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Impact of the earthworm Lumbricus terrestris on the degradation of Fusarium-infected and deoxynivalenol-contaminated wheat straw
Soil Biology & Biochemistry 40 (2008) 3049–3053
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Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Impact of the earthworm Lumbricus terrestris on the degradation of Fusarium-infected and deoxynivalenol-contaminated wheat straw Elisabeth Oldenburg a, *,1, Susanne Kramer b, Stefan Schrader b, Joachim Weinert c a
¨ hn-Institute, Federal Research Centre for Cultivated Plants, Bundesallee 50, D-38116 Braunschweig, Germany Institute for Crop and Soil Science, Julius Ku ¨ nen-Institute, Federal Research Institute for Rural Areas, Forestry and Fishery, Bundesallee 50, D-38116 Braunschweig, Germany Institute of Biodiversity, Johann Heinrich von Thu c ¨ ttingen, Grisebachstraße 6, D-37077 Go ¨ttingen, Germany Department of Crop Science, Plant Pathology and Plant Protection, University of Go b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 May 2008 Received in revised form 2 September 2008 Accepted 4 September 2008 Available online 10 October 2008
When conservation tillage is practised in agriculture, plant residues remain on the soil surface for soil protection purposes. These residues should be widely decomposed within the following vegetation period as microbial plant pathogens surviving on plant litter may endanger the currently cultivated crop. Important soil-borne fungal pathogens that preferably infect small grain cereals belong to the genus Fusarium. These pathogens produce the mycotoxin deoxynivalenol (DON), a cytotoxic agent, in infected cereal organs. This toxin frequently occurs in cereal residues like straw. So far it is unclear if DON degradation is affected by members of the soil food web within decomposing processes in the soil system. For this purpose, a microcosm study was conducted under controlled laboratory conditions to investigate the degradation activity of the earthworm species Lumbricus terrestris when exposed to Fusarium-infected wheat straw being contaminated with DON. Highly Fusarium-infected and DON-contaminated straw seemed to be more attractive to L. terrestris because it was incorporated faster into the soil compared with straw infected and contaminated at low levels. This is supported by a greater body weight gain (exposure time 5 weeks) and smaller body weight loss (exposure time 11 weeks) of L. terrestris, respectively, when highly contaminated straw was offered for different time periods. Furthermore, L. terrestris takes part in the efficient degradation of both Fusarium biomass and DON occurring in straw in close interaction with soil microorganisms. Consequently, earthworm activity contributes to the elimination of potentially infectious plant material from the soil surface. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Degradation Fusarium-infected straw Deoxynivalenol DON-contamination Earthworm activity
1. Introduction Different conservation tillage techniques are practised in agriculture in order to protect soil from erosion and to maintain soil fertility and productivity (Hobbs, 2007). When soils are mulched, residues of the pre-crop remain on the soil surface or in low depth of the topsoil. This organic layer stimulates decomposition processes by the soil biota thus improving the humus balance of the soil (Berg and McClaugherty, 2003). However, pathogens surviving on plant residues before decomposing may endanger the health of the following crop by increasing the infection risk for specific plant diseases (Pereyra et al., 2004). * Corresponding author. Institute for Plant Protection in Field Crops and Grassland, Julius Ku¨hn-Institute, Federal Research Centre for Cultivated Plants, Messeweg 11/12, D-38104 Braunschweig, Germany. Tel.: þ49 0531 2994545; fax: þ49 0531 2993008. E-mail address: [email protected] (E. Oldenburg). 1 Present address: Institute for Plant Protection in Field Crops and Grassland, Julius Ku¨hn-Institute, Federal Research Centre for Cultivated Plants, Messeweg 11/12, D-38104 Braunschweig, Germany. 0038-0717/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2008.09.004
One of the most important soil-borne fungal diseases in small grain cereals worldwide is Fusarium head blight, often caused by Fusarium graminearum, Fusarium culmorum and Fusarium avenaceum (Parry et al., 1995). These Fusarium species are able to produce mycotoxins in infected plant organs. The trichothecene mycotoxin deoxynivalenol (DON) is frequently detected in cereals that have been infected with F. graminearum or F. culmorum (Mirocha et al., 1994). Contamination with DON is often detected not only in the kernels of cereals, but also in harvest residuals such as straw (Paulsen et al., 2004; Brinkmeyer et al., 2006). DON is not only a potent phytotoxin, but also induces toxic effects in animals and humans (Ueno, 1983). At the cellular level, DON inhibits DNA, RNA and protein synthesis (Feinberg and McLaughlin, 1989) and has adverse effects on the immune system (Harvey et al., 1991; Rotter et al., 1994). Studies on DON affecting invertebrates are very rare and so far seem restricted to some insects and aquatic invertebrates suggesting cytotoxic effects of the mycotoxin (Fornelli et al., 2004; Supamattaya et al., 2005). Earthworms are important members of the soil food web playing a major role as primary and secondary decomposers of
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with chopped wheat straw of the control, at 15 1 C for 10 days. Before being introduced into the microcosms the earthworms were washed three times with water to remove adhesive residues and mucus.
