CN115851495B - Biocontrol bacterium for preventing and treating wheat scab, application thereof and biocontrol bacterium agent - Google Patents
Biocontrol bacterium for preventing and treating wheat scab, application thereof and biocontrol bacterium agent Download PDFInfo
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- CN115851495B CN115851495B CN202211098603.0A CN202211098603A CN115851495B CN 115851495 B CN115851495 B CN 115851495B CN 202211098603 A CN202211098603 A CN 202211098603A CN 115851495 B CN115851495 B CN 115851495B
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- gladioli
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- graminearum
- fusarium
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to a biocontrol bacterium for preventing and treating wheat scab, an application thereof and a biocontrol microbial agent, wherein the biocontrol bacterium is B.gladioli7-1, the strain is classified and named as Burkholderia gladioli (Burkholderiagladioli), and the strain is preserved in China general microbiological culture collection center (CGMCC) for 25 th month 07 in 2022. And B, the gladioli7-1 can colonize in wheat bodies, has a good biocontrol effect, is a potential biocontrol preparation for preventing and treating wheat scab, has broad-spectrum antibacterial activity, can inhibit the occurrence of scab by 100% when being mixed with fusarium graminearum PH-1 spore liquid for inoculation, and also remarkably reduces the accumulation of DON toxin.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a biocontrol bacterium for preventing and treating wheat scab, application thereof and a biocontrol bacterium agent.
Background
Wheat scab (Fusarium head blight, FHB) is a destructive fungal disease caused by multiple fusarium species. Wheat scab in China is concentrated in middle and downstream of the Yangtze river where the climate is humid, in Huaihe river and in the Trigonella Foenum-graecum. In recent years, diseases tend to spread in the north due to the influence of factors such as adjustment of agricultural structures and climate warming. In addition to the reduction of yield, fusarium graminearum can produce Deoxynivalenol (DON) toxin when infecting wheat, and is a tertiary carcinogen according to European classification standards, so that the quality of the wheat is affected, and acute poisoning symptoms such as vomiting and diarrhea can be caused.
Effectively control the occurrence and the popularity of wheat scab and is related to the food safety of grains and foods in China. If continuous overcast and rainy weather is encountered in the flowering period of wheat, the disease is easy to occur in a large area, so that the spraying of benzimidazole bactericides such as carbendazim and the like in the flowering period of wheat is a key measure for preventing and treating scab. Due to the problems of increased pathogen resistance and the like caused by the large-scale use of chemical agents, people pay more attention to environmental problems, and the exploration of green and safe biological control resources is important to effectively control scab.
The most widely used biological agents of Pseudomonas and Bacillus are currently produced, and the preventive and therapeutic effects are good in timeliness but the duration is not long. Endophytes can stably colonize in plants for a long time, are a type of biocontrol resource worthy of excavation, but lack the biocontrol resource at present.
Disclosure of Invention
In order to solve the technical problems, the invention provides a biocontrol bacterium for preventing and treating wheat scab, application thereof and a biocontrol bacterium agent.
The biocontrol bacterium for preventing and treating wheat scab is characterized in that the biocontrol bacterium is B.gladioli 7-1, and the strain is classified and named as Burkholderia tangutica (Burkholderia gladioli) which is preserved in the China general microbiological culture Collection center of the China general microbiological culture Collection center, and the preservation address is shown as 25 th month of 2022: the preservation number of the Beijing Chaoyang area North Star Xiyu No.1 of China is CGMCC No. 25395.
A biocontrol microbial agent for preventing and treating wheat scab comprises the B.gladioli 7-1.
Preferably, the concentration of B.gladioli 7-1 is 1X 10 6-1×109 CFU/mL.
The application of the B.gladioli 7-1 in preparing a biocontrol microbial agent for preventing and treating wheat scab.
Preferably, said b.gladioli 7-1 is used for inhibiting fusarium graminearum.
The application of the B.gladioli 7-1 in preventing and treating wheat scab.
The application of the gladioli 7-1 in inhibiting the fusarium graminearum, the wheat variety of the top capsule shell, the fusarium asiaticum, the rhizoctonia graminearum, the colletotrichum asiaticum, the colletotrichum siamensis and the fusarium pseudograminearum.
Compared with the prior art, the invention has the beneficial effects that:
1. The endophytic strain B.gladioli 7-1 can colonize in wheat, has good biocontrol effect, is a potential biocontrol agent for preventing and treating wheat scab, widely exists on the surface or rhizosphere of plants, and has a plurality of functions beneficial to the plants and the environment, such as growth promotion, plant resistance induction and the like.
