Bio-control inoculant for improving medicinal active ingredients and wilt resistance of longya lilium and preparation method of bio-control inoculant

Abstract

The invention discloses a biocontrol inoculant for improving medicinal active ingredients and wilt resistance of lilium brownii and a preparation method of the biocontrol inoculant, belongs to the technical field of agricultural microorganisms, and particularly relates to a biocontrol inoculant for improving medicinal active ingredients and wilt resistance of lilium brownii and a preparation method of the biocontrol inoculant. Comprising the following components in percentage by mass: 20-30% of conidium wettable powder of penicillium citrinum and 70-80% of bacillus velezensis wettable powder, and the prepared biocontrol microbial inoculum is relatively good in control effect, non-toxic, environment-friendly, powder-shaped, convenient to apply, efficient, green and environment-friendly, has wide market application potential, is worthy of popularization and application, and has a wide application prospect. Meanwhile, the content of medicinal active ingredients of polysaccharide, total flavone, saponin and total phenol of lilium brownii can be remarkably increased, the change of a microbial community structure in rhizosphere soil is influenced, the systematic resistance of plants to wilt is enhanced, and the application effect is good.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and in particular relates to a biocontrol agent capable of improving the medicinal active ingredients and wilt resistance of lily of the valley and a preparation method thereof. Background Art

[0002] Lilium longituba is a perennial bulbous plant of the genus Lilium in the family Liliaceae. It is a popular health product with medicinal and edible properties. It is an important economic crop that integrates food, medicine and ornamental purposes. It is naturally distributed in the mountains of Shanxi, Henan, Hubei, Hunan, Jiangxi, Anhui and Zhejiang provinces in my country at an altitude of 300-920m. The underground bulb of Lilium longituba is not only one of the three major edible lilies in my country, but also one of the lily-based raw drugs designated by the Chinese Pharmacopoeia. It has the effects of moistening the lungs and relieving coughs, clearing away heat and calming the nerves. Its bulb mainly contains steroidal saponins, colchicine, phenolic glycerol glycosides, polysaccharides, amino acids, phospholipids, and rich trace elements such as copper, zinc, magnesium, and calcium. It is a popular health product with medicinal and edible properties. In addition, Lilium longituba has white flowers and rich fragrance. It can be used as a spice and is also widely planted as a cut flower around the world. Therefore, Lilium longituba is an important economic crop that integrates food, medicine and ornamental purposes. It is one of the "Ten Flavors of Jiangxi Food" of traditional Chinese medicine in Jiangxi Province. At present, dragon tooth lilies are cultivated on a large scale in Wanzai, Taihe, Yongfeng in Jiangxi, Longhui, Anhua, Longshan in Hunan, Yongfu, Ziyuan, Liuzhou in Guangxi and other cities and counties in China. Among them, the history of dragon tooth lily cultivation in Wanzai, Jiangxi has been as long as 500 years.

[0003] During the cultivation process, Lilium longituba is prone to diseases such as wilt. From May to June every year, it is very common for lilies to be infected with diseases such as wilt, which seriously affects their yield and quality, causing great economic losses to growers. Effective prevention and control of such diseases is of great significance. With the rapid development of the Lilium longituba industry, it is becoming increasingly difficult to implement a strict crop rotation system. Fusarium wilt has occurred on a large scale, causing the entire Lilium longituba plant to wilt and the bulbs to deteriorate and rot, resulting in a significant reduction in yield. The disease rate of seriously affected fields reached 91.42%, and the disease index was 88.50. The average yield reduction of the affected fields was more than 46%, which often led to reduced yields and even total crop failures due to lily disease. The rampant pests and diseases seriously restricted the development of the Lilium longituba industry. Lily diseases are serious, with more than 40 kinds of diseases occurring, including fungal diseases, bacterial diseases, nematode diseases, physiological diseases, etc. In order to prevent and control diseases, growers use a large amount of pesticides. According to analysis, 29 kinds of pesticides are currently used in Longya lilies in Jiangxi Province, including insecticides, fungicides, herbicides and growth regulators, which also poses a huge hidden danger to product safety. At present, the research on Longya lily is mostly focused on cultivation and reproduction, extraction and identification of effective ingredients, and pest and disease control, while the research on biological control of wilt disease is extremely rare.

[0004] Therefore, it is of great significance to study the biocontrol agents that can improve the medicinal active ingredients and resistance to wilt of Lilium truncatum. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a biocontrol fungicide for improving the medicinal active ingredients and wilt resistance of Lilium argentea, which has good control effect, is non-toxic and environmentally friendly, and is in the form of powder for easy application. The biocontrol fungicide comprises the following components in mass percentage: 20-30% of conidia wettable powder of Penicillium citrinum and 70-80% of Bacillus velezii wettable powder.