plant residues (Lee, 1985). One of the primary decomposers of crop residues in temperate regions is the deep-burrowing earthworm species Lumbricus terrestris. This detritivorous species incorporates plant litter into the soil thus removing potentially infectious material from the soil surface. Interacting with soil microorganisms, L. terrestris directly decomposes the incorporated plant material by ingestion and digestion (Devliegher and Verstraete, 1995). It has been shown that the activity of earthworms in soils is enhanced when tillage intensity is reduced (Tebru¨gge and Du¨ring, 1999; Hangen et al., 2002). However, higher DON concentrations were observed in wheat grain when the soil was mulched compared with ploughing due to higher mass of infectious plant residues remaining on the soil surface (Oldenburg et al., 2007). The decomposing activities of earthworms can therefore be regarded as an important mechanism to reduce plant pathogens surviving on mulch layers. Food selection experiments with earthworms and soil fungi demonstrated a clear preference of L. terrestris for Fusarium species (Bonkowski et al., 2000). However, no information is available so far if Fusarium mycotoxins occurring in plant residues affect the decomposing activity of earthworms. Furthermore, it is still an open question if earthworm activity has an impact on DON degradation. The objective of this study was to investigate the degradation activity of the earthworm L. terrestris exposed to Fusarium-infected and DON-contaminated wheat straw in a microcosm study under controlled laboratory conditions. Special attention was focused on the determination of Fusarium biomass and DON concentrations in the straw in order to assess the decomposing efficiency of L. terrestris.
Forty microcosms were prepared by filling 700 g of soil (moisture: 23%) equally into rectangular plastic jars (dimensions: 11 15.5 6.5 cm [width length height]), which were closed with plastic lids to prevent earthworms from escaping. The lids were perforated with some pinholes to allow air circulation. The soil was covered with 8 g air dried wheat straw (DM: 94%) per microcosm, and in each case 20 microcosms received C-straw or AIstraw. The straw was subsequently moistened and incorporated slightly into the surface area to initiate microbial activity. Microcosms were randomly positioned in a climate chamber and incubated at 15 1 C in the dark. After four days, two earthworms of 10.15 1.07 g total body weight were introduced into each of 10 microcosms containing C-straw and 10 microcosms containing AIstraw, no worms were placed into the remaining microcosms. In the course of incubation, microcosms were sprayed several times with water to prevent soil from desiccation. Thus a mean percentage of 22.4 3.7 of soil moisture could be maintained throughout the whole experiment. After 5 and 11 weeks of incubation, half of the microcosms from each treatment (C or AI-straw with or without worms) were terminated to gather samples for quantitative analysis. The number of replicates of each treatment was n ¼ 5.
2. Materials and methods
2.5. Determination of soil surface cover
2.1. Production of straw
The soil surface cover was determined by scanning top view photographs of the microcosms at the start and after 5 and 11 weeks of incubation. The specific areas of uncovered soil and those covered with straw were evaluated with the colour analysis program WinRHIZO, 2002 (Re´gent Instruments Inc.).
Winter wheat (Triticum aestivum) of the cultivar ‘Tommi’ was cultivated at an experimental field located near Go¨ttingen, Germany. At the time of flowering, wheat covering an area of 4 m2 was artificially infected by top–down spraying of three toxigenic Fusarium culmorum strains (F.c. 34, F.c. 35 and F.c. 36 from culture collection of the Department of Crop Science, Plant Pathology and Plant Protection, University of Go¨ttingen, Germany) suspended in 400 ml water containing Tween 20 (0.5 ml l1). The conidiospore concentration of the suspension was 3 105 ml1. At harvest time the ears of the wheat were removed and the straw was manually chopped to a length of approximately 2.5 cm (hereafter referred as AI-[artificially infected]-straw). For comparison, straw of the same cultivar originating from the same experimental field, but not artificially infected was used as control (hereafter referred as C-[control]-straw).
2.4. Experimental microcosms
2.6. Sampling procedure At first, the residual straw on the soil surface was removed from the microcosms. When sampling the straw, great care was taken to mechanically separate adhesive soil, but washing was avoided to prevent elution of DON, which is water soluble. Soil samples were taken from areas that visually appeared unaffected by earthworm activities and earthworm casts were also sampled. Finally the earthworms were removed, carefully cleaned off soil particles and weighed. The total wet weight of all samples was determined gravimetrically. The samples were then stored at 20 C in readiness for analytical preparation.
2.2. Preparation of soil 2.7. Sample preparation About 30 kg of a Luvisol characterized by a soil texture of 12% clay, 85% silt and 3% sand was taken from the Ap horizon of a site near Braunschweig, Germany, and was stored at 4 C until further treatment. Seven days before filling the microcosms, the soil was defaunated by cyclic freezing (twice for 24 h) interrupted by thawing for 24 h. Crude organic plant residues like straw or roots were manually removed and the soil was stored at 15 1 C until use.
All samples including the parent materials at the start of the microcosms were dried by lyophilisation. The straw was ground to pass through a 1-mm sieve using of an ultra centrifugal mill (ZM 200, Retsch GmbH, Haan, Germany). Samples of soil and earthworm casts were manually homogenized with a mortar to obtain a fine powder (<0.5 mm). 2.8. Determination of Fusarium Protein Equivalents
2.3. Adaptation of L. terrestris Adult individuals of L. terrestris, purchased from a commercial supplier, were adapted to the experimental conditions by keeping them in plastic jars containing the soil described above, covered
Fusarium Protein Equivalents (FPE) were quantified with a double antibody sandwich (DAS) ELISA according to the method as previously described (Tian et al., 2005) by using Fusarium specific antibodies and protein standards.
