Biocontrol and Growth Promotion Potential of Bacillus subtilis CTXW 7-6-2 against Rhizoctonia solani that Causes Tobacco Target Spot Disease

The Guizhou Province Tobacco Company (Guiyang, Guizhou province, China) provided a tobacco variety Yunna 87. B. subtilis CTXW 7-6-2 was previously isolated from kiwifruit and strongly inhibits R. solani (GenBank accessions: OK287282) associated with tobacco target spot. The biocontrol strain and pathogenic fungi were preserved at the Kiwifruit Engineering Technology Research Center of Guizhou University, stored in glycerol at −80°C. The isolation method and in vitro antifungal activity assay, as described by Chen et al. (2022), were employed in the study. The cell concentration of the CTXW 7-6-2 culture medium was adjusted to 1 × 10⁷ CFU/ml before application. The culture media were nutrient broth NB: 5 g beef extract, 10 g peptone, 5 g NaCl, and 1000 ml water) and potato dextrose agar (PDA) (200 g potatoes, 10 g glucose, and 1,000 ml water).

Domańska’s (2019) method of determining bacterial growth was used and modified slightly to 2% (v/v) of the 1.0 × 10⁷ CFU/ml CTXW 7-6-2 bacterial suspension inoculation in NB medium. The cultivation conditions were as follows: fluid volume 100 ml/250 ml, temperature 28°C, and shaking at 180 rpm. The OD₆₀₀ was measured using a Thermo Scientific (USA) UV-VIS spectrophotometer at 6, 12, 24, 48, 72, 96, and 120 h.

The CTXW 7-6-2 strain was activated, transferred to NB medium, and incubated at 28°C with shaking at 180 rpm for 48 h as a seed solution. R. solani of tobacco target spot was transferred to a PDA plate for cultivation for 4 d (cultured in the dark at 28°C). Subsequently, holes were created in the mycelium cake using a 6-mm hole punch placed in the PDA plate’s center. Then, 5 μl CTXW 7-6-2 seed solution was inoculated at four points 25 mm from the plate. Plates containing R. solani alone were used as the control group. After culturing at 28°C for 4 d, the colony diameter was measured, and the inhibition rate was calculated using the formula below: Inhibition rate (%)=(diameterofcontrolcolony−diamdeter of treated colony)diameterofcontrolcolony×100 $$Inhibition{\rm{ }}rate(\% ) = {{\matrix{ {(diameterofcontrolcolony - diamdeter{\rm{ }}of{\rm{ }}treated{\rm{ }}colony)} \hfill \cr } } \over {diameterofcontrolcolony}} \times 100$$

The antagonistic effect of sterile fermentation broth against R. solani was determined according to the method described by Xia et al. (2017), with slight modifications. Briefly, 1 ml of CTXW 7-6-2 seed solution was inoculated in 150 ml NB medium in a conical flask (specification 250 ml), and the fermentation broth was obtained via shaking culture at 28°C and 220 rpm for 96 h. The 150-ml fermentation broth was centrifuged at 4,000 rpm for 30 min in a low-speed benchtop centrifuge, and the supernatant was filtered through a 0.22-μm filter to remove the bacteria to produce a sterile fermentation broth without high-temperature treatment. To further verify that the antifungal substances in the fermented broth are resistant to high temperatures, a sample fermentation broth was treated at a high temperature (120°C) for 20 min. The fermentation broth (5 ml) with or without high-temperature treatment was added to 45-ml PDA medium at 40°C, mixed, and poured into a sterile petri dish. After coagulation, a 6-mm diameter R. solani cake was placed in the center of the plate, and a PDA plate without fermentation broth was used as the control group. The experiment was repeated in triplicate. After the colonies in the control group covered 23{}^{2}\diagup{}_{3} of the culture dish, the colony diameter on each treatment plate was measured, recorded, and the inhibition rate was calculated.

The crude lipopeptide was extracted using acid precipitation (Nair et al. 2020). Briefly, 600 ml of CTXW 7-6-2 sterile fermentation broth was added to 6 mol/l HCl acid, pH 2.0, sealed at 4°C, and allowed to stand for 16 h. The above mixture was centrifuged at 10,000 rpm at 4°C for 15 min, the precipitate was collected and dissolved using methanol solution, and the supernatant was collected via centrifugation at 10,000 rpm at 4°C for 15 min. The supernatant was then evaporated at 55°C and 60 rpm. The crude lipopeptide extract was collected, weighed, recorded, and stored in the refrigerator at 4°C until further use.

The mycelium growth rate method was used to determine the inhibitory effect of the crude lipopeptide on R. solani. Briefly, crude lipopeptide (0.12 g) was dissolved in 4-ml methanol solution, and the excess impurities were removed using a 0.22-μm filter membrane to obtain 30,000 mg/l mother liquor. Then, 0.15 ml of mother liquor was added to 45 ml of PDA medium, mixed evenly, and poured into a sterile petri dish. The crude lipopeptide concentration was 100 mg/l, and the methanol content was 0.33%. A 6-mm diameter R. solani cake was placed in the center of the plate. A PDA medium plate containing 0.33% methanol was used as the control group, and the experiment was repeated in triplicate. After the colony in the control group covered 2/3 of the culture dish, the colony diameter on each treatment plate was measured, recorded, and the inhibition rate was calculated.

