Multifunctional paenibacillus terrae capable of degrading organophosphorus pesticide, resisting diseases and promoting growth and application of multifunctional paenibacillus terrae

Abstract

The invention provides a multifunctional paenibacillus terrae capable of degrading organophosphorus pesticides, resisting diseases and promoting growth, the strain is paenibacillus terrae and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.30973. The invention further provides a preparation method of the paenibacillus terrae. The strain can degrade organophosphorus pesticides: profenofos, malathion, diazinon, phoxim and fenthion, wherein the degradation effect on the diazinon is the best; the growth and development of meloidogyne incognita can be inhibited, and tomato rhizobium caused by the meloidogyne incognita Fusarium oxysporum can be antagonized, and tomato fusarium wilt caused by the fusarium oxysporum can be prevented and treated; the fertilizer can increase the supply of rapidly available phosphorus in soil and promote the growth of leaf vegetables.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and relates to a multifunctional Paenibacillus terrae capable of degrading organophosphorus pesticides, resisting diseases and promoting growth, and its application. Background Art

[0002] The large-scale production and use of organophosphorus pesticides have led to their accumulation in the soil environment, thus endangering human health. For highly concentrated organophosphorus pesticide pollution, it is impossible to safely utilize the polluted soil in a short time under natural conditions. Therefore, the development of artificial enhanced technologies for organophosphorus pesticide degradation is also an important field in the research of environment-friendly agricultural technologies. Phosphorus-dissolving bacteria can absorb and utilize both organic phosphorus and inorganic phosphorus. The former can effectively degrade organophosphorus pesticides and achieve the ecological restoration of pesticide-polluted soil. Chlorpyrifos, omethoate, diazinon, etc. are synthetic organophosphorus insecticides that are widely used in agricultural production. The accumulation of these pesticides in the soil and the excessive pesticide content in agricultural products endanger the health of consumers. Artificially isolating and culturing various phosphorus-dissolving bacteria existing in the soil can produce microbial agents for repairing pesticide-polluted soil and improving 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. The phosphorus absorbed by plants is generally hydrogen phosphate or dihydrogen phosphate. The phosphorus absorbed by plants mainly comes from the phosphorus in the soil solution. Since some metal elements in the soil will react with phosphorus to form metal complexes, such as Ca, Al, etc., which precipitate or adsorb the phosphorus in the soil, the content of available phosphorus that can be absorbed by plants is reduced. Therefore, how to decompose the phosphorus fixed in the soil into phosphorus that can be absorbed and utilized by plants to improve the yield and quality of crops is an important field in the research of environment-friendly agricultural technologies. Phosphorus-dissolving bacteria can create an acidic environment by secreting various organic acids, inorganic acids or enzymes, activate insoluble or poorly soluble phosphorus, and improve the transformation and absorption of phosphorus by crops, thereby promoting crop growth.

[0004] Meloidogyne incognit and Fusarium oxysporum are the main pathogenic bacteria that cause root-knot disease and fusarium wilt in plants respectively. The accumulation of Meloidogyne incognit and Fusarium oxysporum in continuous cropping soil can aggravate the occurrence of soil-borne diseases and intensify the continuous cropping obstacle. There are various antagonistic microorganisms against the above pathogens in the soil. By artificially isolating and culturing them, microbial agents can be produced for controlling soil-borne diseases and alleviating pathogenic continuous cropping obstacles.

[0005] An important member of plant growth-promoting rhizobacteria such as Paenibacillus terrae. The inventor isolated Paenibacillus terrae from the rhizosphere soil of continuously cropped Lilium davidii var. unicolor. After identification, strain P34 belongs to Paenibacillus terrae, and this strain has the functions of phosphorus degradation, nematode resistance inhibition, and growth promotion. The Paenibacillus terrae strain involved in the present invention is isolated from the rhizosphere soil of Lilium davidii var. unicolor, can stably colonize in the soil, has the effects of decomposing organic phosphorus, degrading soil organic phosphorus pesticides to improve the safety of agricultural products, improving the soil phosphorus supply capacity to improve the phosphorus utilization rate of plants, inhibiting Meloidogyne incognita and antagonizing Fusarium oxysporum to control soil-borne diseases, and has a good growth-promoting effect on Chinese cabbage. Summary of the Invention

[0006] To solve the problems existing in the prior art, the present invention provides a multifunctional Paenibacillus terrae that can degrade organic phosphorus pesticides, resist diseases and promote growth. This strain can degrade organic phosphorus pesticides well and promote the growth of crops at the same time.

[0007] The first object of the present invention is to provide a multifunctional Paenibacillus terrae that can degrade organic phosphorus pesticides, resist diseases and promote growth. The Paenibacillus terrae is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 30973.

[0008] The present invention isolates Paenibacillus terrae strain P34 from the rhizosphere soil of continuously cropped Lilium davidii var. unicolor. After identification, the taxonomic name of this strain is Paenibacillus terrae. It was preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 17, 2024. The preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is: CGMCC No. 30973.

