Microbial combination for preventing and treating corn stalk rot and having growth promotion function and application thereof

Abstract

The application discloses a kind of microbial combination for preventing and treating corn stem rot and with the function of promoting growth and application thereof, and relates to the technical field of biotechnology.The microbial combination includes Paenibacillus peoriae HFF-0-C5 and Bacillus licheniformis HFF-0-C43.The two strains are used in combination, can produce synergistic disease resistance, effectively improve the inhibition effect on corn stem rot pathogenic fungus, Fusarium pseudograminearum.Utilizing the two strains provided by the application to prepare composite microbial inoculant can effectively enhance soil fertility, improve soil environment, induce plants to produce disease resistance, so as to better achieve the comprehensive prevention and control effect of promoting growth and disease resistance, realize the quality improvement, yield increase and income increase of corn.The composite microbial inoculant is safe, efficient, pollution-free, harmless to human and livestock, and has good application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a microbial ensemble for preventing and controlling corn stalk rot and having growth-promoting functions, and its application. Background Technology

[0002] To date, over 100 corn diseases have been reported, causing yield losses of 10%-30%. Corn stalk rot is one of the most significant threats to corn production, and its incidence is steadily increasing. With climate change and increased planting density, the incidence of corn stalk rot has significantly worsened, and the affected area is expanding. In normal years, the incidence rate is 15%-20%, but in severe years it can reach over 50% or even result in total crop failure, causing incalculable losses to corn production and becoming a major factor limiting high yields and income.

[0003] Currently, with the long-term and extensive use of chemical pesticides, various drawbacks have gradually become apparent. Therefore, exploring and developing safe, efficient, and environmentally friendly control methods is of paramount importance.

[0004] Microbial inoculants, as alternatives to chemical fertilizers and pesticides, have become a research hotspot for plant disease researchers in recent years. Biological control is environmentally friendly, pathogens are less likely to develop resistance, and the production process of biocontrol agents is relatively simple, promoting crop growth and showing broad application prospects. Compared to single biocontrol strains, biocontrol flora have better disease control effects. Single-flora biocontrol agents are affected by factors such as different application environments and competition from environmental microorganisms, making their control effects unstable. Furthermore, factors such as production costs and shelf life further contribute to their inferior biocontrol efficacy compared to biocontrol flora. Combining strains with different biological functions can compensate for the shortcomings of single strains, allowing for the selection of biocontrol agents with more significant control effects and improving biocontrol efficacy. Based on this, this invention aims to develop a microbial combination for controlling maize stalk rot with growth-promoting functions, thereby improving biocontrol efficacy and increasing maize yield. Summary of the Invention

[0005] The purpose of this invention is to provide a microbial combination for controlling maize stalk rot and possessing growth-promoting functions, and its application, to solve the problems existing in the prior art. This invention has found that the combined use of *Paenibacillus peoriae* HFF-0-C5 and *Bacillus licheniformis* HFF-0-C43, which has potassium-solubilizing functions, can produce synergistic disease resistance, effectively improving the inhibitory effect on the maize stalk rot pathogen *Fusarium verticillata*, thereby achieving a comprehensive control effect of growth promotion and disease resistance.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a microbial combination for preventing and controlling maize stalk rot and having growth-promoting function, including Paenibacillus peoriae HFF-0-C5 and Bacillus licheniformis HFF-0-C43;

[0008] The *Bacillus pilaris* HFF-0-C5 was deposited on October 23, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 28717.

[0009] The Bacillus licheniformis HFF-0-C43 was deposited on October 23, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 28718.

[0010] Furthermore, the ratio of the number of *Bacillus pilaris* HFF-0-C5 to the number of the number of *Bacillus licheniformis* HFF-0-C43 is 2:(1-3).

[0011] Preferably, the ratio of the number of *Bacillus pilaris* HFF-0-C5 to the number of the number of *Bacillus licheniformis* HFF-0-C43 is 1:1.

[0012] The present invention also provides the application of the above-mentioned microbial combination in the preparation of a compound microbial agent with functions of preventing and controlling corn stalk rot and promoting growth.

[0013] Furthermore, the compound microbial agent is a liquid microbial agent, a coating agent, or granules.

[0014] The present invention also provides a compound microbial agent with functions of preventing and controlling corn stalk rot and promoting growth, the active ingredients of which include the above-mentioned microbial combination.

[0015] Furthermore, the compound microbial agent also includes excipients.

