Salt-alkali-resistant bacillus paralicheniformis hmf14 and application thereof

Abstract

The present application relates to a kind of parabacillus (Bacillus paralicheniformis) Bacillus paralicheniformis ) HMF14, the preservation number is CGMCC No.30480.The parabacillus HMF14 of the present application can resist salt and alkali, can simultaneously antagonize fusarium oxysporum, microdochium nivale var. avenae and xanthomonas campestris pv. tridii, also has toxic effect to pratylenchus penetrans and tetranychus cinnabarinus, can effectively prevent and treat wheat take-all disease and wheat black foot disease.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, specifically to Bacillus paralicheniformis HMF14 and its applications. Background Technology

[0002] As an important food crop widely grown globally, wheat has always faced challenges in achieving high yields and quality due to disease problems throughout its planting and growth cycle.

[0003] Take-all disease of wheat, also known as wheat damping-off or black foot disease, is mainly caused by a variety of wheat with hulled tops ( Gaeumannomyces graminis var. tritici Take-all disease is a soil-borne fungal disease caused by wheat. It mainly affects the roots and stem base of wheat, causing blackening and rotting of the root and stem base tissues. This severely hinders the wheat's absorption of water and nutrients, ultimately leading to plant wilting and death, resulting in "white ears." In affected areas, yields are reduced by 10%–70%, or even total crop failure. The occurrence of wheat take-all disease is closely related to the soil environment, making its control difficult. Current control methods include using 250–300 mL of 12% silthiophanate-methyl seed treatment suspension or 150–200 mL of 60 g / L tebuconazole suspension seed dressing agent, adding an appropriate amount of water, mixing 100 kg of wheat seeds, drying the seeds, and then sowing; or using 0.2% of the seed weight of 2% tebuconazole seed dressing before sowing.

[0004] Wheat black rot is caused by Xanthomonas aureus wheat-specific strain (Xanthomonas cucumeroides). Xanthomonas Campestris pv. translucens f. sp. unclulosa This disease is caused by a wheat-specific pathogen. It primarily affects the ear, but can also infect the stem and leaves. In high humidity, yellow bacterial ooze develops on the affected areas. Wheat black glume is mainly distributed in parts of Northeast, North, and Northwest China. Infected wheat develops streaks on the leaves, and severe cases can result in shriveled grains, significantly impacting wheat yield and quality. According to reports from various regions, the incidence of wheat black glume has been increasing year by year in recent years, with mild cases resulting in yield reductions of about 10% and severe cases exceeding 30%. Current control methods include a variable-temperature seed soaking method: soaking the seeds at 28-32℃ for 4 hours, followed by soaking in 53℃ water for 7 minutes. Alternatively, treat seeds with 0.2% of their weight of 40% seed dressing solution; or soak seeds in 3000 mg / kg of 15% chlorothalonil suspension for 12 hours, then spread them out to dry before sowing; or spray with chlorothalonil or agricultural streptomycin sulfate during the wheat heading stage, which is the early stage of disease, once every 7-10 days, for 2-3 consecutive times. These are the more effective chemical control methods.

[0005] Currently reported control methods are mostly based on chemical agents. The application of microbial inoculants generally yields limited effectiveness and fails to meet the requirements of safe, high-quality, high-yield, and effective prevention of multiple diseases. Summary of the Invention

[0006] The purpose of this invention is to provide a Bacillus paralicheniformis HMF14 that can effectively antagonize wheat variant of wheat husk and wheat-specific Xanthomonas aureus, the causal agent of black rot in cabbage, and its applications.

[0007] The present invention adopts the following technical solution:

[0008] A type of Bacillus paralicheniformis ( Bacillus paralicheniformis HMF14 was deposited on April 30, 2024, at the China General Microbiological Culture Collection Center, Beijing, China, with accession number CGMCC No. 30480.

[0009] Furthermore, the *Bacillus paralichrysiformis* HMF14 is resistant to salt and alkali.

[0010] Furthermore, the *Bacillus paralicheniformis* HMF14 can antagonize *Fusarium oxysporum*, *Triticum aestivum* variant, and / or *Xanthomonas aureum* wheat-specific variant.

[0011] Furthermore, the *Bacillus paralichrysogenum* HMF14 has a toxic effect on *Aeromonas nigra*.

[0012] Furthermore, the *Bacillus paralichrysogenum* HMF14 has a toxic effect on *Cercospora leptospira*.

