Co-Composted Biochar Compost for Improving Juvenile Growth After Replanting Copper-Contaminated Hop Gardens

Co-Composted Biochar Compost for Improving Juvenile Growth After Replanting Copper-Contaminated Hop Gardens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Co-Composted Biochar Compost for Improving Juvenile Growth After Replanting Copper-Contaminated Hop Gardens Johannes Görl, Dieter Lohr, Elke Meinken, Kurt-Jürgen Hülsbergen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7215331/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Dec, 2025 Read the published version in Plant and Soil → Version 1 posted 6 You are reading this latest preprint version Abstract Background and Aims The long-term application of copper-based fungicides in hop cultivation has led to substantial copper accumulation in the topsoil, potentially impairing the early growth of newly planted hop plants and affecting soil biota. To reduce copper bioavailability in soil, co-composted biochar compost was evaluated as a remediation strategy. Methods Two biochar composts, produced by co-composting 5 and 20 vol% biochar with chopped hop bines, and a biochar-free hop bine compost were applied into the planting holes during replanting of a copper-contaminated hop garden. A limed treatment and an unamended control were included. Remediation effects were assessed over two growing seasons based on visual damage ratings, copper concentrations in leaves and roots, biomass production, and soil respiration. Results During both growing seasons, leaf chlorosis and necrosis were observed, but were associated with copper toxicity only in the year of planting. In the second year, Mo deficiency was the primary cause of leaf damage. Biochar compost, particularly the one co-composted with 5 vol% biochar, significantly reduced these symptoms and increased biomass production by about 30%. In contrast, liming and biochar-free compost were less effective. Soil respiration was significantly enhanced by up to 81% with biochar-free compost showing the strongest effect due to higher microbial degradability. Conclusions Based on these findings, co-composted biochar compost can be recommended for farmers to improve juvenile growth after replanting copper-contaminated hop gardens. However, biochar-free compost also showed beneficial effects, particularly on microbial respiration, and may serve as a cost-effective alternative on less challenging sites. hop cultivation phytotoxicity co-composting molybdenum deficiency remediation ecotoxicology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction The use of copper-based fungicides in agriculture has a long history, dating back to the late 19th century when the discovery of Bordeaux mixture - a combination of copper sulfate and lime - marked a milestone in crop protection, particularly in combating downy mildew in grapevines (Tamm et al. 2022 ). This innovation provided the first effective measure for controlling various phytopathogenic fungi, leading to its widespread application across diverse crops, including vegetables, fruits, and hops. Despite the development of effective synthetic alternatives, copper-based fungicides are still used not only in organic but also in conventional crop production, particularly in viticulture as well as fruit and hop cultivation. This is due to their high efficacy against a broad spectrum of fungal pathogens, resilience under adverse weather conditions, minimal risk of resistance development, relatively low toxicity to terrestrial vertebrates and low costs (Lamichhane et al. 2018 ; Panagos et al. 2018 ; Speiser et al. 2018 ). Especially in organic farming, the absence of copper-based fungicides would result in yield losses ranging from 50% to total failure in hops, grapes, apples and other fruit crops (Burandt et al. 2023 ). Despite their numerous benefits, copper-based fungicides have a major limitation. Due to the low mobility and non-degradability of copper in soil, their long-term application leads to a creeping accumulation of copper in topsoil (Kabata-Pendias and Pendias 2010 ). After several decades of continuous spraying, concentrations of total copper can reach up to 450 mg kg -1 in topsoil of hop gardens (Schramel et al. 2000 ) and 1,500 mg kg -1 in vineyards and orchards (Burandt et al. 2023 ; Wightwick et al. 2008 ). This is 15 to 50 times higher than the average copper content in unpolluted soils (Vlček and Pohanka 2018 ). Such elevated copper concentrations in soil impair plant growth by inhibiting germination, reducing root development and negatively affecting nutrient and water uptake. Furthermore, copper toxicity reduces photosynthetic activity. As a result, total biomass and agricultural productivity decreases significantly (Mir et al. 2021 ). In hop cultivation, symptoms of copper toxicity typically do not occur in plants that have been established for several years, as they root to a depth of at least 2 m (Brant et al. 2020 ), where copper concentrations are considerably lower than in the topsoil (Schramel et al. 2000 ). However, in hop gardens with high copper concentrations in the topsoil, in the first few years after replanting, when the root system is still limited to the contaminated soil layer, symptoms of copper toxicity are frequently reported by hop farmers, impairing the juvenile growth and establishment of the hop plants (Portner 2009 ). Although such symptoms are limited to the early growth phase, they may still affect long-term productivity, as initial plant development is crucial for achieving stable yields in perennial crops (Pan et al. 2023 ). Indeed, quantitative studies addressing the economic relevance of copper toxicity on yield losses in hop production are currently lacking and should be further investigated. In addition to plant growth, high copper concentrations in the soil negatively affect soil biota such as microflora or earthworms. Their abundance decreases with increasing copper contamination, accompanied by shifts in community structure and a reduction in species diversity (Naveed et al. 2014 ). Consequently, the activity of essential soil enzymes (e.g., phosphatase, β-glucosidase and dehydrogenase), which play a crucial role in nutrient cycling, is significantly reduced (Fernández-Calviño et al. 2010 ). Results from permanent monitoring plots in hop gardens across Bavaria confirm that copper accumulation in the soil significantly reduces the abundance and diversity of earthworms (Walter and Burmeister 2022 ). In particular, no endogeic earthworm species, which live in the top 30 cm of soil and are most sensitive to copper pollution, were detected on two of the three monitoring plots during the entire observation period from 1985 to 2019. In view of the decline in soil health caused by the impairment of soil biota as well as the risk of damage to new plantings, there is a need for effective solutions against copper toxicity in contaminated hop gardens. Due to the concentration of cultivation in specific regions and the resulting limited availability of suitable land, as well as the high costs associated with establishing new trellis systems, the abandonment of contaminated sites is not a viable alternative (Rossini et al. 2021 ; Sawicka et al. 2021 ). Moreover, conventional remediation methods such as soil replacement, soil washing or phytoextraction are neither economically viable nor feasible within a reasonable period of time or they have significant ecological consequences (Kaparwan et al. 2020 ; Robinson et al. 2015 ). A more promising approach for mitigating copper toxicity is to reduce the bioavailability of existing copper by liming (Ambrosini et al. 2015 , 2017 ; Chatzistathis et al. 2015 ) or by applying highly sorptive organic amendments as compost (Huang et al. 2016 ) and biochar (Wang et al. 2022 ), respectively. Compared to liming, compost and biochar - especially co-composted biochar - offer additional benefits, e.g. water retention and long-term carbon sequestration (Antonangelo et al. 2021 ). Görl et al. ( 2023a ) demonstrated that the application of co-composted biochar compost to artificially and freshly copper-spiked soils improved the growth of Chinese cabbage while mitigating copper-induced avoidance behavior of earthworms and increasing soil oxygen consumption as an indicator of microbial activity. In a related study, in which co-composted biochar composts were also applied to long-term copper-contaminated soils from hop gardens, Görl et al. ( 2023b ) reported improvements in plant growth, along with a reduction in copper phytoavailability. The biochar composts used in this study were produced from hop bines - an agricultural by-product arising annually in large quantities during harvest. Thus, co-composting hop bines with biochar could potentially offer hop farmers a promising strategy to close local material cycles and to mitigate the toxic effects of high copper loads in their soils. Furthermore, it prevents the spread of diseases like Verticillium , which is regularly found in hop bines, as the addition of biochar at the beginning of the composting process intensified decomposition (Görl et al. 2023b ), which likely eliminated these pathogens (Hagemann et al. 2024 ). However, the limitations of the above-mentioned studies to avoid Cu toxicity include their conduction under controlled environmental conditions in a greenhouse or laboratory as well as the short trial duration of at most a few weeks. To our knowledge, no long-term field trials on this topic have yet been conducted on-site in hop gardens. To address this research gap, a field trial was established on a copper-contaminated soil, previously used for hop production. The field was replanted with young hop plants and the effect of biochar compost, derived from co-composting of hop bines and biochar, as well as biochar-free hop bine compost on the hop plants and the microbial activity in the soil was studied over a period of two growing seasons. We hypothesized that the application of co-composted biochar compost at the time of planting (i) improves the juvenile development and growth of hop plants, (ii) enhances microbial activity in the soil, and (iii) is more effective than biochar-free compost. 2 Materials and Methods 2.1 Study Site Conditions The field experiment was conducted in 2022 and 2023 on a soil that had been used as a hop garden between 1970 and 2019. The site is located in Hüll (48°36'10"N, 11°40'28"E), about 50 km north of Munich in the center of the Hallertau region, which is the world´s largest hop-growing area with about 17,000 ha (Kubeš 2025 ). The area is part of the Bavarian Tertiary Hill Country, approximately 445 m above sea level with soils classified as Cambisol. Soil properties, analyzed according to the methods of the Association of German Agricultural Inspection and Research Institutes, are listed in Table 1 (VDLUFA 2016a, b). Table 1 Soil properties of the experimental area (sampling depth: 30 cm). Values are presented as means, with the range from grid sampling (minimum to maximum) in parentheses. n.a. = not analyzed. Soil type pH (CaCl 2 ) P (CAL) K (CAL) Cu total Cu (CAT) mg kg − 1 mg kg − 1 mg kg − 1 mg kg − 1 silty loam 5.4 (5.0-5.8) 82 (52–105) 118 (100–133) 154 (n.a.) 68 (31–104) Compared to the average weather conditions of previous years (1991–2020), the experimental years 2022 and 2023 were significantly warmer (Table 2 ). Furthermore, the 2022 growing season was characterized by a dry spring and particularly hot and dry weather from June to August. While higher rainfall was recorded in the spring of the 2023 growing season, precipitation during the hot months of June and July again remained below the long-term average. Table 2 Monthly and annual values of mean air temperature (2 m height) and total precipitation during the experimental years 2022 and 2023, compared to the long-term average (1991–2020) (DWD 2024). Month / Year Temperature (°C) Precipitation (mm) 1991–2020 2022 2023 1991–2020 2022 2023 Jan -0.3 1.1 2.5 59.6 42.0 20.2 Feb 0.7 3.9 2.3 49.3 30.8 30.9 Mar 4.6 4.4 6.1 61.9 9.1 45.3 Apr 9.2 7.7 7.4 53.6 48.0 64.2 May 13.6 15.2 14.0 93.3 66.7 59.8 Jun 17.0 19.3 19.0 100.0 88.4 30.5 Jul 18.7 19.9 19.8 103.2 43.3 79.5 Aug 18.3 19.4 18.9 98.9 68.5 159.2 Sep 13.7 12.8 16.7 70.6 77.3 16.0 Oct 9.0 11.8 11.6 65.8 73.1 45.6 Nov 3.9 5.2 5.4 63.0 53.6 154.1 Dec 0.7 1.4 2.6 64.6 58.9 126.0 Year 9.1 10.2 10.5 883.8 659.7 831.3 2.2 Biochar and Compost Characterization The biochar (BC) used in this experiment was produced from untreated wood chips from certified sustainable and regional forestry in a Syncraft reactor (Huber et al. 2016 ) and pyrolyzed at a temperature of 850°C. It complied with all thresholds of the European Biochar Certificate for feed classification (EBC 2017 ). The most important physico-chemical properties are presented in Table 3 . Table 3 Physico-chemical properties of the biochar used for composting, as provided by the manufacturer. Properties Unit Values pH (CaCl 2 ) 9.3 Specific surface area (BET) m 2 g − 1 DM 282 Bulk density (< 3 mm) kg m − 3 DM 275 N total g kg − 1 DM 5 P total g kg − 1 DM 1 K total g kg − 1 DM 11 Ca total g kg − 1 DM 27 Mg total g kg − 1 DM 3 C org g kg − 1 DM 849 Molar H/C org ratio 0.1 The biochar was co-composted with freshly chopped hop bines derived from hop plants of the cultivar ‘Mandarina Bavaria’, which were harvested and processed according to common agricultural practice. The biochar was applied aligning with previous studies (e.g., Kammann et al. 2015 ) at rates of 5% and 20% by volume (5BC and 20BC), corresponding to 17% and 46% on dry weight basis, respectively. Additionally, chopped hop bines were composted without the addition of biochar (0BC). After an intensive rotting period of 65 days, including a total of five turnings, and a subsequent maturation phase of five months, composting was completed. Temperature measurements in the compost heaps showed that the addition of biochar extended the thermophilic phase by nearly 30 days, leading to a higher degree of sanitization. For further details on the composting procedure, refer to Görl et al. ( 2023b ). The physico-chemical properties of the three mature composts (Table 4 ) were analyzed according to the guidelines of the German compost quality association (BGK 2016 ). Table 4 Physico-chemical properties of the three mature composts used as soil amendments. Properties Unit 0BC 5BC 20BC pH (CaCl 2 ) 8.0 8.0 8.0 Salts (H 2 O) g KCl L − 1 2.3 3.5 2.7 NH 4 -N (CaCl 2 ) mg L − 1 4 4 1 NO 3 -N (CaCl 2 ) mg L − 1 164 285 138 N (CaCl 2 ) mg L − 1 168 289 139 P (CAL) mg L − 1 107 234 137 K (CAL) mg L − 1 1487 2462 2134 N total g kg − 1 DM 27 25 13 P total g kg − 1 DM 2.5 2.2 1.6 K total g kg − 1 DM 17 19 14 C total g kg − 1 DM 376 466 629 C/N 14 19 48 2.3 Experimental Setup and Plant Cultivation Considering the spatial heterogeneity of soil properties in the Hallertau region as a part of the Tertiary Hill Country (Fiener et al. 2019 ), and the strong effect of topography on copper distribution in soil (Jurisic et al. 2012 ), the entire experimental area was divided into a 20.8 m x 6.4 m grid, and soil samples were collected from the former hop row area to a depth of 20 cm and analyzed for pH, CAL-extractable P and K as well as CAT-extractable copper (VDLUFA 2016a, b). The results revealed considerable variability in soil pH and CAT-extractable copper content. Consequently, the experimental area was subdivided into six blocks (replicates), each measuring 20.8 m × 12.8 m and characterized by relatively uniform values regarding pH and plant-available copper concentration (Fig. 1 in the Appendix). To ensure a high concentration of compost in the root zone at reliable costs, compost was applied targeted around the planting holes, as suggested by Schmidt et al. ( 2015 ). Consequently, based on a planting density of 2,400 hop plants per hectare, the two biochar composts and the biochar-free compost were applied at rates of 1.25 and 2.5 kg of dry matter per planting hole, corresponding to application rates of 3 and 6 t ha -1 of dry matter, respectively. For this purpose, 20 cm deep planting holes were drilled in the former hop rows with a distance of 1.3 m within the rows and 3.2 m between the rows, and composts were applied over an area of 0.4 m² around each planting hole. Two control treatments without compost were included: one without lime and one with lime, as the pH of the field, with an average value of 5.4, was below the optimal range of 6.2 to 6.5 for a silty loam. The lime requirement was 5.5 t CaO ha -1 to achieve the optimal pH range (Knöferl et al. 2022 ). Liming was carried out using natural limestone, which was applied in one-meter wide strips along the respective planting holes. In total, the experiment resulted in eight treatments arranged in a completely randomized block design with six replicates (48 plots), each plot consisting of eight plants. To ensure uniform growth conditions across the experimental area, additional border rows were established all around. After the application of amendments, potted plants of the variety 'Hallertauer Mittelfrüh' were planted into the prepared planting holes at the end of May 2022. To ensure rapid establishment despite the hot weather conditions, the young plants were regularly irrigated by hand. Fertilization, plant protection and general cultivation measures such as soil management and training of hop plants were conducted according to the guidelines of the Bavarian State Research Center for Agriculture ( 2024 ). To avoid additional copper input, the application of copper-based fungicides and fertilizers was omitted. 