A microbial agent and its use in preventing and treating citrus canker

Abstract

The present invention relates to a microbial agent and its use in preventing and treating citrus canker, and belongs to the field of microbial agents. The microbial agent of the present invention mainly comprises Beauveria bassiana and Bacillus polymyxa, and also contains citrus peel and tangerine peel fermentation products, sodium alginate and polyvinyl alcohol. The microbial agent provided by the present invention can effectively inhibit the reproduction of citrus canker pathogens, and the prevention and treatment effect of citrus canker is more than 80%, and the effect is remarkable.

Description

Technical Field

[0001] The invention belongs to the field of microbial agents and relates to a microbial agent and application thereof in preventing and treating citrus canker. Background Art

[0002] Citrus, also known as tangerine or orange, is a small tree plant of the genus Citrus in the Rutaceae family. China is one of the important origins of citrus, with rich citrus resources and a wide variety of fine varieties, and a cultivation history of more than 4,000 years. In recent years, the scale of the citrus industry has continued to expand, but citrus diseases and pests have also brought significant negative impacts on its development. Among them, citrus canker is a pathological factor that seriously affects citrus production, causing immeasurable losses to the citrus industry.

[0003] Citrus canker is a serious bacterial disease in citrus producing areas. It can harm the leaves, branches and fruits of citrus, and it is very difficult to prevent and control. There are three main pathogens that cause citrus canker, namely Xanthomonas citrus subsp. citrus, Xanthomonas rufipogon subsp. citrus and Xanthomonas lucerne subsp. citrus. The distribution areas of these three pathogens are different. In China, Xanthomonas citrus is mainly caused by Xanthomonas citrus. The pathogen overwinters in diseased leaves, branches or fruits, and overflows from the diseased parts when it encounters water in the following spring. It spreads through rainwater, insects, seedlings, artificial tools and contact with branches and leaves, and invades from the host's stomata, lenticels or wounds. The disease can infect and cause disease from April to November, with the highest incidence in September. Summer shoots are most susceptible to the disease, followed by autumn shoots, early autumn shoots and late spring shoots, and spring shoots and winter shoots are not susceptible to the disease. The occurrence and prevalence of the disease are closely related to climatic conditions, cultivation management measures and varieties. High temperature, high humidity and heavy rain are conducive to the occurrence and spread of the disease. It is easy to get sick after storms and typhoons. Improper cultivation and management, insufficient fertilization, excessive nitrogen fertilizer application, and increased leggy branches will lead to severe disease. Young trees and seedlings are more seriously ill than mature trees and old trees.

[0004] When citrus canker damages leaves and branches, it will cause leaf fall and branch dieback, affecting tree vigor. When it damages fruits, in mild cases, the fruits will bear scars and cannot be stored, and the fruit will rot, greatly reducing the commercial rate. In severe cases, it will cause early fruit drop and a significant reduction in yield. At the same time, it will increase the cost of disease and pest control and damage economic benefits.

[0005] In order to prevent the citrus canker from causing the citrus industry to stop or even decline, it is urgent to prevent and control the disease. Chemical methods are currently the most popular means of prevention and control. Although this method is effective, its negative impact on soil and water bodies, such as damage and pollution, cannot be ignored.

[0006] Microorganisms are an important part of biological control. Some microorganisms can directly inhibit the growth of pathogens by secreting secondary metabolites such as antibiotics; some microorganisms can also compete with pathogens for iron ions by secreting iron carriers, thereby indirectly inhibiting the growth of pathogens; some microorganisms can enhance the disease resistance of plants by inducing plants to produce systemic resistance. However, the microbial agents in the prior art are basically single bacteria, and the effect of preventing and controlling citrus canker is poor. Summary of the invention

[0007] The main purpose of the present invention is to provide a microbial agent, which contains multiple microorganisms, and the synergistic effect of the microorganisms greatly improves the effect of preventing and treating citrus canker.

[0008] The present invention adopts the following technical solutions to achieve the above purpose:

[0009] A microbial agent, wherein the preparation method of the microbial agent comprises the following steps:

[0010] Step A, grinding tangerine peel aged for 2-3 years into powder, and setting aside the tangerine peel powder; taking fresh citrus peel, adding water, and preparing slurry by high pressure homogenization, and setting aside the slurry; adding tangerine peel powder, brown algae oligosaccharide, galacto-oligosaccharide, and calcium lactate to the slurry, and fermenting anaerobically at 25-30° C. for 1-1.5 days, centrifuging the fermentation liquid, and setting aside the supernatant;

[0011] Step B, adding Beauveria bassiana, Bacillus polymyxa and yeast extract powder to the fermentation liquid obtained in step A, culturing at 25-30° C. for 2.5-3 days, then adding sodium alginate and polyvinyl alcohol, mixing well, to obtain a microbial agent.

