Two strains of Sphingomonas and their application

Abstract

The present invention discloses two strains of Sphingomonas and applications thereof. Sphingomonas sp.MMSM20, whose deposit number is: GDMCC No: 62519. Sphingomonas sp.MMSM24, whose deposit number is: GDMCC No: 62520. The rhizosphere growth-promoting bacteria Sphingomonas sp. MMSM20 and MMSM24 can produce iron carriers and secrete IAA, and have a dual growth-promoting effect compared with the reported plant rhizosphere growth-promoting bacteria. Therefore, the two new species of Sphingomonas sp. MMSM20 and MMSM24 disclosed in the present invention can be used to improve the stress resistance of plants and promote plant growth, and have broad application prospects in the preparation of plant stress-resistant and growth-promoting microbial agents, organic bio-fertilizers, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and specifically relates to two new species of Sphingomonas with growth-promoting functions and applications thereof. Background Art

[0002] There are a large number of beneficial microorganisms in the rhizosphere soil of plants, which play an important role in promoting plant growth, increasing crop yields and preventing and controlling plant diseases. These beneficial microorganisms are called growth-promoting rhizobacteria (PGPR). The growth-promoting function of PGPR mainly involves three aspects. The first is to promote the absorption of nutrients by plants by producing iron carriers, solubilizing phosphorus, potassium and fixing nitrogen; the second is to regulate plant growth by producing auxin (IAA), gibberellins, ACC deaminase or cytokinins; the third is to prevent and control plant diseases by secreting antibiotics, extracellular enzymes or lipopolysaccharides. PGPR can promote plant growth and increase crop yields through one or more mechanisms, reduce the use of chemical fertilizers and pesticides and production costs, and is of great significance to promoting the green development of ecological agriculture.

[0003] Iron is an essential mineral nutrient element in the growth of plants and microorganisms, and plays an important role in plant photosynthesis, respiration and nitrogen metabolism. 3+ ) has a very low solubility, and the iron in the soil is in the form of Fe 3+ and insoluble oxidation states, soluble Fe 2+ The content is very low and difficult to be absorbed by plants and microorganisms. Most plants in agricultural production are generally in a state of iron deficiency. In order to adapt to the low-iron environment, microorganisms have evolved several high-affinity iron uptake systems to solve the problem of low iron content available to microorganisms in the environment. Among them, iron carriers are an important way for microorganisms to absorb iron.

[0004] Siderophores are a class of low-molecular-weight substances secreted by plant rhizosphere microorganisms. They can efficiently bind to iron in the soil environment, converting insoluble iron in the soil into effective iron that can be used by plants, thereby enhancing the tolerance of plants to environmental conditions. In addition, in addition to chelating iron to meet the plant's iron needs, siderophores secreted by plant rhizosphere bacteria can also reduce the amount of iron that can be used by pathogenic microorganisms, thereby promoting plant growth. Therefore, in agricultural production, the purpose of promoting plant growth and increasing crop yields can be achieved by inoculating siderophore-producing bacteria in the rhizosphere of plants. Continuously exploring new PGPR resources with multiple growth-promoting mechanisms and using them to prepare microbial fertilizers are of great significance for improving soil fertility and promoting the sustainable development of green agriculture. Summary of the invention

[0005] The first object of the present invention is to provide two new species of Sphingomonas sp. MMSM20 and MMSM24 that can efficiently produce siderophores and IAA. Sphingomonas sp. MMSM20 was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on June 7, 2022, with an address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, zip code: 510070, and its accession number is: GDMCC No: 62519. Sphingomonas sp. MMSM24 was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on June 7, 2022, with an address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, zip code: 510070, and its accession number is: GDMCC No: 62520.

[0006] The second object of the present invention is to provide the use of Sphingomonas MMSM20 or MMSM24 in the preparation of biological preparations that promote plant growth and / or improve plant stress resistance.

[0007] The third object of the present invention is to provide the use of Sphingomonas MMSM20 or MMSM24 in producing siderophore and / or IAA.

[0008] The fourth object of the present invention is to provide a biological preparation for promoting plant growth and / or improving plant stress resistance, which contains Sphingomonas MMSM20 and / or MMSM24 as main active ingredients.

[0009] Preferably, the biological agent can be a microbial agent or an organic biological fertilizer.

[0010] The present invention has the following advantages over the prior art:

[0011] 1) The two new species of Sphingomonas provided by the present invention are new species of the genus Sphingomonas that are different from other plant growth-promoting bacteria.

[0012] 2) The ability of the rhizosphere growth-promoting bacteria, Sphingomonas MMSM20 and MMSM24, to produce siderophores in R2A liquid medium for 2 days was 68.8% and 64.9%, respectively, which was stronger than that of most reported bacteria.