2. The culture filtrate of the endophytic strain B.gladioli 7-1 has broad-spectrum antibacterial activity, and can not only inhibit the occurrence of scab by 100% but also remarkably reduce the accumulation of DON toxin when being mixed with fusarium graminearum PH-1 spore liquid for inoculation. 21 metabolite synthesis gene clusters are predicted after sequencing the genome of the strain B.gladioli 7-1, so that the antibacterial compound of the strain culture filtrate is separated and identified, and the method has good development and application prospects and is hopeful to excavate new secondary metabolites.
3. Description of biological preservation information:
Biological material: 7-1, taxonomic nomenclature: burkholderia tangutica, latin name: burkholderia gladioli, the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.25395 at 25 of 2022 and 07.
Drawings
FIG. 1 is a preliminary screening of bacteria antagonizing Fusarium graminearum (A); gladioli 7-1 colony map (B); graph (C) of the results of experiment of gladioli 7-1 against Fusarium graminearum plates; a statistical plot of gladioli 7-1 effect on fusarium graminearum conidium yield (D), t-test for significant difference analysis (p=0.05), representing p <0.001;
FIG. 2 is a graph showing the effect of bacteria colonization after root-irrigation treatment of wheat with a B.gladioli bacterial solution;
FIG. 3 is a schematic drawing of the bacteriostasis spectrum of B.gladioli 7-1 (A); a statistical graph (B) of inhibition rates for different pathogenic bacteria;
FIG. 4 is a schematic diagram of bacteriostasis spectrum (A) of culture filtrate of B.gladioli 7-1; a statistical graph (B) of inhibition rates for different pathogenic bacteria;
FIG. 5 is a graph (A) and a statistical graph (B) of inhibition effect of the filtrate of the shaking culture of different culture media of gladioli 7-1 on fusarium graminearum;
FIG. 6 is a schematic diagram (A) of the results of inoculating gladioli 7-1 and its culture filtrate with F.graminearum PH-1 spore liquid; statistical plot of the results of the above-mentioned inoculation procedure on wheat head morbidity (B), t-test with significant difference analysis (p=0.05), p <0.001;
fig. 7 is a statistical plot (a) of the effect of gladioli 7-1 and culture filtrate on the DON content of fusarium graminearum, with t-test for significant difference analysis (p=0.05), representing p <0.001; schematic representation of gladioli 7-1 affecting expression of Fusarium graminearum TRI1, TRI5 and TRI12 (B).
Detailed Description
The following detailed description of specific embodiments of the invention is, but it should be understood that the invention is not limited to specific embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental methods described in the examples of the present invention are conventional methods unless otherwise specified.
The media used in the examples are as follows:
The CM solid culture medium comprises the following components in parts by weight (per 1L): 10g of glucose, 20g of peptone, 1g of yeast extract, 1g of acid hydrolyzed casein, 6g of sodium nitrate, 0.5g of potassium chloride, 1.023g of magnesium sulfate heptahydrate, 0.5g of potassium dihydrogen phosphate, 15g of agar, 1L of deionized water and adjusting the pH to 6.5 by using sodium hydroxide liquid, wherein the culture medium is obtained by mixing the components and sterilizing at high temperature and high pressure at 121 ℃ for 20 min;
The CMC culture medium comprises the following components in proportion (per 1L): 15g of sodium carboxymethylcellulose, 1g of ammonium nitrate, 1g of monopotassium phosphate, 1g of yeast extract, 0.5g of magnesium sulfate heptahydrate and 1L of deionized water, wherein the culture medium is obtained by mixing the components and sterilizing the mixture at the high temperature and the high pressure of 121 ℃ for 20 min;
the components and the proportions (per 1L) of the LB liquid medium are as follows: 10g of tryptone, 5g of yeast extract, 5g of sodium chloride and 1L of deionized water, wherein the culture medium is obtained by mixing the components and sterilizing the mixture at the high temperature and the high pressure of 121 ℃ for 20 min;
The components and the proportions (per 1L) of the LB solid medium are as follows: 10g of tryptone, 5g of yeast extract, 5g of sodium chloride, 15g of agar powder and 1L of deionized water, wherein the culture medium is obtained by mixing the components and sterilizing the mixture at the high temperature and the high pressure of 121 ℃ for 20 min;
the components and proportions (per 1L) of the YEPD medium are: 3g of yeast extract, 10g of peptone, 20g of glucose and 1L of deionized water, wherein the culture medium is obtained by mixing the components and sterilizing at the high temperature and the high pressure of 121 ℃ for 20 min;
