Rhodococcus bacteria X-20 and application thereof

Abstract

The invention relates to the technical field of environmental microbiological technology and agricultural bioremediation, and discloses a rhodococcus bacterium X-20 and application thereof. The Rhodococcus sp. X-20 can efficiently degrade various pesticides in liquid and soil environments, including carbendazim, dinotefuran, atrazine, pendimethalin and cyhalothrin, and especially shows specific efficient degradation ability to carbendazim. In addition, the strain has various plant probiotic functions: the strain can secrete indole-3-acetic acid (IAA), generate iron carriers, dissolve inorganic phosphorus and antagonize phytophthora nicotianae, and the growth of tobacco seedlings can be remarkably promoted. Field experiments show that the strain can relieve growth inhibition of compound pesticide pollution on tobacco, and the strain can be used for preparing microbial agents and applied to bioremediation of pesticide-polluted soil and green agriculture.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental microorganism technology and agricultural bioremediation technology, in particular to a strain of Rhodococcus bacteria X-20 and its application. BACKGROUND

[0002] Pesticide residues are a major environmental problem in current agricultural production, which poses a threat to soil ecosystems and crop safety. The existing physical and chemical remediation methods are high in cost and easy to cause secondary pollution, while microbial remediation technology has gradually become a research hotspot due to its environmental friendliness, low cost, and no residues. In addition, most of the reported pesticide-degrading bacteria have single function and can only degrade specific types of pesticides, making it difficult to cope with the actual complex pollution scene in the field. At present, some strains of Rhodococcus have been reported to have the ability to degrade carbendazim, but most of the strains have limited degradation efficiency and single function, and lack systematic field application verification. SUMMARY

[0003] In order to solve the problems existing in the prior art, one of the purposes of the present application is to provide a strain of Rhodococcus bacteria (Rhodococcus sp.) X-20. Rhodococcus sp. The strain has been deposited with the Guangdong Microbial Culture Collection Center on September 17, 2025, at the address of No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard, and the strain is classified and named as Rhodococcus sp. Rhodococcus sp. , with the preservation number of GDMCC No: 66973.

[0004] The second aspect of the present application is to provide the application of the Rhodococcus bacteria X-20 in any one of the following: (1) application in degrading complex pesticides.

[0005] (2) application in preparing indole acetic acid.

[0006] (3) application in preparing siderophores.

[0007] (4) application in preparing a reagent for inhibiting Phytophthora nicotianae.

[0008] (5) application in preparing a reagent for inhibiting tobacco black shank.

[0009] Preferably, the complex pesticide is one or more of dinotefuran, carbendazim, atrazine, pendimethalin, or lambda cyhalothrin.

[0010] Compared with the prior art, the present application provides a strain of Rhodococcus bacteria X-20 and its application, which has the following beneficial effects: (1) Broad-spectrum and high-efficiency degradation ability: the strain X-20 described in the application can simultaneously degrade dinotefuran, atrazine, pendimethalin, lambda-cyhalothrin and carbendazim in soil or water, wherein the degradation efficiency of the soil carbendazim is the highest, up to 97.78%.

[0011] (2) Strong plant growth promotion and stress resistance function: X-20 can secrete IAA, produce siderophores, and antagonize Phytophthora parasitica, and can effectively alleviate the toxic effects of pesticides on plants from the two dimensions of “growth promotion” and “stress resistance”, and improve the health level of plants.

[0012] (3) The strain X-20 described in the application has multiple effects, and provides a microorganism with multiple effects for agricultural production. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is a colony morphology diagram of the strain X-20 on R2A agar medium.

[0014] Figure 2 It is an evolutionary tree analysis of the strain X20.

[0015] Figure 3 It is the degradation rate of the strain X-20 on five kinds of pesticides in liquid medium.

[0016] Figure 4 It is the degradation efficiency of the strain X-20 on mixed pesticides in contaminated soil.

