A strain of Pseudomonas defensinus QNF1 and its application in microbial agents

Abstract

The invention discloses a Pseudomonas protegens QNF1, a microbial agent and an application thereof. The Pseudomonas protegens QNF1 is an endophyte isolated from the root system of leek and is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee with a deposit number of CGMCC No. 28587. The fermentation broth, cell-free supernatant and fermentation broth volatiles of the strain have significant antagonistic effects on Monilinia fructicola and have good inhibitory effects on peach fruit brown rot caused by Monilinia fructicola, wherein the inhibition rate of the fermentation broth on peach fruit brown rot is as high as 85.58%, and the inhibition rate of the fermentation broth volatiles on peach fruit brown rot is as high as 98.76%. Pseudomonas protegens QNF1 is a potential strain of biological control with good control effect and safe ecology, and its control agent has a simple fermentation process, low production cost and broad market application prospects.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a defense system against Pseudomonas QNF1 and application of a microbial agent thereof. Background Art

[0002] Brown rot caused by the ascomycete Monilinia spp. is an important infectious disease of postharvest drupes, which can cause up to 80% yield loss under conditions that are favorable for the disease. The main species causing brown rot in my country are Monilinia fructicola, Monilinia mumecola, Monilinia Lara, and Monilinia vunnanen, among which Monilinia fructicola is the dominant species causing brown rot in my country, also known as M. fructicola. Brown rot fungus (M.fructicola) is a multi-host pathogen that can infect not only stone fruit trees such as peach, cherry, plum, apricot, and plum, but also pome fruit trees such as apple, pear, sand apple, and hawthorn. It mainly harms fruits in the ripening and storage periods, and is prone to re-infection, which in turn causes significant economic losses.

[0003] In production applications, brown rot fungi (M.fructicola) are mainly controlled by various fungicides. However, excessive application of fungicides will cause many problems such as toxic residues, environmental pollution, and pathogen resistance. Therefore, it is urgent to explore new green, efficient, and environmentally friendly prevention and control measures. The use of antagonistic microorganisms to control plant diseases has the advantages of low toxicity, less pollution, and not easy to produce resistance. It is a safer and sustainable plant disease control strategy. In recent years, there have been many studies on antagonistic bacteria for brown rot (M.fructicola), such as Bacillus 14b published in the patent with announcement number CN 105820981 B and Bacillus 12a published in the patent with announcement number CN 105907669 B. Both are antagonistic bacteria isolated from the rhizosphere soil of peach trees, which can effectively prevent and control peach brown rot; but there are relatively few reports on the isolation of endogenous antagonistic bacteria from plants. At the same time, there are currently few antagonistic bacteria resources with commercial application potential that meet both ecological safety and food safety requirements, which limits the production application of antagonistic bacteria biological control technology in the control of post-harvest fruit brown rot.

[0004] Therefore, the prior art needs to be further improved. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the present application provides an endophyte defense Pseudomonas Protegens QNF1 isolated for the first time from the root system of leek. The fermentation broth, cell-free supernatant and fermentation broth volatiles have significant antagonistic effects on brown rot fungi (M.fructicola), and at the same time have a good inhibitory effect on peach fruit brown rot caused by brown rot fungi (M.fructicola), and can significantly maintain the fruit hardness and fruit quality.

[0006] In order to solve the above technical problems, this application provides the following technical solutions:

[0007] In the first aspect, the present invention provides a Pseudomonas protegens QNF1, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on October 8, 2023, with a deposit number of CGMCC No. 28587 and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. In biological classification, the bacteria belong to the Pseudomonas protegens species of the genus Pseudomonas.

[0008] In a second aspect, the present invention provides the use of the above-mentioned Pseudomonas protegens QNF1 in antagonizing brown rot fungus (M.fructicola), apple rot fungus (Valsa mali), pear rot fungus (Valsa pyri), apple ring rot fungus (Botryosphaeria dothidea) or apple anthracnose leaf blight fungus (Glomerella cingulata). Experiments have shown that the Pseudomonas protegens QNF1 provided in the present application has excellent antibacterial effects on the above-mentioned pathogens.

[0009] In a third aspect, the present invention provides the use of Pseudomonas protegens QNF1 in preventing and controlling fruit brown rot caused by brown rot fungus (M. fructicola).

[0010] Optionally, the fruit trees are stone fruit trees such as peach, apricot, cherry, plum, etc., or pome fruit trees such as apple, pear, etc.

[0011] In a fourth aspect, the present invention provides a microbial agent for antagonizing brown rot fungus (M. fructicola), wherein the active ingredient includes the aforementioned Pseudomonas protegens QNF1.

