CN110564647B - Bacillus amyloliquefaciens for promoting germination and growth of axillary buds of regenerated rice and application thereof - Google Patents
Bacillus amyloliquefaciens for promoting germination and growth of axillary buds of regenerated rice and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of plant microorganisms, and particularly relates to bacillus amyloliquefaciens ZSY-3 and application thereof as a growth promoting strain of rhizosphere of ratoon rice. The invention takes the tillering number of the regenerated season rice as a main index, obtains a strain of bacillus amyloliquefaciens ZSY-3 by screening, and stores the strain in a China center for type culture collection, and experiments prove that the strain of bacillus amyloliquefaciens ZSY-3 screened by the invention has certain nitrogen fixation, phosphorus and potassium dissolving and IAA (International IAA) production capacities, can be effectively used as a rhizosphere growth promoting strain, promotes axillary buds of the regenerated season rice to germinate and grow into ears, and is beneficial to improving the yield of the regenerated season rice.
Description
Technical Field
The invention belongs to the technical field of microbial strains and application thereof, and particularly relates to a growth-promoting bacterium and the technical field of promotion of the growth-promoting bacterium in germination and growth of axillary buds of ratoon rice.
Background
Rice is one of three main grain crops in China, and the stability and the improvement of the total yield of the rice are of great importance to the maintenance of the grain safety in China. There are 3 ways to increase the total crop yield: (1) the yield per season of unit area is improved, namely the yield per unit area is increased; (2) increasing the cultivated land area; (3) and (4) improving the multiple cropping index. However, China still faces some practical problems in rice production: the consumption demand of the rice by everyone is increased rigidly, and the yield per unit of the rice is increased, but the yield increase amplitude is smaller and smaller, so that great breakthrough is difficult to occur; the agricultural production cost is continuously increased, the grain comparison benefit is low, the agricultural labor force structure is changed, the rural labor force is in short supply, the rice seeding area is in a descending trend, the phenomenon of 'double changing single' or even abandoning is particularly generated in partial rice areas in south China, and the possibility of increasing the rice planting area is extremely low; therefore, increasing the harvest area by increasing the multiple cropping index becomes a main way to increase the total yield of the rice.
The ratoon rice is a resource-saving rice cropping mode which adopts certain cultivation management measures to ensure that dormant buds on rice stakes after first-season rice is harvested germinate and grow into ears and then harvest the same, has the characteristics of 'seven-saving and two-increasing and one-excellent' (saving seeds, labor, time, water, fertilizer, medicine, seedling fields, increasing yield, increasing income and high rice quality), and becomes an important rice cropping system which is characterized in that the light and temperature resources in one season in south China are insufficient and the 'two-changing and single' rice area improves multiple cropping indexes, stabilizes the total yield of rice, optimizes the structure of the rice, improves the rice quality, implements structural reform on the agricultural supply side and improves the benefit of the rice under the double pressure of continuous rising of the current grain planting cost, and the 'two-changing and single' and continuous increase of the abandoned area.
The yield of the ratoon rice is important for playing the role of the ratoon rice as a rice working system; numerous studies have shown that the yield of the regenerated rice is most closely related to the number of regenerated tillers per unit area, and the amount of regenerated buds, seedlings and spikes directly affects the yield of the regenerated rice (Li Jing Yong, 1997, Ling Zhong, 1989, Ningzhi, 1993, Liu Bao, 1998, Zheng Jing Sheng, 2004). Therefore, the improvement of the number of the regeneration tillers per unit area becomes the key to play the role of the rice cropping system of the regenerated rice.
At present, the improvement of the tillering quantity of the regenerated rice mostly depends on the application of chemical fertilizers, but the long-term application of the chemical fertilizers can aggravate the environmental pollution and cause damage to the structure and the physicochemical property of soil, so that the problems of nutrient imbalance, hardening and the like of the soil are caused.
Plant growth-promoting rhizobacteria (PGPR) refers to a kind of beneficial bacteria which can freely live in soil or attached to plant roots, can promote plant growth and absorption and utilization of mineral nutrition, and can inhibit harmful organisms, and can promote plant growth by synthesizing certain substances (such as auxin and the like) having direct effects on plant growth and development and/or changing the forms of certain ineffective elements in soil, so that the substances are activated to facilitate plant absorption (such as nitrogen fixation, phosphorus removal and the like) to promote plant growth, and can also inhibit or reduce the adverse effects of certain plant diseases on plant growth and development and yield. The plant rhizosphere growth-promoting bacteria has the characteristics of low carbon, pure nature, no toxicity, no harm and no pollution, is increasingly widely applied to crops, and has good growth-promoting or biocontrol effects, which are separated from cotton, wheat, tobacco, hot pepper, tomatoes, potatoes and other crops by researchers, but no research report on the rhizosphere growth-promoting bacteria for the germination and growth of axillary buds of ratory rice is found at present.
