Construction and application of a complex microbial agent for preharvest preservation of litchi

Abstract

The application discloses a kind of compound microbial inoculants for preharvest preservation of litchi and application thereof.The application is directed to the key enzymes of litchi pericarp browning and its pathogenic bacteria, and directional screening of microbial strains tolerating high concentration of metal ions, high yield of lignin-degrading enzyme and antifungal, etc. The compound microbial inoculants for preharvest preservation of litchi is formed and applied to litchi fruit growth period. After litchi is picked, the fruit size becomes larger, the shell skin is harder and not easy to be damaged, the inner wall white film is thicker, the natural oxidation speed is slower, the pulp is solid and crystal, the fruit core and pulp are connected tightly, in addition, the pericarp browning degree is delayed, the fruit disease rate and water loss rate are low. The application does not need any physical, chemical treatment or biological preservative after litchi is picked, which can prolong the natural preservation period after picking while keeping the original taste of the product. The application innovatively solves the problem of litchi preservation, and will produce important economic and social benefits after industrial application.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, and particularly relates to construction and application of a compound microbial agent for pre-harvest preservation of litchi. BACKGROUND

[0002] Litchi is rich in nutrients and bioactive substances, and its taste is deeply loved by consumers. However, litchi matures in high temperature in summer, and the physiological metabolism of the fruit after harvesting is vigorous, the peel is prone to browning, the pulp is prone to change and rot, and then the nutritional value and economic value of litchi are affected.

[0003] At present, scientific research institutions and enterprises at home and abroad all take cold chain technology as the entry point, and focus on key technology links such as precooling, bacteriostasis, packaging, cold storage and the like. However, these technologies have the disadvantages of limited capacity, high cost, and difficulty in large-scale promotion, and once the litchi leaves the cold chain environment and is placed at room temperature, the litchi will start to change color, taste and quality in a short time, and the problem of litchi preservation in the whole industry chain cannot be fundamentally solved. The present application aims to solve the technical problems of litchi preservation, and develop a revolutionary green biological technology of litchi "preservation before picking". That is, by applying a compound microbial preservation agent to the soil within a certain period of time before picking litchi, the microbial community and its density of the soil are changed to affect the biological synthesis of the fruit skin, so that the fruit can be placed at room temperature for a longer time after picking, and is not easy to rot and deteriorate, and the original nutrition, taste and appearance of the fruit are not changed. SUMMARY

[0004] The present application provides construction and application of a compound microbial agent for pre-harvest preservation of litchi, a plurality of microbial strains are selected by targeting the key enzymes of litchi peel browning and pathogenic bacteria to form a compound microbial agent, which is applied to the soil during the growth of litchi fruit, and the preservation period is prolonged without any treatment after picking litchi, solving the problems of limited capacity, high cost and short preservation period caused by the use of litchi post-harvest preservation process in the prior art.

[0005] The present application is realized by the following technical scheme:

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

[0007] Preferably, the complex microbial inoculant is prepared by mixing the complex bacterial inoculant and the complex fungal inoculant at a weight ratio of 2:1, the complex bacterial inoculant has a volume ratio of S. marcescens, B. pseudomycoides and L. sphaericus of 1:1:1, and the complex fungal inoculant has a volume ratio of T. koningii and Coriolopsis sp. of 1:1.

[0008] Preferably, the S. marcescens and B. pseudomycoides are heavy metal and fungus resistant double antibiotics with high tolerance to copper, iron and manganese and inhibition to litchi pathogenic fungi; the L. sphaericus, T. koningii and Coriolopsis sp. are lignin-decomposing bacteria capable of producing laccase or / and lignin peroxidase.

[0009] Further preferably, the S. marcescens is S. marcescens X1, the B. pseudomycoides is B. pseudomycoides X8, the L. sphaericus is L. sphaericus 38-6, the T. koningii is T. koningii Z15-3-2, and the Coriolopsis sp. is Coriolopsis sp. 9-3-19.

