Strains

B. cinerea, Fusarium graminearum, Fusarium oxysporum, Cylindrocarpon destructans, Pestalotiopsis paeoniicola, Trichothecium roseum, Fusarium proliferatum, Fusarium semitectum, and Alternaria panax were provided by the Plant Pathology Laboratory, Institute of Special Animal and Plant Sciences of CAAS, China. Each pathogen was re-cultured in a Petri dish containing potato dextrose agar (PDA) and incubated at 28°C for seven days in the dark. After this, each sample was transferred to a PDA slant in a test tube and stored at 4°C until use.

Screening of antagonistic strains

The bacteria strains isolated from 20 mL LB culture medium were transferred to 50 mL triangle bottle and cultured under 28°C, 220 r·min^-1 condition. The bacteria fermented in 1 mL of sterile water were poured into a PDA plate containing the grey mould. The bacterial spores were blown and placed in a bacterial suspension (10⁵ spores·mL^-1). It (tablet form) was mixed with PDA medium in a Petri dish containing B. cinerea. The tablet was uniformly punched with injection. It was placed in 50 μL fermented liquid, at 28°C and cultured for 3 days based on the inhibitory effect of bacteriostatic ring size of the strain (Zhou, 2021) (Figure 1).

Morphological identification

The morphological characteristics of the antagonistic strains were observed after incubating them on BPA (The Reagent Company of Shiao, China) at 28°C for 3 days. They were identified using light microscopy according to the morphological characteristics of their color.

Analysis of 16 s rRNA sequence

The 16 s rRNA sequence was amplified using polymerase chain reaction (PCR); the PCR was amplified using the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (Weisburg et al., 1991). The amplified PCR products were cloned and sequenced according to the Pan’s protocol, and nucleic acid was determined using NCBI BLAST (http://www.ncbi.nlm.nih.gov). Phylogenetic analysis was conducted using maximum likelihood in MEGA 5.10. The topology of the phylogenetic tree was evaluated by 1,000 re-samplings.

Bacteriostatic spectrum determination

The antagonist bacteria strains were inoculated in LB medium at 28°C, 220 r·min^-1 for 24 h. They  were  fermented  in  liquid  at  4°C, 10000 r·min^-1 after centrifuging for 10 min. The fermented liquid was sterilized, using 0.45 μm microporous filtration membrane. The liquid was fermented at 1, 3, 5, 7 and 9% concentrations, and cooled to 40 ~ 45°C, respectively with melt blending. The PDA medium containing the tablet, and the PDA medium without sterilized and fermented liquid (control) developed 6 kinds of pathogenic fungi cakes (5 mm in diameter) for 7 days. The mycelium was placed upside down in the medium plate, at a constant temperature of 25°C and cultured for 7 days. The diameter of the colonies was measured using cross over method. The antibacterial rate of the pathogenic bacteria was calculated. Each of these pathogens was set at three repetitions.

Bacteriostatic rate (%) = (controlled colony diameter - processing colony diameter) /(controlled colony diameter - 5) by 100.

Determination of nitrogen fixation ability

The antagonistic bacteria were inoculated on Ashby's medium (Cui et al., 2016), liquid medium, sterile water (control) 3 times. They were cultured at a constant temperature of 28°C in an incubator, and liquid medium at 120 r/min. The culture was shaken after 7 days. The tablet on the colony and liquid medium is cloudy and positive.

Extracellular protease qualitative detection

The antagonistic bacteria strains were inoculated on casein medium at a constant temperature of 28°C in an incubator. They were cultivated for 48 h, and observed whether they produced a transparent circle.

Screening of antagonistic bacterial strains

A total of 56 endophytic bacteria strains were isolated through two rounds of leaf sample screening, according to the morphological characteristics of the colonies. Fifteen of these showed prominent antagonistic activities against B. cinerea in vitro. Strain FS-1 had 18 cm inhibition zone against B. cinerea in the dual-culture test (Table 1), which was the largest inhibitory activity among the isolates.

Morphological identification of antagonistic bacteria

The colonies of FS-1 strains were like candle. They had the shape and quality of a candle; no pigment, and their diameter was 5 mm (Figure 2A). They were placed on the BPA medium at a constant temperature of 37°C, for 24 h and then they were cultured. The FS-1 strains had rod shape with size of 0. 5 to 0.8 µm × 1.0 to 1.5 μm. They were Gram positive (Figure 2B). FS - 1 strains  needed oxygen to grow at temperature ranges between 30 and 38°C, pH 5.6 - 7.2, maltose, sucrose and salicin, enzyme, determination of V - P, oxidation of glucose fermentation, gelatin liquefaction, nitrate reduction, malonic acid, urea enzyme,  hydrogen  sulfide,  egg   yolk   lecithin  enzyme, motility, hydrolyzed starch, citrate utilization, and methyl red (Table 1). The strain FS - 1 was identified as Bacillus spp. according to the morphological characteristics combined with the physiological and biochemical test results.

