CN113388549A

CN113388549A - Bacillus solitarius and application thereof

Info

Publication number: CN113388549A
Application number: CN202110791190.3A
Authority: CN
Inventors: 张亚波; 张威; 滕莹; 王迪; 高岩; 李志红; 舒金平; 王浩杰
Original assignee: Research Institute of Subtropical Forestry of Chinese Academy of Forestry
Current assignee: Research Institute of Subtropical Forestry of Chinese Academy of Forestry
Priority date: 2021-07-13
Filing date: 2021-07-13
Publication date: 2021-09-14
Anticipated expiration: 2041-07-13
Also published as: CN113388549B

Abstract

A Bacillus sonoralis desert and application thereof belong to the technical field of microorganisms. The invention provides a Bacillus sonorensis (Bacillus sonorensis) PCYB09 with the preservation number of CGMCC No. 21571; on the other hand, the application of the 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

Bacillus solitarius and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to Bacillus solitarius and application thereof.

Background

Bacillus spp is a group of gram-positive bacteria that produce spores and is widely distributed in nature. Most of the bacillus is not pathogenic to animals and plants, and many varieties of the bacillus also have the activity of inhibiting pathogenic bacteria of the animals and plants and can produce various bioactive products such as enzymes, antibiotics and the like, so the bacillus is widely applied to industry and agriculture. In 1879, a strain of bacillus (bacillus subtilis) was developed by friedrichs-bayer corporation into bacterial manure products, which can improve plant nutrition, promote crop health, and increase yield by up to 40%. Since then, new strains of bacillus have been developed and utilized. From the middle of the last century, bacillus bactericides developed in the united states have been registered on at least 7 crops, and the application area exceeds 200 million hectares, opening the era of large-scale application of biopesticides. Because of spore protection, the vitality of the bacillus is relatively tenacious and generally exists in the environment, a large number of strains with biocontrol potential are separated, and nearly 300 strains of bacillus with biocontrol potential are reported or commercially applied in China.

Although bacillus products are the most important biopesticide varieties, the development of the bacillus products has a plurality of problems, and the development of the bacillus products is greatly hindered. Firstly, the homogenization is serious, most strains used by the strains are bacillus subtilis, and the action effect is greatly the same and slightly different, so that other types of bacillus are urgently developed in production. The existing biocontrol bacteria have a single action mechanism, although bacillus has a multiple biocontrol mechanism as a typical biocontrol bacterium, most of the biocontrol bacteria mainly play a competitive role and produce bacteriostatic substances, and few bacillus strains with induced resistance are reported.

As a living microbial bactericide, survival and colonization in the environment are prerequisites for the bacillus subtilis to realize the function. The main production areas of hickory and rice are in subtropical or tropical areas, strong sunlight and hot air are the main climatic features of the areas, and the ultraviolet rays in the sunlight are most unfavorable for colonization. Ultraviolet light destroys the DNA of bacillus, denatures the mycoprotein and makes the strain lose activity. Ultraviolet resistance is undoubtedly an advantageous trait for fungi control, but bacillus, as a true bacterium, is far from ultraviolet resistance and actinomycetes, and other kinds of microorganisms such as fungi, and ultraviolet resistant strains of bacillus are rarely reported, and screening of ultraviolet resistant strains and use thereof for biological control are valuable innovative results from both academic and commercial perspectives.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to design and provide a technical scheme of Bacillus sonolavae and application thereof.

The invention is realized by the following technical scheme:

on the one hand, the invention provides the Bacillus somnolia desert PCYB09 with the preservation number of CGMCC No.21571 and the preservation time of 2020, 12 months and 30 days, which is suggested to be classified and named as Bacillus sonoreensis, the preservation unit is China general microbiological culture Collection center (CGMCC) with the short name, and the address is the institute of microbiology of China academy of sciences No. 3, North West Lu No. 1 of the sunward area, Beijing.

On the other hand, the invention provides the application of the bacillus sonoralis PCYB09 in the prevention and control of plant diseases.

Further, the plant diseases include: rice blast, wheat scab, rice sheath blight, rice false smut, rice bakanae disease, camellia oleifera bud disease, carya illinoensis black spot disease, carya illinoensis anthracnose disease, carya illinoensis black spot disease and phyllostachys pubescens blight.

The invention also provides application of the Bacillus sonoralis PCYB09 in inducing plant resistance.

In another aspect, the invention provides a plant bactericide, which is prepared from Bacillus sonoralis PCYB 09.

