CN112920975B - Bacillus belgii YL94 and application thereof - Google Patents
Bacillus belgii YL94 and application thereof Download PDFInfo
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Abstract
The invention discloses a Bacillus belgii YL94 and application thereof. The strain is preserved in Guangdong province microorganism culture collection center in 12 months and 08 days in 2020, and the preservation number is GDMCC No: 61355. the Bacillus belgii YL94 capable of effectively controlling anthracnose of oncidium is obtained by separation and screening, has good antagonistic action and prevention and control effects on plant fungal diseases caused by the infection of oncidium plants by various pathogenic fungi, is safe and effective when the strain or fermentation liquor thereof is used for carrying out biological prevention and control on the oncidium pathogenic fungi, can avoid various problems of soil environment deterioration, pathogenic drug resistance enhancement and the like caused by chemical pesticides, provides an excellent antagonistic strain for the prevention and control of the anthracnose of the oncidium and other various diseases, and thus improves the yield and ornamental value of the oncidium.
Description
Technical Field
The invention belongs to the technical field of plant disease prevention and control. More particularly, relates to a Bacillus belezii YL94 strain and application thereof.
Background
Orchid is a flower with great ornamental and collection value, is rare, elegant and popular with people, but is attacked by various pathogenic bacteria in the cultivation process, so that the ornamental and collection value is greatly reduced. With the development of orchid industry, orchid diseases are highly valued by people. Wen Xin lan, also called dancing orchid, dancing girl orchid and golden butterfly orchid, is very popular because its flowers are fantastic and lovely and shaped like flying golden butterfly. The oncidium has dense planting, poor ventilation, water imbalance or wound and other susceptible anthracnose, leaves generate brown concave small points at the early stage of the disease, and then the brown concave small points are expanded into round or irregular spots, and gangrene is generated in the center of the spots in serious cases.
Orchid has various diseases, mainly including fungi, bacteria and viruses, wherein Colletotrichum gloeosporioides is a common plant pathogen and is reported as the eighth plant pathogen in the world. Orchid anthracnose is caused by Colletotrichum and Colletotrichum sp, orchid wilt is caused by two fusarium sp, stem rot and the like of orchid can be caused by fusarium sp, and the germs greatly influence the yield of orchid and cause huge economic loss for orchid production. At present, the disease is mainly controlled by a chemical agent, and compared with chemical control, biological control of the disease is more and more popular with people due to the characteristics of no pollution, difficult induction of drug resistance, lasting control effect and the like, so that the biological control becomes an important method for controlling plant diseases.
Nowadays, chemical control pollution is increasingly serious, and the phenomena of crop resistance, pesticide residue and disease regeneration are more and more common. Therefore, screening of novel biocontrol strains and research of antibacterial substances thereof have become a hotspot. The antagonistic bacteria is utilized to prevent and treat plant diseases, has the advantages of safety, environmental protection, high specificity to pathogenic bacteria and the like, and is beneficial to sustainable development. Among biocontrol bacteria for plant diseases, Trichoderma spp, Bacillus spp, Pseudomonas spp and the like are common. However, the researches on orchid biocontrol bacteria are few at present, and the antagonistic mechanism is not clear. Researches show that the solid-liquid preparation of T2-16 trichoderma strains can effectively reduce the morbidity degree of southern blight, stem rot and anthracnose of cymbidium ensifolium, and the control effect of trichoderma agent soil treatment on 3 diseases is superior to that of a control chemical medicament carbendazim (Weilin, Dillenware, Lvgang, and the like. Trichoderma harzianum T2-16 microbial agent on the disease prevention and growth promotion effects of cymbidium ensifolium [ J ]. China gardening abstracts, 2017(2) ]; the inhibition rates of bacillus LV5 and Trichoderma harzianum HG on pathogenic substances of leaf brown spot of oncidium hyalopecuroides are respectively 82.10% and 72.82%, and the application potentials of the microorganisms in preventing diseases of orchids are shown (Wang culture, Jing, Chun willow, and the like). However, at present, a novel biocontrol bacterium which can effectively control anthracnose of oncidium and other various oncidium pathogenic bacteria is lacked.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the existing orchid disease control and provides a Bacillus belgii YL94 strain for controlling anthracnose of oncidium and other various oncidium pathogenic bacteria.
