Compound bacteria and application thereof in prevention and treatment of postharvest diseases of tomatoes and storage and preservation

Abstract

The invention belongs to the field of biological prevention and control of postharvest diseases of fruits and vegetables, and relates to compound bacteria and application thereof in prevention and control of postharvest diseases of tomatoes and storage and preservation. The compound bacterium provided by the invention is prepared by mixing the pantoea agglomerans B10 (CCTCC M20241322) and the Caribik Yarrowia mayer yeast Y9 (CCTCC M 20241324), and the compound bacterium is prepared by mixing the Pantoea agglomerans B10 and the Caribik Yarrowia mayer yeast Y9. The compound bacteria are used for preventing and treating postharvest diseases of tomatoes and for storage and preservation, the natural condition of the tomatoes is kept, the tomatoes are soaked in the compound bacteria, the soaked tomatoes are taken out and naturally aired, and the tomatoes are stored in a certain environment, so that the prevention and treatment of postharvest diseases of the tomatoes and the application of storage and preservation can be realized. According to the present invention, the occurrence of gray speck disease, black spot and leaf spot disease after tomato harvesting can be significantly inhibited, the natural rotting rate after tomato harvesting can be reduced, the significant adverse effect on the main quality index of the harvested tomato can be avoided, and the deterioration of the storage quality can be relieved; the method is convenient to operate, safe and environment-friendly, and has wide market application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control of postharvest diseases, and specifically relates to a composite bacteria constructed by Pantoea agglomerans and Meyer yeast of Kalibik, and uses thereof in controlling postharvest diseases of tomatoes and in storage and preservation. Background Art

[0002] Tomato, a plant of the genus Solanaceae, is rich in lycopene, minerals, vitamin C, etc., and has high nutritional value and health functions. my country is the world's largest tomato producer. As a fruit and vegetable, tomatoes can be eaten raw or made into various tomato products according to consumer preferences and needs, and are deeply loved by people. However, tomato fruits are soft and juicy, and are easily mechanically damaged during picking, transportation and storage, providing a pathway for infection for various pathogens, thereby causing post-harvest diseases. Post-harvest diseases of tomatoes are mainly fungal diseases, the most common of which include gray mold, black spot and soft rot, which can cause serious rot of tomatoes. Therefore, effective methods must be taken to control post-harvest diseases of tomatoes.

[0003] At present, the use of chemical fungicides is the most effective method to control tomato postharvest diseases, such as thiabendazole, benzimidazole and fludioxonil. However, excessive use of chemical fungicides can easily cause ecological pollution, increase the residual amount of toxic chemicals, and cause serious harm to the health of consumers. In addition, drug-resistant strains may also be produced. Biological control uses antagonistic microorganisms naturally present in fruits and vegetables to control postharvest diseases of fruits and vegetables. In recent years, it has become a hot topic in academic research and is expected to become an effective method to replace chemical fungicides to control postharvest diseases of tomatoes. The acquisition of antagonistic microorganisms is the premise and basis for studying biological control methods. At present, many strains of microorganisms have been reported to have the effect of controlling postharvest diseases of tomatoes, such as Trichoderma, Pichia guilliermongensis, Rhodotorula glutinosus, Cryptococcus albus and Glechoma glutinosus. However, with the deepening of biological control research, researchers have found that the antibacterial spectrum of a single antagonistic bacterium is narrow and highly dependent on the living environment, and it cannot achieve the same efficacy as chemical fungicides. Therefore, new methods need to be studied to improve the biological control efficacy of antagonistic bacteria. Summary of the invention

[0004] The invention provides a composite bacteria constructed by Pantoea agglomerans B10 and Meyerozyma caribbica Y9. The invention uses tomatoes as raw materials for research, and research proves that the composite bacteria of Pantoea agglomerans B10 and Meyerozyma caribbica Y9 can significantly reduce the incidence and rot diameter of gray mold, black spot and leaf spot of postharvest tomatoes, can be used to control the occurrence of postharvest diseases of tomatoes and the storage and preservation of tomatoes, can significantly reduce the losses caused by postharvest diseases, achieves substantial effects, and has potential application value.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention first provides a Pantoea agglomerans B10, which is deposited in China Center for Type Culture Collection (CCTCC) on June 20, 2024, and has a deposit number of CCTCC NO: M20241322; and a Meyerozyma caribbica Y9, which is deposited in China Center for Type Culture Collection (CCTCC) on June 20, 2024, and has a deposit number of CCTCC NO: M 20241324.

[0007] Secondly, the present invention provides a composite bacteria, which is formed by mixing the Pantoea agglomerans B10 and the Meyer yeast of Kalibik Y9; when the Pantoea agglomerans B10 and the Meyer yeast of Kalibik Y9 are mixed, both bacterial suspensions are used, wherein the concentration of the bacterial suspensions is 1×10 8 CFU / mL.