Disclosure of Invention
The invention aims to provide a rhizosphere growth-promoting bacterium bacillus amyloliquefaciens for germination and growth of axillary buds of regenerated rice and application thereof.
In order to achieve the aim, the rhizosphere growth-promoting bacterium bacillus amyloliquefaciens capable of promoting germination and growth of axillary buds of regenerated rice provided by the invention is bacillus amyloliquefaciens (bacillus amyloliquefaciens) ((Bacillus amyloliquefaciens) ZSY-3 has been preserved in China general microbiological culture Collection center (CGMCC) in 2018, 12 months and 27 months, and the preservation number is CGMCC No. 17041. The general microbiological center of China Committee for culture Collection of microorganisms is institute of microbiology, No. 3 Xilu No.1 of Shangyang province, China, Beijing.
Further, the bacillus amyloliquefaciens strain is applied to promoting germination and growth of axillary buds of regenerated rice.
Further, the bacillus amyloliquefaciens strain is applied to preparation of a microbial fertilizer or a microbial inoculum for promoting germination and growth of axillary buds of the regenerated rice.
The bacillus can be cultured and fermented in a bacillus universal culture medium. The components of the culture medium: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH = 7.0.
Observing the shape of the bacterial colony under a microscope, wherein the bacterial colony is rough in surface, raised in the middle and white and opaque; the strain is rod-shaped, gram-positive, and determined as bacillus amyloliquefaciens by combining morphology and 16S rDNA molecular biology identificationBacillus amyloliquefaciens）。
The strain can hydrolyze starch and casein with xylose, lactose, raffinose, maltose, mannose and arabinose, and without galactose.
The invention has the beneficial effects that: the bacillus amyloliquefaciens has IAA producing, phosphate dissolving, potassium dissolving and nitrogen fixing capacities, wherein the nitrogen fixing capacity is 4.71 mg/L, the phosphate dissolving capacity is 3.18 mg/L, the potassium dissolving capacity is 15.17 mg/L, and the IAA producing capacity is 0.99 mg/L. Can be effectively used as a rhizosphere growth promoting strain to promote the germination and growth of axillary buds of the regenerated season rice into ears, improve the tiller number and the effective ear number of the regenerated season rice and further improve the yield.
Drawings
FIG. 1 shows Bacillus amyloliquefaciens (B) of the present inventionBacillus amyloliquefaciens) The ZSY-3 has the growth promoting effect on the ratooning rice.
FIG. 2 shows Bacillus amyloliquefaciens (B.amyloliquefaciens)Bacillus amyloliquefaciens) Colony morphology of ZSY-3.
FIG. 3 shows Bacillus amyloliquefaciens (B) of the present inventionBacillus amyloliquefaciens) Gram staining pattern of ZSY-3.
Detailed Description
In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.
Example 1
1. Isolation and purification of Bacillus amyloliquefaciens
Collecting a soil sample: rhizosphere soil samples of rice regeneration seasons with strong regenerability were collected from the experimental field of the teaching base of the university of agriculture and forestry in Fujian in 2015. And (3) uniformly mixing by adopting a 5-point sampling method to obtain a sample. Separating and purifying soil sample microorganism by dilution separation method, weighing 10g soil, adding into a triangular flask containing sterile water 90 mL, 120r/min, oscillating for 30min to obtain soil suspension, sequentially diluting the obtained soil suspension, and making into 10-1~10-6And the soil solution with different dilution degrees is obtained. Preparing a culture medium by using 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride, and adjusting the pH value of the culture medium to 7.0-7.4 by using NaOHAdding 10-15 g of agar powder per liter, and taking 10g of agar powder-3~10-6And (3) smearing 0.1 mL of each dilution gradient sample on a flat plate made of the culture medium, repeating each concentration for 3 times, inverting the flat plate in an incubator at 30 ℃ for culturing for one day, selecting a flat plate with an appropriate colony number, picking out a single colony in the flat plate, purifying by adopting a parallel scribing method, continuously transferring for 3 times, confirming to be a single bacterium, and obtaining 3 strains.
