CN103371041A - Method for screening bio-control bacteria of cucumber fusarium wilt - Google Patents

Abstract

The invention discloses a method for screening biocontrol bacteria of cucumber wilt. The invention provides a method for identifying the biocontrol properties of the bacteria to be tested to the pathogenic bacteria of cucumber wilt, including experimental treatment; the experimental treatment comprises the following steps in turn: (1) accelerating the germination of the sterilized cucumber seeds to obtain Lubai seeds; (2) After soaking the Lubai seeds in the suspension of the bacteria to be tested, continue to cultivate until the radicle length reaches 1.5-2.0cm; (3) inoculate the cucumber seedlings with the suspension of cucumber wilt pathogenic bacteria by dipping the radicle (4) cucumber seedlings are transferred to MS solid medium for cultivation, by investigating the disease index and/or observing the pathogenesis phenotype identification test bacteria to the biocontrol properties of cucumber wilt pathogenic bacteria.Described method can be used for screening cucumber Fusarium wilt biocontrol bacteria. In the present invention, cucumber seedlings are used as experimental samples, which can more truly reflect the disease prevention effect of biocontrol bacteria, and can screen out bacterial strains with various biocontrol mechanisms.

Description

A method for screening biocontrol bacteria of cucumber wilt

technical field

The invention relates to a method for screening biocontrol bacteria of cucumber wilt.

Background technique

It is the key to obtain the biocontrol strains with high efficiency to control plant diseases successfully to select the appropriate strain selection method. Ideally, the screening of biocontrol bacteria on plants is more effective than the biocontrol strains obtained by cell culture or other screening methods in vitro. However, performing large-scale screening efforts on a large number of plants is time-consuming and labor-intensive. Therefore, the traditional double culture method has been one of the very popular screening methods. In this method, the strains with antagonistic activity can form an obvious inhibition zone with pathogenic bacteria on the PDA plate, but those strains that have no obvious antagonistic activity but may have better control effect in the actual control process pass this screening. Method not available.

Aseptic seedlings have been used in the laboratory to test the effect of strains on plant disease control. The main method is to use tissue culture seedlings to inoculate pathogenic bacteria and biocontrol bacteria under sterile conditions. This method is one of the ideal screening methods for biocontrol strains. Especially for the screening of those bacterial strains that have no obvious resistance to pathogenic bacteria on the PDA plate. However, not all plants can obtain tissue culture seedlings, and the cultivation of tissue culture seedlings is time-consuming and laborious, and the nutrient substrates involved are different according to different plants, which is not suitable for wide application.

Contents of the invention

The object of the present invention is to provide a method for identifying the biocontrol properties of the bacteria to be tested against the pathogenic bacteria of cucumber wilt, and the method can be used to screen the biocontrol bacteria of cucumber wilt to obtain the biocontrol bacteria of cucumber wilt with good effect.

The invention provides a method for identifying the biocontrol properties of the bacteria to be tested to the pathogenic bacteria of cucumber wilt, including experimental treatment; the experimental treatment comprises the following steps in turn: (1) accelerating the germination of the sterilized cucumber seeds to obtain Lubai seeds; (2) soak the Lubai seeds obtained in step (1) in the bacteria suspension to be tested and continue to cultivate until the radicle length reaches 1.5-2.0cm; (3) the cucumber seedlings obtained in step (2) Inoculate the cucumber wilt pathogen suspension by dipping the radicle; (4) transfer the cucumber seedlings treated in step (3) to MS solid medium for cultivation, and identify the disease index and/or observe the disease phenotype to be tested Biocontrol properties of fungus against cucumber wilt pathogen.