[0013] A microbial inoculant comprising the above-mentioned Bacillus paralicheniformis HMF14 cells, spores, and / or extracellular metabolites.

[0014] In the aforementioned microbial inoculant, the number of spores of Bacillus paralichrysum HMF14 is not less than 4.0 × 10⁻⁶. 8 cfu / g.

[0015] The microbial agent also includes soluble starch.

[0016] The microbial agent is a soluble powder.

[0017] Application of the above-mentioned Bacillus paralichrysiformis HMF14 in the control of wheat take-all disease.

[0018] Application of the above-mentioned Bacillus paralichrysiformis HMF14 in the control of wheat black luster disease.

[0019] The application of the above-mentioned Bacillus paralichrysiformis HMF14 in the control of Agniformis brevis.

[0020] Application of the above-mentioned Bacillus paralicheniformis HMF14 in the control of wheat round leaf claw mite.

[0021] An application of the aforementioned Bacillus paralichrysogenus HMF14 in the remediation of heavy metal contamination of wheat in saline-alkali land.

[0022] In the application of remediating heavy metal pollution in wheat grown on saline-alkali land, the heavy metal is Cd.

[0023] The beneficial effects of this invention are as follows: The *Bacillus paralichrysum* strain HMF14 of this invention is tolerant to saline-alkali soils and can effectively antagonize *Triticum aestivum* var. *grass* and *Xanthomonas aureum* wheat-specific strain of black rot fungus. Using *Bacillus paralichrysum* strain HMF14 of this invention can effectively control wheat take-all and wheat black glume. Compared with the blank control, seed soaking with a microbial agent containing *Bacillus paralichrysum* strain HMF14 of this invention showed a control efficacy of over 80.82% against wheat take-all at the jointing, heading, and milk stages, increasing wheat yield by 6.71% per mu (approximately 0.067 hectares), with both efficacy and yield increase exceeding those of conventional chemical agents. Compared with the blank control, seed soaking with a microbial agent containing *Bacillus paralichrysum* strain HMF14 of this invention showed an 81.40% control efficacy against wheat black glume, increasing wheat yield by 3.99% per mu (approximately 0.067 hectares), with both efficacy and yield increase exceeding those of conventional chemical agents. The Bacillus paralichrysogenum HMF14 microbial inoculant has a significantly better control effect than commercially available chemical pesticides and can be promoted as a replacement for chemical pesticides.

[0024] In addition, the Bacillus paralichrysogenum HMF14 strain of the present invention can also kill and control agile short-bodied nematodes and wheat round-leaved claw mites, and at the same time has a significant effect on remediating heavy metal Cd pollution in wheat in saline-alkali land. Attached Figure Description

[0025] Figure 1 The results of a confrontation experiment between strain HMF14 and wheat variety 'Hedyotis diffusa'.

[0026] Figure 2 The colony morphology of strain HMF14 is shown.

[0027] Figure 3 The image shows the microscopic morphology of strain HMF14.

[0028] Figure 4 Phylogenetic tree of strain HMF14.

[0029] Figure 5 The results of a confrontation experiment between strain HMF14 and the wheat-specific Xanthomonas aureus, the causal agent of black rot in cabbage.

[0030] Figure 6 The results of the confrontation experiment between strain HMF14 and Fusarium oxysporum.

[0031] Figure 7 The toxicity test of fermentation broth of strain HMF14 against Agile Short-bodied Nematode was conducted. Detailed Implementation

[0032] The present invention will be further described below with reference to the embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the embodiments, and any modifications made by those skilled in the art within the scope defined by the claims also fall within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the reagents used in the following examples were all purchased from conventional biochemical reagent stores.

[0034] Example 1: Isolation and purification of salt-tolerant strains

[0035] In October 2022, six soil samples (100g each) were collected from saline-alkali land in Gaotuo Village, Liutaizhuang Town, Changli County, Hebei Province. The six soil samples were then mixed evenly.

[0036] Preparation of solid Gibson's modified medium: 5.0 g casein, 3.0 g sodium citrate, 10.0 g yeast extract, 2.0 g KCl, 5.0 g peptone, 2.0 g MgSO4·7H2O, 60 g NaCl, 18 g agar, pH 8.5, 1000 mL distilled water. Sterilize at 121℃ for 20 min and set aside. Liquid Gibson's modified medium is prepared without agar.

