Petroleum neorhizobium CTR46 and application thereof in prevention and treatment of cotton verticillium wilt

Abstract

The invention belongs to the technical field of microorganisms, and discloses novel rhizobium petroleum CTR46 and application thereof in prevention and treatment of cotton verticillium wilt. According to the present invention, the new petroleum rhizobium having good inhibition effect on cotton verticillium wilt pathogenic bacteria is separated from the healthy cotton stem tissue, specifically, the new petroleum rhizobium CTR46 is obtained, and the plate buckling test and the pot experiment show that the new petroleum rhizobium CTR46 strain has good verticillium dahliae growth inhibition function, and can be used for preparing the new petroleum rhizobium CTR46 strain, such that the new petroleum rhizobium CTR46 can provide the good cotton verticillium wilt pathogenic bacteria inhibition effect, and can provide the good cotton verticillium wilt pathogenic bacteria inhibition effect on cotton verticillium wilt pathogenic bacteria inhibition effect on cotton verticillium wilt pathogenic bacteria inhibition effect on cotton verticillium wilt pathogenic bacteria inhibition effect on cotton verticillium wilt pathogenic bacteria. The composition can be used for preventing and treating cotton verticillium wilt. The novel rhizobium petroleum CTR46 also has the effect of inhibiting the growth of sclerotinia sclerotiorum and botryosphaeria dothidea, and has broad-spectrum antibacterial activity. The invention provides a new strain resource for biological prevention and control of fungal diseases of cotton.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to petroleum new rhizobium CTR46 and application thereof in the prevention and treatment of cotton Verticillium wilt. Background Art

[0002] Cotton Verticillium Wilt is a fungal soil-borne disease caused by Verticillium dahliae. The pathogen infects the vascular tissue of cotton, resulting in stunted growth of cotton plants, which in turn leads to reduced yield and quality. Verticillium dahliae lurks in the soil and diseased residues in the form of microsclerotia for a long time. It has strong infectivity and soil persistence. It usually invades from the roots of cotton and spreads upward through the vascular bundles, causing the leaves of cotton plants to turn yellow and wilt. In severe cases, the entire plant dies. Due to factors such as continuous cotton cropping and returning aid to the fields, Verticillium dahliae accumulates and increases in the soil year by year, posing a threat to the development of the cotton industry and the production of raw materials for the textile industry.

[0003] At present, the prevention and control methods of cotton Verticillium wilt include: selecting disease-resistant varieties, field management, scientific fertilization, chemical control, biological control, etc. Among them, biological control has the advantages of low toxicity, pollution-free and sustainability. Plant endophytes are fungi or bacteria that live inside plant tissues and organs. They are commonly found in higher plants. They are safe to the host and not easy to develop resistance. They can effectively inhibit the infection of pathogens through mechanisms such as competing for nutrients, producing antibiotics and promoting resistance of receptor plants. They are a high-quality source of biological control agents. Using endophytes for biological control of cotton Verticillium wilt can not only reduce the use of chemical pesticides and protect the ecological environment, but also improve the disease resistance and quality of cotton. Therefore, exploring a plant endophyte for cotton Verticillium wilt is of great significance for the biological control of cotton Verticillium wilt. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides Neorhizobium petrolearium CTR46 and its application in the prevention and treatment of cotton Verticillium wilt. The present invention isolates a rhizobium with good inhibitory effect on cotton Verticillium wilt pathogens from healthy cotton stem tissue, specifically Neorhizobium petrolearium CTR46. Neorhizobium petrolearium CTR46 also has the effect of inhibiting the growth of Sclerotinia sclerotiorum and Botrytis cinerea, and has a broad-spectrum antibacterial property. The present invention provides a new strain resource for the biological control of cotton fungal diseases.

[0005] To achieve the technical purpose of the present invention, on the one hand, the present invention provides a strain of Neorhizobium petroliferum CTR46, the nucleotide sequence of 16S rRNA of Neorhizobium petroliferum CTR46 is shown in SEQ ID NO: 1. Neorhizobium petroliferum CTR46 appears as white circular colonies with rounded protrusions on the surface on an LB plate, and can reduce nitrate.

