CN112458021A

CN112458021A - Biocontrol pseudomonas fluorescens and culture method and application thereof

Info

Publication number: CN112458021A
Application number: CN202011428350.XA
Authority: CN
Inventors: 周冬梅; 魏利辉; 何亮亮; 赵敏; 冯辉
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2021-03-09
Anticipated expiration: 2040-12-07
Also published as: CN112458021B

Abstract

The invention discloses a biocontrol pseudomonas fluorescens and a culture method and application thereof, wherein the preservation number of the pseudomonas fluorescens MF11 is CGMCC No.20594, and the application of the pseudomonas fluorescens MF11 can be specifically the application in the aspect of preventing and treating root knot nematode disease or vegetable seedling damping-off. The biocontrol bacterium, namely Pseudomonas fluorescens MF11(Pseudomonas fluorescens), provided by the invention can effectively prevent and treat root knot nematode diseases and vegetable seedling damping-off diseases, simultaneously shows broad-spectrum plant disease resistance, is non-toxic to people and animals, does not pollute the environment, has no residue, is beneficial to sustainable development of ecological environment, and also provides high-quality biological resources for researching and developing new environment-friendly biocontrol microbial inoculum.

Description

Biocontrol pseudomonas fluorescens and culture method and application thereof

Technical Field

The invention relates to the technical field of biological control of crops, in particular to biocontrol bacteria, namely pseudomonas fluorescens, a culture method thereof and application thereof in controlling plant diseases.

Background

Root-knot nematodes (Meloidogyne spp) are one of the main pathogens which are distributed worldwide and threaten agricultural production, and plant tissues can be changed after infection, so that hosts are more susceptible to infection of pathogenic bacteria such as bacterial wilt, wilting and root rot, and complex diseases are caused, the disease speed is increased, and the occurrence severity of the soil-borne diseases is increased. Due to wide hosts, the occurrence and prevalence rules of root-knot nematodes in different ecological environments are different, so that the prevention and control of the root-knot nematode disease of crops are very difficult.

Pythium spp is an important pathogen for many plants and its disease is very diverse (dry soldiers, 2002). Hosts of pythium include plants of cucurbitaceae, solanaceae, leguminosae, and gramineae, which cause serious damage to host plants (Zhang Bo et al, 2008). In recent years, pythium has been widely reported as an important pathogenic bacterium for seedling diseases of many crops. Pythium can cause seedling damping-off, rhizoctonia, root rot, stem rot, fruit rot, etc. (to Xinhua, 2012), which causes severe losses to agricultural production. Seedling damping-off, rootstock and melon and fruit rot caused by pythium aphanidermatum are the most serious. Pythium damping-off is a disease which is difficult to control, is often caused by melons, beans, hot peppers, eggplants and other melon and fruit vegetables, particularly has the most serious damage to cucumbers and tomatoes (Dimitrios F, 2011), and the disease symptoms of the pythium damping-off mainly cause that cotyledons are not withered or are suddenly inverted after the basal parts or the middle parts of plant embryonic stems are soaked in water (Wangdong et al, 2006).

The use of chemical agents is a main means for preventing and controlling root-knot nematodes and seedling damping-off in crop production at present, but due to the fact that misuse and abuse of high-toxicity chemical agents cause pollution to soil and water bodies and threaten safety of people and livestock, development of certain efficient, low-toxicity, low-residue and high-selectivity agents is needed to control the occurrence and damage of the root-knot nematodes and seedling damping-off.

Biological control is a hot point of attention because of the advantages of environmental friendliness, no toxicity or harm to non-target organisms and the like. The control of disease occurrence and the induction of improvement of crop stress tolerance by utilizing environment-friendly microorganisms are hot spots of research in recent years, particularly, the microorganisms have no pollution to the environment, can overcome the defects caused by chemical agent control, and arouse the interest of scientists in controlling diseases and coping with adverse environments by utilizing biological control means; particularly, the method has higher economic benefit and environmental benefit in agricultural production, accords with the development trend of protecting ecological environment and maintaining human health and safety, and can provide wide prospect for national economy of China and sustainable development of future agriculture.

Therefore, it is very important to find a safe and economic method for effectively controlling the root knot nematode and pythium disease, and more urgent is to select a microorganism which can effectively control the root knot nematode disease or the crop seedling damping-off disease so as to meet the requirement of green agriculture.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention screens a biocontrol bacterium, namely pseudomonas fluorescens, optimizes the culture method of the biocontrol bacterium, tests the biocontrol bacterium by measuring the control effect of a greenhouse potted plant, and researches the disease control effect of the biocontrol bacterium on the saprolegnia rot of melons and fruits; meanwhile, the plate antagonism test is carried out on other plant pathogenic bacteria, the antagonistic spectrum is preliminarily known, and a solid foundation is laid for the popularization and the application of the plant pathogenic bacteria in the prevention and the treatment of plant diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a biocontrol bacterium, which is Pseudomonas fluorescens MF11, is classified and named as Pseudomonas fluorescens (Pseudomonas fluorescens), has a preservation number of CGMCC No.20594, a preservation date of 2020 and 09 and 03 days, a preservation unit of China general microbiological culture Collection center (CGMCC), and a preservation unit address of China institute of academy of sciences, China institute of microbiology, No. 3, North Cheng Lu No. 1 institute of North West No. 3, Chao Yang, Beijing.

