Composition containing long-chain fatty acid, short-chain fatty acid and oleaginous yeast and application of composition in inhibiting soil-borne diseases of panax notoginseng and relieving continuous cropping obstacles of panax notoginseng

Abstract

The invention relates to the technical field of ecological agriculture, and provides a composition prepared by compounding a basidiomycetes oleaginous yeast S.T (with the CGMCC (China General Microbiological Culture Collection Center) preservation number of 2.1816) and a fatty acid composite solution, a preparation of the composition and application of the composition in the aspects of inhibiting soil-borne diseases of panax notoginseng and relieving continuous cropping obstacles of panax notoginseng. The composition is prepared by compounding basidiotype oleaginous yeast S.T (the preservation number of CGMCC (China General Microbiological Culture Collection Center) is 2.1816) and a fatty acid composite solution according to the volume ratio of 1: 1, and compared with a contrast, the composition can reduce the relative abundance of pathogenic bacteria in panax notoginseng planting soil, increase the seedling rate and plant height of panax notoginseng and increase the content of flavone, polysaccharide and other plant resistance related substances in underground main roots. The composition and the preparation thereof disclosed by the invention are used for biological prevention and control of soil-borne root rot of panax notoginseng and can be used for relieving continuous cropping obstacles of panax notoginseng, inhibiting root rot and promoting the quality of panax notoginseng. The method has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of ecological agriculture technology, specifically to a composition comprising long-chain fatty acids, short-chain fatty acids and oil-producing yeast, and its use in inhibiting soil-borne diseases of Panax notoginseng and alleviating continuous cropping obstacles of Panax notoginseng. Background Technology

[0002] The Chinese medicinal herb industry is a core component of traditional Chinese medicine. In recent years, with the upgrading of health consumption and policy support (such as the implementation of the Traditional Chinese Medicine Law), the industry scale has continued to expand. China is the world's largest producer and consumer of Chinese medicinal herbs, with a planting area of ​​over 30 million mu (approximately 2 million hectares), encompassing thousands of varieties. The industrial chain covers agriculture, processing, pharmaceuticals, and the broader health industry, holding strategic significance for rural revitalization, increasing farmers' income, and the internationalization of traditional Chinese medicine. Continuous cropping obstacles (referring to soil degradation, increased pests and diseases, and decreased yield and quality caused by continuous planting of the same medicinal herb on the same plot) are a key issue restricting the sustainable development of the industry. The causes include soil nutrient imbalance, microbial community disorder, and accumulation of allelochemicals. For example, continuous cropping obstacles have led to the relocation of planting sites or reduced yields for ginseng, Panax notoginseng, and other traditional Chinese medicinal herbs, increasing production costs and threatening stable resource supply. Solving continuous cropping obstacles requires technological breakthroughs through crop rotation, microbial remediation, and soil improvement to ensure the quality and safety of Chinese medicinal herbs and the green transformation of the industry. The contradiction between growing market demand and resource bottlenecks caused by continuous cropping highlights the urgency of technological innovation and ecological planting models.

[0003] The core factor contributing to continuous cropping obstacles is the large-scale accumulation of soil pathogenic microorganisms, leading to a high incidence of root rot. Soil microbial regulation plays a crucial role in controlling soil-borne root rot and alleviating continuous cropping obstacles. Targeted regulation of soil microbial community structure and enrichment of pathogen-antagonistic microorganisms can counteract root rot pathogens by secreting antibiotics, competing for nutrients, or inducing plant resistance, thereby reducing the incidence of root rot. Simultaneously, regulating the microbial community structure, particularly the rhizosphere microbiota, can restore soil microbial diversity, promote the colonization of beneficial bacteria, improve soil physicochemical properties, and break the vicious cycle of pathogen-toxin accumulation. Furthermore, microbial metabolites, such as lipopeptides and siderophores, can induce systemic resistance in plants, enhance root vitality and nutrient absorption capacity, and alleviate the accumulation of autotoxic substances and nutrient imbalances caused by continuous cropping.

