A compound microbial agent for preventing and treating cypress needle blight and application thereof

Abstract

The application discloses a compound microbial agent for preventing and treating cypress leaf blight, which is composed of the following components in mass percentage: 20-40% of Paecilomyces variotii spore powder, 10-20% of Paecilomyces variotii fermentation broth extract melanin, 15-25% of dispersing agent, and the balance of liquid fermentation medium, with the total mass of the compound microbial agent being 100%; the Paecilomyces variotii fermentation broth extract melanin and the Paecilomyces variotii spore powder are both prepared by fermenting Paecilomyces variotii with a preservation number of CGMCC3.24355; the effective viable cell number of the Paecilomyces variotii spore powder is greater than 6.5*10 7 g ‑1 Compared with existing chemical agents, the compound microbial agent has low toxicity, small environmental pressure, remarkable specific inhibition effect on the cypress green cup fungus which is the pathogenic bacterium of the cypress leaf blight, good prevention and treatment effect on the cypress leaf blight, and good development and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of arborvitae disease control technology, specifically relating to a compound microbial agent for controlling arborvitae leaf blight and its application. Background Technology

[0002] Chinese arborvitae (Platycladus orientalis), also known as fragrant cedar, is the most widely distributed coniferous tree species in my country. It is highly adaptable, drought-tolerant, and thrives in poor soil, making it a pioneer species for afforestation of barren mountains. However, Chinese arborvitae leaf blight is prevalent in Hebei, Jiangsu, Shaanxi, and Gansu provinces. Infection with the pathogen can range from mildly affecting growth to severe enough to cause the entire tree to die, posing a serious threat to the achievements of years of forestry ecological construction projects.

[0003] The pathogen causing leaf blight in Chinese arborvitae is *Chloros cyphaplatycladus* Daisp. nov., belonging to the Ascomycota, Discomycetes, Heliomycetes, and *Chloros cyphaplatycladus*. It primarily infects the scale leaves and twigs of the Chinese arborvitae, a plant in the Cupressaceae family. Severe disease occurs in the inner and lower parts of the crown. Within approximately 15 days, affected plants will experience leaf drop in large numbers, branch dieback with a scorched appearance, and ultimately die.

[0004] Currently, chemical fungicides are generally used to control arborvitae leaf blight. However, this method is not conducive to ecological sustainability. Therefore, there is an urgent need to develop an environmentally friendly, efficient, and low-toxicity agent for the control of arborvitae leaf blight. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies that use chemical fungicides to control arborvitae leaf blight, and to provide a melanin-based compound fungicide for controlling arborvitae leaf blight.

[0006] The technical solution of this invention is as follows:

[0007] A compound microbial agent for the prevention and control of arborvitae leaf blight, comprising, by weight percentage, 100% of the total mass of the compound microbial agent: 20-40% of Paecilomyces wani spore powder, 10-20% of melanin extracted from Paecilomyces wani fermentation broth, 15-25% of dispersant, with the remainder being liquid fermentation culture medium.

[0008] The melanin extracted from the fermentation broth of *Paecilomyces wani* and the spore powder of *Paecilomyces wani* were both prepared by fermentation culture of *Paecilomyces wani* with accession number CGMCC3.24355. The effective viable count of the *Paecilomyces wani* spore powder was greater than 6.5 × 10⁻⁶. 7 g -1 .

[0009] Furthermore, the preparation method of the *Penicillium wani* spore powder includes the following steps:

[0010] (1) Inoculate *Paecilomyces wani* into *Paecilomyces wani* seed culture medium and culture at 25℃ and 120r / min for 4 days to obtain *Paecilomyces wani* seed liquid;

[0011] (2) After soaking the rice, it is steam-sterilized and cooked to 5-6 maturity. After cooling, a rice culture medium is prepared. The seed liquid of *Penicillium wanyi* is added to the rice culture medium at a volume mass ratio of 10%. The medium is cultured at 25℃ for 6-7 days. After the spores are fully grown, the fermentation is stopped and the rice is dried in a ventilated place for 1-2 days to obtain the final product.

