CN114015624B - Bacillus and application thereof in chickpea seed cultivation - Google Patents
Bacillus and application thereof in chickpea seed cultivation Download PDFInfo
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- CN114015624B CN114015624B CN202111531849.8A CN202111531849A CN114015624B CN 114015624 B CN114015624 B CN 114015624B CN 202111531849 A CN202111531849 A CN 202111531849A CN 114015624 B CN114015624 B CN 114015624B
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- 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
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- 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
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
Abstract
Bacillus strain classified asBacillus sp.Wyccwf10080=1c-6, CCTCC No. M20191042, 12 months 13 days 2019, china center for type culture collection, and university of armed Han and armed Han. The invention discloses a bacillus strain, which can greatly improve the rooting speed and the germination quantity of chickpea in the process of culturing chickpea sterile potted seedlings, so as to improve the seedling rate of chickpea, greatly reduce the cost of seedling replacement and death, and improve the germination rate of chickpea culture groups treated by the bacillus by nearly two times to 91.25 percent compared with control groups; the root length is increased by 84%, the average dry weight is increased by 101.2%, and the chickpea seed-emergence growth-promoting high-efficiency microbial inoculum can be used.
Description
Technical Field
The invention belongs to the field of plant production, relates to cultivation of chickpea seeds, and in particular relates to bacillus and application thereof in chickpea seed cultivation.
Background
Chickpea, academic name:Cicer arietinumthe peaches, chickpeas and the like are also called as one of important vegetables of India and Pakistan, are also very common in Europe, and are also common medicinal materials in Uygur medicine. This name is called as the tip of the tip is like the olecranon because of its peculiar shape. Chickpea is a leguminous herb, originating in the western asia and in the near east. Is a leguminous plant with larger cultivation area in the world, about one hundred million mu, wherein the planting area of two countries of India and Pakistan accounts for more than 80% of the world, and only sporadically distributed in China. The starch of chickpea has chestnut flavor. The chickpea powder and milk powder are added to prepare soybean milk powder, which is easy to absorb and digest, and is a nutritional food for infants and the elderly. Chickpea can also be made into various snack and fried bean. The seed can be used as diuretic, lactation promoting agent, and can be used for treating insomnia, preventing skin diseases and preventing and treating gallbladder diseases. Starch is widely used in the paper industry and textile industry.
However, chickpea seeds have a particularly low germination rate, which is mainly due to the fact that the wet environment of chickpea roots after germination is extremely easy to cause mildew and death, and the emergence of seedlings is affected. The present inventors have made an effort to improve the germination rate and cultivation direction of chickpeas.
Disclosure of Invention
The invention provides bacillus and application thereof in chickpea seed cultivation, and solves the problem of low chickpea emergence rate.
The technical scheme of the invention is realized as follows:
bacillusThe bacillus strains are classified asBacillus sp.Wyccwf10080=1c-6, CCTCC No. M20191042, 12 months 13 days 2019, china center for type culture collection, and university of armed Han and armed Han.
The bacillus is applied to improving the germination rate of chickpea seeds and increasing root biomass.
The application of the bacillus comprises the following steps:
(1) Inoculating bacillus strains into an LB liquid culture medium, and shake culturing overnight at 37 ℃ to obtain bacillus liquid;
(2) Sterilizing the surface of chickpea seeds by using sodium hypochlorite solution, washing the seeds for 5-8 times by using sterile water, and placing the washed chickpea seeds on a 1% water agar plate for culture to obtain chickpea seeds with germination of 1-2 cm;
(3) Filling the nutrient medium into a culture dish, and then opening a small hole on the upper part of the nutrient medium by using sterile forceps;
(4) Taking 1-5mL of bacillus bacteria liquid obtained in the step (1), uniformly adding the bacillus bacteria liquid to the bottom of a small hole of a nutrition matrix, then placing chickpea seeds obtained in the step (2) into the bacteria liquid at the bottom of the small hole, covering the seeds by 2-5cm with the nutrition matrix, growing in the dark in a constant-temperature illumination incubator until buds come out, and performing illumination-darkness cyclic culture to improve the germination rate of the chickpea seeds and increase the root biomass;
the bacillus strain in the step (1) is inoculated in an amount of 1-10 hundred million bacteria per seed.
The concentration of the sodium hypochlorite solution in the step (2) is 1.5-3.0%.
The culture condition in the step (2) is that the culture is carried out in an incubator at 25-30 ℃ for 2-3 days under the dark condition.
The specification of the small holes in the step (3) is 2-2.5cm in diameter and 4-5cm in depth.
The light-dark circulation culture in the step (4) is to perform repeated circulation culture for a plurality of times by taking 16h light-8 h dark as one circulation.