2.4 Data Collection After a short establishment phase of three weeks, hop plants were visually rated weekly for symptoms of copper toxicity in both growing seasons as long as significant differences between treatments occurred. To minimize subjective variability, the ratings were always conducted by the same person. Plants without symptoms received a score of 0, those with moderate symptoms a score of 1, and those with severe symptoms a score of 2. Subsequently, a damage index per plot was calculated for each treatment as the sum of scores of the eight plants per plot. From calendar week 25 to 29 in the first growing season, in calendar week 23 of the second growing season as well as at the end of both growing seasons when cones were ripe for picking (BBCH 89), the concentrations of copper and other essential nutrients in the hop leaves were analyzed. Except of the final analyses, the youngest fully developed leaf of each plant was collected as plants had not yet reached the top wire at a height of 7 m (BBCH 38; Rossbauer et al., 1995 ). Since sampling at this height was technically unfeasible, at the end of the growing seasons, older leaves were collected at a height of 1.5 m. These were the first intact leaves located above the common defoliation zone in hop cultivation. The collected leaves were dried at 60°C in a forced-air oven until constant weight and grinded in a centrifugal mill (ZM 200, Retsch, Haan, Germany). Subsequently, microwave-assisted digestion (Multiwave ECO, Anton Paar, Graz, Austria) in HNO 3 + H 2 O 2 was done (VDLUFA 2016b) and concentrations of copper as well as other essential macro- and micronutrients (P, K, Ca, Mg, S, Fe, Mn, B, Zn, Mo) in the digestion solutions were measured via ICP-OES (iCAP 6000 DV, Thermo Fisher Scientific, Dreieich, Germany). To avoid insufficient establishment of the plants and to align with common practice, harvest took place only at the end of the second growing season in August 2023 (calendar week 35). To minimize the influence of adjacent plots, only the four central plants per plot were cut off just above soil surface and the fresh biomass of cones and the remaining bines were determined for each plant. Additionally, in calendar week 41, several hop roots from each plant were carefully removed from the soil and washed with deionized water. Cones, bines, and roots were dried at 60°C in a forced-air oven until constant weight and the dry biomass of cones and bines was recorded. Finally, the concentrations of copper as well as other essential macro- and micronutrients (P, K, Ca, Mg, S, Fe, Mn, B, Zn, Mo) in the roots were analyzed as described above. All analyses were performed in duplicate and control samples (e.g. reference material, blanks) were part of each analytical run. In addition to the plant experiment, microbial respiration in soil as an indicator of microbial activity was determined at the end of both growing seasons using the OxiTop® respirometry system (WTW, Germany). The system consists of glass jars with a volume of 2.7 L, which were equipped with a pressure measuring head. Sodium hydroxide solution (1 M NaOH, 50 mL) was placed in the headspace as a carbon dioxide trap to absorb CO₂ produced by microbial respiration. This absorption results in a pressure drop, which is continuously recorded by the measuring head. When the pressure dropped below − 100 hPa, the jar was ventilated to prevent O 2 depletion and the NaOH solution was renewed. For the soil respiration test, 1 kg of field-moist topsoil was collected to a depth of 20 cm from the planting hole areas of each plot using a soil corer, homogenized and filled into a jar. O 2 consumption was then measured at 25 ± 0.5°C in a climate chamber for five days and expressed as respiration rate in mg O 2 per kg dry matter (DM) per day, following the method described by Platen and Witz ( 1999 ). At the end of both growing seasons, soil samples were collected from the areas between the planting holes of each plot to a depth of 20 cm, in order to assess the effect of liming while avoiding the sampling of compost-amended soil. The samples were analyzed for pH and CAT-extractable copper as well as other essential micronutrients (Fe, Mn, B, Zn, Mo) according to VDLUFA (2016a, b). 2.5 Statistical Analyses The fresh and dry biomass of cones, bines and total plants, the copper concentration of leaves and roots, as well as the respiration rate were first subjected to a one-way ANOVA including all treatments. The significance of the means of the compost-amended and limed treatments was then tested pairwise against the unamended control using a Dunnett test. In a second step, the compost-amended treatments were additionally compared to the limed control using the same test. Subsequently, the two control treatments were removed from the dataset and a two-way ANOVA was conducted with the type of compost as first and the application rate as second factor. Since there were no interactions between both factors, a Tukey test was performed for each single factor. Additionally, the damage indices from the visual ratings were analyzed using a Kruskal-Wallis test. In case of significant differences, the rank sums were compared using a Nemenyi test. The level of significance for all statistical tests was set to 5%. Data pre-processing and visualization were performed using MS Excel 2016 (Microsoft Corporation, Redmond, WA, USA), while statistical analyses were conducted with Minitab V22 (Minitab Inc., State College, PA, USA). 3 Results 3.1 Plant Evaluation First Growing Season after Planting Already three weeks after planting, at the beginning of shoot elongation in week 25 (BBCH 31-32), the first symptoms of copper toxicity appeared on hop leaves, characterized by chlorosis and necrosis starting from the top, as well as by reduced growth (Figure 2). Until week 27, the number of plants with moderate and severe symptoms and consequently the cumulative damage indices were lower in treatments with biochar compost (5BC and 20BC) than in both treatments without compost (unlimed and limed). A similar trend was observed for the biochar-free compost (0BC) at the high application rate, whereas at the lower rate, a significant reduction of symptoms compared to both control treatments was only found in week 25. In contrast to compost application, liming had no mitigating effect on toxicity symptoms, but even led to a significantly higher proportion of affected plants in week 26. For the biochar-free compost (0BC) and the compost co-composted with 5 vol% biochar (5BC), increasing the application rate from 3 to 6 t DM ha -1 resulted in a significant reduction in damage indices until week 27. In contrast, for the compost co-composted with 20 vol% biochar (20BC), a positive effect of doubling the application rate was restricted to week 25. Among all treatments, the compost with 5 vol% biochar (5BC) applied at the high application rate of 6 t DM ha -1 was most effective in reducing toxicity symptoms during the first three assessment weeks. Regardless of the amendment type, symptoms of copper toxicity continuously declined over the growing season, and thus, from week 28 onwards, significant differences between treatments were no longer detectable. Consequently, visual ratings were stopped at the end of week 29. In contrast to the visual ratings, the copper analyses of the youngest fully developed leaves revealed only negligible differences among the eight treatments (Figure 3). Accordingly, compost application did not differ significantly from liming, and none of the amendments reduced the copper concentration of the leaves compared to the unlimed control. Moreover, no significant interaction was observed between compost type and application rate. Except for week 25, in which the biochar-free compost (0BC) led to a lower copper concentration than the compost co-composted with 20 vol% biochar (20BC), no significant differences were detected among the three compost types. Furthermore, doubling the application rate from 3 to 6 t DM ha -1 also had no effect on the copper concentration of the leaves. The copper concentration of older hop leaves at the end of the 2022 growing season in week 36 ranged from 5.6 to 6.0 mg kg -1 DM on average. As observed in the previous leaf analyses from week 25 to 29, no significant differences were detected between the treatments, neither in the Dunnett tests nor in the two-way ANOVA. While the macronutrient concentrations of the leaves in the first growing season showed no irregularities and were within optimal ranges for hop (Table 5 in the Appendix), the micronutrient analyses revealed elevated iron levels and a molybdenum deficiency from week 28 to 36 (Table 6 in the Appendix), which was also reflected in the low soil molybdenum levels (Table 7 in the Appendix). Second Growing Season after Planting Leaf chlorosis and necrosis also occurred on the hop plants in the second growing season (Figure 4). However, in contrast to the previous year, these symptoms appeared at a later stage of development during advanced shoot elongation (BBCH 33-35), and they were restricted to the lower part of the hop plants and rather atypical for copper toxicity (Portner 2009; Zorn et al. 2016). Nonetheless, compared to the unlimed control, both liming and compost application - regardless of application rate and biochar content - resulted in a significant reduction of damage symptoms in week 23. Except for the low application rate of compost co-composted with 20 vol% biochar (20BC), the effect of compost application was significantly greater than that of liming. In contrast to the biochar-free compost (0BC), where doubling the application rate had no effect, a clear benefit of increased application was observed for both biochar composts (5BC and 20BC). As in the first growing season, the compost co-composted with 5 vol% biochar (5BC), applied at 6 t DM ha -1 , consistently resulted in the lowest damage index. As symptom intensity remained almost constant until the end of the second growing season and no significant differences between treatments were observed from week 24 onwards, visual ratings were stopped after calendar week 25. As in the first growing season, copper analysis of the youngest fully developed leaves in week 23 of the second growing season (Figure 5a) revealed no significant differences between the compost-amended treatments (0BC, 5BC, and 20BC) and both control treatments (unlimed and limed). Due to this lack of differences, which was also reflected in the visual ratings from week 24 onwards, further leaf analyses were omitted until cone ripening (BBCH 89) at the end of the growing season. Similar to the young leaves, copper concentrations of the older leaves in week 35 (Figure 5c), as well as of the hop roots collected in week 41 (Figure 5e), showed no significant amendment effects. Furthermore, no significant interaction between the type of compost and the application rate was observed at any of the three sampling dates and the type of compost had no significant effect on the copper concentration of young leaves (b), old leaves (d) and roots (f), respectively. While doubling the application rate from 3 to 6 t DM ha -1 resulted in a significant 6% increase in copper concentration of young leaves (b), such an effect was not observed in old leaves (d) or roots (f). In contrast to the copper concentration of the youngest fully developed leaves, the copper concentration of the older leaves was on average about half lower, whereas it was more than ten times higher in hop roots. As in the first growing season, no notable irregularities were observed in the macronutrient concentrations of leaves and roots during the second season (Table 5 in the Appendix). Micronutrient analyses again revealed partly elevated iron levels and a significant molybdenum deficiency (Table 6 in the Appendix), consistent with the low levels of molybdenum in the soil (Table 7 in the Appendix). Additionally, a marked manganese surplus was detected in the leaves and roots. 3.2 Biomass Production in the Second Growing Season after Planting After two growing seasons, the hop plants were harvested in week 35, and both fresh and dry biomass of hop cones and bines were determined. Neither liming nor application of biochar-free compost (0BC) increased the yield of fresh and dry cones and bines compared to the control treatment without any amendment (Figure 6a, c). In contrast, the application of compost co-composted with 5 vol% biochar (5BC), especially at the higher application rate of 6 t DM ha -1 , generally increased the fresh and dry biomass of bines by up to 32% and 34% and the total fresh and dry biomass by up to 27% and 29%, respectively, compared to both control treatments. Furthermore, the compost co-composted with 20 vol% biochar (20BC) applied at a rate of 3 t DM ha -1 was more effective than liming. No significant interaction was observed between the type of compost and the application rate, and doubling the application rate had no significant effect on the fresh and dry biomass of cones and bines as well as on total fresh and dry biomass (Figure 6b, d). However, composts differed significantly in their effects on the fresh and dry biomass of bines as well as on total dry biomass. Yields were significantly higher for the compost co-composted with 5 vol% biochar (5BC) compared to the biochar-free compost (0BC). In contrast, no significant differences were found between both biochar composts (5BC and 20BC). 3.3 Soil Respiration Oxygen consumption in soils from the planting hole area of each treatment was analyzed at the end of both growing seasons. With the exception of the 20 vol% biochar compost (20BC) at 3 t DM ha -1 , compost application resulted in a significant increase in soil respiration rate of up to 81% in the year of planting compared to both control treatments (Figure 7a). In contrast, liming had no effect on the respiration rate. The two-way ANOVA revealed significant differences in the respiration rate between the type of compost (0 BC > 5BC ≈ 20BC) and a significant increase of more than 20% by doubling the application rate (Figure 7b). In contrast to the first growing season, a significant increase in the respiration rate was only observed for the biochar-free compost (0BC) at 6 t DM ha -1 at the end of the second growing season (Figure 7c). As in the first growing season, liming had no effect on the respiration rate. Moreover, no significant interaction was found between the type of compost and the application rate, and none of the two factors had a significant effect on the respiration rate (Figure 7d). 