[0012] In step (1), the seed culture medium formula of *Penicillium wani* is as follows: soluble starch 20 g / L, glucose 20 g / L, yeast powder 2 g / L, NaCl 4 g / L, K2HPO3 0.5 g / L, MgSO4·H2O 0.5 g / L, CaCO3 2 g / L;

[0013] In step (2), the rice culture medium formula is as follows: 100g rice, 10g soybean flour, 15g wheat bran, 0.5g NaCl, 0.5g K2HPO3, 1g CaCO3; the material-to-water ratio is 10:6.

[0014] Furthermore, the preparation method of melanin from the fermentation broth of *Paecilomyces wanensis* is as follows: *Paecilomyces wanensis* is inoculated into potato broth medium and cultured at 28℃ and 120 r / min for 7 days. Then, it is centrifuged to remove the precipitate. The obtained fermentation supernatant is concentrated, anhydrous ethanol is added, centrifuged again, and the precipitate is removed. The obtained supernatant is concentrated, Sevage reagent is added, and after mixing evenly, it is centrifuged again. The upper layer solution is collected, concentrated under reduced pressure, and freeze-dried to obtain crude extract powder of *Paecilomyces wanensis* fermentation broth. Finally, it is separated and purified to obtain melanin from the fermentation broth of *Paecilomyces wanensis*. The separation and purification are performed using high-speed countercurrent chromatography, with a mobile phase of methanol, n-hexane, and water in a volume ratio of 4:2:3.

[0015] Furthermore, the dispersant is sodium lignosulfonate.

[0016] Furthermore, the formula of the liquid fermentation medium is as follows: 20 g / L soybean meal powder, 2 g / L soybean peptone, 20 g / L glucose, 5 g / L soluble starch, 2 g / L yeast powder, 4 g / L NaCl, 0.5 g / L K2HPO3, 0.5 g / L MgSO4·H2O, and 2 g / L CaCO3.

[0017] The preparation method of the above-mentioned compound microbial agent for the prevention and control of arborvitae leaf blight is as follows: according to the above-mentioned mass percentages, take the spore powder of Paecilomyces wani, the melanin extracted from the fermentation broth of Paecilomyces wani and the dispersant, add them to the liquid fermentation culture medium, mix them evenly, and the compound microbial agent is obtained.

[0018] The above-mentioned compound microbial agent is used to control arborvitae leaf blight. When applying, the compound microbial agent is sprayed on the leaves once a week for 10 consecutive weeks.

[0019] The beneficial effects of this invention are:

[0020] This invention discloses a compound microbial agent for the prevention and control of arborvitae leaf blight. Its active ingredients are composed of melanin extracted from the fermentation broth of *Paecilomyces wani* and *Paecilomyces wani* liquid. Compared with existing chemical agents, this compound microbial agent has low toxicity, low environmental stress, and a significant specific inhibitory effect on *Chloroscyphaplatycladus* Daisp.nov., the pathogen of arborvitae leaf blight. It has a good control effect on arborvitae leaf blight and has good prospects for development and application. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that in the following embodiments, the Paecilomyces variotii used has the accession number CGMCC3.24355, which was deposited on December 6, 2023, at the China General Microbiological Culture Collection Center, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0022] The culture media and formulations involved in the following examples are as follows:

[0023] Seed culture medium for *Penicillium wani*: soluble starch 20 g / L, glucose 20 g / L, yeast extract 2 g / L, NaCl 4 g / L, K2HPO3 0.5 g / L, MgSO4·H2O 0.5 g / L, CaCO3 2 g / L.

[0024] Rice culture medium: 100g rice, 10g soybean flour, 15g wheat bran, 0.5g NaCl, 0.5g K2HPO3, 1g CaCO3; material-to-water ratio 10:6.

[0025] Liquid fermentation medium: 20 g / L soybean meal, 2 g / L soybean peptone, 20 g / L glucose, 5 g / L soluble starch, 2 g / L yeast powder, 4 g / L NaCl, 0.5 g / L K2HPO3, 0.5 g / L MgSO4·H2O, 2 g / L CaCO3.

