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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

A SID 5A form must be completed where a project is paid on a monthly basis or against quarterly invoices. No SID 5A is required where payments are made at milestone points. When a SID 5A is required, no SID 5 form will be accepted without the accompanying SID 5A.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code HH3218 (CSA6138)

2. Project title

Disease management in ornamental & vegetable crops using low-cost protection systems incorporating photoselective claddings

3. Contractororganisation(s)

Department of Biological Sciences,Lancaster University,LA1 4YQ andStockbridge Technology CentreYO8 3TZ

54. Total Defra project costs £ 367,562

5. Project: start date................ 01 November 2002

end date................. 31 October 2005

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

Executive summary “Smart plastics” are horticultural cladding materials that alter the light spectrum reaching the

crop. This project focussed on the potential use of “smart plastics” for disease control in crops produced under protection in the UK, whether throughout growth or during an initial propagation phase prior to transplanting to the field. The model crop was lettuce, and the primary focus was on lettuce downy mildew caused by the fungus Bremia lactucae. Grey mould disease of lettuce caused by the fungus Botrytis cinerea was also investigated.

The project linked crop experiments with research in to the fundamental responses of the host plant and the pathogens to their light environment. The understanding of the basic biology of this system achieved by combining these approaches aimed to deliver a basis for predicting the effects of smart plastics under a range of conditions and locations.

The project compared a standard commercial plastic typical of those widely used in UK horticulture with three “smart plastics” that either (a) had increased transmission of ultraviolet light (= UV-transparent) so that almost all solar UV reached the crop, (b) had reduced transmission of UV (= UV-opaque) so that the crop received almost zero solar UV or (c) had modified transmission of visible light so that the crop environment was enriched in blue light compared with green and red (= blue).

The properties of “smart plastics” were stable over the course of the project, but it was notable that their effects was more wide ranging than their “headline” claims. Notably, the blue plastic was also highly opaque to UV light and achieved its blue shift by attenuating other wavelengths so much that light required for crop photosynthesis and growth was substantially reduced. The blue plastic also tended to reduce temperature and increase humidity. These broader effects of plastics are important in understanding their effects and the claims made by manufacturers.

The data obtained from this contract allows recommendations on the commercial use of “smart plastics” in the UK to be based on a much more solid foundation of understanding than existing previously. From a commercial perspective, all three smart plastics showed some potential to contribute to the control of both downy mildew and grey mould of lettuce. The most consistent disease control was delivered by the blue plastic but, as a result of its wider effects on the crop environment, it produced lettuce that were unmarketable, and it clearly cannot not be recommended for commercial production. UV-opaque and UV-transparent films

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gave significant control of grey mould and downy mildew in some experiments, but not others. While none of the three smart plastics studied here gave sufficiently consistent disease control

to be recommended for commercial use solely on that effect, UV-transparent and UV-opaque have other effects on crop yield and/or quality that may be valuable for UK production of a range of crops. For example UV-transparent improves colour and taste in lettuce, and improves the quality of seedlings propagated under protection. Commercial decisions on the use of these plastics should include effects on disease control as one of many possible effects.

These recommendations on the commercial use of the smart plastics are based on data obtained in this contract from both (a) crop studies and (b) model predictions derived from the improved understanding of the basis biology of the systems. There was good agreement between the model predictions and the results of the crop studies. The models produced allow predictions of the effects of “smart plastics” to be made across locations and seasons. One observation is that the effects of a specific plastic will vary substantially between the UK and other growing regions.

The models derived from the mechanistic studies conducted during this contract predicted that the effects of smart plastics on both B. lactucae and B. cinerea would vary with season and weather conditions, as was observed. A further prediction is that the UV-transparent film used here would give consistently good disease control in more Mediterranean climates where solar UV is higher than in the UK.

Lettuce exposed to UV light becomes more resistant to infection by B. lactucae, but there is no such effect with B. cinerea. However, even with B. lactucae, the effects of UV on host resistance make a smaller contribution to overall changes in crop disease that the direct effects of UV on the fungi.

The direct effects of UV on B. lactucae or B. cinerea can be divided into (a) damage and (b) the stimulation of spore production.

Both B. lactucae and B. cinerea were sensitive to damage by UV radiation, especially UV-B (280-315nm), but B. lactucae was more sensitive to longer wavelength UV-A (315-400nm) than B. cinerea. Spore germination in B. lactucae but not B. cinerea was also inhibited by high doses of blue light (400-500nm).

The definition of the contrasting responses of these two fungi to light of different wavelengths was an important observation from this contract, and also formed an important step in defining model to predict the effects of smart plastics on crop disease. Both fungi are predicted to suffer high spore mortality under the high UV conditions of summer, but to be relatively unaffected by sunlight during winter.

Dispersed spores of both B. lactucae and B. cinerea were significantly more vulnerable to UV damage that spores still attached to their parent colonies.

In B. cinerea UV damaged spores and inhibited mycelial growth at relatively high doses, but at low doses induced an increase in spore production. The dose response for the effects of UV on sporulation showed that induction would be saturated even by the low UV exposure that occurs in the UK during winter. Doses sufficient to inhibit germination would only occur in summer in the UK.

The mechanistic studies also showed that the effect of the blue plastic was due to its extremely low UV transmission, not its visible colour.

The contract also investigated the UV responses of (a) a range of micro-organisms living on the leaf surface (phylloplane) and (b) two agents (Trichoderma harzianum and Bacillus subtilis) that may act as biocontrol agents for crop diseases caused by fungi like B. lactucae and B. cinerea. Most leaf surface organisms were more resistant to UV than B. lactucae or B. cinerea, but the biocontrol agents were at least as sensitive as their potential targets.

Although the quantitative responses of the host plant and microbes studied in this contract are unlikely to be identical to those of other crops or pathogens, the basic principles defined here provide a solid basis for future research and for commercial assessments of the use of “smart plastics” in a range of crops within UK horticulture.

Project Report to Defra

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8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

1. INTRODUCTION AND SCIENTIFIC OBJECTIVESThis contract investigated the effectiveness of plastic cladding materials that modify the light spectrum reaching the crop (“smart plastics”) on disease in crops grown under protection either throughout production or during propagation. The primary focus was the effect on obligate downy mildew pathogens, and the necrotrophic pathogen Botrytis cinerea, using lettuce as a model system. The contrast was organised around seven objectives. Objective 1To quantify the effectiveness of low-cost protection systems using a range of wavelength selective plastics for the control of downy mildews in vegetables and ornamentals. Objective 2To define the broader effects of spectral filters on the incidence and severity of other diseases, primarily B. cinerea. Natural pest invasion were observed alongside various aspects of crop quality to enable an overall cost-benefit appraisal of spectral filters in the propagation of vegetable and ornamental crops.Objective 3To define the effects of photoselective filters on the microclimate within protected structures, with particular reference to the light environment. Objective 4To characterise the photobiological mechanisms, in both pathogen and host, that underlie the effects of altered light spectrum on downy mildews, using Bremia lactucae on lettuce as a model, B. cinerea and disease biocontrol agents and so ii) to make predictions of the effects of wavelength selective filters under a range of field conditions (different seasons, cloud etc.).Objective 5To study the impact of spectral filters on the establishment and survival of phylloplane micro-organisms and to investigate their effect on the application and successful use of selected bio-control agents in vegetable and ornamental crops.Objective 6To integrate mechanistic understanding of the mechanisms of light spectrum effects on downy mildews into a simple model to provide a mechanistic basis from which to interpret and ultimately predict responses at the crop scale. Objective 7To communicate the results of the project to the vegetable and ornamentals industry, including suppliers, consumer and environmental groups and other interested parties in the form of a workshop organised in the final year of the project.