Further, the plant bactericide comprises at least one of thallus and metabolite of the bacillus sonolatus PCYB 09.

On the other hand, the invention provides the application of the plant bactericide in preventing and controlling plant diseases.

The Bacillus is obtained by separating the leaf part of the hickory nut, the Bacillus is identified as the Bacillus Sonoralis (Bacillus sonorensis) by a reliable multiple identification method, the Bacillus shows strong indoor antagonism and field biocontrol action on various rice and hickory nut diseases, and the Bacillus also shows strong induced plant resistance capability and strong ultraviolet resistance, which is an innovative academic discovery and makes the Bacillus have more application value.

Drawings

FIG. 1 shows the cell morphology of the PCYB09 strain;

FIG. 2 shows a phylogenetic tree of antagonistic bacteria PCYB09 constructed from gene sequences of 16SrDNA, rpoB and secY and a reference strain;

FIG. 3 shows the effect of PCYB09 bacterial liquid treatment on rice defense-related gene expression level;

FIG. 4 shows the effect of the PCYB09 strain on rice defense-related enzyme activity;

FIG. 5 shows the resistance of the strain PCYB09 and its control strain to UV light.

Detailed Description

The present invention is further illustrated by the following examples.

Example 1: isolation and identification of Bacillus sonola desert Strain PCYB09

Picking up hickory leaves in the Linan area, taking 3-5 hickory leaves, washing with tap water, drying residual water on the surface of the hickory leaves by toilet paper, soaking the hickory leaves in 70% alcohol solution for 30 seconds, washing for 3 times by using sterile water, homogenizing at low speed by using a homogenizer, coating 100 mu l of homogenate on a 9cmNA (polypeptone 5g, yeast powder 1g, beef extract 3g, sucrose 15g, NaOH to adjust the pH value to 7.0, adding 20 g of agar powder, sterilizing at 121 ℃ for 20 minutes) plate, placing the plate on an incubator at 25 ℃ until bacterial colonies grow out, and selecting a single bacterial colony to transfer to a new 4cm NA plate. Uses pecan and pecan anthracnose bacteria (Caryaillinoensis) as target, screens antagonistic strain by opposite culture method, selects strain with most strong antagonistic action, and is named as strain PCYB 09.

The sterilized water is dropped on a clean cover glass, a small amount of PCYB09 bacterial lawn is dipped, the bacterial lawn is continuously stirred in sterilized water and evenly smeared on the cover glass, the cover glass is dried at 40 ℃, the bacterial lawn is dyed by 0.1 percent (w/v) crystal violet solution and washed by deionized water, the shape of the bacterial lawn is observed by a laser confocal microscope (Chuiss, LSM700), and the PCYB09 bacterial lawn is found to be in a typical rod shape, and the size is 0.9-1.1 multiplied by 1.2-3.8 mu m, as shown in figure 1. Gram staining reaction is positive.

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

TABLE 1 available carbon sources for PCYB09

The fatty acid composition of the strain is obtained by analyzing a fatty acid analysis system of MIDI (results are shown in table 2), and the content of 15: 0 anteiso accounts for the majority of fatty acids and is as high as 42.71% in terms of the fatty acid composition; secondly, the 15: 0 ISO ratio also reached 13.9%, secondly 9.84% of 17: 0 anteiso and 7.58% of 17: 0 ISO, the strain being a Bacillus in view of the fatty acid composition characteristics.

TABLE 2 PCYB09 fatty acid composition

Type of fatty acid	Ratio of occupation of
		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 finds that the material can resist the high temperature of 55 ℃ at most. The salt tolerance is poor and growth is not possible at sodium chloride concentrations above 5%. Amylase and protease activity can be produced.

Extracting strain DNA, amplifying to obtain 16SrDNA, rpoB and secY gene sequences, performing phylogenetic tree analysis by using various similar corresponding genes as reference genes and using Bacillus cereus as OUTGROUP, calculating by using Maximum Likelihood Method (ML) and self-development test method (Bootstrap)1000 times of repeated calculation of branch support rate by adopting Mega6.0 software, and manufacturing a phylogenetic tree. The results showed that the PCYB09 and b.

Thus, the PCYB09 strain was identified as bacillus sonorensis (b.sonorensis) as a result of a combination of morphological, physiobiochemical and three-gene phylogenetic tree analyses.

