CN110205273B - Bacillus amyloliquefaciens with growth promoting and disease resisting effects and application thereof - Google Patents
Bacillus amyloliquefaciens with growth promoting and disease resisting effects and application thereof Download PDFInfo
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Abstract
The disclosure belongs to the technical field of microorganisms, and particularly relates to bacillus amyloliquefaciens with growth promoting and disease resisting effects and application thereof. In the prior art, microbial preparations related to bacillus amyloliquefaciens have certain use defects, such as poor field colonization capacity, single disease prevention effect and the like. The present disclosure provides a bacillus amyloliquefaciens (b. amyloliquefaciens)44, which has the effects of inhibiting various plant pathogenic bacteria and promoting crop growth, and has a wide application range. In addition, the application also provides a composite microbial inoculum which is composed of bacillus amyloliquefaciens (B.amyloliquefaciens)44, rahnella aquatilis (R.aquatilis)27 and bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619, and has good antibacterial effect and growth promoting capability and also has good colonization capability.
Description
Technical Field
The disclosure relates to the technical field of microorganisms, in particular to Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)44 with growth promoting and disease resisting functions and application thereof in growth promoting and disease resisting.
Background
The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The plant root system has the functions of absorbing, synthesizing, conducting water and fixing nutrients, can directly utilize mineral nutrients in the external environment, and has the functions of storing nutrients and propagating for some plants. The enhancement of the root system activity of the plants is beneficial to accelerating the photosynthesis of the overground parts of the plants and accelerating the metabolism of the crops, thereby achieving the effects of promoting the growth of the plants, delaying the aging of the leaves of the plants and the like. The importance of plant roots is more and more emphasized by people. However, in recent years, with the increasing use amount of fertilizers and pesticides, the negative effects of the fertilizers and the pesticides are increasingly obvious. Soil structure and microflora diversity suffer to destroy, soil pollution, soil harden etc. a series of problems make plant roots fungus crowd receive serious influence, and plant roots fungus crowd microecological balance is destroyed, and the root system protection barrier is weak, and is relatively poor to the resistance of soil-borne disease pathogen, and then influences the normal growth of plant roots, influences the plant health.
Plant growth-promoting bacteria (PGPB) are beneficial bacteria capable of promoting plant growth, can colonize plant roots, and can obtain nutrients required for growth and propagation of the plants through ablation substances of the roots. The plant growth promoting bacteria can protect plants from being damaged by plant pathogenic bacteria and reduce the damage degree of the plant pathogenic bacteria through mechanisms such as site competition, antibiotic generation, induction system resistance and the like, and indirectly promote the growth of the plants. Bacillus spp, many strains have the ability to inhibit phytopathogens, and some are able to promote plant growth. The main bacteriostatic activities of currently studied bacillus are: bacillus subtilis (b), bacillus amyloliquefaciens (b), bacillus atrophaeus (b), and the like. Among them, bacillus amyloliquefaciens (b. amyloliquefaciens) can secrete extracellular metabolites with antibacterial activity in the growth process thereof, can well inhibit various plant pathogenic bacteria, the extracellular substances play an important role in controlling plant diseases, and some strains can control the diseases and promote the growth of plants. Bacillus amyloliquefaciens is widely distributed in nature, is easy to separate, is mostly separated from the vicinity of plant root system soil, is environment-friendly and is not easy to generate resistance, thereby being widely concerned by scientific researchers at home and abroad.
The inventor considers that related researches aiming at the development of a biological control preparation of bacillus amyloliquefaciens (B. amyloliquefaciens) are numerous, and the products of the biological control microecological preparation on the market are various, but still have certain use defects, including unstable biological control effect and poor field colonization capacity; the single microbial inoculum has single disease prevention and poor comprehensive disease prevention effect; the microecological preparation product is influenced by conditions such as storage, transportation, environment and the like, and the difference of viable count is large.
Disclosure of Invention
In view of the above technical background, the inventors believe that the development of a bacterial strain which has a broad-spectrum inhibitory effect on plant pathogens and can promote the growth of various crops is beneficial to reducing the usage amount of pesticides and fertilizers in the crop planting process and reducing the damage of chemical reagents to soil. In order to achieve the above technical object, the present disclosure provides the following technical solutions:
in a first aspect of the present disclosure, a bacillus amyloliquefaciens (b. amyloliquefaciens)44 is provided, which has been deposited in the chinese typical culture collection center at 23/8/2018, abbreviated as CCTCC, and has the address: the biological preservation number of the Wuchang Lojia mountain in Wuhan city of Hubei province is as follows: CCTCC M2018564.
The Bacillus amyloliquefaciens strain of the first aspect has a 16S rDNA sequence shown in SEQ ID NO. 1.
The strain is separated from a soil sample in Ningyang county of Shandong province, has 99% similarity with 16S rDNA sequences of other known bacillus species, and is determined as bacillus amyloliquefaciens by comprehensive sequencing and physiological and biochemical reaction results, and is named as bacillus amyloliquefaciens (B. amyloliquefaciens) 44.
