CN113151099A - Pseudomonas aeruginosa and application thereof - Google Patents

Abstract

The invention discloses Pseudomonas aeruginosa and application thereof, wherein the Pseudomonas aeruginosa is classified and named as Pseudomonas aeruginosa YY322 with a preservation number of CCTCC NO: m2021208, deposited at the China center for type culture Collection on 03/08 d 2021. The research result shows that: the pseudomonas aeruginosa and the volatile gas generated by the pseudomonas aeruginosa have high-efficiency antagonistic action on saffron pathogenic bacteria, and can be used for preventing and treating the saffron diseases caused by the saffron pathogenic bacteria; moreover, the experimental results of the invention also prove that: the pseudomonas aeruginosa of the invention has the functions of producing protease, glucanase, fixing nitrogen,Dissolving phosphorus, producing siderophores and producing NH₃The growth promoting performance is good; therefore, the pseudomonas aeruginosa can be expected to be used for preparing a soil conditioner or/and a fertilizer for preventing and treating saffron diseases, and has wide application prospect.

Description

Pseudomonas aeruginosa and application thereof

Technical Field

The invention relates to pseudomonas aeruginosa and application thereof, in particular to pseudomonas aeruginosa YY322 and application thereof in preventing and treating crocus sativus pathogenic bacteria, and belongs to the technical field of microorganisms.

Background

Saffron (Crocus sativus) is a perennial flower of Crocus of iridaceae, is a world famous dye perfume, is also an important medicinal plant, has the effects of promoting blood circulation to remove blood stasis, cooling blood to remove toxic substances, relieving depression and soothing nerves, is listed in the quality list of medicine and food congeners in China, and has great development and utilization values. Meanwhile, the medicinal part is only dry stigma, the yield is extremely low, the market price is extremely high, about 50 yuan per gram of the superior is known as 'red gold', and the research on the cultivation and planting of the red gold is particularly important.

The saffron is originally produced in Iran, Greek, India, Spain and the like, China starts to introduce seeds in the last 80 th century, Zhejiang, Shanghai and Anhui are the main producing areas at present, and the saffron is also cultivated in Jiangsu, Henan, Hebei, Jiangxi and the like. With the continuous cropping obstacle problem of saffron, the planting area of saffron is continuously enlarged, the continuous cropping obstacle problem of saffron is continuously aggravated, and although some places adopt crop rotation such as rice, corn, soybean and the like to overcome the problem, the corm rot condition of saffron still exists in many places every year, the incidence rate of mild people is several hundreds, and severe people even the whole army do not cover the situation, so that great loss is brought to growers.

At present, the research on the disease of saffron is relatively few, reported saffron pathogenic bacteria include Aspergillus brasiliensis (Aspergillus brasiliensis), Fusarium oxysporum (Fusarium oxysporum), Fusarium solani (Fusarium solani), Penicillium citrinum (Penicillium citrinum) and the like, and the subject group finds that the disease condition of the saffron of chongming island in Shanghai is mainly divided into three types after investigation and analysis and separation and identification of pathogenic bacteria: red rot: the main pathogenic bacteria causing red rot are fusarium oxysporum which is the most pathogenic bacterium with strongest pathogenicity and most widely distributed, and the main pathogenic bacteria are black rot or yellow white from one side or the whole base of the bulb; ② white rot: is characterized in that white villi are grown on outer membranous scaly leaves, bulbs in the outer membranous scaly leaves are shriveled and wrinkled, the surfaces of the outer membranous scaly leaves are uneven, the textures of the outer membranous scaly leaves are hard, some inner parts of the outer membranous scaly leaves are hollow and are grey white or black, the quality of the outer membranous scaly leaves is obviously reduced compared with healthy people, and the main pathogenic bacteria causing the grey disease is fusarium solani; ③ pockmark disease: the characteristic is that the bottom groove of the bulb is blackened, which is obviously different from the normal person, the black spots scattered and distributed can be seen after the membranous scaly leaves are peeled off, the periphery of the black spots is brownish, the black spots are connected into pieces in severe persons, the inside of the black spots is more and more browned along with the deepening of the pathological change degree, the damage of the black spots to the saffron is not fatal, the yield is only reduced, and the main pathogenic bacteria causing the pockmark disease are Penicillium citrinum sulfate (Penicillium citrinum) and citrinum aurantium.

