Disclosure of Invention
In order to solve the problems, the invention provides bacillus tropicalis NB379, application thereof and a microbial inoculum.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a bacillus tropicalis (Bacillus tropicus) NB379 which is deposited in China center for type culture collection (China, with a deposit number of CCTCCNo:M 2024509) on 18 th year of 2024.
The invention also provides application of the bacillus tropicalis NB379 in preventing and controlling spider mites, wherein the spider mites comprise one or more of spider mites, panonychus citri and tetranychus cinnabarinus.
The invention also provides application of the bacillus tropicalis NB379 in promoting plant growth.
The invention also provides application of the bacillus tropicalis NB379 in alleviating plant heavy metal stress, wherein the heavy metal comprises one or more of copper, manganese, chromium, lead, cadmium and nickel.
The invention also provides application of the tropical bacillus NB379 in inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi comprise one or more of rice sheath blight pathogenic bacteria, wheat scab pathogenic bacteria, wheat glume blight pathogenic bacteria, tomato wilt pathogenic bacteria, tobacco black shank pathogenic bacteria, cotton verticillium wilt pathogenic bacteria, pear black spot pathogenic bacteria and gray mold.
The invention also provides application of the bacillus tropicalis NB379 in indole-3-acetic acid production.
The invention also provides application of the bacillus tropicalis NB379 in dissolving phosphorus and/or producing ammonium nitrogen.
The invention also provides application of the bacillus tropicalis NB379 in producing catalase.
The invention also provides application of the bacillus tropicalis NB379 in the technical scheme in the production of the mesophilic ferrite, wherein the mesophilic ferrite is catechol type mesophilic ferrite.
The invention also provides a microbial inoculum, which contains the bacillus tropicalis NB379 according to the technical scheme, and the microbial inoculum content is 1 multiplied by 106 CFU/mL.
The single colony of the tropical bacillus NB379 strain growing on the LB agar plate is in a milky round shape, the edge of the single colony is protruded from the center (figure 1), and the cell morphology is observed to be rod-shaped by microscopic examination. The strain was found to have 96.72% ANI similarity to the model strain (N24) of Bacillus tropicalis (Bacillus tropicus) by genome-Average Nucleotide Identity (ANI) alignment analysis. By combining ANI analysis and physiological and biochemical identification, NB379 is defined as bacillus tropicalis, and the strain is preserved in China Center for Type Culture Collection (CCTCCNO) with the preservation number of M2024509 and the preservation date of 2024 and 03 months and 18 days.
The half-lethal concentrations (LC50) of the fermentation broth of Bacillus tropicalis NB379 on Tetranychus urticae, tetranychus citri and Tetranychus cinnabarinus were 9.8. Mu.L/mL, 22.7. Mu.L/mL and 16.3. Mu.L/mL, respectively.
Bacillus tropicalis NB379 has good environmental tolerance, shows good viability in the pH range 5.0-10.0, in the heavy metal concentration range 0.5mM to 64mM, and in saline-alkaline environment (NaCl: na2SO4:NaHCO3:Na2CO3 = 1:9:9:1, salt concentration range 40mM-200 mM).
The tropical bacillus NB379 can effectively inhibit plant pathogenic bacteria, including rhizoctonia solani (rhizoctonia solani), fusarium graminearum (fusarium graminearum), fusarium graminearum (septoria glume), fusarium solani (fusarium oxysporum tomato specialization), phytophthora nicotianae (phytophthora nicotianae), verticillium cotton (verticillium dahliae), pear black spot bacteria (alternaria alternate) and gray mold.
The tropical bacillus NB379 has the capability of promoting the growth and development of plants, such as the production of ferrite, IAA, phosphorus dissolution, ammonium nitrogen production and the like, has a certain growth promoting potential for the growth of tomatoes and arabidopsis thaliana, and relieves the stress influence of heavy metals such as Cu2+、Mn2+、Zn2+、Cr3+、Fe2+、Pb2+、Cd2+、Ni2+ on plants.
Therefore, the bacillus tropicalis NB379 provides a novel and efficient microbial resource for solving the problems of mites, plant diseases and heavy metal pollution in agricultural production. The application of the NB379 strain is expected to reduce the use of chemical pesticides and fertilizers, improve the crop yield and quality, promote the sustainable development of agriculture, and have important practical application and popularization values.
