CN113388549B - Bacillus solitarius and application thereof - Google Patents

Abstract

A Bacillus sonoralis desert and application thereof belong to the technical field of microorganisms. The invention provides a Bacillus sonoralis PCYB09 with the preservation number of CGMCC No. 21571; on the other hand, the application of Bacillus sonorensis (Bacillus sonorensis) PCYB09 is provided. The strain shows strong indoor antagonism and field biocontrol effects on various rice and hickory diseases, and also shows strong plant resistance induction capability and strong ultraviolet resistance, which is discovered by innovative academia and also makes the strain have higher application value.

Description

A kind of Sonora desert bacillus and application thereof

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to a Sonora desert bacillus and applications thereof.

Background technique

Bacillus spp. is a class of Gram-positive bacteria that can produce spores and is widely distributed in nature. Most Bacillus are not pathogenic to animals and plants, and many of them have the activity of inhibiting animal and plant pathogenic bacteria, and can produce a variety of enzymes, antibiotics and other biologically active products, so they are widely used in industry and agriculture. As early as 1879, Friedrich-Bayer developed a Bacillus bacterium (Bacillus subtilis) into a bacterial fertilizer product, which can improve plant nutrition, promote crop health, and increase yields by up to 40%. Since then, new strains of Bacillus have been continuously developed and utilized. Since the middle of the last century, Bacillus fungicides developed in the United States have been registered on at least 7 crops, with an application area of more than 2 million hectares, ushering in the era of large-scale application of biological pesticides. Due to the protection of spores, the vitality of Bacillus is relatively tenacious, and it is ubiquitous in the environment. A large number of strains with biocontrol potential have been isolated. There are nearly 300 biocontrol potential Bacillus strains reported or commercially used in my country.

Although Bacillus products are the most important biological pesticides, there are many problems in their development, which greatly hinder their development. First of all, the homogeneity is serious. The strains used in most varieties are Bacillus subtilis, and the effect is similar. Therefore, it is urgent to develop other types of Bacillus in production. Furthermore, the existing biocontrol bacteria have a single mechanism of action. Although Bacillus as a typical biocontrol bacteria has multiple biocontrol mechanisms, most of them are mainly based on competition and the production of bacteriostatic substances. A report of Bacillus strains that induce resistance.

As a live microbial fungicide, survival and colonization in the environment is a prerequisite for Bacillus subtilis to achieve its function. The main producing areas of both pecans and rice are in subtropical or tropical regions. Strong sunlight and hot temperatures are the main climatic characteristics of these regions, and the ultraviolet rays in sunlight are the most unfavorable for their colonization. Ultraviolet light will damage the DNA of Bacillus, denature the bacterial protein, and make the strain inactive. For biocontrol bacteria, UV resistance is undoubtedly a favorable trait, but as a eubacteria, its UV resistance is far from actinomycetes and other types of microorganisms such as fungi. The screening of UV-resistant strains and their use in biological control is a valuable and innovative achievement from both an academic and a commercial point of view.

SUMMARY OF THE INVENTION

Aiming at the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for Bacillus sonora and its application.

The present invention is specifically realized through the following technical solutions:

On the one hand, the present invention provides Bacillus sonora desert Bacillus PCYB09, the preservation number is CGMCC No. 21571, the preservation time is December 30, 2020, and the proposed classification name is Bacillus sonorensis (Bacillus sonorensis), and the preservation unit is The General Microbiology Center of China Microbial Culture Collection Management Committee, referred to as CGMCC, is located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.

Another aspect of the present invention provides the application of Bacillus sonora deserti PCYB09 in preventing and controlling plant diseases.

Further, the plant diseases include: rice blast, wheat blast, wheat scab, rice sheath blight, rice smut, rice bakanae, camellia oleifera bud disease, pecan black spot, husk Hickory anthracnose, pecan black spot and bamboo dieback.

Another aspect of the present invention provides the use of Bacillus sonora deserti PCYB09 in inducing plant resistance.

Another aspect of the present invention provides a plant fungicide, which is prepared from Bacillus sonora desertification PCYB09.

Further, the plant fungicide includes at least one of the bacterial cells and metabolites of Bacillus sonora desertification PCYB09.

