CN116640679A

CN116640679A - Bacillus subtilis, biocontrol microbial agent and application thereof

Info

Publication number: CN116640679A
Application number: CN202310254957.8A
Authority: CN
Inventors: 刘吉平; 张兴楠; 罗龙辉; 吴晓梅
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2023-03-15
Filing date: 2023-03-15
Publication date: 2023-08-25
Anticipated expiration: 2043-03-15
Also published as: CN116640679B

Abstract

The application discloses bacillus subtilis and a biocontrol microbial agent and application thereof. The application separates a bacillus subtilis RSTC-DS07 strain from mulberry leaf tissue, the strain is preserved in the microorganism strain collection of Guangdong province in 2022, 12 months and 15 days, and the preservation number is GDMCC No:63053. the research of the application shows that the strain has good antagonism to various plant pathogenic fungi and/or pathogenic bacteria, and can prevent and control various mulberry diseases, such as Sang Heban disease, mulberry ring rot, mulberry bacterial wilt, sang Tanju disease, sang Qing blight, sang Yibing and the like; meanwhile, lipopeptid substances in the fermentation product of the strain are extracted, so that the incidence rate of the diseases can be obviously reduced. The strain and the fermentation product thereof are used for biological prevention and control, are safe and effective, can avoid various problems of soil environment deterioration, pathogen resistance enhancement and the like caused by chemical pesticides, and lay a foundation for biological prevention and control of mulberry diseases.

Description

Bacillus subtilis, biocontrol microbial agent and application thereof

Technical Field

The application belongs to the technical field of plant disease prevention and control. More particularly relates to bacillus subtilis and a biocontrol microbial agent and application thereof.

Background

The mulberry (Morus) is a perennial woody economic plant and has the value of both medicine and food; the theory of 'Lisang as industry and multiple development' is held. However, the disease of mulberry has been affecting the healthy and stable development of the mulberry industry, and in recent years, the fungal disease and bacterial disease occurring on mulberry have also been showing a tendency to be significantly aggravated.

The mulberry diseases mainly comprise: fungal diseases, bacterial diseases, viruses and mycoid diseases, wherein the fungal diseases are various in species and various in hazard positions, such as Sang Heban diseases, mulberry leaf scald, sang Tanju diseases, sang Chi rust diseases, sang Li powdery mildew, sang Wushe diseases and purple hairline diseases, and the pathogenic bacteria conidia are spread along with wind and rain, so that the spread speed is high, the spread range is wide, and the hazard is serious. Bacterial diseases of mulberry mainly include bacterial wilt of mulberry, sang Qing blight, sang Yibing and the like, and the bacterial diseases of mulberry have a plurality of pathogenic bacteria incidence periods and strong infectivity, are frequently exploded in a plurality of mulberry producing areas, and generally cause the wilt death of the whole plant. Since many diseases are caused by infection of various pathogenic bacteria, various pathogenic bacteria exist on the mulberry at the same time, and various diseases exist on the mulberry at the same time; therefore, control of mulberry diseases is difficult.

At present, for the prevention and treatment of diseases, the main prevention and treatment measures are to use disease-resistant varieties, but due to the diversity of pathogens and pathogenic instability, the disease-resistant varieties of mulberry cannot be completely resisted when being infected and endangered by a plurality of diseases. And is beneficial to chemical method for prevention and control, and chemical pesticide with quick effect and low cost is mainly adopted. However, these chemical pesticides are few in variety and single in control method, and excessive use of chemical pesticides not only results in enhanced pathogen resistance, but also negatively affects the environment and human health. Therefore, an effective and green method is urgently needed to replace or reduce excessive use of chemicals, exploration of biological control technology is one of the environmental protection and effective prevention and control measures, and searching for beneficial microorganisms for biological control of mulberry diseases is of great significance.

In recent years, research on plant disease prevention and control by utilizing plant endophytes is attracting attention, wherein bacillus is used as one of the plant endophytes, and can generate various antibacterial active substances, such as lipopeptide antibiotics, which have strong antibacterial activity and relatively high antibacterial activity, play an important role in plant disease prevention and control, and have potential application value in the field of drug development research; however, at present, there are few antibacterial active substances for preventing and controlling mulberry diseases, and few biocontrol bacteria for preventing and controlling various mulberry pathogenic bacteria. Therefore, research and development of more antagonistic bacteria with excellent properties and strong control effects on various plant pathogenic bacteria are needed for prevention and control research of plant diseases.

