Pseudomonas palustris QNF3 and application thereof in prevention and control of fruit and vegetable diseases

Abstract

The invention relates to the field of microorganisms, in particular to pseudomonas pale yellow QNF3 and application thereof to prevention and control of fruit and vegetable diseases. The pseudomonas palustris QNF3 is preserved in China General Microbiological Culture Collection Center on August 26, 2024, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.31739. The pseudomonas palustris QNF3 disclosed by the invention has the advantages that the pseudomonas palustris QNF3 can be used for preparing the pseudomonas palustris QNF3; according to the present invention, the Pseudomonas palustris QNF3 can significantly inhibit the growth of pathogenic bacteria such as Botryosphaeria dothidea, Monilinia fruticosa, Alternaria alternata, Fusarium oxysporum, Aspergillus niger, Penicillium digitatum, Botrytis cinerea and the like, and can efficiently prevent and control the occurrence of related fruit and vegetable diseases, for example, the prevention and control effect of the fermentation broth of the Pseudomonas palustris QNF3 on the apple ring spot caused by Botryosphaeria dothidea reaches up to 94.59%; and the fruit quality, especially the fruit hardness, can be improved, and an important role in prolonging the storage period of the fruits is played.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a light yellow Pseudomonas QNF3 and an application thereof in preventing and controlling fruit and vegetable diseases. Background Art

[0002] In the fruit industry, fungal pathogens such as Botryosphaeria dothidea, Monilinia fructicola, Alternaria alternata, Fusarium Oxysporum, Aspergills niger, Penicillum digitatum and Botrytis cinerea pose a serious threat to the growth, quality and yield of fruits. The diseases they cause, such as apple ring rot, peach brown rot, pear black spot, citrus green mold and various fruit tree gray mold, not only cause fruit rot and quality decline, but also affect fruit storage and market value. Therefore, developing effective prevention and control measures is the key to ensuring the health of fruit trees and improving economic benefits.

[0003] At present, these important diseases mainly rely on chemical control. However, the long-term use of chemical agents can lead to environmental pollution, drug resistance, residue problems, harm to non-target organisms and other disadvantages. Biological control, as a sustainable solution, can reduce the impact on the environment, reduce chemical pesticide residues, improve food safety, maintain ecological balance, and effectively reduce the risk of resistance of pathogens by utilizing natural pests and microbial agents. Therefore, the use of biological control methods is an important way to ensure the health and sustainable development of the fruit industry. Summary of the invention

[0004] In order to solve the above technical problems, the present invention isolates and screens an endophyte Pseudomonas dothidea QNF3 from leek, which can significantly inhibit the growth of fungal pathogens such as Botryosphaeria dothidea and Monilinia fructicola, thereby effectively preventing and controlling the occurrence of related fruit and vegetable diseases such as apple ring rot, peach brown rot, peach gray mold, pear black spot, citrus green mold, tomato wilt, grape black aspergillus disease, etc.; and can also improve fruit quality.

[0005] The present invention provides a light yellow Pseudomonas QNF3 (Pseudomonas lurida QNF3), which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on August 26, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.31739.

[0006] The present invention also provides the use of the pale yellow Pseudomonas QNF3 for inhibiting the growth of pathogens such as Botryosphaeria dothidea, Monilinia fructicola, Alternaria alternata, Fusarium Oxysporum, Aspergills niger, Penicillum digitatum, and Botrytis cinerea.

[0007] The present invention also provides the use of the pale yellow Pseudomonas QNF3 for preventing and controlling fruit and vegetable related diseases caused by pathogens such as Botryosphaeria dothidea, Monilinia fructicola, Alternaria alternata, Fusarium Oxysporum, Aspergills niger, Penicillum digitatum, and Botrytis cinerea.

[0008] Preferably, the fruits and vegetables are apples, peaches, pears, tomatoes, grapes, citrus and other important horticultural crops.

[0009] Preferably, the fruit and vegetable diseases are apple ring rot, peach brown rot, pear black spot, tomato wilt, grape black aspergillus disease, citrus green mold, or peach gray mold.

[0010] The present invention also provides application of the Pseudomonas aureus QNF3 in improving the quality of apple fruits.

[0011] Preferably, the apple fruit quality refers to apple fruit firmness, apple fruit soluble solids content, titratable acid content, solid-acid ratio (ratio of soluble solids content to titratable acidity) and sugar-acid ratio (ratio of soluble sugar content to titratable acidity).

[0012] The present invention also provides a microbial preparation, comprising the Pseudomonas aureus QNF3; preferably, comprising the fermentation broth of the Pseudomonas aureus QNF3.

[0013] Compared with the prior art, the beneficial effects of the present invention include at least:

[0014] 1. The present invention obtains a novel strain of Pseudomonas aureus QNF3, which can significantly inhibit the growth of plant pathogens such as Botryosphaeria dothidea, Monilinia fructicola, Alternaria alternata, Fusarium oxysporum, Aspergills niger, Penicillum digitatum, and Botrytis cinerea, and effectively prevent and control the occurrence of related diseases.

