Alcaligenes faecalis AF12 as well as fungicide and application thereof

Abstract

The invention relates to alcaligenes faecalis AF12 as well as a fungicide and application thereof, and belongs to the technical field of microorganisms. The alcaligenes faecalis AF12 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.37190, the preservation date is December 25, 2025, and the address of the preservation institution is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The invention further provides application of the alcaligenes faecalis AF12 in inhibition of harmful bacteria in soil, prevention and treatment of root-knot nematode and promotion of plant growth. The alcaligenes faecalis AF12 provided by the invention can be applied to any plant species or plant habitat, realizes multiple effects of plant growth promotion, prevention and control of plant fungal diseases and bacterial diseases and prevention and control of plant root-knot nematode, and has extremely high application value and popularization prospect in the field of plant cultivation.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Alcaligenes faecalis AF12, its inoculum, and its applications. Background Technology

[0002] Vegetables, as an important economic crop, are cultivated at a high degree of intensification, resulting in severe and frequent outbreaks of pests and diseases. This not only causes enormous economic losses but also leads to a series of problems, including soil microecological imbalance, increased pesticide resistance, environmental pollution, and food safety risks, seriously hindering the sustainable development of high-efficiency agriculture. To effectively control pests and diseases, professionals from various fields have implemented a series of effective measures, including agricultural control, physical control, chemical pesticide control, and biological control. However, with the increasing intensification of vegetable cultivation, pest and disease control remains a serious challenge, urgently requiring efficient and stable control technologies and related products.

[0003] Microbial control, as a form of biological control, can maintain soil ecological balance and sustainable development. It is characterized by being green, environmentally friendly, inexpensive, and highly efficient, making it a current hot topic in pest and disease control. Currently, a large number of functional microorganisms have been isolated and identified, and some microbial products have been registered as microbial pesticides or microbial fertilizers, with Bacillus and Trichoderma accounting for a significant proportion. These microorganisms inhibit and kill pathogenic microorganisms through antimicrobial, competitive, and parasitic mechanisms, while also promoting crop growth and enhancing crop immunity.

[0004] Alcaligenes faecalis ( Alcaligenes faecalis Alcaligenes are widely found in soil, and related studies have shown that these strains possess certain functions such as promoting plant growth, antagonizing pathogens, degrading organic matter, tolerating heavy metals, and desulfurization. However, most of these strains have limited functions. For example, patent CN121286904A discloses a strain of Alcaligenes faecalis A18, which only controls root rot of Schisandra chinensis caused by Fusarium. Patent CN120866120A discloses a strain of Alcaligenes faecalis X4, which can only efficiently degrade flumorph and tolerate cadmium. The application of Alcaligenes faecalis in controlling cucumber target spot disease has not been disclosed. Summary of the Invention

[0005] To address the issue that existing biological pesticides have not yet found an application of Alcaligenes faecalis in controlling cucumber target spot disease, this invention provides a strain of Alcaligenes faecalis AF12, its inoculant, and its application to solve the aforementioned problem.

[0006] In a first aspect, the present invention provides a strain of Alcaligenes faecalis AF12, wherein the Alcaligenes faecalis ( Alcaligenes faecalisAF12 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37190, on December 25, 2025. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] Furthermore, the 16S rDNA sequence of the *Alcaligenes faecalis* AF12 is shown in SEQ ID NO.3.

[0008] Secondly, the present invention provides a bacterial agent containing Alcaligenes faecalis AF12, wherein the bacterial agent is a liquid bacterial agent.

[0009] A method for preparing the above-mentioned liquid bacterial agent, wherein the preparation method of the liquid bacterial agent is as follows: Alcaligenes faecalis AF12 is inoculated into a test tube containing LB liquid medium, grown overnight at 30°C and 180 rpm, and then inoculated into TSB liquid medium at a 1% inoculation rate, and cultured at 30°C and 180 rpm with shaking for 48 hours to obtain a bacterial suspension containing Alcaligenes faecalis AF12; the concentration of Alcaligenes faecalis AF12 in the bacterial suspension is adjusted to 1×10⁻⁶. 7 ~1×10 8 A liquid bacterial agent of Alcaligenes faecalis AF12 was prepared by using CFU / mL.

