Bacillus subtilis QNF2 and application thereof in prevention and control of fruit and vegetable diseases

Abstract

The invention relates to the field of microorganisms, in particular to bacillus subtilis QNF2 and application of the bacillus subtilis QNF2 to prevention and control of fruit and vegetable diseases. The bacillus subtilis QNF2 disclosed by the invention is preserved in the 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.31740. The bacillus subtilis QNF2 disclosed by the invention has the advantages that the bacillus subtilis QNF2 disclosed by the invention can be used for preparing the bacillus subtilis QNF2; according to the present invention, with the application of the bacillus subtilis QNF2, the growth of pathogenic bacteria such as Monilinia fucicola, Botryosphaeria dothidea, Alternaria alternata, Fusarium oxysporum, Aspergillus niger, Penicillium digitatum, Botrytis cinerea and the like can be significantly inhibited, and the occurrence of related fruit and vegetable diseases can be efficiently prevented and controlled, for example, the prevention and control effect of the volatile matter VOCs of the bacillus subtilis QNF2 on the peach brown rot caused by the Monilinia fucicola can be up to 98%; 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 Bacillus subtilis QNF2 and an application thereof in preventing and controlling peach brown rot and other fruit and vegetable diseases. Background Art

[0002] Plant diseases are one of the main limiting factors in agricultural production, seriously affecting the yield and quality of crops. According to statistics, diseases caused by various plant pathogenic microorganisms can lead to a 10%-30% reduction in crop yield each year. Traditional plant disease prevention and control mainly relies on chemical pesticides. However, with the extensive use of chemical pesticides, the resistance of pathogens continues to increase, and the negative impact of chemical pesticides on the environment and human and animal health is becoming increasingly prominent. Therefore, it has become particularly urgent to develop safe, environmentally friendly and effective biological control measures.

[0003] Using antagonistic microorganisms to control plant diseases has the advantages of high safety, environmental friendliness, no chemical residues, reduced use of chemical pesticides, reduced agricultural production costs, and improved food safety. For example, CN117660201A discloses an Indian Glycosporium strain Yc10, which can be used to inhibit post-harvest diseases of fruits and vegetables, especially for Wenzhou mandarin oranges and London late navel oranges. CN110292051B discloses an Aspergillus tubingensis QF05, which has an inhibitory effect on tomato gray mold, pepper anthracnose, cucumber wilt, potato early blight, and cucumber bacterial angular leaf spot on fruit and vegetable crops, and effectively increases the yield of cucumbers.

[0004] However, there are some urgent problems in the field of plant disease biological control, such as insufficient strain diversity, which leads to the reliance of biological control agents on a few common strains on the market, limiting the diversity and adaptability of products; the existing strains are not capable of dealing with pathogen changes and drug resistance, and the scope of prevention and control is limited; some biological control agents may affect fruit quality while preventing and controlling diseases, affecting the market competitiveness and safety of agricultural products; many measures can only target specific pathogens, requiring a combination of multiple methods to increase costs and complexity, and long-term use of specific microbial control agents may still lead to tolerance or resistance of pathogens, reducing the effectiveness of prevention and control, etc. These factors have jointly restricted the widespread application and application effect of biological control technology.

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention is proposed. Summary of the invention

[0006] In order to solve the above technical problems, the present invention isolates and screens an endophyte Bacillus subtilis QNF2 from leek, which can significantly inhibit the growth of pathogens such as Monilinia fructicola, Botryosphaeria dothidea, Alternaria alternata, Fusarium Oxysporum, Aspergills niger, Penicillum digitatum, Botrytis cinerea, etc., and effectively prevent and control the occurrence of related fruit and vegetable diseases; it can also improve the quality of fruits, especially the hardness of fruits, and play an important role in prolonging the storage period of fruits.

[0007] The present invention provides a Bacillus subtilis QNF2, which was deposited in the China General Microbiological Culture Collection Center on August 26, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.31740.

[0008] The present invention also provides the use of the Bacillus subtilis QNF2 for inhibiting the growth of pathogens such as Monilinia fructicola, Botryosphaeria dothidea, Alternaria alternata, Fusarium Oxysporum, Aspergills niger, Penicillum digitatum, and Botrytis cinerea.

[0009] The present invention also provides the use of the Bacillus subtilis QNF2 for preventing and controlling fruit and vegetable related diseases caused by pathogens such as Monilinia fructicola, Botryosphaeria dothidea, Alternaria alternata, Fusarium Oxysporum, Aspergills niger, Penicillum digitatum, and Botrytis cinerea.

[0010] Preferably, the fruits and vegetables are peaches, apples, pears, tomatoes, grapes, citrus and other important horticultural crops.

[0011] Preferably, the fruit and vegetable diseases are peach brown rot, apple ring rot, pear black spot, tomato wilt, grape black aspergillus disease, citrus green mold, or peach gray mold.

[0012] The present invention also provides the use of the Bacillus subtilis QNF2 for improving the quality of peach fruit.

[0013] Preferably, the peach fruit quality refers to peach fruit hardness, peach fruit solid-acid ratio or peach fruit sugar-acid ratio.

[0014] The present invention also provides a microbial preparation, comprising the Bacillus subtilis QNF2; preferably, comprising the fermentation broth of the Bacillus subtilis QNF2.

