Synthetic bacterial agent for preventing and controlling tobacco black shank and relieving successive cropping obstacles, preparation method and application

Abstract

The invention discloses a synthetic bacterial agent for preventing and controlling tobacco black shank and relieving successive cropping obstacles as well as a preparation method and application thereof, and belongs to the technical field of biological prevention and control of plant diseases. The synthetic bacterial agent is prepared by mixing Aspergillus terreus JD-6, Aspergillus terreus JD-8, Aspergillus fumigatus JD-33, Actinomucor elegans JD-25, Mucor circinelloides JD-27 and Penicillium chrysogenum JD-38 according to the proportion of biomass and the like, and the synthetic bacterial agent is prepared by mixing Aspergillus terreus JD-6, Aspergillus terreus JD-8, Aspergillus fumigatus JD-33, Actinomucor elegans JD-25, Mucor circinelloides JD-27 and Penicillium chrysogenum JD-38 according to the proportion of biomass and the like. The synthetic microbial agent provided by the invention has no antagonistic effect among microbial members, can significantly inhibit the growth of phytophthora nicotianae, reduces the incidence rate of tobacco and promotes the growth of continuous cropping tobacco, and has an effect superior to that of a single microbial strain.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology of plant diseases, specifically relating to a synthetic microbial agent for controlling tobacco black shank disease and alleviating continuous cropping obstacles, its preparation method and application. Background Technology

[0002] Tobacco (Nicotiana tabacum), as a cash crop, is an important source of income for tobacco farmers in tobacco-growing areas, as well as for local government revenue and national tax revenue. However, tobacco is a typical crop that is susceptible to continuous cropping problems, particularly the occurrence of soil-borne diseases. Among these, black shank disease, caused by the parasitic fungus *Phytophthora nicotianae*, has a wide outbreak area and causes significant economic losses. *Phytophthora nicotianae* can exist in the soil environment for extended periods, infecting tobacco plants at any stage of growth, and is more likely to spread rapidly in humid and hot environments, making effective control of black shank disease extremely difficult.

[0003] Currently, the use of chemical pesticides is the most effective measure for controlling tobacco pests and diseases, such as metalaxyl and mancozeb. The large-scale application of chemical pesticides not only increases the production input costs for tobacco farmers, but also causes environmental pollution in tobacco fields, leading to pathogen resistance and the 3R problem of pesticides (Residue, Resistance, Resurgence).

[0004] Rhizosphere microorganisms play a crucial role in crop resistance to various stresses and maintenance of normal life activities. Numerous studies have confirmed that plant growth-promoting bacteria and biocontrol bacteria can promote plant growth, health, and yield. Therefore, biological control is an environmentally friendly way to alleviate soil-borne diseases in crops and achieve sustainable, high-quality tobacco production.

[0005] Currently, research on plant growth-promoting bacteria largely focuses on exploring single microbial strains. However, when these single strains are extended from laboratory conditions to field environments, their actual effects often fall short of expectations. Further research indicates that the stable disease-suppressing capacity of soil ecosystems is more likely due to close cooperation among microbial communities than the effect of a single strain. However, a synthetic microbial community system with good control effects against tobacco black shank is still lacking. Strengthening the exploration of novel microbial strains and constructing highly efficient and stable synthetic microbial communities with disease-resistant functions will provide a green and reliable biocontrol strategy for soil ecological environment health and sustainable agricultural development. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and to provide a synthetic microbial agent for controlling tobacco black shank disease and alleviating continuous cropping obstacles, its preparation method and application.

[0007] The specific technical solution adopted in this invention is as follows:

[0008] In a first aspect, the present invention provides a synthetic microbial agent for controlling tobacco black shank disease and alleviating continuous cropping obstacles. The synthetic microbial agent is a mixed microbial agent of Aspergillus terreus JD-6, Aspergillus terreus JD-8, Aspergillus fumigatus JD-33, Actinomucor elegans JD-25, Mucor circinelloides JD-27 and Penicillium chrysogenum JD-38.