[0006] Preferably, the Penicillium citrinum has a strain number of Qing 6, a preservation date of July 29, 2024, and a preservation number of CGMCC NO:41479.

[0007] Preferably, the Bacillus velezensis has a strain number of Hx182, a preservation date of July 29, 2024, and a preservation number of CGMCC NO:31478.

[0008] Preferably, the formula of the conidia wettable powder of Penicillium citrinum is as follows: the conidia powder content of Penicillium citrinum is 8-10%; the wetting agent is 1-1.25% sodium dodecyl sulfate; the dispersant is 5-6% sodium lignosulfonate; the ultraviolet protective agent is 0.3-0.4% xanthan gum; the spore promoter is 0.1-0.2% calcium chloride; the carrier is 82-85% kaolin; the spore content of the conidia wettable powder of Penicillium citrinum is 3.7×10 11 cfu / g.

[0009] Preferably, the formula of the Bacillus Velez wettable powder is: 40-50% fermentation liquid of Bacillus Velez, 40-45% diatomaceous earth, 9-12% saponin powder, 2-4% sodium tripolyphosphate, and 2-3% xanthan gum. The live bacteria content of the Bacillus Velez wettable powder is 2.65×10 11 cfu / g.

[0010] The present invention also provides a method for preparing a biocontrol agent for improving the medicinal active ingredients and wilt resistance of Lilium truncatum, comprising the following steps:

[0011] 1) preparing a conidia wettable powder of Penicillium citrinum;

[0012] 2) preparing Bacillus Velez wettable powder;

[0013] 3) Mix the conidia wettable powder of Penicillium citrinum and the wettable powder of Bacillus velez according to a certain proportion.

[0014] Preferably, the method for preparing the conidia wettable powder of Penicillium citrinum is:

[0015] a. Isolation and purification of antagonistic bacteria: The soil dilution method was used to isolate the strains from the rhizosphere soil of healthy lily. 10 g of fresh rhizosphere soil of lily was added to 90 mL of sterile water and shaken on a shaker at 200 r / min for 30 min to obtain a soil suspension. The soil suspension was diluted to 10 3 , 10 4 , 10 5 , 10 6 times, take 100uL of soil dilution and spread it on PDA medium, repeat three times for each concentration, culture at 25℃ for 1-3 days, pick single colonies with obvious morphological differences, inoculate them into PDA medium, observe the morphology of the separated colonies, check whether they contain foreign bacteria, and continue to purify the colonies with obvious morphological differences for 3-4 times;

[0016] b. Identification of Qing 6 strains: The strains with good antagonistic effects that were screened out were identified using morphological characteristics and molecular biology methods;

[0017] c. Solid fermentation and spore powder harvesting: The strain Qing 6 cultured on PDA medium for 7 days was rinsed with sterile water to obtain a spore suspension and adjust its concentration to 1.0×10 6 cfu / ml, added to PDB medium at a rate of 1%, cultured at 28°C and 170r / min for 7 days for subsequent experiments, 50g of glutinous rice flour was placed in a 15cm culture dish, sterilized in a high-pressure steam sterilizer at 121°C for 30min, 30ml of the collected seed liquid was drawn into the culture dish in a clean bench, stirred evenly, repeated 3 times, placed in a 28°C incubator and cultured at a constant temperature for 7 days, cultured for 7 days until the glutinous rice flour grew dark green spore powder, dried in a 35°C forced air drying oven for 5 days, took out the culture dish and ground it with a mortar to obtain spore powder;

[0018] d. Screening of the formulation of wettable powder of Penicillium 6;

[0019] e. The conidia powder of Penicillium citrinum, a wetting agent, a dispersant, an ultraviolet protective agent, a spore promoter and a carrier are mixed and stirred in proportion, and then continuously dried in an electric constant temperature blast drying oven, and finally ground and pulverized to obtain a wettable powder.

[0020] Preferably, the method for preparing Bacillus Velez subtilis wettable powder is:

[0021] a. Isolation and purification of bacteria: The soil dilution method was used to isolate the strains from the rhizosphere soil of healthy lily. 10 g of fresh rhizosphere soil of lily was added to 90 mL of sterile water and shaken on a shaker at 200 r / min for 30 min to obtain a soil suspension. The soil suspension was diluted to 10 3 , 10 4 , 10 5 , 10 6 times, take 100 μL of soil dilution and spread it on PDA medium, repeat three times for each concentration, culture at 25℃ for 1-3 days, pick single colonies with obvious morphological differences, inoculate them into PDA medium, observe the morphology of the separated colonies, check whether they contain foreign bacteria, and continue to purify the colonies with obvious morphological differences for 3-4 times;

[0022] b. Identification of Hx182 strains: The strains with good antagonistic effects that were screened were identified using morphological characteristics and molecular biology methods;

