A complex microbial agent composition and its application in the prevention and treatment of potato soil-borne diseases

Abstract

The application discloses a kind of composite microbial inoculant composition and its application in potato soil-borne disease prevention and treatment.The active ingredient of the composite microbial inoculant composition of the application includes Paenibacillus polymyxa ZF129, Paenibacillus peoriae ZF390 and Bacillus subtilis ZF517.The composite microbial inoculant composition further includes carrier and adjuvant.The application uses Paenibacillus polymyxa ZF129, Paenibacillus peoriae ZF390 and Bacillus subtilis ZF517 as effective components, through the screening of carrier and dispersing agent, and potting test verification, determines the composite microbial inoculant composition formula as potato seed treatment agent, and the yield-increasing effect is obvious.

Description

Technical Field

[0001] This invention relates to a compound microbial agent composition in the field of biotechnology and its application in the control of soil-borne diseases of potatoes. Background Technology

[0002] Potatoes are a high-yield and nutritious food crop. In recent years, continuous cropping has led to a year-on-year increase in soil-borne diseases such as potato soft rot, black scurf, and scab, seriously affecting potato yield and quality.

[0003] Potato soft rot is mainly caused by *Pectinobacterium*. Pectobacterium spp. and Digibacterium spp. Dickeya This disease is caused by *Rhizoctonia solani*, a bacterium that can cause black rot symptoms in the underground stems, tubers, and base of the above-ground stems of potato plants. Potato black scurvy is caused by *Rhizoctonia solani*. Rhizoctonia solani This fungal disease is caused by pathogens that typically reside in the soil as mycelia and sclerotia, primarily affecting the underground stems and tubers of potatoes, forming reddish-brown lesions and black sclerotia, thus reducing the marketability of the potatoes. Potato scab (PCS) is caused by various pathogenic Streptomyces fungi. Streptomyces Scab disease is a soil-borne disease caused by spp. infection. The pathogen infects the epidermal tissue of the tuber, forming scabs on the tuber surface, which leads to a decline in the appearance and quality of the tubers. In production, traditional methods such as timely irrigation, adjusting soil pH, crop rotation, breeding resistant varieties, and using chemical agents are commonly used to control scab disease. Although these methods have some effect, they suffer from problems such as poor resistance in the main cultivated varieties, short-term effectiveness of chemical control, significant environmental pollution, and easy development of drug resistance, which are detrimental to the sustainable development of the potato industry. Biological control, due to its environmental friendliness and compliance with the requirements of green production, is gradually gaining attention.

[0004] Huang Xun et al. found that the culture medium of Bacillus laterosporus JYC 688 could significantly inhibit Streptomyces acidicus, which causes potato scab. Streptomyces acidiscabiesThe growth of Rhizoctonia solani was investigated (Huang Xun, Feng Jiawen, Jin Chunlin, et al. Identification and antibacterial and growth-promoting characteristics of Bacillus laterosporus JYC 688 [J]. Plant Protection. 2024, 50(02): 153-162.). Shang Shuyan et al. used Bacillus belye BEV2 and Bacillus amyloliquefaciens BAM7 to achieve stable control effects against various Streptomyces scabies of potatoes. Zhang Xiaoyun et al. screened a strain of Bacillus amyloliquefaciens PHOODG36, whose fermentation broth achieved a control effect of 56.8% against potato black scurf (Zhang Xiaoyun, Cong Rong, Yu Wenqian, et al. Functional analysis of phosphorus solubilization, growth promotion and control of potato black scurf by Bacillus amyloliquefaciens PHOODG36 [J]. Journal of Hebei Agricultural University. 2023, 46(03): 83-90.). This indicates that Bacillus has good application prospects in the control of soil-borne diseases of potatoes.

[0005] Previous reports on biocontrol have mostly focused on single-strain control of diseases. Soil physicochemical properties and plant growth status can easily affect the control efficacy, leading to problems such as reduced efficacy or short duration of effect. Studies have shown that combining biocontrol strains of the same or different genera can significantly improve biocontrol ability and the stability of biocontrol effect. Zou Liwen et al. (Zou Liwen, Li Tingting, Fu Bo, et al. Field control effect of Trichoderma-Bacillus mixture on rice sheath blight [J]. Journal of Shanghai Jiaotong University (Agricultural Science Edition). 2019, 37(06): 1-5.) applied Trichoderma-Bacillus mixture twice (100 g / 667 m) before or at the early stage of rice sheath blight. 2 The control efficacy and yield increase rate can reach 53.61% and 28.9%, respectively, which exceed the control efficacy and yield increase rate of single Trichoderma and Bacillus agents (47.8% and 12.5%; 32.2% and 26.2%) and the residual effect is better (Zou Liwen, Li Tingting, Fu Bo, et al. Field control effect of Trichoderma and Bacillus mixture on rice sheath blight [J]. Journal of Shanghai Jiaotong University (Agricultural Science Edition). 2019, 37(06): 1-5.). Tian Yongyong et al. controlled melon wilt by using a mixed fermentation broth of Bacillus F-1 and D-3, achieving a control efficacy of 91.8%, which was higher than the control efficacy of F-1 and D-3 fermentation broth against melon wilt (85.7% and 81.6%, respectively), and significantly higher than the control efficacy of carbendazim (64.6%) (Tian Yongyong, Chen Li, Xie Yun, et al. Screening of antagonistic bacteria against melon wilt and field control efficacy test [J]. Chinese Agricultural Science Bulletin. 2011, 27(5): 367-371.).

[0006] As of October 2024, there were no registered biological agents for the control of potato soft rot and black scurf, while only Bacillus amyloliquefaciens QST713 suspension (PD20211364) had been registered as a pesticide in China for potato scab. Under these circumstances, it is essential to carry out research on the development and application technology of highly efficient and green control products for the above three diseases.

[0007] Vectors constitute the largest proportion of microbial preparations; therefore, determining the biocompatibility between vectors and strains is extremely important. Chiou et al. (Chiou AL, Wu W S. Formulation of...) Bacillus amyloliquefaciens B190 for control of lily grey mould (Botrytis elliptica)[J]. Journal of Phytopathology. 2003, 151(1): 13-18.) found that using kaolin as a carrier can improve the spore survival rate in Bacillus preparations. Sun Lili et al. (Sun Lili, Cao Chuanwang, Xue Xuting, et al. Development and fungicidal activity determination of wettable powder of Trichoderma echinosporum[J]. Journal of Beijing Forestry University. 2015, 37(6): 45-52.) found that the spore survival rate of the preparation was higher when using diatomaceous earth as a carrier. However, Chen Ru et al. (Chen Ru, Cao Xuemei, Wu Haixia, et al. Development of wettable powder of marine polymyxa[J]. Plant Protection. 2020, 46(3): 62-69.) showed that when diatomaceous earth was used as the carrier of WP, its spore germination rate was inhibited. This indicates that different strains have different adaptability to the same vector, and the biocompatibility of the same vector to different strains also varies.

[0008] Microbial preparations have become a research hotspot due to their environmental friendliness, safety, and high efficiency. However, their short duration of action and susceptibility to environmental influences hinder their widespread application in production. Adjuvants are indispensable in pesticide formulation development, with the main purpose of improving efficacy, extending shelf life, reducing dosage, enhancing stability, and reducing environmental pollution (Zhao Weisong, Lu Xiuyun, Guo Qinggang, et al. Development of Bacillus subtilis BAB-1 dust for controlling tomato gray mold [J]. Chinese Journal of Biological Control. 2018, 34(1): 99-108.). There are already reports on the screening and optimization of carriers and adjuvants for microbial preparations. Shen Yunxin et al. (Shen Yunxin, Li Minggang, Shi Zhufeng, et al. Development of Bacillus vesiculosus SH-1471 wettable powder and its control effect on tomato wilt [J]. Chinese Journal of Biological Control. 2023, 39(4): 904-914.) determined the formula and ratio of Bacillus vesiculosus SH-1471 wettable powder WP after screening as follows: 87% masterbatch powder with kaolin as carrier, 2% sodium lignosulfonate, 8% NNO, 2% xanthan gum, and 1% ascorbic acid (VC); Guo Zhuangyuan et al. (Guo Zhuangyuan, Yang Chengde, Jin Mengjun, et al. Development of Bacillus subtilis 262XY2′ wettable powder [J]. Chinese Journal of Biological Control. 2022, 38(02): (414-420.) The optimal formulation of Bacillus subtilis 262XY2′ wettable powder was determined to be 87% masterbatch, 10% sodium dodecyl sulfate, 2% potassium dihydrogen phosphate and 1% ascorbic acid.

[0009] This invention uses Bacillus polymyxa ZF129, Bacillus pilaris ZF390, and Bacillus subtilis ZF517 as active ingredients, and screens carriers and adjuvants to develop a potato seed treatment agent. It is hoped that this invention can provide a new biocontrol product for controlling soil-borne diseases of potatoes and increasing potato yield. Summary of the Invention

[0010] The purpose of this invention is to explore how to utilize microorganisms for the integrated control of soil-borne diseases such as potato soft rot, black spore, and scab.

[0011] This invention provides a compound microbial agent composition, the active ingredient of which includes Bacillus polymyxa (B. polymyxa). Paenibacillus polymyxa ZF129, Piperella salina ( Paenibacillus peoriae ZF390 and Bacillus subtilis ( Bacillus subtilis ZF517.

[0012] Polymyxin Bacillus ( Paenibacillus polymyxaZF129 was deposited on April 25, 2019, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No: 17631.

[0013] Piperella spp. ( Paenibacillus peoriae ZF390 was deposited on July 8, 2020, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No: 20322.

[0014] Bacillus subtilis ( Bacillus subtilis ZF517 was deposited on March 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No: 30137.

[0015] Specifically, in the compound bacterial agent composition, *Bacillus polymyxa* (… Paenibacillus polymyxa ZF129, Piperella salina ( Paenibacillus peoriae ZF390 and Bacillus subtilis ( Bacillus subtilis The quantity ratio of ZF517 is (1-10): (1-10): (1-10). Specifically, in the compound bacterial agent, Bacillus polymyxa (… Paenibacillus polymyxa ZF129, Piperella salina ( Paenibacillus peoriae ZF390 and Bacillus subtilis ( Bacillus subtilis The quantity ratio of ZF517 is 1:1:1. The quantity ratio refers to the mass ratio.

[0016] In the compound microbial agent composition, the active ingredient may exist in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate.

[0017] As needed, the composite microbial agent composition may further include a carrier. Preferably, a carrier commonly used in the field of microbial agents and biologically inert is preferred. The carrier may be a solid carrier or a liquid carrier. The solid carrier is at least one selected from silica, diatomaceous earth, attapulgite, and talc.