[0016] Furthermore, the formulation of the compound microbial agent is a liquid microbial agent, a coating agent, or granules.

[0017] Furthermore, when the formulation of the compound microbial agent is a liquid microbial agent, the excipients are gum arabic powder, Tween 80, and tea polyphenols;

[0018] When the formulation of the compound microbial agent is a coating agent, the excipient is a film-forming agent;

[0019] When the compound microbial agent is in granule form, the excipients are kaolin and tyrosine.

[0020] The present invention also provides the application of the above-mentioned microbial combination or compound inoculant in the prevention and control of corn stalk rot and / or the increase of corn yield.

[0021] The present invention discloses the following technical effects:

[0022] This invention isolates two strains from maize rhizosphere soil: *Paenibacillus peoriae* HFF-0-C5, which antagonizes *Fusarium verticillatum*, the pathogen of maize stalk rot, and *Bacillus licheniformis* HFF-0-C43, which has potassium-solubilizing function. Combining these two strains produces synergistic resistance, effectively enhancing the inhibitory effect against *Fusarium verticillatum*, the pathogen of maize stalk rot.

[0023] The compound microbial agent prepared using the two strains provided in this invention can effectively enhance soil fertility, improve the soil environment, and induce plant disease resistance, thereby achieving a better integrated prevention and control effect of promoting growth and resisting disease, and realizing improved quality, increased yield, and increased income for corn. This compound microbial agent is safe, efficient, and pollution-free, and has great application value. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Phylogenetic tree diagram of strain HFF-0-C5;

[0026] Figure 2 This is a phylogenetic tree diagram of strain HFF-0-C43. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] The *Fusarium verticilliodes* used in this invention were provided by the Laboratory of Fungal Toxins and Plant Molecular Pathology, Hebei Agricultural University.

[0033] Example 1

[0034] 1. Strains Isolation

[0035] This invention isolated four bacterial strains from maize rhizosphere soil, named KF5, HFF-0-C5, K43, and HFF-0-C43, respectively. KF5 and HFF-0-C5 are antagonistic strains that inhibit the fungus *Fusarium verticillatum*, the pathogen of maize stalk rot, screened using a plate confrontation experiment; K43 and HFF-0-C43 are strains with potassium-solubilizing function screened using potassium-solubilizing medium.

[0036] Among them, strains KF5 and K43 have been disclosed in the literature "Screening and Identification of Biocontrol Strains for Maize Fusarium Stem Rot and Development of Inoculants".

[0037] Potassium-solubilizing medium: sucrose 10 g / L, yeast extract 0.5 g / L, ammonium sulfate 1 g / L, disodium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, calcium carbonate 1 g / L, potassium feldspar powder 1 g / L, and agar powder 15 g / L.

[0038] 2. Strain identification

[0039] (1) Physiological and biochemical identification

[0040] On LB solid medium, both HFF-0-C5 and HFF-0-C43 colonies were white with irregular surfaces. Gram staining confirmed that both HFF-0-C5 and HFF-0-C43 were Gram-positive strains. Physiological and biochemical identification revealed that HFF-0-C5 and HFF-0-C43 were positive for methyl red, nitrate reduction, starch hydrolysis, and lactose decomposition; however, they were negative for VP assay and indole production.

[0041] (2) Molecular biological identification

[0042] Using molecular biology techniques, the 16S rDNA of the strains was amplified separately using universal primers. After sequencing, phylogenetic trees were constructed using BLASTN alignment. The results are shown below. Figure 1 and Figure 2 .

[0043] The results showed that strain HFF-0-C5 was most closely related to *Paenibacillus peoriae*, with a similarity exceeding 99.9%. Based on physiological and biochemical characteristics, strain HFF-0-C5 was identified as *Paenibacillus peoriae*. Strain HFF-0-C43 was most closely related to *Bacillus licheniformis*, and based on physiological and biochemical characteristics, it was identified as *Bacillus licheniformis*.

[0044] 3. Preservation of microbial strains

[0045] Paenibacilluspeoriae HFF-0-C5 was deposited on October 23, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 28717.

[0046] Bacillus licheniformis HFF-0-C43 was deposited on October 23, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 28718.

[0047] Example 2

[0048] Strains HFF-0-C5 and HFF-0-C43 were cultured together on LB medium for 2 days. The results showed that the growth of strain HFF-0-C43 on LB medium containing strain HFF-0-C5 was the same as that of the control, indicating that the two bacteria do not affect each other's growth when cultured at the same time. Therefore, HFF-0-C5 and HFF-0-C43 can be used together.

