Microbial agent for preventing and treating typical diseases of tomato and preparation method and application thereof

Abstract

The application discloses a kind of microbial inoculants for preventing and treating typical diseases of tomato, and belongs to the technical field of microorganism.The microbial inoculants include the following components: Trichoderma virens, Trichoderma hamatum, Bacillus licheniformis and Azotobacter chroococcum, and the volume ratio of the four is 1:1:1:1.The fungi and bacteria with disease resistance and growth promotion are reasonably combined and used, which effectively improves various typical diseases of tomato.By combining and using the fungi and bacteria in the microbial inoculants, the practical problems of unstable prevention and weak persistence of single biocontrol strain are solved, the biocontrol effect is greatly improved, and the typical diseases of tomato, such as root rot, gray mold, wilt and early blight, have significant prevention and treatment effect.In addition, the microbial inoculants are compatible with soil ecology, which is beneficial to fully exert the advantages of the strain, effectively ensures and improves the yield and quality of tomato.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a microbial agent for controlling typical diseases of tomatoes and its preparation method. Background Technology

[0002] Tomato (Lycopersicon esculentum Mill.), belonging to the genus Lycopersicon in the family Solanaceae. Native to South America, tomatoes contain various vitamins, minerals, and fiber beneficial to the human body. They have anti-aging and cancer-reducing effects, and show great promise in the fields of medicine, food, and health care.

[0003] Tomatoes are now widely cultivated across various regions, with the planting area expanding year by year. During cultivation, numerous and severe diseases arise. These diseases reduce tomato yield and quality, causing significant economic losses for farmers. Common tomato diseases include gray mold, early blight, leaf mold, viral diseases, bacterial wilt, late blight, nematode disease, and bacterial canker. With the continuous expansion of planting areas and years of continuous cropping, disease incidence has gradually worsened, new diseases have emerged, and multiple diseases often occur simultaneously, increasing the difficulty of prevention and control.

[0004] Microbial inoculants are products of microorganisms and their life activities. They can directly or indirectly improve soil, restore soil fertility, prevent various plant diseases, maintain the balance of rhizosphere microbiota, and degrade toxic substances. Using microbial inoculants can reduce the drug resistance of pathogens, and they are safe and healthy for fruits and do not pollute the environment. Currently, most microbial inoculants researched for tomato diseases are effective against a single common tomato disease and cannot simultaneously achieve high-efficiency control against multiple diseases and pests. For example, Chinese patent CN104087541A discloses a biocontrol strain for controlling early blight of tomatoes and its application, and Chinese patent CN103952352A discloses a Bacillus circulans microbial inoculant and its application, which mainly show good control effects against gray mold of tomatoes. Although such microbial strains have significant control effects on a certain type of tomato disease, in actual production, various tomato diseases do not occur in isolation; multiple diseases may occur simultaneously during the growth process. Therefore, these types of microbial inoculants have limited functions and are difficult to effectively utilize and promote in actual agricultural production. Therefore, developing a new microbial agent for the prevention and control of various tomato diseases is a technical problem that urgently needs to be solved. Summary of the Invention

[0005] This invention provides a microbial agent for the prevention and control of typical tomato diseases and its preparation method. By rationally combining the microbial agent, fungi and bacteria can work synergistically to effectively prevent and control a variety of typical tomato diseases.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0007] A microbial inoculant for controlling typical tomato diseases comprises the following components: Trichoderma viridescens, Trichoderma hamatum, Bacillus licheniformis, and Azotobacter chroococcum, in a volume ratio of 1:1:1:1.

[0008] The *Trichoderma viride* strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), strain number CGMCC No. 5.1040; the original deposit date was August 8, 2005.

[0009] The *Trichoderma hookeri* strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), strain number CGMCC No. 3.7249; the original deposit date was July 8, 2005.

[0010] The Bacillus licheniformis strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), with the strain accession number CGMCC No. 1.7677; the original accession date was June 24, 2008.

[0011] The *Azotocinobacter chrysogenum* strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), strain number CGMCC No. 1.5803; the original deposit date was June 27, 2005.

[0012] All four microbial strains used in this invention can be purchased by searching the strain catalog of the China General Microbiological Culture Collection Center, without the need for repeated biological preservation.

[0013] Preferably, the microbial agent is a liquid.

