A Multifunctional Paenibacillus terrae and Its Application

Abstract

The present invention provides a multifunctional Paenibacillus terrae and its application, relating to the technical field of microorganisms and their applications. The multifunctional Paenibacillus terrae is the Paenibacillus terrae strain KY834, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC No. 24708. From a large number of microbial strains, the present invention obtained a strain KY834 with the ability to kill the pathogens of apple leaf spot disease and pear black spot disease through high-throughput screening. Further research found that this strain has broad-spectrum bactericidal activity against the pathogens of other plant diseases, and also has the functions of killing algae and activating silicon, showing good application prospects in the biological control of plant (especially fruit tree) diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms and their applications, and particularly to a multifunctional Paenibacillus terrae strain and its applications. Background Art

[0002] Fruit tree production occupies an important position in agricultural production in most countries of the world. With the rapid development of social economy and the improvement of the national living standard, the market demand for fruits has correspondingly increased, which has put forward higher requirements for the yield of fruit trees, the quality and appearance of fruits. However, due to the perennial influence of global climate and environmental conditions on fruit trees, coupled with the susceptibility of fruit trees during the growth cycle, the spread rate of diseases is increasing explosively, endangering the safety of agricultural production (Man Zhonghe, 2021). Therefore, the prevention and control of fruit tree diseases and algae have become one of the key research topics in the development of the fruit industry.

[0003] Among fruit tree diseases, apple leaf spot and pear black spot have a relatively high incidence rate. Apple Alternaria leaf spot, also known as brown spot disease, mainly harms young leaves, and also harms young leaves and fruits (Jin Ying, 2021). Apple Alternaria leaf spot can cause early leaf fall, affecting the tree vigor and yield, and occurs in all apple-producing areas. In the initial stage of leaf infection, brown dots appear, which then expand into reddish-brown, the leaf margin is purple-brown, and there is often a dark small dot in the center of the diseased part, which is extremely likely to cause leaf distortion and shrinkage, and the diseased part is scorched and incomplete. When the fruit is infected, small black shiny spots or rust spots appear on the fruit surface in the initial stage. In the later stage, the diseased part of the fruit sometimes shows grayish-brown scab-like patches, with cracks at the junction of the diseased and healthy parts, and the diseased spots do not peel off. This disease can not only cause a large number of early leaf falls of fruit trees, but also infect the fruit surface to form spots, affecting the quality of apples and the yield of the following year, greatly threatening the development of the fruit industry (Yu Xiaoli et al., 2016). Pear black spot also occurs widely in China, especially in Xuehua pears and crisp pears with the most serious incidence. This disease is mainly caused by the fungal infection of Alternaria alternate (Fr.) Keissl. The flowering period and young fruit period of pear trees are potential infection periods of pear black spot, especially during storage, when it is most susceptible to disease and difficult to control and prevent.

[0004] At present, the main control measures for the major diseases of fruit trees in production mainly include grafting, intercropping, rotation cropping, relay cropping techniques, breeding of disease-resistant varieties, chemical control and biological control, etc. Grafting may have certain limitations as it may make the taste of the product worse (Rodríguez-negrete E A et al., 2020). The promotion of intercropping, rotation cropping, and relay cropping techniques is restricted by factors such as region and cultivated land area. The breeding of disease-resistant varieties has become more difficult due to a series of problems such as long breeding cycles, high costs, complex disease-resistant inheritance laws, and the continuous emergence of new physiological races (Gimeno A et al., 2020). The chemical pesticides used in chemical control have properties such as high toxicity and high residues, which not only endanger the health of humans and livestock, but also pollute the environment and damage the ecological balance; in addition, chemical pesticides can also cause drug resistance problems, increasing the difficulty of their control (Wang R et al., 2021). Biological control, due to its characteristics of being green, environmentally friendly, efficient, and safe, can better serve the sustainable development of agriculture and achieve the unity of social, economic, and ecological benefits (Li Y et al., 2020). Therefore, biological control has become a key control measure that people focus on. The research and development of biocontrol bacteria is an important aspect of biological control, and currently it is widely studied for the control of plant fungal diseases. Therefore, developing microorganisms that have a control effect on fruit tree diseases has important economic and social significance.

[0005] In addition to fruit tree diseases threatening the yield of fruit trees, the frequent occurrence of algae also poses a threat to the growth of fruit trees. Algae often exist in the form of moss on fruit trees. Among them, citrus moss disease is a typical example of algae harming fruit trees, and it has become a common and severely harmful disease (Yang L et al., 2019). This disease occurs on the main trunks, branches, and perennial leaves of citrus. Eventually, the moss will surround the entire trunk and branches, or cover the entire leaf. Due to the obstruction of the moss, the photosynthesis of plants is blocked, resulting in poor growth of the tree body, curling or shedding of leaves, withering of branches, decline of tree vigor, resulting in smaller fruits, and seriously affecting the yield and quality of citrus (Yang L et al., 2020). Secondly, moss also grows on the ground, affecting the air permeability of the soil at the same time. In addition, the moss parasitizes on the branches and trunks of fruit trees, absorbing the water and nutrients in the branches to provide sufficient water and fertilizer for its own reproduction, resulting in the weakening of the tree body (Yu X L et al., 2018). At present, the main method for controlling moss (algae) is to use copper sulfate (Kang X B et al., 2021). However, this often involves problems such as drug residues, drug resistance, and harm to human health. At the same time, to a certain extent, the moss blocks the contact between drugs, fertilizers and the tree body, affecting the effectiveness and indirectly increasing the application costs of drugs and fertilizers (Yu X L et al., 2018). Based on the current dilemmas in chemical algae control and the characteristics of algae, we can choose an environmentally friendly biological control method to solve the algae problem on fruit trees.

