Application of lysobacter capsici in preventing and treating alfalfa root rot and leaf spot disease

Abstract

The invention discloses application of lysobacter capsici FX-17 in preventing and treating alfalfa root rot and leaf spot disease, the preservation number of the lysobacter capsici FX-17 is GDMCCNO: 64592, and experiments prove that the lysobacter capsici FX-17 can effectively inhibit growth of alfalfa root rot fungi, rhizoctonia solani, phoma medicaginis, pseudomonas palmi and mould, and can be used for preventing and treating alfalfa root rot and leaf spot disease, and the lysobacter capsici FX-17 can be used for preventing and treating alfalfa root rot and leaf spot disease. The effect of preventing and treating alfalfa root rot and leaf spot disease is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to the application of a Lonsdalea quasivorata in preventing and treating root rot and leaf spot of alfalfa. BACKGROUND

[0002] Alfalfa is an important high-quality forage, known as the "king of forage", and is the main food source for livestock around the world, with extremely high nutritional value and extremely rich protein and fat content. Root rot and leaf spot are one of the main causes of alfalfa diseases, and have become an important factor affecting the quality and yield of alfalfa. Due to the continuous single planting of alfalfa for many years, if not timely eradication, the incidence of root rot and leaf spot will also increase with the increase of the cultivation period.

[0003] In recent years, more and more attention has been paid to the protection of the ecological environment worldwide, and biological control of crop diseases has gradually replaced chemical pesticides and is increasingly attracting attention. Rational use of biocontrol agents can reduce and avoid the negative effects of chemical pesticides, thereby preventing disease occurrence and reducing ecological damage, and thus ensuring safe and high-quality production of crops.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] The purpose of the present application is to provide the application of a Lonsdalea quasivorata in preventing and treating root rot and leaf spot of alfalfa, which can effectively prevent and treat root rot and leaf spot of alfalfa.

[0006] The present application is implemented as follows: In a first aspect, the present application provides a Lonsdalea quasivorata, which is named FX-17 in the present application, and its taxonomic name is Lonsdalea quasivorata (L. Lysobacter capsici ), its accession number is GDMCC NO: 64592, the preservation time is May 08, 2024, and it is preserved in the Guangdong Microbial Culture Collection Center, located at No. 59 Building, 5th Floor, Guangdong Institute of Microbiology, 100 Middle Martyrs Road, Guangzhou.

[0007] In a second aspect, the present application provides the application of the above-mentioned Lonsdalea quasivorata in preventing and treating plant root rot.

[0008] In some embodiments, the above-mentioned plant includes alfalfa.

[0009] In some embodiments, the above-mentioned application includes preventing and treating root rot caused by Phymatotrichopsis omnivorum and Rhizoctonia solani.

[0010] In a third aspect, the present application provides the application of the above-mentioned Lonsdalea quasivorata in preventing and treating plant leaf spot.

[0011] In some embodiments, the above-mentioned plant comprises alfalfa.

[0012] In some embodiments, the above-mentioned application comprises preventing and treating leaf spot caused by Phoma medicaginis and Pseudomonas palustris.

[0013] In a fourth aspect, the present application provides a use of the L. chilense in inhibiting growth of mold.

[0014] In a fifth aspect, a microbial agent, wherein the effective component comprises the L. chilense and fermentation product or fermentation broth thereof.

[0015] In a sixth aspect, the present application provides a method for preventing and treating alfalfa root rot and leaf spot, comprising: applying the above-mentioned microbial agent to the diseased part of the alfalfa.

