CN114514833B - Application of bacillus in rice cadmium pollution repair - Google Patents

Abstract

The invention discloses application of bacillus to rice cadmium pollution repair, which can effectively promote rice growth and mainly aims at improving rice plant height and increasing single plant yield and brown rice yield. The bacillus can effectively repair rice cadmium pollution, mainly aims at reducing rice rootstock and brown rice cadmium content, and can effectively improve brown rice quality, mainly aims at improving brown rice crude protein percentage content and the like; belonging to the technical field of plant rhizosphere growth promotion and pollution repair bacteria.

Description

Application of bacillus in rice cadmium pollution repair

Technical Field

The invention relates to the technical field of plant rhizosphere growth-promoting and pollution-repairing bacteria, and application of bacillus in promoting growth of rice and repairing cadmium pollution.

Background

Cadmium is a metal with strong biological enrichment capability, and when cadmium passes through food chain to human or animal and plant body, persistent toxicity is generated, and serious harm is caused to various organs and immune systems of human, since 50 th century, the mass water is caused by eating cadmium out of standard rice by residents in Shentong river basin in Fushan county, japan, and the problem of cadmium pollution gradually attracts worldwide attention (dolina et al 2012). With the acceleration of industrialization and urbanization processes, the heavy metal pollution condition of soil is increasingly serious. 2014. Annual national soil surveys show that 19.4% of the cultivated land soil has an excessive heavy metal. Wherein the cadmium point position exceeding rate is 7.0 percent. At present, the soil heavy metal restoration technology is mainly divided into three categories of physical restoration, chemical restoration and biological restoration. While the mechanisms of repair can be divided into two broad categories: one is to remove cadmium in the polluted soil by heavy metal enrichment plant measures; the other is immobilization, i.e. reducing the availability of heavy metals in the soil, making them difficult to migrate and bioaccumulate (Li Xingjie, 2019).

The biological fertilizer has the great advantages of activating soil nutrients, improving fertilizer utilization rate, promoting growth, improving yield and quality and the like, has the unique advantages of rapid propagation, strong metabolism, low repair cost, no secondary pollution and the like, and is of daily importance in repairing soil pollution. Such as Bacillus, streptomyces, pseudomonas putida (Pseudomonas putida), staphylococcus (Staphylococcus), thiobacillus, etc., show great potential in heavy metal pollution remediation applications.

The bacillus gorilla has the functions of promoting growth, resisting heavy metal toxicity and the like, has the advantages of simple industrial production process, long shelf life, easy survival and colonization in soil environment and the like, and is used as a novel strain resource, but has not been excavated and researched in rice cadmium pollution repair.

Disclosure of Invention

Aiming at the problems, the invention aims to provide the application of the bacillus gorilla which can effectively promote the growth of rice, effectively repair the cadmium pollution of the rice and effectively improve the quality of brown rice.

Therefore, the technical scheme provided by the invention is as follows: an application of bacillus gorilla in rice cadmium pollution repair is provided, wherein the bacillus gorilla is used for improving rice plant height.

The application of the bacillus gorilla strain in the repair of cadmium pollution of rice is provided, wherein the bacillus gorilla strain is used for improving the yield of a single rice plant.

The application of the bacillus gorilla strain in the repair of cadmium pollution of rice is provided, wherein the bacillus gorilla strain is used for improving the yield of brown rice of rice.

The application of the bacillus gorilla strain in the repair of rice cadmium pollution is provided, wherein the bacillus gorilla strain is used for reducing the content of brown rice cadmium.

The application of the bacillus gorilla strain in the repair of rice cadmium pollution is provided, wherein the bacillus gorilla strain is used for reducing the content of rice root cadmium.

The application of the bacillus gorilla strain in the repair of rice cadmium pollution is that the bacillus gorilla strain is used for rice plant stem and leaf cadmium content.

The application of the bacillus gorilla strain in the repair of rice cadmium pollution is that the bacillus gorilla strain is derived from bacillus gorilla strain with deposit number GDMCCNo of 60979, which is deposited in microorganism strain collection GDMCC (building 5 No. 59 of Qingzhou university 100 in Guangzhou, guangdong province) at 16 days of 2020.

