Degradable organophosphorus pesticide, anti-disease growth-promoting multifunctional paenibacillus terrae and application thereof

Abstract

The application provides a multifunctional Aenibacillus terrae capable of degrading organophosphorus pesticides and resisting diseases, the strain is Aenibacillus terrae, and is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 30973. The strain can degrade organophosphorus pesticides, including profenofos, malathion, diazinon, phoxim and fenthion, and the degradation effect on diazinon is the best; the strain can inhibit the growth and development of southern root-knot nematodes, and prevent tomato root knot disease caused by the nematodes; the strain can antagonize fusarium oxysporum, and prevent tomato fusarium wilt caused by the fusarium oxysporum; and the strain can increase the supply of soil available phosphorus, and promote the growth of leafy vegetables.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a multifunctional soil-borne Bacillus that can degrade organophosphorus pesticides, resist diseases and promote growth, and its applications. Background Technology

[0002] The large-scale production and use of organophosphorus pesticides leads to their accumulation in the soil environment, thus endangering human health. For high concentrations of organophosphorus pesticide pollution, the safe utilization of contaminated soil cannot be achieved naturally in a short period. Therefore, developing artificial enhancement technologies for organophosphorus pesticide degradation is an important area of ​​research in environmentally friendly agricultural technologies. Phosphate-solubilizing bacteria can absorb and utilize both organic and inorganic phosphorus. The former can effectively degrade organophosphorus pesticides, achieving ecological restoration of pesticide-contaminated soil. Chlorpyrifos, dimethoate, and diazinon are synthetic organophosphorus insecticides widely used in agricultural production. The accumulation of these pesticides in the soil leads to excessive pesticide content in agricultural products, endangering consumer health. Artificially isolating and culturing various organophosphorus phosphate-solubilizing bacteria present in the soil can produce microbial agents for the remediation of pesticide-contaminated soil and improve the safety of agricultural products.

[0003] On the other hand, phosphorus is one of the three major mineral elements required for plant growth and development. Plants typically absorb phosphorus as hydrogen phosphate or dihydrogen phosphate. The phosphorus absorbed by plants mainly comes from the soil solution. However, some metal elements in the soil react with phosphorus, such as Ca and Al, forming metal complexes that precipitate or adsorb phosphorus, thus reducing the amount of available phosphorus that plants can absorb. Therefore, how to decompose fixed phosphorus in the soil into phosphorus that can be absorbed and utilized by plants to improve crop yield and quality is an important area of ​​research in environmentally friendly agricultural technology. Phosphorus-solubilizing bacteria can create an acidic environment by secreting various organic and inorganic acids or enzymes, activating insoluble or poorly soluble phosphorus, improving the conversion and absorption of phosphorus by crops, and thus promoting crop growth.

[0004] Southern root-knot worm (Meloidogyne incognit) and Fusarium oxysporum are the main pathogens causing root-knot disease and wilt disease in plants, respectively. Accumulation of these pathogens in continuously cropped soils can exacerbate soil-borne diseases and intensify continuous cropping obstacles. Various antagonistic microorganisms against these pathogens exist in the soil. Through artificial isolation and cultivation, these microbial agents can be prepared for the control of soil-borne diseases and the alleviation of pathogenic continuous cropping obstacles.

[0005] Paenibacillus terrae is an important member of plant rhizosphere growth-promoting bacteria. The inventors isolated Paenibacillus terrae from the rhizosphere soil of continuously cropped Lanzhou lily (Lilium davidii var. unicolor). After identification, strain P34 belongs to Paenibacillus terrae, which has the functions of degrading phosphorus, inhibiting nematode disease resistance, and promoting growth. The Paenibacillus terrae strain involved in this invention, isolated from the rhizosphere soil of Lanzhou lily, can stably colonize in the soil and has the functions of decomposing organic phosphorus, degrading soil organophosphorus pesticides to improve agricultural product safety, improving soil phosphorus supply capacity to improve plant phosphorus utilization, inhibiting root-knot nematodes, and antagonizing Fusarium oxysporum to prevent soil-borne diseases. It also has a very good growth-promoting effect on heading Chinese cabbage. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides a multifunctional soil-borne Bacillus strain that can degrade organophosphorus pesticides and promote crop growth while resisting disease.

[0007] The first objective of this invention is to provide a multifunctional terrestrial Bacillus terrae that can degrade organophosphorus pesticides, resist diseases, and promote growth. The terrestrial Bacillus terrae is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 30973.

[0008] This invention isolates Paenibacillus terrae P34 from the rhizosphere soil of continuously cropped Lanzhou lily. After identification, the strain is classified and named Paenibacillus terrae. It was deposited on June 17, 2024, at the China General Microbiological Culture Collection Center (CGMCC), at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30973.

[0009] The second objective of this invention is to provide a culture of Paenibacillus terrae, obtained by expanding the culture of the aforementioned Paenibacillus terrae CGMCC No.30973 strain.

[0010] Preferably, the expanded culture specifically involves inoculating the *Paenibacillus terrae* CGMCC No. 30973 strain into a culture medium and culturing it in a shaker at 28±1℃; preferably, the culture medium is LB liquid medium.