[0009] The *Aspergillus terreus* JD-6 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 26, 2025, with accession number CGMCC 3.29341; the *Aspergillus terreus* JD-8 is deposited at the CGMCC on September 26, 2025, with accession number CGMCC 3.29342; the *Aspergillus fumigatus* JD-33 is deposited at the CGMCC on September 26, 2025, with accession number CGMCC 3.29345; the *Actinomucor*... The following fungi are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession date of September 26, 2025, and accession number CGMCC 3.29343: *Mucorcircinelloides* JD-27; *Penicillium chrysogenum* JD-38; and *Penicillium chrysogenum* JD-38.

[0010] Preferably, the synthetic microbial agent contains Aspergillus terreus JD-6, Aspergillus terreus JD-8, Aspergillus fumigatus JD-33, Mucor radiata JD-25, Mucor truncatula JD-27, and Penicillium chrysogenum JD-38 mixed in equal proportions of biomass.

[0011] Secondly, the present invention provides a method for preparing a synthetic microbial agent for controlling tobacco black shank disease and alleviating continuous cropping obstacles, as detailed below:

[0012] Mycelia of Aspergillus terreus JD-6, Aspergillus terreus JD-8, Aspergillus fumigatus JD-33, Mucor radiata JD-25, Mucor truncatula JD-27, and Penicillium chrysogenum JD-38, as described in the first aspect, were selected and placed in Bengal red liquid medium. Each culture was incubated at 150-180 rpm and 30-32℃ for 3-4 days to obtain mycelia of each strain. The mycelia of each strain were broken and diluted to prepare spore suspensions. Finally, equal volumes of spore suspensions were mixed evenly to obtain the synthetic inoculum.

[0013] Preferably, the concentration of fungal spores in the spore suspension is 10. 6 ~10 8 spores / mL.

[0014] Thirdly, the present invention provides a synthetic bacterial agent prepared according to the preparation method described in the second aspect.

[0015] Fourthly, this invention provides the application of the synthetic microbial agent described in the third aspect in controlling tobacco black shank disease and alleviating tobacco continuous cropping obstacles. The synthetic microbial agent is applied by root irrigation during the tobacco seedling growth stage, with an application rate of 10... 6 ~10 7 spores / g soil.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The synthetic microbial agent provided by this invention exhibits no antagonistic effect among its various microbial members, significantly inhibiting the growth of Phytophthora tobaccois, reducing tobacco disease incidence, and promoting the growth of continuously cropped tobacco, with effects superior to those of single microbial strains. The method provided by this invention is universally applicable and can be further applied to the construction of disease-resistant synthetic microbial communities for other crops. Attached Figure Description

[0018] Figure 1 This is a graph showing the comparison of the inhibition rates of the initial screening microorganisms against Phytophthora tobaccois in Example 1;

[0019] Figure 2 This refers to the growth-promoting and disease-resistant functions of the microorganisms initially screened in Example 1;

[0020] Figure 3 The following is a comparison of the results of the pot experiment of tobacco with a single strain in Example 2, where (a) is a comparison of plant height and (b) is a comparison of wet biomass.

[0021] Figure 4 This is a comparison chart of the incidence rates of tobacco black shank in pot experiments with different treatments in Example 4 and the comparative example;

[0022] Figure 5This is a comparison chart of tobacco plant height in pot experiments with different treatments in Example 4 and the comparative example;

[0023] Figure 6 This is a comparison diagram of the wet biomass of tobacco in pot experiments with different treatments in Example 4 and the comparative example. Detailed Implementation

[0024] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.