[0015] Compared with the prior art, the beneficial effects of the present invention include at least:

[0016] 1. The present invention obtains a novel strain Bacillus subtilis QNF2, which can significantly inhibit the growth of Monilinia fructicola, Botryosphaeria dothidea, Alternaria alternata, Fusarium Oxysporum, Aspergillsniger, Penicillum digitatum, Botrytis cinerea, etc., and effectively prevent and control the occurrence of related diseases.

[0017] 2. The volatiles of Bacillus subtilis QNF2 fermentation broth have a prevention and control effect of up to 98% on peach brown rot.

[0018] 3. The Bacillus subtilis QNF2 of the present invention can also improve fruit quality, especially fruit hardness, and plays an important role in extending the storage period of fruits.

[0019] 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 peach brown rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Phylogenetic tree analysis of the endophytic bacterium Bacillus subtilis QNF2;

[0021] Figure 2The endophytic bacterium Bacillus subtilis QNF2 inhibits the growth of M. fructicola on PDA medium. A: The growth of the control M. fructicola after 6 days; B: The growth of M. fructicola treated with endophytic bacterium Bacillus subtilis QNF2 after 6 days; C: Comparison of colony diameters of M. fructicola treated with endophytic bacterium Bacillus subtilis QNF2 and control M. fructicola on the 6th day; D: The inhibition rate of endophytic bacterium Bacillus subtilis QNF2 on the growth of M. fructicola on the 6th day. ****P<0.0001

[0022] Figure 3 The inhibitory effect of endophytic Bacillus subtilis QNF2 on the mycelial growth of M. fructicola in PDB medium. Control mycelium (A) and cultures treated with 0.001% (B), 0.01% (C), 0.1% (D), 1% (E), and 10% (F) endophytic Bacillus subtilis QNF2 for 48 h. The weights of mycelial cultures treated with different treatments were different (G). ns: not significant; **, P < 0.01; *** P < 0.001; **** P < 0.0001.

[0023] Figure 4 The inhibitory effect of the cell-free supernatant (CFS) of the endophytic fungus Bacillus subtilis QNF2 on the mycelial growth of M. fructicola. On the 6th day, the control colony (A) and the colonies treated with 10% (B), 20% (C), 30% (D), 40% (E) and 50% (F) CFS. The diameters of mycelial colonies under different treatments were different (G), ns: not significant; **, P < 0.01; **** P < 0.0001.

[0024] Figure 5 The inhibitory effect of endophytic Bacillus subtilis QNF2 VOCs on the mycelial growth of M. fructicola. A: Control mycelia grown on PDA medium for 6 days. B: Mycelia treated with endophytic Bacillus subtilis QNF2 VOCs grown on PDA medium for 6 days. C: Colony diameters of control mycelia and mycelia treated with endophytic Bacillus subtilis QNF2 VOCs. ****, P<0.0001.

[0025] Figure 6 The control effect of endophytic bacteria Bacillus subtilis QNF2 on brown rot of peach fruit inoculated with M. fructicola. A, C: control peach fruit 3 days after inoculation; B, D: peach fruit treated with endophytic bacteria Bacillus subtilis QNF2 3 days after inoculation. E: Comparison of lesion diameters on treated and control fruits 3 days after inoculation.

[0026] Figure 7 The control effect of endophytic bacteria Bacillus subtilis QNF2 on brown rot of post-harvest peach fruit soaked in M. fructicola culture solution. A, C: Disease symptoms of control fruit 3 days after inoculation; B, D: Disease symptoms of fruit treated with endophytic bacteria Bacillus subtilis QNF2 3 days after inoculation; E: Comparison of the number of lesions between fruit treated with endophytic bacteria Bacillus subtilis QNF2 and control fruit during the test period.

[0027] Figure 8 The inhibitory effect of endophytic Bacillus subtilis QNF2 VOCs on brown rot of peach fruit 3 days after inoculation with M. fructicola. A: Lesions on control fruit. B: Diseases on fruit treated with endophytic Bacillus subtilis QNF2 VOCs. C: Comparison of the diameter of lesions on control mycelium and endophytic Bacillus subtilis QNF2 VOCs-treated fruit. ****, P<0.0001.

[0028] Fig. 9 Effects of endophyte Bacillus subtilis QNF2 on postharvest peach fruit quality. Soluble solids (A), soluble sugars (B), titratable acid (C), solid-acid ratio (D), sugar-acid ratio (E), vitamin C (F), and fruit firmness (G). Control: untreated peach fruit; QNF2: fruit treated with endophyte Bacillus subtilis QNF2; Mf: peach fruit inoculated with M. fruiticola; QNF2+Mf: peach fruit treated with endophyte Bacillus subtilis QNF2 and then inoculated with M. fruiticola. ns: not significant; *, P<0.05; **, P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] Example 1: Isolation, screening and identification of Bacillus subtilis QNF2 strain

[0032] Fresh single-root red leek roots were taken from the campus base of Qingdao Agricultural University. They were washed with tap water to remove dirt, then rinsed with distilled water, and placed in a clean bench after disinfecting the outer surface with 75% alcohol. The leek fibrous roots were cut off, and the outer skin 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 bacteria attached to the surface, then rinsed with sterile distilled water 3 times to remove sodium hypochlorite, and then rinsed with distilled water once, and the distilled water was retained for plate control. The disinfected leek material was placed in a sterile mortar, and 10 mL of sterile distilled water was added to grind it into a fine slurry. It was allowed to stand for 10 minutes. 100 μL of supernatant was drawn from the upper layer and added to the LB plate. 100 μL of the retained sterile rinsing solution was also drawn as a control. They were evenly coated with a coating rod, sealed, and placed in a 28°C constant temperature incubator for inversion culture until a single colony grew. The plate confrontation method was used to screen endophytes with potential antibacterial effects on Monilinia fructicola. The isolated endophytes with significant antibacterial potential against brown rot pathogens were cultured in single colonies, and Shanghai Bioengineering Technology Co., Ltd. was commissioned to sequence their 16S rDNA.