The diseased stigma croci Sativi corm can cause the leaves of the plants to yellow, brown and wither to die, thus seriously affecting the quality and yield of the stigma croci Sativi corm and filament and seriously reducing the economic value and use value of the stigma croci Sativi. In order to improve the yield and quality of stigma croci Sativi corm and filament, so as to achieve the purpose that stigma croci Sativi is rare but can be popularized and enjoyed, the rot of stigma croci Sativi corm becomes a problem which needs to be solved urgently in production and cultivation.

At present, the disease control of saffron mainly depends on chemical fertilizers and pesticides, and although the saffron has the advantages of quick response and lasting effect, the problems of pesticide residue, environmental pollution and the like can be caused, so that the quality of medicinal materials can be influenced, and the strategy is not in accordance with the strategy of national green development and ecological agriculture. Biological control is a widely studied technology in recent years, is advocated due to the characteristics of environmental protection, safety and effectiveness, and attracts the attention of many scholars at home and abroad, so that the research on antagonistic bacteria is needed to be carried out on pathogenic bacteria of saffron.

Disclosure of Invention

Aiming at the problems and the requirements in the prior art, the invention aims to provide pseudomonas aeruginosa and application thereof in preventing and treating crocus sativus pathogenic bacteria.

The Pseudomonas aeruginosa of the invention is classified and named as Pseudomonas aeruginosa (Pseudomonas aeruginosa) YY322, and has been preserved in China Center for Type Culture Collection (CCTCC), the preservation address is Wuhan university, the preservation date is 2021, 03 and 08 days, and the preservation number is CCTCC NO: m2021208.

Furthermore, the pseudomonas aeruginosa of the invention has the following main morphological and biological characteristics: the single colony is nearly round, irregular in edge, relatively flat, yellowish green, not smooth and opaque, and gram staining reaction is negative, and the 16Sr DNA sequence analysis of the strain shows that the strain has 100 percent of consistency with Pseudomonas aeruginosa (Pseudomonas aeruginosa) with GenBank number of MN 611437.

Furthermore, the pseudomonas aeruginosa can produce protease and glucanase, but does not produce chitinase and cellulase; has the functions of fixing nitrogen, dissolving phosphorus, producing iron carrier and producing NH₃But not potassium, indoleacetic acid (IAA) and ACC deaminase producing ability.

Experiments prove that: the pseudomonas aeruginosa has an inhibition rate of 73.17% on fusarium oxysporum, an inhibition rate of 62.20% on fusarium solani, an inhibition rate of 23.16% on penicillium citrinum and an inhibition rate of 49.14% on penicillium citrinum, and volatile gas generated by the pseudomonas aeruginosa has inhibition rates of 90.24% and 89.02% on fusarium oxysporum and fusarium solani respectively, so that the pseudomonas aeruginosa and the volatile gas generated by the pseudomonas aeruginosa can be used for preparing an antagonist for pathogenic saffron so as to prevent and treat the saffron, and the pathogenic saffron comprises at least one of fusarium oxysporum, fusarium solani, penicillium citrinum and penicillium citrinum.

Compared with the prior art, the invention has the following remarkable beneficial effects:

the research result of the invention shows that: the pseudomonas aeruginosa and the volatile gas generated by the pseudomonas aeruginosa have high effect on saffron pathogensEffective antagonism, can be used for preventing and treating stigma croci Sativi diseases caused by stigma croci Sativi pathogenic bacteria; moreover, the experiment of the invention also proves that: the pseudomonas aeruginosa of the invention has the functions of producing protease, glucanase, nitrogen fixation, phosphorus dissolution, siderophore and NH₃The pseudomonas aeruginosa can be expected to be used for preparing a soil conditioner or/and a fertilizer for preventing and treating saffron diseases, and has wide application prospect.