The invention has the beneficial effects that:
The strain NB379 is a bacillus tropicalis (Bacillus tropicus), has acaricidal activity and stability under various environmental stresses, and can stably grow under the extreme pH value range, saline-alkali condition and heavy metal stress. Compared with the prior report, the strain has unique spider mite killing capability, can synthesize various bioactive substances such as antibiotics, enzymes, plant hormones and the like, has remarkable antibacterial effect on various pathogenic bacteria, shows growth promotion effect on plants such as tomatoes, arabidopsis thaliana and the like, and can reduce toxic action of heavy metals on the plants. The strain has wider application prospect in agricultural production, can effectively control agricultural diseases and insect pests, improve crop yield and quality, and can also adapt to the crop growth requirements under different soil and environmental conditions. Meanwhile, the environmental tolerance of the strain also provides possibility for the application of the strain in the fields of soil restoration, ecological restoration and the like.
Detailed Description
The invention provides a bacillus tropicalis (Bacillus tropicus) NB379 which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2024509 in the year 2024 and the month 18.
The invention also provides application of the bacillus tropicalis NB379 in preventing and controlling tetranychus urticae, wherein the tetranychus urticae includes one or more of tetranychus urticae, panonychus citri and tetranychus cinnabarinus.
The invention also provides application of the bacillus tropicalis NB379 in promoting plant growth. In the present invention, the plant preferably comprises tomato and/or arabidopsis thaliana.
The invention also provides application of the bacillus tropicalis NB379 in alleviating plant heavy metal stress, wherein the heavy metal comprises one or more of copper, manganese, chromium, lead, cadmium and nickel.
The invention also provides application of the tropical bacillus NB379 in inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi comprise one or more of rice sheath blight pathogenic bacteria, wheat scab pathogenic bacteria, wheat glume blight pathogenic bacteria, tomato wilt pathogenic bacteria, tobacco black shank pathogenic bacteria, cotton verticillium wilt pathogenic bacteria, pear black spot pathogenic bacteria and gray mold.
The invention also provides application of the bacillus tropicalis NB379 in indole-3-acetic acid production.
The invention also provides application of the bacillus tropicalis NB379 in dissolving phosphorus and/or producing ammonium nitrogen. In the present invention, the phosphorus preferably comprises organic phosphorus, preferably comprising calcium phosphate, and/or inorganic phosphorus, preferably comprising lecithin.
The invention also provides application of the bacillus tropicalis NB379 in producing catalase.
The invention also provides application of the bacillus tropicalis NB379 in the technical scheme in the production of the mesophilic ferrite, wherein the mesophilic ferrite is catechol type mesophilic ferrite.
The invention also provides a microbial inoculum, which contains the bacillus tropicalis NB379 according to the technical scheme, and the microbial inoculum content is 1 multiplied by 106 CFU/mL. In the present invention, the microbial agent further preferably comprises a carrier, and the carrier is preferably a solid carrier or a liquid carrier. In the invention, the dosage form of the microbial inoculum is preferably liquid, emulsion, suspending agent, powder, granule, wettable powder or water dispersible granule.
In the invention, the microbial inoculum is preferably applied to the roots, leaves or soil of plants, and the application amount is preferably 1X 107 CFU per liter of soil after the microbial inoculum is applied to the roots or soil.
The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.
The medium composition referred to in the examples:
(1) LB medium, tryptone 10.0g, yeast extract 5.0g,NaCl 10.0g,pH 7.2, fixed volume to 1L.
(2) Fermentation medium 1 comprises (by mass) soybean meal 3%, corn steep liquor 1.5%, corn starch 1.5%, and pH 7.0-7.5.
(3) SPA culture medium, peptone 5.0g, sucrose 20.0g, KH2PO40.5 g,MgSO4 0.25.25 g, was fixed to 1L.
(4) PDA culture medium is prepared from potato 200g, glucose 20.0g, agar 15.0g, and pH is not regulated.
(5) MSA liquid culture medium, sucrose 20.0g, L-asparagine 2.0g, K2HPO41.0 g,MgSO4·7H2 O0.5 g, pH 7.0, was fixed to 1L.
(6) NBRIP culture medium glucose 10g,MgCl25.0 g,MgSO4·7H2O 0.25g,KCl0.2g,(NH4)2SO41.0 g,pH 7.0, to 1L, agar 15g.