[0014] Preferably, the number of spores of the *Trichoderma viride* is 2 × 10⁻⁶. 8 cfu / mL; the number of spores of the *Trichoderma hookeri* was 2 × 10⁻⁶. 8 cfu / mL; the viable count of Bacillus licheniformis in the microbial agent is 2×10⁻⁶. 8 cfu / mL, viable count of Azotobacter brownii was 2×10⁻⁶. 8 cfu / mL.

[0015] A method for preparing a microbial inoculant for controlling typical tomato diseases includes the following preparation steps:

[0016] (1) Activate the slant cultures of *Trichoderma viride* and *Trichoderma hookeri* in test tubes. Pick out the cultures and place them into 100 ml of potato dextrose liquid culture medium. Incubate at 170 r / min and 25-28 ℃ for 48 h to obtain seed culture. Inoculate the seed culture into the fermentation broth at a 10% inoculation rate and incubate at 170 r / min and 25-28 ℃ for 96 h to obtain culture medium. At this time, the culture medium is covered with mycelia and spores of *Trichoderma viride* and *Trichoderma hookeri*. Wash the spores with sterile water, filter through four layers of gauze, and then dilute to obtain *Trichoderma viride* and *Trichoderma hookeri* solutions respectively. Ensure that the spore concentration of the two *Trichoderma* solutions obtained after dilution is 2 × 10⁻⁶. 8 cfu / mL;

[0017] (2) After thawing, *Azotocinobacter chrysogenum* was inoculated into PDA medium and activated at 28°C. After activation, 1% of the inoculum was added to the fermentation broth and fermented at 25-28°C and 150 r / min until a viable count of 2 × 10⁻⁶ was obtained. 8 Fermentation broth with cfu / mL;

[0018] (3) After thawing, Bacillus licheniformis was inoculated into LB solid medium and cultured at 28-30℃ for 1 day to activate the strain; the activated Bacillus licheniformis was then inoculated into LB liquid medium and fermented at 28-30℃ and 220 r / min until a viable count of 2×10⁻⁶ was obtained. 8 Fermentation broth with cfu / mL;

[0019] (4) After mixing the four fermentation liquids in a volume ratio of 1:1:1:1, the final product microbial agent is obtained.

[0020] LB solid medium uses conventional culture medium.

[0021] PDA culture medium preparation method: Boil 200g of potatoes in water until soft, filter and take the filtrate, then add 20g of glucose and 15-20g of agar, add water to 1000ml, and adjust to natural pH.

[0022] The fermentation broth, or potato glucose liquid culture medium (PDB), consists of: 300 g / L potato, 20 g / L glucose, and distilled water to 1000 mL, with a pH of 5.6–5.8.

[0023] The microbial agent of this invention has good control effects on tomato root rot, gray mold, wilt and early blight.

[0024] The microbial agent of this application can be used during the seed stage, or as a root drench during the mature or seedling stage. The prepared microbial agent should be diluted 100 times, with 25 ml used per plant. Apply twice consecutively, with a 10-day interval between applications.

[0025] The present invention has the following beneficial effects:

[0026] Firstly, this invention combines *Trichoderma viride* and *Trichoderma hookeri*, which exhibit antagonistic effects against various tomato pathogens and can control a variety of typical tomato diseases. The *Trichoderma viride* and *Trichoderma hookeri* selected in this invention can secrete various extracellular enzymes, such as cellulase and chitosanase. These enzymes can degrade the cell walls of pathogens, thereby inhibiting their growth. Simultaneously, they can produce various volatile and non-volatile metabolites, which have significant antibacterial effects against pathogens.

[0027] Secondly, *Bacillus licheniformis* and *Azotobacter chrysogenum* were introduced. *Bacillus licheniformis* can produce various antibacterial substances, directly inhibiting the growth and reproduction of pathogens. Simultaneously, it can induce systemic resistance in plants, significantly reducing the incidence of plant diseases. Furthermore, the selected *Bacillus licheniformis* and *Azotobacter chrysogenum* possess excellent nitrogen-fixing capabilities, improving the soil microecological environment and increasing soil nutrient content, thus providing a better growth environment for other plant growth-promoting bacteria. They can also produce indoleacetic acid, exhibit phosphorus-solubilizing activity, and generate siderophores, promoting plant growth and helping to alleviate the damage of diseases to tomato yield, thereby improving both yield and quality. Additionally, these nitrogen-fixing bacteria have a certain inhibitory effect on the pathogens of gray mold and wilt diseases in fruits and vegetables.

