Priestia megaterium KW16: A Novel Plant Growth-Promoting and Biocontrol Agent Against Rhizoctonia solani in Oilseed Rape (Brassica napus L.)—Functional and Genomic Insights

Abstract

Plant diseases caused by Rhizoctonia solani present a significant challenge in agriculture. While chemical pesticides remain a common control strategy, their use leads to health and environmental problems. In contrast, endophytic bacteria with plant growth-promoting (PGP) activity offer a promising, sustainable alternative. In this context, a novel endophytic Priestia megaterium strain, KW16, originated from the bluegrass (Poa pratensis L.), demonstrated distinct biocontrol potential against R. solani. in vitro assays showed that KW16 inhibited R. solani growth by up to 58%, primarily by releasing volatile compounds. In planta experiments further highlighted KW16′s ability to colonize oilseed rape internal tissues, significantly enhancing its growth and development. In the presence of the pathogen, KW16 abolished the negative impact of R. solani and promoted plant growth, increasing shoot and root biomass by 216% and 1737%, respectively, when compared to the plants grown in fungal-infested soil. Biochemical and genome analyses confirmed the strain’s metabolic versatility, resistance to biotic and abiotic factors, and a whole spectrum of PGP and biocontrol traits such as biofilm formation, production of phytohormones, and synthesis of lytic enzymes, siderophores, and volatiles, alongside its ability to survive in the presence of autochthonous soil microflora. These findings position KW16 as a potent biological alternative to synthetic fungicides, with significant potential for sustainable crop protection.

1. Introduction

Pathogenic fungi are the most numerous plant pathogens, using plants as a habitat and source of nutrients and posing a threat to their growth and development. Plant diseases caused by these pathogens are a serious problem in agriculture, resulting in yield losses in all globally important crops, such as wheat, rice, corn, potato, soybean, and oilseed rape, and a significant threat to food security [1,2]. A ubiquitous soil necrotroph, Rhizoctonia solani, poses a serious risk [3]. In the pathogenesis of the fungus infection, the secretion of reactive oxygen species, detoxifying enzymes, lignocellulosic enzymes, and host-selective phytotoxins plays an essential role. For example, a specific toxin against rice (Oryza L.) is a carbohydrate consisting of mannose, N-acetylgalactosamine, N-acetylglucosamine, and glucose. Other biologically active molecules synthesized by R. solani are oxalic acid or phenylacetic acid and their derivatives [4]. Characteristic symptoms of R. solani infection are gray water-soaked spots on leaves and leaf sheaths, and rotting of hypocotyls, leaves, young seedlings, and roots. Chlorosis and leaf deformation are also frequently observed. The earliest lesions are observed 24–72 h after infection. Rhizoctoniosis is very difficult to control due to the broad host range of R. solani and the transmission of mycelial hyphae in soil, water, and infected plants [4,5,6].

Plant protection against this pathogen is overwhelmingly based on chemical pesticides. However, the widespread use of such compounds is increasingly disputed, as it leads to serious health and environmental problems, including pollution, destruction of ecosystems, and ecological imbalances [7]. Chemical residues in food are also a problem. For example, a report prepared on behalf of Greenpeace indicated that as many as 50% of tested rapeseed honey samples had exceeded limits for the pesticides used to cultivate this crop [8]. Pesticide contamination of many other bee products has also been demonstrated by Swiatly-Blaszkiewicz et al. [9]. In addition, regular and long-term use of pesticides results in increased resistance in target organisms and the spread of genes that determine resistance to the applied pesticide into the environment [10].

A valuable option to reduce the use of pesticides and their detrimental impacts on humans and benefit microbial diversity, soil chemistry, and underground water lies in biological methods. Therefore, there is a constant search for microorganisms that effectively control pests and plant pathogens and could be used as active ingredients in biopesticides as well as biofertilizers [11,12]. In recent years, special attention has been paid to endophytic bacteria, which inhabit the same ecological niche as phytopathogens. Their constant direct contact with plant cells facilitates their beneficial effects on the host and contributes to systemic plant immunity [13].

Among various microbial candidates, Priestia megaterium (formerly Bacillus megaterium), a Gram-positive, spore-forming bacterium, holds significant promise as a potential biocontrol agent and has been reported to inhibit bacterial and fungal diseases in various plants. For example, P. megaterium KD7, is a known antagonist of Erwinia amylovora, a plant pathogen that causes fire blight disease in Rosaceous plants [14]. P. megaterium T3 reduces leaf spot disease symptoms induced by Xanthomonas vesicatoria [15], and the B. megaterium strain NBAII-63 suppressed bacterial wilt caused by Ralstonia solanacearum in tomato plants [16]. In addition, B. megaterium AB4 controlled the disease symptoms caused by Alternaria japonica [17], while B. megaterium BM344-1 inhibited growth and mycotoxin production in Aspergillus flavus, A. carbonarius, Penicillium verrucosum, and Fusarium verticillioides [18]. However, only a few studies deal with combating R. solani infections by Priestia strains. Zheng et al. [19] described the positive effect of B. megaterium strain B153-2-2 on suppressing Rhizoctonia root rot in soybeans, and Solanki et al. [20] reported the protective effect of P. megaterium on root rot in tomatoes. Nevertheless, the antagonistic efficacy of different Priestia representatives against pathogens varies, making exploring their in-depth disease-inhibitory potential crucial.

In addition to its ability to inhibit the growth and development of pathogens, P. megaterium was also reported as a plant growth promoter. For instance, Nascimento et al. [21] demonstrated the significant impact of the STB1 strain on the development of roots, shoots, and leaves in tomatoes, which resulted in an expressive increase in plant total dry biomass (295.8 mg) relative to that of the non-inoculated control (81.9 mg). In turn, the P. megaterium JR48 strain described by Li et al. [22] notably promoted the growth of Arabidopsis (0.25-fold increase in fresh weight), Chinese cabbage (0.29-fold increase in fresh weight), and tomato plants (increase in fresh weight of 17.29%), when compared to the control.

Biological activity, colonization, and promotion of plant growth are determined by the presence of many genes involved in PGP and biocontrol mechanisms, including nitrogen, phosphorus, and sulfur metabolism; biofilm formation; and the production of phytohormones, siderophores, volatile compounds, lytic enzymes, and antibiotics, which may participate in one or both of these activities [23,24,25,26]. Understanding the molecular basis of these mechanisms, and thus the potential of the studied strain along with its response to the pathogen at the level of gene expression and revealed activity, provides a more comprehensive view, allowing for future application of the microorganism in agriculture.

Due to the lack of data related to control rhizoctonia-related diseases in oilseed rape by P. megaterium and the continuous search for new strains with high efficiency, a wide range of biocontrol characteristics, modes of action, and plant growth promotion (PGP) potential, the aims of our study are as follows: (i) examine biological activity of a new endophytic Priestia megaterium strain KW16 isolated from the bluegrass (Poa pratensis L.) against R. solani, (ii) evaluate the effect of the strain on the growth and protection of oilseed rape (Brassica napus L.) against the fungal pathogen, (iii) characterize the KW16 strain in terms of traits necessary for plant colonization, endophytic–host interactions, and biocontrol mechanisms, (iv) identify genes crucial for plant growth promotion and antifungal activity in the genome of the strain, and (v) to determine the effect of R. solani on the expression of genes potentially involved in the biocontrol process in the KW16 strain, and its ability to form a biofilm and produce siderophores, the main traits engaged in effective plant colonization and biological activity.

2. Materials and Methods

2.1. Bacterial and Fungal Strains, Growth Media, and Culture Conditions

Priestia megaterium KW16 was isolated from the surface-sterilized roots of the bluegrass (Poa pratensis L.) overgrowing soils in the vicinity of Kalina Pond in Świętochłowice, Poland (50.278758 N 18.928032 E), according to the standard protocol [27], and deposited in the Polish Collection of Microorganisms (Wrocław, Poland) under deposit no. B/00441. Bacteria were cultivated routinely in Luria–Bertani broth (LB Broth) at 30 °C with shaking (140 rpm), or on LB agar (LBA) at 30 °C.

Rhizoctonia solani W70, isolated from the tissues of the grapevine (Vitis vinifera L.), was derived from the Microbial Culture Collection of the Institute of Biology, Biotechnology, and Environmental Protection (Faculty of Natural Sciences, University of Silesia in Katowice, Poland) and incubated on PDA (A&A Biotechnology, Gdańsk, Poland) at 28 °C.

2.2. In Vitro Antifungal Activity of KW16

The antifungal activity of the KW16 strain towards R. solani was tested by a dual-culture in vitro assay on PDA and LBA medium, according to the method described by Chlebek et al. [27]. The 5 mm agar disks of actively growing fungal mycelium were inoculated separately on one side of the Petri dishes with PDA or the LBA medium. A loop of overnight KW16 culture was then streaked 30 mm away from the disk of the pathogen. As a control, plates inoculated only with the fungus were used. Samples were incubated for 3 days at 28 °C. After incubation, the distance between the center of the agar disk and the edge of the actively growing mycelium was measured in both the control plate and those inoculated with the fungus and the bacterium. The percent growth inhibition (PGI) was calculated using the following formula:

$$\mathrm{PGI}={\displaystyle \frac{\mathrm{KR}-\mathrm{R}1}{\mathrm{KR}}} \times 100\%$$

(1)

where KR represents the distance (in mm) from the point of fungal inoculation to the mycelium growing edges on control dishes; and R1 represents the distance of fungal growth towards the antagonist, from the point of fungal inoculation to the fungal colony margin on plates inoculated with bacteria [28]. The experiment was performed in triplicate.

