Plant growth-promoting rhizobacteria (PGPR) have been reported to exhibit biocontrol processes including competition, antibiosis, growth promotion, and the induction of systemic resistance in plants [1
]. The Bacillus
genus is one of the most frequently reported and promising PGPR with various biocontrol mechanisms. The nematicidal activity of the guanidine compound from B. pumilus
LYMC-3 and its high potential in the biocontrol of Bursaphelenchus xylophilus
was demonstrated [7
]. Bacillus velezensis
FZB42, a model strain for excellent plant growth promoting and biocontrol rhizobacteria, has been studied on its genes and expression involved in biocontrol process and the bacteria-plant interaction [8
Due to colonizing the root of plants persistently, PGPR can effectively promote the growth of plants and inhibit the pathogens [10
]. Persistent colonization of PGPR on roots is essential for suppressing different plant diseases, and biofilm formation is considered a key factor for efficient root colonization [13
]. Biofilm formation is regulated by aggregation and quorum sensing, which influences wide activities of the bacteria [15
]. Therefore, exploring the distribution, expansion and colonization of bacterial biofilm in the plant rhizosphere is an important aspect to clarify the biocontrol mechanisms of PGPR.
Bacterial biofilms are formed by multicellular communities that are embedded in a self-produced matrix of extracellular substances such as extracellular polysaccharide, protein, and nucleic acids, which enhance the resistance of bacteria to adverse environments and facilitate bacterial interactions with plants [15
]. Most cells in multilayered biofilm experience contact between cells and then aggregates formed, either in surface-attached biofilms or in flocs. The biofilm phenotypes were often observed on the solid-gas or liquid-gas surface [15
]. Swarming motility in bacteria is a social behavior that enables rapid movement and microbial biofilm development, allowing bacteria to colonize nutrient-rich environments [4
]. It has been reported that nonribosomally synthesized antibiotics, especially surfactin, play a significant role in suppressing diseases and are involved in regulating the process of biofilm formation [20
]. However, the role of biofilm by B. pumilus
involved in biocontrol efficacy against plant diseases is rarely reported.
HR10 was isolated from the rhizosphere of Pinus thunbergii
. The strain was found to significantly promote mycorrhizal symbionts and the growth of P. thunbergii
]. In vitro testing, B. pumilus
HR10 showed excellent inhibitory activity against Rhizoctonia solani
, causing pine seedling damping-off disease, implying that B. pumilus
HR10 has excellent biocontrol potential [24
]. Pinus massoniana
was chosen for the study because it is one of the most popular and native tree species in southern China. However, varieties of pathogens and insect pests’ diseases occurred during the afforestation of P. massoniana
including seedling damping-off disease. The occurrence of the disease caused the death of a large number of P. massoniana
seedlings, which seriously threatened the sustainable development of the P. massoniana
seedlings forestry [25
]. Currently, the role of biofilm formation by B. pumilus
HR10 in inhibiting pathogens is not understood, restricting further studies on the biocontrol mechanism of B. pumilus
HR10. We propose that the biocontrol efficacy of B. pumilus
HR10 depends on the coordinated action of inhibitory activity, induced systemic resistance and excellent colonization. Under this hypothesis, we mutated B. pumilus
HR10 and screened biofilm-deficient mutants. Then, we compared the biofilm-related properties and gene expression, and root colonization and activity against R. solani
of the strains.
2. Materials and Methods
2.1. Strains and Culture Conditions
Bacillus pumilus HR10 was isolated from the rhizosphere of Pinus thunbergii, and now it is preserved in the China Center for Type Culture Collection (CCTCC, NO: NO.M2010143). It was routinely grown at 30 °C in Luria-Bertani (LB) medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L). The pathogenic fungus of pine seedling damping-off disease, Rhizoctonia solani was grown in potato dextrose (PDA) medium at 25 °C.
