CN114196585B - Burkholderia for preventing and treating tomato bacterial wilt and application thereof - Google Patents
Burkholderia for preventing and treating tomato bacterial wilt and application thereof Download PDFInfo
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Abstract
The invention discloses Burkholderia for preventing and treating tomato bacterial wilt and application thereof, and belongs to the technical field of agricultural microorganisms. The Burkholderia (Burkholderia anthina) MMYH13 of the invention has the deposit number of: GDMCC No:61895. the strain is easy to culture, the strain can grow well on a LB, NA, TTC flat plate, strain cells are in a short rod shape, and more extracellular polysaccharide is easy to generate in colony when the strain is cultured on a TTC flat plate; the bacterial wilt has high activity, can effectively inhibit the growth of bacterial wilt, and the fermentation liquor can effectively reduce the incidence rate of the bacterial wilt of tomatoes to the bacterial wilt of tomatoes, thereby being a potential biocontrol bacterium.
Description
Technical Field
The invention relates to the technical field of agricultural microorganisms, in particular to Burkholderia for preventing and treating tomato bacterial wilt and application thereof.
Background
Tomato bacterial wilt (Tomato bacteria wilt) is a destructive soil-borne vascular bundle disease in worldwide distribution caused by infection with the strain Laurella (Ralstonia solanacearum, also known as Rhizoctonia solani). The disease is serious in the south China, the middle China and the southwest, and the occurrence of the disease forms a great threat to agricultural production. The bacterial wilt can be transmitted by soil or water, and pathogenic bacterial wilt can survive for more than a few years in the soil without host plants, so that how to efficiently realize the prevention and control of soil-borne diseases is extremely difficult.
Bacterial wilt is a gram-negative bacterium belonging to the genus Burkholderia (Ralstonia) of the family Burkholderiaceae (Burkholderiales) belonging to the class beta-Proteus, is a complex species (RSSC), has obvious physiological differentiation and genetic diversity, has a very wide host range, and can infect more than 50 plants of the family 200, including tomatoes, tobacco, eggplants, potatoes, bananas, peppers and the like. The south China is high temperature and rainy throughout the year, and the plants grow and reproduce throughout the year, which creates a very favorable condition for the occurrence of bacterial wilt. The latest investigation and monitoring results show that at present, at least 35 plants in Guangdong area have bacterial wilt, and the occurrence of diseases is in an ascending trend year by year, so that huge economic losses are caused for 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 at present.
The traditional methods for preventing and treating soil-borne diseases are physical prevention (such as insolation) and chemical prevention based on medicament fumigation, the methods can effectively reduce the quantity of rhizosphere pathogenic bacteria and reduce the occurrence of diseases in a short period, but due to the non-pertinence of the physical prevention and treatment methods, the quantity and the types of rhizosphere beneficial bacteria are reduced while the pathogenic bacteria are reduced, the plant rhizosphere microbial community structure and the microecological balance are damaged, and the sustainable development of plant rhizosphere and soil and disease prevention and control are not facilitated in the long term. Rhizosphere microbiome is known as the "second genome" of crops and plays a very important role in maintaining crop health. The biological control based on the rhizosphere beneficial bacteria can effectively protect the plant rhizosphere from being infected by pathogenic bacteria, can effectively maintain the stability of plant rhizosphere microbial communities, and has no pollution to the environment. Therefore, the prevention and treatment of plant diseases by utilizing rhizosphere beneficial microorganisms or gene products thereof is becoming an important direction of research hotspots and application research. The scientific agreement in 2021 will also "how to efficiently utilize agricultural microbial germplasm resources? "is one of 10 engineering technical problems. In addition, the biocontrol bacteria used as patent protection in China has fewer resources at present, and the patent protection on the separated biocontrol bacteria has important significance for realizing disease prevention and control based on the current situation.
Disclosure of Invention
The primary aim of the invention is to solve the problem of difficult control of the existing soil-borne diseases, overcome the defects and shortcomings of the existing control technology, and provide a Burkholderia (Burkholderia anthina) MMYH13 strain capable of controlling tomato bacterial wilt, which is preserved in the microorganism strain preservation center of Guangdong province for 8 months and 27 days in 2021, and has the address of building 5 of No. 59 of the first university of China, the Va district of Guangzhou, guangdong province, and the post code is: 510070, which has the accession number: GDMCC No:61895.
the 16S rDNA sequence of the strain MMYH13 has homology of 99.65% with Burkholderia anthina (Burkholderia), and has morphological characteristics most similar to Burkholderia anthina (Burkholderia). Gram staining results are negative, the strain grows well in a culture medium such as LB, NA, TTC, bacterial strain cells are short rods and have flagella, and colonies are easy to generate more extracellular polysaccharide when cultured on a TTC plate. Based on the 16S rDNA sequence and morphological feature results, the strain MMYH13 was classified as Burkholderia (Burkholderia anthina).
