CN115369064B - Development and application of multifunctional composite microbial inoculum for enhancing rhizosphere colonization and control effect of biocontrol bacillus bailii - Google Patents
Development and application of multifunctional composite microbial inoculum for enhancing rhizosphere colonization and control effect of biocontrol bacillus bailii Download PDFInfo
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Abstract
The invention relates to development and application of a multifunctional composite microbial inoculum for enhancing rhizosphere colonization and prevention effect of biocontrol bacillus beijerinckii. The composite microbial inoculum consists of bacillus belgium HNU and bacillus fraxinus HNU-13. The two strains are separated from the rhizosphere soil of the eggplant stock, the two strains have a metabolic and mutual culture relationship, the Fulankton bacillus HNU-13 can obviously enhance the formation of a biological film of the Bacillus berensis HNU, enhance the antagonism effect of the Bacillus berensis on the Ralstonia solani, and the two strains can stably colonize the rhizosphere of the tomato, and can reduce the incidence rate of bacterial wilt under the synergistic effect. The compound flora can also obviously enhance antagonism effect on fusarium and phomopsis, and has a certain biocontrol prospect in preventing and treating banana wilt and betel nut coconut leaf spot.
Description
Technical Field
The invention belongs to the field of agricultural microorganisms, relates to development of a plant growth promoting composite microbial inoculum based on rhizosphere biomembrane co-colonization excited by mixed flora metabolism and resistance, and has application prospects for preventing and treating tomato bacterial wilt, banana wilt and betel nut coconut leaf spot.
Background
Crop bacterial wilt caused by solarenia (Ralstonia solanacearum, abbreviated as bacterial wilt) is a worldwide plant disease, and new diseases caused by bacterial wilt are continuously reported, and statistics show that bacterial wilt is the second largest plant bacterial disease in the world. Bacterial wilt is one of the most serious diseases in agricultural production in China all the time, and is reported to occur in at least 30 provinces in China according to statistics, especially in more than 90 crops such as tomatoes, eggplants, peppers, tobacco, peanuts, ginger and the like in the provinces of the south of Yangtze river, and serious losses are caused to economic crops such as tomatoes, eggplants, peppers, tobacco and the like. Therefore, the deep research on the infection mechanism and pathogenicity regulation of the bacterial wilt has important significance for preventing and controlling diseases.
The microorganism is a natural abundant resource, and in the aspect of agriculture, the microbial agent has the advantages of green, high efficiency, no pollution, no effect, strong pertinence and the like. In recent years, synthetic biology has become a research hotspot in the related field, and two or more microorganisms are artificially combined for a certain purpose from a natural microorganism resource library according to a certain design principle, so that a synthetic microorganism community with a certain specific function is formed. Compared with a single microbial agent, the synthesized microbial community has high controllability and good stability. The use of synthetic microbial communities in agriculture is in the beginning and will also be the main research direction in the biological arts of synthesis over a period of time in the future.
At present, in the agricultural field, the most studied and developed microorganism species are bacillus, and more than 70% of the microorganism fertilizers registered in our country belong to the species. Most of the bacillus species are beneficial microorganisms capable of secreting large amounts of different types of secondary metabolites and which are capable of producing spores that are more easily maintained active in complex environments. The bacillus is used as a center, an exciting agent of the bacillus is searched in a wide microbial resource library, the characteristics of the bacillus in the aspect of biocontrol are amplified, the colonization capability of the bacillus in plant rhizosphere is improved, the strong growth promotion and antibacterial activities of the bacillus are fully exerted, and the bacillus microbial preparation is of great significance in the aspects of disease resistance and growth promotion.
In order to solve the problem of green prevention and control of bacterial wilt of solanaceae crops, the invention provides a bacterial liquid for preventing and controlling tomato bacterial wilt by using a double-strain combination and an application method thereof. In addition, the combined flora is found to have better antagonism effect on pathogenic bacteria such as fusarium oxysporum which causes banana vascular wilt and phomopsis which causes betel nut coconut leaf spot. In view of the serious hazard of banana vascular wilt, the invention has important application value because of the importance of bananas in commercial crops and grain crops and the fact that betel nuts and coconuts are also used as important commercial crops in Hainan area.
