Search Results (710)

Salinity is a major abiotic stress limiting black gram (Vigna mungo) productivity, particularly in arid and semi-arid regions. Saline soils negatively impact plant growth, nodulation, nitrogen fixation, and yield. This study evaluated the efficacy of co-inoculating salt-tolerant plant growth-promoting bacteria Paenibacillus sp. SPR11 and Bradyrhizobium yuanmingense PR3 on black gram performance under saline field conditions (EC: 8.87 dS m⁻¹; pH: 8.37) with low organic carbon (0.6%) and nutrient deficiencies. In vitro assays demonstrated the biocontrol potential of SPR11, inhibiting Fusarium oxysporum and Macrophomina phaseolina by 76% and 62%, respectively. Germination assays and net house experiments under 300 mM NaCl stress showed that co-inoculation significantly improved physiological traits, including germination rate, root length (61.39%), shoot biomass (59.95%), and nitrogen fixation (52.4%) in nitrogen-free media. Field trials further revealed enhanced stress tolerance markers: chlorophyll content increased by 54.74%, proline by 50.89%, and antioxidant enzyme activities (SOD, CAT, PAL) were significantly upregulated. Electrolyte leakage was reduced by 55.77%, indicating improved membrane stability. Agronomic performance also improved, with co-inoculated plants showing increased root length (7.19%), grain yield (15.55 q ha⁻¹; 77.04% over control), total biomass (26.73 q ha⁻¹; 57.06%), and straw yield (8.18 q ha⁻¹). Pod number, seed count, and seed weight were also enhanced. Nutrient analysis showed elevated uptake of nitrogen, phosphorus, potassium, and key micronutrients (Zn, Fe) in both grain and straw. To the best of our knowledge, this is the very first field-based report demonstrating the synergistic benefits of co-inoculating Paenibacillus sp. SPR11 and Bradyrhizobium yuanmingense PR3 in black gram under saline, nutrient-poor conditions without external nitrogen inputs. The results highlight a sustainable strategy to enhance legume productivity and resilience in salt-affected soils. Full article

Sulfur-containing fertilizers increase production costs, which leads to low utilization of this nutrient. Thus, evaluating how the absence of sulfur influences the early development of Urochloa brizantha is essential. Study was conducted in a greenhouse at the Federal University of Rondonópolis in a completely randomized design, with six treatments in a 3 × 2 factorial scheme, and eight replications. Three cultivars of U. brizantha (Marandu, Xaraés and Piatã) were evaluated under two fertilization strategies: with or without sulfur fertilization. Sufur presence increased the number of leaves and forage mass, in which cultivar Xaraés presented the greatest means. Piatã was the cultivar most sensitive to sulfur deficiency at establishment, which reduced forage mass, number of leaves and number of tillers by 42%, 32%, and 45%, respectively. Despite these differences between cultivars, sulfur efficiently increased the forage yield. Sulfur fertilization increased the concentrations of nutrients in the plants without significantly affecting the uptake of nitrogen, phosphorus, potassium, calcium and magnesium. Sulfur omission resulted in increased phosphorus uptake in all grass. In contrast, Marandu grass exhibited the greatest reduction in sulfur uptake. Therefore, the use of sulfur in the fertilization of grasses is recommended, it is important to evaluate the responses of each cultivar to better adjust the fertilization management. Full article

Glycyrrhiza uralensis (G. uralensis), a leguminous plant, is an important medicinal and economic plant in saline–alkaline soils of arid regions in China. Its main bioactive components include liquiritin, glycyrrhizic acid, and flavonoids, which play significant roles in maintaining human health and preventing and adjuvantly treating related diseases. However, the cultivation of G. uralensis is easily restricted by adverse soil conditions in these regions, characterized by high salinity, high alkalinity, and nutrient deficiency. This study investigated the impacts of four multistrain microbial inoculants (Pa, Pb, Pc, Pd) on the growth performance and bioactive compound accumulation of G. uralensis in moderately saline–sodic soil. The aim was to screen the most beneficial inoculant from these strains, which were isolated from the rhizosphere of plants in moderately saline–alkaline soils of the Hexi Corridor and possess native advantages with excellent adaptability to arid environments. The results showed that inoculant Pc, comprising Pseudomonas silesiensis, Arthrobacter sp. GCG3, and Rhizobium sp. DG1, exhibited superior performance: it induced a 0.86-unit reduction in lateral root number relative to the control, while promoting significant increases in single-plant dry weight (101.70%), single-plant liquiritin (177.93%), single-plant glycyrrhizic acid (106.10%), and single-plant total flavonoids (107.64%). Application of the composite microbial inoculant Pc induced no significant changes in the pH and soluble salt content of G. uralensis rhizospheric soils. However, it promoted root utilization of soil organic matter and nitrate, while significantly increasing the contents of available potassium and available phosphorus in the rhizosphere. High-throughput sequencing revealed that Pc reorganized the rhizospheric microbial communities of G. uralensis, inducing pronounced shifts in the relative abundances of rhizospheric bacteria and fungi, leading to significant enrichment of target bacterial genera (Arthrobacter, Pseudomonas, Rhizobium), concomitant suppression of pathogenic fungi, and proliferation of beneficial fungi (Mortierella, Cladosporium). Correlation analyses showed that these microbial shifts were linked to improved plant nutrition and secondary metabolite biosynthesis. This study highlights Pc as a sustainable strategy to enhance G. uralensis yield and medicinal quality in saline–alkali ecosystems by mediating microbe–plant–nutrient interactions. Full article

