CN113801816B

CN113801816B - Bacillus and application thereof in promoting growth and degrading chlorpyrifos in rice

Info

Publication number: CN113801816B
Application number: CN202111112565.5A
Authority: CN
Inventors: 葛静; 余向阳; 王亚; 黄博闻
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2021-09-18
Filing date: 2021-09-18
Publication date: 2023-01-03
Anticipated expiration: 2041-09-18
Also published as: CN113801816A

Abstract

The invention relates to a Bacillus (Bacillus sp.) with a preservation number of CGMCC No.22887 and application thereof in promoting growth, promoting rice enrichment and degrading chlorpyrifos; the bacterium can be prepared into a bacterium agent, and can effectively promote the absorption of rice roots to chlorpyrifos and promote rice plants to degrade the chlorpyrifos after being colonized in rice. The Bacillus aryabhattai belongs to plant endophytic bacteria, can be colonized on rice plants for a long time, can promote the growth of rice under the stress of chlorpyrifos, can effectively promote the enrichment and degradation of the chlorpyrifos in the rice, is favorable for reducing pesticide residues in the environment and crops, and is favorable for ensuring the safety of the environment and agricultural products.

Description

Bacillus and application thereof in growth promotion and degradation of chlorpyrifos in rice

Technical Field

The invention relates to Bacillus aryabhattai and application thereof in promoting plant growth and degrading chlorpyrifos in rice, and belongs to the fields of microbiology, environmental health and safety.

Technical Field

Chlorpyrifos is one of the commonly used insecticides in rice fields, and has important effect on preventing and controlling rice pests and ensuring the rice yield. Chlorpyrifos, a representative product of organophosphorus pesticides, has efficient and broad-spectrum insecticidal properties. The pesticide can also affect the growth of crops while achieving the effects of killing pests and preventing diseases, and in addition, pesticide residues can also have negative effects on farmland soil and water ecological environment. Under the stress of pesticides, the stress resistance of plants is improved, and pesticide residues in soil environment are removed in time, so that the problem which needs to be solved in agricultural production is urgent.

Aiming at the microbial degradation of chlorpyrifos, the method mainly aims at the soil or plant chlorpyrifos, and discloses the application of bacillus in degrading the chlorpyrifos in the environment, such as the research on the degradation kinetics of two strains of bacillus cereus to the chlorpyrifos and the research on the influence factors of the bacillus cereus HY-1 for degrading the methyl parathion and the chlorpyrifos in documents; chinese patent "CN 112226385A" discloses a bacillus megaterium with IAA secretion ability, phosphate-solubilizing ability and ACC deaminase activity, but none of the strains is related to promoting rice to absorb and degrade chlorpyrifos.

Disclosure of Invention

The invention provides a Bacillus aryabhattai strain, which can be colonized in rhizosphere and plants of rice, relieve the stress of chlorpyrifos on the rice, promote the growth of the rice, promote the enrichment and degradation of the rice, and reduce the residual of the chlorpyrifos in crops and environment. The strain can also effectively improve the rice biomass by releasing growth promoting substances such as ACC deaminase, IAA and the like under the duress of chlorpyrifos, thereby serving green agriculture.

Specifically, the method is realized by the following technical scheme:

firstly, the application provides a Bacillus (Bacillus sp.) with the preservation number of CGMCC No.22887, the strain does not contain gram-positive bacteria, and the thallus is straight rod-shaped and has endogenous spores. Applicant self-names it DJ2. The strain is separated from the rice plants planted in the soil polluted by the chlorpyrifos and is harmless to animals and plants.

Secondly, the application of the bacillus with the preservation number of CGMCC No.22887 in the degradation of chlorpyrifos comprises the application of chlorpyrifos in a degradation environment and rice plants.

Further, the application of the bacillus in degrading chlorpyrifos in crop plants, wherein the crop plants are preferably rice. The method comprises the following specific steps: carrying out root soaking treatment on crops by using a bacillus agent to enhance the degradation of residual chlorpyrifos in rice; the bacillus agent comprises: MSM Medium and final concentration of 10 ¹⁰ cfu/mL of Bacillus aryabhattai.

Furthermore, the root soaking treatment of the crops refers to the root soaking treatment of the rice in the three-leaf one-heart stage for 6 hours by using the bacillus agent.

Thirdly, the application also provides the application of the bacillus with the preservation number of CGMCC No.22887 in promoting the growth of crops and improving the biomass; the crops are preferably rice. The method comprises the following specific steps: the bacillus agent is used for soaking roots of rice for 6 hours, and is used for coping with the stress of pesticide chlorpyrifos and secreting growth promoting substances and active enzymes at the same time, so that the growth of the rice is promoted, and the biomass of the rice is increased.

