CN117229970A - Bacillus bailii for resisting various plant pathogenic bacteria and application thereof - Google Patents
Bacillus bailii for resisting various plant pathogenic bacteria and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus belgium lk5-1 resistant to various plant pathogenic bacteria and application thereof. The strain is classified and identified as bacillus beijerinus (Bacillus velezensis), and the preservation number is as follows: GDMCC No:63132. the bacillus belgium lk5-1 disclosed by the invention has obvious inhibition effect on 3 plant pathogenic bacteria such as melon bacterial fruit blotch, rice bacterial brown streak and citrus canker, and the disease index of the melon bacterial fruit blotch can be obviously reduced by a foliar spraying inoculation mode, and the control effect is as high as 86.57%. Therefore, the bacillus beleiensis lk5-1 disclosed by the invention can effectively prevent bacterial fruit blotches, can be used for preparing microbial preparation products for preventing and treating various plant pathogenic bacteria such as melon bacterial fruit blotches and the like, and reduces the use of chemical medicines in agricultural production.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to bacillus belicus for resisting various plant pathogenic bacteria such as melon bacterial fruit blotch and application thereof.
Background
Bacterial fruit blotch (Bacterial Fruit Blotch, BFB) is a destructive disease caused by watermelon acidovorous bacteria (Acidovorax citrulli) and seriously damaging leaves and fruits of various cucurbitaceae plants such as watermelons, melons, cantaloupes and the like, and has the characteristics of rapid onset, quick transmission, strong explosiveness and the like. In order to prevent and treat bacterial fruit blotch, a large amount of antibiotics such as kasugamycin, streptomycin sulfate, kasugamycin, copper, and copper preparation chemical medicines are often used in production, so that on one hand, the drug resistance of pathogenic bacteria is gradually enhanced and the prevention effect is reduced, and the dosage of the medicines is forced to be increased, so that vicious circle is formed; on the other hand, the pesticide residue seriously affects the quality safety of melons, and also seriously damages the ecological environment of the production area, so that a more green and environment-friendly control technology needs to be developed.
The control technology based on the biocontrol microorganism has the advantages of low cost, high efficiency, no pollution and the like, plays an important role in reducing the use of chemical medicines and improving the quality of agricultural products, and receives more and more attention in agricultural production. Microorganisms for preventing and treating bacterial fruit blotch are reported at present and comprise groups of saccharomycetes, pseudomonas, bacillus and the like. The bacillus has the characteristics of wide distribution, strong stress resistance, rapid propagation, stable effect and the like, and is the most widely applied group in agricultural production. The bacillus belicus can generate rich and diverse active metabolites, and has great development value in the aspect of controlling different types of bacterial and fungal plant diseases, and the application and research of the bacillus belicus in the control of melon bacterial fruit blotch are still in the starting stage at present.
Disclosure of Invention
The invention aims to provide bacillus beijerinckii (Bacillus velezensis) lk5-1 with biological control effect on plant diseases, which is preserved in the microorganism strain preservation center (GDMCC) of Guangdong province at the 8 th month 4 th year of 2023, address: the university of first, middle road 100, building 59, the university of Guangdong, guangzhou, guangdong, university of Guangdong, microbiological institute, post code: 510070, accession number: GDMCC No:63132.
the second object of the invention is to provide the application of bacillus belicus lk5-1 in preparing biological agent for preventing and treating plant diseases.
The biological agent is microbial agent, microbial fertilizer, microbial pesticide or microbial additive.
The biological agent takes the bacillus belgium lk5-1 or a culture or a metabolite thereof as an active ingredient.
Pathogenic bacteria of the plant diseases comprise melon bacterial fruit blotch bacteria (Acidovorax citrulli), rice bacterial brown stripe bacteria (Acidovorax oryzae) and citrus canker bacteria (Xanthomonas citri).
A third object of the present invention is to provide a method for controlling plant diseases, which comprises the step of contacting Bacillus belicus lk5-1 or a culture or metabolite thereof with plants.
The bacillus beleiensis lk5-1 culture or metabolite is obtained by inoculating bacillus beleiensis lk5-1 into a culture medium for culture.
The plant diseases comprise melon bacterial fruit blotch, rice bacterial brown streak and citrus canker.