[0010] A second object of the present application is to provide a preparation method of the complex microbial inoculant for preharvest preservation of litchi, which specifically comprises the following steps: inoculating L. sphaericus, S. marcescens and B. pseudomycoides bacterial liquid with OD 600 0.5 into LB culture medium for culture, then mixing the bacterial liquids in equal volume to obtain a complex bacterial inoculant; crushing T. koningii and Coriolopsis sp. blocks with a diameter of 1 cm, then inoculating them into PDA culture medium for culture, then mixing the bacterial liquids in equal volume to obtain a complex fungal inoculant; solidifying the complex bacterial inoculant and the complex fungal inoculant to obtain a complex bacterial inoculant and a complex fungal inoculant, then mixing them at a weight ratio of 2:1 to obtain the complex microbial inoculant.

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

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

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

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

[0015] A third object of the present application is to provide the use of the above-mentioned complex microbial agent in pre-harvest preservation of litchi.

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

[0017] A fourth object of the present application is to provide a pre-harvest preservation method for litchi, which specifically applies the above-mentioned complex microbial agent to the soil during the growth period of the litchi fruit, so that the fruit after being picked does not need to be treated in any way and can be placed for a longer time at room temperature indoors without being easily rotten and deteriorated.

[0018] The present application aims to screen microbial strains that can tolerate high concentrations of metal ions, produce high yields of lignin-degrading enzymes, resist fungi and harmful bacteria, enhance water retention and stress resistance of litchi, etc., for litchi peel browning key enzymes and their pathogenic bacteria. The strains are assembled into a complex microbial agent for pre-harvest preservation of litchi, and a unique technology of the team is used for low-cost, high-density cultivation. The complex microbial agent is applied during the growth period of the litchi fruit, 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 pathogenic bacteria, affecting the biological synthesis of the fruit skin and pulp, and ultimately making the fruit after being picked not need to be treated in any way, can be placed for a longer time at room temperature without being easily rotten and deteriorated, and does not change the original nutrition, taste and appearance of the fruit.

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

[0020] 1. Currently, the preservation of litchi at home and abroad is based on the concept of post-harvest preservation, i.e. using cold storage, vacuum preservation film, chemical preservatives, biological preservatives, etc. for "post-harvest preservation" technology. The present application proposes a "pre-harvest preservation" technology, i.e. applying a complex microbial agent during the growth period of the litchi fruit. The complex microbial agent for pre-harvest preservation of litchi contains functional strains that can tolerate high concentrations of metal ions, produce high yields of lignin-degrading enzymes, and resist litchi pathogenic fungi, etc.

[0021] 2. Compared with the control group (without applying the complex microbial inoculant), the fruit of the test group (applying the complex microbial inoculant) is larger in size and has a harder pericarp epidermis, so that the exocarp is not easily damaged. The inner wall white membrane is thicker, so that the natural oxidation speed of the endocarp is slowed down. The fruit flesh is solid, crystal, juicy and sweet, and the fruit core is tightly connected with the fruit flesh. In addition, the browning index, disease index and water loss rate of the test group are lower than those of the control group after the litchi is picked and placed at room temperature for 7 days without any pretreatment. This shows that applying the complex microbial inoculant of the application during the growth of litchi fruit can effectively improve the fruit quality, improve the hardness and thickness of the pericarp, delay the browning degree of the pericarp, and reduce the disease rate of the fruit. Therefore, the technology can prolong the natural shelf life of the fruit after picking, and does not need to use any physical treatment or chemical and biological preservative method to maintain the original taste of the product.

