Strain NJSH56 for promoting plant growth and application thereof

Abstract

The invention discloses a strain NJSH56 for promoting plant growth and application thereof, the strain NJSH56 is identified as bacillus pumilus, the strain NJSH56 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation time is November 14, 2024, and the strain preservation number is CGMCC NO.32614. The strain NJSH56 can promote plant growth. The dendrobium officinale endophytic growth-promoting strain NJSH56 for promoting plant growth is obtained through separation and screening, growth of dendrobium officinale tissue culture seedlings can be effectively promoted, and the fresh weight, the rooting number and the leaf number are increased; greenhouse tests show that the number of new buds of dendrobium officinale plants in a strain NJSH56 treatment group reaches 4.67 and is obviously higher than that of a control group; meanwhile, the NJSH56 is used for treating the non-host plant Chinese cabbage, so that the growth of the overground part of the Chinese cabbage can be remarkably promoted, and the biomass accumulation of the Chinese cabbage is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a plant growth-promoting strain NJSH56 and an application thereof. Background Art

[0002] Dendrobium officinale (Kimura et Migo), a perennial epiphytic herb, is a traditional and valuable Chinese medicine. Modern pharmacological research has shown that it has the effects of strengthening the spleen and stomach, nourishing yin and tonifying the kidneys, and moistening the lungs and promoting fluid production.

[0003] Dendrobium officinale uses its dense fibrous root system to cling to tree bark or gravel, absorbing moisture and nutrients from the rock. Roots exposed to the air absorb moisture from mist and dew, relying on its own chlorophyll for photosynthesis. Consequently, its distribution is severely restricted by its microclimate, particularly humidity, and its distribution is extremely limited. Due to its unique growing environment, the plant's difficulty in reproducing, and excessive harvesting, its wild resources are dwindling.

[0004] In order to effectively protect resources and take into account market demand, the artificial cultivation industry of Dendrobium officinale came into being. Although the planting area has been expanding year by year, it is still a drop in the bucket compared to the annual demand for "Dendrobium Fengdou", which increases by 10%-15% each year. Therefore, without destroying the wild resources of Dendrobium officinale, it is imperative to vigorously improve the yield and quality of Dendrobium officinale. As a medicinal plant of the orchid family, the life history of Dendrobium officinale is closely related to endophytes. Endophytic bacteria can directly affect the growth and metabolism of plants by providing substances that are lacking in plants through means such as nitrogen fixation, phosphorus solubilization, iron solubilization, and production of plant hormones (auxin, gibberellins, cytokinins and ethylene). Screening for microbial factors that promote the growth of Dendrobium officinale will help develop its cultivation industry. Summary of the Invention

[0005] Purpose of the Invention: To address the long growth period and slow growth of Dendrobium officinale, the present invention provides a plant growth-promoting strain, NJSH56, which can be used as an endophytic growth-promoting bacterium in Dendrobium officinale to promote its budding and growth. The use of this strain can effectively promote the growth of Dendrobium officinale tissue culture seedlings, increase fresh weight, root count, and leaf count, and increase the number of buds after transplanting. Application of this strain to non-host plants such as Chinese cabbage can significantly promote the growth of the aboveground part of the cabbage.

[0006] Another object of the present invention is to provide a growth-promoting bacterial agent of the strain NJSH56 for promoting plant growth and its application.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the plant growth-promoting strain NJSH56 described in the present invention has been identified as Bacillus pumilus and has been deposited in the General Microbiology Center of the China Culture Collection Administration. The deposit date is November 14, 2024, and the strain deposit number is CGMCC NO.32614.

[0008] The plant growth-promoting strain NJSH56 of the present invention is used as an endophytic growth-promoting bacterium of Dendrobium officinale.

[0009] The plant growth promoting strain NJSH56 of the present invention is used to promote the growth of Dendrobium officinale and Chinese cabbage.

