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Article

A Differential Diagnostic Tool for Identifying the Causes of Clover Decline

by
Beatrice Francisco Menezes
1,†,
Annika Kühnl
1,*,†,
Kristin Steinfurth
1,
Ulrike Hakl
2,
Irene Jacob
3,
Jürgen Müller
1,
Christel Baum
1 and
Christine Struck
1
1
Faculty of Agricultural and Environmental Sciences, University of Rostock, 18051 Rostock, Germany
2
Landwirtschaftskammer Nordrhein-Westfalen, 48147 Münster, Germany
3
Öko-BeratungsGesellschaft mbH, 85411 Hohenkammer, Germany
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(7), 1566; https://doi.org/10.3390/agronomy15071566
Submission received: 30 April 2025 / Revised: 2 June 2025 / Accepted: 6 June 2025 / Published: 27 June 2025
(This article belongs to the Special Issue Grass and Forage Diseases: Etiology, Epidemic and Management)
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Abstract

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.

1. Introduction

Forage legumes play a critical role in cropping and livestock production systems. The inclusion of legumes in crop rotations can significantly impact chemical, physical, and biological soil properties, contributing to the maintenance of soil fertility and productivity, especially in organic farming systems [1]. Due to their symbiotic association with rhizobacteria, which enable the fixation of atmospheric nitrogen, legumes are a valuable source of nitrogen in low input systems and a fundamental component of organic farming practices [2,3]. Along with their contribution to soil fertility, forage legumes are an important source of energy, fiber, and protein in livestock farming [4]. Their capacity of increasing herbage production together with their high voluntary intake and nutritive value are often associated with the improvement of animal performance [5,6]. Legume decline in crop stands is a phenomenon in which they lose vitality and performance, often after repeated cultivation. In fields with otherwise healthy and well-growing plants, sudden nests presenting growth depression appear. Plants start showing stunted growth and, often, symptoms of disease, such as chlorosis and necrosis. However, plant diseases are frequently not pronounced enough to be the sole source of the crop decline. The causes for growth depressions are often unclear since they can be part of a complex system of biotic and abiotic factors and their interactions. Biotic limitations in clover and alfalfa crops are often associated with diseases caused by seed- and soil-borne pathogens [7,8,9]. Among these, the root and foot rot complex—including species such as Fusarium spp., Ascochyta medicaginicola, and Didymella pinodella—plays a critical role in legume decline, also due to their broad host range and persistence in the soil [9,10]. Additional biotic stressors contributing to reduced crop performance include infestations with plant-parasitic nematodes [11,12] and weeds [13,14,15,16]. Furthermore, a disruption in the symbiotic association between legumes and rhizobia and alterations in the soil microbial community are factors associated with decline in crop productivity [17,18,19].
The accumulation of allelochemicals or the presence of heavy metals in the soil can also result in a suboptimal crop development. The release of phytotoxic compounds that limit crop establishment is another factor in the decline of legume-based pastures [20,21]. Heavy metal toxicity can lead to impaired physiological processes, nutrient deficiency, and compromised Rhizobium–legume symbiosis [22].
Next to heavy metal toxicity, nutrient availability is a further abiotic factor associated with yield depression in legumes. In addition to their importance for a plant’s general development, macro- and micronutrients play a fundamental role in the nitrogen fixation in legumes, and their shortage can affect the functioning of rhizobia symbiosis and lead to the development of nodules, as well as the production and regulation of enzymes involved in N2 fixation [17,23,24,25]. In addition, plant nutrition is known to affect disease resistance [26,27,28]. Soil compaction has been associated with hampered growth and reduced nutrient uptake in Egyptian clover (Trifolium alexandrinum L.) [29] and with impaired internal soil drainage favoring the infection of root rot (Aphanomyces euteiches) in pea (Pisum sativum L.) [30]. Ref. [31] found reduced root growth, lower nutrient reserves, and more patchy stands of lucerne (Medicago sativa L.) in the presence of soil compaction.
The optimal development of forage legumes crops is fundamental to the success of crop rotation and to the production of high-quality forage. In the case of legume decline, however, farmers and advisors need to be aware of the causes of these phenomena to make the right management decisions. Therefore, the development of a test that could identify causes of forage legume decline would be a great help for targeted cultivation interventions aimed at enhancing and stabilizing legume performance. Such a differential diagnostic test to investigate the possible causes of legume depression was first developed by [32] for the pulse crop, pea (Pisum sativum L.). The applicability of such a test on other legumes cannot be guaranteed, since vegetative depression may have a species-specific soil–plant relationship and requires specific soil–plant studies to be uncovered [33]. Thus, the aim of this study was to develop a diagnostic tool that can be applied to narrow down potential causes of decline in red clover. For this purpose, the differential diagnostic test developed by [32] was modified. In the present study, the test consisted of a one-level instead of a two-level diagnostic system, and the test plant used was red clover (Trifolium pratense L.). The sterilization through γ-irradiation was replaced by heat sterilization at 70 °C, and the nutrient solution containing all macro- and micronutrients was substituted by a Knop’s solution, which contains all the macronutrients and Fe and Cl as micronutrients. The diagnostic test efficiency was evaluated through a bioassay using soil from three organically managed farms where forage legume decline occurred. Plant and soil samples from the same sites were tested in the laboratory for fungal diseases, the presence of nematodes, and nutrient levels. As bacterial and viral diseases are not a major concern in Central Europe, the focus was on investigating fungal diseases only. The findings of the laboratory analyses were compared with the results of the diagnostic test. We used these comparisons to test the following hypotheses: (a) If the presence of detrimental organisms in the soil is the cause of legume depression, soil sterilization might inactivate those organisms, improving the growth of red clover. (b) If nutrient imbalance is the cause for hindered legume development, fertilization with nutrient solution can enhance plants’ growth. Additionally, a third hypothesis was tested in the bioassay: (c) If the presence of toxic substances in the soil is the cause for legume decline, amendment with activated charcoal will positively affect the development of red clover.

