Achromobacter ip1 and application thereof

Abstract

The application discloses a strain of achromobacter IP1 and application thereof, and belongs to the technical field of microorganisms. The achromobacter IP1 has a preservation number of CGMCC NO.34256, and is isolated from peanut rhizosphere soil in a peanut and corn intercropping system. The achromobacter IP1 provided in the application has strong abilities of producing an iron carrier and preventing and controlling a root-knot nematode, the iron carrier secreted by the achromobacter IP1 can effectively inhibit egg hatching of the root-knot nematode, and has lethality to larvae of the root-knot nematode. The achromobacter IP1 provided in the application and / or a fermentation product thereof are prepared into a bacterial agent, so that the bacterial agent can reduce infection of the root-knot nematode on peanuts, promote absorption of trace elements iron by plants, especially can successfully colonize in peanut rhizosphere soil, improve photosynthetic capacity of peanuts and promote growth, and has great application potential in integration of a medicine and a fertilizer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a strain of achromobacter IP1 and its application. BACKGROUND

[0002] During the cultivation of peanuts, the synergistic occurrence of iron deficiency chlorosis and root-knot nematode disease seriously restricts the improvement of crop yield and quality. In the northern calcareous soil, iron elements are easily fixed in an invalid state, leading to typical iron deficiency symptoms in peanuts, and the photosynthetic efficiency and metabolic function of peanuts are significantly reduced. At the same time, the imbalance of soil micro-ecology caused by continuous cropping leads to the rapid increase of the population of root-knot nematodes (Meloidogyne spp.), which form root knot structures after invading the roots, further hindering the absorption and transport of nutrients such as iron by plants, forming a vicious cycle of iron deficiency-nematode damage. Traditional solutions mostly use the strategy of separate application of iron fertilizer and chemical pesticide, but there are problems such as low utilization rate of iron fertilizer, poor targeting of pesticide, and inability to inhibit egg hatching, which not only makes the prevention and control effect unstable, but also easily causes resource waste and environmental pollution.

[0003] In the environment of mineral nutrient deficiency, microorganisms have formed a special mechanism for the activation, absorption and transport of insoluble nutrients during long-term evolution, i.e. secreting microbial iron carriers to activate soil trace elements to ensure the effective absorption of soluble nutrients. The application of bacteria that activate soil nutrients not only can alleviate the negative effects of nutrient deficiency on plants and directly or indirectly promote the growth of plants, but also can reduce various soil-borne diseases through iron competition mechanism. However, the existing microbial control technology usually only has single functional characteristics: some rhizosphere growth-promoting bacteria can activate soil iron elements by secreting iron carriers, but have no significant inhibitory effect on root-knot nematodes; while some biocontrol strains can inhibit the activity of nematode adults, but lack the ability to secrete iron carriers, and have limited effect on blocking egg hatching. This functional fragmentation phenomenon makes it difficult for microbial preparations to simultaneously solve the problem of iron deficiency and nematode complex damage, which restricts the practical application effect of the integration technology of fertilizer and pesticide.

[0004] In view of this technical bottleneck, it is urgent to explore microbial resources with both iron carrier secretion and broad-spectrum nematode inhibition functions. The key is to obtain strains that can not only efficiently chelate soil iron elements to improve plant iron nutrition, but also can simultaneously target and kill root-knot nematode adults and inhibit egg hatching. SUMMARY

[0005] The application discloses an Achromobacter xylosoxidans IP1 and application thereof, and aims to solve the problems in the prior art.

[0006] To achieve the above object, the application provides the following scheme.

[0007] The application provides an Achromobacter xylosoxidans IP1, and the preservation number of the Achromobacter xylosoxidans IP1 is CGMCC NO.34256.

[0008] The application further provides an iron carrier secreted by the Achromobacter xylosoxidans IP1.

[0009] The application further provides a microbial agent, which comprises the Achromobacter xylosoxidans IP1 and / or a fermentation product thereof.

[0010] Optionally, the fermentation product comprises the iron carrier.

[0011] Optionally, in the microbial agent, the bacterial activity of the Achromobacter xylosoxidans IP1 is 1x10 9 CFU / mL.

[0012] Optionally, in the microbial agent, the concentration of the iron carrier is 40-1000 mu M.

