CN116179446B - Microbial agent for preventing and treating citrus root rot yellowing disease, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a microbial agent for preventing and treating citrus root rot yellowing disease, a preparation method and application thereof, wherein the microbial agent comprises bacillus aryabhattai and bacillus berryis, and the effective viable count of the bacillus aryabhattai and the bacillus berryis is more than or equal to 2.0x10 ¹⁰ Each/g. The microbial agent has obvious antagonism effect on Fusarium (Fusarium) causing citrus xanthomonas, and the bacillus aryabhattai also has the effects of decomposing heavy metal pollution such as cadmium in soil, phosphate and potassium dissolving, silicon dissolving and secreting plant growth hormone to promote plant growth. Through a large number of experiments in the field, the microbial agent disclosed by the invention can prevent or reduce the occurrence of citrus yellowing, promote stable yield and income increase of citrus, and improve and enhance the yield and quality of citrus.

Description

Microbial agent for preventing and treating citrus root rot yellowing disease, and preparation method and application thereof

Technical Field

The invention relates to the technical field of citrus yellowing disease control, in particular to a microbial agent for controlling citrus root rot yellowing disease, and a preparation method and application thereof.

Background

The leaves of citrus grow to lose green and yellow, and dead trees are caused when the leaves are serious, which is called as 'yellowing disease'. Citrus yellowing occurs to varying degrees in many citrus parks. The photosynthesis of the leaves is blocked, so that the yield of fruits is low, the quality is poor, and the method is a serious problem to be solved in citrus production. The cause of citrus yellowing problems is numerous and mainly focused on the following aspects: 1. the nutrient elements are deficient. And the yellow pigment is lack of large, medium and trace elements such as nitrogen, potassium, magnesium, calcium, sulfur, iron, zinc, manganese, boron, copper and the like. 2. Excessive or improper fertilization causes yellowing. The improper use of chemical fertilizers leads to excessive elements such as chlorine, boron, copper, sulfur and the like, or after the organic fertilizer which is not fermented and decomposed is applied, the organic fertilizer is caused to ferment at high temperature again at the plant root, the plant root is damaged, the timely supply of moisture and nutrition is hindered, and yellow leaves appear due to malnutrition of the above-ground and underground parts. 3. Environmental climatic conditions cause yellowing of the blades. The soil has sticky and heavy texture, hardened soil, excessive or low water content, and yellowing of leaves caused by freeze injury or weak tree vigor. 4. The disease and insect damage causes citrus yellowing. Root rot, rhizome epidemic disease and the like caused by plant pathogenic bacteria, and red spiders, aphids and the like cause leaf blight and falling off, and even the whole fruit tree dies.

At present, the problem of citrus yellowing is mainly solved by aiming at the problem finding reason, scientific fertilization, reasonable irrigation, chemical pesticide spraying and other measures to prevent and treat diseases and insect pests. The use of chemical fertilizers or pesticides can cause environmental pollution, agricultural chemical residues, and the quality of agricultural products is reduced, which is unfavorable for the sustainable development of the green agriculture.

In recent years, microbial agents are increasingly developed and applied, and are favored by people. The microorganism in the microbial inoculum has the effect of inhibiting plant diseases, can reduce the use of chemical fertilizers and pesticides, and increases and improves beneficial microorganism flora structures around rhizosphere. According to the search of technicians of the company, no report of preventing and treating pathological root rot yellowing of citrus is currently available for bacillus aryabhattai (Bacillus aryabhattai) and bacillus berryis (Bacillus velezensis).

Disclosure of Invention

The invention aims at solving the technical defects of environmental pollution, agrochemical residue, agricultural product quality reduction and adverse to the green sustainable development of agriculture existing in the prior art, and provides a microbial agent for preventing and treating citrus root rot yellowing disease, a preparation method and application thereof.

The technical scheme adopted for realizing the purpose of the invention is as follows:

a microbial agent for preventing and treating citrus root rot yellowing disease comprises bacillus aryabhattai (Bacillus aryabhattai) and bacillus berryis (Bacillus velezensis), wherein the effective viable count of the bacillus aryabhattai and the bacillus berryis is more than or equal to 2.0x10 ¹⁰ Each/g.

