CN114015592B - Endophytic control strain HN075 for preventing and treating litchi frosted epidemic disease and application thereof - Google Patents

Abstract

The invention discloses an endophytic control strain HN075 for preventing and treating a lychee frosting epidemic disease and application thereof. The biocontrol strain is Bacillus spp HN075 and has been registered and preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 11 months and 30 days in 2020, and the strain preservation number is CGMCC No.21270. The strain has good control effect on litchi frost epidemic diseases. The biocontrol strain bacillus HN075 is obtained by separating from the inside of litchi leaves, belongs to endophytic biocontrol bacteria, is nontoxic and nonpathogenic, and has good environmental compatibility, thus having good application prospect in biological control of litchi frost epidemic diseases.

Description

Endophytic control strain HN075 for preventing and treating litchi frosted epidemic disease and application thereof

Technical Field

The invention belongs to the technical field of crop disease control, and particularly relates to an endophytic control strain HN075 for controlling a lychee frosting epidemic disease and application thereof.

Background

The litchi frost epidemic disease caused by the litchi frost phytophthora (Peronophythora litchii Chen ex Ko et al) seriously threatens the healthy development of the litchi industry in China, and is the disease with the most serious harm and the most extensive influence after litchi production and picking. The disease can infect tender leaves, twigs, spikes and fruits, cause a large amount of rot of the spikes and fruits, can cause 10% -30% yield loss in general year, can cause 80% yield loss in popular year, and is particularly serious in Guangdong, guangxi, fujian, hainan and other places, thereby seriously affecting the development of litchi industry in China and causing great economic loss.

At present, the variety for preventing and treating the litchi frost epidemic disease is lacking, and chemical agents are mainly used for preventing and treating the litchi frost epidemic disease, wherein the cultivated disease-resistant variety is the most economical, effective and simple method for preventing and treating the litchi frost epidemic disease, but the breeding of the new variety for preventing and treating the litchi frost epidemic disease in a short time is very difficult. While pesticides play an important role in the development of agriculture, safety and environmental problems associated with chemical pesticides are increasingly prominent with the development of society. The use of chemical pesticides has not only lacked long-term effectiveness, but also has been increasingly questioned as to pollution to food and environment, potential harm to human health, influence on non-target organisms, and the occurrence of pathogenic bacterial drug resistance. Therefore, the use of biological control, especially antagonism microorganism, is paid attention to and paid attention to, and the exploration of effective biocontrol ways clearly has great theoretical significance and practical value.

Bacillus spp is a dominant biological population of soil and plant micro-ecological systems, and has high stress resistance and antibacterial and disease-preventing effects. Bacillus not only can be widely existed in the external environments of soil, plant rhizosphere body surface and the like, but also is common endophytic bacteria in plants, especially in the roots and stems of plants. At present, the strain has good disease control effect on crops such as rice, soybean, cotton, wheat, chilli, tomato, corn and the like. The bacillus subtilis has the outstanding characteristics of fast growth, simple nutrition, heat resistance, stress resistance and the like, so that the bacillus subtilis is not only beneficial to the survival, colonization and reproduction of the biocontrol microbial inoculum in production, dosage form processing and environment, but also has simple mass production process, lower cost, convenient application and long storage period, and becomes an ideal biocontrol bacterium. Many natural isolates with excellent traits have been successfully used for biological control of plant diseases. The bacillus successfully colonizes the rhizosphere, the body surface or the body of the plant, competes with pathogenic bacteria for nutrition around the plant, secretes antibacterial substances to inhibit the growth of the pathogenic bacteria, and induces a plant defense system to resist the invasion of the pathogenic bacteria, so that the purpose of biocontrol is achieved. The main control object is plant diseases caused by filamentous fungi and oomycetes. The bacillus is a mesophilic, aerobic and spore-producing rod-shaped bacterium, has various physiological characteristics and wide distribution, and is extremely easy to separate and culture. The strain is widely existed in the nature, is nontoxic and harmless to people and livestock, does not pollute the environment, can produce various antibiotics and enzymes, and has extremely strong stress resistance.