[0012] Optionally, the active ingredient of the microbial agent is the fermentation liquid, fermentation liquid supernatant or volatiles of the aforementioned Pseudomonas defense QNF1. Experiments have shown that the fermentation liquid, fermentation liquid supernatant or volatiles of Pseudomonas defense QNF1 have significant inhibitory effects on brown rot fungi. Among them, the inhibition rate of the fermentation liquid on peach fruit brown rot is as high as 85.58%, and the inhibition rate of the fermentation liquid volatiles on peach fruit brown rot is as high as 98.76%.

[0013] The microbial agent can be directly applied using the fermentation broth, fermentation broth supernatant or volatiles of Pseudomonas defensis QNF1, or it can be used as an active ingredient and supplemented with a carrier and other auxiliary agents (such as stabilizers, antioxidants, etc.) to make various types of preparations (such as solid granular preparations, liquid suspensions and gaseous sprays, etc.) that are more stable or easy to carry and transport, so as to be better used in actual production.

[0014] In a fifth aspect, the present invention also provides a method for preparing the aforementioned active ingredient of the microbial agent, wherein the active ingredient of the microbial agent is a fermentation broth of Pseudomonas defense QNF1, and the preparation method is:

[0015] The colony or bacterial liquid of Pseudomonas defense QNF1 was inoculated into the liquid culture medium and incubated at 26-29°C at 180-250 r·min -1 The culture was shaken under the conditions for 30 to 45 hours to obtain the fermentation liquid of Pseudomonas defense QNF1.

[0016] Preferably, the preparation method of the fermentation broth of Pseudomonas defense QNF1 is as follows: inoculate the colony or bacterial solution of Pseudomonas defense QNF1 into 200 mL LB liquid culture medium, and incubate at 28°C for 200 r·min -1 The culture was shaken for 36 h, the initial pH value of the culture medium was 7.0, and the concentration of the microbial solution was 1×10 10 cfu·mL -1 Defensins of Pseudomonas QNF1 fermentation broth.

[0017] In a sixth aspect, the present invention also provides a method for preparing the active ingredient of the microbial agent, wherein the active ingredient of the microbial agent is a cell-free supernatant of Pseudomonas defense QNF1, and the preparation method thereof is:

[0018] The colony or bacterial liquid of Pseudomonas defense QNF1 was inoculated into the liquid culture medium and incubated at 26-29°C at 180-250 r·min -1 The culture was shaken for 10 to 15 hours under the conditions, and the bacterial solution was rotated at 5000 to 7000 r / min. -1 The mixture was centrifuged at a speed of 8 to 15 minutes, the supernatant was taken, and then the supernatant was filtered with a sterile filter to finally obtain the cell-free supernatant of Pseudomonas defense bacteria QNF1.

[0019] Preferably, the preparation method of the cell-free supernatant of Pseudomonas defensin QNF1 is: inoculating the colony or bacterial solution of Pseudomonas defensin QNF1 into 200 mL LB liquid culture medium, placing it at 28°C and 200 r·min -1 The culture was shaken for 12 h under the same conditions. -1 The mixture was centrifuged at a speed of 10 min, the supernatant was taken, and then filtered three times with a 0.22 μm sterile filter to obtain the Pseudomonas defensis QNF1 cell-free supernatant.

[0020] In addition, the preparation method of the fermentation broth volatiles of the bacteria is as follows: 10 mL of LB solid culture medium and 10 mL of PDA solid culture medium are poured into a culture dish cover and a culture dish with a diameter of 9 cm, respectively, and after solidification, a certain concentration of Pseudomonas defensis QNF1 fermentation broth is added to the culture dish cover (LB culture medium), and evenly spread with an applicator, dried, and the culture dish is sealed. The space inside the culture dish is the volatiles of the fermentation broth of Pseudomonas defensis QNF1.

[0021] In a seventh aspect, the present invention also provides the use of the aforementioned Pseudomonas defense QNF1 or microbial agent in maintaining the firmness of peach fruit.

[0022] Preferably, the application method is: soaking the fruit in the fermentation liquid of Pseudomonas defense QNF1, drying it, and then soaking it again in the bacterial liquid of brown rot fungus (M. fructicola) (1×10 4 cfu·mL -1 ), optionally, the soaking time is 10 to 30 minutes.

[0023] Experiments have shown that in the defense Pseudomonas QNF1+Mf treatment group, 9 days after inoculation, the hardness of the fruit in this treatment group was 12.63, which was 124.41% higher than that of the control (Mf) fruit. The difference was extremely significant, which indicates that the defense Pseudomonas QNF1 treatment significantly maintains the hardness of peach fruit and has certain market application prospects.