Manganese (Mn) excess and low pH often coexist in some citrus orchard soils. Little information is known about the underlying mechanism by which raising pH reduces Mn toxicity in citrus plants. ‘Sour pummelo’ (Citrus grandis (L.) Osbeck) seedlings were treated with 2 (Mn2) or 500 (Mn500) μM Mn at a pH of 3 (P3) or 5 (P5) for 25 weeks. Raising pH mitigated Mn500-induced increases in Mn, iron, copper, and zinc concentrations in roots, stems, and leaves, as well as nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, copper, iron, and zinc distributions in roots, but it mitigated Mn500-induced decreases in nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, and boron concentrations in roots, stems, and leaves, as well as nutrient imbalance. Raising pH mitigated Mn500-induced necrotic spots on old leaves, yellowing of young leaves, decreases in seedling growth, leaf chlorophyll concentration, and CO₂ assimilation (A_CO2), increase in root dry weight (DW)/shoot DW, and alterations of leaf chlorophyll a fluorescence (OJIP) transients and related indexes. Further analysis indicated that raising pH ameliorated Mn500-induced impairment of nutrient homeostasis, leaf thylakoid structure by iron deficiency and competition of Mn with magnesium, and photosynthetic electron transport chain (PETC), thereby reducing Mn500-induced declines in A_CO2 and subsequent seedling growth. These results validated the hypothesis that raising pH reduced Mn toxicity in ‘Sour pummelo’ seedlings by (a) reducing Mn uptake, (b) efficient maintenance of nutrient homeostasis under Mn stress, (c) reducing Mn excess-induced impairment of thylakoid structure and PEPC and inhibition of chlorophyll biosynthesis, and (d) increasing A_CO2 and subsequent seedling growth under Mn excess. Full article

Background/Objectives: Nitrogen (N) is an essential macronutrient that strongly influences maize growth and metabolism. While many studies have focused on nitrogen responses during later developmental stages, early-stage physiological and metabolic responses remain less explored. This study investigated the effect of different nitrogen-deficient levels on maize seedling growth and primary metabolite profiles. Methods: Seedlings were treated with N-modified nutrient solution, which contained 0% to 120% of the standard nitrogen level (8.5 mM). Results: Nitrogen starvation (N0) significantly reduced plant height (by 11–14%), shoot fresh weight (over 30%) compared to the optimal N supply (N100). Total leaf nitrogen content under N0–N20 was less than half of that in N100, whereas moderate N deficiency resulted in moderate reductions in growth and nitrogen content. Metabolite analysis revealed that N deficiency induced the accumulation of soluble sugars and organic acids (up to threefold), while sufficient N promoted the synthesis of amino acids related to nitrogen assimilation and protein biosynthesis. Statistical analyses (PCA and ANOVA) showed that both genotypes (MB and TYC) and tissue type (upper vs. lower leaves) influenced the metabolic response to nitrogen, with MB displaying more consistent shifts and TYC exhibiting greater variability under moderate stress. Conclusions: These findings highlight the sensitivity of maize seedlings to early nitrogen deficiency, with severity influenced by nitrogen level, tissue-specific position, and genotype; thus underscore the close coordination between physiological growth and primary metabolic pathways in response to nitrogen availability. These findings expand current knowledge of nitrogen response mechanisms and offer practical insights for improving nitrogen use efficiency in maize cultivation. Full article

