CN111690578A

CN111690578A - Salt and alkali resistant Siamese bacillus and production method and application of viable bacteria preparation thereof

Info

Publication number: CN111690578A
Application number: CN202010761301.1A
Authority: CN
Inventors: 孙中涛; 辛寒晓; 刘丽英; 侯汉学; 赵升远; 孙国科; 陈君君; 韩志真; 徐艳平
Original assignee: Shandong Zoeticland Biological Technology Co ltd; Shandong Agricultural University
Current assignee: Shandong Zoeticland Biological Technology Co ltd; Shandong Agricultural University
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2020-09-22
Anticipated expiration: 2040-07-31
Also published as: CN111690578B

Abstract

The invention discloses a salt and alkali tolerant Siamese bacillus and a production method and application of a living bacterium preparation thereof. The strain is separated from rhizosphere soil of rice, is preserved in China general microbiological culture Collection center (CGMCC) at 22 months 6 and 2020, and has a preservation number of CGMCC No. 20119. Siam bacillus PAPM-15 strain has strong saline-alkali resistance and NH production₃The active bacteria preparation can effectively promote the growth of rice, has higher prevention effect on the rice bakanae disease caused by fusarium graminearum, and can be used for producing biological fertilizers, particularly saline-alkali fertilizersA special biofertilizer for paddy rice.

Description

Salt and alkali resistant Siamese bacillus and production method and application of viable bacteria preparation thereof

Technical Field

The invention relates to a Siamese bacillus and a production method and application of a viable bacteria preparation thereof, belonging to the technical field of agricultural biology.

Background

Rice is one of the main grain crops in China, has wide planting area, and has other purposes besides being applied to staple food. Therefore, the quality and yield of rice are inseparable from national grain safety. However, from the current practical situation of rice planting, a plurality of factors reduce the yield of rice, wherein bakanae disease is a common factor influencing the yield of rice, the disease incidence rate is high, the threat to stable yield and high yield of rice is large, the yield is generally reduced by 10% -20%, and the yield can be reduced by more than 50% seriously.

The bakanae disease of rice is also called as spindly disease, and can occur from seedling stage to heading stage, and is mainly spread by means of germ-carrying seeds. Fusarium oxysporum (Fusarium fujikuroi) is a main pathogenic bacterium of rice bakanae disease, and Fusarium graminearum (Fusarium graminearum), Fusarium sambucinum (Fusarium sambucinum), Fusarium equiseti (Fusarium equiseti) and the like have weak pathogenic effects in different degrees. Rice bakanae disease often results in rice seeds not germinating or not emerging after sowing. The diseased rice seedlings are usually withered due to water shortage of the plants. Rice plants in the growth period wither and die if damaged by rice bakanae disease, and most of the rice plants are small in spike and few in grain and are not full even though individual plants can sprout and fruit, so that the yield of rice is seriously influenced.

For a long time, the bakanae disease of rice is mainly controlled chemically, and technical measures such as breeding disease-resistant varieties and optimizing cultivation management are combined. The propagation way of the bakanae disease of rice is mainly that seeds carry bacteria for propagation, and medicaments such as bakanae disease, captan, carbendazim and the like are usually adopted for carrying out disinfection treatment on the rice seeds for production. The wide application of chemical pesticides creates great value for human beings, but meanwhile, due to the long-term use of the chemical pesticides, the problems of drug resistance and toxicity of germs, environmental pollution, threat to human and livestock health and the like are increasingly obvious, people also realize the importance of pollution-free ecological agriculture at present, develop biological control technology, reduce the use of the chemical pesticides and are more and more valued by people.

Biocontrol is a method of controlling pests using beneficial organisms or their metabolites. Its advantages are no environmental pollution, high safety to human body and other living things, saving energy and keeping ecological balance, and it is an important means for developing green food and protecting health. At present, research shows that beneficial microorganisms such as trichoderma harzianum, paenibacillus polymyxa, actinomycetes and the like have certain effects on the prevention and treatment of the bakanae disease of rice, but the prevention and treatment effects are limited, so that the method cannot be popularized in large areas in agricultural production.