[0016] The present application has the following beneficial effects: The present application screens a L. chilense in scientific research practice, and verifies that the bacteria can effectively inhibit growth of alfalfa root rot mold, Rhizoctonia solani, Phoma medicaginis, Pseudomonas palustris and mold, and achieve the effect of preventing and treating alfalfa root rot and leaf spot. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below, and it should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0018] Figure 1 Colony morphological characteristics and bacterial morphological observation of FX-17; Figure 2 Evolution tree of 16S rDNA sequence of FX-17 of the present application; Figure 3 Position map of bacterial liquid in point plate in Example 2; Figure 4 Different antagonistic effects of FX-17 on Py in early and late growth time periods on combined nitrogen-fixing medium and 1 / 2 combined + 1 / 2 PDA medium in Example 2; Figure 5 Different antagonistic effects of FX-17 on Rki in early and late growth time periods on combined nitrogen-fixing medium and 1 / 2 combined + 1 / 2 PDA medium in Example 2; Figure 6Different antagonistic effects of FX-17 on Pm in early and late growth periods on the joint nitrogen-fixing medium and 1 / 2 joint + 1 / 2 PDA medium in Example 2; Figure 7 Different antagonistic effects of FX-17 on Ppi in early and late growth periods on the joint nitrogen-fixing medium and 1 / 2 joint + 1 / 2 PDA medium in Example 2; Figure 8 Preventive effect of FX-17 on Root 1 infected alfalfa in Example 3; Figure 9 Preventive effect of FX-17 on Root 26 infected alfalfa in Example 3; Figure 10 Preventive effect of FX-17 on leaf spot disease infected alfalfa in Example 3. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments of the present application will be clearly and completely described below. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased in the market.

[0020] The features and performances of the present application will be further described in detail below in combination with the embodiments.

[0021] The pathogenic bacteria Py provided in the present application is alfalfa root rot fungus Pythium coloratum The pathogenic bacteria Rki is Rhizoctonia solani Rhizoctonia solani Both of the strains are given by Ms. Fang Xiangling of Lanzhou University; the pathogenic bacteria Pm is alfalfa phoma stem spot Phoma medicaginis given by Mr. Duan Tingyu of Lanzhou University; the pathogenic bacteria Ppi is Pseudomonas palmae isolate Pseudopithomyces palmicola isolate, which is alfalfa leaf spot disease bacteria; the mold Vs is alfalfa variant Medicago sativa isolate 7628 Vicia sativa var ; the pathogenic bacteria Root 1 is Fusarium solani, and Root 26 is Fusarium decemcellulare, which are both isolated from the field by the research group.

[0022] The control bacteria FX-18 is Pseudomonas aceris strain LA-29 Rhod pseudomonas sp ; the control bacteria Aq is Agropine engineering strain ARqual of Agrobacterium, which is given by Mr. Liu Zhigeng of Lanzhou University.

[0023] Example 1 The present embodiment is the separation and identification of FX-17, as follows: (1) Selection of culture medium Joint nitrogen fixation medium: Qingdao Haibo Biological Company's joint nitrogen fixation medium was selected, product number HB8541.

[0024] PDA medium: PDA medium produced by Wuhan Puint Biological Company was selected, product number WS1039.

[0025] 1 / 2 joint nitrogen fixation + 1 / 2 PDA medium: the joint nitrogen fixation medium and the PDA medium were mixed half by half.

[0026] (2) Bacterial culture isolated from soil Plate isolation method was used to isolate strains from soil samples collected from the experimental site of the Loess Plateau Experimental Station in Qingyang.

[0027] Soil was collected and naturally air-dried, and sieved. Plate dilution method was used to culture the bacterial flora in the soil. The specific method is as follows: The pretreated soil sample was diluted with sterile water to 10 -1 , 10 -2 , 10 -4 , 10 -6 , 10 -8 , 10 -10 times suspension, and placed in a 30°C, 200 rpm shaker for 24 h. After the culture was completed, 100 μL of bacterial solution was dropped on the joint nitrogen fixation medium, and evenly smeared with a spreader, and placed in a 30°C constant temperature incubator for culture for 2-3 d, and the growth of the colonies was observed. The single colonies with good growth were picked into centrifuge tubes for purification, and the purified single colonies were transferred to the joint nitrogen fixation medium for culture, and numbered and stored in the freezer tube, a total of 100 strains of bacteria were obtained.