[0010] Among them, the strain NJSH56 is used in promoting the growth of Dendrobium officinale tissue culture seedlings, increasing fresh weight, root number, leaf number and sprouting growth of Dendrobium officinale.

[0011] The strain NJSH56 is used to promote the growth of the aboveground part of cabbage and increase the biomass accumulation of cabbage.

[0012] The present invention discloses a growth-promoting bacterial agent containing the strain NJSH56.

[0013] The preparation method of the growth-promoting bacterium agent of the present invention comprises the following steps:

[0014] The strain NJSH56 was cultured in a culture medium, and then centrifuged to obtain the strain and prepare a bacterial agent with a sodium chloride solution.

[0015] Among them, the total concentration of live bacteria in the finished product of the microbial agent is 1×10 9 ~1×10 12 cfu / ml.

[0016] Preferably, the strain NJSH56 that promotes the budding growth of Dendrobium officinale is cultured in LB culture medium at 30°C and 180rpm for 20 to 24 hours, and then centrifuged at 6000rpm for 10 minutes. The wet bacteria of NJSH56 are mixed with 0.85% sodium chloride solution to prepare a microbial agent. The total concentration of live bacteria in the finished microbial agent is 1×10 9 ~1×10 12 The application method is to dilute the NJSH56 strain preparation and use it to soak the roots or irrigate the roots during the rooting period of plant tissue culture and transplanting.

[0017] The growth-promoting bacterial agent of the present invention is used in promoting the growth of Dendrobium officinale and Chinese cabbage.

[0018] The growth-promoting agent is used to promote the growth of Dendrobium officinale tissue culture seedlings, increase fresh weight, root number, leaf number, sprouting growth of Dendrobium officinale, growth of the aboveground part of cabbage, and improve cabbage biomass accumulation.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0020] The present invention obtains the plant growth-promoting endophytic growth-promoting strain NJSH56 of Dendrobium officinale through isolation and screening. The NJSH56 strain exhibits phosphate-solubilizing ability on an inorganic phosphorus (NPA) medium and can turn a nitrogen-fixing medium blue, indicating its nitrogen-fixing ability. Further tissue culture seedling inoculation experiments show that the strain NJSH56 can effectively promote the growth of Dendrobium officinale tissue culture seedlings, increasing fresh weight, number of roots, and number of leaves. Greenhouse experiments show that the number of new buds of Dendrobium officinale plants in the NJSH56-treated group reached 4.67, significantly higher than the control group (1.33). NJSH56 treatment of non-host plant Chinese cabbage can significantly promote the growth of the aboveground part of the cabbage and increase the biomass accumulation of the cabbage.

[0021] Furthermore, the growth-promoting agent prepared from the strain of the present invention can be produced using common fermentation equipment in the fermentation industry, with low production cost and ease of use. It is suitable for promoting the growth of Dendrobium officinale and Chinese cabbage. Therefore, strain NJSH56 has great potential for development into a commercial live bacterial growth-promoting agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are the tissue culture seedlings of Dendrobium officinale in group CK1 (A), group CK2 (B), group NJSH56 (C), and group DX (D).

[0023] Figure 2 Figure 3. Fresh weight (A), number of sprouts (B), number of roots (C), and number of leaves (D) of tissue-cultured Dendrobium officinale seedlings in each group. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. Each data represents the mean (± standard error) of three replicates.

[0024] Figure 3 Effects of Bacillus sp. NJSH56 on the dry weight (A) and number of new shoots (B) of Dendrobium officinale. Different letters indicate significant differences at the p < 0.05 level according to Duncan's multiple-range test. Each data point represents the mean (± standard error) of three replicates.

[0025] Figure 4 This is the growth-promoting effect of Bacillus NJSH56 on cabbage plants. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the accompanying drawings and examples.

