Bacterial composition for promoting growth of panax notoginseng and accumulation of saponin

Abstract

The invention discloses a bacterial composition for promoting growth of panax notoginseng and accumulation of saponin, the bacterial composition is composed of three strains, the three strains comprise Arthrobacter nicotinovorans SQ 15, Bacillus velezenis SQ 24 and Pseudomonas nitroreducens SQ 63, the strains are preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation numbers are respectively CGMCC No.31366, CGMCC No.31369 and CGMCC No.31367. The invention further discloses a preparation method of the bacterial composition for promoting growth of panax notoginseng and accumulation of saponin. Three beneficial bacteria with a disease-resistant or growth-promoting function are provided, growth of panax notoginseng tuberous roots is promoted, the saponin accumulation amount in panax notoginseng roots is increased, panax notoginseng root rot can be prevented, and the quality of panax notoginseng is improved.

Description

Technical Field

[0001] The invention relates to the field of strain technology and Chinese medicinal material cultivation, and in particular to a bacterial composition for promoting the growth of Panax notoginseng and the accumulation of saponins. Background Art

[0002] Panax notoginseng is a perennial herbaceous plant of the genus Panax, Araliaceae. Its dried roots and rhizomes are used as medicine. It has the effects of promoting blood circulation, removing blood stasis, reducing swelling and relieving pain. It is a precious and authentic Chinese medicinal material unique to my country. The size of the main root of Panax notoginseng is usually used as one of the indicators for evaluating the quality of Panax notoginseng. In the market, the number of Panax notoginseng heads (the number of Panax notoginseng in 500 grams) represents the grade and medicinal value. The larger the Panax notoginseng, the higher the price. Saponins are the main active ingredients of Panax notoginseng for its medicinal effects. Modern pharmacological studies have shown that saponins have the effects of protecting cardiovascular and cerebrovascular, anti-thrombotic, regulating blood sugar and blood lipids, anti-tumor, enhancing immunity, and delaying aging. The clinical application range is very wide. Panax notoginseng root is the main extraction site of saponins. Therefore, increasing the accumulation of saponins in the root of Panax notoginseng is of great significance to the cultivation of high-quality Panax notoginseng. However, the growth of Panax notoginseng has strict environmental requirements. Its yield and quality are easily restricted by factors such as soil and climate. The quality decline caused by the expansion of the planting area has begun to appear. In addition, the growth cycle of Panax notoginseng is relatively long, and it can only be harvested after three years in the field. Continuous cropping is a serious obstacle, and soil-borne diseases are very likely to occur during the growth cycle, which seriously affects the yield and quality of Panax notoginseng. Therefore, how to improve the yield and quality is one of the difficulties faced in Panax notoginseng cultivation.

[0003] Rhizosphere soil microorganisms are crucial to the growth and health of crops. Studies have shown that rhizosphere soil microorganisms can promote the growth of medicinal plants, improve the resistance of medicinal plants to stress and disease, and promote the synthesis and accumulation of effective ingredients of medicinal plants. In recent years, the relationship between the rhizosphere microecology of Chinese medicinal materials and the quality of Chinese medicinal materials has received more and more attention. Many probiotics have been used in the cultivation of medicinal plants and have been proven to have the effect of improving yield or quality. The application of microbial agents has become an important means to alleviate the abuse of pesticides and chemical fertilizers in the cultivation of medicinal plants and improve the effectiveness and safety of Chinese medicinal materials. IAA is indoleacetic acid. The strains that produce IAA are currently applied to plants, which is recorded in the existing patent (CN202111311796.9), but it has not been used in Panax notoginseng. In addition, the microbial agents developed specifically for Panax notoginseng are still very limited, and it is difficult for a single agent to comprehensively solve the problems of diseases, poor growth, and quality degradation in Panax notoginseng production. Summary of the invention

[0004] The purpose of the present invention is to provide a bacterial composition that promotes the growth and saponin accumulation of Panax notoginseng. In view of the slow growth, easy occurrence of diseases and poor quality of Panax notoginseng, beneficial strains that can antagonize Panax notoginseng pathogens and produce IAA are screened out from the rhizosphere soil of Panax notoginseng and combined, and the effects on the main pathogens of Panax notoginseng, biomass, root growth and saponin accumulation of Panax notoginseng are verified under laboratory and field conditions. The purpose is to lay a foundation for the development of beneficial microbial agents that promote the growth and saponin accumulation of Panax notoginseng, promote the growth of Panax notoginseng tubers, increase the accumulation of saponins in Panax notoginseng roots, prevent Panax notoginseng root rot, and improve the quality of Panax notoginseng.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A kind of Arthrobacter nicotinovorans, wherein the Arthrobacter nicotinovorans is Paenarthrobacter nicotinovorans SQ 15, which is used to produce IAA and inhibit Fusarium oxysporum;