Disclosure of Invention

The application aims to overcome the defects and shortcomings of the problems and provide a bacillus subtilis and biocontrol microbial inoculum and application thereof.

The application aims to provide a bacillus subtilis RSTC-DS07 strain.

Another object of the present application is to provide the use of a strain of Bacillus subtilis RSTC-DS 07.

It is still another object of the present application to provide a biocontrol agent which inhibits plant pathogenic fungi and/or pathogenic bacteria.

It is still another object of the present application to provide a method for controlling plant diseases.

The above object of the present application is achieved by the following technical scheme:

the application separates a Bacillus RSTC-DS07 strain (hereinafter, referred to as DS07 strain) from mulberry leaf tissue, the strain is preserved in the microorganism strain preservation center of Guangdong province in 12 months and 15 days of 2022, and the preservation number is GDMCC No:63053. the application identifies that the DS07 strain belongs to the mulberry endophyte bacillus subtilis (Bacillus subtilis), and the colony of the DS07 strain is dirty white or yellowish, opaque, dry on the surface, rough and wrinkled and irregular in edge; belongs to gram positive, rod-shaped, spore-shaped and round at two ends; the contact enzyme, the V-P test and the nitrate reduction reaction are positive, so that starch and gelatin can be degraded; the optimal medium for extracting the antibacterial active substances for the DS07 strain on the BYP medium is suitable.

The research of the application shows that the DS07 strain has antagonism on different mulberry pathogenic bacteria and fungi such as Sang Xi Arthrobacter, sang Xin brown spot fusarium, fusarium oxysporum, fusarium solani, anthracnose of Siamese, laurella of Solanaceae, klebsiella oxytoca, and Pseudomonas syringae, can inhibit a plurality of pathogenic bacteria, and is used for preventing and treating the mulberry diseases such as Sang Heban disease, mulberry ring rot, mulberry bacterial wilt, sang Tanju disease, sang Qing blight, sang Yibing and the like. Meanwhile, lipopeptid substances in fermentation products of the strain are extracted, and the incidence rate of the diseases can be obviously reduced by adopting lipopeptid substance crude extract solution of the strain.

Accordingly, the present application provides the following uses of the bacillus subtilis RSTC-DS07 strain and/or a fermentation broth thereof:

use in inhibiting plant pathogenic fungi and/or pathogenic bacteria.

The application of the composition in preparing the biocontrol microbial inoculum for inhibiting plant pathogenic fungi and/or pathogenic bacteria.

The application of the microbial inoculum in preventing and controlling plant diseases or preparing a biocontrol microbial inoculum for preventing and controlling plant diseases.

Further, the plant disease is a mulberry disease.

The application provides a biocontrol microbial inoculum for inhibiting plant pathogenic fungi and/or pathogenic bacteria, which contains RSTC-DS07 strain or fermentation liquid thereof.

The application also provides a method for preventing and controlling plant diseases, which adopts the biocontrol microbial inoculum to treat plants.

Preferably, the fermentation liquid is lipopeptid substance crude extract solution with concentration not lower than 1000mg.L ^-1 。

Preferably, the plant pathogenic fungi and/or pathogenic bacteria are: sang Xi Arthrobacter (Gonatophragmimmori), sang Xin Fusarium brown spot (Neophloeospora maculans), fusarium equisetum (Fusarium equiseti), fusarium oxysporum (Fusarium oxysporum), fusarium solani (Fusarium solani), siamese anthrax (Colletotrichum siamense), lauraceae (Ralstonia solanacearum), klebsiella oxytoca (Klebsiella oxytoca), and Pseudomonas syringae (Pseudomonas syringae).

Particularly, the DS07 lipopeptid substance crude extract solution adopted by the application is a compound or complex of lipid and amino acid (lipopeptid substance) in a strain fermentation product, and the extraction of the lipopeptid substance of the strain is obtained by adopting a kit or a conventional extraction method in the field. The lipopeptides of DS07 have good antagonism to a plurality of plant pathogenic fungi and/or pathogenic bacteria, so that RSTC-DS07 strain and fermentation products thereof can be used for preventing and controlling plant diseases caused by the infection of plants by the plant pathogenic fungi and/or pathogenic bacteria, and have certain effect.