[0015] 2. The control effect of the fermentation liquid of Pseudomonas aureus QNF3 on apple ring rot is 55.23-94.59%, and the control effect of its volatile organic substances VOCs on apple fruit ring rot is 53.00-74.00%. Disease prevention methods include spraying or soaking, and fumigation using its volatiles, which can achieve significant antibacterial effects and efficient control effects.

[0016] 3. The Pseudomonas flavus QNF3 of the present invention can also improve the quality of fruits, especially the hardness of fruits, and plays an important role in extending the storage period of fruits.

[0017] 4. The present invention provides new candidate biocontrol strains for green prevention and control of plant diseases, enriches the strain resources for biological control of plant diseases, and develops efficient prevention and control measures for specific diseases such as apple ring rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Phylogenetic tree analysis of Pseudomonas aureus QNF3.

[0019] Figure 2 The inhibition of Pseudomonas aureus QNF3 on the growth of Botryosphaeria dothidea (PDA medium). A: Growth of control colonies 3 days after inoculation; B: Growth of colonies treated with Pseudomonas aureus QNF3 3 days after inoculation; C: Comparison of the diameters of colonies treated with different treatments; D: Inhibition rate of Pseudomonas aureus QNF3 on the growth of Botryosphaeria dothidea.

[0020] Figure 3The inhibition effect of Pseudomonas aureus QNF3 on the growth of Botryosphaeria dothidea (PDB medium). A: control mycelial culture after 48 hours of treatment; BD: mycelial culture treated with 0.02mL / L, 2mL / L, and 200mL / L Pseudomonas aureus QNF3, respectively; E: weight of Botryosphaeria dothidea culture treated with different treatments; F: inhibition rate of Pseudomonas aureus QNF3 on the growth of Botryosphaeria dothidea.

[0021] Figure 4 The control effect of Pseudomonas aureus QNF3 on apple fruit ring rot inoculated with Botryosphaeria dothidea. A: control fruit 3 days after inoculation; B: fruit treated with Pseudomonas aureus QNF3 3 days after inoculation; C: comparison of lesion diameters of fruits treated with different treatments; D: inhibition rate of Pseudomonas aureus QNF3 on apple fruit ring rot.

[0022] Figure 5 The control effect of Pseudomonas aureus QNF3 on apple fruit ring rot soaked in Botryosphaeria dothidea. A: Control fruit 8 days after inoculation; B: Fruit treated with Pseudomonas aureus QNF3 8 days after inoculation; C: Comparison of the number of lesions in fruits treated with different treatments; D: Inhibition rate of Pseudomonas aureus QNF3 on apple fruit ring rot

[0023] Figure 6 The figure shows the inhibitory effect of volatile organic compounds (VOCs) of Pseudomonas flavus QNF3 on the growth of Botryosphaeria dothidea. A: Growth of control colonies 3 days after inoculation; B: Growth of colonies treated with volatile organic compounds (VOCs) of Pseudomonas flavus QNF3 3 days after inoculation; C: Comparison of the diameters of colonies treated with different treatments; D: Inhibition rate of volatile organic compounds (VOCs) of Pseudomonas flavus QNF3 on the growth of Botryosphaeria dothidea.

[0024] Figure 7The control effect of VOCs of Pseudomonas aureus QNF3 on apple ring rot caused by Botryosphaeria dothidea inoculation. A: Control fruit 3 days after inoculation; B: Fruit treated with VOCs of Pseudomonas aureus QNF3 3 days after inoculation; C: Comparison of lesion diameters of treated and control fruits; D: Inhibition rate of VOCs of Pseudomonas aureus QNF3 on fruit diseases.

[0025] Figure 8 The control effect of VOCs of Pseudomonas aureus QNF3 on apple ring rot caused by Botryosphaeria dothidea. A: Control fruit 8 days after inoculation; B: Fruit treated with VOCs of Pseudomonas aureus QNF3 3 days after inoculation; C: Comparison of the number of lesions between treated and control fruits; D: Inhibition rate of VOCs of Pseudomonas aureus QNF3 on fruit diseases

[0026] Fig. 9 Effects of Pseudomonas aureus QNF3 on apple fruit quality parameters. A: total soluble solids (TSS); B: soluble sugars (SS); C: titratable acidity (TA); D: TSS / TA; E: SS / TA; F: vitamin C; G: fruit firmness. *p<0.05, **p<0.01, ****p<0.0001, ns indicates no significant difference. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0028] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0029] Example 1: Isolation, screening and identification of Pseudomonas aureus QNF3 strain

[0030] Fresh single-root red leek roots were taken from the campus base of Qingdao Agricultural University. They were washed with tap water to remove soil, then rinsed with distilled water, and placed in a clean bench after disinfection of the outer surface with 75% alcohol. The leek fibrous roots were cut off and the outer epidermis of the shortened stems was scraped off with a sterile scalpel. The leek fibrous roots and peeled shortened stems were disinfected: first soaked in 75% alcohol for 40 seconds, then soaked in 3% sodium hypochlorite for 5 minutes to kill the bacteria attached to the surface, and then rinsed with sterile distilled water for 4 times, and the last distilled water was retained for plate control. The disinfected materials were placed in a sterile mortar, 10mL of sterile distilled water was added to grind into fine slurry, and allowed to stand for 10 minutes. 100μL of supernatant was drawn from the upper layer and added to the LB plate. 100μL of the same retained sterile rinse solution was also drawn as a control. They were evenly coated with a coating rod, sealed, and placed in a 28℃ constant temperature incubator for inverted culture until a single colony grew. The plate confrontation method was used to screen endophytes with potential antibacterial effects on Botryosphaeria dothidea. The isolated endophytes with significant inhibitory potential on Botryosphaeria dothidea were cultured in single colonies and 16S rDNA sequencing was commissioned to Shanghai Bioengineering Technology Co., Ltd.

