Complex microbial inoculant for preventing and treating garlic diseases as well as preparation method and application of complex microbial inoculant

Abstract

The invention relates to the technical field of microbial pesticides, in particular to a complex microbial inoculant for preventing and treating garlic diseases and a preparation method and application thereof.The complex microbial inoculant is composed of seven strains including bacteria and fungi T4, JR14, BR55, A25, BR33, BR43 and JR22, experiments prove that the complex microbial inoculant has excellent garlic pathogenic bacterium hypha inhibition and sporulation inhibition effects, and the complex microbial inoculant can be used for preventing and treating garlic diseases. All bacterial and fungal candidate strains participate in inhibition of fungal pathogens through synergistic interaction. The potting effect verifies that the growth of garlic plants is severely influenced under the stress of T9 pathogenic bacteria, but the complex microbial inoculant disclosed by the invention not only can keep the normal growth of the garlic plants and has no obvious difference with the plant height of the plants growing in normal soil, but also has a certain effect of promoting the growth of the garlic plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial pesticides, and particularly relates to a compound microbial agent for preventing and treating garlic diseases, a preparation method and application thereof. BACKGROUND

[0002] Under the background of highly intensive agriculture, the perennial single crop cultivation and the use of a large amount of chemicals pose a serious threat to the soil health and plant growth of the rice-garlic rotation system, especially as the planting years increase, the complexity of the rhizosphere microbial network decreases, the number of beneficial populations such as bacteria and actinomycetes decreases, and the number of harmful pathogenic fungi increases. The colonization of pathogenic fungi and beneficial microorganisms in the rhizosphere competes with each other, leading to an imbalance of the garlic microecological structure, and the continuous cropping obstacle problem is increasingly prominent.

[0003] The formation mechanism of continuous cropping obstacle is very complex, and the imbalance of soil microbial community is one of the most key factors. The rhizosphere microbiome, as the "second genome" of plants, plays a crucial role in maintaining plant growth and health. Although a variety of rhizosphere microorganisms have been found to play a good role in plants and soil, the role of a single strain is often limited. For a long time, microorganisms have been used as soil inoculants, but their field performance often fluctuates due to climate, soil type and competition of local microbiome. Especially when introducing foreign single strains, their colonization success rate may be greatly reduced due to the inability to adapt to the complex rhizosphere microenvironment.

[0004] As an emerging technology, SynCom (Synthetic Microbiome) is constructed by combining microorganisms with clear functions and synergistic interactions in a specific ratio, and a stable and efficient microbial community is constructed. Compared with single strain inoculation, SynCom can more effectively colonize the rhizosphere of plants, and play a more sustainable and efficient role in promoting plant growth, improving yield and disease resistance. SUMMARY

[0005] The present application aims to overcome the deficiencies of the prior art, and provides a compound microbial agent for preventing and treating garlic diseases, a preparation method and application thereof.

[0006] The object of the application is achieved by the following technical scheme: a compound microbial agent for preventing and treating garlic diseases, wherein the effective components of the compound microbial agent include Ensifer sesbaniae BR33, Sphingomonas caeni BR55, Pseudomonas alloputida A25, Bacillus zanthoxyli JR22, Flavobacterium humicola BR43, Bacillus subtilis JR14 and Trichoderma hamatum T4:

[0007] The Ensifer sesbaniae BR33 is preserved in the China General Microbiological Culture Collection Center, and the preservation date is July 30, 2025, and the preservation number is CGMCC No. 35451.

[0008] The Sphingomonas caeni BR55 is preserved in the China General Microbiological Culture Collection Center, and the preservation date is July 30, 2025, and the preservation number is CGMCC No. 35455.

[0009] The Pseudomonas alloputida A25 is preserved in the China General Microbiological Culture Collection Center, and the preservation date is July 30, 2025, and the preservation number is CGMCC No. 35452.

[0010] The Bacillus zanthoxyli JR22 is preserved in the China General Microbiological Culture Collection Center, and the preservation date is July 30, 2025, and the preservation number is CGMCC No. 35453.

[0011] The Flavobacterium humicola BR43 is preserved in the China General Microbiological Culture Collection Center, and the preservation date is July 30, 2025, and the preservation number is CGMCC No. 35456.

[0012] The Bacillus subtilis JR14 is preserved in the China General Microbiological Culture Collection Center, and the preservation date is July 30, 2025, and the preservation number is CGMCC No. 35454.