The second object of the invention is to provide the application of Burkholderia MMYH13 in preventing and treating bacterial wilt.
Preferably, the bacterial liquid of Burkholderia is applied to the soil where crops are planted.
Preferably, the crop is a crop not infected by bacterial wilt or a crop infected by bacterial wilt.
Preferably, the crop is a bacterial wilt susceptible crop; more preferably, the crop is tomato.
A third object of the present invention is to provide a biocontrol microbial agent comprising burkholderia MMYH13 according to claim 1.
It is a fourth object of the present invention to provide a method for culturing said Burkholderia MMYH13 by inoculating it in LB, NA, NB or TTC medium.
Preferably, the burkholderia MMYH13 is inoculated in NB medium under the following culture conditions: culturing at 28-30deg.C and 130-200rpm for 1-2d.
Compared with the current situation of biological control of tomato bacterial wilt, the invention has the following advantages: the invention provides the burkholderia with good antagonism to the bacterial wilt, the bacterial wilt is easy to culture, the bacterial wilt growth can be effectively inhibited, the incidence of tomato bacterial wilt can be effectively reduced by the fermentation liquor, and a new method approach is provided for preventing and treating the tomato bacterial wilt.
The Burkholderia anthina MMYH of the invention is preserved in the microorganism strain collection center of Guangdong province at 8 months and 27 days of 2021, and has the address of building 5 of No. 59 of Xianzhonglu 100 of Xiuzhou district of Guangzhou province, and the post code is: 510070, which has the accession number: GDMCC No:61895.
drawings
FIG. 1 is a graph showing the antagonistic effect of the strain MMYH13 of the present invention on bacterial wilt on a plate; of these, NB (nutrient broth) was the negative control and Gm (25. Mu.g/mL gentamicin) was the positive control.
FIG. 2 is a view of the MMYH13 cell of the strain of the present invention by transmission electron microscopy on a scale of 5. Mu.m.
Fig. 3 is a graph showing the statistical result 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 invention and are not intended to be limiting thereof.
The present invention will be further described with reference to examples, in which the test methods are conventional test methods unless otherwise specified, and the test reagents and consumables described in the examples are from conventional Biochemical reagent company unless otherwise specified.
EXAMPLE 1 isolation and purification of strains
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 are dissolved in distilled water to a constant volume of 1L and a pH of 7.2. Sterilizing with steam at 115deg.C for 20min.
2. Experimental procedure
(1) Sample collection: selecting a field with serious bacterial wilt disease in the Maoque area of Guangdong province, removing surface soil, carefully pulling out tomato plants, shaking off and collecting rhizosphere soil, filling the soil into a sampling bag, and taking 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 shake culturing for 30min on a shaking table at 30 ℃ at 200 rpm. Standing for 5min, collecting 1mL supernatant, adding into test tube containing 9mL sterilized water, and sequentially performing gradient dilution to 10 -2 -10 -7 g/mL of the soil suspension was ready for use.
(3) Bacterial isolation: 100. Mu.L of diluted Agrobacterium suspension (10) -2 -10 -7 g/mL) was added to TTC plates and spread evenly with a disposable plastic spreading bar, three replicates were set for each concentration, and after spreading was completed the plates were placed upside down in a 30℃incubator. And observing the growth condition of colonies on the flat plate every other day, picking single colonies with inconsistent colony morphology, streaking on the TTC flat plate, and purifying the strain.
(4) Separation results: a total of 184 bacteria were isolated.
Example 2 plate antagonism test of isolated strains against Ralstonia
1. Culture medium
NA solid medium: is prepared by using a nutrient broth of a synthetic medium.
2. Experimental procedure
2.1 plate antagonistic assay-preliminary screening
2.1.1 activation of Strain
The 184 bacteria isolated from the bacterial strain GMI1000 and example 1 were transferred to TTC solid plates and NA solid plates, respectively, and cultured at 30℃for 1-2d for use.
