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Communication

Biological Traits of Vertically Seed-Transmitted Bacillus mojavensis in Triticum aestivum L.

by
Roderic Gilles Claret Diabankana
1,2,3,*,
Daniel Mawuena Afordoanyi
1,4,
Maria Nikolaevna Filimonova
2,
Shamil Zavdatovich Validov
1 and
Radik Ilyasovich Safin
3
1
Laboratory of Molecular Genetics and Microbiology Methods, Kazan Scientific Centre Russian Academy of Sciences, 420111 Kazan, Russia
2
Academic and Research Centre, Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia
3
Centre of Agroecological Research, Kazan State Agrarian University, 420015 Kazan, Russia
4
Tatar Research Institute of Agriculture, Kazan Scientific Center of Russian Academy of Sciences, 420111 Kazan, Russia
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2024, 15(4), 2369-2380; https://doi.org/10.3390/microbiolres15040159
Submission received: 27 September 2024 / Revised: 11 November 2024 / Accepted: 19 November 2024 / Published: 22 November 2024
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Abstract

:
Seed-borne endophytic bacteria can influence host responses to biotic and abiotic stress conditions. Their presence in seeds is related to their ability to colonize plant tissues and to pass from parent plants to offspring. In this study, we investigated the ability of Bacillus mojavensis PS17 to pass into the next generation of spring wheat plants via seeds and the effect of the transmission mode on the functional traits of seed-transmitted colonies of PS17. The rifampicin-resistant PS17 strain at 100 µg/mL was used to track PS17 effectively throughout the wheat growth cycle. The results demonstrated the successful colonization of B. mojavensis PS17 and its ability to pass into the next plant generation through seeds. During plant development, the PS17 cell population was almost higher in the rhizosphere than in the aboveground parts of plants, including seeds at the grain-filling stage. The seed-transmitted B. mojavensis PS17 colonies exhibited identical biological traits to those of the parental PS17 strain. Bacillus mojavensis PS17 retained its ability to suppress the growth of pathogens, such as Fusarium oxysporum and Alternaria alternata, and produce hydrolases, including protease, lipase, amylase, and cellulase. These results highlight the potential of vertical transmission through seeds as a mode of spreading bacterial biocontrol agents in future plants.

1. Introduction

Biotic stresses caused by phytopathogens constrain sustainable agricultural production and affect global food security [1,2,3]. Seeds, as reproductive organs necessary for species survival, harbor diverse microbial communities, including beneficial symbionts and pathogenic microorganisms that significantly affect the development of next-generation plants. Chemical and physical seed pretreatments enhance seed traits and promote plant growth [4,5]. However, exploring sustainable green technologies is crucial [6,7]. To date, studies have highlighted the potential of seed-borne endophytic microorganisms as an alternative approach for managing seed-borne pathogens and abiotic factors that affect seeds at emergence and during early growth stages [8,9,10]. Seed-borne endophytic microorganisms can improve a plant’s physiological and morphological characteristics via both direct and indirect mechanisms, including the production of siderophores and various phytohormones, as well as by inducing systemic acquired resistance and induced systemic resistance [11,12,13,14,15,16]. In addition, the competition between endophytic microorganisms and plant pathogens for the same ecological niche within plants makes them potential instruments for plant disease management and crop yield enhancement [17,18,19]. According to Frank et al. [20], seed endophytes can originate from the surrounding environment or be inherited from parent plants through seed transmission. The transmission mode by which beneficial microorganisms can be transmitted from parent plants to offspring through seeds provides a range of advantages. This transmission mode protects plants against biotic and abiotic stress during the early stages of plant development [20,21,22,23]. Successful vertical transmission promotes the preservation of beneficial microorganisms in seeds for many generations, creating a self-sustaining method of pest and disease control [24]. Moreover, vertically transmitted microorganisms may eventually become dominant in seed microbiota, further increasing their efficiency and stability in plant ecosystems [25,26]. Vertical transmission of endophytic bacteria can be influenced by the following factors: the host plant characteristics, including genotype, growth stage, and immune response; the interaction with seed microbial communities present within their tissues; and the biological traits of microorganisms. For example, resistance to high osmotic pressure is characteristic of microorganisms that develop in seeds during the ripening period when starch accumulates and the water content decreases [27,28,29]. Bacillus mojavensis PS17 is a biological agent isolated from the wheat (Triticum aestivum L.) variety ‘’Sadokat” (Tatdjik Republic). Its ability to suppress the growth of phytopathogenic fungi, such as Fusarium graminearum, Fusarium oxysporum, Fusarium chlamydosporum, Ascochyta pisi, Alternaria alternata, Sclerotinia sclerotiorum, Verticillium dahliae, and Epicoccum nigrum, to promote plant development and produce hydrolytic enzymes, such as chitinase, β-glucanase, cellulase, lipase, and protease, was reported in our previous study [30]. Considering the potential benefits of vertical transmission of endophytic bacteria, this study examined the ability of B. mojavensis PS17, a facultative endophyte, to be vertically transmitted to the next generation of plants via the seeds. We also evaluated the biological traits of seed-transmitted PS17 colonies in both soil and plant tissue. To ensure efficient tracking and establish their effectiveness under field conditions, we employed a rifampicin-resistant mutant strain, which is a well-established marker in such studies [31].

