Application of Mongolian white mushroom LY9 strain in tobacco planting

Abstract

The invention discloses an application of a Leucocallis mongolica LY9 strain in tobacco planting, the strain is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC (China General Microbiological Culture Collection Center) No.63678. The invention further discloses a preparation method of the Leucocallis mongolica LY9 strain. According to the application, the LY9 strain is prepared into a transformation matrix and then applied to tobacco. The application can comprehensively promote the growth of tobacco plants, enhance the photosynthetic ability and stress resistance, and systematically optimize the leaf quality. By adjusting the application amount of the transformation substrate, the proportion and the total amount of amylose and amylopectin in the leaves can be precisely and directionally regulated and controlled, so that the flavor and the quality of the tobacco are optimized. The invention provides an innovative solution for green cultivation and accurate quality improvement of tobacco.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, and relates to the application of a Mongolian white mushroom strain LY9 in tobacco cultivation. Background Technology

[0002] Tobacco is an important leaf crop, and its industrial value depends heavily on the physical properties of its leaves and the harmony of their internal chemical composition. Current cultivation practices, in pursuit of yield and appearance, rely heavily on chemical fertilizers and regulators, which can easily lead to soil degradation, root microecological imbalance, and may cause carbon and nitrogen metabolism disorders in the leaves, affecting the final quality and safety of tobacco.

[0003] Microbial fertilizers have become a research hotspot due to their green, environmentally friendly, and sustainable advantages, but their application in tobacco cultivation still has significant limitations. On the one hand, the product types are relatively limited, mainly consisting of Bacillus species (such as Bacillus subtilis and Bacillus licheniformis), accounting for more than 70% of the total amount of microbial fertilizers used in tobacco. On the other hand, their functions are not targeted enough, with most products focusing on promoting growth or preventing single diseases. Effective technical means are still lacking in systematically and synergistically optimizing multiple quality indicators of tobacco leaves and achieving targeted regulation according to industrial needs.

[0004] The applicant's prior innovative research pioneered the application of the Leucocalocybe mongolica LY9 strain in the agricultural field, and disclosed through patents (application numbers: 202410517671.9, 202410517649.4) the excellent effects of this strain and its transformation substrate in promoting rice growth and development, specifically manifested in significantly stimulating chlorophyll synthesis, promoting root development, and increasing effective tillering. This prior art provides a novel microbial resource for green crop production. However, the interaction mechanism between microorganisms and plants is highly complex and exhibits significant species specificity. The quality formation of tobacco involves a unique and complex secondary metabolic network, which is fundamentally different from that of conventional grain or vegetable crops. Applying a microbial resource to tobacco, expecting it to simultaneously and positively regulate leaf number, photosynthetic efficiency, stress resistance physiology, and numerous chemical components related to flavor, aroma, and safety, presents significant uncertainties and unpredictability in terms of technology. Currently, no existing technology has disclosed or implied that the Mongolian white mushroom LY9 strain can be used for tobacco cultivation, nor has it revealed that it can precisely affect the composition of the comprehensive traits and quality components of tobacco leaves.

[0005] Therefore, exploring and developing new applications of the LY9 strain in tobacco production to overcome the dependence on chemical inputs and the shortcomings of existing tobacco cultivation techniques in precise quality control is of great industrial significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an application of Leucocalocybemongolica strain LY9 in tobacco cultivation.

[0007] The aforementioned Leucocalocybe mongolica LY9 strain is deposited at the Guangdong Microbial Culture Collection Center, with accession number GDMCC No. 63678 and deposit date of August 22, 2023.

[0008] The application is to promote the growth of tobacco plants, improve the stress resistance of tobacco plants, or improve the quality of tobacco leaves.

[0009] The promotion of tobacco plant growth includes: increasing the chlorophyll content of tobacco leaves, increasing the height and stem diameter of tobacco plants, increasing the leaf length, leaf width, and mid-leaf length of tobacco plants, increasing the number of effective leaves of tobacco plants, and promoting the root growth of tobacco plants.