[0013] The application further provides application of the Achromobacter xylosoxidans IP1 or the microbial agent in inhibiting root-knot nematodes.

[0014] Optionally, the inhibition of root-knot nematodes comprises inhibition of egg hatching of root-knot nematodes and lethality to larvae of root-knot nematodes.

[0015] The application further provides application of the Achromobacter xylosoxidans IP1 or the microbial agent in preparation of a medicine-fertilizer integrated product, and the medicine-fertilizer integrated product is used for preventing and controlling soil root-knot nematode diseases, promoting plants to absorb iron nutrition, promoting plant growth and improving plant yield.

[0016] Optionally, the plants comprise peanuts.

[0017] The application discloses the following technical effects:

[0018] The present application isolates an achromobacter IP1 from the rhizosphere soil of peanuts in a peanut-corn intercropping system. The achromobacter IP1 provided by the present application is a gram-negative bacterium without spores, has a strong ability to produce siderophores and control root-knot nematodes, the siderophores secreted by the achromobacter IP1 can effectively inhibit the hatching of root-knot nematode eggs, and have lethality to root-knot nematode larvae. The achromobacter IP1 provided by the present application can convert insoluble mineral nutrients in the soil into available nutrients, at the same time, can reduce the infection of root-knot nematodes on peanuts, promote the absorption of trace element iron by plants, especially can successfully colonize in the rhizosphere soil of peanuts, improve the photosynthetic capacity of peanuts and promote the growth of peanuts, and has great application potential in the integration of medicine and fertilizer. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0020] Figure 1 It is a colony phenotype diagram of the strain IP1;

[0021] Figure 2 It is a phylogenetic tree constructed according to 16S rDNA;

[0022] Figure 3 It is an effect diagram of the achromobacter IP1 inhibiting root-knot nematodes, wherein a is an observation diagram under a stereomicroscope after treatment with different concentrations of microbial siderophores; b is a statistical diagram of the lethality of root-knot nematodes to different concentrations of microbial siderophores; c is a statistical diagram of the chemotaxis index of root-knot nematodes to different concentrations of microbial siderophores; d is the inhibitory effect of different days of treatment with microbial siderophores at the optimal nematode inhibition concentration of 1000 μM on the hatching of root-knot nematode eggs;

[0023] Figure 4 It is an effect diagram of the achromobacter IP1 reducing peanut root-knot nematode disease, wherein a is a phenotype diagram of peanuts in different treatment groups; b is the number of root-parasitic nematodes of peanuts in different treatment groups; c is the number of root knots of peanuts in different treatment groups;

[0024] Figure 5 It is an effect diagram of the achromobacter IP1 improving the iron nutrition of peanuts, wherein a is the SPAD value of new leaves of peanuts in different treatment groups; b is the active iron content of new leaves of peanuts in different treatment groups; c is the effective iron content of soil of peanuts in different treatment groups;

[0025] Figure 6The graph shows the effect of Achromobacterium IP1 on peanut growth, where a represents the peanut plant height in different treatment groups; b represents the fresh weight of peanut roots in different treatment groups; and c represents the fresh weight of peanut plants in different treatment groups. Detailed Implementation

[0026] This invention provides a strain of *Achromobacter xylosoxidans*, isolated from the rhizosphere soil of peanuts in a peanut-maize intercropping system, named *Achromobacter xylosoxidans* IP1 (hereinafter referred to as IP1). 16S rRNA gene sequence analysis confirmed that strain IP1 belongs to the genus *Achromobacter xylosoxidans*. This strain was deposited on April 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCCNO.34256.

[0027] The colony and cell characteristics of strain IP1 are as follows: colonies are round with a raised center, pale yellow in color, and have smooth, regular edges; the cells are moist and translucent. Microscopic observation reveals rod-shaped, single-celled cells that are Gram-negative and do not form spores.

[0028] The physiological and biochemical characteristics of strain IP1 are as follows: aerobic bacteria, gelatin liquefaction capability, siderophore production, catalase production, starch hydrolysis capability, citrate utilization capability, phosphorus solubility, potassium solubility, and auxin production capability.

[0029] The present invention provides a microbial agent containing the aforementioned Achromobacterium IP1 and / or its fermentation products.

[0030] Furthermore, the fermentation product includes siderophores secreted by the achromobacterium IP1.

