Introduction

Rapeseed (Brassica napus L.) is an important cash crop used for edible oil and biofuel production, as well as a break crop between cereal crop rotations (Friedt et al., 2018). Clubroot disease, caused by the protist Plasmodiophora brassicae Woronin, is one of the top 10 diseases threatening rapeseed production and yields in the five major rapeseed growing zones, including Australia, Canada, China, Europe, and India (Javed et al., 2023; Zamani-Noor et al., 2022a). Due to the absence of effective chemical treatments against the pathogen, clubroot demands precise cultural management. This involves practices such as field sanitation, extended crop rotations, strategic fertilizer application, and prioritizing cultivar resistance (Struck et al., 2022).

Currently, clubroot-resistant cultivars are the most successful strategy for rapeseed growers against clubroot losses (Diederichsen et al., 2014; Struck et al., 2022). In resistant cultivars, clubroot infection and development are significantly slower than in susceptible cultivars, which decrease the pathogen propagation inside the infected roots and can lower the pathogen load in the soil (Czajka et al., 2020; Zamani-Noor et al., 2021). Nevertheless, the clubroot resistance in all current resistant rapeseed cultivars relies on one major gene resistance that can be overcome when new aggressive pathotypes evolve (Czajka et al., 2020; Řičařová et al., 2016; Zamani-Noor, 2017; Zamani-Noor et al., 2022a). Additionally, the presence of susceptible Brassica weeds and volunteer rapeseeds in fields contributes to the buildup of P. brassicae inoculum over time leading to clubroot outbreaks in succeeding rapeseed cultivars (Botero-Ramirez et al., 2021; Hwang et al., 2012; Wallenhammar et al., 2021; Zamani-Noor & Rodemann, 2018; Zamani-Noor, et al., 2022b).

The demand for biocontrol options in integrated crop management has increased in recent years, partly due to the public concern about the effect of chemical plant protection products on the environment as well as human health (Köhl et al., 2019; Latz et al., 2018). While a higher usage of such biocontrol options is desirable, one common challenge is the inconsistent efficacy of many biocontrol strains under high pathogen pressure which demands further investigation into the potential use of these strains as well as the search for additional biocontrol candidates.

Several studies showed good efficacy against clubroot disease when bacteria or fungi were applied as a biocontrol agent against the disease (Auer & Ludwig-Müller, 2023). Effective microbes include bacterial strains from Bacillus spp., Lysobacter spp. and Streptomyces spp. (Arif et al., 2021; Fu et al., 2018; Hu et al., 2021; Peng et al., 2011; Wang et al., 2012; Zhou et al., 2014; Zhu et al., 2020) and fungal strains from Clonostachys spp. (formerly Gliocladium), Cladophialophora spp. (formerly Heteroconium), Phoma spp. and Trichoderma spp. (Arie et al., 1998; Cheah & Page, 1997; Lahlali & Peng, 2014; Narisawa et al., 2005; Yu et al., 2015). Here we investigated the endophytic fungus Acremonium alternatum which can reduce clubroot symptoms in Arabidopsis thaliana, Chinese cabbage, and rapeseed plants (Auer & Ludwig-Müller, 2014; Doan et al., 2010; Jäschke et al., 2010). A. alternatum colonizes whole cruciferous plants through roots and could be detected with quantitative PCR in leaves of Chinese cabbage within four days after inoculation (Auer & Ludwig-Müller, 2015; Doan et al., 2010; Jäschke et al., 2010). So far, it remains uncertain whether this fungus exhibits antibiosis or produces compounds that adversely affect P. brassicae. However, A. alternatum did not have a negative effect on the spore germination of P. brassicae (Jäschke et al., 2010). Instead, it appears to decelerate the progression of clubroot disease (Auer & Ludwig-Müller, 2014).

Based on previous studies, A. alternatum has shown promising potential in reducing clubroot disease symptoms by up to 20% in the rapeseed cultivar Ability, but not in Visby, in greenhouse trials (Auer & Ludwig-Müller, 2014). However, the performance of this fungus under field-like conditions or with other rapeseed cultivars remains unknown. Therefore, in order to gain further insight into whether a potential use of this species in agriculture could be worthwhile, we conducted additional trials incorporating different growing conditions, field isolates of P. brassicae, and additional rapeseed cultivars. We aimed to emulate varying disease pressures in order to investigate the scenarios in which the application of A. alternatum could be advantageous, as well as those where the use of this biocontrol agent might not yield optimal results. To encompass a range of field situations, we employed a spectrum of resting spore concentrations of P. brassicae to simulate disease pressures categorized as low (103–105 spores mL−1 inoculum or g−1 soil), average (106), or high (107). The principal objective of this study was to evaluate the effectiveness of A. alternatum in mitigating clubroot disease induced by different pathotypes of P. brassicae, characterized by varying levels of virulence. This assessment was conducted on one clubroot-resistant and four clubroot-susceptible rapeseed cultivars.

