A kind of endogenous polypsiformis of leek and its application

Abstract

The present invention discloses an endophytic Paenibacillus polymyxa of leek and its application, and belongs to the field of microbial technology. The Paenibacillus polymyxa (Paenibacillus polymyxa) to which the present invention relates was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on September 15, 2022. The strain preservation name is: KLBMP0582, and the preservation number is: CGMCC No. 25720. It has a significant effect on promoting the germination of leek seeds and the accumulation of leek secondary metabolites, and can be artificially cultivated, reproduces quickly, and is easy to mass produce. The bacterium can provide excellent strain resources for the development of microbial fertilizers specifically for leeks, and has good application development prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to endophytic Paenibacillus polymyxa of leek and application thereof. Background Art

[0002] Leek (Allium tuberosum) is a perennial herbaceous plant of the genus Allium in the lily family. It has a unique spicy smell and is green throughout except for the white roots. It is also known as "aphrodisiac grass". Leek is one of the most commonly used medicinal and edible plants in the world and occupies a certain position in people's daily diet. Because the conditions required for its growth are not harsh, it has good adaptability to the environment, and is cold-resistant and heat-resistant, the leek planting area in my country is very large. Leek contains a variety of active ingredients, including flavonoids, sulfur-containing compounds, saponins, polysaccharides and other substances, and has a variety of pharmacological effects, such as anti-inflammatory, anti-cancer, antioxidant, antibacterial and anti-hypertensive effects. Its seeds, leek seeds, can also be directly used as traditional Chinese medicine. Most of the leeks that people eat daily come from wild leeks that have been domesticated and then cultivated artificially. However, in order to increase the yield of leeks and shorten the growth cycle, artificially cultivated leeks are subject to the application of a large amount of chemical fertilizers and plant growth regulators, resulting in the types and contents of bioactive substances in domesticated leeks being much lower than those in wild leeks. Therefore, how to reasonably reduce the use of chemical fertilizers, find green and ecological ways to promote the growth of leek, and increase the content of its secondary metabolites is of great significance to improving the quality of artificially cultivated leek.

[0003] There are a large number of microbial groups closely related to the growth of leek plants, which affect the absorption of nutrients, the synthesis of effective active substances, and the regulation of the immune system. Therefore, applying beneficial growth-promoting microorganisms to artificially cultivated leeks is an effective biological means to improve the microecological environment of the leek rhizosphere, promote leek growth, and synthesize and accumulate secondary metabolites. At present, there is no research on using leek endophytes to improve the quality of artificially cultivated leeks. Artificially cultivated leeks still rely mainly on traditional chemical fertilizers to promote the growth of leeks. Summary of the invention

[0004] The purpose of the present invention is to: aim at the phenomenon that a large amount of chemical fertilizers, plant growth regulators and the like are applied in artificial cultivation of leeks in order to pursue high yield and fast growth rate, resulting in the quality of artificially cultivated leeks being worse than that of wild leeks, and the large amount of use of chemical fertilizers also causing environmental pollution and other problems. The present invention provides an endophytic Paenibacillus polymyxa of leek and an application thereof, which can effectively promote the germination of leek seeds and the accumulation of leek secondary metabolites.

[0005] To achieve the above-mentioned purpose, the present invention provides an endophytic polymyxa bacillus of leek, and the classification name of the endophytic polymyxa bacillus of leek is Paenibacillus polymyxa (Paenibacilluspolymyxa), the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the strain preservation name is: KLBMP0582, the preservation number is: CGMCC No.25720, and the preservation date is: September 15, 2022.

[0006] A method for preparing a bacterial agent from endophytic Paenibacillus polymyxa of leek, using Paenibacillus polymyxa for the following treatment:

[0007] The strain KLBMP0582 was inoculated into fresh LB liquid medium and cultured overnight at 28±2°C and 220 rpm / min shaking to activate the strain;

[0008] The activated bacterial solution was inoculated into LB liquid medium at a ratio of 1:100 and cultured at 28±2℃ and 220rpm for 12h.