[0028] II. Strain screening The mold Vs cake was placed in the center of the joint nitrogen fixation medium, and after a certain size circle was formed, 5 μL of bacteria FX-17 was spotted beside it to observe whether the strain had antagonistic effect on the mold. After the mold grew on the medium, it was found that one of the strains had a zone of inhibition around it. The single strain was selected for repeated experiments, and the experimental results were the same as those in the screening, and it was named FX-17.

[0029] III. Observation of colony morphology and bacterial morphology The colony morphology and bacterial morphology of FX-17 are shown in Table 1 and Figure 1 .

[0030] Table 1 Bacterial morphology

[0031] The colonies of FX-17 grown on the nitrogen-fixing medium were smooth, yellow, round, and the cell morphology was rod-shaped, as shown in FIG. 1. Figure 1

[0032] IV. Physiological and biochemical characteristics of the strain 1. Culture medium and reagents (1) Sugar fermentation medium: a. 10.0 g of proteose peptone, 5.0 g of sodium chloride, 1.0-2.0 mL of 1.6% bromocresol purple ethanol solution, 1000.0 mL of distilled water, pH 7.6; b. 20.0 g of sugar (D-mannose, D-xylose, D-glucose, and L-arabinose, respectively), 100.0 mL of distilled water.

[0033] Preparation: a and b were sterilized separately (112°C, 30 min), and mixed before use according to the ratio of 0.5 mL of b per 10.0 mL of a.

[0034] (2) Starch medium: 2.0 g of soluble starch, 5.0 g of beef extract, 10.0 g of proteose peptone, 5.0 g of sodium chloride, 15.0-20.0 g of agar, 1000.0 mL of distilled water.

[0035] (3) Casein medium: a. 100.0 g of skimmed milk powder, 1000.0 mL of distilled water (112°C, 30 min); b. 30.0 g of agar, 1000.0 mL of distilled water.

[0036] Preparation: a and b were sterilized separately, and mixed when cooled to 45-50°C.

[0037] (4) Tyrosine medium: a. 5.0 g of L-tyrosine, 100.0 mL of distilled water (112°C, 30 min); b. 30.0 g of beef extract, 5.0 g of proteose peptone, 15.0-20.0 g of agar, 1000.0 mL of distilled water.

[0038] Preparation: a and b were sterilized separately, and mixed when cooled to warm.

[0039] (5) Gelatin medium: 3.0 g of beef extract, 5.0 g of proteose peptone, 10.0 g of sodium chloride, 120.0-180.0 g of gelatin, 1000.0 mL of distilled water, pH 7.2-7.4.

[0040] (6) Glucose proteose peptone medium (for V-P test): 5.0 g of glucose, 5.0 g of proteose peptone, 2.0 g of potassium phosphate dibasic, 1000.0 mL of distilled water, pH 7.0-7.2 (112°C, 30 min).​

[0041] (7) Proteose peptone medium (for indole formation test): Proteose peptone 10.0 g, sodium chloride 5.0 g, distilled water 1000.0 mL, pH 7.6.

[0042] (8) Nitrate medium: Potassium nitrate 0.2 g, proteose peptone 5.0 g, distilled water 1000.0 mL, pH 7.4.

[0043] (9) 3% hydrogen peroxide solution (for contact enzyme test): Take 10.0 mL of 30% hydrogen peroxide solution and dilute to 100.0 mL with distilled water.

[0044] 2. Experimental method (1) Sugar fermentation test: The test strains were inoculated into test tubes containing 4 kinds of sugar fermentation medium, 3 replicates for each strain, and the pure medium without inoculation was used as a control, and placed in a 37°C incubator. After 24 h, observation was taken, if the medium turned yellow, it indicated that the strain produced acid, marked as positive; if the medium was purple, it indicated that the strain produced alkali, marked as negative. In addition, if there were bubbles in the Durham tube in the D-glucose fermentation medium, it indicated that the strain produced gas.

[0045] (2) Starch hydrolysis test: The test strains were inoculated on the surface of starch medium, 3 replicates for each strain, and placed in a 37°C incubator. After 24 h, they were taken out, a small amount of Lugol's iodine solution was added to the surface of the medium, and gently rotated to make the iodine solution evenly cover the entire culture dish. If a colorless transparent circle appeared around the colony, it indicated that starch had been hydrolyzed, indicating that the bacteria had the ability to decompose starch, marked as positive, otherwise marked as negative.