2.2. Mutagenesis of B. pumilus HR10 and Screening of Biofilm-Deficient Mutants
The B. pumilus
HR10 wild-type strain was treated with 0.4 mg/mL N
-nitrosoguanidine (MNNG), and incubated in the dark for 30 min. After diluting to the appropriate concentration, the suspension was placed on an LB plate. The mutants were screened for different biofilm defects [27
2.3. Colony Morphology, Growth Curve and Spore Formation
The colony morphologies of the wild-type strain and mutants were observed with a Carl Zeiss stereomicroscope (SteREO Discovery V.20, Germany). The growth curves were measured at 600 nm with a microbial growth curve tester. The suspensions (OD600 = 1.0) of the wild-type strain and mutants were transferred into new media and cultured at 30 °C with shaking at 200 rpm. After cultivating for 12, 18, 24, 48, 66 and 72 h, 100 μL of culture of each strain was diluted with sterilized water to the appropriate concentration to measure the number of live bacteria. The sterile tubes containing 100 μL of each culture were incubated at 85 °C for 10 min, and the number of spores were measured. Moreover, the growth curves of cells of 4 strains were also detected by the automatic growth curve analyzer for microorganisms (Bioscreen M, Shanghai, China).
2.4. Analysis of Biofilm Formation and Swarming Motility
The biofilm formation assay of strains was carried out following a previously described method with few modifications [29
]. For quantitative analysis of biofilm formation, 10 μL of liquid culture (OD600
= 1.0) of each strain was inoculated into a well of a 24-well microliter plate containing 1 mL of LB and was incubated without agitation at 30 °C for 96 h. The medium was carefully removed, and the wells were washed with sterilized water. Crystal violet solution (1%) was used to stain the biofilm for 15 min, and the excess stain was rinsed off with water. Pure ethanol was used to extract the dye bound to the biofilm structure, and the absorbance at 570 nm was visualized with a Multiskan Spectrum microplate reader (Thermo, Waltham, MA, USA). The biofilm formation assays were conducted 6 times.
Swarming motility assays of wild-type B. pumilus
HR10 and biofilm mutants were performed according to Chen et al. [29
]. One milliliter of fresh culture of each strain (OD600
= 0.5) was collected by centrifugation at 6000 rpm for 5 min, washed with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2
, and 2 mM KH2
), and resuspended in 100 μL of PBS. Subsequently, 3 μL of each culture was separately spotted onto the center of the swarming motility plates containing 0.7% agar that were dried for 20 min. The plates were placed upside down in a 30 °C incubator for 15 h. The swarming motilities of cells were observed and took pictures to record every 5 h.
2.5. Quantitative Estimation of Biofilm Polysaccharides
Standard curve of the glucose: the glucose (1 mL) concentrations were set as 0, 40, 80, 120, 160 and 200 μg/mL. Anthrone (0.2 g) was dissolved in 100 mL of sulfuric acid, and then 2 mL of liquid was slowly added into the glucose solutions. The tubes were incubated in boiling water for 10 min and then in ice-cold water for 10 min. Subsequently, the solutions were placed at room temperature for 2 min, and the absorbances were measured at 620 nm with a Multiskan Spectrum microplate reader.
Biofilm polysaccharide extraction and measurement: the cultures of B. pumilus
HR10 wild strain and biofilm-deficient mutants were harvested at OD600
= 1.0. For quantitative analysis of biofilm polysaccharides, 10 μL of liquid culture of each strain was used to inoculate into the well with 1 mL LB of 24-well microliter plates and was incubated without agitation at 30 °C for 48 h. The medium was carefully removed, and the wells were washed with sterilized water. Three milliliters of ethanol were added into each well. The biofilm polysaccharides were precipitated at 4 °C overnight. Subsequently, biofilm polysaccharides were collected by centrifugation (10,000 rpm, 4 °C, 10 min). The polysaccharide pellet was washed three times with ice-cold 75% ethanol and centrifuged. Similarly, two washing steps with distilled water were performed. The biofilm polysaccharides of the wild-type strain and mutants were dissolved, and the absorbances were measured as described above [8
2.6. Quantitative Estimation of Biofilm Proteins
Standard curve of bovine serum albumin (BSA): the BSA solution (1 mL) concentrations were set as 0, 20, 40, 60, 80 and 100 μg/mL. Coomassie brilliant blue G250 (0.1 g) was dissolved in 1000 mL of distilled water containing 50 mL of 95% ethanol and 100 mL of 85% phosphoric acid. The BSA solution was stained with the dye liquid (5 mL) respectively for 5 min and then the absorbance at 595 nm was measured with a Multiskan Spectrum microplate reader.