[0027] Unless otherwise specified, the raw materials and reagents in the present invention are all commercially available. The Dendrobium officinale pods and Dendrobium officinale tissue culture seedlings in the present invention are conventional cultivated Dendrobium officinale, provided by Nanjing Normal University.

[0028] Example 1

[0029] Isolation and preservation: Fresh wild Dendrobium officinale plants were collected from their place of origin, Fujian. The plants were first rinsed with water, cut into three parts: roots, stems, and leaves with scissors, weighed, and soaked in a 2.5% sodium hypochlorite aqueous solution for 10 minutes. The plants were repeatedly rinsed with sterile water in a sterile petri dish three times (0.1 mL of the aqueous solution from the last rinse was evenly applied to R2A culture medium. If no sterile growth was observed after 48 hours, the surface was considered to be cleanly disinfected). The surface-disinfected plant materials were placed in a sterile mortar and ground into powder after adding 9 times the weight of the plant material with sterile water. 0.1 mL of the grinding solution was applied to R2A medium (0.5 g yeast powder, 0.25 g casein peptone, 0.25 g meat peptone, 0.5 g casein hydrolyzate, 0.5 g glucose, 0.5 g starch, 0.3 g sodium pyruvate, 0.3 g potassium dihydrogen phosphate, 0.024 g hydrated magnesium sulfate, 15 g agar, 1000 ml distilled water, pH 7.2, sterilized at 121°C / 100 kPa for 20 min before use). The medium was incubated at 28°C for 1-3 days. After colonies grew, colonies of different morphologies were selected and streaked onto LB medium plates for purification. A single purified colony was selected and cultured in LB medium until the logarithmic phase. 40% glycerol (1:1 volume ratio) was added and stored in an ultra-low temperature freezer (-80°C). The strain was named NJSH56.

[0030] Screening: Prepare organophosphorus medium (OPA): 10g glucose, 0.3g KCl, 0.3g NaCl, 0.03g FeSO4·7H2O, 0.3g MgSO4·7H2O, 1.0g CaCO3, 0.03g MnSO4·4H2O, 0.2g lecithin, 0.5g (NH4)2SO4, 0.5g yeast powder, 15-18g agar, dissolve in 1000mL deionized water, adjust pH to 7.0-7.2, and sterilize at 121℃ for 20min. Prepare inorganic phosphate medium (NPA): Dissolve 10g glucose, 0.3g KCl, 0.3g NaCl, 0.03g FeSO4·7H2O, 0.3g MgSO4·7H2O, 5.0g calcium phosphate, 0.03g MnSO4·4H2O, 15-18g agar, and 0.5g (NH4)2SO4 in 1000mL deionized water, adjust the pH to 7.0-7.2, and sterilize at 121°C for 20 minutes. Pipette 10μL of the bacterial culture medium that has reached logarithmic phase onto an NPA / OPA plate and incubate at 28°C for 48 hours. Observe the NPA / OPA plate for the presence of a clearing zone and record the radius of the clearing zone. If the clearing zone is faint and difficult to measure, record "W"; if no clearing zone is present, record "0." Test each strain in triplicate.

