in the field (by using lateral flow devices) the system developed in this project provides the background for the development and use of lateral flow tests. These tests meet the criteria necessary for its uptake by the brassica industry. However to develop a full system for vegetable brassicas will require further work to include measures of inoculum for all diseases which affect this crop. The information will also be of value within organic production systems. Key outputs New technology for rapid measurement of airborne spores of fungal pathogens. Sampling techniques for detecting airborne ascospores of Mycosphaerella brassicicola and conidia of Alternaria brassicae. Optimised assay formats for rapid detection of ascospores of Mycosphaerella brassicicola and conidia of Alternaria brassicae. New information on the role of air-borne transmission of fungal spores in disease epidemic development in vegetable brassica crops.

Project title


DEFRA project code

HH1759SFV

CSG 15 (9/01) 5

Scientific report (maximum 20 sides A4) 1. INTRODUCTION In areas of intensive vegetable brassica production transmission of airborne inoculum between brassica crops causes disease early in the season and can lead to complete crop loss despite heavy usage of eradicant fungicides. The presence of a single lesion on Brussels sprout buttons can incur downgrading of crop quality. Air-borne fungal diseases affecting vegetable brassicas have become increasingly prevalent in all major areas of production. Dark leaf spot (Alternaria brassicae) and ringspot (Mycosphaerella brassicicola) occur as a complex on Brussels sprouts where they have the potential to cause major losses. In other areas where oilseed rape crops (an alternate host for both Mycosphaerella and Alternaria) are grown a build up of disease on these crops (mostly unsprayed) can be transmitted to Brussels sprout crops. Both diseases are common in many seasons. The main objective of this work is to develop methods for measuring the transmission of ringspot and dark leaf spot and describe environmental and host factors which affect the degree of windborne dispersal of inoculum by these pathogens. Traditionally growers have used 6 7 applications of fungicide in favourable disease years to control air-borne fungal diseases. However many existing forecasting systems assume that airborne inoculum is not limiting. This leads to mistiming of sprays and the over usage of fungicides. By developing systems, which can ascertain, the presence or absence and quantity of pathogenic inoculum reductions in pesticide usage can be achieved without increased risk of crop loss. With this strategic information on inoculum availability a rationale for use of existing disease forecasting systems spatially can be ascertained. This will reduce the need for meteorological information from every field in cropping areas by using information on inoculum availability to ascertain variability in disease pressure within areas of production. 2. OBJECTIVES OF THE PROJECT 1) Develop rapid assay formats for the airborne detection of M. brassicicola (ringspot) and A. brassicae / A. brassicicola

(dark leaf spot) using appropriate antibody technology. 2) Identify and test appropriate trapping technology which could be used to investigate pathogen transmission. 3) Determine positional and environmental factors which affect quantitative measurements of spore transmission. 4) Identify key environmental factors which affect quantitative measurements of spore transmission. 5) Identify key environmental factors, cropping patterns and disease levels in unsprayed crops influencing the

transmission of ringspot and dark leaf spot under natural conditions. 6) Evaluate incorporation of the knowledge gained for prediction of disease risk in different crops/areas and different years. Determine disease risk to freshly transplanted crops. 7) To identify microtiter well coatings, which could be used to optimise spore catches of pathogenic and non-pathogenic

field fungi and reduce interference in rapid assays by reducing spore germination. 3. EXPERIMENTS 3.1 Develop rapid assay formats for the airborne detection of M. brassicicola (ringspot) and A. brassicae / A. brassicicola (dark leaf spot) using appropriate antibody technology (Objective 1) 3.1.1 Production of a monoclonal antiserum to the airborne stage of M. brassicicola Materials and Methods A 30ml ascospore suspension of M. brassicicola (5x104 ascospores per ml) was prepared in sterile distilled water (Kennedy et al., 1999). Using a Soniprep 150 (MSE, Crawley) the ascospore preparation was intermittently sonicated to a micron amplitude of 20 and concentrated by freeze-drying. The sonicated freeze-dried ascospore preparation was rehydrated with a polyclonal antiserum (Pab) (Pab raised in a female New Zealand White rabbit to mycelial immunogen of Mycosphaerella pinodes and used at a dilution of 1:400 phosphate buffered saline solution (PBS pH 7.5)) to a final volume of 10ml PBS and, mixed for one hour at 22C. Five female Balb C mice were immunised (by intraperitoneal injection) each with 50l of the co-immunogen ascospore preparation mixed with an equal volume of Titermax adjuvant. The mice were immunised twice more at 4 weekly intervals without the adjuvant. Three of the mice each received a final pre-fusion boost four days before a fusion was carried out. Using a modified method of Kennett et al., (1978) hybridoma cell lines were produced. Cell culture supernatants were screened 14 days after cell fusion for the presence of antibodies which recognised ascosporic epitopes of M. brassicicola both by immunofluorescence (IF) and plate trapped antigen enzyme-linked immunosorbent assay (PTA ELISA). All selected cell lines recognised only soluble epitopes of M. brassiciciola and proved highly non-specific when tested with a wide range of airborne fungal spora. A cell line which recognised ascosporic components of M. brassicicola (coded Mab EMA 185) was cloned and selected for further co-immunisation studies.