Preferably, the mulberry disease is one or more of Sang Heban disease, mulberry leaf scald disease, mulberry bacterial wilt, sang Tanju disease, sang Qing blight and mulberry epidemic disease.

The application has the following beneficial effects:

the application separates and identifies a Bacillus subtilis (Bacillus sp.) RSTC-DS07 strain which has good antagonism to plant pathogenic fungi and/or pathogenic bacteria such as Sang Xi section mould, sang Xin brown spot aschersonia, fusarium equiseti, fusarium oxysporum, fusarium solani, colletotrichum sium, solanaceae Laurella, klebsiella acidi, pseudomonas syringae and the like, and can be used for preventing and controlling plant diseases caused by the plant pathogenic fungi and/or pathogenic bacteria infection plants, such as: sang Heban disease, mulberry leaf scald disease, morus bacterial wilt, sang Tanju disease, sang Qing blight, sang Yibing, etc.; meanwhile, the application also researches the active ingredients of the fermentation product of the RSTC-DS07 strain, extracts the lipopeptid substances thereof, and researches show that the lipopeptid substance crude extract solution of the fermentation product of the RSTC-DS07 strain can be used on mulberries, and reduces the incidence rate of the mulberries diseases.

The RSTC-DS07 strain or the fermentation liquor thereof provided by the application is used for biological prevention and control of plant diseases, is safe and effective, can avoid various problems of soil environment deterioration, pathogen resistance enhancement and the like caused by chemical pesticides, and lays a foundation for biological prevention and control of mulberry diseases. Therefore, the bacillus subtilis RSTC-DS07 strain provided by the application has good application prospect in preventing and controlling plant diseases or preparing a preparation for preventing and controlling plant diseases.

Drawings

FIG. 1 is a graph of the screening results of the optimal fermentation medium for the strain (panel inhibition zone graph on the left and data on the right).

FIG. 2 is a graph showing growth conditions of strains on LB medium.

FIG. 3 is a graph of the gram stain results of the strain.

FIG. 4 is a graph of the scanning electron microscope (7000X) results of the strain.

FIG. 5 is a phylogenetic tree diagram of the construction of gyrB sequences of strains.

FIG. 6 is a graph showing the results of antagonistic activity of DS07 lipopeptides on the results of antagonistic activity of Sang Xi Arthrobacter, sang Xin Fusarium brown spot, fusarium equisetosum, fusarium oxysporum, fusarium solanacearum, klebsiella oxytoca, and Pseudomonas syringae.

Detailed Description

The application is further illustrated in the following drawings and specific examples, which are not intended to limit the application in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

The samples used in the examples below were derived from healthy, strong mulberry 1 mulberry plant leaves collected by the mulberry training center in the asia-tai area of agricultural university in south China.

The source of the test strain in the following examples: the following pathogenic bacteria are all common pathogenic bacteria in the prior art research, are stored and researched by the inventor laboratory after being separated by commercial or prior art;

sang Xi section mould (Gonatophragmium mori): gao Qiaoxing Ji, temple Cumin, hu Xingping, et al. Litsea. Phellinus (Gonatophragmium mori) life history and Classification [ J ]. Foreign agro-silkworm, 1988 (01): 27-30;

sang Xin brown aschersonia (Neophloeospora maculans): da Costa C A, veloso J S, de Oliveira B F, et al first report of neophloeospora maculans causing leaf spots in morus nigra and m. Alba in brazil [ J ]. Journal of Plant Diseases and Protection,2021,128 (1): 317-321.