[0008] Finally, the present invention provides a use of a composite bacteria for preventing and controlling post-harvest diseases of tomatoes and for storage and preservation, and the steps are as follows:

[0009] (1) First, Pantoea agglomerans B10 and Myersinia californica Y9 were inoculated into LB and NYDB medium, respectively, for the first activation culture to obtain the first activation culture solution; then, the first activation culture solution was aspirated and transferred into LB and NYDB medium, respectively, at an inoculation amount of 1% to 2% by volume, for the second activation culture to obtain the second activation culture solution, which was centrifuged to obtain bacterial sludge, which was washed by centrifugation with sterile saline, and the washed bacterial sludge was resuspended with sterile saline to obtain Pantoea agglomerans B10 bacterial suspension and Myersinia californica Y9 bacterial suspension; wherein the concentrations of the Pantoea agglomerans bacterial suspension and the Myersinia californica Y9 bacterial suspension were both 1×10 8 CFU / mL; finally, the suspension of Pantoea agglomerans B10 and the suspension of Mycosaccharomyces calibicolor Y9 were mixed to obtain a composite bacterium;

[0010] (2) selecting tomatoes without diseases, mechanical damage, uniform color and size, performing surface disinfection treatment, and then washing with clean water, placing the washed tomatoes in a sterilized plastic basket to dry; punching holes on the surface of the dried tomatoes to form wounds of a certain size and depth, adding the composite bacteria prepared in step (1) to each wound, letting it stand for a period of time, and then inoculating a spore suspension of Botrytis cinerea, Alternaria tenuissima or Cladosporium cladosporium in an equal volume to the composite biocontrol bacteria suspension, sealing it with plastic wrap after natural drying, and placing it in a constant temperature and humidity incubator for cultivation, so as to achieve control of corresponding post-harvest diseases of tomatoes;

[0011] Alternatively, disease-free, mechanically-free, uniformly colored, and similarly sized tomatoes are selected, and the tomatoes are kept in their natural state without any disinfection treatment. The tomatoes are then soaked in the composite bacteria prepared in step (1), and the tomatoes are taken out after soaking and naturally dried, and stored under a certain environment, thereby achieving the purpose of preventing and controlling post-harvest diseases of tomatoes and storing and preserving them.

[0012] Preferably, the LB medium described in step (1) comprises, per liter: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and distilled water to a volume of 1000 mL, with a natural pH and sterilized at 115° C. for 20 min;

[0013] NYDB medium, per liter: 5g yeast extract, 8g beef extract, 10g glucose, distilled water to 1000mL, natural pH, sterilized at 115℃ for 20min.

[0014] Preferably, the conditions for the first activation culture and the second activation culture in step (1) are: the culture temperature of Pantoea agglomerans B10 is 37°C and the time is 12 hours; the culture temperature of Mayer yeast Y9 is 28°C and the time is 24 hours; the centrifugation conditions are: 4°C, 8000rpm, and the time is 10-15min.

[0015] Preferably, in step (2), the volume ratio of the mixture of the Pantoea agglomerans B10 suspension and the Meyer yeast Y9 suspension is 1-3:1-3.

[0016] Preferably, the operation of the tomato disinfection treatment in step (2) is as follows: immersing the tomato in a solution containing 1% sodium hypochlorite for disinfection for 2 to 3 minutes; the hole diameter of the punched hole is 5 mm and the depth is 4 mm; 20 μL of the composite bacterial suspension and the spore suspension of Botrytis cinerea, Alternaria tenuissima or Cladosporium cladosporium are added to each wound; the standing period is 2 hours; the concentration of the spore suspension of Botrytis cinerea, Alternaria tenuissima or Cladosporium cladosporium is 1×10 5 Pieces / mL.

[0017] Preferably, the storage operation under a certain environment in step (2) is as follows: first, the naturally dried tomatoes are placed in a sterilized plastic basket, which is sealed with plastic wrap and placed in a constant temperature and humidity incubator at a temperature of 25° C. and a relative humidity of 90% for storage; wherein the plastic basket is sterilized by cleaning it and then drying it, and then spraying it evenly with 75% alcohol for sterilization.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) The Pantoea agglomerans B10 and the Mayer yeast Y9 used in the present invention are isolated and purified in our laboratory and have been verified to be non-toxic by acute toxicity tests on mice and are safe and harmless to the human body.

[0020] (2) The composite bacteria of Pantoea agglomerans B10 and Mycosaccharomyces calibicolor Y9 used in the present invention can effectively control tomato postharvest diseases, and the control effect is better than that of a single strain, thereby reducing the losses caused by tomato postharvest diseases and having good market application prospects.