[0013] The Trichoderma hamatum T4 is preserved in the China General Microbiological Culture Collection Center, and the preservation date is July 30, 2025, and the preservation number is CGMCC No. 42151.

[0014] Further, the complex microbial agent is a fermentation liquor of the bacteria and fungi T4, JR14, BR55, A25, BR33, BR43 and JR22.

[0015] Further, the complex microbial agent is a fermentation liquor of the bacteria and fungi T4, JR14, BR55, A25, BR33, BR43 and JR22.

[0016] Further, the garlic diseases include root rot and soft rot.

[0017] Further, the preparation method comprises the following steps:

[0018] First, inoculate the Flavobacterium BR43 and Sphingomonas BR55 into the culture medium for fermentation, and then inoculate the remaining strains into the culture medium for fermentation, so that all the strains enter the logarithmic growth phase at the same time, until the fermentation is completed.

[0019] The application further provides the use of the complex microbial agent for preventing and treating garlic diseases for inhibiting garlic pathogenic bacteria.

[0020] Further, the inhibition of garlic pathogenic bacteria includes mycelium inhibition and spore inhibition of garlic pathogenic bacteria.

[0021] Further, the garlic pathogenic bacteria include Fusarium fungi.

[0022] The application has the following beneficial effects: the complex microbial agent for preventing and treating garlic diseases provided by the application is composed of seven strains of bacteria and fungi T4, JR14, BR55, A25, BR33, BR43 and JR22, and experiments prove that the complex microbial agent has excellent mycelium inhibition and spore inhibition effects on garlic pathogenic bacteria, and all the candidate strains of bacteria and fungi participate in the inhibition of fungal pathogens through synergistic interaction. Through potting effect verification, under the stress of T9 pathogenic bacteria, the growth of garlic plants is seriously affected, but the complex microbial agent can not only maintain the normal growth of garlic plants, but also has no significant difference with the plant height grown in normal soil, and has a certain effect of promoting the growth of garlic plants. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 To construct the phylogenetic trees of cultivable bacteria (a) and fungi (b) based on 16S rRNA / ITS gene sequences;

[0024] Figure 2 for the interaction effect between candidate strains;

[0025] Figure 3 for the growth curve of candidate strains;

[0026] Figure 4 for the mycelium inhibition effect of SynCom on T9;

[0027] Figure 5 for the mycelium (a) and sporulation (b) inhibition rate % of SynCom on T9;

[0028] Figure 6 for the growth status of garlic treated with different proportions of T9 spore suspension;

[0029] Figure 7 for the growth index and disease index of each treatment garlic; (a) aboveground plant height, (b) aboveground fresh weight, (c) underground fresh weight, (d) disease index;

[0030] Figure 8 for the growth status of 50-hole plug garlic plants at 0-21d;

[0031] Figure 9 for the aboveground plant height of 50-hole plug garlic at each period; (a) 0d, (b) 7d, (c) 14d, (d) 21d;

[0032] Figure 10 for the test results of 28d 50-hole plug garlic plants;

[0033] Figure 11 for the disease index of 28d 50-hole plug garlic plants;

[0034] Figure 12 for the growth index of 28d 50-hole plug garlic; (a) aboveground plant height, (b) aboveground fresh weight, (c) underground fresh weight, (d) disease index. DETAILED DESCRIPTION

[0035] The technical solutions of the present application will be described in further detail below in combination with the accompanying drawings, but the protection scope of the present application is not limited to the following description.

[0036] Example 1 Strain source and screening

[0037] The bacterial strains involved in the present application are mainly obtained by screening from 1 year and 10 years of fresh garlic rhizosphere soil samples stored in a 4℃ refrigerator. The fungal strains are mainly obtained by screening from 10 years of fresh garlic rhizosphere soil and plant tissues at the disease-healthy boundary of diseased garlic stored in a 4℃ refrigerator. (from a rice-garlic rotation base in Wenjiang, Chengdu, Sichuan Province)

[0038] (1) Isolation, purification, identification and preservation of bacterial strains

[0039] Weigh 10 g of fresh soil sample into 90 mL of sterile water, and shake at 4°C and 150 rpm / min for 30 min to obtain a soil suspension. Take 1 mL of the soil suspension and dilute it with sterile water in a gradient manner to 10-6. Take 100 μL of each gradient dilution and evenly spread it on each culture plate, with three repeats for each gradient. Place the plates in a 30°C constant temperature incubator and incubate them upside down for 3-5 days. Pick single colonies with different morphological characteristics and re-streak them for culture until pure strains are obtained.