[0009] The second object of the present invention is to provide a Paenibacillus terrae bacterial liquid, which is obtained by expanding the culture of the above-mentioned Paenibacillus terrae CGMCC No. 30973 strain.

[0010] Preferably, the expansion culture is specifically: inoculating the Paenibacillus terrae CGMCC No. 30973 strain into a medium and culturing it on a shaker at 28±1°C; preferably, the medium is LB liquid medium.

[0011] The third object of the present invention is to provide the application of the above-mentioned Paenibacillus terrae or the bacterial liquid of Paenibacillus terrae in the preparation of a phosphorus-solubilizing microbial inoculant product.

[0012] The fourth object of the present invention is to provide the application of the above-mentioned Paenibacillus terrae or the bacterial liquid of Paenibacillus terrae in the preparation of a microbial inoculant product for ecological restoration of organophosphorus pesticide-contaminated soil.

[0013] Preferably, it is at least one of the organophosphorus pesticide-contaminated soils.

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

[0015] The fifth object of the present invention is to provide the application of the above-mentioned Paenibacillus terrae or the bacterial liquid of Paenibacillus terrae in the preparation of a product for biological control of soil-borne diseases such as plant root-knot nematodes.

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

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

[0018] Paenibacillus terrae or the bacterial liquid of Paenibacillus terrae can be used for controlling soil-borne diseases and alleviating 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 solanaceous vegetables, including tomatoes.

[0021] The seventh object of the present invention is to provide the application of the above-mentioned Paenibacillus terrae or Bacillus terrae or the bacterial liquid of Paenibacillus terrae or Bacillus terrae in the preparation of products for improving the physical and chemical properties of soil and promoting the growth of crops.

[0022] The crops are preferably leafy vegetables, including Chinese cabbage.

[0023] Beneficial effects:

[0024] The number of colonies of the Bacillus terrae strain P34 of the present invention is relatively stable, and it can be effectively colonized in the rhizosphere soil. The available phosphorus content in the culture solution after 2 days of culture is 45.38 μg / mL, and it has good phosphorus-solubilizing ability. It can activate insoluble or poorly soluble phosphorus in the soil and improve the transformation and absorption of phosphorus by crops. Secondly, the Bacillus terrae strain P34 can degrade organophosphorus pesticides: profenofos, malathion, diazinon, phoxim, fenthion, among which the degradation effect on diazinon is the best. Thirdly, the Bacillus terrae strain P34 inhibits the growth and development of Meloidogyne incognita and controls tomato root-knot disease caused by this nematode. Fourthly, the Bacillus terrae strain P34 antagonizes Fusarium oxysporum and controls tomato wilt disease caused by Fusarium oxysporum, reducing the occurrence and harm of soil-borne diseases. Fifthly, the Bacillus terrae strain P34 increases the supply of available phosphorus in the soil and promotes the growth of leafy vegetables. Description of the drawings

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

[0026] Figure 2 Phosphorus-solubilizing ability of the Bacillus terrae P34 strain.

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

[0028] A is the primary screening medium; B is the culture on the LB medium; C is the culture on the Meng Jinna organic phosphorus bacteria medium; D is the Gram staining.

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

[0030] Figure 5 Molecular phylogenetic tree of the Bacillus terrae P34 strain.

[0031] Figure 6 Effect of the Bacillus terrae P34 strain on soil colonization ability.

[0032] Figure 7 Degradation ability of the Bacillus terrae P34 strain to organophosphorus pesticides;

[0033] A represents the proliferation ability of bacteria on the culture medium containing 4 kinds of organophosphorus pesticides; B represents the phosphorus accumulation in the culture medium.

[0034] Figure 8 is the effect of Bacillus terrae strain P34 on Meloidogyne incognita under in vitro conditions;

[0035] A is CK; B is the micrograph of the P34 treatment; the stiffened worm body is the dead nematode.

[0036] Figure 9 is the effect of Bacillus terrae strain P34 on Meloidogyne incognita in cultivated tomatoes;

[0037] A is a diseased tomato plant infected with nematodes in CK, with root knots on the roots; B is the tomato plant after the P34 treatment.

[0038] Figure 10 is the effect of Bacillus terrae strain P34 on Fusarium oxysporum.

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

[0040] Figure 11 is the effect of different amounts of Bacillus terrae strain P34 on Fusarium oxysporum;

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

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

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

[0044] Figure 13 is the effect of Bacillus terrae strain P34 on the growth of Chinese cabbage plants.

[0045] Figure 14 is the effect of Bacillus terrae strain P34 on the roots of Chinese cabbage. Detailed implementation methods

[0046] The following embodiments facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The test materials used in the following embodiments are purchased from conventional biochemical reagent companies unless otherwise specified.