[0004] Fatty acids (FA) are an important component of soil organic matter, serving as both a carbon and energy source for microorganisms and influencing the composition and function of microbial communities through their structural characteristics (such as chain length and saturation). However, there is currently little research on utilizing fatty acids to control soil-borne root rot diseases associated with continuous cropping obstacles. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a composition comprising long-chain fatty acids, short-chain fatty acids, and oil-producing yeast, and its use in inhibiting soil-borne diseases of Panax notoginseng and alleviating continuous cropping obstacles of Panax notoginseng.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a composition for inhibiting soil-borne diseases of Panax notoginseng and alleviating continuous cropping obstacles of Panax notoginseng, the composition comprising an LSs7:3-Mix complex solution and a spore suspension of basidiomycete oil-producing yeast ST (Solicoccozymaterrea, CGMCC strain preservation number 2.1816), wherein the LSs7:3-Mix complex solution comprises a mixture of long-chain fatty acids and a mixture of short-chain fatty acids.

[0008] The basidiomycete oil-producing yeast ST is also known as terrestrial ball yeast.

[0009] Alternatively, in the above composition, the long-chain fatty acid mixture is composed of palmitic acid (CAS: 57-10-3), palmitoleic acid (CAS: 373-49-9), nonadecanoic acid (CAS: 646-30-0), pentadecanoic acid (CAS: 1002-84-2), and erucic acid (CAS: 112-86-7), and the short-chain fatty acid mixture is composed of 2-methylbutyric acid (CAS: 116-53-0), aminohexanoic acid (CAS: 1319-82-0), 3-methylthiopropionic acid (CAS: 646-01-5), valeric acid (CAS: 109-52-4), and fumaric acid (CAS: 110-17-8).

[0010] Alternatively, in the above composition, the concentration of each long-chain fatty acid and short-chain fatty acid mixture is 0.20 mM.

[0011] Alternatively, in the above composition, the mixture of long-chain fatty acids and the mixture of short-chain fatty acids are mixed at a volume ratio of 7:3.

[0012] Alternatively, in the above composition, the concentration of the basidiomycete oleogenetic yeast ST is 10. 7 -10 8 spores / mL.

[0013] Alternatively, in the above composition, the volume ratio of the LSs7:3-Mix composite solution to the ST spore suspension of the basidiomycete oil-producing yeast is 1:1.

[0014] In a second aspect, the present invention provides a biological control agent for inhibiting soil-borne diseases of Panax notoginseng and alleviating continuous cropping obstacles of Panax notoginseng, the agent comprising the composition described in the first aspect above.

[0015] In a third aspect, the present invention provides the use of the composition described in the first aspect or the biocontrol agent described in the second aspect for the prevention and control of soil-borne root rot disease in Panax notoginseng.

[0016] Alternatively, in the above-mentioned uses, the soil-borne root rot disease of Panax notoginseng is root rot caused by the root rot fungus Ilyonectriadestructans.

[0017] In a fourth aspect, the present invention provides the use of the composition described in the first aspect or the biocontrol agent described in the second aspect for alleviating the obstacle of continuous cropping of Panax notoginseng, promoting the growth of Panax notoginseng and / or improving the quality of Panax notoginseng.

[0018] The beneficial effects of this invention are as follows:

[0019] The invention includes the basidiomycete oil-producing yeast ST (CGMCC strain accession number 2.1816) at 1×10⁻⁶. 7 spores / mL and 1×10 8At a spore concentration of 1 spore / mL, the aboveground and underground dry weights of annual Panax notoginseng were significantly increased, with increases of 0.23 g and 0.3002 g for the aboveground and underground dry weights per plant, respectively; and increases of 0.67773 g and 0.35998 g for the underground dry weights per plant, respectively. Simultaneously, at these concentrations, the basidiomycete oleotrophic yeast ST (CGMCC strain preservation number 2.1816) exhibited an inhibition rate of over 39.775% against *Ilyonectriadestructans*, the pathogen causing root rot in Panax notoginseng. Adding a 3:7 volume ratio of a long-chain fatty acid mixture (1 mM) and a short-chain fatty acid mixture (1 mM) to PDA significantly inhibited the root rot pathogen in Panax notoginseng while significantly promoting the growth of the oleotrophic yeast ST (CGMCC strain preservation number 2.1816). Based on the above research, the inventors simultaneously applied LSs7:3-Mix compound solution, along with the basidiomycete oil-producing yeast ST (CGMCC strain preservation number 2.1816) and the root rot pathogen of Panax notoginseng, Ilyonectriadestructans I1, to one-year-old Panax notoginseng seedlings in pots. The results showed that compared to the control treatment which only received Ilyonectriadestructans I1, the combined application of LSs7:3-Mix compound solution (a 3:7 volume ratio of long-chain fatty acid mixture and short-chain fatty acid mixture) with the basidiomycete oil-producing yeast ST and Ilyonectriadestructans I1 significantly increased the plant height and seedling survival rate of Panax notoginseng, increased the diversity of the fungal community structure in the potted soil, and significantly inhibited the growth of Ilyonectriadestructans I1. Simultaneously, the content of flavonoids and polysaccharides, two types of secondary metabolites, increased in the main root of Panax notoginseng, improving its quality. The increase in these two substances has great potential for enhancing the resistance of Panax notoginseng plants. Attached Figure Description