The first purpose of the invention is to provide a Bacillus velezensis (Bacillus velezensis) YL94 strain.
The second purpose of the invention is to provide the application of the Bacillus belgii YL94 or the fermentation liquor thereof in inhibiting phytopathogen.
The third purpose of the invention is to provide the application of the Bacillus belgii YL94 or the fermentation liquor thereof in preventing and controlling plant fungal diseases or preparing a preparation for preventing and controlling the plant fungal diseases.
It is a fourth object of the present invention to provide a formulation for controlling fungal plant diseases.
The above purpose of the invention is realized by the following technical scheme:
the method takes colletotrichum gloeosporioides (C.gloeosporioides) as a target bacterium, separates and screens an endophyte with antagonistic action on 9 oncidium pathogenic bacteria at different degrees, and determines that the endophyte is Bacillus subtilis (Bacillus velezensis) with the serial number of YL94 by combining morphological, physiological and biochemical characteristics and 16S rDNA, gyrA and gyrB polygene joint identification.
The invention provides a Bacillus belgii (Bacillus velezensis) YL94, which is preserved in Guangdong province microorganism strain preservation center in 12 months and 08 days in 2020, with the preservation number being GDMCC No: 61355.
the application of the Bacillus belgii YL94 and/or the fermentation liquor thereof in inhibiting phytopathogen or preparing the bacteria for inhibiting the phytopathogen is within the protection scope of the invention.
Preferably, the phytopathogen is an oncidium pathogenic fungus.
The application of the Bacillus belgii YL94 and/or the fermentation liquor thereof in the prevention and control of plant fungal diseases or the preparation of preparations for preventing and controlling the plant fungal diseases also belongs to the protection scope of the invention.
Preferably, the plant fungal disease is a pathogenic fungal disease of oncidium.
More preferably, the pathogenic fungal disease of oncidium is a disease caused by any one or more of anthrax fungus, fusarium fungus, coccidioides fungus, pseudotheca fuliginosum fungus, curvularia mounts or chaetotheca megaterium fungus.
More preferably, the pathogenic fungal disease of oncidium is a disease caused by any one or more of colletotrichum gloeosporioides, colletotrichum tropicalis, fusarium oxysporum, fusarium solani, fusarium oxysporum, alternaria alternata, pseudotheca fuliginosa, curvularia leaf spot or chaetomium giganteum.
Still more preferably, the pathogenic fungal disease of oncidium is a disease caused by any one or more of colletotrichum gloeosporioides, colletotrichum tropicalis, mangifera indica, fusarium oxysporum or fusarium oxysporum.
More preferably, the oncidium pathogenic fungal disease is a disease caused by colletotrichum gloeosporioides and/or mangifera globispora.
Preferably, the oncidium pathogenic fungal disease is oncidium anthracnose.
In addition, the invention also provides a preparation for preventing and controlling plant fungal diseases, which contains the Bacillus belgii YL94 and/or fermentation liquor thereof.
Preferably, the plant fungal disease is a pathogenic fungal disease of oncidium.
More preferably, the oncidium pathogenic fungal disease is oncidium anthracnose.
