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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.

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Project identification

1. Defra Project code HH3204

2. Project title

Integrated use of biological control agents for sustainable control of Allium white rot

3. Contractororganisation(s)

Warwick HRIUniversity of WarwickWellesbourneWarwickCV35 9EF     

54. Total Defra project costs £ 293294

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

end date................. 31 March 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 In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain.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.

Allium white rot (AWR) is caused by the fungal pathogen Sclerotium cepivorum and is the major soilborne disease of onions and other Allium crops in the UK. The fungus forms durable resting structures (sclerotia) which can survive in the soil for over 20 years. Tebuconazole is currently the only fungicide available for use against AWR and there are no resistant onion varieties. Control of AWR is therefore difficult but a previous Defra project (HH1813SFV) identified Trichoderma isolates (Trichoderma viride isolates L4 and S17A; Trichoderma pseudokoningii 99-27) as potential biological control agents (BCAs) which could be used as a new control treatment in an integrated control strategy. The first aim of this project was to establish the soil temperature and moisture range which would support the disease control activity of T. viride. A further aim was to determine whether AWR control by Trichoderma could be integrated and enhanced by combining with a) onion accessions which may have partial resistance to AWR, b) tebuconazole treated seed and c) composted onion waste (also shown to suppress AWR; HortLINK220). T. viride parasitises and degrades S. cepivorum sclerotia which results in the reduction of AWR disease. A sclerotial degradation assay was therefore used as a measure of T. viride L4 and S17A activity at soil temperatures of 5, 10, 15, 20 and 25°C and soil moisture levels between 2.5% (dry soil) and 24% (saturated soil). T. viride was added to S. cepivorum sclerotia in soil and the number of soft and degraded sclerotia assessed after 8 weeks. Results showed that L4 and S17A could degrade sclerotia at moisture content as low as 5% but was optimum at ≥ 10% for L4 and ≥ 15% for S17A. Degradation also occurred for temperatures as low as 5°C but was greatest for temperatures above 10°C. T. viride L4 and S17A were therefore shown to be active against S. cepivorum for the range of temperature and soil moisture levels commonly found

To determine whether Trichoderma could be integrated with other potential AWR control methods, experiments were carried out in the glasshouse and field testing different treatments

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and combinations in soil infested with S. cepivorum sclerotia and monitoring subsequent AWR disease levels on onion plants. Treatments consisted of onion accessions, tebuconazole seed treatment and composted onion waste.

In glasshouse and field tests, new onion accessions derived from lines previously thought to be resistant to AWR from the Warwick HRI gene bank did not show any potential resistance compared to commercial onion varieties. However, T. viride S17A consistently reduced AWR when combined with different onion accessions or varieties demonstrating that efficacy was independent of plant genotype.

Tebuconazole seed treatment and composted onion waste were both found to be effective control methods for AWR in glasshouse experiments. When these treatments were combined with applications of either T. viride L4 and S17A, disease control was almost always enhanced compared to using T. viride alone. This was also demonstrated in the field for T. viride S17A combined with the tebuconazole seed treatment. These results demonstrate that biological control of AWR by T. viride can be improved by combining with both tebuconazole seed treatment and use of composted onion waste.

Trichoderma, tebuconazole and onion compost therefore show great potential in an integrated control strategy for effective and sustainable control of AWR disease, but further work is required for effective development and exploitation. In particular, the efficacy of Trichoderma combined with composted onion waste must be tested in the field as well as the full combination of Trichoderma, tebuconazole and onion compost. In addition, the project results have identified the need for further work on the biology of both T. viride and S. cepivorum in order to fully understand their interaction and how this might further be exploited in the future.

This research has made considerable impact on both scientific and grower / industry communities in the form of numerous scientific and industry publications and presentations.

Project Report to Defra8. 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).

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Introduction and background

Allium white rot (AWR) caused by the fungal pathogen Sclerotium cepivorum is the most important and damaging soilborne disease of Allium crops. A disease loss of only 15% due to AWR in the UK onion crop valued in 2003 at £46 M (Defra Horticultural Statistics) equates to a reduction in revenue of £ 7M. This does not take into account further financial loss due to land taken out of production because pathogen levels are too high. This is estimated at between 20-50% of the land suitable for onion cultivation.

S. cepivorum forms extremely durable resting structures (sclerotia) which can remain viable for at least 20 years and therefore disease control is very difficult. In the UK, there are no chemicals with full approval for AWR control although tebuconazole (Folicur) can be applied under the off-label scheme. Soil fumigants are difficult to apply, expensive, often unreliable and are becoming environmentally unacceptable. Steam sterilisation on a field scale is still unrealistic. Furthermore, there are no resistant bulb onion varieties yet identified which are suitable for commercial production. Consequently, there is a need to identify alternative control measures for this disease.

Previous Defra funded work (HH1813SFV) identified Trichoderma spp. as effective fungal biological control agents (BCAs) which reduced AWR in both field and glasshouse tests. However, activity of these BCAs needed to be confirmed over a range of temperatures and water availabilities to provide information on whether control by the BCAs is likely to be reliable under UK onion growing conditions. As the BCAs do not give complete control of AWR, and in order to develop a sustainable, integrated control strategy for the disease, the potential of combining Trichoderma with other control measures also required investigation. These included the use of a) onion accessions which may have partial resistance to AWR, b) a standard tebuconazole seed treatment and c) composted onion waste which has been shown to decrease sclerotial survival (HortLINK22).