The strain PCYB09 is preserved, the preservation number is CGMCC No.21571, the preservation time is 30 days 12 months 2020, the strain is suggested to be classified and named as Bacillus Sonorensis, the preservation unit is China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, CGMCC for short, and the address is the institute of microbiology, China academy of sciences, No. 3, Naciwa Xilu, North Cheng Yang, Beijing.

Example 2: fermentation medium and fermentation method of bacillus sonolatus strain PCYB09 and preparation method of microbial inoculum

In order to reduce the cost and improve the fermentation efficiency, the invention creates and improves the formula of the fermentation medium for the strain: the fermentation medium per liter comprises the following components: 10-20 g of cane sugar, 1-2 g of yeast powder, 3-5 g of beef extract, 5-10 g of peptone, 1g of potassium phosphate, 0.5 g of magnesium chloride, 0.1 g of calcium chloride, 0.01 g of ferric chloride and 0.5 g of sodium chloride, and the pH is adjusted to 5.5-6.5.

The specific fermentation method comprises the following steps: inoculating PCYB09 strain into NB liquid culture medium (5 g of polypeptone, 1g of yeast powder, 3g of beef extract and 15g of cane sugar) and shaking for 6-8h at 37 ℃ to obtain seed liquid, adding the seed liquid into a fermentation culture medium according to the proportion of 0.5-1: 1000, shaking for 36h at 26-28 ℃, and standing and culturing at the same temperature until the fermentation is finished.

By using the fermentation method, the fermentation can be started in logarithmic growth phase within 15-18h, the fermentation can be finished within 48-60 h, and the content of the bacteria can reach 1-5 × 10 after the fermentation is finished¹⁰cfu/ml。

Example 3: antagonistic capacity of Bacillus sonolaraensis strain PCYB09 against common pathogenic bacteria

The control effect of the PCYB09 strain on various harmful pathogenic bacteria of hickory, bamboo, tea-oil tree, rice and other forest trees and field diseases is detected by adopting a confrontation culture method. The specific method comprises the following steps: inoculating the PCYB09 strain to NB culture medium, shaking and culturing at 37 ℃ to logarithmic growth phase, taking 3 microliter of bacterial liquid, placing the bacterial liquid at a position 1.5cm away from the midpoint of a PDA (personal digital assistant) plate, drying under an ultra-clean workbench, placing a 0.5 cm-diameter hypha block at a position 1.5cm away from the midpoint of the plate along a straight line, slightly pressing the hypha surface to be tightly attached to the PDA plate by using an inoculating needle, culturing at 28 ℃ in the dark until the radius of the bacterial colony at a non-opposing end of the pathogenic bacteria exceeds 3cm, respectively measuring the growth length of the pathogenic bacteria at the opposing end in the direction of biocontrol bacteria and the distance between the inoculating points, and calculating 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 pathogenic fungi

Pathogenic bacteria	Cause disease	Inhibition ratio (%)
			Magnaportheoryzae	Blast of rice and wheat	75.7-83.6
Fusariumgraminearum	Scab of wheat	78.9-87.6
			Rhizoctoniasolani	Sheath blight of rice	85.3-92.1
Ustilaginoideaoryzae	False smut of rice	80-89.8
			Fusariumfujikuroi	Bakanae disease of rice	83.9-88.1
Exobasidiumgracile	Bud disease of Camellia oleifera	76.1-86.1
			Botryosphaeriadothidea	Black spot of apocarya	78.8-82.8
Colletotrichumnymphaeae	Anthracnose of thin-shelled mountain walnut	83.6-84.6
			Neopestalotiopsisclavispora	Black spot of apocarya	76.5-88.1
Ceratosphaeriahyllostachydis	Blight of moso bamboo	66.4-79.4

As can be seen from Table 3, the strain PCYB09 shows strong antagonism to various pathogenic bacteria, wherein the antagonism to rice sheath blight is the most obvious, and the inhibition rate is 85.3-92%; the inhibition rate of the rhizoctonia solani on the moso bamboo is slightly low, but the highest inhibition rate can also reach 79.4, which indicates that the bacterial strain has a wider antibacterial spectrum, and is beneficial to popularization and application.

Example 4: field control effect of bacillus sonolatus strain PCYB09 on rice sheath blight disease

The test arrangement is carried out in a transgenic field of a test base of a Chinese rice research institute in the Hangzhou area of Zhejiang, the planting system is single-season middle-late rice all the year round, the test rice is a conventional japonica rice variety 'Xiushui 09', the field is divided into cells by ditches, each cell is 25 square meters, the operation is repeated for 3 times, and the test rice is arranged according to a random block grouping method. The control agents were commercially available 1000 billion spores/gram of Bacillus subtilis and 24% thifluzamide SC (Fumeishi).

