CN114916563A - Bacillus amyloliquefaciens and application of synergist thereof - Google Patents

Bacillus amyloliquefaciens and application of synergist thereof Download PDF

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CN114916563A
CN114916563A CN202210479336.5A CN202210479336A CN114916563A CN 114916563 A CN114916563 A CN 114916563A CN 202210479336 A CN202210479336 A CN 202210479336A CN 114916563 A CN114916563 A CN 114916563A
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bacillus amyloliquefaciens
essential oil
tea tree
tree essential
sdtb009
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CN114916563B (en
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王红艳
杨秀
王开运
朱天生
宋应华
姜莉莉
武玉国
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Shandong Agricultural University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/28—Myrtaceae [Myrtle family], e.g. teatree or clove
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides a bacillus amyloliquefaciens strainBacillus amyloliquefaciens) The preservation number is CGMCC No. 19213. The bacillus amyloliquefaciens and the fermentation liquid thereof can be used for inhibiting plant pathogenic bacteria and promoting plant growth, and meanwhile, the tea tree essential oil is used as a synergist to show synergistic effect on disease control effect. The bacillus amyloliquefaciens has the biological control and growth promotion effects, can effectively reduce the occurrence of soil-borne diseases of crops such as tomatoes, strawberries and the like, can effectively prevent and treat the diseases of dry fruit trees such as alternaria leaf spot and black date leaf spot, and reduces the using amount of chemical agents. The preparation prepared from the bacillus amyloliquefaciens strain and the synergist prepared from the tea tree essential oil have stable storage performance, good prevention and treatment effect, no adverse effect on plant growth and development, convenient processing and lower production and transportation costs.

Description

Bacillus amyloliquefaciens and application of synergist thereof
Technical Field
The invention belongs to the technical field of biological pesticides, and particularly relates to bacillus amyloliquefaciens and application of a synergist thereof.
Background
The walnut planting area and the walnut yield in China all account for the first place in the world. The pathogenic bacteria of Alternaria Juglandis leaf spot is Alternaria tenuissima of Alternaria of Deuteromycotina ()Alternaria tenuissima) Can damage the leaves, fruits, buds and young tips of the walnut tree. The disease causes black brown spots on walnut leaves, necrosis and scorching, insufficient walnut grouting and yield reduction, and has potential threat to the development of walnut industry. The alternaria leaf spot of walnut is used as a new disease, and an effective prevention and control green high-efficiency pesticide is lacked in production.
Tomato and strawberry soil-borne diseases become more serious and become important factors for restricting production, wherein the tomato neck rot and strawberry rhizoctonia solani root rot are new diseases in recent years in facility greenhouses. The neck rot and root rot of the tomato are serious in the winter-heating greenhouse and are attacked at low temperature. The tomato neck rot and root rot disease is characterized by fusarium oxysporum tomato neck rot and root rot specialization type (Fusarium oxysporumf. sp, radces-lycopersici, FORL) infection, and the two diseases are caused by fusarium oxysporum as well as tomato blight, but due to the difference of specialization types, the two diseases have great difference in the disease period and disease symptoms. Strawberry blight and root rot are important diseases in strawberry production, and are mainly caused by pathogenic bacteria, Fusarium oxysporum: (A)Fusarium oxysporum) And Rhizoctonia solani (F.), (Rhizoctonia solani) The infection of AG-4 fusion group is developed. Due to the long infection period of soil-borne diseases, difficult control and no registered medicament, great economic loss is caused to the production of tomatoes and strawberries.
At present, the control of fruit and vegetable diseases in China is mainly based on chemical agents, and the frequent and unreasonable use of bactericides not only easily causes the outbreak of drug resistance of pathogenic bacteria, but also has serious threats to the ecological environment and human health. The beneficial microorganisms are developed and utilized as biological antagonistic preparations, so that abundant resources and methods are provided for biological control of fruit and vegetable diseases.
Bacillus amyloliquefaciens (A), (B), (C), (B) and (C)Bacillus amyloliquefaciens) It was first discovered by Priest and classified from bacillus subtilis. The bacillus amyloliquefaciens can generate various substances with bacteriostatic effects, such as antibacterial proteins, lipopeptides, cyclic dipeptides and other substances, has the effects of promoting plant growth and resisting pathogenic microorganisms in agricultural production, has broad-spectrum antibacterial activity, and is a microorganism with great potential in developing biological medicaments.