[0020] Figure 1 The antagonistic effect of the basidiomycete oil-producing yeast ST (CGMCC strain preservation number 2.1816) on the root rot pathogen of Panax notoginseng, Ilyonectriadestructans;

[0021] Figure 2 The interaction between the complex solution of long-chain fatty acids and short-chain fatty acids, the basidiomycete yeast ST (CGMCC strain preservation number 2.1816), and the pathogenic fungus Destructive Cyclospora I1;

[0022] Figure 3 The effects of different volume ratios of long-chain fatty acid mixtures and short-chain fatty acid mixtures in fatty acid complexes on the combined application of the root rot pathogen *Cyclophorus spp.* I1 and the basidiomycete oil-producing yeast ST (CGMCC strain preservation number 2.1816) on the plant height and seedling survival rate of Panax notoginseng plants were investigated.

[0023] Figure 4The effects of applying fatty acid complex and basidiomycete oil-producing yeast ST (CGMCC strain preservation number 2.1816) spore suspension alone and in combination on the survival rate of Panax notoginseng seedlings were investigated.

[0024] Figure 5 The effects of single and combined application of fatty acid complex and basidiomycete oil-producing yeast ST (CGMCC strain preservation number 2.1816) spore suspension on the diversity and abundance of rhizosphere soil fungi in Panax notoginseng;

[0025] Figure 6 The effects of single and combined application of fatty acid complex and basidiomycete oil-producing yeast ST (CGMCC strain preservation number 2.1816) spore suspension on the total flavonoid and polysaccharide content in the main root of Panax notoginseng. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0027] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0028] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0029] Example 1: Effect of basidiomycete oil-producing yeast ST (CGMCC strain preservation number 2.1816) on the growth-promoting effect of Panax notoginseng.

[0030] Preparation of ST spore suspension of basidiomycete oil-producing yeast: Basidiomycete yeast ST was inoculated into PDB (potato dextrose broth) and cultured at 25℃ for 2-3 days. The bacterial suspension was centrifuged at 8000 r / min for 5 min, the supernatant was discarded, and the precipitate was resuspended in sterile deionized water. The suspension was then serially diluted to obtain 10-1 spores. 7 10 spores / mL and 10 8 A yeast spore suspension with a concentration of 1 spore / mL.

[0031] Growth-promoting effect: Select one-year-old Panax notoginseng potted plants, and apply 50 mL of the above two spore concentration bacterial solutions to the soil of the Panax notoginseng potted plants. The control is treated with an equal volume of sterile water. The treatment is repeated every 7 days, and each concentration treatment is set up with 10 replicates. After 8 treatments, the survival rate is counted, and Panax notoginseng plants are taken, dried at 60℃ to constant weight, and the dry weight of the above-ground and underground parts is measured.

[0032] Survival rate (%) = (Number of Panax notoginseng seedlings after spore suspension treatment - Number of seedlings in control treatment) / Number of seedlings in control treatment × 100

[0033] like Figure 1 As shown, 10 7 10 spores / mL 8 A spore suspension with a concentration of 1 spore / mL significantly improved the survival rate of Panax notoginseng seedlings, and at the same time, the dry weight of both the aboveground and underground parts of one-year-old Panax notoginseng was significantly increased. The basidiomycete oil-producing yeast ST had a significant growth-promoting effect on Panax notoginseng.

[0034] Example 2: Interactions among a complex solution of long-chain and short-chain fatty acids, basidiomycete oleogenous yeast ST (CGMCC strain preservation number 2.1816), and the pathogen *Strombospora destructicai* I1.

