CN111019856A

CN111019856A - Cockerella rosea SDB9 and preparation method and application thereof

Info

Publication number: CN111019856A
Application number: CN201911272326.9A
Authority: CN
Inventors: 王建武; 王永利; 相微微; 尚爱军; 严加坤; 陈花; 张长现; 艾银婷; 李刚; 冯光惠
Original assignee: Yulin Zhongtai Agricultural Technology Co ltd; Yulin University
Current assignee: Yulin Zhongtai Agricultural Technology Co ltd
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2020-04-17
Anticipated expiration: 2039-12-11
Also published as: CN111019856B

Abstract

The invention relates to Cockerella rosea SDB9, a preparation method thereof and application thereof in promoting plant growth, belonging to the technical field of microorganism application. The Cockerella rosea SDB9 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number: CGMCC No.17922, preservation date: 12/6/2019, taxonomic name: kocuria rosea strain SDB 9. The invention successfully separates and obtains the saline-alkali-resistant root endophytic bacteria with the plant growth promoting effect from the root of natural sabina vulgaris with very strong stress resistance in northern Shaanxi: cockerella rosea SDB 9. The Cockerella rosea SDB9 has high saline-alkali resistance, can remarkably promote plant growth, develops plant root systems, obviously increases new lateral roots, and has a potassium-dissolving effect, thereby laying a foundation for improving saline-alkali soil, promoting plant growth and improving the soil quick-acting potassium technology by a microbiological method.

Description

Cockerella rosea SDB9 and preparation method and application thereof

Technical Field

The invention relates to the technical field of microorganism application, in particular to Cockerella rosea SDB9, a preparation method thereof and application thereof in promoting plant growth.

Background

Plant Growth-Promoting Rhizobacteria (PGPR) is a general term for useful Rhizobacteria that live in the rhizosphere micro-area of plants and antagonize pathogenic bacteria or promote Plant Growth. The growth-promoting rhizobacteria can promote the absorption of plants to nutrients and improve the resistance of a host system by secreting hormones such as indoleacetic acid and the like so as to improve the growth condition of the plants. China is a big country with saline-alkali soil, and the third is the top 10 countries with saline-alkali soil. The total area of saline-alkali wasteland and saline-alkali land affecting cultivated land exceeds 5 hundred million acres, wherein the saline-alkali land with agricultural development potential accounts for more than 10 percent of the total area of the cultivated land in China. The large area of saline-alkali soil and salinized soil makes it difficult for some crop varieties to exert yield potential due to the influence of different degrees of salt damage. Due to natural conditions and human activities, over 6% of the land area and about 20% of cultivated land in the world are threatened by salt damage and increase year by year. Soluble salts in soil are accumulated continuously, and when the salt content reaches a certain amount, the soil is salinized. The method is an environment-friendly and sustainable method, and can improve the variety and the quantity of plant rhizosphere microorganisms by utilizing rhizosphere growth promoting bacteria, improve the property of soil and promote the growth of plants, thereby improving the utilization efficiency of saline-alkali soil.

According to the current fertilization situation of China, potassium is generally in a loss state in the balance of three elements of a farmland. The deficiency of soil potassium already constitutes the limiting factor of the improvement of the yield and the quality of crops in China. But the potassium in the soil is in a mineral potassium form, mainly exists in potassium feldspar and mica, the content of quick-acting potassium which can be absorbed and utilized by crops is less than 2% of total potassium, and the mineral potassium cannot be directly absorbed and utilized by crops. The data show that the production of the potash fertilizer in China only accounts for 0.34 percent of the world yield, the consumption accounts for 14.7 percent, and although about 200 ten thousand tons of potash fertilizer are imported every year, the demand of agricultural production on the potash fertilizer cannot be met. Under the condition that the supply of chemical potash fertilizer is not in short supply, a new microbial fertilizer with good effect, low cost and no pollution is researched and developed, potential potassium resources in soil are excavated, and the method has important research and application significance.

At present, experts pay more and more attention to the research of PGPR on improving the saline-alkali tolerance of plants, but the research result of the PGPR on the influence of the plant rhizosphere microenvironment under the saline-alkali stress is less. Although researchers in recent years have isolated a large number of rhizosphere growth-promoting bacteria which have one or more of the characteristics of hormone secretion, nitrogen fixation, phosphorus dissolution, potassium dissolution, host system resistance improvement and the like, the bacteria have poor stress resistance and have growth-promoting functions under proper cultivation conditions, and under poor growth environments, the bacteria may survive but lose growth-promoting characteristics such as no hormone secretion and the like. Therefore, it is highly desirable to screen out PGPR bacteria having excellent stress resistance and growth promoting effect.

Disclosure of Invention

The invention aims to solve the technical problem of providing a bacterium which has high saline-alkali resistance and can obviously promote the growth of plants, wherein the Cockera roseum SDB9 can tolerate the stress of 1.5mol/L NaCl, is a facultative alkali-resistant bacterium, can grow on a culture medium with the pH of 7-12, has the potassium-dissolving function, and solves the problems that the stress resistance of microbial strains is insufficient and the improvement of the plant growth and development and the soil quick-acting potassium cannot be considered in the prior art.

In order to solve the technical problems, the invention aims to realize the following technical scheme:

the Cockerella rosea SDB9 is preserved in China general microbiological culture Collection center (CGMCC) with the address as follows: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation number is: CGMCC No.17922, preservation date: 12/6/2019, taxonomic name: kocuria rosea strain SDB 9.

The sequence of the 16S rDNA of the Cockera rosea SDB9 is shown as SEQ ID NO: 1 is shown.

The genome of the Cockera roseoflavus SDB9 has obvious variation, and the sequence of an aldehyde dehydrogenase gene ALDH4 which can be used as a bar code sequence specific to the Cockera roseoflavus SDB9 is shown as SEQ ID NO: 2, respectively.

