CN107736379B

CN107736379B - Application of bacillus amyloliquefaciens in preventing and treating plant fungal diseases

Info

Publication number: CN107736379B
Application number: CN201711060132.3A
Authority: CN
Inventors: 孙龙华; 黄华枝; 阮琳; 毕可可; 邓嘉茹; 张劲蔼; 吴超; 唐立鸿; 胡彦辉; 邓从双; 候亚会
Original assignee: Guangzhou Institute Of Forestry And Landscape Architecture
Current assignee: Guangzhou Institute Of Forestry And Landscape Architecture
Priority date: 2017-11-01
Filing date: 2017-11-01
Publication date: 2020-04-03
Anticipated expiration: 2037-11-01
Also published as: CN107736379A

Abstract

The invention discloses application of bacillus amyloliquefaciens in preventing and treating plant fungal diseases. The research of the invention finds that the bacillus amyloliquefaciens, in particular the bacillus amyloliquefaciens strain BSY3 separated and screened from marigold by the inventor has obvious antagonistic action on common plant fungi such as brown root pathogen, canna indica pestis, sclerotinia sclerotiorum, colletotrichum album anthracnose pathogen, alternaria alternata leaf spot pathogen, fusarium oxysporum and the like, and has obvious biological control action and potential application value in preventing and treating fungal diseases caused by the pathogenic bacteria. Moreover, the bacillus amyloliquefaciens is derived from endophytic bacteria in plants, and has safer biocontrol effect on fungal diseases compared with the traditional chemical agent control method; and the biological control agent can establish a harmonious, co-located and stably developed survival relationship with plants for a long time in the plants, and can more effectively and stably exert the biological control function compared with chemical agents.

Description

Application of bacillus amyloliquefaciens in preventing and treating plant fungal diseases

Technical Field

The present invention belongs to the field of biological plant disease preventing and controlling technology. More particularly, it relates to the application of bacillus amyloliquefaciens in the control of plant fungal diseases.

Background

The tree fungus disease is caused by pathogenic fungi, such as brown root disease, and is caused by harmful Phellinus (A)Phellinus noxius) Soil-borne diseases causing destructive damages to cash crops, fruit trees, ancient and famous trees, landscaping plants and the like are called tree cancer. At present, prevention and control measures such as cutting off diseased trees, digging ditches to block pathogen transmission, fumigating diseased soil and planting disease-free nursery stocks are mainly adopted, but no ideal prevention and control agent and method can effectively control the occurrence of brown root diseases of trees. In addition, although some chemical agents can effectively inhibit brown root germs at present, the chemical pesticide is applied for a long time, but the cost is high, and simultaneously, the soil and the ecological environment are damaged. Meanwhile, in recent years, with the attention and importance of people on personal health, food safety and ecological environment, the reduction of the dosage of chemical agents, especially traditional high-toxicity and high-residue chemical pesticides, is imperative in agricultural production. Biological control is the first and inevitable trend for plant disease control measures.

Although the research on plant endophytes has been developed later, the biological control of plant diseases by using plant endophytes has become a hot point of research. Endophytic bacteria are a group of microorganisms that can colonize living in healthy plant tissues and organs, but do not cause substantial damage to plants, and can form a mutually restricted, harmonious, combined, and mutually beneficial relationship with plants (Istvan F,et al., 1995). From early studies to the 90's of the 20 th century, endophytic bacteria of nearly 60 genera, of which about 2/3 is a gram-negative bacterium, have been isolated from nearly 30 plants according to incomplete literature statistics, so that a population of microorganisms of the type commonly occurring endophytic bacteria is present in most higher plants (Kobayashi D Y,et al.,1976）. Under the action of long-term life in plants, endophytic bacteria have beneficial direct or indirect biological promotion effects on host plants. The direct growth promotion of endophytic bacteria in plants is mainly based on nitrogen fixation, promotion or synthesis of plant-associated growth hormones, or synthesis of siderophores to assist the host plant in absorbing iron ions from the soil (Lazarovits G,et al., 1997). While the indirect growth promotion effect on host plants mainly means that the endophytic bacteria can form a complex disease resistance mechanism to play a role of a plant disease control factor after interacting with plants and the environment, thereby reducing or preventing the occurrence of diseases and the like (Sturz av,et al., 2000）。

