CN108265015B - Multifunctional potassium-decomposing bacterium and application thereof - Google Patents
Multifunctional potassium-decomposing bacterium and application thereof Download PDFInfo
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Abstract
The invention provides a multifunctional potassium-dissolving bacterium which is classically named Burkholderia (Burkholderia tripica) H1 and has the preservation number as follows: CCTCC NO. M2017281. The invention also provides application of the multifunctional potassium-solubilizing bacteria in soil potassium solubilization, phosphorus solubilization and nitrogen fixation. The invention also provides a microbial inoculum for potassium dissolution, phosphorus dissolution and nitrogen fixation of soil, namely, the dry powder microbial inoculum is obtained by fermenting the multifunctional potassium-dissolving bacteria to obtain fermentation liquor and performing spray drying. The multifunctional potassium-dissolving bacteria can effectively dissolve potassium, dissolve phosphorus and fix nitrogen in soil, after the multifunctional potassium-dissolving bacteria are applied, the content of the quick-acting nutrients in the soil is increased, the using amount of various fertilizers is reduced, the crop yield is improved, the water and soil loss is reduced, the multifunctional potassium-dissolving bacteria have important significance and application value in the aspects of cultivating and exerting the ecological fertility of the soil and keeping the ecological balance of agriculture, and are low in cost, simple to operate, free of pollution and harmless to the environment.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a multifunctional potassium-dissolving bacterium, an effect of the multifunctional potassium-dissolving bacterium in soil potassium-dissolving phosphorus-dissolving and nitrogen-fixing, and a microbial inoculum for soil potassium-dissolving phosphorus-dissolving and nitrogen-fixing.
Background
Nitrogen, phosphorus and potassium are three important nutrient elements necessary for the growth of crops. Nitrogen is a component of protein, nucleic acid and chlorophyll in crops, phosphorus plays an important role in improving the yield and quality of crops, and potassium can improve the capability of crops in adapting to external adverse environments. However, the total nitrogen content of soil in most cultivated lands in China is below 0.2 percent (namely nitrogen-deficient soil), the area of phosphorus-deficient soil is as much as 10.09 hundred million acres, the potassium content in the soil can be directly utilized and only accounts for 2 to 10 percent of the total potassium, and more than 90 percent of the potassium exists in silicate minerals such as feldspar, mica and the like, and the minerals have stable chemical properties and are difficult to be absorbed and utilized by crops.
In order to meet the requirement of crops on nutrient elements, most cultivated lands in China need to be supplemented with various fertilizers rich in nitrogen, phosphorus and potassium. The environmental problems of environmental pollution, soil hardening, land capability decline, agricultural product quality reduction, reduction of soil microorganism quantity, ecological deterioration, farmland soil ecosystem imbalance and the like can be caused by applying a large amount of fertilizers. Therefore, it is a strategic subject to improve and increase the utilization rate of nutrient elements, especially potassium, phosphorus and nitrogen in the soil of the cultivated field in China. From the existing literature reports, the burkholderia strains, the phosphorus dissolving strains and the nitrogen fixing strains are various and complex in variety and are different, the strains with the functions of dissolving potassium, dissolving phosphorus and fixing nitrogen are few, the survival rate in soil is low, the strains applied to actual production are few in variety, and innovation is lacked. From the perspective, the work of screening the wild strains with multiple functions, high potassium dissolving capacity and good phosphorus dissolving and nitrogen fixing effects is still significant.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a multifunctional potassium-dissolving bacterium and an application technology for dissolving potassium, dissolving phosphorus and fixing nitrogen in soil by using the multifunctional potassium-dissolving bacterium. The technology not only can effectively dissolve potassium, dissolve phosphorus and fix nitrogen for soil, but also has the advantages of low cost, simple operation, no pollution and no harm to the environment.
