CN115851476A - Rice root endophytic bacillus altitudinis 258R-7 and biological agent and application thereof - Google Patents
Rice root endophytic bacillus altitudinis 258R-7 and biological agent and application thereof Download PDFInfo
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Abstract
The invention discloses a rice root endophytic geobacillus 258R-7 and a biological preparation and application thereof, belonging to the field of biological pesticides, and the key points of the technical scheme are as follows: the collection number of the bacillus altitudinis 258R-7 is GDMCC No: 62070A composition for treating diabetes (ii) a; bacillus altitudinis 258R-7 is applied to preventing and controlling rice blast; inoculating the Geobacillus altivelis 258R-7 into an LB liquid culture medium for culture, thus obtaining the biological agent. The invention is mainly used for inhibiting rice blast germs, enhancing the rice blast resistance of in vitro leaves of rice, having excellent potential biological prevention and control effects for preventing and controlling the diseases, and the biological source is environment-friendly and nontoxic, and the biological source is sprayed on the surfaces of the rice leaves for preventing and controlling the diseases, has little influence on the ecological environment, and is safer and more reliable for the eating of rice and the processed products thereof; the culture condition requirement is low, and the method has good development and application prospects.
Description
Technical Field
The invention relates to the field of biopesticides, and particularly relates to a rice root endophytic geobacillus 258R-7 and a biological preparation and application thereof.
Background
Rice (Oryza sativa l.) is used as one of the three staple grains in the world, is an important guarantee for human survival and development. However, various rice regions in the world suffer from various degrees of diseases and pests every year are major factors that restrict rice production, with rice blast caused by Magnaporthe oryzae (Magnaporthe oryzae) being the most devastating disease in rice production. At present, chemical pesticide spraying is combined with field water and fertilizer management and disease-resistant variety breeding and the like in rice production as main methods for disease control. However, the long-term use of a single chemical agent or a compound chemical agent easily causes the problems of drug resistance of rice, pesticide residue, environmental pollution, food safety and the like, and the problems of long breeding time, easy loss of disease resistance due to the change of physiological race of rice blast germs and the like are often caused although the problems can not be caused when breeding disease-resistant varieties. Therefore, the search for a rapid, effective and safe way for preventing and treating diseases is of great significance for realizing the sustainable development of the rice industry.
Biological control refers to a technology for effectively preventing and controlling crop diseases and insect pests by using beneficial organisms and/or metabolites thereof, and is a great development trend in the field of agricultural disease and insect pest control research internationally due to the advantages of high selection (safety to human, animals and the like, small influence on the environment, strong inhibition effect only on pathogenic substances), difficult resistance generation, abundant resources, easy development and the like. Studies have shown that 30% of beneficial organisms and/or their metabolites are derived from microorganisms and their related products, most of which are isolated from different microenvironments such as soil, ocean or air, and although these microorganisms also have inhibitory effects on many pathogenic bacteria, they are difficult to colonize and express in host plants, resulting in poor disease control. Therefore, it is of great significance to excavate the microorganisms in the plant tissues which have the inhibiting effect on pathogenic bacteria and research the disease prevention mechanism of the microorganisms.
Research reports that antagonistic bacteria which are separated and screened from various microenvironments and have the best inhibitory effect on rice blast are Bacillus, for example, lipopeptide compounds synthesized and secreted by Bacillus subtilis Bs916 are key factors for resisting various diseases including rice blast of rice; the fungicide developed by Bacillus megaterium has good rice blast field control effect and can be applied in large area; the control effect of Bacillus firmus (Bacillus adamantium) B-332 on rice blast is as high as more than 70 percent; the control effect of the fermentation filtrate of the brevibacillus laterosporus (Bacillus brachyspora) BPM3 separated from the Indian hot spring soil on the rice blast is 30-67 percent; bacillus licheniformis (BS-3) achieves the effect of efficiently inhibiting rice blast bacteria by producing a 31kDa antifungal polypeptide.
At present, the research and utilization of Bacillus altitudinis (Bacillus altitudinis) as a biocontrol bacterium are mostly focused on the control of rice bacterial leaf streak and root-knot nematode, but the inhibition of rice blast bacteria by Bacillus altitudinis is rarely reported.
In order to solve the problems, a rice root endophytic Bacillus altivelis 258R-7, a biological agent and application thereof are provided on the basis of the prior art.