In a second aspect of the invention there is provided a bacterial formulation comprising pseudomonas fluorescens MF11 or a sterile fermentation broth obtained from fermentation thereof.

Further, the above-mentioned bacterial preparation also includes agriculturally and pharmaceutically acceptable auxiliary agents, including but not limited to: dispersing agents, stabilizers, carriers, and the like. The dosage forms of the fungal preparation include but are not limited to: liquid preparations, solid preparations (such as powders), and the like.

The third aspect of the present invention provides a method for culturing biocontrol bacteria, which comprises the steps of: activating and culturing the strain of pseudomonas fluorescens MF11, and sequentially performing primary fermentation culture, secondary fermentation culture and centrifugation on the activated strain to obtain sterile fermentation liquor.

Further, in the above culture method, the step of activation culture comprises: growing the strain on an LB culture medium, and carrying out dark culture in a constant-temperature incubator at the temperature of 25-30 ℃ for 0.5-3 days to obtain an activated strain; preferably, the activated strain is obtained by dark culture in a constant temperature incubator at 28 ℃ for 1 to 2 days.

Further, in the above culture method, the primary fermentation culture step comprises: performing primary fermentation on the activated strain in an LB culture medium to obtain a seed solution, wherein the culture conditions are as follows: carrying out shake fermentation culture for 18-30 h at the temperature of 25-30 ℃ and the rotating speed of 120-240 r/min; preferably, the culture conditions are: carrying out oscillatory fermentation culture at the temperature of 28-30 ℃ and the rotation speed of 170-; more preferably, the culture conditions are: the temperature is 28 ℃, and the rotation speed is 180r/min, and the shaking fermentation culture is carried out for 24 hours.

Further, in the above culture method, the secondary fermentation culture step comprises: inoculating the seed solution obtained by the primary fermentation culture into an LB culture medium in an inoculation amount of 4-12 wt% for secondary fermentation to obtain a culture solution, wherein the culture conditions are as follows: carrying out oscillatory fermentation culture for 18-30 h at the temperature of 25-30 ℃ and the rotating speed of 120-240 r/min; preferably, the inoculation amount is 6-10 wt%, and the culture conditions are as follows: carrying out oscillatory fermentation culture at the temperature of 28-30 ℃ and the rotation speed of 170-; more preferably, the inoculum size is 8 wt%, and the culture conditions are: the temperature is 28 ℃, and the rotation speed is 180r/min, and the shaking fermentation culture is carried out for 24 hours.

Further, in the above culture method, the centrifugation step comprises: centrifuging the culture solution obtained by the secondary fermentation culture at the rotating speed of 4000-6000 r/min, and filtering the supernatant fermentation solution through a 0.1-0.3 mu m bacterial filter to obtain sterile fermentation solution; wherein the viable bacteria concentration in the sterile fermentation liquid is 5 × 10⁹～1 ×10¹⁰CFU/mL. Preferably, the culture solution obtained by the secondary fermentation culture is centrifuged at the rotating speed of 5000r/min, and the supernatant fermentation solution is filtered by a 0.2 mu m bacterial filter to prepare the sterile fermentation solution.

Further, in the above culture method, the preparation step of the LB medium comprises: using 10g of tryptone, 5g of yeast extract and 10g of sodium chloride in 1000ml of LB culture medium, adjusting the pH value to 6.0-7.5, using distilled water to fix the volume to 1000ml, and sterilizing for 15-30 min at the temperature of 110-130 ℃; preferably, the pH is adjusted to 7.2 and the mixture is sterilized at 121 ℃ for 20 min. The method comprises the following steps of activation culture, wherein an LB solid culture medium is adopted in the activation culture step, and 10-15g of agar is added in the preparation process; the primary fermentation culture and the secondary fermentation culture both adopt LB liquid culture medium.

The fourth aspect of the invention provides a method for preventing and controlling plant diseases, which adopts biocontrol bacteria-pseudomonas fluorescens MF11 or a mycodrug preparation prepared from pseudomonas fluorescens MF11 to carry out root irrigation treatment on crops.

Further, in the above method for controlling plant diseases, the step of root irrigation treatment comprises: performing root irrigation treatment on a fungus preparation obtained by fermenting biocontrol bacteria on the same day as the crop transplantation; before the crops are attacked, the transplanted crops are irrigated with the fungicide preparations every 30-45 days according to the growth cycle of the crops. Preferably, the microbial preparation can be sterile fermentation liquor or a composition of the sterile fermentation liquor and an auxiliary agent, and the sterile fermentation liquor needs to be diluted before root irrigation, and is preferably diluted by 100-200 times.