Project title


MAFF project code HH1759S

FV

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Three months after the initial immunisations the two remaining mice were boosted twice more at four weekly intervals this time using a co-immunogen of a sonicated freeze-dried ascospore preparation (as above) mixed with Mab EMA 185. The mice received a final pre-fusion boost four days before a fusion was carried out and the selection and screening of positive hybridomas were as described above. A cell line which recognised an ascospore wall epitope was selected and cloned (coded Mab EMA 187). In IF reactivity studies, where a wide range of fungi were tested, Mab EMA has proved highly specific (Table 1) cross-reacting only with the ascosporic stage of Pyrenopeziza brassicae (Wakeham et al., 2000). Table 1. Reactivity MAb EMA 187 to a range of fungi employing immunofluorescence (IF) Ascospore Reactivity of MAb EMA 187 Mycosphaerella brassicicola + Mycosphaerella pinodes Not tested Mycosphaerella cryptica - Mycsophaerella nubilosa - Sclerotinia sclerotiorum - Pyrenopeziza brassicae + Gaeumannomyces graminis var. tritici - Conidia Mycosphaerella pinodes - Pyrenopeziza brassicae - Ascochyta fabae - Ascochyta rabiei - Ascochyta lentis - Ascochyta allii - Botrytis cinerea - Botrytis squamosa - Alternaria brassicae - Alternaria brassicicola - Aspergillus flavus - Conclusions In a previous DEFRA study (HH1713SFV) a polyclonal antiserum was raised to ascosporic inoculum of M. brassicicola. However this antiserum lacked the specificity required for field usage as it cross-reacted with a wide range of fungal spore types (Kennedy et al., 1999). Barclay and Smith (1986) established that co-immunisation was a valuable method for raising MAbs specific to bacteria. Using a co-immunogenic approach enabled more specific antisera to be raised for M. brassicicola. By using non-specific antisera which cross-reacts with sites shared by both the target pathogen and closely related species, it may be possible to block common immunodominant sites and elicit an immune response to a less immunodominant but specific epitope. The results indicate fundamental differences in wall components between asexual and sexual stages of fungi. 3.1.2 Production of monoclonal antiserum to airborne stage of Alternaria brassicae / A. brassicicola Materials and Method A 10ml suspension of A .brassicae and A. brassicicola (1x104 per ml) was prepared in sterile distilled water and concentrated by freeze-drying. The conidial material was resolubalised with MAb EMA 185 (monoclonal raised to M. brassicicola with reactivity to many fungal spore types including Alternaria spp.) in PBS to a volume of 5ml and mixed for 1hr at room temperature prior to immunisation. A female New Zealand white rabbit was given an intramuscular injection of 500l Freunds complete adjuvant mixed with 500l of the co-immunogen of Alternaria /MAb EMA 185. Four further injections were then administered at weekly intervals with 500l of Freunds incomplete adjuvant mixed with 500l of the Alternaria co-immunogen. All further stages were as described by Kennedy et al., 1999.In PTA-ELISA the polyclonal antiserum proved non-specific cross-reacting with a wide range of fungal species tested Using a Soniprep 150, conidia of a 10ml suspension of A. brassicae and A. brassicicola ( 1x104 per ml) were disrupted. The soluble fraction was separated from the particular material by centrifugation. After which the soluble fraction was

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freeze-dried and then re-hydrated with MAb EMA 185 in PBS to a volume of 5ml and, mixed for 1hr at room temperature, prior to immunisation. This was designated immunogen A. The remaining particular conidial material was re-solublised with a 5ml volume of PBS and mixed with MAb EMA 185 as described above for immunisation (immunogen B). A final immunogen preparation was prepared by mixing MAb EMA 185 with a conidial suspension of A. brassicae / A. brassicicola as described above (immunogen C). For each of the immunogen preparations 3 female Balb C mice were immunised (by intraperitoneal injection) as described above. Using a modified method of Kennet et al., (1978) hybridoma cell lines were produced. Cell culture supernatants were screened 14 days after cell fusion for the presence of antibodies which recognised conidial wall epitopes of A. brassicae / brassicicola both by immunofluorescence (IF) and plate trapped antigen enzyme-linked immunosorbent assay (PTA ELISA). All cell lines recognised only soluble epitopes of A. brassicae and proved highly non-specific when tested with a wide range of airborne fungal spora. A further three mice were immunised with preparation C but without MAb EMA 185 as a co-immunogen. Following the immunisation process and the hybridoma screen as described above selected cell lines were tested for reactivity to other fungal plant pathogens. Two cell lines EMA 190 and 191 proved highly specific only reacting to germinating conidia of Alternaria brassicae (Table 2). However further tests revealed that both were A. brassicae isolate specific and did not recognise that pathogen when cultured on host material. Following this last set of fusions three further immunogen preps were prepared. Conidia of a range of single spore isolates of Alternaria brassicae (AA3,4,5,10,11) were produced in vitro, as described above, and collected from inoculated Brussels sprout plant material. Again a range of Alternaria brassicae isolates were used as inoculum to induce conidial production on host plant material. For each of the three immunogen preps a total of 9 mice were immunised. From three fusions only one cell line was selected (coded MAb EMA 6C7) which in PTA ELISA reactivity screening tests exhibited a level of sensitivity and specificity that may be useful for field detection studies (Table 2). Results Table 2. Reactivity of Alternaria PAb and MAbs in PTA ELISA Fungal species MAb 6C7 MAb 190 191 PAb Alternaria brassicae (AA11-1) + - - + Alternaria brassicae (AA11-2) + - - + Alternaria brassicae (AA10-1) + + + + Alternaria brassicae (AA3-1) + + - + Alternaria brassicae (AA5) + Not tested Not tested + Alternaria brassicae (AA3-2) Not tested + - + Alternaria brassicae (AA4) Not tested + - + Alternaria brassicicola + - - + Alternaria dauci -/+ - - + Alternaria alternata -/+ - + + Botrytis squamosa - - - -/+ Botrytis cinerea - - - -/+ Fusarium culmorum - - - + Aspergillus - - - -/+ Mycosphaerella brassicicola - - Conclusions Although of high specificity MAbs 190 and 191 were unsuitable for use in in-field detection systems recognising only in vitro produced germinating conidia of a limited number Alternaria brassicae single spore isolates (Table 2). Alternatively MAb EMA 6C7 may prove useful in determining viable field inoculum of A. brassicae / brassicicola in that it recognises an epitope present only at the point of germination. With the modification of the PTA ELISA to include a biotin streptavidin amplification system (Kennedy et al., 2000), the sensitivity and specificity attained employing both MAb EMA 187 (M. brassicicola) and EMA 6C7 (Alternaria brassicae / brassicicola) can be further improved for use in monitoring airborne transmission of target propagules. It is unlikely that the cross- reactivity of EMA 187 to Pyrenopeziza brassicae will not be of epidemiological significance due to temporal differences in ascospore production by these pathogens. 3.2 Identify and test appropriate trapping technology which could be used to investigate pathogen

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transmission (Objective 2) 3.2.1 Development of spore trapping equipment for integration within rapid immunoassay formats Materials and Methods In conjunction with Burkard Manufacturing Company a personal volumetric sampler (Burkard Manufacturing Co. Ltd. Rickmansworth, Hertfordshire, UK) was modified to sample air particulates directly into microtiter wells (Figure 1a). The microtiter immunospore trap (MTIST) is operated by a standard Burkard turbine suction unit and air is drawn through the system at a constant rate of 57 litres of air per minute (Figure 1b). The volume of air can be increased or decreased depending on the requirements of the test. Particulates in the airstream are channelled through delivery trumpet nozzles and directed across the base of each collection well of 4 microtiter well strips (4 by 8 wells). After which the microtiter strips are removed and the contents of which may be immunoquantified using specific antisera by PTA- ELISA (Figure 1c). Tests were carried out to determine the collection characteristics of the MTIST for ascosporic inoculum of M. brassicicola. Using a TMS inverted NIKON binocular microscope at a magnification of x 200 impacted ascospores were viewed by bright field microscopy.