[0009] The culture product was then centrifuged at 6000 rpm for 10 min, the supernatant was discarded to collect the bacteria, and the bacteria were resuspended in sterile water to adjust the bacterial solution concentration to OD 600 =0.5;

[0010] The LB liquid culture medium: 10 g of tryptone; 5 g of yeast extract; 10 g of sodium chloride; 1000 mL of distilled water; PH=7.

[0011] Application of an endophytic polymyxa bacillus agent in leek: promoting leek seed germination and the accumulation of leek secondary metabolites.

[0012] Furthermore, liquid inoculants were supported by Bacillus polymyxa, and the inoculants were used to treat leek seeds to improve the germination rate of leek seeds; the inoculants were used to irrigate the roots of leek potted seedlings to promote the accumulation of secondary metabolites such as total flavonoids, total polysaccharides and total triterpenes of leek.

[0013] Furthermore, the specific method of treating leek seeds with the microbial agent is as follows: first, the leek seeds are surface-sterilized and then placed in a centrifuge tube, sterile water is added to immerse the seeds, and the seeds are soaked in a 50°C water bath for 6 hours, and then spread on a sterile culture dish, and then 3 mL of Paenibacillus polymyxa microbial agent is added to the culture dish with the leek seeds.

[0014] Furthermore, the specific method of using the bacterial agent to irrigate the roots of the potted leek seedlings is as follows: the potted leek seedlings are irrigated with the Paenibacillus polymyxa bacterial agent at 0h, 6h, and 24h, respectively, with 5 leek seedlings in each pot and 100 μL of the agent applied to the roots of each leek seedling.

[0015] Furthermore, the bacterial agent is a microbial fertilizer.

[0016] A composition comprises endophytic Paenibacillus polymyxa from leek.

[0017] The Paenibacillus polymyxa KLBMP0582 of the present invention is obtained by separation and purification of endophytic bacteria from the roots of wild leeks in Yangshanzhai, Anhui Province, and the specific separation and purification method is as follows:

[0018] (1) Take wild leek collected from Yangshanzhai, Anhui Province, weigh 1 g of root tissue, and rinse it with running water for 30 minutes to wash away dust and gravel, then place the rinsed root tissue in an ultrasonic cleaning machine for ultrasonic cleaning for 15 minutes to further clean the dust and sand remaining on the surface of the leek root tissue;

[0019] After that, the rinsed root tissue was soaked in 75% ethanol for 1 min in a clean bench, rinsed once with sterile water, then soaked in 2% sodium hypochlorite solution for 8 min, and rinsed three times with sterile water. The root tissue after surface disinfection was put into a mortar, and 9 mL of sterile water was added to grind it evenly. The grinding liquid was diluted with sterile water in a gradient manner (10 -1 , 10 -2 ), then grind the stock solution and dilution (10 -1 , 10 -2 ) were spread on LB solid medium and cultured at 28±2°C for 2-5 days until a single colony grew;

[0020] (2) Pick a single colony and streak it on an LB plate for purification, then culture it at 28±2℃ for 2-5 days;

[0021] (3) Repeat step (2) until a pure culture is obtained to obtain Paenibacillus polymyxa KLBMP0582.

[0022] The culture medium used in the present invention comprises:

[0023] LB liquid medium: tryptone 10 g; yeast extract 5 g; sodium chloride 10 g; distilled water 1000 mL; PH = 7;

[0024] LB solid medium: 10 g tryptone; 5 g yeast extract; 10 g sodium chloride; 20.0 g agar; 1000 mL distilled water; PH = 7. The above culture media are all conventional culture media and need to be sterilized by conventional high temperature and high pressure before use.

[0025] Beneficial effects: Compared with the prior art, the advantages of the present invention are:

[0026] (1) The Paenibacillus polymyxa KLBMP0582 isolated and obtained by the present invention can significantly improve the germination rate of leek seeds and promote the accumulation of leek secondary metabolites. The strain can be used as a microbial fertilizer to improve the quality of artificially planted leeks, and at the same time can alleviate the environmental pollution caused by the large-scale use of chemical fertilizers to a certain extent, so it is more green and environmentally friendly.

