CN115612649A

CN115612649A - Salt-tolerant growth-promoting disease-resistant bacillus of plant and application thereof

Info

Publication number: CN115612649A
Application number: CN202211348031.7A
Authority: CN
Inventors: 刘开辉; 刘月; 丁小维; 陈妮; 张智维; 何帅; 王国亮; 刘婉婷
Original assignee: Shaanxi University of Science and Technology
Current assignee: Shaanxi University of Science and Technology
Priority date: 2022-10-31
Filing date: 2022-10-31
Publication date: 2023-01-17

Abstract

The invention provides a plant salt-tolerant growth-promoting and disease-resistant Bacillus and application thereof, wherein the Bacillus belongs to Bacillus (Bacillus swezeyi) and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.25411. The Bacillus (Bacillus swezeyi) provided by the invention can simultaneously relieve double stresses of biotic stress (pathogenic bacteria) and abiotic stress (salinity), and can also prevent and control the black shank of rape.

Description

Salt-tolerant growth-promoting and disease-resistant bacillus of plant and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a plant salt-tolerant growth-promoting disease-resistant bacillus and application thereof.

Background

Rape is an important oil crop and is planted in various places in a large area at present. Under the influence of soil salinization, rape crops are weak in growth vigor and poor in disease resistance and stress resistance, are easily attacked and infected by various diseases in the growth process, seriously affect the yield and the quality of the rape crops, easily cause yield reduction and even absolute harvest, and cause great economic and social losses (Cao Feng, 2020). Therefore, the development of rape disease control technology and the improvement of salt tolerance become important directions for agricultural development.

The rape has the diseases of Sclerotinia sclerotiorum, black shank, downy mildew, white rust and the like, wherein the Sclerotinia sclerotiorum caused by Sclerotinia sclerotiorum can cause the yield reduction of the rape to reach 80 percent. Secondly, researches find that rape black shank caused by Leptosphaeria biglobosa (L.biglobosa) type closely related species occurs in most rape producing areas, can cause damage to rape leaves, stems, siliques, grains and other parts, can cause leaf spots, stem rot and pod rot, and seriously affect the rape growth and the rape seed yield.

The biological control has the characteristics of high efficiency, no toxicity, no harm, no pollution and the like, and becomes a hot point for researching plant disease control in recent years. The biocontrol bacillus is the dominant biological population of soil and plant rhizosphere, has strong stress resistance and disease prevention and growth promotion effects, and is an important microbial source biopesticide. For example, the Jade substitute finds that the Bacillus subtilis RSS-1 separated from the rape field has obvious inhibition effect on the hypha of sclerotinia sclerotiorum (S.sclerotiorum); the Sunli et al discover that the Bacillus beleisi separated from healthy rape plants is prepared into wettable powder to act on rape, and has inhibition effect on the formation of sclerotinia sclerotiorum hyphae and the germination of conidia of black shank bacteria; jiangshijing et al disclose a strain of Bacillus mobilis (B.mobilis) which can promote the growth of rape seedlings by inoculating the B.mobilis under the salt stress; suzhongtao et al disclose a Paenibacillus polymyxa separated from rhizosphere soil of cotton in saline-alkali land, which can promote growth of cotton in saline-alkali land and reduce continuous cropping obstacles. At present, the research on promoting the growth of crops and improving the stress resistance and disease resistance of crops by using biocontrol bacteria is many, but the biocontrol bacteria applied to rape mostly focus on relieving single stress, and reports on relieving biotic stress (pathogenic bacteria) and abiotic stress (such as salinity) are few. Secondly, no report is made on the research of the Bacillus (Bacillus swezeyi) for relieving the double stress of plants. In addition, biocontrol bacteria in saline-alkali soil for preventing and controlling rape black shank are rarely reported.

Disclosure of Invention

The invention aims to provide a salt-tolerant growth-promoting and disease-resistant bacillus strain ZM96 of a plant and application thereof, aiming at the problems of biotic stress (pathogenic bacteria) and abiotic stress (salinity) of rape in the growth cycle.

The invention is realized by the following technical scheme:

a salt-tolerant growth-promoting and disease-resistant Bacillus of plants belongs to Bacillus (Bacillus swezeyi), is preserved in China general microbiological culture Collection center (CGMCC), and has a preservation number of CGMCC No.25411.

Preferably, the bacillus has an antagonistic effect on salt stress.