Figure 1. (A,B)The microtiter immunospore trap (MTIST) and (C) immunoassay test for detection and immunoquantification of trapped spores. Results Ascospores were distributed evenly throughout the base of each microtiter well and, as the ascospore concentration increased, the spores remained spatially separated. Employing a single factor analysis of variance it was determined that, with the exception of wells 1 and 8, there was significant variation in the mean percent ascospore distribution within a microtiter strip; greater numbers of ascopores were collected in the inner four wells of each microtiter strip (Kennedy et al., 2000). However there was no significant difference in the collection of M. brassicicola between the strips (Figure 2).

Air particulates are impacted onto the base of each microtiter well

Air movement througheach microtiter well

microtiter well

Target fungal spore

Specific antibody

Anti-species antibodybiotin complex

Streptavidin / biotinamplification system

A B C

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Figure 2. Mean percentage of M. brassicicola ascospore distribution across each of the 4x8 well microtiter strips Conclusions The MTIST spore trap device impacted ascospores of M. brassicicola in the base of microtiter wells. At high concentrations (>1,500 ascospores per microtiter well) ascospores remained spatially separated avoiding important clumping / masking of spores which could have affected the accuracy of the immunoquantification of spore numbers. In addition, the difference in collection of ascospores of M. brassicicola within a microtiter strip (Figure 2) is of importance indicating that for each target pathogen one complete strip can be used. With four microtiter strips the MTIST device could be used to assess the presence or absence and quantity of several target air spores. However this would depend on the availability of suitable antibody probes. 3.2.2 Investigate the potential of new spore trapping system for detection and immunoquantification of

ascosporic inoculum of M. brassicicola in controlled environment Materials and Methods Ten sporulating cultures of M. brassicicola (Kennedy et al., 1999) were place in a controlled environment cabinet (Sanyo Gallenkamp SGC70/C/RO-HFL) which was operating at 94% relative humidity under continuous light with intermittent wetting for 0.3min per 60 min period. Using the MTIST spore trapping device discharged ascospores were collected in 4 x 8 well microtiter strips during 3,4, and 12 hr trapping periods. A Burkard 7-day volumetric trap was also used with ascospores immunoquantified by immunofluorescence. Following each collection period microtiter strips were removed and stored at -20C before enumeration of spore numbers in each of the exposed microtiter wells. This was determined using a Nikon model TMS inverted binocular microscope. The MTIST trapped ascospores were immunoquantified by ELISA (employing Mab EMA 187) using a DAKO duet amplification system. Experiments were repeated under controlled conditions using Alternaria spp. without comparison to plant infection. Results Observations on the exposed microtiter wells of the MITST confirmed that impacted ascospores were distributed throughout the base of each microtiter well however the greatest numbers occurred in the central region of the well. A correlation of r2 = 0.8886 was determined between the number of ascospores per well and the absorbance figures recorded in PTA ELISA (Figure 3). Employing a polyclonal antiserum (raised to M. brassicicola and coded 96/10/3) a correlation of r2 > 0.943 has been observed between no. MTIST trapped ascospores and B7 day IF trapped ascospores of M. brassicicola. This relationship was unnafected in a mixed spore population of Erysiphe cruciferarum (Figure 4). Employing MAb EMA 212 a close correlation was observed between increasing numbers of trapped viable conidia of Alternaria and corresponding PTA ELISA absorbance values (r2 = 0.991) (Figure 5). An association was also observed between the number of MTIST trapped ascospores of M. brassicicola per liter of air sampled either as visual count which

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represents the conventional method of measurement or the absorbance value and the total number of of ringspot lesions that developed on exposed B. oleracea plants under controlled environment conditions (Kennedy et al., 2000) (Figure 6).

Figure 3. Immunoquantification of MTIST trapped ascospores of M. brassicicola in a controlled- environment cabinet, as determined with MAb EMA 187

Figure 4. Relationship of MTIST trapped ascospores of M. brassicicola and conidia of Erysiphe cruciferarum in a PTA- ELISA analysis, as determined with PAb 96/10/3.

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Figure 5. Immunoquantification of MTIST trapped viable conidia of A. brassicae as determined using MAb EMA 212 by PTA ELISA

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200

250

300

350

400

450

0.0 0.2 0.4 0.6 0.8

ELISA Absorbance 450nm

Mea

n no

. MTI

ST tr

appe

d ge

rmin

ated

con

idia

of A

. bra

ssic

ae p

er m

icro

titer

wel

l

MTIST trapped ascospores

Ringspot lesions

12 hr4 hr

4 hr

0

1

2

3

4

5

6

0

1400

700

No.

MTI

ST tr

appe

d as

cosp

ores

of

M. b

rass

icic

ola

/ Li

treai

r sam

pled

Tota

l no.

ring

spot

lesi

ons

on 1

0 B

rass

ica

oler

acea

pla

nts

Sampling period (hr)

Figure 6. Number of MTIST trapped ascospores of M. brassicicola per Litre air sampled in a mixied fungal population and, the total number of ringspot lesions on 10 exposed Brassica oleracea c.v. Golfer seedlings.

Project title



FV

12

Conclusions The results obtained demonstrate a clear correlation between the number of ascospores / conidia trapped by the MTIST spore trapping device and the absorbance values generated by ELISA. The amount of ringspot disease, which appeared on exposed trap plants placed inside the controlled environment cabinet, and the number of MTIST device-trapped ascospores of M. brassicicola also demonstrate a clear relationship between PTA ELISA values and lesion numbers on plants. The device can be used to determine the potential risk of infection at specific locations. Mixed spore population studies suggest that there is likely to be little interaction between large and small propagules in the trap. This is important if the accuracy of the system was not to be compromised. 3.2.3 Comparison of MTIST with the modified Burkard 7 day immunofluorescence test for collection of M.

brassicicola Materials and Methods Sporulating plate cultures of M. brassicicola were placed in a controlled environment cabinet which was operating as described in section 2.2. Employing an MTIST spore trap discharged ascosporic inoculum was collected over 30 min, 1,2,3,4,6,12, and 36 hr sampling periods. The total number of ascospores in each well of each microstrip were counted using an inverted Nikon microscope. To determine the collection efficiency of the MTIST in relation to conventional spore trapping systems a Burkard 7 day volumetric spore trap (Burkard 7 day volumetric spore traps are used routinely in epidemiological studies of airborne fungal diseases) was positioned adjacent to the MTIST. The B-7 day spore trap operated at a sample flow rate of 10 L air per minute. Following enumeration spores in wells were immunoquantified as described above. Ascospores trapped on the Melinex spore tape of the B-7 day spore trap were immunolabelled with MAb EMA 187 and an anti-species fluorosecein conjugate (Kennedy et al., 1999) and counted by using a fluorescence microscope. Results A correlation of r2 = 0.968 was determined between the number of ascospores per well or conidia of Alternaria spp. per well and the absorbance figures derived by PTA-ELISA. Ascospores trapped using the the B-7day spore trap were identified on the treated Melinex tape by immunofluorescence of the ascospore wall (Figure 7a). Only viable conidia of Alternaria brassicae were identified with MAb EMA 212 with antisera binding solely to an epitope present at the point of germination (Figure 7b). The relative collection efficiency for the MTIST in controlled plant-growth chambers with no airflow was 1.7 times greater than that of the B-7day spore trap.