Material and methods

To assess the efficacy of the biological control agent A. alternatum in controlling clubroot disease across diverse environmental conditions, multiple experiments were conducted at two different research institutes: TU Dresden, Department of Plant Physiology, Faculty of Biology, Germany (TUD), and Julius Kühn-Institute, Institute for Plant Protection in Field Crops and Grassland, Braunschweig, Germany (JKI).

Plants, microorganisms and inoculum production

In the current study, two winter rapeseed cultivars susceptible to clubroot, namely Visby and Jenifer, and two spring rapeseed cultivars susceptible to clubroot, Ability and Jumbo, were selected from the German Plant Variety Catalogue in 2016. Additionally, a clubroot-resistant winter rapeseed cultivar, Mentor, was included. This selection aimed to evaluate the efficacy of the biological control treatment across a spectrum of cultivar susceptibility/resistance.

Seeds of cvs. Visby, Ability and Jumbo were obtained from Sächsisches Landesamt für Umwelt, Landwirtschaft und Geologie, while seeds of cv. Jenifer were provided by Bayer Crop Science. Additionally, seeds of clubroot-resistant rapeseed cv. Mentor were provided by Norddeutsche Pflanzenzucht Hans-Georg Lembke KG.

Plasmodiophora brassicae single spore isolate SSI e3 (Fähling et al., 2003) and two field isolates, P1 and P1 ( +), were used in the current study. The SSI e3 isolate was propagated on Chinese cabbage (Brassica rapa ssp. pekinensis) and the resulting galls were stored at -20 °C until use.

Two field isolates were chosen based on their virulence, as described in Zamani-Noor (2017). The aggressive isolate P1 was identified as pathotype 1 according to the classification system of Somé et al. (1996), or 16/31/12 using the European Clubroot Differential (ECD) set (Buczacki et al., 1975). The second isolate P1 ( +), a highly aggressive pathotype that could overcome the resistance in clubroot-resistant cultivars such as Mentor, was classified as 17/31/31 on the ECD set or pathotype 1 using the system of Somé et al. (1996). The P1 isolate was originally obtained from a naturally infested rapeseed field in Schleswig–Holstein, Germany in 2012 and the P1 ( +) isolate collected from a field in Hessen, Germany in 2013 (Zamani-Noor, 2017). Both isolates were preserved at -20 °C as frozen galls until use.

The clubroot inoculum was prepared following the protocol outlined by Zamani-Noor (2017). Briefly, the resting spores of each P. brassicae isolate were released from frozen clubbed roots by homogenizing 100 g clubbed roots in 200 mL of sterile deionized water in a laboratory blender (Vital Mixer Pro, Hollenstedt, Germany) running at 20,000 rpm for 5 min. The resulting solution was filtered multiple times through fine layers of cheesecloth until it was devoid of any plant debris. The quantity of resting spores was determined by using a Fuchs haemocytometer slide (Hecht-Assistent, Sondheim, Germany) under a light microscope. Resting spore suspensions were stored in microcentrifuge tubes at -20 °C at a concentration of 1 × 109 spores mL−1 for one year (field isolates) or longer (single spore isolate SSI e3). We ensured the viability of resting spores by routinely inoculating susceptible Chinese cabbage varieties with the stored spores and assessing disease symptoms. All spore suspensions used here were confirmed to be still viable. Prior to each experiment, the resting spore suspensions were thawed and appropriately diluted with buffer (KH2PO4, pH 5.5; pot experiments) or water (raised-bed containers), resulting in concentrations ranging from 1 × 103, 1 × 104, 1 × 105, 1 × 106, to 1 × 107 spores mL−1.

Acremonium alternatum strain MUCL 12012 (Mycotheque de L´Universite Catholique de Louvain, Belgium; Jäschke et al., 2010) was used for all experiments. Conidia of this strain were propagated in Erlenmeyer flasks filled with potato dextrose broth (Carl Roth, Germany), cultivated on a rotary shaker at 150 rpm at 26 °C in darkness. Three-week-old cultures were filtered through polyester gauze with a pore size of 20 µm (neoLab Migge GmbH, Germany) to harvest the conidia. The conidia suspension was centrifuged for 10 min at 5000 × g, after which the supernatant, along with the medium, was discarded. The conidia suspension was subsequently washed with sterile tap water three times. Conidia were counted with a haemocytometer under the light microscope. The concentration was adjusted to 1 × 109 conidia mL−1 with water and the conidia suspension was stored at – 20 °C for up to 1 year. The viability of the conidia was assessed several times per year by microscopy and preparation of new liquid cultures with the stored inoculum.