[0033] The results of 16S rDNA sequencing showed that the 16S rDNA sequence of the isolated endophyte was 1443 bp, as shown in SEQ ID NO: 1. 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 Bacillus subtilis strain NCIB 3610 (T) (NCBI Accession: ABQL01000001) (e.g. Figure 1 The isolated endophyte was determined to belong to the genus Bacillus and was named Bacillus subtilis QNF2.

[0034] Example 2: Inhibitory effect of Bacillus subtilis QNF2 on the mycelial growth of M. fructicola on PDA medium

[0035] 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 M. fructicola cake at the intersection, and inoculate a 0.7cm diameter endophytic bacterium Bacillus subtilis QNF2 cake at 1.5cm away from the intersection on the intersection line. The M. fructicola that does not confront the endophytic bacterium Bacillus subtilis QNF2 of the biocontrol bacteria is used as the control. All culture dishes are inverted in an incubator at 28℃ for culture, and the experiment is repeated 3 times. The cross method is used to measure the colony diameter of M. fructicola on the 5th day after inoculation to evaluate the inhibitory effect of the endophytic bacterium Bacillus subtilis QNF2 cake on the growth of M. fructicola.

[0036] The results showed that the endophytic bacterium Bacillus subtilis QNF2 had a significant inhibitory effect on the mycelial growth of M. fructicola on PDA medium. Figure 2 By the fifth day, the diameter of the control colony was 3.12 cm, while the diameter of the colony treated with the endophytic bacterium Bacillus subtilis QNF2 was 1.49 cm ( Figure 2 C), 67.17% less than the control ( Figure 2 (middle D).

[0037] Example 3: Inhibitory effect of Bacillus subtilis QNF2 on hyphae growth of M. fructicola on PDB medium

[0038] 5000 μL, 500 μL, 50 μL and 5 μL of endophytic bacteria Bacillus subtilis QNF2 fermentation broth (1×10 10 CFU / mL) were added to 100mL sterile flasks, and then different volumes of PDB medium were added to the flasks to make the total volume reach 50mL, and the final concentrations were 10%, 0.1%, 1%, 0.1% and 0.01%, respectively. 50ml PDB medium was used as a control. Then, two fresh M. fructicola cakes (0.053g) with a diameter of 0.7cm were placed in the flask. Subsequently, all flasks were cultured at 28°C and 200rpm. The experiment was repeated three times. After 48 hours, the hyphae were collected and weighed to evaluate the inhibitory effect of the endophytic bacterium Bacillus subtilis QNF2 on the growth of M. fructicola in PDB medium.

[0039] The results showed that different concentrations of endophyte Bacillus subtilis QNF2 had different inhibitory effects on the growth of M. fructicola ( Figure 3After 48 h, the weight of the control culture was 790.04 mg, while the weights of the cake cultures treated with 0.001%, 0.01%, 0.1%, 1% and 10% endophyte Bacillus subtilis QNF2 were 617.91 mg, 477.33 mg, 377.16 mg, 263.44 mg and 105.51 mg, respectively, which were 23.35%, 42.43%, 56.02%, 71.45% and 92.88% lighter than the control, respectively. Figure 3 Middle G).

[0040] Example 4: Inhibitory effect of Bacillus subtilis QNF2 cell-free supernatant (CFS) on the mycelial growth of M. fructicola

[0041] The overnight endophyte Bacillus subtilis QNF2 was prepared into 1×10 10 CFU / mL. Centrifuge at 6000rpm for 10min, filter 3 times with 0.22μm filter membrane to obtain cell-free supernatant (CFS). Mix 5mL, 10mL, 15mL, 20mL, and 25mL of CFS with 45mL, 40mL, 35mL, 30mL, and 25mL of liquid PDA medium to make the final concentration of CFS 10%, 20%, 30%, 40%, and 50%, respectively. Pour the mixed solution into a culture dish with a diameter of 9cm, and use the PDA medium without cell-free supernatant as the control. After the PDA medium becomes solid, a fresh M. fructicola cake (diameter 0.7cm) is inoculated in the center. The experiment was performed 5 times. All culture dishes were cultured in the dark at 28℃. The colony diameter was measured on the 6th day of inoculation to evaluate the inhibitory effect of the cell-free supernatant of endophytic bacterium Bacillus subtilis QNF2 on the mycelial growth of M. fructicola.

[0042] The results showed that on the 6th day, the growth of M. fructicola colonies treated with different concentrations of endophytic bacteria Bacillus subtilis QNF2 cell-free supernatant was significantly different ( Figure 4 The colony diameters of M. fructicola treated with 10%, 20%, 30%, 40% and 50% cell-free supernatant were 4.56cm, 4.52cm, 3.87cm, 1.71cm and 0.81cm, which were 7.79%, 7.06%, 23.3%, 75.49%, 97.4% and 10.6% smaller than those of the control group, respectively. Figure 4 Middle G).

