Pseudomonas mediterranea with mildew-proof effect and application thereof

Abstract

The application belongs to the technical field of biocontrol bacteria, and particularly relates to a Pseudomonas mediterranea with a mildew-proof effect and application thereof. Specifically, a Pseudomonas mediterranea with a potential biocontrol function is isolated from tobacco rhizosphere soil. The full-length sequence of the bacteria is obtained through 16S rRNA gene sequencing, and a phylogenetic tree is constructed to determine the taxonomic status and genetic relationship thereof. Further research shows that the strain not only has a high self-inhibition rate, but also can inhibit the growth of Aspergillus niger, Aspergillus flavus, filamentous fungi and Talaromyces variabilis through producing volatile substances and fermentation products. In addition, the antibacterial effect of the strain on dry tobacco leaves is verified, and the strain shows good application potential, so the strain has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biocontrol bacteria technology, specifically relating to a type of Pseudomonas thaliana with antifungal properties and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Tobacco diseases are not limited to the plant's growth stage; mold growth during storage is also a significant concern. Mold growth in tobacco leaves during storage is a complex biological deterioration process, primarily caused by environmental factors and the inherent characteristics of the material itself. Under high humidity storage conditions, tobacco leaves readily absorb moisture from the air, providing a breeding ground for mold. A suitable temperature range, especially between 20°C and 30°C, further accelerates mold reproduction. Furthermore, inadequate or poorly managed warehouse ventilation can obstruct airflow, hindering moisture dissipation and exacerbating the risk of mold growth. The moisture content of the tobacco leaves themselves is also crucial; leaves that have not undergone sufficient drying are more susceptible to mold growth during storage. Warehouse hygiene is also critical; residual mold and dust can become breeding grounds for mold. Finally, improper packaging and stacking methods can lead to excessively high local humidity or poor ventilation, further promoting mold growth.

[0004] Currently, chemical control methods are commonly used to prevent mold growth in tobacco leaves during storage. However, these chemical agents tend to leave persistent residues in the tobacco leaves, leading to the degradation of polyphenols, loss of aroma components, and deterioration of combustion characteristics, directly reducing commercial value and negatively impacting the environment. Therefore, biological control, as an emerging environmental trend, is gaining increasing attention. By screening for microorganisms that can inhibit these pathogens and developing biological agents that suppress their growth, this method can effectively protect stored tobacco leaves from damage. However, antagonistic strains against tobacco mold are still rarely reported. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a *Pseudomonas mediterranea* strain with antifungal properties and its applications. Specifically, this invention isolates and screens a strain of *Pseudomonas mediterranea* Cas656, which exhibits a high self-inhibition rate and can also inhibit the growth of *Aspergillus niger*, *Aspergillus flavus*, filamentous fungi *Talaromyces verruculosus*, and *Talaromyces variabilis* by producing volatile substances and their fermentation products. Its antifungal effect on dried tobacco leaves has also been verified. Based on the above research results, this invention is thus completed.

[0006] To achieve the above-mentioned technical objectives, the present invention relates to the following technical solutions:

[0007] In a first aspect, the present invention provides a strain of Pseudomonas mediterranea Cas656, which was deposited on February 26, 2025, at the China General Microbiological Culture Collection Center (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China), with the biological accession number CGMCC No. 33664.

[0008] A second aspect of the present invention provides a fermentation production method for the above-mentioned Pseudomonas thaliana, the fermentation production method comprising: inoculating the Pseudomonas thaliana into a fermentation medium for fermentation culture.

[0009] A third aspect of the present invention provides a microbial inoculant containing the aforementioned Pseudomonas thaliana or its ferments or metabolites.

[0010] The aforementioned microbial agents can specifically be fungal inhibitors or plant mold inhibitors;

[0011] Furthermore, the fungus can be a mold that mediates mold growth in plants (especially tobacco), including but not limited to Aspergillus niger, Aspergillus flavus, filamentous fungi (Talaromyces verruculosus), and Talaromyces variabilis.

