Acetobacter luviangensis and application thereof in shortening growth period of crops in rice-wheat rotation system

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to acetobacter luviangensis and application thereof in shortening the growth period of crops in a rice-wheat rotation system. The invention discloses an Acetobacter luvianense JY1053 strain, a preparation method of a microbial agent of the Acetobacter luvianense JY1053 strain, and application of the Acetobacter luvianense JY1053 strain in shortening the growth period of crops in a rice-wheat crop rotation system. The microbial agent can significantly shorten the growth periods of rice and wheat and increase the yield of rice and wheat, compared with conventional fertilization, the growth period in the rice season is shortened by 9 days, the yield is increased by 8.97%, the growth period in the wheat season is shortened by 5 days, and the yield is increased by 6.35%. In addition, the Acetobacter luwanensis JY1053 has a relatively good antagonistic effect on fusarium oxysporum cucumber specialized type, botrytis cinerea, alternaria tenuissima and fusarium solani, and the Acetobacter luwanensis JY1053 has a relatively good antagonistic effect on fusarium oxysporum cucumber specialized type, botrytis cinerea, alternaria tenuissima and fusarium solani.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a strain of Acetobacter rhodopsinus and its application in shortening the crop growth period in a rice-wheat rotation system. Background Technology

[0002] Rice-wheat rotation is a core planting system for achieving high yields year-round and ensuring national food security. However, this model has long faced a key contradiction: the excessively long rice growing season often leads to late wheat sowing, which in turn affects wheat seedling growth and final yield, creating a "time bottleneck" for year-round production. Existing solutions, such as selecting short-growing-season rice varieties or adjusting the sowing date, often come at the cost of sacrificing single-season yield or varietal adaptability. Meanwhile, soil-borne diseases (such as root rot and Fusarium head blight caused by Fusarium and Botrytis) accumulate year by year in the rotation system. Chemical control not only increases costs and poses residue risks but may also disrupt the soil microecological balance. Therefore, the market urgently needs a green biotechnology that can "effectively" shorten the field growth cycle of crops through physiological regulation without shortening the actual number of days of crop growth (avoiding the sacrifice of photosynthetic product accumulation), and create a favorable soil environment and growth window for subsequent crops. Summary of the Invention

[0003] The purpose of this invention is to solve the aforementioned technical problems by providing a strain of *Acetobacter lovaniensis* JY1053 and a microbial agent prepared from *Acetobacter lovaniensis* JY1053. By utilizing this microbial agent, the rice growing season can be shortened and yield increased in rice-wheat rotation, gaining valuable time for timely wheat sowing. Simultaneously, it continues to shorten the growing season and promote yield during the wheat season. Furthermore, *Acetobacter lovaniensis* JY1053 exhibits antagonistic activity against various fungal pathogens. The application of this microorganism can systematically protect the health of double-cropping crops, alleviate the dual constraints of "time-disease" in rice-wheat rotation, and provide a novel microbial technology pathway for improving year-round productivity.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] First, we provide a strain of Acetobacter lovaniensis JY1053, which was deposited on December 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36824. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0006] The *Acetobacter lovaniensis* JY1053 strain was isolated from healthy rice roots collected in Dushan County, Qiannan Buyi and Miao Autonomous Prefecture, Guizhou Province.

[0007] Based on the above scheme, the 16S rDNA sequence of Acetobacter lovaniensis JY1053 is shown in SEQ ID No.3.

[0008] This invention also provides a method for preparing a microbial inoculant of *Acetobacter lovaniensis* JY1053, comprising the following steps:

[0009] Acetobacter lovaniensis JY1053 was inoculated onto MRS solid medium for activation to obtain activated Acetobacter lovaniensis JY1053.

[0010] Activated Acetobacter lovaniensis JY1053 was inoculated into MRS liquid medium for primary fermentation to obtain primary seed culture of Acetobacter lovaniensis JY1053.

[0011] The primary seed culture of Acetobacter lovaniensis JY1053 was inoculated into a secondary fermentation medium for secondary fermentation to obtain the secondary seed culture of Acetobacter lovaniensis JY1053.