[0031] The results of 16S rDNA sequencing showed that the 16S rDNA sequence of the isolated endophyte was 1431 bp, as shown in SEQ ID No: 1 (see the sequence table for details). The 16S rDNA sequence was imported into the ezbiocloud database for online comparison analysis, and it was found that the bacterium was most closely related to Pseudomonas lurida strain LMG 21995 (NCBI Accession: PDJB01000001) (see Figure 1 Thus, the endophyte was determined to belong to Pseudomonas and was named Pseudomonas lurida QNF3.

[0032] Example 2: Inhibitory effect of Pseudomonas aureus QNF3 on hyphae growth of Botryosphaeria dothidea on PDA medium

[0033] Pour 10ml of PDA medium into the culture dish. Draw two cross lines on the back of the culture dish after solidification. Inoculate a 0.7cm diameter Botryosphaeria dothidea cake at the intersection, and inoculate a 0.7cm diameter Pseudomonas QNF3 cake at 1.5cm from the intersection on the intersection line. Botryosphaeria dothidea facing Pseudomonas QNF3 was used as the control. All culture dishes were inverted in an incubator at 28℃ for culture, and the experiment was repeated 6 times. After inoculation, the cross method was used to measure the colony diameter of Botryosphaeria dothidea to evaluate the inhibitory effect of Pseudomonas QNF3 on the growth of Botryosphaeria dothidea.

[0034] The results showed that on PDA medium, Pseudomonas aureus QNF3 had a significant inhibitory effect on the mycelial growth of Botryosphaeria dothidea ( Figure 2 ). At 1d, 2d and 3d after inoculation, the colony diameters of Pseudomonas aureus QNF3-treated strains were 1.47cm, 1.93cm and 2.22cm, respectively, which were reduced by 26.06%, 58.27% and 68.83% compared with the control, respectively.

[0035] Example 3: Inhibitory effect of Pseudomonas aureus QNF3 on hyphae growth of Botryosphaeria dothidea in PDB medium

[0036] 10 mL, 0.1 mL and 0.001 mL of Pseudomonas aureus QNF3 (1×10 9 CFU / L) was added to a 100mL Erlenmeyer flask, and the total volume was adjusted to 50mL with PDB medium to make the final concentrations of 200ml / L, 2ml / L and 0.02ml / L. PDB medium without Pseudomonas aureus QNF3 was used as a control. Two Botryosphaeria dothidea cakes with a diameter of 0.7cm (weight 0.03g) were added to each Erlenmeyer flask. The experiment was performed three times. All Erlenmeyer flasks were shaken at a speed of 150rpm in a constant temperature shaking incubator at 28°C. After 48h, the culture was collected and weighed by centrifugation at 4000rpm for 5min to evaluate the inhibitory effect of Pseudomonas aureus QNF3 on the growth of Botryosphaeria dothidea.

[0037] The results showed that Pseudomonas aureus QNF3 had a significant inhibitory effect on the growth of Botryosphaeria dothidea ( Figure 3 After 48 hours, the weight of the culture in the control group was 5.875 g, and the weight of the culture treated with 0.02 mL / L, 2 mL / L, and 200 mL / L of Pseudomonas aureus QNF3 were 0.327 g, 0.165 g, and 0.012 g, respectively, and the inhibition rates were 94.42%, 97.19%, and 99.80%, respectively.

[0038] Example 4: Effect of Pseudomonas aureus QNF3 on the prevention and control of apple fruit ring rot caused by Botryosphaeria dothidea bacterial cake

[0039] A sterile needle was used to make a wound at the equator of the apple fruit, with a diameter of 3 mm and a depth of 2 mm. 20 μL of Pseudomonas aureus QNF3 (1×10 9 CFU / L). 20μL LB medium was used as control. After 1 hour, the Botryosphaeria dothidea cake with a diameter of 0.7cm was divided into four equal parts, and one portion was inoculated into the wound of the fruit. All fruits were placed at 28℃. Ten fruits were treated for each treatment, and the experiment was repeated 3 times. The diameter of the lesion was measured every day to evaluate the control effect of Pseudomonas dothidea QNF3 on apple ring rot.

[0040] The results showed that Pseudomonas aureus QNF3 had a significant control effect on apple fruit ring rot inoculated with Botryosphaeria dothidea fungus cakes. Figure 4 ). The diameters of the lesions on the control fruits were 0.05 cm, 0.63 cm and 1.84 cm at 1d, 2d and 3d after inoculation, which were 88.93%, 68.04% and 55.23% less than those of the control, respectively.