[0047] Example 1 Basic characteristics of the Paenibacillus terrae strain P34 of the present invention, CGMCC No. 30973 1.1 Obtaining the Paenibacillus terrae P34 strain of the present invention:

[0048] Take a soil sample from the 5 cm depth of the cultivated layer of Lilium davidii var. unicolor. Add 99 mL of sterile water to a conical flask, take 1 g of rhizosphere soil and put it into the flask, vortex for 1 - 2 min, and then place it in a constant temperature shaker (37 °C, 180 rpm) for shaking culture for 1 h. From the prepared soil suspension, pipette 1 mL of the supernatant and dilute it successively to concentrations of 10 -3 g / mL, 10 -4 g / mL, 10 -5 g / mL, 10 -6 g / mL, 10 -7 g / mL. Pipette 100 μL of each concentration and spread it on a plate (Mengjina organophosphorus bacteria medium, which uses lecithin as the sole phosphorus source) with a sterile spreader. Invert the plate and place it in an incubator at 37 °C for 48 h, repeating 3 times. Pick single colonies with a phosphorus solubilization circle formed from the medium with an appropriate dilution factor, and purify them 4 - 5 times on LB medium using the three-line method to obtain pure single colonies, thereby screening out strains that can solubilize phosphorus.

[0049] Prepare the strain into a bacterial suspension for subsequent determination. The method for preparing the bacterial suspension is as follows: Inoculate strain P34 in LB liquid medium and culture it overnight at 37 °C, 180 rpm; take out the cultured fermentation broth, centrifuge at 11000 rpm for 5 min, pour out the supernatant, and then mix it evenly with sterile distilled water; measure the OD600 value with a UV-visible spectrophotometer and prepare a bacterial suspension with OD600 = 1.

[0050] After identification, the taxonomic name of this strain is Paenibacillus terrae. It was deposited in the China General Microbiological Culture Collection Center (CGMCC) on June 17, 2024. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is: CGMCC No. 30973.

[0051] 1.2 Determination of the phosphorus solubilization ability of the Paenibacillus terrae P34 strain of the present invention

[0052] Inoculate the purified strain into LB liquid medium and shake culture overnight at 37°C and 180 rpm. Then centrifuge at 11,000 r / min for 6 minutes and prepare a bacterial suspension with an OD 600 of 1 using sterile water. Take 2 mL of the above bacterial suspension and inoculate it into 100 mL of Meng Jina organophosphorus bacteria liquid medium (using lecithin as the sole phosphorus source), and shake culture at a constant temperature on a shaker (37°C, 180 rpm) for 48 h. Take 20 mL of the fermentation broth and place it in a 50 mL centrifuge tube. Break it in an ultrasonic cleaner for 20 min to release the available phosphorus in the cells. Then centrifuge at 4°C and 11,000 r / min for 6 minutes. Finally, take 10 mL of the supernatant and filter it through a 0.22 μm filter membrane. Pipette 5 mL into a 50 mL volumetric flask, add about 20 mL of sterile water, add 2 drops of 2,4-dinitrophenol indicator, adjust the pH to slightly yellow with calcium carbonate solution and sulfuric acid solution, add one drop of dilute sulfuric acid until the reaction solution is colorless, accurately add 5 mL of molybdenum antimony anti-color reagent, mix it evenly and make up the volume, let it stand for 30 min, and perform colorimetry at a wavelength of 660 nm. Measure the pH value of the remaining supernatant with a pH meter. Use the non-inoculated medium as a control, adjust the absorbance value to 0, and measure the value of the control with sterile water. Calculate the content of available phosphorus according to the standard curve. The standard curve equation is y = 0.376x + 0.001.

[0053] Results: From the polyphosphate-solubilizing bacteria obtained by primary screening ( Figure 1 ), strain P34 with high phosphorus-solubilizing efficiency was preferably selected. The phosphorus-solubilizing amount of this strain was between 26.94 - 49.66 μg / ml between 12 - 72 h, and reached the peak at 48 h. After that, the phosphorus-solubilizing amount decreased slightly, but it still had strong phosphorus-solubilizing ability ( Figure 2 ). It can be seen that strain P34 degrades the insoluble organic phosphorus in the medium and increases the content of soluble phosphorus in the medium.

[0054] 1.3 The Paenibacillus terrae P34 of the present invention, CGMCC No. 30973, has the following biological characteristics:

[0055] 1.3.1 Morphological and cultural characteristics: As Figure 3 shown, colonies can be observed to be round on the LB plate, slightly yellow in color, with a raised surface and neat edges; colonies can be observed to be round, milky white in color, in the shape of half a glass bead, slightly wet on the surface, and with uneven colony edges on the Meng Jina organophosphorus bacteria medium; under the microscope with Gram staining, strain P34 can be seen to be rod-shaped and Gram-negative. Other physiological and biochemical reactions are as follows: The diacetyl (V-P) reaction and starch hydrolysis test are positive, and the 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 measured in the present invention and is as follows, with a full length of 1434 bp.