2. Materials and Methods

2.1. Microbial Strains and Growth Conditions

The microbial strains used in this study are listed in Table 1. Bacterial strains were cultivated on King’s B (KB) medium and incubated at 28 ± 1 °C, while fungal strains were grown on Sabouraud medium (Merck, Darmstadt, Germany) and incubated at 25 ± 1 °C. Bacillus mojavensis PS17 was deposited in the All-Russia Collection of Industrial Microorganisms (NRC “Kurchatov Institute”, Moscow, Russia) under number VKPM B-13415. The genomic sequence of B. mojavensis PS17 was deposited in GenBank under the accession number CP066516.1. The spring wheat variety “Ulianovsky 105” (Tatarstan, Kazan, Russia) was provided by the Centre of Agroecological Research, Kazan State Agrarian University (Kazan, Russia).

2.2. Rifampicin-Resistant B. mojavensis PS17 Preparation

Rifampicin-resistant Bacillus mojavensis PS17 [PS17 (Rif100)] was obtained using a stepwise gradient method [31]. For this purpose, B. mojavensis PS17 wild-type was plated onto KB agar containing 0.1 μg/mL rifampicin and incubated overnight at 30 ± 1 °C. Rifampicin-resistant colonies were then selected and re-streaked onto KB agar containing a higher concentration (0.5 μg/mL) of rifampicin. This process of selection and re-streaking at increasing rifampicin concentrations (with 0.5 μg/mL increments) was repeated until a mutant was stably resistant to 100 μg/mL rifampicin.

2.3. Verification of Rifampicin Resistance Specificity

To ensure that the rifampicin resistance marker at the concentration of 100 μg/mL was specific to B. mojavensis PS17 (Rif100) and that no other rifampicin resistance at this concentration was present in the soil or seed endophytic bacteria, a screening analysis was performed. The presence of pre-existing rifampicin-resistant bacterial strains was assessed at 100 μg/mL from garden soil and seed endophytes before use. For this purpose, 1 g of garden soil or wheat seed was serially diluted. After dilution, aliquots (100 μL) of 105 dilutions were plated onto KB agar amended with rifampicin (100 μg/mL) and nystatin (50 μg/mL). All plates were incubated at 30 ± 1 °C.

2.4. Bacterial Cell Suspension Preparation

The bacterial suspension was prepared from the bacterial culture of B. mojavensis PS17 (Rif100) grown for 24 h in lysogeny broth (LB) [g/L: tryptone, 10 g; yeast extract, 5 g; and NaCl, 10 g] at 30 ± 1 °C. Cells were pelleted by centrifuging for 5 min at 4000 rpm and 4 ± 1 °C. The resulting supernatant was removed, and the precipitate was washed twice with sterile phosphate-buffered saline (PBS) [140 mM NaCl, 5 mM KH2PO4, 1 mM NaHCO3, and pH 7.4]. After washing, the pellet was resuspended in PBS buffer to a final optical density (OD) of 0.1 at 595 nm, corresponding to a bacterial concentration of approximately 106 CFU/mL.