[0041] Example 5: Effect of Pseudomonas aureus QNF3 on the prevention and control of apple fruit ring rot caused by Botryosphaeria dothidea bacterial solution

[0042] Apple fruits were immersed in Pseudomonas aureus QNF3 (1×10 9CFU / L) for 15 min. After 1 h, the fruits were immersed in Botryosphaeria dothidea for 15 min. The apple fruits were immersed in LB broth as a control. The experiment was repeated three times with 5 biological replicates. All fruits were placed in plastic boxes (29 cm × 20 cm × 10 cm), covered with lids, and kept in a humid environment at 28 ° C. Disease symptoms were observed and the number of lesions on the fruits was recorded to evaluate the control effect of Pseudomonas aureus QNF3 on apple fruit ring rot.

[0043] The results showed that Pseudomonas aureus QNF3 had a significant control effect on apple fruit ring rot soaked in Botryosphaeria dothidea bacterial solution ( Figure 5 ). Obvious lesions appeared on the fruit starting from the 4th day after inoculation. From 4 to 8 days, the number of lesions on the control fruit was 2.46, 6.13, 9.40, 13.60, and 18.53, respectively, while the number of lesions on the fruit treated with Pseudomonas aureus QNF3 was 0.13, 0.40, 1.33, 2.26, and 4.26, respectively, which were 94.59%, 93.48%, 85.82%, 83.33%, and 76.98% less than the control, respectively.

[0044] Example 6: Inhibitory effect of volatile organic compounds (VOCs) from Pseudomonas aureus QNF3 on the growth of Botryosphaeria dothidea

[0045] Add 10 mL PDA medium to a 9 cm diameter culture dish, and add 10 mL LB medium to the top of the culture dish. Inoculate a 0.7 cm diameter Botryosphaeria dothidea cake in the center of the PDA medium, and add 100 μL of Pseudomonas dothidea QNF3 (1×10 9 CFU / L), and spread it evenly with a coating rod. The plate without Pseudomonas aureus QNF3 was used as the control. The culture dish was placed in a constant temperature incubator at 28°C. The colony diameter of Botryosphaeria dothidea was measured every day to evaluate the inhibitory effect of VOCs of Pseudomonas aureus QNF3 on the growth of Botryosphaeria dothidea.

[0046] The results showed that the volatile organic compounds (VOCs) of Pseudomonas aureus QNF3 had a significant inhibitory effect on the growth of Botryosphaeria dothidea. Figure 6). At 1d, 2d, and 3d after inoculation, the colony diameters of Pseudomonas aureus QNF3 treated with VOCs were 0.63cm, 1.32cm, and 2.24cm, respectively, which were 84.12%, 71.54%, and 62.26% smaller than those of the control.

[0047] Example 7: Effect of VOCs of Pseudomonas aureus QNF3 on the prevention and control of apple ring rot caused by Botryosphaeria dothidea inoculation cake

[0048] Pour 200 mL of LBA medium into the prepared plastic box (29 cm × 20 cm × 10 cm). Add 1 mL of Pseudomonas aureus QNF3 (1 × 10 9 CFU / L) and evenly spread it on LBA medium. Then, use a sterile needle to make a wound (3mm in diameter and 2mm in depth) at the equator of each apple fruit. Inoculate each hole with 1 / 4 of a 0.7cm diameter Botryosphaeria dothidea cake. Then, put the apple fruit in a plastic box, cover it with a lid, and seal it with plastic wrap. Place a box containing no Pseudomonas aureus QNF3 (1×10 9 Apples in plastic boxes with 100 CFU / L were used as controls. All plastic boxes were placed at 28°C. Each treatment was repeated 10 times, and the experiment was conducted three times. After 3 days, the lesion diameter was measured to evaluate the control effect of VOCs of Pseudomonas aureus QNF3 on apple fruit ring rot.

[0049] The results showed that the VOCs of Pseudomonas aureus QNF3 had a significant control effect on apple ring rot caused by Botryosphaeria dothidea inoculation cakes. Figure 7 ). On the third day, the average lesion diameter of the control fruit was 4.29 cm, while the lesion diameter of the fruit infected with Pseudomonas aureus QNF3 VOCs was 2.02 cm, which was 53.00% smaller than that of the control fruit.

[0050] Example 8: Effect of VOCs of Pseudomonas aureus QNF3 on the prevention and control of apple ring rot caused by Botryosphaeria dothidea bacterial solution

[0051] Pour 200 mL of LB medium into the prepared plastic box (29 cm × 20 cm × 10 cm). Add 1 mL of Pseudomonas aureus QNF3 (1 × 10 9CFU / L) and evenly spread on LB medium. A plastic box without Pseudomonas aureus QNF3 was used as a control. Then, the apple fruit was immersed in the bacterial solution of Botryosphaeria dothidea for 15 minutes for inoculation. Subsequently, the apple fruit was placed in a plastic box and treated at 28°C. Each treatment had 5 biological replicates and the experiment was conducted three times. The disease symptoms were observed and the diseases were recorded to evaluate the control effect of volatile organic compounds (VOCs) of Pseudomonas aureus QNF3 on the ring rot of apple fruit immersed in the bacterial solution of Botryosphaeria dothidea.