2.1.2 preparation of the bacterial plates
Inoculating single colony of bacterial wilt GMI1000 strain into 2-5mL NB medium (NA medium without agar), culturing at 30deg.C and 200rpm for 24 hr to obtain bacterial wilt GMI1000 strain 10 And sucking 1mL of the bacterial suspension from cfu/mL, adding the bacterial suspension into 100mL of NA culture medium cooled to about 45 ℃, and pouring the bacterial wilt after shaking uniformly.
2.1.2 antagonistic bacteria Pre-screening
And (3) respectively streaking and activating the strains separated and purified in the earlier stage on an NA plate, picking the activated strains by using sterilized bamboo sticks, inoculating 8 tested strains to each plate, culturing at 30 ℃ for 48 hours, observing the existence of a bacteriostasis zone, measuring the size of the bacterial strain by using a spiral micrometer, and storing the bacterial strain with the bacteriostasis zone for rescreening.
2.2 plate antagonistic assay-rescreening
2.2.1 Strain activation
And 2.1.1, activating the strain with antagonistic effect on the primary screening.
2.2.2 liquid fermentation of isolated strains
And (3) picking single colony of the antagonistic strain obtained by primary screening, inoculating into 100mL of NB culture medium (placed in a 250mL triangular flask), and culturing at 28 ℃ and 130rpm for 48 hours to obtain antagonistic bacterial fermentation liquor.
2.2.3 Complex sieves with plate coating method
100 mu L of bacterial suspension of bacterial wilt GMI1000 strain is evenly coated on NA solid culture medium, a yellow gun head is used for punching on a flat plate, then 100 mu L of antagonistic bacteria fermentation liquor is added into the hole, the culture is carried out for 1-3d at 28 ℃, whether a bacteriostasis ring appears or not is observed, and a cover is opened in an ultra clean bench to record a result by photographing.
2.2.4 re-screening by decantation
Preparing 100 mL/bottle of NA solid culture medium, cooling the culture medium to about 45 ℃, adding 1mL of bacterial suspension according to a proportion of 1%, preparing a double-layer flat plate with oxford cups, taking out the oxford cups after the flat plate is dried, adding 100 mu L of antagonistic bacteria fermentation liquor into the holes, culturing for 1-3d at 28 ℃, observing whether a bacteriostasis ring appears, and opening a cover in an ultra-clean bench to record a result by photographing.
3. Experimental results
The 28 strains with antagonistic effect are obtained through a preliminary screening test, and 6 strains with obvious antagonistic effect are obtained through flat plate rescreening and pouring rescreening, wherein MMYH13 is one strain with excellent antagonistic effect.
FIG. 1 is a graph showing the antagonism effect of the strain MMYH13 on the bacterial wilt GMI1000 on a flat plate, and the obvious inhibition zone appears, which shows that the strain MMYH13 has obvious antagonism on the bacterial wilt.
Example 3: identification of isolated strains
Isolated strains were identified by reference to the handbook for identification of common bacterial systems (scientific press, dongxiu bead). The test method is as follows: DNA of the strain MMYH13 is extracted by a water boiling method, a 16S rDNA gene sequence of the strain MMYH13 is amplified by utilizing a 16S rDNA specific primer 27F and 1492R of bacteria and Taq enzyme, an amplified product is subjected to electrophoresis analysis to generate a band of about 1500b, the band is cut, recovered by gel cutting and sent to Soviet Jin Weizhi biotechnology limited company for sanger sequencing, and the sequenced sequence is spliced by seqman software to obtain a 16S rDNA sequence, the nucleotide sequence of which is shown as SEQ ID NO.1 and submitted 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 homology of 99.65% with Burkholderia anthina (Burkholderia), and has morphological characteristics most similar to Burkholderia anthina (Burkholderia). Gram staining was negative, and the strain grew well in media such as LB, NA, TTC, and the strain cells were short rods with flagella (FIG. 2), and colonies were prone to more exopolysaccharides when cultured on TTC plates. Based on the 16S rDNA sequence and morphological feature results, the strain MMYH13 was classified as Burkholderia (Burkholderia anthina).
According to the identification result, the strain MMYH13 is named as Burkholderia anthina MMYH and is preserved in the microorganism strain collection center of Guangdong province at the 8 th month of 2021, and the address is the 59 th building 5 of the first China center for road 100 in the View district of Guangzhou province, and the post code is as follows: 510070, which has the accession number: GDMCC No:61895.
example 4: biocontrol effect test of strain MMYH13 on tomato bacterial wilt
The strain MMYH13 shows obvious antagonism to the bacterial wilt on a flat plate, and in order to verify the biocontrol effect of the bacterial wilt of tomatoes, the biocontrol effect of fermentation liquor of the strain MMYH13 to the bacterial wilt of tomatoes is evaluated through a greenhouse potting test.
