Control of Allium white rot (Sclerotium cepivorum) with composted onion waste

Soil Biology & Biochemistry 34 (2002) 1037±1045

www.elsevier.com/locate/soilbio

Control of Allium white rot (Sclerotium cepivorum) with composted onion waste E. Coventry a, R. Noble a,*, A. Mead b, J.M. Whipps a a

Department of Plant Pathology and Microbiology, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK b Department of Biometrics, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK Received 2 October 2001; received in revised form 21 January 2002; accepted 6 February 2002

Abstract We investigated the possibility of composting onion waste to destroy any sclerotia present and then applying the composted waste to soil to stimulate sclerotial germination to disinfest the soil of the white rot fungus. Three mixtures of dry (shale-skins or onion tops) and wet (peelings or chopped whole bulbs) onion waste were incubated for 7 d at 50 8C with aeration to simulate a large-scale composting process. Under these conditions, a mixture of 10:1 (w/w) wet/dry (80% moisture content) produced the desired characteristics of minimal run-off and no unpleasant odour. This ratio of onion wastes was then inoculated with sclerotia of S. cepivorum and incubated for 3 or 7 d at a range of temperatures (18±60 8C). The pathogen was destroyed after 3 d at temperatures of 48 8C and above. Gas chromatography±mass spectrometry (GC±MS) analysis of raw onion waste and waste incubated for 3 or 7 d at 42 or 54 8C (referred to as `composted waste') revealed the presence of the sclerotia germination stimulant, di-n-propyl disulphide (DPDS). Concentrations of DPDS decreased during incubation. A pot bioassay, with sclerotia buried in sandy loam soil mixed with different rates (1, 10 and 50% w/w) of raw onion waste and waste which had been composted, indicated the composted waste was more effective (suppressive) than the raw waste in reducing the viability of sclerotia. Onion waste which had been incubated at the higher temperature (54 8C) was most active despite a reduction in DPDS content compared with the raw waste. The 50% incorporation rate of this waste was most effective with a reduction in viability of sclerotia recorded after 1 month exposure. Both 1 and 10% rates reduced viability of sclerotia but after a longer exposure than for the 50% rate. The results indicate that other factors are more important in the suppressive effect of composted onion waste on S. cepivorum sclerotia than germination stimulant activity. The results demonstrate the potential use of composted onion waste as a method for white rot control, and as a means of disposing of packhouse onion wastes. q 2002 Published by Elsevier Science Ltd. Keywords: Sclerotium cepivorum; Sclerotia; Allium white rot; Composting; Onion

1. Introduction The soil-borne fungus, Sclerotium cepivorum Berk. is the causal agent of Allium white rot (Entwistle, 1990a), one of the most serious diseases of onions (Allium cepa L.) (Merriman et al., 1980; Metcalf and Wilson, 1999). The disease can reduce yields to uneconomic levels in 4 years of successive onion crops (Coley-Smith, 1987). The fungus penetrates the root epidermis then invades the cortical parenchyma (Abd El-Razik et al., 1973). Infected plants suffer from water stress and often die prior to harvest or rot in storage (Entwistle, 1990b). The pathogen persists in the soil in the absence of host plants as sclerotia and can survive in this form for more than 20 years (Coley-Smith and Par®tt, 1986; Coley-Smith et al., 1990). Following their * Corresponding author. Tel.: 144-1789-470382; fax: 144-1789472076. E-mail address: [email protected] (R. Noble). 0038-0717/02/$ - see front matter q 2002 Published by Elsevier Science Ltd. PII: S 0038-071 7(02)00037-8

production, sclerotia are constitutively dormant for 1±3 months and will then only germinate in the presence of host plants (Coley-Smith et al., 1987). The stimulus for germination is the exudation of alk(en)yl cysteine sulphoxides by the roots of Allium species (Coley-Smith and Cooke, 1971). These sulphoxides are metabolised by the soil micro¯ora to produce volatile thiols and sulphides which trigger the dormant sclerotia to germinate (Coley-Smith and Par®tt, 1986). Much work has focused on the potential of germination stimulants to control Allium white rot (Coley-Smith and King, 1969; Coley-Smith and Par®tt, 1986; Coley-Smith, 1990a) of which the most active are di-allyl disulphide (DADS) and di-propyl disulphide (DPDS), breakdown products of thiosul®nates and their precursors, alk(en)yl cysteine sulphoxides in garlic and onion, respectively (Coley-Smith, 1990a; Block et al., 1992a). Land free of Allium white rot in onion-growing areas of the UK and other countries is decreasing, forcing onion production into structurally poorer soils (Entwistle, 1990b;

1038

E. Coventry et al. / Soil Biology & Biochemistry 34 (2002) 1037±1045

Couch and Kohn, 2000). Control of white rot and the return of infested land to onion production are clearly major concerns of the onion-growing industry, particularly with the loss of methyl bromide as a soil sterilant (Merriman et al., 1980; Coley-Smith, 1990b; De Ceuster and Hoitink, 1999). A number of control methods have been researched including fungicide application (Davies and Savinelli, 1994; Stewart et al., 1994b), soil fumigants (Entwistle, 1990a), soil solarisation (Porter and Merriman, 1983; Melero-Vara et al., 2000), use of biological control agents (Stewart et al., 1994a; Gerlagh et al., 1996) and germination stimulants (Coley-Smith and Par®tt, 1986). Levels of disease control vary with the method used and the time of year (ColeySmith and Par®tt, 1986; Melero-Vara et al., 2000), with no one method offering complete control. Composted waste has been shown to provide some degree of control of a number of soil-borne plant pathogens due to a suppressive effect of the microbiota in the compost (De Ceuster and Hoitink, 1999). Composts have been shown to have a suppressive effect on the related soil-borne pathogen Sclerotium rolfsii (Hadar and Gorodecki, 1991). In addition, onion waste contains the compounds capable of inducing S. cepivorum sclerotia to germinate and germinated sclerotia are unable to survive in the absence of a living host (Entwistle, 1990a). Over 20,000 tonnes of waste onions are produced annually in the UK (Anon, 2000). Skin and peelings waste from imported and homegrown onions also accumulates at packhouses, so that in total over 30,000 tonnes of onion waste are produced annually in the UK, with associated land®ll disposal costs (Noble et al., 2000). In addition, the accumulation of vegetable crop wastes in growing areas and processing centres poses a risk of infestation from crop pests and pathogens, as well as a source of odour and run-off pollution. One possible option for disposal of the waste is to compost it and return it to the land as a potential biological control process. The aims of this study were to identify the optimum controlled composting regimes for onion-based waste material to produce composts with limited or no ef¯uent run-off and odour, free of S. cepivorum and with white rot sclerotia germination stimulant or control activity.