[0007] The strain is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No.31366; the deposit time of the deposit number CGMCC No.31366 is July 19, 2024, and it is deposited at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0008] A Bacillus velezenis, wherein the Bacillus velezenis is Bacillus velezenis SQ 24, used for inhibiting Fusarium oxysporum, Fusarium solani and Cylindrospermum destructum;

[0009] The strain is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No.31369. The deposit date of the deposit number CGMCC No.31369 is July 19, 2024, and it is deposited at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] A nitroreducing Pseudomonas, wherein the nitroreducing Pseudomonas is Pseudomonas nitroreducens SQ 63, used to inhibit Fusarium oxysporum, Fusarium solani, and Cylindrospermum destructum;

[0011] The strain is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 31367. The deposit date of the deposit number CGMCC No. 31367 is July 19, 2024, and it is deposited at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. A bacterial composition for promoting the growth and saponin accumulation of Panax notoginseng, comprising: Paenarthrobacter nicotinovorans SQ 15, Bacillus velezenis SQ24, and Pseudomonas nitroreducens SQ 63 as claimed in claim 1.

[0013] Application of a bacterial composition that promotes the growth and saponin accumulation of Panax notoginseng to promoting the growth of Panax notoginseng.

[0014] Application of a bacterial composition that promotes the growth and saponin accumulation of Panax notoginseng to promote the accumulation of saponins in Panax notoginseng roots.

[0015] Further: The method applied is:

[0016] Take the single strain seed liquid of SQ 15, SQ 24 and SQ 63 and inoculate it into the culture medium, and then culture it on a shaker. After the culture, centrifuge to remove the culture medium, resuspend it in sterile water, and then adjust the OD value according to the OD value. 600 The dilution factor is determined by the value, and the concentration is configured to be 10 8 cfu / mL of mixed bacterial solution of SQ 15, SQ 24, and SQ 63;

[0017] After Panax notoginseng seedlings emerge, the plants are irrigated with mixed bacterial solution.

[0018] Further: the culture medium is selected from LB liquid culture medium, including: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, and 15g / L agar powder.

[0019] Furthermore: 200 mL of the mixed bacterial solution is added to each Panax notoginseng plant, and then added again after an interval of 1 month, and the mixed bacterial solution is added continuously for 3 times.

[0020] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0021] (1) The present invention is based on rhizosphere soil microorganisms of Panax notoginseng, uses multiple culture media to isolate multiple soil bacteria as much as possible, screens the antibacterial activity and IAA production activity of Panax notoginseng root rot pathogens, and finally identifies SQ 15, SQ 24, and SQ63. At the same time, SQ 15, SQ 24, and SQ63 suspensions are prepared for potted and field tests, and a bacterial combination that can promote the growth of Panax notoginseng roots, increase the content of Panax notoginseng saponins, and prevent Panax notoginseng root rot is obtained.

[0022] (2) The present invention can effectively promote the growth of Panax notoginseng root tubers, increase the accumulation of Panax notoginseng saponins, and avoid the problem of the abuse of growth regulators in Panax notoginseng cultivation leading to the rapid growth of Panax notoginseng root tubers and the reduction of active ingredients; it can improve the beneficial microbial population in the soil and enhance the protection of the ecological environment; it can antagonize the pathogens of Panax notoginseng root rot, have a preventive and therapeutic effect on Panax notoginseng root rot, reduce the investment cost of Panax notoginseng disease management, and at the same time provide new materials for the development of medicinal microbial agents.