Project Objectives

1. Determine the effect of soil water potential on activity of potential biocontrol agents of Allium white rot2. Assess the effect of temperature on the activity of potential BCAs of Allium white rot3. Examine the effect on white rot disease of combining known BCAs with both onion accessions exhibiting resistance to white rot and similar commercially available onion cultivars.4. Examine the effect on white rot disease of combining known BCAs with tebuconazole treatment.5. Examine the effect on white rot disease of combining known BCAs with onion waste compost.

General Materials and Methods

Trichoderma isolates and formulationsThe three Trichoderma isolates used in this project (codes L4, S17A, 99-27) originated from roots and bulbs from white rot infected UK onion fields and parasitised sclerotia of S. cepivorum. Their ability to degrade S. cepivorum sclerotia and control AWR in glasshouse and field tests was confirmed in a previous DEFRA project (HH1828SFV; Clarkson et al., 2002). Two species were represented: T. viride (S17A, L4; IMI 386638 and IMI 386639) and T. pseudokoningii (99-27; IMI 386640). Trichoderma isolates were cultured on potato dextrose agar (PDA) at 20°C, and stored in liquid nitrogen. Trichoderma wheat bran cultures were obtained by inoculating 250 ml flasks containing sterile wheat bran (12 g) and water (30 ml) with spore suspensions (5 ml) and incubating for 3 days at 20°C. Trichoderma alginate pellets were made by combining 30 g Trichoderma biomass grown in yeast molasses medium (15g molasses, 2.5g brewers yeast, 500 ml water) for 9 days at 20°C and 110 rpm with 750 ml sodium alginate solution (26.6 g L-1) water) and 250 ml ground wheat bran suspension (200 g L-1) and pumping this mixture drop wise into a calcium chloride solution (5 g L-1). The resulting pellets were dried under airflow and stored at 5°C until used.

S. cepivorum isolate and production of sclerotiaA single isolate of S. cepivorum (code: Kirton) was used in this study and was stored as stock sclerotia at 5°C and in liquid nitrogen. Working cultures were obtained after first surface sterilising stock sclerotia (washing in sodium hypochlorite solution (15% available chlorine)) for 90 s followed by washing three times in sterile distilled water (SDW)) and then squashing onto PDA. Cultures were incubated at 20°C. To obtain large numbers of sclerotia for experiments, mushroom spawn bags each containing an autoclaved mixture of washed sand (1920 g, <2mm particle size), ground maizemeal (80 g), and water (175 ml) were inoculated with a diced PDA Petri dish culture. Bags were heat-sealed and incubated at 20°C for 6 weeks after which sclerotia had formed. Sclerotia were harvested by floatation in water and retrieval in a sieve (212 μm mesh diameter). They were then dried in an airflow for 12 h before storage at 5°C. The sclerotia used in onion seedling bioassays were further subjected to ‘conditioning’ in order to overcome dormancy.

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This consisted of burial in mesh bags for at least 12 weeks in the field (Coley-Smith et al., 1987). After this period, sclerotia were washed, sieved and dried as before and then rolled in filter paper to remove any that were soft and degraded.

Systems used to test Trichoderma and other control treatments for control of AWR

The following methodologies for testing fungal BCAs against AWR were developed as part of Defra project HH1828SFV and full details have been published (Clarkson et al., 2002).

a) Sclerotial degradation assay: this assay was used to assess the ability of T. viride L4 and S17A to degrade sclerotia of S. cepivorum in a silty clay soil at different temperature and water potentials. Sclerotia were placed in mesh bags (150 μm mesh diameter) with 10 g soil (100 sclerotia per bag). T. viride was then added to each bag as wheat bran inoculum (1 g in 100 g soil).The bags were then tied and buried in clear plastic boxes containing 175 g soil. Water was added as appropriate to give the desired water potential and boxes were incubated at the required temperature for 8 weeks. Sclerotia were then retrieved by floatation and sieving and assessed for degradation (soft or collapsed) by squeezing with forceps under a low power binocular microscope (x 120). In all experiments each treatment tested consisted of three replicate boxes arranged as a randomised complete block design. Control treatments (sclerotia only, no Trichoderma added) were also included.

b) Onion seedling bioassay: this assay was used to test the ability of Trichoderma either alone or in combination with different onion accessions, composted onion waste or tebuconazole seed treatments to reduce white rot disease on onion seedlings in glasshouse pot trials. The method was adapted as appropriate depending on the test treatments. Conditioned sclerotia of S. cepivorum were mixed thoroughly with soil (1.5 sclerotia g-1 soil) and Trichoderma added as wheat bran cultures (1 g in 100 g soil). The amended soil was mixed well and then added to 7 cm pots (220 g per pot) and onion seed (one seed per pot) planted. In all experiments there were 10 pots for every test treatment within each of five replicate blocks (total 50 plants per treatment) arranged in a randomised complete block design in the glasshouse (min. temperature 15°C). Appropriate control treatments were included. All watering was from below and the emerging onion plants were assessed for symptoms of AWR every week until no further disease occurred.

