CN108753638B

CN108753638B - Nitrogen-fixing blue algae for producing salicylic acid in rice field and application of nitrogen-fixing blue algae in rice field

Info

Publication number: CN108753638B
Application number: CN201810397659.3A
Authority: CN
Inventors: 贺鸿志; 包江桥; 周伊薇; 卢玉真; 陈桂葵; 黎华寿; 秦俊豪
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2018-04-28
Filing date: 2018-04-28
Publication date: 2020-04-17
Anticipated expiration: 2038-04-28
Also published as: CN108753638A

Abstract

The invention discloses a paddy field nitrogen-fixing blue algae for producing salicylic acid and application thereof in a paddy field. The name of the strain is Nostoc piscinale SCAU-003, the preservation number is CCTCC NO: m2018194, the preservation date is 2018, 4 and 11, and the preservation unit is the China center for type culture Collection, located at the university of Wuhan, China. The strain can produce high-concentration plant hormones such as salicylic acid and the like, grows fast, has strong nitrogen and carbon fixing capacity, can be used as a biological fertilizer in a new application mode in a paddy field, can replace half of chemical nitrogen fertilizers, and simultaneously increases the lodging resistance and disease resistance of paddy rice.

Description

Nitrogen-fixing blue algae for producing salicylic acid in rice field and application of nitrogen-fixing blue algae in rice field

Technical Field

The invention relates to the field of microorganisms and the field application of microbial fertilizers, in particular to a paddy field nitrogen-fixing blue-green alga for producing salicylic acid and application thereof in a paddy field.

Background

After 1939 Indian scientists report that blue-green algae is used for fertilizing the field for the first time, related researches are carried out in many countries successively, and the researches prove that the yield can be increased by inoculating the blue-green algae in a rice field. The rice field algae cultivation test is carried out in partial provinces of south China at the beginning of Li Shanghao in the last 50 th century, and the result shows that the rice yield is increased by 10-30%. The nitrogen-fixing blue algae bred in the rice field not only can provide nitrogen and organic matters for crops, improve the yield, reduce the fertilizer consumption, improve the phosphate solubility and reduce water and soil pollution, but also has beneficial ecological functions of secreting phytohormones to promote the growth of the crops and the like.

Phytohormones are active substances produced by plant cells under the induction of specific environmental signals and capable of regulating physiological reactions at very low concentrations, and are simple small molecular organic compounds and classified into six major groups, namely auxin, cytokinin, abscisic acid, gibberellin, ethylene, brassinosteroid and the like. The plant hormone has important regulation and control effect on the growth and development of plants. The indoleacetic acid, the salicylic acid and the like can promote the growth of plants and simultaneously improve the stress resistance of crops, and the salicylic acid has an important role in inducing the disease resistance of the crops. However, studies at home and abroad show that the nitrogen-fixing blue algae can generate various plant hormones including auxin, cytokinin and the like, but no report about the generation of salicylic acid by the nitrogen-fixing blue algae in the rice field is provided. Under the current big background that the country requires the reduction of both fertilizer and pesticide, if the nitrogen-fixing blue algae which can grow rapidly, fix nitrogen and fix carbon and produce phytohormone to induce plant disease resistance can be screened, the nitrogen-fixing blue algae has important significance for being used as biological fertilizer and biological pesticide in agricultural production.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the nitrogen-fixing blue algae for the rice field for producing the salicylic acid.

The invention also aims to provide the application of the salicylic acid-producing paddy field nitrogen-fixing blue algae in paddy fields.

The purpose of the invention is realized by the following technical scheme: a paddy field nitrogen-fixing blue algae for producing salicylic acid is named as Nostoc piscicola SCAU-003, and the preservation number is CCTCC NO: m2018194, the preservation date is 2018, 4 and 11 months, and the preservation unit is the China center for type culture Collection, located at the university of Wuhan, China.

The paddy field nitrogen-fixing blue algae for producing salicylic acid can be used as a biological fertilizer to be applied in paddy fields, and preferably comprises the following steps: and (3) putting the paddy field nitrogen-fixing blue algae for producing the salicylic acid into the paddy field at a proper time.