[0012] The plant mold inhibitor can specifically act on mold growth in plants (especially tobacco) mediated by mold.

[0013] In a fourth aspect, the use of the aforementioned *Pseudomonas thaliana* Cas656 and / or the aforementioned microbial agents in all or part of the following a)-c) is also within the scope of protection of this invention:

[0014] a) Inhibit mold or prepare mold inhibitors;

[0015] b) Inhibit mold growth or prepare mold inhibitors;

[0016] c) Prevent plant rot.

[0017] The molds mentioned are those that mediate mold growth in plants (especially tobacco), including but not limited to Aspergillus niger, Aspergillus flavus, filamentous fungi (Talaromyces verruculosus), and Talaromyces variabilis.

[0018] The mold refers specifically to plant mold.

[0019] The plant can specifically be a plant or a part of a plant; in one specific embodiment of the present invention, the plant is tobacco; further, it can be harvested and stored tobacco leaves.

[0020] A fifth aspect of the present invention provides a method for preventing and controlling the rotting of tobacco leaves, the method comprising applying the aforementioned Pseudomonas thuringiensis Cas656 or the aforementioned microbial agent to tobacco (such as tobacco leaves) to inhibit the growth of mold and achieve prevention and control of rotting of tobacco leaves, especially stored tobacco leaves.

[0021] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0022] The above-mentioned technical method isolated a strain of *Pseudomonas mediterranea* with potential biocontrol function from tobacco rhizosphere soil. The full-length sequence of the bacteria was obtained through 16S rRNA gene sequencing, and a phylogenetic tree was constructed to determine its taxonomic position and phylogenetic relationships. Further studies showed that this strain not only has a high self-inhibition rate but also inhibits the growth of *Aspergillus niger*, *Aspergillus flavus*, *Basilella vulgaris*, and filamentous fungi by producing volatile substances and their fermentation products. Furthermore, the antibacterial effect of this strain on dried tobacco leaves was also verified, demonstrating good application potential and therefore broad application prospects. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 This is the colony morphology of strain Cas656 in Example 1 of the present invention.

[0025] Figure 2 In Example 1 of this invention, a phylogenetic tree was constructed using the Neighbour-Joining method to build 16S rDNA sequences of Cas656 and other standard bacteria of Pseudomonas thaliana.

[0026] Figure 3 The plate plate antibacterial ability of strain Cas656 in Example 1 of the present invention was tested, wherein A is Aspergillus niger, B is Aspergillus flavus, C is Aspergillus variantans, and D is filamentous fungus.

[0027] Figure 4 This invention illustrates the inhibitory effect of volatile organic compounds from strain Cas656 on Aspergillus niger in Example 1. In Example 1, A is a diagram of the sample treatment process, with the left side representing the treatment group and the right side representing the control group; in Example 2, B is a diagram with the left side representing the treatment group and the right side representing the control group.

[0028] Figure 5 This invention illustrates the inhibitory effect of volatile organic compounds from strain Cas656 on Aspergillus flavus in Example 1. In Example 1, A is a diagram of the sample treatment process, with the left side representing the treatment group and the right side representing the control group; in Example 2, B also shows the left side representing the treatment group and the right side representing the control group.

[0029] Figure 6 This invention illustrates the inhibitory effect of volatile organic compounds from strain Cas656 on *Bacillus mutans* in Example 1 of this invention. In Example 1, A is a diagram of the sample processing, with the left side representing the treatment group and the right side representing the control group; in Example 2, B also shows the left side representing the treatment group and the right side representing the control group.

[0030] Figure 7 This invention illustrates the inhibitory effect of volatile organic compounds from strain Cas656 on filamentous fungi in Example 1. In Example 1, A is a diagram of the sample treatment process, with the left side representing the treatment group and the right side representing the control group; in Example 2, B also shows the left side representing the treatment group and the right side representing the control group.

[0031] Figure 8 The supernatant of the Cas656 strain fermentation strain in Example 1 of this invention inhibits Aspergillus niger; wherein, A is the control group, B is 2% supernatant, C is 5% supernatant and D is 10% supernatant.

