Construction and application of compound microbial agent for fresh keeping of litchis before picking

Abstract

The invention discloses construction and application of a compound microbial agent for fresh keeping of litchis before picking. According to key enzymes and pathogenic bacteria of litchi peel browning, microbial strains which can tolerate high-concentration metal ions, high-yield lignin degrading enzymes, resist fungi and the like are directionally screened, and the microbial strains are combined into the compound microbial agent for fresh keeping of litchis before picking and applied to the growth period of litchis fruits, so that the litchis fruits become larger after being picked, and the fresh-keeping effect of the litchis is improved. The peel of the fruit shell is harder and not easy to damage, the white film on the inner wall is thicker, the natural oxidation speed is slowed down, the pulp is firm and glittering, the kernel and the pulp are tightly connected, the browning degree of the peel is delayed, and the lesion rate and the water loss rate of the fruit are low. According to the method, any physical or chemical treatment or biological fresh-keeping agents are not needed after the litchis are picked, and the natural fresh-keeping period after picking is prolonged while the original taste and flavor of the litchis are kept. The method creatively solves the problem of fresh keeping of the litchis, and important economic and social benefits are generated after industrial application.

Description

Technical field:

[0001] The invention relates to the field of agricultural biotechnology, and in particular to the construction and application of a composite microbial agent for pre-harvest preservation of litchi. Background technology:

[0002] The rich and diverse nutrients and bioactive substances of litchi as well as its taste are very popular among consumers. However, litchi matures in the high temperature of midsummer, and the physiological metabolism of the fruit after harvest is vigorous. The peel is very easy to turn brown, and the flesh will deteriorate and rot, which in turn affects the nutritional value and economic value of litchi.

[0003] At present, domestic and foreign research institutes and enterprises are all using cold chain technology as the entry point, focusing on key preservation technologies such as pre-cooling, antibacterial, packaging, and cold storage. However, the disadvantages of these technologies are limited capacity, high cost, and difficulty in large-scale promotion. Moreover, once litchi leaves the cold chain environment and is placed at room temperature, it will begin to change color, taste, and deteriorate in a short period of time, and it has not fundamentally solved the problem of preservation of the entire industrial chain of litchi. The present invention aims to develop a subversive and innovative green biotechnology for "pre-harvest preservation" of litchi in response to the technical difficulties of litchi preservation. That is, by applying a composite microbial preservative agent to the soil within a certain period of time before litchi is picked to change the soil microbial community and its density, thereby affecting the biological synthesis of the fruit epidermis, so that it can be placed in the open air at room temperature for a longer time after picking, and it is not easy to rot and deteriorate, and the original nutrition, taste, and appearance of the fruit are not changed. Summary of the invention:

[0004] The present invention provides the construction and application of a composite microbial agent for pre-harvest preservation of litchis. A plurality of microbial strains are selectively screened for key enzymes and pathogens causing browning of litchi peel to form the composite microbial agent, which is applied to soil during the growth period of litchi fruits. After litchis are picked, the shelf life can be extended without any treatment, thereby solving the problems of limited capacity, high cost and short shelf life caused by the post-harvest preservation process of litchis in the prior art.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first object of the present invention is to provide a composite microbial agent for pre-harvest preservation of litchi, which comprises Serratia marcescens, Bacillus pseudomycoides, Lysinibacillus sphaericus, Trichoderma koningii and Coriolopsis sp.

[0007] Preferably, the composite microbial agent is formed by mixing a composite bacterial agent and a composite fungal agent in a weight ratio of 2:1, wherein the volume ratio of the bacterial solutions of Serratia marcescens, Bacillus pseudomycoides and Bacillus sphaeroides in the composite bacterial agent is 1:1:1, and the volume ratio of the bacterial solutions of Trichoderma koningii and Porcini sphaeroides in the composite fungal agent is 1:1.

[0008] Preferably, the Serratia marcescens and Bacillus pseudomycoides are dual antibacterials that are resistant to heavy metals and fungi and have high tolerance to copper, iron and manganese and inhibitory properties against litchi pathogenic fungi; the Lysinibacillus sphaeroides, Trichoderma koningii and Lepidium indica are lignin decomposing bacteria that can produce laccase and / or lignin peroxidase.