Fusarium equisetum (Fusarium equiseti): han Zhongming, sun Zhuo, wang Yan, etc. screening and identification of wind-proof root rot antagonistic fungi and research on biocontrol action [ J ]. Chinese journal of biological control, 2022,38 (05): 1288-1295;

fusarium oxysporum (Fusarium oxysporum): zhao Zhixiang, ling, yan Wanrong, et al isolation and identification of pepper fusarium wilt bacteria and evolutionary analysis [ J ]. Molecular plant breeding, 2019,17 (19): 6383-6389;

fusarium solani (Fusarium solani): shen Baoyu, sun Wensong, zhang Tianjing, et al. Isolation and identification of root rot pathogen of Dictamni [ J ]. Molecular plant breeding: 1-14;

siamese anthrax (Colletotrichum siamense): yang Xiaoqi infection pathogenic study of Siamese anthrax (Colletotrichum siamense) on strawberry stem base [ D ] university of agricultural and forestry Zhejiang, 2022;

the accession number of the Lauraceae bacteria (Ralstonia solanacearum) is GDMCC No:1.1616 Klebsiella oxytoca (Klebsiella oxytoca) accession number GDMCC No:1.1603 Pseudomonas syringae (Pseudomonas syringae) accession number GDMCC No:61844.

the formulation of the fermentation medium used in the following examples was:

NYD medium: 8g/L of beef extract, 3g/L of yeast extract and 1g/L of glucose;

YPG medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L;

YPD medium: yeast extract 10g/L, tryptone 20g/L, glucose 20g/L;

YSB medium: sucrose 20g/L, yeast extract 20g/L, beef extract 15g/L, mgSO ₄ ·7H ₂ O0.06g/L，FeSO ₄ ·7H ₂ O 0.009g/L；

BYP medium: beef extract 5g/L, peptone 10g/L, yeast extract 5g/L, glucose 10g/L, naCl 5g/L.

The bacterial DNA extraction kit and PCR reagents used in the following examples were purchased from the division of bioengineering (Shanghai); potato dextrose agar medium, beef extract peptone agar medium, physiological and biochemical medium, LB broth medium were all purchased from Guangdong CycloKai microorganism technologies Co.

EXAMPLE 1 isolation and identification of strains

1. Isolation of strains

Collecting fresh mulberry leaf tissue, washing with sterile water, and cutting into pieces of about 1×3cm ² Placing in a sterile glass dish, adding 75% alcohol into the sterile glass dish on an ultra-clean workbench, soaking for about 15s, draining alcohol, treating with mercuric chloride for 5min, washing with sterile water for 3 times, placing leaf tissue in a sterile mortar, grinding to obtain juice, and extracting juice with a sterile gun head for gradient dilution of 10 ³ After that, 25. Mu.L of the dilution was aspirated and spread evenly on LB medium, and the plating was repeated 3 times for each dilution. After 24 hours, single colony is picked up on LB culture medium for streak expansion culture, single colony is picked up every other day and inoculated on LB inclined plane and flat culture medium, and stored in glycerol and placed in a refrigerator at-80 ℃ for one portion.

2. Screening of strains

The method comprises the steps of taking Sang Heban disease bacteria Sang Xin brown spot aschersonia aleyrodis N.maculons as indicator bacteria, testing bacteriostatic activity by adopting an agar diffusion method, placing the indicator bacteria which are cultivated for 15d on a PDA culture medium into a sterile mortar, removing redundant agar, adding 10mL of sterile water, grinding the indicator bacteria by using a sterile grinding rod, uniformly smearing 500 mu L of indicator bacteria on the PDA culture medium, uniformly punching 5mm small holes in a culture dish coated with a tested strain by using a puncher, taking 10 mu L of fermentation liquor by using a micropipette, adding the fermentation liquor into the punched small holes, standing and cultivating for 5d in a 25 ℃ incubator, detecting whether a bacteriostatic circle exists or not, and measuring the diameter of the antibacterial circle.

The results show that: the new brown spot aschersonia is taken as indicator bacteria, and an endophytic antagonistic strain with good antibacterial effect is separated from the endophytic bacteria of the mulberry leaves and is named DS07.

3. Screening of optimal fermentation Medium for Strain

Picking single colony of DS07 strain, inoculating into 6 culture mediums such as NYD, YPG, YPD, YSB, BPY and LB, culturing at 28deg.C and 180r/min for 48 hr, and adjusting concentration to 10 ⁸ cfu/mL. Centrifuging the antagonistic fermentation liquor at 10000r/min for 15min, removing precipitate, collecting supernatant, filtering the supernatant with 0.2 μm bacterial filter, and the method is the same as above, the bacteriostasis test is carried out by using Sang Heban disease germ Sang Xin aschersonia aleyrodis N.maculons as indicator bacteria and adopting an agar diffusion method, each culture medium is repeated 3 times, and the diameter of a bacteriostasis circle is measured.

