CN116254192B - Pacific bacillus rice strain for producing siderophore and application thereof - Google Patents
Pacific bacillus rice strain for producing siderophore and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/20—Cereals
- A01G22/22—Rice
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a Pacific bacillus rice strain producing siderophores and application thereof. Pacific bacillus rice ORL1-5 strain producing siderophores of the invention is deposited in the Guangdong province microorganism strain collection at the year 2022, 11 and 13, and the deposit number is GDMCC No:62966. the ORL1-5 strain has the capability of producing siderophores, can promote the absorption of iron elements by rice, improve the chlorophyll content of leaves, promote the growth of the rice, promote the tillering and grouting of the rice and further improve the single plant yield of the rice, can be used for preparing products for improving the yield of the rice, such as microbial fertilizers and the like, and is beneficial to guaranteeing the grain safety.
Description
Technical Field
The invention belongs to the technical field of endophytes. More particularly relates to a Pacific bacillus rice strain producing siderophore and application thereof.
Background
Rice (Oryza sativa L.) is one of the most important grains for human beings, and the population taking rice as main food in the world accounts for more than half of the total population, so that the rice production is important to guaranteeing the grain safety.
In the normal growth and development process of rice, the necessary mineral nutrient elements are continuously absorbed from the outside besides the conditions of sunlight, moisture, air and the like. Among mineral nutrient elements required for rice, iron occupies an important position, participates in a plurality of physiological and biochemical reaction processes, and has an indispensable effect on the whole life history of plants. Iron is also an important nutrient element affecting chloroplast function, and researches show that 80% of iron in the leaf is located in chloroplasts and plays an important role in maintaining the integrity of chloroplast structures. The structural damage of chloroplast caused by iron deficiency can influence chlorophyll synthesis, so that photosynthesis of plants is reduced, synthesis of electron transfer chain complex is blocked, enzyme activity and a series of oxidation-reduction reactions are also influenced, oxidation resistance of plants is reduced, respiration is weakened, metabolic activity in plant cells is disturbed, green-losing symptoms of young leaves are shown, and crops are reduced in yield and quality.
Iron deficiency and green loss of plants is a worldwide problem of plant malnutrition. Although the abundance of iron element in soil is very high, the available iron which can be absorbed and utilized by plants is very small, so that the plants are very easy to be deficient in iron, and particularly in arid and semiarid calcareous soil, the phenomenon of iron deficiency is more serious. And the area of the global arid region is continuously increased along with the influence of factors such as global warming, increasing greenhouse gas emission, enhancing human activities and the like, and the iron deficiency and green loss of plants are serious. Although the application of the iron fertilizer is a common method for correcting iron deficiency of plants, the iron fertilizer is mainly inorganic iron fertilizer, namely ferrous sulfate heptahydrate, and when the iron fertilizer is excessively applied, the acidity in soil can be increased, so that element deficiency diseases of elements such as potassium, calcium, magnesium, phosphorus and the like are easily caused, and plants are withered and even necrotized. In view of the abundant content of iron elements in soil, the absorption of iron elements by plants is promoted or the iron deficiency and green loss of plants can be effectively solved.
It was found that part of the microorganisms can secrete soluble organics, together with Fe 3+ Specific binding creates stable complexes that help to increase iron concentration in the soil, promoting iron absorption by the root system, a substance known as siderophores. Many plants can absorb siderophores in the soil, so microorganisms secreting siderophores in the soil are important for plants to absorb and utilize the siderophores. The plant endophyte can colonize in cells or cells of various tissues and organs of the plant, establishes a harmonious symbiotic relationship with the plant, is an important component part of a plant micro-ecological system, and has important ecological effect and agricultural application value. The endophytic bacteria of the plants have rich biodiversity, can colonize and conduct in the plants for a long time, are not easily influenced by external environment conditions, and are ideal biocontrol and biological fertilizer resources.
Although report about plant endophyte capable of producing siderophores exists at present, the method has the advantages that the absorption of iron elements by rice can be promoted, less endophyte for improving the yield of rice is provided, and the plant endophyte library capable of continuously enriching siderophores is of great value for the development of biological bacterial fertilizers.