(7) Peptone water peptone 10.0g,NaCl 5.0g,pH 7.0, constant volume to 1L.
(8) CAS detection solution, chrome azure 1.0mM, feCl3 0.1mM, hexadecyl trimethyl ammonium bromide 4.0mM, phosphate buffer 0.1M, pH 6.8.
Example 1
Screening of Bacillus tropicalis NB379
(1) Isolation of bacillus strains:
The applicant collects samples from the sludge of the island lake and selects about 100g of fertilizer soil in the area of 5-10cm of the surface layer of the sludge. The collected silt soil sample is transferred into a triangular flask containing 45mL of water, and after shaking and mixing, the soil sample is subjected to heat treatment at 85 ℃ for 15min to inactivate non-spore bacteria. After treatment, the suspension was allowed to stand for 5min to precipitate large particulate matter, then 1mL of supernatant was subjected to gradient dilution, and each dilution gradient was incubated overnight at 30℃with 0.1mL of coated LB agar plate. Single colony with obvious morphological characteristics is selected from a flat plate for streak purification, and is transferred into 5mL of LB liquid culture medium after being confirmed to be pure bacillus species through colony observation and microscopic examination, and 0.5mL of bacterial liquid is taken after being cultured at 30 ℃ and 220rpm overnight, and is stored at-80 ℃ after 60% glycerol with equal volume is added. Through this procedure, 17 strains of Bacillus were isolated in total.
(2) Screening of strains with acaricidal and bacteriostatic Activity:
① Acaricidal Activity assay
The isolated bacillus strains were inoculated into 5mL of LB liquid medium, respectively, activated overnight at 30 ℃ and 220rpm, and transferred to fermentation medium No.1, respectively, and cultured for 48 hours at 30 ℃ and 220 rpm. The spore concentration in the fermentation broths was determined by dilution counting and adjusted to 1×106 CFU/mL using fresh fermentation medium No. 1. Subsequently, according to the spider mite slide dipping method in the national agricultural industry standard (NY/T1154.12-2008), the mite killing activity of the strain fermentation broth is evaluated, and the pest mite is spider mite (Tetranychus urticae). The biological measurement steps are as follows:
Cutting double-sided adhesive tape into small blocks of about 2cm2, adhering the small blocks to clean glass slides, and selecting healthy female mites, so that the backs of the healthy female mites are adhered to the double-sided adhesive tape, and each glass slide is adhered to about 30 heads; rapidly immersing a mite-bearing slide into a fermentation broth, taking out the slide after gently shaking for 5 seconds, sucking redundant liquid by using water-absorbing paper, placing the slide in a white magnetic disk filled with wet sponge, covering a plastic film with good light transmittance, setting 4 times of repetition for each treatment, taking untreated fresh fermentation medium as a blank control, standing a container with test insects in an environment with the light period of L: D=16 h:8h at 25 ℃ for 24 hours, counting the death rate of two-spotted spider mites in each treatment group, and correcting the death rate according to a formula if the death rate in a control group is less than 5% and does not need to be corrected, wherein the death rate of the treatment group is = (the death rate of the treatment group-the death rate of the control group)/(the death rate of the 1-control group)/(100) is corrected according to the formula, and carrying out a test again if the death rate of the control group is more than 20%.
② Determination of bacteriostatic Activity
The inhibition of bacillus against specific pathogenic bacteria was tested by plate diffusion. The pathogenic bacteria include Erwinia carotovora, xanthomonas nigricans, pseudomonas, escherichia coli, pantoea agglomerans, aeromonas hydrophila, aeromonas veronii, bacillus cereus, staphylococcus aureus, and Micrococcus luteus. The method comprises the steps of culturing the separated bacillus strain in an LB liquid medium to a logarithmic growth medium, centrifugally collecting a supernatant, utilizing the SPA liquid medium to activate pathogenic bacteria overnight, mixing a bacterial suspension with an SPA agar medium with the temperature of about 50 ℃ to prepare a solid flat plate, enabling the final concentration of the pathogenic bacteria to be 1 multiplied by 108 CFU/mL, after the culture medium is solidified, using a 6mm sterile puncher to punch, digging out a bacterial cake, adding the collected supernatant of a fermentation broth of the strain to be tested into a hole, placing the flat plate in a refrigerator with the temperature of 4 ℃ for 2 hours, placing the inoculated flat plate in a constant temperature incubator for culturing overnight according to the optimal growth temperature of an indicator bacterium, and after the culturing is finished, observing whether a clear antibacterial circle is formed around the hole on the flat plate, and recording an experimental result. Sterile water was used as a control group and each experiment was repeated at least 3 times.