[0006] In view of this, the present invention is specifically proposed. The purpose of the present invention is to develop a microorganism that has a control effect on fruit tree diseases and also has an algae-removing function. Summary of the Invention

[0007] The purpose of the present invention is to provide a multi-functional Paenibacillus terrae strain and its application. The strain of the present invention not only has the activities of killing the pathogens of apple leaf spot disease and pear black spot disease at the same time, but also has a broad-spectrum bactericidal activity against the pathogens of other plant diseases, and has the functions of killing algae and activating silicon, and has good application prospects in the biological control of plant (especially fruit tree) diseases.

[0008] The technical solution provided by the present invention is as follows:

[0009] In one aspect, the present invention provides a multi-functional Paenibacillus terrae strain, and the multi-functional Paenibacillus terrae strain is Paenibacillus terrae strain KY834, which is deposited in the China General Microbiological Culture Collection Center, and the deposit number is CGMCC No. 24708.

[0010] Through high-throughput screening, the present invention obtains a multi-functional microorganism KY834 that can broadly kill plant disease pathogens, efficiently kill algae, and activate silicon. According to the determination results of its 16s DNA sequence (SEQ ID No. 1), and at the same time, sequence alignment with the 16sRNA database recognized by the International Committee on Bacteriology and combined with literature analysis to determine the taxonomic status of the target microorganism. The results show that this strain is determined to be Paenibacillus terrae.

[0011] Although there are some reports on the biocontrol functions of Paenibacillus terrae in the existing literature, none of them involve the reports on the functions of Paenibacillus terrae strain KY834 of the present invention in killing plant disease pathogens (including apple leaf spot disease, pear black spot disease, wheat root rot disease, wheat sheath blight disease, tomato early blight disease, cotton verticillium wilt disease) and killing algae. The report on the functions of strain KY834 of the present invention is the first in the world.

[0012] In another aspect, the present invention provides a microbial inoculum, and the microbial inoculum contains the fermentation broth, bacterial suspension and / or supernatant of the aforementioned Paenibacillus terrae strain KY834. That is, Paenibacillus terrae strain KY834 is used as an active ingredient.

[0013] In the present invention, the microbial inoculant can be a liquid preparation or a solid preparation. In a specific embodiment, the microbial inoculant can be prepared by mixing the fermentation broth, bacterial suspension and / or supernatant of the strain KY834 with other excipients to form an aqueous or oily suspension, powder, emulsion, oil dispersion, paste, ointment or granule, etc. The microbial inoculant of the present invention can be used as an antibacterial agent against plant pathogens.

[0014] In one embodiment, silica, light calcium carbonate, kaolin, bentonite or straw powder can be added to the microbial inoculant; glycerol, vegetable oil, sodium alginate or chitosan can also be added, as long as the function of the strain is not affected.

[0015] In one embodiment, in the microbial inoculant, the viable cell concentration of the strain KY834 is 10 6 cfu / mL or 10 6 cfu / g or more, preferably 10 8 cfu / mL or 10 8 cfu / g or more.

[0016] In another aspect, the present invention provides a biological fertilizer containing the aforementioned Paenibacillus terrae strain KY834 or its metabolites.

[0017] In another aspect, the present invention provides the application of the Paenibacillus terrae strain KY834, the microbial inoculant or the biological fertilizer in one or more of the following aspects:

[0018] (a) inhibiting plant pathogens or controlling plant diseases; (b) algicidal; (c) activating silicon.

[0019] In one embodiment, the plant diseases include apple leaf spot and / or pear black spot.

[0020] In one embodiment, the plant diseases also include one or more of gray mold, tomato early blight, cotton wilt, watermelon wilt, banana wilt, rice sheath blight, wheat sheath blight, cotton verticillium wilt and wheat root rot.

[0021] In one embodiment, the strain KY834 has antibacterial activity against three different special forms of banana wilt (banana wilt special form 1, banana wilt special form 2, banana wilt special form 3) and two different special forms of cotton verticillium wilt (cotton verticillium wilt special form Vd991, cotton verticillium wilt special form OD08047).

[0022] Compared with the biocontrol microorganism Bacillus amyloliquefaciens DSM7 widely used in the market at present, the strain KY834 of the present invention exhibits a broader spectrum of bactericidal activity and excellent bactericidal intensity. Compared with the biocontrol microorganism Paenibacillus polymyxa, which performs well and has a high recognition rate in the current market, the performance of KY834 is not inferior to that of Paenibacillus polymyxa. In addition, KY834 has the ability to kill algae, and its algae-killing ability is not worse than that of Paenibacillus polymyxa. KY834 has the ability to activate silicon. Under the same conditions, on a silicon-containing medium, no clear zone was produced near the colonies of the commercial strains 92068 and DSM7.