2.3. Effect of VOCs Produced by KW16 on the Growth of R. solani

100 µL of overnight bacterial culture was spread on one half of a BI plate with LBA medium, and a 5 mm agar disk of R. solani mycelium was placed at the center of the other half with PDA medium. BI plates inoculated only with the fungus were used as controls. The plates were immediately tightly sealed with Parafilm and incubated for 3 days at 28 °C. The inhibition of fungal growth was calculated as in Section 2.2. The experiment was performed in triplicate.

2.4. In Planta Growth Promotion and Antifungal Activity of KW16

2.4.1. Soil Samples

The soil for planting was collected from an oilseed rape field near Pszczyna, Poland (49.988546 N, 18.848113 E), from the top (0–20 cm) surface layer. Physico-chemical analysis of the sampled soil was performed by the external certified laboratory i2 Analytical Ltd., Poland (Table S1 Supplementary Materials).

2.4.2. Generation of a Rifampicin-Resistant Mutant of KW16

Spontaneous rifampicin-resistant mutants of the strain were obtained to monitor its presence and survival in soil and plant tissues. The KW16 strain was inoculated into LB and grown overnight with shaking (130 rpm) at 30 °C. Later, 100 µL of the overnight culture was plated onto an LBA medium supplemented with 2 μg mL⁻¹ of rifampicin and incubated at 30 °C for 24 h. The grown colonies were transferred to an LBA medium with 5 μg mL⁻¹ of rifampicin and incubated at 30 °C for 24 h. In each subsequent round, the colonies were transferred onto an LBA medium with increasing antibiotic concentration until colonies growing in the presence of 150 µg mL⁻¹ rifampicin were obtained. To confirm the persistence of the acquired resistance, obtained mutants grown in the presence of the highest antibiotic concentration were cultured five times in LB medium without antibiotic selection and then re-cultured on LBA medium with rifampicin at a concentration of 150 µg/mL.

2.4.3. The Inoculum Preparation and Experiment Design

The rifampicin-resistant mutant KW16^RIF was inoculated into LB medium supplemented with rifampicin (150 µg/mL) and grown overnight with shaking (130 rpm) at 30 °C. The cells were harvested by centrifugation at 4415× g for 15 min, and the pellet was washed three times with sterile 0.9% NaCl, following the cell suspension with a concentration of 10⁹ CFU mL⁻¹ preparation. Subsequently, four different treatments were designed as follows:

(1)

non-inoculated soil (control);

(2)

soil inoculated with the KW16^RIF strain (KW16^RIF),

(3)

soil inoculated with R. solani (RS);

(4)

soil inoculated with the KW16^RIF strain and R. solani (RS + KW16^RIF).

The study was conducted in a randomized design. Before starting the experiment, the soil was sieved (2 mm) to remove clods and stones. Then, 750 g of the soil was weighed into separate pots, and five seeds of oilseed rape (B. napus L.) were randomly sawn in every pot at a depth of 1 cm. In the R. solani system, the soil was infested with fungus by introducing 5 mm agar disks of actively growing fungal mycelium. Then, 50 mL of the prepared inoculum was introduced into the pots except for the control and soil inoculated only with R. solani (RS). Cultures were carried out in the culture room at a temperature of 21 °C, constant soil moisture, and a 16 h of light photoperiod. On the 14th, 28th, and 35th day of culture, 10 plants were taken from each of the test systems, and the length and weight of the roots and shoots were measured. Additionally, the survival rate of the bacterium in plants (roots and shoots) and their presence in soil were determined. The chlorophyll (Chl), NB, flavonol (Flav), and anthocyanin (Anth) indices were measured on the 35th day of the experiment using a Dualex Scientific+^TM sensor (Force-A, Orsay, France) to analyze plants physiological status.

2.4.4. Evaluation of KW16^RIF Strain Survival in Plant Tissues and Soil

To verify the endophytic colonization of the oilseed rape with strain KW16^RIF, 1 g of plant mass was taken, and isolation of the bacteria was carried out under sterile conditions. Root and shoot fragments of selected plants were surface sterilized by successively immersing in 70% ethanol (roots—30 s, shoots—20 s) and 5% ACE (4–5 min), followed by washing three times with sterile distilled water for 5 min. Sterile samples were homogenized in sterile mortars, and 9 mL of sterile saline (0.9% NaCl) was added. A series of decimal dilutions (10⁻¹–10⁻⁵) of the resulting macerate were prepared, and 20 µL of each dilution was plated, in duplicate, onto an LBA medium supplemented with rifampicin (150 µg/mL). The plates were incubated at 30 °C for 48 h. A sterility control was also prepared by inoculating 100 µL of water from the last wash of the plant organ onto an LBA medium with rifampicin (150 µg/mL).

To assess the survival of the KW16^RIF strain in soil, 10 g of soil from every treatment was weighed, suspended in 90 mL of 0.9% NaCl with 1% Tween 80, and shaken for 30 min (120 rpm, 28 °C). From the resulting soil solutions, a series of decimal dilutions (10⁻¹–10⁻⁵) were prepared, and 20 µL of each dilution was plated, in duplicate, onto LBA medium supplemented with rifampicin (150 µg/mL). Plates were incubated for 24 h at 30 °C.

The pot experiments were carried out from March to April 2022.

2.5. Physiological and Biochemical Characterization of KW16

2.5.1. Colonization and Plant Growth-Promoting Features

Swimming, swarming, and twitching motility assays and oxidase and catalase activity were tested as described by Naveed et al. [29]. Production of exopolysaccharides (EPS) was assessed by cultivating the KW16 on Congo Red Agar (CRA) [30], while solubilization of phosphate was detected on the Pikovskay’a medium containing insoluble Ca₃(PO₄)₂ [31]. The Phosphate Solubilization Index (PSI) was calculated according to Edi-Premono [32]. The ability of the KW16 strain to produce indole-3-acetic acid (IAA), ammonia, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase was determined according to the method of Szilagyi-Zecchin et al. [33], Cappuccino and Sherman [34], and Sandhya et al. [35], respectively. Biofilm formation was analyzed using the crystal violet (CV) method in 96-well microtiter plates by staining bacteria with 0.1% CV for 20 min, following a triple wash with 200 μL of PBS buffer and fixation with 200 μL of methanol. Further, 150 μL of ethanol-detained CV was transferred into a new microtiter plate, and the absorbance was measured at 590 nm (modified from Naveed et al. [29]). The ability of the KW16 strain to utilize 1 mM glucose, arabinose, rhamnose, mannose, trehalose, succinic acid, 4-hydroxyphenylacetic acid, fumaric acid, benzoic acid, mannitol, citric acid, or p-coumaric acid as the sole source of carbon and energy was studied using the 96-well microplates as described by Chlebek et al. [27]. All experiments were performed in three biological replicates.

2.5.2. Biocontrol Traits

Production of proteases, cellulases, and lipases was verified, as described by Vijayalakshmi et al. [36], and chitinase production was verified following the method of Kuddus and Ahmad [37]. The ability of the KW16 strain to produce acetoin, 2,3-butanediol, and hydrogen cyanide (HCN) was analyzed according to Johnston-Monje and Raizada [38], Syamala and Sivaji [39], and Ahmad et al. [40], respectively. All experiments were performed in three biological replicates.

2.5.3. Siderophore Production

The siderophore production was evaluated using both qualitative and quantitative methods. For the qualitative assay, the Chrome Azurol Blue (CAS) agar plate method was used [41]. The microtiter plate method was used to quantify the strain’s siderophore activity. Briefly, the KW16 strain was inoculated into LB and grown overnight with shaking (130 rpm) at 30 °C. The cells were harvested by centrifugation at 4415× g for 15 min. Then, 100 µL of the culture supernatant was added into the separate wells of the 96-well microplates, followed by the addition of 100 µL CAS reagent. After 30 min incubation, the absorbance of each sample was measured at 630 nm using TECAN SPARK 10M Multimode Microplate Reader, and the percentage of siderophore was calculated according to Kumar et al. [42]. Eight replicates for each biological treatment were performed.

Biochemical assays and microbial tests were conducted between 2022 and 2025.

2.6. R. solani Filtrates Preparation

The liquid medium was inoculated with plugs taken from actively growing fungus cultures and incubated for 14 days at 28 °C in darkness. The fungal biomass was collected by centrifugation (4415× g, 20 min), and the obtained supernatant was filtered through a 0.22 μm pore size syringe filter to remove fungal cells.

2.7. Influence of R. solani on the Expression Level of Selected Genes of the KW16 Strain

In detail, 25 mL of LB medium was inoculated with 1 mL of overnight LB culture of the KW16 strain, and 5 mL of fungal filtrate was added. The KW16 strain grown in LB medium without the filtrate served as a control. Cultures were incubated with shaking (130 rpm) at 30 °C for 72 h. After 24 h, 48 h, and 72 h of incubation, total RNA was isolated from the control and the filtrate-treated cultures using a GeneMATRIX Universal RNA Purification Kit (EURx, Gdansk, Poland) and purified with TURBO DNA-free™ DNase (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). The concentration and purity of obtained RNA were assessed using an ND-1000 NanoDrop spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). The synthesis of a single-stranded cDNA was carried out using a RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA) with 1 µg of the total RNA and random hexamer primers. The generated cDNA was used as a template in qPCR reactions performed in a 10 μL reaction volume with 5 µL LightCycler^® 480SYBR Green I Master, 2 µL PCR-grade water, 0.5 μM of each gene-specific primer, and 2 μL cDNA as a template, in LightCycler^®480 Multiwell Plates 96 under the following conditions: 10 min at 95 °C and 45 cycles of 15 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C (LightCycler^® 480 Real-Time PCR System; Roche, Basel, Switzerland). Two biological and two technical replicates were performed for each treatment. The expression levels of the following genes were analyzed: gltB, iucC, katA, ilvB, bdhA, and bglA. In order to increase the stability of the measurements, two genes whose expression was stable across the treatments, pyk and ftsZ, were used as the internal controls [43]. Each gene-specific primer was designed using Geneious Prime (version 2022.0.1; Table S2—Supplementary Materials). The amplification efficiency of the primers was checked according to Taylor et al. [44]. The relative expression level was calculated according to Livak and Schmittgen [45].