The preparation method of the cocklebur SDB9 comprises the following steps:

(1) thoroughly disinfecting the root surface of sabina vulgaris;

(2) culturing, separating and purifying sapphium arietinum root endophytic bacteria;

(3) culturing, separating and identifying saline-alkali tolerant bacteria;

(4) observing the growth characteristics and performing physiological and biochemical identification on the Alcaligenes halodurans SDB 9;

(5) 16S rDNA sequencing and genome sequencing of the Alcaligenes halodurans SDB9 isolate;

(6) identification of Cockera roseoflavus SDB 9.

The Cocker roseochracea SDB9 is spherical and gram-positive, the colony is rose after being cultured for a long time, the result of an environmental scanning electron microscope shows that the strain has a thick capsule layer, and the diameter range of the strain can be seen to be 784.8nm-890.6nm from a high-resolution scanning microscopic picture.

The Cockerella rosea SDB9 can grow normally on NaCl culture medium with concentration of 0-1.5mol/L and culture medium with pH value of 7-12. The saline-alkali composite stress is similar to the single stress result, which indicates that the Cockerella rosea SDB9 has strong saline-alkali tolerance.

Similarly, the research finds that the Cockera roseum SDB9 has a remarkable promoting effect on plant growth, and particularly still has the capability of remarkably promoting plant growth and improving oxidation resistance under the saline-alkali stress.

The bacterial liquid of the Cockerella rosea SDB9 has the effects of dissolving potassium and promoting absorption of nitrogen and phosphorus of plants.

The application of the Cockerella rosea SDB9 in promoting plant growth comprises the following specific steps:

inoculating the Cocker roseum SDB9 into a culture medium, culturing for 20-28h at 25-30 ℃ to obtain a culture solution, centrifuging the culture solution to obtain Cocker roseum SDB9 thalli, re-suspending the Cocker roseum SDB9 thalli with water to obtain a bacterial solution of the Cocker roseum SDB9, and diluting the bacterial solution of the Cocker roseum SDB9 to be used for seed soaking, root pouring and inoculation of plants. After the plants are planted in the field, the Cockerella rosea SDB9 begins to propagate by itself along with the growth of root systems, and the effect of obviously promoting the growth of the plants is shown when the plants are planted for about 80 days, which is specifically embodied in that the main root systems are developed, and the new lateral roots are obviously increased.

Wherein the concentration of the bacterial liquid is in a relatively wide range of 1 × 10⁶-1×10¹⁰CFU/mL, too high concentration needs to be diluted and used. Gradient tests show that the effect of promoting plant growth can be achieved only by inoculating the bacterial liquid of the Coccocus roseus SDB9 to the roots of plants, but seed soaking at ultrahigh concentration can inhibit seed germination.

Preferably, the use concentration of the bacterial liquid of the Cockera rosea SDB9 is 1 × 10⁷CFU/mL。

The invention has the beneficial effects that:

the invention successfully separates and obtains saline-alkali-resistant root endophytic bacteria with plant growth promoting effect from natural sabina vulgaris roots with very strong stress resistance in northern Shaanxi, the bacteria is identified as Cockerella rosea SDB9, and is preserved in China general microbiological culture Collection center (CGMCC) in 2019 and 6 and 12 months, and the addresses are as follows: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation number is: CGMCC No.17922, taxonomic name: kocuriarosea strain SDB 9. The Cockerella rosea SDB9 has high saline-alkali tolerance, can remarkably promote plant growth, develops plant root systems, obviously increases new lateral roots, and has a potassium-dissolving effect, thereby laying a foundation for improving saline-alkali soil, promoting plant growth and improving the soil quick-acting potassium technology by a microbiological method.

Drawings

FIG. 1A is a salt tolerance analysis of Cockera roseoflavus SDB 9;

FIG. 1B is an alkali resistance analysis of Cockera roseoflavus SDB 9;

FIG. 2 shows the results of scanning electron microscopy of Cockera roseum SDB9 in an environment;

FIG. 3 shows the high resolution electron microscopy results of Cockera roseoflavus SDB 9;

FIG. 4A is a comparison of the results of the length measurements of the first nodes of mung bean plants;

FIG. 4B is a comparison of green gram plant leaf length measurements;

FIG. 4C is a comparison of green gram plant leaf width measurements;

FIG. 4D is a comparison of root weight measurements from mung bean plants;

FIG. 4E is a comparison of the results of the overground weight measurements of mung bean plants;

FIG. 5A is a comparison of the measured results of the root activity of mung beans;

FIG. 5B is a comparison of the results of the chlorophyll content measurement of green beans;

FIG. 5C is a comparison of the results of mung bean SOD activity measurements;

FIG. 5D is a comparison of the results of mung bean POD activity assays;

FIG. 5E is a comparison of CAT activity assay results for mung beans;

FIG. 5F is a comparison of the results of mung bean MDA assay;

FIG. 5G is a comparison of the results of mung bean proline assay;

FIG. 6A is a comparison of the results of corn plant height measurements;

FIG. 6B is a comparison of fresh weight corn measurements;

FIG. 6C is a comparison of the results of the chlorophyll content measurements of corn;

FIG. 6D is a comparison of the results of a corn POD activity assay;

FIG. 6E is a comparison of the results of the corn CAT activity assay;

FIG. 6F is a comparison of the results of corn SOD activity assays;

FIG. 6G is a comparison of results of corn MDA content determinations;

FIG. 6H is a comparison of results of corn root activity measurements;

FIG. 6I is a comparison of the results of the corn proline content assay;

FIG. 7A is a comparison of the ammoniacal nitrogen content of corn rhizosphere soil;