The fifth aspect of the invention provides the application of a biocontrol bacterium, namely pseudomonas fluorescens MF11 or a bacterial preparation prepared from pseudomonas fluorescens MF11 in the aspect of preventing and treating plant diseases.

Further, in the above method for controlling a plant disease or the application in controlling a plant disease, the plant disease includes a plant disease caused by a root knot nematode, pythium oligandrum, pythium aphanidermatum, pythium wakame, phytophthora citri-fusca, rhizoctonia solani or fusarium oxysporum.

Further, in the above application, the plant disease is root knot nematode disease or crop seedling damping-off disease.

Compared with the prior art, the invention has the following beneficial effects by adopting the technical scheme:

the biocontrol bacterium CGMCC No.20594 (Pseudomonas fluorescens) provided by the invention can effectively prevent and treat root knot nematode disease and seedling damping-off disease, simultaneously shows broad-spectrum plant disease resistance, is nontoxic to human and livestock, does not pollute the environment, has no residue, is beneficial to sustainable development of ecological environment, and also provides high-quality biological resources for researching and developing new environment-friendly biocontrol bacterium agents.

Drawings

FIG. 1 is a schematic diagram showing the result of nematode aggregation at the root tip after tomato roots are treated by the biocontrol bacterium Pseudomonas fluorescens MF11 in one embodiment of the present invention;

FIG. 2 is a schematic diagram showing the results of the nematode entering the roots after the tomato roots are treated by the biocontrol bacterium Pseudomonas fluorescens MF11 in one embodiment of the present invention.

FIG. 3 is a schematic diagram showing the effect of Pseudomonas fluorescens MF11 on the control of root-knot nematodes and the effect of reproductive ability in one embodiment of the present invention.

The biocontrol bacterium, Pseudomonas fluorescens MF11, is deposited and named as pseudomonad fluorescens (Pseudomonas fluorescens) in classification, the deposition number is CGMCC No.20594, the deposition date is 09 months and 03 days in 2020, the deposition unit is China general microbiological culture Collection center (CGMCC), and the deposition unit address is China institute of microbiology, institute of sciences, China institute of sciences, No. 3, North Chen Lu, No. 1 of south China, Chaoyang, Beijing.

Detailed Description

The following description of the embodiments of the present invention will be made with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby. Materials, reagents and the like used in the following embodiments are commercially available unless otherwise specified; wherein the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature of the art (for example, refer to J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, scientific Press, translated by Huang Petang et al) or according to the product instructions.

Example 1

This example shows the selection and identification process (separation and selection from natural environment) of a biocontrol bacterium MF11(Pseudomonas fluorescens).

Screening of biocontrol bacterium MF11

(1) Isolation of potential biocontrol bacteria present in vitro

1) Preparation of soil suspension: weighing 10g of soil sample (from rhizosphere soil of tomatoes in a greenhouse of a bamboo town tomato in Liuhe district of Nanjing City, Jiangsu province) in a triangular flask (containing sterilized glass beads) containing 90ml of sterile 0.85% NaCl, and performing shake culture for 30min at 150rpm of a shaking table;

2) 10-fold gradient dilution: standing for 5min, taking 1ml of soil suspension from triangular flask, placing in test tube containing 9ml of sterile 0.85% NaCl (diluted by 10 times), shaking, mixing, further diluting in gradient to obtain 10^-2、10^-3、10^-4、10^-5、10^-6The soil dilution of (1);

3) plating culture: 0.1ml of each of the above-mentioned dilutions was uniformly applied to R2A medium (Reasoner & Geldreich, 1985) in 3 replicates per gradient, and the resulting mixture was inverted at 28 ℃ for about 2 days;

4) selecting a plate with the colony number between 50 and 300, counting, picking all colonies, purifying on a solid LB culture medium, picking a single colony by using an inoculating loop, inoculating the single colony into a test tube containing 5ml of LB culture medium (liquid), shaking the bacteria at the temperature of 28 ℃ and the speed of 200rpm for culturing for 24 to 48 hours, uniformly mixing the bacterial suspension with an equal volume of 80 percent glycerol solution, and storing the mixture at the temperature of-70 ℃ for later use (Berg et al, 2000).

(2) Enzyme production activity determination for preliminary screening of biocontrol strain

Strains with enzyme-producing activities such as protease, chitinase, glucanase, cellulase and the like are often used as the standard for primary screening of biocontrol bacteria. Selecting a strain in a vigorous growth stage, putting the strain on a protease enzyme activity determination culture medium, a culture medium (Chi-adsorbents) taking colloidal chitin as a unique carbon source, a beta-1, 3-glucanase enzyme activity determination culture medium and a cellulase enzyme activity determination culture medium, culturing at 28-30 ℃ for 3 days after inoculation, observing the existence of a transparent ring, and respectively recording the inner diameter and the outer diameter of the transparent ring. Strains having one or more of the above enzyme-producing activities are retained for further screening.

