Bacillus velezensis ZKY08 for preventing composite infection of banana wilt and soft rot and application of bacillus velezensis ZKY08

Abstract

The invention discloses bacillus velezensis ZKY08 for preventing composite infection of banana wilt and soft rot and application of the bacillus velezensis ZKY08. The Bacillus velezensis ZKY08 is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on July 8, 2024, the address is the fifth floor, the building 59, the No.100, the Xianlie Middle Road, the Xiaoxian District, Guangzhou City, Guangdong Province, the postal code is 510070, and the preservation number is GDMCC No: 64844. The bacillus velezensis ZKY08 disclosed by the invention can be used for effectively preventing and controlling composite diseases of banana soft rot and banana fusarium wilt, the prevention and control effect can reach 64.7%, and the bacillus velezensis ZKY08 has the effects of increasing the plant height, stem girth and overground fresh weight of bananas after being applied. The microbial agent can effectively promote banana growth while preventing and controlling diseases in a green manner, and is a biological prevention and control microbial agent with a good development prospect.

Description

Technical Field

[0001] The invention belongs to the field of biological pesticides and relates to a strain of Bacillus velez ZKY08 for preventing and controlling a combined disease of banana soft rot and banana wilt and an application thereof. Background Art

[0002] Banana (Musa spp.) is one of the four major fruits in the world. Its production areas are widely distributed around the world. Bananas are grown in about 127 countries and regions, and China is one of the oldest countries in the world to cultivate bananas. Banana is a Lingnan fruit with distinctive varieties, obvious regional advantages, a long planting history, and great development potential in Guangdong Province. Guangdong Province ranks first in the country in banana production and is the largest banana producing area in my country. Banana wilt is a devastating soil-borne disease. The disease is severe in traditional banana planting areas in Guangdong Province. At present, the banana wilt pathogen has evolved, and traditional prevention and control measures have little effect. Now traditional banana orchards have begun to be infected with banana soft rot and banana wilt. The incidence rate in some orchards even exceeds 50%, causing devastating disasters. As a result, Foshan City, Enping City, Yangjiang City, Zhaoqing City and other places have seen a trend of declining planting area and yield. At present, the prevention and control of banana diseases mainly focuses on the selection and breeding of disease-resistant varieties, chemical control and biological control. Due to defects such as high mutation rate and unstable disease resistance, it is very difficult to make breakthroughs in the selection and breeding of disease-resistant varieties. Chemical control is not ideal for banana wilt disease, and there are pesticide residue problems. As people's awareness of food safety and environmental protection increases, we have begun to study the use of biological control to replace chemical control. It has been reported that many microbial antagonists have antagonistic activity against plant pathogens. Endophytes are known as the second line of defense for plants. They are a type of microorganisms that live in healthy plant tissues and do not cause obvious symptoms of disease. Studies have found that endophytes can not only enhance plant resistance to diseases and pests, but also enhance the host plant's resistance to environmental stress. Bacillus is widely present in plants and in the rhizosphere. It has been developed into a variety of biocontrol products for production practice, causing good stress resistance and is an excellent commercial biocontrol species. Summary of the invention

[0003] The invention aims to provide a strain of Bacillus Velezii and use the strain in preventing and controlling the combined diseases of banana soft rot and banana wilt.

[0004] The Bacillus velezensis ZKY08 of the present invention was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on July 8, 2024, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, Deposit Number: GDMCC No: 64844.

[0005] The invention finds through experiments that Bacillus velezensis ZKY08 has the functions of producing protease, producing cellulase, decomposing potassium, decomposing inorganic phosphorus, decomposing organic phosphorus and fixing nitrogen.

[0006] The present invention also provides application of the Bacillus Velezii ZKY08 in biological control.

[0007] Preferably, Bacillus velezensis ZKY08 is used to inhibit Ralstonia solanacearum, Klebsiella variegata, Fusarium asiatica, Sclerotium truncatum, Cladosporium, Aspergillus niger, Dickie's bacillus, and Fusarium oxysporum.

[0008] Preferably, Bacillus velezensis ZKY08 is used for improving plant immune activity.

[0009] Preferably, Bacillus Velez ZKY08 is used for preventing and controlling banana soft rot or banana wilt or their combined diseases.