Gene expression studies were conducted in 2023.

2.8. Influence of R. solani on Auto-Aggregation, Biofilm Formation and Siderophore Production by KW16 Strain

Auto-aggregation assays were conducted following the method of Chlebek et al. [27]. Two biological and four technical replicates were performed.

The effect of R. solani on the biofilm formation activity of the KW16 strain was analyzed in 96-well microtiter plates. 180 µL of LB medium mixed with the fungal filtrate in the ratio of 5:1 (v:v) was introduced into separate wells of the plates and inoculated with 20 µL of the overnight LB bacterial culture. As a control, 180 µL of LB medium inoculated with 20 µL of the bacterial culture was used. The abiotic control was 200 µL of sterile LB medium. The plates were protected from evaporation and incubated for 24, 48, and 72 h at 30 °C. Thereafter, biofilm formation was evaluated, as described in Section 2.5.1. Two biological and eight technical replicates were performed.

To determine the effect of R. solani on the siderophore activity of the KW16 strain, 1 mL of the overnight LB bacterial culture was introduced into 25 mL of fresh LB medium, and 5 mL of the phytopathogen filtrate was added. The KW16 strain grown in LB medium without the filtrate served as a control. Cultures were incubated with shaking (130 rpm) at 30 °C for 72 h. After 24 h, 48 h, and 72 h of incubation, cells were harvested by centrifugation at 4415× g for 15 min, and siderophore production was quantified as described in Section 2.5.3. Two biological and eight technical replicates were performed.

2.9. Genome Sequencing and Sequence Analysis

Genomic DNA of P.megaterium KW16 was extracted using the GeneMatrix Bacterial and Yeast Genomic Purification Kit (EURx). The sequencing was performed with MicrobesNG using the Illumina MiSeq platform with 2 × 250 bp paired-end reads. The results of the sequencing were put through a standard MicrobesNG analysis pipeline and were submitted to the GenBank database under the accession number JAHTKR000000000.1. Functional annotation of the genes was performed using a multitude of tools and databases, such as the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (www.ncbi.nlm.nih.gov/genome/annotation_prok/, accessed on 24 November 2020) and the eggNOG orthology prediction pipeline (http://eggnog5.embl.de, accessed on 24 November 2020) [46]. The genes that were assigned to multiple COG (clusters of orthologous groups) categories were counted as being present in each of these categories. For the annotation of gene function, genes were compared to the KEGG database (Functional Kyoto Encyclopedia of Genes and Genomes database) (http://www.genome.jp/kegg/, accessed on 24 November 2020) [47]. The identification of gene clusters responsible for the biosynthesis of secondary metabolites was performed using antiSMASH (https://antismash.secondarymetabolites.org/, accessed on 17 December 2020) [48]. The CAZy database (Carbohydrate Active Enzymes database, http://www.cazy.org/, 15 June 2021) was used to classify cell wall degrading enzymes (CWDEs) and divide them into different families. CAZy families were identified with dbCAN2 according to the DIAMOND database [49,50]. Sequencing data and assembly were submitted to a public NCBI database under the BioProject accession number PRJNA529642.

2.10. Statistical Analyses

All data presented in this study are expressed as the mean values and were analyzed using Statistica^®13.3 PL (StatSoft^® Inc., Tulsa, OK, USA). A Student’s t-test or a one-way analysis of variance (ANOVA) was performed to evaluate the effect of KW16 and/or RS application on bacteria or plants. For samples with normal distributions, homogeneity of variance was assessed using Levene’s or Brown Forsythe test, depending on sample size. Tukey Honestly Significant Difference (HSD) test at the 5% level of significance (p ≤ 0.05) was used for the comparisons of means. The correlation between plant fitness indices (shoots and roots length, mass, and pigments) and the application of microorganisms was determined via Principal Component Analysis (PCA). For data without a normal distribution, the non-parametric U Mann–Whitney test was used. For all data, the different lowercase letters indicate statistical significance between the samples at p ≤ 0.05 level.

3. Results and Discussion

Microorganisms capable of degrading xenobiotics have been studied at our institute for years. The P. megaterium strain KW16 was isolated from the roots of the bluegrass (Poa pratensis L.) growing in the soil around the Kalina reservoir contaminated with various toxic compounds, including aromatic hydrocarbons and heavy metals. This strain showed the ability to degrade phenol and its chlorinated derivatives, as well as benzoate and catechol. It also showed resistance to high concentrations of zinc, copper, cadmium, and nickel. Nowadays, there is increasing scientific interest in searching for microorganisms that combine the characteristics of a good plant growth promoter and biocontrol agent and show survival ability in extreme and contaminated environments. Therefore, we attempted to find out whether strain KW16, as an endophyte, is capable of colonizing plants other than grasses, whether it significantly promotes their growth and development, and whether it can assist plants in their fight against pathogens.

3.1. Priestia megaterium KW16 as an Effective Biocontrol Agent

In the first stage of the study, we tested the ability of strain KW16 to inhibit the growth of R. solani, a common pathogen of oilseed rape. Many studies indicate that vast genera of strains with confirmed PGP traits may lack the ability to control the growth of phytopathogenic fungi in culture, depending on the identity of competing fungi, the mode of transmission of antifungal substances in the environment, and the composition of the growth medium, which may affect the interaction outcomes by modulating antifungal molecules [51]. That is why experiments were conducted on PDA and LBA solid media to assess the effect of diffusible compounds secreted by bacteria. In addition, the impact of microbial volatile compounds was studied on bipartite plates where bacteria and fungi and the media in which they grew did not contact each other.

When microorganisms were grown separately on the LBA or PDA medium, a significant 30% difference was found in fungal growth (Figure 1a,d) in favor of the PDA (Figure 1a). The media also affected mycelial morphology. Similarly, differences in bacterial growth were observed, with the ability of the bacteria to move (motility) and enhanced expansion visible solely on LBA (Figure 1e). Next, dual-culture assays were performed, and it was revealed that the endophyte inhibited the fungus growth in each system relative to the control (Figure 1a,d). This effect was evident to the greatest extent on the LBA medium (58%) and to the least on the PDA medium (21%), as shown in Figure 1b and Figure 1e, respectively.

The almost three-times more potent inhibition on LBA was most likely caused by the fact that the KW16 growing on this medium secreted more antifungal substances and/or they had a different composition than those produced on the PDA medium. The bacteria not only reduced the growth of the fungus but also induced changes in mycelial morphology and color. A lower (50.5%), though statistically insignificant, inhibition than that observed for LBA was exerted by the volatile compounds secreted by the bacteria (Figure 1c). It can, therefore, be assumed that strain KW16 controls R. solani mainly through the secretion of volatile compounds. The ability to produce such substances gives the bacteria a significant advantage as microbial volatile compounds are effective even at low concentrations and do not require physical contact with pathogens since they can be transferred over long distances.

In contrast, the lesser inhibition of pathogen expansion observed on the PDA medium may have been due to several factors. First, the production of volatile organic compounds by bacteria may require specific ingredients in the medium, as many studies indicate. For example, Huang et al. [52] observed significantly higher (up to 90%) growth inhibition of R. solani and Pythium phanidermatum (Edson) by culturing B. mycoides on protein-rich media, while on PDA, the bacteria showed no biocontrol activity. A similar relationship was observed by Prigigallo et al. [53] in a study in which bacteria did not show biocontrol ability when cultured on media without peptone or tryptone. These components were necessary to convert amino acids into the antifungal biogenic ammonia, which can act directly on the fungal pathogen or indirectly by increasing the pH of the agar, a phenomenon also observed in other studies [54]. The outstanding antifungal activity of KW16 cultured on LBA medium may also be related to the production of specific volatile organic compounds (VOCs) dependent on the composition of the medium, e.g., when growing on M9 medium, different strains of B. amyloliquefaciens produced 2,3-butanediol and acetoin. In contrast, TSA and LBA media favored the synthesis of the antifungal 5-methylheptanone, 2-methylpyridine, and 2-pentanone [55].

The inhibition of fungal growth observed on PDA may be related to the ability of P. megaterium KW16 to secrete diffusible antifungal agents (e.g., enzymes, soluble bioactive secondary metabolites—see later sections), which can be produced on PDA, the standard medium used to test biocontrol activity against fungi. However, the effect may be less pronounced than on LBA, a medium that favors the synthesis of volatile compounds, because diffusible compounds are only transported over short distances between organisms, also requiring higher concentrations of signaling molecules [56]. Significant differences in the degree of pathogen inhibition on different media are illustrated in Figure 1f.

3.2. Priestia megaterium KW16 as Pronounced Plant Growth Promoter

The next step was to evaluate the effect of inoculation of the KW16 strain into the soil on plant growth and development. For the pot study, we chose oilseed rape as a crop of great economic importance and with higher nitrogen, phosphorus, and sulfur requirements than other crops [57]. The increase in the length and weight of the shoots and roots of this plant was studied over 35 days. To further assess whether the effect of KW16 introduction into the soil on plants is due to colonization of plant internal tissues, it was necessary to use spontaneous rifampicin-resistant mutants, as this approach allowed their selective isolation from soil and plants [58]. The colonization of plant internal tissues had to be confirmed because, according to Burns et al. [59], plant species and specific root exudates are among the most critical factors for successful PGPB colonization, and KW16 was isolated from bluegrass, not oilseed rape.

