A light yellow Pseudomonas sp. QNF3 and its application in preventing and controlling fruit and vegetable diseases

Abstract

The present invention relates to the field of microorganisms, and particularly to a Pseudomonas lutea QNF3 and its application in preventing and controlling fruit and vegetable diseases. The Pseudomonas lutea QNF3 of the present invention was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 26, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 31739. The Pseudomonas lutea QNF3 can significantly inhibit the growth of pathogenic bacteria such as Botryosphaeria dothidea, Monilinia fructicola, Alternaria alternata, Fusarium oxysporum, Aspergillus niger, Penicillum digitatum, Botrytis cinerea, etc., and efficiently prevent and control the occurrence of related fruit and vegetable diseases. For example, the control effect of its fermentation broth on apple ring rot caused by Botryosphaeria dothidea is as high as 94.59%; it can also improve the fruit quality, especially the fruit hardness, which plays an important role in extending the fruit storage period.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly to a Pseudomonas lurida QNF3 and its application in preventing and controlling fruit and vegetable diseases. Background Art

[0002] In the fruit tree industry, fungal pathogens such as Botryosphaeria dothidea, Monilinia fructicola, Alternaria alternata, Fusarium oxysporum, Aspergillus niger, Penicillium 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 diseases, not only lead to 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] Currently, these important diseases mainly rely on chemical control. However, the long-term use of chemical agents can cause disadvantages such as environmental pollution, drug resistance, residue problems, and harm to non-target organisms. As a sustainable solution, biological control can reduce the impact on the environment, lower chemical pesticide residues, improve food safety, maintain ecological balance, and effectively reduce the risk of pathogen drug resistance by using natural enemies and microbial agents. Therefore, adopting biological control methods is an important way to ensure the health and sustainable development of the fruit tree industry. Summary of the Invention

[0004] To solve the above technical problems, the present invention isolated and screened an endophytic bacterium, Pseudomonas lurida QNF3, from Chinese chives, 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, and grape Aspergillus niger disease; it can also improve fruit quality.

[0005] The present invention provides a Pseudomonas lurida QNF3, which was deposited with the China General Microbiological Culture Collection Center on August 26, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 31739.

[0006] The present invention also provides the use of the light yellow Pseudomonas QNF3 for inhibiting the growth of pathogenic bacteria such as Botryosphaeria dothidea, Monilinia fructicola, Alternaria alternata, Fusarium oxysporum, Aspergillus niger, Penicillium digitatum, Botrytis cinerea, etc.

[0007] The present invention also provides the use of the light yellow Pseudomonas QNF3 for controlling fruit and vegetable-related diseases caused by pathogenic bacteria such as Botryosphaeria dothidea, Monilinia fructicola, Alternaria alternata, Fusarium oxysporum, Aspergillus niger, Penicillium digitatum, Botrytis cinerea, etc.

[0008] Preferably, the fruits and vegetables are apples, peaches, pears, tomatoes, grapes, citrus fruits 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 Aspergillus black rot, citrus green mold, or peach gray mold.

[0010] The present invention also provides the use of the light yellow Pseudomonas QNF3 for improving the quality of apple fruits.

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

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

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

[0014] 1. The present invention has obtained a new strain, Pseudomonas luteola QNF3, which can significantly inhibit the growth of plant pathogenic fungi such as Botryosphaeria dothidea, Monilinia fructicola, Alternaria alternata, Fusarium oxysporum, Aspergillus niger, Penicillum digitatum, and Botrytis cinerea, and effectively prevent and control the occurrence of related diseases.

[0015] 2. The control effect of the fermentation broth of Pseudomonas luteola QNF3 against apple ring rot is 55.23 - 94.59%, and the control effect of its volatile organic compounds (VOCs) against apple ring rot is 53.00 - 74.00%. The disease prevention methods can be spraying or soaking, or fumigation using its volatiles, all of which can achieve significant antibacterial effects and high control efficiency.

[0016] 3. The Pseudomonas luteola QNF3 of the present invention can also improve fruit quality, especially fruit firmness, which plays an important role in extending the fruit storage period.

[0017] 4. The present invention provides a new candidate biocontrol strain for the green control of plant diseases, enriches the strain resources for the biological control of plant diseases, and develops efficient control measures for specific diseases such as apple ring rot. Description of the Drawings

[0018] Figure 1 It is the phylogenetic tree analysis of Pseudomonas luteola QNF3.

[0019] Figure 2 It is the inhibitory effect of Pseudomonas luteola QNF3 on the growth of Botryosphaeria dothidea (PDA medium). Among them, A: The growth of the control colony after 3 days of inoculation; B: The growth of the colony treated with Pseudomonas luteola QNF3 after 3 days of inoculation; C: Comparison of the diameters of different treatment colonies; D: The inhibition rate of Pseudomonas luteola QNF3 on the growth of Botryosphaeria dothidea.

