Mechanism of Brevibacillus brevis strain TR-4 against leaf disease of Photinia×fraseri Dress

Experimental material

The C. siamense strain was obtained from the Laboratory of Forest Protection, Nanjing Forestry University, Nanjing, Jiangsu Province, China, and was stored in the China Forestry Microbial Strain Preservation and Management Center under the preservation number CFCC54215.

The strain was cultured on PDA medium and subsequently cultured in an incubator at 25 °C. The bacterial strains were isolated from healthy Photinia rubra rhizosphere soil, cultured on NA medium and subsequently cultured in a 30 °C incubator.

The TR-4 fermentation liquid broth was the bacterial broth of TR-4 added to 100 ml of LB liquid medium and incubated at 30 °C for 3 days with an OD₆₀₀ of about 5.

The P. × fraseri leaves used in the experiment were obtained from Yaping Nursery in Nanjing, two-year-old seedlings, which were transplanted and cultured at 28 °C under natural light.

Isolation and screening of antagonistic bacteria

A total of 50 g of soil was taken from five randomly selected points in the inter-root soil of healthy P. × fraseri plants by random sampling method and diluted using sterile water to obtain dilutions at concentrations of 10⁻¹ 10⁻², 10⁻³, 10⁻⁴ and 10⁻⁵, respectively (Ambardar & Vakhlu, 2013). The dilutions were spread on LB solid medium by spreading method and incubated in an incubator at 30 °C for 3 days. Single strains were isolated after labeling based on colonies with different morphological and color characteristics. Counts were taken and the inhibitory effect of the isolated antagonistic bacteria on C. siamense was determined using the plate antagonism method. The antagonistic effect on fungal mycelium was calculated as percentage growth inhibition (% GI). The formula for growth inhibition was 1-(experimental/control) × 100%. Data were obtained from three different experiments.

Effect of TR-4 fermentation liquid broth on the germination of C. siamense spores

A total of 500 µl of 0.1% glucose aqueous solution was added to the C. siamense spores suspension (spore suspension concentration is 10⁶/ml) and TR-4 fermentation liquid in a 2 ml aseptic centrifuge tube, and the concentration was adjusted to the EC₅₀ (median effective concentration), 10 EC₅₀, and EC₉₀ according to the ratio, with a final volume of 500 µl. LB liquid medium was used instead of TR-4 bacterial solution as a control. The spores were cultured in a dark incubator at 25 °C, after which 5 µl was extracted from the test tube every 12 h and placed on a glass plate, until the control spores had fully germinated. Spore germination was observed under a Zeiss microscope.

In vivo antagonism experiment

The experimental samples was divided into three groups. The first group was the biocontrol group. The spore solution of C. siamense 10 µl (spore suspension concentration of 10⁶/ml) was inoculated first, and the bacterial solution of TR-4 fermentation liquid was inoculated 24 h later to observe the control effect of TR-4 on C. siamense on plant leaves of P. × fraseri. In second group (the protection group), plants were sprayed with TR-4 fermentation liquid; they were completely dried and inoculated with C. siamense spore solution to observe whether TR-4 could help plants resist C. siamense infection on the leaves. The third group (the control group) was inoculated with only the C. siamense spore solution, and each experiment was repeated 3 times.

Molecular identification of TR-4

The universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CGGCTACCTTGTTACCAC-3′) of the bacterial 16S rRNA gene were used for PCR amplification (Johnson, Bowman & Dunlap, 2020). The PCR mixture was as follows: 2 × Taq PCR Master Mix 25 µL, 2 µL of F primer, 2 µL of R primer (the primer working solution concentration was 10 µM), 2 µL of template DNA (DNA extraction was performed using Vazyme’s DNA extraction kit), and ddH₂O to a total volume of 50 µL. The PCR procedure was as follows: predenaturation at 94 °C for 5 min; denaturation at 94 °C for 1 min; annealing at 58 °C for 1 min; and denaturation at 72 °C for 2 min. Thirty cycles were repeated and finally extended for 10 min at 72 °C. The PCR products were sequenced by Nanjing Bioengineering. After NCBI BLAST comparison, MEGA7 software was used for sequence analysis, and the neighbor-joining (NJ) method was used to construct a phylogenetic tree.