[0024] The present invention has the following beneficial effects:

[0025] (1) The present invention screened and isolated a plant-derived endophyte, Pseudomonas protegens QNF1 strain, from the root system of leek for the first time. Experiments showed that the fermentation broth, cell-free supernatant and fermentation broth volatiles of the bacteria had significant antagonistic effects on brown rot fungi (M. fructicola), and had a good inhibitory effect on peach fruit brown rot caused by brown rot fungi (M. fructicola); at the same time, the fermentation broth of the bacteria also had significant antagonistic effects on apple rot fungi (Valsa mali), pear rot fungi (Valsa pyri), apple ring rot fungi (Botryosphaeria dothidea) and apple anthracnose leaf blight fungi (Glomerella cingulata); in addition, the fermentation broth treatment had no adverse effects on the quality of the fruit, and the fermentation broth was an easily available microbial antagonistic bacteria resource for brown rot fungi (M. fructicola) with both ecological safety and food safety, and was helpful in maintaining the firmness of the fruit and extending the shelf life.

[0026] (2) Defense Pseudomonas QNF1 strain has a significant antagonistic effect on brown rot fungus (M.fructicola). The examples show that the inhibition rate of its bacterial cake on the growth of brown rot fungus (M.fructicola) mycelium can be as high as 67.11%, the inhibition rate of its fermentation broth on the biomass of brown rot fungus (M.fructicola) can be as high as 95.93%, the inhibition rate of its cell-free supernatant on the growth of brown rot fungus (M.fructicola) mycelium can be as high as 97.95%, and the inhibition rate of its fermentation broth volatiles on the growth of brown rot fungus (M.fructicola) mycelium can be as high as 95.35%.

[0027] (3) The Pseudomonas defense strain QNF1 has a good inhibitory effect on peach brown rot caused by M. fructicola. The examples show that the inhibition rate of its fermentation broth on peach brown rot is as high as 85.58%, and the inhibition rate of its fermentation broth volatiles on peach brown rot is as high as 98.76%.

[0028] (4) Defense Pseudomonas QNF1 strain has a significant antagonistic effect on other fruit tree pathogens. The examples show that the inhibition rates of its fermentation broth on the biomass of apple rot pathogen (Valsa mali), pear rot pathogen (Valsa pyri), apple ring rot pathogen (Botryosphaeria dothidea), and apple anthracnose leaf blight pathogen (Glomerella cingulata) are 92.45%, 88.69%, 95.06%, and 89.72%, respectively.

[0029] (5) The microbial inoculant based on the Pseudomonas defense QNF1 strain has a simple fermentation process and low production cost, which is conducive to its production and application in the control of postharvest fruit brown rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Phylogenetic tree constructed based on the 16S rDNA of strain Pseudomonas defensinus QNF1.

[0031] Figure 2 To prevent the inhibition of Pseudomonas QNF1 on the hyphae growth of brown rot fungus (M. fructicola); A is the control, B is the inhibition effect, C is the inhibition effect on colony diameter, and D is the inhibition effect on hyphae growth; *P<0.05, **P<0.01, ***P<0.001;

[0032] Figure 3 The inhibition effect of the fermentation broth of Pseudomonas QNF1 on the biomass of brown rot fungus (M.fructicola); A is the control, BF are the inhibition effects of 0.001%, 0.01%, 0.1%, 1% and 10% treatment concentrations, G is the mycelium weight of brown rot fungus (M.fructicola) under each treatment concentration, H is the inhibition rate of Pseudomonas QNF1 on the biomass of brown rot fungus (M.fructicola) under each treatment concentration; ***P<0.001, ****P<0.0001;

[0033] Figure 4 The inhibitory effect of the cell-free supernatant of Pseudomonas QNF1 on the hyphae growth of brown rot fungus (M. fructicola); A is the control, BF are the inhibitory effects of 10%, 20%, 30%, 40% and 50% treatment concentrations, G is the colony diameter of brown rot fungus (M. fructicola) under each treatment concentration, and H is the inhibition rate of the cell-free supernatant of Pseudomonas QNF1 at each treatment concentration on the hyphae growth of brown rot fungus (M. fructicola); ***P<0.001;

[0034] Figure 5 To prevent the inhibitory effect of volatiles from the fermentation broth of Pseudomonas sp. QNF1 on the mycelial growth of brown rot pathogen (M. fructicola); A is the control, and BD is 1×10 6 , 1×10 8 and 1×10 10 cfu·mL -1Inhibitory effect of treatment concentration, E is the colony diameter of brown rot fungus (M. fructicola) at each treatment concentration, F is the inhibition rate of Pseudomonas defense QNF1 volatiles at each treatment concentration on the mycelial growth of brown rot fungus (M. fructicola); **P<0.01, ***P<0.001, ****P<0.0001;

