Eradication of Plasmodiophora brassicae during composting of wastes

Authors


Abstract

Survival of infectious inoculum of the clubroot pathogen Plasmodiophora brassicae was assessed following bench-scale flask composting experiments and large-scale composting procedures. Clubroot-affected material was provided by artificial inoculation of Chinese cabbage or naturally infected Brussels sprout and cabbage roots. Both sets of diseased material were used in flask experiments, and the latter in large-scale windrow and aerated tunnel experiments. Municipal green wastes, onion waste and spent mushroom compost were evaluated in flask experiments with varying temperature, aeration and moisture conditions. Green wastes were used in larger-scale composts. Within the limits of a Chinese cabbage seedling bioassay, both temperature and moisture content were critical for eradication of P. brassicae spores extracted from composted clubroot-affected residues. Incubation in compost at 50°C for 7 days or 1 day at 60°C with high moisture levels (= −5 kPa matric potential or 60% w/w moisture content) eradicated inoculum from artificially inoculated Chinese cabbage roots. In large-scale windrows and aerated tunnels, the pathogen was eradicated from naturally infected brassica wastes after 6–7 days at 54–73°C.

Introduction

Currently, landfill is the most common route for disposal of vegetable wastes in Europe. With the decrease in the availability of landfill sites, and the need to reduce the quantity of biodegradable waste disposed of in this way in accordance with the EU Landfill Directive, an alternative to landfill disposal is required. One possible option is to compost the waste and incorporate the composted waste into soil or growing media for subsequent crop production. To avoid disease risks for subsequent crops from composted agronomic wastes, the composts must be free of plant pathogens. Large volumes of cabbage, cauliflower and other brassica residues are available for composting, and the products could be incorporated in fields used for cruciferous crops. As the sampling techniques and detection methods available do not enable a reliable analysis of the presence or absence of pathogens in compost (Noble & Roberts, 2004), one must define the composting conditions required for sanitizing crop residues. Noble & Roberts (2004) defined eradication as a reduction in the levels of a pathogen to below the limit of detection of the specific method used, in this case a plant-based bioassay.

Plasmodiophora brassicae, the causal agent of clubroot of crucifers, is a biotrophic, soilborne pathogen that is resistant to degradation and difficult to eradicate from crop residues. In infected cells, the plasmodium gives rise to numerous resting spores that are resistant to adverse conditions and can survive for very long periods in soil. Bollen et al. (1989) and Ryckeboer et al. (2002) demonstrated that P. brassicae could survive for long periods at low temperatures during the maturation phase of composting. Lopez-Real & Foster (1985) concluded that P. brassicae survived in cool composts (max. 35°C), but was not detected after 96 h exposure to an average compost temperature of 54°C, or after 24 h exposure to high temperatures (65–75°C). Bollen et al. (1989) and Ryckeboer (2001) found that a peak temperature of 54–60°C and a composting duration of 1–21 days eradicated P. brassicae. Christensen et al. (2001) eradicated P. brassicae by using an average compost temperature of 49°C for 14 days. Ylimaki et al. (1983) observed that exposure to 70°C for c. 1 week eradicated P. brassicae spores provided the moisture content and pH were optimal (60–80% w/w and alkaline, respectively), but a 3-week exposure to 60–65°C was less effective. Bruns et al. (1993) found that a temperature of c. 60°C held for 14 weeks eradicated P. brassicae if the compost had adequate moisture (50%), but not if the compost was mixed solely with wood chips. Bollen & Volker (1996) stated that the occurrence of dry pockets in composting material is probably the main cause of pathogen survival in heaps where eradication was expected on the basis of compost temperatures. They recommended a minimum compost moisture content of 40%.

This study reconsiders the composting conditions (temperature, aeration and moisture) required to eradicate the clubroot pathogen from inoculated and naturally infected hosts, involving a variety of waste plant material in both flask and large-scale composting procedures.

Materials and methods

Compost feedstock wastes

The wastes used in the flask composting experiments throughout this study were: green waste (GW), predominantly municipal green wastes from Dijon district, France, referred to as GW(F), and spent mushroom compost (SMC) from Cultures France Champignon, Chace, France, referred to as SMC(F). In addition, two other composts were used in one experiment: onion waste from a processing factory (Goldwood Ltd, Moulton, Lincolnshire, UK) and SMC from Warwick HRI, referred to as SMC(UK). Two further sources of GW from the UK were used for the large-scale composting tests.

Moisture, nitrogen and ash contents, electrical conductivity and pH of the wastes were determined according to the methods of Anonymous (1986, 1999, 2000a, 2000b, 2000c). Water-release curves of the waste materials were determined using a modified sand-table method according to Noble & Dobrovin-Pennington (2005). This determined the matric potentials of the waste materials at particular gravimetric moisture contents.