[0022] 3. The complex microbial inoculant provided by the application has simple raw material preparation, easy operation and is convenient for industrial production, and innovatively solves the problem of litchi 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. The accession numbers in NCBI are OR342587, OR342592, PQ113570, PQ113582 and PQ113583 respectively. The applicant also holds the five strains, and guarantees to issue them to the public for 20 years from the application date. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a screening result diagram of the metal-resistant strains and the anti-fungal strains in Example 1, wherein (A) is the metal-resistant strains; (B) is the copper ion tolerance result; (C) is the iron ion tolerance result; (D) is the manganese ion tolerance result; and (E) is the anti-fungal strains.

[0025] Figure 2 is a screening result diagram of the lignin-decomposing strains in Example 1.

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

[0027] Figure 4 is a diagram of the soluble sugar and titratable acid content of the litchi fruit flesh of the test group and the control group on the day of picking in Example 3.

[0028] Figure 5 This is a graph showing the malondialdehyde (MDA) content of litchi pulp from the experimental group and the control group on days 0 and 7 of harvest in Example 3.

[0029] Figure 6 This is a graph showing the browning index of the lychee peel after being harvested and stored at room temperature for several days in the experimental and control groups in Example 3.

[0030] Figure 7 This is a graph showing the lesion index results of litchi fruits stored at room temperature for several days after harvesting in the experimental and control groups in Example 3.

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

[0032] Figure 9 This is a comparison chart of lychees from the experimental group and the control group that were left at room temperature for 7 days after harvesting without any pretreatment, as shown in Example 3. Detailed implementation method:

[0033] Those skilled in the art will understand that the techniques disclosed in the following embodiments represent those discovered by the inventors that have proven effective in the practice of this invention. However, many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, still yielding the same or similar results. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional in the art.

[0034] Example 1: Screening and Identification of Functional Strains of Compound Microbial Agents

[0035] (1) Screening of strains with dual resistance to metals and fungi

[0036] Rhizosphere soil samples from banana, sugarcane, and lychee trees were collected from agricultural land in Guangzhou. 5g of soil sample was weighed and prepared into a bacterial suspension with sterile water, then diluted 10g. -3 and 10 -4 (w / v). 100 μL of diluted bacterial suspension was spread onto LB solid medium containing 200 mg / L CuSO4, 200 mg / L Fe2(SO4)3, and 200 mg / L MnSO4, respectively (1 L of medium was prepared with: 10 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, 15.0 g agar, and the remainder water; all components were mixed thoroughly, the pH was adjusted to 7.0 with NaOH solution, and the medium was autoclaved at 121°C for 15 min before use). The medium was incubated at 30°C for 2 days. Single colonies were screened based on different colony morphologies. Single colonies were purified and cultured using the same medium. Pure cultures were stored at -80°C with 20% glycerol.

[0037] The screening results are from Figure 1 As shown in Figure A, 16 metal-resistant bacterial strains with different colony morphologies were isolated and purified from rhizosphere soil samples of bananas and sugarcane.

[0038] To determine the maximum metal tolerance concentration of metal-resistant strains, 16 metal-resistant strains were inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 1 day. The OD values ​​of the bacterial culture were then measured. 600 And adjust the concentration of each strain to OD 600 =0.5. Take 3 μL of bacterial suspension from each strain and inoculate it into LB solid medium containing 100-1000 mg / L CuSO4, 100-1000 mg / L Fe2(SO4)3 and 200-2000 mg / L MnSO4 respectively. After incubation at 30℃ for 3 days, observe the survival of each strain.

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

[0040] To investigate the inhibitory effect of the screened strains on the growth of pathogens causing litchi fruit, we selected *Geotrichum sp.*, the fungal pathogen causing litchi fruit sour rot, and *Cylindrocladium sp.*, the fungal pathogen causing litchi fruit white mold, as the pathogens to be tested. Sixteen metal-resistant strains were inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 1 day. The OD values ​​of the bacterial culture for the metal-resistant strains were then measured. 600 And adjust the concentration of each strain to OD 600 =0.5. 10 μL of each strain was inoculated into LB solid medium containing Geotrichum sp. and Cylindrocladium sp. that had been cultured for 3 days, respectively. After incubation at 30°C for 3 days, the inhibitory effect of each strain on the growth of the pathogen was observed.