TABLE 4 test design for field control of rice sheath blight disease

14 days after the application of the pesticide, the disease condition of the rice sheath blight disease is investigated and the control effect is calculated according to the regulation of the GB/T17980.20-2000 field pesticide effect test criterion (I) bactericide control on the rice sheath blight disease. The results are shown in Table 4.

TABLE 5 prevention and control effects of PCYB09 fermentation broth on rice sheath blight disease

As can be seen from the results in Table 5, PCYB09 is at 1X 10⁹cfu mL^-1The control effect of the bacillus subtilis reaches 71.40 percent under the working concentration, is second to the thifluzamide which is a chemical bactericide control product, and is obviously higher than that of a bacillus subtilis commercial product, and the strain is 1 multiplied by 10⁸cfu mL^-1The control effect of the strain reaches 68.31% under the working concentration, and is also obviously higher than that of a bacillus subtilis control product with the same concentration, so that the PCYB09 strain has excellent control effect on rice sheath blight.

Example 5: field control effect of Bacillus sonolatus strain PCYB09 on rice blast

The test arrangement is carried out in a rice blast test base of Linan China Rice research institute in Zhejiang province, the planting system is single-season middle-late rice all the year round, the test rice is a conventional japonica rice variety 'Nanjing 46', the field is divided into cells by ditches, each cell is 25 square meters, the operation is repeated for 3 times, and the test rice is arranged according to a random block grouping method. The control agents are commercial wettable powder of 1000 hundred million spores per gram of Bacillus subtilis and 75% tricyclazole WP (Dow Yiwei)Agriculture). As shown in Table 6, the experimental design was carried out at 675L ha at the initial stage of rice blast (vigorous tillering stage of rice)^-1The application dosage of the pesticide is that each treatment agent is sprayed.

TABLE 6 test design for field control of rice blast

12 days after the pesticide is applied, the disease condition of the rice leaf blast in each cell is investigated according to the regulation of GB/T17980.20-2000 field efficacy test criterion (I) bactericide for controlling the rice leaf diseases, and the related control effect is calculated. The results are shown in Table 7:

TABLE 7 prevention and treatment effects of PCYB09 fermentation broth on rice leaf blast

As shown in Table 7, PCYB09 is at 1X 10⁸cfu mL^-1And 1X 10⁹cfu mL^-1The control effect on the rice blast of rice respectively reaches 70.1 percent and 68.73 percent under the working concentration, is almost equivalent to that of 69.21 percent of a contrast chemical bactericide, and is obviously higher than 61.69 percent of that of a contrast medicament, namely a commercial bacillus subtilis microbial inoculum. The strain is 1 × 10⁷cfu mL^-1The prevention effect of the composition can still reach 57.76 percent under low concentration. The test results show that the PCYB09 has excellent control effect on rice blast.

Example 6: prevention and treatment effect of bacillus sonolatus strain PCYB09 on black spot disease of carya illinoensis

The experimental design is shown in table 8, 3 carya illinoensis trees at the initial stage of disease onset are randomly selected for each treatment, the spraying is carried out according to the experimental design, after the disease state is stable, the disease rate of the black spot disease is investigated, and the prevention and treatment effect is counted according to the disease rate.

TABLE 8 Experimental design and prevention and treatment effects of Bacillus sonolavae strain PCYB09 on prevention and treatment of black spot disease

As can be seen from Table 8, PCYB09 is no matter at 1X 10⁸cfu mL^-1Or 1 × 10⁹cfu mL^-1The bacillus subtilis can show excellent prevention and control effects on apocarya black spot disease under the working concentration, the prevention effects are equivalent under the two concentrations, namely 63.45 and 64.71 percent respectively, the prevention effects are remarkably superior to those of bacillus subtilis sold in the market, and the prevention effects are between prochloraz and thiophanate-methyl. Therefore, the results show that the fermentation product of the PCYB09 strain has excellent control effect on the black spot disease of the carya illinoensis.

Example 7: prevention and control effect on other diseases

According to the results of indoor bioassay, the field prevention and control effects of the fermentation product of the strain on various diseases are evaluated, and the results show that PCYB09 has obvious prevention and control effects on rice false smut, rice bakanae disease, wheat scab, pecan anthracnose, moso bamboo blight and other diseases, part of the PCYB09 is close to the prevention and control effects of chemical pesticides, and the strain has huge application potential in the prevention and control of various diseases in consideration of the advantages of low cost and environmental protection of biological pesticides.