the 5 XYEG culture medium comprises the following components in proportion (per 1L): 5g of yeast extract, 10g of glucose and 1L of deionized water, wherein the culture medium is obtained by mixing the components and sterilizing at the high temperature of 121 ℃ for 20 min;
The components and the proportions (per 1L) of the M9 culture medium are as follows: 12.98g of disodium hydrogen phosphate heptahydrate, 3g of sodium dihydrogen phosphate, 0.5g of sodium chloride, 1g of ammonium chloride, 2mL of 1M magnesium sulfate, 100 mu L of 1M calcium chloride, 20mL of 20% glucose and 1L of deionized water, wherein the culture medium is obtained by mixing the components and sterilizing at the temperature of 121 ℃ for 20min under high temperature and high pressure;
The PDB culture medium comprises the following components in parts by weight (per 1L): 200g of potato, 20g of glucose and 1L of deionized water, wherein the culture medium is obtained by mixing the components and sterilizing the mixture at the high temperature and the high pressure of 121 ℃ for 20 min;
The CM liquid culture medium comprises the following components in proportion (per 1L): 10g of glucose, 20g of peptone, 1g of yeast extract, 1g of acid hydrolyzed casein, 6g of sodium nitrate, 0.5g of potassium chloride, 1.023g of magnesium sulfate heptahydrate, 0.5g of potassium dihydrogen phosphate, 1L of deionized water and adjusting the pH to 6.5 by using sodium hydroxide liquid, wherein the culture medium is obtained by mixing the components and sterilizing at the temperature of 121 ℃ for 20min under high temperature and high pressure;
The 2 XCM solid culture medium comprises the following components in proportion (per 1L): 10g of glucose, 20g of peptone, 1g of yeast extract, 1g of acid hydrolyzed casein, 6g of sodium nitrate, 0.5g of potassium chloride, 1.023g of magnesium sulfate heptahydrate, 0.5g of potassium dihydrogen phosphate, 30g of agar, 1L of deionized water, and regulating the pH to 6.5 by using sodium hydroxide liquid, wherein the culture medium is obtained by mixing the components and sterilizing at high temperature and high pressure at 121 ℃ for 20 min;
the PDA solid culture medium comprises the following components in parts by weight (per 1L): 200g of potato, 20g of glucose, 15g of agar powder and 1L of deionized water, wherein the culture medium is obtained by mixing the components and sterilizing the mixture at the high temperature and the high pressure of 121 ℃ for 20 min;
the TBI culture medium comprises the following components (per 1L) of 30g of sucrose, 1g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 0.5g of potassium chloride, 0.01g of ferrous sulfate heptahydrate, 0.871g of arginine, 0.2mg of citric acid, 0.2mg of zinc sulfate heptahydrate, 0.002mg of manganese sulfate monohydrate, 0.01mg of copper sulfate pentahydrate, 0.002mg of boric acid, 0.002mg of sodium permanganate dihydrate, 0.032mg of putrescine, 0.3g of gel, 1L of deionized water and adjusting the pH to 5.5 by using sodium hydroxide liquid, and is obtained by mixing the components and sterilizing the mixture at the high temperature and the high pressure at 121 ℃ for 20 min;
The species used were selected from the group consisting of Leptosporum graminearum, fusarium asiaticum, rhizoctonia cerealis, leptoanthrax siamensis and Fusarium pseudograminearum as taught by the university of Henan technology Xu Jianjiang.
Example 1
Isolation, screening and identification of biocontrol bacteria B.gladioli 7-1
Separating: corn root systems collected from Yang Lingou Cao Xin fields of Shaanxi province were sterilized. Washing with sterile water, soaking in 75% ethanol for 3min, soaking in 2% NaClO for 15min to remove surface bacteria, washing with sterile water, grinding plant root system with mortar, adding sterile water, and oscillating to obtain endophyte-containing suspension.
Screening and storing: mixing the CM culture medium cooled to 50 ℃ after thawing with spore liquid of fusarium graminearum (stored in functional genomics subject group of crop pathogenic fungi of the university of agricultural and forestry science and technology in northwest) of CMC shaking culture for 4d until the spore liquid concentration is 1 multiplied by 10 5/mL, pouring the mixture into a flat plate, dipping the separated endophyte into the single colony after the flat plate is solidified, shaking culture for 24h by using LB to obtain bacterial liquid, blowing 10 mu L of bacterial liquid drop on the flat plate, culturing for 3d at 25 ℃ and observing the result, and storing bacteria with antagonism on fusarium graminearum. The inhibition rate of fusarium graminearum is determined by a plate counter experiment.
