Endophytic bacterium pantoea agglomerans SE16 and application thereof

Abstract

The pantoea agglomerans SE16 is classified and named as pantoea agglomerans, and is preserved in the China General Microbiological Culture Collection Center on October 17, 2024, the address is Institute of Microbiology, Chinese Academy of Sciences, No.3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 32246. The bacterial strain is applied to strengthening phytoremediation of heavy metal pollution, improving soil nutrients, promoting plant growth and strengthening phytoremediation of pollutants. The strain has the functions of dissolving phosphorus, fixing nitrogen, producing iron carriers, producing indoleacetic acid, ACC deaminase and cellulase, resisting acid, resisting salt and the like, and also can tolerate various heavy metals, polycyclic aromatic hydrocarbons and combined pollution of the heavy metals and the polycyclic aromatic hydrocarbons; by promoting the growth of the sudangrass, improving the photosynthesis of the sudangrass, relieving the oxidation pressure of the sudangrass and changing the composition of soil microbial communities, the toxicity of chromium to the sudangrass is relieved, and the method has practical popularization and application values.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a strain of endophytic bacteria, Pantotheca acuminata (…). Pantoea agglomerans SE16 and its applications. Background Technology

[0002] With the rapid development of industrial and agricultural production and the accelerating pace of urbanization, heavy metal pollution in soil has become one of the most harmful forms of environmental pollution. Phytoremediation technology for heavy metal-contaminated soil has become a hot topic in soil remediation due to its low cost and environmental friendliness. However, several problems exist in its implementation, such as the low bioavailability of heavy metals in soil, which hinders plant absorption; and the slow growth and small biomass of super-heavy plants, which severely affect the efficiency of phytoremediation. Therefore, promoting the growth of remediation plants and improving their remediation efficiency are key aspects of phytoremediation technology.

[0003] Microorganisms can enhance plant remediation of heavy metals through mechanisms such as altering the activity of heavy metals, promoting plant growth, and increasing plant resistance to heavy metals. Endophytic bacteria, existing around plant roots or within plant tissue cells, can more effectively coordinate population size and adapt to the environment and host. In particular, endophytic bacteria with growth-promoting functions can enhance the plant's ability to remediate heavy metal pollution through symbiotic systems with plants. Plant endophytic bacteria can promote plant growth under heavy metal stress through biological nitrogen fixation, phosphorus solubilization, secretion of plant hormones, organic acids, chelates, biosurfactants, siderophores, specific enzymes (ACC deaminase and chitinase, etc.), antibiotics, and by enhancing plant photosynthesis and biomass, thereby reducing the toxicity of heavy metals to plants and enhancing their resistance to heavy metals.

[0004] Currently, compound pollution is a prevalent form of soil pollution. Due to the complexity of the soil environment and the compound nature of soil pollution, achieving breakthroughs in the remediation of compound-polluted soils through the integration of various remediation technologies and methods has become a current research hotspot. Soil salinization leads to a significant reduction in soil fertility and is a major obstacle to increasing plant planting area and yield, posing a significant challenge to agricultural and pastoral development. Therefore, screening plant growth-promoting bacteria tolerant to multiple pollutions and resilient to harsh environments can further expand their role in promoting plant growth and colonization under abiotic stress, thereby improving plant resistance, promoting plant growth, and even remediating and improving stressed environments. However, no publicly reported studies have been published to date. Summary of the Invention

[0005] In view of the above situation and to overcome the shortcomings of the prior art, the purpose of this invention is to provide an endophytic bacterium that clumps together (Pantotheca acuminata). Pantoea agglomerans SE16 and its applications; this strain is classified and named Pantotheca cumulus. Pantoea agglomeransIt was deposited on October 17, 2024, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32246.

[0006] This strain has been applied in enhancing phytoremediation of heavy metal pollution.

[0007] Applications of this strain in soil improvement, plant growth promotion, and enhanced phytoremediation of pollutants.

[0008] This strain is an endophytic bacterium with functions including phosphorus solubilization, nitrogen fixation, siderophore production, indoleacetic acid production, ACC deaminase production, cellulase production, acid and salt tolerance, and can also tolerate multiple heavy metals (Cr). 6+ Pb 2+ Mn 2+ As 3+ This strain can mitigate the toxicity of chromium to Sudan grass by promoting its growth, increasing its photosynthesis, reducing its oxidative stress, and altering the composition of the soil microbial community. It is expected to be developed into a biological agent with broad application prospects in soil improvement, plant growth promotion, and enhanced phytoremediation of pollutants, and has practical application value. Attached Figure Description

[0009] Figure 1 This is a comparative diagram showing the effect of the present invention on the chlorophyll content of Sudan grass; Figure 2 This is a comparative diagram showing the effect of the present invention on the malondialdehyde (MDA) content (aerial parts) of Sudan grass; Figure 3 This is a comparative diagram showing the effect of the present invention on the malondialdehyde (MDA) content (underground part) of Sudan grass. Detailed Implementation

[0010] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances.