Example 8: induction of plant resistance by the PCYB09 strain

Culturing 39 seeds of rice to 4 leaf stage, and culturing at 1 × 10 concentration⁸cfu mL^-1The PCYB09 bacterial liquid is uniformly sprayed on the surfaces of rice seedling plants, 24 hours and 48 hours after spraying are respectively carried out, a leaf sample is taken and put into liquid nitrogen for quick freezing, and the leaf sample is preserved at the low temperature of minus 80 ℃.

Meanwhile, sample RNA is extracted, and expression quantities of rice defense related genes such as PR1a, PR1b, PR3, PR4, PR5, PR10 and NH1 are examined after reverse transcription. As shown in FIG. 3, it was found that the expression level of most defense-related genes was significantly increased in addition to NH1, and the increase in expression level was more significant with PR 4. The result shows that the PCYB09 strain treatment can induce plant disease resistance by improving the expression quantity of defense related genes.

After the collection of the sample, the sample was homogenized by ice bath, and the activities of polyphenol oxidase (PPO), Peroxidase (POD), superoxide dismutase (SOD) and Catalase (CAT) were analyzed using an enzyme activity kit (sozhou ke mingming biotechnology limited, china), and the operation method was strictly referred to the specification. Relative enzyme activities were calculated according to the following formula based on the enzyme activities per unit mass of the treated and control samples, as shown in FIG. 4.

Polyphenol Oxidase (PPO), Peroxidase (POD), superoxide dismutase (SOD) and Catalase (CAT) are the most important defense-related enzymes of plants. Polyphenol Oxidase (PPO) and Peroxidase (POD) are positively correlated with plant resistance, while superoxide dismutase (S0D) and Catalase (CAT) are negatively correlated with plant resistance. After the PCYB09 strain is used for treating the rice for 24h and 48h, the enzyme activities of polyphenol oxidase (PPO) and Peroxidase (POD) can be obviously improved, and superoxide dismutase (SOD) and Catalase (CAT) are inhibited, and the effect of 48h is obviously stronger than 24h, which shows that the PCYB09 strain can improve the resistance of the rice to pathogenic bacteria by regulating the activity of defense-related enzymes.

From the results of the two tests, the PCYB09 strain can induce the generation and the improvement of the disease resistance of plants by improving the expression quantity of defense-related genes and regulating the activity of defense-related enzymes.

Example 9: UV resistance of the PCYB09 Strain

30ml of PCYB09 strain and contrast bacillus subtilis strain fermentation liquor are placed in a 9cm culture dish, a magnetic rotor is placed in the culture dish, stirring is carried out continuously, ultraviolet lamp tubes are used for respectively irradiating for 1, 2, 4, 6 and 10s, then the culture dish is diluted to a certain multiple, a colony counter is used for counting the number of colonies, and the survival rate (%) of each strain in different time periods is calculated by taking the bacteria liquid which is not irradiated with ultraviolet rays as contrast. As shown in FIG. 5, the survival rate of the strain PCYB09 was significantly higher than that of the control strain at each time period, and after 10s of ultraviolet irradiation, Bacillus subtilis was completely inactivated, while the strain PCYB09 still had a survival rate of approximately 20%. Thus, the PCYB09 strain was more resistant to uv light than the control strain, bacillus subtilis.

Claims

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

2. The use of the bacillus sonolatus PCYB09 of claim 1 in the prevention and control of plant diseases.

3. Use according to claim 2, characterized in that the plant diseases comprise: rice blast, wheat scab, rice sheath blight, rice false smut, rice bakanae disease, camellia oleifera bud disease, carya illinoensis black spot disease, carya illinoensis anthracnose disease, carya illinoensis black spot disease and phyllostachys pubescens blight.

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

5. The plant bactericide is characterized by being prepared from the Bacillus sonoralis PCYB09 as claimed in claim 1.

6. The plant fungicide according to claim 5, which comprises at least one of the fungus body and metabolite of Bacillus cereus PCYB 09.

7. Use of the plant fungicide according to claim 5 or 6 for controlling plant diseases.

8. Use according to claim 7, characterized in that the plant diseases comprise: rice blast, wheat scab, rice sheath blight, rice false smut, rice bakanae disease, camellia oleifera bud disease, carya illinoensis black spot disease, carya illinoensis anthracnose disease, carya illinoensis black spot disease and phyllostachys pubescens blight.