[0035] Figure 6 The control effect of the fermentation liquid of Pseudomonas QNF1 on the brown rot of peach fruit caused by brown rot fungus (M. fructicola) (punch inoculation cake method); A and C are controls, B and D are the control effects of the treatments, E is the diameter of the fruit lesions, and F is the inhibition rate of Pseudomonas QNF1 on the brown rot of peach fruit caused by brown rot fungus (M. fructicola); ****P<0.0001;

[0036] Figure 7 The control effect of the fermentation liquid of Pseudomonas QNF1 on the brown rot of peach fruit caused by brown rot fungus (M. fructicola) (immersion inoculation method); A and C are controls, B and D are the control effects of the treatments, E is the diameter of the fruit lesions, and F is the inhibition rate of Pseudomonas QNF1 on the brown rot of peach fruit caused by brown rot fungus (M. fructicola); *P<0.05, ***P<0.001;

[0037] Figure 8 The control effect of volatiles of Pseudomonas QNF1 fermentation liquid on peach brown rot caused by brown rot fungus (M. fructicola); A and C are controls, B and D are the control effects of treatments, E is the diameter of fruit lesions, and F is the inhibition rate of volatiles of Pseudomonas QNF1 fermentation liquid on peach brown rot caused by brown rot fungus (M. fructicola); ***P<0.001;

[0038] Fig. 9 Effects of treatment with fermentation broth of Pseudomonas defense QNF1 on peach fruit quality; A is soluble solids (TSS) content, B is soluble sugar (SS) content, C is titratable acidity (TA) content, D is solid-acid ratio (TSS / TA), E is sugar-acid ratio (SS / TA), F is vitamin C content, and G is fruit firmness; CK is the control, QNF1 is the fruit treated with Pseudomonas defense QNF1, Mf is the fruit soaked in the bacterial solution of brown rot fungus (M.fructicola), and QNF1+Mf is the fruit first treated with Pseudomonas defense QNF1 and then soaked in the bacterial solution of brown rot fungus (M.fructicola); ns: no statistical difference, ***P<0.001. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0040] Example 1 Isolation, Screening and Identification of Pseudomonas protegens QNF1 Strain

[0041] 1. Experimental methods

[0042] The roots of the leek variety "Fuljiu No. 12" were collected from the experimental base of Qingdao Agricultural University, the outer skin was removed after cleaning, and the surface was thoroughly disinfected, then the root tissue was ground. The ground homogenate was diluted with sterile distilled water and cultured with NA medium at 28°C using the conventional gradient dilution spreading separation method. Single colonies with obvious differences in colony morphology were picked, purified and preserved on NA medium; the plate confrontation method was used to screen endophytes with potential antibacterial effects on brown rot pathogen (M. fructicola).

[0043] The isolated endophyte with significant antibacterial potential against brown rot pathogen (M. fructicola) was cultured as a single colony and commissioned to Shanghai Bioengineering Technology Co., Ltd. for 16S rDNA sequencing.

[0044] 2. Experimental results and analysis

[0045] The results of 16sRNA sequencing showed that the 16sRNA sequence of the isolated endophyte was 1420bp, as shown in SEQ ID No: 1 (see sequence table for details). The 16S rDNA sequence was imported into the ezbiocloud database for online comparison analysis, and it was found that the bacteria had the closest evolutionary relationship with the Pseudomonas protegens strain MN959751 (NCBI Accession: MH813535.1), with a similarity of 99.93% ( Figure 1 ), thus determining that the isolated endophyte belongs to Pseudomonas protegens, and named it Pseudomonas protegens QNF1.

[0046] The strain was then deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 8, 2023, with the deposit number CGMCC No. 28587 and the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0047] Example 2 Inhibition test of Pseudomonas defense QNF1 on brown rot fungus (M. fructicola) 1. Inhibition test of Pseudomonas defense QNF1 strain on the hyphae growth of brown rot fungus (M. fructicola) (1) The plate confrontation method was used for determination:

[0048] A fresh cake of brown rot pathogen (M. fructicola) with a diameter of 7 mm was inoculated in the center of a culture dish containing PDA medium, and a fresh cake of Pseudomonas defense QNF1 of the same size was inoculated 1.5 cm around the cake. The pathogen culture dish without the cake of Pseudomonas defense QNF1 was used as the control. The experiment was repeated 6 times. All culture dishes were inverted and cultured at 28℃ for 5 days. The diameter of the colony was measured by cross-crossing. The inhibition rate of Pseudomonas defense QNF1 on the mycelial growth of brown rot pathogen (M. fructicola) was calculated according to formula (1).