Preferably, the bacillus has antifungal activity.

Further, the fungus is phytophthora parasitica or sclerotinia sclerotiorum.

Preferably, the bacillus has a phosphorus solubilizing ability.

Preferably, the bacillus has siderophore capacity.

Preferably, the bacillus is separated from saline-alkali soil in the saline-alkali desert in northern Shaanxi of China.

The salt-tolerant growth-promoting and disease-resistant bacillus of the plant is applied to the preparation of the crop antibacterial agent.

Preferably, the crop is oilseed rape.

Compared with the prior art, the invention has the following beneficial effects:

the Bacillus (Bacillus swezeyi) provided by the invention is separated from saline-alkali soil, and has the capabilities of promoting the growth and development of rape under salt stress and preventing and treating rape diseases. The growth promoting activity function test shows that the strain has the characteristics of dissolving phosphorus and producing siderophores; saline-alkali soil cultivation test research shows that the biomass (root length, plant height and fresh weight) of a plant inoculated with the strain under salt stress is obviously higher than that of an uninoculated plant, which indicates that the strain can promote the growth and development of rape while relieving the salt stress; the antagonistic experiment shows that the strain has stronger bacteriostatic activity on sclerotinia rot and phytophthora parasitica of rape. Therefore, the Bacillus (Bacillus swezeyi) provided by the invention can simultaneously relieve double stresses of biotic stress (pathogenic bacteria) and abiotic stress (salinity), and can also prevent and control rape black shank. The strain can be used for the production of live bacteria preparations, has low production cost, is an effective substitute of chemical pesticides, and has important significance for the sustainable development of agriculture and the restoration of saline-alkali soil.

Drawings

FIG. 1 is a (a) colony morphology map and (b) strain microscopic map of Bacillus ZM 96;

FIG. 2 is a phylogenetic tree of the 16S rDNA gene of Bacillus ZM 96;

FIG. 3 shows the identification of the phosphorus solubilizing ability of Bacillus ZM96 (a) and the ability of siderophore production (b);

FIG. 4 is a comparison graph of the growth vigor of the rape plants after the root irrigation treatment; (a) day 0, (b) day 40;

FIG. 5 is a comparison of the root system of rape plants after root irrigation treatment;

FIG. 6 is the effect of the inoculum strain ZM96 on the root length of rape plants;

FIG. 7 is the effect of plant height of the inoculated strain ZM96 rape plant;

FIG. 8 is the effect of the inoculum strain ZM96 on the fresh weight of rape plants;

FIG. 9 shows the inhibition of pathogenic bacteria of Brassica napus by strain ZM 96; the bacillus subtilis comprises (a) phytophthora parasitica and (b) sclerotinia sclerotiorum.

Detailed Description

For a further understanding of the invention, reference will now be made to the following examples, which are provided to illustrate further features and advantages of the invention, and are not intended to limit the scope of the invention as set forth in the following claims.

The salt-tolerant growth-promoting and disease-resistant Bacillus of the plant is a Bacillus strain ZM96 which is classified and named as Bacillus (Bacillus swezeyi), is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation date of 2022 years, 7 months and 27 days, and has the preservation number of CGMCC No.25411.

The salt-tolerant growth-promoting and disease-resistant bacillus strain ZM96 of the plant is obtained by culturing a diluent prepared from salinized soil collected from the saline-alkaline desert of Shaanxi province in an Ashby culture medium and a beef extract peptone plate at a constant temperature of 32 ℃ for 2-3d, separating and purifying.

The bacterial colony and the thallus characteristics of the bacillus strain ZM96 are as follows: the bacterial colony can grow well on an LB culture medium, is cultured for 48 hours at 28 ℃, is pink, opaque, moist, viscous and glossy, has longer culture time and drier surface, and has wrinkles and central bulges after being cultured for 2 to 3 days, and the single bacterial colony is circular; the thallus is in the shape of short rod and is gram-positive.

The Bacillus strain ZM96 is subjected to 16S rDNA gene amplification and sequencing analysis, and the result shows that the strain ZM96 has homology of 99.86% with Bacillus swezeyi (NR _ 157608.1) and is far away from other Bacillus.

The bacillus ZM96 has the capability of dissolving phosphorus and producing siderophores and has strong growth promoting capability, and the application of the bacillus ZM96 bacterial liquid in promoting plant growth is provided. Preferably, under salt stress, healthy growth of rape seedlings is promoted.