Tea Tree essential Oil (Tea Tree Oil, TTO) is Melaleuca alternifolia (Melaleuca alternifolia) of Melaleuca (Myrtaceae) speciesMeloleuca alternifolia) The fresh branches and leaves are distilled to obtain the aromatic essential oil. The tea tree essential oil is transparent in color, extremely low in viscosity and strong in volatility. The tea tree essential oil has the effects of efficiently inhibiting the growth of fungal hyphae and spore germination, and achieves the antibacterial effect by destroying the cell wall or cell membrane structure of microorganisms, influencing the morphological structure of the hyphae and the like. In addition, tea tree essential oil has the function of inducing plant disease resistance.
Disclosure of Invention
Aiming at the problems of poor prevention and control efficiency, low prevention and control range and insufficient colonization capacity on plants in the prior art, the invention provides the bacillus amyloliquefaciens SDTB009 which can be used together with tea tree essential oil, can prevent and control diseases on various crops such as walnuts, jujube trees, strawberries, tomatoes and the like, and improves the prevention and control effect.
In order to achieve the purpose, the invention adopts the following technical scheme.
Tea tree essential oil as bacillus amyloliquefaciens (B)Bacillus amyloliquefaciens) SDTB009 synergist application.
The tea tree essential oil meets the requirements of the standard GB/T26514-2011 melaleuca alternifolia (essential) oil and terpinene-4-ol [ tea tree (essential) oil ].
Bacillus amyloliquefaciens (A), (B), (C), (B) and (C)Bacillus amyloliquefaciens) SDTB009 for preventing and treating Alternaria tenuissima (A), (B) and (C)Alternaria tenuissima) Fusarium oxysporum (F.), (Fusarium oxysporumf. sp, fragrae), fusarium oxysporum neck rot root rot specialization type (f: (f)Fusarium oxysporumf. sp, radics-lycopersici) and Rhizoctonia solani: (Rhizoctonia solani) Use of AG-4 fusion group.
The above Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) The preservation number of the SDTB009 is CGMCC No. 19213.
The application is that the bacillus amyloliquefaciens SDTB009 is used alone or in combination with tea tree essential oil.
Preferably, in the application, the use concentration of the tea tree essential oil is 100-500 mg/L.
The applied crops are selected from walnuts, jujubes, tomatoes or strawberries.
A composition comprises Bacillus amyloliquefaciens SDTB009 and tea tree essential oil.
The invention has the following advantages:
the invention provides a bacillus amyloliquefaciens strain capable of inhibiting plant pathogenic bacteria and promoting plant growth and a fermentation liquid thereof, and simultaneously, tea tree essential oil is used as a synergist, so that the bacillus amyloliquefaciens strain has an obvious synergistic effect on a disease control effect. The bacillus amyloliquefaciens has the biological control and growth promotion effects, can effectively reduce the occurrence of soil-borne diseases of crops such as tomatoes, strawberries and the like, can effectively prevent and treat the diseases of dry fruit trees such as alternaria leaf spot and black jujube leaf spot, and reduces the use amount of chemical agents. The preparation prepared from the bacillus amyloliquefaciens strain and the synergist prepared from the tea tree essential oil have stable storage performance, good prevention and treatment effect, no adverse effect on plant growth and development, convenient processing and lower production and transportation costs.
Biological preservation information
Bacillus amyloliquefaciens (A), (B), (C), (B) and (C)Bacillus amyloliquefaciens) SDTB009, which is deposited in China general microbiological culture Collection center (CGMCC) at 12 months and 16 days in 2019, is deposited at the institute of microbiology, institute of academy of sciences of China, No. 3 of Xilu No.1, Beijing, Chaoyang, Beicheng, and Beicheng,the preservation number is CGMCC number 19213.