Another aspect of the present invention provides the application of a plant fungicide in preventing and controlling plant diseases.

Further, the plant diseases include: rice blast, wheat blast, wheat scab, rice sheath blight, rice smut, rice bakanae, camellia oleifera bud disease, pecan black spot, husk Hickory anthracnose, pecan black spot and bamboo dieback.

In the present invention, a Bacillus strain is isolated from the leaves of pecan, and the strain is identified as Bacillus sonorensis by a reliable multiple identification method. Indoor antagonism and field biocontrol effects, the bacteria also showed strong ability to induce plant resistance and strong UV resistance, which is an innovative academic discovery and makes it more valuable for application.

Description of drawings

Fig. 1 is the thalline morphology of PCYB09 strain;

Fig. 2 is the phylogenetic tree of antagonistic bacteria PCYB09 and reference strain constructed by 16SrDNA, rpoB and secY gene sequences;

Figure 3 shows the effect of PCYB09 bacterial solution treatment on the expression of rice defense-related genes;

Figure 4 shows the effect of PCYB09 strain on rice defense-related enzyme activities;

Figure 5 shows the UV resistance of PCYB09 strain and its control strains.

Detailed ways

The present invention will be further described below in conjunction with the embodiments.

Example 1: Isolation and identification of Bacillus sonora desert strain PCYB09

Pick pecan leaves from Lin'an area, take 3-5 pieces of pecan leaves, wash under running water, absorb the residual water on the surface with toilet paper, soak in 70% alcohol solution for 30S, wash 3 times with sterile water, and use a homogenizer at low speed Homogenize, take 100 μl of homogenate and spread it on 9cmNA (polypeptone 5g, yeast powder 1g, beef extract 3g, sucrose 15g, NaOH adjust the pH value to 7.0, add 20g agar powder, sterilize at 121 ℃ for 20 minutes) on the plate , placed in a 25°C incubator until colonies grow, and single colonies were selected and transferred to a new 4cm NA plate. Taking American pecan and pecan anthracnose (Caryaillinoensis) as the target, the antagonistic strains were screened by the confrontation culture method, and the strain with the strongest antagonistic effect was selected and named as the strain PCYB09.

Drop sterilized water on the clean coverslip, dip a small amount of PCYB09 moss, keep stirring in sterilized water, spread it evenly on the coverslip, dry at 40°C, and use 0.1% (w/v) After staining with crystal violet solution and washing with deionized water, the morphology of the bacteria was observed with a laser confocal microscope (Zeiss, LSM700). 1 shown. Gram stain was positive.

Physiological and biochemical tests show that the strain can utilize most carbon sources such as glucose, raffinose, mannose, gentiobiose, lactose, aspartic acid, fructose, and alanine, but cannot utilize 3-methyl-D - Glucose, D-galactose, L-lactic acid, propionic acid, saccharic acid, D-malic acid, mucic acid, L-serine, L-malic acid, D-glucose carboxylic acid, creatinine, pectin, Tween 40 , D-maltose, D-methyl lactate, d-hydroxybutyric acid, D-arabitol and acetoacetic acid and other carbon sources (Table 1).

Table 1 Available carbon sources of PCYB09

Using MIDI's fatty acid analysis system, the fatty acid composition of the strain was analyzed (the results are shown in Table 2). From the perspective of fatty acid composition, the content of 15:0 anteiso accounted for most of the fatty acids, up to 42.71%; followed by The proportion of 15:0 ISO also reached 13.9%, followed by 9.84% of 17:0 anteiso and 7.58% of 17:0 ISO. From the perspective of fatty acid composition characteristics, the strain is Bacillus.

Table 2 PCYB09 fatty acid composition

fatty acid type proportion 13:0 iso 0.22 14:0 iso 1.51 14:00 1.19 15:0 iso 13.9 15:0 anteiso 42.17 15:00 ---- 16:0 iso 3.25 16:1 w11c 1.24 16:00 17.7 17:1 isow10c 0.53 17:0 iso 7.58 17:0 anteiso 9.84 18:00 0.87

The temperature tolerance test found that it can withstand a high temperature of up to 55 ℃. It has poor salt tolerance and cannot grow at concentrations above 5% sodium chloride. Amylase and protease activity can be produced.