[0010] Improving the quality of tobacco leaves includes: increasing the content of total sugar, reducing sugar, total alkaloids, and total phenols in tobacco leaves; increasing the protein and cellulose content in tobacco leaves; increasing the total starch content in tobacco leaves; and regulating the ratio and content of amylose, amylopectin, and total starch in the leaves.

[0011] The above-mentioned method involves using an inoculum containing the Leucocalocybe mongolica LY9 strain to ferment the culture medium, obtaining a transformation substrate, and then using the transformation substrate for tobacco cultivation.

[0012] The beneficial effects of this invention are:

[0013] This invention applies the transformation substrate of Leucocalocybe mongolica LY9 strain to tobacco cultivation, achieving the following synergistic and beneficial effects:

[0014] 1. The LY9 conversion substrate can significantly improve key agronomic traits of tobacco plants such as plant height, stem diameter, number of effective leaves and leaf area, and strongly promote root biomass and vitality, comprehensively promote plant growth and development, and build strong individual plants.

[0015] 2. The LY9 conversion substrate can significantly increase the chlorophyll content of tobacco leaves and enhance photosynthetic capacity; at the same time, it can reduce malondialdehyde content, improve the plant's antioxidant and stress resistance levels, and ensure normal growth under stress conditions.

[0016] 3. LY9 conversion matrix treatment can synergistically enhance flavor components such as total sugar, reducing sugar, total alkaloids and total phenols, improve the sweetness and aroma coordination of flue gas; at the same time, it increases the protein and cellulose content, optimizes the combustion characteristics and physical toughness of leaves, and systematically optimizes the overall quality of leaves.

[0017] 4. The most prominent effect of this invention is that by adjusting the application amount of LY9 conversion matrix, the ratio and content of amylose, amylopectin and total starch in the leaves can be controlled in a targeted manner, thereby achieving predictable and precise optimization of tobacco energy storage metabolism and final flavor style. Attached Figure Description

[0018] Figure 1 This is a field experiment site for tobacco cultivation.

[0019] Figure 2 A graph showing the analysis of growth and developmental traits of tobacco.

[0020] Figure 3 A diagram illustrating the regulation and analysis of physiological characteristic indicators in tobacco leaves;

[0021] Figure 4 A graph showing the analysis of soil enzyme indicators in tobacco root zone; Detailed Implementation

[0022] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present 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 present invention.

[0023] Example 1: Application of Leucocalocybe mongolica LY9 strain in tobacco cultivation

[0024] 1. Preparation of transformation substrate for Leucocalocybe mongolica LY9

[0025] (1) Propagation of Leucocalocybe mongolica LY9

[0026] Prepare PDA solid medium, inoculate Leucocalocybe mongolica LY9 strain onto PDA solid medium, and place the inoculated medium in a constant temperature incubator at 25℃ for 20-30 days.

[0027] (2) Preparation of transformation matrix of Leucocalocybe mongolica LY9

[0028] To prepare PDA liquid culture medium, transfer mycelia from PDA solid culture medium into the PDA liquid culture medium and incubate in a shaker (25℃, 120 rpm) in the dark for 20 days to obtain LY9 liquid culture. Dilute the humus soil with distilled water to a moisture content of over 80%, pack it into edible mushroom inoculation bags, and autoclave at 121℃ for 2 hours. After sterilization and cooling, inoculate each bag with 10 ml of LY9 liquid culture and incubate at room temperature (25℃) in the dark for 30-60 days until the mycelia have fully colonized the bags.

[0029] 2. Tobacco Field Cultivation Experiment Design

[0030] (1) Test materials

[0031] Experimental crop and variety: Common agricultural tobacco variety (Yunyan 87), selecting healthy, disease-free seedlings.

[0032] Fertilizers used in the experiment: Leucocalocybe mongolica LY9 strain transformation substrate as a novel potato bio-fertilizer, compound fertilizer produced by BASF AG, and peat soil produced by Pinstop Horticulture (Shanghai) Co., Ltd.

[0033] (2) Experimental site and field test method Experimental site: Field test site of College of Agronomy and Life Sciences, Zhaotong University, Zhaoyang District, Zhaotong City, Yunnan Province.

[0034] Experimental period: Sowing on May 11, 2025, and sampling on July 13.

