CN114196585A - Burkholderia for preventing and treating tomato bacterial wilt and application thereof - Google Patents
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Abstract
The invention discloses burkholderia for preventing and treating tomato bacterial wilt and application thereof, belonging to the technical field of agricultural microorganisms. Burkholderia (Burkhorderia anthina) MMYH13 of the present invention was deposited under the accession number: GDMCC No: 61895. the strain is easy to culture, can grow well on LB, NA and TTC plates, has short rod-shaped cells, and is easy to generate more extracellular polysaccharide when cultured on the TTC plate; the activity is high, the growth of the ralstonia solanacearum can be effectively inhibited, the tomato bacterial wilt disease incidence rate can be effectively reduced by the fermentation liquid, and the ralstonia solanacearum is a potential biocontrol bacterium.
Description
Technical Field
The invention relates to the technical field of agricultural microorganisms, and particularly relates to burkholderia for preventing and treating tomato bacterial wilt and application thereof.
Background
Tomato bacterial wilt (Tomato bacterial wilt) is a worldwide destructive soil-borne vascular bundle disease caused by infection with Laurella sp. The disease is generated in east China, south China, China and southwest China, wherein the disease is particularly serious in the south China, and the generation of the disease poses a great threat to agricultural production. Bacterial wilt can be spread by soil or water, pathogenic ralstonia solanacearum can survive in soil for more than years when no host plant exists, and how to efficiently realize the control of soil-borne diseases is extremely difficult.
The Ralstonia solanacearum is a gram-negative bacterium belonging to burkholderia (Burkholderiales) burkholderia (Ralstonia) in beta-proteobacteria, is a composite species (RSSC), has obvious physiological differentiation and genetic diversity, has a very wide host range, and can infect more than 200 plants of more than 50 families, including tomatoes, tobaccos, eggplants, potatoes, bananas, peppers and the like. The southern China is always in high temperature and rainy days, and plants grow and propagate all the year round, which creates very favorable conditions for the occurrence and prevalence of bacterial wilt. The latest investigation and monitoring results show that at least 35 plants in Guangdong area have bacterial wilt at present, the generation of diseases is on the rising trend year by year, huge economic loss is caused to agricultural production, and how to realize green and efficient prevention and control of the bacterial wilt of crops is a major problem to be solved urgently at present.
The traditional methods for preventing and treating soil-borne diseases comprise physical prevention and treatment (such as insolation) and chemical prevention and treatment mainly based on medicament fumigation, the methods can effectively reduce the number of pathogenic bacteria at the rhizosphere in a short time and reduce the occurrence of diseases, but due to the non-pertinence of the physical prevention and treatment methods, the number and the variety of beneficial flora at the rhizosphere are reduced while the pathogenic bacteria are reduced, the microbial community structure and the microecological balance of the rhizosphere of plants are destroyed, and the methods are not beneficial to the sustainable development and the disease prevention and control of the rhizosphere and the soil of the plants in the long run. The rhizosphere microbiome is known as the "second genome" of crops and plays a very important role in maintaining crop health. By utilizing biological control based on the rhizosphere beneficial bacteria, the rhizosphere of the plant can be effectively protected from being infected by pathogenic bacteria, the stability of a microbial community of the rhizosphere of the plant can be effectively maintained, and the environment is not polluted. Therefore, the use of rhizosphere beneficial microorganisms or gene products thereof for preventing and treating plant diseases gradually becomes an important direction for research hotspots and application research. In 2021, the Chinese science will also "how to efficiently utilize the germplasm resources of agricultural microorganisms? "lists one of 10 engineering technical problems. In addition, at present, China has fewer biocontrol bacteria resources as patent protection, and the patent protection of separated biocontrol bacteria has important significance for realizing disease control based on the current situation.