Azospirillum is a well-studied genus of plant growth-promoting rhizobacteria (PGPR) and one of the most extensively researched diazotrophs. This genus can colonize rhizosphere soil and enhance plant growth and productivity by supplying essential nutrients to the host. Azospirillum–plant interactions involve multiple mechanisms, including nitrogen fixation, the production of phytohormones (auxins, cytokinins, indole acetic acid (IAA), and gibberellins), plant growth regulators, siderophore production, phosphate solubilization, and the synthesis of various bioactive molecules, such as flavonoids, hydrogen cyanide (HCN), and catalase. Thus, Azospirillum is involved in plant growth and development. The genus Azospirillum also enhances membrane activity by modifying the composition of membrane phospholipids and fatty acids, thereby ensuring membrane fluidity under water deficiency. It promotes the development of adventitious root systems, increases mineral and water uptake, mitigates environmental stressors (both biotic and abiotic), and exhibits antipathogenic activity. Biological nitrogen fixation (BNF) is the primary mechanism of Azospirillum, which is governed by structural nif genes present in all diazotrophic species. Globally, Azospirillum spp. are widely used as inoculants for commercial crop production. It is considered a non-pathogenic bacterium that can be utilized as a biofertilizer for a variety of crops, particularly cereals and grasses such as rice and wheat, which are economically significant for agriculture. Furthermore, Azospirillum spp. influence gene expression pathways in plants, enhancing their resistance to biotic and abiotic stressors. Advances in genomics and transcriptomics have provided new insights into plant-microbe interactions. This review explored the molecular mechanisms underlying the role of Azospirillum spp. in plant growth. Additionally, BNF phytohormone synthesis, root architecture modification for nutrient uptake and stress tolerance, and immobilization for enhanced crop production are also important. A deeper understanding of the molecular basis of Azospirillum in biofertilizer and biostimulant development, as well as genetically engineered and immobilized strains for improved phosphate solubilization and nitrogen fixation, will contribute to sustainable agricultural practices and help to meet global food security demands. Full article

Understanding the interplay between plant leaf functional traits and plant and soil factors under different soil thicknesses is significant for quantifying the interaction between plant growth and the environment. However, in the context of ecological restoration of vegetation in mining areas, there has been a lot of research on trees, shrubs, and grasses, but the characteristics and correlations of leaf functional traits of vines have not been fully studied to a large extent. Here, we report the differences in leaf functional traits of six vine plants (Parthenocissus quinquefolia, Pueraria lobata, Hedera nepalensis, Campsis grandiflora, Mucuna sempervirens, and Parthenocissus tricuspidata) with distinct growth forms in different soil cover thicknesses (20 cm, 40 cm, and 60 cm). In addition, soil factor indicators under different soil cover thicknesses were measured to elucidate the linkages between leaf functional traits of vine plants and soil factors. We found that P. lobata showed a resource acquisition strategy, while H. nepalensis demonstrated a resource conservation strategy. C. grandiflora and P. tricuspidata shifted toward more conservative resource allocation strategies as the soil cover thickness increased, whereas M. sempervirens showed the opposite trend. In the plant trait–trait relationships, there were synergistic associations between specific leaf area (SLA) and leaf nitrogen content (LNC); leaf moisture content (LMC) and leaf nitrogen-to-phosphorus ratio (LN/P); and leaf specific dry weight (LSW), leaf succulence degree (LSD), and leaf dry matter content (LDMC). Trade-offs were observed between SLA and LSW, LSD, and LDMC; between leaf phosphorus content (LPC) and LN/P; and between LMC, LSW, and LDMC. In the plant trait–environment relationships, soil nutrients (pH, soil total phosphorus content (STP), and soil ammonium nitrogen content (SAN)) and soil enzyme activities (cellulase (CB), leucine aminopeptidase (LAP), enzyme C/N activity ratio, and enzyme N/P activity ratio) were identified as the primary drivers of variation in leaf functional traits. Interestingly, nitrogen deficiency constrained the growth of vine plants in the mining area. Our study revealed that the responses of leaf functional traits of different vines under different soil thicknesses have significant species specificity, and each vine shows different resource acquisition and conservation strategies. Furthermore, soil cover thickness primarily influences plant functional traits by directly affecting soil enzyme activities and nutrients. However, the pathways through which soil thickness impacts these traits differ among various functional traits. Our findings provide a theoretical basis and practical reference for selecting vine plants and optimizing soil cover techniques for ecological restoration in mining areas. Full article