[0010] Preferably, the LB liquid culture medium comprises the following components: 10g tryptone, 5g yeast extract, 10g NaCl, and sterile water to make up to 1L, with a pH of 7.0~7.2.

[0011] Preferably, the TSB liquid culture medium comprises the following components: 17g casein digested with trypsin, 3g soy protein, 2.5g glucose, 5g NaCl, 2.5g K2HPO4, and sterile water to make up to 1L; pH=7.3.

[0012] Thirdly, the present invention provides an application of Alcaligenes faecalis AF12 in promoting cucumber growth.

[0013] Furthermore, the promotion of cucumber growth refers to promoting the growth and development of the root system of cucumber seedlings.

[0014] Fourthly, the present invention provides the application of Alcaligenes faecalis AF12 in the prevention and control of Alternaria alternata, Cladosporium multiflorum or Anthrax sicca.

[0015] Fifthly, the present invention provides the application of Alcaligenes faecalis AF12 in inhibiting root-knot nematodes.

[0016] The beneficial effects of this invention are as follows: The *Alcaligenes faecalis* AF12 provided by this invention can be applied to any plant species or plant habitat, achieving multiple effects such as promoting plant growth, controlling fungal and bacterial diseases, and controlling root-knot nematodes. Addressing the lack of existing reports on strains that simultaneously promote plant growth, control diseases, and manage root-knot nematodes, the *Alcaligenes faecalis* AF12 of this invention significantly promotes plant growth and effectively inhibits the growth of *Alternaria alternata* and *Alternaria spp.*, the pathogens of cucumber target spot disease, as well as the growth of root-knot nematodes. It also significantly inhibits the growth and pathogenicity of *Alternaria alternata* within cucumber plants. Furthermore, indoor pot experiments have verified that *Alcaligenes faecalis* AF12 exhibits excellent control effects against both cucumber target spot disease and root-knot nematodes, making it highly valuable and promising for application in the field of plant cultivation. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a colony morphology diagram of Alcaligenes faecalis AF12 in Example 1 of the present invention.

[0019] Figure 2 This is a morphological image of Alcaligenes faecalis AF12 under an optical microscope in Example 1 of the present invention.

[0020] Figure 3 This is a phylogenetic tree diagram of the AF12 strain constructed based on the proximity method in this invention.

[0021] Figure 4 This is a detection chart of growth-promoting indicators of Alcaligenes faecalis AF12 in Example 2 of the present invention, wherein (a) is the detection result chart of nitrogen fixation plate, (b) is the detection result chart of IAA production, and (c) is the detection result chart of motility.

[0022] Figure 5 This is a graph showing the detection results of biofilm formation of Alcaligenes faecalis AF12 in three different culture media in Example 2 of the present invention.

[0023] Figure 6 This is a graph showing the antagonistic activity of Alcaligenes faecalis AF12 against four pathogens in Example 3 of the present invention. (a) is a plate confrontation graph, and (b) is a bar graph comparing the inhibition rates of Alcaligenes faecalis AF12 against the mycelial growth of the four pathogens. Different lowercase letters above the bars indicate significant differences at the 0.05 level.

[0024] Figure 7This is a comparison diagram of the growth promotion of cucumber seedlings in Example 5 of the present invention.

[0025] Figure 8 This is a comparison of the above-ground parts of cucumber in the experiment of controlling cucumber root-knot nematodes with Alcaligenes faecalis AF12 in Example 6 of the present invention. Detailed Implementation

[0026] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0027] The components of the various culture media used in the embodiments of this invention are as follows: LB solid medium: tryptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L, agar powder 15 g / L, pH=7.0~7.2.

[0028] LB liquid medium: 10g tryptone, 5g yeast extract, 10g NaCl, sterile water to make up to 1L, pH=7.0~7.2.

[0029] TSB liquid culture medium: trypsin digested casein 17g, soy protein 3g, glucose 2.5g, NaCl 5g, K2HPO4 2.5g, sterile water to make up to 1L; pH=7.3.