[0032] Figure 9 shows the inhibitory effect of the fermentation supernatant of Cas656 strain on Aspergillus flavus in Example 1 of the present invention; where A is the control group, B is 2% supernatant, C is 5% supernatant and D is 10% supernatant.

[0033] Figure 10The above describes the inhibitory effect of the fermentation supernatant of Cas656 strain on *Trametes versicolor* in Example 1 of this invention; wherein A is the control group, B is 2% supernatant, C is 5% supernatant, and D is 10% supernatant.

[0034] Figure 11 The supernatant of the Cas656 strain fermentation in Example 1 of this invention inhibits filamentous fungi; wherein, A is the control group, B is 2% supernatant, C is 5% supernatant, and D is 10% supernatant.

[0035] Figure 12 This invention illustrates the effect of Cas656 bacterial suspension on mold-induced mildew in tobacco leaves in Example 1. In Example A, the left side represents the Aspergillus niger control group, and the right side represents the treatment group; in Example B, the left side represents the Aspergillus flavus control group, and the right side represents the treatment group; in Example C, the left side represents the *Agrocytogenes* control group, and the right side represents the treatment group; and in Example D, the left side represents the filamentous fungi control group, and the right side represents the treatment group. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0038] In a typical embodiment of the present invention, a strain of Pseudomonas mediterranea Cas656 is provided. This strain was deposited on February 26, 2025, at the China General Microbiological Culture Collection Center (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China), with the biological accession number CGMCC No. 33664.

[0039] In another specific embodiment of the present invention, a fermentation production method for the above-mentioned Pseudomonas thaliana Cas656 is provided, the fermentation production method comprising: inoculating the Pseudomonas thaliana Cas656 into a fermentation medium for fermentation culture.

[0040] In this invention, no specific limitation is made to the fermentation production method; any conventional bacterial fermentation culture method can be used for cultivation.

[0041] The fermentation medium can be any common bacterial culture medium, such as LB medium in one specific embodiment of the present invention, and fermentation culture at 28°C for 24 hours.

[0042] In another specific embodiment of the present invention, a microbial inoculant is provided, which contains the aforementioned Pseudomonas thaliana Cas656 or its fermentation product or its metabolites.

[0043] In this invention, the term "fermentation product" is used to refer to fermentation products. The corresponding fermentation product can be a liquid obtained from the fermentation culture of *Pseudomonas thaliana* Cas656, and therefore can also be called a fermentation broth; the liquid may contain bacteria (cells), but is not necessarily required to contain bacteria. The liquid preferably contains metabolites produced by *Pseudomonas thaliana* Cas656 of this invention. Furthermore, the fermentation product also contains metabolites with volatile properties.

[0044] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is separated from the liquid by centrifugation, filtration, sedimentation, or other means known in the art. The liquid remaining after removing the bacterial cells is called the "supernatant," and in the present invention, the supernatant contains extracellular metabolites of Pseudomonas thaliana Cas656. In embodiments of the present invention, the bacterial agent may also contain this supernatant.

[0045] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is separated from the liquid by centrifugation, filtration, sedimentation, or other means known in the art to obtain bacterial cells. The bacterial cells can be disrupted to obtain bacterial fragments, which can be achieved by ultrasound (e.g., ice bath ultrasound to disrupt cells) or other means known in the art. Alternatively, the bacterial fragments can be centrifuged to collect the supernatant, which is designated as the cell-free extract. In this invention, the bacterial fragments or cell-free extract contain intracellular metabolites of *Pseudomonas thaliana* Cas656. In embodiments of the present invention, the bacterial agent may also contain the bacterial fragments or cell-free extract.

[0046] Furthermore, in embodiments of the present invention, for ease of storage and transportation, and to improve the survival rate of the bacterial strain, the bacterial agent may also be a solid, and more preferably a lyophilized powder. That is, it is obtained by further freeze-drying the aforementioned *Pseudomonas thaliana* Cas656 or its fermentation products or their metabolites. The freeze-drying technology (including vacuum freeze-drying technology) can be carried out using conventional methods, and will not be elaborated further here.