[0012] The secondary seed culture of Acetobacter lovaniensis JY1053 was inoculated into a tertiary fermentation medium for tertiary fermentation to obtain the microbial agent.

[0013] Based on the above scheme, the MRS solid culture medium is purchased from the Chindenuo brand, with the following formula: peptone: 10 g / L, beef extract: 5 g / L, yeast extract: 4 g / L, glucose: 20 g / L, dipotassium hydrogen phosphate: 2 g / L, triammonium citrate: 2 g / L, sodium acetate: 5 g / L, magnesium sulfate: 0.2 g / L, manganese sulfate: 0.05 g / L, Tween 80: 1 g / L, and agar: 15 g / L, with a pH of 6.2 ± 0.2.

[0014] The activation conditions for Acetobacter lovaniensis JY1053 are: temperature 28-32℃, time 2-3 days.

[0015] Based on the above scheme, the MRS liquid culture medium is a product of the Chindenuo brand, with the following formula: peptone: 10 g / L, beef extract powder: 5 g / L, yeast extract powder: 4 g / L, glucose: 20 g / L, dipotassium hydrogen phosphate: 2 g / L, triammonium citrate: 2 g / L, sodium acetate: 5 g / L, magnesium sulfate: 0.2 g / L, manganese sulfate: 0.05 g / L, and Tween 80: 1 g / L, with a pH of 6.2±0.2.

[0016] The process conditions for the primary fermentation are as follows: liquid volume 40%-60%, inoculum 1%-3%, culture temperature 28-32℃, and shaking culture at 180-200 rpm for 36-48 h.

[0017] Based on the above scheme, the secondary fermentation culture medium is the same as that of the tertiary fermentation culture medium, and the formula is: yeast extract 15 g / L, glucose 10 g / L, KH2PO4 4 g / L, and ethanol 30 mL / L (added after sterilization).

[0018] The process conditions for the secondary fermentation are as follows: liquid volume 50%-70%, inoculum 2%-10%, culture temperature 28-32℃, sodium hydroxide added to control the pH of the fermentation broth to 5.5-6.0, and shaking culture at 180-200 rpm for 16-24 h.

[0019] The three-stage fermentation process conditions are as follows: liquid volume 50%-70%, inoculum 2%-10%, culture temperature 28-32℃, sodium hydroxide added to control the pH of the fermentation broth to 5.5-6.0, and simultaneously adding 0.2%-1% of bitter melon polypeptide solution (6 g / L) by volume of the liquid, and shaking culture at 180-200 rpm for 24-48 h.

[0020] The present invention also provides an application of the aforementioned Acetobacter lovaniensis JY1053 or a microbial agent containing it in shortening the crop growth period in a rice-wheat rotation system.

[0021] The microbial agent disclosed in this invention can significantly shorten the growth period of rice and wheat and increase yield. Compared with conventional fertilization control, the growth period of rice is shortened by 9 days and the yield is increased by 8.97%, while the growth period of wheat is shortened by 5 days and the yield is increased by 6.35%, effectively improving the annual productivity of rice-wheat rotation.

[0022] The present invention also provides the efficacy of Acetobacter lovaniensis JY1053 in inhibiting plant fungal pathogens.

[0023] Based on the above scheme, the plant fungal pathogens include any one of Fusarium oxysporum cucumber-specific type, Botrytis cinerea, Alternaria alternata, and Fusarium solanum.