And (3) identification: the species of bacteria are identified by sequencing the amplified bacterial 16s sequences. A pair of primers 27F (SEQ ID No. 2) and 1492R (SEQ ID No. 3) was designed to amplify the bacterial 16S fragment (primer sequence 27F:5'-AGAGTTTGATCCTGGCTCAG-3'; 14992R: 5 '-TACGACTTAACCCCAATCGC-3'). The antagonistic bacteria were activated by LB plate, and after single colony was grown on the plate, the 16S fragment was amplified by the above primer, and the PCR reaction system was (25. Mu.L): 10X EasyTaq Buffer 2.5.5. Mu.L, dNTPs 2. Mu.L, template DNA 1.5. Mu.L, upstream primer 27F 0.5. Mu.L, downstream primer 149910.5. Mu.L, ddH 2 O17.7. Mu.L. PCR reaction procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 58℃for 40s, elongation at 72℃for 120s for 35 cycles; and extending at 72 ℃ for 10min.
The PCR product is detected by 1% agarose gel electrophoresis, and the size of a target band is about 2000 bp. The PCR product was recovered and sent to the Productivity company for sequencing, the sequencing result being shown in SEQ ID No.1. According to the sequencing result of company splice, sequence alignment is carried out in NCBI (https:// www.ncbi.nlm.nih.gov /) database, and the strain is identified according to the homology of the sequence.
As shown in FIG. 1, among the isolated strains, B.gladioli 7-1 showed the most remarkable inhibitory effect on Fusarium graminearum (FIG. 1A), and the experiment of plate opposition gave that B.gladioli 7-1 had an inhibitory rate of 50% (FIG. 1C). Alignment on NCBI website by 16S sequencing results identified the B.gladioli 7-1 strain as Burkholderia gladioli (Burkholderia gladioli), the colony morphology is shown in FIG. 1B.
Example 2
Effect of gladioli 7-1 on Fusarium graminearum spore production
Endophyte B.gladioli 7-1 strain stored in a refrigerator at-80 ℃ is dipped with toothpick, and streaked on LB plate for activation. Selecting single colony, inoculating in liquid LB culture medium, shake culturing for 12h, regulating the concentration of strain B.gladioli 7-1 to 1× 8 CFU/mL with CMC culture medium to obtain B.gladioli 7-1 bacterial liquid, inoculating activated fusarium graminearum cake in CMC culture medium, supplementing CMC to 50mL, culturing at 25deg.C and 175rpm for 4d, and counting spore yield of fusarium graminearum with blood cell counting plate under microscope.
As shown in fig. 1, the significant difference analysis (p=0.05) was performed on the calculated control group and the b.gladioli 7-1-treated fusarium graminearum by t-test, and p <0.001 was expressed, and a significant decrease in the b.gladioli 7-1-treated fusarium graminearum was observed (fig. 1D).
Example 3
Evaluation of the ability of gladioli 7-1 to colonise wheat
(1) Wheat culture
Soaking the dried wheat seeds in clear water for 5min, soaking the wheat seeds in 75% alcohol for 2min, and washing the seeds with clear water. The seeds are placed on wet filter paper with the abdominal furrows facing downwards, the seeds are transplanted into soil after germination in a 30 ℃ incubator for 2 days, and B.gladioli 7-1 bacterial liquid is poured into the wheat regularly.
(2) After wheat is treated by root irrigation, the colonization effect of the strain is detected
With reference to example 2, a B.gladioli 7-1 bacterial solution was prepared, and the B.gladioli 7-1 bacterial solution was poured into the pots for wheat seeds every two weeks 100mL each time, and wheat was poured with liquid LB as a control, and three replicates were set for both the treatment group and the control group. Taking the root, stem, leaf and soil in the flowerpot of the wheat after one month, and separating endophytic bacteria of each tissue by adopting a dilution coating method after sample collection. Gladioli 7-1 was resistant to chloramphenicol, so 100. Mu.L of wheat root, stem, leaf and soil mill solution were aspirated, respectively, and the mixture was spread on LB plates containing chloramphenicol, and colonies on the plates were amplified and detected by using primers specific to B.gladioli 7-1 after 2d cultivation in an incubator at 25 ℃.
