Application of thymol ZJLQ024 in promoting rapeseed growth

Abstract

This invention discloses the application of *Mucor* ZJLQ024 in promoting rapeseed growth, belonging to the field of microbial technology. *Mucor* ZJLQ024 is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 2863. This invention provides a novel use of *Mucor* ZJLQ024 in promoting rapeseed growth and / or increasing rapeseed yield. Applying *Mucor* ZJLQ024 by root dipping before transplanting can promote root growth in rapeseed seedlings, improve transplant survival rate; significantly increase rapeseed plant height and aboveground growth; improve pod traits, increasing pod length and width; increase yield components such as thousand-grain weight; and achieve stable yield increases under field conditions.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to the application of *Mucor* ZJLQ024 in promoting rapeseed growth. Background Technology

[0002] A vast number of beneficial microorganisms exist in natural ecosystems, among which plant endophytic microorganisms are an important component. Plant endophytic microorganisms generally refer to a class of microorganisms that can colonize healthy plant tissues at least for a part of their life cycle without causing obvious disease symptoms in the host. They are ubiquitous in soil-plant systems and can form relatively stable long-term interaction relationships with host plants.

[0003] In the mutually beneficial symbiotic relationship between plants and endophytic microorganisms, endophytic microorganisms obtain carbon sources, mineral nutrients, and a suitable living environment from the host plant. On the other hand, endophytic microorganisms promote plant growth, increase crop yield, and enhance their adaptability to biotic and abiotic stresses through nitrogen fixation, phosphorus and potassium solubilization, secretion of plant hormone-like substances, production of stress-resistance-related metabolites, and inhibition of pathogens. With the deepening of research on plant microecology, the mechanisms of plant-microorganism interactions and their agricultural applications have become a research hotspot both domestically and internationally.

[0004] Rapeseed is one of my country's important oilseed and economic crops, and rapeseed is a significant source of edible vegetable oil and protein feed. Achieving stable and high yields and improving the quality of rapeseed is of great significance for ensuring the security of edible oil supply and promoting green agricultural development. Recent studies have found that in addition to being influenced by its own genetic characteristics and cultivation practices, the growth and development of rapeseed are also significantly regulated by the rhizosphere and endophytic microbial communities.

[0005] Constructing a "microbial yield-enhancing and efficiency-enhancing" pathway for rapeseed by utilizing beneficial endophytic microorganisms is expected to reduce the input of chemical fertilizers and pesticides, while improving the rapeseed's tolerance to adverse environments such as low temperature, drought, and salt stress, and reducing the risk of diseases such as sclerotinia rot and clubroot, thereby improving the overall and ecological benefits of rapeseed production.

[0006] In production practice, rapeseed requires high seedling quality and stress resistance from sowing and emergence to overwintering and greening (or from seedling stage to bolting and flowering). Due to problems such as large sowing period, frequent low-temperature freezing damage, insufficient soil nutrient availability, continuous cropping obstacles, and disease pressure in some areas, uneven emergence, weak seedlings, poor root development, insufficient population size before winter, or slow greening often occur. This leads to a reduction in effective branches, a decrease in the number of pods, and insufficient grain filling, ultimately resulting in a decrease in yield and oil content.

[0007] Current methods for improving rapeseed yield and quality mainly rely on chemical fertilizers, plant growth regulators, and chemical control measures. However, these methods suffer from high input costs, increased environmental risks, potential soil microecological imbalance, and increased pesticide resistance. Therefore, developing a safe, stable, and effective endophytic microorganism that can colonize rapeseed or its rhizosphere and significantly promote robust growth, enhance stress resistance, and increase yield and oil content, along with its application methods, is of significant practical importance and has broad application prospects.

[0008] Currently, there is still a relative shortage of functional strains of rapeseed endophytic microorganisms that can improve seedling vigor, stress resistance, yield, and quality. Furthermore, the formulation and field application methods for their large-scale application in rapeseed production still require further research and improvement.

[0009] Therefore, it is urgent to screen and obtain an endophytic microorganism that has a significant growth-promoting and yield-increasing effect on rapeseed, and to establish its application method in rapeseed cultivation in order to meet the needs of green and efficient rapeseed production. Summary of the Invention

[0010] The purpose of this invention is to provide a microorganism that can promote the growth of rapeseed, and to develop it into a bio-fertilizer for application in rapeseed cultivation, so as to achieve stable and high yield of rapeseed through symbiotic interaction with rapeseed.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides thymol ( Muscodor The application of sp.) ZJLQ024 in promoting rapeseed growth and / or increasing rapeseed yield, wherein sp. ZJLQ024 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 2863.