[0024] Beneficial effects of the present invention

[0025] This invention provides a strain of *Acetobacter lovaniensis* JY1053 and a method for preparing a microbial inoculant of *Acetobacter lovaniensis* JY1053. Through year-round field application in rice-wheat rotation, it was found that the microbial inoculant *Acetobacter lovaniensis* JY1053 significantly shortens the field growth period of rice from transplanting to maturity and increases yield during the rice season, thus gaining valuable time for timely wheat sowing. During the wheat season, it continues to shorten the growth period and promote yield, similarly providing sufficient time for the next rice production season. Furthermore, *Acetobacter lovaniensis* JY1053 exhibits antagonistic effects against various pathogens, including *Fusarium oxysporum* (specific strain of cucumber), *Botrytis cinerea*, *Alternaria alternata*, and *Fusarium solani*. The application of Acetobacter lovaniensis JY1053 can ensure the health of double-cropping crops, resolve the dual constraints of "time-disease" in rice-wheat rotation, and provide a new microbial technology path for year-round high and stable yields. Attached Figure Description

[0026] Figure 1 The colony morphology of Acetobacter lovaniensis JY1053 on the front of a plate;

[0027] Figure 2 Phylogenetic tree of the 16S rDNA sequence of Acetobacter lovaniensis JY1053 constructed by the neighbor-joining method;

[0028] Figure 3 The effects of Acetobacter lovaniensis JY1053 on the ripening stage of Japonica rice (46-year maturity) are shown in the left figure, which represents conventional fertilization, and the right figure represents conventional fertilization supplemented with Acetobacter lovaniensis JY1053 microbial agent.

[0029] Figure 4The image shows the antagonistic effect of Acetobacter lovaniensis JY1053 against Fusarium oxysporum cucumber-specific strain, Botrytis cinerea, Alternaria alternata, and Fusarium solani. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, other embodiments obtained by those skilled in the art without creative effort are all within the scope of protection of this invention.

[0031] Example 1: Isolation, purification, and identification of Acetobacter rosenbergii

[0032] (1) Isolation and purification of strains

[0033] Healthy rice roots were collected from Dushan County, Qiannan Buyi and Miao Autonomous Prefecture, Guizhou Province. The roots were rinsed with tap water, then disinfected with 5% sodium hypochlorite and 75% alcohol before grinding. Endophytic bacteria were isolated from the roots using the dilution plating method and cultured on MRS medium at 30°C for 2 days. The strain with high abundance was isolated and purified using the streak plating method and numbered JY1053. The colony morphology is shown in the figure. Figure 1 .

[0034] (2) Identification of strains

[0035] Species identification of JY1053 was performed using 16S rDNA identification technology. The 16S rDNA sequence of strain JY1053 was amplified using universal bacterial primers (sequences shown in SEQ ID No. 1 and SEQ ID No. 2). After PCR amplification, the PCR products were detected by electrophoresis on a 1% agarose gel. The gene sequence obtained after sequencing (nucleotide sequence of the 16S rDNA gene shown in SEQ ID No. 3) was compared and analyzed in NCBI. The comparison analysis showed that strain JY1053 had the highest homology with *Acetobacter lovaniensis* strain JCM 17121, with a sequence similarity of 99.85%. Multiple sequence alignment was performed using MEGA 7.0 software, and a phylogenetic tree was constructed, as shown below. Figure 2As shown, strain JY1053 clustered on the same branch as *Acetobacter lovaniensis* strain JCM 17121. Based on the combined colony morphology and 16S rDNA gene sequence determination, strain JY1053 was found to be most closely related to *Acetobacter lovaniensis*, and was named *Acetobacter lovaniensis* JY1053. *Acetobacter lovaniensis* was deposited on December 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36824, located at Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0036] SEQ ID No.1: 27F 5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ;

[0037] SEQ ID No.2: 1492R 5ʹ-TACGGCTACCTTGTTACGACTT-3ʹ;

[0038]

[0039] Example 2: Preparation of Acetobacter laurentii microbial inoculant

[0040] This embodiment will describe the preparation of the microbial inoculant, and the specific process is as follows:

[0041] (1) Activation of strain: Acetobacter lovaniensis JY1053 was inoculated on MRS solid medium and cultured at 30℃ for 2 days to obtain activated Acetobacter lovaniensis. The MRS medium used was Chinok brand, with the following formula: peptone: 10 g / L, beef extract: 5 g / L, yeast extract: 4 g / L, glucose: 20 g / L, dipotassium hydrogen phosphate: 2 g / L, triammonium citrate: 2 g / L, sodium acetate: 5 g / L, magnesium sulfate: 0.2 g / L, manganese sulfate: 0.05 g / L, Tween 80: 1 g / L and agar: 15 g / L, pH 6.2±0.2.