The invention has the following beneficial effects:
according to the invention, a strain capable of effectively controlling anthracnose of oncidium is obtained by separation and screening, and the bacteriostatic property of biocontrol bacteria for controlling various pathogenic fungi and fermentation liquor is analyzed, so that the fact that the strain fermentation liquor contains rich bacteriostatic active substances is found, the biocontrol bacteria has good antagonistic action on the anthracnose of the oncidium, black spot mango tee bacteria, chickpea fusarium oxysporum, fusarium solani and the like, the biocontrol bacteria has good control effect on orchid plant diseases (especially the anthracnose of the oncidium) caused by the orchid infection by the various fungi, and the biocontrol bacteria is determined to be Bacillus belief (Bacillus velezensis) by combining morphological and physiological and biochemical characteristics and multiple gene joint identification of 16S rDNA, gyrA and gyrB and has the number of YL 94;
the Bacillus belgii YL94 or the fermentation liquid thereof is used for biologically preventing and controlling the anthracnose of oncidium, is safe and effective, can avoid various problems of soil environment deterioration, pathogen drug resistance enhancement and the like caused by chemical pesticides, lays a foundation for the biological prevention and control of the oncidium diseases, and provides excellent antagonistic strains for the prevention and control of the anthracnose of the oncidium and other various diseases, thereby improving the yield and the ornamental value of the oncidium. Therefore, the Bacillus belgii YL94 or the fermentation liquid thereof has good application prospect in preventing and controlling plant fungal diseases or preparing preparations for preventing and controlling plant fungal diseases.
Drawings
FIG. 1 shows the inhibitory effect of different treatments of strain YL94 on anthracnose of oncidium; wherein, A: the antagonistic effect of the strain YL94 and pathogenic bacteria; c: bacterial strain YL94 fermentation liquor is bacteriostatic; B. d: blank control.
FIG. 2 shows the results of morphological, physiological and biochemical identification of strain YL 94; wherein, A: a front view of a colony of strain YL 94; b: gram stain pattern (positive); c: spore staining pattern.
FIG. 3 is a phylogenetic tree of strain YL94 based on the 16S gene sequence.
FIG. 4 is a phylogenetic tree of strain YL94 based on the gyrA gene sequence.
FIG. 5 is a phylogenetic tree of strain YL94 based on the gyrB gene sequence.
FIG. 6 is greenhouse pricking inoculation diagram of oncidium plants and morbidity in tissue culture seedlings; wherein, A: inoculation strain YL 94; b: control group (sterile PDA cake).
FIG. 7 shows the antagonism of strain YL94 against various pathogenic fungi; wherein, A: fusarium solani (WB 11); b: fusarium solani (WB 23); c: fusarium solani (WG 1); d: fusarium solani (W47); e: fusarium oxysporum Fusarium delphinidinides (WB 6); f: fusarium oxysporum (W38); g: fusarium oxysporum (WB 1); h: black spot mango sphaericus guignandiangieriae (WB 35); i: pseudocercospora septoriae, Paraconiothyrium thysanolaenae (WG 2); j: curvularia angustifolia (WG3) of Curvularia angustifolia; k: colletotrichum tropicalis (WB 19); l: muyocopropron alcornii (WG4) which is a fungus of the family Macrothecaceae; the left side of each numbering drawing is a picture of the confronting side of the strain YL94 and the pathogen, and the right side is a control group pathogen.
FIG. 8 is a graph of growth rates of different pathogenic bacteria antagonised by strain YL 94; wherein, A: colletotrichum gloeosporioides, B: sphaerotheca nigrospora Guignardia mangiferae (WB35), C: fusarium oxysporum Fusarium delphinidinides, D: fusarium solani, E: fusarium oxysporum (WB38), F: fusarium solani (W47); (WB6), G: fusarium solani (WB11), H: curvularia angustifolia, I: pseudocercospora septoriae, fungi Paraconiothyrium thysanolaenae, J: colletotrichum tropicalis, K: fusarium solani (WG1), L: fusarium oxysporum (WB1), M: the fungus Muyocopropron alcornii of the family Macrospermaceae.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Culture medium: LB culture medium is adopted for the isolation culture of the conventional bacteria, and PDA culture medium is adopted for the isolation culture of the conventional fungi. The PDA culture medium comprises the following components: 200g of potato, 2g of glucose, 15g of agar and 1000mL of distilled water, and the pH value is (6.5 +/-0.5).
EXAMPLE 1 isolation, purification and preservation of endophytes
Materials and methods
1. Test materials
The test material is a 'Boda No. 1' variety of oncidium industrial park of the Hainan Boda orchid science and technology Limited company, normal oncidium plants which are healthy and disease-free in 3 years and have the same growth vigor are collected in the oncidium industrial park, and the oncidium plants are taken back to a laboratory for microbial separation.