[0012] The strain *Mucor* ZJLQ024 is a publicly available biological material. It was isolated from the leaves of a broad-leaved tree in the Fengyangshan Nature Reserve, Zhejiang Province, and belongs to the kingdom Fungi. Fungi Ascomycota ( Ascomycota ), Scale-class ( Sordariomycetes ), Chlorophycetes ( Xylariales ), Carbonaceae ( Xylariaceae ), genus *Mucor* ( Muscodor ), Fengyang Musk Mold ( Muscodor fengyangensis ).

[0013] This invention has found that applying *Tetrandibella thymol* ZJLQ024 to the roots of rapeseed plants and interacting symbiotically with the rapeseed promotes rapeseed growth and significantly improves the quality of rapeseed seedlings. This is manifested in: promoting root growth and increasing transplant survival rate; significantly increasing plant height and above-ground growth; and improving pod characteristics, increasing pod length and width. Furthermore, it has a yield-increasing effect on rapeseed, as evidenced by: significantly increasing yield components such as thousand-seed weight; compared to the control group, rapeseed treated with *Tetrandibella thymol* ZJLQ024 under field conditions showed an actual yield increase of 11.9%.

[0014] Furthermore, the indicators for rapeseed growth include at least one of the following: plant height, stem diameter, number of tillers, chlorophyll content, pod length, and pod width.

[0015] Furthermore, the application includes applying thymol ZJLQ024 to the roots of rapeseed seedlings by root dipping before transplanting.

[0016] Furthermore, the application includes: inoculating activated *Mucor* ZJLQ024 into a liquid fermentation medium for fermentation culture, collecting the fermentation broth, and preparing a viable cell count concentration ≥2×10⁻⁶. 8 A treatment solution of CFU / mL was prepared; then, thymol ZJLQ024 was applied to the roots of rapeseed seedlings by root dipping.

[0017] The liquid fermentation medium consists of: 4 g / L soybean meal, 10 g / L corn flour, 0.5 g / L magnesium sulfate, and 1 g / L potassium dihydrogen phosphate.

[0018] Another object of the present invention is to provide a method for promoting rapeseed growth and increasing rapeseed yield using *Tetrandex thymol* ZJLQ024, the method comprising the following steps: (1) The *Mucor* strain ZJLQ024 with preservation number CGMCC No. 2863 was inoculated into liquid fermentation medium for fermentation culture, and the fermentation broth was collected to prepare a viable bacterial count concentration ≥2×10⁻⁶. 8 The treatment solution contains CFU / mL; the liquid fermentation medium consists of: 4 g / L soybean meal, 10 g / L corn flour, 0.5 g / L magnesium sulfate, and 1 g / L potassium dihydrogen phosphate. (2) The roots of rapeseed seedlings are immersed in the treatment solution for root dipping treatment, and then the rapeseed seedlings are transplanted to the field and cultivated until harvest. During this process, the bacterial strain interacts symbiotically with rapeseed, thereby promoting rapeseed growth and achieving stable and high yield of rapeseed.

[0019] Further, in step (1), the activated thymol ZJLQ024 is inoculated into liquid fermentation medium and cultured to the exponential phase to obtain seed liquid; then the seed liquid is inoculated into liquid fermentation medium for secondary propagation culture; then the fermentation liquid is concentrated and filtered to remove soybean meal and corn flour, and the filtrate is collected and diluted to obtain the treatment liquid.

[0020] Furthermore, the activation conditions are as follows: thymol ZJLQ024 is inoculated into potato dextrose agar (PDA) medium and cultured in the dark at 22-25℃ for 7-10 days. The mycelia in the medium are white or pale yellow and grow vigorously.

[0021] Furthermore, the seed liquid is prepared under the following conditions: cultured at a temperature of 22-25℃ and a rotation speed of 100-150 rpm for 7-10 days.

[0022] Furthermore, the conditions for the secondary propagation culture are as follows: the seed liquid is inoculated into the liquid fermentation medium at a volume ratio of 5%, and cultured at 25-28°C with continuous stirring and oxygen supply for 48-72 hours.

[0023] Furthermore, the fermentation broth was sequentially filtered through filters with pore sizes of 0.45 μm and 0.22 μm to collect the filtrate and prepare the treatment solution.