[0031] Weigh 11.2g of KOH and dissolve it in 1000mL of deionized water to make a 0.2mol / L KOH solution. Weigh 0.05g of bromothymol blue and dissolve it in 10mL of 0.2mol / L KOH solution to make a 0.5% bromothymol blue solution. Weigh 100mg of biotin and 200mg of pyridoxine hydrochloride and dissolve them in 1000mL of deionized water to make a vitamin C mixed solution. Weigh 1.64g of Fe-EDTA and dissolve it in 100mL of distilled water to make a 1.64% Fe-EDTA solution. Weigh 0.4 g CuSO4, 1.5 g MnSO4·4H2O, 0.12 g ZnSO4·7H2O, 4 mL 1.64% Fe-EDTA solution, 1.4 g H3BO3, 4.5 g KOH, and 1.0 g Na2MoO4·2H2O and dissolve them in 1000 mL deionized water. Adjust the pH to 6.8 to prepare a trace element solution. Weigh 5.0 g malic acid, 0.5 g K2HPO4, 0.2 g MgSO4·7H2O, 0.1 g NaCl, 0.02 g CaCl2, and 17 g agar. Weigh 2 mL 0.5% bromothymol blue solution, 1 mL Vc mixed solution, and 2 mL trace element solution and dissolve them in 1000 mL deionized water. Adjust the pH to 7.0-7.2 and sterilize at 121°C for 20 min to prepare nitrogen fixation test medium (NFb). Pipette 10 μL of NJSH56 bacterial suspension cultured to the logarithmic growth phase onto a NFb plate and incubate at 28°C for 48 hours. Bacteria that grow on nitrogen-fixing medium and turn the medium blue are considered to have nitrogen-fixing activity. Observe the NFb plate for the presence of a clearing zone and measure the radius of the clearing zone. If the clearing zone is faint and difficult to measure, record it as "W"; if no clearing zone is present, record it as "0". Repeat the test three times for each strain.

[0032] NJSH56 produces single colonies after 10 hours of incubation on LB medium. After 24 hours of incubation, the colonies are milky white, uniform, round, moist, with a central bulge and a smooth surface. They stain Gram-positive. This strain has nitrogen-fixing activity and the ability to dissolve inorganic phosphate. The radius of its nitrogen-fixing zone on nitrogen-fixing medium is 2.00±0.00 mm, and the radius of its phosphate-dissolving zone on inorganic phosphate medium is 3.00±1.00 mm.

[0033] Identification: The genomic DNA of strain NJSH56 was extracted, and PCR amplification was performed using the genomic DNA as a template and the universal primers of the bacterial 16SrRNA gene as primers to obtain an amplified product of length. The sequence obtained by sequencing is shown in SEQ ID NO.1. The sequence was aligned in the Nucleotide Sequence Database of the NCBI website using the Basic Local Alignment Search Tool (BLAST) tool. The results showed that the 16S rDNA sequence of the strain had a high similarity with the sequences of Bacillus pumilus strains GR23 and HN-10. Based on the physiological and biochemical characteristics and the phylogenetic tree analysis constructed based on the 16srDNA sequence, the strain NJSH56 was identified as Bacillus pumilus. The strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on November 14, 2024. The culture collection number is CGMCC NO.32614. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0034] Example 2

[0035] Growth promotion experiment of Dendrobium officinale tissue culture seedlings

[0036] The tissue culture seedlings of Dendrobium officinale in the strong seedling stage with consistent growth were selected and transferred to MS medium and cultured in a tissue culture room at a temperature of 25±1℃ and a photoperiod of 12h / 12h. Ten days after the transfer, two experimental groups and two control groups were set up in the experiment, namely NJSH56 experimental group, DX experimental group, CK1 control group and CK2 control group. Each group contained 3 replicates, and each replicate contained 3 bottles of tissue culture seedlings. The treatments of each group are shown in Table 1. In the NJSH56 experimental group, the strain NJSH56 was cultured to the logarithmic phase and centrifuged. The supernatant was the secondary metabolite of Bacillus NJSH56; the bacteria were resuspended in sterile water and prepared with 1×10 9 cfu / ml of bacterial solution. 2 mL of bacterial solution was applied to the roots of each flask of tissue culture seedlings. The DX experimental group used an equal amount of filter-sterilized secondary metabolites of Bacillus sp. NJSH56. 55 days after inoculation, stem length, fresh weight, number of roots, number of leaves, and number of buds were measured in both the experimental and control groups to investigate the effects of Bacillus sp. NJSH56 on their growth.