2. Materials and methods 2.1. Composting of onion waste Onion wastes were collected from packhouses in eastern England and analysed for moisture, N and ash contents as described by Noble and Gaze (1994). Small-scale ¯ask composting experiments were conducted using various mixtures of dry (shale-skins or onion tops) and wet (peelings or chopped whole bulbs) onion waste. Wet waste and two different wet to dry onion waste mixtures (16:1 and 10:1 w/w) providing 87, 84 and 80% moisture content, respectively,

were prepared to identify the optimum waste composition to achieve minimal run-off and odour during composting. The wet and dry wastes had average bulk densities of 593 and 55 kg m 23, respectively; mixtures with greater amounts of dry waste than the 10:1 wet-to-dry ratio were considered to be too bulky for transportation and large-scale composting. Three replicates of each mixture were composted in 2 l `Quick®t' multiadapter ¯asks (Fischer Scienti®c, Loughborough, UK) immersed in thermostatically controlled waterbaths (Noble et al., 1997). Urea was added to the waste (4 g kg 21 waste) to achieve a N content of 1.4% of dry matter. The waste mixtures (ca. 700 g) were placed on a perforated stainless steel platform within each ¯ask and the ¯asks immersed in the waterbaths such that the water level was above the level of the enclosed waste. Each ¯ask was connected to ancillary equipment to aerate the waste for 2 min in every 30 min at a ¯ow rate of 250 ml min 21 controlled by means of ¯ow meters. The temperature of the waste in the ¯asks was monitored with Squirrel multipoint temperature loggers (Grant Instruments Ltd, Cambridge, UK). Ammonia, carbon dioxide and oxygen concentrations in the ¯asks were monitored using a Draeger Gas Detector (Draegerwerk, LuÈbeck, Germany) with appropriate sample tubes (CH20501, CH31401, 8101991 and 6728081). The waste was incubated at 50 8C, a temperature shown to be suitable for composting and eradicating pathogens by Bollen et al. (1989). After 7 d any run-off below the platform in the ¯asks was measured and pH, weight loss, and N, dry matter and ash contents of the materials determined as described by Noble and Gaze (1994). 2.2. Effect of incubation temperature on survival of sclerotia Sclerotia from two 14 d old potato dextrose agar (PDA) plate cultures of S. cepivorum (strain from Kirton, Lincs., UK) were removed with a sterile spatula and added to 10 ml sterile distilled water (SDW). The sclerotia suspension was homogenised for 30 s then added to 500 ml SDW. This suspension (100 ml) was used to inoculate mushroom spawn bags (Van Leer Packaging Systems Ltd, Dorset, UK). Each bag contained 1920 g sand (Dried Silica Sand, Hepworth Minerals and Chemicals Ltd, Cheshire, UK), 80 g ¯aked maize (Midland Shires Farmers Ltd, Worcester, UK) which had been ground in a blender to ,1 mm dia particle size, and 175 ml water, which had been autoclaved at 121 8C for 15 min. The bags were heat sealed after inoculation and incubated for 6 weeks at 20 8C. To harvest the sclerotia, water was added to the sand-maize-sclerotia mixes and the sclerotia decanted into a 212 mm mesh size sieve. The sclerotia were left to dry in a laminar ¯ow cabinet then mixed to 50% with sand, enclosed within ®ne polyester mesh bags (150 mm pore size) (Lockertex Ltd, Warrington, UK) and buried outside in soil ca. 150 mm deep for 12 weeks to condition the sclerotia. This conditioning period is necessary to overcome the constitutive dormancy of

E. Coventry et al. / Soil Biology & Biochemistry 34 (2002) 1037±1045

the sclerotia (Coley-Smith et al., 1987). After 12 weeks, the sclerotia were retrieved as previously described for the sand-maize-sclerotia mixes. This served as the stock of conditioned sclerotia which was stored at room temperature (18±20 8C) for a maximum of 1 year. Conditioned sclerotia (100) and 2 g of onion waste were enclosed within ®ne mesh polyester bags (prepared from 140 mm dia circles and closed with a tie wrap), placed in the centre of onion waste mixtures in ¯asks and incubated at temperatures ranging from 18 to 60 8C with 6 8C intervals for up to 7 d. There were three replicate ¯asks per treatment with each replicate ¯ask incubated in a separate waterbath. One bag of sclerotia was removed from each composting ¯ask after 3 and 7 d. Sclerotia were washed from the onion waste with water, collected on a 212 mm mesh size sieve, and retrieved using forceps under a binocular microscope. They were surface sterilised in sodium hypochlorite (.5% but ,16% available chlorine, Hays Chemical Distribution Ltd, Leeds) for 1.5 min using a hand-held pipette with a modi®ed 10 ml tip which held the sclerotia on a mesh platform (Williams et al., 1998). Sclerotia were then rinsed in SDW four times and plated on to PDA containing 20 mg chlortetracycline l 21 (Williams et al., 1998). Viability of the sclerotia was assessed as percentage germination after 14 d. This experiment was arranged following a split-plot design, with the three replicates of each incubating temperature completely randomised to ¯asks (main plots), and with harvest date randomly allocated to bags within ¯asks (subplots). However, for the purposes of analysis, a completely randomised design was assumed, as there was little evidence of the within-¯ask variability being different from the between-¯ask variability. The proportion of non-germinating sclerotia was related to incubation temperature within a generalised linear model framework, assuming a binomial error distribution and logit link function, conventionally referred to as a `probit analysis'. In the model used, an additional `control mortality' parameter was included (Finney, 1971) to allow the estimation of the proportion of sclerotia that would not have germinated at any incubation temperature. From this model for the proportion of non-germinating sclerotia, an equivalent model for the proportion of germinating sclerotia (Pg) was constructed Pg ˆ Pmax ‰e2b…t2LT50 † =1 1 e2b…t2LT50 † Š