[0049]

[0050] (2) Test results and analysis:

[0051] On the first day of inoculation, the colonies of brown rot pathogen (M. fructicola) on all plates began to grow, and there was no significant difference in colony diameter; from the third day to the fifth day, the control colonies grew rapidly, while the colonies treated with Pseudomonas defense QNF1 grew relatively slowly, and the colony diameter was significantly different from the control ( Figure 2 ).

[0052] The test results showed that the Pseudomonas defense QNF1 strain could significantly inhibit the mycelial growth of brown rot fungus (M. fructicola), and the inhibition rate was as high as 67.11% 5 days after inoculation.

[0053] 2. Inhibition test of Pseudomonas defense QNF1 fermentation broth on the biomass of brown rot fungus (M. fructicola)

[0054] (1) Experimental methods:

[0055] PDB medium was prepared, and fermentation liquid of Pseudomonas defense QNF1 was added thereto to make the final concentrations 0.001%, 0.01%, 0.1%, 1%, and 10%. The blank control was set without the fermentation liquid of Pseudomonas defense QNF1. A fresh cake of brown rot fungus (M. fructicola) with a diameter of 7 mm was added to 50 mL of the above bacterial liquid, and the mixture was incubated at 28°C and 200 r / min. -1 After 2 days of constant temperature oscillation culture, the culture solution was filtered through two layers of lens paper, and the precipitate was collected and air-dried on sterile filter paper and weighed. Six replicates were set for each treatment. The inhibition rate of the fermentation liquid of Pseudomonas defense QNF1 on the biomass of brown rot fungus (M. fructicola) was calculated according to formula (2).

[0056]

[0057] (2) Experimental results and analysis:

[0058] The results showed that the fermentation broth of Pseudomonas defenses QNF1 at all concentrations had a significant or extremely significant inhibitory effect on the biomass of brown rot fungus (M. fructicola), and the inhibition rate of 10% QNF1 fermentation broth was as high as 95.93% ( Figure 3 ).

[0059] 3. Inhibition test of the cell-free supernatant of Pseudomonas defense QNF1 on the hyphae growth of brown rot fungus (M. fructicola)

[0060] (1) Experimental methods:

[0061] Preparation of cell-free supernatant of Pseudomonas defensin QNF1: A fresh bacterial cake of Pseudomonas defensin QNF1 with a diameter of 7 mm was inoculated into 200 mL LB liquid medium and placed at 28°C and 200 r·min -1 The culture was shaken for 12 h under the same conditions. -1 The mixture was centrifuged at a speed of 10 min, the supernatant was taken, and then filtered three times with a 0.22 μm sterile filter to obtain the Pseudomonas defensis QNF1 cell-free supernatant.

[0062] 2, 4, 6, 8, and 10 mL of the cell-free supernatant of Pseudomonas defense QNF1 were added to a 9 cm culture dish, and sterile PDA medium was poured in to make up to a total volume of 20 mL. The dish was shaken and allowed to stand until it solidified for use. The PDA medium without QNF1 cell-free supernatant was used as a control. That is, 0% (blank control), 10%, 20%, 30%, 40%, and 50% of the cell-free supernatant of Pseudomonas defense QNF1 were obtained. A fresh bacterial cake of brown rot pathogen (M. fructicola) with a diameter of 7 mm was inoculated in the center of the culture medium. All culture dishes were placed in a 28°C constant temperature incubator and inverted in the dark. The plaque diameter was measured by the cross method, and photographed and recorded every day until the blank control grew to the edge of the culture medium. Six replicates were set for each treatment. The inhibition rate of the cell-free supernatant of Pseudomonas defense QNF1 on the mycelial growth of brown rot pathogen (M. fructicola) was calculated according to formula (1).

[0063] (2) Experimental results and analysis

[0064] The test results showed that the inhibition rate of mycelial growth of brown rot fungus (M. fructicola) by 50% QNF1 cell-free supernatant treatment was 93.42% and 95.55% on the 4th and 5th day after inoculation. After 6 days of inoculation, the inhibition rates of mycelial growth of brown rot fungus (M. fructicola) by 10%, 20%, 30%, 40%, and 50% QNF1 cell-free supernatant treatment were 33.22%, 18.71%, 12.29%, 59.68%, 97.95% ( Figure 4 The above results indicate that the cell-free supernatant of Pseudomonas defense QNF1 can significantly inhibit the growth of mycelium of brown rot fungus (M. fructicola).

[0065] 4. Inhibition of the growth of mycelium of brown rot fungus (M. fructicola) by volatiles of fermentation broth of Pseudomonas sp. QNF1

[0066] (1) Experimental methods:

[0067] Pour 10 mL of PDA solid medium into a 9 cm diameter culture dish, wait for it to solidify, and then inoculate a 7 mm diameter brown rot fungus (M. fructicola) cake in the center of the culture dish; pour 10 ml of LB solid medium into the lid of the culture dish, wait for it to solidify, and then add 100 μL of 1×10 6 cfu·mL -1 , 1×10 8 cfu·mL -1 , 1×10 10 cfu·mL -1The fermentation liquid of Pseudomonas defense QNF1 with a concentration of 1.5-2.5 μg / cm2 was evenly spread with a spreader, and the plate was sealed after drying. The plate without the fermentation liquid of Pseudomonas defense QNF1 was used as the control.