FIG. 7B is a comparison of the nitrate nitrogen content of corn rhizosphere soil;

FIG. 7C is a comparison of the available phosphorus content of corn rhizosphere soil;

FIG. 7D is a comparison of the rapid-acting potassium content in corn rhizosphere soil;

FIG. 8A is a comparison of nitrogen content in maize plants;

FIG. 8B is a comparison of phosphorus content in maize plants;

FIG. 8C is a comparison of potassium content in maize plants;

FIG. 9A is a comparison of rice stem bases;

FIG. 9B is a comparison of the base of the rice panicle;

FIG. 9C is a comparison of rice panicle lengths;

FIG. 9D shows a comparison of under shoot of rice ears;

FIG. 9E shows a comparison of rice plant height;

FIG. 10A is a comparison of root lengths of rice roots;

FIG. 10B is a comparison of root surface areas of rice roots;

FIG. 10C is a comparison of root volumes of rice roots;

FIG. 11A is a comparison of rice spikelets;

FIG. 11B is a comparison of rice grain numbers;

FIG. 11C is a comparison of rice blight rate;

FIG. 11D is a comparison of fresh weight of overground part of rice;

FIG. 11E is a comparison of fresh weights of rice.

Detailed Description

The present invention will be described in further detail below: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation is given, but the scope of the present invention is not limited to the following embodiments.

The Cockerella rosea SDB9 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number: CGMCC No.17922, preservation date: 12/6/2019, taxonomic name: kocuria roseastrainen SDB 9.

The sequence of the aldehyde dehydrogenase aldehydodehydrogenasee gene ALDH4 which can be used as a unique barcode sequence of the Cockera roseoflavus SDB9 is shown as SEQ ID NO: 2, respectively.

The preparation method of the cocklebur SDB9 comprises the following steps:

(1) thoroughly disinfecting the root surface of sabina vulgaris;

(3) culturing, separating and identifying saline-alkali tolerant bacteria;

(6) identification of Cockera roseoflavus SDB 9.

inoculating the Cocker roseum SDB9 into a culture medium, culturing for 24h at 28 ℃ to obtain a culture solution, centrifuging the culture solution to obtain Cocker roseum SDB9 thalli, re-suspending the Cocker roseum SDB9 thalli with water to obtain a bacterial solution of the Cocker roseum SDB9, and using the bacterial solution of the Cocker roseum SDB9 for seed soaking, root irrigation and inoculation of plants. After the plants are planted in the field, the Cockerella rosea SDB9 begins to propagate by itself along with the growth of root systems, and the effect of obviously promoting the growth of the plants is shown when the plants are planted for about 80 days, which is specifically embodied in that the main root systems are developed, and the new lateral roots are obviously increased.

Wherein the culture medium is an LB culture medium and comprises the following components: 10g tryptone, 5g yeast extract, 10g NaCl and 1000mL distilled water, pH 8.0.

The use concentration of the bacterial liquid of the Cockerella rosea SDB9 is 1 multiplied by 10⁷CFU/mL。

Example 1 isolation and identification of saline-alkali tolerant bacteria

(1) Thorough disinfection of the root surface of sabina vulgaris

Washing the soil on the root surface of herba Selaginellae Doederleinii with tap water, placing into a super clean bench, ultraviolet sterilizing for 20min, and washing with sterile water for 3-4 times. Soaking in 75% ethanol for 10min, sequentially washing with sterile water for 3 times, soaking in 3% sodium hypochlorite for 5min, and washing with sterile water for 4-5 times. The sterile water of the last washing is reserved for disinfection and inspection. Uniformly coating the sterilized water for the last time on an LB culture medium flat plate, placing the flat plate in an environment with the temperature of 28 ℃ for culturing for 3-5 days, and checking whether colonies are formed. And (4) disinfection inspection results: if the germ-free colony is generated, the disinfection is complete.

(2) Cultivation, separation and purification of sapphium arietinum root endophytic bacteria

The operation is carried out in a clean bench, ultraviolet lamp sterilization is carried out for 20min, the root of sabina vulgaris is cut into small pieces by a sterilized scalpel, and the small pieces are placed in a mortar for grinding. Grinding into slurry, standing for 30min, and collecting supernatant with pipette. The supernatant was diluted in three different concentrations, 10-fold, 100-fold, and 1000-fold in this order, and the three concentrations of tissue fluid were uniformly applied by the application method to LB (10g tryptone, 5g yeast extract, 10g NaCl, 15g/L agar, and 1000mL distilled water, pH 7.0) and TSA (5g/L soybean peptone, 15g/L tryptone, 5g/L NaCl, 15g/L agar, and 1000mL distilled water, pH 7.0) medium plates. And (3) inversely placing the culture medium in an incubator at 28 ℃, waiting for bacterial colonies to grow on a culture medium plate, and separating and purifying the bacterial strains by a plate streaking method according to the shape and the color of the bacterial strains. This is repeated until a single colony can be isolated and purified. Then placing the mixture into an incubator with the set temperature of 28 ℃ for continuous culture.

(3) Culture, separation and identification of saline-alkali tolerant bacteria

Adopting a drop plate experiment to continuously dilute all strains separated in the step (2) into 1, 10 and 10 respectively²Three bacterial liquids with different multiples are dripped onto culture media with different pH values and different NaCl concentrations, the growth conditions of the three bacterial liquids are observed, and finally, a bacterial strain SDB9 capable of growing on a NaCl culture medium with the concentration of 1.5mol/L and a solid LB culture medium with the pH value of 12 is identified and obtained, as shown in figure 1.