Bacillus alfasin MES85801 classified as Bacillus alfasin (Bacillus aryabhattai) was deposited in China general microbiological culture Collection center, CGMCC, at the following address: the preservation number of the Beijing city, the Chaoyang district, north Chen Xili No.1 and 3 is CGMCC No.26404. Bacillus beliae MES861, named Bacillus belicus (Bacillus velezensis), was deposited in China general microbiological culture Collection center, CGMCC, at the address: the preservation number of the Beijing city, the Chaoyang district, the North Chen Xili No.1 and the North Chenli No. 3 is CGMCC No.16857.

In the technical scheme, the mass ratio of the bacillus aryabhattai to the bacillus beijerinus is 1:1.

In another aspect of the invention, a preparation method of the microbial agent is provided, which comprises the steps of preparing fermentation liquor of bacillus aryabhattai and bacillus beijerinus, preparing a liquid preparation by the fermentation liquor and a stabilizer, and spray drying, wherein the stabilizer is preferably xanthan gum.

In the technical scheme, fermentation broths of the bacillus aryabhattai and the bacillus beijerinus are prepared by the following methods:

step 1, primary seed culture: culturing the strain activated by the inclined surface of the test tube to obtain first-stage seeds, wherein the culture medium is a first-stage seed culture medium;

step 2, fermentation, namely filling a liquid fermentation medium in an expansion fermentation tank, sterilizing the liquid fermentation medium, and inoculating the first-stage seeds obtained in the step 1 into the liquid fermentation medium for fermentation culture;

and 3, ending fermentation when the spore formation ratio is more than or equal to 90%, and placing the fermentation tank to obtain fermentation liquor.

In the technical scheme, the primary seed culture medium comprises 8-13 parts by weight of peptone, 2-5 parts by weight of beef extract, 3-5 parts by weight of sodium chloride, 19.5 parts by weight of agar and 1000 parts by weight of water, wherein the pH value of the primary seed culture medium is 7.0-7.5;

the liquid fermentation culture medium comprises 2-4% of glucose, 0.5-1% of yeast extract powder and 0.5-0.75% of potassium chloride, and the pH value of the liquid fermentation culture medium is 7.0-7.5.

In the technical scheme, the fermentation culture condition in the step 2 is that the pressure of the expansion fermentation tank is controlled to be 0.02Mpa-0.07 Mpa, and the fermentation is carried out for 26 hours at 35 ℃.

In another aspect, the invention provides an application of the microbial agent in preventing and treating citrus root rot yellowing, wherein the microbial agent is diluted by water and uniformly poured on plant roots or after ditching to apply organic fertilizer, a proper amount of water is poured on the plant roots.

In the technical scheme, the dilution ratio by adding water is 400-800 times.

In the technical scheme, the microbial agent has an effect of preventing citrus yellowing disease of not less than 85%.

Compared with the prior art, the invention has the beneficial effects that:

the microbial agent has obvious antagonism effect on Fusarium (Fusarium) causing citrus xanthomonas, and the bacillus aryabhattai also has the effects of decomposing heavy metal pollution such as cadmium in soil, phosphate and potassium dissolving, silicon dissolving and secreting plant growth hormone to promote plant growth. Through a large number of experiments in the field, the microbial agent disclosed by the invention can prevent or reduce the occurrence of citrus yellowing, promote stable yield and income increase of citrus, and improve and enhance the yield and quality of citrus.

Drawings

FIG. 1 is a graph showing the antagonistic effect of the microbial agent of the present invention on citrus yellow root rot.

FIG. 2 shows the control effect of the microbial agent of the invention on citrus yellowing root rot, wherein a is before applying the microbial agent, and b is after applying the microbial agent.

Bacillus alfasin MES85801 classified as Bacillus alfasin (Bacillus aryabhattai) was deposited in China general microbiological culture Collection center, CGMCC, at the following address: the preservation number of the Beijing city, the Chaoyang district, north Chen Xili No.1 and 3 is CGMCC No.26404. Bacillus beliae MES861, named Bacillus belicus (Bacillus velezensis), was deposited in China general microbiological culture Collection center, CGMCC, at the address: the preservation number of the Beijing city, the Chaoyang district, the North Chen Xili No.1 and the North Chenli No. 3 is CGMCC No.16857.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Bacillus aryabhattai (Bacillus aryabhattai) is obtained by separating a research staff of my department from soil in Tianjin Wuqing district, and has a preservation date: 2023, 01 and 06 are preserved in China general microbiological culture collection center with a preservation number of CGMCC No.26404. Bacillus aryabhattai belongs to the genus Bacillus, and belongs to gram-positive bacteria and bacillus, and the bacillus aryabhattai is positive in contact enzyme and smooth in colony.