[0068] All culture dishes were placed in a 28°C constant temperature incubator and cultured upside down in the dark.

[0069] The colony diameter was measured by the cross method and photographed. Each group was repeated 6 times. The inhibition rate of the volatiles of the fermentation broth of Pseudomonas defense QNF1 on the mycelial growth of brown rot fungus (M. fructicola) was calculated according to formula (1).

[0070] (2) Experimental results and analysis

[0071] The test results showed that the colonies of brown rot fungus (M. fructicola) in the control group began to grow on the first day of inoculation. On the sixth day, the number of colonies was large and dense, and the average diameter of the plaque was about 4.14 cm. 6 cfu·mL -1 , 1×10 8 cfu·mL -1 In the concentration treatment group, the colonies of brown rot fungus (M. fructicola) began to grow on the second day after inoculation; while in the 1×10 10 cfu·mL -1 In the concentration treatment group, the colonies of brown rot fungus (M. fructicola) began to grow on the 5th day after inoculation. On the 6th day after inoculation of brown rot fungus (M. fructicola), 1×10 6 cfu·mL -1 , 1×10 8 cfu·mL -1 , 1×10 10 cfu·mL -1 The average diameter of the plaque of brown rot fungus (M.fructicola) in the concentration treatment group was reduced by 71.51%, 83.43% and 95.35% respectively compared with the control group. The above results show that the volatiles of the fermentation broth of Pseudomonas defense QNF1 can significantly inhibit the growth of brown rot fungus (M.fructicola). Figure 5 ).

[0072] Example 3 Inhibitory Effects of Pseudomonas QNF1 Fermentation Broth on Various Fruit Tree Pathogenic Fungi 1. Experimental Methods

[0073] Referring to the experimental method of Example 2, the inhibitory effect of the fermentation liquid of Pseudomonas defense QNF1 on various major pathogenic fungi of fruit trees was determined.

[0074] 2. Experimental results and analysis

[0075] The experimental results of the inhibition rate at 6 days of culture are shown in Table 1. It can be seen from the table that the defensive Pseudomonas QNF1 has a high antagonistic effect on major fruit tree pathogenic fungi such as apple rot, apple ring rot, anthracnose leaf blight, and pear rot, showing good broad-spectrum antibacterial properties.

[0076] Table 1 Inhibitory effect of Pseudomonas defense QNF1 on major pathogenic fungi of fruit trees

[0077]

[0078] Example 4 Application of Pseudomonas defense QNF1 in the prevention and control of peach fruit brown rot 1. Experiment on the prevention and control of fruit brown rot by fermentation liquid of Pseudomonas defense QNF1

[0079] Fruit preparation: Select healthy fruits of uniform size and without obvious lesions as test materials. Sterilize the surface of the fruits with 1.5% NaClO solution for 5 min, rinse with sterilized distilled water for 3 times, and then air-dry for later use.

[0080] (1) Punch inoculation method:

[0081] A sterile needle was used to make a wound (0.3 cm wide × 0.3 cm deep) at the equator of the fruit, and 20 μL of Pseudomonas defense QNF1 fermentation liquid (1 × 10 10 cfu·mL -1 ), and 20 μL LB liquid medium was used as a blank control. After 1 hour, a piece of brown rot fungus (M. fructicola) cake with a diameter of 0.3 cm was inoculated into the wound. Ten fruits were treated for each treatment, and the experiment was repeated 6 times.

[0082] All fruits were placed on plastic trays and cultured at 28°C in the dark for 3 days. The diameter of the fruit lesions was observed and measured every day. The inhibition rate of the fermentation liquid of Pseudomonas defense QNF1 on the occurrence of fruit brown rot was calculated according to formula (3).

[0083]

[0084] The test results showed that the fruit of the control group showed symptoms 1 day after inoculation, and the lesions expanded rapidly. On the 3rd day, the lesions had spread to the entire fruit surface, with an average diameter of 2.76 cm, and the rot symptoms penetrated into the core of the fruit in a funnel shape ( Figure 6 A, 6C). However, for fruits treated with the fermentation liquid of Pseudomonas defense bacteria QNF1, only a few fruits showed slight symptoms at the inoculation port 1 day after inoculation, and the development speed was relatively slow. On the 3rd day, the diameter of the lesion was evaluated to be 0.51 cm, and the inhibition rate was 81.57% ( Figure 6 B, 6D).