[0026] Potato glucose liquid culture medium: 200g potato (peeled), 20g glucose, 1000mL distilled water, natural pH.

[0027] Potato glucose solid plate medium: 200g potato (peeled), 20g glucose, 20g agar, 1000mL distilled water, natural pH.

[0028] Example 1

[0029] Preparation of Penicillium wani spore powder:

[0030] (1) Inoculate *Paecilomyces wani* into *Paecilomyces wani* seed culture medium and culture at 25℃ and 120 r / min for 4 days to obtain *Paecilomyces wani* seed liquid (effective bacterial count > 1×10⁻⁶). 9 );

[0031] (2) After soaking the rice, it was steam-sterilized and matured to 5-6 maturity. After cooling, a rice culture medium was prepared. *Paecilomyces wani* seed solution was added to the rice culture medium at a volume-to-mass ratio of 10%. The medium was incubated at 25℃ for 6-7 days. Once the spores were fully grown, fermentation was stopped, and the rice was dried in a ventilated area for 1-2 days. The spores were then harvested using a vibrating sieve. The effective viable count of *Paecilomyces wani* spores was measured to be 10. 8 / g.

[0032] Preparation of melanin from the fermentation broth of *Penicillium wani*:

[0033] Prepare 1000 mL of potato dextrose liquid culture medium, sterilize at 115 °C for 30 min, cool, and inoculate *Penicillium wanyi* into the medium. Incubate at 28 °C with shaking at 120 rpm for 7 days. Centrifuge the fermentation product at 4000 rpm to remove the precipitate. Concentrate the obtained fermentation supernatant to 200 mL under reduced pressure at 50 °C, add 600 mL of anhydrous ethanol, centrifuge at 5000 rpm for 20 min to remove the precipitate, concentrate the supernatant to 200 mL, and add 50 mL of Seva. The solution was prepared with GE reagent (chloroform: n-butanol solution (4:1 v / v)), shaken for 20 min, centrifuged at 5000 r / min for 20 min, and the supernatant was collected. This process was repeated until no protein emulsion was produced in the middle layer. The supernatant was concentrated under reduced pressure to obtain an extract. The extract was freeze-dried to obtain the crude extract powder of *Penicillium wanyi* fermentation broth. The crude extract powder was separated and purified by high-speed countercurrent chromatography (mobile phase: methanol: n-hexane: water = 4:2:3 v / v / v) to obtain melanin from the *Penicillium wanyi* fermentation broth extract.

[0034] A compound microbial agent was prepared by distributing Paecilomyces wani spore powder, melanin extracted from Paecilomyces wani fermentation broth, dispersant (sodium lignin sulfonate), and liquid fermentation medium according to the mass percentages in Table 1. The preparation method was as follows: Paecilomyces wani spore powder, melanin extracted from Paecilomyces wani fermentation broth, and dispersant were added to the liquid fermentation medium and mixed thoroughly to obtain the compound microbial agent. A total of nine compound microbial agents (compound microbial agents 1a-3c) were prepared according to the formulations in Table 1.

[0035] Table 1. Preparation Table of Compound Microbial Agent

[0036]

[0037] Application testing:

[0038] 1. Inhibitory effect test on *Platycladus orientalis* var. *mongolica*

[0039] Experimental methods:

[0040] (1) Four pathogens (Thuja stenoptera green cup fungus, Populus spp. black spot fungus, Pine trunk base brown rot fungus and Phytophthora camphorata fungus, all four pathogens were derived from diseased plants and were purified and identified by strains) were inoculated into potato glucose solid plate medium and cultured at 28°C. When the mycelium covered the culture dish, the plate was removed to obtain four pathogen mycelium cakes.

[0041] (2) Nine compound microbial agents prepared in Example 1 were added to melted potato glucose solid medium to form nine experimental groups. The control groups were treated with Paecilomyces wani spore powder (2 mL spore suspension), Paecilomyces wani fermentation broth extract melanin (2 mL), 40% cymoxanil suspension (2 mL), and 50% tebuconazole (2 mL). The cultured pathogenic fungal cakes were cut with a 5 mm diameter punch and inoculated in the center of the plates of the experimental and control groups. The plates were placed at a constant temperature of 28 °C and each treatment was repeated three times. The medium without the above agents was used as the blank control group.