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

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

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

[0044] Banana, sugarcane and litchi rhizosphere soil samples were collected from agricultural land in Guangzhou City. 5g of soil sample was weighed and suspended in sterile water to prepare a bacterial suspension and diluted 10 -3 and 10 -4 (w / v). 100μL of diluted bacterial suspension was spread on LB solid medium and PDA solid medium (1L of medium raw material preparation: 300g potato extract leaching powder, glucose 20.0g, 15.0g agar, the rest is water, mix the components uniformly, natural pH, and sterilize at 115℃ under high pressure for 20min, then reserve) containing guaiacol, and cultured at 30℃ for 3-7d. Different single colonies were screened according to the color change of the medium. The single colonies were purified and cultured with the same medium. The pure culture was stored in -80℃ with 20% glycerol.

[0045] In order to detect the lignin-decomposing enzyme production of each single bacterium, the colonies with color change were inoculated on LB solid medium (or PDA solid medium) containing guaiacol and aniline blue for pure culture, and cultured at 30℃ for 3-7d. The color change of the medium was observed to qualitatively determine whether laccase was produced by whether the guaiacol medium turned red, and whether lignin peroxidase was produced by whether the aniline blue medium faded.

[0046] From the above results, it can be seen that the 16 strains screened by the present application can resist high concentrations of metals such as copper, iron and manganese, and in particular, the strain X1 has very significant resistance to metals such as copper, iron and manganese. In addition, 9 strains have inhibitory effect on litchi fruit pathogens. Among them, the inhibitory effect of strain X8 on litchi fruit pathogens is the most significant. Figure 2As shown, strains 38-6, Z15-3-2 and 9-3-19 all show red in the guaiacol-containing medium, thus it can be qualitatively explained that strains 38-6, Z15-3-2 and 9-3-19 all can produce laccase, and the red color of strains Z15-3-2 and 9-3-19 in the guaiacol-containing 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-containing medium is weaker, indicating that the amount of laccase produced by the strain is low. However, the fading of strain 38-6 in the aniline blue-containing medium is particularly obvious, thus it can be qualitatively explained that strain 38-6 can produce lignin peroxidase and the amount of enzyme produced is high. From the above results, it can be known 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 the complex microbial inoculant

[0048] The present application is aimed at the key enzymes of litchi pericarp browning and its pathogenic bacteria, and strains capable of tolerating high-concentration metal ions (such as copper, iron and manganese ions), producing high-yield lignin-degrading enzymes (such as laccase and lignin peroxidase) and resisting fungi are screened. The strains are assembled into a complex microbial inoculant for pre-harvest preservation of litchi. According to the above results, strains X1, X8, 38-6, Z15-3-2 and 9-3-19 are selected as the functional strains of the pre-harvest complex microbial preservation inoculant of litchi.

[0049] The functional strains X1, X8, 38-6, Z15-3-2 and 9-3-19 screened above are identified by 16s rRNA and ITS genes. The 16s rRNA gene of bacteria is amplified by primers 27F and 1492R, and the ITS gene of fungi is amplified by primers ITS1 and ITS4, and then sent to Qike Biotechnology Co., Ltd. for sequencing. The measured 16s rRNA and ITS gene sequences are subjected to sequence alignment by BLAST on the NCBI website, and sequences with high homology to the measured 16s rRNA and ITS gene sequences are screened, so as to identify the functional strains. The results are shown in Table 1.

