Rahnella aquatica DG-1, fungicide and application of Rahnella aquatica DG-1 in relieving lily yellowing and / or reducing lily continuous cropping obstacles

Abstract

The invention provides rahnella aquatica DG-1, a bacterial agent and application of the rahnella aquatica DG-1 in relieving lily yellowing and / or reducing lily continuous cropping obstacles, and belongs to the technical field of microbial agents. The invention provides Rahnella aquatica DG-1, and the collection number of the Rahnella aquatica DG-1 is CGMCC (China General Microbiological Culture Collection Center) No. 28906. The rahnella aquatica DG-1 can interact with plants to influence the physiological and biochemical states of lilies and rhizosphere soil microbial communities and functions, and not only can relieve lily yellowing, but also can reduce successive cropping obstacles, promote lily growth, improve the disease resistance of lilies and increase the yield of lilies.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial inoculants, and particularly relates to a Rahnella aquatilis DG-1, an inoculant and application of the Rahnella aquatilis DG-1 and the inoculant in relieving lily yellowing and / or reducing lily continuous cropping obstacles. BACKGROUND

[0002] Lanzhou lily (Lilium davidii var.unicolor salisb) is a variety of Lilium davidii var.unicolor salisb of the genus Lilium of the lily family (Liliaceae), and is a perennial bulbous plant. Lanzhou lily is mainly propagated asexually. When commodity lilies are harvested, small daughter bulbs attached to underground stems are picked for the propagation of commodity mother bulbs. Generally, 3 years are needed for commodity mother bulbs to be transplanted and then commodity lilies to be harvested after 3 years. Continuous cropping for many years leads to degradation of varieties, and viral diseases, bacterial diseases and fungal diseases are serious. Soil nutrient imbalance occurs, and the whole plant turns yellow soon after germination in mid-May every year, growth vigor is weakened, photosynthetic efficiency is greatly reduced, and bulb yield and quality are reduced, which seriously restricts the sustainable development of the planting industry.

[0003] Existing research shows that there are many reasons for the yellowing of Lanzhou lily, such as soil pH imbalance, extreme climate conditions such as high temperature and drought, invasion of pests and diseases, and excessive planting density. In actual production, measures such as increasing the application of organic fertilizer and biological bacterial fertilizer to improve soil structure and fertility, using dazomet soil fumigation to reduce the content of pathogenic bacteria, foliar application of trace element water-soluble fertilizer to supplement nutrients, and reasonable adjustment of planting density and strengthening of field ventilation and light management are used to reduce lily yellowing. However, the above-mentioned methods have the defect of single action and cannot stably relieve the yellowing of Lanzhou lily.

[0004] In addition, many microbial products on the market are mainly solid root application, and the application effect depends on the strain colonization and persistence period and the disturbance degree of indigenous communities due to the influence of environmental factors, soil types and indigenous microorganisms in the soil. In addition, plastic mulching is generally used in Lanzhou lily planting. In addition to the convenience of root application during soil preparation, the operation is difficult in the later period. SUMMARY

[0005] The present application aims to provide a Rahnella aquatilis DG-1, an inoculant and application of the Rahnella aquatilis DG-1 and the inoculant in relieving lily yellowing and / or reducing lily continuous cropping obstacles, which can be sprayed and applied.

[0006] The present application provides a Rahnella aquatilis DG-1, with a preservation number of CGMCC No.28906.

[0007] The present application also provides an inoculant containing the Rahnella aquatilis DG-1 described in the above scheme.

[0008] Preferably, the effective component of the bacterial agent comprises Raoultella aquatilis DG-1 and / or the fermentation liquor of Raoultella aquatilis DG-1.

[0009] Preferably, the effective viable cell concentration of Raoultella aquatilis DG-1 in the bacterial agent is 2×(10 8 ~ 10 9 )CFU / mL.

[0010] The present application also provides the use of Raoultella aquatilis DG-1 or the bacterial agent as described in the above-mentioned scheme in at least one of the following:

[0011] 1) improving the disease resistance of lilies;

[0012] 2) increasing the abundance of probiotics and / or reducing the abundance of pathogenic bacteria;

[0013] 3) preventing and / or alleviating the yellowing of lilies;

[0014] 4) increasing the chlorophyll content of lily leaves and the ratio of chlorophyll to flavonoids;

[0015] 5) improving the transport and metabolism of amino acids in lilies;

[0016] 6) promoting the absorption and utilization of nitrogen and / or potassium by lilies;

[0017] 7) promoting the metabolism and activation of iron in lilies;

[0018] 8) increasing the content of endogenous salicylic acid hormones in lilies;

[0019] 9) promoting the growth of lilies and / or increasing the yield of lilies;

[0020] 10) promoting photosynthesis in lilies and / or increasing the photosynthetic nitrogen use efficiency of lilies;

[0021] 11) alleviating the continuous cropping obstacles of lilies.

[0022] Preferably, the lilies comprise Lanzhou lilies.