On day 14, after sowing, no significant effect of the bacterial strain on the tested plant parameters was observed. This effect became apparent in the following weeks when all parameters of the control and inoculated plants differed significantly (p < 0.05). The most significant differences, even at p < 0.01, were observed after 35 days in plant weight when the shoots and roots of inoculated plants were about 30 and 60% longer and about 130% heavier than the control plants (Figure 2c–f). As can be seen from Figure 2a,b, after 35 days of the experiment, leaf numbers, total leaf areas, and development of lateral roots (LR) were significantly higher than the control plants.

The positive effect of bacteria of the genera Bacillus and Priestia on plant length and weight has long been indicated. Among others, Priestia sp. LWS1 increased the biomass of rice shoots and roots by 63% and 47%, respectively [60]. Li et al. [22] also showed that plant inoculation with the P. megaterium strain JR48 significantly increased the fresh weight of Arabidopsis, Chinese cabbage (Brassica rapa ssp. chinensis), and tomato (Solanum lycopersicum) seedlings in a dose-dependent manner. Up to a 0.3-fold increase in the fresh weight of plant seedlings was observed. However, a clear answer regarding what factor induces the changes is still being sought. It is known that auxin synthesis is responsible for plants’ morphogenesis and root/shoots development. A study by Hwang et al. [61] showed that inoculation of Arabidopsis and pak choi plants with IAA producing P. megaterium strain BP-R2 resulted in heavier, taller, and larger plants. This was also confirmed in studies on the B. megaterium strain CDK25, which induced the growth of Capsicum annum L. (chili plant) [62]. In addition to modulating phytohormones, PGPB increases external plant nutrient availability and improves plant biomass yield. As the main bacterial target of PGP, the root is the first organ to show morphological and functional changes after bacterial infection. The most prominent effect of PGPB is the increase in lateral root development, which facilitates gas diffusion, water, ion, and nutrient uptake, thus contributing significantly to plant growth [63]. For example, readily available iron can stimulate LR emergence and elongation when locally available to plants [64] through bacterial siderophore production. In the case of nitrogen supply, root length increases under mild conditions and decreases under severe nitrogen deficiency. Interestingly, deficiency in other nutrients results in a progressive reduction in total root length [65,66]. In our study, a more than 100% increase in the roots’ biomass, with a significantly higher number of additional lateral roots in oilseed rape, was observed as compared to the control. Such changes in the overall root morphology can also result from altered nutrient balance induced, e.g., by biogenic ammonium, which stimulates branching [66,67]. The role of VOCs produced by Bacillus resulting in an increase in lateral roots and root hair was also reported by Fincheira et al. [68] and Gutiérrez-Luna et al. [69].

3.3. Priestia megaterium KW16 as an Efficient Plant Protector

The search for ever-new strains beneficial in reducing plant fungal diseases has led to the selection of many strains from the genera Bacillus and Priestia. Among them are B. amyloliquefaciens LMR2, B. halotolerans (SF3 and SF4), and B. mojarvensis SF16, with high efficacy in reducing fire blight of apples [70]; B. velezensis FZB42 (commercial product), inhibiting lower lettuce rot caused by R. solani or B. amyloliquefaciens SF14; and B. amyloliquefaciens SP10, inhibiting Monilinia fructigena and M. laxa, the efficacy of which was only slightly lower than that of commercial synthetic fungicides [71,72,73]. The effectiveness of endophytes against various diseases depends on many factors. For example, Liu et al. [74] found that the growth of endophytic bacteria depends on the source plants and their secondary metabolites and activities; in this study, P. megaterium P-NA14 and P. megaterium D-HT207 showed higher efficacy against pathogens associated with the host plant and much lower efficacy against fungal pathogens associated with other plant species. Therefore, further studies assessed whether the endophytic strain KW16 derived from the bluegrass has a protective effect on oilseed rape sown in R. solani-infested soil, since in vitro assays do not necessarily reflect the strength of antagonistic interactions of bacteria with fungi in planta [75,76]. One of the reasons might be that the amount of secondary metabolites produced by microorganisms in the in vitro systems is likely much higher than that reached in natural habitats [77].

Our previous work confirmed the ability of R. solani W70 to damage oilseed rape seedlings from the very early stages of plant development in sterile, soilless cultures [27]. In this study, statistically significant differences between control plants (sown in RS-infested soil) and plants sown in RS-infested soil and inoculated with KW16^RIF were observed in the second half of the experiment. The most significant differences (at p < 0.01) for both groups were shown in the shoot and root weights on the 35th day of cultivation, which are also visualized in the photo (Figure 3a,b).

In addition, a comparison of the shoot and root lengths of the control plants (C) (Figure 2a) and plants grown in R. solani-infested soil (RS), as visible in Figure 3a (both groups not inoculated with bacteria), showed that oilseed rape infected with RS had 15% shorter shoots but 46% longer roots.

There are some potential explanations for this phenomenon. The first is based on the “cry for help” hypothesis, by which plants recruit beneficial microorganisms from the soil that can help fight the pathogen after initial contact and disease outbreak. Higher amounts of beneficial microorganisms improve nutrient uptake and activate the plant defense system, which may, at that moment, influence infected plants’ growth [78,79]. Thus, in our experiment, root growth-stimulating, yet unknown, microorganisms that plants recruited in response to the presence of the pathogen may have been present in the soil from the oilseed rape fields used in pot experiments. The second explanation is based on the theory that some pathogens manipulate auxin signaling, encouraging plants to develop wounds with emerging lateral roots that can be an entry point for pathogens [80]. An interesting experiment on Brassica rapa showed that pathogenic fungi R. solani produce VOCs that attract the roots of plants growing in soil, gaining an advantage over them [81]. Also, others reported that fungal volatiles promoted root biomass in vitro. Research by Cordovez et al. [82] revealed that VOCs released from R. solani mycelium and sclerotia enhance A. thaliana root biomass growth, thus predisposing plants to infection. As a result, the authors observed significant increase in shoot weight of 96%.

Inoculation with KW16^RIF abolished the adverse effect of R. solani on the oilseed rape shoot length and enhanced root growth. A comparison of the average shoot and root lengths of RS-infested plants with those inoculated with RS + KW16^RIF showed that the addition of the bacteria supported shoot length growth by 66% and root growth by 30% (Figure 3c,d), while shoot and root weights increased by 216% and 1737%, respectively (Figure 3e,f). The most likely explanation is related to the increased recruitment of inoculated bacteria from the soil under the influence of signals sent by the fungus, whose increased ‘rescue’ signal significantly stimulated the growth of plant roots and shoots.

3.4. Priestia megaterium KW16 as a Facultative Endophyte

Despite many years of work on the effects of endophytic bacteria on plants, only a few papers have dealt with tracking bacteria in the rhizosphere and plants, mainly because of the complex procedures involved in the study [83]. In this work, we used stable rifampicin mutants, whose isolation on a selective medium allowed us to trace the effect of the presence of R. solani on KW16^RIF behavior related to the protective impacts on oilseed rape. The addition of rifampicin into the LBA medium effectively inhibited the growth of soil and endophytic bacteria other than the KW16^RIF mutant strain, allowing only rifampicin-resistant KW16 to be recovered from the inoculated soil and plants. To assess whether the endophytic strain introduced into the soil can survive directly in the soil and/or whether it can efficiently colonize plant tissues to exhibit long-term effective action, its survival in soil, as well as shoots and roots of oilseed rape, was tested. These relationships were tested for both the system in which the soil was inoculated solely with bacteria (KW16^RIF) and the system infested with R. solani and inoculated with bacteria (RS + KW16^RIF). It was found that on day 14 after sowing, in the KW16^RIF system, bacteria were only found in the soil (0.9 log CFU/g soil), whereas, in the presence of the fungus, bacteria colonized not only the soil (0.94 log CFU/g soil) but above all the interior of plant root tissues, where they multiplied to 37 log CFU/g roots fw (Figure 4a).

On day 28 of the experiment in the bacterium system, microorganisms colonized both the soil and the roots of the oilseed rape to amounts of 0.72 CFU/g soil and 2.65 log CFU/g roots fw, respectively. In the system with the fungus (RS + KW16^RIF), the presence of bacteria was recorded in quantities similar not only in the soil and roots but also in the shoots (2.77 log CFU/g shoots fw) (Figure 4b). On day 35, the number of microorganisms and their location in the bacterial system did not change significantly. The presence of RS did not affect the number of bacteria in the soil but stimulated bacterial proliferation in the roots, where the number of bacteria increased to 3.5 log CFU/g roots fw. No bacteria were isolated from oilseed rape shoots (Figure 4c).

In our experiment, a distinct translocation of bacteria from the soil to the roots and shoots of plants was observed in a fungus–bacteria system. The number of bacteria in the soil did not change significantly, irrespective of whether the soil was infested with fungus, which meant that after 2 weeks, the equilibrium of the amount of introduced strain in the soil had been established. In contrast, a fascinating pattern was observed when colonizing the interior of plants. For plants grown in non-infested soil, a constant equilibrium of the amount of KW16^RIF inside the roots was established. However, the number of bacteria was lower than indicated in the literature for sub-populations inhabiting roots ranging from 5 to 7 log CFU/g fresh weight [84]. In contrast, the significantly higher number of bacteria in the interior of plants growing in the presence of R. solani was probably related, as mentioned earlier, to their increased recruitment by the plants to protect themselves from the pathogen. The intensified recruitment on day 14 of cultivation probably also resulted in the entry of some bacteria, e.g., via the transpiration stream or intercellular spaces to aerial parts, which was only observed after 28 days of cultivation. According to Compant et al. [85] and Afzal et al. [86], PGPB can establish stem and leaf population densities between 3 and 4 log CFU/g fw under natural conditions. The decrease in bacterial counts in plants on day 35 of the experiment to amounts comparable to the uncontaminated system was most likely due to the activation of systemic plant resistance (ISR), which not only allowed plants to fight off the pathogen but also prevented over-colonization by P. megaterium KW16. Zinniel et al. [87] reported that contrary to pathogenic bacteria found in plant tissues in amounts around 7–10 log CFU/g fw, maintaining low cell densities (between 2 and 6 log CFU/g fw) by endophytic bacteria is crucial for avoiding being detected by the plant.