[0023] (3) The method of the present invention is scientific, reliable and stable; the strains SQ 15, SQ 24 and SQ 63 are derived from the rhizosphere soil of Panax notoginseng, have strong adaptability to the soil environment of Panax notoginseng, and can improve the soil microbial ecological environment. The use of the mixed bacterial solution can effectively promote the growth of the fresh weight of Panax notoginseng seedlings by 23.45%, promote the growth of the dry weight of 3-year-old Panax notoginseng tubers by 9.57% to 23.87%, increase the accumulation of saponins by 8.17% to 26.52%, and reduce the risk of quality decline caused by blindly increasing production and abusing growth regulators; the inhibition rate of SQ 24 and SQ 63 strains on the growth of various Panax notoginseng root rot pathogens in the culture medium reaches 12.38% to 65.95%. The bacterial composition can also prevent the occurrence of Panax notoginseng root rot, further ensuring the increase of Panax notoginseng production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The plate confrontation diagram of the strains SQ 15, SQ 24, SQ63 of the present invention and Fusarium oxysporum, Fusarium solani, and Cylindrospermum destructum.

[0025] Figure 2 This is a verification diagram of the production of IAA by the strains SQ 15, SQ 24, and SQ63 of the present invention.

[0026] Figure 3 The morphological diagrams are of the strains SQ 15, SQ 24 and SQ63 of the present invention.

[0027] Figure 4 The 16S sequence homology phylogeny diagram of the strains SQ 15, SQ 24 and SQ63 of the present invention.

[0028] Figure 5 This is a picture of the growth of Panax notoginseng seedlings after the bacterial composition of the present invention is applied. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0030] Embodiment 1:

[0031] 1. Isolation of Rhizosphere Soil Bacteria of Panax notoginseng

[0032] Soil bacteria were isolated using a variety of media, including the following 5 media:

[0033] (1) LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder;

[0034] (2) NA medium: peptone 10 g / L, beef extract powder 3 g / L, sodium chloride 5 g / L, agar powder 15 g / L;

[0035] (3) TSA medium: trypticase 17.0 g / L, soybean papain hydrolysate 3.0 g / L, sodium chloride 5.0 g / L, agar powder 15 g / L;

[0036] (4) R2A medium: yeast extract powder 0.5 g / L, peptone 0.5 g / L, casein hydrolysate 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, potassium dihydrogen phosphate 0.3 g / L, anhydrous magnesium sulfate 0.024 g / L, sodium pyruvate 0.3 g / L, agar 15.0 g / L;

[0037] (5) Improved Gao's medium No. 1: soluble starch 20 g / L, sodium chloride 0.5 g / L, potassium nitrate 1 g / L, dimethyl phosphate 0.5 g / L, magnesium sulfate 0.5 g / L, ferrous sulfate 0.01 g / L, agar powder 15 g / L.

[0038] Weigh 5g of rhizosphere soil of Panax notoginseng and mix it with 45mL of sterilized water, place it in a shaker at 25℃ and 180r / min for 30min to make a soil suspension, let it stand for 1h, and after stratification, take 1mL of the upper soil suspension and dilute it 1000 times, 10000 times, and 100000 times with sterile water. Take 100μL of the soil suspension diluted 1000 times and evenly apply it on the above-mentioned LB, NA, TSB, R2A and improved Gao's No. 1 medium; take 100μL of the soil suspension diluted 10000 times and evenly apply it on the above-mentioned LB, NA, TSB, R2A and improved Gao's No. 1 medium; take 100μL of the soil suspension diluted 10000 times and evenly apply it on the above-mentioned LB, NA, TSB, R2A and improved Gao's No. 1 medium. The medium after applying the soil suspension. The culture medium diluted with sterile water was inverted and cultured in a 37°C incubator for 24 hours to obtain a single colony. When diluted with sterile water, it was found that the concentrations of 10,000 times and 100,000 times were too low, and only a few to more than 10 colonies grew on the plate; a concentration of 1,000 times was used for single bacterial purification, and dozens to more than 100 colonies grew on the plate. Single colonies of NA, TSB, R2A and modified Gao's No. 1 can all grow on LB culture medium, so LB culture medium is used in the purification process. The same culture medium is convenient for observing the morphology of colonies. Single colonies with different morphologies are selected and streaked on a new LB culture medium for purification. After multiple streaking purifications, purified single strain colonies are obtained for use.

[0039] From the purified culture medium, a purified single colony was picked and inoculated into LB liquid culture medium (tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L), and cultured at 37°C and 220r / min for 1 day. 1mL of bacterial solution was mixed with 1mL of 50% sterilized glycerol and frozen in a -80°C refrigerator as a single strain seed solution. The single strain seed solution was prepared, and the seed solution was used for the screening of pathogen antagonists and IAA and for inoculation each time a mixed bacterial solution was prepared.