[0018] The formulation of the compound microbial agent composition can be in various forms, such as liquid, emulsion, suspension, powder, granules, wettable powder, or water-dispersible granules. Specifically, it can be a wettable powder.

[0019] The compound microbial agent composition may also contain adjuvants, including one or more of wetting agents, dispersants, and colorants.

[0020] The wetting agent may be one or more of sodium dodecylbenzene sulfonate (SDBS), Morwet EFW, Morwet IP, LT-9004, LT-DK3, sodium lignosulfonate (NA), etc.; specifically, the wetting agent may be sodium lignosulfonate (NA).

[0021] The dispersant may be one or more of sodium salt of polynaphthalene sulfonic acid (NNO), Morwet D-425, Morwet D-450, etc.; specifically, the dispersant may be sodium salt of polynaphthalene sulfonic acid (NNO).

[0022] Specifically, the colorant may be Acid Red.

[0023] The compound microbial agent composition is made by mixing the following raw materials in parts by weight: 4-10 parts by weight of mixed bacterial strain mother drug, 3 parts by weight of sodium methylene bis(naphthalene) sulfonate, 4 parts by weight of sodium lignin sulfonate, 0.1 parts by weight of Acid Red, and 82.9-88.9 parts by weight of talc.

[0024] The mixed strain parent drug was composed of ZF129, ZF390, and ZF517 strain parent drugs according to the *Bacillus polymyxa* (… Paenibacillus polymyxa ZF129, Piperella salina ( Paenibacillus peoriae ZF390 and Bacillus subtilis ( Bacillus subtilis It is a mixture of ZF517 in a specific ratio;

[0025] The parent drug for the ZF129 strain is Bacillus polymyxa (… Paenibacillus polymyxa The fermentation broth obtained from ZF129 fermentation is mixed with attapulgite soil at a mass ratio of 10%, and then repeatedly filtered until the filtrate is clear. The filter cake is collected, dried in the shade, and then crushed.

[0026] The parent drug for the ZF390 strain is *Bacillus pilaris* (Pierreotype bacillus). Paenibacillus peoriaeThe fermentation broth obtained from ZF390 fermentation is mixed with attapulgite soil at a mass ratio of 10%, and then repeatedly filtered until the filtrate is clear. The filter cake is collected, dried in the shade, and then crushed.

[0027] The parent drug for the ZF517 strain is Bacillus subtilis (… Bacillus subtilis The fermentation broth obtained from ZF517 fermentation is mixed with attapulgite soil at a mass ratio of 10%, and then repeatedly filtered until the filtrate is clear. The filter cake is collected, dried in the shade, and then crushed.

[0028] Specifically, in this embodiment of the invention, the number of viable bacteria per gram of the ZF129 strain mother drug is 4.32 × 10⁻⁶. 7 The CFU / g of the ZF390 strain mother drug was 4.14 × 10⁻⁶ CFU / g. 8 The CFU / g of the ZF517 strain mother drug was 4.86 × 10⁻⁶ CFU / g. 9 CFU / g.

[0029] Specifically, the compound microbial agent composition is made by mixing the following raw materials in parts by weight: 8 parts by weight of mixed bacterial strain mother drug, 3 parts by weight of sodium methylene bis(naphthalene) sulfonate, 4 parts by weight of sodium lignin sulfonate, 0.1 parts by weight of Acid Red, and 84.9 parts by weight of talc.

[0030] The present invention also provides a method for preparing the composite microbial agent composition as follows:

[0031] The ZF129 strain, ZF390 strain, and ZF517 strain parent drugs were used according to the *Bacillus polymyxa* (… Paenibacillus polymyxa ZF129, Piperella salina ( Paenibacillus peoriae ZF390 and Bacillus subtilis ( Bacillus subtilis The mixed strain mother drug was obtained by mixing ZF517 in a specific ratio.

[0032] The parent drug for the ZF129 strain is Bacillus polymyxa (… Paenibacillus polymyxa The fermentation broth obtained from ZF129 fermentation is mixed with attapulgite soil at a mass ratio of 10%, and then repeatedly filtered until the filtrate is clear. The filter cake is collected, dried in the shade, and then crushed.

[0033] The parent drug for the ZF390 strain is *Bacillus pilaris* (Pierreotype bacillus). Paenibacillus peoriae The fermentation broth obtained from ZF390 fermentation is mixed with attapulgite soil at a mass ratio of 10%, and then repeatedly filtered until the filtrate is clear. The filter cake is collected, dried in the shade, and then crushed.

[0034] The parent drug for the ZF517 strain is Bacillus subtilis (… Bacillus subtilis The fermentation broth obtained from ZF517 fermentation is mixed with attapulgite soil at a mass ratio of 10%, and then repeatedly filtered until the filtrate is clear. The filter cake is collected, dried in the shade, and then crushed.

[0035] Sodium methylene bis(naphthalene) sulfonate, sodium lignin sulfonate, Acid Red, and talc were added to the mixed bacterial strain mother drug in parts by weight and mixed evenly to obtain the composite bacterial agent composition.

[0036] In the above method, the air drying is carried out at 35°C and 100% wind speed (in an electronic blower dryer, the wind speed is set to 100%).

[0037] This invention also protects the application of any of the above-described compound microbial agent compositions in the control of soil-borne diseases of potatoes.

[0038] In the above applications, the soil-borne diseases of potatoes are any one, two, or three of potato soft rot, potato black scurf, and potato scab.

[0039] In the above applications, the compound microbial agent composition is applied at a rate of 22.5 kg / hm². 2 -56.25 kg / hm 2 For potato seed dressing, the preferred dosage is 45 kg / hm². 2 Dosage.

[0040] This invention uses a compound of *Bacillus polymyxa* ZF129, *Bacillus pilaris* ZF390, and *Bacillus subtilis* ZF517 as active ingredients. Through screening of carriers and dispersants, and verification via pot experiments, the formula (mass fraction) for a potato seed treatment agent was determined as follows: 8% of the strain mother drug (mZF129 : mZF390 : mZF517 strain mother drug = 1:1:1), 3% of the dispersant NNO, 4% of the wetting agent NA, 0.1% of Acid Red, and 84.9% of talc. The average spore concentration of this formulation is between 1.5 × 10⁻⁶. 8 -2.0×10 8 The average concentration of CFU / g of the seed treatment agent was 2.8%, the average bacterial count was 2.8%, the average pH was 6.7, the weight loss on drying was 0.9%, the wetting time was 114.3 s, and the suspension rate was 77.8%. Greenhouse pot experiments showed that 45 kg / hm² of seed treatment agent was effective. 2 Seed treatment ensures safe potato emergence and promotes growth. It achieves a 66.81% control rate against potato soft rot, a 97.51% control rate against potato black scurf, and a 65.05% control rate against potato scab. Field demonstration trials in different regions show that this seed treatment significantly improves the marketable yield of potatoes, with yield increases ranging from 14.92% to 26.11%. These findings demonstrate that potato seed treatments effectively control soil-borne potato diseases and significantly improve marketable yield, providing a basis for their application in production.

[0041] Instructions for the Preservation of Biological Materials

[0042] Preservation instructions for Bacillus polymyxa ZF129

[0043] Classification and nomenclature of biological materials: Polymyxin Bacillus

[0044] Latin scientific name of biological material: Paenibacillus polymyxa

[0045] Strain number of the biological material: ZF129

[0046] Full name of the depository: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0047] Abbreviation of depositary institution: CGMCC

[0048] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Postcode: 100101

[0049] Date of preservation: April 25, 2019

[0050] Accession number: CGMCC No: 17631

[0051] Preservation instructions for Bacillus pilaris ZF390

[0052] Classification and nomenclature of biological materials: Bacillus repens

[0053] Latin scientific name of biological material: Paenibacillus peoriae

[0054] Strain number of the biological material: ZF390

[0055] Full name of the depository: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0056] Abbreviation of depositary institution: CGMCC

[0057] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Postcode: 100101

[0058] Date of preservation: July 8, 2020

[0059] Accession number: CGMCC No: 20322

[0060] Preservation instructions for Bacillus subtilis ZF517

[0061] Classification and nomenclature of biological materials: Bacillus subtilis

[0062] Latin scientific name of biological material: Bacillus subtilis

[0063] Strain number of the biological material: ZF517

[0064] Full name of the depository: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0065] Abbreviation of depositary institution: CGMCC

[0066] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Postcode: 100101

[0067] Deposit date: March 26, 2024

[0068] Accession number: CGMCC No: 30137 Attached Figure Description

[0069] Figure 1 This is the result of determining whether ZF129, ZF390 and ZF517 strains are antagonistic to each other using the cross-stamping method in Example 1 of the present invention. Figure 1 In A, the vertical line is ZF129 and the horizontal line is ZF390; Figure 1 In B, the vertical line is ZF129; the horizontal line is ZF517. Figure 1 In the letter C, the vertical line is ZF390; the horizontal line is ZF517. Figure 1 D represents the antagonistic strain control, the vertical line represents ZF129, and the horizontal line represents ZF516.

[0070] Figure 2 The strains *Bacillus polymyxa* ZF129, *Bacillus pilaris* ZF390, and *Bacillus subtilis* ZF517 used in Example 1 of this invention were cultured on LB solid medium containing 1% precipitated silica, 2% precipitated silica, 3% precipitated silica, 1% diatomaceous earth, 2% diatomaceous earth, and 3% diatomaceous earth, respectively. The number of viable bacteria cultured on LB solid medium containing 3% diatomaceous earth, 1% attapulgite, 2% attapulgite, 3% attapulgite, 1% talc, 2% talc, and 3% talc were compared, with LB medium without carrier as the blank control (CK).

[0071] Figure 3 This illustrates the effect of different proportions of adjuvants on the growth viability of the bacterial strain in Example 1 of the present invention. Figure 3 A represents the effect of A on the growth activity of strain ZF129; Figure 3 B represents the effect on the growth activity of strain ZF390. Figure 3 C represents the effect of C on the growth activity of strain ZF517.

[0072] Figure 4 The results show the screening of potato seed dressing agent carriers and the optimal dosage of adjuvants in Example 1 of this invention. Figure 4 A represents the screening result of the seed dressing agent carrier for strain ZF129. Figure 4 Screening results of seed dressing agent carrier for strain B ZF390 technical grade; Figure 4 C represents the screening result of the seed dressing agent carrier for strain ZF517 technical material; Figure 4 D represents the results of suspension rate and wetting time determination for different proportions of additives.