The invention has the following beneficial effects:
the invention discloses a bacillus strain, which can greatly improve the rooting speed and the germination quantity of chickpea in the process of culturing chickpea sterile potted seedlings, so as to improve the seedling rate of chickpea, greatly reduce the cost of seedling replacement and death, and improve the germination rate of chickpea culture groups treated by the bacillus by nearly two times to 91.25 percent compared with control groups; the root length is increased by 84%, the average dry weight is increased by 101.2%, and the chickpea seed-emergence growth-promoting high-efficiency microbial inoculum can be used.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing germination percentage experiments of chickpeas cultured with Bacillus sp.1C-6 and a control.
FIG. 2 is a root system pattern of chick pea cultured with Bacillus sp.1C-6 and a control.
FIG. 3 is a dendrogram of 16S rRNA phylogenetic development of Bacillus 1C-6 of the present application.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.
Bacillus strain classified asBacillus sp.Wyccwf10080=1c-6, CCTCC No. M20191042, 12 months 13 days 2019, china center for type culture collection, and university of armed Han and armed Han.
Disclosed formula of bacterial LB solid medium:
10g of tryptone, 5g of yeast extract, 10g of NaCl, 20g of agar powder, 1L of distilled water and pH to 7.0.
1. Bacillus spBacillus sp.Isolation and screening of wyccwf10080=1c-6
Soil samples are collected at the places such as river sides, lawns, pine forests, outside experimental buildings and the like in the university campus of Zhengzhou light industry in the high new area of Zhengzhou in Henan province, five-point sampling methods are adopted, 5-10cm of fertile soil is collected from the river sides, the lawns and the pine forests for multiple years respectively, and the fertile soil is stored in a sterile sealing bag, recorded and immediately brought back to a laboratory. 10g of soil sample is randomly taken and placed into a triangular flask which is sterilized in advance and contains 90mL of sterile water and glass beads, and the triangular flask is placed into a shaking table for shaking and uniformly mixing for 10 minutes under the conditions of 28 ℃ and the rotating speed of 150 r/min. Then heating the triangular flask in 90 deg. water bath for 20 min, shaking and mixing at 28deg.C and rotation speed of 150 r/min for 5 min, standing for 10 min, and taking supernatant for 10 times gradient dilution (10 -1 ~10 -9 ) Then coating the bacillus strain on a solid plate of an LB culture medium, coating 200 mu L of each gradient, putting the plate into a constant temperature incubator, and culturing at 37 ℃ reversely overnight to initially obtain the bacillus strain.
Further streaking and separating out the colony with better selected form, and repeatedly streaking, separating and purifying until no impurity colony appears on the flat plate. 6 single colonies are screened, the single colonies are picked and connected into a 5mL LB liquid test tube, shaking culture is carried out at 37 ℃ and 180rpm for 12 h, the OD600 of the bacterial liquid is adjusted to 1.0, and the bacterial liquid is prepared according to the following steps: taking 0.5 to mL according to the proportion of 100, putting the mixture into 50 mL of LB liquid medium, and culturing the mixture in a shaking table at 37 ℃ and 180rpm for 12 h; then according to each seed 10 6 -10 8 Inoculating the strain to the surface of chickpea seed to be germinated after disinfection, culturing in dark at 28deg.C for 5 days, screening out a strain with obvious germination promoting rate, and identifying as Bacillus by 16S rRNA sequencing and phylogenetic analysis, and naming as BacillusBacillus sp.Wyccwf10080=1c-6. The 16S rRNA sequence of 1C-6 is shown as SEQ ID No. 1, and the phylogenetic tree is shown as figure 3.
Application example
The method for culturing chickpeas by using the bacillus comprises the following steps:
(1) The bacillus strain was used in an amount of 10 per seed 0 -10 9 Inoculating the bacterial strain proportion into LB liquid culture medium, shake culturing overnight at 37 ℃ to obtain bacillus bacterial solution;
(2) Sterilizing the surface of chickpea seeds with 1.5-3.0 wt% sodium hypochlorite solution, washing the seeds with sterile water for 5-8 times, placing the washed chickpea seeds on a 1% water agar plate, culturing in a constant temperature oven at 25-30 ℃ for 2-3 days under dark condition to obtain chickpea seeds with germination of 1-2 cm;
(3) Filling the nutrient medium into a culture dish, and then opening a small hole with the diameter of 2-2.5cm and the depth of 4-5cm at the upper part of the nutrient medium by using sterile forceps;
(4) Taking 1-5mL of bacillus bacteria liquid obtained in the step (1), uniformly adding the bacillus bacteria liquid to the bottom of a small hole of a nutrient medium, then putting chickpea seeds obtained in the step (2) into the bacteria liquid at the bottom of the small hole, covering the seeds by 2-5cm with the nutrient medium, growing in the dark in a constant-temperature illumination incubator until buds are unearthed, and then carrying out repeated cyclic culture for a plurality of times by taking 16h illumination-8 h darkness as a cycle, thereby improving the germination rate of the chickpea seeds and increasing the root biomass;
(5) After 50 days of growth, each plant was carefully removed and the root-attached vermiculite was carefully washed off with clean water, then the root length was measured, and the root was dried to constant weight and measured for dryness.