[0009] More preferably, the Serratia marcescens is Serratia marcescens X1, the Bacillus pseudomycoides is Bacillus pseudomycoides X8, the Lysinibacillus sphaericus is Lysinibacillus sphaericus 38-6, the Trichoderma koningii is Trichoderma koningii Z15-3-2, and the Coriolopsis sp. is Coriolopsis sp. 9-3-19.

[0010] The second object of the present invention is to provide a method for preparing the composite microbial agent for preserving litchi before harvest, which specifically comprises the following steps: 600 The bacterial liquids of spherical Lysinibacillus, Serratia marcescens and Bacillus pseudomycoides with a concentration of 0.5 were inoculated into LB culture medium for culture, and then the bacterial liquids of equal volumes were combined to obtain a composite bacterial liquid; the bacterial blocks of Trichoderma koningii and Lepomistra spp. with a diameter of 1 cm were broken up and inoculated into PDA culture medium for culture, and then the bacterial liquids of equal volumes were combined to obtain a composite fungal liquid; the composite bacterial liquid and the composite fungal liquid were solidified to obtain a composite bacterial agent and a composite fungal agent, and then they were mixed at a weight ratio of 2:1 to obtain a composite microbial agent.

[0011] Preferably, the formula of the LB medium is: 10 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L NaCl, 15.0 g / L agar, and the balance is water; the formula of the PDA medium is: 300 g / L potato extract powder, 20.0 g / L glucose, 15.0 g / L agar, and the balance is water.

[0012] Preferably, the conditions for culturing in LB medium are: culturing at 37° C. and 180 rpm for 24-48 h; and the conditions for culturing in PDA medium are: culturing at 30° C. and 180 rpm for 72-96 h.

[0013] Preferably, the solidification step is: mixing the composite bacterial liquid with the solid material in a solid fermentation reactor, reacting for 24 hours, then adding LB liquid culture medium, reacting for 24 hours, and immobilizing to obtain a composite bacterial agent; mixing the composite fungal liquid with the solid material in a solid fermentation reactor, reacting for 24 hours, then adding PDA liquid culture medium, reacting for 24 hours, and immobilizing to obtain a composite fungal agent.

[0014] Further preferably, the mass volume ratio of the solid material to the composite bacterial liquid or the composite fungal liquid is 2 Kg / L; the solid material is sawdust, bagasse or a mixture thereof; and the reaction temperature is 30-40°C.

[0015] The third object of the present invention is to provide the use of the above-mentioned composite microbial agent in pre-harvest preservation of litchi.

[0016] Preferably, the composite microbial agent is evenly spread on the surface soil of the litchi tree during the growth period of the litchi fruit.

[0017] The fourth object of the present invention is to provide a method for preserving litchi before harvest, specifically, applying the above-mentioned composite microbial agent to the soil during the growth period of litchi fruit, so that the fruit does not need any treatment after picking, can be placed at room temperature for a longer time, and is not easy to rot and deteriorate.

[0018] The purpose of the present invention is to target the key enzymes and pathogens of litchi peel browning, and screen microbial strains that can tolerate high concentrations of metal ions, high-yield lignin-degrading enzymes and antifungal and harmful bacteria, and can enhance the water retention and stress resistance of litchi. It is assembled into a composite microbial agent for pre-picking litchi, and the invention uses the unique technology of this team for low-cost, high-density cultivation. During the growth of litchi fruit, the composite microbial agent is applied, and the functional strains in the agent change the indigenous microbial community and its density in the orchard soil, adjust the metal ion, carbon, and nitrogen content in the soil, and inhibit the growth of pathogens, affecting the biological synthesis of the fruit skin and pulp, so that the fruit does not need any treatment after picking, and can be placed at room temperature for a longer time without being easy to rot and deteriorate, and does not change the original nutrition, taste and appearance of the fruit.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1. At present, the preservation of litchi at home and abroad is based on the concept of post-harvest preservation without exception, that is, the "post-harvest preservation" technology such as refrigeration, vacuum preservative film, chemical preservatives, biological preservatives, etc. is used after the fruit is picked. The "pre-harvest preservation" technology proposed in the present invention is to apply a composite microbial agent during the growth period of litchi fruit. The composite microbial agent for pre-harvest preservation of litchi contains functional strains that can tolerate high concentrations of metal ions, high-yield lignin-degrading enzymes, and resistance to litchi pathogenic fungi.