2. Materials and Methods

2.1. Bioassay Setup

Soil samples were collected in May/June 2023 from three organic farms where a decline in vitality and productivity of clover or alfalfa has been reported. The soil and location characteristics are shown in Table 1. At each site, 16 L soil samples were taken from at least 10 randomly chosen spots from the field patches presenting clover decline. Soil samples were taken close to the root zone, up to a depth of 20 cm, and stored in the dark and under aerobic conditions in a climate cabinet at 5 °C until further use.
A modified first-level assay of the two-level differential diagnostic presented by [32] was carried out to investigate nutrient deficiency, the presence of toxic compounds, and the presence of pathogens and parasites in the soil as possible causes for clover decline. The soil sample from each site was sieved to 1 cm, hand homogenized, and subjected to four distinct treatments, namely, (i) untreated control, (ii) fertilized weekly with a 5% diluted Knop’s solution (g L−1) (1 Ca(NO3)2, 0.25 MgSO4 × 7 H2O, 0.25 KH2PO4, 0.25 KNO3, 0.12 KCl, trace FeSO4) [34], (iii) mixed with activated charcoal (10 g L−1 soil) [35], and (iv) sterilized at 70 °C/24 h and allowed to cool down for 12 h [36]. The soil was distributed into 1 L pots and sown with five seeds of red clover (Trifolium pratense L., variety “Diplomat”) per pot. Each treatment had four replicates, resulting in a total of 16 pots per site. The experiment was conducted in a growth chamber with a 16 h day and an 8 h night cycle at a constant temperature of 18 °C [32]. To guarantee five plants in each pot, extra seeds were germinated on filter paper in two Petri dishes, and after two weeks, the sprouts were transplanted to pots containing less than five seedlings. Weeds growing in the pots were not plucked until the day of the measurement. After eight weeks, the plants were carefully removed from the pots. The roots were washed to eliminate the soil and dried with a paper towel. The shoot and root fresh weight (g), as well as plant height (cm) were measured. The dry weight (g) of the shoots and roots were determined after drying at 60 °C for 72 h.