[0031] Preferably, the microbial agent is a plant root-knot nematode control agent, a plant growth-promoting microbial agent, or a plant mineral nutrition improver.

[0032] Preferably, the bacterial agent contains an effective viable count of Achromobacterium IP1 of not less than 1 × 10⁻⁶. 9 CFU / mL.

[0033] Furthermore, the fermentation of the aforementioned Achromobacterium IP1, yielding a microbial siderophore, also falls within the scope of protection of this invention.

[0034] In specific embodiments of the present invention, experiments have confirmed that *Achromobacterium IP1* possesses high iron-carrier function, and has effects such as controlling peanut root-knot nematode disease, improving peanut mineral nutrition, and promoting peanut production. The growth-promoting agent prepared from *Achromobacterium IP1* can activate available iron in the peanut rhizosphere soil, promote root growth, enhance the root system's ability to absorb iron, and increase plant dry weight.

[0035] Specifically, the control of peanut root-knot nematode disease is achieved by inhibiting the hatching of root-knot nematode eggs, killing root-knot nematodes, and repelling root-knot nematodes, thus suppressing root-knot nematodes throughout their entire life cycle.

[0036] Furthermore, the Achromobacterium IP1 and / or its fermentation products provided by this invention can reduce root-knot nematode infection of the root system, reduce the root knot index, and eliminate root-knot nematode disease in crops during the crop growth period.

[0037] Furthermore, the Achromobacterium IP1 and / or its fermentation products provided by this invention can chelate insoluble iron in the soil, increase the available iron content in the soil, enhance the absorption and utilization of iron by crops, improve crop iron nutrition, and promote plant growth.

[0038] Preferably, in the above applications, the plant includes peanuts. Those skilled in the art will understand that applying the Achromobacterium IP1 of the present invention to the growth of other crops also has similar disease control and growth-promoting effects as described above.

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0041] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0042] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0043] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0044] The culture medium, formulation, reagents, and preparation methods used in the embodiments of this invention are as follows:

[0045] Standard succinic acid medium (SSM): (NH4)2SO4 1g, MgSO4·7H2O 0.2g, succinic acid 4g, K2HPO4 6g, KH2PO4 3g, add distilled water to 1L, and adjust pH to 7 with KOH.

[0046] Preparation of CAS blue detection solution: 1 mM ferric chloride stock solution: 0.2703 g ferric chloride hexahydrate dissolved in 1 L of 10 mM HCl; CAS stock solution: 0.2421 g CAS (chromium azurite) dissolved in 200 mL of deionized water; HTDMA solution (hexadecyltrimethylammonium bromide): 0.0219 g HTDMA dissolved in 50 mL of water; piperazine buffer: 4.3079 g anhydrous piperazine dissolved in 30 mL of water, adjusted to pH 5.6 with hydrochloric acid. After preparing the above solutions, add 1.5 mL of 1 mM ferric chloride stock solution to 7.5 mL of CAS stock solution, then add 50 mL of HTDMA solution while stirring, followed by 30 mL of piperazine buffer, and finally add deionized water to bring the volume to 100 mL.

[0047] Example 1: Isolation, Screening and Identification of Strains

[0048] 1. Isolation and screening of strains

[0049] Soil samples were taken from the rhizosphere of intercropped peanuts in a peanut-corn intercropping system. The soil samples were resuspended in sterile water and cultured on CAS solid medium. Strains with strong siderophore production capacity were selected using CAS blue detection solution. After further screening and breeding, a purified strain was obtained and named IP1.

[0050] 2. Morphological and physiological-biochemical characteristics of strain IP1

[0051] (1) The colony characteristics and cell morphology of strain IP1 are as follows: the colonies are round with a raised center, pale yellow in color, and smooth and regular edges. The cells are moist and translucent. Microscopic observation shows that the cell morphology is rod-shaped, single-celled, Gram-negative, and without spores. Figure 1 ).

[0052] (2) Determination of physiological and biochemical characteristics of strain IP1

[0053] Aerobicity test: The aerobicity of bacteria can be determined by whether they can survive in an anaerobic environment. After culturing strain IP1 to the logarithmic growth phase in LB medium, bacterial suspension was punctured with a needle and inserted into LB solid medium. The medium was then incubated at 28°C for 2 days. If growth occurs only on the surface of the medium, the bacteria are aerobic; if growth occurs only along the puncture line, the bacteria are anaerobic; and if growth occurs on both the surface and along the puncture line, the bacteria are facultative anaerobic.

