Bacillus subtillis ZKY07 for preventing and controlling bacterial diseases of pachyrhizua angulatus and application of bacillus subtillis ZKY07

Abstract

The invention discloses bacillus subtillis ZKY07 for preventing and controlling bacterial diseases of pachyrhizua angulatus and application of the bacillus subtillis ZKY07. According to the invention, a strain ZKY07 which has an antagonistic effect on two pathogenic bacteria of the Pachyrhizua angulatus bacterial wilt is screened from strains separated from dendrobium leaves and is used for biological control of the Pachyrhizua angulatus bacterial wilt, and the preservation number of bacillus subtillis ZKY07 is GDMCC No: 64618. Through determination, the ZKY07 has a good antagonistic effect on ralstonia solanacearum causing pogostemon cablin bacterial wilt, ralstonia solanacearum causing peanut bacterial wilt, ralstonia solanacearum causing tomato bacterial wilt, Klebsiella variicola causing banana sheath rot, fusarium asiaticum causing peanut fruit rot and sclerotium rolfsii causing peanut southern blight. The strain ZKY07 has the effects of producing protease, producing cellulase, dissolving potassium, dissolving organophosphorus and fixing nitrogen. The invention provides an excellent biological control means for the bacterial wilt of the pueraria thomsonii.

Description

Technical Field

[0001] The invention belongs to the field of biological pesticides and relates to a Bacillus subtilis ZKY07 for preventing and controlling kudzu vine bacterial diseases and an application thereof. Background Art

[0002] Pueraria lobata is the general name for plants of the genus Pueraria in the Leguminosae family, and Pueraria root is its tuberous root. There are about 20 species of Pueraria plants worldwide, mainly distributed in tropical, subtropical and temperate regions, and distributed in Guangdong, Guangxi, Sichuan, Yunnan and other provinces in China. According to the Flora of China, China's Pueraria plants include 8 species and 2 varieties, among which wild Pueraria (Pueraria lobata) and its variety Pueraria lobata var.thomsoni are the most commonly used species in my country. They are both included in the catalog of medicinal and edible plants approved by the National Health Commission, and are known as "millennium ginseng" and "plant gold". The main part of Pueraria lobata is the tuberous root. The isoflavone compounds such as puerarin, daidzein, daidzin, genistein, etc. in Pueraria root are effective medicinal ingredients, which have the effects of lowering blood pressure, lowering blood sugar, clearing heat, detoxifying, and anti-oxidation. Pueraria lobata prefers a warm and humid environment, and its stems, leaves and roots can grow rapidly at a temperature of 20℃ to 30℃. However, with the large-scale production of kudzu, pests and diseases that affect kudzu production have also occurred. According to reports, kudzu rust, kudzu pseudorust and kudzu root rot are the main diseases of kudzu. During a survey in Zhushan Village, Fogang County, Qingyuan City, Guangdong Province, a new bacterial disease of kudzu was discovered. It mainly harms the stem base and leaves. The symptoms are browning of leaf veins, rot and browning of the stem base, and in severe cases, cracking. Root nutrients cannot be transported to the branches above the ground, and the whole kudzu plant begins to wither and die. It has a great impact on the production and quality of kudzu, and it is named kudzu bacterial wilt. There are currently no reports on prevention and control measures for this disease.

[0003] Biological control, chemical control, and screening of resistant varieties are the main means used in my country to prevent and control bacterial plant diseases. Chemical control is difficult to work when the disease breaks out in large numbers and has great pollution to the environment. The process of screening resistant varieties is long and long-term cultivation will lead to plant mutations, creating favorable conditions for the proliferation and accumulation of pathogens, and ultimately leading to the loss of variety resistance. Biological control is an efficient and environmentally friendly prevention and control method that can effectively prevent large-scale outbreaks of diseases before they occur, and effectively avoid environmental pollution and the development of pathogen resistance. Bacillus has become an excellent material for biological control due to its antagonistic activity against plant pathogens, good colonization ability, and harmlessness to the environment. Microbial agents made from Bacillus have been used in major diseases of crops such as bananas, rice, and peanuts, and have achieved good prevention effects. However, no effective biological control methods have been reported for bacterial wilt of kudzu. The development of green and efficient biocontrol bacteria to prevent bacterial wilt of kudzu has become an important issue to ensure the healthy development of the kudzu industry. Summary of the invention

[0004] The invention aims to provide a Bacillus subtilis ZKY07 for preventing and controlling bacterial diseases of Pueraria lobata and application thereof.