Experimental trials for evaluating the efficacy of A. alternatum in controlling clubroot

Pot experiments

The seeds of rapeseed cultivars Ability, Jenifer, Jumbo and Visby were pre-germinated over a period of 2–3 days in glass Petri dishes filled with moist filter paper (using sterile tap water) until radicle emergence (BBCH-scale 05; Lancashire et al., 1991). A standard soil (Einheitserde Type P, pH 5.8; Hermina Samen, Germany) was mixed with sand (Sahara Spielsand; Hornbach Germany) at a 4:1 soil:sand ratio. The mixture was steam-sterilized for 120 min at 100 °C. Subsequently, plastic pots (11 × 11 cm diameter, 12 cm height) were filled with the soil-sand mixture to the top. Within each pot, two seedlings were placed at 1 cm depth and covered with soil. To facilitate watering, the pots were placed in plastic trays and kept under greenhouse conditions at 27/18 °C, 20–70% relative humidity, and a 16/8 h day/night regime with a light intensity of 150 µmol m−2 s−1. Plants from each treatment were grouped together in a single tray and watered from below. During the first four weeks, while the plants were still small, these trays were rotated weekly in the greenhouse to ensure comparable conditions for all groups. The rotation was discontinued once the plants grew larger and became intertwined to prevent potential damage to leaves or stems. A minimum of 30 plants was used for each treatment group.

Inoculation with P. brassicae resting spore solutions and A. alternatum conidia solution was carried out using KH2PO4 buffer (50 mM, pH 5.5; Carl Roth). The pathogen resting spores and endophyte conidia were thawed and added to the buffer either alone or together at the desired end concentration of P. brassicae spores and A. alternatum conidia per mL, according to the conidia and spore counts that were made prior to storing the inoculum. The thoroughly mixed solution was then pipetted beside the hypocotyl of each seedling directly onto the soil. In each treatment, a 2 mL suspension was used and KH2PO4 buffer alone was utilized as a mock inoculation.

Experiments under semi-field conditions

The clubroot-resistant rapeseed cv. Mentor and the clubroot susceptible cv. Visby were used in this study. To stimulate crop growth under field conditions, the seeds of both cultivars were sown in 2 cm deep holes within portable raised-bed containers (200 × 100 × 25 cm) containing a mix of potting soil, sand, and peat (5:1:1; pH < 6.5; FloraSelf®, Germany). Seeds were sown at a 7.5 cm spacing in a row spaced 11.5 cm apart from each other. In total, 17–20 seeds per row were sown; seedlings were thinned on emergence to leave 13 plants, and 5 rows per rapeseed cultivar (in total 65 plants). Plants were grown under greenhouse conditions at 20/16 °C, 70% relative humidity, and a 16/8 h day/night regime with a light intensity of 150-µmol m−2 s−1. Plants were inoculated at growth stages 11–12 (BBCH-scale; young seedling). Adequate irrigation was provided prior to inoculation, which involved watering the plants sufficiently to maintain moist soil conditions, ensuring that the soil was evenly moist but not waterlogged or saturated.

In this experiment, plants were inoculated with the same isolate of P. brassicae-P1 or P1 ( +) at concentrations of 105, 106, or 107 spores mL−1. The preparation of the inoculum followed the same procedure as previously described. Inoculations were conducted by first injecting 2 × 1 mL of spore suspension of A. alternatum (1 × 107 spores mL−1) into the soil at two locations near the root zone of each seedling at a depth of around 2 cm. Subsequently, P. brassicae isolates with varying concentrations were inoculated into the soil separately, one day after the initial A. alternatum inoculation, following the same procedure. Control plants were mock-inoculated with water. The plants were not irrigated for 72 h post inoculation to avoid washing the inoculum from the root area. Additionally, the temperature was maintained at 24 °C to create optimal conditions for infection. Following this period, plants were grown in greenhouse conditions as described above and irrigated every other day to maintain soil moisture, without reaching water-saturation levels. Throughout the experiment, the plants were maintained without the addition of any fertilizer.

Experiments under semi-field conditions with clubroot infected soil

A. alternatum and P. brassicae spore suspensions were prepared as described before.

The greenhouse assays were conducted using large greenhouse trays (200 × 100 × 25 cm), each containing a 160 kg mix of potting soil, sand, and peat (Einheitserde, 5:1:1; pH < 6.5; FloraSelf®, Germany). The spore suspension of P. brassicae-isolate P1 was incorporated into the soil at the appropriate proportion to achieve an inoculum density of 106 and 107 resting spores per g−1 soil. Likewise, the conidia suspension of A. alternatum was incorporated into the soil at the same time at the appropriate proportion to achieve an inoculum density of 107 resting spores per g−1 soil.

