Biocontrol bacteria CL95 for preventing ginseng rust and promoting ginseng growth

Abstract

The invention discloses a biocontrol fungus group CL95 for preventing and controlling ginseng rust and promoting ginseng growth; the biocontrol fungus group CL95 comprises: methylotrophic Bacillus NJ13, Bacillus Velezii NT35, Bacillus amyloliquefaciens FG14, Pseudomonas chlororaphis TU26 with a deposit number of CCTCC M 2024220, Pseudomonas severvale JI6, and Arthrobacter nicotinicus JI39‑2; the biocontrol fungus group CL95 significantly resists I. robusta It can reduce the infection of ginseng, reduce the content of pathogens in ginseng, increase soil enzyme activity, expression of defense enzyme genes, and promote ginseng growth, showing better disease prevention effects. The colonization in plants and soil is more stable. The microorganisms enriched in ginseng roots and soil can affect plant health through direct antagonism, inducing systemic resistance, or indirectly regulating pathogens through soil microorganisms. The ginseng rhizosphere bacteria Pseudomonas chlororaphis TU26 strain has a certain nitrogen fixation ability and a strong ability to dissolve phosphorus and potassium. It can promote the growth and development of ginseng and increase ginseng yield; the effect is more significant when used in combination with biocontrol bacteria.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a biocontrol bacterial group CL95 for preventing and controlling ginseng rust and promoting ginseng growth. Background Art

[0002] Ginseng, a perennial medicinal plant of the genus Panax in the Araliaceae family, has high medicinal value. Due to long-term continuous cropping and long-term unreasonable use of drugs, ginseng diseases have increased, directly affecting ginseng yield and quality. Among them, ginseng rust can harm all stages of ginseng's growth period and is the most serious root disease of ginseng. The average incidence rate is 15% - 30%, and the incidence rate in some serious places is more than 70%, and the yield loss rate is as high as 20% - 50%. Leading to serious economic losses. The disease has a strong latent infection ability and a long disease cycle. According to statistics, the field incidence rate is 15% - 30%, and the incidence rate in some serious places is more than 70%, and the yield loss rate is as high as 20% - 50%. At present, chemical control is the main method, but due to the perennial characteristics of ginseng and the strong latent infection characteristics of the disease, coupled with the short duration of chemical agents, the control effect is not good, and long-term use is prone to environmental pollution, excessive pesticide residues and the emergence of drug resistance, which seriously affects the safety and commodity value of ginseng production. To solve this problem, biological control has received more and more attention. There have been reports on the use of Bacillus, Pseudomonas and Trichoderma in the control of ginseng rust, and there are also biological control agents based on single strains. However, in practical applications, there are problems such as unstable control effects, single mechanism of action, and poor colonization ability. In recent years, the emergence of synthetic bacterial communities has received more and more attention and attention, especially in the control of soil-borne root diseases such as pea sclerotinia, tomato wilt, tomato nematode disease, and sweet pepper blight. The effect is significantly better than that of single strains, providing new research ideas and development potential for biological control. However, there are currently no relevant reports on the use of biological control communities in the control of ginseng diseases. Summary of the invention

[0003] The purpose of the present invention is to solve the above problems and provide a biocontrol fungus group CL95 for preventing and controlling ginseng rust and promoting ginseng growth.

[0004] Pseudomonas chlororaphis ( Pseudomonas chlororaphis ) TU26 strain, deposited in China Center for Type Culture Collection, and its deposit number is CCTCC M 2024220.

[0005] The biocontrol bacteria group CL95 for preventing and controlling ginseng rust and promoting ginseng growth comprises: a methylotrophic Bacillus sp. ( Bacillus methylotrophicus )NJ13, Bacillus Velezii ( B. velezensis )NT35, Bacillus amyloliquefaciens ( B. amyloliquefaciens ) FG14, Pseudomonas chlororaphis with the accession number of CCTCC M2024220 ( Pseudomonas chlororaphis )TU26, Pseudomonas severvale JI6 ( P. thivervalensis ), Arthrobacter nicotinicus with the accession number of CCTCC M 2015667 ( Arthrobacter nicotinovorans )JI39-2;

[0006] Their volume ratio is 1:1:1:1:1:1;

[0007] The concentration of each strain was 1×10 9 cfu / mL.

[0008] The present invention provides a biocontrol bacterial group CL95 for preventing and controlling ginseng rust and promoting ginseng growth, which comprises: NJ13+NT35+FG14+TU26+JI6. The biocontrol bacterial group CL95 significantly resists I. robusta It can reduce the infection of ginseng, reduce the content of pathogens in ginseng, increase soil enzyme activity and defense enzyme gene expression, significantly promote ginseng growth, and show better disease prevention effect. Compared with single strains, the colonization in plants and soil is more stable. The microorganisms enriched in ginseng roots and soil can affect plant health through direct antagonism, inducing systemic resistance, or indirectly regulating pathogens through soil microorganisms. Pseudomonas chlororaphis ) TU26 strain has a certain nitrogen fixing ability and a strong ability to dissolve phosphorus and potassium. It can promote the growth and development of ginseng and increase ginseng yield. It has a significant effect when used in combination with biocontrol bacteria.

[0009] The problem that the selected biocontrol strains are mainly based on the interaction between individual microbial strains and plants has been solved, resulting in the existing biocontrol agents being mainly single strains, with problems such as a single mechanism of action, poor colonization ability in the field, and unstable control effect. Different biocontrol bacteria are combined to construct a biocontrol bacterial community, and the composite bacterial community has the characteristics of multi-effect stability. The optimal bacterial community combination is screened out through indoor and outdoor screening methods to prevent and control ginseng rust rot, and the mechanism of action of the biocontrol bacterial community is preliminarily clarified, laying a theoretical foundation and providing solutions for overcoming the problems of unstable biocontrol effects and single functions of single strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Antagonistic test of TU26 strain, A: ginseng rust rot pathogen, B: ginseng gray mold pathogen, C: ginseng sclerotinia pathogen, D: ginseng blight pathogen, E: ginseng damping-off pathogen, F: ginseng black spot pathogen, G: ginseng root rot pathogen

[0011] Figure 2Analysis of growth-promoting functions of TU26 strain, A: nitrogen fixation potential, B: phosphorus solubilization characteristics, C: potassium solubilization characteristics, D: IAA production, E: siderophore production;