[0020] Figure 3Inhibitory effect of Pseudomonas luteola QNF3 on the growth of Botryosphaeria dothidea (PDB medium). Among them, A: control mycelial culture after 48 h of treatment; B - D: mycelial cultures treated with 0.02 mL / L, 2 mL / L, and 200 mL / L of Pseudomonas luteola QNF3, respectively; E: weights of Botryosphaeria dothidea cultures under different treatments; F: inhibition rate of Pseudomonas luteola QNF3 on the growth of Botryosphaeria dothidea.

[0021] Figure 4 Control effect of Pseudomonas luteola QNF3 on ring rot of apple fruits inoculated with Botryosphaeria dothidea mycelial disks. Among them, A: control fruits after 3 days of inoculation; B: fruits treated with Pseudomonas luteola QNF3 after 3 days of inoculation; C: comparison of lesion diameters of fruits under different treatments; D: inhibition rate of Pseudomonas luteola QNF3 on the occurrence of ring rot of apple fruits.

[0022] Figure 5 Control effect of Pseudomonas luteola QNF3 on ring rot of apple fruits soaked in Botryosphaeria dothidea bacterial solution. Among them, A: control fruits after 8 days of inoculation; B: fruits treated with Pseudomonas luteola QNF3 after 8 days of inoculation; C: comparison of the number of lesions of fruits under different treatments; D: inhibition rate of Pseudomonas luteola QNF3 on the occurrence of ring rot of apple fruits

[0023] Figure 6 Inhibitory effect of volatile organic compounds (VOCs) of Pseudomonas luteola QNF3 on the growth of Botryosphaeria dothidea. Among them, A: growth of control colonies after 3 days of inoculation; B: growth of colonies treated with VOCs of Pseudomonas luteola QNF3 after 3 days of inoculation; C: comparison of colony diameters under different treatments; D: inhibition rate of VOCs of Pseudomonas luteola QNF3 on the growth of Botryosphaeria dothidea.

[0024] Figure 7The control effect of volatile organic compounds (VOCs) of Pseudomonas luteola QNF3 on apple ring rot caused by inoculating Botryosphaeria dothidea mycelial cakes. Among them, A: Control fruits 3 days after inoculation; B: Fruits treated with VOCs of Pseudomonas luteola QNF3 for 3 days 3 days after inoculation; C: Comparison of lesion diameters of treated and control fruits; D: Inhibition rate of VOCs of Pseudomonas luteola QNF3 on fruit diseases.

[0025] Figure 8 The control effect of volatile organic compounds (VOCs) of Pseudomonas luteola QNF3 on apple ring rot caused by soaking in Botryosphaeria dothidea bacterial solution. Among them, A: Control fruits 8 days after inoculation; B: Fruits treated with VOCs of Pseudomonas luteola QNF3 for 3 days 8 days after inoculation; C: Comparison of the number of lesions on treated and control fruits; D: Inhibition rate of VOCs of Pseudomonas luteola QNF3 on fruit diseases

[0026] Figure 9 The effects of Pseudomonas luteola QNF3 on apple fruit quality parameters. Among them, A: Total soluble solids (TSS); B: Soluble sugar (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 implementation manners

[0027] To make the objectives, technical solutions and beneficial effects of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners. Examples of the embodiments are shown in the drawings. It should be understood that the specific embodiments described in the following implementation manners of the present invention are only illustrative descriptions of the specific implementation manners of the present invention, intended to explain the present invention, and do not constitute a limitation on the present invention.

[0028] The endpoints and any values disclosed in this article are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges.

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

[0030] Fresh single-root red Chinese chive roots were taken from the campus base of Qingdao Agricultural University. The soil on them was washed with tap water, then rinsed with distilled water, surface-sterilized with 75% alcohol, and placed in a laminar flow hood. The fibrous roots of the Chinese chives were cut off, and the outer epidermis of the shortened stem was scraped off with a sterile scalpel. The fibrous roots and peeled shortened stems of the Chinese chives were disinfected: first, soaked in 75% alcohol for 40 s, then soaked in 3% sodium hypochlorite for 5 min to kill the bacteria attached to the surface, and then rinsed 4 times with sterile distilled water. The last rinse of distilled water was retained for use as a control for plating. The disinfected materials were placed in a sterile mortar, added with 10 mL of sterile distilled water and ground into a fine and homogeneous slurry. After standing for 10 min, 100 μL of the supernatant was aspirated from the upper layer and added to the LB plate. Similarly, 100 μL of the same retained sterile rinsing solution was used as a control. They were respectively spread evenly with a spreader, sealed, and placed in an incubator at 28 °C for inverted culture until single colonies grew. The plate confrontation method was used to screen for endophytes with potential antibacterial effects against Botryosphaeria dothidea. The endophytes isolated with significant inhibitory potential against Botryosphaeria dothidea were cultured as single colonies, and Shanghai Bioengineering Technology Company was entrusted to perform 16S rDNA sequencing on them.