2. Isolation, purification and preservation of endophytes
And (3) separating the oncidium endophyte by adopting a tissue block method. The oncidium plants were cut and rinsed with tap water for 15 minutes for future use. Placing the leaves in a super clean workbench, cleaning the leaves for 3-5 times by using sterile water, sequentially soaking the leaves in 75% ethanol solution for 30s, soaking the leaves in 2.5% NaClO solution for 5min, and finally rinsing the leaves for 4 times by using the sterile water. And uniformly spreading the sterile water rinsed at the last time on a culture medium by using a triangular coating rod, and placing the culture medium in an incubator for 3-5 days at 28 ℃ in a dark place to check whether the surface of the leaf tissue is thoroughly disinfected.
After bacteria grow out from the edge of the section of the leaf tissue, inoculating the leaf tissue to an LB culture medium in a streaking mode to purify the bacteria, and culturing the purified bacteria in a 28 ℃ culture box. The purified bacteria were inoculated in LB liquid medium and cultured on a shaker at a rotation speed of 180r/min for 1 day. Mixing the bacteria solution and sterilized 50% glycerol at a ratio of 1:1, placing in a centrifuge tube, sealing and storing in an ultra-low temperature refrigerator of-80 deg.C.
3. Screening of antagonistic oncidium anthracnose strains
Screening by adopting a confronting culture method, taking Colletotrichumgloeosporides which is pathogenic bacteria of anthracnose of oncidium anthracnose as indicating bacteria, punching an activated indicating bacteria cake by using a 5mm sterilization puncher, firstly inoculating the bacteria cake in the center of a fresh PDA (personal digital assistant) flat plate as the indicating bacteria, then inoculating the bacteria to be detected at a position (4 position) 3cm away from the bacteria cake, and using the flat plate only inoculated with the bacteria cake as a contrast. Putting the mixture into a constant-temperature incubator at 28 ℃ for inverted culture for 5d, measuring the antibacterial zone, and screening out a strain with a wider antibacterial zone, wherein the strain is numbered as a strain WB 75. Placing the activated fungus cake into a 500mL triangular flask containing 200mLPDA culture medium, 2-3 blocks, performing shake culture at 28 deg.C and 180r/min for 7d, centrifuging the fermentation liquid at 10000r/min for 5min, filtering with gauze sterilized at 121 deg.C, filtering the fermentation liquid with 8 layers of gauze, centrifuging the filtrate at 10000r/min for 15min, and collecting the supernatant. One part was sterilized by filtration through a 0.22 μm bacterial filter to obtain a sterile fermentation broth. Sterilizing the other part of fermentation broth at 121 deg.C for 30 min. 10mL of fermentation broth was mixed with 50mLPDA medium to make a toxin-containing plate, and the center of the plate was inoculated with pathogenic bacteria, as compared with PDA without fermentation broth, and the treatment was repeated 3 times. And (4) observing and recording the growth vigor of the colonies after culturing for 3 days at 28 ℃, measuring the diameter of the colonies until the control colonies grow to fill the plate, and calculating the bacteriostasis rate.
The bacteriostatic ratio (%) - (control colony diameter-treated colony diameter)/control colony diameter × 100%.
4. Classification and identification of antagonistic bacteria
(1) Morphological identification
Firstly, activating a strain WB75 on an LB flat plate, inoculating two rings of activated lawn into 100mL of liquid LB culture solution, carrying out constant-temperature shaking culture at 35 ℃ and 150r/min for 24h to prepare a bacterial suspension, diluting the bacterial suspension, carrying out gram staining, carrying out spore staining after 36h, and recording the characteristics of the colony such as shape, color, texture, colony size and the like after 72 h.
(2) Physiological and biochemical identification
Physiological and biochemical identification is carried out according to the characteristics in bacillus in the manual for identifying common bacteria systems.