3.5. Priestia megaterium KW16 as an Active Colonizer

The active penetration of plants by bacteria requires a set of complex machinery involving, as the first step, biofilm formation and the secretion of cell wall-degrading enzymes at the level that prevent triggering the plant defense system. Insufficient rhizosphere colonization by PGPB is commonly related to variable and inadequate biocontrol activity in field tests [12]. In our work, when assessing the effect of P. megaterium KW16 on oilseed rape growth, it was noted that the bacterium has a promising ability to colonize its roots and shoots, particularly in the presence of R. solani (Figure 4), which may be due to its ability to move towards plant-specific exudates. The ability of the KW16 strain to form a biofilm and characteristics related to this process, such as aggregation and siderophore synthesis, were further investigated. Since substances secreted by pathogenic fungi can affect the mentioned features, the effect of filtrates containing R. solani exudates after 24 h, 48 h, and 72 h of contact was also assessed (Figure 5).

Generally, the ability to auto aggregate may be fixed or reveal itself phenotypically under the influence of an inducer, which may be an environmental abiotic or biotic factor or the need to occupy and persist in a new ecological niche [88], since rapidly aggregating bacteria are more resistant to adverse environmental conditions [89]. After the first 24 h of the experiment, the aggregation of the bacteria recorded after 4 h from the start of the measurement did not change significantly in the control system (KW16) compared to the bacteria in contact with the fungal filtrate. In contrast, after 48 h, a sharp 3-fold increase in aggregation was observed for the control system and a 2-fold increase for the bacteria incubated with the filtrate. After 72 h, a proportional reduction in this capacity was observed in both systems, with the control system still showing faster auto-aggregation (Figure 5a). The reduced auto-aggregation capacity of strain KW16 under the influence of fungal filtrates and the substances contained therein may have been due to the presence of biosurfactants, which, by adhering to bacterial cells, may alter their physicochemical properties, including hydrophobicity. It may have favored cell retention in suspension, limiting the capacity for aggregation [90]. The biosurfactants may have been the metabolic products of both R. solani and the bacterium, since P. megaterium is known for its efficient production [91,92]. It was also reported that through intercellular communication, bacterial biosurfactants facilitate plant colonization by affecting motility, virulence, and biofilm formation [93].

Biofilm formation on plant roots has several functions. Firstly, by remaining in close contact with the plant, bacteria reduce losses in the mutual exchange of nutrients and hormones. Secondly, by enveloping plant roots, bacteria protect them from invasion by other organisms through antimicrobial compounds secreted in various effective local concentrations. Thirdly, bacteria in the biofilm are more resistant to biotic and abiotic factors, which provides an advantage when occupying a new niche. Checking this ability is, therefore, crucial in identifying a strain with PGP properties. The tests that were conducted showed that KW16 was capable of biofilm formation. The ability of the culture to form a biofilm increased with time. In the control culture, this was because the bacteria entered a metabolic state in which cells produced more extracellular polymeric substances, which are crucial for establishing the bacterial biofilm. The bacteria incubated for 72 h with the fungal filtrate produced a significantly more abundant biofilm than the control cultures (Figure 5b). This may indicate that the previously observed inhibition of planktonic cell auto-aggregation did not reduce the ability to form a biofilm.

Root exudates drive another factor that influences plant colonization. Siderophores secreted by PGPB recruited in the root zone contribute to changes in the social motility of bacterial cells and promote biofilm formation. In addition, they exhibit indirect antagonism against pathogens via competition for nutrients and for the occupation of niches [12]. Our results regarding the secretion of siderophores by KW16 (control) agree with the work of Santos et al. [94], where B. megaterium continuously produced siderophores throughout the stationary growth phase. In turn, in the presence of fungal filtrates (RS + KW16), a significant (p < 0.01) increase of more than 200% in their concentration relative to the control, was already observed after the first 24 h of incubation. Over the next two days, we observed fluctuations in their concentration, with a maximum after 48 h (Figure 5c). This capability for detecting fungi and their metabolites and the overproduction of certain compounds, including siderophores, can affect the pathogen by destroying it. Bacteria of the Bacillaceae family can synthesize a mixture of siderophores and other secondary metabolites, the composition and action of which depend on the fungal competitors [95]. An example is bacillibactin—a siderophore secreted by all members of the B. subtilis species complex, whose synthesis seems dependent on a fungal competitor and is linked with direct antifungal activity. One of many examples might be the reaction of B. amyloliquefaciens, for which R. solani triggered the overproduction of bacillomycin, while the opposite response was observed upon contact with Rhizomucor variabilis [96,97].

Endophytic bacteria may enter plants passively through root tips, cracks, or lesions. However, the determining factor for the effective colonization and spread in plant tissues is the production of CWDEs, including cellulase. Tests carried out on plates containing cellulose derivative showed that KW16 was capable of producing and secreting this enzyme directly to the medium. In addition, the enzyme showed high activity, as can be seen by the significant clarity visible in the image (Figure 5d). The presence and activity of cellulose-hydrolyzing enzymes in endophytic strains belonging to different genera, including Bacillus and Priestia, have been confirmed by numerous studies [98,99].

3.6. Priestia megaterium KW16 as a Plant Metabolism Booster

It is known from the literature that the quantity and quality of pigments synthesized by plants in leaves vary with environmental conditions and the physiological state of the plants. Since the colonization of plants by growth-promoting bacteria and the presence and attack of pathogens can affect their synthesis or degradation, plant metabolite parameters such as NBI, Chl, Flav, and Anth indices were studied after 35 days of plant growth in the tested systems (Figure 6).

There were no statistically significant differences observed in the chlorophyll index in any of the experimental setups (Figure 6b). It has been reported that R. solani, as a necrotrophic fungus, can hijack some plant components, known as susceptible genes, to induce host susceptibility to fungal infection. Upon infection, plants often respond by cell death and production of ROS, a strategy effective against biotrophic pathogens [100,101]. Unfortunately, for necrotrophs producing additional phytotoxic compounds to exacerbate cell death further, such signals might promote infection [102]. Therefore, most studies on fungus-infected plants indicate that the amount of chlorophyll in infected plants decreases significantly after contact with pathogens [3,103,104]. In a study by Cao et al. [100] on rice and maize with greater resistance to R. solani, it was shown that their ability to inhibit chlorophyll degradation induced by contact with the pathogen prevented senescence in these plants and thus limited the spread of fungal infection. Since our previous study [27] showed a destructive effect of R. solani on B. napus seedlings, we were confident that the plants were not resistant to the fungus. In our current study, therefore, there must have been a factor that prevented chlorophyll degradation in plants incubated in R. solani-infected soil. Thus, the only reasonable explanation for the lack of chlorophyll degradation in oilseed rape leaves could be the presence of other indigenous microorganisms in the soil, as previously mentioned, which had a protective effect on germinating and growing plants.

Flavonoids are another group of plant pigments important in the context of pathogen invasion. They control plant cell wall synthesis, participate in mutual interactions between plants and symbiotic microorganisms, and also act as phytoalexins that modulate plant defensive mechanisms, e.g., sakuranetin, which improved the plant’s resistance to R. solani [105]. Moreover, catechins and rutin directly inhibit pathogen invasion by preventing biofilm formation, disrupting the fungal cell wall, or uncoupling the electron transport chain [106]. In our study, a higher flavonol index was observed in all experimental setups compared to the control. However, these differences were not statistically significant (p > 0.05) (Figure 6c). Interestingly, the Anth index was significantly lower in the plants treated with the fungus and bacterium than in the other plants. Reduced anthocyanin concentrations with increased flavonoid synthesis in tomato plants after R. solani contamination were also observed in previous work [3]. Confirmation of the protective role of flavonoids can be found in a study conducted by [107], which showed a correlation between plant infection and increased expression of genes coding for isoflavonoid-specific enzymes involved in the biosynthesis of phytoalexins. Also, PGP bacteria may induce flavonoid production in plants, increasing the plant defense system [105]. For example, Li et al. [107] inoculated Nanguo pear fruit with P. megaterium strain PH3 and observed an enhanced and prolonged production of phenolics and flavonoids. A similar trend was observed in their previous study [22] on the protective role of P. megaterium strain JR48 upon infection of A. thaliana by the pathogenic bacterium Xanthomonas campestris. Strain JR48 induced the biosynthesis of lignin and secondary metabolites such as flavonoids and salicylic acid in planta. As a result, the enforced plant cell wall more efficiently hinders pathogen entry, while phenolic compounds can be converted to quinones with antimicrobial activity.

Nitrogen balance index (NBI) reflects the ratio of chlorophyll to epidermal flavonoids (Chl/Flav) [108]. It is widely used for diagnosing plants’ N nutrition, crucial for better crop yield prediction. Our study showed no statistically significant differences between the NBI in all four systems (Figure 6b).