Analysis of 16S rRNA sequence

PCR products of 1472-bp were obtained by amplifying the  16S   rRNA   of   the   genomic   RNA  of  FS-1 strain. Sequence analysis showed that FS-1 strain shared 100% identity with a number of B. cereus in the NCBI database (Accession No. MN746190). Phylogenetic tree was constructed by using 16S rRNA sequences, and it clearly showed that FS-1 strain was clustered with members of the genus Bacillus (Figure 3). FS-1 strain was identified as B. cereus based on the results of the 16S rRNA sequence analysis and the morphological characterization.

Bacteriostatic spectrum

The bacteriostatic spectrum test results showed that different concentrations of liquid fermented   FS-1 strains (8 kinds of plant pathogenic fungi) have certain inhibitory effect. Their bacteriostatic spectrum ranges changed with the fermented liquid concentration; the inhibition rate of the pathogens changed. When the fermented liquid concentration was 9%, the bacteriostatic rate of T. roseum was highest; at 91.47%, the bacteriostatic rate of F. oxysporum was the lowest; at 47.91%, the bacteriostatic rate of the other 6 kinds of pathogens was between 49.26 and 90.02% (Table 2).

Nitrogen fixation ability

The colony of FS-1 strain formed was inoculated on the solid Ashby's medium, at a constant temperature of 28°C. It was placed in an incubator for 3 days. The FS-1 strain was inoculated on the liquid Ashby's medium at a constant temperature of 28°C and cultured in an incubator for 5 days. It became turbid. The experimental results showed that FS-1 strain had ability to fix nitrogen.

Extracellular protease

FS-1  strain  can  form  a  transparent  circle   around  the colonies (Figure 4) inoculated on the casein medium plate at a constant temperature of 28°C in an incubator for 2 days. Its diameter was 18.53 mm. Extracellular protease qualitative test results showed that FS-1 strain can produce extracellular protease.

At present, these chemical pesticides were used to prevent and control ginseng gray mold disease such as difenoconazole, propiconazole, mancozeb, carbendazim, kresoxim-methyl, cyprodinil, azoxystrobin, polyoxins, fluazinam, hymexazol, copper hydroxide, etc. But studies have shown that with long-term use of chemical pesticides, pathogens will develop resistance (Saito et al., 2016). Therefore, there is need to seek new methods to prevent and control ginseng grey mould to slow down the drug resistance of pathogenic bacteria. Endophytic bacteria in plant body can form stable colonization for long, and they are not susceptible to the influence of external environment. Endogenous bacteria and pathogens have mutual reciprocity and benefit; they are a kind of important resources (Aly et al., 2011). It has been reported that B. amyloliquefaciens, Bacillus methylotrophicus, Paenibacillus polymyxa, and Enterobacter cloacae can be used to prevent and control ginseng disease (Wang et a., 2016, Jiang et al., 2013, Li et al., 2013 and Jiang et al., 2019), but it has not been reported that B. cereus can be used to prevent and treat ginseng grey mould.

Endophytic bacillus is a kind of non-pathogenic bacteria that can produce spores, a variety of antibacterial substances, such as antibiotics and antimicrobial proteins. Bacillus has strong resistance, reproduces fast, can survive in an environment, forms colonization fast, is an important   resource  of   bio-control   bacteria,   has  wide application in the prevention and control of green plant disease. In this study, a strain isolated from the leaves of P. ginseng was found to have very good inhibitory effect against ginseng gray mould bacterium. The endophytic bacterium identified is B. cereus strain; its bacteriostatic spectrum is more extensive. FS - 1 to 8 kinds of common plant pathogenic fungi are highly bacteriostasis. Zhou et al. (2017)’s studies showed that B. cereus metabolites can effectively restrain the growth of the pathogen of cotton yellow dwarf. B. cereus is a kind of bio-control bacterium. This study found that FS - 1 strain can fix nitrogen in plants. Extracellular protease test results showed that the strain can produce extracellular protease, and degrade the cell wall of enzymes. This has laid a foundation for its further use and application in the production of new bio-control bacteria resources for preventing ginseng mould.

The authors have not declared any conflict of interests.

This research was supported by the Chinese Academy of Agricultural Science and Technology Innovation Project (No. CAAS - ASTIP - 2020 - ISAPS).