[0035] 1) Preparation of fatty acid-containing culture medium: Prepare an LSs7:3-Mix mixture, in which the concentration of each of the five compounds in the long-chain fatty acid mixture is 2.0 mM, and the concentration of each of the five compounds in the short-chain fatty acid mixture is 2.0 mM. Then, mix the long-chain fatty acid mixture (L) and the short-chain fatty acid mixture (S) at a volume ratio of 3:7 to obtain the LSs7:3-Mix mixed solution. Prepare PDA medium and autoclave at 121℃ for 20 minutes. When the medium cools to 60℃, add the LSs7:3-Mix mixed solution to the medium at a volume ratio of 9:1 (medium medium volume to LSs7:3-Mix volume). Gently shake to mix the compounds evenly, then pour into petri dishes to prepare a solid drug-loaded culture medium containing LSs7:3-Mix as the sole carbon source. The concentration of each compound in the long-chain fatty acid in the drug-loaded plate is 0.2 mM, and the concentration of each compound in the short-chain fatty acid is 0.20 mM. Use PDA medium with an equal volume of sterile water as the control medium.

[0036] 2) Preparation of ST spore suspension of basidiomycete oil-producing yeast: Inoculate ST into PDB (potato dextrose broth), incubate at 25℃ for 2-3 days, centrifuge the bacterial suspension at 8000 r / min for 5 min, remove the supernatant, add sterile deionized water for reprecipitation, and perform serial dilutions to obtain 10 7 -10 8 A yeast spore suspension with a concentration of 1 spore / mL.

[0037] 3) Antagonistic effect of basidiomycete oil-producing yeast ST on pathogen *Strombosporium demisotomyces* I1: A 5mm diameter mycelial disc of *Strombosporium demisotomyces* I1 was inoculated onto the control medium from step 1) 1cm from the edge of the culture dish. Then, a line was drawn 5cm vertically from the center of the culture dish to the mycelial disc using 20μL of yeast spore suspension. For the control group, the 20μL yeast spore suspension was replaced with sterile deionized water. All other procedures were the same. Each treatment was repeated in 5 replicates. The mixture was incubated at 25℃ for 5-7 days, and the inhibition rate was calculated.

[0038] Inhibition rate (%) = (D Y / D0)×100

[0039] (D Y (D0 represents the diameter of the pathogen after the yeast is drawn, where D0 is the diameter of the pathogen in the control medium without yeast inoculation.)

[0040] 4) Antagonistic effect of LSs7:3-Mix mixed solution on the pathogen *Streptococcus oxydegenes* I1: 5 mm *Streptococcus oxydegenes* I1 mycelial pellets were inoculated into PDA medium containing the compound from step 1) and control medium, with 8 replicates per treatment. The pellets were incubated at 25°C for 5 days, and the colony diameter was measured to calculate the inhibition rate.

[0041] Inhibition rate (%) = (Control colony diameter - Colony diameter containing compound PDA) / Control colony diameter × 100

[0042] As shown in Table 1 and Figure 2 As shown, firstly, the effects of different volume ratios of long-chain fatty acid mixtures and short-chain fatty acid mixtures in the fatty acid complex on the growth of *Strombospora destructicai* I1, a root rot pathogen, and *ST*, were investigated. Figure 2 B indicates that LSs7:3-Mix has the best effect. And if... Figure 2 A and Figure 2 As shown in Figure C, the combined effect of fatty acid LSs7:3-Mix mixed solution and basidiomycete oleogenous yeast ST spore suspension on the inhibitory effect of destroying columnar spore I1 is the strongest.

[0043] Table 1: Inhibition rate of mixtures of long-chain and short-chain fatty acids and basidiomycete oleogen ST on *Panax notoginseng* root rot pathogen *Cyclocarya spp.* I1 in PDA medium.

[0044]

[0045] Example 3: Effects of different volume ratios of long-chain fatty acid mixtures and short-chain fatty acid mixtures in a fatty acid complex on the combined application of *Strombospora globosum* I1 (a root rot pathogen) and *Saccharomyces cerevisiae* ST (CGMCC strain preservation number 2.1816) on the plant height and seedling survival rate of *Panax notoginseng* plants.

[0046] 1) Preparation of fatty acid complexes with different volume ratios: According to the method in 1) of Example 2, fatty acid complexes with volume ratios of 10:0, 9:1, 7:3, 5:5, 3:7, 9:1 and 0:10 were prepared.

[0047] 2) Preparation of ST spore suspension of basidiomycete oil-producing yeast: The method is the same as that in Example 2).

[0048] 3) Spore suspension of *Cyclophorus spp.* I1 (the pathogen destroyer): Inoculate 5mm diameter mycelial discs onto PDA medium and incubate at 25℃ for 7-10 days. Once the colonies have fully colonized the petri dishes, add 15mL of sterile water to each dish. Scrape the mycelium with a glass slide, or use the spore suspension to filter the mycelium through gauze. Perform serial dilutions to obtain 10... 7 -10 8 A spore suspension of pathogenic columnar spore I1 at a concentration of 1 spore / mL.

