Bacillus pseudomycoides BN59 and application thereof in wheat growth promotion

Abstract

The invention belongs to the technical field of microbial fertilizers, and particularly relates to bacillus pseudomycoides BN59 and application thereof in wheat growth promotion. The invention discloses a strain of bacillus pseudomycoides BN59, and the preservation number of the bacillus pseudomycoides BN59 is CGMCC (China General Microbiological Culture Collection Center) No.36454. The bacillus pseudomycoides BN59 provided by the invention has a remarkable growth promoting effect on root length, plant height and fresh weight of wheat in a seedling stage, and has a remarkable promoting effect on plant height and yield of potted wheat, and the field growth promoting and yield increasing effect can reach 19.21%. Meanwhile, the BN59 is a natural microorganism and has high safety to plants, the environment, people and livestock, toxic and harmful substances cannot be generated in the fermentation and bacterium liquid preparation process, and theoretical support and a technical approach are provided for development and production of the wheat microbial fertilizer.

Description

Technical Field

[0001] This invention belongs to the field of microbial fertilizer technology, specifically relating to Bacillus pseudomycetes BN59 and its application in promoting wheat growth. Background Technology

[0002] wheat( Triticum aestivum L. is an important staple crop worldwide, serving as the food source for 30% of the population. It provides humans with 8-20% of their protein and 20% of their energy.

[0003] To increase wheat yields, people mainly rely on the application of chemical fertilizers. Long-term, excessive use of chemical fertilizers has caused a series of production and environmental problems, such as declining land productivity, heavy metal accumulation (mainly from chemical phosphate fertilizers), eutrophication of water bodies, reduced biodiversity, and food safety issues. Currently, the main measures to reduce chemical fertilizer use in my country's agricultural production include traditional techniques such as soil testing and formula fertilization, precise fertilization based on soil nutrient supply and crop nutrient requirements, and organic-inorganic fertilizer combinations. Under conditions of continuous cropping and high yields, relying solely on traditional techniques to reduce fertilizer use and increase efficiency is far from sufficient. There is an urgent need to develop new ideas, new technologies, and new products. Microbial biotechnology is one of the important means to activate soil nutrients, promote plant growth, enhance crop resistance, improve fertilizer efficiency, and reduce chemical fertilizer use.

[0004] Plant growth-promoting rhizobacteria (PGPR) possess diverse physiological and ecological functions and are safe, non-toxic, resource-saving, and environmentally friendly. Currently, over 10,000 PGPR microbial fertilizers are registered in my country, with live bacteria primarily consisting of Bacillus subtilis, Bacillus mucilaginosa, and Bacillus megaterium. However, only about 500 PGPR microbial fertilizers are registered for use on wheat, and these exhibit unsatisfactory effects and high costs. Therefore, there is an urgent need to breed superior wheat PGPR strains to provide reliable microbial resources for increasing yields and reducing fertilizer application in wheat cultivation. Summary of the Invention

[0005] The purpose of this invention is to provide the application of Bacillus pseudomycosis BN59 in wheat growth promotion, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The first objective of this invention is to provide a strain of *Bacillus pseudomycosis* BN59, which has the accession number CGMCC No. 36454.

[0007] A second objective of this invention is to provide the application of the aforementioned Bacillus pseudomycosis BN59 in promoting wheat growth.

[0008] Furthermore, the wheat growth promotion includes: promoting wheat root length, bud length, plant height, fresh weight, ear length, and number of grains per ear.

[0009] A third objective of this invention is to provide a method for promoting wheat growth using the aforementioned *Bacillus mycoides* BN59, specifically: treating wheat seeds with the fermentation broth of *Bacillus mycoides* BN59; wherein the viable count of the *Bacillus mycoides* BN59 is not less than 1.0 × 10⁻⁶. 7 CFU / mL.

[0010] Furthermore, the fermentation broth of Bacillus pseudomycosis BN59 is diluted 10-100 times.