One day after soil inoculation, the seeds of two rapeseed cultivars (cv. Visby and cv. Mentor) were sown in 2 cm deep holes. In total, 12 rows, each comprising 13 plants, were sown. Rows were alternately sown with seeds of the clubroot susceptible and resistant cultivars to ensure a balanced and systematic comparison of the two cultivar types under identical environmental conditions. This arrangement helps minimize potential variations in soil properties, microclimate, and inoculum distribution within the trays, allowing for more accurate and reliable assessment of the differential responses of the susceptible and resistant cultivars to the treatments. Plants were grown under the same conditions as the previous experiment for eight weeks. Throughout the experiment, plants were irrigated daily to maintain soil moisture but the soil was not water-saturated.

Clubroot disease assessment

Root samples were collected 45 days post inoculation (dpi) for pot experiments and semi-field experiments, and 56 days after sowing (das) for semi-field experiments conducted with infested soil. Subsequently, each sample was thoroughly washed under tap water to remove soil particles and was visually evaluated for clubroot severity. In the pot experiments at TU Dresden, we used a scale ranging from 0 (no symptoms) to 4 (severe swelling or decay of roots), according to the method described by Yoshikawa et al. (1977). In the context of clubroot research at TU Dresden, disease severity in the model plant Arabidopsis thaliana is assessed using a scale ranging from 0 to 4, as established by Klewer et al. (2001). To facilitate comparisons between Arabidopsis and crop plants, a similar rating scale was adopted. Conversely, research conducted at JKI focused on agriculture-related studies and utilized a scale ranging from 0 to 3, following the methodology outlined by Kuginuki et al. (1999). This scale categorizes disease severity as: 0 for no galling, 1 for a few small galls, 2 for moderate galling, and 3 for severe galling.

Both rating systems yield a Disease Severity Index (DSI), which is standardized across laboratories, ensuring comparability of results. Each disease category is assessed based on its impact and severity on the plant.

The DSI was calculated for each treatment using the following formula:

$$\text{Disease Severity Index}=\frac{\sum \left(\text{Class frequency}\times \text{score of rating class}\right)}{\text{Total number of plants}\times \text{no}.\text{classes with symptoms}}\times 100$$

Efficacy (%) of A. alternatum treatment for reducing clubroot symptoms was calculated using the following formula:

$$\mathrm{Efficacy }\left(\mathrm{\%}\right)=100-\left(\frac{\mathrm{AaPb}}{\mathrm{Pb}}\times 100\right)$$

AaPb and Pb in the equation represent the treatment group co-inoculated with A. alternatum and the group infected with only P. brassicae, respectively.

Subsequent to disease rating, the roots were excised from the shoots, and the shoot weight (in grams) was measured in experiments conducted with raised-bed containers utilizing the two different inoculation methods (soil inoculation with pipettes and infested soil). This assessment was carried out to evaluate the treatment's influence on the fresh shoot weight.

Statistical analyses

For the disease rating data, the non-parametric Kruskal–Wallis test (α = 0,05) was used on the ordinally scaled disease categories for two-group comparisons between treatment groups infected with P. brassicae and those inoculated with P. brassicae and A. alternatum simultaneously for each experiment. The biomass measurements were examined for major outliers using boxplots and the interquartile range method: biomass measurements of each experiment were visualized with BoxplotR (after Tukey; Spitzer et al., 2014) and all single data points were removed manually that were far outside of the outer fences (whiskers) of the boxplot. Data points just outside of the boxplot whiskers were kept to not further reduce the sample size. The cleaned-up data sets were used for further analyses. To estimate the effect of A. alternatum on the biomass of plants we calculated the effect size estimate Cohen´s d as multiple two-group comparisons for each experiment with estimation statistics (Ho et al., 2019). The accelerated and bias-corrected 95% CI of the two-group mean differences were calculated by bootstrap resampling (5000 resamples).

Results

In this study we investigated the effects of the biocontrol fungus A. alternatum on various rapeseed cultivars exhibiting different degrees of resistance to clubroot under diverse growth conditions and with varying levels of P. brassicae-inoculum virulence and inoculum densities.

