A soil microbial probiotic combination for increasing resistance of banana rhizosphere pathogens and its application

Abstract

The invention discloses a soil microbial probiotic combination for increasing the resistance of banana rhizosphere pathogens and its application. The soil microbial probiotic combination consists of Brevundimonas huaxiensis S6 and Pseudomonas defluvii S30. The combined bacterial agent of the invention is used to inhibit the colonization of rhizosphere pathogens in banana seedlings. The bacterial strain of the invention inhibits the proliferation of Rhizospora under the condition of mixed co-culture with Oxyspora, and inoculation under greenhouse conditions can significantly reduce the number of rhizosphere pathogens in banana seedlings. In summary, the addition of the combined bacterial agent of Brevundimonas and Pseudomonas resists the invasion of pathogens and reduces the incidence of banana seedlings.

Description

Technical Field

[0001] The invention belongs to the field of agricultural microorganisms, and specifically relates to a combined bacterial agent of Brevundimonas huaxiensis S6 and Pseudomonas defluvii S30 for effectively inhibiting the colonization of rhizosphere pathogens of bananas at the seedling stage and an application thereof. Background Art

[0002] Banana (scientific name Musanana Lour) is a perennial herb. Banana cultivation originated in tropical and subtropical areas of Southeast Asia. It is the main food source for many developing countries in tropical and subtropical regions of the world, and is also one of the most important export cash crops in the world. China is one of the earliest countries in the world to cultivate and grow bananas on a large scale. It has now become the most important banana producer in the world. Among them, Guangdong, Hainan, Guangxi, Fujian, Yunnan and Taiwan are the main areas for growing bananas in my country. In recent years, the banana planting industry has developed rapidly in my country, and bananas have become the fourth largest fruit in China after apples, grapes and citrus. With the continuous expansion of the planting area, my country has now become the world's second largest banana producer, and its banana production is second only to India. However, wilt diseases caused by Fusarium oxysporum have been found in all major banana planting areas in my country, which seriously endangers the development of my country's banana industry.

[0003] Banana Fusarium wilt disease is a highly lethal soil-borne vascular fungal disease caused by the soil-borne necrotic fungus Fusarium oxysporum f.sp.cubense (FOC), and is one of the most destructive soil-borne pathogens. Soil-borne diseases cause serious plant diseases worldwide, seriously hindering global food security under the general trend of global warming. Modern intensive agricultural planting patterns cause pathogens to accumulate continuously in the soil. When the number of pathogens exceeds the threshold that the soil ecological environment can bear, soil-borne diseases will break out and begin to spread rapidly. The disease is difficult to effectively control in the short term after it occurs. It is the most serious disease that endangers the world's banana production and has caused extremely serious restrictions on the sustainable development of the global banana industry. Exploring effective management to overcome banana wilt is a prerequisite for supporting sustainable banana production.

[0004] Banana can be infected with pathogens and develop banana wilt disease at all stages of growth and development, and its transmission pathways are also diverse and complex. Once Fusarium oxysporum contaminates healthy banana soil, it will be difficult to completely eliminate and eradicate the pathogen in a short period of time through traditional methods. Conventional physical and chemical treatment methods have caused many adverse effects on the soil, water, atmosphere and other environments. At the same time, the problem of drug resistance has become increasingly prominent, and the residues of drugs in plants will indirectly cause harm to humans. With people's pursuit of green, organic and healthy food, microorganisms as a green and efficient treatment method have attracted more and more attention from scholars. Therefore, it is necessary to develop environmentally friendly and non-toxic microbial preparations to humans and animals. The most promising biological control method is to use bacterial strains isolated from banana disease-suppressing soil that can increase the activity of banana seedling disease-resistant enzymes and antagonize Fusarium oxysporum to prevent and control banana soil-borne wilt. Screening antagonistic strains with good effects and stable activity is a prerequisite for ensuring the success of biological control. Synthetic microbial communities (Synthetic microbial communities, SynComs) are artificial microbial systems that combine and co-cultivate multiple species with known classifications and functions. Compared with single bacterial preparations, synthetic bacterial communities have greater potential to play plant growth-promoting functions in situ in the rhizosphere. Compared with single bacteria, synthetic microbial communities contain members of multiple species, and there is communication and exchange between strains. Compared with single species, they have higher robustness and stability, and are more capable of resisting interference from the external environment. The composition of multiple species makes microbial communities have more diverse functions and can complete complex tasks that a single strain cannot complete. This type of microorganism is non-toxic to humans and animals, friendly to the environment, and can be developed as a bacterial resource for plant disease biocontrol. It is in line with my country's agricultural development strategy of "less input, more output, and environmental protection."