[0028] The combined use of *Trichoderma viride* and *Trichoderma hookeriana*, and *Bacillus licheniformis* and *Azotobacter chrysogenum*, exhibits synergistic inhibitory effects against pathogens causing tomato root rot, gray mold, wilt, and early blight. This synergistic effect of fungi and bacteria not only solves the practical problems of unstable and weak control by single biocontrol strains, but also greatly improves the biocontrol efficacy, effectively preventing diseases such as root rot, gray mold, wilt, and early blight. Furthermore, this microbial agent is compatible with the soil ecosystem, which helps to fully utilize the advantages of the strains and effectively guarantee and increase tomato yield. Attached Figure Description

[0029] Figure 1 Figures showing the growth status of different strains on LB medium;

[0030] Figure 2 This is a diagram showing the inhibitory effect of the bacterial solution in Example 1 of the present invention on different pathogens, where A0: water + Pythium, A1: Example 1 + Pythium; B0: water + Botrytis cinerea, B1: Example 1 + Botrytis cinerea; C0: water + Fusarium oxysporum, C1: Example 1 + Fusarium oxysporum; D0: water + Alternaria alternata, D1: Example 1 + Alternaria alternata.

[0031] Figure 3 The graph shows the inhibitory effect of Example 1 and Comparative Examples 1-4 on Fusarium oxysporum. Detailed Implementation

[0032] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0033] Example 1

[0034] A microbial inoculant for controlling typical tomato diseases comprises the following components: Trichoderma viridescens, Trichoderma hamatum, Bacillus licheniformis, and Azotobacter chroococcum, in a volume ratio of 1:1:1:1.

[0035] The *Trichoderma viride* strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), strain number CGMCC No. 5.1040; the original deposit date was August 8, 2005.

[0036] The *Trichoderma hookeri* strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), strain number CGMCC No. 3.7249; the original deposit date was July 8, 2005.

[0037] The Bacillus licheniformis strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), with the strain accession number CGMCC No. 1.7677; the original accession date was June 24, 2008.

[0038] The *Azotocinobacter chrysogenum* strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), strain number CGMCC No. 1.5803; the original deposit date was June 27, 2005.

[0039] All four microbial strains used in this embodiment can be purchased by searching the strain catalog of the China General Microbiological Culture Collection Center, without the need for repeated biological preservation.

[0040] The microbial agent is a liquid.

[0041] The number of spores of the *Trichoderma viride* is 2 × 10⁻⁶. 8 cfu / mL; the number of spores of the *Trichoderma hookeri* was 2 × 10⁻⁶. 8 cfu / mL; the viable count of Bacillus licheniformis in the microbial agent is 2×10⁻⁶. 8 cfu / mL, viable count of Azotobacter brownii was 2×10⁻⁶. 8 cfu / mL.

[0042] A method for preparing a microbial inoculant for controlling typical tomato diseases includes the following preparation steps:

[0043] (1) Activate the slant cultures of *Trichoderma viride* and *Trichoderma hookeri* in test tubes. Pick out the cultures and place them into 100 ml of potato dextrose liquid culture medium. Incubate at 170 r / min and 25-28 ℃ for 48 h to obtain seed culture. Inoculate the seed culture into the fermentation broth at a 10% inoculation rate and incubate at 170 r / min and 25-28 ℃ for 96 h to obtain culture medium. At this time, the culture medium is covered with mycelia and spores of *Trichoderma viride* and *Trichoderma hookeri*. Wash the spores with sterile water, filter through four layers of gauze, and then dilute to obtain *Trichoderma viride* and *Trichoderma hookeri* solutions respectively. Ensure that the spore concentration of the two *Trichoderma* solutions obtained after dilution is 2 × 10⁻⁶. 8 cfu / mL;

[0044] (2) After thawing, *Azotocinobacter chrysogenum* was inoculated into PDA medium and activated at 28°C. After activation, 1% of the inoculum was added to the fermentation broth and fermented at 25-28°C and 150 r / min until a viable count of 2 × 10⁻⁶ was obtained. 8 Fermentation broth with cfu / mL;

[0045] (3) After thawing, Bacillus licheniformis was inoculated into LB solid medium and cultured at 28-30℃ for 1 day to activate the strain; the activated Bacillus licheniformis was then inoculated into LB liquid medium and fermented at 28-30℃ and 220 r / min until a viable count of 2×10⁻⁶ was obtained. 8 Fermentation broth with cfu / mL;

[0046] (4) After mixing the four bacterial solutions in a volume ratio of 1:1:1:1, the final product microbial inoculant is obtained.