[0011] Furthermore, the wheat seeds are treated by the Bacillus pseudomycosis BN59 fermentation broth through irrigation, soaking, and / or seed coating.

[0012] A fourth objective of this invention is to provide a wheat microbial fertilizer, wherein the wheat microbial fertilizer comprises the Bacillus pseudomycosis BN59 or its fermentation broth.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The *Bacillus pseudomycosis* BN59 provided by this invention has a significant growth-promoting effect on root length, plant height, and fresh weight of wheat seedlings, and a significant promoting effect on plant height and yield of potted wheat. The field growth-promoting and yield-increasing effect can reach 19.21%, effectively promoting wheat growth and increasing yield. Furthermore, BN59 is a natural microorganism derived from the rhizosphere soil of wheat, exhibiting high safety for plants, the environment, and humans and animals, and does not produce toxic or harmful substances during fermentation and bacterial solution preparation. Biological Preservation Instructions

[0014] Bacillus pseudomycosis ( Baclllus pseudomycoides BN59 was deposited on December 31, 2025, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 36454. Attached Figure Description

[0015] Figure 1 The image shows the morphology of a single colony of BN59 on an LB plate (a) and the scanning electron microscope image of the bacterial cells (b). Figure 2 Phylogenetic tree of BN59 16S rRNA sequence; Figure 3The image shows the growth-promoting effect of BN59 on wheat under plate filter paper conditions. From left to right, the images are: sterile water (blank control), BN59 stock solution, BN59 diluted 10 times, BN59 diluted 100 times, and BN59 diluted 1000 times. Figure 4 The image shows the growth-promoting effect of BN59 on wheat under fine sand conditions in a petri dish. From left to right, the control group (CK) is sterile water, BN59 diluted 10 times, and BN59 diluted 100 times. Figure 5 The image shows the growth-promoting effect of BN59 on wheat under pot cultivation conditions. From left to right, the images show sterile water (blank control), LB diluted 10 times (culture medium control), and BN59 diluted 10 times. Figure 6 The images show the effects of manual seed dressing of BN59, with the results from left to right: seed dressing with BN59 diluted 10 times and seed dressing with 1.5‰ xanthan gum (control). Figure 7 Figures (a), (b), and (c) show the wheat greening period, grain-filling period, and harvest period under field conditions of BN59. Detailed Implementation

[0016] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0017] 1. Materials and Methods 1.1 Strain screening The *Bacillus pseudomycosis* BN59 strain of this invention was screened from the topsoil of dryland farmland in wheat-maize rotation in Laowo Town, Zhaoling District, Luohe City, Henan Province. The specific method is as follows: Bacillus spores in wheat rhizosphere soil samples were isolated using a water bath method and a serial dilution method. 10g of soil sample was added to 100mL of sterile water, vortexed for 5 minutes, and then incubated in an 85℃ water bath for 10 minutes. The soil suspension was then resuspended and serially diluted 100 times. -1 -10 -2 The extract was evenly spread on LB solid medium plates (5g yeast extract, 10g tryptone, 10g sodium chloride, 20g agar, deionized water to 1L, pH adjusted to 7.0) and cultured in triplicate. After 36 hours, single colonies were observed, and colonies with different morphology, color and size were picked for streak plating purification culture.

[0018] After the above steps, 105 spore-forming bacteria of varying colors, morphologies, and sizes were selected for use. Using the filter paper disc method, the spore-forming bacteria with the best wheat-promoting effect was selected from these 105 strains and designated as BN59.

[0019] 2.1 Identification of strain BN59 2.1.1 Morphological identification See results Figure 1 .

[0020] Depend on Figure 1 As can be seen from this, strain BN59 on LB medium exhibits single colonies that are off-white, nearly round, with almost regular edges, a rough and uneven surface, regular wrinkles, and are opaque, with a single colony diameter of approximately 6 mm; Figure 1 b indicates that strain BN59 has a bacillus morphology with a size of 20-30 μm.

