Penicillium baylii and purpureocillium lilacinum complex microbial inoculant and application thereof in preventing and treating green onion and garlic root rot

Abstract

The invention discloses a Penicillium beijeri and Purpureocillium lilacinum complex microbial inoculant and application thereof in preventing and treating green onion and garlic root rot, and relates to the technical field of microorganisms, and the technical scheme is characterized in that the Penicillium beijeri and Purpureocillium lilacinum complex microbial inoculant comprises Purpureocillium lilacinum LS003 and Penicillium beijeri LS004; according to the purpureocillium lilacinus LS003, the Latin name is Paecilomyces lilacinus, and the preservation number of the purpureocillium lilacinus LS003 is CGMCC (China General Microbiological Culture Collection Center) NO.41613; the Latin name of the penicillium bilaiae LS004 is Penicillium bilaiae, and the preservation number of the penicillium bilaiae LS004 is CGMCC (China General Microbiological Culture Collection Center) NO.41614. The disease-preventing and growth-promoting compound microbial agent provided by the invention has good phosphorus-dissolving and growth-promoting functions, and is a good microbial agent which can be used for efficiently promoting growth of lycoris crops.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, more particularly, it relates to a complex microbial agent of Penicillium bilaiae and Sporichthrus anguillarum and application of the complex microbial agent in preventing and treating onion and garlic root rot. BACKGROUND

[0002] Cultivated land is the foundation of agricultural development, however, China has long been faced with the serious problem of insufficient cultivated land per capita, in order to meet people's basic survival needs, it is necessary to continuously improve yield per unit and increase farmland utilization rate, and it is necessary to continuously increase the multiple cropping index of farmland, resulting in overdevelopment of cultivated land soil, causing soil infertility, serious occurrence of soil-borne diseases and other consequences. The use of chemical fertilizers and pesticides has to a great extent alleviated the plight of soil fertility deficiency and disease damage, and has made outstanding contributions to agricultural income increase. However, in agricultural production, due to people's lack of understanding of soil fertilization and scientific use of fertilizers and pesticides, in order to pursue yield increase, the application amount of chemical fertilizers and pesticides is continuously increased, thereby causing soil acidification, soil compaction, water eutrophication, crop quality decline, food safety threat and a series of problems.

[0003] With the continuous deepening of social development and the gradual improvement of people's living standards, the demand for food is also changing and improving, from basic subsistence to food safety, which continuously puts forward new requirements for agricultural production. Ecological agriculture and green agriculture have become the current focus of people's attention, and under the premise of guaranteeing soil ecological function, effectively improving the fertility of cultivated land will be the most critical link. In the soil environment, especially in the rhizosphere microenvironment, there are a large number of microbial populations, which form a microecosystem with plant roots and soil, create a suitable environment for plant growth and regulate plant growth, disease resistance, stress resistance and other functions. The popularization and application of microbial inoculants can meet the urgent needs of the current microbial inoculant market, and is an important component of promoting agricultural green development. In some developed countries, microbial inoculants have played a crucial role in agricultural development. In the past few decades of practice in the United States, it has proposed an important conclusion that microorganisms can feed the world, and the application of microbial inoculants and related preparations in the United States has reduced the use of chemical fertilizers and pesticides by about 20%. China has also proposed a "double reduction and efficiency increase" strategy as early as 2015, and there is no doubt that the large-scale application of microbial preparations is one of the alternative means to guarantee this strategy. Therefore, vigorously developing microbial inoculants is an important strategy to realize efficient use of agricultural resources and sustainable development of agriculture.

[0004] Although microbial inoculants are rich in functions, eco-friendly, and meet the needs of agricultural green and high-quality development. However, in production practice, there is a problem of unstable field effect. On the one hand, the fermentation system of the strain needs to be further optimized in the production process; most importantly, the in-situ and ex-situ effects of the strain in the microbial inoculant. Therefore, it is of great value to develop and create functional preparations by mining specific in-situ microorganisms of crops to maximize the functions of microbial preparations and ensure stable effects.