The DS07 strain was inoculated into a sterile culture solution of 6 common media, and the result is shown in FIG. 1, which shows that the sterile culture solution after DS07 is fermented in BYP medium has the largest inhibition zone, so BYP medium is selected as a standby medium for extracting antibacterial active substances from DS07 strain.

4. Identification of strains

The endophytic antagonistic bacteria DS07 obtained by screening are inoculated in LB agar medium and cultured for 24 hours at 28 ℃, and the colony morphology, growth condition and the like are observed and recorded. The measurement is carried out by gram staining, morphological observation (scanning electron microscope), and relevant physicochemical parameters such as the activity of the contact enzyme and the oxidase, and the specific measurement is described in the general bacterial System identification Manual and the Berger bacterial identification Manual.

And carrying out gyrB gene sequencing on the endophytic antagonistic DS07 strain, carrying out multi-sequence comparison on the gyrB sequence obtained and the gyrB sequence of a target species closely related species downloaded from NCBI by adopting MUSCLE v.3.8.31 (http:// www.drive5.com/MUSCLE /) software, and then constructing a phylogenetic tree by using the compared sequences through Mega6 software and setting a Bootstrap value to 1000 by using a maximum likelihood method (ML method: maximum Likelihood method).

The growth of the endophytic antagonistic strain DS07 on the LB medium is shown in FIG. 2, which shows that the endophytic antagonistic strain is dirty white or yellowish, opaque, dry, rough and wrinkled and uneven in edge on the LB medium.

The result of gram staining of the endophytic antagonistic strain DS07 is shown in FIG. 3, which shows that the endophytic antagonistic strain is gram-positive, rod-shaped and has spores.

The scanning electron microscope (7000×) results of the endophytic antagonistic strain DS07 are shown in FIG. 4, which shows that the endophytic antagonistic strain is a rod-shaped strain with rounded ends.

The physiological and biochemical characteristic results of the endophytic antagonistic strain DS07 are shown in the following table 1, and show that the endophytic antagonistic strain is positive in contact with enzyme, V-P test and nitrate reduction reaction, and can degrade starch and gelatin.

TABLE 1 physiological Biochemical characterization results of strains

Note that: "+" represents positive reaction and "-" represents negative reaction

The phylogenetic tree is constructed based on the full length of the endogenous antagonistic gyrB sequence, and the result is shown in figure 5, wherein the DS07 strain and the bacillus subtilis (Bacillus subtilis) are found to be one, and the confidence coefficient is close to 100%. And determining the strain as bacillus subtilis (Bacillus subtilis) according to the morphological identification, the physiological and biochemical characteristics and the homology comparison result, wherein the classification name is as follows: bacillus sp.sp.designated RSTC-DS07 strain and deposited at the Cantonese microorganism strain collection at 12.15 2022 under the accession number GDMCC No:63053. the preservation address is Guangzhou City first China No. 100 college 59 building 5 Guangdong province microbiological institute.

EXAMPLE 2 lipopeptides antibacterial Activity of Bacillus subtilis DS07

1. Extraction of lipopeptides secreted by bacterial strain

After the antagonistic bacteria were obtained by preliminary screening of the strain broth in example 1, the strain broth was shown to have an antibacterial effect, and the strain identification result showed that the strain was bacillus subtilis (Bacillus subtilis), and according to the prior art, the bacillus bacteria were shown to produce various antibacterial active substances, in which lipopeptides compounds play a major role (Morikawa M, ito M, imanaka T.isolation of a new surfactin producer bacillus pumilus a-1,and cloning and nucleotide sequence of the regulator gene,psf-1[ J ]. Journal of Fermentation and Bioengineering,1992,74 (5): 255-261), and bacillus subtilis was able to produce one antibacterial lipopeptide, which acts as an antibacterial active substance. Therefore, the antagonistic experiment is carried out again by extracting lipopeptid substances of the bacillus subtilis DS07 strain, so that the antagonistic effect of antibacterial active substances of a fermentation product is verified, and technical support is provided for the synthesis development of plant disease prevention and control by replacing chemical pesticides in the later period.