When the paddy field nitrogen-fixing blue-green algae for producing the salicylic acid is put into a paddy field at a proper time, the environmental conditions are favorable for the survival of the paddy field nitrogen-fixing blue-green algae for producing the salicylic acid; preferably, the temperature is 25-30 ℃, the sunshine is sufficient, and the environmental condition that no rain exists within 5 days after the algae is put in, or the environmental condition that the temperature is lower than 15 ℃.

The nitrogen-fixing blue algae in the paddy field for producing the salicylic acid is preferably in a high-density state and floats to form a film when being put into the paddy field.

The paddy field nitrogen-fixing blue algae for producing the salicylic acid is preferably obtained by culturing the following steps:

(1) culturing the salicylic acid-producing paddy field nitrogen-fixing blue algae to logarithmic growth phase under aseptic condition to obtain algae liquid;

(2) inoculating the algae liquid obtained in the step (1) into a sterilized culture medium, and culturing in a natural environment to obtain the salicylic acid-producing nitrogen-fixing cyanobacteria algae liquid in the rice field.

The culture in the step (1) is preferably carried out at the temperature of 25-30 ℃, the illumination is 2500-3500 LX, and the light-dark time is 14-18 h: culturing for 6-10 h; more preferably, the culture is carried out at 28 ℃ under 3000lx light for 16h to 8h in dark time.

The culture medium used in the culture in the step (1) is preferably BG11₀A liquid culture medium;

BG11₀the composition of the liquid medium was as follows: k₂HPO₄.3H₂O 0.04g/L、MgSO₄.7H₂O 0.075g/L、CaCl₂.2H₂O0.036 g/L, citric acid 0.006g/L, ferric ammonium citrate 0.006g/L, EDTA 0.001.001 g/L, Na₂CO₃0.02g/L, trace element A₅1mL；

Trace element A₅The composition of (A) is as follows: h₃BO₃2.860g/L、NaMoO₄.2H₂O 0.021g/L、ZnSO₄.7H₂O0.222g/L、CuSO₄.5H₂O 0.079g/L、MnCl₂.4H₂O 1.810g/L、NiSO₄.6H₂O 0.479g/L。

The amount of the seed solution of the alga to be inoculated in the step (2) is preferably 0.5% by volume of the culture medium.

The culture medium in the step (2) is preferably BG11 liquid culture medium (BG 11) containing no sodium nitrate₀)。

The natural environment in the step (2) is preferably the open space in the front and the back of the farmer house.

The vessel used in the culture described in step (2) is preferably an open vessel; more preferably a plastic tub.

The step of culturing in step (2) is preferably: covering a mosquito net or screen window cloth at the opening of the culture container to prevent the mosquitoes from laying eggs; culturing in a place directly sunned in the daytime, and moving into a room at night; and (5) growing the algae until an algae membrane is formed.

Compared with the prior art, the invention has the following advantages and effects:

the nitrogen-fixing blue algae in the rice field, which can generate high-concentration plant hormones such as salicylic acid and the like, has the advantages of fast growth and strong nitrogen and carbon fixing capability, can be used as a biological fertilizer in the rice field in a new application mode, can replace half of chemical nitrogen fertilizers, and simultaneously increases the lodging resistance and disease resistance of rice.

Drawings

FIG. 1 is a graph showing the results of the cultivation of rice, nitrogen-fixing cyanobacteria co-culture and inoculation in the micro-chamber at the seedling stage of sheath blight; wherein, Panel A is a photograph of a culture in a beaker; FIG. B shows rice seedlings obtained by different treatments; from left to right, the blank control group, the SCAU-003 group, the SCAU-010 group, the SCAU-012 group and the FACHB119 group are arranged in sequence.

FIG. 2 is a diagram of nitrogen-fixing cyanobacteria under an optical microscope; in the figure, a is a photograph magnified 300 times, and B is a photograph magnified 1500 times (oil mirror).

FIG. 3 is a BLAST alignment chart of the nitrogen-fixing cyanobacteria 16srDNA gene sequence.

FIG. 4 is a BLAST alignment chart of the nitrogen-fixing cyanobacteria rbcLX gene sequence.

FIG. 5 is a graph showing the results of the growth performance test of SCAU-003; wherein, the graph A is a growth curve graph, and the graph B is a specific growth rate calculation graph.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1: SCAU-003 separation, purification and identification of nitrogen-fixing cyanobacteria strain in rice field

(1) Algae seed separation and purification

Surface soil sample: collected from the rice field in the city-increasing and western-style base of southern China university of agriculture.

