CN110564646B

CN110564646B - Bacillus belgii CY30 and application thereof in prevention and treatment of tea round spot

Info

Publication number: CN110564646B
Application number: CN201910891472.3A
Authority: CN
Inventors: 黄大野; 曹春霞; 刘芳; 杨丹; 姚经武; 廖先清; 饶犇; 周荣华
Original assignee: Hubei Biopesticide Engineering Research Center
Current assignee: WUHAN CHUQIANG BIOLOGICAL TECHNOLOGY Co.,Ltd.
Priority date: 2019-09-20
Filing date: 2019-09-20
Publication date: 2020-05-05
Anticipated expiration: 2039-09-20
Also published as: CN110564646A

Abstract

The invention belongs to the field of agricultural microorganisms and discloses a Bacillus belgii CY30 and application thereof, wherein the Bacillus belgii is Bacillus belgii (Bacillus (R) (R))Bacillus velezensis) CY30, accession number: m2018710, the strain is reported for the first time to be used for preventing and treating tea tree alternaria leaf spot by foliar spray, and the growth promoting and yield increasing effects of the irrigated root on tea trees, and has wide application prospect in agricultural production.

Description

Bacillus belgii CY30 and application thereof in prevention and treatment of tea round spot

Technical Field

The invention belongs to the field of agricultural microorganisms, and particularly relates to a Bacillus beili (Bacillus velezensis) CY30 and application thereof in prevention and control of tea round spot.

Background

Tea trees are important economic crops in China, and the tea industry is rapidly developed since the establishment of new China. According to FAO statistics, the area of the tea garden is 2.22 multiplied by 10 and the cut-off is 2017⁶Hectare, yield 2.47X 10⁶Ton, tea garden area and total tea yield all live in the first place in the world. The tea plays a great role in poverty alleviation and export foreign exchange of the industry in poor mountainous areas in China.

Tea trees mostly grow in warm and humid tropical and subtropical regions, and the climate of the regions is also favorable for breeding tea tree diseases. Tea leaf spot disease is an important disease of tea, is reported in tea planting areas all over the world, is also commonly generated in tea areas in China, and mainly damages grown leaves and old leaves, influences tree vigor and causes yield reduction. The chemical bactericide is still a main means for preventing and treating the tea round spot, but the application of the chemical bactericide is easy to cause the drug resistance of pathogenic bacteria, and can cause pesticide residue and environmental pollution.

In tea planting, a large amount of chemical fertilizer is usually applied to stabilize and improve the yield, soil hardening can be caused by long-term application of the chemical fertilizer, the nutrient absorption and utilization rate of tea is insufficient, soil acidification can be caused, the decline of continuous cropping tea trees is accelerated, and the yield reduction of tea is caused, so that the problem of tea production needs to be solved.

Bacillus, an aerobic or facultative anaerobic bacillus, which can produce stress-resistant endospore, has broad-spectrum antifungal activity and good stability due to being capable of producing antifungal substances such as lipopeptide, protein and the like, and becomes an important agricultural biocontrol bacterium. Meanwhile, the fertilizer can secrete growth promoting substances, improve soil microbial flora, promote absorption of nitrogen, phosphorus and potassium and increase yield, and is a safe, environment-friendly and long-acting measure with high economic benefit.

Bacillus belgii is a new species of bacillus, and can be used for preventing and treating diseases such as pear gray mold, penicillium disease, rice blast, early blight of tomato and the like at present.

According to the invention, a strain of Bacillus beiLeisi CY30 is separated and screened from the rhizosphere soil of tea trees, the prevention and treatment effect, the colonization ability and the growth promotion effect of the tea trees of tea tree leaf spot are researched, and the purpose of the invention is to lay a foundation for the later application of CY30 in the tea trees.

Disclosure of Invention

The invention aims to provide an isolated Bacillus belgii strain, which is named as Bacillus belgii CY30 and has the preservation number as follows: CCTCC NO: M2018710.

The application of the separated Bacillus belgii can be used for preparing microbial preparations with the functions of preventing diseases and promoting growth of tea leaves, including preventing and treating tea leaf spot and promoting growth of the tea leaves.

In order to achieve the purpose, the invention adopts the following technical measures:

an isolated bacillus belgii strain is obtained by screening alternaria alternata, is further determined to be bacillus belgii by comparing 16s rDNA and gyrA genes and combining physiological and biochemical identification, is delivered to a China center for type culture collection for collection in 2018, 10 and 25 months, and is classified and named: bacillus thuringiensis CY 30; the preservation number is: CCTCC NO of M2018710; a place: china, wuhan university.