The saline-alkali soil is an important land resource in China, the total area of the saline-alkali soil and saline-alkali obstacle cultivated land in China exceeds 5 hundred million acres, wherein the total area of the saline-alkali soil and the saline-alkali obstacle cultivated land has the agricultural utilization potential of 2 hundred million acres, and accounts for about 10 percent of the area of the cultivated land in China. The rice has strong saline-alkali resistance and is one of the crops widely cultivated in saline-alkali soil. The biological prevention and control of the bakanae disease of the rice in the saline-alkali soil requires that the biological prevention strain has better disease-resistant growth-promoting effect and stronger saline-alkali tolerance and can be planted in the saline-alkali soil. Therefore, the screening of the saline-alkali tolerant disease-resistant growth-promoting strain has important significance.

Disclosure of Invention

The invention aims to provide a Bacillus siamensis (PAPM-15) strain separated from rhizosphere soil of rice and a strain thereofA preparation method and application of a live bacteria preparation. Siam bacillus PAPM-15 strain has strong saline-alkali resistance and NH production₃And ACC deaminase has strong capability, the viable bacteria preparation can effectively promote the growth of rice, and has higher prevention effect on the rice bakanae disease caused by fusarium graminearum.

A salt and alkali resistant Siamese Bacillus (Bacillus siamensis) PAPM-15 separated from rhizosphere soil of rice, wherein the preservation number of the strain in China general microbiological culture Collection center (CGMCC) is CGMCC No. 20119; the preservation address is microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, and the preservation date is 2020, 6 months and 22 days.

Colony and thallus characteristics of Siamese bacillus PAPM-15 strain: culturing in NA culture medium at 37 deg.C for 48h to obtain colony diameter of about 0.5-0.8mm, white, opaque, round, irregular edge, rough surface, and ridge; the thallus is rod-shaped. The NA culture medium is a nutrient agar culture medium, and the formula of the NA culture medium is as follows: 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar, 1000mL of water and pH7.0.

The physiological and biochemical characteristics of the Siamese bacillus PAPM-15 strain are as follows: a gram-positive bacillus; positive catalase test, positive starch hydrolysis test, positive nitrate reduction test, positive malonic acid utilization test, positive gelatin liquefaction test, negative methyl red test, positive D-glucose fermentation test, positive D-mannitol fermentation test and positive lactose fermentation test.

The preparation method of the Siamese Bacillus PAPM-15 strain viable bacteria preparation is characterized in that after being subjected to expanded culture, Siamese Bacillus (Bacillus simensis) PAPM-15 is inoculated in (the inoculation amount is 1-3%) fermentation culture medium, the Siamese Bacillus (Bacillus simensis) PAPM-15 is cultured for 48-72h at the temperature of 32-37 ℃, then 0.01-0.02% (w/w) of Propyl Gallate (PG), 3-5% (w/w) of polyglutamic acid and 0.05-0.1% (w/w) of carbazone pine are added, and the mixture is uniformly stirred, so that the viable bacteria preparation is obtained.

The method specifically comprises the following steps:

1) activating strains: transferring the low-temperature preserved Siamese bacillus PAPM-15 strain to the test tube inclined plane of an NA culture medium, and culturing at 32-37 ℃ for 24-36h for activation;

2) preparing seeds in a triangular flask: scraping the activated Siamese bacillus PAPM-15 lawn by using an inoculating ring, inoculating the activated Siamese bacillus PAPM-15 lawn in an NB culture medium, and culturing for 24-36h at the temperature of 32-37 ℃;

3) preparing strains in a seeding tank: transferring the seeds in the triangular flask into a seeding tank according to the inoculation amount of 1-3%, and culturing at 32-37 ℃ for 20-24 h;