The inhibition ability of bacillus to specific pathogenic fungi was tested by plate counter method against the pathogenic fungi. Pathogenic fungi include Rhizoctonia solani (Rhizoctonia solani), rhizoctonia cerealis (Fusarium graminearum), rhizoctonia cerealis (Septoria nodorum), fusarium solani (Fusarium oxysporum, tomato specialization), nicotiana tabacum (Phytophthora nicotianae), verticillium cotton (Verticillium dahliae), pyricularia pomonensis (Alternaria alternata) and Botrytis cinerea. The method comprises inoculating each pathogenic fungus to PDA culture medium, culturing until colony growth is vigorous, usually for 2-3 days, punching on colony of pathogenic fungus with 6mm sterile puncher, digging out bacterial cake, transferring to new PDA plate center, lightly pressing to make bacterial cake well contacted with culture medium, culturing bacillus to be tested in LB culture medium to logarithmic growth medium, collecting supernatant for use, when pathogenic fungus grows to a certain extent (for example, colony diameter reaches 5-10 mm), punching equidistantly on both sides of bacterial cake, adding fermentation broth supernatant of strain to be tested and fresh LB liquid culture medium (as control) of equal volume, placing inoculated plate in 28 deg.C constant temperature incubator for 3-7 days, periodically observing and recording growth condition and change of pathogenic fungus in the opposite area. At least 3 replicates were performed for each experiment.
Combining the results of a bioassay experiment and a bacteriostasis experiment of the two-spotted spider mites, screening out a bacillus strain, and naming the bacillus strain as NB379. When the spore concentration of the fermentation liquor of the strain reaches 1X 106 CFU/mL, the corrected death rate of the tetranychus urticae reaches 97.4% (+/-1.7%) (figure 2), and NB379 shows good antibacterial activity on various pathogenic fungi and pathogenic bacteria to be tested (figure 3).
Example 2
Identification of bacillus tropicalis NB 379:
(1) Colony morphological feature detection
Strain NB379 was cultivated on LB solid medium at 30 ℃ for 36h, the colony was circular, milky white, the edge protruded more than the center, and the cell morphology was rod-shaped by microscopic examination, and the specific colony morphology is shown in fig. 1 a.
Scanning Electron Microscope (SEM) observations a large number of bacterial samples were obtained by centrifugation and the supernatant removed. Then bacteria were washed three times with 1 XPBS buffer (pH 7.2-7.4) to remove impurities, bacteria were fixed with 2.5% glutaraldehyde for 3 hours, centrifuged, washed again with PBS buffer and supernatant removed, samples were dehydrated stepwise with a series of increasing concentrations of aqueous ethanol (30%, 50%, 70%, 80%, 90%), each about 15min, dehydrated with 100% ethanol twice, each 15min, continued treatment with a 1:1 mixture of ethanol and t-butanol for 15min, then 2 substitutions with pure t-butanol for 15min, coverslips containing bacterial-t-butanol suspensions were placed in a-80℃refrigerator and subsequently transferred to a lyophilizer for lyophilization, samples were attached to a sample stage on which conductive tape was attached and gold plated, and the treated samples were placed on SEM sample stages for observation and imaging. Strain NB379 was rod-shaped under scanning electron microscope (see B in fig. 1), 1.1-3.2 μm in length, and approximately 0.6 μm in diameter.
(2) Analysis of physiological and biochemical characteristics
The physiological and biochemical characteristics were examined by BIOLOG (Table 1, table 2)
TABLE 1 physiological and biochemical characteristics of strain NB 379-enzyme activity and carbon assimilation
Positive reaction + & lt- & gt negative reaction and weak positive reaction.
TABLE 2 physiological and biochemical characteristics of strain NB 379-acid production Using carbon sources
Positive reaction + & lt-negative reaction.