2. THE EXTENT TO WHICH THE OBJECTIVES SET-OUT IN THE CONTRACT HAVE BEEN METDiscussions with DEFRA near the mid-point of this contract led to an agreement that the scope of the contract should be focussed more specifically on delivering a thorough understanding of fewer model crop diseases, rather than the wider range of downy mildews originally proposed. The modified objectives agreed in that revised contract have all been substantially met. The contract has certainly delivered its fundamental objectives of assessing the potential of plastics that modify the light spectrum reaching the crop for disease control under UK conditions, and doing so on the basis of understanding the basic photobiology of the underlying processes. The published literature existing prior to this contract contained, on the one hand, good agronomic research in to spectral modification and crop disease (e.g. 37-41), and on the other hand, equally good fundamental research in to the effects of light spectrum on components of crop-pathogen interactions (e.g. 7, 8, 11, 17, 22, 23, 34, 42, 48, 50, 55). However, in proposing this programme of research we argued that these two aspects of research had not been fully integrated and that this limited understanding of the use of spectral modification. This contract successfully delivered co-ordinated crop-scale and fundamental research. Of course, the research carried-out

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under the contract has highlighted areas that need further research to improve our understanding. For example, the quantification of the induction of sporulation in Bremia lactucae at very low UV doses could be improved (see Results). In addition, the complexity of the spectral responses of B. lactucae made producing predictive models for lettuce downy mildew more complex than initially expected, and this delayed the completion of the final report. Nonetheless, the contract has delivered a substantial advance in the integrated understanding of the mechanisms of the effects of light, especially UV wavelengths, on crop-disease interactions. This biological understanding was combined with a simple radiation transfer model of sunlight under different conditions, previously used largely in the context of understanding stratospheric ozone depletion. This integration produced simple models of the effects of spectral modification on two major crops diseases that broadly predict the responses observed in crops across season, etc. These integrated models could be used to predict the effect of any plastic with known spectral properties at any location and time of year, something that would be impossible had research been limited to crop-scale evaluations of specific plastics at certain sites. This allows clear recommendations to the industry on the potential, and limits, of “smart plastic” technology for disease control in commercial production in the UK.

3. MATERIALS AND METHODSThe contract was a collaboration between Lancaster University and Stockbridge Technology Centre (STC). Experiments related to Objectives 1-3 took place at STC using an array of 16 polytunnels arranged in a 4x4 Latin Square with four treatments (= four plastics) and four replicate tunnels per treatment. The four plastics were chosen to deliver contrasting spectral properties that might be expected to reduce crop disease. Within each tunnel, disease was monitored on 10-30 replicate plants or were the occurrence of infection across populations was measured, the replicates where trays holding 20-30 modules. For all statistical analyses, the mean of replicate measurements from each tunnel were calculated and these means used in analyses of variance with n=4. The tunnels were also used to assess crop microclimate, with temperature, relative humidity and the light environment monitored continuously using a data-logger with appropriate probes.

Experiments related to Objectives 4, 5 and 6 took place at Lancaster using a range of controlled environment facilities. Most have a well-established track-record of use with light effects on plant-disease interactions, and the techniques were well-established (see for example 13, 16, 28, 31, 36). Microbial responses to UV in vitro were studied using custom-built lamp arrays in a temperature controlled dark room held at 20 + 2C. The lamp systems also incorporated a cooling base-plate which held the temperature of the agar at 18 + 2C, and the base plate had sixteen defined locations, each capable of holding a 50mm Petri dish. By using combinations of plastic films and metal mesh filters which attenuated the UV without altering the spectrum, a series of treatments were provided simultaneously, and these were used to generate dose response relationships. All experiments also included foil-wrapped Petri dish as an internal dark control. For UV-B irradiation two Philips TL40/12–RS tubes were used. In these experiments seven doses (plus a dark control) from two filter treatments were provided simultaneously. Filters for UV-B tubes were either 0.13mm cellulose diacetate (Clarifoil, Courtaulds Ltd., Derby, UK), clear polyester (Lee filters, Andover, UK), or unfiltered. For UV-A irradiations four unfiltered Philips TLD-30/12 tubes (Starna Ltd, Romford, UK) were used, and white light and blue treatments were provided using standard cool-white fluorescent tubes, with or without a blue filter. Following irradiation, Petri dishes were sealed and foil wrapped, then incubated as per initial culturing procedures for each organism for a period of 24 hours. Spore germination was measured by examining each Petri dish surface under a light microscope at 20 times magnification.

In planta responses to UV were carried out in controlled environment rooms. Lactuca sativa cv. Rex RZ and Constance RZ (as at STC) were sown in 40 cell tray inserts with Levington M3 compost and were propagated under a light/dark regime of 16hr/8hr at 25 + 2°C and a PAR background of 500 µmol m-2 s-1 provided from 400W metal halide lamps (Osram HQI-BT 400W: Osram Ltd., St Helens, UK). Six days after initial sowing, when the first leaf had emerged, plants were exposed to experimental UV treatments, provided much as we have described before (32). Briefly, background UV-A was provided by the metal halide lamps plus six Q Panel UVA-340 tubes (Q-Panel Laboratory Products, Bolton, UK) filtered with clear polyester (Lee filters, Andover, UK). UV-B was provided by six UV-B tubes (Philips TL40/12–RS: Starna Ltd, Romford, UK) filtered with 0.13mm thick cellulose diacetate (Clarifoil, Courtaulds Ltd., Derby, UK). In dose response experiments the UV-B irradiance was manipulated by wrapping UV tubes with a white cotton fabric which altered irradiance but not the UV spectrum. Plants were harvested after 18 days total growth and leaf areas determined using a LI-COR LI-3000A area meter.

All treatments were quantified using a double scanning spectroradiometer (model SR991-v7 Macam Photometrics, Livingston, UK) and UV treatments were weighted using biological weightings function (BSWF- see below). Standard analyses of data were performed using SPSS v 11.5 and dose responses were fitted using non-linear regression in GraphPad Prism v4.02. Models of the effects of the various “smart plastics” in the field were based on the integration of experimental doses responses and a simplified radiation transfer model for solar ultraviolet radiation (1 as used before by Paul, 30).

4. RESULTS AND DISCUSSION 4.1. The effects of the “smart plastics” on disease in experimental crops (Objectives 1 and 2)

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4.1.a Lettuce downy mildew caused by Bremia lactucae (Objective 1)

Experiments investigating downy mildew infection of lettuce in the replicated tunnels clad with the four plastics were repeated between late summer 2003 and autumn 2005, using two different cultivars. While there was considerable variation in the effects of plastics between experiments, some underlying patterns are revealed when the whole dataset is analysed. Across 12 repeat experiments where downy mildew was assessed as the percentage of leaf area infected, the overall effect of both the UV-opaque and blue plastics was to significantly reduce infection (Figure 1a). The mean reduction compared with plants under the standard plastic was 25% for UV-opaque and 40% for blue. The UV-transparent film had no overall effect on downy mildew infection, but caused a significant reduction in one individual experiment (August 2004). There were some indications that any reduction in downy mildew under the UV-T film was greater in summer, but this was not clear (Figure 1b). However, the same seasonal variation in effect was apparent with UV-opaque and blue, with the most marked changes occurring between late spring and early autumn (Figure 1c,d). The blue plastic produced statistically significant reductions in downy mildew compared with standard plastic in six out of seven individual experiments conducted between April and October (six out of 12 in total). Significant reductions in downy mildew under the UV-opaque plastic occurred in three individual experiments (April, May and August). No plastic caused a significant increase in the percent of leaf area of lettuce infected by downy mildew in any individual experiment.

Figure 1 The effects of “smart plastics” on downy mildew of lettuce caused by Bremia lactucae in crop-scale experiment. (a) the overall effects of the four plastics across 12 repeat experiments between 2003 and 2005, each separate experiment is treated as a replicate in one-way ANOVA. Data are means + 1SEM, and those not sharing the same letter are significantly different at p=0.05. (b-d) Data for individual experiments for (b) UV-transparent, (c) UV-opaque and (d) blue films expressed relative to disease in the standard film, illustrating the range of response across season. The number of significant results obtained in individual experiments is also shown for each plastic.

4.1.b Lettuce grey mould caused by Botrytis cinerea (Objective 2)

Experiments investigating Botrytis cinerea infection of lettuce in the replicated tunnels clad with the four plastics were repeated between early spring and autumn 2005, using two different cultivars. As with B. lactucae, there was considerable variation in the effects of plastics between experiments, but also some underlying patterns that became apparent when the whole dataset is analysed. Across fifteen repeat experiments where B. cinerea infection was investigated the overall effect of all the “smart plastics” was to significantly reduce infection (Figure 2a). The mean reductions compared with plants under the standard plastic were 25% for both UV-opaque and UV-transparent, and 45% for the blue plastic. All three “smart plastics” caused significant reductions in at least two individual experiments (Figure 2b-d). The blue film was the most consistent in reducing B. cinerea infection (7 out of 15 experiments, while both UV-opaque and UV-transparent caused significant reductions in two experiments. UV-opaque was exceptional in causing significant increases in B. cinerea over the standard plastic in two individual experiments (Figure 2d). In general, the responses of B. cinerea infection to spectral modification appeared to be more variable than those of B. lactucae during the period when comparison is possible (April-October). Because experiments with B. cinerea were not conducted over the winter months it is not possible to assess any broader seasonal variation in the effects of smart plastics on disease caused by this pathogen.