[0043] Example 5: Inhibitory effect of volatile organic compounds (VOCs) from Bacillus subtilis QNF2 on the mycelial growth of M. fructicola

[0044] The in vitro dual culture method was used for determination. 10 mL of PDA culture medium was poured into a 9 cm diameter culture dish. After the culture medium solidified, a 0.7 cm diameter M. fructicola cake was inoculated in the center. 10 mL of LB solid culture medium was poured into the inner lid of the culture dish. After it solidified, 100 μL of 1×10 10 CFU / mL of endophytic bacteria Bacillus subtilis QNF2. 100μl sterile distilled water was used as control. All culture dishes were sealed and inverted at 28℃ in the dark. The colony diameter was measured by the cross method every day. The experiment was repeated 5 times in total.

[0045] The VOCs of endophytic Bacillus subtilis QNF2 significantly inhibited the mycelial growth of endophytic Bacillus subtilis QNF2. On the 6th day, all control mycelia grew tightly on the surface of the culture medium ( Figure 5 However, the mycelium of the endophytic fungus Bacillus subtilis QNF2 treated with VOCs was sparse and almost all grew into the culture medium, making it difficult to see ( Figure 5 (B). On days 1-6, the diameters of mycelial colonies of endophytic bacteria Bacillus subtilis QNF2 treated with VOCs were 0.70 cm, 0.70 cm, 0.71 cm, 0.73 cm, 0.92 cm, and 1.41 cm, respectively. Figure 5 (C), which were 100.00%, 100.00%, 98.92%, 98.11%, 89.86% and 73.53% smaller than the control, respectively.

[0046] Example 6: Control effect of Bacillus subtilis QNF2 on brown rot of peach fruit inoculated with M. fructicola

[0047] Use a sterile needle to make a 3 mm wide × 2 cm deep hole at the equator of each peach fruit. Add 20 μL of 1 × 10 10 CFU / mL endophyte Bacillus subtilis QNF2. 20 μL LB medium was used as control. Cultured at 28℃ for 24 h. Then, a quarter of a fresh M. fructicola cake with a diameter of 0.7 cm was inoculated in the wells. All fruits were incubated at 28℃. Five fruits were treated for each experiment and repeated 3 times. The lesion diameter was measured every day to evaluate the control effect of endophyte Bacillus subtilis QNF2 on peach fruit brown rot.

[0048] The results showed that: 3 days after inoculation, the control fruit ( Figure 6 A, C) and fruits treated with endophytic bacteria Bacillus subtilis QNF2 ( Figure 6The diameters of the lesions on the fruits treated with endophyte Bacillus subtilis QNF2 were 0.41 cm, 0.73 cm and 1.58 cm on the 1st, 2nd and 3rd day, respectively. Figure 6 (E), which were reduced by 88.67%, 83.95% and 76.47% respectively compared with the control.

[0049] Example 7: Control effect of Bacillus subtilis QNF2 on brown rot of peach fruit soaked in M. fructicola

[0050] Peach fruit was fermented in the endophytic bacteria Bacillus subtilis QNF2 (1×10 10 CFU / mL) for 15 min, and then soaked in M. fructicola solution (1×105 CFU / mL) for 15 min after 1 h. Peach fruits that were soaked in LB medium for 15 min, and then soaked in M. fructicola solution (1×105 CFU / mL) for 15 min after 1 h were used as controls. Five fruits were treated in each treatment, and the experiment was repeated 3 times. All peach fruits were placed in plastic trays and cultured at 28°C for 3 days. The number of lesions on the fruits was recorded to evaluate the control effect of endophytic bacterium Bacillus subtilis QNF2 on peach fruit brown rot.

[0051] The results showed that 3 days after inoculation, the control peach fruit ( Figure 7 A, C) and endophytic bacteria Bacillus subtilis QNF2 treatment ( Figure 7 B, D) showed disease symptoms. However, the disease symptoms of peach fruits treated with endophytic bacteria Bacillus subtilis QNF2 were milder than those of the control fruits. On the 1st, 2nd and 3rd day, the number of lesions on the control fruits was 3.67, 5.20 and 6.33, respectively, while the number of lesions on the fruits treated with endophytic bacteria Bacillus subtilis QNF2 was 0.13, 0.87 and 1.33, respectively. Figure 7 E), which were reduced by 96.36%, 83.33% and 78.95% compared with the control, respectively.

[0052] Example 8: Effect of volatile organic compounds (VOCs) of Bacillus subtilis QNF2 on the prevention and control of peach brown rot

[0053] 200 mL of LB medium was added to the bottom of the plastic box (volume 4.5 L). After the medium solidified, 1 mL of P.protegens QNF1 (1×10 10CFU / mL) was evenly spread on the surface of LB culture medium. A plastic box without endophyte Bacillus subtilis QNF2 was used as a control. After making a small hole (3 mm in diameter and 3 mm in depth) at the equator of the peach fruit, the fruit was placed in a plastic box. In order to avoid direct contact with the culture medium, a mineral water bottle cap was placed on the culture medium, and then the fruit was placed on the bottle cap. All plastic boxes were sealed and cultured at 28°C for 24 hours. The fruit was removed and a 3 mm diameter M.fructicola cake was inoculated in the small hole on the fruit. The plastic box was sealed again and cultured under the same conditions for 3 days. The lesion diameter was measured by the cross method. Each treatment was repeated 5 times, and the experiment was repeated 3 times.

