CN108396002B - Bacillus licheniformis and application thereof in preventing and treating sweet melon fusarium wilt - Google Patents

Abstract

The invention discloses bacillus licheniformis and application thereof in preventing and treating fusarium wilt of sweet melons. The strain is Bacillus licheniformis (Bacillus licheniformis) strain LT4-3, is separated from canal mud of Jining city in Shandong province, and has been preserved in China general microbiological culture Collection center in 2017, 9 and 26 months, with the preservation number of CGMCC No. 14745. The test result shows that: the bacillus licheniformis strain LT4-3 wettable powder has obvious effect of preventing and treating the sweet melon fusarium wilt, the prevention and treatment effect reaches 74.31%, the melon has good growth vigor, and no phytotoxicity is generated.

Description

Bacillus licheniformis and application thereof in preventing and treating sweet melon fusarium wilt

Technical Field

The present invention belongs to the field of biological plant disease preventing and controlling technology. Specifically, the invention relates to bacillus licheniformis and application thereof in preventing and treating sweet melon fusarium wilt, and a biological control preparation prepared from the bacillus licheniformis.

background

The muskmelon is also called muskmelon, cooked muskmelon, Hami melon and the like, has a long cultivation history in China, has the functions of relieving summer heat, inducing diuresis, protecting liver, supplementing vitamins and the like due to higher nutritive value of muskmelon pulp, and is well loved by the public due to the excellent color, aroma and taste. The muskmelon is widely cultivated in the world as an annual vine plant, and the muskmelon yield is the top of China. In recent years, with the increase of melon planting density and multiple cropping index, the incidence frequency of melon diseases and insect pests is higher and higher, wherein the melon blight is one of the main diseases affecting melon agricultural production. Sweet melon wilt disease is also known as dead seedling, dead vine, wilting disease and vine cutting disease, can occur in open field and protected field cultivation, is an important fungal disease in melon production, and often causes melon seedling withering and death. Sweet melon Fusarium wilt is caused by Fusarium oxysporum melon specialization (Fusarium oxysporum f.sp.melonis). Sweet melon fusarium wilt is a typical soil-borne fungal disease and can occur from a seedling stage to a plant-forming stage. Among them, the most serious diseases occur in the flowering and fruit setting stage, which often causes the withering and death of melon seedlings.

At present, the main method for preventing and treating the fusarium wilt of sweet melons is chemical prevention and treatment, pathogenic bacteria of the fusarium wilt of sweet melons generate drug resistance due to long-term use of medicines, the prevention and treatment effect is not ideal, in addition, a large amount of use of chemical agents causes many negative effects on the quality safety and the environment of the melons, and biological prevention and treatment has the advantages of safety to people and animals, good environmental compatibility, difficulty in generation of drug resistance of the pathogenic bacteria and the like, so that green and pollution-free biological prevention and treatment of the fusarium wilt of sweet melons are more and more concerned by.

The bacillus licheniformis is one of the strains which are widely applied and researched at present and have great potential development value, and the bacillus licheniformis can generate antibacterial active substances, has a unique biological oxygen-deprivation action mechanism, inhibits the growth and the reproduction of pathogenic bacteria, and can generate various growth-promoting related substances to promote the growth of plants.

Disclosure of Invention

The invention aims to provide a novel microorganism, namely bacillus licheniformis LT4-3, for efficiently preventing and treating fusarium wilt of sweet melons. The bacillus licheniformis strain disclosed by the invention has the characteristics of high growth speed, high yield, strong stress resistance, capability of fast and massively colonizing on the surface of a plant (especially melon) and the like, and therefore, the bacillus licheniformis strain has a good application prospect.

