NL2028299B1

NL2028299B1 - Bacillus velezensis (b. velezensis) strain and use thereof in control of plant downy mildew

Info

Publication number: NL2028299B1
Application number: NL2028299A
Authority: NL
Inventors: Wang Bo; Liang Chen; Xiao Qian; Li Shouwang
Original assignee: Univ Qingdao Agricultural
Priority date: 2020-11-06
Filing date: 2021-05-26
Publication date: 2022-06-24
Also published as: CN112195135A; CN112195135B

Abstract

The present disclosure provides a Bacillus velezensis (B. velezensis) strain HlVIQAU19044, and the strain was deposited in the China General Microbiological Culture Collection Center (CGMCC) on January 19, 2020, with deposit registration No: CGMCC No.19420. The B. velezensis strain HlVIQAU19044 provided in the present disclosure shows a significant control effect on cucumber downy mildew. Studies in the examples show that a fermentation filtrate of the strain HlVIQAUl9044 can significantly inhibit the release of zoospores of Pseudoperonospora cubensis (P. cubensis); a fermentation broth of the strain HlVIQAU19044 can significantly inhibit the release of zoospores of P. cubensis; the fermentation filtrate of the strain HlVIQAU19044 can significantly inhibit the resting spore germination and germ tube elongation of P. cubensis; the fermentation filtrate of the strain HlVIQAU19044 can inhibit the lesion expansion on diseased cucumber leaves to some extent; and the fermentation filtrate and fermentation broth of the strain HlVIQAUl9044 of the present disclosure exhibit prominent control effect on cucumber downy mildew.

Description

BACILLUS VELEZENSIS (B. VELEZENSIS) STRAIN AND USE THEREOF IN CONTROL OF PLANT DOWNY MILDEW TECHNICAL FIELD

[01] The present disclosure belongs to the technical field of biological control of plant diseases, and specifically relates to a Bacillus velezensis (B. velezensis) strain and use thereof in the control of plant downy mildew.

BACKGROUND ART

[02] Cucumber is one of the most important staple vegetables in China, but the occurrence of downy mildew causes heavy loss to cucumbers cultivated in open and protected fields all year round. Cucumber downy mildew 1s a severe disease caused by the infection of Pseudoperonospora cubensis (P. cubensis). Cucumber downy mildew has a strong epidemic, a faster transmission rate and a higher incidence. Under suitable environmental conditions, cucumber downy mildew can cause all leaves on cucumber plants to wither and die within just 1 to 2 weeks, which will directly affect the cucumber’s fruiting. Generally, downy mildew can cause a decrease of 30% to 50% in cucumber yield and a decrease of more than 70% in serious cases, and even may lead to no harvest. Therefore, downy mildew is one of the most serious diseases in cucumber production and cultivation, and the impact of the disease is huge and the loss is serious. .

[03] Cucumber varieties are generally not resistant to downy mildew, and pathogen is prone to pesticide resistant leading to pesticide residues exceeding the standard. Therefore, it is particularly critical to explore new control method.

[04] B. velezensis is a new species of Bacillus and a new biocontrol bacterium, which newly named by Ruiz-Garcia et al. in 2005. As a new biocontrol microbial factor, B. velezensis has attracted much attention in plant disease control, plant growth promotion, and the like. For example, Chinese patent CN201811068345.5 discloses a biocontrol B. velezensis strain and use thereof in the control of Sphaerotheca fuliginea;, Chinese patent CN201910453041.9 discloses a B. velezensis strain and use thereof in the control of root- knot nematodes (RKNs); Chinese patent CN201910891472.3 discloses a B. velezensis strain CY30 and use thereof in the control of Pestalotiopsis theae;, and Chinese patent CN201910297167.1 discloses a B. velezensis strain that inhibits viruses and promotes plant growth, and use thereof.

[05] However, there is currently no report of B. velezensis to control cucumber downy mildew at home and abroad. How to solve the above technical problem is a technical problem that needs to be solved in the field of microbiological technology.

SUMMARY

[06] The present disclosure is intended to provide a B. velezensis strain and use thereof in the control of plant oomycete diseases in view of the shortcomings in the prior art. The strain shows a prominent inhibitory effect on P. cubensis, which provides a new resource for using microorganisms instead of chemically-synthesized fungicides to control cucumber downy mildew by biological control and can be developed and utilized as a biological pesticide.

[07] The present disclosure provides a B. velezensis strain HMQAU19044, which was deposited in the China General Microbiological Culture Collection Center (CGMCC) on January 19, 2020, with deposit registration No.: CGMCC No.19420. The China General Microbiological Culture Collection Center (CGMCC) is located at No. 3, Courtyard 1, West Beichen Road, Chaoyang District, Beijing.

[08] The present disclosure also provides a microbial agent prepared from the B. velezensis strain HMQAU 19044 described above, and the microbial agent includes the strain HMQAU 19044 and/or metabolites of the strain HMQAU 19044.

[09] Preferably, an active ingredient of the microbial agent provided in the present disclosure is a fermentation broth or a fermentation filtrate of the strain HMQAU 19044.

[10] Preferably, the microbial agent provided in the present disclosure may be a liquid microbial agent and may have a live bacterium and/or spore number > 1.5 x 10°/mL to 2.0 x 10°/mL.

[11] The present disclosure also provides a method for preparing the microbial agent, specifically including the following steps:

[12] (1) preparing a solid LB medium;

[13] (2) preparing a liquid LB medium;

[14] (3) strain activation: picking up a loopful of bacterial colonies of the strain HMQAUI19044, inoculating in 50 mL of the liquid LB medium, and cultivating at 200 rpm and 30°C for 24 h to obtain a starter culture;

[15] (4) preparation of a fermentation broth: inoculating 1/50 of the prepared starter culture into 50 mL of the liquid LB medium, and cultivating at 200 rpm and 30°C for 48 h to obtain the fermentation broth of the strain HMQAU 19044; and

[16] (5) preparation of a fermentation filtrate: centrifuging the prepared fermentation broth at 4°C and 8,000 r/min for 15 min, and filtering a resulting supernatant with a 0.22 um microporous membrane to obtain the fermentation filtrate of the strain HMQAUI19044.

[17] The solid LB medium provided in the present disclosure may be prepared by the following method: dissolving 10 g of tryptone, 10 g of sodium chloride, 5 g of yeast extract, and 15 g to 20 g of agar powder in deionized water, with pH 6.8; diluting a resulting solution to 1 L with deionized water; and subjecting a resulting solution to moist-heat sterilization at 121°C for 25 min.

[18] The liquid LB medium provided in the present disclosure may be prepared by the following method: dissolving 10 g of tryptone, 10 g of sodium chloride, and S g of yeast extract in deionized water, with pH 6.8; diluting a resulting solution to 1 L with deionized water; and subjecting a resulting solution to moist-heat sterilization at 121°C for 25 min.

[19] The present disclosure also provides use of the microbial agent. Preferably, the microbial agent may be used for the control of cucumber downy mildew.