[0023] The present application also provides a method for alleviating the yellowing of lilies and / or reducing the continuous cropping obstacles of lilies, comprising at least one of the following steps:

[0024] Soaking lily seedlings with the bacterial agent as described in the above-mentioned scheme before sowing;

[0025] Soaking lily commercial seed balls with the bacterial agent as described in the above-mentioned scheme before transplanting;

[0026] Spraying the aerial parts of lilies with the bacterial agent as described in the above-mentioned scheme.

[0027] Preferably, when the lily seedling or commodity seed ball is soaked, the effective viable concentration of the Rahnella aquatilis DG-1 in the bacterial agent is 5×(10 6 ~10 7 ) CFU / mL; and the soaking time is 30-60 min.

[0028] Preferably, the soaking time is 30-45 min.

[0029] Preferably, when the lily aboveground part is foliar sprayed, spraying is performed once every 4 weeks, and spraying is performed 3-4 times in total; the spraying amount of the Rahnella aquatilis DG-1 is 4×(10 11 ~10 12 ) CFU / acre each time.

[0030] The present application provides a Rahnella aquatilis DG-1, and the preservation number is CGMCC No. 28906. The Rahnella aquatilis DG-1 of the present application can affect the lily physiological and biochemical state, the rhizosphere soil microbial community and function through interaction with plants, not only can relieve lily yellowing, but also can reduce continuous cropping obstacles, promote lily growth, improve lily disease resistance and increase lily yield. In addition, the Rahnella aquatilis DG-1 of the present application can be applied through seed soaking or spraying, which is easy to operate and conducive to popularization.

[0031] Biological preservation

[0032] The Rahnella aquatilis DG-1 is biologically classified as Rahnella aquatilis, and was preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 8, 2023, the preservation address is No. 1, Xibaheyi, Chaoyang District, Beijing, China, and the preservation number is CGMCC No. 28906. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0034] Figure 1 Evolutionary branch diagram for fungal LEfSe analysis;

[0035] Figure 2 Evolutionary branch diagram for bacterial LEfSe analysis. DETAILED DESCRIPTION

[0036] The application provides a Rahnella aquatilis DG-1, and the preservation number is CGMCC No. 28906.

[0037] The Rahnella aquatilis DG-1 is isolated from medicinal plants rhubarb growing in an alpine and humid area, and is identified as Rahnella aquatilis through colony characteristics and 16S rDNA sequence identification. The colony characteristics of the Rahnella aquatilis DG-1 on LB solid culture medium are white, round, semi-transparent, smooth and moist. The 16S rDNA sequence of the Rahnella aquatilis DG-1 is shown as SEQ ID NO. 1, and the strain has 99.79% similarity with a standard strain Rahnella aquatilis YL-245 strain through NCBI database blast comparison.

[0038] The strain of the Rahnella aquatilis DG-1 has strong activities of phosphorus release, ferroxidase production, can utilize citrate, has strong nitrification, is positive in methyl red experiment, positive in ammonification experiment, positive in contact enzyme experiment, has strong abilities of acid production, protein decomposition and hydrogen peroxide decomposition. Meanwhile, the strain has activities of ACC deaminase, peroxidase, neutral xylanase, cellulase and laccase, and can degrade self-toxic substances such as phenolic acid, ferulic acid and anthraquinone. The Rahnella aquatilis DG-1 can affect physiological and biochemical states of lilies, rhizosphere soil microbial community and functions through interaction with plants, so as to promote lily growth, disease resistance, yield increase, effectively relieve lily yellowing and reduce lily continuous cropping obstacles.

[0039] The application further provides a microbial agent containing the Rahnella aquatilis DG-1.

[0040] As an embodiment, the effective component of the microbial agent comprises the Rahnella aquatilis DG-1 and / or fermentation liquor of the Rahnella aquatilis DG-1. As another embodiment, the effective component of the microbial agent can be Rahnella aquatilis DG-1 or fermentation liquor of the Rahnella aquatilis DG-1; as another embodiment, the effective component of the microbial agent can be fermentation liquor of the Rahnella aquatilis DG-1. As an embodiment, the effective viable concentration of the Rahnella aquatilis DG-1 in the microbial agent is 2x(10 8 ~ 10 9 )CFU / mL.

[0041] As an embodiment, the method for preparing the fermentation broth of the Raenella aquatilis DG-1 provided by the present application comprises inoculating the Raenella aquatilis DG-1 into a liquid fermentation medium for fermentation culture to obtain the fermentation broth of the Raenella aquatilis DG-1. As an embodiment, the liquid fermentation medium can be PDB medium. As an embodiment, the temperature of the fermentation culture can be 25-30℃; as another embodiment, the temperature of the fermentation culture can be 28℃. As an embodiment, the time of the fermentation culture can be 1-3d; as another embodiment, the time of the fermentation culture can be 2d. As an embodiment, the shaking frequency of the fermentation culture can be 150-200r / min; as another embodiment, the shaking frequency of the fermentation culture can be 180r / min.