4) fermentation culture: inoculating the strain in the seeding tank into a fermentation tank according to the inoculation amount of 1-3%; culturing for 48-72h at 32-37 ℃ to obtain a fermentation liquor of Siamese bacillus PAPM-15;

the formula of the fermentation medium is as follows: 15g of soybean meal powder, 10g of starch, 5g of glucose, 1g of yeast extract powder, 1.0g of monopotassium phosphate, 1.0g of magnesium sulfate, 0.5g of zinc sulfate, 0.2g of complex enzyme preparation and 1000mL of water, wherein the initial pH value is 7.5. The complex enzyme preparation comprises the following components: 70% of alkaline protease and 30% of cellulase according to the mass ratio.

5) Adding 0.01-0.02% (w/w) of Propyl Gallate (PG), 3-5% (w/w) of polyglutamic acid and 0.05-0.1% (w/w) of carbazone into the fermentation liquor, and uniformly stirring to obtain the viable bacteria preparation.

The use method of the Siamese bacillus PAPM-15 viable bacteria preparation comprises the following steps: diluting a Siamese bacillus PAPM-15 viable bacteria preparation by 200-300 times with water, dipping roots when soaking seeds or transplanting seedlings or spraying with water when watering.

The invention also provides the salt and alkali resistant Siam bacillus PAPM-15 in NH production₃And the application of producing ACC deaminase and antagonizing Fusarium oxysporum.

The invention also provides the application of the live bacteria preparation in the aspects of promoting the growth of rice, preventing and treating rice bakanae disease caused by fusarium graminearum and the like.

The invention has the beneficial effects that:

the Siamese bacillus PAPM-15 is separated from saline-alkali soil, has stronger saline-alkali resistance and produces NH₃The microbial fertilizer has strong ACC deaminase capacity, promotes the growth of rice, has strong inhibiting effect on pathogenic bacteria of rice bakanae disease, can be used for producing biofertilizer, particularly a microbial fertilizer special for saline-alkali soil rice, can reduce the occurrence of rice bakanae disease, and improves the yield and the quality of rice.

The production method of the Siamese bacillus PAPM-15 viable bacteria preparation is scientific, the viable bacteria number of the fermentation liquor is high, and the production cost is low. The complex enzyme preparation is added into the fermentation medium, and the raw materials such as soybean meal and the like are hydrolyzed in the temperature rise process during sterilization, so that the delay period can be shortened, the utilization rate of the raw materials can be improved, and the concentration of viable bacteria in the fermentation liquor can be improved. The antioxidant propyl gallate and the polyglutamic acid with the protection effect on the viable bacteria are added into the fermentation liquor, so that the loss of the viable bacteria of the microbial inoculum in the storage process can be reduced, and the biological stability of the product is improved.

Drawings

FIG. 1 is a morphology diagram of strains of Bacillus siamensis PAPM-15;

FIG. 2 is a phylogenetic tree constructed based on partial 16S rRNA sequences;

FIG. 3 is a picture of growth of Bacillus siamensis PAPM-15 strain on a culture medium with ACC as a unique nitrogen source, and it can be seen from the picture that: the Siamese bacillus PAPM-15 strain can grow on a culture medium with ACC as a unique nitrogen source, so that the Siamese bacillus PAPM-15 strain has the capability of producing ACC deaminase;

FIG. 4 is a photograph of a culture of the Siamese Bacillus strain PAPM-15 with the addition of Nessler's reagent, from which it can be seen that: a yellow precipitate resulted after addition of Nessler's reagent.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1: screening of Siam bacillus PAPM-15