[0057]

[0058] 1.3.3 Molecular systematic identification of the strain

[0059] Inoculate strain P34 into LB liquid medium and shake the bacteria overnight at 180 rmp. The bacterial genomic DNA extraction kit was purchased from Beijing Tsingke Biotechnology Co., Ltd., and the DNA extraction steps were carried out according to the method described in the reagent kit. Use the universal bacterial primers 27F (5’-AGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) for PCR amplification. After appropriately diluting the extracted DNA sample, use it as the PCR template and amplify with Tsingke 1×TSE101 Gold Mix. The components of the amplification system are as follows:

[0060]

[0061] The PCR reaction procedure is as follows:

[0062]

[0063] Use agarose gel electrophoresis to detect the amplified PCR products (2 ul sample + 6 ul bromophenol blue) at 300 V for 12 minutes to obtain the identification gel image. The sequencing work was carried out at the Chengdu Branch of Beijing Tsingke Biotechnology Co., Ltd. Finally, the obtained 16S rDNA sequence was aligned in NCBI, and a phylogenetic tree was constructed using MEGA7.0 to identify the phosphate-solubilizing bacterial strain.

[0064] The results showed that: a target gene fragment of 1500 bp was obtained by PCR amplification. By aligning with the homologous sequences in NCBI, strain P34 was identified as Paenibacillus terrae ( Figure 4 , 5).

[0065] 1.4 Soil colonization ability of Paenibacillus terrae strain P34 of the present invention

[0066] Whether strain P34 can exert its phosphate-solubilizing effect depends on its ability to effectively colonize in the rhizosphere soil. Inoculate the bacterial suspension (the preparation method is shown in step 1.1 of Example 1) into the sterilized soil. Monitor the number of bacteria in the soil at different times (obtained by measuring the OD value of soil bacteria).

[0067] The results showed that: 40 days after inoculation, it can be seen that the number of bacteria of strain P34 in the soil decreased with the extension of time. Among them, the number of bacteria decreased rapidly within 2 days of cultivation, and the decrease was relatively slow in the later stage. It can be seen that the number of bacteria in the strain P34 at 12 d, 20 d, and 32 d increased compared with the previous day, and finally stabilized at 2.4×105 CFU / g. Generally speaking, the population of strain P34 is relatively stable and the colonization effect is good.

[0068] ( Figure 6 )

[0069] Example 2 Remediation function of Bacillus terrae strain P34 (CGMCC No. 30973) of the present invention on soil contaminated with organophosphorus pesticides

[0070] 2.1 Under in vitro conditions, strain P34 has a degradation effect on organophosphorus pesticides

[0071] Four organophosphorus pesticides, malathion, diazinon, phoxim, and fenthion, were selected. Strain P34 was inoculated in a beef extract peptone liquid medium added with organophosphorus pesticides. By measuring the OD value of the bacterial solution and the available phosphorus content in the medium, its degradation effect on organophosphorus pesticides under in vitro conditions was evaluated. Phosphorus accumulation was measured by the NaHCO3 extraction - molybdenum antimony anti - colorimetric method.

[0072] Figure 7 Figure A shows that between 12 - 72 h, the OD values of Bacillus terrae strain P34 treated with each pesticide were between 0.96 - 2.19, generally higher than those of the CK, and higher than those of the CK after 24 h. Bacterial proliferation reached a peak between 24 - 36 h and then decreased slightly. Figure 7 Figure B shows that the accumulated amounts of available phosphorus in each pesticide treatment were between 2.40 - 3.14 mg / kg, all significantly higher than those of the CK. Generally speaking, it can be seen that Bacillus terrae strain P34 can effectively degrade organophosphorus pesticides in the medium, increase the content of soluble phosphorus in the medium, and promote the proliferation of phosphorus - solubilizing bacteria. Among them, the degradation effect on diazinon is the best.

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

[0074] The technical principle of this case is as follows: Excessive application of organophosphorus pesticides can cause soil pollution, produce phytotoxicity to crops, and inhibit plant growth; at the same time, pesticides accumulate in plants, damaging the safety and quality of agricultural products. Bacillus terrae strain P34 has a degradation function for organophosphorus pesticides. Therefore, it is very likely that this strain can repair contaminated soil by degrading organophosphorus pesticides, relieve pesticide phytotoxicity, and reduce the residue of organophosphorus pesticides in agricultural products.