2.2. Isolation and Identification of Pathogenic Fungi

In the same area and period where the soil for the bioassay was collected, plant blocks (10 × 10 × 10 cm) containing the whole symptomatic plant were taken. The infestation intensity was also recorded. For this purpose, the proportion of symptomatic plants out of 100 plants was counted in the field. After removal, the plant blocks were stored at 4 °C for a maximum of 48 h. The roots, stem bases, and leaves were examined for fungal pathogens. Symptomatic plant parts were washed with water and, afterwards, surface-sterilized. For this purpose, the samples were placed in 1% NaClOH and 70% ethanol, each for 1 min, and then rinsed in sterile water three times. The plant tissue was dried on sterile filter paper. Sections of plant tissue were placed on Petri dishes with potato dextrose agar (PDA) and incubated at 20 °C for 4–7 days. Afterwards, subcultures were prepared on PDA medium and incubated at 20 °C for 14–28 days. The pure fungal isolates were flooded with sterile water and gently rubbed with a scalpel. The resulting suspensions were washed with sterile water three times, and the fungal mycelium was mechanically digested using a Retsch MM 301 mixer mill. For the mechanical digestion of the mycelium, metal beads were placed in the vessel and homogenized for 4 min with an oscillation frequency of 30 1/s.
Fungal DNA was isolated using an E.Z.N.A Fungal DNA Mini Kit (Omega-Bio-Tek, Norcross, GA, USA) according to the manufacturer’s instructions. The fungal internal transcribed spacer (ITS) region was amplified using the primers ITS4 and ITS1F [37,38]). For PCR, an AccuStar Master Mix (Quantabio, Beverly, MA, USA) was used. The PCR products were purified using Ron’s PCR Pure Mini Kit (Bioron, Römerberg, Germany), according to the manufacturer’s instructions. The DNA concentration was quantified with a NanoDrop spectrophotometer (Life Technologies GmbH, Darmstadt, Germany). The sequencing was performed by Eurofins Genomic, Ebersberg, Germany. Fungal identification was carried out by BLAST 2.15.0 searches [39] using the ITS sequences and based on holotype material when needed.

2.3. Nematode Isolation and Identification

The soil samples taken from the root zone of the plants were used to determine nematode infestation. The methods for the extraction of plant parasitic nematodes from plant and soil samples are currently included in [40], Diagnostic PM 7/119 (1) nematode extraction. The extraction of free-living nematodes was carried out using the Oostenbrink dish (modified Baermann funnel) method [40]. The nematodes were identified to the genus level, and the infestation intensity was recorded as the number of nematodes per 100 mL of soil.

2.4. Soil and Plant Nutrient Analyses

Nutrient analyses were carried out by certified laboratories on air-dried soil samples (0–10 cm depth) and oven-dried (55 °C) biomass samples (in each case, bulk samples from throughout the area). For the soil samples, the double-lactate method was used for P, K, and Mg according to the VDLUFA methods A 6.2.1.2 and A 6.2.4.2 [41] and the CAT method according to the VDLUFA methods A 6.4.1 and A 13.1.1 for B, Cu, Fe, Mn, and Zn [41]. The soil texture was assessed via a finger test. The soil pH was determined in a solution of CaCl2. The biomass contents of P, K, Mg, B, Cu, Fe, Mn, and Zn were determined according to VDLUFA methods 10.8.1.2 and 10.8.2 (for B) [42].

2.5. Mycorrhizal Colonization and Nodule Formation with Rhizobia

To investigate the mycorrhizal colonization of the fine roots, the roots were cleaned under running tap water, then the nodules were counted and partly cut, and, subsequently, the fine roots were cut into 1 cm pieces, bleached with 10% KOH, and strained with Chlorazol Black E. The stained root sections were analyzed microscopically for their percentage of mycorrhizal root length [43]. To record nodule formation by rhizobia, the washed root systems were quantified under a stereomicroscope using a handpiece counter (Roth). At the same time, the vitality (color of the sections), distribution, and size of the nodules were evaluated.