[0042] (2) Primary fermentation (50 mL): The liquid volume was 50%, and the inoculum size was 1%. Activated Acetobacter lovaniensis JY1053 was inoculated into MRS liquid medium for primary fermentation at 30℃ and shaken at 180 rpm for 36 h. The primary seed culture of Acetobacter lovaniensis JY1053 was obtained. The formula of MRS liquid medium was as follows: peptone: 10 g / L, beef extract powder: 5 g / L, yeast extract powder: 4 g / L, glucose: 20 g / L, dipotassium hydrogen phosphate: 2 g / L, triammonium citrate: 2 g / L, sodium acetate: 5 g / L, magnesium sulfate: 0.2 g / L, manganese sulfate: 0.05 g / L, and Tween 80: 1 g / L, pH 6.2±0.2.

[0043] (3) Secondary fermentation (20 L): The liquid volume was 70%. The primary seed culture of Acetobacter lovaniensis JY1053 was inoculated into the seed tank for secondary fermentation at an inoculation amount of 5%. The culture temperature was 30℃, and sodium hydroxide was added to control the pH of the fermentation broth to 5.5-6.0. The culture was carried out at 200 rpm for 16 h to obtain the secondary seed culture of Acetobacter lovaniensis JY1053. The culture medium formula used for secondary fermentation was: yeast extract 15 g / L, glucose 10 g / L, KH2PO4 4 g / L and ethanol 30 mL / L (added after sterilization).

[0044] (4) Tertiary fermentation (200 L): The liquid volume was 70%. The secondary seed culture of Acetobacter lovaniensis JY1053 was inoculated into the fermenter for tertiary fermentation at an inoculation volume of 5%. The culture temperature was 30℃. Sodium hydroxide was added to control the pH of the fermentation broth to 5.5-6.0. At the same time, 0.5% of bitter melon polypeptide solution (6 g / L) was added by volume. The culture was carried out at 200 rpm for 32 h. The culture medium formula used for tertiary fermentation was: yeast extract 15 g / L, glucose 10 g / L, KH2PO4 4 g / L, and ethanol 30 mL / L (added after sterilization). The effective viable count of Acetobacter lovaniensis JY1053 obtained from fermentation was 6 billion CFU / mL. After the fermentation in the production tank was completed, the culture medium was directly dispensed into liquid form, which is the microbial agent containing Acetobacter lovaniensis JY1053.

[0045] Example 3: Application of Acetobacter rewanensis in increasing yield in rice-wheat rotation

[0046] This embodiment provides the application of *Acetobacter laurentii* microbial inoculant in rice-wheat rotation to increase yield, and the implementation site is Gangoutou Village, Wujin District, Changzhou City, Jiangsu Province.

[0047] (1) Rice season

[0048] Rice variety: Nanjing 46.

[0049] Planting method: manual transplanting, with a row spacing of 25 cm and a plant spacing of 13 cm.

[0050] Experimental treatments: Treatment group 1: Conventional fertilization (according to local rice planting habits: nitrogen: 15 kg / mu, phosphorus pentoxide: 4.2 kg / mu, potassium oxide: 5.7 kg / mu; of which nitrogen fertilizer accounts for 60% of the base fertilizer, tillering fertilizer and panicle fertilizer each account for 20%, and phosphorus fertilizer and potassium fertilizer are applied as base fertilizer in one application); Treatment group 2: Conventional fertilization (same as treatment group 1) + spraying of Acetobacter laurentii microbial agent (base fertilizer: 2 L / mu of agent from Example 2, tillering fertilizer: 1 L / mu of agent from Example 2, panicle fertilizer: 1 L / mu of agent from Example 2).