[0042] The present application also provides the use of the Raenella aquatilis DG-1 or the microbial agent as described in the above-mentioned scheme in at least one of the following:

[0043] 1) improving the disease resistance of lilies;

[0044] 2) increasing the abundance of probiotics and / or reducing the abundance of pathogenic bacteria;

[0045] 3) preventing and / or relieving lily yellowing;

[0046] 4) improving the chlorophyll content of lily leaves and the ratio of chlorophyll to flavonoids;

[0047] 5) improving the transport and metabolism of amino acids in lilies;

[0048] 6) promoting the absorption and utilization of nitrogen and / or potassium by lilies;

[0049] 7) promoting the metabolism and activation of iron in lilies;

[0050] 8) increasing the endogenous salicylic acid content in lilies;

[0051] 9) promoting the growth of lilies and / or increasing the yield of lilies;

[0052] 10) promoting photosynthesis in lilies and / or increasing the photosynthetic nitrogen use efficiency of lilies;

[0053] 11) relieving lily continuous cropping obstacles

[0054] As an embodiment, the lilies include Lanzhou lilies.

[0055] In the present application, increasing the endogenous salicylic acid content in lilies can reduce fungal and bacterial diseases.

[0056] As an implementation form, the probiotics comprise rhizosphere soil probiotic fungi and rhizosphere soil probiotic bacteria; the rhizosphere soil probiotic fungi comprise Chaetomella cupreata and / or Bacteroidetes.

[0057] As an implementation form, the pathogenic bacteria comprise pathogenic fungi and pathogenic bacteria; the pathogenic fungi comprise at least one of Lectera sambuci, Alternaria nepalensis, Fusarium domesticum and Bisifusarium tonghuanum; the pathogenic bacteria comprise Unclassified Xanthomonadales.

[0058] In the present application, the Raenii aquatilis DG-1 or the bacterial agent can alleviate leaf yellowing, promote photosynthesis, significantly increase the content of chlorophyll in leaves, the ratio of chlorophyll and flavonoids, photosynthetic rate and photosynthetic nitrogen use efficiency.

[0059] The present application also provides a method for alleviating lily yellowing and / or reducing lily continuous cropping obstacles, comprising at least one of the following steps:

[0060] The bacterial agent described in the above scheme is used to soak lily small seedlings before sowing;

[0061] The bacterial agent described in the above scheme is used to soak lily commodity seed balls before transplanting;

[0062] The bacterial agent described in the above scheme is used to spray the aboveground part of lily.

[0063] As an implementation form, when the lily small seedlings or commodity seed balls are soaked, the effective viable concentration of Raenii aquatilis DG-1 in the bacterial agent is 5×(10 6 ~10 7 )CFU / mL; the soaking time is 30-60 min.

[0064] As an implementation form, the soaking time is 30-45 min.

[0065] As an implementation form, when the aboveground part of lily is sprayed, spraying is performed once every 4 weeks, and spraying is performed for 3-4 times; the spraying time is June-September of the solar calendar; the spraying amount of each time is 4×(10 11 ~10 12 )CFU / mu in terms of the number of viable Raenii aquatilis DG-1. As another implementation form, the spraying amount of each time is 4×(10 11~10 12 )CFU / acre, sprayed once every 4 weeks, a total of 4 times. As an embodiment, the bacterial agent of the present application can be a fermentation broth of Raoultella aquatilis DG-1, which has been limited in the above technical solutions and will not be repeated here.

[0066] In the present application, the application means of soaking and foliar spraying are convenient to operate and conducive to popularization.

[0067] In order to further illustrate the present application, a strain of Raoultella aquatilis DG-1, a bacterial agent and its application in relieving lily yellowing and / or reducing lily continuous cropping obstacles provided by the present application are described in detail below in combination with the drawings and examples, but they cannot be understood as limiting the protection scope of the present application.

[0068] The lily seedlings used in the examples were picked from lily plants when the inventors' research group dug commercial lilies. The test was conducted in 2024 in Mapo Town, Yuzhong County, Gansu Province. The soil type was mountain calcareous soil, and the pH value was 7.3. The base fertilizer was organic fertilizer 1 ton / acre (organic matter ≥40%, N+P2O5+K2O ≥4%), balanced compound fertilizer (17-17-17) 20 kg / acre.

[0069] The reagents and culture media used in the test were of chemical purity. Cellulase, laccase, xylanase, peroxidase, ACC deaminase, glutamine synthetase, glutamate synthase, nitrite reductase, nitrate reductase, phenylalanine ammonia lyase activity detection kit, and ABTS, DPPH, siderophore, malondialdehyde, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugar, chlorophyll, flavonoid, nitrite nitrogen, and nitrate nitrogen content detection kit were purchased from Beijing Boxi Shengong Technology Co., Ltd.

[0070] The composition of the culture medium used in the following examples is as follows:

[0071] PDB culture medium: potato 200 g / L, glucose 20 g / L, natural pH.

[0072] LB culture medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH value adjusted to 7.0-7.4.

[0073] Modified Stephenson culture medium: ammonium sulfate 2 g / L, manganese sulfate 0.01 g / L, sodium dihydrogen phosphate 0.25 g / L, magnesium sulfate 0.03 g / L, calcium carbonate 0.5 g / L, dipotassium hydrogen phosphate 0.75 g / L, pH value adjusted to 8.2.