[0021] 2. Compared with the control group (without the application of the composite microbial agent), the litchi fruit of the test group (with the composite microbial agent) became larger and the skin of the fruit shell became harder, making the outer peel less likely to be damaged. The inner wall white film was thicker, which slowed down the natural oxidation rate of the inner peel. The flesh was firm, crystal clear, juicy and sweet, and the core and the flesh were tightly connected. In addition, the litchis in the test group and the control group were placed at room temperature for 7 days without any pretreatment after picking. The browning index, lesion index and water loss rate of the litchi in the test group were lower than those in the control group. This shows that the application of the composite microbial agent of the present invention during the growth of litchi fruit can effectively improve the quality of the fruit, improve the hardness and thickness of the peel, delay the degree of browning of the peel, and reduce the fruit lesion rate. It can be seen that this technology can extend the natural fresh-keeping period of the fruit after picking, and no physical treatment or chemical and biological preservative methods are required after picking to keep the original flavor of the product.

[0022] 3. The composite microbial agent raw material provided by the present invention is simple to prepare, easy to operate, and convenient for industrial production. It innovatively solves the problem of lychee preservation and will produce important economic and social benefits after industrial application.

[0023] S. marcescens X1, B. pseudomycoides X8, L. sphaericus 38-6, T. koningii Z15-3-2 and Coriolopsis sp. 9-3-19. They are published in NCBI with accession numbers OR342587, OR342592, PQ113570, PQ113582 and PQ113583, respectively. The applicant also holds the five strains and guarantees that they will be released to the public within 20 years from the date of application. Description of the drawings:

[0024] Figure 1 The figure is a screen result diagram of the metal-resistant strain and the antifungal strain in Example 1, wherein: (A) metal-resistant strain; (B) copper ion tolerance result; (C) iron ion tolerance result; (D) manganese ion tolerance result; and (E) antifungal strain.

[0025] Figure 2 This is a diagram showing the screening results of the lignin decomposing strains in Example 1.

[0026] Figure 3 It is a comparison diagram of litchi fruits in the test group and the control group in Example 3.

[0027] Figure 4 This is a graph showing the soluble sugar and titratable acid content of litchi pulp in the test group and the control group on the day of picking in Example 3.

[0028] Figure 5 Graph showing the malondialdehyde content in litchi pulp of the test group and the control group at the 0th and 7th day after picking in Example 3.

[0029] Figure 6 This is a graph showing the results of the peel browning index of litchis in the test group and the control group in Example 3 after being picked and stored at room temperature for several days.

[0030] Figure 7 This is a graph showing the fruit lesion index results of the test group and the control group in Example 3 after the lychees were picked and stored at room temperature for several days.

[0031] Figure 8 This is a graph showing the water loss rate of litchi in the test group and the control group in Example 3 after being picked and stored at room temperature for several days.

[0032] Fig. 9 This is a comparison diagram of litchis in the test group and the control group in Example 3, which were placed indoors at room temperature for 7 days without any pretreatment after picking. Specific implementation method:

[0033] It will be appreciated by those skilled in the art that the disclosed techniques in the following examples represent techniques that the inventors have found to work well in the practice of the present invention. However, many changes may be made in the disclosed specific embodiments, and still the same or similar results may be obtained without departing from the spirit and scope of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0034] Example 1 Screening and identification of functional strains of composite microbial agents

[0035] (1) Screening of dual-resistant strains with anti-metal and anti-fungal properties