Figure 7a. Immunofluorescence of ascospore of M. brassicicola employing MAb EMA 187

Project title



FV

13

UV microscopy bright field microscopy Figure 7b. Immunofluorescence of germinated conidium of Alternaria brassicae employing MAb EMA 212 as viewed by UV and bright field microscopy. Conclusions The correlation between the B-7 Day test and the PTA ELISA indicates a useful bench mark for assessing the efficiency of other more rapid systems. However the results can be obtained by PTA-ELISA in a few hours. The B-7day test required 1 week to enable the collection of the same amount of information and a further week to process. 3.2.4 Selective detection of ascosporic inoculum of M. brassicicola and Alternaria in the same sample. Materials and Methods Ascosporic inoculum of M. brassicicola was produced and collected using the MTIST spore trap, as described in Section 2.2. Microtiter wells were examined by light microscopy and ascospore numbers determined. After which the microtiter strips were stored at -20C prior to PTA ELISA. From a 1 week old culture of A. brassicae, which had been growing on Potato Dextrose Agar at 25C, a 5 cm cube of actively growing mycelium was removed and disrupted in 5ml of sterile distilled water. A 0.8 ml suspension of the disrupted Alternaria material was transferred and mixed with 5ml of sterile V8 juice in a 9cm Petri dish. Following incubation at 25C for 7 days the conidia produced were collected by adding 5ml of distilled water to the plate and agitating the surface of the culture with a sterile glass rod. This was repeated for A. brassicicola and the collected material of both Alternaria species was mixed and aliquoted at 100l per well to microtiter strips and incubated overnight at 18C to promote germination. Unbound material was removed and the plates air-dried before storage at -20C. Leaf material was collected in Lincolnshire from a commercial brassica crop which was exhibiting ringspot and dark leaf spot symptoms. The collected leaf material was air-dried before exposure to intermittent wetting and drying conditions in a controlled environment as described above. A 7-day Burkard volumetric spore trap adapted to take glass slides was operated throughout the test with airborne inoculum impacted directly onto a glass slide. Following exposure the glass slide was removed and processed by immunofluorescence employing both MAb EMA 187 (produced to M. brassicicola)and MAb EMA212 (produced to Alternaria). To differentiate between the two antisera a fluoroscein and a rhodamine red conjugate were used within the IF assay format. A field modified MTIST spore trap was also operated throughout. However exposed microtiter strips (4 by 8 wells) included one pre-coated germinated conidial microtiter strip (prepared as described above) and a separate microtiter strip of impacted ascospores of M. brassicicola (see above). The two remaining strips had not been pre-exposed prior to sampling. Following exposure the microtiter strips were removed and processed by PTA ELISA employing both MAb EMA 187 and MAb 6C7 (Section 3.2.1).

Project title



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14

Results Using two monoclonal antibodies (Mab EMA 187 and EMA 6C7) it was possible to selectively detect and differentiate between M. brassicicola and Alternaria brassicae in a mixed sample employing immunofluorescence and PTA ELISA (Figure 8). In immunofluorescence studies (IF) ascospores were identified by a red fluorescence (rhodamine fluorchrome) of the ascospore wall however germinating conidia of Alternaria brassicae were identified by an area of green fluorescence (fluoroscein fluorochrome) around the conidium and germ tube. Employing bright field microscopy many other spore types were present on the exposed glass slides. However with the exception of MAb EMA 212, where a very low level of recognition was observed, no cross-reactivity was observed with the other spore types present . Employing PTA ELISA the absorbance values generated confirmed those results observed using IF with each monoclonal antiserum binding only to its homologous spore type only (Figure 8A, Mycosphaerella brassicicola; Figure 8B, Alternaria).

Figure 8. Selective detection of M. brassicicola (A) and Alternaria (B) in a mixed spore sample employing monoclonal antisera in PTA-ELISA Conclusions Both Mabs (EMA 187 and EMA212) proved useful in the detection and discrimination of target fungal spores in a mixed sample. Both MAbs can be employed within existing spore trapping technology using immunofluorescence. However employing the new microtiter immunospore trap (MTIST) enables the end-user to rapidly multitest within the same sample for two target pathogens. The ability to detect and quantify airborne inoculum of M. brassicicola both rapidly and reliably will proved useful both in research programmes and within disease forecasting systems. Studies have also demonstrated that the MTIST spore trap has proved useful in the collection and retention of Alternaria conidia. However germination of the conidia is a pre-requisite of this. Nevertheless this may enable quantitative measurements of viable inoculum to be made which can be used to forecast onset of disease in vegetable brassica crops.

A - Alternaria pre-coated microtitre strips MTIST exposed to sporulating Ringspot (M. brassicicola) and Dark Leaf Spot (Alternaria) infected plant materialB - M. brassicicola pre-coated microtitre strips MTIST exposed to sporulating Ringspot and Dark Leaf Spot infected plant materialC - Alternaria pre-coated microtiter strips D - M. brassicicola pre-coated microtiter stripsE - No spores

0100200300400500600700800900

1000

A B C D E0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

No M. brassicicola ascospores

Mab EMA 187

0102030405060708090

100

A B C D E00.10.20.30.4

0.50.60.70.80.9

No. Alternaria conidia per microtitre well

Mab 6C7

PTA-

ELIS

A (A

bsor

banc

e 45

0nm

)

No

Alte

rnar

ia c

onid

ia p

er

mic

rotit

re w

ell

No

M. b

rass

icic

ola

coni

dia

per

mic

rotit

re w

ell

A B

Project title



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15

3.3 Determine postional and environmental factors which affect quantitative measurements of spore transmission (Objective 3) 3.3.1 Adaptation of MTIST for field usage Materials and Methods Using the MTIST and the Burkard 7-day volumetric spore trap (B-7 day), field trials were established at HRI to monitor ringspot disease inoculum from a point source. Factors which effect spatial and temporal numbers of spores caught were investigated (Table 3). However earlier studies were carried in controlled environments where little or no air-flow was observed(Figure 9a). For field usage with variable wind speeds the sampling efficiency was improved by adding a 90 inlet (Figure 9b) mounted with a wind vane ensuring that the MTIST samples in the direction of the wind at all times. Periods of heavy rainfall resulted in water droplets entering the delivery trumpet wells of the MTIST preventing airflow. Extension of the 90 inlet adaptation corrected this problem. Table 3. Factors which were investigated which effect the spatial and temporal spore numbers caught Environmental Factor