A fourth object of the present invention is to provide a biological agent comprising the above bacillus beleiensis lk5-1 or a culture or metabolite thereof as an active ingredient.
The preparation forms of the biological agent comprise powder, wettable powder, soluble powder, emulsifiable concentrate, water aqua, suspending agent, microemulsion, granules or water dispersible granules.
Compared with the prior art, the invention has the following advantages:
(1) the strain lk5-1 is a novel strain of bacillus belicus which can generate various bacteriostatic activity metabolites and has growth promoting potential through accurate genome identification;
(2) the prevention and treatment effect of the pre-inoculated bacillus belicus lk5-1 on melon bacterial fruit blotch is up to 86.57 percent, and is superior to most of reported similar strains, and the strain has huge development and application values.
Therefore, the bacillus belgium lk5-1 disclosed by the invention can be used for preventing the occurrence of melon bacterial fruit blotch, effectively reducing the incidence rate of the melon bacterial fruit blotch, and has important application potential in the aspects of preparing biocontrol microbial preparations and the like.
Bacillus belicus (Bacillus velezensis) lk5-1, deposited at the Guangdong province microbiological bacterial collection center (GDMCC) at 4/2023, address: the university of first, middle road 100, building 59, the university of Guangdong, guangzhou, guangdong, university of Guangdong, microbiological institute, post code: 510070, accession number: GDMCC No:63132.
drawings
FIG. 1 is a core genome-based strain lk5-1 phylogenetic tree constructed using software UBCG.
FIG. 2 shows the inhibitory effect of Bacillus belicus lk5-1 on 3 plant pathogenic bacteria such as melon bacterial fruit blotch.
FIG. 3 shows the biocontrol effect of Bacillus belicus lk5-1 on melon bacterial fruit blotch.
Detailed Description
The following examples are illustrative of the present invention and are not intended to limit the scope of the invention. The following embodiments are all reagents and method steps conventional in the art, except as specifically described.
Example 1: accurate identification and genome analysis of strain lk5-1
16S rRNA Gene analysis: the total DNA of strain lk5-1 was extracted using a bacterial DNA extraction kit. Bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTA ACCCCAATCGC-3') were used for PCR amplification of the 16S rRNA gene of the strain. The amplification procedure was: pre-denaturation at 94℃for 5min;35 cycles (94 ℃ denaturation 30s,56 ℃ annealing 30s,72 extension 90 s); extending at 72℃for 10min. The PCR products were detected by 1% (w/v) agarose gel electrophoresis, after which sequencing was completed by Jin Weizhi. The 16S rRNA gene sequence similarity alignment of the strains was performed using an EzBiocloud online platform (https:// www.ezbiocloud.net /). The 16S rRNA gene sequencing obtains a sequence with the fragment length of 1420bp, and the similarity is higher than that of the 16 model strains of the strain lk5-1 and Bacillus (Bacillus), which shows that the strain lk5-1 is a strain of the Bacillus, but the strain in the Bacillus cannot accurately judge the classification status only through similarity or systematic evolution analysis due to the excessively high sequence similarity of the 16S rRNA gene, and more accurate identification is needed by means of comparison of genome and the like.
Genome analysis: the total DNA of the bacterial strain lk5-1 extracted by using a genome extraction kit is sent to PE library construction and genome sequencing by Shanghai Meijia biological medicine science and technology Co., ltd, and a sequencing platform is Illumina Hiseq2500. Splicing and assembling the off-machine data by adopting software SPades v3.15.0, downloading published genome sequences of different model strains of bacillus from a GenBank database, and constructing a phylogenetic tree based on a core genome by adopting software UBCG (Phylogenomic tree inference using bacterial core genes) so as to determine the model strains of different species in bacillus with the closest genetic relationship of the strain lk 5-1. The strain lk5-1 and its kindred model strain were subjected to comparative genomic analysis using on-line software ANI Calculator (http:// www.ezbiocloud.net/tools/ANI) and GGDC 2.1 (http:// GGDC. Dsmz. De/home. Php), respectively, to calculate Average Nucleotide Identity (ANI) and simulated DNA-DNA hybridization values (dDDH). To gain more insight into and grasp the functional properties of strain lk5-1, the genome of strain lk5-1 was annotated with online software anti smash 7.0.1 (https:// anti map. Second ymethylabsolite. Org/# |/start) and RAST (https:// RAST. Nmddr. Org /).