[0027] (2) The Paenibacillus polymyxa KLBMP0582 of the present invention has simple culture conditions, is genetically stable, is easy to industrialize, and has good development and application prospects.

[0028] The present invention uses the leek endophyte KLBMP0582 from the perspective of plant endophytes to promote leek seed germination and effectively increase the content of leek secondary metabolites. The strain provided by the present invention can be used as a microbial fertilizer to replace or reduce the use of traditional fertilizers such as chemical fertilizers, and can be used for artificially planted leeks to improve the quality of planted leeks, while being able to reduce environmental pollution problems caused by the abuse of chemical fertilizers, so it is more green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a morphological diagram of the strain KLBMP0582 of the present invention on LB solid medium; Figure 1 A is the front view of the tablet. Figure 1 B is the reverse side of the plate;

[0030] Figure 2 The present invention shows the influence of the strain KLBMP0582 on the germination of leek seeds. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with specific embodiments to help better understand the present invention, but the protection scope of the present invention is not limited thereto:

[0032] The present invention provides a Paenibacillus polymyxa strain, the strain number of which is KLBMP0582, which has been deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 25720 and a deposit date of September 15, 2022. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0033] The morphological, physiological and biochemical characteristics of Paenibacillus polymyxa KLBMP0582:

[0034] (1) Morphological characteristics of strains

[0035] The strain grew well on LB solid medium. The colonies were nearly round, milky white, opaque, moist, slightly wrinkled, and had neat edges (such as Figure 1 ).

[0036] (2) Physiological and biochemical characteristics of strains

[0037] Strain KLBMP0582 can utilize glucose, hydrolyze starch, urea, mannitol, xylose, and gelatin, and its oxidase reaction and VP reaction are negative. It can dissolve phosphorus, degrade cellulose, and produce indoleacetic acid. It grows in 10% NaCl but not in 12% NaCl. Its optimum growth temperature is 30°C.

[0038] Example 1: Isolation, purification, preservation and identification of strain KLBMP0582

[0039] (1) Wild leek collected from Yangshanzhai, Anhui Province was taken, 1 g of root tissue was weighed, and it was rinsed with running water for 30 min to wash away dust and gravel. The rinsed sample was then placed in an ultrasonic cleaner for ultrasonic cleaning for 15 min.

[0040] (2) The cleaned leek samples were surface disinfected in an ultra-clean workbench. They were first soaked in 75% ethanol for 1 min, rinsed once with sterile water, and then soaked in 2% sodium hypochlorite solution for 8 min. They were rinsed three times with sterile water. The sterile water from the third rinse was retained for subsequent reliability testing. The surface disinfected samples were placed on sterile filter paper to absorb the moisture for later use.

[0041] (3) Place the surface disinfected sample in a sterile mortar, add 9 mL of sterile water and grind evenly, and then dilute the grinding solution with sterile water in a gradient manner (10 -1 , 10 -2 ), then take the grinding stock solution and dilution (10 -1 , 10 -2) 100 μL of each was spread on LB solid culture medium and cultured at 28±2°C for 2-5 days until a single colony grew. At the same time, the sterile water used for the last rinse in step (2) was subjected to routine sterility testing to ensure that the surface of the sample was thoroughly disinfected.

[0042] (4) Pick a single colony grown on the LB plate and inoculate it into the same culture medium for streak purification and separation. Incubate it at 28±2℃ for 2-5 days, and continue to pick a single colony. Repeat the separation, purification and culture several times until a pure culture is obtained.

[0043] (5) The purified single colony was inoculated into LB liquid culture medium, cultured in a shaking incubator at 28±2°C for 12 h, and then added into a seed tube in a ratio of 1:1 of bacterial liquid and 50% glycerol, mixed well, and placed in a -80°C ultra-low temperature refrigerator for long-term storage.