Disclosure of Invention

In view of the defects of the prior art, the invention provides an endophytic control strain HN075 for preventing and treating the litchi frosting epidemic diseases and application thereof, aiming at the problems that the existing litchi frosting epidemic diseases seriously occur, the area is enlarged year by year, and the chemical prevention and treatment cause environmental pollution.

The technical scheme of the invention mainly comprises the following contents:

the endophytic control strain HN075 for preventing and treating the lychee frosted epidemic disease is identified as Bacillus (Bacillus spp.) by morphological observation, culture characteristic observation and molecular biological research, and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 11 months and 30 months in 2020, and has the following addresses: the collection number of the strain is CGMCC No.21270 at the institute of microbiological study, china academy of sciences, north Chen Xiu Lu No. 1, 3, the Korean region of Beijing city.

The invention also provides a biocontrol microbial agent prepared by fermenting the endophytic biocontrol strain HN075, and the preparation method comprises the following steps:

inoculating seed liquid of an endophytic fungus strain HN075 into a fermentation medium for fermentation to obtain fermentation liquid, centrifuging the fermentation liquid, and filtering to obtain the biocontrol microbial agent; the fermentation conditions are as follows: the inoculation amount is 5% (v/v), the pH value of the fermentation medium is 7.0-7.2, the culture temperature is 37 ℃, the rotating speed of a shaking table is 180r/min, and the culture time is 48h.

Preferably, the fermentation medium is: each 1000mL of fermentation medium contains 10g of tryptone, 5g of yeast powder, 5g of sodium chloride, 15g of agar powder and the balance of distilled water.

Preferably, the use method of the biocontrol microbial agent is as follows: spraying on the branches, leaves or fruits of litchi; the medicine is used for 2 to 3 times continuously, and each time is 7 to 10 days.

The invention also provides application of the endophytic biocontrol strain HN075 in preventing and treating litchi downy mildew.

On the other hand, the invention also discovers that the optimized fermentation medium has the effect of preventing and treating the lychee frost epidemic disease, and the fermentation medium is as follows: every 1000mL of fermentation medium contains 10g of tryptone, 5g of yeast powder, 5g of sodium chloride, 15g of agar powder and the balance of distilled water; the pH value of the fermentation medium is 7.0-7.2.

The invention has the following effects:

the biocontrol bacteria are separated from litchi leaves, so that the advantages of the strains are fully exerted, the colonization capability is strong in litchi leaves and litchi fruits, and the control effect is good and stable.

The biocontrol strain HN075 screened by the invention has strong antibacterial effect and can obviously inhibit the growth of downy mildew. Because the litchi seed extract is endophytic beneficial bacteria separated from litchi leaves, a series of problems caused by the use of chemical pesticides are avoided, agricultural pollution is reduced, and the litchi frost epidemic disease can be effectively prevented and treated.

The biocontrol strain disclosed by the invention has the advantages of simple culture conditions, easiness in industrial production and good development and application prospects.

The invention discovers that the optimized fermentation medium has control effect on the litchi frost epidemic disease.

Drawings

FIG. 1 is a graph showing the effect of direct inhibition test on biocontrol bacteria.

Detailed Description

In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.

Example 1: isolation of endophyte-control strains

Washing litchi leaves with tap water, washing the litchi leaves with sterilized water for 3-5 times, washing the surfaces of the litchi leaves, rinsing the litchi leaves with 75% alcohol for 3min, sterilizing the surfaces of the litchi leaves, and washing the litchi leaves with sterilized water for 3 times; mercuric chloride is treated for 10 to 15 minutes, washed 3 times by sterile water and then dried. Adding 10mL of sterile water and a little sterilized quartz sand into a sterile mortar, grinding the surface-treated leaf tissue into homogenate in a sterile environment of an ultra-clean workbench, covering and standing for 30min, diluting the homogenate into 10, 100 and 1000 times of liquid, respectively sucking 50 mu L of the liquid onto a prepared NA flat plate by a liquid transfer device, uniformly coating the liquid onto a culture medium by a coating rod for growth, picking single bacterial colonies after 48h, and purifying and preserving the flat plate to obtain the endophyte strain HN075.