(1) Screening of saline-alkali tolerant strains

Siamese bacillus PAPM-15 is separated from rhizosphere soil of rice. The soil was sampled in 2018 in 9 months and was in moderate saline-alkali soil in the coastal city region of Binzhou, Shandong province. The specific separation method comprises the following steps: the soil sample was mixed well, 5g was weighed and placed in a triangular flask containing 95mL of sterile water and 10 beads, and shaken at 37 ℃ and 180rpm for 30 min. Taking 1mL of soil suspension for 10^-1-10^-7Serial concentration gradient dilutions were made and then 10 taken^-5、10^-6、10^-7Three dilutions were plated onto plates containing selection medium and cultured in an inverted format at 37 ℃ for 2 d. Selecting single colony and streaking to selective mediumOn the plate, the plate was cultured in an inverted state at 37 ℃ for 2 days. Picking single colony, transferring to the slant of the preservation culture medium test tube, culturing at 37 deg.C for 2d, growing with thallus Porphyrae, and storing in 4 deg.C refrigerator. 182 strains of bacteria were co-selected.

The formula of the selective culture medium is as follows: 10g of peptone, 5g of yeast extract, 15g of NaCl, 20g of agar, 1000mL of distilled water, pH8.0, and sterilizing at 121 ℃ for 20 min.

The formula of the preservation culture medium is as follows: 10g of peptone, 5g of yeast extract, 5g of NaCl, 20g of agar, 1000mL of distilled water, pH7.5, and sterilizing at 121 ℃ for 20 min.

(2) Screening of high-saline-alkali-resistant bacterial strains

And (3) inoculating the strains screened in the step (1) on a high-saline-alkali flat plate, carrying out inverted culture at 37 ℃ for 3-7 d, and observing the growth condition. 72 strains of bacteria were co-selected.

The formula of the high-saline-alkali culture medium is as follows: 10g of peptone, 5g of yeast extract, 100g of NaCl, 20g of agar, 1000mL of distilled water, pH10.0, and sterilizing at 121 ℃ for 20 min.

(3) Production of NH₃Screening of strains

The bacterial strain with high saline-alkali tolerance degree screened in the step (2) is transferred into a test tube containing 10mL peptone water (10g/L) and cultured for 72h at 37 ℃. A test tube containing only 10mL peptone water was used as a control. Addition of 0.5mL Nessler's reagent per tube produced a yellow or brown precipitate indicating NH₃Generated (as shown in fig. 4). Co-screening to yield NH₃20 strains of the strain.

The peptone water formula comprises: 10.0g of peptone and 1000mL of distilled water; sterilizing at 121 deg.C for 20 min; pH 7.0. The preparation method of the Nessler's reagent comprises the following steps: 50.0g of HgI and 40.0g of KI were weighed out and dissolved in 200mL of ammonia-free water, and the solution was poured into 700mL of NaOH solution (210g/L), diluted to 1000mL and left to stand, and the supernatant was used.

(4) Screening of strains producing ACC deaminase

The NH product obtained by screening in the step (3)₃The high saline-alkali tolerant strain is inoculated into a DF salt culture medium plate which takes ACC as a unique nitrogen source, and the strain which can normally grow has the capability of producing ACC deaminase. 12 strains of ACC deaminase producing strains are co-screened.

The DF salt culture medium comprises the following components in percentage by weight: KH (Perkin Elmer)₂PO₄4.0g；NaH₂PO₄6.0g；MgSO₄·7H₂O 0.2g；FeSO₄·7H₂O0, lg; 2.0g of glucose; 2.0g of sodium gluconate; 2.0g of citric acid; trace element 0.1mL, pH 7.5. (trace elements 100 mL: MoO)₃l 0.0mg；H₃BO₃10.0mg；MnSO₄·H₂O 11.19mg；CuSO₄·5H₂O 78.22mg；ZnSO₄·7H₂O124.6 mg). The prepared ACC solution was sterilized by suction filtration through a 0.2 μm filter and then added to DF medium containing no ammonium sulfate at a final concentration of 3mmo 1/L.

