Does non-inversion tillage positively affect earthworm communities and soil structure in crop rotations including root crops?

Does non-inversion tillage positively affect earthworm communities and soil structure in crop rotations including root crops? Bas Oudshoorn April 2013 Supervisors: Dr. M.M. Pulleman & S. Crittenden MSc Examiner: Prof. dr. L. Brussaard Soil Biology and Biological Soil Quality Department of Soil Quality Wageningen University The Netherlands

Wageningen University Does non-inversion tillage positively affect earthworm communities and soil structure in crop rotations including root crops? MSc thesis Soil Quality Bas Oudshoorn April 2013 Supervisors: Dr. M.M. Pulleman S. Crittenden MSc Examiner: Prof. dr. L. Brussaard Student number: 890521635120 Course code: SOQ-81836 Soil Biology and Biological Soil Quality Department of Soil Quality Wageningen University The Netherlands

Table of Contents Abstract... 4 1 Introduction... 5 1.1 Effect of earthworms on soil structure... 5 1.2 Soil structure stability... 5 1.3 Effect of tillage on soil structure... 5 1.4 Effect of tillage on earthworms... 6 1.5 Objectives and hypotheses... 7 2 Materials and methods... 8 2.1 Description of the field experiment... 8 2.1.1 Site description... 8 2.1.3 Crop management... 8 2.1.2 Soil management... 8 2.2 Sampling, data collection and analysis... 8 2.2.1 Earthworm abundance... 8 2.2.2 Water infiltration... 9 2.2.3 Aggregate stability... 10 2.2.4 Soil organic matter content... 10 2.2.5 Penetration resistance... 11 2.2.6 Yields... 11 2.2.7 Statistics... 11 3 Results... 12 3.1 Earthworm community... 12 3.1.1 Earthworm abundance and biomass... 12 3.1.2 Earthworm diversity... 12 3.1.3 Comparison with previous years.... 13 3.2. Soil physical properties... 14 3.2.1 Water infiltration in autumn 2012... 14 3.2.2 Water infiltration in spring 2013... 14 3.2.3 Aggregate stability... 15 3.2.4 Soil organic matter content... 16 3.2.5 Penetration resistance... 16 3.3. Yield... 17 3.4 Correlations... 17 4 Discussion... 19 4.1 Effect of tillage on earthworm communities... 19 4.2 Effect of tillage on soil physical properties... 19 4.3 Insight in measurements... 21 5 Conclusions... 22 6 Acknowledgments... 23 References... 24 2

Appendix I. Layout experimental fields... 28 Appendix II Crop rotations and crop management practices... 29 Appendix III Means and P-values... 32 Appendix IV Data Crittenden... 36 Appendix V. Correlations... 38 Appendix VI. Photos soil profiles... 41 3

Abstract Agricultural intensification, including mechanical soil disturbance, has increased over the last 50 years. Research has shown that mechanical soil disturbance can have negative effects on earthworm communities and soil structure. In contrast, no-till systems have been shown to increase soil structural stability and earthworm abundance. However, no-till systems are not considered a feasible option in The Netherlands, due to the economic importance of root crops. Non-inversion tillage has been proposed as an intermediate system. The aim of my study was to quantify the effects of non-inversion tillage versus conventional tillage on earthworm abundance and diversity and soil physical properties. The study was executed in the fall of 2012 and spring of 2013 in the BASIS field experiment in Lelystad, The Netherlands. The field experiment compared conventional, non-inversion and minimal tillage systems in a randomized complete block design. It was found that non-inversion tillage resulted in higher total earthworm abundance, in particular due to an increase of A. caliginosa populations. Additionally, noninversion tillage resulted in a higher soil organic matter content at 0-10 cm depth and higher aggregate stability at 10-20 cm depth. However, conventional tillage had a higher infiltration rate in one out of three fields, and a lower soil penetration resistance in one out of two fields. Beneficial effects of NIT were found in 2012 on earthworm abundance but comparison with data from 2009-2012 showed that this effect was not consistent across the years. Based on these results one can argue if implementation of non-inversion tillage alone will be sufficient to obtain improvements in soil structural conditions and soil physical quality, in crop rotations that include root crops. Keywords: Non-inversion tillage, earthworms, A. caliginosa, soil structure, soil organic matter, aggregate stability, water infiltration. 4