[0017] Figure 5 It is a beneficial function verification diagram of the strain X-20 (a: siderophore production; b: phosphorus solubilization ring; c: IAA yield; d, e: antagonizing Phytophthora parasitica).

[0018] Figure 6 It is the effect of the strain on tobacco growth.

[0019] Figure 7 It is the effect of the strain on tobacco growth in compound pesticide-contaminated field soil.

[0020] Figure 8 It is the growth status statistics of tobacco in each group in the field experiment. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, not all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0022] Unless otherwise specified, the reagents used in the present application are all routinely commercially available analytical pure.

[0023] Example 1 Isolation, screening and identification of strain X-20 Strain X-20 was isolated from the stem tissue of tobacco plants in Yunnan Province, Kunming City, Yiliang County, and the specific steps are as follows: (1) After the sample was surface sterilized and ground, it was used as an inoculum to be inoculated into an inorganic salt medium (MSM) with dinotefuran, carbendazim, atrazine, pendimethalin and lambda-cyhalothrin (initially 50 mg / L each) as the only carbon source, and cultured at 30°C with 160 rpm shaking for 7 days to obtain a bacterial liquid.

[0024] (2) Then the bacterial liquid was transferred to MSM with increasing pesticide concentrations (50 mg / L to 300 mg / L) in multiple rounds of directional acclimation with a 5% inoculation amount. After acclimation, the culture liquid was gradiently diluted and plated on R2A plates, and after two purifications, the pure culture strain X-20 was obtained. The colony morphology of strain X-20 on R2A agar medium is shown in Figure 1 from which it can be seen that the colony is light yellow, round, smooth and moist.

[0025] (3) After 16S rRNA gene sequence (about 1500 bp) analysis of strain X-20, the 16S rRNA gene sequence of strain X-20 is shown as SEQ ID NO: 1, and the similarity of strain X-20 with Rhodococcus qingshengii strain djl-6-2 (NR_115708) is 99.70%, as shown in Figure 2 phylogenetic analysis shows that it is clustered in the same evolutionary branch with the model strain, indicating that it belongs to Rhodococcus genus bacteria.

[0026] Example 2 Evaluation of pesticide degradation efficiency of strain X-20 1. Liquid degradation experiment: Strain X-20 was inoculated in inorganic salt medium (MSM) containing mixed pesticides (mixed pesticides including: dinotefuran 16.00 mg / L, carbendazim 15.84 mg / L, atrazine 18.90 mg / L, pendimethalin 5.05 mg / L or lambda-cyhalothrin 13.25 mg / L), and cultured at 30°C with 160 rpm shaking for 8 days. After that, the residual concentrations of various pesticides were determined by high performance liquid chromatography (HPLC), and the results are shown in Figure 3As shown, the concentration of each pesticide in the X-20 treatment group decreased to varying degrees. Among them, the degradation rate of dinotefuran was 55.41% (from 16.00 mg / L to 7.14 mg / L), the degradation rate of carbendazim was 86.42% (from 15.84 mg / L to 2.15 mg / L), the degradation rate of atrazine was 30.85% (from 18.90 mg / L to 13.07 mg / L), the degradation rate of pendimethalin was 27.41% (from 5.05 mg / L to 3.67 mg / L), and the degradation rate of lambda-cyhalothrin was 18.27% (from 13.25 mg / L to 10.83 mg / L).

[0027] 2. Soil degradation experiment: mixed pesticides (including dinotefuran, carbendazim, atrazine, pendimethalin and lambda-cyhalothrin) were added to the soil, and X-20 bacterial solution (10% v / w, OD 600 ≈1.0) was inoculated, and after 10 days of culture at 25°C, the residual concentration of each pesticide was determined by high performance liquid chromatography (HPLC). As shown in the figure, compared with the control group without inoculation of the strain, the residual concentration of each pesticide in the X-20 treatment group was significantly reduced, among which the degradation rate of dinotefuran was 40.94%, the degradation rate of carbendazim was 97.78%, the degradation rate of atrazine was 38.60%, the degradation rate of pendimethalin was 21.19%, and the degradation rate of lambda-cyhalothrin was 14.26%.