[0006] The petroleum new rhizobium CTR46 of the present invention was collected from the stem tissue of cotton plants in the field of Alaer City, Xinjiang Uygur Autonomous Region. The deposit information is as follows:

[0007] Taxonomic nomenclature: Neorhizobium petrolearium CTR46;

[0008] Deposit date: December 4, 2024;

[0009] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0010] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0011] Deposit number: CGMCC No.31497.

[0012] On the other hand, the present invention seeks to protect the use of the above-mentioned New Rhizobium petroleum CTR46 in the prevention and treatment of cotton Verticillium wilt, wherein the pathogen of cotton Verticillium wilt is Verticillium dahliae.

[0013] Furthermore, the present invention found through a plate matching test that the petroleum new rhizobium CTR46 has a strong antagonistic effect on Verticillium dahliae. The petroleum new rhizobium CTR46 is an antagonistic strain of Verticillium dahliae, and its antibacterial rate is 62.86%.

[0014] On the other hand, the present invention requests to protect the application of the volatile substances of the above-mentioned new petroleum rhizobium CTR46 under solid culture in the prevention and control of cotton wilt, and the volatile substances of the new petroleum rhizobium CTR46 under solid culture are any one of dimethyl trisulfide, furfural, butyric acid, and terpinene-4-ol.

[0015] Furthermore, the present invention cross-cultures the pure volatile substances obtained by petroleum rhizobium CTR46 under solid culture conditions with Verticillium dahliae and finds that the volatile substances of petroleum rhizobium CTR46 have a strong antagonistic effect on Verticillium dahliae, wherein dimethyl trisulfide can achieve a 100% inhibition rate on Verticillium dahliae at an addition amount of 5 μL, furfural can achieve a 100% inhibition rate on Verticillium dahliae at an addition amount of 20 μL, butyric acid can achieve a 100% inhibition rate on Verticillium dahliae at an addition amount of 10 μL, and terpinene-4-ol can achieve a 100% inhibition rate on Verticillium dahliae at an addition amount of 20 μL.

[0016] On the other hand, the present invention claims to protect the use of the above-mentioned New Rhizobium petroleum CTR46 in inhibiting the growth of Sclerotinia sclerotiorum or inhibiting the growth of Botryosphaeria dothidea.

[0017] Furthermore, the present invention cultured the new rhizobium CTR46 with Sclerotinia sclerotiorum and Botrytis cinerea respectively and found that the colonies in the control group covered the entire culture dish, and the colony diameter of the group treated with the new rhizobium CTR46 was significantly smaller than that of the control group, indicating that the new rhizobium CTR46 has a good inhibitory effect on both Sclerotinia sclerotiorum and Botrytis cinerea, and has a broad-spectrum antibacterial effect.

[0018] On the other hand, the present invention claims protection for a microbial agent comprising the above-mentioned Neorhizobium petroleum CTR46.

[0019] In addition, the present invention seeks to protect a method for preventing and controlling cotton Verticillium wilt, using the above-mentioned microbial agent.

[0020] Furthermore, the present invention found through potted plant experiments that the new rhizobium CTR46 has a good control effect on cotton Verticillium wilt, and the growth state of cotton plants is better after inoculation with the new rhizobium CTR46. The control effect of the new rhizobium CTR46 on cotton Verticillium wilt is 55%±1%.

[0021] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0022] (1) The present invention isolates a rhizobium strain having a good inhibitory effect on the pathogen of cotton Verticillium wilt from healthy cotton stem tissue, specifically, Neorhizobium petrolearium CTR46. The present invention found through a plate-to-plate test that Neorhizobium petrolearium CTR46 has a strong antagonistic effect on Verticillium dahliae. Neorhizobium petrolearium CTR46 is an antagonistic strain of Verticillium dahliae, and its inhibition rate is 62.86%.

[0023] (2) The volatile substances produced by the petroleum rhizobium CTR46 of the present invention under solid culture have a good inhibitory effect on the pathogenic bacteria of cotton Verticillium wilt. The present invention cultured the pure volatile substances obtained by the petroleum rhizobium CTR46 under solid culture conditions with Verticillium dahliae and found that the volatile substances of the petroleum rhizobium CTR46 had a strong antagonistic effect on Verticillium dahliae, among which dimethyl trisulfide could achieve a 100% inhibition rate on Verticillium dahliae at an addition amount of 5 μL, furfural could achieve a 100% inhibition rate on Verticillium dahliae at an addition amount of 20 μL, butyric acid could achieve a 100% inhibition rate on Verticillium dahliae at an addition amount of 10 μL, and terpinene-4-ol could achieve a 100% inhibition rate on Verticillium dahliae at an addition amount of 20 μL.