Biofilm protein extraction and measurement: the biofilms of wild-type (WT) B. pumilus HR10 and biofilm-deficient mutants were harvested as described above. Ammonium sulfate was added to the supernatant liquid to a final concentration of 70%. The biofilm proteins were precipitated at 4 °C overnight. Subsequently, extracellular proteins were collected by centrifugation (10,000 rpm, 4 °C, 15 min). The protein pellet was washed three times with ice-cold 50% ethanol and centrifuged. Similarly, two washing steps with distilled water were performed. The extracellular proteins obtained from WT and mutants were dissolved, and the absorbances were measured as described above.
2.7. qRT-PCR Assay of Biofilm-Related Gene Expression
The biofilms of WT and biofilm mutants were collected after 12 h, 24 h, 48 h and 72 h of incubation. The biofilms were collected by centrifugation and ground into powder with liquid nitrogen. Then, 1 mL of TRIzol (Invitrogen, Waltham, MA, USA) was added to the powder (100 mg) of each sample. The 1.5 mL tubes with powder were shaken and the samples were mixed and then incubated for 5 min at 25 °C. The solution was extracted with 200 μL of trichloromethane (99%), mixed and centrifugated. The supernatant was collected and washed 2 times with 75% ethanol. The extracted RNA was dried and dissolved with RNase-Free H2
O. The quantity and purity of isolated RNA were measured by ultraviolet absorbance NanoDrop 2000C at A260/280 (Thermo Scientific, Waltham, MA, USA) and assessed by electrophoresis on a 1% agarose gel. The first-strand cDNA was synthesized from 1 μg total RNA using the HiScript IIqRT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China), which could completely remove the residual genomic DNA in the RNA template.qRT-PCR was performed using a HiScript IIqRT reagent kit (Vazyme, Nanjing, China). The cDNA samples were diluted to 90 ng/μL. A solution of cDNA was prepared with 2 × ChamQ SYBR qPCR Master Mix. The reaction system (20 μL) included 10 μL of SYBR qPCR Master Mix (2×), 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 2 μL of cDNA sample, and 7.2 μL of ddH2
O. Thermal conditions were as follows: 30 s at 95 °C for initial denaturation and 40 cycles of 10 s at 95 °C, followed by 34 s at 60 °C. The relative expression levels of epsA
(extracellular polysaccharide-related gene), tasA
(extracellular protein-related gene), fliG
(flagellum motility-related gene), and sfp
(surfactin synthesis-related gene) were measured. The B. pumilus gyrB
gene was used as an internal reference. Experiments were performed in triplicate with three biological replicates on an ABI Prism 7500 (Applied Biosystems, Foster City, CA, USA). The relative gene expression levels were analyzed using ABI Prism 7500 software and the 2−ΔΔCt
method. The primers used to amplify these genes are listed in Table 1
2.8. Antifungal Activity Assay
plate was grown on PDA plates for the assay. The antifungal activity of the fermentation liquid of B. pumilus
HR10 culture against R. solani
was also measured. The cultures of B. pumilus
HR10 WT and mutants were harvested to OD600
= 1.0 in 20 mL LB respectively. Subsequently, fermentation liquid was collected by centrifugation (10,000 rpm, 10 min) and the further removing cells with a 0.22 μm filter. One milliliter of the fermentation culture of WT B. pumilus
HR10 and mutants was added into a melted PDA plate respectively. The plates were incubated and observed, and the colony diameter for each treatment was recorded [6
2.9. Cultivation of P. massoniana Seedlings
The 300 seeds of P. massoniana were surface-sterilized with 30% H2O2, after which they were placed in water agar medium with 1% agar. After growing to the cotyledon periods, 100 seedlings were grown in sterilized soil matrix for the root colonization assay. The remaining 200 seeds of P. massoniana were for reduced-plant-resistance assay and biocontrol assay against pine seedling damping-off disease.