Compared with the prior art, the bacillus gorilla YC9L has high yield of auxin IAA, has phosphate and potassium dissolving and heavy metal resisting functions, has obvious promoting effect on the growth of rice, and obviously restores cadmium pollution. The concrete steps are as follows:

(1) The technical scheme provided by the invention utilizes the capability of the bacillus gorilla for efficiently producing IAA, promotes the synthesis of genetic materials and proteins in plant cells, accelerates the division and differentiation of cells, has the capability of efficiently promoting the growth of rice plants, can obviously improve the plant height of the rice plants, and effectively increases the yield of rice single plants and brown rice.

(2) The technical scheme provided by the invention utilizes the function of restoring heavy metal pollution by the bacillus gorilla to reduce the cadmium content of root systems, the cadmium content of plant stems and leaves and the cadmium content of seeds of rice.

(3) According to the technical scheme provided by the invention, the function of improving the quality of rice grains by utilizing bacillus gorilla is utilized to improve the iron-manganese-zinc content of brown rice and the crude protein content of brown rice.

Drawings

FIG. 1 is an aerial view of a field test of the promotion of rice growth by YC9L bacteria

FIG. 2 is a photograph of a field test of YC9L bacterium promoting rice growth

FIG. 3 shows the effect of YC9L bacteria on promoting rice plant height

FIG. 4 is a graph showing the effect of YC9L bacteria on the effective Cd content of soil

FIG. 5 shows the effect of YC9L bacteria on reducing Cd content in rice root system

FIG. 6 shows the effect of YC9L bacteria on reducing Cd content in the stem and leaf of rice plants

FIG. 7 shows the effect of YC9L bacteria on reducing the heavy metal Cd content of brown rice

FIG. 8 shows the effect of YC9L bacteria on reducing the Pb content of heavy metals in brown rice

FIG. 9 shows the effect of YC9L bacteria on increasing the Fe content of brown rice

FIG. 10 shows the effect of YC9L bacteria on increasing Cu content of brown rice

FIG. 11 shows the effect of YC9L bacteria on increasing the Mn content of brown rice

FIG. 12 shows the effect of YC9L bacteria on increasing Zn content in brown rice

FIG. 13 is a graph showing the effect of YC 9L-strain on amylose content of brown rice

FIG. 14 shows the effect of YC9L bacteria on increasing the crude protein content of brown rice

FIG. 15 shows the effect of YC9L bacteria on improving rice individual yield

FIG. 16 shows the effect of YC9L bacteria on increasing the brown rice content of a single rice plant

FIG. 17 shows IAA secretion by YC9L bacterium

FIG. 18 is a micrograph of YC9L bacteria.

Detailed Description

The detailed description further details the claims of the invention do not constitute any limitation of the invention, any limited number of modifications made by anyone within the scope of the claims of the invention remain within the scope of the claims of the invention.

The following examples are, unless otherwise indicated, all the procedures and procedures used in the art.

The gorilla bacillus described in the present application is derived from the gorilla bacillus deposited at the Guangdong province microorganism strain collection center (GDMCC) on month 03 and 16 of 2020 under accession number GDMCC No. 60979.

Example 1 field test of YC9L Rice growth promoting Effect

Preparing a bacterial suspension: (1) activating the strain, inoculating the strain into an LB liquid culture medium, culturing overnight, transferring the strain into a fresh LB liquid culture medium, and shaking until the OD600 = 1.0; (2) centrifuging the bacterial liquid in a centrifuge at 8000rpm for 5min; (3) the strain is resuspended in an equal amount of sterile water and applied by dipping roots when the rice is transplanted. Test site: the Shaoguan city, the Humihua county silver farm cadmium pollutes the farmland, north latitude N: 25°05' 44.04 ", east longitude E: 113 deg. 37' 51.51 ". Test time: 2021. transplanting 8 months and 16 days of year. The plant height of rice and the soil effective Cd content were measured at day 8 and 26, rice was harvested at day 11 and 25, and root, stem and leaf and grain samples were collected for analysis (FIGS. 1 and 2). Test treatment: split into 2 treatments, treatment 1: applying a peasant household conventional fertilizer; treatment 2: the YC9L microbial inoculum is applied, and the specific operation is as follows, the YC9L microbial inoculum is added with water for 1000 times, then seedlings are directly transplanted in a conventional way after being dipped in root for 1 second, each treatment is repeated for 3 times, and the area of each cell is 12 m < 2 >. The microbial inoculum remarkably promotes the growth of rice: has remarkable effect on reducing cadmium in rice. The YC9L microbial inoculum significantly increased the rice plant height by 10 days after application of the microbial inoculum, and YC9L microbial inoculum treatment increased the plant height by 8.5% as compared to the control (fig. 3).