[0052] The results showed that the VOCs of Pseudomonas aureus QNF3 had a significant control effect on apple fruit ring rot caused by Botryosphaeria dothidea. Figure 8 ). On the 8th day after inoculation, the number of lesions on the fruits treated with VOCs of Pseudomonas aureus QNF3 was 3.93, which was 74% less than that of the control.

[0053] Example 9: Effect of Pseudomonas aureus QNF3 on Apple Quality

[0054] The experimental design was divided into four treatments: (1) immersing the fruit in LB medium for 15 min (CK); (2) fermenting the fruit in Pseudomonas aureus QNF3 broth (1×10 9 CFU / L) for 15 min (QNF3). (3) The fruit was soaked in LB medium for 15 min, dried at room temperature for 1 h, and then soaked in the culture medium of Botryosphaeria dothidea for 15 min (Bd). (4) The fermentation liquid of Pseudomonas aureus QNF3 (1×10 9 CFU / L) for 15 minutes, dried at room temperature for 1 hour, and then immersed in the bacterial solution of Botryosphaeria dothidea for 15 minutes (QNF3+Bd). All fruits were placed in plastic trays and placed in an incubator at 28°C. After 3 days, the fruit hardness was measured at the equator of the peeled fruit using a GY-4 fruit hardness tester. The soluble solids content (TSS) in the juice was analyzed using a PAL-1 digital refractometer. The vitamin C (VC) content was determined using the molybdenum blue colorimetric method. The soluble sugar (SS) content was determined using the anthrone colorimetric method. The titratable acid (TA) content was determined using the acid-base titration method. The experiment was repeated three times.

[0055] The results showed that Pseudomonas aureus QNF3 had a certain improvement effect on the quality of apple fruit under the condition of disease susceptibility or non-susceptibility. Fig. 9). In the absence of disease, Pseudomonas buffa QNF3 significantly reduced the TA content of apple fruit and increased the TSS / TA ratio. The TA content of fruits treated with Pseudomonas buffa QNF3 was 35.66% lower than that of the control, while TSS / TA increased by 34.98%. In the case of diseased fruit, Pseudomonas buffa QNF3 significantly increased the TSS content and SS / TA ratio in apple fruit. The TSS content of fruits treated with Pseudomonas buffa QNF3 increased by 22.48% and SS / TA increased by 32.04% compared with the control. In addition, Pseudomonas buffa QNF3 significantly maintained the firmness of apple fruit, which was 92.64% higher than that of control fruit.

[0056] Example 10: Inhibitory effect of Pseudomonas aureus QNF3 on the growth of Monilinia fructicola

[0057] 0.5ul, 50ul, and 5ml of Pseudomonas aureus QNF3 (1×10 10 CFU / L) were added to 100ml sterile conical flasks, and then appropriate amounts of PDB culture medium were added to the conical flasks to a final volume of 50ml, with concentrations of 0.001%, 0.1%, and 10%. 50ml PDB liquid culture medium was used as a control. Two Monilinia fructicola cakes (0.053g) with a diameter of 0.7cm were inoculated into each conical flask. The experiment was repeated three times. After all conical flasks were placed in a shaker at 28°C and 200rpm for 48h, the culture was collected and weighed by centrifugation at 5000rpm for 4min to evaluate the degree of inhibition of the growth of the pathogen by the biocontrol bacteria fermentation broth.

[0058] The results showed that different concentrations of Pseudomonas aureus QNF3 had a significant inhibitory effect on Monilinia fructicola. After 48 hours, the weight of the control culture was 503.7 mg, while the weight of the culture treated with 0.001%, 0.1% and 10% Pseudomonas aureus QNF3 was 188.6 mg, 138.01 mg and 65.53 mg, respectively, which was 62.51%, 72.6% and 87.02% less than the control, respectively.

[0059] Example 11: Effect of Pseudomonas aureus QNF3 on the control of peach brown rot caused by Monilinia fructicola inoculation

[0060] Use a sterile needle to make a 3 mm wide × 3 cm deep hole at the equator of the peach fruit. Add 20 ul of Pseudomonas aureus QNF3 (1×10 10CFU / L). 20ul LB medium was used as control. Culture was carried out at 28℃ for 24h. Then, one eighth of a fresh Monilinia fructicola cake with a diameter of 0.7cm was inoculated in the well. All fruits continued to be cultured at 28℃. Five days after inoculation, the diameter of the lesions was measured to evaluate the control effect of Pseudomonas aureus QNF3 on peach fruit brown rot.

[0061] The results showed that Pseudomonas aureus QNF3 had a significant control effect on peach brown rot caused by Monilinia fructicola. Five days after inoculation, both the control fruit and the fruit treated with Pseudomonas aureus QNF3 showed typical disease symptoms. However, the diameter of the lesions on the fruit treated with Pseudomonas aureus QNF3 was significantly smaller than that on the control fruit. On the fifth day, the diameter of the lesions on the fruit treated with Pseudomonas aureus QNF3 was 1.03 cm, which was 87.78% less than that of the control.