(3) Strain genome analysis and taxonomic identification
The genome DNA of the strain NB379 is extracted by adopting a Qiagen genome extraction kit, and the genome of the strain NB379 is subjected to sequencing analysis by adopting a second-generation high-throughput sequencing technology and combining with an Oxford Nanopore third-generation sequencing technology. To further clarify the taxonomic status of strain NB379, the applicant uploaded the sequenced genomic data to a bacterial identification platform EzBioCloud (https:// www.ezbiocloud.net /), and compared the genomic sequence of strain NB379 to known model strains using the genomic average nucleotide identity alignment tool ANI Calculator. The analysis result shows that the ANI of the strain NB379 and the tropical bacillus (Bacillus tropicus) model strain N24 is 96.72%, and the ANI values of the model strains similar to the rest are less than 92%. Based on the results that ANI values between strains of the same species are 95% or more, strain NB379 was identified as Bacillus tropicalis (Bacillus tropicus) according to the criteria of microbiological classification.
Example 3
Determination of acaricidal Activity of Strain NB379
A fermentation broth of Bacillus tropicalis NB379 was prepared using the fermentation broth of example 1, and fermentation broths with different spore concentrations were prepared by mixing different volumes of fresh fermentation broth. The poisoning activity of these fermentation broths against spider mites was examined by a spider mite slide dipping method, and the results obtained are summarized in table 3. The insecticidal probability of fermentation broths with different concentrations is calculated by using SPSS 26.0 data processing software, and a unit model equation of PROBIT (P) = -2.631+2.652X (variable X is converted by using a logarithm with a base of 10) is obtained, and a Pearson model fitting goodness test of X2 = 0.490 and P = 0.135 shows that the model fitting is good. The experimental result shows that the half lethal concentration LC50 of the fermentation liquor of the tropical bacillus NB379 on the tetranychus urticae is 9.8 mu L/mL.
TABLE 3 poisoning Activity of Bacillus tropicalis NB379 fermentation broths against Tetranychus urticae
According to the experimental steps, the results of the biological measurement of the fermentation liquid of the tropical bacillus NB379 with different concentrations on the Panonychus citri (panonyichus citri) and the tetranychus cinnabarinus (Tetranychus cinnabarinus) are counted, and the LC50 values of the fermentation liquid of the NB379 on the Panonychus citri and the tetranychus cinnabarinus are respectively 22.7 mu L/mL and 16.3 mu L/mL by using SPSS26.0 data processing software.
Example 4
Growth-promoting ability assay of strain NB379
(1) Ability to produce indole-3-acetic acid
The NB379 strain was inoculated into LB liquid medium containing 5mM tryptophan and cultured in a shaker at 30℃and 200rpm for 12 hours, the culture broth was taken out, centrifuged at 10,000rpm for 5 minutes, the supernatant was separated, the supernatant was mixed with Salkowski colorimetric solution (50 mL 15% HCIO4 mixed with 1mL 0.5M FeCl3) at a ratio of 1:4, and after standing for 30 minutes in the dark, the absorbance at 530nm was measured using a spectrophotometer, and the blank medium was used as a control. The absorbance was converted to the actual IAA concentration (g/L) according to the IAA standard curve prepared in advance. The results showed that the NB379 strain broth was pink after reaction with Salkowski reagents and had IAA yields of 68.4.+ -. 5.0mg/L.
(2) Phosphorus dissolving ability
Strain NB379 was inoculated on NBRIP medium plates to which 0.5% Ca3(PO4)2 (inorganic phosphorus source) or lecithin (organic phosphorus source) was added, and the plates were placed in a 30 ℃ incubator for 10 days, and colony growth was observed. The results showed that a transparent halo formed around the colony, indicating that the NB379 strain had phosphorus-dissolving ability for both inorganic and organic phosphorus.
(3) Capability of producing ammonium nitrogen
Strain NB379 was inoculated into a test tube containing 10mL of peptone water, cultured at 30 ℃ for 3 days, 500 μl of nessler reagent was added to the culture broth, and a color change was observed. After the Neschler reagent is added, the NB379 strain culture solution presents brown yellow, which shows that the strain has the capability of producing ammonium nitrogen.