…1†

where Pmax is the estimated maximum proportion of sclerotia that would germinate whatever the incubation temperature, t is the incubation temperature, LT50 is the estimated temperature at which 50% of those sclerotia can be germinated, and b is the ®tted linear slope of the response on the logit scale (a larger value indicates a more rapid decrease in Pg as t increases). A modi®cation of this model was also considered, ®tting parallel lines on the logit scale by allowing the LT50 parameter to vary with harvest date.

1039

2.3. Sclerotia stimulant and control activity of composted onion waste To determine the effect of incubation temperature on the sclerotial germination stimulant di-n-propyl disulphide (DPDS) and other sulphurous compounds present in onions, a mixture of 10:1 (w/w) wet/dry onion waste (80% moisture content) was incubated at 42 or 54 8C for 3 or 7 d. There were three replicate ¯asks per treatment and each replicate ¯ask was incubated in a separate waterbath. A freeze-dried sample of each of the composted waste mixtures produced was then analysed for sulphur-containing compounds using GC±MS. A 2 g sample was heated to 70 8C for 5 min, the headspace gases autosampled on to the head of the GC column (type Porabond Q; Varian, Bergen op Zoom, Netherlands) and detected by mass spectrometry (Mountainheath Services Ltd, Stevenage, Hertfordshire, UK). There were three replicates per sample. The composted waste produced was also used to set up a glasshouse pot bioassay (glasshouse heating set points were 14 8C day, 12 8C night; ventilation set points were 18 8C day, 16 8C night). Three rates (1, 10 and 50% w/w) of each of the four incubated onion waste treatments (onion waste which had been incubated at 42 or 54 8C for 3 or 7 d) were incorporated into sieved sandy loam soil (Cople, Bedfordshire, UK), 16% w/w moisture, with 20% v/v vermiculite to prevent clumping of the soil. Soil containing the same rates of the raw waste was also included in the bioassay, together with untreated soil. The incubation temperature of 54 8C was chosen since results showed that a temperature of 48 8C for 3 d was necessary to ensure eradication of sclerotia of S. cepivorum from the waste (Bollen et al., 1989: Results Section 3.2). The lower incubation temperature of 42 8C was chosen since it was less likely to degrade the sclerotia germination stimulants in the waste. Square pots (70 £ 70 £ 80 [deep] mm Optipots (LBG Ltd, Evesham, UK)) were ®lled with the soil±waste mixtures (220 g) together with four polyester mesh bags (20 £ 20 mm) containing 2 g of 50:50 (w/w) sand/soil and 100 sclerotia. Pots were watered from the bottom to maintain a moisture content of ca. 40% w/w. There were three replicate pots for each of the 15 treatment combinations including onion waste, plus nine replicate pots of the untreated soil. The mesh bags were retrieved at 1-month intervals for the ®rst 3 months and then at 6 months (August±January), and the sclerotia assessed for viability. Viability was assessed both in terms of percentage soft sclerotia, which is indicative of decay or the early stages of germination (Entwistle and Smith, 1994) and hence imminent death in the absence of a host, and percentage germination of the remaining hard sclerotia, which are potentially viable. This experiment was arranged as a split-plot design. The three replicates of each of the 15 onion waste treatments, comprising the three rates of the ®ve waste or compost types, and the nine replicates of the untreated control

1040

E. Coventry et al. / Soil Biology & Biochemistry 34 (2002) 1037±1045

(soil alone) were allocated to pots (main plots) following a completely randomised design. Retrieval time was randomly allocated to bags within pots (sub-plots). Again, for the purposes of analysis, a completely randomised design was assumed as there was little evidence of the within-pot variability differing from the between-pot variability. Numbers of soft sclerotia, as a proportion of the number of sclerotia recovered, and of viable (germinating) sclerotia, as a proportion of the number tested (up to 50 where available) were analysed within a generalised linear model framework, assuming a binomial error distribution and logit link function. Pots, where the number of sclerotia recovered was signi®cantly fewer than the mean recovery rate, were omitted from the analysis. The analysis allowed the assessment of differences between the onion waste treatments and the untreated control, between the different rates of incorporation, between raw and composted waste, and between the temperatures and durations of composting, and of the interactions between these factors. From the analyses, there was evidence of over-dispersion, i.e. there was more variation in the data than would be expected from a binomial distribution of the numbers of soft or germinating sclerotia. The signi®cance of these effects was therefore assessed using approximate F-tests, and predicted mean proportions for treatment combinations of interest were calculated from the ®tted models. 2.4. Sclerotia stimulant activity of DPDS Five concentrations of the sclerotia germination stimulant DPDS (4.55 £ 10 22 to 455 mg g 21 soil), covering the range found in onion waste (0.3±11 mg g 21, Artacho MartinLagos et al., 1992) and the concentrations used by ColeySmith and King (1969) (0.1±100 mg g 21), were prepared and added to 220 g sandy loam soil (16% w/w moisture) with 20% v/v vermiculite in square pots (70 £ 70 £ 80 [deep] mm Optipots). The range in concentrations was made by 1:10 serial dilution of a 98% solution of DPDS (Sigma-Aldrich Company Ltd, Poole, Dorset, UK) using 10% ethanol. Aliquots (10 ml) of solutions from the dilution series were added to the soil in each pot, followed by thorough mixing with a clean spatula. Two untreated controls (water and 10% ethanol in water) were included in the experiment. Single mesh bags of 100 sclerotia, as described previously, were buried in the soil in each pot, and the individual pots were covered with clear plastic bags to help retain the volatile DPDS within their vicinity. The pots were left outside under cover for 1 month (July), and watered from the bottom to maintain a moisture content of ca. 40% w/w, after which time the sclerotia were removed and assessed for viability. There were three replicate pots of each of the six treatments, arranged in a completely randomised design. The numbers of soft sclerotia, as a proportion of the number recovered, and of germinating sclerotia, as a proportion of the number tested (30 for all pots), were analysed within a