[0062] Example 12: Effect of Pseudomonas aureus QNF3 on the prevention and control of brown rot in peach fruit caused by Monilinia fructicola bacterial solution

[0063] Peach fruits were immersed in Pseudomonas aureus QNF3 (1×10 9 CFU / L) for 15 min. After 1 h, the fruits were immersed in Monilinia fructicola for 15 min. The apple fruits were immersed in LB broth as a control. The experiment was repeated three times with 5 biological replicates. All fruits were placed in plastic boxes (29 cm × 20 cm × 10 cm), covered with lids, and kept in a humid environment at 28 ° C. Disease symptoms were observed and the number of lesions on the fruits was recorded to evaluate the control effect of Pseudomonas aureus QNF3 on peach fruit brown rot.

[0064] The results showed that Pseudomonas aureus QNF3 had a significant control effect on the brown rot of peach fruit soaked in Monilinia fructicola. Obvious lesions appeared on the fruit from the second day of inoculation. At 2-4 days, the number of lesions on the control fruit was 3.66, 5.2 and 6.3, respectively, while the number of lesions on the fruit treated with Pseudomonas aureus QNF3 was 0.86, 1.00 and 1.13, respectively, which was 76.5%, 80.77% and 82.06% less than the control, respectively.

[0065] Example 13: Inhibitory effect of Pseudomonas aureus QNF3 on the growth of Alternaria alternata

[0066] 0.5ul, 50ul, and 5ml of Pseudomonas aureus QNF3 (1×10 10 CFU / L) were added to 100ml sterile conical flasks, and then appropriate amounts of PDB culture medium were added to the conical flasks to a final volume of 50ml, with concentrations of 0.001%, 0.1%, and 10%. 50ml PDB liquid culture medium was used as a control. Two Alternaria alternata cakes (0.053g) with a diameter of 0.7cm were inoculated into each conical flask. The experiment was repeated three times. After all conical flasks were placed in a shaker at 28°C and 200rpm for 48h, the culture was collected and weighed by centrifugation at 5000rpm for 4min to evaluate the degree of inhibition of the growth of the pathogen by the biocontrol bacteria fermentation broth.

[0067] The results showed that different concentrations of Pseudomonas aureus QNF3 had a significant inhibitory effect on Alternaria alternata. After 48 hours, the weight of the control culture was 123.16 mg, while the weight of the culture treated with 0.001%, 0.1% and 10% Pseudomonas aureus QNF3 was 71.46 mg, 23.00 mg and 4.73 mg, respectively, which was 41.9%, 81.29% and 96.17% less than the control, respectively.

[0068] Example 14: Control effect of Pseudomonas aureus QNF3 on black spot disease of pear fruit caused by Alternaria alternata

[0069] Use a sterile needle to make a 3 mm wide × 3 cm deep hole at the equator of the pear fruit. Add 20 ul of Pseudomonas aureus QNF3 (1 × 10 10 CFU / L). 20ul LB medium was used as control. Culture was carried out at 28℃ for 24h. Then, one eighth of a fresh Alternaria alternata cake with a diameter of 0.7cm was inoculated in the well. All fruits continued to be cultured at 28℃. Five days after inoculation, the diameter of the lesions was measured to evaluate the control effect of Pseudomonas aureus QNF3 on black spot disease of pear fruit.

[0070] The results showed that Pseudomonas aureus QNF3 had a significant control effect on black spot disease of pear fruit caused by Alternaria alternata. Five days after inoculation, both the control fruit and the fruit treated with Pseudomonas aureus QNF3 showed typical disease symptoms. However, the diameter of the lesions on the fruit treated with Pseudomonas aureus QNF3 was significantly smaller than that on the control fruit. On the fifth day, the diameter of the lesions on the fruit treated with Pseudomonas aureus QNF3 was 1.15 cm, which was 70.56% less than that of the control.

[0071] Example 15: Inhibitory effect of Pseudomonas aureus QNF3 on the growth of Fusarium Oxysporum

[0072] 0.5ul, 50ul, and 5ml of Pseudomonas aureus QNF3 (1×10 10 CFU / L) were added to 100ml sterile conical flasks, and then appropriate amounts of PDB culture medium were added to the conical flasks to a final volume of 50ml, with concentrations of 0.001%, 0.1%, and 10%. 50ml PDB liquid culture medium was used as a control. Two Fusarium Oxysporum cakes (0.053g) with a diameter of 0.7cm were inoculated into each conical flask. The experiment was repeated three times. After all conical flasks were placed in a shaker at 28°C and 200rpm for 48h, the culture was collected and weighed by centrifugation at 5000rpm for 4min to evaluate the degree of inhibition of the growth of the pathogen by the biocontrol bacteria fermentation broth.

[0073] The results showed that different concentrations of Pseudomonas aureus QNF3 had a significant inhibitory effect on Fusarium Oxysporum. After 48 hours, the weight of the control culture was 115.3 mg, while the weight of the culture treated with 0.001%, 0.1% and 10% Pseudomonas aureus QNF3 was 44.3 mg, 21.36 mg and 4.6 mg, respectively, which was 61.51%, 81.52% and 96.18% less than the control, respectively.