(4) Catalase Activity
A loop of fresh bacterial culture was mixed with 50. Mu.L of 3% (v/v) hydrogen peroxide solution on a sterile slide and the reaction was observed after incubation for 1min at room temperature. The NB379 strain culture forms bubbles after mixing with hydrogen peroxide, indicating that oxygen evolution occurs, which is a catalase-positive reaction, demonstrating that the NB379 strain is capable of producing catalase.
(5) Capacity to produce mesophilic ferrite
Inoculating single colony of strain NB379 into MSA liquid culture medium, culturing at 30deg.C and 200rpm for 48 hr, taking culture solution, centrifuging at 10,000r/min for 10min, separating supernatant, mixing supernatant with CAS detection solution at volume ratio of 1:1, standing at 30deg.C under dark condition for 1 hr, and measuring OD630. The absorbance (Ar) of the mixture of the uninoculated blank medium and the CAS test solution was used As a control, and the absorbance (As) of the mixture of the strain fermentation broth supernatant and the CAS test solution was compared with that of the strain fermentation broth, whereby the lower the ratio of As/Ar, the higher the siderophilic producing ability of the strain was evaluated. In general, bacteria with a high ferriphilic capacity have an As/Ar value of less than 0.5. The calculation formula of the ferrite activity unit (Siderophore Unit, SU) is as follows:
The results showed that the MSA medium of NB379 strain had a SU of 0.952.+ -. 0.027, an As/Ar value of 0.048.+ -. 0.012, and a SU of 48h fermentation of 0.901.+ -. 0.019, and an As/Ar value of 0.099.+ -. 0.024. The NB379 strain showed progressively lower ferrite-producing ability with increasing time, but its ferrite-producing ability remained at a higher level even after 48 h.
(6) Testing of the ferrite-producing species:
① Shenker's test (detection of Carboxylic acid type siderophores) 1mL of strain NB379 fermentation broth supernatant was taken, 1mL of CuSO4 solution (750. Mu.M) and 2mL of acetate buffer (pH 4.0) were added, after mixing well, scanning was performed in the 190-280nm wavelength range using an ultraviolet spectrophotometer, and whether a specific absorption peak occurred was recorded.
② Arnow's experiment (detection of catechol type of mesophilic) 1mL of strain NB379 broth supernatant was taken, 1mL of 0.5M HCl and 1mL of 10% sodium molybdate-sodium nitrite solution were added, the color change of the solution was observed, if yellow color appeared, indicating the possible catechol structure was present, then 1mLNaOH solution (1M) was added, if the solution became red and the solution was not discolored for at least 1h, indicating the presence of catechol type of mesophilic. The characteristic absorption peak at 510nm was detected using an ultraviolet spectrophotometer.
③FeCl3 Experiments (differentiating between hydroxamic acid and catechol type of mesophilic) were carried out by taking 1mL of the supernatant of the strain NB379 fermentation broth, adding 1-5mL of FeCl3 solution (2%), observing the colour change of the solution, which may be hydroxamic acid type of mesophilic if it became red immediately, and catechol type of mesophilic if it became red or violet after adding more than 1mL of FeCl3 solution.
The results indicate that the ferrite produced by strain NB379 is catecholic.
(7) Experiment of saline-alkali tolerance
① Salt tolerance determination
The method comprises the steps of taking an LB liquid culture medium without NaCl as a basic culture medium, respectively adding NaCl to the final concentration of 1%, 3%, 5%, 7% and 9%, inoculating bacterial liquid with the OD600 of 0.6-0.8 into LB culture media with different salt concentration gradients according to the inoculation amount of 3%, setting 3 parallel samples for each salt concentration, culturing for 12 hours in a shaking table with the speed of 180rpm at 30 ℃, observing the growth condition of the bacterial liquid, and measuring the OD600. The results show that the strain NB379 grows well in the concentration range of 1% -9% NaCl, and shows stronger salt tolerance.
② Determination of pH tolerance
Adjusting the pH value of LB liquid culture medium to 3.0-12.0, setting 3 parallel samples for each pH value, inoculating bacterial liquid with OD600 of 0.6-0.8 into LB culture medium with different pH gradients according to 3% inoculum size, culturing in a shaking table with speed of 180rpm at 30 ℃ for 12h, and measuring the pH values of OD600 and the culture liquid. The results showed that strain NB379 grew well in the pH range 5.0-10.0, exhibiting good pH tolerance.