1 Introduction 1.1 Effect of earthworms on soil structure Earthworms have long been known to be important soil animals due to their positive effect on soil properties related to plant production, including soil structure, aggregate stability, soil organic matter content and soil fertility (De Oliveira et al., 2012; Marinissen, 1994; Ulrich et al., 2010). Soil structure is affected by earthworms by their burrowing and feeding activities (Langmaack et al., 1999) and through the production of casts (Blanchart et al., 1997). This has an effect on soil water dynamics (Ernst et al., 2009). Laboratory studies also showed that earthworms can regenerate compacted zones in agricultural soils by making burrows (Langmaack et al., 1999). However, earthworms could not physically avoid compacted zones in the laboratory experiment. Additional research showed that earthworms preferentially try to avoid these compacted zones (Capowiez et al., 2009). In addition, it is known that soil compaction (i.e. increasing soil bulk density) can decrease earthworm abundance (Beylich et al., 2010; Kretzschmar, 1991). Soil structure is affected differently by different earthworm species and functional groups (Ernst et al., 2009; Haynes et al., 2003). Earthworms can be divided into three ecological or functional groups, i.e. epigeic, anecic and endogeic (Bouché, 1972). They are categorised based on their habitat in the soil, and their primary feeding behaviour. Epigeic species live at the soil surface and feed on fresh organic material (e.g. plant residues). They mainly contribute to decomposition by breakdown of litter and organic matter and stimulation of soil microbes (Keith and Robinson, 2012). Anecic species make deep permanent vertical burrows in the soil and feed at the soil surface. Therefore they incorporate organic matter into the soil profile. However, they mainly contribute to the soil structure by increasing porosity and pore continuity with their vertical burrows (Keith and Robinson, 2012). Endogeic species live in the mineral layers in the soil where they also feed. Their main contribution to soil processes is the formation of soil aggregates (Capowiez et al., 2012; Keith and Robinson, 2012). All earthworm species contribute to nutrient cycling and therefore improve plant nutrient uptake and plant growth (Keith and Robinson, 2012). 1.2 Soil structure stability Soil structure can have a considerable effect on crop growth by facilitating root growth, water uptake and nutrient uptake due to porosity of the soil. Low soil porosity can restrict root growth. However, too high soil porosity can decrease water and nutrient uptake because the roots do not make enough contact with the soil (Passioura, 1991). Stable soil aggregates contribute to the maintenance of soil productivity (Álvaro-Fuentes et al., 2008) by affecting root density, root elongation and nutrient adsorption (Amézketa, 1999; Six et al., 2004). Additionally, they physically protect soil organic matter against rapid decomposition (Pulleman and Marinissen, 2004; Six et al., 2004). Aggregates can break down by rapid wetting (slaking), which can result in clogging of the soil pores and reduced water infiltration (Tisdall and Oades, 1982). Soil aggregation is affected by many factors including soil organic matter, texture, clay mineralogy, crop type, root development, climate, soil life and tillage (Six et al., 2000; Wakindiki and Ben-Hur, 2002). It is known that tillage affects both the dry and wet aggregate size distribution (Eynard et al., 2004; Unger et al., 1998). Soil aggregates should contain enough large pores (>75 µm diameter) to remain aerobic but also should contain small pores (30-0.2 µm diameter) to retain water for crop growth. The presence of large pores between the soil aggregates is needed to allow for rapid water infiltration and drainage (Tisdall and Oades, 1982). 1.3 Effect of tillage on soil structure Tillage is the most common way to change the soil structure. It affects the porosity, workability of the soil, C and N dynamics and soil water characteristics and thereby affects the growth conditions of crops (Andruschkewitsch et al., 2012; Castellini and Ventrella, 2012). Another objective of soil tillage is mechanical weed control. In recent years land use intensity and soil disturbance (e.g. ploughing) have increased in order to achieve higher yields per hectare and maintain soil workability, respectively. Increased soil disturbance, however, can have a negative effect on earthworm communities (Capowiez et al., 2009) and thereby reduce their positive effects on soil properties. Besides this indirect negative effect, conventional tillage can also directly affect the soil structure and soil aggregation. The effects of soil tillage on soil structure have been studied widely. Many studies have compared no-tillage versus conventional tillage, mainly addressing grain crops in Australia and North and South America (Abid and Lal, 2009; Azooz and Arshad, 1996; Benjamin, 1993; Munkholm et al., 2013). Less research has been 5

done on non-inversion tillage (sub soiling) in a temperate climate with crop rotations including both tuber and grain crops. Conventional tillage can compact the soil below the plough layer (i.e. plough pan), disrupt soil pores and increase the decomposition rate of SOM (Azooz and Arshad, 1996; Carter and Colwick, 1971; Gregorich et al., 1993; Roth et al., 1988; Shukla et al., 2003). Additionally, conventionally tilled soils tend to become less porous over time (Abid and Lal, 2009; Voorhees and Lindstrom, 1984). This could have a negative effect on soil water dynamics (Abid and Lal, 2009). Conventional tillage methods leave a bare soil surface and can deplete the soil organic matter content which makes the soil more sensitive to soil erosion (Celik and Ersahin, 2011). Similarly, infiltration of water into the soil is linked to stability of the soil structure, the bulk density (Celik and Ersahin, 2011) and the distribution of pores (Ankeny et al., 1990). By disruption of surface-vented pores, an increase in the breakdown of residues (lower organic matter content), surface sealing (Roth et al., 1988) and reducing aggregate stability, infiltration is reduced (Abid and Lal, 2009). Compared to conventional tillage, no-till soils tend to have a higher bulk density. However, the infiltration rate in no-till soils is often higher compared to conventional-tillage soils (Azooz and Arshad, 1996; Lal and Vandoren Jr, 1990; Shukla et al., 2003). This is caused by a more stable soil structure and more continuous macropores which are connected to the soil surface. These macropores are formed by soil cracks, old root channels and earthworms. Although there are also studies which reported higher infiltration rates in conventional tillage soils, e.g. in Southern Turkey in a Mediterranean climate (Celik and Ersahin, 2011), Lampkin (1990) stated that non-inversion tillage is less harmful for soil life and concentrates the soil organic matter in the top layer of the soil. Research on the effects of non-inversion tillage showed contrasting results. Non-inversion tillage is a form of reduced tillage. Reduced tillage is a general term and includes tillage systems that are considered less intensive or less frequent used compared to ploughing. The term can refer to many types of tillage systems without giving much specific information on the actual practices. In this report the term non-inversion tillage is used for a system that uses sub soiling. For other methods the term reduced tillage will be used. Celik & Ersahin (2011) found that reduced tillage (rotary tiller) increased penetration resistance and reduced the water infiltration rate compared to conventional tillage. However, Capowiez et al. (2009) did not find any significant difference in water infiltration caused by reduced tillage (rotary or disc harrow). Both found an increased bulk density. Capowiez et al. (2009) suggested that a higher number of macropores and increased continuity in reduced-tilled soil eliminated the effect of an increased bulk density. Munkholm et al. (2001) found that non-inversion tillage still showed remains of a plough pan 2 years after conversion to non-inversion tillage. 1.4 Effect of tillage on earthworms Earthworms in agricultural fields are affected by tillage. Not only by mechanical damage and predation during ploughing but also due to changes in the habitat of the earthworm (e.g. soil structure, organic matter content and vertical organic matter and moisture distribution) (Capowiez et al., 2009). Many studies concerning the effect of tillage on earthworms have been conducted. The outcomes, however, show different results. Additionally, many studies are about the abundance of earthworms in general and not at species level. Some studies show a decrease in total earthworm numbers in conventionally tilled soils compared to no-till soils and other showed the opposite. In addition, the effect of tillage may differ depending on species or functional group. Anecic earthworms are relatively less abundant in soils under conventional tillage compared to soils under reduced tillage (rotary or disc harrow) (Capowiez et al., 2009). This could be related to the fact that anecic earthworms run a higher risk of mechanical damage due to their bigger size (Ernst et al., 2009). They also have a slower reproduction compared to other functional groups. Endogeic earthworms are more abundant in ploughed soil compared to reducedtill soils. This could be caused by the incorporation of organic matter into the soil, reduced compaction of the top 10-20 cm s of the soil (Ernst et al., 2009) and a decrease in competition with anecic and epigeic species. There are also not many studies on the simultaneous effect of tillage on earthworm communities and soil structure in both conventional and organic farming systems (Crittenden, in press). In organic farming systems soil is a more determining factor for crop growth because of less external factors (e.g. fertilizers and pesticides) that can affect plant growth conditions (Munkholm et al., 2001). In organic farming systems the input of animal manure which increases the OM, mineralization, earthworm activity and aggregate stability is higher (Berry and Karlen, 1993; Pulleman et al., 2003). 6