[0023] The strain KY834 of the present invention can be used to prepare products for inhibiting plant pathogens, especially agricultural preparations for inhibiting broad-spectrum plant pathogens, and has broad application prospects in the field of biological control of plant diseases.

[0024] In one embodiment, the algae include cyanobacteria, preferably Microcystis aeruginosa. Given the algae-killing function of this strain, in addition to being used on fruit tree varieties with partial algae occurrence, it can also be used in the treatment of aquaculture wastewater.

[0025] In one embodiment, the plants include vegetables, food crops or fruit trees; preferably fruit trees. The food crops include but are not limited to wheat, rice, cotton, tomatoes, watermelons, etc.

[0026] In one embodiment, the fruit trees include but are not limited to apple trees, pear trees, banana trees and citrus trees, etc.

[0027] In another aspect, the present invention provides a method for planting fruit trees to prevent and control the harm of pathogens and algae, and the method includes using the strain KY834 of Paenibacillus terrae, the microbial inoculum or the biological fertilizer in the planting of fruit trees.

[0028] In one embodiment, the method includes applying the fermentation broth, bacterial suspension, supernatant of the strain KY834, the microbial inoculum or the biological fertilizer containing the strain KY834 to the rhizosphere soil of fruit tree plants. In one embodiment, the method includes inoculating the fermentation broth, bacterial suspension, supernatant of the strain KY834, the microbial inoculum containing the strain KY834 onto the roots, stems, branches and leaves of fruit trees.

[0029] Due to the broad-spectrum plant pathogen-killing, algae-killing and silicon-activating multifunctions of the strain KY834, it can be used in plant planting, especially in fruit tree planting, to prevent and control plant diseases and promote better growth of fruit trees.

[0030] Preservation information: Paenibacillus terrae strain KY834, which was deposited on April 18, 2022 at the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, with the deposit number: CGMCC No. 24708; the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101. It was detected as a viable strain by the preservation center on April 18, 2022.

[0031] Beneficial effects:

[0032] The Paenibacillus terrae strain KY834 provided by the present invention has broad-spectrum activity against pathogenic bacteria of plant diseases and strong antagonistic effects. It is a microorganism that is environmentally friendly and beneficial to the growth and production of crops, and has great development potential.

[0033] The Paenibacillus terrae strain KY834 provided by the present invention has multiple functions. In addition to having broad-spectrum resistance to pathogenic bacteria, it also has algicidal / algae-removing functions, which also has important application significance for eliminating the impact of algae on fruit tree crops;

[0034] The ability of the Paenibacillus terrae strain KY834 provided by the present invention to activate silicon can help solve the problem of nutritional imbalance caused by excessive application of chemical fertilizers and pesticides at present, and achieve green production;

[0035] The Paenibacillus terrae strain KY834 of the present invention and products containing the same are safe and efficient, can replace conventional and easily resistant pesticides, and achieve better control effects; the present invention has good application prospects in the prevention and control of plant diseases, especially in the prevention and control of diseases of fruit trees and vegetable crops. Description of the drawings

[0036] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0037] Figure 1The microorganisms obtained by high-throughput screening with the function of killing the pathogen of apple leaf spot are shown in the figure as the boxes (the radius of the inhibition zone produced by the bacteria in the boxes is ≥1 mm, among which the radius of the inhibition zone produced by bacterium A is 2 mm, and the radius of the inhibition zone produced by bacterium B is 1-2 mm);

[0038] Figure 2 This is the morphology of strain KY834 provided by the embodiment of the present invention under a microscope (100× oil immersion lens);

[0039] Figure 3 This is the test result of the broad-spectrum bactericidal activity of KY834 and commercial strains (note: successively for apple leaf spot, pear black spot, cotton fusarium wilt, watermelon fusarium wilt, banana fusarium wilt race 1, banana fusarium wilt race 2, banana fusarium wilt race 3, wheat root rot, rice sheath blight, gray mold, wheat sheath blight, tomato early blight, cotton verticillium wilt race Vd991, cotton verticillium wilt race OD08047);

[0040] Figure 4 This is the test result of the algae-lysing ability of strain KY834 of the present invention and commercial strains;

[0041] Figure 5 This is the test result of the ability of strain KY834 of the present invention and commercial strains to activate silicon. Detailed implementation manners

[0042] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] Example 1. High-throughput screening of microorganisms for killing the pathogen of apple leaf spot

[0044] Apple leaf spot is a serious fruit tree disease, which can cause fruit tree defoliation, poor fruit appearance, and fruit yield reduction. Screening microorganisms that can kill the pathogen of apple leaf spot is of great significance for preventing and controlling this disease. Therefore, the present invention uses the pathogen of apple leaf spot as the indicator bacterium for the primary screening of this study to obtain microorganisms that can kill the pathogen of apple leaf spot. The specific methods and results are as follows:

[0045] 1.1 Soil sample collection

[0046] A total of 87 soil samples were collected from all over the country, including black soil, clay, red soil and other soil samples, which came from forests, grasslands, wheat fields, rice fields, etc. These soil samples were marked with the collection place (province, city, county), collection time, and collection source (forest, grassland, wheat field, rice field, etc.).