[0036] Banana, sugarcane and litchi rhizosphere soil samples were collected from agricultural land in Guangzhou. 5 g of soil sample was weighed and made into a suspended bacterial solution with sterile water and diluted 10 -3 and 10 -4 (w / v). Take 100 μL of diluted bacterial solution and apply it to LB solid medium containing 200 mg / L CuSO4, 200 mg / L Fe2(SO4)3 and 200 mg / LMnSO4 respectively (1L of culture medium raw materials: 10g tryptone, 5.0g yeast extract, 10.0g NaCl, 15.0g agar, the rest is water, mix all ingredients evenly, adjust pH to 7.0 with NaOH solution, and sterilize at 121℃ for 15min before use), and culture at 30℃ for 2 days. Single colonies are selected according to different colony morphology. Single colonies are purified and cultured with the same culture medium. Pure culture is stored at -80℃ with 20% glycerol.

[0037] Filter results by Figure 1 As shown in A, 16 metal-resistant strains with different colony morphologies were isolated and purified from banana and sugarcane rhizosphere soil samples.

[0038] In order to detect the highest metal tolerance concentration of metal-resistant strains, 16 metal-resistant strains were inoculated into LB liquid culture medium and cultured at 30°C and 180 rpm for 1 day. 600 , and adjust the concentration of each strain to OD 600 =0.5. Take 3 μL of bacterial solution of each strain and inoculate it in LB solid medium containing 100-1000 mg / L CuSO4, 100-1000 mg / L Fe2(SO4)3 and 200-2000 mg / L MnSO4, and observe the survival of each strain after culturing at 30°C for 3 days.

[0039] Depend on Figure 1 BD learned that in the copper ion tolerance analysis, except for strain X26, the resistance of other strains to CuSO4 was ≥200mg / L, among which strains X1 and X6 could tolerate up to 1000mg / L CuSO4 ( Figure 1 B). In the analysis of iron tolerance, 16 strains were resistant to Fe2(SO4)3 ≥ 600 mg / L, of which strains X1, X4 and X33 could tolerate up to 1000 mg / L of Fe2(SO4)3 ( Figure 1 C). In the tolerance analysis of manganese ions, except for strain X33, all other strains could tolerate up to 2000 mg / L of MnSO4 ( Figure 1 D). The above results show that among the 16 metal-resistant strains, X1 is resistant to multiple metals at high concentrations, and its resistance to copper, iron and manganese is higher than 1000 mg / L.

[0040] In order to detect the inhibitory effect of the selected strains on the growth of litchi fruit pathogens, we selected Geotrichum sp., the fungal pathogen of litchi fruit sour rot, and Cylindrocladium sp., the fungal pathogen of white mold, as the test pathogens. The 16 metal-resistant strains were inoculated into LB liquid culture medium and cultured at 30°C and 180rpm for 1 day. The OD value of the bacterial liquid of the metal-resistant strains was detected. 600 , and adjust the concentration of each strain to OD 600 =0.5. 10 μL of each strain was inoculated in LB solid medium containing Geotrichum sp. and Cylindrocladium sp. that had been cultured for 3 days, and the inhibition of each strain on the growth of pathogens was observed after culturing at 30°C for 3 days.

[0041] Depend on Figure 1As shown in Figure E, strain X17 inhibited the growth of Geotrichum sp., the fungal pathogen of litchi sour rot, while strains X1, X4, X5, X6, X8, X19, X33 and G2 all inhibited the growth of Geotrichum sp., the fungal pathogen of litchi sour rot and Cylindrocladium sp., the fungal pathogen of white mold. Among them, X8 had the most significant inhibitory effect on the growth of litchi fruit pathogens.

[0042] From the above results, it can be seen that the 16 strains screened by the present invention can resist high concentrations of metals such as copper, iron and manganese, especially strain X1 has very significant resistance to metals such as copper, iron and manganese. In addition, 9 strains have inhibitory effects on litchi fruit pathogens. Among them, strain X8 has the most significant inhibitory effect on litchi fruit pathogens.