Efficacy of Acremonium alternatum on clubroot development in pots

The endophytic fungus A. alternatum reduced the disease severity index in five out of eight pot greenhouse trials conducted, and the reduction was dependent on the cultivar used (Fig. 1, Table 1). In the majority of rapeseed cultivars, except for cv. Jumbo, treatment with A. alternatum resulted in a decrease in the number of root samples exhibiting severe clubroot symptoms (class 3 and 4) and an increase in root samples showing either no evident clubroot symptoms (class 0) or only minor galls (class 1). This effect was more prominent when the plants were inoculated with 106 spores mL−1 of P. brassicae-e3, compared to 107 spores mL−1 (Fig. 1). The most significant reduction in disease symptoms was observed at a moderate inoculum density of P. brassicae-e3 (106 P. brassicae spores mL−1) where A. alternatum decreased DSI by 70% in cv. Ability and 54% in cv. Jenifer (Table 1). The highest reduction of clubroot symptoms occurred in cv. Visby, reaching 55% at a high P. brassicae-e3 inoculum density (107 P. brassicae spores mL−1) while cv. Ability exhibited an 11% reduction. As expected, cv. Mentor demonstrated complete resistance to P. brassicae-e3 (data not displayed).

Fig. 1
figure 1

Assessment of the performance of Acremonium alternatum as a biological control agent in reducing clubroot symptoms in pot experiments. Clubroot-susceptible rapeseed cultivars Ability, Jenifer, Jumbo and Visby were inoculated at BBCH 10 with either 106 or 107 mL−1 resting spores of P. brassicae-e3 along with107 conidia mL−1 of A. alternatum. A Clubroot disease severity index and B the percentage of plants categorized into distinct symptom classes (ranging from 0–4) were assessed 45 days post-inoculation (dpi). Asterisks indicate statistically significant differences between P. brassicae treated and co-inoculated plants (P. brassicae + A. alternatum)

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Table 1 Percentage efficacy of Acremonium alternatum in suppression of clubroot disease
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In pot experiments, where the clubroot susceptible rapeseed cv. Visby and the resistant cv. Mentor were inoculated with varying spore concentrations of the field isolate of P. brassicae-P1, the outcomes of the A. alternatum treatment were inconsistent. At a low inoculum density of 103 spores mL−1 P. brassicae-P1 a moderate dosage of A. alternatum (106 conidia mL−1) reduced the DSI by 9%. However, applying a higher dose of A. alternatum treatment (107 conidia mL−1) led to a 24% increase in DSI. While the number of plants in disease category 4 remained consistent in the treatment without A. alternatum, there was an increase in the number of plants in category 3. This increase contributed to the overall increase of the DSI (Fig. 2A, Table 1). Unlike cv. Visby, cv. Mentor displayed minimal (DSI < 10) clubroot symptoms when inoculated by 103 spores mL−1 P. brassicae-P1. However, treating plants with A. alternatum did not result in a significant reduction in the DSI. The DSI in cv. Visby varied between 93 and 98 when plants were inoculated with 104 or 105 spores mL−1 P. brassicae-P1. However, treating plants with 106 or 107 conidia mL−1 A. alternatum resulted in a reduction of the DSI by 15% and 8%, respectively (Fig. 2A, Table 1). The DSI in cv. Mentor remained consistently low even as the P. brassicae-P1 inoculum increased to 104 or 105 spores mL−1 (Fig. 2A). Treatment with A. alternatum had minimal to no effect on reducing the disease (Fig. 2A, Table 1).

Fig. 2
figure 2

Assessment of the performance of Acremonium alternatum as a biological control agent in reducing clubroot symptoms in pot experiments. Clubroot susceptible rapeseed cv. Visby and clubroot resistant cv. Mentor were co-inoculated with 103 to 106 mL−1 resting spores of P. brassicae isolates P1 and P1 ( +) and different concentrations of A. alternatum (106, 107 or 108 conidia mL −1) at growth stage BBCH 10. Inoculation occurred simultaneously for both organisms. A-C Clubroot disease severity index and D-F the percentage of plants categorized into distinct symptom classes (ranging from 0–4) were assessed 45 days post-inoculation (dpi); n.d.: not determined. Asterisks indicate statistically significant differences between P. brassicae treated and co-inoculated plants (P. brassicae + A. alternatum)