Disclosure of Invention
The invention aims to provide a rice root endophytic geobacillus 258R-7 and a biological preparation and application thereof, wherein the strain is derived from rice roots and can be well colonized in rice tissues; the biological source is environment-friendly and nontoxic, and the biological source is sprayed on the surfaces of rice leaves for preventing and treating diseases, has little influence on the ecological environment and is safer and more reliable for the eating of rice and processed products thereof; the culture condition requirement is low, and the method has good development and application prospects.
The technical purpose of the invention is realized by the following technical scheme:
the rice root endophytic geobacillus 258R-7 is preserved in Guangdong province microbial strain preservation center in 2021 at 11 th and 17 th, and the preservation address is Guangzhou city Middy 100 # 59 th building 5 th Guangdong province microbial research institute, and the preservation number of the geobacillus 258R-7 is GDMCC No:62070.
further, the Bacillus altitudinis 258R-7 is applied to control rice blast.
Further, pathogenic strains of the rice blast comprise rice blast fungus Guy11, rice blast fungus B157, rice blast fungus Y7, rice blast fungus 98-06, rice blast fungus Y34 and rice blast fungus 70-15.
Through the technical scheme, the bacillus altitudinis 258R-7 is obtained by separation and purification from rice roots, and the bacillus altitudinis 258R-7 endogenously cultured on an LB culture medium for 24 hours at the temperature of 28-30 ℃, so that bacterial colonies are white, dry in surface, rough and opaque, have spores, are oval in spores, and are mesogenic or subterminal.
The invention also provides a biological agent which is prepared on the basis of the bacillus altitudinis 258R-7.
Further, the biological agent is prepared by fermenting the bacillus altitudinis 258R-7 with liquid.
Further, the geobacillus 258R-7 is inoculated into an LB liquid culture medium for culture, and the biological agent is obtained.
Further, the pH of the LB liquid medium was 7.0.
Further, the culture is carried out for 24 hours under the conditions that the temperature is 28-30 ℃ and the shaking speed of a shaking table is 180-200 rpm.
Further, the biological agent is applied to control rice blast.
Further, pathogenic strains of the rice blast comprise rice blast fungus Guy11, rice blast fungus B157, rice blast fungus Y7, rice blast fungus 98-06, rice blast fungus Y34 and rice blast fungus 70-15.
In conclusion, the invention has the following beneficial effects:
1. the obtained highland bacillus 258R-7 comes from rice roots and can be well colonized in rice tissues (or surfaces); in addition, the antagonistic endophyte screened from the rice roots is used for preventing and treating rice blast, only shows that the number of certain microorganisms among specific populations is increased or reduced, the types of the microorganisms inside and outside the rice tissues are not changed, so that the population stability is influenced, in addition, as the antagonistic endophyte coexists with the rice for a long time and mutually grows, a series of survival mechanisms similar to the antagonistic endophyte are formed, and the antagonistic endophyte is sprayed on the surfaces of the rice tissues for preventing and treating diseases, so that the antagonistic endophyte is safer and more reliable for rice and processed products thereof.
2. The bacillus altitudinis has strong inhibition effect on a plurality of rice blast bacterial strains, has excellent effect of preventing and treating rice blast, and is environment-friendly and nontoxic in biological source and small in influence on ecological environment.
3. The Bacillus altitudinis obtained by the invention has low requirement on culture conditions and has good development and application prospects.
Drawings
FIG. 1 is a diagram of a single colony of Bacillus altitudinis 258R-7 on LB medium of example 1 of the present invention;
FIG. 2 is a graph showing the inhibitory effect of different forms of Bacillus hyperviscosity 258R-7 and its biological agents (thallus, fermentation broth, supernatant filtrate) on a plurality of rice blast fungus strains (70-15, guy11, B157, 98-06, Y7, Y34) in example 2 of the present invention and a control (blank control and LB control);
FIG. 3 is a graph showing the control effect of Bacillus altitudinis 258R-7 of example 3 of the present invention on Pyricularia oryzae Guy11 based on detached leaves of rice, "-" indicates no inoculation in treatment and "+" indicates inoculation.