[0047] In another specific embodiment of the present invention, the microbial agent may further include excipients acceptable to the agent.

[0048] In another specific embodiment of the present invention, the excipients are selected from one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents, thickeners, fillers, and solvents. The present invention does not impose any special restrictions on the sources of acceptable excipients for the bacterial agent; commercially available products are generally sufficient.

[0049] In another specific embodiment of the present invention, the above-mentioned microbial agent may specifically be a fungal inhibitor or a plant mold inhibitor.

[0050] The active ingredient of the above-mentioned plant pathogen inhibitor may be Pseudomonas thaliana Cas656 or its fermentation product or its metabolite. The active ingredient of the above-mentioned plant pathogen inhibitor may also contain other biological or non-biological components. Other active ingredients of the above-mentioned plant pathogen inhibitor can be determined by those skilled in the art based on the inhibitory effect on plant pathogens.

[0051] Furthermore, the fungus can be a mold that mediates mold growth in plants (especially tobacco), including but not limited to Aspergillus niger, Aspergillus flavus, filamentous fungi (Talaromyces verruculosus), and Talaromyces variabilis.

[0052] The plant mold inhibitor can specifically act on mold growth in plants (especially tobacco) mediated by mold.

[0053] In another specific embodiment of the present invention, the application of the above-mentioned Pseudomonas thaliana Cas656 and / or the above-mentioned microbial agent in all or part of the following a)-c) is also within the scope of protection of the present invention:

[0054] a) Inhibit mold or prepare mold inhibitors;

[0055] b) Inhibit mold growth or prepare mold inhibitors;

[0056] c) Prevent plant rot.

[0057] The molds mentioned are those that mediate mold growth in plants (especially tobacco), including but not limited to Aspergillus niger, Aspergillus flavus, filamentous fungi (Talaromyces verruculosus), and Talaromyces variabilis.

[0058] The mold refers specifically to plant mold.

[0059] The plant can specifically be a plant or a part of a plant; in one specific embodiment of the present invention, the plant is tobacco; further, it can be harvested and stored tobacco leaves.

[0060] In another specific embodiment of the present invention, a method for preventing and controlling the rotting of tobacco leaves is provided. The method includes applying the above-mentioned Pseudomonas thaliana Cas656 or the above-mentioned microbial agent to tobacco (such as tobacco leaves) to inhibit the growth of mold and achieve the prevention and control of rotting of tobacco leaves, especially stored tobacco leaves.

[0061] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. The pathogens tested in the examples were all isolated and preserved by the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences.

[0062] Example 1

[0063] 1. Materials and Methods

[0064] 1.1 Materials

[0065] Culture media: Nutrient agar (NA) solid medium, potato dextrose agar (PDA) solid medium, LB broth, and potato dextrose agar (NB) liquid medium.

[0066] The four molds, namely Aspergillus niger, Aspergillus flavus, Talaromyces verruculosus, and Talaromyces variabilis, were isolated and identified from moldy tobacco leaves.

[0067] 1.2 Methods

[0068] 1) Isolation and culture of strains

[0069] Environmental samples were collected from the root surface of healthy tobacco plants. Soil adhering to the tobacco plant roots was cleaned, and the root tissue samples were then immersed in physiological saline (0.85% NaCl) and sonicated until no obvious soil was visible. Pre-cooled deionized water (4°C) was added to an ultrasonic cleaner, and centrifuge tubes were placed in the water for two ultrasonic treatments, each lasting 20 seconds with a 5-second interval between treatments. Fine roots were removed from the centrifuge tubes using sterile forceps and discarded. 1 mL of the soil suspension was added to 9 mL of physiological saline, serially diluted 10-fold, and spread onto NA or other culture media. The media were incubated at 28°C for 48 hours. Based on growth characteristics such as colony color, size, elevation, transparency, hardness, and edge regularity, single colonies were picked and streaked three times on NA medium to obtain pure cultured strains.