[0013] 3) The rhizosphere growth-promoting bacteria Sphingomonas MMSM20 and MMSM24 can produce both siderophores and secrete IAA, and have a dual growth-promoting effect compared with the reported plant rhizosphere growth-promoting bacteria. Therefore, the two new species of Sphingomonas MMSM20 and MMSM24 disclosed in the present invention can be used to improve the stress resistance of plants and promote plant growth, and have broad application prospects in the preparation of plant stress resistance and growth-promoting microbial agents, organic biofertilizers, etc.

[0014] Sphingomonas sp.MMSM20 was deposited on June 7, 2022 in the Guangdong Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and its deposit number is: GDMCC No: 62519.

[0015] Sphingomonas sp.MMSM24 was deposited on June 7, 2022 in the Guangdong Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and its deposit number is: GDMCC No: 62520. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figures show the morphologies of two new species of Sphingomonas MMSM20 (A) and MMSM24 (B) of the present invention on CPG plates.

[0017] Figure 2 The images are transmission electron microscopic observations of cells of two new species of Sphingomonas MMSM20 (A) and MMSM24 (B) of the present invention, with a scale of 5 μm.

[0018] Figure 3 This is the phylogenetic tree of the 16S rRNA gene of two new species of Sphingomonas, MMSM20 and MMSM24.

[0019] Figure 4 This is the phylogenetic tree of the genomes of two new species of Sphingomonas, MMSM20 and MMSM24.

[0020] Figure 5 The transparent zones produced by two new species of Sphingomonas MMSM20 (A) and MMSM24 (B) of the present invention on a CAS plate.

[0021] Figure 6 The ability of two new Sphingomonas strains, MMSM20 and MMSM24, to produce siderophores and IAA was analyzed. DETAILED DESCRIPTION

[0022] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0023] The present invention is further described below in conjunction with the embodiments. The experimental methods in the following embodiments are conventional experimental methods unless otherwise specified. The experimental reagents and consumables described in the following embodiments are from conventional biochemical reagent companies unless otherwise specified.

[0024] Example 1 Isolation and purification of two new species of Sphingomonas MMSM20 and MMSM24

[0025] 1. Isolation and screening culture medium

[0026] CPG agar medium: 10.0 g of bacteriological peptone, 1.0 g of acid hydrolyzed casein, 5.0 g of glucose (or 5 mL of glycerol), dissolved in distilled water to 1 L, pH 6.5-7.0, sterilized by wet heat at 121°C for 20 min, and set aside.

[0027] 2. Experimental steps

[0028] (1) Sample collection: Soil samples from a perennial tomato-growing area were collected from Maoming City, Guangdong Province, placed in sampling bags, and brought back to the laboratory to air-dry naturally at room temperature.

[0029] (2) Preparation of soil gradient dilution: Weigh 10 g of soil and add it to a sterile Erlenmeyer flask with glass beads and 90 mL of sterile water. Incubate at 200 rpm on a shaker at 30°C for 30 min. After standing for 5 min, take 1 mL of the supernatant and add it to a test tube with 9 mL of sterile water. Then perform gradient dilutions to 10. -2 -10 -7 g / mL soil suspension for later use.

[0030] (3) Bacterial isolation: Take 100 μL of the diluted soil bacterial suspension (10 -2 -10 -7 g / mL) was evenly coated on the CPG medium plate with a disposable plastic coating rod, and three replicates were set for each concentration. After coating, the plate was inverted and placed in a 30°C constant temperature incubator for culture. The growth of the colonies on the plate was observed every other day, and strains of different forms that produced transparent circles were selected and streaked on the CPG solid plate, and the strains were purified, and finally strains MMSM20 and MMSM24 were isolated.

[0031] Example 2. Classification and Identification of Strain MMSM20 and MMSM24

[0032] The isolated strains were identified with reference to the Manual of Identification of Common Bacterial Systems (Edited by Dong Xiuzhu, Science Press).

[0033] (1) Phenotypic characteristics:

[0034] The strains MMSM20 and MMSM24 were inoculated on CPG medium using the three-zone streak method, and their colony morphology was observed after culturing at 30°C for 3 days. The cultured colonies were picked for Gram staining and morphological observation using an optical microscope. Transmission electron microscopy samples were prepared and the cell morphology of the strains was observed using a Hitachi H7650 transmission electron microscope. Tests such as nitrate reduction, gelatin hydrolysis, indole production, β-glucosidase, arginine dihydrolase, urease, and glucose oxidation were performed using API20NE reagent strips (Mérieux Biotech, France).