The morphological characteristics of the bacillus amyloliquefaciens (b. amyloliquefaciens)44 are as follows:
the characteristics of the thallus are as follows: gram-positive bacteria, a bacterial straight rod, with a size of 0.6 × 1.0-1.4 μm, having rounded ends, grouped individually or in pairs to form endospores; the spores are oval with a central or sub-apical orientation; there was no swelling of the sporangia.
Colony characteristics: colonies were smooth, opaque, irregular, gray, 2-4mm in diameter, with serrated edges and flat profile, slightly raised in the center, and viscous in texture after 48h incubation with NA plate solid medium.
The physiological and biochemical characteristics of the bacillus amyloliquefaciens (b. amyloliquefaciens)44 are as follows:
positive in oxidase, catalase, gelatin hydrolysis test, Prischol test, nitrate reduction test, malonate utilization test, starch hydrolysis test and casein enzymolysis test; indole detection, H2S test, NH3Tests, urea decomposition tests, citrate utilization tests, methylation tests, lecithinase and phenylalanine negatives.
Preferably, the culture medium of the above bacillus amyloliquefaciens (B. amyloliquefaciens)44 comprises 2.0-3.5% of soluble starch/molasses, 1.5-3.0% of peptone/soybean meal/corn steep liquor dry powder and K2HPO4×3H2O 0.5-1.0％、KH2PO4 0.1-0.5％、(NH4)2SO40.1-0.3 percent of sodium citrate multiplied by 2H2O 0.01-0.05％、MgSO4×7H20.01 percent of O and the balance of water.
Preferably, the culture medium of the above bacillus amyloliquefaciens (b. amyloliquefaciens)44 has 1-3% of molasses as a carbon source and 1-4% of soybean meal as a nitrogen source.
More preferably, the molasses culture medium of the bacillus amyloliquefaciens (B.amyloliquefaciens)44 comprises 2.5-3.5% of molasses, 1.5-2.5% of soybean meal and K2HPO4×3H2O 0.5-1.0％，KH2PO4 0.1-0.5％，(NH4)2SO40.1-0.3%, sodium citrate x 2H2O 0.01-0.05％，MgSO4×7H20.01 percent of O and the balance of water, and the pH value is 6.6-7.0.
The research of the disclosure shows that the method adopts the following steps: under the condition that the proportion of the carbon source to the nitrogen source in the culture medium is different, the number of live bacteria and the number of spores in the culture medium are influenced, the number of live bacteria, the number of spores and the proportion between the number of live bacteria and the number of spores are changed in a disordered way along with the increase of the proportion of the nitrogen source in the culture medium, and researches show that the culture medium with the proportion is beneficial to obtaining the maximum number of live bacteria and spores.
The culture medium is used for culturing the bacillus amyloliquefaciens (B. amyloliquefaciens)44, and is favorable for obtaining the maximum viable count and spore count.
Preferably, the culture temperature of the bacillus amyloliquefaciens (b. amyloliquefaciens)44 is 10 to 40 ℃, and more preferably 30 to 40 ℃; the pH value of the culture is 3.5 to 8.5, and more preferably 6.5 to 7.5.
In a second aspect of the present disclosure, there is provided a microbial inoculum comprising a culture of bacillus amyloliquefaciens (b. amyloliquefaciens)44 and/or bacteria of the first aspect.
In a third aspect of the present disclosure, a composite microbial inoculum is provided, which includes the bacillus amyloliquefaciens (b.amyloliquefaciens)44, Rahnella aquatica (Rahnella aquatilis)27, bacillus amyloliquefaciens (b.amyloliquefaciens) B-1619 microbial inoculum of the first aspect and their respective cultures, wherein the Rahnella aquatica (r.aquatilis)27 is deposited in the china type culture collection center of the university of wuhan on 8-23 m 2018, abbreviated as CCTCC, and the address is: the preservation number of the Wuchang Lojia mountain in Wuhan city of Hubei province is as follows: CCTCC M2018566; the bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619 is purchased from China general microbiological culture collection center.
Preferably, in the complex microbial inoculum, the ratio of bacillus amyloliquefaciens (b. amyloliquefaciens) 44: rahnella aquatica (r. aquatilis) 27: the ratio of Bacillus amyloliquefaciens B-1619 is 0.8-5.2:0.8-3: 0.8-2.
Further preferably, in the composite microbial inoculum, the ratio of the three bacteria is 1:1: 1.
Preferably, the number of effective viable bacteria in the composite microbial inoculum is more than or equal to 10 hundred million/mL.