And finally, separating a strain from tomato rhizosphere soil, and confirming the strain as a potential biocontrol strain through detecting the activity of the produced enzyme and performing a plate antagonism test, wherein the strain is named as biocontrol bacterium MF 11.

Identification of biocontrol bacterium MF11

(1) Morphological feature observation

Morphological characteristic observation is carried out by using an insertion method, and the method comprises the following specific operations: the sterilized coverslip was inserted obliquely at an angle of 45 degrees into an LB medium plate coated with the above-mentioned selected strains, and cultured at a constant temperature of 28 ℃ while observing the culture characteristics and morphological characteristics with an optical microscope.

Morphological characteristics: the single colony is round, smooth in edge, smooth and transparent in surface and light yellow.

(2) Analysis of physiological and biochemical properties

Physiological and biochemical traits of the screened biocontrol bacteria were analyzed according to the handbook of identification of common bacteria systems (Dongxiu pearl, 2001).

1) Gram staining method

A clean glass slide is taken and a drop of sterile water is dripped on the glass slide, then a small amount of bacterial colonies are picked by a sterile gun head and coated on the periphery of the drop, and the smear is fixed. And (4) dyeing the crystal violet dye liquor by ammonium oxalate for 1 minute, and washing the crystal violet dye liquor by using distilled water. Then iodine solution is dripped to cover the coating surface for dyeing for 1 minute, and water is absorbed by absorbent paper after water washing. Several drops of 95% alcohol were added dropwise for decolorization, and after about 20 seconds, the solution was washed with water, and finally stained with safranin staining solution for 1 minute, and then dried for microscopic examination. The colonies stained red by microscopic examination and were gram-negative bacteria.

2) Physiological and biochemical characteristic identification of strain

A. Catalase test

Single colonies were picked with a sterile tip and placed on a clean glass slide, and a drop of 3% H was added₂O₂On the colony; the presence or absence of bubble generation was immediately observed. The results show that MF11 reacted positively with the enzyme.

B. Salt tolerance test

Respectively inoculating 100 μ l of biocontrol bacteria liquid into 10ml LB liquid culture medium containing NaCl (0-10%) with different concentrations; the strain growth was observed after culturing in a shaker at 28 ℃ and 180rpm for 1 day using LB liquid medium without inoculation as a control. The results show that MF11 can grow normally in 1% and 4% NaCl culture solution, but not in 8% NaCl culture solution.

C. Acid resistance test

Inoculating 2ml of biocontrol bacteria liquid into LB liquid culture medium with pH 5 and LB liquid culture medium with pH 6 respectively, taking the LB liquid culture medium without bacteria inoculation as a control, culturing for 3-7 days at constant temperature in an incubator at 28 ℃, and observing the growth condition of the strains. The results show that MF11 can grow normally in liquid culture medium with pH of 5 or 6.

D. Oxidase test

A small piece of filter paper is taken to dip a small number of bacterial colonies, then a proper amount of 1% tetramethyl-p-phenylenediamine hydrochloride solution is dripped, if bacteria are in deep purple within 10 seconds of contacting with the reagent, the bacteria are positive, and the bacteria are negative (Liangzheng et al, 2012). The results showed that MF11 oxidase reaction was positive.

E. Aerobic type experiment

And (3) the NA culture medium is subpackaged in test tubes, after the inoculating loop picks the bacteria to be detected to perform puncture inoculation at the bottom of the test tube, the test tube is placed in an incubator at 28 ℃ to be cultured for 2 days and the change of bacterial colonies is observed, and if the bacteria grow along the puncture line of the culture medium, the bacteria are anaerobic bacteria. Growing along the surface is aerobic. The results showed that MF11 was an aerobic type bacterium.

(3) Molecular biological identification

The biocontrol bacteria MF11 obtained by screening is cultured on a Gao's first culture medium at 28 ℃ to logarithmic phase, and the bacteria are collected by centrifugation at 5000r/min for 5 min.

The total genomic DNA of the collected thalli is extracted by adopting a bacterial genomic DNA rapid extraction kit of Kangji century Co.

And then the 16S rRNA sequence and the gyrB gene of the biocontrol MF11 are amplified by adopting a PCR amplification kit of Kangji century Co. The total genomic DNA extracted above was used as a template, and the primers used for amplification were 16S rRNA universal primer 27F/1492R (Yamamoto & Harayama, 1995) and gyrB primer UP-1S/UP2Sr (Galkiewicz & Kellogg, 2008), respectively. The specific PCR amplification operation was performed according to the kit instructions.

And (3) carrying out 1% agarose gel electrophoresis on the PCR product, observing under an ultraviolet lamp, and recovering the PCR product with a target band. Then, the PCR product is sent to the company of Biotechnology engineering (Shanghai) for sequencing.