(5) Antagonistic bacteria for screening pathogenic bacteria of bakanae disease of rice by counter culture method

Inoculating Fusarium oxysporum blocks with the diameter of 5mm to the center of a PDA culture medium plate, inoculating the screened strains at equal distances of 2.5cm from the center of the plate, repeating the treatment for 3 times by taking the plate without inoculating the screened strains as a control, culturing for 10 days at 28 ℃, selecting the strains with larger antibacterial circle diameter, and storing for later use.

The PDA culture medium formula comprises: 200g of potato, 20g of glucose, 20g of agar and 1000mL of distilled water, and the pH value is natural.

Through the multiple screening, the growth speed, the saline-alkali tolerance and the NH yield of the strain are comprehensively considered₃The obtained strain has strong saline-alkali resistance and NH production capacity₃And the antagonistic bacteria of pathogenic bacteria of the rice bakanae disease with the capability of producing ACC deaminase are numbered as PAPM-15.

Example 2 identification of Bacillus siameses PAPM-15

(1) Morphological and physiological biochemical characteristics

The Siamese bacillus PAPM-15 strain has the morphological characteristics that: culturing in NA culture medium at 37 deg.C for 48h to obtain colony diameter of about 0.5-0.8mm, white, opaque, round, irregular edge, rough surface, and ridge; the bacterial cells are in the form of rods, as shown in FIG. 1. The NA culture medium is a nutrient agar culture medium, and the formula of the NA culture medium is as follows: 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar, 1000mL of water and pH 7.0.

(2)16S rDNA sequence analysis

The PAPM-15 strain is inoculated into an LB culture medium, shake culture is carried out for 24h at 37 ℃ and 180r/min, thalli are collected, total DNA is extracted, and then PCR amplification of the 16S rRNA gene is carried out under the guidance of universal primers F27: 5'-AGA GTT TGA TCATGG CTC AG-3' and F27: 5'-AGA GTT TGA TCA TGG CTC AG-3' of the prokaryotic 16S rRNA gene by taking the strain as a template. After the amplification product is separated by 1% agarose gel electrophoresis, the amplification product is recovered by a gel recovery kit and handed over to Shanghai Biotechnology Limited company for sequencing. The 16S rDNA sequence (SEQ No.1) was aligned with the sequences in the GenBank database, and multiple sequence homology analysis was performed using MEGA 7.0 software, and a phylogenetic tree was constructed, as shown in FIG. 2.

Through morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, the strain is Siamese Bacillus and named as Siamese Bacillus (Bacillus simensis) PAPM-15. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 22.6.2020, with the preservation number of CGMCC No. 20119.

Example 3 preparation of Bacillus siamensis PAPM-15 microbial inoculum

The preparation method of the Siamese bacillus PAPM-15 strain microbial inoculum comprises the following steps:

1) activating strains: transferring the strains of Bacillus siamensis PAPM-15 preserved at low temperature to the test tube inclined plane of the NA culture medium, and culturing at 32-37 ℃ for 24-36h for activation. The NA culture medium is a nutrient agar culture medium, and the formula of the NA culture medium is as follows: 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar, 1000mL of water and pH7.0.

2) Preparing seeds in a triangular flask: scraping the activated Siamese bacillus PAPM-15 lawn by using an inoculating ring, inoculating the activated Siamese bacillus PAPM-15 lawn in an NB culture medium, and culturing for 24-36h at the temperature of 32-37 ℃. The NB culture medium is a nutrient broth culture medium, and the formula of the NB culture medium is as follows: 3g of beef extract, 10g of peptone, 5g of NaCl, 1000mL of water and pH7.0.

3) Preparing strains in a seeding tank: inoculating 2% of the seed in a triangular flask into a 10L seeding tank containing 6L NB culture medium, culturing at 32-37 deg.C for 20-24h, stirring at 200rpm, ventilating at 0-6 h/min and 6L/min for 6-20 h.