2. Screening of rhizosphere growth-promoting bacteria and determination of growth-promoting effect
The test adopts a greenhouse potting method, the site is arranged in a greenhouse of a teaching test site of the university of agriculture and forestry in Fujian, plastic barrels with the upper caliber of 30cm, the lower caliber of 23cm and the height of 30cm are adopted as containers, the soil weight of each pot is 12kg, each pot is treated and contrasted with 6 pots, and the three times of treatment and contrasting are repeated for 90 pots. The treatment is started 3 days after harvesting, during the treatment, roots are irrigated with bacterial suspension, each strain is irrigated with 500 ml of bacterial suspension, and the concentration is 106-108CFUs/ml were watered 1 more times every 3 days for 2 times to enhance colonization of the rhizosphere with bacteria, and the same volume of sterile medium was watered in contrast, and the treatment was the same.
Preparing a bacterial suspension: preparing LB liquid culture medium (NaCl 10g, yeast extract 5g, peptone 10 g), dissolving with distilled water and supplementing to 1000mL, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min. Respectively inoculating the 3 strains obtained by separation and purification into LB liquid culture medium, and performing shaking culture at 34 ℃ and 220 r/min for 48h to obtain bacterial suspension.
Growth promotion and investigation: in the mature period of the ratooning rice, 3 plants are taken for each treatment to examine the ear number, the grain number, the seed setting rate, the thousand grain weight and the yield of each plant.
TABLE 1 regeneration season yields and their constitutive factors under treatment with different strains
Therefore, the screened strain ZSY-3 can be effectively used as a rhizosphere growth promoting strain, remarkably promotes the germination and spiking of axillary buds of the ratoon rice, and is favorable for improving the yield of the ratoon rice. (see FIG. 1)
3. Identification of strains
Gram staining and morphological identification: the strain is observed to be rod-shaped, the surface of the colony is rough, the middle part is raised, and the strain is white and opaque (see figure 2), and the strain ZSY-3 obtained by the screening is positive by gram staining (see figure 3).
And (3) molecular identification: to further identify the strain, the total DNA of the strain was extracted, PCR amplified using universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGA CTT-3'), analyzed for 16S rDNA sequence, and compared to the CNBI database to identify it as B.amyloliquefaciens.
16S rDNA sequence:
tgcagtcgagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttgtttgaaccgcatggttcagacataaaaggtggcttcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctcaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaagaacaagtgccgttcaaatagggcggcaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggcgtaaagggctcgcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtctgtaactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaa。
EXAMPLE 2 determination of the growth-promoting potential of the Strain
(1) Determination of nitrogen fixation ability of strain
Preparing nitrogen-fixing bacteria seed liquid: using nitrogen-free liquid culture medium of arbutus(glucose 10.0g, KH)2PO40.2g，CaCO35.0g，MgS04·7H2O 0.2g，NaCl 0.2g,，CaSO40.2g, agar 15g, distilled water 1000mL, pH 7.0) were dispensed into test tubes (10 mL/tube), sterilized, and the strain was inoculated into the above medium and cultured by shaking at 150rpm at 28 ℃ for 2 days. Adding the prepared nitrogen-free liquid culture medium into a 250mL triangular flask in a volume of 50mL per flask, sterilizing, inoculating 1mL of nitrogen-fixing strain seed culture solution, and setting a blank control without inoculation. After inoculation, the cells were cultured at 28 ℃ for 7d with shaking at 150 rpm. And (3) respectively metering the cultured azotobacter fermentation liquid to 50mL, and centrifuging at 500rpm for 10min to remove insoluble substances in the azotobacter fermentation liquid. 10mL of supernatant is digested and boiled and then used for measuring the nitrogen content by a Kjeldahl method. As shown in Table 2, the nitrogen-fixing ability of the strain ZSY-3 was measured to be 4.71 mg/L. Therefore, the strain ZSY-3 has the nitrogen fixation capacity.
(2) Determination of phosphate solubilizing ability of Strain
Preparing phosphate solubilizing bacteria seed liquid: phosphate-solubilizing liquid medium (glucose 10.0g, (NH)4）2SO40.5g，KCl0.3g,，NaCl 0.3g， MgSO4·7H2O 0.3g ，MnSO4·4H2O 0.03g,，FeSO4·7H2O 0.03g ，Ca3(PO4)28.0g, 1000mL of distilled water, 15g of agar, pH 7.0-7.5) are subpackaged into test tubes (10 mL/tube) and sterilized, and the strain is inoculated into the culture medium, and is subjected to shake culture at 150rpm for 2d at 28 ℃.