Bacillus beleiensis (Bacillus velezensis), date of preservation: 12.03 in 2018, the name of the preservation unit and short: china general microbiological culture collection center (CGMCC), number of collection: CGMCC No.16857. The bacteria are gram positive cells of Bacillus and endophytic spore ellipsoids of Bacillus. Catalase positive, contact enzyme positive, gelatin liquefaction positive, no exercise, indole positive, starch decomposition and cellulose decomposition. Has broad-spectrum antibacterial activity and the characteristic of promoting plant growth, and has great development and research values.

Example 1

A microbial agent for preventing and treating citrus root rot yellowing disease is prepared by the following steps:

(1) primary seed culture: the bacillus albopictus (Bacillus aryabhattai) and bacillus bezier (Bacillus velezensis) activated by the inclined surface of the test tube are respectively subjected to medium bottle culture, and the primary seed culture medium is a nutrient agar culture medium. The formula of the primary seed culture medium is as follows: 8-15 g of peptone, 3-6 g of beef extract, 3-5 g of sodium chloride, 19.5 g of agar, 1000 ml of ultrapure water and pH value of 7.0-7.5. The culture conditions are as follows: culturing at 22-37 deg.C and 24-h.

(2) Fermentation at 30 m ³ The fermentation tank is filled with 20L liquid fermentation medium, the temperature of 118-122 ℃ is reduced to 35 ℃ after being sterilized by damp heat for 35-40 min, the first-level seeds obtained in the step (1) are respectively inoculated into the liquid fermentation medium, the tank pressure is controlled to be 0.02Mpa-0.07 Mpa, and the fermentation culture is carried out at 35 ℃ for 26 h; the culture medium used for fermentation is prepared from glucose 2-5%, yeast extract powder 0.2-0.6%, potassium chloride 0.5-0.8% and pH 7.0-7.5.

(3) Ending fermentation when the spore formation proportion of the fermentation culture in the step (2) is more than or equal to 90%, and placing the fermentation tank to obtain fermentation liquor;

(4) compounding the fermentation liquor obtained in the step (3) with xanthan gum to prepare a liquid preparation:

(5) concentrating and spray drying the liquid preparation in a spray drying tower to obtain the microbial agent, and filling the microbial agent into a packaging bag, wherein the mass ratio of the bacillus aryabhattai to the bacillus berliner is 1:1.

Detecting and counting, wherein the effective viable Count (CFU) of bacillus bailii and bacillus aryaensis products respectively reaches more than or equal to 2.0X10 ¹⁰ Each/g.

Example 2

Bacteriostasis test of citrus root rot etiological agent Using the microbial agent obtained in example 1

Test time and place: is performed in a laboratory of a company development center.

(II) test method:

(1) Source of test strain

Bacteria causing citrus root rot yellowing diseaseBYJ-19 is separated from citrus orchard in Shanglin county of Guangxi nan Ning, and is separated, purified and cultured by company research personnel, and purified strain is finally identified as fusarium oxysporum through 16S rDNAFusarium oxysporum) The disease is identified as strong disease by pathogenicity measurement.

The microbial agent is prepared in example 1.

(2) Plate antagonism experiment

Preparing a PDA solid flat plate, picking a small amount of root rot pathogenic bacteria hypha by using a sterile inoculating needle, placing the root rot pathogenic bacteria hypha in the middle of a culture dish, burning the inoculating needle, dipping a small amount of microbial agent around 2.0 cm away from the inoculated pathogenic bacteria bacterial plaque after the inoculating needle is cooled, setting the treatment of inoculating only the root rot pathogenic bacteria without inoculating functional antagonistic bacteria as a blank control, and setting three times of repetition for each treatment. When the whole culture dish is fully grown with the blank control mycelium, the control colony area (colony radius) inoculated with root rot only and the colony area (colony radius inhibition) inoculated with microbial inoculum are measured, and the antagonistic effect is expressed as (antagonistic effect (%) = (control colony area-treated colony area)/control colony area×100).

(3) Antagonistic effect

The experiment shows that the antagonism effect of bacillus belicus MES861 and bacillus aryabhattai MES85801 on citrus yellow root rot reaches 80.2%; the average colony radius for the control was 4.5. 4.5 cm and for the inoculated Bacillus belicus and Bacillus aryabhattai was 2.1. 2.1 cm (as shown in FIG. 1). The bacillus belicus and the bacillus aryabhattai have obvious inhibition effect on the citrus yellowing root rot and have biocontrol potential for preventing and treating the citrus yellowing root rot.