When different strains are in a common environment, multiple action factors which are synergistic or antagonistic among the strains can occur due to the extracellular secretion of the strains. When the proportion of the strain is changed, the action factors are also changed, and finally, various effects such as mutual inhibition, mutual promotion, increase of viable count, increase of spore count, increase of content of certain secretion and the like can be shown, and certain unpredictability is provided. The research of the present disclosure shows that the compound product of the three bacteria in a certain proportion has good growth promoting and disease preventing effects, can effectively reduce the dosage of pesticides and fertilizers in the crop planting process, and has obvious economic benefits.
Preferably, the culture medium of the Rahnella aquatilis (R.aquatilis)27 is 3.0% of corn starch, 2.0% of peptone and K2HPO4×3H2O 0.7％，KH2PO4 0.3％，(NH4)2SO40.15 percent of sodium citrate multiplied by 2H2O 0.05％，MgSO4×7H20.01 percent of O; the balance being water. And/or the fermentation condition is pH 6.8; culturing at 37 deg.C and stirring rate of 150rpm/min for 48 hr to obtain Rahnella aquatilis (R.aquatilis)27 fermentation liquid.
Preferably, the culture medium of the bacillus amyloliquefaciens B-1619 is 5% of glucose, 5% of tryptone, 5% of yeast extract and the balance of water. And/or the fermentation conditions are pH 7.0; culturing at 28 deg.C and stirring speed of 180rpm/min for 48 hr to obtain fermentation liquid of Bacillus amyloliquefaciens B-1619.
Preferably, the formulation of the microbial inoculum according to the second aspect or the complex microbial inoculum according to the third aspect is liquid microbial inoculum, powder or granules; further is a water suspending agent, a dispersible oil suspending agent, a wettable powder or a water dispersible granule.
Preferably, the microbial inoculum according to the second aspect or the composite microbial inoculum according to the third aspect further comprises an agriculturally and pharmaceutically acceptable auxiliary material, and the agriculturally and pharmaceutically acceptable auxiliary material is selected from one or more of a dispersing agent, a wetting agent, a disintegrating agent, a binder, an antifoaming agent, an antifreeze agent, a thickening agent, a filler and a solvent. The sources and the like of the agriculturally and pharmaceutically acceptable auxiliary materials are not particularly limited in the present disclosure, and a commercially available product is generally used.
In a fourth aspect of the present disclosure, there is provided a use of the strain of the first aspect, the microbial inoculum of the second aspect, and/or the complex microbial inoculum of the third aspect in any one of the following aspects:
(1) inhibiting plant pathogenic bacteria, including plant fungal pathogenic bacteria and plant bacterial pathogenic bacteria;
(2) promoting the growth of plants;
preferably, the plant fungal pathogens include, but are not limited to, fusarium oxysporum, rhizoctonia solani, alternaria, sclerotinia sclerotiorum, wheat root rot pathogens;
preferably, the plant bacterial pathogens include, but are not limited to, erwinia, agrobacterium tumefaciens, xanthomonas, pseudomonas syringae, tomatose bacteria, phytophthora parasitica;
preferably, the plant growth promoting means promotes plant seedling growth and/or weight gain; such plants include, but are not limited to, garlic, tomato, radish, lettuce, cucumber, and wheat.
Compared with the prior art, the beneficial effect of this disclosure is:
1. the bacillus amyloliquefaciens (B.amyloliquefaciens)44 provided by the disclosure has good inhibition effect on various plant pathogenic bacteria causing crop diseases, has inhibition zones of 13-23mm and 20-31mm on plant fungal pathogens and plant bacterial pathogens respectively, and can promote the growth of various crops such as tomatoes, radishes, cucumbers, wheat and the like.
2. The disclosure also provides a composite microbial inoculum of bacillus amyloliquefaciens (B.amyloliquefaciens)44, Rahnella aquatilis (R.aquatilis)27 and bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619, wherein the composite microbial inoculum shows good promotion effect on the growth of plants under a certain proportion, and effectively improves the germination rate of seeds, the weight of seedlings and the length of seedlings.
3. In addition, the composite microbial inoculum also has a good biocontrol effect, and the probability of disease occurrence can be effectively reduced by the composite microbial inoculum after the radish seeds inoculated with plant pathogenic bacteria are co-cultured with the composite microbial inoculum under the laboratory condition; under the condition of a greenhouse, the compound microbial inoculum is used for planting tomatoes, after the compound microbial inoculum is applied for four weeks, the promotion effect of the compound microbial inoculum on tomato plants is obviously higher than that of a control group, the compound microbial inoculum is proved to have a good colonization effect, can adapt to the field environment, and is expected to be applied as the growth of crops to replace part of fertilizer to promote the growth of the crops.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.