Disclosure of Invention
The invention aims to overcome the defect of the existing plant endophytic bacteria which lack the capability of promoting the iron element absorption of rice, and provides a Pacific bacillus strain ORL1-5 for producing siderophores and application thereof.
The first object of the present invention is to provide a Pacific Bacillus Pacific rice ORL1-5 strain which is a siderophore-producing strain.
A second object of the present invention is to provide the use of said Pacific Bacillus Pacific ORL1-5 strain for promoting rice growth.
A third object of the present invention is to provide the use of said Pacific bacillus rice ORL1-5 strain in the preparation of a product for promoting rice growth.
The fourth object of the invention is to provide the application of the Pacific bacillus rice ORL1-5 strain in improving rice yield.
The fifth object of the invention is to provide the application of the Pacific bacillus Pacific rice ORL1-5 strain in the preparation of products for improving rice yield.
The sixth object of the invention is to provide the application of the Pacific bacillus strain ORL1-5 in promoting iron absorption of rice and increasing chlorophyll content of rice.
The seventh object of the invention is to provide the application of the Pacific bacillus rice ORL1-5 strain in preparing products for promoting iron absorption of rice and improving chlorophyll content of rice.
An eighth object of the present invention is to provide a preparation for improving rice yield.
A ninth object of the present invention is to provide a method for improving rice yield.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a Pacific bacillus (Bacillus pacificus) ORL1-5 strain of paddy rice for producing siderophores, which is deposited in the microorganism strain collection of Guangdong province at the year 2022, 11 and 13, and the deposit number is GDMCC No:62966 the preservation address is building No. 100 and building No. 59 in the Mitsui of Guangzhou City of Guangdong.
The Pacific bacillus rice ORL1-5 strain provided by the invention can promote the absorption of iron elements by rice, improve the chlorophyll content of rice leaves, promote the growth of rice seedlings and improve the grain yield of rice. Thus, the present application protects the following uses of the Pacific Bacillus oryzae ORL1-5 strain:
the invention claims the application of the Pacific bacillus strain ORL1-5 in promoting the growth of rice.
The invention also claims the application of the Pacific bacillus strain ORL1-5 in preparing products for promoting rice growth.
The invention claims the application of the Pacific bacillus strain ORL1-5 in improving the yield of rice.
The invention also claims the application of the Pacific bacillus strain ORL1-5 in preparing the products for improving the rice yield.
The invention claims the application of the Pacific bacillus strain ORL1-5 in promoting the absorption of iron element by rice and improving chlorophyll content of rice.
The invention also claims the application of the Pacific bacillus strain ORL1-5 in preparing the product for promoting the absorption of iron element by rice and improving the chlorophyll content of rice.
The Pacific bacillus rice ORL1-5 strain can produce siderophores and promote host plants to absorb iron, and can be used for preventing and/or treating iron deficiency and greening of plants and preparing products for preventing and/or treating iron deficiency and greening of plants.
Alternatively, the product may comprise a seed soaking agent or a microbial fertilizer, etc.
Specifically, the rice used in the embodiment of the invention is Chu japonica 28, and Pacific bacillus strain ORL1-5 is isolated from common wild rice, which indicates that the strain ORL1-5 can be colonized in different rice.
The invention also provides a preparation for improving the yield of rice, which takes the Pacific bacillus ORL1-5 strain or bacterial liquid thereof as an active ingredient.
The invention also provides a method for improving the yield of rice, namely, before rice seeds germinate, seed soaking treatment is carried out by using the bacterial liquid of the Pacific bacillus ORL1-5 strain of rice.
Specifically, the concentration of the bacterial liquid is 1.0X10 7 ～1.0×10 9 cfu/mL, soaking seeds or soaking treatment time is10～14h。
Specifically, the rice is japonica rice.
More specifically, the rice is Chu japonica 28.
The invention has the following beneficial effects:
the invention provides a Pacific bacillus rice ORL1-5 strain for producing siderophores, which is preserved in the microorganism strain collection of Guangdong province at the year 2022, month 11 and 13, and the preservation number is GDMCCNo:62966. the ORL1-5 strain not only has the capability of producing siderophores, can promote the absorption of rice to iron elements and improve the chlorophyll content of leaves, but also can promote the growth of rice, promote the tillering and grouting of the rice so as to improve the single plant yield of the rice, can be used for preparing products for improving the yield of the rice, such as microbial fertilizers, and has wide application prospects in the aspects of improving the yield of the rice, and the like, is beneficial to stabilizing or improving the yield of the rice and guaranteeing the grain safety.