[0037] Table 1. Group settings for growth promotion experiments

[0038]

[0039] The results showed that the average stem lengths of the CK1, CK2, NJSH56, and DX groups were 1.00, 0.97, 0.90, and 0.77 cm, respectively. One-way ANOVA showed that the P values ​​among the four groups were all greater than 0.05, indicating that there was no significant difference in the stem lengths of Dendrobium officinale among the groups. However, there were significant differences in the number of buds and roots among the groups, and extremely significant differences in the fresh weight and number of leaves among the groups ( Figure 1 and Figure 2 The average fresh weights of the CK1, CK2, NJSH56, and DX groups were 0.0947, 0.1034, 0.1348, and 0.0481 g, respectively; the average number of buds was 1.0, 0.7, 2.0, and 0.7, respectively; the average number of roots was 5.0, 3.3, 4.7, and 4.0, respectively; and the average number of leaves was 4.7, 5.3, 11.7, and 5.0, respectively. Figure 2 The fresh weight, number of buds, number of roots, and number of leaves of the Bacillus NJSH56 experimental group were significantly higher than those of the CK2 group, and were less contaminated by other microorganisms. However, the growth indicators of the filter-sterilized strain secondary metabolite (DX) experimental group did not show a significant increase. On the contrary, the fresh weight of the DX group was lower than that of the control group, and the seedlings burned. This proves that the NJSH56 bacterial solution of the present invention can effectively promote the growth of Dendrobium officinale tissue culture seedlings, increasing the fresh weight, number of roots, and number of leaves.

[0040] Example 3

[0041] Greenhouse growth promotion experiment of Dendrobium officinale

[0042] Preparation of NJSH56 bacterial suspension: Activate NJSH56 strain on a plate, pick a single colony and inoculate into 5 ml of LB culture medium (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.2), shake at 180 rpm at 28°C for 20 h, transfer to 50 ml of LB culture medium, shake at 180 rpm at 28°C for 24 h, and adjust the bacterial suspension concentration to OD 600 =1.00(approximately 10 9 cells / mL).

[0043] Preparation of Dendrobium officinale seedlings: Dendrobium officinale seeds were placed on MS culture medium for germination, growth, and rooting. The culture conditions were 25°C with a 16 / 8h photoperiod. The seedlings were transplanted when they had 5 true leaves (about 30 days, with a seedling height of 3-5 cm).

[0044] The tissue-cultured seedlings were transplanted into a 520×260 mm (32 holes, each 60×60×50 mm). Each hole was filled approximately two-thirds of its volume with pine bark and sphagnum moss (1:1 volume ratio) and then transplanted with three Dendrobium officinale seedlings. On the day of transplanting, the roots of the seedlings in each hole in the treatment group were watered with 5 mL of bacterial culture solution; the roots of the seedlings in each hole in the control group were watered with 5 mL of LB broth. After one week, each hole was watered with 5 mL of water every three days. The control group and each treatment group each contained three replicates, each consisting of 24 Dendrobium officinale seedlings. The experiment was conducted using a completely randomized design. Plants were grown under the following conditions: a temperature of 22–24°C, a relative humidity (RH) of 70–80%, and a 12h / 12h photoperiod.

[0045] Three months after transplanting, the dry weight of the seedlings (fresh plants were dried at 53°C for 5 days) and the number of new shoots were measured and recorded. The results showed that the dry weight of the plants in both the treatment and control groups was approximately 46 mg, indicating that NJSH56 had no effect on the dry weight of the plants ( Figure 3 However, the number of new buds in the NJSH56-treated group reached 4.67, which was significantly higher than that in the control group (1.33), indicating that the strain had an effect on the differentiation of Dendrobium officinale plants ( Figure 3 ).

[0046] Example 4

[0047] Greenhouse growth promotion experiment of non-host cabbage

[0048] The test soil was Pelei Flower Cultivation Medium, produced by Jiangsu Pelei Cultivation Medium Technology Development Co., Ltd. Conventional commercial vegetable cultivation medium can also be used. The cabbage variety used was "Yuexia," produced by Beijing Junchuan Seed Technology Co., Ltd. Any commercially available cabbage variety can also be used.