[0012] Furthermore, in step A of the above-mentioned method for preparing the microbial agent, the mass-volume ratio of citrus peel to water, in kg / L, is 1:(1.5-2).

[0013] Furthermore, in step A of the above-mentioned microbial agent preparation method, the weight ratio of citrus peel, tangerine peel powder, brown algae oligosaccharide, galacto-oligosaccharide and calcium lactate is 1:(0.1-0.3):(0.03-0.05):(0.07-0.08):(0.005-0.008).

[0014] Furthermore, in step B of the above-mentioned method for preparing the microbial agent, the amount of Beauveria bassiana added is 0.03-0.05 g / L, and the amount of Bacillus polymyxa added is 0.1-0.3 g / L.

[0015] Furthermore, the effective viable count of the above-mentioned Beauveria bassiana is 10 billion cfu / g, and the effective viable count of Bacillus polymyxa is 2 billion cfu / g.

[0016] Furthermore, in step B of the method for preparing the microbial agent, the amount of yeast extract powder added is 15-20 g / L; the amount of sodium alginate added is 25-30 g / L, and the amount of polyvinyl alcohol added is 12-15 g / L.

[0017] Furthermore, the total number of effective live bacteria contained in the above-mentioned microbial agent is ≥50 billion cfu / L.

[0018] The present invention also provides a specific application of the microbial agent, that is, the microbial agent can be used to prevent and treat citrus canker.

[0019] The present invention has the following beneficial effects:

[0020] 1. During the research and development process, the present invention found that the product of co-fermentation of fresh citrus peel and tangerine peel has an inhibitory effect on citrus canker pathogens. Therefore, fresh citrus peel and tangerine peel fermentation products are creatively used in the microbial agent. The oil bag in the oil chamber of fresh citrus peel contains a large number of various types of components, such as flavonoids, volatile components, etc., which have high biological activity. After high-pressure homogenization, the components in the oil chamber and cells are released; tangerine peel is citrus peel after aging. During the aging process, the substances in the citrus peel are converted by bacterial colonies into a large number of beneficial and biologically active substances. The microorganisms contained in tangerine peel can utilize the substances in fresh citrus peel and tangerine peel to grow and reproduce during the fermentation process, and can decompose the macromolecular substances such as polysaccharides therein into small molecular active substances. Meanwhile, metabolites are produced in the process of their growth, reproduction and metabolism, thereby increasing the types of active ingredients in the fermentation liquid that have an inhibitory effect on the citrus canker pathogen. The ingredients such as brown algae oligosaccharides, oligogalactose, and calcium lactate added during the fermentation process are beneficial to the growth and metabolism of microorganisms and promote the generation of metabolites. The increase of microbial metabolites enhances the inhibitory effect of the fermentation liquid on the citrus canker pathogen.

[0021] 2. During the aging process of tangerine peel, the bacterial flora and colonies on its surface and inside will change, leaving behind beneficial bacteria that continue to grow and multiply during the fermentation process. The growth of beneficial bacteria in tangerine peel can compete with citrus disease pathogens for living space, limiting the living space of the pathogens and having a good inhibitory effect on citrus canker pathogens.

[0022] 3. Beauveria bassiana and Bacillus polymyxa have good inhibitory effects on citrus canker pathogens. When inoculated into the fermentation liquid, they will produce antagonistic effects with the small amount of canker pathogens remaining in the fresh citrus peel. The presence of canker pathogens will induce Beauveria bassiana and Bacillus polymyxa to enhance their biological activity and produce metabolites with antibacterial effects. At the same time, when used on citrus trees, they produce highly active antibacterial substances through highly active bacterial flora, and inhibit the growth and reproduction of pathogens by competing for nutrients.

[0023] 4. The microbial agent prepared by the present invention has a good preventive and therapeutic effect on citrus canker. The preventive and therapeutic effect on canker of fruit trees that were sick in the previous year is more than 80%, and the preventive and therapeutic effect on ordinary fruit trees is more than 90%. It has a significant effect and can avoid environmental pollution caused by excessive use of chemical agents. It is suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : The situation of navel orange leaves in experiment 1, among which Figure 1 (a) is a group of navel orange leaves, Figure 1 (b) is the blank group navel orange leaves, Figure 1 (c) are 4 groups of navel orange leaves, Figure 1 (d) 7 groups of navel orange leaves;

[0025] Figure 2 : The situation of navel orange fruit in experiment 1, among which Figure 2 (a) is a group of navel orange fruits, Figure 2 (b) are 4 groups of navel orange fruits, Figure 2 (c) are 7 groups of navel orange fruits, Figure 2 (d) is the navel orange fruit of the blank group. DETAILED DESCRIPTION

[0026] The present invention is further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent modifications to the present invention by those skilled in the art all fall within the scope of protection of the claims of this application.