2.10. Root Colonization Assay
The assay of root colonization was carried out following a described method with few modifications [32
]. The overnight-grown bacterial cultures (OD600
= 1.0) of wild-type B. pumilus
HR10 and biofilm mutants were collected by centrifugation at 6000 rpm for 5 min and diluted to 1 × 108
CFU/mL with PBS for use. P. massoniana
seeds were soaked in water for 18 h. The seeds were disinfected with 30% H2
, washed with sterilized water and sown on medium with 1% agar. The 10-day-old seedlings were then transplanted into sterilized bottles containing sterile soil matrix. Forty-day-old seedlings grown in sterilized bottles were inoculated with five milliliters of diluted culture. Subsequently, 1 cm of root below the base of the stem was collected at 2, 4, 6, 8, 10 and 12 dpi. The roots were gently shaken in sterilized water. Crystal violet solution (1%) was used to stain the roots for 15 min, and the excess stain was rinsed off with sterilized water. The 5 mL of pure ethanol was used to extract the dye bound to the roots, and the absorbance at 570 nm was visualized with a Multiskan Spectrum microplate reader. Simultaneously, other gently shaken roots were placed into the tube with 5 mL sterilized water. Afterwards, the tubes were prepared with shaking at 200 rpm for 20 min, and 100 μL liquid was collected, diluted, and coated on an LB plate. The number of colonies was counted two days later. Subsequently, the roots with a length of 1 cm were taken at the time with most colonies. After drying with a critical point dryer (K850, Laughton, East Sussex, UK), colonization of the HR10 WT strain and mutants on the surface of pine roots was observed under a scanning electron microscope (Quanta 200, Hillsboro, OR, USA). Each treatment contains 3 seedlings in the assays of colony counting, crystal violet staining, and SEM observation.
2.11. Assay of Plant Defense Gene Expression
seeds were pregerminated as described above and sown in a sterilized soil matrix. Fifteen of the fifty-day-old P. massoniana
seedlings grown in plastic holes containing sterilized soil matrix were inoculated with two milliliters of B. pumilus
HR10 and mutant cultures diluted to 108
CFU/mL with sterilized water. After 4 days, the seedlings were carefully removed, flash-frozen in liquid nitrogen separately and ground. RNA extraction was performed using the Fast Plant RNA Kit for Polysaccharides and Polyphenolics-Rlch (Zomanbio, Beijing, China). The measurement of RNA quantity, removing gDNA, synthesis of cDNA, and qRT-PCR were conducted as described above (2.7). The relative expression levels of PR2
(regulation of salicylic acid-related gene), COI
(cytochrome oxidase-related gene), and GPX
(stress tolerance-related gene) were measured. The P. massoniana
splicing factor U2af
gene was used as an internal actin reference. Experiments were performed in triplicate with three biological replicates on an ABI Prism 7500 (Applied Biosystems, Foster City, CA, USA). The relative gene expression levels were analyzed using ABI Prism 7500 software and the 2−ΔΔCt
method. The primers used to amplify these genes are listed in Table 2
2.12. Biocontrol Assay
seedlings were cultured as described in Section 2.11
and inoculated with two milliliters of solution (108
CFU/mL) of B. pumilus
HR10 and mutants dissolved with sterilized water. Four days later, each seedling was inoculated with R. solani
in the soil at the same time of sampling in Section 2.11
]. One week after inoculation with the pathogen, disease severity and biocontrol efficacy were recorded and calculated according to the following formula:
Disease incidence (%) = (the number of damping-off diseased seedlings in each treatment/
total number of seedlings in each treatment) × 100.
Biocontrol efficacy (%) = ((disease incidence of untreated seedlings–disease incidence of treated seedlings)/
disease incidence of untreated seedlings) × 100. Each treatment contained 16 P. massoniana seedlings.
2.13. Statistical Analyses
One-way analysis of variance (ANOVA) was carried out with SPSS software, and different letters indicate significant differences (p ˂ 0.05).
spp. have been identified as biological control agents against fungal and bacterial diseases. Bacillus subtilis
and Bacillus amyloliquefaciens
exert growth-promoting and biocontrol functions by competition, antibiosis, and the induction of plant systemic resistance [38
]. Successful colonization of PGPR is considered a crucial step for biocontrol [41
]. Swarming motility and biofilm formation are major adaptive strategies for Bacillus
in colonization [43
]. The results of this study demonstrated that B. pumilus
HR10 produces a mature biofilm structure, which contributes to its colonization on the roots of pine seedlings. The biocontrol efficacy of B. pumilus
HR10 was proposed to be the result of multiple causes. This study explains the mechanisms of the biocontrol efficacy from many aspects.