[0074] Example 16: Control of tomato fruit rot caused by Fusarium Oxysporum by Pseudomonas aureus QNF3

[0075] Use a sterile needle to make a 3 mm wide × 3 cm deep hole at the equator of the tomato fruit. Add 20 ul of Pseudomonas aureus QNF3 (1 × 10 10 CFU / L). 20ul LB medium was used as control. Culture was carried out at 28℃ for 24h. Then, one eighth of a fresh Fusarium Oxysporum cake with a diameter of 0.7cm was inoculated in the well. All fruits continued to be cultured at 28℃. Five days after inoculation, the diameter of the lesions was measured to evaluate the control effect of Pseudomonas aureus QNF3 on tomato fruit rot.

[0076] The results showed that Pseudomonas aureus QNF3 had a significant control effect on tomato fruit rot caused by Fusarium Oxysporum. Five days after inoculation, both the control fruit and the fruit treated with Pseudomonas aureus QNF3 showed typical disease symptoms. However, the diameter of the lesions on the fruit treated with Pseudomonas aureus QNF3 was significantly smaller than that on the control fruit. On the fifth day, the diameter of the lesions on the fruit treated with Pseudomonas aureus QNF3 was 0.98 cm, which was 83.52% less than that of the control.

[0077] Example 17: Inhibitory effect of Pseudomonas aureus QNF3 on the growth of Aspergills niger

[0078] 0.5ul, 50ul, and 5ml of Pseudomonas aureus QNF3 (1×10 10 CFU / L) were added to 100ml sterile conical flasks, and then appropriate amounts of PDB culture medium were added to the conical flasks to a final volume of 50ml, with concentrations of 0.001%, 0.1%, and 10%. 50ml PDB liquid culture medium was used as a control. Two Aspergills niger cakes (0.053g) with a diameter of 0.7cm were inoculated into each conical flask. The experiment was repeated three times. After all conical flasks were placed in a shaking incubator at 28°C and 200rpm for 48h, the culture was collected and weighed by centrifugation at 5000rpm for 4min to evaluate the degree of inhibition of the growth of the pathogen by the biocontrol bacteria fermentation broth.

[0079] The results showed that different concentrations of Pseudomonas aureus QNF3 had a significant inhibitory effect on Aspergills niger. After 48 hours, the weight of the control culture was 170.13 mg, while the weight of the culture treated with 0.001%, 0.1% and 10% Pseudomonas aureus QNF3 was 71.1 mg, 51.96 mg and 21.96 mg, respectively, which was 58.02%, 69.29% and 87.03% less than the control, respectively.

[0080] Example 18: Control effect of Pseudomonas aureus QNF3 on grape fruit rot caused by Aspergills niger

[0081] Use a sterile needle to make a 3 mm wide × 3 cm deep hole at the equator of the grape fruit. Add 20 ul of Pseudomonas aureus QNF3 (1×10 10CFU / L). 20ul LB medium was used as control. Culture was carried out at 28℃ for 24h. Then, one eighth of a fresh Aspergills niger cake with a diameter of 0.7cm was inoculated in the well. All fruits continued to be cultured at 28℃. Five days after inoculation, the diameter of the lesions was measured to evaluate the control effect of Pseudomonas aureus QNF3 on grape fruit rot.

[0082] The results showed that Pseudomonas aureus QNF3 had a significant control effect on grape fruit rot caused by Aspergills niger. Five days after inoculation, both the control fruit and the fruit treated with Pseudomonas aureus QNF3 showed typical disease symptoms. However, the diameter of the lesions on the fruit treated with Pseudomonas aureus QNF3 was significantly smaller than that on the control fruit. On the fifth day, the diameter of the lesions on the fruit treated with Pseudomonas aureus QNF3 was 1.55 cm, which was 32.1% less than that of the control.

[0083] Example 19: Inhibitory effect of Pseudomonas aureus QNF3 on the growth of Penicillum digitatum

[0084] 0.5ul, 50ul, and 5ml of Pseudomonas aureus QNF3 (1×10 10 CFU / L) were added to 100ml sterile conical flasks, and then appropriate amounts of PDB culture medium were added to the conical flasks to a final volume of 50ml, with concentrations of 0.001%, 0.1%, and 10%. 50ml PDB liquid culture medium was used as a control. Two Penicillium digitatum cakes (0.053g) with a diameter of 0.7cm were inoculated into each conical flask. The experiment was repeated three times. After all conical flasks were placed in a shaker at 28℃ and 200rpm for 48h, the culture was collected and weighed by centrifugation at 5000rpm for 4min to evaluate the degree of inhibition of the growth of the pathogen by the biocontrol bacteria fermentation broth.

[0085] The results showed that different concentrations of Pseudomonas aureus QNF3 had a significant inhibitory effect on Penicillum digitatum. After 48 hours, the weight of the control culture was 84.56 mg, while the weight of the culture treated with 0.001%, 0.1% and 10% Pseudomonas aureus QNF3 was 52.73 mg, 37.73 mg and 12.5 mg, respectively, which was 36.72%, 55.46% and 84.82% less than the control, respectively.

[0086] Example 20: Control effect of Pseudomonas aureus QNF3 on green mold disease of citrus fruits caused by Penicillum digitatum

[0087] Use a sterile needle to make a 3 mm wide × 3 cm deep hole at the equator of the citrus fruit. Add 20 ul of Pseudomonas aureus QNF3 (1 × 10 10 CFU / L). 20ul LB medium was used as control. Culture was carried out at 28℃ for 24h. Then, one eighth of a fresh Penicillum digitatum cake with a diameter of 0.7cm was inoculated in the well. All fruits continued to be cultured at 28℃. Five days after inoculation, the diameter of the lesions was measured to evaluate the control effect of Pseudomonas aureus QNF3 on grape fruit rot.