[0049] The HFF-0-C5 strain was inoculated into fermentation medium A and fermented at 30℃ and 160 rpm for 2 days to obtain the fermentation broth. The fermentation broth was centrifuged at 4000 rpm for 10 min to enrich the bacterial cells, and then resuspended in PBS buffer to a bacterial concentration of 10. 8 The concentration of cfu / mL was increased to obtain HFF-0-C5 bacterial suspension.

[0050] Strain KF5 was inoculated into fermentation medium A and fermented at 30℃ and 160 rpm for 2 days to obtain the fermentation broth. The fermentation broth was centrifuged at 4000 rpm for 10 min to enrich the bacterial cells, and then resuspended in PBS buffer to a bacterial concentration of 10. 8 CFU / mL was used to obtain a KF5 bacterial suspension.

[0051] Strain HFF-0-C43 was inoculated into fermentation medium B and fermented at 30℃ and 160 rpm for 2 days to obtain the fermentation broth. The fermentation broth was centrifuged at 4000 rpm for 10 min to enrich the bacterial cells, and then resuspended in PBS buffer to a bacterial concentration of 10. 8 The concentration of cfu / mL was increased to obtain HFF-0-C43 bacterial suspension.

[0052] Strain K43 was inoculated into fermentation medium B and fermented at 30℃ and 160 rpm for 2 days to obtain the fermentation broth. The fermentation broth was centrifuged at 4000 rpm for 10 min to enrich the bacterial cells, and then resuspended in PBS buffer to a bacterial concentration of 10. 8 CFU / mL was used to obtain a K43 bacterial suspension.

[0053] The fermentation medium A has the following formula: yeast extract 5 g / L, ammonium sulfate 7.5 g / L and potassium dihydrogen phosphate 5 g / L; the fermentation medium B has the following formula: yeast extract 7.5 g / L, tryptone 7.5 g / L and magnesium sulfate 5 g / L.

[0054] Five mixed bacterial agents were obtained by mixing HFF-0-C5 bacterial suspension and HFF-0-C43 bacterial suspension at volume ratios of 0:1, 1:0, 1:1, 1:2, 2:1, and 2:3. Five mixed bacterial agents were also obtained by mixing KF5 bacterial suspension and K43 bacterial suspension at volume ratios of 1:0, 1:1, 1:2, 2:1, and 2:3. These mixed bacterial agents were then subjected to plate confrontation culture with Fusarium oxysporum (the amount of compound bacterial solution added to the Oxford cups was the same in each group). The results are shown in Table 1.

[0055] Table 1. Diameter of inhibition zone in each group of plate confrontation experiments

[0056] volume ratio The diameter of the inhibition zone (cm) of the HFF-0-C5 and HFF-0-C43 combination. The diameter of the inhibition zone (cm) of the KF5 and K43 combination. 0:1 7.8±0.1 7.8±0.1 1:0 1.35±0.21 1.26±0.14 1:1 1.62±0.09 1.44±0.08 1:2 1.22±0.28 1.00±0.13 2:1 1.39±0.14 1.00±0.24 2:3 1.50±0.17 0.94±0.12

[0057] The results showed that different ratios of HFF-0-C5 and HFF-0-C43 bacterial suspensions could produce inhibition zones against *Fusarium verticillatum*. Comparing the antibacterial abilities, the strongest antibacterial effect was observed when HFF-0-C5 and HFF-0-C43 were mixed at a 1:1 volume ratio, with an inhibition zone diameter of 1.62 cm, which was higher than the inhibition zone diameter when the antagonistic bacterium HFF-0-C5 was used alone. This indicates that the mixture of the two bacterial suspensions has a better inhibitory effect on *Fusarium verticillatum* than HFF-0-C5 alone. Furthermore, the combination of HFF-0-C5 and HFF-0-C43 showed a better antibacterial effect than the combination of KF5 and K43.

[0058] The results above indicate that the antagonistic bacteria HFF-0-C5 and potassium-solubilizing bacteria HFF-0-C43 can be combined, and the combination effect is best when HFF-0-C5:HFF-0-C43 = 1:1.