Trap Position

Spore Release

Vertical Sampling height

X

NA

Windspeed

X

NA

Humidity

NA

X

Wetness

NA

X

Light

NA

X

Rainfall

X

X

Temperature

NA

X

NA - Not applicable

Figure 9. MTIST spore trap without 90 inlet (A) and with attached inlet (B)

A B

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16

3.3.2 Comparison of MTIST with conventional spore trapping technology under a range of environmental

conditions in controlled environments Materials and Methods The collection efficiency of the modified field spore trap was tested using four different spore types at variable windspeeds. The collection efficiency of the MTIST for each spore type was compared with the Burkard 7-day volumetric spore trap. Studies to determine the trapping efficiency of the modified MTIST spore trap were carried out in the wind tunnel at IACR-Rothamsted. The four fungal spore types used were Lycopodium clavatum (>40m diamater), Erysiphe crucifierarum (30x15m), Botrytis cinerea (18x12m) and Penicillium roqueforti (4-6m diameter). The MTIST and three B-7day suction traps were placed 3m downwind of the spore source. Three different spore sources were used. Source A consisted of a frame (43 x 21cm), centred in the wind tunnel at right angles to the air-flow, on which wicks containing sporulating cultures of C. cladosporioides, B. cinerea, P. roqueforti and Brussels sprout florets infected with E. cruciferarum were suspended. Spores were released into the tunnel by wind action. Spores source B was a mesh cylinder, 60cm long and 30cm in diameter orientated so that its axis was at right angles to the air flow and placed in the centre of the tunnel with its axis 30cm above the floor of the tunnel. The cylinder was connected to an electric motor that could be rotated at 5 rpm. Wicks and Brussel sprout florets were place in the cylinder and the cylinder was rotated during experiments. Spores were released into the tunnel by the tumbling action of the wicks and florets. The third source, C, was used to release dry L. clavatum spores into the tunnel. Spores were placed in five boiling tubes placed to the right, left, above, below and in the centre of the frame. L. clavatum spores were released into the tunnel by passing air through the boiling tubes. Source C was used in conjunction with either source A or source B. The three sources released spores from an area larger than the sampling area of the MTIST sampler. Experiments were carried out at wind speeds of 1, 2 and 3.5 ms-1 with the numbers of spores of different types collected by the MTIST and compared with the numbers collected by the B-7day spore traps. For each trapping period the spore traps were activated simultaneously to the release of the five different types of spores. The traps were operated for different periods of time to vary the numbers of spores collected and at different windspeeds. After each test period the wind tunnel fan and the traps were simultaneously switched off when the wind speed fell below about 10cm s-1. Following each trapping period the number and type of spores collected by each trap type was determined by bright field microscopy. A log10+1transformation was carried out prior to statistical analysis. Results There was a significant difference (p

Project title



FV

17

Figure 10. Relationship of trapped spores per cubic metre of air sampled at windspeed of 2 ms-1 employing the MTIST and B-7day spore trap 3.3.3 Key environmental parameters of release and transmission of M. brassicicola Materials and Methods Experiments were established to monitor environmental factors required for transmission of M. brassicicola over a range of environmental conditions. Humidity, temperature, light and wetness were monitored throughout the experiment. To determine ascospore release a B-7 day spore trap was positioned adjacent to collected leaf material which exhibited severe ringspot infection. Prior to exposure the leaf material had been dried as described in Section 2.4 Results M. brassicicola ascospore release and transmission was related to a number of key environmental parameters. Intermittent periods of wetness, humidity at of above 80% (Figure 11a,b) and a light intensity at or above 0.03 kwm2 are required (Figure c). Ascospore release is inhibited when temperature falls below 1C. Optimal release was observed at a temperature of 17C. During periods of rainfall ascospore release was reduced (Figure 11c). Conclusions Information derived has enabled optimisation of the MTIST spore trap for collection of field inoculum of M. brassicicola ascospores. Employing a Delta T data logger (Delta T Devices Ltd) the MTIST can be activated when a light intensity of >0.003kwm2 is observed together with a relative humidity (r.h.) of > 80 %. The MTIST is deactivated when one or both of these environmental parameters are not fulfilled.

Transformation (Log 10+1) of no. MTIST trapped sporesper m3 air sampled

0

2

3

4

0 1 3 4

1

5

2 5

Tran

sfor

mat

ion

(Log

10+1

) of n

o B-

7day

trapp

ed s

pore

s pe

r m3 a

ir sa

mpl

ed

LycopodiumErysiphe

BotrytisPenicillium

Project title



FV

18

Figure 11. Key environmental parameters of release and transmission of M. brassicicola; Relative humidity (A), Light intensity (B) and rainfall/wetness (C).

No

B-7

day

IF tr

appe

das

cosp

ores

0

5

10

15

20

25

30

35

75

80

85

90

95

100

Ascospores

relative humidity

Rel

ativ

e H

umid

ity (%

)

A15

:00

21:0

0

3:00

9:00

15:0

0

21:0

0

3:00

9:00

15:0

0

21:0

0

3:00

9:00

Diurnal periodicity

0

5

10

15

20

25

30

35

00.050.10.150.20.250.30.350.40.45

Light intensity

Ligh

t int

ensi

ty k

w/m

2B

15:0

0

21:0

0

3:00

9:00

15:0

0

21:0

0

3:00

9:00

15:0

0

21:0

0

3:00

9:00

00.5

11.5

22.5

33.5

44.5

0

5

10

15

20

25

30

35

Rainfall

Rai

nfal

l (m

m)