This study aimed to evaluate the impact of intercropping Brassica oleracea. with three perennial grasses (Poa pratensis L., Lolium perenne L., and Festuca rubra L.) under varying levels of iron (Fe) availability (Fe0, Fe1 and Fe5) in nutrient solutions. The research focused on biomass accumulation, photosynthetic efficiency, root development, nutrient uptake, and oxidative stress response. In the absence of Fe, Brassica sp. exhibited chlorosis, reduced biomass, and increased ferric chelate reductase (FCR) enzyme activity as an adaptive response. Brassica plants intercropped with Poa sp. maintained higher chlorophyll (Chl) levels and photosystem II efficiency (F_v/F_m values), mitigating Fe deficiency effects. Catalase activity and polyphenol production varied with intercropping species, indicating differential stress response mechanisms. Intercropping improved Zn, Mn, and P accumulation, with Poa sp. facilitating greater Zn and Mn uptake. Intercropping Brassica sp. with specific grass species offers potential agronomic benefits by improving Fe use efficiency, mitigating stress, and enhancing nutrient uptake. Future research should focus on optimizing intercropping combinations for sustainable agricultural practices. Full article

Shaker K⁺ channel proteins are responsible for potassium (K⁺) uptake and transport, playing a critical role in plant growth, development, and adaptation to K⁺ deficiency. Cassava, a key tropical root crop, is known for its characteristic of resilience to nutrient-poor soil and drought stress. However, the Shaker K⁺ channel gene family in cassava has not yet been characterized. In this study, 13 Shaker channel genes were identified from the near telomere-to-telomere (T2T) cassava genome using bioinformatics analysis. Phylogenetic relationships classified these genes into five distinct subfamilies, and all encoded proteins contained the conserved GYGD/GYGE motif typical of Shaker channels. Protein interaction network predictions revealed potential interactions among the Shaker family, as well as with the potassium transporter HAK5. Tissue-specific expression pattern analysis showed that MeGORK and MeAKT1.2 were expressed in all tissues. Furthermore, quantitative real-time PCR (qRT-PCR) analysis was conducted to examine the transcriptional levels of Shaker K⁺ channel gene family members in the roots and leaves of two cassava germplasms with different low-potassium tolerance after one month of low-potassium treatment. The results revealed that MeAKT1.2, MeAKT2.2, and MeKAT1 exhibited distinct expression patterns between the two germplasms, with higher expression levels observed in the potassium-tolerant germplasm. Therefore, these three genes may serve as important candidate genes for potassium stress tolerance in cassava. In summary, this study provides valuable insights into the characteristics and biological functions of the Shaker K⁺ channel gene family in cassava and identifies potential candidate genes for breeding or engineering potassium-efficient cassava cultivars. Full article

Calcareous soils, characterized by high pH and calcium carbonate content, often limit the availability of essential nutrients for crops such as rice (Oryza sativa L.), reducing yield and nutritional quality. In this study, we evaluated the effect of the halotolerant yeast Debaryomyces hansenii on the growth, nutrient uptake, and phosphorus acquisition mechanisms of rice plants cultivated in calcareous soil under controlled greenhouse conditions. Plants inoculated with D. hansenii, particularly via root immersion, exhibited significantly higher SPAD chlorophyll index, plant height, and grain yield compared to controls. A modest increase (~4%) in dry matter content was also observed under sterilized soil conditions. Foliar concentrations of Fe, Zn, and Mn significantly increased in plants inoculated with D. hansenii via root immersion in non-sterilized calcareous soil, indicating improved micronutrient acquisition under these specific conditions. Although leaf phosphorus levels were not significantly increased, D. hansenii stimulated acid phosphatase activity, as visually observed through BCIP staining, and upregulated genes involved in phosphorus acquisition under both P-sufficient and P-deficient conditions. At the molecular level, D. hansenii upregulated the expression of acid phosphatase genes (OsPAP3, OsPAP9) and a phosphate transporter gene (OsPTH1;6), confirming its influence on P-related physiological responses. These findings demonstrate that D. hansenii functions as a plant growth-promoting yeast (PGPY) and may serve as a promising biofertilizer for improving rice productivity and nutrient efficiency in calcareous soils, contributing to sustainable agricultural practices in calcareous soils and other nutrient-limiting environments. Full article