Drawings

FIG. 1 is a graph showing the inhibitory effect of YY322 strain selected in example 1 on pathogenic bacteria of saffron, wherein: FIG. A is a graph showing the inhibitory effect on Fusarium solani, FIG. B is a graph showing the inhibitory effect on Fusarium oxysporum, FIG. C is a graph showing the inhibitory effect on Penicillium citrinum, and FIG. D is a graph showing the inhibitory effect on Penicillium citrinum;

FIG. 2 is a morphological feature diagram of the YY322 strain according to the present invention;

FIG. 3 is a graph showing the results of producing degradative enzymes by the YY322 strain of the present invention, wherein: panel A shows the results of protease production, panel B shows the results of glucanase production, panel C shows the results of chitinase production, and panel D shows the results of cellulase production;

FIG. 4 is a graph showing the results of evaluating the growth promoting performance of the YY322 strain according to the present invention, wherein: a is a graph showing nitrogen fixation ability, B is a graph showing organic phosphorus dissolution ability, C is a graph showing inorganic phosphorus dissolution ability, D is a graph showing siderophore production ability, and E is a graph showing NH production ability₃The ability, F is the ability to release potassium, G is the ability to produce IAA, and H is the ability to produce ACC deaminase;

FIG. 5 is a graph showing the effect of volatile gases produced by the YY322 strain of the present invention on the inhibition of Fusarium oxysporum, wherein: panel A shows a treatment group, and panel B shows a control group;

fig. 6 is a graph showing the effect of the volatile gas generated by the YY322 strain of the present invention on the inhibition of fusarium solani, wherein: panel A shows the treatment group and panel B shows the control group.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and equipment used in the present invention are conventional in the art unless otherwise specified, and reagents and materials used in the following examples are commercially available unless otherwise specified.

Example 1: isolation and screening of YY322 Strain

1) Collection of samples

Collecting healthy saffron rhizosphere soil of Shanghai Chongming island (east longitude 121 degrees 09 '30' to 121 degrees 54 '00', north latitude 31 degrees 27 '00' to 31 degrees 5l '15'), sealing in a sterile self-sealing bag, transporting the sterile self-sealing bag back to a laboratory by using an ice box, and drying in the shade for two weeks in a clean and ventilated place for later use.

2) Isolation and purification of bacteria

Grinding the naturally dried soil in the shade by using a mortar, sieving by using a 20-mesh sieve, weighing 5g of soil sample, placing the soil sample into a triangular flask filled with 45mL of sterile water, adding a little glass beads into the triangular flask, placing the triangular flask on a 150rmp shaking table for oscillation for 2 hours and standing for 1 hour, sucking 1mL of soil from the triangular flask, placing the soil sample into a test tube filled with 9mL of sterile water and a plug, fully oscillating the test tube, and preparing into 10mL of soil sample^-2The soil dilution was made into 10 by analogy^-3、10^-4、10^-5The soil suspension is ready for use;

sucking 200 mu L of each suspension with different concentrations by using a sterile pipette, respectively adding the suspension into an NA culture medium (10g of peptone, 5g of NaCl, 3g of beef extract and 18g of agar, adding distilled water to 1000mL, uniformly stirring by using a glass rod, and sterilizing at 121 ℃ for 20min to obtain), an LB culture medium (10g of peptone, 5g of yeast extract, 5g of NaCl and 18g of agar, adding distilled water to 1000mL, uniformly stirring by using a glass rod, and sterilizing at 121 ℃ for 20min to obtain the final product), uniformly coating the suspension by using sterile coating beads, then inversely placing the culture medium at 30 ℃ for culturing for 24h, selecting bacterial single colonies with different colony forms and colors, carrying out streaking purification for multiple times, transferring to an LB inclined plane culture medium, culturing for 24h, and storing in a refrigerator at 4 ℃ for later use.

3) Primary screen for antagonistic bacteria

Activating and culturing fusarium oxysporum for 5d by adopting a plate confronting method, then punching holes in the edge area of a colony by using a puncher to prepare a pathogenic bacteria cake with the diameter of 5mm, placing fungi on one side of a PDA (personal digital assistant) plate, placing bacteria obtained through separation and purification on the other side of the PDA plate at a distance of about 2cm, culturing for 5 days in a culture box at 28 ℃ by taking the PDA plate only with the pathogenic bacteria cake as a contrast, and observing whether an antagonistic zone appears on the PDA plate to determine the antagonistic action on the pathogenic bacteria.