the above object of the present invention is achieved by the following technical solutions: the strain provided by the invention is a Bacillus licheniformis (Bacillus licheniformis) strain LT4-3 which is separated from canal and river mud of Jining City in Shandong province, is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of West Lu No.1 of the sunward area in Beijing, institute of microbiology in China institute of academy of sciences) on 26 months and 9 months in 2017, and has the preservation number of CGMCC No. 14745. It has the following biological properties: the strain is in a rod shape on a beef extract peptone agar medium or an LB (Luria-Bertani) medium, the flagellum characteristic is obvious, a single colony is circular, the stain is white, the stain is opaque, the center is raised, the edge is neat, and wrinkles exist in the later period; culturing on beef extract peptone agar medium at 28 deg.C for two days, and examining under microscope to obtain rod-shaped thallus cell capable of moving. Gram-positive (E.coli control). After spore dyeing, the spores are observed to be oval, and the sacs are not expanded when the spores are in the middle or near middle.

the culture method or the propagation method of the bacillus licheniformis (B.licheniformis) strain LT4-3 comprises the following steps:

(1) An LB culture medium is adopted for ordinary culture and preservation, and the formula is as follows: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 15-20 g of agar and 1000mL of distilled water, and the pH value is adjusted to 7.0-7.2.

(2) The laboratory liquid culture adopts an LB liquid culture medium, and the formula is as follows: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water, and adjusting the pH value to 7.0-7.2.

(3) The solid culture medium formula comprises: comprises a solid material and inorganic salt, wherein the mass ratio of the solid material to the inorganic salt is 98.6: 0.4; the solid materials are rice hulls, corn flour, soybean meal and bran in a mass ratio of 54: 30: 10: 6; the inorganic salt comprises the following components in percentage by mass: 21% of monopotassium phosphate, 5% of magnesium sulfate, 9% of ammonium sulfate and 65% of light calcium carbonate.

(4) The formula of the mass fermentation culture comprises: the formula of the solid culture medium in the step (3).

The invention also provides a biological control preparation for controlling the fusarium wilt of sweet melons, which comprises the bacillus licheniformis (B.licheniformis) strain LT 4-3.

Also, the present invention provides a biocontrol method for controlling blight of sweet melon, which comprises applying the above bacillus licheniformis (b. licheniformis) strain LT4-3 or the above biocontrol agent to a melon plant having blight.

The invention also provides application of the bacillus licheniformis strain LT4-3 or the biological control preparation in preventing and treating fusarium wilt of sweet melons.

The preparation method of the biological control preparation comprises the following steps:

(1) Preparing a seed solution of said bacillus licheniformis strain LT 4-3;

(2) inoculating the seed liquid prepared in the step (1) into a solid culture medium, and culturing at a constant temperature of 27-29 ℃;

(3) And (3) adding sterile water into the culture cultured in the step (2), mixing, filtering, inoculating the filtrate into a large amount of fermentation medium, and performing fermentation culture in a fermentation chamber with the room temperature of 27-29 ℃ and the relative humidity of more than 85%.

in one embodiment of the present invention, the preparation method comprises the steps of:

(1) Transplanting spores of the bacillus licheniformis strain LT4-3 into an LB liquid culture medium, and performing shake culture on a shaker at the temperature of 27-29 ℃ for 3-5 days to obtain a seed solution;

(2) Inoculating the seed solution prepared in the step (1) into a solid culture medium according to the mass ratio of 10%, and performing shake culture at 27-29 ℃ for 3-5 days;

(3) Mixing the culture cultured in the step (2) with sterile water according to the mass ratio of 1: 15, filtering, inoculating the filtrate to a large amount of fermentation medium according to the volume ratio of 1: 6, and performing fermentation culture in a fermentation chamber with the room temperature of 27-29 ℃ and the relative humidity of more than 85% for 8-9 days.

Wherein the LB liquid culture medium in the step (1) has the formula: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water, and adjusting the pH value to 7.0-7.2.

The solid culture medium in the step (2) comprises a solid material and inorganic salt, wherein the mass ratio of the solid material to the inorganic salt is 98.6: 0.4; the solid materials are rice hulls, corn flour, soybean meal and bran in a mass ratio of 54: 30: 10: 6; the inorganic salt comprises the following components in percentage by mass: 21% of monopotassium phosphate, 5% of magnesium sulfate, 9% of ammonium sulfate and 65% of light calcium carbonate.