[0011] A third objective of this invention is to provide the application of the above-mentioned Paenibacillus terrae or Paenibacillus terrae bacterial solution in the preparation of phosphate-solubilizing microbial inoculant products.

[0012] The fourth objective of this invention is to provide the application of the above-mentioned Paenibacillus terrae or Paenibacillus terrae bacterial solution in the preparation of microbial inoculants for the ecological remediation of organophosphorus pesticide-contaminated soil.

[0013] Preferably, the soil is contaminated with at least one of the organophosphorus pesticides.

[0014] Preferably, the organophosphorus pesticides include malathion, diazinon, phoxim, and fenthion.

[0015] The fifth objective of this invention is to provide the application of the above-mentioned Paenibacillus terrae or Paenibacillus terrae bacterial solution in the preparation of products for the biological control of soil-borne diseases such as root-knot nematodes.

[0016] Preferably, the plant root-knot nematode is the southern root-knot nematode (Meloidogyne incognit).

[0017] The sixth object of the present invention is to provide the application of the above-mentioned Paenibacillus terrae or Paenibacillus terrae bacterial solution in the preparation of products for the biological control of soil-borne diseases such as plant wilt.

[0018] The liquid of *Paenibacillus terrae* or *Paenibacillus terrae* can be used to control soil-borne diseases and alleviate pathogenic continuous cropping obstacles.

[0019] The continuous cropping obstacles include at least one of plant pathogenic continuous cropping obstacles and continuous cropping obstacles caused by plant allelopathic autotoxicity;

[0020] Preferably, the pathogen is *Fusarium oxysporum*. The plant is preferably a solanaceous vegetable, including tomato.

[0021] The seventh object of the present invention is to provide the use of the above-mentioned Paenibacillus terrae or soil-borne Bacillus or Paenibacillus terrae or soil-borne Bacillus bacterial solution in the preparation of products that improve soil physical and chemical properties and promote crop growth.

[0022] Leafy vegetables, including head cabbage, are preferred crops.

[0023] Beneficial effects:

[0024] The soil-borne Bacillus strain P34 of this invention exhibits a relatively stable bacterial population and can effectively colonize in the rhizosphere soil. After 2 days of cultivation, the available phosphorus content in the culture medium is 45.38 μg / mL, demonstrating good phosphorus solubilization ability. It can activate insoluble or poorly soluble phosphorus in the soil, improving the conversion and absorption of phosphorus by crops. Secondly, soil-borne Bacillus strain P34 can degrade organophosphorus pesticides: profenofos, malathion, diazinon, phoxim, and fenthion, with the best degradation effect on diazinon. Thirdly, soil-borne Bacillus strain P34 inhibits the growth and development of southern root-knot nematodes, preventing tomato root-knot disease caused by these nematodes. Fourthly, soil-borne Bacillus strain P34 antagonizes Fusarium oxysporum, preventing tomato wilt caused by Fusarium oxysporum and reducing the occurrence and damage of soil-borne diseases. Fifthly, soil-borne Bacillus strain P34 increases the supply of available phosphorus in the soil, promoting the growth of leafy vegetables. Attached Figure Description

[0025] Figure 1 The phosphorus-solubilizing ability of different phosphate-solubilizing bacteria strains.

[0026] Figure 2 The phosphorus solubilizing ability of Bacillus subtilis P34 strain.

[0027] Figure 3 Colony morphology of Bacillus subtilis P34 strain;

[0028] A is the initial screening medium; B is cultured on LB medium; C is cultured on Mongkina organophosphate bacteria medium; D is Gram staining.

[0029] Figure 4 PCR amplification of the 16S rDNA gene fragment of Bacillus subtilis P34 strain.

[0030] Figure 5 A molecular phylogenetic tree for Bacillus subtilis strain P34.

[0031] Figure 6 The effect of Bacillus subtilis P34 strain on soil colonization.

[0032] Figure 7 The degradation ability of Bacillus subtilis strain P34 for organophosphorus pesticides;

[0033] A represents the bacterial proliferation capacity on a culture medium containing four organophosphorus pesticides; B represents the amount of phosphorus accumulated in the culture medium.

[0034] Figure 8 The effects of Bacillus subtilis strain P34 on southern root-knot nematodes under in vitro conditions;

[0035] A represents the control group (CK); B represents a photomicrograph of the P34-treated nematode; the rigid worms are dead nematodes.

[0036] Figure 9 The effect of Bacillus subtilis strain P34 on root-knot nematodes in tomato under cultivation conditions;

[0037] A represents the CK tomato plant infected with nematodes, with root knots at the base; B represents the tomato plant treated with P34.

[0038] Figure 10 The effect of Bacillus subtilis strain P34 on Fusarium oxysporum.

[0039] A represents the antagonistic effect between strain P34 and Fusarium oxysporum; B represents the hyphae of normal Fusarium oxysporum; C represents the hyphae of abnormal Fusarium oxysporum.

[0040] Figure 11 The effects of different amounts of Bacillus subtilis strain P34 on Fusarium oxysporum;

[0041] A: P34 bacterial solution 0.5 μg / ml; B: P34 bacterial solution 1.0 μg / ml.