4 Discussion 4.1 Plant Evaluation Shortly after the replanting of the copper-contaminated hop garden, significant intercostal leaf chlorosis appeared on the hop plants. They started from the top and in case of severely affected individuals, leaves bleached completely and became necrotic. These findings align with observations from the Hop Research Center in Hüll that toxicity symptoms in copper-contaminated hop gardens typically appear shortly after replanting and in subsequent years at the beginning of shoot growth. Furthermore, symptoms similar to those observed in the present study have also been reported in various plant species, including brassicaceae such as Indian mustard, rapeseed, stone-head cabbage and Chinese cabbage (Feigl et al. 2013 ; Ali et al. 2015 ; Görl et al. 2023b ; Shahbaz et al. 2010 ), cereals like maize, durum wheat and barley (Ali et al. 2002 ; Bravin et al. 2010 ; Vassilev et al. 2003 ), as well as legumes, e.g. beans (Miyazawa et al. 2002), and have been attributed to copper toxicity. The application of the three composts (0BC, 5BC, and 20BC), particularly those containing biochar, significantly reduced these symptoms. A similar effect of the same composts was previously observed under controlled environmental conditions in a pot experiment with Chinese cabbage, where copper-induced leaf chlorosis was prevented in a soil artificially spiked with 240 mg Cu kg -1 (Görl et al. 2023b ). However, there are two arguments in the present study, which contradict the assumption that copper toxicity was the primary cause of the observed symptoms. First, the absence of a liming effect in the first year: According to Ambrosini et al. ( 2015 , 2017 ) and Chatzistathis et al. ( 2015 ), increasing pH by liming effectively reduces copper bioavailability and its phytotoxic effects. Thus, if copper toxicity had been the main driver, a mitigating effect from liming would have been expected. Indeed, at the end of the first growing season, a significant increase of pH up to 1.0 unit was found in limed plots (Table 7 in the Appendix). However, superficial application of lime in combination with dry weather conditions (Table 2 ) may have restricted the vertical movement of alkaline compounds in the soil and consequently an increase of pH in the root zone right after planting until the symptoms appeared (Azam and Gazey 2020 ). Second, the Cu concentration of leaves: Although the total copper content in the soil exceeded the toxicity threshold of 100 mg kg -1 DM (Mengel and Kirkby 2001 ), leaf concentrations only ranged from 3 to 10 mg Cu kg -1 DM. This is far below the threshold for excessive or toxic levels of copper, which are between 20 and 100 mg kg -1 DM for various plant species (Kabata-Pendias 2001). Moreover, in contrast to the severity of damage, no differences in leaf concentrations could be detected between the treatments. However, although increased copper availability in soil is generally reflected by higher copper uptake, a gap between copper concentrations in roots and aerial parts of the plants – especially leaves – is well documented in other species, e. g. grapevines, apples or peaches (Juang et al. 2012 ; Wang et al. 2016 ; Hammerschmidt et al. 2020). This may be explained by a protective mechanism: plants can limit the translocation of Cu from roots to shoots by retaining it in root cell walls, vacuoles or the apoplast to avoid damage to sensitive aerial tissues (Adrees et al. 2015 ). In the year of planting, this mechanism could not be confirmed by measurements, as root sampling was not possible without permanently damaging the young plants. However, the presence of such a mechanism is strongly supported by the Cu concentrations in hop roots at the end of the second growing season after planting. These ranged from 72 to 96 mg kg -1 DM, which is 10 to 30 times higher than the concentrations in leaves at the same sampling date. Although copper is strongly retained in the roots, it still may disrupt essential physiological processes, particularly those related to Fe uptake and translocation. Excess Cu can compete with Fe for shared uptake pathways such as IRT1 and inhibit the activity of ferric chelate reductase, both are critical for efficient Fe acquisition and translocation (Perea-García et al. 2013 ). In addition, copper-induced oxidative stress can impair intracellular Fe homeostasis, and thus hinder the proper allocation of Fe to key sites, e.g. the chloroplasts (Thomas et al. 2008 ). Consequently, even if sufficient Fe is present in the plant, it may remain physiologically unavailable. Both, copper-related reduction in Fe uptake and translocation as well as disruptions of Fe homeostasis may have contributed to the development of chlorosis, which were typical for Fe deficiency in the year of planting (Zorn et al. 2016 ), even in the absence of elevated Cu concentrations in leaf tissue. Leaf chlorosis also occurred in the second growing season, whereby a significant positive effect of compost application as well as of liming was observed. However, symptoms appeared during advanced shoot elongation (BBCH 33–35), and thus considerably later than at the beginning of shoot growth (BBCH 07–09), as typically reported for copper toxicity in hop gardens (Portner 2009 ). Furthermore, the symptoms were restricted to the lower part of the plants and atypical for copper toxicity (Portner 2009 ; Zorn et al. 2016 ). Both makes copper toxicity quite unlikely to be the sole cause of symptom development in the second growing season. The same applies to the macronutrients analyzed in leaves and roots, which showed no irregularities during the entire experimental period (Table 5 in the Appendix). In contrast, molybdenum (Mo) concentrations in leaves were clearly below the sufficiency range both at the end of the first growing season and especially during the second growing season (Table 6 in the Appendix). Moreover, Mo deficiency was not only evident in the leaves, but also in the roots and the soil (Tables 6 , 7 and 8 in the Appendix). These findings strongly support Mo deficiency as the main cause of symptom expression in the second growing season after planting. In line with this, previous studies reported similar leaf concentrations and symptom development in hop grown under Mo deficiency (Askew et al. 1958 ; Watson and Askew 1956 ). Since soil pH is generally positively correlated with Mo uptake by plants (Kabata-Pendias and Pendias 2010 ), the low soil pH in the present study was likely the main reason for the limited Mo availability. As a result, liming significantly reduced symptom expression due to an increase of pH during the second growing season. The same likely applied to the application of compost, which had a high pH of 8.0 (Table 4 ). In addition, compost application may have supplied additional Mo into the planting holes, which could explain its stronger effect on both symptom reduction and Mo uptake compared to liming. This assumption is supported by the positive effect of the increasing application rate. In contrast to Mo, Fe and Mn concentrations in leaves frequently exceeded the sufficiency range during both growing seasons (Table 6 in the Appendix). This pronounced uptake likely resulted from increased solubility and mobility under low pH (Kabata-Pendias and Pendias 2010 ), as indicated by the reduction of Mn concentrations after liming (Table 6 in the Appendix). However, the lack of correlation between leaf Fe and Mn concentrations and the severity of visual symptoms as well as the elevated but generally subtoxic Fe levels in hop leaves do not support Fe or Mn excess as the primary cause of the observed leaf damage (Afonso et al. 2020 ). Moreover, Watson ( 1960 ) reported a significant increase of phosphorus and potassium concentration in leaf tissues of hop plants suffering from Mn toxicity which was not observed at any time point of the current research (Table 5 in the Appendix). Indeed, this is not a sound evidence against Mn excess as the primary cause of the observed leaf damage, but a further indication. 4.2 Biomass Production While neither liming nor the application of biochar-free compost had a significant effect on biomass production, the addition of both biochar-containing composts - particularly the compost co-composted with 5 vol% biochar at high application rate - resulted in an increase of about 30% in bine biomass and total biomass compared to the treatments without compost. Notably, the compost co-composted with 5 vol% biochar showed the most beneficial impact on plant growth not only in the present field trial, but also in a previous pot experiment with fresh and aged copper-contaminated soils. This might be due to the most intensive composting process and thus the highest degree of maturity achieved with this compost (Görl et al. 2023b ), which is also reflected in its higher levels of plant-available N, P, and K (Table 4 ). The growth-promoting effect of co-composted biochar compost can be attributed not only to a potential reduction in the phytoavailability of heavy metals in soil, but also to other positive impacts on soil properties. These include increases in soil organic carbon and available nutrients, improved water retention and holding capacity, and enhancements in soil aggregation, structural stability, aeration, cation exchange capacity and pH, as well as stimulation of microbial activity (Antonangelo et al. 2021 ). Although biochar-free compost also exhibits similar effects on soil properties, these were generally less pronounced than those observed with biochar compost in our trial, which fits to results of others (Fischer and Glaser 2012 ; Liu et al. 2012 ). In contrast to bine and total biomass, cone yield was not significantly increased by compost application despite the reductions in symptom expression. This suggests that the existing copper contamination in the soil did not negatively affect cone production, possibly due to the copper level of 154 mg kg -1 being too low to noticeably impact cone yield. On sites with higher contamination levels, as frequently observed in the Hallertau region (Schramel et al. 2000 ), stronger effects on cone yield can be expected. 4.3 Soil Respiration The addition of compost, both with and without biochar, increased respiration rates in the soil of the planting holes areas at the end of the first growing season after planting. This increase in soil respiration can be attributed to higher microbial biomass and the stimulation of microbial activity due to enhanced resource availability, as well as to shifts in microbial community composition (Iovieno et al. 2009 ) and improved physical soil properties (Tejada et al. 2009 ). The strongest stimulation of soil respiration by the biochar-free compost is likely due to its higher degradability, as biochar is largely resistant to microbial decomposition (Wang et al. 2015 ). Moreover, the addition of biochar at the beginning of the composting process has shown to intensify the composting process (Görl et al. 2023b ), further reducing the proportion of degradable carbon in the biochar-containing composts. A comparable difference in respiration rates between soils amended with co-composted biochar composts and those amended with biochar-free compost was already observed in a previous pot experiment using artificially and freshly copper-spiked soils (Görl et al. 2023a ). With the exception of the high-rate application of biochar-free compost, no clear effect of compost addition on respiration rates was observed in the second growing season after planting. This lack of effect was likely due to the advanced microbial consumption of easily degradable carbon fractions in the composts (Pane et al. 2013 ), as well as their redistribution from the initially concentrated placement in the planting holes into the surrounding soil as a result of soil management practices within the cultivation period of about 20 months. 4.4 Recommendations for Field Application Based on our present and previous findings (Görl et al. 2023 a, b), the use of co-composted biochar compost can generally be recommended for farmers to improve juvenile development and growth of hop plants after replanting copper-contaminated hop gardens. If the biochar compost is produced from hop bines, as in the present study, the addition of 5 vol% biochar at the beginning of composting is already sufficient and even preferable to the higher rate of 20 vol%, as this reduces biochar costs while maximizing positive effects on plant growth and achieving improvements in the composting process. As doubling the application rate from 1.25 to 2.5 kg per planting hole generally improved the effectiveness of the compost amendments, the higher rate should be preferred, particularly on sites where impairments in the juvenile growth of hop plants are to be expected. As even the more cost-effective biochar-free compost showed positive effects in reducing leaf symptom expression and particularly in stimulating microbial activity, its use can also be recommended to farmers. However, in the present experiment, biomass production could not be enhanced without the addition of biochar. Nevertheless, this effect was limited to higher bine and total biomass, while cone yields remained unchanged. For this reason, it should be carefully considered whether the additional costs of biochar are justified, and decisions should always be made depending on the specific site conditions. While on less challenging sites, such as in the present experiment, the biochar-free compost might already be sufficient, on highly contaminated soils, the use of co-composted biochar compost should be preferred. Moreover, when using biochar-free compost, the absence of biochar costs could allow a compost application across the entire area instead of a planting hole-specific application, which covers only about 10% of the total area at a planting density of 2,400 plants per hectare. However, even when biochar is not used, compost availability and cost may still represent limiting factors. Furthermore, without biochar, additional benefits such as improved composting efficiency and long-term carbon sequestration in the soil cannot be expected. Beside the positive effects on plant growth and soil biota, further positive effects of applying (co-composted biochar) compost to planting holes should be mentioned here: Due to higher infiltration rates, irrigation of plants was easier in the year of planting and less superficial run-off was observed. Furthermore, planting into compost-amended soil was considerably easier than into bare soil, particularly under the dry planting conditions of the present study. This could be a strong argument for hop growers, especially in view of the expected increase in spring drought due to climate change (Hänsel et al. 2019 ). In contrast to biochar-free compost, liming may not be a suitable alternative to the application of co-composted biochar compost, as its effects on plant growth and soil respiration were either minimal or absent in this study. 