The sequencing results obtained were submitted to the GenBank database for BLAST analysis and alignment. The result shows that the homology of the 16S rRNA sequence of the biocontrol bacterium MF11 and the Pseudomonas fluorescens sequence reaches 99.93 percent, and the biocontrol bacterium MF11 obtained by screening is finally identified as the Pseudomonas fluorescens (Pseudomonas fluorescens) by combining the results of morphological characteristics, culture characteristics and physiological and biochemical characteristics of the strain.

The biocontrol bacterium MF11 belongs to Pseudomonas fluorescens (Pseudomonas fluorescens), is preserved in the China general microbiological culture Collection center (address: West Lu No. 1, Ministry of microbiology, China academy of sciences, North Chen, south China, Beijing), has the preservation date of 2020, 09 and 03 days, and has the preservation number of CGMCC No. 20594.

Example 2

This embodiment is a preferred cultivation method and preparation method of the bacterial agent for pseudomonas fluorescens MF11 obtained by screening in example 1, and the method comprises the following steps:

the biocontrol bacterium MF11 was grown on LB medium (plate streaking) and cultured in an incubator at 28 ℃ for 1-2 days in the dark to grow a single colony (to obtain an activated bacterial strain). The preparation method of the LB culture medium comprises the following steps: 10g of tryptone, 5g of yeast extract powder, 10g of sodium chloride and 10-15g of agar are used for preparing 1000ml of LB culture medium, the pH is adjusted to 7.2, the volume is fixed to 1000ml by using distilled water, and the LB culture medium is sterilized for 20min at the temperature of 121 ℃;

selecting a single colony (activated strain) and inoculating the single colony into a 250ml triangular flask filled with 50ml LB culture medium, and carrying out shaking fermentation culture for 24h at the temperature of 28 ℃ and the rotating speed of 180r/min to obtain a seed solution. The preparation method of the fermentation culture medium used in the step is an LB culture medium;

inoculating the obtained seed liquid into LB culture medium with an inoculation amount of 8 wt% for secondary fermentation under the same conditions to obtain a culture solution, and storing at 4 ℃ for later use.

Centrifuging the obtained culture solution at a rotation speed of 5000r/min, discarding bottom precipitate, filtering supernatant fermentation solution with 0.2 μm bacterial filter to obtain sterile fermentation solution (viable bacteria concentration of 5 × 10)⁹～1×10¹⁰CFU/ml), stored at 4 ℃ until use.

Example 3

The embodiment is a method and application of pseudomonas fluorescens MF11 obtained by screening in embodiment 1 in the aspect of preventing and treating plant diseases, and the specific application steps comprise: diluting the sterile fermentation liquor obtained in the embodiment 2 by 100-200 times, and performing root irrigation treatment on the same day as crop transplanting; and then, before the crops are attacked, diluting the sterile fermentation liquor obtained in the example 2 by 100-200 times according to the growth period of the crops every 30-45 days to irrigate roots of the transplanted crops. The application process can also adopt the bacterial preparation taking the pseudomonas fluorescens MF11 as the active ingredient, such as powder, liquid preparation and the like, and the specific application mode can be adjusted according to the specific preparation formulation.

The plant diseases comprise: plant diseases caused by root-knot nematode, pythium oligandrum, pythium forest, pythium aphanidermatum, pythium gordonii, phytophthora citri, rhizoctonia solani or fusarium oxysporum.

Example 4

The embodiment is a plate antagonism test of biocontrol bacteria-pseudomonas fluorescens MF11 on phytopathogens, and the specific test method comprises the following steps:

pythium aphanidermatum (Pythium aphanidermatum), Pythium gorgonioides (Pythium sponens), Pythium linnensis (Pythium sylvaticum), Phytophthora citri (Phytophthora citri), Pythium Oligandrum (Pythium Oligandrum), Rhizoctonia solani (Rhizoctonia solani), and Fusarium oxysporum (Fusarium oxysporum) were each cultured in 10% V8 solid medium for 1-4 days for use.

The above 10% V8 solid medium was cultured in the following ratio of 1: 100(w/V) ratio CaCO added to V8 vegetable juice₃Centrifuging at 5000r/min for 10min, collecting supernatant, removing precipitate, adding 9 times of distilled water into the supernatant to obtain 10% V8 vegetable juice culture medium, adding agar in a proportion of 1.5%, sterilizing at 121 deg.C for 20min, and cooling.

For the above plant pathogens, the part close to the edge of each colony was taken, a sterilized punch was used to punch a bacterial dish with a diameter of 6mm, the hypha side of the bacterial dish was inoculated downwards to the center of a 10% V8 solid medium plate, 10. mu.l of the MF11 sterile fermentation solution prepared in example 2 was inoculated at a distance of 5mm from the edge of the plate, the plate was dried in an ultra-clean bench and cultured in the dark at 25 ℃ for 1-5 days, and 3 treatment settings were repeated for each group. The colony diameter is measured by a cross method, and the inhibition rate of the hypha growth is calculated by the formula:

the inhibition rate (%) was (control colony diameter-treatment colony diameter)/(control colony diameter-6) × 100%.