[0074] NBRIP solid medium: glucose 10 g / L, calcium phosphate 5 g / L, magnesium chloride 5 g / L, magnesium sulfate heptahydrate 0.25 g / L, potassium chloride 0.2 g / L, ammonium sulfate 0.1 g / L, agar 15 g / L, pH 7.0 ± 0.2.

[0075] Simon's citrate agar medium: sodium chloride 5.0 g / L, magnesium sulfate 0.2 g / L, ammonium dihydrogen phosphate 1.0 g / L, dipotassium hydrogen phosphate 1.0 g / L, sodium citrate 5.0 g / L, agar 20 g / L, 0.2% bromothymol blue solution 40 mL / L, adjust pH to 6.8 ± 0.2.

[0076] Glucose peptone water medium: glucose 0.5 g / L, peptone 0.5 g / L, dipotassium hydrogen phosphate 0.2 g / L, pH adjusted to 7.2-7.4.

[0077] Peptone ammonium medium: peptone 5 g / L, dipotassium hydrogen phosphate 0.5 g / L, sodium chloride 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, and ferrous sulfate 0.01 g / L, adjust pH to 7.2.

[0078] DF medium: KH2PO4 4.0 g / L, Na2HPO4 6.0 g / L, MgSO4·7H2O 0.2 g / L, FeSO4·7H2O 0.1 g / L, CaCl2·2H2O 0.02 g / L, (NH4)2SO4 2.0 g / L, H3BO3 0.3 g / L, CuSO4·5H2O 0.04 g / L, MnSO4·H2O 0.04 g / L, ZnSO4·7H2O 0.12 g / L, Na2MoO4·2H2O 0.025 g / L, and glucose 2.0 g / L.

[0079] ADF medium: KH2PO4 4.0 g / L, Na2HPO4 6.0 g / L, MgSO4·7H2O 0.2 g / L, FeSO4·7H2O 0.1 g / L, CaCl2·2H2O 0.02 g / L, H3BO3 0.3 g / L, CuSO4·5H2O 0.04 g / L, MnSO4·H2O 0.04 g / L, ZnSO4·7H2O 0.12 g / L, Na2MoO4·2H2O 0.025 g / L, glucose 2.0 g / L, and ACC (1-aminocyclopropane-1-carboxylic acid) 3.0 mM.

[0080] M9 medium: Na2HPO4·7H2O 12.8 g / L, KH2PO4 3.0 g / L, NaCl 0.5 g / L, NH4Cl 1 g / L, MgSO4·7H2O 0.492 g / L, CaCl2·6H2O 0.022 g / L, and glucose 4.0 g / L.

[0081] Example 1

[0082] The isolation and identification of Ralstonia aquatilis DG-1 is as follows:

[0083] Ralstonia aquatilis DG-1 is isolated from a medicinal plant Rheum officinale grown in a high-cold and humid area in Gansu Province.

[0084] The colony characteristics of the strain are as follows: the isolated strain is inoculated into LB solid medium and cultured at 30°C for 2 days, and the colony morphology of the strain is white, round, semi-transparent, and smooth and moist on the surface.

[0085]

[0086] Example 2

[0087] The function detection of Ralstonia aquatilis DG-1 is as follows:

[0088] 1) Phosphorus dissolving property determination: inoculate the Ralstonia aquatilis DG-1 strain into LB medium and activate at 28°C and 180 r / min for 2 days. Inoculate 10 μL of the Ralstonia aquatilis DG-1 liquid into NBRIP solid medium and place in a constant temperature incubator at 30°C. Observe the phosphorus dissolving ring and its size on the medium periodically within 7 days, and determine the phosphorus dissolving ability of the strain according to the size of the phosphorus dissolving ring.

[0089] The results show that the Ralstonia aquatilis DG-1 can dissolve phosphorus.

[0090] 2) Citrate utilization: inoculate the Ralstonia aquatilis DG-1 into the center of the Simon citrate agar medium with a loop, and repeat the inoculation three times to make the colonies evenly distributed. Invert the inoculated plate into a constant temperature incubator at 30°C and incubate for 24-48 h. Observe whether there is a color change around the colonies on the plate, and the blue color is positive.

[0091] The results show that the Ralstonia aquatilis DG-1 can utilize citrate.

[0092] 3) Nitration determination: inoculate the Ralstonia aquatilis DG-1 into LB medium, and incubate at 30°C and 180 r / min for 2 days. Centrifuge to collect the bacterial cells, resuspend in sterile water to OD 600 1.0, and then inoculate into sterilized modified Stephenson medium, and incubate at 30°C and 180 r / min for 2 days. Measure the OD 600 value, detect the content according to the method described in the nitrite nitrogen and nitrate nitrogen kit, calculate the nitration according to the formula 1, divide the calculation result by the turbidity of the corresponding bacterial liquid, and standardize the nitration of the unit turbidity bacterial liquid. Repeat 3 times for each strain, and calculate the average value.