[0059] Example 3

[0060] To verify the growth-promoting effects of compound microbial agent A (a 1:1 volume mixture of HFF-0-C5 and HFF-0-C43 bacterial suspensions from Example 1) and compound microbial agent B (a 1:1 volume mixture of KF5 and K433 bacterial suspensions from Example 1) on maize, a greenhouse pot experiment was conducted. At the three-leaf stage of maize, the compound microbial agent was applied to the roots, with 20 mL applied to each plant. An equal volume of PBS buffer was used as a control. After 15 days of continued cultivation, maize plant height, stem diameter, root length, and fresh weight were recorded. The results are shown in Table 2.

[0061] Table 2. Statistical table of indoor growth-promoting effects in different treatment groups.

[0062]

[0063] The results showed that the root irrigation treatment with compound microbial agent A resulted in better growth than that with compound microbial agent B and the control. The root length, plant height, leaf length, and fresh weight of maize treated with compound microbial agent A were significantly better than those treated with compound microbial agent B and the control. The study indicated that the compound microbial agent prepared from strains HFF-0-C5 and HFF-0-C43 had a good growth-promoting effect on maize seedlings.

[0064] Example 4

[0065] To verify the control efficacy of compound microbial agent A (a 1:1 volume ratio mixture of HFF-0-C5 and HFF-0-C43 bacterial suspensions from Example 1) and compound microbial agent B (a 1:1 volume ratio mixture of KF5 and K433 bacterial suspensions from Example 1) against maize stalk rot induced by *Fusarium oxysporum*, a greenhouse pot experiment was conducted, detailed as follows:

[0066] After the corn stalks reached a diameter of 2 cm, a compound microbial agent was applied to the roots (with an equal volume of PBS buffer as a control). 20 mL of the agent was applied to each plant. Following the root drench treatment, *Fusarium verticillatum* was inoculated into the corn stalks via needle pricking (i.e., a concentration of 1×10⁻⁶ was injected at a 45° angle downwards from the second node at the base of the corn stalk). 6 The disease incidence was observed after 7 days using 2 mL of a suspension of Fusarium spores per mL of compound fungicide A and compound fungicide B. The results are shown in Table 3. The results showed that the expansion area of ​​maize stalk lesions was significantly inhibited after treatment with compound fungicide A and compound fungicide B, and compound fungicide A had a better inhibitory effect.

[0067] Table 3. Statistical table of the expanded area of ​​maize stalk lesions in different treatment groups.

[0068]

[0069] Example 5

[0070] The preparation methods of HFF-0-C5 and HFF-0-C43 bacterial suspensions involved in this embodiment are the same as in Example 2. The HFF-0-C5 and HFF-0-C43 bacterial suspensions are mixed evenly at a volume ratio of 1:1 to obtain a composite bacterial solution.

[0071] After preparing compound microbial agents in different formulations, field trials were conducted:

[0072] 1. Liquid bacterial agent: Gum arabic powder, Tween 80, and tea polyphenols were added to the compound bacterial solution to achieve final concentrations of 0.3 wt%, 0.2 wt%, and 0.03 wt%, respectively, resulting in liquid bacterial agents (HFF-0-C5 and HFF-0-C43, both with a bacterial concentration of 10). 8 (cfu / mL). The prepared liquid bacterial agent was used for root irrigation, with 20 mL applied to each plant.

[0073] 2. Granules: The bacterial concentration of both HFF-0-C5 and HFF-0-C43 in the compound bacterial solution is 10... 8 Kaolin and tyrosine were added to a solution of cfu / mL to achieve final concentrations of 5 wt% and 3 wt%, respectively, to obtain a mixed suspension. This suspension was then mixed thoroughly with whole wheat flour at a volume-to-mass ratio of 1:2. The mixture was granulated using a granulator and dried in a cool, well-ventilated place to obtain granules. The prepared granules were then applied to individual plants using the same dosage per plant as the liquid inoculant, based on the viable bacterial count.

[0074] 3. Coating agent: The compound bacterial solution and the finished film-forming agent (purchased from Beinong Haili Company) are mixed uniformly at a volume ratio of 1:5 to obtain the coating solution, ensuring that the final concentrations of HFF-0-C5 and HFF-0-C43 are both 10. 8After sterilizing healthy corn seeds (cfu / mL), add them to the prepared coating solution and stir quickly with a glass rod to ensure uniform coating. Let the mixture air dry. Sow the prepared coated seeds.

[0075] After treatment with different formulations of compound microbial agents (with no compound microbial agent as the control), various growth indicators of maize, including aboveground plant height, leaf length, and leaf width, were investigated at the four-leaf and one-heart stage. The results are shown in Table 4.