[0040] Pick single colonies from the purified plates for PCR identification. The universal primers 27F (5'-AGAGTTTGATCCTGGCTCACAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACT-3') are used for bacterial amplification. The PCR reaction system and program are set as shown in Tables 1 and 2. The PCR stock solution is sent to Beijing Qikong Biotechnology Co., Ltd. for 16S rRNA gene sequencing. The obtained sequence is uploaded to the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) for homologous sequence comparison to obtain species information. The purified single colonies are inoculated in the corresponding liquid medium and cultured to the logarithmic phase. They are mixed with 60% glycerol solution at a volume ratio of 1:2, aliquoted in cryotubes, and stored in a -80°C ultra-low temperature freezer for preservation.

[0041] Table 1 Bacterial PCR reaction system

[0042]

[0043] Table 2 Bacterial PCR reaction program

[0044]

[0045] (2) Isolation, purification, identification and preservation of fungal strains

[0046] Take the diseased garlic leaf at the healthy and diseased interface, cut it into small pieces of 5 mm x 5 mm with a sterile blade, and reserve it for use. Soak the diseased tissue in 75% alcohol and 5% NaClO solution for 30 s, and then wash it with sterile water for 3 times. Transfer the diseased tissue to a sterile filter paper and dry it. Place the diseased tissue on PDA medium containing streptomycin (30 mg / mL), with 4 pieces of diseased tissue per dish. Incubate them upside down for 7 days, pick the mycelium of the colonies that grow, and transfer them to PDA medium for purification culture. The soil dilution separation is performed according to the bacterial dilution separation described above.

[0047] The mycelium was picked from the purification plate for PCR identification, and primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used. The PCR reaction system and procedure were set as shown in Table 3 and Table 4. The PCR original solution was sent to Beijing Qikexin Biotechnology Co., Ltd. for ITS gene sequencing, and the obtained sequence was uploaded to the NCBI website for homologous sequence comparison to obtain the species information. The color and morphology of the fungal colonies were observed and recorded, and the mycelium was picked to make a slide, and the morphology of the mycelium and spores was observed under a microscope. The mycelium of the purified fungal colonies was picked and inoculated in a slant test tube, which was cultured in a 30°C incubator for 7 days and then stored in a 4°C refrigerator for standby.

[0048] Table 3 Fungal PCR reaction system

[0049]

[0050] Table 4 Fungal PCR reaction procedure

[0051]

[0052] (3) Screening results

[0053] Using R2A and LB medium (R2A medium: soluble starch 0.5 g / L, casein hydrolysate 0.5 g / L, potassium phosphate dibasic 0.3 g / L, yeast extract powder 0.5 g / L, peptone 0.5 g / L, glucose 0.5 g / L, anhydrous magnesium sulfate 0.024 g / L, sodium pyruvate 0.3 g / L, agar powder 20 g / L. LB medium: yeast extract 5.0 g / L, agar powder 20 g / L, sodium chloride 10.0 g / L, tryptone 10.0 g / L.), 150 strains of bacteria were isolated and identified from the rhizosphere soil of 1-year and 10-year garlic, covering 6 phyla and 57 genera Figure 1 a) Among them, the strains isolated in larger quantities were Bacillus, Pseudomonas, Pseudoxanthomona, Lysobacter, Flavobacterium, Stenotrophomonas, Chryseobacterium and Microbacterium. Bacillus was isolated in the largest quantity in the rhizosphere soil, and a total of 13 different species of strains were isolated.

