Bacterivorous nematodes correlate with soil fertility and improved crop production in an organic minimum tillage system

Organic farming systems are generally based on intensive soil tillage for seed bed preparation and weed control, which in the long-term often leads to reduced soil fertility. To avoid this, organic farming systems need to adopt conservation agriculture practices, such as minimum tillage and diligent crop rotations. However, minimum tillage generally delays soil warming in spring causing reduced nitrogen mineralization and thus poor plant growth. This negative effect needs to be compensated. We hypothesize that, in a diverse crop rotation, organic minimum tillage based on frequent cover cropping and application of dead mulch will improve soil fertility and thus crop production as confirmed by a number of chemical and biological soil indicators. We made use of two long-term field experiments that compare typical organic plough-based systems (25 cm) with minimum tillage systems (<15 cm) including application of transfer mulch to potatoes. Both tillage systems were either fertilized with compost or equivalent amounts of mineral potassium and phosphate. In 2019, soil samples from both fields were collected and analyzed for soil pH, organic carbon, macro-, micronutrients, microbial biomass, microbial activity and total nematode abundance. In addition, performance of pea in the same soils was determined under greenhouse conditions. Results from the field experiments showed an increase of macronutrients (+52%), micronutrients (+11%), microbial biomass (+51%), microbial activity (+86%), and bacterivorous nematodes (+112%) in minimum tillage compared with the plough-based system. In the accompanying greenhouse bioassay, pea biomass was 45% higher under minimum than under plough tillage. In conclusion, the study showed that under organic conditions, soil fertility can be improved in minimum tillage systems by intensive cover cropping and application of dead mulch to levels higher than in a plough-based system. Furthermore, the abundance of bacterivorous nematodes can be used as a reliable indicator for the soil fertility status.

Introduction Organic farming systems are generally based on intensive soil tillage for seed bed preparation and 39 weed control, which in the long run often leads to reduced soil fertility [1]. Although intensive soil 40 tillage increases microbial turnover rates and thus nutrient availability required for plant growth, long-41 term intensive soil tillage can cause depletion of the soil organic carbon content and thus reduced soil 42 fertility [2]. For a long-term improvement of soil fertility and its maintenance at a sustainable level, 43 organic production systems need to reduce the frequency and intensity of soil tillage and increase the 44 organic matter supply to the soil. The resulting accumulation of organic carbon will likely increase the 45 microbial activity and thus result in accelerated nutrient cycles [3][4][5]. However, minimum tillage 46 generally tends to delay soil warming in spring, and therefore N-mineralization rates are often too low 47 to meet the demand of the crops, especially in temperate climates [6]. That's why applying 48 conservation agriculture methods, i.e. the simultaneous application of minimum tillage, crop rotations,

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and residue retention, to organic farming may not necessarily improve soil fertility, even after 10 years 50 of adaptation to the system [7]. Similary, Krauss et al. [5] reported that yield of winter wheat, silage 51 maize, and spelt in an organic long-term experiment was still 10% lower even more than 10 years after 52 transition to reduced tillage compared to standard moldboard ploughing, even though manure compost 53 and slurry had been frequently applied Although nutrient levels and biological soil components were 54 generally higher under reduced tillage compared to plough tillage in the top 10 cm soil, the massively 55 enhanced weed competition under reduced tillage likely reduced crop yields. Thus, organic minimum 56 tillage systems need to be modified in order to provide sufficient levels of nutrients and weed control 57 at the same time [8].

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Two options to achieve this might be the use of legume and non-legume cover crops and mulches.

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Cover crops are known among others to conserve the nutrients of the previous crop for the following 60 crop, increase the organic matter content, stimulate microbial activity and suppress weeds [1].

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Especially leguminous cover crops and cover crop mixtures of brassicas with legumes have shown 62 positive effects on microbial biomass and activities as well as specific enzyme activities independent 63 of the climatic region and weather conditions [9]. Furthermore, the use of cover crops can reduce weed 64 seed banks in minimum tillage systems similar to levels in plough systems [10]. Organic mulch applications, referred here as the harvest of cover crops and their subsequent application to a specific crop or field, have been shown to contribute substantially to soil fertility in organic minimum tillage systems [6]. All those measures also protect the soil from a range of environmental impacts, such as 68 drought, wind and water erosion or even plant diseases [11].

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In combination with a long-term organic fertilizer strategy, such cropping systems should result in 70 more sustainable cropping systems in which nutrient cycles are almost closed. For example,

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application of high quality and certified composts that are free of pathogens, weeds, and toxic 72 compounds can contribute to a better plant performance in minimum tillage systems. Besides 73 nutrients, composts introduce additional microorganisms to the systems that may contribute to the 74 suppression of soil-borne diseases and should therefore enhance the overall soil fertility [12].