[0073] Figure 5 The results of the safety test of the seed dressing agent on Kexin No. 1 potato in Example 1 of this invention are shown. Figure 5 A represents seed treatment with A1 seed dressing agent; Figure 5 B represents seed treatment with A2 seed dressing agent; Figure 5 C represents seed treatment with A3 seed dressing agent; Figure 5 D represents seed treatment with A4 seed dressing agent; Figure 5 E is the blank control; Figure 5 F represents the naked control; Figure 5 G represents the effect of different treatments on the plant height of Kexin No. 1 potato. Figure 5 H represents the effect of different treatments on the stem diameter of Kexin No. 1 potato. Among them, A1 represents the A1 seed dressing treatment, A2 represents the A2 seed dressing treatment, A3 represents the A3 seed dressing treatment, A4 represents the A4 seed dressing treatment, Talc represents the talc powder seed dressing control, and CK represents the bare seed control. There are significant differences among the treatments labeled with different lowercase letters (Talc: talc powder).

[0074] Figure 6 The results show the safety test results of the seed dressing agent in Example 1 of this invention on Dutch 15 potatoes. Figure 6 A represents seed treatment with A1 seed dressing agent; Figure 6 B represents seed treatment with A2 seed dressing agent; Figure 6 C represents seed treatment with A3 seed dressing agent; Figure 6 D represents seed treatment with A4 seed dressing agent; Figure 6 E is the blank control; Figure 6 F represents the naked control; Figure 6 G represents the effect of different treatments on the plant height of Dutch potato 15. Figure 6H represents the effect of different treatments on the stem diameter of Dutch 15 potatoes. Among them, A1 represents the A1 seed dressing treatment, A2 represents the A2 seed dressing treatment, A3 represents the A3 seed dressing treatment, A4 represents the A4 seed dressing treatment, Talc represents the talc seed dressing control, and CK represents the bare seed control. There are significant differences among the treatments labeled with different lowercase letters.

[0075] Figure 7 The results of the safety test of the seed dressing agent on Xufeng No. 1 potato in Example 1 of this invention are shown. Figure 7 A represents seed treatment with A1 seed dressing agent; Figure 7 B represents seed treatment with A2 seed dressing agent; Figure 7 C represents seed treatment with A3 seed dressing agent; Figure 7 D represents seed treatment with A4 seed dressing agent; Figure 7 E is the blank control; Figure 7 F represents the naked control; Figure 7 G represents the effect of different treatments on the plant height of Xufeng No. 1 potato. Figure 7 G represents the effect of different treatments on the stem diameter of Xufeng No. 1 potato. Among them, A1 represents the A1 seed treatment, A2 represents the A2 seed treatment, A3 represents the A3 seed treatment, A4 represents the A4 seed treatment, Talc represents the blank control, and CK represents the naked seed control. There are significant differences among the treatments labeled with different lowercase letters.

[0076] Figure 8 The results of the safety test of the seed dressing agent on Shisen 6 potato in Example 1 of this invention are shown. Figure 8 A represents seed treatment with A1 seed dressing agent; Figure 8 B represents seed treatment with A2 seed dressing agent; Figure 8 C represents seed treatment with A3 seed dressing agent; Figure 8 D represents seed treatment with A4 seed dressing agent; Figure 8 E is the blank control; Figure 8 F represents the naked control; Figure 8 G represents the effect of different treatments on the plant height of the Hissen 6 potato variety. Figure 8 G represents the effect of different treatments on the stem diameter of Xisen 6 potato. Among them, A1 represents the A1 seed treatment, A2 represents the A2 seed treatment, A3 represents the A3 seed treatment, A4 represents the A4 seed treatment, Talc represents the blank control, and CK represents the naked seed control. There are significant differences among the treatments labeled with different lowercase letters.

[0077] Figure 9 This is a photograph of the pot experiment results for screening the optimal dosage of potato seed dressing agent A3 in Example 1 of the present invention. Figure 9 The value of A is 22.5 kg / hm. 2 ; Figure 9 The B value is 33.75 kg / hm.2 ; Figure 9 The C is 45 kg / hm 2 ; Figure 9 The D is 56.25 kg / hm. 2 ; Figure 9 E is the commercial microbial agent Qiwei; Figure 9 F is a commercial microbial agent, Greenland Gold. Figure 9 G represents the commercial fungicide Jin Dian; Figure 9 H represents the commercial microbial agent, Tiandaoluotu. Figure 9 I is the commercial inoculant Potato Steward; Figure 9 J is the commercial bacterial agent Qiangshi; Figure 9 K represents a healthy control group.

[0078] Figure 10 This is a bar chart showing the results of a pot experiment to screen the optimal dosage of potato seed dressing agent A3 in Example 1 of this invention. 1 represents 22.5 kg / hm². 2 ;2 is 33.75 kg / hm 2 3 is 45 kg / hm 2 ;4 is 56.25 kg / hm 2 ; 5 is the commercial inoculant Qiwei; 6 is the commercial inoculant Lvdichengjin; 7 is the commercial inoculant Jindian; 8 is the commercial inoculant Tiandaoletu; 9 is the commercial inoculant Shuguanjia; 10 is the commercial inoculant Qiangshi; 11 is the healthy control group.

[0079] Figure 11 These are photographs showing the control effects of different treatments on potato soft rot in Example 1 of this invention. Figure 11 A and Figure 11 'a' represents seed treatment with A1 seed dressing agent; Figure 11 B and Figure 11 b represents seed treatment with A2 seed dressing agent; Figure 11 C and Figure 11 c represents seed treatment with A3 seed dressing agent; Figure 11 D and Figure 11 d represents seed treatment with A4 seed dressing agent; Figure 11 E and Figure 11 e is a commercial microbial agent for seed treatment of Greenland Gold; Figure 11 F and Figure 11 f represents the commercial inoculant "Potato Steward" seed treatment; Figure 11 G and Figure 11 g represents the disease control group; Figure 11 H and Figure 11 h represents a healthy control group.

[0080] Figure 12This is a bar chart showing the control effects of different treatments on potato soft rot in Example 1 of this invention. In the chart, A represents seed treatment with A1 seed dressing agent, B represents seed treatment with A2 seed dressing agent, C represents seed treatment with A3 seed dressing agent, D represents seed treatment with A4 seed dressing agent, E represents seed treatment with the commercial inoculant "Greenland Gold", F represents seed treatment with the commercial inoculant "Potato Steward", and G represents the disease control group. Significant differences exist between treatments labeled with different lowercase letters.

[0081] Figure 13 These are photographs showing the control effects of different treatments on potato black scurf in Example 1 of this invention. Figure 13 A and Figure 13 'a' represents seed treatment with A1 seed dressing agent; Figure 13 B and Figure 13 b represents seed treatment with A2 seed dressing agent; Figure 13 C and Figure 13 c represents seed treatment with A3 seed dressing agent; Figure 13 D and Figure 13 d represents seed treatment with A4 seed dressing agent; Figure 13 E and Figure 13 e is a commercial microbial agent for seed treatment of Greenland Gold; Figure 13 F and Figure 13 f represents the commercial inoculant "Potato Steward" seed treatment; Figure 13 G and Figure 13 g represents the disease control group; Figure 13 H and Figure 13 h represents a healthy control group.

[0082] Figure 14 This is a bar chart showing the control effects of different treatments on potato black scurf in Example 1 of this invention. In the chart, A represents seed treatment with A1 seed dressing agent, B represents seed treatment with A2 seed dressing agent, C represents seed treatment with A3 seed dressing agent, D represents seed treatment with A4 seed dressing agent, E represents seed treatment with the commercial inoculant "Greenland Gold", F represents seed treatment with the commercial inoculant "Potato Steward", and G represents the disease control group. Significant differences exist between treatments labeled with different lowercase letters.

[0083] Figure 15 These are photographs showing the control effects of different treatments on potato scab disease in Example 1 of the present invention. Figure 15 A represents seed treatment with A1 seed dressing agent; Figure 15 B represents seed treatment with A2 seed dressing agent; Figure 15 C represents seed treatment with A3 seed dressing agent; Figure 15 D represents seed treatment with A4 seed dressing agent; Figure 15 E represents the commercial microbial agent Greenland Gold Seed Treatment; Figure 15 F represents the commercial inoculant "Potato Steward" seed treatment; Figure 15 G represents the disease control group; Figure 15 H represents a healthy control group.

[0084] Figure 16 This is a bar chart showing the control effects of different treatments on potato scab in Example 1 of this invention. In the chart, A represents seed treatment with A1 seed dressing agent, B represents seed treatment with A2 seed dressing agent, C represents seed treatment with A3 seed dressing agent, D represents seed treatment with A4 seed dressing agent, E represents seed treatment with the commercial inoculant "Greenland Gold", F represents seed treatment with the commercial inoculant "Potato Steward", and G represents the disease control group. Significant differences exist between treatments labeled with different lowercase letters.

[0085] Figure 17 The results show the efficacy of the compound microbial seed dressing agent and the single-strain seed dressing agent against potato soft rot in Example 1 of this invention. Figure 17 A represents seed treatment with ZF129 single-strain seed dressing agent; Figure 17 B is the seed treatment with ZF390 single-strain seed dressing agent; Figure 17 C represents seed treatment with ZF517 single-strain seed dressing agent; Figure 17 D represents seed treatment with A3 compound microbial seed dressing agent; Figure 17 E represents the commercial microbial agent Greenland Gold Seed Treatment; Figure 17 F represents the seed treatment with the commercial microbial agent Zhongnong Lvkang; Figure 17 G is the commercial inoculant Potato Steward Seed Treatment; Figure 17 H represents the disease control group; Figure 17 I represents the healthy control group.

[0086] Figure 18 This is a bar chart showing the results of the efficacy tests of compound microbial seed dressing agents and single-strain seed dressing agents against potato soft rot in Example 1 of this invention. In the chart, ZF129 represents the seed treatment with ZF129 single-strain seed dressing agent, ZF390 represents the seed treatment with ZF390 single-strain seed dressing agent, ZF517 represents the seed treatment with ZF517 single-strain seed dressing agent, A3 represents the seed treatment with A3 compound microbial seed dressing agent, LDCJ represents the seed treatment with the commercial inoculant "Greenland Gold", ZNLK represents the seed treatment with the commercial inoculant "Zhongnong Green Health", SGJ represents the seed treatment with the commercial inoculant "Potato Steward", and CK represents the disease control. Significant differences exist between treatments labeled with different lowercase letters.

[0087] Figure 19 The results show the efficacy of the compound microbial seed dressing agent and the single-strain seed dressing agent against potato black scurf in Example 1 of this invention. Figure 19 A represents seed treatment with ZF129 single-strain seed dressing agent; Figure 19 B is the seed treatment with ZF390 single-strain seed dressing agent; Figure 19 C represents seed treatment with ZF519 single-strain seed dressing agent; Figure 19 D represents seed treatment with A3 compound microbial seed dressing agent; Figure 19 E represents the commercial microbial agent Greenland Gold Seed Treatment; Figure 19 F represents the seed treatment with the commercial microbial agent Zhongnong Lvkang; Figure 19 G is the commercial inoculant Potato Steward Seed Treatment; Figure 19 H represents the disease control group; Figure 19 I represents the healthy control group.