Adding 50mL of prepared phosphate solubilizing liquid culture medium into a 250mL triangular flask per bottle, inoculating 1mL of phosphate solubilizing seed culture medium after sterilization, and setting a blank control without inoculation. After inoculation, the cells were cultured at 28 ℃ for 7d with shaking at 150 rpm. And measuring the phosphorus content by a spectrophotometer by adopting a molybdenum blue colorimetric method. As shown in Table 2, the phosphorus-solubilizing ability of the strain ZSY-3 was measured to be 3.18 mg/L. Therefore, the strain ZSY-3 has the phosphate-solubilizing capability.
(3) Determination of potassium-decomposing ability of bacterial strain
Preparing a potassium bacteria seed solution: adding potassium-dissolving liquid culture medium (sucrose 5g, FeCl)30. 005 g，MgS04·7H2O0. 5 g,，CaCO30. 1 g ,，Na2HPO42g, potassium feldspar powder 2g, agar 15g, pH 7.0) were dispensed into test tubes (10 mL/tube), sterilized, and the strain was inoculated into the above medium and shake-cultured at 28 ℃ and 150rpm for 2 d. Adding 50mL of prepared potassium-dissolving liquid culture medium into a 250mL triangular flask per flask, inoculating 1mL of phosphorus-dissolving seed culture medium after sterilization, and setting a blank control without inoculation. After inoculation, the cells were cultured at 28 ℃ for 7d with shaking at 150 rpm. And (3) metering the volume of the fermentation liquor in the triangular flask to 50mL, centrifuging 10mL of bacterial liquid at 10000rpm for 10min, and measuring the potassium content of the supernatant by using a flame spectrophotometer. As shown in Table 2, the potassium-solubilizing ability of the strain ZSY-3 was measured to be 15.17 mg/L. Therefore, the strain ZSY-3 has potassium-dissolving capacity.
(4) Determination of indolylacetic acid-producing ability of strains
Picking fresh thallus Porphyrae in MM medium (KH)2PO450mM， K2HPO450mM，MgSO45mM，(NH4)2SO425mM, 1% glucose, 0.05w/v% L-tryptophan added, 1000mL distilled water), cultured at 25 ℃ and 180r/min for 7 d. The volume of the fermentation liquid in the triangular flask is increased to 50mL, the fermentation liquid is centrifuged at 10000rpm for 10min, and 1mL of supernatant is taken and added into 2 mL of Salkowski's reaction liquid. Mixing, reacting at room temperature in dark for 30min, centrifuging to obtain blank culture medium, and measuring the light absorption value of the reaction solution at 530 nm. As shown in Table 2, the IAA-producing ability of the strain ZSY-3 was measured to be 0.99 mg/L. Therefore, the strain ZSY-3 has the capability of producing IAA.
TABLE 2 determination of nitrogen fixation, phosphorus and potassium dissolution, IAA production ability of the strain
Therefore, the bacillus amyloliquefaciens ZSY-3 obtained by screening has good nitrogen fixation, phosphorus dissolving, potassium dissolving and IAA production capabilities.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> bacillus amyloliquefaciens for promoting germination and growth of axillary buds of regenerated rice and application thereof
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tgcagtcgag cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa 60
cacgtgggta acctgcctgt aagactggga taactccggg aaaccggggc taataccgga 120
tggttgtttg aaccgcatgg ttcagacata aaaggtggct tcggctacca cttacagatg 180
gacccgcggc gcattagcta gttggtgagg taacggctca ccaaggcgac gatgcgtagc 240
cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag 300
gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg 360
atgaaggttt tcggatcgta aagctctgtt gttagggaag aacaagtgcc gttcaaatag 420
ggcggcacct tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg 480
gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagggct cgcaggcggt 540
ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcattgg aaactgggga 600
acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt 660
ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggagcgaaag 720
cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa 780
gtgttagggg gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg 840
agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900
atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaat 960
cctagagata ggacgtcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag 1020
ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga tcttagttgc 1080
cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga aggtggggat 1140
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Claims (3)
1. Bacillus amyloliquefaciens strainBacillus amyloliquefaciens) ZSY-3 is preserved in China general microbiological culture Collection center (CGMCC) at 27 months 12 and 2018, and the preservation number is CGMCC No. 17041.
2. A Bacillus amyloliquefaciens strain according to claim 1 for promoting germination and growth of axillary buds of ratoon rice.
3. The application of the bacillus amyloliquefaciens strain disclosed by claim 1 in preparation of a microbial fertilizer or a microbial inoculum for promoting germination and growth of axillary buds of regenerated rice.
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