[0012] Figure 3 TU26 strain, colony morphology (A) and Gram staining (B);

[0013] Figure 4 Molecular identification clustering profile of TU26 strain;

[0014] Figure 5 The depth and diameter of lesions on the surface of ginseng in vitro inoculated in the ginseng room; the dots in the figure represent the diameter of lesions in vitro inoculated with ginseng of different bacterial flora combinations;

[0015] Figure 6 Diameter of transparent circle of biocontrol bacteria for indoor nitrogen fixation, phosphorus dissolution and potassium dissolution; Note: Different colors represent bacterial combinations with different richness. The triangle symbol in the figure represents the diameter and depth of lesions of ginseng in vitro inoculated with different bacterial combinations;

[0016] Figure 7 Determination of the mycelial growth of CBLJ-3 rust and rot pathogen by biocontrol bacteria;

[0017] Figure 8 Determination of spore production of CBLJ-3 rust pathogen by biocontrol bacteria;

[0018] Fig. 9 Effects of single biocontrol strains and biocontrol bacterial groups on ginseng field growth;

[0019] (A): Disease index; (B): Prevention and treatment effect;

[0020] Fig.10 Effects of single biocontrol strains and biocontrol bacterial groups on ginseng field growth;

[0021] Fig.11 Effects of single biocontrol strains and biocontrol bacterial groups on ginseng yield increase rate;

[0022] Fig.12 Changes in the colonization of single biocontrol strains and biocontrol bacterial groups in ginseng; (A): leaves; (B): stems; (C): epidermis; (D): root core; (E): lateral roots; (F): fibrous roots; (G): rhizosphere soil;

[0023] Fig.13 Effects of single biocontrol strains and biocontrol bacterial groups on enzyme activities in rhizosphere soil of ginseng at different stages; (A): catalase; (B): urease; (C): sucrase; (D): phosphatase;

[0024] Fig.14 Changes in relative expression of ginseng defense enzyme genes induced by single biocontrol strains and biocontrol bacterial strains;

[0025] (A): β-1,3 glucanase; (B): phenylalanine ammonia lyase; (C): peroxidase; (D): superoxide dismutase; (E): chitinase;

[0026] Fig.15 The composition of fungal taxa at different growth stages at the genus level;

[0027] AC is a bar graph of relative abundance of species in fungal communities; DF is a heat map of species abundance clustering in fungal communities. DETAILED DESCRIPTION

[0028] The pathogenic fungus strain is Panax ginseng rust CBLJ-3, which was collected by the College of Plant Protection of Jilin Agricultural University and was isolated and purified and identified as Ilyonectria robusta (Ginseng rust pathogen Ilyonectria robusta Screening and verification of reference genes for real-time quantitative PCR infecting ginseng roots, Journal of Jilin Agricultural University, Zhang Yalin, Jilin Agricultural University);

[0029] NJ13 ( Bacillus methylotrophicus ), accession number: CGMCC No.6679;

[0030] FG14 ( B. amyloliquefaciens ) ("Preparation of wettable powder of Bacillus amyloliquefaciens FG14 and its field control of ginseng rust disease" Journal of Jilin Agricultural University. 2020, 42(04));

[0031] NT35( B. velezensis ) (Master's thesis Bacillus velezensis Study on the Control Effect of NT35 Strain on Ginseng Rust Disease, Wang Jiarui, Jilin Agricultural University).

[0032] JI39-2 ( Arthrobacter nicotinovorans ), accession number: CCTCC M 2015667);

[0033] TJ23-2 ( B. subtilis ), accession number: CCTCC M 2022750

[0034] TU26 ( Pseudomonas chlororaphis ), accession number: CCTCC M 2024220 JI6 ( P. thivervalensis ), (Master's thesis "Optimization of fermentation process of strain JI6 and its effect on promoting ginseng growth", Liu Tingting, Jilin Agricultural University)

[0035] JA38( Burkholderia cepacia ), (Master's thesis Burkholderia cepaciaOptimization of fermentation process of JA38 strain and its biocontrol effect on ginseng rust pathogen, Zhang Ziheng, Jilin Agricultural University)

[0036] JJ5-2( B. thuringiensis )("Screening and identification of bacteria producing indoleacetic acid in a ginseng strain"[J]. Chinese Journal of Traditional Chinese Medicine, Jiang Yun, Jilin Agriculture)

[0037] JA26( Acinetobacter sp. Strain).

[0038] The above strains are preserved in the Microbial Resources Development and Utilization Laboratory of the College of Life Sciences of Jilin Agricultural University.

[0039] .

[0040] Culture medium: LB liquid medium; LB solid medium; potato dextrose medium (PDA);

[0041] Strain NT35 fermentation medium: 2.5 g yeast extract powder, 1.5 g sucrose, 1.5 g potassium dihydrogen phosphate, 2.5 g ammonium sulfate, pH 7.0;

[0042] Fermentation medium of strain TJ23-2: glucose 1 g, yeast extract 1 g, sodium chloride 0.6 g, magnesium sulfate 0.01 g, pH 7.0;

[0043] Fermentation medium for strain FG14: 2.03 g yeast extract, 0.52 g sucrose, 0.33 g potassium dihydrogen phosphate, 0.55 g ammonium oxalate, pH 7.0;

[0044] Strain NJ13 fermentation medium: 3 g glucose, 1.5 g starch, 1.5 g yeast extract powder, 0.1 g potassium dihydrogen phosphate, 0.5 g sodium chloride, pH 7.0;

[0045] Strain JA38 fermentation medium: sodium citrate 1.0 g, beef extract 1.0 g, ferrous sulfate 0.15 g, NaCl 0.9 g, pH 7.0;

[0046] Strain JI39-2 fermentation medium: glucose 2.0 g, potassium nitrate 1.75 g, sodium chloride 1.5 g, magnesium sulfate 0.08 g, pH 7.0;

[0047] Fermentation medium of strain JI6: sodium citrate 1.79 g, yeast extract powder 1.99 g, potassium chloride 0.318 g, magnesium sulfate 0.11 g, pH 7.0;

[0048] Fermentation medium for strains TU26, JJ5-2, and JA26: yeast extract powder 1 g, glucose 1 g, NaCl 0.5 g, magnesium sulfate 0.075 g, pH 7.0.