[0054] Ten candidate strains with good functions were screened, including Ensifer sesbaniae BR33, Sphingomonas caeni BR55, Pseudomonas alloputida A25, Bacillus zanthoxyli JR22, Rhodanobacterspathiphylli JR6, Flavobacterium humicola BR43, Lysobacter panacisoli BR29, Bacillus subtilis JR14, Bacillus velezensis JR1A and Trichoderma hamatum T4. Among them, the IAA concentration of Ensifer sesbaniae BR33 was the highest, reaching 51.92 mg / mL, which was significantly different from other strains (P<0.05), followed by Bacillus zanthoxyli JR22, with a concentration of 42.43 mg / mL. Bacillus zanthoxyli JR22 and Pseudomonas alloputida A25 showed strong phosphorus solubilizing ability (D / d>2) and IAA producing ability (37.97-42.43 mg / mL). Ensifer sesbaniae BR33, Pseudomonas alloputida A25 and Bacillus zanthoxyli JR22 had multiple growth-promoting functions such as phosphorus solubilization, siderophore production and IAA production. The mycelial inhibition rates of JR14 and JR1A on Fusarium T9 were 77.44% and 84.41%, respectively, and on Chaetomium C1 were 83.41% and 90.87%, respectively. The highest inhibition rate was on Stemphylium C3, almost reaching 100%, which was significantly higher (P<0.05) than other isolated strains. The mycelial inhibition rates of Trichoderma T4 on the three pathogenic fungi were 48.24%, 66.44% and 61.70%, respectively. Sphingomonas caeni BR55, Flavobacterium humicola BR43, Rhodanobacterspathiphylli JR6 and Lysobacter panacisoli BR29 were representative bacterial species.

[0055] S. amylotrophicum BR33 was preserved in China General Microbiological Culture Collection Center on July 30, 2025, and the preservation number was CGMCC No. 35451;

[0056] S. limax BR55 was preserved in China General Microbiological Culture Collection Center on July 30, 2025, and the preservation number was CGMCC No. 35455;

[0057] P. alternanivorans A25 was preserved in China General Microbiological Culture Collection Center on July 30, 2025, and the preservation number was CGMCC No. 35452;

[0058] B. faggenese JR22 was preserved in China General Microbiological Culture Collection Center on July 30, 2025, and the preservation number was CGMCC No. 35453;

[0059] F. terricola BR43 was preserved in China General Microbiological Culture Collection Center on July 30, 2025, and the preservation number was CGMCC No. 35456;

[0060] B. subtilis JR14 was preserved in China General Microbiological Culture Collection Center on July 30, 2025, and the preservation number was CGMCC No. 35454;

[0061] T. hook T4 was preserved in China General Microbiological Culture Collection Center on July 30, 2025, and the preservation number was CGMCC No. 42151.

[0062] A total of 13 fungal strains were isolated and identified, covering 2 phyla and 9 genera Figure 1b). Among them, Fusarium and Stemphylium were isolated in more strains. Fusarium was isolated in most in rhizosphere soil; Stemphylium, Chaetomium and Pleospora were isolated in most in diseased tissues. The isolated fungal strains were observed by morphological observation with medium plate and microscope. Four pathogenic fungi of common garlic diseases, Fusarium foetens T9, Chaetomium globosum C1, Stemphylium eturmiunum C3, Pleospora rosae-caninae C2 and one common beneficial fungus Trichoderma hamatum T4 were isolated. Fusarium, Stemphylium, Chaetomium and Pleospora are the reported garlic pathogenic fungi, which can cause garlic root rot and leaf blight in large-scale field infection. T9 Fusarium is the main pathogenic fungus of root diseases in the early growth stage of garlic, and C3 Stemphylium is the main pathogenic fungus of leaf diseases in the late growth stage of garlic.

[0063] Experimental Example 2 Interaction and growth curve of candidate strains

[0064] (1) Experimental method

[0065] Candidate strain interaction test method: the candidate bacterial seed liquid was diluted to 10 -4 μL of diluted bacterial liquid was uniformly coated on the plate as the bottom bacteria, and then 3 μL of diluted bacterial liquid was spotted on the corresponding position of the plate, and each group was repeated three times. After incubation at 30°C for 48-72h, whether the interaction between the candidate bacterial strains was determined by observing whether the inhibition zone was formed between the test strains and the bottom bacteria on the plate. Take 8mm fungus cake in the center of the plate, and spot the diluted bacterial liquid at a distance of 2cm, and observe whether there is an inhibition zone between the test fungus and the spotted bacterial liquid on the plate to determine the interaction between the candidate fungus and the candidate bacterial strain.

[0066] Candidate strain growth curve determination: the candidate bacterial strain seed liquid was inoculated into 100mL LB liquid medium at 1% inoculation amount, and incubated at 30°C. The OD 600 of the culture liquid at each time node within 36h was measured every 3h using ultraviolet spectrophotometer.