The results of the above tests are given in the following table:

as can be seen from the results in the table above, the biocontrol bacterium MF11 has a pythium aphanidermatum inhibition rate of more than 88% and has obvious antagonistic activity (inhibition effect). Therefore, the biocontrol bacterium MF11 and the microbial inoculum thereof screened in the example 1 can be used for preventing and treating the plant seedling damping-off caused by pythium aphanidermatum.

Meanwhile, as can be seen from the results in the table above, the biocontrol bacterium MF11 has an inhibition rate of more than 60% on Pythium oligandrum, Pythium lincolnense, Pythium gordonii, Phytophthora citri, Fusarium oxysporum and Rhizoctonia solani, which indicates that the biocontrol bacterium MF11 can inhibit the growth of the Pythium and Fusarium. From this, it is inferred that the biocontrol bacterium MF11 screened in example 1 and its microbial inoculum can also be used for controlling plant diseases caused by Pythium oligandrum, Pythium linzii, Pythium gordonii, Phytophthora citri, Fusarium oxysporum and Rhizoctonia solani.

Example 5

The embodiment is a pot culture control effect determination method of biocontrol bacteria, namely pseudomonas fluorescens MF11 pythium aphanidermatum, and the specific test method comprises the following steps:

(1) preparation of pythium aphanidermatum zoospore suspension: activating and growing the preserved fruit and fruit pythium aphanidermatum strain on a 10% V8 culture medium, and culturing in a constant temperature incubator at 25 ℃ in the dark, wherein the culturing time is 1-2 days for later use; cutting the strain into 10mm × 15mm mycelium blocks (10-20 blocks) with scalpel, placing in 15ml sterilized tap water with mycelium side facing upward, changing water every 30min, and repeating for 3 times; finally adding about 8ml of sterilized tap water, placing and culturing in a constant-temperature incubator at 25 ℃, inducing to generate zoospores after culturing for about 24 hours, and adjusting the zoospore suspension to the concentration of about 1 × 10 by using sterile water⁴One per ml.

(2) Determination of potted plant control effect: cucumber seedlings growing for 1 week are inoculated with 100-fold diluted biocontrol bacterium MF11 sterile fermentation broth (prepared in example 2) at the root, and each cucumber seedling is inoculated with 5ml (the cucumber seedling is inoculated with the same volume of fermentation medium supernatant of non-inoculated bacterium MF11 at the root position as a control). And after 3 days of root irrigation, irrigating 2ml of zoospores of pythium aphanidermatum into roots of the cucumber seedlings, repeating 3 groups of treatment, and inoculating 36 ginger seedlings in total for 12 seedlings in each group. Culturing in a constant temperature incubator at 25 deg.C, and performing statistics on plant lodging conditions and calculating control effect 11 days after inoculation. The calculation formula is as follows:

disease index is 100 × Σ (number of diseased plants × representative value of each stage)/(total number of investigated plants × representative value of highest stage);

0 ═ the plants remained green and healthy; 1 ═ leaf sheath ring discolours and lower leaves turn yellow; 2-plants survived but leaves were completely yellow or dead; the whole plant died.

The preventing and treating effect (%) is (contrast disease index-treatment disease index)/contrast disease index is multiplied by 100

The results of the assay are shown in the following table.

The disease index results in the above table are significantly different at the 0.01 level.

From the results in the table, the disease index of the cucumber seedling treated by root irrigation with the biocontrol bacterium MF11 microbial inoculum (fermentation broth) prepared in example 2 is 13.15, and the control effect is 75.01%, which shows that the biocontrol bacterium MF11 microbial inoculum can be used for controlling crop seedling damping-off.

Example 6

The embodiment is a method for measuring the lethality of a biocontrol bacterium, namely pseudomonas fluorescens MF11, to second-instar larvae of meloidogyne incognita, and the specific test method comprises the following steps:

washing tomato roots infected by nematodes for more than 8 weeks, cutting into pieces, bottling, adding 10% sodium hypochlorite, shaking for 3min, immediately pouring into a separation sieve of 20, 170 and 500 meshes, washing off sodium hypochlorite with a large amount of clear water, and collecting eggs on a 500-mesh standard sieve. Suspending the collected eggs with 35% sucrose, collecting upper layer eggs, adding 10% sodium hypochlorite solution, oscillating and suspending for 5min, centrifuging to collect eggs, sterilizing, and washing with water for 3 times to remove sodium hypochlorite. After incubating for 2-3 days under the aseptic condition at room temperature, collecting the second-instar larvae of the meloidogyne incognita for later use.

1ml of the sterile fermentation broth obtained in example 2 and 50 instar larvae were sequentially added to a 24-well cell culture plate, and the cell culture plate was placed in a 25 ℃ incubator; observing the activity of the nematodes after 2h, 4h, 8h, 12h and 24h respectively, counting the number of dead heads of the nematodes, and repeating the treatments for 3 times by taking LB culture solution and sterilized water as controls. The result shows that the mortality rate of MF11 to the second-instar larvae is 94% (see the table below), and the pesticide can be used for controlling plant diseases caused by root-knot nematodes.