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Figure 2 The effects of “smart plastics” on grey mould of lettuce caused by Botrytis cinerea in crop-scale experiment. (a) the overall effects of the four plastics across 12 repeat experiments between 2003 and 2005, each separate experiment is treated as a replicate in one-way ANOVA. Data are means + 1SEM, and those not sharing the same letter are significantly different at p=0.05. (b-d) Data for individual experiments for (b) UV-transparent, (c) UV-opaque and (d) blue films expressed relative to disease in the standard film, illustrating the range of response across season. The number of significant results obtained in individual experiments is also shown for each plastic.

4.1.c Other pests and pathogens (Objective 2)

In the initial 18 months of this contract the effects of “smart plastics” on downy mildew of brassicas (Peronospora parasitica, Hebe (Peronospora grisea) and pansy (Peronospora violae). As a result of (a) the increasingly evident complexity of responses to smart plastics and (b) the difficulty in establishing consistent and reliable experimental infections with these pathogens, it was agreed with Dr Robert Bradbourne that the contract should be revised to focus on lettuce downy mildew and grey mould. However, in the initial experiments, there were no clear or consistent effects of any plastic on the other downy mildews, beyond some hints of reduced disease on Hebe under the UV-transparent and UV-opaque films (data not presented).

In addition, the occurrence of other pests and pathogens on experimental crops was recorded throughout the contrast, and the effects of “smart plastics” quantified where the frequency and/or severity of attack was sufficient. In practice, there were no significant attacks of any disease other than those being specifically manipulated, and only naturally occurring pest problems with the lettuce crops were occasional attacks of aphids (three occasions), molluscs (once) and caterpillars (once). These were pest attacks were quantified and analysed, but there were no significant differences between the four plastics used (data not shown).

4.1.d. The effects of “smart plastics” on crop growth and yield.

Crop growth (leaf area, biomass etc.) and commercial yield (including elements such as loss to trimming etc.) were assessed in parallel with disease. While UV-opaque and UV-transparent films both produced crops that were more or less comparable to the standard film, it is notable that the blue film greatly reduced growth and consistently produced plants that were not commercially acceptable. This poor growth, attributable the reduction in photosynthetic radiation, makes this plastic unviable for commercial use. It is worth noting that in related research in to “smart plastics”, funded by HDC, the UV-T was shown to have substantial commercial benefits for specific lettuce crops since it delivered (a) improved colour and taste in cultivars such as “lollo rosso” and (b) improved quality in seedlings propagated under protection (see Section 5).

4.2. The effects of the “smart plastics” on the crop environment (Objective 3)

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4.2.a The effects of “smart plastics” on the light environment.

4.1.a.i Laboratory measurements of the spectral properties of the plastics

Spectral measurements made under laboratory conditions confirmed that the initial properties of the plastics were as specified by the manufacturers (Figure 3 and Table 1a). The properties of UV-transparent and UV-opaque are effectively summarised by their names, although it is notable that “UV-opaque” actually transmits a measurable fraction of UV-A (Figure and Table 1a). Neither of the UV-modifying plastics have any substantial effects on PAR or far red. By contrast, the blue film has a number of features that are worth comment. This plastic actually has a lower UV transmission than the UV-opaque film, i.e. the blue film is both blue and UV opaque. In addition, it achieves its blue shift by attenuating red light by around 70% (Table 1), so that its total PAR transmission (61%) is substantially lower than any other plastic used here. However, since the far red transmission of the blue film is even lower than that in the red, it causes a clear increase in R:FR ratio.

These inherent properties of the films were broadly stable over the three years of the project. All plastics showed a progressive reduction in the transmission of all wavelengths between approx 330 and 750nm as they aged. This change was small, in the order of 5% for all films, and appeared to be largely a function of the accumulation of dust, algae etc. At wavelengths shorter than 330nm the properties of the UV-modifying films were stable over three years, i.e. the UV-transparent continued to transmit 85-90% of solar UV-B while the UV-opaque transmitted less than 2%, even after three years. There was no measurable UV-B transmission through the blue film in samples taken at any stage in the experiments. By contrast, the standard film showed a progressive increase in its transmission of shorter UV wavelengths over time.

Figure 3. Spectral transmissions for the four plastics used in the field experiments. Data are for new films at the start of the project. Spectral transmissions were determined for triplicate samples (minimum 4cm2) using a 75W Xenon arc lamp (LOT Oriel, Leatherhead, UK) and 10 cm integrating, and a double scanning spectroradiometer (Macam Photometrics, Livingston, UK).

4.1.a.ii Measurements of light transmission within polythene tunnels clad with “smart plastics”Measurements made within the polythene tunnels confirm the contrasting transmission properties of the plastics observed the laboratory, and that the treatments being imposed on the crops are as planned (Table 1b). In general, broad-band transmissions measured in the field were similar to, or a little lower, than measured in laboratory, but some reduction was expected due to presence of the structure of the tunnels etc. Some transmission of UV-B (approx 1%) through UV-opaque and blue films was observed in the field. One other difference from laboratory measurements was that field data suggested a general small reduction in R:FR ratio in all but the blue film (where R:FR was increased, but less than expected from the laboratory measurements).

a) Laboratory Standard UV opaque Blue UV transparentUV-B 3% (9%) 0% (0%) 0% (0%) 84% (80%)UV-A 40% (37%) 8% (9%) 3% (1%) 94% (89%)PAR 93% (89%) 96% (91%) 61% (59%) 95% (90%)blue (400-500) 92% (90%) 93% (89%) 75% (69%) 94% (88%)red (600-700) 93% (88%) 96% (91%) 32% (30%) 91% (86%)

b) Polythene tunnels Standard UV opaque Blue UV transparentUV-B 7% (11%) 1% (0%) 1% (0%) 77% (75%)UV-A 42% (40%) 11% (11%) 3% (1%) 78% (75%)PAR 84% (79%) 82% (78%) 53% (51%) 83% (80%)blue (400-500) 80% (78%) 79% (75%) 70% (67%) 80% (78%red (600-700) 89% (85%) 89% (85%) 35% (31%) 87% (85%)Table 1. Measurements of transmission properties of the four plastics under study made a) under laboratory conditions (measured as described in Figure 3) and b) in the polythene tunnels at STC measured in situ using two

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double scanning spectroradiometers (Macam Photometrics, Livingston, UK) making simultaneous measurements of light spectrum in open sunlight and in a tunnel. Data are for new plastics and, in brackets, after 33 months use in the field, ____________________________________________________________________________________________________________________

4.2 The effects of “smart plastics” on temperature and humidity Temperature and relative humidity in the tunnels were recorded throughout the experiments. There were no marked differences in either temperature of relative humidity between tunnels clad with the standard, UV-opaque or UV-transparent plastics. However, the blue plastic consistently reduced maximum day temperature and increased minimum relative humidity. Taking the 2005 growing season as an example (Figure 4a,b), maximum temperature under the blue plastic averaged 3C lower than under the other plastics, and under bright conditions in summer was as much as 8C lower. The minimum relative humidity averaged 6% lower under the blue film than the other plastics.

Figure 4 Seasonal variation in (a) maximum daily temperature and (b) minimum daily relative humidity recorded in tunnels clad with four different plastics. Data are weekly means of data recorded at 5 minute intervals.

4.3. Experimental investigation of the photobiological mechanisms of the effects of light spectrum on downy mildews, using Bremia lactucae /lettuce as a model system, and Botrytis cinerea / grey mould (Objective 4)4.3.a The mechanisms of Bremia lactucae - lettuce responses to light spectrum

4.3.a.i The responses of dispersed sporangiospores of B. lactucae to the light environment

The dose responses for the germination of dispersed sporangiospores of B. lactucae determined across a range of different light spectra all fitted well to an inverse sigmoidal dose response with a shoulder at low doses, followed by a sharp fall (Figure 5a). As expected, the inhibition of germination was greater for light spectra with the greatest content of shorter wavelengths, and complete inhibition only occurred with spectra including UV-B (Figure 5a). Mylar filtered UV-B tubes (peak wavelength 325-330nm) had a greater effect than UV-A tubes (peak wavelength 365-370nm), which had a very similar effect to light from the blue-filtered tubes (peak c. 445nm). Only white light caused no measurable inhibition (Figure 5a). Although these dose responses showed the expected increase in effect with decreasing wavelength, none of the established biological weighting functions allowed the data for all lamp-filter combinations to be integrated in to a single dose response.