The colony of the strain grows well in the NA culture medium, and the colony is milky white (figure 1).

The application of the separated Bacillus belgii comprises the step of preparing a crop microbial preparation by using the Bacillus, wherein the preparation has the effects of preventing and treating tea round spot and/or promoting growth of tea trees.

Compared with the prior art, the invention has the following advantages:

(1) the invention provides a Bacillus beilesiensis strain which has strong stress resistance and is easy to ferment in large quantities, can colonize tea leaves and has lasting bactericidal activity on tea round spot disease in the first report.

(2) The strain has the growth-promoting activity on tea trees, is convenient to use, has no residue and pollution, and can effectively reduce the application of chemical fertilizers and pesticides.

Drawings

FIG. 1 is a schematic representation of the colony morphology of strain CY30 on a plate.

FIG. 2 is a schematic diagram showing the effect of 5-fold dilution of CY30 fermentation liquor on preventing tea round spot.

Detailed Description

In order to better explain the invention, the following further illustrate the main content of the invention in connection with specific examples, but the content of the invention is not limited to the following examples. The technical solutions of the present invention, if not specifically mentioned, are conventional in the art, and the reagents or materials, if not specifically mentioned, are commercially available.

Example 1:

acquisition and characterization of bacillus belgii CY 30:

bacillus belgii CY30, is separated from the rhizosphere soil of tea trees in Enshi Zhongfeng county of Hubei province, and is obtained by screening the bacteriostatic activity of killing tea round spot. The strain is subjected to 16s rDNA and gyrA gene sequencing, is combined with physiological and biochemical identification to further determine the taxonomic status (table 1 and table 2), is delivered to the China center for type culture collection for collection in 2018, 10 and 25 months, and is classified and named: bacillus velezensis CY 30; the preservation number is CCTCC NO of M2018710; a place: china, wuhan university.

In the present invention, Bacillus velezensis CY30 or simply CY 30.

TABLE 1 physiological and biochemical characteristics of Strain CY 30-enzyme Activity, carbon Source Oxidation

+: positive reaction; -: negative reaction; w: weak positive reaction

TABLE 2 physiological and biochemical characteristics of Strain CY 30-production of acid Using a carbon Source

+: positive reaction; -: negative reaction; w-weakly positive reaction

Example 2:

fermentation of bacillus belgii CY30 (the percentages in the following culture medium formula are mass fractions):

1) first-order seed culture: 200ml of first-level seed culture medium is filled in a 500ml triangular flask, moist heat sterilization is carried out for 30min at the temperature of 121 ℃, live bacteria freeze-dried tube strain of Bacillus beiLeisi CY30 is inoculated in the first-level seed culture medium, and culture is carried out for 8-10 h at the temperature of 30-35 ℃; the raw materials and the dosage of the culture medium used for the first-stage seed culture are as follows (mass fraction): 2% of glucose, 2% of beef extract, 1% of peptone, 0.35% of sodium chloride and 7% of culture medium pH;

2) secondary seed culture: placing 240L of secondary seed culture medium in 400L fermentation tank, performing moist heat sterilization at 121 deg.C for 30min, and inoculating 200ml of primary seed into the secondary seed culture medium; culturing at 35 deg.C for 10 hr; controlling the tank pressure to be 0.05Mpa, the stirring speed to be 150rpm, the aeration ratio to be 1:0.8, and the raw materials and the dosage of the culture medium used for the secondary seed culture to be: 2% of glucose, 2% of yeast extract, 1% of peptone, 0.02% of dipotassium hydrogen phosphate, 0.5% of sodium chloride, 0.05% of magnesium sulfate and the pH value of a culture medium is adjusted to 7;

3) fermentation: at 40m³The fermentation tank is filled with 30m³Sterilizing live Bacillus belgii CY30 fermentation culture medium at 121 deg.C for 30min, inoculating 240L of secondary seeds into live Bacillus belgii CY30 fermentation culture medium, controlling tank pressure at 0.05Mpa, stirring at 120rpm, and ventilating at a ratio of 1:0.8, and culturing at 35 deg.C for 36 hr; the raw materials and the dosage of the culture medium used for fermentation are as follows: 3% of sweet potato powder, 2% of peanut meal, 1% of industrial peptone, 1% of yeast extract, 0.015% of monopotassium phosphate, 0.02% of magnesium sulfate, 0.1% of calcium carbonate and 7% of culture medium pH;

4) the fermentation is finished when the spore falls off by 20 percent, and the number of the spores is about 100 hundred million cfu & mL by measurement^-1The following examples were conducted using the fermentation broth.