2.4.1. Vertical Transmission of B. mojavensis PS17

The vertical transmission of B. mojavensis PS17 was evaluated in a climate-controlled chamber (HPP 750 Memmert, Memmert, Germany) at 25 °C with 75% humidity and a 16 h light/8 h dark cycle. For this purpose, surface-sterilized wheat seeds were used, as studied by Simon et al. [32], alongside garden soil. Seeds were pretreated with a cell suspension of B. mojavensis PS17 using the semi-dry method of 1 L of cell bacterial culture suspension per 100 kg of seeds. Pretreated seeds (10 seeds per pot) were immediately sown in pots containing garden soil amended with sterile plant nutrient solution (PNS) [1.25 mM Ca(NO3)2; 1.25 mM KNO3; 0.50 mM MgSO4; 0.25 mM KH4PO4; 0.75 mg/L KI; 3.00 mg/L H3BO3; 10.0 mg/L MnSO4·H2O; 2.0 mg/L ZnSO4·5H2O; 0.25 mg/L Na2MoO4·2H2O; 0.025 mg/L CuSO4·5H2O; 0.025 mg/L CoCI2·6H2O; and pH 5.8] to 60% of its water-holding capacity. Pots were watered twice daily with sterile tap water to maintain the soil moisture. Plants were cultivated up to the grain-filling stage. Samples were collected from different plant parts (leaves, stems, roots, and grains) at each growth stage (seedling and tillering, stem elongation, and milk development) to assess the endophytic colonization of PS17. Plants without bacterial treatment (pretreated only with water) were maintained as a control group to ensure the absence of any bacterial strain that could acquire spontaneous rifampicin resistance at 100 μg/mL in the soil and growing plants during the plant cultivation process. For statistical analysis, the experiment was repeated twice, and four pots per group were maintained. A significant difference between pretreatments was evaluated using one-way ANOVA and post hoc Tukey’s test at p < 0.05.

2.4.2. Screening for B. mojavensis PS17 in Wheat

We screened for the presence of B. mojavensis PS17 in different wheat tissues, including soil, rhizosphere, roots, stems, leaves, and grains. Plant tissues were surface sterilized as described by Simon et al. [32]. After sterilization, the tissues were homogenized in PBS buffer using a mortar and pestle, then 100 μL of obtained solutions were plated onto LB agar amended with rifampicin at a 100 μg/mL concentration and nystatin at 50 μg/mL to inhibit the growth of non-rifampicin-resistant bacteria and fungi. The plates were incubated at 30 ± 1 °C. Colonies growing on selective medium were presumed to be PS17 colonies. Subsequently, single-cell colonies were randomly selected and compared with the original PS17 strain using BOX-PCR and 16S rRNA, recA, rpoB, and gyrA gene sequence analyses.

2.5. DNA Fingerprinting Analysis

The total chromosomal DNA from bacterial strains was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. BOX-PCR was performed in a 25 μL volume, which included 2.5 µL of 10× PCR buffer, 0.4 µL of a 10 µM mixture of dNTPs, 0.5 µL of 10 µM primer BOXA1R (5′-CTACGGCAAGGCGACGCTGACG-3′), 5.0 µL of DNA template (50 ng), 1.0 μL of Taq DNA polymerase (1U), and nuclease-free water. Polymerase chain reaction (PCR) was performed using a thermocycler (Bio-Rad, Hercules, CA, USA) under initial denaturation at 95 °C for 2 min, followed by 30 cycles at 94 °C for 30 s. The primer annealing and elongation cycles were set to 58 °C for 30 s and 72 °C for 8 min, respectively. The final cycle was followed by a cycle at 72 °C for 10 min. The resulting PCR product was subjected to electrophoresis in 1% agarose gel.