Plant material

Brassica sp. roots infected with P. brassicae obtained from Brittany, France were incorporated into steam-treated soil. Seeds of the clubroot-susceptible Chinese cabbage (Brassica rapa ssp. pekinensis) cv. Granaat were sown in pots (300 mL) of the P. brassicae-infested soil and maintained in a growth chamber at 20°C day, 18°C night. After 6 weeks the roots were washed and the galls harvested. Galls were stored at −20°C and served as the stock inoculum for the flask experiments.

Naturally clubroot-infected cabbage and Brussels sprout (Brassica oleracea) roots with ‘woody galls’ obtained from Kirton, Lincolnshire, UK, were also used in the flask experiments, and served as the inoculum source for the tests in windrows and tunnels.

Composting systems

The effects of composting conditions on the survival of P. brassicae in clubroot-affected plant material were examined in: (i) bench-scale composting flasks; (ii) composting windrows; and (iii) aerated bulk-composting tunnels.

Bench-scale flask experiments

Galls (3–5 g) from young plants were enclosed within fine mesh polyester sample bags and placed in the centre of the compost feedstocks in 2-L Quickfit multiadapter flasks (Fischer Scientific) and immersed in thermostatically controlled water baths or chambers (Noble et al., 1997). Where required, the flasks were aerated for 2 min in every 30 min at a flow rate of 250 mL min−1. The remaining flasks were not aerated, i.e. no positive air flow was used. The temperature of the waste was monitored with Squirrel multipoint data loggers (Grant Instruments). Single sample bags were removed successively from the composting wastes after 1, 3 and 7 days’ incubation. The viability of the inoculum in the bags was assessed using the bioassay described below.

Effect of compost type and temperature on eradication

Flasks filled with the four wastes described above, SMC(F), GW(F), SMC(UK) and onion waste, were incubated at 40, 50 and 60°C for 7 days without aeration.

Effect of compost temperature and aeration on eradication

Flasks filled with SMC(F) were incubated at 40, 50 and 65°C for 7 days, with and without aeration.

Effect of compost temperature and moisture on eradication

Samples of SMC(F) and GW(F) were standardized to three matric potentials in the range −2·3 to −32·1 kPa by adding water. Flasks were filled with composts (two types × three moisture levels) and incubated at 50 and 60°C for 7 days. The experiment was repeated three times. Laboratory-prepared Chinese cabbage inoculum was used for all the above experiments.

Effect of compost temperature and moisture on eradication using natural inoculum

Flasks were filled with SMC(F) (matric potential −2·3 or −29·9 kPa) or GW(F) (−3·6 or −32·1 kPa) and incubated at 50°C for 7 days. Naturally infected cabbage root material with woody galls was used.

Composting windrows

Roots of Brussels sprout and cabbage with woody galls were enclosed within mesh bags filled with GW, predominantly fruit and vegetable wastes referred to as GW(UK1), and buried 0·5 m deep in windrows of composting GW (Organic Recycling Ltd, Lincolnshire, UK). A depth of 0·5 m was considered sufficient to avoid the effects of low ambient air temperatures (Christensen et al., 2001, 2002).

The windrows had dimensions of 30 × 5 × 3 (high) m and contained c. 150 t GW.

The temperature of the waste and infected plant material in the mesh bags was monitored with Squirrel multipoint data loggers. Woody galls were removed from the windrow after 7 days and the viability of the pathogen was assessed by bioassay.

Aerated bulk-composting tunnels

Aerated bulk-composting tunnels (Hensby Composts, Woodhurst, Cambridgeshire, UK), similar to those described by Noble & Gaze (1998), were used. The tunnels had dimensions of 20 × 4 × 3 (high) m and held c. 100 t GW. A controlled flow of air was blown through a slatted floor beneath the GW and maintained a minimum oxygen concentration of 6% v/v. Infected plant material, similar to that used in the windrows, was buried 0·5 m deep in GW, predominantly tree prunings and grass clippings referred to as GW(UK2), similarly to the procedure for windrows. The temperature of the plant material was monitored. After 6 days the material was removed from the bulk-composting tunnels and the viability of the pathogen was assessed by bioassay.