[0047] 1.2 High-throughput screening of microorganisms capable of killing apple leaf spot pathogens

[0048] (1) Take 5 soil samples (0.2 g of each soil sample), mix them and evenly disperse them in 50 mL of sterile water. Take a small amount of liquid and dilute them 10 times, 100 times, and 1000 times respectively. Take 100 μL of each and evenly spread them on R2A solid culture medium and culture them at 30°C for 2-4 days.

[0049] (2) The colonies in the above culture dishes were picked and inoculated into a 96-well plate containing R2A solid medium and cultured at 30°C for 2 days. (The preparation method of solid R2A medium is as follows: 0.5g peptone, 0.5g yeast, 0.5g tryptone, 0.5g glucose, 0.5g soluble starch, 0.3g sodium pyruvate, 0.3g potassium dihydrogen phosphate, 0.05g magnesium sulfate, 15.0g agar, 1000mL H 2 O, 121.0℃ high pressure sterilization for 30min).

[0050] (3) Scrape the hyphae of the pathogen of apple leaf spot disease and evenly disperse them in sterile water to prepare a bacterial suspension; then take an appropriate amount of the bacterial suspension and evenly spread it on the R2A solid culture medium to obtain a selective culture medium A for standby use.

[0051] (4) Use a sterilized microplate replicator (96 wells) to dip the microorganisms in the 96-well microplate and copy them onto selective medium A. Culture at 30°C for 2-3 days and observe the growth of the microorganisms and the formation of inhibition zones. If a bactericidal zone is formed around the colony, it indicates that the microorganism has obvious fungicidal activity. The specific phenomenon is as follows: Figure 1 The microorganisms producing sterilization zone radius ≥ 1 mm were selected and streaked on R2A solid medium for culture and purification to obtain microbial strains capable of killing apple leaf spot pathogens.

[0052] 1.3 Repeat verification

[0053] The obtained microbial strain having the function of killing the pathogen of apple leaf spot disease is inoculated again on the selective culture medium A to exclude false positive microorganisms that cannot produce sterilization circles on the specific culture medium, and finally obtain microorganisms having the function of killing the pathogen of apple leaf spot disease.

[0054] 1.4 Results

[0055] 87 soil samples (nearly 35,000 microorganisms) were subjected to high-throughput screening and functional tests for killing the pathogen of apple leaf spot disease. A total of 1,201 microorganisms with the function of killing the pathogen of apple leaf spot disease were obtained. The classification of these functional microorganisms is shown in Table 1 as follows:

[0056] Table 1. Statistical table of microorganisms with the function of killing the pathogen of apple leaf spot disease (unit: mm)

[0057]

[0058] Example 2. Screening of microorganisms that can kill Alternaria alternata

[0059] Alternaria alternata is also a common fruit tree disease in China, which will seriously damage the appearance of fruits and is also an important object for the prevention and control of fruit tree diseases. Therefore, the research on the prevention and control of Alternaria alternata is particularly important for fruit production. To achieve the purpose of preventing and controlling Alternaria alternata, the present invention further screens the above 309 strains with good bactericidal ability against the pathogen of apple leaf spot disease and conducts bactericidal activity tests (the pathogen of Alternaria alternata). The specific methods and results are as follows:

[0060] 2.1 Obtaining microorganisms with bactericidal activity against the pathogen of Alternaria alternata

[0061] The above microorganisms with the function of killing the pathogen of apple leaf spot disease and the pathogen of Alternaria alternata were simultaneously inoculated on R2A solid medium and cultured at 30 °C for 6 d. Observe the strength of antagonism of the strains against plant pathogens, measure the bactericidal circle and the colony radius of the test strains, and calculate the bactericidal intensity.

[0062]

[0063] 2.2 Results

[0064] The above 309 microorganisms with good ability to kill the pathogen of apple leaf spot disease were tested by bactericidal activity tests (against the pathogen of Alternaria alternata), and 165 microorganisms that can simultaneously kill the pathogens of apple leaf spot disease and Alternaria alternata were obtained.

[0065] Table 2. Statistical table of microorganisms with the function of killing the pathogen of Alternaria alternata and algae killing ability

[0066]

[0067] (Note: A indicates that the microorganism has good ability to kill the pathogen of apple leaf spot, and B indicates that the microorganism has the ability to kill the pathogens of both apple leaf spot and Alternaria alternata.)