[0043] (2) Screening of lignin-degrading strains

[0044] Banana, sugarcane and litchi rhizosphere soil samples were collected from agricultural land in Guangzhou. 5 g of soil sample was weighed and made into a suspended bacterial solution with sterile water and diluted 10 -3 and 10 -4 (w / v). Take 100 μL of diluted bacterial solution and apply it to LB solid medium and PDA solid medium containing guaiacol respectively (1L of culture medium raw materials: 300g potato extract powder, 20.0g glucose, 15.0g agar, the rest is water, mix all ingredients evenly, natural pH, and sterilize at 115℃ high pressure for 20min before use), and culture at 30℃ for 3-7d. Different single colonies are selected according to the color change of the culture medium. Single colonies are purified and cultured with the same culture medium. Pure culture is stored at -80℃ with 20% glycerol.

[0045] In order to detect the production of lignin decomposing enzymes by each single bacterium, the colonies with color change circles were inoculated into LB solid medium (or PDA solid medium) containing guaiacol and aniline blue for pure culture, and cultured at 30°C for 3-7 days. The color change of the medium was observed. Whether laccase was produced was determined by whether the guaiacol medium turned red, and whether lignin peroxidase was produced was determined by whether the aniline blue medium faded.

[0046] Depend on Figure 2As shown, strains 38-6, Z15-3-2 and 9-3-19 all show red in the medium containing guaiacol, which can qualitatively indicate that strains 38-6, Z15-3-2 and 9-3-19 can all produce laccase, among which the red color of strains Z15-3-2 and 9-3-19 in the guaiacol medium is particularly obvious, indicating that strains Z15-3-2 and 9-3-19 produce high laccase. The red color of strain 38-6 in the guaiacol medium is weaker, indicating that the amount of laccase produced by the strain is low. However, the fading of strain 38-6 in the medium containing aniline blue is particularly obvious, which can qualitatively indicate that strain 38-6 can produce lignin peroxidase, and the enzyme production is high. From the above results, it can be seen that strains 38-6, Z15-3-2 and 9-3-19 are high-yield lignin degrading enzyme strains.

[0047] (3) Identification of functional strains of composite microbial agents

[0048] The present invention targets the key enzymes and pathogens of litchi peel browning, and selectively screens strains that can tolerate high concentrations of metal ions (such as copper, iron and manganese ions), high-yield lignin-degrading enzymes (such as laccase and lignin peroxidase) and antifungal strains. The strains are then assembled into a composite microbial agent for pre-harvest preservation of litchi. Based on the above results, strains X1, X8, 38-6, Z15-3-2 and 9-3-19 are selected as the functional strains of the composite microbial preservative agent for litchi before harvesting in this project.

[0049] The present invention identifies the above-screened functional strains X1, X8, 38-6, Z15-3-2 and 9-3-19 by 16s rRNA and ITS genes. The 16s rRNA gene of bacteria was amplified with primers 27F and 1492R, and the ITS gene of fungi was amplified with primers ITS1 and ITS4, and then sent to Qingke Biotechnology Co., Ltd. for sequencing. The measured 16s rRNA and ITS gene sequences were compared by BLAST on the NCBI website, and sequences with high homology to the measured 16s rRNA and ITS gene sequences were screened out, thereby identifying the functional strains. The results are shown in Table 1.

[0050] Table 1 16s rRNA / ITS gene similarity between the strains of the present invention and homologous strains

[0051]

[0052] From the 16s rRNA and ITS gene similarities in Table 1, it can be seen that the functional strains X1, X8, 38-6, Z15-3-2 and 9-3-19 of the present invention are highly homologous to Serratia marcescens C3, Bacillus pseudomycoides SM1, Lysinibacillus sphaericus HBUM07034, Trichoderma koningii ACCC32835 and Coriolopsis sp. Melodinus suaveolens, respectively, and their homologies are all higher than 99.7%. For this purpose, the functional strains X1, X8, 38-6, Z15-3-2 and 9-3-19 of the present invention are named as Serratia marcescens X1, Bacillus pseudomycoides X8, Lysinibacillus sphaericus 38-6, Trichoderma koningii Z15-3-2 and Coriolopsis sp. 9-3-19, respectively. These 5 strains are disclosed in NCBI, and their accession numbers are OR342587, OR342592, PQ113570, PQ113582 and PQ113583, respectively.