The microbial inoculum obviously reduces the cadmium content of rice, the effective cadmium content of soil is not changed by YC9L microbial inoculum (figure 4), the Cd content of root system of rice is obviously reduced by YC9L microbial inoculum (figure 5), the Cd contents of the microbial inoculum treatment and control root system in the harvest period are respectively 11.56mg/kg and 5.45mg/kg, and the cadmium content of the root system is obviously reduced by 53% by microbial inoculum. The Cd content of the stems and leaves of the fungus agent treatment and the control plants in the harvest period is 1.87 mg/kg and 0.94mg/kg respectively, and the Cd content of the stems and leaves of the plants is obviously reduced by 49.7% by the fungus agent (figure 6). The Cd content of the microbial inoculum treatment and the Cd content of the control brown rice are respectively 0.50 mg/kg and 0.26mg/kg, and the cadmium reduction rate of the microbial inoculum on the brown rice is 48% (figure 7). In addition, the Pb content of the brown rice is obviously reduced by the microbial inoculum, and the Pb content of the brown rice treated by the microbial inoculum and the Pb content of the control brown rice are respectively 0.18 mg/kg and 0.15mg/kg (figure 8).

The microbial inoculum obviously improves the contents of beneficial elements such as iron, copper, manganese, zinc and the like of the rice brown rice, and compared with a control, 26.2%, 16.7%, 21.3% and 16.0% of the microbial inoculum are respectively obtained. The microbial inoculum obviously improves the crude protein content of the rice brown rice, and compared with a control, the microbial inoculum treated crude protein content is improved by 13.9 percent, but the amylose content is reduced by 12.5 percent (figures 9-14).

The microbial inoculum significantly improved rice yield, the individual plant yields of YC9L microbial inoculum treatment and control treatment were 34.7 and 27.4 g/plant, respectively, and the brown rice yields of YC9L microbial inoculum treatment and control treatment were 33.6 and 21.2 g/plant, respectively, thus demonstrating that YC9L microbial inoculum significantly improved rice yield (fig. 15-16).

By combining the above, the YC9L microbial inoculum can obviously promote the growth of rice plants, improve the yield of rice and increase the contents of crude protein, zinc and iron in seeds. The cadmium content of the rice is obviously reduced, and the method has a huge application prospect in the aspect of safe utilization of the cadmium-polluted soil of the rice.