[0031] The 16S rDNA sequencing results showed that the 16S rDNA sequence of the isolated endophyte was 1431 bp, as shown in SEQ ID No: 1 (see the sequence listing for details). After importing the 16S rDNA sequence into the ezbiocloud database for online alignment analysis, it was found that this bacterium had the closest genetic relationship with Pseudomonas lurida strain LMG 21995 (NCBI Accession: PDJB01000001) (as Figure 1 shown). Thus, it was determined that this endophyte belonged to Pseudomonas, and it was named Pseudomonas lurida QNF3.

[0032] Example 2: Inhibitory effect of Pseudomonas lurida QNF3 on the mycelial growth of Botryosphaeria dothidea on PDA medium

[0033] Pour 10 ml of PDA medium into a petri dish. After solidification, draw two cross lines on the back of the petri dish. Inoculate a Botryosphaeria dothidea fungal cake with a diameter of 0.7 cm at the intersection point, and inoculate a Pseudomonas lutea QNF3 fungal cake with a diameter of 0.7 cm at a distance of 1.5 cm from the intersection point on each cross line. Use Botryosphaeria dothidea confronted with Pseudomonas lutea QNF3 as the control. Invert all petri dishes and incubate them in an incubator at 28 °C. The experiment is repeated 6 times. After inoculation, use the cross method to measure the colony diameter of Botryosphaeria dothidea to evaluate the inhibitory effect of Pseudomonas lutea QNF3 on the growth of Botryosphaeria dothidea.

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

[0035] Example 3: Inhibitory effect of Pseudomonas lutea QNF3 on the mycelial growth of Botryosphaeria dothidea in PDB medium

[0036] Add 10 mL, 0.1 mL, and 0.001 mL of Pseudomonas lutea QNF3 (1×10 9 CFU / L) into 100 mL Erlenmeyer flasks, and adjust the total volume to 50 mL with PDB medium to make the final concentrations 200 ml / L, 2 ml / L, and 0.02 ml / L. Use PDB medium without adding Pseudomonas lutea QNF3 as the control. Add two Botryosphaeria dothidea fungal cakes with a diameter of 0.7 cm (weight 0.03 g) into each Erlenmeyer flask. The experiment is carried out three times. All Erlenmeyer flasks are shaken at a speed of 150 rpm in a constant temperature shaking incubator at 28 °C. After 48 h, collect the culture by centrifuging at 4000 rpm for 5 min and weigh it to evaluate the inhibitory effect of Pseudomonas lutea QNF3 on the growth of Botryosphaeria dothidea.

[0037] The results showed that Pseudomonas luteola QNF3 had a significant inhibitory effect on the growth of Botryosphaeria dothidea( Figure 3 ). After 48 h, the weight of the culture in the control group was 5.875 g, and the weights of the cultures treated with Pseudomonas luteola QNF3 at concentrations of 0.02 mL / L, 2 mL / L, and 200 mL / L 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: Control effect of Pseudomonas luteola QNF3 on apple ring rot caused by inoculating Botryosphaeria dothidea fungal cakes

[0039] Wound the apple fruits at the equator with a sterile needle, with a wound diameter of 3 mm and a depth of 2 mm. Add 20 μL of Pseudomonas luteola QNF3 (1×10 9 CFU / L) to the wound. Use 20 μL of LB medium as the control. After 1 h, divide a Botryosphaeria dothidea fungal cake with a diameter of 0.7 cm into four equal parts, and take one part to inoculate into the wound of the fruit. All fruits were placed at 28°C. There were 10 fruits in each treatment, and the experiment was repeated 3 times. Measure the lesion diameter every day to evaluate the control effect of Pseudomonas luteola QNF3 on apple ring rot.

[0040] The results showed that Pseudomonas luteola QNF3 had a significant control effect on apple ring rot caused by inoculating Botryosphaeria dothidea fungal cakes( Figure 4 ). At 1 d, 2 d, and 3 d after inoculation, the lesion diameters of the control fruits were 0.05 cm, 0.63 cm, and 1.84 cm, respectively, which were reduced by 88.93%, 68.04%, and 55.23% compared with the control.