[20] The present disclosure also provides a method for controlling cucumber downy mildew, including: using the B. velezensis strain HMQAU19044 described above or the microbial agent described above for controlling cucumber downy mildew.

[21] The present disclosure has the following beneficial effects:

[22] 1. The B. velezensis strain HMQAU19044 provided in the present disclosure shows a significant control effect on cucumber downy mildew. Studies in the examples show that a fermentation filtrate of the strain HMQAU19044 can significantly inhibit the release of zoospores of P. cubensis, where, an original filtrate shows an inhibition rate up to 100% on the release of zoospores, and still shows an inhibition rate up to

92.99% on the release after being diluted by 12.5 times; a fermentation broth of the strain HMQAU19044 can significantly inhibit the release of zoospores of P. cubensis, where, an original fermentation broth shows an inhibition rate up to 100% on the release of zoospores, and still shows an inhibition rate up to 94.16% on the release after being diluted by 12.5 times; the fermentation filtrate of the strain HMQAUI19044 can significantly inhibit the resting spore germination and germ tube elongation of P.

cubensis; the fermentation filtrate of the strain HMQAU19044 can inhibit the lesion expansion on diseased cucumber leaves to some extent; and the fermentation filtrate and fermentation broth of the strain HMQAU19044 of the present disclosure exhibit prominent control effect on cucumber downy mildew.

[23] 2. The B. velezensis strain HMQAU19044 provided in the present disclosure shows a stable control effect, is environmentally friendly, and only requires a simple fermentation process for large-scale production, with low production cost.

[24] 3. The microbial agent of the present disclosure can be used to control various plant downy mildew diseases, including but not limited to cucumber downy mildew, spinach downy mildew, lettuce downy mildew, welsh onion downy mildew, grape downy mildew, or litchi downy mildew.

BRIEF DESCRIPTION OF DRAWINGS

[25] FIG. 1 shows the colonial morphology and Gram staining result of the strain HMQAU19044;

[26] FIG. 2 shows the effects of a fermentation broth of the strain HMQAU19044 on the release of zoospores of P. cubensis at different dilution factors,

[27] where, FIG. 2A shows the effect of an original fermentation broth, FIG. 2B shows the effect of a fermentation broth diluted 50 times, FIG. 2C shows the effect of sterile water control, and FIG. 2D shows the morphology of zoospores;

[28] FIG. 3 shows the effects of a fermentation filtrate of the strain HMQAU19044 on the resting spore germination of P. cubensis at different dilution factors,

[29] where, FIG. 3A shows the effect of an original fermentation filtrate, FIG. 3B shows the effect of a fermentation filtrate diluted 100 times, and FIG. 3C shows the effect of sterile water control; [B0] FIG. 4 shows the control efficiency of a bacterial suspension, fermentation filtrate, and fermentation broth of the strain HMQAU19044 on downy mildew of detached cucumber leaves,

[31] where, in FIG. 4 Aa, the pathogen is inoculated 24 h after the bacterial suspension is sprayed; in FIG. 4 Ab, the pathogen is inoculated after the bacterial suspension is sprayed and the leaves are slightly dried; in FIG. 4 Ac, the pathogen is inoculated 24 h before the bacterial suspension is sprayed; in FIG. 4 Ba, the pathogen is inoculated 24 h after the fermentation filtrate is sprayed; in FIG. 4 Bb, the pathogen is inoculated after the fermentation filtrate is sprayed and the leaves are slightly dried; in

FIG. 4 Bc, the pathogen is inoculated 24 h before the fermentation filtrate is sprayed; in FIG. 4 Ca, the pathogen is inoculated 24 h after the fermentation broth is sprayed; in FIG. 4 Cb, the pathogen is inoculated after the fermentation broth is sprayed and the leaves are slightly dried; in FIG. 4 Cc, the pathogen is inoculated 24 h before the 5 fermentation broth is sprayed; in FIG. 4D, the sterile water is sprayed as a control; and in FIG. 4E, the sterile liquid LB medium is sprayed as a control;

[32] FIG. 5 shows the control efficiency of a fermentation filtrate of the strain HMQAU 19044 on downy mildew of detached cucumber leaves,

[33] where, in FIG. 5 A, the fermentation filtrate of the strain HMQAU19044 is sprayed O h after the pathogen is inoculated; in FIG. 5 B, the fermentation filtrate of the strain HMQAU19044 is sprayed 1 h after the pathogen is inoculated; in FIG. 5 C, the fermentation filtrate of the strain HMQAU19044 is sprayed 2 h after the pathogen is inoculated; in FIG. 5 D, the fermentation filtrate of the strain HMQAU19044 is sprayed 3 h after the pathogen is inoculated; in FIG. 5 E, the fermentation filtrate of the strain HMQAUI19044 is sprayed 4 h after the pathogen is inoculated; and in FIG. 5 F, the sterile water is prayed as a control;

[34] FIG. 6 shows the control efficiency of a fermentation broth of the strain HMQAU19044 on downy mildew of detached cucumber leaves,

[35] where, in FIG. 6 A, the fermentation broth of the strain HMQAU19044 is sprayed O h after the pathogen is inoculated; in FIG. 6 B, the fermentation broth of the strain HMQAU19044 is sprayed 1 h after the pathogen is inoculated, in FIG. 6 C, the fermentation broth of the strain HMQAU19044 is sprayed 2 h after the pathogen is inoculated; in FIG. 6 D, the fermentation broth of the strain HMQAU19044 1s sprayed 3 h after the pathogen is inoculated; in FIG. 6 E, the fermentation broth of the strain HMQAUI19044 is sprayed 4 h after the pathogen is inoculated; and in FIG. 6 F, the sterile water is prayed as a control; and

[36] FIG. 7 shows the control efficiency of the strain HMQAU19044 on cucumber downy mildew of indoor potted plants,

[37] where, FIG. 7 A shows the treatment 1 in Example 6; FIG. 7 B shows the treatment 2 in Example 6; and FIG. 7 C shows the treatment 3 in Example 6.

DETAILED DESCRIPTION OF THE EMBODIMENT

[38] The technical solutions of the examples of the present disclosure are clearly and completely described below with reference to the content in the examples of the present disclosure. Apparently, the described examples are merely some rather than all of the examples of the present disclosure. All other examples obtained by a person of ordinary skill in the art based on the examples of the present disclosure without creative efforts shall fall within the protection scope of the present disclosure.

[39] Unless defined otherwise, all technical and scientific terms used in the specification have the same meanings as those commonly understood by those skilled in the art of the present disclosure.

[40] In the present disclosure, the B. velezensis strain HMQAU19044 can also be simply referred to as strain HMQAU19044.

[41] Example 1 Isolation and screening of an antagonistic strain

[42] 1. Isolation and preliminary screening of a strain

[43] (1) Isolation of an antagonistic strain

[44] In July 2019, tomato roots with root knots of RKNs were collected in He'an Village, Liugezhuang Town, Haiyang City, Shandong Province. About 1 g of the tomato roots with root knots were subjected to surface disinfection and then chopped with sterilized scissors, then 1 mL of sterilized water was added, and a resulting mixture was ground with a sterilized mortar and pestle. The dilution-plate method was used to isolate a bacterial strain from a liquid obtained from the grinding, and the medium used was an LB solid medium. After bacterial colonies grew, single colonies were picked up and streaked on an LB solid plate for purification, and stored.