Therefore, the application adopts a method of combining hydrochloric acid precipitation with acetone extraction to extract lipopeptides substances of DS07 strain, single colony of DS07 strain is selected and inoculated in YPG fermentation culture medium, and is cultured for 72 hours under the condition of 28 ℃ and 180r/min, after centrifugation for 15min at 8000r/min, precipitate is removed, supernatant fluid is collected, HCl is slowly added into the supernatant fluid, and the mixture is stood overnight under the condition of adjusting pH to 2.0,4 ℃. Centrifuging at 8000r/min for 15min, discarding supernatant, collecting precipitate, adding 5 times of acetone, mixing, suspending, centrifuging at 8000r/min for 15min, collecting acetone organic phase, and repeatedly collecting twice. Mixing the acetone extract for 2 times, placing in a fume hood for air drying, dissolving with methanol, sterilizing with an organic filter membrane filter of 0.2 μm to obtain DS07 strain lipopeptide substance crude extract solution, and measuring the mass concentration with Abbkine BCA protein quantitative kit.

2. Antagonism experiment of lipopeptid substance of bacterial strain on pathogenic bacteria

The antagonistic activity of DS07 strain lipopeptid substances on pathogenic bacteria (Lauraceae, klebsiella and Pseudomonas syringae) is tested by adopting an agar diffusion method: respectively inoculating pathogenic bacteria into LB liquid culture medium, shake culturing at 28deg.C and 180r/min to D ₆₀₀ nm about 0.3, at 1:30 volume ratio of pathogenic bacteria bacterial liquid is added into sterile LB agar medium at about 40 ℃, after being mixed evenly, 15mL of culture is poured into each culture dish, and perforation is used after solidificationUniformly punching 5mm holes in a culture dish, taking 10 mu L of DS07 bacterial strain lipopeptide substance crude extract solution by a micropipette, adding the solution into the punched holes, standing and culturing for 24 hours at 25 ℃, detecting whether a bacteria inhibition zone exists or not, and measuring the diameter of the bacteria inhibition zone.

3. Antagonism experiment of lipopeptid substance of strain on pathogenic fungi

The antagonistic activity of the lipopeptid substances of the DS07 strain on pathogenic fungi (Sang Xin brown spot aschersonia, sang Xi festival mould, fusarium equiseti, fusarium oxysporum, eggplant disease fusarium, siamese anthracnose) was measured by a plate counter method, wherein Sang Xin brown spot aschersonia, sang Xi festival mould and other pathogenic fungi are treated by an agar diffusion method, the treatment method of the pathogenic fungi is the same as in example 1, and 10 mu L of antagonistic lipopeptid substances are added into the punched holes. The antibacterial activity of other fungi is measured by adopting a plate counter method, a bacterial cake is placed at the center of a PDA plate, 4 small holes with the size of 5mm are drilled at equal intervals around the bacterial cake by using a puncher, and 10 mu L of DS07 bacterial strain lipopeptid substance crude extract solution is added.

The inhibition effect of the DS07 bacterial strain lipopeptide substance crude extract solution on pathogenic bacteria and pathogenic fungi is shown in figure 6, and specific antagonism experimental results are shown in table 2, which show that the DS07 bacterial strain lipopeptide substance has better inhibition effect on fungal pathogens, wherein the antagonism effect on Sang Xin brown spot aschersonia and Sang Xi festival mould is most obvious, and the inhibition zone size can be respectively 27.67mm and 27.00mm. The antibacterial rate of the DS07 strain lipopeptid substances can reach 76.20%, 48.57%, 50.49% and 26.63% for fusarium equiseti, fusarium oxysporum, fusarium solani and Siamese anthracnose, so that the DS07 strain lipopeptid substances can be used for preventing and treating Sang Heban diseases and mulberry leaf scald. The lipopeptid substance of the DS07 strain has good antibacterial effect on bacterial pathogens such as Laurella of Solanaceae, klebsiella and Pseudomonas syringae, and the diameters of the antibacterial rings can reach 19.67mm, 9.00mm and 10.33mm respectively, wherein the effect on the Laurella of Solanaceae is most obvious, so that the DS07 strain can be suitable for preventing and treating bacterial wilt of mulberry.