[0005] Therefore, the patent of this invention uses a combination of functional microorganisms, Brevundimonas and Pseudomonas, screened from banana disease-suppressing soil to study its effects on the colonization of rhizosphere pathogens in banana seedlings, providing technical support for resisting the invasion of pathogens in the banana seedling stage, thereby reducing the incidence of banana wilt disease and ultimately providing support for the sustainable development of the banana industry. Summary of the invention

[0006] The purpose of the present invention is to provide a combined bacterial agent of Brevundimonas and Pseudomonas for effectively inhibiting the colonization of rhizosphere pathogens of banana seedlings in response to actual problems and needs in production practice.

[0007] Another object of the present invention is to provide the application of the plant in resisting pathogenic bacteria.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A soil microbial probiotic combination for increasing the resistance of banana seedlings to pathogens. The soil microbial probiotic combination consists of Brevundimonas huaxiensis S6 and Pseudomonas defluvii S30. Brevundimonas huaxiensis S6 was deposited in the General Microbiology Center of the China National Microbiological Culture Collection on February 20, 2023, with a preservation number of CGMCCNO.26597; Pseudomonas defluvii S30 was deposited in the General Microbiology Center of the China National Microbiological Culture Collection on February 20, 2023, with a preservation number of CGMCCNO.26598.

[0010] The soil microbial probiotic combination of the present invention is used in inhibiting the pathogen Fusarium oxysporum f.sp. cubense from colonizing the rhizosphere during the banana seedling stage.

[0011] The soil microbial probiotic combination of the present invention is used in preparing a bacterial agent for inhibiting the colonization of pathogenic bacteria Fusarium oxysporum f.sp. cubense in the rhizosphere during the banana seedling stage.

[0012] The combined bacterial agent prepared by Brevundimonas S6 and Pseudomonas S30 described in the present invention.

[0013] As a preferred embodiment of the present invention, the bacterial agent is prepared by the following method: a single colony of Brevundimonas S6 and a single colony of Pseudomonas S30 are simultaneously inoculated into LB liquid culture medium, and placed at 30°C and 170rmin -1 The mixture was shaken on a shaker for 1 day to obtain the fermentation liquid of Brevundimonas S6 and Pseudomonas S30, which was the combined bacterial agent.

[0014] As a further preferred embodiment of the present invention, the Brevundimonas S6 single colony is obtained by streaking and activating the Brevundimonas S6 glycerol strain on a 1 / 10 TSB solid medium (the amount added is 1 / 10 of the TSB medium), and then culturing the strain in a 30°C incubator overnight. The Pseudomonas S30 single colony is obtained by streaking and activating the Pseudomonas S30 glycerol strain on a 1 / 10 TSB solid medium, and then culturing the strain in a 30°C incubator overnight.

[0015] The combined bacterial agent of the present invention is used for inhibiting the colonization of rhizosphere pathogens of banana seedlings.

[0016] A method for inhibiting the colonization of rhizosphere pathogens in banana seedlings, wherein a fermented functional microbial combination agent is inoculated into a seedling culture medium of transplanted banana seedlings at an inoculation ratio of 10 7 CFU / g substrate dry weight, the bacteria were poured three times, with an interval of 10 days between each pouring.

[0017] As a preferred embodiment of the present invention, after three times of watering with bacteria in the greenhouse, the cultivation is continued for a period of time, and all the potted plants are moved to the field for another watering with bacteria treatment.

[0018] The invention discloses an application of the combined bacterial agent of Brevundimonas sinensis S6 and Pseudomonas septicagen S30 in the process of transplanting and domesticating banana seedlings.