[0027] Example 1 Preparation of microbial agent

[0028] Step A, grinding 3-year-old tangerine peel into powder, and setting aside the tangerine peel powder; taking 1 kg of fresh citrus peel, adding 2 L of water, and preparing slurry by high-pressure homogenization, and setting aside the slurry; adding 0.1 kg of tangerine peel powder, 0.05 kg of brown algae oligosaccharide, 0.08 kg of galacto-oligosaccharide, and 0.005 kg of calcium lactate to the slurry, and fermenting anaerobically at 30° C. for 1 day, centrifuging the fermentation liquid, and setting aside the supernatant;

[0029] Step B, adding 0.05 g / L of Beauveria bassiana with an effective live bacteria count of 10 billion cfu / g, 0.1 g / L of Bacillus polymyxa with an effective live bacteria count of 2 billion cfu / g, and 20 g / L of yeast extract powder to the supernatant obtained in step A, culturing at 25° C. for 3 days, then adding 30 g / L of sodium alginate and 12 g / L of polyvinyl alcohol, mixing well, to obtain a microbial agent.

[0030] After testing, the total number of effective live bacteria contained in the microbial agent prepared above is 53.8 billion cfu / L.

[0031] Example 2 Preparation of microbial agent

[0032] Step A, grinding 2-year-old tangerine peel into powder, and using the tangerine peel powder for later use; taking 1 kg of fresh citrus peel, adding 1.5 L of water, and preparing a slurry by high-pressure homogenization, and using the slurry for later use; adding 0.3 kg of tangerine peel powder, 0.03 kg of brown algae oligosaccharide, 0.07 kg of galacto-oligosaccharide, and 0.008 kg of calcium lactate to the slurry, and fermenting anaerobically at 25° C. for 1.5 days, centrifuging the fermented liquid, and taking the supernatant for later use;

[0033] Step B, adding 0.03 g / L of Beauveria bassiana with an effective live bacteria count of 10 billion cfu / g, 0.3 g / L of Bacillus polymyxa with an effective live bacteria count of 2 billion cfu / g, and 15 g / L of yeast extract powder to the supernatant obtained in step A, culturing at 30° C. for 2.5 days, then adding 25 g / L of sodium alginate and 15 g / L of polyvinyl alcohol, mixing well, to obtain a microbial agent.

[0034] After testing, the total number of effective live bacteria contained in the above-prepared microbial agent is 76.3 billion cfu / L.

[0035] Example 3 Preparation of microbial agent

[0036] Step A, grinding 3-year-old tangerine peel into powder, and using the tangerine peel powder for later use; taking 1 kg of fresh citrus peel, adding 1.8 L of water, and preparing a slurry by high-pressure homogenization, and using the slurry for later use; adding 0.2 kg of tangerine peel powder, 0.04 kg of brown algae oligosaccharide, 0.08 kg of galacto-oligosaccharide, and 0.006 kg of calcium lactate to the slurry, and fermenting anaerobically at 30° C. for 1.5 days, centrifuging the fermented liquid, and taking the supernatant for later use;

[0037] Step B, adding 0.04 g / L of Beauveria bassiana with an effective live bacteria count of 10 billion cfu / g, 0.2 g / L of Bacillus polymyxa with an effective live bacteria count of 2 billion cfu / g, and 18 g / L of yeast extract powder to the supernatant obtained in step A, culturing at 30° C. for 3 days, then adding 28 g / L of sodium alginate and 13 g / L of polyvinyl alcohol, mixing well, to obtain a microbial agent.

[0038] After testing, the total number of effective live bacteria contained in the above-prepared microbial agent is 103.6 billion cfu / L.