[0050] Table 1 Similarity of 16s rRNA / ITS genes of strains of the present application and homologous strains

[0051]

[0052] From the 16s rRNA and ITS gene similarity of Table 1, the functional strains X1, X8, 38-6, Z15-3-2 and 9-3-19 of the application are highly homologous to Serratia marcescens C3, Bacillus pseudomycoides SM1, Lysinibacillus sphaericus HBUM07034, Trichoderma koningii ACCC32835 and Coriolopsis sp. Melodinus suaveolens respectively, and the homology is all higher than 99.7%. Therefore, the functional strains X1, X8, 38-6, Z15-3-2 and 9-3-19 of the application are respectively named as Serratia marcescens X1, Bacillus pseudomycoides X8, Lysinibacillus sphaericus 38-6, Trichoderma koningii Z15-3-2 and Coriolopsis sp. 9-3-19. The five strains are disclosed in NCBI, and the accession numbers are OR342587, OR342592, PQ113570, PQ113582 and PQ113583 respectively.

[0053] Example 2 Preparation of the compound microbial inoculant

[0054] (1) Cultivation of the functional strains of the compound microbial inoculant

[0055] The 10 mL bacterial liquid OD 600 The 10 mL bacterial liquid OD

[0056] The 5 pieces of Trichoderma koningii Z15-3-2 and Coriolopsis sp. 9-3-19 mycelial blocks with a diameter of 1 cm were crushed and inoculated into the 1L liquid PDA medium respectively, and cultivated at 30℃, 180 rpm for 72-96h. The compound fungal liquid was obtained by combining equal volumes of 1:1.

[0057] (2) Solidification of the functional strains of the compound microbial inoculant

[0058] Add 10 Kg of sawdust (or bagasse) solid material to the solid fermentation reactor, then add 5 L of complex bacterial inoculum and mix with the solid material, react at 30-40°C for 24 h, then add 3 L of LB liquid medium, react at 30-40°C for 24 h, and immobilize the complex bacterial inoculum.

[0059] Add 10 Kg of sawdust (or bagasse) solid material to the solid fermentation reactor, then add 5 L of complex bacterial inoculum and mix with the solid material, react at 30-40°C for 24 h, then add 3 L of LB liquid medium, react at 30-40°C for 24 h, and immobilize the complex bacterial inoculum.

[0060] Mix the complex bacterial inoculum and the complex fungal inoculum in proportion to obtain the complex microbial inoculum.

[0061] Example 3 Application of the complex microbial inoculum to pre-harvest preservation of litchi

[0062] (1) Litchi orchard test

[0063] In early May, when the litchi trees began to bear small fruits, the complex microbial inoculum (complex bacterial inoculum mixed with complex fungal inoculum at a weight ratio of 2:1) was applied to the litchi trees. The leaves on the surface of the litchi trees were pushed aside, and the complex microbial inoculum (15 Kg per tree) was evenly spread on the surface soil of the litchi trees, and then the pushed aside leaves were covered on the surface of the litchi trees to cover the complex microbial inoculum (test group). The litchi trees without the application of the complex microbial inoculum were used as the control. The daily management of the litchi trees in the test group and the control group in the litchi orchard was as usual. In early July, when the litchi fruits of the Guiwei variety were mature, the litchi fruits in the test group and the control group were picked.

[0064] (2) Detection of litchi preservation effect

[0065] The picked litchi fruits in the test group and the control group were not pretreated and were stored at room temperature indoors. The soluble sugar and titratable acid contents of the litchi pulp on the day of picking were detected. Every 2 days, the changes of the litchi fruits were observed, and the malondialdehyde content, browning index, disease index (disease index), and water loss rate of the litchi fruits were detected. The results are shown in Table 1. Figures 3-9 .

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

[0067] Titration of acid content: The pulp of litchi was smashed and centrifuged at 5000 rpm for 5 min. The supernatant was taken and 20.0 mL of the supernatant was transferred into a flask. 2 drops of 1% phenolphthalein indicator was added and the solution was titrated with the calibrated NaOH solution. The end point was reached when the solution initially turned pink and did not fade within 0.5 min. The amount of NaOH solution used was recorded. The titration was repeated three times. The titration was also performed with distilled water instead of the supernatant as a blank control.