In order to assess the mutual correlations between the determined plant parameters, Principal Component Analysis (PCA) was used. Three main components described 85.5% of the variability, with the most significant contribution from principal component 1 (PCA1), which accounted for 43.99% of the total variability, and second principal component (PCA2), which accounted for 24.18% of the total variability. In total, the two components explained 68.17% of the variability. PCA1 can be characterized as a factor depicting plant growth promotion, and PCA2 as a factor representing plant metabolic status.

As one can see from Figure 6d,e, plants can be divided into four clusters depending on the experimental setup. The group of plants treated with RS emerged as a separate cluster due to differences in metabolic status relative to the control group. On the other hand, the two groups treated with bacteria were separated primarily due to the intensity of the plant growth promotion effect, although some plants manifested changes similar to the pathogen-treated group. This was probably because the presence of R. solani in the soil did not induce plant disease, despite the detrimental effect previously found on oilseed rape [27]. However, the fungus and its diffusible and volatile secondary metabolites may have stimulated the plant defense system by acting similarly to vaccines. A growing body of evidence suggests that adequately severe disruption of plant homeostasis can result in increased plant fitness and resistance to a wide range of pathogens [109]. Ethylene, salicylic acid, and jasmonic acid—phytohormones playing a central role in plants’ defense systems—are orchestrated by such disturbances triggered by abiotic and biotic factors. The resultant resistance is determined by the interplay between these pathways [110]. For example, research conducted by Trapet et al. [111] shows that Fe deficiency arising from the activity of fungal siderophores induces plants’ defense against B. cinerea through the ethylene-signaling pathway. In turn, B. velezensis VOCs triggered a salicylic acid-mediated defense signaling pathway, resulting in improved plant biomass, chlorophyll content, and biocontrol effectiveness in pepper seedlings [112]. Sharifi and Ryu [113] have found that the application of B. subtilis GB03 volatiles elicited 90% of ISR and prevented the invasion of B. cinerea into A. thaliana plants, even though the bacteria themselves had not shown any antifungal activity in vitro.

3.7. Genetic Potential and Biochemical Features of KW16 as a Comprehensive PGP and Biocontrol Factor

The ability of bacteria to invade plant internal tissues and fight pathogens is determined by chance features and bacterial genetic determinants that enable mutual crosstalk between plants and bacteria and between bacteria and pathogens [83,86]. Therefore, upon demonstrating the positive effect of the endophytic P. megaterium KW16 on plant growth and protection against the fungal pathogen, its genome was explored, and biochemical tests were carried out to shed light on the potential of the strain for PGP and biocontrol activity at the molecular level.

The general properties of the sequenced genome are presented in Table S3 (Supplementary Materials). As a result, 6.167.380 bp were assembled into 141 contigs with an average G + C content of 64%. In total, 6402 genes, of which 6231 were annotated as coding DNA sequences (CDSs), 123 tRNAs, and 40 rRNAs, have been predicted. Additionally, 5323 out of the 6231 predicted CDSs (85.40%) were, assigned to a cluster of orthologous groups (COG). The genome of the KW16 strain was enriched with genes belonging to five COG categories: transcription (K category, 10.05%), amino acid transport, and metabolism (E category, 10.84%), inorganic ion transport and metabolism (P category, 6.73%), carbohydrate transport and metabolism (G category, 7.50%), cell wall, membrane, envelope biogenesis (M category, 4.72%), and energy production and conversion (C category, 5.56%). Specifically, the number of genes participating in the G and E categories indicates the ability of KW16 to metabolize the wide carbon and nitrogen sources. It is worth noting that the metabolism class was the most significant functional group, which assigned 2262 predicted genes, followed by the poorly characterized class (1381, 25.90%), information storage and processing class (943, 17.70%), and cellular process and signaling class (737, 13.82%). The KEGG functional analysis identified 3125 genes (50.20% of predicted CDS). The most significant fraction of these genes were involved in the metabolism of carbohydrates (343; 10.98%), amino acids (282, 9.09%), cofactors and vitamins (171, 5.47%), and energy metabolism (137, 4.38%). Figure S1 and Table S4 (Supplementary Materials) present the CDS numbers allocated to the KEGG and COG. Furthermore, we analyzed the potential production of secondary metabolites using the antiSMASH tool, finding possible gene clusters involved in the biosynthesis of a siderophore, terpene, lassopeptide, phosphonate, and lanthipeptide (Table S5—Supplementary Materials). The functional annotation of proteins based on the eggNOG and KEGG database revealed crucial genes contributing to plant-beneficial and biocontrol activity of the KW16 strain, and were grouped according to their general roles.

It is well documented that the first step of effective plant root colonization involves the coordinated expression of many genes engaged in bacterium motility, chemotaxis, and adherence [114]. Bacterial motility, which depends mainly on the presence of flagella or pili, is one of the key factors promoting adhesion to various surfaces and biofilm formation [115]. The genome of the KW16 strain showed the presence of a number of genes involved in flagella biosynthesis and folding, such as genes of the flb, flg, and fli operons (Table S6—Supplementary Materials).

The results of tests on solid media with increasing agar content confirmed its ability to swim and express swarm motility (Table S7—Supplementary Materials), which is powered by rotating flagella. Twitching motility, a slow surface movement not requiring the locomotion structure [116], was also observed.

Swarming is described as a social form of motility that allows flagellated bacteria to travel rapidly toward a nutrient-rich environment, and it is supposed to be crucial for plant colonization by bacteria [117]. This is confirmed by the results of Gao et al. [118], among others, on tomato root colonization by mutants of B. subtilis SWR01 defective in motility and chemotaxis. The active movement of bacteria oriented by chemotaxis is determined by the presence of genes responsible for transmitting external environmental signals, modulating the activity and direction of rotation of the bacterial flagellum. An essential role in this process is played by methyl-accepting chemotaxis proteins (MCPs) belonging to trans-membrane receptors found in the inner cell membrane of many bacteria [119]. In silico analyses confirmed the presence of genes crucial for chemotaxis in the KW16 genome (Table S6—Supplementary Materials). Apart from genes for the MCPs, che, and mot genes encoding response regulators, chemotaxis, signal transduction, and flagellar motor proteins were identified. The importance of chemotaxis in plant colonization was reported for B. subtilis FB17 in A. thaliana [120] and B. subtilis N11 in cucumber and banana roots [121]. The studies on B. subtilis mutants also confirmed the significant role of genes involved in chemotaxis. Allard-Massicotte et al. [122] showed that mutants in 10 genes encoding chemoreceptors exhibited a reduced ability to colonize Arabidopsis roots compared to the wild strain.

The auto-aggregation and biofilm formation ability confirmed by biochemical tests and genetic analysis (Figure 5, Table S6 and S7—Supplementary Materials) highlights KW16’s potential to colonize plants and thus express its capacity to promote and protect plant hosts against biotic and abiotic stresses. One crucial component of biofilms are EPSs, affecting bacteria aggregation efficiency and attachment to the root surface [123]. These polymeric compounds also contribute to the survival of bacteria in the plant or soil by acting as a barrier to plant defense mechanisms or protecting against environmental factors such as drought or predation [124]. The production of polysaccharides by KW16 was confirmed by plating the strain on the CRA medium, while the genome analyses revealed the presence of algA involved in the biosynthesis of alginate and the hxlBAR cluster engaged in the biosynthesis of polysaccharides (Table S6—Supplementary Materials).

Plant host colonization is associated with osmotic stress, which bacteria can cope with by synthesizing and accumulating osmoprotectants. Similarly to P. megaterium ZS-3 [24], in the KW16 genome, we identified key genes involved in the mitigation of osmotic stress, including genes for the biosynthesis of highly soluble molecules such as proline (pro) and glutamine (gln), along with genes involved in glycine betaine/proline transport (proPVWX). In addition, the des genes encoding fatty acid desaturase and the clsB gene for cardiolipin synthase, and enzymes modulating lipid and fatty acid composition in the lipid membrane, thus playing a central role in the tolerance to salt and to cold stress, were also found (Table S6—Supplementary Materials).

To successfully colonize a plant, bacterial endophytes must be adapted to survive in an environment rich in reactive oxygen or nitrogen species, which plants produce as a mechanism protecting against viral, bacterial, or fungal infections, or during colonization by endophytic organisms [98,123]. Therefore, plant-colonizing organisms are equipped with mechanisms to overcome oxidative stress. A wide range of genes responsible for the oxidative/nitrosative stress response have been identified in the genome of P. megaterium KW16, including catalase (catE, catX, catA), superoxide dismutase (sodA), thiol peroxidase (tpx), or nitric oxide dioxygenase (hmp) genes (Table S6—Supplementary Materials). Similarly to other known plant colonizers and biocontrol agents [125], KW16 produced catalases and oxidases (Table S7—Supplementary Materials), enzymes capturing toxic free radicals generated by abiotic and biotic stresses and enabling them to enter and settle in B. napus tissues and protect the plant from the oxidative damage caused by R. solani infection. Since ROS are essential for vegetative hyphal growth and the differentiation of conidial anastomosis tubes, in addition to their protective activity, catalases may also have a crucial role in the biocontrol process. For example, Srikhong et al. [126] observed that the Bacillus sp. strain M10 produced a protein similar to vegetative catalase, the activity of which induced abnormal elongation of the shoots and swelling and cracking of C. capsici conidia.