FIG. 1 is a microscopic image of the bacterial cells of Bacillus amyloliquefaciens (B. amyloliquefaciens)44 of example 1;
FIG. 2 is a morphological diagram of a colony of Bacillus amyloliquefaciens (B. amyloliquefaciens)44 in example 1;
FIG. 3 is a graph showing the antagonistic effect of Bacillus amyloliquefaciens (B. amyloliquefaciens)44 on Pseudomonas syringae and Fusarium oxysporum in example 1;
FIG. 4 is a graph showing the root growth of garlic sprouts in the compound preparation group and the control group in example 1;
FIG. 5 is a graph showing the growth of tomato plants in the combined preparation group and the control group in example 1.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As introduced in the background art, many related researches are conducted on a bacillus amyloliquefaciens (b. amyloliquefaciens) biocontrol preparation, but the existing biocontrol preparation has certain defects, such as poor field colonization capability, single disease prevention effect, poor comprehensive disease prevention effect and the like. In order to solve the above technical problems, the present disclosure provides a bacillus amyloliquefaciens (b. amyloliquefaciens)44 having good effects of inhibiting phytopathogens and promoting plant growth; in addition, the disclosure also provides a composite microbial inoculum, which comprises bacillus amyloliquefaciens (B.amyloliquefaciens)44, rahnella aquatilis (R.aquatilis)27 and bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619, and the composite microbial inoculum also has good effects of inhibiting phytopathogen and promoting plant growth.
In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.
The general culture medium and its components involved in the experimental procedure were as follows:
NB medium: peptone-1%, beef powder-0.3%, and sodium chloride-0.5%; agar-2% is NA medium.
PDA culture medium: potato-2%, glucose-0.2%, agar-2%.
Example 1
Screening and purifying of bacterial strains
Collecting soil sample from Ningyang county of Shandong province, weighing 1g of the sample, inoculating into 100ml of sterile water, shake culturing at 30 deg.C in incubator for 5-7d, and subjecting the soil sample solution to 105-106The dilution was applied to a plate containing NA solid medium containing 2% agar and incubated overnight at 30 ℃. Separating and purifying the strain from the plate with the grown colony, and inoculating the strain into an NA slant culture medium for preservation.
II, identifying strains
The purified strain was observed under a microscope for its characteristics (fig. 1, 2): bacterial straight rods with the size of 0.6 multiplied by 1.0 to 1.4 mu m and round ends, which are grouped singly or in pairs to form endospores; the spores are oval with a central or sub-apical orientation; there was no swelling of the sporangia. After 48h incubation the colonies were smooth, opaque, irregular, grey, 2-4mm in diameter, with serrated edges and flat profile, slightly raised in the centre, and thick in texture.
The objective strain is further verified by referring to Bergey's manual of bacteriology and identification methods for physiological and biochemical experiments, and the reaction is as follows:
TABLE 1 physiological and biochemical Properties of the strains
And (3) selecting a single colony, inoculating the single colony into an NB liquid culture medium, performing shake culture at 37 ℃ and 180rpm for 24 hours, and extracting the genomic DNA of the strain from 1-5ml of bacterial liquid by using a bacterial genomic DNA extraction kit. The 16S rRNA universal primers 8f (5'-agagtttgatcctggctcag-3') and 1492r (5'-ggttaccttgttacgactt-3') amplified the genomic DNA of the strain and sent to Shanghai Biotech for sequencing. The BLAST software compares and analyzes that the identity of the strain can reach 99 percent with the following strains: bacillus amyloliquefaciens subsp. plantarum FZB42 (99%), Bacillus subtilis 168 (99%), Bacillus vallisort IAM 12118 (99%); the accuracy coefficient of MALDI-TOF mass spectrometry and B.amyloliquefaciens is 2.294.
The strain is determined to belong to bacillus amyloliquefaciens by the comprehensive sequencing and physiological biochemical reaction results, and is delivered into China center for type culture Collection of Wuhan university for preservation with the preservation number of CCTCC M2018564.
Third, optimization of culture medium and culture conditions
In order to optimize the composition of the main nutrient medium for the cultivation of strain 44, cane molasses (1-3% concentration) and soybean meal (1-4%) were selected as carbon and nitrogen source, respectively (table 2). And counting the bacterial liquid by a multiple dilution counting method.
TABLE 2 viable count of Bacillus amyloliquefaciens (B. amyloliquefaciens) on nutrient media of different composition
And (4) surface note:
1. the 1# culture medium comprises 3.0% of cane molasses; k2HPO4 0.7％；KH2PO4 0.3％；(NH)2SO40.15 percent; 0.05% of sodium citrate; MgSO (MgSO)4·7H20.01 percent of O and the balance of water; the pH value is 7.0-7.2.
2. 1# culture medium + 1.0% of soybean meal
3. 1# culture medium + soybean meal 1.5%
4. 1# Medium + Bean pulp 1.5%, omitting (NH)2SO4
5. 1# culture medium + soybean meal 2.0%
6. 1# Medium + Soy meal 2.0%, omitted (NH)2SO4
According to the comprehensive analysis of the viable count and the spore count, the components of the culture medium are finally determined as follows: molasses 3.0%, soybean meal 2.0%, K2HPO4×3H2O 0.7％，KH2PO4 0.3％，(NH4)2SO40.15 percent of sodium citrate multiplied by 2H2O 0.05％，MgSO4×7H20.01 percent of O and the balance of water.
