A multifunctional microbial agent and its application

Abstract

The present invention discloses a multifunctional microbial agent and its application. The microbial agent comprises synergistic Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Paecilomyces lilacinus E16, Trichoderma viride H06, a carrier and a synergist, and the agent has disease prevention and growth promotion effects on various plant diseases such as wilt, root knot nematode, heart rot, powdery mildew, anthrax, rust, bacterial wilt, ulcer and Huanglongbing. The present invention solves the problem of unstable field application of previous microbial agents due to unscientific defects in quality composition and application method, and has the advantages of being green, environmentally friendly and efficient.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease biological control and microbial fermentation, and particularly relates to a multifunctional microbial agent and its application. Background Art

[0002] Destructive soil-borne diseases such as crop wilt, bacterial wilt, root rot and root-knot nematode diseases caused by Fusarium oxysporum, Phytophthora nicotianae, Ralstonia solanacearum and Meloidogyne spp.; citrus Huanglongbing (HLB), caused by the Gram-negative bacterium Candidatus Liberibacter Asiaticus (CLas) that specifically parasitizes the phloem sieve tube tissue of citrus, is regarded as the "citrus cancer" and has seriously restricted the healthy development of the citrus industry. As well as rice blast, melon powdery mildew, mango anthracnose, mulberry red rust, citrus canker and other foliar diseases caused by Magnaporthe oryzae, Sphaerotheca fuliginea, Colletotrichum gloeosporioides, Aecidium mori and Xanthomonas axonopodis pv.citri, they have caused huge losses to my country's agricultural production. Traditional measures based on chemical control have not only failed to curb the rising trend of the above major diseases, but also suppressed beneficial microorganisms in the soil and plants, leading to drug resistance and resurgence of pathogens, while causing harmful substances to remain, resulting in a decline in the quality of agricultural products. The use of highly toxic and high-residue pesticides will also cause major safety issues for agricultural products.

[0003] There are many research papers and products on the market on the application of microbial agents to overcome soil-borne diseases, but the overall control effect is unstable and unsatisfactory. The reasons are mainly two aspects: (1) quality composition defects: poor synergy of functional strains, unreasonable ratio of adjuvants and carriers; (2) application technology defects, direct dilution and application according to traditional chemical fertilizers, ignoring the dormant state of effective bacteria in the microbial agents, and application without pre-cultivation, resulting in insufficient effective application of bacteria and active substances.

[0004] Although there is a paper "Shao Xuefeng. Prevention and control of soil-borne diseases Research and Application of Field Activation Technology of No. 41 Bacterial Fertilizer. 2020. Huazhong Agricultural University. "It was reported that the field activation technology of compound Bacillus fertilizer containing amino acids can increase its effective live bacteria by 13.8 times, the diameter of the inhibition zone by 70.7%, and the IAA content by 81.4%, and its antagonistic effect on banana wilt and tomato wilt pathogens. However, this technology still has defects: single dilution multiple (50 times), narrow temperature range (25-37℃), high cost (continuous ventilation of the oxygen pump for 36 hours); narrow antibacterial spectrum and single antagonistic mechanism (for two soil-borne fungal diseases, banana wilt and tomato wilt), which makes it susceptible to Contamination by foreign bacteria in nature inhibits the reproduction of functional bacteria in the product, thus affecting the application activity of the product; at the same time, the above-mentioned technology does not involve how microorganisms with different antagonistic mechanisms work together, and how to expand the antibacterial spectrum after compounding with carriers, etc., which not only controls the contamination of foreign bacteria after pre-culture or application, but also effectively prevents and treats a variety of soil-borne fungi, bacteria and root-knot nematode diseases, leaf fungi and bacterial diseases, systemic diseases citrus Huanglongbing and other key application technologies. Therefore, it is necessary to select a synergistic combination of excellent strains with different functions, supplement with reasonably proportioned carriers and enhancers, and develop multifunctional microbial agents with a broad antibacterial spectrum and high efficiency and scientific application technology. Summary of the invention

[0005] In view of the shortcomings of the existing technology, the present invention selects excellent biocontrol strains with complementary functions to construct a microbial flora, develops multifunctional microbial agents and scientific field application technologies, and provides effective technical support for the green and efficient prevention and control of plant diseases.

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

[0007] A multifunctional microbial agent comprises Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Paecilomyces lilacinus E16 and Trichoderma viride H06, wherein the effective viable bacteria count per gram of the multifunctional microbial agent are: Bacillus subtilis 1.2×10 9 -1.8×10 10 Bacillus amyloliquefaciens 1.2×10 9 -1.8×10 10 1.2×10 8 -1.8×10 9 Trichoderma viride 1.2×10 8 -1.8×10 9 indivual.

[0008] The four strains were provided by the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, and were all deposited in the China General Microbiological Culture Collection Center. The deposit number of the Bacillus subtilis BLG010 is CGMCC No.5953, and the deposit date is April 6, 2012; the deposit number of the Bacillus amyloliquefaciens HW05 is CGMCC No.10273, and the deposit date is January 4, 2015; the deposit number of the Paecilomyces lilacinus E16 is CGMCC No.5951, and the deposit date is April 6, 2012; the deposit number of the Trichoderma viride H06 is CGMCC No.6229, and the deposit date is June 15, 2012.

[0009] Preferably, the multifunctional microbial agent also includes a carrier; the carrier is amino acid chelated calcium magnesium zinc boron powder (total free amino acid content = 35.6%, chelated calcium content = 10.7%, chelated magnesium content = 3.1%, zinc content = 1.54%, boron content = 0.52%).

[0010] Furthermore, the present invention provides a method for preparing the multifunctional microbial agent, comprising the following steps:

[0011] (1) inoculating Bacillus subtilis BLG010 into 1 / 2LB liquid culture medium, culturing for 48-72 hours, and spray drying to obtain Bacillus subtilis powder;

[0012] (2) inoculating Bacillus amyloliquefaciens HW05 into 1 / 2LB liquid culture medium, culturing for 48-72 hours, and spray drying to obtain Bacillus amyloliquefaciens powder;

[0013] (3) inoculating Paecilomyces lilacinus E16 into a liquid fermentation medium and culturing for 48-72 hours, inoculating 20% ​​by mass into a double-layer gauze bag containing a solid fermentation medium, culturing for 5-7 days, drying for 24-48 hours, taking the fermentation product and shaking and sieving it to obtain Paecilomyces lilacinus powder;

[0014] (4) Inoculate green Trichoderma H06 into 1 / 2LB liquid culture medium and culture for 48-72 hours, inoculate into a double-layer gauze bag filled with solid fermentation medium at a mass ratio of 20%, culture for 5-7 days, dry for 24-48 hours, take the fermentation product and shake and sieve to obtain green Trichoderma powder;

[0015] (5) According to the weight ratio, 10-30 parts of Bacillus subtilis powder, 10-30 parts of Bacillus amyloliquefaciens powder, 1-5 parts of Paecilomyces lilacinus powder, and 1-5 parts of Trichoderma viride powder are taken, the powders are mixed to obtain a composite powder, and the composite powder is mixed with a carrier according to a mass ratio of 1:10-20 to obtain a multifunctional microbial agent.

[0016] Preferably, the 1 / 2LB liquid medium contains, by mass percentage, 0.1-0.5% sucrose or glucose, 0.5-1% tryptone, 0.1-0.5% yeast extract and 0.5-1% NaCl; the 1 / 2LB solid medium contains: 0.1-0.5% sucrose or glucose, 0.5-1% tryptone, 0.1-0.5% yeast extract, and 0.5-1% NaCl. 0.5-1% and agar 1.2-1.5%; the liquid fermentation medium of Paecilomyces lilacinus contains: 2% sucrose, 1% chitin and 0.001% manganese sulfate; the solid fermentation medium of Paecilomyces lilacinus E16 includes cassava residue: corn flour: coconut bran: chitosan: water in a mass ratio of 20-40:30-60:30-40:1-5:10-30; the solid fermentation medium of Trichoderma viride H06 includes cassava residue: corn flour: coconut bran: water in a mass ratio of 20-40:30-60:30-40:20-40.

[0017] On the other hand, the present invention provides the use of the multifunctional microbial agent in preventing and controlling plant diseases caused by pathogens, wherein the diseases include: wilt, root-knot nematodes, heart rot, bacterial wilt, rice blast, powdery mildew, rust, anthracnose, ulcer and Huanglongbing.

[0018] Preferably, the diseases include: banana wilt, banana root-knot nematodes, tomato root-knot nematodes, celery root-knot nematodes, pepper root-knot nematodes, pineapple heart rot, rice blast, melon powdery mildew, mango anthracnose, mulberry red rust, citrus canker, and citrus Huanglongbing.

[0019] Preferably, the pathogens include: Fusarium oxysporum, root-knot nematodes, Phytophthora nicotianae, Ralstonia solanacearum, Pyricularia grisea, Erysiphe gloeosporioides, Colletotrichum gloeosporioides, Puccinia mulariae, Bacillus subtilis, and Xanthomonas aeruginosa.