[0088] Figure 20 This is a bar chart showing the results of the efficacy tests of compound microbial seed dressing agents and single-strain seed dressing agents against potato black scurf in Example 1 of this invention. In the chart, ZF129 represents the seed treatment with ZF129 single-strain seed dressing agent, ZF390 represents the seed treatment with ZF390 single-strain seed dressing agent, ZF517 represents the seed treatment with ZF517 single-strain seed dressing agent, A3 represents the seed treatment with A3 compound microbial seed dressing agent, LDCJ represents the seed treatment with the commercial inoculant "Greenland Gold", ZNLK represents the seed treatment with the commercial inoculant "Zhongnong Green Health", SGJ represents the seed treatment with the commercial inoculant "Potato Steward", and CK represents the disease control. Significant differences exist between treatments labeled with different lowercase letters.

[0089] Figure 21 The results show the efficacy of the compound microbial seed dressing agent and the single-strain seed dressing agent against potato scab in Example 1 of this invention. Figure 21 A represents seed treatment with ZF129 single-strain seed dressing agent; Figure 21 B is the seed treatment with ZF390 single-strain seed dressing agent; Figure 21 C represents seed treatment with ZF521 single-strain seed dressing agent; Figure 21 D represents seed treatment with A3 compound microbial seed dressing agent; Figure 21 E represents the commercial microbial agent Greenland Gold Seed Treatment; Figure 21 F represents the seed treatment with the commercial microbial agent Zhongnong Lvkang; Figure 21 G is the commercial inoculant Potato Steward Seed Treatment; Figure 21 H represents the disease control group; Figure 21 I represents the healthy control group.

[0090] Figure 22 This is a bar chart showing the results of the efficacy tests of compound microbial seed dressing agents and single-strain seed dressing agents against potato soft rot in Example 1 of this invention. In the chart, ZF129 represents the seed treatment with ZF129 single-strain seed dressing agent, ZF390 represents the seed treatment with ZF390 single-strain seed dressing agent, ZF517 represents the seed treatment with ZF517 single-strain seed dressing agent, A3 represents the seed treatment with A3 compound microbial seed dressing agent, LDCJ represents the seed treatment with the commercial inoculant "Greenland Gold", ZNLK represents the seed treatment with the commercial inoculant "Zhongnong Green Health", SGJ represents the seed treatment with the commercial inoculant "Shuguanjia", and CK represents the disease control. Significant differences exist between treatments labeled with different lowercase letters.

[0091] Figure 23 The effect of A3 seed dressing agent in the potato field of Dananying Village in Example 1 of this invention (2023). Figure 23 A is A3, used for seed dressing of potatoes. Figure 23 B is the blank control.

[0092] Figure 24 The effect of A3 seed dressing agent in Example 1 of this invention in potato fields in Zhaobizhuangzi Village (2023). Figure 24 A is A3, used for seed dressing of potatoes. Figure 24 B is the blank control.

[0093] Figure 25 The effect of A3 seed dressing agent in Example 1 of this invention in potato fields in Yongsheng Village (2024). Figure 25 A is A3, used for seed dressing of potatoes. Figure 25 B is the blank control.

[0094] Figure 26 The effect of A3 seed dressing agent in the potato field of Daxuzhuang Village in Example 1 of this invention (2024). Figure 26 A is A3, used for seed dressing of potatoes. Figure 26 B is the blank control.

[0095] The figure shows the application effect of A3 seed dressing agent in potato fields in Gedinggai Village in Example 1 of the present invention (2024). Figure 27 A is A3, used for seed dressing of potatoes. Figure 27 B is the blank control. Detailed Implementation

[0096] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0097] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0098] The polymyxobacterium ZF129 in the following examples is a polymyxobacterium ( Paenibacillus polymyxa ), belongs to the genus Bacillus ( Paenibacillus The bacteria, with its registration number at the China General Microbiological Culture Collection Center (CGMCC) as CGMCC No: 17631, was deposited on April 25, 2019, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0099] The *Bacillus pilaris* ZF390 in the following examples is *Bacillus pilaris* (… Paenibacillus peoriae ), belongs to the genus Bacillus ( Paenibacillus The bacteria, with the registration number CGMCC No: 20322 at the China General Microbiological Culture Collection Center (CGMCC), was deposited on July 8, 2020, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0100] The Bacillus subtilis ZF517 in the following examples is Bacillus subtilis ( Bacillus subtilis ), belongs to the genus Bacillus ( Bacillus The bacteria, with its registration number at the China General Microbiological Culture Collection Center (CGMCC) as CGMCC No: 30137, was deposited on March 26, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0101] The tested strains, Bacillus polymyxa ZF129, Bacillus pilaris ZF390, and Bacillus subtilis ZF517, were all isolated and preserved by the Vegetable Disease Integrated Prevention and Control Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0102] The adjuvants used in the following examples are as follows:

[0103] Wetting agents: Sodium dodecylbenzene sulfonate (SDBS), Morwet EFW, Morwet IP, LT-9004, LT-DK3, and sodium lignosulfonate (NA). Among them, sodium dodecylbenzene sulfonate (SDBS) is a product of Shanghai Xinyu Biotechnology Co., Ltd., catalog number XY-B1016. Morwet EFW and Morwet IP are both products of Nanjing Jierun Technology Co., Ltd. LT-9004 is a product of Shenzhen Langtai Biotechnology Co., Ltd., catalog number: Langtai LT-9004 wetting and dispersing agent. LT-DK3 is a product of Shenzhen Langtai Biotechnology Co., Ltd., catalog number: Darun Disintegrating Suspension DK3. Sodium lignosulfonate (NA) is a product of Sinopharm Chemical Reagent Beijing Co., Ltd., catalog number: XW01806151601.

[0104] Dispersants: Sodium salt of polynaphthalene sulfonic acid (NNO), Morwet D-425, and Morwet D-450. Sodium salt of polynaphthalene sulfonic acid (NNO) is a product of Beijing Caster Technology Development Co., Ltd., item number: YSHX0713. Morwet D-425 and Morwet D-450 are products of Nanjing Jierun Technology Co., Ltd.

[0105] In the following examples, the control seed dressing agent Qiwei Microbial Seed Dressing Agent is a product of China Agricultural University, Greenland Golden is a product of Guangzhou Mu'en Biotechnology Co., Ltd., Jindian Potato Special Seed Dressing Agent is a product of Jinyinong Microbial Products Co., Ltd., Tiandaoletu Potato Seed Treatment Agent is a product of Letu Biotechnology Co., Ltd., Shuguanjia Potato Seed Dressing Special Agent is a product of Shandong Fengren Agricultural Technology Co., Ltd., and Qiangshi Potato Special Biological Seed Dressing Agent is a product of Qiangshi Agricultural Technology Co., Ltd.

[0106] The potato varieties Kexin No. 1, Holland 15, Xufeng No. 1, Xisen No. 6, and Wotu in the following examples were all purchased from Inner Mongolia Xufeng Agricultural Technology Co., Ltd., Wuchuan County, Hohhot City, Inner Mongolia Autonomous Region.

[0107] The tested pathogen, Rhizoctonia solani, in the following examples Rhizoctonia solani Brazilian pectinobacterium Pectobacterium brasiliense Streptomyces scabbingus Streptomyces scabies All of them were separated and preserved by our laboratory, and the literature records are as follows:

[0108] Rhizoctonia solani Rhizoctonia solani The results have been published in the literature “Liu Ruichi, Cheng Youpu, Chai Ali, Shi Yanxia, ​​Xie Xuewen, Patiguli, Li Baoju. Establishment and application of triple PCR detection system for soil-borne pathogens in vegetables. Chinese Journal of Agricultural Science, 2019, 52(12):2069-2078” and are available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0109] Pectobacterium brasiliense has been published in the literature “Zhao Yurong, Xie Xuewen, Xu Shuai, Xie Hua, Shi Yanxia, ​​Chai Ali, Li Lei, Li Baoju. Control effect of Pectobacterium brasiliense ZF390 on bacterial soft rot of cucumber. Chinese Journal of Biological Control, 2022, 38(02):476-486.” and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0110] The species *Streptomyces scabies* has been published in the literature “Chen Lida, Xie Xuewen, Shi Yanxia, ​​Chai Ali, Pan Haoqin, Li Lei, Li Baoju. Construction and application of real-time fluorescence quantitative PCR detection system for *Streptomyces scabies*. Journal of Agricultural Biotechnology, 2020, 28(07):1314-1321.” and is available to the public from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0111] In the following examples, the culture media were prepared as follows: NA medium: 10 g peptone, 3 g beef powder, 5 g NaCl, 15 g agar, and distilled water to a final volume of 1000 mL. LB medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar, and distilled water to a final volume of 1000 mL. PDA medium: 200 g potato, 20 g D+ anhydrous glucose, and 15 g agar. Oatmeal agar (OMA): 30 g oat grains / flakes, 20 g agar, and distilled water to a final volume of 1000 mL.

[0112] In the following examples, the performance indicators of the potato seed treatment agent were determined according to relevant national standards. Spore content was determined using the plate count method according to industry standard NY / T2293.1—2012 (Bacterial Microbial Pesticides Bacillus subtilis Part 1: Bacillus subtilis Technical Grade [S]). On the plates used for spore content determination, the number of contaminating bacteria other than those of strains ZF129, ZF390, and ZF517 was recorded; the percentage of contaminating bacteria in the total bacterial count was the contamination rate. pH value was determined according to national standard GB / T1601—1993 (Guangxi Tianyuan Biochemical Co., Ltd., Jiangsu Dongbao Agrochemical Co., Ltd., Anhui Shanghe Woda Biotechnology Co., Ltd., et al. Method for Determination of pH Value of Pesticides [S]). Drying loss was determined according to industry standard NY / T2293.1—2012. Wetting time was determined according to national standard GB / T5451—2001 (Shenyang Chemical Research Institute). Method for determining the wettability of wettable powder pesticides [S].); Suspension rate was determined according to national standard GB / T14825—2006 (Crop Protection Co., Ltd. Syngenta Suzhou, Jiangsu Dongbao Agrochemical Co., Ltd., Jiangsu Longdeng Chemical Co., Ltd., et al. Method for determining the suspension rate of pesticides [S].).

[0113] In the following examples, the experimental data were statistically analyzed using Excel and SPSS software.