[0054] The results showed that after the fruit was inoculated with M. fructicola and cultured at 28℃ for 3 days, the control fruit showed typical disease symptoms, with gray hyphae growing on the lesions ( Figure 8 (A). Some fruits treated with volatile organic substances from the endophyte Bacillus subtilis QNF2 also showed symptoms of brown rot ( Figure 8 The diameter of the lesions on the control fruits was 1.56 cm, while the diameter of the lesions on the fruits treated with volatile organic substances of the endophyte Bacillus subtilis QNF2 was 0.47 cm on average, which was 86.74% smaller than that of the control ( Figure 8 Middle C).

[0055] Example 9: Bacillus subtilis QNF2 improves peach fruit quality

[0056] The experimental design was divided into four treatments: (1) immersing the fruit in LB medium for 15 min (control); (2) immersing the fruit in the fermentation broth of endophytic bacillus subtilis QNF2 (1×10 10 CFU / mL) for 15 min (endophyte Bacillus subtilis QNF2). (3) The fruit was soaked in LB medium for 15 min, dried at room temperature for 1 h, and then soaked in M. fructicola culture medium (1×105 spores / mL) for 15 min (MF). (4) The fermentation liquid of endophyte Bacillus subtilis QNF2 (1×10 10 CFU / mL) for 15 min, dried at room temperature for 1 h, and then soaked in M. fructicola solution (1×105 spores / mL) for 15 min (endophytic bacteria Bacillus subtilis QNF2+MF). 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 total soluble solids (TSS) content in the juice was analyzed using a PAL-1 digital refractometer. The vitamin C (VC) content was determined using the molybdenum blue colorimetry. The soluble sugar content was determined using the anthrone colorimetry. The titratable acid (TA) content was determined using the acid-base titration method. The experiment was repeated three times.

[0057] The results showed that the effect of endophyte Bacillus subtilis QNF2 on the quality of peach fruit infected with M. fructicola and not infected with M. fructicola was determined on the third day after inoculation. When the fruit was not infected with M. fructicola, the effect of endophyte Bacillus subtilis QNF2 on the quality of the fruit was not significant ( Fig. 9 AG). However, when the fruit was infected with M. fructicola, the endophyte Bacillus subtilis QNF2 significantly reduced the titratable acid content, thereby increasing the solid-acid ratio and sugar-acid ratio. Compared with the control, the titratable acid content in the fruit treated with endophyte Bacillus subtilis QNF2 was reduced by 50.00% ( Fig. 9 C), the solid-acid ratio and sugar-acid ratio increased by 130.59% ( Fig. 9 D) and 133.62% ( Fig. 9 In addition, the endophyte Bacillus subtilis QNF2 significantly maintained the fruit firmness. The fruit firmness after treatment with the endophyte Bacillus subtilis QNF2 was 4.67 kg / cm2, 49.77% higher than the control ( Fig. 9 Middle G).

[0058] Example 10: Bacillus subtilis QNF2 significantly inhibits the growth of Botryosphaeria dothidea

[0059] Endophytic bacteria Bacillus subtilis QNF2 (1×10 9 CFU / L) 10ml, 0.1ml and 0.001ml were poured into a 100ml triangular flask. Then PDB medium was used to volume up to 50mL, so that the final concentrations of endophytic bacteria Bacillus subtilis QNF2 were 20%, 0.2% and 0.002%, respectively. Two B. dothidea cakes with a diameter of 7mm and a weight of 0.03g were placed in the triangular flask. The culture without endophytic bacteria Bacillus subtilis QNF2 was used as a control. The experiment was repeated three times. All cultures were cultured at a temperature of 28°C and at 150rpm. After 48h, the culture was collected, centrifuged at 6000rpm for 10min, weighed, and the inhibitory effect of endophytic bacteria Bacillus subtilis QNF2 on the hyphae growth of B. dothidea was evaluated.

[0060] The results showed that the endophytic bacterium Bacillus subtilis QNF2 had a significant inhibitory effect on the growth of B. dothidea. The mycelium of the control increased significantly after 48 hours of inoculation, and the mycelium weight reached 5.875g. The mycelium growth was not significant when treated with different concentrations (0.002%, 0.2%, and 20%) of the endophytic bacterium Bacillus subtilis QNF2, and the mycelium weight was 0.029g (P<0.0001), 0.211g (P<0.0001), and 1.216g (P<0.0001), respectively, and the inhibition rates were 79.0%, 96.4%, and 99.5%, respectively.

[0061] Example 11: Effect of Bacillus subtilis QNF2 on apple fruit ring rot inoculated with B. dothidea

[0062] A small hole (3 mm wide and 2 mm deep) was cut at the equator of the prepared apple fruit. 20 μL of endophytic bacteria Bacillus subtilis QNF2 (1×109 CFU / L) was added to the hole. After 1 hour, a 7 mm diameter cake of B. dothidea was evenly divided into 4 parts. One quarter was inoculated into the small hole. All fruits were placed at 28°C. The diameter of the lesions was measured every day to evaluate the control effect of endophytic bacteria Bacillus subtilis QNF2 on post-harvest apple fruit ring rot.