The strain lk5-1 obtained by sequencing had a genome size of 3.89Mb and a GC content of 46.43mol%. UBCG phylogenetic tree constructed based on core genome clearly showed strain lk5-1 and Bacillus belicus (Bacillus velezensis NRRL B-41580) T ) Has the closest relationship (FIG. 1), followed by Bacillus siamensis (Bacillus siamensis KCTC 13613) T ) Bacillus amyloliquefaciens (Bacillus amyloliquefaciens DSM 7) T ) Bacillus clathratus (Bacillus nakamurai NRRL B-41091) T ). Comparative genome analysis the results are shown in Table 1, strain lk5-1 was closest to the related Bacillus bailii (Bacillus velezensis NRRL B-41580) T ) ANI and dDDH values of 98.25% and 84.4%, respectively, and model strain ANI values of 86.40% -94.36% for other 3 closely related species, and dDDH values of 30.7% -56.6%. The above results clearly indicate that strain lk5-1 should be identified as Bacillus belicus (Bacillus velezensis) according to internationally recognized species discrimination thresholds (ANI 95% -96%; dDDH 70%) as a criterion.
The anti-SMASH annotation result of the genome sequence shows that the strain lk5-1 has different types of active secondary metabolite synthesis gene clusters such as surface active agents (surfactin), difficile agents (difficidin), fengycin (fengycin), bacillus lysin (bacilysin), bacillaene, macrolactin H, bacilebactin and the like, and has important application potential in preventing and treating different types of pathogenic bacteria. As a result of RAST annotation of the genome sequence, the strain lk5-1 has the plant growth promoting capability of producing siderophores, secreting auxins, dissolving inorganic phosphorus and the like, and has certain application potential in preparing microbial agents for promoting plant growth.
Thus, the strain lk5-1 of the present invention belongs to Bacillus velezensis and is designated Bacillus belicus (Bacillus velezensis) lk5-1, which was deposited at the Guangdong province microorganism strain collection (GDMCC) on month 8 and 4 of 2023, accession number GDMCC No:63132, deposit address: the university of first, middle road 100, building 59, the university of Guangdong, guangzhou, guangdong, university of Guangdong, microbiological institute, post code: 510070.
TABLE 1 comparative genomic analysis of strain lk5-1 and model strains of its closely related species
Example 2: the inhibition effect of bacillus belgium lk5-1 on various plant pathogenic bacteria such as melon bacterial fruit blotch and the like selects 3 plant pathogenic bacteria such as melon bacterial fruit blotch (Acidovorax citrulli GDMCC 803619), rice bacterial brown streak disease (Acidovorax oryzae GMDCC 805499), citrus canker (Xanthomonas citri KY 1) and the like as targets, and the plant pathogenic bacteria and bacillus belgium lk5-1 are respectively inoculated into a TSA liquid culture medium and subjected to shaking culture for 48 hours at 28 ℃ and 180 r/min. TSA solid medium plates were prepared, and 100. Mu.L of cultured plant pathogenic bacteria were uniformly spread on the TSA plates, respectively. And (3) testing the inhibition effect of bacillus beijerinus lk5-1 culture solution on different plant pathogenic bacteria by adopting a punching method. Centrifuging the culture solution 12000r/min of the strain lk5-1 for 10min, taking 100 mu L of supernatant into holes drilled on TSA plates coated with different pathogenic bacteria, taking the sterilized TSA culture solution without inoculating lk5-1 as a reference, standing and culturing at 28 ℃ for 72h, and observing and measuring the size of the bacteriostasis zone of bacillus belicus lk5-1 to different pathogenic bacteria. As shown in Table 2 and FIG. 2, bacillus belicus lk5-1 showed a strong inhibition effect on the above 3 plant pathogenic bacteria, wherein the diameter of the inhibition zone on melon bacterial fruit blotch (Acidovorax citrulli GDMCC 803619) was maximum, 40.8+ -3.3 mm, and then the disease of citrus canker (inhibition zone diameter 39.8+ -2.1 mm) and rice bacterial brown streak (inhibition zone diameter 27.5+ -2.5 mm) were observed.
TSA medium: 15.0g of peptone, 5.0g of soybean peptone, 5.0g of NaCl, 15.0g of agar and ddH 2 O1000 mL, pH 7.3+ -0.2, mixing the above materials uniformly, 12Sterilizing at 1deg.C for 20 min.