When the bacillus ZM96 bacterial liquid is specifically applied, the root of a plant under salt stress is irrigated by the bacillus ZM96 bacterial liquid. The bacillus ZM96 bacterial liquid is a bacterial suspension, and the preparation method is preferably as follows: the bacillus strain ZM96 is cultured in liquid, preferably LB liquid culture medium, in a shaker at 28 ℃ and 120rpm for 48-96 h to obtain fermentation liquor as a thallus suspension, and more preferably, the fermentation liquor is diluted to 10 ℃ by sterile distilled water ⁸ cfu·mL ^-1 The suspension of the cells was prepared.

The invention further provides application of the bacillus ZM96 bacterial liquid in prevention and control of sclerotinia rot of colza and black shank of colza. Preferably, the bacillus ZM96 bacterial liquid has strong bacteriostatic ability on sclerotinia rot of rape, and sclerotinia sclerotiorum is completely inhibited from growing on an antagonistic flat plate; the bacteriostatic activity on rape black shank is also strong, and the bacteriostatic rate is 70.76-88.36%.

Example 1: isolation of Bacillus Strain ZM96

Preparing 10 salinized soil collected from the saline-alkali desert of northern Shaanxi in China ^-1 -10 ^-7 Concentration gradient soil suspension. Take 200. Mu.l of the above 10 in a sterile bench ^-5 、10 ^-6 、10 ^-7 And (3) coating the soil suspension on a beef extract peptone plate and an Ashby culture medium, culturing for 2d at a constant temperature of 32 ℃, respectively selecting single colonies, streaking the single colonies onto a plate of an LB solid culture medium, and performing inverted culture for 2d at 32 ℃. Selecting single colony, streaking, transferring to LB culture medium test tube slant, culturing at 32 deg.C for 2d, growing with thallus Porphyrae, and storing in 4 deg.C refrigerator. And screening antagonistic bacteria for the next step.

The beef extract peptone culture medium formula: 3g of beef extract, 10g of peptone, 5g of NaCl, 18g of agar and distilled water, wherein the volume is determined to be 1000mL, and the beef extract is sterilized by high-pressure steam at 121 ℃ for 20min.

The formula of the Ashby culture medium is as follows: KH (natural Kill) ₂ PO ₄ 0.2g，MgSO ₄ 0.2g，NaCl 0.2g，CaCO ₄ 5g, mannitol 10g, caSO ₄ 0.1g, 15g of agar and distilled water to 1000mL, the pH is 7.0, and the mixture is sterilized by steam at 121 ℃ for 20min.

LB solid medium formula: 10g of NaCl, 10g of tryptone, 5g of yeast extract powder, 18g of agar powder and 1000mL of distilled water, wherein the pH value is 7.0-7.5.

Example 2: identification of Bacillus Strain ZM96

(1) Morphological identification

When cultured on LB medium at 28 ℃ for 48h, the colonies were opaque, moist, sticky, shiny, and the surface of the colonies was drier the longer the culture time, wrinkles and central protrusions appeared for 4 days, and the single colonies were round and gram-positive (FIG. 1).

(2) 16S rDNA sequence analysis

The purified strain ZM96 is inoculated in LB slant culture medium, and is placed at the constant temperature of 32 ℃ for 2d, and cell culture is collected under the aseptic environment.

The genome DNA of the strain ZM96 is extracted by adopting an Ezup column type bacteria genome DNA extraction kit, and the extracted DNA is stored at the temperature of minus 20 ℃ for later use.

Bacterial 16S rDNA universal primer sequences were used:

27F(5’-AGAGTTTGATCCTGGCTCAG-3’)

1492R(5’-GGTTACCTTGTTACGACTT-3’)；

PCR amplification was performed using the bacterial genomic DNA as a template.

And detecting the purity and the size of the amplified fragment by electrophoresis, and recovering the target fragment by using the gel.

The recovered product was sent to Shanghai Bioengineering Co., ltd for bidirectional sequencing, and Mega 4.0 was used to construct a NJ phylogenetic tree of 16S rDNA gene, as shown in FIG. 2. According to the phylogenetic relationship analysis of 16S rDNA, the strain ZM96 is determined to be Bacillus (Bacillus swezeyi) bacteria.