[0021] (3) The composite bacteria of Pantoea agglomerans B10 and Mycosaccharomyces calibicolor Y9 used in the present invention have not been reported to be used in the prevention and control of post-harvest diseases of tomatoes, and are original; they can replace or reduce the use of chemical fungicides, reduce the harm of chemical fungicides to consumer health and the environment, and have the advantage of being green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figure 2 shows the affinity of Pantoea agglomerans B10 and Mycosaccharomyces calibikensis Y9, where (A) shows Pantoea agglomerans B10 as the indicator strain and Mycosaccharomyces calibikensis Y9 as the opposing strain; (B) shows Mycosaccharomyces calibikensis as the indicator strain and Pantoea agglomerans B10 as the opposing strain.

[0023] Figure 2 Figure 2 The control effect of Pantoea agglomerans B10 and Mycosaccharomyces calibacterium Y9 complex bacteria in different proportions on gray mold of postharvest tomatoes; (A): incidence, (B): lesion diameter; CK is the control group, tomatoes treated with sterile saline, Pa is 1×10 8 CFU / mL Pantoea agglomerans B10 treated tomatoes; Mc is 1×10 8 CFU / mL of tomatoes treated with Mycobacterium californicum Y9; 1:1, 1:2, 1:3, 2:1, 3:1 refer to tomatoes treated with a composite bacteria mixture of Pantoea agglomerans B10 and Mycobacterium californicum Y9 at a volume ratio of 1:1, 1:2, 1:3, 2:1, 3:1, respectively; the concentration of pathogen spores was 1×10 5 / mL; Different lowercase letters represent significant differences (p<0.05).

[0024] Figure 3 The control effect of Pantoea agglomerans B10 and Mycosaccharomyces calibicolor Y9 complex bacteria in different proportions on postharvest black spot disease of tomatoes; (A): incidence, (B): lesion diameter; CK is the control group, tomatoes treated with sterile saline, Pa is 1×10 8 CFU / mL Pantoea agglomerans B10 treated tomatoes; Mc is 1×10 8CFU / mL of tomatoes treated with Mycobacterium californicum Y9; 1:1, 1:2, 1:3, 2:1, 3:1 refer to tomatoes treated with a composite bacteria mixture of Pantoea agglomerans B10 and Mycobacterium californicum Y9 at a volume ratio of 1:1, 1:2, 1:3, 2:1, 3:1, respectively; the concentration of pathogen spores was 1×10 5 / mL; Different lowercase letters represent significant differences (p<0.05).

[0025] Figure 4 Figure 2 The control effect of different ratios of Pantoea agglomerans B10 and Mycosaccharomyces calibicolor Y9 on postharvest leaf spot disease of tomatoes; (A): incidence, (B): lesion diameter; CK is the control group, tomatoes treated with sterile saline, Pa is 1×10 8 CFU / mL Pantoea agglomerans B10 treated tomatoes; Mc is 1×10 8 CFU / mL of tomatoes treated with Mycobacterium californicum Y9; 1:1, 1:2, 1:3, 2:1, 3:1 refer to tomatoes treated with a composite bacteria mixture of Pantoea agglomerans B10 and Mycobacterium californicum Y9 at a volume ratio of 1:1, 1:2, 1:3, 2:1, 3:1, respectively; the concentration of pathogen spores was 1×10 5 / mL; Different lowercase letters represent significant differences (p<0.05). DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art of the present invention generally understand. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and / or materials related to the documents. In the event of conflict with any incorporated document, the content of this specification shall prevail.

[0028] The Pantoea agglomerans B10 and Meyerozyma caribbica Y9 provided by the present invention are separated and purified in this laboratory (Zhenjiang, China); they are currently deposited in the China Center for Type Culture Collection, and the strain preservation time is June 20, 2024, and the preservation numbers are CCTCC NO: M20241322 and CCTCC NO: M 20241324, respectively.

[0029] The culture procedures of Pantoea agglomerans B10 and Meyer yeast Y9 were as follows:

[0030] (1) First activation: 2 rings of Pantoea agglomerans B10 and Mycosaccharomyces californica Y9 were picked and inoculated into 50 mL of LB and NYDB medium, respectively. Pantoea agglomerans B10 was cultured at 37° C. and 180 rpm for 12 h, and Mycosaccharomyces californica Y9 was cultured at 25° C. and 180 rpm for 24 h for the first activation to obtain the first activated culture solution;

[0031] (2) Second culture: Use a sterilized pipette tip to transfer 1 mL of the first activated culture solution to new LB and NYDB medium (the volume of the culture medium is 50 mL). Cultivate Pantoea agglomerans B10 at 37°C and 180 rpm for 12 h, and cultivate Meyer yeast Y9 at 25°C and 180 rpm for 24 h to obtain the second activated culture solution.

[0032] (3) Centrifugal separation and resuspension: The second activated culture solution obtained in step (2) was centrifuged at 4°C and 8000 rpm for 10 min, and the obtained bacterial precipitate was washed twice with sterile saline to remove the culture matrix, and then centrifuged again to obtain bacterial sludge, which was resuspended with sterile saline. The final concentration of the bacterial suspension was adjusted to 1×10 8 CFU / mL.