[0035] The strains MMSM20 and MMSM24 were milky white on CPG medium, and the single colonies were round, smooth, with neat edges and no fluidity ( Figure 1 The strains MMSM20 and MMSM24 were Gram-negative, and the cells were short rod-shaped and without flagella under transmission electron microscopy ( Figure 2 ). The hydrolysis of gelatin, β-glucosidase, arginine dihydrolase and urease were positive, while nitrate reduction, indole production and glucose oxidation were negative (Table 1).

[0036] Table 1 Phenotypic characteristics of strains MMSM20 and MMSM24

[0037]

[0038]

[0039] (2) System evolution analysis:

[0040] DNA of strains MMSM20 and MMSM24 was extracted by boiling method, and the 16S rDNA gene sequences of MMSM20 and MMSM24 were amplified using bacterial 16S rDNA specific primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGACTTAACCCCAATCGC-3′) and Taq enzyme. The amplified products were analyzed by electrophoresis to produce a band of about 1500b. After gel cutting and recovery, the band was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for Sanger sequencing. The sequenced sequences were spliced ​​by seqman software to obtain 16S rDNA sequences and submitted to the EzBioClode website (https: / / www.ezbiocloud.net / ) for identification. MEGA 10 software was used to construct the NeighborJoining phylogenetic evolutionary tree based on 16S rRNA sequences, and the calculation model was Kimura 2-paremeter. The full length of the 16S rRNA gene of strains MMSM20 and MMSM24 obtained by Sanger sequencing was 1338 bp and 1357 bp respectively (SEQ ID NO. 1 and 2). Sequence comparison revealed that the similarity between strains MMSM20 and MMSM24 and the published model bacteria sequences was lower than the critical value of 98.65% for species differentiation, and the model bacteria with the highest similarity was Sphingomonas dokdonensis DSM 21029 T The NJ phylogenetic tree constructed based on the 16S rRNA gene sequence showed that strains MMSM20 and MMSM24 belonged to the genus Sphingomonas and were closely related to Sphingomonas dokdonensis DSM 21029. T , Sphingomonas jeddahensisG39 T , Sphingomonas mucosissima DSM 17494 T The closest relatives of the model bacteria are Figure 3 In summary, based on the 16S rDNA sequence and morphological characteristics, strains MMSM20 and MMSM24 were potential new species of the genus Sphingomonas.

[0041] >MMSM20 (SEQ ID NO.1)

[0042]

[0043] >MMSM24(SEQ ID NO.2)

[0044]

[0045] (3) Comparative genomics analysis:

[0046] The genomic DNA of strains MMSM20 and MMSM24 was extracted using a bacterial genomic DNA extraction kit (Magen), and then the genome sequencing was completed by Shanghai Meiji Biotechnology Co., Ltd. after verification by 16S rRNA gene amplification. The construction of paired-end libraries and Illumina Hiseq 2500 sequencing were commissioned to this company. The off-machine data were assembled using SPAdes (3.13.0) software for genome sequence splicing. The genome sequences of the published model species to be used for comparative analysis were downloaded from the NCBI database, and the average nucleotide identity (ANI) of each strain was calculated based on the genome sequence using FastANI. The DNA-DNA hybridization value (dDDH) based on the genome sequence was calculated using the online tool GGDC 2.1 (http: / / hhdc.dsmz.de / home.php). The genome sizes of strains MMSM20 and MMSM24 were approximately 5.03Mb and 5.16Mb, respectively, and the GC contents were 66.62 and 66.36mol%, respectively. The results of the analysis based on the genome sequence are shown in Table 2. The ANI values ​​and dDDH values ​​of strains MMSM20 and MMSM24 compared with the closest published type strains were 78.6% to 81.1% and 19.5% to 21.7%, respectively, which are significantly lower than the currently recognized thresholds of 95% to 96% ANI and 70% dDDH as species boundaries ( Figure 4 ). These results indicate that strains MMSM20 and MMSM24 represent two different new species of the genus Sphingomonas. The present invention named the strain MMSM20 as Sphingomonas sp. MMSM20, and it was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on June 7, 2022, with an address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, with a zip code of 510070, and its accession number is: GDMCC No: 62519. The present invention named the strain MMSM24 as Sphingomonas sp. MMSM24, and it was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on June 7, 2022, with an address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, with a zip code of 510070, and its accession number is: GDMCC No: 62520.

[0047] Table 2 Comparative genome analysis of strains MMSM20 and MMSM24 with 30 published closely related model species

[0048]

[0049]

[0050]

[0051] Example 3: Analysis of the ability of two new Sphingomonas strains, MMSM20 and MMSM24, to produce siderophores and IAA

[0052] (1) Qualitative determination of the ability of strains MMSM20 and MMSM24 to produce siderophores

[0053] Two new strains of Sphingomonas MMSM20 and MMSM24 were activated on CPG solid plates and cultured at 30°C for 2 days. The activated strains were inoculated onto R2A plates with a sterilized cotton swab. Three colonies were inoculated on each plate and cultured at 30°C for 2 days. 15-20 mL of CAS detection solution was added to the cultured colony plates. The color changes around the colonies were observed within 1 hour after solidification, and the iron carrier secretion was recorded.