5 Conclusions The targeted application of co-composted biochar compost to planting holes during the replanting of a copper-contaminated hop garden significantly reduced the occurrence of visible damage symptoms and increased biomass production until the end of the second growing season, thus confirming our first hypothesis. However, indications of copper toxicity were only observed in the year of planting, whereas Mo deficiency was identified as the primary cause of leaf symptoms in the second year. In addition to plant growth, the application of co-composted biochar compost significantly enhanced microbial activity in the soil of the planting hole area, at least in the first growing season, partly confirming our second hypothesis. While co-composted biochar compost was more effective than biochar-free compost in promoting plant growth, the opposite was observed for soil respiration, thus rejecting our third hypothesis with respect to soil biota. Since the present findings demonstrated consistent or even enhanced effects with reduced biochar amounts in co-composting, future research should focus on evaluating the impact of biochar additions below 5 vol%. If further reductions in cost-intensive biochar prove feasible without compromising efficacy, the acceptance among farmers and consequently the prospects for using co-composted biochar compost could be significantly enhanced. Abbreviations BC – biochar; BET – specific surface area; B – boron; Ca – calcium; CaCl 2 – calcium chloride; CAL – calcium acetate lactate extraction; CAT – CaCl₂/DTPA extraction; C org – organic carbon; Cu – copper; DM – dry matter; Fe – iron; H – hydrogen; K – potassium; Mg – magnesium; Mn – manganese; Mo – molybdenum; N – nitrogen; NH 4 -N – ammonium nitrogen; NO 3 -N – nitrate nitrogen; P – phosphorus; R B – basal respiration rate; S – sulfur; Zn – zinc Declarations Competing Interests: The authors have no competing interests to declare that are relevant to the content of this article. Funding: This research was funded by the Bavarian State Ministry for Food, Agriculture and Forestry (Grant No. A/21/08) and further supported by the Bavarian Academic Forum—BayWISS. Author Contributions: Conceptualization, methodology, investigation, formal analysis, visualization, and the initial drafting of the manuscript were conducted by Johannes Görl. Supervision of laboratory analyses, project administration, and funding acquisition were carried out by Dieter Lohr. Review and editing were performed by Dieter Lohr, Elke Meinken, and Kurt-Jürgen Hülsbergen, with overall supervision provided by Elke Meinken and Kurt-Jürgen Hülsbergen. All authors read and approved the final manuscript. Acknowledgments The authors express their gratitude to the technical staff of the Institute of Horticulture for their careful assistance in conducting the experiments. They also wish to thank the staff of the Hop Research Center in Hüll for their valuable technical and scientific support, with special thanks to Florian Weiß for his dedicated and tireless assistance. Data availability: The data supporting this study’s findings are available from the corresponding author upon reasonable request. 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Bulg J Plant Physiol 29:33–43 VDLUFA-Verlag, Method Book VDLUFA (2016a) I: Analysis of Soils, 4th ed.; with 1–7 Suppl.; VDLUFA-Verlag: Darmstadt, Germany, ; ISBN 978-3-941273-13-9 VDLUFA-Verlag, Method Book VDLUFA (2016b) VII: Environmental Analysis, 4th ed.; with 1–7 Suppl.; VDLUFA-Verlag: Darmstadt, Germany, ; ISBN 978-3-941273-10-8 Vlček V, Pohanka M (2018) Adsorption of copper in soil and its dependence on physical and chemical properties. Acta Univ Agric Silvic Mendel Brun 66:219–224. https://doi.org/10.11118/actaun201866010219 Walter R, Burmeister J (2022) 35 Jahre Bodendauerbeobachtung landwirtschaftlich genutzter Flächen in Bayern: Band 5 – Regenwürmer. Bavarian State Research Center for Agriculture (LfL), Freising, Germany Wang JY, Xiong ZQ, Kuzyakov Y (2015) Biochar stability in soil: meta-analysis of decomposition and priming effects. Glob Change Biol Bioenergy 8:512–523. https://doi.org/10.1111/gcbb.12266 Wang QY, Liu JS, Hu B (2016) Integration of copper subcellular distribution and chemical forms to understand copper toxicity in apple trees. Environ Exp Bot 123:125–131 Wang Y, Wang HS, Tang CS, Gu K, Shi B (2022) Remediation of heavy metal contaminated soils by biochar: a review. Environ Geotech 9:135–148. https://doi.org/10.1680/jenge.18.00091 Watson GA (1960) The effect of soil pH and manganese toxicity upon the growth and mineral composition of the hop plant. J Hortic Sci 35:136–145. https://doi.org/10.1080/00221589.1960.11513979 Watson J, Askew HM (1956) Molybdenum deficiency in hops. Nature 178:1302–1303. https://doi.org/10.1038/1781302a0 Wightwick A, Mollah M, Partington D, Allinson G (2008) Copper fungicide residues in Australian vineyard soils. J Agric Food Chem 56:2457–2464. https://doi.org/10.1021/jf0727950 Zorn W, Marks G, Heß H, Bergmann W (2016) Handbuch zur visuellen Diagnose von Ernährungsstörungen bei Kulturpflanzen. Springer-, Berlin Supplementary Files Appendix.docx GraphicalAbstract.pdf Cite Share Download PDF Status: Published Journal Publication published 27 Dec, 2025 Read the published version in Plant and Soil → Version 1 posted Editorial decision: Major revisions 21 Aug, 2025 Reviewers agreed at journal 29 Jul, 2025 Reviewers invited by journal 29 Jul, 2025 Editor invited by journal 28 Jul, 2025 Editor assigned by journal 28 Jul, 2025 First submitted to journal 25 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7215331","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":492750852,"identity":"034fb5db-33f4-4c9f-abaf-ec784d4a53ca","order_by":0,"name":"Johannes Görl","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYBACxgYeZG6BTQKY5sGmFrsWgzSgFmb8WtBkDQ4T1sLc3nvwc+EOG3sG/sUHH1cYnM+Tn5F/gOFNBR6H9ZxLlp55Ji2xQeJZsuEZg9vFBjeSGRjnnMGjZUaOgTRvG9A9EmfMJBsMbidukE5mYOZtw6vF+Ddv2397oBbznw0G5xLnzwZp+YdXixnQlgOMDfw9ZowNBgcSG26DtDTg88sZM2vetuTENgm2ZKDDkhM33H9scHDOMdxaDNt7jG/zttnZ8/MfPvixocIucX7PwYcP3tTg0QJzAZtEAkL0AG4NDAzycBY/XnWjYBSMglEwkgEAR9NQbcbxQCsAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0006-4200-5565","institution":"TUM: Technische Universitat Munchen","correspondingAuthor":true,"prefix":"","firstName":"Johannes","middleName":"","lastName":"Görl","suffix":""},{"id":492750853,"identity":"7edc1ab8-ff82-4c02-8fb8-597816ec3d04","order_by":1,"name":"Dieter Lohr","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dieter","middleName":"","lastName":"Lohr","suffix":""},{"id":492750854,"identity":"f12972d2-0063-4bd8-be2c-bc519a330a9d","order_by":2,"name":"Elke Meinken","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Elke","middleName":"","lastName":"Meinken","suffix":""},{"id":492750855,"identity":"828194ce-082e-4929-86c0-75748e29cd81","order_by":3,"name":"Kurt-Jürgen Hülsbergen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kurt-Jürgen","middleName":"","lastName":"Hülsbergen","suffix":""}],"badges":[],"createdAt":"2025-07-25 14:48:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7215331/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7215331/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11104-025-07990-7","type":"published","date":"2025-12-27T15:58:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88033390,"identity":"dbb6c7b1-5966-4830-8579-066e4c53fafc","added_by":"auto","created_at":"2025-07-31 15:54:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":68546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 2.\u003c/strong\u003e Results of the visual ratings for symptoms of copper toxicity from calendar week 25 to 29 in the first growing season. Treatments not sharing a letter within a week differ significantly (Nemenyi test with p ≤ 0.05; n.s. = no significant effect in the Kruskal-Wallis test). The image shows a hop plant from week 25 with severe symptoms, rated with a score of 2.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215331/v1/d512982efb9e87911d018c31.jpg"},{"id":88032245,"identity":"b222ec29-00cc-43b7-8674-0965a6ffd211","added_by":"auto","created_at":"2025-07-31 15:46:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 3.\u003c/strong\u003e Copper (Cu) concentration of hop leaves dry mass (DM) from calendar week 25 to 29 in the first growing season (n.s. = no significant effect in the Dunnett test or ANOVA; n.a. = no samples taken due to unfavorable weather conditions). Treatments not sharing a letter within the type of compost (0BC, 5BC, and 20BC) differ significantly (Tukey test with p ≤ 0.05).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215331/v1/a21e306145d05f62d4d5299c.jpg"},{"id":88032254,"identity":"b58a8ae1-63bd-4554-b545-b39258bd6e94","added_by":"auto","created_at":"2025-07-31 15:46:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4.\u003c/strong\u003e Results of the visual ratings for symptoms of copper toxicity from calendar week 23 to 25 in the second growing season. Treatments not sharing a letter within a week differ significantly (Nemenyi test with p ≤ 0.05; n.s. = no significant effect in the Kruskal-Wallis test). The image shows a hop plant from week 23 with severe symptoms, rated with a score of 2.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215331/v1/a6f3f1b86112a23827ca9e68.jpg"},{"id":88032248,"identity":"dc53e3a7-8f75-4461-8ab4-4c26e6605cc2","added_by":"auto","created_at":"2025-07-31 15:46:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":59074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 5.\u003c/strong\u003e Copper (Cu) concentration of hop leaves dry mass (DM) in calendar week 23 (a, b) and 35 (c, d), as well as of hop roots in calendar week 41 (e, f) of the second growing season. n.s. = no significant effect in the Dunnett test (a, c, e) or ANOVA (b, d, f). Treatments not sharing a letter within the application rate (3 and 6 t ha\u003csup\u003e-1\u003c/sup\u003e) differ significantly (Tukey test with p ≤ 0.05). Error bars represent the standard error of the mean (n = 6 (a, c, e); n = 12 for compost type and n = 18 for application rate (b, d, f)).\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215331/v1/47fb95bb7a182a3b63b35ebe.jpg"},{"id":88032252,"identity":"22443525-fc5f-4e43-83f7-f0fe6335f7fc","added_by":"auto","created_at":"2025-07-31 15:46:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":61648,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 6.\u003c/strong\u003e Fresh (a, b) and dry biomass (c, d) of hop cones as well as the remaining bines in calendar week 35 of the second growing season. Asterisks indicate significant differences in bine biomass (symbols within the respective bars) as well as in total biomass (symbols above the bars) compared to the unlimed control, while plus signs indicate significant differences compared to the limed control (Dunnett test with p ≤ 0.05 (a, c)). Treatments not sharing a letter within the type of compost (0BC, 5BC, and 20BC) differ significantly in bine biomass (letters within the respective bars) as well as in total biomass (letters above the bars; Tukey test with p ≤ 0.05 (b, d)). n.s. = no significant effect in the ANOVA. Error bars represent the standard error of the mean for total biomass (n = 6 (a, c); n = 12 for compost type and n = 18 for application rate (b, d)).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215331/v1/630463fb8cc86c429ee08961.jpg"},{"id":88032251,"identity":"c1df0254-b848-4914-841d-5f8656a9f1c5","added_by":"auto","created_at":"2025-07-31 15:46:26","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":43139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 7. \u003c/strong\u003eRespiration rate (R\u003csub\u003eB\u003c/sub\u003e) of soil samples taken from the planting hole areas at the end of the first (a, b) and second growing season (c, d). Asterisks indicate significant differences compared to the unlimed control, while plus signs indicate significant differences compared to the limed control (a, c; Dunnett test with p ≤ 0.05). Treatments not sharing a letter within the type of compost (0BC, 5BC, and 20BC) or application rate (3 and 6 t ha\u003csup\u003e-1\u003c/sup\u003e) differ significantly (b, d; Tukey test with p ≤ 0.05; n.s. = no significant effect in the ANOVA). Error bars represent the standard error of the mean (n = 6 (a, c); n = 12 for compost type and n = 18 for application rate (b, d)).\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215331/v1/c4fbe425251ec90ab3f4a253.jpg"},{"id":99172454,"identity":"cbe92488-348e-4966-998c-526c9168150a","added_by":"auto","created_at":"2025-12-29 16:09:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1319123,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7215331/v1/f4de9281-cadd-402c-b2a3-023bbedb80fe.pdf"},{"id":88032246,"identity":"25cf3b06-a6c3-4469-9815-db43dd30a168","added_by":"auto","created_at":"2025-07-31 15:46:26","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":369868,"visible":true,"origin":"","legend":"","description":"","filename":"Appendix.docx","url":"https://assets-eu.researchsquare.com/files/rs-7215331/v1/c6cfeffbfd2e24ffa656b0a3.docx"},{"id":88033392,"identity":"1effd13c-5c61-4de1-ae21-2ef82a25df9a","added_by":"auto","created_at":"2025-07-31 15:54:26","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":571452,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7215331/v1/e530f3495cf04e1e17f540a7.pdf"}],"financialInterests":"","formattedTitle":"Co-Composted Biochar Compost for Improving Juvenile Growth After Replanting Copper-Contaminated Hop Gardens","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe use of copper-based fungicides in agriculture has a long history, dating back to the late 19th century when the discovery of Bordeaux mixture - a combination of copper sulfate and lime - marked a milestone in crop protection, particularly in combating downy mildew in grapevines (Tamm et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This innovation provided the first effective measure for controlling various phytopathogenic fungi, leading to its widespread application across diverse crops, including vegetables, fruits, and hops.\u003c/p\u003e\u003cp\u003eDespite the development of effective synthetic alternatives, copper-based fungicides are still used not only in organic but also in conventional crop production, particularly in viticulture as well as fruit and hop cultivation. This is due to their high efficacy against a broad spectrum of fungal pathogens, resilience under adverse weather conditions, minimal risk of resistance development, relatively low toxicity to terrestrial vertebrates and low costs (Lamichhane et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Panagos et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Speiser et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Especially in organic farming, the absence of copper-based fungicides would result in yield losses ranging from 50% to total failure in hops, grapes, apples and other fruit crops (Burandt et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite their numerous benefits, copper-based fungicides have a major limitation. Due to the low mobility and non-degradability of copper in soil, their long-term application leads to a creeping accumulation of copper in topsoil (Kabata-Pendias and Pendias \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). After several decades of continuous spraying, concentrations of total copper can reach up to 450 mg kg\u003csup\u003e-1\u003c/sup\u003e in topsoil of hop gardens (Schramel et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) and 1,500 mg kg\u003csup\u003e-1\u003c/sup\u003e in vineyards and orchards (Burandt et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wightwick et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This is 15 to 50 times higher than the average copper content in unpolluted soils (Vlček and Pohanka \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Such elevated copper concentrations in soil impair plant growth by inhibiting germination, reducing root development and negatively affecting nutrient and water uptake. Furthermore, copper toxicity reduces photosynthetic activity. As a result, total biomass and agricultural productivity decreases significantly (Mir et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn hop cultivation, symptoms of copper toxicity typically do not occur in plants that have been established for several years, as they root to a depth of at least 2 m (Brant et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), where copper concentrations are considerably lower than in the topsoil (Schramel et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). However, in hop gardens with high copper concentrations in the topsoil, in the first few years after replanting, when the root system is still limited to the contaminated soil layer, symptoms of copper toxicity are frequently reported by hop farmers, impairing the juvenile growth and establishment of the hop plants (Portner \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Although such symptoms are limited to the early growth phase, they may still affect long-term productivity, as initial plant development is crucial for achieving stable yields in perennial crops (Pan et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Indeed, quantitative studies addressing the economic relevance of copper toxicity on yield losses in hop production are currently lacking and should be further investigated.