generalised linear model framework, assuming a binomial error distribution and logit link function. One pot was omitted from the analysis of the proportion of soft sclerotia due to a low recovery rate. The quantitative effect of the DPDS dose was assessed by ®tting a `controlled mortality' probit model (logit link function) for the proportions of soft or dead (i.e. not germinating) sclerotia against loge(dose). Differences between doses were also assessed by ®tting dose as a factor, rather than as a quantitative explanatory variable. The effect of the solvent used for DPDS (ethanol), which is naturally found in composted wastes (Noble et al., 2001), on the viability of sclerotia was determined in a similar manner. Aliquots of 100% ethanol (10 ml) were added to pots of sandy loam soil containing bags of sclerotia. Water (10 ml) was added to control pots. Sclerotia were removed from the pots after 1 month and assessed for viability as previously described. 3. Results 3.1. Composting of onion waste The dry matter content of the onion waste was 69±80% for the dry wastes and 10±15% for the wet wastes. The dry wastes were found to have a higher ash content (6.8±14.9% of dry matter) than the wet wastes (4.5±8.0% of dry matter) but lower total N (dry 0.4±0.7; wet 1.0±1.4% of dry matter). The wet onion waste alone was unsuitable for composting because of the volume of run-off (50±100 ml per ¯ask) and the strong odours produced. An onion waste mixture with a moisture content of 80% (10:1 w/w wet/dry waste) was optimal in terms of minimising run-off (0±3 ml) and odours produced when incubated at 50 8C for 7 d. Under the temperature and aeration conditions used, the waste mixture softened and darkened in colour although it was still recognisable as onion waste mixture after 7 d. Gas measurements con®rmed the composting waste was well aerated (15.0± 19.5% O2; 1.5±1.9% CO2). No ammonia was detected. This was presumably due to the acidic nature of the waste mixture (pH 3.82 ^ 0.01 [mean ^1 standard error of the mean for three replicates]), preventing hydrolysis of ammonium ions with subsequent loss of ammonia. A 4±15% loss in weight was recorded after 7 d. After composting, the waste mixtures had a moisture content of 80±84% and N and ash contents of 1.4±2.2% and 9.8±16.2% of dry matter, respectively. 3.2. Effect of incubation temperature on survival of sclerotia Germination of sclerotia of S. cepivorum was affected by incubation temperature, with percentage germination decreasing as incubation temperature increased (Fig. 1). However, there was no additional reduction in percentage germination when sclerotia were exposed for 7 d compared to those exposed for 3 d (P . 0.05 for approximate F-test

E. Coventry et al. / Soil Biology & Biochemistry 34 (2002) 1037±1045

Fig. 1. Effect of incubation temperature and period (B 3 d, O 7 d) on the percentage germination of sclerotia held in composting onion waste. The curve ®tted to all data is of the form shown in Eq. (1). The ®tted model includes an additional `control germination' parameter to estimate the maximum percentage of sclerotia (93%, shown by the dashed line) that would germinate when not exposed to composting onion waste.

for additional deviance explained when ®tting parallel lines model allowing LT50 parameter to vary between harvest dates). There was no germination for either exposure time at temperatures of 48 8C and above. 3.3. Sclerotia stimulant and control activity of composted onion waste GC±MS analysis of the onion waste revealed the presence of the white rot sclerotia germination stimulant DPDS in both dry and wet raw waste as well as composted waste (Table 1). The raw onion wastes contained more DPDS than the composted wastes. The maximum concentration of DPDS detected in the waste incubated for 3 d at 42 8C was greater than that detected in the waste incubated for 7 d at 42 8C or 3 and 7 d at 54 8C. Methyl mercaptan and n-propyl mercaptan were also detected in the raw and composted wastes at concentrations above 10 mg kg 21

1041

Fig. 2. Effect of incubation period (d) and temperature (8C) of onion waste during preparation on the percentage of soft sclerotia retrieved from soil with which the waste had subsequently been mixed. Values shown (mean ^1 standard error of the mean) were obtained from predicted mean proportions, averaged across incorporation rate, exposure time and replicate for the waste treatments, and across exposure time and replicate for the untreated control. There were no soft sclerotia in the initial stock of conditioned sclerotia.

(Table 1). Other sulphurous compounds detected (dimethyl disulphide and di-n-propyl sulphide) were present in both the raw and composted wastes at a concentration of ,10 mg kg 21 waste. No DADS was detected in the raw or composted waste. Acetaldehyde, ethanol and ethyl acetate were also detected in the raw and composted wastes. Acetic acid was also present in the raw dry and composted wastes. The results of the glasshouse pot bioassay showed that the viability of sclerotia in sandy loam soil was in¯uenced by the presence and condition of onion waste (Fig. 2). Incorporation of raw waste, or of waste incubated at 42 8C, had no effect on the percentage of soft sclerotia retrieved relative to the control, whereas the percentage of soft sclerotia was increased in the presence of waste incubated at 54 8C (P , 0.001) (means of all exposure times) (Fig. 2). There was an interaction between the effects of incubation

Table 1 Di-n-propyl disulphide (DPDS), methyl mercaptan and n-propyl mercaptan content (mg kg 21 onion waste) of raw onion waste and waste incubated for 3 or 7 d at 42 or 54 8C (composted waste). Values from at least three samples of each waste Compound (mg kg 21)