[0030] MKB medium: 5g casein amino acids, 2.5g K2HPO4, 2.5g MgSO4·7H2O, 15mL glycerol, and sterile water to make up to 1L.

[0031] Example 1 Isolation, screening and identification of Alcaligenes faecalis AF12 (1) Sampling: On April 27, 2025, rhizosphere soil was collected from a greenhouse in Qudi Village, Jiyang District, Jinan City, Shandong Province, where cucumbers are grown, and stored in a self-sealing bag for later use.

[0032] (2) Separation: Take the rhizosphere soil collected in step (1), and perform a 10-fold serial dilution with physiological saline. Select a dilution of 10. -2 10 -3 10 -4Soil suspensions of 100–200 µL were spread onto LB agar plates. The plates were incubated upside down at 30 °C for 24 h, and colony growth was observed. Colonies of different morphologies and colors were selected for streak plating purification to obtain pure cultures. The purified strains were then tested for growth-promoting indicators and antagonistic properties. The strain exhibiting strong antagonism and growth-promoting characteristics was designated AF12.

[0033] (3) Morphological identification: Stable colonies of strain AF12 grown to the logarithmic phase were described by single-colony characteristics, mainly including colony size, color, edge regularity, and transparency. The results showed that after culturing strain AF12 on LB solid medium at 30℃ for 24 h, the colonies were pale yellow with irregular edges, a smooth surface, and were opaque. See the detailed colony morphology diagram below. Figure 1 The bacteria are short rod-shaped, measuring 0.5–0.9 × 0.7–3.2 µm. See the image for detailed morphological images of the bacteria under an optical microscope. Figure 2 .

[0034] Molecular identification: 16S rDNA gene amplification was performed using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3' SEQ ID NO.1) and 1492R (5'-CGGTTACCTTGTTACGACTT-3' SEQ ID NO.2). The PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The 16S rDNA sequence of strain AF12 is shown in SEQ ID NO.3. The sequenced sequences were aligned with NCBI, and homology sequences were downloaded. A phylogenetic tree was constructed using the nearest neighbor method. The phylogenetic tree of strain AF12 is shown below. Figure 3 As shown.

[0035] The sequences obtained from sequencing were submitted to GenBank for homology comparison. Strains AF12 and... Alcaligenes faecalis strain AFS055854 With 100.00% homology, the strain was preliminarily identified as *Alcaligenes faecalis*. Alcaligenes faecalis Alcaligenes faecalis AF12 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37190, on December 25, 2025. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0036] Example 2 Detection of growth-promoting characteristics of Alcaligenes faecalis AF12 (1) Nitrogen fixation Alcaligenes faecalis AF12 was streaked onto Ashby solid medium and incubated at 30°C for 4 days. The growth of the strain was observed. If it could grow normally on the plate, it indicated that it had nitrogen-fixing ability.

[0037] Ashby medium consists of the following components: mannitol 10.0 g / L, KH2PO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, CaCl2 0.1 g / L, CaCO3 5.0 g / L, and agar 15.0 g / L.

[0038] Four days later, AF12 was observed to grow on Ashby plates, as shown in the following figures. Figure 4 As shown in (a), it has a certain nitrogen-fixing ability.

[0039] (2) IAA (indoleacetic acid) production capacity test Take a single colony of Alcaligenes faecalis AF12 and place it in a test tube containing LB liquid medium. Incubate overnight at 30°C with shaking at 180 rpm. Take a small amount to adjust the bacterial concentration to OD. 600 =1. Inoculate 1% of the culture medium into 10 mL of King-B liquid medium and incubate at 30°C with shaking at 180 rpm for 120 h. Take 1 mL samples at 48 h, 72 h, 96 h, and 120 h of culture, centrifuge at 8000 rpm for 5 min, and transfer 50 µL of the supernatant to a 96-well plate. Add 2 µL of 83% orthophosphate and 100 µL of Salkowski reagent. Incubate at room temperature in the dark for 30 min, and observe. A pink color change indicates a positive result. If the solution turns pink, measure the absorbance at 530 nm using a microplate reader to determine the IAA concentration produced by strain AF12 at different time points.