And (3) placing the culture medium inoculated with the strain into shaking tables at different temperatures for 48h, and confirming that the strain can grow in an environment of 10-40 ℃ and grows fastest in an environment of 30-40 ℃ according to the turbidity degree of the bacterial liquid and no foreign bacteria are detected by microscopic examination. And (3) placing the culture medium inoculated with the strain into different initial pH value environments for shake cultivation for 48 hours, wherein the strain can grow in the environment with the pH value ranging from 3.5 to 8.5 and grows fastest in the environment with the pH value ranging from 6.5 to 7.5.
Culturing at 37 deg.C and pH 6.8 with shaking at 120rpm, 150rpm, and 180rpm, and collecting culture solution at 12 th, 24 th, 36 th, 48 th, and 60 th positions for counting. As shown in tables 3 and 4:
TABLE 3 optimization of culture conditions for Bacillus amyloliquefaciens (B. amyloliquefaciens)44 1
TABLE 4 optimization of culture conditions for Bacillus amyloliquefaciens (B. amyloliquefaciens)44 2
Finally, the optimal culture conditions of the bacillus amyloliquefaciens (B. amyloliquefaciens)44 are determined as follows: the growth number is 6.8; the temperature is 37 ℃, the stirring speed is 180r/min, and the culture time is 48 h.
Growth promotion experiment of Bacillus amyloliquefaciens strain 44
The growth promoting effect of the bacillus amyloliquefaciens (b. amyloliquefaciens)44 strain was tested under laboratory conditions according to the following method (table 5):
1. soaking the seeds in 70% ethanol for two minutes
2. Rinsing with sterile tap water
3. Sterile seeds were soaked in the bacterial formulation for 30 minutes and then placed in a sterile wet chamber on the surface of filter paper in a petri dish under test tube conditions. For each variant, ten seeds were placed in each wet chamber in triplicate. The seeds in the control group were soaked in tap water. The seedlings are cultivated for 7-10 days at 28 ℃.
After 7-10 days, the length and weight of the seedlings were measured.
Level of growth stimulating activity (PGP,%): calculated by the following formula
PGP＝(B-A)/A*100
A is the arithmetic mean of the length (mass) of control seedlings;
b is the arithmetic mean of the length (mass) of the seedlings in the experiment.
Seedling growth was estimated within 2 weeks (see table 5): the application of 2% of culture solution of bacillus amyloliquefaciens (B.amyloliquefaciens)44 increases the length and the weight of seedlings by 20.19% and 26.7% respectively, and the bacillus amyloliquefaciens (B.amyloliquefaciens)44 has a certain growth promoting effect.
TABLE 5 growth promoting action of Bacillus amyloliquefaciens strain 44
Fifth, bacteriostasis experiment
The inhibition effect of bacillus amyloliquefaciens (B.amyloliquefaciens)44 on main pathogenic bacteria and fungi in agriculture is measured by adopting a plate punching method, a lower layer culture medium is a 2% NA solid culture medium, 1ml of pathogenic bacteria and 5ml of 1.2% NA culture medium (or 1.2% PDA culture medium) are fully and uniformly mixed to be used as an upper layer culture medium after solidification, a puncher punches holes after solidification, 100 mu l of bacillus amyloliquefaciens (B.amyloliquefaciens)44 bacterial liquid is added, the mixture is kept still for 2h in a refrigerator, and the mixture is vertically cultured for 48h at 37 ℃ (bacteria)/28 ℃ (fungi). And detecting the bacteriostatic ability of the bacteria to be detected by measuring the diameter of the bacteriostatic zone. The results are shown in tables 6 and 7:
TABLE 6 inhibition of plant fungal pathogens by Bacillus amyloliquefaciens (B. amyloliquefaciens)44
TABLE 7 inhibition of plant bacterial pathogens by Bacillus amyloliquefaciens (B. amyloliquefaciens)44
Bacillus amyloliquefaciens (b. amyloliquefaciens)44 has good inhibitory action on plant fungi pathogenic bacteria (fusarium oxysporum, rhizoctonia solani, alternaria alternata, sclerotinia sclerotiorum and wheat root rot pathogenic bacteria) and plant bacteria pathogenic bacteria (erwinia bacillus, agrobacterium tumefaciens, xanthomonas campestris, pseudomonas syringae, tomato canker and potato phytophthora parasitica), and the inhibition zones are respectively 13-23mm and 20-31 mm.
Growth promotion experiment of compound preparation
Two strains of Rahnella aquatilis (R.aquatilis)27 and Bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619 with better rooting promoting effect are screened from an existing strain bank in a laboratory, and are compounded with the Bacillus amyloliquefaciens (B.amyloliquefaciens)44 to prepare the microecological preparation.