[0020] Preferably, the application method comprises the following steps:

[0021] (1) Preparing a pre-culture solution: The multifunctional microbial agent is applied at a dosage of 0.1-0.3 g / kg of soil for each potting application, and 1-10 kg / mu for each field application. After diluting 10-30 times with water according to the mass multiple, it is placed at an ambient temperature of 15-40° C. for 24-72 hours, allowed to stand or ventilated with a water pump or manually stirred once every 2-12 hours to obtain a pre-culture solution, which is then diluted 50-100 times, and a synergist is added at a mass ratio of 0.1-0.5% to obtain a pre-culture solution. The pre-culture solution is divided into two parts, 70%-90% of the volume of the pre-culture solution is sprayed on the roots, and 10%-30% of the volume of the pre-culture solution is applied to the leaves;

[0022] (2) Applying the pre-culture solution to the roots: After 4 p.m. on a sunny day or on a cloudy day, take the pre-culture solution in step (1), or the pre-culture solution to which 0.1% to 0.5% compound fertilizer is added according to the mass ratio and sprayed on the roots;

[0023] (3) Applying the pre-culture solution to the leaves: After 4:00 p.m. on a sunny day or on a cloudy day, take the pre-culture solution in step (1), or add 0.1% to 0.5% compound fertilizer and / or common foliar insecticide in a mass ratio and spray it on the front and back of the leaves.

[0024] This application method can increase the effective amount of bacteria and the content of active substances in the multifunctional microbial agent, overcome the hydrophobicity of the wax layer on the leaf surface and the soil adhesion, promote the rapid penetration and diffusion of the multifunctional microbial agent into the interior of the plant and the diseased parts after application, and improve the disease prevention and growth promotion effect after application.

[0025] Preferably, the synergist includes 10%-30% glycerol, 10%-70% Antrodia cinnamomea polysaccharide, 20%-80% methylcellulose, and 10%-20% sodium dodecylbenzene sulfonate by mass percentage. Its main function is to assist the effective bacteria in the multifunctional microbial agent to quickly attach, grow, spread and penetrate into the diseased parts of the plant body on the soil and plant surface, and remove the pathogens in the diseased parts.

[0026] Beneficial effects of the present invention:

[0027] Biological control has the advantages of being green and environmentally friendly, without any pesticide residues and key problems. Bacillus subtilis has a broad antimicrobial spectrum, antagonistic to a variety of soil-borne wilt, blight fungi and bacterial wilt bacteria, and has strong stress resistance; Bacillus amyloliquefaciens has a broad antimicrobial spectrum, antagonistic to systemic citrus Huanglongbing, a variety of soil-borne pathogenic fungal diseases (wilt, blight, etc.) and soil-borne bacterial diseases (bacterial wilt, etc.), leaf diseases (powdery mildew, ulcer, rust and anthracnose, etc.); Paecilomyces lilacinus is an excellent biocontrol bacterium for the prevention and control of root-knot nematodes, and has an inhibitory effect on wilt. Trichoderma's ability to antagonize soil-borne pathogens is not as good as that of antagonistic Bacillus, and its nutrient competition and persistence ability in the soil are longer, and its root-spherical growth-promoting effect is better. The four strains can be combined with carriers and enhancers to form a synergistic bacterial community application combination, and the effective live bacteria content in the bacterial agent can extend the shelf life. At the same time, it can supplement the key nutrients needed for field bacterial germination and secretion of active substances. It has a broad antibacterial spectrum and is highly effective. Pre-cultivation before field application can not only avoid contamination by foreign bacteria, but also quickly promote the transformation of the bacterial flora from a dormant structure to a germination state, greatly increasing the number of bacteria and the content of active substances. Combined with the integrated application of water and fertilizer, with the assistance of carriers and enhancers, it can quickly attach, grow, diffuse and penetrate into the diseased parts of the plant body on the soil and plant surface, eliminate pathogens in the diseased parts, and achieve the purpose of significantly increasing the disease prevention and growth promotion efficacy and stability in field applications.

[0028] The invention is simple to operate, highly efficient, and has a short cycle, with significant advantages. At the same time, the coordinated application of microbial agents and / or compound fertilizers and pesticides reduces the application cost, allows beneficial microorganisms to spread and reproduce in the soil and plants after application, and can effectively eliminate the content of pathogens in the soil and plants, greatly reduce the incidence of major diseases such as wilt, root-knot nematodes, bacterial wilt, heart rot, and citrus Huanglongbing, as well as leaf diseases such as rice blast, powdery mildew, canker, anthracnose, rust, and canker, and improve plant yield and quality. DETAILED DESCRIPTION

[0029] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] Bacillus subtilis BLG010 is deposited with CGMCC No.5953; Bacillus amyloliquefaciens HW05 is deposited with CGMCC No.10273; Paecilomyces lilacinus E16 is deposited with CGMCC No.5951; and Trichoderma viride H06 is deposited with CGMCC No.6229, which are provided by the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences. The above strains are all publicly available strains that can be obtained by the public from public channels.

[0033] Public literature on Bacillus amyloliquefaciens HW05: Wang Jun, Huang Junsheng, Liang Changcong, Zhou You, Liu Lei, Yang Laying, Guo Lijia. A highly efficient Bacillus amyloliquefaciens strain and its bacterial agent and application (ZL201810578016.9).

[0034] Public literature on Bacillus subtilis BLG010: Huang Junsheng, Liang Changcong, Zhu Lilin, Wang Guofen, Liu Lei. A Bacillus subtilis strain and its application (ZL201210248300.2).

[0035] Public literature on Paecilomyces lilacinus E16: Huang Junsheng, Wang Jun, Liang Changcong, Deng Guoping, Ren Wenbin, Guo Lijia. A strain of Paecilomyces lilacinus and its application (ZL201210358765.3).

[0036] Public literature on Trichoderma viride H06: Huang Junsheng, Liang Changcong, Yang Laying, Wu Lin, Wang Ya. A strain of Trichoderma viride and its application (ZL201210323738.2).

[0037] The pathogens such as Fusarium oxysporum, tobacco phytophthora, pepper phytophthora, Ralstonia solanacearum, root-knot nematodes, Pyricularia grisea, powdery mildew, Colletotrichum gloeosporioides, Puccinia mulberry, citrus canker, and Escherichia coli involved in the embodiments are all provided by the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, and can be obtained by conventional methods in the prior art.

[0038] The carrier described in the embodiment of the present invention: amino acid chelated calcium magnesium zinc boron powder (total amount of free amino acids = 35.6%, chelated calcium content = 10.7%, chelated magnesium content = 3.1%, zinc content = 1.54%, boron content = 0.52%), purchased from Sichuan Shihong Technology Co., Ltd.

[0039] The Fusarium selective culture medium in the embodiments of the present invention: see the literature (Jing Xiaohui, Wu Lunying, Qu Xiaoling, Zhu Lilin, Xue Yuxiao, Wu Lin, Huang Junsheng. A selective culture medium for simply isolating banana wilt pathogens [J]. Journal of Tropical Crops, 2009, 30(11): 1671-1673.).

[0040] The Bacillus subtilis BLG010 in the embodiment of the present invention is induced to have resistance to streptomycin sulfate by antibiotic labeling experiment, and can stably grow and maintain the activity of antagonizing pathogens such as Fusarium oxysporum on a medium such as 1 / 2LB containing 300 μg / mL of streptomycin sulfate. Due to its resistance to streptomycin sulfate, it can be used to detect the survival ability of BLG010 in the field.

[0041] The selective culture medium of Paecilomyces lilacinus E16 and the chitinase activity assay in the examples of the present invention: see the literature (Ren Wenbin, Jiang Guifang, Zhang Shiqing, Huang Junsheng. Effects of fermentation conditions on chitinase production by Paecilomyces lilacinus E7 strain [J]. Journal of Agro-Environment Science, 2006.25(3), 812-816.)

[0042] The selective culture medium of Trichoderma viride H06 in the embodiment of the present invention is red Bengal culture medium.

[0043] The selective culture medium of the control strain D34 in the embodiment of the present invention is provided in the literature (Cao Zhichun. Construction of a multiple screening system for banana wilt antagonistic Bacillus and research on its prevention effect [D]. Hainan University, 2016.)

[0044] For information about Bacillus amyloliquefaciens bwa7, see the literature: (Xue Yuxiao. Preliminary study on the colonization of Bacillus amyloliquefaciens bwa7 on banana and its biocontrol mechanism [D]. Hainan University, 2012).

[0045] Control bacterial agent in the embodiment of the present invention No. 41 bacterial fertilizer (amino acid 100g / L, organic matter ≥40%, total nutrients N+P2O5+K2O ≥12%, total effective live bacteria count: 50x108 cfu / g, of which Bacillus subtilis BLG010 viable count: 28×10 8 cfu / g, viable count of Bacillus amyloliquefaciens HC200: 22×10 8 cfu / g, powder. ) provided by the Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences. Public literature: (Shao Xuefeng. Prevention and control of soil-borne diseases Research and Application of Field Activation Technology of No. 41 Bacterial Fertilizer[D]. Huazhong Agricultural University, 2020).