[0053] Example 2 Preparation of composite microbial agent

[0054] (1) Cultivation of functional strains of composite microbial agents

[0055] 10 mL of bacterial solution OD 600 The bacterial liquids of spherical Lysinibacillus 38-6, Serratia marcescens X1 and Bacillus pseudomycoides X8 were inoculated into the above 1L liquid LB medium respectively, and cultured at 37°C and 180rpm for 24-48h. The composite bacterial liquid was obtained by combining equal volumes at a ratio of 1:1:1.

[0056] Five 1 cm diameter Trichoderma koningii Z15-3-2 and Psora 9-3-19 fungus blocks were broken up and inoculated into the 1 L liquid PDA culture medium, respectively, and cultured at 30° C. and 180 rpm for 72-96 hours. The composite fungus culture solution was obtained by combining equal volumes in a ratio of 1:1.

[0057] (2) Solidification of functional strains of composite microbial agents

[0058] 10 kg of sawdust (or bagasse) solid material was added to the solid fermentation reactor, and then 5 L of composite bacterial liquid was added to mix with the solid material, reacted at 30-40 ° C for 24 hours, and then 3 L of LB liquid culture medium was added, and reacted at 30-40 ° C for 24 hours to immobilize the composite bacterial agent.

[0059] 10 kg of sawdust (or bagasse) solid material was added to the solid fermentation reactor, and then 5 L of composite fungal liquid was added to mix with the solid material, reacted at 30-40 ° C for 24 hours, and then 3 L of PDA liquid culture medium was added, reacted at 30-40 ° C for 24 hours to immobilize the composite fungal agent.

[0060] The composite bacterial agent and the composite fungal agent are mixed in proportion to obtain a composite microbial agent.

[0061] Example 3 Application of composite microbial agent in preserving litchi before harvesting

[0062] (1) Litchi orchard experiment

[0063] In early May, when Guiwei began to produce primary small fruits, composite microbial agents were applied to the litchi trees (composite bacterial agents and composite fungal agents were mixed at a weight ratio of 2:1). The leaves on the surface of the litchi trees were spread apart, and the composite microbial agents (15Kg / tree) were evenly spread on the surface soil of the litchi trees. Then the spread leaves were spread back on the surface of the litchi trees and covered with the composite microbial agents (experimental group). The litchi trees without composite microbial agents were used as controls. The litchi orchard maintained daily management of the litchi trees in the experimental and control groups as usual. When Guiwei matured in early July, the litchi fruits of the experimental and control groups were picked.

[0064] (2) Lychee preservation effect test

[0065] The picked litchis of the test group and the control group were stored at room temperature without any pretreatment. The soluble sugar and titratable acid content of the litchi pulp on the day of picking were tested. The changes of litchi fruits were observed every 2 days, and the malondialdehyde content, browning index, disease susceptibility index (lesion index) and water loss rate of litchi were tested. The results are shown in Figure 3-Figure 9 .

[0066] According to the instructions of the plant soluble sugar content detection kit (PYFA4-M96) and the malondialdehyde content detection kit (PYHA4-M96) (Mofan Biotechnology), the soluble sugar and malondialdehyde contents of litchi pulp were detected respectively.

[0067] Titratable acid content detection: Crush the litchi pulp, centrifuge at 5000rpm for 5min, take the supernatant extract, transfer 20.0mL of the extract into a conical flask, add 2 drops of 1% phenolphthalein indicator, and titrate with a calibrated sodium hydroxide solution. Titrate until the solution begins to turn pink and does not fade within 0.5min, record the amount of sodium hydroxide titration solution, and repeat three times. Then titrate with distilled water instead of the supernatant as a blank control.

[0068] According to the consumption of NaOH titration solution, the titratable acid content in litchi pulp is calculated. Calculation formula:

[0069] Titratable acid content = V × c × (V1-V0) × 0.064 × 100% / Vs

[0070] V is the total volume of sample extract, mL;

[0071] Vs is the volume of the extract taken during titration, mL;

[0072] c is the concentration of NaOH titration solution, mol / L;

[0073] V1 is the volume of NaOH solution consumed in titrating the extract, mL;

[0074] V0 is the volume of NaOH solution consumed in titrating distilled water, mL;

[0075] 0.064 is the number of grams of citric acid equivalent to 1mL of 0.1mol / LNaOH solution.