[0042] Figure 12 Antagonistic effect of Bacillus subtilis P34 strain on tomato wilt caused by Fusarium oxysporum under hydroponic conditions;

[0043] A represents a normally growing tomato plant; B represents a tomato plant infected with Fusarium oxysporum after inoculation with Fusarium oxysporum; C represents the mitigating effect of inoculation with strain P34 on tomato wilt; D represents the observed yellowing of leaf tips after inoculation with Fusarium oxysporum.

[0044] Figure 13 The effects of Bacillus subtilis strain P34 on the growth of heading Chinese cabbage plants.

[0045] Figure 14 The effects of Bacillus subtilis strain P34 on the root system of Chinese cabbage. Detailed Implementation

[0046] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0047] Example 1. Basic characteristics of the *Bacillus terrestrialus* strain P34 (CGMCC No. 30973) of the present invention: 1.1 Obtaining the *Bacillus terrestrialus* strain P34 of the present invention:

[0048] Soil samples were collected from the top 5 cm layer of the *Lilium lanzhouense*. 99 mL of sterile water was added to an Erlenmeyer flask, and 1 g of rhizosphere soil was added. The mixture was vortexed for 1–2 min, then placed in a constant-temperature shaker (37℃, 180 rpm) for 1 h. 1 mL of the supernatant from the prepared soil suspension was pipetted and diluted sequentially to a 10⁻⁶ concentration. -3 g / mL, 10 -4 g / mL, 10 -5 g / mL, 10 -6 g / mL, 10 -7 The concentration was calculated as g / mL. 100 μL of each concentration was aseptically spread onto agar plates (Monginna organophosphate bacterial culture, using lecithin as the sole phosphorus source). The plates were inverted and incubated at 37°C for 48 h, repeated three times. Single colonies with phosphate-solubilizing zones were picked from appropriately diluted media and purified 4–5 times on LB agar using the triple-line plotting method to obtain pure single colonies, thus screening for phosphate-solubilizing strains.

[0049] The bacterial strain was prepared into a bacterial suspension for subsequent assays. The preparation method was as follows: strain P34 was inoculated into LB liquid medium and cultured overnight at 37°C and 180 rpm; the cultured fermentation broth was taken out, centrifuged at 11,000 rpm for 5 min, the supernatant was discarded, and then mixed thoroughly with sterile distilled water; the OD600 value was measured using a UV-Vis spectrophotometer to prepare a bacterial suspension with OD600 = 1.

[0050] The strain was identified and classified as Paenibacillus terrae. It was deposited on June 17, 2024, at the China General Microbiological Culture Collection Center (CGMCC), at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30973.

[0051] 1.2 Determination of phosphorus solubilization ability of the P34 strain of *Bacillus thuringiensis* in this invention

[0052] The purified bacterial strain was inoculated into LB liquid medium (37°C, 180 rpm) and incubated overnight by shaking. Then, it was centrifuged at 11000 rpm for 6 minutes, and the OD value was adjusted with sterile water. 600 The bacterial suspension was prepared as shown in Figure 1. 2 mL of the suspension was inoculated into 100 mL of *Bacillus mongholicus* liquid culture medium (using lecithin as the sole phosphorus source) and cultured on a shaker (37℃, 180 rpm) for 48 h. 20 mL of the fermentation broth was transferred to a 50 mL centrifuge tube and ultrasonically cleaned for 20 min to release the available phosphorus from the cells. The tube was then centrifuged at 4℃ and 11000 rpm for 6 min. 10 mL of the supernatant was filtered through a 0.22 μm filter, and 5 mL was transferred to a 50 mL volumetric flask. Approximately 20 mL of sterile water and 2 drops of 2,4-dinitrophenol indicator were added. The pH was adjusted to a slightly yellow color using calcium carbonate and sulfuric acid solutions. One drop of dilute sulfuric acid was added until the reaction solution became colorless. 5 mL of molybdenum antimony anti-chromic reagent was accurately added, and the mixture was thoroughly mixed and brought to a final volume. The mixture was allowed to stand for 30 min, and colorimetric analysis was performed at 660 nm. The pH of the remaining supernatant was measured using a pH meter. Using an uninoculated culture medium as a control, the absorbance was adjusted to 0, and the control value was measured using sterile water. The available phosphorus content was calculated based on the standard curve. The standard curve equation is y = 0.376x + 0.001.

[0053] Results: Polyphosphate-solubilizing bacteria obtained from the initial screening ( Figure 1 From this strain, the most efficient phosphorus-solubilizing strain P34 was selected. This strain exhibited phosphorus solubilization capacity ranging from 26.94 to 49.66 μg / ml between 12 and 72 hours, reaching a peak at 48 hours, after which the capacity slightly decreased but still maintained strong phosphorus-solubilizing ability. Figure 2 It is evident that strain P34 degrades insoluble organic phosphorus in the culture medium, thereby increasing the content of soluble phosphorus in the culture medium.

[0054] 1.3 The *Bacillus terrestrialus* P34 CGMCC No. 30973 of the present invention has the following biological characteristics:

[0055] 1.3.1 Morphological and cultural characteristics: such as Figure 3 As shown, on LB agar plates, colonies are observed to be round, slightly yellow in color, with a raised surface and regular edges; on Monkina organophosphate bacteria agar, colonies are observed to be round, milky white in color, resembling half a glass bead, with a slightly moist surface and irregular edges; Gram staining under a microscope reveals that strain P34 is rod-shaped and Gram-negative. Other physiological and biochemical reactions are as follows: diacetyl (VP) reaction and starch hydrolysis test are positive, while hydrogen peroxide test, methyl red reaction, and indole reaction are negative.