[0044] (6) The 16S rRNA gene of the purified single colony was amplified (by conventional PCR method) and sequenced, and the sequencing results were compared with the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). After similarity comparison, the strain was identified as Paenibacillus polymyxa and named KLBMP0582. The sequencing results of the 16S rRNA gene of strain KLBMP0582 are shown in SEQ ID NO.1.

[0045] The culture medium used in the present invention includes: LB liquid culture medium: 10 g of tryptone; 5 g of yeast extract; 10 g of sodium chloride; 1000 mL of distilled water; PH = 7; LB solid culture medium: 10 g of tryptone; 5 g of yeast extract; 10 g of sodium chloride; 20.0 g of agar; 1000 mL of distilled water; PH = 7. The above culture media are all conventional culture media and need to be sterilized by conventional high temperature and high pressure before use.

[0046] Example 2: Effect of strain KLBMP0582 on leek seed germination

[0047] (1) Bacterial strain activation and bacterial solution preparation

[0048] The strain KLBMP0582 was inoculated into fresh LB liquid medium, and cultured overnight at 28±2℃ and 220rpm to activate the strain; the activated bacterial solution was transferred to LB liquid medium at a ratio of 1:100, and cultured at 28±2℃ and 220rpm for 12h; centrifuged at 6000rpm for 10min, the supernatant was discarded, and the bacterial cells were resuspended with sterile water to adjust the bacterial solution concentration to OD 600 =0.5 for standby use.

[0049] (2) Surface disinfection of leek seeds and bacterial solution KLBMP0582 to promote leek seed germination

[0050] Select leek seeds with full and round particles and uniform size, soak the seeds in 75% ethanol for 1 minute, rinse with sterile water twice; then soak in 2% sodium hypochlorite solution for 5 minutes, and rinse with sterile water 4 times; place the surface-sterilized seeds on sterile filter paper to dry, put them in a sterile centrifuge tube, add an appropriate amount of sterile water to submerge the seeds, soak them in a 50℃ water bath for 6 hours, and conduct experiments after the seeds absorb water and swell; take the sterilized filter paper and place it in a culture dish, and take 1mL of sterile water to soak the filter paper; After absorbing water and swelling, leek seeds were spread in culture dishes, with 50 seeds in each dish, a total of 10 culture dishes, divided into two groups: an experimental group and a control group (5 in each group); 3 mL of the KLBMP0582 bacterial solution in (1) was added to the culture dishes of the experimental group on which leek seeds were spread, and 3 mL of sterile water was added to the culture dishes of the control group on which leek seeds were spread, and each group of culture dishes was placed in a 28±2°C light incubator for 15 days; the water volume in the culture dishes was observed every day, and 1 mL of sterile water was added to each group of culture dishes every 2 days.

[0051] After 15 days of cultivation, the germination rates of the two groups of seeds were counted. Germination rate (%) = (number of seeds germinated on 15 days / total number) × 100. The statistical results showed that the germination rate of the control group was 50%, and the germination rate of the experimental group was 64.67%. In addition, the germination length of the leek seeds after 15 days of cultivation was also measured. The germination length of the seeds in the experimental group was significantly higher than that in the control group (Table 1). The germination of some seeds is shown in Figure 2 Therefore, strain KLBMP0582 has the effect of promoting the germination of leek seeds.

[0052] Table 1 Effect of strain KLBMP0582 on leek seed germination

[0053]

[0054] Note: ** indicates P<0.01

[0055] Example 3: Strain KLBMP0582 promotes the accumulation of leek secondary metabolites

[0056] The same as Example 2 was used to select potted leek seedlings with the same growth state, and the KLBMP0582 bacterial solution was used for root irrigation at 0h, 6h, and 24h, respectively. Each leek seedling was irrigated with 100 μL of root (5 leek seedlings per pot), and sterile water was used as the control group in the same way. The experimental group and the control group were repeated for 5 pots per group. The leek seedlings were placed in a greenhouse for cultivation (12h / d light, 25°C), and the contents of secondary metabolites such as total brass, total polysaccharides, and total triterpenes in the experimental group and the control group were detected after 7d.