The application also provides a microbial inoculum comprising the bacillus and the MSM culture medium. In the microbial inoculum, the content of bacillus is 10 ¹⁰ cfu/mL. The MSM medium comprises the following components: mgSO (MgSO) ₄ ·7H ₂ O 0.4g、FeSO ₄ ·7H ₂ O 0.2g、K ₂ HPO ₄ 0.2g、(NH ₄ ) ₂ SO ₄ 0.2g、CaSO ₄ 0.08g, deionized water to 1L, pH 7.0.

According to the application, a bacillus is found in rice plants planted in chlorpyrifos-polluted soil for the first time, and is researched and preserved. In subsequent developments by the applicant, further detection was made that either the strain was Bacillus aryabhattai (Bacillus aryabhattai). Experiments prove that the strain can be colonized in rhizosphere and plants of rice, so that the rice is promoted to absorb and degrade chlorpyrifos, and chlorpyrifos residues in crops and the environment are reduced; meanwhile, by releasing growth promoting substances such as ACC deaminase, IAA and the like, the biomass of the rice is effectively increased, and the growth of the rice is promoted.

Drawings

FIG. 1 is an electron micrograph of Bacillus aryabhattai DJ2.

FIG. 2 is a graph showing fluorescence of Bacillus aryabhattai DJ2 after colonization in rice.

FIG. 3 is a schematic diagram showing the in vitro degradation function verification result of Bacillus aryabhattai DJ2 on chlorpyrifos.

FIG. 4 shows the repairing function of the microbial inoculum on residual chlorpyrifos in rice.

FIGS. 5-7 show the growth promoting function of Bacillus aryabhattai DJ2.

FIG. 8 shows that Bacillus aryabhattai DJ2 enhances the activity of glutathione-S-transferase in plants.

Figure 9 is a display of the enhanced biological yield of bacillus aryabhattai DJ2 after colonization of rice.

Detailed Description

Media referred to in the examples:

LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and pH 7.0;

inorganic salt liquid medium (MSM): mgSO (MgSO) in vitro ₄ ·7H ₂ O(0.4g)、FeSO ₄ ·7H ₂ O(0.2g)、K ₂ HPO ₄ (0.2g)、(NH4)2SO ₄ (0.2 g) and CaSO ₄ (0.08 g) deionized water to 1L, pH 7.0;

inorganic salt solid medium: adding 20g/L agar into an inorganic salt liquid culture medium;

king B medium (gold culture medium): 1000mL of deionized water, 32g of tryptone, 1.15g of dipotassium phosphate, 1.5g of magnesium sulfate heptahydrate, 10mL of glycerol and 1g of L-tryptophan.

NBRIP medium: 1000mL of deionized water, 10g of glucose, 5g of calcium phosphate, 5g of magnesium chloride, 0.25g of magnesium sulfate, 0.2g of potassium chloride and 0.1g of ammonium sulfate.

Asibi (Ashby) nitrogen-free medium: 1000mL of deionized water, 10g of glucose, 0.2g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate heptahydrate, 0.2g of sodium chloride, 0.1g of calcium sulfate and 5g of calcium carbonate.

The reagents referred to in the following examples, unless otherwise specified, were purchased commercially.

Example 1 isolation and identification of Bacillus aryabhattai DJ2 and verification of degradation function

1. Isolation and identification of strain DJ2 (Bacillus aryabhattaistrain DJ 2)

The applicant collected rice planted in soil contaminated by chlorpyrifos in experimental fields (E118 degrees 86', N32 degrees 03') of agricultural academy of sciences of Jiangsu province in 2016 and collected rice, and screened endophytes with the function of chlorpyrifos degradation. Endophyte screening mode: plant samples 60s immersed in 75% alcohol and 120s immersed in 1% sodium hypochlorite, and sterile water rinsing 3 times after discarding the solution each time. And finally, coating the flat plate with sterile water for the last time, and verifying the sterilization effect. Adding a sterilized steel ball and 200 mu L of sterilized water into a sample tube with a sterilized surface, beating for 20min at 750rpm/min by a shaker, and coating 100 mu L of the solution in the tube on an inorganic salt solid culture medium containing chlorpyrifos as the only carbon-nitrogen source, wherein the bacterial strain is named DJ2 by the applicant, is rod-shaped and has endophytic spores. The electron micrograph of the strain is shown in FIG. 1.