[0056] 1.3.2 Genetic characteristics (16S rRNA sequence of the strain): The 16S rRNA sequence of the strain was determined in this invention, as shown below, with a full length of 1434 bp.

[0057]

[0058] 1.3.3 Molecular systematics identification of the strain

[0059] Strain P34 was inoculated into LB liquid medium and incubated overnight at 180 rpm. Bacterial genomic DNA extraction kits were purchased from Beijing Qingke Biotechnology Co., Ltd., and DNA extraction was performed according to the reagent kit's instructions. Universal bacterial primers 27F (5'-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used for PCR amplification. The extracted DNA sample was diluted appropriately and used as a PCR template. Amplification was performed using Qingke 1×TSE101 Gold Mix, and the components of the amplification system are as follows:

[0060]

[0061] The PCR reaction procedure is as follows:

[0062]

[0063] The amplified PCR products (2 μL sample + 6 μL bromophenol blue) were detected by agarose gel electrophoresis at 300V for 12 minutes to obtain the identification gel image. Sequencing was performed at the Chengdu branch of Beijing Qingke Biotechnology Co., Ltd. The final 16S rDNA sequence was compared with NCBI, and a phylogenetic tree was constructed using MEGA 7.0 to identify phosphate-solubilizing bacterial strains.

[0064] The results showed that PCR amplification yielded a 1500 bp target gene fragment. Through sequence alignment with homologous sequences in NCBI, strain P34 was identified as *Paenibacillus terrae*. Figure 4 ,5).

[0065] 1.4 Soil colonization ability of the soil-borne Bacillus p34 strain of the present invention

[0066] For strain P34 to effectively solubilize phosphorus, the primary condition is its ability to colonize the rhizosphere soil. The bacterial suspension (preparation method as described in step 1.1 of Example 1) was inoculated into sterilized soil. The number of bacteria in the soil was monitored at different time points (obtained by measuring the OD value of soil bacteria).

[0067] The results showed that 40 days after inoculation, the number of bacteria of strain P34 in the soil decreased over time, with a sharp decline within the first 2 days of culture, followed by a slower decline in the later stages. The number of bacteria of strain P34 increased compared to the previous day at 12, 20, and 32 days, eventually stabilizing at 2.4 × 10⁻⁶.5 CFU / g. Overall, the bacterial population of strain P34 was relatively stable, and the colonization effect was good.

[0068] ( Figure 6 )

[0069] Example 2: Remediation function of organophosphorus pesticide contaminated soil by Bacillus thuringiensis P34 strain CGMCC No. 30973 of the present invention.

[0070] 2.1 Under in vitro conditions, strain P34 exhibits degradation activity against organophosphorus pesticides.

[0071] Four organophosphorus pesticides—malathion, diazinon, phoxim, and fenthion—were selected. Strain P34 was inoculated into beef extract peptone broth containing these organophosphorus pesticides. The degradation effect of the strain on organophosphorus pesticides under in vitro conditions was evaluated by measuring the OD value of the bacterial suspension and the available phosphorus content in the broth. Phosphorus accumulation was determined using the NaHCO3 leaching-molybdenum antimony colorimetric method.

[0072] Figure 7 A indicates that, between 12 and 72 hours, the OD values ​​of the *Bacillus thuringiensis* P34 strain under each pesticide treatment ranged from 0.96 to 2.19, which were generally higher than the control (CK) and remained higher than the CK after 24 hours. Bacterial proliferation reached its peak between 24 and 36 hours, and then decreased slightly. Figure 7 B indicates that the available phosphorus accumulation in each pesticide treatment ranged from 2.40 to 3.14 mg / kg, all significantly higher than that in the control (CK). Overall, it is evident that *Bacillus terrestris* strain P34 can effectively degrade organophosphorus pesticides in the culture medium, increasing the content of soluble phosphorus in the medium and promoting the proliferation of phosphate-solubilizing bacteria, with the best degradation effect on diazinon.

[0073] 2.2 Strain P34 has a remediation effect on soil contaminated with organophosphorus pesticides.

[0074] The technical principle behind this case is as follows: Excessive application of organophosphorus pesticides can cause soil pollution, phytotoxicity to crops, and inhibit plant growth; simultaneously, pesticides accumulate in plants, compromising the safety and quality of agricultural products. The *Bacillus thuringiensis* P34 strain has the ability to degrade organophosphorus pesticides. Therefore, it is highly probable that this strain can degrade organophosphorus pesticides to remediate contaminated soil, alleviate pesticide damage, and reduce organophosphorus pesticide residues in agricultural products.