[0047] LB solid medium uses conventional culture medium.

[0048] PDA culture medium preparation method: Boil 200g of potatoes in water until soft, filter and take the filtrate, then add 20g of glucose and 15-20g of agar, add water to 1000ml, and adjust to natural pH.

[0049] The fermentation broth, or potato glucose liquid culture medium (PDB), consists of: 300 g / L potato, 20 g / L glucose, and distilled water to 1000 mL, with a pH of 5.6–5.8.

[0050] The microbial inoculant in this embodiment has a good control effect on tomato root rot, gray mold, wilt and early blight.

[0051] Verification of antagonistic effects of strains:

[0052] 1. After activating the frozen Trichoderma viride, Trichoderma hookeriana, Bacillus licheniformis, and Azotobacter chrysogenum, streak them onto LB agar plates and incubate at 25-28℃ for 3-5 days.

[0053] Three different strains were streaked onto a single culture medium to fully demonstrate that there was no mutual inhibition between the strains:

[0054] horizontal line vertical line diagonal line LB1 Trichoderma chloroticum Trichoderma hookeri Bacillus licheniformis LB2 Trichoderma hookeri Bacillus licheniformis Brown azotocin LB3 Trichoderma chloroticum Bacillus licheniformis Brown azotocin LB4 Trichoderma chloroticum Trichoderma hookeri Brown azotocin

[0055] Observe the growth of the four tested bacterial strains on LB medium and whether there are any antagonistic effects among them. The culture and growth status is as follows: Figure 1 As shown, there is no obvious inhibition zone among the four types of bacteria, indicating that there is no obvious inhibitory effect between them, and they can be subjected to compound fermentation or the bacterial solutions can be mixed.

[0056] Comparative Example 1

[0057] A method for preparing a microbial inoculant for controlling tomato diseases includes the following preparation steps:

[0058] (1) Activate the slant cultures of *Trichoderma viride* and *Trichoderma hookeri* in test tubes. Pick out the cultures and place them into 100 ml of potato dextrose liquid culture medium. Incubate at 170 r / min and 25-28 ℃ for 48 h to obtain seed culture. Inoculate the seed culture into the fermentation broth at a 10% inoculation rate and incubate at 170 r / min and 25-28 ℃ for 96 h to obtain culture medium. At this time, the culture medium is covered with mycelia and spores of *Trichoderma viride* and *Trichoderma hookeri*. Wash the spores with sterile water, filter through four layers of gauze, and then dilute to obtain *Trichoderma viride* and *Trichoderma hookeri* solutions respectively. Ensure that the spore concentration of the two *Trichoderma* solutions obtained after dilution is 2 × 10⁻⁶. 8 cfu / mL;

[0059] (2) After mixing the two spore cultures at a volume ratio of 1:1, the final product microbial inoculant is obtained.

[0060] The difference in this comparative example is that only equal volumes of *Trichoderma viride* solution and *Trichoderma harzianum* solution obtained in step (1) of the preparation method in Example 1 are mixed to obtain the microbial inoculant. All other raw materials and preparation methods are the same as in Example 1.

[0061] Comparative Example 2

[0062] A method for preparing a microbial inoculant for controlling typical tomato diseases includes the following preparation steps:

[0063] (1) After thawing, *Azotocinobacter chrysogenum* was inoculated into PDA medium and activated at 28°C. After activation, 1% of the inoculum was added to the fermentation broth and fermented at 25-28°C and 150 r / min until a viable count of 2 × 10⁻⁶ was obtained. 8Fermentation broth with cfu / mL;

[0064] (2) After thawing, Bacillus licheniformis was inoculated into LB solid medium and cultured at 28-30℃ for 1 day to activate the strain. Activated Bacillus licheniformis was then picked and inoculated into LB liquid medium and fermented at 28-30℃ and 220 r / min until a viable count of 2 × 10⁻⁶ was obtained. 8 Fermentation broth with cfu / mL;

[0065] (3) After mixing the two fermentation liquids at a volume ratio of 1:1, the final product microbial agent is obtained.