[0021] 2.1.2 Physiological and biochemical identification Physiological and biochemical identification was performed in accordance with the "Handbook for Systematic Identification of Common Bacteria" (Dong Xiuzhu and Cai Miaoying, 2001), and the specific results are shown in Table 1.

[0022] Table 1. Physiological and biochemical test results of strain BN59 Measurement items result Measurement items result Gram staining + Methyl red test - Starch hydrolysis test + Indole test + Gelatin liquefaction test + Salt tolerance 2% NaCl Oxidase reaction + Salt tolerance 4% NaCl + VP reaction - Salt tolerance 6% NaCl + Catalase reaction + Salt tolerance of 8% NaCl + Nitric acid reduction reaction + Salt tolerance of 10% NaCl - Phenylalanine dehydrogenase - -- -- As shown in Table 1, strain BN59 was positive for Gram staining, starch hydrolysis, oxidase reaction, catalase reaction and indole test; it was negative for methyl red, VP and phenylalanine dehydrogenase test; it has a certain salt tolerance and can grow in <8% NaCl medium.

[0023] Preliminary identification based on morphological and physiological biochemical tests has confirmed that BN59 belongs to Bacillus.

[0024] 2.1.3 Analytical biological identification Genomic DNA from BN59 was extracted using the Ezup column-based bacterial genomic DNA rapid extraction kit and then amplified by PCR. The 16S rRNA amplification primers are shown in Table 2, and the PCR amplification system is shown in Table 3.

[0025] Table 2 Primer Sequences reagents Base sequence (5'-3') UP-27F AGAGTTTGATCMTGGCTCAG UP-1495R GGTTACCTTGTTACGACTT Table 3 PCR reaction system reagents Amount added (µL) 10 X PCR Buffer 2.5 dNTPs (each 10mM) 1 Taq Plus DNA Polymerase (5U / µL) 1 50mM MgSO4 1 Primer F 1 Primer R (10µM) 1 Template (DNA) 1 <![CDATA[ddH2O]]> 16.5 The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 90 s, 30 cycles; 72℃ reaction for 10 min.

[0026] After amplification, the PCR product was sequenced, yielding a 1147 bp sequence, as shown in SEQ ID NO. 1. The sequenced 16S rRNA gene sequence was then compared with nucleic acid data in GenBank using BLAST for homology analysis. When constructing a phylogenetic tree using the 16S rRNA gene sequence, *E. coli* was used as the primary gene source. E. coli K-12 (J01695) was selected as the outgroup. A phylogenetic tree was constructed using the Neighbour-joining method; the results are shown below. Figure 2 .

[0027] Depend on Figure 2 It can be seen that homology comparison of BN59 using 16S rRNA sequence shows that BN59 has a homology of 99.95% with Bacillus pseudomycosis XZPGS4JN999843.1, and the phylogenetic tree results are consistent.

[0028] In summary, based on morphological, physiological, biochemical, and molecular biological identification, strain BN59 of this invention has been identified as *Bacillus pseudomycosis*. Bacillus pseudomycoides .

[0029] 3. The growth-promoting effect of strain BN59 on wheat seedlings was determined using the plate filter paper disc method. The *Bacillus pseudomycosis* BN59 strain screened in Example 1 was fermented and cultured, as follows: (1) Activation of microbial strains BN59 was activated using the streak plate method on LB solid medium. A small amount of BN59 inoculum was dipped into a sterile bacterial inoculation loop and streaked evenly in a zigzag pattern on an LB plate. The plate was then incubated in the dark at 37°C for 24 hours. The LB solid medium formulation was as follows: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 20 g agar, and deionized water was added to a final volume of 1 L and the pH was adjusted to 7.0. (2) Preparation of seed culture for growth-promoting bacteria Select activated BN59 single colonies and transfer them to LB liquid medium. Incubate at 35°C and 150 rpm for 24 hours. The LB liquid medium formula is: 5g yeast extract, 10g tryptone, 10g sodium chloride, and deionized water to a final volume of 1L. Adjust the pH to 7.0. (3) Preparation of fermentation broth 2.5 mL of BN59 seed culture was added to 250 mL of LB medium and incubated at 35℃ and 180 rpm for 120 h. The viable cell count in the fermentation broth was determined to be 1.18 × 10⁻⁶ using the plate dilution method. 9 CFU / mL.