[0046] (3) Casein hydrolysis test: The test strains were inoculated on the surface of casein medium, 3 replicates for each strain, and placed in a 37°C incubator. After 24 h, observation was taken to see if there was a transparent circle around the colony, if there was, it indicated that casein had been hydrolyzed, marked as positive, otherwise marked as negative.

[0047] (4) Tyrosine hydrolysis test: The test strains were inoculated on the surface of tyrosine medium, 3 replicates for each strain, and placed in a 37°C incubator. After 24 h, observation was taken to see if there was a transparent circle around the colony, if there was, it indicated that the strain could hydrolyze tyrosine, marked as positive, otherwise marked as negative.

[0048] (5) Gelatin liquefaction test: the test strain was inoculated in a large amount into the test tube containing gelatin medium, the depth of puncture was about 2 / 3 of the height of gelatin, 3 repeats for each strain, and the medium without inoculation was used as a control, and was placed in a 37°C incubator. The results were observed every day. If the gelatin was liquefied, it indicated that the strain had gelatinase which could make the gelatin lose the gel property, and was marked as positive, otherwise was marked as negative. Note: In order to avoid the situation that the gelatin itself is liquefied due to high culture temperature, the test tube should be placed in the refrigerator for 20-30 min before observation.

[0049] (6) Acetyl methyl carbinol test (V-P test for short): the test strain was inoculated into the test tube containing glucose peptone medium, 3 repeats for each strain, and the pure medium without inoculation was used as a control, and was placed in a 37°C incubator for 24 h. After being taken out, 10-20 drops of 40% sodium hydroxide solution were added after fully shaking for 2 min, and then an equal amount of α-naphthol was added, the test tube was shaken to dissolve oxygen in the air, and was placed in a 37°C incubator for 15-30 min. After being taken out, if the medium was red, the V-P test was recorded as positive, otherwise was negative.

[0050] (7) Indole formation test: the test strain was inoculated into the test tube containing peptone medium, 3 repeats for each strain, and the pure medium without inoculation was used as a control, and was placed in a 37°C incubator for 24 h. After being taken out, 1.0-2.0 mL of diethyl ether was added in the medium, and the generated indole was extracted into diethyl ether by fully shaking, and was placed for a while until the diethyl ether layer floated on the upper layer of the medium. At this time, 5-10 drops of indole reagent were slowly added along the wall of the test tube. If indole existed, the diethyl ether layer showed rose red, and the test result was marked as positive, otherwise was marked as negative.

[0051] (8) Nitrate reduction test: the test strain was inoculated into the test tube containing nitrate medium, 3 repeats for each strain, and the pure medium without inoculation was used as a control, and was placed in a 37°C incubator for 24 h. After being taken out, 1 drop of methanol (0.8 g of p-aminobenzenesulfonic acid was dissolved in 100.0 mL of 2.5 mol / L acetic acid solution) and 1 drop of ethyl solution (0.5 g of methylnaphthalene was dissolved in 100.0 mL of 2.5 mol / L acetic acid solution) were added in the medium. Red color appeared immediately or within a few minutes, and the test result was marked as positive, otherwise was negative.

[0052] (9) Contact enzyme test (also known as hydrogen peroxidase test): 1 piece of clean glass slide was taken, 1 drop of 3% hydrogen peroxide solution (freshly prepared) was dropped on it, and 1 ring of strain was picked and smeared in the hydrogen peroxide solution. If bubbles appeared, it indicated that the strain could produce hydrogen peroxidase, and the test result was positive. No bubbles appeared, and the test result was negative.