[0075] This case is designed for tomatoes (variety: TOM cherry tomatoes). Two organophosphorus pesticides are selected, and a single-factor randomized block experiment is designed, with 4 treatments, namely: Treatment 1: Malathion + P34 bacterial liquid; CK1: Malathion + clear water; Treatment 2: Parathion + P34 bacterial liquid; CK2: Parathion + clear water. The treatment method is as follows: Sow TOM cherry tomato seeds in nutrient pots (8 cm × 10 cm), with 1 seed sown in each pot, place them in an artificial climate chamber, and cultivate them under a photoperiod of 25°C, 85% relative humidity, and 12 h day / 12 h night. When the tomatoes have three true leaves and one heart leaf, dilute the pesticides malathion and parathion at a ratio of 1:500 and irrigate them by the root irrigation method, with 50 mL irrigated per pot, once every 15 days, for 2 times. 5 days after the last pesticide treatment is completed, irrigate the Paenibacillus terrae P34 bacterial liquid of the present invention, with 50 mL per pot, once every 5 days, for 2 times. The preparation method of the Paenibacillus terrae P34 bacterial liquid is the same as that in Step 1.1 of Example 1, and it is suspended with sterile water and the concentration of the Paenibacillus terrae P34 bacterial liquid is adjusted to OD600 = 1. Measure the pesticide accumulation in tomato fruits 5 days after the last treatment. The detection of pesticide residues in tomato fruits is carried out by the gas chromatography method of NY / T 761—2008 "Determination of Multi-Residues of Organophosphorus, Organochlorine, Pyrethroid and Carbamate Pesticides in Vegetables and Fruits". Randomly select 5 plants for each treatment, select the first fruit, and measure it at the color-changing stage. The experimental results are shown in Table 1. The results show that malathion was not detected in the tomato fruits in Treatment 1, and the detected amount of malathion in the soil decreased by 90.07% compared with the control; the detected amount of parathion in the tomato fruits in Treatment 2 decreased by 48.13% compared with the control, and the detected amount of parathion in the soil decreased by 70.67% compared with the control.

[0076] Table 1 Alleviating effect of P34 strain treatment on phytotoxicity of organophosphorus pesticides in TOM tomato fruits

[0077]

[0078] Note: ND means not detected; According to GB2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Foods", the limit values of malathion and parathion in foods are 50 μg·kg -1 , 10 μg·kg -1 .

[0079] Conclusion: The Paenibacillus terrae P34 strain of the present invention can degrade organophosphorus pesticides in the soil, reducing the residue of organophosphorus pesticides in the substrate. At the same time, due to the decrease in the content of organophosphorus pesticides in the soil, the enrichment degree of this pesticide in plants decreases, so the detection rate of this type of pesticide in tomato fruits decreases, and the safety quality of vegetables is significantly improved.

[0080] Example 3 Nematocidal function of Bacillus terrae strain P34 (CGMCC No. 30973) of the present invention 3.1 Inhibitory effect of strain P34 on Meloidogyne incognita under in vitro conditions

[0081] Meloidogyne incognita was selected for in vitro experiments. The nematode was propagated on Solanum lycopersicum 'Qiemen' peppers to prepare a suspension of Meloidogyne incognita eggs (1000 eggs / ml). At the same time, second-stage larvae of Meloidogyne incognita were collected to prepare a nematode suspension (the egg masses were hatched at 28 °C, and the hatched second-stage larvae were collected after 24 hours, 1000 larvae / ml). The bacterial suspension of strain P34 (OD value = 1, 100 μL was added to each well) was co-cultured with nematode eggs and second-stage larvae in a 96-well cell culture plate (50 μL of nematode egg suspension or nematode suspension was added to each well), with 100 μL of sterile water as a control. Each treatment was repeated 6 times, and there were 3 biological replicates in total. It was found that after 6 days, the bacterial suspension treatment had a significant inhibitory effect on the hatching of Meloidogyne incognita eggs, and there was a significant difference in nematode mortality after 72 hours. The results are shown in Table 2. Figure 8 。

[0082] The bacterial suspension of P34 had a significant inhibitory effect on Meloidogyne incognita. The hatching rate of second-stage larvae after 6 days was 65.61% lower than that of the control (Table 2); after culturing for 72 hours, it had a significant lethal effect on nematodes ( Figure 8 ), and the nematode mortality rate treated with P34 was 1.86 times that of the control (Table 2). Therefore, it was shown that strain P34 produced certain toxic substances against Meloidogyne incognita, which inhibited the growth and development of nematodes.

[0083] Table 2 Lethal effect of P34 bacterial suspension treatment on the hatching of Meloidogyne incognita eggs and larvae

[0084]

[0085] Lethality rate = number of dead nematodes / total number of test nematodes

[0086] 3.2 Strain P34 has a control effect on Meloidogyne incognita

[0087] The technical principle of this case is as follows: The infection of Meloidogyne incognita can cause root knots to form on the roots of tomatoes, resulting in poor plant growth. Strain P34 has an inhibitory effect on Meloidogyne incognita. Therefore, by irrigating with the bacterial solution of P34, the incidence of Meloidogyne incognita can be alleviated.