Example 2 screening of YC9L Strain, (1) isolation of soil sample from root soil of vegetation in Guangzhou Tianhe greenbelt, 10g of soil sample was weighed and put into a 250mL triangular flask (glass beads) containing 90mL of sterile water, and shaking for 30min. And fully dispersing bacteria, and standing for 20-30 s. Then, the supernatant was subjected to 10-fold incremental gradient dilution in a water bath at 90℃for 10min, 100. Mu.L of each gradient was applied to LB solid plates at 10-3 to 10-4 dilutions, and the culture was inverted at 37℃for 24 hours. Observing and picking the bacterial colonies according to the growth characteristics of the bacterial colony, such as morphology, size, color and the like, purifying the bacterial colonies on an LB solid medium, and then preserving the separated bacteria for later use. (2) Screening the isolated strains were assayed for IAA-producing ability using Salkowski colorimetry. The isolated bacteria were inoculated into 4mL of LB liquid medium containing L-tryptophan (100 mg/L), and cultured in a shaking table at 30℃and 180 r/mm for 24 hours, to thereby perform qualitative and quantitative analysis. 200. Mu.L of the bacterial suspension was dropped onto a white ceramic plate, while 200. Mu.L of Salkowski broth (500mL 35%HClO4+10mL 0.5mol/L FeCl 3) was added. 200. Mu.L IAA standard (50 mg/L) was used as positive control and 200. Mu.L of non-inoculated LB liquid medium containing L-tryptophan was used as a blank. After 30min of standing at room temperature in the dark, the strain with reddening color was recorded (FIG. 17A). IAA quantification is carried out on bacterial suspension of the strain, OD600 value of bacterial liquid is measured by spectrophotometry, then the bacterial liquid is centrifuged for 10min at 10000rpm/min, supernatant fluid is taken and added into equal volume Salkowski colorimetric liquid, and the obtained product is kept stand for 30min in dark place, and OD530 value of the obtained product is measured. And drawing a standard curve according to an IAA gradient dilution measuring method, and calculating the IAA content in the fermentation broth in unit volume. Strains with high IAA yield, YC9L with higher IAA yield and IAA secretion up to 46.72mg/L were screened out (FIG. 17B).

Example 3 YC9L gram-positive bacteria, YC9L single colony was picked up and cultured in liquid LB medium for 24h, 4. Mu.L of sterile water was sucked up on a glass slide, 1. Mu.L of bacterial liquid was sucked up in the added sterile water, and then mixed well and dried naturally. Fixing after passing flame for 2-3 times; performing crystal violet primary dyeing for 1 min, washing with water, and naturally airing; mordant dyeing for 1 min, washing with water, and air drying; decolorizing with 95% ethanol by 20 s, washing with water, and air drying; safranine counterstain for 1 min, water washing, air drying, and observing under 100 times oil microscope, wherein the thallus is blue-violet (figure 18A), and YC9L is gram positive bacteria.

Example 4 YC9L bacteria can produce spores, YC9L single colonies are picked up and cultured for 24 hours in a liquid LB culture medium, 4 mu L of sterile water is sucked up on a glass slide, 1 mu L of bacteria liquid is sucked up in the added sterile water, and then the mixture is uniformly mixed and naturally dried. And (5) fixing after passing through the flame for 2-3 times. Dripping the malachite green dye liquor onto the fixed smear, clamping the glass slide by forceps, heating on the flame of the alcohol lamp, and allowing the dye liquor to emit steam for 3-4 times in 30 s; washing with water and airing; counterstaining with safranin aqueous solution for 1 min, washing with water, and air drying. The spores were green and red with 100-fold oil microscopy (FIG. 18B), and the YC9L bacteria produced spores.

Claims (5)

1. The application of the bacillus to the repair of the cadmium pollution of the rice is characterized in that the bacillus is used for improving the plant height of the rice; the bacillus is the gorilla bacillus deposited with the Guangdong province microorganism strain collection center (GDMCC) with the deposit number GDMCC No. 60979.

2. The application of the bacillus to the repair of the cadmium pollution of the rice is characterized in that the bacillus is used for improving the single plant yield of the rice; the bacillus is the gorilla bacillus deposited with the Guangdong province microorganism strain collection center (GDMCC) with the deposit number GDMCC No. 60979.

3. The application of the bacillus to the repair of the cadmium pollution of the rice is characterized in that the bacillus is used for improving the yield of brown rice of the rice; the bacillus is the gorilla bacillus deposited with the Guangdong province microorganism strain collection center (GDMCC) with the deposit number GDMCC No. 60979.

4. The application of the bacillus to the repair of the cadmium pollution of the rice is characterized in that the bacillus is used for reducing the cadmium content of brown rice of the rice; the bacillus is the gorilla bacillus deposited with the Guangdong province microorganism strain collection center (GDMCC) with the deposit number GDMCC No. 60979.

5. The application of the bacillus to the repair of the cadmium pollution of the rice is characterized in that the bacillus is used for improving the crude protein content of the brown rice; the bacillus is the gorilla bacillus deposited with the Guangdong province microorganism strain collection center (GDMCC) with the deposit number GDMCC No. 60979.

Images