[0040] 2. Screening and Identification

[0041] 2.1 Screening of antagonistic bacteria against pathogens

[0042] 2.11 Screening of antagonistic bacteria against pathogens

[0043] PDA medium (potato extract powder 12g / L, glucose 20g / L, agar powder 15g / L) was used to target the pathogens of Panax notoginseng root rot, Fusarium oxysporum, Fusarium solani, and Columnar spores of destruction. The target bacteria were inoculated in the center of the PDA medium, and the above-mentioned isolated and purified single strain seed liquid was inoculated 2cm away from the center of the plate. The plate without bacteria inoculation was used as the control, and each treatment was repeated 5 times. All plates of Fusarium oxysporum and Fusarium solani were inverted in a 30°C incubator, and all plates of Columnar spores of destruction were inverted in a 25°C incubator. After 7 days of growth, the diameter of the target pathogen was measured.

[0044] 2.12 Screening results of pathogen antagonists

[0045] The screening of purified single colony pathogen antagonists based on naked eye morphology showed that only SQ 15, SQ 24 and SQ 63 had significant antagonistic effects on Panax notoginseng pathogens, and the results are shown in Table 1.

[0046] Table 1 Antagonistic effect of strains on pathogenic fungi of Panax notoginseng root rot

[0047]

[0048]

[0049] Note: Lowercase letters in the same column in the table represent significant differences in the antagonistic effects of strains on pathogens, P<0.05.

[0050] Depend on Figure 1 As shown in Table 1, after 7 days of growth, the colony diameters of the three pathogens on the plates inoculated with SQ 24 and SQ 63 were significantly (P<0.05) smaller than those on the CK plates, indicating that SQ 24 and SQ 63 had significant inhibitory effects on the three pathogens. In addition, the colony diameters of the three pathogens on the plates inoculated with SQ 24 were also significantly (P<0.05) smaller than those on the plates inoculated with SQ 63. The inhibition rate of antagonistic bacteria on pathogens was calculated using [(pathogen diameter on CK plates - pathogen diameter on plates inoculated with antagonistic bacteria) / pathogen diameter on CK plates]. The average inhibition rates of SQ 24 on Fusarium oxysporum, Fusarium solani and Cylindrospermum destructum were 47.52%, 65.95% and 49.52%, respectively, while SQ The average inhibition rates of SQ 63 against the three pathogens were 20.21%, 31.45% and 12.38%, respectively, indicating that the inhibitory effect of SQ 24 was significantly better than that of SQ 63; the colony diameter of Fusarium oxysporum on the plate inoculated with SQ15 was 2.52 cm, which was significantly (P<0.05) lower than that on the CK plate (2.82 cm), indicating that SQ 15 had a slight inhibitory effect on Fusarium oxysporum.

[0051] 2.2 Screening of IAA-producing strains

[0052] 2.21 Screening of IAA-producing strains

[0053] The isolated and purified single strain seed liquid was inoculated into LB liquid medium containing L-tryptophan (100 mg / L), and cultured at 37°C and 220 r / min for 1 day. 50 μL of bacterial liquid was dropped onto a white ceramic plate, and 50 μL of pre-prepared Salkowski colorimetric solution (50 mL of 35% perchloric acid mixed with 1 mL of 0.5 mol / L FeCl3 solution is the Salkowski colorimetric solution) was added. LB liquid medium containing L-tryptophan (100 mg / L) without bacteria was used as a negative control. The white ceramic plate was placed at room temperature and dark conditions for 30 minutes and then observed. The color turned red, indicating that the strain was able to produce IAA.

[0054] 2.22 Screening results of IAA-producing strains

[0055] When L-tryptophan is added to the culture medium as a precursor for the synthesis of IAA, the IAA-producing strain can convert it into IAA through the tryptophan pathway; indole compounds can react with Fe in the presence of perchloric acid. 3+ A color reaction, the Salkowski color reaction, occurs, in which indoleacetic acid (IAA) reacts to form a pink to red color, while tryptophan reacts to form a yellow color. Figure 2 As shown, the SQ 15 bacterial solution turned pink after adding the Salkowski colorimetric solution, indicating that SQ 15 can produce IAA by using tryptophan; while the SQ 24 and SQ 63 bacterial solutions and the negative control showed light yellow, indicating that SQ 24 and SQ 63 could not produce IAA through the tryptophan pathway.