As shown in FIG. 2, B.gladioli7-1 was detected in both root and soil; 12 colonies were detected on LB plates coated with the stem polishing solution, and 9 colonies were amplified to the target band; presumably, the time to harvest is somewhat short and the strain is not currently detected in the leaves. The results show that the strain B.gladioli7-1 can be transported in wheat after root colonization, thereby exerting the biocontrol effect of the strain and being a potential biological agent for preventing and treating wheat scab.
Example 4
Statistics of bacteriostasis spectrum and bacteriostasis rate of gladioli 7-1
The antagonism of B.gladioli 7-1 strain against other pathogens was determined by plate-facing assays. The selected pathogens are selected from the group consisting of alternaria tenuis, wheat variety of gramineae, fusarium asiaticum, rhizoctonia cerealis, colletotrichum asiaticum and colletotrichum siamensis, and the pathogens are activated on a PDA dish three days in advance; activating strain B.gladioli 7-1 on LB plate, growing single colony, picking single colony, inoculating in liquid LB culture medium, shaking culture to bacterial concentration of 1×10 8 CFU/mL; a few filter paper strips of 4 cm. Times.1.5 cm were cut and sterilized for use. Pouring 15mL of CM solid culture medium into each 90mm disposable plastic dish, placing the sterilized filter paper strip at the center of the dish by forceps, dripping 20 μl of B.gladioli 7-1 bacterial liquid with the concentration of 1×10 8 CFU/mL on the filter paper strip, inoculating the wheat cakes of Gramineae cavity bacteria and Gramineae shell wheat varieties, asian Fusarium bacteria and Rhizoctonia cerealis, asian anthracis bacteria and Siamese anthracis bacteria on two sides of the filter paper strip respectively, and culturing in an incubator at 25 ℃ for three days to count the antibacterial rate. The calculation formula of the bacteriostasis rate is as follows: antibacterial ratio (%) = [ (control colony diameter-diameter of pathogenic bacterial colony expanding toward bacterial liquid)/control colony diameter ] ×100.
As shown in FIG. 3, B.gladioli 7-1 has antibacterial effects on Leptosporum graminearum, fusarium asiaticum, rhizoctonia graminearum, america anthracis and Amanopsis siamensis (FIG. 3A), wherein the inhibition effect on the pathogenic bacteria Leptosporum graminearum wheat variety of wheat take-all disease is best and can reach 56.3% (FIG. 3B). The above results demonstrate that B.gladioli 7-1 has a broad spectrum of bacteriostatic activity.
Example 5
Evaluation of the bacteriostatic Effect of the culture filtrate B.gladioli 7-1 on other pathogenic bacteria of wheat diseases
The B.gladioli 7-1 was streaked on LB plates. After streaking, the cells were cultured in an incubator at 25℃for 2d. Single colonies were inoculated in liquid LB medium overnight and shaken to a bacterial concentration of 1X 10 8 CFU/mL, and then each inoculated in 50mL YEPD liquid medium at an inoculum size of 1% and shaken. Culturing at 25deg.C, 175 r.min -1 for 3d, and filtering with 0.22 μm microporous membrane bacterial filter to obtain culture filtrate. 2 XCM and culture filtrate were mixed at 1:1, inoculating activated fusarium graminearum, wheat variety of top-coat wheat, asian fusarium, rhizoctonia graminearum, asian anthrax and Fusarium pseudograminearum in advance in the center of a plate after solidification, taking the treatment of the culture filtrate without B.gladioli 7-1 as a control, carrying out colony diameter measurement after culturing in a culture box at 25 ℃ for three days, and calculating the bacteriostasis rate.
As shown in FIG. 4, the culture filtrate of B.gladioli 7-1 has antibacterial effect on Fusarium graminearum, wheat variety of Potop-coat wheat, fusarium asiaticum, rhizoctonia graminearum, anthrax asiatica and Fusarium pseudograminearum (FIG. 4A), wherein the inhibition effect on Fusarium pseudograminearum is preferably 60.3% (FIG. 4B). The inhibiting effect on the pathogenic bacteria of wheat take-all disease, graminis, can reach 48.7 percent, and is similar to the opposite culture result. The above results demonstrate that the culture filtrate of B.gladioli 7-1 also has a broad spectrum of bacteriostatic activity.