[0046] The method for detecting the effective viable count of BLG010, HW05, H06, and E16 adopts the dilution plate method, referring to the literature (Li Fudi, Yu Ziniu, He Shaojiang. Agricultural Microbiology Experimental Technology [M]. Beijing: China Agriculture Press, 1996: 32-34, 305-306.).

[0047] The detection method of pineapple heart rot pathogen Phytophthora nicotianae in soil refers to the literature (Qin Haili. Isolation and identification of the pathogenic bacteria of Hainan golden pineapple heart rot and research on its biocontrol effect [D]. Haikou: Master's thesis of Hainan University, 2015).

[0048] References for chitinase activity and protease activity (Deng Guoping. Study on biological characteristics of Paecilomyces lilacinus E7 strain and its application in controlling plant root-knot nematodes [D]. Danzhou: Master's thesis of South China University of Tropical Agriculture, 2007).

[0049] Reference for analysis of lipopeptide active substances (Isolation and identification of endophytic antagonistic bacteria in mulberry and preliminary study on their biocontrol of mulberry leaf rot disease [J]. Acta Microbiologica Sinica. 2019, 59(11): 2130-2143).

[0050] The leaf sample processing method involved in the detection of endophytic colonization of antagonistic strains and control strain D34 in citrus mesophyll and veins can be found in the literature (Zhu Lilin. Screening of antagonistic Bacillus against banana wilt and study on its colonization characteristics [D]. Haikou: Hainan University, 2012).

[0051] The PCR detection method for citrus Huanglongbing refers to the literature (Comparative study of PCR detection primers for citrus Huanglongbing. Plant Quarantine. 2022, 36(02): 1-6).

[0052] Compound fertilizer refers to fertilizer with an effective content ≥ 45%, of which N, P2O and K2O contents are 15% each and are commercially available.

[0053] The 1 / 2LB liquid culture medium of the present invention contains: 0.1-0.5% sucrose or glucose, 0.5-1% tryptone, 0.1-0.5% yeast extract and 0.5-1% NaCl; the 1 / 2LB solid culture medium contains: 0.1-0.5% sucrose or glucose, 0.5-1% tryptone, 0.1-0.5% yeast extract, 0.5-1% NaCl and 1.2-1.5% agar; the 1 / 2LB liquid culture medium used in the embodiment contains: 0.5% sucrose or glucose, 0.5% tryptone, 0.5% yeast extract and 0.1% NaCl; the 1 / 2LB solid culture medium contains: 0.5% sucrose or glucose, 0.5% tryptone, 0.5% yeast extract, 0.1% NaCl and 1.5% agar.

[0054] The solid fermentation medium of Paecilomyces lilacinus E16 of the present invention comprises cassava residue: corn flour: coconut bran: chitosan: water in a mass ratio of 20-40:30-60:30-40:1-5:10-30; the solid fermentation medium of Trichoderma viride H06 comprises cassava residue: corn flour: coconut bran: water in a mass ratio of 20-40:30-60:30-40:20-40. The solid fermentation medium of Paecilomyces lilacinus E16 used in the embodiment comprises cassava residue: corn flour: coconut bran: chitosan: water in a mass ratio of 40:30:40:5:10; the solid fermentation medium of Trichoderma viride H06 comprises cassava residue: corn flour: coconut bran: water in a mass ratio of 40:30:30:20.

[0055] The synergist of the present invention (by mass percentage) includes 10%-30% glycerol, 10%-70% Antrodia cinnamomea polysaccharide, 20%-80% methylcellulose, and 10%-20% sodium dodecylbenzene sulfonate. The synergist ① used in the embodiment is 20% glycerol, 20% Antrodia cinnamomea polysaccharide, 65% methylcellulose, and 15% sodium dodecylbenzene sulfonate. Its main function is to assist the effective bacteria in the multifunctional microbial agent to quickly attach, grow, diffuse and penetrate into the plant body on the soil and plant surface. In the following specific embodiments, if not specifically marked, the synergist refers to synergist ①. Synergist ② is 20% glycerol, 20% chitosan, 65% methylcellulose, and 15% sodium dodecylbenzene sulfonate.

[0056] The Antrodia cinnamomea polysaccharide is a commercially available product with a polysaccharide content of 30%.

[0057] In the embodiment of the present invention, the dilution water is a clean water source such as clean water or tap water.

[0058] Example 1: Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Paecilomyces lilacinus E16, and Trichoderma viride H06 have good synergy and are compatible with carriers and synergists

[0059] Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Paecilomyces lilacinus E16, and Trichoderma viride H06 were selected, and Bacillus subtilis BSA-6, Bacillus amyloliquefaciens C10-1, Bacillus amyloliquefaciens bwa7, and Bacillus amyloliquefaciens D34 were used as control strains to prepare bacterial suspensions of each strain, and the concentration was adjusted to 1.2×10 8 / mL, confrontation culture to determine compatibility: prepare bacterial suspensions containing the above strains, prepare 1 / 2LB solid culture medium, pour into a 6cm diameter culture dish, and take 0.1g of different strains of bacterial suspensions, enhancers, and carriers according to the treatment in Table 1, dilute 1000 times, shake well, and pipette 5 microliters. Take the center of each culture dish as the midpoint, and inoculate 2 different test bacterial solutions at an equal distance of 0.5cm from both ends of the center of the circle in a straight line direction. Use equal volumes of clean water, carriers and enhancers as controls, and culture in a 28℃ incubator for 2-3d, and count the width of the inhibition zone between the two. Each experiment was repeated 3 times. The results are shown in Table 1.

[0060] Among them, - represents no inhibition; + represents weak antagonism, and the width of the inhibition zone is 1-5mm; ++ represents strong antagonism, and the diameter of the inhibition zone is 5-10mm; +++ represents strong antagonism, and the width of the inhibition zone is >10mm.

[0061] Table 1

[0062]

[0063]

[0064] The results showed that in the pairwise confrontation culture, Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Paecilomyces lilacinus E16, and Trichoderma viride H06 had no obvious antibacterial bands with each other, and there was no antibacterial band between the carrier and the synergist and all the strains, while the four control strains BSA-6, C10-1, bwa7 and D34 had antibacterial bands with each other, and also with Paecilomyces lilacinus E16, bwa7 had an antibacterial band with Bacillus subtilis BLG010 and Bacillus amyloliquefaciens HW05, and bwa7 and D34 had an antibacterial band with Trichoderma viride H06. In summary, Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Paecilomyces lilacinus E16, and Trichoderma viride H06 had good synergy and were also compatible with the synergist and carrier, indicating that this combination has the prospect of preparing multifunctional microbial agents.

[0065] Example 2: Preparation method of a multifunctional microbial agent

[0066] The composite microbial agent comprises Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Paecilomyces lilacinus E16, Trichoderma viride H06 and a carrier, and the specific preparation method is as follows:

[0067] (1) Preparation method of Bacillus amyloliquefaciens HW05 powder: inoculate Bacillus amyloliquefaciens in 1 / 2LB liquid culture medium, culture for 48 hours, and spray dry to obtain Bacillus amyloliquefaciens powder;

[0068] (2) A method for preparing Bacillus subtilis BLG010 powder: Bacillus subtilis is inoculated into 1 / 2LB liquid culture medium, cultured for 48 hours, and spray-dried to obtain Bacillus amyloliquefaciens powder;

[0069] (3) Preparation method of Paecilomyces lilacinus E16 powder: inoculate the Paecilomyces lilacinus E16 slant strain into the Paecilomyces lilacinus selective culture medium, culture it in a biochemical incubator at 28°C for 40 hours, and after a single colony grows, inoculate it into a liquid culture medium for 60 hours to obtain a seed solution, inoculate it into a double-layer gauze bag filled with a solid culture medium at a mass ratio of 20%, culture it for 5 days, dry it for 36 hours, take the fermentation product and shake and sieve it to obtain Paecilomyces lilacinus powder;

[0070] (4) Preparation method of green Trichoderma HW06 powder: inoculate the green Trichoderma H06 slant strain into a selective culture medium, culture it in a biochemical incubator at 28°C for 40 hours, and after a single colony grows, inoculate it into a solid liquid culture medium and culture it for 72 hours to obtain a seed solution, inoculate it into a double-layer gauze bag of a paper solid culture medium at a mass ratio of 20%, culture it for 5 days, dry it for 36 hours, take the fermentation product and shake and sieve it to obtain green Trichoderma powder.

[0071] According to the mass ratio, 20 parts of Bacillus subtilis, 20 parts of Bacillus amyloliquefaciens, 3 parts of Paecilomyces lilacinus and 3 parts of Trichoderma viride were uniformly mixed to obtain a composite microbial powder. The mass ratio of the composite microbial powder to the carrier was 1:15 to obtain a multifunctional microbial agent. The effective live bacteria count was measured once every month for 18 consecutive months.

[0072] After 18 months of storage, there was no significant change in the number of effective live bacteria. The number of effective live bacteria per gram of multifunctional microbial agent was: Bacillus subtilis 1.8×10 10 Bacillus amyloliquefaciens 1.5×10 10 1.6×10 9 Trichoderma viride 1.2×10 9 indivual.