[0037] Take 10g of the mixed soil sample, add it to 100mL of distilled water and stir well. After standing for 1 minute, take 1mL of the supernatant and add it to a test tube containing 9mL of sterile water to obtain 1×10⁻⁶ ppm. -1 Diluent, serially diluted, successively diluted to 1×10 -4 1×10 -5 1×10 -6 Concentration, from 1×10 -4 1×10 -5 1×10 -6 0.1 μL of the diluted solution was evenly spread onto solid Gibson's modified medium plates and incubated at 30°C for 2–4 days, with regular observation of colony growth. After colony growth, the culture was purified three times on solid Gibson's modified medium to select six salt-tolerant bacterial strains, which were numbered 1, 2, 3, 4, 5, and 6 and stored at 4°C for later use.

[0038] Example 2: Screening of antagonistic bacteria against pathogens of wheat scab var. gracilis

[0039] Using the mycelial growth inhibition method, with *Triticum aestivum* as the indicator fungus, a confrontation experiment was conducted on PDA medium plates with six different strains. The diameter of the inhibition zone was measured, and the strain with the largest inhibition zone diameter was screened out.

[0040] The specific method is as follows: ① Remove the *Eriocaulon buergerianum* strain from the refrigerator and streak it on a PDA medium plate for 2 days to activate it; ② Use a 1000 μL pipette tip to create 5 mm diameter mycelial cakes from the activated *Eriocaulon buergerianum* strain and inoculate them in the center of the PDA medium plate; ③ Create two mycelial cakes for each isolated and purified numbered strain and symmetrically inoculate them 3 cm from the center on the above PDA medium plate. Each treatment is repeated 3 times. The plate containing only the pathogen serves as a control. Incubate at 28℃ and observe daily; ④ Once the control mycelia have fully grown on the plate, measure the diameter of the inhibition zone using the cross-hatching method.

[0041] Determination method: The larger the diameter of the inhibition zone of the numbered strain, the stronger the inhibitory effect on the wheat variety with scabra.

[0042] PDA solid culture medium preparation method: 200g fresh potatoes (peeled), boil for 20min, filter, add 20g glucose and 20g agar powder, add distilled water to make up to 1000mL, sterilize at 121℃ for 30min, and set aside.

[0043] As shown in Table 1, strain number 2 has the largest inhibition zone diameter against *Triticum aestivum* var. *gracilaria* (e.g., *Triticum aestivum*). Figure 1 As shown), followed by strain number 4.

[0044] Table 1. Results of inhibition zone diameter determination of strain numbered wheat variety *Triticum aestivum*.

[0045] .

[0046] Example 3: Determination of the genetic stability of antagonistic strains

[0047] LB solid medium preparation method: 10.0g tryptone, 5.0g yeast extract, 10.0g NaCl, 18g agar, 1000mL distilled water, adjust pH to 7.2~7.4, sterilize at 121℃ for 20min before use.

[0048] Strain No. 2 was subcultured on LB solid medium using the streak method. After 15 generations, the inhibition rate of the subcultured strain No. 2 against the wheat variety *Triticum aestivum* was determined using the inhibition rate method. The results showed that, compared with the original strain No. 2 as a control, the strain after 15 generations had a similar inhibition rate to the original strain, indicating that the strain has good genetic stability. The strain was then cultured on LB liquid medium, and the bacterial culture was aspirated and stored in glycerol at -80℃, and named HMF14.

[0049] Example 4 Identification of strain HMF14

[0050] (1) Microbiological characteristics

[0051] The colony morphology of strain HMF14 after incubation at 37°C on LB agar plates for 3 days is shown below. Figure 2 As shown, single colonies of this strain are white, with a rough surface and irregular, serrated edges. Under an optical microscope, the bacteria are rod-shaped, measuring 0.5 μm × (1.5–3.5 μm). The spores are elliptical, centrally located, and swollen at the end into sporangia. Gram staining is positive. Figure 3 .

[0052] Based on the morphological identification of the strain in Bergey's Manual of Bacterial Identification, strain HMF14 was preliminarily identified as belonging to the genus Bacillus.

[0053] (2) Physiological and biochemical characteristics

[0054] The physiological and biochemical characteristics of strain HMF14 were identified, and the results are shown in Table 2.

[0055] Table 2 Physiological and biochemical characteristics of strain HMF14

[0056] .