4) Compound sieve for antagonistic bacteria

Inoculating fusarium oxysporum and fusarium solani to a PDA culture medium (200 g of potatoes are taken, a proper amount of water is added for decocting for half an hour after peeling, eight layers of gauze are filtered, 20g of glucose and 18g of agar are added into the filtrate, distilled water is added to 1000mL, the mixture is uniformly stirred by a glass rod and is sterilized at 121 ℃ for 20min) and then is activated for 5 days in an incubator at 30 ℃, and penicillium citrinum sulfate are inoculated on the PDA culture medium by adopting a zone scribing method and are activated for 2 days in the incubator at 30 ℃ to obtain single colonies;

inoculating the preliminarily screened antagonistic bacteria on an LB culture medium, culturing for 24h in an incubator at 30 ℃, then punching a pathogenic bacteria cake with the diameter of 5mm by using a puncher, placing the pathogenic bacteria cake in the center of a PDA (personal digital assistant) plate, picking bacteria by using an aseptic toothpick, dotting the pathogenic bacteria cake around the bacteria cake at a distance of 2.5cm by using a cross method, repeating the steps for 3 times by using the PDA plate only provided with the pathogenic bacteria cake as a control, culturing for 7 days in the incubator at 28 ℃, and then calculating an inhibition rate (the inhibition rate is (the diameter of the control colony-the diameter of a treated colony)/the diameter of the control colony x 100%) to determine the target antagonistic bacteria;

the target antagonistic bacteria obtained by the method are named as YY322 strain, the inhibition rate of the strain on fusarium oxysporum is 73.17%, the inhibition rate on fusarium solani is 62.20%, the inhibition rate on penicillium citrinum sulfate is 23.16%, the inhibition rate on penicillium citrinum is 49.14%, and the specific inhibition effect is shown in a graph in figure 1, wherein: FIG. A is a graph showing the inhibitory effect on Fusarium solani, FIG. B is a graph showing the inhibitory effect on Fusarium oxysporum, FIG. C is a graph showing the inhibitory effect on Penicillium citrinum, and FIG. D is a graph showing the inhibitory effect on Penicillium citrinum.

The strain YY322 is preserved in China Center for Type Culture Collection (CCTCC) at 03/08 th 2021, the preservation address is Wuhan university, and the preservation number is CCTCC NO: m2021208.

Example 2: identification of YY322 Strain

1) Physiological and biochemical identification

According to the ' handbook of identifying common bacteria system ' and ' Bergey ' handbook of identifying bacteria ', the YY322 strain obtained by screening in example 1 is inoculated on an LB culture medium for culturing for 24h, and then the growth condition, colony morphology and color, gram stain and other morphological and physiological and biochemical characteristics of antagonistic bacteria are observed.

FIG. 2 is a morphological feature diagram of the YY322 strain, as can be seen from FIG. 2: the strain grows rapidly, and a single colony is nearly circular, has irregular edges, is relatively flat, light yellow green, not smooth and opaque.

The specific physiological and biochemical identification results are shown in table 1:

TABLE 1 physiological and biochemical characteristics of YY322 Strain

It can be seen from the results shown in table 1: the YY322 strain has positive physiological and biochemical tests such as oxidase reaction, catalase reaction, urease reaction, lipase reaction, gelatin liquefaction, milk coagulation, nitrate reduction, citrate utilization and the like, and has gram staining reaction, MR test, V-P test, starch hydrolysis, H test and the like₂S production and other physiological and biochemical tests are negative.

The results of morphological characteristics and physiological and biochemical characteristics of the YY322 strain are combined, so that the YY322 strain can be preliminarily identified as the Pseudomonas (Pseudomonas).

2) Molecular identification

Extracting DNA of antagonistic bacteria by using a bacterial DNA extraction kit (Nanjing Nuojingzu Biotechnology Co., Ltd.), and selecting 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-CTACGGCTACCTTGTTACGA-3') to perform PCR amplification on the antagonistic bacteria by using the extracted DNA as a template so as to obtain a target fragment, wherein a specific PCR reaction system and PCR reaction conditions are respectively shown in tables 2 and 3:

TABLE 2 PCR reaction System

Composition of	Volume (μ L)
		27F(10μmol/L)	2
1492R(10μmol/L)	2
		2×Phanta Max Master Mix	25
Template DNA	2
		ddH2O	19

TABLE 3 PCR reaction conditions

The PCR products were detected by 1% agarose gel electrophoresis, sent to Panno Biotechnology Co., Ltd for sequencing, and then subjected to BLAST homology search on NCBI website (National Center for Biotechnology Information) to show that: the consistency of the strain and Pseudomonas aeruginosa (Pseudomonas aeruginosa) with GenBank number of MN611437 is 100%; combining the above morphological and molecular identification results, the YY322 strain was thus identified as Pseudomonas aeruginosa (Pseudomonas aeruginosa).