[0056] 2.3 Identification of antagonistic and growth-promoting functional bacteria

[0057] 2.31 Identification of antagonistic and growth-promoting functional bacteria

[0058] Strains SQ 15, SQ 24 and SQ 63 were inoculated into LB solid medium, respectively, and cultured at 37°C and 220 r / min for 7 days, and the morphology of single colonies was observed.

[0059] Strains SQ 15, SQ 24 and SQ 63 were inoculated into LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L), respectively, and cultured at 37°C and 220 r / min for 1 day. 1 mL of the bacterial solution was taken and the bacterial genomic DNA was extracted using a bacterial extraction kit (TIAN Bamp Bacteria DNA Kit, Tiangen Biochemical Technology Co., Ltd., Beijing). The bacterial DNA was amplified using 16S universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TACGGCTACCTTGTACGACTT-3'). The PCR reaction system included: 2×Master Mix 25μL, 2μL of forward and reverse primers, 19μL of dd water, and 2μL of DNA template; the PCR reaction conditions were: 95℃ pre-denaturation for 5min; 95℃ melting for 40s, 52℃ low-temperature annealing and extension for 1min, 72℃ extension for 90s, 35 cycles; 72℃ for 10min. The obtained PCR products were sent to Sangon Biotech Co., Ltd. for sequencing. The obtained strain 16S sequences were analyzed by NCBI-BLAST for homology sequence comparison, and the sequences with higher similarity were obtained. The phylogenetic tree was constructed using MEGA6.0 software.

[0060] 2.32 Identification results of antagonistic and growth-promoting functional bacteria

[0061] On LB solid medium, the colony of SQ 15 was yellow, opaque, with a convex and moist surface and neat edges. Its 16S nucleic acid sequence was identified as Paenarthrobacter nicotinovorans by NCBI-BLAST comparison. The colony of SQ24 had neat edges, a convex and moist surface, a viscous texture, a milky white color, and was opaque. Its 16S nucleic acid sequence was identified as Bacillus velezensis by NCBI-BLAST comparison. The colony of SQ 63 was light yellow and translucent, with a smooth surface and neat edges. Its 16S nucleic acid sequence was identified as Pseudomonas nitroreducens by NCBI-BLAST comparison. Figure 3 and Figure 4 ).

[0062] In summary, colonies were selected for purification based on naked eye morphology observation, and the purified single strains were screened for antagonism against the pathogens of Panax notoginseng root rot and IAA function screening, and single strains SQ 15, SQ 24 and SQ 63 with the ability to prevent and control Panax notoginseng root rot or promote Panax notoginseng growth were obtained. The PCR products of these three single strains were sequenced, and the 16S sequences of the strains obtained by sequencing were analyzed by homology sequence alignment using NCBI-BLAST. The phylogenetic tree sequencing results were constructed using MEGA6.0 software, and the species of these three single strains were identified. Finally, the single strains SQ 15, SQ 24 and SQ 63 were identified as: Paenarthrobacter nicotinovorans SQ 15, Bacillus velezenis SQ 24, Pseudomonas nitroreducens SQ 63.

[0063] 3. Application

[0064] The application method of the combination of SQ 15, SQ 24 and SQ 63 strains in promoting the growth and saponin accumulation of Panax notoginseng is as follows: the single strain seed liquid of SQ15, SQ 24 and SQ 63 strains is inoculated into LB liquid medium, and cultured on a shaker at 37°C and 220 r / min for 2 days. The bacterial liquid is centrifuged at a speed of 5000 r / min to remove the medium, and the bacteria are resuspended with an appropriate amount of sterile water. The OD value is calculated. 600 The dilution multiple was determined by the value, and the final concentration of the three strains was 10 8 After the emergence of Panax notoginseng, each Panax notoginseng plant was irrigated with 200 mL of 10 8 cfu / mL of SQ 15, SQ 24, and SQ 63 mixed bacterial solution (about 4200L-4500L per mu) was added again in the above manner after an interval of 1 month, and used continuously for 3 times.