[0023] c. Solid fermentation and spore powder harvesting: The strain Hx182 cultured on PDA medium for 7 days was rinsed with sterile water to obtain a spore suspension and adjust its concentration to 1.0×10 6 cfu / ml, added to PDB medium at a rate of 1%, cultured at 28°C and 170r / min for 7 days for subsequent experiments, 50g of glutinous rice flour was placed in a 15cm culture dish, sterilized in a high-pressure steam sterilizer at 121°C for 30min, 30ml of the collected seed liquid was drawn into the culture dish in a clean bench, stirred evenly, repeated 3 times, placed in a 28°C incubator and cultured at a constant temperature for 7 days, cultured for 7 days until the glutinous rice flour grew dark green spore powder, dried in a 35°C forced air drying oven for 5 days, took out the culture dish and ground it with a mortar to obtain spore powder;

[0024] d. Preparation of fermentation broth of Hx182 strain: inoculate the cultured Hx182 strain into LB medium by streak culture, culture at 25°C for 24 h to obtain a single colony, pick a single colony and inoculate it into a 250 ml conical flask containing 30 ml seed medium, shake culture for 24 h to obtain seed liquid, take Hx182 seed liquid, inoculate it into fermentation medium at an inoculation rate of 1%, shake culture at 28°C, 170 r / min for 24 h to obtain strain fermentation broth;

[0025] e. Screening of Hx182 wettable powder formulation;

[0026] f. Mix and stir the Hx182 strain fermentation liquid, diatomaceous earth, saponin powder, sodium tripolyphosphate and xanthan gum according to a proportion, then continue to dry in an electric constant temperature blast drying oven, and finally grind and crush to make a wettable powder.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] The biocontrol agent prepared by the invention has good control effect, is non-toxic and environmentally friendly, and the product is in the form of powder, which is convenient to apply. It is a highly efficient, green and environmentally friendly biocontrol agent, has broad market application potential, and is worthy of popularization and use. At the same time, it can significantly increase the content of polysaccharides, total flavonoids, saponins and total phenols, which are medicinal active ingredients of lily of the valley, affect the changes in the microbial community structure in the rhizosphere soil, and enhance the systemic resistance of the plant to wilt disease, and has a good application effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The morphology of Qing 6 and Hx182 colonies and their spores;

[0030] Figure 2 This is a comparison chart of the antibacterial effect of the composite bacterial agent on wilt bacteria;

[0031] Figure 3 This is the effect of the composite biocontrol agent treatment on the polysaccharides, total flavonoids, total saponins and total phenols of Lilium longituba;

[0032] Figure 4 This is the simper analysis chart of species and genus levels of differences among the groups treated with the compound bacterial agent. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1

[0035] 1) Preparation of Qing 6 bacteria agent:

[0036] A. Isolation and purification of bacterial colonies: The soil dilution method was used to isolate the bacterial strains from the rhizosphere soil of healthy lilies. 10 g of fresh lily rhizosphere soil was added to 90 mL of sterile water and shaken on a shaker at 200 r / min for 30 min to obtain a soil suspension. The soil suspension was diluted to 10 3 , 10 4 , 10 5 , 10 6 times, draw 100uL of soil dilution and apply it on PDA medium, repeat three times for each concentration, culture at 25℃ for 1-3 days, pick single colonies with obvious morphological differences, and inoculate them into PDA medium. Observe the morphology of the isolated colonies and check whether they contain foreign bacteria. Purify the colonies with obvious morphological differences for 3-4 times.

[0037] B. Identification of Qing 6 strain: The strains with good antagonistic effects that were screened were identified using morphological characteristics and molecular biology methods.

[0038] C. Solid fermentation and spore powder harvesting: The strain Qing 6 cultured on PDA medium for 7 days was rinsed with sterile water to obtain a spore suspension and adjust its concentration to 1.0×10 6 cfu / ml, added to PDB medium at a rate of 1%, cultured at 28°C and 170r / min for 7 days for subsequent experiments. Place glutinous rice flour (50g) in a 15cm culture dish, sterilize in a high-pressure steam sterilizer at 121°C for 30min, draw 30ml of the collected seed liquid in a clean bench and add it to the culture dish, stir evenly, and repeat 3 times. Place in a 28°C incubator and culture at a constant temperature for 7 days. After culturing for 7 days until the glutinous rice flour grows dark green spore powder, dry it in a 35°C forced air drying oven for 5 days, take out the culture dish and grind it with a mortar to obtain crude spore powder.