[45] (2) Preparation of a bacterial fermentation supernatant

[46] A bacterial strain kept as a slant was streaked on an LB solid medium and cultivated at 30°C for 24 h. Cultivated colonies were transferred by a transfer needle to a250 mL Erlenmeyer flask with 125 mL of a liquid LB medium, and cultivated at 30°C and 200 r/min for 48 h under shaking to obtain a bacterial fermentation broth. The bacterial fermentation broth was centrifuged at 5,000 r/min for 5 min, and a resulting supernatant was collected and stored in a refrigerator at 4°C.

[47] (3) Acquisition of a large number of second-instar larvae of Meloidogyne incognita

[48] Plant root knots propagated from RKNs in the laboratory were collected and sterilized with 0.5% NaClO for 2 min, and rinsed 3 times with sterile water. Egg masses were collected and placed in a sterilized petri dish with sterilized tweezers, and hatched in a 28°C incubator. Hatched second-instar larvae (J2) were collected and stored in a refrigerator at 4°C for later use.

[49] (4) Effects of fermentation supernatants of biocontrol bacteria on Meloidogyne incognita J2

[50] 1 mL of the fermentation supernatant was taken, and 30 Meloidogyne incognita J2 were added and cultivated in a 28°C incubator. The posture method and normal saline stimulation method (reference: Fang Zhongda. Plant disease research methods, Beijing: China Agriculture Press, 1998: 307-320.) were adopted, and the death of J2 was observed at 6 h and 12 h respectively under a stereoscopic microscope. The number of J2 deaths was recorded, and the mortality and corrected mortality were calculated for J2. [S1] Mortality of RK Ns J2 = number of dead J2/number of test J2 * 100%

[52] Corrected mortality of RKNs J2 = [(J2 mortality in treatment - J2 mortality in control)/(1 - J2 mortality in control)] [S3] A bacterial fermentation broth was prepared from the isolated and purified bacteria by streaking activation and transferring. A supernatant was collected and a nematode-killing activity thereof on Meloidogyne incognita was determined. In the experiment, a total of 3 strains exhibiting a lethality of more than 70% on Meloidogyne incognita J2 at 12 h were screened out, namely, strain 4-2, strain HMQAU19044, and strain 4-5.

[54] Table 1 Effects of fermentation supernatants of biocontrol bacteria on Meloidogyne incognita J2

[55] Strain No. 6h 12h 31 937+815cdefe 40.63x4274efech 3-2 10.5743. 74cdety 47 87419 26def 3-3 3.57+6.42defg 13.68+8.95ghi 3-5 9.90+8 94cdefg 50.40+28.57bcdefg 3-6 12.93+11.12ecdef 85.23+3.69ab 4-1 17.50+7.35bed 48.93+30.51cdef 4-2 14.23+3.77bcdef 79.80118.53abecd

Strain HMQAU19044 24 77+15.34ab 78.87+6.21abed 4-4 0.00+0.00g 1.07+1.851 4-5 16.28+3.89bcde 75.67+8.84abcde 4-6 5.53+4.99defg 9.70+5.56hi

[56] Notes: In each column, those with the same letter show a difference not reaching a significant level (P > 0.05), and those with different letters show a difference reaching a significant level (? < 0.05).

[57] 2. Screening of antagonistic bacteria against cucumber downy mildew

[58] The preliminarily-screened strains 4-2, HMQAU19044, and 4-5 were separately cultivated at 37°C and 200 r/min for 2 d under shaking in LB, and then a bacterial concentration was adjusted to 1 x 10%/mL with a hemacytometer for later use. Fresh downy mildew sporangia were collected, and a concentration of the sporangia was adjusted to 5 x 10*/mL with a hemacytometer for later use. The bacterial strains 4-2 and 4-5 were initially identified as Bacillus cereus (B. cereus) and had the same origin as the strain HMQAU 19044.

[59] Cucumber leaves of the same size were selected, petioles were wrapped with absorbent cotton, and then the leaves were placed in a petri dish with wet filter paper, with a back side of the leaves facing upward. The bacterial fermentation broth was first evenly spotted on the cucumber leaves, and 24 h later, a prepared pathogenic spore suspension was spotted on the back side of the leaves, with 5 spots for each leaf and 10 uL for each spot. The dish was covered and incubated at 25°C in a 12 h light-dark cycle for 7 d, and then results were observed. Sterile water instead of the bacterial fermentation broth was sprayed as a control, and 3 leaves were used for each treatment.

[60] The grading criteria of Technical Identification Rules for Cucumber Resistance to Downy Mildew (2010) in the national agricultural industry standards were adopted as the investigation grading criteria for cucumber disease index, as follows:

[61] grade 0: asymptomatic;

[62] grade 1: with a diseased spot area less than 1/10 of a total leaf area;

[63] grade 3: with a diseased spot area 1/10 to 1/4 of a total leaf area,

[64] grade 5: with a diseased spot area 1/4 to 1/2 of a total leaf area;

[65] grade 7: with a diseased spot area 1/2 to 3/4 of a total leaf area; and

[66] grade 9: with a diseased spot area more than 3/4 of a total leaf area, even withered.

[67] A calculation formula of the disease index:

[68] disease index = > (number of diseased leaves at all grades x relative grade) x 100/[total number of investigated leaves x 9]

[69] Table 2 Screening of antagonistic bacteria against cucumber downy mildew

[70] {a}

CK JE Fermentation broth of 4-2 183 39.3 Fermentation broth of 4-5 223 35 Fermentafion breth of strain © GG

BMOAT ISS

[71] It can be seen from Table 2 that detached leaves sprayed with the fermentation broth of the strain HMQAU19044 had a disease index of 0 and were not attacked by the disease, so the strain would be used for the follow-up in-depth study.

[72] Example 2 Identification of the antagonistic bacterial strain HMQAU19044 against cucumber downy mildew

[73] 1. Morphological identification

[74] The strain HMQAU19044 on a slant was transferred to an LB medium plate by three-zone streaking, and cultivated in a 30°C incubator for 48 h. After distinct single colonies grew, the colony morphology was observed and photographed, including the shape, size, color, surface, edge, transparency, and bulge shape of bacterial colonies. The bacterial strain cultivated for 24 h was subjected to Gram staining microscopy by a method with reference to the Manual for Identification of Common Bacterial Systems.

[75] After the strain HMQAU19044 was cultivated on an LB solid medium at 30°C for 2 d, formed colonies were bulged, milky-white, opaque, and round and had a diameter of 2.5 mm to 5 mm, irregular edges, and wrinkled surfaces, and the bacteria were Gram-positive and short rod-shaped and could produce oval spores, as shown in FIG. 1.