[0033] The bacterial suspensions of Pantoea agglomerans B10 and Mycosaccharomyces calibicolor Y9 were prepared into composite bacteria at volume ratios of 1:1, 1:3 and 2:1.

[0034] Example 1: Affinity of Pantoea agglomerans B10 and Meyer yeast Y9;

[0035] 1. Experimental plan

[0036] Pantoea agglomerans B10 was spread on the PDA medium as an indicator strain, a sterilized filter paper was placed in the center of the plate coated with Pantoea agglomerans B10, and 10 μL of Kalibiker Y. meyeri Y9 was injected on the filter paper as a counter strain, and cultured at 25°C for 1 day. Then, in the same way, Kalibiker Y. meyeri Y9 was spread on the PDA medium as an indicator strain, a sterilized filter paper was placed in the center of the plate coated with Kalibiker Y. meyeri Y9, and 10 μL of Kalibiker B10 was injected on the filter paper as a counter strain, and cultured at 25°C for 1 day.

[0037] 2. Test results

[0038] According to the above test steps, the affinity of Pantoea agglomerans B10 and Mycobacterium californicum Y9 was investigated. The results are as follows Figure 1 Whether Pantoea agglomerans B10 or Meyer yeast Y9 is used as the indicator strain, Figure 1 It can be seen from (A)-(B) that there is no inhibition zone around the central filter paper, indicating that there is no mutual inhibition between Pantoea agglomerans B10 and Mycobacterium calibicolor Y9, and the affinity is very good, which can be used for the construction of composite bacteria; because there will be antagonism between different bacteria, they cannot be used in combination directly.

[0039] Example 2: Safety of Pantoea agglomerans B10 and Meyer yeast Y9;

[0040] 1. Experimental plan

[0041] Preparation of Pantoea agglomerans B10 slurry: Pantoea agglomerans B10 was inoculated in a flask containing 50 mL of LB medium, cultured in a shaking incubator at 37°C for 12 h, and then centrifuged to obtain bacterial slurry. After washing and centrifugation, the bacterial slurry was diluted with pure water to a concentration of 503 mg / mL. The bacterial slurry for each oral administration of mice was diluted from fresh bacterial slurry.

[0042] Preparation of Y. meyeri Y9 bacterial slurry: Y. meyeri Y9 was inoculated into a triangular flask containing 50 mL of NYDB medium, cultured in a shaking incubator at 25°C for 24 hours, and then centrifuged to obtain bacterial slurry, which was washed and diluted with pure water to a concentration of 503 mg / mL. Each oral administration of mice was from the dilution of fresh bacterial slurry.

[0043] The experimental species was SPF-grade KM mice. Twenty KM mice with a weight range of 18g to 22g were selected, half of which were male and half were female. Before the experiment, the mice were pre-raised in the laboratory barrier environment animal room for 6 days to adapt to the environment and quarantine observation, and fasted for 4 hours before the experiment. The test substance was given once, with an oral volume of 20mL / kg·bw, and then observed for 14 days. Throughout the experiment, the activity of the mice, abnormal behavior, poisoning or death were fully observed and recorded. If abnormalities and death occurred, the time, degree and duration of the animal changes were recorded to evaluate possible toxic target organs. The animal weight was weighed and recorded at the beginning of the experiment, and the animal weight was weighed once a week during the observation period.

[0044] 2. Test results

[0045] According to the above test steps, the statistical results of the safety test of Bacillus amyloliquefaciens are as follows:

[0046] The mice gavaged with Pantoea agglomerans B10 and Kalibiker Y. meyersin Y. 9 did not die within 14 days, and the mice did not show poisoning or other abnormal phenomena during the whole experiment. As can be seen from Table 1, the acute oral LD ​​50 of the strain for female and male KM mice in the experiment was greater than 10060 mg / kg·bw. According to the acute toxicity classification standard, Pantoea agglomerans B10 and Kalibiker Y. meyersin Y. 9 are both safe and non-toxic, and can be used in the control of postharvest diseases and storage and preservation of tomatoes.

[0047] Table 1 Results of oral acute toxicity test of Pantoea agglomerans B10 and Meyer yeast Y9 on mice

[0048]

[0049]

[0050] Embodiment 3:

[0051] Take tomatoes as an example: the control effect of Pantoea agglomerans B10 and Mycosaccharomyces calibicolor Y9 on postharvest gray mold, black spot, and leaf spot of tomatoes;

[0052] 1. Experimental plan

[0053] (1) Botrytis cinerea inhibition:

[0054] Preparation of composite bacterial suspension: adjust to 1×10 8 The bacterial suspensions of Pantoea agglomerans B10 and Mycobacterium californicum Y9 with a volume ratio of 1:1, 1:2, 1:3, 2:1, and 3:1 were compounded and then vortexed to prepare a composite bacterial suspension.