[0088] The results showed that Pseudomonas aureus QNF3 had a significant control effect on green mold disease of citrus fruit caused by Penicillum digitatum. Five days after inoculation, both the control fruit and the fruit treated with Pseudomonas aureus QNF3 showed typical disease symptoms. However, the diameter of the lesions on the fruit treated with Pseudomonas aureus QNF3 was significantly smaller than that on the control fruit. On the fifth day, the diameter of the lesions on the fruit treated with Pseudomonas aureus QNF3 was 0.67 cm, which was 88.4% less than that of the control.

[0089] Example 21: Inhibitory effect of Pseudomonas aureus QNF3 on the growth of Botrytis cinerea

[0090] 0.5ul, 50ul, and 5ml of Pseudomonas aureus QNF3 (1×10 10 CFU / L) were added to 100ml sterile conical flasks, and then appropriate amounts of PDB culture medium were added to the conical flasks to a final volume of 50ml, with concentrations of 0.001%, 0.1%, and 10%. 50ml PDB liquid culture medium was used as a control. Two Botrytis cinerea cakes (0.053g) with a diameter of 0.7cm were inoculated into each conical flask. The experiment was repeated three times. After all conical flasks were placed in a shaker at 28°C and 200rpm for 48h, the culture was collected and weighed by centrifugation at 5000rpm for 4min to evaluate the degree of inhibition of the growth of the pathogen by the biocontrol bacteria fermentation broth.

[0091] The results showed that different concentrations of Pseudomonas aureus QNF3 had a significant inhibitory effect on Botrytis cinerea. After 48 hours, the weight of the control culture was 126.56 mg, while the weight of the culture treated with 0.001%, 0.1% and 10% Pseudomonas aureus QNF3 was 101.5 mg, 75.73 mg and 21.56 mg, respectively, which was 19.91%, 39.58% and 82.83% less than the control, respectively.

[0092] Example 22: Control effect of Pseudomonas aureus QNF3 on gray mold of peach fruit caused by Botrytis cinerea

[0093] Use a sterile needle to make a 3 mm wide × 3 cm deep hole at the equator of the peach fruit. Add 20 ul of Pseudomonas aureus QNF3 (1×10 10 CFU / L). 20ul LB medium was used as control. Culture was carried out at 28℃ for 24h. Then, one eighth of a fresh Botrytis cinerea cake with a diameter of 0.7cm was inoculated in the well. All fruits continued to be cultured at 28℃. Five days after inoculation, the diameter of the lesions was measured to evaluate the control effect of Pseudomonas aureus QNF3 on gray mold of peach fruit.

[0094] The results showed that Pseudomonas aureus QNF3 had a significant control effect on gray mold of peach fruit caused by Botrytis cinerea. Five days after inoculation, both the control fruit and the fruit treated with Pseudomonas aureus QNF3 showed typical disease symptoms. However, the diameter of the lesions on the fruit treated with Pseudomonas aureus QNF3 was significantly smaller than that on the control fruit. On the fifth day, the diameter of the lesions on the fruit treated with Pseudomonas aureus QNF3 was 0.83 cm, which was 77.85% less than that of the control.

[0095] The present invention relates to the sequence:

[0096] SEQ ID NO: 1

[0097]

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A Pseudomonas lurida QNF3 (Pseudomonas lurida QNF3), characterized in that: It was deposited in the China General Microbiological Culture Collection Center on August 26, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.31739.

2. Use of the Pseudomonas aureus QNF3 according to claim 1 for inhibiting the growth of Botryosphaeria dothidea, Monilinia fructicola, Alternaria alternata, Fusarium Oxysporum, Aspergills niger, Penicillum digitatum, and Botrytis cinerea.

3. The use of the light yellow Pseudomonas QNF3 according to claim 1 for preventing and controlling fruit and vegetable related diseases caused by Botryosphaeria dothidea, Monilinia fructicola, Alternaria alternata, Fusarium Oxysporum, Aspergills niger, Penicillum digitatum, and Botrytis cinerea.

4. The use according to claim 3, characterized in that The fruits and vegetables are apples, peaches, pears, tomatoes, grapes, citrus and other important horticultural crops.

5. The use according to claim 3, characterized in that The fruit and vegetable diseases are apple ring rot, peach brown rot, pear black spot, tomato wilt, grape black aspergillus disease, citrus green mold, or peach gray mold.

6. Use of the Pseudomonas flavus QNF3 according to claim 1 for improving the quality of apple fruit.

7. The use according to claim 6, characterized in that The apple fruit quality refers to the apple fruit firmness, soluble solids content, titratable acid content, solid-acid ratio or sugar-acid ratio.

8. A microbial preparation, characterized in that: The method comprises the Pseudomonas aureus QNF3 according to claim 1.

9. A microbial preparation according to claim 8, characterized in that: A fermentation broth comprising the Pseudomonas aureus QNF3.

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