Drawings
FIG. 1 is a drawing (A) of streaked plate culture of Bacillus amyloliquefaciens, a drawing (B) of action of hydrolyzed starch of the culture, and a drawing (C) of transmission electron microscopy of the cells;
FIG. 2 is a graph showing the effect of the addition of tea tree essential oil to a final concentration of 0.5mg/L on the growth of 2 strains of Bacillus amyloliquefaciens strain LB medium, wherein A is Bacillus amyloliquefaciens strain SDTB009 and B is Bacillus amyloliquefaciens JM-3;
FIG. 3 is a graph of the effect of the addition of tea tree essential oil at a final concentration of 0.5mg/L on the fermentation of 2 strains of Bacillus amyloliquefaciens, wherein treatment 1 is negative control LB medium, 2 is Bacillus amyloliquefaciens SDTB009 strain shake-cultured, 3 is Bacillus amyloliquefaciens SDTB009 strain shake-cultured after the addition of essential oil, 4 is Bacillus amyloliquefaciens strain JM-3 shake-cultured, and 5 is Bacillus amyloliquefaciens JM-3 strain shake-cultured after the addition of essential oil;
FIG. 4 is an antagonistic effect of Bacillus amyloliquefaciens on target pathogenic bacteria, wherein A is an antibacterial effect of confronting a tomato neck rot root rot plate and adding tea tree essential oil, B is an antibacterial effect of confronting a strawberry wilt bacterium plate and adding tea tree essential oil, C is an antibacterial effect of confronting a walnut alternaria alternata leaf spot plate and adding tea tree essential oil, D is an antibacterial effect of confronting a jujube black spot bacterium and adding tea tree essential oil, and E is an antibacterial effect of confronting a Rhizoctonia solani AG-4 fusion group plate and adding tea tree essential oil.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
Example 1 isolation and characterization of SDTB009
In 8 months 2017, collecting soil of a facility greenhouse with a good effect of solving continuous cropping obstacles in a Dayue district of Taian city, Shandong, separating bacterial strains from the soil, wherein the specific separation and purification processes are as follows: weighing 10g of sieved soil sample, placing into a 250mL sterilized triangular flask containing 90mL sterile water and a small amount of glass beads, placing the triangular flask on a shaker at 37 ℃ and 180rpm for shaking for 30min to fully suspend, and then performing water bath at 80 DEG CTreating for 10min, diluting the bacterial suspension with sterile water to 10% -4 、10 -5 、10 -6 And (4) grading. Respectively sucking 100 mu L of each gradient solution, uniformly coating the gradient solution on an NA culture medium flat plate, repeating each gradient for 3 times, placing the gradient solution in a constant temperature incubator at 28 ℃ for culturing for 1-2d, and then picking different single colonies for streak purification by the flat plate. The purified strains are selected from the group consisting of tomato rhizoctonia solani (A)Fusarium oxysporumf. sp, radis-lycopersici) as a target bacterium to obtain a target strain with the largest inhibition zone, wherein the number of the target strain is SDTB 009. Morphological identification and physiological and biochemical characteristic tests are carried out on the strain, 16s rDNA sequencing is carried out on the strain, and a phylogenetic tree is constructed.
TABLE 1 physiological and biochemical characteristics of the strains
Figure 446950DEST_PATH_IMAGE001
The culture morphology and the transmission electron microscope photograph are shown in figure 1, after the target strain is cultured on an NA solid medium for 24 hours at 37 ℃, the bacterial colony is milky white, opaque, irregular and round in edge and gram-positive in staining. The rod-shaped thallus cells are observed under a transmission electron microscope, have thicker capsules and periphytic flagella and are about 1.2-2.5 mu m in length. Combining the morphological, physiological and biochemical characteristics and the 16s rDNA sequence homology comparison result of the target strain to identify the strain as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) And the strain is preserved in the China general microbiological culture Collection center on 16 th 12 th 2019, with the preservation number of CGMCC 19213.
Example 2 tea tree essential oil synergizing action on Bacillus amyloliquefaciens SDTB009
1. Effect of tea Tree essential oil on growth of Bacillus amyloliquefaciens SDTB009
Plates were prepared by pouring 15mL of LB medium into the filter paper-attached dish on a clean bench. The single colony of Bacillus amyloliquefaciens described in example 1 was streaked using a streaking ring, and the antagonistic bacterium, Bacillus amyloliquefaciens JM-3 (accession number: CGMCC number 15444), was used as a control. Dripping tea tree essential oil with final concentration of 0.5mg/L onto the filter paper sheet, and rapidly sealing. After 24h of dark culture at 30 ℃, the streaking effect is observed. As shown in FIG. 2, the tea tree essential oil showed no inhibition of the growth of Bacillus amyloliquefaciens strain SDTB009, but showed significant inhibition of the growth of Bacillus amyloliquefaciens strain JM-3.