[0040] The King-B medium consists of the following components: peptone 20 g / L, K2HPO4 1.5 g / L, magnesium sulfate heptahydrate 1.5 g / L, L-tryptophan 0.5 g / L, glycerol 15 mL / L, pH=7.2.

[0041] The test results showed that the solution turned pink after color development, such as... Figure 4 As shown in (b), Alcaligenes faecalis AF12 produces IAA. The concentration of IAA produced at different culture times is detailed in Table 1. Table 1 - IAA Concentrations (mg / L) Produced by Alcaligenes faecalis AF12

[0042] As can be seen from the results in Table 1, IAA accumulated in the culture medium over time, reaching 12.07 mg / L after 5 days.

[0043] (3) Exercise Prepare a culture medium for both modes of bacterial motility: Swimming medium: 1% tryptone, 0.5% sodium chloride, 0.3% agar.

[0044] Swarming medium: 1% tryptone, 0.5% yeast extract, 1.0% sodium chloride, 0.4% glucose, 0.45% agar.

[0045] Methods: After sterilization at 121℃ for 20 minutes, plates were laid out, and Alcaligenes faecalis AF12 was inoculated in the center of the plates. The plates were incubated at 30℃ for 5 days, and the motility and movement patterns of Alcaligenes faecalis AF12 were observed.

[0046] For detailed results, please see [link to results]. Figure 4 (C) The motility of Alcaligenes faecalis AF12 differed significantly between the two culture media. On swimming medium, it covered the plate in 5 days, and the motility was significantly faster than on swarming medium, indicating that the motility of the bacteria was mainly characterized by individual bacterial movement.

[0047] (4) Biofilm formation Preparation of bacterial culture: Inoculate a single colony of Alcaligenes faecalis AF12 into a test tube containing LB liquid medium. Incubate overnight at 30°C and 180 rpm. Centrifuge 1 mL of the culture at 8000 rpm for 5 minutes, discard the supernatant, resuspend the bacterial cells in an equal volume of sterile water, and then dilute 10-fold with sterile water. Measure the OD of the diluted bacterial culture. 600 Adjust the bacterial solution concentration to OD0.05 600 =0.2, reserved.

[0048] Take a sterile 24-well plate, add 1 mL of TSB liquid medium to 3 wells, 1 mL of MKB liquid medium to 3 wells, and 1 mL of LB liquid medium to 3 wells. Inoculate 10 µL of the prepared bacterial culture into each of the above 9 wells. The wells without bacterial culture, containing only the three different media, serve as controls. Place the plate in an incubator and incubate statically at 37°C for 72 h. Remove the 24-well plate, aspirate the bacterial culture, wash three times with sterile water, stain with 0.1% crystal violet for 20 min, aspirate the crystal violet, wash three more times with sterile water, dissolve thoroughly in 95% ethanol for at least 30 min, and measure the A590 absorbance of each well using a microplate reader.

[0049] After the culture was completed, the A590 absorbance values ​​in each culture medium were compared as follows: Figure 5 As shown in the figure, *Alcaligenes faecalis* AF12 showed the best growth in TSB liquid medium, with obvious biofilm formation on the surface of the medium in each well, and the highest A590 absorbance (4.226) among TSB liquid media. It exhibited the worst biofilm formation in LB liquid medium, with an A590 absorbance of 1.589.

[0050] Example 3 Antibacterial experiment of Alcaligenes faecalis AF12 The inhibitory effect of Alcaligenes faecalis AF12 on the mycelial growth of pathogenic bacteria was determined using the confrontation culture method. The determination method is as follows: Experimental group: A pathogenic bacterial cake with a diameter of 5 mm was inoculated in the center of a PDA plate culture medium. Alcaligenes faecalis AF12 was inoculated on both sides of the bacterial cake and 2 cm away from the bacterial cake. The plate was incubated at 28℃ for 6 days, with three replicates.

[0051] Control group: The only difference between the control group and the experimental group was that the control group was not inoculated with Alcaligenes faecalis AF12.

[0052] After the constant temperature incubation was completed, the colony radius of the pathogen in the experimental group and the control group was measured respectively, and the inhibition rate of Alcaligenes faecalis AF12 on the mycelial growth of the pathogen was calculated. The calculation formula is as follows: .