2.6. Molecular Identification of Selected Bacterial Isolates

The identity of the selected bacterial strains was established by comparing the target genes presented in Table 2 with those of their parental B. mojavensis PS17 deposited in the National Center for Biotechnology Information (NCBI) GenBank.
PCR amplification of the target genes was performed using a QuantStudio 5 thermocycler (Applied Biosystems, Forester City, CA, USA). The PCR master mix contained 2.5 µL of 10× PCR buffer, 0.4 µL of a mixture of dNTPs (10 µM), 1.25 µL of each primer (10 µM), 2.5 µL of DNA sample (100 ng), 1.0 μL Taq DNA polymerase (1U), and nuclease-free water. The conditions included an initial denaturation at 95 °C for 3 min, followed by 36 cycles of denaturation at 95 °C for 15 s. The annealing temperature was set for 30 s, as described in Table 2, and the extension cycle was set at 72 °C for 40 s. The final extension cycle was performed at 72 °C for 10 min. After PCR amplification, the products were fragmented by electrophoresis in 1% agarose gel. Subsequently, DNA fragments were purified from the agarose gel using a cleanup kit (Evrogen, Moscow, Russia) according to the manufacturer’s recommendations. Amplified fragments were determined by Evrogen (Moscow, Russia) using both forward and reverse primers. The resulting chromatograms were evaluated using the Clone Manager 9 software package (Sci Ed Software, Cologne, Germany) and blasted on NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 May 2024).

2.7. The Antagonistic Activity of the Selected Bacterial Isolates Against Phytopathogens

The ability of isolated bacterial colonies transmitted through seeds to inhibit the growth of the phytopathogenic fungi F. oxysporium and A. alternata was assayed using a dual culture method. For this purpose, a plug of phytopathogenic fungi was inoculated into the center of agar plates and allowed to grow for two days. Subsequently, 2 µL of the bacterial cell suspensions from the selected strains were co-inoculated at a distance around the periphery of the fungal inoculum. Plates were incubated at 28 ± 1 °C for up to ten days. The antagonistic activity against the fungi was determined by the formation of inhibition zones, which are clear areas around the bacterial colonies in which fungal growth is absent.

2.8. Hydrolytic Enzyme Production by Selected Bacterial Isolates

The ability of selected isolates to produce protease, cellulase, and amylase was determined by inoculating 2 μL of overnight bacterial culture onto basal medium (g/L: K2HPO4—5.8; KH2PO4—3; NH4SO4—1.0; MgSO4·7H2O—0.2) amended with 1% skim milk powder (HiMedia, Mumbai India), carboxymethylcellulose sodium salt (HiMedia, India), and starch soluble AR (HiMedia, India), respectively. Lipase activity was assessed on lipase agar medium (peptone—10 g; NaCl—5 g; CaCl2·2H2O—0.1 g; agar—18 g, and 10 mL (v/v) Tween-80). Subsequently, all plates were incubated at 30 ± 1 °C for up to four days. Cellulase and amylase were detected by flooding plates with 0.1% Congo red and 0.5% iodine solutions for 5 min, respectively. The plates were then destained with 0.1 N NaCl solution. The presence of a clearance zone (protease, amylase, and cellulase) or crystallite bubbles around the growing colonies indicated enzymatic activity.

3. Results

3.1. The Ability of B. mojavensis PS17 to Pass into Next-Generation Plants

The ability of PS17 to pass into next-generation plants through seeds was evaluated under laboratory conditions. The obtained results are presented in Figure 1. The presence of B. mojavensis PS17 was assessed in various wheat plant parts during tillering, stem elongation, heading, and grain-filling stages. The colony-forming units of PS17 after seed pretreatment were 6.37 ± 0.15 × 104 cfu/g of plant tissue (Figure 1A). The presence of B. mojavensis PS17 remained relatively high in the rhizosphere during plant development. The colony-forming units of B. mojavensis PS17 in the rhizosphere were assayed as 3.87 ± 0.14 × 107, 4.58 ± 0.17 × 106, and 3.14 ± 0.36 × 105 CFU/g of soil at the seedling and tillering (Figure 1B), stem elongation (Figure 1C), and grain-filling stages (Figure 1D), respectively. However, a decrease in PS17 colonies in the aboveground parts of the plants was observed only at the grain-filling stage, mostly in seeds. In contrast, the density of PS17 cells in the aboveground parts throughout the entire period of plant development did not exceed 107 CFU/g. While PS17 was detected in the aboveground plant parts, the population density was significantly lower than 104 CFU/g, particularly in seeds (Figure 1). In addition, the absence of bacterial growth in the control group confirmed the use of rifampicin as a selective marker under the tested conditions.