1.5 Objectives and hypotheses The aim of this study was to quantify the effects of non-inversion tillage versus conventional tillage in a marine loam soil on (i) earthworm abundance and diversity; and (ii) soil physical properties. Soil physical properties refer to water infiltration, soil resistance, aggregate stability, and vertical soil organic matter distribution. Additionally, correlations between those soil physical properties and yield data were assessed. We hypothesized that non-inversion tillage compared to conventional tillage will: Result in a higher earthworm abundance in terms of biomass and numbers. Increase earthworm diversity in terms of earthworm species richness and a higher relative abundance of anecic earthworms. Result in a higher water infiltration, higher soil penetration resistance, higher aggregate stability and higher SOM in the top soil layer (0-10 cm). 7

2 Materials and methods 2.1 Description of the field experiment 2.1.1 Site description This study was conducted within the BASIS project. The BASIS project is a study of PPO Lelystad in the Netherlands comparing three tillage systems both under conventional and organic farming. The experimental fields are located in the Flevopolder near Lelystad (52 54 N, 5 56 E). The site has been used for arable production since 1960-1961 shortly after the Flevopolder was reclaimed from the sea. The BASIS project started in the autumn of 2008 and the fields were last ploughed during the autumn in 2007. The fields that are part of the organic farming system have been organically certified since 2004. The soil type is a marine clay loam which consists of 22.4% clay, 10.0 % silt and 67.6% sand. The soil has a ph of 7.9 (Crittenden, In press). The climate is temperate with an average annual temperature of 9.7 C an average rainfall of 850 mm per year (2011). 2.1.3 Crop management The conventional farming system has a four year crop rotation. The organic farming system has a five year crop rotation. Rotations include root crops and cereal crops with green manures during the fallow period in those years where the crop was harvested early enough (see table 1 in Appendix II). In 2012 sugar beet was grown on J9-2b, spring wheat was grown on J10-3 and potatoes were grown in J10-6 followed by grass clover. Each field is at a different point in the crop rotation. Crop management was preformed according to the crop type and farming system (i.e. conventional and organic). When cover crops were used, they were applied in all tillage treatments. However, there were some years were cover crops were only grown in non-inversion plots due to a short time gap between the harvest of the main crop and autumn ploughing. The cover crop stays on the field until spring seed bed preparation in case of minimal tillage and non-inversion tillage, but is ploughed under in November or December in case of conventional tillage (see table 2 Appendix II). The field operations and crop management practices (i.e. tillage, sowing date, harvest date, residues and fertilization) are shown in Appendix II (table 2). Crop residues were chopped and either incorporated into the soil by ploughing in the conventional plots or left on the soil surface in the non-inversion plots and minimal tillage plots and superficially incorporated later by cultivation for seedbed preparation. 2.1.2 Tillage treatments Three fields of the BASIS field experiment were used, one conventional field (J9-2b) and two organic fields (J10-3 & J10-6). Three different tillage treatments were compared in each field; (i) conventional tillage which consists out of mouldboard ploughing to a depth of 25 cm in the autumn and shallow cultivation to a depth of 8 cm in the spring for seedbed preparation using a rotary harrow and a tine cultivator, (ii) non-inversion tillage which consists of sub soiling to a depth of 10-20 cm in the autumn using a paragrubber and shallow cultivation to a depth of 8 cm in the spring for seedbed preparation, and (iii) minimum tillage (MT) which consists of annual shallow cultivation to a depth of 8 cm and occasional sub soiling (once in J9-2b). The experimental design used for all fields was a randomized complete block design with four replications. The layout of the all three experimental field can be found in figure 1, 2 and 3 in Appendix I. The plot dimensions are 85 m long and 12.5 m width. Each plot consists out of four beds with a width of 3.125 m. All the fields in the BASIS project have permanent tracks using GPS operated tractors. These tracks are used for all traffic except for ploughing and harvesting. 2.2 Sampling, data collection and analysis 2.2.1 Earthworm abundance This study was conducted from the autumn of 2012 until spring of 2013. In November 2012 monolith samples of 20 x 20 x 20 cm were taken to determine earthworm densities and species composition in all three fields and all tillage treatments. Three monolith samples were taken within one bed of each plot. The earthworms in the upper 20 cm of the soil were collected by hand sorting. The earthworms below 20 cm depth were extracted using formaldehyde solution (0.185%). Formaldehyde solution was poured into the hole left by the monolith. The hole was monitored for 20 minutes for earthworms coming to the surface. The monolith samples were collected within a period of two weeks during/after a period of heavy 8