An increasing number of soils, including those in EU countries, are affected by organic matter deficiency and the deterioration of nutrients, and using mineral fertilizers is often associated with negative environmental impacts. One of the basic recommendations for sustainable agriculture is to increase the proportion of organic fertilizers in crop production and preserve soil biodiversity. An increasingly common organic fertilizer is biogas plant digestate, the physical and chemical properties of which depend primarily on the waste material used in biogas production. However, the fertilizer value of this additive and its effects on the soil environment, including beneficial organisms, remain insufficiently studied. Soil macrofauna, particularly earthworms, play a crucial role in soil ecosystems, because they significantly impact the presence of plant nutrients, actively participate in forming soil structures, and strongly influence organic matter dynamics. The present study was undertaken to determine the effects of fertilizing a silt loam soil with the solid fraction of digestate in monoculture crop production on earthworm community characteristics and the resulting changes in selected soil physicochemical properties. The research was conducted at a single site, so the original soil characteristics across the experimental plots were identical. Plots were treated annually (for 3 years; 2021–2023) with different levels of digestate: DG100 (100% of the recommended rate; 30 t ha⁻¹), DG75 (75% of the recommended rate; 22.5 t ha⁻¹), DG50 (15 t ha⁻¹), DG25 (7.5 t ha⁻¹), and CL (a control plot without fertilizer). An electrical method was used to extract earthworms. Those found at the study site belonged to seven species representing three ecological groups: Dendrodrilus rubidus (Sav.), Lumbricus rubellus (Hoff.), and Dendrobaena octaedra (Sav.) (epigeics); Aporrectodea caliginosa (Sav.), Aporrectodea rosea (Sav.), and Octolasion lacteum (Örley) (endogeics); and Lumbricus terrestris (L.) (anecics). Significant differences in the abundance and biomass of earthworms were found between the higher level treatments (DG100, DG75, and DG50), and the lowest level of fertilization and the control plot (DG25 and CL). The DG25 and CL plots showed an average of 24.7% lower earthworm abundance and 22.8% lower biomass than the other plots. There were no significant differences in the earthworm metrics between the plots within each of the two groups (DG100, DG75, and DG50; and DG25 and CL). The most significant influence on the average abundance and average biomass of Lumbricidae was probably exerted by soil moisture and the annual dosage of digestate. A significant increase in the abundance and biomass of Lumbricidae was shown at plots DG100, DG75, and DG50 in the three successive years of the experiment. The different fertilizer treatments were found to have different effects on selected soil parameters. No significant differences were found among the values of the analyzed soil traits within each plot in the successive years of the study. Full article

Peanut productivity is severely restricted by soil salinization and associated nutrient deficiency in saline soil. The quinoa–peanut relay intercrop pattern (IP) is a promising planting system that utilizes the biological advantages of quinoa to improve soil ecological functions and productivity. However, the effects of IP on soil physicochemical and biological properties and the yield formation of the combined peanut crop are still unclear. Two-year field experiments in coastal saline soil were conducted to explore the effects of IP on peanut growth and pod yield, soil physicochemical properties, and microbial community characterization at different growth stages of peanut based on the traditional monocrop pattern (MP). The results show that IP promoted peanut pod yield, although there was the disadvantage of plant growth at an early stage. Soil water content, electrical conductivity (EC), and Na⁺ content in the peanut rhizosphere were lower, whereas K⁺, NH₄⁺, and total organic carbon (TOC) contents were higher in IP systems at both the vegetative and reproductive stages. The pod yield of peanut was significantly negatively correlated with soil EC and Na⁺ contents at the vegetative stage, but positively correlated with K⁺, NO₃⁻, NH₄⁺, PO₄³⁻, and TOC contents at the reproductive stage. IP rebuilt the composition of the soil bacterial community in the peanut rhizosphere and increased the abundance of the beneficial bacterial community, which were positively correlated with soil TOC, K⁺, NH₄⁺, NO₃⁻, and PO₄³⁻ contents. These findings suggest that IP can increase peanut pod yield through optimizing soil physicochemical properties and microbial community composition, and it is a promising planting system for improving agricultural production in coastal saline lands. Full article