[0039] Comparative Example 1 Preparation of microbial agent

[0040] Step A, grinding 3-year-old tangerine peel into powder, and setting aside the tangerine peel powder; taking 0.15 kg of tangerine peel powder, 0.04 kg of brown algae oligosaccharide, 0.08 kg of galacto-oligosaccharide, and 0.006 kg of calcium lactate, mixing them, adding 1 L of water, and fermenting them anaerobically at 30° C. for 1.5 days, centrifuging the fermentation liquid, and setting aside the supernatant;

[0041] Step B, adding 0.04 g / L of Beauveria bassiana with an effective live bacteria count of 10 billion cfu / g, 0.2 g / L of Bacillus polymyxa with an effective live bacteria count of 2 billion cfu / g, and 18 g / L of yeast extract powder to the supernatant obtained in step A, culturing at 30° C. for 3 days, then adding 28 g / L of sodium alginate and 13 g / L of polyvinyl alcohol, mixing well, to obtain a microbial agent.

[0042] After testing, the total number of effective live bacteria contained in the above-prepared microbial agent is 82.9 billion cfu / L.

[0043] Comparative Example 2 Preparation of microbial agent

[0044] To a culture medium containing 18 g / L yeast extract, 50 g / L brown algae oligosaccharide, 80 g / L galacto-oligosaccharide and 6 g / L calcium lactate, add 0.04 g / L Beauveria bassiana with an effective live bacteria count of 10 billion cfu / g and 0.2 g / L Bacillus polymyxa with an effective live bacteria count of 2 billion cfu / g, and culture at 30°C for 3 days. Then, add 28 g / L sodium alginate and 13 g / L polyvinyl alcohol, and mix well to obtain a microbial agent.

[0045] After testing, the total number of effective live bacteria contained in the above-prepared microbial agent is 60.3 billion cfu / L.

[0046] Comparative Example 3 Preparation of microbial agent

[0047] Step A, taking 1 kg of fresh citrus peel, adding 1.8 L of water, and preparing a slurry by high-pressure homogenization, and the slurry is set aside; adding 0.4 kg of brown algae oligosaccharide, 0.8 kg of galacto-oligosaccharide, and 0.06 kg of calcium lactate to the slurry, and fermenting anaerobically at 30° C. for 1.5 days, centrifuging the fermentation liquid, and taking the supernatant for later use;

[0048] Step B, adding 0.04 g / L of Beauveria bassiana with an effective live bacteria count of 10 billion cfu / g, 0.2 g / L of Bacillus polymyxa with an effective live bacteria count of 2 billion cfu / g, and 18 g / L of yeast extract powder to the supernatant obtained in step A, culturing at 30° C. for 3 days, and mixing well to obtain a microbial agent.

[0049] After testing, the total number of effective live bacteria contained in the above-prepared microbial agent is 91.2 billion cfu / L.

[0050] Experiment 1: The preventive and therapeutic effect of the microbial agent of the present invention on citrus canker

[0051] 1.1 Test methods

[0052] Sixteen navel orange trees with a tree age of 5 years and severe ulcer disease in the previous year were selected and randomly divided into 8 groups, each with 2 trees. The microbial agents obtained in Examples 1-3, the microbial agents obtained in Comparative Examples 1-3, and 45% copper quinoline were sprayed at the symptom-prone stage of spring shoot ulcer disease, the late stage of flower falling, and the young fruit stage, respectively. One group was sprayed with water as a blank group. 30 days after the last application of the drug, the ulcer disease condition of the navel orange trees was investigated by visual inspection. For each navel orange tree, the five-point sampling method of east, south, west, north, and center was used. Two autumn shoots of the current year were randomly selected from each direction as the investigation objects, and the total number of leaves on the shoots, i.e., the number of diseased leaves at each level, was recorded; when investigating the fruits, 10 fruits were randomly selected from each direction, and the number of diseased fruits at each level was counted.

[0053] Leaf (fruit) grading according to the number of lesions:

[0054] Grade 0, no lesions;

[0055] Level 1: The number of lesions on each leaf (fruit) is 1-5;

[0056] Level 3: The number of lesions on each leaf (fruit) is 6-10;

[0057] Level 5: The number of lesions on each leaf (fruit) is 11-15;

[0058] Level 7: The number of lesions on each leaf (fruit) is 16-20;

[0059] Level 9: The number of lesions on each leaf (fruit) is more than 21.

[0060] The disease index and control effect are calculated according to the following formula.

[0061]

[0062] 1.2 Test results

[0063] From the results in Table 1 and Table 2, it can be seen that spraying the microbial agent obtained by the present invention on navel orange trees with serious ulcer disease can effectively prevent and control the ulcer disease, and the prevention and control effect on leaves and fruits is more than 80%.