[76] 2. Physiological and biochemical identification

[77] With reference to the Manual for Identification of Common Bacterial Systems, the strain HMQAU19044 was subjected to aerobism, growth temperature, salt tolerance, growth pH, citrate utilization, contact enzyme, oxidase, glucose oxidative fermentation, indole reaction, nitrate reduction, denitrification, fermentation of sugar and alcohol, methyl red (MR) reaction, V-P determination, starch hydrolysis, gelatin liquefaction, and other tests.

[78] With reference to relevant contents of the Manual for Identification of Common Bacterial Systems and Berger's Bacterial Identification Manual, the physiological and biochemical experimental results were analyzed for the strain HMQAU19044, and identification results were shown in Table 3. It can be seen from Table 3 that the strain HMQAU19044 was an aerobic strain; could grow in a 2% to 10% (mass fraction) NaCl medium at pH 5.5 and 20°C or 45°C, but cannot grow at pH 9.0 and 4°C; could decompose and utilize glucose, sucrose, maltose, and mannitol, but cannot ferment fructose, lactose, and mannose; could hydrolyze starch and gelatin, but cannot produce indole; could produce acids with glucose; and showed positive results in contact enzyme reaction, V-P reaction, and nitrate reduction reaction, but negative results in oxidase reaction, citrate reaction, denitrification reaction, and MR reaction. According to the morphological characteristics and physiological and biochemical identification results of the strain HMQAU 19044, the strain HMQAU 19044 had characteristics similar to that of B. velezensis, so the strain was preliminarily identified as B. velezensis.

[79] Table 3 Physiological and biochemical identification results of the strain HMQAU19044

[80] Test name Result Aerobism + Salt tolerance 2% + 5% + 10% + Temperature 4°C - 20°C + 45°C + pH 55 +

9.0 - Starch hydrolysis + Oxidase - Contact enzyme + Indole reaction -

Citrate utilization - Glucose oxidative fermentation + Nitrate reduction + Denitrification - MR - V-P + Gelatin liquefaction + Fermentation of sugar and alcohol Maltose + Mannitol + Glucose + Sucrose + Fructose - Lactose - Mannose -

[81] 3. Molecular biological identification

[82] The gyrB sequence analysis is a molecular biological identification method for the identification of Bacillus. The extraction of genomic DNA (gDNA) of the strain can refer to the chelex100 method of Xia Han et al. (reference: Xia Han, Fu Weiling, Chen Ming, Huang Qing, Zhao Meng, Zhao Yuhui, Wang Feng, and Luo Yang. Study on Rapid Extraction method of Bacterial DNA [J]. Modern Preventive Medicine, 2005, 32 (5): 571-573).

[83] gB primers UPI (5'- GAAGTCATCATGACCGTTCTGCAY GCNGGNGGNAARTT YGA-3') and UP2r (5'- AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCA T-3') were used to amplify a genome of the strain.

[84] Reaction system: 25 pL in total: 2.5 uL of 10 x Buffer, 2 pL of 5 mmol/L dNTP, 1 pL of each of 10 pmol/L primers UP1 and UP2r, 0.5 pL of PremixTaq (5 U/uL), 2.5 pL of template DNA, and the balance of water.

[85] Reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 98°C for 10 s, annealing at 65°C for 1 min, extension at 72°C for 2 min, 30 cycles; final extension at 72°C for 8 min. 5 uL of an amplification product was taken for the amplification effect determination by 1% agarose gel electrophoresis, and the remaining amplification product was recovered with a gel recovery kit. A recovered amplification product was subjected to T-A cloning and transformation with a pMD 18-T vector, and a product was subjected to bacterial-liquid PCR and then sent for bi-directional sequencing.

[86] In the present disclosure, the gyrB sequencing results were automatically assembled by the software Sequencher 5.0 to derive a contig, and BLAST analysis was conducted in the NCBI (http://www.ncbi.nlm.gov) database.

[87] In summary, a taxonomic status of the strain was further determined through gene sequence alignment and physiological and biochemical identification, and the strain HMQAU19044 was identified as B. velezensis. The strain was deposited in the China General Microbiological Culture Collection Center (CGMCC) on January 19, 2020, with deposit registration No.: CGMCC No.19420. The China General Microbiological Culture Collection Center (CGMCC) is located at No. 3, Courtyard 1, West Beichen Road, Chaoyang District, Beijing.

[88] Example 3 Inhibitory effect of the strain HMQAU19044 on P. cubensis at different developmental stages

[89] 1. Preparation of a strain HMQAU19044 and microbial agents thereof

[90] A solid LB medium was prepared as follows: 10 g of tryptone, 10 g of sodium chloride, 5 g of yeast extract, and 15 gto 20 g of agar powder were dissolved in deionized water, with pH 6.8; a resulting solution was diluted to 1 L with deionized water; and a resulting solution was subjected to moist-heat sterilization at 121°C for 25 min.

[91] Aliquid LB medium was prepared as follows: 10 g of tryptone, 10 g of sodium chloride, and 5 g of yeast extract were dissolved in deionized water, with pH 6.8; a resulting solution was diluted to 1 L with deionized water; and a resulting solution was subjected to moist-heat sterilization at 121°C for 25 min.

[92] Strain activation: a loopful of bacterial colonies was picked up and inoculated in 50 mL of the liquid LB medium, and cultivated at 200 rpm and 30°C for 24 h to obtain a starter culture.

[93] Preparation of a fermentation broth: 1/50 of the prepared starter culture was inoculated into SO mL of the liquid LB medium, and cultivated at 200 rpm and 30°C for 48 h to obtain the fermentation broth of the strain HMQAU19044. The bacterial liquid had a live bacterium and/or spore number > 1.5 x 10°/mL to 2.0 x 10°/mL.

[94] Preparation of a fermentation filtrate: the prepared fermentation broth was centrifuged at 4°C and 8,000 r/min for 15 min, and a resulting supernatant was filtered with a 0.22 um microporous membrane to obtain the fermentation filtrate of the strain HMQAU19044.

[95] Preparation of bacteria: A precipitate obtained from the centrifugation of the fermentation broth was washed with sterile water, and a resulting mixture was centrifuged at 8,000 r/min for 5 min. The operations were repeated three times. The bacteria were diluted with sterile water and prepared into a bacterial suspension at 1 x 10°/mL with a hemacytometer.

[96] 2. Inhibitory effect of the strain HMQAU19044 on zoospores of P. cubensis

[97] (1) Effect of bacteria on the release and movement of zoospores of pathogens

[98] A P cubensis sporangium suspension with a concentration of 1 x 10°/mL was prepared with sterilized tap water. The bacterial suspension of the strain HMQAU19044 was serially diluted to obtain bacterial suspensions with concentrations of 1 = 10°%/mL, 1 x 10%mL, 1 x 107/mL, 1 x 10%mL, 1 x 10%mL, and 1 x 10%mL, and the obtained bacterial suspensions each were mixed with the same volume of the sporangium suspension. A mixture of sterilized tap water and the sporangium suspension in equal volumes was adopted as a control. For each treatment, 40 uL of a sample was added dropwise on a concave slide, then the concave slide was put in a petri dish covered with wet filter paper, and the dish was incubated in the dark for 1 h in a refrigerator at 4°C and RH > 80% and then incubated for 1 h in a 20°C incubator. After more than 80% of the sporangia in the control released zoospores, the emptying rate of 100 sporangia and the movement frequency of zoospores were observed under a microscope, and microphotographs were taken. 3 replicates were set for each treatment, and the experiment was repeated 3 times.