[0065] 3.1 Potted experiment on promoting the growth of Panax notoginseng seedlings by SQ 15, SQ 24 and SQ 63

[0066] 3.11 Experimental location: Panax notoginseng cultivation greenhouse, School of Geographical Sciences, Nanjing Normal University

[0067] 3.12 Pre-treatment of the test: Vermiculite and perlite were mixed in a ratio of 4:1, sterilized, and filled into small flower pots with a diameter of 8 cm and a height of 12 cm. Selected full and uniform-sized Panax notoginseng seeds and planted them in the substrate, 5 seeds per pot, with a planting depth of 1-1.5 cm. After planting, the substrate was covered. A total of 40 pots were planted, and then each pot was poured with 150 mL of 1 / 4 Hoagland nutrient solution.

[0068] 3.13 Preparation of mixed bacterial liquid of SQ 15, SQ 24 and SQ63: The isolated single strains of SQ 15, SQ 24 and SQ63 were inoculated into LB liquid medium, and cultured on a shaker at 37°C and 220 r / min for 2 days. The bacterial liquid was centrifuged at 5000 r / min to remove the medium, and the bacteria were resuspended in an appropriate amount of sterile water. The OD values ​​were calculated. 600 The dilution multiple was determined by the value, and the final concentration of the three strains was 10 8 cfu / mL of mixed bacterial solution.

[0069] 3.14 Experimental treatment: One week after the Panax notoginseng seeds were planted, 40 pots were randomly divided into 20 control groups and 20 experimental groups. Each pot in the experimental group was watered with the concentrations of the three strains in 3.12, respectively. 8 cfu / mL mixed bacterial solution 50mL, the control group was watered with the same amount of sterilized water 50mL, and the same method was repeated after the Panax notoginseng seedlings emerged. During the planting period, the same amount of 1 / 4 Hogland nutrient solution was added in time according to the dryness and wetness of the substrate. After 2 months of planting, the fresh weight of the Panax notoginseng seedlings and the growth of the root system were counted.

[0070] 3.15 Test results: as shown in Table 2 and Figure 5 shown.

[0071] Table 2 Effect of bacterial composition on the growth of Panax notoginseng seedlings

[0072]

[0073] Note: The lowercase letters in the same row in the table represent significant differences between the experimental group and the control group, P<0.05

[0074] The experimental group with added bacterial combination can significantly increase the fresh weight of Panax notoginseng seedlings by 23.45%, increase the maximum leaf width by 11.86% and leaf length by 9.79%. The root analysis results show that the addition of bacterial combination has no significant effect on the root growth of Panax notoginseng seedlings. The total root length, root surface area, average root diameter and root volume of Panax notoginseng seedlings in the experimental group are slightly increased, indicating that the addition of bacterial combination can make the roots of Panax notoginseng thick to a certain extent, while the number of root tips and forks are slightly reduced.

[0075] 3.2 Application of SQ 15, SQ 24 and SQ 63 in promoting the growth of Panax notoginseng in the field in Qiubei

[0076] 3.21 Experimental location: Panax notoginseng planting base, Xiaoxinzhai Village, Shupi Township, Qiubei County, Wenshan Prefecture (104°09′E, 23°51′N)

[0077] 3.22 Preparation of mixed bacterial solution of SQ 15, SQ 24 and SQ 63: Inoculate the single strain seed solution of SQ 15, SQ 24 and SQ 63 into LB liquid medium respectively, and shake at 37℃ and 220r / min for 2 days; then, centrifuge the bacterial solution at 5000r / min to remove the medium, and resuspend the bacteria in an appropriate amount of sterile water. 600 The standard curve of the three strains was drawn based on the value and the bacterial concentration obtained by the plate count method. 600 The dilution multiple was determined by the value, and the final concentration of the three strains was 10 8 cfu / mL of mixed bacterial solution for later use.

[0078] 3.23 Experimental method: The experiment was set up in Qiubei County, and the three-year-old Panax notoginseng was tested. After the three-year-old Panax notoginseng seedlings were all grown, plots with good growth and basically the same size were selected, and a control group and an experimental group with added bacteria (SQ 15, SQ 24, SQ 63) were set up. Each group was repeated 3 times, and each replicate had 300 Panax notoginseng plants. The mixed bacterial solution was added by root irrigation, and the addition amount was 200mL of mixed bacterial solution per Panax notoginseng plant. The control group was added with an equal amount of clean water. After an interval of 1 month, it was added again in the above manner, and used continuously for 3 times. When the three-year-old Panax notoginseng was harvested, the Panax notoginseng plants were collected to determine the content of Panax notoginseng biomass (plant height, fresh weight of the aboveground part, dry weight of the underground part) and saponins (notoginseng saponin R1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd).