2.6. Data Analysis

A linear mixed model was applied to evaluate the relation between plant growth and different soil treatments, defining soil treatments as fixed effects and pots as random effects. Skewed distributions of length and weight were normalized using natural logarithmic transformations. The fulfillment of the model’s requirements was tested using the DHARMa package (version 0.4.6) [44]. DHARMa applies the KS test for the residual’s normal distribution [45] and the Levene test for residual’s homogeneity of variance [46]. A multiple comparison Dunnett test was used to compare each treatment to the control group and identify possible significant differences. A p value of <0.05 was considered significant. Statistical analyses were performed using R statistical Software (version 4.1.1) [47] embedded in the RStudio environment (1.4.17.17).

3. Results

3.1. Bioassay

Red clover cultivated in the soil from the three sites exhibited different levels of yield depression. The shoot fresh mass of the plants growing in non-treated soil from site 3 was 4.9-fold and 5.5-fold heavier than those from sites 1 and 2, respectively (Figure 1). Site 3 showed also the highest plants (Figure 2) and the largest shoot and root weight, both fresh and dry, of all the sites for each treatment (Figure 1 and Figure 3).
The soil sterilization treatment was successful and improved the growth of red clover at all the sites. Overall, the plants from the sterilized soil were higher than those from the other treatments (Figure 2) and exhibited heavier shoot and root mass, both fresh and dry (Figure 1 and Figure 3). This was especially remarkable in the soil from site 2, where the difference between the non-treated and sterilized soil was statistically significant (p < 0.05) for all the measured parameters. Although soil sterilization improved the development of red clover in the soil from site 1, the difference between the non-treated and sterilization treatment was not statistically significant, whereas in the soil from site 3, a statistically significant difference was found only for the root dry weight (p < 0.05) (Figure 3).
The supplement with a nutrient solution resulted in a slight enhancement in plant growth at site 1 and significantly hampered growth in the plants from site 3 (p < 0.05, Figure 1, Figure 2 and Figure 3). The positive effect of nutrient addition in the soil from site 1 was observed only on shoot growth, both length and mass, while the impaired growth at site 3 affected the whole plant. The nutrient solution treatment also had a negative impact on the shoot and root mass of red clover from site 2 (Figure 1 and Figure 3), which were marginally lighter, but not significantly, than those growing in non-treated soil.
A minor positive effect of activated charcoal was found on both the shoot and root growth in the soil from site 1 (Figure 1 and Figure 3). At site 3 soil, the shoot length and mass were significantly lower in the activated charcoal treatments than in comparison to the non-treated soil (p < 0.05). In the soils from site 2 and site 3, both the root fresh and dry mass were lower in the activated charcoal treatments than in the non-treated soil. At site 3 this difference was significant for the root dry mass (p < 0.05, Figure 3).

3.2. Plant and Soil Field Screening

Pathogens of the Ascochyta complex (Didymella pinodella var. Phoma pinodella and Ascochyta medicaginicola var. Phoma medicaginis) were found at all the sites (Table 2). The plants exhibited pronounced symptoms of foot rot, as well as brown to black lesions and scorch on the leaves and stems on red clover and alfalfa, indicating a moderate level of infestation. At sites 1 and 3, low levels of infections with Alternaria infectoria were also detected. The lower leaves showed circular black spots (Figure 4).
Additionally, there was evidence of clover rust (Uromyces trifolii-repentis var. fallans) at site 3; however, the infestation level was low. Nevertheless, red-brown uredospore deposits were visible on the leaves of some red clover plants.
There was no notable nematode infestation at site 1. Conversely, elevated levels of the genera Heterodera spp. and Pratylenchus spp. were found at site 2, the last one also being present in increased levels at site 3.
The degree of mycorrhization was in the low range for sites 1 and 3, while it was in the upper range for site 2. Sites 2 and 3 showed considerably higher numbers of nodules compared to site 1. At Site 1, deficiencies in iron, manganese, and zinc were found; at site 2 there were deficiencies in phosphorus and copper. The nutrient values of site 3 were within the normal range (Table 2).