[0057] (3) Molecular biological characteristics

[0058] Genomic DNA was extracted from strain HMF14 and used as a template for PCR amplification using universal primers for bacterial 16S rDNA. The amplification product was recovered and sequenced to obtain a DNA sequence containing 729 bp (as shown in SEQ ID No. 1).

[0059] The sequencing results were input into the GeneBank database for BLAST alignment analysis. Comparison with the 16S rDNA sequence in the NCBI database revealed that HMF14 showed 99% identity with *Bacillus paralichrysogenum*. The phylogenetic tree was constructed as follows: Figure 4 As shown. Based on the morphological, sequencing analysis, and physiological and biochemical detection results, HMF14 was identified as Bacillus paralicheniformis (…). Bacillus paralicheniformis ).

[0060] Bacillus paralicheniformis HMF14 was deposited on April 30, 2024 at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with the accession number CGMCC No. 30480.

[0061] Example 5: Antagonistic test between strain HMF14 and wheat-specific Xanthomonas aureus, the causal agent of black rot in cabbage.

[0062] The sensitivity method was used, with Xanthomonas aureus wheat-specific strain as the indicator bacterium for black rot of cabbage. A confrontation test was conducted on LB agar plates, and the diameter of the inhibition zone was measured to determine its sensitivity.

[0063] The specific method is as follows: ① Remove *Xanthomonas aureus* wheat-specific strain, the causal agent of black rot in cabbage, from the refrigerator and streak it on an LB agar plate at 24℃ for 2 days to activate it; ② First, scrape one loop of the activated *Xanthomonas aureus* wheat-specific strain using an inoculation loop, add it to a test tube containing 1 mL of sterile water, and then repeatedly pipette and mix it to prepare a bacterial suspension; ③ Add the bacterial suspension to LB agar that has not solidified but is not hot to the touch, shake well, immediately pour it onto a plate, and let it cool and solidify. Then, place a mycelial cake of strain HMF14 in the center of the LB agar plate, repeating 3 times; ④ Incubate the culture plates in a 30℃ incubator for 3-5 days, measure the diameter of the inhibition zone using the cross-sectional method, and take the average value to determine the sensitivity.

[0064] Judgment criteria: An inhibition zone diameter ≥ 15 mm indicates extremely sensitive; 10 mm ≤ inhibition zone diameter < 15 mm indicates moderately sensitive; 6 mm ≤ inhibition zone diameter < 10 mm indicates lowly sensitive; no inhibition zone indicates no sensitive.

[0065] From Table 3 and Figure 5 It is evident that strain HMF14 exhibits an inhibition zone diameter greater than 15.1 mm against the wheat-specific Xanthomonas aureus of cabbage black rot, indicating extreme sensitivity.

[0066] Table 3. Antagonistic test results of strain HMF14 against wheat-specific Xanthomonas aureus, the causal agent of black rot in cabbage.

[0067] .

[0068] Example 6 Antagonism test between strain HMF14 and Fusarium oxysporum

[0069] Fusarium oxysporum HMB-1 ( Fusarium oxysporum (This item is preserved in our laboratory.)

[0070] The preserved Bacillus paralichrysogenum HMF14 bacterial suspension was streaked on LB plates and cultured for 48 h. Four mycelial cakes were then made using a 1 mL pipette tip and placed symmetrically 3 cm above, below, to the left, and to the right of the center of a PDA plate. The plates were incubated at 28 °C for 72 h. Then, mycelial cakes were made on a pre-activated Fusarium oxysporum plate using a 1 mL pipette tip and inoculated into the center of the PDA plate. A separate PDA plate was used as a control, with only Fusarium oxysporum mycelial cakes inoculated into the center. The plates were incubated upside down in a 28 °C incubator. Once the control mycelium had fully colonized the plate, the colony diameter of the treatment groups was measured using the cross-crossing method, and the inhibition rate was calculated.

[0071] Inhibition rate (%) = (mycelial growth diameter of control group - mycelial growth diameter of treatment group) / mycelial growth diameter of control group × 100.

[0072] The results are as follows Figure 6As shown, the average mycelial diameter of the treatment group was 45.8 mm, the plate diameter was 90 mm, and the calculated inhibition rate was 50.5%.

[0073] Example 7: Virulence test of strain HMF14 against the agile short-bodied nematode.