the endophytic bacteria of the plant can form a long-term cooperative relationship with the plant, which can show that the endophytic bacteria occupy stable ecological niches in the plant, are not easily influenced by external environment, are easy to colonize the interior of each tissue in the plant body, and the characteristic of the stable ecological niche of the endophytic bacteria is beneficial to the effect of the endophytic bacteria on the aspect of biocontrol. In addition, the endophytic bacteria of plants have a plurality of disease prevention action mechanisms, which mainly comprise secretion of antibacterial active substances, competition of nutrition and ecological niches, enhancement of the stress resistance of the plants, induction of systemic resistance of the plants and the like. When the endophytic bacteria in the plant tissue can sense that the plant is hurt by the pathogenic bacteria or directly act with the pathogenic bacteria, the pathogenic bacteria can directly or indirectly promote the endophytic bacteria to generate antagonistic substances to inhibit the growth of the pathogenic bacteria or weaken the pathogenicity of the pathogenic bacteria. From the perspective of microorganisms, endophytic bacteria are used as carriers of exogenous genes, and exogenous insecticidal and disease-resistant genes can be accidentally transferred into plants on the premise of not changing the natural properties of the plants, so that the disease resistance of the plants is improved, and the biocontrol effect is played.

At present, no report of applying endophytic bacteria to prevent and treat tree brown root diseases and other fungal diseases exists, and the corresponding selection of the bacteria and antagonistic bacteria thereof is also an important factor and index for selecting biocontrol endophytic bacteria.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the existing tree brown root disease and other fungal disease control technology, and provides an application of an endophyte antagonistic bacterium from marigold in tree brown root disease and other fungal diseases control.

The invention aims to provide application of bacillus amyloliquefaciens in preventing and treating plant fungal diseases.

The invention also aims to provide a biological agent containing the bacillus amyloliquefaciens for preventing and treating plant fungal diseases.

The above purpose of the invention is realized by the following technical scheme:

the present invention provides Bacillus amyloliquefaciens (B.amyloliquefaciens)Bacillus amyloliquefaciens) Application in preventing and treating plant fungal diseases.

Preferably, the bacillus amyloliquefaciens is bacillus amyloliquefaciens (A), (B), (CBacillus amyloliquefaciens) The strain BSY3 is preserved in the China center for type culture Collection in 2017, 8 and 31 months, and the preservation number is CCTCCNO: M2017463.

The strain is separated from leaves of a herbal plant marigold by the inventor, is marigold endophytic bacteria, has obvious antagonistic action on brown root pathogen, canna edulis Leporis, Sclerotinia sclerotiorum, Alternaria albus, Blackberella tabacis and Sclerotinia oxysporum, and has certain biocontrol effect and potential application value in preventing and treating fungal diseases caused by the pathogenic bacteria.

Therefore, the biological agent for preventing and treating plant fungal diseases, which contains the bacillus amyloliquefaciens and/or the bacillus amyloliquefaciens fermentation liquor, and the application of the biological agent in preventing and treating plant fungal diseases are both within the protection scope of the invention.

Specifically, the biological agent takes bacillus amyloliquefaciens strain BSY3 and/or bacillus amyloliquefaciens strain BSY3 fermentation liquor as active ingredients.

Preferably, the plant fungal diseases are plant fungal diseases caused by brown root pathogen, canna plague pathogen, sclerotinia sclerotiorum, colletotrichum album, alternaria leaf spot pathogen and/or banana vascular wilt pathogen.

In addition, preferably, the preparation method of the bacillus amyloliquefaciens fermentation liquid comprises the following steps: culturing bacillus amyloliquefaciens for 36-50 h, and then obtaining a crude extract from a fermentation supernatant through a filter membrane, wherein the culture medium used for culture comprises the following components in proportion: 8-12 g of glucose or sucrose, 4-6 g of yeast extract, 5g of peptone and MgSO₄Or MgCl₂4-6 g, and fixing the volume of water to 1L; the pH value is 6-7.

More preferably, the preparation method of the bacillus amyloliquefaciens fermentation liquid comprises the following steps: culturing bacillus amyloliquefaciens for 48 hours, and then, passing the fermentation supernatant through a 0.22 mu m filter membrane to obtain a crude extract, wherein the culture medium used for culture comprises the following formula: 10g of glucose, 5g of yeast extract, 5g of peptone and MgSO₄5g, and fixing the volume of water to 1L; the pH was 6.5.

