CN115786210A - A strain of multifunctional Paenibacillus terreus and its application - Google Patents

Abstract

The invention provides a multifunctional Paenibacillus terrae and application thereof, and relates to the technical field of microorganisms and application thereof. The multifunctional Paenibacillus terrae is a Paenibacillus terrae (Paenibacillus terrae) strain KY834 which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.24708. According to the invention, a strain KY834 capable of killing pathogenic bacteria of apple alternaria leaf spot and pear black spot is obtained from a large number of microbial strains through high-throughput screening, and further research shows that the strain has broad-spectrum bactericidal activity on pathogenic bacteria of other plant diseases, has functions of killing algae and activating silicon, and has good application prospects in biological control of plant (especially fruit trees) diseases.

Description

A strain of multifunctional Paenibacillus terreus and its application

technical field

The invention relates to the technical field of microorganisms and their applications, in particular to a strain of multifunctional Paenibacillus terreus and its applications.

Background technique

Fruit tree production occupies an important position in the agricultural production of most countries in the world. With the rapid development of social economy and the improvement of national living standards, the market demand for fruits has also increased accordingly, which has higher requirements for the yield of fruit trees, the quality and appearance of fruits. However, because fruit trees are affected by changes in global climate and environmental conditions all year round, coupled with the susceptibility of fruit trees during the growth cycle, the spread of diseases is growing explosively, endangering agricultural production safety (Manzhonghe, 2021). Therefore, the prevention and control of fruit tree diseases and algae has become one of the key tasks for the development of the fruit industry.

Among fruit tree diseases, apple leaf spot and pear black spot are the most common diseases. Alternaria alternaria is called brown spot disease, which mainly damages young leaves, and also damages young leaves and fruits (Jin Ying, 2021). Apple spot defoliation can cause early fall, affecting tree vigor and yield, and it occurs in all apple producing areas. Brown dots appear on the leaves at the beginning of the disease, and then expand to reddish brown, the edge of the leaf is purple-brown, and there is often a dark spot in the center of the diseased part, which can easily cause the leaves to twist and shrink, and the diseased part is scorched and incomplete. The fruit is infected, and there are small black spots or rust spots on the fruit surface in the early stage. In the later stage, the diseased part of the fruit sometimes shows gray-brown scab-like plaques, and there are 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 defoliation of fruit trees, but also infect the fruit surface to form spots, affecting the quality of apples, and affecting the yield of the next year, which greatly threatens the development of the fruit industry (Yu Xiaoli et al., 2016). Pear black spot also commonly occurs in my country, among which snow pear and crisp pear are the most serious. The disease is mainly caused by fungal Alternaria alternate (Fr.) Keissl infection. The flowering period and young fruit period of pear trees are all potential infection periods of pear black spot, especially during storage, which is the most susceptible and difficult to control and prevent.

At present, the methods of prevention and control of major diseases of fruit trees in production mainly include grafting, intercropping, rotation and intercropping techniques, breeding of disease-resistant varieties, chemical control and biological control, etc. Grafting has certain limitations that may make the product less palatable (Rodríguez-negrete E A et al., 2020). Space, rotation and intercropping techniques are limited by factors such as region and cultivated land area, which limits their promotion. The selection of disease-resistant varieties has a series of problems such as long breeding cycle, high cost, complex genetic rules of disease resistance, and the continuous emergence of new physiological races, making disease-resistant breeding technology more difficult (Gimeno A et al., 2020). The chemical pesticides used in chemical control are highly toxic and highly residual, which not only endangers the health of humans and animals, but also pollutes the environment and destroys the ecological balance; in addition, chemical pesticides can also cause drug resistance problems, increasing the difficulty of their governance (Wang Rui et al., 2021). Due to the characteristics of green, environmental protection, high efficiency, and safety, biological control can better serve the sustainable development of agriculture and realize the unity of social, economic, and ecological benefits (Li Yang et al., 2020). Therefore, biological control has become a means of prevention and control that people focus on. The research and development of biocontrol bacteria is an important aspect of biological control, and it is widely studied in the control of plant fungal diseases. Therefore, it is of great economic and social significance to develop microorganisms that can prevent and control fruit tree diseases.