Analysis of TR-4 secretions

Determination of ferriphilin

Iron is a micronutrient widely found in the Earth’s crust; a small amount of iron is necessary for plants, and iron deficiency is a plant nutrient disorder. Iron forms iron oxide hydrates in the environment, resulting in a lower concentration of free iron and reduced bioavailability. The CAS test solution is a bright blue compound consisting of chromium, cetyltrimethyl ammonium bromide, and iron ions. When the iron ions in the blue test solution are removed by the ferritin secreted by microorganisms, the CAS test solution changes from blue to orange, so the CAS liquid medium can be used to detect the production of ferritin by microorganisms (Pérez-Miranda et al., 2007). The light absorption (As) of the supernatant after centrifugation was measured at 630 nm and adjusted to zero using double steaming water as a control. Another blank medium was mixed with the CAS test solution in equal amounts, and its light absorption value was taken as the reference ratio (Ar). The experimental method was performed according to the CAS assay kit instructions.

Determination of cellulase activity (3, 5-dinitrosalicylic acid method)

Cellulase hydrolyzes cellulose to produce cellobiose, glucose and other reducing sugars, which can reduce the nitro in 3, 5-dinitrosalicylic acid to orange amino compounds, and use a colorimetric method to determine the generation of reducing compounds to indicate the activity of the enzyme (Song et al., 2016). The experimental method refers to the assay of cellulase by Song et al.

Determination of chitosanase activity

The modified Schales method was used to determine the enzyme activity. The principle behind this process is that soluble chitosan undergoes enzymolysis and releases reducing sugars, which react with the Schales reagent to change color. With N-acetylglucosamine as the standard sugar, the light absorption value of the reducing sugars was determined via a spectrophotometer at 420 nm. The amount of enzyme that breaks down 1 µ/mol NAG per minute is defined as one unit of activity (U) (Mojumdar et al., 2019).

Microscopic analysis of the inhibitory effect of strain TR-4 on C. siamense

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) samples were obtained from fresh PDA plates. The samples were divided into two categories:(1) no inhibition on fungal growth, such as control group; (2) obvious inhibition of fungal growth, such as the experimental group.

The samples were fixed with 2.5% glutaraldehyde and 1% cesium tetroxide at room temperature, dehydrated with ethanol, critically dried, covered with gold, and observed with a scanning electron microscope (JEM 2100) .

The TEM samples were prepared by a similar method. The C. siamense are cut into 2 × 3 mm slices. The specimens were placed in 2.5% glutaraldehyde solution, fixed at room temperature for 5∼6 h, cleaned with 0.1M phosphate buffer (PBS, pH 7.2) for 5 times, fixed with 1% cesium tetroxide for 1.5 h, cleaned with PBS for eight times, dehydrated with ethanol and acetone, coated with SPUS resin, sliced 10 µm, stained, and observed under transmission electron microscope.

Data analysis

In this research, all the experiments were carried out in triplicate and repeated three times to get accurate and reliable data. A completely randomized design was used for the greenhouse experiment, and its data were examined with analysis of variance (ANOVA) followed by the least significant difference (LSD) tests at p < 0.05 using the DPS v9.5 statistical software package.

Soil sample collection and screening of antagonistic bacteria

The experiment isolated 68 soil bacteria from five different concentrations of soil solutions. After the antagonism experiment of the 68 isolated bacteria against C. siamense, it was concluded that a total of 13 strains of bacteria had an effect on C. siamense. The strain with the strongest inhibitory effect was selected as the experimental strain and named TR-4(Fig. 1).