[0053] Mycelial cakes containing the pathogenic bacteria *Corynebacterium multiflorum* (Cc), *Alternaria alternata* (Aa), *Fusarium oxysporum* (FOC), and *Anthrax sicca* (CM9) were prepared and the experiment was repeated using the method described above. The results are as follows: Figure 6 As shown in (a) and (b).

[0054] The test results show that at 6 days, *Alcaligenes faecalis* AF12 has a certain inhibitory effect on the mycelial growth of the four pathogenic fungi mentioned above. Among the four pathogenic fungi tested, *Alcaligenes faecalis* AF12 showed the highest inhibition rate against *Alternaria alternata*, the pathogen of cucumber target spot disease, reaching 67.5%. Its inhibition rates against *Clostridium multiflorum* and *Anthracnose sicca* were comparable, at 62.86% and 61.04% respectively, while its inhibition rate against *Fusarium oxysporum* was the lowest, at only 33.96%.

[0055] Example 4 The toxic effect of Alcaligenes faecalis AF12 on southern root-knot nematodes (J2) Infected cucumber plants were collected from severely affected areas infested with root-knot nematodes. After rinsing with running water to remove surface dirt, the egg sacs were peeled off from the roots using tweezers. The egg sacs were then surface-sterilized in a 0.5% sodium hypochlorite solution for 90 seconds, followed by rinsing with sterile water 3–5 times. The sterilized egg sacs were transferred to 12-well cell culture plates, with 2 mL of sterile water added to each well, and incubated at 28°C in the dark. Newly hatched second-instar larvae (J2) were collected using sterile pipettes for subsequent virulence assays.

[0056] Preparation of fermentation filtrate of Alcaligenes faecalis AF12: First, a single colony was picked from LB solid medium and inoculated into 100 mL of TSB liquid medium. The culture was then incubated at 30°C and 180 rpm with shaking for 48 hours. After incubation, the bacterial culture was centrifuged at 8000 rpm for 5 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane for sterilization. This yielded the sterile fermentation filtrate of Alcaligenes faecalis AF12, which was diluted to 5× and 10× with sterile LB liquid medium for use in the J2 nematode toxicity test.

[0057] 400 μL of sterile fermentation filtrate of Alcaligenes faecalis AF12 at different dilutions and 100 μL of suspension containing approximately 50 J2 larvae were added to 24-well plates. Six biological replicates were set up for each treatment, and negative controls (sterile water) and culture medium controls (TSB liquid medium) were set up simultaneously. The culture plates were placed at 28°C in the dark and the viability of nematodes was observed under a stereomicroscope after 12 hours and 24 hours. The criteria for determining nematode mortality were as follows: (1) The nematode was considered dead if it was stiff and immobile; (2) If the nematode was not stiff and did not respond after repeated touching with a fine needle, it was considered dead; (3) If the nematode was stiff or slightly stiff and did not move after stimulation with 1% (w / v) NaOH solution, it was considered dead. The formula for calculating the corrected mortality rate is as follows: Corrected mortality rate (%) = [(treatment group mortality rate - control group mortality rate) ÷ (1 - control group mortality rate)] × 100%.

[0058] The mortality rate of Alcaligenes faecalis AF12 against Southern Root-knot Nematode (J2) is shown in Table 2.

[0059] Table 2 - Lethality of Alcaligenes faecalis AF12 against Southern Root-knot Nematode (J2)

[0060] Note: Different lowercase letters in the table represent significant differences at the P<0.05 level.

[0061] As shown in Table 2, the toxicity of different concentrations of Alcaligenes faecalis AF12 fermentation filtrate against Southern Root-knot Nematode (J2) increased with increasing concentration. One hour after treatment, the corrected mortality rate of the sterile fermentation stock was 83.64%, reaching 93.27% after 24 hours, significantly higher than the 5× and 10× dilution gradients. The corrected mortality rate of the TSB control group was only 3.21% after 24 hours, indicating that the toxicity of Alcaligenes faecalis AF12 fermentation broth against Southern Root-knot Nematode (J2) originates from metabolites produced during the fermentation process rather than from interference from the culture medium.