[0076] Peel browning index: According to the size of the browning area on the fruit surface, it is graded as follows: Grade 0: no browning; Grade 1: browning area less than 5%; Grade 2: browning area 5% to 25%; Grade 3: browning area 25% to 50%; Grade 4: browning area more than 50%. Formula: Browning index = ∑ (browning level × number of fruits in this level) × 100% / (total number of fruits × highest grade number).

[0077] Fruit susceptibility index (lesion index): According to the size of the lesions on the fruit surface, it is graded as follows: 0: no susceptibility; 1: slight susceptibility; 2: lesion area 5% to 25%; 3: lesion area 25% to 50%; 4: lesion area more than 50%. Formula: susceptibility index = ∑ (number of diseased fruit grades × number of fruits in that grade) × 100% / (total number of fruits × highest grade number).

[0078] Water loss rate: Weigh and record the weight of each group of litchi on an electronic balance every 2 days. Formula: Weight loss rate (%) = (m0-m1) / m0×100%; where m0 is the initial mass of litchi before treatment (g); m1 is the mass of litchi after several days of storage at room temperature (g).

[0079] Depend on Figure 3 As shown, compared with the control group (without composite microbial agent), the experimental group (with composite microbial agent) had larger litchi fruits (20 fruits in the experimental group weighed 368±5g; 20 fruits in the control group weighed 290±4g, with an average fruit weight increase of 26.9%), and the yield increased. The fruit shell was harder, making the outer peel less susceptible to damage. The inner wall white film was thicker, which slowed down the natural oxidation of the inner peel. The flesh was firm and crystal clear. Figure 4 is the content of soluble sugar and titratable acid in litchi pulp on the day of picking, Figure 4 As shown, on the day of picking, the soluble sugar content in the litchi pulp of the experimental group (478 mg / g) was higher than that of the control group (450 mg / g), while the titratable acid content in the litchi pulp of the experimental group (0.166 g / 100 mL) was lower than that of the control group (0.173 g / 100 mL), indicating that the litchi in the experimental group was sweeter, less sour and astringent, and had a better taste than the litchi in the control group. Figure 3 and Figure 4 The results show that the application of composite microbial agents during the small fruit stage of litchi fruit can improve the size of litchi fruit, the hardness and thickness of the peel, and the taste of the flesh. Figure 5 It can be seen that after the litchis in the experimental group and the control group were picked and stored for 7 days, the malondialdehyde content in the litchi flesh of the experimental group and the control group increased compared with the malondialdehyde content on the day of picking, but the malondialdehyde content in the litchi flesh of the experimental group (2.53nmol / g) was lower than that of the control group (3.33nmol / g), indicating that the degree of cell membrane peroxidation in the litchi flesh of the experimental group was lower, and the flesh damage and decay were lower. Figure 6 It can be seen that after the litchis in the experimental group and the control group were stored at room temperature for 7 days without any pretreatment after picking, the browning index of the litchis in the control group reached 71.67%, while that in the experimental group was only 43.75%, which shows that the application of composite microbial agents during the litchi small fruit stage can delay the browning degree of litchi peel after picking. Figure 7 It can be seen that after 7 days of storage at room temperature, more than half of the litchi fruits in the control group were diseased (lesion index 57.03%), while only 27.91% of the fruits in the test group were diseased. After 11 days of storage, all the litchi fruits in the control group were diseased, and the disease index of the litchi fruits in the test group was 79.58%, which shows that the application of composite microbial agents during the litchi fruiting period can reduce the degree of disease in litchi fruits after picking. Figure 8 It can be seen that after 11 days of storage at room temperature, the water loss rate of litchi in the experimental group (14.19%) was significantly lower than that in the control group (20.93%), indicating that the application of composite microbial agents during the small fruit stage of litchi can reduce the water loss rate of litchi fruit after picking.