[0088] This case study was designed for tomatoes (TOM cherry tomatoes). It was a single-factor experiment with two treatments, 15 plants in each treatment group, and three replicates: the two treatments were: (1) CK (control): soil infected with root-knot nematodes + clean water. (2) Treatment: soil infected with root-knot nematodes + P34 bacterial solution. The method for obtaining the soil infected with root-knot nematodes was to inoculate the second-instar larvae of southern root-knot nematodes (500 per plant) when the tomatoes had two leaves and one heart. Three consecutive crops of tomatoes were planted in this soil (60 days each crop). Significant symptoms of root-knot nematode disease were observed in the roots of tomatoes, and the soil was collected for research. TOM cherry tomatoes were planted in the infected soil collected by the above method, and cultivated in nutrient pots (8 cm infected soil). One seed was sown in each pot, placed in an artificial climate chamber, and cultured at 25°C, 85% relative humidity, and a photoperiod of 12h day / 12h night. When the first true leaf grows, water the pot with 50 mL of P34 bacterial suspension, once every 5 days, and continuously water for 8 times; the preparation method of P34 bacterial suspension is the same as step 1.1 of Example 1 (bacterial liquid OD600 = 1). After 40 days of treatment, the root root knot nematode disease symptoms 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 nodules ( Figure 9 A), while the tomato root system developed normally after treatment with P34 suspension, with no visible root knots ( Figure 9 B); At the same time, the treatment with P34 bacterial suspension significantly improved various growth indicators of the plants. The plant height, stem diameter, leaf area, fresh weight of the aboveground part, and fresh weight of the underground part increased by 28.52%, 6.93%, 77.31%, 89.71%, and 8.94% respectively compared with the control. Therefore, the inhibitory effect of diseased soil on plant growth was significantly alleviated. (Table 3).

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

[0091]

[0092] Conclusion: The Paenibacillus terrestris P34 strain of the present invention can inhibit southern root-knot nematodes, reduce the incidence of nematode diseases, alleviate the pathogenic continuous cropping obstacles of tomatoes caused by nematode damage, and promote tomato growth. After being treated with phosphate-solubilizing bacteria (i.e., Paenibacillus terrestris P34 of the present invention), the growth of the plants was improved to varying degrees, and the overall growth was significantly improved.

[0093] Example 4 Disease resistance of the Paenibacillus terrestris P34 strain CGMCC No. 30973 of the present invention 4.1 Antagonistic effect of the P34 strain on Fusarium oxysporum under in vitro conditions

[0094] Our laboratory isolated Fusarium oxysporum that causes Fusarium wilt in Lilium davidii var. unicolor. Strain P34 and Fusarium oxysporum were inoculated together on a PDA plate to observe their antagonistic effect. As Figure 10 shown, compared with the control, strain P34 caused Fusarium oxysporum to grow poorly, with yellowing of the mycelium and a smaller colony size. Under the microscope, it can be observed that the hyphae of normal Fusarium oxysporum are smooth and unsegmented, while the hyphae of Fusarium oxysporum under the antagonistic effect are abnormal, with multiple breaks, deformities, and twisted swelling. Figure 11 Different amounts of strain P34 bacterial liquid and Fusarium oxysporum were inoculated together on a PDA plate, and the inhibitory effect of P34 on Fusarium oxysporum was determined by the plate confrontation method. The inhibition rate of the 1.0 μg / ml P34 bacterial liquid was 70.93%, and the inhibition rate of the 0.5 μg / ml P34 bacterial liquid was 18.60%. The results showed that strain P34 produced certain resistance substances that damaged the hyphae of Fusarium oxysporum, inhibiting the growth of Fusarium oxysporum.

[0095] 4.2 Under hydroponic conditions, strain P34 has a disease-resistant effect on tomato Fusarium wilt caused by Fusarium oxysporum

[0096] The technical principle of this case is as follows: Fusarium oxysporum can cause tomato Fusarium wilt, and Bacillus terrae strain P34 has an antagonistic function against Fusarium oxysporum. Therefore, it is very likely that strain P34 can reduce the incidence of tomato Fusarium wilt by inhibiting the proliferation of Fusarium oxysporum.

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

[0098] (1) Treatment 1: Fusarium oxysporum + clear water. The inoculation method of Fusarium oxysporum was as follows: The Fusarium oxysporum strain was cultured in a PDA medium in an incubator at 28°C for 7 days. 12 cakes were placed in each Erlenmeyer flask, and glass beads were added. It was shaken at 28°C and 180 rpm for 24 h. The filtrate was collected by suction filtration, diluted with sterile water, and the spore count was determined by a hemocytometer. The spore suspension concentration was diluted to 10 7 spores / mL as the Fusarium oxysporum bacterial liquid for standby. When the tomato had two true leaves and one heart leaf, 50 mL of the Fusarium oxysporum bacterial suspension was added to the nutrient solution, and 50 mL of clear water was added 2 days later.

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

[0100] (3) CK: Treat with equal volumes of clear water instead of Fusarium oxysporum and P34 bacterial suspension respectively, and the treatment time is the same as that of Treatment 2.

[0101] Sow TOM cherry tomato seeds in hydroponic nutrient pots (8.7 cm × 12.7 cm × 11.4 cm), sowing 6 seeds in each pot, place them in an artificial climate chamber, and cultivate them under a photoperiod of 25°C, 85% relative humidity, and 12 h day / 12 h night. When the plants grow to two leaves and one heart, start the treatment. Measure the plant height, stem diameter, and biomass of each plant 5 days after the last treatment. Table 4 shows the growth of TOM tomatoes after treatment with Fusarium oxysporum and P34.