Figure 5. (a) Dose responses for the inhibition of sporangiospore germination in B. lactucae exposed to a range of different lamp spectra, with treatments expressed as unweighted dose. The lines are fitted inverse sigmoidal

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dose responses. All data are the means of five (UV treatments) or three (blue and white light) independent repeated experiments. Error bars are + 1SE, where no error bar is visible it is within the plotted symbol. (b) The biological spectral weighting function of inhibition on sporangiospore germination in B. lactucae determined from the data shown in Figure 1. A range of widely used BSWFs for plants of microbes are plotted for comparison (4, 10, 32, 33, 49).

A specific Bremia weighting function was calculated using the dose responses to all five lamp/filter combinations covering the UV and blue wavebands (Figure 5b). Comparison between this BSWF and those used previously for plant and/or microbial responses highlights the following points:-

This Bremia BSWF has a relatively small response to decreasing wavelength in the UV-B, with the relative effectiveness of radiation at 290nm being approximately 10 fold higher than that at 320nm.

In the shorter UV-A wavelengths (approx. 320-350nm) the change in effectiveness per increase in wavelength is similar to or slightly greater than that in the UV-B (Figure 4b). This is comparable to the response in the Quaite plant action spectrum(33), although the Bremia BSWF is “flatter” than the Quaite BSWF.

The Bremia BSWF has a plateau of response at longer UV-A wavelengths. This has parallels in the recent plant action of Flint and Caldwell (10), but the plateau response in the Bremia BSWF is approximately an order of magnitude lower than that plant BSWF (Figure 5b). The other BSWFs used widely applied to plant or microbes have responses to longer wavelength 2-3 orders of magnitude lower than that determined here for Bremia ((4, 10, 32, 33, 49).

When treatments from all five lamp-filter combinations were expressed together using doses weighted according to the Bremia BSWF (UVBremia) a single inverse sigmoidal dose response was obtained which explained 85% of the variation in this whole dataset (Figure 6a). In addition, the comparable data on UV inhibition of B. lactucae spore germination published by Wu et al (56) fits the dose response curve well, both data for UV-A and UV-B (Figure 6a). This is strong corroborative evidence for the validity of both the dose response and action spectrum determined here. It also suggests that the dose response has broad applicability across strains from different genotypes from contrasting locations.

Based on the integrated dose response to UVBremia, the calculated UVBremia dose required to reduce spore germination by 50% (ED50) is 3.84 kJ m-2 (with 95% confidence limits of 3.31 and 4.37 kJ m-2). The estimated ED90 and ED99 values are 11.4 and 34.7 kJ m-2 UVBremia, both well within the ambient range of daily doses for the UK (see below).

Figure 6. Dose responses (a) for the inhibition of germination of dispersed sporangiospores of B. lactucae and (b) undispersed sporangiospores of B. lactucae exposed to a range of different lamp spectra, with doses weighted using the Bremia BSWF shown in Figure 4b. All data are the means of three - six independent repeated experiments. Error bars are + 1SE, where no error bar is visible it is within the plotted symbol. The line is the fitted inverse sigmoidal dose response fitted to all data. In Figure 5a includes two comparable datasets published by Wu et al.,56, which conform to the same dose response.

4.3.a.ii The responses of undispersed sporangiospores of B. lactucae to the light environment

The response of undispersed sporangiospores to a range of lamp and filter combinations could be integrated to a single dose response using the Bremia BSWF discussed above (Figure 6b). There is substantial overlap between the responses of dispersed and undispersed sporangiospores at UVBremia doses which caused relatively little inhibition of germination (up to approx 30-40%, caused by doses less than approx 2.5 kJ m-2), but at higher

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doses undispersed sporangiospores are less affected by radiation treatments (Figure 6b). The major difference between dispersed and undispersed sporangiospores in these experiments appears to be that there is a population of undispersed spores, perhaps up to 20% of the total, that survive even at very high doses. The fitted dose response for undispersed sporangiospores is significantly different from that for dispersed sporangiospores (F2,58 = 12.20: p<0.0001). The predicted ED50 for undispersed sporangiospores (10.3 kJ m-2 with 95% confidence limits of 6.7 and 13.9 kJ m-2) is significantly higher (F1,58 = 17.05: p<0.001) than that for dispersed sporangiospores. The estimated ED90 value for undispersed sporangiospores is greater than 60 kJ m-2 UVBremia, approximately 6 times higher than that for dispersed sporangiospores, and above the ambient range of daily doses for the UK (see below).

4.3.a.iii Responses of disease to exposure of the host of light treatments

When the host was exposed to light treatments prior to inoculation there was a trend for the latent period, the time to first sporulation, to be delayed by approximately one day at the higher UV-B doses, although this was not quite statistically significant (p=0.07). There were indications of a progressive increase in latent period with increasing dose, but the data were too variable to define a precise dose response (Figure 7a). This apparent delay had longer term consequences, for example in the early stages of disease development incidence showed a clear dose response, decreasing with increasing dose (Figure 7b). Similarly, the effect of UV on the area of infection was largely confined to the early stages of disease development and the maximum area of infection was not significantly affected by UV exposure. However, measured over three weeks after inoculation, the integrated severity of infection, measured as the area under the disease progress curve (AUDPC) based on the extent of sporulation, was significantly reduced by higher doses (Figure 7c). This response could be fitted by an inverse sigmoid dose response, explaining 86% of the variation in the data across two repeated experiments. This modelled response predicts no significant reduction in AUDPC until UV dose approaches 10 kJ m-2 d-1 UVQuaite (Figure 6c) and the calculated ED50 is 19.0 kJ m-2 d-1 UVQuaite (with 95% confidence limits of 15 and 22 kJ m-2 d-1). The quantity of sporangiospores produced per unit sporulating areas is also reduced by increasing UV radiation (Figure 7d). The combined data from three repeat experiments could be fitted by a dose response that explained 64% of the variation, with a predicted ED50 for reduced sporangiospore production being 12.1 kJ m-2 d-1 UVQuaite (with 95% confidence limits of 8.9 and 15.2 kJ m-2 d-1).

Figure 7

The effects of UV exposure of plants prior to inoculation on the subsequent development of downy mildew disease / Bremia lactucae. Data for 2-3 independent repeat experiments are shown. Individual data points are means of 10-12 plants per treatment in each repeat experiment. Lines are the dose responses fitted to the full dataset from all experiments, with the 95% confidence limits on the fitted curve.

Although the effects of pre-inoculation light environment were significant, when treatments were provided both before and after exposure, it was treatment post-inoculation that had the dominant effect. When plants were inoculated with B. lactucae and then exposed to a range of UV doses there were no significant effects on latent period, disease incidence or the progress of sporulation. However, the quantity of B. lactucae sporangiospores produced per unit sporulating areas was significantly reduced in plants exposed to higher doses of UV radiation after inoculation (Figure 8a). This post-inoculation response might be due to UV effects on the pathogen, the host or a combination of the two. Assuming a host response, and expressing UV doses in terms of UVQuaite, the combined data from three repeat experiments could be fitted by a dose response that explained 68% of the

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variation. This dose response has no clear shoulder at low doses, and the predicted ED50 of 5.1 kJ m-2 d-1 UVQuaite (with 95% confidence limits of 2.9 to 7.2 kJ m-2 d-1). If the post-inoculation response were due to direct UV effects on B.lactucae, then it would be expected to conform to the BSWF determined in vitro (see above). Expressed in this way, the dose response predicted an ED50 of 6.5 kJ m-2 d-1 UVBremia (with 95% confidence limits of 4.2 to 10.1 kJ m-2 d-1). It is notable that this dose response is not significantly different from that obtained in vitro for undispersed sporangiospores (Figure 8b). This good agreement between experiments dealing with very different elements of this pathosystem and under very different experimental conditions suggests (i) that the observed effects of post-inoculation UV environment was most likely dominated by direct fungal responses to UV, and (ii) that the BSWF and overall dose response for this pathogen is robust across a range of conditions.