rain. The crops in all three fields had been harvested. In J10-6 grass\clover had been sown and had covered the field. The monolith samples of fields J9-2b and part of J10-6 were hand sorted in the field. The monolith samples from field J10-3 and part of J10-6 were stored in a cold room before hand sorting in the laboratory due to planned tillage practices. Around the monolith for the earthworm sampling, soil moisture samples were taken. 5 soil cores of 20 cm were taken close to the monolith and combined into one sample. In the laboratory the samples were weighed, dried for 48 hours at 40 C and weighed again. The gravimetric soil moisture content (u) mass was calculated with the formula: u Where u is the M w is the mass of water and M t is the total mass. This process was repeated in April 2013 for the soil penetration resistances measurements (see next section). The collected earthworms were stored in a cold room until they were weighed. They were stored in a jar with some soil to prevent weight loss. The weighing took place every 2 or 3 days. The earthworms were cleaned with water to remove soil and dried on tissue paper to remove excess water from cleaning. The earthworms were counted and weighed to determine the earthworm abundance per sample before they were fixed in ethanol (70%). The earthworms were identified at the species level and classified into different functional groups (Sims and Gerard, 1985). 2.2.2 Water infiltration In November 2012 the effect of different tillage methods on water infiltration was measured by using a double ring infiltrometer (Eijkelkamp) according to Reynolds (2002). Measurements were conducted in all three fields in CT and NIT plots only. The inner ring and outer ring had a diameter of 28 and 50 cm, respectively. The rings were pressed 5 cm into the soil and were placed in representative spots in the fields. In field J9-2b machinery had completely covered the field with wheel tracks during the harvest of the sugar beets. Therefore the rings were also placed over wheel tracks. In fields J10-3 and J10-6 wheel tracks were avoided. Vegetation that could block or hamper the float was cut off at ground level to prevent soil surface disturbance. The soil was saturated first by filling the rings with water and letting in infiltrated into the soil. During the measurements the water level in the inner and outer ring were kept at similar level to prevent lateral flow. The drop in water level was noted at an interval depending on the infiltration rate (1 minute for J9-2b and 30 seconds for J10-3 and J10-6). Water levels were recorded until the infiltration rate was stable. Three consecutive readings with less than 10% variation between each other were considered stable. The principle behind the double ring method is that the water infiltration from the outer ring prevents a lateral flow from water in the inner ring. However, it is very likely that a plough pan or other disturbing layer (if present) could prevent vertical infiltration and water will flow lateral. Therefore additional infiltration measurements with stained water were conducted to test the double ring method in April 2013. This test was conducted once for conventional tillage and non-inversion tillage in field J9-2b and was only used to give a qualitative insight in the pattern of water infiltration. No quantitative analysis was conducted bassed on the results. The double ring method was applied as follows: after the rings were placed, they were filled with water to saturate the soil. When the water had completely infiltrated the outer ring was filled with water and the inner ring with stained water. Brilliant Blue FCF (Fast colours) was used as dye. A dye used for studying soil water flow should meet three criteria: (i) it should be distinctly visible in the soil, (ii) it should have similar transport properties as water, (iii) and it should not be toxic (Flury and Fluhler, 1994; Ketelsen and Meyer-Windel, 1999). Brilliant Blue meets these criteria. The mobility of brilliant blue is almost the same as water (Flury and Fluhler, 1995). The absorption of Brilliant Blue depends on soil type. The clay fraction and in particular the clay minerals absorb Brilliant Blue. Organic tissues does not absorb Brilliant Blue (Flury and Fluhler, 1995; Ketelsen and Meyer-Windel, 1999). The concentration used was 4g/l (Flury and Fluhler, 1994; van Schaik, 2009). One day after infiltration one vertical soil profile was prepared and five horizontal soil profiles. The profile pit was dug to a depth of 120 cm. The horizontal profiles were prepared at 10, 20, 30, 40 and 50 cm. Of each soil profile a photograph was taken with centimetre scales along two adjacent sides and the total horizontal distribution of the stained water was measured. 9

Fig. 2.1 Schematic top view (A) and side view (B) of the infiltration ring (1), vertical soil profile (2) and horizontal soil profile (3). 2.2.3 Aggregate stability Aggregate stability is a measure of the ability of the soil to resist aggregate break down due to wetting. Samples from early May 2012 were used for fields J9-2b and J10-3. Per plot 3 samples at 0-10 cm depth and 3 samples at 10-20 cm depth were taken. A wet sieving method, according to Elliot (1986), was used to determine the aggregate stability. Immediately after sampling in May 2012, the samples had been broken up along natural cracks, passed through a 10 mm sieve and dried at room temperature. Of each sample one subsample of 50 g of air dry soil was taken and manually sieved through a series of 3 sieves (2 mm, 250 µm and 53 µm respectively) to obtain 4 size fractions. Soils were submersed in water for 5 minutes on the largest sieve before sieving started. Soils were sieved by moving the sieve 50 times up and down (3 cm in vertical direction) in water during 2 minutes. Material remaining on the sieve was collected in an aluminium tray and dried at 105 C for 1 day. Material that passed though the sieve was transferred to the next sieve and this process was repeated. After drying the four fractions were weighed to determine the stable aggregate size distribution. The stable aggregate size distribution was expressed firstly as the mean weight diameter (MWD) (Pulleman et al., 2003): Where X is the mean diameter of each fraction size, W is the proportion of the total sample weight occurring in the size fraction and n is the number of size fractions. Secondly the aggregate stability was expressed as the percentage of water-stable macro aggregate (WSM) fractions > 250 µm after wet sieving. 2.2.4 Soil organic matter content Soil organic matter (SOM) content was determined in fields J9-2b and J10-3 in all tillage treatments in order to study the effect of tillage on soil SOM and its possible effect on earthworms. The same samples from aggregates stability were used. Per plot 3 samples at 0-10 cm depth and 3 samples at 10-20 cm depth were used. The SOM was determined by the loss on ignition method according to Nelson et al.(1996). The samples were air dried before they were sieved with a 10 mm sieve. A subsample was taken, put into a crucible and oven dried for 24 hours at 105 C and cooled in a desiccator. The dried samples were weighed and combusted for at least 3 hours at 550 C in a muffle furnace. Subsamples were cooled in a desiccator after combustion and weighed. The soil loss-on-ignition in % (LOI) was determined with the following formula (Lim C. H., 1982): 100% Where m 0 is the mass of the empty crucible in g, m 2 is the mass of the crucible with the oven dried soil in g and m 3 is the mass of the crucible with the ignited soil in g. The SOM in % was determined according to: 0.07 10