[0102] The results show that: compared with CK ( Figure 12 A), after the tomato is inoculated with Fusarium oxysporum, typical symptoms of fusarium wilt appear in the plants 3 - 4 days later: from the lower part to the upper part of the tomato plant, the leaf tips of the leaves turn yellow, the growth is inhibited, and some plants wilt and die. Figure 12 B), after adding the P34 bacterial solution to the plants inoculated with the pathogen, wilting and death of the tomato plants only occur sporadically, and the symptom of yellowing leaf tips is 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 Classification criteria for the disease level of TOM tomato fusarium wilt

[0104]

[0105]

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

[0107] Table 5 Incidence of TOM tomato fusarium wilt after treatment with Fusarium oxysporum and P34

[0108]

[0109] Note: The data of the incidence rate and disease index are statistically processed for Treatment 1 and Treatment 2.

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

[0111] Disease index (DI):

[0112] Where: DI - disease index; s - representative value of each disease level; n - number of plants at each disease level; N - total number of surveyed plants; S - representative value of the highest disease level

[0113] Conclusion: The Paenibacillus terrae strain P34 of the present invention can reduce the incidence rate of fusarium wilt of solanaceous vegetables caused by Fusarium oxysporum, reduce the disease index, and promote plant growth. After treatment with the Paenibacillus terrae strain P34, the growth of tomatoes is improved to varying degrees, and the overall growth is promoted.

[0114] 4.3 Under the conditions of substrate cultivation, the P34 strain promotes the growth of tomatoes by overcoming the pathogenic continuous cropping obstacle of fusarium wilt

[0115] The technical principle of this case is as follows: Fusarium oxysporum is the main fungus that causes fusarium wilt in plants. The increase of Fusarium oxysporum leads to the occurrence of fusarium wilt in plants, exacerbating the continuous cropping obstacle. The Paenibacillus terrae strain P34 has an antagonistic function against Fusarium oxysporum. Therefore, by irrigating the Paenibacillus terrae strain P34 bacterial solution, the pathogenic continuous cropping obstacle is alleviated.

[0116] This case is designed for tomatoes (variety: TOM cherry tomatoes), which is a single-factor experiment. There are 2 treatments, 15 plants in each group, and 3 replicates:

[0117] (1) Treatment 1: Fusarium oxysporum + clear water. The inoculation method of Fusarium oxysporum is as follows: The Fusarium oxysporum strain is cultured in a PDA medium in an incubator at 28°C for 7 days. 12 cakes are placed in each Erlenmeyer flask, and glass beads are added. It is shaken at 28°C and 180 rpm for 24 hours. The filtrate is collected by suction filtration, and the filtrate is diluted with sterile water. The number of spores is counted by a hemocytometer, and the spore suspension concentration is diluted to 10 7 cells / mL and used as the Fusarium oxysporum bacterial solution for standby. When the tomatoes have three true leaves and one heart leaf, Fusarium oxysporum is irrigated by the root irrigation method, 50 mL is irrigated per pot, and it is irrigated once every 5 days for 4 times. After the plants are diseased, clear water is irrigated, 50 mL is irrigated per pot, and it is irrigated once every 5 days for 4 times.

[0118] (2) Treatment 2: Fusarium oxysporum + P34 bacterial liquid. The inoculation method of Fusarium oxysporum is the same as that in step 4.2 of Example 4. When the tomato has three leaves and one heart, Fusarium oxysporum is watered by the root irrigation method, 50 mL is watered per pot, and it is watered once every 5 days for 4 times. After the plant gets sick, the Paenibacillus terrae P34 bacterial liquid of the present invention is watered, 50 mL is watered per pot, and it is watered once every 5 days for 4 times. The preparation method of the Paenibacillus terrae P34 bacterial liquid is the same as that in step 1.1 of Example 1, and it is suspended with sterile water and the concentration of the Paenibacillus terrae P34 bacterial liquid is adjusted to OD600 = 1.

[0119] Sow TOM cherry tomato seeds in nutrient pots (8 cm × 10 cm), sow 1 seed per pot, place them in an artificial climate chamber, and cultivate them under a photoperiod of 25°C, 85% relative humidity and 12 h day / 12 h night. When the plants grow to three leaves and one heart, start the treatment. The plant height, stem diameter and biomass of each plant are measured 5 days after the last treatment. Table 6 shows the growth of TOM tomatoes after treatment with Fusarium oxysporum and P34.

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

[0121]

[0122] Conclusion: The Paenibacillus terrae P34 strain of the present invention can antagonize Fusarium oxysporum, can overcome pathogenic continuous cropping obstacles, and promote plant growth. After treatment with phosphate-solubilizing bacteria (i.e., the Paenibacillus terrae P34 of the present invention), the growth of tomatoes is improved to varying degrees, and the overall growth is promoted.

[0123] Example 5 Growth promotion effect of the Paenibacillus terrae strain P34 strain CGMCC No. 30973 of the present invention

[0124] By watering the P34 bacterial liquid, the growth of Chinese cabbage is promoted under the condition of complete nutrient substrate cultivation. In this case, the Paenibacillus terrae strain P34 bacterial suspension is watered to Chinese cabbage to study its growth promotion effect on Chinese cabbage.