[0114] Example 1

[0115] 1. Biocompatibility determination of biocontrol strains, vectors, and adjuvants

[0116] The cross-streaking method was used to determine whether there was mutual antagonism among strains ZF129, ZF390 and ZF517. LB solid medium was used as the culture medium. Figure 1 In A, the vertical line is ZF129 and the horizontal line is ZF390; Figure 1 In B, the vertical line is ZF129; the horizontal line is ZF517. Figure 1 In the letter C, the vertical line is ZF390; the horizontal line is ZF517. Figure 1 D represents the antagonistic strain control. The vertical line represents ZF129, and the horizontal line represents ZF516 (Wei Xinchen, Zhao Zixuan, Xie Xuewen, et al. Isolation, identification and biocontrol effect of ZF516, an antagonistic bacterium against tomato bacterial canker [J / OL]. Acta Phytopathologica Sinica, 1-13 [2025-01-14].). The results show that there is no antagonistic effect between strains ZF129, ZF390 and ZF517, and the three strains can be used in combination.

[0117] 1.1 Effect of the vector on the growth viability of the strain by the toxicity plate method

[0118] The toxicity plate method was used (refer to "Bacillus amyloliquefaciens BA"). 12. Development of wettable powder and its effect on the control of walnut root rot [J].” The biocompatibility of the adjuvant with three biocontrol bacteria was evaluated as follows:

[0119] Silica, diatomaceous earth, attapulgite, and talc were mixed with LB solid medium at weight percentages of 1%, 2%, and 3% respectively (carrier as a percentage of the medium's mass), sterilized, and then poured into plates to obtain LB solid medium containing 1% silica, 2% silica, 3% silica, 1% diatomaceous earth, 2% diatomaceous earth, 3% diatomaceous earth, 1% attapulgite, 2% attapulgite, 3% attapulgite, 1% talc, 2% talc, and 3% talc. LB medium without carrier was used as a blank control.

[0120] Single colonies of strains ZF129, ZF390, and ZF517 were inoculated into 14 mL sterile shaking tubes containing 3 mL of liquid LB medium. The seed culture, obtained by shaking at 28°C and 180 rpm for 24 h, was then transferred to 250 mL Erlenmeyer flasks containing 100 mL of liquid LB medium. The fermentation broth, obtained by shaking at 28°C and 180 rpm for 36 h, was then serially diluted using the fermentation broths of strains ZF129, ZF390, and ZF517. 10 μL of each broth was then used as the final product. -4 10 -5 10 -6 10 -7 100 μL of each sample was plated separately, with 3 replicates per group. The samples were incubated at 37°C for 12 h, and the number of colonies was recorded.

[0121] See results Figure 2 The results indicate that diatomaceous earth inhibits the growth of the three strains. Talc, attapulgite, and silica can enhance the growth activity of strain ZF129, but have no significant inhibitory effect on the growth of ZF390 and ZF517. Considering all factors, attapulgite has the least impact on the growth activity of the three strains, and increasing the carrier concentration has the least impact on the viability of the strains, showing the most stability. Therefore, it can be used as a carrier for the parent drug of the strains.

[0122] 1.2 Effect of adjuvants on bacterial growth viability determined by toxicity plate method

[0123] Six wetting agents were tested: sodium dodecylbenzenesulfonate (SDBS), Morwet EFW, Morwet IP, LT-9004, LT-DK3, and sodium lignosulfonate (NA).

[0124] Three dispersants were tested: sodium salt of polynaphthalene sulfonate (NNO), Morwet D-425, and Morwet D-450.

[0125] The biocompatibility of adjuvants with three biocontrol bacteria strains was evaluated using the toxicity plate method. The tested adjuvants (6 wetting agents and 3 dispersants, a total of 9 adjuvants) were mixed with LB medium at mass ratios of 1%, 2%, and 3% to prepare solid culture plates. For example, mixing NNO with LB medium at mass ratios of 1%, 2%, and 3% yielded LB solid media containing 1% NNO, 2% NNO, and 3% NNO; similarly, mixing NA with LB medium at mass ratios of 1%, 2%, and 3% yielded LB solid media containing 1% NA, 2% NA, and 3% NA. Other media containing adjuvants were prepared using similar methods.

[0126] Single colonies of strains ZF129, ZF390, and ZF517 were inoculated into 14 mL sterile shaking tubes containing 3 mL of liquid LB medium. The seed culture, obtained by shaking at 28°C and 180 rpm for 24 h, was then transferred to 250 mL Erlenmeyer flasks containing 100 mL of liquid LB medium. The fermentation broth, obtained by shaking at 28°C and 180 rpm for 36 h, was then serially diluted using the fermentation broths of strains ZF129, ZF390, and ZF517. 10... -4 10 -5 10 -6 10 -7 100 μL of each culture was plated separately, with LB medium without the carrier as a blank control. Each group was in triplicate and incubated at 37°C for 12 h. The number of colonies was recorded.

[0127] Within the tested concentration range (mass fraction 1%-3%), all five wetting agents except for NA prevented the growth of the three biocontrol bacteria at different concentrations. Dispersants D-425 and D-450 also prevented the growth of the three biocontrol bacteria at different concentrations (see Table 1). With increasing dosage of wetting agent NA, the viable count of the mother powder slightly decreased. NNO dispersant showed good compatibility with the three strains at dosages of 1%, 2%, and 3% (see Table 1). Figure 3 Therefore, dispersant NNO and wetting agent NA were selected as additives, and their optimal dosages were screened.

[0128] Table 1. Effects of different proportions of adjuvants on the growth viability of bacterial strains.

[0129]

[0130] Note: "+" indicates that the tested Bacillus strain is growing normally; "" indicates that the growth of the tested Bacillus was completely inhibited; "±" indicates that the growth of the tested Bacillus was partially inhibited.

[0131] 2 Screening of carriers and adjuvants for potato seed treatment

[0132] 2.1 Preparation of strain parent drug

[0133] Single colonies of strains ZF129, ZF390, and ZF517 were inoculated into 14 mL sterile shake tubes containing 3 mL of liquid LB medium. The seed culture, obtained by shaking at 28°C and 180 rpm for 24 h, was transferred to 3 L Erlenmeyer flasks containing 1.5 L of liquid LB medium. The fermentation broth, obtained by shaking at 28°C and 180 rpm for 36 h, was then collected from the fermentation broths of strains ZF129, ZF390, and ZF517. 10% (w / w) of the optimal carrier (attapulgite clay) was added to each flask, and the mixture was stirred thoroughly for 6 h. The mixture was then filtered four times using a vacuum filter until the filtrate was clear. The filter cake was collected and air-dried at 35°C with 100% airflow. The air-dried product was then added to a high-speed grinder and thoroughly pulverized to obtain the strain mother drug. The viable count was determined by dilution and spread. Specifically, the ZF129 strain mother drug yielded 4.32 × 10⁻⁶ cells / mL. 7 CFU / g), ZF390 strain mother drug (4.14×10) 8 CFU / g), ZF517 strain mother drug (4.86×10) 9 CFU / g).

[0134] 2.2 Screening of Potato Seed Treatment Carriers

[0135] The biocompatibility of the seed dressing agent carrier and the mycelium powder was determined using a dry mixing method. Representative carriers, including silica, diatomaceous earth, attapulgite, and talc, were selected for compatibility testing. Different carriers were mixed with the technical grade ZF129, ZF390, and ZF517 strains at a 1:1 (m / m) (mass ratio) and left to stand for 21 days. Viable cell counts were measured every 7 days. The technical grade strains without carriers served as a blank control. Each treatment was repeated three times. After thorough mixing and standing, the plate count method was used to analyze the effect of the seed dressing agent carrier on the number of viable cells in the mycelium powder.

[0136] See results Figure 4 A, Figure 4 B and Figure 4As storage time increases, the number of viable bacteria in the inoculum powder decreases to some extent. From 0 to 14 days, the number of viable bacteria after mixing talc, diatomaceous earth, attapulgite, and silica with the strain mother drug all decreased significantly. At 21 days, the number of viable bacteria after mixing diatomaceous earth carrier with the strain mother drug still showed a decreasing trend. The number of viable bacteria after mixing talc, attapulgite, silica with the inoculum powder was not significantly different from that at 14 days. Based on experience in actual potato production, talc can be given priority as the carrier for potato seed dressing agent.

[0137] 2.3 Optimization of the types and amounts of wetting agents and dispersants

[0138] Mixed strain mother drugs (compound microbial powder) obtained by mixing ZF129 strain mother drugs, ZF390 strain mother drugs, and ZF517 strain mother drugs in a mass ratio of m:m:m=1:1:1 were added to a mixture of different types and proportions of wetting agents and dispersants (see Table 2 for specific types and proportions). The mixture was then made up to 100% with talc powder to obtain different wettable powders. Seed dressing agents without adjuvants were used as a control. The wetting time and suspension rate of each treatment were measured.

[0139] Table 2 Screening of Optimal Dosage of Potato Seed Treatment Dispersant

[0140]

[0141] See results Figure 4 As the amount of NNO and NA used increases, the wetting time decreases and the suspension rate increases. When the amount of NNO added is 3%-5% and the amount of NA added is 4%-5%, both the suspension rate and wetting time meet the national standards. The wetting time is less than 120 s (national standard), and the suspension rate is greater than 75.00% (national standard).

[0142] 3. Processing and quality testing of potato seed treatment agents

[0143] Based on the research results, the formulations of four potato seed treatment agents were determined:

[0144] A1: Mixed strain mother drug (m ZF129 strain mother drug : m ZF390 strain mother drug : m ZF517 strain mother drug = 1:1:1) 4% (mass fraction, the same below), dispersant NNO 3%, wetting agent NA 4%, Acid Red 0.1%, talc to make up to 100%.

[0145] A2: Mixed strain mother drug (m ZF129 strain mother drug : m ZF390 strain mother drug : m ZF517 strain mother drug = 1:1:1) 6%, dispersant NNO 3%, wetting agent NA 4%, Acid Red 0.1%, talc to make up to 100%.

[0146] A3: Mixed strain mother drug (m ZF129 strain mother drug : m ZF390 strain mother drug : m ZF517 strain mother drug = 1:1:1) 8%, dispersant NNO 3%, wetting agent NA 4%, Acid Red 0.1%, talc to make up to 100%.

[0147] A4: Mixed strain mother drug (m ZF129 strain mother drug : m ZF390 strain mother drug : m ZF517 strain mother drug = 1:1:1) 10%, dispersant NNO 3%, wetting agent NA 4%, Acid Red 0.1%, talc to make up to 100%.

[0148] The processing methods for A1, A2, A3, and A4 are all the same: adding optimized and screened wetting agents and dispersants to the mixed bacterial strain mother drug, supplementing with talc to 100%, mixing thoroughly with a mixer, and then pulverizing with an air jet mill to obtain potato seed treatment agents A1, A2, A3, and A4, which are Bacillus wettable powders.

[0149] The potato seed treatment agents A1, A2, A3, and A4 were tested for quality according to relevant standards. The results showed that the average spore concentration of each potato seed treatment agent was between 1.5 × 10⁻⁶. 8 -2.0×10 8 The average CFU / g, average contamination rate of 2.8%, average pH value of 6.7, loss on drying of 0.9%, wetting time of 114.3 s, and suspension rate of 77.8% all met the relevant industry standards for wettable powders. The prepared seed treatment agent was a pink powder without lumps. After being stored in a glass bottle with a ground glass stopper at (54±2)℃, the sample did not show any clumping or stickiness.