Statistical Analyses

The efficacy of the Trichoderma isolates in degrading sclerotia was calculated as the number of degraded (soft) sclerotia recovered plus the number of unrecovered sclerotia (assumed to be totally degraded) as a proportion of the total number of sclerotia (100) used in each experimental plot. These proportions were analysed using a generalised linear model (GLM) assuming a binomial distribution and logit link function. Predicted proportions were estimated from each analysis. One-sided t-tests were performed on logit coefficients to determine significant increases in the proportions of degraded/lost sclerotia for Trichoderma isolates relative to the control. The effect of changing water content or temperature on sclerotial degradation was quantified by fitting linear regressions within the GLM resulting in sigmoidal curves when plotted against proportion of degraded sclerotia.

For onion seedling bioassays testing the effects on AWR of different treatments either individually or in combination, the efficacy in controlling white rot was estimated by comparison with appropriate controls. The final number of seedlings with white rot at the last assessment date as a proportion of the number emerged was analysed for each treatment using the GLM approach and significant reductions in the proportions of seedlings with white rot compared to appropriate control treatments determined. The same approach was used in the analyses of field experiments.

Results

Objective 1: Determine the effect of soil water potential on activity of potential biocontrol agents of Allium white rot

Experiment: The ability of T. viride isolates L4 and S17A to degrade S. cepivorum sclerotia in a silty clay soil at 20°C and six soil water potentials was tested using the sclerotial degradation assay. The soil in the mesh bags and in the boxes was adjusted to 2.5, 5, 10, 15, 21 and 24% water content corresponding to water potentials of -55.2, -4.03, -0.022, -0.00012, -2.17 x 10-7, and -9.40 x 10-9 MPa respectively. Control treatments consisted of sclerotia only (no Trichoderma) in soil at 5, 15 and 24 % water content.

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Results: For both T. viride isolates there was a clear relationship between log10 water potential and the proportion of sclerotia lost or degraded at 20°C as described by the fitted regression models (Fig. 1). Degradation occurred for both L4 and S17A at water potentials as low as -4.03 MPa (5% water content) and was optimum at ≥ -0.022 MPa for L4 and ≥ -0.00012 MPa for S17A (Fig. 1). There was no degradation of sclerotia by either T. viride isolate for the lowest soil water potential of -55.2 MPa. To compare the two T. viride isolates, the soil water potential required to produce 50% and 90% degradation of S. cepivorum sclerotia at 20°C after 8 weeks was calculated. T. viride isolate L4 required a water potential of -1.26 MPa to achieve 50% degradation compared to -0.056 MPa for S17A whereas for 90% degradation of sclerotia, L4 required a water potential of -0.0137 MPa compared with -0.00001 MPa for S17A. Soil water potential also affected S. cepivorum sclerotia in the absence of T. viride. At the greatest water potential (-9.40 x 10-9 MPa; 24% soil water content), 97% of S. cepivorum control sclerotia not treated with T. viride were degraded in the experiment with L4 and 87% in the experiment with S17A (Fig. 1). Degradation of control sclerotia was 25-35% in both experiments at -0.00012 MPa (15% soil water content) compared with 2-7% degradation at -4.03 MPa (5% soil water content).

Figure 1: Effect of soil water potential on the degradation of S. cepivorum sclerotia by T. viride L4 (a) and S17A (b) after 8 weeks at 20°C in silty clay soil. Lines for L4 and S17A fitted through regression within the GLM analysis. Bars represent standard errors.

Objective 1 conclusions Both T. viride isolates degraded the majority of S. cepivorum sclerotia at soil moisture levels

of 10% or above (-0.022 MPa) but efficacy decreased in drier soil. S. cepivorum sclerotia degrade when soil is saturated in the absence of Trichoderma.

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Objective 2: Assess the effect of temperature on the activity of potential BCAs of Allium white rot

Experiment: The ability of T. viride isolates L4 and S17A to degrade S. cepivorum sclerotia in a silty clay soil at 5, 10, 15, 20 and 25°C for 5, 10 and 15% water content (-4.03, -0.0012, -0.022 MPa) was tested using the sclerotial degradation assay as before. Control treatments consisted of sclerotia only (no Trichoderma) in soil at each temperature at 15% water content.

Results: For both T. viride isolates there was a clear relationship between sclerotial degradation and temperature as described by the fitted regression models (Fig. 2). Percentage degradation of sclerotia increased for both T. viride isolates with increasing temperature at -0.00012 and -0.022 MPa from 17-33% at 5°C to 60-89% at 25°C. However at -4.03 MPa there was < 17% degradation at any temperature for T. viride L4 and degradation increased from 5% to 48% between 5 and 25°C for S17A (Fig. 2). When the proportion of sclerotia degraded by the T. viride isolates was compared with control treatments at -0.00012 MPa, degradation increased (P ≤ 0.01) at all temperatures for L4 and at 10-25°C for S17A. For control sclerotia without addition of T. viride at -0.00012 MPa, degradation increased from 4% at 5°C to 36% at 25°C in the experiment with S17A but this did not occur for the experiment with L4 (Fig. 2).

Figure 2: Effect of temperature on the degradation of S. cepivorum sclerotia by T. viride isolates L4 (a) and S17A (b) at three soil water potentials after 8 weeks at 20°C in silty clay soil. Lines for L4 and S17A fitted through regression within the GLM analysis. Bars represent standard errors.

Objective 2 conclusion When there is sufficient soil moisture, degradation of S. cepivorum sclerotia by T. viride

increases with increasing temperature between 5 and 25°C.