Drawings
FIG. 1 shows the morphological characteristics and gram staining characteristics of single colonies of the Pacific Bacillus Pacific ORL1-5 strain of Pacific rice.
FIG. 2 shows the result of electrophoresis detection of the 16S rRNA gene fragment of amplified Pacific bacillus strain ORL 1-5; m in the figure is a DNA marker;1 is the 16S rRNA gene of the amplified ORL1-5 strain.
FIG. 3 is a phylogenetic tree of the strain ORL1-5 of Pacific bacillus oryzae.
FIG. 4 shows the total iron content comparison of Chu japonica 28 seedlings after seed soaking treatment of Pacific bacillus strain ORL1-5 with Control (CK); in the figure, p < 0.01 is represented.
FIG. 5 shows the chlorophyll content comparison result of Chu japonica 28 seedlings after seed soaking treatment of Pacific bacillus strain ORL1-5 with Control (CK); in the figure, p < 0.01 is represented.
FIG. 6 shows the phenotypic comparison of Chu japonica 28 seedlings after seed soaking treatment of Pacific bacillus strain ORL1-5 with Control (CK) plants.
FIG. 7 shows the plant height and root length comparison results of Chu japonica 28 seedlings after seed soaking treatment of Pacific bacillus strain ORL1-5 with Control (CK); in the figure, p < 0.01 is represented.
FIG. 8 is the effect of Pacific bacillus strain ORL1-5 on Chu japonica 28 yield property index; A-E in the graph are the influence results of ORL1-5 strain on the setting rate of Chu japonica 28, the number of grains per ear (seed), thousand grain weight, effective spike number and single plant yield in sequence; CK is an untreated control, and Treatent is Chu japonica 28 plants after seed soaking Treatment by using ORL1-5 strain; in the figure, p < 0.05 is represented.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The ORL1-5 strain in the embodiment of the invention is Pacific bacillus (Bacillus pacificus) ORL1-5 strain of rice, which is deposited in the microorganism strain collection of Guangdong province at 11/13 of 2022, with the deposit number of GDMCC No:62966 the preservation address is building No. 100 and building No. 59 in the Mitsui of Guangzhou City of Guangdong.
NA medium: 10g of peptone, 3g of beef extract, 5g of NaCl, 20g of agar, constant volume of distilled water to 1L, pH adjustment to 7.0-7.5 and sterilization at 121 ℃ for 20min.
NB medium: 10g of peptone, 3g of beef extract, 5g of NaCl, distilled water to 1L, regulating the pH to 7.0-7.5, and sterilizing at 121 ℃ for 20min.
CAS detection solution: 6mL of 10mM cetyltrimethylammonium bromide was added to a 100mL volumetric flask, and 1.5mL of 1mM FeCl was taken 3 The mixture of the solution and 7.5mL of 2mM Chromium Azurin (CAS) was dissolved in 30mL of sterile water to give 4.3g of anhydrous piperazine, and 6.25mL of 12mM HCl was added to give a buffer solution of pH=5.6, which was added to the above-mentioned volumetric flask, and the volume was fixed to 100mL, and mixed well.
EXAMPLE 1 isolation and purification of Pacific Bacillus Pacific ORL1-5 Strain of Rice
The Pacific bacillus Pacific ORL1-5 strain of the invention is separated from leaf tissues of common wild rice, and the separation and purification process is as follows:
and (3) taking healthy leaves of ordinary wild rice, washing surface stains with tap water, and then sucking surface moisture with filter paper to perform surface disinfection. The surface disinfection procedure is as follows: soaking in 75% alcohol for 2min 30s, rinsing with sterile water for 1 time, soaking in 2.5% sodium hypochlorite for 2min, and rinsing with sterile water for 3 times.