[0041] Example 5: Control effect of Pseudomonas luteola QNF3 on apple ring rot caused by soaking in Botryosphaeria dothidea fungal liquid

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

[0043] The results showed that Pseudomonas syringae pv. syringae QNF3 had a significant control effect on ring rot of apple fruits immersed in the Botryosphaeria dothidea bacterial solution ( Figure 5 ). Starting from the 4th day after inoculation, obvious lesions appeared on the fruits. From 4 to 8 days, the number of lesions on the control fruits were 2.46, 6.13, 9.40, 13.60, and 18.53 respectively, while the number of lesions on the fruits treated with Pseudomonas syringae pv. syringae QNF3 were 0.13, 0.40, 1.33, 2.26, and 4.26 respectively, which were reduced by 94.59%, 93.48%, 85.82%, 83.33%, and 76.98% respectively compared with the control.

[0044] Example 6: Inhibitory effect of volatile organic compounds VOCs of Pseudomonas syringae pv. syringae QNF3 on the growth of Botryosphaeria dothidea

[0045] Add 10 mL of PDA medium to a petri dish with a diameter of 9 cm, and add 10 mL of LB medium to the lid of the petri dish. Inoculate a Botryosphaeria dothidea fungal cake with a diameter of 0.7 cm in the center of the PDA medium, and add 100 μL of Pseudomonas syringae pv. syringae QNF3 (1×10 9 CFU / L) to the LB medium, and spread it evenly with a spreader. Use the plate without adding Pseudomonas syringae pv. syringae QNF3 as a control. All petri dishes were placed in an incubator at a constant temperature of 28°C. The diameter of the Botryosphaeria dothidea colony was measured every day to evaluate the inhibitory effect of volatile organic compounds VOCs of Pseudomonas syringae pv. syringae QNF3 on the growth of Botryosphaeria dothidea.

[0046] The results showed that volatile organic compounds VOCs of Pseudomonas syringae pv. syringae 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 the apples treated with volatile organic compounds (VOCs) of Pseudomonas luteola QNF3 were 0.63 cm, 1.32 cm, and 2.24 cm respectively, which were 84.12%, 71.54%, and 62.26% smaller than those of the control.

[0047] Example 7: Control effect of volatile organic compounds (VOCs) of Pseudomonas luteola QNF3 on apple ring rot caused by inoculation with Botryosphaeria dothidea mycelial discs

[0048] Pour 200 mL of LBA medium into the prepared plastic box (29 cm × 20 cm × 10 cm). Add 1 mL of Pseudomonas luteola QNF3 (1×10 9 CFU / L) and spread it evenly on the LBA medium. Then, use a sterile needle to make wounds (diameter 3 mm, depth 2 mm) at the equator of each apple fruit. Inoculate each hole with 1 / 4 of a Botryosphaeria dothidea mycelial disc with a diameter of 0.7 cm. Then, place the apple fruits in the plastic box, cover the lid, and seal it with plastic wrap. Use the apples placed in the plastic box without Pseudomonas luteola QNF3 (1×10 9 CFU / L) as the control. All plastic boxes are placed at a temperature of 28°C for treatment. There are 10 replicates for each treatment, and the experiment is carried out three times. After 3d, measure the disease diameter to evaluate the control effect of the volatile substances (VOCs) of Pseudomonas luteola QNF3 on apple fruit ring rot.

[0049] The results showed that: The volatile organic compounds (VOCs) of Pseudomonas luteola QNF3 had a significant control effect on apple ring rot caused by inoculation with Botryosphaeria dothidea mycelial discs ( Figure 7 ). On the 3rd day, the average lesion diameter of the control fruits was 4.29 cm, while the lesion diameter on the fruits treated with the volatile organic compounds (VOCs) of Pseudomonas luteola QNF3 was 2.02 cm. Compared with the control fruits, the lesion diameter decreased by 53.00%.

[0050] Example 8: Control effect of volatile organic compounds (VOCs) of Pseudomonas luteola QNF3 on apple ring rot caused by soaking in 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 luteola QNF3 (1×10 9(CFU / L) and evenly spread on the LB medium. Use the plastic box without adding Pseudomonas lutea QNF3 as the control. Then, soak the apple fruits in the Botryosphaeria dothidea bacterial solution for 15 min for inoculation. Subsequently, put the apple fruits into the plastic box and place them under the condition of 28 °C. There are 5 biological replicates for each treatment, and the experiment is carried out three times. Observe the disease symptoms, record the diseases, and evaluate the control effect of the volatile organic compounds (VOCs) of Pseudomonas lutea QNF3 on the ring rot of apple fruits soaked in the Botryosphaeria dothidea bacterial solution.

[0052] The results showed that: The volatile organic compounds (VOCs) of Pseudomonas lutea QNF3 had a significant control effect on the ring rot of apple fruits caused by soaking in the Botryosphaeria dothidea bacterial solution ( Figure 8 ). On the 8th day after inoculation, the number of lesions on the fruits treated with the volatile organic compounds (VOCs) of Pseudomonas lutea QNF3 was 3.93, which was 74% less than that of the control.