[0049] Before sowing, soak the cabbage seeds in water for 24 hours. Place 500g of growing medium in each pot and evenly distribute the germinated cabbage seeds throughout the pot, planting 10 seeds per pot. Water the pot every two days. Repeat three times for each treatment, ensuring that each treatment contains 24 cabbage plants.

[0050] The NJSH56 strain was inoculated into 150 mL of LB liquid medium and cultured at 28°C and 180 rpm until the logarithmic phase. After centrifugation, the concentration was adjusted to 5 × 10 8 CFU / mL. Cabbage was inoculated when it reached the 4-leaf stage. The bacterial solution was evenly poured over the roots of the cabbage, 20 mL per pot, for a total of 3 pots. The control group was watered with 20 mL of LB liquid medium. Continue watering every two days.

[0051] Thirty days after inoculation with the NJSH56 strain, the cabbages were removed from the pots, the plant culture medium at the roots was washed, and the fresh weight, above-ground length, and underground length of the cabbages were measured. The cabbages were then placed in an oven at 75°C for 1080 minutes, and their dry weight was measured. The growth-promoting effect of the endophytes was calculated using the following formula:

[0052] Growth promotion effect = (treated biomass - control biomass) / control biomass × 100%

[0053] The results are as follows Figure 4 As shown in Table 2, the seedling height, fresh weight, and dry weight of the NJSH56-treated cabbage reached 16 cm, 5.19 g, and 0.31 g, respectively, significantly higher than those in the control group (seedling height 13.67 cm, fresh weight 3.05 g, and dry weight 0.20 g). Furthermore, strain NJSH56 had no effect on cabbage root growth.

[0054] Table 2 Growth promotion experiment of Bacillus NJSH56 on Chinese cabbage

[0055]

[0056] Note: Different letters indicate significant differences at the p < 0.05 level according to Duncan's multiple-range test.

Claims

1. A plant growth-promoting strain NJSH56, identified as Bacillus pumilus, has been deposited in the General Microbiology Center of the China Culture Collection Administration on November 14, 2024, with the culture collection number CGMCC NO.32614.

2. Use of the plant growth-promoting strain NJSH56 according to claim 1 as an endophytic growth-promoting bacterium of Dendrobium officinale.

3. Use of the plant growth promoting strain NJSH56 according to claim 1 in promoting the growth of Dendrobium officinale and Chinese cabbage.

4. The use according to claim 3, characterized in that The strain NJSH56 is preferably used in promoting the growth of Dendrobium officinale tissue culture seedlings, increasing the fresh weight, the number of roots, the number of leaves, and the sprouting growth of Dendrobium officinale.

5. The use according to claim 3, characterized in that The strain NJSH56 is used to promote the growth of the aboveground part of cabbage and increase the biomass accumulation of cabbage. A growth-promoting agent containing the strain NJSH56 according to claim 1.

7. A method for preparing the growth-promoting bacterium agent according to claim 6, characterized in that: The steps include: The strain NJSH56 is cultured in a liquid culture medium, centrifuged, and then the strain is taken out and mixed with a sodium chloride solution to prepare a bacterial agent.

8. The method for preparing the growth-promoting bacterium agent according to claim 7, characterized in that: The total concentration of live bacteria in the finished product of the inoculant is 1×10 9 ~1×10 12 cfu / ml.

9. Use of the growth-promoting bacterial agent according to claim 6 in promoting the growth of Dendrobium officinale and Chinese cabbage.

10. The use according to claim 9, characterized in that The growth-promoting microbial agent is used to promote the growth of Dendrobium officinale tissue culture seedlings, increase fresh weight, the number of roots, the number of leaves, the sprouting growth of Dendrobium officinale, the growth of the aboveground part of cabbage, and improve the biomass accumulation of cabbage.

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