[0049] Ashby medium: sucrose 1 g, potassium dihydrogen phosphate 0.02 g, magnesium sulfate heptahydrate 0.02 g, sodium chloride 0.02 g, calcium carbonate 0.5 g, calcium sulfate dihydrate 0.01 g, agar powder 1.5 g, pH 7.0;

[0050] Pikovaskaia's medium: tricalcium phosphate 0.4 g, sucrose 1 g, ammonium sulfate 0.05 g, sodium chloride 0.02 g, magnesium sulfate heptahydrate 0.01 g, potassium chloride 0.02 g, yeast powder 0.05 g, manganese sulfate 0.1 mL (0.004 g / L), ferrous sulfate 0.01 mL (0.002 g / L), agar powder 1.5 g, pH 7.0;

[0051] Silicate bacterial culture medium: potassium feldspar powder 0.5 g, disodium hydrogen phosphate 0.2 g, calcium carbonate 0.01 g, magnesium sulfate heptahydrate 0.05 g, ferric chloride 0.05 g, sucrose 0.5 g, trehalose 0.1 g, agar powder 1.8 g, pH 7.0.

[0052] The strain TU26 was isolated from the rhizosphere soil of ginseng planting area in Tonghua City, Jilin Province. It has the effect of antagonizing a variety of plant pathogenic fungi, and has the growth-promoting properties of nitrogen fixation, phosphorus and potassium solubilization, and indoleacetic acid production. It was identified as Pseudomonas chlororaphis ( Pseudomonas chlororaphis ).

[0053] 1. Determination of antibacterial activity of ginseng rhizosphere bacteria TU26

[0054] The plate confrontation method was used to screen the antagonistic strains of ginseng. The pathogenic fungus tested was ginseng rust ( Cylindrocarpon destructans )、Ginseng Botrytis cinerea( Botrytis cinerea )、Sclerotinia ginseng( Sclerotinia schinseng )、Phytophthora ginseng( Phytophthora cactorum )、Panax ginsengii( Alternaria panax )、Ginseng root rot fungus( Fusarium solani ), Panax ginseng fungus ( Rhizoctonia solani ) 7 kinds of pathogens. Use a puncher to punch out a fungus cake with a diameter of 8 mm and place it in the center of the PDA plate. Then place TU26 fungus cakes with a diameter of 8 mm evenly on both sides of the fungus. Incubate inverted in a 25 ℃ constant temperature incubator for 12-72 h, and repeat the experiment 3 times. ( Figure 1 ).

[0055] 2. Determination of the Growth-Promoting Properties of Ginseng Rhizobacteria TU26

[0056] 1. Determination of nitrogen fixation capacity

[0057] The strain TU26 was streaked on Ashby solid medium and cultured at 30°C to observe the growth of the strain. The results showed that the strain TU26 grew well under nitrogen-free conditions and had nitrogen fixation potential ( Figure 2 A).

[0058] 2. Determination of phosphate solubilization capacity

[0059] Initial screening: strain TU26 was inoculated on PVK solid medium and cultured at 30°C to observe the size of the phosphate solubilization circle. Strain TU26 had an obvious phosphate solubilization circle ( Figure 2 B); then the molybdenum antimony scandium colorimetric method was used to quantitatively determine the dissolved phosphorus, and the soluble phosphorus content could reach 94.78µg / mL.

[0060] 2. Determination of potassium-solubilizing capacity

[0061] In the initial test, strain TU26 was inoculated on silicate solid medium and cultured at 30℃ to observe the size of the potassium-dissolving zone. Strain TU26 had an obvious potassium-dissolving zone ( Figure 2 C); Then, the strain TU26 was inoculated into LB medium by flame photometry, and the absorbance was measured after 12 hours of culture. The potassium solution content was quantitatively measured by flame photometer and reached 11.09 µg / mL.

[0062] 3. Determination of IAA production capacity

[0063] Inoculate strain TU26 into liquid LB medium and culture for 12 hours. Dilute the bacterial solution to an absorbance of about 0.8 under bench operation. Take 0.2 ml and inoculate it into a medium containing 100 mg / L tryptophan for culture, then centrifuge at 12000 rpm for 5 minutes, take 2 ml of the supernatant and add 4 ml of colorimetric solution, place it in the dark for 0.5 hours, and then measure the absorbance at 540 nm. It was determined that the strain TU26 can produce IAA up to 1.98 µg / mL ( Figure 2 D).

[0064] 4. Determination of Siderophore Production Capacity

[0065] Use an inoculation loop to pick up a loop of fresh bacterial moss and culture it in MKB culture medium on a shaker for 2 days. Take the bacterial solution and centrifuge it at 10000r / min for 10 minutes to obtain 3ml of supernatant in a test tube. Add 3ml of CAS detection solution and mix it. Then react it at 20℃ for 1h, and measure the absorbance value As at 630nm; take another 3ml of CAS detection solution and mix it with 3ml of uninoculated MKB culture medium, and measure the absorbance value Ar in the same way. Calculate the amount of iron carrier produced according to the formula ((Ar-As) / Ar)× 100). The results showed that the TU26 strain has the activity of producing iron carriers, and its activity is as high as 85.2% ( Figure 2 E).

[0066] 3. Identification of strain TU26

[0067] The TU26 strain was streaked on NA medium and cultured at 28°C. The strain TU26 appeared as yellow opaque round small colonies on the NA medium with neat edges ( Figure 3 A). Gram-positive bacteria ( Figure 3 B)

[0068] The strain 16S rDNA sequence was compared with the sequence in the GenBank database by BLAST analysis, and the accession number was KX951467. Pantoea agglomerans The similarity between strain SSF (KT075194) and strain WSE (KT075212) reached 99.80%; the phylogenetic tree was constructed ( Figure 4 ) shows that TU26 and P. agglomerans Based on the morphological, cultural, physiological and biochemical characteristics and molecular biological identification results, strain TU26 was identified as Pantoea agglomerans ( P. agglomerans TU26 strain was deposited in China Center for Type Culture Collection on January 25, 2024, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number: CCTCC M 2024220.