[0075] This case study applied a design for tomatoes (TOM cherry tomatoes), selecting two organophosphorus pesticides and designing a single-factor randomized block experiment with four treatments: Treatment 1: malathion + P34 bacterial solution; CK1: malathion + water; Treatment 2: parathion + P34 bacterial solution; CK2: parathion + water. The treatment methods were as follows: TOM cherry tomato seeds were sown in nutrient pots (8cm × 10cm), one seed per pot, placed in an artificial climate chamber, and cultured under a photoperiod of 25°C, 85% relative humidity, and 12h day / 12h night. When the tomato plants have three leaves and one bud, malathion and parathion pesticides were diluted 1:500 and applied as a root drench, 50 mL per pot, once every 15 days, for two applications. Five days after the last pesticide treatment, 50 mL of *Bacillus thuringiensis* P34 bacterial solution was applied per pot, once every 5 days, for two applications. The preparation method of *Bacillus thuringiensis* P34 bacterial solution was the same as in step 1.1 of Example 1, suspending the solution in sterile water and adjusting the concentration of *Bacillus thuringiensis* P34 bacterial solution to OD600 = 1. The pesticide accumulation in the tomato fruit was measured five days after the last treatment. Pesticide residues in tomato fruit were detected using gas chromatography according to NY / T761—2008 "Determination of Multiple Residues of Organophosphorus, Organochlorine, Pyrethroid and Carbamate Pesticides in Vegetables and Fruits". Five plants were randomly selected from each treatment, and the first fruit was selected for testing during the color-changing stage. The experimental results are shown in Table 1. The results showed that malathion was not detected in tomato fruits in treatment 1, and the amount of malathion detected in the soil was reduced by 90.07% compared with the control; in treatment 2, the amount of parathion detected in tomato fruits was reduced by 48.13% compared with the control, and the amount of parathion detected in the soil was reduced by 70.67% compared with the control.

[0076] Table 1. The mitigating effect of strain P34 treatment on organophosphorus pesticide damage in TOM tomato fruit.

[0077]

[0078] Note: ND indicates not detected; according to GB2763-2021 "National Food Safety Standard - Maximum Residue Limits for Pesticides in Food", the limits for malathion and parathion in food are 50 μg·kg⁻¹. -1 10 μg·kg -1 .

[0079] Conclusion: The *Bacillus thuringiensis* P34 strain of this invention can degrade organophosphorus pesticides in soil, thereby reducing organophosphorus pesticide residues in the substrate. Simultaneously, the reduced content of organophosphorus pesticides in the soil decreases their accumulation in plants, resulting in a lower detection rate of these pesticides in tomato fruits and a significant improvement in the safety and quality of vegetables.

[0080] Example 3 Nematicidal Function of Bacillus subtilis P34 Strain CGMCC No. 30973 of the Present Invention 3.1 Inhibitory Effect of P34 Strain on Southern Root-knot Nematode under In Vitro Conditions

[0081] Southern root-knot nematode (Meloidogyne incognit) was used for in vitro experiments. The nematode was propagated on peppers (eggs), and a suspension of nematode eggs (1000 eggs / ml) was prepared. Simultaneously, second-instar larvae were collected and a nematode suspension was prepared (egg masses were incubated at 28℃, and hatched second-instar larvae were collected after 24 hours, 1000 larvae / ml). A bacterial suspension of strain P34 (OD value = 1, 100 μL per well) was co-cultured with the nematode eggs and second-instar larvae in a 96-well cell culture plate (50 μL of nematode egg suspension or nematode suspension per well), with 100 μL of sterile water as a control. Each treatment was repeated 6 times, for a total of 3 biological replicates. The results showed that after 6 days, the bacterial suspension treatment significantly inhibited the hatching of southern root-knot nematode eggs, and the nematode mortality rate differed significantly after 72 hours. The results are shown in Table 2. Figure 8 .

[0082] P34 bacterial suspension significantly inhibited root knot in southern nematodes, reducing the hatching rate of second-instar larvae by 65.61% after 6 days compared to the control (Table 2); after 72 hours of culture, it showed significant lethality against nematodes. Figure 8 The mortality rate of nematodes treated with P34 was 1.86 times that of the control (Table 2). This indicates that strain P34 produces certain substances toxic to root-knot nematodes, inhibiting their growth and development.

[0083] Table 2. Effects of P34 bacterial suspension treatment on hatching and larval mortality of Southern root-knot nematodes.

[0084]

[0085] Mortality rate = Number of dead nematodes / Total number of tested nematodes

[0086] 3.2 Strain P34 has a controlling effect against root-knot nematodes.

[0087] The technical principle behind this case is as follows: Root-knot nematode infection can cause root knots to form in tomato roots, leading to poor plant growth. The P34 bacterial strain has an inhibitory effect on root-knot nematodes. Therefore, irrigating with P34 bacterial solution can alleviate the incidence of root-knot nematodes.

[0088] This case study applied a single-factor experiment to tomatoes (the TOM cherry tomato variety), with two treatments, each with 15 plants and three replicates: (1) CK (control): soil infected with root-knot nematodes + water. (2) Treatment: soil infected with root-knot nematodes + P34 bacterial solution. The method for obtaining soil infected with root-knot nematodes was as follows: when the tomato had two leaves and one heart, it was inoculated with second-instar larvae of the southern root-knot nematode (500 larvae / plant). Tomatoes were continuously planted in this soil for three crops (60 days per crop), and significant symptoms of root-knot nematode disease were observed in the tomato roots. This soil was collected for research. TOM cherry tomatoes were planted in the infected soil collected by the above method, and cultivated in nutrient pots (8cm of infected soil). One seed was sown in each pot, and the pots were placed in an artificial climate chamber and cultured under a photoperiod of 25°C, 85% relative humidity, and 12h day / 12h night. When the first true leaf appeared, the plants were irrigated with P34 bacterial suspension, 50 mL per pot, once every 5 days, for a total of 8 applications. The preparation method of P34 bacterial suspension was the same as step 1.1 in Example 1 (OD600 = 1). After 40 days of treatment, the symptoms of root-knot nematode disease were observed, and growth indicators such as plant height, stem diameter, leaf area, and biomass were measured to evaluate the control effect. The results are shown in Table 3. Figure 9 .