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Objective 3: Examine the effect on white rot disease of combining known BCAs with both onion accessions exhibiting resistance to white rot and similar commercially available onion cultivars

In a previous Defra project (HH1828SFV) eight Rijnsburger type bulb onion accessions from the Warwick-HRI gene bank (92006, 92010, 92016, 92026, 92038, 92059, 92061, 92064) were tested for resistance to AWR. These had previously been reported to exhibit some resistance by other workers, but results from Defra project HH1828SFV showed that there was little difference in disease levels between these accessions and the commercial variety Hysam. Nevertheless, at the start of the current project, these accessions were openly crossed with one another to produce progeny accession lines listed in Table 1 to determine if AWR resistance could be detected in these new lines.

New Accession Parent Accession

New Accession

Parent Accession

New Accession

Parent Accession

2001 92006 2023 92038 2046 920612002 92006 2024 92038 2053 920612006 92006 2028 92038 2055 920612008 92006 2031 92038 2061 920642009 92010 2033 92059 2063 920642010 92010 2037 92059 2066 920642011 92010 2035 92059 2068 920642015 92010 2039 92059

Table 1: List of new bulb onion accessions tested against AWR

Onion seedling bioassays: Eight of the new bulb onion accessions (2010, 2015, 2023, 2035, 2046, 2053, 2055, 2061) and five combinations of accessions (2002&2006, 2009&2011, 2024&2031, 2033&2037, 2066&2068) were tested with and without T. viride S17A added to infested soil as wheat bran cultures for control of AWR in three replicate onion seedling bioassays and compared with the commercial variety Hystar. Combinations of accessions were used where only small amounts of seed were available and were from the same parent accession. Six commercial onion varieties (White Lisbon (salad onion), Hysam, Red Baron, Summit, Renate, Supasweet (bulb onions)) were also tested for control of AWR with and without T. viride S17A and L4 in two onion seedling bioassays.

Seedling bioassay results: The new bulb onion accessions showed a wide range of AWR disease levels within each experiment (proportion infected plants 0 - 0.90) but there was no consistent effect of any of the accessions over all the experiments (data not shown). Briefly, the only accessions or combination of accessions which resulted in significantly less AWR than the control commercial variety Hystar were 2053 in experiment 1, and 2015, 2061 and 2009+20011 in experiment 3. The majority of accessions were therefore at least as susceptible to AWR as Hystar. When the accessions were treated with T. viride S17A, the BCA consistently reduced AWR compared to untreated plants and over all the accessions, this effect was significant in each of the three experiments (P ≤ 0.01). In the experiments with the commercial onion varieties, T. viride S17A and L4 significantly reduced the final proportion of plants with AWR in both experiments, with the exception of L4 with White Lisbon and Hysam in experiment 1 and S17A with Summit and Renate in experiment 1 and Supasweet in experiment 2, although disease was still reduced in these treatments (Fig. 3). The reduction in AWR disease compared to untreated plants for each variety for S17A and L4 ranged from 29-71% and 44-73% respectively over both experiments. In the absence of T. viride S17A or L4 there was no significant difference between disease levels for any of the commercial varieties.

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Figure 3: Final proportion of onion plants infected with AWR for commercial bulb onion varieties with and without T. viride S17A or L4 for experiments 1 (a) and 2 (b). Significance from untreated controls (no Trichoderma) for each variety shown as *** P ≤ 0.001, ** P ≤ 0.01, *P ≤ 0.05, NS=not significant.

Field experiments: Field experiments were carried out in 2003 and 2004 to assess commercial bulb onion varieties and WHRI onion accessions combined with T. viride S17A and / or L4 for control of AWR. Experiments were carried out in a quarantine field at WHRI Wellesbourne which was infested with sclerotia of S. cepivorum at a rate of approx. 50,000 per m2 each year.

In 2003, four commercial bulb onion varieties (Hystar, Red Baron, Renate and Supasweet) were tested in the field with and without T. viride S17A and L4. Trichoderma was applied as wheat bran cultures suspended in a guar gum gel (240 g wheat bran culture, 50 g guar gum, 1 L water) at sowing using a fluid drill apparatus. This system delivered approx. 7-10 ml T. viride suspension per m row of bulb onion seed. A tebuconazole seed treatment for Hystar was included as a fungicide control. There were four replicate plots per treatment, each consisting of four 6 m rows 35 cm apart in beds 1.83 m wide. Each week, all onion plants in the middle two rows of each plot were assessed for typical AWR symptoms of wilting or severe leaf yellowing and infection was confirmed by examining the roots and stem base for mycelium or sclerotia.

In 2004, 13 onion accessions (2001, 2008, 2010, 2015, 2023, 2028, 2035, 2039, 2046, 2053, 2055, 2061, 2063) and five onion accession combinations (2002&2006, 2009&2011, 2024&2031, 2033&2037, 2066&2068) were tested in the field with and without T. viride S17A for control of AWR. This time the Trichoderma was applied as alginate pellets in furrow at a rate of 115 g m-2 at sowing. Seed and pellets were delivered using a cone drill. There were three replicate rows 3.05 m long for each treatment randomised within beds 1.83 m wide of four rows 35 cm apart. Hystar was included as a commercial control variety (six replicate rows, with and without T. viride S17A). Each week, all plants were assessed for symptoms of AWR as before until harvest.

Field experiment results 2003: Environmental conditions resulted in little AWR disease in the field experiment and hence differences in treatments were not large enough to be of significance. Generally however, for the three commercial varieties tested, T. viride S17A and L4 reduced the final proportion of AWR infected plants compared to the untreated controls and tebuconazole was also effective. The commercial varieties were therefore tested again in 2004 with T. viride S17A (see Objective 4).