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The isolate P1 ( +) developed on the resistant cv. Mentor on fields in Germany (Zamani-Noor et al., 2021). Therefore, this cultivar was not tested at very low concentrations since no gall formation was expected to occur, but the susceptible cv. Visby showed also low DSI (Fig. 2B). Higher spore concentrations of P. brassicae led to higher DSI in cv. Visby compared to cv. Mentor (Fig. 2C). The DSI in clubroot-susceptible cv. Visby plants inoculated only with P. brassicae-P1 ( +), ranged between 28 and 38, depending on the density of the inoculum of P. brassicae (Fig. 2C). Unexpectedly, treating plants with A. alternatum at all densities (106 up to 108 conidia mL1) had a synergetic impact on clubroot development. The majority of galls fell into disease classes 3 and 4, causing the DSI to increase to values between 50 and 70, depending on the spore concentration of both microorganisms (Fig. 2C). The DSI in clubroot-resistant cv. Mentor showed a range between 7 and 23 when inoculated with 105 and 106 spores per mL of P. brassicae-P1 ( +), respectively (Fig. 2C). Treating cv. Mentor plants with A. alternatum consistently decreased the clubroot DSI across all P. brassicae spore densities, except in one case. The exception occurred when plants were simultaneously exposed to 106 spores per mL of P. brassicae-P1 ( +) and 107 A. alternatum conidia mL−1, resulting in a slight increase in the DSI to 29 (Fig. 2C).

Effect of A. alternatum on clubroot development under semi-field conditions in raised-bed containers

The winter rapeseed cultivars, Visby and Mentor, exhibited differences for all evaluated disease parameters in response to both inoculum concentrations that were administered to the plants after sowing and the pathotype of P. brassicae. Within the clubroot-resistant cv. Mentor, there was an increase in the number of root samples showing no apparent clubroot symptoms (class 0) or only a few minor galls (class 1) when plants were exposed to A. alternatum. This effect was more pronounced when plants were inoculated with P. brassicae-P1 ( +) (Fig. 3B). Conversely, in the clubroot-susceptible cv. Visby, a reduction in the number of root samples exhibiting severe galling (class 3) was observed when plants were inoculated with a lower concentration of P. brassicae-P1 and concurrently treated with A. alternatum (Fig. 3A).

Fig. 3
figure 3

Assessment of the performance of Acremonium alternatum as a biological control agent in reducing clubroot symptoms in clubroot susceptible rapeseed cv. Visby and clubroot resistant cv. Mentor. Rapeseed plants were inoculated with 2 × 107 spores mL−1 A. alternatum at growth stage BBCH 11–12. One day after treatment, plants were inoculated with either P. brassicae-P1 (A) or P. brassicae-P1 ( +) (B) isolate with varying concentrations (2 × 105, 2 × 106 or 2 × 107 spores mL−1). Clubroot disease severity index (values above the histograms) and the percentage of plants categorized into distinct symptom classes (ranging from 0–3) were assessed 42 days post-inoculation (dpi). There were no statistically significant differences found

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The Disease severity index (DSI) of clubroot-susceptible cv. Visby plants inoculated with P. brassicae-P1 ranged from 44 to 100, depending on the inoculum concentration, reaching 100 with all concentrations of P. brassicae-P1 ( +) (Fig. 3). However, a very low to no reduction in DSI was observed between A. alternatum-treated and untreated plants in the clubroot susceptible cultivar Visby (Fig. 3). In contrast to cv. Visby, the DSI in cv. Mentor ranged from 0 to 3 when inoculated by P. brassicae-P1 and from 12 to 36 by P. brassicae-P1 ( +) (Fig. 3). A low to average reduction in clubroot disease severity was observed in the resistant cultivar, Mentor (Fig. 3, Table 1). We assume that a reduction in DSI of 20% or higher when plants were co-inoculated with both organisms may indicate potential for biocontrol in the field. These values, although not statistically significant, are presented in bold in Table 1.

In a second set of experiments, the inoculation method was altered to simulate field-like conditions, wherein the spores were mixed with the soil before sowing the seeds. Consistent with previous experiments in the current study, the rapeseed cultivars Visby and Mentor exhibited differences in their response to inoculum density in soils infected with P. brassicae-P1 (Fig. 4). In the clubroot-resistant cv. Mentor, a slight decrease was observed in the number of root samples displaying either moderate galling (class 2) or severe galling (class 3) when the plants were exposed to A. alternatum. No differences in clubroot symptom severity were observed in the clubroot-susceptible cv. Visby.

Fig. 4
figure 4

Assessment of the performance of Acremonium alternatum as a biological control agent in reducing clubroot disease on rapeseed cultivars. Clubroot susceptible rapeseed cv. Visby and resistant cv. Mentor were sown in soil infested with 107 spores per mL A. alternatum and either 106 or 107 spores per g soil of P. brassicae-P1. Clubroot disease severity index (values above the histograms) and the percentage of plants categorized into distinct symptom classes (ranging from 0 to 3) were assessed 56 days after sowing (das). There were no statistically significant differences found

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The DSI in cv. Visby ranged from 96–100 when grown in infested soil with 106 or 107 spores per g soil, respectively (Fig. 4). In cv. Mentor the DSI was very low (8–9) as expected for the resistant cultivar and dependent on the spore concentration in the soil (Fig. 4). However, A. alternatum did not show any effect in reducing the clubroot severity index in the clubroot-susceptible cultivar Visby, sown in clubroot-infected soil with either 106 or 107 spores per g soil (Fig. 4). In contrast, A. alternatum reduced the DSI in the clubroot-resistant cv. Mentor by up to 15% (Fig. 4).