Measuring 5mL of LB liquid culture medium on a clean bench, adding the LB liquid culture medium into a sterilization test tube, picking out a single colony of the Bacillus amyloliquefaciens described in the embodiment 1 by using a sterilization toothpick, adding the single colony into the test tube, sticking a filter paper sheet on the inner side of a test tube plug, dripping tea tree essential oil with the final concentration of 0.5mg/L on the filter paper sheet, and quickly sealing. After culturing at 30 ℃ in dark at 180rpm for 24h with shaking, detecting the OD of the fermentation liquid 600 Values were negative control with LB medium, positive control with Bacillus amyloliquefaciens SDTB009 without essential oil treatment, and control with Bacillus amyloliquefaciens JM-3. The results are shown in fig. 3, the tea tree essential oil has no inhibition on the fermentation of bacillus amyloliquefaciens SDTB009 strain, and has significant inhibition effect on the fermentation of bacillus amyloliquefaciens JM-3 strain. The 0.5mg/L tea tree essential oil has no inhibiting effect on the bacillus amyloliquefaciens SDTB009 strain, and can be used together with the bacillus amyloliquefaciens SDTB009 for preventing and treating target pathogenic bacteria.
2. Synergism of tea tree essential oil on bacillus amyloliquefaciens SDTB009
The method comprises the steps of mixing the rhizoctonia solani (fusarium oxysporum tomato rhizoctonia solani) with a carrier,Fusarium oxysporumf. sp, radis-lycopersici), strawberry blight bacterium (F. oxysporumf. sp, fragrae), alternaria Juglandis blotch (alternaria tenuis,Alternaria tenuissima) Jujube black spot pathogen (alternaria tenuissima,A. tenuissima) And rhizoctonia solani of strawberry (rhizoctonia solani AG-4 fusion group,Rhizoctonia solani) Activating for standby. Selecting a culture dish with the diameter of 9cm, pasting 1 filter paper sheet with the diameter of 5mm in the center of the dish cover, and sterilizing for later use.
On a clean bench, 15mL of PDA medium was poured into a sterile plate to prepare a PDA solid medium plate. 15mL of PDA medium was poured into the dish with the filter paper attached, and a PDA plate was prepared. On a clean bench, pre-activated vigorous growing tomato root-rot fungus, strawberry fusarium wilt fungus, walnut alternaria leaf spot fungus, jujube black spot fungus and strawberry rhizoctonia solani fungus cakes with the diameter of 7mm are respectively inoculated in the centers of two PDA culture medium flat plates. Meanwhile, 0.5mg/L tea tree essential oil is dripped on the filter paper sheet, and the opening is rapidly sealed. After culturing for 24h in the dark at 25 ℃, picking the activated single colony of the bacillus amyloliquefaciens described in the example 1 by using a sterilizing toothpick, inoculating bacteria at a position 2cm away from the center of a fungus cake, quickly sealing by using a sealing film, culturing for 3-7d in the dark at 20-28 ℃, measuring the diameter of a bacteriostatic zone and calculating the efficiency increase.
TABLE 2 synergistic effect of Bacillus amyloliquefaciens on the zone of inhibition of target pathogenic bacteria and tea tree essential oil
Figure 563198DEST_PATH_IMAGE002
The antagonistic effect of bacillus amyloliquefaciens SDTB009 against target pathogenic bacteria and the synergistic effect of tea tree essential oil on bacillus amyloliquefaciens are shown in FIG. 4, and the inhibition zone results are shown in Table 2. As can be seen from FIG. 4 and Table 2, the inhibition effect of Bacillus amyloliquefaciens on various pathogenic bacteria is very obvious, and the inhibition zone is clearly visible. After the tea tree essential oil is added, the synergistic effect on the bacillus amyloliquefaciens is remarkable. Therefore, the bacillus amyloliquefaciens is effective on various fungal diseases, and the bacteriostatic action of the tea tree essential oil on the bacillus amyloliquefaciens is unexpected synergistic effect.