The biofilm-deficient mutants of B. pumilus HR10 were screened by colony phenotype. The aggregate structure of three mutants, such as the middle or colony edge, exhibited different degrees of defects. The WT B. pumilus HR10 strain was found to be most complicated. The defects of MA15 and MA1330 were more significant than those of MA4828. The mutants formed fewer floating pellicles at the liquid air interface of liquid cultures compared to WT, consistent with their simpler colony morphology than that of WT on the solid–air interface. MA15 appeared the sparsest, and MA1330 degraded the quickest. The results suggested that the defectiveness of biofilm substances and swarming motility result in defective pellicle formation and rapid degradation.
Surfactin, a typical lipopeptide that antagonizes fungal and bacterial pathogens, is also known to be involved in swarming motility and biofilm formation [20
]. For example, the inability of B. subtilis
9407 to produce surfactin led to a reduction in antagonistic activity, swarming motility and biofilm formation [4
]. In accordance with qRT-PCR measurements, we found that the expression levels of sfp
at 48 h in MA15 and MA1330 were lower than that of WT, indicating that surfactin may play an important role in the swarming motility and biofilm formation of B.pumilus
HR10 (Figure 3
Biofilm formation facilitates the colonization of Bacillus
on the roots or leaves of plants [45
]. Similarly, in our study, WT B. pumilus
HR10 effectively colonized on the roots of Pinus massoniana
seedlings, while the biofilm-deficient mutants showed significantly decreased in the colonization on the roots (Figure 4
). These results demonstrated that biofilm formation by B. pumilus
HR10 contributes to efficient colonization.
The biological control of Bacillus
depends on its inhibitory activity against R. solani
to a large extent [3
]. The inhibitory activities of MA1330 and MA4828 showed no significant differences compared with that of WT, and MA15 exhibited the weakest activity. Surfactin is a major lipopeptide compound that suppresses pathogens [4
]. According to the results of Figure 3
D and Figure 5
, we proposed that sfp
expression of B. pumilus
HR10 is related to biocontrol against R. solani
Previous studies have reported that biofilm formation is associated with rhizobacteria-induced systemic resistance [29
]. However, the function of biofilm formation in the induction of systemic resistance is not very evident. In this study, we conducted qRT-PCR measurements of defense-related genes in P. massoniana
seedlings in response to inoculation with WT or mutants. We found that the expression levels of PR2
exhibited obvious differences, whereas the expression levels of other genes were not significant. The typical gene of the salicylic acid (SA) signaling pathway of seedlings, PR2
, exhibited high levels when seedlings were inoculated with WT HR10 or mutants. The PR2
transcription level increased approximately fivefold after inoculation with WT. These results demonstrated that biofilm formation by B. pumilus
HR10 contributes its ability to induce seedling resistance by promoting the SA signaling pathway of P. massoniana
seedlings. Deficiency in biofilm formation of the mutants significantly weakened the induced seedling resistance.
There were obvious differences in the effects of WT B. pumilus
HR10 and mutants on the biocontrol against pine seedling damping-off disease. The biocontrol results suggested that biofilm formation plays a significant role during the biocontrol process mediated by the WT and mutants. This finding was consistent with a previous study by Luo et al., who reported that biofilm formation further strengthens the colonization of B. subtilis
916 and biocontrol efficacy against rice sheath blight [20
]. Indeed, chemotaxis of PGPR and subsequent biofilm formation are very important for root colonization and for providing beneficial functions for plants and PGPR [47
]. Interestingly, the biocontrol effect of MA1330, which produced defective biofilm, was equal to that of MA4828. This may be due to MA1330 triggering higher expression of SA signaling pathway-related genes, which is necessary for the resistance of plants and biocontrol of PGPR.
Overall, the results reported here indicate that the antagonistic activity of B. pumilus HR10 suppresses R. solani, and biofilm formation also contributes to the promotion of colonization, induction of plant resistance and biocontrol against seedling damping-off disease. The results of our study may provide a theoretical basis for the field application of B. pumilus HR10, which is beneficial for promoting the growth of pine trees, symbiosis of mycorrhizae and biocontrol against pine diseases. A major challenge for the future will be to explore the regulatory pathway of biofilm formation of B. pumilus HR10 and the role of lipopeptides in the formation process. It will be interesting and necessary to determine how multiple mechanisms are synergistically involved in regulating biocontrol.