[0068] Example 3. Screening of microorganisms that can kill algae

[0069] In addition to fungal diseases that harm fruit trees (apple leaf spot, pear black spot), algae can also directly harm fruit trees by competing for nutrients, robbing sunlight, etc., threatening the safe production of fruits. Developing microorganisms that can simultaneously kill pathogenic bacteria of fruit tree diseases and algae is of great significance. Therefore, the present invention further screens the above 165 strains with good bactericidal ability against the pathogenic bacteria of apple leaf spot and pear black spot, and conducts algicidal activity tests. The specific methods and results are as follows:

[0070] 3.1 Obtaining microorganisms with algicidal ability

[0071] 3.1.1 Scraping an appropriate amount of algae (Microcystis aeruginosa) and placing it in sterilized water, shaking well to make a uniform algal solution; then sucking a small amount of the algal solution and dispersing it evenly in a mixed solid medium of BG11 and R2A (volume ratio BG11:

[0072] R2A = 1:1) to make a selective medium B for standby. (Among them, the components of the BG11 solid medium are as follows: 1.5 g sodium nitrate, 0.04 g dipotassium hydrogen phosphate trihydrate, 0.075 g magnesium sulfate heptahydrate, 0.036 g calcium chloride dihydrate, 0.006 g citric acid, 0.006 g ferric ammonium citrate, 0.001 g EDTA, 0.02 g sodium carbonate, 0.00286 g boric acid, 0.00181 g manganese chloride monohydrate, 0.000222 g zinc sulfate heptahydrate, 0.000079 g copper sulfate pentahydrate, 0.00039 g sodium molybdate dihydrate, 0.000049 g cobalt nitrate hexahydrate, 1000 mL water, 15.0 g agar; pH = 7.1, autoclaved at 121.0 °C for 30 min.)

[0073] 3.1.2 Inoculating the microorganisms obtained in step "2.2" with the function of killing the pathogenic bacteria of apple leaf spot and pear black spot on the selective medium B, and culturing at 28 °C for 6 d. Observe the size of the algicidal zone produced by the strains, measure the radius of the bactericidal zone and the colony of the test strains, and calculate the algicidal intensity.

[0074]

[0075] 3.2 Results

[0076] The above 165 strains of microorganisms with the ability to kill the pathogenic bacteria of apple leaf spot and pear black spot were tested for algicidal activity, and 76 strains of microorganisms that can simultaneously kill the pathogenic bacteria of apple leaf spot, pear black spot and Microcystis aeruginosa were obtained.

[0077] Table 3. Statistical table of microorganisms with the ability to kill the pathogenic bacteria of pear black spot and algicidal ability

[0078]

[0079] (Note: B indicates the ability of the microorganism to kill the pathogenic bacteria of apple leaf spot and pear black spot simultaneously, and C indicates the ability of the microorganism to kill the pathogenic bacteria of apple leaf spot, pear black spot and algae simultaneously.)

[0080] Example 4. Obtaining microorganisms with broader-spectrum plant pathogenic bacteria killing activity

[0081] In the process of agricultural production, in addition to the relatively widespread apple leaf spot and pear black spot that will harm fruit production, other diseases occurring in the leaves and fruits (such as gray mold, tomato early blight, etc.), root rot occurring in the roots, and wilt diseases caused by blocking the vascular bundles (such as cotton wilt, watermelon wilt, banana wilt, etc.) will also have an adverse impact on the growth of fruit trees, the appearance of fruits and the fruit yield. In addition, rice sheath blight, wheat sheath blight, and cotton verticillium wilt are also key targets for prevention and control in agricultural production. Therefore, in order to better prevent and control fruit tree diseases and develop microorganisms with a broader application scenario, the present invention conducts a broad-spectrum plant disease pathogenic bacteria test on the above 76 microorganisms that can simultaneously kill the pathogenic bacteria of apple leaf spot, pear black spot and algae (including: three transformation types of cotton wilt, watermelon wilt, banana wilt, wheat root rot, rice sheath blight, wheat sheath blight, gray mold, tomato early blight, two special forms of cotton verticillium wilt). The specific methods and results are as follows:

[0082] 4.1 Obtaining microorganisms with broader-spectrum fungal disease pathogenic bacteria killing activity

[0083] Set the pathogenic bacteria of three special forms of cotton wilt, watermelon wilt, banana wilt, wheat root rot, rice sheath blight, wheat sheath blight, gray mold, tomato early blight, and two special forms of cotton verticillium wilt as indicator strains, and the 76 microorganisms that can simultaneously kill the pathogenic bacteria of apple leaf spot, pear black spot and algae in step "3.2" as test strains, and conduct a broad-spectrum plant disease pathogenic bacteria test. The specific steps refer to "2.1". Cultivate at 30°C for 6 days (where the two special forms of cotton verticillium wilt need to be cultivated for 10 days), observe the strength of the antagonism of the strains against plant disease pathogenic bacteria, measure the bactericidal circle and colony radius, and calculate the bactericidal intensity.

[0084] 4.2 Results

[0085] Conduct a broad-spectrum plant disease pathogenic bacteria test on the 76 microorganisms that can simultaneously kill the pathogenic bacteria of apple leaf spot, pear black spot and algae, and find 5 excellent microorganisms, named KY834, J157, J162, J167, J203. The results of the broad-spectrum plant disease pathogenic bacteria test of these 5 microorganisms are shown in Table 4. It can be seen from Table 4 that KY834 has good bactericidal activity against the 12 plant disease pathogenic bacteria tested.