4. Discussion

4.1. The Use of Nutrient Solution for Detecting Nutrient Deficiencies

The supplementation of nutrients could provide an indication of a nutrient deficiency in the soil [32]. The soil from site 1 presented a shortage of three micronutrients, namely, iron, zinc, and manganese. The requirement for iron in leguminous plants is especially high due to the symbiotic association with nitrogen-fixing bacteria, and its deficiency can impact the plant and the rhizobacteria individually, as well as their symbiosis [50,51]. Therefore, the observed slight enhancement on shoot growth could be attributed to the increase in the level of iron in the soil through the nutrient solution application. It is interesting to note that, in a preliminary experiment (Figure A1 and Figure A2), the addition of a nutrient solution and amendment with activated charcoal had a positive impact on plants growing in the soil from site 1, as in the present study.
One unanticipated result of this study was the significant stunted growth of plants cultivated in the soil treated with a nutrient solution from site 3. Knop’s solution contains nitrate, which can reduce the nodulation and competitiveness of legumes [52,53]. Nitrogen availability at the sites was not tested, since nitrogen nutrition usually is not a problem for legumes. However, site 3 had only recently been converted to organic farming. Therefore, it is possible that nitrogen availability in this soil was quite high and that the further increase by nitrate application inhibited the growth of red clover. Other possible explanations are related to nutrient interactions. There were signs of nutrient imbalances at site 3, which could have been aggravated by the application of Knop’s solution. While the soil boron contents were sufficient at site 3, the biomass contents were low, a possible explanation being inhibited absorption because of high abundance of zinc at this site [54]. Another antagonist of boron absorption is phosphate, which was applied via Knop’s solution, maybe causing further boron deficiency in the plants. In the samples from site 3, the analyses of soil nutrients also showed high levels of potassium, which was additionally provided weekly through fertilization. Elevated levels of potassium in the soil experiment could interfere with magnesium uptake by plants [5].

4.2. The Effect of Activated Charcoal on Plant Growth

Several studies have shown different effects of activated charcoal on plant growth. Usually, a positive impact on plant development has been attributed to absorption of allelopathic compounds in the soil [55,56].
Previous studies have reported the impact of allelochemicals from other species on the growth of forage legumes, indicating that hindered growth caused by allelopathy is a common phenomenon in forage legumes. For example, the release of self-inhibitory compounds by white clover (Trifolium repens L.) has been considered as a possible cause for clover decline in organic farming crops [57], whereas extracts from tall fescue (Festuca arundinacea Schreb.) have been shown to inhibit the seedling growth of red clover [58]. However, the plants grown in the soil of site 3, treated with activated charcoal, showed inhibited growth. One possible reason for the growth-inhibiting effect could be that activated charcoal may also be able to bind signaling substances that are important for plant–microbe interactions [55]. Suppressing beneficial plant–microbe interactions can reduce plant growth [59]. In addition, activated charcoal reduces the concentrations of organic molecules used by microbes as substrates or as signals to promote their growth, which may also reduce microbial activity [60]. Finally, activated carbon can reduce the rate of N and P mineralization and thereby reduce the availability of nutrients to plants [61].