E. Oldenburg et al. / Soil Biology & Biochemistry 40 (2008) 3049–3053
100
a
C-straw
90
Soil surface cover [%]
Samples of 0.5 g of straw or 1.0 g of soil were suspended in PBSTBuffer (0.01 M phosphate buffered saline þ 0.05% [v/v] Tween 20 containing 1% [w/v] polyvinylpyrolidone, pH 7.2) at a ratio of 1:20 [w/v] and shaken on a horizontal shaker at 4 C for approximately 24 h. After centrifugation at 12,000 g for 10 min and dilution (1:25 [v/v]) with PBST-buffer, the extracts were applied in two replicates on a 96-well convex-bottomed plastic microplate (Immuno Plate Maxisorb, Nunc International, Denmark) pre-coated with antibody. The ELISA procedure was then continued according to Tian et al. (2005). The absorbance of the resulting dye was measured at 405 nm (reference: 592 nm) with a microplate photometer (SLT Spectra, TECAN GmbH, Crailsheim, Germany). The absorbance values of sample extracts free of Fusarium were subtracted from the absorbance values of the Fusarium-positive sample extracts. The values were then converted to Fusarium Protein Equivalents (FPE) using a standard protein curve, and the final FPE value was defined as the amount of Fusarium protein in the corresponding samples. The limits of quantification were 30 mg FPE kg1 for straw and earthworm casts, and 120 mg FPE kg1 for soil samples.
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AI-straw
80 70
b
60
c
50
d
40
e
30 20 10 0
0
5
11
5
11
Incubation time [weeks] Fig. 1. Change of soil surface cover with C-straw or AI-straw during an incubation period of 5 or 11 weeks due to incorporation activity of L. terrestris. Various letters (a, b, c, d, e) are significantly different between the columns (P < 0.05). C, control; AI, artificially infected.
2.9. Determination of deoxynivalenol
2.10. Statistics The Kolmogorov–Smirnov-test confirmed that all data were normally distributed. For this reason, analysis of variance (ANOVA) and the student’s t-test were used to compare treatment effects. All statistical analyses were undertaken using the software package SPSS for Windows Version 13. 3. Results 3.1. Soil surface cover In the presence of L. terrestris, the soil surface area of the microcosms covered by either C-straw or AI-straw decreased significantly within both incubation periods of 5 and 11 weeks (Fig. 1). The AI-straw was more efficiently incorporated into the soil, as the soil surface cover was reduced faster from the start until 5 weeks (56%) and 11 weeks (74%) compared with C-straw (38% and 53% until 5 and 11 weeks, respectively). ANOVA revealed a significant influence of Fusarium infection (F ¼ 24.98; P < 0.001) and experimental time (F ¼ 17.47; P < 0.01) on straw litter incorporation.
3.2. Body weight of L. terrestris All individuals in all treatments had survived to sampling at either 5 or 11 weeks. The body weight of L. terrestris increased over 5 weeks when both C-straw and AI-straw were provided (Fig. 2), but the weight gain was larger when the earthworms ingested AIstraw (þ6.5% versus þ1.4%). After 11 weeks of incubation, the body weight of L. terrestris decreased more when C-straw was offered (14%) as compared to AI-straw (8%). Only in the case of C-straw after 11 weeks, the body weight difference of L. terrestris compared to the initial body weight was significant (P < 0.01). 3.3. FPE concentration in straw The Fusarium biomass in the C-straw on the soil surface of about 0.33 0.14 mg FPE kg1 was degraded to about 85% after 5 weeks of incubation without L. terrestris. After 11 weeks of incubation only one microcosm contained straw with positive FPE, so that degradation of the Fusarium biomass was nearly complete. In the microcosms containing L. terrestris, no FPE could be detected in the straw after 5 weeks of incubation, but slight amounts of FPE (0.05 0.01 mg kg1) were observed in the straw after 11 weeks of incubation. The Fusarium biomass present in AI-straw of about 132.4 21.5 mg FPE kg1 at the start of the microcosms was reduced significantly to about 98.8 and 98.9% after 5 and 11 weeks
10
Change of body weight [%]
Deoxynivalenol (DON) was determined by use of the competitive ELISA test kit ‘Ridascreen DON’, product No. 5906 from R-Biopharm, Darmstadt, Germany. The sample extraction procedure was carried out as follows: 1.0 g straw, 2.5 g of soil or earthworm casts, respectively, were suspended with distilled water at a ratio of 1:10 or 1:20 [w/v] and shaken for 15 min using a horizontal shaker at 160 motions per minute. The extracts were first filtered through a fluted filter and then centrifuged at 15,300 rev min1 at 10 C for 5 min to remove solid particles. The resulting supernatants, occasionally diluted with distilled water, were directly applied in the ELISA test (two replicates), which was performed according to the manufacturer’s procedure. The absorbance of the resulting dye was measured at 450 nm using a microplate reader (PowerWave, Biotek Instruments GmbH, Bad Friedrichshall, Germany). The DON concentrations were calculated by use of the KC4 operating Software (Biotek Instruments) of the reader. Analysis was repeated when the standard deviation of the resulting mean concentration of the two replicates exceeded 10%. The limits of quantification were 74 mg DON kg1 for straw and 37 mg DON kg1 for soil and earthworm casts.
L. terrestris fed with C-straw L. terrestris fed with AI-straw
5 0 -5 -10 -15
5 weeks
11 weeks
Fig. 2. Mean relative change of body weight of L. terrestris as influenced by type of straw and incubation time in comparison with initial body weight (set as 0%) at experimental start. C, control; AI, artificially infected.