[0053] 3. Observation of culture characteristics The results of the physiological and biochemical determination of the strains were compared and analyzed for positive (+) and negative (-) results, and the results are shown in Table 2: Table 2 FX-17 physiological and biochemical characteristics determination results

[0054] V. Molecular biology and phylogenetic analysis of strain FX-17 Total DNA of the isolated bacteria was extracted, and 16S rDNA was amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO. 2), 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO. 3). The PCR amplification product was separated and detected by 1.0% agarose gel electrophoresis, and then sent to a sequencing company for bidirectional full-length sequence determination. The sequencing sequence was first corrected and spliced using Mega11 software, then compared with the standard mode strain by BLAST program on the NCBI website (while downloading the 16S rDNA sequence of the related standard mode strain), and finally the Mega11 software was used to construct a phylogenetic tree (using the Neighbor-joining algorithm).

[0055] The results of the phylogenetic analysis are shown in Figure 2 It is concluded that the strain FX-17 is a new subspecies of the L. capsici strain YC5194.

[0056] The 16S rDNA sequencing results of the FX-17 strain are shown in SEQ ID NO. 1.

[0057] Example 2 This example is the determination of the antibacterial status of FX-17, which is as follows: (1) The strain FX-17, which has been isolated and purified from soil, was picked into 500 μL of LB medium and placed in a constant temperature shaker at 30°C and 200 rpm for 36 h of shaking culture; (2) The control strains FX-18 and Aq, which have been previously purified, were picked into 500 μL of LB medium and placed in a constant temperature shaker at 30°C and 200 rpm for 36 h of shaking culture; (3) The disease bacteria Py, Rki, Pm, and Ppi, which have grown on PDA plates, were punched with a puncher to select well-grown places, and the bacterial cakes were placed on joint nitrogen fixation plates and 1 / 2 joint nitrogen fixation + 1 / 2 PDA plates, which had been punched in the center, and placed in a 33°C incubator for 48 h of growth; (4) After the bacteria grow to a certain size, 5 μL of the culture completed bacterial liquid of strains Fx-17, FX-18 and Aq is taken by a pipette gun and spotted around the bacteria to form an equilateral triangle around the bacteria, as shown in Figure 3 ; (5) After the initial antagonistic effect between the strain and the bacteria is shown, a photograph is taken. After the bacteria grow on the culture medium, a photograph is taken again.

[0058] The detection results are shown in Figure 4-7 . FX-17 has antagonistic effects on Py, Rki, Pm and Ppi in two culture media, but the effect is better in the combined nitrogen-fixing culture medium than in the 1 / 2 combined + 1 / 2 PDA culture medium.

[0059] Example 3 This example is a verification experiment of the prevention and treatment effect of FX-17 on alfalfa with root rot and leaf spot, which is as follows: 1. Prevention and treatment effect on alfalfa root rot Root 1 and Root 26 (1) The root rot fungi Root 1 and Root 26 are inoculated on potato dextrose agar (PDA) culture medium plates, and incubated at 30°C for 5-7 days for preparation of inoculum. An appropriate amount of millet is weighed, washed and soaked for 8 hours, and then the excess water is poured out and autoclaved at 121°C. The colonies on the PDA culture medium are cut into 2 mm x 2 mm blocks, inoculated into a conical flask containing millet, and then placed in a 30°C incubator for culture. The conical flask is shaken every two days to make the mycelium grow evenly on the grains. After 14 days, the pathogenic bacteria are inoculated; (2) A single colony of FX-17 is selected and placed in a 50 mL tube containing 10 mL of combined nitrogen-fixing culture solution, and placed in a constant temperature shaker at 30°C and 300 rpm for 24 hours. Then 1 mL of the bacterial liquid is taken and placed in a conical flask containing 150 mL of combined nitrogen-fixing culture solution, and placed in a constant temperature shaker at 30°C and 300 rpm for 24 hours until the OD value is 0.4; (3) The sterilized grass charcoal soil and vermiculite are mixed in a ratio of 2:1 to form the soil substrate. The cuttings of alfalfa with consistent growth are transplanted into pots, with 2 seedlings per pot; (4) The millet inoculum of the root rot fungus is mixed into the soil at a ratio of 5% of the soil weight, and then the soil is placed in the pots and watered to saturation. Two treatments of inoculating the bacteria and inoculating the bacteria with FX-17 bacterial liquid are set up, and the treatment without inoculating the root rot fungus is used as a control. Each treatment is repeated three times, and FX-17 bacterial liquid is inoculated every three days; (5) Photographs are taken after 15 days of inoculation; The detection results are shown in Figure 8-9As shown in Table 1, the plant height of the treatment without inoculation of the pathogen was the highest, and after inoculation of the FX-17 bacterial liquid, the plant height was significantly higher than that of the plant inoculated with the root rot pathogen alone, indicating that the inoculation of the FX-17 bacterial liquid had better prevention and treatment effects on the root rot Root 1 and Root 26 of alfalfa.