[0074] Preparation of suspension of *Agile spicata* larvae: Thirty female *Agile spicata* larvae were isolated, disinfected with 0.3% streptomycin sulfate solution for 8 h, washed 5 times with sterile water, and then inoculated onto carrot callus. Second-instar larvae (J2) of *Agile spicata* were obtained by incubation at 25°C in the dark. The second-instar larvae (J2) propagated on carrot callus were isolated using a modified Behmann funnel method. The collected nematodes were placed in beakers to prepare a suspension of 500 larvae / mL.

[0075] The specific steps for preparing the fermentation broth are as follows:

[0076] (1) Preparation of LB liquid culture medium: LB culture medium formula: 5g yeast extract, 10g peptone, 10g sodium chloride, put into a 1000mL beaker, add 900mL distilled water and heat to dissolve, adjust pH to 7.2 ~ 7.4, make up to 1L with distilled water, sterilize at 121℃ for 30 minutes, and set aside.

[0077] (2) Activation of strain: Pick one loopful of Bacillus paralicheniformis HMF14 colony, inoculate it into a 150mL Erlenmeyer flask containing 50mL LB liquid medium, and activate it by constant temperature shaking at 160rpm and 37℃ for 24h.

[0078] (3) Preparation of seed culture: Take 4 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 200 mL of LB liquid culture medium. Incubate at 160 rpm and 37 °C for 24 h to obtain seed culture.

[0079] (4) Preparation of fermentation broth: 180 mL of the prepared seed culture was inoculated into a 6 L fermenter containing 3.5 L of LB liquid medium and cultured at 160 rpm and 37 °C for 48 h with constant temperature shaking to obtain Bacillus paralichrysiformis HMF14 fermentation broth with an effective viable count of 3.41 × 10⁻⁶ cells / mL. 9 cfu / mL.

[0080] (5) Preparation of fermentation broth: The fermentation broth was filtered through a 0.22 μm filter membrane to obtain the fermentation broth of Bacillus paralichrysum HMF14.

[0081] Add 1 mL of a suspension of agile short-bodied nematode larvae to one well of a 24-well plate, followed by 1 mL of supernatant. Mix well. Use sterile water as a control. Each treatment is repeated three times and incubated in a dark incubator at 25°C. Observe the viability of the nematodes after 48 hours. Dead nematodes are rigid, while live nematodes are bent. Count the number of dead nematodes and calculate the corrected mortality rate.

[0082] Corrected mortality rate (%) = [(treatment mortality rate - control mortality rate) / (1 - control mortality rate)] × 100.

[0083] The toxic effects of fermentation broth from strain HMF14 on the larvae of the agile short-bodied nematode are shown in Table 4. Figure 7 As shown.

[0084] Table 4. Statistical analysis of the toxicity of fermentation broth from strain HMF14 to larvae of the agile short-bodied nematode.

[0085] .

[0086] Example 8: Virulence test of Bacillus paralichrysiformis HMF14 against Cervus nippon mites.

[0087] The preparation steps of the fermentation broth were the same as in Example 7. The acaricidal activity of the fermentation broth was evaluated using the spider mite slide immersion method, with the target mite being *Tetranychus hygroscopicus*.

[0088] Cut the double-sided tape into 2cm pieces. 2 Small pieces of the mite were attached to a clean glass slide. Healthy female adult mites were selected and their backs were attached to double-sided tape, with about 30 mites attached to each slide. The slide containing the mites was quickly immersed in the fermentation broth, gently shaken for 5 seconds, and then removed. Excess liquid was absorbed with absorbent paper. The slide was placed on a white dish with a damp sponge and covered with a transparent plastic film. Each treatment was repeated 4 times. Fresh, untreated fermentation medium was used as a blank control. The container containing the test mites was placed in an environment of 25°C with a photoperiod of L:D = 16h:8h and left to stand for 24 hours. The number of dead mites was checked under a stereomicroscope. Mites that could not move when touched with the tip of a small brush were considered dead.

[0089] Fermentation broths with different spore concentrations were prepared by mixing different volumes of fresh fermentation broth. The toxic activity of the fermentation broth concentrations against *Tetranychus spp.* was determined using the spider mite slide immersion method, as shown in Table 5. The insecticidal probability of different concentrations of fermentation broth was calculated using SPSS 27.0 software, yielding the unit equation model PROBIT(P) = -0.08 + 0.014X. The experimental results show that the median lethal concentration (LC50) of HMF14 fermentation broth against *Tetranychus spp.* 50 The concentration was 5.6 μL / mL, indicating that it has a high toxicity to the wheat scab.

[0090] Table 5. Toxicity of different concentrations of HMF14 fermentation broth against *Ceratophyllum demersum*.