[0062] Example 5 Growth-promoting experiment of Alcaligenes faecalis AF12 on cucumber (1) Cucumber seed germination test Preparation of Alcaligenes faecalis AF12 liquid inoculum: Single colonies of Alcaligenes faecalis AF12 were inoculated into test tubes containing LB liquid medium and grown overnight at 30°C and 180 rpm. Then, a 1% inoculum was transferred to TSB liquid medium and cultured at 30°C and 180 rpm with shaking for 48 hours. The bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL was used to obtain Alcaligenes fecal microbial agent AF12.

[0063] Select healthy, plump cucumber seeds of uniform size. First, surface disinfect them with 75% (volume fraction) alcohol for 30 seconds, then with 5% NaClO for 3 minutes. Wash them six times with sterile water and air-dry the surface moisture. Soak the seeds in Alcaligenes faecalis AF12 liquid inoculant for 20 minutes, air-dry them, and then place 20 seeds per petri dish containing sterile, moistened filter paper. Seeds soaked in an equal volume of sterile water and TSB liquid medium served as the control group. Each treatment was replicated four times. The seeds were placed at 28℃ in the dark. Germination was defined as the length of the sprouting white root ≥ 1 / 2 of the seed length. After 5 days, the germination rate and the number of lateral roots were counted, and the root length was measured. The results after 5 days are shown in Table 3.

[0064] Table 3 - Effects of Alcaligenes faecalis AF12 on cucumber seed germination

[0065] Table 3 shows that *Alcaligenes faecalis* AF12 significantly promoted the length of the coxspinous root and the number of lateral roots in cucumber seeds. Compared with the sterile water control group, the *Alcaligenes faecalis* AF12 treatment group showed a significant increase in coxspinous root length and lateral root number, by 33.01% and 31.54%, respectively. Compared with the TSB liquid culture medium control group, the *Alcaligenes faecalis* AF12 treatment group showed a significant increase in coxspinous root length and lateral root number, by 41.15% and 26.84%, respectively. However, there was no significant difference in germination rate among the groups. These results indicate that *Alcaligenes faecalis* AF12 has no effect on cucumber seed germination but promotes coxspinous root growth and lateral root number.

[0066] (2) Cucumber seedling growth promotion experiment Select healthy seeds of uniform size, disinfect them with 5% NaClO for 3 minutes, wash them 5-6 times with sterile water, and then place them in petri dishes containing sterile moistened filter paper and germinate at 28℃. Sow one seed per pot (8.5cm×8cm×10cm) in sterile soil (substrate:garden soil volume ratio 1:1) when all seeds have germinated. When the first true leaf appears, select seedlings of uniform growth for treatment. Three treatment groups were established: control group: watered with 10mL of water per pot; TSB culture medium group: watered with 10mL of TSB culture medium per pot; bacterial solution group: watered with Alcaligenes faecalis AF12 liquid bacterial agent (concentration 1×10⁻⁶). 8 10 mL of CFU / mL solution was applied per pot. Five pots were treated, with three replicates, and placed in an artificial climate chamber (28°C under light / 25°C in darkness, 75% relative humidity, 16 hours of light). One month later, the plant height, root length, plant fresh weight, and root fresh weight of the cucumber seedlings were measured.

[0067] After the experiment, the growth of cucumber seedlings in each treatment group was as follows: Figure 7As shown, plants treated with Alcaligenes faecalis AF12 liquid inoculant grew vigorously, significantly better than the control group (1#, irrigated with plain water). The statistical results of each indicator are shown in Table 4.

[0068] Table 4 - Results of the growth-promoting experiment of Alcaligenes faecalis AF12 on cucumber seedlings

[0069] Note: Different lowercase letters in the table represent significant differences at the P < 0.05 level.