[0073] Example 3: The multifunctional microbial agent prepared in Example 2 has a synergistic antagonistic effect on multiple pathogens

[0074] The method of Example 2 was used to prepare single strain powder and carrier, and single Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Paecilomyces lilacinus E16, Trichoderma viride H06 inoculum, D34 inoculum, and microbial inoculum of different single inoculum combinations were used as controls. Fertilizer No. 41, carrier, synergist and clean water were used as controls to determine the synergistic antagonistic effect of the multifunctional microbial agent on multiple pathogens. The specific treatments are shown in Table 2.

[0075] 1. Determination of the antagonistic effect of multifunctional microbial agents on various pathogens

[0076] 1 / 2LB solid culture medium containing suspensions of Fusarium oxysporum, Phytophthora capsici, Pyricularia gloeosporioides, Colletotrichum gloeosporioides, Xanthomonas gloeosporioides, and Ralstonia solanacearum was prepared respectively, poured into a 9 cm diameter culture dish, and pre-cultured at 28°C for 12 hours. According to the treatment in Table 2, 0.1 g of each multifunctional microbial agent, different combinations of control agents, enhancers, and carriers of the same mass were taken, diluted 1000 times with clean water, shaken well, and 1000 times dilution was obtained. 5 microliters were pipetted, and 6 test bacterial solutions were inoculated at equal distances with the center of each culture dish as the center of the circle and 2.5 cm from the center of the circle. Equal volumes of clean water, carriers, and enhancers were used as controls. The culture was carried out in a 28°C incubator for 3 days, and the inhibition zone was observed. Each experiment was repeated 3 times. The width of the inhibition zone between the edge of the test bacterial colony and the pathogen was measured (the results are shown in Table 2).

[0077] 2. Determination of the egg hatching inhibition rate of root-knot nematodes using multifunctional microbial agents

[0078] Take 1 mL of the root-knot nematode egg suspension with a concentration of 120 eggs / mL and dispense it into 2 mL centrifuge tubes. Then add 200 μL of the 1000-fold dilution of the multifunctional microbial agent, different combinations of control agents, enhancers and carriers prepared in step 1 into the centrifuge tubes.

[0079] The banana root-knot nematode egg suspension is composed of southern root-knot nematode (Meloidogyne incongnita) and Javan root-knot nematode (Meloidogyne javanica) in a ratio of 1:1.

[0080] Each experiment was repeated three times, and the egg hatching rate and egg hatching inhibition rate were calculated on the third day. The results are shown in Table 2.

[0081] Egg hatching rate (%) = [number of hatched larvae / (number of hatched larvae + number of unhatched eggs)] × 100

[0082] Egg hatching inhibition rate (%) = [(control egg hatching rate - treatment egg hatching rate) / control egg hatching rate] × 100

[0083] The results in Table 2 show that the multifunctional microbial agent has antagonistic activity against multiple pathogens of the above-mentioned pathogens, which is superior to the powder of a single strain. This shows that the microbial agent of the combination of BLG010+HW05+E16+H06 is superior to other combinations of agents, has a broad antibacterial spectrum, and has the potential to prepare multifunctional microbial agents.

[0084] Table 2

[0085]

[0086]

[0087] Example 4: Potted plant protection effect of the multifunctional microbial agent prepared in Example 2 on banana wilt

[0088] In this embodiment, the amount of soil for each treatment is divided into 20 flower pots, 3 kg / pot, and a total of 60 kg is required. With the carrier and clean water as the control, the multifunctional microbial agent and the control agent of Example 3 are calculated at the same dosage of 0.1 g / kg soil, and each treatment requires 6 g of agent, which is divided into pre-culture application and direct application without pre-culture; the same mass of carrier is taken, directly diluted 1000 times and applied according to the root and leaf application method of the multifunctional microbial agent, and spraying an equal amount of clean water is used as a control, and banana seedlings planted in disease-free soil are used as a blank control. A total of 37 treatments are shown in Table 3. The details are as follows:

[0089] Hainan Danzhou Banana Base Greenhouse, take disease-free soil, press 10 5 cfu / g soil concentration was added to banana wilt spore suspension, stirred evenly to obtain banana wilt disease soil, and 35 experimental groups were arranged, and the diseased soil was divided into plastic pots according to the mass of 3kg / pot. The blank group was also divided into plastic pots according to 3kg / pot, with 20 pots in each group, and 5-leaf Brazilian bananas were transplanted. Each experimental group had 20 plants, and repeated 3 times. The above experimental plants were placed in a greenhouse, with conventional water and fertilizer management, and the disease index and incidence rate were calculated after the control showed symptoms of the disease on the 34th day.

[0090] Pre-culture application:

[0091] (1) Preparing a pre-culture solution: The temperature during the pre-culture is a natural temperature of 20-28° C. Take 6 g of the multifunctional microbial agent, dilute it 10 times with clean water or tap water, place it in a container such as a plastic bucket for 48 hours, stir it manually once every 12 hours to obtain a pre-culture solution, dilute it 100 times, add a synergist at a mass ratio of 0.1%, and obtain a pre-culture solution; divide it into two parts according to the volume percentage according to the amount required for root application and foliar application, with 80% of the volume used for root spraying and 20% of the volume used for foliar application.

[0092] (2) Applying the pre-culture solution to the roots: After 4 p.m. on a sunny day or on a cloudy day, take the pre-culture solution in step (1), add 0.1% compound fertilizer, mix well, and spray evenly on the roots of each replicate;

[0093] (3) Applying the pre-culture solution to the leaves: After 4 p.m. on a sunny day or on a cloudy day, take the pre-culture solution prepared in step (1), add 0.1% compound fertilizer, mix well, and evenly spray the solution on the front and back of each replicate leaf.

[0094] Administration without pre-cultivation:

[0095] (1) Preparation of dilution: Take 1 kg of multifunctional microbial agent, dilute 1000 times, and add synergist at a ratio of 0.1%. Divide the required amount for root application and leaf application into two parts by volume percentage, with 80% of the volume used for root spraying and 20% of the volume used for leaf application.

[0096] (2) Spraying the diluted solution on the roots: After 4 p.m. on a sunny day or on a cloudy day, take the diluted solution in step (1), add 0.1% compound fertilizer, mix well, and spray evenly on the roots of each replicate;

[0097] (3) Applying the diluted solution to the leaves: After 4 p.m. on a sunny day or on a cloudy day, take the diluted solution in step (1), add 0.1% compound fertilizer, mix well, and spray evenly on the front and back of each replicate leaf.

[0098] The incidence of banana wilt is divided into the following five standards:

[0099] Level 0: The plants have no yellowing symptoms.

[0100] Level 1: The lower leaves of the plant show slight yellowing symptoms, the young leaves are intact, a small part of the roots are slightly brown, and the stems show water-soaked browning.

[0101] Level 2: The lower leaves of the plant show obvious symptoms of yellowing, but the young leaves are intact, the roots show browning, and the stems and pseudostems show water-soaked browning.

[0102] Level 3: The whole plant shows symptoms of yellowing and withering, the roots turn brown and rot, the stems and pseudostems turn brown in patches, and a few petioles turn reddish-brown.

[0103] Level 4: The whole plant shows symptoms of withering and death, and the roots are severely browned and rotten.

[0104] Disease index = 100 × ∑ (number of diseased plants at each level × number of representatives at that level) / (total number of plants surveyed × highest representative value)

[0105] Control effect = (control disease index - experimental disease index) / control disease index × 100%

[0106] Table 3

[0107]

[0108]

[0109] The results showed that the multifunctional microbial agent had a good disease prevention and growth promotion effect on banana Fusarium wilt. All indicators of the multifunctional microbial agent were better than those of a single strain agent or other control strain combination agents, indicating that the four strain groups of BLG010+HW05+E16+H06 had a synergistic effect on the prevention and control of banana Fusarium wilt. The same microbial agent had a better disease prevention and growth promotion effect after pre-cultivation than the treatment without pre-cultivation.

[0110] Example 5: Field control effect of the multifunctional microbial agent prepared in Example 2 on banana wilt

[0111] Take the same quality of multifunctional microbial agent and control agent The application method of No. 41 bacterial fertilizer and carrier is as described in Example 4.

[0112] In the banana planting area of ​​Banqiao Town, Dongfang City, Hainan Province, the incidence of banana wilt was over 90% before the experiment. The soil was evenly plowed before planting, with a depth of 0.5m. The concentration of Fusarium in the soil detected by selective culture medium reached 4.2×10 5 Each treatment used 2.5 kg / mu of microbial agent or carrier, which was applied 7 days before banana planting and every 30 days after planting for 8 consecutive times, totaling 20 kg / mu, and water and fertilizer were sprayed in an integrated manner. There were 6 treatments in total: (1) multifunctional microbial agent, pre-cultured; (2) multifunctional microbial agent, not pre-cultured; 3) No. 41 bacterial fertilizer, applied after pre-cultivation; (4) No. 41 bacterial fertilizer, sprayed without pre-culture; (5) carrier, directly diluted 1000 times and applied; (6) clean water control: spray the same amount of clean water. Conventional water and fertilizer management, investigation of disease index, disease prevention effect, plant variety, tree vigor, tree age and management level were all consistent. The results are shown in Table 4. Random arrangement, repeated 3 times, a total of 18 plots, each plot area of ​​1 mu. Ditches were opened between plots and plastic film was used for isolation. Banana planting density was 170 plants / mu, row spacing was 1.6m, plant spacing was 1.5m, and zigzag planting was used. All the test fertilizers for conventional fertilization were used as topdressing, and base fertilizer, other topdressing and field management were carried out uniformly according to local normal levels.