In addition, the invention also provides a method for preventing and treating brown root disease of trees, which is to spray a biological agent containing bacillus amyloliquefaciens and/or fermentation liquor thereof on an affected part. The invention provides a novel method for preventing and treating brown root disease called tree cancer.

The invention has the following beneficial effects:

(1) the bacillus amyloliquefaciens is used as a pathogenic fungus antagonistic bacterium, has obvious control effect on common fungal diseases including brown root disease germs of trees, and provides a new biological control method for controlling plant fungal diseases such as brown root disease of trees and the like.

(2) The pathogenic fungi antagonistic bacteria is derived from endophytic bacteria in plants, and has safer biocontrol effect on fungal diseases compared with the traditional chemical agent control method.

(3) The pathogenic fungi antagonistic bacterium is derived from endophytic bacteria in plants, can establish a harmonious, co-located and stably developed survival relationship with plants for a long time in the plants, and can more effectively and stably exert the biological control function of the endophytic bacteria compared with chemical agents.

Drawings

FIG. 1 shows the antagonistic effect of Bacillus amyloliquefaciens strain BSY3 on Fusarium oxysporum.

FIG. 2 shows the colony morphology of Bacillus amyloliquefaciens strain BSY 3.

FIG. 3 shows the effect of different carbon sources on the growth and bacteriostatic activity of Bacillus amyloliquefaciens strain BSY 3.

FIG. 4 shows the effect of different nitrogen sources on the growth and bacteriostatic activity of Bacillus amyloliquefaciens strain BSY 3.

FIG. 5 shows the effect of different inorganic salts on the growth and bacteriostatic activity of Bacillus amyloliquefaciens strain BSY 3.

FIG. 6 shows the effect of different pH on the growth and bacteriostatic activity of Bacillus amyloliquefaciens strain BSY 3.

FIG. 7 shows the effect of different culture times on the growth and bacteriostatic activity of Bacillus amyloliquefaciens strain BSY 3.

FIG. 8 shows the antagonistic effect of Bacillus amyloliquefaciens strain BSY3 on 6 common pathogenic fungi.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 isolation and characterization of Bacillus amyloliquefaciens strain BSY3

1. The strain is separated from marigold plants collected from Guangzhou white cloud nursery, and the specific separation method comprises the following steps:

(1) isolation of Single colony

1) Cleaning collected fresh marigold leaf materials with tap water;

2) washing with sterile water, soaking in 75% alcohol for 5min, soaking in 2% sodium hypochlorite solution for 2 min, washing with sterile water for 4 times, and coating the sterile water on a flat plate to obtain a blank control;

3) placing the sterilized fresh plant material into a sterilization mortar, adding a small amount of sterile water, grinding into homogenate, and diluting into grinding liquids with different gradients to be respectively coated on a flat plate;

4) and (3) placing the plate in an incubator at 30 ℃ for culture, observing the growth condition of bacterial colonies, picking bacterial colonies with different forms, performing purification culture on the target bacterial strain, and storing for later use.

(2) The above-mentioned strain isolated from marigold plants and purified and cultured was cultured overnight in 2 mL of LB medium at 280 rpm in a shaker at 30 ℃.

Performing antagonism test on a WA culture medium flat plate to complete a primary screening experiment of antagonistic bacteria, wherein the specific method comprises the following steps: the method comprises the steps of inoculating brown root germ target bacteria into the center of a WA culture medium plate, inoculating endophytic bacterial strains separated from marigold plants at two sides of a position 2cm away from the target bacteria, and screening out bacterial strains with bacteriostatic action on the target bacteria (the result is shown in figure 1).

Wherein the formula of the WA medium is as follows: 5 g/L of peptone, 10 g/L of glucose, 3 g/L of yeast extract, 5 g/L of sodium chloride, 20 g/L of agar and pH 6.8.

(3) Rescreening by a face-off method: re-screening the strains purified by the primary screening by using a PDA culture medium:

the purified strain was cultured overnight in 35 mL of LB medium at 250 rpm in a shaker at 30 ℃ for 17 hours for 2 days, and the supernatant was collected by centrifugation and filtered through a 0.22 μm bacterial filter.