4) Inoculating seed tank strains into a 500L fermentation tank by transferring inoculum size of 2% in volume ratio, filling 300L fermentation medium in the fermentation tank, culturing for 48-72h at 32-37 ℃ to obtain Siamese bacillus PAPM-15 fermentation liquor, stirring at the full-process rotating speed of 180rpm, ventilation of 0-6h of 200L/min and ventilation of 6-28h of 300L/min, and after fermentation is finished, the viable count of Siamese bacillus PAPM-15 in the fermentation liquor is (1-2) × 10¹⁰cfu/ml。

The formula of the fermentation medium in the step 4) is as follows: 15g of soybean meal powder, 10g of starch, 5g of glucose, 1g of yeast extract powder, 1.0g of monopotassium phosphate, 1.0g of magnesium sulfate, 0.5g of zinc sulfate, 0.2g of complex enzyme preparation and 1000mL of water, wherein the initial pH value is 7.5. The compound enzyme preparation comprises 70% of alkaline protease and 30% of cellulase. The alkaline protease and the cellulase are both produced by Taian Xindeli biological technology limited company, and the product specification is 10 ten thousand U/g. The unit definition and detection method of the activity of the alkaline protease execute the national standard GB/T23527-.

The preparation method of the fermentation medium in the step 4) comprises the following steps: weighing raw materials according to the formula, dissolving in water, heating to 50-55 deg.C, maintaining the temperature for 1-2h, heating to 121 deg.C, maintaining the temperature for 20-30min, and sterilizing.

5) Preparation of the viable bacteria preparation: adding 0.01% (w/w) of Propyl Gallate (PG), 3% (w/w) of agricultural polyglutamic acid powder and 0.05% of carbazone (w/w) into the Siamese bacillus PAPM-15 fermentation liquor obtained in the step 4), and uniformly stirring to obtain a viable bacteria preparation, wherein the viable bacteria content of the viable bacteria preparation isPreferably (1.0-2.0) × 10¹⁰cfu/g. The agricultural polyglutamic acid powder is produced by Nanjing Xuan Kai biological technology Limited, and the content of polyglutamic acid is 25%.

Example 4 prevention and treatment Effect of Bacillus siamensis PAPM-15 microbial inoculum on Rice bakanae disease

Soil for potting experiments is taken from the coastal urban area of Binzhou city in Shandong province, the soil type is moderate saline-alkali soil, potting experiments are carried out according to the table 1, and a rice bakanae bacterium agent and a PAPM-15 bacterium agent are sprayed after thinning rice seedlings. Spraying a rice bakanae disease bacterium agent, and spraying a Siamese bacillus PAPM-15 bacterium agent after 2 days. Thinning is carried out when the height of the rice seedlings reaches 5cm, 5 seedlings are reserved in each pot (200g of soil), 10 pots are treated in each pot, and the process is repeated for 3 times. The management is carried out in a conventional mode, the number of the disease strains and the relative lesion height of 20d, 40d and 60d are observed and recorded, and the disease index and the relative prevention effect are calculated.

TABLE 1 design of the potting experiment

The preparation method of the agent comprises inoculating pathogenic bacteria of rice bakanae disease (Fusarium oxysporum) into PDA plate, culturing at 28 deg.C for 8-10 days, and making spore suspension with spore concentration of 1 × 10⁶cfu/mL. The PDA culture medium formula comprises: 200g of potato, 20g of glucose, 20g of agar and 1000mL of distilled water, and the pH value is natural.

The preparation method of the PAPM-15 microbial inoculum comprises the steps of diluting the Siamese bacillus PAPM-15 microbial inoculum prepared in the embodiment 3, and adjusting the concentration of the Siamese bacillus PAPM-15 microbial inoculum to 1 × 10⁸cfu/mL, i.e., PAPM-15 microbial inoculum.

The disease grading standard takes the whole plant as an index, and the grading is as follows: level 0: no symptoms; level 1: the base of the stem is slightly browned, and the rice seedling grows basically normally; and 3, level: the base of the stem turns markedly brown with softening and slight decay; and 5, stage: the basal part of the stem is obviously browned and rotten, and the heart leaves are withered and curled; and 7, stage: the base of the stem turns brown and decays, and the whole plant is withered or yellow brown and withered; and 9, stage: the whole plant is rotten.