The ability of KW16 to produce phytohormones, including IAA, is also a vital characteristic of the strain, positively affecting the growth and development of oilseed rape roots (Figure 1). The results of the biochemical tests show that strain KW16 produced this hormone at a concentration of 17.42 ± 0.9 μg/mL (Table S7—Supplementary Materials). However, since the strain’s ability to produce IAA was evaluated in a tryptophan-supplemented medium, it is impossible to assess the amount of IAA secreted by this strain in the plant. IAA production by endophytic strains in vitro is widely discussed in the literature. In a study by Kukla et al. [127], all strains tested were able to produce between 0.7 ± 0.1 and 132.0 ± 1.3 μg/mL of IAA, while a study conducted by Amaresan et al. [128] showed that endophytic bacteria isolated from tomatoes and chilies produced IAA at concentrations ranging from 15.0 to 59.2 μg/mL. However, since plant growth is determined by plant-synthesized auxin, IAA produced by the bacteria can only enhance shoot and root development in cases of low plant auxin levels. Therefore, it is difficult to determine at what concentration bacterial IAA can positively affect the plant. Analysis of the KW16 genome revealed genes encoding proteins related to auxin biosynthesis and modulation, including genes for tryptophan biosynthesis (trpABCDE) and conversion to IAA intermediates (yod, yug), indicating trp-dependent IAA production. The presence of the patB, yclB/yclC, and dhaS encoding tryptophan transaminase, indole-3-pyruvate decarboxylase, and indole-3-acetaldehyde dehydrogenase, respectively, suggests that in the studied strain, IAA synthesis proceeds through the indole-3-pyruvate (IPyA) pathway. However, the ycl genes might also be engaged in the transformation of tryptophan to tryptamine, a substrate characteristic of the TAM pathway of IAA biosynthesis [129]. Genes involved in IAA biosynthesis were also identified in the B. megaterium strain STB1, which increased the biomass of tomato plants [21], and the B. megaterium strain RmBm31, which significantly improved Arabidopsis seedling growth [25]. Apart from the genes engaged in IAA synthesis, several genes for polyamine metabolism (put, spd, spm) and transport (puuP, potABCD) were also identified in the genome of the strain. Polyamines such as putrescine, spermidine, and spermine play essential roles in plant growth promotion as they are among the synthetic precursors of GABA [130]. As an example, the GABA-producing B. velezensis strain FX-6 increased biomass in tomato plants by improving nutrient absorption and influencing hormone levels, promoting the elongation and differentiation of plant cells [131].

Another important mechanism that promotes plant growth is the ability of endophytes to produce ACC deaminase, reducing ethylene levels in the plant [132]. At low concentrations, ethylene regulates plant growth and various metabolic processes. However, under stress conditions, the ethylene concentration in plants increases significantly, adversely affecting root growth and the metabolism of the entire plant. The strain KW16 grown on DF medium, supplemented with 3 mM ACC as a nitrogen source, confirmed its ability to produce the enzyme (Table S7—Supplementary Materials) and suggested its role in reducing ethylene levels during rapeseed growth in soil infested with R. solani and in alleviating the adverse effects of the pathogen (Figure 3).

Genome analysis also confirmed the potential of the KW16 strain for ammonia production and acquisition. The presence of genes for urea degradation and transport (ureABCDEFGH), the amtB gene for ammonia transport, and a number of genes encoding enzymes involved in ammonia synthesis (Table S6—Supplementary Materials) were found. We hypothesize that ammonium produced by the strain might have contributed to promoting the growth of oilseed rape roots and shoots, thus increasing the fresh weight of the inoculated plants (Figure 2), and also helped the plants to minimize the effect of R. solani (Figure 3). Moreover, this inorganic volatile compound could negatively affect the growth of the pathogen (Figure 1).

Phosphorus is the primary nutrient mandated for plant growth and increased plant immune and defense mechanisms. The presence of genes of the pstABSC operon involved in phosphate transport, genes phoA, phoD encoding phosphatases, and phoR regulating phosphate uptake, together with genes involved in phosphate metabolism, e.g., phoQ, pqq, murF, pepM, phnW, confirms the ability of the strain KW16 to manage phosphate efficiently (Table S6). Indeed, the bacterium showed the ability to solubilize phosphate (PSI 1.3 ± 0.08) (Table S7), as evidenced by clear zones around the bacterial colonies on a medium containing tricalcium phosphate as the sole source of phosphorus (Figure S1a—Supplementary Materials). However, it is suggested that in vitro P solubilization by microorganisms might not be associated with the promotion of plant growth [133], and tricalcium phosphate is supposed to be unreliable as a universal selection factor for isolating phosphate-solubilizing bacteria (PSB) for enhancing plant growth [134]. Since PSB-gathering phosphates in the plant tissues deplete the nutrient pool for pathogens, phosphate solubilization may also be considered a pathogen control mechanism [135]. Therefore, the genes discussed above are also included in Table S7 (see further paragraphs).

Genes involved in sulfur metabolism in the KW16 strain are also noteworthy. Many bacteria can use sulfate as a primary sulfur source under sulfur starvation [136]. The assimilatory reduction in sulfate and the formation of cysteine comprise a sequence of enzymatic reactions catalyzed by adenosine 5′-phosphosulfate kinase, 3′-phosphoadenosine 5′-phosphosulfate (PAPS) sulfotransferase, and sulfite reductase (among others), encoded by cysC, sat, and cysIJ, respectively (Table S6). These genes were identified in the KW16 genome along with cysPW, which is responsible for the transport of sulfonates. In turn, the presence of ssuD and ssuE genes (Table S6) encoding alkanesulfonate monooxygenase indicates its ability to obtain sulfur from the environment by organosulfonate or sulfonate-derived compound utilization. Moreover, sulfur-oxidizing bacteria improve sulfur availability for many plants, including oilseed rape promoting their growth and inducing resistance against various pathogens such as R. solani [137].

Root exudates abundant in sugars, organic acids, amino acids, polyamines, fatty acids, purines, vitamins, and other potent sources of nutrients for PGPB have a massive impact on shaping the plant microbiome, especially since their composition varies according to the plant genotype and growth stage and can be modified in the presence of pathogens [98,138]. The positive chemotaxis toward plant exudates and the ability of bacteria to utilize them are crucial for the successful colonization of the root surface and competition with other microorganisms [139]. Biochemical tests showed that the strain KW16 exhibits metabolic activity and nutrient diversity, utilizing a wide range of organic compounds such as organic acids, phenolic compounds, and sugars (Table S7—Supplementary Materials). Furthermore, the COG and KEGG analyses of the KW16 genome (Figure S2 and Table S4—Supplementary Materials) confirmed the presence of many genes involved in the metabolic transformation of these compounds. We speculate that this metabolic diversity supported strain adaptability to the new environment during plant colonization and increased its potential to compete with microorganisms present in the soil. Moreover, this may explain the fact that the KW16 strain originated from the bluegrass was able to colonize the roots of the oilseed rape.

Except for the above-described potential of the KW16 strain for plant promotion, the tested strain holds tremendous potential as a biocontrol agent. The biochemical assays and genome analyses revealed many traits and mechanisms engaged in combating pathogens. An exciting feature is a spectrum of genes for hydrolytic enzymes (cel, yqeZ, amyY, ypmRS) (Table S8—Supplementary Materials) and the activity of cellulases, proteases, amylases, and lipases confirmed by biochemical tests (Table S7, and Figure S2b—Supplementary Materials). A wide array of hydrolytic enzymes produced by the strain may have the significant advantage of suppressing multiple pathogens present in the host rhizosphere, since it is believed that the hydrolysis of the cell wall of pathogenic fungi is the result of the coordinated action of the lytic enzyme complex. For example, the production of chitinase, β-1,3-glucanase, and protease by four strains of the genus Bacillus was an important mechanism responsible for inhibiting the growth of R. solani [19]. Similarly, the B. subtilis strain 330-2 isolated from rapeseed, which produced β-1,3-glucanase, β-1,4-glucanase, and proteases, strongly inhibited the growth of fungi from A. alternata, B. cinerea, C. heterostrophus, F. oxysporum, R. solani AG1-IA, and Nigrospora oryzae [140], and the cellulase along with extracellular pectinase and chitinase from the B. subtilis strain EG21 inhibited the growth of P. infestans and lowered zoospore germination and infection. In turn, in S. marcescens B2 and S. proteamaculans 568, the antagonistic effect correlated with high chitinase activity [141,142]. Strain KW16 did not exhibit chitinolytic activity. However, as shown in studies by Kamenski et al. [143], chitinase was not essential for the biocontrol of B. cinerea and Sclerotinia sclerotiorum by Serratia plymuthica IC14 in synthesis of other enzymes, such as proteases. Similarly, in Paenibacillus sp. B2 activity against Phytophthora parasitica among various hydrolytic enzymes, only proteases were responsible for inhibition of the mycelial growth [144].

Competition for nutrients, especially iron, is an essential antagonistic trait against plant pathogens. It has been frequently described that siderophore-producing bacteria can reduce iron availability to phytopathogens, inhibiting their growth and development. In addition, they can sequester it around plant roots to toxic levels, preventing pathogens from surviving in such an environment. The vital role of siderophores produced by various antagonistic microorganisms has been proven to inhibit the growth of fungi from the genera Colletotrichum, Fusarium, Rhizoctonia, and Sclerotinia [27,145]. Many genes involved in iron transport and the production and transport of siderophores have also been identified in the genome of the KW16 strain. The analyses revealed the presence of feoA and feoB genes in a Feo system, which are common, especially for Gram-negative bacteria, and dedicated to iron [146]; yusV, yfhA, and yfiZ genes involved in the import of iron-hydroxamate and catecholate siderophores [147]; and genes involved in siderophore synthesis (rhbA, iucC). KW16’s ability to produce siderophores (Table S7—Supplementary Materials) was proved by the change in CAS medium color from blue to orange (Figure S2c—Supplementary Materials). The P. megaterium species is known to be a producer of hydroxamate siderophores. One is schizokinen and its derivatives, with a citrate backbone and a high affinity for iron, aluminum, and gallium ions [145]. Recently, Zhu et al. [148] isolated and characterized another, Ferrioxamine E [M + Fe-2 H], with the structure of a hexadentate octahedral complex. The siderophore engaged in the biological activity of KW16 could be rhizobactin 1021, a siderophore similar to the aerobactin commonly found in Enterobacteriaceae, as indicated by the presence of the rhbA and iucC genes.