[0030] Wheat hybrid seeds that are plump, undamaged, and uniform in size were rinsed three times with sterile water, then soaked in sterile water for 5 hours to allow them to swell, and then placed in the dark at 25°C for 24 hours to germinate until they show white sprouts. Ten white sprouted wheat seeds of similar size were selected and evenly placed in sterile petri dishes (Ф=9cm) with sterile filter paper at the bottom. Then, 10 mL of the original fermentation broth, a 10-fold dilution, a 100-fold dilution, and a 1000-fold dilution were added, respectively. Simultaneously, 10 mL of sterile water, LB broth, a 10-fold dilution, a 100-fold dilution, and a 1000-fold dilution were added as parallel control groups. Each treatment was repeated seven times. Finally, the petri dishes were placed in a constant temperature incubator and cultured in the dark at 25°C for 5 days. The number of roots, the longest root length, and the shoot length of the wheat were then examined. The results are shown in Table 4 and [Table data would be inserted here]. Figure 3 .

[0031] Table 4. Growth-promoting effects of strain BN59 on wheat root number, root length, and shoot length. Note: Lowercase letters in the table indicate significant differences between columns (α=0.05), and uppercase letters indicate highly significant differences between columns (α=0.01). The same applies to the following tables.

[0032] From Table 2 and Figure 3 It was found that the 10-fold dilution of BN59 had the most significant effect on promoting wheat growth compared with other treatments and the control group, with the number of roots and the length of shoots reaching extremely significant maximum values ​​of 5.46 mm and 85.46 mm, respectively. The root length of 59.29 mm was not significantly different from other treatments. The 100-fold dilution of BN59 had a significant effect on promoting wheat shoot length compared with the control group, reaching 76.93 mm. At the same time, both the original fermentation broth and the original LB modified medium could inhibit wheat growth by 100%, indicating that the original solutions had a toxic effect on wheat growth. There were no significant differences in the growth-promoting indicators of root number, root length, and shoot length between the sterile water control and the LB modified medium control, indicating that the growth-promoting factor was strain BN59, rather than the LB modified medium.

[0033] 4. The growth-promoting effect of strain BN59 on wheat seedlings was determined using the fine sand plate method. As mentioned above, 10-fold and 100-fold dilutions of BN59 fermentation broth have a growth-promoting effect on wheat seedlings, independent of the LB modified medium. Therefore, this example was designed as follows: 20 mL of 10-fold and 100-fold dilutions of BN59 fermentation broth and sterile water (control) were added to 70 g of sterile, dry fine sand (Ф=9 cm) that had passed through a 40-mesh sieve. After the bacterial solution completely moistened the fine sand, 10 uniformly sized, germinated wheat seeds with white tips were evenly planted in the culture medium. Each treatment was repeated 4 times. The culture dishes were then placed in a 25℃ constant temperature and light incubator (L:D=14:10) for 10 days. The root length, shoot length, fresh weight, and dry weight of the wheat were recorded. Simultaneously, from the second day onwards, a certain amount of sterile water was added daily to ensure sufficient water supply, which is beneficial for normal wheat growth. The experimental results are shown in Tables 5 and 6. Figure 4 .