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of incubation in the absence of L. terrestris, respectively (Fig. 3). In the presence of L. terrestris activity, FPE degradation was significantly enhanced to about 99.4 and 99.5% after 5 and 11 weeks of incubation, respectively. 3.4. DON concentration in straw At the start of the microcosms, the C-straw on the soil surface contained 0.39 0.07 mg DON kg1. After 5 weeks of incubation, the primary DON concentration in the layer was reduced about 27% in the microcosms without L. terrestris, but was no longer detectable when L. terrestris was active. An approximately 50% increase of DON concentration was observed at 11 weeks of incubation without L. terrestris. However, again no DON was detected in the C-straw after 11 weeks in the microcosms containing L. terrestris. The initial DON concentration in the AI-straw on the soil surface of 146.7 18.1 mg DON kg1 was reduced to about 77% in the microcosms without L. terrestris, and to about 99.7% in microcosms containing L. terrestris after 5 weeks of incubation (Fig. 4). After 11 weeks the reduction of the primary DON concentration amounted to 88% in the microcosms without L. terrestris, and was nearly complete (about 99.9%) in the microcosms containing L. terrestris (Fig. 4). 3.5. FPE and DON concentration in soil and earthworm casts Neither in the undigested soil nor in the earthworm casts could positive FPE concentrations (120 mg kg1, 30 mg kg1, respectively) or DON concentrations (37 mg kg1) be detected at the start and after 5 and 11 weeks of incubation. 4. Discussion It appears that wheat straw highly infected with Fusarium is more attractive as a food source for L. terrestris than less infected wheat straw, as lower amounts of residual AI-straw compared to C-straw remained on the soil surface of earthworm containing microcosms. This is also confirmed by the greater body weight gain and smaller weight losses of L. terrestris when AI-straw was ingested over the course of 5 weeks and 11 weeks, respectively. It is well documented that earthworms do not feed at random (Bonkowski et al., 2000), but generally select leaf litter with high N-content (Hendriksen, 1990). In addition, soil fungi are regarded as an important food source for earthworms (Tiwari and Mishra, 1993; Edwards and Fletcher, 1988). In selection experiments L. terrestris
106
Without L. terrestris
FPE concentration[µg kg-1]
a
With L. terrestris
105 104 10
b
b
c
c
5
11
3
102 101 100
0
5
11
Incubation time [weeks] Fig. 3. Logarithmic presentation of FPE concentration (Mean and SD of n ¼ 5) in artificially infected straw during an incubation period of 5 or 11 weeks. Various letters (a, b, c) are significantly different between the columns (P < 0.05).
106
DON concentration [µg kg-1]
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Without L. terrestris
a 10
5
b c
With L. terrestris
104 d
103 10
e
2
101 100
0
5
11
5
11
Incubation time [weeks] Fig. 4. Logarithmic presentation DON concentration (mean and SD of n ¼ 5) in artificially infected straw during an incubation period of 5 and 11 weeks. Various letters (a, b, c, d, e) are significantly different between the columns (P < 0.05).
clearly preferred Fusarium sp. compared with other genera like Mucor, Trichoderma and Rhizoctonia (Moody et al., 1995; Bonkowski et al., 2000). Plant residues colonized by plant pathogens may indicate nutrient rich organic matter for earthworms and are therefore attractive for consumption (Bonkowski et al., 2000). It is likely that the highly Fusarium-infected straw is preferred as food because the fungus enhances the N-content thus increasing the nutrient quality of the straw (Weichert et al., 1991). Furthermore, due to its cellulolytic activity (Manka, 1988) the fungus probably makes hardly-digestible straw compounds more available for earthworm digestion. L. terrestris is involved in the degradation of Fusarium biomass in the wheat straw layer, because in the presence of earthworm activity FPE reduction was more efficient than in its absence both after 5 and 11 weeks of incubation. Furthermore, L. terrestris accelerates the degradation of DON in the straw layer. This seems to happen in close interaction with the soil microorganisms, which were able to decrease DON to some extent in the absence of the earthworm. It is therefore uncertain if L. terrestris possesses its own specific metabolic pathway for DON or rather acts as a catalyst enhancing microbial activity. This interactive breakdown of DON seems to be efficient enough to prevent adverse effects on the decomposition/incorporation activity and the body weight gain of L. terrestris. L. terrestris seems to take part in the degradation of residual Fusarium biomass in ingested straw, as no positive concentrations of FPE could be detected in the casts. This is comparable with results of a study showing a reduced viability of Fusarium spores when passed through the guts of L. terrestris (Moody et al., 1996). As no positive concentrations of DON were detected in the earthworm casts it is suggested that L. terrestris, probably promoted by the gut microorganisms, is also involved in the breakdown of residual traces of DON in ingested straw. As leaching of DON into the soil was not observed in this study, DON applied with the Fusarium-infected straw was probably metabolized in an interactive process between the soil microorganisms and the earthworms. However, the products to which DON might have been transformed in the course of the degradation process remain to be elucidated, as only little information is available at time giving indications about the structure of degradation compounds produced from DON by soil microorganisms (Bata and Lasztity, 1999). A bacterium isolated from soil was able to degrade DON into 3-keto-4-deoxynivalenol, a compound of less toxicity relative to DON (Shima et al., 1997). In conclusion, detritivorous earthworm species promote both the disappearance of Fusarium plant pathogens and the mycotoxin