[0005] Therefore, the present application aims to isolate microorganisms from the rhizosphere of Amaryllidaceae crops, and to provide a composite microbial inoculant, wherein the microbial species in the composite microbial inoculant are Paecilomyces lilacinus and Penicillium bilaiae, in order to provide a green means for efficient production of Amaryllidaceae crops. SUMMARY

[0006] The present application focuses on the problem of green synergism of Amaryllidaceae crops, and provides a composite microbial inoculant with disease prevention and growth promotion function, which is compounded by the fermentation products of Paecilomyces lilacinus LS003 and Penicillium bilaiae LS004. The strains Paecilomyces lilacinus LS003 and Penicillium bilaiae LS004 were submitted to the China General Microbiological Culture Collection Center (CGMCC) for patent preservation on November 1, 2024, and the registration numbers are CGMCC NO.41613 and CGMCC NO.41614, respectively, and the preservation address is No.3, Beichen West Road, Chaoyang District, Beijing. The strains P. lilacinus LS003 and P. bilaiae LS004 have good phosphorus solubilization function, and the fermentation seed liquid of the two strains can effectively promote seed germination and inhibit the interference of miscellaneous bacteria during germination; the composite microbial inoculant prepared by the present application can significantly inhibit the occurrence of garlic root rot and reduce the damage of root rot at the seedling stage; in addition, the microbial inoculant can significantly promote the growth of Chinese chives, onions and garlic after soil mixing, and compared with the blank control, the composite microbial fertilizer treatment makes the plant height, root length and fresh weight of Chinese chives, onions and garlic increase by at least 17.8%, 10.1% and 25%, respectively.

[0007] The above technical purpose of the present application is achieved by the following technical scheme:

[0008] The present application provides a composite microbial inoculant of Penicillium bilaiae and Paecilomyces lilacinus, which comprises Paecilomyces lilacinus LS003 and Penicillium bilaiae LS004;

[0009] The Paecilomyces lilacinus LS003 has a Latin name of Paecilomyces lilacinus, and a preservation number of CGMCC NO.41613.

[0010] The Penicillium bilaiae LS004 has a Latin name of Penicillium bilaiae, and a preservation number of CGMCC NO.41614.

[0011] In combination with the first aspect, the application further provides that the number of conidia of the single strain of the Paecilomyces lilacinus LS003 and the Penicillium bilaiae LS004 in the complex microbial agent is greater than or equal to 1.0×10 10 CFU / g.

[0012] The second aspect of the application further provides the application of any of the above complex microbial agents in any of the following aspects:

[0013] A1, inhibiting and / or killing Fusarium oxysporum;

[0014] A2, preparing a biopesticide for inhibiting and / or killing Fusarium oxysporum;

[0015] A3, inhibiting and / or treating root rot;

[0016] A4, preparing a biopesticide for inhibiting and / or treating root rot;

[0017] A5, preparing a microbial agent for promoting seed germination of herbaceous plants;

[0018] A6, preparing a microbial agent for promoting growth of herbaceous plants;

[0019] A7, dissolving inorganic phosphorus.

[0020] In combination with the second aspect, the application further provides that the herbaceous plant is a plant of the Amaryllidaceae family.

[0021] In combination with the second aspect, the application further provides that the plant of the Amaryllidaceae family is a plant of the Allium genus.

[0022] In combination with the second aspect, the application further provides that the plant of the Allium genus is Allium fistulosum, Allium cepa and Allium sativum.

[0023] The third aspect of the application further provides a preparation method of the complex microbial agent, characterized by comprising the following steps:

[0024] S1. preparing a fermentation seed solution of the Paecilomyces lilacinus strain LS003 and the Penicillium bilaiae strain LS004;

[0025] S2. configuring a fermentation medium, wherein the medium comprises a carbon source, a nitrogen source and inorganic salts;

[0026] S2. The two strains are fermented by solid fermentation to prepare fermentation products of the two strains, and the mixed bacteria rate is less than 0.5%;

[0027] S4. A compound microbial inoculant is prepared.