20 mL of PDA and 2 mL of the filtered supernatant of the purified strain were mixed and poured into a tray, a target strain block of about 4mm was placed in the center of a PDA plate, each treatment was performed 3 times, the plate was cultured in an incubator at 30 ℃, and the confrontation results were observed and recorded on the 7 th and 14 th days.

An endophytic bacterial strain with obvious antagonism to brown root pathogenic bacteria is obtained through screening, and is named as BSY 3.

2. Identification of strains

(1) Morphological identification

As shown in FIG. 2, the bacterial strain BSY3 appeared pale yellowish white, nearly circular, wrinkled and opaque on LB medium plates. The thalli of the strain BSY3 is rod-shaped, produces spores and has no capsule when observed by a microscope. The gram reaction was positive.

(2) Physiological and biochemical experiments of strain BSY3

The basic biological properties of the strain BSY3, such as physiology, biochemistry, etc., are shown in Table 1.

TABLE 1 basic biological Properties of Strain BSY3

Note: positive reaction, or can grow and use; negative reaction, or growth and utilization are not possible.

(3) 16S rRNA sequence alignment

The 16S rRNA sequence of the strain BSY3 is obtained through clone sequencing, and BLAST comparison analysis is carried out on the sequence on NCBI website, so that the similarity with the 16S rRNA of the bacillus amyloliquefaciens reaches 99 percent.

Meanwhile, in conclusion, the morphological characteristics and physiological and biochemical characteristics of the strain are most similar to those of the bacillus amyloliquefaciens, so that the strain can be classified as the bacillus amyloliquefaciens according to the comprehensive results of 16S rRNA gene sequence determination and the morphological characteristics and physiological and biochemical characteristicsBacillus amyloliquefaciens）。

In conclusion, the endophytic bacterial strain with obvious antagonism to brown root pathogenic bacteria, namely the Bacillus amyloliquefaciens strain BSY3, is obtained by separation and screening and is preserved in China center for type culture collection (CCTCC NO: M2017463) in 2017, 8 and 31 days, and the preservation address is Wuhan university in China.

Example 2 optimization of optimal culture conditions for Bacillus amyloliquefaciens strain BSY3

1. Optimal culture medium for strain growth

(1) The optimization method comprises the following steps: the strain BSY3 is streaked and activated on LB culture medium (30 ℃), inoculated into LB liquid culture medium and activated under the conditions of 280 rpm and 30 ℃. The activated strain BSY3 was inoculated to a shake flask fermentation basal medium YGN [ 10g yeast extract (in different nitrogen sources), 10g glucose (in different carbon sources), 5g NaCl (in different inorganic salts) ] at an inoculum size of 0.5%, to a constant volume of 1L, pH 7.0 ]. Inoculating, placing in a shaking table at 30 ℃ and 160rpm for culturing for 2-5 days, then centrifuging fermentation liquor of different determination substances (carbon source, nitrogen source and inorganic salt) at 12000rpm for 15min, filtering supernate with a 0.22-micron microporous filter membrane to obtain original fermentation liquor, preparing a flat plate (9 cm) from the original fermentation liquor and a PDA culture medium according to a ratio of 1:10, placing the phellinus igniarius harmful phellinus, which is pathogenic bacteria of brown root disease, in the center of the PDA flat plate, and determining the growth diameter of the target fungi on the flat plate with different fermentation liquors to react with the bacteriostatic activity when the hypha of the target fungi harmful phellinus igniarius is on the control PDA flat plate is full of the disc (day), namely the influence of the culture of different nutrient sources on the bacteriostatic activity of the BSY3 strain. The blank was prepared in equal proportions of LB broth, three replicates per treatment.

(2) Best C source

As above, 1% soluble starch, sucrose, lactose, maltose and corn flour, and no sugar added as a control, were used as media in place of glucose in the basal medium in equal amounts, and the target bacteria were cultured in the center of the plate in an incubator at 30 ℃. The results show (as shown in FIG. 3) that the bacterial strain BSY3 has the best bacteriostatic activity when glucose and sucrose are used as carbon sources.

(3) Optimum N source

Respectively adding 1% peptone, yeast extract, beef extract, soybean powder, (NH) into basic fermentation medium₄)₂SO₄And NH₄Cl and a culture medium prepared by replacing yeast extract in a basic culture medium in an equal amount without adding any nitrogen source as a control, placing the target bacteria in the center of a flat plate, and culturing in an incubator at 30 ℃. The results show (see FIG. 4) that the bacteriostatic activity of the fermentation filtrate of strain BSY3 was the best when yeast extract powder and peptone were used as nitrogen sources.