[0079] 3.24 Test results: as shown in Table 3.

[0080] Table 3 Effects of bacterial composition on the growth and saponin accumulation of Qiubei Panax notoginseng

[0081]

[0082] Note: The lowercase letters in the same row in the table represent significant differences between the experimental group and the control group, P<0.05.

[0083] As shown in Table 3, the selected bacterial combination (SQ 15, SQ 24, SQ 63) can significantly (P<0.05) increase the aboveground and underground biomass of Panax notoginseng, among which the plant height of Panax notoginseng in the experimental group increased by 16.69% compared with the control group, the aboveground fresh weight increased by 31.23%, and the underground fresh weight and dry weight increased by 23.15% and 23.87%, respectively, indicating that the bacterial combination can effectively promote the growth of Panax notoginseng; although the bacterial combination relatively reduced the content of total saponins in Panax notoginseng, due to its significant increase in the underground biomass of Panax notoginseng, the single-plant cumulative amounts of notoginseng saponin R1, ginsenoside Rb1, ginsenoside Rd and total saponins in the experimental group were 5.02%, 28.72%, 22.80% and 8.71% higher than those in the control group, respectively, indicating that the bacterial combination can improve the accumulation of notoginseng saponins to a certain extent.

[0084] 3.3 Application of SQ 15, SQ 24 and SQ 63 in promoting the growth of Panax notoginseng in Shilin field

[0085] 3.31 Test location: Panax notoginseng planting base, Shitouzhai Village, Nuohei Village Committee, Guishan Town, Shilin County, Kunming City (103°32′E, 24°41′N)

[0086] 3.32 Preparation of mixed bacterial solution of SQ 15, SQ 24 and SQ 63: Inoculate the single strain seed solution of SQ 15, SQ 24 and SQ 63 into LB liquid medium respectively, and shake at 37℃ and 220r / min for 2 days; then, centrifuge the bacterial solution at 5000r / min to remove the medium, and resuspend the bacteria in an appropriate amount of sterile water. 600 The standard curve of the three strains was drawn based on the value and the bacterial concentration obtained by the plate count method. 600 The dilution multiple was determined by the value, and the final concentration of the three strains was 10 8 cfu / mL of mixed bacterial solution for later use.

[0087] 3.33 Test method:

[0088] The experiment was set up in Shilin County, and the three-year-old Panax notoginseng was tested. After the three-year-old Panax notoginseng seedlings were fully grown, plots with good growth and basically the same size were selected, and a control group and an experimental group with the addition of bacterial composition (SQ 15, SQ 24, SQ 63) were set up. Each group was repeated 3 times, and each repeat had 300 Panax notoginseng plants. The mixed bacterial solution was added by root irrigation, and the addition amount was 200mL of mixed bacterial solution per Panax notoginseng plant. The control group added an equal amount of clean water, and added again in the above manner after an interval of 1 month, and used it continuously for 3 times. When the three-year-old Panax notoginseng was harvested, the Panax notoginseng plants were collected, and the content of Panax notoginseng biomass (plant height, fresh weight of the aboveground part, dry weight of the underground part) and saponins (notoginseng saponin R1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd) was determined.

[0089] 3.34 Test results: as shown in Table 4.

[0090] Table 4 Effects of bacterial composition on growth and saponin accumulation of Panax notoginseng

[0091]

[0092]

[0093] Note: The lowercase letters in the same row in the table represent significant differences between the experimental group and the control group, P<0.05.

[0094] The bacterial combination (SQ 15, SQ 24, SQ 63) can significantly (P<0.05) increase the aboveground and underground biomass of Shilin Panax notoginseng, and increase the aboveground fresh weight, underground fresh weight and underground dry weight of Panax notoginseng by 12.7%, 9.57% and 11.05%, respectively. In addition, the bacterial combination can also increase the content and accumulation of Panax notoginseng saponins, among which the contents of Panax notoginseng ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd and total saponins in the experimental group increased by 20.91%, 14.22%, 21.34% and 14.04% respectively compared with the control group, and their accumulation increased by 33.66%, 26.86%, 34.60% and 26.52%, respectively. The results showed that the bacterial combination of SQ 15, SQ 24 and SQ 63 can promote the growth of Panax notoginseng and the synthesis and accumulation of effective components saponins.