Raw waste Wet

Composted waste (10:1 w/w wet/dry mixture) Dry

3 d at 42 8C

7 d at 42 8C

3 d at 54 8C

7 d at 54 8C

DPDS

Min Max

6.0 46.8

7.5 37.7

1.6 15.6

,1.5 1.8

,1.5 7.7

,1.5 10.2

Methyl mercaptan

Min Max

,10 40.8

,10 753.0

,10 353.1

,10 23.9

,10 63.7

,10 69.4

n-Propyl mercaptan

Min Max

,10 14.4

,10 301.2

,10 110.7

,10 ,10

,10 40.5

,10 34.7

1042

E. Coventry et al. / Soil Biology & Biochemistry 34 (2002) 1037±1045

Fig. 3. Effect of rate of incorporation of onion waste and duration of exposure on the percentage of soft sclerotia retrieved from soil mixed with waste which had previously been incubated at 54 8C. Values shown (mean ^1 standard error of the mean) were obtained from predicted mean proportions for the 54 8C incubation temperature treatment, averaged across incubation period and replicate, and for the untreated control, averaged across replicate only. There were no soft sclerotia in the initial stock of conditioned sclerotia.

temperature and incubation period (P ˆ 0.001) on the viability of sclerotia: waste incubated for 7 d was slightly more effective than that incubated for 3 d for the 54 8C incubation temperature, but there was no difference in effect between the waste incubated for 3 or 7 d at 42 8C (Fig. 2). In addition to the effect of incubation temperature, the rate of waste incorporation (P , 0.001) and the duration of exposure of the sclerotia to the waste (P , 0.001) both had effects on viability (Fig. 3). As only the high incubation temperature had a signi®cant effect on the percentage of soft sclerotia retrieved (Fig. 2), the effects of incorporation rate and exposure duration are only shown for waste incubated at 54 8C (Fig. 3). Relative to the control, the 1% rate of the waste incubated at 54 8C only increased the percentage of soft sclerotia retrieved after 6 months exposure, whilst the 10% rate increased this percentage after both 3 and 6 months exposure (Fig. 3). The 50% rate was the most effective in terms of the speed of effect, with an increase in the percentage of soft sclerotia retrieved recorded at all sample dates (Fig. 3). While all three rates of incorporation increased the percentage of soft sclerotia retrieved after 6 months burial relative to the control, the 10 and 50% rates showed the largest increase. The 10 and 50% rates of the raw waste and the waste incubated at the lower temperature (42 8C) only increased the percentage of soft sclerotia retrieved after 6 months exposure (results not shown). The 1% rate of these wastes had no signi®cant effect on the percentage of soft sclerotia.

Fig. 4. Effect of rate of incorporation and condition of onion waste on percentage germination of sclerotia retrieved from soil±waste mixtures. Values shown (mean ^1 standard error of the mean) were obtained from the predicted mean proportions averaged across exposure time and replicate for all treatments, and across incubation period and temperature for previously composted waste treatments.

In addition to affecting the percentage of soft sclerotia, the rate of incorporation of the waste had an effect on the germination of hard sclerotia retrieved. The 50% rate of the raw and composted wastes reduced germination (P , 0.001) relative to the control (Fig. 4). There was an interaction between rate of incorporation and the condition of the waste (P , 0.001): the 50% incorporation rate of both the raw and composted wastes reduced viability, with the composted waste considerably more effective than the raw waste (Fig. 4). In contrast, the 1 and 10% rates of the raw and composted wastes had no effect on the germination of retrieved sclerotia (Fig. 4). Unlike the percentage of soft sclerotia, there was no signi®cant effect of duration of exposure to waste on the germination of hard sclerotia retrieved. The mean glasshouse air temperature during the pot bioassay was 17 8C. 3.4. Sclerotia stimulant activity of DPDS The effect of addition of DPDS to soil on sclerotia viability after 1 month exposure was minimal (Table 2). The highest concentration applied (455 mg g 21 soil) signi®cantly increased the number of soft sclerotia retrieved compared with the control (P ˆ 0.002), although there was no effect on the germination of hard sclerotia. Lower concentrations of DPDS, similar to those found in raw onion waste (Table 1), had no effects on the number of soft sclerotia or on the germination of hard sclerotia. Addition of ethanol, which was detected in the onion waste, to the pots at 4.5 mg g 21 soil (10% ethanol) had no effect on sclerotia viability compared with water (Table 2), whereas addition at 45 mg g 21 soil (100% ethanol) was

E. Coventry et al. / Soil Biology & Biochemistry 34 (2002) 1037±1045 Table 2 Percentage of soft sclerotia and germination of hard sclerotia of S. cepivorum retrieved after a 1-month burial in pots of soil treated with different concentrations of DPDS (mg g 21 soil). Values shown were obtained from predicted mean proportions, with standard errors of means shown in parentheses DPDS (mg g 21 soil)

% Soft sclerotia

% Germination

0 (water) 0 (10% ethanol) 4.55 £ 10 22 4.55 £ 10 21 4.55 4.55 £ 10 1 4.55 £ 10 2

7.3 (^1.33) 6.0 (^1.37) 8.0 (^1.55) 6.5 (^1.73) 6.3 (^1.41) 6.0 (^1.34) 14.0 (^2.00)