[0093]

[0094] The results show that the nitration of the Ralstonia aquatilis DG-1 is 0.645 ± 0.03%.

[0095] 4) Relative content determination of siderophore: centrifuge the Ralstonia aquatilis DG-1 after inoculation in LB medium, take the fermentation supernatant (SCS), mix the SCS and the two solutions of chrome azurol S (CAS) at a volume ratio of 1:1, and measure the OD 630, the relative content of siderophore of the strain was calculated by the following formula: relative content of siderophore of the strain = (Ar-As) / Ar x 100%, wherein Ar is the OD 630 of the reference substance 630 As is the OD of the sample (mixture of the strain SCS and CAS). The calculated result was divided by the turbidity of the corresponding bacterial liquid, and was standardized as the relative content of siderophore per unit turbidity of the bacterial liquid.

[0096] The results showed that the relative content of siderophore of the Raenella aquatica DG-1 was 43.1%.

[0097] 5) ACC deaminase activity determination of the strain: the Raenella aquatica DG-1 was cultured in the LB medium at 30°C and 180 r / min for 1 d, then was centrifuged at 4°C and 8000 r / min for 10 min, the supernatant was discarded, the bacterial body was washed twice with the ADF medium without (NH4)2SO4, and was resuspended in the ADF medium after centrifugation, and was cultured at 30°C and 180 r / min for 1 d. Then, the bacterial body was collected by centrifugation at 4°C and 8000 r / min for 10 min after the supernatant was discarded, was washed twice with the 0.1 mol / L Tris-HCl buffer solution with pH value of 7.6, was resuspended in 0.2 mL Tris-HCl buffer solution with the same concentration and pH value of 8.5, a small amount of toluene was added, the cells were broken by ultrasonic, 20 μL 0.5 mol / L ACC was added, and after mixing, the mixture was placed in a water bath at 30°C for 15 min, then 0.3 mL of dinitrophenylhydrazine was added, the lid was covered, and the mixture was placed in a water bath at 30°C for 0.5 h; then, 2 mL of 2 mol / L NaOH was added to terminate the reaction, and the absorbance at 540 nm was determined. The enzyme activity of ACC deaminase was the content of α-ketobutyric acid produced per minute (μmol / min).

[0098] The results showed that the ACC deaminase activity of the Raenella aquatica DG-1 was 0.545 μmol / min.

[0099] 6) Enzyme activity determination of the strain: the Raenella aquatica DG-1 was inoculated in 50 mL of sterilized LB medium, was cultured at 30°C and 180 r / min for 2 d, was filtered, and the bacterial body was collected and resuspended in sterile water to a turbidity OD 600 of 1 to obtain the bacterial liquid to be tested. The bacterial liquid to be tested and the enzyme extract were mixed in a volume ratio of 1:10 in an ice bath, the cells were broken by ultrasonic, and the mixture was centrifuged at 4°C and 8000 r / min for 10 min, and the supernatant was determined on ice. The enzyme activities of peroxidase, cellulase, neutral xylanase and laccase were determined by the spectrophotometric method according to the instructions of the enzyme activity kit, and the determination was repeated for 3 times. The determination result was divided by the turbidity of the corresponding bacterial liquid, and was standardized as the enzyme activity per unit turbidity of the bacterial liquid.

[0100] The results show that the peroxidase activity of the Raoultella planticola DG-1 is 10.554±0.345 U / mL, the neutral xylanase activity is 22.59±0.032 U / mL, the cellulase activity is 21.962±0.258 U / mL, and the laccase activity is 1.033±0.256 U / mL.

[0101] 7) Methyl red test: the Raoultella planticola DG-1 is inoculated into a sterile glucose peptone water culture medium by using a sterile inoculation loop, and the medium without inoculation is used as a control, and then the mixture is incubated at 30°C for 2-5 days, 5 drops of methyl red reagent are added into the medium, and the color change is observed, and the red color indicates the positive result.

[0102] The results show that the Raoultella planticola DG-1 has strong ability to produce acid by decomposing glucose.

[0103] 8) Ammonification test: the fermentation liquor of the Raoultella planticola DG-1 is inoculated into a peptone ammonification culture medium, and 6 dilution degrees (10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ) are used, and 3 tubes are inoculated for each dilution degree, and a tube of the medium is inoculated with sterile water as a control. The mixture is cultured in a constant temperature incubator at 30°C. The turbidity of the medium is checked on the 3rd day and the 5th day after the culture. On the 7th day of the culture, 5 drops of the culture liquid are taken on a white porcelain colorimetric plate, 2 drops of Nessler's reagent are added, and whether the brownish color appears is checked to determine whether the ammonia is produced.

[0104] The results show that the Raoultella planticola DG-1 has strong ammonification ability.

[0105] 9) Contact enzyme test: the Raoultella planticola DG-1 colonies are picked and placed on a clean glass slide, and an appropriate amount of 3% hydrogen peroxide solution is added dropwise. A large number of bubbles are generated within 1 min, which is positive.

[0106] The results show that the Raoultella planticola DG-1 has strong hydrogen peroxidase activity.