[0113] Morbidity rate: 300 days after banana transplanting, measure the morbidity rate, observe the growth of bananas, record the number of diseased plants, and calculate the cumulative morbidity rate and yield per mu. Morbidity rate (%) = number of diseased plants / (total number of plants) × 100%.

[0114] Table 4

[0115]

[0116] The results showed that the disease prevention and growth promotion effect of the multifunctional microbial agent applied after pre-cultivation under field conditions was better than that of the agent applied directly without cultivation, and was also better than the control agent applied by the same method.

[0117] Example 6: Potted plant protection effect of the multifunctional microbial agent prepared in Example 2 on banana root knot nematode disease

[0118] In this embodiment, the amount of soil for each treatment is divided into 50 flower pots, 5 kg / pot, and a total of 250 kg of soil is required. With the carrier and clean water as the control, the multifunctional microbial agent and the control agent of Example 3 are calculated at the same dosage of 0.1 g / kg soil, and each treatment requires 25 g of the agent, which is divided into pre-culture application and direct application without pre-culture; the same mass of carrier is taken, directly diluted 1000 times and applied according to the root and leaf application method of the multifunctional microbial agent, and spraying an equal amount of clean water is used as a control, and banana seedlings planted in disease-free soil are used as a blank control. A total of 37 treatments are shown in Table 5. The details are as follows:

[0119] Banana root-knot nematode-infected soil was collected from the banana planting area of ​​Baodao New Village, Danzhou City. The diseased roots were chopped and mixed with the diseased soil for later use. The insect population density reached 214 per 100g of soil. At the Puzi base in Danzhou, Hainan, each experimental group had 50 pots, each pot was filled with 5kg of diseased soil, and 5-leaf Brazilian bananas were transplanted, and repeated 3 times. The above experimental plants were placed in a greenhouse and managed with conventional water and fertilizer. After the control showed symptoms of the disease on the 42nd day, the disease index and prevention effect were calculated.

[0120] The incidence of banana root-knot nematodes is statistically divided into the following five standards:

[0121] Level 0: No root knots or egg cysts

[0122] Level 1: 1-10 root knots or egg cysts / plant

[0123] Level 2: 10-20 root knots or egg cysts / plant

[0124] Level 3: 20-40 root knots or egg cysts / plant

[0125] Level 4: More than 40 root knots or oocysts / plant

[0126] Disease index = 100 × ∑ (number of diseased plants at each level × number of representatives at that level) / (total number of plants surveyed × highest representative value)

[0127] Control effect = (control disease index - experimental disease index) / control disease index × 100%

[0128] The results in Table 5 show that the multifunctional microbial agent has a good disease prevention and growth-promoting effect on banana root-knot nematodes. All indicators of the multifunctional microbial agent are better than those of a single strain agent or other control strain combination agents, indicating that the four-strain combination of BLG010+HW05+E16+H06 in the multifunctional microbial agent has a synergistic effect on the prevention and control of banana wilt. The same agent, applied after pre-cultivation, has a better disease prevention effect than the treatment of direct application without pre-cultivation.

[0129] Table 5

[0130]

[0131]

[0132] Example 7: Field control effect of the multifunctional microbial agent of Example 2 on root-knot nematodes of four plants

[0133] The method for applying the multifunctional microbial agent is similar to that of Example 4, and the commercially available chemical agent 10% thiazolyl granules is used as a comparative agent.

[0134] In the banana, tomato, pepper and celery planting areas with a root-knot nematode incidence rate of more than 80% in Dongfang City, Hainan Province, the multifunctional microbial agent was used at a dosage of 2.5 kg / mu each time, of which bananas were applied 7 days before planting and once every 30 days after planting, for 8 consecutive times, for a total of 20 kg / mu; tomatoes, peppers and celery were applied 3 times at planting, 15 days and 30 days after planting, each time 2.5 kg / mu, for a total of 7.5 kg / mu, and water and fertilizer were sprayed in an integrated manner, and pre-cultivation application and direct application without pre-cultivation were referred to the method of Example 4. The dosage of the comparative agent 10% thiathiazolyl granules was 1.5 kg / mu, and applied once before planting. Four experimental treatments were designed for banana, tomato, pepper and celery: (1) multifunctional microbial agent, applied after pre-cultivation; (2) multifunctional microbial agent, applied without pre-cultivation; (3) 10% thiazolyl granules 1.5 kg / mu, applied once before planting, the granules and fine soil were mixed evenly (1.5 kg 10% thiazolyl granules mixed with 15 kg fine soil), evenly spread on the ground surface, and then use a rotary tiller or hand tools to fully mix the agent and soil; (4) clear water control: spray the same amount of clear water. Randomly arranged, repeated 3 times, a total of 12 plots, each plot area of ​​1 mu. Ditches were opened between plots, and plastic film was used for isolation. Conventional water and fertilizer management, refer to the sixth example method to investigate the disease index, disease prevention effect, plant variety, tree vigor, tree age and management level, etc. The yield increase rate was investigated and the prevention effect was calculated in accordance with the example.

[0135] The results in Table 5 show that the prevention and growth promotion effects of the multifunctional microbial agent after pre-culture are better than those of direct application, and are comparable to those of the chemical agent 10% thiazolyl granules.

[0136] Table 6

[0137]

[0138] Example 8: The multifunctional microbial agent prepared in Example 2 is effective in preventing pineapple heart rot in potted plants

[0139] Take the same quality of multifunctional microbial agent and control agent No. 41 bacterial fertilizer and carrier, the application method is as in Example 4. In this example, the amount of soil for each treatment is divided into 30 flower pots, the amount of soil is 5 kg / pot, and a total of 150 kg is required. The multifunctional microbial agent and the control agent are calculated at the same amount of 0.1 g / kg soil. Each treatment requires 15 g of the agent, which is divided into pre-culture application and direct application without pre-culture; take the same mass of carrier, directly dilute 1000 times and apply it according to the root and leaf application method of the multifunctional microbial agent, and spray an equal amount of clean water as a control.

[0140] The soil from a plot of pineapple planted in Lingao, Hainan for three consecutive years was collected and the pathogen content was ≥5×10 3 CFU / g, the experimental design had 6 treatments: (1) multifunctional microbial agent, applied after pre-culture; (2) multifunctional microbial agent, applied without pre-culture; 3) No. 41 bacterial fertilizer, applied after pre-cultivation; (4) No. 41 bacterial fertilizer, applied without pre-culture; (5) Carrier, directly diluted 1000 times and applied; (6) Clear water control: spray the same amount of clear water. Each treatment was repeated 30 times, and each plastic pot was filled with 5.0 kg of soil. Before potting, organic fertilizer was applied at 1% (mass fraction) of the soil weight, and the difference in nitrogen, phosphorus and potassium content between chemical fertilizer treatments was supplemented with chemical fertilizer. Seedlings were planted 5 days after potting, with one plant planted in each pot. The fertilizer effect was kept equal throughout the growth period, and conventional water and fertilizer management was adopted.

[0141] The pineapple variety tested was "Golden Pineapple MD-2". After the pineapple seedlings were planted, the number of diseased plants was observed and recorded every day. Pineapple plants with yellow leaves, short stature, and leaves that were easy to fall off were counted as diseased plants. After 90 days of the test, the incidence rate was calculated.

[0142] Incidence rate (%) = 100 × number of infected plants / total number of planted pineapple plants

[0143] Biocontrol effect (%) = 100 × (CK incidence rate - treatment incidence rate) / CK incidence rate

[0144] The number of pineapple leaves was recorded 140 days after pineapple planting. The longest leaf length (pineapple leaf length) and width of the pineapple plant leaves when they were bundled were measured with a tape measure, and the pathogen content was detected using a selective culture medium.

[0145] The results in Table 7 show that the multifunctional microbial agent has better control effect and growth promotion effect after pre-culture than direct application, and is also better than the same method of application. No. 41 bacterial fertilizer.

[0146] Table 7

[0147]

[0148] Example 9: Field control effect of the multifunctional microbial agent prepared in Example 2 on melon powdery mildew

[0149] Take the same mass of multifunctional microbial agent and control agent The application method of No. 41 bacterial fertilizer and carrier is as described in Example 4.