Detailed Description
The invention is described in further detail below with reference to the following figures and embodiments:
example 1: the separation, purification and preservation of the rice root endophytic geobacillus 258R-7 comprises the following steps:
(1) Preparation of LB medium: weighing 10g of Tryptone (Tryptone, oxoid LTD LP0042, england), 5g of Yeast extract (Yeast extract, oxoid LTD LP0021, england), 10g of sodium chloride (NaCl, national drug group chemical reagent Co., ltd., 10019318), adding 1000mL of water, stirring uniformly, adding 15g of agar when preparing a solid culture medium, fully heating and dissolving, subpackaging, sterilizing at 121 ℃ for 20min, and storing for later use.
(2) Separation and purification of rice root endophytic geobacillus:
the root system of rice for separating the endogenous microorganism is collected from the area Lv Tianzhen Lianma village in Guangzhou city, guangdong province.
The separation method of the endophytic bacteria specifically comprises the following operations: washing rice blast resistant rice root system with tap water, drying in the air, weighing root tissue 10g, soaking in 70 vol% alcohol for 2min, surface sterilizing with 2.5 vol% sodium hypochlorite for 10min, washing with sterile water for 4 times, sucking water on sterile filter paper, transferring the sample into a sterile mortar, adding 10mL of sterile water, grinding, standing for 15min, coating 100 microliter with LB plate, repeating the test treatment for 3 times, culturing at 28 deg.C for 48-72 h, and observing the result.
The disinfection effect of the tissue surface is tested by adopting a rinsing liquid test method (Schulz et al, 1993), 200 mu L of rinsing liquid of the disinfection material rinsed at the last time is coated on an LB solid plate for culture, and aseptic colonies are observed to grow out after the culture, so that the surface of the material is completely sterilized, otherwise, the separation result cannot be used.
The homology of the 16S rRNA sequence of the strain isolated from the Guangzhou city derivation area Lv Tianzhen Lianma village rice blast-resistant rice root in Guangdong province and the 16S rRNA sequence of the Bacillus altitudinis 19RS3 strain 16S with the accession number of MH883312.1 in GenBank reaches 99.93 percent, and the classification and the designation of the strain are proved to be Bacillus altitudinis (Bacillus altitudinis) and Bacillus altitudinis (Bacillus altitudinis) 258R-7 by combining the colony morphology characteristics and the related physiological and biochemical characteristics of the strain on an LB culture medium plate. Sequence of fragments the following were used:
CACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTTGAACCGCATGGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCAAGAGTAACTGCTTGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCTGCGAGACCGCAAGGTTTAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCTTT。
the Geobacillus altivelis 258R-7 is cultured on LB culture medium at 28 ℃ for 24 hours, and the colony is white, dry in surface, rough and opaque, has spores, is oval in shape, and is mesogenic or subterminal, as shown in figure 1.
(3) Preservation of Bacillus altivelis 258R-7: the strain is preserved in Guangdong province microorganism culture collection center at 11 months and 17 days in 2021, and the preservation number is GDMCC No:62070.
example 2: determination of bacteriostatic activity of Bacillus altitudinis 258R-7 by opposite culture method
(1) LB medium was prepared as in example 1; the LB liquid culture medium is prepared by the same formula as the LB solid culture medium except that agar is not added, the weighed medicament is fully dissolved and then is subpackaged into conical flasks (each flask is 100mL of culture solution), the conical flasks are plugged and bound, sterilized at 121 ℃ for 20min, and the conical flasks are cooled and stored for later use.
(2) Preparation of CM medium: weighing 6g of Yeast extract, 6g of enzymatically hydrolyzed casein Casamino acid and 10g of Sucrose in a measuring cup, fixing the volume to 1000mL, fully heating to dissolve, subpackaging in conical flasks (each flask is 100mL of culture solution), adding 1% agar powder, sterilizing at 121 ℃ for 20min, cooling and storing for later use.
(3) Preparation of a single colony of Bacillus altitudinis: and (3) streaking and inoculating the highland bacillus onto an LB culture medium plate for activation culture for 24h, and then picking out a single colony for streaking and storing.
(4) Preparation of geobacillus fermentation broth (biological agent) and supernatant filtrate: inoculating the single bacterial colony of the bacillus plateau prepared in the step (3) into the LB liquid culture medium conical flask prepared in the step (1), and culturing at 28 ℃ and at the shaking table speed of 200rpm for 24 hours to obtain a bacillus plateau fermentation liquid; the obtained fermentation liquid was centrifuged at 12000rpm for 10min, and the supernatant was collected and filtered through a 0.2 μm filter to remove the cells, thereby obtaining a Bacillus altitudinis supernatant filtrate.