Where A is the content of particles < 2 µm in % (clay fraction, see section 2.1.1) 2.2.5 Penetration resistance The soil penetration resistance was measured in November 2012. However, the penetrologger did not save the data and the measurements had to be repeated in April 2013 fall ploughing and seedbed preparation. The soil penetration resistance was measured in field J9-2b and J10-6. A digital penetrologger (penetrologger 1.00, Eijkelkamp) was used to measure the penetration resistance till 60 cm depth. A cone with a base area of 1 cm 2 and a 60 angle was used. In each plot 10 measurements were taken resulting in 360 measurements. 2.2.6 Yields The yields from 2012 were collected from PPO Lelystad for all fields and tillage treatments. The yield was determined per plot. The harvested crops were unloaded in storage boxes. The boxes were weighed with and without the crop to determine the weight of the crop. A subsample of 1 kg from the wheat yield was taken from each storage box to determine the moisture content and the dry weight of the crop. The wheat yield was corrected to a standard moisture content of 16%. The potato yield was determined by the size and weight of the potatoes. Potatoes smaller than 28mm (diameter) and larger than 55 mm were not included into the total yield because farmers do not get paid for it. 2.2.7 Statistics Data from the different fields (J9-2b, J10-3 and J10-6 respectively) were treated as different datasets because of the different crop rotations and place in crop rotation and therefore there was no replication. The data sets were therefore analysed separately with software program IBM SPSS 19. The data used to make a comparison with previous years was collected from Crittenden (In press). The effects of tillage treatments on soil properties were analysed using one way ANOVA. When more than two tillage methods were used and the one way ANOVA showed a significant effect a post hoc multiple comparison test (LSD) was performed to explore which treatments were significantly different from each other. For the analysis of aggregate stability and SOM, Depth was included as a second independent factor in a two-way ANOVA. Data from samples within plots were pooled to calculate one value per plot (experimental unit). The Levene s test was used to test the homogeneity of variance and the Shapiro-wilk test was used determine whether the data was normally distributed. P-values <0.05 were considered to be significant. In case of violation of the Levene s test or Shapiro-wilk test, data were transformed (log, square, or square root) and tested again. If the data was still not normally distributed or homogeneous after transformation, the non-parametric test Kruskal-Wallis was used to analyse the data. Pearson product-moment correlation coefficient (Pearson s correlation) was used to measure the correlation between the dependent variables earthworm species, total abundance, biomass, OM, MWD and WSM. P-values <0.05 were considered to be a significant correlation. 11

3 Results 3.1 Earthworm community 3.1.1 Earthworm abundance and biomass The earthworm biomass did not show any significant effect of tillage in any of the three fields. In field J9-2b (conventional) the total number of earthworms was significantly different between tillage treatments. Figure 3.1 shows that MT and NIT (263 m -2 and 308 m -2 respectively) had a significantly higher earthworm abundance than CT (123 m -2 ). No difference was found between MT and NIT. When the total abundance is split up into adults and juveniles there were no differences in total adult abundance. The total abundance of juveniles was significantly different between CT (119 m -2 ) and NIT (265). MT (233 m - 2 ) did not differ significantly compared to CT and NIT. In field J10-3 (organic) the total earthworm abundance was significantly lower in CT (427 m -2 ) compared to MT and NIT (702 m -2 and 660 m -2 respectively). There was no significant difference between MT and NIT. The same pattern was found for total abundance of juveniles (CT 349 m -2, MT 606 m -2 and NIT 564 m -2 respectively). No differences were found in total adult abundance between tillage treatments. Field J10-6 shows the same trends as field J10-3. However, the differences are smaller and not significant. Table 3 in appendix III gives an overview of the means of biomass, total abundance, total abundance of adults and juveniles and percentage of juveniles. Abundance (m -2 ) 1000 900 800 700 600 500 400 300 200 100 0 b b a b b a CT MT NIT CT MT NIT CT MT NIT J9-2b J10-3 J10-6 Adult Juvenile Fig. 3.1. Total adult and juvenile earthworm abundances in fields J9-2b (conventional farming system), J10-3 and J10-6 (organic farming systems) for conventional tillage (CT), minimal tillage (MT) and non-inversion tillage (NIT). Means followed by different letter are significantly different between tillage treatments for total abundance respectively (P < 0.05). Error bars represent standard errors. 3.1.2 Earthworm diversity The diversity, expressed as species richness, did not show any significant effect of tillage in any of the three fields. Four different earthworm species were found in field J9-2b (see table 4 in appendix III). The population was dominated by endogeic species, especially Aporrectodea caliginosa (88%) (Fig. 3.2). Table 4 in appendix III shows the means and P-values for species richness of all earthworms found per treatment. No anecic species were found. Significantly less earthworms of the species A. caliginosa were found in the CT (115 m -2 ) plots compared to the MT (237.50 m -2 ) plots and the NIT (247.92 m -2 ) plots. There were no differences between MT and NIT. Less Lumbricus rubellus were found in the CT (2.08 m -2 ) plots compared to the NIT (45.83 m -2 ) plots. The MT (18.75 m -2 ) plots did not differ significantly compared to the CT plots and NIT plots. 12