(3) Molecular biological identification
Bacterial DNA extraction kit was used to extract the genome of strain WB75 as a template for PCR amplification. Designed for amplification:
performing PCR amplification using a pair of universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') of bacterial 16S rDNA sequences;
carrying out PCR amplification on the gyrA gene by using bacterial gyrA gene primers p-gyrA-f (5'-CAGTCAGGAAATGCGTACGTCCTT-3') and p-gyrA-r (5'-CAAGGTAATGCTCCAGGCATTGCT-3');
the DNA of the strain was PCR amplified using bacterial gyrB gene degenerate primers UP1S (5'-GAAGTCATCATGACCGTTCTGC-3') and UP2Sr (5'-AGCAGGGTACGGATGTGCGAGCC-3').
The 3 PCR amplification products are detected by 1% agarose gel electrophoresis, and the recovered products are sent to Huada Gene Co., Ltd for sequence determination after electrophoresis detection.
The obtained sequences were subjected to BLAST similarity analysis at NCBI, and multiple alignments of the sequences were performed using ClustalX2.0, followed by construction of a phylogenetic tree (bootstrap 1000) using Neighbor-Joining method using MEGA7.0 software package. The sequencing results are all submitted to a GenBank database to obtain sequence accession numbers.
Second, experimental results
1. Separation and screening of antagonistic bacteria
The method comprises the steps of separating endophytes of oncidium plants to obtain 119 endophytes, screening the separated endophytes by taking Colletotrichum gloeosporioides, a pathogenic bacterium of oncidium anthracnose, as an indicator bacterium, screening an YL94 strain, wherein the bacteriostatic rate of the YL94 strain reaches 54.43%, and performing bacteriostatic activity determination on a fermentation liquor of an YL94 strain, wherein the inhibition rate of a stock solution of the fermentation liquor is 67.11%, the inhibition rate is greatly reduced to 25.90%, and the results are shown in fig. 1 and table 1.
TABLE 1 inhibitory Effect of each treatment with Strain YL94 on anthracnose of oncidium
2. Results of strain morphology and physiological and biochemical experiments
The results are shown in FIG. 2. Morphological observation of the YL94 strain revealed that on LB medium, the bacteria were milky white, with rounded surface wrinkles, rough colonies and slightly irregular edges (fig. 2A). The strain YL94 was gram-positive and sporulated, cells were short rod-shaped, single or paired (fig. 2B), the above morphology and gram-staining, spore-staining similar to morphological features of Bacillus (Bacillus) (fig. 2C).
A series of physiological and biochemical experiments were also performed on the strain, and the results are shown in Table 2. The strain is identified according to the manual of identifying common bacteria systems, and the strain is preliminarily judged to be Bacillus subtilis with the number of YL 94.
TABLE 2 physiological and biochemical Properties of Strain YL94
Note: "+" and "-" indicate positive and negative, respectively
3. Molecular biological identification results
Using total DNA as a template, carrying out PCR amplification by using bacterial 16S rDNA universal primers 27F and 1492R to obtain a PCR amplification product with the length of 1421bp, and obtaining a PCR amplification product with a sequence shown as SEQ ID NO: 1 is shown. The Sequence of this PCR amplification product (16S rDNA of YL 94) was compared with the Sequence of the bacterial 16S rDNA registered in GenBank by NCBI (http:// www.ncbi.nlm.nih.gov /) for nucleotide homology, and found to have the highest similarity to Bacillus velezensis (Sequence ID: MT372157) and Bacillus velezensis (Sequence ID: MK445133), several known strains were selected and mapped into phylogenetic trees with the Sequence repeats of the selected strains using the software MEGA7, as shown in FIG. 3.
A gyrA gene sequence is amplified by using bacterial gyrA gene primers to obtain a 947bp gene fragment, and the gyrA gene sequence of YL94 is subjected to BLAST analysis in NCBI to find that the identity of the gyrA gene sequence with bacterial velezensis (CP021011), bacterial velezensis (MN648416), bacterial velezensis (CP061704) and bacterial velezensis (CP023320) strains can reach 98%, and the support rate is high. The sequence and phylogenetic analysis are shown in FIG. 4.