Field experiment results 2004: In the onion accession experiment, the final proportion of plants with AWR varied between 0.54 and 0.80 for untreated accessions but none of them reduced disease significantly compared to the commercial variety Hystar. T. viride S17A significantly reduced the final proportion of plants with AWR for every accession or combination of accessions compared to untreated control plants with the exception of 2008 (Fig. 4). Disease reduction compared to untreated plants ranged from 17 (Hystar) to 53% (2066&2068).

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Figure 4: Final proportion of onion plants infected with AWR for different onion accessions with and without T. viride S17A in the field in 2004. Significance from untreated controls (no Trichoderma) for each accession shown as *** P ≤ 0.001, ** P ≤ 0.01, *P ≤ 0.05, NS=not significant.

Objective 3 conclusions No AWR resistance was detected either in new bulb onion accessions or in existing

commercial onion varieties. T. viride consistently reduced AWR in glasshouse and field experiments.

Objective 4: Examine the effect on white rot disease of combining known BCAs with tebuconazole treatment.

Onion seedling bioassays: The effect on AWR of Trichoderma (T. viride S17A or L4 or T. pseudokoningii 99-27) and tebuconazole seed treatment alone or in combination was tested using the bulb onion variety Hysam in two onion seedling bioassays. T. viride was incorporated as wheat bran cultures into soil infested with S. cepivorum sclerotia in pots either six weeks before or at sowing of the untreated or fungicide-treated seed. Pots of soil infested with S. cepivorum sclerotia and treated with Trichoderma were watered regularly during the 6 weeks before sowing. An untreated control (no Trichoderma, no tebuconazole) was included for both application times of the Trichoderma. The inclusion of the six week pre-sowing treatments was to allow for the possibility that tebuconazole might inhibit the performance of the Trichoderma isolates when applied at sowing.

Field experiment: In 2004, three commercial bulb onion varieties (Hystar, Red Baron and Renate) were tested against AWR in combination with T. viride S17A and/or a tebuconazole seed treatment in the field. T. viride S17A was applied as alginate pellets in furrow at 115 g m-2. There were four replicate plots per treatment each consisting of four 3.05 m rows 35 cm apart in beds 1.83 m wide. Each week, all onion plants in the middle two rows of each plot were assessed for AWR symptoms until harvest.

Seedling bioassay results: In the absence of tebuconazole, all three Trichoderma isolates significantly reduced the final proportion of onion plants with AWR whether applied at 6 weeks pre-sowing or at sowing (Fig. 6). Disease reduction compared to the untreated controls (no Trichoderma, no tebuconazole) was greater in both experiments when Trichoderma was applied at sowing (L4, 68-96% and S17A, 70-84%) than when applied 6 weeks before (L4, 35-68% and S17A, 46-48%). In the absence of Trichoderma, the tebuconazole seed treatment significantly reduced AWR compared to the untreated control at sowing (47 and 51% disease reduction for experiments 1 and 2 respectively) and also when soil and sclerotia were left for 6 weeks pre-sowing (63 and 54% disease reduction for experiments 1 and 2 respectively).

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2001 2008 2010 2015 2023 2028 2035 2039 2046 2053 2055 2061 2063 2002 2009 2024 2033 2066 Hystar &2006 &2011 &2031 &2037 &2068

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Figure 6: Final proportion of onion plants (Hysam) infected with AWR for Trichoderma (T. viride L4 or S17A or T. pseudokoningii 99-27) applied 6 weeks pre-sowing or at sowing either alone or with tebuconazole treated seed for two experiments. Significance from untreated control (no tebuconazole, no Trichoderma) shown as *** P ≤ 0.001, ** P ≤ 0.01, *P ≤ 0.05.

When Trichoderma and tebuconazole were combined, the final proportion of plants with AWR was significantly reduced compared to the untreated controls (no Trichoderma, no tebuconazole) in both experiments for all Trichoderma isolates and for both application times (53-94% disease reduction). The use of tebuconazole with Trichoderma generally improved AWR control compared to using the BCAs alone and this was significant for the treatments shown in Table 2. The application of Trichoderma also significantly improved AWR control compared to using tebuconazole alone for the treatments shown in Table 3.

Experiment 1 Experiment 299-27 applied at sowing ( P≤ 0.05) L4 applied at sowing (P ≤ 0.05)L4 applied 6 weeks pre-sowing (P ≤ 0.05) L4 applied 6 weeks pre-sowing (P ≤ 0.001)S17A applied 6 weeks pre-sowing (P ≤ 0.001) 99-27 applied 6 weeks pre-sowing (P ≤ 0.001)

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Table 2: Trichoderma treatments where control of AWR was better when combined with tebuconazole seed treatment than using Trichoderma alone.

Experiment 1 Experiment 2S17A at sowing (P ≤ 0.05) L4 at sowing (P ≤ 0.01)99-27 at sowing (P ≤ 0.05) S17A at sowing (P ≤ 0.01)L4 applied 6 weeks pre-sowing (P ≤ 0.01) L4 applied 6 weeks pre-sowing (P ≤ 0.01)S17A applied 6 weeks pre-sowing (P ≤ 0.01) 99-27 applied 6 weeks pre-sowing (P ≤ 0.01)99-27 applied 6 weeks pre-sowing (P ≤ 0.05)

Table 3: Trichoderma treatments where control of AWR was better when combined with tebuconazole seed treatment than using tebuconazole alone.