And (3) separation and purification of algal species: taking a soil sample, adding 20mL BG11 into the soil sample according to 1g₀Pre-culturing in liquid culture medium at 28 deg.C under 3000lx light for 16h and 8h in light-dark incubator for 7 days. Collecting supernatant containing algae, and spreading on 9cm BG11₀Plates of solid medium. After the macroscopic bacteria grow out, carefully selecting out the bacteria and scratching the plates again, performing microscopic examination, and repeating the process for more than 3 times to obtain the nitrogen-fixing blue algae cultured singly. Finally, the sterile algae strain is obtained by the combined treatment of ultraviolet irradiation and various antibiotics. The specific method comprises the following steps: 20. mu.l of algal solution with OD680 adjusted to about 0.05 was carefully spread on the inner wall of a quartz cuvette and irradiated under an ultraviolet lamp for 20 to 30 minutes. After treatment 40. mu.l BG11 were used₀The algae cells are washed off by the culture medium and then spread to the culture medium containing BG11₀Agar solid plates of medium. After the bacteria can be seen by naked eyes, the bacteria are picked out and placed in BG11₀Culturing in liquid culture medium to logarithmic growth phase. Sequentially passes through BG11 containing 10mg/L penicillin₀Treating with liquid culture medium for 3 days, centrifuging to obtain algae cells, and treating with BG11₀After the suspension is washed by liquid culture medium, the suspension is treated by 1mg/L gentamicin and 1mg/L kanamycin according to the penicillin treatment method in turn, and finally the suspension is suspended in BG11₀The culture medium is the sterile algae cell.

BG11₀The formulation of the medium is as follows:

TABLE 1 BG11₀Media drug list

TABLE 2 Trace elements A₅List of items

(2) Evaluation of disease resistance of nitrogen-fixing cyanobacteria-rice co-culture

Selecting three separated sterile algae strains for detection, and respectively naming the three sterile algae strains as SCAU-003, SCAU-010 and SCAU-012. Meanwhile, nitrogen-fixing cyanobacteria strain FACHB119 purchased from freshwater algae seed repository (Wuhan Hubei) of Chinese academy of sciences was used as a control.

According to the literature (slow)National azadir, etc., chinese rice science 2015,29 (1): 97-105) and establishes a rice-algae co-culture and rhizoctonia solani micro-chamber inoculation technology of rice seedling stage semisolid culture-algae liquid culture. The method comprises the following specific steps: after germination, 3 rice seeds (Huanghuazhan, purchased from Shenzhen Longjingu breed Co., Ltd.) were placed in a 1L open glass beaker containing 400mL 1/2MS semisolid medium, and cultured in an artificial climate chamber at 29 ℃ under 85% humidity, 3000lx under illumination and 16h in darkness for 8 h. When the rice seedlings grow to 2 leaves and 1 heart, 40mL of BG11 solution is added₀The nitrogen-fixing blue algae solution (SCAU-003, SCAU-010, SCAU-012 and FACHB119 respectively) growing to logarithmic phase in the culture medium is cultured for 7 days, and then a rhizoctonia solani (bacterial seed bank of Guangdong institute of microbiology, Guangdong, Guangzhou, China, number GIM3.512) bacterial cake is placed in a rice leaf sheath. At this time, a 2.5L empty plastic barrel with the bottom cut off is sleeved on the glass beaker to form a relatively closed environment to cause the banded sclerotial blight to occur. The treatment cup without added algae was used as a blank control, and the treatment and control were each repeated three times. The resistance of the algae and rice co-culture to rice sheath blight was evaluated and the results are shown in table 3 and fig. 1. As can be seen from Table 3 and FIG. 1, the plant height and disease inhibition rate of SCAU-003-treated rice were higher than those of the control group and the FACHB 119-treated group. Thus, SCAU-003 was selected for identification.

TABLE 3 evaluation results of algal-rice co-culture-Rhizoctonia solani micro-chamber inoculation technique

(3) Identification of algal species

The strain form of SCAU-003 is shown in FIG. 2, and is filamentous nitrogen-fixing blue algae with heteromorphic cells.