Described method can also comprise control treatment; Described control treatment comprises described step (1), described step (3) and described step (4) successively; Said step ( 4) all identify the biocontrol properties of the bacteria to be tested by investigating the disease index to the pathogenic bacteria of cucumber wilt; if the disease index of the experimental treatment is lower than the disease index of the control process, the bacteria to be tested are candidates The biocontrol bacteria of the pathogen of cucumber wilt; if the disease index of the experimental treatment is equal to or higher than the disease index of the control treatment, the bacteria to be tested are candidates for the pathogen of cucumber wilt Non-biocontrol bacteria. The calculation method of the disease index is as follows: remove a plant with the heaviest disease and a plant with the lightest disease, and the disease index of the remaining plants is calculated according to the following formula: disease index=∑(number of diseased plants at all levels×the disease level value)/(total number of investigated plants×highest level value)×100.

Described method can also comprise control treatment; Described control treatment comprises described step (1), described step (3) and described step (4) successively; Said step ( 4) all identify the biocontrol properties of the bacteria to be tested by observing the pathogenic phenotype of the pathogenic bacteria of cucumber wilt; Described test bacterium is the biocontrol bacterium of the described cucumber wilt pathogenic bacterium of candidate; If the pathogenesis phenotype of plant in described experimental treatment is consistent with the pathogenesis phenotype of plant in described control treatment, described test bacterium is Candidate non-biocontrol bacteria of the cucumber wilt pathogen; if the pathogenesis phenotype of the plants in the experimental treatment is more severe than that of the plants in the control treatment, the bacteria to be tested are the candidates. Non-biocontrol fungi of cucumber wilt pathogen.

The cucumber wilt pathogenic bacterium suspension may specifically be a cucumber-specific spore suspension of Fusarium oxysporum with a concentration of 1×10 ⁶ CFU ml ⁻¹ .

The soaking time may specifically be 1 hour.

In the step (4), the timing can be started from the completion of the step (3), and the disease index and/or the onset phenotype can be observed after 5 days.

Any of the methods described above can be used to screen the biocontrol bacteria against the pathogenic bacteria of cucumber wilt.

Any one of the above-mentioned cucumber wilt pathogenic bacteria can specifically be Fusarium oxysporum cucumber-specific type.

In the method, all adopt aseptic operation.

Cucumber wilt can occur in various stages of cucumber growth, and the prevention and control of cucumber wilt should start from the seedling stage, which can not only reduce the incidence of fusarium wilt, but also effectively improve the disease resistance of cucumber. In the present invention, cucumber seedlings are used as experimental samples, first soaked with the suspension of bacteria to be tested, and then inoculated with pathogenic bacteria, and the biocontrol characteristics of the bacteria to be tested are judged according to the onset phenotype and disease index, thereby screening biocontrol strains, which can be more realistic. It embodies the disease prevention effect of birth control bacteria, and can screen out strains with various biological control mechanisms.

The traditional double culture method can only screen out strains that have antagonistic activity against pathogenic bacteria, and it is difficult to screen out strains that have no antagonistic activity on the PDA plate but are also potential plant disease biocontrol strains, and the use of the sterile seedling system has strong limitations sex. And the screening method in the present invention can well avoid the generation of above-mentioned problem, and accurate, rapid screening obtains more potential biocontrol bacterial strains, and the biocontrol mechanism of the biocontrol bacterial strains obtained by screening will not be affected by the screening method. It is conducive to the in-depth study of the theoretical mechanism of biological control.

In the present invention, a strain of Bacillus subtilis is screened and named as B201. Bacillus subtilis (Bacillus subtilis) B201 was preserved in the General Microbiology Center of China Committee for Microbial Culture Collection (CGMCC for short) on February 22, 2012, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences Institute of Microbiology, postal code 100101), the preservation number is CGMCC No.5786. Bacillus subtilis (Bacillus subtilis) B201CGMCC No.5786 is referred to as Bacillus subtilis B201. Bacillus subtilis B201 has a good biocontrol effect, and the control effect on cucumber seedling stage cucumber wilt in the greenhouse reaches 58.66%, but when it is confronted with the cucumber wilt pathogen on the PDA plate, no obvious inhibition zone is formed.