Figure 8. (a) The effects of UV exposure of plants after inoculation on the sporulation of Bremia lactucae. Data for 3 independent repeat experiments are shown. Individual data points are means of 10-12 plants per treatment in each repeat experiment. Lines are the dose responses fitted to the full dataset from all experiments, with the 95% confidence limits on the fitted curve. (b) the full dose responses for sporulation in planta (with the fitted dose response) and the inhibition of sporangiospore germination in vitro with dose plotted on a log scale.

Figure 9. Dose responses for the inhibition of germination of dispersed conidiospores in Botrytis cinerea exposed to a range of different lamp spectra, with treatments expressed as (a) unweighted dose and (b) doses weighted using the BSWF of Quaite et al., (33) . The lines are fitted inverse sigmoidal dose responses. All data are the means of five independent repeated experiments. Error bars are + 1SE, where no error bar is visible it is within the plotted symbol.

4.3.b The mechanisms of Botrytis cinerea - lettuce responses to light spectrum

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4.3.b.i The effects of exposure of dispersed conidiospores to light treatmentsUV-A and longer wavelengths had no significant effects on the germination of dispersed conidiospores of B. cinerea but all UV-B treatments inhibited germination and showed an inverse sigmoidal dose response with a shoulder at low doses, followed by a sharp fall (Figure 9a). In contrast to the data obtained with Bremia lactucae, the increase in effect with decreasing wavelength, could be accounted for using the BSWF of Quaite et al., (33), which produced a fitted dose response curve that explained approximately 90% in the variation in the data across all treatments (Figure 9b). Across the UV-B and shorter UV-A wavelengths this BSWF is broadly similar to the Bremia BSWF determined here, but longer wavelengths have markedly lower effectiveness (by 2-3 orders of magnitude) than with B. lactucae (compare Figures 5 and 9b). The integrated dose response of B. cinerea to UVQuaite gives a calculated ED50 of 5.1 kJ m-2 UVQuaite (with 95% confidence limits of 4.68 and 5.45 kJ m-2). The estimated ED90 and ED99 values are 8.5 and 12.1 kJ m-2 UVQuaite both well within the ambient range of daily doses for the UK (see below).

Figure 10. Dose responses for the inhibition of germination of Botrytis cinerea conidiospores exposed prior to dispersal to a range of different lamp spectra, with treatments expressed as doses weighted using the BSWF of Quaite et al., (33) . The line is the fitted inverse sigmoidal dose response. All data are the means of five independent repeated experiments. Error bars are + 1SE, where no error bar is visible it is within the plotted symbol.

4.3.b.ii The effects of exposure of undispersed conidiospores to light treatmentsThe response of undispersed conidiospores to a range of lamp and filter combinations could be integrated to a single dose response based on UVQuaite (Figure 10), explaining 60% of the variation in the data. Undispersed spore showed little response to doses less than approx. 20 kJ m-2 UVQuaite., a dose that causes almost complete kill of dispersed conidiospores The fitted dose response for undispersed conidiospores is highly significantly different from that for dispersed conidiospores (F2,276 = 691: p<0.0001). The predicted ED50 for undispersed conidiospores (50.5 kJ m-2 UVQuaite with 95% confidence limits of 49.2 and 57.6 kJ m-2) is significantly higher (F2,276 = 2292: p<0.001) than that for dispersed conidiospores. This is above the ambient range of daily doses for the UK (see below).

3.2.a.iii The effects of light treatments on sporulation in B. cinerea

When B. cinerea was cultured under light treatments from the first light period after plates were inoculated, sporulation after 7 days showed a biphasic dose response (Figure 11a). Cultures grown with no exposure to UV, whether in the dark or with white or blue light, showed variable sporulation, ranging from no sporulation to a maximum of approximately 105 conidiospores per plate. By contrast, even low doses of UV induced the production of 107-108 conidiospores per plate, with an ED50 for the induction of sporulation determined from the fitted biphasic dose response is 0.4 kJ m-2 UVQuaite. The fitted plateau of maximum sporulation is 107.7 conidiospores per plate (95% confidence limits 107.2 to 108.3), i.e. two orders of magnitude greater than the highest measured in the absence of UV. Induction reaches 99% of its maximum at a dose of approx. 1.3 kJ m-2 UVQuaite and remains near this maximum over a relatively narrow range of doses, falling to below 95% of the maximum at around 4 kJ m-2 UVQuaite. The ED50 for suppression of sporulation is 12 kJ m-2 UVQuaite with 95% confidence limits of 7.3 and 19.5 kJ m-2. However, this suppression of sporulation appears to be a function of the reduction of mycelial growth by UV exposure (data not shown), and does not occur when B. cinerea is exposed to light treatments only after mycelial growth had filled the Petri dish. B. cinerea exposed in this way showed the same induction of sporulation (ED50 was not significantly changed: p=0.71) but sporulation then remains near constant up to doses equivalent to the maximum daily dose received in the UK (Figure 11b).

As noted above, blue light alone had no effect on sporulation in B. cinerea. In addition, when colonies were exposed to a combination of UV and blue light the presence of blue had no effect on the induction of sporulation (Figure 11a). Statistical analysis of the fitted dose responses of sporulation in the presence and absence of blue light showed that neither the overall dose response nor the ED50 for the induction of sporulation were significantly different (p=0.578 and p=0.782 respectively). In terms of the links to the crop-scale studies, these data show that any effect of blue plastic on B. cinerea would be function of its very low UV transmission, not due to any specific effect of its transmission in the blue.

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Figure 11 Induction of conidiospore production in B. cinerea, (a) where the fungus is exposed to UV treatments throughout development and (b) where the fungus is exposed to UV treatments only after the colony is well established. Data for 2-3 independent repeat experiments are shown, and in Figure 11a data shown in red are for treatments were UV was provided against a background of blue light (not significantly different from UV alone). Individual data points are means of 10-12 plants per treatment in each repeat experiment. Lines are the dose responses fitted to the full dataset from all experiments.

Figure 12 The effect of the light environment of the host of grey mould disease in lettuce. (a) The percentage of plants infected 12 days after inoculation and (b) the percentage of those infected plants having severe symptoms such as tissue collapse and B. cinerea sporulation, Data are means + 1 SEM of two independent repeat experiments (with disease incidence assessed on populations of 50-60 plants in each experiment)are shown. In Figure 11b the fitted dose response for B. cinerea sporulation determined in vitro (see Figure 10) is shown for comparison.

4.3.b.iii Responses of disease to exposure of the host to light treatmentsIn contrast to Bremia lactucae, we found no clear evidence that exposure of the host influenced infection by B. cinerea or the subsequent development of disease. For example, across two repeated experiments between 65% and 85% of plants showed symptoms of B.cinerea infection 12 days after inoculation, but there was no significant effect of UV dose (Figure 12a). There were some indications that when plants were exposed to different light environments after inoculation severe B. cinerea infection developed more quickly at intermediate UV doses, leading to transient differences in disease expressed in this way. Thus, six days after inoculation the percentage of infected plants showing severe symptoms such as tissue collapse and B.cinerea sporulation tended to be greater between 1.5 and 7 kJ m-2 UVQuaite (Figure 12b). Although these differences were not significant, they show an interesting parallel with the dose response determined for B. cinerea sporulation in vitro (Figure 11a and 12b). While there are risks in over-interpreting these parallels, the broadly similar forms of these dose responses suggest that (i) the data obtained in vitro are indicative of the responses occurring in planta, but (ii) that the dose required to produce a response is somewhat higher in planta than in vitro.

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4.4. Experimental investigation of the effects of light spectrum on phylloplane micro-organisms and disease biocontrol agents (Objective 5) The basic photobiology of two biocontrol agents, Trichoderma harzianum and Bacillus subtillis, and a range of phylloplane micro-organisms were investigated in vitro using the same techniques as for the two pathogens. The results are summarised here in terms of ED50s determined from dose responses (Table 2), which also includes ED50s for the range of responses of Bremia lactucae sporangiospores and Botrytis cinerea conidiospores discussed above.