[0067] (2) Interaction results between candidate strains

[0068] As shown in Table 5 and Figure 2By observing the presence or absence of a clear zone around the coated colonies, it can be seen that most candidate strains can coexist with each other; however, several strains exhibited negative interactions with other strains. JR1A showed a general negative interaction, inhibiting the growth of other candidate strains to varying degrees. Secondly, BR14 also showed slight inhibition of JR6, BR43, BR29, JR1A, and T4, but the inhibitory effect was far less than that of JR1A. JR6 and BR29 also showed negative interactions with JR14 and JR1A. Therefore, strains JR6 and BR29 were no longer considered as candidate strains for constructing SynCom. (Note: Although JR1A showed a general negative interaction, this strain exhibited extremely strong inhibitory activity against common garlic pathogens in individual experiments, far exceeding the inhibitory effects of all existing microorganisms, with an inhibition rate as high as 84%–100%. The relevant patent is 202511137811.0, "A strain of Bacillus belye JR1A and its application in inhibiting garlic pathogenic fungi").

[0069] Table 5 Interactions among candidate strains

[0070] Note: "++" indicates strong antagonism, "++" indicates moderate antagonism, "+" indicates antagonism, and "-" indicates no antagonism.

[0071] (3) Growth curve results of candidate strains

[0072] To more accurately assess the growth dynamics and rate changes of the strains in the culture medium, the growth curves of seven candidate strains—BR33, BR55, A25, JR22, BR43, JR14, and T4—were measured. Figure 3 A25 entered the logarithmic growth phase immediately after inoculation, exhibiting a rapid growth rate. BR33, JR14, and JR22 initially had lower growth rates than A25, but entered the logarithmic growth phase after 3 hours and reached a plateau after 15 hours. The final OD of the bacterial culture... 600 The values ​​were not significantly different. BR43 and BR55, however, had slower growth rates, entering the logarithmic growth phase from 6 hours onwards and reaching a plateau after 21 hours. 600 The value is around 1.5, which is much lower than that of other strains. Therefore, when preparing the bacterial culture to construct SynCom, BR43 and BR55 need to be inoculated in advance due to their slower growth rate so that they can enter the logarithmic growth phase at the same time as the other strains for subsequent SynCom preparation.

[0073] Example 3: Construction of Synthetic Microbiome and Preliminary Inhibition Effect in Indoor Environment

[0074] (1) Experimental methods

[0075] Preparation method of synthetic microbiome: the candidate bacterial seed liquid was inoculated into a 500 mL conical flask containing 300 mL TSB medium at an inoculation amount of 2%, and cultured at 30°C, 180 rpm / min until OD 600 = 1.00 to obtain a bacterial fermentation broth, which was centrifuged at 4°C, 4000 rpm / min for 15 min, and the upper fermentation supernatant was discarded, and the bacterial cells were retained and resuspended with sterile water to adjust the concentration of the bacterial liquid to OD 600 = 1.00. Five to six activated fungal strain bacterial cakes on the PDA plate were taken using a puncher, inoculated into a 500 mL conical flask containing 300 mL PDB medium, and cultured at 30°C, 180 rpm / min for 7 d. After filtration through 4 layers of gauze, the concentration of the spore suspension was adjusted to 1x10 6 cfu / mL under a microscope using ddH2O and a hemocytometer. The above bacterial cell suspension and spore suspension were mixed in equal proportions to complete the preparation of SynCom.

[0076] Plate confrontation of synthetic microbiome and Fusarium T9: 100 μL of each of the prepared different SynComs were evenly inoculated on sterile filter paper placed on one side of the plate, and an 8 mm bacterial cake containing T9 fungal mycelium was placed 2 cm away from the filter paper. After co-culturing at 30°C for 7 d, the diameter of the fungal mycelium was measured, and filter paper inoculated with ddH2O was used as a CK control to calculate the mycelium inhibition rate. Mycelium inhibition rate (%) = (T9 diameter in CK - T9 diameter after adding SynCom) / T9 diameter in CK x 100%.

[0077] Liquid co-culture of synthetic microbiome and Fusarium T9: 1 mL of T9 spore suspension and 1 mL of SnyCom were added to a 250 mL conical flask containing 100 mL of liquid antagonistic medium in equal volumes, and after co-culturing at 30°C, 180 rpm / min for 7 d, the number of spores was counted under a microscope using a hemocytometer, and an equal volume of ddH2O was inoculated as a CK control to calculate the spore production inhibition rate. Spore production inhibition rate (%) = (T9 spore suspension concentration in CK - T9 spore suspension concentration after adding SynCom) / T9 spore suspension concentration in CK x 100%.