Example 3: detecting the performance of the YY322 strain for producing degrading enzyme

3.1 proteases

The activated YY322 strain is picked by a sterile toothpick and spotted on a protease detection plate (15 g of skimmed milk powder, 18g of agar, 1L of distilled water, and sterilization is carried out for 15min at 115 ℃), the steps are repeated for 3 times, and the strain is cultured in an incubator at 30 ℃ for 2 days and then observed, and a clear transparent ring is positive.

3.2 chitinase

Selecting activated YY322 strain with sterile toothpick, and inoculating to chitinase detection plate (NH)₄H₂PO₄ 1g，KCl 0.2g，MgSO₄·7H₂0.2g of O, 10g of colloidal chitin, 18g of agar and 1L of distilled water, sterilizing for 20min at 121 ℃), repeating for 3 times, culturing in an incubator at 30 ℃ for 2 days, and observing to obtain a clear transparent ring, namely positive.

3.3 cellulase

Selecting the activated YY322 strain by using a sterile toothpick and inoculating the strain to a cellulase detection plate ((NH)₄)₂SO₄ 2g， MgSO₄·7H₂O 0.5g，KH₂PO₄1g, 0.5g of NaCl, 2g of CMC-Na, 0.4g of Congo red, 18g of agar and 1L of distilled water, sterilizing for 20min at 121 ℃), repeating for 3 times, culturing for 2 days in an incubator at 30 ℃, adding a proper amount of 1mg/mL Congo red solution into a culture dish, dyeing for 1h, discarding the dye solution, adding a proper amount of 1mol/L NaCl solution, washing for 1h, pouring 1mol/L HCl, and observing, wherein a clear transparent circle is positive.

3.4 dextranase

The activated YY322 strain is picked by a sterile toothpick and spotted on a dextranase detection plate (20 g of oat is decocted, dregs are removed to obtain juice, 0.1g of yeast extract, 0.1g of Congo red, 0.1g of NaCl, 18g of agar and 1L of distilled water are sterilized for 20min at 121 ℃), and the steps are repeated for 3 times, and the subsequent steps are the same as 3.3.

The results of the above tests show that: the YY322 strain can produce protease and glucanase, but does not produce chitinase and cellulase, and the specific detection result is shown in figure 3, wherein: panel A shows the results of protease production, panel B shows the results of glucanase production, panel C shows the results of chitinase production, and panel D shows the results of cellulase production.

Example 4: evaluation of growth promoting Properties of YY322 Strain

4.1, nitrogen fixation ability

Picking activated YY322 strain with sterile toothpick and inoculating on nitrogen-free culture medium (KH)₂PO₄ 0.2g，CaCO₃0.5g， MgSO₄·7H₂O 0.2g，NaCl 0.2g，CaSO₄0.1g, 10g of glucose, 15g of agar and 1L of distilled water, sterilizing for 20min at 121 ℃), repeating for 3 times, culturing in an incubator at 30 ℃ for 5 days, and observing to obtain positive growth.

4.2 phosphorus dissolving ability

The activated YY322 strain is picked up by a sterile toothpick and inoculated on an inorganic phosphorus bacteria culture medium (10g of glucose, (NH)₄)₂SO₄ 0.5g，NaCl 0.3g，Ca₃(PO₄)₂ 5g，MgSO₄ 0.3g，K₂SO₄ 0.3g，MnSO₄ 0.03g，FeSO₄0.03g, 15g of agar, 1L of distilled water, sterilization at 121 ℃ for 20min and organophosphorus bacteria culture medium (10g of glucose, 0.5g of yeast extract, (NH)₄)₂SO₄ 0.5g，NaCl 0.3g，KCl 0.3g，MgSO₄ 0.3g，FeSO₄ 0.03g，MnSO₄ 0.03g，CaCO₃1g of lecithin, 0.2g of agar, 15g of agar and 1L of distilled water, sterilizing for 20min at 121 ℃), repeating for 3 times, culturing in an incubator at 30 ℃ for 5 days, and observing to obtain a phosphorus-dissolving ring which is positive, wherein the larger the ratio of the diameter of the phosphorus-dissolving ring to the diameter of a bacterial colony, the better the phosphorus-dissolving effect is.