\u003c/p\u003e\u003cp\u003eIn addition to plant growth, high copper concentrations in the soil negatively affect soil biota such as microflora or earthworms. Their abundance decreases with increasing copper contamination, accompanied by shifts in community structure and a reduction in species diversity (Naveed et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Consequently, the activity of essential soil enzymes (e.g., phosphatase, β-glucosidase and dehydrogenase), which play a crucial role in nutrient cycling, is significantly reduced (Fern\u0026aacute;ndez-Calvi\u0026ntilde;o et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Results from permanent monitoring plots in hop gardens across Bavaria confirm that copper accumulation in the soil significantly reduces the abundance and diversity of earthworms (Walter and Burmeister \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In particular, no endogeic earthworm species, which live in the top 30 cm of soil and are most sensitive to copper pollution, were detected on two of the three monitoring plots during the entire observation period from 1985 to 2019.\u003c/p\u003e\u003cp\u003eIn view of the decline in soil health caused by the impairment of soil biota as well as the risk of damage to new plantings, there is a need for effective solutions against copper toxicity in contaminated hop gardens. Due to the concentration of cultivation in specific regions and the resulting limited availability of suitable land, as well as the high costs associated with establishing new trellis systems, the abandonment of contaminated sites is not a viable alternative (Rossini et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sawicka et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, conventional remediation methods such as soil replacement, soil washing or phytoextraction are neither economically viable nor feasible within a reasonable period of time or they have significant ecological consequences (Kaparwan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Robinson et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). A more promising approach for mitigating copper toxicity is to reduce the bioavailability of existing copper by liming (Ambrosini et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chatzistathis et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) or by applying highly sorptive organic amendments as compost (Huang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and biochar (Wang et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), respectively. Compared to liming, compost and biochar - especially co-composted biochar - offer additional benefits, e.g. water retention and long-term carbon sequestration (Antonangelo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eG\u0026ouml;rl et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e) demonstrated that the application of co-composted biochar compost to artificially and freshly copper-spiked soils improved the growth of Chinese cabbage while mitigating copper-induced avoidance behavior of earthworms and increasing soil oxygen consumption as an indicator of microbial activity. In a related study, in which co-composted biochar composts were also applied to long-term copper-contaminated soils from hop gardens, G\u0026ouml;rl et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e) reported improvements in plant growth, along with a reduction in copper phytoavailability. The biochar composts used in this study were produced from hop bines - an agricultural by-product arising annually in large quantities during harvest. Thus, co-composting hop bines with biochar could potentially offer hop farmers a promising strategy to close local material cycles and to mitigate the toxic effects of high copper loads in their soils. Furthermore, it prevents the spread of diseases like \u003cem\u003eVerticillium\u003c/em\u003e, which is regularly found in hop bines, as the addition of biochar at the beginning of the composting process intensified decomposition (G\u0026ouml;rl et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e), which likely eliminated these pathogens (Hagemann et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, the limitations of the above-mentioned studies to avoid Cu toxicity include their conduction under controlled environmental conditions in a greenhouse or laboratory as well as the short trial duration of at most a few weeks. To our knowledge, no long-term field trials on this topic have yet been conducted on-site in hop gardens. To address this research gap, a field trial was established on a copper-contaminated soil, previously used for hop production. The field was replanted with young hop plants and the effect of biochar compost, derived from co-composting of hop bines and biochar, as well as biochar-free hop bine compost on the hop plants and the microbial activity in the soil was studied over a period of two growing seasons. We hypothesized that the application of co-composted biochar compost at the time of planting (i) improves the juvenile development and growth of hop plants, (ii) enhances microbial activity in the soil, and (iii) is more effective than biochar-free compost.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Study Site Conditions\u003c/h2\u003e\u003cp\u003eThe field experiment was conducted in 2022 and 2023 on a soil that had been used as a hop garden between 1970 and 2019. The site is located in H\u0026uuml;ll (48\u0026deg;36'10\"N, 11\u0026deg;40'28\"E), about 50 km north of Munich in the center of the Hallertau region, which is the world\u0026acute;s largest hop-growing area with about 17,000 ha (Kubeš \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The area is part of the Bavarian Tertiary Hill Country, approximately 445 m above sea level with soils classified as Cambisol. Soil properties, analyzed according to the methods of the Association of German Agricultural Inspection and Research Institutes, are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (VDLUFA 2016a, b).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSoil properties of the experimental area (sampling depth: 30 cm). Values are presented as means, with the range from grid sampling (minimum to maximum) in parentheses. n.a. = not analyzed.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSoil type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003epH (CaCl\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eP (CAL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eK (CAL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCu\u003csub\u003etotal\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCu (CAT)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003emg kg\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003emg kg\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003emg kg\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003emg kg\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esilty loam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c2\"\u003e\u003cp\u003e5.4 (5.0-5.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e82 (52\u0026ndash;105)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e118 (100\u0026ndash;133)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e154 (n.a.)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e68 (31\u0026ndash;104)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eCompared to the average weather conditions of previous years (1991\u0026ndash;2020), the experimental years 2022 and 2023 were significantly warmer (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, the 2022 growing season was characterized by a dry spring and particularly hot and dry weather from June to August. While higher rainfall was recorded in the spring of the 2023 growing season, precipitation during the hot months of June and July again remained below the long-term average.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMonthly and annual values of mean air temperature (2 m height) and total precipitation during the experimental years 2022 and 2023, compared to the long-term average (1991\u0026ndash;2020) (DWD 2024).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMonth / Year\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eTemperature (\u0026deg;C)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003ePrecipitation (mm)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1991\u0026ndash;2020\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2022\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2023\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1991\u0026ndash;2020\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2022\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2023\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e59.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e42.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e20.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFeb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e49.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e30.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e30.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMar\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e61.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e9.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e45.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eApr\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e53.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e48.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e64.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e14.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e93.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e66.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e59.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJun\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e88.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e30.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJul\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e19.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e103.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e43.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e79.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAug\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e98.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e68.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e159.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSep\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e16.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e70.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e77.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e16.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOct\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e65.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e73.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e45.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNov\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e63.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e53.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e154.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e64.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e58.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e126.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYear\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e883.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e659.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e831.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Biochar and Compost Characterization\u003c/h2\u003e\u003cp\u003eThe biochar (BC) used in this experiment was produced from untreated wood chips from certified sustainable and regional forestry in a Syncraft reactor (Huber et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and pyrolyzed at a temperature of 850\u0026deg;C. It complied with all thresholds of the European Biochar Certificate for feed classification (EBC \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The most important physico-chemical properties are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePhysico-chemical properties of the biochar used for composting, as provided by the manufacturer.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProperties\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnit\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eValues\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH (CaCl\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecific surface area (BET)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003em\u003csup\u003e2\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e282\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBulk density (\u0026lt;\u0026thinsp;3 mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ekg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e275\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN\u003csub\u003etotal\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP\u003csub\u003etotal\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eK\u003csub\u003etotal\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCa\u003csub\u003etotal\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMg\u003csub\u003etotal\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC\u003csub\u003eorg\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e849\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMolar H/C\u003csub\u003eorg\u003c/sub\u003e ratio\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe biochar was co-composted with freshly chopped hop bines derived from hop plants of the cultivar \u0026lsquo;Mandarina Bavaria\u0026rsquo;, which were harvested and processed according to common agricultural practice. The biochar was applied aligning with previous studies (e.g., Kammann et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) at rates of 5% and 20% by volume (5BC and 20BC), corresponding to 17% and 46% on dry weight basis, respectively. Additionally, chopped hop bines were composted without the addition of biochar (0BC). After an intensive rotting period of 65 days, including a total of five turnings, and a subsequent maturation phase of five months, composting was completed. Temperature measurements in the compost heaps showed that the addition of biochar extended the thermophilic phase by nearly 30 days, leading to a higher degree of sanitization. For further details on the composting procedure, refer to G\u0026ouml;rl et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). The physico-chemical properties of the three mature composts (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) were analyzed according to the guidelines of the German compost quality association (BGK \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePhysico-chemical properties of the three mature composts used as soil amendments.