[0070] As shown in Table 4, compared with the control group, the cucumber plants treated with the bacterial solution showed significant differences in plant height, root length, plant fresh weight, and root fresh weight, increasing by 23.91%, 18.91%, 17.56%, and 41.79%, respectively. Compared with the TSB medium group, the bacterial solution group only showed significant differences in root length and root fresh weight. This indicates that *Alcaligenes faecalis* AF12 primarily promotes root growth and development, enabling plants to better absorb water and nutrients, thereby promoting the growth of the above-ground parts.

[0071] Example 6 Pot experiment on the control of cucumber target spot disease and root-knot nematode disease by Alcaligenes faecalis AF12 (1) Control of cucumber target spot disease Preparation of spore suspension of the pathogen *Cyclocarya polycarpa*: Spores of the pathogen *Cyclocarya polycarpa* were inoculated onto PDA plates and incubated in the dark at 28°C for 10 days. The spores were scraped off with sterile water and passed through four layers of sterile lens paper to obtain a spore suspension. The concentration of the spore suspension was adjusted to 1×10⁻⁶. 5 CFU / mL, ready for use.

[0072] Cucumber seeds were disinfected and sown in seed trays. The experiment was conducted when the seeds reached the two-leaf-one-heart stage. Three treatment groups were established: a blank control (no treatment); a pathogen control group (10 mL of pathogen spore suspension was sprayed first, followed by 10 mL of water after 24 hours); a chemical treatment group (10 mL of pathogen spore suspension was sprayed first, followed by 10 mL of a 1000-fold diluted solution of 75% chlorothalonil (wettable powder); and an AF12 treatment group (10 mL of pathogen spore suspension was sprayed first, followed by the Alcaligenes faecalis AF12 liquid inoculant prepared in Example 5 (concentration adjusted to 1×10⁻⁶). 7 10 mL of 10 mL (CFU / mL) was used. Each treatment was replicated three times, with 18 cucumber seedlings per replicate. Treatments were separated using isolation hoods to avoid cross-contamination. The seedlings were placed in an artificial climate chamber (32°C in light / 28°C in darkness, 80% relative humidity, 16-hour light cycle). After 7 days, the disease incidence was assessed in each treatment, and the disease index and control efficacy were calculated. The formula for calculating the disease index is as follows: Disease severity grading standards: Grade 0: No lesions; Grade 1: Lesions cover less than 5% of the total leaf area; Grade 3: Lesions cover 5% to 25% of the total leaf area; Grade 5: Lesions cover 26% to 50% of the total leaf area; Grade 7: Lesions cover 51% to 75% of the total leaf area; Grade 9: Lesions cover more than 75% of the total leaf area.

[0073] Disease index = [∑(number of diseased leaves at each level × grade value of each disease level) ÷ (total number of leaves surveyed × 9)] × 100; The formula for calculating the prevention and control effect is as follows: Prevention and treatment effect (%) = [(disease index of control group)] [Disease index of the treatment group ÷ disease index of the control group] × 100.

[0074] The control effects of different treatment groups on cucumber target spot disease are shown in Table 5 below.

[0075] Table 5 - Control effects of different treatment groups on cucumber target spot disease

[0076] As shown in Table 5, the control effect of spraying Alcaligenes faecalis AF12 liquid inoculant reached 62.67%, which is comparable to the control effect of spraying pesticides. This indicates that Alcaligenes faecalis AF12 has a good control effect on cucumber leaf target spot disease caused by Corynebacterium multiflorum.

[0077] (2) Experiment on the control of cucumber root-knot nematode disease Select cucumber seedlings with two leaves and one bud and uniform growth, and transplant them into flower pots (16cm×12cm×20cm), with 3-4 seedlings per pot. Drip 1mL of root-knot nematode egg suspension (preparation method is the same as in Example 4) around the roots of the plants to ensure that each cucumber seedling is inoculated with 1000 nematode eggs.