TABLE 2 Flat-plate bacteriostatic Effect of Bacillus bailii lk5-1 on different plant pathogens
Example 3: control effect of bacillus belicus lk5-1 on melon bacterial fruit blotch
Taking bacterial fruit blotch pathogenic bacteria-watermelon acidovorax (Acidovorax citrulli) GDMCC 803619 as a test strain, picking up purified single colony, inoculating into an R2A liquid culture medium, culturing at 30deg.C under 200R/min shaking for 36h, and regulating the concentration of bacterial liquid to 10 with sterile water 8 cfu/mL is reserved. Picking single colony of Bacillus bailii lk5-1 cultured on the plate, shake culturing at 30deg.C and 200r/min in nutrient broth agar medium (NA) for 48 hr, and regulating bacterial liquid concentration to 10 with sterile water 8 cfu/mL is reserved.
The Hami melon seeds are used as test plants, the Hami melon seeds are sowed in a seedling raising matrix after surface disinfection and room temperature germination acceleration, the seedlings are planted in a large plastic basin (with an upper caliber phi of 12cm and a lower caliber phi of 10cm and a height of 10 cm) when the seedlings grow to two leaves and one core, and planting conditions are set to be 16 hours of illumination and 8 hours of darkness. The bacterial liquid is uniformly sprayed on the leaves of the whole Hami melon by adopting a positive and negative leaf surface spraying inoculation method, sterile water is used as non-inoculation contrast, and the disease condition of the Hami melon is observed and recorded every day. The experiment was set up with a total of 4 treatments, each treatment being set up with 5 replicates. Treatment T1 is to spray 5mL to adjust the concentration to 10 8 After 24h of airing, spraying pathogenic bacteria with the equal volume concentration of 10 per mL 8 cfu/mL biocontrol bacteria lk5-1; treatment T2 is that 5mL of the solution is sprayed at first with a concentration of 10 8 cfu/mL biocontrol bacteria lk5-1, and spraying 10 with equal volume after 24 hours of airing 8 cfu/mL of pathogenic bacteria; treatment T3 is that 5mL of the solution is sprayed at first with the concentration of 10 8 cfu/mL of biocontrol bacteria lk5-1, and spraying an equal volume of sterile water after 24 hours of airing; the CK is sprayed 5mL to a concentration of 10 8 cfu/mL of pathogenic bacteria, and spraying an equal amount of sterile water after 24 hours of airing. After 3 days of spraying, plants begin to attack, and on 5 days, disease observation under different treatments is carried outAnd counting and analyzing the control effect of the bacterial fruit blotch.
Bacterial fruit blotch grading criteria on Hami melon plants: grade 0, no lesions; stage 1, the area of the disease spots accounts for less than 5% of the whole blade; stage 3, the area of the disease spots accounts for 5% -25% of the whole blade; grade 5, the area of the disease spots accounts for 26% -50% of the whole blade; grade 7, the area of the disease spots accounts for 51% -75% of the whole blade; grade 9, the area of the disease spots accounts for 76-100% of the whole blade.
As shown in FIG. 3 and Table 3, the results of the potting experiment showed that no disease was found on the leaves of T3 cantaloupe treated with biocontrol bacteria lk5-1 alone without bacterial fruit blotch pathogenic bacteria. The disease conditions of different degrees appear on the cantaloupe leaves in different treatments of spraying inoculation pathogenic bacteria, but the disease indexes among different treatments are greatly different, wherein the disease spot number, spot area and the like on the cantaloupe leaves in two treatments (T1 and T2) inoculated with the biocontrol bacteria lk5-1 are obviously smaller than those of the control treatment CK without the biocontrol bacteria lk 5-1. The control effect of the treatment T2 of firstly spraying and inoculating biocontrol bacteria lk5-1 and then inoculating pathogenic bacteria on the Hami melon is up to 86.57 percent, which is obviously higher than that of the treatment T1 of firstly spraying and inoculating pathogenic bacteria and then inoculating biocontrol bacteria lk5-1 (the control effect is only 7.47 percent). The bacillus belicus lk5-1 has an important prevention effect on the occurrence of bacterial fruit blotch of melons, and the bacterial fruit blotch can be obviously reduced by pre-spraying and inoculating the strain culture solution, so that the Hami melon plants are protected to the greatest extent. When the cantaloupe is infected with bacterial fruit spot disease and then inoculated with lk5-1, the control effect is greatly reduced, which is far lower than that of the treatment mode of pre-spraying inoculation. Therefore, it is suggested that the occurrence of diseases is reduced by pre-spraying and inoculating bacillus beijerinus lk5-1 in the production season in which bacterial fruit blotch is easy to occur, and healthy growth of plants is protected.