The DNA of the strain was extracted and amplified to obtain its 16SrDNA, rpoB and secY gene sequences. The corresponding genes of each similar species were used as reference genes, and B. cereus was used as the OUTGROUP for phylogenetic tree analysis. Mega6.0 software was used for phylogenetic tree analysis. , using the maximum likelihood method (Maximum Likelihood, ML) calculation, with the Bootstrap method (Bootstrap) 1000 times repeated calculation of branch support rate, making a phylogenetic tree. The results showed that the related genes of PCYB09 and B.sonorensis had the highest similarity (Figure 2).

Therefore, combining the results of morphological, physiological and biochemical and three-gene phylogenetic tree analysis, PCYB09 strain was identified as B. sonorensis.

This strain PCYB09 is preserved, its preservation number is CGMCC No.21571, and the preservation time is December 30, 2020. It is proposed to be classified and named as Bacillus sonorensis, and the preservation unit is Chinese microbial strains. The General Microbiology Center of the Preservation and Management Committee, referred to as CGMCC, is located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Example 2: Fermentation medium, fermentation method and inoculum preparation method of Bacillus sonora desert strain PCYB09

In order to improve the fermentation efficiency while reducing the cost, the present invention creates and improves the fermentation medium formula for the strain: the formula composition of each liter of the fermentation medium is: 10-20 grams of sucrose, 1-2 grams of yeast powder, beef extract 3-5 g, peptone 5-10 g, potassium phosphate 1 g, magnesium chloride 0.5 g, calcium chloride 0.1 g, ferric chloride 0.01 g, sodium chloride 0.5 g, pH adjusted to 5.5-6.5.

The specific fermentation method is as follows: PCYB09 strain is inoculated with NB liquid medium (polypeptone 5g, yeast powder 1g, beef extract 3g, sucrose 15g) and shake cultured at 37°C for 6-8h, which is the seed liquid. The proportion is added to the fermentation medium, and after shaking at 26-28 °C for 36 hours, it is allowed to stand at the same temperature until the fermentation is completed.

Using this fermentation method, the logarithmic growth phase can be entered in 15-18 hours, and the fermentation can generally be completed in 48-60 hours. After the fermentation, the bacterial content can reach 1-5×10 ¹⁰ cfu/ml.

Example 3: Antagonistic ability of Bacillus sonora deserti strain PCYB09 against commonly used pathogenic bacteria

The inhibitory effect of PCYB09 strain on a variety of pathogenic bacteria harmful to pecan, bamboo, camellia oleifera, rice and other forest trees and fields was tested by the confrontation culture method. The specific method is: inoculate PCYB09 strain in NB medium at 37°C and shake to the logarithmic growth phase, take 3 μL of bacterial solution and place it on the PDA plate 1.5cm away from the midpoint, blow dry under the ultra-clean workbench, and place the midpoint of the plate along a straight line on the PDA plate. Place a 0.5cm diameter mycelium block 1.5cm on the other side of the midpoint, press lightly with an inoculating needle to make the hyphal surface close to the PDA plate, and cultivate it in the dark at 28°C until the colony radius of the non-opposing end of the pathogen exceeds 3cm. Measure the growth length of the pathogenic bacteria at the opposite end and the distance between the inoculation points in the direction of the biocontrol bacteria, and calculate the inhibition rate according to the following formula.

The test results are shown in Table 3:

Table 3 Inhibitory effect of PCYB09 on common plant pathogens

Pathogens cause disease Inhibition rate(%) Magnaportheoryzae Rice blast, wheat blast 75.7-83.6 Fusariumgraminearum Wheat scab 78.9-87.6 Rhizoctoniasolani Rice sheath blight 85.3-92.1 Ustilaginoideaoryzae Rice smut 80-89.8 Fusariumfujikuroi rice bakanae disease 83.9-88.1 Exobasidiumgracile Camellia bud disease 76.1-86.1 Botryosphaeriadothidea Shell Pecan Black Spot 78.8-82.8 Colletotrichumnymphaeae Pecan anthracnose 83.6-84.6 Neopestalotiopsisclavispora Shell Pecan Black Spot 76.5-88.1 Ceratosphaeriahyllostachydis Phyllostachys pubescens 66.4-79.4

It can be seen from Table 3 that the strain PCYB09 has a strong antagonistic effect on a variety of pathogens, among which the antagonistic effect on rice sheath blight is the most obvious, with an inhibition rate of 85.3-92.%; Low, but the highest can reach 79.4, indicating that the strain has a broad antibacterial spectrum, which will be beneficial to its promotion and application.