[0011] The present invention is illustrated by the following embodiments.

[0012] This invention relates to an endophytic bacterium, Pantotheca acuminata (Pantotheca acumin Pantoea agglomerans SE16 and its applications; this strain is classified and named Pantotheca cumulus. Pantoea agglomerans It was deposited on October 17, 2024, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32246.

[0013] Preparation of NB medium: 3 g beef extract powder, 10 g peptone, 5 g sodium chloride, 1000 mL distilled water, pH 7.2±0.2, sterilized at 121℃ for 20 min; Preparation of NA medium: 3 g beef extract powder, 10 g peptone, 5 g sodium chloride, 15-20 g agar powder, 1000 mL distilled water, pH 7.2±0.2, sterilized at 121℃ for 20 min; Preparation of LB liquid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1000 mL distilled water, pH 7.2±0.2, sterilized at 121℃ for 20min; Preparation of culture medium for organophosphate bacteria: 10g glucose, 0.5g ammonium sulfate, 0.3g sodium chloride, 0.3g magnesium sulfate, 0.03g manganese sulfate, 0.3g potassium sulfate, 0.03g ferrous sulfate, 5g calcium phosphate, 0.2g lecithin, 15g agar, 1000mL distilled water, pH 7.0-7.5; Preparation of Assumption nitrogen-fixing medium: 0.2 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, 0.2 g sodium chloride, 5 g calcium carbonate, 10 g mannitol, 0.1 g calcium sulfate, 15 g agar, 1000 mL distilled water, pH 7.0±0.1; Preparation of CAS detection medium: Chromium azurite (CAS) 60.5 mg, hexadecyltrimethylammonium bromide (HDTMA) 72.9 mg, ferric chloride hexahydrate 2.465 mg, sodium dihydrogen phosphate dihydrate 295.25 mg, disodium hydrogen phosphate dodecahydrate 1213.5 mg, ammonium chloride 125 mg, potassium dihydrogen phosphate 37.5 mg, sodium chloride 62.5 mg, agar 9000 mg, distilled water 1000 mL, pH 6.8±0.2; Preparation of cellulase plate medium: 10 g peptone, 10 g yeast extract, 10 g sodium carboxymethyl cellulose, 5 g sodium chloride, 1 g potassium dihydrogen phosphate, 15 g agar powder, 1000 mL distilled water, pH 7.0; Preparation of inorganic salt culture medium: MgSO4·7H2O 0.2 g, CaCl2·2H2O 0.01 g, FeSO4·7H2O 0.01 g, K2HPO4 0.4 g, MnSO4·H2O 0.02 g, NH4NO3 1.0 g, Na2HPO4 0.6 g, distilled water 1000 mL, pH 7.2-7.4;

[0014] Endophytic bacteria SE16 were isolated from the leaves of healthy Sudan grass plants growing in farmland in Xingyang City, Henan Province. After washing the soil off the plant surface with sterile water in a laminar flow hood, the plants were separated according to different tissues. The plant organ surfaces were disinfected, and each organ was then ground separately in a sterile mortar with a small amount of sterile water. The resulting mortar was the stock solution. The stock solution was then serially diluted, with 100 μL of each diluted solution. -3 10 -4 10 -5 The diluted solution was spread on NA solid plates and incubated at 30°C for 24 hours. Colonies with different morphologies were picked and streaked for purification. The pure single colonies were then transferred to test tube slant culture medium and stored at 4°C for later use.

[0015] Endophytic bacteria strain SE16 was cultured on NA medium for 24 h. The colonies were pale yellow, Gram staining was negative, methylation was positive, VP test was positive, oxidase was negative, catalase was positive, starch hydrolysis was negative, citrate utilization test was positive, glucose oxidation fermentation produced acid, and gelatin liquefaction test was negative.

[0016] The amplified 16S rDNA sequence of SE16 (see SEQ ID No. 1) was compared with the sequence in GenBank using BLAST. The strain with the highest similarity to the 16S rDNA sequence of SE16 was... Pantoea agglomerans The consistency reached 100%. Homologous sequences and determined sequences obtained from alignment were analyzed for multiple sequence homology using Clustal X software, and then a phylogenetic tree was constructed using MEGA 6.05 software. The constructed phylogenetic tree shows that SE16 and... Pantoea agglomerans They belong to the same genetic branch. Based on bacterial morphological characteristics and physiological and biochemical tests, strain SE16 was ultimately identified as *Pantotheca cumulus* (…). Pantoea agglomerans ), classified and named Pantotheca acuminata Pantoea agglomerans It was deposited on October 17, 2024, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32246.