[0066] This comparative example differs in that only equal volumes of the *Azotobacter globosum* solution and *Bacillus licheniformis* solution obtained in Example 1 are mixed to obtain the microbial inoculant. All other raw materials and preparation methods are the same as in Example 1.

[0067] Comparative Example 3

[0068] A method for preparing a microbial inoculant for controlling typical tomato diseases includes the following preparation steps:

[0069] (1) Activate the slant cultures of *Trichoderma viride* and *Trichoderma hookeri* in test tubes. Pick out the cultures and place them into 100 ml of potato dextrose liquid culture medium. Incubate at 170 r / min and 25-28 ℃ for 48 h to obtain seed culture. Inoculate the seed culture into the fermentation broth at a 10% inoculation rate and incubate at 170 r / min and 25-28 ℃ for 96 h to obtain culture medium. At this time, the culture medium is covered with mycelia and spores of *Trichoderma viride* and *Trichoderma hookeri*. Wash the spores with sterile water, filter through four layers of gauze, and then dilute to obtain *Trichoderma viride* and *Trichoderma hookeri* solutions respectively. Ensure that the spore concentration of the two *Trichoderma* solutions obtained after dilution is 2 × 10⁻⁶. 8 cfu / mL;

[0070] (2) After thawing, *Azotocinobacter chrysogenum* was inoculated into PDA medium and activated at 28°C. After activation, 1% of the inoculum was added to the fermentation broth and fermented at 25-28°C and 150 r / min until a viable count of 2 × 10⁻⁶ was obtained. 8 Fermentation broth with cfu / mL;

[0071] (3) After thawing, Bacillus licheniformis was inoculated into LB solid medium and cultured at 28-30℃ for 1 day to activate the strain; the activated Bacillus licheniformis was then inoculated into LB liquid medium and fermented at 28-30℃ and 220 r / min until a viable count of 2×10⁻⁶ was obtained. 8 Fermentation broth with cfu / mL;

[0072] (4) After mixing the four bacterial solutions in a volume ratio of 2:2:1:1, the final product microbial inoculant is obtained.

[0073] This comparative example differs in that the volume ratio of the four bacterial cultures is changed. All other raw materials and preparation methods are the same as in Example 1.

[0074] Comparative Example 4

[0075] A method for preparing a microbial inoculant for controlling typical tomato diseases includes the following preparation steps:

[0076] (1) Activate the slant cultures of *Trichoderma viride* and *Trichoderma hookeri* in test tubes. Pick out the cultures and place them into 100 ml of potato dextrose liquid culture medium. Incubate at 170 r / min and 25-28 ℃ for 48 h to obtain seed culture. Inoculate the seed culture into the fermentation broth at a 10% inoculation rate and incubate at 170 r / min and 25-28 ℃ for 96 h to obtain culture medium. At this time, the culture medium is covered with mycelia and spores of *Trichoderma viride* and *Trichoderma hookeri*. Wash the spores with sterile water, filter through four layers of gauze, and then dilute to obtain *Trichoderma viride* and *Trichoderma hookeri* solutions respectively. Ensure that the spore concentration of the two *Trichoderma* solutions obtained after dilution is 2 × 10⁻⁶. 8 cfu / mL;

[0077] (2) After thawing, *Azotocinobacter chrysogenum* was inoculated into PDA medium and activated at 28°C. After activation, 1% of the inoculum was added to the fermentation broth and fermented at 25-28°C and 150 r / min until a viable count of 2 × 10⁻⁶ was obtained. 8 Fermentation broth with cfu / mL;

[0078] (3) After thawing, Bacillus licheniformis was inoculated into LB solid medium and cultured at 28-30℃ for 1 day to activate the strain; the activated Bacillus licheniformis was then inoculated into LB liquid medium and fermented at 28-30℃ and 220 r / min until a viable count of 2×10⁻⁶ was obtained. 8 Fermentation broth with cfu / mL;

[0079] (4) After mixing the four bacterial solutions in a volume ratio of 1:1:2:2, the final product microbial inoculant is obtained.