(4) Growth characteristic observation and physiological and biochemical identification of Alcaligenes halodurans SDB9

As shown in FIGS. 2 and 3, the SDB9 colony is a rose gram-positive coccoid, and contains a thick film, the diameter of the coccoid is 784.8nm-890.6nm, the colony is small, and the colony is round and convex, and is neat, moist and opaque. The optimal growth medium of the SDB9 colony is LB medium (10g tryptone, 5g yeast extract powder, 10g NaCl, 1000mL distilled water, pH 8.0), the optimal growth temperature is 28 ℃, and the results of the liquid phase mass spectrometry determination of the hormone synthesis capacity show that: the IAA (auxin) content in the SDB9 strain is 7748.1ng/g, the cytokinins are mainly isopentenyl adenosine (iPR) and isopentenyl adenine (iP), the contents of the IAA and the isopentenyl adenine are respectively 28.9ng/g and 1.41ng/g, and the trans-zeatin cytokinins are very low.

By usingThe Salkowski colorimetric method was used to measure the secretion of plant growth hormone (IAA) by the SDB9 strain. 100. mu.L of a suspension (1X 10) of the test strain was added⁷one/mL) was inoculated into 5mL of L-tryptophan growth medium and cultured in the dark at 28 ℃ for 2 d; centrifuging 1mL of bacterial liquid at 10000r/min for 5min, and taking supernatant as a sample to be detected. IAA stock solutions at 1g/L were diluted to 0, 20, 40, 60, 80 and 100. mu.g/mL IAA series of standards. 150 μ L of the standard sample and the sample to be tested were added to a Costar 96 well plate, and then 100 μ L of Salkowski's reagent (0.5mmol/L FeCl) was immediately added to all the wells₃，35％HClO₄) And developing the color at 30 ℃ for 30min, observing the result of the color development reaction, measuring an absorbance OD530 by using a SpectraMax spectrophotometer, and obtaining the secretion of the IAA of the SDB9 strain to be 722 mu g/mL according to a standard curve.

50mL of the culture medium was placed in a 250mL Erlenmeyer flask, sterilized at 121 ℃ for 30min, and cooled to room temperature. The SDB9 treatment group is inoculated according to the inoculum size of 4 percent of volume fraction, the same amount of inactivated bacterial liquid is added into the control group, and the culture is carried out for 72 hours in a full-temperature shaking incubator at 28 ℃ and 200 r/min. Transferring all the culture solution into an evaporating dish, concentrating in water bath to about 10mL, adding 6% H₂O₂5mL, stirring. Repeating the steps until the mucus is completely ashed, removing the excessive hydrogen peroxide, adding water, filtering the solution into a 50mL volumetric flask, and measuring the content of the water-soluble potassium by using a flame spectrophotometer method, wherein the result shows that the SDB9 treatment group can increase the effective potassium by 47.2%.

(5) 16S rDNA sequencing and genome sequencing of Alcaligenes halodurans SDB9 isolate

And (3) streaking the strains of the target bacteria in an LB culture medium, and culturing for 24h in a constant-temperature incubator at 28 ℃ to obtain single colonies.

Bacterial 16S rDNA universal primers were used:

27F (5 '-AGAGTTTGATCMTGGCTCAG-3') and

1942R (5 '-TACGGHTACCTTGTTACGACTT-3') amplifies the 16S rDNA gene sequence.

The reaction system is as follows: 10 XPCR buffer 5. mu. L, MgCl₂(25mmol/L) 2.0. mu. L, dNTP (4mmol/L), Taq DNA polymerase 1. mu.L, primers 1. mu.L each, template 2. mu.L, and deionized water to 50. mu.L. The reaction conditions are as follows: 5min at 95 ℃; 30s at 94 ℃; 530s at 0 ℃; 1min at 72 ℃; 33 cycles, and finally 5min of incubation at 72 ℃. After the reaction is finished, 2 mu L of PCR product is taken to carry out electrophoresis on 1% agar gel, the electrophoresis result of the amplified product is observed by a gel imaging system, and the 16S rDNA PCR amplified product is sequenced by the company of Biotechnology engineering (Shanghai) GmbH. BLAST sequence alignment was performed according to the sequencing results registered in ncbi (national Center for biotechnology) database, and the results showed that the sequence of 16SrDNA of alcaligenes halodurans SDB9 has the highest homology with cockleboxera rosea (Kocuria rosea), and therefore alcaligenes halodurans SDB9 was identified as cockleboxera rosea (Kocuria rosea).

The genome sequencing of the Alcaligenes halodurans SDB9 isolate is completed by Shanghai Meiji biological medicine science and technology limited company, and the deep excavation and analysis of the genome sequencing result shows that: 4 aldehyde dehydrogenase aldehydydehydrogenase genes exist on the genome of Alcaligenes halodurans SDB9, the genes belong to an IPyA/TAM/TSO channel synthesized by auxin (IAA), wherein the ALDH4 gene is special, the highest homology with the sequence in an NCBI database is Arthrobacter sp.U41 of Arthrobacter, the homology is 80.39% (NCBI accession number: CP015732.1), and the homology with the Cockella is lower, so the gene sequence can be regarded as a unique sequence of the screened Cockia halodurans SDB9 bacteria, and is a bar code of the Alcaligenes halodurans SDB9 so as to be different from other Cockia rosea. The bacteria of Arthrobacter can often synthesize IAA, which is a relatively deep genus for the research of the IAA synthesis mechanism of the bacteria at present, and considering that the Alcaligenes SDB9 can synthesize IAA in a large amount, other IAA synthesis related genes in the Alcaligenes SDB9 have no specificity, but the ALDH4 sequence is relatively special and has the highest homology with the Arthrobacter, so the ALDH4 is presumed to be a key gene which endows the Alcaligenes SDB9 with a large amount of IAA synthesis capability.