A gene fragment with a gyrB conserved gene sequence of 1147bp is determined by using bacterial gyrB gene universal primers, and the gene fragment and Bacillus amyloliquefaciens (CP017953) and Bacillus amyloliquefaciens (MH428832) form the same branch with 67% support rate. The sequence and phylogenetic analyses are shown in FIG. 5.
By integrating the identification and analysis of the thallus morphology, physiological and biochemical characteristics and gene sequence of the endogenous antagonistic bacteria, the strain YL94 is identified as Bacillus subtilis velezensis, the strain is named as Bacillus subtilis velezensis YL94 and is preserved in Guangdong province microbial strain preservation center at 12 months and 08 days 2020, and the preservation number is GDMCC No: 61355 the preservation address is Guangzhou institute of microorganisms of Guangdong province, No. 59 building, No. 5 building, Middledo, No. 100 college, Middleway, Guangzhou, China.
EXAMPLE 2 endophytic Strain YL94 pathogenicity verification experiment
First, experiment method
The pathogenicity of endophyte strain YL94 was verified on sterile tissue culture seedlings and potted seedlings, respectively.
1. Sterile tissue culture seedling of oncidium
Firstly, inoculating the bacterial strain to an LB culture medium, culturing for 4-5 days at 28 ℃ in the dark, and then beating a bacterial cake for later use by using a sterile puncher (with the inner diameter of 5 mm). Inoculating 3 sterile tissue culture seedlings of oncidium with consistent growth in an improved DE culture medium. Culturing for 7 days, inoculating 1 bacterial cake in the middle of 3 seedlings, inoculating sterile PDA block with the same size as Control (CK), repeating each treatment for 5 times, sealing, and illuminating for 10 hr (10/14h L/D) each day with intensity of 40 μmol. m-2·s-1Culturing at 25 +/-1 deg.C and RH 70% -75%And (5) nourishing. Periodically observing the symbiotic condition of the bacterins, and recording whether the bacterins are pathogenic or lethal.
2. Potted seedling of oncidium
The pathogenicity of bacteria on oncidium plants was verified by inoculation with a needle punch. The test adopts 8-month old potted seedling of oncidium hybridum provided by Boda orchid science and technology Limited, after wiping leaves with 75% alcohol, scrubbing the leaves with sterile water for 3 times, after drying the leaves, slightly stabbing the leaves with a disposable sterile acupuncture needle on the front surface, performing several times of stabbing on each wound (the occupied area is smaller than the bottom area of a fungus cake), treating 6-8 wounds on one leaf, then sticking the fungus cake on the wound, paving a wet sterile filter paper on the inoculated leaves for moisturizing, inoculating sterile PDA fungus cakes with the same size in contrast, and setting 3 times of repetition of the test treatment. All oncidium plants were grown at 28 ℃ and a relative humidity of about 60% and leaf development was observed and recorded.
Secondly, the pathogenicity determination result of the strain
The isolated YL94 strain was subjected to pathogenicity determination, as shown in fig. 6, and observed after 7 d: no lesion is generated when the puncture inoculation and the wounds of the aseptic PDA bacterial cakes of the control group are consistent; by using the bacterin symbiotic DE culture medium, the plants are cultured for two months in the tissue culture seedlings, and the disease phenomenon does not appear in the plants, which indicates that the YL94 strain has no pathogenic effect on the plants.
Example 3 broad-spectrum assay for the inhibition of endophyte strain YL94
First, experiment method
For testing oncidium disease pathogenic fungi strains:
various pathogenic bacteria collected in the laboratory, such as Colletotrichum tropicalis, Fusarium oxysporum, Fusarium solani, Fusarium oxysporum, Irish spot mango tee fungus Guignardia mangiferae, Pseudotheca septoriae fungus Paraconiothyrium chrysanolinaceae, leaf spot Curvularia angustifolia Curvularia grossedi, and Microcoproprion alconii, are all separated from the pathogenic lesion tissue of the onchavicum wenshuehne in the early stage of the laboratory, and are collected in the important laboratory of the department of genetics and germplasm education of the special forest of Hainan university.