[0068] The titratable acid content in the pulp of litchi was calculated according to the amount of NaOH solution consumed. The calculation formula was as follows:

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

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

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

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

[0073] V1 is the volume of NaOH solution consumed for titration of the extract, mL;

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

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

[0076] Browning index of the peel: The browning area of the peel was graded as follows: 0 grade: no browning; 1 grade: browning area less than 5%; 2 grade: browning area 5% to 25%; 3 grade: browning area 25% to 50%; and 4 grade: browning area more than 50%. The formula was: browning index = ∑(browning grade x number of fruits in the grade) x 100% / (total number of fruits x highest grade number).

[0077] Disease index (disease index) of the fruit: The disease area of the fruit was graded as follows: 0 grade: no disease; 1 grade: slight disease; 2 grade: disease area 5% to 25%; 3 grade: disease area 25% to 50%; and 4 grade: disease area more than 50%. The formula was: disease index = ∑(disease grade x number of fruits in the grade) x 100% / (total number of fruits x highest grade number).

[0078] Water loss rate: The weight of each group of litchi was measured and recorded on an electronic balance every 2 days. The formula was: weight loss rate (%) = (m0-m1) / m0 x 100%; wherein m0 is the initial weight (g) of litchi before treatment; and m1 is the weight (g) of litchi after storage at room temperature for a number of days.

[0079] ByFigure 3 As shown, compared with the control group (without applying the complex microbial inoculant), the test group (applying the complex microbial inoculant) had larger litchi fruits (20 fruits of 368 ± 5 g; 20 fruits of 290 ± 4 g, an average fruit weight increase of 26.9%), increased yield. The shell epidermis was harder, making it difficult for the outer pericarp to be damaged. The inner wall white film was thicker, making the natural oxidation rate of the inner pericarp slower. The flesh was firm and crystal. Figure 4 The soluble sugar and titratable acid contents in the litchi pulp on the day of picking were determined by Figure 4 As shown, the soluble sugar content in the litchi pulp of the test group (478 mg / g) was higher than that of the control group (450 mg / g), and the titratable acid content in the litchi pulp of the test group (0.166 g / 100 mL) was lower than that of the control group (0.173 g / 100 mL), indicating that the test group litchi had higher sweetness and lower astringency than the control group litchi, and the taste was better. Figure 3 and Figure 4 The results show that applying the complex microbial inoculant during the small fruit stage of litchi can improve the fruit size, pericarp hardness and thickness, and the taste of the pulp. From Figure 5 It can be seen that after the litchi of the test group and the control group was stored for 7 days, the malondialdehyde content in the litchi pulp of the test group and the control group was higher than that on the day of picking, but the malondialdehyde content in the litchi pulp of the test group (2.53 nmol / g) was lower than that of the control group (3.33 nmol / g), indicating that the test group litchi pulp cell membrane had lower degree of lipid peroxidation, lower damage and rot. From Figure 6 It can be seen that after the litchi of the test group and the control group was stored for 7 days at room temperature without any pretreatment, the browning index of the control group litchi reached 71.67%, while that of the test group was only 43.75%, which indicates that applying the complex microbial inoculant during the small fruit stage of litchi can delay the browning degree of the litchi pericarp after picking. From Figure 7 It can be seen that after being stored for 7 days at room temperature, more than half of the litchi fruits of the control group were diseased (disease index 57.03%), while only 27.91% of the litchi fruits of the test group were diseased. After being stored for 11 days, all the litchi fruits of the control group were diseased, and the disease index of the litchi fruits of the test group was 79.58%, which indicates that applying the complex microbial inoculant during the small fruit stage of litchi can reduce the disease degree of the litchi fruits after picking. From Figure 8 It can be seen that after being stored for 11 days at room temperature, the water loss rate of the litchi of the test group (14.19%) was significantly lower than that of the control group (20.93%), indicating that applying the complex microbial inoculant during the small fruit stage of litchi can reduce the water loss rate of the litchi fruits after picking.