[0080] This comparative example differs in that the volume ratio of the four bacterial cultures is changed. All other raw materials and preparation methods are the same as in Example 1.

[0081] The antibacterial effect of the bacterial solution:

[0082] The microbial cultures prepared in the examples and comparative examples were tested.

[0083] Tomato root rot (Pythium), gray mold (Botrytis cinerea), wilt (Fusarium oxysporum), and early blight (Alternaria alternata) pathogens were selected. A plate confrontation method was used to conduct growth inhibition experiments. The size of the antagonistic zone was observed to detect the growth-inhibiting activity of the target bacterial solution against the plant pathogens. The method is as follows: Freshly activated, healthy pathogens were selected, and mycelial cakes with a diameter of 5 mm were punched. The mycelial cake was placed in the center of a PDA plate, and 10 μL of the test bacterial solution was added to each side approximately 22 mm away. An equal volume of sterile water was used as a control. Each treatment was repeated three times, and the plates were incubated at 28-30℃ for 3 days. The antibacterial effect was then observed. Figure 2 To illustrate the inhibitory effect of the bacterial suspension on four pathogens in Example 1, [the following was observed]. Figure 2 We can see that the bacterial solution of this invention has a good inhibitory effect on four pathogenic bacteria.

[0084] Taking *Fusarium oxysporum*, a major pathogen of tomato, as an example, following the experimental method described above, a *Fusarium oxysporum* mycelial cake was placed in the center of a PDA plate, with circular filter paper placed approximately 22 mm on either side. 10 μL of bacterial suspensions from Example 1 and Comparative Examples 1-4 were added, and the plate was incubated at a constant temperature of 28-30℃ for 3 days. The antibacterial effect was then observed. Figure 3 The figures show the inhibitory effects of Example 1 and Comparative Examples 1-4 on Fusarium oxysporum. As can be seen from the figures, compared with the examples, Comparative Examples 1-2, which only used fungi or bacteria, showed a significant decrease in antibacterial effect. In Comparative Examples 3-4, which changed the ratio of fungal and bacterial dosages, the synergistic balance between the two was broken, resulting in a weakening of the antibacterial effect.

[0085] Planting Trial

[0086] Disease prevention efficacy trial:

[0087] Disinfect the surface of crop seeds, germinate them in a petri dish with multiple layers of wet filter paper, and keep them moist with sterile water for several days. After the seed coat is removed, transplant them into sterile soil at the usual planting density. When the tomato seedlings grow to three leaves and one heart, irrigate the roots of the seedlings with the corresponding pathogen suspension.

[0088] After inoculation with the pathogen, tomato seedlings were cultured for another 2 days. Then, the inoculant of the present invention diluted 100 times and the microbial inoculant prepared in the comparative example were respectively applied to the roots of the seedlings, with a dosage of 25 ml per seedling. The control group was treated with water. Each treatment consisted of 20 tomato seedlings, and the results were repeated three times. The average value was taken. The control effect on tomatoes was observed after 25 days. The results of the control experiment are shown in Table 1.

[0089] The classification standards for tomato root rot disease are as follows:

[0090] Grade 0: Asymptomatic, no lesions at the base of the stem;

[0091] Grade 1: Cotyledons turn yellow or the base of the stem turns slightly brown;

[0092] Grade 2: Cotyledons wilted or less than 50% of the stem base turned brown;

[0093] Grade 3: Plants wilt or 50%-75% of the stem base turns brown;

[0094] Level 4: The vascular bundles of the plant turn brown, and the roots die.

[0095] Disease index = Σ(Disease grade × Number of plants at that disease grade) / (Highest disease grade × Total number of plants) × 100% Control efficacy = (Disease index of control group - Disease index of treatment group) / Disease index of control group × 100%

[0096] The classification standards for tomato gray mold disease are as follows:

[0097] Grade 0: No lesions;

[0098] Grade 1: The lesion area accounts for less than 5% of the total leaf area;

[0099] Grade 2: Lesions cover 5%-25% of the total leaf area;

[0100] Grade 3: Lesions cover 25%-50% of the total leaf area;

[0101] Grade 4: The lesion area accounts for more than 50% of the total leaf area.