[0086] Table 4. Bactericidal intensity of 5 strains of microorganisms in the broad-spectrum bactericidal activity test

[0087]

[0088] (Note: The greater the bactericidal effect shown in the table, the stronger the bactericidal activity of the strain against the pathogen of the corresponding disease; "0" indicates that the strain has no bactericidal activity against the pathogen of the corresponding disease).

[0089] Meanwhile, after comparing the data in Table 4, it was found that KY834 had good bactericidal activity against all 12 plant disease pathogens tested and had a stronger bactericidal intensity against most plant disease pathogens than other tested strains, showing the best performance among the 5 tested strains. To sum up, the present invention takes the strain KY834 as the research object and conducts strain identification and further research on it.

[0090] Example 5. Identification of strain KY834

[0091] 5.1 Preparation of DNA template

[0092] Pick a purified single colony to the bottom of an EP tube, add 200 μL of 5% (w / v) BT-chelex 100 (prepared with distilled water, sterilized at 121 °C for 30 min). Boil in a boiling water bath for 15 min, quickly place it at -20 °C or -80 °C for quick freezing, then thaw at room temperature, centrifuge at 6000 r / min for 3 min, and take 2 μL of the supernatant as the template. Amplify the 16S gene according to the 16S amplification system.

[0093] 16S PCR amplification system: Green taqMix, 12.5 μL; DDH 2 O, 9.5 μL; 27F, 0.5 μL; 1492R, 0.5 μL; DNA template, 2.0 μL; Total: 25 μL;

[0094] PCR amplification program: (1) 95 °C, 5 min; (2) 94 °C, 1 min; 55 °C, 1 min; 72 °C, 1.5 min, 35 cycles; (3) 72 °C, 10 min; 4 °C forever.

[0095] 5.2 16sDNA sequence determination results of KY834

[0096] The 16S rDNA sequence (SEQ ID No.1) of KY834 was used to search for homologous sequences in GenBank and perform homologous sequence analysis and comparison. At the same time, sequence alignment was carried out with the 16S RNA database (NBCI) recognized by the International Committee on Bacteriology and combined with literature analysis to determine the taxonomic status of the target microorganism (Yoon, S.H., Ha, S.M., Kwon, S., Lim, J., Kim, Y., Seo, H. and Chun, J. (2017). Introducing EzBioCloud: A taxonomically united database of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol. 67:1613-1617). The results showed that the 1443-base sequence of this strain was highly homologous to that of Paenibacillus terrae, with a similarity of up to 100%, and KY834 was determined to be Paenibacillus terrae.

[0097] The sequencing result of the 16S rDNA sequence of strain KY834 (SEQ ID No.1) is as follows: GGGTGCCTAATACATGCAAGTCGAGCGGGGTTGTTGTGGAAGCTTGCTTCTACAACAACCTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTATCAGACTGGGATAACTACCGGAAACGGTAGCTAATACCGGATACATCCTTTCCCTGCATGGGGAGGGGAGGAAAGACGGAGCAATCTGTCACTGATGGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGCCAGGGAAGAACGTCTTGTAGAGTAACTGCTACAAGAGTGACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCTCTTTAAGTCTGGTGTTTAATCCCGAGGCTCAACTTCGGGTCGCACTGGAAACTGGGGAGCTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGATATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGGCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTTAACACATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCCCTGATCGGTCTAGAGATAGATCTTTCCTTCGGGACAGGGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGCTTAGTTGCCAGCAGGTCAAGCTGGGCACTCTAAGCAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCCGGTACAACGGGAAGCGAAAGAGCGATCTGGAGCGAATCCTAGAAAAGCCGGTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTACAACACCCGAAGTCGGTGGGGTAACCCGCAAGGGAGCCAGCCGCCCA。

[0098] Example 6. Observation of Strain Morphology

[0099] 6.1 Operation

[0100] Inoculate the screened strain onto an R2A plate and culture it at 30 °C for 2 days. Observe the size, shape, color, glossiness, viscosity, elevation shape, transparency, edge characteristics, and presence or absence of spores of the colony.

[0101] Observation results of the morphology of strain 6.2

[0102] After observation, strain KY834 (Paenibacillus terrae) was cultured on R2A medium for 2 days. The colonies were milky white, round, slightly irregular in shape, flat to low convex, and translucent. Its cell length was measured by microscope to be about 3.20 - 4.16 μm (as Figure 2 ).

[0103] Example 7. Comparison with commercial strains

[0104] After the previous tests, KY834 has been verified to have the ability to broadly kill plant disease pathogens and algae. To better understand the functions and application scenarios of KY834, the commercial strain Paenibacillus polymyxa (which has good disease prevention effect and high recognition in the market) and the commercial strain DSM7 (Bacillus amyloliquefaciens, used for disease prevention in the market) were set as controls to compare the activities of broadly killing plant disease pathogens and the ability to kill algae; the commercial strain 92068 (Bacillus subtilis, commonly used for promoting plant growth, disease prevention, potassium and phosphorus solubilization in the market) and the commercial strain DSM7 (Bacillus amyloliquefaciens, used for disease prevention in the market) were set as controls to compare the multifunction (ability to activate silicon).