The conventional culture method without adding bacillus bacteria liquid is used as a control.
The germination rate of chickpeas is shown in fig. 1, and the number of germination rates is shown in table 1:
table 1: germination rate statistics of added bacillus culture group and control group
From the above, the germination rate of the chick pea culture group treated with the bacillus of the present application was improved by nearly two times relative to the control group.
Bacillus warpBacillus sp.Wyccwf10080=1c-6 treated chickpea plants and chickpea plants of the control group, roots of chickpeas cultivated for 50 days are shown in fig. 2, and dry weight statistics after drying the roots to constant weight are shown in table 2:
table 2: root data statistics of bacillus culture and control
| Treatment of | Average root length (cm) of 10 plants | 10 average root dry weight (g) |
| The bacillus liquid is added | 13.8 | 32.4 |
| Adding LB culture medium | 7.5 | 16.1 |
As can be seen from FIG. 2 and Table 2, the root length increased by 84% and the average dry weight increased by 101.2% using Bacillus 1C-6 of the present application. The effect is obvious.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
<110> university of light industry in Zhengzhou
<120> a bacillus and its use in chickpea seed cultivation
<141> 2021-12-15
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1401
<212> DNA
<213> Bacillus sp
<400> 1
ctgcaagtcg agcggacaga agggagcttg ctcccggatg ttagcggcgg acgggtgagt 60
aacacgtggg taacctgcct gtaagactgg gataactccg ggaaaccgga gctaataccg 120
gatagttcct tgaaccgcat ggttcaagga tgaaagacgg tttcggctgt cacttacaga 180
tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggcg acgatgcgta 240
gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg 300
aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag 360
tgatgaaggt tttcggatcg taaagctctg ttgttaggga agaacaagtg caagagtaac 420
tgcttgcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480
ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540
tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactggga 600
aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660
tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720
gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780
agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840
gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900
catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960
ccctagagat agggctttcc cttcggggac agagtgacag gtggtgcatg gttgtcgtca 1020
gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080
ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140
tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacagaa 1200
caaagggctg cgagaccgca aggtttagcc aatcccacaa atctgttctc agttcggatc 1260
gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320
cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgcaaca 1380
cccgaagtcg gtgaggtaac c 1401
Claims (8)
1. A bacillus wyccwf10080=1c-6 strain, characterized in that: the bacillus strain classification name isBacillus sp.The preservation number is CCTCC NO: M20191042, the preservation time is 12 months 13 days of 2019, the preservation unit is China center for type culture Collection, and the address is China university of Wuhan.
2. The use of bacillus according to claim 1, characterized in that: the bacillus is applied to improving the germination rate of chickpea seeds and increasing root biomass.
3. The use of bacillus according to claim 2, characterized by the steps of:
(1) Inoculating bacillus strains into an LB liquid culture medium, and shake culturing overnight at 37 ℃ to obtain bacillus liquid;
(2) Sterilizing the surface of chickpea seeds by using sodium hypochlorite solution, washing the seeds for 5-8 times by using sterile water, and placing the washed chickpea seeds on a water agar plate with the concentration of 1% -5% for culturing to obtain chickpea seeds with the germination of 2-5 cm;
(3) Filling the nutrient medium into a culture dish, and then opening a small hole on the upper part of the nutrient medium by using sterile forceps;
(4) Taking 1-5mL of bacillus bacteria liquid obtained in the step (1), uniformly adding the bacillus bacteria liquid to the bottom of a small hole of a nutrition matrix, then placing chickpea seeds obtained in the step (2) into the bacteria liquid at the bottom of the small hole, covering the seeds by 2-5cm with the nutrition matrix, growing in the dark in a constant-temperature illumination incubator until buds come out, and then performing illumination-darkness cyclic culture to improve the germination rate of the chickpea seeds and increase the root biomass.
4. The use of bacillus according to claim 3, wherein: the bacillus strain in the step (1) is inoculated in an amount of 1-10 hundred million bacteria per seed.
5. The use of bacillus according to claim 3, wherein: the concentration of the sodium hypochlorite solution in the step (2) is 1.5-3.0%.
6. The use of bacillus according to claim 3, wherein: the culture condition in the step (2) is that the culture is carried out in an incubator at 33-39 ℃ for 2-3 days under the dark condition.
7. The use of bacillus according to claim 3, wherein: the specification of the small holes in the step (3) is 2-2.5cm in diameter and 4-5cm in depth.
8. The use of bacillus according to claim 3, wherein: the light-dark circulation culture in the step (4) is to perform repeated circulation culture for a plurality of times by taking 16h light-8 h dark as one circulation.
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