Example 4 potted plant control Effect on Rhizoctonia solani of strawberry
Activated single colonies of Bacillus amyloliquefaciens SDTB009 of example 1 were picked, inoculated into a 500mL Erlenmeyer flask containing about 300mL of sterilized LB liquid medium, shake-cultured at 37 ℃ and 150rpm for 24 hours, and the cell concentration was adjusted to 10 7 cfu/mL of Bacillus amyloliquefaciens bacterial suspension. Adding emulsifier and solvent into tea tree essential oil to prepare 10% mother liquor, and diluting to 1% emulsion.
Soil is taken from a land where the strawberry rhizoctonia root rot occurs seriously, the land is filled in a plastic flowerpot with the diameter of 15cm, and the cultured rhizoctonia solani AG-4 group is artificially inoculated. Transplanting strawberry seedlings, 1 plant/pot, and filling each pot of strawberry with biocontrol bacteria treatment groupRoot treatment 30mL concentration of 10 7 cfu/mL of Bacillus amyloliquefaciens SDTB009 suspension, and the control group replaced the bacterial liquid with clear water. Synergist treatment group 10mL of 1% tea tree essential oil emulsion was treated per pot of strawberry root irrigation, except the same procedure as above. Each treatment was set up to 4 replicates, each 10 pots. The disease incidence of each treatment group was investigated every 15d by using conventional cultivation management, and the relative control effect (%) = (control group disease incidence-treatment group disease incidence)/control group disease incidence x 100%) was calculated and continuously investigated for 2 times; at 30d, 10 plants in each of the treatment group and the control group were taken for root length measurement.
TABLE 3 potted plant test results of Bacillus amyloliquefaciens bacterial liquid on strawberry rhizoctonia root rot
Figure 892417DEST_PATH_IMAGE003
The data in the table are counted by a new repolarization method, and the difference of the P <0.05 level is obvious by different lower case letters after the data.
The results are shown in table 3: the disease rate of the control group is 100% 15d and 30d after transplanting. The control effects of the bacillus amyloliquefaciens SDTB009 on the rhizoctonia solani root rot are respectively 58.5% and 55.6%, and the control effects are obvious. The control effect of the treatment group added with the tea tree essential oil synergist on the rhizoctonia solani of strawberries is 72.5 percent and 74.9 percent respectively, is obviously higher than that of the treatment group added with the bacillus amyloliquefaciens used alone, and the synergistic effect is obvious (P is less than 0.05). The heights of the strawberry plants are measured 30 days after treatment, the heights of all treatment groups are obviously higher than those of a clear water control group, and the bacillus amyloliquefaciens SDTB009 has an obvious growth promoting effect (P is less than 0.05) on the strawberry plants.
Example 4 preparation of Bacillus amyloliquefaciens microbial agent and tea tree essential oil synergist
(1) Preparing bacterial liquid: a single colony of the Bacillus amyloliquefaciens SDTB009 purified in example 1, which had been activated in advance on a LB medium solid plate, was picked up, inoculated into a 500mL Erlenmeyer flask containing about 300mL of sterilized LB liquid medium, and shake-cultured at 37 ℃ and 150rpm for 24 hours to prepare a strain seed solution. Transferring the seed solution into 100L fermentation tank containing 50L sterilized LB liquid culture medium, culturing at 37 deg.C and 300rpmCulturing for 16h to obtain thallus concentration of 10 8 -10 9 cfu/mL of fermentation broth;
(2) and (2) centrifuging the fermentation liquor prepared in the step (1) for 15min at 4000rpm, and removing the supernatant to leave a precipitate. Uniformly mixing 1kg of precipitate, 0.5kg of potassium fulvate, 0.5kg of diatomite and 1kg of humic acid, drying at 60 ℃ until the water content is less than 15%, crushing and sieving to obtain the powdery microbial agent with the viable bacteria content of 5 hundred million cfu/g.
(3) Preparing the tea tree essential oil synergist: preparing tea tree essential oil into 10% tea tree essential oil missible oil: 10% of tea tree essential oil, 84% of dimethylbenzene and 6% of pesticide emulsion 2201 emulsifier; mixing the above materials, and storing in refrigerator at 4 deg.C.