[0039] D. Screening of Penicillium 6 wettable powder formulation:

[0040] Vector screening:

[0041] The crude spore powder was mixed with melted PDA medium at a ratio of 5% (w / v). After sterilization, the plate was prepared. The spore suspension was prepared and adjusted to 1.0×10 6 cfu / mL, and perform gradient dilution 1000 times. Take 0.1ml of the diluted spore suspension and spread it on plates containing different carriers (kaolin, diatomaceous earth, calcium carbonate, white carbon black, talcum powder), and use PDA plates without carriers as controls. Set up 3 replicates for each treatment. Incubate all treated plates at 25±1℃ for 3 days. Observe the number of colony forming units (cfu) of Acremonium spores grown on each plate.

[0042] Prepare plates containing various carriers according to the above method. Take 2 μl of spore suspension and inoculate it on the culture medium of different carriers. Measure the diameter of each colony on the 7th day and record it as dl. Set up 3 replicates for each treatment. Calculate the daily growth of colony diameter: daily growth of colony diameter (mm / d) = d1 / days of growth, and select suitable carriers through calculation.

[0043] Screening of wetting agents:

[0044] Accurately weigh 1.0g of spore powder (w1) and prepare samples with different wetting agent contents and types with wetting agent powder (w2) at a ratio of 1%, 3% and 5% (w2 / w1). Accurately weigh 0.1g of sample and pour it evenly into the liquid surface of a 150mL beaker containing 100mL of standard water at the level of the beaker mouth. Use a stopwatch to record the time when adding the sample until the sample is completely wetted. Record the wetting time. Repeat this 5 times and take the average value as the wetting time of each sample. The sample without wetting agent is used as the control.

[0045] Wetting agent (sodium dodecylbenzene sulfonate, sodium dodecyl sulfate) powder was mixed with PDA medium at a concentration ratio of 125ug / mL, 250ug / mL, 500ug / mL, and 1000ug / mL, and plated after sterilization. Prepare and adjust the spore suspension to 1.0*10 6 cfu / mL, perform gradient dilution 1000 times, take 0.1ml of spore suspension, and spread it on plates with different wetting agents, with PDA plates without wetting agents as controls. Set up 3 replicates. Incubate all treated plates at 25±1℃ for 3 days. Observe the number of blue 6 colony forming units (cfu) grown on each plate to screen out the appropriate wetting agent.

[0046] The test on colony diameter growth is the same as above.

[0047] Screening of dispersants:

[0048] Dispersant (sodium lignosulfonate, sodium tripolyphosphate) powder was mixed with melted potato dextrose agar (PDA) medium at a concentration ratio of 125ug / mL, 250ug / mL, 500ug / mL, 1000ug / mL, and 2500ug / mL, and plated after sterilization. Prepare and adjust the spore suspension to 1.0*10 6 cfu / mL, perform gradient dilution 1000 times, take 0.1mL of spore suspension, and spread it on each plate containing different carriers. Use PDA plate without dispersant as control. Set up 3 replicates for each treatment. Incubate all treated plates at 25±1℃ for 4 days. Observe the number of colony forming units (cfu) of Acremonium spores grown on each plate.

[0049] The test on colony diameter growth is the same as above.

[0050] The above selected better dispersants were prepared into mother solutions of 125ug / mL, 250ug / mL and 500ug / mL concentrations. 10mL of Acremonium spore suspension (10 7spores / mL) were added to 90mL of mother solution, and samples were taken from the upper, middle and lower layers of the suspension with an inoculating gun at 0h, 2h and 4h after shaking, and counted with a hemocytometer. The sample without dispersant was used as the control. The dispersion index (I) was calculated according to the following formula: I = σ2 / ā, σ is the standard deviation; ā is the sample mean; when I < 1, the spores are distributed in clusters; I = 1, the spores are randomly distributed; I > 1, the spores are distributed in clusters.

[0051] Screening of UV protectants:

[0052] Various UV protectants (sodium alginate, xanthan gum, dextrin, humic acid) were mixed with melted potato dextrose agar (PDA) medium at a final concentration of 0.3%, and the plates were prepared after sterilization. The spore suspension was prepared and adjusted to 1.0×10 6 cfu / mL, perform gradient dilution 1000 times, take 0.1mL of spore suspension, and spread it on each plate containing different carriers. Use PDA plate without UV protectant as control. Set up 3 replicates for each treatment. Incubate all treated plates at 25±1℃ for 4 days. Observe the number of colony forming units (cfu) grown on each plate.

[0053] Each tested UV protectant was mixed with PDB liquid culture medium at a final mass concentration of 0.3%, 0.010g of conidia powder was weighed and added to 1mL of the prepared solution, and the conidia suspension was obtained by mixing evenly. Then 100μL of the suspension was aspirated with a pipette and dropped on the center of a sterile glass slide, and then placed under an ultraviolet lamp (30w, 120Lux) for 10min and 1h, and then the concave glass slide was placed flat in a culture dish, and the culture dish was moisturized with moist sterile filter paper. The control without UV protectant was used as the control and CK was recorded, and 3 replicates were set for each treatment. The spore germination rate was determined to screen out the UV protectant.