[0080] From Figure 9And the above results can be known, during the growth of litchi fruit, the application of the compound microbial preservative bacteria agent developed in this project can effectively improve the hardness, thickness of the fruit skin and the taste of the pulp, improve the fruit quality, delay the browning degree of the fruit skin, and reduce the fruit disease rate and the water loss rate. Finally, the fruit after picking does not need to be treated, and can be placed for a longer time at room temperature in the room, and is not easy to rot and deteriorate.

Claims

1. The application of a compound microbial agent in pre-harvest preservation of litchi, characterized in that, During the growth period of lychee fruit, compound microbial agents are evenly spread on the surface soil of lychee trees. The compound microbial agent is composed of a compound bacterial agent and a compound fungal agent mixed at a weight ratio of 2:

1. The compound bacterial agent is composed of Serratia marcescens (…). Serratia marcescens X1, Pseudomycium-like Bacillus ( Bacillus pseudomycoides X8 and Bacillus spheroidae ( Lysinibacillus sphaericus The 38-6 bacterial solution was composed of Trichoderma koningii in a volume ratio of 1:1:

1. Trichoderma koningii Z15-3-2 and F. geraniol ( Coriolopsis sp. The bacterial suspension (9-3-19) was composed of bacteria in a volume ratio of 1:

1. The *Serratia marcescens* and *Bacillus pseudofungus* are dual-resistant bacteria that are highly resistant to copper, iron, and manganese and inhibit litchi pathogenic fungi; the *Bacillus spheroidans*, *Trichoderma koningii*, and *Gynostemma pentaphyllum* are lignin-decomposing bacteria that can produce laccase and / or lignin peroxidase.

2. The application according to claim 1, characterized in that, The preparation method of the compound microbial agent specifically includes the following steps: [The text abruptly shifts to a different topic] 600 Spherical lysine-containing Bacillus, Serratia marcescens, and Bacillus pseudofungiformis bacterial suspensions (0.5 g / L) were inoculated into LB medium and cultured separately. Then, equal volumes of the bacterial suspensions were combined to obtain a composite bacterial suspension. Trichoderma koningii and Pterygomycetes mycelial blocks (1 cm in diameter) were crushed and inoculated into PDA medium and cultured separately. Then, equal volumes of the bacterial suspensions were combined to obtain a composite fungal suspension. The composite bacterial suspension and the composite fungal suspension were solidified separately to obtain a composite bacterial inoculum and a composite fungal inoculum. They were then mixed at a weight ratio of 2:1 to obtain a composite microbial inoculum.

3. The application according to claim 2, characterized in that, The LB medium is formulated as follows: 10 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L sodium chloride, 15.0 g / L agar, with the remainder being water; the PDA medium is formulated as follows: 300 g / L potato extract powder, 20.0 g / L glucose, 15.0 g / L agar, with the remainder being water.

4. The application according to claim 2, characterized in that, The conditions for inoculation in LB medium are: incubation at 37℃ and 180 rpm for 24-48 h; the conditions for inoculation in PDA medium are: incubation at 30℃ and 180 rpm for 72-96 h.

5. The application according to claim 2, characterized in that, The solidification steps are as follows: in a solid-state fermentation reactor, the compound bacterial liquid is mixed with solid materials and reacted for 24 h, then LB liquid culture medium is added and reacted for 24 h to obtain the compound bacterial agent; in a solid-state fermentation reactor, the compound fungal liquid is mixed with solid materials and reacted for 24 h, then PDA liquid culture medium is added and reacted for 24 h to obtain the compound fungal agent.

6. The application according to claim 5, characterized in that, The mass-to-volume ratio of the solid material to the composite bacterial or fungal inoculum is 2 kg / L; the solid material is sawdust, bagasse, or a mixture thereof; and the reaction temperature is 30-40°C.

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