[0025] The strains used in the following examples are specifically listed below. These strains can all be obtained from the official website of the China General Microbiological Culture Collection Center:

[0026] Aspergillus terreus JD-6 is deposited at the China General Microbiological Culture Collection Center on September 26, 2025, with accession number CGMCC 3.29341;

[0027] Aspergillus terreus JD-8 is deposited at the China General Microbiological Culture Collection Center on September 26, 2025, with accession number CGMCC 3.29342;

[0028] Aspergillus fumigatus JD-33 is deposited at the China General Microbiological Culture Collection Center on September 26, 2025, with accession number CGMCC 3.29345;

[0029] Actinomucor elegans JD-25 is deposited at the China General Microbiological Culture Collection Center on September 26, 2025, with accession number CGMCC 3.29343;

[0030] Mucor circinelloides JD-27 is deposited at the China General Microbiological Culture Collection Center on September 26, 2025, with accession number CGMCC 3.29344;

[0031] Penicillium chrysogenum JD-38 is deposited at the China General Microbiological Culture Collection Center on September 26, 2025, with accession number CGMCC 3.29346.

[0032] The culture medium formulations used in the following examples are as follows:

[0033] Beef extract peptone solid medium (BPM, for bacterial isolation): 3 g beef extract, 10 g peptone, 5 g NaCl, 20 g agar, 1000 mL deionized water, pH 7.0~7.2.

[0034] Bengal Red Solid Medium (RB, for fungal isolation): Peptone 5.0 g, KH2PO4 1.0 g, MgSO4·7H2O 0.5 g, Glucose 10.0 g, Chloramphenicol 0.1 g, Bengal Red 0.033 g, Agar 20.0 g, Deionized Water 1000 mL.

[0035] Oatmeal solid medium (OA, for culturing Phytophthora tobaccois): 30.0 g oatmeal, 18.0 g agar, 1000 mL deionized water, pH 6.8. Weigh a measured amount of oatmeal, heat in boiling water for 20 min, filter through two layers of gauze, and bring to a final volume.

[0036] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, commercially available or prepared according to conventional methods in the art, and are of laboratory purity. Unless otherwise specified, the experimental methods and conditions used in the following examples are conventional experimental methods and conditions in the art, and can be found in relevant experimental manuals, public literature, or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] Example 1 Screening of antagonistic strains of Phytophthora indicum

[0038] (1) Microbial isolation

[0039] In Chengjiang City, Yuxi City, Yunnan Province, fields where tobacco was continuously cropped and black shank disease occurred were selected. Rhizosphere soil from healthy tobacco plants was collected using the root-shaking method and brought back to the laboratory. Microorganisms were isolated and screened using the dilution plating method.

[0040] (2) Preliminary screening of antagonistic microorganisms of Phytophthora tobaccois

[0041] Antagonistic microorganisms against *Phytophthora tobaccota* were screened using the plate confrontation method. *Phytophthora tobaccota* was inoculated onto OA medium and cultured for 4 days. 6 mm diameter circular mycelial blocks were punched out and transferred to the center of a fresh OA medium. Candidate single colonies were picked up with a pipette tip and symmetrically plotted with a length of 3 cm on each side, 2.5 cm away from the *Phytophthora tobaccota* mycelial block. A blank control was prepared by inoculating only *Phytophthora tobaccota* without candidate mycelia. All colonies were cultured at 30°C for 5 days. Three replicates were set up for each candidate mycelium. The colony diameters of the pathogens in the control and treatment groups were measured to calculate the inhibition rate. Inhibition rate (%) = [(Diameter of pathogens in the control group d0 - Diameter of pathogens in the treatment group d) / Diameter of pathogens in the control group d0] × 100%.

[0042] Nine fungal strains with strong inhibitory effects against *Phytophthora indicum* were finally screened, including three strains from *Aspergillus*, three strains from *Mucor*, two strains from *Trichoderma*, and one strain from *Penicillium*. Their inhibition rates against *Phytophthora indicum* ranged from 34.0% to 60.0%. Figure 1 As shown.

[0043] (3) Biological identification

[0044] Nine fungal strains obtained from the initial screening were sent to Beijing Qingke Biotechnology Co., Ltd. for strain identification. Universal primers were used to amplify the ITS gene fragment of the fungi, and the amplified products were subjected to Sanger sequencing. The sequencing results were compared with the NCBI database using BLAST. The specific comparison results are shown in Table 1 below.

[0045] Primer ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′;

[0046] Primer ITS4: 5′-TCCTCCGCTTATTGATATGC-3′.