4.3. The Potential of Sterilization for Detecting Detrimental Organisms

In all the tested soils, the sterilization treatment improved the growth of plants, indicating the presence of harmful organisms in the soil as the main reason for clover yield depression. This improvement was statistically significant for all the measured parameters at site 2 and for root dry weight at site 3, while no significant difference was found at site 1. The results of the diagnostic test are supported by the plant and soil screening, which reported pathogenic fungi at all the sites and plant-parasitic nematodes at two sites. These findings reflect those of [32], who also observed a significantly higher weight for peas grown in sterilized soil compared to those in untreated soil in most soil samples, suggesting biotic factors as the primary cause of unexplained legume depression in arable organic farming in Germany. The presence of plant-parasitic nematodes and fungi in leguminous crops is frequent. This is exemplified in the work undertaken by [62], in which the occurrence of plant pathogenic nematodes and fungi was observed in more than 40 areas of forage legumes cultivation in Germany.
Several phytoparasitic nematodes and plant pathogenic fungal species are associated with diseases that affect the growth and persistence of clover and alfalfa [7,11], which could explain the enhanced growth of red clover after a possible pathogen inactivation by soil sterilization in this study. The species of the genus Pratylenchus spp. present at moderate levels at sites 2 and 3 (110 and 153/per 100 mL of soil, respectively) are common pathogens in forage legume crops and have been shown to reduce leaf yield in red clover and alfalfa [12,63]. The infestation with cyst nematodes Heterodera spp. at site 2 was high (461/per 100 mL of soil). According to [12], Heterodera trifolii is the predominant cyst nematode impacting forage legume cultivation in the world. An example of this is the study carried out by [64], in which the presence of clover cyst nematode (H. trifolii) was reported in 77% of the studied sites of clover cultivation in Australia.
In the case of fungal infections, studies have shown that foliar and stem diseases caused by Didymella pinodella and Uromyces trifolii-repentis, present at sites two and three, respectively, can result in a decrease in yield and nutritive value and persistence of red clover [65,66]. Previous studies have noted the importance of the pathogen Ascochyta medicaginicola to forage legume cultivation. Ascochyta black stem is considered one of the most important sicknesses that affects alfalfa in Europe, reducing its crop productivity and quality [67,68,69]. The damage potential of the fungi reported in the results does not affect forage legumes exclusively. A. medicaginicola and D. pinodella, together with Fusarium spp., play a major role in crop depression of other legumes grown in Germany, such as peas (Pisum sativum) and faba beans (Vicia faba) [70,71,72].
A possible side effect of the heating during sterilization is a higher nutrient availability, especially evident for phosphorus [73,74]. This effect might have contributed to the increased growth after sterilization, at least for sites with nutrient deficiencies.

4.4. Multifactorial Effects Causing Legume Decline

Several factors causing red clover decline can simultaneously play a role in legume depression. Consequently, it is not surprising that the diagnostic test carried out in the soil from site 1 showed an improvement on the growth of red clover in more than one treatment; the differences were not significant, though. Overall, the plants cultivated in the soil from site 1 were smaller than those from the other two sites. It seems possible that in addition to the already mentioned factors of plant pathogens, nutrient deficiency, and the presence of potentially phytotoxic compounds in the soil, other factors play a role in clover decline at site 1. Silty loams, as present at site 1, are prone to soil compaction [75], which can lead to reduction in root growth and aboveground biomass in red clover [76]. Therefore, soil compaction might have an influence on the hampered growth at this site. While a strong form of soil compaction caused by heavy equipment in the field is not to be expected in a pot experiment, the soil texture of silty loam might still have led to a reduction in soil pore volume and, thus, to stunted root development compared to the other soils. Furthermore, from the three study sites, C. album was found exclusively in the soil from site 1, and it grew in all the treatments. Thus, competition could have an influence on red clover growth in pots with soil from site 1. Additionally, suboptimal symbiosis formations have been found in the soil from site 1; therefore, the low mycorrhization level, as well as the low number of root nodules could be another factor contributing to the limited performance of clover crops in the soil of site 1.

5. Conclusions

The results of this study indicate that the diagnostic test successfully identified the presence of noxious organisms in the soil as the primary cause for limited clover performance at the three studied organic farms. Additionally, the diagnostic test can also indicate nutrient deficiencies or toxic compounds in the soil, though the corresponding results were less clear in this study. These findings suggest that the diagnostic tool is a reliable tool to detect the presence of detrimental organisms in the soil as a possible cause for clover decline and might be an indicator for nutrient deficiencies. However, further research with a more optimal nutrient solution for forage legumes and trials with other forage legumes and soil types would help to improve the diagnostic test. Overall, our results show that the diagnostic test is a promising tool, and it provides farmers and advisors with a low-cost method to detect site-specific causes of clover decline and thus improve the management of forage legume crops.