[0150] The experiment was conducted in a greenhouse micro-plot in Sanya, Hainan. The dosage of the microbial agent or carrier used in each treatment was 2.5 kg / mu each time. The melon plants at the early stage of disease were sprayed, and then sprayed once again after 7 days, for a total of 5 kg / mu. The experimental design included 6 treatments: (1) multifunctional microbial agent, applied after pre-culture; (2) multifunctional microbial agent, applied without pre-culture; (3) No. 41 bacterial fertilizer, applied after pre-cultivation; (4) No. 41 bacterial fertilizer, applied without pre-culture; (5) Carrier, directly diluted 1000 times and applied; (6) Clear water control: spray the same amount of clear water. Each treatment was 1 mu, repeated 3 times. Placed in a greenhouse, conventional water and fertilizer management, the disease index, disease prevention effect, plant height, and sugar content were investigated after 7 days. The results are shown in Table 8.

[0151] The incidence of melon powdery mildew is divided into the following five standards

[0152] Level 0: no disease;

[0153] Level 1: The lesion area accounts for less than 20% of the entire leaf area;

[0154] Level 2: The lesion area accounts for 20-50% of the entire leaf area;

[0155] Level 3: The lesion area accounts for 50%-70% of the entire leaf area;

[0156] Level 4: The lesion area accounts for more than 70% of the entire leaf area.

[0157] Disease index = 100 × ∑ (number of diseased plants at each level × number of representatives at that level) / (total number of plants surveyed × highest representative value)

[0158] Control effect = (control disease index - experimental disease index) / control disease index × 100%

[0159] Table 8

[0160]

[0161] The results showed that the multifunctional microbial agent had better protective effect and growth promotion after pre-culture than direct application, and was also better than the control agent. No. 41 bacterial fertilizer.

[0162] Example 10: Field control effect of the multifunctional microbial agent prepared in Example 2 on mango anthracnose

[0163] Take the same mass of multifunctional microbial agent and control agent The application method of No. 41 bacterial fertilizer and carrier is as described in Example 4.

[0164] Mango trees at the early stage of anthracnose disease were selected from a mango planting area in Sanya, Hainan. The dosage of the microbial agent or carrier used in each treatment was 2.5 kg / mu. Plants at the early stage of disease were sprayed, and then sprayed again 7 days later, for a total of 5 kg / mu. The experimental design included 6 treatments: (1) 2.5 kg of multifunctional microbial agent, applied after pre-culture; (2) multifunctional microbial agent, applied without pre-culture; (3) No. 41 bacterial fertilizer, applied after pre-cultivation; (4) No. 41 bacterial fertilizer, applied without pre-culture; (5) Carrier, directly diluted 1000 times, applied; (6) Clean water control: spray the same amount of clean water. Each treatment is 1 mu, repeated 3 times. After 7 days of the above treatment, the incidence rate was investigated and the control effect was calculated according to the method of Example 9. The results are shown in Table 9.

[0165] Table 9

[0166]

[0167] The results showed that after 7 days of application of the multifunctional microbial agent, the lesions of mango anthracnose basically disappeared, with less recurrence, and the prevention efficiency reached 90.16%. This shows that the prevention effect and growth promotion of the multifunctional microbial agent after pre-culture are better than those of direct application, and are also better than those of the same method of application. No. 41 bacterial fertilizer.

[0168] Example 11: Field control effect of the multifunctional microbial agent prepared in Example 2 on mulberry red rust

[0169] Take the same mass of multifunctional microbial agent and control agent The application method of No. 41 bacterial fertilizer and carrier is as described in Example 4.

[0170] In the mulberry planting area in Qiongzhong, Hainan, the dosage of the microbial agent or carrier used in each treatment was 2.5 kg / mu each time. Mulberry trees were sprayed at the early stage of red rust. Seven days after spraying, the agent was sprayed again, for a total of 5 kg / mu. The experimental design included 6 treatments: (1) multifunctional microbial agent, applied after pre-culture; (2) multifunctional microbial agent, applied without pre-culture; (3) No. 41 bacterial fertilizer, applied after pre-cultivation; (4) No. 41 bacterial fertilizer, applied without pre-culture; (5) The carrier was directly diluted 1000 times and applied; (6) Clean water control: spray the same amount of clean water. Each treatment was 1 mu, repeated 3 times. After 10 days of the above treatment, the incidence rate was investigated and the control effect was calculated according to the method of Example 9. The results are shown in Table 10.

[0171] Table 10

[0172]

[0173] The results showed that 10 days after the application of the multifunctional microbial agent, the lesions of mango anthracnose basically disappeared and recurrence was rare, indicating that the multifunctional microbial agent had better prevention and growth promotion effects after pre-culture than direct application, with a prevention effect of 88.27%. No. 41 bacterial fertilizer.

[0174] Example 12: The prevention and control effect of the multifunctional microbial agent prepared in Example 2 on citrus Huanglongbing

[0175] Take the same mass of multifunctional microbial agent and control agent The application method of No. 41 bacterial fertilizer and carrier is the same as that of Example 4.

[0176] Citrus fruits (Qiongzhong green orange) that had been infected for 6 years were selected from the citrus planting area in Qiongzhong, Hainan. Fertilization began at the spring shoot germination stage, and then was applied once every month. The amount of microbial agent or carrier applied in each treatment was 5 kg / mu each time, and was applied 6 times in succession, totaling 30 kg / mu. Water and fertilizer were sprayed in an integrated manner, divided into 6 treatments: (1) multifunctional microbial agent, applied after pre-cultivation; (2) multifunctional microbial agent, applied without pre-cultivation; (3) No. 41 bacterial fertilizer, pre-culture application; (4) No. 41 bacterial fertilizer, applied without pre-culture; (5) Carrier, directly diluted 1000 times, reference; (6) Clean water control: applied according to the method of treatment 4, spraying an equal amount of clean water. Each treatment was 1 mu, repeated 3 times. Conventional water and fertilizer management. Regularly collect plant branch and leaf samples, select 5 citrus plants in the 5 directions of east, west, south, north and center for each treatment, randomly collect 1 leaf sample from each citrus plant in the 5 directions of east, west, south, north and center, mix them, and determine the starch content of the leaves. After DNA is extracted from the collected samples, the PCR method is used to determine the Huanglongbing bacteria carrying status of the plants; collect fruits in the same way during the harvest period to determine the fruit yield and juice yield.

[0177] Table 11

[0178]

[0179] Table 12

[0180]

[0181] Table 13

[0182]

[0183] The results showed that compared with the control, the application of multifunctional microbial agents reduced the starch content in diseased leaves, eliminated pathogens in the diseased parts, and increased yield, juice yield and sugar content. This shows that the multifunctional microbial agents had better prevention and growth promotion effects after pre-culture than those directly applied, and were also better than those applied by the same method. No. 41 bacterial fertilizer.

[0184] Example 13: Field control effect of the multifunctional microbial agent prepared in Example 2 on citrus canker

[0185] Take the same mass of multifunctional microbial agent and control agent The application method of No. 41 bacterial fertilizer and carrier is as described in Example 4.

[0186] In the citrus planting area in Qiongzhong, Hainan, the dosage of the microbial agent or carrier used in each treatment was 5 kg / mu each time. It was sprayed at the early stage of citrus canker disease, and then applied once after an interval of 1 month for 5 consecutive times, totaling 25 kg / mu, and sprayed with integrated water and fertilizer. After that, it was applied once every 1 month for 6 consecutive times, each time 5 kg / mu, totaling 30 kg / mu, and sprayed with integrated water and fertilizer. There were 6 treatments: (1) multifunctional microbial agent, applied after pre-cultivation; (2) multifunctional microbial agent, applied without pre-cultivation; (3) No. 41 bacterial fertilizer, pre-culture application; (4) No. 41 bacterial fertilizer, applied without pre-culture; (5) Carrier, directly diluted 1000 times and applied; (6) Clean water control: spray the same amount of clean water. Each treatment was 1 mu, repeated 3 times. After 30 days of treatment, the disease situation of the plants was observed and recorded, and the control effect was statistically analyzed.

[0187] The disease index and prevention and treatment effect are calculated according to the following grading standards:

[0188] Level 0: disease-free; Level 1: 1-5 disease spots on each leaf; Level 3: 6-10 disease spots on each leaf; Level 5: 11-15 disease spots on each leaf; Level 7: 16-20 disease spots on each leaf; Level 9: more than 21 disease spots on each leaf.

[0189] Disease index = ∑ [number of diseased leaves at each level × relative level] / [total number of leaves surveyed × highest relative level] × 100

[0190] Control effect (%) = [1-(disease index after treatment in treatment area - disease index before treatment in treatment area) / (disease index after treatment in control area - disease index before treatment in control area)] × 100

[0191] Table 13

[0192]

[0193]

[0194] The results showed that the multifunctional microbial agent had a control effect of 88.09%. No. 41 bacterial fertilizer.

[0195] Example 14: Field control effect of the multifunctional microbial agent of Example 2 on tomato bacterial wilt

[0196] Take the same mass of multifunctional microbial agent and control agent The application method of No. 41 bacterial fertilizer and carrier is as described in Example 4.