TABLE 2 antibacterial Activity of lipopeptides of DS07 Strain on pathogenic bacteria

EXAMPLE 3 Mulberry pot control experiment of lipopeptid substance of Strain on Sang Heban disease

Selecting a potted mulberry with good field growth, cleaning the surface and puncturing surface tissues with a needle; setting 3 treatments: (1) spraying 1.00 g.L ^-1 Inoculating N.maculons after the DS07 strain lipopeptides substance crude extract solution; (2) inoculating N.macus; (3) after spraying methanol, N.maculons were inoculated. A crude extract solution of the lipopeptid substance of DS07 strain was prepared as in example 2.

Inoculating N.maculons, puncturing 4 points on both sides of a main vein on each leaf, repeating for 3 times, sleeving a sterile bag to protect the leaf, culturing for 5-7 days at room temperature, investigating the disease condition of the leaf, calculating the disease rate, and measuring the size (cm multiplied by cm) of the disease spots by using a crisscross method.

Morbidity = number of incidents/total number of puncture wounds x 100%

The results are shown in Table 3 below, which shows the use of 1.00 g.L ^-1 The crude extract solution of lipopeptid substances is treated and inoculated with the brown spot pathogen of the mulberry, so that the inhibition effect on the brown spot pathogen of the mulberry is optimal, the incidence rate is lowest, the disease spots are smaller, the average incidence rate is 45.80%, and the average disease spot size is 0.44cm multiplied by 0.34cm.

TABLE 3 inhibition of crude extracts of Bacillus subtilis DS07 lipopeptides at different concentrations against Brown spot in mulberry

Treatment group	Average lesion size (cm. Times.cm)	Incidence of disease
			1000mg·L ^-1 lipopeptides+N.maculons	(0.44x0.34)d	45.80％
CK1	—	—
			N.maculans	(0.97x0.81)a	96.50％
methanol+N.maculons	(0.98x0.80)a	97％

Note that: lower case letters differing from the same column indicate significant differences between treatments at P <0.05 levels.

The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present application should be made in the equivalent manner, and the embodiments are included in the protection scope of the present application.

Claims

1. A Bacillus subtilis (Bacillus sp.) RSTC-DS07 strain, which has been deposited at the cantonese province microorganism strain collection at 12 months and 15 days 2022 under the accession number GDMCCNo:63053.

2. use of the RSTC-DS07 strain and/or its fermentation broth according to claim 1 for inhibiting plant pathogenic fungi and/or pathogenic bacteria.

3. Use of the RSTC-DS07 strain and/or its fermentation broth according to claim 1 for the preparation of a biocontrol agent for inhibiting phytopathogenic fungi and/or pathogenic bacteria.

4. The use of RSTC-DS07 strain and/or its fermentation broth according to claim 1 for controlling plant diseases or for preparing biocontrol bacterials for controlling plant diseases.

5. The use according to claim 4, wherein the plant disease is a mulberry disease.

6. A biocontrol agent for inhibiting plant pathogenic fungi and/or pathogenic bacteria comprising the RSTC-DS07 strain and/or a fermentation broth thereof according to claim 1.

7. A method for controlling plant diseases, which is characterized in that the biocontrol microbial agent of claim 6 is used for treating plants.

8. The biocontrol agent of claim 6 or the method of claim 7, wherein the fermentation broth is a lipopeptides crude extract solution, preferably the concentration of the lipopeptides crude extract solution is not less than 1000 mg-L ^-1 。

9. The use according to claim 2 or 3, or the formulation according to claim 6, wherein the phytopathogenic fungi and/or bacteria are: sang Xi Arthrobacter (Gonatophragmium mori), sang Xin Fusarium brown spot (Neophloeospora maculans), fusarium equisetum (Fusarium equiseti), fusarium oxysporum (Fusarium oxysporum), fusarium solani (Fusarium solani), siamese anthrax (Colletotrichum siamense), laurella solanaceae (Ralstonia solanacearum), klebsiella oxytoca (Klebsiella oxytoca), and Pseudomonas syringae (Pseudomonas syringae).

10. The use according to claim 4 or 5, or the method according to claim 7 or 8, wherein the plant disease is a plant disease caused by a pathogenic fungus and/or a pathogenic bacterium according to claim 9.