[0089] The observation results showed that the control group had obvious root knot formation. Figure 9 A), while the tomato root development was basically normal after treatment with P34 bacterial suspension, and no root knots were observed to the naked eye. Figure 9 B); Meanwhile, treatment with P34 bacterial suspension significantly improved various plant growth indicators. Plant height, stem diameter, leaf area, aboveground fresh weight, and underground fresh weight increased by 28.52%, 6.93%, 77.31%, 89.71%, and 8.94%, respectively, compared with the control. Therefore, it significantly alleviated the inhibitory effect of diseased soil on plant growth (Table 3).

[0090] Table 3. Effects of P34 treatment on the growth of tomato plants infected with root-knot nematodes.

[0091]

[0092] Conclusion: The *Bacillus terrestris* strain P34 of this invention can inhibit southern root-knot nematodes, reduce the severity of nematode disease, alleviate the pathogenic continuous cropping obstacles in tomatoes caused by nematode damage, and promote tomato growth. After treatment with phosphate-solubilizing bacteria (i.e., *Bacillus terrestris* strain P34 of this invention), plant growth was improved to varying degrees, and overall growth was significantly improved.

[0093] Example 4: Disease resistance function of the *Bacillus terrestrialus* P34 strain CGMCC No. 30973 of the present invention. 4.1 The P34 strain exhibits antagonistic activity against *Fusarium oxysporum* under in vitro conditions.

[0094] Our laboratory isolated *Fusarium oxysporum*, the bacterium that causes wilt disease in Lanzhou lilies. We inoculated strain P34 and *Fusarium oxysporum* together on PDA plates and observed their antagonistic effects. Figure 10 As shown, compared with the control, strain P34 caused *Fusarium oxysporum* to grow poorly, with yellowing of the cells and smaller colonies. Under a microscope, normal *Fusarium oxysporum* hyphae were observed to be smooth and unsegmented, while the hyphae of the antagonized *Fusarium oxysporum* showed abnormalities, with multiple breaks, deformities, twists, and swellings. Figure 11 Different amounts of P34 bacterial suspension and Fusarium oxysporum were inoculated together on PDA plates. The inhibitory effect of P34 on Fusarium oxysporum was determined by the plate confrontation method. The inhibition rate of 1.0 μg / ml P34 bacterial suspension was 70.93%, and the inhibition rate of 0.5 μg / ml P34 bacterial suspension was 18.60%. The results indicate that strain P34 produced certain resistance substances that have a destructive effect on Fusarium oxysporum hyphae, thereby inhibiting the growth of Fusarium oxysporum.

[0095] 4.2 Under hydroponic conditions, strain P34 exhibits resistance to tomato wilt caused by Fusarium oxysporum.

[0096] The technical principle behind this case is as follows: Fusarium oxysporum can cause wilt disease in tomatoes. The P34 strain of Bacillus subtilis has an antagonistic function against Fusarium oxysporum. Therefore, it is highly probable that the P34 strain can reduce the incidence of wilt disease in tomatoes by inhibiting the proliferation of Fusarium oxysporum.

[0097] This case study applies a design for tomatoes (TOM cherry tomatoes). The experiment is a single-factor completely randomized design with 3 treatments, 30 plants in each treatment group, and 3 replicates:

[0098] (1) Treatment 1: Fusarium oxysporum + water. The inoculation method for Fusarium oxysporum was as follows: The Fusarium oxysporum strain was cultured in PDA medium at 28°C in a constant temperature incubator for 7 days. 12 discs were placed in each Erlenmeyer flask, and glass beads were added. The flask was shaken at 28°C and 180 rpm for 24 hours. The filtrate was collected by suction filtration, diluted with sterile water, and the spore count was determined by hemocytology. The spore concentration was then diluted to 10-1. 7 50 mL of *Fusarium oxysporum* suspension was added to the nutrient solution when the tomato plant had two leaves and one bud. After two days, 50 mL of water was added.

[0099] (2) Treatment 2: Fusarium oxysporum + P34 bacterial suspension. The Fusarium oxysporum inoculation method is the same as above. When the tomato has two leaves and one bud, 50 mL of Fusarium oxysporum bacterial suspension is added to the nutrient solution. Two days later, 50 mL of the Bacillus terrestris P34 bacterial suspension of this invention is added. The preparation method of the Bacillus terrestris P34 bacterial suspension is the same as step 1.1 in Example 1, suspending it in sterile water and adjusting the concentration of the Bacillus terrestris P34 bacterial suspension to OD600 = 1.

[0100] (3) CK: Use equal volumes of clean water to replace the Fusarium oxysporum and P34 bacterial suspensions respectively for treatment, and the treatment time is the same as that for treatment 2.