The present invention has the following innovations: (1) use MS nutrient solution as the culture substrate of cucumber seedlings, the composition and pH of the nutrient solution can be artificially regulated, so that the influence of some unknown factors brought by soil cultivation can be eliminated, It can also provide the necessary growth elements for plant growth; (2) adopt the bacterium-limited vaccine system, that is, all the operation steps are carried out under sterile conditions, so that it can basically ensure that in the process of disease occurrence, the main pathogenic bacteria inoculated and colonized A certain reaction between the treated endophytes eliminates the interference of miscellaneous bacteria; (3) The operation is simple, the materials are easy to obtain, and it is easy to operate.

Description of drawings

FIG. 1 is a phylogenetic tree of strain B201 constructed in Example 4.

Fig. 2 is the Biolog system identification result of bacterial strain B201 in embodiment 4.

Fig. 3 is the result of antagonism experiment between bacterial strain B201 and Fusarium oxysporum cucumber-specific type (pathogen) in Example 5 on a PDA plate.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Fusarium oxysporum f.sp.cucumerinum: Publicly available from China Agricultural University; Reference: Mandeel, Q., and Baker, R. Mechanisms involved in biological control of Fusarium of cucumber with strains of nonpathogenic Fusarium oxysporum. Phytopathology. 1991. 81: 462-469.

Embodiment 1, the preparation of culture medium

1. MS solid medium

MS solid medium (pH6.2) was obtained by adding 0.45 g of agar per 100 mL of MS liquid medium.

MS liquid medium is obtained by dissolving the solute and distilling the volume to 1 L with water; see Table 1 for the solute and its concentration.

The use concentration of each solute in table 1MS liquid culture medium

Two, the preparation of PDA liquid culture medium

Get 200g potato and cut into small pieces and add water and boil until soft and not rotten, filter and collect the filtrate with four layers of gauze, then add 20 grams of glucose, and finally settle to 1000 milliliters with distilled water to obtain PDA liquid medium (natural pH).

3. PDA solid medium

Add 15-20 grams of agar per liter of PDA liquid medium to form a PDA solid medium.

Embodiment 2, the acquisition of bacteria to be tested

In June 2011, the soil was taken from the cucumber experimental field of China Agricultural University, and 400 purely cultured strains were obtained by gradient plate dilution method. Among them, 51 strains were randomly selected as the bacteria to be tested, and the screening in Example 3 was carried out respectively.

Embodiment 3, screening of cucumber fusarium wilt biocontrol bacteria

The variety name of cucumber seeds is Xintaimici, which was purchased from Xintaimici Cucumber Farm in Shandong Province.

1. Preparation of pathogen spore suspension

1. Use a puncher with a diameter of 0.5cm to punch the Cucumber specialization of Fusarium oxysporum cultured on the PDA solid medium into a cake, inoculate 5-8 pieces of the cake into 250mL PDA liquid medium, and keep at 26°C Shake culture for 4 days, filter with three layers of gauze under aseptic conditions, and collect the filtrate (ie spore liquid, relatively uniform sickle-shaped megaspores can be observed by microscopy).

2, the filtrate of step 1 is centrifuged (5000rpm centrifugal 5 minutes), collects precipitation (being spore), washes spore with sterile water, then by microscopic counting of hemocytometer, spore is made into concentration with sterile water and is 2 * 10 ⁸ CFU ml ^-1 of pathogenic bacteria spore suspension mother solution, temporarily stored at 4°C for later use.

2. Screening of biocontrol bacteria for cucumber wilt

The bacteria to be tested were subjected to the following experiment respectively (each bacteria to be tested was repeated three times):

1. Treatment of cucumber seeds

(1) Sterilization

Pick cucumber seeds with uniform size and plumpness, disinfect with 10g/100mL sodium hypochlorite aqueous solution for 10 minutes, and then rinse with sterile water several times until there is no sodium hypochlorite smell.

(2) Soak the cucumber seeds treated in step (1) in sterile water for 8 hours, place them in a sterile petri dish, keep them moist with filter paper, cover them with two layers of gauze, and culture them in a dark incubator at 28°C until the seeds turn white.