E. Oldenburg et al. / Soil Biology & Biochemistry 40 (2008) 3049–3053
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Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Impact of the earthworm Lumbricus terrestris on the degradation of Fusarium-infected and deoxynivalenol-contaminated wheat straw Elisabeth Oldenburg a, *,1, Susanne Kramer b, Stefan Schrader b, Joachim Weinert c a
¨ hn-Institute, Federal Research Centre for Cultivated Plants, Bundesallee 50, D-38116 Braunschweig, Germany Institute for Crop and Soil Science, Julius Ku ¨ nen-Institute, Federal Research Institute for Rural Areas, Forestry and Fishery, Bundesallee 50, D-38116 Braunschweig, Germany Institute of Biodiversity, Johann Heinrich von Thu c ¨ ttingen, Grisebachstraße 6, D-37077 Go ¨ttingen, Germany Department of Crop Science, Plant Pathology and Plant Protection, University of Go b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 May 2008 Received in revised form 2 September 2008 Accepted 4 September 2008 Available online 10 October 2008
When conservation tillage is practised in agriculture, plant residues remain on the soil surface for soil protection purposes. These residues should be widely decomposed within the following vegetation period as microbial plant pathogens surviving on plant litter may endanger the currently cultivated crop. Important soil-borne fungal pathogens that preferably infect small grain cereals belong to the genus Fusarium. These pathogens produce the mycotoxin deoxynivalenol (DON), a cytotoxic agent, in infected cereal organs. This toxin frequently occurs in cereal residues like straw. So far it is unclear if DON degradation is affected by members of the soil food web within decomposing processes in the soil system. For this purpose, a microcosm study was conducted under controlled laboratory conditions to investigate the degradation activity of the earthworm species Lumbricus terrestris when exposed to Fusarium-infected wheat straw being contaminated with DON. Highly Fusarium-infected and DON-contaminated straw seemed to be more attractive to L. terrestris because it was incorporated faster into the soil compared with straw infected and contaminated at low levels. This is supported by a greater body weight gain (exposure time 5 weeks) and smaller body weight loss (exposure time 11 weeks) of L. terrestris, respectively, when highly contaminated straw was offered for different time periods. Furthermore, L. terrestris takes part in the efficient degradation of both Fusarium biomass and DON occurring in straw in close interaction with soil microorganisms. Consequently, earthworm activity contributes to the elimination of potentially infectious plant material from the soil surface. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Degradation Fusarium-infected straw Deoxynivalenol DON-contamination Earthworm activity
1. Introduction Different conservation tillage techniques are practised in agriculture in order to protect soil from erosion and to maintain soil fertility and productivity (Hobbs, 2007). When soils are mulched, residues of the pre-crop remain on the soil surface or in low depth of the topsoil. This organic layer stimulates decomposition processes by the soil biota thus improving the humus balance of the soil (Berg and McClaugherty, 2003). However, pathogens surviving on plant residues before decomposing may endanger the health of the following crop by increasing the infection risk for specific plant diseases (Pereyra et al., 2004). * Corresponding author. Institute for Plant Protection in Field Crops and Grassland, Julius Ku¨hn-Institute, Federal Research Centre for Cultivated Plants, Messeweg 11/12, D-38104 Braunschweig, Germany. Tel.: þ49 0531 2994545; fax: þ49 0531 2993008. E-mail address: [email protected] (E. Oldenburg). 1 Present address: Institute for Plant Protection in Field Crops and Grassland, Julius Ku¨hn-Institute, Federal Research Centre for Cultivated Plants, Messeweg 11/12, D-38104 Braunschweig, Germany. 0038-0717/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2008.09.004
One of the most important soil-borne fungal diseases in small grain cereals worldwide is Fusarium head blight, often caused by Fusarium graminearum, Fusarium culmorum and Fusarium avenaceum (Parry et al., 1995). These Fusarium species are able to produce mycotoxins in infected plant organs. The trichothecene mycotoxin deoxynivalenol (DON) is frequently detected in cereals that have been infected with F. graminearum or F. culmorum (Mirocha et al., 1994). Contamination with DON is often detected not only in the kernels of cereals, but also in harvest residuals such as straw (Paulsen et al., 2004; Brinkmeyer et al., 2006). DON is not only a potent phytotoxin, but also induces toxic effects in animals and humans (Ueno, 1983). At the cellular level, DON inhibits DNA, RNA and protein synthesis (Feinberg and McLaughlin, 1989) and has adverse effects on the immune system (Harvey et al., 1991; Rotter et al., 1994). Studies on DON affecting invertebrates are very rare and so far seem restricted to some insects and aquatic invertebrates suggesting cytotoxic effects of the mycotoxin (Fornelli et al., 2004; Supamattaya et al., 2005). Earthworms are important members of the soil food web playing a major role as primary and secondary decomposers of
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with chopped wheat straw of the control, at 15 1 C for 10 days. Before being introduced into the microcosms the earthworms were washed three times with water to remove adhesive residues and mucus.