[0042] (3) Results Analysis:

[0043] When the mycelial diameter in the blank control group reached approximately half the diameter of the petri dish, the colony diameter for each treatment was measured, and the average diameter for each treatment was calculated. The inhibition rate was then calculated using the following formula:

[0044] I = [(D0-D t ) / D O ]×100

[0045] I – Inhibition rate.

[0046] D0 – Colony growth diameter in the blank control group.

[0047] Dt — Diameter of colony growth after chemical treatment.

[0048] An antibacterial rate greater than 60% indicates a significant effect, while a rate greater than 90% indicates a remarkable effect.

[0049] The experimental results are shown in Table 2:

[0050] Table 2. Antibacterial effects of different treatments on *Platycladus orientalis* and other pathogens.

[0051]

[0052]

[0053] As shown in Table 2, among the four forest tree pathogens, the compound microbial agent of this invention exhibits superior inhibitory effects against *Pleurotus ostreatus* compared to the other three, with inhibition rates exceeding 70%. It shows no significant inhibitory effect on the other three pathogens. Furthermore, the compound microbial agent of this invention is more effective in inhibiting *Pleurotus ostreatus* than the chemical control agents 40% carbendazim and 50% tebuconazole. This indicates that the compound microbial agent of this invention has a significant and specific inhibitory effect on *Pleurotus ostreatus*.

[0054] 2. Field efficacy test of compound microbial agents

[0055] (1) Preparation of spore suspension of *Platycladus orientalis* var. *mongolica*: *Platycladus orientalis* var. *mongolica* was inoculated into potato dextrose liquid medium and cultured at 28°C with shaking for 6 days. The spore suspension was obtained by filtration through sterile gauze, and the spore concentration was diluted to 10⁻⁶. 6 CFU / mL was used to obtain a spore suspension.

[0056] (2) Setting up drug treatment groups:

[0057] 1) No treatment (control, CK);

[0058] 2) Treatment of Penicillium wani spore powder suspension (25 g / L): Penicillium wani spore powder prepared in Example 1 was used to prepare a 25 g / L suspension with distilled water;

[0059] 3) Melanin treatment (1g / L): The melanin extracted from the fermentation broth of *Paecilomyces wani* prepared in Example 1 was used to make a suspension of 1g / L with distilled water.

[0060] 4) Compound microbial agent treatment group: 9 compound microbial agents 1a-3c prepared in Example 1, a total of 9 groups;

[0061] 5) Treatment with 40% methomyl suspension;

[0062] 6) Treatment with 50% chlorhexidine.

[0063] (3) Three replicates were set up for each drug treatment group. Undamaged healthy arborvitae leaves were taken and wounds were made directly on the leaves with a dissecting needle. Spores of *Platycladus orientalis* spores were inoculated into the wounds on the arborvitae leaves. The arborvitae leaves were sprayed with the treatment solutions every three days for a total of three applications. The disease incidence was observed two days after the last application, and the control efficacy of each treatment was calculated.

[0064] The disease severity index is graded as follows: Grade 1: No symptoms on leaves; Grade 2: Diseased parts cover less than one-quarter of the leaves; Grade 3: Diseased parts cover one-quarter to one-half of the leaves; Grade 4: Diseased parts cover one-half to three-quarters of the leaves; Grade 5: Diseased parts cover more than three-quarters of the entire leaf or are basically dead.

[0065] Incidence rate = (Number of infected plants / Total number of plants surveyed) × 100%

[0066] The experimental results are shown in Table 3:

[0067] Table 3. Morbidity and disease index under different drug treatments

[0068]

[0069] 'a' indicates a comparison with the control group (<0.05); '*' indicates a comparison with 40% carbendazim suspension (<0.05); '#' indicates a comparison with 50% tebuconazole (<0.05).