Author Contributions

Conceptualization, B.F.M. and A.K.; data curation, A.K. and C.S.; funding acquisition, I.J., J.M., C.B., and C.S.; investigation, A.K.; methodology, B.F.M., A.K., K.S., and U.H.; project administration, I.J., J.M., C.B., and C.S.; supervision, I.J., J.M., and C.S.; validation, B.F.M. and A.K.; writing—original draft, B.F.M.; writing—review and editing, A.K., K.S., I.J., U.H., and C.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Federal Ministry of Food and Agriculture (BMEL), based on a decision of the parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE) under the federal program Protein Crop Strategy (Grant No. 2818EPS039).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

This work is part of the project TriSick, funded by the Federal Ministry of Food and Agriculture (BMEL), based on a decision of the parliament of the Federal Republic of Germany via the Federal Office for Agriculture and Food (BLE) under the federal program Protein Crop Strategy (Grant No. 2818EPS039). The authors thank Ingolf Gliege and Rosa Minderlen for their help in the greenhouse and Timo Seibert for assistance with soil sample collection.

Conflicts of Interest

Irene Jacob is employed by the company Öko-BeratungsGesellschaft mbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Appendix A

Appendix A.1

Agronomy 15 01566 g0a1
Figure A1. Plant length (cm) of red clover grown in soil of site 1 (preliminary experiment). The soils were non-treated, treated with charcoal or nutrients, and heat-sterilized. For the preliminary test, an ANOVA followed by a Tukey HSD post-hoc test (α = 5%) was conducted. Different letters (a–c) indicate significant differences (p < 0.05).
Figure A1. Plant length (cm) of red clover grown in soil of site 1 (preliminary experiment). The soils were non-treated, treated with charcoal or nutrients, and heat-sterilized. For the preliminary test, an ANOVA followed by a Tukey HSD post-hoc test (α = 5%) was conducted. Different letters (a–c) indicate significant differences (p < 0.05).
Agronomy 15 01566 g0a1

Appendix A.2

Agronomy 15 01566 g0a2
Figure A2. Plant weight (g) of red clover grown in soil of site 1 (preliminary experiment). The soils were non-treated, treated with charcoal or nutrients, and heat-sterilized. For the preliminary test, an ANOVA followed by a Tukey HSD post-hoc test (α = 5%) was conducted. Different letters (a and b) indicate significant differences (p < 0.05).
Figure A2. Plant weight (g) of red clover grown in soil of site 1 (preliminary experiment). The soils were non-treated, treated with charcoal or nutrients, and heat-sterilized. For the preliminary test, an ANOVA followed by a Tukey HSD post-hoc test (α = 5%) was conducted. Different letters (a and b) indicate significant differences (p < 0.05).
Agronomy 15 01566 g0a2