91.3 (^2.96) 89.0 (^3.31) 96.7 (^1.89) 87.8 (^3.45) 88.7 (^3.31) 90.0 (^3.16) 91.3 (^3.00)

found to have a deleterious effect on sclerotia viability (results not shown). The mean air temperature during the experiment was 16 8C. 4. Discussion 4.1. Composting of onion waste In our study onion waste was kept aerated during composting and no strong odours, typically associated with anaerobic onion waste, were produced. In addition, the moisture content of the waste mixture was found to be important in minimising the volume of run-off and odours produced during composting, with 80% shown to be optimal for onion waste. A 7 d incubation period was chosen for practical reasons with the aim of achieving a quick turnover of waste, necessary in a commercial situation. 4.2. Effect of incubation temperature on survival of sclerotia Incubation in onion waste at temperatures of 48 8C and above for 3 d was shown to kill sclerotia of S. cepivorum (Fig. 1). These results agree with those of Adams (1987) who found that only 48 min at 50 8C were required to kill 50% of S. cepivorum sclerotia in soil and Porter and Merriman (1983), who found a 6 h daily temperature of 50 8C for 14 d destroyed all sclerotia of S. cepivorum in soil. Entwistle (1990c) found temperatures ranging from 42 to 62 8C over a period of 2±25 d in a heap of salad onions and cereal straw suf®cient to kill sclerotia after 1 month exposure. The position of the sclerotia in the heap dictated whether they were killed. Those in the inner, warmer part of the heap were killed, whereas those on the cooler, outer edges were still viable. Other pathogens have similarly been shown to be destroyed during composting (Yuen and Raabe, 1984; Bollen et al., 1989). However, the problem with uncontrolled composting is the non-uniformity of temperature throughout the waste (Entwistle, 1990c). Thus, it cannot be assumed that all pathogens present have been destroyed, even in a turned windrow (Miller,

1043

1991). This problem can be overcome with the use of aerated bulk composting tunnels. Waste for composting is housed on a slatted ¯oor above a plenum through which a controlled ¯ow of air is blown. This maintains a uniform temperature throughout the waste and ensures that all pathogens with a thermal death point below the waste temperature are destroyed (Noble and Gaze, 1994; Noble et al., 2000). 4.3. Sclerotia stimulant activity of composted onion waste Allium species produce volatile compounds which stimulate germination of sclerotia of S. cepivorum (Coley-Smith and King, 1969). High activity has been shown to be present in mercaptans, monosulphides and disulphides possessing an n-propyl or an allyl radical whereas methyl, ethyl and butyl derivatives are relatively inactive (Coley-Smith and King, 1969). The stimulatory properties of volatiles from Allium species have been utilised in white rot control methods, although the level of control achieved may be inconsistent (Entwistle, 1994). Coley-Smith and Par®tt (1986) found the response of sclerotia to DADS treatment to be seasonal in effect with poorer results obtained with summer than with autumn, winter or spring applications. The control achieved was related to the rate at which DADS disappeared from treated soil, which was more rapid in the summer when the soil temperature was higher. In agreement with the results of Coley-Smith and King (1969) and Block et al. (1992a,b), DADS, which has been detected in garlic (Lawson et al., 1991), was not detected in onion waste. The most active sclerotia germination stimulant to be detected in signi®cant concentrations in onion waste was DPDS, although ethanol, which was found to have a detrimental effect on sclerotia viability (Section 3.4, results not shown) was also detected. The concentrations of DPDS in the onion waste (Table 1) were about 10% of those found by Artacho Martin-Lagos et al. (1992) in fresh onions. Despite the partial loss of DPDS during composting (Table 1), the higher temperature composts were the most effective in reducing viability of sclerotia in the pot bioassay (Fig. 2). In addition, the raw waste with the highest DPDS content (Table 1) had no effect on sclerotia viability (Fig. 2). Results from the pot bioassay with the addition of DPDS to soil showed that the DPDS concentration which stimulated germination to give soft sclerotia (455 mg g 21 soil, Table 2) was much higher than that present in the onion waste (2±47 mg kg 21, Table 1). Coley-Smith and King (1969) found that DADS and DPDS solutions or garlic and onion extracts to be more effective in stimulating S. cepivorum sclerotia germination, than either the DPDS solution or raw onion waste added to soil in our study. They found that DADS or DPDS concentrations of between 1 and 10 mg ml 21 in aqueous solution caused signi®cant stimulation of germination of sclerotia, while a 100 mg ml 21 solution of DADS or DPDS, or 10% garlic or onion juice extracts increased germination to 37±98%.

1044

E. Coventry et al. / Soil Biology & Biochemistry 34 (2002) 1037±1045

Although several workers have detected DPDS in onions (Coley-Smith and King, 1969; Artacho Martin-Lagos et al., 1992) using GC and GC±MS analyses, disulphides were not detected in signi®cant quantities using either HPLC or low temperature GC±MS analysis by Block et al. (1992a,b). Block et al. (1992a,b) detected thiosulphinates at concentrations of about 250 mg g 21 in fresh onion and suggested that disulphides may be secondary products of the precursor thiosulphinates resulting from high temperature GC±MS analysis. However, as well as being the products of high temperature decay, DADS and DPDS may be metabolites of soil micro¯ora in contact with Allium plants (ColeySmith and Cooke, 1971). However, the results of this present study, which showed composted waste to be lower in DPDS and mercaptans than raw waste but more effective in reducing sclerotia viability, suggest that this effect was mainly due to factors other than germination stimulants released from onion waste. Linderman and Gilbert (1969) and Linderman and Gilbert (1973) found that the germination of Sclerotium rolfsii sclerotia was in¯uenced by other volatiles from plant residues (alfalfa hay), both directly and indirectly through soil microbial activity.

large-scale bulk composting tunnels, to ensure uniform pasteurisation (Noble et al., 2000), for ®eld scale application and white rot control needs to be examined. Although high rates of waste (50%) would probably be needed to achieve effective control, this only applies to the root depth of onion crops. Sclerotia of S. cepivorum germinate in response to volatiles from onion roots, and remain dormant outside of the root zone (Entwistle, 1994). It is possible that other vegetable and organic wastes (Noble et al., 2000) could be used in addition to onion waste. Following such large applications, it is likely that there would need to be a delay before subsequent onion crops to avoid phytotoxity. This should also increase the effectiveness of the treatment by lengthening the exposure of sclerotia to the waste (Fig. 3). Acknowledgements This research was funded by the Department for Environment, Food and Rural Affairs, the Horticultural Development Council and the other consortium members of Horticulture LINK Project CSA 4802.