The plant tissue with the surface sterilized is dried by using sterilizing filter paper, put into a sterile mortar, added with a proper amount of sterile water for grinding, and the grinding liquid is diluted (the dilution concentration is 10) -2 、10 -3 、10 -4 And 10 -5 ) Sucking 100 mu L of the culture medium to NA, and uniformly coating the culture medium by using a sterile coater; simultaneously sucking 100 mu L of 3 rd rinsing liquid coating plate to detect whether the tissue surface is thoroughly disinfected; inverting the NA culture medium, culturing in a dark incubator at 37 ℃ for 1-2 d until colonies grow out, observing colony morphology, and picking bacterial colonies with different morphologies for purification; after numbering, glycerol preservation is adopted to preserve strains at-80 ℃.
The culture temperature of the Pacific bacillus strain ORL1-5 of rice is 28-37 ℃, and the activation culture method comprises the following steps: taking out the strain of the ORL1-5 strain stored at the temperature of minus 80 ℃, dipping a loop of bacteria liquid on the surface of the NA flat plate by using an inoculating loop, streaking, culturing for 1-2 d at the temperature of 37 ℃ in an inverted way, picking single bacterial colony, inoculating the single bacterial colony to 800 mu L of NB culture medium, culturing for 16-24 h at the temperature of 37 ℃ at 200rpm in an oscillating way.
EXAMPLE 2 identification of Pacific Bacillus Pacific ORL1-5 Strain of Rice
1. Morphological identification
(1) Colony morphology characterization
After the preserved ORL1-5 strain was activated, streaking was performed on the NA plate, and after culturing in a constant temperature incubator at 28℃for 24 hours, the single colony morphology of the strain was observed using a stereoscope.
(2) Gram staining characteristics of colonies single colonies of ORL1-5 strain were picked from NA plate and inoculated in NB medium at 37℃and 200rpm, cultured for 16-24 hours with shaking, and gram staining was performed with an appropriate amount of bacterial liquid and observed for the morphological characteristics under a microscope.
The morphological characteristics and gram staining characteristics of single colonies of the Pacific Bacillus Pacific ORL1-5 strain of Pacific rice are shown in FIG. 1; wherein, the left graph shows the morphological characteristics of single colonies of the ORL1-5 strain, and the right graph shows the gram staining characteristics of the ORL1-5 strain. As shown in FIG. 1, the strain ORL1-5 is a gram-positive bacterium, and the cell size is 0.5-0.8. Mu.m.times.4.0-6.0. Mu.m; the single colony is white, round, irregular in edge, smooth and moist in surface, raised and opaque.
2. Molecular biological identification
(1) Amplification of 16S rRNA Gene fragment
DNA of ORL1-5 strain was extracted and PCR amplification was performed on the 16S rRNA gene fragment thereof using the amplification primers:
16S rRNA(27F)：5’-AGAGTTTGATCCTGGCTCAG-3’
16S rRNA(1492R)：5’-GGTTACCTTGTTACGACTT-3’
the PCR conditions were as follows:
PCR reaction system: 2 XPCR Buffer 12.5. Mu.L, 2mM dNTPs 5. Mu.L, 10pmoL/mL 27F0.75. Mu.L, 10pmoL/mL 14992R 0.75. Mu.L, KOD FX (1.0U/. Mu.L) 0.5. Mu. L, DNA 1.0.0. Mu. L, ddH 2 O 4.5μL。
PCR amplification procedure: pre-denaturation at 94℃for 5min, denaturation at 98℃for 10s, annealing at 55℃for 30s, elongation at 68℃for 90s, total of 35 cycles, and elongation at 68℃for 5min.
The PCR amplified products were examined by 1% agarose gel electrophoresis, and the electrophoresis results are shown in FIG. 2. As can be seen from FIG. 2, the 16S rRNA gene fragment of the ORL1-5 strain was successfully amplified by the present invention.
(2) Sequencing and construction of phylogenetic trees
The PCR products were sequenced, the sequences obtained from the sequencing were aligned at NCBI, and phylogenetic trees were established using mega X.
The 16S rRNA sequence of the ORL1-5 strain is shown as SEQ ID NO. 1.
The phylogenetic tree of Pacific bacillus strain ORL1-5 is shown in FIG. 3. Through homology alignment and phylogenetic tree analysis, the 16S rRNA sequence of the ORL1-5 strain was found to be closest (99.79% similar) to that of the Pacific bacillus (Bacillus pacificus) HBUM207168 strain (GenBank accession No: MT 239514.1) and aggregated into the same evolutionary branch. Comprehensive morphological and molecular identification shows that the ORL1-5 strain is Pacific bacillus (Bacillus pacificus).