[0047] Table 1. Classification of 9 candidate fungal strains

[0048]

[0049] (4) Determination of functions related to growth promotion and disease resistance

[0050] Eight representative strains were selected, and various growth-promoting and disease-resistant functions were measured, including phosphorus solubilization capacity, heparin secretion capacity, and the production capacity of hydrolytic enzymes such as chitinase, protease, cellulase, and β-glucanase. At the same time, the secretion of indoleacetic acid (IAA) was also detected.

[0051] Each candidate strain was inoculated either by spot inoculation or streak plating onto the corresponding functional identification media: phosphate-solubilizing microbial medium (for detecting phosphate-solubilizing zones), CAS medium (for detecting siderophiles), chitin-based medium (for detecting chitinase), skim milk medium (for detecting proteases), cellulose medium (for detecting cellulase), β-1,3-glucan medium (for detecting β-glucanase), and medium supplemented with precursor substances (for detecting IAA). After an appropriate incubation period, the formation of a clear zone or a characteristic discolored halo around the colony was observed to determine whether the corresponding function was positive or negative.

[0052] The results are as follows Figure 2 As shown, all candidate strains can produce β-glucanase, and some strains can produce cellulase, heptaphosphate, and lysophosphatase. "×" in the figure indicates that they do not produce these enzymes.

[0053] Example 2: Pot experiment of a single strain of tobacco

[0054] (1) Preparation of bacterial culture

[0055] The fungal strains selected in Example 1 were used. Mycelia from these nine strains were placed in Erlenmeyer flasks containing Bengal red liquid medium and incubated at 150 rpm and 30°C for 3-4 days. After incubation, the mycelial clumps were broken up using a tissue homogenizer to prepare homogeneous spore suspensions. Fungal spores were counted under a microscope using a hemocytometer, and the concentration of each suspension was adjusted to approximately 10% with sterile water. 7 1 spore / mL, stored at 4℃ for later use.

[0056] (2) Pot experiment

[0057] The experimental soil was collected from tobacco fields in Chengjiang City, Yuxi City, Yunnan Province. The pot experiment included 10 treatments: one control group (CK, treated with sterile water) and nine experimental groups (each treated with a fungal inoculum of one of nine different strains). Each treatment was replicated five times, with each pot containing 500 g of soil. The tested tobacco variety was K326.

[0058] Select tobacco seedlings with uniform growth at the 4-5 leaf stage and pre-culture them in an artificial climate chamber (photoperiod 12 h / 12 ​​h, temperature 25℃, humidity 50%). After their growth stabilizes, transplant them into experimental pots, one seedling per pot. Water regularly to maintain the soil moisture content at about 50% of the maximum field capacity.

[0059] Root irrigation was performed on the 7th and 15th days after transplanting the tobacco seedlings: 10 mL of sterile water was added to each pot in the control group, while 10 mL of the corresponding bacterial solution was added to each experimental group, so that the final spore concentration in the soil reached approximately 1×10⁻⁶. 6 Spores / g soil. Sampling was conducted 20 days after transplanting tobacco seedlings to measure plant height and aboveground wet biomass. Results are as follows: Figure 3As shown.

[0060] according to Figure 3 It can be seen that, compared with the control group, except for *Mucor* JD-28 which had a certain inhibitory effect on tobacco growth, the other 8 fungal strains could increase the plant height and wet biomass of tobacco plants. Among them, the 3 *Aspergillus* strains had the best effect, especially *Aspergillus terreus* JD-8 and *Aspergillus fumigatus* JD-33. Different letters in the figure indicate significant differences between different single strain treatments (Duncan test, P < 0.05, n = 5).

[0061] Example 3: Study on inter-fungal antagonistic effects

[0062] To assess whether two or more fungal strains exhibit mutual inhibition (antagonism) during co-growth, the plate confrontation method was used to study the antagonistic effect. Specifically, Bengal Red solid medium was selected, and mycelia of two fungal strains were streaked symmetrically on both sides of an RB medium plate using a sterilized pipette tip, with a streak length of 3 cm. All plates were incubated at 30°C for one week, and the antagonistic growth between the strains and the formation of obvious inhibition zones were observed.