[0024] (3) The new rhizobium CTR46 of the present invention has a broad-spectrum antibacterial property, which can not only inhibit the growth of cotton Verticillium wilt pathogens, but also inhibit the growth of Sclerotinia sclerotiorum and Botrytis cinerea. The present invention cultured the new rhizobium CTR46 with Sclerotinia sclerotiorum and Botrytis cinerea respectively, and found that the colonies in the control group were all covered with the culture dish, and the colony diameter of the group treated with the new rhizobium CTR46 was significantly smaller than that of the control group, indicating that the new rhizobium CTR46 has a good inhibitory effect on Sclerotinia sclerotiorum and Botrytis cinerea, and has a broad-spectrum antibacterial effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a plate culture picture of the new rhizobium for petroleum CTR46.

[0026] Figure 2 This is the average nucleotide similarity analysis diagram of the genome of Neorhizobium petroleum CTR46.

[0027] Figure 3 This is the morphology of the new rhizobium for petroleum CTR46 under a scanning electron microscope.

[0028] Figure 4 This is the result of the nitrate reduction test of the new rhizobium for petroleum CTR46. Figure 4 Figure a is the result of the nitrate reduction test of Neorhizobium petroleum CTR46; Figure 4 b in the figure is the negative control.

[0029] Figure 5 This is a plate-matching image of Neorhizobium petroleum CTR46 and Verticillium dahliae. Figure 5 a in the figure is a plate cross-section diagram of Verticillium dahliae in the control group; Figure 5 Figure b is a cross-plating image of the petroleum neorhizobium CTR46 and Verticillium dahliae.

[0030] Figure 6This is a plate cross-plot of volatile substances from the solid culture of Neorhizobium petroleum CTR46 and Verticillium dahliae.

[0031] Figure 7 This is the result of a potted test of the new petroleum rhizobium CTR46 against cotton Verticillium wilt. Figure 7 a in the figure is the cotton plant of the control group; Figure 7 Figure b shows cotton plants in the group treated with Neorhizobium petroleum CTR46.

[0032] Figure 8 This is the disease index result of the potted test of New Rhizobium petroleum CTR46 against Verticillium wilt in cotton.

[0033] Fig. 9 This is a plate-matching image of Neorhizobium petroleum CTR46 and Sclerotinia sclerotiorum. Fig. 9 a in the figure is a plate-button diagram of Sclerotinia sclerotiorum in the control group; Fig. 9 Figure b is a plate-matching image of Neorhizobium petroleum CTR46 and Sclerotinia sclerotiorum.

[0034] Fig.10 This is a plate-matching image of the new rhizobium of petroleum CTR46 and Botrytis cinerea. Fig.10 a in the figure is a plate cross-section of the control group of Botrytis cinerea; Fig.10 Figure b is a plate-to-plate image of Neorhizobium petroleum CTR46 and Botrytis cinerea.

[0035] Fig.11 This is the statistical result of colony diameter after the plate matching test of New Rhizobium Petroleum CTR46 with Sclerotinia sclerotiorum and Botrytis cinerea. Fig.11 a in the figure is the statistical result of colony diameter after the plate matching test of Neorhizobium petroleum CTR46 and Sclerotinia sclerotiorum; Fig.11 Figure b is the statistical result of colony diameter after the plate matching test between Neorhizobium petroleum CTR46 and Botrytis cinerea. DETAILED DESCRIPTION

[0036] The technical scheme of the present invention is now described in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0037] Potato dextrose agar (PDA) medium, including: peeled potato 200 g / L, glucose 20 g / L, agar 20 g / L, autoclaved at 115°C for 20 min.

[0038] Nitrate reduction test medium: 0.2 g potassium nitrate, 5 g peptone, 1 L distilled water, pH = 7.0, dispense 5 mL into test tubes, and sterilize at 121°C for 20 min.

[0039] Griess reagent: Solution A (0.8 g p-aminobenzenesulfonic acid dissolved in 100 mL 2.5 mol / L acetic acid solution) and Solution B (0.5 g naphthylamine dissolved in 100 mL 2.5 mol / L acetic acid solution).