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However, the evidence of disease suppression and the resulting soil fertility improvement through the 76 use of composts often failed under field conditions in temperate climates due to variable 77 environmental conditions and inadequate application rates of composts [13,14]. Thus, long-term field 78 trials are required for a deeper understanding of the importance of compost in disease suppression and 79 soil fertility improvement [15].

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Soil fertility, which in this context is used synonymous for soil quality and soil health, can be assessed 81 through chemical and biological indicators, such as organic carbon, pH, micro-and macronutrients, 82 microbial biomass, or microbial respiration [16]. Furthermore, free-living nematodes are considered 83 important indicators of soil quality [17][18][19][20]. Different feeding types of nematodes occupy different 84 niches within the soil food web and hence, their classification and enumeration can determine certain 85 carbon pathways. In a recent review, Bünemann et al. [16] pointed out that biological indicators are 86 rarely used to assess soil health and quality and that most of the commonly used indicators are "black 87 box" indicators, such as C mic and microbial respiration. They further criticize that such assessments are 88 rarely linked to specific ecosystem services, which impedes the evaluation of their suitability as soil 89 quality and health indicators.

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Here we investigated two long-term experiments that were set up in adjacent fields in 2010 and 2011 91 to assess the effects of an organic minimum tillage system on chemical and biological soil properties 92 over time. The study specifically addressed the question, whether a crop rotation that includes cover 93 crops and mulch applications can maintain or even improve soil fertility and if this can even be further improved by the regular application of compost. Furthermore, the study investigated which chemical 95 and biological parameters were best linked with biomass production in a pea (Pisum sativum L.)

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bioassay and therefore could serve as indicator for soil fertility. The study compared a typical 97 plough-based system (25 cm) with a minimum tillage system (max. 15 cm), whereas the minimum

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The microbial respiration as indicator for microbial activity, was determined from the sieved soils in 175 2019. Soils were moistened to 50% water-holding capacity for seven days prior to analysis. Two 70 g 176 sub-samples of each soil were then filled into glass beakers placed in preserving jars that contained 20 177 ml water to prevent drying of the soils. Glass beakers with 15 ml of 0.5 mol NaOH were additionally 178 placed in the jars. Six blinds without soil were used as controls. Jars were closed hermetically and 179 incubated for seven days at 20°C. After incubation, glass beakers with NaOH were stored in vacuum 180 desiccators filled with soda-lime to avoid evaporation of the CO 2 . The total CO 2 concentration in the 181 NaOH was assessed via HCl titration. For this, a solution containing 3 ml of the NaOH, 30 ml water, 3 182 ml 0.5 mol BaCl 2 , and two drops of phenolphthalein was stirred and titrated with 0.1 mol HCl until 183 color change to rose. This back-titration will titrate the excessive NaOH. The soil respiration was 184 calculated according to the formula: VB and VS are the volumes of HCl titrated to the blinds and samples, respectively, F is the dilution 187 factor (3 ml aliquot of the 15 ml NaOH samples means F = 5), 2.2 corresponds to the amount of CO 2

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(mg) that refers to 1 ml of the titrated 0.1 mol HCl, FM and DM are the fresh matter (g) and dry matter 189 (%) of the soil samples, respectively, and d is the incubation time (days) of the samples at 20°C. For nematode analysis, 250 ml soil aliquots were processed with the Oostenbrink elutriator [26]. sub-samples with 700 ml soil each were filled in 11x11x12 cm pots and the pots were organized as a 207 randomized complete block (160 pots). Five surface-sterilized (70% ethanol for 5 minutes) and pre-208 germinated (2 days) pea seeds were planted per pot and reduced to three plants per pot after one week.

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At the day of plant reduction, mixed stages of P. penetrans (males, females, juveniles, and eggs) were 210 inoculated in all soils at densities of 1000 nematodes and eggs 100 ml soil -1 . The inoculation density 211 was based on repeated pre-experiments with inoculation densities of 0, 500, 1000, 2000, and 3000 212 nematodes 100 ml soil -1 of the experiment 1 field. The pea biomass reduction was 11% and 12% in the 213 pre-trial 1 and 2, respectively, after inoculation with 1000 P. penetrans 100 ml soil -1 .
For the data analysis of the greenhouse experiment, the random term was extended to "random = ~ 244 1|experiment/ replicate/ (greenhouse replicate/ tillage)".

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Spearman's ρ rank correlations were used to study the relationship of chemical and biological 246 indicators as well as their correlation with pea biomass production, root disease severity, and the 247 number of P. penetrans in roots by using the 'rcorr' function of the R-package 'Hmisc' [37]. Results

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were visualized for each field experiment separately using the R function 'corrplot' of the 'Hmisc' 249 package based on the P < 0.05 significance level.