The mass fermentation medium in the step (3) is synchronous with the solid medium in the step (2).

Experiments show that the bacillus licheniformis strain has the characteristics of high growth speed, large sporulation amount, strong stress resistance, capability of fast and massively colonizing on the surface of a plant (especially melon) and the like, thereby having good application prospect. The biological control preparation prepared from the bacillus licheniformis can effectively control melon wilt disease and effectively promote melon growth, and is a biological control preparation with a great application prospect. The microbial preparation can be used as a biological pesticide or a biological fertilizer for preventing and treating the fusarium wilt of sweet melons. The test result shows that: the bacillus licheniformis (B.licheniformis) strain LT4-3 wettable powder has obvious effect of preventing and treating the fusarium wilt of the sweet melons, the prevention and treatment effect reaches 74.31%, and the melons have good growth vigor and no phytotoxicity.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications are within the scope of the invention.

Example 1

1. Isolation and purification of Bacillus licheniformis (B. licheniformis) LT4-3

the bacillus licheniformis (B.licheniformis) LT4-3 is obtained by separating river mud by a dilution plate method and a plate marking method, and the separation method comprises the following steps:

(1) Isolation of Bacillus

The river mud sample was taken from the canal of Jining city, Shandong, 5 months and 15 days in 2014. Weighing 1g of river mud in 100mL of sterile water, placing the river mud in a shaking table at 30 ℃ for 10min with shaking at 150rpm, then placing the river mud in a water bath kettle at 60 ℃ for incubation for 30min, taking 100 mu L of 10-2, 10-3 and 10-4 diluent to coat on an LB (LB) culture medium plate, coating three layers of diluent in parallel in each gradient, picking out microbial strains with different forms on the LB culture medium after 2d of culture at 30 ℃ to lay lines, and regularly observing the growth condition of bacterial colonies. Then adopting a plate marking method to purify the bacillus strains, numbering and storing.

(2) Screening of sweet melon fusarium wilt high-efficiency antagonistic strain

Firstly, primary screening: preparing a PDA (potato Dextrose agar) plate by adopting a contraposition culture method, taking fungus cakes with the diameter of 5mm from the edges of pathogenic bacteria of the bacillus and the sweet melon fusarium wilt by using a puncher, respectively transplanting the fungus cakes into the centers of two opposite sides of the plate, culturing at constant temperature of 25 ℃, and observing the inhibition effect of the bacillus on the pathogenic bacteria day by day.

Secondly, re-screening: and (2) re-screening the screened bacillus strains with high-efficiency antagonistic activity, mainly screening bacillus strains with better tolerance through temperature resistance, acid and alkali resistance and drug resistance tests, performing pot control tests and field tests, and identifying the screened strain LT 4-3.

The inventor obtains a bacillus licheniformis LT4-3 capable of efficiently preventing and treating fusarium wilt of sweet melons through a large amount of screening work. Experiments prove that the bacillus licheniformis raw powder shows a very high-efficient prevention and treatment effect in preventing and treating the melon fusarium wilt, so that the melon grows well. Therefore, the bacillus licheniformis provided by the invention is a new bacillus licheniformis strain with wide application prospect, and can be used for preparing a biological control preparation for preventing and treating fusarium wilt of sweet melons.

2. Identification of strains

(1) Microbiological characteristics: the strain is rod-shaped on a beef extract peptone agar medium or an LB medium, the flagellum characteristic is obvious, a single colony is round, the stain is white, the single colony is opaque, the center is raised, the edge is neat, and wrinkles are formed in the later period; culturing on beef extract peptone agar medium at 28 deg.C for two days, and examining under microscope to obtain rod-shaped thallus cell capable of moving. Gram-positive (E.coli control). After spore dyeing, the spores are observed to be oval, and the sacs are not expanded when the spores are in the middle or near middle.