[99] Inhibition rate of zoospore release (%) = (release rate of zoospores in the control group - release rate of zoospores in the treatment group)/release rate of zoospores in the control group x 100%

[100] Inhibition rate of zoospore movement (%) = (movement frequency of zoospores in the control group - movement frequency of zoospores in the treatment group)/movement frequency of zoospores in the control group * 100%

[101] Table 4 Effects of bacterial suspensions of the strain HMQAU19044 on the release and movement of zoospores of P. cubensis

[102]

Concentration of Release rate of Inhibition rate Mlevement Ealtbifion rte bacterial Tonspores {95} of release (36) frequency of of movement SUSpEnsIon zoospores {3} is) fbactens/mL] CE 5inZ2 - S143 3 - 1195 25751 1.46 TI28hH 17.66 ste £352 ab 3.58 LBS h 18.18 ix 74 70d id 1 5433 abs FIR Ls Aided 13.18 53.230 ab GIN 1a 1° Tass d 14.33 TR20h 14 47 twig? TG 74 be 8.37 7834 b 13.32

[103] Note: The letters after the numbers in the same column indicate significant differences at the p < 0.05 level (Duncan's new multiple range method).

[104] It can be seen from experimental results shown in Table 4 that the bacterial suspensions of the strain HMQAU19044 exhibited no significant inhibitory effects on the release and movement of zoospores of P cubensis, with the highest release inhibition rate only of 14.33% and the highest movement inhibition rate only of 18.12%.

[105] (2) Effects of fermentation filtrates on the release and movement of zoospores of pathogens

[106] A sporangium suspension with a concentration of 1 x 105/mL was prepared with sterilized tap water. The fermentation filtrate was diluted at the dilution factors of 0,

12.5, 25, 50, 100, 150 and 200 times, separately, and obtained fermentation filtrates each were mixed with the same volume of the sporangium suspension. A mixture of sterilized tap water and the sporangium suspension in equal volumes was adopted as a control. For each treatment, 40 uL of a sample was added dropwise on a concave slide, then the concave slide was put in a petri dish covered with wet filter paper, and the dish was incubated in the dark for 1 h in a refrigerator at 4°C and RH > 80% and then incubated for 1 hin a 20°C incubator. After more than 80% of the sporangia in the control released zoospores, the emptying rate of 100 sporangia and the movement frequency of zoospores were observed under a microscope, and microphotographs were taken. 3 replicates were set for each treatment, and the experiment was repeated 3 times,

[107] Table 5 Effects of fermentation filtrates of the strain HMQAU19044 on the release and movement of zoospores of P. cubensis

[108]

Erlunon factor of Release rate of Thabo Movement Inhibition rate fermentation Hiwate Zoospares {Ye} gate of frequency of of movement release (5) zoospares {Sel £3 Ongiaal LED 105.568 G80 100 48 fermentston flvate 135% S08e S299 T&R ab R68 23x 18.704 TE 44 B IIHT Six JEE 44 68 F251 ab 1471 200% 86.493 0.2% ETI 26.85

[109] Note: The letters after the numbers in the same column indicate significant differences at the p < 0.05 level (Duncan's new multiple range method).

[110] It can be seen from experimental results shown in Table 5 that the fermentation filtrates of the strain HMQAU19044 exhibited a significant inhibitory effect on the release of zoospores of P. cubensis, where, the original filtrate exhibited an inhibition rate as high as 100% on zoospore release, and a filtrate diluted by 12.5 times still exhibited a release inhibition rate as high as 92.99%. A regression curve (y = -

3.4722x+10.567 (R? = 0.9480)) was obtained from the inhibition rates of fermentation filtrates at different dilution factors on the zoospore release, with a median effective inhibitory concentration (ECso value) of 40.11. However, the fermentation filtrates did not show a significant inhibitory effect on the movement of zoospores.

[111] (3) Effects of fermentation broths on the release and movement of zoospores of pathogens

[112] A sporangium suspension with a concentration of 1 = 105/mL was prepared with sterilized tap water. The fermentation broth was diluted at the dilution factors of 0, 12.5, 25, 50, 100, 150 and 200 times, separately, and obtained fermentation broths each were mixed with the same volume of the sporangium suspension. A mixture of sterilized tap water and the sporangium suspension in equal volumes was adopted as a control. For each treatment, 40 pL of a sample was added dropwise on a concave slide, then the concave slide was put in a petri dish covered with wet filter paper, and the dish was incubated in the dark for 1 h in a refrigerator at 4°C and RH > 80% and then incubated for 1 hin a 20°C incubator. After more than 80% of the sporangia in the control released zoospores, the emptying rate of 100 sporangia and the movement frequency of zoospores were observed under a microscope, and microphotographs were taken. 3 replicates were set for each treatment, and the experiment was repeated 3 times.

[113] Table 6 Effects of fermentation broths of the strain HMQAU19044 on the release and movement of zoospores of P. cubensis

[114] Elston Hotor of Release rte of Inhibition me Movement Inhsbifion rate fenmenishon broth zoospores (55) of relesae fs} frequency of of moversem zoospores {Ye} ie Original 2.00 MOD 4.80 100.00 fermentation broth 13 5x 338¢c 8116 84 88 cd 2131 Ide 53903 303 F288 abe 11.72 EK SLT 3 - F256a - [IIS] Note: The letters after the numbers in the same column indicate significant differences at the p < 0,05 level (Duncan's new multiple range method).

[116] It can be seen from experimental results shown in Table 6 and FIG. 2 that the fermentation broths of the strain HMQAU 19044 exhibited a significant inhibitory effect on the release of zoospores of P. cubensis, where, the original fermentation broth exhibited an inhibition rate as high as 100% on zoospore release, and a fermentation broth diluted by 12.5 times still exhibited a release inhibition rate as high as 94.16%. A regression curve (y =-3.3227 x + 10.473 (R2= 0.9586)) was obtained from the inhibition rates of fermentation broths at different dilution factors on the zoospore release, with a median effective inhibitory concentration (ECso value) of 44.38. However, the fermentation broths did not show a significant inhibitory effect on the movement of zoospores.

[117] 3. Effects of fermentation filtrates on the germination and germ tube elongation of cystospores of P. cubensis

[118] A sporangium suspension with a concentration of 1 < 105/mL was prepared with sterilized tap water. 20 pL of the sporangium suspension was added dropwise to a concave slide and then incubated for 2 h in a dark incubator at 13°C and RH > 80%. After more than 80% of the sporangia in the control released zoospores, 20 pL of each of fermentation filtrates at the dilution factors of 100, 50, 25, and 12.5 times and an original fermentation filtrate was added, and a resulting mixture was incubated for 4 h to 5 hina dark incubator at 13°C and RH > 80%. After more than 80% of the zoospores became cystospores, the mixture was further cultivated for 4 h, and when a germination rate of cystospores in the control was greater than 45%, a germ tube length was recorded and measured by photographing.