Disclosure of Invention
The invention aims to solve the problem of difficult prevention and control of the current soil-borne diseases, overcome the defects and shortcomings of the existing prevention and control technology, and provide Burkholderia (Burkhorderia anthina) MMYH13 capable of preventing and controlling tomato bacterial wilt, which is preserved in the microbial strain preservation center of Guangdong province in 2021, 27 th, with the address of Michelia Tokyo No. 100 Dazhou No. 59 building 5 of Guangzhou province, Guangzhou City, Guangzhou, and the postal code is as follows: 510070, having a deposit number of: GDMCC No: 61895.
the 16S rDNA sequence of the strain MMYH13 of the invention has 99.65% homology with Burkhorderia anthina (Burkholderia), and the morphological characteristics of the strain are most similar to Burkhorderia anthina (Burkholderia). The gram staining result is negative, the strain grows well in LB, NA, TTC and other culture media, the strain cell is in a short rod shape and has flagella, and the bacterial colony is easy to produce more extracellular polysaccharide when cultured on a TTC plate. The strain MMYH13 was classified as Burkholderia (Burkhorderia anthina) according to the results of 16S rDNA sequence and morphological characteristics.
The second purpose of the invention is to provide the application of Burkholderia MMYH13 in the control of bacterial wilt.
Preferably, the bacterial liquid of Burkholderia is applied to the soil where the crops are planted.
Preferably, the crop is a crop not infected by ralstonia solanacearum, or a crop already infected by ralstonia solanacearum.
Preferably, the crops are crops susceptible to bacterial wilt; more preferably, the crop is tomato.
The third object of the present invention is to provide a biocontrol microbial inoculum containing Burkholderia plantarii MMYH13 as set forth in claim 1.
The fourth purpose of the invention is to provide a method for culturing the Burkholderia plantarii MMYH13, which is to inoculate the Burkholderia plantarii MMYH13 into LB, NA, NB or TTC culture medium.
Preferably, the Burkholderia plantarii MMYH13 is inoculated in NB medium under the following culture conditions: culturing at 28-30 ℃ and 130-200rpm for 1-2 d.
Compared with the current biological control situation of tomato bacterial wilt, the invention has the following advantages: the invention provides a burkholderia with good antagonistic action on ralstonia solanacearum, the bacterial strain is extremely easy to culture, the growth of the ralstonia solanacearum can be effectively inhibited, the fermentation liquid of the bacterial strain can effectively reduce the incidence of the tomato bacterial wilt, and a new method way is provided for preventing and treating the tomato bacterial wilt.
The Burkhorderia anthina MMYH13 is preserved in the microbial strain preservation center of Guangdong province in 2021, 8 and 27 days, and is addressed to No. 59 building and No. 5 building of Jie Tou Dazhou No. 100 Yao of Virginia city of Guangdong province, and is coded by the following postal code: 510070, having a deposit number of: GDMCC No: 61895.
drawings
FIG. 1 is a photograph showing the antagonistic effect of the strain MMYH13 of the present invention against Ralstonia solanacearum on a plate; among them, NB (nutrient broth) was a negative control, and Gm (gentamicin at 25. mu.g/mL) was a positive control.
FIG. 2 is a transmission electron microscope image of the strain MMYH13 of the invention, with a scale of 5 μm.
FIG. 3 is a diagram showing the results of statistics of the biocontrol effect of the strain MMYH13 of the invention on tomato bacterial wilt.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The present invention will be further described with reference to the following examples, which are all conventional test methods unless otherwise specified, and the test reagents and consumables described in the following examples, which are all available from conventional biochemical reagents companies, unless otherwise specified.
EXAMPLE 1 isolation and purification of the Strain
1. Isolation medium
TTC agar medium (TTC solid medium): 10.0g of bacteriological peptone, 1.0g of acid hydrolyzed casein, 5.0g of glucose, 0.5g of 2,3, 5-triphenyltetrazolium chloride and 15.0g of agar powder, and dissolving the components with distilled water to a constant volume of 1L and a pH value of 7.2. Sterilizing with 115 deg.C steam for 20 min.
2. Experimental procedure
(1) Collecting samples: selecting a field with serious tomato bacterial wilt disease in the famous region of Guangdong province, removing surface soil, carefully pulling out tomato plants, shaking off and collecting rhizosphere soil, putting the tomato plants into a sampling bag, and bringing the sampling bag back to a laboratory for natural air drying at room temperature.
(2) Preparing a soil gradient diluent, weighing 10g of soil, adding the soil into a sterilized triangular flask which is added with glass beads and 100mL of sterile water in advance, and performing shake culture on a shaker at 30 ℃ and 200rpm for 30 min. Standing for 5min, adding 1mL supernatant into test tube containing 9mL sterilized water, and sequentially performing gradient dilution to 10-2-10-7g/mL of the soil suspension is ready for use.