[0005] The Bacillus subtilis ZKY07 for preventing and controlling bacterial diseases of kudzu vine of the present invention is deposited in the Guangdong Microbial Culture Collection Center (GDMCC), the address of which is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, the deposit date is May 13, 2024, and the deposit number is GDMCC No: 64618.

[0006] The second object of the present invention is to provide a bacterial agent, which includes the above-mentioned Bacillus subtilis ZKY07.

[0007] The third object of the present invention is to provide the use of the above-mentioned Bacillus subtilis ZKY07 or the above-mentioned bacterial agent in the preparation of a preparation for inhibiting Ralstonia solanacearum, Klebsiella variegata, Fusarium asiatica, Sclerotium uniformis, Pseudomonas oryzae and / or Pantoea dispersa.

[0008] Preferably, the invention is used in the preparation of a composition for treating patchouli bacterial wilt, peanut bacterial wilt, tomato bacterial wilt, banana sheath rot, peanut fruit rot, peanut white rot, banana wilt, banana soft rot and / or kudzu bacterial wilt.

[0009] The present invention also provides a method for preventing and controlling patchouli bacterial wilt, peanut bacterial wilt, tomato bacterial wilt, banana sheath rot, peanut fruit rot, peanut white rot, banana wilt, banana soft rot and / or kudzu bacterial wilt, which uses the above-mentioned Bacillus subtilis ZKY07 for prevention and control.

[0010] Preferably, the bacterial solution of Bacillus subtilis ZKY07 is inoculated into the root system of the corresponding plant or the surrounding soil.

[0011] The plants are patchouli, peanut, tomato, banana and kudzu.

[0012] The present invention collects rotten samples of kudzu from Zhushan Village, Fogang County, Qingyuan City, Guangdong Province for separation, and obtains strains FG5-1 and y1, which are pathogens verified by Koch's postulates, and the two pathogens are identified as pseudomonas oryzihabitans and Pantoea dispersa. From the strains isolated from the leaves of Dendrobium orchid, a strain ZKY07 having antagonistic effects on two pathogens of bacterial wilt of kudzu is screened out for biological control thereof. It is determined that ZKY07 has good antagonism against Ralstonia solanacearum causing patchouli bacterial wilt, Ralstonia solanacearum causing peanut bacterial wilt, Ralstonia solanacearum causing tomato bacterial wilt, Klebsiella variegata causing banana sheath rot, Fusarium asiatica causing peanut fruit rot, and Sclerotium uniformis causing peanut white rot. The strain is identified as Bacillus subtilis by ANI average nucleotide consistency comparison using the whole genome sequence, and is named ZKY07. In a potted plant experiment, the strain had a control effect of 72.96% on banana varieties infected with banana wilt and banana soft rot, which could effectively reduce the occurrence of banana compound diseases and increase banana yield. In addition, compared with the blank control group, plant height, stem girth and aboveground fresh weight were significantly increased after the strain was applied. The physical and chemical properties of the strain were determined, and it was found that the strain had the functions of producing protease, producing cellulase, decomposing potassium, decomposing organic phosphorus and fixing nitrogen. The present invention provides an excellent biological control method for kudzu bacterial wilt.

[0013] Collection Instructions

[0014] Bacillus subtilis ZKY07 is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), the address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, the deposit date is May 13, 2024, and the deposit number is GDMCC No: 64618. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the colony morphology of strain ZKY07;

[0016] Figure 2 is the biofilm morphology of strain ZKY07;

[0017] Figure 3 This is the identification result of strain ZKY07;

[0018] Figure 4 This is the result of back-inoculation of strains FG5-1 and y1;

[0019] Figure 5The antagonistic effect diagram of ZKY07 on the pathogen of kudzu vine bacterial wilt; A, the antagonistic effect diagram of strain ZKY07 on kudzu vine pathogen Pseudomonas oryzae FG5-1, B, the antagonistic effect diagram of strain ZKY07 on pathogen Pantoea dispersa y1;