[0197] In a tomato planting area in Qionghai, Hainan, the incidence of bacterial wilt was over 90% after three consecutive years of planting. The dosage of the microbial agent or carrier used in each treatment was 2.5 kg / mu. The multifunctional microbial agent was applied when the tomato was established, and then applied once every 20 days for three consecutive times, a total of 7.5 kg / mu. Water and fertilizer were sprayed in an integrated manner. There were six treatments: (1) multifunctional microbial agent, applied after pre-cultivation; (2) multifunctional microbial agent, applied without pre-cultivation; (3) No. 41 bacterial fertilizer, pre-culture application; (4) No. 41 bacterial fertilizer, applied without pre-culture; (5) Carrier, directly diluted 1000 times and applied; (6) Clear water control, sprayed with the same amount of clear water. Each treatment was 1 mu, repeated 3 times. After 30 days of treatment, the disease status of the plants was observed and recorded, the soil nutrient content was measured, and the control effect was statistically analyzed.

[0198] Table 14

[0199]

[0200] The results showed that the application of multifunctional microbial agents had a control effect of 81.59% and improved soil nutrient indicators, which was better than the control agent applied in the same way. No. 41 bacterial fertilizer.

[0201] Example 15: Effects of root application and leaf application on the field control of banana wilt by the multifunctional microbial agent of Example 2

[0202] In this example, clean water is used as a control, and the same mass of the multifunctional microbial agent and the control agent D34 prepared in Example 3 are taken, and the application method is referred to Example 4.

[0203] In the banana planting area of ​​Banqiao Town, Dongfang City, Hainan Province, the incidence of banana wilt was over 90% before the experiment. The soil was evenly plowed before planting, with a depth of 0.5m. The concentration of Fusarium in the soil detected by selective culture medium reached 4.2×10 4 Each treatment used 2.5 kg / mu of microbial agents or carriers, 7 days before banana planting and every 30 days after planting, for 8 consecutive times, totaling 20 kg / mu, and spraying water and fertilizer in an integrated manner, with a total of 7 treatments: (1) application after pre-cultivation of multifunctional microbial agents; (2) application to roots after pre-cultivation of multifunctional microbial agents; (3) application to leaves after pre-cultivation of multifunctional microbial agents; (4) application after pre-cultivation of D34 agents; (5) application to roots after pre-cultivation of D34 agents; (6) application to leaves after pre-cultivation of D34 agents; (7) clear water control: spraying the same amount of clear water. Conventional water and fertilizer management, investigation of disease index, disease prevention effect, plant varieties, tree vigor, tree age and management level were all the same. Random arrangement, repeated 3 times, a total of 21 plots, each plot area of ​​1 mu. Ditches were opened between plots, and plastic film was used for isolation. Banana planting density was 170 plants / mu, row spacing was 1.6m, plant spacing was 1.5m, and zigzag planting was used. All the fertilizers tested in conventional fertilization were used as topdressing, and base fertilizer, other topdressing and field management were carried out uniformly according to the local normal level.

[0204] 300 days after banana transplanting, the banana wilt pathogens in the banana roots, the colonization of BLG010, HW05, E16, H06, and D34 in the banana roots and leaves, the incidence rate, the banana growth, the number of diseased plants, and the cumulative incidence rate and yield per mu were calculated. Incidence rate (%) = number of diseased plants / (total number of plants) × 100%.

[0205] The results in Tables 15, 16 and 17 show that simultaneous root and leaf application increased the colonization of BLG010, HW05, E16, H06 and the control strains in the roots and leaves, and reduced the content of pathogens, which was significantly better than single root or leaf application. This shows that leaf application can promote the penetration of effective strains BLG010 and HW05 from the leaves to the roots.

[0206] Table 15

[0207]

[0208] Table 16

[0209]

[0210]

[0211] Table 17

[0212]

[0213] Example 16: Effects of carriers and synergists on the multifunctional microbial agent of Example 2 in field control of banana wilt

[0214] Take the same mass of the multifunctional microbial agent and the control agent D34 prepared in Example 3, the multifunctional microbial agent without a synergist and a carrier, the carrier and the synergist, and refer to Example 4 for the application method.

[0215] In the banana planting area of ​​Banqiao Town, Dongfang City, Hainan Province, the incidence of banana wilt was over 90% before the experiment. The soil was evenly plowed before planting, with a depth of 0.5m. The concentration of Fusarium in the soil detected by selective culture medium reached 4.2×10 4Each treatment used 2.5 kg / mu of microbial agents, carriers or synergists, 7 days before banana planting and every 30 days after planting, for 8 consecutive times, a total of 20 kg / mu, water and fertilizer were sprayed in an integrated manner, with a total of 13 treatments: (1) pre-cultured application of multifunctional microbial agents (including carriers and synergists ①); (2) pre-cultured application of multifunctional microbial agents (including carriers and synergists ②); (3) pre-cultured application of multifunctional microbial agents (without carriers); (4) Multifunctional microbial agent (without synergist) pre-cultured and applied; (5) Multifunctional microbial powder (without carrier and synergist) pre-cultured and applied; (6) D34 agent pre-cultured and applied; (7) D34 (without carrier) pre-cultured and applied; (8) D34 (without synergist) pre-cultured and applied; (9) D34 powder (without carrier and synergist) pre-cultured and applied; (10) carrier; (12) synergist ①; (12) synergist ②; (13) Water control: spray the same amount of water. Conventional water and fertilizer management, investigation of disease index, disease prevention effect, plant varieties, tree vigor, tree age and management level were all consistent. Random arrangement, repeated 3 times, a total of 39 plots, each plot area of ​​1 mu. Ditches were opened between plots and plastic film was used for isolation. Banana planting density was 170 plants / mu, row spacing was 1.6m, plant spacing was 1.5m, and zigzag planting was used. All the test fertilizers for conventional fertilization were used as top dressing, and base fertilizer, other top dressing and field management were carried out uniformly according to local normal levels.

[0216] 300 days after banana transplanting, the number of banana wilt pathogens in the banana roots, the number of BLG010, HW05, E16, H06, and D34 colonization in the banana roots and leaves, the incidence rate, the growth of bananas, the number of diseased plants, and the cumulative incidence rate and yield per mu were calculated. Incidence rate (%) = number of diseased plants / (total number of plants) × 100%.

[0217] The results in Tables 18 and 19 show that the carrier and synergist increased the colonization of BLG010, HW05, E16, H06 and the control strains in the roots and leaves, and reduced the content of pathogens, which was significantly better than the single bacterial powder, and also significantly better than the multifunctional microbial agent containing only carriers or synergists and the control agent. This shows that the synergist can promote the effective strains BLG010, HW05, E16, and H06 to diffuse, penetrate and conduct from the leaves and soil to the leaves and roots respectively. In addition, the effect of synergist ① is significantly better than that of synergist ②.

[0218] Table 18

[0219]

[0220] Table 19

[0221]

[0222]

[0223] Table 20

[0224]

[0225] Example 17: Effect of application on the front and back of leaves on the multifunctional microbial agent prepared in Example 2 for controlling citrus Huanglongbing

[0226] The same mass of multifunctional microbial agent and Example 3 were used to prepare the control agent D34. In the citrus planting area in Qiongzhong, Hainan, the amount of the agent used in each treatment was 5 kg / mu each time. It was sprayed at the early stage of citrus Huanglongbing disease, and water and fertilizer were sprayed in an integrated manner. After that, it was applied once every month for 6 consecutive times, 5 kg / mu each time, a total of 30 kg / mu, divided into 7 treatments: (1) multifunctional microbial agent, applied on the front and back of the leaves; (2) multifunctional microbial agent (applied on the front of the leaves); (3) multifunctional microbial agent (applied on the back of the leaves); (4) D34 agent, applied on the front and back of the leaves; (5) D34 agent, applied on the front of the leaves); (6) D34 agent, applied on the back of the leaves; (7) clear water control: spray the same amount of clear water. Each treatment was 1 mu, and repeated 3 times. After 30 days of treatment, the disease conditions of the plants were observed and recorded. The leaf sample collection method was referred to Example 15. PCR was used to detect the presence of Huanglongbing pathogens on the leaves. Selective culture medium was used to determine the colonization of antagonistic bacteria on the leaf surface, mesophyll, and veins.

[0227] The results showed that spraying the fungicide on both the front and back of leaves significantly reduced the content of pathogens (Table 21) and increased the number of antagonistic bacteria BLG010, HW05, E16, and H06 colonizing the veins and mesophyll (Tables 22-23), which was significantly better than the colonization effect of spraying the front or back of leaves alone, and improved the movement and colonization of antagonistic bacteria in citrus.

[0228] Table 21

[0229]

[0230] Table 22

[0231]

[0232] Table 23

[0233]

[0234] Example 18: Effects of pre-culture and no pre-culture on the bacterial count and activity of the multifunctional microbial agent prepared in Example 2

[0235] Effect of temperature on pre-cultivation of multifunctional microbial agents under simulated field natural conditions:

[0236] (1) Preparation of pre-culture dilution: 1 kg of multifunctional microbial agent was diluted 10 times and then divided into 4 plastic barrels, which were placed in 4 biochemical incubators at different temperatures for 48 hours, with manual stirring once every 6 hours to obtain pre-culture solution, which was then diluted 100 times to obtain a pre-culture dilution solution with a final concentration of 1000 times.