[0060] 2, the prevention and treatment effect on leaf spot disease Pm of alfalfa (1) All the stem point fungi growing on the joint nitrogen fixation medium were transferred into a 50 mL tube containing 30 mL of sterilized joint nitrogen fixation culture solution, and placed in a constant temperature shaking incubator (300 r / min, 30°C) for 20 min. Filtration, blood plate counting, and adjustment of the number of conidia to 10 7 CFU / mL with sterilized distilled water; (2) Single colony of FX-17 was selected in a 50 mL tube containing 10 mL of joint nitrogen fixation culture solution, and placed in a constant temperature shaker at 30°C and 300 rpm for 24 h. Then 1 mL of the bacterial liquid was taken into a conical flask containing 150 mL of joint nitrogen fixation culture solution, and placed in a constant temperature shaker at 30°C and 300 rpm for 24 h until the OD value was 0.4 for use; (3) The sterilized grass charcoal soil and vermiculite were mixed in a ratio of 2:1 to form the soil substrate, and the cuttings of alfalfa with consistent growth were transplanted into pots, with 3 seedlings per pot; (4) The pathogen inoculation was performed by spraying method, and the conidial suspension of the pathogen Pm was uniformly sprayed on the plant leaf surface, and the proportion of the conidial suspension and the FX-17 bacterial liquid was 4:1, 50 mL per pot. Two treatments of inoculation of the pathogen and inoculation of the pathogen and FX-17 bacterial liquid were set, and the treatment without inoculation was sprayed with the same amount of sterile water as the control. Each treatment was repeated 3 times, and the conidial suspension of Pm and the FX-17 bacterial liquid were inoculated every 3 days; (5) Photographing was performed 21 days after inoculation; The detection results are shown in Table 2. Figure 10 As shown in Table 2, there were no disease spots on the leaves of the plants without inoculation of the pathogen, and the disease spots on the leaves of the plants sprayed with the FX-17 bacterial liquid were significantly less than those of the plants inoculated with the conidial suspension of the stem point fungus alone, indicating that the inoculation of the FX-17 bacterial liquid had better prevention and treatment effects on the leaf spot disease Pm of alfalfa.

[0061] The above only describes the preferred embodiments of the present application and is not used to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of Xanthomonas campestris, characterized in that, The xanthomonas campestris Lysobacter capsici with the accession number: GDMCC NO: 64592.

2. Use of Lysobacter capsici according to claim 1 for controlling plant root rot.

3. Use according to claim 2, characterized in that, The plant includes alfalfa.

4. Use according to claim 3, characterized in that, The use includes controlling root rot caused by P. alfalfae, R. solani.

5. Use of Lysobacter capsici according to claim 1 for controlling plant leaf spot.

6. Use according to claim 5, characterized in that, The plant includes alfalfa.

7. Use according to claim 6, characterized in that, The use includes controlling leaf spot caused by P. alfalfae, P. palmivora.

8. Use of Lysobacter capsici according to claim 1 for inhibiting growth of mold.

9. A microbial inoculant, characterized in that, The effective component of the microbial agent includes Lysobacter capsici according to claim 1 and its fermentation product or fermentation broth.

10. A method of controlling Aphanomyces euteiches and leaf spot diseases in alfalfa, comprising applying to the locus of the plant, applying to the plant, or applying to the seed of the plant, a fungicidally effective amount of a compound of Claim 1. Comprising: Applying the microbial agent according to claim 9 to the diseased part of alfalfa.

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