[0150] 4. Safety test of potato seed treatment agent

[0151] 4.1 Safety test of potato seed treatment agents on different potato varieties

[0152] The method described in the reference "Zhao Weisong, Li Shezeng, Lu Xiuyun, et al. Development and application of seed treatment agent for controlling potato Verticillium wilt [J]. Chinese Journal of Biological Control. 2019, 35(5): 759-767" was used to evaluate the effects of potato seed treatment agent on the growth of different potato varieties using a greenhouse pot experiment.

[0153] Healthy seed potatoes were cut into 35-50 g pieces (1-2 eyes / piece) and placed on a plastic film. Potato seed treatment agents (A1, A2, A3, and A4) were sprinkled on the surface of the pieces, and the pieces were stirred to ensure even coating. They were then planted in flowerpots (16 cm in diameter, 13 cm deep) filled with seedling substrate and cultivated in a greenhouse. The experimental setup used 45 kg / hm² of potato seed treatment agent. 2 At 45 kg / hm 2 Talc seed treatment was used as a blank control, and untreated potatoes were used as a naked seed control. The tested potato varieties were Kexin No. 1, Holland 15, Xufeng No. 1, and Xisen No. 6. The number of seedlings in each treatment was investigated 30 days after sowing, the seedling rate was calculated, and the plant height and stem diameter of potatoes were investigated to evaluate the effect of seed treatment agents on the plant height and stem diameter of different potato varieties.

[0154] Emergence rate (%) = (Number of seedlings emerged / Total number of seedlings) × 100

[0155] Corrected germination rate (%) = (germination rate / blank control germination rate) × 100

[0156] Data showed that all seed treatment agents posed no safety risks to potato emergence and exhibited varying degrees of growth-promoting effects. There was no significant difference in plant height and stem diameter between the naked seed control and the blank control, indicating that talcum powder had minimal impact on potato growth. The Kexin No. 1 potato plants treated with seed treatment agent A3 had a plant height of 185.22 mm and a stem diameter of 9.73 mm, significantly higher than the blank control (see...). Figure 5 The Dutch 15 potato plants treated with seed treatment agent A3 had a plant height of 217.14 mm and a stem diameter of 8.49 mm, significantly higher than the control group (see...). Figure 6 The Xufeng No. 1 potato plants treated with seed treatment agent A3 had a plant height of 259.65 mm and a stem diameter of 9.56 mm, significantly higher than the blank control (see...). Figure 7 The Xisen 6 potato plants treated with seed treatment agent A3 had a plant height of 220.11 mm and a stem diameter of 7.96 mm, significantly higher than the control group (see...). Figure 8 In summary, among the four formulations A1, A2, A3, and A4, the seed treatment agent in formulation A3 is safe for the emergence of Kexin No. 1, Holland 15, Xufeng No. 1, and Xisen No. 6 potatoes and has a significant growth-promoting effect.

[0157] 4.2 Effects of different dosages of seed treatment agents on seed potato emergence

[0158] The method described in the reference "Zhao Weisong, Li Shezeng, Lu Xiuyun, et al. Development and application of seed treatment agent for controlling Bacillus wilt of potato [J]. Chinese Journal of Biological Control. 2019, 35(5): 759-767" was used to evaluate the effect of different dosages of potato seed treatment agent on potato growth using a greenhouse pot experiment.

[0159] Healthy seed potatoes were cut into 35-50g pieces (1-2 eyes / piece) and placed on a plastic film. Potato seed treatment agent A3 (the optimal formula selected in section 4.1) was sprinkled on the surface of the pieces, and after stirring to ensure even coating, the pieces were planted in pots (16 cm in diameter, 13 cm deep) filled with seedling substrate and cultivated in a greenhouse. The experiment used 22.5 kg / hm² of potato seed treatment agent. 2 33.75 kg / hm 2 45 kg / hm 2 and 56.25 kg / hm 2 Four doses were administered, with each treatment replicated three times. Each replicate consisted of 12 pots, with one seed potato per pot. The production yield was approximately 2250 kg / hm² of seed potatoes (cut into pieces). 2 The dosage of pesticides used in the pot experiment was calculated based on the recommended dosage of commercial seed dressing agents (Qiwei Microbial Seed Dressing Agent, Lvdichengjin, Jindian Potato-Specific Seed Dressing Agent, Tiandaole Potato Seed Treatment Agent, Shuguanjia Potato Seed Dressing Agent, and Qiangshi Potato-Specific Biological Seed Dressing Agent) served as the control, while seed dressing with 45 kg of talcum powder served as the blank control. Twenty days after sowing, the number of seedlings in each treatment was investigated, the seedling rate was calculated, and the plant height of potatoes was also investigated to evaluate the effect of different dosage treatments on potato plant height.

[0160] The results of the safety test of different dosages of seed treatment agent A3 on potatoes are shown in the figure. Figure 9 and Figure 10 When the control group's potatoes emerged 7 days after emergence, the emergence rate of all treatments was 100%, with no inhibition of emergence. When the control group's potatoes emerged 10 days after emergence, the application rate was 45 kg / hm². 2 Potato plants treated with seed treatment agent A3 reached a height of 142.13 mm and a stem diameter of 10.35 mm, both significantly higher than the control group and the commercial seed dressing group, demonstrating a clear growth-promoting effect. These results indicate that 22.5 kg / hm² of potato seed dressing agent is effective. 2 33.75 kg / hm 2 45 kg / hm 2 and 56.25 kg / hm 2 Four dosages showed safety and no negative effects on seed potatoes, and also exhibited a certain growth-promoting effect, with 45 kg / hm² being the most effective. 2The treatment has the strongest growth-promoting effect.

[0161] Pot test on the disease prevention effect of potato seed treatment agent

[0162] 5.1 Pot Experiment on the Effect of Potato Seed Treatment on the Control of Potato Soft Rot

[0163] Potato soft rot was artificially inoculated using the soil-mixing and root-drenching inoculation method. The inoculum suspension of the soft rot pathogen (concentration 1×10⁻⁶) was prepared. 8 cfu·mL -1 Mix the sterilized seedling substrate soil (substrate:sand = 2:1) at a volume ratio of 1:40, adjust the soil moisture, and make sure the substrate soil can be squeezed into a ball without dripping water. Cut healthy potato seed tubers into 35-50 g pieces (1-2 eyes / piece) and place them on a plastic film. Sprinkle potato seed treatment agent on the surface of the tubers, stir and turn to ensure even coating, and then plant them in diseased soil. Cultivate in a greenhouse. Use seed treatment with commercial seed dressing agents (Greenland Golden, Shuguanjia potato seed dressing special bacteria agent) at the recommended dosage as a control, 45 kg of talcum powder seed dressing as a disease control, and 45 kg of talcum powder seed dressing planted in pathogen-free soil as a healthy control.

[0164] Emergence time for each treatment was investigated. Seven days after the healthy controls had fully emerged, the number of seedlings in each treatment was counted, and the emergence rate and corrected emergence rate were calculated. Fourteen days after emergence, a root drench was applied with *Bacillus brasiliensis* (1×10⁻⁶). 8 cfu·mL -1 50 mL per plant. After the diseased control group has fully developed the disease, investigate the incidence of soft rot in the plants, calculate the disease index and control effect.

[0165] The severity level is based on the tobacco black shank disease severity level. The severity is divided into 5 levels: Level 0, no disease on the entire plant; Level 1, stem lesions not exceeding 50% of the stem circumference, or less than 50% of the leaves slightly wilted, or a few lower leaves showing lesions; Level 2, stem lesions exceeding approximately 50% of the stem circumference, or 50% of the leaves wilted; Level 3, stem lesions encircling the stem circumference, or more than 66% of the leaves wilted; Level 4, all leaves of the diseased plant wilted or died.

[0166] Disease incidence rate (%) = Number of diseased plants / Total number of plants surveyed × 100;

[0167] Disease index = ∑ (number of diseased plants at each level × disease level) / (total number of plants surveyed × highest representative value) × 100

[0168] Prevention and control effect (%) = ((Disease index of disease control - Disease index of each treatment) / Disease index of disease control) × 100

[0169] The test results are shown in Figure 11 and Figure 12 After 45 days of growth, the control group showed full disease development, with wilting of the above-ground leaves, blackening of the stems, and rotting of the underground stems and tubers. In severe cases, the entire potato plant withered and died. The disease index of the disease control group reached 69.05, and the tuber rot rate was 90.48%. The disease incidence of potatoes treated with seed treatment agents was significantly reduced. Among them, the disease index of soft rot in potatoes treated with A3 was significantly reduced to only 22.92, and the tuber rot rate was 33.33%, with a control effect of 66.81%, which was significantly higher than that of seed treatment agents Greenland City Gold and Potato Stem Manager.

[0170] 5.2 Pot experiment on the effect of potato seed treatment on the control of potato black scurf.

[0171] Potato black scurvy pathogen was artificially inoculated using the root-wound inoculation method. After activating the preserved pathogen on PDA plates for 6 days, 10 mycelial discs (5 mm) of the activated *Rhizoctonia solani* were inoculated into the PDA culture medium at 28°C and 180 r·min. -1 After culturing for 7 days, the colonies were broken up using a cell wall blender and the concentration was adjusted to 1×10⁻⁶. 5 cfu·mL -1 Prepare for use. Cut healthy potato seed tubers into 35-50 g pieces (1-2 eyes / piece) and place them on a plastic film. Sprinkle potato seed treatment agent on the surface of the tubers, stir to ensure even coating, and then plant them in flowerpots (16 cm in diameter, 13 cm deep) filled with seedling substrate. Cultivate in a greenhouse. Use commercial seed dressing agents (Greenland Golden, Shuguanjia potato seed dressing agent) at the recommended dosage as a control, and use 45 kg of talcum powder as a healthy control. After the potatoes have emerged for 14 days, damage the roots and inoculate the roots with 100 mL of potato black scurf fungus per pot. After the diseased control group has fully developed the disease, investigate the incidence of potato black scurf, calculate the disease index, and assess the control effect.

[0172] The representative values ​​for potato underground stem disease are determined according to the underground stem disease grading standard: 0, no lesions on the underground stem; 1, 0 < the proportion of lesion area to the underground stem area ≤ 5%; 2, 5% < the proportion of lesion area to the underground stem area ≤ 25%; 3, 25% < the proportion of lesion area to the underground stem area ≤ 50%; 4, 50% < the proportion of lesion area to the underground stem area ≤ 75%; 5, 75% < the proportion of lesion area to the underground stem area ≤ 100%.