[0107] 10) Self-toxic substance degradation: the M9 medium without sugar is used as a basic medium, and 50 mg / L of ferulic acid or anthraquinone is added as a sole carbon source. The Raoultella planticola DG-1 is inoculated into the above-mentioned medium. The inoculated medium is cultured at 30°C and 180 r / min for 3-5 days, the color change of the medium is observed, and the OD 600 value is measured.

[0108] The results show that the Raoultella planticola DG-1 can degrade ferulic acid and anthraquinone.

[0109] Example 3

[0110] The effect verification of Ralstonia aquatilis DG-1 in alleviating continuous cropping obstacles was carried out as follows:

[0111] The test was set as follows:

[0112] Treatment group (T): The fermentation liquor of Ralstonia aquatilis DG-1 cultured in PDB medium at 28℃ and 180 r / min for 2 days was diluted 40 times with water to an effective viable bacterial count of 5×(10 6 ~ 10 7 ) CFU / mL. 40 L was sprayed per mu.

[0113] Positive control group (CK): PDB medium without bacteria was diluted by the same multiple as the treatment group, and 40 L was sprayed per mu.

[0114] The test adopted single factor complete random design, and the land of harvested commodity lily (i.e. planted for 3 years) was used to continue commodity seedling breeding. T and CK were set as two treatments, each with 3 replicates, and the plot area was 30 m 2 (4 m × 7.5 m). The seedlings were sowed in late April 2024, and foliar spraying was carried out 4 times from May to September, once every 4 weeks. The spraying volume was equal in different plots.

[0115] In late April 2024, before transplanting the lily seedlings, the corresponding seedlings were soaked in the treatment group T and the positive control group CK for 30 min, and then transplanted after draining the surface water. After the seedlings were transplanted, foliar spraying was carried out 4 times from May to September, once every 4 weeks. The spraying volume was equal in different plots. The field management was carried out according to the conventional measures.

[0116] In late August, 3 days after the 4th treatment, 100 lily seedlings were randomly selected from each plot, the leaves were removed, and the rhizosphere soil of the corresponding lily was collected and mixed, and then taken back to the laboratory for standby.

[0117] In late September, the lily was dug up to count the root number and root length, the root system was removed, the lily surface soil was washed and dried, and then the weight of 100 plants was measured.

[0118] The average weight of 100 plants, the average root number, and the root length index of different treatments are shown in Table 1.

[0119] Table 1 Average weight of 100 plants, average root number, and root length of lily in different treatments

[0120] Mean FC (T vs CK) Mean weight per 1000 seeds (g) Positive control group CK 416±0.75b 1.40 Treatment group T 583±0.56a Mean root number (pieces) Positive control group CK 7.31±0.53b 1.40 Treatment group T 10.22±0.41a Mean root length (cm) Positive control group CK 5.01±0.39b 1.32 Treatment group T 6.63±0.42a

[0121] Lowercase letters represent multiple comparisons P<0.05.

[0122] Compared with the positive control group CK, the treatment group T of the present application significantly increased the average 100-seed weight, average root number and average root length. It is shown that the treatment group T of the present application effectively alleviates the lily continuous cropping obstacles.

[0123] Example 4

[0124] Effects of Raoultella aquatilis DG-1 on endogenous hormones and physiological and biochemical indexes in lily leaves

[0125] The test setting and test scheme are the same as those in Example 3

[0126] The leaves of different samples are sent to a third party, and 109 substances of nine kinds of plant hormone-related metabolites, such as auxin, cytokinin (CK), abscisic acid (ABA), jasmonate (JAs), salicylic acid (SA), gibberellin (GAs), 1-aminocyclopropanecarboxylic acid (ACC), strigolactone (SLs) and melatonine (MLT), are determined by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS). The most important substances (VIP>1) that can effectively distinguish the differences between the T group and the CK group are screened by the OPLS-DA (a kind of supervised pattern recognition multivariate statistical analysis) method, and the difference degrees of different substances between the two groups are compared by the FC value, as shown in Table 2.

[0127] Table 2 Endogenous hormone content (ng / g) of leaves in different treatments

[0128]

[0129] Log2FC: difference ratio taking logarithm with base 2, the same below.

[0130] Compared with the positive control group CK, the treatment group T of the present application significantly reduces the contents of cytokinin, gibberellin and jasmonic acid; significantly increases the contents of auxin and salicylic acid, especially the content of salicylic acid, with a Log2FC value of 3.5, and the absolute value of the content of salicylic acid SAG is very large. It is shown that the treatment group T of the present application significantly improves the disease resistance of lily.

[0131] Determination of lily leaf ABTS, DPPH, total phenol, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugar, chlorophyll, flavonoids, and malondialdehyde content by spectrophotometry as described in the kit; glutamine synthetase, glutamate synthetase, nitrite reductase, nitrate reductase, and phenylalanine ammonia lyase (PAL) activity. The photosynthetic rate, stomatal conductance, intercellular carbon dioxide concentration, and transpiration rate were detected by a photosynthesis detector. The photosynthetic nitrogen use efficiency was calculated (photosynthetic nitrogen use efficiency = photosynthetic rate / (ammonium nitrogen + nitrate nitrogen + amino nitrogen)). The indicators significantly different by T test (P < 0.05) are shown in Table 3, and the FC values compare the difference degree between the two groups of different substances.