The DJ2 strain is preliminarily identified as Bacillus (Bacillus sp.) by comparing the physiological and biochemical characteristics with the 16SrDNA conserved sequence, and is preserved in 7-13 months in 2021 to China general microbiological culture Collection center (CGMCC) with the address: west road No. 1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101, the preservation number is CGMCC No.22887, and the classification is named as Bacillus (Bacillus sp.). The applicant, after submitting the deposit, further carried out a taxonomical study of the strain DJ2, or its taxonomical name Bacillus (Bacillus sp.) genus Bacillus aryabhattai (Bacillus aryabhattai)

2. Functional verification of strain DJ2 (Bacillus aryabhattaistraining DJ 2) for degrading chlorpyrifos

Firstly, culturing the DJ2 strain obtained by screening in the step 1 by using an LB culture medium for 12 hours, centrifuging the bacterial liquid (6000rpm, 5min,20 ℃), then, re-suspending and cleaning the bacterial liquid by using a sterilized inorganic salt solution (pH 7.0) for three times, and adjusting (OD) ₆₀₀ = 1.0); then 2 percent by volumeRespectively adding DJ2 bacterial suspension (10 mu g/mL) into an inorganic salt liquid culture medium which takes chlorpyrifos as a unique carbon source to culture for 1, 3, 6, 9, 12 and 15 days; detecting chlorpyrifos residue in inorganic salt liquid culture medium at different culture times by Gas Chromatography (GC), comparing with a blank group (CK), calculating the degradation rate of DJ2 strain:

C＝(A-B)/A*100％

a = chlorpyrifos concentration in CK, mg/kg;

b = concentration of chlorpyrifos in experimental group, mg/kg;

c = rate of chlorpyrifos degradation

The in vitro chlorpyrifos degradation capacity of DJ2 is determined by calculation of the formula. Gas Chromatography (GC) parameters are described in literature:

Feng Fayun et al.Enhanced degradation ofchlorpyrifos in rice(Oryza sativaL.)by five strains ofendophytic bacteria and theirplant growth promotional ability.[J].Chemosphere,2017,184:505-513.

the detection results are shown in FIG. 3, and DJ2 bacterial liquid (OD) was detected within 15 days ₆₀₀ = 1.0) rate of chlorpyrifos degradation 21.6% at a concentration of 10 μ g/mL. The degradation property of DJ2 on chlorpyrifos is verified.

Example 2 preparation of DJ2 inoculum

The DJ2 microbial inoculum is obtained by the following steps:

a) Inoculating DJ2 strain as defined in claim 1 to LB culture medium containing 10 μ g/mL chlorpyrifos for enrichment, streaking MSM solid plate containing 10 μ g/mL chlorpyrifos and LB solid plate successively, picking out single colony from the plates to LB liquid culture medium, shake-culturing at 30 ℃,150-220rpm for 12h, and continuously activating twice to obtain activated bacterial liquid;

b) Placing the activated bacteria liquid in a 50ml sterilized centrifuge tube in a clean bench, centrifuging at 20 deg.C and 6000rpm for 5min to obtain precipitated thallus, rinsing with sterilized MSM liquid culture medium for 3 times, adding MSM to regulate bacteria liquid OD ₆₀₀ =1 (bacterial liquid concentration about 10) ¹⁰ cfu/mL), and obtaining the DJ2 microbial inoculum.

Example 3 DJ2 colonization experiment for promoting rice to absorb and degrade chlorpyrifos

The soil is collected from the field of agricultural science college of Jiangsu province, is dried and then is hammered, and is sieved by a 20-mesh sieve to remove impurities for later use. Composition (normalization): 0.03 parts of organic matters; 0.50 parts of sludge; 0.21 parts of clay; contains 0.26 of sand.

Chlorpyrifos treatment: the leaves of the rice were sprayed manually with 45% chlorpyrifos emulsifiable concentrate at 2 times the recommended dose (initial deposition on rice of about 20 ppm). An inoculation group and a control group are set, and n =3 is processed in parallel.

Colonization of rice by endophytes: the rice variety Nanjing 9108 (grain crop institute of agricultural science, jiangsu province). Adopting the DJ2 microbial inoculum obtained in the example 2, selecting three-leaf and one-heart rice with consistent growth vigor, washing the root of the rice clean with deionized water, immersing the root of the rice into the bacterial liquid, inoculating for 6h, transferring the rice into a rice nutrient solution for culturing, taking a rice plant without bacteria as a control, collecting a rice sample of 6d after colonization, observing the distribution of the green fluorescent protein-converted strain in different tissues of the rice by using a fluorescence confocal microscope, and observing the result as shown in figure 2. The root (root), stem (stems) and leaf (leaf) parts of the colonized rice emit obvious green fluorescence, which indicates that DJ2 can be successfully colonized in each tissue of the rice.