4.3 Potassium-dissolving ability

Picking activated YY322 strain with sterile toothpick, inoculating to silicate bacteria culture medium (sucrose 5g, Na)₂HPO₄2g， MgSO₄·7H₂O 0.5g，CaCO₃ 0.1g，FeCl₃·6H₂0.005g of O, 1g of potassium feldspar powder, 15g of agar and 1L of distilled water, sterilizing for 20min at 121 ℃), repeating for 3 times, culturing in an incubator at 30 ℃ for 5 days, and observing to obtain a positive result after a smooth transparent ring appears.

4.4 siderophore production ability

Picking activated YY322 strain with sterile toothpick and inoculating on CAS culture medium (60.5 mg cromolyn dissolved in 50mL Fe³⁺Solution (1mmol/L FeCl₃·6H₂O, 10mmol/L HCl) was mixed well and while stirring, cetyltrimethylammonium bromide solution (72.9mg in 40mL H) was added slowly₂O), adjusting the pH value to 7.0, and then fixing the volume to 100 mL; and then mixing the solution with 900mL of culture medium (containing 20g of agar, 4.5g of peptone, 9g of glucose, 2.7g of beef extract, 4.5g of NaCl and 7.0 of pH) uniformly after sterilizing at 115 ℃ for 20min, repeating the steps for 3 times, and observing after culturing in an incubator at 30 ℃ for 5 days, wherein the positive result is that orange halos appear.

4.5 production of NH₃Capability of

The YY322 strain is inoculated into a 50mL triangular flask containing 20mL peptone water (10g/L), after 2 days of culture in a shaker 200r/min at 30 ℃, 2mL are taken out and put into a test tube, 0.5mL Nessler's reagent is added, observation is carried out after shaking up, the peptone water is used as a control, 2 times of repetition are carried out, and the positive is obtained when the color is changed from brown to yellow.

4.6 IAA-producing ability

The YY322 strain was inoculated into a liquid medium containing 20mL of LB liquid medium (containing 100 mg. L of L-tryptophan)^-1) Culturing in 50mL triangular flask at 30 deg.C for 1 day at 200r/min, centrifuging, collecting supernatant 2mL, adding into test tube, and adding Salkowski colorimetric solution (50mL 35% HClO) in the same volume₄+1mL 0.5mol·L^-1FeCl₃) After shaking and mixing uniformly, observing, taking the LB liquid culture medium without bacteria and the mixed solution of the isometric colorimetric solution as a control, repeating for 2 times, and obtaining the positive property after the color turns red.

4.7 ACC deaminase producing ability

YY322 strain was inoculated into a medium containing 20mL of nitrogen-free liquid medium (MnSO)₄·7H₂O 0.2g，CaCO₃1g of cane sugar, 10g of cane sugar, K₂HPO₄·3H₂0.5g of O, 0.12g of NaCl, 1L of distilled water, pH7.2) in a 50mL Erlenmeyer flask, shaking and culturing at 30 ℃ for 2 days in a shaker at 200r/min, sucking 1mL of the culture medium, and inoculating to a medium containing 20mL of DF (KH)₂PO₄ 4g， Na₂HPO₄6g，MgSO₄·7H₂O 0.2g，(NH₄)₂SO₄2g of glucose, 2g of sodium gluconate, 2.0g of citric acid and 0.1mL of solutions of the first component and the second component respectively, and then distilled water is used for fixing the volume to 1L and the pH value is 7.2; the component one: MnSO₄·H₂O 11.19mg， CuSO₄·5H₂O 78.22mg，ZnSO₄·7H₂O 124.6mg，H₃BO₃ 10.0mg，MoO₃10.0mg dissolved in 100mL sterile water; and (2) component two: FeSO₄·7H₂O100.0 mg in 10mL of sterile water), shaking and culturing at 30 ℃ for 2 days in a shaker at 200r/min, sucking 1mL of the culture solution, inoculating to 20mL of ADF medium (ACC dissolved in deionized water, filter-sterilizing, and adding to None (NH)₄)₂SO₄And the final concentration of ACC in the sterilized DF culture medium is 3.0mmol/L), shaking, uniformly mixing, sucking 200 mu L to measure the OD of the mixture₆₀₀Then, the culture was continued for 2 days, and whether turbidity appeared in the medium was observed and OD was measured₆₀₀All the above steps are repeated for 3 times, and if the difference between the two is more than 0.1, the result is positive.