[0028] Example 3 Study on the probiotic properties of strain X-20 Salkowski colorimetric method, strain X-20 was inoculated in LB liquid medium containing L-tryptophan (100 mg / L), 28°C, 180 r / min shaking culture for 3d, 2mL bacterial solution was centrifuged (10,000 r / min, 10min), 1mL supernatant was added with equal volume of Salkowski reagent (35% HClO4 solution containing 0.5 mol / L FeCl3), and the OD 530 was measured after 30min of dark standing. The standard curve was drawn with IAA standard, and the IAA yield was calculated as 20.23μg / mL (as shown in Figure 5 c, CK is the blank control without adding bacteria).

[0029] Strain X-20 was inoculated on PVK (Pikovskaya) solid medium (containing insoluble calcium phosphate) and incubated at 28°C for 7 days, and the phosphorus solubilization ring was observed. The CAS (Chrome Azurol S) agar plate method was used, and strain X-20 was inoculated and incubated at 28°C for 4 days, and the orange halo was observed. The iron carrier secretion level was quantitatively evaluated by the ratio of halo diameter to colony diameter. Strain X-20 formed an obvious phosphorus solubilization ring on the PVK plate, and an obvious orange halo was produced on the CAS plate, indicating that the strain had the ability to produce iron carriers and solubilize phosphorus (as shown in Figure 5 a and 5b).

[0030] The plate confrontation method was used, and a P. nicotianae ( Phytophthora nicotianae ) cake (Φ = 5 mm) was inoculated in the center of the PDA medium, and strain X-20 was inoculated symmetrically at a distance of 2 cm from the cake, and incubated at 28°C for 6 days. The results showed that X-20 had significant inhibitory ability against P. nicotianae ( Phytophthora nicotianae ) (as shown in Figure 5 d and Figure 5 e).

[0031] The above results show that X-20 has multiple plant probiotic functions.

[0032] Example 4 Biocontrol pot experiment of strain X20 on tobacco black shank In order to confirm the prevention and treatment effect of strain X20 on tobacco black shank, a tobacco pot experiment was conducted. The variety name of the tested tobacco ( Nicotiana tabacum ) was Yunyan 87, which was purchased from a local agricultural products company and was self-preserved by the laboratory.

[0033] 1. Preparation of P. nicotianae grain Rice grains were placed in distilled water and boiled at high temperature until 2 / 3 of the grains were blooming. After draining the water with gauze, they were placed in a 500 mL conical flask and autoclaved at 121°C for 1 h. Meanwhile, P. nicotianae ( Phytophthora nicotianae ) was inoculated on a PDA plate and incubated at 28°C for 7 days. P. nicotianae mycelium was picked up with an inoculation loop and inoculated into the autoclaved grain medium, and incubated at 28°C until the mycelium covered the grain medium, obtaining P. nicotianae grain.

[0034] 2. Tobacco pot experiment Strain X20 was inoculated in LB liquid medium and cultured at 28°C for 7 days to obtain strain X20 fermentation broth. The test soil was collected from the tobacco field in Yiliang, Yunnan (red soil). The P. parasitica was added to the soil at a ratio of 1000 g of soil to 10 g of P. parasitica and mixed evenly, as the diseased soil. The Yunyan 87 seedlings with 5 true leaves were transplanted into pots containing 500 g of diseased soil, with 1 plant per pot. The control group and the treatment group were set up, with 4 replicates in each group, and 6 pots in each replicate. The control group: no strain X20 fermentation broth was applied. The treatment group: 5 mL of strain X20 fermentation broth was poured into the soil around the roots of the tobacco seedlings on the day of transplanting. All the tobacco seedlings were placed in a 28°C, 70% relative humidity environment for conventional cultivation. The incidence and incidence rate of tobacco black shank were counted on the 9th day after inoculation of P. parasitica (transplanting to diseased soil), and the disease index and disease prevention effect were calculated. The experimental results are shown in Table 1, and the prevention effect of strain X20 on tobacco black shank reached 48.30%.