3.2. Biological Traits of B. mojavensis PS17 Seed-Transmitted Colonies

Bacillus mojavensis PS17(Rif100) was observed in all plant parts throughout the seedling, head differentiation, and grain-filling stages of spring wheat. All isolated bacterial colonies had identical morphologies, which were uniformly round in shape with a convex profile, similar to the original B. mojavensis PS17(Rif100). Seed-transmitted colonies isolated from seeds (Figure 2C) were opaque, white, and homogeneous, exhibiting a slightly folded and scalloped edge with a hint of transparency compared to those isolated from roots (Figure 2A) and leaves (Figure 2B).

3.2.1. The Antagonistic Activity of the Isolated Bacteria Against Phytopathogens

The abilities of seed-transmitted colonies of PS17(Rif100) to inhibit the growth of phytopathogens compared with the parental strain are presented in Figure 3. Vertical transmission from seed to seed in spring wheat did not affect the ability of PS17 to produce antimicrobial compounds that inhibit the growth of the phytopathogenic fungi F. oxysporium (Figure 3A) and A. alternata (Figure 3B) used in this study.

3.2.2. Hydrolytic Enzyme Production by Selected Bacterial Strains

We evaluated hydrolytic enzyme production in selected PS17 mutant strains. All tested strains formed clear zones around them on media containing protease (Figure 4A), cellulase (Figure 4B), and amylase (Figure 4D) substrates, indicating their ability to produce these enzymes. In addition, among the isolated bacterial colonies transmitted through seeds, some did not exhibit lipase activity, which was observed by the absence of a transparent zone on the lipase medium (Figure 4C).

3.3. DNA Fingerprinting Analysis and Molecular Identification of Seed-Isolated Bacterial Strains

To confirm the taxonomy of PS17 isolates recovered from seeds and the rhizosphere at the grain-filling stage, 50 colonies were randomly selected. These selected colonies were compared with the original B. mojavensis PS17 strain using BOX-PCR fingerprinting. As shown in Figure 5, all selected isolates from seedling (Figure 5(AI)), tillering, and head differentiation (Figure 5(AII)) generated identical banding DNA patterns to those of the original PS17(Rif100). Notably, all isolates generated the same number of box fragments, ranging from 500 to 1500 base pairs (bp).
An identical BOX-PCR profile was obtained at the grain-filling stage (Figure 6). All randomly selected bacterial colonies isolated from soil, the rhizosphere, and surface-sterilized roots, stems, leaves, and seeds, which grew on LB medium amended with Rifampicin, showed a fingerprint profile that was indistinguishable from that of their parent B. mojavensis PS17. Moreover, the generated profile boxes differed from those generated by related bacteria B. amyloliquefaciens, B. halotolerans, and Bacillus sp. used as control bacterial strains.
Additionally, specific genes (rpoB, gyrA, 16S rRNA, and RecA) were sequenced and compared to the reference parental sequences available in the NCBI database to confirm the identity of the isolated bacterial strains. The 16S rRNA gene sequences showed a high-scoring segment pair, demonstrating 100% identity with B. mojavensis PS17 (accession number MW350040.1). Likewise, the coding sequences (CDS) for the rpoB, gyrA, and RecA genes were also identical, showing 100% similarity with the corresponding sequences of B. mojavensis PS17 (accession numbers PQ044378, PQ044379, and PQ044380, respectively). Based on the high sequence similarity to the reference strain, we concluded that all isolates belonged to B. mojavensis.