[0051] Table 1. Growth period and yield of Nanjing 46 under different treatments

[0052]

[0053] The growth periods of rice in each treatment are shown in Table 1. After applying *Acetobacter laurentii* microbial agent, the entire growth period of Nanjing 46 was 9 days shorter than that after conventional fertilization. Phenotypic comparison is shown in Table 1. Figure 3Meanwhile, the application of Acetobacter lovaniensis microbial inoculant significantly increased the actual yield of Nanjing 46, by 8.97% compared to conventional fertilization. This indicates that Acetobacter lovaniensis JY1053 can significantly shorten the growth period of Nanjing 46 while increasing its yield.

[0054] (2) Wheat season

[0055] Wheat variety: Yangmai 25.

[0056] Planting method: machine row sowing, seeding amount is shown in Table 2.

[0057] Experimental treatments: Treatment group 1: Conventional fertilization (according to local wheat planting practices: nitrogen: 12 kg / mu, phosphorus pentoxide: 8 kg / mu, potassium oxide: 6.7 kg / mu; of which nitrogen fertilizer accounts for 60% of the base fertilizer, jointing fertilizer accounts for 40%, and phosphorus fertilizer and potassium fertilizer are applied as base fertilizer in one application); Treatment group 2: Conventional fertilization (same as treatment group 1) + spraying of Acetobacter laurentii microbial agent (base fertilizer: 2 L / mu of agent from Example 2, jointing fertilizer: 1 L / mu of agent from Example 2).

[0058] Table 2. Growth period and yield of Yangmai 25 under different treatments

[0059]

[0060] The land used for treatment group 1 was the same as that used for treatment group 1 during the rice season, and the land used for treatment group 2 was the same as that used for treatment group 2 during the rice season. The wheat growth period for each treatment is shown in Table 1. Applying *Acetobacter lovaniensis* microbial inoculant shortened the growth period of Yangmai 25 by 5 days compared to conventional fertilization; the actual yield increased by 6.35% compared to conventional fertilization. This indicates that *Acetobacter lovaniensis* JY1053 can shorten the growth period of Yangmai 25 and increase yield.

[0061] The combined results from both rice and wheat seasons indicate that *Acetobacter lovaniensis* JY1053 can shorten the growth period of rice and wheat, increase the yield of both rice and wheat, and improve the annual productivity of rice and wheat.

[0062] Example 4: Antagonistic experiment of Acetobacter lovaniensis JY1053 against Fusarium oxysporum cucumber-specific strain, Botrytis cinerea, Alternaria alternata, and Fusarium solani.

[0063] The plate confrontation method was used: Fusarium oxysporum cucumber-specific strain, Botrytis cinerea, Alternaria alternata, and Fusarium solanum were respectively inoculated into the center of PDA medium plates. Acetobacter lovaniensis JY1053 was inoculated into the medium at a distance of 2.5 cm from the center using a cross-inoculation method. The control group was not inoculated with Acetobacter lovaniensis JY1053. The plates were incubated at 25°C for 7 days, and the diameter of the pathogen colonies was measured. Each treatment was repeated 3 times.

[0064] Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / Control colony diameter * 100%

[0065] Table 3. Inhibitory effects of Acetobacter lovaniensis JY1053 on Fusarium oxysporum (specific strain of cucumber), Staphylococcus aureus, Alternaria alternata, and Fusarium solani.

[0066]

[0067] The results are as follows Figure 4 As shown in Table 3, Acetobacter lovaniensis JY1053 has good antagonistic effects against Fusarium oxysporum cucumber-specific type, Botrytis cinerea, Alternaria alternata, and Fusarium solani, with inhibition rates of 44.52%, 42.95%, 34.63%, and 34.48%, respectively.

Claims

1. A strain of Acetobacter lovaniensis JY1053, characterized in that, The described *Acetobacter rhovenensis* was deposited on December 1, 2025, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 36824, address: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. A microbial inoculant, characterized in that, The microbial agent contains Acetobacter lovaniensis JY1053 as described in claim 1.