[0019] The invention discloses an application of the combined bacterial agent in the process of transplanting and domesticating banana seedlings.

[0020] The application effects of the present invention are as follows:

[0021] The present invention screened for the first time a strain of Sinohyperdactyla S6 and Pseudomonas spp. S30 that can inhibit the colonization of rhizosphere pathogens in banana seedlings. Compared with the control of adding clean water, adding 5×10 7 The combined bacterial agent of CFU of Shortwave Mononas S6 and Pseudomonas aeruginosa S30 significantly reduced the number of rhizosphere pathogens in banana seedlings, and both showed significant disease inhibition. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Photos of strains and their taxonomic tree

[0023] Figure 2 The number of pathogens in the co-culture system of strains and pathogens in liquid PDB medium

[0024] Figure 3 Effects of inoculation with different strains on the abundance of rhizosphere pathogens in banana seedlings in the greenhouse Note: Lowercase letters indicate differences between treatments with different functional strains (p = 0.05). (The same below)

[0025] Biomaterial Deposit Information

[0026] S6, classified and named Brevundimonas huaxiensis, was deposited in the General Microbiology Center of China Culture Collection Administration on February 20, 2023, with the preservation number CGMCCNO.26597. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing.

[0027] S30, classified and named Pseudomonas defluvili, was deposited in the General Microbiology Center of China Culture Collection Administration on February 20, 2023, with the preservation number CGMCCNO.26598. DETAILED DESCRIPTION

[0028] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0029] Unless otherwise specified, the raw materials used in the following examples are as follows:

[0030] Ordinary seedling medium: It is an ordinary seedling medium made of imported peat and coconut bran as the main raw materials, and is added with natural minerals and humic acid organic fertilizer, as well as biological fermentation and harmless treatment. This medium is produced and provided by Jiangsu Xingnong Matrix Technology Co., Ltd. and can also be purchased.

[0031] Example 1. Isolation and identification of functional strains

[0032] Weigh 5 g of banana disease-inhibiting soil and place it in a triangular flask containing 45 mL of sterile water (the flask contains 4-6 glass beads) at 30°C and 170 rmin. -1 After shaking for 30 minutes, take out and dilute in gradient. Take 100 μL of soil suspension with different concentration gradients and place it on 1 / 10 TSB solid medium for coating, then place the coated plate in a 30℃ incubator for 2 days. When the colonies grow, select the differential colonies for purification, inoculate the purified strains on TSB plates and store them in a 4℃ refrigerator for use, and store the purified single colonies in a glycerol tube in a -80℃ refrigerator.

[0033] Two target strains were finally obtained through screening: one was numbered S6, which grew faster on TSB solid medium, and the bacteria were short rod-shaped and yellow. The phylogenetic tree analysis constructed by the strain 16SrDNA sequence showed that strain S6 was located on the Brevundimonashuaxiensis branch, and S6 was classified as Brevundimonas huaxiensis, which was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee with a preservation number of CGMCCNO.26597; the other was numbered S30, which grew faster on TSB solid medium, and the bacteria were short rod-shaped and light yellow. The phylogenetic tree analysis constructed by the strain 16SrDNA sequence showed that strain S30 was located on the Pseudomonasdefluvii branch, and S30 was classified as Pseudomonasdefluvii, which was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee with a preservation number of CGMCCNO.26598. Other treated strains were also screened out at the same time as Brevundimonas S7.

[0034] Solid culture medium formula: TSB medium, i.e. tryptone soy broth medium (1L): 30g tryptone soy broth, dilute to 1L with deionized water, 20g agar powder, sterilize at 115℃ for 30min. Note: 1 / 10TSB means 1 / 10 of the amount of TSB culture medium added.

[0035] Liquid culture medium formula: LB medium (1L): peptone 10g, yeast powder 5g, NaCl 10g, dilute to 1L with deionized water, sterilize at 115℃ for 30min.