[0070] 2) Identification of strain species

[0071] The isolated strain was streaked and cultured onto fresh NA medium, incubated at 28°C for 24 h, and then single colonies were picked for further purification and cultured on new plates. The gene sequence of the strain was determined using colony PCR. An appropriate amount of colony was placed in 50 μL of sterile water, microwaved for 5–7 min, centrifuged at 5000 rpm for 5 min, and the supernatant was used as the subsequent genomic DNA sample. Using the genomic DNA as the PCR template, the 16S rRNA gene of the strain was amplified using universal bacterial primers (27F / 1492R: 5'-AGAGTTTGATCCTGGCTCAG-3'; 5'-GGTTACCTTGTTACGACTT-3'). The PCR system is shown in Table 1. After agarose gel electrophoresis analysis of the PCR products, the fluorescently displayed fragments were subjected to gene sequencing analysis. The gene sequences obtained from sequencing were aligned on the bacterial data website EzBioCloud (http: / / www.ezbiocloud.net / ), and a phylogenetic tree was constructed using MEGA 11 software. The strain name was finally determined based on the sequence similarity and its position in the phylogenetic tree.

[0072] Table 1. PCR reaction system

[0073]

[0074] 3) Preservation of microbial strains

[0075] The purified strain was streaked onto NA plates and incubated at 28°C for 24 hours. The mature bacteria were then sealed and sent to the China General Microbiological Culture Collection Center (CGMCC), with the preservation number CGMCC No. 33664.

[0076] 4) Antibacterial ability test

[0077] Select a mature single colony and inoculate it into 10 mL of NB medium, then incubate at 28°C for 24 h. Observe that the bacterial suspension becomes significantly turbid, and adjust the OD of the bacterial suspension accordingly. 600 The value was 0.3. 10 μL of bacterial suspension was inoculated into two locations 1.5 cm from the center of PDA solid medium. After absorption, the medium was placed in an incubator at 28°C and inverted for 48 hours. The pathogenic fungus was then prepared into an OD value. 420The value was 0.3. The solution was evenly sprayed onto a plate containing antagonistic bacterial colonies in the center of the culture medium. The plate was then transferred to a 28°C fungal incubator and incubated upside down for 2-3 days. The size of the inhibition zone was measured, and the antibacterial ability of the strain was calculated. The inhibition rate was calculated using the formula: Inhibition rate (%) = (center to fungal edge - center to bacterial edge) / center to fungal edge × 100%.

[0078] 5) Determination of the inhibitory effect of volatile organic compounds on mold.

[0079] After applying activated antagonistic bacterial colonies using an inoculation loop, streak them evenly and densely onto a normal NA (NA) plate to ensure the bacteria cover the entire plate. Separately, inoculate a pathogenic bacterial block (Φ = 8 mm) into the center of a PDA (Pulse Diameter A) plate. Incubate both plates upside down at 28°C in the dark, using a blank NA plate as a control. Observe and measure the colony diameter on both the treated and control plates at 2-day intervals, continuing until the control plate is fully colonized. Each treatment is repeated three times. The growth inhibition rate is calculated using the following formula to compare the antagonistic effects of volatiles: Inhibition rate (%) = (Control group colony diameter - Treated group colony diameter) / Control group colony diameter × 100%.

[0080] 6) Inhibitory effect of fermentation supernatant of the strain on pathogens

[0081] Mature single colonies were picked and inoculated into 10 mL of LB medium and incubated at 28°C for 24 h. Then, 1% colonies were inoculated into 30 mL of LB medium and incubated at 28°C. Samples were taken at 36 h and 72 h, centrifuged at 8000 rpm for 5 min, and the supernatant was filtered through a sterile membrane and added to 40-50°C PDA medium. After thorough mixing, the mixture was poured into Petri dishes. 8 mm diameter mold cakes were inoculated in the center of the medium and incubated at 28°C for 12 h. Colony growth was continuously observed.