Disease index ═ Σ (number of diseased rice × number of disease grades)/(total number of rice plants × number of highest disease grades) × 100%

Relative control effect (%) - (CK2 disease index-PAPM-15 treatment disease index)/CK 2 disease index x 100%

The test results are shown in table 2, during the experimental investigation, the incidence index of CK1 is 0, and the incidence index of CK2 is as high as 50.23-68.91%, which shows that the pathogenic bacteria of rice bakanae disease adopted in the experiment have strong pathogenicity to the tested rice. Compared with CK2, the morbidity index of the inoculated PAPM-15 microbial inoculum is only 14.01-27.55%, and the average relative prevention effect is up to 67.00%, which shows that the Siamese bacillus strain PAPM-15 has better prevention and treatment effect on the rice bakanae disease.

TABLE 2 prevention and treatment of Rice bakanae disease by Siamese Bacillus strain PAPM-15 bacterium preparation

Note: the data in the table are the average of 3 replicates. "-" indicates none. Different lower case letters indicate significant differences (P <0.05, Duncan's new double pole difference method).

Example 5 growth promoting action of Bacillus siamensis PAPM-15 microbial inoculum on rice

Soil for pot culture experiments is taken from the coastal urban area of Binzhou in Shandong province, and the soil type is moderate saline-alkali soil. The rice seeds are selected by a saline water floating method, washed for 3 times by clear water after selection, the salinity of the rice shells is removed, and the rice seeds are put into a beaker to be soaked in sterile water for germination acceleration at the temperature of 30 ℃.

Respectively taking 50.0mL of 100-fold diluent of the Siamese bacillus strain PAPM-15 viable bacteria preparation prepared in the embodiment 3, adding the Siamese bacillus strain PAPM-15 viable bacteria preparation into a cylindrical nutrition pot (the diameter is 10cm, and the height is 10cm) filled with 200.0g of potted seedling raising soil, selecting seeds with consistent growth vigor and respectively sowing the seeds into the nutrition pot when the germ of the rice seeds is about 0.5cm long, and sowing 5 seeds in each pot. The control group was set as a nutrition pot to which 50.0mL of a 100-fold diluted solution of sterilized viable preparations of the Siamese Bacillus strain PAPM-15 was added. Each treatment was 10 pots, repeated 3 times. Pouring sterile water into the nutrition pot during the test period, putting the nutrition pot with the planted rice into an intelligent artificial climate incubator, wherein the illumination time is 12h, and the illumination grade is set to be 3 grade; setting the light level to be 0 in the dark time of 12 h; the temperature and the humidity were both 30 ℃. And after 20 days of seedling culture, measuring related physiological indexes of the rice.

The test results are shown in table 3, the height of rice is increased by 41.38%, the stem thickness is increased by 83.21%, the ground dry mass is increased by 79.17%, and the ground fresh mass is increased by 80.00% after 20 days by adding the Siamese bacillus strain PAPM-15 viable bacteria preparation. Therefore, compared with the soil added with the Siamese bacillus strain PAPM-15 viable bacteria preparation, all indexes of the rice are improved, and the PAPM-15 strain has the growth promoting effect on the growth of the rice.

TABLE 3 growth promoting effect of Siamese bacillus strain PAPM-15 bacterium preparation on rice

Note: the data in the table are the average of 3 replicates. Different lower case letters indicate significant differences (P <0.05, Duncan's new double pole difference method).