The strain was subjected to sequence analysis by 16SrDNA method: PCR amplification with 16S pair of primers 27F (AGAGAGTTTGATCTMTGGCTCAG) and 1492R (TACGGYTACCTTGTTACGACTT) determined the 16srDNA sequence by sequencing (as shown below) and Blast comparison analysis by Gene bank (FIG. 3) indicated that the algae has the closest affinity to the known Nostoc sp. PCC9426, indicating that the algae species belongs to the genus Nostoc.

16srDNA sequence:

CCTTGGCGGTTGCTTACCATGCAGTCGAACGGAATCTTAGGATTTAGTGGCGGACGGGTGAGTAACGCGTGAGAATCTAGCTTCAGGTCGGGGATAACTACTGGAAACGGTGGCTAATACCGGATGTGCCGAAAGGTGAAAGGCTTGCTGCCTGAAGATGAGCTCGCGTCTGATTAGCTAGTTGGTGTGGTAAGAGCGCACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAATGGGCGAAAGCCTGACGGAGCAATACCGCGTGAGGGAGGAAGGCTCTTGGGTTGTAAACCTCTTTTCTCAAGGAATAAGTTCTGAAGGTACTTGAGGAATAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCAAGCGTTATCCGGAATGATTGGGCGTAAAGCGTCCGCAGGTGGCTGTGTAAGTCTGCTGTTAAAGAGTCTAGCTTAACTAGATAAAAGCAGTGGAAACTACATAGCTAGAGTACGTTCGGGGTAGAGGGAATTCCTGGTGTAGCGGTGAAATGCGTAGAGATCAGGAAGAACACCGGTGGCGAAGGCGCTCTACTAGGCCGTAACTGACACTGAGGGACGAAAGCTAGGGGAGCGAATGGGATTAGATACCCCAGTAGTCCTAGCCGTAAACGATGGATACTAGGCGTTGCGAGTATCGACCCTCGCAGTGCCGGAGCCAACGCGTTAAGTATCCCGCCTGGGGAGTACGCACGCAAGTGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCAAGACTTGACATGTCGCGAATCCTCTTGAAAGGGAGGAGTGCCTTCGGGAGCGCGAACACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTTTTAGTTGCCAGCATTAAGTTGGGCACTCTAGAGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCAGCATGCCCCTTACGTCTTGGGCTACACACGTACTACAATGCTCCGAACAGAGGGCAGCGAGCTAGCGATAGCAAGCAAATCCCGGAAATCGGAGCTCAGTTCAGATCGAAGGCTGCAACTCGCCTTCGTGAAGGAGGAATCGCTAGTAATTGCAGGTCAGCATACTGCAGTGAATTCGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGAAGCTGGCAACGCCCGAAGTCATTACCCCAACTTTCGAGAGGGGGATGCCTAAGGCA。

to further identify the algal strain, after extracting the genomic DNA of the algae, the genomic DNA was extracted using two primer pairs: CX (5'-GGCGCAGGTAAGAAAGGGTTTCGTA-3') and CW (5'-CGTAGCTTCCGGTGGTATCCACGT-3') amplified and sequenced the cyanobacteria azotobacter specific gene rbcLX (shown below). As shown in FIG. 4 based on BLAST analysis, it was found that the alga has very high homology (99%) with the known Nostoc piscaline CENA 21. Namely, the algal species was identified as Nostoc piscicaleCEA 21.

rbcLX gene sequence:

GGTGATGACTCCGTACTACAATTTGGTGGTGGTACACTCGGACACCCCTGGGGTAATGCTCCTGGTGCAACCGCTAACCGTGTAGCTTTGGAAGCGTGTATCCAAGCTCGTAACGAAGGCCGCAACTTGGCGCGTGAAGGTAACGATGTTATTCGTGAAGCTGCTAAGTGGTCTCCTGAACTAGCTGCTGCTTGCGAACTGTGGAAAGAAATCAAGTTCGAGTTTGAGGCAATGGATACCGTCTGATCATCAAGTAAAAGGTAAAAAGTTAAAAGGTAAAAGAAAATCTTTTTACTTATTACTTTTTACTTTTAACTTAAATTTGGGCTGGGTCAAGCATGAATCTCAAGCAAATTGCGAAAGATACAGCCAAAACTCTCCAAAGCTACCTAACATATCAGGCGCTAAGGATTGTACTGGCACAGCTAGGCGAAACGAATCCACCGTTAGCTGTTTGGCTGCATAACTTTTCTGCCGGAAAAGTTCAGGATGGAGAAGCTTATATTGAACAACTTTTCCAACACAAACCTGACTTAGCACAGCGAATTATGACAGTTAGAGAACATATAGCCGAAGAAGTGGCAGAGTTCTTACCAGAAATGGTTCGCATTGGTATTAAGCAAGCCAATATGGAACAGCGTCGCCAACACTTAGAACGCATCACGCAGCTGAGTTTAACTAACCCCAGTCCTGAATCAGAACAACAGCAATTTTCCGATCCTGACTGGGATAACTTAGCCAGTTAGGAAACATCCAAGTCGTCACTCAATAGCAAACCTTTATTATTAGCTATGC。