[0035] The experiment employed a randomized block design with eight treatment groups: ①CK1 (blank control, no fertilizer applied), ②CK2 (blank control 2) cultivated in unconverted substrate (peat soil), ③CK3: compound fertilizer (30g per plant), ④CK4: 50g LY9 + compound fertilizer (50g LY9 conversion substrate + 30g compound fertilizer per plant), ⑤CK5: 100g LY9 + compound fertilizer (100g LY9 conversion substrate + 30g compound fertilizer per plant), ⑥ 30g LY9 (30g LY9 conversion substrate per plant), ⑦ 50g LY9 (50g LY9 conversion substrate per plant), and ⑧ 100g LY9 (100g LY9 conversion substrate per plant). Each treatment plot had a cultivation area of ​​25m². 2 The row and plant spacing were set according to conventional tobacco planting standards, with 10 tobacco plants planted in each plot, arranged completely randomly. Fertilization was carried out by furrow application or root irrigation depending on the treatment type. At sowing, basal fertilizer was applied between the tobacco plants, and topdressing was applied in early July. Other field management measures remained consistent.

[0036] (3) Field trial management

[0037] When the tobacco seedlings reach a height of 10-15cm, perform the first weeding and loosening of the soil. Subsequent weeding should be done as needed based on weed growth and soil compaction. After sowing and fertilization, perform the first deep watering. During the growing season, keep the soil moist but not waterlogged. During key growth stages (such as the seedling stage and vigorous growth stage), maintain soil moisture at 70-80%. Reduce watering in the later stages of growth to avoid waterlogging. Throughout the growing season, focus on pest and disease control, especially against tobacco mosaic virus and black shank. Other management practices are the same as in conventional field operations.

[0038] (4) Field trial design

[0039] A random interval design is adopted, with 8 processing groups:

[0040] CK1 group: Purple sandy loam soil without any fertilizer applied;

[0041] Group CK2: Cultivated with 100g of peat moss per plant;

[0042] CK3 group: Apply 30g of compound fertilizer per tobacco plant;

[0043] CK4 group: Apply 50g of LY9 conversion substrate + 30g of compound fertilizer per tobacco plant;

[0044] CK5 group: Apply 100g of LY9 conversion substrate + 30g of compound fertilizer per tobacco plant;

[0045] 30g treatment group: 30g of LY9 conversion substrate was applied to each tobacco plant;

[0046] 50g treatment group: 50g of LY9 conversion substrate was applied to each tobacco plant;

[0047] 100g treatment group: 100g of LY9 conversion substrate was applied to each tobacco plant;

[0048] Cultivation experiment site Figure 1 As shown, Figure 1 The photo was taken 47 days after planting. The tobacco plants in the 30g and 50g groups are visibly growing the best.

[0049] 3. Methods for determining key indicators

[0050] (1) Determination of agronomic trait indicators

[0051] During the tobacco maturity stage, the following measurements were taken from 10 tobacco plants in each plot: stem diameter (measured at the base of the stem using calipers), longest leaf length, longest leaf width, mid-leaf length, mid-leaf width (leaf length from the petiole to the leaf tip and width of the widest part of the leaf were measured using a ruler), natural plant height (measured from the base of the stem to the top of the plant using a soft measuring tape), and number of effective leaves. The average values ​​were calculated. Simultaneously, the fresh weight, dry weight, and water content of the tobacco roots were measured at maturity. Root activity was determined using a kit. The results are as follows: Figure 2 As shown,

[0052] from Figure 2 The agronomic trait data show that the LY9 conversion substrate treatment has a significant promoting effect on tobacco plant height (natural plant height and absolute plant height), stem diameter, leaf indicators (length / width of the longest leaf and length / width of the middle leaf) and number of effective leaves. Among the indicators, the natural plant height of the 30 g / plant treatment group was significantly higher than that of CK1 and CK5; the absolute plant height of the 100 g / plant treatment group was significantly higher than that of CK1 (+76.8%) and CK2 (+14.2%); the longest leaf length of the 30 g / plant, 50 g / plant, and 100 g / plant treatment groups was significantly higher than that of CK1 and CK2; the longest leaf width of the 30 g / plant, 50 g / plant, and 100 g / plant treatment groups was significantly higher than that of CK1 (+51.9%, +51.3%, and +55.0%, respectively); the waist leaf length of the 30 g / plant and 100 g / plant treatment groups was significantly higher than that of CK1, CK2, and CK5; the waist leaf width of the 100 g / plant treatment group was significantly higher than that of each CK control group; and the number of effective leaves of the 30 g and 50 g treatment groups was significantly higher than that of CK1 (+22.5%) and CK2 (+32.4%). The LY9 treatment group showed significantly higher results than the control group, indicating that the LY9 conversion substrate can effectively promote the growth and development of the aboveground parts of tobacco and improve the overall lushness of the plants.