C

15:0

0

21:0

0

3:00

9:00

15:0

0

21:0

0

3:00

9:00

15:0

0

21:0

0

3:00

9:00

Project title



FV

19

3.3.4 Monitoring vertical variation of disease transmission of ringspot ascospores from a point source. Materials and Methods Three field-modified MTIST spore traps were positioned within a 5m x 5m ringspot inoculated Brussels sprout crop (Brassica oleracea) cv. Golfer. The MTIST traps were positioned at a sampling height of 8 cm, 1m 30 cm , and 2m 20cm from the ground. Operation of the spore traps was limited to periods optimal for release of ascosporic inoculum of the ringspot pathogen. A Delta T data logger (Delta T Devices) was used to activate the MTIST during these time periods when a r.h. of 80 % or greater was recorded and a light intensity of 0.03 kw/m2 was observed. Following 24hr field exposure periods 4 x 8 well microtiter of each of the MTIST spore traps were removed and stored at 20 C until they were examined by PTA-ELISA. Two microtiter strips were selected from each exposure. To each well of each microtiter strip, 200l of 1% Casein buffer was added and following an incubation period of 30 min at 37C unbound casein blocking material was removed. Wells were washed twice with 200l PBSTinc Tw 0.1% after which 100l MAb EMA 187 (1:80 PBSTinc) or 100 l per well of PBSTincTW 0.1% Casein alone was added to wells of each microtiter strip. All further stages of the PTA-ELISA process were as described by Kennedy et al., 2000. Following the ELISA process ascospore numbers were determined using a Nikon model TMS inverted binocular microscope. Enumeration of M. brassicicola ascospores was determined following the ELISA process. To act as reference traps three B-7day volumetric spore traps were positioned within the ringspot inoculated Brussels sprout crop adjacent to and at the same height as the three MTIST spore traps. Following 7 day exposure periods the Melinex tape of each Burkard spore trap was removed and processed by immunofluorescence employing MAb EMA 187 (Wakeham et al., 2000). Ascospores trapped using the B-7day spore traps were identified on the treated Melinex tape by immunofluorescence. Results Fitting a polynomial curve a relationship of 0.948 was derived from PTA ELISA absorbance values and number MTIST trapped ascospores of M. brassicicola. Vertical variation of spore numbers was observed for both the B-7day spore trap and the MTIST spore trap (Figure 12).

Figure 12. Vertical gradient of M. brassicicola inoculum form a point source Conclusions Monitoring inoculum over a vertical gradient of 2m suggests that in a small experimental plot a significant population of ringspot inoculum will remain within the crop canopy. To determine disease transmission potential monitoring at a vertical distance of >1m would be appropriate. This could need be adjusted if very high amounts of disease were present in the crop.

0200400600800

100012001400160018002000

Sampling distance from ground level (m)

00.10.20.30.40.50.60.70.80.9

MTI

ST P

TA E

LISA

A

bsor

banc

e (4

50nm

)

No. B-7 day IFascosporesMTIST PTA-ELISA

No.

B7d

ay IF

tra

pped

asc

ospo

res

0.08 1.3 2.0

Project title



FV

20

3.4 Identify key environmental factors which affect airborne transmission of M. brassicicola at different stages in disease development in unsprayed cauliflower crops (Objective 4) 3.4.1 Field detection of M. brassicicola ascospores employing new novel MTIST spore trapping system and conventional spore trapping technology Materials and Methods A B7 day volumetric spore trap and a field modified MTIST spore trap were positioned adjacent to each other and 6 m outside a 24m x 12m B. oleracea (Brussels sprouts) plot (c.v. Golfer). A 300 x 300 mm2 Tygan gauze sachet sectioned in to 9 compartments, with each compartment filled with 10g of air-dried M. brassicicola infected leaf material, was positioned a further 3 m outside of the plot. The base of the Tygan gauze sachet was positioned 100 mm above ground level. The B-7day spore trap was operated continuously over a 5 week period (Melinex tape changed at 7 day intervals). A Delta T data logger (Delta T Devices Ltd) was used to activate the MTIST when relative humidities (r.h.) of > 80 % were recorded. The MTIST stopped sampling when the r.h. fell below 80 %. During a 5 week sampling period ( Sept to October ) the microtiter strips in the MTIST (4 x 8 wells) were removed for processing after either 1, 2 or 3 days field exposure. Microtiter well strips were processed by PTA-ELISA (Wakeham et al., 1999) employing MAb EMA 187 and an anti-mouse IgG SEEKit (Harlan Sera Lab Ltd, U.K.). For each of these sampling periods, 4 seedlings of B. oleracea (Brussels sprouts c.v. Golfer, 3 true leaves) which had been grown in the absence of disease were positioned adjacent to the position of the two spore traps. Following each of the sampling periods the plants were removed and placed in an environment of 100 % humidity for 24 hrs. The plants were then removed dried and retained in a glasshouse, at a temperature of 12 - 14 C for 21 days. Plants were visually examined for expression of ringspot lesions and confirmatory isolations made on to sprout leaf decoction agar after this time period had elapsed (Kennedy et al., 1999) Results Field MTIST absorbance values as derived from the PTA-ELISA show a high level of correlation between the level of ringspot observed on exposed trap plants (r2=0.9947) (Figure 13a) and the numbers of ascospores trapped using the B-7day spore trap (r2=0.8613) (Figure 13b). On some days over 800 lesions were recorded on the trap plants. This was equivalent to an absorbance value of ~0.2.

Figure 13. Relationship of MTIST PTA-ELISA (A) ringspot lesions on exposed Brassica oleracea trap plants and (B) the number of B-7day field trapped IF ascospores of M. brassicicola. Conclusions Using a modified version of the MTIST spore trap results demonstrated that epidemiologically significant levels of M. brassicicola inoculum in the air can be detected both reliably and rapidly in the field. There was a highly

No.

B-7

day

fiel

d tra

pped

IF

asco

spor

es o

f M.

bras

sici

cola

0

50

100

150

200

250

300

350

0 0.05 0.1 0.15 0.2 0.25

PTA ELISA - Absorbance 450 nm

0

100

200

300

400

500

600

700

800

900

0 0.05 0.1 0.15 0.2 0.25

No.

ring

spot

lesi

ons

on e

xpos

ed tr

ap p

lant

s

A B

Project title



FV

21

significant relationship between the results of the ELISA and the number of ringspot lesions. The reasons for this difference are unclear however it may be possible to over estimate ascospore numbers using immunofluorescence. The difference might also reflect the trapping efficiencies of the systems. As the MTIST trap is sampling with a 1.7 times greater efficiency the results of this trap may be more accurate. 3.5 Identify key environmental factors, cropping patterns and disease levels in unsprayed crops influencing the transmission of ringspot and dark leaf spot under natural conditions (Objective 5) 3.5.1 Field experiments to determine the transmission of ringspot and dark leaf spot in Brussels sprout and

cauliflower crops. Materials and Methods Field experiments were established at 3 sites at HRI. The distance between each site was approximately 0.8 km and 1.6km. At each of two sites a 5m x 5m plot of Brussels sprouts (c.v. Golfer) and an overwintered cauliflower crop (c.v. Jerome) (5m x 5m) were planted. At the remaining site 5mx5 m plots of Brussels sprouts and overwintered cauliflower crop were sown 5m distance from an established ringspot inoculated 5m x 5m Brussels sprout plot (Figure 14). No other ringspot crops were present in the locality during the experiment.