(3) And (3) bacteria separation: 100 μ L of diluted soil bacteria suspension (10) was taken separately-2-10-7g/mL) was added to a TTC plateThe medium is uniformly coated by a disposable plastic coating rod, each concentration is set to be three times, and after the coating is finished, the flat plate is placed in a 30 ℃ constant temperature incubator to be cultured in an inverted mode. Observing the growth condition of colonies on the plate every other day, selecting single colonies with inconsistent colony morphology, streaking on a TTC plate, and purifying the strains.
(4) And (3) separating results: a total of 184 bacteria were isolated.
Example 2 plate antagonism of isolated strains against Ralstonia solanacearum
1. Culture medium
NA solid medium: prepared by using synthetic culture medium nutrient broth.
2. Experimental procedure
2.1 Flat antagonism test-preliminary screening
2.1.1 activation of the Strain
The ralstonia solanacearum GMI1000 and 184 strains of bacteria isolated in example 1 were transferred to TTC solid plates and NA solid plates, respectively, and cultured at 30 ℃ for 1-2 days for further use.
2.1.2 preparation of plates containing bacteria
Selecting single colony of Ralstonia solanacearum GMI1000 strain, inoculating into 2-5mLNB culture medium (NA culture medium without agar), culturing at 30 deg.C and 200rpm for 24 hr to obtain about 1010And (3) sucking 1mL of bacterial suspension into a 100mLNA culture medium cooled to about 45 ℃, and pouring the bacterial suspension into a plate after the ralstonia solanacearum is shaken up.
2.1.2 preliminary screening for antagonistic bacteria
Respectively marking and activating the strains separated and purified in the early stage on an NA plate, picking the activated strains by using a sterilized bamboo stick, carrying out point grafting on the activated strains on a strain-containing plate, carrying out point grafting on 8 test strains on each plate, culturing for 48h at 30 ℃, observing whether a bacteriostatic circle exists or not, measuring the size of the bacteriostatic circle by using a spiral micrometer, and storing the strains with the bacteriostatic circle for re-screening.
2.2 Flat antagonism test-rescreening
2.2.1 Strain activation
And (3) activating the strains with antagonistic effect after primary screening as in 2.1.1.
2.2.2 liquid fermentation of isolated strains
And (3) selecting a single colony of the antagonistic strain obtained by primary screening, inoculating the single colony into 100mL of NB culture medium (placed in a 250mL triangular flask), and culturing at 28 ℃ and 130rpm for 48h to obtain antagonistic bacterium fermentation liquor.
2.2.3 rescreening with Flat coating
Uniformly coating 100 mu L of bacterial suspension of the ralstonia solanacearum GMI1000 strain on a NA solid culture medium, punching a hole on a flat plate by using a yellow gun head, then adding 100 mu L of antagonistic bacteria fermentation liquid into the hole, culturing for 1-3d at 28 ℃, observing whether a bacteriostatic ring appears, and opening a cover in a super clean bench to photograph and record a result.
2.2.4 pour method rescreening
Preparing 100 mL/bottle of NA solid culture medium, cooling the culture medium to about 45 ℃, adding 1mL of ralstonia solanacearum suspension according to the proportion of 1%, manufacturing a double-layer flat plate with an Oxford cup, taking out the Oxford cup after the flat plate is dried, adding 100 mu L of antagonistic bacteria fermentation liquid into a hole, culturing at 28 ℃ for 1-3d, observing whether a bacteriostatic zone appears, and opening a cover in an ultraclean bench to photograph and record the result.
3. Results of the experiment
28 strains of bacteria with antagonistic effect are obtained through a primary screening test, 6 strains of bacteria with obvious antagonistic effect are obtained through a flat plate secondary screening and a pouring method secondary screening, and the MMYH13 strain is one strain with excellent antagonistic effect.
FIG. 1 is a graph showing the antagonistic effect of strain MMYH13 on Ralstonia solanacearum GMI1000 on a plate, and it can be seen that a significant inhibition zone appears, which indicates that strain MMYH13 has a significant antagonistic effect on Ralstonia solanacearum.