(2) Molecular biological Properties

The sequence determination result of the 16s rDNA gene of the strain is as follows (SEQ-1):

TCCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACG TATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGA ACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTG TAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTA GAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACA CGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGA TGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCA ATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGC GGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACG CTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTT CACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAG CCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCA CCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTAT TTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCC GTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGT GTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGC CGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTAT TAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACA TCAGGGAGCAAGCTCCCATCG。

(3) Results of cell morphology and physicochemical experiments

TABLE 1 cell morphology and results of physicochemical experiments for Bacillus licheniformis (B. licheniformis) LT4-3

Experimental project	results	Experimental project	results
				gram stain	Positive for	Acid production from carbohydrates	+
cell shape	Rod-shaped	Glucose	+
				The diameter of the cells is more than 1 mu m	－	xylose	+
form spores	+	L-arabinose	+
				Enlargement of spore	－	mannitol	－
Spore is round	－	Lactose	－
				Parasporal crystal	－	Fermentation of glucose to produce gas	+
Contact enzyme	+	Using citric acid salts	+
				Oxidase enzyme	+	growth at 50 deg.C	+
Anaerobic growth	－	Growth at pH5.7	+
				VP assay	+	Growth with 7% NaCl	+
VP﹤pH 6	+	Starch hydrolysis	+
				VP﹥pH 7	－	Decomposition of casein	+
Nitrate reduction	+	Hydrolyzed gelatin	+

the strain is identified as Bacillus licheniformis (Bacillus licheniformis), which has been preserved in China general microbiological culture Collection center in 2017, 9 and 26 months, and the preservation number is CGMCC No. 14745.

Example 2

1. Fermentation process of bacillus licheniformis strain

The LB liquid culture medium formula is: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water, and adjusting the pH value to 7.0-7.2.

The solid culture medium formula comprises: the solid material, inorganic salt and water, wherein the mass ratio of the solid material to the inorganic salt is 98.6: 0.4; the solid materials are rice hulls, corn flour, soybean meal and bran in a mass ratio of 54: 30: 10: 6; the inorganic salt comprises the following components in percentage by mass: 21% of monopotassium phosphate, 5% of magnesium sulfate, 9% of ammonium sulfate and 65% of light calcium carbonate. The preparation method comprises the following steps: adding a proper amount of water (10-15 times of the mass) to dissolve inorganic salt to obtain an inorganic salt solution; mixing inorganic salt solution and solid material, adding water to adjust water content of the solid culture medium to 40-60%, and grasping the solid culture medium in hand with water in finger joint without dripping.

A bulk solids fermentation process for bacillus licheniformis (b. licheniformis) strain LT 4-3:

Culture of strain seed liquid

A small amount of spores of a bacillus licheniformis strain (B.licheniformis) LT4-3 are picked from a test tube slant, transferred into an LB liquid culture medium, and subjected to shaking culture for 3-5 days at 28 ℃ in a shaking table, wherein the spores are used as seed liquid.

② culture of solid production strain

inoculating the seed solution into a solid culture medium (500mL triangular flask) according to the proportion of 10%, culturing at the constant temperature of 28 ℃ for 3-5 days, and shaking for multiple times in the middle.

③ a large amount of solid fermentation:

Diluting the culture obtained by solid culture in step two with sterile water according to the ratio of 1: 15, filtering with sterile gauze, removing coarse residue to obtain production bacterial liquid, and inoculating the production bacterial liquid in a mass fermentation culture medium according to the inoculation ratio of 1: 6. Placing the inoculated raw materials in a fermentation chamber (at 28 ℃ and relative humidity of more than 85%) for fermentation culture for 8-9 days; further drying to obtain raw powder of Bacillus licheniformis (B. licheniformis) strain LT4-3 with active bacteria content of 150 hundred million/g.

Wettable powder

The raw material ratio is as follows: 10% of raw powder of bacillus licheniformis LT4-3, 45% of glucose, 10046% of wetting agent, 2.0% of dispersant NNO, 1% of CMC and the balance of light calcium carbonate.