[0055] Tomatoes without diseases, mechanical damage, uniform color and size were selected, soaked in 1% sodium hypochlorite for 2 minutes, then rinsed with running water, and the washed tomatoes were placed in a sterilized plastic basket to dry. A 5mm×4mm (diameter×depth) wound was made at the equatorial part of the dried tomatoes with a sterile puncher; then the tomatoes were divided into seven groups, 20μL of a single suspension of Pantoea B10 was added to the first group of wounds, 20μL of a single suspension of Kalibiker Y. meyeri Y9 was added to the second group of wounds, and 20μL of a composite bacterial suspension compounded at a volume ratio of 1:1, 1:2, 1:3, 2:1, and 3:1 was added to the third to seventh groups of wounds.

[0056] After 2 h at room temperature, 20 μL of Botrytis cinerea suspension (concentration 1×10 5 / mL); after drying at room temperature, place in a constant temperature and humidity incubator (25°C, relative humidity 90%), and record the incidence of tomatoes and the diameter of lesions. Tomatoes treated with sterile saline instead of bacterial suspension were used as controls. Each treatment was repeated 3 times, with 12 tomatoes in each parallel.

[0057] Incidence rate = number of rotten holes / total number of holes × 100%

[0058] (2) Black spot inhibition:

[0059] The operation is the same as the operation of gray mold inhibition in step (1) above, except that the Botrytis cinerea suspension is replaced by Alternaria alternata suspension.

[0060] (3) Leaf spot disease inhibition:

[0061] The operation is the same as the operation of gray mold inhibition in step (1) above, except that the Botrytis cinerea suspension is replaced by Cladosporium cladocerum suspension.

[0062] 2. Test results

[0063] Effects of different ratios of Pantoea agglomerans B10 and Mycosaccharomyces calibacterium Y9 on the control of postharvest gray mold in tomatoes Figure 2 As shown by Figure 2 It can be seen in (A) that after 3 days of storage, the incidence of gray mold in tomatoes treated with all compound bacteria was significantly lower than that in the control group and the single antagonistic strain treatment group (p<0.05). Among them, the compound bacteria treatment group with a ratio of 1:3 had the lowest incidence (rot rate of 14.81%), followed by the compound bacteria treatment group with a ratio of 2:1 (rot rate of 24.07%); followed by the compound bacteria treatment groups with a ratio of 1:1, 1:2 and 3:1 (rot rates of 34.57%, 35.80%, and 35.80%). Figure 2As shown in (B), the tomato gray mold lesions in the treatment with the composite bacteria in a ratio of 2:1 were the lowest, followed by the 3:1 and 1:3 composite bacteria treatment groups, and then the 1:1 and 1:2 composite bacteria treatment groups.

[0064] Effects of different ratios of Pantoea agglomerans B10 and Mycosaccharomyces calibicolor Y9 on the control of postharvest black spot disease in tomatoes Figure 3 As shown. Figure 3 (A) It can be seen that after 4 days of storage, the incidence of black spot disease in tomatoes treated with all compound bacteria was significantly lower than that in the control group and the single antagonistic strain treatment group (p<0.05) (except for the 1:2 compound bacteria treatment group). Among them, the incidence of black spot disease in tomatoes treated with the 2:1 compound bacteria was the lowest (rot rate was 18.52%), followed by the 3:1 compound bacteria treatment group (rot rate was 27.78%), but there was no significant difference between the two; then came the 1:1 and 1:3 compound bacteria treatment groups (rot rates were 35.19% and 38.89%), and the worst effect was the 1:2 compound bacteria treatment group (rot rate was 50.00%). According to Figure 3 (B) It can be seen that the diameter of tomato black spot disease treated with 2:1 composite bacteria was the smallest, significantly lower than that of the control group and the single Kalibikmycin Y9 treatment group, but had no significant difference with the single Pantoea agglomerans B10 treatment group (p<0.05), followed by 3:1, 1:1 and 1:3 composite bacteria treatment groups. There was no significant difference between the 1:2 composite bacteria treatment group and the two antagonistic bacteria single treatment groups.

[0065] Effects of different ratios of Pantoea agglomerans B10 and Mycosaccharomyces calibicolor Y9 on postharvest leaf spot disease in tomatoes Figure 4 As shown. After 5 days of storage, the tomato leaf spot disease incidence rate of the 1:1 compound bacteria treatment was the lowest (rot rate was 22.22%), which was significantly lower than that of other treatment groups (p<0.05). Next were the 2:1, 3:1 and 1:2 compound bacteria treatment groups (rot rates were 33.33%, 33.89% and 35.19%). The 1:3 compound bacteria treatment group had the worst effect, and the incidence rate of the treated tomatoes was significantly lower than that of the control and the single Kalibikerella Y9 treatment group, but there was no significant difference with the single Pantoea agglomerata B10 treatment group (p<0.05) (rot rate was 41.67%). Figure 4 B shows that the diameter of leaf spot disease in the 1:1 compound bacteria treatment group was the smallest, significantly lower than that in other treatment groups (p<0.05). The diameter of lesions in other compound bacteria treatment groups was also lower than that in the control group and the single antagonistic bacteria treatment group, and was significantly different from the control group, but not significantly different from the single antagonistic bacteria.