Example 3: identification of isolated strains
The isolated strains were identified with reference to "Manual of identification of common bacteria systems" (scientific Press, eds. of Dongxu Zhu Shuizhiu). The test method is as follows: extracting DNA of the strain MMYH13 by a water boiling method, amplifying a 16S rDNA gene sequence of MMYH13 by using 16S rDNA specific primers 27F and 1492R of bacteria and Taq enzyme, carrying out electrophoresis analysis on an amplification product to generate a band about 1500b, cutting gel, recovering the band, sending the band to Sanger Biotech limited Suzhou for sanger sequencing, splicing the sequence obtained by sequencing by seqman software to obtain a 16S rDNA sequence, wherein the nucleotide sequence is shown as SEQ ID NO.1 and submitting the 16S rDNA sequence to an EzBioClode website (https:// www.ezbiocloud.net /) for identification. The identification result is as follows: the 16S rDNA sequence of the strain MMYH13 has 99.65% homology with Burkhorderia anthina (Burkholderia), and the morphological characteristics of the strain are most similar to Burkhorderia anthina (Burkholderia). Gram staining results are negative, the strain can grow well in LB, NA, TTC and other culture media, the strain cells are short rod-shaped and have flagella (figure 2), and the bacterial colony is easy to generate more extracellular polysaccharide when cultured on a TTC plate. The strain MMYH13 was classified as Burkholderia (Burkhorderia anthina) according to the results of 16S rDNA sequence and morphological characteristics.
According to the identification result, the strain MMYH13 is named Burkhorderia anthina MMYH13, which is preserved in Guangdong province microorganism strain preservation center at 27.8.2021, and the address is No. 59 building 5 of Zhan Zhou Yao Jun district of Guangzhou city, Guangdong province, with the postal code: 510070, having a deposit number of: GDMCC No: 61895.
example 4: biocontrol effect test of strain MMYH13 on tomato bacterial wilt
The strain MMYH13 has obvious antagonistic effect on ralstonia solanacearum on a flat plate, and in order to verify the biocontrol effect on the tomato bacterial wilt, the biocontrol effect of the fermentation liquor of the strain MMYH13 on the tomato bacterial wilt is evaluated through a greenhouse pot experiment.
Test procedure
1. Seedling growing method for tomato variety' Zhongshu four
1.1 seed pregermination and sowing
Soaking tomato seeds in water bath at 37 ℃ for 2h, soaking in 75% alcohol for 30s, washing with deionized water twice, sterilizing the surfaces with 5% NaClO solution for 15min, and then washing with deionized water. Uniformly placing in a culture dish paved with two layers of gauze, adding appropriate amount of distilled water, keeping the seeds and the gauze moist, and placing in a constant temperature incubator at 28 ℃ for germination acceleration for 2 d. After the seeds germinate and grow about 1-2mm radicles, the seeds are sowed in a seedling tray filled with perlite, 1 seed is sowed in each hole, and the seeds are placed in a greenhouse for culturing.
1.2 transplanting of seedlings
The matrix soil is mixed with water to be wet, half of the mixed matrix soil is filled in a flowerpot (the diameter is 9cm), tomato seedlings are carefully taken out of a seedling tray and put in the flowerpot, half of the matrix soil is added, the plants are stabilized by lightly pressing with fingers, watering is carried out, the water content is controlled to be about 30 percent, and the tomato seedlings are cultured in a greenhouse for about 28 days for later use.