[0085] The experimental results showed that the fermentation broth of Pseudomonas defense QNF1 can effectively inhibit the occurrence of fruit brown rot.

[0086] (2) Immersion test determination:

[0087] The fruits were divided into two groups and processed as follows:

[0088] Control group: The fruits were immersed in LB medium for 15 min, air-dried at room temperature for 1 h, and immersed again in the culture medium of brown rot fungus (M. fructicola) (1×10 4 cfu·mL -1 ) for 15 minutes.

[0089] Treatment group: The fruit was incubated in the fermentation broth of Pseudomonas defense QNF1 (1×10 10 cfu·mL -1 ) for 15 min, dried at room temperature for 1 h, and then immersed in the bacterial solution of brown rot fungus (M. fructicola) (1×10 4 cfu·mL -1 ) for 15 minutes.

[0090] All treated fruits were placed in plastic boxes and placed in an incubator at 28°C for 3 days. Ten fruits were used for each treatment, and the experiment was repeated 6 times. The disease of the fruits was observed every day, and the inhibition rate of the fermentation liquid of Pseudomonas defense QNF1 on the occurrence of fruit brown rot was calculated according to formula (3).

[0091] The test results showed that: 1 day after inoculation, small water-soaked spots appeared on the surface of the control fruit, forming rot spots of varying sizes. As time went on, the number of spots increased, the area gradually expanded, and gradually penetrated into the center of the fruit ( Figure 7 A, 7D). However, the lesions on fruits treated with the fermentation liquid of Pseudomonas defense QNF1 were smaller and expanded slowly. At 3 days, the average number of lesions on the control fruits was 6.93, while the average number of lesions on fruits treated with the fermentation liquid of Pseudomonas defense QNF1 was 1, with an inhibition rate of 85.58% (see Figure 7 EF).

[0092] 2. Inhibition test of volatiles from fermentation broth of Pseudomonas defense bacteria QNF1 on the occurrence of brown rot in fruits

[0093] (1) Experimental methods:

[0094] Healthy peach fruits of uniform size and without obvious lesions were selected as test materials. The fruits were surface sterilized with 1.5% NaClO solution for 5 min, rinsed with sterilized distilled water 3 times, and then air-dried for later use. During the test, a sterile needle was used to make a wound (0.3 cm wide × 0.3 cm deep) on the equator of the fruit. 200 mL of LB solid culture medium was poured into a 4.5 L plastic box. After the culture medium solidified, 1 mL of Pseudomonas defense QNF1 fermentation liquid (1 × 10 10 cfu·mL -1 ), spread evenly, and after incubation at 28℃ for 12h, the fruits with wounds were placed in plastic boxes. A plastic box without the fermentation liquid of Pseudomonas defensinus QNF1 was used as a control.

[0095] After the treatment and control plastic boxes were placed under the same conditions for another 24 hours, the fruits were taken out and a brown rot fungus (M. fructicola) cake with a diameter of 0.3 cm was inoculated at the wound and placed back into the plastic box. After 6 days of culture under the same conditions, the diameter of the lesions was measured by the cross method. Ten fruits were treated in each case and the experiment was repeated 6 times. The inhibition rate of the volatiles of the fermentation liquid of Pseudomonas defense QNF1 on the occurrence of brown rot in fruits was calculated according to formula (3).

[0096] (2) Experimental results and analysis

[0097] The test results showed that 3 days after inoculation, the control fruit began to show symptoms, the lesions began to soften and rot, the lesions turned brown, and grayish white hyphae appeared, and then the lesions began to expand with the passage of time; on the 6th day, the average diameter of the lesions was about 2.16cm. The fruits treated with volatiles from the fermentation liquid of Pseudomonas defense QNF1 began to show signs of disease on the 6th day, and the average diameter of the lesions was about 0.33cm, with an inhibition rate of 98.76% ( Figure 8 ).

[0098] Example 5 Experiment on the effect of Pseudomonas QNF1 fermentation liquid on peach fruit quality (1) Experimental method:

[0099] Healthy fruits of uniform size and without obvious lesions were selected as test materials. The fruits were surface sterilized with 1.5% NaClO solution for 5 minutes, rinsed with sterilized distilled water for 3 times, and then air-dried for later use. The fruits were randomly divided into four groups and treated according to the following methods:

[0100] CK: Soak the fruit in LB medium for 15 min;

[0101] QNF1: The fruits were incubated in the fermentation broth of Pseudomonas defensinus QNF1 (1×10 10 cfu·mL -1 ) for 15 min;

[0102] Mf: The fruits were immersed in LB medium for 15 min, air-dried at room temperature for 1 h, and immersed again in a solution of brown rot fungus (M. fructicola) (1×10 10 cfu·mL -1 )15min;

[0103] QNF1+Mf: The fruit was fermented in the Pseudomonas defensinus QNF1 broth (1×10 10 cfu·mL -1 ) for 15 min, dried at room temperature for 1 h, and then immersed in the bacterial solution of brown rot fungus (M. fructicola) (1×10 4 cfu·mL -1 )15min.