ED50 (UVQuaite)Biocontrol agents (as dispersed propagules) Mean SETrichoderma harzianum 4.37a,b 0.42Bacillus subtilis 3.49a 0.84Phylloplane microorganisms (as dispersed propagules)Sporobolomyces roseus 13.9c 0.61Bullera alba 3.22a 0.26Aureobasidum pullulans 19.7d,e 0.88Cladosporium sp. 23.1eBremia lactucaeDispersed spore germ. 4.47b 0.35Undispersed spore germ. 14.9c,d 2.64Botrytis cinereaDispersed spore germ. 5.07b 0.20Undispersed spore germ. 53.4 2.13

Table 2. The calculated UVQUAITE dose (kJ m-2) required to reduce spore germination by 50% (ED50) in two biocontrol agents (T. harzianum and Bacillus subtilis), four representative phylloplane microbes (Bullera alba, Sporobolomyces roseus, Aureobasidum pullulans and Cladosporium sp.) and, for comparison, the two fungal pathogens of lettuce that were the main focus of this contract. ED50s were calculated from inverse sigmoidal dose responses fitted to data obtained using a range of lamp and filter combinations. Note that UVQUAITE was used as a default BSWF in the absence of precise spectral response data for most of the organisms studied, although this may underestimate the responses of several of these species to UV-A (see text). ED50s that do not share the same letter are significantly different at p<0.05.

Expressed in terms of UVQUAITE, the calculated ED50s for T. harzianum was not significantly different from those of B. lactucae or B cinerea, while B. subtilis was significantly more UV-sensitive than either pathogen (Table 2). Of the phylloplane organisms, Bullera alba was highly sensitive to UV treatments, with an ED50 not significant different from that of Bacillus subtilis. However, Sporobolomyces roseus, Aureobasidum pullulans and Cladosporium sp. were all substantially more UV-tolerant, with ED50s significantly greater than for the dispersed propagules of the other micro-organisms that were investigated (Table 2). On this basis these three phylloplane organisms would be expected to be relatively unaffected by exposure to solar UV, while B. alba, T. harzianum and Bacillus subtilis, like B. lactucae and B. cinerea, would all the expected to suffer high degrees of mortality if exposed to summer sunlight. Although the effects of the different “smart plastics” on phylloplane microbiology was not investigated in detail in the tunnel facility at STC, small-scale experiments were carried-out at Lancaster using B. alba and Sporobolomyces roseus as marker species. These experiments revealed that populations of both, but especially B. alba, were significantly reduced under UV-T compared with standard plastic, while populations under UV-O tended to be higher than under the standard film (data not presented). These data are broadly similar to those obtained using these “phylloplane yeasts” in previous studies at Lancaster (16). Although the spectral responses of the biocontrol and phylloplane organisms were not investigated in detail, it was notable for both T. harzianum and B. subtilis that UVQUAITE underestimated responses to UV-A wavelengths. The contrasting spectral responses of B. cinerea and T. harzianum may have practical implications since the greater sensitivity of the biocontrol agents to long wave UV-A leaves them more vulnerable than their target, B. cinerea, to radiation transmitted through standard horticultural plastic. We would predict that switching from standard plastics to UV-opaque film would be favourable for biocontrol of B. cinerea. However, experiments carried out in the tunnels at STC did not confirm this prediction for biocontrol using T. harzianum largely because the degree of control achieved was inconsistent under all plastics (data not presented).

4.5. Models for the effects of spectral modification using filter plastics on disease caused by Bremia lactucae and Botrytis cinerea. (Objective 4 and 6)4.5.a Models for Bremia lactucae

Modelling the responses of B. lactucae / lettuce downy mildew to altered spectral challenges posed particular challenges since the experimental data had revealed the complexity of the underlying photobiology. In particular, the pathogen itself required a specific biological weighting function distinct from any that could be applied to the host. Thus, host and pathogen responses needed to be modelled independently using different BSWFs that have different responses to both plastics and natural variation in UV with season, latitude etc. Modelling of the component responses of this pathosystem was relatively straight-forward. Based on laboratory studies, all the

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stages investigated were more or less sensitive to UV within the ambient range (Figure 13). As evident in the ED50s (Table 2) damage to dispersed spores and the suppression of sporulation were more sensitive to UV than damage to undispersed spores and the reduction in AUDPC caused by pre-inoculation exposure of the host plant. Thus, under UV-transparent film in mid-summer germination of dispersed spores and sporulation are both predicted to be reduced by approximately 80% compared with the near zero UV environment provided under UV-opaque or blue films (Figure 13a,d). By contrast, germination of undispersed spores would be reduced by no more than 60% and AUDPC no more than 40%. The responses of all processes are predicted to be highly seasonal, with minimal effect of plastics in winter. The standard film would be expected to cause small reductions in the germination of dispersed spores in mid-summer, but to have little effect on other processes (Figure 13). The blue and UV-opaque films both provide sufficiently low UV environments to support maximum germination, AUDPC and sporulation (Figure 13).

Integrating the predicted changes in the component processes show in Figure 13a-d leads to the overall expectation that the UV-transparent film would deliver a good control of B. lactucae under summer conditions, while UV-opaque and blue would tend to increase this disease. It was this last point that highlighted the clear difference between the predictions of these models and the results obtained in the experimental tunnels, where UV-opaque and blue often produced good control of B. lactucae (see section 4.1). In seeking to resolve the contradiction between the initial model predictions and the crop results, attention was focussed on the effects of the light environment on sporulation. In B. cinerea, studies of sporulation in vitro confirmed that substantial induction occurred in response to very low UV (Figure 11). Since B. lactucae can not be grown in vitro, directly comparable experiments to those conducted with B. cinerea were not possible. There were indications from both laboratory studies and at the crop scale that sporulation was suppressed at low UV, but our efforts to study the effects of very low UV in the sporulation of B. lactucae in planta were limited by its BSWF, which made it very difficult to deliver near zero UV whilst maintaining adequate light for proper plant growth. In order to assess the potential role of this poorly defined induction of B. lactucae sporulation at low UV doses, we used the data obtained for B. cinerea as an analogue. In that pathogen induction was near 100% even at doses occurring in mid-winter, and inhibition only occurred at relatively high ambient doses (Figure 10), comparable to the dose response seen in B. lactucae. Assuming the same form of “bell-shaped” dose response applies in B. lactucae at doses below the minimum achieved experimentally generated the predictions shown in Figure 13e. This predicts that the low doses of UV penetrating the standard film would promote sporulation in summer compared with the baseline that occurs in winter. The far higher UV penetrating UV-transparent film would cause an induction of sporulation of approximately one order of magnitude over that predicted under the standard film. Conversely, the blue film maintains UV so low as to hold sporulation at its dark baseline. With the long UV-A tail of the Bremia BSWF the low UV transmission of the “UV-opaque” film is sufficient to cause some induction during summer, although this is predicted to be at least one order of magnitude lower than with the standard film (Figure 13e).

Figure 13. Model predictions for the effects of spectral modification using “smart plastic” on components of the interaction between lettuce and Bremia lactucae. (a) germination of dispersed sporangiospores, (b) germination of undispersed sporangiospores, (c) disease development (AUDPC) and (d) sporulation. Figure 12e is sporulation assuming induction by low UV doses (see text). All data were derived from fitted dose responses to experimental data and UV conditions modelled using the model of Bjorn and Murphy (1) assuming clear sky conditions. The data was run for each day of the year, the data presented are the monthly means.

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When the induction of sporulation at low UV doses is included then the modelled overall effects of spectral modification on B. lactucae are far closer to the results obtained in the crop experiments. All three smart plastics are expected to deliver some degree of control of downy mildew (Figure 14). The population of surviving sporangiospores under UV-T (or in the field) is predicted to reach a maximum between autumn and spring (October-March) when UV is sufficient to induce maximum sporulation but not to cause significant spore mortality (Figure 14a,b). The surviving spore population declines during the higher UV conditions of the summer months, although the magnitude of this seasonal variation is dependent on cloud, with survival in summer increasing by around an order of magnitude under cloudy conditions (Figure 14b). The attenuation of UV by the standard plastic is predicted to have very major effects on the surviving spore population of B. lactucae. In winter (November-February) this attenuation reduces UV doses sufficiently to suppress sporulation and reduce the spore population compared with the field or UV-T (Figure 14a). However, in summer the reduction in UV under the standard plastic reduces spore mortality, and the model predicts an increase in the spore population by as much as two orders of magnitude under sunny conditions (Figure 14a), but less under cloud (Figure 14b). The suppression of sporulation under UV-O and, especially, blue films (the latter due to its very low UV transmission) is predicted to deliver consistent control of B.lactucae compared with the standard film. Indeed, the effectively zero UV environment provided by the blue plastic holds the B. lactucae population at a minimum throughout the year, although is not expected to halt sporulation completely (Figure 14a,b). By contrast, the penetration of some solar UV through the UV-O film is predicted to lead to some increase in sporulation during summer (Figure 14a). However, overall UV-O and blue are predicted to give good control compared with standard film for much of the year, but especially during summer. This predicted pattern is as observed in the tunnel experiments at STC (compare Figure 14 with Figure 1).