[0173] Disease index = ∑ (number of diseased plants at each level × disease level) / (total number of plants surveyed × highest representative value) × 100

[0174] Prevention and control effect (%) = ((Disease index of disease control - Disease index of each treatment) / Disease index of disease control) × 100

[0175] The test results are shown in Figure 13 and Figure 14 After 45 days of growth, the control group showed full disease development, with typical reddish-brown lesions and black sclerotia appearing on the underground stems. The disease index of the disease control group reached 89.17. The disease incidence of potatoes treated with seed treatment agents was significantly reduced, and no potato rot occurred. Among them, the disease index of potatoes treated with A3 was the lowest, at only 2.22, with a control effect of 97.51%, which was significantly better than the control seed treatment agents Greenland City Gold and Potato Stem Manager.

[0176] 5.3 Pot Experiment on the Control Effect of Potato Seed Treatment on Potato Scab

[0177] First, *Streptomyces scabica* was inoculated onto solid OM medium. After 10 days of growth, the spores were washed off with sterile water, and the spore concentration was adjusted to 1×10⁻⁶. 8 The pathogen inoculum was prepared by mixing the pathogen inoculum with field soil at a volume ratio of 1:40 to make diseased soil. The diseased soil was then mixed with seedling substrate (field soil: seedling substrate = 1:1) and divided into flower pots with a diameter of 17.5 cm and a depth of 17 cm. Healthy potato seed tubers were cut into 35-50 g pieces (1-2 eyes / piece) and placed on a plastic film. Potato seed treatment agent was sprinkled on the surface of the tubers, and the tubers were stirred to ensure even coating. They were then planted in the flower pots containing the diseased soil. Commercial seed dressing agents (Greenland Golden, Shuguanjia potato seed dressing agent) were used as controls, and seeds treated with talcum powder and planted in healthy soil (field soil: seedling substrate = 2:1) served as healthy controls. The incidence of tuber diseases was investigated after the potatoes had grown for 65 days.

[0178] Based on the percentage of scabs covering the potato tuber area, potatoes are classified into five grades: Grade 0, no scabs; Grade 1, scabs covering 0% of the tuber area. Grade 15%; Grade 2, diseased area accounts for 15% of the tuber area. 30%; Level 3, diseased area accounts for 30% of the tuber area. 50%; Level 4, the area of ​​diseased spots accounts for more than 50% of the tuber area.

[0179] Disease index = ∑ (number of diseased plants at each level × disease level) / (total number of plants surveyed × highest representative value) × 100

[0180] Prevention and control effect (%) = ((Disease index of disease control - Disease index of each treatment) / Disease index of disease control) × 100

[0181] The test results are shown in Figure 15 and Figure 16 After 65 days of growth, the control group showed full disease development, with typical scabs and sunken lesions appearing on the tubers. The disease index of the control group reached 45.31. The disease index of the tubers treated with seed dressing was reduced in all cases. Among them, the tubers treated with A3 seed dressing agent had the lowest disease index, with a disease index of only 15.83, and a control efficacy of 65.05%. The control effect was significantly better than that of the control seed dressing agents Greenland City Gold and Potato Stem.

[0182] 5.4 Determination of the efficacy of compound microbial seed dressing agents and single-strain seed dressing agents against potato soft rot

[0183] Potato soft rot was artificially inoculated using the soil-mixing and root-drenching inoculation method. The inoculum suspension of the soft rot pathogen (concentration 1×10⁻⁶) was prepared. 8 cfu·mL -1 Mix the sterilized seedling substrate soil (substrate:sand = 2:1) at a volume ratio of 1:40, adjust the soil moisture, and make sure the substrate soil can be squeezed into a ball without dripping water. Cut healthy potato seed tubers into 35-50 g pieces (1-2 eyes / piece) and place them on a plastic film. Sprinkle the surface of the tubers with a single-strain potato seed dressing agent (the active ingredients are ZF129 single-strain biocontrol bacteria, ZF390 single-strain biocontrol bacteria, and ZF517 single-strain biocontrol bacteria) and compound microbial seed dressing agent A3. Turn and stir to ensure the surface of the tubers is evenly coated with the agent, and then plant them in diseased soil. Cultivate them in a greenhouse. Use seed dressing treatment with the recommended dosage of commercial seed dressing agents (Greenland Golden, Zhongnong Green Health, and Shuguanjia potato seed dressing special microbial agent) as a control, seed dressing with 45 kg of talcum powder as a disease control, and seed dressing with 45 kg of talcum powder planted in soil free of pathogens as a healthy control.

[0184] The severity level is based on the tobacco black shank disease severity level. The severity is divided into 5 levels: Level 0, no disease on the entire plant; Level 1, stem lesions not exceeding 50% of the stem circumference, or less than 50% of the leaves slightly wilted, or a few lower leaves showing lesions; Level 2, stem lesions exceeding approximately 50% of the stem circumference, or 50% of the leaves wilted; Level 3, stem lesions encircling the stem circumference, or more than 66% of the leaves wilted; Level 4, all leaves of the diseased plant wilted or died.

[0185] Disease incidence rate (%) = Number of diseased plants / Total number of plants surveyed × 100;

[0186] Disease index = ∑ (number of diseased plants at each level × disease level) / (total number of plants surveyed × highest representative value) × 100

[0187] Prevention and control effect (%) = ((Disease index of disease control - Disease index of each treatment) / Disease index of disease control) × 100

[0188] The test results are shown in Figure 17 and Figure 18 After 55 days of growth, the control group showed full disease development, with symptoms including wilting of leaves, blackening of stems, missing seedlings, and poor growth. In severe cases, the entire potato plant wilted and died, with a disease index of 78.33. The disease incidence was significantly reduced in the seed-treated potatoes. In particular, the disease index of soft rot in potatoes treated with A3 was significantly reduced to only 27.16, with a control effect of 65.32%, which was significantly higher than that of single-strain potato seed treatment agents and other commercial seed treatment agents.

[0189] 5.5 Determination of the control efficacy of compound microbial seed dressing agents and single-strain seed dressing agents against potato black scurf

[0190] Potato black scurvy pathogen was artificially inoculated using the root-wound inoculation method. After activating the preserved pathogen on PDA plates for 6 days, 10 mycelial discs (5 mm) of the activated *Rhizoctonia solani* were inoculated into the PDA culture medium at 28°C and 180 r·min. -1 After culturing for 7 days, the colonies were broken up using a cell wall blender and the concentration was adjusted to 1×10⁻⁶. 5 cfu·mL -1 Prepare for use. Cut healthy potato seed tubers into 35-50 g pieces (1-2 eyes / piece) and place them on a plastic film. Sprinkle the surface of the tubers with a single-strain potato seed dressing agent (the active ingredients are ZF129 single-strain biocontrol bacteria, ZF390 single-strain biocontrol bacteria, and ZF517 single-strain biocontrol bacteria) and compound microbial seed dressing agent A3. Stir and turn the tubers to ensure even coating. Then plant them in flower pots (16 cm in diameter and 13 cm in depth) filled with seedling substrate and cultivate them in a greenhouse. Use commercial seed dressing agents (Greenland Golden, Zhongnong Green Kang, and Shuguanjia potato seed dressing special microbial agent) at the recommended dosage as a control, and use 45 kg of talcum powder as a healthy control. After the potatoes have emerged for 14 days, damage the roots and inoculate the roots with potato black scurf fungus, 100 mL per pot. After the diseased control group has fully developed the disease, investigate the incidence of potato black scurf, calculate the disease index and control effect.

[0191] The representative values ​​for potato underground stem disease are determined according to the underground stem disease grading standard: 0, no lesions on the underground stem; 1, 0 < the proportion of lesion area to the underground stem area ≤ 5%; 2, 5% < the proportion of lesion area to the underground stem area ≤ 25%; 3, 25% < the proportion of lesion area to the underground stem area ≤ 50%; 4, 50% < the proportion of lesion area to the underground stem area ≤ 75%; 5, 75% < the proportion of lesion area to the underground stem area ≤ 100%.

[0192] Disease index = ∑ (number of diseased plants at each level × disease level) / (total number of plants surveyed × highest representative value) × 100

[0193] Prevention and control effect (%) = ((Disease index of disease control - Disease index of each treatment) / Disease index of disease control) × 100

[0194] The test results are shown in Figure 19 and Figure 20 After 45 days of growth, the control group showed full disease development, with typical reddish-brown lesions and black sclerotia appearing on the potato rhizomes. The disease index of the disease control group reached 68.00. The disease incidence of potatoes treated with seed treatment agents was significantly reduced, and no potato rot occurred. Among them, the disease index of potatoes treated with A3 was the lowest, at only 26.67, with a control effect of 60.78%, which was significantly higher than that of single-spore potato seed dressing agents and other commercial seed dressing agents.

[0195] 5.6 Determination of the control efficacy of compound microbial seed dressing agents and single-strain seed dressing agents against potato scab.

[0196] First, *Streptomyces scabica* was inoculated onto solid OM medium. After 10 days of growth, the spores were washed off with sterile water, and the spore concentration was adjusted to 1×10⁻⁶. 8 The inoculum was prepared by mixing the pathogen inoculum with field soil at a volume ratio of 1:40 to create diseased soil. This diseased soil was then mixed with seedling substrate (field soil:seedling substrate = 1:1) and dispensed into pots with a diameter of 17.5 cm and a depth of 17 cm. Healthy potato seed tubers were cut into 35-50 g pieces (1-2 eyes / piece) and placed on a plastic film. A single-strain potato seed dressing agent (active ingredients ZF129 single-plant biocontrol bacteria, ZF390 single-plant biocontrol bacteria, and ZF517) was sprinkled on the surface of the tubers. Single-plant biocontrol bacteria), compound microbial seed dressing agent A3, after turning and stirring to ensure the potato tubers are evenly coated with the agent, plant them in flowerpots containing diseased soil. Use commercial seed dressing agents (Greenland Golden, Zhongnong Green Health, Shuguanjia potato seed dressing agent) as a control, and use talcum powder-treated seeds planted in healthy soil (field soil: seedling substrate = 2:1) as a healthy control. After the potatoes have grown for 65 days, investigate the tuber disease situation.

[0197] Based on the percentage of scabs covering the potato tuber area, potatoes are classified into five grades: Grade 0, no scabs; Grade 1, scabs covering 0% of the tuber area. Grade 15%; Grade 2, diseased area accounts for 15% of the tuber area. 30%; Level 3, diseased area accounts for 30% of the tuber area. 50%; Level 4, the area of ​​diseased spots accounts for more than 50% of the tuber area.