[0054] Liquefaction Capacity Assay: Bacteria capable of liquefying gelatin can hydrolyze gelatin into amino acids for utilization by producing extracellular gelatinase, simultaneously transforming semi-solid gelatin culture medium into a fluid state. Strain IP1 was cultured in SSM medium to the logarithmic growth phase. Bacterial suspension was then punctured into the gelatin culture medium using a puncture needle and incubated at 28°C for 2 days. The medium was then placed in a 4°C refrigerator for 30 minutes. Afterward, the test tube was tilted and observed. Significant liquefaction indicates a positive gelatin liquefaction capacity; no significant liquefaction indicates a negative result.

[0055] Siderophore production capacity determination: The supernatant of the fermentation broth of strain IP1 was mixed with CAS detection solution at a 1:1 ratio and incubated in the dark for two hours. The absorbance was measured at 630 nm using an ELISA reader. The absorbance values ​​of the mixture of control and CAS detection solution (denoted as Ar) and the mixture of treatment solution and CAS detection solution (denoted as As) were measured separately. A standard curve was constructed using the commercially available microbial siderophore desferrioxamine B (DFOB) to accurately determine the siderophore production capacity. The siderophore concentration was calculated using the formula: Siderophore concentration SP (μM) = 32.99 × (Ar - As) / Ar + 0.2746.

[0056] Catalase production capacity assay: Bacteria with catalase activity can catalyze hydrogen peroxide into water and molecular oxygen, forming bubbles. After culturing strain IP1 in LB medium to the logarithmic growth phase, the liquid culture medium is dipped into a sterile test tube using an inoculation loop, and 2 mL of 3% hydrogen peroxide solution is added. If bubbles are produced within half a minute, the catalase production capacity is positive; if no bubbles are produced, the capacity is negative.

[0057] Starch hydrolysis ability test: Some bacteria can hydrolyze starch into dextrin, maltose, glucose, and other substances for utilization. After culturing strain IP1 in LB medium to the logarithmic growth phase, 100 μL was inoculated into the center of a starch-hydrolyzing solid medium and incubated at 28°C for 2 days. A small amount of iodine solution was dropped onto the plate, and the plate was rotated until the iodine solution was uniform. If the medium turned dark blue and a colorless transparent zone appeared around the colony, the starch hydrolysis ability was positive; if no colorless transparent zone appeared, the result was negative.

[0058] Citrate utilization ability test: Some bacteria can utilize citrate as a carbon source and decompose it into carbon dioxide. During this process, the pH of the culture medium becomes alkaline due to the presence of sodium ions. After culturing strain IP1 in LB medium to the logarithmic growth phase, a loop is used to streak the bacterial suspension onto citrate-based solid medium and incubate at 28°C for 2 days. If the medium changes from green to dark blue, the citrate utilization ability is positive; if it remains green after another 7 days of incubation, the ability is negative.

[0059] Phosphate-solubilizing capacity determination: Strain IP1, in its logarithmic growth phase, was inoculated at a 1% inoculum into phosphate-solubilizing medium and cultured at 28°C and 180 rpm for 2 days. The bacterial culture was then diluted to an OD value. 600 = 1 After centrifugation at 10000 rpm for 30 min, the supernatant was diluted 10 times, and 2 mL of the diluted solution was added to 2 mL of molybdenum antimony colorimetric solution. After standing for 30 min, the wavelength at 880 nm was measured, and a phosphorus standard curve was plotted using phosphorus standard solution prepared with KH2PO4. The phosphorus-solubilizing ability of the bacteria was calculated based on the phosphorus standard curve and the wavelength of the bacterial solution at 880 nm.

[0060] Potassium solubilizing ability determination: Strain IP1, in its logarithmic growth phase, was inoculated at a 1% inoculum into potassium-solubilizing medium and cultured at 28°C and 180 rpm for 2 days. The bacterial culture was then diluted to OD0.05. 600 = 1 After centrifugation at 10000 rpm for 30 min, the supernatant was collected and the concentration of water-soluble potassium was determined by flame photometry. A potassium standard solution was prepared with KCl and a potassium standard curve was plotted. The potassium solubilization ability of the strain was calculated based on the potassium standard curve and the potassium concentration of the supernatant.