Example 6
Evaluating the inhibition effect of the culture filtrate on fusarium graminearum under different culture medium shaking culture conditions
Culture filtrate shake-cultured in 5X YEG, LB, YEPD, M, PDB and CM liquid medium was prepared according to example 5.2 XCM and culture filtrate were mixed at 1:1, inoculating a pre-activated fusarium graminearum bacterial cake which is pre-activated in the center of a flat plate after solidification, taking the treatment of culture filtrate without B.gladioli7-1 as a control, culturing for three days in a 25 ℃ incubator, measuring the colony diameter, and calculating the bacteriostasis rate.
As shown in FIG. 5, the inhibition effect of B.gladioli 7-1 filtrate shake-cultured with 5 XYEG on Fusarium graminearum was the best (FIG. 5A), and the inhibition rate could reach 79.4% (FIG. 5B), so that the inhibition effect of the strain filtrate could be improved by changing the culture medium used for preparing the culture filtrate.
Example 7
Evaluation of the control Effect of gladioli 7-1 and its culture filtrate on wheat scab
Fusarium graminearum is activated on a PDA dish, cultured for 3 days at 25 ℃, and then inoculated into CMC culture medium, and shake-cultured for 3-5 days at 25 ℃ and a shaking table at 175 rpm. Filtering the spore liquid by using filter cloth, centrifuging at 3500rpm for 7min, and discarding the supernatant to obtain the spore liquid of Fusarium graminearum. A B.gladioli 7-1 strain solution was prepared as in example 2, and a culture filtrate was prepared as in example 5. The spore solution concentration of Fusarium graminearum was adjusted to 2X 10 5/mL with B.gladioli 7-1 broth and culture filtrate. In the flowering period of wheat, 10 mu L of mixed solution of fusarium graminearum spore liquid and B.gladioli 7-1, 10 mu L of mixed solution of fusarium graminearum spore liquid and culture filtrate are respectively inoculated on the fourth spike of each wheat spike from top to bottom, marks are made, and the bags are removed after bagging and moisture preservation are carried out for 48 hours. Counting the occurrence of wheat ears after 14d inoculation, and performing significant difference analysis (p=0.05) on the occurrence grain numbers treated by singly inoculating fusarium graminearum and B.gladioli 7-1 and culture filtrate thereof by using a t test, wherein p <0.001 is expressed.
As shown in FIG. 6, both the B.gladioli 7-1 bacterial liquid and the culture filtrate can obviously inhibit the disease of wheat ears (FIG. 6A) after mixed inoculation with fusarium graminearum, and the disease of the inoculated wheat ears only has single seed at an inoculation point (FIG. 6B), so that the disease seed number is obviously reduced compared with that of the fusarium graminearum singly inoculated. Ma Dongfang, lu Tao, zhan Chuang, et al, isolation and identification of wheat scab antagonistic bacteria XG-6 and application thereof [ J ]. University of Yangtze river (Natl sciences edition), 2021,18 (04): 105-113 discloses a control effect of Bacillus amyloliquefaciens XG-6 on wheat scab, which is specifically as follows: distilled water + fusarium graminearum treated wheat had a morbidity of 82.67% at day 25, whereas wheat treated with XG-6+ fusarium graminearum had a morbidity of 51.67% at day 25; deng Yun identification of an antagonistic Paenibacillus polymyxa and field control effect of the antagonistic Paenibacillus polymyxa on wheat scab [ J ]. Fujian university of agriculture and forestry (Nature science edition), 2022,51 (01): 21-26 discloses control effect of Paenibacillus polymyxa DY04 on wheat scab, and the specific method is as follows: the wheat treated by the ear spraying stock solution and the fermentation liquor is 52.64 percent and 42.24 percent respectively. The incidence of wheat treated with gladioli 7-1 bacteria solution and culture filtrate was 6.5% and 6.8%, respectively. Therefore, the control effect of the invention is better than that of the prior art. The result shows that the B.gladioli 7-1 and the culture filtrate can effectively prevent and treat wheat scab during mixed inoculation, and the effect of inhibiting the occurrence of wheat ears is very stable.
Example 8
Effects of gladioli 7-1 and culture filtrate on Fusarium graminearum DON toxin production
(1) QRT-PCR detection of expression of Fusarium graminearum TRI
Fusarium graminearum spore liquid is regulated to 10 6/mL, a single bacterial colony is picked into the spore liquid by a treatment group, and no treatment is carried out by a control group. 150 microliters of spore liquid was pipetted into a conical flask with 50mL TBI and the mycelia were collected after 4d of dark shaking. Hyphal RNA was extracted and measured for OD and DNA digestion and RNA reverse transcription were performed. And designing quantitative primers of the TRI gene and the reference gene for qRT-PCR, and finally analyzing and processing data.