Figure 14. Model predictions for the overall effects of spectral modification using “smart plastic” on downy mildew of lettuce caused by Bremia lactucae. Data were derived from the component processes (see Figure 12) assuming induction of sporulation by low UV doses (Figure 12e) and under either clear sky (a,c) or 8 octas cloud (b,d). Figure 13a,b assume all spores are exposed to solar UV throughout the day. Figures 13c,d assume a peak spore release in late morning, and that 30% of spores escape UV exposure.

The model predicts that UV-T has the potential to give a high degree of control of B. lactucae dependent on high light conditions, but that effects will be variable in the UK due to variation in solar UV due to cloud, season etc. (Figure 14a,b). Whilst experiments at STC confirmed the variability of responses to UV-T, the very substantial reductions in B. lactucae that the model predicts for this film were not observed. A possible factor in this discrepancy that could not be fully explored within this contract was the interaction between diurnal variations in (a) UV exposure and (b) sporangiospore release by the pathogen. It is known that sporangiospore release in B. lactucae is influenced by temperature and humidity during the preceding night (5, 47). Even when a well-defined morning peak occurs, in excess of 25% of sporangiospores are produced at other times of day, and spores produced later in the day or at night will escape much of the potential damaging effect of solar UV. In addition, some fraction of the sporangiospore population will almost certainly avoid exposure to solar UV, due for example to shading within a canopy etc. Although hard to quantify, and likely to be variable, such escape will clearly limit the effects of any mechanism that depends on direct exposure of the fungus to incident UV. In an attempt to assess the consequences of “escape” from UV exposure, the model runs presented in Figures 14a and b were

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repeated assuming (i) the diurnal pattern of sporulation is such that on average the sporangiospore population is exposed to only 50% of potential daily UV dose and (ii) that 25% of the sporangiospore population escapes UV exposure completely. Adding these assumptions (Figure 14c,d) has relatively little effect on the predicted effects of UV-T between October and March under either clear skies (compare Figures 14a and 14c) or cloud (Figure 14b,d). However, between April and September, taking to account “escape” increases the predicted population of surviving sporangiospores by as much as an order of magnitude under clear skies (Figure 14a,c) and approximately four-fold even under 8 octas cloud (Figure 14b,d). Such correction, although to some extent arbitrary in terms of the values selected for “escape”, produces a model that matches the experimental data obtained at STC in that UV-T is, at its best, as effective in limiting B. lactucae as UV-O or blue, but far more variable than either of those films.

Adding “escape” to the modelling component of this contract is less directly based on our own experimental data than other components, it is useful in highlighting that any response dependent on direct exposure to high UV conditions will be more vulnerable to inconsistency than responses mediated by the attenuation of UV. Another factor is that maximum UV exposure will occur during bright, sunny conditions which are likely also to be warm and dry, which will constrain B. lactucae due to effects of spore germination and dispersal (44-47, 54-56). Thus, the potential benefits of UV-T films may often be masked because they occur when other factors are limiting B. lactucae regardless of UV. By contrast, suppression of sporulation by attenuating solar UV is less dependent on variation in incident UV and is expected to be effective under conditions that are otherwise favourable to the development of downy mildew. Thus, although (i) B. lactucae is clearly vulnerable to direct damage from UV from the time of sporangiophore emergence until the resultant sporangiospores have successfully infected the host, and (ii) host resistance is stimulate under high UV conditions, the resultant disease control under UV-T is unlikely to more than a contribution to the control of B. lactucae. Commercial decisions about the use of UV-T, and indeed any plastic, are likely to be based on a wider suite of effects than disease control alone.

4.5 b Models for Botrytis cinerea

Figure 15 Model predictions for the effects of spectral modification using “smart plastics” on components of the interaction between lettuce and Botrytis cinerea under either clear sky (a,c,e) or 8 octas cloud (b,d,f). (a,b) sporulation, (c,d) germination of dispersed conidiospores and (e,f) overall production of viable spores. All data were derived from fitted dose responses to experimental data and UV conditions modelled using the model of Bjorn and Murphy (1). The model was run for each day of the year, the data presented are the monthly means.

Given that there was no clear host responses that influence the interaction between lettuce and Botrytis cinerea the model for this pathosystem is simpler than that for Bremia lactucae, and can be based on the Quaite BSWF. Even so, the relatively simple photobiology of this system predicts complex responses to spectral modification (Figure 15). Sporulation under standard plastic would be suppressed in winter because the plastic reduces UVQuaite below the threshold for induction, while in summer sporulation is near its maximum because the plastic prevents doses sufficiently high to suppress sporulation (Figure 15a,b). By contrast, sporulation under UV-T would be near maximum in winter, but would be suppressed by high UV during mid-summer, although this

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suppression would be largely confined to bright sunlight conditions (Figure 15a,b). The low UV transmission of the UV-opaque film would reduce sporulation to a minimum in winter, but in summer would allow sufficient UV both to induce sporulation and to protect against high UV damage, leading to increased sporulation compared with the UV-T environment (Figure 15a,b). The blue film, with no measurable UV transmission, would be expected to consistently prevent any induction of sporulation above the dark baseline, regardless of season and weather conditions (Figure 15a,b). The effects of spectral modification on spore germination are simpler. The only plastic predicted to have major effects is UV-T, where exposure to solar UV is predicted to reduce conidiospore germination, with the magnitude of this response varying greatly with season and cloud cover. Only bright sunlight conditions during summer (May-August) would be expected to cause suppression greater than 99% (Figure 15c,d).

Clearly, in practice, the overall effects of plastics (Figure 15e,f) will be the result of changes in both sporulation and spore germination. In summer UV-T, UV-O and blue would all be expected to reduce B. cinerea compared with standard plastic. The effect of UV-T would be greatest under bright sunlight conditions (Figure 15e), while the effectiveness of the UV-O film would be greatest under less intense sunlight (Figure 15f). Blue, based on its immeasurable low transmission of UVQuaite always provides a UV environment too low to induce any increase in sporulation above the dark baseline, and is expected to be the most consistent film (Figure 15e,f). UV-O and blue should also be effective under the lower UV conditions of winter. In spring and autumn UV-T is expected to have little effect on B. cinerea compared with the standard plastic, while in winter it may increase B. cinerea by allowing sufficient UV to reach the crop to induce sporulation. Overall, under UK conditions both UV-O and UV-T are expected to deliver some decrease in disease caused by B. cinerea, but neither are likely to give consistently high degrees of control (Figure 15e,f). While UV-O is expected to be consistently preferable to standard throughout the year, there is some risk that UV-T could increase infection during mid-winter. Blue, due to its very low UV transmission, is expected to give the most consistent control.

5. Main implications of the results of the projectThe work of CSA6138 at Lancaster and STC took place in parallel with research in to smart plastics funded by HDC. The two funding streams were highly complementary since the HDC research dealt with the broader commercial, agronomic aspects of the use of smart plastics in the UK. Crops included lettuce, but also the ornamental crops that were initially included in this contract. Thus, assessment of the prospects for future work benefits from inputs from both the current contract and research funded by HDC.

This DEFRA contract showed that only the blue plastic delivered generally consistent control of lettuce diseases caused by Bremia lactucae and Botrytis cinerea. However, this plastic highlighted a number of points that are vital in the assessment of commercially produced “smart plastics”. Firstly the “blue” plastic had far wider effects on the crop environment than selective transmission of the blue waveband. Its reduced transmission of red and longer wavelengths led to reduced photosynthetic radiation reaching the crop, and lower temperatures coupled with higher relative humidity, especially under high insolation during summer. In addition, the film was highly UV-opaque. All of these changes could have major effects on crops, and their associated pests and pathogens that would not be anticipated simply by the label of “blue”. Secondly, developing from the first point, the reduction of almost 40% in photosynthetic radiation transmitted by the blue film led to lettuce crops that were not commercially viable, so commercially the blue film is not usable in its present form. However, the current contract has shown that the effect of the blue plastic was not due to its effect on the balance of “visible light”, but rather to its exceptionally low UV transmission, which was lower than the commercial “UV-opaque” film. Understanding of this mechanism is founded on both the increased knowledge of dose responses and the BSWF determined for Bremia, where the substantial tail in the UV-A places a specific requirement for low transmission at longer wavelengths. The effect of very low UV transmission was due to induction of sporulation by low UV doses, but in practice control via this route requires a film to have near zero UV transmission. Providing it had such low UV transmission, an uncoloured film with good transmission of photosynthetic radiation should deliver good disease control and good crop growth. Our assessment of currently available commercially films suggest that none delivers these properties, and preliminary discussions with film producers suggest there may be technical limits on producing plastics that retain very low UV transmission over a commercial life-span of three years or more. Nonetheless, the data obtained in this contract suggests that such films would be worth pursuing given the increasing commercial pressures for alternatives to control disease control.