Example 4: Field control effect of Bacillus sonora deserti strain PCYB09 on rice sheath blight

The experiment is arranged in the transgenic field of the China Rice Research Institute Experimental Base in Fuyang District, Hangzhou, Zhejiang Province. The planting system is single-season mid-late rice all year round. The rice tested is the conventional japonica rice variety "Xiushui 09". , repeated 3 times, and arranged according to the random block method. Control agents were commercially available 100 billion spores/gram of Bacillus subtilis and 24% Thiefamide SC (FMC).

Table 4. Design of field control experiment of rice sheath blight

14 days after application, according to the provisions of "GB/T 17980.20-2000 Field Efficacy Test Guidelines (1) Fungicides for the Control of Rice Sheath Blight", the incidence of rice sheath blight was investigated and the control effect was calculated. The results are shown in Table 4.

Table 5 Control effect of PCYB09 fermentation broth on rice sheath blight

From the results in Table 5, it can be seen that the control effect of PCYB09 at the working concentration of 1×10 ⁹ cfu mL ^-1 reached 71.40%, second only to the chemical fungicide control product thifuramide, and significantly higher than that of Bacillus subtilis commercially available. The control effect of this strain reached 68.31% at the working concentration of 1×10 ⁸ cfu mL ^-1 , which was also significantly higher than that of the Bacillus subtilis control product at the same concentration. Therefore, it shows that the PCYB09 strain has excellent control of rice sheath blight Effect.

Example 5: Field control effect of Bacillus sonora deserti strain PCYB09 on rice blast

The test is arranged in the rice blast test base of China Rice Research Institute in Lin'an, Zhejiang Province. The planting system is single-season mid-late rice all year round. The rice tested is the conventional japonica rice variety "Nanjing 46". m, repeated 3 times, arranged according to the random block method. The control agents were commercially available 100 billion spores/g Bacillus subtilis wettable powder and 75% tricyclazole WP (Dow AgroSciences). The experimental design is shown in Table 6. At the initial stage of rice blast onset (rice tillering period), each treatment agent was sprayed according to the application rate of 675 L ha ^-1 .

Table 6 The experimental design of rice blast field control

12 days after the treatment, the investigation refers to the provisions of "GB/T 17980.20-2000 Field Efficacy Test Guidelines (1) Fungicides for the Control of Rice Leaf Diseases" to investigate the incidence of rice leaf blast in each plot, and calculate the relevant control effects. The results are shown in Table 7:

Table 7 Control effect of PCYB09 fermentation broth on rice leaf blast

As shown in Table 7, the control efficacy of PCYB09 against rice blast at working concentrations of 1×10 ⁸ cfu mL ^-1 and 1×10 ⁹ cfu mL ^-1 reached 70.1 and 68.73%, respectively, and the control chemical fungicide was 69.21%. % control effect is almost the same, significantly higher than 61.69% of the commercial Bacillus subtilis inoculum of the control agent. At the low concentration of 1×10 ⁷ cfu mL ^-1 , the control effect of this strain could still reach 57.76%. The above test results show that PCYB09 has excellent control effect on rice blast.

Example 6: Control effect of Bacillus sonora deserti strain PCYB09 on pecan black spot

The experimental design is shown in Table 8. For each treatment, 3 pecan trees at the initial stage of the disease were randomly selected and sprayed according to the experimental design. After the disease was stable, the incidence of black spot disease was investigated and the control effect was calculated according to the incidence.

Table 8 The experimental design and control effect of Bacillus sonora deserti strain PCYB09 in the prevention and control of black spot

It can be seen from Table 8 that PCYB09 can show excellent control effect of pecan black spot disease at the working concentration of 1×10 ⁸ cfu mL ^-1 or 1×10 ⁹ cfu mL ^-1 . The control effects at the two concentrations were comparable, reaching 63.45 and 64.71%, respectively, which were significantly better than those of commercially available Bacillus subtilis, and their control effects were between prochloraz and thiophanate-methyl. Therefore, the above results show that the fermentation product of PCYB09 strain has excellent control effect on pecan black spot.