[0010] Preferably, the Ralstonia solanacearum is the Ralstonia solanacearum that causes patchouli bacterial wilt, the Ralstonia solanacearum that causes peanut bacterial wilt, and the Ralstonia solanacearum that causes tomato bacterial wilt; the Klebsiella variegata is the Klebsiella variegata that causes banana sheath rot; the Fusarium asiatica is the Fusarium asiatica that causes peanut fruit rot, the Sclerotium solani is the Sclerotium solani that causes peanut white rot, the Cladosporium is the Cladosporium that causes blueberry gray mold, and the Aspergillus niger is the Aspergillus niger that causes peanut stem rot.

[0011] The present invention also provides a biocontrol preparation, which comprises the above-mentioned Bacillus Velezii ZKY08 as an active ingredient.

[0012] Beneficial effects:

[0013] The Velez's bacillus ZKY08 discovered by the present invention can effectively prevent and control banana soft rot and banana wilt combined diseases, with a control effect of up to 64.7%, and has the effect of increasing banana plant height, stem girth, and aboveground fresh weight after application. It can effectively promote banana growth while greenly preventing and controlling diseases, and is a biological control agent with good development prospects.

[0014] Bacillus velezensisZKY08 was deposited on July 8, 2024 in the Guangdong Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, Deposit Number: GDMCC No: 64844. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the colony morphology of strain ZKY08;

[0016] Figure 2 It is the biofilm morphology of strain ZKY08;

[0017] Figure 3 This is the identification result of strain ZKY08;

[0018] Figure 4 The diagram shows the antagonistic effect of ZKY08 on banana pathogens; A, the antagonistic effect of ZKY08 on FOC004; B, the antagonistic effect of ZKY08 on FOC009; C, the antagonistic effect of ZKY08 on MS2;

[0019] Figure 5 This is a potted plant control effect diagram of ZKY08 on the combined disease of banana soft rot and banana wilt; A only applied banana soft rot and banana wilt pathogens; B applied ZKY08 and then banana soft rot and banana wilt pathogens;

[0020] Figure 6 This is the liquid chromatogram of lipopeptide substances of strain ZKY08;

[0021] Figure 7 It is the activity determination of components 1 and 2 of lipopeptide substances of strain ZKY08 against MS2;

[0022] Figure 8 The antagonistic effect of the bacterial liquid and supernatant of strain ZKY08 on MS2; A, ZKY08 bacterial liquid, B, ZKY08 supernatant;

[0023] Fig. 9 It is the color change result of the supernatant of strain ZKY08 at different times after co-culture with CV017 for 12 h;

[0024] Fig.10 This is the field situation after applying ZKY08;

[0025] Fig.11 This is the field situation without ZKY08 application;

[0026] Fig.12 Damage caused by diseased plants in the field;

[0027] Fig.13 It is the change of BR level in plants caused by the application of ZKY08;

[0028] Fig.14 is the change in SA level in plants caused by the application of ZKY08;

[0029] Fig.15 It is the change of JA level in plants caused by the application of ZKY08. DETAILED DESCRIPTION

[0030] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0031] Embodiment 1:

[0032] 1. Use the plate antagonism method to screen the strain ZKY08 that has an inhibitory effect on pathogens

[0033] From the strains isolated from the leaves of 52 Dendrobium orchids, a strain ZKY08 was screened that was antagonistic to both banana wilt pathogens and banana soft rot pathogens. The colony morphology of strain ZKY08 was as follows: Figure 1 As shown, the biofilm morphology of strain ZKY08 is as follows Figure 2 As shown. PDA and LB mixed culture medium was used to screen banana wilt pathogens Fusarium oxysporum FOC004 and Fusarium oxysporum FOC009, and CPG culture medium was used to screen banana soft rot pathogen Dirketia MS2. A single colony was picked into 5 mL LB culture medium to prepare a secondary bacterial culture. After overnight culture at 37°C and 180 rpm, the culture was continued for 4 hours at a 1% inoculation rate to prepare a tertiary bacterial culture. Take a 5 mm diameter block of Fusarium oxysporum FOC004 and FOC009 in the middle of the plate, and take 10 μL of the bacterial solution and spot it 2.5 cm away from the center of the plate. Shake Dirketia MS2 to OD 600 =1 and then diluted 10 times, 100μL of the diluted MS2 bacterial solution was spread on the culture medium, and 10μL of the bacterial solution was spotted 2.5cm away from the center of the plate. Each plate was replicated 4 times, biologically replicated 3 times, and a blank control was set. Fusarium oxysporum was cultured at 25℃ for 7d and Dickie's bacteria was cultured at 28℃ for 48h. The results showed that the inhibition rate of ZKY08 on FOC004 reached 71.4%, the inhibition rate on FOC009 reached 75.3%, and the radius of the transparent circle of inhibition on MS2 was 0.31cm (such as Figure 4 shown).