(4) The most preferred inorganic salt

Respectively using 0.5% NaCl and CuSO in basic fermentation medium₄、CaCl₂、MgSO₄、ZnCl₂、MgCl₂And MnSO₄And a culture medium prepared by replacing NaCl in the basic culture medium in an equal amount without adding inorganic salt as a blank control, placing the target bacteria in the center of the flat plate, and culturing in an incubator at 30 DEG CAnd (5) nourishing. The results showed (see FIG. 5), as MgSO₄And MgCl₂The bacterial strain BSY3 has the best bacteriostatic activity when the bacterial strain is inorganic salt.

The results show that when glucose and sucrose are used as carbon sources, the bacteriostatic activity of the fermentation filtrate is highest; when yeast extract powder and peptone are used as nitrogen sources, the bacteriostatic activity of the fermentation filtrate is the best; MgSO (MgSO)₄And MgCl₂Two inorganic salts are most preferred. Therefore, the screening result of the combined component ratio shows that the optimal shake flask fermentation medium component of the strain BSY3 is as follows: 10g of glucose, 5g of yeast extract, 5g of peptone and MgSO₄5g, constant volume to 1L, and initial pH value of 7.0.

2. Optimum pH value for bacterial strain growth

The test strain BSY3 was cultured in an optimum liquid medium (glucose 10g, yeast extract 5g, peptone 5g, MgSO 5 g)₄5g, constant volume is set to 1L, initial pH is 5.5-8.0 respectively), the mixture is cultured in a shaking table at 30 ℃ and 160rpm for 2 days, the inoculated mixture is placed in a shaking table at 30 ℃ and 160rpm for culturing for 2-5 days, fermentation liquor with different pH values (5.5-8.0) is centrifuged at 12000rpm for 15min, supernate is filtered by a 0.22 mu m microporous filter membrane to obtain original fermentation liquor, a flat plate (9 cm) is prepared by the original fermentation liquor and a PDA culture medium according to the proportion of 1:10, phellinus igniarius as pathogenic bacteria of the brown root disease is placed in the center of a PDA flat plate, and when hypha of the phellinus igniarius of the target fungus on the flat plate with different pH values is full of the plate, the growth diameter of the target fungus on the flat plate with different pH values is measured to react the bacteriostatic activity. PDA medium was used as a blank and each treatment was repeated 3 times.

The results show (as shown in fig. 6) that the difference of the bacteriostatic effect is not obvious when the pH is 6-7, and the bacteriostatic effect is best when the pH is 6.5. When the pH value exceeds 7.5, the bacteriostatic activity is obviously reduced.

3. Optimal cultivation time for growth of the strain

Subjecting the biocontrol bacteria BSY3 to shake culture in optimal liquid medium (glucose 10g, yeast extract 5g, peptone 5g, MgSO 5)₄5g, constant volume to 1L and optimum pH value of 6.5), shake culturing at 30 deg.C and 160rpm for 24-120 h, sampling every 24h, and collecting the above samples for different time (24-12)0h) The fermentation liquor is centrifuged at 12000rpm for 15min, the supernatant is filtered by a 0.22 mu m microporous filter membrane to obtain the original fermentation liquor, a flat plate (9 cm) is prepared by the original fermentation liquor and a PDA culture medium according to the proportion of 1:10, the phellinus igniarius harmful phellinus igniarius is taken as target fungi and placed in the center of the PDA flat plate, and when the hypha of the target fungi harmful phellinus igniarius is full of the plate on a control PDA flat plate, the growth diameter of the target fungi on the flat plate in different time is measured to react the bacteriostatic activity. PDA medium was used as a blank and each treatment was repeated 3 times.

The results show (as shown in fig. 7) that the bacteriostatic activity reaches the best effect after 48h of culture, and the bacteriostatic activity cannot be obviously enhanced by prolonging the fermentation time.

Example 3 detection of the antagonistic Effect of the Bacillus amyloliquefaciens Strain BSY3 on common pathogenic fungi

1. The BSY3 strain is respectively cultured with several pathogenic fungi together, and the antagonism to the pathogenic fungi is detected.