[0078] 72 hours after cucumber seedlings were inoculated with nematode eggs, the following three treatments were tested: Control group: 40 mL of water per pot; Pesticide group: 40 mL of 500-fold diluted thiazophos solution per pot; Inoculum group: 1 × 10⁻⁶ calcipophyllum AF12 liquid inoculum per pot. 8 40 mL (CFU / mL). Five pots per treatment, three replicates. Placed in an artificial air chamber (28℃ light / 25℃ darkness, 70% relative humidity, 14-hour light / dark cycle), all treatments were managed at the same level. After one month of cultivation, a disease survey was conducted, and the disease index and control effect were calculated. The disease index was graded according to the number of root knots and the degree of root damage using the following criteria: Level 0: No root knots; Level 1: Root knots are present in more than 0% and less than or equal to 20% of the root system; Level 3: Root knots are present in more than 20% and less than or equal to 40% of the root system; Level 5: Root knots are present in more than 40% and less than or equal to 60% of the root system; Level 7: Root knots are present in more than 60% and less than or equal to 80% of the root system; Level 9: Root knots are present in more than 80% of the root system.

[0079] Disease index = [∑(cucumber trees at each level × disease level value) ÷ (total number of cucumber trees surveyed × 9)] × 100; Prevention and treatment effect (%) = [(Control group disease index)] [Treatment group disease index ÷ Control group disease index] × 100.

[0080] For a detailed comparison of the aboveground growth of cucumber seedlings treated with different methods after one month of cultivation, please see the following images. Figure 8 The control effects of different treatment groups on cucumber root-knot nematode disease are shown in Table 6 below.

[0081] Table 6 - Control effects of different treatment groups on cucumber root-knot nematode disease

[0082] Note: Different lowercase letters in the table represent significant differences at the P < 0.05 level.

[0083] Depend on Figure 8 It can be seen that one month after inoculation with root-knot nematode eggs, the growth of cucumbers in the control group was inhibited, and some plants wilted and died, while the growth of plants in the pesticide group and the bacterial solution group was not affected. Table 6 shows the disease survey results: the disease index of the control group reached 64.47, while the disease indices of the pesticide group and the bacterial solution group were 23.52 and 25.63, respectively, with no significant difference between the two groups. The control efficiencies were 63.52% and 60.25%, respectively.

[0084] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A strain of Alcaligenes faecalis AF12, characterized in that, The alkalobacterium faecalis ( Alcaligenes faecalis AF12 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37190, on December 25, 2025. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The *Alcaligenes faecalis* AF12 as described in claim 1, characterized in that, The 16S rDNA sequence of the *Alcaligenes faecalis* AF12 is shown in SEQ ID NO.

3.

3. A bacterial agent containing *Alcaligenes faecalis* AF12 as described in claim 1, characterized in that, The bacterial agent is a liquid bacterial agent.

4. The microbial agent as described in claim 3, characterized in that, The preparation method of the liquid bacterial agent is as follows: Alcaligenes faecalis AF12 is inoculated into a test tube containing LB liquid medium and grown overnight at 30°C and 180 rpm. Then, it is inoculated into TSB liquid medium at a 1% inoculation rate and cultured at 30°C and 180 rpm with shaking for 48 hours to obtain a bacterial suspension containing Alcaligenes faecalis AF12. The concentration of Alcaligenes faecalis AF12 in the bacterial suspension is adjusted to 1 × 10⁻⁶. 7 ~1×10 8 A liquid bacterial agent of Alcaligenes faecalis AF12 was prepared by using CFU / mL.

5. The microbial agent as described in claim 4, characterized in that, The LB liquid culture medium comprises the following components: 10g tryptone, 5g yeast extract, 10g NaCl, and sterile water to make up to 1L, with a pH of 7.0~7.

2.

6. The microbial agent as described in claim 4, characterized in that, The TSB liquid culture medium comprises the following components: 17g casein digested with trypsin, 3g soy protein, 2.5g glucose, 5g NaCl, 2.5g K2HPO4, and sterile water to make up to 1L; pH=7.

3.

7. The application of Alcaligenes faecalis AF12 as described in claim 1 in promoting cucumber growth.

8. The application as described in claim 7, characterized in that, The promotion of cucumber growth refers to promoting the growth and development of the root system of cucumber seedlings.

9. The use of Alcaligenes faecalis AF12 as described in claim 1 in the prevention and control of Alternaria alternata, Cladosporium multiflorum or Anthrax sicca.

10. The application of Alcaligenes faecalis AF12 as described in claim 1 in the inhibition of root-knot nematodes.

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