The results of the above evaluation experiments show that: the YY322 strain has the functions of fixing nitrogen, dissolving phosphorus, producing siderophores and producing NH₃But not potassium, IAA and ACC deaminases, as shown in fig. 4, wherein: a is a graph showing nitrogen fixation ability, B is a graph showing organic phosphorus dissolution ability, C is a graph showing inorganic phosphorus dissolution ability, D is a graph showing siderophore production ability, and E is a graph showing NH production ability₃The capacity, F plot represents potassium-resolving capacity, G plot represents IAA-producing capacity, and H plot represents ACC-producing capacity.

Example 5: investigating the bacteriostasis effect of volatile gas generated by YY322 strain

Adding an LB culture medium into a dish cover of the same plate, carrying out streak inoculation on a YY322 strain, adding a PDA culture medium into a culture dish, respectively inoculating fusarium oxysporum and fusarium solani with the diameters of 5mm at the center of the plate, sealing the plate without inoculating the YY322 strain by using a sealing film, sealing the culture dish by using a preservative film, repeating the steps for 3 times, inversely placing the plate in a 28 ℃ incubator for culturing for 7 days, observing the inhibition condition of the growth of pathogenic bacteria in an experimental group, and calculating the inhibition rate which is (the diameter of a control bacterial colony-the diameter of a treated bacterial colony)/the diameter of the control bacterial colony multiplied by 100%.

The experimental results show that: the inhibition rates of volatile gases generated by the YY322 strain on fusarium oxysporum and fusarium solani are respectively 90.24% and 89.02%, but pathogenic bacteria hypha of the two bacteria slightly spread to the whole flat plate, and specific inhibition effect graphs are respectively shown in figures 5 and 6, and figure 5 is an inhibition effect graph of the volatile gases generated by the YY322 strain on fusarium oxysporum, wherein: panel A shows a treatment group, and panel B shows a control group; FIG. 6 is a graph showing the effect of volatile gases produced by the YY322 strain of the present invention on the inhibition of Fusarium solani, wherein: panel A shows the treatment group and panel B shows the control group.

The results of the above studies of the present invention show that: the pseudomonas aeruginosa and the volatile gas generated by the pseudomonas aeruginosa have high-efficiency antagonistic action on saffron pathogenic bacteria, and can be used for preventing and treating the saffron diseases caused by the saffron pathogenic bacteria; and, the experiment of the invention also proves that: the pseudomonas aeruginosa of the invention has the functions of producing protease, glucanase, nitrogen fixation, phosphorus dissolution, siderophore and NH₃The growth promoting performance is good; therefore, the pseudomonas aeruginosa can be used for preparing a soil conditioner or/and a fertilizer for preventing and treating saffron diseases, and has a wide application prospect.

Finally, it must be said here that: the above embodiments are only used for further detailed explanation of the technical solutions of the present invention, and should not be interpreted as a limitation of the scope of the present invention, and the non-essential modifications and adjustments made by those skilled in the art according to the above descriptions of the present invention are included in the scope of the present invention.

Claims (5)

1. Pseudomonas aeruginosa is characterized in that: the classification and the name are Pseudomonas aeruginosa YY322, the preservation number is CCTCC NO: m2021208, deposited at China Center for Type Culture Collection (CCTCC) on 03/08 at 2021.

2. The use of pseudomonas aeruginosa according to claim 1 for controlling saffron diseases.

3. Use of volatile gases produced by pseudomonas aeruginosa according to claim 1 for the control of saffron disease.

4. The use according to claim 2 or 3 for the preparation of an antagonist of the pathogenic bacterium crocus sativus.

5. The use according to claim 2 or 3 for the preparation of soil conditioners and/or fertilizers antagonistic to pathogenic bacteria of saffron.

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