[119] Table 7 Effects of fermentation filtrates on the resting spore germination and germ tube elongation of P. cubensis

[120] fermentation limite rate of germination {Se} fram} Tate {20} eystospores {463 Ceamal DDG 100.40 Da TLD fermentation filtrate

12.5% 1.554 97.83 18.8¢ 4 FIR Fix 2628 SES ZE 72 ed 85.00 G0 58548 i128 81540 25 88 LE S228 3 - E2053 -

[121] Note: The letters after the numbers in the same column indicate significant differences at the p < 0.05 level (Duncan's new multiple range method).

[122] It can be seen from Table 7 and FIG. 3 that the fermentation filtrates of the strain HMQAUI19044 exhibited a significant inhibitory effect on the germination and germ tube elongation of cystospores of P. cubensis. The original fermentation filtrate exhibited an inhibition rate up to 100% on both the germination and the germ tube elongation of cystospores. Except that the treatment with a fermentation filtrate diluted by 100 times exhibited no significant difference from the control in terms of the cystospore germination rate, and the other treatments all exhibited a significant difference from the control. A regression curve (y = -3.343 x + 10.391 (R2=0.9437)) was obtained from the inhibition rates of fermentation filtrates at different dilution factors on cystospore germination, with a median effective inhibitory concentration (ECso) of 40.98. A regression curve (y =-1.5097 x + 7.4736 (R2=0.9488)) was obtained from the inhibition rates of fermentation filtrates at different dilution factors on germ tube elongation, with a median effective inhibitory concentration (EC) of 43.50.

[123] 4. Effects of fermentation filtrates on the diseased spot expansion and spore production of P. cubensis

[124] In this experiment, the spot method was adopted, and detached leaves were used instead of leaf disks. Cucumber leaves of the same size were placed in a petri dish with wet filter paper, with a back side of the leaves facing upward. A sporangium suspension with a concentration of 1 x 105/mL was prepared with sterilized tap water and inoculated on the back side of the leaves, with 5 spots for each leaf and 10 pL for each spot. Then the leaves were incubated in an incubator with a 12 h light-dark cycle (22°C in the day and 18°C at night) for 4 d, at which time, diseased spots occurred, but no spores were produced. Then the initial diseased spot area was investigated. Then fermentation filtrates diluted by 100, 50, 25, and 12.5 times and an original fermentation filtrate were evenly sprayed on the surface of the leaves with a small watering can until the filtrate ran off. Sterile water was sprayed as a blank control. The leaves were incubated for 3 d to 6 d until spores were produced. A final diseased spot area was investigated. An inhibition rate on the expansion of diseased spot area was calculated according to initial and final diseased spot areas under treatments at different dilution factors. A spore production per unit diseased spot area was investigated as follows: the mould layer on the leaves of each treatment was washed off with 1 mL of sterile water to obtain a sporangium suspension, the sporangium suspension was well shaken, and 10 uL was taken for determining the number of sporangia under a 10 x 10 microscope; and the number of sporangia per mL of the sporangium suspension was converted into the spore production per unit diseased spot area. The spore productions of leaves in the treatments and control were compared to calculate inhibition rates of the fermentation filtrates at different dilution factors on spore production.

[125] Diseased spot expansion = average value of final diseased spot area - average value of initial diseased spot area

[126] Inhibition rate of diseased spot expansion (%) = [(average diseased spot expansion in the control - average diseased spot expansion in the treatment)/average diseased spot expansion in the control] x 100%

[127] Inhibition rate on spore production per unit diseased spot area (%) = [(spore production per unit diseased spot area in the control - spore production per unit diseased spot area in the treatment)/spore production per unit diseased spot area in the control] x 100%

[128] Table 8 Effects of fermentation filtrates of the strain HMQAU19044 on the diseased spot expansion and spore production of P. cubensis

[129]

Dilution facker Diseased spot expansion Spore production per Tut of fermentation Average expansen Inhibusm Spore producine ver ua: Inhstbon filtrate tom} rate Dd (spores'cn aL} rate {3a} fermentation filtrate 135% Gost ab 31.57 25 ed 58.440 Thx 041 ab 31.57 88 od 58.42 ix C885 a 3.33 13 bk JAT CE {3 064 ab - 2342 -

[130] Note: The letters after the numbers in the same column indicate significant differences at the p < 0.05 level (Duncan's new multiple range method).

[131] It can be seen from Table 8 that the fermentation filtrates of the strain HMQAUI19044 could inhibit the expansion of diseased spots on diseased cucumber leaves to some extent, but there was no significant difference between each treatment and the control. A regression curve (y = - 0.3985 x + 5.025 (R? = 0.9867)) was obtained from the inhibition rates of fermentation filtrates at different dilution factors, with a median effective inhibitory concentration (ECso) of 1.16.

[132] However, the fermentation filtrates of the strain HMQAU19044 could effectively inhibit the spore production of F cubensis, the spore production per unit diseased spot area increased with the increase of the dilution factor, and there was a significant difference between each treatment and the control. A regression curve (y = -

0.4662 x + 5.7866 (R2 = 0.9335)) was obtained from the inhibition rates of fermentation filtrates at different dilution factors on spore production per unit diseased spot area, with a median effective inhibitory concentration (ECso) of 48.67.

[133] Example 4 Control effects of the strain HMQAU19044 sprayed in different ways on downy mildew of detached cucumber leaves

[134] Cucumber leaves of the same size were selected and rinsed with tap water, and water on the leaves was removed. Then the leaves were put in a petri dish with wet filter paper for later use, with a back side of the leaves facing upward. A sporangium suspension with a concentration of 1 x 10°/mL was prepared with sterilized tap water. 9 treatments were set in this experiment: combinations of each of 3 spraying times (a. the sporangium suspension was inoculated 24 h after the agent was sprayed; b. the sporangium suspension was inoculated immediately after the agent was sprayed and the leaves were slightly dried; and c. the sporangium suspension was inoculated 24 h before the agent was sprayed) with each of 3 spraying agents (A. bacterial suspension; B. fermentation filtrate; and C. fermentation broth).

[135] The inoculation was conducted by the spot method, with S spots for each leaf and 10 pL for each spot. Sterile water was inoculated as a control for A and B, and a sterile LB medium was inoculated as a control for C. The cultivation was conducted in a 12 h light-dark cycle (22°C in the day and 18°C at night). 7 d later, the size was determined for diseased spots, and the disease index and control efficiency were calculated according to the diseased spot size.