[0063] The results showed that two Trichoderma strains readily parasitized the mycelia of other fungi, thereby inhibiting the growth of other strains, while no antagonistic effect was found among the other candidate strains. Therefore, Aspergillus terreus JD-6, Aspergillus terreus JD-8, Aspergillus fumigatus JD-33, Mucor radiata JD-25, Mucor truncatula JD-27, and Penicillium chrysogenum JD-38 can be used in combination in the future.

[0064] Example 4: Preparation of Synthetic Microbial Agent and Tobacco Pot Experiment

[0065] (1) Preparation of synthetic microbial agents

[0066] Mycelia of *Aspergillus terreus* JD-6, *Aspergillus terreus* JD-8, *Aspergillus fumigatus* JD-33, *Mucor radiata* JD-25, *Mucor truncatula* JD-27, and *Penicillium chrysogenum* JD-38 were selected and placed in Bengal red liquid medium. Each culture was incubated at 150 rpm and 30°C for 3-4 days to obtain the mycelia of each strain. The mycelia of each strain were then broken up and diluted with sterile water to a concentration of 10. 7 A spore suspension of 1 spore / mL was prepared; finally, equal volumes of spore suspensions were mixed evenly to obtain a synthetic inoculum, which was designated as fungal synthetic microbial group 1.

[0067] (2) Pot experiment

[0068] The conditions for potted plants, experimental environment, and the method and frequency of bacterial suspension addition were the same as in Example 2. Sampling began 20 days after transplanting the tobacco seedlings to measure the disease incidence, plant height, and wet biomass. Results are as follows: Figures 4-6 As shown.

[0069] Comparative Example 1: Tobacco pot experiment with added Bacillus subtilis

[0070] (1) This comparative example has a total of 7 treatment groups:

[0071] 1) Control group (CK): Sterile water was applied;

[0072] 2) Bacillus subtilis single-strain treatment group: only Bacillus subtilis bacterial suspension was applied;

[0073] 3) Aspergillus fumigatus JD-33 single-strain treatment group: only Aspergillus fumigatus JD-33 bacterial suspension was applied;

[0074] 4) Two groups of fungal synthetic microorganisms: a mixed bacterial solution of Aspergillus terreus JD-6, Aspergillus terreus JD-8, Aspergillus fumigatus JD-33, Trichoderma longifolia JD-19, Trichoderma longifolia JD-20, Mucor radiata JD-25, Mucor truncatula JD-27, and Penicillium chrysogenum JD-38 was applied.

[0075] 5) Group 1 of cross-species synthetic microbial community: A mixed bacterial solution of Bacillus subtilis and Aspergillus fumigatus JD-33 was applied;

[0076] 6) Two groups of cross-species synthetic microbial communities: a mixed bacterial solution of Bacillus subtilis, Aspergillus terreus JD-6, Aspergillus terreus JD-8, Aspergillus fumigatus JD-33, Mucor radiata JD-25, Mucor truncatula JD-27, and Penicillium chrysogenum JD-38 was applied.

[0077] 7) Three groups of cross-species synthetic microbial communities: A mixed bacterial solution of Bacillus subtilis, Aspergillus terreus JD-6, Aspergillus terreus JD-8, Aspergillus fumigatus JD-33, Trichoderma longifolia JD-19, Trichoderma longifolia JD-20, Mucor radiata JD-25, Mucor truncatula JD-27, and Penicillium chrysogenum JD-38 was applied.

[0078] The Bacillus subtilis used in this embodiment was purchased from the China Agricultural Microbial Culture Collection Center (http: / / www.accc.org.cn / ), and the strain preservation number is ACCC 19742.

[0079] (2) Preparation of bacterial culture

[0080] The method for preparing the fungal suspension is the same as in Example 2, and each spore suspension is diluted to a concentration of 10. 7 Mix after each spore / mL.