Test strains: phellinus igniarius (harmful phellinus igniarius) ((P. noxius) Pathogenic bacteria of anthrax of white butterfly: (Colletotrichumsp.), pathogenic bacteria of canna indica pestis (Canna indica)Pyricularia cannaecola) Pathogenic bacteria of black pine leaf spot disease: (Pestalotiopsissp., pathogenic bacteria of banana vascular wilt (Fusarium oxysporumf. sp.cubense) And sclerotinia rot of colza pathogen: (Sclerotinia sclerotiorum）。

2. Confrontation culture detection method:

(1) inoculating the bacterial liquid with the ratio of 1% (v/v) into a 150 mL triangular flask filled with 40 mL LB culture medium, and culturing at 28 ℃ and 200 rpm for 2.5 days;

(2) centrifuging at 8000 rpm for 10 min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain crude extract;

(3) taking 1 mL of crude extract to mix with 9 mL of PDA medium to prepare a bioassay plate, taking LB medium only added in the same amount as a control group, measuring the diameter of a colony at 7 d and 14 d, calculating the relative inhibition rate according to the following formula, and repeating the experiment for 3 times:

relative inhibition = (control colony diameter-treated colony diameter)/control colony diameter × 100%.

3. The results are shown in Table 2 and FIG. 8

TABLE 2 antagonistic action of the strain BSY3 on 6 pathogenic fungi

The result shows that the bacillus amyloliquefaciens strain BSY3 obtained by screening has strong antagonistic action on common pathogenic fungi, inhibits the growth of pathogenic bacteria of fungal diseases and has obvious biological control effect on the fungal diseases.

4. In addition, the bacillus amyloliquefaciens strain BSY3 can be correspondingly modified by a gene process means, so that the bacillus amyloliquefaciens strain BSY3 has more remarkable effect of preventing and treating fungal plant diseases and is more suitable for industrial application.

Claims

1. The application of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) in preventing and treating plant fungal diseases is characterized in that the Bacillus amyloliquefaciens is a Bacillus amyloliquefaciens strain BSY3 which is preserved in a China Center for Type Culture Collection (CCTCC) in 2017 and 31 months, wherein the preservation number is M2017463; the plant fungal diseases are plant fungal diseases caused by brown root pathogen, canna plague pathogen, white butterfly anthracnose pathogen and/or black pine leaf spot pathogen.

2. A biological agent for preventing and treating plant fungal diseases is characterized in that the biological agent takes Bacillus amyloliquefaciens and/or Bacillus amyloliquefaciens fermentation liquor as an active ingredient; the Bacillus amyloliquefaciens is a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain BSY3, and the strain is preserved in a China Center for Type Culture Collection (CCTCC) No. M2017463 in 2017, 8 and 31 months; the preparation method of the fermentation liquor comprises the following steps: culturing bacillus amyloliquefaciens for 36-50 h, and then obtaining a crude extract from a fermentation supernatant through a filter membrane, wherein the culture medium used for culture comprises the following components in proportion: 8-12 g of glucose or sucrose and 4-6 g of yeast extractg, peptone 5g, MgSO₄Or MgCl₂4-6 g, and fixing the volume of water to 1L; the pH value is 6-7.

3. The biological agent as claimed in claim 2, wherein the preparation method of the fermentation liquid comprises the following steps: culturing bacillus amyloliquefaciens for 48 hours, and then, passing the fermentation supernatant through a 0.22 mu m filter membrane to obtain a crude extract, wherein the culture medium used for culture comprises the following formula: 10g of glucose, 5g of yeast extract, 5g of peptone and MgSO₄5g, and fixing the volume of water to 1L; the pH was 6.5.

4. The use of a biological agent according to claim 2 for controlling plant fungal diseases, wherein the plant fungal diseases are plant fungal diseases caused by brown root pathogen, canna indica pest pathogen, colletotrichum album and/or alternaria leaf spot pathogen.

5. A method for preventing and treating tree brown root disease is characterized in that a biological agent containing bacillus amyloliquefaciens and/or fermentation liquor thereof is sprayed on an affected part; the bacillus amyloliquefaciens is a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain BSY3, and the strain is preserved in China Center for Type Culture Collection (CCTCC) in 2017, 8 and 31, with the preservation number of M2017463.