[0082] 7) The effect of biocontrol bacteria on the growth and reproduction of molds

[0083] Take equal areas of dried tobacco leaves and place them in sealed bags. Pick mature single colonies and inoculate them into 10 mL of NB medium, incubating at 28°C for 24 hours. Adjust the OD ratio of the antagonistic bacterial suspension and the suspension of moldy fungal spores with sterile water. 600 The concentration was 0.3. Antagonistic bacteria: A suspension of mold-causing fungi was mixed at a ratio of 1:5, and 1 mL was sprayed onto the tobacco leaf samples. The samples were incubated at 28℃ for 4-6 days, and colony growth was observed to determine the antagonistic effect of the antagonistic strains on tobacco leaf mold. A control group was prepared by inoculating a suspension of mold-causing fungal spores.

[0084] 2. Experimental Results

[0085] 2.1 Identification of strain morphology and species

[0086] The antagonistic bacteria were inoculated onto NA medium and incubated at 28°C for 24 hours. Single colonies of the strain were milky white, opaque, and had a rough, dull surface. Figure 1 The full-length 16S rDNA sequence was obtained, totaling 1405 bp. Comparison with the EZBioCloud database revealed that Cas656 shared the highest similarity (99.57%) with *Pseudomonas mediterranea*. A phylogenetic tree was constructed using the 16S rDNA sequences of Cas656 and other standard strains of *Pseudomonas mediterranea*. Figure 2 ), and found that Cas656 and Pseudomonas thaliana clustered together, and finally identified Cas656 as Pseudomonas thaliana.

[0087] 2.2 Antibacterial ability of the strain

[0088] To determine the antibacterial activity of strain Cas656 against Aspergillus niger, Aspergillus flavus, Talaromyces verruculosus, and Talaromyces variantabilis, suspensions of the four fungi were adjusted to OD0.05. 420 The concentration was 0.3, and the mixture was evenly sprayed onto PDA medium containing antagonistic bacteria. After incubation at 28°C for 2-3 days, the inhibition zone was observed, and the inhibition rate could be calculated using the formula. This yielded the inhibition rate of strain Cas656 against Aspergillus niger. Figure 3 A) The left side represents the treatment group, and the right side represents the control group. The inhibition rate was 43.52%; against Aspergillus flavus (… Figure 3 B) The left side represents the treatment group, and the right side represents the control group. The inhibition rate was 35.20%; against *Trametes versicolor* (… Figure 3 C) The left side represents the treatment group, and the right side represents the control group. The inhibition rate was 29.85%; against filamentous fungi ( Figure 3 D) The left side represents the treatment group, and the right side represents the control group. The inhibition rate was 19.40%. Among them, strain Cas656 showed the most significant inhibitory effect on Aspergillus niger and Aspergillus flavus, while its inhibitory effect on Aspergillus variantans and filamentous fungi was relatively small, but a significant inhibition range could be observed.

[0089] 2.3 Determination of the inhibitory effect of volatile organic compounds on mold

[0090] Experiments revealed that volatile substances produced by the Cas656 strain also affect mold growth. To determine the specific antibacterial effect of these volatile substances, two interacting strains were sealed in the same space and cultured to observe mold growth. Figure 4The images show the growth of Aspergillus niger and its antagonistic bacteria in the culture medium at the same time. Group A shows the sample treatment process, with the treatment group on the left and the control group on the right; Group B shows the treatment group on the left and the control group on the right. It can be seen that the growth area in the treatment group is significantly smaller than that in the control group, and the calculated growth inhibition rate is 54.69%. Figure 5 The images show the growth of Aspergillus flavus and antagonistic bacteria in the culture medium at the same time. Group A shows the sample treatment process, with the treatment group on the left and the control group on the right; Group B shows the treatment group on the left and the control group on the right. It can be seen that the growth area in the treatment group is significantly smaller than that in the control group, and the calculated growth inhibition rate is 54.69%. Figure 6 The images show the growth of variant basket bacteria and antagonistic bacteria in the culture medium at the same time. Group A shows the sample treatment process, with the treatment group on the left and the control group on the right; Group B shows the treatment group on the left and the control group on the right. It can be seen that the colonies in the treatment group show almost no growth. Figure 7 The images show the growth of filamentous fungi and antagonistic bacteria in the culture medium at the same time point. Group A shows the sample treatment process, with the treatment group on the left and the control group on the right; Group B shows the treatment group on the left and the control group on the right. It can be seen that the treated group showed almost no growth. This indicates that strain Cas656 can produce volatile compounds and inhibits the growth of Aspergillus niger, Aspergillus flavus, Aspergillus mutans, and filamentous fungi.