Mannaa and Kim’s protocol (2018), was used to determine the effects of CTXW 7-6-2 volatile compounds on fungal mycelia, with slight modifications. The specific protocol was as follows: a 6-mm hole punch was used to obtain the cake containing R. solani, which was placed in the center of a PDA plate for later use. A total of 150 μl of CTXW 7-6-2 seed solution was coated evenly on nutrient agar plates, air-dried naturally, and the two plates were aligned and sealed together, with the solution containing no CTXW 7-6-2 being used as the control group. The experiment was repeated in triplicate. The colony diameters and inhibition rate were calculated.

An antifungal area on the edge of the hyphae was selected to evaluate the restraining effect of CTXW 7-6-2. Hyphal morphology and cell ultrastructure were observed using the SU8100 scanning electron microscope (SEM) (Hitachi, Japan) and JEM1200EX transmission electron microscope (TEM) (Mo et al. 2021; Chen et al. 2022).

Yunyan 87 seeds were cultured in floating seedling trays with sterilized substrates. The seedlings with four leaves were selected as the test plants, with a consistent single spraying of CTXW 7-6-2 bacterial suspension at a concentration of 1.0 × 10⁷ CFU/ml. NB liquid medium was used as a blank group. Seven days after treatment, the maximum leaf length, leaf width, whole plant fresh weight, root length, root dry weight, and root number of the seedlings were measured. Each process was repeated thrice, each repeat containing five seedlings.

Real-time PCR was used to examine the tobacco’s CTXW 7-6-2 treatment time course-related gene expression. Yunyan 87 was used as the test plant with six leaves at the seedling stage. The mycelium of R. solani was cultured in a 50-ml PD medium with shaking for four days, and the fresh mycelium was collected by filtering with gauze. Mycelial suspensions were collected by breaking the mycelium and filtering with two layers of gauze, and sterile water was used to adjust the mycelium to 15–20 strips (100 ×) under a light microscope. It was then sprayed uniformly onto the surface of tobacco leaves, and the suspension of CTXW 7-6-2 (1.0 × 10⁷ CFU/ml) was sprayed after the onset of symptoms. Samples were collected at 0, 6, 12, 24, 48, and 72 h, with three replicates per treatment and three seedlings per replicate, to determine the disease-related genes, including pathogenesis-related gene1 (NPR1), plant defensin (PDF1.2), chitinase (chit), phenylalanine ammonialyase (PAL), peroxidase (POD), and polyphenol oxidase (PPO) genes.

Referring to the primer sequence published by Jiao et al. (2019), Shanghai Bioengineering Co., Ltd. (China) was commissioned to synthesize the required primers, and the tubulin gene was used as the reference gene for tobacco. Each tobacco sample was weighed, and 100 mg was placed in a pre-cooled mortar. Liquid nitrogen was added, and the tobacco sample was quickly ground into a powder. The ground sample was transferred to a 1.5-ml centrifuge tube. Total RNA was extracted using the RNAprep Pure Plant Total RNA extraction kit (TIANGEN Biotech (Beijing) Co., Ltd., China), following the manufacturer’s instructions. Genomic DNA was removed using DNase I. RNA integrity and DNA contamination were analyzed using 1.5% agarose gel electrophoresis. RNA was reverse transcribed according to the instructions of the FastKing one-step (TIANGEN Biotech (Beijing) Co., Ltd., China) method. The reaction system consisted of 2 μg RNA, 4 μl 5 × FastKing-RT SuperMix, and RNase-free ddH₂O up to a 20-μl solution volume. The experiment was performed at 42°C for 15 min, 95°C for 3 min, and finally stored at −20°C. The RT-qPCR reaction system was configured according to the instructions described by the Talent Fluorescence Quantitative Detection kit (TIANGEN Biotech (Beijing) Co., Ltd., China), and the two-step reaction program was set for detection. Twenty microliters of the reaction mixture comprised 10 μl 2× Talent qPCR PreMix, 0.6 μl reverse primers (10 μM), 0.6 μl forward primers (10 μM), 0.5 μl cDNA template (0.1 μg/μl), and 8.3 μl RNase-free ddH₂O. Amplification was performed using a BIO-RAD (USA) Bole real-time PCR instrument with a reaction program of one cycle at 95°C for 3 min, 40 cycles at 95°C for 5 s, and 60°C for 15 s. Three technical replicates of the target gene were performed for each sample, and relative expression was calculated.

All experimental data were analyzed using SPSS^® 17.0 (SPSS Inc., USA) and the Duncan multiple range test. Statistical significance was set at p < 0.05.