3. A method for preparing a microbial inoculant containing *Acetobacter repens*, characterized in that, The preparation method includes the following steps: The Acetobacter lovaniensis JY1053 of claim 1 was inoculated onto MRS solid medium for activation to obtain activated Acetobacter lovaniensis JY1053. Activated Acetobacter lovaniensis JY1053 was inoculated into MRS liquid medium for primary fermentation to obtain primary seed culture of Acetobacter lovaniensis JY1053. The primary seed culture of Acetobacter lovaniensis JY1053 was inoculated into a secondary fermentation medium for secondary fermentation to obtain the secondary seed culture of Acetobacter lovaniensis JY1053. The secondary seed culture of Acetobacter lovaniensis JY1053 was inoculated into a tertiary fermentation medium for tertiary fermentation to obtain the microbial agent.

4. The method for preparing microbial inoculants according to claim 3, characterized in that, The formulation of the MRS solid culture medium is as follows: peptone: 10 g / L, beef extract: 5 g / L, yeast extract: 4 g / L, glucose: 20 g / L, dipotassium hydrogen phosphate: 2 g / L, triammonium citrate: 2 g / L, sodium acetate: 5 g / L, magnesium sulfate: 0.2 g / L, manganese sulfate: 0.05 g / L, Tween 80: 1 g / L and agar: 15 g / L, pH 6.2±0.

2.

5. The method for preparing microbial inoculants according to claim 3, characterized in that, The activation conditions are: temperature 28-32℃, time 2-3 days.

6. The method for preparing microbial inoculants according to claim 3, characterized in that, The formulation for the MRS liquid culture is as follows: peptone: 10 g / L, beef extract: 5 g / L, yeast extract: 4 g / L, glucose: 20 g / L, dipotassium hydrogen phosphate: 2 g / L, triammonium citrate: 2 g / L, sodium acetate: 5 g / L, magnesium sulfate: 0.2 g / L, manganese sulfate: 0.05 g / L and Tween 80: 1 g / L, pH 6.2±0.2; The conditions for primary fermentation are as follows: liquid volume 40%-60%, inoculum amount 1%-3% of the volume of primary fermentation medium, culture temperature 28-32℃, and shaking culture at 180-200 rpm for 36-48 h.

7. The method for preparing microbial inoculants according to claim 3, characterized in that, The formulation of the secondary and tertiary fermentation media is as follows: yeast extract 15 g / L, glucose 10 g / L, KH2PO4 4 g / L, and ethanol 30 mL / L.

8. The method for preparing microbial inoculants according to claim 3, characterized in that, The conditions for the secondary fermentation are as follows: liquid volume 50%-70%, inoculum amount 2%-10% of the volume of the secondary fermentation medium, culture temperature 28-32℃, sodium hydroxide added to control the pH of the fermentation broth to 5.5-6.0, and shaking culture at 180-200 rpm for 16-24 h. The conditions for the tertiary fermentation are as follows: liquid volume 50%-70%, inoculum 2%-10% of the volume of the tertiary fermentation medium, culture temperature 28-32℃, sodium hydroxide added to control the pH of the fermentation broth to 5.5-6.0, and simultaneously adding 0.2%-1% of bitter melon polypeptide solution by volume of liquid, wherein the concentration of bitter melon polypeptide is 6 g / L, and cultured with shaking at 180-200 rpm for 24-48 h.

9. The application of the *Acetobacter lovaniensis* JY1053 as described in claim 1, or the microbial agent as described in claim 3, or the microbial agent prepared by the method described in claims 3-7, in a rice-wheat rotation system to shorten the crop growth period or increase yield.

10. The application of a microbial agent prepared by the method described in claims 1 (Acetobacter lovaniensis JY1053) or claim 2, or claims 3-7, in inhibiting plant fungal pathogens.

11. The use of a microbial agent prepared by the method described in claim 1 (Acetobacter lovaniensis JY1053), claim 2, or claims 3-7, in the preparation of a product that inhibits plant fungal pathogens.

12. The application according to claim 10 or 11, characterized in that, The plant fungal pathogens include at least one of Fusarium oxysporum cucumber-specific strain, Botrytis cinerea, Alternaria alternata, and Fusarium solanum.

Images