[136] Table 9 Control efficiency of the strain HMQAU19044 on downy mildew of detached cucumber leaves

[137] Treatment Disease index Control efficiency {%) Bacterial Fermentation Faymentation Bacterial Fermentation Fermentation suspension filtrate broth suspension filtrate broth Inoqulation 25.934 5.005 133b -133.39 76.72 93.81 24 b after application Inoculation 17.04 a 3.00 kh 0,00 kh -53.38 1090.00 100.00 while application Application 22.982 17.78 a 12.78 a -108.66 17.23 40.30 24 h after inoculation CK ilila 2148 3 2148 3 - - -

[138] Note: The letters after the numbers in the same column indicate significant differences at the p < 0.05 level (Duncan's new multiple range method).

[139] It can be seen from Table 9 and FIG. 4 that, among the three treatments using A (bacterial suspension), there was no significant difference among the control efficiency of a (inoculation 24 h after application), b (inoculation while application), and c (application 24 h after inoculation), and the three treatments basically exhibited no control efficiency on the disease and even tended to aggravate the disease; among the treatments using B (fermentation filtrate), there was no significant difference between the high control efficiency of a (inoculation 24 h after application) and b (inoculation while application), and the b treatment exhibited a control efficiency as high as 100%, but the control efficiency of the c (application 24 h after inoculation) treatment was significantly lower than that of the other two treatments, which was only of 17.23%; and among the treatments using C (fermentation broth), there was no significant difference between the control efficiency of a (inoculation 24 h after application) and b (inoculation while application), which both were higher than 90%, and the b treatment exhibited the optimal effect, but the control efficiency of the c (application 24 h after inoculation) treatment was significantly lower than that of the other two treatments, which was only of 40.50%, as shown in FIG. 4. It can be seen that the application of the fermentation broth and fermentation filtrate exhibited a high control efficiency on the disease; the application of the bacterial suspension exhibited no control efficiency on the disease; and the treatment of first application and then inoculation exhibited a better control efficiency than the treatment of first inoculation and then application.

[140] Example 5 Treating effects of the strain HMQAU19044 sprayed at different times on downy mildew of detached cucumber leaves

[141] In order to further explore the treating effects of the biocontrol bacterium strain HMQAU19044 on cucumber downy mildew, cucumber leaves of the same size were selected and rinsed with tap water, and water on the leaves was removed. Then the leaves were put in a petri dish with wet filter paper for later use, with a back side of the leaves facing upward. A sporangium suspension with a concentration of 1 = 10°/mL was prepared with sterilized tap water. The inoculation was conducted by the spot method, with 5 spots for each leaf and 10 uL for each spot. The fermentation filtrate and fermentation broth of the strain HMQAU19044 were evenly sprayed at O h, 1 h, 2 h, 3 h, and 4 h after the pathogens were inoculated, and sterile water was evenly sprayed as a control. The cultivation was conducted in a 12 h light-dark cycle (22°C in the day and 18°C at night). 7 d later, the size was determined for diseased spots, and the disease index and control efficiency were calculated according to the diseased spot size.

[142] Table 10 Treating effects of the strain HMQAU19044 on downy mildew of detached cucumber leaves

[143] ~ Diseaseindex(%) Relative control efficiency (%) Time “Fermentation Fermentation ~~ Fermentation Fermentation filtrate broth filtrate broth oh 000 000 10000 10000 Ih 0.00 0.00 100.00 100.00 2h 8.89 ¢ 6.06 c 77.36 84.23

3h 14.07 be 14.82 be 64.16 64.90 4h 21.48b 25.93 b 45.29 38.58 CK 39.26 a 42.224 - -

[144] Note: The letters after the numbers in the same column indicate significant differences at the p < 0.05 level (Duncan's new multiple range method).

[145] It can be seen from Table 10, FIG. 5, and FIG. 6 that the fermentation filtrate and fermentation broth of the strain HMQAU19044 sprayed at 1 h after the pathogens were inoculated exhibited excellent treating effects on the disease, with relative control efficiency up to 100%; in the case where the fermentation broth and fermentation filtrate of the strain HMQAU19044 were sprayed at 2 h after the pathogens were inoculated, diseased spots began to appear on the leaves, and the relative control efficiency gradually decreased over time, as shown in FIG. 5 and FIG. 6; and the fermentation filtrate and fermentation broth of the strain HMQAU19044 sprayed at 4 h after the pathogens were inoculated exhibited relative control efficiency only of 45.29% and 38.58%, respectively.

[146] Example 6 Determination of control efficiency of the strain HMQAU19044 on cucumber downy mildew of indoor potted plants

[147] A sporangium suspension with a concentration of 1 x 10*/mL was prepared with sterilized tap water for spray inoculation. The improved Lu cucumber No. 3 variety was adopted, and a mixture of sand and nutrition soil in a ratio of 1:1 was used as a seedling medium. Cucumber seeds were sown on a seedling tray; seedlings, when growing to have about 2 to 3 true leaves, were transplanted into 5 x 5 x 7 cm flowerpots; and when the seedlings grew to have 4 to 5 true leaves, the control efficiency test was conducted.

[148] Treatment 1: the fermentation filtrate of the strain HMQAU19044 was first evenly sprayed on the back side of the leaves, with 2 mL per pot, and the sporangium suspension was evenly sprayed on the back side of the leaves after the filtrate was slightly dried, with 2 mL per pot.

[149] Treatment 2: the fermentation broth of the strain HMQAU19044 was first evenly sprayed on the back side of the leaves, with 2 mL per pot, and the sporangium suspension was evenly sprayed on the back side of the leaves after the fermentation broth was slightly dried, with 2 mL per pot.

[150] Treatment 3: pathogen control: sterilized tap water was first evenly sprayed on the back side of the leaves, with 2 mL per pot, and the sporangium suspension was evenly sprayed after the water was slightly dried, with 2 mL per pot.

[151] 4 replicates were set for each treatment. After the pathogens were inoculated in each treatment, the plants were covered with black plastic bags and then moisturized in an 18°C incubator for 24 h. The plastic bags were removed, and the plants were cultivated in an incubator with a 12 h light-dark cycle (22°C in the day and 18°C at night) for 7 d. Then the disease incidence was investigated in each treatment, as shown in FIG. 7.

[152] Table 11 control efficiency of the strain HMQAU19044 on cucumber downy mildew of indoor potted plants

[153] Treatment Disease index (%) Relative control efficiency (%) EO 94s 2 31.25b 59.82 3(CK) 77.78 a -

[154] Note: The letters after the numbers in the same column indicate significant differences at the p < 0.05 level (Duncan's new multiple range method).

[155] As shown in Table 11, the strain HMQAU19044 exhibited a prominent control efficiency on cucumber downy mildew. The fermentation filtrate treatment showed a relative control efficiency of 79.45%, and the fermentation broth treatment also showed a relative control efficiency of 59.82%. According to statistical analysis, there was a significant difference between the control efficiency of the two treatments, and the disease indexes of the experimental groups also showed a significant difference from that of the pathogen control group.

[156] The above descriptions are merely preferred examples of the present disclosure, and are not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, and the like made within the spirit and principle of the present disclosure shall be all included in the protection scope of the present disclosure.