(2) Detection of wheat Shan Zi grain toxigenic DON toxin production
Fusarium graminearum spore liquid, B.gladioli7-1 and culture filtrate thereof were prepared as in examples 2 and 5, and the experimental method of inoculating wheat ears was as described in example 4, and the morbidity of wheat ears was counted after 14d inoculation. And taking down the single seed after inoculation, drying, weighing and recording the quality. Putting into a centrifuge tube, adding 3mL of acetonitrile/water (84/16), grinding and smashing by a grinding instrument, oscillating on a cradle for 24h, centrifuging at 12000rpm for 1min, passing the supernatant through a PE small column, collecting filtrate, transferring 1mL of the filtrate after passing the column into a new centrifuge tube, concentrating by rotation, adding 50 mu L of TMS (TMSI/TMCS=100/1) silanization reagent, swirling to make a sample fully contact with TMS, oscillating on an oscillator for 10min, adding 800 mu L of chromatographic grade isooctane, mixing uniformly upside down, adding 800 mu L of ultrapure water, mixing uniformly upside down, standing and layering; the supernatant was transferred to a GC-MS loading flask. The content of DON toxins was determined by GC-MS, after which the DON toxin content after treatment with Fusarium graminearum and B.gladioli7-1 alone and with the culture filtrate was analyzed for significant differences by t-test (p=0.05), with p <0.001 indicated.
As shown in fig. 7, the content of single grain DON toxin was significantly reduced compared to the control after b.gladioli 7-1 and culture filtrate mixed inoculation with fusarium graminearum (fig. 7A); the qRT-PCR results of liquid toxigenic showed (FIG. 7B) that the expression level of Fusarium graminearum TRI1, TRI5 and TRI12 genes was reduced after B.gladioli 7-1 treatment. The results show that the strain can remarkably reduce accumulation of fusarium graminearum DON toxin and reduce threat to food safety.
In conclusion, the endophyte B.gladioli 7-1 can colonize in wheat bodies, has a good biocontrol effect, is a potential biocontrol preparation for preventing and treating wheat scab, and the culture filtrate of the endophyte B.gladioli 7-1 has broad-spectrum antibacterial activity, can inhibit the occurrence of scab by 100% when being mixed with fusarium graminearum PH-1 spore liquid for inoculation, and also remarkably reduces the accumulation of DON toxin. 21 metabolite synthesis gene clusters are predicted after sequencing the genome of the strain B.gladioli 7-1, so that the antibacterial compound of the strain culture filtrate is separated and identified, and the method has good development and application prospects and is hopeful to excavate new secondary metabolites.
It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (7)
1. The biocontrol bacterium for preventing and treating wheat scab is characterized by being B.gladioli 7-1, and is classified and named as Burkholderia tangutica (Burkholderia gladioli), and is preserved in China general microbiological culture collection center (CGMCC) No. 25395) at 25 months of 2022.
2. A biocontrol microbial agent for preventing and treating wheat scab, which is characterized by comprising the b.gladioli 7-1 as claimed in claim 1.
3. The biocontrol agent of claim 2, wherein said b.gladioli 7-1 concentration is 1 x 10 6-1×109 CFU/mL.
4. Use of b.gladioli 7-1 as claimed in claim 1 for the preparation of a biocontrol agent for controlling wheat scab.
5. Use of b.gladioli 7-1 as claimed in claim 1 for controlling wheat scab.
6. The use according to claim 5, wherein b.gladioli 7-1 is used for inhibiting fusarium graminearum (Fusarium graminearum).
7. Use of b.gladioli 7-1 as claimed in claim 1 for inhibiting c.graminearum (Cochliobolus sativus), c.graminearum varieties (Gaeumannomyces graminis var. Tritici), c.asiaticum (Fusarium asiaticum), c.graminearum (Rhizoctonia cerealis), c.asiaticum (Colletotrichum asianum), c.siamensis (Colletotrichum siamense) and c.pseudograminearum (Fusarium pseudograminearum).