Disclosure of Invention
The invention provides a Furanacter HNU-13 strain, which is characterized by the strain preservation information: preservation unit name: the collection of microorganism strains in Guangdong province; deposit unit address: building 5, guangzhou city, first, china, no. 100, university, no. 59, guangdong national academy of sciences of microbiology; preservation date: 2022, 5, 30; preservation number: GDMCC No.62498; classification naming: franconibacter pulveris.
Another embodiment of the present invention provides the use of Furanacter freundii HNU-13 for controlling bacterial wilt and/or banana vascular wilt in tomato plants. Enhancing the application of plant rhizosphere colonisation.
Another embodiment of the invention provides the use of Furanobacter freundii HNU-13 in the synergistic enhancement of Bacillus belicus HNU for the control of bacterial wilt and/or banana vascular wilt in tomato plants. Enhancing the application of plant rhizosphere colonisation.
Another embodiment of the present invention provides a composite microbial inoculant, wherein the active ingredients of the composite microbial inoculant comprise bacillus belicus HNU and bacillus fraxinus HNU-13. Wherein the ratio of the bacterial load is preferably HNU24: HNU 6.6-13=2:1 to 10:1.
The invention provides a composite microbial agent, which is characterized in that the preparation method of the composite microbial agent comprises the following steps:
the culture obtained stroma-removed thalli HNU and HNU6-13 are respectively resuspended to bacterial liquid OD by using sterile water 600 And (3) uniformly mixing until the ratio is 1.0-2.0, and obtaining the composite microbial inoculum according to the ratio of HNU24: HNU 6-13=2:1 to 10:1.
Another embodiment of the present invention provides a method for preparing the composite microbial inoculum, which is characterized by comprising the following steps:
the culture obtained stroma-removed thalli HNU and HNU6-13 are respectively resuspended to bacterial liquid OD by using sterile water 600 And (3) uniformly mixing until the volume ratio is 1.0-2.0, and obtaining the composite microbial inoculum according to the volume ratio of HNU24: HNU 6-13=2:1 to 10:1.
Another embodiment of the invention provides the use of the above-described composite microbial inoculant in the control of tomato plant bacterial wilt and/or banana vascular wilt.
Another embodiment of the present invention provides the use of the above-described complex microbial agents for enhancing plant rhizosphere colonization.
The culture obtained de-matrixed thalli HNU and HNU6-13 according to the invention are obtained according to the conventional seed liquid culture method in the field, and can be obtained by the following method: inoculating seed solutions of the activated strains HNU and HNU6-13 into LB liquid culture medium, and culturing at 28 ℃ and 200rpm for 24 hours; centrifuging and cleaning to obtain the substrate-removed thalli. The preparation method of the LB liquid medium is that 1L of medium is taken as an example: 10g of peptone, 5g of yeast extract, 10g of sodium chloride, 15g of agar and the balance of water. Sterilizing at 121deg.C for 20min.
The strain preservation information of the bacillus beijerinckii HNU is as follows: preservation unit name: the collection of microorganism strains in Guangdong province; deposit unit address: building 5, guangzhou city, first, china, no. 100, university, no. 59, guangdong national academy of sciences of microbiology; preservation date: 10 months and 15 days 2020; preservation number: GDMCC No.61231; classification naming: bacillus sp.
Compared with the prior art, the invention has the advantages that: the combination of the strain HNU screened from the rhizosphere of healthy tomato plants and HNU6-13 screened from the rhizosphere of a stock which has high resistance to bacterial wilt, i.e. a solanum lycopersicum rootstock No. 3, and the strain HNU6-13 and HNU24 is found to be capable of remarkably reducing the incidence rate of bacterial wilt of tomatoes, forming a biological film structure with higher biomass together and being capable of stably colonizing the rhizosphere of tomatoes.