[0057] Specific method for determining the total flavonoid content: Take the fresh leek sample to be tested, rinse it, cut it into pieces, grind it thoroughly with liquid nitrogen, weigh 2g into a 50mL centrifuge tube, add 80% ethanol, the solid-liquid ratio is 1:25 (g / mL), mix well, and then ultrasonically extract at 35℃ for 15min. Then add the ultrasonic solution to a 50mL centrifuge tube, add 1mL 5% NaNO2 solution, shake well, let it stand for 6min, then add 1mL 10% Al(NO3)3, shake well, let it stand for 6min, then add 8mL 1mol / LNaOH solution, add to the scale line with deionized water, mix well, take 0.3mL sample solution, OD 510 Detect absorbance. Make a standard curve: dry the rutin standard to constant weight, weigh 0.012g of the dried standard into a 50mL volumetric flask, dilute to the mark with methanol, mix well, take 0, 0.5, 1.0, 2.0, 4.0, 6.0mL of the rutin standard solution into a 25mL volumetric flask, add 6mL of deionized water in sequence, and then detect the OD according to the above method. 510 Absorbance value.

[0058] OD 510 The absorbance value is the ordinate, and the concentration of the standard is the abscissa. The standard curve is drawn to obtain the linear regression equation y=12.426x+0.0007, R 2 =0.9996, indicating a good linear relationship. Based on the standard curve, the total flavonoid content of the experimental group and the control group was calculated. The results are shown in Table 2. The total flavonoid content of the leek treated with KLBMP0582 bacterial solution increased by 36.12% compared with the control group.

[0059] The specific method for determining the total polysaccharide content is as follows: take the fresh leek sample to be tested, rinse it, cut it into pieces and grind it thoroughly with liquid nitrogen, weigh 1g of the sample into a centrifuge tube, add 50mL of deionized water, soak it for 5min, mix it well and then extract it with ultrasound at 35℃ for 40min; take 1mL of the extract, add 2.0mL of 5% phenol solution, and then immediately add 7.0mL of concentrated sulfuric acid, mix it well and heat it in a 40℃ water bath for 20min, cool it to room temperature and OD 490 Detect the absorbance. Make a standard curve: Prepare the glucose standard into a 1 mg / mL solution and dilute it in a gradient manner. Then, calculate the OD value according to the above method. 490 Detect the absorbance value.

[0060] OD 490 The absorbance value is the ordinate, and the concentration of the standard is the abscissa. The standard curve is drawn to obtain the linear regression equation y=7.1804x+0.0077, R 2=0.9951, indicating a good linear relationship. According to the standard curve, the total polysaccharide content of the experimental group and the control group was calculated. The results showed that compared with the control group, the total polysaccharide of leek treated with KLBMP0582 bacterial solution increased by 53.30% (see Table 2).

[0061] The specific method for determining the total triterpene content is as follows: take the fresh leek sample to be tested, rinse it, cut it into pieces and grind it thoroughly with liquid nitrogen; weigh 0.2g of the sample into a 50mL centrifuge tube, add 10mL of methanol and mix well, extract it with ultrasound at 35℃ for 2h and leave it overnight; take 0.1mL of the ultrasonic extract into a 50mL centrifuge tube, add 0.2mL of vanillin solution and 0.8mL of perchloric acid solution, mix well and heat it in a 70℃ water bath for 15min, cool it at room temperature, take 0.2mL, add 5.0mL of glacial acetic acid, OD 545 Detect the absorbance. Make a standard curve: weigh 0.02g of ursolic acid and add it to a 100mL volumetric flask, dilute to the mark with methanol and mix. Vanillin-glacial acetic acid standard: weigh 5g of vanillin powder in a 100mL volumetric flask, dilute to the mark with glacial acetic acid and mix. Pipette 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2mL of ursolic acid standard solution respectively, use 1.2mL of methanol as the control group, add 0.2mL of vanillin-glacial vinegar standard, 0.8mL of perchloric acid solution, mix well, heat in a 70℃ water bath for 15min, cool to room temperature, add 5.0mL of glacial acetic acid, OD 545 Detect the absorbance value.