At present, the UV-opaque and UV-transparent plastics may make a contribution to disease control alongside other measures, but are too inconsistent to be considered as “stand-alone” controls. Clearer commercial benefits of these two plastics were evident from the HDC projects, and these may drive commercial assessment of their use, with short-term disease control a secondary benefit. Specifically for crops investigated under CSA6138 two points are highly significant commercially. Firstly, for crops propagated under protection for subsequent transplantation to the field, including lettuce and a range of Brassicas, there are clear advantages of using UV-transparent film. Plants propagated under this plastic are consistently assessed to have a higher commercial quality, and HDC data shows that they can go on to give significantly higher yields (by up to 40%) than those propagated conventionally under glass. At least two major propagators, one of lettuce and one of brassicas have already adopted UV-transparent plastics for large-scale production facilities, based on the results of the DEFRA and HDC projects. The experience of these producers and data from CP19 suggests that one element of the

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benefit of UV-transparent films may be reduced disease, consistent with the results on this project, but the nature of any “carry-over” of this effect in to the field is still unclear. Secondly, for some crops there are trade-offs between yield and quality regardless of any effects on disease. For example, in coloured lettuce cultivars such as lollo rosso, production under UV-T film produces a crop with more intense colour and distinct taste that is considered preferable to crops from standard plastic. By contrast, when lollo rosso is grown under UV-O it produces large heads but these have very little anthocyanin and are considered to be of poor commercial quality. Similar trade-offs between yield and quality have been observed in a range of other crops.

Thus, two overall conclusions can be drawn. Firstly, while “smart plastics” can deliver a range of commercially desirable end-points, including disease control, no single film is suitable for all crops, whether assessment is based on disease control or other responses to spectral modification. Choice of film will depend on the type of crop being grown and the overall commercial priorities of the grower. Secondly, plastics developed to deliver specific commercial outcomes in other regions of horticultural production, such as the Mediterranean rim, may not deliver the same benefits under UK conditions, and vice versa. The modelling approach adopted here can be applied to any location and predicts that under the high radiation environments typical of growing areas such as Israel, southern Spain or California, the UV-T film would be expected to have a clear advantage over standard film under a wide range of conditions (data not presented). This is consistent with the major role of solar UV in the epidemiology of B. lactucae in high light climates (54). In such climates, even though UV-O would also be expected to reduce B. cinerea under many conditions, UV-T would be expected to give a higher degree of control, exceeding that even of a completely UV-opaque film such as blue under many conditions. Caution is clearly required when extrapolating field data, or moving cladding materials, from one climate to another.

6. Future workAs noted in Section 5, the work of CSA6138 at Lancaster and STC took place in parallel with complementary research in to smart plastics funded by HDC. Thus, assessment of the prospects for future work benefits from inputs from both the current contract and research funded by HDC. In particular, the interactions between the HDC and DEFRA funded research contracts allowed integration between fundamental mechanistic investigations and commercial, agronomic assessments. Such integration has been extremely valuable and forms a good model for further work into smart plastics. In terms of the priorities for future work, the principle conclusions from across all our inter-related work on smart plastics is that their commercial use will be driven by their overall effects on crop yield and quality, with contributions to pest and disease control one component of many. Regardless of the specific plastic used there are clear benefits of protection for many crops, for example in terms of keeping the crop dry (which in itself has many benefits in terms of disease control), preventing physical damage and that due to extremes of temperature. This clearly improves quality and reduces wastage in a wide range of crops that have not conventionally been grown under protection. Spectral modification using smart plastics offers additional benefits for specific crops. i) Improved quality through altered crop chemistry. A general conclusion is that exclusion of UV using the

UV-O increases the bulk of above-ground tissues, and this may have yield benefits in some crops. However, increased growth under UV-O may come at the expense of quality, and our experience across all projects suggests that quality may be major driver for the commercial adoption of smart plastics. Quality benefits are specific to specific plastics and crops, but UV-T appears to have particular potential. Key areas appear to be (a) improved quality of propagation crops and (b) improved flavour, colour and, for herb crops, oil yield. The HDC projects have also provided initial evidence that choice of plastic can influence shelf life, including that of lettuce. At present both the fundamental mechanisms of these responses and the full scope for their exploitation remain poorly understood. Changes in crop chemistry appear to be a central mechanism for many of the responses, but the details remain unknown. Equally, the potential value of altered crop chemistry is not yet clear, in terms not only of flavour and colour but also concentrations of phytochemicals known to be important in the human diet, for example flavonoids (3, 9, 14, 15, 18, 21, 24, 29, 51, 57). Based on our preliminary data, such “metabolic engineering” through the use of smart plastics may also be important for crops grown for oil or pharmaceuticals.

ii) Improved quality in propagation crops. In this contract, the effects of UV in inducing host resistance to B. lactucae and B. cinerea were significant, but this effect appeared small in our models of the overall impact of plastics for crops grown permanently under protection. However, it may make a contribution to the benefits of propagation under UV-transparent film. At present, the mechanism of this response, and its links to the molecular biology of UV responses (e.g. 2, 19, 20, 27) remains unknown.

iii) Spectral modification and disease control: a wider perspective. This contract focussed on downy mildew and B. cinerea. HDC funded research suggests that smart plastics also reduce powdery mildew infection in some crops. As well as UV modifying films, this included a response to a film that increases R:FR ratio that was not included in the current contract. R:FR films are used largely as a contribution to growth regulation rather than for pest or disease control (6, 12, 25, 26, 43, 53). However, effects on disease are broadly consistent with some recent fundamental research (23, 35, 42, 48), and their practical exploitation merits further exploration.

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7. Actions resulting from the work. Communication of the results of the project to the vegetable and ornamentals industry was a formal objective of the contract (Objective 7). This “extension” element of the contract benefited greatly from the links between this contract and that funded by HDC that was carried-out in parallel. The HDC projects used commercial scale plastic-clad structures clad with “smart plastics” for the production of a range of crops, including lettuce, under plastics that shared the standard, UV-opaque and UV-transparent films with the current project. These tunnels alongside those used here were visited on a regular basis by grower’s groups representing a range of crop sectors. For example, in the final growing season the facilities were visited by several dozen growers during a meeting focussing on leafy salads, who received specific information on the data on disease control obtained under CSA6138, presented by staff employed on the contract. In addition, results from CSA6138 were referred to in articles on Lancaster-STC research on smart plastics published in HDC News. As noted above, the good links between the research and commercial producers has already led to the results being exploited by growers through their adoption of UV-transparent films for propagation crops. Results from the project were also presented at the International Symposium on Horticultural Lighting and the Meeting of the American Photobiological Society, resulting in peer reviewed papers in “Photochemistry and Photobiology” (31) and “Acta Horticulturae” (52).

References to published material1. Bjorn, L. O. and Murphy, T. M. 1985. Computer Calculation of Solar Ultraviolet-Radiation at Ground-Level.

Physiologia Plantarum 64: A23-A232. Brosche, N. and Strid, A. 2003. Molecular events following perception of ultraviolet-B radiation by plants. Physiologia

Plantarum 117: 1-103. Brown, D. M., Kelly, G. E. and Husband, A. J. 2005. Flavonoid compounds in maintenance of prostate health and

prevention and treatment of cancer. Molecular Biotechnology 30: 253-2704. Caldwell, M. M., Camp, L. B., Warner, C. W. and Flint, S. D. 1986 Action spectra and their key role in assessing

biological consequences of solar UV-B radiation change in Stratospheric Ozone Reduction, Solar Ultraviolet Radiation and Plant Life; Workshop on the Impact of Solar Ultraviolet Radiation Upon Terrestrial Ecosystems, 1. Agricultural Crops. (eds. Worrest, R. C. and Caldwell, M. M.) 87-111 (Springer, New York).

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