[0125] Two treatment groups are designed for each plant: CK group (sterile water), P34 group (P34 bacterial liquid of the present invention). There are 15 plants in each treatment, with 3 replicates.

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

[0127] Disinfect and sterilize the plug tray with 75% alcohol in advance, then put in the seedling medium (commercial seedling medium, complete nutrition), then plant the cabbage seeds in the plug tray, place it in an artificial climate box (light temperature / dark temperature is 25℃ / 20℃, light time / dark time is 16h / 8h), water it regularly, and maintain it. After growing 3 to 4 true leaves, select the cabbage seedlings with consistent growth and transplant them into the disinfected pots, one plant per pot. Pour 50mL of the above bacterial suspension on the roots of the plants, repeat 8 times, add an equal amount of sterile water for the control, then place it in an artificial climate box (light temperature / dark temperature is 25℃ / 20℃, light time / dark time is 16h / 8h), and water it regularly and quantitatively. Observe and record the situation after 40 days. Observe and record the morphological indicators such as plant root length, plant height, stem thickness, leaf area, aboveground weight and underground weight. The experimental results are shown in Tables 7-8 and Figures 13 - 14 .

[0128] Determination of physical and chemical properties and biological properties of the matrix: The method of collecting soil samples is: dig out the plant roots and gently shake the plant roots to shake off the attached matrix, wear sterile gloves and collect the fallen matrix as rhizosphere soil in a sterile sample bag. Five plants were randomly selected for each treatment for determination. Each index was determined 30 days after the plants were treated with the bacterial solution. The EC value of soil physical and chemical properties was determined using a DDS-307A conductivity meter according to V soil: V water = 1:10. Alkaline hydrolysis nitrogen, available potassium, and available phosphorus were determined using alkaline diffusion method, 1.0 mol / L ammonium acetate extraction-flame photometry, and 0.5 mol / L sodium bicarbonate extraction-molybdenum antimony colorimetric method. The experimental results are shown in Table 9.

[0129] Figure 13 and Figure 14 It can be seen that the cabbage treated with strain P34 for 40 days was taller, had more and larger leaves, and longer roots than the control group CK. The plant height, root length, and leaf area were significantly increased by 22.83%, 82.87%, and 67.31% respectively compared with the control, and the stem diameter was not significantly different from the control, but increased by 6.21% (Table 7). As shown in Table 8, the aboveground fresh weight, aboveground 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 Effect of strain P34 on the growth of cabbage

[0131]

[0132] Table 8 Effect of strain P34 on the biomass of cabbage

[0133]

[0134] Table 9 Effects of Strain P34 on the Physicochemical Properties of the Substrate for the Seedling Stage of Chinese Cabbage

[0135]

[0136] Table 9 shows that there were no significant differences in the EC value and available potassium between the P34 treatment and the CK (P>0.05); however, the content of alkaline hydrolyzable nitrogen in the P34 treatment was significantly higher than that in the CK treatment by 61.92%; the available phosphorus content in the P34 treatment reached 61.94 mg / kg, which was significantly higher than that in the CK treatment by 224.96%.

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

Claims

1. A multifunctional Paenibacillus terrae that can degrade organophosphorus pesticides, resist diseases and promote growth, characterized in that, The multifunctional Paenibacillus terrae is Paenibacillus terrae. The strain is Paenibacillus terrae and is deposited in the General Microbiological Center of the China General Microbiological Culture Collection Center with the deposit number of CGMCC No. 30973.

2. Paenibacillus terrae bacterial liquid, characterized in that: It is obtained by enlarged cultivation of the Paenibacillus terrae CGMCC No. 30973 strain described in claim 1.

3. The Paenibacillus terrae bacterial solution according to claim 2, characterized in that: The enlarged cultivation is specifically as follows: inoculating the Paenibacillus terrae CGMCC No. 30973 strain into an LB liquid medium and culturing it on a shaker at 28±1°C.

4. Use of the Paenibacillus terrae described in claim 1 or the Paenibacillus terrae bacterial liquid described in claim 2 or 3 in the preparation of a product for phosphorus solubilization.

5. Use of the Paenibacillus terrae described in claim 1 or the Paenibacillus terrae bacterial liquid described in claim 2 or 3 in the preparation of a microbial inoculant product for ecological restoration of soil polluted by organophosphorus pesticides.

6. Use of the Paenibacillus terrae described in claim 1 or the Paenibacillus terrae bacterial liquid described in claim 2 or 3 in the preparation of a disease-resistant product.

7. The application according to claim 6, wherein The pathogen includes Fusarium oxysporum.

8. Use of the Paenibacillus terrae described in claim 1 or the Paenibacillus terrae bacterial liquid described in claim 2 or 3 in the preparation of a product for biological control of plant root-knot nematodes or plant fusarium wilt.

9. Use of the Paenibacillus terrae described in claim 1 or the Paenibacillus terrae bacterial liquid described in claim 2 or 3 in the preparation of a product for improving soil physical and chemical properties and promoting crop growth.

10. The application according to claim 9, characterized in that: The crop is leafy vegetable balls.

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