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 is a typical example of algal damage to fruit trees, and it has become a common and serious disease (Yang Lei et al., 2019). The disease occurs on citrus trunks, branches, and perennial leaves, and eventually moss will surround the entire trunk and branches, or cover the entire leaf. Because of the cover of moss, the photosynthesis of plants is hindered, resulting in poor tree growth, leaf curling or falling off, branches and tips dying, and tree vigor declining, resulting in smaller fruits, which seriously affect the yield and quality of citrus (Yang Lei et al., 2020 ). Secondly, moss will also grow on the ground, which will affect the air permeability of the soil. In addition, mosses parasitize on the branches of fruit trees to absorb water and nutrients in the branches to provide sufficient water and fertilizer supply for their own reproduction, resulting in tree weakness (Yu Xiaoli et al., 2018). At present, copper sulfate is mainly used to control moss (algae) (Kang Xiaobo et al., 2021). But this often involves drug residues, drug resistance, and harm to human health. At the same time, moss blocks the contact between medicines and fertilizers and trees to a certain extent, which affects the effectiveness and indirectly increases the application cost of medicines and fertilizers (Yu Xiaoli et al., 2018). According to the current difficulties encountered in chemical algae removal and the characteristics of algae, we can choose environmentally friendly biological control methods to solve algae on fruit trees.

In view of this, the present invention is particularly proposed, and the present invention aims to develop microorganisms that have a control effect on fruit tree diseases and have algae removal functions.

Contents of the invention

The object of the present invention is to provide a strain of multifunctional Paenibacillus terreus and its application. The bacterial strain of the present invention not only has the activity of killing apple leaf spot pathogen and killing pear black spot pathogen simultaneously, but also has broad-spectrum bactericidal activity for the pathogen of other plant diseases, and has the function of killing algae and activating silicon, and can be used in plants (especially It has a good application prospect in the biological control of fruit tree) diseases.

The technical scheme provided by the invention is as follows:

In one aspect, the present invention provides a strain of multifunctional Paenibacillus terrae, said multifunctional Paenibacillus terrae is Paenibacillus terrae (Paenibacillus terrae) strain KY834, preserved in the General Microorganism Center of China Microorganism Culture Preservation Management Committee, The deposit number is CGMCC No.24708.

Through high-throughput screening, the invention obtains a multifunctional microorganism KY834 capable of broad-spectrum killing plant disease pathogenic bacteria, efficiently killing algae and activating silicon. According to its 16s DNA sequence (SEQ ID No.1) determination results, at the same time, sequence comparison with the 16sRNA database approved by the International Bacteriology Committee and combined with literature analysis to determine the taxonomic status of the target microorganism, the results showed that the strain was identified as land Paenibacillus terrae.

Although existing literature has some biocontrol function reports about Paenibacillus terrae (Paenibacillus terrae), all do not involve Paenibacillus terrae (Paenibacillus terrae) bacterial strain KY834 of the present invention in killing plant disease pathogenic bacteria (comprising apple leaf spot, pear leaf spot). Black spot, wheat root rot, wheat sheath blight, tomato early blight, cotton verticillium wilt) and algicidal function, the present invention is the first report on the function of strain KY834 in the world.

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

In the present invention, the microbial agent can be a liquid preparation or a solid preparation. In a specific embodiment, the microbial bacterial agent can be formulated into an aqueous or oily suspension, powder, emulsion, oil dispersion, Paste, ointment or granule etc. The microbial agent of the present invention can be used as an antibacterial agent for plant pathogens.

In one embodiment, silicon dioxide, light calcium carbonate, kaolin, bentonite or straw powder can be added to the microbial agent; and glycerin, vegetable oil, sodium alginate or chitosan can be added, as long as it does not affect The function of the strain can be.

In one embodiment, in the microbial agent, the viable bacterial concentration of the strain KY834 is 10 ⁶ cfu/mL or above 10 6 cfu/g, preferably ^{10 8 cfu} /mL or above 10 ⁸ cfu/g.

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

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

(a) inhibiting plant pathogenic bacteria or preventing and controlling plant diseases; (b) killing algae; (c) activating silicon.

In one embodiment, the plant disease comprises apple leaf spot and/or pear black spot.

In one embodiment, the plant diseases also include Botrytis cinerea, tomato early blight, cotton blight, watermelon wilt, banana wilt, rice sheath blight, wheat sheath blight, cotton verticillium wilt and wheat root rot one or more of the diseases.