[0132] Table 3 Physiological and biochemical indicators of different treatments (mean ± se)

[0133] Indicator T CK P log2FC ABTS scavenging equivalent quantification value (mmol / L) 2.355±0.036 2.210±0.041 0.002 0.092 Total phenols (mg / g) 7.099±0.167 6.211±0.298 0.002 0.193 Ammonium nitrogen (pg / g) 48.340±0.615 49.672±0.530 0.017 -0.039 Amino nitrogen (pg / g) 48.340±0.615 49.672±0.530 0.017 -0.039 Soluble sugar (mg / g) 13.448±0.238 12.626±0.097 0.001 0.091 Chlorophyll (mg / g) 2.466±0.068 1.028±0.189 0.000 1.262 PAL (U / g) 39.228±4.793 30.362±5.129 0.045 0.370 Flavonoids (mg / g) 1.725±0.083 3.054±0.256 0.000 -0.824 Chlorophyll / flavonoids 1.430 0.337 - 2.085 Total phenols / flavonoids 4.115 2.034 - 1.016 Photosynthetic rate (pmol.m"2s"1) 2.942±0.089 2.169±0.035 0.000 0.440 Photosynthetic nitrogen use efficiency 0.040 0.0267 - 0.583

[0134] Compared with the positive control group CK, the treatment group T of the present application significantly increased the chlorophyll content, the ratio of chlorophyll and flavonoids, and the ratio of total phenol and flavonoids, with log2(FC) being 1.262, 2.085, and 1.016, respectively, effectively alleviating the yellowing of the leaves. The photosynthetic rate and photosynthetic nitrogen use efficiency also increased to a certain extent, indicating that the treatment group T of the present application effectively promoted photosynthesis. In addition, the increase in PAL activity was consistent with the significant increase in salicylic acid content.

[0135] Example 5

[0136] Effect of Raoultella aquatilis DG-1 on soil nutrients and enzyme activity of lilies

[0137] The test setting and test scheme were the same as in Example 3

[0138] The soil sucrase, urease, β-glucosidase, xylanase, alkaline phosphatase, and catalase activities were determined by spectrophotometry as described in the kit; the ammonium nitrogen and nitrate nitrogen contents were determined; the alkaline hydrolysis nitrogen was determined by alkaline hydrolysis diffusion method; the available potassium was determined by flame photometry after ammonium acetate dissolution and extraction; the total iron was determined by inductively coupled plasma spectrometry after four-acid digestion; and the available phosphorus was determined by molybdenum-antimony anti-colorimetry after sodium bicarbonate solution extraction. The indicators significantly different by T test (P < 0.05) are shown in Table 4, and the FC values compare the difference degree between the two groups of different substances.

[0139] Table 4 Soil nutrient and enzyme activity content (mean ± se)

[0140]

[0141]

[0142] Compared with the positive control group CK, the treatment group T of the application significantly increased the soil urease, alkali hydrolytic nitrogen content, and decreased the nitrate nitrogen and ammonium nitrogen content, indicating that the treatment group T of the application promoted the circulation and utilization of nitrogen; the total iron content was significantly increased; the available potassium content was significantly reduced, indicating that the treatment group T of the application absorbed more. The absolute value of alkaline phosphatase activity was large, and the absolute value of available phosphorus content belonged to the level of phosphorus-rich soil, indicating that lilies had a large demand for phosphorus.

[0143] Example 6

[0144] Rhizosphere soil microbial diversity and function detection:

[0145] The test setting and test scheme are the same as those in Example 3

[0146] After sample collection, dry ice was used for cooling and sent to a third party to carry out 16S and ITS amplicon sequencing analysis. Bacteria use 16sv3+v4 region, and fungi use its1. Based on the Illumina NovaSeq sequencing platform, the method of paired-end sequencing is used to construct a small fragment library for sequencing. Bioinformatics analysis: LEFSE biomarker analysis (see Figure 1 and Figure 2 ), species-level significant difference species analysis (see Tables 5 and 6), and functional prediction analysis (see Tables 7 and 8).

[0147] The rhizosphere soil of the treatment group T (LNS) of the application has 8 types of marker fungi, 2 markers annotated to the genus level: Brunneochlamydosporium, unclassified Chaetomiaceae; 2 markers annotated to the species level: Brunneochlamydosporium nepalense, unclassified Chaetomiaceae. The rhizosphere soil of the positive control group CK has 16 types of marker fungi, 3 markers annotated to the genus level: Alternaria, Fusarium, Mortierella; 3 markers annotated to the species level: Plectosphaerella niemeijerarum, (Fusarium domesticum) Fusarium domesticum, (unclassified Mortierella) unclassified Mortierella. It is shown that the treatment group T of the application mainly enriches probiotic Chaetomiaceae; the positive control group CK mainly enriches Alternaria and Fusarium pathogenic bacteria.