(5) And (3) activity determination:
(1) the inhibition effect of the high-upland bacillus on the growth of a plurality of rice blast germs (70-15, guy11, B157, 98-06, Y7 and Y34) is determined: folding the filter paper with the diameter of 9cm in half, turning the filter paper for 90 degrees, folding the filter paper in half again, and fixing four points of the culture dish. Inoculating the magnaporthe grisea dish to a spot, and after 4 days, streaking and inoculating the geobacillus thallus prepared in the step (3) to a spot opposite to the spot. The experiment was conducted in 3 replicates each of the test samples without inoculating Bacillus altitudinis (first row in FIG. 2: blank control) and with inoculating LB liquid medium (second row in FIG. 2: LB control), and incubated at a constant temperature of 28 ℃. After 4 days, the growth of Pyricularia oryzae was observed, and the results are shown in the third column of FIG. 2.
(2) The inhibition effect of the fermentation liquor of the geobacillus on the growth of a plurality of rice blast germs (70-15, guy11, B157, 98-06, Y7 and Y34) is determined: and (2) replacing the geobacillus bacteria in the step (1) with 200 mu L of geobacillus fermentation liquor filled in an Oxford cup, and keeping the rest steps unchanged. Preparing fermentation liquor: a single colony of Bacillus altitudinis was inoculated into 5mL of LB liquid medium, cultured at 28 ℃ for 24 hours at a shaker speed of 200rpm, and then placed in a refrigerator at 4 ℃ for further use, and the results are shown in the fourth column of FIG. 2.
(3) The inhibition effect of the supernatant filtrate of the high-upland bacillus on the growth of a plurality of rice blast germs (70-15, guy11, B157, 98-06, Y7 and Y34) is determined: and (3) replacing the fermentation liquor of the geobacillus in the step (2) with 200 mu L of the supernatant of the geobacillus in an Oxford cup, and keeping the rest steps unchanged. Preparation of the supernatant: and (3) putting the fermentation liquor of the bacillus altitudinis in the step (2) into a centrifuge, centrifuging at 12000rpm for 10min, sucking the supernatant, and filtering by using a 0.2-micron filter to remove thalli, namely the supernatant filtrate, wherein the result is shown in the fifth column of the figure 2.
Example 3: method for determining control effect of bacillus 258R-7 on rice blast fungus Guy11 based on rice in-vitro leaves
(1) And (3) cultivation of rice seedlings: selecting healthy and plump rice seeds, drying to break dormancy, soaking and disinfecting with a sodium hypochlorite solution, washing with sterile deionized water, continuously soaking the seeds with the sterile deionized water for germination acceleration, transferring the seeds into a 96-hole PCR plate with the bottom cut off in advance with a sterilized forceps after the seeds are exposed to white, firstly culturing with the sterile deionized water until the first leaf and the first heart stage, then culturing with a 1/4 final concentration international rice formula nutrient solution until the second leaf and the first heart stage, then culturing with a 1/2 final concentration international rice formula nutrient solution until the third leaf and the first heart stage, and finally culturing seedlings with a 1 multiplied international rice formula nutrient solution until the fourth leaf and the first heart stage for later use;
(2) Preparation of a rice blast fungus dish: activating and culturing Magnaporthe grisea Guy11 in PA medium, culturing in dark at 28 deg.C for 3d, culturing alternately in light and dark for 4 days (light/dark =12h/12 h), and perforating the edge of colony with a perforator with diameter of 8 mm;
(3) Preparing a geobacillus fermentation liquid: the procedure is as in example 2 (4), one drop of tween 20 is added before use;
(4) And (3) determining the control effect:
(1) preparation of leaves of rice to be inoculated: adhering double-sided adhesive tapes to two opposite sides of a square culture dish (side length is 13 cm), shearing healthy rice leaves (12 leaves) with consistent culture time, arranging and placing the rice leaves in the culture dish (the head and the tail of each leaf are respectively adhered to the double-sided adhesive tapes), marking the rice leaves with the numbers of 1-12, and pressing sterile water-soaked absorbent cotton at two ends of each leaf (the adhesion part of the double-sided adhesive tapes) for moisturizing.