[0102] Disease index = Σ(Number of diseased leaves × Disease grade) / (Total number of leaves surveyed × Highest disease grade) × 100%

[0103] Prevention efficacy = (Control disease index - Treatment disease index) / Control disease index × 100%

[0104] The disease severity grading criteria for tomato wilt are as follows:

[0105] Level 0: Asymptomatic;

[0106] Grade 1: 1-2 true leaves turn yellow or the true leaves wilt and droop;

[0107] Grade 2: 3-4 true leaves turn yellow or wilt and droop;

[0108] Grade 3: 5-6 true leaves turn yellow or wilt and droop.

[0109] Level 4: The entire plant is severely wilted.

[0110] Disease index = Σ(number of diseased plants at each level × relative level value) / (total number of plants surveyed × highest disease level) × 100%; Control efficacy = (1 - disease index of experimental area / disease index of control area) × 100%.

[0111] The disease severity classification of early blight in tomatoes is as follows:

[0112] Grade 0: No lesions

[0113] Grade 1: The lesion area accounts for less than 25% of the total leaf area;

[0114] Grade 2: Lesions cover 25% to 50% of the total leaf area;

[0115] Grade 3: Lesions cover 51% to 75% of the total leaf area;

[0116] Grade 4: The lesion area accounts for more than 75% of the total leaf area.

[0117] Disease index = Σ(Number of diseased leaves × Disease grade) / (Total number of leaves surveyed × Highest disease grade) × 100%

[0118] Prevention efficacy = (Disease index of control group - Disease index of treatment group) / Disease index of control group × 100%

[0119] Table 1. Control efficacy (%) against typical tomato diseases

[0120] Pathogens Tomato root rot Tomato gray mold Tomato Fusarium wilt Early blight of tomatoes Example 1 88.3 81.3 89.2 86.5 Comparative Example 1 48.7 52.8 51.4 46.2 Comparative Example 2 50.9 49.5 47.0 53.4 Comparative Example 3 64.2 64.2 68.5 57.8 Comparative Example 4 67.5 62.1 56.3 69.7 control group -- -- -- --

[0121] As shown in Table 1, the microbial agents of the present invention have significant control effects on typical tomato diseases, with control effects on multiple diseases exceeding 81%. Furthermore, the control effect of the microbial agents composed of four different fungal and bacterial strains is significantly higher than that of microbial agents composed of fungi or bacteria alone. In particular, Example 1, in which the ratio of the four strains of the present invention is 1:1:1:1, shows the best control effect on typical tomato diseases.

[0122] Tomato growth status and quality testing:

[0123] Ten plots of land with similar terrain and soil conditions were selected and planted with tomato seedlings of the same quality. These were divided into ten experimental groups, with 20 seedlings planted in each group. After the tomato seedlings had recovered from acclimatization for one week, they were irrigated every 10 days with 25 ml of the microbial agent prepared according to the examples and comparative examples, for two consecutive times. The control group was treated with sterile water. After harvest, the net weight of each tomato and the number of fruits per plant were measured. The data are shown in Table 2 below. The quality of the tomatoes was also measured, and the data are shown in Table 3 below.

[0124] Table 2. Net weight of a single tomato and number of fruits per plant

[0125] Net weight of a single fruit (g) Number of fruits per plant Example 1 0.302 8.6 Comparative Example 1 0.219 7.4 Comparative Example 2 0.206 7.2 Comparative Example 3 0.253 7.9 Comparative Example 4 0.248 7.7 control group 0.197 7.1

[0126] Table 3 Tomato Quality

[0127]

[0128]

[0129] Experimental results show that the microbial agent of this invention can not only increase the net weight of a single tomato fruit but also increase the number of fruits set on the tomato plant. Detection of the vitamin C content and soluble protein content of tomatoes reveals that, compared to the control group, the microbial agent of this invention significantly increases the vitamin C content and soluble protein content of tomatoes, thus improving tomato quality.

[0130] Soil physicochemical property testing:

[0131] Experimental group: Sterile soil was filled into 20cm diameter flowerpots, and tomato seedlings of uniform size and good growth were selected for transplanting, one seedling per pot, with 6 pots per group. The prepared microbial agent was diluted 100 times with sterile water according to Example 1. After planting, 25ml of microbial agent was used to drench the roots, twice consecutively, with an interval of 10 days between each application.

[0132] Control group: The control group was irrigated with sterile water.