2. Soak the lubai seeds in the suspension of bacteria to be tested (for the bacteria that produce spores, use 1×10 ⁸ CFU ml ^-1 spore suspension) for 1 hour, and then culture them in the dark in a 28°C incubator until the radicle The length is 1.5-2.0cm.

3. Inoculate the cucumber seedlings treated in step 2 with the pathogen spore suspension by dipping the radicle (dilute the pathogen spore suspension mother liquor prepared in step 1 with sterile water to obtain a pathogen spore suspension of 1×10 ⁶ CFU ml ⁻¹ ).

4. Transfer the cucumber seedlings treated in step 3 to culture bottles (the diameter of the culture bottle is 9cm × 9cm, containing aseptic MS solid medium, each culture bottle is inoculated with 13 cucumber seedlings), and carry out in the tissue culture room Cultivate (cultivation conditions are: dark culture for 8 hours, light culture for 16 hours; temperature 23°C-25°C; humidity 50%-60%), and observe the phenotype after 5 days.

Set up a parallel experiment without step 2 as a positive control. Parallel experiments without steps 2 and 3 were set up as negative controls.

There are 5 grades for the severity of cucumber wilt disease at the seedling stage, specifically as follows: 0, the roots, stems and leaves of the plants grow normally; 1, less than one quarter of the roots turn yellow, and the plants are slightly dwarfed; 2, one quarter to two One of the rhizomes turns yellow, and the lower veins fade; 3. One-half to three-quarters of the rhizomes turn yellow, and the stem base is longitudinally split; 4. More than three-quarters of the rhizomes turn yellow, or the plant directly withers and dies.

Remove one plant with the most serious disease and one plant with the lightest disease, and the disease index of the remaining plants is calculated according to the following formula: disease index = ∑ (number of diseased plants at all levels × value of the disease level) / (total number of plants under investigation × highest level value) × 100.

Control effect (%) = (condition index of the positive control group - condition index of the experimental group) / condition index of the positive control group × 100.

The results of the 51 tested bacteria are shown in Table 2.

Table 2 Screening results of cucumber wilt biocontrol bacteria (data is the average value of three repetitions)

Different letters indicate significant differences.

The bacteria with the best control effect was strain B201.

Embodiment 4, identification of bacterial strain B201

1. Morphological identification and physiological and biochemical identification

Morphological identification: including cell shape, motility, whether spores are formed, etc.

Physiological and biochemical identification: including methyl red test, catalase activity, Voges-Proskauer test, anaerobic culture, pH, temperature and sodium salt depth range suitable for strain growth, etc., the operation method refers to (Roberts et al., 1994). Except for the temperature resistance experiment, the cultivation temperature of other experiments was 30°C.

The results of morphological identification and physiological and biochemical identification of strain B201 are shown in Table 3. The results showed that strain B201 was a Gram-positive, spore-forming, motile rod-shaped bacterium, positive for V-P assay, aerobic and able to utilize citric acid, and capable of rapidly forming a robust biofilm.

Table 3 Morphological identification results and physiological and biochemical identification results of bacterial strain B201

2. Cell total fatty acid analysis

Utilize Sherlock Microbial Identification System (MIS) TSBA6.0 system, refer to the standard operation method to work (Microbial ID, Newark, DE, USA). Firstly, the cells cultured overnight at 30°C on TSBA medium were collected, and the total fatty acids of strain B201 cells were extracted by gas chromatography (model 6850, Agilent). The Agilent capillary column model was 19091B-102E (25m×200μm×0.33μm). The injection temperature was 170°C, the final temperature was 260°C, and the heating rate was 5°C/min. Then rapidly rise to 310°C at a rate of 40°C/min. The carrier gas is nitrogen, and the flow rate is 0.469 mL/min. Data analysis and strain classification were carried out through the MIS database.

According to the results of cell fatty acid analysis, comparison was made in the TSBA66.10 and CLIN66.10 information bases for identifying bacteria based on bacterial cell fatty acid composition. The main fatty acids of strain B201 are 13-methyltetradecanoic acid (iso-15:0), 12-methyltetradecanoic acid (anteiso-15:0), methyl palmitate (iso-17:0) ,, identified as Bacillus subtilis by computer.