The inhibition effect of the strain YL94 on the pathogenic bacteria of the above 8 12 oncidium plants is determined by adopting a confrontation culture method:
inoculating the above pathogenic bacteria to PDA culture medium, activating, punching bacterial cake at the edge of bacterial colony with a puncher with inner diameter of 5mm, inoculating to the center of PDA plate, and inoculating single bacterial colony of purified and separated bacterial strain YL94 to four ends with equal distance of 3cm from the center by cross method. And (3) taking a flat plate without adding endophytes as a negative control, repeating the test for 3 times, placing the flat plate in dark culture at 28 ℃ for 4-6 d, measuring the diameter of a bacterial colony by using a cross method every day, observing the morphological change of the bacterial colony of the pathogenic bacteria of the experimental group and calculating the hypha growth inhibition rate when the pathogenic bacteria of the control plate grow over the dish.
Inhibition (inhibition rate, IR) — (control colony diameter-treated colony diameter)/control colony diameter × 100%
Secondly, the result of the broad spectrum determination of the bacterial strain YL94
1. Bacteriostatic effect and rate
The bacteriostatic spectrum of strain YL94 is shown in FIG. 7 and Table 3, and the results of experiments on the opposing show that it has inhibitory effects on 12 strains of Erwinia ensiformis, i.e., Fusarium solani Colles, Fusarium solani Delphinii, Fusarium solani Solani, Fusarium oxysporum, Fusarium nigrum Magnaporta Magnifolium, Pseudocercospora septoriae, Curvularia angustifolia agrostis, and Microprocopron cornii, which are obtained by separation, and the bacteriostatic map of different pathogenic bacteria is shown in FIG. 7.
Wherein, the inhibition rate of the strain YL94 to 4 pathogenic bacteria is over 50%: the inhibitor has the strongest inhibition effect on the coccobactium nigrospora guignandii mangiferae, and the inhibition rate reaches 82.84% (fig. 7-H, WB 35). Secondly, the inhibition rate of Fusarium degerminum Fusarium delphinides reaches 56.31% (FIG. 7-E, WB6), and the inhibition rate of Fusarium solani reaches 51.40% (FIG. 7-E, WG1) and 50.1% (FIG. 7-B, WB 23). The results show that YL94 can inhibit different pathogenic fungi and shows broad-spectrum bacteriostatic activity.
TABLE 3 inhibition of different pathogenic bacteria by the strain YL94
Note: fusarium solani (WB 11); fusarium solani (WB 23); fusarium solani (WG 1); fusarium solani (W47); fusarium oxysporum Fusarium delphinidinides (WB 6); fusarium oxysporum (W38); fusarium oxysporum (WB 1); iregnia melanosporum Guignardia mangiferae (WB 35); pseudothecaceae fungus Paraconiothyriumthysanolanea (WG 2); curvularia angustifolia (WG3) of Curvularia angustifolia; colletotrichum tropicalis (WB 19); the fungus Muyocopropron alcornii of the family Macrothecaceae (WG 4).