[0063] The results showed that the endophytic bacterium Bacillus subtilis QNF2 had a significant inhibitory effect on the ring rot of apple fruit inoculated with the fungus cake of B. dothidea. On the first and second days, the diameters of the lesions on the fruits treated with the endophytic bacterium Bacillus subtilis QNF2 were 0.05cm (P<0.0001) and 0.63cm, respectively, which were 88.52% and 67.84% smaller than those in the control group. On the third day, the lesions in the control group almost extended to the entire fruit, with a diameter of 4.11cm. The lesions on the fruits treated with the endophytic bacterium Bacillus subtilis QNF2 were 1.95cm, which was 52.61% smaller than the control group.

[0064] Example 12: Effect of Bacillus subtilis QNF2 on ring rot of apple fruit soaked in B. dothidea

[0065] Apple fruits were immersed in the endophytic bacteria Bacillus subtilis QNF2 (1×10 9 CFU / L) for 15 min. After 1 h, the apple fruits were immersed in the solution of B. dothidea for 15 min, and the apples were immersed in LB broth medium as a control. The experiment was carried out with 5 biological replicates and repeated 3 times. All fruits were placed in a plastic box (29 cm × 20 cm × 10 cm), covered with a lid, sealed with a film, and incubated at 28 ° C. The disease situation was recorded and the control effect of endophytic fungus Bacillus subtilis QNF2 on postharvest apple fruit ring rot was evaluated.

[0066] The results showed that the endophytic bacterium Bacillus subtilis QNF2 also had a significant inhibitory effect on the ring rot of apple fruits soaked in Botrytis cinerea. On the 4th day after inoculation, lesions gradually appeared on the surface of most of the control fruits. The number of lesions on the 4th to 8th days was 2.4, 6.13, 9.40, 13.60, and 18.53, respectively. On the 4th day, some fruits treated with the endophytic bacterium Bacillus subtilis QNF2 showed lesions, and the number of lesions on the 4th to 8th days was 0, 0.46, 1.33, 2.53, and 4.2, respectively, which was reduced by 100.00, 92.39, 85.82, 81.37, and 77.34% compared with the control, respectively.

[0067] Example 13: Bacillus subtilis QNF2 inhibits the growth of Alternaria alternata

[0068] 5000 μL, 50 μL and 0.5 μL of endophytic bacteria Bacillus subtilis QNF2 fermentation broth (1×10 10 CFU / mL) were added to 100mL sterile flasks, and then different volumes of PDB medium were added to the flasks to make the total volume reach 50mL, and the final concentrations were 10%, 0.1% and 0.001%, respectively. 50ml PDB medium was used as a control. Then, two fresh A. alternata cakes (0.053g) with a diameter of 0.7cm were placed in the flask. Subsequently, all flasks were cultured at 28°C and 200rpm. The experiment was repeated three times. After 48 hours, the hyphae were collected and weighed to evaluate the inhibitory effect of the endophytic bacterium Bacillus subtilis QNF2 on the growth of A. alternata in PDB medium.

[0069] The results showed that after 48 h, the weight of the control culture was 122.63 mg, while the weights of the cultures treated with 0.001%, 0.1% and 10% endophyte Bacillus subtilis QNF2 were 106.46 mg, 51.5 mg and 14.3 mg, respectively, which were 13.19%, 58.25% and 88.33% lighter than the control.

[0070] Example 14: Bacillus subtilis QNF2 inhibits black spot on pear fruit caused by A. alternata

[0071] Use a sterile needle to make a 3 mm wide × 2 mm deep hole at the equator of the pear fruit. Add 20 μL of 1 × 10 10CFU / mL endophyte Bacillus subtilis QNF2. 20 μL LB medium was used as control. Cultured at 28℃ for 24 h. Then, one eighth of a fresh A. alternata cake with a diameter of 0.7 cm 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 endophyte Bacillus subtilis QNF2 on black spot disease of pear fruit.

[0072] The results showed that 5 days after inoculation, both the control fruit and the fruit treated with endophytic bacteria Bacillus subtilis QNF2 showed typical disease symptoms. The diameter of the lesions on the fruit treated with endophytic bacteria Bacillus subtilis QNF2 was significantly smaller than that on the control. On the 5th day, the diameter of the lesions on the fruit treated with endophytic bacteria Bacillus subtilis QNF2 was 1.065 cm, which was 73.35% less than that of the control.

[0073] Example 15: Endophyte Bacillus subtilis QNF: Inhibition of Fusarium Oxysporum hyphae growth

[0074] 5000 μL, 50 μL and 0.5 μL of endophytic bacteria Bacillus subtilis QNF2 fermentation broth (1×10 10 CFU / mL) were added to 100mL sterile flasks, and then different volumes of PDB medium were added to the flasks to make the total volume reach 50mL, and the final concentrations were 10%, 0.1% and 0.001%, respectively. 50ml PDB medium was used as a control. Then, two fresh Fusarium oxysporum cakes (0.053g) with a diameter of 0.7cm were placed in the flask. Subsequently, all flasks were cultured at 28°C and 200rpm. The experiment was repeated three times. After 48 hours, the hyphae were collected and weighed to evaluate the inhibitory effect of the endophytic bacterium Bacillus subtilis QNF2 on the growth of Fusarium oxysporum in PDB medium.