4.4. Other potential mechanisms for suppression of S. cepivorum with composted waste

References

A number of workers have reported control of phytopathogens with composted wastes (Theodore and Toribio, 1995; Hoitink and Boehm, 1999). Compost prepared from grape waste (skins, seeds, stalks) was shown to reduce the viability of sclerotia of S. rolfsii in vitro, with the antagonistic microbial community of the compost implicated as the cause of the suppression (Hadar and Gorodecki, 1991). Composts support a rich diversity of microorganisms and hence a potentially antagonistic community to phytopathogens. Previous work has shown Trichoderma spp. and Trichoderma (Gliocladium) virens in compost have a suppressive effect on soil-borne pathogens (Hoitink and Fahy, 1986). Such a community of antagonistic microorganisms in the composted onion waste may have contributed to the control of S. cepivorum observed in this study. Further work is needed to determine if antagonistic microorganisms are involved in decreasing the inoculum of S. cepivorum sclerotia in soil amended with compost, and if so, which species are responsible. Application of composts which eradicate white rot sclerotia from infested soils would be an environmentally friendly method of disease control, compatible with organic onion production. Our study demonstrated the potential of composted onion waste for decreasing the inoculum density of soil (sandy loam) infested with S. cepivorum on a smallscale basis. The effects of composted onion waste on sclerotia in other soil types should be determined. Further work is also needed to determine the effects of controlled composted onion-based wastes on white rot control in planta. The feasibility of composting onion waste in

Abd El-Razik, A.A., Shatla, M.N., Rushdi, M., 1973. Studies on the infection of onion plants by Sclerotium cepivorum Berk. Phytopathologische Zeitschrift 76, 108±116. Adams, P.B., 1987. Effects of soil temperature, moisture and depth on survival and activity of Sclerotium minor, Sclerotium cepivorum, and Sporidesmium sclerotivorum. Plant Disease 71, 170±174. Anon, 2000. Monthly report on the fruit and vegetable crops in England and Wales. Report No. 863ÐVegetable Section, 31 March 2000. Ministry of Agriculture, Fisheries and Food, York. pp. 19. Artacho Martin-Lagos, R., Olea Serrano, M.F., Ruiz-Lopez, M.D., 1992. Comparative study of gas chromatography±mass spectrometry of methods for the extraction of sulfur compounds in Allium cepa L. Food Chemistry 44, 305±308. Block, E., Naganathan, S., Putman, D., Zhao, S.-H., 1992a. Allium chemistry: HPLC analysis of thiosul®nates from onion, garlic, wild garlic (Ramsons), leek, scallion, shallot, Elephant (Great-Headed) garlic, chive, and Chinese chive. Uniquely high allyl to methyl ratios in some garlic samples. Journal of Agricultural and Food Chemistry 40, 2418±2430. Block, E., Putman, D., Shu-Hai Zhao, 1992b. Allium chemistry: GC±MS analysis of thiosul®nates and related compounds from onion, leek, scallion, shallot, chive, and Chinese chive. Journal of Agricultural and Food Chemistry 40, 2431±2438. Bollen, G.J., Volker, D., Wijnen, A.P., 1989. Inactivation of soil-borne plant pathogens during small-scale composting of crop residues. Netherlands Journal of Plant Pathology 95 (Supplement 1), 19±30. Coley-Smith, J.R., 1987. Alternative methods of controlling white rot disease of Allium. In: Chet, I. (Ed.). Innovative Approaches to Plant Disease Control. Wiley, New York, pp. 161±177. Coley-Smith, J.R., 1990a. Control of Allium white rot with diallyl disulphide. In: Entwistle, A.R., Mattusch, P. (Eds.), Proceedings of the Fourth International Workshop on Allium White Rot. Biologische Bundesanstalt fuÈr Land- und Forst-wirtschaft, Braunschweig. pp. 133±139. Coley-Smith, J.R., 1990b. White rot disease of Allium: problems of soilborne diseases in microcosm. Plant Pathology 39, 214±222.