[0053] from Figure 2In terms of tobacco root indicators, the results of root fresh weight, dry weight, water content, and root activity measurements showed that the LY9 conversion substrate alone had a significantly better promoting effect on the underground parts of tobacco: In terms of fresh weight and dry weight (core indicators of root growth and material accumulation), the fresh weight and dry weight of the 30g / plant LY9 treatment were much higher than those of the control group CK1, and also better than the compound fertilizer, humus soil, and compound fertilizer + LY9 combined treatment groups; the fresh weight and dry weight of the 50g / plant and 100g / plant LY9 treatments were also significantly higher than those of CK1 and CK2; and in terms of root activity (core indicator of root nutrient absorption efficiency), the average root activity of the 30g / plant and 50g / plant LY9 treatments not only exceeded that of CK1, but also exceeded that of CK2, CK3, and various combined treatment groups. These data together demonstrate the positive promoting effect of the LY9 conversion substrate on the growth scale, material accumulation, and physiological absorption capacity of the underground parts of tobacco.

[0054] In summary, the LY9 conversion substrate comprehensively promotes tobacco growth: In the aboveground parts, it significantly increases plant height, stem diameter, leaf indicators, and the number of effective leaves; the relevant indicators in all LY9 conversion substrate treatment groups are generally higher than those in the control groups (CK), with significant increases. In the underground parts, its individual treatment is more effective, with root fresh weight and dry weight superior to CK1, CK2, and CK3 treatments; the root activity of the 30g / plant and 50g / plant treatments is also significantly stronger. Overall, the LY9 conversion substrate not only promotes the vigorous growth of the aboveground parts of tobacco but also enhances the growth and nutrient absorption capacity of the underground parts, comprehensively improving its growth and development level.

[0055] (2) Determination of leaf quality indicators

[0056] Fresh leaves were randomly selected from three tobacco plants at maturity for each treatment. The leaves should be from similar parts and have similar color. Fresh tobacco leaf samples were weighed and extracted with 95% ethanol. The absorbance of the sample solution at 649 nm and 665 nm was measured using a UV-Vis spectrophotometer, and the chlorophyll concentration was calculated based on the absorbance. Fresh tobacco leaf samples were then weighed and analyzed using the barbituric acid colorimetric method. Subsequently, fresh leaf samples were weighed again, and the contents of chlorophyll a, chlorophyll b, total chlorophyll, malondialdehyde, protein, total phenols, total alkaloids, total sugar, amylopectin, amylose, total starch, cellulose, and reducing sugar were determined using corresponding kits. The results are shown below. Figure 3 As shown;

[0057] ①Photosynthesis-related indicators

[0058] from Figure 3It can be seen that after treatment with the LY9 conversion substrate, the number of effective leaves increased, and the contents of chlorophyll a, chlorophyll b, and total chlorophyll significantly improved. The 30g / plant treatment group showed significantly higher levels of chlorophyll a, chlorophyll b, and total chlorophyll compared to all other treatment groups. Specifically, the CK5 treatment group showed increases of 12.83%, 10.37%, and 12.26% in chlorophyll a, chlorophyll b, and total chlorophyll contents compared to the CK3 treatment group, respectively. This indicates that the conversion substrate promotes the effects of chemical fertilizers, demonstrating the synergistic effect of LY9 and compound fertilizers in enhancing chlorophyll synthesis. This enhances leaf photosynthetic efficiency, increases the accumulation of photosynthetic products such as sugars and starches, provides a sufficient material basis for tobacco growth, development, and the synthesis of quality components, resulting in more vigorous plant growth.