③ Determination of saline-alkali tolerance
Adopting an LB liquid culture medium without NaCl as a basic culture medium, adding a mixed salt solution of NaCl, na2SO4、NaHCO3 and Na2CO3 according to a molar mass ratio of 1:9:9:1, setting 5 total salt concentration gradients of 40mM-200mM, inoculating bacterial liquid with OD600 of 0.6-0.8 into culture media with different total salt concentration gradients according to an inoculation amount of 3%, setting 3 parallel samples for each gradient, and after culturing for 12 hours in a shaking table with the speed of 180rpm at 30 ℃, observing the growth condition of the bacterial liquid and measuring the pH values of the OD600 and the culture liquid. The result shows that the strain NB379 grows well in the saline-alkali concentration range of 40mM-200mM, and has better saline-alkali resistance.
(8) Heavy metal resistance test
Adding metal ions with different concentrations into MSA culture medium, wherein the final concentrations are 0, 0.5, 1,2, 4, 8, 16, 32 and 64mM respectively, taking a treatment group without adding metal ions as a blank control, inoculating strain NB379, culturing for 24 hours at 30 ℃ and 180rpm, streaking the culture solution on a solid plate, culturing for 12 hours at a constant temperature of 30 ℃, and observing the growth state of the strain. The metal ions include Cu2+、Mn2+、Zn2+、Cr3+、Fe2+、Pb2+、Cd2+、Ni2+ and the like.
The experimental results show (as shown in fig. 4) that strain NB379 shows a significant difference in the tolerance to different heavy metal ions in MSA medium, a maximum tolerance concentration of 4.0mM for copper ions (Cu2+) at which the growth of strain NB379 is slightly affected but still active, a maximum tolerance concentration of 64mM for manganese ions (Mn2+) well above the other tested heavy metal ions, a maximum tolerance concentration of 2mM for chromium ions (Cr3+), a heavy metal toxic to many organisms, and a maximum tolerance concentration of NB379 at which growth is maintained for both lead ions (Pb2+) and cadmium ions (Cd2+) of 32mM, indicating that strain NB379 has a high tolerance to both heavy metals, and a maximum tolerance concentration of 12mM for nickel ions (Ni2+) shows a good tolerance.
Example 5
Living promotion of strain NB379 on tomato and Arabidopsis thaliana
(1) Tomato seed-derived positive effect of strain NB379
Selecting tomato seeds with consistent and full shape and size, soaking the tomato seeds in 15% hypochlorous acid solution for 15min for surface disinfection, soaking the seeds in a culture dish containing sterile water for 3 times, selecting vermiculite as a culture medium, sowing the tomato seeds to the surface of the sterilized vermiculite for less than 2 cm, keeping the water content of the medium to be 80% by weighing method every 3 grains of the culture medium, ensuring proper water condition to promote seed germination and seedling growth, selecting seedlings with uniform growth condition after about two weeks of seedling raising, transplanting the seedlings into a new flowerpot (18.8 cm, 13.5cm and 18.5cm of inner diameter and height of the upper bottom and lower bottom of the pot respectively), keeping 1 seedling in each pot, starting root irrigation treatment on 7 days after transplanting, wherein the sample groups are respectively NB379 strain MSA fermentation liquor 5 times diluent (treatment group 1#), 15 times diluent (treatment group 2#), 50 times diluent (treatment group 3#), and deionized water as a control group, and 100mL of each pot treatment liquid every 6 pot is irrigated for 7 days. After 4 weeks of root irrigation treatment, the growth index of tomato seedlings, including plant height, fresh weight of plants, dry weight of plants, etc. was determined.
The results showed (as shown in fig. 5) that the tomato seedling height of treatment group 1# was significantly higher than that of the control group and other treatment groups, 46% higher than that of the control group, and the stem thickness of all treatment groups was increased, wherein the stem thickness of treatment group 1# was roughly higher than that of the other treatment groups and 1.14 times that of the control group, and the fresh weight (45.85.+ -. 0.77 g) and dry weight (0.86.+ -. 0.03 g) of tomato seedling of treatment group 1# were 1.38 times and 2.03 times that of the control group, respectively. Overall, strain NB379 broth at 5-fold dilution, i.e. 1 x 106 CFU/mL broth, has a significant promoting effect on tomato seedling growth, in particular in terms of increase in plant height, stem thickness, fresh weight and dry weight.