Abundance (m -2 ) 350 300 250 200 150 100 50 0 J9-2b b b a (b) (ab) (a) A. caliginosa A. rosea L. rubellus CT MT NIT Abundance (m -2 ) 600 500 400 300 200 100 a b b J10-3 Abundance (m -2 ) 800 700 600 500 400 300 200 100 J10-6 a b b 0 A. caliginosa A. rosea E. tetraedra L. rubellus 0 A. caliginosa A. rosea E. tetraedral. rubellus Fig. 3.2. Earthworm abundance per species in fields J9-2b (conventional farming system), J10-3 and J10-6 (organic farming systems) for conventional tillage (CT), minimal tillage (MT) and non-inversion tillage (NIT). Species with an abundance < 2% of the total abundance are not included. Means followed by different letters are significantly different (P < 0.05) between tillage treatments. Error bars represent standard error. (...) indicates that the data was not distributed normally according to the Shapiro-Wilk test and a non-parametric test (Kruskal-Wallis) was performed. In field J10-3 eight species were found including anecic species A. longa and L. terresteris. These were not found in the other fields. In the NIT plots both species were found. In the CT plots only A. longa was found and in the MT plots Lumbricus terresteris was found. Figure 3.2 shows that the community was dominated by A. caliginosa (71%). Significantly less earthworms of the species A. caliginosa were found in the CT (285.42 m -2 ) plots compared to the MT (502.08 m -2 ) and NIT (497.92 m -2 ) plots. The difference between MT and NIT was not significant. Seven different earthworm species were found in field J10-6. A. caliginosa dominates the population with a relative abundance of 75%. Significantly less earthworms of the species L. rubellus were found in the CT (64.58 m -2 ) plots compared to the MT (177.08 m -2 ) and NIT (168.75 m -2 ) plots. No significant difference was found between MT and NIT. 3.1.3 Comparison with previous years. When the data of 2009, 2010, 2011 and 2012 (Crittenden, in press) are compared (see table 3.1 and table 7 and 8 in appendix IV) we can see differences between fields. In J9-2b no significant differences were found prior to fall 2012. In fall 2012 CT had the lowest total abundance. In J10-6 significant difference were found prior to fall 2012 but no differences were found in fall 2012. In contrast with J9-2b, CT showed significantly higher abundance than NIT and MT in those cases where significant differences were found. 13

Table 3.1. Total abundance for fields J9-2b (conventional farming system and J10-6 (organic farming system) for conventional tillage (CT), minimal tillage (MT) and non-inversion tillage (NIT). Means followed by different letters were significantly different between tillage treatments (P<0.05) Sampling date 9-2b J10-6 Tillage system CT MT NIT CT MT NIT Spring 2009 41 68 38 375 a 195 b 236 ab Fall 2009 95 110 169 389 415 289 Fall 2010 279 208 358 357 a 159 b 104 b Spring 2011 29 60 61 75 28 21 Fall 2011 192 245 127 841 a 560 b 555 b Spring 2012 143 188 204 543 457 446 Fall 2012 123 a 263 b 308 b 723 797 804 3.2. Soil physical properties 3.2.1 Water infiltration in autumn 2012 In field J9-2b no significant differences in infiltration rate were found between the CT plots (0.24 cm min - 1 ) and NIT plots (0.05 cm min -1 ) (Fig 3.3). The CT plots in field J10-3 had a significantly higher infiltration (8.31 cm/min -1 ) compared to the NIT plots (5.34 cm/min -1 ). In field J10-no significant differences in infiltration rates were found between the CT plots (0.87 cm/min -1 ) and NIT plots (0.70 cm/min -1 ). Although the tillage effect in fields J9-2b and J10-6 was not significant, they showed the same trend as J10-3. Table 5 in appendix III shows the mean infiltration rates and P-values for all tillage treatments. Infiltration rate (cm/min -1 ) 10 9 8 7 6 5 4 3 2 1 0 a b J9-2b J10-3 J10-6 CT NIT Fig. 3.3 Effects of tillage on the infiltration rate in fields J9-2b (conventional farming system), J10-3 and J10-6 (organic farming systems) for conventional tillage (CT) and non-inversion tillage (NIT).. Letters indicate that means differ significantly (P < 0.05) between tillage treatments. Error bars indicate standard errors. 3.2.2 Water infiltration in spring 2013 The infiltration with brilliant blue showed that the water from the inner ring did not only infiltrate vertically. The vertical profile (Fig 3.3) shows that the water only flowed through the soil matrix at the first 5 to 10 cm and used preferential flow paths afterwards. The water column of the outer ring could not prevent lateral flow. Already in the first 5 cm brilliant blue was found outside the inner ring. Water from the inner ring infiltrated to a depth of at least 1 m and the lateral flow was at least 50 cm. Figures 4 and 5 in appendix VI show how the infiltrating water used old root chancels as preferential flow paths for both vertical and horizontal distribution in both fields. It was observed that the soil was very compact and no distinct plough pan was found. At 40 cm a sand layer was found which had a high water infiltration in horizontal direction. 14

Figure 3.3 Vertical soil profile of 1 m deep in CT plot. The blue sticks represents the placement of the inner ring. The horizontal soil profiles confirm the results obtained from the vertical soil profile. Lateral flow of brilliant blue could already be seen at 20 cm depth (Fig 3.4A) and a large lateral flow at 50 cm (Fig 3.4B). The horizontal soil profile at 50 cm also showed that the sandy layers were more dyed compared to the clay layers. Although the infiltration rate was not measured, it was noticed that the second infiltration rate with brilliant blue after the first infiltration without brilliant blue was slower. However, this was also noticed in the outer ring which contained water without brilliant blue. Additionally, during the digging of the pit in the NIT plot it was observed that there was substantial lateral flow through the cracks left by the tillage equipment (paragrubber). Figure 3.4 horizontal soil profile in CT plot with scale at 20 cm (A) and 50 cm (B). The point where the blue stick and the edge of the vertical soil profile meet, indicates the centre of the inner ring. 3.2.3 Aggregate stability Two indices of aggregate stability were calculated: mean weight diameter (MWD) and water stable macro-aggregation (WSM) (Table 3.2). No significant interaction was found between the effects of tillage and depth for both indices. The MWD was significantly affected by tillage at 10-20 cm depth in field J9-2b (conventional). CT (0.45 mm) had a lower MWD compared to NIT (0.71 mm). The same trend was found in field J10-6 at 10-20 cm; the effect was marginally significant (P=0.052). There was no effect at 0-10 cm in both fields. Depth had no significant effect on MWD. 15