[0054] R 2 A agar culture medium (g / L Qingdao Haibo Biological HB0167-5): 0.5g yeast extract powder, 0.5g peptone, 0.5g casein hydrolysate, 0.5g glucose, 0.5g agar powder, 0.5g soluble starch, 0.3g dipotassium hydrogen phosphate, 0.024g anhydrous magnesium sulfate, 0.3g sodium pyruvate, 15g agar. When used, weigh 15.2g of the above culture medium, heat and dissolve it in 1L distilled water, sterilize it with high-pressure steam at 121℃ for 15min, and set aside.

[0055] CAS detection solution (1L): Chrome azurol S (chrome azurol) 60.5 mg, CTAB 72.9 mg, PIPES (piperazine-1,4-diethanesulfonic acid), 30.24 g, FeCl 3 6H 2 O 1 mM, HCl, 10 mM, agarose 0.9% (w / v).

[0056] (2) Quantitative determination of the ability of strains MMSM20 and MMSM24 to produce siderophores

[0057] Single colonies of strains MMSM20 and MMSM24 on the CPG solid plate were picked and inoculated into a conical flask containing 50 mL of CPG liquid culture medium, and then placed on a horizontal shaker at 30°C and 200 rpm for shaking culture for 2 days. The sterilized CPG culture medium without inoculation of MMSM20 and MMSM24 was used as a control to quantitatively determine the amount of iron carriers produced by the strains. Three biological replicates were set up for the experiment.

[0058] The iron carrier determination method is to take 1mL of cultured bacterial liquid into an EP tube, centrifuge at 12000rpm for 5min, take 100μL of supernatant into a 96-well plate, add an equal volume of CAS detection solution and mix evenly, let it stand for 1h, and then use an enzyme marker to measure the absorbance value of 630nm wavelength As, and add ddH 2 O is the control and adjusted to 0. Take an equal volume of uninoculated sterilized CPG medium and mix it with an equal volume of CAS detection solution. The absorbance value measured is the reference value Ar. Finally, the amount of iron carrier produced is calculated according to the formula. The amount of iron carrier produced (%) = (Ar-As) / Ar*100. If the calculated result is less than 10%, it is considered that the iron carrier secretion is negative.

[0059] The IAA content of the strains was determined using R2A liquid medium containing L-tryptophan (100 mg / mL). The specific method was to inoculate strains MMSM20 and MMSM24 into R2A liquid medium containing L-tryptophan (100 mg / mL) and culture them on a shaker at 28°C and 180 rpm for 2 days. 1 mL of the cultured bacterial solution was centrifuged at 10000 rpm for 5 min in an EP tube, 50 μL of the supernatant was taken into a 96-well plate, and 50 μL of Salkowski colorimetric solution (50 mL 35% HClO 4 +1mL 0.5M FeCl 3 ). Add 50 μL of 50 mg / L IAA colorimetric solution as a positive control. Place the 96-well plate at room temperature and away from light for 30 minutes, then use an ELISA reader to measure the OD530 absorbance. Use gradient dilutions of 50 mg / L IAA as a standard to make a standard curve, and finally calculate the IAA content of the strain according to the standard curve equation.

[0060] The results of the siderophore production of the new Sphingomonas species MMSM20 and MMSM24 on plates are shown in Figure 5 , it can be seen that the color around the colony changes from blue to yellow, forming an obvious halo, which indicates that strains MMSM20 and MMSM24 have the ability to produce siderophores. The ability of the two strains to produce siderophores and IAA was further quantitatively evaluated, and the results are shown in Figure 6 After 2 days of inoculation and culture, the iron carrier production capacity of strains MMSM20 and MMSM24 was 68.8% and 64.9%, respectively, and the amount of IAA produced was 18.5 mg / L and 18.2 mg / L, respectively.

Claims

1. Sphingomonas Sphingomonas sp.) MMSM20, the deposit number is: GDMCC No: 62519.

2. Sphingomonas Sphingomonas sp.) MMSM24, the deposit number is: GDMCC No: 62520.

3. Use of the Sphingomonas MMSM20 described in claim 1 or the Sphingomonas MMSM24 described in claim 2 in producing siderophores and / or IAA.

4. A biological agent for promoting plant growth and / or improving plant stress resistance, comprising the Sphingomonas MMSM20 according to claim 1 or the Sphingomonas MMSM24 according to claim 2 as a main active ingredient.

5. The biological agent according to claim 4, characterized in that The biological agent is a microbial agent or an organic biological fertilizer.

Images