[0104] All treated fruits were cultured in an incubator at 28°C. Then, on the third day, the fruit was peeled near the equator with a sterilized peeling knife, and the fruit hardness was measured with a GY-4 hardness tester. The pulp tissue was then cut with a peeling knife and stored in a -80°C ultra-low temperature refrigerator for testing fruit quality. Soluble solids were measured using a PAL-1 saccharimeter; vitamin C content was measured using the molybdenum blue colorimetric method; soluble sugars were measured using the anthrone colorimetric method; and titratable acid was measured using the acid-base neutralization method.

[0105] (2) Experimental results and analysis

[0106] The experimental results show:

[0107] ① Endophyte defense against Pseudomonas QNF1 helps maintain peach fruit firmness

[0108] When the fruit was inoculated with brown rot fungus (M.fructicola), the firmness of the fruit treated with endophytic defense pseudomonas QNF1 (QNF1+Mf group) was 12.63, which was 124.41% higher than that of the fruit treated with Mf, and the difference was extremely significant; when the fruit was not inoculated with fungus, the firmness of the fruit treated with QNF1 was also higher than that of the control (CK), but the difference was not significant (see Fig. 9 ). This indicates that the endophyte defense Pseudomonas QNF1 treatment can significantly maintain the firmness of peach fruit, which is beneficial to prolong its shelf life and has certain market application prospects.

[0109] ② Endophyte defense Pseudomonas QNF1 treatment had no adverse effects on other qualities of peach fruit

[0110] Without inoculation of brown rot pathogen (M.fructicola), the soluble sugar, titratable acid, vitamin C content and SS / TA of fruits treated with endophytic defense Pseudomonas QNF1 increased by 36.44%, 17.57%, 105.45% and 13.73% respectively, compared with the control (CK), but the difference was not significant. The vitamin C content, TSS / TA and SS / TA of fruits treated with QNF1+Mf were 16.28%, 26.42% and 17.83% higher than those treated with Mf, respectively (see Fig. 9 The above experimental results show that the treatment with endophyte defense Pseudomonas QNF1 has no adverse effects on other qualities of peach fruit and can be promoted and applied in the market.

[0111] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A strain of Pseudomonas Defensinus ( Pseudomonas Protegens )QNF1, characterized in that It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No. 28587.

2. A microbial agent, characterized in that: The active ingredient comprises the Pseudomonas defensinus QNF1 as claimed in claim 1.

3. A microbial agent, characterized in that: The active ingredient is the fermentation broth, cell-free supernatant or fermentation broth volatiles of the Pseudomonas defensinus QNF1 according to claim 1.

4. The defensive Pseudomonas QNF1 according to claim 1 or the microbial agent according to claim 2 or 3 is used to antagonize brown rot fungi M. fructicola , apple rot bacteria Valsa mali , Pear rot Valsa pyri , Apple ring rot Botryosphaeria dothidea Colletotrichum indica Glomerella cingulata Application in.

5. The use of the Pseudomonas defense QNF1 as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3 in preventing and treating brown rot of peach fruit, wherein the brown rot of peach fruit is caused by M. fructicola Caused by infection.

6. The method for preparing the microbial agent according to claim 3, characterized in that: The active ingredient of the microbial agent is the fermentation broth of Pseudomonas defense QNF1 as claimed in claim 1, and the preparation method thereof is as follows: Inoculate the colony or bacterial suspension of Pseudomonas defense QNF1 into the liquid culture medium and incubate at 26-29°C at 180-250 r / min -1 The culture was shaken under the conditions for 30 to 45 hours to obtain the fermentation broth of Pseudomonas defense QNF1.

7. The method for preparing the microbial agent according to claim 3, characterized in that: The active ingredient of the microbial agent is the cell-free supernatant of Pseudomonas defense QNF1 as claimed in claim 1, and the preparation method thereof is as follows: the colony or bacterial liquid of Pseudomonas defense QNF1 is inoculated into a liquid culture medium, and the culture medium is incubated at 26-29° C. and 180-250 r·min -1 The culture was shaken for 10-15 h under the conditions of 5000-7000 r·min. -1 The mixture was centrifuged at a speed of 8 to 15 minutes, the supernatant was taken, and then filtered with a sterile filter to obtain the cell-free supernatant of Pseudomonas defense bacteria QNF1.

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