[0028] In combination with the third aspect, the application is further provided as follows: in step S2,

[0029] The carbon source is one or a mixture of several of corn powder, bran, brown rice, soluble starch and sucrose;

[0030] The nitrogen source is one or a mixture of several of soybean powder, soybean meal powder, cottonseed cake powder, peanut cake powder, corn steep liquor and rice bran;

[0031] The inorganic salt is one or several of CaCO3, K2HPO4, KCl and MgSO4·7H2O.

[0032] In combination with the third aspect, the application is further provided as follows: the fermentation medium of P.lilacinus: bran 30-35%, brown rice 40-45%, soybean powder 10-15%, corn steep liquor 5-10%, sucrose 3-5%, CaCO3 1-2%, K2HPO4 0.5-1%, the ratio of material to water is 1:0.5-0.7, the inoculation amount is 5%, and the fermentation conditions are as follows: the fermentation temperature is 26-30℃, and the fermentation time is 8d.

[0033] The fermentation medium of P.bilaiae: corn powder 15-25%, sucrose 3-5%, soybean meal powder 25-30%, peanut cake powder 15-20%, rice bran 30-35%, KCl 0.5-1%, MgSO4·7H2O 0.5-1%, the ratio of material to water is 1:0.5-0.7, the inoculation amount is 5%, and the fermentation conditions are as follows: the fermentation temperature is 26-30℃, and the fermentation time is 8d.

[0034] In combination with the third aspect, the application is further provided as follows: in step S4, the fermentation products of P.lilacinus LS003 and P.bilaiae LS004 are configured in a ratio of 1:1.

[0035] In summary, the application has the following beneficial effects:

[0036] 1. The strains P.lilacinus LS003 and P.bilaiae LS004 involved in the application are in-situ microbial strains of Amaryllidaceae and have good phosphorus dissolution function;

[0037] 2. The P.lilacinus LS003 and P.bilaiae LS004 are fermented and compounded innovatively in the application to form a new compound microbial inoculant, and the compound microbial inoculant shows significant effects in seed germination and seedling growth of Amaryllidaceae crops. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Phosphate solubilizing activity of strains LS003 and LS004. Left is strain LS003, right is strain LS004;

[0039] Figure 2 Culture morphology and micro-morphology of conidia of Purpureocillium lilacinum LS003 and Penicillium bilaiae LS004 on PDA plates. Top left is the culture morphology of strain LS003, bottom left is the conidial morphology of strain LS003; top right is the culture morphology of strain LS004, bottom right is the conidial morphology of strain LS004;

[0040] Figure 3 Phylogenetic tree of Purpureocillium lilacinum LS003 and Penicillium bilaiae LS004 based on ITS sequence;

[0041] Figure 4 Effect of fermentation seed liquid of strains P. lilacinum LS003 and P. bilaiae LS004 on seed germination of Allium fistulosum;

[0042] Figure 5 Effect of compound microbial inoculant on plant height of seedlings of Allium fistulosum, Allium cepa and Allium sativum;

[0043] Figure 6 Effect of compound microbial inoculant on root length of seedlings of Allium fistulosum, Allium cepa and Allium sativum;

[0044] Figure 7 Effect of compound microbial inoculant on fresh weight of seedlings of Allium fistulosum, Allium cepa and Allium sativum. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0046] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0047] The experimental methods used in the following embodiments are all conventional methods, unless otherwise specified.

[0048] The materials, reagents and the like used in the following examples, unless otherwise specified, can be obtained commercially.

[0049] The culture medium used in the examples and the preparation method are as follows:

[0050] Inorganic phosphorus culture medium: 10 g glucose, 0.5 g (NH4)2SO4, 0.2 g NaCl, 0.2 g KCl, 5.0 g Ca3(PO4)2, 0.03 g MgSO4·7H2O, 0.03 g MnSO4, 0.003 g FeSO4, 0.5 g yeast extract, 16 g agar, distilled water to 1000 mL, pH 6.8-7.0, 121 ℃ high pressure steam sterilization for 30 min.