In one embodiment, the bacterial strain KY834 is resistant to three different specializations of banana wilt (banana fusarium wilt specialization 1, banana fusarium wilt specialization 2, banana fusarium wilt specialization 3) and cotton yellow Two different specializations of wilt (cotton Verticillium wilt specialization Vd991 and cotton Verticillium wilt specialization OD08047) had pathogenic killing activity.

Compared with the currently widely used biocontrol microorganism Bacillus amyloliquefaciens DSM7 in the market, the bacterial strain KY834 of the present invention exhibits a wider spectrum of bactericidal activity and excellent bactericidal intensity. Compared with Paenibacillus polymyxa, which is an excellent and highly recognized biocontrol microorganism currently on the market, the performance of KY834 is not inferior to Paenibacillus polymyxa. In addition, KY834 has the algicidal ability and the algicidal ability is not inferior to that of Paenibacillus polymyxa. KY834 has the ability to activate silicon. Under the same conditions, on the silicon-containing medium, there is no transparent circle near the colonies of the commercial strain 92068 and DSM7.

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

In one embodiment, the algae comprise blue-green algae, preferably Microcystis aeruginosa. In view of the algicidal function of the strain, it can also be used in the treatment of aquaculture wastewater in addition to being used on some fruit tree species where algae occurs.

In one embodiment, the plants comprise vegetables, food crops or fruit trees; preferably fruit trees. The food crops include but not limited to wheat, rice, cotton, tomato, watermelon and the like.

In one embodiment, the fruit trees include, but are not limited to, apple trees, pear trees, banana trees, citrus trees, and the like.

In another aspect, the present invention provides a method for planting fruit trees for the prevention and treatment of pathogenic bacteria and algae damage, the method comprising using the Paenibacillus terrae bacterial strain KY834, the microbial agent or The biological fertilizer.

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

Due to the broad-spectrum killing of phytopathogens and the multi-functionality of killing algae and activating silicon, the bacterial strain KY834 can be used in planting, especially in fruit tree planting, to prevent and control plant diseases and promote better growth of fruit trees.

Preservation information: Paenibacillus terrae (Paenibacillus terrae) strain KY834, which was deposited in the General Microorganism Culture Collection Center of China Microbiology Culture Collection Management Committee on April 18, 2022, with the preservation number: CGMCC No.24708; Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, 100101. It was detected as a viable strain on April 18, 2022 by the preservation center.

Beneficial effect:

The Paenibacillus terrae strain KY834 provided by the present invention has broad-spectrum activity of killing plant disease pathogenic bacteria and strong antagonism, is an environmentally friendly microorganism that is beneficial to the growth and production of crops, and has great potential for development .

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 the function of killing algae/algae, and is also important for eliminating the impact of algae on fruit tree crops. application meaning;

The silicon activation ability of the Paenibacillus terrae strain KY834 provided by the present invention can help solve the nutritional imbalance problem caused by the excessive application of chemical fertilizers and pesticides at present, and realize green production;

The Paenibacillus terrae strain KY834 of the present invention and the products containing it are safe and efficient, can replace conventional and easily resistant pesticides, and achieve better control effects; the present invention is especially effective in plant disease control. It has a good application prospect in the prevention and control of fruit tree and vegetable crop diseases.

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work.

Fig. 1 is the microorganism with the function of killing the pathogenic bacteria of apple leaf spot disease obtained by the high-throughput screening provided by the embodiment of the present invention as shown in the box (the radius of the inhibition zone produced by the bacteria in the box is all ≥ 1mm, wherein A bacterium The radius of the inhibition zone produced by bacteria B is 2 mm, and the radius of the inhibition zone produced by B bacteria is 1-2 mm);

Fig. 2 is the morphology of bacterial strain KY834 provided by the embodiment of the present invention under a microscope (100 times oil lens);

Figure 3 shows the broad-spectrum fungicidal activity test results of KY834 and commercial strains (note: followed by apple leaf spot, pear black spot, cotton wilt, watermelon wilt, banana wilt specialization 1, banana wilt specialization Type 2, banana fusarium wilt specialized type 3, wheat root rot, rice sheath blight, gray mold, wheat sheath blight, tomato early blight, cotton Verticillium wilt specialized Vd991, cotton Verticillium wilt specialized OD08047);

Fig. 4 is the algae-dissolving ability test result of bacterial strain KY834 of the present invention and commercial bacterial strain;

Figure 5 is the test results of silicon activation ability of the inventive strain KY834 and commercial strains.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1. High-throughput screening of microorganisms that kill the pathogenic bacteria of apple leaf spot