[0101] TOM cherry tomato seeds were sown in hydroponic nutrient pots (8.7cm×12.7cm×11.4cm), with 6 seeds sown per pot. The pots were placed in an artificial climate chamber and cultured under a photoperiod of 25°C, 85% relative humidity, and a 12h day / 12h night. Treatment began when the plants reached the two-leaf stage and one bud. Plant height, stem diameter, and biomass were measured 5 days after the last treatment. Table 4 shows the growth of TOM tomatoes after Fusarium oxysporum and P34 treatments.

[0102] The results showed that compared with CK ( Figure 12 A) After tomatoes were inoculated with Fusarium oxysporum, typical symptoms of wilt appeared in the plants 3-4 days later: yellowing of leaf tips, stunted growth, and wilting and death of some plants occurred from the lower part of the plant upwards. Figure 12 B) After adding P34 bacterial solution to plants inoculated with the pathogen, wilting and death of tomato plants occurred only sporadically, and the yellowing of leaf tips was significantly improved. Figure 12 C). Compared with treatment 1, the incidence rate and disease index of treatment 2 decreased significantly by 29.92% and 86.41%, respectively (Table 5).

[0103] Table 4. TOM Classification Standards for Fusarium Wilt Disease in Tomatoes

[0104]

[0105]

[0106] Note: The results of the survey on the number of diseased plants include treatment 1 (Fusarium oxysporum) and treatment 2 (Fusarium oxysporum + FS-20).

[0107] Table 5. Incidence of Fusarium oxysporum and P34 in TOM tomatoes after treatment with these fungi.

[0108]

[0109] Note: Data from the statistical processing of incidence rate and incidence index 1 and processing 2.

[0110] Disease incidence rate (DR): DR = (Number of infected plants / Number of plants surveyed) × 100%

[0111] Disease Index (DI):

[0112] In the formula: DI—disease index; s—representative value for each disease level; n—number of plants at each disease level; N—total number of plants surveyed; S—representative value for the highest disease level.

[0113] Conclusion: The *Bacillus terrestris* P34 strain of this invention can reduce the incidence of Fusarium wilt in solanaceous vegetables caused by *Fusarium oxysporum*, lower the disease index, and promote plant growth. After treatment with *Bacillus terrestris* P34 strain, tomato growth was improved to varying degrees, and overall growth was promoted.

[0114] 4.3 Under substrate cultivation conditions, strain P34 promoted tomato growth by overcoming the continuous cropping barrier caused by Fusarium wilt pathogens.

[0115] The technical principle behind this case is as follows: *Fusarium oxysporum* is the main fungus causing wilt disease in plants. An increase in *Fusarium oxysporum* leads to wilt disease and exacerbates continuous cropping obstacles. *Bacillus oryzae* strain P34 has an antagonistic effect against *Fusarium oxysporum*. Therefore, irrigating with *Bacillus oryzae* strain P34 can alleviate pathogenic continuous cropping obstacles.

[0116] This case study applied a single-factor experiment design for tomatoes (TOM cherry tomatoes), with two treatments, 15 plants in each treatment group, and three replicates:

[0117] (1) Treatment 1: Fusarium oxysporum + water. The inoculation method for Fusarium oxysporum was as follows: The Fusarium oxysporum strain was cultured in PDA medium at 28°C in a constant temperature incubator for 7 days. 12 discs were placed in each Erlenmeyer flask, and glass beads were added. The flask was shaken at 28°C and 180 rpm for 24 hours. The filtrate was collected by suction filtration, diluted with sterile water, and the spore count was determined by hemocytology. The spore concentration was then diluted to 10-1. 7 The concentration of *Fusarium oxysporum* was set at 50 mL / ml for later use as a fungal inoculum solution. When the tomato plant had three leaves and one bud, the *Fusarium oxysporum* fungus was applied by root irrigation, with 50 mL per pot, once every 5 days, for a total of 4 times. After the plant developed symptoms, the solution was applied by watering with clean water, with 50 mL per pot, once every 5 days, for a total of 4 times.

[0118] (2) Treatment 2: Fusarium oxysporum + P34 bacterial solution. The inoculation method of Fusarium oxysporum is the same as in step 4.2 of Example 4. When the tomato has three leaves and one heart, Fusarium oxysporum is irrigated by root irrigation, 50 mL per pot, once every 5 days, for 4 times. After the plant becomes diseased, the soil-borne Bacillus P34 bacterial solution of the present invention is irrigated, 50 mL per pot, once every 5 days, for 4 times. The preparation method of soil-borne Bacillus P34 bacterial solution is the same as in step 1.1 of Example 1. The soil-borne Bacillus P34 bacterial solution is suspended in sterile water and the concentration of soil-borne Bacillus P34 bacterial solution is adjusted to OD600 = 1.

[0119] TOM cherry tomato seeds were sown in nutrient pots (8cm×10cm), one seed per pot, and placed in an artificial climate chamber under a photoperiod of 25°C, 85% relative humidity, and 12h day / 12h night. Treatment began when the plants reached the three-leaf stage. Plant height, stem diameter, and biomass were measured 5 days after the last treatment. Table 6 shows the growth of TOM tomatoes after Fusarium oxysporum and P34 treatments.