[0237] (2) Preparation of non-pre-cultured dilution solution: Take 1 g of the multifunctional microbial agent and directly dilute it to 1000 times to obtain a non-pre-cultured dilution solution.

[0238] (3) Index determination: The effective viable counts of 1000-fold dilutions of the multifunctional microbial agents of BLG010, HW05, E16, and H06 at different temperatures were determined using selective culture medium; the antagonistic effect of the pre-culture dilutions on different pathogens was determined using the non-pre-cultured dilutions as a control according to the method in Example 3; 2 mL of the pre-cultured dilutions was placed in a centrifuge tube, centrifuged at 8000 r / min for 5 minutes, and then filtered with a 0.22 μm microporous filter membrane to obtain the sterile metabolites of the pre-cultured dilutions, and the sterile metabolites of the non-pre-cultured dilutions were obtained in the same manner. The sterile metabolites of the non-pre-cultured dilutions were used as a control to determine the inhibitory effect of the sterile metabolites of the pre-cultured dilutions on different pathogens; the 10-fold dilutions of the agents were taken and the content of active substances was determined according to the method in the literature.

[0239] The results show that, as shown in Table 24, in the temperature range of 15-40°C, the effective bacterial count of antagonistic bacteria in the pre-cultured multifunctional microbial agent dilution before application is more than 100 times higher than that in the non-pre-cultured dilution;

[0240] As shown in Table 25, in the temperature range of 15-40°C, the pre-cultured dilution of the multifunctional microbial agent before application has a more significant effect on inhibiting pathogenic fungi, bacteria and root-knot nematodes than the non-pre-cultured dilution, and can also significantly inhibit the common pathogenic bacteria Escherichia coli in the environment;

[0241] It can be seen from Table 26 that in the temperature range of 15-40°C, the sterile metabolites of the pre-cultured dilution before application of the multifunctional microbial agent have a more significant effect on inhibiting pathogenic fungi, bacteria and root-knot nematodes than the sterile metabolites of the non-pre-cultured dilution, and can also significantly inhibit the common pathogenic bacteria Escherichia coli in the environment.

[0242] It can be seen from Table 27 that in the temperature range of 15-40°C, the sterile bacterial metabolites in the non-precultured multifunctional microbial agent dilution were not detected, while the sterile metabolites in the precultured multifunctional microbial agent dilution contained active substances lipopeptides Iturin A and Bacillomycin L, deacetylchitinase, protease and chitinase.

[0243] In summary, it is directly proved that the pre-culture technology before application can improve the reproduction of effective bacteria and the secretion of active substances in multifunctional microbial agents, which helps to avoid contamination by miscellaneous bacteria in the environment during the application process, thereby significantly improving the antagonistic effect on various types of diseases such as pathogenic fungi, bacteria and root-knot nematodes.

[0244] Table 24

[0245]

[0246] Table 25

[0247]

[0248]

[0249] Table 26

[0250]

[0251] Table 27

[0252]

[0253] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. A multifunctional microbial agent, characterized in that: The microorganisms are composed of Bacillus subtilis BLG010, Bacillus amyloliquefaciens HW05, Paecilomyces lilacinus E16 and Trichoderma viride H06, and the effective live bacteria counts per gram of the multifunctional microbial agent are: Bacillus subtilis 1.2×10 9 ~1.8×10 10 Bacillus amyloliquefaciens 1.2×10 9 ~1.8×10 10 1.2×10 8 ~1.8×10 9 Trichoderma viride 1.2×10 8 ~1.8×10 9 indivual; The four strains are all deposited in the China General Microbiological Culture Collection Center. The deposit number of the Bacillus subtilis BLG010 is CGMCC No.5953, and the deposit date is April 6, 2012; the deposit number of the Bacillus amyloliquefaciens HW05 is CGMCC No.10273, and the deposit date is January 4, 2015; the deposit number of the Paecilomyces lilacinus E16 is CGMCC No.5951, and the deposit date is April 6, 2012; and the deposit number of the Trichoderma viride H06 is CGMCC No.6229, and the deposit date is June 15, 2012.

2. The multifunctional microbial agent according to claim 1, characterized in that: It also includes a carrier; the carrier is amino acid chelated calcium magnesium zinc boron powder.

3. The method for preparing the multifunctional microbial agent according to claim 2, characterized in that: The following steps are involved: (1) Inoculate Bacillus subtilis BLG010 into 1 / 2 LB liquid culture medium and culture for 48-72 hours, and spray dry to obtain Bacillus subtilis powder; (2) inoculating Bacillus amyloliquefaciens HW05 into 1 / 2 LB liquid culture medium and culturing for 48-72 hours, and spray drying to obtain Bacillus amyloliquefaciens powder; (3) Inoculate Paecilomyces lilacinus E16 into a liquid fermentation medium and culture it for 48-72 hours, then inoculate it into a double-layer gauze bag containing a solid fermentation medium at a mass ratio of 20%, culture it for 5-7 days, dry it for 24-48 hours, take the fermentation product and shake and sieve it to obtain Paecilomyces lilacinus powder; (4) Inoculate green Trichoderma H06 into 1 / 2 LB liquid culture medium and culture for 48-72 hours, then inoculate 20% by mass into a double-layer gauze bag containing solid fermentation medium, culture for 5-7 days, dry for 24-48 hours, take the fermentation product and shake and sieve it to obtain green Trichoderma powder; (5) According to weight parts, 10-30 parts of Bacillus subtilis powder, 10-30 parts of Bacillus amyloliquefaciens powder, 1-5 parts of Paecilomyces lilacinus powder, and 1-5 parts of Trichoderma viride powder are taken, and the powders are mixed to obtain a composite powder, and the composite powder is mixed with a carrier at a mass ratio of 1:10-20 to obtain a multifunctional microbial agent.

4. The preparation method according to claim 3, characterized in that: In terms of mass percentage, the 1 / 2 LB liquid culture medium contains: 0.1-0.5% sucrose or glucose, 0.5-1% tryptone, 0.1-0.5% yeast extract and 0.5-1% NaCl; the liquid fermentation medium of Paecilomyces lilacinus E16 contains: 2% sucrose, 1% chitin and 0.001% manganese sulfate; The solid fermentation medium of Paecilomyces lilacinus E16 includes cassava residue: corn flour: coconut bran: chitosan: water in a mass ratio of 20-40:30-60:30-40:1-5:10-30; the solid fermentation medium of Trichoderma viride H06 includes cassava residue: corn flour: coconut bran: water in a mass ratio of 20-40:30-60:30-40:20-40.

5. Use of the multifunctional microbial agent according to any one of claims 1 to 2 in preventing and treating plant diseases caused by pathogens, characterized in that: The diseases are one or more of wilt, root-knot nematode, heart rot, rice blast, bacterial wilt, powdery mildew, rust, anthracnose, canker and Huanglongbing disease.

6. The use according to claim 5, characterized in that: The diseases are one or more of banana wilt, banana root-knot nematode, tomato root-knot nematode, celery root-knot nematode, pepper root-knot nematode, pineapple heart rot, rice blast, tomato bacterial wilt, melon powdery mildew, mulberry red rust, mango anthracnose, citrus canker, and citrus Huanglongbing.

7. The use according to claim 5, characterized in that: The pathogen is one or more of Fusarium oxysporum, root-knot nematode, tobacco phytophthora, Pyricularia grisea, Ralstonia solanacearum, Erysiphe leucoderma, Puccinia mulberry, Colletotrichum gloeosporioides, Xanthomonas carpetii, and Bacillus cereus.

8. The use according to claim 7, characterized in that: The application method comprises the following steps: (1) Preparing pre-culture solution: The multifunctional microbial agent is applied at a dosage of 0.1-0.3 g / kg soil for each potting application and 1-10 kg / mu for each field application. After diluting 10-30 times with water according to the mass multiple, place it at an ambient temperature of 15-40°C for 24-72 hours, let it stand or ventilate it with a water pump or stir it manually every 2-12 hours to obtain pre-culture solution 1, then dilute it 50-100 times, add a synergist at a mass ratio of 0.1-0.5% to obtain pre-culture solution 2, divide pre-culture solution 2 into two parts, spray 70%-90% of the volume of pre-culture solution 2 on the roots, and apply 10%-30% of the volume of pre-culture solution 2 on the leaves; In terms of mass percentage, the synergist includes 10%-30% glycerol, 10%-70% Antrodia cinnamomea polysaccharide, 20%-80% methylcellulose and 10%-20% sodium dodecylbenzene sulfonate; (2) Applying pre-culture solution 2 to the roots: After 4:00 p.m. on a sunny day or on a cloudy day, take the pre-culture solution 2 in step (1), or add 0.1% to 0.5% of compound fertilizer to the pre-culture solution 2 by mass ratio and spray it on the roots; (3) Applying pre-culture solution 2 to leaves: spray the pre-culture solution 2 prepared in step (1) or the pre-culture solution 2 to which 0.1% to 0.5% compound fertilizer and / or common foliar insecticides have been added on a mass basis on the front and back sides of leaves after 4 p.m. on a sunny day or on a cloudy day.

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