In order to determine the control effect of the bacillus licheniformis (B.licheniformis) strain LT4-3 powder on the fusarium wilt of sweet melons, a basis is provided for popularization and application of the bacillus licheniformis strain LT4-3 powder. We performed the test in the chapter-dune region of Jinan, Shandong, from 2 to 3 months in 2016, and the test results are reported as follows:

1 test materials and methods

1.1 test agent

Bacillus licheniformis (B. licheniformis) strain LT4-3 powder (15 hundred million/gram spore powder WP) produced by the fermentation method of example 2 of the present invention; 60% carbendazim wettable powder (produced by Jinliang Fine chemical Co., Ltd., Henan province, sold in markets).

1.2 test materials and control objects

The test material is muskmelon, and the variety is Jingtian 5; the control object is Fusarium oxysporum f.sp.melonis.

1.3 conditions of the test

The test site is arranged in a greenhouse of a high official village and town melon planting base in a hilly region in Jinan province of Shandong province. In recent years, due to the fact that melons are planted continuously in the area, blight of the melons is common. The test soil type is moist soil, the organic matter content is 1.72%, the pH value is 7.5, the cultivation conditions and management measures of all test districts are consistent, and the blight of the sweet melons is in the early stage of disease development when the pesticide is applied.

1.4 test design and arrangement

The experiment was repeated 4 times for 3 treatments, i.e., 20 times the powder of Bacillus licheniformis (B. licheniformis) strain LT4-3, 800 times the wettable powder of 60% carbendazim, and 140 treatments without application of clear water. The roots of the diseased plants and the nearby plants are irrigated once for 10 days and continuously for 3 times at the early stage of disease attack, and each diseased plant is irrigated with 0.25kg of the medicine liquid. Irrigating the roots once every 2016 every 2 months and 25 days, 3 months and 5 days, and 3 months and 15 days.

1.5 test investigation and calculation method

1.5.1 weather conditions

The medicine for the 1 st application (2 months and 25 days) is cloudy in the day, the wind power is 3 grades, the highest air temperature is 9 ℃, the lowest air temperature is-2 ℃, and the relative humidity is 51%. The medicine application for the 2 nd time (3 months and 5 days), the day is cloudy, the wind power is 3 grades, the highest temperature is 13 ℃, the lowest temperature is 3 ℃, and the relative humidity is 50%; the 3 rd application (3 months and 15 days) is carried out on the sunny day, the wind power is 3 grades, the highest air temperature is 15 ℃, the lowest air temperature is 5 ℃, and the relative humidity is 52%.

1.5.2 drug efficacy and safety investigation

And (3) drug effect investigation: investigation was performed 14d after the last application. 120 plants were investigated, all leaves were investigated, incidence was investigated, progression of disease was recorded and disease index was calculated.

Safety investigation: the safety of the melon is observed at 7d and 14d after the first application, if the phytotoxicity occurs, the phytotoxicity symptoms are described in detail, and the phytotoxicity degree is determined according to the phytotoxicity degree grading standard.

The melon fusarium wilt classification standard is as follows:

Level 0: the whole plant has no disease symptoms.

Level 1: the leaves below plant 1/4 showed wilting symptoms.

And 2, stage: the leaves of the plants 1/4-1/2 show wilting symptoms.

And 3, level: the leaves of the plant 1/2 show wilting symptom.

4, level: the plants died due to illness.

1.5.3 method for calculating drug effect

The preventing and treating effect is calculated according to the formulas (1) and (2):

In the formula: CK 0-pre-drug disease index for placebo;

CK 1-disease index after drug administration in placebo zone;

PT 0- — index of disease before administration before treatment with the agent;

PT 1-disease index after application after treatment with the agent.

1.5.4 direct Effect on melon

observing whether the pesticide has phytotoxicity on the melons, and recording the type and the degree of the phytotoxicity. In addition, the effect on the growth of the melons was also recorded.

Phytotoxicity was recorded in the following manner:

(a) If the phytotoxicity can be measured or calculated, it is expressed in absolute terms, for example, as plant height.

(b) In other cases, the extent and frequency of phytotoxicity can be estimated in two ways:

Recording the phytotoxicity degree of each cell according to a phytotoxicity grading method, and representing the phytotoxicity degree by-, + + + + + +, +.