(2) Protoffee of strain NB379 on Arabidopsis thaliana
Placing Arabidopsis seeds in 15% hypochlorous acid solution, soaking for 15min for surface disinfection, washing with sterile water, placing the seeds at 4deg.C for vernalization for 3 days, pouring MS solid culture medium on one side of a cell culture dish, uniformly placing 30 Arabidopsis seeds subjected to vernalization thereon after the culture medium is left, and placing appropriate amount of strain NB379 bacterial blocks on the other side of the cell culture dish to release Volatile Organic Compounds (VOCs). Dishes without bacterial blocks were set up as a control group to evaluate the effect of VOCs of strain NB379 on arabidopsis growth. The dishes were sealed with a sealing film and placed in a 25 ℃ greenhouse for cultivation, and photoperiod treatment of 16h light and 8h darkness per day was performed to simulate natural light dark cycle. Each group was repeated 3 times. After 2 weeks of cultivation, the germination and growth of the Arabidopsis seeds were observed and recorded.
As shown in FIG. 6, the Arabidopsis plants in the treated group had a higher density, indicating that the seed germination rate was improved under the effect of the VOCs of strain NB 379. Meanwhile, the leaf area of the Arabidopsis thaliana in the treatment group is larger, the color is fresh green, which indicates that the Arabidopsis thaliana grows healthier and more vigorous,
(3) Strain NB379 relieves stress of heavy metals on arabidopsis seed germination
MS solid culture medium is added into a 24-well plate, and after the solid culture medium is solidified, 1 arabidopsis seed subjected to vernalization is planted in each well. The sample groups are respectively a control group without adding any treatment substance, NB379 strain MSA fermentation broth treatment groups with different dilutions for evaluating the direct influence on seed germination, heavy metal ion stress groups with different concentrations including Cr3+、Cd2+、Ni2 < + > and the like for simulating heavy metal pollution environment, and a strain NB379 and heavy metal ion co-treatment group for evaluating the relieving effect of the strain NB 379. Each set of samples was set up in 4 sets of parallelism. The treated plates were placed in a greenhouse for 2 weeks of continued culture, and the germination and growth conditions of arabidopsis seeds of each sample group were observed and recorded. According to the results shown in FIG. 7, the heavy metal ions inhibit the germination of Arabidopsis seeds in the concentration range of 0.5mM-8.0mM, strain NB379 can relieve the stress of low concentration heavy metal ions (0.5 mM-1.0 mM) on seed germination, and the germination rate and growth rate of Arabidopsis seeds are improved with the increase of the concentration of the fermentation supernatant of strain NB 379.
(4) Strain NB379 relieves stress of heavy metals on arabidopsis seedling growth
Adding MS solid culture medium into 24-well plate, after it is solidified, sowing 1-2 pieces of spring-treated Arabidopsis seeds in each well, sealing 24-well plate, placing them in 25 deg.C greenhouse, culturing, implementing photoperiod treatment of 16 hr light and 8 hr darkness every day, after culturing for 1 week, removing excess Arabidopsis seedlings, ensuring that 1 seedling with similar growth vigor is placed in each well, and making several treatments including control group, NB379 strain MSA fermentation liquor treatment group with different dilutions, heavy metal ion (Cr3+、Cd2+、Ni2 + etc.) stress group with different concentrations and common treatment group of strain NB379 and heavy metal ion, every treatment group is set into 4 groups. The treated plates were placed in a greenhouse for 2 weeks and the growth of arabidopsis was observed and recorded for each treatment group. As a result, as shown in FIG. 8, in the control group, the vigor of Arabidopsis seedlings was decreased with the increase of the concentration of heavy metal ions, with the stress effect of 8mM heavy metal ions being most remarkable. After being treated by NB379, the growth condition of arabidopsis seedlings under heavy metal stress conditions is obviously improved compared with that of untreated stress groups, particularly, the growth condition of high-concentration NB379 fermentation liquor supernatant, namely treated group 1#, is similar to that of seedlings without aggravated metal ions in leaf area and color of arabidopsis under the stress concentration of 1.0-4.0 mM of heavy metal ions, and the strain NB379 can effectively relieve the negative influence of heavy metals such as Cr3+、Cd2 + on the growth of seedlings.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.