4. Discussion

Preserving the beneficial endophytic bacteria transmitted through seeds is a promising strategy for introducing microbiological agents into seed microbiota because successful colonization often leads to mutually beneficial interactions [37,38,39]. The efficacy of plant colonization by endophytic bacteria requires an initial adherence to seed surfaces and tolerance to natural defense compounds secreted by plants [37,40,41]. This suggests that B. mojavensis PS17 is closely associated with spring wheat. Moreover, a high PS17 cell density was recovered from various plant parts (roots, stems, and leaves) during the early seedling stage on LB amended with rifampicin (100 μg/mL). These findings indicated the ability of B. mojavensis PS17 to act as an endophyte. Successful plant colonization may be facilitated by the release of root exudates from spring wheat, which are known to play crucial roles in attracting and establishing beneficial microbial communities [42,43,44,45]. Bacillus mojavensis PS17 was detected in all analyzed plant tissues at different plant growth phases (germination, tillering, and head formation), as well as in seeds at the grain-filling stage (milk development), despite a slight decrease in the PS17 cell population at the grain-filling stage. The decreased PS17 population in seeds may be due to the seed coat forming a protective layer over the endosperm and embryo, which provides a safe environment that may affect the colonization ability of microorganisms [26,46,47]. Endophytic behavior is affected by complex interactions such as nutrient availability, host plant species, microbial communities, and environmental conditions [48]. The persistence of seed-transmitted endophytes is crucial for elucidating their ecological roles and potential applications in agriculture, particularly in crop protection. Several studies have reported that under stressful conditions, microorganisms undergo phenotypic variation, resulting in the formation of colonies with different phenotypic characteristics [49,50,51]. Likewise, Troxler et al. [52] reported that the root endophyte Pseudomonas fluorescens strain CHAO isolated from maize exhibited distinct biocontrol abilities compared with strains isolated from the rhizosphere. Similarly, Barnett et al. [53] observed functional differences in antagonistic activities among various colonies of the endophytic bacteria Pseudomonas corrugate 2140 after long-term residence within the plant. In this study, B. mojavensis PS17 demonstrated the ability to inhibit the growth of phytopathogenic fungi and produce lytic enzymes such as amylase and protease after its long-term survival in seed wheat, indicating its phenotypic persistence. BOX-PCR fingerprinting is a well-established method that seems to be able to identify bacteria at the strain level [54]. In a study conducted by Tacão et al. [55], BOX-PCR was used to discriminate various Aeromonas species, with most of the analyzed strains displaying unique banding patterns. In our study, all isolated bacterial strains exhibited identical banding patterns (Figure 5), indicating their genetic relatedness to B. mojavensis PS17.

5. Conclusions

Beneficial seed-borne endophytic microorganisms play a key role in the early seedling growth of plants and constantly affect the host plant’s interaction with abiotic and biotic conditions. In this study, B. mojavensis PS17 demonstrated the ability to colonize the non-native host plant spring wheat and undergo vertical transmission to subsequent generations through seeds. Overall, our findings highlight the potential for using seed-mediated vertical transmission as a method for spreading biocontrol agents in future plants, which may eventually become dominant. However, field studies are required to validate the efficiency of vertical transmission and its persistence under varying soil and environmental conditions because these factors may affect this mode of transmission.

Author Contributions

Conceptualization, R.G.C.D.; methodology., M.N.F., R.I.S. and R.G.C.D.; data acquisition, R.G.C.D. and D.M.A.; software, R.G.C.D.; investigation, R.G.C.D.; analysis, R.G.C.D.; writing—original draft preparation, D.M.A. and R.G.C.D.; writing—review and editing, D.M.A., R.I.S., R.G.C.D., M.N.F. and S.Z.V.; supervision, R.G.C.D. All authors have read and agreed to the published version of the manuscript.