Apple leaf spot disease is a serious disease of fruit trees, which will cause the leaves of fruit trees to fall, the appearance of fruits to be poor, and the yield of fruits to be reduced. Screening out microorganisms that can kill the pathogen of apple leaf spot disease is of great significance for the control of the disease. Therefore, the present invention uses the pathogenic bacteria of apple leaf spot disease as the indicator bacteria of the primary screening in this study, and obtains microorganisms that can kill the pathogenic bacteria of apple leaf spot disease. The specific methods and results are as follows:

1.1 Soil sample collection

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 are marked with the collection place (province, city, county), collection time, and collection source (forest, grassland, wheat field, rice field, etc.).

1.2 High-throughput screening of microorganisms with the function of killing the pathogen of apple leaf spot disease

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

(2) The colonies in the above petri dishes were picked and inoculated into a 96-well plate containing R2A solid medium, and cultured at 30° C. for 2 days. (Solid R2A medium preparation method 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, 15.0 g agar, 1000mLH ₂ O, autoclaved at 121.0°C for 30min).

(3) Scrape the hyphae of the pathogenic bacteria of apple leaf spot disease and evenly disperse them in sterilized water to make a bacterial suspension; then absorb an appropriate amount of bacterial suspension and evenly spread it on the R2A solid medium to obtain a selective medium A for use.

(4) Dip the microorganisms in the 96-well microplate with a sterilized microplate replicator (96 wells) and copy them to selective medium A, culture them at 30°C for 2-3 days, and observe the growth of the microorganisms and the formation of the inhibition zone. If a bactericidal zone is formed around the colony, it indicates that the microorganism has obvious fungicidal activity, and the specific phenomenon is shown in Figure 1. Microorganisms with a bactericidal zone radius ≥ 1 mm were selected and cultured and purified by streaking on R2A solid medium to obtain microbial strains with the function of killing the pathogenic bacteria of apple leaf spot disease.

1.3 Repeat verification

The obtained microbial strains with the function of killing the pathogenic bacteria of apple leaf spot disease are inoculated again on the selective medium A to eliminate the false positive microorganisms that cannot produce a bactericidal zone on the specific medium, and finally obtain the microorganisms with the function of killing the pathogenic bacteria of apple leaf spot disease .

1.4 Results

87 soil samples (nearly 35,000 microorganisms) were screened through high-throughput and tested for the function of killing the pathogen of apple leaf spot disease, and a total of 1201 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:

Table 1. The microbial statistics table with the function of killing the pathogenic bacteria of apple leaf spot disease (unit: mm)

Embodiment 2. screening can kill the microorganism of pear black spot

Pear black spot is also a common fruit tree disease in my country, which can seriously damage the appearance of fruit, and is also a key target for fruit tree disease control. Therefore, research on the control of pear black spot is particularly important for fruit production. In order to achieve the purpose of preventing and treating pear black spot, the present invention further screens the above-mentioned 309 strains that have better bactericidal ability to the pathogen of apple leaf spot, and carries out a bactericidal activity test (pathogen of pear black spot). The specific methods and results are as follows:

2.1 Obtaining microorganisms with bactericidal activity against pear black spot pathogen

The above-mentioned microorganisms with the function of killing the pathogenic bacteria of apple leaf spot and the pathogenic bacteria of pear black spot were respectively inoculated on the R2A solid medium at the same time, and cultured at 30° C. for 6 days. Observe the strength of the strain's antagonism to the plant pathogenic bacteria, measure the sterilizing zone and the colony radius of the test strain, and calculate the bactericidal intensity.

2.2 Results

The above-mentioned 309 strains of microorganisms with good ability to kill the pathogenic bacteria of apple leaf spot were tested by the bactericidal activity test (for the pathogenic bacteria of pear black spot), and 165 strains were obtained. Microorganisms that can simultaneously kill apple leaf spot and pear black spot pathogens.

Table 2. The statistical table of microorganisms with the ability to kill pear black spot pathogen and algae killing ability

(Note: A means that the microorganism has the ability to kill the pathogenic bacteria of apple leaf spot and B means that the microorganism has the ability to kill the pathogens of apple leaf spot and pear black spot.)