[0034] Table 5. Effects of strain BN59 on root number, root length, and shoot length of wheat seedlings grown in sand culture. Table 6. Effects of strain BN59 on the fresh and dry weight of wheat seedlings grown in sand culture. From Table 5, Table 6 and Figure 4 It can be seen that the number of roots in the water control group was 4.61, the root length was 89.46 mm, the shoot length was 101.36 mm, the fresh weight was 0.176 g, and the dry weight was 0.027 g. The number of roots in the wheat treated with strain BN59 diluted 10 times was 7.32, the root length was 112.93 mm, the shoot length was 153.50 mm, the fresh weight was 0.301 g, and the dry weight was 0.045 g. The number of roots in the wheat treated with strain 59-7 diluted 100 times was 6.82, the root length was 90.14 mm, the shoot length was 128.00 mm, the fresh weight was 0.235 g, and the dry weight was 0.039 g.

[0035] There were highly significant differences in root number, shoot length, fresh weight, and dry weight among wheat treated with CK and BN59 at dilutions of 10-fold and 100-fold, but no significant difference in root length among the three treatments. (Based on the data in the table...) Figure 4 It can be seen that, compared with the CK group, both the 10-fold and 100-fold dilutions of the strain promoted the growth of wheat, and the 10-fold dilution of strain BN59 had the most significant effect on promoting the growth of wheat seedlings.

[0036] 5. The growth-promoting effect of strain BN59 on wheat seedlings was determined using the seed soaking pot method. As mentioned above, a 10-fold dilution of BN59 fermentation broth had the most significant effect on promoting wheat seedling growth. Therefore, this example was designed as follows: Sixty uniformly sized, plump wheat hybrid seeds were selected and soaked for 5 hours in a 10-fold dilution of BN59 fermentation broth, sterile water (blank control), and a 10-fold dilution of LB broth (culture medium control). These seeds were then evenly planted in flowerpots containing 4.5 kg of soil (total soil volume 6 L: bottom diameter: 15.7 cm, height: 17 cm, mouth diameter: 26.5 cm), with each treatment repeated four times. The flowerpots were then randomly placed in an open area, with water replenished quantitatively as needed until the wheat matured. The number of plants, plant height, thousand-grain weight, and yield of the potted wheat were recorded. The results are shown in Tables 7 and 8. Figure 5 .

[0037] Table 7. Effects of strain BN59 on the number and height of potted wheat plants. Different treatments Dilution factor Number of plants (plants) Plant height (mm) CK — 46.75±4.50a 382.40±8.57Aa LB medium 10 47.75±4.99a 432.50±25.94Bb BN59 fermentation broth 10 51.25±4.35a 443.70±23.00Bb Table 8. Effects of strain BN59 on thousand-grain weight and yield of potted wheat. Different treatments Dilution factor Number of grains per pot (grains) 1000-grain weight (g) Production increase rate (%) CK — 41.50±5.07 Aa 33.83±1.96 Aa — LB medium 10 42.75±4.57 Aa 34.30±1.48 Aa 3.43 BN59 fermentation broth 10 123.25±6.99Bb 40.17±1.83Ab 254.06 From Table 7, Table 8 and Figure 5 It was found that, under pot cultivation conditions, BN59 diluted 10 times significantly promoted the plant height and number of grains per pot of wheat compared to the two controls of sterile water and LB diluted 10 times, with values ​​of 443.70 mm and 123.25 grains, respectively. The thousand-grain weight increased significantly by 40.17 g, and the yield increase rate was as high as 254.06%. Meanwhile, there was no significant difference in emergence rate among the three.

[0038] 6. Field trials 6.1 Test Site Wheat field in Yinzhuang Village, Hongqi District, Xinxiang City, Henan Province.

[0039] 6.2 Test Varieties Low resistance 58.

[0040] 6.3 Test strains Strain BN59.

[0041] 6.4 Test Methods After BN59 was activated to produce single colonies, a single colony was picked and inoculated into LB medium. The culture was incubated at 37°C and 150 rpm for 5 days with shaking. Wheat seeds were then treated with a 10-fold dilution of 1.5‰ sterile xanthan gum (seed weight to bacterial solution volume: 20 kg: 1 L). 5 kg of wheat seeds and 0.25 L of the diluted bacterial solution were placed in a sealed plastic bag and manually shaken to mix the seeds (see attached image for seed treatment results). Figure 6 The seeds were then spread out and allowed to air dry naturally for 5 days before being sown by machine at a rate of 15 kg / mu.