Microelement deficiencies, often termed “hidden hunger”, represent a significant global health challenge. Optimal human health relies on adequate dietary intake of essential microelements, including selenium (Se), zinc (Zn), copper (Cu), boron (B), manganese (Mn), molybdenum (Mo), iron (Fe), nickel (Ni), and chlorine (Cl). In recent years, there has been a growing focus on vitality and longevity, which are closely associated with the sufficient intake of essential microelements. This review focuses on these nine elements, whose bioavailability in the food chain is critically determined by their geochemical behavior in soils. There is a necessity for an understanding of the sources, soil–plant transfer, and plant uptake mechanisms of these microelements, with particular emphasis on the influence of key soil properties, including pH, redox potential, organic matter content, and mineral composition. There is a dual challenge of microelement deficiencies in agricultural soils, leading to inadequate crop accumulation, and the potential for localized toxicities arising from anthropogenic inputs or geogenic enrichment. A promising solution to microelement deficiencies in crops is biofortification, which enhances nutrient content in food by improving soil and plant uptake. This strategy includes agronomic methods (e.g., fertilization, soil amendments) and genetic approaches (e.g., marker-assisted selection, genetic engineering) to boost microelement density in edible tissues. Moreover, emphasizing the need for advanced predictive modeling techniques, such as ensemble learning-based digital soil mapping, enhances regional soil microelement management. Integrating machine learning with digital covariates improves spatial prediction accuracy, optimizes soil fertility management, and supports sustainable agriculture. Given the rising global population and the consequent pressures on agricultural production, a comprehensive understanding of microelement dynamics in the soil–plant system is essential for developing sustainable strategies to mitigate deficiencies and ensure food and nutritional security. This review specifically focuses on the bioavailability of these nine essential microelements (Se, Zn, Cu, B, Mn, Mo, Fe, Ni, and Cl), examining the soil–plant transfer mechanisms and their ultimate implications for human health within the soil–plant–human system. The selection of these nine microelements for this review is based on their recognized dual importance: they are not only essential for various plant metabolic functions, but also play a critical role in human nutrition, with widespread deficiencies reported globally in diverse populations and agricultural systems. While other elements, such as cobalt (Co) and iodine (I), are vital for health, Co is primarily required by nitrogen-fixing microorganisms rather than directly by all plants, and the main pathway for iodine intake is often marine-based rather than soil-to-crop. Full article

In personalized nutrition, dietary guidelines must be adapted to the physiological and developmental needs of individuals across the lifespan, especially during childhood and adolescence. These should account for nutritional status, health conditions, and early-life risk factors, including those that emerge during pregnancy. This narrative review synthesizes recent evidence (2020–2025) on the biological value of protein sources in supporting pediatric growth and development. While adequate protein intake is essential for physical and cognitive development in individuals under nineteen, excessive intake may accelerate growth and increase the long-term risks of overweight and obesity. Compared to animal-based proteins (ABPs), plant-based proteins (PBPs) carry a higher risk of nutrient deficiencies in vulnerable populations due to lower digestibility and incomplete amino acid profiles. Although plant-based diets are encouraged for environmental reasons—particularly to reduce the ecological impact of livestock—protein intake must remain appropriate for age, sex, health status, and context. Nutritional strategies must ensure an adequate supply of essential amino acids and proper micronutrient supplementation, regardless of whether children follow diets rich in ABPs, PBPs, or a combination of both. Attention to these factors is vital to balancing nutritional adequacy with long-term health and sustainability goals. Full article

Forage legumes often show poor growth, the cause of which is not always immediately apparent to the farmer. The aim of the present study was the development of a diagnostic tool to identify possible causes for unexplained forage legume decline. A modified diagnostic test was carried out under a controlled environment using red clover (Trifolium pratense L.) as a test plant. Soil samples from three organic farms in Germany showing decline in productivity of forage legumes were tested using (i) an untreated control, (ii) application of a nutrient solution to investigate nutrient deficiencies, (iii) amendment with activated charcoal to immobilize toxic compounds, and (iv) heat sterilization to eliminate detrimental organisms. In addition, plant and soil samples from the three study sites were analyzed for pathogens and nutrient levels in the laboratory. At all the sites, plants growing in the sterilized soil showed an improvement in growth, indicating the presence of pathogens in the soil as the main cause for red clover decline. Hints at nutrient deficiency and a minor effect of phytotoxic compounds were found in addition to detrimental organisms at one studied site, indicating an interaction of abiotic and biotic factors as the cause of clover decline. The fertilization with a nutrient solution led to stunted growth at one site, which could be associated with a negative effect of nitrogen application on red clover and nutrient imbalances. The results of the bioassay were corroborated by the plant screening for pathogens and nutrient levels analyses. The diagnostic test proved to be a reliable tool for identifying possible causes of red clover decline, such as harmful organisms or, to a lesser extent, nutrient deficiencies. Full article