Example 3. Screening for algae-killing microorganisms

In addition to fungal diseases that harm fruit trees (apple leaf spot, pear black spot), algae can also directly cause damage to fruit trees by competing for nutrients and robbing sunlight, threatening the safe production of fruits. It is of great significance to develop microorganisms that can kill fruit tree disease pathogens and algae at the same time. Therefore, the present invention further screens the above-mentioned 165 strains that have better bactericidal ability to the pathogenic bacteria of apple leaf spot and pear black spot, and performs algicidal activity test. The specific methods and results are as follows:

3.1 Obtaining microorganisms with algicidal ability

3.1.1 Scrape an appropriate amount of algae (Microcystis aeruginosa) and place in sterilized water, shake well to make a uniform algae liquid; then absorb a small amount of algae liquid to evenly disperse it in the mixed solid medium of BG11 and R2A (volume ratio BG11:

R2A=1:1) to prepare selective medium B for use. (Wherein, the components of BG11 solid medium are as follows: 1.5g sodium nitrate, 0.04g dipotassium hydrogen phosphate trihydrate, 0.075g magnesium sulfate heptahydrate, 0.036g calcium chloride dihydrate, 0.006g citric acid, 0.006g citric acid Iron ammonia, 0.001g EDTA, 0.02g sodium carbonate, 0.00286g boric acid, 0.00181g manganese chloride monohydrate, 0.000222g zinc sulfate heptahydrate, 0.000079g copper sulfate pentahydrate, 0.00039g sodium molybdate dihydrate, 0.000049g hexahydrate Cobalt nitrate, 1000mL water, 15.0g agar; pH=7.1, autoclave at 121.0°C for 30min.)

3.1.2 Inoculate the microorganisms obtained in step "2.2" with the function of killing apple leaf spot and pear black spot pathogens into selective medium B, and culture at 28°C for 6 days. Observe the size of the algicidal circle produced by the strain, measure the bactericidal zone and the colony radius of the test strain, and calculate the algicidal intensity.

3.2 Results

The above-mentioned 165 microorganisms capable of killing apple leaf spot and pear black spot pathogenic bacteria passed the algicidal test, and obtained 76 microorganisms that could simultaneously kill apple leaf spot disease, pear black spot pathogen and Microcystis aeruginosa.

Table 3. Statistics of microorganisms with the ability to kill pear black spot pathogen and algicide

(Note: B means that the microorganism has the ability to kill the pathogenic bacteria of apple leaf spot and pear black spot at the same time, and C means that the microorganism has the ability to kill the pathogens of apple leaf spot and pear black spot and algae.)

Example 4. Obtaining microorganisms with broader spectrum phytopathogenic activity

In the process of agricultural production, in addition to the widespread occurrence of apple leaf spot and pear black spot that will harm fruit production, other diseases that occur on leaves and fruits (grey mold, tomato early blight, etc.), and root diseases Root rot and blights caused by clogging vascular bundles (cotton wilt, watermelon wilt, banana wilt, etc.) can also adversely affect fruit tree growth, fruit appearance and fruit yield. In addition, rice sheath blight, wheat sheath blight, and cotton verticillium wilt are also key control targets in agricultural production. Therefore, in order to better prevent and control fruit tree diseases and develop microorganisms with wider application scenarios, the present invention performs broad-spectrum killing of plant diseases on the above-mentioned 76 strains that can simultaneously kill apple leaf spot, pear black spot pathogens and algae. Pathogen test (including: cotton fusarium wilt, watermelon fusarium wilt, three transformation types of banana fusarium wilt, wheat root rot, rice sheath blight, wheat sheath blight, gray mold, tomato early blight, cotton verticillium wilt two specialized type). The specific methods and results are as follows:

4.1 Microorganisms to obtain broader spectrum fungicidal disease pathogens

There are three specialized types of cotton wilt, watermelon wilt, and banana wilt, and two specialized types of wheat root rot, rice sheath blight, wheat sheath blight, gray mold, tomato early blight, and cotton verticillium wilt The pathogenic bacteria in the test are indicator strains. In step "3.2", 76 strains of microorganisms that can kill apple leaf spot, pear black spot pathogens and algae at the same time are test strains for broad-spectrum killing of plant disease pathogenic bacteria. For specific steps, refer to "2.1". Cultivate at 30°C for 6 days (the two specialized types of cotton Verticillium dahliae need to be cultivated for 10 days), observe the strength of the strain's antagonism against plant disease pathogens, measure the sterilization zone and colony radius, and calculate the sterilization intensity.