Example 3:

application of bacillus beilesiensis CY30 in preventing and treating tea round spot disease:

activating tea leaf spot bacteria (Pestalotiopsis theta) to PDA plate for 5d for backup, collecting tea leaves (Zhongcha 108) without bactericide and with consistent size for in vitro leaf test. CY30 fermentation broth with a spore count of about 100 hundred million cfu mL was prepared as in example 2^-1The fermentation broth was diluted 5 and 10 times with sterile water, respectively, for use. Uniformly spraying 5 times of the diluted solution and 10 times of the diluted solution of the fermentation liquor on the tea leaves by using an electric sprayerLeaves, 2mL per leaf, blank control sprayed with sterile water. After each treatment is air-dried, inoculating tea wheel spot fungus cakes by using a needle-prick inoculation method, carrying out moisture-keeping culture at 28 ℃ for 12d, counting the diameters of control and treatment spots, calculating the control effect, repeating each treatment for 3 times, repeating each treatment for 15 leaves, and multiplying the control effect (the diameter of the control spot-the diameter of the treatment spot/the diameter of the control spot) by 100.

The Bacillus belgii CY30 is subjected to resistance screening in a medium containing rifampicin with different concentrations to obtain rifampicin resistant mutant strain (the strain has no significant difference from CY30 in physiological and biochemical characteristics except rifampicin resistance after detection), and the strain can contain 300 mug. mL^-1Normal growth in LB of rifampicin.

The rifampicin resistant mutant strain was fermented as in example 2, the fermentation broth was sprayed on the leaves of tea and the control tomato, after 10 days the leaf surface was sterilized with 75% water and washed 3 times with sterile water. Air drying, weighing 1g of folium Camelliae sinensis and fructus Lycopersici Esculenti, adding 1mL of sterile water, grinding in mortar, standing, collecting supernatant, diluting by gradient dilution method, and coating on a substrate containing 300 μ g/mL^-1The number of the rifampicin in LB plate was counted after culturing at 37 ℃ for 24 hours, and the colonization ability of CY30 in tea leaves and tomato leaves was determined.

Tests show that the Bacillus belgii CY30 has good prevention effect on tea-leaf spot, the diameters of the disease spots after 5-time fermentation liquid treatment and 10-time fermentation liquid treatment are obviously lower than the control diameter, and the prevention effects are 77.58% and 60.38% respectively (Table 3, figure 2). CY30 separated from tea plant rhizosphere has specific colonization effect on tea leaves, and the colonization amount is 6.5 × 10⁵cfu g, while there is no colonization in tomato leaves, this specific colonization effect of CY30 can persistently control the infestation of tea round spot.

TABLE 3 prevention and treatment of tea leaf spot with CY30 fermentation broth

Treatment of	Diameter of lesion (mm)	Control effect (%)
			CY 305 times fermentation broth	6.033	77.59
CY 3010 times fermentation liquor	10.68	60.38
			Control	26.97	-

Example 3:

growth promoting effect of Bacillus belgii CY30 on tea trees

The test field is selected from the black fungus mountain base of tea agriculture, Tan and Xin, Wan, Hubei province, Enshi, He Feng county, CY30 fermentation stock solution (100 hundred million cfu. mL)^-1) Diluting by 20 times, performing root irrigation treatment on the tea trees, wherein the water consumption per mu is 600 jin, and irrigating clear water in contrast. Once on 7 days, three times in total. And (4) counting the germination density at the two-leaf stage of the bud after 20 days, investigating the germination density (in a box of 33.3cm multiplied by 33.3 cm), and measuring the weight of the bud.

Tests show that CY30 can obviously improve the germination density of tea trees, the weight of hundreds of buds is reduced, but the mass of one bud and two leaves tea in unit area is increased, and the yield is obviously increased (Table 4).

TABLE 4 CY30 Effect on growth promotion and yield increase of tea trees

Treatment of	Germination density (volume)	Baiya weight (g)
			CY30	191.00	40.59
CK	136.33	42.33

Claims

1. An isolated Bacillus belgii strain, wherein the Bacillus belgii strain is Bacillus belgii (Bacillus belgii: (B))Bacillusvelezensis) CY30, accession number: CCTCC NO: M2018710.

2. Use of bacillus beijerinckii according to claim 1 for the preparation of a formulation for promoting tea tree production.

3. Use of the bacillus beilesensis of claim 1 for the preparation of a formulation for controlling tea round spot.

4. Use of the bacillus beilesensis of claim 1 in the preparation of a medicament for controlling tea round spot while promoting tea plant yield increase.