Sequence Listing-GWP202103411

SEQUENCE LISTING <110> Qingdao Agricultural University <120> BACILLUS VELEZENSIS (B. VELEZENSIS) STRAIN AND USE THEREOF IN

CONTROL OF PLANT DOWNY MILDEW <130> GWP202193411 <150> 202011230642.2 <151> 2020-11-06 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> gyrB primer UP1 <220> <221> misc feature <222> (24)..(24) <223> y can be "t" or "c" <220> <221> misc feature <222> (27)..(27) <223> n can be "a", "g", "Cc" or "tn" <220> <221> misc feature <222> (30)..(30) <223> n can be "a", "g", "Cc" or "tn" <220> <221> misc feature <222> (33)..(33) <223> n can be "a", "g", "Cc" or "tn" <220> <221> misc feature <222> (36)..(36) <223> r can be "g" or "a" <220> <221> misc feature Pagina 1

Sequence Listing-GWP202103411 <222> (39)..(39) <223> y can be "t" or "c" <400> 1 gaagtcatca tgaccgttct gcaygcnggn ggnaarttyg a 41 <2105 2 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> gyrB primer UP2r <220> <221> misc feature <222> (24)..(24) <223> r can be "g" or "a" <220> <221> misc feature <222> (27)..(27) <223> n can be "a", "g", "Cc" or "tn" <220> <221> misc feature <222> (30)..(30) <223> r can be "g" or "a" <220> <221> misc feature <222> (33)..(33) <223> n can be "a", "g", "Cc" or "tn" <220> <221> misc feature <222> (36)..(36) <223> r can be "g" or "a" <220> <221> misc feature <222> (39)..(39) <223> n can be "a", "g", "Cc" or "tn" <400> 2 agcagggtac ggatgtgcga gccrtcnacr tcngcrtcng tcat 44 <2105 3 <211> 1259

Pagina 2

Sequence Listing-GWP202103411 <212> DNA <213> Artificial Sequence <220> <223> gene GyrB of Bacillus velezensis <400> 3 gaagtcatca tgaccgttct gcacgcgggc ggtaagttcg acggaagcgg atataaagta 60 tccggcggtc ttcacggtgt aggggcgtcc gtcgtaaacg ccttgtcgac cactcttgac 120 gttacggttc atcgtgacgg aaaaatccac tatcaggcgt acgagcgcgg tgtgcctgtg 180 gccgatcttg aagtgatcgg tgatactgat aagaccggaa cgattacgca cttcgttccg 240 gatccggaaa tcttcaaaga aacaactgta tacgactatg atctgctttc aaaccgtgtc 300 cgggaattgg ccttcctgac aaaaggcgta aacatcacga ttgaagacaa acgtgaagga 360 caagaacgga aaaacgagta ccactacgaa ggcggaatca aaagctatgt tgagtactta 420 acccgttcca aagaagtcgt tcatgaagag ccgatttata tcgaaggcga gaaagacggc 480 ataacggttg aagttgcatt gcaatacaac gacagctata caagcaatat ttattctttc 540 acaaataata tcaacacata cgaaggcggg acgcacgagg ccggatttaa aactggtctg 600 acccgtgtca taaacgacta tgcaagaaga aaagggattt tcaaagaaaa tgatccgaat 660 ttaagcgggg atgatgtgag agaagggctg actgccatta tttcaattaa gcaccctgat 720 ccgcaattcg aagggcagac gaaaaccaag ctcggcaact ctgaagcgag aacgatcact 780 gacacgctgt tttcttctgc gctggaaaca ttccttcttg aaaatccgga ctcagcccgc 840 aaaatcgttg aaaaaggttt aatggccgca agagcgcgga tggcagcgaa aaaagcgcgg 900 gaattgatcc ggcgcaaaag tgcgcttgag atttccaatc tgccgggcaa actggcgggc 960 tgttcttcta aagatccgag catttccgag ctgtatatcg tagagggtga ctctgcgggc 1020 ggatcagcga aacagggacg ggaccgtcat ttccaagcca ttctgccgct gcgcggtaag 1080 attctgaacg tcgagaaagc tagacttgat aagattctct caaacaatga ggtcagatca 1140 atgatcacgg ccctcggaac aggaatcggc gaagatttta atcttgaaaa agcgcgttat 1200 cataaagtgg tcatcatgac ggatgctgat gtagacggct cgcacatccg taccctgct 1259

Pagina 3

Claims

“24 - Conclusions

1. Strain HMQAU 19044 of Bacillus Velezensis (B. Velezensis), wherein the strain was deposited in the China General Microbiological Culture Collection Center (CGMCC) on January 19, 2020 with Deposit Registration Number: CGMCC No. 1920.

The microbial agent prepared from the HMQAU19044 strain of B. Velezensis according to claim 1, wherein the microbial agent comprises the HMQAU19044 strain and/or metabolites of the HMQAU19044 strain.

The microbial agent according to claim 2, wherein an active ingredient of the microbial agent is a fermentation broth or a fermentation filtrate of the strain HMQAU19044.

The microbial agent of claim 2, wherein the microbial agent is a liquid microbial agent and has a live bacteria and/or spore number of > 1.5 x 10°/mL to 2.0 x 10°/mL.

A method for preparing the microbial agent of claim 2, specifically comprising the steps of: (1) preparing a solid LB medium; (2) preparing a liquid LB medium; (3) strain activation: taking a loopful of bacterial colonies of the strain HMQAU 19044, inoculating into 50 mL of the liquid LB medium, and culturing at 200 rpm and 30°C for 24 hours to obtain a starter culture; (4) preparing a fermentation broth: inoculating 1/50 of the prepared starter culture in 50 mL of liquid LB medium, and culturing at 200 rpm and 30°C for 48 hours to obtain the fermentation broth of strain HMQAU 19044 ; and (5) preparing a fermentation filtrate: centrifuging the prepared fermentation broth at 4°C and 8,000 r/min for 15 min, and filtering a resulting supernatant with a 0.22 µm microporous membrane to remove the fermentation filtrate from the strain HMQAU19044 available.

25.

The method for preparing the microbial agent according to claim 5, wherein the solid LB medium is prepared by the following method: dissolving 10 g tryptone, 10 g sodium chloride, 5 g yeast extract and 15 g-20 g agar powder in deionized water, with pH 6.8; diluting a resulting solution to 1 L with deionized water; and subjecting a resulting solution to moist heat sterilization at 121°C for 25 min.

The method for preparing the microbial agent according to claim 5, wherein the liquid LB medium is prepared by the following method: dissolving 10 g tryptone, 10 g sodium chloride, and 5 g yeast extract in deionized water, pH 6, 8; diluting a resulting solution to 1 L with deionized water; and subjecting a resulting solution to moist heat sterilization at 121°C for 25 min.

Use of the microbial agent according to claim 2, wherein the microbial agent is used for the control of cucumber downy mildew.

A method for controlling cucumber downy mildew comprising using the HMQAU19044 strain of B. Velezensis according to claim 1 or the microbial agent according to claim 2 for controlling cucumber downy mildew.