Growth chamber pot bioassay for P. brassicae viability

Following removal from the composting systems, galls full of resting spores were ground in sterile distilled water to provide a suspension of resting spores. The density of the resting spore suspension was measured with a haemocytometer and diluted to 1 × 105 spores mL−1. Resting spore suspension (20 mL) was added to steam-treated soil in 300-mL pots. Five Chinese cabbage seeds (cv. Granaat) were sown per pot, with a minimum of five pots per treatment. The pots were incubated for 6 weeks, after which time plants were uprooted and the roots washed and scored for pathogen infection. These were assessed according to symptom expression: 0 = no gall; 1 = one or two small galls; 2 = more than two galls but some roots still symptomless; 3 = entire root system distorted. A disease index was calculated for each pot: DI = [(N0 × 0 +N1 × 0·25 + N2 × 0·5 + N3 × 1)/5] × 100, where Ni is the number of plants in class i (Doublet et al., 1988).

For each of the above experiments, resting spore suspension from noncomposted, diseased plant inoculum was added to five bioassay pots (positive controls). Five bioassay pots without spore suspension (negative controls) were also included.

Results

Analysis of compost feedstock wastes

The wastes used for the tests differed significantly in chemical properties, moisture content and matric potential (Table 1). The N content of the wastes ranged from <1% of dry matter for GW(UK2) and onion waste to >2% of dry matter for SMC(UK). The onion waste had the lowest ash content and pH, but the highest moisture content. GW(F) was the most alkaline waste, had the highest ash content and, together with GW(UK2), had the lowest moisture content and electrical conductivity. SMC(UK) had the highest electrical conductivity. Matric potential, which was lowest for GW(F) and GW(UK2) and highest for GW(UK1), depended on both the moisture content of the material and its water retention.

Table 1.  Physical and chemical properties of French and UK compost waste feedstocks used in bench-scale flask and large-scale composting experiments
WasteN (% DM)Ash (% DM)pHEC (mS m−1)Moisture (% w/w)Matric potential (kPa)
  1. Each value is the mean of three replicate samples.

French municipal green wastes [GW(F)]1·71608·711043·1−30·1
UK fruit and vegetable wastes [GW(UK1)]1·33377·318359·8 −4·0
UK tree prunings and grass clippings [GW(UK2)]0·68496·815143·3−29·9
French spent mushroom compost [SMC(F)]1·73517·836151·3−22·6
UK spent mushroom compost [SMC(UK)]2·08366·954959·0−15·9
UK onion waste0·86 74·345379·5−15·6

Eradication of P. brassicae in bench-scale flask experiments

In all the experiments using laboratory-prepared Chinese cabbage inoculum, the disease index of bioassay plants inoculated with spore suspension from noncomposted plant material (positive controls) was >95. No disease was detected in the negative controls in any of the experiments.

Effect of compost type and temperature on eradication

The bioassays revealed that there was a very high survival of P. brassicae in the four compost types after composting at 40°C (disease index >95), and only partial eradication of the inoculum under the other conditions, even after incubation at 60°C for 7 days (Fig. 1). All the wastes used in the experiment had a matric potential less than −15 kPa (Table 1).

Figure 1.

Effect of compost temperature and composting duration on eradication of Plasmodiophora brassicae from inoculum in four composts. Control, original noncomposted inoculum. After composting at 40°C for 7 days in all composts, the disease index of test plants was >90. Each value is the mean of at least five replicate test plants; error bars, 95% CI.

Effect of compost temperature and aeration on eradication

Eradication of P. brassicae was achieved in SMC(F) composted at 65°C for ≥1 day, i.e. disease index of bioassay test plants = 0. The pathogen survived in compost at 40°C for 1 and 3 days; intermediate results were obtained in the compost at 50°C for 1 and 3 days (Fig. 2), but eradication was achieved after 7 days at 40 and 50°C (data not shown). Survival of the pathogen under aerated and nonaerated conditions was not significantly different (P = 0·05).

Figure 2.

Effect of compost temperature, aeration and composting duration on eradication of Plasmodiophora brassicae from inoculum in French (F) spent mushroom compost. Control, original noncomposted inoculum. There was no survival after composting at 65°C for 1 day or at 40°C for 7 days. Each value is the mean of at least five replicate test plants; error bars, 95% CI.

Effect of compost temperature and moisture on eradication

Eradication of P. brassicae was achieved in both SMC(F) and GW(F) composts at 60°C for 1 day or at 50°C for 7 days. At 50°C, for shorter incubation periods, there was an influence of compost moisture content on P. brassicae survival. As there was no significant difference between the three repeat experiments, Fig. 3 represents the mean values of the disease indices of these experiments. Eradication of the pathogen was achieved at the highest moisture contents (least negative matric potentials) of the GW after 3 days at 50°C, whereas the pathogen survived in the driest GW (Fig. 3). Survival of P. brassicae also declined in SMC with increasing moisture content (Fig. 3). Results of an anova performed on the data from the three experiments identified significant effects (P = 0·05) of compost temperature, type, moisture content and incubation time on survival of P. brassicae. At equivalent matric potentials, eradication was more successful in GW than in SMC.