[0061] Assay for tryptophan production capacity: 100 mg of tryptophan was filtered through a 0.2 μm filter and sterilized, then added to 1 L of autoclaved LB medium. Strain IP1, in the logarithmic growth phase, was inoculated at a 1% inoculum and cultured at 28°C and 180 rpm for 2 days. The bacterial culture was then diluted to OD0.05. 600 = 1. After centrifugation at 10000 rpm for 30 min, take 1 mL of the supernatant and mix it with Salkowski colorimetric solution (containing 50 mL of 35% HClO4 solution and 0.5 mol·L⁻¹). -1Mix equal volumes of FeCl3 (1 mL) and incubate at 40°C for 30 min. Measure the absorbance of the mixture at 530 nm. Prepare a standard solution with indoleacetic acid and plot a standard curve. Calculate the strain's ability to produce auxin based on the standard curve and the absorbance at 530 nm.

[0062] The test results showed that strain IP1 is an aerobic bacterium that can liquefy gelatin, produce siderophores, produce catalase, hydrolyze starch, utilize citrate, and has the ability to solubilize phosphorus, potassium, and produce auxin.

[0063] 3. 16S rDNA sequence determination and analysis of strain IP1

[0064] The 16S rDNA sequence of strain IP1 was amplified by PCR, and the 16S rDNA sequence is as follows:

[0065] IP1(5'-3'):

[0066]

[0067] The BLAST alignment of its 16S rDNA sequence yielded the following results: Figure 2 As shown, Figure 2 The results showed that IP1 had a similarity of over 98% with several different strains of Achromobacter xylosoxidans. Based on the morphology, culture characteristics and physiological and biochemical analysis results of the strain, strain IP1 was identified as Achromobacter xylosoxidans.

[0068] This strain was deposited on April 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and was classified as Achromobacter xylosoxidans, with accession number CGMCC No. 34256.

[0069] Example 2: Inhibitory effect of siderophores secreted by strain IP1 on root-knot nematodes.

[0070] 1. Determination of the siderophore production capacity of strain IP1

[0071] Strain IP1 was cultured in SSM medium at 28°C and 180 rpm for 2 days. After culturing, the culture was centrifuged at 9500 rpm for 25 min to remove bacterial cells. The supernatant was mixed with CAS detection solution at a volume ratio of 1:1, and the absorbance was measured at 630 nm. A standard curve was constructed using deferoxamine B (DFOB) to evaluate the relative yield of siderophores. Simultaneously, the microbial siderophores in the supernatant were purified and collected by column chromatography for later use.

[0072] 2. Determination of the inhibitory rate of siderophores secreted by strain IP1 on the hatching of root-knot nematode eggs.

[0073] Root-knot nematode egg masses were collected into 10 mL centrifuge tubes, 1% to 5% sodium hypochlorite was added, and the mixture was left to stand for 3 minutes. The sodium hypochlorite was then removed by pipetting, and the mixture was washed repeatedly with deionized water 3 to 5 times. The mixture was then vortexed for 3 to 5 minutes. The vortexed suspension was then passed through a double sieve (200 mesh upper sieve, 500 mesh lower sieve). The upper sieve was used to filter out impurities such as plant tissue residues, while the lower sieve was used to collect the eggs. The egg suspension was rinsed with deionized water, and the density of the egg suspension was adjusted to 100 eggs / mL.

[0074] The siderophore secreted by strain IP1 was prepared at a concentration of 1000 μM, mixed with the egg suspension, and incubated for 5 days with continuous observation. The number of hatched nematodes was counted, and the inhibition rate of microbial siderophore on nematode egg hatching was calculated.

[0075] 3. Determination of the lethality of the siderophores secreted by strain IP1 against root-knot nematodes.