plant residues (Lee, 1985). One of the primary decomposers of crop residues in temperate regions is the deep-burrowing earthworm species Lumbricus terrestris. This detritivorous species incorporates plant litter into the soil thus removing potentially infectious material from the soil surface. Interacting with soil microorganisms, L. terrestris directly decomposes the incorporated plant material by ingestion and digestion (Devliegher and Verstraete, 1995). It has been shown that the activity of earthworms in soils is enhanced when tillage intensity is reduced (Tebru¨gge and Du¨ring, 1999; Hangen et al., 2002). However, higher DON concentrations were observed in wheat grain when the soil was mulched compared with ploughing due to higher mass of infectious plant residues remaining on the soil surface (Oldenburg et al., 2007). The decomposing activities of earthworms can therefore be regarded as an important mechanism to reduce plant pathogens surviving on mulch layers. Food selection experiments with earthworms and soil fungi demonstrated a clear preference of L. terrestris for Fusarium species (Bonkowski et al., 2000). However, no information is available so far if Fusarium mycotoxins occurring in plant residues affect the decomposing activity of earthworms. Furthermore, it is still an open question if earthworm activity has an impact on DON degradation. The objective of this study was to investigate the degradation activity of the earthworm L. terrestris exposed to Fusarium-infected and DON-contaminated wheat straw in a microcosm study under controlled laboratory conditions. Special attention was focused on the determination of Fusarium biomass and DON concentrations in the straw in order to assess the decomposing efficiency of L. terrestris.
Forty microcosms were prepared by filling 700 g of soil (moisture: 23%) equally into rectangular plastic jars (dimensions: 11 15.5 6.5 cm [width length height]), which were closed with plastic lids to prevent earthworms from escaping. The lids were perforated with some pinholes to allow air circulation. The soil was covered with 8 g air dried wheat straw (DM: 94%) per microcosm, and in each case 20 microcosms received C-straw or AIstraw. The straw was subsequently moistened and incorporated slightly into the surface area to initiate microbial activity. Microcosms were randomly positioned in a climate chamber and incubated at 15 1 C in the dark. After four days, two earthworms of 10.15 1.07 g total body weight were introduced into each of 10 microcosms containing C-straw and 10 microcosms containing AIstraw, no worms were placed into the remaining microcosms. In the course of incubation, microcosms were sprayed several times with water to prevent soil from desiccation. Thus a mean percentage of 22.4 3.7 of soil moisture could be maintained throughout the whole experiment. After 5 and 11 weeks of incubation, half of the microcosms from each treatment (C or AI-straw with or without worms) were terminated to gather samples for quantitative analysis. The number of replicates of each treatment was n ¼ 5.
2. Materials and methods
2.5. Determination of soil surface cover
2.1. Production of straw
The soil surface cover was determined by scanning top view photographs of the microcosms at the start and after 5 and 11 weeks of incubation. The specific areas of uncovered soil and those covered with straw were evaluated with the colour analysis program WinRHIZO, 2002 (Re´gent Instruments Inc.).
Winter wheat (Triticum aestivum) of the cultivar ‘Tommi’ was cultivated at an experimental field located near Go¨ttingen, Germany. At the time of flowering, wheat covering an area of 4 m2 was artificially infected by top–down spraying of three toxigenic Fusarium culmorum strains (F.c. 34, F.c. 35 and F.c. 36 from culture collection of the Department of Crop Science, Plant Pathology and Plant Protection, University of Go¨ttingen, Germany) suspended in 400 ml water containing Tween 20 (0.5 ml l1). The conidiospore concentration of the suspension was 3 105 ml1. At harvest time the ears of the wheat were removed and the straw was manually chopped to a length of approximately 2.5 cm (hereafter referred as AI-[artificially infected]-straw). For comparison, straw of the same cultivar originating from the same experimental field, but not artificially infected was used as control (hereafter referred as C-[control]-straw).
2.4. Experimental microcosms
2.6. Sampling procedure At first, the residual straw on the soil surface was removed from the microcosms. When sampling the straw, great care was taken to mechanically separate adhesive soil, but washing was avoided to prevent elution of DON, which is water soluble. Soil samples were taken from areas that visually appeared unaffected by earthworm activities and earthworm casts were also sampled. Finally the earthworms were removed, carefully cleaned off soil particles and weighed. The total wet weight of all samples was determined gravimetrically. The samples were then stored at 20 C in readiness for analytical preparation.
2.2. Preparation of soil 2.7. Sample preparation About 30 kg of a Luvisol characterized by a soil texture of 12% clay, 85% silt and 3% sand was taken from the Ap horizon of a site near Braunschweig, Germany, and was stored at 4 C until further treatment. Seven days before filling the microcosms, the soil was defaunated by cyclic freezing (twice for 24 h) interrupted by thawing for 24 h. Crude organic plant residues like straw or roots were manually removed and the soil was stored at 15 1 C until use.
All samples including the parent materials at the start of the microcosms were dried by lyophilisation. The straw was ground to pass through a 1-mm sieve using of an ultra centrifugal mill (ZM 200, Retsch GmbH, Haan, Germany). Samples of soil and earthworm casts were manually homogenized with a mortar to obtain a fine powder (<0.5 mm). 2.8. Determination of Fusarium Protein Equivalents
2.3. Adaptation of L. terrestris Adult individuals of L. terrestris, purchased from a commercial supplier, were adapted to the experimental conditions by keeping them in plastic jars containing the soil described above, covered
Fusarium Protein Equivalents (FPE) were quantified with a double antibody sandwich (DAS) ELISA according to the method as previously described (Tian et al., 2005) by using Fusarium specific antibodies and protein standards.