Test procedure
1. Seedling raising of tomato variety "medium and vegetable No. four
1.1 seed germination and seeding
The tomato seeds are soaked in water bath at 37 ℃ for 2 hours and 75% alcohol for 30 seconds, washed twice with deionized water, surface sterilized with 5% NaClO solution for 15 minutes, and then rinsed with deionized water. Uniformly placing in a culture dish paved with two layers of gauze, adding a proper amount of distilled water, keeping seeds and gauze moist, and accelerating germination in a constant temperature incubator at 28 ℃ for 2d. After the seeds germinate and grow radicle of about 1-2mm, sowing the seeds into a seedling tray filled with perlite, sowing 1 seed in each hole, and culturing in a greenhouse.
1.2 seedling transplanting
The substrate soil is mixed with water, half of the mixed substrate soil is filled in a flowerpot (diameter is 9 cm), tomato seedlings are carefully taken out from a seedling tray, put into the flowerpot, half of the substrate 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%, and the tomato seedlings are cultured in a greenhouse for about 28 days for later use.
2. Bacterial wilt and antibacterial MMYH13 activation and fermentation broth preparation
The bacterial wilt GMI1000 and antagonistic bacteria MMYH13 were activated by the same method as in 2.1.1 of example 1, and then single colonies were picked up and respectively inoculated into triangular flasks containing TM liquid medium (TTC medium without TTC and agar) and NB medium (NA medium without agar), and shake-cultured at 200rpm on a shaking table at 28℃for 2d (OD 600 ＝2.5)。
3. Inoculating tomato plants with bacterial wilt and inoculating antibacterial fermentation liquor
The test set 7 treatments, respectively, (1) a blank control without any bacteria added with sterile water, (2) a negative control of a TM liquid culture medium, (3) a positive control inoculated with a bacterial wilt fermentation broth, (4) a control inoculated with an antagonistic bacteria MMYH13 fermentation broth, (5) an antagonistic bacteria MMYH13 and bacterial wilt in a volume ratio of 1:1, mixed inoculation treatment; (6) the antagonistic bacteria MMYH13 and the bacterial wilt are in a volume ratio of 3:1, mixing and inoculating, (7) antagonistic bacteria MMYH13 and bacterial wilt in a volume ratio of 5:1 treatment of mixed inoculation. Five replicates were inoculated per treatment, 10 seedlings per replicate. The inoculation amount of the bacterial liquid is 5mL per basin, the bacterial liquid root irrigation method is adopted for inoculation, the bacterial liquid is placed in a glass greenhouse for culture for 14d (the temperature is 28 ℃ and the humidity is 40%) after inoculation is finished, and watering is carried out for 1 time every day the next day after inoculation. Disease severity of tomato seedlings was observed and recorded daily as described in Kempe published paper, and the incidence of each treatment was calculated according to the following formula.
Plant incidence = (number of plants developed/total number of plants) x 100%
4. Test results
As shown in fig. 3, the incidence of tomato treated with sterile water blank, TM broth negative control, and antagonistic alone was 0 after inoculation; all the tomatoes inoculated with the bacterial wilt fungus GMI1000 fermentation broth die, and the morbidity is 100%; meanwhile, the average morbidity of tomatoes in treatment groups (1:1, 3:1 and 5:1) inoculated with the antagonistic bacteria MMYH13 fermentation liquor and the bacterial wilt 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 tomato bacterial wilt, and the tomato bacterial wilt is a potential biocontrol bacterium.
Claims (7)
1. A strain of burkholderia (Burkholderia anthina) MMYH13, characterized by having a deposit number of: GDMCC No:61895.
2. the use of burkholderia MMYH13 according to claim 1 for controlling bacterial wilt.
3. The use according to claim 2, wherein said burkholderia MMYH13 is a use in the control of bacterial wilt in tomatoes.
4. The use according to claim 2 or 3, characterized in that the bacterial liquid of burkholderia MMYH13 is applied to the soil in which the crop is planted.
5. A biocontrol microbial agent comprising the Burkholderia MMYH13 of claim 1.
6. A method of culturing burkholderia MMYH13 according to claim 1, characterized by inoculating it in LB, NA, NB or TTC medium.
7. The method of claim 6, wherein the burkholderia MMYH13 is inoculated into NB medium under the following conditions: culturing at 28-30deg.C and 130-200rpm for 1-2d.
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