[0069] Preparation of bacteria-containing plates: prepare 27 mL of LB solid culture medium per bottle, punch 2 bacterial cakes on the bacterial plate with a 6 mm puncher, inoculate in LB liquid culture medium, shake and culture for 24 h, and set aside. Take 3 mL of the fermentation broth of the strain and add it to 27 mL of LB culture medium, mix thoroughly and pour into the culture dish to make a bacterial plate. Punch a bacterial cake of the strain to be tested with a 6 mm puncher, invert it in the center of the prepared bacterial plate, keep it in close contact with the bacterial plate, and then put it in a 28 ℃ incubator for 2-3 days. After the strain grows out, observe whether there is a transparent antibacterial zone near the central bacterial cake.

[0070] The results showed that the compatibility of 10 bacterial strains tested in the laboratory was determined by the agar block method. The results are shown in Table 2. It was found that strains FG14, JA38 and JI39-2 had antagonistic effects, and the diameters of the inhibition zones were all 6.0 mm; strains NT35, JA38 and JI39-2 had antagonistic effects, and the diameters of the inhibition zones were 8 mm and 6 mm; strains TJ23-2 and JA38 had the strongest antagonistic effect, and the inhibition diameter was 10 mm. There was no antagonism between the other strains, and they had good compatibility and were suitable for strain combination. In summary, strains FG14, NJ13, JJ5-2, TU26, JI6, JA26; strains NJ13, JI39-2, TJ23-2, TU26, JI6, JJ5-2; strains NJ13, JA38, TU26, JI6, JJ5-2 were combined to construct biocontrol bacteria.

[0071] .

[0072] After the biocontrol bacteria were cultured on LB solid medium at 28°C for 24 h, they were transferred to LB liquid culture medium and cultured at 28°C to make seed culture medium. The seed culture medium was inoculated into the fermentation medium in proportion and cultured at 28°C. The OD was then measured using an enzyme marker. 600 , dilute the bacterial solution with sterile water to a uniform concentration of 1×10 9 cfu / mL, and then the diluted single-bacteria fermentation broth was used in a 1:1 ratio for full combination compound design, and a total of 98 biocontrol bacterial community treatments were constructed (see Table 3).

[0073] Table 3 Full combination of biocontrol bacteria

[0074] ;

[0075] ;

[0076] ;

[0077] ;

[0078] 1. Antagonistic activity assay

[0079] The plate confrontation method was used to punch the ginseng rust pathogens that had been activated and cultured for 7 days into round bacterial blocks with a diameter of 6 mm from the outer edge of the colony using a sterilized puncher. The bacterial blocks (6 mm) were inoculated in the center of the PDA medium, and 50 µL of the fermentation liquid of the biocontrol single strain and the biocontrol bacterial group (Table 3) were inoculated at a distance of about 27 mm from the center of the medium. The plates were cultured at 25°C for 10 days. After the control bacteria grew all over the plate, the diameter of the pathogens was recorded by the cross method. Each treatment was repeated 3 times.

[0080] The results showed that in order to preliminarily screen out the biocontrol bacteria with better control of rust and rot in dishes, a bacteria-pathogenic fungus system was established on agar medium in this study in an artificially controlled environment. Under the same conditions, it was found that different biocontrol strains and biocontrol bacteria groups had different abilities to inhibit the growth of rust and rot pathogens, and the antibacterial effect of the biocontrol bacteria group was much higher than that of the single biocontrol strain. As the richness of the biocontrol bacteria group increased, the antibacterial effect increased synchronously. The antibacterial group composed of six biocontrol strains had the best effect on inhibiting the growth of rust and rot pathogens, and the antibacterial diameter was the smallest (Table 4). Different single biocontrol strains and biocontrol bacterial groups had significant inhibitory effects on rust rot hyphae. Among the single strain treatments, CL1 had the best inhibitory effect on ginseng rust rot hyphae, with an inhibition diameter of 43 mm and an inhibition rate of 28%. Among the biocontrol bacterial groups composed of two strains, CL11 and CL12 had the best inhibitory effect on ginseng rust rot hyphae, with inhibition diameters of 39 mm and 40 mm and inhibition rates of 35% and 33.33%, which were 1.19 times and 1.25 times higher than those of the single strain treatment CL1. Among the biocontrol bacterial groups composed of three strains, CL43 and CL44 had the best inhibitory effect on ginseng rust rot hyphae, with inhibition diameters of 28 mm and 33 mm and inhibition rates of 53% and 45%, which were 1.89 times and 1.61 times higher than those of the single strain treatment CL1. Among the biocontrol bacterial groups composed of four strains, CL69 and CL77 had the best inhibitory effect on ginseng rust rot hyphae, with inhibition diameters of 23 mm and 22 mm. mm, and the inhibition rates were 63% and 62%, which were 2.25 times and 2.21 times higher than those of the single strain treatment CL1; among the five strains of biocontrol bacteria, CL95 and CL94 had the best inhibitory effect on the mycelium of ginseng rust rot, with the inhibition diameter of 16.33 mm and 16.40 mm and the inhibition rate of 73%, which was 2.6 times higher than that of the single strain treatment CL1; the six strains of biocontrol bacteria, CL98 and CL97, had the best inhibitory effect on the mycelium of ginseng rust rot, with the inhibition diameter of 15.30 mm and 15.60 mm and the inhibition rate of 75% and 74%, which was 2.67 times and 2.64 times higher than that of the single strain treatment CL1. In summary, with the increase of the richness of the biocontrol bacteria, the growth rate of ginseng rust rot pathogen weakened, and the smaller the mycelium diameter, the stronger the inhibition effect.

[0081] ;

[0082] 2. In vitro inoculation of ginseng with biocontrol bacteria

[0083] Take healthy 4-year-old ginseng of the same growth condition, clean and disinfect it with 75% ethanol (EtOH) for 2 min, then disinfect it with 2% sodium hypochlorite (NaClO) for 2 min, rinse it with sterile water, and dry it.

[0084] Four holes were made on the main root of ginseng using a sterilized inoculation needle as the inoculation area. The preventive effects of the biocontrol strains and the biocontrol bacterial consortium on the infection of ginseng rust were evaluated. In the preventive treatment, the fermentation supernatant of the biocontrol strains and the bacterial consortium (Table 3) was inoculated into ginseng 24 hours later. I. robusta CBLJ-3 was inoculated on ginseng roots. Three replicates were set for each treatment, and the roots were dried naturally. Then the treated ginseng roots were placed on filter paper soaked in sterile water to maintain sufficient moisture. They were cultured in an incubator at 25°C for 1 d, 3 d, 5 d, 7 d, 9 d, and 11 d, and the size and depth of ginseng lesions were measured and photographed. The positive control was ginseng infected with CBLJ-3 strains, and the blank control was sterile water.