[0024] Furthermore, in step (2), the root dipping treatment time is 15-20 minutes. Studies have shown that under the above bacterial solution concentration and soaking conditions, it is possible to ensure that the rapeseed roots are fully in contact with the bacterial solution.

[0025] The beneficial effects of this invention are as follows: This invention provides a novel use of thymol ZJLQ024 in promoting rapeseed growth and / or increasing rapeseed yield. By applying thymol ZJLQ024 through root dipping before transplanting, it can promote the root growth of rapeseed seedlings and improve the transplant survival rate; significantly increase rapeseed plant height and aboveground growth level; improve rapeseed pod traits, increasing pod length and pod width; increase yield components such as thousand-grain weight; and achieve a stable increase in rapeseed yield under field conditions. Attached Figure Description

[0026] Figure 1 The liquid fermentation agent of *Mucor* ZJLQ024 (MF024) prepared in Example 1.

[0027] Figure 2 Photographs showing the effects of the liquid fermentation agent of *Mucor* ZJLQ024 on rapeseed growth in a field experiment.

[0028] Figure 3A bar chart comparing rapeseed plant height in the thymol ZJLQ024 treatment group and the control group. Significance was determined using a t-test, with **** indicating statistical significance. P <0.0001.

[0029] Figure 4 A bar chart comparing the stem diameter of rapeseed treated with *Mucor* ZJLQ024 with the control group. Significance was determined using a t-test, with **** indicating statistical significance. P <0.0001.

[0030] Figure 5 A bar chart comparing the chlorophyll content (spad) of rapeseed leaves between the *Tetrandrine thymol* ZJLQ024 treatment group and the control group. Significance was determined using a t-test, with ** indicating statistical significance. P <0.01.

[0031] Figure 6 A bar chart comparing the number of rapeseed tillers in the *Mucor* ZJLQ024 treatment group and the control group. Significance was determined using a t-test, with **** indicating statistical significance. P <0.0001.

[0032] Figure 7 Photographs showing the effects of the thymol ZJLQ024 treatment group on rapeseed plant height and tiller number.

[0033] Figure 8 Photographs showing the effects of the thymol ZJLQ024 treatment group on the growth of rapeseed pods.

[0034] Figure 9 A bar chart comparing the length of rapeseed pods in the *Mucor* ZJLQ024 treatment group and the control group. Significance was determined using a t-test, with **** indicating statistical significance. P <0.0001.

[0035] Figure 10 A bar chart comparing the width of rapeseed pods in the *Mucor* ZJLQ024 treatment group and the control group. Significance was determined using a t-test, with **** indicating statistical significance. P <0.0001.

[0036] Figure 11 A bar chart comparing the thousand-grain weight of rapeseed in the *Mucor* ZJLQ024 treatment group and the control group. Significance was assessed using a t-test; *** indicates statistical significance. P <0.001. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0039] The fungal strain *Mucor* ZJLQ024 (MF024) involved in the following examples is a publicly available biological material. See Chinese Patent Application No. 200910153512.0, or the article (Fungal Biology, 2010, 114(10):797-808). This strain was isolated from the leaves of a broad-leaved tree in the Fengyangshan Nature Reserve, Zhejiang Province, during the inventors' research group's previous studies. It belongs to the Kingdom Fungi (Fungalidae). Fungi Ascomycota ( Ascomycota ), Scale-class ( Sordariomycetes ), Chlorophycetes ( Xylariales ), Carbonaceae ( Xylariaceae ), genus *Mucor* ( Muscodor ), Fengyang musk mold ( Muscodor fengyangensis This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 2863. Its morphological characteristics are as follows: it grows slowly on PDA medium, with transparent hyphae; after 10 days of growth at 25°C, the colony diameter reaches 35 mm. The outer ring of the colony is white, with a yellowish-brown center; white hyphae are present on the colony surface. During growth, the mycelia intertwine, and a distinctive odor is produced when growing on PDA. The mycelial balls are large and light brown; no spores or other reproductive structures are produced when growing on either PDA or PDB.

[0040] Example 1: Preparation of liquid inoculant of *Mucor* ZJLQ024 Fermentation equipment: 500 L industrial fermenter, using 25℃ for initial fermentation and 28℃ for secondary propagation culture.