[0095] The laboratory pot experiment found that the application of the bacterial composition of SQ 15, SQ 24 and SQ 63 of the present invention (mixed bacterial solution 10 8 cfu / mL) can effectively promote the fresh weight growth of Panax notoginseng seedlings by 23.45%, increase the maximum leaf width by 11.86% and the leaf length by 9.79%; field experiments further proved that the application of the bacterial composition of the present invention (after the emergence of Panax notoginseng, 4200L-4500L of concentration of 10 per mu was used every 1 month ... 8 cfu / mL of mixed bacterial liquid once, used continuously for 3 times) can promote the growth of three-year-old Panax notoginseng root dry weight by 9.57% to 23.87%, and increase the accumulation of saponins by 8.17% to 26.52%; in addition, the strains SQ 24 and SQ 63 of the present invention have an inhibition rate of 12.38% to 65.95% on the growth of various Panax notoginseng root rot pathogens in the culture medium. In summary, the bacterial composition of the present invention has the potential to promote the growth of Panax notoginseng, increase the accumulation of Panax notoginseng saponins, and ensure the high quality and high yield of Panax notoginseng. It may also be able to prevent the occurrence of Panax notoginseng root rot, and further ensure the quality and yield of Panax notoginseng.

[0096] Although the present invention is described herein with reference to a number of illustrative embodiments of the present invention, it will be appreciated that those skilled in the art may devise many other modifications and implementations that fall within the scope and spirit of the principles disclosed herein. More specifically, within the scope of the present disclosure, drawings, and claims, a variety of variations and modifications may be made to the components and / or layout of the subject combination layout. In addition to the variations and modifications made to the components and / or layout, other uses will also be apparent to those skilled in the art.

Claims

1. A bacterium called Arthrobacter nicotianae, characterized in that: The Arthrobacter nicotinovorans SQ 15 is used to produce IAA to inhibit Fusarium oxysporum; The strain is deposited in China General Microbiological Culture Collection Center with the deposit number of CGMCC No.31366.

2. A Bacillus Velez, characterized in that: The Bacillus velezenis is Bacillus velezenis SQ24, which is used to inhibit Fusarium oxysporum, Fusarium solani, and Cylindrospermum destructum; The strain is deposited in China General Microbiological Culture Collection Center with the deposit number of CGMCC No.31369.

3. A nitroreducing Pseudomonas, characterized in that: The nitroreducing Pseudomonas is Pseudomonas nitroreducens SQ 63, which is used to inhibit Fusarium oxysporum, Fusarium solani, and Cylindrospermum destructum; The strain is deposited in China General Microbiological Culture Collection Center with the deposit number of CGMCC No.31367.

4. A bacterial composition for promoting the growth of Panax notoginseng and the accumulation of saponins, characterized in that: The bacterial composition comprises: Paenarthrobacter nicotinovorans SQ 15 as claimed in claim 1; Bacillus velezenis SQ 24 as claimed in claim 2; and Pseudomonas nitroreducens SQ 63 as claimed in claim 3.

5. Use of the bacterial composition for promoting the growth and saponin accumulation of Panax notoginseng as described in claim 4 in promoting the growth of Panax notoginseng.

6. Use of the bacterial composition for promoting the growth and saponin accumulation of Panax notoginseng as claimed in claim 5 to promote the accumulation of saponins in Panax notoginseng roots.

7. The application method according to claim 6, characterized in that: The methods applied are: Take the single strain seed liquid of SQ 15, SQ 24 and SQ 63 and inoculate it into the culture medium, and then culture it on a shaker. After the culture, centrifuge to remove the culture medium, resuspend it in sterile water, and then adjust the OD value according to the OD value. 600 The dilution factor is determined by the value, and the concentration is configured to be 10 8 cfu / mL of mixed bacterial solution of SQ 15, SQ 24, and SQ 63; After Panax notoginseng seedlings emerge, the plants are irrigated with mixed bacterial solution.

8. The application method according to claim 6, characterized in that: The culture medium is selected from LB liquid culture medium, including: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder.

9. The application method according to claim 6, characterized in that: 200 mL of the mixed bacterial solution was added to each Panax notoginseng plant, and the mixed bacterial solution was added again after an interval of 1 month, and the mixed bacterial solution was added continuously for 3 times.

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