Example 3

Test of the control Effect of the microbial agent obtained in example 1 on citrus yellow root rot

Test Agents

(1) Microbial agent

The microbial agent prepared in example 1 was diluted 500 times with water;

(2) Chemical agent

3% metalaxyl-hymexazol 700 times solution (50 ml drug in 35 kg water) (Texas lucky dragon Biochemical Co., ltd.).

(3) Blank control

The clear water treatment was set as a blank.

(II) test time and place

The test was performed in the citrus orchard in Shanglin county, guangxi nan Ning.

(III) test method

Processing and setting: the disease plants were treated in 4 treatments, respectively: 1. bacterial manure is not applied to the diseased plants; 2. administering a chemical agent; 3. the composite microbial agent is applied and diluted 500 times with water.

The experimental method comprises the following steps: and weighing each treatment agent, adding water to dilute to the corresponding concentration, uniformly pouring the water on the plant roots or ditching, applying the organic fertilizer, and pouring a proper amount of water on the plant roots. The effect was observed after 15-30 days.

(IV) results

The test results are shown in Table 1, and it can be seen from the results that the disease index (10.12) of the treatment with Bacillus belicus MES861 and Bacillus aryabhattai composite powder is significantly lower than that of the blank (85.35) and higher than that of the treatment with the chemical agent (8.38). The microbial agents added with bacillus belicus MES861 and bacillus aryaensis MES85801 can be used as microbial agents for preventing and treating citrus root rot yellowing (shown in figure 2), and have wide popularization and utilization values.

TABLE 1 prevention and treatment effects of different treatments on citrus yellowing diseases

Treatment of	Index of disease condition	Preventing effect (%)
			Nail creamHymexazol 700 times liquid	8.38b	90.28
500 times of composite microbial agent	10.12ab	87.12
			Blank control	85.35a	0.00

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (5)

1. The application of the microbial agent in preventing and treating citrus root rot yellowing disease is characterized in that the microbial agent is diluted by 400-800 times by adding water and uniformly poured on the root of a plant, and the prevention effect of the microbial agent on the citrus root rot yellowing disease is not less than 85%;

the microbial agent consists of bacillus aryabhattai (Bacillus aryabhattai) and bacillus behenii (Bacillus velezensis), and the effective viable count of the bacillus aryabhattai and the bacillus behenii is more than or equal to 2.0x10 ¹⁰ Individual/g;

the bacillus aryabhattai is preserved in China general microbiological culture Collection center, and the preservation number is: CGMCC No.25404, date of preservation: 2023, 01, 06; the bacillus belicus is preserved in China general microbiological culture Collection center, and the preservation number is: CGMCC No.16857, date of preservation: 12 months of 2018;

the mass ratio of the bacillus aryabhattai to the bacillus beijerinus is 1:1.

2. A process for the preparation of a microbial agent for use according to claim 1, comprising the preparation of a fermentation broth of Bacillus aryabhattai and Bacillus bailii, the preparation of a liquid formulation of the fermentation broth with a stabilizer, and spray drying.

3. The preparation method according to claim 2, wherein the fermentation broths of bacillus aryabhattai and bacillus beijerinus are each prepared by the following method:

step 1, primary seed culture: culturing the strain activated by the inclined surface of the test tube to obtain first-stage seeds, wherein the culture medium is a first-stage seed culture medium;

step 2, fermentation, namely filling a liquid fermentation medium in an expansion fermentation tank, sterilizing the liquid fermentation medium, and inoculating the first-stage seeds obtained in the step 1 into the liquid fermentation medium for fermentation culture;

and 3, ending fermentation when the spore formation ratio is more than or equal to 90%, and placing the fermentation tank to obtain fermentation liquor.

4. The method of claim 3, wherein the primary seed medium comprises 8-13 parts by mass of peptone, 2-5 parts by mass of beef extract, 3-5 parts by mass of sodium chloride, 19.5 parts by mass of agar and 1000 parts by mass of water, and the pH of the primary seed medium is 7.0-7.5;

the liquid fermentation culture medium comprises 2-4% of glucose, 0.5-1% of yeast extract powder and 0.5-0.75% of potassium chloride, and the pH value of the liquid fermentation culture medium is 7.0-7.5.

5. The method according to claim 3, wherein the fermentation culture in the step 2 is carried out under a pressure of 0.02MPa to 0.07 MPa in a fermentation tank, and at 35℃for 26 hours.