[0053] Example 9: The effect of Pseudomonas lutea QNF3 on the quality of apples

[0054] The experimental design was divided into 4 treatments, namely (1) soak the fruits in the LB medium for 15 min (CK). (2) Soak in the fermentation broth of Pseudomonas lutea QNF3 (1×10 9 CFU / L) for 15 min (QNF3). (3) Soak the fruits in the LB medium for 15 min, dry at room temperature for 1 h, and then soak them in the Botryosphaeria dothidea bacterial solution for 15 min (Bd). (4) Soak in the fermentation broth of Pseudomonas lutea QNF3 (1×10 9 CFU / L) for 15 min, dry at room temperature for 1 h, and then soak the fruits in the Botryosphaeria dothidea bacterial solution for 15 min (QNF3 + Bd). All fruits were placed in plastic trays and put into an incubator at 28 °C. After 3 d, at the equator of the peeled fruits, use a GY-4 type fruit hardness tester to measure the fruit hardness. Use a PAL-1 digital refractometer to analyze the soluble solid content (TSS) in the fruit juice. The vitamin C (VC) content was determined by the molybdenum blue colorimetric method. The soluble sugar (SS) content was determined by the anthrone colorimetric method. The titratable acid (TA) content was determined by the acid-base titration method. The experiment was repeated three times.

[0055] The results showed that: In the case of being infected or not infected with diseases, Pseudomonas lutea QNF3 had a certain improvement effect on the quality of apple fruits ( Figure 9)。In the case of no disease infection, Pseudomonas lutea QNF3 significantly reduced the TA content of apple fruits and increased the TSS / TA ratio. The TA content of fruits treated with Pseudomonas lutea QNF3 decreased by 35.66% compared to the control, while the TSS / TA increased by 34.98%. In the case of fruit disease infection, Pseudomonas lutea QNF3 significantly increased the TSS content and SS / TA ratio in apple fruits. The TSS content of fruits treated with Pseudomonas lutea QNF3 increased by 22.48% compared to the control, and the SS / TA increased by 32.04%. In addition, Pseudomonas lutea QNF3 significantly maintained the hardness of apple fruits, which was 92.64% higher than that of control fruits.

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

[0057] 0.5 μl, 50 μl, and 5 ml of Pseudomonas lutea QNF3 (1×10 10 CFU / L) were respectively added into 100-ml sterile conical flasks, and then an appropriate amount of PDB medium was added to each conical flask to a final volume of 50 ml, making the concentrations 0.001%, 0.1%, and 10%. 50 ml of PDB liquid medium was used as the control. Two Monilinia fructicola fungal discs (0.053 g) with a diameter of 0.7 cm were inoculated into each conical flask. The experiment was repeated three times. After culturing all conical flasks in a shaker at 28°C and 200 rpm for 48 h, the cultures were centrifuged at 5000 rpm for 4 min to collect the cultures and weighed to evaluate the inhibitory effect of the biocontrol bacteria fermentation broth on the growth of this pathogen.

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

[0059] Example 11: Control effect of Pseudomonas lutea QNF3 on brown rot of peach fruits caused by inoculation with Monilinia fructicola fungal discs

[0060] A 3-mm wide × 3-cm deep hole was made at the equator of the peach fruit with a sterile needle. 20 μl of Pseudomonas lutea QNF3 (1×10 10(CFU / L). 20 μL of LB medium was used as a control. Incubate at 28 °C for 24 h. Then, inoculate an eighth of a mycelial disc with a diameter of 0.7 cm of fresh Monilinia fructicola into the wells. All fruits were continued to be cultured at 28 °C. After 5 days of inoculation, measure the diameter of the lesion to evaluate the control effect of Pseudomonas luteola QNF3 on brown rot of peach fruits.

[0061] The results showed that Pseudomonas luteola QNF3 had a significant control effect on brown rot of peach fruits caused by Monilinia fructicola. After 5 days of inoculation, both the control fruits and the fruits treated with Pseudomonas luteola QNF3 showed typical disease symptoms. However, the diameter of the lesion on the fruits treated with Pseudomonas luteola QNF3 was significantly smaller than that of the control fruits. On the 5th day, the diameter of the lesion on the fruits treated with Pseudomonas luteola QNF3 was 1.03 cm, a reduction of 87.78% compared to the control.