[0078] (2) Preliminary indoor inhibition results of SynCom on Fusarium T9

[0079] In order to maximize the beneficial effects of the strains, the present application is based on the non-antagonistic relationship between candidate strains, and five SynComs are constructed. The SC1 group is composed of Bacillus velezensis JR1A which has good antagonistic effect on multiple garlic pathogenic fungi, but has inhibitory effect on other strains. The SC2 group is composed of Trichoderma hamatum T4 which has good antagonistic effect on multiple garlic pathogenic fungi and has multiple beneficial effects on plants. The SC3 group is composed of three strains of JR14, A25 and BR55 which are single key microbial groups, antagonistic and growth-promoting functional species. The SC4 group is composed of six strains of JR14, A25, BR55, BR33, BR43 and JR22. The cross-border microbial community of bacteria and fungi has been found to have excellent effect, so the SC5 group is composed of seven strains of T4, JR14, BR55, A25, BR33, BR43 and JR22.

[0080] Under indoor test conditions, the mycelial inhibition and sporulation inhibition ability of SynComs composed of different strains on Fusarium T9 were tested. Compared with CK, different SynComs all produced universal inhibitory effect on T9 mycelium Figure 4 ). Among them, SC5 showed the highest mycelial inhibition rate, inhibiting 51.07% of mycelial growth, which was significantly higher (P<0.05) than other SynComs, and the mycelial inhibition rates of SC1, SC2, SC3 and SC4 groups were 45.35%, 44.32%, 35.77% and 43.58% respectively Figure 5 a) Different SynComs all showed excellent sporulation inhibition effect, and the sporulation inhibition rate was between 88.12-98.80%. Among them, the sporulation inhibition rate of SC2 group was the lowest, 88.12%, which was significantly lower (P<0.05) than other groups Figure 5 b) Based on the above results, SC5 has the relatively best preliminary inhibitory effect on T9 in the laboratory, and this combination is composed of seven rhizosphere microorganisms of all candidate bacterial and fungal microorganisms, which indicates that all bacterial and fungal candidate strains participate in the inhibition of fungal pathogens through synergistic interaction.

[0081] Example 4 Verification of the greenhouse potting effect of synthetic microbial community

[0082] (1) Experimental method

[0083] Fusarium T9 pathogenicity determination method to garlic: garlic seeds were soaked in 5% NaClO and 75% ethanol respectively for 10 min, and finally washed with ddH2O for 3 times to complete the disinfection treatment. The disinfected seeds were placed in the seedling tray with wet filter paper to accelerate germination. After germination, they were planted in 50-hole seedling trays. The experiment was carried out in pots with a radius of 12 cm and a height of 13 cm, each pot containing 300 g of soil. Three treatments were set up, each with 6 replicates. Treat 1: CK control, add 100 mL H2O per pot; Treat 2: 10 mL T9 spore suspension + 90 mL H2O; Treat 3: 50 mL T9 spore suspension + 50 mL H2O. Select garlic seedlings with uniform size and growth potential and transplant them into the soil of each treatment. After 21 days of cultivation in the greenhouse, measure the aboveground height, fresh weight, underground root length, root weight and disease index.

[0084] Greenhouse pot experiment of SC5: The pot experiment was conducted from December 2024 to January 2025 in a greenhouse in Chengdu, Sichuan Province (26°C). The pre-treatment and cultivation method of garlic seeds was as follows: garlic seeds were soaked in 5% NaClO and 75% ethanol respectively for 10 min, and finally washed with ddH2O for 3 times to complete the disinfection treatment. The disinfected seeds were placed in the seedling tray with wet filter paper to accelerate germination. After germination, they were planted in 50-hole seedling trays. The experiment was carried out in 50-hole seedling trays with a size of 54 cm x 28 cm. Three treatments were set up, each with 30 replicates. Treat 1 was the control group CK: 10 mL H2O was applied per hole; Treat 2 was the T9 application group: 1 mL T9 spore suspension and 9 mL H2O were applied per hole; Treat 3 was the T9 and SC5 application group: 1 mL T9 spore suspension and 9 mL SC5 were applied per hole. After 28 days of experiment, the growth indicators and disease index of garlic were measured. There was no significant difference in the growth potential of the original garlic plants among the three treatments, except for the different application methods. Other management was consistent.