[0059] ② Antioxidant-related indicators

[0060] Malondialdehyde (MDA) content showed a decreasing trend, with a significant reduction in MDA content in the 100g / plant treatment group compared to the control CK1. This decrease in MDA content indicates a reduction in cell membrane lipid peroxidation, enhanced activity of the tobacco's antioxidant system, and improved stress resistance (such as drought and heat resistance). This helps ensure that leaves maintain normal physiological functions under stress conditions and reduces the occurrence of quality deterioration.

[0061] ③ Flavor and texture related indicators

[0062] In the 50g / plant treatment group, total sugar increased by 23.67% compared to CK1 and by approximately 28.3% compared to CK2; reducing sugar increased by approximately 27.2% compared to CK1 and by approximately 21.5% compared to CK2. These increases in both can balance the spiciness of the smoke, resulting in a smoother and sweeter taste. Total alkaloids increased by 17.2% compared to CK1 and by approximately 13.5% compared to CK2, enhancing the characteristic flavor and aroma of tobacco, serving as a core source of flavor. Total phenols increased by approximately 18.1% compared to CK1, improving the taste of the smoke, increasing aroma harmony, reducing harmful substances in the smoke, and improving smoking safety.

[0063] ④ Physicochemical and combustion-related indicators

[0064] The protein content of the 30g / plant, 50g / plant, and 100g / plant treatment groups was significantly higher than that of each control group. The moderate increase in protein can optimize the combustion characteristics of tobacco, making combustion more complete and the amount of smoke more stable. The cellulose content of the 50g / plant treatment group was significantly higher than that of CK1 (+214.2%), CK2 (+122.4%), and CK3 (+73.3%), which can enhance the stability of the physical structure of the leaves, improve the toughness of the leaves, and facilitate harvesting, processing, and storage.

[0065] ⑤ Components related to carbohydrate metabolism

[0066] The appropriate ratio and content of amylose, amylopectin, and total starch are key to optimizing tobacco quality: when the ratio of the two is coordinated and the content is appropriate, it can promote the efficient conversion of starch to sugar during the curing process and significantly improve the sweetness of the smoke; if the content is too high, it will lead to stiff leaf texture, and starch flavor will easily remain after curing, which will destroy the flavor harmony; if the content is too low, the tobacco will not have enough energy storage, which will easily result in thin and small leaves and poor internal quality.

[0067] The application rate of LY9 conversion substrate allows for precise control of the ratio and content of the three starches: as the application rate increases from 30g / plant, 50g / plant to 100g / plant, the total starch content and proportion gradually increase. The 30g / plant treatment group showed the most optimal ratio of amylose, amylopectin, and total starch, with the content also falling within the suitable range. Therefore, by adjusting the application dosage of LY9 conversion substrate, the ratio and content of the three types of starch in tobacco leaves can be precisely controlled, thereby achieving precise optimization of tobacco flavor and quality.

[0068] In summary, the LY9 conversion substrate can comprehensively improve the photosynthetic physiological function and core quality indicators of tobacco leaves. In particular, the 50g / plant treatment group showed the best results. It not only enhances the material accumulation capacity by optimizing the photosynthetic system, but also improves the stress resistance by regulating the antioxidant system. At the same time, it significantly improves the content of key components related to flavor and combustion characteristics, achieving multi-dimensional regulation of the physiological function and quality formation of tobacco. The overall quality is superior to the conversion substrate treatment and compound fertilizer treatment.

[0069] (3) Determination of soil indicators

[0070] Tobacco leaves matured on July 13, 2025. Tobacco was harvested according to the treatment group. The soil from the roots was dried, passed through a 60-mesh sieve, and the corresponding reagent kits were used to determine the levels of α-glucosidase, alkaline phosphatase, cellulase, sucrase, β-glucosidase, amylase, alkaline protease, acidic protease, and urease in the soil.