[0036] Example 2: Liquid propagation culture of functional strains

[0037] The glycerol tubes of Sinohyperdactyly S6 and Pseudomonas aeruginosa S30 isolated from banana disease-suppressing soil were taken out of the -80℃ refrigerator and placed on ice. A sterile inoculation loop was dipped on the ice residue in the glycerol tube, and then streaked on 1 / 10 TSB solid culture medium for activation. The tubes were placed in a 30℃ incubator for overnight culture. After the colonies grew out, the single colonies were inoculated into LB liquid culture medium and placed at 30℃, 170rmin. -1 After shaking and culturing overnight on a shaker, 200 μL of the culture fluid of the two single colonies were respectively pipetted into new LB liquid medium for mixed culture, and then placed at 30°C and 170 rmin. -1 After shaking on a shaker for 1 day, the fermentation liquid of the combination of Brevundimonas huaxiensis S6 and Pseudomonas septicagensis S30 was obtained for later use. The culture method of strain S7 was the same as above.

[0038] Example 3: Antagonistic test of co-culture of functional strains with Oxysporium in liquid shake flasks

[0039] Each strain was co-cultured with the pathogen FocTR4 in potato dextrose broth (PDB) medium at 28°C and 170 rpm, and glass beads were added to prevent the pathogen hyphae from agglomerating. After 5 days, the fermentation broth was serially diluted 10 times, and the appropriate dilution was spread on Bengal red medium and incubated at 28°C for 36 hours to count the number of FocTR4.

[0040] Solid culture medium formula: Red Bengal medium: glucose 10g, peptone 5g, magnesium sulfate 0.5g, Red Bengal 10ml1 / 300, agar 20g, deionized water 1000mL, natural pH, sterilization at 115℃ for 30min.

[0041] Liquid culture medium formula: PDB culture medium: Peel and weigh 200 g of potatoes, cut into 1 cm small pieces, boil for 20 min, filter the filtrate through gauze, add 20 g of glucose, and distilled water to 1 L, pH natural, and sterilize at 115°C for 30 min.

[0042] Depend on Figure 2 It can be seen that after 5 days of co-culture of the strains with the pathogens, the results of applying the pathogens showed that compared with the control (FOC culture alone), strains S6, S30 and S6+S30 showed lower abundance of Oxysporium. S7 had no significant difference in inhibiting pathogens compared with the control.

[0043] Example 4: Disease resistance test of functional bacteria in banana seedling cultivation

[0044] Prepare the above-mentioned common seedling medium and plastic pots, and take 200g of the medium into each pot for standby use. The test tissue culture seedlings are commercial sterile seedlings of Brazilian varieties, which are pre-cultured in sterile culture solution in a light incubator for 15 days. After the matrix and tissue culture seedlings are fully prepared, the tissue culture seedlings with similar growth and intact root systems are transplanted into the plastic pots filled with the matrix. After transplanting, water the plastic pots and ensure that the moisture content in the pots is maintained between 65% and 70%.

[0045] The banana seedlings transplanted into plastic pots were set up with seven treatments, namely T1 (CK), T2 (S6), T3 (S7), T4 (S30), T5 (S6 + S7), T6 (S6 + S30) and T7 (S7 + S30). After the banana seedlings grew stably for 30 days after transplantation, they were inoculated with fermented functional microbial agents at an inoculation ratio of 10 7 CFU / g substrate dry weight. 5 The pathogen FocTR4 was inoculated at a final concentration of CFU / g substrate dry weight. After three inoculations in the greenhouse, the cultivation was continued for a period of time to monitor the typical symptoms of Fusarium wilt. When the wilting symptoms were observed in the control-treated banana seedlings, all the roots in each pot were collected, and then gently shaken by hand to remove the loosely adhering soil and cut into pieces of about 5 cm in length. For each sample, about 200 g of roots were placed in a 500 mL conical flask pre-filled with 200 mL of sterile saline. After shaking at 170 rpm for 30 min at ambient temperature, the roots and plant residues were removed by sterile tweezers, and then the soil slurry was centrifuged at 4000 × g for 5 min, and the sediment was collected as the rhizosphere soil. The genomic DNA of the rhizosphere soil was extracted, and then the abundance of FocTR4 in the rhizosphere soil was quantified. At the same time, the abundance of culturable FocTR4 in the rhizosphere soil was determined by spreading the soil dilution on K2 medium.