[0105] 7.1 Comparison of the activities of KY834 and commercial strains in broadly killing plant disease pathogens

[0106] The pathogens of apple marssonina leaf spot, pear black spot, cotton fusarium wilt, watermelon fusarium wilt, banana fusarium wilt race 1, banana fusarium wilt race 2, banana fusarium wilt race 3, wheat root rot, rice sheath blight, wheat sheath blight, gray mold, tomato early blight, cotton verticillium wilt race Vd991, and cotton verticillium wilt race OD08047 were set as indicator bacteria, KY834 as the test strain, and Paenibacillus polymyxa and DSM7 as control strains for the test of broadly killing disease pathogens. The specific steps refer to "2.1". They were cultured at 30°C for 6 days (among which cotton verticillium wilt race Vd991 and OD08047 need to be cultured for 10 days). Observe the strength of the antagonism of the strains against plant disease pathogens, measure the bactericidal circle and colony radius, calculate the bactericidal intensity, and fill in Table 5.

[0107] 7.2 Comparison of the algae-killing ability of KY834 and commercial strains

[0108] (1) Activation of strains

[0109] Take appropriate amounts of KY834, Paenibacillus polymyxa, and DSM7 from an -80°C refrigerator and inoculate them onto solid R2A medium, then culture at room temperature for 2 days. Among them, KY8334 is the test strain, and Paenibacillus polymyxa and DSM7 are the control strains.

[0110] Take an appropriate amount of Microcystis aeruginosa and inoculate it onto solid BG11 medium, then culture in a light incubator for 15 days.

[0111] (2) Inoculation

[0112] Inoculate the microorganisms in “(1)” into selective medium B, culture at 28°C for 6 days, take out and observe, and compare the sizes of the clear algicidal zones produced.

[0113] 7.3 Compare the silicon-activating abilities of KY834 and commercial strains

[0114] Silicon is an important element in the process of plant growth. Exploring the silicon-activating ability of KY834 can help solve the problem of nutritional imbalance caused by excessive application of chemical fertilizers and pesticides at present and achieve green production. Therefore, in this invention, KY834 is set as the test strain, and 92068 and DSM7 are the control strains for the silicon-activation test, which is specifically as follows:

[0115] (1) Strain activation

[0116] Take appropriate amounts of KY834, 92068, and DSM7 from an -80°C refrigerator and inoculate them onto solid R2A medium, then culture at room temperature for 2 days. Among them, KY8334 is the test strain, and 92068 and DSM7 are the control strains.

[0117] (2) Preparation of silicon-containing solid medium

[0118] 5.0 g MgSiO 4 , 0.5 g peptone, 0.5 g yeast, 0.5 g tryptone, 0.5 g glucose, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.13 g potassium dihydrogen phosphate, 0.05 g magnesium sulfate, 1000 mL H 2 O, 15 g agar, autoclave at 121.0°C for 30 min.

[0119] (3) Inoculation

[0120] Inoculate the microorganisms in “(1)” into the silicon-containing solid medium described in “(2)”, culture at 30°C for 6 days, take out and observe, and compare the sizes of the clear zones produced.

[0121] 7.4 Results

[0122] From Table 5, Figure 3It can be seen that compared with the biocontrol microorganism Bacillus amyloliquefaciens DSM7 which is widely used in the market at present, KY834 exhibits a broader spectrum of bactericidal activity and excellent bactericidal intensity. Compared with the biocontrol microorganism Paenibacillus polymyxa which performs well and has a high recognition in the current market, the performance of KY834 is not inferior to that of Paenibacillus polymyxa. KY834 shows strong bactericidal activity against 14 kinds of pathogenic bacteria of plant diseases including apple leaf spot and pear black spot.

[0123] From Table 6, Figure 4 it can be seen that on the algae-containing medium, obvious transparent algicidal zones can be formed near the colonies of KY834 and Paenibacillus polymyxa, and no transparent algicidal zone is produced near the colony of DSM7. At the same time, the algicidal zone produced by KY834 is significantly larger than that produced by Paenibacillus polymyxa, and the algicidal intensity of KY834 is also significantly stronger than that of Paenibacillus polymyxa. It can be judged from this that KY834 has the ability to kill algae and its algicidal ability is not worse than that of Paenibacillus polymyxa.

[0124] From Table 7, Figure 5 it can be seen that on the silicon-containing medium, a transparent zone can be produced near the colony of KY834, and no transparent zone is produced near the colonies of 92068 and DSM7. KY834 has the ability to activate silicon.

[0125] Table 5. Comparison results of the broad-spectrum bactericidal activity of KY834, DSM7 and Paenibacillus polymyxa against pathogenic bacteria of plant diseases

[0126]

[0127] (Note: The greater the bactericidal intensity shown in the table, the stronger the bactericidal activity of the strain against the pathogenic bacteria of the corresponding disease; "0" indicates that the strain does not have bactericidal activity against the pathogenic bacteria of the corresponding disease).