Table 3.2. Mean weight diameter (MWD) and water-stable macro-aggregates (WSM) after wet sieving for fields J9-2b (conventional farming system) and J10-3 (organic farming systems) for conventional tillage (CT), minimal tillage (MT) and non-inversion tillage (NIT) followed by the standard errors (n=4). Means followed by different letters capital letter were significantly different between tillage treatment or depth respectively (P < 0.05). Field Tillage MWD (mm) WSM (%) 0-10 cm 10-20 cm 0-10 cm 10-20 cm J9-2b CT 0.43 (0.05) 0.45 (0.02) a 34.9 (2.75) 40.0 (2.04) a Conventional NIT 0.65 (0.10) 0.71 (0.05) b 39.8 (4.30) 50.2 (1.68) b J10-3 CT 0.57 (0.03) 0.56 (0.03) 42.1 (2.18) 45.4 (2.16) a Organic NIT 0.57 (0.05) 0.72 (0.06) 40.5 (3.13) A 52.0 (1.22) bb WSM was significantly affected by tillage at 10-20 cm depth. In field J9-2b CT (40.0%) had a lower WSM compared to NIT (50.2%). The same effect was observed in field J10-6 (45.4% and 52.0% respectively). Depth had a significant effect in field J10-6 in the NIT plots were 0-10 cm (40.5%) had a significant lower WSM compared to 10-20 cm (52.0%). No significant effects were found in the CT plot of field J10-6 or in field J9-2b. Table 6 in appendix III shows the P-values for MWD and WSM per treatment. 3.2.4 Soil organic matter content A significant interactive effect on SOM content was found for the factors tillage and depth in fields J9-2b and J10-3 (Fig. 3.5). The lowest soil organic matter content (Nelson) in J9-2b was found in the CT plots at 0-10 cm (2.85%). It was significantly lower compared to the NIT plots at both 0-10 cm and 10-20 cm (3.23% and 3.10% respectively) but not compared to the CT plots at 10-20 cm (2.96%). In field J10-3 the NIT plots at 0-10 (4.11%) had the highest SOM content. There were no significant differences between the other treatments and depths (NIT 10-20 3.59, CT 0-10 3.52 and CT 10-20 3.42). SOM % 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 a CT ab 0 10 4.5 10 20 4 a b b 3.5 3 2.5 2 1.5 1 0.5 0 NIT SOM % CT a b NIT a Fig. 3.5 Means of organic matter content (OM) affected by the interaction of depth and tillage for fields J9-2b (A) (conventional farming system) and J10-3 (B) (organic farming system) for conventional tillage (CT), minimal tillage (MT) and non-inversion tillage (NIT). Error bars indicate standard error. Means followed by different letters differ significantly between tillage treatments and depth. 3.2.5 Penetration resistance Soil penetrations resistance was measured in J9-2b and J10-6. There was no significant difference between treatments in J10-6. In J9-2b significant differences were found from 5 to 30 cm. The penetration resistance of CT plots was lower from 5 to 30 cm and 10 to 30 cm compared to MT and NIT, respectively (Fig 3.6). MT and NIT differed significantly at 5 cm. Table 7 in appendix III shows the P- values of soil penetrations for J9-2b and J10-6. 16

Penetration resistance MPa 0 0 100 200 300 400 0 0 100 200 300 400-10 CT MT 10-20 NIT 20-30 30 Depth cm -1-40 -50 Depth cm -1 40 50-60 60-70 70-80 80-90 90 Fig. 3.6. Soil penetration resistance as affected by tillage in field fields J9-2b (A) (conventional farming system) and J10-6 (B) (organic farming system) for conventional tillage (CT), minimal tillage (MT) and non-inversion tillage (NIT). Error bars indicate standard errors. Means followed by an asterisk differ significantly between CT and reduced tillage (MT and NIT) treatments. 3.3. Yield The different tillage treatments did not have any significant effect on yield in all three fields. Table 5 in appendix III shows the yields per treatment. 3.4 Correlations The Pearson correlation test, executed with data from both J9-2b and J10-3, shows that there were three earthworm species which had a significant positive correlation with the total abundance, A. caliginosa, L. rubellus and E. tetraedra (Table 3.3). An overview of all species can be found in Table 9 in appendix V. These species, including total abundance, also have a positive correlation with SOM at 10-20 depth, but not with SOM at 0-10 cm. A. caliginosa and L. rubellus have a positive correlation with WSM and MWD at 10-20 cm. Additionally, L. rubellus also has an positive correlation with WSM at 0-10 cm and a marginally significant positive correlation with MWD at 0-10 (P=0.076). Table 9 in appendix V shows the correlations and their significance of all earthworm species. 17