[0051] PDA culture medium: 200 g potato, 20 g glucose, 18 g agar, distilled water to 1000 mL, 121 ℃ high pressure steam sterilization for 30 min.

[0052] PDB culture medium: 200 g potato, 20 g glucose, distilled water to 1000 mL, 121 ℃ high pressure steam sterilization for 30 min.

[0053] Example 1, Isolation and screening of strains Purpureocillium lilacinum LS003 (CGMCC NO. 41613) and Penicillium bilaiae LS004 (CGMCC NO. 41614)

[0054] 1. Isolation of rhizosphere phosphorus-solubilizing fungi

[0055] Strain LS003 and strain LS004 were isolated from garlic field soil in Qi County, Kaifeng City, Henan Province.

[0056] Healthy plant rhizosphere soil samples were collected from garlic, Chinese chive, and onion planting fields in various regions of Qi County, Henan Province. The collected soil samples were crushed and filtered through a 50-mesh sieve to remove large pieces of soil. 10 g of soil sample was weighed and added to 90 mL of sterile physiological saline, and shaken at 200 rpm for 30 min at 25 ℃ to uniformly disperse it in the sterile physiological saline. After standing for 3-5 min, 1 mL of supernatant was taken for 10-fold gradient dilution, i.e., original liquid, 10 -1 -2 -3 -4 ​​​Different gradient soil suspensions, 100 μL of each concentration of soil suspension was spread on inorganic phosphorus culture medium plates containing streptomycin sulfate with a final concentration of 50 μg / mL, 3 plates were spread for each concentration, and after air-drying, they were placed in an inverted culture in a 25°C incubator. The growth of the colonies was observed every day, and single colonies with obvious differences in phenotype such as mycelial growth morphology and colony color and with phosphorus-dissolving function were further purified and cultured, and the purified strains were inoculated again in inorganic phosphorus culture medium to verify the phosphorus-dissolving activity. Finally, 2 strains with better phosphorus-dissolving effect were obtained and named LS003 and LS004 Figure 1 ), and stored in a -80°C refrigerator

[0057] Example 2, Identification of Purpureocillium lilacinum LS003 (CGMCC NO. 41613) and Penicillium bilaiae LS004 (CGMCC NO. 41614)

[0058] Strain LS003 can grow normally on PDA medium at 25°C, the aerial mycelium is white, and with the increase of culture time, it begins to produce conidia, and then the colony shows pink, the single colony is regular and round, raised, dry surface, and no exudate is produced; under microscopic observation, the mycelium is smooth and non-septate, the tip forms a conidial phialide, the phialide is swollen at the end to form a bottleneck, and the conidia are borne on it, the conidia are single spores, oval to short rod-shaped Figure 2 (left upper and lower).

[0059] Strain LS004 can grow normally on PDA medium at 25°C, the initial colony is white, and with the increase of culture time, it forms a colony with white edge and dark green middle, the single colony is regular and round, the aerial mycelium is short, the colony is villous, the center is slightly raised, and it produces a diffusible light yellow to yellow pigment in the culture medium; under microscopic observation, the mycelium is thick and colorless, the surface is smooth but has internal septa, the conidia are colorless, transparent, and nearly spherical Figure 2 (right upper and lower).