One of the purposes of the invention is to provide a multifunctional potassium-solubilizing bacterium, which is classified and named as Burkholderia (Burkholderia tripica) H1, China Center for Type Culture Collection (CCTCC) NO. M2017281. The inventor screens a strain of bacteria from corn rhizosphere soil, proves that the strain of bacteria has the effects of soil potassium dissolution, phosphorus dissolution and nitrogen fixation, the bacteria is Burkholderia (Burkholderia tripica) H1 through morphological identification, biochemical identification and sequencing analysis, wherein the colony morphology result is shown in figure 1, and the biochemical identification result is shown in example 2.
The second purpose of the invention is to provide the application of the multifunctional potassium-solubilizing bacteria in soil potassium solubilization, phosphorus solubilization and nitrogen fixation. The multifunctional potassium-dissolving bacteria are prepared into a microbial inoculum for potassium dissolving, phosphorus dissolving and nitrogen fixation of soil according to the following method:
activating the multifunctional potassium-decomposing bacteria stored on the inclined plane, inoculating the multifunctional potassium-decomposing bacteria on an activation culture medium, putting the activated multifunctional potassium-decomposing bacteria into a constant-temperature incubator, and performing constant-temperature culture for 3-5 days at the temperature of 30 ℃; selecting strains on an activation culture medium, inoculating the strains in a fermentation culture medium, and performing shaking culture at 30 ℃ for 10d at 180r/min to obtain a fermentation liquid for potassium dissolution of soil, or drying the fermentation liquid to obtain a dried substance for potassium dissolution, phosphorus dissolution and nitrogen fixation of soil.
The activation medium is an Alexander bauov solid medium, the fermentation medium is an Alexander bauov liquid medium, and the Alexander bauov liquid medium comprises the following components in parts by weight: 5g of cane sugar, 2g of sodium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 0.005g of ferric chloride, 0.1g of calcium carbonate, 1.5g of potassium feldspar powder (a sample is dried at 50 ℃ after being washed for 5 times by deionized water), and 1000ml of distilled water; adjusting the pH value of the culture medium to 7.0-7.5, and sterilizing at 121 ℃ for 30min for later use; the Alexander bauov solid culture medium is prepared by adding 20g of agar on the basis of the components of the Alexander bauov liquid culture medium.
The invention also aims to provide a microbial inoculum for soil potassium dissolution, phosphorus dissolution and nitrogen fixation, which is dry powder microbial inoculum obtained by fermenting the multifunctional potassium dissolution bacteria to obtain fermentation liquor and performing spray drying.
The invention has the beneficial effects that: the screened multifunctional potassium-solubilizing bacteria have high potassium-solubilizing capability, good phosphorus-solubilizing capability and nitrogen-fixing capability, can increase the contents of available potassium, available phosphorus and available nitrogen in soil, and improve the utilization efficiency of insoluble potassium and phosphorus in soil; after the fermentation broth obtained by fermenting the multifunctional potassium-decomposing bacteria or the dry powder microbial inoculum obtained by spray drying the fermentation broth is applied, the use amount of various fertilizers can be reduced, the crop yield is improved, the water and soil loss is reduced, the application prospect is wide, the operation is simple, no pollution is caused, and no harm is caused to the environment.
The preservation date of the strain is 2017, 5 and 24 months, and the preservation number is CCTCC NO. M2017281. The classification name is Burkholderia (Burkholderia tripica) H1, the name of the preservation unit is China center for type culture Collection, and the address is Wuhan university, Wuhan City, China, zip code: 430072.
Drawings
FIG. 1 is a colony morphology diagram of the multi-functional potassium-solubilizing bacteria provided in example 2 of the present invention in a plate streaking during identification;
FIG. 2 is a diagram showing the potassium-releasing effect of the multi-functional potassium-releasing bacterium provided in example 3 of the present invention;
FIG. 3 is a diagram showing the phosphorus solubilizing effect of the multi-functional potassium solubilizing bacteria provided in example 4 of the present invention;
FIG. 4 is a diagram illustrating the nitrogen fixation effect of the multi-functional potassium bacteria provided in embodiment 5 of the present invention;
FIG. 5 is a diagram of the effect of the multi-functional potassium-solubilizing bacteria on sugar beet in a potting experiment, according to an application example of the invention.