Table 3.3. Pearson correlations between species, total abundance, OM, WSM and MWD. Correlations followed by * or ** are significant at the level 0.05 and 0.01 respectively (2-tailed) Total abundance SOM 0-10 cm SOM 10-20 cm WSM 0-10 cm WSM 10-20 cm MWD 0-10 cm MWD 10-20 cm A. caliginosa 0.958** 0.171 0.800** 0.322 0.634** 0.313 0.559* L. rubellus 0.826** 0.190 0.777** 0.551* 0.588* 0.456 0.541* E. tetraedra 0.662** 0.044 0.687** 0.115 0.144 0.005 0.173 Total abundance - 0.183 0.863** 0.383 0.626** 0.336 0.570* SOM 0-10 cm 0.183 - -0.047 0.311 0.259 0.274 0.312 SOM 10-20 cm 0.863** -0.047-0.346 0.527* 0.303 0.460 WSM 0-10 cm 0.383 0.331 0.346-0.489 0.868** 0.609* WSM 10-20 cm 0.626** 0.259 0.527* 0.489-0.579* 0.890** MWD 0-10 cm 0.336 0.274 0.303 0.868** 0.579* - 0.763** MWD 10-20 cm 0.570* 0.312 0.460 0.609* 0.890** 0.763** - The WSM at 0-10 cm showed a correlation with MWD at 0-10 cm and MWD at 10-20 cm (P<0.01 and P<0.05 respectively). Same trend can be seen at WSM at 10-20 cm (stronger effect same depth). The SOM only has a significant correlation with WSM 10-20 cm (OM 10-20) and there is a weak correlation between SOM 10-20 and MWD 10-20 (P=0.073). When the correlations are analysed separately for each field (see table 10 and 11 in Appendix V) the correlations did not all hold and some became less strong. A. caliginosa still showed a strong correlation with the total abundance but no correlations with SOM, WSM and MWD except for WSM at 10-20 cm in J10-3. The correlations between L. rubellus and the total abundance became weaker in J9-2b and there was no correlation in J10-3. E. tetraedra did not show any correlation in J9-2b with the total abundance and a weaker correlation in J10-3. SOM at 0-10 cm did not show any correlation when both field were analysed together but did show correlations when J10-3 was analysed separately with A. caliginosa, total abundance and MWD at 10-20 cm. Most correlations of SOM at 10-20 cm did not hold except for WSM 18

4 Discussion 4.1 Effect of tillage on earthworm communities A positive effect of reduced tillage on the total earthworm abundance was found for J10-3 and J92b. In accordance with the hypotheses, NIT and MT had a significantly higher total abundance compared to CT. J10-6, although not significantly, showed the same trend. However there was no significant difference in biomass due to tillage treatment. The results indicate that A. caliginosa had the highest abundance and that the effect of tillage followed the same trend as total abundance in field J9-2b and J10-3. The same effect of A. caliginosa was found by Crittenden (in press) in the same fields during a 4 year study. However, the negative effect of CT on A. caliginosa compared to MT and NIT during this study does not correspond with other studies (Crittenden, in press; Ernst and Emmerling, 2009). CT incorporates SOM into the soil which benefits endogeic species (Ernst et al., 2009). Table 3.3 also shows a strong correlation between A. caliginosa and SOM at 10-20 cm depth. However, this was not found when both fields were analysed for correlations separately. This also indicates farming system has an effect. Additionally, CT also increases the breakdown of SOM (Celik and Ersahin, 2011). CT does not show any differences in depth where NIT does show a significantly higher SOM at 0-10 cm depth in field J10-3. However, although the SOM in CT is more evenly distributed with depth, the SOM in NIT is similar or higher at 10-20 cm and 0-10 cm depth respectively in both fields. Therefore, endogenic species did not have higher food resources which could explain why the NIT and MT plots had a higher earthworm abundance compared to the CT plots. L. rubellus had a higher abundance in NIT and MT compared to CT. Unlike endogeic species, which benefit from incorporation of organic matter by inversion tillage, epigeic species do not benefit because they live close to the soil surface where they feed on organic-rich material (Keith and Robinson, 2012). The SOM at 0-10 cm was higher in the NIT plots compared to the CT plots which could explain the higher abundance of L. rubellus in NIT and MT compared to CT. Surprisingly, the SOM at 10-20 cm shows a correlation with L. rubellus but only when both fields are analysed together. The SOM at 0-10 cm does not (P=0.480) show a correlation, indicating an effect of farming system. Lowe and Butt (2002) found that L. rubellus does not only feed at the soil surface but also forages within the soil unlike the majority of epigeic species. They stated that L. rubellus is a pioneer species which is able to adapt to environmental changes. The earthworms were sampled close after harvest. This could have had an effect on L. rubellus, especially in J9-2b and J10-6 since the harvest of sugar beet and potato caused major soil disturbance. The incorporation of soil organic matter by ploughing in the CT plots can also be a reason for L. rubellus to feed in the lower soil layers. When the results of the present study are compared to results of previous years (Crittenden, In press) (see table 7 and 8, J9-2b and J10-6 respectively, in appendix IV) we can see that in conventional field J9-2b there were no significant differences in total abundance prior to 2012. Additionally, there was no obvious recurring trend. This shows that the earthworm population is not stable and is probably also influenced by factors other than tillage such as climate and position in crop rotation. Crittenden found CT resulted in a significant higher abundance in J10-6. The results of the current research do not show any significant effects in J10-6. However, after the potato harvest in fall 2012 the soil was not cultivated according to the different treatments (i.e. ploughing and sub soiling). Only seed bed preparation was applied and this may explain why no tillage effects were found in fall 2012. Fall 2012 was the first season in which significant differences in earthworm numbers were found in J9-2b where CT had a negative effect on total earthworm abundance. The contrasting results between J9-2b and J10-6 suggests that there is an effect of farming system. It seems that CT increased the earthworm abundance in organic systems where there is a higher input of organic matter. Ct resulted in a decrease in earthworm abundance in the conventional farming system where organic matter input is lower compared to the organic system. This is however in contrast with the results found in J10-3. Further research is needed in order to clarify the differences in earthworm response to tillage treatments in J10-3 and J 10-6. It was hypothesized that NIT would increase species richness and have a higher abundance of anecic species. However, no significant differences were found in species richness, nor in the abundance of anecic species. Anecic species were only found in J10-3 in negligible numbers. This could be caused by a history of continuous mouldboard ploughing (Crittenden, in press) which negatively affects the large anecic species by injuries, predation and destruction of their vertical burrows (van Capelle et al., 2012). 4.2 Effect of tillage on soil physical properties The infiltration rates only differed significantly in J10-3 where NIT had a significantly lower infiltration rate compared to CT. These results do not correspond with earlier research (Abid and Lal, 2009; Azooz 19