[0066] The above test results show that different ratios of Pantoea agglomerans B10 and Kalibikerella meyeri Y9 composite bacteria have significant control effects on major post-harvest diseases of tomatoes, including gray mold, black spot and leaf spot. In terms of their control effects on the three major post-harvest diseases of tomatoes, the 2:1 composite bacteria is particularly effective, followed by 1:1, 1:3 and 3:1 composite bacteria, and finally 1:2 biocontrol bacteria.

[0067] The above results indicate that the composite bacteria prepared by the present invention can be used to prevent and treat gray mold, black spot and leaf spot.

[0068] Embodiment 4:

[0069] Use of a composite bacteria of Pantoea agglomerans B10 and Mycosaccharomyces calibicolor Y9 (2:1) for controlling postharvest natural decay of tomatoes and preserving them during storage;

[0070] 1. Experimental plan

[0071] Take the effect of 2:1 composite bacteria of Pantoea agglomerans B10 and Kalibikerella meyeri Y9 on the natural decay and storage quality of tomatoes as an example. Select tomatoes without diseases, mechanical damage, uniform color and size, do not disinfect, keep the tomatoes in their natural state, and directly soak the tomatoes in the composite bacteria suspension. After soaking for 2 minutes, take out the tomatoes and place them in a sterilized plastic basket. After the tomatoes are naturally dried, seal the plastic basket with plastic wrap and place it in a constant temperature and humidity incubator at 20℃ (relative humidity 90%) for 18 days. Count the incidence of tomatoes, and measure the weight loss rate, hardness, soluble solids content, soluble sugar content, titratable acid content, Vc content and lycopene content of tomatoes. Tomatoes soaked in sterile saline were used as controls. Each treatment had 3 parallels, and each parallel had 12 fruits.

[0072] Incidence rate (%) = number of rotten fruits / total number of fruits × 100%

[0073] The specific determination methods of quality indicators are as follows:

[0074] 1. Weight loss rate: Weight loss rate (%) = (mass before storage - mass after storage) / mass before storage × 100%

[0075] 2. Hardness: Use TA-XT2i physical property analyzer to measure the hardness of tomatoes. Use P5 probe, test speed of 2mm / s, test depth of 10mm. Select three points at equal distances at the equator of the tomato for testing. The maximum resistance encountered when the probe is inserted into the tomato is recorded as hardness (N).

[0076] 3. Soluble solid content: 5.0 g of fruit sample was ground into a homogenate under low temperature conditions, centrifuged at 4°C and 4000 rpm for 10 min, and the supernatant was aspirated. The soluble solid content of tomatoes was determined using a handheld refractometer.

[0077] 4. Soluble sugar content: Determine it using the anthrone reagent method. Weigh about 0.5g of sample, add 16mL of distilled water, grind into a homogenate, and transfer to a graduated test tube. Boil with a stopper for 30 minutes, cool to room temperature, and transfer to a 50mL centrifuge tube. Centrifuge at 4°C, 8000rpm for 5 minutes. Transfer the supernatant to a 100mL volumetric flask and dilute to the scale. Pipette 0.5mL of the sample solution into a 25mL graduated test tube, add 1.5mL of distilled water, 0.5mL of anthrone-ethyl acetate and 5mL of concentrated sulfuric acid, immediately shake thoroughly, and accurately keep warm in a boiling water bath for 1min each. After taking it out, cool it naturally to room temperature, and measure its absorbance at 630nm. Distilled water is used as a blank control, and the results are expressed in mass fraction (%), calculated using the following formula:

[0078] Soluble sugar content (%) = (m'×V×N) / (Vs×m×10 6 )×100%

[0079] Where m' is the mass of sucrose obtained from the standard curve (μg), V is the total volume of the extract (mL), N is the dilution factor, Vs is the volume of the sample solution taken during the measurement (mL), and m is the sample mass (g).

[0080] 5. Titratable acid content: Determine by sodium hydroxide titration. Weigh about 1.0g sample, add appropriate amount of distilled water, grind into a mortar into a homogenate, transfer to a 50mL centrifuge tube and let stand for 30min, shake once every 10min. Centrifuge at 4℃, 8000rpm for 10min, aspirate the supernatant into a 25mL volumetric flask and dilute to the scale. Aspirate 20mL sample solution into a conical flask, add 3 drops of 1% phenolphthalein, and titrate with 0.1mol / L NaOH. Titrate until the solution begins to show light pink and does not fade within 0.5min as the end point. Distilled water is used as the blank control, and the results are expressed as mass fraction (%).