[0054] Screening of Qing 6 spore germination promoter:

[0055] The spore germination promoters (zinc sulfate, calcium chloride, potassium chloride) were mixed at a ratio of 0.05%, 0.1% and 0.2% (w / v) to prepare a mother solution. 6 The spore suspension with cfu / mL was diluted 1000 times, and 0.1 ml of the mother solution was evenly applied on the PDA plate with a pipette. Each treatment was repeated 3 times, and the blank control was a spore suspension without spore germination promoter. After 4 days of cultivation, the number of colony forming units (cfu) of Acremonium spores grown on each plate was observed to select the optimal germination promoter.

[0056] The formula of the obtained Qing 6 conidia wettable powder is as follows: the active ingredient is 10% of Qing 6 conidia powder; the wetting agent is 1.25% of sodium dodecyl sulfate; the dispersant is 5% of sodium lignosulfonate; the ultraviolet protective agent is 0.3% of xanthan gum; the spore promoter is 0.10% of calcium chloride; the carrier is kaolin, the content of which is 83.35%. The spore content of the final spore powder is 3.7×10 11 cfu / g.

[0057] 2) Preparation method of Hx182 bacterial agent:

[0058] A. Isolation and purification of bacteria: The soil dilution method was used to isolate the strains from the rhizosphere soil of healthy lilies. 10 g of fresh lily rhizosphere soil was added to 90 mL of sterile water and shaken on a shaker at 200 r / min for 30 min to obtain a soil suspension. The soil suspension was diluted to 10 3 , 10 4 , 10 5 , 10 6 times, take 100 μL of soil dilution and apply it on PDA medium, repeat three times for each concentration, culture at 25℃ for 1-3 days, pick single colonies with obvious morphological differences, and inoculate them into PDA medium. Observe the morphology of the isolated colonies and check whether they contain foreign bacteria. Purify the colonies with obvious morphological differences for 3-4 times.

[0059] B. Identification of Hx182 strains: The strains with good antagonistic effects that were screened were identified using morphological characteristics and molecular biology methods.

[0060] C. Solid fermentation and spore powder harvesting: The strain cultured on PDA medium for 7 days was rinsed with sterile water to obtain a spore suspension and its concentration was adjusted to 1.0×10 6 cfu / ml, added to PDB medium at a rate of 1%, cultured at 28°C and 170r / min for 7 days for subsequent experiments. Place glutinous rice flour (50g) in a 15cm culture dish, sterilize in a high-pressure steam sterilizer at 121°C for 30min, draw 30ml of the collected seed liquid in a clean bench and add it to the culture dish, stir evenly, and repeat 3 times. Place in a 28°C incubator and culture at a constant temperature for 7 days. After culturing for 7 days until the glutinous rice flour grows dark green spore powder, dry it in a 35°C forced air drying oven for 5 days, take out the culture dish and grind it with a mortar to obtain crude spore powder.

[0061] D. Screening of Hx182 wettable powder formula:

[0062] Preparation of seed solution: The cultured Hx182 strain was inoculated into LB medium by streak culture, and cultured at 25°C for 24 hours to obtain a single colony. A single colony was picked and inoculated into a 250 ml conical flask containing 30 ml of seed culture medium, and cultured with shaking for 24 hours to obtain seed solution.

[0063] Fermentation culture: Take Hx182 seed liquid, inoculate it into fermentation medium at an inoculum rate of 1%, and culture it at 28°C and 170r / min with shaking for 24h to obtain the strain fermentation liquid.

[0064] Screening of wettable powder carriers and adjuvants:

[0065] (1) Selection of carrier: The carrier (kaolin, diatomaceous earth, calcium carbonate, white carbon black, talc) was mixed evenly with sterilized LB medium at a ratio of 5% (mass fraction) to prepare a plate. Take 1 ml of the fermentation liquid (adjust OD600 = 0.8) and dilute it with sterile water to 1.0 × 10 7 cfu / ml, take 0.1mL of the dilution and spread it evenly on plates containing different vectors. The plates without vectors were used as blank controls. Each treatment was repeated 3 times. After culturing at 25℃ for 2 days, the number of colonies on the plates was counted to screen out the vectors that had no effect on the bacteria.