[0085] The results showed that in a fully controlled environment, in order to screen for a combination of microorganisms with better biocontrol effects, a microscopic system representing natural bacterial derivation was established in ginseng, simplifying the system into a plant-microorganism-pathogen microcosm. Under the same conditions, the pattern observed in the plant-microorganism-pathogen system was verified. Ginseng treated with biocontrol bacteria and pathogens grew for 11 days, and it was found that compared with roots inoculated with pathogens only (IF), the lesions in the treatment group inoculated with biocontrol bacteria were smaller, lighter in color, and shallower in infection depth ( Figure 5 ). At 11 dpi, the ginseng in the control group (IF) was severely rotten. In the treatment of inoculation with biocontrol bacteria, the depth and diameter of ginseng lesions gradually decreased with the increase of strain richness, and there were no serious infection symptoms. There were significant differences in the ability to control rust rot between single strains and different bacterial groups ( Fig. 9 ), the ability of the biocontrol bacteria to control rust and rot may have a strain diversity effect. Among the single strain treatments, CL1 had the best preventive effect, with the smallest lesion, and the size and depth of the lesion were 6.50 mm. Among the biocontrol bacteria composed of two strains, CL15 had the best preventive effect, with the smallest lesion, and the size and depth of the lesion were 5.47 mm and 5.58 mm. Among the biocontrol bacteria composed of three strains, CL43 had the best preventive effect, with the smallest lesion, and the size and depth of the lesion were 3.00 mm. Among the biocontrol bacteria composed of four strains, CL69 had the best preventive effect, with the smallest lesion, and the size and depth of the lesion were 3.83 mm. Among the biocontrol bacteria composed of five strains, CL94 had the best preventive effect, with the smallest lesion, and the size and depth of the lesion were 2.00 mm and 2.3 mm. Among the biocontrol bacteria composed of six strains, CL98 had the best preventive effect, with the smallest lesion, and the size and depth of the lesion were 2.00 mm and 1.67 mm. In summary, through indoor ginseng in vitro inoculation, we screened 98 combinations of biocontrol bacteria and selected 40 combinations with better disease prevention effects (see Table 5).

[0086] ;

[0087] Based on the in-dish antagonism test and ginseng in vitro inoculation test, 40 bacterial community combinations with better biocontrol effects were preliminarily screened (see Table 5). The 40 optimized bacterial communities were inoculated into Ashby medium plates, silicate bacterial medium plates and Pikovaskaia's medium plates, respectively. After 5 days, the size of the transparent zone was measured by the cross method, and the nitrogen fixation, potassium solubilization and phosphorus solubilization effects of single strains and biocontrol bacterial communities were compared.

[0088] The results showed that all 40 bacterial combinations screened had the ability to dissolve potassium, 30 of them had the ability to fix nitrogen, and 31 of them had the ability to dissolve inorganic phosphorus. Fig.10 ). Moreover, the ability of the selected biocontrol bacteria to dissolve potassium, fix nitrogen, and dissolve phosphorus is higher than that of a single strain, indicating that the growth-promoting effect of the biocontrol bacteria is better than that of a single strain. As the richness of the biocontrol bacteria increases, the growth-promoting effect is enhanced simultaneously. The six biocontrol strains constitute the biocontrol bacteria group with the best growth-promoting transparent circle and the largest diameter. In summary, 40 combinations of biocontrol bacteria were screened, and 29 combinations with better growth-promoting and disease-preventing effects were selected (see Table 6).

[0089] ;

[0090] Biocontrol bacteria I. robusta Determination of the inhibitory effect on the mycelial growth of CBLJ-3

[0091] Two bacterial cakes with a diameter of 8 mm were picked from the LB plate, inoculated into LB medium and cultured for 12 h to obtain seed solution, which was then injected into the biocontrol strain fermentation solution and cultured in a shake flask at 28°C and 160 rpm to obtain the strain fermentation solution, and the concentration was adjusted to 10 using a spectrophotometer. 9 cfu / mL, and the diluted single-bacteria fermentation broth was prepared into a biocontrol bacterial group in a 1:1 ratio and stored at 4°C for later use.

[0092] Will cultivate good I. robusta CBLJ-3 strain was inoculated into PDB medium and cultured at 25℃ and 150 rpm for 48h. Then, the biocontrol bacteria (Table 6) with different treatments were added to the medium for further culture. The PDB medium without biocontrol bacteria was used as the control. Samples were taken after 7 days. The mycelium was filtered through 8-layer sterilized gauze and washed with deionized water for 3 times. The mycelium was picked into an aluminum dish, dried at 80℃, and weighed. Three replicates were set for each treatment to calculate the bacterial growth inhibition rate.

[0093] The calculation formula of the rate is as follows:

[0094] Inhibition rate (%) = (control mycelium dry weight - treatment mycelium dry weight) / control mycelium dry weight × 100%

[0095] The results show that: Figure 7It can be seen that during the shake flask co-cultivation process, the biocontrol bacteria group had an inhibitory effect on the CBLJ-3 strain, causing the latter's yield to decrease. As shown in the figure, among the single strain treatments, CL7 had the best inhibitory effect on the growth of ginseng rust rot mycelium, with an inhibition rate of 43.42%. Among the biocontrol bacteria group composed of two strains, CL13 had the best inhibitory effect on the growth of ginseng rust rot mycelium, with an inhibition rate of 68.51%, which was 1.6 times higher than that of the single strain treatment CL7; among the biocontrol bacteria group composed of three strains, CL43 had the best inhibitory effect on the growth of ginseng rust rot mycelium, with an inhibition rate of 98.53%, which was 2.27 times higher than that of the single strain treatment CL7; the four strains Among the biocontrol bacteria groups composed of 5 strains, CL80 had the best inhibitory effect on the growth of ginseng rust mycelium, with an inhibition rate of 96.67%, which was 2.2 times higher than that of the single strain treatment CL7; the biocontrol bacteria group composed of 5 strains had a good inhibitory effect on the growth of ginseng rust mycelium, with an inhibition rate of more than 98%, which was 2.3 times higher than that of the single strain treatment CL7; the biocontrol bacteria group composed of 6 strains had the best inhibitory effect on the growth of ginseng rust mycelium, with an inhibition rate of 100%, which was 2.3 times higher than that of the single strain treatment CL7. In summary, with the increase of strain diversity, the growth rate of ginseng rust pathogen weakened, and the greater the growth inhibition rate, the stronger the inhibitory effect.