Funding

This study was conducted with financial support provided by the Ministry of Science and Higher Education of the Russian Federation, Grant # 075-15-2021-1395, 25 October 2021 (15.IP.21.0020), and from the government assignment (№124050300050-4) for FRC Kazan Scientific Center of RAS.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Colony-forming units of B. mojavensis PS17 screened in different parts of wheat tissues from seed inoculation (A), seedling and tillering (B), stem elongation (C), and grain-filling (D) growth stages. Statistical differences at p-value < 0.05 between groups are indicated by different lowercase letters.
Figure 1. Colony-forming units of B. mojavensis PS17 screened in different parts of wheat tissues from seed inoculation (A), seedling and tillering (B), stem elongation (C), and grain-filling (D) growth stages. Statistical differences at p-value < 0.05 between groups are indicated by different lowercase letters.
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Figure 2. Screening B. mojavensis PS17 as an endophyte in wheat at the grain-filling stage. Bacterial colonies isolated from surface-sterilized roots (A), leaves (B), and seeds (C) of spring wheat. Bacterial strains were grown on LB agar medium amended with Rifampicin (100 μg/mL) for 48 h at 30 ± 1 °C.
Figure 2. Screening B. mojavensis PS17 as an endophyte in wheat at the grain-filling stage. Bacterial colonies isolated from surface-sterilized roots (A), leaves (B), and seeds (C) of spring wheat. Bacterial strains were grown on LB agar medium amended with Rifampicin (100 μg/mL) for 48 h at 30 ± 1 °C.
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Figure 3. The antimicrobial activities of seed-transmitted colonies of PS17 against F. oxysporium (A) and Alternaria alternata (B) after seven days of incubation at 28 ± 1 °C. a–b—parental strain PS17; c–h—vertical transmitted bacterial colonies isolated from next-generation seeds.
Figure 3. The antimicrobial activities of seed-transmitted colonies of PS17 against F. oxysporium (A) and Alternaria alternata (B) after seven days of incubation at 28 ± 1 °C. a–b—parental strain PS17; c–h—vertical transmitted bacterial colonies isolated from next-generation seeds.
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Figure 4. The abilities of seed-transmitted B. mojavensis PS17 colonies to produce the hydrolytic enzymes protease (A), cellulase (B), lipase (C), and amylase (D). The black and white arrows indicate the formation of crystals and halo zones surrounding the bacterial colonies, respectively. Parental strain PS17 (Rif100) (a) and bacterial colonies isolated from next-generation seeds (b–d).
Figure 4. The abilities of seed-transmitted B. mojavensis PS17 colonies to produce the hydrolytic enzymes protease (A), cellulase (B), lipase (C), and amylase (D). The black and white arrows indicate the formation of crystals and halo zones surrounding the bacterial colonies, respectively. Parental strain PS17 (Rif100) (a) and bacterial colonies isolated from next-generation seeds (b–d).
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Figure 5. 1% agarose gel electrophoresis of the BOX-PCR products of bacterial strains isolated from spring wheat grown during seedling (AI) and tillering and head differentiation (AII). Line M—DNA ladder 1 kb; line A1–A3—bacterial strains isolated from rhizosphere; line B1–B3—bacterial strain isolated from surface-sterilized roots; line C1–C3—bacterial strain isolated from stems; line D1–D3—bacterial strain isolated from leaves; line E1–E3—parental B. mojavensis strain PS17(Rif100); line F—B. amyloliquefaciens; line G—B. halotolerans; and line H—negative control.
Figure 5. 1% agarose gel electrophoresis of the BOX-PCR products of bacterial strains isolated from spring wheat grown during seedling (AI) and tillering and head differentiation (AII). Line M—DNA ladder 1 kb; line A1–A3—bacterial strains isolated from rhizosphere; line B1–B3—bacterial strain isolated from surface-sterilized roots; line C1–C3—bacterial strain isolated from stems; line D1–D3—bacterial strain isolated from leaves; line E1–E3—parental B. mojavensis strain PS17(Rif100); line F—B. amyloliquefaciens; line G—B. halotolerans; and line H—negative control.
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Figure 6. 1% agarose gel electrophoresis of the BOX-PCR products of bacterial strains isolated from spring wheat grown at the grain-filling stage. Line M—DNA ladder 1 kb; line A1–A4—bacterial strains isolated from the rhizosphere; line B1–B4—bacterial strain isolated from surface-sterilized roots; line C1–C4—bacterial strain isolated from surface-sterilized stems; line D1–D5—bacterial strain isolated from surface-sterilized leaves; Line E1–E5—bacterial strain isolated from surface-sterilized seeds; line J1–J5—parental B. mojavensis PS17(Rif100); line F—B. amyloliquefaciens; line G—B. halotolerans; line I—Bacillus sp; and line H—negative control.
Figure 6. 1% agarose gel electrophoresis of the BOX-PCR products of bacterial strains isolated from spring wheat grown at the grain-filling stage. Line M—DNA ladder 1 kb; line A1–A4—bacterial strains isolated from the rhizosphere; line B1–B4—bacterial strain isolated from surface-sterilized roots; line C1–C4—bacterial strain isolated from surface-sterilized stems; line D1–D5—bacterial strain isolated from surface-sterilized leaves; Line E1–E5—bacterial strain isolated from surface-sterilized seeds; line J1–J5—parental B. mojavensis PS17(Rif100); line F—B. amyloliquefaciens; line G—B. halotolerans; line I—Bacillus sp; and line H—negative control.
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Table 1. The microbial strains used in this study.
Table 1. The microbial strains used in this study.
Microbial StrainsSourceReference
B. mojavensis PS17From wheat seed[30]
B. mojavensis PS17 (Rif100)Rifampicin-resistant PS17 at 100 μg/mLIn this study
Bacillus amyloliquefaciensFrom rhizosphere winter wheat*
Bacillus halotolerans*
Bacillus sp.*
F. oxysporiumFrom onion (Allium cepa L.)*
A. alternataFrom spring wheat seed*
* Microbial collection from the Centre of Agroecological Research, Kazan State Agrarian University.
Table 2. Primers used to identify isolated bacterial strains.
Table 2. Primers used to identify isolated bacterial strains.
Target GenesOligonucleotideAnnealing TemperatureReference
recA5′-GATCGTCAAGCAGCCTTAGAT-3′55 °C[33]
5′-TTACCGACCATAACGCCGAC-3′
16S rRNA5′-AGAGTTTGATCMTGGCTCAG-3′58 °C[34]
5′-AAGGAGGTGATCCAGCCGCA-3′
rpoB5′-ATCGAAACGCCTGAAGGTCCAAACAT-3′58 °C[35]
5′-ACACCCTTGTACCGTGACGACC-3′
gyrA5′-CAGTCAGGAAATGCGTACGTCCTT -3′58 °C[36]
5′-CAAGGTAATGCTCCAGGCATTGCT -3′
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MDPI and ACS Style