Example 2: identification of Strain HN075

1. Morphological feature observations

The strain HN075 is streaked and inoculated in LB culture medium (5 g yeast powder, l 0g tryptone, 5g sodium chloride and 15g agar powder are added with distilled water to 1000mL, dissolved and pH is adjusted to 7.0-7.5, and the mixture is sterilized under high pressure for standby). Colony characteristics and morphological characteristics were observed after 48h incubation at 37 ℃.

Colony characteristics: the bacterial colony is round, milky white, opaque, neat in edge, convex in the center and moist and slightly sticky in surface.

Morphological characteristics: the strain HN075 thallus is in a rod shape, gram staining is positive, both ends are round, endophytic spores are elliptical, the spores are not expanded, and the spores grow from the middle to the secondary. The strain HN075 was initially judged to belong to the genus Bacillus (Bacillus sp) based on morphological characteristics.

2. Molecular biological identification

After genomic DNA of strain HN075 was extracted using the genomic extraction kit, PCR amplification of 16S rDNA was performed using the universal primers L1:5-AGTCGTAACAACGTAGCCGT-3 'and L2:5-GTGCCAAGGCATCCACC-3'. And (3) recovering PCR amplification products, carrying out positive clone sequencing after connection, transformation and identification, submitting the obtained sequences to a GenBank database for BLAST analysis and comparison, and finding that the sequences have more than 99.8% of sequence homology with bacillus subtilis. The strain HN075 was identified as Bacillus spp, in combination with morphological and culture characteristics.

Example 3: inhibition of strain HN075 on Phytophthora litchi

Biocontrol strain direct inhibition test: after the downy mildew of litchi is activated, a downy mildew fungus block (5 mm) of litchi is inoculated at the center of a culture dish, fungus liquid of a strain HN075 is dipped, two sides which are 2.5cm away from the center of the fungus block are symmetrically streaked, 3 times of experiments are repeated, and after the litchi fungus block is placed in a constant temperature incubator at 25 ℃ for dark culture for 5 days, the size of a bacteria inhibition zone is observed (figure 1).

Biocontrol bacterium fermentation liquor inhibition test: the strain HN075 is inoculated on an LB plate for activation for 20 hours, a single colony is picked up in an LB liquid culture medium (200/500 mL triangular flask) by an inoculating loop, and the strain HN075 is placed on a shaking table for culturing for 48 hours at a constant temperature of 37 ℃ and 180r/min, so that fermentation liquor is obtained for standby. 20mL of aseptic fermentation liquor of strain HN075 is taken and respectively added into melted 200mL of PDA culture medium cooled to about 45 ℃ to be mixed and poured into a flat plate, so as to prepare an antibacterial flat plate of the aseptic fermentation liquor with different concentrations, the center of the flat plate is inoculated with mycelium blocks with the diameter of 5mm, and the aseptic water is used for replacing the fermentation liquor as a control. Each treatment was repeated 3 times and incubated in an incubator at 37℃for 5-7 days, and the hypha growth diameter was measured by the crisscross method.

Example 4: optimization of fermentation process of strain HN075

The strain HN075 is inoculated in an LB liquid culture medium at 37 ℃,180r/min and cultured for 24 hours to prepare seed liquid, and 10mL of the seed liquid is inoculated in a fermentation culture medium for culture. The formula of the fermentation medium obtained after optimization is as follows: the formula of the fermentation medium is as follows: 10g of tryptone, 5g of yeast powder, 5g of sodium chloride and 15g of agar powder are added with distilled water to reach 1000mL, dissolved and adjusted to pH 7.0-7.2. By fumbling the culture conditions, the optimal fermentation conditions are: the inoculation amount is 5% (v/v), the bottling amount is 100mL/250mL, the pH value of the fermentation medium is 7.0-7.2, the temperature is 37 ℃, the speed is 180r/min, and the culture is carried out for 48 hours, so that the concentration is about 10 ⁸ CFU/mL of strain HN075 broth. Centrifuging the fermentation liquor at 4 ℃ for 20min at 8000r/min, and filtering with a 0.22 mu m bacterial filter to obtain a sterile fermentation liquor (namely HN075 biocontrol microbial agent).