[0060] The genomic DNA of strains LS003 and LS004 was obtained by using Tian Gen Fungus DNA Extraction Kit, and the DNA concentration and quality were detected by microspectrophotometer. The ITS sequences of the two strains were amplified by using fungal ITS universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR reaction system was 20 μL: 2 × PCR premix 10 μL, 10 μM upstream and downstream primers 1 μL each, DNA template 2 μL, ddH2O 6 μL. The PCR reaction program was 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 55 °C annealing for 20 s, 72 °C extension for 30 s, 35 cycles; 72 °C extension for 5 min. After the PCR product was detected by agarose gel electrophoresis, the sequencing work was completed by Baisheng Bioengineering (Dalian) Co., Ltd., and the ITS sequences of strains LS003 and LS004 were obtained as shown in Sequence 1 and Sequence 2. Online BLAST comparison was performed by using NCBI website, and phylogenetic analysis was performed by MEGA-X based on the sequences of closely related strains. The results showed that strain LS003 and strain Purpureocillium lilacinum clustered in the same branch, proving that the strain had the closest homologous relationship with Purpureocillium lilacinum, and the strain was named Purpureocillium lilacinum LS003. Strain LS004 had the highest homology with Penicillium bilaiae, so strain LS004 was identified as Penicillium bilaiae LS004. The two strains were preserved in the China General Microbiological Culture Collection Center on November 1, 2024, and the registration numbers were CGMCC NO.41613 and CGMCC NO.41614, and the preservation address was No. 3, Beichen West Road, Chaoyang District, Beijing. Hereinafter referred to as P. lilacinum LS003 and P. bilaiae LS004 Figure 3 ).

[0061] Example 3, Preparation of Fermentation Seed Liquid of Purpureocillium lilacinum LS003 and Penicillium bilaiae LS004

[0062] The frozen strains were taken out from the -80℃ refrigerator, and a single bacterial cake was inoculated on a PDA plate, and the plate was placed in a 25℃ constant temperature incubator for 2-3d. After fresh mycelium was generated, the edge mycelium was picked and re-inoculated on a new PDA plate for secondary activation of the strain, and the plate was placed in the same culture conditions for 7d. 3mL of sterile distilled water was added to the culture plates of the strains LS003 and LS004 after secondary purification, respectively, and the conidiospores on the surface of the colonies were repeatedly blown and washed by using a pipette to prepare a spore suspension, and the concentration of the spore suspension was calculated by using a hemocytometer. The concentrations of the conidiospore suspensions of the two strains were adjusted to 1×10 8 CFU / mL. According to a 1:100 ratio, the conidiospore suspensions were added to PDB liquid medium separately, and the fermentation seed liquid of the strains LS003 and LS004 was obtained under the conditions of 25℃ and 200rpm for 5d, and the purity of the seed liquid was verified by plate coating.

[0063] Example 4, Scale-up fermentation of the strains P. lilacinum LS003 and P. bilaiae LS004 and preparation of a compound microbial inoculant

[0064] The fermentation medium includes a carbon source, a nitrogen source and inorganic salts. The carbon source is one or a mixture of several of corn powder, bran, brown rice, soluble starch and sucrose; the nitrogen source is one or a mixture of several of soybean meal, soybean meal powder, cottonseed cake powder, peanut cake powder, corn syrup and rice bran; and the inorganic salts are one or a mixture of several of CaCO3, K2HPO4, KCl and MgSO4·7H2O.

[0065] The optimized fermentation medium for P. lilacinum is as follows: bran 30-35%, brown rice 40-45%, soybean meal 10-15%, corn syrup 5-10%, sucrose 3-5%, CaCO3 1-2%, K2HPO4 0.5-1%, and the ratio of material to water is 1:0.5-0.7, the inoculation amount is 5%, the fermentation temperature is 26-30℃, and the fermentation time is 8d. Under the above conditions, the concentration of the conidiospores of P. lilacinum is about 1.8-2.2×10 10 CFU / g.

[0066] The optimized fermentation medium for P. bilaiae is as follows: corn powder 15-25%, sucrose 3-5%, soybean meal powder 25-30%, peanut cake powder 15-20%, rice bran 30-35%, KCl 0.5-1%, MgSO4·7H2O 0.5-1%, and the ratio of material to water is 1:0.5-0.7, the inoculation amount is 5%, the fermentation temperature is 26-30℃, and the fermentation time is 8d. Under the above conditions, the concentration of the conidiospores of P. bilaiae is about 1.5-2.0×10 10 CFU / g.