The phytotoxicity grading method comprises the following steps:

And (2) preparing: no chemical injury;

+: mild phytotoxicity, no influence on plant growth;

++: moderate phytotoxicity, can be recovered, and does not reduce the biomass of plants;

+++: moderate phytotoxicity affects the normal growth of plants and reduces the biomass of the plants to a certain extent;

++++: serious phytotoxicity, plant growth retardation and serious reduction of plant biomass.

and secondly, comparing the medicament treatment area with the blank control area to evaluate the phytotoxicity percentage. At the same time, phytotoxicity symptoms (dwarfing, chlorosis, malformations, etc.) of plants are to be described accurately.

2 results

2.1 prevention and treatment effects of the test agent on blight of sweet melon

the control effect of the test agent on the fusarium wilt of sweet melons is shown in tables 2-3. The results in table 3 show that the bacillus licheniformis (b. licheniformis) strain LT4-3 wettable powder and the 60% carbendazim wettable powder both have good control effects on the fusarium wilt of sweet melons, wherein the control effect of the bacillus licheniformis (b. licheniformis) strain LT4-3 wettable powder is more than 70%.

From the disease index of field test of the sweet melon fusarium wilt (table 3), 14 days after the last application, the control effect of the bacillus licheniformis (b. licheniformis) LT4-3 is 74.31%, which is obviously higher than that of 55.42% of 60% carbendazim wettable powder.

TABLE 2 blight disease status of sweet melon before three agents treatment

TABLE 3 preventive and therapeutic effects of three kinds of drug treatment on fusarium wilt of sweet melon

2.2 melon safety survey

And through observation of application of 7d and 14d, compared with the control area, the melon grows normally and has no phytotoxicity in each agent treatment area, which indicates that the bacillus licheniformis powder is safe for the melon.

3 small knot

3.1 from the view of disease index and prevention and treatment effect, the Bacillus licheniformis (B.licheniformis) strain LT4-3 (with the preservation number of CGMCC No.14745) has better prevention and treatment effect on the fusarium wilt of sweet melons, and the prevention and treatment effect of 14 days after the last application reaches more than 70%.

3.2 from the aspect of melon growth, the bacillus licheniformis (B. licheniformis) strain LT4-3 has the growth promoting effect on melons, has no phytotoxicity, and is safe and reliable.

SEQUENCE LISTING

<110> Shandong Xinhe Feng crop Nutrition Co Ltd

<120> bacillus licheniformis and application thereof in preventing and treating sweet melon fusarium wilt

<130> 0

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1381

<212> DNA

<213> 16s rDNA Gene sequence of Bacillus licheniformis (B. licheniformis) Strain LT4-3