[0064] Table 1 Statistical results of the incidence of navel orange leaf canker in each group

[0065]

[0066]

[0067] Table 2 Statistical results of the incidence of navel orange fruit canker in each group

[0068]

[0069] Experiment 2: The preventive and therapeutic effect of the microbial agent of the present invention on citrus canker

[0070] 2.1 Test methods

[0071] 80 navel orange trees with a tree age of 5 years, a tree height of about 2m, and basically the same growth were selected and randomly divided into 10 groups, with 10 trees in each group. The microbial agents obtained in Examples 1-3 of the present invention, the microbial agents obtained in Comparative Examples 1-3, and 45% spring thunder quinoline copper were sprayed for treatment, and one group was treated with clean water as a blank group. The first application of the drug was when the summer shoots were just drawn, and the drug was applied twice in total, with an interval of 10 days in between; the second application of the drug was during the young fruit stage of citrus. 30 days after the second application of the drug, 2 navel orange trees were selected from each group as observation plants, and the condition of navel orange tree canker was investigated by visual inspection. Each navel orange tree was sampled by the five-point sampling method of east, south, west, north, and center. Three summer shoots of the year were randomly selected from each direction as the investigation objects, and the total number of leaves on the shoots and the number of diseased leaves at all levels were recorded; when investigating the fruits, 10 fruits were randomly selected from each direction, and the number of diseased fruits at all levels was counted.

[0072] Leaf (fruit) grading according to the number of lesions:

[0073] Grade 0, no lesions;

[0074] Level 1: The number of lesions on each leaf (fruit) is 1-5;

[0075] Level 3: The number of lesions on each leaf (fruit) is 6-10;

[0076] Level 5: The number of lesions on each leaf (fruit) is 11-15;

[0077] Level 7: The number of lesions on each leaf (fruit) is 16-20;

[0078] Level 9: The number of lesions on each leaf (fruit) is more than 21.

[0079] The disease index and control effect are calculated according to the following formula.

[0080]

[0081] 2.2 Test results

[0082] From the results in Table 3 and Table 4, it can be seen that spraying the microbial agent obtained by the present invention can effectively prevent and treat ulcer disease, and the prevention and control effect on leaves and fruits is basically above 90%.

[0083] Table 3 Statistical results of the incidence of navel orange leaf canker in each group

[0084]

[0085] Table 4 Statistical results of the incidence of navel orange fruit canker in each group

[0086]

[0087]

Claims

1. A microbial agent, characterized in that: The preparation method of the microbial agent comprises the following steps: Step A, grinding tangerine peel aged for 2-3 years into powder, and setting aside the tangerine peel powder; adding water to fresh citrus peel, and preparing slurry by high pressure homogenization, and setting aside the slurry; adding tangerine peel powder, brown algae oligosaccharide, galacto-oligosaccharide, and calcium lactate to the slurry, and fermenting anaerobically at 25-30° C. for 1-1.5 days, centrifuging the fermentation liquid, and setting aside the supernatant; Step B, adding Beauveria bassiana, Bacillus polymyxa and yeast extract powder to the fermentation liquid obtained in step A, culturing at 25-30° C. for 2.5-3 days, then adding sodium alginate and polyvinyl alcohol, mixing well, to obtain a microbial agent.

2. The microbial agent according to claim 1, characterized in that The mass-volume ratio of citrus peel to water in step A of the microbial agent preparation method is 1:(1.5-2) in kg / L.

3. The microbial agent according to claim 1, characterized in that In step A of the method for preparing the microbial agent, the weight ratio of citrus peel, tangerine peel powder, brown algae oligosaccharide, galacto-oligosaccharide and calcium lactate is 1:(0.1-0.3):(0.03-0.05):(0.07-0.08):(0.005-0.008).

4. The microbial agent according to claim 1, characterized in that In step B of the method for preparing the microbial agent, the amount of Beauveria bassiana added is 0.03-0.05 g / L, and the amount of Bacillus polymyxa added is 0.1-0.3 g / L.

5. The microbial agent according to claim 4, characterized in that: The effective viable bacteria count of the Beauveria bassiana is 10 billion cfu / g, and the effective viable bacteria count of Bacillus polymyxa is 2 billion cfu / g.

6. The microbial agent according to claim 1, characterized in that: In step B of the microbial agent preparation method, the added amount of yeast extract powder is 15-20 g / L; the added amount of sodium alginate is 25-30 g / L; and the added amount of polyvinyl alcohol is 12-15 g / L.

7. The microbial agent according to claim 1, characterized in that: The total number of effective live bacteria contained in the microbial agent is ≥50 billion cfu / L.

8. Use of the microbial agent according to any one of claims 1 to 7 in preventing and treating citrus canker.

Images