Gathering and degrading chlorpyrifos: the concentration of the chlorpyrifos enriched at the roots of the inoculated rice is 37.2 percent higher than that of the control rice at 3d, and after 9d, the concentration of the chlorpyrifos enriched at the roots of the inoculated rice and the control rice tends to be consistent, which shows that the inoculation DJ2 can promote the enrichment and the degradation of the rice to the chlorpyrifos; in the stem and leaf of the rice, the degradation half-life period of chlorpyrifos in the inoculated rice is reduced from 3.5d to 2.7d, which indicates that the endophyte colonization can promote the degradation of the chlorpyrifos in the rice; periodically sampling and determining the degradation dynamics of chlorpyrifos in rice roots, stems and leaves by using a GC. The experimental result is shown in fig. 4, compared with the control, the chlorpyrifos concentration in the rice stem leaves of the inoculation group is reduced by 45.8% at most in the 15d experimental period.

Example 4 DJ2 growth promoting characteristics and Rice growth promoting Effect experiment under Chlorpyrifos stress

DJ2 growth promoting properties:

1-IAA: taking DJ2 microbial inoculum to inoculate King B culture medium containing L-tryptophan (1 g) (IAA synthesis precursor), taking King B culture medium without bacteria as a control group, and performing shake culture at 32 ℃ and 180r/min for 12h. The cells were centrifuged at 8000rpm for 5min, and the supernatant was mixed well with Salkowski reagent (50 mL of 35% perchloric acid plus 1mL of 0.5mol/L ferric chloride) at a ratio of 1.

2-phosphorus solubility: DJ2 microbial inoculum is inoculated in NBRIP solid culture medium and cultured for 7d at the constant temperature of 32 ℃, the result is shown in figure 6, transparent rings are generated around three endophytes, and DJ2 is shown to have phosphorus dissolution property.

3-ACC deaminase Activity DJ2 inoculum was inoculated in Ambizaro medium containing ACC and cultured at 30 ℃ at 180r/min for 14d in a medium without ACC as a control. As a result, as shown in FIG. 7, it was observed that the Ashbya medium containing ACC in the DJ2 group became turbid, indicating that DJ2 was able to produce ACC deaminase and grow using ACC as a nitrogen source.

4-glutathione-S-transferase activity: glutathione-S-transferase is an enzyme which is ubiquitous in animals and plants and has multiple functions, and can degrade certain external toxins and endogenous toxic metabolites. Soaking roots of the three-leaf one-heart stage rice for 6 hours by using DJ2 microbial inoculum, spraying 45% chlorpyrifos missible oil to leaf surfaces of the rice in 2 times of recommended dose after the colonization is finished, and weighing three rice plants as a group.

The result of figure 8 shows that the activity of glutathione-S-transferase is significantly increased after the rice colonized by endophyte DJ2 is stressed by chlorpyrifos, and the activity of the glutathione-S-transferase in the leaves of the rice colonized by endophyte DJ2 reaches 74.50U/kg under the stress of chlorpyrifos, compared with a CK treatment group stressed by chlorpyrifos, the activity of the glutathione-S-transferase is improved by 16.50%, and the enzyme is relevant to the degradation of the chlorpyrifos.

5-DJ2 colonization promotes rice biomass, as shown in FIG. 9, the DJ2 colonization rice under chlorpyrifos stress is promoted by 29.6% in the 30d compared with the CK group and the non-inoculated group.

The experimental result shows that the strain has a good growth promoting effect in vitro, and can promote the increase of rice biomass under the stress of chlorpyrifos after being colonized in rice.

Claims

1. Bacillus (b)Bacillus sp.) The preservation number is CGMCC No.22887; the bacillus is gram-positive bacteria, the thallus is straight rod-shaped and has endogenous spores.

2. A bacterial agent comprising the Bacillus of claim 1.

3. The use of the bacillus of claim 1 to promote the absorption and degradation of chlorpyrifos residues in rice.

4. Use of the bacillus of claim 1 for promoting growth of rice.

5. The microbial inoculum of claim 2, further comprising MSM media.

6. The use according to claim 3, wherein the use is carried out with a bacteria content of 10 ¹⁰ The cfu/mL microbial inoculum is used for carrying out root soaking treatment on the rice so as to promote the rice to absorb and degrade chlorpyrifos residues.

7. The use of claim 4, wherein the use is of a bacteria content of 10 ¹⁰ cfu/mL microbial inoculum is used for root soaking treatment of rice to promote the growth of crops.