[0081] Titratable acid content (%) = (V×c×(V1-V0)×0.067) / (Vs×m)×100%

[0082] Where V is the total volume of the sample extract (mL), c is the NaOH concentration (mol / L), V1 is the volume of sodium hydroxide solution consumed in titrating the sample solution (mL), V0 is the volume of sodium hydroxide solution consumed in titrating distilled water (mL), Vs is the volume of the sample solution taken during titration (mL), m is the sample mass (g), and 0.067 is the malic acid conversion coefficient (g / mmol).

[0083] 6. Vc content: Determine by 2,6-dichlorophenol indophenol titration method. Weigh about 1.0g tomato tissue sample and place it in a mortar, add an appropriate amount of 1% oxalic acid solution, place it in a mortar in a dark ice bath and grind it into a homogenate, and transfer it to a 50mL centrifuge tube. Centrifuge at 4℃, 8000rpm for 10min, transfer the supernatant to a 50mL brown volumetric flask and dilute to the scale, shake well. Take 5mL of sample solution in a conical flask, titrate the sample solution with a calibrated 2,6-dichlorophenol indophenol solution until a slightly reddish color appears and does not fade for 30s, which is the titration end point. At the same time, use 5mL of 1% oxalic acid solution as a blank control. According to the amount of 2,6-dichlorophenol indophenol, calculate the Vc content in tomatoes, expressed as the mass of Vc contained in every 100g sample, that is, mg / 100g.

[0084] Titer T = (c × V) / (V1-V0)

[0085] Where c is the mass concentration of Vc standard solution (mg / mL), V is the volume of Vc standard solution absorbed (mL), V1 is the volume of 2,6-dichlorophenol indophenol solution consumed when titrating the standard liquid (mL), and V0 is the volume of 2,6-dichlorophenol indophenol solution consumed when titrating the blank (1% oxalic acid) (mL).

[0086] Vc content (mg / 100g) = (V×(V1-V0)×T) / (Vs×m)×100

[0087] Where V is the total volume of the sample extraction solution (mL), V1 is the volume of the dye consumed in the sample titration (mL), V0 is the volume of the dye consumed in the blank titration (mL), Vs is the volume of the sample solution taken during the titration (mL), and m is the sample mass (g).

[0088] 7. Lycopene: petroleum ether extraction method. Weigh about 5.0g sample, add 25mL distilled water and place in a mortar, grind to homogenate in a light-proof ice bath, transfer to a separatory funnel, add 25mL petroleum ether, mix well, let stand for 30min, separate, take the upper layer, add 25mL petroleum ether, mix well again, let stand for 30min, separate again and take the upper layer, and measure its absorbance (A) at 472nm.

[0089] Lycopene content (mg / 100g) = (c'×V) / (m×10)

[0090] where c' is the lycopene concentration (μg / mL) obtained from the standard curve, V is the total volume of the extract (mL), and m is the sample mass (g).

[0091] 2. Test results

[0092] As can be seen from Table 2, the incidence of tomatoes treated with the 2:1 composite bacteria was only 10.42% after being stored at 20°C for 18 days, which was 45.83% lower than the control group (56.25%), and 17% and 19.27% ​​lower than the single antagonistic bacteria treatment group (27.42%, 29.69%), respectively, with significant differences (p<0.05). It can be seen that the composite bacteria of Pantoea agglomerans B10 and Kalibikerella meyeri yeast Y9 have a significant control effect on the natural decay of post-harvest tomatoes. Therefore, the composite bacteria prepared by Pantoea agglomerans B10 and Kalibikerella meyeri yeast Y9 of the present invention has a very significant control effect on the natural decay of post-harvest tomatoes, and has achieved unexpected significant results.

[0093] The tomato quality indicators measured according to the above steps are shown in Table 2.

[0094] As can be seen from Table 2, the weight loss rate of tomatoes treated with the composite bacteria was significantly lower than that of the control group, and there was no significant difference with the single antagonistic strain treatment group; the hardness was significantly higher than that of the control group and the single Pantoea agglomerans B10 treatment group, and there was no significant difference with the single Kalibik Y9 treatment group; the lycopene content was significantly increased compared with the treatment group and the single Kalibik Y9 treatment group (p<0.05), and there was no significant difference with the single Pantoea agglomerans B10 treatment group; there was no significant difference in titratable acid, soluble solids and Vc content among the groups. It can be seen that the composite bacteria of Pantoea agglomerans B10 and Kalibik Y9 can not only prevent and control the natural decay of tomatoes after harvest, but also alleviate the deterioration of fruit quality to a certain extent. Table 2 Effect of the composite bacteria of Pantoea agglomerans B10 and Kalibik Y9 (2:1) on the natural decay rate and quality of tomatoes during storage

[0095]

[0096] Note: 0d refers to tomatoes before storage; CK refers to the control group, i.e. tomatoes treated with sterile saline and stored for 18d; 2:1 refers to tomatoes treated with a composite bacteria of Pantoea agglomerans B10 and Mycobacterium calibicolor Y9 at a volume ratio of 2:1 and stored for 18d. Different lowercase letters represent significant differences (p<0.05).