[0085] Measurement of garlic growth indicators and disease index: Disease index was scored according to the 0-4 rating: 0, no disease symptoms; 1, less than 1 / 4 of the plants wilted, but growing normally; 2, 1 / 4-1 / 2 of the plants wilted, root disease; 3, more than 1 / 2 of the plants wilted, root necrosis; 4, whole plant wilted, plant death or very small and wilted. Garlic growth indicators were evaluated according to the five-level disease grading standard table: 0, no leaf necrosis; 1, 1≤ leaf necrosis pieces <3; 2, 3≤ leaf necrosis pieces <5; 3, 5≤ leaf necrosis pieces <7; 4, leaf necrosis pieces ≥7. Disease index calculation formula: Disease index = ∑(number of each level of disease x value of that level) / (total number of plants surveyed x highest level value) x 100.

[0086] (2) Fusarium T9 pathogenicity to garlic

[0087] Through the indoor pathogen inhibition experiment, it is comprehensively concluded that SC5 has good inhibition effect on Fusarium T9. Next, SC5 will be further applied to explore its influence on garlic plants. The disease of garlic mainly occurs in the root at the seedling stage, and at this time Fusarium is the main pathogenic fungus in the rhizosphere soil. Therefore, in this pot experiment, Fusarium foetens T9 is used as the test pathogenic fungus. Fusarium fungus invades plants, first attracts attention by yellowing of old leaves, and gradually develops to tender leaves and finally leads to plant death.

[0088] The pathogenicity of T9 to garlic is studied to explore the most suitable pathogenic fungus addition ratio. After irrigating garlic plants with different volumes of T9 spore suspension, the growth status of garlic after 21 days is as follows Figure 6 It can be seen that there are obvious differences between different T9 addition ratios of garlic plants, and the higher the addition ratio, the more serious the disease of garlic. Treat 2 (10 mL T9) plant leaf tip appears yellow, atrophy, and even leaf lodging, indicating that the plant is infected with disease; Treat 3 (50 mL T9) plant leaf completely atrophies and dies, root rots, and the whole plant shows wilting and death, and the disease is serious. The physiological indexes and disease indexes of each treatment of garlic plants are measured, and Treat 3 shows the least aboveground plant height and underground root length, the lightest aboveground fresh weight and underground root weight, and the most serious disease; while Treat 2 is affected in each index, but the damage is not as serious as Treat 3, at this time the plant is infected with disease and not immediately wilted and died Figure 7 Therefore, the present application selects 10 mL T9 spore suspension addition ratio as the pathogenic fungus stress concentration in the subsequent pot experiment.

[0089] (3) Effect of applying SC5 on 50-hole plug garlic plants

[0090] The growth status of garlic plants every week after applying SC5 to 50-hole plug is as follows Figure 8 It can be seen that the plants in each treatment do not show disease characteristics in the initial growth stage, and until the 21st day of garlic growth, the leaves of Treat 2 (only adding T9 spore suspension) begin to yellow and atrophy, showing the characteristics of disease infection. Treat 1 (CK) and Treat 3 (applying T9 spore suspension and SC5 at the same time) do not show significant differences in appearance at each period.

[0091] The height of the garlic plants in each growth period (0, 7, 14, 21d) was measured. Figure 9 It can be observed that there is no difference in the height of the garlic plants in each treatment in the initial state 0d. After 7d, the height of the garlic plants in each treatment starts to change, and there is no significant difference between Treat 1 and Treat 3, but compared with Treat 2, Treat 1 and Treat 3 are significantly (P<0.05) increased by 10.05% and 10.75%, respectively. In the following 14d and 21d growth periods, the height of Treat 1 and Treat 3 continues to increase, and the gap with Treat 2 gradually widens, and compared with Treat 2, Treat 1 and Treat 3 are significantly (P<0.05) increased by 24.25%, 32.50% and 30.97%, 41.82%, respectively. The above results show that the growth of the garlic plants is affected under the stress of T9 pathogenic bacteria, and the application of SC5 can maintain the normal growth of the garlic plants, and there is no significant difference in the height of the plants grown in the normal soil.