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100
a
C-straw
90
Soil surface cover [%]
Samples of 0.5 g of straw or 1.0 g of soil were suspended in PBSTBuffer (0.01 M phosphate buffered saline þ 0.05% [v/v] Tween 20 containing 1% [w/v] polyvinylpyrolidone, pH 7.2) at a ratio of 1:20 [w/v] and shaken on a horizontal shaker at 4 C for approximately 24 h. After centrifugation at 12,000 g for 10 min and dilution (1:25 [v/v]) with PBST-buffer, the extracts were applied in two replicates on a 96-well convex-bottomed plastic microplate (Immuno Plate Maxisorb, Nunc International, Denmark) pre-coated with antibody. The ELISA procedure was then continued according to Tian et al. (2005). The absorbance of the resulting dye was measured at 405 nm (reference: 592 nm) with a microplate photometer (SLT Spectra, TECAN GmbH, Crailsheim, Germany). The absorbance values of sample extracts free of Fusarium were subtracted from the absorbance values of the Fusarium-positive sample extracts. The values were then converted to Fusarium Protein Equivalents (FPE) using a standard protein curve, and the final FPE value was defined as the amount of Fusarium protein in the corresponding samples. The limits of quantification were 30 mg FPE kg1 for straw and earthworm casts, and 120 mg FPE kg1 for soil samples.
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AI-straw
80 70
b
60
c
50
d
40
e
30 20 10 0
0
5
11
5
11
Incubation time [weeks] Fig. 1. Change of soil surface cover with C-straw or AI-straw during an incubation period of 5 or 11 weeks due to incorporation activity of L. terrestris. Various letters (a, b, c, d, e) are significantly different between the columns (P < 0.05). C, control; AI, artificially infected.
2.9. Determination of deoxynivalenol
2.10. Statistics The Kolmogorov–Smirnov-test confirmed that all data were normally distributed. For this reason, analysis of variance (ANOVA) and the student’s t-test were used to compare treatment effects. All statistical analyses were undertaken using the software package SPSS for Windows Version 13. 3. Results 3.1. Soil surface cover In the presence of L. terrestris, the soil surface area of the microcosms covered by either C-straw or AI-straw decreased significantly within both incubation periods of 5 and 11 weeks (Fig. 1). The AI-straw was more efficiently incorporated into the soil, as the soil surface cover was reduced faster from the start until 5 weeks (56%) and 11 weeks (74%) compared with C-straw (38% and 53% until 5 and 11 weeks, respectively). ANOVA revealed a significant influence of Fusarium infection (F ¼ 24.98; P < 0.001) and experimental time (F ¼ 17.47; P < 0.01) on straw litter incorporation.
3.2. Body weight of L. terrestris All individuals in all treatments had survived to sampling at either 5 or 11 weeks. The body weight of L. terrestris increased over 5 weeks when both C-straw and AI-straw were provided (Fig. 2), but the weight gain was larger when the earthworms ingested AIstraw (þ6.5% versus þ1.4%). After 11 weeks of incubation, the body weight of L. terrestris decreased more when C-straw was offered (14%) as compared to AI-straw (8%). Only in the case of C-straw after 11 weeks, the body weight difference of L. terrestris compared to the initial body weight was significant (P < 0.01). 3.3. FPE concentration in straw The Fusarium biomass in the C-straw on the soil surface of about 0.33 0.14 mg FPE kg1 was degraded to about 85% after 5 weeks of incubation without L. terrestris. After 11 weeks of incubation only one microcosm contained straw with positive FPE, so that degradation of the Fusarium biomass was nearly complete. In the microcosms containing L. terrestris, no FPE could be detected in the straw after 5 weeks of incubation, but slight amounts of FPE (0.05 0.01 mg kg1) were observed in the straw after 11 weeks of incubation. The Fusarium biomass present in AI-straw of about 132.4 21.5 mg FPE kg1 at the start of the microcosms was reduced significantly to about 98.8 and 98.9% after 5 and 11 weeks
10
Change of body weight [%]
Deoxynivalenol (DON) was determined by use of the competitive ELISA test kit ‘Ridascreen DON’, product No. 5906 from R-Biopharm, Darmstadt, Germany. The sample extraction procedure was carried out as follows: 1.0 g straw, 2.5 g of soil or earthworm casts, respectively, were suspended with distilled water at a ratio of 1:10 or 1:20 [w/v] and shaken for 15 min using a horizontal shaker at 160 motions per minute. The extracts were first filtered through a fluted filter and then centrifuged at 15,300 rev min1 at 10 C for 5 min to remove solid particles. The resulting supernatants, occasionally diluted with distilled water, were directly applied in the ELISA test (two replicates), which was performed according to the manufacturer’s procedure. The absorbance of the resulting dye was measured at 450 nm using a microplate reader (PowerWave, Biotek Instruments GmbH, Bad Friedrichshall, Germany). The DON concentrations were calculated by use of the KC4 operating Software (Biotek Instruments) of the reader. Analysis was repeated when the standard deviation of the resulting mean concentration of the two replicates exceeded 10%. The limits of quantification were 74 mg DON kg1 for straw and 37 mg DON kg1 for soil and earthworm casts.
L. terrestris fed with C-straw L. terrestris fed with AI-straw
5 0 -5 -10 -15
5 weeks
11 weeks
Fig. 2. Mean relative change of body weight of L. terrestris as influenced by type of straw and incubation time in comparison with initial body weight (set as 0%) at experimental start. C, control; AI, artificially infected.