Example 3: bacillus strain ZM96 growth promoting activity function screening

(1) Activity function determination of bacillus strain ZM96 phosphorus-dissolving and siderophore

The strain is respectively inoculated in a phosphate solubilizing culture medium (organophosphorus PKO) and a CAS detection culture medium by a streaking method to identify the phosphorus solubilizing capacity and the siderophore producing capacity, and the culture temperature is as follows: at 28 ℃.

The formulation of each medium was as follows:

phosphate solubilizing medium: 10.0g of glucose and 10.0g of Ca ₃ (PO ₄ ) ₂ 5g，(NH ₄ ) ₂ SO ₄ 0.5g，NaCl 0.2g，KCl 0.2g，MgSO ₄ ·7H ₂ O 0.3g，MnSO ₄ ·H ₂ O 0.03g，FeSO ₄ ·7H ₂ O0.03g, adjusting pH to 6.8-7.0, and sterilizing at 121 deg.C for 20min.

CAS assay medium: 2.645mg of ferric chloride hexahydrate, 72.9mg of hexadecyl trimethyl ammonium bromide, 900mg of agar powder, 60.5mg of chrome azure, 1213.5mg of disodium hydrogen phosphate tridecylate, 295.25mg of sodium dihydrogen phosphate dihydrate, 125mg of ammonium chloride, 1000mL of deionized water and pH7.0.

As a result, as shown in FIG. 3, the strain ZM96 can grow on the phosphate solubilizing medium and produce a transparent phosphate solubilizing band, indicating that the strain has the phosphate solubilizing ability; on the CAS detection medium plate, a transparent ring appears around the colony, which indicates that the strain can produce iron carrier chelated iron ions, so that the medium becomes transparent and the strain has the capacity of producing the iron carrier. Therefore, the strain has certain growth promoting characteristics, is a potential excellent strain resource for preparing the microbial fertilizer, and has good application and development prospects.

Example 3: bacillus strain ZM96 salt tolerance growth promotion test

(1) Sterilization of seeds

Selecting full and uniform rape seeds, soaking and disinfecting the seeds for 5min by using a 5% NaClO solution, fully shaking the seeds in the soaking period, fully soaking the seeds in liquid, and washing the seeds for 5 times by using sterile water.

(2) And soaking the disinfected seeds for 24 hours at room temperature, placing the seeds on a culture dish paved with wet gauze, and accelerating germination for 2 days at the temperature of 27 ℃. Selecting seeds with consistent growth vigor, sowing 8 seeds in each plastic pot (water is respectively mixed with nutrient soil till water can be wrung out of the soil), watering periodically until the seedlings grow to four leaves, reserving 4 seedlings with consistent growth vigor in each pot, beginning to add salt (160 mmol/L NaCl), slowing seedling for 3 days, irrigating roots with bacterial liquid, watering 3 pots in each of an experimental group and a control group with equal amount of salt water, and culturing in a constant-temperature illumination incubator with the temperature of 18-20 ℃, the light-dark period of 16h/8h and the illumination intensity of 18000 Lux. Observing the growth vigor of the rape, and measuring the biomass (indexes such as root length, stem length, plant height, chlorophyll content and the like) of the rape in 40 days.

Preparing bacterial liquid: the thalli is inoculated into an LB liquid culture medium and cultured in a shaking table at the temperature of 28 ℃ and the rpm of 120 for 48 to 96 hours to obtain fermentation liquor as thalli suspension. Pouring the bacterial liquid into a centrifuge tube, centrifuging at 6000rpm for 2min, collecting thallus, pouring out the supernatant, adding saline water to the original volume, pouring the mixture to the roots of rape seedlings after fully shaking up, pouring 20mL of bacterial liquid in each pot, and pouring the same amount of saline water in a control group. Watering every 7 days, and simultaneously supplementing equal amount of saline water or water to the two groups of seedlings according to soil moisture and rape growth conditions.

The results show that the rape plants to which the bacillus ZM96 bacterial liquid is applied have vigorous growth, thick leaves and developed root systems compared with the control group (without the bacterial liquid) under the stress of 160mmol/L NaCl (FIGS. 4 and 5). Compared with the control group, the root length of the rape treated by root irrigation with the bacterial liquid is increased by 42.35%, the plant height is increased by 27.24%, and the fresh weight is increased by 105.61%, which indicates that the inoculated bacillus ZM96 can relieve salt stress and promote the growth of the rape (fig. 6, 7 and 8).