[0035] Table 1 Example 5 Hydroponic experiment The hydroponic experiment was carried out in a greenhouse, using 1L black hydroponic boxes, each containing 0.8L Hoagland full nutrient solution (pH 6.0±0.2), 1 tobacco seedling with uniform growth at 30d after transplanting, the root was fixed with sterilized sponge and immersed 2cm below the liquid level; the test was divided into 2 groups: ① CK, only nutrient solution; ② X-20 treatment, adding 20ml of strain X-20 suspension with a concentration of 1×10 8 CFU / mL to the nutrient solution. Each treatment had 4 boxes, which were completely randomized. After 15 days of culture, samples were taken to measure plant height, root length and fresh weight. The results showed that (Table 2) Figure 6 ), X-20 had a significant growth-promoting ability on plants. Specifically, compared with the control group, the inoculation treatment significantly increased the plant height, fresh weight and root length of tobacco: the plant height increased from 22.67 cm to 29.13 cm (an increase of 28.50%), the fresh weight increased from 11.57 g to 16.87 g (an increase of 45.81%), and the root length increased from 16.07 cm to 22.67 cm (an increase of 41.07%).

[0036] Example 6 Effect of strain on tobacco growth in compound pesticide-contaminated field soil

[0037] The same field of tobacco was divided into 3 groups, and the soil in each group was detected to ensure that the growth environment of each group of tobacco was the same, and the following treatment groups were set: (1) blank control (CK): tobacco plants growing normally; (2) pesticide control group (PC): tobacco plants planted in mixed pesticide contaminated soil; (3) treatment group (T): tobacco plants planted in mixed pesticide contaminated soil and inoculated with bacterial liquid. The specific operation is as follows: 100 mL of mixed pesticide solution with a concentration of 85 mg / L is added to the root soil of each tobacco plant, and after the pesticide reaches a stable state in the soil, the treatment group is irrigated with 100 mL of bacterial suspension with OD 600 =1.0, and all growth indicators are uniformly measured at the 30th day of the experiment.

[0038] After applying strain X-20 bacterial liquid in the mixed pesticide contaminated tobacco farmland, the growth status of the experimental group of tobacco was significantly better than that of the mixed pesticide control group ( Figure 7 ). Specifically, the tobacco inoculated with X-20 bacterial liquid was significantly better than the control group in morphological indicators such as plant height, leaf length and leaf width ( Figure 8 ). Specifically, compared with the mixed pesticide control group, the leaf length of the X-20 inoculated treatment group increased from 32.72 cm to 62.06 cm (increased by 89.67%), the leaf width increased from 30.56 cm to 36.94 cm (increased by 20.88%), and the leaf width increased from 20.83 cm to 22.44 cm (increased by 7.73%).

Claims

1. A strain of Rhodococcus bacteria ( Rhodococcus sp. X-20, characterized in that: The accession number is: GDMCCNo:66973.

2. The application of the Rhodococcus bacteria X-20 as described in claim 1 in the degradation of compound pesticides.

3. The application according to claim 2, characterized in that: The compound pesticide is one or more of the following: fipronil, atrazine, pendimethalin, lambda-cyhalothrin, or carbendazim.

4. The use of the Rhodococcus bacteria X-20 as described in claim 1 in the preparation of indoleacetic acid.

5. The use of the Rhodococcus bacteria X-20 as described in claim 1 in the preparation of siderophores.

6. The use of the Rhodococcus bacteria X-20 as described in claim 1 in the preparation of a reagent to inhibit Phytophthora intoxin.

7. The use of the Rhodococcus bacteria X-20 of claim 1 in the preparation of a reagent to inhibit tobacco black shank disease.

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