[0042] The control group was treated with a 1.5‰ xanthan gum solution diluted 10 times with sterile water. After wheat was sown, the plots were divided into 40m plots per treatment. 2 Four replicates were set up. Normal fertilization and field management were implemented throughout the growth period. After wheat maturity, a five-point sampling method was used in each plot, with a 50cm long row of wheat sampled at each sampling point. Plant height, ear length, number of grains per ear, thousand-grain weight, and total weight of wheat sampled from each plot were recorded. Results are shown in Tables 9 and 10. Figure 7 .

[0043] Table 9 Effects of strain BN59 on plant height, spike length, and grain number per spike in wheat in the field. Different treatments Plant height (cm) Ear length (cm) Number of grains per ear (grains) 1.5‰ Xanthan Gum (CK) 63.4±1.74a 7.0±0.10a 35.35±2.73Aa BN59 fermentation broth 65.3±1.47a 7.3±0.22a 44.35±2.42Bb Table 10 Effects of strain BN59 on wheat plant number and plant height in the field. Different treatments 1000-grain weight (g) Yield (g) Production increase rate (%) 1.5‰ Xanthan Gum (CK) 45.72±2.98a 218.75±17.95a — BN59 fermentation broth 48.56±1.87a 270.75±30.69b 19.21 From Table 9, Table 10 and Figure 7 It was found that, under field conditions, the 10-fold dilution of strain BN59 did not show significant differences in plant height, spike length, and thousand-grain weight of wheat compared to the control group. However, there were extremely significant differences in the number of grains per spike, resulting in a significant increase in yield, with values ​​of 44.35 grains and 270.75 g, respectively, representing a yield increase rate of 19.21%. This indicates that strain BN59 has a significant effect on promoting growth and increasing yield of wheat under field conditions.

[0044] In summary, the *Bacillus pseudomycosis* BN59 provided by this invention has a significant promoting effect on root length, plant height, and fresh weight of wheat seedlings, and a significant promoting effect on plant height and yield of potted wheat. The field growth-promoting and yield-increasing effect can reach 19.21%, effectively promoting wheat growth and increasing yield. Furthermore, BN59 is a natural microorganism derived from the rhizosphere soil of wheat, exhibiting high safety for plants, the environment, and humans and animals, and does not produce toxic or harmful substances during fermentation and bacterial solution preparation.

[0045] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A strain of *Bacillus pseudomycosis* BN59, characterized in that, The Bacillus pseudomycosis BN59 has the accession number CGMCC No. 36454.

2. The application of Bacillus pseudomycosis BN59 as described in claim 1 in promoting wheat growth.

3. The application according to claim 2, characterized in that, The wheat growth promotion includes: promoting wheat root length, bud length, plant height, fresh weight, ear length, and number of grains per ear.

4. A method for promoting wheat growth using *Bacillus pseudomycosiscinus* BN59 as described in claim 1, characterized in that, Specifically, wheat seeds are treated with the fermentation broth of *Bacillus pseudomycosis* BN59; wherein the viable count of *Bacillus pseudomycosis* BN59 is not less than 1.0 × 10⁻⁶. 7 CFU / mL.

5. The method according to claim 4, characterized in that, The fermentation broth of Bacillus pseudomycosis BN59 was diluted 10-100 times.

6. The method according to claim 4, characterized in that, The Bacillus pseudomycosis BN59 fermentation broth is used to treat wheat seeds through irrigation, soaking, and / or seed coating.

7. A wheat microbial fertilizer, characterized in that, Includes the Bacillus pseudomycosis BN59 or its fermentation broth as described in claim 1.

Images