[0017] 4.1 Determination of SE16's salt and alkali resistance and pH sensitivity: SE16 seed culture was inoculated at a 1% inoculum into LB liquid medium containing NaCl (0, 1, 2, 4, 8, 10, 11, 12, 13, 14, 15%) at pH 7, and cultured at 30°C and 180 rpm for 24 h. The OD600 value of the bacterial culture was then measured. The results showed that SE16 could grow under 0-12% NaCl stress, proving that this strain can be used in the improvement of saline-alkali land. SE16 seed culture was inoculated at a 1% inoculum into LB liquid medium at pH 4, 5, 6, 7, 8, 9, 10, 11, and 12, and cultured at 30°C and 180 rpm for 24 h on a shaker. The OD600 value of the bacterial culture was then measured. It was found that SE16 could grow normally within a pH range of 4-9, indicating that this strain has a wide pH tolerance.

[0018] Single colonies of SE16 were spot-inoculated onto organic phosphorus-solubilizing solid medium, Assumption nitrogen-fixing medium, CAS medium, and cellulase medium, respectively, and incubated at 30°C for 48–72 h. The phosphorus-solubilizing ability was represented by calculating the ratio of the clear zone diameter D to the colony diameter d. Measurements showed ratios of 1.25–1.33, 1.34–1.52, 1.42–1.45, and 1.25–1.34, respectively, indicating that SE16 possesses good abilities in phosphorus solubilization, nitrogen fixation, siderophore production, and cellulase production.

[0019] 4.3 Determination of ability to produce indoleacetic acid (IAA): The SE16 strain was inoculated into LB liquid medium containing L-tryptophan and incubated at 30°C and 180 rpm. -1 The bacteria were cultured on a shaker for 24 hours. The Salkowski reaction was used to detect whether SE16 produced IAA; the results showed that the bacteria produced IAA at a yield of 73.7 mg / L.

[0020] The ACC deaminase activity of endophytic bacteria was assessed using the 2,4-dinitrophenylhydrazine colorimetric method. ACC deaminase activity was measured as mmol / (mg·min) of α-butanone produced per mg of bacteria per minute. The results showed that the ACC deaminase activity of this bacterium was 14.86 mmol / (mg·min).

[0021] 4.5 Determination of tolerance to different heavy metals and polycyclic aromatic hydrocarbons: SE16 seed culture was inoculated into plants containing different concentrations of Pb. 2+ (1000, 1100, 1200, 1300, 1400, 1500, 1600 mg / L), Mn 2+ The concentrations were set to (500, 1000, 1500, 2000, 2500 mg / L), As 3+ The concentrations were set to (5, 10, 15, 20, 25 mg / L), Cd 2+ The concentrations were set to (5, 10, 15, 20, 25 mg / L), Cr 6+In LB liquid medium (0, 20, 40, 60, 80, 100 mg / L), the culture was carried out at 30°C and 180 rpm on a shaker for 2-3 days. LB medium containing different concentrations of heavy metals without inoculation was used as a control to observe whether the bacterial solution became turbid.

[0022] SE16 seed culture was inoculated into inorganic salt medium containing 50 mg / L pyrene and 50 mg / L benzo[a]pyrene, respectively, and cultured on a shaker at 30°C and 180 rpm for 24-48 h. An uninoculated inorganic salt medium containing 50 mg / L pyrene and 50 mg / L benzo[a]pyrene was used as a control to observe whether the bacterial culture was turbid.

[0023] The results showed that SE16 could withstand 1200 mg / Lb. 2+ 1500 mg / LMn 2+ 10 mg / LAs 3+ 10 mg / LCd 2+ 60mg / LCr 6+ 50 mg / L pyrene and 50 mg / L benzo[a]pyrene.

[0024] 4.6 Tolerance test for two or more mixed pollutants: SE16 seed culture was inoculated into plants containing different concentrations of Pb. 2+ (800, 1000, 1200, 1400 mg / L), Cd 2+ The concentrations were set to (5, 10 mg / L), Cr 6+ SE16 was cultured in inorganic salt medium containing 30, 40, and 50 mg / L of pyrene, benzo[a]pyrene, and benzo[a]pyrene at 30°C and 180 rpm for 24–48 h on a shaker. An uninoculated inorganic salt medium containing the corresponding mixture was used as a control. Turbidity of the bacterial culture was observed. Results showed that SE16 could be cultured at 800 mg / L Pb. 2+ It can grow in 50 mg / L pyrene or 50 mg / L benzo[a]pyrene; it can grow in 60 mg / L pyrene. 6+ It can grow in 50 mg / L pyrene or 50 mg / L benzo[a]pyrene; it can grow in 10 mg / L LCd 2+ It grows in 50 mg / L pyrene or 50 mg / L benzo[a]pyrene.