Effect of A. alternatum on plant growth promotion under clubroot pressure

A visible impact of A. alternatum on plant fresh biomass was observed across all rapeseed cultivars. The rapeseed plants co-inoculated with both organisms exhibited an enhanced overall survival rate and increased weight of aboveground plant parts compared to those infected solely with P. brassicae. In 19 out of 21 trials, co-inoculation with A. alternatum led to an increase in the fresh weight of rapeseed cultivars by 2% to 40%. However, in two trials, the biomass was reduced by 9% and 56% compared to plants inoculated with P. brassicae only (Table 2). Co-inoculation with A. alternatum resulted in an average increase in green plant biomass of 17% in trials involving the single spore isolate P. brassicae-e3 and biomass was increased by 12% (P1) and 15% (P1 ( +)) in trials with the field isolates (Table 2).

Table 2 Effect of Acremonium alternatum on average (avg.) fresh aboveground biomass (without roots) of clubroot susceptible and resistant rapeseed cultivars inoculated with different isolates and resting spore concentrations of Plasmodiophora brassicae. A Cohen´s d larger than 0.200 and P values smaller than 0.05 are indicated in bold
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Discussion

The management of clubroot has persistently posed a difficult challenge for farmers and pathologists due to the intracellular nature of the pathogen and its soil-borne attributes (Javed et al., 2023; Zamani-Noor et al., 2022a). Controlling and eradicating clubroot disease from infested fields proves exceptionally difficult. Traditional chemical and agronomic methods have demonstrated limited efficacy in managing the disease. Endophytic fungi, which inhabit plant tissues without manifesting visible disease symptoms, represent a promising strategy for eliciting beneficial effects in plants by stimulating immune responses, fostering plant growth, or suppressing other plant pathogens (Card et al., 2016; Van Wees et al., 2008). An effective biological control candidate for controlling clubroot disease should initially aim to suppress the germination of resting spores and prevent the primary infection of root hairs and the secondary infection of the root cortex. Additionally, it should demonstrate antagonistic or competitive capabilities against the developing pathogen within the host root tissue (Struck et al., 2022).

Acremonium alternatum is a ubiquitous endophytic fungus with promising potential as a biocontrol agent against various plant pathogens. Several studies have reported its antagonistic effects against fungal and bacterial pathogens, including Plasmodiophora brassicae (Auer & Ludwig-Müller, 2014, 2015, 2023; Jäschke et al., 2010; Kasselaki et al., 2006; Kiss, 2003). Moreover, A. alternatum has been observed to induce systemic resistance in plants, enhancing their natural defence mechanisms against diseases (Kasselaki et al., 2006). However, given the widespread threat of clubroot disease in rapeseed cultivations, there is an urgent need for fundamental research focused on identifying effective biocontrol agents to control this disease. Relying solely on a single biocontrol agent or biopesticide is insufficient to effectively reduce clubroot disease to a tolerable level. Therefore, a comprehensive approach involving multiple strategies is necessary to achieve satisfactory control of the clubroot disease.

In the current study, we present the findings of multiple trials conducted with five rapeseed cultivars and three P. brassicae inoculum sources, encompassing two field isolates that are commonly found in German arable fields (Zamani-Noor et al., 2022a) and one aggressive single spore isolate (SSI e3) that is well characterized (e.g., Schwelm et al., 2015; Siemens et al., 2002). The control efficiency of A. alternatum varied between the pot experiments and semi-field trials, potentially influenced by factors such as the storage and preparation of inoculum and biological agents, differences in greenhouse conditions across institutes and variations in the microbial community present in different soils. However, the results clearly show that, in principle, the application of A. alternatum is capable of reducing clubroot disease symptoms and promoting the growth of various rapeseed cultivars under different greenhouse conditions. Nevertheless, the effectiveness of biocontrol against clubroot can vary greatly under natural field conditions due to factors such as disease pressure and spore load, aggressiveness of the pathotype, soil properties like fertility and soil structure and environmental factors such as soil moisture, temperature and crop variety (Ahmed et al., 2020; Auer & Ludwig-Müller, 2023; Struck et al., 2022; Zamani-Noor et al., 2021). In the present study, the efficacy of A. alternatum treatments varied considerably among clubroot-susceptible rapeseed cultivars. Pot experiments conducted in the greenhouse using a single spore isolate, P. brassicae-e3, exhibited promising results, suggesting the potential of A. alternatum in controlling clubroot disease. However, when the susceptible cultivar Visby was inoculated with field isolates of P. brassicae, the application of A. alternatum showed minimal to no effect on clubroot development, as observed in both pot experiments and semi-field trials. This could be attributed to the presence of different pathotypes within a single gall of a field isolate, potentially affecting the performance of A. alternatum. Therefore, it is important to expand future studies to include additional rapeseed cultivars and several P. brassicae field isolates with varying levels of virulence, especially under diverse field conditions.