[0066] (2) Selection of adjuvants: Wetting agent: Sodium dodecylbenzene sulfonate, sodium dodecyl sulfate, and saponin powder were mixed with melted LB medium at a concentration of 250 mg / L, and then sterilized to make a plate; Dispersant: Sodium lignin sulfonate, sodium tripolyphosphate, and sodium carboxymethyl cellulose were mixed with melted LB medium at a concentration of 1.5 g / L, and then sterilized to make a plate; Protective agent: Sodium alginate, xanthan gum, dextrin, and humic acid were mixed with melted LB medium at a concentration of 50 mg / L, and then sterilized to make a plate. 0.1 ml of the fermentation liquid of strain Hx182 diluted in "1.4.2" was added to the above treatments and spread on plates containing different adjuvants. The LB plate without adjuvants was used as the control, and each treatment was repeated 3 times. After culturing at 25°C for 3 days, the number of colonies of strain Hx182 on each plate was recorded, and the strain that had no effect on the bacteria was selected.

[0067] (3) Using the selected carriers and adjuvants, a 4-factor 3-level orthogonal experimental design (L9(34)) was conducted to detect the effects of different ratios on the number of viable bacteria and determine the optimal ratio of the carrier and adjuvant. The experimental design is shown in Table 1.

[0068] Table 1 Orthogonal test table of carrier and auxiliary agent ratio

[0069]

[0070] Processing of wettable powder of strain Hx182: Hx182 (4.3×10 9cfu / ml) of fermentation liquid (50ml) was mixed and stirred at the same time according to the optimized ratio of carrier and adjuvant, and then dried in an electric constant temperature blast drying oven, and finally ground and crushed to make a wettable powder. Weigh 1.0g and dilute and apply it with sterile water, repeat 3 times, and measure the bacterial count after culturing at 25℃ for 48h.

[0071] The best ratio is 45% of Hx182 fermentation liquid, 40% of diatomaceous earth, 10% of saponin powder, 3% of sodium tripolyphosphate, and 2% of xanthan gum. 11 cfu / g.

[0072] 3) The conidia wettable powder of Penicillium citrinum and the wettable powder of Bacillus velez were mixed in a ratio of 3:7 to prepare a biocontrol agent.

[0073] Antagonistic bacteria mutual antagonism test

[0074] A. Preparation of bacterial liquid: To avoid antagonism between the two bacterial strains caused by the compound bacteria, which will lead to poor control effect of the bacterial strains themselves, inoculate Qing 6 into PDB medium and Hx182 into LB medium. Incubate the culture at 25°C, 180r / min and shake for 24h. Centrifuge the fermentation liquid at 8000r / min for 10min. Take the supernatant and filter it through a 0.22μm filter membrane (bacteria) for later use. Filter the fungi with sterile cotton.

[0075] B. Plate confrontation method: After the plate is cooled, place the sterilized filter paper with a diameter of 5 mm on the plate, with four filter papers on each plate, and take 1 μl of the corresponding bacterial solution and drop it on the filter paper, with 1 μl on each filter paper. Repeat each treatment 3 times and place it at a constant temperature of 25°C for incubation. After 1-2 days, observe the presence of the inhibition zone to determine whether the two bacteria can be compounded.

[0076] Potted Plant Experiment of Lilium argentea

[0077] The bulbs of Lilium longituba weigh 14-18 grams. Each treatment has 12 pots, with the following settings: bulbs not inoculated with bacteria ck and bulbs inoculated with pathogenic bacteria Fusarium oxysporum, soaked for 30 minutes + homemade bacterial agent Hx182 + Qing 6, 50 ml per plant, 2 treatments in total, 3 repetitions, 72 pots in total, 3 bulbs planted in each pot, all using normal stubble soil.

[0078] Bulb pretreatment: dense planting with substrate in October 2023, and planting of bulbs with uniform germination and no diseases in February 2024. The rest of the management is the same as usual. The bulbs of Lilium longituba were sampled on June 7 (second phase) and July 4 (third phase) in 24 for the determination of the active medicinal ingredients of Lilium longituba.

[0079] Methods for determining the contents of total phenols, total flavonoids, total saponins and polysaccharides in Lilium argentea: The total phenol content was determined by the Folin phenol colorimetric method, the total saponins were determined by the perchloric acid-vanillin colorimetric method, the total flavonoids content was determined by the sodium nitrite-aluminum nitrate method, and the polysaccharide content was determined by the anthrone sulfate method.

[0080] Statistical method for the incidence of lily wilt disease: The method of Ren Shenglin et al. was used to measure the yellow and withered leaves, stem rot, scale and root disease grading of Lilium longituba. The severity of the scale disease was divided into: 0 for health, 1 for 1-2 scales rot, 2 for 3-5 scales rot, 3 for 1 / 2-3 / 4 scales rot, and 4 for more than 3 / 4 scales rot. The disease index and control effect were also measured.

[0081] Disease index = Σ(disease level × number of plants at that level) / (total number of plants × highest disease level) × 100.

[0082] Control effect = (control disease index - treatment disease index) / control disease index × 100%.