2. Peak value of bacteriostasis
The peak inhibitory results are shown in fig. 8. Inhibition by YL94 strain on Guignardia mangiferae (WB35) peaked at 24h to 48h, inhibition by Colletotrichum gloeosporioides, Fusarium delphinioides (WB6), Fusarium solani (WB23), Fusarium oxysporum (W38), Fusarium solani (W47), Fusarium solani (WB11), Curvularia erasitidis and Paraconiothyrium thysanolana (WG2) peaked at 96h to 120h, inhibition by Colletotrichum tropicolia (19), Fusarium solani (WG1) peaked at 120h to 144h, inhibition by Fusarium oxysporium (1) and Fusarium 168 peak, and growth by pathogen was no longer controlled at 192h to 168 h. The inhibition effect on Muyocoprogen alcornii (WG4) is relatively poor, but the Muyocoprogen alcornii (WG4) still has a certain inhibition effect under the influence of YL94 strain relative to a control group.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> university of Hainan
<120> Bacillus belezii YL94 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1421
<212> DNA
<213> Bacillus velezensis(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
tgcagtcgag cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa 60
cacgtgggta acctgcctgt aagactggga taactccggg aaaccggggc taataccgga 120
tggttgtttg aaccgcatgg ttcagacata aaaggtggct tcggctacca cttacagatg 180
gacccgcggc gcattagcta gttggtgagg taacggctca ccaaggcgac gatgcgtagc 240
cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag 300
gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg 360
atgaaggttt tcggatcgta aagctctgtt gttagggaag aacaagtgcc gttcaaatag 420
ggcggcacct tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg 480
gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagggct cgcaggcggt 540
ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcattgg aaactgggga 600
acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt 660
ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggagcgaaag 720
cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa 780
gtgttagggg gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg 840
agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900
atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaat 960
cctagagata ggacgtcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag 1020
ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga tcttagttgc 1080
cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga aggtggggat 1140
gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggacagaac 1200
aaagggcagc gaaaccgcga ggttaagcca atcccacaaa tctgttctca gttcggatcg 1260
cagtctgcaa ctcgactgcg tgaagctgga atcgctagta atcgcggatc agcatgccgc 1320
ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac 1380
ccgaagtcgg tgaggtaacc tttatggagc cagccgccga a 1421
Claims (7)
1. Bacillus belgiiBacillus velezensis) YL94, wherein Bacillus belgii YL94 was deposited at Guangdong province culture Collection, 12/08/2020, with accession numbers GDMCC No: 61355.
2. use of Bacillus belgii YL94 and/or its fermentation broth according to claim 1 for inhibiting a plant pathogenic bacterium or for preparing a preparation for inhibiting a plant pathogenic bacterium, wherein the plant pathogenic bacterium is a pathogenic fungus of oncidium, wherein the pathogenic fungus of oncidium is selected from the group consisting of Colletotrichum gloeosporioides, Colletotrichum tropicalis, Fusarium Cicerinum, Fusarium solani, Fusarium oxysporum, Iris gabonensis, Pseudocercospora septoria, and combinations thereofParaconiothyrium thysanolaenaeLeaf spot of curvularia lunata or Sphaerothecaceae fungusMuyocopron alcorniiAny one of orSeveral kinds of them.
3. Use according to claim 2, wherein the pathogenic fungus of oncidium is the anthracnose of oncidium.
4. Use of Bacillus belgii YL94 and/or its fermentation broth according to claim 1 for controlling plant fungal diseases or for preparing a formulation for controlling plant fungal diseases, wherein the plant fungal diseases are pathogenic fungal diseases of oncidium, wherein the pathogenic fungal diseases of oncidium are fungal diseases of Colletotrichum gloeosporioides, Fusarium tropicalis, Fusarium Cicerinum, Fusarium putrescens, Fusarium oxysporum, Fusarium nigrosporum, Pseudocercospora dissovis, and mixtures thereofParaconiothyrium thysanolaenaeLeaf spot of curvularia lunata or Sphaerothecaceae fungusMuyocopron alcorniiAny one or more of them.
5. The use according to claim 4, wherein the pathogenic fungal disease of oncidium is oncidium anthracnose.
6. A preparation for controlling a plant fungal disease comprising Bacillus belgii YL94 and/or a fermentation broth thereof according to claim 1, wherein the plant fungal disease is a pathogenic fungal disease of oncidium, wherein the pathogenic fungal disease of oncidium is a fungal disease selected from the group consisting of Colletotrichum gloeosporioides, Colletotrichum tropicalis, Fusarium Cicerinum, Fusarium solani, Fusarium oxysporum, Irpex negoticola, Pseudocercospora dissovirida, and combinations thereofParaconiothyrium thysanolaenaeLeaf spot of curvularia lunata or Sphaerothecaceae fungusMuyocopron alcorniiAny one or more of them.
7. The formulation of claim 6, wherein the pathogenic fungal disease of oncidium is oncidium anthracnose.
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