(2) And (3) dipping sterile water (a drop of Tween 20 is added in advance) by using a brush, uniformly brushing the surface of the rice leaf with the number of 1-6, dipping the prepared fermentation liquor of the bacillus altitudinis in addition, uniformly brushing the surface of the rice leaf with the number of 7-12, spraying a proper amount of sterile water on a culture dish cover, covering the culture dish cover, putting the whole culture dish into a tray, adding a proper amount of sterile water into the tray (no water seepage can be caused, the culture dish can be filled with the sterile water), sealing the tray by using a preservative film, and placing the tray in the dark at 28 ℃ for culturing for 24 hours.
(3) Taking out the tray which is cultured in the dark for 24 hours, tearing off the preservative film, opening the cover of the culture dish, inoculating the rice blast fungus plate upside-down (the hypha surface is attached to the surface of the rice leaf) to rice leaves with the numbers of 4-6 and 10-12, inoculating the PA culture medium block to the rice leaves with the numbers of 1-3 and 7-9 as blank control, covering the dish cover, placing the dish cover in the tray (ensuring a proper amount of sterile water to achieve the moisturizing effect), sealing the tray by the preservative film, placing the tray in the dark at 28 ℃ for 24 hours, transferring the tray into an incubator with light/dark alternation for 12 hours, and culturing the tray at 28 ℃ for 5 days, wherein the result is shown in figure 3.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications without inventive contribution to the present embodiment as required after reading the present specification, but all of them are protected by patent law within the scope of the present invention.
Claims (10)
1. An endophytic geobacillus altitudinis 258R-7 of rice roots is characterized in that: the collection number of the bacillus altitudinis 258R-7 is GDMCC No:62070.
2. the application of the Bacillus mucilaginosus 258R-7 in rice roots according to claim 1, wherein the Bacillus mucilaginosus 258R-7 comprises the following components in percentage by weight: the bacillus altitudinis 258R-7 is applied to preventing and treating rice blast.
3. The application of the Bacillus mucilaginosus 258R-7 strain of rice roots as claimed in claim 2, wherein the Bacillus mucilaginosus is characterized in that: the pathogenic strains of the rice blast comprise rice blast germs Guy11, rice blast germs B157, rice blast germs Y7, rice blast germs 98-06, rice blast germs Y34 and rice blast germs 70-15.
4. A biological agent characterized by: the biological agent is prepared based on Bacillus altitudinis 258R-7.
5. A biological agent according to claim 4, wherein: the biological agent is prepared by fermenting bacillus altitudinis 258R-7 with liquid.
6. A biological agent according to claim 4, wherein the biological agent is prepared by a method comprising: inoculating the Geobacillus altivelis 258R-7 into an LB liquid culture medium for culture, and obtaining the biological agent.
7. A biological agent according to claim 6, wherein: the pH value of the LB liquid culture medium is 7.0.
8. A biological agent according to claim 6 or 7, wherein: the culture is carried out for 24 hours under the conditions that the temperature is 28-30 ℃ and the shaking speed of a shaking table is 180-200 rpm.
9. Use of a biological agent according to any of claims 4 to 7, wherein: the biological agent is applied to control rice blast.
10. The use of a biological agent as claimed in claim 9, wherein: the pathogenic strains of the rice blast comprise rice blast germs Guy11, rice blast germs B157, rice blast germs Y7, rice blast germs 98-06, rice blast germs Y34 and rice blast germs 70-15.
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| CN117165449A (en) * | 2023-09-06 | 2023-12-05 | 广东省农业科学院水稻研究所 | Rice endophytic cladosporium cucumerinum JZ4, biological preparation thereof and application of biological preparation in preventing and controlling rice blast bacteria |
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| KR20150057083A (en) * | 2013-11-18 | 2015-05-28 | 대한민국(농촌진흥청장) | Antimicrobial bacillus subtilis HC57 for cladobotryum mycophilum, and process for culturing thereof |
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| CN117165449A (en) * | 2023-09-06 | 2023-12-05 | 广东省农业科学院水稻研究所 | Rice endophytic cladosporium cucumerinum JZ4, biological preparation thereof and application of biological preparation in preventing and controlling rice blast bacteria |
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