[0062] Example 12: Control effect of Pseudomonas luteola QNF3 on brown rot of peach fruits caused by soaking in Monilinia fructicola bacterial solution

[0063] Soak peach fruits in Pseudomonas luteola QNF3 (1×10 9 CFU / L) for 15 min. After 1 h, soak the fruits in Monilinia fructicola for 15 min. Immerse apple fruits in LB broth medium as a control. This experiment was conducted with 5 biological replicates and repeated three times. All fruits were placed in plastic boxes (29 cm × 20 cm × 10 cm), covered with lids, and kept in a moist environment inside the boxes. Incubate at a constant temperature of 28 °C. Observe the disease symptoms and record the number of lesions on the fruits to evaluate the control effect of Pseudomonas luteola QNF3 on brown rot of peach fruits.

[0064] The results showed that Pseudomonas luteola QNF3 had a significant control effect on brown rot of peach fruits soaked in Monilinia fructicola. Obvious lesions appeared on the fruits starting from the 2nd day after inoculation. On the 2nd - 4th day, the number of lesions on the control fruits were 3.66, 5.2, and 6.3 respectively, while the number of lesions on the fruits treated with Pseudomonas luteola QNF3 were 0.86, 1.00, and 1.13 respectively, a reduction of 76.5%, 80.77%, and 82.06% respectively compared to the control.

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

[0066] 0.5 μl, 50 μl, and 5 ml of light yellow Pseudomonas sp. QNF3 (1×10 10 CFU / L) were respectively added into 100-ml sterile conical flasks. Then, appropriate amounts of PDB medium were added to the conical flasks to a final volume of 50 ml, making the concentrations 0.001%, 0.1%, and 10%. 50 ml of PDB liquid medium served as the control. Two Alternaria alternata fungal discs (0.053 g) with a diameter of 0.7 cm were inoculated into each conical flask. The experiment was repeated three times. After culturing all the conical flasks in a shaker at 28°C and 200 rpm for 48 h, the cultures were centrifuged at 5000 rpm for 4 min to collect the cultures and weighed to evaluate the inhibitory effect of the biocontrol bacteria fermentation broth on the growth of this pathogen.

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

[0068] Example 14: Control effect of light yellow Pseudomonas sp. QNF3 on Alternaria alternata - caused black spot disease of pear fruits

[0069] A 3-mm-wide × 3-cm-deep small hole was made at the equator of the pear fruit with a sterile needle. 20 μl of light yellow Pseudomonas sp. QNF3 (1×10 10 CFU / L) was added into the small hole. 20 μl of LB medium served as the control. The fruits were cultured at 28°C for 24 h. Then, an eighth of a fresh Alternaria alternata fungal disc with a diameter of 0.7 cm was inoculated into the hole. All the fruits were continued to be cultured at 28°C. After 5 d of inoculation, the lesion diameter was measured to evaluate the control effect of light yellow Pseudomonas sp. QNF3 on Alternaria alternata - caused black spot disease of pear fruits.

[0070] The results showed that light yellow Pseudomonas sp. QNF3 had a significant control effect on Alternaria alternata - caused black spot disease of pear fruits. After 5 d of inoculation, both the control fruits and the fruits treated with light yellow Pseudomonas sp. QNF3 showed typical disease symptoms. However, the lesion diameter of the fruits treated with light yellow Pseudomonas sp. QNF3 was significantly smaller than that of the control fruits. On the 5th day, the lesion diameter of the fruits treated with light yellow Pseudomonas sp. QNF3 was 1.15 cm, which was 70.56% less than that of the control.

[0071] Example 15: Inhibitory effect of Pseudomonas luteola QNF3 on the growth of Fusarium oxysporum

[0072] 0.5 μl, 50 μl, and 5 ml of Pseudomonas luteola QNF3 (1×10 10 CFU / L) were added into 100-ml sterile conical flasks respectively. Then, appropriate amount of PDB medium was added into the conical flasks to make the final volume 50 ml, so that the concentrations were 0.001%, 0.1%, and 10%. 50 ml of PDB liquid medium was used as the control. Two mycelial discs (0.053 g) of Fusarium oxysporum with a diameter of 0.7 cm were inoculated into each conical flask. The experiment was repeated three times. After culturing all the conical flasks in a shaker at 28°C and 200 rpm for 48 h, the cultures were collected by centrifugation at 5000 rpm for 4 min and weighed to evaluate the inhibitory degree of the biocontrol bacteria fermentation broth on the growth of this pathogen.

[0073] The results showed that: Different concentrations of Pseudomonas luteola QNF3 had significant inhibitory effects on Fusarium oxysporum. After 48 h, the weight of the control culture was 115.3 mg, while the weights of the cultures treated with 0.001%, 0.1%, and 10% Pseudomonas luteola QNF3 were 44.3 mg, 21.36 mg, and 4.6 mg respectively, which were reduced by 61.51%, 81.52%, and 96.18% respectively compared with the control.