[0080] Depend on Fig. 9The above results show that applying the composite microbial preservative agent developed in this project during the growth period of litchi fruit can effectively improve the hardness and thickness of the peel and the taste of the flesh, improve the fruit quality, delay the browning degree of the peel, and reduce the fruit lesion rate and water loss rate. Ultimately, the fruit does not need any treatment after picking, and can be placed at room temperature for a longer time without being easily rotten or deteriorated.

Claims

1. A composite microbial agent for preserving litchi before harvest, characterized in that: The composite microbial agent comprises Serratia marcescens, Bacillus pseudomycoides, Lysinibacillus sphaericus, Trichoderma koningii and Coriolopsis sp.

2. The composite microbial agent according to claim 1, characterized in that: The composite microbial inoculant is prepared by mixing a composite bacterial inoculant and a composite fungal inoculant in a weight ratio of 2:1, wherein the volume ratio of the bacterial liquids of Serratia marcescens, Bacillus pseudomycoides and Bacillus sphaeroides in the composite bacterial inoculant is 1:1:1, and the volume ratio of the bacterial liquids of Trichoderma koningii and Porcinotrachea rapae in the composite fungal inoculant is 1:

1.

3. The composite microbial agent according to claim 1, characterized in that: The Serratia marcescens and Bacillus pseudomycoides are anti-heavy metal and anti-fungal dual antibacterials that have high tolerance to copper, iron and manganese and have inhibitory effects on litchi pathogenic fungi; the Lysinibacillus sphaeroides, Trichoderma koningii and Lepidium indica are lignin decomposing bacteria that can produce laccase and / or lignin peroxidase.

4. The method for preparing the composite microbial agent according to claim 1, 2 or 3, characterized in that: The specific steps include: The bacterial solution OD 600 The bacterial liquids of spherical Lysinibacillus, Serratia marcescens and Bacillus pseudomycoides with a concentration of 0.5 were respectively inoculated into LB culture medium for cultivation, and then the bacterial liquids of equal volumes were combined to obtain a composite bacterial liquid; the fungal blocks of Trichoderma koningii and Leptospira rapae with a diameter of 1 cm were broken up and inoculated into PDA culture medium for cultivation, and then the bacterial liquids of equal volumes were combined to obtain a composite fungal liquid; the composite bacterial liquid and the composite fungal liquid were respectively solidified to obtain a composite bacterial agent and a composite fungal agent, which were mixed at a weight ratio of 2:1 to obtain a composite microbial agent.

5. The preparation method according to claim 4, characterized in that: The formula of the LB medium is: 10 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L sodium chloride, 15.0 g / L agar, and the balance is water; the formula of the PDA medium is: 300 g / L potato extract powder, 20.0 g / L glucose, 15.0 g / L agar, and the balance is water.

6. The preparation method according to claim 4, characterized in that: The conditions for culturing in the LB medium are: culturing at 37° C. and 180 rpm for 24-48 h; the conditions for culturing in the PDA medium are: culturing at 30° C. and 180 rpm for 72-96 h.

7. The preparation method according to claim 4, characterized in that: The solidification step is: mixing the composite bacterial liquid with the solid material in a solid fermentation reactor, reacting for 24 hours, then adding LB liquid culture medium, reacting for 24 hours, and immobilizing to obtain a composite bacterial agent; mixing the composite fungal liquid with the solid material in a solid fermentation reactor, reacting for 24 hours, then adding PDA liquid culture medium, reacting for 24 hours, and immobilizing to obtain a composite fungal agent.

8. The preparation method according to claim 7, characterized in that: The mass volume ratio of the solid material to the composite bacterial liquid or the composite fungal liquid is 2 Kg / L; the solid material is sawdust, bagasse or a mixture thereof; and the reaction temperature is 30-40°C.

9. Use of the composite microbial agent according to claim 1, 2 or 3 in preserving litchi before harvest, characterized in that: The method is to evenly spread the composite microbial agent on the surface soil of the litchi tree during the litchi fruit growth period.

10. A method for preserving litchi before harvest, comprising applying the composite microbial agent of claim 1, 2 or 3 to the soil during the growth period of litchi fruit.

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