Example 5: strain HN075 fermentation liquor field anti-effect application

The litchi variety is Feizixiao, and the experimental land is a perennial litchi frost epidemic disease perennial land. The test soil is loam, the organic matter content is general, and normal fertilizer and water management is performed. Planting density: plant spacing is 4m, row spacing is 6m, tree age is 12 years, and growth period is flowering to fruiting period. The test is carried out by setting 5 treatments, 2 trees are processed in each treatment cell, 20 cells are added, and the test is repeated for 4 times. And (3) determining clusters, carrying out comprehensive pesticide spraying treatment after marking, marking 10 clusters of each plant, and uniformly spraying the whole plant by using a swing type high-pressure sprayer until the pesticide spraying amount is equal to that of the leaf surface water. The pesticide is sprayed for 1 time and 3 times respectively at the second flowering stage of litchi, the initial stage of physiological fruit dropping of litchi and about 0.5 cm in fruit diameter. Investigation was performed when the condition was substantially stable.

The marked clusters were investigated for total number of clusters, number of diseased clusters, and disease grade of clusters, respectively. The disease grade of the fruits is divided according to the proportion of the disease spot area to the whole fruit surface area.

The disease classification criteria were as follows: level 0: no disease spots on fruits; stage 1: 1-2 brown small lesions are formed on the fruits; 3 stages: 3-4 brown small lesions are formed on the fruits; 5 stages: the surface area of the disease spots on the fruits reaches 1/4;7 stages: the surface area of the disease spots on the fruits reaches 1/3, and part of the disease spots are connected; stage 9: the disease spots on the fruits reach 1/2 of the surface area of the fruits, and decay occurs. Calculating the prevention effect according to formulas (1) and (2):

disease index = Σ (number of diseased fruits at each stage×number of diseased fruits at each stage)/(number of total fruits investigated×9) ×100%

Prevention effect (%) = [ (CK disease index-treatment disease index)/(CK disease index ] ×100%)

Experimental results show that (Table 1), HN075 biocontrol microbial inoculum (see example 4) can obviously reduce the disease index of the litchi frost epidemic disease, the control effect is up to 72.20%, and the control effect is only slightly lower than the control effect 82.79% of 50% metalaxyl wettable powder 600 times liquid of chemical pesticide. In addition, as can be seen from the table, the disease index of the fermentation medium (see example 4) is slightly lower than that of the clear water control treatment, and the fermentation medium also plays a role in preventing and treating. Experimental results show that the strain HN075 can effectively prevent and treat the litchi downy mildew, and has good application potential.

TABLE 1 prevention and treatment effects of bacterial strain HN075 fermentation broth on litchi downy mildew

Note that: the difference between the values represented by the lower case letters in the same column is significant (P < 0.05)

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims (6)

1. The endophytic control strain HN075 for preventing and treating the downy mildew of litchis is characterized in that the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 11 months and 30 days in 2020, and the strain preservation number is CGMCC No.21270.

2. A biocontrol microbial agent fermented by the endophytic biocontrol strain HN075 of claim 1.

3. The method for preparing the biocontrol microbial agent as claimed in claim 2, which is characterized by comprising the following steps:

inoculating seed liquid of an endophytic control strain HN075 into a fermentation medium for fermentation to obtain fermentation liquid, centrifuging the fermentation liquid, and filtering to obtain aseptic fermentation liquid, thus obtaining the biocontrol microbial agent; the fermentation conditions are as follows: the inoculation amount is 5% by volume, the pH value of the fermentation medium is 7.0-7.2, the culture temperature is 37 ℃, the rotation speed of a shaking table is 180r/min, and the culture time is 48h.

4. A method of preparation according to claim 3, wherein the fermentation medium is: each 1000mL of fermentation medium contains 10g tryptone, 5g yeast powder, 5g sodium chloride, 15g agar powder and the balance of distilled water.

5. The method for using the biocontrol microbial agent as claimed in claim 2, wherein the biocontrol microbial agent is sprayed on litchi branches, leaves or fruits; the usage is carried out for 2-3 times continuously, and each time is 7-10 days apart.

6. The use of the endophytic biocontrol strain HN075 according to claim 1 for controlling litchi downy mildew.