[0081] Bacillus subtilis was added to beef extract peptone (BPM) liquid medium and cultured at 150 rpm and 30°C for 3-4 days. The OD was measured. 600 The bacterial concentration is determined by using sterile water to adjust the OD value. 600The value is adjusted to 1.0, so the concentration of the bacterial solution is approximately 10. 8 cells / mL, store at 4℃ for later use.

[0082] (3) Pot experiment

[0083] The conditions for potting soil, experimental environment, bacterial solution addition method and frequency, etc., were the same as in Example 2. The amount of bacteria added to the soil was approximately 10. 7 The number of cells / g soil and the amount of fungi added to the soil are approximately 10. 6 spores / g soil. Sampling began 20 days after tobacco seedling transplanting to measure disease incidence, plant height, and wet biomass. Results are as follows: Figures 4-6 As shown.

[0084] according to Figures 4-6 It can be seen that although all microbial treatments can effectively reduce the incidence of tobacco black shank and promote tobacco plant growth, the synthetic inoculant containing 6 fungi (group 1 of fungal synthetic flora) provided by this invention exhibits the best synergistic disease prevention and growth promotion ability due to the absence of antagonistic effects: compared with the control group, the incidence of tobacco disease in Example 4 decreased by 58.3%, and the biomass increased by 42.1%. Different letters in the figure indicate significant differences between different treatments (Duncan test, P < 0.05, n = 5).

[0085] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A synthetic microbial agent for controlling tobacco black shank disease and alleviating continuous cropping obstacles, characterized in that, The synthetic microbial agent is a mixture of Aspergillus terreus JD-6, Aspergillus terreus JD-8, Aspergillus fumigatus JD-33, Actinomucor elegans JD-25, Mucor circinelloides JD-27, and Penicillium chrysogenum JD-38. The *Aspergillus terreus* JD-6 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 26, 2025, with accession number CGMCC 3.29341; the *Aspergillus terreus* JD-8 is deposited at the CGMCC on September 26, 2025, with accession number CGMCC 3.29342; the *Aspergillus fumigatus* JD-33 is deposited at the CGMCC on September 26, 2025, with accession number CGMCC 3.29345; the *Actinomucor*... The following fungi are deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession date of September 26, 2025, and accession number CGMCC 3.29343: *Mucorcircinelloides* JD-27; *Penicillium chrysogenum* JD-38; and *Penicillium chrysogenum* JD-38.

2. The synthetic microbial agent for controlling tobacco black shank disease and alleviating continuous cropping obstacles according to claim 1, characterized in that, The synthetic microbial agent contains Aspergillus terreus JD-6, Aspergillus terreus JD-8, Aspergillus fumigatus JD-33, Mucor radiata JD-25, Mucor truncatula JD-27, and Penicillium chrysogenum JD-38 mixed in equal proportions of biomass.

3. A method for preparing a synthetic microbial agent for controlling tobacco black shank disease and alleviating continuous cropping obstacles, characterized in that, Specifically as follows: Mycelia of Aspergillus terreus JD-6, Aspergillus terreus JD-8, Aspergillus fumigatus JD-33, Mucor radiata JD-25, Mucor truncatula JD-27, and Penicillium chrysogenum JD-38 as described in claim 1 were selected and placed in Bengal red liquid medium. Each culture was incubated at 150-180 rpm and 30-32℃ for 3-4 days to obtain mycelia of each strain. The mycelia of each strain were broken and diluted to prepare spore suspensions. Finally, equal volumes of spore suspensions were mixed evenly to obtain the synthetic inoculum.

4. The method for preparing the synthetic microbial agent for controlling tobacco black shank disease and alleviating continuous cropping obstacles according to claim 3, characterized in that, The concentration of fungal spores in the spore suspension was 10. 6 ~10 8 spores / mL.

5. A synthetic bacterial agent prepared according to the preparation method described in claim 3 or 4.

6. The application of the synthetic microbial agent according to claim 5 in controlling tobacco black shank disease and alleviating tobacco continuous cropping obstacles, characterized in that, Synthetic inoculants were applied via root irrigation during the tobacco seedling growth stage, with an application rate of 10... 6 ~10 7 spores / g soil.

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