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of incubation in the absence of L. terrestris, respectively (Fig. 3). In the presence of L. terrestris activity, FPE degradation was significantly enhanced to about 99.4 and 99.5% after 5 and 11 weeks of incubation, respectively. 3.4. DON concentration in straw At the start of the microcosms, the C-straw on the soil surface contained 0.39 0.07 mg DON kg1. After 5 weeks of incubation, the primary DON concentration in the layer was reduced about 27% in the microcosms without L. terrestris, but was no longer detectable when L. terrestris was active. An approximately 50% increase of DON concentration was observed at 11 weeks of incubation without L. terrestris. However, again no DON was detected in the C-straw after 11 weeks in the microcosms containing L. terrestris. The initial DON concentration in the AI-straw on the soil surface of 146.7 18.1 mg DON kg1 was reduced to about 77% in the microcosms without L. terrestris, and to about 99.7% in microcosms containing L. terrestris after 5 weeks of incubation (Fig. 4). After 11 weeks the reduction of the primary DON concentration amounted to 88% in the microcosms without L. terrestris, and was nearly complete (about 99.9%) in the microcosms containing L. terrestris (Fig. 4). 3.5. FPE and DON concentration in soil and earthworm casts Neither in the undigested soil nor in the earthworm casts could positive FPE concentrations (120 mg kg1, 30 mg kg1, respectively) or DON concentrations (37 mg kg1) be detected at the start and after 5 and 11 weeks of incubation. 4. Discussion It appears that wheat straw highly infected with Fusarium is more attractive as a food source for L. terrestris than less infected wheat straw, as lower amounts of residual AI-straw compared to C-straw remained on the soil surface of earthworm containing microcosms. This is also confirmed by the greater body weight gain and smaller weight losses of L. terrestris when AI-straw was ingested over the course of 5 weeks and 11 weeks, respectively. It is well documented that earthworms do not feed at random (Bonkowski et al., 2000), but generally select leaf litter with high N-content (Hendriksen, 1990). In addition, soil fungi are regarded as an important food source for earthworms (Tiwari and Mishra, 1993; Edwards and Fletcher, 1988). In selection experiments L. terrestris
106
Without L. terrestris
FPE concentration[µg kg-1]
a
With L. terrestris
105 104 10
b
b
c
c
5
11
3
102 101 100
0
5
11
Incubation time [weeks] Fig. 3. Logarithmic presentation of FPE concentration (Mean and SD of n ¼ 5) in artificially infected straw during an incubation period of 5 or 11 weeks. Various letters (a, b, c) are significantly different between the columns (P < 0.05).
106
DON concentration [µg kg-1]
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Without L. terrestris
a 10
5
b c
With L. terrestris
104 d
103 10
e
2
101 100
0
5
11
5
11
Incubation time [weeks] Fig. 4. Logarithmic presentation DON concentration (mean and SD of n ¼ 5) in artificially infected straw during an incubation period of 5 and 11 weeks. Various letters (a, b, c, d, e) are significantly different between the columns (P < 0.05).
clearly preferred Fusarium sp. compared with other genera like Mucor, Trichoderma and Rhizoctonia (Moody et al., 1995; Bonkowski et al., 2000). Plant residues colonized by plant pathogens may indicate nutrient rich organic matter for earthworms and are therefore attractive for consumption (Bonkowski et al., 2000). It is likely that the highly Fusarium-infected straw is preferred as food because the fungus enhances the N-content thus increasing the nutrient quality of the straw (Weichert et al., 1991). Furthermore, due to its cellulolytic activity (Manka, 1988) the fungus probably makes hardly-digestible straw compounds more available for earthworm digestion. L. terrestris is involved in the degradation of Fusarium biomass in the wheat straw layer, because in the presence of earthworm activity FPE reduction was more efficient than in its absence both after 5 and 11 weeks of incubation. Furthermore, L. terrestris accelerates the degradation of DON in the straw layer. This seems to happen in close interaction with the soil microorganisms, which were able to decrease DON to some extent in the absence of the earthworm. It is therefore uncertain if L. terrestris possesses its own specific metabolic pathway for DON or rather acts as a catalyst enhancing microbial activity. This interactive breakdown of DON seems to be efficient enough to prevent adverse effects on the decomposition/incorporation activity and the body weight gain of L. terrestris. L. terrestris seems to take part in the degradation of residual Fusarium biomass in ingested straw, as no positive concentrations of FPE could be detected in the casts. This is comparable with results of a study showing a reduced viability of Fusarium spores when passed through the guts of L. terrestris (Moody et al., 1996). As no positive concentrations of DON were detected in the earthworm casts it is suggested that L. terrestris, probably promoted by the gut microorganisms, is also involved in the breakdown of residual traces of DON in ingested straw. As leaching of DON into the soil was not observed in this study, DON applied with the Fusarium-infected straw was probably metabolized in an interactive process between the soil microorganisms and the earthworms. However, the products to which DON might have been transformed in the course of the degradation process remain to be elucidated, as only little information is available at time giving indications about the structure of degradation compounds produced from DON by soil microorganisms (Bata and Lasztity, 1999). A bacterium isolated from soil was able to degrade DON into 3-keto-4-deoxynivalenol, a compound of less toxicity relative to DON (Shima et al., 1997). In conclusion, detritivorous earthworm species promote both the disappearance of Fusarium plant pathogens and the mycotoxin
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