[0075] The results showed that after 48 hours, the weight of the control culture was 116.83 mg, while the weights of the cake cultures treated with 0.001%, 0.1% and 10% endophytic bacteria Bacillus subtilis QNF2 were 68.06 mg, 46.26 mg and 8.5 mg, respectively, which were 41.82%, 60.61% and 92.71% lighter than the control.

[0076] Example 16: Bacillus subtilis QNF2 inhibits tomato fruit diseases caused by Fusarium oxysporum

[0077] A 3 mm wide × 2 cm deep wound was made at the equator of each tomato fruit using a sterile needle. 20 μL of 1×1010 CFU / mL endophytic bacterium Bacillus subtilis QNF2 was added. 20 μL of LB medium was used as a control. The wells were incubated at 28°C for 24 h. Then, a quarter of a fresh cake of Fusarium oxysporum with a diameter of 0.7 cm was inoculated in the wells. All fruits were further incubated at 28°C. The diameter of the lesions was measured on the 5th day after inoculation to evaluate the control effect of endophytic bacillus subtilis QNF2 on tomato wilt.

[0078] The results showed that 5 days after inoculation, both the control fruit and the fruit treated with endophytic bacteria Bacillus subtilis QNF2 showed typical disease symptoms. The diameter of the lesions on the fruit treated with endophytic bacteria Bacillus subtilis QNF2 was smaller than that of the control fruit. On the 5th day, the diameter of the lesions on the tomato fruit treated with endophytic bacteria Bacillus subtilis QNF2 was 1.02 cm, which was 82.75% less than that of the control.

[0079] Example 17: Endophytic Bacillus subtilis QNF2 significantly inhibits the growth of Aspergills niger

[0080] 5000 μL, 50 μL and 0.5 μL of endophytic bacteria Bacillus subtilis QNF2 fermentation broth (1×10 10 CFU / mL) were added to 100mL sterile flasks, and then different volumes of PDB medium were added to the flasks to make the total volume reach 50mL, and the final concentrations were 10%, 0.1% and 0.001%, respectively. 50ml PDB medium was used as a control. Then, two fresh Aspergillus niger cakes (0.053g) with a diameter of 0.7cm were placed in the flasks. Subsequently, all flasks were shaken and cultured at 200rpm at 28°C: the experiment was repeated three times. After 48 hours, the hyphae were collected and weighed to evaluate the inhibitory effect of endophytic bacterium Bacillus subtilis QNF2 on the growth of Aspergillus niger in PDB medium.

[0081] The results showed that after 48 hours, the weight of the control culture was 165.3 mg, while the weight of the cake culture treated with 0.001%, 0.1% and 10% endophytic bacteria Bacillus subtilis QNF2 were 102.16 mg, 65.33 mg and 18.83 mg, respectively, which were 38.25%, 60.56% and 88.57% lighter than the control.

[0082] Example 18: Endophyte Bacillus subtilis QNF: Inhibition of grape fruit diseases caused by Aspergillus niger

[0083] Use a sterile needle to make a 3 mm wide × 2 cm deep hole at the equator of each grape fruit. Add 20 μL of 1 × 1010 CFU / mL endophytic bacterium Bacillus subtilis QNF2. 20 μL LB medium was used as control. Cultured at 28℃ for 24 h. Then, one eighth of a fresh Aspergillus niger cake with a diameter of 0.7 cm was inoculated in the well. All fruits were cultured at 28℃, and the diameter of the lesions was measured every day to evaluate the control effect of endophytic bacillus subtilis QNF2 on grape black aspergillus disease.

[0084] The results showed that 5 days after inoculation, both the control fruit and the fruit treated with endophyte Bacillus subtilis QNF2 showed typical disease symptoms. The diameter of the lesions on the fruit treated with endophyte Bacillus subtilis QNF2 was smaller than that of the control fruit. On the 5th day, the diameter of the lesions on the grape fruit treated with endophyte Bacillus subtilis QNF2 was 1.605 cm, which was 28.8% less than that of the control fruit.

[0085] Example 19: Bacillus subtilis QNF2 inhibits the growth of Penicillum digitatum

[0086] 5000 μL, 50 μL and 0.5 μL of endophytic bacteria Bacillus subtilis QNF2 fermentation broth (1×10 10 CFU / mL) were added to 100mL sterile flasks, and then different volumes of PDB medium were added to the flasks to make the total volume reach 50mL, and the final concentrations were 10%, 0.1% and 0.001%, respectively. 50ml PDB medium was used as a control. Then, two fresh P.digitatum cakes (0.053g) with a diameter of 0.7cm were placed in the flasks. Subsequently, all flasks were cultured at 28°C and 200rpm. The experiment was repeated three times. After 48 hours, the hyphae were collected and weighed to evaluate the inhibitory effect of the endophytic bacterium Bacillus subtilis QNF2 on the growth of P.digitatum in PDB medium.

[0087] The results showed that different concentrations of endophytic bacteria Bacillus subtilis QNF2 had different inhibitory effects on the growth of Penicillium digitatum. After 48 hours, the weight of the control culture was 86.96 mg, while the weight of the cake culture treated with 0.001%, 0.1% and 10% endophytic bacteria Bacillus subtilis QNF2 was 68.83 mg, 60.86 mg and 3.76 mg, respectively, which were 18.95%, 28.02% and 95.66% lighter than the control, respectively.