Example 4: inhibition of bacillus ZM96 on rape pathogenic bacteria

The bacillus strain ZM96 is reactivated on an LB plate, and respectively inoculated into an LB liquid culture medium containing 5mL, and cultured in a shaker at 28 ℃ and 120rpm for 48h to obtain bacterial suspension. Activating rape pathogenic bacteria on a PDA (potato) plate (200 g, glucose 20g and agar 20g, constant volume is 1000mL, pH is natural), culturing at 28 ℃ for 7d, selecting a pathogenic fungus cake about 5mm, inoculating the pathogenic fungus cake to the center of the PDA plate, inoculating 5uL of antagonistic bacteria suspension at a position 1.5cm away from the pathogenic bacteria by taking the center of the plate as a round point, and taking the plate without inoculating bacteria as a blank control. Each group of experiments is repeated for 3 times, and the experiments are cultured at constant temperature of 28 ℃, observed and recorded with antagonism. When the colony of the control group grows over the whole container, the radius of the colony is measured by a cross method, and the bacteriostasis rate of the colony is calculated.

Bacteriostatic ratio% = [ control pathogenic bacteria colony radius (cm) — treatment group pathogenic bacteria colony radius (cm) ]/control pathogenic bacteria colony radius (cm) × 100%

The test results are shown in table 1:

TABLE 1 inhibitory Effect of Bacillus ZM96 on pathogenic bacteria tested

Multiple test results show that the bacillus ZM96 inoculated on the PDA culture medium plate has obvious inhibition effect on rape pathogenic bacteria (figure 9), particularly has extremely strong inhibition capability on sclerotinia rot, the experimental group antagonizes the sclerotinia rot of rape in the plate to be almost not grown, the control group sclerotinia rot is fully distributed on the plate, and the inhibition rate reaches 97.65%; secondly, the black shank inhibitor has strong inhibition capacity on the black shank of rape, the growth radius of the black shank pathogenic bacteria in the antagonistic plate is 0.39cm, and the inhibition rate reaches 88.36 percent.

In conclusion, the bacillus ZM96 has the growth promoting capabilities of dissolving phosphorus, producing siderophores and the like, has strong colonization capability, and promotes the healthy growth of rape crops under the salt stress; the strain shows strong bacteriostatic activity on sclerotinia rot of colza and black shank of colza, and can be used for preventing and controlling plant soil-borne diseases; the effects of improving the crop yield and improving the quality are very obvious, resources are provided for developing novel biological bacterial fertilizers, and the biological bacterial fertilizers are perfect substitutes for chemical pesticides; the salt stress can be relieved, the soil structure of the saline-alkali soil can be effectively improved, the fertility is enhanced, and the method has important significance for soil remediation, maintenance of the balance of an ecological system, sustainable development of agricultural green food and human health; is a safe, green and efficient multifunctional disease-resistant growth-promoting salt-tolerant strain and has wider industrial application prospect.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A salt-tolerant growth-promoting and disease-resistant Bacillus of plants is characterized in that the Bacillus belongs to Bacillus (Bacillus swezeyi), is preserved in the China general microbiological culture Collection center (CGMCC), and has a preservation number of CGMCC No.25411.

2. The plant salt tolerant growth-promoting and disease-resistant bacillus according to claim 1, wherein said bacillus has an antagonistic effect on salt stress.

3. The plant salt-tolerant growth-promoting and disease-resistant bacillus of claim 1, wherein said bacillus has antifungal activity.

4. The plant salt-tolerant growth-promoting and disease-resistant Bacillus according to claim 3, wherein the fungus is a Heptosphaeria maculans or Sclerotinia sclerotiorum.

5. The plant of claim 1, wherein said bacillus has the ability to solubilize phosphorus.

6. The plant salt-tolerant growth-promoting and disease-resistant bacillus of claim 1, wherein said bacillus has siderophore production capability.

7. The plant bacillus for salt tolerance, growth promotion and disease resistance of the plants according to claim 1, wherein the bacillus is separated from salinized soil in Shanxi saline desert of China.

8. Use of the salt-tolerant growth-promoting and disease-resistant bacillus of any one of claims 1-7 in the preparation of an antimicrobial agent for crops.

9. Use according to claim 8, wherein the crop plant is oilseed rape.