[0067] Solid fermentation process of the strains: Fermentation tanks for LS003 and LS004 were prepared according to the above fermentation medium components, and the fermentation tanks were subjected to sufficient sterilization treatment to reduce contamination by miscellaneous bacteria during fermentation. The seed liquid prepared in Example 3 was inoculated into the corresponding fermentation medium at an inoculation amount of 5%, and the mixture was stirred uniformly. The temperature and humidity of the fermentation environment were monitored during the entire fermentation process, and the environmental temperature was controlled at 26-30°C. The mixture was stirred once every other day, and then once a day from the 6th day. The fermentation temperature and oxygen supply were controlled. After the fermentation was completed, the fermentation product was dried in the shade or dried at 35°C until the water content was about 10-15%, and the number of viable bacteria and the rate of miscellaneous bacteria were detected. The number of viable bacteria reached the above standard, and the rate of miscellaneous bacteria was less than 0.5%.

[0068] Preparation of the complex microbial inoculant: In order to maintain the number of conidia of a single strain in the complex microbial inoculant at 1.0 x 10 10 CFU / g, the fermentation products of the two strains were mixed in appropriate proportions according to the results of each quality inspection after the fermentation of LS003 and LS004 was completed. Generally, the fermentation products can be mixed at a ratio of 1:1 to meet the requirements of a conidia number of 1.0 x 10 10 CFU / g and a ratio of the two strains of approximately 1:1, and the complex microbial inoculant can be obtained.

[0069] Example 5, Effect of the complex microbial inoculant on garlic root rot

[0070] Preparation of the pathogenic spore suspension: Seven bacterial cakes were taken from the edge of a F. oxysporum colony using a 7 mm diameter punch, inoculated into PDB medium, and cultured at 25°C and 220 rpm for 5 days. The mycelium was removed by sterile gauze filtration to obtain a conidial suspension. The concentration of the conidial suspension was calculated by microscopic counting using a hemocytometer, and the concentration was adjusted to 1 x 10 4 conidia / mL using distilled water.

[0071] Preparation and sterilization of the cultivation substrate: Natural soil and vermiculite were mixed at a ratio of 1:3, and then subjected to dry heat sterilization at 160°C for 2 h. After natural cooling, the mixture was ready for use.

[0072] Uniform and plump garlic seeds were selected, washed, and then evenly planted in pots. After sufficient watering, the pots were placed in an artificial greenhouse with a light / dark cycle of 14 / 10 h and day / night temperatures of 20°C and 16°C, respectively. The growth of the garlic seeds was observed every day, and appropriate watering was performed according to the actual situation. After germination, the garlic seeds were divided into two groups, each with 10 pots. The two groups corresponded to two treatments, i.e., root irrigation with the complex microbial inoculant and a blank control. Each treatment was repeated three times. The root irrigation with the complex microbial inoculant treatment group ensured that the number of viable bacteria in each gram of soil was about 1.0 x 10 6CFU, the blank control was irrigated with the same amount of water. After 24 hours of root irrigation, the prepared pathogen conidia suspension was inoculated into the garlic seedlings in the form of root irrigation, with an inoculation concentration of 1×10 4 30 mL of a Fusarium oxysporum spore suspension with 10 conidia / mL was added. No other treatments were applied to the garlic plants except for normal watering during the growth period. Thirty days after inoculation with the pathogen, the number of diseased plants and the severity of the disease were counted, and the incidence rate, disease index, and control efficacy were calculated.

[0073] Garlic root rot disease is graded as follows:

[0074] Level 0, no disease;

[0075] Level 1: a few fibrous roots rot, and there is no obvious difference in plant growth;

[0076] Level 2: Most of the fibrous roots are rotten, the above-ground parts are yellow and short, and the garlic bulbs are easy to fall off after being pulled out;

[0077] Level 3: The fibrous root system is completely rotten, the garlic cloves are softened and water-soaked, have a fishy smell, and the plant is dead.