[0083] Pot test results:

[0084] Table 2 Statistics on the incidence of Lilium truncatum potted plant trials

[0085]

[0086] Figure 1 The morphology of Qing 6 and Hx182 colonies and their spores. A is the Qing 6 colony; B is the Hx182 colony; C is the conidiophore of the Qing 6 strain; D is the conidia of the Qing 6 strain; E is the spores of the Hx182 colony.

[0087] Figure 2 The comparison of the antibacterial effect of the composite bacterial agent on Fusarium wilt pathogen. Figure 2 The left 1 in the middle is a leaf of Lilium oleraceum inoculated with Fusarium oxysporum; the left 2 is a leaf inoculated with Fusarium oxysporum and a composite bacterial agent; the right 2 is a PDA plate inoculated with Fusarium oxysporum; the right 1 is the growth of Fusarium oxysporum inoculated on a PDA plate coated with a composite bacterial agent. It can be seen that the composite biocontrol agent has a good antibacterial effect.

[0088] like Figure 3 As shown, Figure 3 This is the effect of composite biocontrol agent treatment on polysaccharides, total flavonoids, total saponins and total phenols in Lilium argentea. It can be clearly seen that the content of polysaccharides, total flavonoids, total saponins and total phenols in Lilium argentea after using the agent is higher than that in Lilium argentea grown normally.

[0089] Figure 4The difference species (genus level) among the groups treated with the composite bacterial agent. Simper analysis showed that the dominant bacterial community was Bacillus, which increased significantly.

[0090] Therefore, the biocontrol agent of the present invention has good control effect, is non-toxic, and is environmentally friendly. The product is in the form of powder, which is convenient to apply. It is an efficient, green and environmentally friendly biocontrol agent with wide market application potential and is worthy of promotion and use. The composite bacterial agent can significantly increase the content of its medicinal active ingredients, polysaccharides, total flavonoids, saponins and total phenols, affect the changes in the microbial community structure in the rhizosphere soil, and enhance the systemic resistance of plants to wilt disease, and has a good application effect.

[0091] The above descriptions are merely embodiments of the present invention. It should be pointed out that a person skilled in the art can make various changes, modifications, substitutions and variations to these embodiments without departing from the technical principles of the present invention. These changes, modifications, substitutions and variations should also be regarded as within the scope of protection of the present invention.

Claims

1. A biocontrol agent for improving the medicinal active ingredients and wilt resistance of Lilium truncatum, characterized in that: The invention comprises the following components in percentage by weight: 20-30% of conidia wettable powder of Penicillium citrinum and 70-80% of wettable powder of Bacillus velezii.

2. A biocontrol agent for improving the medicinal active ingredients and wilt resistance of Lilium truncatum according to claim 1, characterized in that: The Penicillium citrinum has a strain number of Qing 6 and a preservation number of CGMCC NO:41479.

3. The biocontrol agent for improving the medicinal active ingredients and wilt resistance of Lilium truncatum according to claim 1, characterized in that: The strain number of the Bacillus velezensis is Hx182, and the deposit number is CGMCC NO:31478.

4. The biocontrol agent for improving the medicinal active ingredients and wilt resistance of Lilium truncatum according to claim 1, characterized in that: The formula of the conidia wettable powder of Penicillium citrinum is as follows: the conidia powder content of Penicillium citrinum is 8-10%; the wetting agent is 1-1.25% sodium dodecyl sulfate; the dispersant is 5-6% sodium lignosulfonate; the ultraviolet protective agent is 0.3-0.4% xanthan gum; the spore promoter is 0.1-0.2% calcium chloride; the carrier is 82-85% kaolin; the spore content of the conidia wettable powder of Penicillium citrinum is 3.7×10 11 cfu / g.

5. The biocontrol agent for improving the medicinal active ingredients and wilt resistance of Lilium truncatum according to claim 1, characterized in that: The formula of the Bacillus Velez wettable powder is: 40-50% fermentation liquid of Bacillus Velez, 40-45% diatomaceous earth, 9-12% saponin powder, 2-4% sodium tripolyphosphate, and 2-3% xanthan gum. The live bacteria content of the Bacillus Velez wettable powder is 2.65×10 11 cfu / g.

6. The method for preparing the biocontrol agent for improving the medicinal active ingredients and wilt resistance of Lilium argentea according to claim 1, characterized in that: The following steps are involved: 1) Preparing a wettable powder of conidia of Penicillium citrinum; 2) preparing wettable powder of Bacillus Velez; 3) Mix the conidia wettable powder of Penicillium citrinum and the wettable powder of Bacillus velezensis in proportion.