[0133] After 90 days, soil samples were taken from the tomato root zone to test the soil's physical and chemical properties. The test results are shown in Table 4.

[0134] Table 4 Soil physicochemical properties

[0135] control group Example 1 Microbial inoculant Available phosphorus (mg / kg) 189.47 285.69 Organic matter (g / kg) 65.43 85.79 Ammonium nitrogen (mg / kg) 76.96 101.28 Nitrate nitrogen (mg / kg) 8.77 29.14

[0136] As shown in Table 4, the soil physicochemical properties treated with the compound microbial agent of this invention are significantly better than those of the control group. The nitrogen content and organic matter content of the soil treated with the microbial agent are significantly higher than those of the control group, and the activity of microorganisms also contributes to the increase of soil organic matter. This indicates that the microbial agent of this invention can increase the nitrogen and phosphorus content and the decomposition of organic matter in the soil, thereby helping to improve soil fertility and promote crop growth.

[0137] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A microbial inoculant for controlling typical diseases of tomatoes, characterized in that, Includes the following components: Trichoderma viride ( Trichoderma viridescens ), Trichoderma hookeri ( Trichoderma hamatum ), Bacillus licheniformis ( Bacillus licheniformis ), Azotocinus brownii ( Azotobacter chroococcum The volume ratio is 1:1:1:1; The *Trichoderma viride* strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), strain number CGMCC No. 5.1040; the original deposit date was August 8, 2005. The *Trichoderma hookeri* strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), strain number CGMCC No. 3.7249; the original deposit date was July 8, 2005. The Bacillus licheniformis strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), with the strain accession number CGMCC No. 1.7677; the original accession date was June 24, 2008. The *Azotocinobacter chrysogenum* strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), strain number CGMCC No. 1.5803; the original deposit date was June 27, 2005.

2. The microbial agent for controlling typical tomato diseases according to claim 1, characterized in that, The microbial agent is a liquid.

3. The microbial agent for controlling typical tomato diseases according to claim 1, characterized in that, The number of spores of the *Trichoderma viride* is 2 × 10⁻⁶. 8 cfu / mL; the number of spores of the *Trichoderma hookeri* was 2 × 10⁻⁶. 8 cfu / mL; the viable count of Bacillus licheniformis in the microbial agent is 2×10⁻⁶. 8 cfu / mL, viable count of Azotobacter brownii was 2×10⁻⁶. 8 cfu / mL.

4. A method for preparing a microbial inoculant for controlling typical diseases as described in any one of claims 1-3, characterized in that, The preparation steps include the following: (1) Activate the test tube slant cultures of Trichoderma chloroticum and Trichoderma hookeriana, pick out the cultures and put them into 100ml of potato glucose liquid culture medium, and incubate at 170r / min, 25-28℃ for 48h to obtain seed liquid; The seed culture was inoculated into the fermentation broth at a rate of 10%, and cultured at 170 r / min and 25-28℃ for 96 h to obtain the culture broth. At this time, the culture broth was confluent with mycelia and spores of *Trichoderma viride* and *Trichoderma hookeriana*. The spores were washed with sterile water, filtered through four layers of gauze, and then further diluted to obtain *Trichoderma viride* and *Trichoderma hookeriana* solutions, ensuring that the spore concentration of both *Trichoderma* solutions obtained after dilution was 2 × 10⁻⁶. 8 cfu / mL; (2) After thawing, *Azotocinobacter chrysogenum* was inoculated into PDA medium and activated at 28°C. After activation, 1% of the inoculum was added to the fermentation broth and fermented at 25-28°C and 150 r / min until a viable count of 2 × 10⁻⁶ was obtained. 8 Fermentation broth with cfu / mL; (3) After thawing, Bacillus licheniformis was inoculated into LB solid medium and cultured at 28-30℃ for 1 day to activate the strain; the activated Bacillus licheniformis was picked and inoculated into LB liquid medium and fermented at 28-30℃ and 220r / min until the viable count was 2×10⁻⁶. 8 Fermentation broth with cfu / mL; (4) After mixing the four bacterial solutions in a volume ratio of 1:1:1:1, the final product microbial inoculant is obtained.

5. The application of the microbial agent according to any one of claims 1-3 in the prevention and control of typical diseases of tomatoes, wherein the typical diseases are root rot, gray mold, wilt and early blight.

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