[0071] The measurement results are as follows Figure 4As shown, LY9 transformation substrate treatment significantly enhanced the activities of key enzymes in soil phosphorus conversion (alkaline phosphatase), key enzymes in carbon cycling (α-glucosidase, β-glucosidase, cellulase, amylase), and key enzymes in nitrogen conversion (alkaline protease, urease) through the mechanism of promoting soil nutrient transformation. Compared with the control group (CK), the activities of these enzymes were increased to varying degrees in the LY9 treatment groups (LY9 concentrations of 30 g / plant, 50 g / plant, and 100 g / plant). Among them, the 100 g / plant LY9 treatment showed particularly significant increases in the activities of alkaline phosphatase (16.2% higher than CK1), cellulase (97.7% higher than CK1), amylase (5.8% higher than CK), alkaline protease (113.7% higher than CK), and urease (45.1% higher than CK). The LY9 treatment with 50 g / plant showed outstanding performance in increasing the activities of α-glucosidase (30.0% higher than CK1) and β-glucosidase (20.0% higher than CK1). The cellulase activity of the LY9 treatment with 50 g / plant was also increased by 86.4% compared with CK1. Compared with the humus soil treatment, the LY9 conversion substrate treatment was more effective in improving soil enzyme activity and subsequent nutrient supply. At the same time, the enzyme activity of the LY9 conversion substrate treatment alone was generally higher than that of the compound treatment (compound fertilizer + LY9). Its effect on improving soil enzyme activity covers multiple nutrient cycles of carbon, nitrogen, and phosphorus. Different concentrations of LY9 have their own advantages in improving the activity of various enzymes. Ultimately, by promoting the conversion of soil nutrients, it provides good conditions for tobacco root growth and forms a synergistic effect with tobacco root growth and physiological enzyme indicators, which together demonstrates excellent treatment effect.

[0072] LY9 conversion substrate treatment enhances the activity of cellulase and sucrase, accelerating the decomposition and conversion of organic carbon in the soil, providing sufficient carbon and energy for tobacco, while improving soil aggregate structure and enhancing soil fertility and water retention capacity. It also increases the content of available phosphorus and readily available nitrogen in the soil, providing sufficient nitrogen and phosphorus nutrients for tobacco, ensuring its continued growth and yield potential, and reducing the need for chemical fertilizers. The increased soil enzyme activity signifies enhanced soil organic matter decomposition and nutrient conversion capacity, continuously providing sufficient nutrients for tobacco, ensuring its yield potential, and improving the soil microenvironment, thus achieving sustainable planting.

[0073] The general increase in the activity of other enzymes (α-glucosidase, β-glucosidase, amylase, etc.) indicates that the LY9 conversion matrix fully activates the soil's material cycle (carbon, nitrogen, phosphorus, etc.), optimizes the structure and function of the soil microbial community, and provides a sustainable soil environment for the long-term stable and high yield and quality of tobacco.

Claims

1. Application of Leucocalocybe mongolica strain LY9 in tobacco cultivation, wherein the Leucocalocybe mongolica strain LY9 is deposited at Guangdong Microbial Culture Collection Center, with accession number GDMCC No. 63678 and accession date of August 22, 2023.

2. The application according to claim 1, characterized in that, The application is to promote the growth of tobacco plants, improve the stress resistance of tobacco plants, or improve the quality of tobacco leaves.

3. The application according to claim 2, characterized in that, The promotion of tobacco plant growth includes: increasing the chlorophyll content of tobacco leaves; increasing the plant height and stem diameter of tobacco plants; increasing the leaf length, leaf width, and mid-leaf length of tobacco plants; increasing the number of effective leaves of tobacco plants; or promoting the root growth of tobacco plants.

4. The application according to claim 2, characterized in that, The improvement of tobacco leaf quality includes: increasing the total sugar, reducing sugar, total alkaloid, and total phenol content of tobacco leaves; increasing the protein and cellulose content of tobacco leaves; or increasing the total starch content of tobacco leaves.

5. The application according to claim 2, characterized in that, The method to improve the quality of tobacco leaves is to regulate the ratio and content of amylose, amylopectin and total starch in the leaves.

6. The application according to any one of claims 1-5, characterized in that, The method involves using an inoculum containing the Leucocalocybe mongolica LY9 strain to ferment the culture medium, obtaining a transformation substrate, and then using the transformation substrate for tobacco cultivation.

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