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProperties\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUnit\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0BC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5BC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20BC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH (CaCl\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSalts (H\u003csub\u003e2\u003c/sub\u003eO)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eg KCl L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e-N (CaCl\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e-N (CaCl\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e164\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e285\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e138\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN (CaCl\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e168\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e289\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e139\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP (CAL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e107\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e234\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e137\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eK (CAL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1487\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2462\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2134\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN\u003csub\u003etotal\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP\u003csub\u003etotal\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eK\u003csub\u003etotal\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC\u003csub\u003etotal\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e376\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e466\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e629\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC/N\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Experimental Setup and Plant Cultivation\u003c/h2\u003e\u003cp\u003eConsidering the spatial heterogeneity of soil properties in the Hallertau region as a part of the Tertiary Hill Country (Fiener et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and the strong effect of topography on copper distribution in soil (Jurisic et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), the entire experimental area was divided into a 20.8 m x 6.4 m grid, and soil samples were collected from the former hop row area to a depth of 20 cm and analyzed for pH, CAL-extractable P and K as well as CAT-extractable copper (VDLUFA 2016a, b). The results revealed considerable variability in soil pH and CAT-extractable copper content. Consequently, the experimental area was subdivided into six blocks (replicates), each measuring 20.8 m \u0026times; 12.8 m and characterized by relatively uniform values regarding pH and plant-available copper concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e in the Appendix).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo ensure a high concentration of compost in the root zone at reliable costs, compost was applied targeted around the planting holes, as suggested by Schmidt et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Consequently, based on a planting density of 2,400 hop plants per hectare, the two biochar composts and the biochar-free compost were applied at rates of 1.25 and 2.5 kg of dry matter per planting hole, corresponding to application rates of 3 and 6 t ha\u003csup\u003e-1\u003c/sup\u003e of dry matter, respectively. For this purpose, 20 cm deep planting holes were drilled in the former hop rows with a distance of 1.3 m within the rows and 3.2 m between the rows, and composts were applied over an area of 0.4 m\u0026sup2; around each planting hole.\u003c/p\u003e\u003cp\u003eTwo control treatments without compost were included: one without lime and one with lime, as the pH of the field, with an average value of 5.4, was below the optimal range of 6.2 to 6.5 for a silty loam. The lime requirement was 5.5 t CaO ha\u003csup\u003e-1\u003c/sup\u003e to achieve the optimal pH range (Kn\u0026ouml;ferl et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Liming was carried out using natural limestone, which was applied in one-meter wide strips along the respective planting holes. In total, the experiment resulted in eight treatments arranged in a completely randomized block design with six replicates (48 plots), each plot consisting of eight plants. To ensure uniform growth conditions across the experimental area, additional border rows were established all around.\u003c/p\u003e\u003cp\u003eAfter the application of amendments, potted plants of the variety 'Hallertauer Mittelfr\u0026uuml;h' were planted into the prepared planting holes at the end of May 2022. To ensure rapid establishment despite the hot weather conditions, the young plants were regularly irrigated by hand. Fertilization, plant protection and general cultivation measures such as soil management and training of hop plants were conducted according to the guidelines of the Bavarian State Research Center for Agriculture (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). To avoid additional copper input, the application of copper-based fungicides and fertilizers was omitted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Data Collection\u003c/h2\u003e\u003cp\u003eAfter a short establishment phase of three weeks, hop plants were visually rated weekly for symptoms of copper toxicity in both growing seasons as long as significant differences between treatments occurred. To minimize subjective variability, the ratings were always conducted by the same person. Plants without symptoms received a score of 0, those with moderate symptoms a score of 1, and those with severe symptoms a score of 2. Subsequently, a damage index per plot was calculated for each treatment as the sum of scores of the eight plants per plot.\u003c/p\u003e\u003cp\u003eFrom calendar week 25 to 29 in the first growing season, in calendar week 23 of the second growing season as well as at the end of both growing seasons when cones were ripe for picking (BBCH 89), the concentrations of copper and other essential nutrients in the hop leaves were analyzed. Except of the final analyses, the youngest fully developed leaf of each plant was collected as plants had not yet reached the top wire at a height of 7 m (BBCH 38; Rossbauer et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Since sampling at this height was technically unfeasible, at the end of the growing seasons, older leaves were collected at a height of 1.5 m. These were the first intact leaves located above the common defoliation zone in hop cultivation.\u003c/p\u003e\u003cp\u003eThe collected leaves were dried at 60\u0026deg;C in a forced-air oven until constant weight and grinded in a centrifugal mill (ZM 200, Retsch, Haan, Germany). Subsequently, microwave-assisted digestion (Multiwave ECO, Anton Paar, Graz, Austria) in HNO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was done (VDLUFA 2016b) and concentrations of copper as well as other essential macro- and micronutrients (P, K, Ca, Mg, S, Fe, Mn, B, Zn, Mo) in the digestion solutions were measured via ICP-OES (iCAP 6000 DV, Thermo Fisher Scientific, Dreieich, Germany).\u003c/p\u003e\u003cp\u003eTo avoid insufficient establishment of the plants and to align with common practice, harvest took place only at the end of the second growing season in August 2023 (calendar week 35). To minimize the influence of adjacent plots, only the four central plants per plot were cut off just above soil surface and the fresh biomass of cones and the remaining bines were determined for each plant. Additionally, in calendar week 41, several hop roots from each plant were carefully removed from the soil and washed with deionized water. Cones, bines, and roots were dried at 60\u0026deg;C in a forced-air oven until constant weight and the dry biomass of cones and bines was recorded. Finally, the concentrations of copper as well as other essential macro- and micronutrients (P, K, Ca, Mg, S, Fe, Mn, B, Zn, Mo) in the roots were analyzed as described above. All analyses were performed in duplicate and control samples (e.g. reference material, blanks) were part of each analytical run.\u003c/p\u003e\u003cp\u003eIn addition to the plant experiment, microbial respiration in soil as an indicator of microbial activity was determined at the end of both growing seasons using the OxiTop\u0026reg; respirometry system (WTW, Germany). The system consists of glass jars with a volume of 2.7 L, which were equipped with a pressure measuring head. Sodium hydroxide solution (1 M NaOH, 50 mL) was placed in the headspace as a carbon dioxide trap to absorb CO₂ produced by microbial respiration. This absorption results in a pressure drop, which is continuously recorded by the measuring head. When the pressure dropped below \u0026minus;\u0026thinsp;100 hPa, the jar was ventilated to prevent O\u003csub\u003e2\u003c/sub\u003e depletion and the NaOH solution was renewed.\u003c/p\u003e\u003cp\u003eFor the soil respiration test, 1 kg of field-moist topsoil was collected to a depth of 20 cm from the planting hole areas of each plot using a soil corer, homogenized and filled into a jar. O\u003csub\u003e2\u003c/sub\u003e consumption was then measured at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C in a climate chamber for five days and expressed as respiration rate in mg O\u003csub\u003e2\u003c/sub\u003e per kg dry matter (DM) per day, following the method described by Platen and Witz (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAt the end of both growing seasons, soil samples were collected from the areas between the planting holes of each plot to a depth of 20 cm, in order to assess the effect of liming while avoiding the sampling of compost-amended soil. The samples were analyzed for pH and CAT-extractable copper as well as other essential micronutrients (Fe, Mn, B, Zn, Mo) according to VDLUFA (2016a, b).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Statistical Analyses\u003c/h2\u003e\u003cp\u003eThe fresh and dry biomass of cones, bines and total plants, the copper concentration of leaves and roots, as well as the respiration rate were first subjected to a one-way ANOVA including all treatments. The significance of the means of the compost-amended and limed treatments was then tested pairwise against the unamended control using a Dunnett test. In a second step, the compost-amended treatments were additionally compared to the limed control using the same test. Subsequently, the two control treatments were removed from the dataset and a two-way ANOVA was conducted with the type of compost as first and the application rate as second factor. Since there were no interactions between both factors, a Tukey test was performed for each single factor. Additionally, the damage indices from the visual ratings were analyzed using a Kruskal-Wallis test. In case of significant differences, the rank sums were compared using a Nemenyi test. The level of significance for all statistical tests was set to 5%. Data pre-processing and visualization were performed using MS Excel 2016 (Microsoft Corporation, Redmond, WA, USA), while statistical analyses were conducted with Minitab V22 (Minitab Inc., State College, PA, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003e3.1 Plant Evaluation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFirst Growing Season after Planting\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlready three weeks after planting, at the beginning of shoot elongation in week 25 (BBCH 31-32), the first symptoms of copper toxicity appeared on hop leaves, characterized by chlorosis and necrosis starting from the top, as well as by reduced growth (Figure 2). Until week 27, the number of plants with moderate and severe symptoms and consequently the cumulative damage indices were lower in treatments with biochar compost (5BC and 20BC) than in both treatments without compost (unlimed and limed). A similar trend was observed for the biochar-free compost (0BC) at the high application rate, whereas at the lower rate, a significant reduction of symptoms compared to both control treatments was only found in week 25. In contrast to compost application, liming had no mitigating effect on toxicity symptoms, but even led to a significantly higher proportion of affected plants in week 26.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the biochar-free compost (0BC) and the compost co-composted with 5 vol% biochar (5BC), increasing the application rate from 3 to 6 t DM ha\u003csup\u003e-1\u003c/sup\u003e resulted in a significant reduction in damage indices until week 27. In contrast, for the compost co-composted with 20 vol% biochar (20BC), a positive effect of doubling the application rate was restricted to week 25. Among all treatments, the compost with 5 vol% biochar (5BC) applied at the high application rate of 6 t DM ha\u003csup\u003e-1\u003c/sup\u003e was most effective in reducing toxicity symptoms during the first three assessment weeks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegardless of the amendment type, symptoms of copper toxicity continuously declined over the growing season, and thus, from week 28 onwards, significant differences between treatments were no longer detectable. Consequently, visual ratings were stopped at the end of week 29.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast to the visual ratings, the copper analyses of the youngest fully developed leaves revealed only negligible differences among the eight treatments (Figure 3). Accordingly, compost application did not differ significantly from liming, and none of the amendments reduced the copper concentration of the leaves compared to the unlimed control. Moreover, no significant interaction was observed between compost type and application rate. Except for week 25, in which the biochar-free compost (0BC) led to a lower copper concentration than the compost co-composted with 20 vol% biochar (20BC), no significant differences were detected among the three compost types. Furthermore, doubling the application rate from 3 to 6 t DM ha\u003csup\u003e-1\u003c/sup\u003e also had no effect on the copper concentration of the leaves.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe copper concentration of older hop leaves at the end of the 2022 growing season in week 36 ranged from 5.6 to 6.0 mg kg\u003csup\u003e-1\u003c/sup\u003e DM on average. As observed in the previous leaf analyses from week 25 to 29, no significant differences were detected between the treatments, neither in the Dunnett tests nor in the two-way ANOVA. While the macronutrient concentrations of the leaves in the first growing season showed no irregularities and were within optimal ranges for hop (Table 5 in the Appendix), the micronutrient analyses revealed elevated iron levels and a molybdenum deficiency from week 28 to 36 (Table 6 in the Appendix), which was also reflected in the low soil molybdenum levels (Table 7 in the Appendix).