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Both field experiments were maintained according to the ceteris paribus principle. However, the 253 severe drought in 2018 required some modifications in experiment 2. Due to the drought and also high 254 weed infestation, the winter wheat was terminated two months earlier than usual (

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In general, soil biological properties were enhanced by minimum tillage compared to the plough 301 tillage systems. For C mic , the differences between minimum and plough tillage as well as in part 302 between the compost and mineral fertilization increased over time (Fig 2). This is reflected by 303 significant interactions of sampling date (year) and tillage in both experiments (F 2,30 > 7.7, P ≤ 0.002).

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The status quo analysis was taken after the first differential tillage and compost application (2012, 305 2013), two years after the start of the experiment. Initial C mic values in experiment 1 and 2 were 60% 306 and 27% higher under minimum tillage with mineral fertilizer than under plough tillage with mineral 307 fertilizer. However, those differences were not statistically significant due to large standard errors (Fig   308  2).

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Four years after the start of the experiment when potatoes had been grown for the first time with 310 mulch application, C mic was already 39% and 62% higher under minimum tillage than under plough 311 tillage in experiment 1 (2014) and 2 (2015), respectively (Fig 2). At that time, compost application had 312 increased C mic consistently (6-20%) in comparison to mineral fertilization in both experiments under 313 minimum tillage.

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In 2019, C mic values were 72% and 35% higher under minimum compared to plough tillage in 315 experiment 1 and 2, respectively. The C mic was 15% higher under plough tillage with compost 316 compared to plough tillage with mineral fertilization (Fig 2). Compost effects under minimum tillage were less pronounced in 2019 than in 2014 and 2015. Similar effects of tillage were observed for the 318 microbial respiration and number of free-living nematodes in both years that were on average 86% and 319 64% higher, respectively, under minimum than plough tillage (Table 2). In particular, the number of 320 bacterivorous nematodes was three-fold and two-fold higher under minimum tillage compared to 321 plough tillage in experiment 1 and experiment 2, respectively, which also explains the significance of 322 the experiment by tillage interaction (   performance under greenhouse conditions Overall, pea yield was similar in both experiments. For example, pea aboveground dry weight was 344 50% and 39% higher under minimum tillage compared to plough tillage in experiment 1 and 2, 345 respectively. Although this effect was less clear for the pea root fresh weight, the highest root weights 346 were generally recorded under minimum tillage (Table 3). Similarly, the number of pods produced per 347 pot was always higher under minimum tillage than under plough tillage.

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Root lesion severity and root lesion length were about 5% higher in experiment 1 compared to 349 experiment 2. In general, both diseases parameters were similar for the two plough tillage systems and 350 the minimum tillage system that had received mineral fertilizer. However, when peas were grown in 351 soil collected from the minimum tillage system that was fertilized with compost root lesion severity 352 and root lesion length were reduced. This effect was only significant in experiment 2, though.

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The number of P. penetrans in roots pot -1 was 6,983 and 1,990 in experiment 1 and 2, respectively.

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Hence, the final population density divided by the inoculation density was 1 and 0.29, respectively. In

Biological soil components as indicators of soil fertility 369
In our study, soil fertility was measured as the potential of the soils for pea biomass production in a 370 greenhouse bioassay after artificial soil inoculation with lesion nematodes (Table 3).

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The growth substrate of pea in the greenhouse and the soil used for the analysis of the soil chemical 372 and biological parameters shown in Table 2 were derived from the same composite samples. This 373 allows to directly link these with data from the greenhouse experiment that were used as ecosystem 374 services (Fig 3). Thus, a number of standard biological "black box" indicators of soil quality, such as 375 C mic , N mic , microbial respiration, and C org were positively correlated with the pea biomass production 376 in soils of both field experiments (Fig 3). The severity of root lesions further affected pea dry matter in 377 both experiments (ρ < -0.51, P < 0.46, Fig 3).

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Besides these indicators, we also used the abundance of free-living nematodes that include free-living 393 herbivorous, bacterivorous, fungivorous and omnivorous/predatory nematodes. In both experiments, 394 the abundance of bacterivorous nematodes was highly correlated with pea dry matter (ρ > 0.61, P < 395 0.012, Fig 3) as well as with C mic and microbial respiration (ρ > 0.7, P < 0.003, Fig 3). The pooled 396 abundance of omnivorous/predatory nematodes as well as the number of P. penetrans in roots pot -1 397 were unaffected by any of the applied parameters. Herbivorous nematodes were positively correlated 398 with pea dry matter in experiment 1 (ρ > 0.78, P < 0.001, Fig 3) but not in experiment 2 (ρ > 0.23, P > 399 0.05, Fig 3). In experiment 2, fungivorous nematodes correlated negatively with microbial biomass,  where the first clear differentiations between tillage systems occurred two years after differential 454 tillage and four years after minimum tillage (Fig 2, year 2014 and 2015). Besides, we found higher 455 microbial quotients (C mic /C org ) under minimum than under plough tillage. These likely indicate a 456 higher C input as well as a higher C quality for C mic production [45].