[0120] Table 6. Growth of TOM tomatoes after treatment with Fusarium oxysporum and P34.

[0121]

[0122] Conclusion: The *Bacillus terrestrialus* strain P34 of this invention can antagonize *Fusarium oxysporum*, overcome pathogenic continuous cropping obstacles, and promote plant growth. After treatment with phosphate-solubilizing bacteria (i.e., *Bacillus terrestrialus* strain P34 of this invention), tomato growth was improved to varying degrees, and overall growth was promoted.

[0123] Example 5: Growth-promoting effect of the present invention's *Bacillus subtilis* strain P34, CGMCC No. 30973.

[0124] This study investigated the growth-promoting effect of Bacillus subtilis strain P34 on head-forming Chinese cabbage by irrigating it with P34 bacterial suspension under complete nutrient substrate cultivation conditions.

[0125] Two treatment groups were designed for each plant species: the CK group (sterile water) and the P34 group (P34 bacterial solution of the present invention). Each treatment consisted of 15 plants, with 3 replicates.

[0126] The preparation method of P34 bacterial solution of the present invention is the same as that in step 1.1 of Example 1, and the concentration of P34 bacterial solution in each treatment group is OD600 = 1.

[0127] Sterilize the seedling trays with 75% alcohol beforehand, then fill them with seedling substrate (commercially available, nutritionally complete), and plant the heads of Chinese cabbage seeds in the trays. Place them in an artificial climate chamber (light / temperature / dark temperature: 25℃ / 20℃, light / dark time: 16h / 8h), and water regularly. Once 3-4 true leaves have emerged, select uniformly growing seedlings and transplant them into sterilized flowerpots, one seedling per pot. Apply 50mL of the above-mentioned bacterial suspension to the roots of the plants, repeating 8 times. For the control group, add an equal amount of sterile water, then place them in an artificial climate chamber (light / temperature / dark temperature: 25℃ / 20℃, light / dark time: 16h / 8h), and water regularly and quantitatively. Observe and record the results after 40 days. Observe and record morphological indicators such as root length, plant height, stem diameter, leaf area, above-ground part weight, and underground part weight. The experimental results are shown in Tables 7-8 and 8-9. Figure 13-14 .

[0128] Determination of the physicochemical and biological properties of the substrate: Soil samples were collected by digging up the plant roots and gently shaking the roots to remove the attached substrate. Wearing sterile gloves, the fallen substrate was collected as rhizosphere soil in sterile sample bags. Five plants were randomly selected from each treatment for testing. All indicators were measured 30 days after the plants were treated with the bacterial solution. The EC value of the soil physicochemical properties was determined using a DDS-307A conductivity meter at a ratio of Vsoil:Vwater = 1:10. Available nitrogen, available potassium, and available phosphorus were measured using the alkaline diffusion method, the 1.0 mol / L ammonium acetate extraction-flame photometry method, and the 0.5 mol / L sodium bicarbonate extraction-molybdenum antimony colorimetric method, respectively. The experimental results are shown in Table 9.

[0129] Figure 13 and Figure 14 It can be seen that, compared with the control group (CK), the Chinese cabbage treated with strain P34 for 40 days had taller plants, more and larger leaves, and longer roots. Plant height, root length, and leaf area were significantly increased by 22.83%, 82.87%, and 67.31% respectively compared with the control, while stem diameter showed no significant difference compared with the control, but increased by 6.21% (Table 7). Table 8 shows that the above-ground fresh weight, above-ground dry weight, underground fresh weight, and underground dry weight were significantly increased by 79.45%, 71.43%, 116.67%, and 100% respectively compared with the control.

[0130] Table 7 Effects of strain P34 on the growth of Chinese cabbage.

[0131]

[0132] Table 8. Effects of strain P34 on the biomass of Chinese cabbage.

[0133]

[0134] Table 9. Effects of strain P34 on the physicochemical properties of the substrate during the seedling stage of Chinese cabbage.

[0135]

[0136] Table 9 shows that the EC value and available potassium under the P34 treatment were not significantly different from those under the CK treatment (P > 0.05); however, the alkaline nitrogen content under the P34 treatment was significantly higher than that under the CK treatment by 61.92%; the available phosphorus content under the P34 treatment reached 61.94 mg / kg, which was significantly higher than that under the CK treatment by 224.96%.

[0137] In summary, phosphate-solubilizing bacteria P34 can affect the physicochemical and biological properties of the substrate, increase the available phosphorus content in the substrate, increase the supply of phosphorus available to plants, and significantly increase the alkaline nitrogen content in the substrate, thereby improving the nutrient level of the substrate. It can effectively improve soil fertility and promote plant growth and development, and has important application value.

Claims

1. The application of a biodegradable organophosphorus pesticide-resistant, disease-resistant, and growth-promoting terrestrial Bacillus terrae or Paenibacillus terrae bacterial solution in the preparation of microbial inoculants for ecological remediation of soil contaminated with malathion, diazinon, phoxim, or fenthion pesticides, characterized in that... The multifunctional terrestrial Bacillus is Paenibacillus terrae, a strain of terrestrial Bacillus, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 30973; The Paenibacillus terrae bacterial culture was obtained by expanding the culture of the Paenibacillus terrae CGMCC No.30973 strain.

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