7. The method for preparing the biocontrol agent for improving the medicinal active ingredients and wilt resistance of Lilium truncatum according to claim 6, characterized in that: The method for preparing the conidia wettable powder of Penicillium citrinum is as follows: a. Isolation and purification of antagonistic bacteria: The soil dilution method was used to isolate the strains from the rhizosphere soil of healthy lily. 10 g of fresh rhizosphere soil of lily was added to 90 mL of sterile water and shaken on a shaker at 200 r / min for 30 min to obtain a soil suspension. The soil suspension was diluted to 10 3 , 10 4 , 10 5 , 10 6 times, take 100uL of soil dilution and spread it on PDA medium, repeat three times for each concentration, culture at 25℃ for 1-3 days, pick single colonies with obvious morphological differences, inoculate them into PDA medium, observe the morphology of the separated colonies, check whether they contain foreign bacteria, and continue to purify the colonies with obvious morphological differences for 3-4 times; b. Identification of Qing 6 strains: The strains with good antagonistic effects that were screened out were identified using morphological characteristics and molecular biology methods; c. Solid fermentation and spore powder harvesting: The strain Qing 6 cultured on PDA medium for 7 days was rinsed with sterile water to obtain a spore suspension and adjust its concentration to 1.0×10 6 cfu / ml, added to PDB medium at a rate of 1%, cultured at 28℃, 170r / min for 7 days for subsequent experiments, 50g of glutinous rice flour was placed in a 15cm culture dish, sterilized in a high-pressure steam sterilizer at 121℃ for 30min, 30ml of the collected seed liquid was drawn into the culture dish in a clean bench, stirred evenly, repeated 3 times, placed in a 28℃ incubator for constant temperature culture for 7 days, cultured for 7 days until the glutinous rice flour grew dark green spore powder, dried in a 35℃ forced air drying oven for 5 days, took out the culture dish and ground it with a mortar to obtain spore powder; d. Screening of the formulation of wettable powder of Penicillium 6; e. The conidia powder of Penicillium citrinum, a wetting agent, a dispersant, an ultraviolet protective agent, a spore promoter and a carrier are mixed and stirred in proportion, and then continuously dried in an electric constant temperature blast drying oven, and finally ground and pulverized to obtain a wettable powder.

8. The method for preparing the biocontrol agent for improving the medicinal active ingredients and wilt resistance of Lilium argentea according to claim 6, characterized in that: The method for preparing the Bacillus Velez wettable powder is as follows: a. Isolation and purification of bacteria: The soil dilution method was used to isolate the strains from the rhizosphere soil of healthy lily. 10 g of fresh rhizosphere soil of lily was added to 90 mL of sterile water and shaken on a shaker at 200 r / min for 30 min to obtain a soil suspension. The soil suspension was diluted to 10 3 , 10 4 , 10 5 , 10 6 times, take 100 μL of soil dilution and spread it on PDA medium, repeat three times for each concentration, culture at 25℃ for 1-3 days, pick single colonies with obvious morphological differences, inoculate them into PDA medium, observe the morphology of the separated colonies, check whether they contain foreign bacteria, and continue to purify the colonies with obvious morphological differences for 3-4 times; b. Identification of Hx182 strains: The strains with good antagonistic effects that were screened were identified using morphological characteristics and molecular biology methods; c. Solid fermentation and spore powder harvesting: The strain Hx182 cultured on PDA medium for 7 days was rinsed with sterile water to obtain a spore suspension and adjust its concentration to 1.0×10 6 cfu / ml, added to PDB medium at a rate of 1%, cultured at 28℃, 170r / min shaking for 7 days for subsequent experiments, 50g of glutinous rice flour was placed in a 15cm culture dish, sterilized in a high-pressure steam sterilizer at 121℃ for 30min, 30ml of the collected seed liquid was drawn into the culture dish in a clean bench, stirred evenly, repeated 3 times, placed in a 28℃ incubator and cultured at a constant temperature for 7 days, and after culturing for 7 days until the glutinous rice flour grew dark green spore powder, dried in a 35℃ forced air drying oven for 5 days, took out the culture dish and ground it with a mortar to obtain spore powder; d. Preparation of fermentation broth of Hx182 strain: inoculate the cultured Hx182 strain into LB medium by streak culture, culture at 25°C for 24 h to obtain a single colony, pick a single colony and inoculate it into a 250 ml conical flask containing 30 ml seed medium, shake culture for 24 h to obtain seed liquid, take Hx182 seed liquid, inoculate it into fermentation medium at an inoculation rate of 1%, shake culture at 28°C, 170 r / min for 24 h to obtain strain fermentation broth; e. Screening of Hx182 wettable powder formulation; f. Mix and stir the Hx182 strain fermentation liquid, diatomaceous earth, saponin powder, sodium tripolyphosphate and xanthan gum according to a proportion, then continue to dry in an electric constant temperature blast drying oven, and finally grind and crush to make a wettable powder.

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