[0091] .

[0092] Example 9 Preparation of Bacillus paralichrysiformis HMF14 microbial inoculant

[0093] (1) LB liquid culture medium formulation: same as in Example 7.

[0094] (2) Activation of strain: Pick two loops of strain HMF14 from the preserved test tube, inoculate them into a test tube containing 20 mL LB liquid medium, and activate them by constant temperature shaking at 180 rpm and 37 ℃ for 12 h.

[0095] (3) Preparation of seed culture: Take 7.5 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 250 mL of LB liquid culture medium. Incubate at 180 rpm and 37 °C for 48 h to obtain seed culture.

[0096] (4) Preparation of fermentation broth and inoculum: The prepared seed culture was inoculated into a 6L fermenter containing 4.2L of LB liquid medium, at a rotation speed of 180rpm and an aeration rate of 0.5m³ / h. 3 Fermentation broth with a spore rate of 92.3% was obtained by constant temperature incubation at 37℃ for 48 hours.

[0097] (5) The fermentation broth was spray-dried, and the spore powder of the bacterium was mixed with soluble starch. The spore count of Bacillus paralichrysiformis HMF14 was found to be 4.66 × 10⁻⁶. 8 cfu / g, which means obtaining the powdered bacterial agent of strain HMF14.

[0098] Example 10: Field efficacy test of HMF14 microbial inoculant against wheat take-all disease

[0099] From October 2023 to June 2024, a field efficacy trial for wheat take-all disease was conducted in an area affected by wheat take-all (Dapuhe Village, Changli County, Hebei Province). The wheat variety used was "Hangmai 247". The trial included three treatments: Treatment 1 consisted of a sample diluted with physiological saline using HMF14 microbial inoculant, with an effective viable count of 2.0 × 10⁻⁶ bacteria. 7 The concentration of cfu / mL was 100 mg / mL. Wheat seeds were soaked in water for 24 hours before sowing, then spread out to dry before sowing. Treatment 2 served as the conventional chemical control (CK1), using 0.2% (by seed weight) of 2% levofloxacin before sowing. Treatment 3 served as the blank control (CK0), using only water for 24 hours, then spreading out to dry before sowing. Each treatment was replicated three times. The incidence and control effects of different treatments on wheat take-all disease were investigated, and wheat yield was assessed at harvest.

[0100] Methods for investigating wheat take-all disease incidence: Root surveys were conducted twice, once at the jointing stage and once at the heading stage. If "black foot" symptoms appeared on the wheat roots, the disease was diagnosed. A five-point diagonal sampling method was used, with 20 plants sampled at each point. The extent of root infection was investigated, and the results were recorded according to the wheat take-all disease grading standard. Disease index and control efficacy were statistically analyzed. If wheat plants withered and "white ears" appeared at the milk stage, the disease was diagnosed. For each treatment, two rows were randomly measured within 1 meter, and the total number of ears and the number of white ears were investigated. The white ear rate and control efficacy were statistically analyzed.

[0101] (a) Calculation method for root system survey and control effect at the jointing and heading stages

[0102] The root system survey grading standards are as follows: Grade 0: No disease. Grade 1: Diseased root area accounts for 1%~5%. Grade 3: Diseased root area accounts for 6%~20%. Grade 5: Diseased root area accounts for 21%~40%. Grade 7: Diseased root area accounts for 41%~60%. Grade 9: Diseased root area >61%.

[0103] Disease index (%) = ∑(each disease level × number of plants at each disease level) / (highest disease level × total number of plants surveyed) × 100;

[0104] Control efficacy (%) = (Disease index of water control - Disease index of drug treatment) / Disease index of water control × 100.

[0105] (b) Calculation method for the control effect of "white ears" at the milk stage

[0106] White ear rate (%) = Number of white ears / Total number of ears surveyed × 100;

[0107] Control efficacy (%) = (White ear rate in water control - White ear rate in pesticide treatment) / White ear rate in water control × 100.

[0108] Production survey method: A 5-point sampling method was used for each treatment, with each point surveyed for 1m. 2 The yield was calculated per mu (667m²). 2 ) Output, calculate the rate of increase in output.

[0109] Yield increase rate (%) = (yield in control area - yield in control area) / yield in control area × 100.