Field experiment results 2004: The final proportion of infected plants for the commercial bulb onion varieties ranged from 0.36-0.57 for untreated plants. For Hystar and Red Baron, this proportion was reduced when T. viride S17A was applied (27% and 37% disease reduction respectively) but this was only statistically significant for Red Baron (Fig. 5). There was no control of AWR for S17A with Renate. Tebuconazole treated seed of each variety resulted in the lowest proportion of plants with AWR (0.07-0.11, 70-85% disease reduction) and for Hystar and Renate, this was comparable with the level of control when tebuconazole was combined with T. viride S17A. These two combination treatments significantly reduced AWR compared to T. viride applied alone (P ≤ 0.05) but this was not the case for Red Baron where the combination treatment of Trichoderma and tebuconazole was less effective but still reduced disease significantly compared to the untreated control. No additional effects of T. viride in combination with tebuconazole compared to tebuconazole alone were detected.

Figure 5: Final proportion of onion plants infected with AWR for different commercial bulb onion varieties in combination with T. viride S17A and / or tebuconazole seed treatment in the field in 2004. Significance from untreated controls for each variety (no tebuconazole, no Trichoderma) shown as *** P ≤ 0.001, ** P ≤ 0.01, *P ≤ 0.05, NS=not significant.

Objective 4 conclusions In seedling bioassays, both T. viride and tebuconazole treated seed reduced AWR when used

alone. Combining T. viride and tebuconazole in seedling bioassays resulted in enhanced control of

AWR and in many cases was better than using either treatment alone. In field tests, T. viride reduced AWR for two commercial bulb onion varieties but the

tebuconazole seed treatment was better. Combining T. viride and tebuconazole in field tests was effective, but was no improvement

on using tebuconazole alone.

Objective 5: Examine the effect on white rot disease of combining known BCAs with onion waste compost

Onion seedling bioassays: The effect on AWR of T. viride S17A or L4 and composted onion waste alone or in combination was tested using the bulb onion variety Hysam in two onion seedling bioassays. Bulb

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onion waste was composted at 50°C for 7 days using a laboratory method developed by Coventry et al., (2002). The composted onion waste was mixed with soil infested with S. cepivorum sclerotia (50% v/v) in polythene bags and incubated for 12 weeks at 15°C. At the end of this period, the soil / compost mix was dispensed into pots and onion seedlings transplanted (one per pot). Trichoderma was added as wheat bran cultures to soil only or the soil/compost mix either in bags 12 weeks pre-planting or at planting. Soil and sclerotia only incubated in bags for 12 weeks at 15°C was included as a control treatment (no compost, no Trichoderma)

Results: In the absence of composted onion waste, T. viride S17A and L4 applied 12 weeks pre-planting or at planting significantly reduced the final proportion of onion plants with AWR (Fig. 7). Disease reduction compared to the untreated control (no Trichoderma, no compost) ranged from 62 to 96% for both application times and both experiments. In the absence of Trichoderma, the composted onion waste also significantly reduced the final proportion of plants with AWR and disease reduction was 91% in experiment 1 and 96% in experiment 2. When Trichoderma and composted onion waste were combined, little or no AWR developed (disease reduced 96-100%) and again disease reduction was significant compared to the untreated control (P ≤ 0.001). The use of composted onion waste combined with Trichoderma generally improved AWR control compared to using the BCAs alone. However, significant additive effects compared to Trichoderma used alone were not detected with the exception of T. viride S17A applied 12 weeks pre-sowing in experiment 1 as disease levels for most of the treatments were very low. Additive effects of Trichoderma when combined with compost compared to using compost alone were also not detected for the same reason.

Figure 7: Final proportion of onion plants (Hysam) infected with AWR for Trichoderma (T. viride L4 or S17A) applied 12 weeks pre-sowing or at sowing either alone or with composted onion waste for two experiments. Significance of treatments compared to the untreated control (no compost, no Trichoderma) shown as *** P ≤ 0.001, ** P ≤ 0.01, *P ≤ 0.05.

Objective 5 conclusions T. viride and composted onion waste reduced AWR when used alone. Combining T. viride and onion compost resulted in further reduction of AWR.

Discussion and implications of results

Trichoderma viride L4 and S17A consistently reduced AWR disease in repeated glasshouse and field experiments when applied without any other treatment. Over all the onion seedling bioassays carried out in this project, disease reduction for both isolates varied between 29 and 96% compared to untreated plants and for T. viride S17A, control was 17-39% in the field. Clearly, these Trichoderma isolates have great potential as biological control agents (BCAs) of AWR but variation in the level of disease control is unpredictable. Experiments also showed that T. viride L4 and S17A can degrade S. cepivorum over a wide range of temperatures and water potentials but that wetter conditions (-0.022Mpa) and temperatures

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greater than 10°C are optimum. Dry conditions in particular reduce the activity of T. viride and this may to some extent explain the observed variability in the degree of AWR control in the field and glasshouse tests where soil moisture can potentially vary over time. However, the results do suggest that if applied in a UK field situation, T. viride would be active when the soil is moist and temperatures increase in Spring and early Summer which coincides with the period when AWR is most likely to occur.