Figure 14. Position of crops in relation to ringspot inoculated source plot Ringspot disease transmission at each site was recorded using both conventional spore trapping equipment (B-7day) and the new MTIST spore-trapping device. The B-7day spore traps operated continuously however the MTIST spore traps were activated only during periods favourable for M. brassicicola ascospore release (r.h. of 80 % or greater was recorded and a light intensity of 0.3 w/m2 was observed) Information on disease incidence was recorded using exposed Brassica bait plants and by field assessments. Relative humidity, temperature, light, rainfall, windspeed and wind direction were

0.8 km

0.8km

Ringspot inoculated

Cauliflower Brussels sprout

5m

Project title



FV

22

monitored throughout. Disease development and progression at each of the three sites was monitored by visual examination of twelve Brussels sprout and cauliflower plants during the production season. Results A high incidence of disease inoculum was monitored at source plot with generally a good correlation observed between the B-7day and the MTIST spore trapping systems (Figure 15). However for one sampling period the B-7day failed to detect ascospores of M. brassicicola. For this period a high level of disease was observed on exposed bait plants (>550 lesions on 5 bait plants) confirming the result of the MTIST PTA ELISA. At an additional sampling period both the MTIST and the B-7day failed to detect ascosporic inoculum of M. brassicicola at source where > 303 lesions were identified on 5 bait plants. However during this period an exceptional level of rainfall (25mm) over a short period of time compromising the trapping efficiency of each trap.

Figure 15. Relationship between the B-7day spore trap and MTIST PTA-ELISA in the collection of airborne inoculum of M. brassicicola. Of the 5 bait plants exposed at each site and with the exception of exposure periods 17th through to the 19th October, where a high level of disease was observed both at the 5m and ringspot inoculated source plot, high level disease incidence was localised to that of the source plot. Nevertheless long range transmission was observed towards the end of October (21-22nd and 27th-29th) at each of the experimental sites (5m, 0.6km and 1.6km) with a significant level of ringspot disease identified on exposed bait plants (Figure 16a). For each of these two periods a prevailing northerly wind was observed (non-inoculated plots were situated North of the ringspot inoculated plot) whereas on all other occasions the prevailing wind was south, south westerly or south east. The MTIST spore trap proved sensitive in monitoring low levels of M. brassicicola inoculum over considerable distances (Figure 16b). Conversely the B-7day spore traps at experimental sites 0.8 and 1.6km from the ringspot source plot failed to detect low inoculum levels of M. brassicicola even though ringspot was identified on exposed trap plants (Figures 16a and c). At the beginning of the trial ringspot was not detected at any of the three non-inoculated sites. However as the trial progressed a significant level of disease was observed on the Brussels sprout plot adjacent to the ringspot inoculated plot (Figure 17). However only a low level of disease was observed on the overwintering cauliflowers at this site. A low level of disease incidence was observed at sites 2 and 3 with no disease present on overwintering cauliflowers at 1.6km from source.

0

50

100

150

200

250

0 0.02 0.04 0.06 0.08

PTA ELISA (Absorbance 450 nm)

No

B-7d

ay IF

asc

ospo

res

Project title



FV

23

Figure 16. Disease transmission studies of M. brassicicola in 2000: (a) Relationship between disease expression of 5 bait plants and MTIST PTA-ELISA at source; (b) Relationship between MTIST at source and MTIST positioned at plot 1.6km from source; (c) M. brassicicola inoculum levels as detected by B-7day spore traps at source, 5m, 0.8 and 1.6km.

At 5m from source

0.8km from source

1.6 km from source

At source

15-16th October

19-20th October

25-26th October

2-3rd November

10-11th November020406080

100120140160180

> 200

No.

ring

spot

lesi

ons

on

5 ba

it pl

ants

A

MTIST PTA ELISA at sourceMTIST PTA ELISA 1.6km from source

19-20th October

00.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

1-16th October

25-26th October

2-3rd November

10-11th November

MTI

ST

PTA

ELI

SA

(Abs

orba

nce

450

nm)

B

15-16th October19-20th October

25-26th October2-3rd November

10-11th November010203040506070

> 80

No.

B-7

day

trapp

ed a

scos

pore

s of

M. b

rass

icic

ola

C

Project title



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Figure 17. Ringspot disease on plants within plots situated at 5m, 0.8km and 1.6km from the source of infection. Conclusion The results show that there was a good relationship in the field between the numbers of ringspot ascospores as detected by ELISA or immunofluorescence. However there were days when weather conditions compromised the efficiency of the spore traps (e.g. heavy rainfall). Despite this large numbers of ascospores could be detected at source and up to 1.6 km away. Given the size of the infected plot 5x5m the results have great epidemiological significance for the control of ringspot and other diseases in the field. Overwintered unsprayed cauliflower plots with heavy levels of infection are common in field vegetable production areas. These can on average be approximately 10 to 20 hectares in size and must represent considerable sources of inoculum for plantings carried out during the new season (May onwards). In addition it is also clear that summer-grown cauliflowers and broccoli which are unsprayed also act as considerable sources of disease for overwintered cauliflower and late season sprouts. Any determination of disease risk should incorporate measures of inoculum as a means of designating the need for fungicide application. In this way unnecessary fungicide applications may be avoided. 3.6 Evaluate incorporation of the knowledge gained for prediction of disease risk in different crops/areas. Determine disease risk to freshly transplanted crops (Objective 6) 3.6.1 Spore trapping in areas of differing spatial patterns of Brassica crops Materials and Methods To determine disease risk to freshly transplanted Brassica crops airborne inoculum of M. brassicicola and Alternaria brassicae/brassicicola was monitored at two commercial sites in Lincolnshire (Figure 18). These sites were located in Brussels sprout crops in Skegness (by kind permission of TA Smith & Co, Wainfleet, Skegness) and in Broccoli and cabbage crops at Frieston Shore (by kind permission of Old Leake Growers, Old Leake, Boston). The crops were uninfected by ringspot or dark leaf spot at the start of the monitoring period. Transplants were disease free at time of transplanting and the crop was thoroughly inspected for evidence of disease before the trial commenced at both sites. At each site both conventional (Burkard 7 day volumetric spore trap) and new spore trapping technology (MTIST spore trap) were used . At each trial site two B-7day traps operated continuously whilst the MTIST spore traps were activated only when a relative humidity of 80 % or greater was recorded and a light intensity of 0.03 Kw/m2 observed (as stated previously see Section 2). The microtiter wells in the MTIST traps were changed at either three day or one week intervals to determine the optimal trapping period for use of the ELISA. The spore trapping equipment was operated throughout the lifetime of the field crop and the power supply was maintained by using a Rutland wind charger which maintained a power supply of 12 volts to all batteries used to drive to spore traps. Employing a Skye data hog 2 weather station recorded environmental data was incorporated within BrassicaSpot (ringspot /dark leaf spot disease forecasting system) and in conjunction with information derived on actual spore load (spore trapping study) a prediction of actual disease risk was ascertained. At each site the grower was responsible for applying spray application as it was not possible to maintain an unsprayed site in these trials due to crop risk. Disease incidence was monitored on both unsprayed and sprayed plants at

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each site. The Frieston Shore trapping site was situated 1.25 km from the East coast however the Skegness trapping site was located 4.5 km inland. The distance between the two sites was approximately 27 km.