the product is named as Nostoc piscinale SCAU-003 with the preservation number of CCTCC NO: m2018194, the preservation date is 2018, 4 and 11 months, and the preservation unit is the China center for type culture Collection, located at the university of Wuhan, China.

Example 2: SCAU-003 growth, nitrogen fixation, carbon fixation and biomass capacity assessment

Using the above BG11₀Inoculating the strain SCAU-003 with liquid culture medium to grow to logarithmic phase, culturing in light incubator at 28 deg.C under 3000lx for 16 hr and 8 hr, wherein the strain SCAU-003 has growth curve shown in FIG. 5 and OD₆₈₀The value increased from 0.08 at inoculation to 0.65 at day 13 by more than 5-fold. The calculation result shows that the algae has higher specific growth rate (mu-0.163 d)^-1)。

Culturing algae in the same way, sampling on day 4, collecting algae liquid, and measuring azotase activity by acetylene reduction-gas chromatography, wherein the azotase activity of the algae is 0.309 + -0.032 μmol C₂H₂·h^-1·(μg Chla)^-1. On day 15, samples were taken and algae cells were collected and lyophilized for total carbon and total nitrogen determination using a TOC instrument. The total carbon and nitrogen contents of the algal cells were found to be 1.457. + -. 0.599% and 0.037. + -. 0.017%, respectively.

In addition, BG11₀Culture medium, temperature 28 deg.C, illumination 30Open culture in a 00lx, 10L basin. The wet weight of the algae can reach 15.384 + -2.251 g/L, and the dry weight reaches 0.413 + -0.047 g/L. The algae has strong biomass accumulation capacity and nitrogen and carbon fixing capacity, and can provide organic matters and nitrogen sources for rice production.

Example 3: assessment of SCAU-003 ability to produce phytohormones such as salicylic acid

SCAU-003 algae in BG11₀Culturing in culture medium for 20 days, centrifuging, collecting algae cells, and lyophilizing. The supernatant was filtered through a 0.22 micron filter. The algal cells were ground in liquid nitrogen to break the cell wall, and then 1ml of Bieleski buffer (60% methanol, 25% chloroform, 10% formic acid and 5% water) containing an internal standard (5mg/L DHZ) was added thereto, and the mixture was subjected to ultrasonic treatment (frequency 20kHz, power 200W, working/batch 30s/30s) for 3min to sufficiently extract the algal cells. Centrifuging to collect supernatant, performing ultrasonic extraction on the precipitate for 3min again by using 0.3mL of solution containing 2% formic acid and 50% methanol, centrifuging to collect supernatant, combining the supernatant with the supernatant obtained in the first extraction, performing rotary evaporation at 40 ℃ under reduced pressure, and performing constant volume measurement to 1mL by using methanol aqueous solution with pH being 3 (methanol and water are mixed according to a volume ratio of 1:1, and the pH is adjusted to 3 by using 1mol/L hydrochloric acid) to prepare UPLC-MS-MS determination. The above culture solution was extracted with a Bieleski buffer, the organic phase was rotary evaporated to near dryness at 40 ℃ and then diluted to 1mL with a 1:1 aqueous methanol solution having pH of 3.