[0088] Example 20: Endophyte Bacillus subtilis QNF: Inhibition of Citrus Fruit Diseases Caused by Penicillium digitatum

[0089] Use a sterile needle to make a 3 mm wide × 2 cm deep hole at the equator of each citrus fruit. Add 20 μL of 1 × 10 10 CFU / mL endophytic bacterium Bacillus subtilis QNF2. 20 μL LB medium was used as control. Cultured at 28℃ for 24 h. Then, one-eighth of a fresh cake of Penicillium digitatum with a diameter of 0.7 cm was inoculated in the well. All fruits were incubated at 28℃. The diameter of the lesions was measured every day to evaluate the control effect of endophytic bacillus subtilis QNF2 on citrus penicillium.

[0090] The results showed that 5 days after inoculation, both the control fruit and the fruit treated with endophytic bacteria Bacillus subtilis QNF2 showed typical disease symptoms. The diameter of the lesions on the fruit treated with endophytic bacteria Bacillus subtilis QNF2 was smaller than that of the control fruit. On the 5th day, the diameter of the lesions on the citrus fruit treated with endophytic bacteria Bacillus subtilis QNF2 was 0.54 cm, which was 92.3% less than that of the control fruit.

[0091] Example 21: Bacillus subtilis QNF2 inhibits the growth of Botrytis cinerea

[0092] 5000 μL, 50 μL and 0.5 μL of endophytic bacteria Bacillus subtilis QNF2 fermentation broth (1×10 10 CFU / mL) were added to 100mL sterile flasks, and then different volumes of PDB medium were added to the flasks to make the total volume reach 50mL, and the final concentrations were 10%, 0.1% and 0.001%, respectively. 50ml PDB medium was used as a control. Then, two fresh B. cinerea cakes (0.053g) with a diameter of 0.7cm were placed in the flasks. Subsequently, all flasks were cultured at 28°C and 200rpm shaking. The experiment was repeated three times. After 48 hours, the hyphae were collected and weighed to evaluate the inhibitory effect of endophytic bacterium Bacillus subtilis QNF2 on the growth of B. cinerea in PDB medium.

[0093] The results showed that different concentrations of endophytic bacteria Bacillus subtilis QNF2 had different inhibitory effects on the growth of Botrytis cinerea. After 48 hours, the weight of the control culture was 126.46 mg, while the weight of the cake culture treated with 0.001%, 0.1% and 10% endophytic bacteria Bacillus subtilis QNF2 was 66.83 mg, 53.23 mg and 6.03 mg, respectively, which were 47.3%, 58.34% and 95.3% lighter than the control, respectively.

[0094] Example 22: Endophyte Bacillus subtilis QNF2 significantly inhibits the occurrence of peach fruit diseases caused by Botrytis cinerea

[0095] Use a sterile needle to make a 3 mm wide × 2 cm deep hole at the equator of each peach fruit. Add 20 μL of 1 × 10 10 CFU / mL endophyte Bacillus subtilis QNF2. 20 μL LB medium was used as control. Cultured at 28℃ for 24 h. Then, one-eighth of a fresh B. cinerea cake with a diameter of 0.7 cm was inoculated in the well. All fruits were incubated at 28℃. The lesion diameter was measured every day to evaluate the control effect of endophyte Bacillus subtilis QNF2 on peach gray mold.

[0096] The results showed that 5 days after inoculation, both the control fruit and the fruit treated with endophytic bacteria Bacillus subtilis QNF2 showed typical disease symptoms. The diameter of the lesions on the fruit treated with endophytic bacteria Bacillus subtilis QNF2 was smaller than that on the control fruit. On the 5th day, the diameter of the lesions on the citrus fruit treated with endophytic bacteria Bacillus subtilis QNF2 was 0.905 cm, which was 74.42% less than that of the control fruit.

[0097] The present invention relates to the sequence:

[0098] SEQ ID NO: 1

[0099]

[0100] 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 Bacillus subtilis QNF2, 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.31740.

2. Use of the Bacillus subtilis QNF2 according to claim 1 for inhibiting the growth of Monilinia fructicola, Botryosphaeria dothidea, Alternaria alternata, Fusarium oxysporum, Aspergills niger, Penicillum digitatum, or Botrytis cinerea.

3. Use of the Bacillus subtilis QNF2 according to claim 1 for preventing and controlling fruit and vegetable related diseases caused by Monilinia fructicola, Botryosphaeria dothidea, Alternaria alternata, Fusarium oxysporum, Aspergills niger, Penicillum digitatum, or Botrytis cinerea.

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

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

6. Use of the Bacillus subtilis QNF2 according to claim 1 for improving the quality of peach fruit infected with M. fructicola.

7. The use according to claim 6, characterized in that The peach fruit quality refers to the peach fruit hardness, the peach fruit solid-acid ratio or the peach fruit sugar-acid ratio.

8. A microbial preparation, characterized in that: The method comprises the Bacillus subtilis QNF2 according to claim 1.

9. A microbial preparation according to claim 8, characterized in that: A fermentation broth comprising the Bacillus subtilis QNF2 according to claim 1.

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