Example 7

The embodiment is a method for determining the influence of biocontrol bacteria, namely pseudomonas fluorescens MF11 on the chemotaxis of nematodes, and the specific test method comprises the following steps:

treating tomato seeds (Moneymoker) with a 10% sodium hypochlorite solution for 15min (Ho et al, 1992), carrying out surface disinfection treatment, and washing the treated tomato seeds for 5-6 times in a super clean bench after vigorous shaking; culturing in dark for 4-5 days. Taking out the tomato roots with the length of about 1.5 cm-1.8 cm for use, respectively soaking the tomato seedlings roots into 100-time diluted biocontrol bacteria MF11 fermentation liquid and LB culture liquid, carrying out shaking culture on a shaking table at 180rpm and 28 ℃ for 30min, and taking out.

20000 head second instar nematodes were obtained according to the procedure of example 6. Under the condition of low-temperature ice bath, concentrating the nematode suspension to 500 heads/ml, adding the nematode suspension into Pluronic F-127 gel, and fully mixing the nematode gel liquid at low temperature (Morishita et al, 2001); a6 cm plate of cell culture was placed on ice and 3ml of Pluronic F-127 gel (containing 1500 head second instar nematodes) was pipetted into the plate to fill the bottom of the plate with gel and carefully remove excess air bubbles. Placing the treated seedlings into a cell culture plate containing Pluronic F-127 gel, placing two seedlings in each hole, placing the seedlings, and placing the cell culture plate at room temperature; each treatment was repeated 3 times; the aggregation of the meloidogyne incognita on the roots after the tomato roots are treated by the biocontrol agent MF11 is observed under a stereoscopic microscope at 1h, 4h, 12h, 18h, 24h and 48h respectively.

Meanwhile, the roots are taken out and dyed and decolored, and the number of nematodes entering the tips of the tomato roots is counted. The experiment was repeated 3 times. Preparing a dyeing liquid mother solution: 3.5g of acid fuchsin powder is weighed, added into 250ml of glacial acetic acid, added with sterilized water to reach the volume of 1000ml and stirred uniformly. Preparing a root tissue decolorizing solution: glacial acetic acid: glycerol: mixing the sterilized water in a volume ratio of 1:1: 1.

The experimental results are shown in fig. 1 and fig. 2, fig. 1 shows the accumulation of nematodes at the root tip after tomato roots are treated by the growth control bacterium MF11, and fig. 2 shows the situation that nematodes enter the roots after tomato roots are treated by the growth control bacterium MF 11. The above experimental results show that: compared with the control treatment, after the tomato plants are treated by the fermentation liquor of the bacterium MF11, the accumulation of root-knot nematodes on the tomato roots can be obviously reduced, the number of the root-knot nematodes entering the tomato roots is obviously reduced, and the statistical result of 48 hours shows that the root-knot nematodes are reduced by 80.65% compared with the control treatment.

Example 8

The embodiment is a greenhouse prevention effect determination of biocontrol bacteria-pseudomonas fluorescens MF11 for preventing and treating root-knot nematodes, and the specific test method comprises the following steps:

tomato seedlings (Moneymoker) germinate and are transplanted into a basin bowl filled with fine sand, and the tomato seedlings grow under the conditions of 25 ℃, 16 hours of light and 8 hours of darkness. When tomato seedlings grow to three weeks, 10ml of biocontrol bacteria MF11 bacterial liquid is inoculated to the tomato roots, and 500J 2 suspension is inoculated after one week. Three weeks after the inoculation of J2, the number of root knots was counted, and the number of egg masses was counted after 8 weeks. Each treatment was 18 seedlings, repeated 3 times.

An egg mass staining method comprises the following steps: weighing 100mg of brilliant blue powder, and dissolving in 100ml of sterilized water to prepare a mother solution; measuring 10ml of mother liquor by using a measuring cylinder, adding the mother liquor into a large beaker containing 500ml of tap water, and uniformly stirring; the obtained solution is the working solution; cleaning tomato root with water, placing the root in the working solution, dyeing for 15min, and counting the number of egg masses.

The experimental result is shown in fig. 3, and fig. 3 shows the effect of the biocontrol bacterium MF11 on the biocontrol effect and reproductive capacity of root-knot nematodes. The above experimental results show that: compared with the control treatment, after tomato plants are treated by the fermentation liquor of the bacteria MF11, the root knot number can be obviously reduced by 86.53%, and the egg mass number is reduced by 70.52%, which indicates that the biocontrol bacteria MF11 can be used for preventing and controlling root-knot nematodes.