[0020] Figure 6 This is the potted plant control effect of ZKY07 on banana wilt and banana soft rot compound diseases. A is the application of banana wilt and banana soft rot pathogens only, B is the application of ZKY07 and then banana wilt and banana soft rot pathogens;

[0021] Figure 7 is the change in ET levels in plants caused by the application of ZKY07;

[0022] Figure 8 is the change in plant BR levels caused by the application of ZKY07;

[0023] Fig. 9 It is the change of SA level in plants caused by the application of ZKY07;

[0024] Fig.10 It is the change of JA level in plants caused by the application of ZKY07. DETAILED DESCRIPTION

[0025] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0026] Embodiment 1:

[0027] (1) Isolation of strains

[0028] The diseased and healthy junctions of the rotten kudzu samples were completely cut off and cut into 0.5×0.5 cm diseased tissues and immersed in 75% alcohol for 15 seconds. Then, they were moved into 2.5% sodium hypochlorite for surface disinfection for 10 seconds, rinsed with sterile water for 4 times, placed on sterile filter paper to dry, and inoculated with NA + Plate. Place at 28℃ for 3-5 days and then transfer the grown bacteria to NA － After multiple purifications, strains FG5-1 and y1 were obtained.

[0029] (2) Inoculation and identification of pathogens

[0030] Single colonies of strains FG5-1 and y1 were picked up in 5mL LB, cultured at 37℃ and 180rpm overnight, and then transferred to 50mL LB with a 1% inoculation amount. After cultured at 37℃ and 180rpm overnight, the supernatant was discarded by centrifugation, and the bacteria were resuspended with sterile water, and the OD was adjusted to 1. The leaves and kudzu heads were selected for back-inoculation. Three leaves of equal size and similar health conditions were taken. After wiping the surface of the leaves with 75% alcohol, a sterile needle was used to stab along the veins, and finally 50μL of bacterial solution was dripped into the puncture site. The leaves were placed in a seedling box, cultured at 28℃, and observed after 48h. Three sections of kudzu heads of equal size and similar health conditions were taken, and the surface was wiped with 75% alcohol. After drying, 50μL of bacterial solution was dripped into the vascular part of the kudzu head, cultured at 28℃, and observed after 48h. According to the experimental results, the results of the grafting of FG5-1 and y1 were similar to the symptoms of Pueraria lobata in the field. The leaves turned black and brown along the veins, and the center of the Pueraria lobata head turned black. After separating the diseased tissues after grafting, two strains, FG5-1 and y1, were obtained. FG5-1 and y1 were determined to be the pathogens of Pueraria lobata bacterial wilt. The bacteria were sent for sequencing to obtain the whole genome sequence, and the strains FG5-1 and y1 were identified as pseudomonasoryzihabitans and Pantoea dispersa. Figure 4 This is the result of back-inoculation of strains FG5-1 and y1.

[0031] (3) Screening of strain ZKY07 with antibacterial effect on pathogens using plate antagonism method

[0032] From the strains isolated from the leaves of 52 Dendrobium orchids, strains that were antagonistic to both FG5-1 and y1, the pathogens of fusarium wilt of Pueraria lobata, were screened. Single colonies were picked into 5 mL LB medium to prepare the secondary bacterial culture. After overnight culture, the third bacterial culture was prepared at a 1% inoculation rate and cultured for 4 hours. The strains FG5-1 and y1 were cultured for 24 hours and adjusted to OD 600 =1.0 and then diluted 3 times, 100 μL of the diluted bacterial solution was spread on LB medium, and 10 μL of the test bacterial solution was spotted 2.5 cm away from the center of the plate. Each plate was replicated 4 times, biologically replicated 3 times, and a blank control was set. After 48 hours of culture, observation was made. The results showed ( Figure 5 ), the average antagonistic transparent zone of strain ZKY07 to pathogen FG5-1 was 0.394 cm, and the average antagonistic transparent zone to pathogen y1 was 0.286 cm.

[0033] (4) Determination of some physical and chemical properties of ZKY07

[0034] Prepare the following culture medium, pick strain ZKY07 into 5mL LB culture medium to make the second-level bacterial solution, and make the third-level bacterial solution at a volume ratio of 1% after overnight culture. After continuing to culture for 4 hours, take 10μL of bacterial solution and inoculate it at 2.5cm from the center of the plate. Repeat four times. After culturing at 28℃ for 5 days, observe whether a transparent circle is formed around the bacterial solution. The formation of a transparent circle indicates that the strain has the corresponding physical and chemical properties.