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSecond Growing Season after Planting\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLeaf chlorosis and necrosis also occurred on the hop plants in the second growing season (Figure 4). However, in contrast to the previous year, these symptoms appeared at a later stage of development during advanced shoot elongation (BBCH 33-35), and they were restricted to the lower part of the hop plants and rather atypical for copper toxicity (Portner 2009; Zorn et al. 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNonetheless, compared to the unlimed control, both liming and compost application - regardless of application rate and biochar content - resulted in a significant reduction of damage symptoms in week 23. Except for the low application rate of compost co-composted with 20 vol% biochar (20BC), the effect of compost application was significantly greater than that of liming. In contrast to the biochar-free compost (0BC), where doubling the application rate had no effect, a clear benefit of increased application was observed for both biochar composts (5BC and 20BC). As in the first growing season, the compost co-composted with 5 vol% biochar (5BC), applied at 6 t DM ha\u003csup\u003e-1\u003c/sup\u003e, consistently resulted in the lowest damage index. As symptom intensity remained almost constant until the end of the second growing season and no significant differences between treatments were observed from week 24 onwards, visual ratings were stopped after calendar week 25.\u003c/p\u003e\n\u003cp\u003eAs in the first growing season, copper analysis of the youngest fully developed leaves in week 23 of the second growing season (Figure 5a) revealed no significant differences between the compost-amended treatments (0BC, 5BC, and 20BC) and both control treatments (unlimed and limed). Due to this lack of differences, which was also reflected in the visual ratings from week 24 onwards, further leaf analyses were omitted until cone ripening (BBCH 89) at the end of the growing season. Similar to the young leaves, copper concentrations of the older leaves in week 35 (Figure 5c), as well as of the hop roots collected in week 41 (Figure 5e), showed no significant amendment effects. Furthermore, no significant interaction between the type of compost and the application rate was observed at any of the three sampling dates and the type of compost had no significant effect on the copper concentration of young leaves\u0026nbsp;(b), old leaves\u0026nbsp;(d) and roots\u0026nbsp;(f), respectively. While doubling the application rate from 3 to 6 t DM ha\u003csup\u003e-1\u003c/sup\u003e resulted in a significant 6% increase in copper concentration of young leaves (b), such an effect was not observed in old leaves (d) or roots (f). In contrast to the copper concentration of the youngest fully developed leaves, the copper concentration of the older leaves was on average about half lower, whereas it was more than ten times higher in hop roots.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs in the first growing season, no notable irregularities were observed in the macronutrient concentrations of leaves and roots during the second season (Table 5 in the Appendix). Micronutrient analyses again revealed partly elevated iron levels and a significant molybdenum deficiency (Table 6 in the Appendix), consistent with the low levels of molybdenum in the soil (Table 7 in the Appendix). Additionally, a marked manganese surplus was detected in the leaves and roots.\u003c/p\u003e\n\u003cp\u003e3.2 Biomass Production in the Second Growing Season after Planting\u003c/p\u003e\n\u003cp\u003eAfter two growing seasons, the hop plants were harvested in week 35, and both fresh and dry biomass of hop cones and bines were determined. Neither liming nor application of biochar-free compost (0BC) increased the yield of fresh and dry cones and bines compared to the control treatment without any amendment (Figure 6a, c). In contrast, the application of compost co-composted with 5 vol% biochar (5BC), especially at the higher application rate of 6 t DM ha\u003csup\u003e-1\u003c/sup\u003e, generally increased the fresh and dry biomass of bines by up to 32% and 34% and the total fresh and dry biomass by up to 27% and 29%, respectively, compared to both control treatments. Furthermore, the compost co-composted with 20 vol% biochar (20BC) applied at a rate of 3 t DM ha\u003csup\u003e-1\u003c/sup\u003e was more effective than liming.\u003c/p\u003e\n\u003cp\u003eNo significant interaction was observed between the type of compost and the application rate, and doubling the application rate had no significant effect on the fresh and dry biomass of cones and bines as well as on total fresh and dry biomass (Figure 6b, d). However, composts differed significantly in their effects on the fresh and dry biomass of bines as well as on total dry biomass. Yields were significantly higher for the compost co-composted with 5 vol% biochar (5BC) compared to the biochar-free compost (0BC). In contrast, no significant differences were found between both biochar composts (5BC and 20BC).\u003c/p\u003e\n\u003cp\u003e3.3 Soil Respiration\u003c/p\u003e\n\u003cp\u003eOxygen consumption in soils from the planting hole area of each treatment was analyzed at the end of both growing seasons. With the exception of the 20 vol% biochar compost (20BC) at 3 t DM ha\u003csup\u003e-1\u003c/sup\u003e, compost application resulted in a significant increase in soil respiration rate of up to 81% in the year of planting compared to both control treatments (Figure 7a). In contrast, liming had no effect on the respiration rate. The two-way ANOVA revealed significant differences in the respiration rate between the type of compost (0 BC \u0026gt; 5BC \u0026asymp; 20BC) and a significant increase of more than 20% by doubling the application rate (Figure 7b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast to the first growing season, a significant increase in the respiration rate was only observed for the biochar-free compost (0BC) at 6 t DM ha\u003csup\u003e-1\u003c/sup\u003e at the end of the second growing season (Figure 7c). As in the first growing season, liming had no effect on the respiration rate. Moreover, no significant interaction was found between the type of compost and the application rate, and none of the two factors had a significant effect on the respiration rate (Figure 7d).\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Plant Evaluation\u003c/h2\u003e\u003cp\u003eShortly after the replanting of the copper-contaminated hop garden, significant intercostal leaf chlorosis appeared on the hop plants. They started from the top and in case of severely affected individuals, leaves bleached completely and became necrotic. These findings align with observations from the Hop Research Center in H\u0026uuml;ll that toxicity symptoms in copper-contaminated hop gardens typically appear shortly after replanting and in subsequent years at the beginning of shoot growth. Furthermore, symptoms similar to those observed in the present study have also been reported in various plant species, including brassicaceae such as Indian mustard, rapeseed, stone-head cabbage and Chinese cabbage (Feigl et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ali et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; G\u0026ouml;rl et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e; Shahbaz et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), cereals like maize, durum wheat and barley (Ali et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Bravin et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Vassilev et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), as well as legumes, e.g. beans (Miyazawa et al. 2002), and have been attributed to copper toxicity.\u003c/p\u003e\u003cp\u003eThe application of the three composts (0BC, 5BC, and 20BC), particularly those containing biochar, significantly reduced these symptoms. A similar effect of the same composts was previously observed under controlled environmental conditions in a pot experiment with Chinese cabbage, where copper-induced leaf chlorosis was prevented in a soil artificially spiked with 240 mg Cu kg\u003csup\u003e-1\u003c/sup\u003e (G\u0026ouml;rl et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, there are two arguments in the present study, which contradict the assumption that copper toxicity was the primary cause of the observed symptoms. First, the absence of a liming effect in the first year: According to Ambrosini et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and Chatzistathis et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), increasing pH by liming effectively reduces copper bioavailability and its phytotoxic effects. Thus, if copper toxicity had been the main driver, a mitigating effect from liming would have been expected. Indeed, at the end of the first growing season, a significant increase of pH up to 1.0 unit was found in limed plots (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e in the Appendix). However, superficial application of lime in combination with dry weather conditions (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) may have restricted the vertical movement of alkaline compounds in the soil and consequently an increase of pH in the root zone right after planting until the symptoms appeared (Azam and Gazey \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSecond, the Cu concentration of leaves: Although the total copper content in the soil exceeded the toxicity threshold of 100 mg kg\u003csup\u003e-1\u003c/sup\u003e DM (Mengel and Kirkby \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), leaf concentrations only ranged from 3 to 10 mg Cu kg\u003csup\u003e-1\u003c/sup\u003e DM. This is far below the threshold for excessive or toxic levels of copper, which are between 20 and 100 mg kg\u003csup\u003e-1\u003c/sup\u003e DM for various plant species (Kabata-Pendias 2001). Moreover, in contrast to the severity of damage, no differences in leaf concentrations could be detected between the treatments. However, although increased copper availability in soil is generally reflected by higher copper uptake, a gap between copper concentrations in roots and aerial parts of the plants \u0026ndash; especially leaves \u0026ndash; is well documented in other species, e. g. grapevines, apples or peaches (Juang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hammerschmidt et al. 2020). This may be explained by a protective mechanism: plants can limit the translocation of Cu from roots to shoots by retaining it in root cell walls, vacuoles or the apoplast to avoid damage to sensitive aerial tissues (Adrees et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the year of planting, this mechanism could not be confirmed by measurements, as root sampling was not possible without permanently damaging the young plants. However, the presence of such a mechanism is strongly supported by the Cu concentrations in hop roots at the end of the second growing season after planting. These ranged from 72 to 96 mg kg\u003csup\u003e-1\u003c/sup\u003e DM, which is 10 to 30 times higher than the concentrations in leaves at the same sampling date.\u003c/p\u003e\u003cp\u003eAlthough copper is strongly retained in the roots, it still may disrupt essential physiological processes, particularly those related to Fe uptake and translocation. Excess Cu can compete with Fe for shared uptake pathways such as IRT1 and inhibit the activity of ferric chelate reductase, both are critical for efficient Fe acquisition and translocation (Perea-Garc\u0026iacute;a et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In addition, copper-induced oxidative stress can impair intracellular Fe homeostasis, and thus hinder the proper allocation of Fe to key sites, e.g. the chloroplasts (Thomas et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Consequently, even if sufficient Fe is present in the plant, it may remain physiologically unavailable. Both, copper-related reduction in Fe uptake and translocation as well as disruptions of Fe homeostasis may have contributed to the development of chlorosis, which were typical for Fe deficiency in the year of planting (Zorn et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), even in the absence of elevated Cu concentrations in leaf tissue.\u003c/p\u003e\u003cp\u003eLeaf chlorosis also occurred in the second growing season, whereby a significant positive effect of compost application as well as of liming was observed. However, symptoms appeared during advanced shoot elongation (BBCH 33\u0026ndash;35), and thus considerably later than at the beginning of shoot growth (BBCH 07\u0026ndash;09), as typically reported for copper toxicity in hop gardens (Portner \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Furthermore, the symptoms were restricted to the lower part of the plants and atypical for copper toxicity (Portner \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Zorn et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Both makes copper toxicity quite unlikely to be the sole cause of symptom development in the second growing season. The same applies to the macronutrients analyzed in leaves and roots, which showed no irregularities during the entire experimental period (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e in the Appendix).\u003c/p\u003e\u003cp\u003eIn contrast, molybdenum (Mo) concentrations in leaves were clearly below the sufficiency range both at the end of the first growing season and especially during the second growing season (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e in the Appendix). Moreover, Mo deficiency was not only evident in the leaves, but also in the roots and the soil (Tables\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and 8 in the Appendix). These findings strongly support Mo deficiency as the main cause of symptom expression in the second growing season after planting. In line with this, previous studies reported similar leaf concentrations and symptom development in hop grown under Mo deficiency (Askew et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1958\u003c/span\u003e; Watson and Askew \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e1956\u003c/span\u003e). Since soil pH is generally positively correlated with Mo uptake by plants (Kabata-Pendias and Pendias \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), the low soil pH in the present study was likely the main reason for the limited Mo availability. As a result, liming significantly reduced symptom expression due to an increase of pH during the second growing season. The same likely applied to the application of compost, which had a high pH of 8.0 (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In addition, compost application may have supplied additional Mo into the planting holes, which could explain its stronger effect on both symptom reduction and Mo uptake compared to liming. This assumption is supported by the positive effect of the increasing application rate.\u003c/p\u003e\u003cp\u003eIn contrast to Mo, Fe and Mn concentrations in leaves frequently exceeded the sufficiency range during both growing seasons (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e in the Appendix). This pronounced uptake likely resulted from increased solubility and mobility under low pH (Kabata-Pendias and Pendias \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), as indicated by the reduction of Mn concentrations after liming (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e in the Appendix). However, the lack of correlation between leaf Fe and Mn concentrations and the severity of visual symptoms as well as the elevated but generally subtoxic Fe levels in hop leaves do not support Fe or Mn excess as the primary cause of the observed leaf damage (Afonso et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, Watson (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1960\u003c/span\u003e) reported a significant increase of phosphorus and potassium concentration in leaf tissues of hop plants suffering from Mn toxicity which was not observed at any time point of the current research (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e in the Appendix). Indeed, this is not a sound evidence against Mn excess as the primary cause of the observed leaf damage, but a further indication.