R2A liquid medium: yeast extract 0.5g, peptone 0.5g, acid hydrolyzed casein 0.5g, glucose 0.5g, soluble starch 0.5g, K 2 HPO 4 0.3g，MgSO 4 ·7H 2 O0.05 g, sodium pyruvate 0.3g, ddH 2 O1000 mL, pH 7.0, and sterilizing at 121deg.C for 20 min.
NA medium: beef extract 3.0g, peptone 5.0g, glucose 2.5g, ddH 2 O1000 mL, pH 7.0, and sterilizing at 121deg.C for 20 min.
TABLE 3 control effect of Bacillus belicus lk5-1 on melon bacterial fruit blotch under different treatments
Note that: CK, spraying pathogenic bacteria, and then spraying sterile water; t1, spraying pathogenic bacteria firstly, and then spraying lk5-1; t2, spraying lk5-1, and then spraying pathogenic bacteria; and T3, spraying biocontrol bacteria lk5-1, and then spraying sterile water.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (10)
1. Bacillus belicus (Bacillus velezensis) lk5-1, accession number: GDMCC No:63132.
2. use of bacillus belgium lk5-1 according to claim 1 for the preparation of a biological agent for controlling plant diseases.
3. The use according to claim 2, wherein the biological agent is a microbial agent, microbial fertilizer, microbial pesticide or microbial additive.
4. The use according to claim 2, wherein the biological agent comprises bacillus beleiensis lk5-1 or a culture or metabolite thereof according to claim 1 as an active ingredient.
5. The use according to claim 2, wherein the pathogenic bacteria of plant diseases include bacterial fruit blotch bacteria (Acidovorax citrulli) of melon, bacterial brown streak bacteria (Acidovorax oryzae) of rice and canker bacteria (Xanthomonas citri).
6. A method for controlling plant diseases, which comprises the step of contacting bacillus beljalis lk5-1 or a culture or metabolite thereof according to claim 1 with a plant.
7. The method according to claim 6, wherein the culture or metabolite of Bacillus belicus lk5-1 is obtained by inoculating Bacillus belicus lk5-1 into a culture medium and culturing.
8. The method of claim 6, wherein the plant disease comprises bacterial fruit blotch of melons, bacterial brown streak of rice, and citrus canker.
9. A biological agent comprising bacillus belgium lk5-1 or a culture or metabolite thereof according to claim 1 as an active ingredient.
10. The biologic of claim 9, wherein the biologic comprises a dosage form of powder, wettable powder, soluble powder, emulsifiable concentrate, aqueous formulation, suspension, microemulsion, granule, or water dispersible granule.
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| CN112746046A (en) * | 2021-02-05 | 2021-05-04 | 中国农业科学院蔬菜花卉研究所 | Bacillus belgii and application thereof in prevention and treatment of cucumber bacterial angular leaf spot |
| CN113755393A (en) * | 2021-10-09 | 2021-12-07 | 河南省科学院生物研究所有限责任公司 | Bacillus beilesensis HP-24 and application thereof in preparation of bacterial liquid for preventing and treating bacterial fruit blotch of melons |
| CN114933995A (en) * | 2022-06-07 | 2022-08-23 | 广西大学 | Biocontrol bacillus belezii ML21 and application thereof |
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| CN112746046A (en) * | 2021-02-05 | 2021-05-04 | 中国农业科学院蔬菜花卉研究所 | Bacillus belgii and application thereof in prevention and treatment of cucumber bacterial angular leaf spot |
| CN113755393A (en) * | 2021-10-09 | 2021-12-07 | 河南省科学院生物研究所有限责任公司 | Bacillus beilesensis HP-24 and application thereof in preparation of bacterial liquid for preventing and treating bacterial fruit blotch of melons |
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