[0096] 2. Biocontrol bacteria I. robusta Determination of the inhibitory effect on the conidia production of CBLJ-3

[0097] Two bacterial cakes with a diameter of 8 mm were picked from the LB plate, inoculated into LB medium and cultured for 12 h to obtain seed solution, which was then injected into the biocontrol strain fermentation solution and cultured in a shake flask at 28°C and 160 rpm to obtain the strain fermentation solution, and the concentration was adjusted to 10 using a spectrophotometer. 9 cfu / mL, and the diluted single-bacteria fermentation broth was prepared into a biocontrol bacterial group in a 1:1 ratio and stored at 4°C for later use.

[0098] Will I. robusta The spore solution of CBLJ-3 strain was diluted with appropriate deionized water to a concentration of 1×10 7 cfu / mL spore suspension. Take different biocontrol bacteria (Table 6) and mix with equal volume of I. robusta The spore suspension of CBLJ-3 strain was mixed onto a water agar medium plate and evenly spread with a disposable applicator. Three replicates were set for each treatment. The spore suspension without biocontrol bacteria was used as the control. The spores were placed upside down at 25°C to germinate. The spore germination was observed after 8 hours. The germination standard was that the length of the germ tube exceeded half of the length of the conidia. When the spore germination rate of the control treatment reached more than 90%, the spore germination inhibition rate was calculated. The inhibition rate formula is as follows:

[0099] Inhibition rate (%) = (control spore production - treatment spore production) / control spore production × 100%

[0100] The results show that: Figure 8 As shown in the results, among the single-strain treatments, CL1 had the best inhibitory effect on the spore production of ginseng rust and rot pathogens, with an inhibition rate of 34%. Among the biocontrol fungi composed of two strains, CL11 and CL13 had the best inhibitory effect on the spore production of ginseng rust and rot pathogens, with an inhibition rate of 40%, which was 1.2 times higher than that of the single-strain treatment CL1. Among the biocontrol fungi composed of three strains, CL44 had the best inhibitory effect on the spore production of ginseng rust and rot pathogens, with an inhibition rate of 58.2%, which was 1.7 times higher than that of the single-strain treatment CL1. The biocontrol fungi composed of four strains Among them, CL79 had the best inhibitory effect on the spore production of ginseng rust rot, with an inhibition rate of 63.6%, which was 1.9 times higher than that of the single strain treatment CL1; the biocontrol bacteria group CL95 composed of five strains had the best inhibitory effect on the spore production of ginseng rust rot, with an inhibition rate of 76%, which was 2.23 times higher than that of the single strain treatment CL1; the biocontrol bacteria group CL97 and CL98 composed of six strains had the best inhibitory effect on the spore production of ginseng rust rot, with inhibition rates of 80% and 80.8%, which were 2.4 times higher than that of the single strain treatment CL1. In summary, the control effect of biocontrol bacteria on rust rot pathogen spores also has a diversity effect, that is, it increases with the increase of the diversity of biocontrol bacteria, but the degree of change is different.

[0101] 3. Determination of the efficacy of biocontrol bacteria on ginseng in the field

[0102] The field test was conducted at the Baixi Base in Songjianghe Town, Fusong County, Jilin Province, where ginseng was continuously planted and rust rot occurred repeatedly. I. robusta The content was 2031.64 fg / μL. The experimental site was divided into 2 m 2 Three plots were randomly selected as a set of replicate treatments, and five-year-old ginseng roots of the same size were planted in the soil at a depth of 5 cm. 9 The field control effect of the biocontrol bacteria (cfu / mL) (Table 6) was evaluated, and the plot without added bacteria was used as the control treatment. The disease occurrence level was investigated when ginseng was harvested at the mature stage.

[0103] The results show that: (the control effect is shown in Fig. 9 ; Increase rate see Fig.11) Ginseng treated with biocontrol bacteria grew for 90 days. The disease index of rust rot in the field of the biocontrol bacteria group was significantly lower than that of the single biocontrol strain and the water control group, and the field control effect of the biocontrol bacteria group was significantly higher than that of the single biocontrol strain and the water control group. The use of biocontrol bacteria in the field can significantly reduce the occurrence of rust rot, and the control effect is higher than that of the single biocontrol strain. There are 6 treatments with a control effect of more than 50%, among which CL95 treatment has the highest control effect, reaching 66.67%, and CL92 treatment has the second best control effect, reaching 62.96%. CL94, CL79, CL80, and CL44 have better control effects, which are 59.26%, 59.26%, 55.56%, and 51.85%, respectively, which are 1.5 times, 1.46 times, 1.37 times, 1.29 times, and 1.2 times higher than the single biocontrol strain treatment CL1 with the best control effect. Compared with the control effect of a single biocontrol bacteria, the biocontrol bacteria significantly reduced the occurrence of rust rot. This indicates that the biocontrol bacteria group is superior to a single bacteria group in resisting pathogen infection. In addition, all bacterial group treatments can promote plant growth. The tested biocontrol bacteria groups all have a certain effect on the ginseng yield increase rate. The biocontrol bacteria group treatments can increase the ginseng yield increase rate, among which the yield increase rate of biocontrol bacteria groups CL95 and CL92 is significantly higher than that of other treatments, reaching more than 300%, which is 3.04 times and 2.78 times higher than that of the best biocontrol single bacteria treatment CL1, respectively.

[0104] The field efficacy test of the Baixi base proved the applicability of the biocontrol bacteria CL95, CL92, CL94, CL79, CL80, and CL44, and the feasibility of the biocontrol bacteria in controlling soil-borne root diseases. Multiple sets of data proved the treatment effects of the two bacteria. It has been proven that these six biocontrol bacteria can not only prevent and control diseases, but also promote growth, greatly increasing the yield of ginseng.

[0105] In summary, this study screened 29 bacterial community treatments in the field and selected 6 bacterial community combinations with better control effects (Table 7).