Drawings
The phylogenetic tree diagram of FIGS. 1HNU and HNU6-13;
FIGS. 2HNU and HNU6-13 are co-cultivation laser confocal maps in which red represents HNU6-13 and green represents HNU24;
FIG. 3HNU and HNU6-13 are biofilm co-cultivation phenotypes, MIX representing the results of equal ratio mixing of two strains;
FIG. 4 is a laser confocal plot of a mixed culture of HNU and HNU6-13 to form a biofilm, wherein red represents HNU6-13 and green represents HNU24;
FIG. 5 Crystal Violet staining was used to examine the biofilm formation capacity of different treatments at the rhizosphere of tomato;
FIG. 6 is a graph of a biological control experiment of composite bacterial antagonism against tomato bacterial wilt, wherein group A represents a clear water treatment; group B represents the inoculation of Ralstonia solani EP1; group C represents the re-inoculation of EP1 after inoculation HNU; group D represents the re-inoculation of EP1 after inoculation HNU6-13; group E represents inoculation HNU followed by HNU-13 with simultaneous inoculation of EP1;
figure 7 statistics of tomato Miao Qing blight incidence under different treatments;
FIG. 8 shows the antagonistic effect of the composite microbial inoculum on banana fusarium wilt bacteria (the intermediate fungus is banana fusarium wilt bacteria strain FOC 4).
Detailed Description
Example 1 isolation and identification of strains
The isolation and purification of biocontrol strains from rhizosphere soil of a stock variety (solanacearum No. 3) having high resistance to solarenergia EP1. Carefully taking out stock seedling from soil, shaking off large soil, cutting root system into 1cm stem segments with sterilized scissors, adding 10mL PBS buffer solution, grinding into homogenate, oscillating for 20min, and diluting to 10 -2 、10 -3 、10 -4 、10 -5 The suspensions with different gradients are coated on LB solid medium and cultured for 48h at 28 ℃. Strains with different forms are selected and purified on a new LB solid medium until a pure culture is obtained, and the pure culture is preserved in 20% glycerol at-80 ℃.
Obtaining a bacterial strain named HNU-13, forming light yellow colony on LB plate, and preserving gram negative bacteria in Guangdong province microorganism strain collection, wherein the preservation date is 2022, 5 months and 30 days, and the preservation number is GDMCC No:62498. the strain is preliminarily identified as the strain of the genus Furanacter through 16S rDNA sequencing and sequence comparison, and the nucleic acid consistency rate of the comparison results table is 99.39% in NCBI database. The results of the sequence clustering analysis are shown in FIG. 1.
A strain of bacteria named HNU was obtained, which was a pale yellow colony on LB plates, and gram-positive bacteria were deposited at the Cantonese microorganism culture Collection, 10/15/2020, under the accession number GDMCC No:61231. and finally identifying bacillus beleiensis through whole genome sequencing and sequence comparison, wherein the original sequence of genome sequencing is submitted to a Sequence Read Archive database, and the authorization number is as follows: SRR16216500. The results of the sequence clustering analysis are shown in FIG. 1.
The preparation method of the PBS buffer solution comprises the following steps: 8.0g NaCl, 0.2g KCl and 1.44g Na are weighed 2 HPO 4 、0.24g KH 2 PO 4 Dissolved in 800mL distilled water, the solution was adjusted to ph=7.4 with HCl, and finally distilled water was added to a volume of 1L at a concentration of 0.01M.
The LB medium is prepared by taking 1L of medium as an example: 10g of peptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar. Sterilizing at 121deg.C for 20min.
Example 2 Co-cultivation of composite microbial agent biofilm
Construction of fluorescent-labeled Strain 1-1.5mL HNU24 bacterial liquid (OD 600 =1.0), after 20min of ice bath, centrifugation was performed at 6000rpm at 4 ℃ for 5min, cells were collected, suspended with 20% glycerol solution, and after 3-5 repetitions, competent cells were prepared with 100 μl of 20% resuspended cells. 10 mu L of GFP fluorescent plasmid (green) is added into 100 mu L of competent cells, after fully and uniformly mixing, the mixed bacterial solution is added into a shock cup with the diameter of 2mm, the shock is carried out under the condition of 2.5KV, 500mL of LB liquid culture medium is added, the culture is carried out for 24 hours under the condition of 28 ℃ and 200rpm, the bacterial solution is evenly coated on an LB plate containing Kan and Amp, the static culture is carried out for 24 hours at 28 ℃, single colony shaking is selected, and whether plasmid introduction is successful or not is detected under a fluorescent microscope. Mcherry fluorescent plasmid (Red) was transferred into HNU6-13 fluorescent marker strain and prepared as described above.