[0078] Incidence rate (%) = number of diseased plants / total number of treated plants × 100%;

[0079] Disease index = ∑(number of diseased plants at each level × corresponding disease level) / (total number of plants surveyed × highest disease level) × 100;

[0080] The control effect (%) = (disease index of the control group - disease index of the treatment group) / disease index of the control group × 100%.

[0081] Results showed that after root irrigation with the composite microbial inoculant, the average incidence of garlic root rot was only 16.67%, significantly lower than that of the blank control group. The disease index was also only 8.33, and the control efficacy against root rot reached 67.55% (Table 1). This demonstrates that the composite microbial inoculant, formed by fermenting the strains Paecilomyces lilacinus LS003 and Penicillium bilaiae LS004, has excellent control efficacy against garlic root rot, reducing plant morbidity and disease severity, and possesses promising market application prospects.

[0082] Table 1 The efficacy of composite microbial agents in preventing garlic root rot

[0083]

[0084] Example 6: Growth-promoting effect of composite microbial agent on various Amaryllis crops

[0085] The composite microbial inoculant created in Example 4 was used for seedling growth tests on garlic, scallion, onion and other crops. The fermentation seed liquid of each strain prepared in Example 3 was used for seed germination tests on scallion and onion at a ratio of 1:1.

[0086] Seed surface sterilization: The scallion and onion seeds were soaked in 75% alcohol for 30-45s, then rinsed with sterile water for 2-3 times after pouring off the alcohol, then soaked in 2% NaClO for 5 min, and rinsed with sterile water for 3-5 times again. The garlic seeds were not sterilized.

[0087] The cultivation substrate was pretreated according to the method in Example 5. The naturally cooled substrate was divided into two groups, one group was mixed with the composite microbial inoculant prepared in Example 4, so that the number of viable bacteria in each gram of soil was about 1.0 x 10 6 CFU; the other group of substrate was not treated as a control group.

[0088] Seed germination test: The surface-sterilized, uniform in size, and full-grain scallion and onion seeds were each divided into two groups, 30 seeds in each group, and soaked in the 1:1 mixed fermentation seed liquid of strains P. lilacinum LS003 and P. bilaiae LS004 and PDB culture solution respectively, and then taken out and air-dried after soaking for 30 min. The dried seeds were placed in sterile culture dishes, sterile filter paper was placed in the culture dishes and moistened with sterile water. 10 seeds were placed in each dish, and each treatment was repeated 3 groups. Then placed in a light and dark 14 / 10h, day and night temperature of 25℃ light incubator, observed the seed germination and recorded the germination rate and growth amount every day, and appropriately supplemented with sterile water for moisturizing every day, and the germination rate and growth amount were counted after 7 days. It was found that the germination rate of scallion and onion seeds was significantly improved after seed liquid soaking treatment, and the germination time was also relatively shortened. The 30 seeds of each crop treated by seed liquid soaking all germinated, and the germination rate reached 100%, while the germination rate of the control group was 87%, and the seedlings grew slowly after germination and were prone to breed bacteria during cultivation Figure 4 ).

[0089] Seedling growth promotion test: The above treated cultivation substrate was divided and placed in 6cm square pots, and surface-sterilized, uniform in size, and full-grain scallion and onion seeds and non-sterilized garlic seeds were planted in the pots, 10-15 seeds of scallion and onion and 8 seeds of garlic were planted in each pot, and then watered uniformly and placed in an artificial greenhouse with light and dark 14 / 10h, day and night temperature of 25℃ and 22℃ respectively for cultivation. During the growth of crops, no other treatment was done except normal watering, and after 4 weeks of growth of scallion and onion, the plant height, root length, and fresh weight were measured, and after 2 weeks of growth of garlic, the plant height, root length, and fresh weight were measured.