3. Molecular biological identification

PCR amplification of 16S rRNA: Bacterial universal primers 63F and 1378R were used (Knapp et al., 2009). Reaction conditions: 95°C for 3min; 95°C for 1min, 55°C for 30s, 72°C for 2min, 30 cycles; 72°C for 10min.

PCR amplification of gyrA gene: the primers p-gyrA-f and p-gyrA-r (Roberts et al., 1994) reported in the literature were used for amplification. Reaction conditions: 95°C for 3min; 95°C for 1min, 60°C for 30s, 72°C for 1min, 30 cycles; 72°C for 10min.

The amplified fragments were connected to the pMD19-T vector (Takara Co. Ltd.), and positive clones were picked and sent to Beijing Sanbo Yuanzhi Biotechnology Company for sequencing. The 16S rDNA and gyrA gene sequences were amplified and sequenced using the extracted genomic DNA, and the lengths of the gene sequence fragments were 1.37kb and 1.025kb, respectively. The 16S rDNA is shown in sequence 1 of the sequence listing. The sequence of the gyrA gene is shown in sequence 2 of the sequence listing.

The 16S rDNA sequence was analyzed by BLAST, and it was found that the strains with the best homology were Bacillus subtilis subsp.subtilis JCM 10629 (99%) and Bacillus licheniformis strain B425 (99%), so the strain B201 could not be identified only based on the 16S rDNA gene. A type of bacillus.

The gyrA gene was compared and analyzed in GenBank, and the closest homologous strain was Bacillus subtilis subsp.subtilis str.168 (99%). The similarity with other published Bacillus subtilis is more than 94%. The Neighbor-joining method was used to construct a phylogenetic tree (see Figure 1) with the Mega 4.1 software package, which completely displayed the evolutionary relationship among the subspecies of Bacillus subtilis. The strain B201 can be accurately identified as Bacillus subtilis by using the gyrA gene, and the genetic relationship between B201 and known Bacillus species can be obtained.

4. Identification of Biolog system

Wet a sterile cotton swab with a single colony of strain B201 in the inoculum in the Biolog kit, tilt the inoculum tube and rotate the swab along the inner wall to disperse the bacteria evenly. Adjust the turbidity value so that it is within 5% of the corresponding standard turbidity value. The turbidity-adjusted bacterial solution was inoculated into the Biolog identification plate with a 100 μL pipette gun, and cultured at 26°C for 24h, 48h and 72h. Turn on the identification system, place the identification plate that has been cultivated on the reader of the strain identification instrument, and the computer reads the data. The results are shown in Figure 2.

3. Preservation of bacterial strain B201

According to the results of cell morphology, physiology and biochemistry, 16S rRNA, gyrA gene sequence analysis, cell total fatty acid analysis and nuclear Biolog system identification, the strain B201 belongs to Bacillus subtilis.

Bacillus subtilis (Bacillus subtilis) B201 was preserved in the General Microbiology Center of China Committee for Microbial Culture Collection (CGMCC for short) on February 22, 2012, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences Institute of Microbiology, postal code 100101), the preservation number is CGMCC No.5786. Bacillus subtilis (Bacillus subtilis) B201CGMCC No.5786 is referred to as Bacillus subtilis B201.

Embodiment 5, confrontation culture of Bacillus subtilis B201 and Fusarium oxysporum cucumber specialized type

One, the preparation of the spore suspension of Bacillus subtilis B201

Beef extract liquid medium (pH7.0-7.2): composed of 3g beef extract, 10g peptone, 5gNaCl and 1000ml water.

Inoculate Bacillus subtilis B201 into beef extract liquid medium, shake and culture at 30°C and 200rpm for 6-7 days, then centrifuge (5000rpm for 5 minutes), collect the precipitate (spores), suspend the spores with sterile water, and obtain the concentration of spores A spore suspension of 1×10 ⁸ CFU ml ^-1 .