Experiment set 3 experimental groups: HNU24 (2. Mu.L HNU 24. Mu.L) in the selected medium, HNU 6.6-13 (2. Mu.L HNU6-13. Mu.L) in the selected medium, and Mix (2. Mu.L of a double bacteria mixed medium in the selected medium, HNU24: HNU 6-13=2:1). 3 replicates of each group, 3 wells of each replicate were incubated at 28℃for 24 hours, photographed, the gas-liquid interface biofilm was collected, the biofilm was oven dried at 60℃and the biofilm dry weight was measured.
Laser confocal observation of the biofilm of the fluorescent marker strain of the double bacteria revealed that the double bacteria formed the biofilm together (fig. 2). At the SOBG liquid medium gas-liquid junction, bacillus belicus HNU and Fulank bacteria HNU-13 formed a more robust biofilm than single bacteria, and the biomass was increased twelve times compared with single bacteria (FIGS. 3, 4). Indicating that the bifidus can exist closely in the form of a biological film.
The preparation method of the SOBG culture medium comprises the following steps: peptone 20g, yeast extract 5g, mgSO 4 ·7H 2 O2.4g,NaCl 0.5g,KCl 0.186g, 20mL of glycerol.
EXAMPLE 3 Co-cultivation of Complex microbial inoculants
The HNU strain and HNU strain 6-13 strain stored in glycerol pipe at-20deg.C are streaked on LB solid medium plate for activation, and cultured in incubator at 28deg.C for 2 days. And respectively picking single colonies, shake-culturing overnight at 30 ℃ and 200rpm in 3mL liquid LB test tubes, respectively taking 2mL of culture bacterial liquid, putting the culture bacterial liquid into 100mL of LB liquid culture medium, culturing at 30 ℃ and 200rpm, and taking the bacterial liquid for observation by a fluorescence confocal microscope after 7 days.
EXAMPLE 4 rhizosphere co-colonization with Complex microbial inoculants
Rhizosphere colonization of the complex microbial agent is performed on tomatoes. The tomato seeds are subjected to surface sterilization by adopting a 2% sodium hypochlorite solution for 10min, soaked in 75% alcohol for 30s, then washed for 5 times by using sterile distilled water, placed on an MS flat plate, germinated at 28 ℃ for 3 days, and then transferred to a 28 ℃ incubator for 7 days for rooting. 16h of light and 8h of darkness. Seedlings with root systems of 5cm are selected, and MS liquid culture medium is added.
The HNU strain and HNU strain 6-13 strain stored in glycerol pipe at-20deg.C are streaked on LB solid medium plate for activation, and cultured in incubator at 28deg.C for 2 days. Picking single colony in 3mL liquid LB test tube, shake culturing at 30deg.C and 200rpm overnight, centrifuging at 4deg.C to collect thallus, washing thallus with sterile water for 3 times, and regulating OD 600 To 1.0, young tomato is obtainedSoaking the seedlings in the bacterial suspension for 20min, transplanting the seedlings in a liquid MS culture medium, culturing for 7 days, taking out the seedlings, washing the seedlings with sterile water for 3 times to remove non-adhering bacteria, soaking the roots in a 0.1% crystal violet solution for 10min, taking out the seedlings, washing the seedlings with flowing sterile water for 1min, cutting the roots at the bases of the seedlings, putting the seedlings in 2mL of a 75% ethanol solution for full oscillation, taking out the roots, and measuring the absorbance value of the ethanol solution at 570 nm.
The results show (FIG. 5) that the amount of biofilm formation by the single bacteria HNU and HNU6-13 was significantly lower in tomato seedlings than in the complex inoculant, indicating that the single bacteria were less able to colonize in biofilm form in the rhizosphere than in the complex inoculant.
Example 5 experiment of plant Probiotics complexing agent on bacterial wilt biocontrol
Experimental setup experiments the following 5 experimental groups were set up: a: treating with clear water; group B: inoculating the forced solarenergia sp.1; group C: inoculating HNU and then inoculating EP1; group D: inoculating HNU-13 and then inoculating EP1; group E: inoculation HNU is followed by inoculation HNU 6.6-13 (the inoculum size of HNU 6.6-13 is 1/10 of HNU 24.24) and inoculation of EP1.