[0041] 1. Culture and liquid fermentation of *Mucor* strain ZJLQ024 The *Mucor* strain ZJLQ024, preserved in cryovials, was inoculated onto potato dextrose agar (PDA) solid medium for activation and culture at 25°C in the dark for 7 days. Five mycelial cakes were then punched out using a 0.5 cm diameter punch and inoculated into Erlenmeyer flasks containing 800 mL of liquid fermentation medium. These flasks were then placed in a shaker (25°C, 150 rpm) and cultured for 7 days to obtain the *Mucor* ZJLQ024 liquid fermentation inoculum seed culture.

[0042] PDA medium: glucose 20 g / L, potato 200 g / L, agar 15 g / L. Weigh the required amount of potato according to the volume of the medium to be prepared, boil in water, mash and dissolve, filter, add glucose and agar, and autoclave at 121℃ for 20 min.

[0043] Liquid fermentation medium: 4 g / L soybean meal, 10 g / L corn flour, 0.5 g / L magnesium sulfate, and 1 g / L potassium dihydrogen phosphate. Weigh each component according to the volume of the medium to be prepared, add water to make up the volume, and sterilize by moist heat at 120℃ for 15 min.

[0044] 2. Preparation of liquid fermentation inoculant ZJLQ024 of *Mucor* The seed culture was transferred to the fermenter (inoculation amount 5%, transfer pipeline pre-sterilized by steam, fermenter sterilization parameters: 121℃, 30 min). Then, the raw materials were fed from the feed tank via a feed conveyor, with the feeding ratio shown in Table 1. Sterile air was continuously introduced at 28℃ and the mixture was stirred for 48-72 hours. The fermentation broth was then transferred to a settling tank for 12 hours for concentration. The remaining soybean meal and corn flour were then filtered through a double-stage filter (0.45 μm + 0.22 μm), and the filtrate was collected in a concentrated bacterial culture storage tank. Finally, the concentrated bacterial culture was transferred to a finished product tank for storage and packaging. The finished product is as follows: Figure 1 As shown.

[0045] Table 1

[0046] Meanwhile, in order to prepare agricultural microbial inoculants, in accordance with the standard GB20287-2006, the inoculants were sent to a professional department for spore concentration testing to ensure that the viable count of *Mucor* ZJLQ024 in the liquid fermentation inoculant was ≥2×10⁻⁶. 9 CFU / mL.

[0047] Example 2: Root dipping treatment before rapeseed transplanting 1. Experimental materials Test plant: rapeseed Brassica napus L., a common variety, Yueyou 1510.

[0048] Test strain: Muscone ZJLQ024, obtained by liquid culture and industrial fermentation propagation, same as in Example 1.

[0049] 2. Root dipping treatment Preparation of bacterial culture: Extract the propagated *Mucor* fungus culture (viable cell count ≥ 2 × 10⁻⁶) from the industrial fermenter. 9 (CFU / mL), dilute it at a ratio of 1:10 to ensure that the bacterial solution concentration is appropriate, harmless to rapeseed seedlings, and can effectively promote root growth.

[0050] Treatment method: Immerse the rapeseed roots in a diluted solution of thymol for 15 minutes, ensuring that each rapeseed root is fully in contact with the solution. After immersion, immediately transplant the treated rapeseed seedlings into the field.

[0051] Example 3: Effect of Thymosporum ZJLQ024 liquid fermentation inoculant on rapeseed yield A field experiment was conducted in Daoxiang Town, Yong'an Village, Yuhang Street, Yuhang District, Hangzhou City, Zhejiang Province. Rapeseed was treated with thymol ZJLQ024 root-dipping before transplanting and compared with the untreated control group.

[0052] 1. Rapeseed seedlings treated with the liquid fermentation agent of *Mucor* ZJLQ024 in Example 2 were evenly sown in the field by manual transplanting. Normal water and fertilizer management was implemented, and no fungicides were applied throughout the entire growth period until harvest. Field growth photos are available. Figure 2 .

[0053] 2. Effects on the physiological indicators of rapeseed growth During the rapeseed maturity period, plants with uniform growth and no obvious pests or diseases were selected as the test subjects, and 15 plants were randomly selected from each treatment for agronomic trait testing.

[0054] Plant height was measured from the ground to the top of the main stem using a ruler; stem diameter was measured at the base of the main stem using a ruler; the number of tillers was counted by direct observation. For pod characteristics, normally developing pods from the middle of each plant were randomly selected, and their length and width were measured using a ruler. Leaf relative chlorophyll content (SPAD value) was measured using a portable chlorophyll meter (SPAD meter), with multiple measurement points selected on functional leaves and the average value taken as the result for that plant.