The culture collection unit of the 27 strains of the Rahnella aquatica (R.aquatilis) is the China center for type culture collection of Wuhan university,the preservation number is as follows: CCTCC M2018566. The culture medium and fermentation conditions of the rahnella aquatilis (r. aquatilis)27 are as follows: corn starch 3.0%, peptone 2.0%, K2HPO4×3H2O 0.7％，KH2PO4 0.3％，(NH4)2SO40.15 percent of sodium citrate multiplied by 2H2O 0.05％，MgSO4×7H20.01 percent of O; the balance being water. The growth number is 6.8; culturing at 37 deg.C and stirring rate of 150rpm/min for 48 hr to obtain Rahnella aquatilis (R.aquatilis)27 fermentation liquid.
The bacillus amyloliquefaciens (B.amyloliquefaciens) B-1619 strain is purchased from China general microbiological culture collection center, and the bacterial liquid preparation method comprises the following steps: 5% of glucose, 5% of tryptone, 5% of yeast extract and the balance of water; pH 7.0; culturing at 28 deg.C and stirring speed of 180rpm/min for 48 hr to obtain fermentation liquid of Bacillus amyloliquefaciens B-1619.
The formula I is as follows: a complex formulation consisting of bacillus amyloliquefaciens (b. amyloliquefaciens) 44: rahnella aquatica (r. aquatilis) 27: bacillus amyloliquefaciens (B. amyloliquefaciens) B-1619 ═ 1:1: 1.
And a second formula: a complex formulation consisting of bacillus amyloliquefaciens (b. amyloliquefaciens) 44: rahnella aquatica (r. aquatilis) 27: bacillus amyloliquefaciens (B. amyloliquefaciens) B-1619 ═ 5:3: 2.
1. Potting experiments were performed under laboratory conditions.
In order to examine the growth promoting capability of the composite microbial inoculum on plants, in the embodiment, garlic is taken as a representative of the plants, healthy garlic with the same size is selected for planting, an experimental group and a control group are arranged, and each group is repeated for 3 times. The experimental group is a micro-ecological preparation, the experimental group is irrigated with products diluted by 300 times and 500 times, the control group is irrigated with clear water, and the garlic is irrigated once 3-5 days after being planted. The total length, root length and root weight of garlic are measured after 15 days, and the data are shown in table 8.
TABLE 8 comparison of growth of garlic in different solutions
And (4) surface note: the method comprises the following steps: a comparison group, namely clear water, a formula II is diluted by 500 times for use; ③ the formula II, the product is diluted by 300 times for use; fourthly, the formula I is diluted by 500 times for use; fifthly, the formula I is diluted by 300 times for use.
As can be seen by comparing each group of data in the table, the garlic applied to the formula one group is superior to other experimental groups in the aspects of root length and weight, the experimental effect of the formula one group is better than that of the formula two group, and the comparison result with the control group data shows that the garlic poured by the formula one group has better growth condition, the total length of the garlic is increased by 13.56%, the root length is increased by 17.64%, and the dry weight of the root is increased by 28.75%. The results demonstrate that the complex formulation has a good root-promoting effect on plants (fig. 4). In conclusion, the addition ratio of the three strains is 1:1:1, and the amount of the three strains is 300 times diluted.
Mixing the three strains of fermentation liquor according to the proportion of 1:1:1 to obtain a compound preparation, diluting the compound preparation by 50 times to prepare the compound preparation with the content of 2 percent, and performing a growth promotion experiment on tomato and lettuce seeds. The seeds were treated according to the experimental method in "growth promotion by bacillus amyloliquefaciens (b. amyloliquefaciens) 44", and the growth of seedlings was counted after 2 weeks, and the results showed that the germination rates of the tomato and lettuce seeds were increased by 23% and 24%, the weight of the seedlings by 22% and 28%, and the length of the seedlings by 29% and 27%, respectively, after treatment. (tables 9 and 10).
Table 9, effect of the complex formulation on germination rate of tomato seeds and seedling growth.
Table 10, effect of the composite formulation on germination rate of lettuce seeds and seedling growth.
2. Biocontrol effect of composite preparation under laboratory conditions
In the embodiment, radish seeds are taken as a representative of plants, and in order to examine the inhibition effect of the composite microbial inoculum on plant pathogenic bacteria, fusarium oxysporum and pseudomonas syringae are respectively selected as representatives of plant fungal pathogenic bacteria and bacterial pathogenic bacteria to examine the bacteriostasis effect of the composite microbial inoculum.