[0091] 2.4 Inhibitory effect of fermentation supernatant of the strain on pathogens

[0092] The fermentation supernatant of Cas656 strain was added to PDA medium at 40-50℃ at concentrations of 2%, 5%, and 10%, respectively. After thorough mixing, the mixture was poured into Petri dishes. Mold cakes with a diameter of 8 mm were inoculated into the center of the medium, and the culture was incubated at 28℃ for 12 hours. Colony growth was then continuously observed. Figure 8 A represents the control group, B represents 2%, C represents 5%, and D represents 10% supernatant addition. From the colony growth area in the graph, it can be seen that the growth area of ​​*Aspergillus niger* in the culture medium with 10% supernatant addition is slightly smaller than that in the control group. Figure 9 A represents the control group, B represents 2%, C represents 5%, and D represents 10% supernatant addition. From the colony growth area in the graph, it can be seen that the Aspergillus flavus growth area in the culture medium with 10% supernatant addition is slightly smaller than that in the control group. Figure 10 A represents the control group, B represents 2%, C represents 5%, and D represents 10% supernatant addition. From the colony growth area in the graph, it can be seen that the growth area of ​​*Variegata* in the 5% supernatant addition culture medium is slightly smaller than that in the control group. Figure 11A represents the control group, B represents 2%, C represents 5%, and D represents 10% supernatant addition. The colony growth area in the graph shows that the growth area of ​​filamentous fungi in the 2% supernatant addition culture medium is slightly smaller than that in the control group. These experimental results indicate that within the same culture time, the colony growth area in the treatment group is slightly smaller than that in the control group, suggesting that the fermentation product of Cas656 strain has a relatively small inhibitory effect on the four types of moldy fungi.

[0093] 2.5 Effects of biocontrol bacteria on fungal growth and reproduction

[0094] After 6 days of cultivation following spraying the bacterial suspension, observation revealed that the area of ​​moldy fungi in the treated group was smaller than that in the control group. Figure 12 A. The left side represents the Aspergillus niger control group, and the right side represents the treatment group; Figure 12 B shows the aspergillus control group on the left and the treatment group on the right; Figure 12 C shows the control group (left side) and the treatment group (right side); Figure 12 D shows the control group (left) and the treatment group (right). It can be observed that the growth area of ​​moldy fungi in the treatment group was smaller than that in the control group, indicating that directly spraying the Cas656 bacterial suspension onto dried tobacco leaves and sealing it for cultivation can also inhibit mold growth.

[0095] Cas656 16S rRNA gene sequence

[0096]

[0097] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A strain of Pseudomonas meliticum ( Pseudomonas mediterranea Cas656, this strain was deposited at the China General Microbiological Culture Collection Center on February 26, 2025, with the biological accession number CGMCC No. 33664.

2. The fermentation production method of Pseudomonas thaliana Cas656 according to claim 1, characterized in that, The fermentation production method includes: inoculating the Mediterranean Pseudomonas Cas656 into a fermentation medium for fermentation culture.

3. A microbial inoculant, characterized in that, It contains the Mediterranean Pseudomonas Cas656 as described in claim 1.

4. The microbial agent as described in claim 3, characterized in that, The microbial agent also includes excipients acceptable to the agent.

5. The use of the Mediterranean Pseudomonas Cas656 of claim 1 and / or the microbial agent of any one of claims 3-4 in the prevention and control of tobacco mold.

6. A method for preventing tobacco leaf mold and rot, characterized in that, The method includes applying the Mediterranean Pseudomonas Cas656 of claim 1 or the microbial agent of any one of claims 3-4 to tobacco leaves.

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