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Biomass Production\u003c/h2\u003e\u003cp\u003eWhile neither liming nor the application of biochar-free compost had a significant effect on biomass production, the addition of both biochar-containing composts - particularly the compost co-composted with 5 vol% biochar at high application rate - resulted in an increase of about 30% in bine biomass and total biomass compared to the treatments without compost. Notably, the compost co-composted with 5 vol% biochar showed the most beneficial impact on plant growth not only in the present field trial, but also in a previous pot experiment with fresh and aged copper-contaminated soils. This might be due to the most intensive composting process and thus the highest degree of maturity achieved with this compost (G\u0026ouml;rl et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e), which is also reflected in its higher levels of plant-available N, P, and K (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe growth-promoting effect of co-composted biochar compost can be attributed not only to a potential reduction in the phytoavailability of heavy metals in soil, but also to other positive impacts on soil properties. These include increases in soil organic carbon and available nutrients, improved water retention and holding capacity, and enhancements in soil aggregation, structural stability, aeration, cation exchange capacity and pH, as well as stimulation of microbial activity (Antonangelo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although biochar-free compost also exhibits similar effects on soil properties, these were generally less pronounced than those observed with biochar compost in our trial, which fits to results of others (Fischer and Glaser \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast to bine and total biomass, cone yield was not significantly increased by compost application despite the reductions in symptom expression. This suggests that the existing copper contamination in the soil did not negatively affect cone production, possibly due to the copper level of 154 mg kg\u003csup\u003e-1\u003c/sup\u003e being too low to noticeably impact cone yield. On sites with higher contamination levels, as frequently observed in the Hallertau region (Schramel et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), stronger effects on cone yield can be expected.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Soil Respiration\u003c/h2\u003e\u003cp\u003eThe addition of compost, both with and without biochar, increased respiration rates in the soil of the planting holes areas at the end of the first growing season after planting. This increase in soil respiration can be attributed to higher microbial biomass and the stimulation of microbial activity due to enhanced resource availability, as well as to shifts in microbial community composition (Iovieno et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and improved physical soil properties (Tejada et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe strongest stimulation of soil respiration by the biochar-free compost is likely due to its higher degradability, as biochar is largely resistant to microbial decomposition (Wang et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, the addition of biochar at the beginning of the composting process has shown to intensify the composting process (G\u0026ouml;rl et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e), further reducing the proportion of degradable carbon in the biochar-containing composts. A comparable difference in respiration rates between soils amended with co-composted biochar composts and those amended with biochar-free compost was already observed in a previous pot experiment using artificially and freshly copper-spiked soils (G\u0026ouml;rl et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWith the exception of the high-rate application of biochar-free compost, no clear effect of compost addition on respiration rates was observed in the second growing season after planting. This lack of effect was likely due to the advanced microbial consumption of easily degradable carbon fractions in the composts (Pane et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), as well as their redistribution from the initially concentrated placement in the planting holes into the surrounding soil as a result of soil management practices within the cultivation period of about 20 months.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e4.4 Recommendations for Field Application\u003c/h2\u003e\u003cp\u003eBased on our present and previous findings (G\u0026ouml;rl et al. 2023 a, b), the use of co-composted biochar compost can generally be recommended for farmers to improve juvenile development and growth of hop plants after replanting copper-contaminated hop gardens. If the biochar compost is produced from hop bines, as in the present study, the addition of 5 vol% biochar at the beginning of composting is already sufficient and even preferable to the higher rate of 20 vol%, as this reduces biochar costs while maximizing positive effects on plant growth and achieving improvements in the composting process. As doubling the application rate from 1.25 to 2.5 kg per planting hole generally improved the effectiveness of the compost amendments, the higher rate should be preferred, particularly on sites where impairments in the juvenile growth of hop plants are to be expected.\u003c/p\u003e\u003cp\u003eAs even the more cost-effective biochar-free compost showed positive effects in reducing leaf symptom expression and particularly in stimulating microbial activity, its use can also be recommended to farmers. However, in the present experiment, biomass production could not be enhanced without the addition of biochar. Nevertheless, this effect was limited to higher bine and total biomass, while cone yields remained unchanged. For this reason, it should be carefully considered whether the additional costs of biochar are justified, and decisions should always be made depending on the specific site conditions. While on less challenging sites, such as in the present experiment, the biochar-free compost might already be sufficient, on highly contaminated soils, the use of co-composted biochar compost should be preferred. Moreover, when using biochar-free compost, the absence of biochar costs could allow a compost application across the entire area instead of a planting hole-specific application, which covers only about 10% of the total area at a planting density of 2,400 plants per hectare. However, even when biochar is not used, compost availability and cost may still represent limiting factors. Furthermore, without biochar, additional benefits such as improved composting efficiency and long-term carbon sequestration in the soil cannot be expected.\u003c/p\u003e\u003cp\u003eBeside the positive effects on plant growth and soil biota, further positive effects of applying (co-composted biochar) compost to planting holes should be mentioned here: Due to higher infiltration rates, irrigation of plants was easier in the year of planting and less superficial run-off was observed. Furthermore, planting into compost-amended soil was considerably easier than into bare soil, particularly under the dry planting conditions of the present study. This could be a strong argument for hop growers, especially in view of the expected increase in spring drought due to climate change (H\u0026auml;nsel et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast to biochar-free compost, liming may not be a suitable alternative to the application of co-composted biochar compost, as its effects on plant growth and soil respiration were either minimal or absent in this study.\u003c/p\u003e\u003c/div\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eThe targeted application of co-composted biochar compost to planting holes during the replanting of a copper-contaminated hop garden significantly reduced the occurrence of visible damage symptoms and increased biomass production until the end of the second growing season, thus confirming our first hypothesis. However, indications of copper toxicity were only observed in the year of planting, whereas Mo deficiency was identified as the primary cause of leaf symptoms in the second year. In addition to plant growth, the application of co-composted biochar compost significantly enhanced microbial activity in the soil of the planting hole area, at least in the first growing season, partly confirming our second hypothesis. While co-composted biochar compost was more effective than biochar-free compost in promoting plant growth, the opposite was observed for soil respiration, thus rejecting our third hypothesis with respect to soil biota.\u003c/p\u003e\u003cp\u003eSince the present findings demonstrated consistent or even enhanced effects with reduced biochar amounts in co-composting, future research should focus on evaluating the impact of biochar additions below 5 vol%. If further reductions in cost-intensive biochar prove feasible without compromising efficacy, the acceptance among farmers and consequently the prospects for using co-composted biochar compost could be significantly enhanced.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBC \u0026ndash; biochar; BET \u0026ndash; specific surface area; B \u0026ndash; boron; Ca \u0026ndash; calcium; CaCl\u003csub\u003e2\u003c/sub\u003e \u0026ndash; calcium chloride; CAL \u0026ndash; calcium acetate lactate extraction; CAT \u0026ndash; CaCl₂/DTPA extraction; C\u003csub\u003eorg\u003c/sub\u003e \u0026ndash; organic carbon; Cu \u0026ndash; copper; DM \u0026ndash; dry matter; Fe \u0026ndash; iron; H \u0026ndash; hydrogen; K \u0026ndash; potassium; Mg \u0026ndash; magnesium; Mn \u0026ndash; manganese; Mo \u0026ndash; molybdenum; N \u0026ndash; nitrogen; NH\u003csub\u003e4\u003c/sub\u003e-N \u0026ndash; ammonium nitrogen; NO\u003csub\u003e3\u003c/sub\u003e-N \u0026ndash; nitrate nitrogen; P \u0026ndash; phosphorus; R\u003csub\u003eB\u003c/sub\u003e \u0026ndash; basal respiration rate; S \u0026ndash; sulfur; Zn \u0026ndash; zinc\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests:\u003c/h2\u003e\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis research was funded by the Bavarian State Ministry for Food, Agriculture and Forestry (Grant No. A/21/08) and further supported by the Bavarian Academic Forum\u0026mdash;BayWISS.\u003c/p\u003e\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e\u003cp\u003eConceptualization, methodology, investigation, formal analysis, visualization, and the initial drafting of the manuscript were conducted by Johannes G\u0026ouml;rl. Supervision of laboratory analyses, project administration, and funding acquisition were carried out by Dieter Lohr. Review and editing were performed by Dieter Lohr, Elke Meinken, and Kurt-J\u0026uuml;rgen H\u0026uuml;lsbergen, with overall supervision provided by Elke Meinken and Kurt-J\u0026uuml;rgen H\u0026uuml;lsbergen. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThe authors express their gratitude to the technical staff of the Institute of Horticulture for their careful assistance in conducting the experiments. They also wish to thank the staff of the Hop Research Center in H\u0026uuml;ll for their valuable technical and scientific support, with special thanks to Florian Wei\u0026szlig; for his dedicated and tireless assistance.\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e\u003cp\u003eThe data supporting this study\u0026rsquo;s findings are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdrees M, Ali S, Rizwan M, Ibrahim M, Abbas F, Farid M, Qayyum MF, Irshad M, Bharwana SA, Malik Z, Sattar A (2015) The effect of excess copper on growth and physiology of important food crops: a review. 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Springer-, Berlin\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"hop cultivation, phytotoxicity, co-composting, molybdenum deficiency, remediation, ecotoxicology","lastPublishedDoi":"10.21203/rs.3.rs-7215331/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7215331/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and Aims\u003c/h2\u003e\u003cp\u003eThe long-term application of copper-based fungicides in hop cultivation has led to substantial copper accumulation in the topsoil, potentially impairing the early growth of newly planted hop plants and affecting soil biota. To reduce copper bioavailability in soil, co-composted biochar compost was evaluated as a remediation strategy.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eTwo biochar composts, produced by co-composting 5 and 20 vol% biochar with chopped hop bines, and a biochar-free hop bine compost were applied into the planting holes during replanting of a copper-contaminated hop garden. A limed treatment and an unamended control were included. Remediation effects were assessed over two growing seasons based on visual damage ratings, copper concentrations in leaves and roots, biomass production, and soil respiration.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eDuring both growing seasons, leaf chlorosis and necrosis were observed, but were associated with copper toxicity only in the year of planting. In the second year, Mo deficiency was the primary cause of leaf damage. Biochar compost, particularly the one co-composted with 5 vol% biochar, significantly reduced these symptoms and increased biomass production by about 30%. In contrast, liming and biochar-free compost were less effective. Soil respiration was significantly enhanced by up to 81% with biochar-free compost showing the strongest effect due to higher microbial degradability.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eBased on these findings, co-composted biochar compost can be recommended for farmers to improve juvenile growth after replanting copper-contaminated hop gardens. However, biochar-free compost also showed beneficial effects, particularly on microbial respiration, and may serve as a cost-effective alternative on less challenging sites.\u003c/p\u003e","manuscriptTitle":"Co-Composted Biochar Compost for Improving Juvenile Growth After Replanting Copper-Contaminated Hop Gardens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 15:46:21","doi":"10.21203/rs.3.rs-7215331/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-08-21T06:11:21+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-07-29T17:46:43+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-29T17:32:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2025-07-29T00:05:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-28T09:50:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2025-07-25T10:47:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"33fcc3df-eb16-4389-9f5e-e0bf99750a33","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T16:05:12+00:00","versionOfRecord":{"articleIdentity":"rs-7215331","link":"https://doi.org/10.1007/s11104-025-07990-7","journal":{"identity":"plant-and-soil","isVorOnly":false,"title":"Plant and Soil"},"publishedOn":"2025-12-27 15:58:01","publishedOnDateReadable":"December 27th, 2025"},"versionCreatedAt":"2025-07-31 15:46:21","video":"","vorDoi":"10.1007/s11104-025-07990-7","vorDoiUrl":"https://doi.org/10.1007/s11104-025-07990-7","workflowStages":[]},"version":"v1","identity":"rs-7215331","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7215331","identity":"rs-7215331","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}