[0092] After 28d of cultivation, the test results of the garlic plants in each treatment are as follows Figure 10 The plants in Treat 2 group show the phenomena of yellowing and wilting of leaves, plant lodging, growth inhibition of roots and severe infection of soft rot, while the plants in Treat 3 group show dark green leaves, straight plants and developed root system, and still maintain relatively healthy and stable growth compared with the plants in Treat 1 group. In order to evaluate the occurrence degree of the diseases during the cultivation, the disease index is calculated from 21d when the diseases appear, and it is found that the disease index of Treat 2 group is significantly higher (P<0.05) than that of Treat 3 and Treat 1 Figure 11 ) groups. In addition, the other growth indexes of the garlic plants in each treatment also show differences. Treat 3 shows the largest height of the aboveground part, root length of the underground part, fresh weight of the aboveground part and fresh weight of the underground part, and compared with Treat 1, Treat 3 is significantly (P<0.05) increased by 12.14%, 18.78%, 38.02% and 49.33%, respectively, and compared with Treat 2, Treat 3 is significantly (P<0.05) increased by 45.37%, 38.34%, 231.18% and 138.20% Figure 12 ) respectively. This shows that the application of SC5 not only can maintain the normal growth of the garlic plants under the stress of T9 pathogenic bacteria, but also can promote the growth of the garlic plants to a certain extent.

[0093] The above only describes the preferred embodiments of the present application, and it should be understood that the present application is not limited to the forms disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concepts described herein by the above teachings or related technical or knowledge. Any modification and change made by the person skilled in the art without departing from the spirit and scope of the present application shall be within the protection scope of the claims of the present application.

Claims

1. A compound microbial agent for controlling garlic diseases, characterized in that, The effective components of the complex microbial agent include Ensifer sesbaniae BR33, Sphingomonas caeni BR55, Pseudomonas alloputida A25, Bacillus zanthoxyli JR22, Flavobacterium humicola BR43, Bacillus subtilis JR14, and Trichoderma hamatum T4. The Ensifer sesbaniae BR33 is preserved in the China General Microbiological Culture Collection Center on July 30, 2025, and has a preservation number of CGMCC No. 35451. The Sphingomonas caeni BR55 is preserved in the China General Microbiological Culture Collection Center on July 30, 2025, and has a preservation number of CGMCC No. 35455. The Pseudomonas alloputida A25 is preserved in the China General Microbiological Culture Collection Center on July 30, 2025, and has a preservation number of CGMCC No. 35452. The Bacillus zanthoxyli JR22 is preserved in the China General Microbiological Culture Collection Center on July 30, 2025, and has a preservation number of CGMCC No. 35453. The Flavobacterium humicola BR43 is preserved in the China General Microbiological Culture Collection Center on July 30, 2025, and has a preservation number of CGMCC No. 35456. The Bacillus subtilis JR14 is preserved in the China General Microbiological Culture Collection Center on July 30, 2025, and has a preservation number of CGMCC No. 35454. The Trichoderma hamatum T4 is preserved in the China General Microbiological Culture Collection Center on July 30, 2025, and has a preservation number of CGMCC No. 42151.

2. The compound microbial agent for preventing and treating garlic diseases according to claim 1, characterized in that, The complex microbial agent is a fermentation liquor of Ensifer sesbaniae BR33, Sphingomonas caeni BR55, Pseudomonas alloputida A25, Bacillus zanthoxyli JR22, Flavobacterium humicola BR43, Bacillus subtilis JR14, and Trichoderma hamatum T4. 3.The compound microbial agent for preventing and treating garlic diseases according to claim 1, characterized in that, The quantity ratio of the number of bacteria of Ensifer sesbaniae BR33, Sphingomonas caeni BR55, Pseudomonas alloputida A25, Bacillus zanthoxyli JR22, Flavobacterium humicola BR43, Bacillus subtilis JR14, and Trichoderma hamatum T4 in the complex microbial agent is 0.5-2:0.5-2:0.5-2:0.5-2:0.5-2:0.5-2:0.5-2.

4. The compound microbial agent for preventing and treating garlic diseases according to claim 1, characterized in that, The types of garlic diseases include root rot and soft rot.

5. The method for preparing the complex microbial agent for preventing and treating garlic diseases according to any one of claims 1 to 3, characterized in that, The preparation method comprises the following steps: First inoculate Flavobacterium indoltheticum BR43 and Sphingomonas sp. BR55 into the culture medium for fermentation, and then inoculate the rest of the strains into the culture medium for fermentation, so that all the strains enter the logarithmic growth phase at the same time until the fermentation is completed.

6. The use of the complex microbial agent for preventing and treating garlic diseases according to any one of claims 1-3 for inhibiting garlic pathogenic bacteria.

7. Use according to claim 6, characterized in that, The inhibition of the garlic pathogenic bacteria includes inhibition of mycelium and inhibition of spore production of the garlic pathogenic bacteria.

8. Use according to claim 6, characterized in that, The garlic pathogenic bacteria include Fusarium fungi.

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