[0076] Siderophores secreted by strain IP1 were prepared at different concentrations (20 μM, 40 μM, 400 μM, 800 μM, and 1000 μM). 200 μL of each siderophore concentration was added to a 24-well cell culture dish, along with 10 μL of a nematode suspension containing approximately 100 second-stage root-knot nematode larvae (J2s). Deionized water was used as a control, and each treatment was performed in quadruplicate. The dishes were incubated at 27°C in the dark. After 24 hours, 10 μL of 1M NaOH was added to stimulate the nematodes. The dishes were gently shaken, and the total number of nematodes and the number of dead nematodes were observed and counted under a stereomicroscope within 3 minutes. Nematodes that still exhibited motility after the addition of NaOH were considered alive, while those that did not respond to NaOH were considered dead. The nematode mortality rate was calculated.

[0077] 4. Analysis of the directional movement of root-knot nematodes

[0078] The root-knot nematode tacticity assay was validated using a petri dish simulation. A 9 cm diameter petri dish was used, with 15 mL of 1.0% agar added. Siderophores secreted by strain IP1 were diluted with sterile water to different concentration gradients (20 μM, 40 μM, 400 μM, 800 μM, and 1000 μM), with sterile water as a control. Two 2 cm diameter circles were marked 1 cm from the center of the bottom of the petri dish. 50 μL of the treatment solution was added to one circle, and 50 μL of sterile water was added to the other circle as a control. The petri dishes were placed at 25°C in the dark to allow the microbial siderophores to fully diffuse on the agar. After 3 h, 20 μL of a J2s suspension containing 100 newly hatched southern root-knot nematodes was added to the center of the petri dish, and the dish was then covered. After being kept in the dark at 25°C for 4 hours, the number of nematodes in the circles on both sides was counted under an optical microscope, and the root-knot nematode chemotaxis index was calculated.

[0079] 5. Results

[0080] The inhibitory effect of Achromobacterium IP1 on root-knot nematodes, such as Figure 3 As shown, by Figure 3 It was found that the inhibitory effect of *Achromobacterium IP1* on root-knot nematodes increased with increasing concentration of its secreted siderophores. An inhibitory effect began to appear at a siderophore concentration of 40 μM, and the inhibitory effect was highest at a concentration of 1000 μM. At a concentration of 1000 μM, the siderophores secreted by *Achromobacterium IP1* had a lethality rate of 68.18% against root-knot nematodes, an egg hatching inhibition rate of 10.9%, and a repulsion index of -0.62.

[0081] Example 3: Effects of Achromobacterium IP1 on reducing peanut root-knot nematode disease

[0082] I. Experimental Methods

[0083] Achromobacterium IP1 was cultured in SSM liquid medium at 28°C until it reached 1×10⁻⁶. 9 The microbial siderophores were centrifuged at 4000 rpm for 10 min in a high-speed centrifuge at cfu / mL. The supernatant was discarded, and the remaining precipitate was added to an equal volume of sterile 10 g / L NaCl solution to prepare a bacterial suspension. At the same time, the microbial siderophores in the supernatant were purified and collected by column chromatography for later use.

[0084] Peanuts of the Luhua 14 variety were transplanted in the greenhouse of the College of Resources and Environment, China Agricultural University. Root-knot nematode inoculation and treatment were initiated in the first week after thinning. The treatment numbers and application methods are shown in Table 1.

[0085] Table 1 Grouping and Processing

[0086]

[0087] Five biological replicates were performed for each treatment. Samples were collected from the control group when root-knot nematode symptoms and iron deficiency chlorosis phenotypes were observed. The number of root knots, root-parasitic nematodes, SPAD value of new leaves, active iron content of new leaves, available iron content in the soil, and plant biomass were measured.

[0088] Peanut root knot number determination: After cleaning the roots, observe and count them directly with the naked eye. Since root knots and root nodules formed by rhizobia that fix nitrogen in peanuts are sometimes difficult to distinguish, when observing, cut open the protrusions on the roots. Those containing pear-shaped milky white female insects are root knots, otherwise they are root nodules.

[0089] Root-parasitic nematode count: Root nematodes were isolated using the shallow dish method. Roots were dried by blotting with absorbent paper, cut into 1 cm lengths, and weighed (5 g). A 20-mesh sieve was placed in a nematode collection dish, lined with a paper towel. The chopped peanut root samples were evenly spread on the paper towel, and 100 mL of deionized water was poured into the collection dish. The dish was then covered with another collection dish and placed in the dark. After 24 hours, the soil sieve was removed, and the nematode suspension in the shallow dish was collected, transferred, and diluted to a 50 mL centrifuge tube. 1 mL of the suspension was taken and the number of root nematodes was counted using a stereomicroscope. A total of 3 mL was counted from each sample, and the average value was used to calculate the root nematode density.