Example 5 field control of Bacillus amyloliquefaciens microbial agent on Alternaria Juglandis blotch
And carrying out a test in an orchard where alternaria juglandis leaf spot occurs for many years continuously. The medicine is applied before or sporadically before the disease occurs, and is continuously applied twice at an interval of 10 days. The microbial inoculum treatment group was sprayed with 500-fold solution of bacillus amyloliquefaciens microbial inoculum prepared in example 4. Each treatment was repeated 4 times, each for 2 walnut trees. And the synergist processing group is used for diluting 10% tea tree essential oil missible oil into 1% aqueous emulsion by adding water before spraying the microbial agent, then adding the aqueous emulsion into the prepared liquid medicine until the dilution times are 20 times and 100 times, uniformly stirring and spraying. The control group was untreated, randomized, block-wise, with guard rows between treatments.
By adopting conventional cultivation management, the disease incidence is investigated once each 10 days after the first drug and 10 days after the second drug. The investigation method is that sampling is carried out according to a diagonal five-point sampling method for each treatment, and 2 plants are investigated respectively. Each tree is sampled according to east, south, west, north and middle 5 directions (taking the upper, middle and lower 3 layers of leaves into consideration when sampling), 100 leaves are investigated (20 leaves are investigated in each direction), the number of diseased leaves is recorded, the disease incidence is calculated, and the control effect (%) = (control group disease incidence-treatment group disease incidence)/control group disease incidence x 100%) is calculated.
TABLE 4 field control of Alternaria Juglandis blotch by Bacillus amyloliquefaciens preparation and synergist
Figure 170339DEST_PATH_IMAGE004
The results are shown in Table 4: compared with a control group, in two result surveys, the relative prevention effect of bacillus amyloliquefaciens microbial inoculum treatment on alternaria walnuts leaf spot is 73.77% and 72.80% respectively, the relative prevention effect of microbial inoculum + tea tree essential oil synergist (100 mg/L) treatment on alternaria walnuts leaf spot is 86.34% and 90.80% respectively, the relative prevention effect of microbial inoculum + tea tree essential oil synergist (500 mg/L) treatment on alternaria walnuts leaf spot is 91.80% and 93.87% respectively, and the prevention effect of 2 treatments added with the tea tree essential oil synergist is obviously higher than that of the control treatment added with the microbial inoculum alone (P < 0.05). The bacillus amyloliquefaciens SDTB009 treated by a microbial agent and added with a tea tree essential oil synergist both remarkably reduce the morbidity of alternaria walnuts leaf spot, and the tea tree essential oil synergist added treatment has good persistence on disease control and remarkable synergism effect.

Claims (6)

1. Tea tree essential oil as bacillus amyloliquefaciens (B)Bacillus amyloliquefaciens) The application of the SDTB009 synergist is characterized in that the preservation number of the Bacillus amyloliquefaciens SDTB009 is CGMCC number 19213.
2. Bacillus amyloliquefaciens (A), (B), (C), (B) and (C)Bacillus amyloliquefaciens) SDTB009 used for preventing and treating Alternaria tenuissima (A), (B), (C) and (C)Alternaria tenuissima) Fusarium oxysporum (F.), (Fusarium oxysporumf. sp, fragrae), fusarium oxysporum neck rot root rot specialization type (Fusarium oxysporumf. sp, radics-lycopersici) and Rhizoctonia solani: (Rhizoctonia solani) The application of AG-4 fusion group is characterized in that the preservation number of the bacillus amyloliquefaciens SDTB009 is CGMCC number 19213.
3. The use as claimed in claim 2, wherein the bacillus amyloliquefaciens SDTB009 is used alone or in combination with tea tree essential oil.
4. The application as claimed in claim 1 or 3, wherein the concentration of tea tree essential oil is 100-500 mg/L.
5. Use according to claim 2, wherein the crops applied are selected from walnuts, jujubes, tomatoes or strawberries.
6. A composition comprising Bacillus amyloliquefaciensBacillus amyloliquefaciens) The composition of SDTB009 and tea tree essential oil, is characterized in that the preservation number of the Bacillus amyloliquefaciens SDTB009 is CGMCC number 19213.
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