[0097] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, a person skilled in the art should understand that the present invention can still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. Pantoea agglomerans B10 was deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M 20241322; Meyerozyma caribbica Y9 was deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M 20241324.

2. A composite bacteria, characterized in that: The method is prepared by mixing the Pantoea agglomerans B10 and the Meyer yeast Y9 described in claim 1.

3. The composite bacteria according to claim 2, characterized in that: When the Pantoea agglomerans B10 and the Mayer yeast Y9 were mixed, bacterial suspensions were used, and the concentration of the bacterial suspensions was 1×10 8 CFU / mL.

4. Use of the composite bacteria according to claim 2 for preventing and controlling postharvest diseases of tomatoes and for storage and preservation.

5. The use according to claim 4, characterized in that Here are the steps: (1) First, Pantoea agglomerans B10 and Myersinia carlibiker Y9 were inoculated into LB and NYDB medium, respectively, for a first activation culture to obtain a first activation culture solution; then, the first activation culture solution was aspirated and transferred into LB and NYDB medium, respectively, at an inoculation amount of 1% to 2% by volume, for a second activation culture to obtain the second activation culture solution, which was centrifuged to obtain bacterial sludge, which was washed by centrifugation with sterile saline, and the washed bacterial sludge was resuspended with sterile saline to obtain a Pantoea agglomerans B10 bacterial suspension and a Myersinia carlibiker Y9 bacterial suspension; The concentrations of the suspensions of Pantoea agglomerata and Mayer's yeast Y9 were both 1×10 8 CFU / mL; finally, the suspension of Pantoea agglomerans B10 and the suspension of Mycosaccharomyces calibicolor Y9 were mixed to obtain a composite bacterium; (2) selecting tomatoes without diseases, mechanical damage, uniform color and size, disinfecting the surface of the tomatoes, then rinsing them with clean water, and placing the washed tomatoes in a sterilized plastic basket to air dry; punching holes on the surface of the air-dried tomatoes to form wounds of a certain size and depth, adding the composite bacteria prepared in step (1) to each wound, letting them stand for a period of time, and then inoculating a spore suspension of Botrytis cinerea, Alternaria tenuissima or Cladosporium cladosporium in an equal volume to the composite bacteria suspension, and sealing them with plastic wrap after natural drying, and placing them in a constant temperature and humidity incubator for cultivation, so as to achieve control of corresponding post-harvest diseases of tomatoes; Alternatively, disease-free, mechanically-free, uniformly colored, and similarly sized tomatoes are selected, and the tomatoes are kept in their natural state without any disinfection treatment. The tomatoes are then soaked in the composite bacteria prepared in step (1), and the tomatoes are taken out after soaking and naturally dried, and stored under a certain environment, thereby achieving the purpose of preventing and controlling post-harvest diseases of tomatoes and storing and preserving them.

6. The use according to claim 5, characterized in that The LB medium described in step (1) is calculated as follows: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and distilled water to 1000 mL, with a natural pH value, and sterilized at 115°C for 20 min; NYDB medium, per liter: 5g yeast extract, 8g beef extract, 10g glucose, distilled water to 1000mL, natural pH, sterilized at 115℃ for 20min.

7. The use according to claim 5, characterized in that The conditions for the first activation culture and the second activation culture in step (1) are as follows: the culture temperature of Pantoea agglomerans B10 is 37°C and the time is 12 hours; the culture temperature of Mayer yeast Y9 is 28°C and the time is 24 hours; the centrifugation conditions are: 4°C, 8000rpm, and the time is 10-15min.

8. The use according to claim 5, characterized in that In step (2), the volume ratio of the mixture of the Pantoea agglomerata B10 suspension and the Meyer yeast Y9 suspension is 1-3:1-3.

9. The use according to claim 5, characterized in that The operation of the tomato disinfection treatment in step (2) is as follows: immersing the tomato in a solution containing 1% sodium hypochlorite for disinfection for 2 to 3 minutes; the hole diameter of the punching is 5 mm and the depth is 4 mm; 20 μL of the composite bacterial suspension and the spore suspension of Botrytis cinerea, Alternaria tenuissima or Cladosporium cladosporium are added to each wound; the standing period is 2 hours; the concentration of the spore suspension of Botrytis cinerea, Alternaria tenuissima or Cladosporium cladosporium is 1×10 5 Pieces / mL.

10. The use according to claim 5, characterized in that The storage operation under a certain environment described in step (2) is as follows: first, the naturally dried tomatoes are placed in a sterilized plastic basket, which is sealed with plastic wrap and then stored in a constant temperature and humidity incubator at a temperature of 25° C. and a relative humidity of 90%. The plastic basket is sterilized by cleaning it, drying it, and then spraying it evenly with 75% alcohol for sterilization.

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