[0055] The results are as follows Figures 3-11 As shown, compared with the control group, the rapeseed plant height, stem diameter, number of tillers, chlorophyll content (spad), pod length, pod width and thousand-grain weight of the thymol ZJLQ024 treatment group were all improved. This indicates that root dipping treatment with thymol ZJLQ024 bacterial solution before rapeseed transplanting can improve the growth physiological indicators and yield components of rapeseed, thereby promoting rapeseed yield.

[0056] 3. Rapeseed yield measurement According to the Ministry of Agriculture and Rural Affairs' methods for yield measurement and acceptance of grain and oil crops, yield measurements were conducted on 100-mu (approximately 6.7 hectares) plots of direct-seeded single-season rice treated with liquid fermentation inoculants. Three plots each were selected from rapeseed fields treated with the *Mucor* ZJLQ024 liquid fermentation inoculant and from a control field. After mechanical harvesting of the entire field and measurement of moisture content, the wet rapeseed was weighed. The yield was calculated using the following formula. Actual yield per mu (kg / mu) of harvested field = W×(1-M)×A÷(1-11%)÷S; Where W represents the weight of wet seeds in kilograms; M represents the moisture content; A represents the conversion factor, A=1; and S represents the area of ​​the field in mu (a Chinese unit of area).

[0057] After deducting impurities by 1.0% and converting the calculations based on a rapeseed moisture content of 11%, the results are shown in Tables 2 and 3.

[0058] Table 2. Yield results of the 100-mu demonstration plot in the control group

[0059] Table 3. Yield measurement results of a 100-mu demonstration plot treated with thymol ZJLQ024

[0060] The results showed that the actual yield of rapeseed treated with root-dipping inoculant ZJLQ024 was 203.22 kg / mu after transplanting, compared with 181.55 kg / mu for the control, representing an actual yield increase of 11.9%.

[0061] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Musk mold ( Muscodor The application of sp.) ZJLQ024 in promoting rapeseed growth and / or increasing rapeseed yield is characterized by, The *Mucor* ZJLQ024 is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 2863. The application includes: inoculating the activated *Mucor* ZJLQ024 into a liquid fermentation medium for fermentation culture, collecting the fermentation broth, and preparing a viable cell count concentration ≥2×10⁻⁶. 8 The treatment solution was prepared at CFU / mL. Then, before transplanting the rapeseed seedlings, the thymol ZJLQ024 was applied to the roots of the rapeseed seedlings by dipping the roots.

2. The application as described in claim 1, characterized in that, The indicators for rapeseed growth include at least one of the following: plant height, stem diameter, number of tillers, chlorophyll content, pod length, and pod width.

3. A method for promoting rapeseed growth and increasing rapeseed yield, characterized in that, Includes the following steps: (1) The *Mucor* strain ZJLQ024 with preservation number CGMCC No. 2863 was inoculated into liquid fermentation medium for fermentation culture, and the fermentation broth was collected to prepare a viable bacterial count concentration ≥2×10⁻⁶. 8 Treatment solution with CFU / mL; (2) The roots of the rapeseed seedlings are dipped in the treatment solution for root treatment, and then the rapeseed seedlings are transplanted to the field and cultivated until harvest.

4. The method as described in claim 3, characterized in that, In step (1), the activated thymol ZJLQ024 was inoculated into liquid fermentation medium and cultured to the exponential phase to obtain seed liquid; The seed culture was then inoculated into a liquid fermentation medium for secondary propagation; the fermentation broth was then concentrated, filtered, and the filtrate was collected and diluted to obtain the treated solution.

5. The method as described in claim 4, characterized in that, In step (1), the activation conditions are as follows: inoculate thymol ZJLQ024 into PDA medium and culture in the dark at 22-25℃ for 7-10 days.

6. The method as described in claim 4, characterized in that, The seed culture was prepared under the following conditions: culturing at 22-25℃ and 100-150 rpm for 7-10 days.

7. The method as described in claim 4, characterized in that, The conditions for the secondary propagation culture are as follows: the seed liquid is inoculated into the liquid fermentation medium at a volume ratio of 5%, and cultured at 25-28℃ with continuous stirring and oxygen supply for 48-72 hours.

8. The method as described in claim 3, characterized in that, In step (2), the root dipping treatment time is 15-20 min.

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