[0128] Table 6. Algicidal zone intensity of KY834, Paenibacillus polymyxa and DSM7

[0129]

[0130] After observing the strain KY834 (Paenibacillus terrae) cultured on R2A medium for 2 days, the colony is milky white, round, slightly irregular in shape, flat to low convex, and semi-transparent. Its cell length is about 3.20 - 4.16 μm as measured by microscope (such as Figure 2 ).

[0131] Table 7. Comparison results of the silicon activation ability of KY834, 92068 and DSM7

[0132]

[0133] (Note: "0" indicates that the microorganism cannot produce a clear zone in the silicon-containing solid medium and does not have the ability to activate silicon; "+" indicates that the microorganism can produce a clear zone in the silicon-containing solid medium and has the ability to activate silicon, but the ability to activate silicon is average; "++" indicates that the microorganism can produce a clear zone in the silicon-containing solid medium and has the ability to activate silicon and the ability to activate silicon is better.)

[0134] Conclusion:

[0135] In the present invention, a microorganism KY834 capable of killing algae and preventing diseases (specifically apple leaf spot and pear black spot) was isolated from soils collected from all over the country (nearly 35,000 strains) by means of high-throughput screening. In addition, through further experiments, the present invention found and confirmed that KY834 also has the functions of broad-spectrum killing of plant disease pathogens and activating silicon. The sequencing result of the 16srDNA sequence of this strain shows that this strain is highly homologous to Paenibacillus terrae. Compared with Paenibacillus polymyxa (which has good disease prevention effect and high recognition in the market) and commercial strain DSM7 (Bacillus amyloliquefaciens, used for disease prevention in the market), KY834 shows strong bactericidal activity and excellent algicidal ability against 14 plant disease pathogens (including apple leaf spot, pear black spot, cotton wilt, watermelon wilt, three special forms of banana wilt, wheat root rot, rice sheath blight, wheat sheath blight, gray mold, tomato early blight and two special forms of cotton verticillium wilt). By using 92068 (Bacillus subtilis, commonly used for promoting plant growth, preventing diseases, decomposing potassium and decomposing phosphorus in the market) and commercial strain DSM7 (Bacillus amyloliquefaciens, used for disease prevention in the market) as controls, a comparison of multifunctions (ability to activate silicon) was carried out, and it was found that KY834 has the ability to activate silicon, which is beneficial to the growth and development of crops.

[0136] In summary, KY834 is a functional microorganism with broad-spectrum killing of plant disease pathogens, high-efficiency algicidal and silicon-activating abilities, and has great application potential in agricultural production and biological control (especially fruit tree disease control).

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multifunctional Paenibacillus terrae Characterized in that The multifunctional Paenibacillus terrae is Paenibacillus terrae Paenibacillus terrae strain KY834, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms with the deposit number of CGMCC No. 24708.

2. A microbial inoculant Characterized in that It contains the fermentation broth and / or bacterial suspension of the Paenibacillus terrae strain KY834 described in claim 1 ( Paenibacillus terrae ).

3. A biological fertilizer Characterized in that It contains the Paenibacillus terrae strain KY834 described in claim 1 ( Paenibacillus terrae ).

4. Use of the Paenibacillus terrae strain KY834 as claimed in claim 1 ( Paenibacillus terrae ), the microbial inoculum as claimed in claim 2, or the biological fertilizer as claimed in claim 3 in one or more of the following aspects: (a)Inhibiting plant pathogenic bacteria or controlling plant diseases; (b)Algaecide; (c)Activating silicon; The plant pathogenic bacteria are one or more of the pathogenic bacteria of apple leaf spot, pear black spot, cotton wilt, watermelon wilt, three special forms of banana wilt, wheat root rot, rice sheath blight, wheat sheath blight, gray mold, tomato early blight or two special forms of cotton verticillium wilt; The plant diseases are one or more of apple leaf spot, pear black spot, gray mold, tomato early blight, cotton wilt, watermelon wilt, banana wilt, rice sheath blight, wheat sheath blight, cotton verticillium wilt and wheat root rot; The alga is Microcystis aeruginosa.

5. The application according to claim 4 Characterized in that The plants include vegetables, food crops or fruit trees.

6. A method for planting fruit trees to control the harm of pathogenic bacteria and algae Characterized in that The method includes using the Paenibacillus terrae strain KY834 ( Paenibacillus terrae ) described in claim 1, the microbial inoculant described in claim 2, or the bio-fertilizer described in claim 3 in the cultivation of fruit trees; The pathogenic bacteria are one or more of the pathogenic bacteria of apple leaf spot, pear black spot, cotton wilt, watermelon wilt, three special forms of banana wilt, wheat root rot, rice sheath blight, wheat sheath blight, gray mold, tomato early blight or two special forms of cotton verticillium wilt; The algae are Microcystis aeruginosa.

7. The planting method according to claim 6 Characterized in that The method includes applying the fermentation broth, bacterial suspension, microbial inoculant containing strain KY834 or biological fertilizer of the strain KY834 to the rhizosphere soil of fruit tree plants.

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