[0040] Example 1

[0041] This example provides the isolation, identification, morphological observation, and physiological and biochemical tests of Neorhizobium petroleum CTR46.

[0042] 1. Isolation of new petroleum rhizobium CTR46

[0043] Samples of cotton plants with good growth and health collected from the field in Alaer City, Xinjiang Uygur Autonomous Region were taken, and 3 g of stem tissue was weighed. The dust on the surface of the tissue was washed with pure water, soaked in anhydrous ethanol for 1 min, and then rinsed with sterile water 3 times, 1 min each time. After wiping dry, the tissue was ground into a homogenate in a sterilized mortar in a clean bench, and 10 mL of sterile phosphate buffer (0.1 mol / L PBS) was added. After fully mixing, gradient dilution was performed, 100 μL of gradient dilution was taken, and it was evenly spread on a tryptone soy agar (TSA) plate with a sterile coating rod, and inverted in a 30 ° C incubator for 3 to 5 days. When colonies grew on the plate, the strains were separated and purified according to the size and morphology of the colonies grown on the culture medium, and they were picked separately with a sterile inoculation loop, and then streaked on a new tryptone soy agar plate for separation and purification until single colonies with different morphologies and obvious separation grew. Pick a single colony grown on the plate and inoculate it into LB liquid medium, culture it at 30℃ and 180rpm / min for 24h, mix the bacterial solution with 50% glycerol in a 1:1 volume ratio, put it into a sterile cryovial, and store it in a -80℃ refrigerator. The growth morphology of CTR46 strains on LB plates is as follows Figure 1 As shown, it appears as white circular colonies with rounded protrusions on the surface.

[0044] 2. Identification of the new petroleum rhizobium CTR46

[0045] The strain CTR46 was sent to Guangdong Meige Technology Co., Ltd. for whole genome second-generation + third-generation sequencing. The 16S rRNA sequence of the strain is shown in SEQ ID NO: 1. After comparison with the NCBI database, the strain was determined to be Rhizobium. The whole genome sequence of strain CTR46 was compared with strains with high similarity to Rhizobium for genome average nucleotide similarity analysis (ANI analysis). The comparison results are shown in Figure 2As shown. Figure 2 It can be seen that the genome similarity between the strain of the present invention and Neorhizobium petrolearium OS53 is 95.34%, indicating that the strain CTR46 of the present invention is Neorhizobium petrolearium, and is named Neorhizobium petrolearium CTR46.

[0046] 3. Morphological observation of Neorhizobium petroleum CTR46 under scanning electron microscope

[0047] The new rhizobium of petroleum CTR46 was inoculated into LB liquid culture medium, cultured in a constant temperature incubator at 37°C for 48h, washed with PBS 3 times, and added an equal volume of PBS to mix and set aside. 300μL 2.5% glutaraldehyde was added to 1mL of bacterial solution, mixed and placed at 4°C overnight for fixation. Washed with PBS and centrifuged at 8000rpm for 10min, washed and centrifuged 3 times, the fixative was removed, and then dehydrated with ethanol gradient (30%, 50%, 70%, 80%, 95%, 100%). After each addition of ethanol, incubated at 4°C for 15min, then centrifuged at 8000rpm for 10min, and finally added 100% anhydrous ethanol for centrifugation and washing twice (5000xg, 10min), and suspended with 200μL anhydrous ethanol for use. Take 10μL of the new rhizobium of petroleum CTR46 suspended in anhydrous ethanol, drop it on the surface of the silicon wafer, and dry it with CO2. The dried samples were separated into front and back sides, and then glued to the conductive glue for further gold spraying and observation. Figure 3 , short rod-shaped.

[0048] 4. Nitrate reduction test of petroleum rhizobium CTR46

[0049] Pick a single colony of Petroleum Rhizobium CTR46 and inoculate it into the sterilized nitrate reduction test medium, and culture it at 30℃ and 220r / min for 5 days. Pour the Petroleum Rhizobium CTR46 cultured in the nitrate reduction test medium into a watch glass, drop one drop of Griess reagent A solution and one drop of Griess reagent B solution respectively, and immediately observe the color change ( Figure 4 ), the appearance of red is a positive reaction, indicating that nitrate is reduced. Figure 4 It can be seen that Neorhizobium petroleum CTR46 can reduce nitrate.