In vitro antagonistic experiment and inhibitory effects of TR-4 on C. siamense spore germination

To further determine the inhibitory effect of TR-4 on C. siamense, the concentrations used were set to 0.001 µl/ml, 0.01 µl/ml, 0.1 µl/ml, 1 µl/ml and 10 µl/ml through C. siamense culture and experiments (Fig. 2). According to DPS v9.5 analysis, the average EC₅₀ was 0.0022 ±0.0013 µl/ml, and the average EC₉₀ was 0.8115 ±0.1024 µl/ml (Table 1).

Moreover, to verify whether the TR-4 strain has an antagonistic effect on C. siamense spore germination, C. siamense spores were treated with sterile TR-4 filtrate, and the final solution EC₅₀, 10 EC₅₀ and EC₉₀ were determined. We found that the spore germination rate of Bacillus anthracis treated with the TR-4 strain fermentation filtrate was significantly lower than that of the control group within 12 h. After 48 h, the spore germination rate of the control was 98%, while the spore germination rate of the TR-4 strain treated with fermentation filtrate was significantly lower than that of the control (Fig. 3 and Table 2). The mycelial length and mycelial branching number after spore germination were significantly lower than those in the control group, and the differences did not decrease with increasing time. Like in other studies, in this study, TR-4 was shown to reduce the germination rate of C. siamense and thus reduce their pathogenicity.

In vivo antagonism experiment

The results of in vivo antagonistic experiments revealed that TR-4 has significant inhibitory effects on C. siamense and protective effects on plants. In the first group of control experiments, the lesion did not expand after spraying the TR-4 bacterial solution, and the control effect was remarkable. In the second group of plant protection experiments, compared with those in the first group of experiments, although the effects were not ideal, some inhibitory effects were detected. The results showed that TR-4 can also be directly inhibit C. siamense growth on leaves of C. siamense plants (Fig. 4). In this study, the phenotype and severity of the disease in the biocontrol group were significantly lower than those in the control group, indicating that TR-4 inhibited the incidence of C. siamense in P. × fraseri.

Molecular identification of TR-4

The 16S rRNA sequences of TR-4 were analyzed. PCR amplification and sequencing revealed that the length of the 16S rRNA gene was 1430 bp. The 16S rRNA nucleotide sequence of TR-4 was registered in the GenBank database under accession number OP658963.1. The 16S rRNA sequence of TR-4 was identified by the National Center for Biotechnology Information (NCBI) database and shared 99% homology with the 16S rRNA gene sequence of B. brevis ON014586. A phylogenetic tree was constructed using MEGA 6 software, and strain TR-4 was identified as B. brevis (Fig. 5).

Analysis of TR-4 secretions

In the determination of ferritin, according to the radical formula [(AR-AS)/Ar] × 100%, the relative content of ferritin was 88.46 ±2.08%.

In the determination of cellulase activity, the absorbance of the color developing solution was determined, and a standard curve was drawn with the absorbance as the vertical coordinate and the glucose content as the horizontal coordinate. The linear regression equation was used to construct the standard curve of the reducing sugars. The standard curve equation was y = 0.16822x + 0.0073 (R² = 0.8802). The absorbances of the blank tube and sample were 0.0264 and 0.0485, respectively. The above standard curve equations for the glucose content of the blank tube and sample were 0.1135 and 0.2449, respectively. The results showed that strain TR-4 could produce cellulase, and the ability to produce cellulase was very strong.

In the chitosan enzyme activity assay experiment, according to the experimental method, the results were obtained as 2.15 µmol of reducing sugars and 0.215 U of enzyme activity units.

Microscopic analysis of the inhibitory effect of strain TR-4 on C. siamense

Under scanning electron microscopy, normal mycelia of C. siamense were found to be evenly distributed, smooth and full. After treatment with TR-4, mycelial growth was abnormal, the mycelial shape and surface were deformed, and the surface was contracted. Under transmission electron microscopy, mitochondria, ribosomes, vacuoles, cell walls and even plasma from normal C. siamense cells were clearly visible. After treatment with the TR-4 strain, the cells of C. siamense exhibited obvious changes and damage. The cell wall was transparent, the organelles disappeared, and the vacuoles were deformed (Fig. 6).