[0074] Example 16: Prevention and control effect of Pseudomonas luteola QNF3 on tomato fruit rot caused by Fusarium oxysporum

[0075] A 3-mm-wide × 3-cm-deep small hole was made at the equator of the tomato fruit with a sterile needle. 20 μl of Pseudomonas luteola QNF3 (1×10 10 CFU / L) was added into the small hole. 20 μl of LB medium was used as the control. The samples were cultured at 28°C for 24 h. Then, one-eighth of a fresh mycelial disc of Fusarium oxysporum with a diameter of 0.7 cm was inoculated into the hole. All the fruits were continuously cultured at 28°C. After 5 d of inoculation, the lesion diameter was measured to evaluate the prevention and control effect of Pseudomonas luteola QNF3 on tomato fruit rot.

[0076] The results showed that Pseudomonas luteola QNF3 had a significant preventive and control effect on tomato fruit rot caused by Fusarium oxysporum. After 5 days of inoculation, both the control fruits and the fruits treated with Pseudomonas luteola QNF3 showed typical disease symptoms. However, the lesion diameter of the fruits treated with Pseudomonas luteola QNF3 was significantly smaller than that of the control fruits. On the 5th day, the lesion diameter of the fruits treated with Pseudomonas luteola QNF3 was 0.98 cm, which was 83.52% less than that of the control.

[0077] Example 17: Inhibitory effect of Pseudomonas luteola QNF3 on the growth of Aspergillus niger

[0078] 0.5 μl, 50 μl, and 5 ml of Pseudomonas luteola QNF3 (1×10 10 CFU / L) were respectively added into 100-ml sterile conical flasks, and then an appropriate amount of PDB medium was added to each conical flask to a final volume of 50 ml, making the concentrations 0.001%, 0.1%, and 10%. 50 ml of PDB liquid medium was used as the control. Two Aspergillus niger agar discs (0.053 g) with a diameter of 0.7 cm were inoculated into each conical flask. The experiment was repeated three times. After culturing all the conical flasks in a shaker at 28 °C and 200 rpm for 48 h, the cultures were collected by centrifugation at 5000 rpm for 4 min and weighed to evaluate the inhibitory effect of the biocontrol bacteria fermentation broth on the growth of the pathogen.

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

[0080] Example 18: Preventive and control effect of Pseudomonas luteola QNF3 on grape fruit rot caused by Aspergillus niger

[0081] A 3-mm wide × 3-cm deep small hole was made at the equator of the grape fruit with a sterile needle. 20 μl of Pseudomonas luteola QNF3 (1×10 10CFU / L). 20 μl of LB medium was used as a control. Incubate at 28 °C for 24 h. Then, inoculate an eighth of a fresh Aspergillus niger fungal disc with a diameter of 0.7 cm into the wells. All fruits were continued to be incubated at 28 °C. After 5 days of inoculation, measure the diameter of the lesion to evaluate the control effect of Pseudomonas luteola QNF3 on grape fruit rot.

[0082] The results showed that Pseudomonas luteola QNF3 had a significant control effect on grape fruit rot caused by Aspergillus niger. After 5 days of inoculation, both the control fruits and the fruits treated with Pseudomonas luteola QNF3 showed typical disease symptoms. However, the diameter of the lesion on the fruits treated with Pseudomonas luteola QNF3 was significantly smaller than that of the control fruits. On the 5th day, the diameter of the lesion on the fruits treated with Pseudomonas luteola QNF3 was 1.55 cm, a 32.1% reduction compared to the control.

[0083] Example 19: Inhibitory effect of Pseudomonas luteola QNF3 on the growth of Penicillium digitatum

[0084] Add 0.5 μl, 50 μl, and 5 ml of Pseudomonas luteola QNF3 (1×10 10 CFU / L) into 100 ml sterile conical flasks respectively, and then add an appropriate amount of PDB medium to each conical flask to a final volume of 50 ml, making the concentration 0.001%, 0.1%, and 10%. 50 ml of PDB liquid medium was used as a control. Inoculate 2 Penicillium digitatum fungal discs (0.053 g) with a diameter of 0.7 cm into each conical flask respectively. The experiment was repeated three times. After culturing all conical flasks in a shaker at 28 °C and 200 rpm for 48 h, centrifuge at 5000 rpm for 4 min to collect the culture and weigh it to evaluate the inhibitory effect of the biocontrol bacteria fermentation broth on the growth of this pathogen.

[0085] The results showed that different concentrations of Pseudomonas luteola QNF3 had a significant inhibitory effect on Penicillium digitatum. After 48 h, the weight of the control culture was 84.56 mg, while the weights of the cultures treated with 0.001%, 0.1%, and 10% Pseudomonas luteola QNF3 were 52.73 mg, 37.73 mg, and 12.5 mg respectively, a 36.72%, 55.46%, and 84.82% reduction compared to the control.