2. Confrontation culture between Bacillus subtilis B201 and Fusarium oxysporum specialized in cucumber

On the first day of the experiment, the following two groups of treatments were carried out respectively:

The first group: the PDA bacterium cake (diameter is 5 millimeters * 5 millimeters) of Fusarium oxysporum cucumber specialized type is placed on the center of PDA plate;

The second group: the PDA bacterium cake of Fusarium oxysporum cucumber specialized type (diameter is 5 millimeters * 5 millimeters) is placed on the center of PDA flat plate, then each drop is respectively on both sides of bacterium cake (position apart from bacterium cake 2cm) Add 1 µl of the spore suspension prepared in step 1.

Three replicates were set up for each treatment. Cultured at 26°C for 7 days.

The photos on the 8th day of the experiment are shown in Figure 3. After confrontation culture of strain B201 and Fusarium oxysporum cucumber-specific type, no obvious zone of inhibition was found.

Claims (8)

1. A method for identifying the biocontrol properties of the bacteria to be tested to the cucumber wilt pathogen, including experimental treatment; Described experimental processing comprises the following steps in turn: (1) accelerating the germination of the sterilized cucumber seeds to obtain dew-white seeds; (2) After soaking the lubai seeds obtained in step (1) in the bacteria suspension to be tested, continue to cultivate until the radicle length reaches 1.5-2.0cm; (3) the cucumber seedling that step (2) obtains is inoculated with the cucumber wilt pathogen suspension by dipping in the radicle; (4) Transfer the cucumber seedlings treated in step (3) to MS solid medium for culture, and identify the biocontrol properties of the test bacteria against the pathogenic bacteria of cucumber wilt by investigating the disease index and/or observing the disease phenotype. 2. The method according to claim 1, characterized in that: also include contrast processing in the described method; described contrast processing comprises step (1) described in claim 1, step (3) described in claim 1 and Step (4) described in claim 1; All in the described step (4) of described experiment process and described control process, identify the biocontrol properties of bacteria to be tested to cucumber wilt pathogenic bacteria by investigating disease index; If the The disease index of the experimental treatment is lower than the disease index of the control treatment, and the bacteria to be tested are the biocontrol bacteria of the candidate cucumber wilt pathogen; if the disease index of the experimental treatment is equal to or higher than the The disease index of the control treatment, the test bacteria is the candidate non-biocontrol bacteria of the cucumber wilt pathogen. 3. The method according to claim 1, characterized in that: said method also includes contrast treatment; said contrast treatment comprises said step (1), said step (3) and said step (4) in sequence ; In the described step (4) of the described experimental treatment and the described control treatment, the biocontrol properties of the bacteria to be tested are identified by observing the pathogenesis phenotype to the pathogenic bacteria of cucumber wilt; Type is milder than the morbidity phenotype of plant in described control treatment, and described test bacterium is the biocontrol bacterium of the described cucumber wilt pathogenic bacterium of candidate; The pathogenesis phenotype of the plants in the treatment is consistent, and the bacterium to be tested is the non-biocontrol bacterium of the described cucumber wilt pathogenic bacteria of the candidate; The phenotype of the disease is severe, and the bacteria to be tested are non-biocontrol bacteria of the candidate cucumber wilt pathogen. 4 . The method according to any one of claims 1 to 3 , characterized in that: the cucumber wilt pathogen is Fusarium oxysporum cucumber-specific type. 5. The method according to claim 4, characterized in that: the cucumber fusarium wilt pathogen suspension is a suspension of Fusarium oxysporum cucumber-specific spores with a concentration of 1×10 CFU ml-1. 6. The method according to any one of claims 1 to 5, characterized in that: in the step (4), start counting from the completion of the step (3), investigate the disease index and/or observe the incidence table after 5 days type. 7. the application of the method described in any one of claims 1 to 6 in screening for the biocontrol bacteria of cucumber wilt pathogenic bacteria. 8. The application according to claim 7, characterized in that: the cucumber wilt pathogen is Fusarium oxysporum cucumber-specific type.

Images