2. Ralstonia solanacearum and inoculated antibacterial MMYH13 activation and fermentation liquor preparation
The method of 2.1.1 of example 1 was followed to activate Ralstonia solanacearum GMI1000 and antagonistic bacterium MMYH13, then single colonies were picked up and inoculated into Erlenmeyer flasks containing TM broth (TTC broth without TTC and agar) and NB broth (NA broth without agar) respectively, and shake-cultured at 200rpm on a 28 ℃ constant temperature shaker for 2d (OD)600＝2.5)。
3. Ralstonia solanacearum and inoculating antibacterial fermentation liquor to inoculate tomato plants
The test is provided with 7 treatments, namely (1) blank control without adding any bacteria and sterile water, (2) negative control of TM liquid culture medium, (3) positive control only inoculating ralstonia solanacearum fermentation liquor, (4) control only inoculating antagonistic bacteria MMYH13 fermentation liquor, (5) antagonistic bacteria MMYH13 and ralstonia solanacearum in a volume ratio of 1:1, treatment of mixed inoculation; (6) antagonistic bacteria MMYH13 and ralstonia solanacearum in a volume ratio of 3:1, mixed inoculation treatment, and (7) antagonistic bacteria MMYH13 and ralstonia solanacearum in a volume ratio of 5:1 treatment of mixed inoculations. Five replicates of 10 seedlings were inoculated per treatment. The inoculation amount of the bacterial liquid is 5mL per pot, the inoculation is carried out by adopting a method of root irrigation by the bacterial liquid, the inoculated bacterial liquid is placed in a glass greenhouse for culture for 14d (the temperature is 28 ℃ and the humidity is 40 percent) after the inoculation, and watering is carried out for 1 time every day on the next day after the inoculation. Disease severity was observed and recorded daily for tomato seedlings as described in the paper published by Kempe and the incidence of each treatment was calculated according to the following formula.
The disease rate of the plants is (number of diseased plants/total number of plants) × 100%
4. Test results
As shown in fig. 3, after the inoculation treatment, the morbidity of the tomatoes treated by the sterile water blank control, the TM liquid medium negative control and the inoculation of the antagonistic bacteria alone was 0; tomatoes inoculated with ralstonia solanacearum GMI1000 fermentation liquor are all withered and dead, and the morbidity is 100%; the average value of the morbidity of tomatoes in treatment groups (1:1, 3:1 and 5:1) inoculated with the antagonistic bacteria MMYH13 fermentation liquor and the ralstonia solanacearum GMI1000 fermentation liquor is 52%, 30% and 18%, so that the treatment of the antagonistic bacteria MMYH13 fermentation liquor can effectively reduce the morbidity of the tomato bacterial wilt, and the tomato bacterial wilt is a potential biocontrol bacterium.
Claims (7)
1. Burkholderia (Burkhorderia anthina) MMYH13, which is deposited under the following accession number: GDMCC No: 61895.
2. the use of Burkholderia plantarii MMYH13 of claim 1 for controlling bacterial wilt.
3. The use of claim 2, wherein the burkholderia MMYH13 is used for preventing and treating tomato bacterial wilt infection.
4. The use of claim 2 or 3, wherein the bacterial liquid of Burkholderia mmYH13 is applied to the soil where crops are planted.
5. A biocontrol bacterial agent comprising Burkholderia plantarii MMYH13 according to claim 1.
6. A method for culturing Burkholderia plantarii MMYH13 as claimed in claim 1, which is characterized in that the Burkholderia plantarii MMYH13 is inoculated into LB, NA, NB or TTC medium.
7. The method of claim 6, wherein the Burkholderia plantarii MMYH13 is inoculated into NB medium under the following conditions: culturing at 28-30 ℃ and 130-200rpm for 1-2 d.
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| CN116218740A (en) * | 2023-04-03 | 2023-06-06 | 上海交通大学 | Burkholderia cepacia and application thereof |
| CN116396889A (en) * | 2023-01-13 | 2023-07-07 | 南京农业大学 | Iron-producing carrier golden fungus for preventing and controlling soil-borne bacterial wilt and promoting tomato growth and application thereof |
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| MX2013011311A (en) * | 2013-09-19 | 2015-03-19 | Univ Politécnica Del Estado De Morelos | Biocide bacterial formulation against bacterial wilt caused by ralstonia solanacearum in tomato crops. |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116396889A (en) * | 2023-01-13 | 2023-07-07 | 南京农业大学 | Iron-producing carrier golden fungus for preventing and controlling soil-borne bacterial wilt and promoting tomato growth and application thereof |
| CN116396889B (en) * | 2023-01-13 | 2024-01-09 | 南京农业大学 | Iron-producing carrier golden fungus for preventing and controlling soil-borne bacterial wilt and promoting tomato growth and application thereof |
| CN116218740A (en) * | 2023-04-03 | 2023-06-06 | 上海交通大学 | Burkholderia cepacia and application thereof |
| CN116218740B (en) * | 2023-04-03 | 2023-09-01 | 上海交通大学 | Burkholderia cepacia and application thereof |
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| CN114196585B (en) | 2023-06-30 |
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