[0076] The results showed that, compared with the blank control, the seed coating treatment group exhibited significantly better growth-promoting effects in terms of plant height, leaf length, leaf width, and stem diameter compared to other treatment groups. The granule treatment group was the second best, followed by the liquid inoculant treatment.

[0077] Table 4. Effects of different formulations of compound microbial agents on maize seedling stage in the field.

[0078]

[0079] After the maize matured, 30 ears were randomly selected from each plot, dried, and then the following indicators were measured: number of rows per ear, number of kernels per row, ear length, ear diameter, thousand-kernel weight, and moisture content. The maize yield for each treatment was calculated. Maize yield (kg / hm²) 2 = Number of plants per mu × Number of rows of ears × Number of grains per row × 1000-grain weight × 8.5 × 10 -1 / (1000×1000).

[0080] As shown in Table 5, the results showed that compared with the blank control, the number of rows and kernels per row of corn were increased to varying degrees after treatment with different formulations of compound microbial agents. Among them, compared with the control group, the corn treatment group treated with antagonistic bacteria HFF-0-C5 and potassium-solubilizing bacteria HFF-0-C43 had a certain increase in yield, and the coating agent had the greatest increase in yield, with a significant yield-increasing effect.

[0081] Table 5. Effects of different compound microbial agents on yield

[0082]

[0083] Example 6

[0084] Similar to Example 5, the only difference is that, at the waxy maturity stage of maize, 30 maize plants inoculated with Fusarium oxysporum were randomly selected from each plot, and the area of ​​lesions was observed using the longitudinal stem-cutting method, and the disease incidence was statistically analyzed. A disease survey was conducted in each area, and the disease severity level of the maize was statistically analyzed. The disease severity grading criteria are shown in Table 6.

[0085] Table 6 Disease Grading Standards

[0086]

[0087] Disease index = [∑(Disease level × Corresponding number of cases) / (Total number of cases surveyed × Highest disease level)] × 100%

[0088] Prevention and control efficacy (%) = [(Disease index of control group - Disease index of treatment group) / Disease index of control group] × 100%

[0089] The control efficacy evaluation results for each treatment group are shown in Table 7. The results show that the application of the compound microbial agent has a better control effect compared with the blank treatment. Therefore, it can be preliminarily concluded that the mixed treatment of antagonistic bacteria HFF-0-C5 and potassium-solubilizing bacteria HFF-0-C43 has a better control effect on stem rot. Among them, the treatment group using the coating agent has the highest control efficacy, reaching 57.48%.

[0090] Table 7 Evaluation of the control effects of different compound microbial agents on maize stalk rot

[0091]

[0092] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A microbial ensemble for controlling maize stalk rot and possessing growth-promoting functions, characterized in that, Including Paenibacillus peoriae HFF-0-C5 and Bacillus licheniformis HFF-0-C43; The *Bacillus pilaris* HFF-0-C5 was deposited on October 23, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 28717. The Bacillus licheniformis HFF-0-C43 was deposited on October 23, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 28718. The ratio of the number of *Bacillus pilaris* HFF-0-C5 to the number of *Bacillus licheniformis* HFF-0-C43 is 1:

1.

2. The application of the microbial combination as described in claim 1 in the preparation of a compound microbial agent with functions of preventing and controlling corn stalk rot and promoting growth.

3. The application according to claim 2, characterized in that, The compound microbial agent is a liquid microbial agent, a coating agent, or granules.

4. A compound microbial agent with functions of preventing and controlling corn stalk rot and promoting growth, characterized in that, The active ingredient includes the microbial ensemble described in claim 1.

5. The compound microbial agent according to claim 4, characterized in that, The compound microbial agent also includes auxiliary materials.

6. The compound microbial agent according to claim 5, characterized in that, The compound microbial agent is available in liquid form, coating form, or granule form.

7. The compound microbial agent according to claim 6, characterized in that, When the compound microbial agent is in the form of a liquid microbial agent, the excipients are gum arabic powder, Tween 80, and tea polyphenols; When the formulation of the compound microbial agent is a coating agent, the excipient is a film-forming agent; When the compound microbial agent is in granule form, the excipients are kaolin and tyrosine.

8. The application of a microbial combination as described in claim 1 or a compound microbial agent as described in any one of claims 4-7 in the prevention and control of maize stalk rot and / or the improvement of maize yield.

Images