E. Coventry et al. / Soil Biology & Biochemistry 34 (2002) 1037±1045 Coley-Smith, J.R., Cooke, R.C., 1971. Survival and germination of fungal sclerotia. Annual Review of Phytopathology 9, 65±92. Coley-Smith, J.R., King, J.E., 1969. The production by species of Allium of alkyl sulphides and their effect on germination of sclerotia of Sclerotium cepivorum Berk. Annals of Applied Biology 64, 289±301. Coley-Smith, J.R., Par®tt, D., 1986. Some effects of diallyl disulphide on sclerotia of Sclerotium cepivorum: possible novel control method for white rot disease of onions. Pesticide Science 37, 587±594. Coley-Smith, J.R., Par®tt, D., Taylor, I.M., Reese, R.A., 1987. Studies of dormancy in sclerotia of Sclerotium cepivorum. Plant Pathology 36, 594±599. Coley-Smith, J.R., Mitchell, C.M., Sansford, C.E., 1990. Long-term survival of sclerotia of Sclerotium cepivorum and Stromatinia gladioli. Plant Pathology 39, 58±69. Couch, B.C., Kohn, L.M., 2000. Clonal spread of Sclerotium cepivorum in onion production with evidence of past recombination events. Phytopathology 90, 514±521. Davies, J.M.L., Savinelli, C., 1994. Control of Allium white rot (Sclerotium cepivorum) in module-raised onions with ¯uazinam. In: Entwistle, A.R., Melero-Vara, J.M. (Eds.). Fifth International Workshop on Allium White Rot. Instituto de Agricultura Sostenible, Cordoba, pp. 117±122. De Ceuster, T.J.J., Hoitink, H.A.J., 1999. Prospects for composts and biocontrol agents as substitutes for methyl bromide in biological control of plant diseases. Compost Science and Utilization 7, 6±15. Entwistle, A.R., 1990a. Root diseases. In: Rabinowitch, H.D., Brewster, J.L. (Eds.). Onions and Allied Crops. Agronomy, Biotic Interactions, Pathology, and Crop Protection, vol. II. CRC Press, Boca Raton, pp. 103±154. Entwistle, A.R., 1990b. Allium white rot and its control. Soil Use and Management 6, 201±209. Entwistle, A.R., 1990c. Aerobic composting, solar heating or combined polythene mulch and dazomet treatment to eradicate Sclerotium cepivorum sclerotia from infected plants. In: Entwistle, A.R., Mattusch, P. (Eds.), Proceedings of the Fourth International Workshop on Allium White Rot. Biologische Bundesanstalt fuÈr Land- und Forst-wirtschaft Braunschweig. pp. 166±173. Entwistle, A.R., 1994. The dynamics of Sclerotium cepivorum inocula: a theoretical framework. In: Entwistle, A.R., Melero-Vara, J.M. (Eds.). Fifth International Workshop on Allium White Rot. Instituto de Agricultura Sostenible, Cordoba, pp. 40±47. Entwistle, A.R., Smith, J.E., 1994. Methods for research on Allium white rot (Sclerotium cepivorum). In: Entwistle, A.R., Melero-Vara, J.M. (Eds.). Fifth International Workshop on Allium White Rot. Instituto de Agricultura Sostenible, Cordoba, pp. 32±37. Finney, D.J., 1971. Probit Analysis. 3rd ed Cambridge University Press, Cambridge. Gerlagh, M., Whipps, J.M., Budge, S.P., Goossen-van de Geijn, H.M., 1996. Ef®ciency of isolates of Coniothyrium minitans as mycoparasites of Sclerotinia sclerotiorum, Sclerotium cepivorum and Botrytis cinerea on tomato stem pieces. European Journal of Plant Pathology 102, 787± 793. Hadar, Y., Gorodecki, B., 1991. Suppression of germination of sclerotia of Sclerotium rolfsii in compost. Soil Biology and Biochemistry 23, 303± 306. Hoitink, H.A.J., Boehm, M.J., 1999. Biocontrol within the context of soil microbial communities: a substrate-dependent phenomenon. Annual Review of Phytopathology 37, 427±446. Hoitink, H.A.J., Fahy, P.C., 1986. Basis for the control of soilborne plant

1045

pathogens with composts. Annual Review of Phytopathology 24, 93± 114. Lawson, L.D., Wang, Z.-Y.J., Hughes, B.G., 1991. Identi®cation and HPLC quantitation of the sul®des and dialk(en)yl thiosul®nates in commercial garlic products. Planta Medica 57, 363±370. Linderman, R.G., Gilbert, R.G., 1969. Stimulation of Sclerotium rolfsii in soil by volatile components of alfalfa hay. Phytopathology 59, 1366± 1372. Linderman, R.G., Gilbert, R.G., 1973. In¯uence of volatile compounds from alfalfa hay on microbial activity in soil in relation to growth of Sclerotium rolfsii. Phytopathology 63, 359±362. Melero-Vara, J.M., Prados-Ligero, A.M., Basallote-Ureba, M.J., 2000. Comparison of physical, chemical and biological methods of controlling garlic white rot. European Journal of Plant Pathology 106, 581± 588. Merriman, P.R., Isaacs, S., MacGregor, R.R., Towers, G.B., 1980. Control of white rot in dry bulb onions with arti®cial onion oil. Annals of Applied Biology 96, 163±168. Metcalf, D.A., Wilson, C.R., 1999. Histology of Sclerotium cepivorum infection of onion roots and the spatial relationships of pectinases in the infection process. Plant Pathology 48, 445±452. Miller, F.C., 1991. Biodegradation of solid wastes by composting. In: Martin, A.M. (Ed.). Biological Degradation of Wastes. Elsevier, New York, pp. 1±30. Noble, R., Gaze, R.H., 1994. Controlled environment composting for mushroom cultivation: substrates based on wheat and barley straw and deep litter poultry manure. Journal of Agricultural Science (Cambridge) 123, 71±79. Noble, R., Fermor, T.R., Evered, C.E., Atkey, P.T., 1997. Bench-scale preparation of mushroom substrates in controlled environments. Compost Science and Utilization 5, 32±43. Noble, R., Whipps, J.M., Coventry, E., 2000. An alternative to land®ll: composting onion and other vegetable wastes to control pests and diseases and avoid pollution. In: McLardy, C. (Ed.), Waste 2000, Waste Conference Limited, Barclay Centre, University of Warwick Science Park. pp. 355±360. Noble, R., Hobbs, P.H., Dobrovin-Pennington, A., Misselbrook, T.H., Mead, A., 2001. Olfactory response to mushroom composting emissions as a function of chemical concentration. Journal of Environmental Quality 30, 760±767. Porter, I.J., Merriman, P.R., 1983. Effects of solarization of soil on nematode and fungal pathogens at two sites in Victoria. Soil Biology and Biochemistry 15, 39±44. Stewart, A., Kay, S.J., Fullerton, R.A., 1994a. Biological control of onion white rot in New Zealand. In: Entwistle, A.R., Melero-Vara, J.M. (Eds.). Fifth International Workshop on Allium White Rot. Instituto de Agricultura Sostenible, Cordoba, pp. 102±104. Stewart, A., Slade, E.A., Fullerton, R.A., 1994b. Chemical control of onion white rot in New Zealand. In: Entwistle, A.R., Melero-Vara, J.M. (Eds.). Fifth International Workshop on Allium White Rot. Instituto de Agricultura Sostenible, Cordoba, pp. 166±168. Theodore, M., Toribio, J.A., 1995. Suppression of Pythium aphanidermatum in composts prepared from sugarcane factory residues. Plant and Soil 177, 219±223. Williams, R.H., Whipps, J.M., Cooke, R.C., 1998. Role of soil mesofauna in dispersal of Coniothyrium minitans: mechanisms of transmission. Soil Biology and Biochemistry 30, 1937±1945. Yuen, G.Y., Raabe, R.D., 1984. Effects of small-scale aerobic composting on survival of some fungal plant pathogens. Plant Disease 68, 134±136.