(6) Identification of Cockera roseoflavus SDB9

The alcaligenes halodurans SDB9 is called as Cockerella rosea SDB9, is preserved in China general microbiological culture Collection center (CGMCC) on 6-month and 12-month in 2019, and has the addresses as follows: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation number is: CGMCC No. 17922.

Example 2 Cockera rosea SDB9 improves the green-keeping function of plants under saline-alkali stress

Saline-alkali lands are mainly formed due to the high content of sodium carbonate in the soil. The invention carries out soda (sodium carbonate) simulated saline-alkali stress green-keeping experiments. The method comprises the steps of taking the Changshan Daming mung beans as test materials, cultivating the test materials by using saline-alkali soil taken from fish and river towns in elm forest city of Shaanxi province, soaking the test materials in a Coccocus roseus SDB9 bacterial solution for 24 hours, then planting the test materials in a flowerpot with the upper caliber of 65mm and the height of 90mm, quantitatively watering 50mL each time, and after cultivating for 2 weeks, watering mung bean seedlings by using 10g/L of soda water, wherein 50mL each time is used. After 2 weeks of stress, Cockera roseum SDB9 was found to improve the green-keeping of mung bean seedlings.

Example 3 Cockera rosea SDB9 plant growth promotion and antioxidant capacity improvement under saline and alkaline stress

(1) The cultivation method of the test material of the mung bean of the Yangshan Daling is the same as that of the example 2, except that the salt and alkali stress simulation with high concentration of sodium carbonate is not carried out. After 80 days of growth, the overground part growth condition is observed, the roots are completely dug out of the soil, and the soil at the roots is cleaned by clean water. The results show that: compared with a control, the mung bean seedlings treated by the Corkspora roseoalba SDB9 bacterial liquid have the advantages that the growth speed of new leaves is high, the root systems are more developed, the lateral roots are more, especially, multi-leaf leafstalks are obviously lengthened, and the fact that the growth of plants can be better promoted by the treatment of the Corkspora roseoalba SDB9 bacterial liquid is proved. As shown in FIG. 4, the mung bean seedlings treated with the Corkspora rosea SDB9 bacterial liquid (SDB9) showed a significant increase in the first node length (FIG. 4A), the root fresh weight (FIG. 4D) and the aerial fresh weight (FIG. 4E) compared to the control group (CK). The mung bean seedlings treated by the bacterial liquid of the Coccocus roseus SDB9 have no obvious change in the length of the overground part and the underground part; it was also observed that there was no significant change in leaf length or leaf width of the mung bean seedlings treated with the bacterial suspension of Coccocus roseus SDB9 (FIG. 4B, C). It is presumed that Cockerella rosea SDB9 may have the effect of promoting the growth of plant roots and make the root system more developed.

(2) The cultivation conditions of the example 2 using the mung beans of the present invention as the test material are as followsThe method is characterized in that the seed soaking and the high-concentration sodium carbonate simulation saline-alkali stress are not carried out, and when the mung bean seedlings grow true leaves, 50ml of the salt with the concentration of 1 multiplied by 10 is utilized⁷The CFU/mL of the bacterial solution of the Cockera rosea SDB9 is treated by root irrigation, and the control group is irrigated with tap water. After the mung beans grow for 60 days, sodium carbonate is used for simulating saline-alkali stress for three times, the concentration of the three times is 0.025mol/L, 0.05mol/L and 0.1mol/L respectively, 50ml of water is poured into each pot, the interval time of the three saline-alkali stress is 1 week, and then the mung bean morphology and the antioxidant physiological indexes are observed and measured. The results show that: compared with the control group (CK), the root activity index of the mung bean treated by the Corkspora roseoalba SDB9 bacterial liquid is not increased greatly, and the difference between the root activity index and the root activity index is not obvious (FIG. 5A). The green bean chlorophyll treated by the bacterial liquid of the cockscomb strain SDB9 is increased by 7.81%, and the green bean leaves treated by the bacterial liquid of the cockscomb strain SDB9 are obviously greener than the control, which shows that the content of the green bean chlorophyll can be increased by the cockscomb strain SDB9, and the result is consistent with the previous result (figure 5B). The mung bean superoxide dismutase (SOD) enzyme activity treated by the Coccocus roseus SDB9 bacterial liquid has no obvious difference with the control group (figure 5C), and the POD content has no obvious change and no obvious difference (figure 5D). However, the mung bean Catalase (CAT) enzyme activity treated by the bacterial liquid of the Coccocus roseus SDB9 is increased by 5 times, and the difference is very obvious (FIG. 5E); the content of mung bean Malondialdehyde (MDA) treated by the Cockerella rosea SDB9 bacterial liquid is reduced by 5.34%, and the difference is not significant (figure 5F); the proline content in mung beans treated by the bacterial liquid of the Coccocus roseus SDB9 is obviously higher than that of a control group, and compared with the control group, the proline content is increased by 41.24%, and the difference between the proline content and the proline content is obvious (figure 5G).

(3) In order to verify the growth promoting effect of Corkspora rosea SDB9 on monocotyledons, the embodiment utilizes corn Zhengdan 958 as a test material, selects about 200 corn seeds which are full, free from plant diseases and insect pests, harmless and small in size difference, washes the selected corn seeds for 2-4 times by tap water, soaks the cleaned corn seeds in clear water for 6 hours, then places the seeds in an incubator at 28 ℃ for germination, prepares a plurality of vermiculite-containing flowerpots with the caliber of 11cm and the height of 14cm, plants the treated corn seeds in the pots, and uses about 8-10 corn seeds in each potThe seeds are sown and then placed in a growth chamber for culture. When the corn seeds grow to form second true leaves, seedlings with poor growth vigor need to be removed intermittently, and finally 3 corn seedlings are kept in each pot. When the height of corn seedlings is 5cm, 150ml of corn seedlings with the concentration of 1 multiplied by 10 is utilized⁷CFU/mL of the Corkspora rosea SDB9 was treated by root irrigation to form a test group (SDB9), and a control group (CK) was irrigated with tap water. After the corn grows for 60 days, sodium carbonate is used for simulating saline-alkali stress for three times, the concentration of each time is 0.025mol/L, 0.05mol/L and 0.1mol/L, each pot is watered for 150ml, the interval time of the saline-alkali stress for three times is 1 week, and then the corn morphology and the antioxidant physiological indexes are observed and measured. The results show that:

the plant heights of the control group corn seedlings and the test group corn seedlings have no significant difference. The test result shows that the plant height of the corn seedlings in the control group is 49.7cm, the plant height of the corn seedlings in the test group is 52cm, and compared with the plant height of the corn seedlings in the control group, the plant height of the corn seedlings in the test group shows a growth trend, the average growth amount is 2.3cm, the growth is about 0.05 times, but the plant height increase effect is not obvious, and the statistical difference is not obvious (fig. 6A).

Compared with the control group, the minute branch roots of the maize seedlings in the test group are more than those of the minute branch roots of the maize seedlings in the control group, and the phenomenon is more obvious particularly on the secondary root parts. Furthermore, the fibrous roots of the maize seedlings of the test group are more slender, the appearance color of the roots is yellowish and whitish, while the fibrous roots of the maize seedlings of the control group are more short and thick, and the appearance color of the roots is darker yellow. Under normal conditions, under the adverse stress of the corn, the root system presents dark yellow, so that the SDB9 endophytic bacteria used in the test have the effect of improving the tolerance of the root of the corn seedling.

The fresh weight of the underground part of the control group of the corn seedlings is 2.33g, the fresh weight of the underground part of the test group of the corn seedlings is 3.09g, the fresh weight of the underground part of the test group of the corn seedlings is increased by 0.76g, and the difference is obvious. Similar results were obtained by comparing and analyzing the fresh weight of the aerial parts of the maize seedlings in the control group, the fresh weight of the aerial parts of the maize seedlings in the test group, and the fresh weight of the aerial parts of the maize.

The chlorophyll content of the control group of maize seedlings was 0.378mg/g, while that of the test group of maize seedlings inoculated with the strain was 0.412mg/g, and the chlorophyll content of the test group of maize seedlings showed a growth phenomenon, with an average increase of 0.034g per gram of leaf, with a smaller amount of increase, with no significant difference (fig. 6C).

The Peroxidase (POD) activity of the test group of maize seedlings was elevated. The peroxidase activity in the control group of maize seedling leaves was 207.778U/mg, whereas the test group was 221.111U/mg, the peroxidase activity in the test group of maize seedling leaves was increased by 0.064 times, the increase was not significant and the difference was not significant (FIG. 6D).

The Catalase (CAT) activity of the test group corn seedlings is obviously higher than that of the control group. The catalase activity of the control group corn seedling leaf is 35.294U/mg, the catalase activity of the test group corn seedling leaf is 402.353U/mg, compared with the control group, the catalase activity of the test group corn seedling leaf is improved by 10 times, the improvement range is large, and the difference is very obvious (fig. 6E).

The activity of superoxide dismutase (SOD) in the control group corn seedling leaves is 917.588U/mg, the activity of SOD in the test group is 921.298U/mg, the content of SOD in the test group corn seedling leaves is increased, but the increase range is small, and no significant difference exists (figure 6F).

The content of Malondialdehyde (MDA) in leaves of maize seedlings in the test group is substantially less than that in the control group. The malondialdehyde content in the control group maize seedling leaves was 32.733nmol/G, whereas the test group was 14.408nmol/G, and the malondialdehyde content in the test group maize seedling leaves was significantly lower than that in the control group maize seedling leaves, with a significant difference (fig. 6G).

The root activity of the control group corn seedlings is 0.112mg/g/H, while the root activity of the test group corn seedlings is 0.129mg/g/H, compared with the root activity of the control group corn seedlings, the root activity of the test group corn seedlings is increased by 0.15 times, the increase range is small, and no significant difference exists (fig. 6H).

The proline content in the control group of maize seedling leaves was 0.001473 μ g/g, whereas the test group was 0.001683 μ g/g, and the content of the test group was slightly increased by 0.14 times without significant difference (fig. 6I).

Example 4 Cockera rosea SDB9 promotes plant growth under field test conditions

Corn (Zhengdan 958) and rice (Jihong No. 6) tests are respectively carried out on an red stone gorge test base in elm city of Shaanxi province and a soldier ditch yellow river bridge saline-alkali soil in Yinchuan city of Ningxia Hui nationality.

The corn is used at a concentration of 1 × 10 when the height of seedling is 5cm⁷The CFU/mL of the Corkspora rosea SDB9 bacterial liquid is subjected to root irrigation treatment, and the result shows that: in the soil without fertilization, after the corns are planted for 80 days, the corns in the test group (SDB9) treated by the bacterial liquid are green, the leaves of the control group (CK) are yellow, the root systems of the test group are more developed, the ears of the corns are larger, the maturity is higher, the seeds of the corns in the control group are still fresh, tender and full, the test group is in a horse-shaped tooth shape, and the sharp points are also very small.

The method is characterized in that ammoniacal nitrogen, nitrate nitrogen, available phosphorus and available potassium are measured on the rhizosphere soil of the corn, the ammonia nitrate nitrogen is measured by a potassium chloride leaching flow analyzer, the available phosphorus is leached by sodium bicarbonate to obtain molybdenum antimony resistance colorimetry, and the available potassium is measured by a spectrophotometer after a nitric acid leaching agent is used. The results show that: the change in the ammoniacal nitrogen and available phosphorus contents of the corn rhizosphere soil of the experimental group was insignificant compared to the control group (fig. 7A, C), but the nitrate nitrogen content of the corn rhizosphere soil of the experimental group decreased by about 21% (fig. 7B) and the available potassium content increased by about 57% (fig. 7D), presumably due to the potassium-solubilizing ability of the cockscomb rosea SDB9, which is also consistent with the conclusion that the confirmed cockscomb rosea SDB9 in example 1 had the potassium-solubilizing ability and promoted the absorption of nitrate nitrogen.