[0035] Protease-producing strain screening culture medium: A: 6.4 g skim milk powder, dissolved in 240 mL of water; B: 6.4 g agar, fixed to 240 mL, A and B are sterilized separately and then mixed for use.

[0036] Cellulase-producing strain screening culture medium: peptone 10 g, yeast powder 10 g, sodium carboxymethyl cellulose 10 g, sodium chloride 5 g, potassium dihydrogen phosphate 1 g, Congo red 0.2 g, agar 18 g, deionized water 1000 mL, sterilize and set aside.

[0037] Potassium-solubilizing strain screening culture medium: sucrose 10g, magnesium sulfate heptahydrate 0.5g, disodium hydrogen phosphate 2g, sodium chloride 0.1g, yeast powder 0.5g, ammonium sulfate 1g, potassium feldspar 10g, agar 15g, deionized water 1000mL, sterilize and set aside.

[0038] Inorganic phosphorus-dissolving strain screening medium: 10g glucose, 0.5g ammonium sulfate, 0.3g sodium chloride, 0.3g magnesium sulfate, 0.03g manganese sulfate, 0.3g potassium sulfate, 0.03g ferrous sulfate, 5.0g calcium phosphate, 15g agar, 1000mL deionized water, sterilize and set aside.

[0039] Organophosphorus-dissolving strain screening medium: 10g glucose, 0.5g ammonium sulfate, 0.5g yeast extract powder, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, 0.2g lecithin, 1g calcium carbonate, 1000mL deionized water, sterilized for later use.

[0040] Nitrogen-fixing strain screening medium: 0.2g potassium dihydrogen phosphate, 0.2g magnesium sulfate, 0.2g sodium chloride, 5g calcium carbonate, 10g mannitol, 0.1g calcium sulfate, 15g agar, 1000mL deionized water, sterilized for later use.

[0041] The test results are shown in Table 1.

[0042] Table 1 Determination of some physical and chemical properties of ZKY07

[0043]

[0044] Note: “-” indicates negative, and “+” indicates positive.

[0045] The colony morphology of strain ZKY07 is as follows Figure 1 As shown, Figure 2 This is the biofilm morphology of strain ZKY07.

[0046] (5) Identification of strain species using whole genome sequences

[0047] The whole genome DNA of the strain was obtained using a bacterial whole genome DNA extraction kit, and the third-generation Nanopore sequencing was performed to obtain the whole genome sequence. The whole genome sequence similar to the strain was obtained by comparison on NCBI, and the average nucleotide consistency analysis of the strain gene sequence was performed using fastANI software to obtain the ANI value between strains. The clustering heat map was drawn online using chiplot, and ZKY07 was identified as Bacillus subtilis (such as Figure 3 It was named Bacillus subtilis ZKY07 and deposited in Guangdong Microbiological Culture Collection Center (GDMCC), the address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, the deposit date is May 13, 2024, and the deposit number is GDMCC No: 64618.

[0048] (6) Antagonism test of Bacillus subtilis ZKY07 against different pathogenic fungi and bacteria

[0049] The method was the same as (3), wherein the pathogens tested included Ralstonia solanacearum, which causes patchouli bacterial wilt, Ralstonia solanacearum, which causes peanut bacterial wilt, Ralstonia solanacearum, which causes tomato bacterial wilt, Klebsiella variegata, which causes banana sheath rot, Fusarium asiatica, which causes peanut fruit rot, and Sclerotium sclerotiorum, which causes peanut white rot. The inhibition rate of pathogenic fungi and the clear zone radius of pathogenic bacteria were recorded, and variance analysis was performed using SPSS (as shown in Tables 2 and 3).