Figure 3.

Effect of compost matric potential and composting duration on eradication of Plasmodiophora brassicae from French (F) spent mushroom compost and green waste composted at 50°C. Control, original noncomposted inoculum. Each value is the mean of three replicate experiments and at least five replicate test plants per experiment; error bars, 95% CI.

Effect of compost temperature and moisture on eradication using natural inoculum

The disease index of the positive controls using the uncomposted, naturally infected cabbage inoculum was 78·3 (±20·2). The pathogen was eradicated from woody gall roots of cabbage naturally infected by P. brassicae by a compost temperature of 50°C for 1 day (disease index of bioassay test plants = 0). Eradication was achieved in SMC(F) and GW(F) at both matric potentials (−2·3 to −32·1 kPa).

Eradication of P. brassicae in large-scale composting systems

The disease index of the positive controls using the naturally infected Brussels sprout and cabbage inoculum was 87·5 (±10·2). Temperature profiles of the windrow and aerated tunnel compost systems, in the location of the inoculum bags, are shown in Fig. 4. In both systems, a compost temperature of 60°C was achieved for at least 2 days. Compost temperatures peaked at 68 and 73°C in the windrow and aerated tunnel, respectively, and remained >54°C for the entire 6–7-day period in both systems. No disease symptoms were detected in any of the plants in the pot bioassay using the brassica residues retrieved from both large-scale systems.

Figure 4.

Compost temperature profile of windrow (green waste UK1) and aerated bulk tunnel (green waste UK2) from the time infected plant material was inserted in the compost at a depth of 0·5 m until the time of retrieval.

Discussion

Symptoms of clubroot occurred on all Chinese cabbage seedlings (positive controls) grown in soils infested with inoculum of P. brassicae from artificially inoculated Chinese cabbage plants. Similar results were reported by Bollen et al. (1989) and Bruns et al. (1993), and in a similar test by Ryckeboer et al. (2002) using cauliflower for the bioassay. However, in the absence of alternative detection techniques these bioassays cannot preclude viable resting spores remaining undected.

Results obtained during this study showed that P. brassicae inoculum does not survive for 7 days at 50°C, or for 1 day at 60°C, provided the moisture content of the compost is high enough (equivalent to a matric potential −5 kPa or c. 60% w/w in GW). The importance of moisture content of the compost in determining eradication has been demonstrated by Ylimaki et al. (1983) and Bruns et al. (1993). The erratic results involving four different wastes and incubation at 40, 50 and 60°C without aeration (Fig. 1), where survival of the pathogen was detected after 7 days at 60°C, could be explained by the low moisture content of these composts (matric potentials between −15 and −30 kPa). This may also explain eradication temperatures as high as 70°C reported by Bollen et al. (1989).

Other factor(s), independent of the effect of moisture, also affect pathogen eradication: Noble & Roberts (2004) list several microbial and chemical processes that may be involved in the inactivation of pathogens during composting. For example, high compost pH did not improve eradication, as suggested by Ylimaki et al. (1983), as onion waste, with the lowest pH of the materials used, was at least as effective as the other wastes in eradicating P. brassicae. In general, the results agree with Bollen & Volker (1996) in that heat generated during the thermophilic high-temperature phase of composting appears to be the most important factor for the elimination of plant pathogens.

The artificially prepared Chinese cabbage inoculum using a French P. brassicae isolate was more temperature-tolerant than the UK inoculum provided by naturally infected cabbage and Brussels sprout plants. Further experiments are needed to determine whether there are differences in temperature tolerance between artificial and natural inocula, and that of different host plants, or between P. brassicae isolates from different localities.

In the large-scale systems at a depth of 0·5 m, the compost temperature was >60°C for at least 2 days and >54°C for 6 days; this eradicated P. brassicae and confirmed the results from the flask composting experiments. However, measurements by Christensen et al. (2001, 2002) showed that there are significant differences in spatial and temporal temperature profiles between different windrow and in-vessel composting facilities. The eradication results obtained here under controlled composting conditions indicated a greater sensitivity to temperature of P. brassicae than was observed by other workers under less well defined composting conditions, using similar bioassays (Ylimaki et al., 1983; Bollen et al., 1989; Christensen et al., 2001).

Acknowledgements

The authors would like to thank the European Union (Project QLRT-01458 ‘RECOVEG’) and the Waste and Resources Action Programme (WRAP), Banbury, Oxon, UK for funding this research.

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