Example 9

The embodiment is a field control effect determination of biocontrol bacteria-pseudomonas fluorescens MF11 for controlling root-knot nematodes, and the specific test method comprises the following steps:

the sterile fermentation broth prepared in example 2 was diluted to 2X 10⁷CFU/ml is reserved, and each seedling is irrigated with 200ml of roots. The control agent was 10% fosthiazate. The test field is divided into 9 small areas with the same area, each small area is 8 square meters, and 32 tomato seedlings are planted in each small area. Set 3 treatments: 10% fosthiazate (chemical agent control group), clear water control group, and biocontrol bacteria MF11 treatment group, wherein the treatment is repeated for 3 times, and protection rows are arranged between adjacent cells and are completely randomly arranged.

After 40 days, all cell tomatoes were graded (Bridge, 1980) to investigate the disease:

level 0: the root system has no insect gall and no disease;

level 1: a small amount of insect gall exists in the root system;

and 3, level: two thirds of root systems are full of insect galls;

and 5, stage: the root system is full of small galls and secondary galls exist;

and 7, stage: the root system forms a fibrous root mass.

Disease index and relative control were calculated according to the formula (Xue Q, 2009):

disease index of 100 × Σ (number of diseased plants x representative value of each stage)/(total number of investigated plants x highest representative value)

As can be seen from the experimental results in the table above, the field control results show that: the disease index of the tomato plant treated by the biocontrol bacterium MF11 is obviously lower than that of a clear water control, and the disease index is not obviously different from that of a 10% fosthiazate treatment effect; the disease index of the tomato treated by MF11 is 19.09, the relative control effect is 66.71%, the field control effect is better, and the tomato can be used for field control of root-knot nematodes.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. The biocontrol bacterium is pseudomonas fluorescens MF11 with the preservation number of CGMCC No. 20594.

2. A microbial preparation comprising the biocontrol bacterium of claim 1 or a sterile fermentation broth obtained from the culture thereof.

3. The microbial drug formulation of claim 2, further comprising an agriculturally acceptable adjuvant.

4. A method of culturing biocontrol bacteria as described in claim 1, comprising the steps of: activating and culturing the strain of pseudomonas fluorescens MF11, and sequentially performing primary fermentation culture, secondary fermentation culture and centrifugation on the activated strain to obtain sterile fermentation liquor.

5. The method of claim 4, wherein the step of activating the culture comprises: growing the strain on an LB culture medium, and carrying out dark culture in a constant-temperature incubator at the temperature of 25-30 ℃ for 0.5-3 days to obtain an activated strain; and/or the presence of a gas in the gas,

the primary fermentation culture step comprises the following steps: performing primary fermentation on the activated strain in an LB culture medium to obtain a seed solution, wherein the culture conditions are as follows: carrying out shake fermentation culture for 18-30 h at the temperature of 25-30 ℃ and the rotating speed of 120-240 r/min; and/or the presence of a gas in the gas,

the secondary fermentation culture step comprises the following steps: inoculating the seed solution obtained by the primary fermentation culture into an LB culture medium in an inoculum size of 4-12 wt% for secondary fermentation to obtain a culture solution, wherein the culture conditions are as follows: carrying out shake fermentation culture for 18-30 h at the temperature of 25-30 ℃ and the rotating speed of 120-240 r/min; and/or the presence of a gas in the gas,

the centrifugation step comprises: centrifuging the culture solution obtained by the secondary fermentation culture at the rotating speed of 4000-6000 r/min, and filtering the supernatant fermentation solution through a 0.1-0.3 mu m bacterial filter to obtain sterile fermentation solution; wherein the viable bacteria concentration in the sterile fermentation liquid is 5 × 10⁹～1×10¹⁰CFU/mL。

6. The method for culturing biocontrol bacteria as in claim 5, wherein the LB medium is formulated by: the method comprises the steps of using 10g of tryptone, 5g of yeast extract and 10g of sodium chloride in 1000ml of LB culture medium, adjusting the pH value to 6.0-7.5, using distilled water to fix the volume to 1000ml, and sterilizing for 15-30 min at the temperature of 110-130 ℃.

7. A method for controlling plant diseases, characterized in that crops are subjected to root irrigation with the biocontrol bacterium as claimed in claim 1 or the mycolic preparation as claimed in claim 2 or 3.

8. The method for controlling plant diseases according to claim 7, characterized in that the step of root irrigation treatment comprises: performing root irrigation treatment on a fungus preparation obtained by fermenting biocontrol bacteria on the same day as the crop transplantation; before the crops are attacked, the transplanted crops are irrigated with the fungicide preparations every 30-45 days according to the growth cycle of the crops.

9. Use of the biocontrol bacterium of claim 1 or the fungal drug preparation of claim 2 or 3 for controlling plant diseases including plant diseases caused by root-knot nematodes, pythium oligandrum, pythium forest, pythium aphanidermatum, pythium gordonii, phytophthora citri-cola, rhizoctonia solani or fusarium oxysporum.

10. The use according to claim 9, wherein the plant disease is root knot nematode disease or crop seedling damping off disease.