Diabankana, R.G.C.; Afordoanyi, D.M.; Filimonova, M.N.; Validov, S.Z.; Safin, R.I. Biological Traits of Vertically Seed-Transmitted Bacillus mojavensis in Triticum aestivum L. Microbiol. Res. 2024, 15, 2369-2380. https://doi.org/10.3390/microbiolres15040159

AMA Style

Diabankana RGC, Afordoanyi DM, Filimonova MN, Validov SZ, Safin RI. Biological Traits of Vertically Seed-Transmitted Bacillus mojavensis in Triticum aestivum L. Microbiology Research. 2024; 15(4):2369-2380. https://doi.org/10.3390/microbiolres15040159

Chicago/Turabian Style

Diabankana, Roderic Gilles Claret, Daniel Mawuena Afordoanyi, Maria Nikolaevna Filimonova, Shamil Zavdatovich Validov, and Radik Ilyasovich Safin. 2024. "Biological Traits of Vertically Seed-Transmitted Bacillus mojavensis in Triticum aestivum L." Microbiology Research 15, no. 4: 2369-2380. https://doi.org/10.3390/microbiolres15040159

APA Style

Diabankana, R. G. C., Afordoanyi, D. M., Filimonova, M. N., Validov, S. Z., & Safin, R. I. (2024). Biological Traits of Vertically Seed-Transmitted Bacillus mojavensis in Triticum aestivum L. Microbiology Research, 15(4), 2369-2380. https://doi.org/10.3390/microbiolres15040159

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