4.2 Results

76 microorganisms that can kill apple leaf spot, pear black spot pathogens and algae at the same time were tested for broad-spectrum killing of plant disease pathogens, and 5 microorganisms with excellent performance were found, named KY834, J157, J162, J167, J203. The results of the broad-spectrum killing of phytopathogenic bacteria of these five strains of microorganisms are shown in Table 4. It can be seen from Table 4 that KY834 has good fungicidal activity against the 12 tested plant disease pathogens.

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

(Note: The greater the bactericidal value shown in the table, the stronger the bactericidal activity of the bacterial strain against the pathogenic bacteria of the corresponding disease; "0" indicates that the bacterial strain has no bactericidal activity against the pathogenic bacteria of the corresponding disease).

At the same time, after comparing the data in Table 4, it was found that KY834 had good bactericidal activity on the 12 tested plant disease pathogens, and the bactericidal intensity on most plant disease pathogens was stronger than other test strains. The best performance among the tested strains. In summary, the present invention takes the strain KY834 as the research object, and carries out strain identification and further research on it.

Example 5. Identification of bacterial strain KY834

5.1 Preparation of DNA template

Pick the purified single colony to the bottom of the EP tube, and add 200 μL of 5% (w/v) BT-chelex 100 (distilled water, sterilized at 121° C. for 30 minutes). Cook in a boiling water bath for 15 minutes, quickly freeze at -20°C or -80°C, then thaw at room temperature, centrifuge at 6000r/min for 3min, and take 2μL of the supernatant as a template. The 16S gene was amplified according to the 16S amplification system.

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;

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

5.2 16sDNA sequence determination results of KY834

According to the obtained 16S rDNA sequence (SEQ ID No.1) of KY834, search for homologous sequences in GenBank and conduct homologous sequence analysis and comparison. At the same time, perform sequence comparison with the 16S RNA database (NBCI) approved by the International Bacteriology Committee and combine 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 the strain had a high degree of homology with the strain Paenibacillus terrae, and the similarity was as high as 100%. It was determined that KY834 was Paenibacillus terrae.

菌株KY834的16S rDNA序列测定结果(SEQ ID No.1)如下：GGGTGCCTAATACATGCAAGTCGAGCGGGGTTGTTGTGGAAGCTTGCTTCTACAACAACCTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTATCAGACTGGGATAACTACCGGAAACGGTAGCTAATACCGGATACATCCTTTCCCTGCATGGGGAGGGGAGGAAAGACGGAGCAATCTGTCACTGATGGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGCCAGGGAAGAACGTCTTGTAGAGTAACTGCTACAAGAGTGACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCTCTTTAAGTCTGGTGTTTAATCCCGAGGCTCAACTTCGGGTCGCACTG GAAACTGGGGAGCTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGATATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGGCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTTAACACATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTC TTGACATCCCCCTGATCGGTCTAGAGATAGATCTTTCCTTCGGGACAGGGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGCTTAGTTGCCAGCAGGTCAAGCTGGGCACTCTAAGCAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCCGGTACAACGGGAAGCGAAAGAGCGATCTGGAGCGAATCCTAGAAAAGCCGGTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTACAACACCCGAAGTCGGTGGGGTAACCCGCAAGGGAGCCAGCCGCCCA。

Embodiment 6. Observation of bacterial strain morphology

6.1 Operation

Inoculate the screened strains on the R2A plate, incubate at 30°C for 2 days, and observe the size, shape, color, gloss, viscosity, bulge shape, transparency, edge characteristics, and presence or absence of spores of the colonies.

6.2 Observation results of strain morphology

It was observed that the bacterial strain KY834 (Paenibacillus terrae, Paenibacillus terrae) was cultured and grown on R2A medium for 2 days, and the colony was milky white, round, slightly irregular in shape, flat to low convex, and translucent. The length of the bacterium was measured by a microscope to be about 3.20-4.16 μm (as shown in Figure 2).

Example 7. Comparison with commercial strains

After previous tests, KY834 has been verified to have broad-spectrum killing of plant disease pathogenic bacteria and algae killing ability. In order to better understand the function and application scenarios of KY834, the commercial strain Paenibacillus polymyxa (good disease prevention and high recognition on the market) and the commercial strain DSM7 (Bacillus amyloliquefaciens, used for disease prevention on the market) are set up. For contrast, carry out the comparison of broad-spectrum killing phytopathogenic bacteria activity and algicidal ability; Set commercial bacterial strain 92068 (Bacillus subtilis, commonly used in the market to promote plant growth, disease prevention, dissolving potassium and phosphorus) and commercial bacterial strain DSM7 (amylolytic Bacillus, which is used in the market for disease prevention) was used as a control to compare the multifunctionality (ability to activate silicon).