Selecting plump radish seeds, washing with sterile water for 3 times, germinating in a climatic chamber at 28 deg.C, and sowing in plastic seedling raising tray (6cm × 6cm × 5.5cm high) when the sprouts grow to 1cm, wherein each hole has 10-15 seeds. Spraying the compound preparation with the concentration of 2% after 21d planting, spraying 15mL in each 4-hole, and treating with clear water as a control. Inoculating Fusarium oxysporum and Pseudomonas syringae suspension after 24h, wherein the concentration of the fusarium oxysporum and the Pseudomonas syringae suspension is 1 × 106CFU/mL. After 7d, the disease occurrence was investigated, and disease index and control effect were calculated (Table 11). Grading the disease condition standard: grade 0, no disease in the whole plant; grade 1, the stem and leaf of the plant have few disease spots and occupy less than 1/5 of the total area; grade 2, the stems and leaves of the plants have a few disease spots, and the total area is 1/5-1/3; grade 3, the stems and leaves of the plants have a plurality of disease spots, the total area is 1/3-2/3, and the leaf surfaces turn yellow; and 4, the whole plant is attacked, and the leaf surface is withered. The control effect of the compound preparation on pseudomonas syringae and fusarium oxysporum is 50.94 percent and 68.75 percent respectively.
TABLE 11 control of Pseudomonas and Fusarium oxysporum by the combination
3. Greenhouse test
The method comprises the steps of renting a greenhouse to plant tomatoes, setting 3 groups in an experiment, wherein each group of tomato seedlings counts 300, an experiment group 1 is a product with a first formula, an experiment group 2 is a product with a second formula, the product is used in two experiment groups before the seedlings are transplanted, the usage amount is 2% after the two experiment groups are flushed with water, and a control group is flushed with chemical fertilizer and normally sprayed with pesticide. The three groups are managed uniformly except for variables. Data were measured once per week (table 12). Since the 4 th week after planting, the tomato stem length in the experimental group is obviously higher than that in the control group, which indicates that the flora in the product starts to colonize and play a role, and the effect of the experimental group 1 is better than that of the experimental group 2.
TABLE 12 tomato plant Stem Length (cm)
Counting the disease condition of tomato plants after the tomato seedlings are planted till the fruits are ripe (table 13), and grading the disease condition standard: grade 0, no disease in the whole plant; grade 1, the stem and leaf of the plant have few disease spots and occupy less than 1/3 of the total area; grade 2, the stems and leaves of the plants have a few disease spots, and the total area is 1/3-2/3; grade 3, the stems and leaves of the plants have disease spots, the total area is more than 2/3, the infection condition of the experimental group 1 is better than that of the control group by the analysis of statistical results, the use effect of the composite preparation is slightly better than that of the pesticide of the control group, and the composite preparation can be used for replacing the pesticide sprayed in the growth process of the tomato plants.
TABLE 13 disease status of greenhouse tomatoes
Compare laboratory conditions and experiment cultivated in a pot, show through big-arch shelter experimental result: the compound microbial inoculum can be well colonized in soil under the condition of greenhouse soil, and the fact that the compound microbial inoculum can be practically applied to the actual planting process is proved. The tomato plant applied with the compound microbial inoculum has fewer diseased conditions, and the growth condition of the plant is proved to have good effects of promoting growth and preventing diseases due to the control group and the use of the agricultural chemicals and the fertilizer group.
The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.
SEQUENCE LISTING
<110> Shandong blue Biotech Ltd
<120> bacillus amyloliquefaciens with growth promoting and disease resisting effects and application thereof
<130> 2010
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1454
<212> DNA
<213> Bacillus amyloliquefaciens 4416S rDNA
<400> 1
tgaggtgggt gctaatacat gcaagtcgag cggacagatg ggagcttgct ccctgatgtt 60
agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg 120
aaaccggggc taataccgga tggttgtctg aaccgcatgg ttcagacata aaaggtggct 180
tcggctacca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca 240
ccaaggcgac gatagcgtag ccgacctgag aaggtgatcg gccacactgg aactgagaaa 300
cggcccagac tcctacggga ggcaggcagt aaggaatctt ccgcaatgga cgaaagtctg 360
accgagcaac gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga 420
agaacaagtg ccgttcaaat agggcggcac cttgacggta cctaaccaga aagccacggc 480
taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg ggaattattg 540
ggcgtaaagg gctcgcaggc ggtttcttaa gtctgatgtg aaagcccccg gctcaaccgg 600
ggagggtcat tggaaactgg ggaacttgag tgcagaagag gagagtggaa ttccacgtgt 660
agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcgact ctctggtctg 720
taactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc ctggtagtcc 780
acgccgtaaa cgatgagtgc taagtgttag ggggtttccg ccccttagtg ctgcagctaa 840
cgcattaagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa ggaattgacg 900
ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 960
aggtcttgac atcctctgac aatcctagag ataggacgtc cccttcgggg gcagagtgac 1020
aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080
gcgcaaccct tgatcttagt tgccagcatt cagttgggca ctctaaggtg actgccggtg 1140
acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac 1200
acacgtgcta caatggacag aacaaagggc agcgaaaccg cgaggttaag ccaatcccac 1260
aaatctgttc tcagttcgga tcgcagtctg caactcgact gcgtgaagct ggaatcgcta 1320
gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380
cacaccacga gagtttgtaa cacccgaagt cggtgaggta acctttagga gccagccgcc 1440
gaagtgacag atgg 1454
Claims (10)
1. Bacillus amyloliquefaciens strainB. amyloliquefaciens）44, wherein the strain has been deposited in China Center for Type Culture Collection (CCTCC) at 23.8.8.2018, with the addresses: the biological preservation number of the Wuchang Lojia mountain in Wuhan city of Hubei province is as follows: CCTCC NO: m2018564.