Example 3 functional identification of siderophores by Bacillus pacific ORL1-5 Strain
The invention utilizes a filter paper sheet method to make sure that the ORL1-5 strain can produce siderophores by inoculating the ORL1-5 strain on a CAS culture medium. On this basis, the method for quantitatively detecting the ferric carrier production capacity of the ORL1-5 strain by using the CAS detection method comprises the following detailed steps: taking culture solution of ORL1-5 strain, centrifuging to obtain supernatant, adding CAS detection solution into the supernatant at a volume ratio of 1:1, standing for 2h, and measuring OD with an enzyme-labeled instrument 630 The value obtained is marked as A; mixing the inoculated sterile culture medium with CAS detection solution at a volume ratio of 1:1, standing for 2 hr, and measuring OD with an enzyme-labeled instrument 630 Value Ar; 3 replicates were set.
The calculation formula of the relative content (SU) of the siderophore is as follows: su=1-a/Ar.
The results of quantitative determination of the capacity of the ORL1-5 strain to produce iron carrier are shown in Table 1:
TABLE 1ORL1-5 Strain iron-producing vector ability
From the quantitative determination results of the siderophore-producing ability of the ORL1-5 strain shown in Table 1, the siderophore relative content (SU) of the ORL1-5 strain was 78.90.+ -. 0.36%, which indicates that the ORL1-5 strain has a high-efficiency siderophore-producing ability.
Example 4 Pacific bacillus ORL1-5 Strain of Rice promotes ferrite uptake in Chu japonica 28 seedlings
Selecting full and consistent Chu japonica 28 seeds, baking the seeds at 50 ℃ for 2 days to break dormancy, cleaning the seeds with 75% wine for 2 times in an ultra-clean workbench for 5min each time and 3 times with 15% sodium hypochlorite each time for 8min each time, and sterilizing. Activating ORL1-5 strain, and collecting bacterial solution (bacterial solution concentration: 1.0X10) of activated ORL1-5 strain 8 cfu/mL) for 12h (the same time as the Control (CK) used NB medium), then placed in a 37 ℃ incubator for germination, sowed in a flowerpot containing sterilized soil after being exposed to white, each treatment was repeated for 3 times, and the total iron content of Chu Jing seedling plants was measured when the seedlings were grown to three leaves and one heart.
The ferrite content was determined and analyzed using the Iron determination Kit Iron Assay Kit (MAK 025-1 KT) from Merck Sigma-Aldrich. The method comprises the following steps:
(1) grinding the sample into powder in liquid nitrogen, weighing 0.01g, putting into a 1.5mL centrifuge tube, adding 100 mu L Iron Assay Buffer, and rapidly and uniformly mixing;
(2) centrifuging at 16,000rpm for 10min at 4deg.C, preparing a new 1.5mL centrifuge tube, transferring supernatant into the centrifuge tube, and mixing thoroughly with Iron Assay buffer to volume of 100 μL;
(3) draw 50 μl of sample into a 96 well clear quartz plate, volume to 100 μl volume using Iron Assay buffer, and add 5 μl of ironreducer, centrifuge for 5min at 2500rpm in a horizontal centrifuge;
(4) incubating for 30min at 25 ℃ in an incubator in the absence of light;
(5) adding 100 mu L of the Iron Probe, and centrifuging for 5min at 2500rpm of a horizontal centrifuge;
(6) incubating in a 25 ℃ incubator in dark for 60min;
(7) colorimetric detection (593 nm) is carried out by using a multifunctional enzyme-labeled instrument, a standard curve is manufactured by using a standard iron sample, and the iron content is calculated by sample colorimetric detection.
As shown in the comparison result of the total iron content of Chu japonica 28 seedlings after seed soaking treatment of Pacific bacillus ORL1-5 strain and the Control (CK), as can be seen from FIG. 4, the total iron content of Chu japonica 28 seedling plants after seed soaking treatment of Pacific bacillus ORL1-5 strain is obviously improved (p is less than 0.01) compared with CK, which indicates that Pacific bacillus ORL1-5 strain promotes the absorption of Chu japonica 28 seedlings to ferrite.