[0090] The results show that the composite microbial inoculant can significantly promote the growth of green onions, onions and garlic after soil mixing, compared with the control group without microbial inoculant, the microbial inoculant treatment makes the green onion plant height increase by 18.6%, the onion plant height increases by 21.2%, and the garlic plant height increases by 17.8%; similarly, it is found that the composite microbial inoculant can also effectively increase the root length and root biomass of the three crops, the green onion root length increases by 19.8%, the onion increases by 17.0%, and the garlic increases by 10.1% relatively less. Figures 5-7

[0091] Finally, it should be noted that: the above only for the preferred embodiments of the present application, and not for limiting the present application, although the foregoing detailed description of the present application is made with reference to the foregoing embodiments, for those skilled in the art, it still can be modified, or part of the technical features of the equivalent replacement of the technical solutions recorded in the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included within the scope of protection of the present application.​

Claims

1. A composite bacterial agent, characterized by: It consists of Pseudomonas lilacinus LS003 and Penicillium bilairum LS004; The lilacinus purpurogenous fungus LS003 has a Latin name of Paecilomyces lilacinus and a deposit number of CGMCC NO.41613; The Penicillium bilaiae LS004 has a Latin name of Penicillium bilaiae and a preservation number of CGMCCNO.41614.

2. The composite bacterial agent according to claim 1, characterized in that: The number of conidia of a single strain of Purpurosporium lilacinum LS003 and Penicillium bilairum LS004 in the composite microbial agent is ≥1.0×10 10 CFU / g.

3. Use of the composite bacterial agent according to any one of claims 1-2 in any of the following: A1. Inhibit and / or kill Fusarium oxysporum; A2. Preparation of biological pesticides for inhibiting and / or killing Fusarium oxysporum; A3. Inhibit and / or treat root rot; A4. Preparation of biopesticides for inhibiting and / or treating root rot; A5. Preparing a fungal agent for promoting herbaceous plant seed germination; A6. Preparation of a bacterial agent for promoting the growth of herbaceous plants; A7. Dissolve inorganic phosphorus.

4. The use according to claim 3, characterized in that: The herb is a plant of the Amaryllidaceae family.

5. The use according to claim 4, characterized in that: The Amaryllis plant is an Allium plant.

6. The use according to claim 5, characterized in that: The allium plants include scallions, onions and garlic.

7. The method for preparing the composite bacterial agent according to claim 1-2, characterized in that: The steps include: S1. Preparation of fermentation seed broth of Pseudomonas aeruginosa strain LS003 and Penicillium bailei strain LS004; S2 configuration fermentation medium, the medium comprising a carbon source, a nitrogen source and an inorganic salt; S2. Both strains were fermented using solid-state fermentation to produce fermentation products with a contaminant bacterial rate of less than 0.5%; S4. Prepare composite microbial agents.

8. The method for preparing the composite microbial agent according to claim 7, characterized in that: In step S2, The carbon source is one or a mixture of corn flour, bran, brown rice, soluble starch, and sucrose; The nitrogen source is one or a mixture of soybean meal, soybean meal, cottonseed meal, peanut meal, corn steep liquor, and rice bran; The inorganic salt is one or more of CaCO3, K2HPO4, KCl, and MgSO4·7H2O.

9. The method for preparing the composite bacterial agent according to claim 8, wherein: The fermentation medium of the Pseudomonas lilacinus comprises: 30-35% bran, 40-45% brown rice, 10-15% soybean powder, 5-10% corn steep liquor, 3-5% sucrose, 1-2% CaCO3, 0.5-1% K2HPO4, a material-water ratio of 1:0.5-0.7, an inoculum size of 5%, and fermentation conditions of: a fermentation temperature of 26-30°C and a fermentation time of 8 days; Penicillium bilaiae fermentation medium: 15-25% corn flour, 3-5% sucrose, 25-30% soybean meal powder, 15-20% peanut cake powder, 30-35% rice bran, 0.5-1% KCl, 0.5-1% MgSO4·7H2O, material-water ratio of 1:0.5-0.7, inoculation amount of 5%, fermentation conditions: fermentation temperature of 26-30°C, fermentation time of 8 days.

10. The method for preparing the composite bacterial agent according to claim 7, wherein: In step S4, the fermentation products of the Pseudomonas lilacinus LS003 and LS004 are prepared in a ratio of 1:1.

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