7.1 Comparison of KY834 and commercial strains for broad-spectrum killing of phytopathogenic bacteria

Set apple spotted leaves, pear black spot, cotton fusarium wilt, watermelon wilt, banana wilt specialization type 1, banana fusarium wilt specialization type 2, banana wilt specialization type 3, wheat root rot, rice sheath blight Blight, wheat sheath blight, gray mold, tomato early blight, cotton Verticillium wilt specialized Vd991, cotton Verticillium wilt specialized OD08047 as indicator bacteria, KY834 as test strain, Paenibacillus polymyxa, DSM7 As a control strain, conduct a broad-spectrum killing pathogenic bacteria test, and refer to "2.1" for specific steps. Cultivate at 30°C for 6 days (of which cotton Verticillium dahliae-specific types Vd991 and OD08047 need to be cultivated for 10 days), observe the strength of the strain’s antagonism against plant disease pathogens, measure the sterilization zone and colony radius, calculate the sterilization intensity, and fill in Table 5.

7.2 Comparison of algicidal ability between KY834 and commercial strains

(1) Strain activation

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

A proper amount of Microcystis aeruginosa was inoculated on solid BG11 medium, and cultured in a light incubator for 15 days.

(2) Vaccination

Inoculate the microorganisms in "(1)" in selective medium B, culture them at 28°C for 6 days, take them out for observation, and compare the sizes of the transparent algicidal circles produced.

7.3 Comparing the ability of KY834 and commercial strains to activate silicon

Silicon is an important element in the process of plant growth. Exploring the ability of KY834 to activate silicon can help solve the problem of nutritional imbalance caused by excessive application of chemical fertilizers and pesticides and realize green production. Therefore, the present invention sets KY834 as the test strain, and 92068 and DSM7 are the control strains, and the activated silicon test is carried out, specifically as follows:

(1) Strain activation

Appropriate amounts of KY834, 92068, and DSM7 were taken out from the -80°C refrigerator, respectively inoculated on solid R2A medium, and cultured at room temperature for 2 days. Among them, KY8334 is the test strain, and 92068 and DSM7 are control strains.

(2) Preparation of silicon-containing solid medium

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

(3) Inoculation

Inoculate the microorganisms in "(1)" into the silicon-containing solid medium described in "(2)", culture them at 30°C for 6 days, take them out for observation, and compare the sizes of the transparent circles produced.

7.4 Results

It can be seen from Table 5 and Figure 3 that, compared with the biocontrol microorganism Bacillus amyloliquefaciens DSM7, which is widely used in the market, KY834 exhibits a broader spectrum of bactericidal activity and excellent bactericidal strength. Compared with Paenibacillus polymyxa, which is an excellent and highly recognized biocontrol microorganism currently on the market, the performance of KY834 is not inferior to Paenibacillus polymyxa. KY834 showed potent fungicidal activity against 14 kinds of plant disease pathogens including apple leaf spot and pear black spot.

It can be seen from Table 6 and Figure 4 that on the algae-containing medium, obvious transparent algicidal circles can be formed near the colonies of KY834 and Paenibacillus polymyxa, and no transparent algicidal circles are formed near the colonies of DSM7. At the same time, the algicidal zone produced by KY834 was significantly larger than that produced by Paenibacillus polymyxa, and the algicidal intensity of KY834 was also significantly stronger than that of Paenibacillus polymyxa. Judging from this, KY834 has algicidal ability and the algicidal ability is not inferior to Paenibacillus polymyxa.

From Table 7 and Figure 5, it can be seen that on the silicon-containing medium, a transparent circle can be formed near the KY834 colony, but there is no transparent circle near the 92068 and DSM7 colonies. KY834 has the ability to activate silicon.

Table 5. Comparison results of KY834, DSM7, and Paenibacillus polymyxa broad-spectrum killing plant disease pathogens

(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" means that the bacterial strain has no bactericidal activity against the pathogenic bacteria of the corresponding disease).