[0034] 2. Determination of some physical and chemical properties of ZKY08

[0035] Prepare the following culture medium, pick strain ZKY08 into 5mL LB culture medium to make secondary bacterial liquid, culture at 37℃, 180rpm overnight and make tertiary bacterial liquid at 1% volume ratio, continue to culture for 4h, take 10μL bacterial liquid and inoculate it at 2.5cm from the center of the plate, repeat four times, culture at 28℃ for 5d and observe whether a transparent circle is formed around the bacterial liquid. The formation of a transparent circle indicates that the strain has the corresponding physical and chemical properties.

[0036] 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.

[0037] Cellulase-producing strain screening culture medium: peptone 10g, yeast powder 10g, sodium carboxymethyl cellulose 10g, sodium chloride 5g, potassium dihydrogen phosphate 1g, Congo red 0.2g, agar 18g, deionized water 1000mL, mix and sterilize for use.

[0038] 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, mix and sterilize for later use.

[0039] Inorganic phosphorus-dissolving strain screening culture 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, mix and sterilize for later use.

[0040] 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, mix and sterilize for later use.

[0041] Nitrogen-fixing strain screening medium: 0.2 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, 0.2 g sodium chloride, 5 g calcium carbonate, 10 g mannitol, 0.1 g calcium sulfate, 15 g agar, 1000 mL deionized water, mix and sterilize for later use.

[0042] The test results are shown in Table 1.

[0043] Table 1 Determination of some physical and chemical properties of ZKY08

[0044]

[0045] Note: “-” indicates negative, and “+” indicates positive.

[0046] 3. Identify strain species using whole genome sequences

[0047] The whole genome DNA of strain ZKY08 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. Chiplot was used to draw a cluster heat map online, and ZKY08 was identified as Bacillus velezsis (such as Figure 3 shown).

[0048] The strain ZKY08 was named Bacillus velezensis ZKY08 and was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on July 8, 2024. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, Deposit Number: GDMCC No: 64844.

[0049] 4. Use AHLs signal degradation indicator bacteria CV017 to verify that strain ZKY08 has a quenching effect on the pathogen MS2.

[0050] Activate ZKY08 strain on NB plate and place incubator at 37℃ for 12h. Select single colony and inoculate into 5mL NB liquid culture medium, incubate at 37℃ and 180rpm for 12h to obtain secondary bacterial solution. Inoculate the secondary bacterial solution into 200mL NB and Landy mixed liquid culture medium (NB and Landy mixed culture medium formula is as follows: glucose 20g, NaCl 5g, beef extract 15g, sodium glutamate 5g, KH2PO4 2g, MgSO4·7H2O 1g, KCl 1g, FeSO4 0.3mg, MnSO410mg, CuSO40.32mg, deionized water 1000mL, pH=7) at 1% inoculation amount, incubate at 37℃ and 180rpm for 72h to obtain fermentation liquid. Centrifuge it at 8000rpm for 8min, collect the supernatant and add HCL to adjust the pH value to 2.0. After being placed in a refrigerator at 4°C overnight, the mixture was centrifuged at 9000rpm for 15min and the precipitate was collected and placed in an oven at 30°C to dry. 100mL of chromatographic grade methanol was added to each 3L fermentation broth to dissolve the precipitate and centrifuged to obtain the supernatant after magnetic stirring for 2h, which was filtered with a 0.22μm organic phase filter. The methanol solution was evaporated to dryness using a rotary evaporator to obtain lipopeptides, which were placed in a refrigerator at 4°C for standby use. The lipopeptides were separated using RP-HPLC under the following conditions: acetonitrile (B, 0.1% TFA)-water (A, 0.1% TFA) as the mobile phase, a flow rate of 1mL / min, a detection wavelength of 210nm, a column temperature of 28°C, a single injection volume of 20μL, and the program settings are shown in Table 2. The LB plate was used to test whether the separated different lipopeptides had antagonistic activity against the pathogen MS2. Place a 6mm filter paper at 2.5cm from the center of the plate, repeat four times, and observe whether a transparent circle is formed around the filter paper after culturing at 28℃ for 48h. The formation of a transparent circle indicates that the component has antagonistic activity against MS2. If there is no antagonistic activity, the AHLs signal degradation indicator bacteria CV017 can be used to verify whether the strain has a quenching effect on the pathogen MS2. When strain CV017 is cultured, it will produce a purple pigment, which can prove that it has produced an AHLs signal. If the purple color fades when the supernatant of other strains is co-cultured with CV017, it means that the strain has produced AIIA enzymes to degrade the AHLs signal.