[0090] Determination of active iron in new leaves: Take 2g of fresh peanut leaves, cut them into pieces, add 10 mL of 1M hydrochloric acid, shake for 5 h, filter with quantitative filter paper, and determine the iron concentration in the filtrate by inductively coupled plasma atomic emission spectrometry. The concentration of active iron in the leaves is then converted.

[0091] Determination of available iron in soil: Dissolve 1.967 g of diethylenetriaminepentaacetic acid (DTPA) in 14.92 g of triethanolamine (TEA), then add 1.47 g of CaCl2·2H2O, adjust the pH to 7.3, and bring the volume to 1 L to obtain the DTPA extractant. Add 20 mL of DTPA extractant to 10 g of soil sample and shake at 200 rpm for 2 h at 25℃. Filter the soil suspension with filter paper and determine the iron content using inductively coupled plasma atomic emission spectrometry (ICP-AES). The calculated available iron content is then obtained.

[0092] II. Experimental Results

[0093] Experimental results are as follows Figures 4-6 As shown.

[0094] Figure 4 The results showed that, compared with the control group treated only with root-knot nematodes, *Achromobacterium IP1* and its fermentation product, the microbial siderophore, significantly reduced the number of root-parasitic nematodes and root knots, decreasing the number of root-parasitic nematodes by an average of 16.7% and 30.7%, and the number of root knots by an average of 17.3% and 32.7%, respectively. Therefore, *Achromobacterium IP1* and its fermentation product, the microbial siderophore, can be applied to the control of root-knot nematode diseases.

[0095] Figure 5 The results showed that, compared with the blank control and the control group treated only with root-knot nematodes, *Achromobacterium* IP1 and its fermentation product, the microbial siderophore, significantly increased the SPAD value, active iron content, and available iron content in peanut new leaves. Specifically, it increased the SPAD value of peanut new leaves by 26.3% and 17.8%, active iron content by 73.4% and 87.6%, and available iron content in the soil by 25.2% and 20.0%, respectively. Therefore, inoculation with *Achromobacterium* IP1 significantly promoted the activation of available iron in the soil, improved iron nutrition in peanuts, and enhanced photosynthesis in leaves.

[0096] Figure 6 This indicates that *Achromobacterium IP1*'s control of root-knot nematodes and improvement of iron nutrition ultimately promoted peanut growth. Compared to the control group treated only with root-knot nematodes, *Achromobacterium IP1* and its fermentation product, the microbial siderophore, significantly increased peanut plant height, root fresh weight, and plant fresh weight. Plant height increased by 22.8% and 22.5%, respectively; root fresh weight increased by 73.8% and 50.5%, respectively; and peanut biomass increased by 20.9% and 24.9%, respectively.

[0097] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of xylose-oxidizing achromobacterium ( Achromobacter xylosoxidans IP1, characterized in that, The preservation number of the xylose-oxidizing achromobacterium IP1 is CGMCC NO.34256; the bacterial suspension of the xylose-oxidizing achromobacterium IP1 or its secreted siderophores can inhibit the hatching of root-knot nematode eggs and kill the larvae of root-knot nematodes.

2. A microbial inoculant, characterized in that, The microbial agent includes Xylose-oxidizing Achromobacterium IP1 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, In the microbial inoculant, the viability of Achromobacterium IP1 is 1×10⁻⁶. 9 CFU / mL.

4. The application of the xylose-oxidizing achromobacterium IP1 as described in claim 1 or the microbial agent as described in claim 2 or 3 in the inhibition of root-knot nematodes.

5. The application according to claim 4, characterized in that, The inhibition of root-knot nematodes includes inhibiting the hatching of root-knot nematode eggs and causing death to root-knot nematode larvae.

6. The application of the xylose-oxidizing achromobacterium IP1 according to claim 1 or the microbial agent according to claim 2 or 3 in the preparation of integrated pesticide-fertilizer products, characterized in that, The integrated pesticide and fertilizer product is used to control soil root-knot nematode disease, promote plant iron absorption, promote plant growth, and increase plant yield. The plant in question is a peanut.

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