[0086] Example 20: Control effect of Pseudomonas luteola QNF3 on green mold of citrus fruits caused by Penicillium digitatum

[0087] Use a sterile needle to make a small hole 3 mm wide × 3 cm deep at the equator of the citrus fruit. Add 20 μl of light yellow Pseudomonas sp. QNF3 (1×10 10 CFU / L) into the small hole. Use 20 μl of LB medium as a control. Incubate at 28 °C for 24 h. Then, inoculate an eighth of a mycelial disc with a diameter of 0.7 cm of fresh Penicillium digitatum into the hole. All fruits were continuously incubated at 28 °C. After 5 days of inoculation, measure the diameter of the lesion to evaluate the control effect of light yellow Pseudomonas sp. QNF3 on the decay of grape fruits.

[0088] The results showed that: Light yellow Pseudomonas sp. QNF3 had a significant control effect on the green mold of citrus fruits caused by Penicillium digitatum. After 5 days of inoculation, both the control fruits and the fruits treated with light yellow Pseudomonas sp. QNF3 showed typical disease symptoms. However, the diameter of the lesion of the fruits treated with light yellow Pseudomonas sp. QNF3 was significantly smaller than that of the control fruits. On the 5th day, the diameter of the lesion of the fruits treated with light yellow Pseudomonas sp. QNF3 was 0.67 cm, which was 88.4% less than that of the control.

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

[0090] Add 0.5 μl, 50 μl, and 5 ml of light yellow Pseudomonas sp. QNF3 (1×10 10 CFU / L) into 100-ml sterile conical flasks respectively, and then add an appropriate amount of PDB medium into the conical flasks to a final volume of 50 ml to make the concentration 0.001%, 0.1%, and 10%. Use 50 ml of PDB liquid medium as a control. Inoculate 2 mycelial discs with a diameter of 0.7 cm (0.053 g) of Botrytis cinerea into each conical flask respectively. The experiment was repeated three times. After incubating all conical flasks in a shaker at 28 °C and 200 rpm for 48 h, centrifuge at 5000 rpm for 4 min to collect the culture and weigh it to evaluate the inhibitory degree of the fermentation broth of the biocontrol bacterium on the growth of this pathogen.

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

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

[0093] Use a sterile needle to make a small hole 3 mm wide × 3 cm deep at the equator of the peach fruit. Add 20 μl of Pseudomonas luteola QNF3 (1×10 10 CFU / L) into the small hole. Use 20 μl of LB medium as the control. Incubate at 28 °C for 24 h. Then, inoculate an eighth of a mycelial disc with a diameter of 0.7 cm of fresh Botrytis cinerea into the hole. All fruits were continuously incubated at 28 °C. After 5 days of inoculation, measure the lesion diameter to evaluate the control effect of Pseudomonas luteola QNF3 on gray mold of peach fruits.

[0094] The results showed that: Pseudomonas luteola QNF3 had a significant control effect on gray mold of peach fruits caused by Botrytis cinerea. After 5 days of inoculation, both the control fruits and the fruits treated with Pseudomonas luteola QNF3 showed typical disease symptoms. However, the lesion diameter of the fruits treated with Pseudomonas luteola QNF3 was significantly smaller than that of the control fruits. On the 5th day, the lesion diameter of the fruits treated with Pseudomonas luteola QNF3 was 0.83 cm, a 77.85% reduction compared to the control.

[0095] Sequences involved in the present invention:

[0096] SEQ ID NO: 1

[0097]

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation to the content of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

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

2. Use of a light yellow Pseudomonas sp. QNF3 as described in claim 1 for inhibiting the growth of Botryosphaeria dothidea, Monilinia fructicola, Fusarium oxysporum, Aspergillus niger, Penicillum digitatum, Botrytis cinerea.

3. Use of a light yellow Pseudomonas sp. QNF3 as described in claim 1 for controlling fruit and vegetable related diseases caused by Botryosphaeria dothidea, Monilinia fructicola, Fusarium oxysporum, Aspergillus niger, Penicillum digitatum, Botrytis cinerea.

4. The application according to claim 3, characterized in that, The fruits and vegetables are apples, peaches, tomatoes, grapes or oranges.

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

6. Use of Pseudomonas luteola QNF3 according to claim 1 for improving the quality of apple fruits, characterized in that, The apple fruit quality refers to apple fruit hardness, soluble solid content, titratable acid content, solid-acid ratio or sugar-acid ratio.

7. A microbial preparation, characterized in that, It contains the light yellow Pseudomonas sp. QNF3 as described in claim 1.

8. A microbial preparation according to claim 7, wherein, It contains the fermentation broth of the light yellow Pseudomonas sp. QNF3.

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