[0050] Example 2

[0051] This example provides a comparison test between Neorhizobium petroleum CTR46 and Verticillium dahliae, the pathogen of cotton Verticillium wilt, to determine the antagonistic ability of Neorhizobium petroleum CTR46 against Verticillium dahliae.

[0052] Preparation of the bacterial solution of Petroleum New Rhizobium CTR46: Inoculate the biocontrol strain stored at -80℃ into LB liquid culture medium, shake and culture at 30℃ and 200r / min for 48h to obtain seed solution. Use a sterile inoculation loop to take the above seed solution and streak it on LB solid culture medium, culture at 30℃ for 48h to obtain a single colony. Pick a single colony and inoculate it into LB liquid culture medium, culture at 30℃ and 220r / min for 48h, wash off the excess LB liquid culture medium with sterile PBS buffer, and resuspend the bacteria with sterile PBS buffer to OD 600 Reaching 1.6-1.7, the new petroleum rhizobium CTR46 bacterial liquid was obtained.

[0053] Take 20 μL of the bacterial solution of New Rhizobium Petroleum CTR46 and spread it evenly on the LB solid culture medium. Pick a bacterial cake (5 mm in diameter) of Verticillium dahliae V592 (provided by the laboratory of Northwest Agriculture and Forestry University) and place it in the center of the PDA culture medium plate. Place the LB solid plate coated with New Rhizobium Petroleum CTR46 on it, and tightly wrap the interface of the two plates with sealing film to seal it. Use sterile phosphate buffer as a negative control. Culture in a 26°C incubator for 5 to 7 days. When the Verticillium dahliae in the control group fills the plate, calculate the antibacterial rate of each test strain. The calculation results are shown in Table 1. The plate overlap diagram is shown in Table 1. Figure 5 shown.

[0054] Inhibition rate = (colony radius of control group - colony radius of treatment group) / colony radius of control group × 100%.

[0055] Table 1: Inhibition rate of Neorhizobium petroleum CTR46 against Verticillium dahliae

[0056] Group Colony diameter Antibacterial rate (%) Control group (CK) 3.50±0.03 - Experimental group (CTR46) 1.3±0.09 62.86

[0057] From Table 1 and Figure 5 It can be seen that the petroleum rhizobium CTR46 has a strong antagonistic effect on Verticillium dahliae. The petroleum rhizobium CTR46 is an antagonistic strain of Verticillium dahliae, and its inhibition rate is 62.86%.

[0058] Example 3

[0059] This example provides the detection of volatile substances of Neorhizobium petroleum CTR46 and the detection of the antibacterial ability of some volatile substances against Verticillium dahliae, the pathogen of cotton Verticillium wilt.

[0060] 1. Detection of volatile substances of petroleum new rhizobium CTR46

[0061] A 20 mL brown headspace injection bottle was sterilized, and 5 mL of LB medium containing agar that had not solidified after sterilization was added to make a solid slope. A single colony of Petroleum Neorhizobium CTR46 was streaked, the bottle mouth was tightened, and the bottle was placed in a 30°C constant temperature incubator for culture for 3 days. The liquid LB streak was used as a negative control, and headspace solid phase microextraction (HS-SPME)-GC-MS was used to detect volatile substances. The differences in volatile substances between Petroleum Neorhizobium CTR46 and the negative control are shown in Table 2.

[0062] HS-SPME extraction conditions: at 60℃ constant temperature, shake for 5min, insert 120μmDVB / CWR / PDMS extraction head into sample headspace bottle, headspace extraction for 15min, analyze at 250℃ for 5min, and then perform GC-MS separation and identification. The extraction head is aged at 250℃ for 5min in Fiber Conditioning Station before sampling. Note: New extraction heads are aged in Fiber Conditioning Station for 2h before extraction. Using SPME Arrow, its sensitivity can reach 10 times that of traditional SPME fiber heads.

[0063] Chromatographic conditions: DB-5MS capillary column (30m×0.25mm×0.25μm, Agilent J&W Scientific, Folsom, CA, USA), carrier gas is high-purity helium (purity not less than 99.999%), constant flow rate 1.2mL / min, injection port temperature 250°C, solvent delay 3.5min. Program temperature rise: 40°C for 3.5min, increase to 100°C at 10°C / min, then increase to 180°C at 7°C / min, and finally increase to 280°C at 25°C / min and hold for 5min.