An interesting finding from our study is that A. alternatum treatment reduced disease severity, specifically in the clubroot-resistant cultivar Mentor. This highlights the synergistic effects of using biocontrol agents with resistant cultivars. This approach helps minimize the selection pressure on resistant cultivars, thereby delaying the overcoming of resistance in fields with highly aggressive P. brassicae isolates (Zamani-Noor et al., 2021). Nonetheless, unfavourable environmental conditions in the field have been observed as a significant factor contributing to the lack of success or inconsistent results in biological control experiments, as these conditions similarly influence biocontrol agents. Therefore, introducing biocontrol agents in greenhouses with precisely controlled environments is expected to produce more reliable and successful outcomes compared to their application in open fields and necessary to first screen for the efficacy potential of a biocontrol agent.

Insights from studies like this one could enhance the effective management of clubroot in Brassica vegetables, typically grown under controlled greenhouse or semi-field conditions. In conjunction with our study, Doan et al. (2010) demonstrated that A. alternatum can mitigate clubroot disease symptoms in Brassica rapa ssp. pekinensis cv. Granaat, suggesting that employing biocontrol fungi could be a promising strategy for clubroot management. Furthermore, co-inoculating A. alternatum with B. rapa and Arabidopsis plants resulted in a significant delay in the development of P. brassicae (Doan et al., 2010).

The results from our study indicate that the application of A. alternatum exhibited potential for promoting plant growth under greenhouse and semi-field conditions even with aggressive P. brassicae field isolates. This discovery supports the hypothesis that numerous antagonistic microbes contribute to plant growth indirectly by inhibiting the detrimental effects of pathogens and reducing the severity of diseases (Beneduzi et al., 2012; Hayat et al., 2010; Natsiopoulos et al., 2022), while simultaneously suggesting that increased plant growth might be attributed to certain microorganisms' ability to induce phytohormones or enhance nutrient uptake (Chowdappa et al., 2013). If these findings can be replicated under field conditions, it is anticipated that clubroot-infected rapeseeds colonized by A. alternatum could potentially yield higher quantities of seeds, maybe accompanied by higher quality. Plants that were able to produce more biomass in the autumn before the winter months, stand a better chance of surviving under severe winter conditions. The anticipated increase in average biomass of 10–20%, as demonstrated here across four cultivars within eight weeks, is likely to confer an advantage in protecting plants against overwinter damage, which needs to be tested in long-term trials. Previous and current evidence points to a higher survival rate of rapeseed cultivars when they are associated with A. alternatum (Auer & Ludwig-Müller, 2014). Increased survival rates of young plants during autumn, even on clubroot-infested fields, can increase the chance for higher yields in the following year when more plants reach maturity and potentially produce more flowers under the fungal influence. Nevertheless, our data cannot distinguish between growth promotion and a reduction in disease severity at the moment. Future testing with a broader range of rapeseed cultivars and under diverse experimental conditions will be essential to deepen our understanding of the underlying mechanisms driving our observations. Such data could further enhance the efficacy of treatments under natural field conditions.

Conclusion

Clubroot, recognized as a globally significant disease, has a lengthy and destructive history in its impact on rapeseed farming worldwide. Various obstacles, such as the long-term survival of dormant spores in the soil, their ability to reproduce on a wide range of hosts, and their genetic diversity, present significant challenges when it comes to managing and containing the spread of clubroot. The findings of the present study highlight that the application of the endophytic fungus Acremonium alternatum represents a sustainable and effective additional treatment for fields with clubroot infestation. Its ability to reduce the severity of clubroot symptoms and promote plant growth makes it an attractive candidate for integrated disease management strategies. However, further research and field trials are necessary to understand the mechanisms of action and optimize its implementation fully. Nevertheless, by using the potential of endophytic fungi like A. alternatum, we can pave the way for more environmentally friendly and sustainable approaches to disease control in rapeseed and other crops.