Detailed Description
Example 1: screening of multifunctional potassium-decomposing bacteria
The screening method of the multifunctional potassium-solubilizing bacteria comprises the following steps:
step 1, taking 10g of corn rhizosphere soil of corn field of Huazhong agriculture university, putting the corn rhizosphere soil into a triangular flask with glass beads, adding 90mL of sterile water,oscillating at constant temperature of 28-30 ℃ for 30-45 min to uniformly disperse the sample in sterile water to obtain the product with dilution factor of 10-1Standing the bacterial suspension, taking supernatant, and sequentially diluting to obtain 10-3、10-5、10-7Multiple concentration gradient sample bacterial suspension, 10-3、10-5、10-7Taking 0.1ml of bacterial suspension with double concentration gradient, sequentially and uniformly coating the bacterial suspension on an Alexander bauov solid culture medium, putting the bacterial suspension into a constant temperature incubator, and performing constant temperature culture for 3-5 days at the temperature of 30 ℃;
and 2, selecting a colony with good growth vigor from the Alexander bauov solid culture medium cultured at the constant temperature in the step 1, separating and purifying the colony, then inoculating the colony into a 250mL conical flask containing 100mL Alexander bauov liquid culture medium, performing shake culture at 30 ℃ for 30d at 180r/min, and repeating 3 bottles.
The Alexander bauov liquid culture medium comprises the following components in parts by weight: 5g of cane sugar, 2g of sodium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 0.005g of ferric chloride, 0.1g of calcium carbonate, 1.5g of potassium feldspar powder (a sample is dried at 50 ℃ after being washed for 5 times by deionized water), and 1000ml of distilled water; adjusting the pH value of the culture medium to 7.0-7.5, and sterilizing at 121 ℃ for 30min for later use; the Alexander bauov solid culture medium is prepared by adding 20g of agar on the basis of the components of the Alexander bauov liquid culture medium.
A strain with the number of H1 is obtained from soil through the separation route, and the strain is proved to have the function of potassium dissolution.
Example 2: identification of multifunctional potassium-decomposing bacteria
The obtained strain with the number of H1 is subjected to morphological identification, biochemical identification and 16S rRNA sequencing analysis to obtain Burkholderia (Burkholderia tripica) H1;
1. the colony morphology result is shown in figure 1, the growth physiological morphology of the strain is that the colony surface is smooth and light yellow, the surface is provided with a transparent clamping film, the edge is neat, and the strain is in a circular convex shape;
2. the biochemical identification result is gram-negative bacteria, and coryneform bacteria with motility and good oxygen are obtained;
3. the strain H1 is amplified and forward sequenced by using the primers 27F and 1492R, the obtained sequence length is 1403bp, and the specific sequence is as follows:
<tgcagtcgaacggcagcacgggtgcttgcacctggtggcgagtggcgaacgggtgagtaatacatcggaacgt gtcctgtagtgggggatagcccggcgaaagccggattaataccgcatacgatctacggatgaaagcgggggatcttcgg acctcgcgctataggggcggccgatggcggattagctagttggtgaggtaaaggctcaccaaggcgacgatccgtagct ggtctgagaggacgaccagccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaattttg gacaatgggcgaaagcctgatccagcaatgccgcgtgtgtgaagaaggccttcgggttgtaaagcacttttgtccggaaa gaaatccctggtcctaatatggccgggggatgacggtaccggaagaataagcaccggctaactacgtgccagcagccg cggtaatacgtagggtgcaagcgttaatcggaattactgggcgtaaagcgtgcgcaggcggtgatgtaagaccgatgtga aatccccgggctcaacctgggaactgcattggtgactgcatcgcttgagtatggcagaggggggtagaattccacgtgta gcagtgaaatgcgtagagatgtggaggaataccgatggcgaaggcagccccctgggtcaatactgacgctcatgcacga aagcgtggggagcaaacaggattagataccctggtagtccacgccctaaacgatgtcaactggttgtcgggtcttcattga cttggtaacgtagctaacgcgtgaagttgaccgcctggggagtacggtcgcaagattaaaactcaaaggaattgacgggg acccgcacaagcggtggatgatgtggattaattcgatgcaacgcgaaaaaccttacctacccttgacatgtacggaattcc gctgagaggtggaagtgcccgaaagggagccgtaacacaggtgctgcatggctgtcgtcagctcgtgtcgtgagatgtt gggttaagtcccgcaacgagcgcaacccttgtccctagttgctacgcaagagcactccagggagactgccggtgacaaa ccggaggaaggtggggatgacgtcaagtcctcatggcccttatgggtagggcttcacacgtcatacaatggtcggaacag agggttgccaagccgcgaggtggagccaatcccagaaaaccgatcgtagtccggatcgcagtctgcaactcgactgcgt gaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggtcttgtacacaccgcccgtcaca ccatgggagtgggttttgccagaagtggctagtctaaccgcaaggaggacgtcaccac>
alignment of the sequences with those in the GenBank database by BLAST gave 99% similarity to Burkholderia tripica.
Implementation 3: determination of potassium-decomposing capacity of multifunctional potassium-decomposing bacterium
Preparing a potassium dissolving culture medium (without water-soluble potassium ions), subpackaging 95mL of the potassium dissolving culture medium, adding 0.5g of accurately weighed potassium mineral powder into a 250mL triangular flask, sterilizing at 121 ℃ for 30min, inoculating the Burkholderia H1 suspension obtained in the step 1 into a shake flask strain in an inoculation amount of 5%, performing three parallels, and simultaneously setting a no-inoculation control. After 30 days of shaking culture in 37 ℃ water bath, the culture is ended, 5ml of fermentation liquor is taken, and 1ml of 6% H is added2O2Digesting in boiling water bath for 1h, centrifuging digestive juice at 5000r/min for 10min, collecting supernatant, and measuring K on flame photometer+And (4) concentration. The potassium-releasing effect of Burkholderia H1 is shown in FIG. 2, and it is understood from FIG. 2 that the highest potassium-releasing amount of Burkholderia H1 is 31.86mg/L, the potassium-releasing rate is 16.04%, and the potassium-releasing ability is strong.
Example 4: determination of phosphorus dissolving capacity of multifunctional potassium-solubilizing bacteria
(1) Preparing an inorganic phosphorus culture medium: the culture medium comprises the following components in parts by weight: glucose 10g, (NH)4)2SO4 0.5g、MgSO4·H2O 0.3g、NaCl 0.3g、KCl 0.3g、FeSO4·7H2O 0.03 g、MnSO4·H20.03g of O, 5g of Morocco natural phosphate rock powder (samples are dried at 50 ℃ after being washed for 5 times by deionized water), 20g of agar and 1000g of distilled water; separately sterilizing the Morocco natural phosphate rock powder and the rest components, mixing, and adjusting the pH value to 7.0-7.5;
(2) and (3) determination of phosphate solubilizing capability: 5mL of the activated Burkholderia H1 bacterial suspension was inoculated into a 250mL Erlenmeyer flask containing 100mL of an inorganic phosphorus liquid medium, and cultured at 30 ℃ for 10 days with shaking at 180r/min, and 3 flasks were repeated. Taking 5ml of fermentation liquor 1, 3, 5 and 7 days after inoculation, centrifuging at 5000r/min for 10min, and determining the content of soluble phosphorus in the supernatant by adopting a molybdenum-antimony colorimetric method. As shown in FIG. 3, the maximum phosphorus-solubilizing amount of Burkholderia H1 was 199.92mg/L, and the phosphorus-solubilizing rate was 23.65%.