[0050] Table 2 Inhibition rate of Bacillus subtilis ZKY07 against pathogenic fungi

[0051]

[0052] Table 3 Transparent zone radius of the inhibitory effect of Bacillus subtilis ZKY07 on pathogenic bacteria

[0053]

[0054] (7) The root inoculation method was used to evaluate the potted plant protection effect. Bacillus subtilis ZKY07 was diluted to 1×10 8 CFU / mL, the banana soft rot pathogen Dickinia MS2 was diluted to 1×10 6CFU / mL, the banana wilt pathogen Fusarium oxysporum FOC009 was diluted to 1×10 6 spores / mL. 100 mL of diluted ZKY07 solution was inoculated at the root of banana seedlings, and diluted Dickie's bacteria solution and Fusarium oxysporum solution were inoculated one week later. A blank control (no Bacillus subtilis ZKY07 solution was inoculated, but water was inoculated) was set up to calculate the biocontrol effect and growth promotion effect (such as Figure 6 , as shown in Tables 4 and 5).

[0055] Table 4 Biocontrol effect after ZKY07 treatment

[0056]

[0057] Table 5 Growth-promoting effect after ZKY07 treatment

[0058]

[0059] The root inoculation method was used to determine the level of plant immune activity changes. The banana seedlings were inoculated at the root, and the experiment set up a water control group (CK) and three treatment groups. FOC treatment: inoculate 50mL of 1.0×10 6 cfu / mL strain FOC004; ZKY07 treatment (R13): inoculate 50mL with a concentration of 1.0×10 8 Strain ZKY07; FOC and ZKY07 treatment (FOC+R13): 50 mL of the culture medium was inoculated 24 h in advance with a concentration of 1.0×10 8 cfu / mL strain ZKY07 and then inoculated with 50 mL of 1.0×10 6 cfu / mL strain FOC004. The changes in ET, BR, SA, and JA hormone levels were measured at 8h, 24h, 48h, and 72h. Figure 7 , 8, 9, and 10). After inoculation with strain ZKY07, the ET and SA hormone levels of banana plants increased rapidly within 48 hours. SA is a low-content plant endogenous immune signal in plants. It accumulates in large quantities when infected by pathogens. After pathogens infect plants, cells quickly synthesize SA, inducing a series of defense reactions, such as the activation of phenylalanine aminase (PAL), polyphenol oxidase (PPO), peroxidase (POD), and the expression of pathogenesis-related proteins (PRS). The increase in SA content may activate the plant's systemic acquired resistance (SAR), that is, the resistance produced by the whole plant when the plant is infected by pathogens. This resistance can improve defense against a variety of pathogens, nematodes, and even parasitic plants. The increase in ET levels indicates that the plant's induced systemic resistance (ISR) may be aroused, and ISR can activate the corresponding cellular defense response before or after the pathogen attacks the host. The activation of ISR will enhance the expression of defense genes, thereby increasing the production and accumulation of related defense proteins and secondary metabolites. After co-treatment with strain ZKY07 and pathogen FOC, the BR hormone level at 72 hours and the JA hormone level at 48 hours were significantly increased compared with other treatments. This indicates that under the co-treatment of biocontrol bacteria and pathogens, the plants will show a stronger immune response to resist the invasion of pathogens and form immune memory, so that they can respond faster when the pathogens invade next time.

Claims

1. Bacillus subtilis ZKY07, deposited with GDMCC No: 64618.

2. A bacterial agent, characterized in that Comprising the Bacillus subtilis ZKY07 according to claim 1.

3. Use of the Bacillus subtilis ZKY07 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a preparation for inhibiting Ralstonia solanacearum, Klebsiella variegata, Fusarium asiatica, Sclerotium uniformis, Pseudomonas oryzae and / or Pantoea dispersa.

4. The use according to claim 3, characterized in that: The invention is used in the preparation of a drug for treating patchouli bacterial wilt, peanut bacterial wilt, tomato bacterial wilt, banana sheath rot, peanut fruit rot, peanut white rot, banana wilt, banana soft rot and / or kudzu bacterial wilt.

5. A method for preventing and controlling patchouli bacterial wilt, peanut bacterial wilt, tomato bacterial wilt, banana sheath rot, peanut fruit rot, peanut white rot, banana wilt, banana soft rot and / or kudzu bacterial wilt, characterized in that: The method comprises using the Bacillus subtilis ZKY07 described in claim 1 for prevention and control.

6. The method according to claim 5, characterized in that The method is to inoculate the bacterial solution of Bacillus subtilis ZKY07 into the root system of the corresponding plant or the surrounding soil.

7. The method according to claim 6, characterized in that The plants are patchouli, peanut, tomato, banana and kudzu.

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