Activating HNU, HNU6-13 on LB culture medium, picking single colony, culturing in 5mL liquid LB culture until OD 600 1.0 as seed solution, inoculating 1mL of seed solution into 500mL of LB liquid medium, culturing at 28deg.C and 200rpm for 24 hr, centrifuging at 6000rpm and 25deg.C, collecting thallus, and adding sterile water to resuspend thallus to OD 600 To 2.0. Tomato seedlings with good growth and uniform growth vigor are selected, grouped according to experimental settings, and 10 seedlings are repeated for each group, and are treated. Counting after inoculating the solarenergia EP1, observing the disease condition of tomato plants every day, and grading disease indexes of the disease tomatoes according to the bacterial wilt disease standard to judge the disease degree.
The results show (figures 6 and 7) that bacillus belicus HNU and bacillus fraxinus HNU-13 can reduce the incidence of bacterial wilt on tomatoes to a certain extent, and the double-bacteria compound biological preparation can reduce the incidence of bacterial wilt to a greater extent by more than 50%.
Example 6 antagonism experiment of plant Probiotics complexing agent against Fusarium oxysporum
Activating HNU, HNU6-13 on LB culture medium, respectively, picking single colony, culturing in 5mL liquid LB culture until OD 600 To 1.0. The banana vascular wilt FOC4 was cultured on the PDA plate for 3 days, and then the biocontrol bacteria were inoculated at a position 1cm away from the edge of the fungal hypha, and then the antagonism was observed after 48 hours, and it was found that the antagonism against banana vascular wilt was superior to HNU cultured alone under the mixed condition of HNU24 and HNU6-13 (HNU 24: HNU 6-13=5:1), and HNU-13 did not exhibit antagonism against banana vascular wilt (FIG. 8).
Claims (8)
1. A strain of langerhans (Franconibacter pulveris) HNU-13, characterized by the information on the preservation of the strain: preservation unit name: the collection of microorganism strains in Guangdong province; deposit unit address: building 5, guangzhou city, first, china, no. 100, university, no. 59, guangdong national academy of sciences of microbiology; preservation date:
2022, 5, 30; preservation number: GDMCC No.62498; classification naming: franconibacter pulveris.
2. Use of the french acetobacter HNU-13 according to claim 1 for controlling bacterial wilt of tomato plants.
3. Use of the french bacillus HNU-13 of claim 1 for the control of bacterial wilt and/or banana wilt in tomato plants by enhancing bacillus belicus (bacillus velezensis) HNU; the accession number of bacillus beijerinus HNU is: the collection of microorganism strains in Guangdong province; deposit unit address: building 5, guangzhou city, first, china, no. 100, university, no. 59, guangdong national academy of sciences of microbiology; preservation date: 10 months and 15 days 2020; preservation number: GDMCC No.61231; classification naming: bacillus sp.
4. A composite microbial agent, which is characterized in that the active ingredients of the composite microbial agent comprise bacillus belicus HNU and bacillus fraxinus HNU-13 as described in claim 1; the accession number of bacillus beijerinus HNU is: the collection of microorganism strains in Guangdong province; deposit unit address: building 5, guangzhou city, first, china, no. 100, university, no. 59, guangdong national academy of sciences of microbiology; preservation date: 10 months and 15 days 2020; preservation number: GDMCC No.61231; classification naming: bacillus sp.
5. The composite microbial inoculum of claim 4, wherein the microbial inoculum size ratio of HNU, HNU, 6-13 is HNU24: HNU6, 6-13=2:1 to 10:1.
6. The composite microbial inoculant of claim 5, wherein the preparation method of the composite microbial inoculant comprises the following steps:
and (3) re-suspending the cultured substrate-removed thalli HNU and HNU6-13 respectively to bacterial liquid OD 600-1.0-2.0 by using sterile water, and uniformly mixing according to the proportion of HNU24: HNU 6-13=2:1-10:1 to obtain the composite microbial inoculum.
7. Use of the composite microbial inoculant according to any one of claims 4-6 for controlling bacterial wilt and/or banana vascular wilt in tomato plants.
8. Use of the complex microbial inoculant of any one of claims 4-6 in tomato rhizosphere colonization.
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| CN108463549A (en) * | 2015-12-14 | 2018-08-28 | 巴斯夫欧洲公司 | The fermentation of enhancing |
| CN111040974A (en) * | 2019-12-31 | 2020-04-21 | 华南农业大学 | Banana endophytic Bacillus belgii and application thereof |
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