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020036265A (en) * | 2000-11-09 | 2002-05-16 | 이재호 | The antagonist, Burkholderia gladioli GB-0999 |
| CN102286401A (en) * | 2011-07-15 | 2011-12-21 | 中国农业科学院农业资源与农业区划研究所 | Rice endotrophic azotobacter for producing activated calcium carbonate (ACC) and realizing antagonistic effect on fusarium graminearum and purpose thereof |
| CN105018371A (en) * | 2015-06-11 | 2015-11-04 | 江西师范大学 | Endophytic bacterium Burkholderia gladioli Fse32 of Dongxiang wild rice and application thereof |
| CN105524871A (en) * | 2016-01-29 | 2016-04-27 | 江苏省中国科学院植物研究所 | Method for separating and screening out antagonistic bacterium from lycoris aurea bulb |
| CN107099478A (en) * | 2017-06-06 | 2017-08-29 | 江苏省中国科学院植物研究所 | One plant of Lycoris aurea endogenetic bacteria bulkholderia cepasea HDXY 02 and its application |
| CN109574719A (en) * | 2018-12-26 | 2019-04-05 | 湖北大学 | A kind of multifunctional composite microbe bacterial manure and its application |
| CN111607537A (en) * | 2020-05-15 | 2020-09-01 | 湖北大学 | Preparation method and application of Burkholderia MEL01 for efficiently antagonizing fusarium graminearum |
| CN112899186A (en) * | 2021-01-29 | 2021-06-04 | 山东省花生研究所 | Burkholderia and application thereof in biological control of fusarium graminearum |
| CN115232776A (en) * | 2022-09-06 | 2022-10-25 | 西北农林科技大学 | Pseudomonas CXZ8 for preventing and treating wheat scab, biocontrol microbial inoculum and application thereof |
| CN116218709A (en) * | 2022-12-16 | 2023-06-06 | 安徽农业大学 | Endophytic Burkholderia gladioli JS-59 and application thereof |
-
2022
- 2022-09-07 CN CN202211098603.0A patent/CN115851495B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020036265A (en) * | 2000-11-09 | 2002-05-16 | 이재호 | The antagonist, Burkholderia gladioli GB-0999 |
| CN102286401A (en) * | 2011-07-15 | 2011-12-21 | 中国农业科学院农业资源与农业区划研究所 | Rice endotrophic azotobacter for producing activated calcium carbonate (ACC) and realizing antagonistic effect on fusarium graminearum and purpose thereof |
| CN105018371A (en) * | 2015-06-11 | 2015-11-04 | 江西师范大学 | Endophytic bacterium Burkholderia gladioli Fse32 of Dongxiang wild rice and application thereof |
| CN105524871A (en) * | 2016-01-29 | 2016-04-27 | 江苏省中国科学院植物研究所 | Method for separating and screening out antagonistic bacterium from lycoris aurea bulb |
| CN107099478A (en) * | 2017-06-06 | 2017-08-29 | 江苏省中国科学院植物研究所 | One plant of Lycoris aurea endogenetic bacteria bulkholderia cepasea HDXY 02 and its application |
| CN109574719A (en) * | 2018-12-26 | 2019-04-05 | 湖北大学 | A kind of multifunctional composite microbe bacterial manure and its application |
| CN111607537A (en) * | 2020-05-15 | 2020-09-01 | 湖北大学 | Preparation method and application of Burkholderia MEL01 for efficiently antagonizing fusarium graminearum |
| CN112899186A (en) * | 2021-01-29 | 2021-06-04 | 山东省花生研究所 | Burkholderia and application thereof in biological control of fusarium graminearum |
| CN115232776A (en) * | 2022-09-06 | 2022-10-25 | 西北农林科技大学 | Pseudomonas CXZ8 for preventing and treating wheat scab, biocontrol microbial inoculum and application thereof |
| CN116218709A (en) * | 2022-12-16 | 2023-06-06 | 安徽农业大学 | Endophytic Burkholderia gladioli JS-59 and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| 1株拮抗真菌的生防细菌Xj11的分离与鉴定;敖武 等;微生物学杂志;20130615;第33卷(第3期);第45-48页 * |
| Burkholderia gladioli, an endophyte with plant growth promoting potential;Aparma Gunjal 等;Journal of Chemo and Biosphere, RAAB Proceeding Issue;20130228;第43-53页 * |
| Toxoflavin Produced by Burkholderia gladioli from Lycoris aurea Is a New Broad-Spectrum Fungicide;Xiaodan Li 等;Applied and Environmental Microbiology;20190418;第85卷(第9期);第1-13页 * |
| 伯克霍尔德氏菌CR7防治小麦赤霉病的作用机制初探;王瑜;西北农林科技大学学位论文;20230501;第1-60页 * |
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