[0070] As shown in Table 3, the compound microbial agent of the present invention can effectively reduce the incidence of arborvitae leaf blight and effectively reduce the disease index, and its effect is better than that of chemical control agents 40% cypermethrin and 50% tebuconazole.

Claims

1. A compound microbial agent for controlling arborvitae leaf blight, characterized in that, Based on the total mass of the compound microbial agent as 100%, it consists of the following components by mass percentage: 20-40% of Paecilomyces wanyi spore powder, 10-20% of melanin extracted from Paecilomyces wanyi fermentation broth, 15-25% of dispersant, and the remainder is liquid fermentation culture medium. The melanin extracted from the fermentation broth of *Paecilomyces wanensis* and the spore powder of *Paecilomyces wanensis* were both obtained using *Paecilomyces wanensis* with the accession number CGMCC3.24355. Paecilomyces variotii The effective viable count of the *Penicillium wani* spore powder obtained after fermentation and culture is greater than 6.5 × 10⁻⁶. 7 g -1 ; The preparation method of melanin from the fermentation broth of *Paecilomyces wani* is as follows: *Paecilomyces wani* is inoculated into potato liquid culture medium and cultured at 28℃ and 120 r / min for 7 days. Then, centrifugation is performed to remove the precipitate. The obtained fermentation supernatant is concentrated and anhydrous ethanol is added. After centrifugation, the precipitate is removed. The obtained supernatant is concentrated and then Sevage reagent is added. After mixing evenly, centrifugation is performed and the upper layer solution is collected. After vacuum concentration and freeze-drying, crude extract powder of *Paecilomyces wani* fermentation broth is obtained. Finally, it is separated and purified to obtain melanin from the fermentation broth of *Paecilomyces wani*. The liquid fermentation medium is formulated as follows: 20 g / L soybean meal, 2 g / L soybean peptone, 20 g / L glucose, 5 g / L soluble starch, 2 g / L yeast powder, 4 g / L NaCl, 0.5 g / L K2HPO3, 0.5 g / L MgSO4·H2O, and 2 g / L CaCO3.

2. The compound microbial agent for controlling arborvitae leaf blight as described in claim 1, characterized in that, The preparation method of the *Penicillium wani* spore powder includes the following steps: (1) Inoculate Paecilomyces wani into Paecilomyces wani seed culture medium and culture at 25℃ and 120 r / min for 4 days to obtain Paecilomyces wani seed liquid; (2) After soaking the rice, steam sterilize and cook it to 5-6 maturity. After cooling, prepare rice culture medium. Add Paecilomyces wani seed liquid to the rice culture medium at a volume mass ratio of 10%. Incubate at 25℃ for 6-7 days. After the spores are fully grown, stop the fermentation and dry in a ventilated place for 1-2 days to obtain the product. In step (2), the rice culture medium formula is as follows: 100 g rice, 10 g soybean flour, 15 g wheat bran, 0.5 g NaCl, 0.5 g K2HPO3, 1 g CaCO3; the material-to-water ratio is 10:

6.

3. The compound microbial agent for controlling arborvitae leaf blight as described in claim 2, characterized in that, In step (1), the seed culture medium for *Penicillium wani* is formulated as follows: soluble starch 20 g / L, glucose 20 g / L, yeast powder 2 g / L, NaCl 4 g / L, K2HPO3 0.5 g / L, MgSO4·H2O 0.5 g / L, and CaCO3 2 g / L.

4. The compound microbial agent for controlling arborvitae leaf blight as described in claim 1, characterized in that, The dispersant is sodium lignosulfonate.

5. The method for preparing the compound microbial agent for controlling Platycladus orientalis leaf blight as described in any one of claims 1 to 4, characterized in that, Add the spore powder of *Penicillium wanyi*, the melanin extracted from the fermentation broth of *Penicillium wanyi*, and the dispersant to the liquid fermentation culture medium, mix well, and the compound bacterial agent is obtained.

6. The application of the compound microbial agent according to any one of claims 1 to 4 in the prevention and control of arborvitae leaf blight.

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