[0046] Solid culture medium formula: K2 medium: 1.0g potassium hydrogen phosphate, 0.5g potassium chloride, 0.5g magnesium sulfate, 0.01g sodium iron EDTA, 2.0g L-asparagine, 20g D-galactose, 16g agar, 1000mL deionized water, sterilized at 115℃ for 30min. Cool to about 60℃, add 1.0g pentachloronitrobenzene (75% WP), 0.5g ox bile, 1.0g sodium tetraborate and 0.3g streptomycin sulfate per liter of culture medium, and finally adjust the pH to 3.8±0.2 with 10% phosphoric acid.

[0047] Depend on Figure 3 It can be seen that after three times of bacterial pouring treatment, the results of the rhizosphere soil pathogen coating plate counts in the greenhouse test showed that, except for the inoculated strain S7, the number of pathogens treated by S6, S30 and their combinations S6+S7, S6+S30, and S7+S30 was significantly lower than that of the control CK, which was consistent with the qPCR results. The number of pathogens in S6 was the lowest and the antagonism was the strongest. We found that the number of rhizosphere pathogens in the S6+S30 treatment of banana functional bacteria in the matrix was significantly lower than that in the CK treatment. This result has guiding significance for the field application of the banana functional bacteria S6+S30 of the present invention in the rhizosphere of banana seedlings to resist pathogen invasion and reduce the incidence of bananas.

Claims

1. A soil microbial probiotic combination for increasing the resistance of banana seedlings to pathogens, characterized in that The soil microbial probiotic combination is composed of Brevundimonas huaxiensis ( Brevundimonas huaxiensis )S6 and Pseudomonas aeruginosa ( Pseudomonas defluvii )S30, Shortwave Mononas S6 in West China was deposited in the General Microbiology Center of China Culture Collection Administration on February 20, 2023, with the preservation number CGMCC NO.26597; Pseudomonas aeruginosa S30 was deposited in the General Microbiology Center of China Culture Collection Administration on February 20, 2023, with the preservation number CGMCCNO.26598.

2. The soil microbial probiotic combination according to claim 1 inhibits the pathogen Fusarium oxysporum Cuban type ( Fusarium oxysporum f.sp. cubense ) in rhizosphere colonization.

3. Use of the soil microbial probiotic combination according to claim 1 in preparing a bacterial agent for inhibiting the colonization of the pathogen Fusarium oxysporum Cuban type in the rhizosphere at the banana seedling stage.

4. A bacterial agent prepared by the soil microbial probiotic combination according to claim 1.

5. The bacterial agent according to claim 4, characterized in that The preparation method is as follows: single colonies of the Chinese Brevundimonas S6 and the sewage Pseudomonas S30 described in claim 1 are inoculated into LB liquid culture medium, placed at 30°C and 170r min -1 After shaking and culturing overnight on a shaker, 200 μL of the culture fluid of the two single colonies were respectively pipetted into new LB liquid medium for mixed culture and placed at 30 °C and 170 r min. -1 After shaking on a shaker for 1 day, a fermentation liquid containing a combination of Brevundimonas huaxiensis S6 and Pseudomonas spp. Sewage S30 was obtained, which was the bacterial agent.

6. Application of the bacterial agent described in claim 4 or 5 in preparing a banana seedling stage matrix additive.

7. A method for inhibiting the colonization of rhizosphere pathogen Fusarium oxysporum Cuban type in banana seedlings at the seedling stage, characterized in that The bacterial agent according to claim 4 is inoculated into the seedling medium of the transplanted banana seedlings at a ratio of 10 7 CFU / g substrate dry weight, the bacteria were poured three times, with an interval of 10 days between each pouring.

8. The method according to claim 7, characterized in that After two waterings in the greenhouse, the cultivation is continued for a period of time, and all the potted plants are moved to the field and then watered once with the bacterial agent described in claim 4.

9. Application of the soil microbial probiotic combination according to claim 1 in the domestication process of banana seedling transplanting.

10. Application of the microbial agent described in claim 4 in the process of transplanting and domesticating banana seedlings.

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