In order to address the potential problem of variability in control of AWR by Trichoderma and to establish compatibility with other control methods, combinations of BCAs with new and potentially resistant bulb onion accessions, tebuconazole seed treatment or composted onion waste were investigated for enhanced AWR control. On their own, none of the new onion accessions showed a consistent effect on AWR or showed reduced AWR levels compared to commercial bulb onion varieties. None of these varieties showed any differences in AWR susceptibility either and hence, combining any onion type with T. viride was not likely to show any additional effects above that of the BCA. However, the results did confirm the consistent suppressive effect of Trichoderma on AWR for all varieties and accessions and this effect was therefore independent of onion genotype.

The use of tebuconazole as a seed treatment was effective on its own in reducing AWR in the glasshouse and field. Combining tebuconazole with Trichoderma gave additional control in onion seedling bioassays, but in the field, the combination was comparable to tebuconazole alone. The onion bioassay results suggest that Trichoderma when applied at sowing is not inhibited by a tebuconazole seed treatment and that the two control methods are compatible. Trichoderma has, however, been shown to be sensitive to tebuconazole (McLean et al., 2001), but it could be that spatial separation of chemical and fungus in soil even within a small pot allows Trichoderma to survive and destroy additional S. cepivorum sclerotia not affected by tebuconazole. In glasshouse experiments, Trichoderma reduced AWR when applied before or at sowing. Control was not improved when Trichoderma was applied 6 weeks pre-sowing which could have had the advantage of allowing the fungus more time to degrade S. cepivorum sclerotia. In fact, more disease developed for untreated control and Trichoderma treatments set up pre-sowing compared to the same treatments at sowing. Sclerotia kept in moist soil for 6 weeks therefore were more infective than those from the same source stored in the laboratory during this period and used to infest soil at sowing. One explanation for this is that the sclerotia kept in moist soil in the glasshouse were being 'conditioned'. S. cepivorum sclerotia require a period of conditioning to overcome dormancy before they will germinate and infect onion plants. Although sclerotia used in all onion seedling bioassays were given a 12 week conditioning period in the field as suggested by other researchers (Coley-Smith et al., 1987), it could be that only a proportion were conditioned during this time and that a further 6 weeks in soil in the glasshouse allowed additional sclerotia to condition and germinate, hence causing more disease.

Composted onion waste was also very effective in reducing AWR disease in onion seedling bioassays when applied alone. When the compost was combined with T. viride, little or no disease developed. Although the results suggested that the combination treatment was better than either treatment alone, the low AWR levels for the individual treatments precluded detection of significant additive effects in the analysis. Further work with reduced compost rates would be required to determine if the two control methods are truly compatible or whether the compost could inhibit Trichoderma. In the absence of composted onion waste, there was no advantage in applying Trichoderma 12 weeks before sowing compared to at sowing suggesting, as in the experiments with tebuconazole, that Trichoderma does not need to be applied in advance in order to reduce AWR.

Overall, it has been demonstrated that the use of Trichoderma, tebuconazole treated seed or composted onion waste are effective control measures for AWR disease. Combining Trichoderma with either tebuconazole or onion compost enhanced control compared with using Trichoderma alone. These three control measures therefore have great potential for use in an integrated control strategy for AWR.

Potential future work

Further work is required if the results from this research and the potential for integrated control of AWR is to be fully developed and exploited. Further treatments and combinations require field testing and in particular the following should be addressed:

testing of onion compost in the field both alone and in combination with T. viride testing of all treatments in combination in the field (T. viride, tebuconazole, onion compost) testing different rates of onion compost with and without T. viride

These tests have been included in a proposal for HortLink funding.

Other important areas concerning the biology of both S. cepivorum and T. viride also require investigation:

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Results indicated that a proportion of S. cepivorum sclerotia used in seedling bioassays may not be fully conditioned and able to germinate despite the recommended field treatment. Therefore the effect of environmental factors such as temperature and soil water potential on conditioning of S. cepivorum sclerotia needs to be examined in relation to their subsequent survival, susceptibility to T. viride and infectivity to onion plants.

T. viride has been applied successfully in this project as wheat bran cultures or alginate pellets incorporated into soil. There is no information on growth or survival of the BCA from these or other formulations which would be useful when considering application rates and times. Studies involving direct colonisation of onion compost by different formulations of BCAs and monitoring populations in soil would also be particularly beneficial.

As the isolates of T. viride used in this project are almost unique in their ability to control white rot in the field, a detailed study of their modes of action would be valuable to help target their optimum method of use and identify how they differ from other Trichoderma. This could involve detailed studies of mycoparasitism by the BCAs against S. cepivorum sclerotia and an examination of their degree of activity from sclerotial germination, through growth in soil to plant colonisation.

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

Publications related to the project

Coley-Smith JR, Parfitt D, Taylor IM, Reese R A, 1987. Studies in dormancy of sclerotia of Sclerotium cepivorum. Plant Pathology 36, 246-257.

McLean et al., 2001. Compatibility of the biocontrol agent Trichoderma harzianum C52 with selected fungicides. New Zealand Plant Protection 54, 84-88.

Publications generated by the project

Refereed papers

Clarkson JP, Mead A, Payne T, Whipps JM, 2002. Selection of fungal biological control agents of Sclerotium cepivorum for control of white rot by sclerotial degradation in a UK soil. Plant Pathology 51, 735–745.

Clarkson JP, Mead A, Payne T, Whipps JM, 2004. Effect of environmental factors and Sclerotium cepivorum isolate on sclerotial degradation and biological control white rot by Trichoderma spp. Plant Pathology 53, 353-362.