[0198] Disease index = ∑ (number of diseased plants at each level × disease level) / (total number of plants surveyed × highest representative value) × 100

[0199] Prevention and control effect (%) = ((Disease index of disease control - Disease index of each treatment) / Disease index of disease control) × 100

[0200] The test results are shown in Figure 21 and Figure 22 After 65 days of growth, the control group showed full disease development, with typical scabs and sunken lesions appearing on the tubers. The disease index of the control group reached 60.13. The disease index of the tubers treated with seed dressing was reduced in all cases. Among them, the tubers treated with A3 seed dressing agent had the lowest disease index, with a disease index of only 20.95, achieving a control efficacy of 65.15%. The control effect was significantly higher than that of single-strain potato seed dressing agents and other commercial seed dressing agents. 6. Evaluation of the field demonstration trial of the effect of potato seed treatment agents on potatoes.

[0201] Based on the pot experiment that determined the optimal dosage of potato seed treatment agent A3, field demonstration trials were conducted in 2023 and 2024 in Dananying Village, Guyuan County, Zhangjiakou City, Hebei Province; Gedinggai Village, Wuchuan County, Hohhot, Inner Mongolia Autonomous Region; Quanzijie Town, Changji Prefecture, Xinjiang Uygur Autonomous Region; and Yongsheng Village, Urumqi County, Urumqi City, to investigate the disease prevention and yield-increasing effects of seed treatment agent on potatoes. Potatoes under normal field management were used as a blank control. The incidence of soft rot, black sclerotium, and scab was investigated at the potato harvest period, and the disease index was calculated. Yield was measured at the potato harvest period, and the yield increase rate was calculated.

[0202] The demonstration results in 2023 at Dananying Village, Guyuan County, Zhangjiakou City, Hebei Province (115°75' E, 41°54' N) are shown below. Figure 23 As shown in Table 3, compared with the blank control, the A3 potato seed treatment agent at 45 kg / hm 2 Seed dressing significantly reduced the disease index of potato scab, with a control effect of 62.23%. In terms of yield, the A3 seed dressing treatment in Dananying Village was significantly higher than the blank control, with a potato marketability rate of 87.26%. Based on multi-point sampling, the yield per mu was 4091.16 kg, while the yield of the blank control was 3389.46 kg, resulting in a yield increase of 20.69%.

[0203] Table 3. Control efficacy of A3 potato seed dressing agent against potato scab in Dananying Village during field plot trials (2023)

[0204]

[0205] The demonstration results in Zhaobizhuangzi Village, Quanzijie Town, Jimusar County, Changji Hui Autonomous Prefecture, Xinjiang Uygur Autonomous Region in 2023 are shown in [reference needed]. Figure 24 According to Table 4, no disease was observed in the potatoes in the field. Regarding yield, compared to the control group, the yield of A3 potato seed treatment agent (45 kg / hm²) was significantly higher. 2 The commercialization rate of seed dressing treatment was 93.18%, and the yield per mu was 3710.03 kg, while the yield of the blank control was 3568.45 kg, with a yield increase rate of 3.97%.

[0206] The demonstration results in 2024 in Yongsheng Village, Yongfeng Town, Urumqi County, Urumqi City, Xinjiang Uygur Autonomous Region (87°26'E, 43°57'N) are shown below. Figure 25 According to Table 4, no disease was observed in the potatoes in the field. Regarding yield, the A3 potato seed treatment agent at 45 kg / hm² was effective. 2 The marketable rate of potatoes treated with seed dressing was 80.94%, with an average yield of 4137.74 kg per mu. The marketable rate of potatoes in the blank control group was 71.84%, with an average yield of 3281.15 kg per mu, representing a yield increase of 26.11%.

[0207] Table 4. Effects of A3 potato seed treatment agent on potato yield in Bizhuangzi Village and Yongsheng Village in field plot trials (2023-2024).

[0208]

[0209] The demonstration results in Daxuzhuang Village, Dongguo Town, Tengzhou City, Zaozhuang City, Shandong Province (117°21' E, 35°17' N) in 2024 are shown below. Figure 26 According to Table 5, no disease was observed in the potatoes in the field. Regarding yield, the A3 potato seed treatment agent at 45 kg / hm² was effective. 2 The commercial rate of seed-treated potatoes was 83.11%, equivalent to a yield of 3672.86 kg per mu (approximately 0.067 hectares), while the yield of the blank control was 3060.72 kg per mu (approximately 0.067 hectares), resulting in a yield increase of 20.00%.

[0210] Table 5. Effects of A3 potato seed dressing agent on potato yield in Daxuzhuang Village during field plot trials (2024)

[0211]

[0212] The demonstration results in Gedinggai Village, Wuchuan County, Hohhot, Inner Mongolia Autonomous Region in 2024 are shown below. Figure 27 According to Table 6, no disease was observed in the potatoes in the field. The A3 potato seed treatment agent was applied at a rate of 45 kg / hm². 2The marketable rate of the seed-treated potatoes was 91.77%, and the yield per mu was 4522.13 kg, while the yield of the blank control was 3935.25 kg, with a yield increase rate of 14.92%.

[0213] Table 6. Effects of A3 potato seed dressing agent on potato yield in Gedinggai Village during field plot trials (2024)

[0214]

[0215] In this invention, attapulgite and talc exhibit the best biocompatibility, while the number of spores is significantly reduced when diatomaceous earth is used as a carrier. Through screening dispersants and wetting agents, this invention preliminarily determined the seed treatment agent formula to be A3, which is prepared according to the following mass fractions: 8% mixed bacterial strain mother drug (mZF129 : mZF390 : mZF517 bacterial strain mother drug = 1:1:1), 3% dispersant NNO, 4% wetting agent NA, 0.1% Acid Red, and 84.9% talc.

[0216] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, contents, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some basic features can be applied within the scope of the following appended claims.

Claims

1. A compound microbial agent composition, characterized in that: The active ingredient in the compound microbial agent composition includes Bacillus polymyxa (Bacillus polymyxa). Paenibacillus polymyxa ZF129, Piperella salina ( Paenibacillus peoriae ZF390 and Bacillus subtilis ( Bacillus subtilis ZF517; The polymyxin Bacillus ( Paenibacillus polymyxa ZF129 has the CGMCC No. 17631 as its accession number at the China General Microbiological Culture Collection Center. The Pirribrium ( Paenibacillus peoriae ZF390 has the CGMCC No. 20322 as its accession number at the China General Microbiological Culture Collection Center. The Bacillus subtilis ( Bacillus subtilis ZF517 has the CGMCC No. 30137 as its preservation registration number at the China General Microbiological Culture Collection Center. Polymyxin Bacillus ( Paenibacillus polymyxa ZF129, Piperella salina ( Paenibacillus peoriae ZF390 and Bacillus subtilis ( Bacillus subtilis The quantity ratio of ZF517 is 1:1:1; The compound microbial agent composition is made by mixing the following raw materials in parts by weight: 4-10 parts by weight of mixed bacterial strain mother drug, 3 parts by weight of sodium methylene bis(naphthalene) sulfonate, 4 parts by weight of sodium lignin sulfonate, 0.1 parts by weight of Acid Red, and 82.9-88.9 parts by weight of talc. The mixed strain parent drug was composed of ZF129, ZF390, and ZF517 strain parent drugs according to the *Bacillus polymyxa* (… Paenibacillus polymyxa ZF129, Piperella salina ( Paenibacillus peoriae ZF390 and Bacillus subtilis ( Bacillus subtilis It is a mixture of ZF517 in a specific ratio; The parent drug for the ZF129 strain is Bacillus polymyxa (… Paenibacillus polymyxa The fermentation broth obtained from ZF129 fermentation is mixed with attapulgite soil at a mass ratio of 10%, and then repeatedly filtered until the filtrate is clear. The filter cake is collected, dried in the shade, and then crushed. The parent drug for the ZF390 strain is *Bacillus pilaris* (Pierreotype bacillus). Paenibacillus peoriae The fermentation broth obtained from ZF390 fermentation is mixed with attapulgite soil at a mass ratio of 10%, and then repeatedly filtered until the filtrate is clear. The filter cake is collected, dried in the shade, and then crushed. The parent drug for the ZF517 strain is Bacillus subtilis (… Bacillus subtilis The fermentation broth obtained from ZF517 fermentation is mixed with attapulgite soil at a mass ratio of 10%, and then repeatedly filtered until the filtrate is clear. The filter cake is collected, dried in the shade, and then crushed.

2. The compound microbial agent composition according to claim 1, characterized in that: The compound microbial agent composition is made by mixing the following raw materials in parts by weight: 8 parts by weight of mixed bacterial strain mother drug, 3 parts by weight of sodium methylene bis(naphthalene) sulfonate, 4 parts by weight of sodium lignin sulfonate, 0.1 parts by weight of Acid Red, and 84.9 parts by weight of talc.

3. A method for preparing the compound microbial agent composition according to any one of claims 1-2, characterized in that: The ZF129 strain, ZF390 strain, and ZF517 strain parent drugs were used according to the *Bacillus polymyxa* (… Paenibacillus polymyxa ZF129, Piperella salina ( Paenibacillus peoriae ZF390 and Bacillus subtilis ( Bacillus subtilis The mixed strain mother drug was obtained by mixing ZF517 in a specific ratio. The parent drug for the ZF129 strain is Bacillus polymyxa (… Paenibacillus polymyxa The fermentation broth obtained from ZF129 fermentation is mixed with attapulgite soil at a mass ratio of 10%, and then repeatedly filtered until the filtrate is clear. The filter cake is collected, dried in the shade, and then crushed. The parent drug for the ZF390 strain is *Bacillus pilaris* (Pierreotype bacillus). Paenibacillus peoriae The fermentation broth obtained from ZF390 fermentation is mixed with attapulgite soil at a mass ratio of 10%, and then repeatedly filtered until the filtrate is clear. The filter cake is collected, dried in the shade, and then crushed. The parent drug for the ZF517 strain is Bacillus subtilis (… Bacillus subtilis The fermentation broth obtained from ZF517 fermentation is mixed with attapulgite soil at a mass ratio of 10%, and then repeatedly filtered until the filtrate is clear. The filter cake is collected, dried in the shade, and then crushed. Sodium methylene bis(naphthalene) sulfonate, sodium lignin sulfonate, Acid Red, and talc were added to the mixed bacterial strain mother drug in parts by weight and mixed evenly to obtain the composite bacterial agent composition.

4. The preparation method according to claim 3, characterized in that: The air-drying process involves air drying at 35°C.

5. The application of the compound microbial agent composition according to any one of claims 1-2 or the preparation method according to claim 3 or 4 in the prevention and control of soil-borne diseases of potatoes; wherein the soil-borne diseases of potatoes are any one, two or three of potato soft rot, potato black scurf, and potato scab.

6. The application according to claim 5, characterized in that: The compound microbial agent composition is prepared at a concentration of 22.5 kg / hm². 2 -56.25kg / hm 2 Use the specified dosage for potato seed dressing.

Images