<400> 1

tcctcaccga cttcgggtgt tacaaactct cgtggtgtga cgggcggtgt gtacaaggcc 60

cgggaacgta ttcaccgcgg catgctgatc cgcgattact agcgattcca gcttcacgca 120

gtcgagttgc agactgcgat ccgaactgag aacagatttg tgggattggc ttaacctcgc 180

ggtttcgctg ccctttgttc tgtccattgt agcacgtgtg tagcccaggt cataaggggc 240

atgatgattt gacgtcatcc ccaccttcct ccggtttgtc accggcagtc accttagagt 300

gcccaactga atgctggcaa ctaagatcaa gggttgcgct cgttgcggga cttaacccaa 360

catctcacga cacgagctga cgacaaccat gcaccacctg tcactctgcc cccgaagggg 420

acgtcctatc tctaggattg tcagaggatg tcaagacctg gtaaggttct tcgcgttgct 480

tcgaattaaa ccacatgctc caccgcttgt gcgggccccc gtcaattcct ttgagtttca 540

gtcttgcgac cgtactcccc aggcggagtg cttaatgcgt tagctgcagc actaaggggc 600

ggaaaccccc taacacttag cactcatcgt ttacggcgtg gactaccagg gtatctaatc 660

ctgttcgctc cccacgcttt cgctcctcag cgtcagttac agaccagaga gtcgccttcg 720

ccactggtgt tcctccacat ctctacgcat ttcaccgcta cacgtggaat tccactctcc 780

tcttctgcac tcaagttccc cagtttccaa tgaccctccc cggttgagcc gggggctttc 840

acatcagact taagaaaccg cctgcgagcc ctttacgccc aataattccg gacaacgctt 900

gccacctacg tattaccgcg gctgctggca cgtagttagc cgtggctttc tggttaggta 960

ccgtcaaggt gccgccctat ttgaacggca cttgttcttc cctaacaaca gagctttacg 1020

atccgaaaac cttcatcact cacgcggcgt tgctccgtca gactttcgtc cattgcggaa 1080

gattccctac tgctgcctcc cgtaggagtc tgggccgtgt ctcagtccca gtgtggccga 1140

tcaccctctc aggtcggcta cgcatcgtcg ccttggtgag ccgttacctc accaactagc 1200

taatgcgccg cgggtccatc tgtaagtggt agccgaagcc accttttatg tctgaaccat 1260

gcggttcaga caaccatccg gtattagccc cggtttcccg gagttatccc agtcttacag 1320

gcaggttacc cacgtgttac tcacccgtcc gccgctaaca tcagggagca agctcccatc 1380

g 1381

Claims (8)

1. Bacillus licheniformis (Bacillus licheniformis) strain LT4-3, wherein the preservation number of the strain is CGMCC No. 14745.

2. Use of the bacillus licheniformis strain LT4-3 according to claim 1 for controlling sweet melon fusarium wilt.

3. A biocontrol formulation comprising bacillus licheniformis strain LT4-3 according to claim 1.

4. a process for the preparation of a biocontrol agent as claimed in claim 3, characterized in that it comprises the steps of:

(1) Preparing a seed solution of Bacillus licheniformis strain LT 4-3;

(2) Inoculating the seed liquid prepared in the step (1) into a solid culture medium, and culturing at a constant temperature of 27-29 ℃;

(3) And (3) adding sterile water into the culture cultured in the step (2), mixing, filtering, inoculating the filtrate into a large amount of fermentation medium, and performing fermentation culture in a fermentation chamber with the room temperature of 27-29 ℃ and the relative humidity of more than 85%.

5. the process for the preparation of a biocontrol agent as claimed in claim 4, characterized in that,

The solid culture medium in the step (2) comprises a solid material and inorganic salt, wherein the mass ratio of the solid material to the inorganic salt is 98.6: 0.4; the solid materials are rice hulls, corn flour, soybean meal and bran in a mass ratio of 54: 30: 10: 6; the inorganic salt comprises the following components in percentage by mass: 21% of monopotassium phosphate, 5% of magnesium sulfate, 9% of ammonium sulfate and 65% of light calcium carbonate;

The mass fermentation medium in the step (3) is synchronous with the solid medium in the step (2).

6. A process for the preparation of a biocontrol agent as claimed in claim 5, characterized in that it comprises the steps of:

(1) transplanting spores of the bacillus licheniformis strain LT4-3 into an LB liquid culture medium, and performing shake culture on a shaker at the temperature of 27-29 ℃ for 3-5 days to obtain a seed solution;

(2) inoculating the seed solution prepared in the step (1) into a solid culture medium according to the mass ratio of 10%, and performing shake culture at 27-29 ℃ for 3-5 days;

(3) mixing the culture cultured in the step (2) with sterile water according to the mass ratio of 1: 15, filtering, inoculating the filtrate to a large amount of fermentation medium according to the volume ratio of 1: 6, and performing fermentation culture in a fermentation chamber with the room temperature of 27-29 ℃ and the relative humidity of more than 85% for 8-9 days.

7. Use of the biocontrol agent of claim 3 for controlling sweet melon fusarium wilt.

8. A biocontrol method for controlling fusarium wilt of sweet melon, characterized in that the bacillus licheniformis strain LT4-3 of claim 1 or the biocontrol formulation of claim 3 is applied to melon plants with fusarium wilt.