[0064] Mass spectrometry conditions: electron impact ion source (EI), ion source temperature 230 °C, quadrupole temperature 150 °C, mass spectrometer interface temperature 280 °C, electron energy 70 eV, scanning mode selected ion detection mode (SIM), qualitative and quantitative ion precise scanning (GB 23200.8-2016).

[0065] Table 2: Analysis of volatile substances in solid culture of Neorhizobium petroleum CTR46

[0066]

[0067]

[0068] 2. Detection of the antibacterial ability of some volatile substances on Verticillium dahliae

[0069] Pick a Verticillium dahliae cake (5 mm in diameter) and place it in the center of the PDA medium plate. Take 2.5 μL, 5 μL, 15 μL, and 20 μL of pure dimethyl trisulfide, furfural, butyric acid, and terpinen-4-ol, respectively, and apply them on the medium cover and buckle them with the PDA plate. Use sealing film to tightly wrap the interface and apply sterile phosphate buffer as a negative control. Culture in a constant temperature incubator at 26°C for 5 to 7 days ( Figure 6 ), when the Verticillium dahliae in the control group fully grew on the plate, the inhibition rate was calculated (Table 3).

[0070] Table 3: Inhibition rate of volatile substances of Neorhizobium petroleum CTR46 against Verticillium dahliae

[0071]

[0072]

[0073] From Table 3 and Figure 6 It can be seen that the volatile substances of the new rhizobium of petroleum CTR46 have a strong antagonistic effect on Verticillium dahliae. Among them, dimethyl trisulfide can achieve a 100% inhibition rate on Verticillium dahliae at an addition amount of 5 μL, furfural can achieve a 100% inhibition rate on Verticillium dahliae at an addition amount of 20 μL, butyric acid can achieve a 100% inhibition rate on Verticillium dahliae at an addition amount of 10 μL, and terpinen-4-ol can achieve a 100% inhibition rate on Verticillium dahliae at an addition amount of 20 μL, which further confirms that the new rhizobium of petroleum CTR46 is an antagonistic species of Verticillium dahliae.

[0074] Example 4

[0075] This example provides a potted plant experiment on the control effect of Neorhizobium petroleum CTR46 on cotton Verticillium wilt.

[0076] Preparation of spore solution of Verticillium dahliae: The culture medium for preparing spore solution is Cha's medium, and its formula is as follows: 30g sucrose, 2g NaNO3, 1.0g K2HPO4, 0.5g MgSO4·7H2O, 0.5g KCl, 0.1g FeSO4·7H2O, water is made up to 1000mL, pH 7.0-7.2. Dispense 300mL of culture medium into 500mL conical flasks and sterilize at 121℃ for 20min. Inoculate 2 pieces of 7d-cultured Verticillium dahliae cakes (5mm in diameter) into a conical flask containing 300mL Cha's medium and culture at 26℃ and 220r / min. After 7d of culture, the number of spores is 5.5×10 7 CFU / mL, and obtain the spore liquid of Verticillium dahliae for later use.

[0077] Take the delinted and full Tahe No. 2 cotton seeds, disinfect them with 75% alcohol for 30s, wash them with sterile water 3 times, disinfect them with 4% sodium hypochlorite for 4min, and wash them with sterile water 3 times. Place the disinfected seeds in a culture dish at 25℃ for moisturizing culture, detect germination after 2d, and plant them. Sow the germinated cotton seeds in a bottomless paper cup (90mm high, 75mm in diameter, cut off the bottom of the paper cup) filled with matrix soil (the mass ratio of sand to vermiculite is 3:2, sterilized at 121℃ for 1h). Cultivate in an artificial climate room with a light-dark ratio of 16:8 and a temperature of 28℃. Pour water into the tray when watering to prevent soil compaction from affecting growth. When the cotton is in the "two leaves and one heart" period, inoculate the seed solution of the new rhizobium CTR46 of petroleum, and use sterile water as a negative control.