Another important biocontrol feature identified in the KW16 genome is the presence of genes determining VOCs production. For instance, Nagrale et al. [149] showed the biocontrol potential of benzene, 1,3-diethyl- and 1,4-diethylbenzene, naphthalene, m-ethylacetophenone, and ethanone, 1-(4-ethylphenyl), produced by three rhizobacterial strains, B. cereus CICR-D3, B. aryabhattai CICR-D5, and B. tequilensis CICR-H3 against Macrophomina phaseolina. In contrast, VOCs secreted by B. megaterium BM344-1 not only inhibited the growth of fungal pathogens of Aspergillus, Penicillium, and Fusarium but also caused complete inhibition of the synthesis of aflatoxins, ochratoxin A, and fumonisin B1 by these pathogens [18]. Notably, in the genome of KW16, ilvB, ilvD, ilvE, ilvC, ilvA, ilvN, and bdhA genes involved in the synthesis of acetoin and butanediol, the two most studied VOCs, were present. As evidenced by many studies, VOCs act not only directly on pathogens but also enhance plant resistance to them and stimulate plant growth by inducing the expression of genes essential in SA, jasmonic acid, and ethylene signaling pathways [150]. Therefore, it can be speculated that the protective effect of the KW16 strain, which significantly inhibited the growth of R. solani by VOCs production (Figure 1c,f), may also be exerted indirectly by the positive influence of these compounds on oilseed rape fitness and the induction of ISR in the colonized plant. Moreover, as the KW16 strain synthesized ammonium (inorganic volatile compound), the pathogen inhibition and spectacular increase in the number of lateral roots in plants inoculated with the strain that was observed in the pot experiments (Figure 3) could be a result of the synergistic action of different volatile compounds.

Antibiosis is considered an essential mechanism for combating plant pathogens. According to many studies, bacteria of the Bacillus and Priestia genera are excellent producers of a wide variety of antimicrobial agents [14,150,151]. Genes determining the synthesis of biologically active secondary metabolites, including lanthionine-containing antibiotics, lipopeptides, polyketides, and phenazines, were also identified in the genome of strain KW16 (Table S8—Supplementary Materials). Of these compounds, documented antifungal activity has been shown for bacillomycin D of the iturin family, responsible for causing damage to the cell wall and membrane of the hyphae and spores of C. gloeosporioides [152], and for polyketides synthesized from carboxylic acids and phenazines—compounds with redox activity, which, apart from their antifungal activity, are known to enable the survival and persistence of bacteria in soil through a key role in biofilm formation and iron reduction [151]. Although the presence of genes for the above-mentioned antibiotics indicates the potential of the strain to synthesize them, confirmation of their contribution to the biological activity of the KW16 strain against R. solani requires further research.

3.8. Rhizoctonia Solani Affects Expression of Selected Genes in Priestia Megaterium KW16

Upon their growth, fungal pathogens produce and release a mixture of secondary metabolites, enzymes, phytotoxins, and volatile compounds that affect plants and nearby microorganisms in the soil. Sequencing and genome analysis of P. megaterium KW16 revealed the presence of many genes potentially involved in plant colonization and biocontrol mechanisms. To verify if the presence of R. solani filtrates influence the KW16 activity, the relative expressions of selected genes, gltB, iucC, katA, ilvB, bdhA, and bglA, were measured using RT-qPCR after 24, 48, and 72 h of culturing bacteria in the presence of fungal filtrates. Changes of at least 2-fold in the expression of a given gene were considered significant.

It was found that the strain KW16 showed significant differences in the expression of most of the genes in response to the presence of the phytopathogen. It was also observed that the expression profiles of individual genes varied significantly depending on the exposure time (Figure 7).

After 24 h of incubation in the presence of R. solani, significant changes were observed in the expression levels of iucC, gltB, and katA genes. The expression of the iucC gene increased by about 13-fold; a 10-fold increase in expression was observed for the gltB gene, while the expression of the katA gene was 2-fold higher compared to the control. In contrast, 48 h incubation of the KW16 strain in the presence of the fungal filtrate resulted in the overexpression of ilvB by about 3.5-fold, while the expression levels of the iucC and gltB genes were significantly lower when compared to the 24 h incubation. After 72 h of culturing the KW16 strain in the presence of fungal filtrates, the expression level of the katA gene was almost 3 times higher compared to the control. Longer incubation with the pathogen did not affect the expression of ilvB. However, the gltB gene was significantly down-regulated (Figure 7). The presence of R. solani has no significant impact on the expression of bglA gene encoding β-glucosidase (GH1), an enzyme involved in the degradation of β-glucosides, and the bdhA gene, encoding an acetoin reductase that catalyzes the conversion of acetoin to 2,3-butanediol.

The gltB gene plays a pivotal role in biofilm formation. As shown in a study by Zhou et al. [153] on B. subtilis Bs916 mutants, the gltB gene product regulates the production of γ-polyglutamate (γ-PGA), which is one of the polysaccharides involved in biofilm formation as well as the synthesis of the lipopeptide antibiotics bacillomycin L and surfactin, thereby affecting the colonization of plant tissues by bacteria. The study also confirmed the importance of the gltB gene in the biocontrol of R. solani. The importance of γ-polyglutamate synthesis in biofilm development was also confirmed in the study by Liu et al. [154], in which a mutant strain of B. amyloliquefaciens C06 defective in γ -PGA production showed lower efficiency in biofilm formation, surface adhesion, and swarming ability, which impaired colonization of apple surfaces. However, the results of this work did not answer the question of whether changes in biofilm synthesis, as a physical barrier against the adverse effects of the pathogen, are induced by the pathogen. Our study clearly indicates the effect of R. solani filtrates on the activity of the gltB gene, as its expression significantly increased after 24 h of the strain’s growth in the presence of fungus filtrate and was maintained on the following day of incubation (Figure 7). This was confirmed by in vitro observations indicating an increase in the biofilm formed by strain KW16 in the presence of the fungus (Figure 5b). The drastic decrease in the level of gltB gene expression after 72 h incubation with KW16 in the presence of the fungus may result from the tight regulation of γ -PGA synthesis in the tested strain, the interaction of other genes involved in biofilm formation, and the metabolic state of the cell as well as substrate availability. The explanation of this phenomenon, however, requires further detailed studies.

On the first day after the exposure of strain KW16 to the phytopathogen, an almost 13-fold increase in expression of the iucC gene was also observed. Overexpression of the gene persisted, but at a lower level for the next 48 h, and biochemical tests conducted in parallel indicated an increased efficiency in the production of siderophores by strain KW16 over time (Figure 5c). The gene iucC, encoding a protein of the IucA/IucC protein family, is associated with iron acquisition, mainly in Enterobacteriaceae. However, the cluster containing three ORFs, identified as the iron-binding IucA/IucC family siderophore biosynthesis proteins, were recently described in P. megaterium AB-S79 [26] and a novel P. megaterium strain MARUCO02 [155]. The siderophore biosynthetic gene clusters are tightly regulated to balance iron acquisition with iron toxicity, and the primary signal for inducing their synthesis is a low iron concentration. To explain our findings, we speculate that the siderophore gene overexpression in the KW16 strain was connected to the rapid decrease in iron concentration due to R. solani siderophores present in its filtrates [156]. The fast response of the bacterium to changing environmental conditions may indicate its significant role in fighting pathogens through siderophore release. In some bacteria, siderophore production is also influenced by population density via quorum sensing signals (QS) [157], which may explain the continuing overexpression and production of siderophores while the culture grows.

KW16 showed a significant increase in katA gene expression in response to treatment with R. solani filtrate. As noted by Faulkner et al., 2012 [158], the increased expression of the major vegetative catalase gene (katA) in Bacillus subtilis depends on the global regulator PerR, which is highly sensitive to H₂O₂. In addition, PerR is involved in the regulation of the Fur protein that controls siderophore synthesis. Therefore, we speculate that the overexpression of the katA gene and the increased siderophore synthesis by the KW16 strain in the presence of a pathogen could be the answer to the presence of fungus elicitors like ROS. The production of these molecules is an essential feature for the development and successful pathogenesis of various necrotrophic fungi [159].

Our study also shows the effect of R. solani on the expression profile of the ilvB gene involved in the synthesis of acetoin and butanediol. The overexpression of ilvB in response to fungal pathogens and the results obtained in the dual-culture assays, where secreted VOCs significantly inhibited the growth of the fungus (Figure 1) by more than 50%, confirm the relevant role of VOCs in the control of R. solani by the KW16 strain.

4. Conclusions

P. megaterium KW16 isolated from the plant environment and devoid of plasmids—major vectors of gene dissemination in the environment—can be used as a replacement for fungicides used in oilseed rape protection. By applying properties such as ability to survive in the presence of soil autochthonous microflora, eligibility to enter plant internal tissues, and distinct biocontrol traits against R. solani, oilseed rape treated with both biotic factors (KW16 and RS) showed more intensive growth and better metabolic fitness than plants in the single-factor systems. This was most likely due to the blockade of pathogen invasion into the plant interior resulting from KW16’s prompt response at the molecular level, with simultaneous stimulation of oilseed rape root density under the influence of R. solani metabolites. This so-called ‘chemical priming’ of biotic stress responses might be used in agriculture in the near future. However, the long-term study and development of formulation for field application require further investigation.

5. Patents

Patent no. PL 246324 B1 “Endophytic strain of Priestia megaterium bacteria and its applications.”