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[0107] 1. Research on colonization and efficiency enhancement of biocontrol bacteria

[0108] Different concentrations of antibiotic culture media were prepared. Rifampicin and streptomycin sulfate stock solutions were added to LB culture media to make the concentrations of rifampicin reach 120 μg / mL, 160 μg / mL and 800 μg / mL, respectively, and the concentrations of streptomycin sulfate reach 250 μg / mL, 400 μg / mL and 2000 μg / mL, respectively, to prepare LB-Rif 120-Stre 250 culture media, LB-Rif 160-Stre 400 culture media and LB-Rif 800 -Stre 2000culture medium, and add 1 mL of FG14-Rif 120 -Stre 250 Glycerol stock, 1 mL NT35-Rif 160 -Stre 400 Glycerol stock and 1 mL NJ13-Rif 800 -Stre 2000 glycerol storage medium, 28℃, 160 rpm shake flask culture for 48 h. Centrifuge the fermentation broth at 12000 rpm, pour off the supernatant, resuspend the cells with sterile water and centrifuge and wash twice, and measure OD with a spectrophotometer 600 The concentration of bacterial suspension was detected by sterile water and diluted to 1×10 9 cfu / mL, stored at 4℃ for future use.

[0109] The antibiotic-labeled strain and the unlabeled strain suspension were mixed in equal proportions to make a concentration of 1×10 6 cfu / mL of dual antimicrobial groups for future use. 6 cfu / mL of the dual-antibiotic bacterial suspension (Table 8) was used to irrigate the roots of four-year-old healthy ginseng cultivated in the field. Each ginseng was irrigated with 100 mL of the suspension at one time. When irrigating the roots, the surface soil above the ginseng was dug up to allow the bacterial solution to be completely irrigated around the roots of the ginseng. Samples were taken at 7 d, 14 d, 35 d, 49 d, and 63 d after inoculation, with clear water irrigating as the control. Each treatment was repeated 3 times.

[0110] The samples taken regularly were disinfected by sodium hypochlorite disinfection. After disinfection, 2 g of each sample was divided into lateral roots, fibrous roots, epidermis, root core, stem, and leaf. 20 mL of sterile water was added to the sample and fully ground in a mortar. After standing for three minutes, the supernatant of the ground tissue was spread on the LB medium containing antibiotics. The spread amount was 100 μL per plate. The sample was cultured in a constant temperature incubator, observed regularly, and the number of colonized colonies was calculated. 2 g of fresh soil samples of ginseng rhizosphere soil taken regularly were weighed and added to a test tube containing 20 mL of sterile water. After culturing at 28°C and 160 rpm for 30 min, the liquid was diluted in a gradient manner, and 100 μL of the dilution was spread on the LB medium containing antibiotics to calculate the number of colonized colonies.

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[0112] The colonization results showed that within 63 days, the antibiotic-labeled strains were able to colonize in ginseng and rhizosphere soil. Bacterial inoculation had a significant effect on the composition of endophytic microbial communities in ginseng roots. Compared with single strains, the antibiotic-labeled strains combined with single strains had a higher colonization ability in ginseng than single strains. The colonization amount of the strains in rhizosphere soil showed a downward trend, while in the leaves, stems, epidermis, root core, lateral roots, and fibrous roots of ginseng, it showed a trend of first increasing and then decreasing ( Fig.12 ). After the labeled strain was inoculated into the roots of ginseng during the leaf expansion stage, the labeled strain was detected in the rhizosphere soil of ginseng at 7 dpi, and the colonization ability of the rhizosphere soil reached its peak. After that, the colonization ability began to decline significantly ( Fig.12 G). The colonization of the labeled strain on the ginseng epidermis was the highest at 7 dpi ( Fig.12 C), which was higher than the colonization amount of ginseng fibrous roots, ginseng lateral roots and ginseng root cores, and showed an upward trend at 14-35dpi, reaching a peak at 35 dpi, but at 35 dpi, the colonization amount of ginseng root cores and lateral roots was significantly higher than that of ginseng epidermis and fibrous roots. The colonization amount dropped sharply from 49 to 63dpi, which was consistent with the colonization trend of rhizosphere soil during the same period. The colonization amount of the labeled strain in the stems and leaves of ginseng was significantly higher than that in the roots. Therefore, the results showed that the colonization dynamics of the labeled strain was to enter the ginseng roots from the rhizosphere soil, then move from the epidermis and fibrous roots to the root core and then to the stem in the ginseng body, and finally colonize in the leaves, completing the entire colonization process. During this stage, the colonization amount of biocontrol bacteria group CL95 in ginseng was significantly better than that of a single strain. The colonization amounts in ginseng leaves, stems, epidermis, root cores, lateral roots, fibrous roots, and soil were 1.5-3.75 times, 1.43-5 times, 3.15-5 times, 2.1-4.3 times, 1.33-3.8 times, 1.33-2 times, and 2-3 times that of a single strain, respectively.

[0113] 2. Effects of biocontrol bacteria on enzyme activities in ginseng rhizosphere soil

[0114] The soil samples tested in the experiment were from field plots. The rhizosphere soil of ginseng was collected at the leaf expansion stage, flowering and fruiting stage, and root expansion stage to determine urease, catalase, phosphatase and sucrase.

[0115] When biocontrol bacteria and single biocontrol strains are inoculated in the field, they can significantly increase the soil enzyme activity levels in the rhizosphere soil of ginseng at various stages ( Fig.13). During the 90-day experiment, the activities of urease, catalase, sucrase and phosphatase in the soil showed an upward trend. The soil enzyme activities treated with single biocontrol bacteria and biocontrol bacteria group were higher than those of the control, indicating that the treatments promoted the activities of the four enzymes in ginseng soil. After the application of bacterial group CL95, the activities of phosphatase, sucrase, catalase and urease in the soil during the root expansion period of ginseng increased significantly compared with the single strain treatment CL1, which were 2 times, 2.4 times, 2.8 times and 2.5 times higher than those of the single strain, respectively. In summary, the biocontrol bacteria group treatment had the best promoting effect on the four enzymes in the soil on the 90th day. The results showed that the biocontrol bacteria group had a long lasting effect and a stable effect.

[0116] 3. Inducible ginseng defense enzyme gene expression assay

[0117] The biocontrol bacteria and biocontrol bacteria groups were used to irrigate the roots of 4-year-old healthy ginseng cultivated in the field. Each ginseng plant was irrigated with 100 mL of the solution at one time. Samples were taken at 1 d, 3 d, 5 d, 7 d, 9 d, and 11 d after inoculation. The water irrigated solution was used as the control. Each treatment was repeated 3 times. The collected ginseng samples were stored in liquid nitrogen and then frozen in a -80℃ refrigerator.