Clarkson JP, Whipps JM, 2002. Control of sclerotial pathogens in horticulture. Pesticide Outlook 13, 97 101.

Coventry E, Noble R, Mead A, Whipps JM, 2002. Control of Allium white rot (Sclerotium cepivorum) with composted onion waste. Soil Biology & Biochemistry 34, 1037-1045.

Coventry E, Noble R, Mead A, Whipps JM, 2005.Suppression of Allium white rot (Sclerotium cepivorum) in different soils using vegetable wastes. European Journal of Plant Pathology 111, 101-112.

Abstracts and Proceedings

Clarkson JP, Coventry E, Noble R, Whipps JM, 2003. Integrated Control of Allium White Rot. Proceedings of the UK Onion and Carrot Conference 2003. HDC, UK, p 22.

Clarkson JP, Mead A, Payne T, Whipps JM, 2003. Biological control of Allium white rot by sclerotial degrading fungi. Proceedings of the 8th International Congress of Plant Pathology, 2-7 February 2003, Christchurch, New Zealand, p 34.

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Clarkson JP, Payne T, Whipps JM, 2002. Biological control of Allium white rot and degradation of sclerotia in different UK soils. Proceedings of the BSPP Presidential Meeting 'Plant Pathology and Global Food Security’, Imperial College, London, 8-10 July 2002. BSPP, UK, p 32.

Clarkson JP, Scruby A, Coventry E, Noble R, Whipps JM, 2004. Integrated control of Allium white rot using biological control agents, composted onion waste and tebuconazole treated seed. In: Integration 2004: Management of plant diseases and arthropod pests by biological control agents and their integration in agricultural systems (eds Y. Elad, I Pertot & A. Enkegaard) IOBC/WPRS Bulletin, 27(8), 71-74.

Whipps JM, 2002. Developments in biological control of soilborne plant pathogens. In: Proceedings of the 2nd International Conference on the alternative control methods against plant pests and diseases, Lille, France, 4-7 March 2002, 18-27.

Whipps JM, 2003. Fungi as plant disease control agents: status and prospects. Proceedings of the SGM meeting, 7 April 2003, Edinburgh, p.36.

Popular Articles

Clarkson JP, Coventry E, Noble R, Whipps JM, 2003. Integrated control of Allium white rot. The Grower 140 (19), 18-19.

Clarkson JP, Coventry E, Noble R, Whipps JM, 2004. Research on integrated control of Allium white rot atWarwick HRI. Onion Market News (BOPA Newsletter), 123, p1 (September 2004).

Clarkson JP, Whipps JM, 2004. Fungicide fight back for Allium. Fresh produce Journal, Nov 2004, p12.

Coventry E, Noble R, Whipps JM, 2002. Control of Allium white rot with composted onion waste. Vegetable Farmer.

Coventry E, Noble R, Whipps JM, 2002. Putting waste to a good use. The Grower, May 23, pp. 16-17.

Coventry E, Noble R, Whipps JM, Banham H, 2002. Waste not ……and control of white rot. HDC News, No. 86, pp. 8-10.

Whipps JM, Noble R, Coventry E, Clarkson JP, 2003. UK research seeks sustainable approach to white rot control. Onions Australia 20, 28-29.

Wood R, Whipps, JM, O'Connor D, 2002. Control of white rot in onions. HDC Factsheet 12/02, Onions - bulb and salad, Project Nos. FV 4a-e. East Malling, Horticultural Development Council, 7 pp.

Scientific presentations

John Clarkson, 2003. Biological Control of Allium White Rot. Allium Diseases Evening Session during the 8th International Congress of Plant Pathology, 2-7th February 2003, Christchurch, New Zealand.

John Whipps, 2002. Concepts and approaches during 50 years of biological control of fungal plant pathogens. 7th International Mycological Congress, Oslo, Norway.

John Whipps, 2002. Biotechnology and the development of biological disease control. British Society for Plant Pathology Meeting, London.

John Whipps, 2003. Composting of Allium and other vegetable wastes for the control of Allium white rot. Allium Diseases Evening Session during the 8th International Congress of Plant Pathology, 2-7th February 2003, Christchurch, New Zealand.

John Whipps, 2003. Prospects and limitations for mycorrhizas in biocontrol of root pathogens. Proceedings of the 4th International Conference on Mycorrhizas, 10-15 August, Montreal, Canada.

Grower / Industry Presentations

John Clarkson, 2004. Biological control of Allium rot research at WHRI. Presentation to British Onion

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Producers Association, July 2004.

John Clarkson, 2005. Research on biological control of Allium white rot at WHRI. Presentation to Irish ADAS advisors, February 2005.

John Whipps, 2002. Application of beneficial micro-organisms to leek seeds. Presentation to British Onion Producers Association.

John Whipps, 2003. Biopesticides for the control of plant diseases. WHRIA Meeting 'Biopesticides - the future' - 18 November, WHRI Wellesbourne.

John Whipps, 2003. Invited presentation to the New Zealand VegFed meeting. White rot. UK progress and developments.

John Whipps, 2003. Plant pathology and microbiology research at WHRI. Presentation to visitors from INRA, November, WHRI Wellesbourne.

John Whipps, 2003. Plant pathology and microbiology research at WHRI. Presentation at the Research Unit Flower Bulbs, Lisse, the Netherlands, November.

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