Example 5: nitrogen fixation capacity determination of multifunctional potassium-decomposing bacteria
(1) Preparing a nitrogen-free culture medium: the culture medium comprises the following components in parts by weight: 10g of glucose, 0.2g of potassium hydrogen phosphate, 0.2g of magnesium sulfate heptahydrate, 0.2g of sodium chloride, 5g of calcium carbonate, 0.1g of calcium sulfate and 1000ml of distilled water; adjusting the pH value of the culture medium to 7.0-7.5, and sterilizing at 121 ℃ for 30min for later use;
(2) and (3) measuring nitrogen fixation capacity: 5mL of the activated Burkholderia H1 bacterial suspension was transferred to a 250mL Erlenmeyer flask containing 100mL of nitrogen-free liquid medium, and cultured at 30 ℃ for 30d with shaking at 180r/min, and the flask was repeated 15 times. Adding concentrated sulfuric acid and 30% H into the whole fermentation liquid at 3, 5, 10, 15, and 30 days after inoculation2O2The nitrogen content was determined by high temperature digestion in an electric furnace. As shown in FIG. 4, it is understood from FIG. 4 that Burkholderia H1 exhibited a relatively good nitrogen-fixing effect, and the nitrogen-fixing amount at 30d was 14.92 mg/L.
This is known from the above. After being identified, the Burkholderia H1 screened by the invention not only has higher potassium-dissolving capability, but also has good phosphorus-dissolving capability and nitrogen-fixing capability, so the Burkholderia H1 is called multifunctional potassium-dissolving bacteria.
Example 6: preparation examples
Activating the multifunctional potassium-decomposing bacteria stored on the inclined plane, inoculating the multifunctional potassium-decomposing bacteria on an activation culture medium, putting the activated multifunctional potassium-decomposing bacteria into a constant-temperature incubator, and performing constant-temperature culture for 3-5 days at the temperature of 30 ℃; selecting strains on an activation culture medium, inoculating the strains in a fermentation culture medium, carrying out shaking culture at the temperature of 30 ℃ for 10d at 180r/min, fermenting to obtain fermentation liquor, and carrying out spray drying to obtain the dry powder microbial inoculum.
The activation medium is an Alexander bauov solid medium, the fermentation medium is an Alexander bauov liquid medium, and the Alexander bauov liquid medium comprises the following components in parts by weight: 5g of cane sugar, 2g of sodium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 0.005g of ferric chloride, 0.1g of calcium carbonate, 1.5g of potassium feldspar powder (a sample is dried at 100 ℃ after being washed for 5 times by deionized water), and 1000ml of distilled water; adjusting the pH value of the culture medium to 7.0-7.5, and sterilizing at 121 ℃ for 30min for later use; the Alexander bauov solid culture medium is prepared by adding 20g of agar on the basis of the components of the Alexander bauov liquid culture medium.
The application example is as follows: verification of growth promoting effect of multifunctional potassium-solubilizing bacteria on beet in pot experiment
The growth promoting effect of the fermentation broth of the multi-functional potassium bacteria or the prepared dry powder microbial inoculum in the embodiment 6 on the beet is verified in a pot experiment, and the experiment is divided into a multi-functional potassium bacteria group (100 ml of H1 bacterial liquid is inoculated in a pot culture) and a CK group (a blank control group without any strain inoculation). And repeating 4 pot plants in each group, sowing 3 germinated beet seeds in each pot, and applying nitrogen fertilizer and phosphate fertilizer according to the application amount of 0.22g/kg of soil urea and 0.42g/kg of soil calcium superphosphate in each pot. And then, placing the potted plants under a rain shelter of an outdoor potting field for culturing, thinning out seedlings by stages after seedling emergence, finally, fixing the seedlings, reserving 2 plants in each pot, pouring equivalent deionized water into each pot at regular intervals, and continuously culturing for 147 days.