[0118] The RNA of the samples was extracted using the RNAiso plus method. The quality of the extracted total RNA was tested by 2.2% agarose gel electrophoresis, and the RNA concentration was tested by NanoDrop 2000. The total RNA was reverse transcribed using M-MLV reverse transcriptase (TaKaRa) to synthesize the first chain of cDNA for fluorescence quantitative PCR.

[0119] According to the research results of NCBI database on ginseng plant defense enzyme genes, β-actinAs the internal reference gene, five ginseng defense enzyme synthesis genes were selected for relative expression analysis in this experiment. The specific primers used for qRT-PCR are shown in Table 9, and the reaction system and reaction conditions are shown in Tables 10 and 11, respectively. Each cDNA sample was repeated 3 times. To analyze the relative expression changes of ginseng plant defense enzyme genes induced by biocontrol single strains and biocontrol bacteria, qRT-PCR reactions were performed using Fast SYBR Mixture (company) on a fluorescence quantitative PCR instrument Mx3000p (STRATAGENE). The PCR reaction system (20 μL) was: cDNA 1 μL, upstream and downstream primers (10 μmol / L) 1 μL, 2×Fast SYBR Mixture 10 μL, ddH2O 7 μL, and the final volume was 20 μL. The qRT-PCR reaction conditions were: 95°C pre-denaturation for 10 min, 95°C denaturation for 10 s, 60°C annealing and extension for 30 s, for a total of 40 cycles. qRT-PCR amplification was performed on the five genes, with each sample repeated 3 times. After the reaction, the Ct value was collected and used 2-△△ The data were analyzed by Ct method.

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[0123] The results showed that after treating ginseng with single biocontrol strains and biocontrol bacteria groups ( Fig.14 ), which has an inducing up-regulation effect on the five defense enzyme synthesis genes of ginseng, and the overall change trend is first increased and then decreased. The activity of defense enzymes in ginseng will be significantly enhanced when stimulated by external organisms. During the infection of ginseng with rust pathogens, the intervention of biocontrol bacteria has a significant induction and enhancement effect on the activities of the five defense enzymes in its body. However, the enhancement time and intensity of the host defense enzyme activities of different biocontrol bacteria are significantly different, among which the bacterial community CL95 has the greatest impact on the activities of the five defense enzymes of ginseng. The five defense enzyme synthesis genes of ginseng treated with bacterial community CL95 were significantly increased compared with those treated with single strains, and the defense enzyme synthesis genes of ginseng treated with single strain NJ13 (CL1) were increased by 1.75 times, 2.41 times, 1.94 times, 2.67 times, and 1.97 times, respectively. When plants are infected by diseases or other stimuli, the activity of defense enzymes will change immediately to resist the damage. The increase in defense enzyme activity can improve the disease resistance of plants to a certain extent. Experiments have shown that both single biocontrol strains and biocontrol fungi can induce an increase in defense enzyme activity to a certain extent, but the biocontrol fungi can induce defense enzyme activity significantly better than single biocontrol strains.

[0124] 4. Effects of biocontrol bacteria on the rhizosphere microbial community of ginseng soil

[0125] NovaSeq was used to determine the ITS2 region sequence of fungal rDNA in the rhizosphere soil of ginseng treated with biocontrol bacteria. The soil samples were from field plots, and the rhizosphere soil of ginseng was collected at the leaf expansion stage, flowering stage and ginseng root expansion stage for microbial community analysis. The control group was treated with clean water without any bacterial agent. Each treatment was repeated 3 times, and a total of 18 soil samples were obtained. After library construction, it was detected and quantified on Agilent 5400, and then paired-end sequencing (2 × 250) was performed on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA).

[0126] The results showed that according to the species annotation and abundance information of each sample at the genus level, the top 10 genera were selected for clustering, and the heat map and bar chart of the soil fungal community were drawn ( Fig.15 The composition of soil microorganisms in ginseng cultivation areas will change significantly due to the addition of biocontrol bacteria. Ilyonectria ) infection also affects the composition of root-associated bacterial communities. Soil fungal communities showed highly dynamic changes in different treatment periods. Compared with the single strain treatment, the biocontrol bacteria group treatment increased the number of Trichoderma ( Ilyonectria ) were significantly enriched. Ilyonectria ) was lower than that in some soils treated with biocontrol bacteria groups at the same time, but in the soils of the control group treated with biocontrol bacteria at the flowering and fruiting stage and the ginseng root expansion stage, the relative abundance of the genus Trichoderma ( Ilyonectria ) was significantly higher than that in the soil treated with biocontrol bacteria in the same period, while the biocontrol bacteria CL92 and CL95 treatments effectively reduced the abundance of ( Ilyonectria ) content, plays a key role in preventing and controlling diseases.

[0127] Through the above experiments, we screened 6 bacterial combinations and finally selected the two groups with the best growth-promoting and disease-preventing effects, namely TJ23-2+JI39-2+NJ13+TU26+JI6 and NJ13+NT35+FG14+TU26+JI6 (Table 12).

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[0129] Note: ★ indicates disease prevention and growth promotion effect, ★★★★ has the best disease prevention and growth promotion effect

Claims

1. The biocontrol bacteria group CL95 for preventing and controlling ginseng rust and promoting ginseng growth is composed of methylotrophic Bacillus ( Bacillus methylotrophicus )NJ13, Bacillus Velezii ( Bacillus velezensis )NT35, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) FG14, Pseudomonas chlororaphis with a deposit number of CCTCCNO: M 2024220 ( Pseudomonas chlororaphis TU26, Pseudomonas severivalensis ( Pseudomonas thivervalensis ) JI6, Arthrobacter nicotinicus with a deposit number of CCTCC NO: M 2015667 ( Arthrobacter nicotinovorans )JI39-2.

2. The biocontrol bacterial group CL95 for preventing and controlling ginseng rust and promoting ginseng growth according to claim 1, characterized in that: The volume ratio of the bacterial suspension of each strain is 1:1:1:1:1:1:

1.

3. The biocontrol bacterial group CL95 for preventing and controlling ginseng rust and promoting ginseng growth according to claim 2, characterized in that: The concentration of each strain was 1×10 9 cfu / mL.

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