Example 7: Control effect on other diseases

According to the results of indoor bioassays, the fermented products of this strain have been evaluated for field control effects against various diseases. Moso bamboo dieback and other diseases have obvious control effects, some of which are close to the control effects of chemical pesticides. Considering the advantages of low cost of biological pesticides and environmental protection, this strain has great application potential in the prevention and control of various diseases.

Example 8: Induction of plant resistance by PCYB09 strain

The seeds of rice Zhongzao 39 were cultivated to about the 4-leaf stage, and the PCYB09 bacterial solution with a concentration of 1×10 ⁸ cfu mL ^-1 was evenly sprayed on the surface of the rice seedlings, and leaf samples were taken 24 and 48 hours after spraying, respectively. , placed in liquid nitrogen for quick freezing, and stored at -80°C.

At the same time, the sample RNA was extracted, and after reverse transcription, the expression levels of rice defense-related genes such as PR1a, PR1b, PR3, PR4, PR5, PR10 and NH1 were investigated. The results are shown in Figure 3. It was found that the expression of most defense-related genes increased significantly except for NH1, and the expression of PR4 increased more significantly. This result indicated that PCYB09 strain treatment could induce plant disease resistance by increasing the expression of defense-related genes.

After the samples were collected, they were homogenized in an ice bath, and polyphenol oxidase (PPO), peroxidase (POD), and superoxide dismutase (SOD) were analyzed using an enzyme activity kit (Suzhou Keming Biotechnology Co., Ltd., China). ) and catalase (CAT) activities, and the operation method strictly refers to the instructions. Based on the enzyme activities per unit mass of the treated and control samples, the relative enzyme activities were calculated according to the following formula, as shown in Figure 4.

Polyphenol oxidase (PPO), peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) are the most important defense-related enzymes in plants. Polyphenol oxidase (PPO) and peroxidase (POD) were positively correlated with plant resistance, while superoxide dismutase (SOD) and catalase (CAT) were negatively correlated with plant resistance. The PCYB09 strain can significantly increase the enzymatic activities of polyphenol oxidase (PPO) and peroxidase (POD), while inhibiting superoxide dismutase (SOD) and catalase (CAT) after 24h and 48h treatment of rice. , and the effect of 48h was significantly stronger than that of 24h, indicating that PCYB09 strain treated rice could improve the resistance of rice to pathogens by regulating the activities of defense-related enzymes.

From the results of the above two experiments, PCYB09 strain can induce and improve plant disease resistance by increasing the expression of defense-related genes and regulating the activities of defense-related enzymes.

Example 9: UV resistance of PCYB09 strain

Put 30ml PCYB09 strain and its control Bacillus subtilis strain fermentation broth into a 9cm petri dish, put a magnetic rotor, keep stirring, and irradiate it with an ultraviolet lamp for 1, 2, 4, 6 and 10s, respectively, and dilute it. When a certain multiple is reached, the number of colonies is counted by a colony counter, and the survival rate (%) of each strain in different time periods is calculated by taking the bacterial liquid without UV irradiation as a control. The results are shown in Figure 5. The survival rate of PCYB09 strain was significantly higher than that of the control strain in each time period. After 10s of UV irradiation, Bacillus subtilis had been completely inactivated, while PCYB09 strain had nearly 20% survival. Therefore, the PCYB09 strain was more resistant to UV light than the control strain Bacillus subtilis.

Claims (3)

1. Bacillus solitarius (Bacillus solitarius)Bacillus sonorensis) The preservation number of the PCYB09 is CGMCC No. 21571.

2. The use of bacillus sonolatus PCYB09 as claimed in claim 1 in the control of plant diseases including: rice blast, wheat scab, rice sheath blight, rice false smut, rice bakanae disease, camellia oleifera leaf bud disease, carya illinoensis anthracnose, carya illinoensis black spot and phyllostachys pubescens blight.

3. The use of Bacillus sonolatus PCYB09 as claimed in claim 1 for inducing plant resistance.

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