[0051] After the lipopeptide substances of strain ZKY08 were separated, two components 1 and 2 were obtained (such as Figure 6 As shown) had no inhibitory effect on the pathogen MS2 (as shown Figure 7 By comparing the inhibitory effects of the bacterial suspension and supernatant of strain ZKY08 on the pathogen MS2, it was proved that it did not exert antagonistic effects by secreting lipopeptides in the supernatant (as shown in Figure 8The supernatant of ZKY08 strain was cultured at 37°C and 180 rpm for 3h, 6h, and 9h and co-cultured with AHLs signal degradation indicator bacteria CV017 at 37°C and 180 rpm for 12h. It was found that the purple color of strain CV017 faded and the OD 600 If the values ​​are not much different, it means that strain ZKY08 destroys the AHLS signal of interspecies communication of pathogen MS2 by secreting AIIA enzyme, thereby producing a quenching effect and preventing further growth of MS2. Fig. 9 , as shown in Table 3)

[0052] Table 2 RP-HPLC procedures

[0053] Time(min) Mobile phase A (%) Mobile phase B (%) 0 40 60 9 40 60 9.1 10 90 36 10 90 36.1 40 60

[0054] Table 3 OD of supernatant of strain ZKY08 at different times after co-culture with CV017 for 12 h 600 value

[0055]

[0056] 5. Antagonistic test of ZKY08 against different pathogenic fungi and bacteria

[0057] The method was the same as (1), wherein the pathogens tested included Ralstonia solanacearum that causes patchouli bacterial wilt, Ralstonia solanacearum that causes peanut bacterial wilt, Ralstonia solanacearum that causes tomato bacterial wilt, Klebsiella variegata that causes banana sheath rot, Fusarium asiatica that causes peanut fruit rot, Sclerotium solani that causes peanut white rot, Cladosporium that causes blueberry gray mold, and Aspergillus niger that causes peanut stem 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 4 and 5).

[0058] Table 4 Inhibition rate of ZKY08 against pathogenic fungi

[0059]

[0060] Table 5 Transparent zone radius of ZKY08 inhibitory effect on pathogenic bacteria

[0061]

[0062] 6. Use root inoculation method to evaluate potted plant protection. Dilute strain ZKY08 to 1×10 8 CFU / mL, the banana soft rot pathogen Dickinia MS2 was diluted to 1×10 6 CFU / mL, the banana wilt pathogen Fusarium oxysporum FOC009 was diluted to 1×10 6Spores / mL. 100 mL of diluted ZKY08 solution was inoculated at the root of banana seedlings, and one week later, 50 mL of diluted Dickie's solution and 50 mL of Fusarium oxysporum solution were inoculated. Only 50 mL of diluted Dickie's solution and 50 mL of Fusarium oxysporum solution were inoculated as blank controls, and the biocontrol effect and growth promotion effect (such as Figure 5 , as shown in Table 6 and Table 7).