[0062] OD 545 The absorbance value is the ordinate, and the concentration of the standard is the abscissa. The standard curve is drawn to obtain the linear regression equation y=4.0474x+0.0423, R 2 =0.9915, indicating a good linear relationship. According to the standard curve, the total triterpene content of the experimental group and the control group was calculated. The test results showed that the treatment of KLBMP0582 bacterial solution can significantly increase the total triterpene content of leek, which is 43.10% higher than that of the control group (see Table 2). Therefore, based on the above test results, the strain KLBMP0582 provided by the present invention can promote the accumulation of secondary metabolites such as total flavonoids, total polysaccharides and total triterpenes in leek to varying degrees, and can be used to improve the quality of artificially cultivated leek.

[0063] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

[0064] Table 2 Effects of strain KLBMP0582 on total flavonoids, total polysaccharides and total triterpenes in leek

[0065]

[0066]

[0067] Note: ** indicates P<0.01

[0068] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An endophytic polymyxa bacillus from leek, characterized in that: The classification name of the endophytic polymyxa of leek is Paenibacillus polymyxa (Paenibacilluspolymyxa), the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration, the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the strain preservation name is: KLBMP0582, the preservation number is: CGMCC No.25720, and the preservation date is: September 15, 2022.

2. A method for preparing a bacterial agent from endophytic polymyxa bacillus of leek, characterized in that: The Paenibacillus polymyxa of claim 1 is used for the following treatment: The strain KLBMP0582 was inoculated into fresh LB liquid medium and cultured overnight at 28±2°C and 220 rpm / min shaking to activate the strain; The activated bacterial solution was inoculated into LB liquid medium at a ratio of 1:100 and cultured at 28±2℃ and 220rpm for 12h. The culture product was then centrifuged at 6000 rpm for 10 min, the supernatant was discarded to collect the bacteria, and the bacteria were resuspended in sterile water to adjust the bacterial solution concentration to OD 600 =0.5; The LB liquid culture medium: 10 g of tryptone; Yeast extract 5g; sodium chloride 10g; distilled water 1000mL; PH=7.

3. An application of the endophytic polymyxa bacillus agent of leek according to claim 2, characterized in that: The application is to promote the germination of leek seeds and the accumulation of leek secondary metabolites, wherein the method for promoting the germination of leek seeds is to use an endogenous Bacillus polymyxa agent of leek, and to treat the leek seeds with the agent to increase the germination rate of the leek seeds; the method for promoting the accumulation of leek secondary metabolites is to use the agent to perform root irrigation treatment on leek potted seedlings to promote the accumulation of total flavonoids, total polysaccharides and total triterpenoid secondary metabolites of leek.

4. The use of the endophytic polymyxa bacillus agent of leek according to claim 3, characterized in that: The specific method of treating leek seeds with the microbial agent is as follows: first, the leek seeds are surface disinfected and then placed in a centrifuge tube, sterile water is added to immerse the seeds, and then the seeds are soaked in a 50°C water bath for 6 hours, and then spread on a sterile culture dish, and then 3 mL of Paenibacillus polymyxa microbial agent is added to the culture dish with the leek seeds.

5. The use of the endophytic polymyxa bacillus agent of leek according to claim 3, characterized in that: The specific method of using the bacterial agent to irrigate the roots of potted leek seedlings is as follows: the potted leek seedlings are irrigated with the Paenibacillus polymyxa bacterial agent at 0h, 6h, and 24h, respectively, with 5 leek seedlings in each pot and 100 μL of the agent applied to the roots of each leek seedling.

6. The use of the endophytic polymyxa bacillus agent of leek according to claim 4, characterized in that: The bacterial agent is a microbial fertilizer.

7. A composition comprising the endophytic Paenibacillus polymyxa of leek according to claim 1.

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