[0110] The control efficacy and yield survey results are shown in Table 6. Seed soaking with HMF14 microbial inoculant showed good control efficacy against wheat take-all disease, with a significant reduction in disease index. Compared with the control group, the control efficacy at the jointing, heading, and milk stages of wheat reached over 80.82%, resulting in a 6.71% increase in wheat yield per mu (667 square meters). The control efficacy at all stages was better than that of conventional chemical agents, increasing wheat yield per mu by 1.91%.

[0111] Table 6. Effects of different treatments on control efficacy and yield of wheat take-all disease.

[0112] .

[0113] Example 11 Field efficacy test of HMF14 microbial inoculant against wheat black glume.

[0114] From October 2023 to June 2024, a field efficacy trial of microbial inoculants against wheat black luster was conducted in an area affected by wheat black luster (Xinlizhuang Village, Changli County, Hebei Province). The wheat variety used was "Hangmai 247". The trial included three treatments: Treatment 1 consisted of a sample diluted with physiological saline using HMF14 microbial inoculant, with an effective viable count of 2.0 × 10⁻⁶. 7 Treatment 1 (CFU / mL) involved soaking wheat seeds in water for 24 hours before sowing, followed by air drying. Treatment 2 (CK1) served as a conventional chemical control, using 15% Yeqing double-coated suspension at 3000 mg / kg for 12 hours, followed by air drying before sowing. Treatment 3 (CK0) served as a blank control, using only water for 24 hours, followed by air drying before sowing. Each treatment was replicated three times. The incidence and control efficacy of wheat black glume were assessed during the grain-filling stage, and wheat yield was assessed at harvest.

[0115] Methods for investigating control efficacy: During the grain-filling stage, a five-point diagonal sampling method was used, with 100 ears sampled at each point, for a total of 500 ears sampled per treatment. If the wheat ears were infected with the disease, and brown to black streaks appeared on the glumes, the disease was considered to have occurred. The total number of ears surveyed and the total number of infected ears were recorded, and the incidence rate and control efficacy were calculated.

[0116] Method for calculating the effectiveness of prevention:

[0117] Incidence rate (%) = Number of infected ears / Total number of ears surveyed × 100;

[0118] Control efficacy (%) = (Incidence rate of water control - Incidence rate of drug treatment) / Incidence rate of water control × 100.

[0119] Production survey method: same as in Example 10.

[0120] The control effects are shown in Table 7. The microbial inoculant has a good control effect on wheat black luster disease. The disease incidence rate of wheat seeds treated with HMF14 microbial inoculant was significantly reduced. Compared with the blank control, the disease control effect reached 81.40%, and the wheat yield per mu increased by more than 3.99%; compared with the conventional chemical agent control, the disease control effect increased by 17.84%, and the wheat yield per mu increased by 1.31%.

[0121] Table 7. Effects of different treatments on control efficacy and yield of wheat black glume.

[0122] .

[0123] The *Bacillus paralichrysogenus* strain HMF14 of this invention exhibits strong antagonistic activity against *Triticum aestivum* var. *gracilaria* and *Xanthomonas aureus* wheat-specific strain, which causes black rot in cabbage. Using *Bacillus paralichrysogenus* HMF14 microbial agent demonstrates excellent control effects against wheat take-all disease caused by *Triticum aestivum* var. *gracilaria* and wheat black glume caused by *Xanthomonas aureus* wheat-specific strain, both exceeding the control effects of conventional chemical agents. The application of this agent can significantly reduce the amount of chemical pesticides used, which is of great significance for environmental protection and human health.

[0124] Example 12: Effect of the composite system of Bacillus paralichrysiforme HMF14 and nano-zero valent iron sulfide (nZVI) on the remediation of heavy metal pollution in wheat fields in saline-alkali land.

[0125] Trial period: October 5, 2023 to June 10, 2024

[0126] The investigation and statistical work on all experimental data shall be completed by June 12, 2024.

[0127] Experimental location: Saline-alkali winter wheat planting area in Qijiawu Township, Huanghua City, Hebei Province. The wheat was sown on October 7, 2023, and harvested on June 10, 2024. The variety was Jiemai 19.

[0128] Set up 4 processing groups:

[0129] CK group: No repair agent added (control);

[0130] S-nZVI group: S-nZVI was added alone, at a rate of 2 kg per acre;

[0131] HMF14 group: Bacillus paralichrysum HMF14 microbial inoculant prepared in Example 9 (Bacillus paralichrysum HMF14 spore count was 4.66 × 10⁻⁶). 8 (cfu / g), application rate: 2 kg / mu;

[0132] Compound group: The Bacillus paralichrysogenum HMF14 microbial agent prepared in Example 9 was thoroughly mixed with S-nZVI at a mass ratio of 1:2, and the application rate was 3 kg per mu.