The growth conditions of the beets are shown in fig. 5, and it can be known from fig. 5 that the height and the root of the beets are obviously larger than those of the blank group after the multifunctional potassium bacteria are added. The influence of the multi-functional potassium-decomposing bacteria on the main growth indexes of the beet, such as plant fresh seeds, root fresh seeds, nitrogen content, phosphorus content, potassium content and sugar yield, is shown in table 1. As can be seen from the table, after the inoculation of the multi-functional potassium-solubilizing bacteria, the fresh beet seeds are increased by 59.24 percent, the plant height is increased by 18.47 percent, the root tuber weight is increased by 81.90 percent, the dry weight is increased by 51.86 percent, the total nitrogen content is increased by 32.45 percent, the total potassium content is increased by 57.60 percent, the total phosphorus content is increased by 38.59 percent, and the sugar yield is increased by 34.64 percent.
TABLE 1 Effect of the Multi-functional Potassium-solubilizing bacteria on the growth index of sugar beet
Note that: the data in table 1 are expressed as mean ± standard deviation of 3 replicates, and the letter differences after the data indicate significant differences between the data.
The application example shows that after the fermentation liquor of the multifunctional potassium-solubilizing bacteria or the prepared dry powder microbial inoculum is applied, phosphorus and potassium in soil are converted into soluble nutrients, the content of the quick-acting nutrients in the soil is increased, the using amount of a chemical fertilizer is reduced, the growth and development of crops are promoted, and the yield is improved. Has important significance and application value in the aspects of cultivating and exerting the ecological fertility of soil and keeping the ecological balance of agriculture.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (5)
1. The taxonomic name of the multifunctional potassium bacteria is Burkholderia tropicalis (Burkholderia tropicalis) H1, and the preservation number is as follows: CCTCC NO. M2017281.
2. The use of the multi-functional potassium solubilizing bacteria of claim 1 in soil potassium solubilization, phosphorus solubilization, and nitrogen fixation.
3. The application of the multi-functional potassium bacteria in soil potassium dissolving, phosphorus dissolving and nitrogen fixing, which is characterized in that the multi-functional potassium bacteria is used for the soil potassium dissolving, phosphorus dissolving and nitrogen fixing according to the following method:
activating the multifunctional potassium-decomposing bacteria stored on the inclined plane, inoculating the activated multifunctional potassium-decomposing bacteria on an activation culture medium, putting the activated multifunctional potassium-decomposing bacteria into a constant temperature incubator, and culturing for 3-5 days at the constant temperature of 30 ℃; selecting strains on an activation culture medium, inoculating the strains in a fermentation culture medium, and performing shaking culture at 30 ℃ for 10d at 180r/min to obtain a fermentation liquid for dissolving potassium and dissolving phosphorus and fixing nitrogen in soil, or drying the fermentation liquid to obtain a dried substance for dissolving potassium, dissolving phosphorus and fixing nitrogen in soil.
4. The application of the multi-functional potassium-solubilizing bacteria in soil potassium solubilizing, phosphorus dissolving and nitrogen fixing according to claim 3, wherein the activation medium is an Alexander bauov solid medium, the fermentation medium is an Alexander bauov liquid medium, and the components of the Alexander bauov liquid medium are calculated according to the parts by weight: 5g of cane sugar, 2g of sodium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 0.005g of ferric chloride, 0.1g of calcium carbonate, 1.5g of potassium feldspar powder and 1000ml of distilled water; adjusting the pH value of the culture medium to 7.0-7.5, and sterilizing at 121 ℃ for 30min for later use; the Alexander bauov solid culture medium is prepared by adding 20g of agar on the basis of the components of the Alexander bauov liquid culture medium.
5. A microbial inoculum for potassium dissolution, phosphorus dissolution and nitrogen fixation of soil, which is characterized in that the multifunctional potassium-dissolving bacterium of claim 1 is fermented to obtain fermentation liquor, and the fermentation liquor is spray-dried to obtain a dry powder microbial inoculum.
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