The method for measuring the content of total nitrogen, total phosphorus and total potassium of different parts of a corn plant comprises the following steps: the total nitrogen is extracted by concentrated sulfuric acid and hydrogen peroxide and then analyzed by a flow analyzer, the total phosphorus is analyzed by a molybdenum-antimony colorimetric method, and the total potassium is analyzed by a flame photometer. The results show that: the corn roots and stems were significantly increased in potassium content (fig. 8C) and facilitated nitrogen uptake in the corn roots and stems of the test group treated with the cocklebur roseum SDB9 broth (SDB9) compared to the control group (CK), and the nitrogen content was significantly increased in the roots, stems and kernels of the corn in the test group (fig. 8A), but also in the phosphorus uptake, the phosphorus content was decreased in the roots, stems and leaves of the corn in the test group, and the phosphorus content was increased in the kernels (fig. 8B).

The rice is soaked for 1 hour by adopting the bacterial solution of the Corksia rosea SDB9 with the same concentration, and then the germination is accelerated and the direct seeding is carried out. After 120 days of growth, the plant height and the ear length of the rice of the test group (SDB9) treated by the Corkspora rosea SDB9 bacterial liquid are both larger than those of the control group (CK), the root system of the test group is more developed, the number of new roots at the root and stem combination part is obviously more than that of the control group, the rice of the test group is grouted ahead of time, the control group does not start to be grouted, and the seeds of the test group are about 3mm long.

After the rice is mature, the plant type analysis shows that: 10 plants are randomly selected from the test group and the control group for statistics, compared with the control group, the plant height of the rice in the test group is remarkably increased by about 2.5cm (figure 9E), the diameter of the stem base is also increased (figure 9A), the diameter of the ear base, the ear length and the length of the stem under the ear are remarkably increased (figure 9B, C, D), and the increase of the plant height is mainly caused by the increase of the stem under the ear.

After scanning the rice root system, the analysis of Win RHIZO PRO 2009 software (Regent inc., Quebec, Canada) shows that: the root length, root surface area and root volume of the rice plants in the test group were significantly increased, about 53%, 61% and 70%, respectively, compared to the control group, as compared to the plant type analysis described above (fig. 10A, B, C).

The analysis of the rice yield related indexes shows that: the sampling is the same as the plant type analysis, compared with the control group, the panicle number and the grain number of the rice in the test group are obviously increased by about 27 percent and 83 percent respectively (FIG. 11A, B), the blight rate is reduced by about 12 percent (FIG. 11C), and the thousand seed weight is not obviously different; sampling 1 square meter, 3 replicates, no significant difference in the aerial part weight (root cut from stem base) of control and test groups (fig. 11D), indicating that there may be no significant increase in ear number per acre, but about a 12% increase in the fresh weight (glume) of the test group rice (fig. 11E). Preliminary analysis shows that the bacterial liquid of the Cockera rosea SDB9 promotes the increase of the ear length of rice, further promotes the increase of the number of small ears and the number of grains of the rice, and achieves the effect of increasing the yield.

The above description is only a preferred embodiment of the present invention, and these embodiments are based on different implementations of the present invention, and the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

<110> Yulin, Zhongtai agricultural science and technology Co., Ltd

Yulin college

<120> Cockerella rosea SDB9, and preparation method and application thereof

<130>20191210

<160>1

<170>SIPOSequenceListing 1.0

<210>3

<211>2919

<212>DNA

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<400>3

atntnvrsng agggtgctac acatgcagtc gaacgatgat gcccagcttg ctgggcggat 60

tagtggcgaa cgggtgagta atacgtgagt aacctgccct tgactctggg ataagcctgg 120

gaaactgggt ctaatactgg atactacctc ttgccgcatg gtgggtggtg gaaagggttt 180

tactggtttt ggatgggctc acggcctatc agcttgttgg tggggtaatg gctcaccaag 240

gcgacgacgg gtagccggcc tgagagggtg accggccaca ctgggactga gacacggccc 300

agactcctac gggaggcagc agtggggaat attgcacaat gggcggaagc ctgatgcagc 360

gacgccgcgt gagggatgac ggccttcggg ttgtaaacct ctttcagtag ggaagaagcg 420

agagtgacgg tacctgcaga agaagcgccg gctaactacg tgccagcagc cgcggtaata 480

cgtagggcgc aagcgttgtc cggaattatt gggcgtaaag agctcgtagg cggtttgtcg 540

cgtctgctgt gaaagcccgg ggctcaaccc cgggtctgca gtgggtacgg gcagactaga 600

gtgcagtagg ggagactgga attcctggtg tagcggtgaa atgcgcagat atcaggagga 660

acaccgatgg cgaaggcagg tctctgggct gttactgacg ctgaggagcg aaagcatggg 720

gagcgaacag gattagatac cctggtagtc catgccgtaa acgttgggca ctaggtgtgg 780

gggacattcc acgttttccg cgccgtagct aacgcattaa gtgccccgcc tggggagtac 840

ggccgcaagg ctaaaactca aaggaattga cgggggcccg cacaagcggc ggagcatgcg 900

gattaattcg atgcaacgcg aagaacctta ccaaggcttg acattcaccg gaccgcccca 960

gagatggggt ttcccttcgg ggctggtgga caggtggtgc atggttgtcg tcagctcgtg 1020

tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc tcgttctatg ttgccagcac 1080