Chromatographic conditions are as follows: the instrument used was UPLC-MS (Waters) and the column was an Acquity UPLC BEH C18 column (2.1X 50mm, 1.7 μm, Waters). Mobile phase 0.1% formic acid water (A)/0.1% formic acid acetonitrile (B) system is used in positive and negative ion determination mode. The flow rate was 0.3 mL/min. The amount of the sample was 10. mu.L. The column temperature was 40 ℃ and the sample temperature was 25 ℃. The gradient elution condition is 0-2 min, 90-75% A; 75-40% of A for 2-3 min; 3-3.5 min, 40% -90% A; 3.5-5 min, 90% A. Mass spectrum conditions: the capillary voltage is 1.50 kV; desolventizing air flow N2 with the flow rate of 800L/h; the desolventizing temperature is 400 ℃; the flow rate of the conical hole airflow N2 is 150L/h; the ion source temperature is 150 ℃; collision chamber pressure 3.1X 10^-3mbar; the collision gas was argon and the mass spectral parameters are as follows.

TABLE 4 Mass Spectrometry parameters

Note: (1) for quantifying ions

The concentrations of phytohormones in the algal cells and the culture solution are shown in Table 5, and the results show that the algal cells can produce auxin IBA, cytokinin TZR and salicylic acid SA at higher concentrations. The yield of salicylic acid from algal cells was at the μ g/g level. The salicylic acid can induce crops to improve stress resistance, and domestic and foreign researches find that the salicylic acid plays an important role in high and low temperature resistance, drought resistance, lodging resistance and insect disease resistance.

TABLE 5 concentration of each hormone in algal cells and culture

Example 4: nitrogen fixation blue algae paddy field plot stocking test

(1) Design of experiments

And in 2017, in 7-12 months, carrying out nitrogen fixation tests on nitrogen-fixing blue algae instead of chemical nitrogen fertilizers and improving stress resistance of rice on a certain farm in the new meeting area of Jiangmen. 3 treatments (the same NPK fertilizer is added as a base fertilizer) are set in the test:

processing one: conventional fertilization control CK (full top dressing)

And (5) processing: nitrogen-fixing blue algae SCAU-003 is applied, and other fertilizers except nitrogen fertilizer are added in the top dressing together with CK (algae addition: three times are respectively added 3-5 days after intertillage, at tillering stage and 5-8 days before closure).

The enlarged culture method of nitrogen-fixing blue algae SCAU-003 comprises the following steps:

1) under laboratory sterile conditions, BG11 was used₀Liquid culture is carried out at 28 deg.C under 3000lx light for 16h in 8h, and the culture is amplified from 20mL to 1L.

2) 100mL BG11₀Diluting a culture medium concentrated nutrient solution (100 times) by using tap water or well water boiled for 10min for 100 times to 10L, placing the culture medium concentrated nutrient solution in a large plastic basin, adding 50mL of algae liquid, covering a mosquito net or a screen window cloth on the basin, covering the periphery of the basin, clamping the basin by using a long tail clamp to prevent mosquitoes from laying eggs, placing the basin in a place directly exposed to the sun for culturing, and moving the basin into a room at night. Culturing for 10-14 days15-20L/mu of algae which is high in density and floats to form a film is released to the rice field under the condition that the algae film is not damaged as much as possible. The field experiments were 5m by 5m (length by width) per cell, with three replicates per treatment.

(2) Test results

The algal strain has strong floating property, is easy to form algal membranes, has strong outdoor stress resistance and can rapidly propagate in large quantities under the conditions of relatively proper temperature and illumination. After the field is successful, the rapid growth and propagation are realized. The lodging of the algae applying rice field is obviously reduced compared with that of the contrast when the typhoon is carried out for three times, and the experiment contrast and the peripheral rice field are damaged in a large area in the later period when the bacterial leaf blight outbreak occurs, but the influence on the algae applying rice field is very small. Finally, the yield is increased, and the actually measured yield increase is over 17 percent as shown in Table 6.

TABLE 6 Effect of algal treatment on Rice yield

(3) Conclusion

The addition of algae in the rice production can replace the addition of nitrogen fertilizer, obviously increase the stress resistance of the rice and increase the yield.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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actttttact tttaacttaa atttgggctg ggtcaagcat gaatctcaag caaattgcga 360