[0148] The rhizosphere soil bacterial marker of the treatment group T (LNS) described in the application is chloroflexi, which mainly plays a role in carbon, nitrogen, sulfur cycle and biodegradation process.

[0149] Table 5 fungi with significant differences in relative abundance at the species level

[0150]

[0151] Compared with the positive control group CK, the treatment group T described in the application significantly increased the abundance of uncultured Chaetomiiaceae of probiotics, and significantly reduced the abundance of pathogenic fungi such as Lectera sambuci, Alternaria nepalensis, Fusarium domesticum and Bisifusarium tonghuanum.

[0152] Table 6 bacteria with significant differences in relative abundance at the species level

[0153]

[0154] Compared with the positive control group CK, the treatment group T described in the application significantly reduced the abundance of pathogenic Xanthomonas.

[0155] Through PICRUSt2 annotation in KEGG and COG databases, the indicators of significant differences in bacterial function abundance are as follows.

[0156] Table 7 bacterial KEGG function prediction

[0157] Function T (mean) CK (mean) P Log2FC Metabolism of cofactors and vitamins 4.043621 4.028056 0.032563 0.0056 Cellular community prokaryotes 1.742365 1.704107 0.034204 0.0320 Infectious diseases: Bacterial 0.347991 0.35213 0.0293 -0.0171 Immune diseases 0.031827 0.033964 0.00718 -0.0938

[0158] Compared with the positive control group CK, the treatment group T described in the application significantly increased the metabolism of cofactors and vitamins, improved the function of prokaryotic cell community, and significantly reduced bacterial pathogen infection and immune-related diseases. It shows that the treatment group T described in the application can reduce the occurrence of bacterial diseases.

[0159] Table 8 bacterial COG function prediction

[0160] Function T (mean) CK (mean) P Log2FC Amino acid transport and metabolism 10.29655 10.17137 0.01904 0.0177 Cytoskeleton 0.01217 0.01386 0.01529 -0.1879

[0161] Compared with the positive control group CK, the treatment group T described in the application significantly improved the transport and metabolism function of amino acids.

[0162] From the above results, it can be concluded that the aquatic raenbacteria DG-1 and the bacterial agent containing the aquatic raenbacteria DG-1 described in the application can relieve lily yellowing, reduce continuous cropping obstacles, promote lily growth, improve lily disease resistance and have significant yield increasing effect.

[0163] Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments. Other embodiments can be obtained based on the above embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A strain of aquatic Rahnella aquatilis DG-1, characterized in that, The accession number is CGMCCNo.28906.

2. A bacterial agent containing the aquatic Ranelia DG-1 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The active ingredient of the microbial agent includes aquatic Ranunculus DG-1 and / or the fermentation broth of aquatic Ranunculus DG-1.

4. The microbial agent according to claim 3, characterized in that, The effective viable bacterial concentration of *Laenia granatum* DG-1 in the bacterial agent is 2 × (10⁻⁶)⁻¹. 8 ~10 9 CFU / mL.

5. The use of the aquatic Ranelia DG-1 according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in at least one of the following: 1) Improve the disease resistance of lilies; 2) Increase the abundance of probiotics and / or decrease the abundance of pathogens; 3) To prevent and / or alleviate yellowing of lilies; 4) Increase the chlorophyll content and the ratio of chlorophyll to flavonoids in lily leaves; 5) Enhances the transport and metabolism of amino acids in lilies; 6) Promotes the absorption and utilization of nitrogen and / or potassium by lilies; 7) Promotes the metabolism and activation of iron in lilies; 8) Increase the content of endogenous salicylic acid hormones in lilies; 9) Promotes lily growth and / or increases lily yield; 10) Promote photosynthesis in lilies and / or improve the photosynthetic nitrogen utilization rate in lilies; 11) Alleviate the obstacle of continuous cropping of lilies.

6. The application according to claim 5, characterized in that, The lilies mentioned include Lanzhou lilies.

7. A method for alleviating lily yellowing and / or reducing lily continuous cropping obstacles, characterized in that, Includes at least one of the following steps: The lily seeds were soaked in the fungal agent described in any one of claims 2 to 4 before sowing. The commercial lily bulbs were soaked in the fungal agent described in any one of claims 2 to 4 before transplanting. The above-ground parts of lilies are sprayed with the fungal agent described in any one of claims 2 to 4.

8. The method according to claim 7, characterized in that, When soaking lily bulblets or commercial seed bulbs, the effective viable concentration of *Laenia granatum* DG-1 in the bacterial agent is 5 × (10⁻⁶)⁻¹. 6 ~10 7 CFU / mL; the soaking time is 30-60 min.

9. The method according to claim 8, characterized in that, The soaking time is 30 to 45 minutes.

10. The method according to claim 7, characterized in that, When foliar spraying the above-ground parts of the lily, spray once every 4 weeks, for a total of 3-4 times; based on the viable count of the aquatic Raenella DG-1, the spraying amount each time is 4 × (10⁻⁶)⁻¹. 11 ~10 12 )CFU / mu.

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