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Agronomy 15 01566 g001
Figure 1. The impact of the diagnostic test on fresh weight of red clover grown in soil from the three sites. The soils were non-treated, treated with nutrients or charcoal, and heat-sterilized. Shown are the means and standard deviations (n = 20). Treatment is statistically significantly different than non-treated soil for this site (Dunett, * p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 1. The impact of the diagnostic test on fresh weight of red clover grown in soil from the three sites. The soils were non-treated, treated with nutrients or charcoal, and heat-sterilized. Shown are the means and standard deviations (n = 20). Treatment is statistically significantly different than non-treated soil for this site (Dunett, * p < 0.05, ** p < 0.01, *** p < 0.001).
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Figure 2. The impact of the diagnostic test on the length of red clover grown in soil from the three sites. The soils were non-treated, treated with nutrients or charcoal, and heat-sterilized. Shown are the means and standard deviations (n = 20). Treatment is statistically significantly different than non-treated soil for this site (Dunnett, * p < 0.05, *** p < 0.001).
Figure 2. The impact of the diagnostic test on the length of red clover grown in soil from the three sites. The soils were non-treated, treated with nutrients or charcoal, and heat-sterilized. Shown are the means and standard deviations (n = 20). Treatment is statistically significantly different than non-treated soil for this site (Dunnett, * p < 0.05, *** p < 0.001).
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Figure 3. The impact of the diagnostic test on dry weight of red clover grown in soil from the three sites. The soils were non-treated, treated with nutrients or charcoal, and heat-sterilized. Shown are the means and standard deviations (n = 20). Treatment is statistically significantly different than non-treated soil for this site (Dunett, * p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 3. The impact of the diagnostic test on dry weight of red clover grown in soil from the three sites. The soils were non-treated, treated with nutrients or charcoal, and heat-sterilized. Shown are the means and standard deviations (n = 20). Treatment is statistically significantly different than non-treated soil for this site (Dunett, * p < 0.05, ** p < 0.01, *** p < 0.001).
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Figure 4. Symptoms of fungal disease on red clover in field: (A,B) Spring black stem disease (D. pinodella var. P. pinodella), (C) spring black stem disease (A. medicaginicola), and (D) black spot disease (A. infektoria).
Figure 4. Symptoms of fungal disease on red clover in field: (A,B) Spring black stem disease (D. pinodella var. P. pinodella), (C) spring black stem disease (A. medicaginicola), and (D) black spot disease (A. infektoria).
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Table 1. Soil and location characteristics.
Table 1. Soil and location characteristics.
SiteCoordinatesCropPrevious CropMonth of SamplingSoil Texture ClasspHMean Annual Temperature (°C)Annual Precipitation (mm)Precipitation April to June 2023 (mm)
Site 149.602433, 8.096633Medicago sativaspring barleyJunesilty loam7.412.255090
Site 254.084617, 12.25405Trifolium pratensespring wheatMayloamy sand5.810.162763
Site 353.648433, 13.3698Trifolium pratenseunknownMayloamy sand6.210.7500130
Table 2. Nematodes, fungal pathogens, and nutrient deficiency reported in plant and soil screening. Nutrient deficiency was defined based on the soil contents according to [48,49] (for iron) (content classes A or B), marked with x. If there was no deficiency in the soil, but the biomass content was below the normal range according to [48], this is marked with (x). No nutrient deficieny is marked with -.
Table 2. Nematodes, fungal pathogens, and nutrient deficiency reported in plant and soil screening. Nutrient deficiency was defined based on the soil contents according to [48,49] (for iron) (content classes A or B), marked with x. If there was no deficiency in the soil, but the biomass content was below the normal range according to [48], this is marked with (x). No nutrient deficieny is marked with -.
Site 1Site 2Site 3
Nematode (number per 100 mL soil)
Pratylenchus spp.
Heterodera spp.		11
-		110
461		153
-
Fungi (Infestation intensity (%))
Ascochyta medicaginicola50--
Alternaria infectoria40-10
Didymella pinodella-3020
Uromyces trifolii-repentis--5
Nutrient deficiency
Phosphorus-x-
Potassium---
Magnesium---
Boron(x)-(x)
Copper-x-
Ironx--
Manganesex--
Zincx(x)
Symbiotic interaction
Number of nodules per plant1.633.873.2
Mycorrhization degree10.132.219.1
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Menezes, B.F.; Kühnl, A.; Steinfurth, K.; Hakl, U.; Jacob, I.; Müller, J.; Baum, C.; Struck, C. A Differential Diagnostic Tool for Identifying the Causes of Clover Decline. Agronomy 2025, 15, 1566. https://doi.org/10.3390/agronomy15071566

AMA Style

Menezes BF, Kühnl A, Steinfurth K, Hakl U, Jacob I, Müller J, Baum C, Struck C. A Differential Diagnostic Tool for Identifying the Causes of Clover Decline. Agronomy. 2025; 15(7):1566. https://doi.org/10.3390/agronomy15071566

Chicago/Turabian Style

Menezes, Beatrice Francisco, Annika Kühnl, Kristin Steinfurth, Ulrike Hakl, Irene Jacob, Jürgen Müller, Christel Baum, and Christine Struck. 2025. "A Differential Diagnostic Tool for Identifying the Causes of Clover Decline" Agronomy 15, no. 7: 1566. https://doi.org/10.3390/agronomy15071566

APA Style

Menezes, B. F., Kühnl, A., Steinfurth, K., Hakl, U., Jacob, I., Müller, J., Baum, C., & Struck, C. (2025). A Differential Diagnostic Tool for Identifying the Causes of Clover Decline. Agronomy, 15(7), 1566. https://doi.org/10.3390/agronomy15071566

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