The strain is soaked in the bacterial solutions of ORL1-5 strains with different concentration gradients for different time periods, and the concentration of the strain soaking solution is 1.0X10 7 ～1.0×10 9 In the cfu/mL range, when the seed soaking time is in the range of 10-14 h, the influence on the ferrite absorption of Chu Jing seedlings is not greatly different, and the effect is not obviousDifferences.
EXAMPLE 5 Pacific bacillus Pacific ORL1-5 Strain of Rice promotes chlorophyll Synthesis in Chu japonica 28 seedlings
Selecting full and consistent Chu japonica 28 seeds, baking the seeds at 50 ℃ for 2 days to break dormancy, cleaning the seeds with 75% wine for 2 times in an ultra-clean workbench for 5min each time and 3 times with 15% sodium hypochlorite each time for 8min each time, and sterilizing. Activating ORL1-5 strain, and collecting bacterial solution (bacterial solution concentration: 1.0X10) of activated ORL1-5 strain 8 cfu/mL) for 12h (the same time as the Control (CK) used NB medium), then placed in a 37 ℃ incubator for germination, sowed in a flowerpot containing sterilized soil after being exposed to white, each treatment was repeated for 3 times, and leaf chlorophyll content data were measured and recorded when seedlings were grown to a three leaf one-heart period. Chlorophyll was measured using a SPAD chlorophyll content meter, the middle of the leaf was selected, measured three times, and the average value was recorded.
As shown in FIG. 5, the chlorophyll content comparison result of the Pacific bacillus ORL1-5 strain seed soaking treated Chu-japonica 28 seedling and the Control (CK) is shown in FIG. 5, and the chlorophyll content of the Pacific bacillus ORL1-5 strain seed soaking treated Chu-japonica 28 seedling plant is obviously improved (p is less than 0.01) compared with the CK, so that the Pacific bacillus ORL1-5 strain promotes the synthesis of chlorophyll by the Chu-japonica 28 seedling.
The concentration of seed soaking bacterial liquid is 1.0X10 7 ～1.0×10 9 In the cfu/mL range, when the seed soaking time is in the range of 10-14 h, the influence on the synthesis of chlorophyll in Chu Jing seedlings is not greatly different.
Example 6 Pacific bacillus Pacific ORL1-5 Strain of Rice promotes growth of Chu japonica 28 seedlings
Selecting full and consistent Chu japonica 28 seeds, baking the seeds at 50 ℃ for 2 days to break dormancy, cleaning the seeds with 75% wine for 2 times in an ultra-clean workbench for 5min each time and 3 times with 15% sodium hypochlorite each time for 8min each time, and sterilizing. Activating ORL1-5 strain, and collecting bacterial solution (bacterial solution concentration: 1.0X10) of activated ORL1-5 strain 8 cfu/mL) soaking seeds for 12h (the same time as the Control (CK) uses NB culture medium to soak seeds), then placing in 37 ℃ for germination accelerating, sowing in a flowerpot containing sterilized soil after exposure to white, each treatment is repeated for 3 times, and measuring root length and plant height data when seedlings grow to three leaves and one heartAnd recorded. And removing abnormal plants, taking 5 plants for measurement and photographing each repetition to record plant phenotype differences, and finally taking an average value for 15 data of three repetitions to record. Root length and plant height are measured by using a ruler, and the ratio scale is as follows: 10cm.
The phenotype comparison result of Chu japonica 28 seedlings after seed soaking treatment of Pacific bacillus ORL1-5 strain of rice and Control (CK) plants is shown in figure 6, and as can be seen from figure 6, the Chu japonica 28 seedlings after seed soaking treatment of Pacific bacillus ORL1-5 strain of rice are thicker, have developed root systems and are obviously more fresh and green. As shown in FIG. 7, the comparison result of the plant height and root length of Chu japonica 28 seedlings after seed soaking treatment of Pacific bacillus ORL1-5 strain of Pacific bacillus is shown in FIG. 7, and the plant height and root length of Chu japonica 28 seedlings after seed soaking treatment of Pacific bacillus ORL1-5 strain of Pacific bacillus are obviously improved (p is less than 0.01) compared with CK. The results shown in FIGS. 6 and 7 indicate that Pacific Bacillus oryzae ORL1-5 strain of rice promotes seedling growth of Chu japonica 28.