[0063] Table 6 Biocontrol effect after ZKY08 treatment

[0064]

[0065] Table 7 Growth-promoting effect after ZKY08 treatment

[0066]

[0067] 7. From June 2024 to September 2024, a demonstration of ZKY08 for preventing and controlling banana soft rot was carried out in Leping Town, Sanshui District, Foshan City, Guangdong Province. A single colony of ZKY08 was picked into 5mL NB culture medium to prepare a secondary strain of bacterial liquid. After overnight culture at 37°C and 180rpm, a tertiary strain of bacterial liquid was prepared at a 1% inoculation rate and cultured for 48 hours to obtain a total of 20L of NB fermentation liquid. Farmers used 10L of NB fermentation liquid of strain ZKY08 each time, diluted 500 times, and then applied it to the Brazilian banana heads. Each banana was sprayed with 2000mL of the diluted liquid for a total of 2 applications, with an interval of about 60 days between the 2 applications. The blank control was not treated with any treatment. A total of 988 Brazilian bananas were applied with ZKY08, of which 49 were infected with a disease incidence rate of 4.9%. There were 196 blank control groups without any treatment, of which 20 were infected with a disease incidence rate of 10.2%. The biological control rate reached 51.96%, and the prevention and control effect was good. (such as Fig.10 , 11 , 12)

[0068] 8. The root inoculation method was used to determine the level of plant immune activity changes. 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; ZKY08 treatment (R20): inoculate 50mL with a concentration of 1.0×10 8 Strain ZKY08; FOC and ZKY08 treatment (FOC+R20): 50 mL of the culture medium was inoculated 24 h in advance with a concentration of 1.0×10 8 After the strain ZKY08, 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 (e.g. Fig.13 , 14, 15). After inoculation with strain ZKY08, the ET hormone level of banana plants increased rapidly at 48h and the JA hormone level increased rapidly at 24h. This indicates that the induced systemic resistance (ISR) of the plant is stimulated, and the activation of the ISR defense mechanism depends on the jasmonic acid (JA) and ethylene (ET) signal transduction pathways in the plant. 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. The SA hormone level also increased rapidly within 24h. SA is a plant endogenous immune signal with a low content in the plant body. It accumulates in large quantities when infected by pathogens. After the pathogen infects the plant, the cell quickly synthesizes 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 protein (PRS). The increase in SA content may activate the plant's systemic acquired resistance (SAR), that is, the resistance of the whole plant when infected by pathogens. This resistance can improve defense against a variety of pathogens, nematodes, and even parasitic plants. After co-treatment with strain ZKY08 and pathogen FOC, the ET hormone level within 72 hours, BR hormone level within 24 hours, and JA hormone level within 48 hours increased significantly compared with other treatments. This shows that under the co-treatment of biocontrol bacteria and pathogens, banana plants show obvious and rapid immune responses to prevent further growth of pathogens and form immune memory, so that the next time the same pathogen invades the plant, it can respond more quickly to protect the plant from damage.

Claims

1. Bacillus velezensis ZKY08, deposit number: GDMCC No: 64844.

2. Use of the Bacillus Velezii ZKY08 described in claim 1 in biological control.

3. The use according to claim 2, characterized in that: The invention relates to the application of Bacillus velezensis ZKY08 in inhibiting Ralstonia solanacearum, Klebsiella variegata, Fusarium asiatica, Sclerotium truncatum, Cladosporium, Aspergillus niger, Dickie's bacillus or Fusarium oxysporum.

4. The use according to claim 2, characterized in that: Application of Bacillus velezensis ZKY08 in improving plant immune activity.

5. The use according to claim 2, characterized in that: Application of Bacillus velez ZKY08 in preventing and controlling banana soft rot or banana wilt or their combined diseases.

6. The use according to claim 2, characterized in that: The Ralstonia solanacearum is the Ralstonia solanacearum that causes patchouli bacterial wilt, the Ralstonia solanacearum that causes peanut bacterial wilt, and the Ralstonia solanacearum that causes tomato bacterial wilt; the Klebsiella variegata is the Klebsiella variegata that causes banana sheath rot; the Fusarium asiatica is the Fusarium asiatica that causes peanut fruit rot; the Sclerotium truncatum is the Sclerotium truncatum that causes peanut white rot; the Cladosporium is the Cladosporium that causes blueberry gray mold; and the Aspergillus niger is the Aspergillus niger that causes peanut stem rot.

7. A biocontrol agent, characterized in that: The invention comprises the Bacillus Velezii ZKY08 described in claim 1 as an active ingredient.

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