[0078] When the cotton has grown one true leaf, inoculate Verticillium dahliae. When inoculating, place a bottomless paper cup on a petri dish, hold the paper cup with your hand and rotate it for two weeks to create wounds on the cotton roots at the bottom, invert the paper cup and add 10mL of Verticillium dahliae spore solution to the bottom of the cup. After the solution is completely absorbed, place the paper cup in a plastic tray covered with a layer of plastic wrap and wetted after sterilization. After 1 hour, pour an appropriate amount of distilled water on the bottom of the tray. Two weeks after infection, the cotton plants began to develop symptoms. 25 days after infection ( Figure 7 ), statistical disease index ( Figure 8 ) and control effects.

[0079] The survey adopts a 5-level grading method, and the grading standards are:

[0080] Level 0: healthy seedlings, no disease symptoms;

[0081] Grade 1: 1-2 cotyledons show disease symptoms, the cotyledons turn yellow, and the true leaves show no disease symptoms;

[0082] Level 2: cotyledons and one true leaf show disease symptoms;

[0083] Level 3: 2 true leaves show disease symptoms;

[0084] Level 4: All leaves show serious symptoms of the disease, the leaves fall off, and the tops die.

[0085] Disease index = 100 × ∑ (number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value);

[0086] The control effect = (disease index of the control group - disease index of the treatment group) / (disease index of the control group) × 100%.

[0087] Depend on Figure 7 and Figure 8It can be seen that the new rhizobium CTR46 has a good control effect on cotton Verticillium wilt, and the growth state of cotton plants is better after inoculation with the new rhizobium CTR46. According to the disease index results, the control effect of the new rhizobium CTR46 on cotton Verticillium wilt is 55%±1%.

[0088] Example 5

[0089] This example provides a broad-spectrum antibacterial test of Neorhizobium petroleum CTR46.

[0090] The antibacterial test method is the same as that in Example 2, except that Verticillium dahliae is replaced by Sclerotinia sclerotiorum and Botryosphaeria dothidea (provided by the laboratory of Northwest Agriculture and Forestry University), three replicates are performed for each group, and when the control group is about to fill the culture dish, the colony diameters of the fungi in the treatment group and the control group are measured. Fig. 9 As shown, the plate matching diagram of Petroleum New Rhizobium CTR46 and Botrytis Cervifolia is as follows Fig.10 As shown in the figure, the statistical results of the colony diameter of the petroleum new rhizobium CTR46 after the plate matching test with Sclerotinia sclerotiorum and Botrytis cinerea are as follows Fig.11 shown.

[0091] Depend on Fig. 9 , Fig.10 and Fig.11 It can be seen that the colonies in the control group all covered the culture dish, and the colony diameter of the group treated with Petroleum Neorhizobium CTR46 was significantly smaller than that of the control group, indicating that Petroleum Neorhizobium CTR46 has a good inhibitory effect on both Sclerotinia sclerotiorum and Botrytis cinerea, and has a broad-spectrum antibacterial effect.

[0092] As described above, the basic principle, main features and advantages of the present invention are well described. The above embodiments and descriptions are only descriptions of the preferred embodiments of the present invention, and the present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the protection scope determined by the present invention.

Claims

1. Petroleum new rhizobium CTR46, characterized in that, It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.31497.

2. The petroleum new rhizobium CTR46 according to claim 1, characterized in that: The nucleotide sequence of 16S rRNA of the petroleum neorhizobium CTR46 is shown in SEQ ID NO:

1.

3. Use of the petroleum rhizobium CTR46 described in claim 1 in the prevention and treatment of cotton Verticillium wilt.

4. The use according to claim 3, characterized in that: The pathogen of cotton Verticillium wilt is Verticillium dahliae.

5. Use of the petroleum neorhizobium CTR46 described in claim 1 in inhibiting the growth of Verticillium dahliae.

6. The use of volatile substances of the petroleum neorhizobium CTR46 cultured in solid culture in the prevention and treatment of cotton Verticillium wilt as claimed in claim 1, characterized in that: The volatile substance of the petroleum new rhizobium CTR46 under solid culture is any one of dimethyl trisulfide, furfural, butyric acid and terpinene-4-ol.

7. Use of the petroleum rhizobium CTR46 described in claim 1 in inhibiting the growth of Sclerotinia sclerotiorum or inhibiting the growth of Botryosphaeria dothidea.

8. A microbial agent, characterized in that it comprises the petroleum neorhizobium CTR46 according to claim 1.

9. A method for preventing and treating cotton Verticillium wilt, characterized in that: Use the microbial agent according to claim 8.

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