The concentration of seed soaking bacterial liquid is 1.0X10 7 ～1.0×10 9 In the cfu/mL range, when the seed soaking time is in the range of 10-14 h, the influence on the plant height and root length of Chu Jing seedlings is not greatly different.
Example 7 Pacific bacillus Pacific ORL1-5 Strain of Rice improves Chu japonica 28 yield
In order to verify whether the Pacific bacillus strain ORL1-5 of rice has a promoting effect on the final yield of Chu japonica 28, the invention transplants the seed soaking treatment (same as in example 4) of the strain ORL1-5 of rice seedlings of Chu japonica 28 in a greenhouse, cultures the seedlings to be mature and harvested, and evaluates the yield property indexes.
The effect of Pacific bacillus strain ORL1-5 on Chu-japonica 28 yield property index is shown in figure 8 (CK is untreated control, treatent is Chu-japonica 28 plant after seed soaking Treatment by using strain ORL 1-5), and A-E in figure 8 are the effect of strain ORL1-5 on Chu-japonica 28 fruiting rate, grain number per spike (seed), thousand kernel weight, effective spike number and single plant yield in sequence. As shown in FIG. 8, the yield of single seed of Chu japonica 28 treated by the Pacific bacillus ORL1-5 strain of rice is obviously improved (p is less than 0.05) compared with CK, and the yield of Chu japonica 28 treated by the Pacific bacillus ORL1-5 strain is improved by about 5.15%. In addition, thousand seed weight, effective spike number and single plant yield of Chu japonica 28 after ORL1-5 strain treatment are all obviously higher than those of untreated control CK, which indicates that the ORL1-5 strain can improve seed yield by promoting tillering and grouting so as to improve single plant yield, and suggests that Pacific bacillus rice ORL1-5 strain can colonize in Chu japonica 28 and regulate in-vivo endophyte systems. In addition, the Pacific bacillus rice ORL1-5 strain is isolated from common wild rice, which shows that the ORL1-5 strain can be colonized in different rice.
The concentration of seed soaking bacterial liquid is 1.0X10 7 ～1.0×10 9 Within the cfu/mL range, when the seed soaking time is within the range of 10-14 h, the influence on the Chu Jing seedling yield property index is not greatly different.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. Pacific bacillus strain producing siderophoreBacillus pacificus) An ORL1-5 strain deposited at the collection of microorganisms and cell cultures, cantonese province, at 2022, 11/13, under the accession number GDMCC No:62966.
2. use of the strain of bacillus pacific ORL1-5 according to claim 1 for promoting rice growth.
3. Use of the strain of bacillus pacific ORL1-5 as claimed in claim 1 for the preparation of a product for promoting rice growth.
4. Use of the strain of bacillus pacific ORL1-5 according to claim 1 for increasing the yield of rice.
5. Use of the strain of bacillus pacific ORL1-5 as claimed in claim 1 for the preparation of a product for increasing the yield of rice.
6. Use of the bacillus pacific ORL1-5 strain according to claim 1 for promoting iron absorption of rice and increasing chlorophyll content of rice.
7. Use of the strain palettacia ORL1-5 as claimed in claim 1 for the preparation of a product for promoting iron absorption in rice and increasing chlorophyll content in rice.
8. A preparation for improving rice yield, which is characterized in that the preparation takes the Pacific bacillus ORL1-5 strain or bacterial liquid thereof as an active ingredient.
9. A method for improving rice yield, characterized in that before rice seeds germinate, seed soaking treatment is carried out by using the bacterial liquid of Pacific bacillus ORL1-5 strain according to claim 1.
10. The method according to claim 9, wherein the concentration of the bacterial liquid is 1.0X10 7 ～1.0×10 9 cfu/mL, the seed soaking or soaking treatment time is 10-14 h.
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Application publication date: 20230613 Assignee: YUNNAN VTAYAN BIOLOGICAL TECHNOLOGY CO.,LTD. Assignor: YUNNAN University Contract record no.: X2024980014831 Denomination of invention: A rice Bacillus subtilis strain producing iron carriers and its application Granted publication date: 20240223 License type: Open License Record date: 20240918 |