SEQUENCE LISTING

<110> Shandong Zongtian Biotech Co., Ltd

<120> saline-alkali-tolerant Siamese bacillus and production method and application of living bacterium preparation thereof

<130>0

<160>1

<170>PatentIn version 3.3

<210>1

<211>1442

<212>DNA

<213> Bacillus siamensis (PAPM-15)

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cgtgctaata catgcaagtc gagcggacag atgggagctt gctccctgat gttagcggcg 60

gacgggtgag taacacgtgg gtaacctgcc tgtaagactg ggataactcc gggaaaccgg 120

ggctaatacc ggatggttgt ttgaaccgca tggttcagac ataaaaggtg gcttcggcta 180

ccacttacag atggacccgc ggcgcattag ctagttggtg aggtaacggc tcaccaaggc 240

gacgatgcgt agccgacctg agagggtgat cggccacact gggactgaga cacggcccag 300

actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct gacggagcaa 360

cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct gttgttaggg aagaacaagt 420

gccgttcaaa tagggcggca ccttgacggt acctaaccag aaagccacgg ctaactacgt 480

gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggaattattg ggcgtaaagg 540

gctcgcaggc ggtttcttaa gtctgatgtg aaagcccccg gctcaaccgg ggagggtcat 600

tggaaactgg ggaacttgag tgcagaagag gagagtggaa ttccacgtgt agcggtgaaa 660

tgcgtagaga tgtggaggaa caccagtggc gaaggcgact ctctggtctg taactgacgc 720

tgaggagcga aagcgtgggg agcgaacagg attagatacc ctggtagtcc acgccgtaaa 780

cgatgagtgc taagtgttag ggggtttccg ccccttagtg ctgcagctaa cgcattaagc 840

actccgcctg gggagtacgg tcgcaagact gaaactcaaa ggaattgacg ggggcccgca 900

caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac 960

atcctctgac aatcctagag ataggacgtc cccttcgggg gcagagtgac aggtggtgca 1020

tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080

tgatcttagt tgccagcatt cagttgggca ctctaaggtg actgccggtg acaaaccgga 1140

ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta 1200

caatggacag aacaaagggc agcgaaaccg cgaggttaag ccaatcccac aaatctgttc 1260

tcagttcgga tcgcagtctg caactcgact gcgtgaagct ggaatcgcta gtaatcgcgg 1320

atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga 1380

gagtttgtaa cacccgaagt cggtgaggta accttttagg agccagccgc cgaaggtgga 1440

ca 1442

Claims

1. A salt and alkali resistant Siamese Bacillus strain is Siamese Bacillus (Bacillus siamensis) PAPM-15, and the preservation number is CGMCC No. 20119.

2. The salt and alkali tolerant Siamese bacillus strain of claim 1 in producing NH₃And the application of producing ACC deaminase and antagonizing Fusarium oxysporum.

3. A viable bacterial preparation containing the Siamese bacillus strain PAPM-15 resistant to salt and alkali as the main effective component.

4. The method for preparing the living bacterial preparation of claim 3, wherein the Siamese bacillus strain PAPM-15 is inoculated in a fermentation medium after the enlarged culture, and cultured for 48 to 72 hours at the temperature of 32 to 37 ℃ to obtain a fermentation liquid, and then the living bacterial preparation is further prepared.

5. The method of preparing the viable bacteria preparation according to claim 4, wherein the fermentation medium is: 15g of soybean meal powder, 10g of starch, 5g of glucose, 1g of yeast extract powder, 1.0g of monopotassium phosphate, 1.0g of magnesium sulfate, 0.5g of zinc sulfate, 0.2g of complex enzyme preparation and 1000mL of water, wherein the initial pH value is 7.5; the complex enzyme preparation comprises the following components: 70% of alkaline protease and 30% of cellulase according to the mass ratio.

6. The preparation method of the viable bacteria preparation according to claim 5, wherein 0.01-0.02% of propyl gallate, 3-5% of polyglutamic acid and 0.05-0.1% of carbazone are added into the fermentation liquor according to the mass ratio, and the mixture is uniformly stirred to obtain the viable bacteria preparation.

7. The use of the live bacterial preparation according to claim 3 for preventing and treating rice bakanae disease caused by Fusarium oxysporum and promoting rice growth.