2. A bacterial agent comprising the Bacillus amyloliquefaciens 44 according to claim 1.
3. A complex microbial preparation comprising the Bacillus amyloliquefaciens 44 and Rahnella aquatica (L) as set forth in claim 1Rahnella aquatilis)27 and bacillus amyloliquefaciens B-1619 microbial inoculum, wherein the Laurencia aquatica 27 is preserved in China center for type culture Collection of Wuhan university at 8.23.8.2018, CCTCC for short, and the addresses are as follows: the preservation number of the Wuchang Lojia mountain in Wuhan city of Hubei province is as follows: CCTCC NO: m2018566; the bacillus amyloliquefaciens B-1619 is purchased from China general microbiological culture collection center.
4. The complex bacterial agent as claimed in claim 3, wherein in said complex bacterial agent, the ratio of said bacillus amyloliquefaciens 44: rahnella aquatilis 27: the ratio of the bacillus amyloliquefaciens B-1619 is 0.8-5.2:0.8-3: 0.8-2.
5. The complex bacterial agent as claimed in claim 3, wherein in said complex bacterial agent, the ratio of said bacillus amyloliquefaciens 44: rahnella aquatilis 27: the ratio of the bacillus amyloliquefaciens B-1619 is 1:1: 1.
6. The microbial inoculum according to claim 2 or the composite microbial inoculum according to claim 3 or 4, wherein the dosage form of the microbial inoculum or the composite microbial inoculum is liquid microbial inoculum, powder or granules.
7. The microbial agent according to claim 2 or the complex microbial agent according to claim 3 or 4, wherein the microbial agent or the complex microbial agent is in the form of an aqueous suspension, a dispersible oil suspension, a wettable powder or a water dispersible granule.
8. The microbial inoculum of claim 2 or the composite microbial inoculum of claim 3 or 4, further comprising an agriculturally and pharmaceutically acceptable auxiliary material, wherein the agriculturally and pharmaceutically acceptable auxiliary material is selected from one or more of a dispersing agent, a wetting agent, a disintegrating agent, a binder, an antifoaming agent, an antifreeze agent, a thickening agent, a filler and a solvent.
9. The use of the bacillus amyloliquefaciens 44 of claim 1 and the microbial inoculum of claim 2 in any one of the following aspects:
(1) inhibiting plant fungal pathogens and plant disease bacterial pathogens, wherein the plant fungal pathogens are fusarium oxysporum, rhizoctonia solani, alternaria alternata, sclerotinia sclerotiorum and wheat root rot pathogens, and the plant bacterial pathogens are erwinia, agrobacterium tumefaciens, xanthomonas, pseudomonas syringae, tomato canker pathogen and potato phytophthora parasitica;
(2) promoting the growth of plants, wherein the plants are tomatoes, radishes, cucumbers and wheat.
10. The use of the complex microbial inoculum of any one of claims 3 to 5 in promoting plant growth: it is characterized in that the plants are tomatoes, lettuce, radishes and garlic.
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| CN111205999B (en) * | 2020-01-13 | 2021-09-07 | 中化农业（临沂）研发中心有限公司 | Bacillus amyloliquefaciens CGMCC No.17842 and application thereof |
| CN111197017B (en) * | 2020-01-13 | 2021-09-17 | 中化农业（临沂）研发中心有限公司 | Bacillus amyloliquefaciens CGMCC No.17844 and application thereof |
| CN111205998B (en) * | 2020-01-13 | 2021-09-07 | 中化农业（临沂）研发中心有限公司 | Bacillus amyloliquefaciens CGMCC No.17841 and application thereof |
| CN111117924B (en) * | 2020-01-13 | 2021-09-17 | 中化农业（临沂）研发中心有限公司 | Compound microbial inoculum and preparation method thereof, fertilizer and method for preventing and treating root rot |
| CN111235076B (en) * | 2020-03-31 | 2020-10-30 | 山东碧蓝生物科技有限公司 | Bacillus anethoides, microbial inoculum thereof and application thereof in heavy metal remediation field |
| CN112481158B (en) * | 2020-11-26 | 2022-04-22 | 元和生物科技(德州)有限公司 | Compound microbial agent for potato black nevus |
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Denomination of invention: A Bacillus amylolyticus with growth promoting and disease resistance and its application Effective date of registration: 20221117 Granted publication date: 20210209 Pledgee: Shandong Ningyang Rural Commercial Bank Co.,Ltd. Pledgor: SHANDONG GREENBLUE BIOTECHNOLOGY CO.,LTD. Registration number: Y2022980022154 |
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