[0133] Each treatment was replicated three times, for a total of 12 experimental plots, each plot being 0.1 acres in size.

[0134] Before winter wheat sowing (October 6, 2023), the materials used in the above treatments (microbial agents and / or S-nZVI) were thoroughly mixed with organic fertilizer, and then the mixture was thoroughly mixed with the soil through deep plowing. Other planting and management measures were carried out in accordance with the local winter wheat production technical specifications.

[0135] The preparation method of sulfide nano-zero valent iron used in this embodiment is referenced in "Liang Jiwei, Mao Wenyan, Wang Xingyu, et al. Study on electrochemical corrosion characteristics based on sulfide nano-zero valent iron [J]. Journal of Nanjing University of Science and Technology, 2023, 47(5):724-730".

[0136] Methods for measuring available cadmium in soil: Soil samples were collected at 5 points before winter wheat sowing and land preparation (October 5, 2023) and at wheat maturity (June 9, 2024), with a depth of 0–20 cm. After natural air drying, the samples were ground and passed through a 10-mesh sieve. The available Cd content in the soil of different treatment groups was determined using inductively coupled plasma mass spectrometry (ICP-MS).

[0137] Table 8 shows the statistical table of available Cd content in soils of different treatment groups. The available Cd content (DTPA extracted) in the composite group was significantly reduced. Before land preparation, the available Cd content was 68.2%. After applying the composite remediation agent, the available Cd in the soil at the winter wheat maturity stage decreased to 22.4%, a reduction of 67.2%, which was significantly higher than that after applying S-nZVI alone (45.3%) and HMF14 alone (31.5%). This is because the organic acids secreted by HMF14 can dissolve the passivation layer (Fe3O4) on the surface of S-nZVI, releasing Fe. 2+ and S 2- This promotes dynamic regeneration of the sulfide layer, while the extracellular polymeric substance (EPS) fixes Cd through chelation. 2+ Therefore, the content of available Cd in soils treated with composite remediation agents decreased most significantly.

[0138] Table 8. Statistics on available Cd content in soils treated with different remediation agents.

[0139] .

[0140] Method for determining cadmium content in wheat grains: Weigh 0.2500 g of winter wheat grains and use concentrated nitric acid and perchloric acid (V... 硝酸 ∶V 高氯酸 The samples were digested using a wet digestion method with a ratio of 4:1, and the cadmium content was determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0141] Table 9 shows the statistical table of Cd content in wheat grains of different treatment groups. The Cd content in wheat grains of the compound treatment group was 0.18 mg / kg, which was 68.8% lower than that of the CK group (0.85 mg / kg). The Cd contents in the grains of the S-nZVI group and the HMF14 group were 0.37 mg / kg and 0.56 mg / kg, respectively, with reductions of 46.4% and 24.4%. This example shows that the combination of HMF14 and S-nZVI can more effectively reduce the Cd content in wheat grains.

[0142] Table 9. Statistics on Cd content in wheat grains after applying different treatment groups of remediation agents.

[0143] .

[0144] The above are merely preferred embodiments of the present invention, and not limitations on its scope of protection. Any improvements made to the present invention by those skilled in the art without requiring creative effort should be considered within the scope of protection of the present invention.

Claims

1. A type of Bacillus paralicheniformis ( Bacillus paralicheniformis HMF14, characterized in that, The accession number is CGMCC No.30480.

2. A microbial inoculant, characterized in that, It includes Bacillus paralicheniformis HMF14 cells and / or spores as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The number of spores of *Bacillus paralichrysogenum* HMF14 is not less than 4.0 × 10⁻⁶. 8 cfu / g.

4. The microbial agent according to claim 2, characterized in that, It also includes soluble starch.

5. The application of Bacillus paralichrysiforme HMF14 as described in claim 1 in the prevention of wheat take-all disease or wheat black glume.

6. The application of Bacillus paralichrysiformis HMF14 as described in claim 1 in the killing of Agile Short-bodied Nematode or Wheat Circotype mite.

7. The application of Bacillus paralichrysiformis HMF14 as described in claim 1 in reducing heavy metal cadmium pollution in wheat grown in saline-alkali land.

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