Figure 18. Trapping locations for ringspot and dark leaf spot used in Lincolnshire in commercial crops of Brussels sprouts, broccoli and cabbage. Results Using conventional spore trapping equipment (Burkard 7day volumetric) results (Figures 19,20) show that in uninfected crops the initial entry of ringspot and dark leaf spot disease could be detected. At the Skegness trial site primary inoculum of both diseases were detected on the 6 July 2001 and lesions observed in this crop on the 14 July 2001. At Frieston Shore ringspot conidia of Alternaria brassice were detected on the 29th June and ascospores of Mycosphaerella brassicicola detected on the 30th June 2001. Disease was first identified in the crop on the 7 July 2001. Further peaks in disease inoculum levels corresponded well with increases in disease levels in both crops at both sites. Fungicide sprays were applied in response to the Brassicaspot model. Fungicide sprays were applied on the 9 and 29 August 2001 at Skegness (further sprays were applied 21 September and 8 October 2001 at Skegness). At Frieston shore fungicide sprays were applied on the 10 July, 23 August, 22 September and 5 October 2001. With increasing fungicide usage there was a poor relationship between airborne spore numbers and disease level in the crop.

Figure 19. Ascospore numbers as detected by immunofluorescence at commercial trial sites in Lincolnshire in 2001.

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Figure 20. Conidial numbers of Alternaria brassicae (dark leaf spot) as detected by bright field microscopy at commercial trial sites in Lincolnshire in 2001. There was a close relationship between ascospore numbers of M. brassicicola as detected by IF and the results of the ELISA using the MTIST at Skegness (Figure 21). A linear relationship (r2 = 0.9054) was observed between the two measures of airborne inoculum. A similar relationship was observed at Frieston Shore (data not presented). However later in the season (August and September) at both trapping sites this relationship was not close due to increasing cross reactivity and higher signals observed in the MTIST ELISA in the field on some occasions. For the dark leaf spot pathogen a correlation of r 2 = 0.8991 (polynomisl) was observed between total predicted weekly inoculum levels (MTIST ELISA) and conidial numbers as determined using a B-7day spore trap.

Figure 21. Relationship between the number of ascospores of M. brassicicola as detected by immunofluorescence and MTIST PTA -ELISA.

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Conclusions The close relationship between immunofluorescence test and the MTIST ELISA test indicates that ELISA test could be used to determine the presence or absence of both the dark leaf spot and ringspot pathogen in the field and quantify inoculum levels. If this was optimised within a lateral flow assay this means that in field tests for inoculum detection of these pathogens could be carried out quickly and simply and the information used with crop protection programmes to eliminate over spraying of fungicides. This would be particularly useful early in the season as a method of preventing disease transfer between over wintered crops and freshly transplanted crops. For Mycosphaerella brassicicola (ringspot) the relationship between conventional spore trapping and that of the ELISA was observed to breakdown later in the season but this was associated with spore germination within the microtiter wells. Methods are needed to reduce this source of error by coating the microtiter wells with agents which prevent spore germination (see Objective 7). 3.6.2 Incorporation of the prediction of disease risk with airborne spore counts Materials and Methods Airborne spore counts were compared with the output from the Brassicaspot infection models using the weather information collected at each site. Measurements of temperature, humidity, leaf surface wetness and rainfall were collected at 30 min intervals from crop transplanting using a SKYE Datahog II 4 channel logger. Leaf wetness sensors were placed within the crop. The data was downloaded automatically using a GSM link and automatically processed through the forecast model Results There was a close relationship between the disease predictions using the Brassicaspot infection models and the onset of disease within the crop at Skegness (Figure 22). Despite numerous infection periods there were only one critical infection period (7-8 July 2001) when there was sufficient ringspot inoculum present to initiate disease development. Had sprays been applied at these times by using information on inoculum (spore detection) and environment (models) it is unlikely that there would have been further disease development in the crop. However the initial inoculum in the crop could not be predicted and using infection risk alone would have resulted in mis-timing of fungicide applications.

Figure 22. Infection risk for Alternaria brassicae and Mycosphaerella brassicicola at Skegness as designated using Brassicaspot

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Conclusions The results clearly show that by incorporating information on inoculum with information on disease risk (using predictive models) it will be possible to designate strategies which can be used to reduce pesticide applications to one or two sprays. The critical date for applying fungicide applications to the crop can be identified. However it is unclear how this information can be applied to bigger cropping areas. One possible route might be to establish networks of traps (3 - 4 traps) which could be applied to cropping in defined areas. With high sampling rates these traps if positioned to reflect prevailing wind patterns could be used to designate to onset of disease risk in different areas and pinpoint specific transmission events affecting different crops within the area. This information could be used to drastically reduce the amounts of pesticide required to control leaf spots of vegetable brassicas. As tests for inoculum can be carried out in the field the system meets the criteria necessary for its uptake by the brassica industry. 3.7 Identify microtiter well coatings, which could be used to optimise spore catches of pathogenic and non- pathogenic field fungi and reduce interference in rapid assays by reducing spore germination. (Objective 7) 3.7.1 Investigation of assay formats which affect collection and retention of spores in spore samplers Materials and Methods Four types of well coating preparations were tested as follows: gelatin, bovine serum albumin (BSA) Poly-L-lysine and a 5:1 mixture of petroleum jelly (Vaseline) and paraffin wax. Gelatin and bovine serum albumin was prepared as a 2% solution in distilled water and the Poly-L-lysine was also dissolved in distilled water (0.1mg ml-1). The petroleum jelly and paraffin wax were melted in a water bath and mixed before being diluted with hexane until the mixture dissolved. This solution was further diluted by adding hexane in the proportions: 1:8, 1:16 and 1:32. These solutions, and a distilled water control were used to coat the interior of microtiter wells as described below. To each of four wells of a microtiter strip 100 l of gelatin coating solution was aliquoted. The remaining four wells of the microtiter strip each received 100 l of BSA coating solution. All remaining solutions were distributed in the same way to each of a further three microtiter strips and incubated at 20 C for 1 hour, after which any unbound material was removed. An inverted binocular microscope was used to check tha

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