gtgatggtgg ggactcatag gagactgccg gggtcaactc ggaggaaggt ggggatgacg 1140

tcaaatcatc atgcccctta tgtcttgggc ttcacgcatg ctacaatggc cggtacaaag 1200

ggttgcgata ctgtgaggtg gagctaatcc caaaaagccg gtctcagttc ggattgaggt 1260

ctgcaactcg acctcatgaa gtcggagtcg ctagtaatcg cagatcagca acgctgcggt 1320

gaatacgttc ccgggccttg tacacaccgc ccgtcaagtc acgaaagttg gtaacacccg 1380

aagccggtgg cctaacccct tgtgggaggg agccgtcgaa ggtgaccgat gaacaccgtg 1440

atccgtcccg ccacgaccgc cgccgaggtg gaccgggccg cccgcgccgc ccaccaggcg 1500

tacctggtgt ccgccgccca gacgcccgcc acccgggcgt cctggctcac ggccctggcc 1560

gaggccctgc gcgcccacga cgacgagctc gtcggcctgg ccgcggagga gacccacctc 1620

gccgaacccc ggctgcgcgg ggagctcaag cgcagcgcct tccaactgga cctgttcgcg 1680

caggaggtcc gcaccggggt ggccctggac gccgtcatcg accacgccga cccggactgg 1740

gggatgggcc cgcgcccgga catccgccgc gtcaacgtgc ccctgggcgt ggtgggcgtc 1800

ttcggcgcct ccaacttccc cttcgccttc agcgtcatgg gcggggacag cgcctcggcc 1860

ctggccgccg ggtgcgccgt cctgcacaag atccacggcg ggcatctgag gctcggtctg 1920

cgcaccgccg agatcgtcgt cgaggcgctg gagaaggtag gcgccccaca gggactgttc 1980

gccgtgctcg agggccggga ggcggccacc gcgctggtcg accacccgtt ggtgaaggcc 2040

atcgggttca ccggttccac cgccggcggc cgggcgctgt tcgatcgggc ggccgcccgg 2100

cccgagccca tccccttcta cggggagctg ggcagcatca accccgtgtt cgtgctcccc 2160

gcagcgtggg agcaccgctg gcaggagatc ctcaccggct acgccgggtc cttcaccatg 2220

ggcatgggcc agttctgcac caagcccggg gtgctcttcg tcccggacac gggcgagacg 2280

gagcaggtcc ggaccaccct gacctcggcc ctggccgatg ccccgctggc gaagcttctc 2340

acccccgggt tgcgtgagtc cttcggccag gcccgtgatg aggtcgccgc gctcgaaggc 2400

gtgcagacgc tcgtggccgg ctctgacgag gaggtgcccg acccgaccgt cctggccgtc 2460

gacgccgagc aggtccggcg gaaccccgag gtgctgcacc gggagatgtt cggcccggcc 2520

acagtgctcg tgcgatatcg ggaggccgct gacctgccgg cccttgccga gctgatggag 2580

ggacagctga cggccaccgt ccacggcgac gaggaggacg acagcgctgc gctggtgggc 2640

accctgaccg gcaaggccgg ccgggtgctg ttcaacggct ggcccaccgg ggtgaccgtc 2700

tcctacgccc agcaccacgg cggtccctac ccggcgacga ccacgaacac cacctcggtg 2760

ggcacggcct cgatcgggcg gttcatgcgc ccggtgtcct accaggacct ccccgacgcc 2820

gcgctgccgc cagccctgca ggaggccaat ccctggggga tcgcccgccg cgtggacggc 2880

cggttcgaac ccgccgagtc ggaaggagcc ggcgcatga 2919

Claims

1. Cockerella rosea SDB9, characterized in that:

the Cockerella rosea SDB9 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number: CGMCC No.17922, preservation date: 12/6/2019, taxonomic name: kocuria rosea strain SDB 9.

2. The Cockera roseochracea SDB9, according to claim 1, wherein:

3. The Cockera roseochracea SDB9, according to claim 1, wherein:

4. The method for preparing the Cockera roseoflavus SDB9 according to claim 1, wherein the method comprises the steps of:

(1) thoroughly disinfecting the root surface of sabina vulgaris;

(3) culturing, separating and identifying saline-alkali tolerant bacteria;

(6) identification of Cockera roseoflavus SDB 9.

5. The method for preparing Cockera roseoflavus SDB9 according to claim 4, wherein:

the Cockerella rosea SDB9 can grow on NaCl culture medium with the concentration of 0-1.5mol/L and culture medium with the pH value of 7-12.

6. The use of the Cockera roseus SDB9 according to any one of claims 1 to 5 for promoting plant growth.

7. The use of Corksia rosea SDB9 according to claim 6, wherein:

the bacterial liquid of the Cockerella rosea SDB9 has obvious capacity of promoting plant growth and improving plant oxidation resistance under the saline-alkali stress environment.

8. The use of Corksia rosea SDB9 according to claim 7, wherein:

9. The use of Corksia rosea SDB9 according to claim 6, wherein:

inoculating the Cocker roseum SDB9 into a culture medium, culturing for 20-28h at 25-30 ℃ to obtain a culture solution, centrifuging the culture solution to obtain Cocker roseum SDB9 thalli, re-suspending the Cocker roseum SDB9 thalli with water to obtain a bacterial solution of the Cocker roseum SDB9, and using the bacterial solution of the Cocker roseum SDB9 for seed soaking, root pouring and inoculation of plants to promote the growth of the plants.

10. The use of Corksia rosea SDB9 according to claim 6, wherein:

the use concentration of the bacterial liquid of the Cockerella rosea SDB9 is 1 multiplied by 10⁶-1×10¹⁰CFU/mL。