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Contrasting to our expectations, the microbial quotient (MR/C mic ), which can be used as indicator of tillage with compost fertilization is overall increased. This was also directly translated into a greater 507 soil fertility in terms of pea biomass production. As pointed out above, under field conditions when 508 there is a lack of rainfall, effects of the mulch and increased soil organic matter contents on water 509 retention may lead to further advantages in practice.

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Especially bacterivorous and fungivorous nematodes contribute to soil fertility as decomposers that 511 release nitrogen to the soil [52,53]. Both feeding types accounted for 50% and 65% of the total 512 nematode community in experiment 1 and 2, respectively, which highlighted their importance for 513 nutrient turnover and thus pea production in our study. Across the globe, organic carbon that is 514 commonly used as indicator of soil fertility was found as one of the main drivers of nematode 515 abundance [15,54]. This was also observed for bacterivorous nematodes in both field experiments in 516 our study. However, the fungivorous:bacterivorous nematode ratio was negatively correlated with pea 517 dry matter yields in experiment 2, which emphasizes that bacterivorous nematodes were more reliable 518 indicators of soil fertility than fungivorous nematodes. This is further expressed by the strong positive 519 correlations of bacterivorous nematodes and pea biomass production in both field experiments (Fig 3). resting stages and dead organisms, under minimum tillage that not only explains the higher microbial 539 respiration in soil but also the higher pea biomass production compared to the plough tillage system.

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We therefore hypothesize that this particular minimum tillage system based on maximum use of cover 541 crops and additional transferred mulch to potatoes fosters microbial nutrient turnover from the labile C 542 pool and thus, improves plant nutrition and plant growth.

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In general, the use of compost as organic fertilizer should be preferred over mineral fertilization as 544 together with compost relevant micronutrients are supplied to the system. However, compost 545 improved only few chemical and biological soil properties in the plough and almost none in the 546 minimum tillage systems. The latter was quite surprising as compost application to the minimum 547 tillage system resulted in the highest pea biomass under controlled conditions. Indeed, compost 548 reduced the disease severity of root and stem root of the peas and, in soils with low organic carbon 549 contents, likely improved the resilience towards the resident pest P. penetrans. Additional physical, 550 chemical, and biological soil properties besides those investigated in our study may also play a role 551 and need to be investigated to fully understand the management-soil-plant interactions studied here. In 552 addition, the overall resilience of the management systems towards important biotic (fungal pathogens) and abiotic (drought, heat) stressors need to be investigated in order to fully understand the 554 ecosystem services provided by the system.

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The simple assessment of free-living nematodes and their classification into feeding types provides a 556 useful indicator for soil fertility. Thus, the bacterivorous nematodes were equivalent indicators of soil 557 fertility than other typically used parameters such as C org , C mic , microbial respiration, and 558 macronutrients. Of course, such analyses could be considerably strengthened by more detailed 559 nematological investigations. The low laboratory equipment cost for simple free-living nematode 560 assessments, e.g. nematodes can easily be obtained from Oostenbrink dishes, Baermann funnels, or 561 Cobb's sieves and counted and classified into feeding types under a microscope at 40x magnification 562 [26], is an additional advantage of using nematodes as bioindicators. Nematode specific indices, such 563 as maturity, channel, and structure indices as well as metabolic footprints will provide more details 564 about the faunal composition that influences the fertility and resilience status of a soil [19,52,56]. The 565 nematodes' key positions in the soil food web will thus allow to track the different carbon pathways in 566 the soil without the use of expensive and specialized equipment, such as needed for phospholipid fatty 567 acid, chloroform fumigation, ergosterol, and other extractions. However, detailed taxonomic 568 knowledge is required to identify the free-living nematodes to the family and genus levels. In addition,

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identification to the species level by using molecular methods will give a detailed overview of the 570 contribution of single species to a certain ecosystem service, such as plant production, disease 571 suppression or resilience. In accordance with many other studies, our results clearly demonstrate that 572 nematodes harbor a great potential for characterization of management effects.
573 574 575 576 support in microbial biomass and activity assessments. Furthermore, the authors would like to thank 580 Stephan Junge for provision of potato yield data and field pictures. 581 582