Table 6. Algicidal zone strength of KY834, Paenibacillus polymyxa, DSM7

It was observed that the bacterial strain KY834 (Paenibacillus terrae, Paenibacillus terrae) was cultured and grown on R2A medium for 2 days, and the colony was milky white, round, slightly irregular in shape, flat to low convex, and translucent. The length of the bacterium was measured by a microscope to be about 3.20-4.16 μm (as shown in Figure 2).

Table 7. Comparison results of KY834, 92068 and DSM7 in activating silicon

(Note: "0" indicates that the microorganism cannot produce transparent circles in silicon-containing solid medium, and does not have the ability to activate silicon; "+" indicates that the microorganism can produce transparent circles in silicon-containing solid medium, and has the ability to activate silicon. ability, but the ability to activate silicon is average; "++" means that the microorganism can produce transparent circles in silicon-containing solid medium, and has the ability to activate silicon and has a good ability to activate silicon.)

in conclusion:

The present invention isolates a microorganism KY834 capable of killing algae and preventing diseases (specifically referring to apple leaf spot and pear black spot) from soil collected from all over the country (nearly 35,000 strains) through a high-throughput screening method. In addition, through further tests, the present invention has discovered and confirmed that KY834 also has the function of broad-spectrum killing plant disease pathogenic bacteria and activating silicon. The results of 16srDNA sequence determination of the strain showed that the strain was highly homologous to Paenibacillus terrae. By comparing with Paenibacillus polymyxa (good disease prevention and high recognition on the market) and the commercial strain DSM7 (Bacillus amyloliquefaciens, used for disease prevention on the market), KY834 is effective against 14 kinds of plant disease pathogens (including apple leaf spot). disease, pear black spot, cotton fusarium wilt, watermelon blight, banana wilt three specialized types, wheat root rot, rice sheath blight, wheat sheath blight, gray mold, tomato early blight and cotton verticillium wilt Disease two specialized types) showed strong fungicidal activity and excellent algicidal ability. By comparing with 92068 (Bacillus subtilis, commonly used in the market to promote plant growth, disease prevention, potassium and phosphorus dissolution) and commercial strain DSM7 (Bacillus amyloliquefaciens, used in the market for disease prevention), the multifunctional (activated silicon Ability) comparison, it is found that KY834 has the ability to activate silicon, which is beneficial to the growth and development of crops.

In summary, KY834 is a functional microorganism with broad-spectrum killing of phytopathogenic bacteria, high-efficiency algae killing, and silicon activation ability, and has great application potential in agricultural production and biological control (especially fruit tree disease control).

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; 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: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for 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 technical solutions of the various embodiments of the present invention. scope.

Claims (10)

1. The multifunctional Paenibacillus terrae strain is characterized in that the multifunctional Paenibacillus terrae strain is a Paenibacillus terrae (Paenibacillus terrae) strain KY834 and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.24708.

2. Microbial agent, characterized in that it contains the fermentation broth, bacterial suspension and/or supernatant of Paenibacillus terreus (Paenibacillus terrae) strain KY834 as defined in claim 1.

3. Biofertilizer characterized in that it contains Paenibacillus terrestris (Paenibacillus terrae) strain KY834 or a metabolite thereof according to claim 1.

4. Use of the Paenibacillus terrestris (Paenibacillus terrae) strain KY834 as claimed in claim 1, the microbial inoculant as claimed in claim 2 or the biofertilizer as claimed in claim 3 in one or more of the following aspects:

(a) Inhibiting plant pathogenic bacteria or preventing and treating plant diseases;

(b) Killing algae;

(c) And activating the silicon.

5. Use according to claim 4, characterized in that the plant diseases comprise apple defoliation and/or pear black spot.

6. The use of claim 5, wherein the plant disease further comprises 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.

7. Use according to claim 4, wherein the algae comprise blue-green algae, preferably microcystis aeruginosa.

8. The use according to claim 4, wherein the plant comprises a vegetable, food crop or fruit tree; preferably fruit trees.

9. A method for growing fruit trees to control pathogenic bacteria and algae hazards, comprising using Paenibacillus terreus strain KY834 of claim 1, microbial agent of claim 2 or biofertilizer of claim 3 in growing fruit trees.

10. The planting method according to claim 9, wherein the method comprises applying fermentation liquor, bacterial suspension, supernatant, microbial agent or biological fertilizer containing the strain KY834 to rhizosphere soil of fruit tree plants.

Images