[0025] The bacterial cells obtained by centrifuging the activated SE16 seed culture were diluted with sterile water to make the bacterial suspension concentration 10. 7 cfu / mL.

[0026] Chromium-contaminated soil was placed in flowerpots (1 kg per pot). 50 mL of bacterial suspension was inoculated into each pot before planting Sudan grass and again after seedling emergence. The control group was inoculated with 50 mL of sterile water. 200 Sudan grass seeds were planted in each pot. After 20 days, relevant indicators of the plants and soil were measured. The results showed that inoculation with the endophytic fungus SE16 promoted the growth of Sudan grass, as evidenced by significantly increased plant height, root length, above-ground fresh weight, and below-ground fresh weight compared to the uninoculated control. Endophytic fungus SE16 increased the chlorophyll content in Sudan grass plants and decreased the malondialdehyde (MDA) content in both above-ground and below-ground parts of Sudan grass. Figure 1-3 After inoculation with endophytic bacteria SE16, the content of exchangeable chromium in the soil was significantly reduced compared to the control, and the chromium content in both the aboveground and underground parts of Sudanese grassland was also significantly lower than the control. High-throughput sequencing analysis of Sudanese grassland rhizosphere soil samples showed that endophytic bacteria inoculation significantly increased the concentration of Delftobacteria (S. spp.) in the Sudanese grassland rhizosphere soil. Delftia ) and Sphingosomalidone spp. Saccharimonadales The relative abundance of the bacteria indicated that the addition of the inoculant promoted the enrichment of Delftobacterium and Sphingomonas, which are recognized as important microorganisms in the bioremediation of pollutants. This suggests that the inoculation of endophytic bacteria SE16 reduces the toxicity of chromium to plants by promoting plant growth, enhancing plant photosynthesis, mitigating oxidative stress in plants, and altering the composition of the soil microbial community, thus possessing the potential to enhance phytoremediation. The results are shown in Tables 1-2. Table 1. Effects of SE16 inoculation on growth of Sudan grass under chromium stress. deal with Plant height (cm) Root length (cm) Fresh weight of above-ground parts (g / plant) Fresh weight of underground parts (g / plant) CK 7.83±1.36b 0.95±0.04b 0.01±0.0003b 0.005±0.0009b SE16 10.97±1.38a 1.98±0.18a 0.03±0.003a 0.016±0.0009a Table 2 Effect of SE16 inoculation on chromium content deal with Chromium content (mg / kg) in Sudanese grasslands Chromium content (mg / kg) in the understory of Sudanese grasslands Exchangeable chromium content in soil (mg / kg) CK 676.80±124.91a 7710.18±737.25a 64.66±14.43a SE16 190.35±72.43b 4506.01±742.34b 24.07±4.36b In summary, the above-mentioned strain is the first endophytic bacterium, Pantotheca acuminata SE16, screened by the applicant, which can enhance the remediation of heavy metals in plants. This endophytic strain also possesses functions such as phosphorus solubilization, nitrogen fixation, siderophore production, indoleacetic acid production, ACC deaminase production, cellulase production, acid and salt tolerance, and can tolerate multiple heavy metals (Cr). 6+ Pb 2+ Mn 2+ As 3+ This strain can mitigate the combined pollution of Sudan grass with polycyclic aromatic hydrocarbons (Pyrene, benzo[a]pyrene) and heavy metals, and can also reduce the toxicity of chromium to Sudan grass by promoting its growth, enhancing its photosynthesis, reducing its oxidative stress, and altering the composition of the soil microbial community. This strain holds promise for development into a bio-agent, with broad application prospects in soil improvement, plant growth promotion, and enhanced phytoremediation of pollutants, and possesses practical application value.

Claims

1. An endophytic bacterium, Pantotheca acuminata SE16, is classified and named Pantotheca acuminata. Pantoea agglomerans It was deposited on October 17, 2024, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32246.

2. The application of the endophytic bacteria clustering pantothecin SE16 as described in claim 1 in enhancing phytoremediation of heavy metal pollution.

3. The application of the endophytic bacteria Pantotheca SE16 as described in claim 1 in soil improvement, plant growth promotion, and enhanced phytoremediation of pollutants.

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