[0049] 4) Treatment of Panax notoginseng potted plants: The control treatment was to apply only 30 mL of spore suspension of the pathogen *Cyclophorus spp. I1* and 60 mL of water (total system: 90 mL). Different volumes of the fatty acid complex prepared above were taken and applied separately or in combination with the spore suspensions of the basidiomycete oil-producing yeast ST and *Cyclophorus spp. I1*. 30 mL of each of the fatty acid complex solution, the basidiomycete oil-producing yeast ST spore suspension, and the *Cyclophorus spp. I1* spore suspension were applied to the soil of Panax notoginseng potted plants. Each treatment was replicated 10 times, and the treatment was carried out once a week for a total of 8 times. The survival rate was then counted and the plant height of Panax notoginseng was measured.

[0050] like Figure 3 The results showed the effects of different volume ratios of fatty acid complex solutions combined with basidiomycete oil-producing yeast ST and pathogenic fungus *Cyclocarya spp.* I1 on the plant height and seedling survival rate of *Panax notoginseng*. Among these, a volume ratio of 7:3 (LSs7:3-Mix) of long-chain fatty acid mixture and short-chain fatty acid mixture had the most significant effect on plant height and seedling survival rate. Figure 4 It can be seen that the combination of LSs7:3-Mix and basidiomycete oil-producing yeast ST has the most significant inhibitory effect on pathogens and the effects of promoting plant height and increasing seedling survival rate.

[0051] Meanwhile, sequencing analysis of rhizosphere microorganisms in potted Panax notoginseng plants and determination of total flavonoids and polysaccharide content in the main root of Panax notoginseng showed that ( Figure 5 and Figure 6Compared with treatment using only the pathogen-destroying columnar spore I1, the combined application of LSs7:3-Mix with the basidiomycete oil-producing yeast ST and the pathogen-destroying columnar spore I1 significantly improved the diversity and abundance of soil fungal communities, which is of great significance for promoting soil health. Furthermore, the increased content of total flavonoids and polysaccharides in the taproot can enhance plant resistance, improve disease resistance, and promote plant health.

[0052] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A composition for inhibiting soil-borne diseases of Panax notoginseng and alleviating continuous cropping obstacles of Panax notoginseng, characterized in that: The composition contains an LSs7:3-Mix complex solution and a spore suspension of the basidiomycete oleogeny yeast ST (Solicoccozymaterrea, CGMCC strain preservation number 2.1816), wherein the LSs7:3-Mix complex solution contains a mixture of long-chain fatty acids and a mixture of short-chain fatty acids.

2. The composition according to claim 1, characterized in that: The long-chain fatty acid mixture consists of palmitic acid, palmitoleic acid, nonadecanoic acid, pentadecanoic acid and erucic acid, and the short-chain fatty acid mixture consists of 2-methylbutyric acid, aminocaproic acid, 3-methylthiopropionic acid, valeric acid and fumaric acid.

3. The composition according to claim 2, characterized in that: In the mixtures of long-chain fatty acids and short-chain fatty acids, the concentration of each long-chain fatty acid and short-chain fatty acid is 0.20 mM.

4. The composition according to claim 2 or claim 3, characterized in that: The mixture of long-chain fatty acids and the mixture of short-chain fatty acids were mixed at a volume ratio of 7:

3.

5. The composition according to claim 1, characterized in that: The concentration of the basidiomycete oleogenetic yeast ST was 10. 7 -10 8 spores / mL.

6. The composition according to claim 1, characterized in that: The volume ratio of the LSs7:3-Mix composite solution to the ST spore suspension of the basidiomycete oil-producing yeast is 1:

1.

7. A biological control agent for inhibiting soil-borne diseases of Panax notoginseng and alleviating continuous cropping obstacles of Panax notoginseng, characterized in that: The formulation comprises the composition of any one of claims 1 to 6.

8. The use of the composition of any one of claims 1 to 6 or the biocontrol agent of claim 7 for the control of soil-borne root rot disease of Panax notoginseng.

9. The use as described in claim 8, characterized in that: The soil-borne root rot disease of Panax notoginseng is caused by the root rot fungus Ilyonectriadestructans.

10. The use of the composition of any one of claims 1 to 6 or the biocontrol agent of claim 7 for alleviating the obstacle of continuous cropping of Panax notoginseng, promoting the growth of Panax notoginseng and / or improving the quality of Panax notoginseng.

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