aagatacagc caaaactctc caaagctacctaacatatca ggcgctaagg attgtactgg 420

cacagctagg cgaaacgaat ccaccgttag ctgtttggct gcataacttt tctgccggaa 480

aagttcagga tggagaagct tatattgaac aacttttcca acacaaacct gacttagcac 540

agcgaattat gacagttaga gaacatatag ccgaagaagt ggcagagttc ttaccagaaa 600

tggttcgcat tggtattaag caagccaata tggaacagcg tcgccaacac ttagaacgca 660

tcacgcagct gagtttaact aaccccagtc ctgaatcaga acaacagcaa ttttccgatc 720

ctgactggga taacttagcc agttaggaaa catccaagtc gtcactcaat agcaaacctt 780

tattattagc tatgc 795

Claims

1. A paddy field nitrogen-fixing blue algae for producing salicylic acid, which is characterized in that: the nitrogen-fixing blue algae in the paddy field for producing the salicylic acid is named as nostoc sphaeroides, (b)Nostoc piscinale) SCAU-003, the preservation number is CCTCC NO: m2018194, the preservation date is 2018, 4 and 11, and the preservation unit is the China center for type culture Collection, located at the university of Wuhan, China.

2. The use of the nitrogen-fixing cyanobacteria in the paddy field for producing salicylic acid as claimed in claim 1, wherein the nitrogen-fixing cyanobacteria comprises: the paddy field nitrogen-fixing blue algae for producing the salicylic acid is applied to paddy fields as a biological fertilizer.

3. The application of the salicylic acid-producing paddy field nitrogen-fixing cyanobacteria as claimed in claim 2, characterized by comprising the following steps: and (3) putting the paddy field nitrogen-fixing blue algae for producing the salicylic acid into the paddy field at a proper time.

4. The application of the salicylic acid-producing nitrogen-fixing cyanobacteria in the paddy field according to claim 3 is characterized by comprising the following steps: the proper time is the environmental condition that the temperature is 25-30 ℃, the sunlight is sufficient, and no heavy rain exists within 5 days after the algae is put in, or the temperature is lower than 15 ℃.

5. The application of the salicylic acid-producing nitrogen-fixing cyanobacteria in the paddy field according to claim 3, characterized in that: the nitrogen-fixing blue algae in the paddy field for producing the salicylic acid is in a floating film forming state when being put into the paddy field.

6. The application of the nitrogen-fixing cyanobacteria in the paddy field for producing salicylic acid according to any one of claims 3 to 5, which is characterized in that: the paddy field nitrogen-fixing blue algae for producing the salicylic acid is obtained by culturing the following steps:

7. The application of the nitrogen-fixing cyanobacteria in the paddy field for producing salicylic acid in claim 6 is characterized in that:

the culture in the step (1) is carried out at the temperature of 25-30 ℃, the illumination is 2500-3500 LX, and the light-dark time is 14-18 h: culturing for 6-10 h;

the culture medium used for the culture in the step (1) is BG11₀A liquid culture medium;

BG11₀the composition of the liquid medium was as follows: k₂HPO₄.3H₂O 0.04 g/L、MgSO₄.7H₂O 0.075 g/L、CaCl₂.2H₂O0.036 g/L, citric acid 0.006g/L, ferric ammonium citrate 0.006g/L, EDTA 0.001.001 g/L, Na₂CO₃0.02g/L, trace element A₅1 mL；

Trace element A₅The composition of (A) is as follows: h₃BO₃2.860 g/L、NaMoO₄.2H₂O 0.021 g/L、ZnSO₄.7H₂O 0.222g/L、CuSO₄.5H₂O 0.079 g/L、MnCl₂.4H₂O 1.810 g/L、NiSO₄.6H₂O 0.479 g/L。

8. The application of the nitrogen-fixing cyanobacteria in the paddy field for producing salicylic acid in claim 7 is characterized in that:

the culture in the step (1) is carried out at 28 ℃, under illumination of 3000lx and in light dark time of 16h:8 h.

9. The application of the nitrogen-fixing cyanobacteria in the paddy field for producing salicylic acid in claim 6 is characterized in that:

the adding amount of the algae liquid in the inoculation in the step (2) is calculated according to 0.5 percent of the volume of the culture medium;

the culture medium in the step (2) is BG11₀A liquid culture medium;

the natural environment in the step (2) is the open space in the front of and behind the farmer house;

the container used in the culture in the step (2) is an open container;

the culturing step in the step (2) is as follows: covering a mosquito net or screen window cloth at the opening of the culture container to prevent the mosquitoes from laying eggs; culturing in a place directly sunned in the daytime, and moving into a room at night; and (5) growing the algae until an algae membrane is formed.