CN113881599A

CN113881599A - Composite microbial fertilizer for bacillus subtilis and tomato, preparation method and application thereof

Info

Publication number: CN113881599A
Application number: CN202111242903.7A
Authority: CN
Inventors: 乔艳; 李双来; 胡诚; 张智; 刘东海; 李世涛; 金利容
Original assignee: Institute of Plant Protection and Soil Fertilizer of Hubei Academy of Agricultural Science
Current assignee: Institute of Plant Protection and Soil Fertilizer of Hubei Academy of Agricultural Science
Priority date: 2021-10-25
Filing date: 2021-10-25
Publication date: 2022-01-04
Anticipated expiration: 2041-10-25
Also published as: CN113881599B

Abstract

The application discloses bacillus subtilis, a composite microbial fertilizer, a preparation method and application thereof. The Bacillus subtilis is preserved in China center for type culture collection, the preservation number is CCTCC No. M2021996, the Bacillus subtilis QK-1 is named by classification, the preservation date is 2021 year 08 month 06, and the preservation address is eight-path Lopa mountain in Wuchang district, Wuhan city, Hubei province. The bacillus subtilis is derived from wormcast, can generate antagonistic action with tomato fusarium oxysporum and pseudomonas solanacearum, and plays a key role in tomato disease resistance; meanwhile, the strain is transplanted in wormcast again for propagation, so that special dominant strains can be formed in the wormcast, and the quality and the quantity of the strains are improved; the compound microbial fertilizer prepared by the method can help tomatoes resist diseases such as blight, bacterial wilt and the like, provide nutrient components required by the growth of the tomatoes and promote the growth of the tomatoes.

Description

Composite microbial fertilizer for bacillus subtilis and tomato, preparation method and application thereof

Technical Field

The application relates to the technical field of tomato crops, in particular to a bacillus subtilis and tomato composite microbial fertilizer, a preparation method and application thereof.

Background

Tomatoes contain a large amount of vitamins and lycopene, wherein each 100 g of tomatoes contains nearly 20 mg of vitamin C, and lycopene also becomes one of the main reasons for the popularity of tomatoes, has very strong oxidation resistance, and has good effects on aging resistance and male prostate diseases. The main diseases of the tomato include fungal wilt, early and late blight, gray mold and leaf mold; viral diseases; bacterial blight, spot blight and physiological diseases have great influence on tomato production, particularly in facility cultivation, chemical disinfection of soil before disease planting can cause environmental risks caused by excessive use of chemical pesticides, and increase of tolerance of pathogenic bacteria is caused, so that disease control is more difficult, and vicious circle is formed.

Disclosure of Invention

In view of this, the present application aims to provide at least one compound microbial fertilizer, which is applied to tomato production, not only provides nutrition for tomatoes, but also has a good prevention effect on tomato diseases, can reduce the environmental pollution risk caused by using chemical pesticides, and can simultaneously alleviate the restriction of soil barrier factors on tomato production.

In a first aspect, the embodiment of the application discloses a strain of Bacillus subtilis which is preserved in China center for type culture Collection, the preservation number is CCTCC No. M2021996, the Bacillus subtilis QK-1 is named in classification, the preservation date is 2021 year 08 month 06, and the preservation address is eight-way Lopa mountain in Wuchang district, Wuhan City, Hubei province.

In a second aspect, the embodiment of the application discloses a compound microbial fertilizer for tomatoes, which comprises the bacillus subtilis with the concentration of not less than 0.2 hundred million/g, organic matter with the concentration of not less than 20 wt%, and N-P with the concentration of not less than 8 wt%₂O₅-K₂O, 1-2 wt% of medium trace elements, 2-5 wt% of CaO and 1-2 wt% of MgO.

In a third aspect, the embodiment of the application discloses a preparation method of a compound microbial fertilizer, which comprises the following steps:

fermenting and culturing the Bacillus subtilis QK-1 until the number of viable bacteria is not less than 7 hundred million/mL and the spore rate reaches 90%, stopping fermentation, collecting fermentation liquor, centrifuging and removing supernatant liquid to obtain concentrated bacterial liquid for later use;

uniformly mixing humic acid, biochar and concentrated bacterial liquid, adding the mixture into wormcast, and continuously culturing for 48 hours to obtain a culture;

and adding amino acid calcium, amino acid magnesium, phosphate fertilizer and potash fertilizer into the culture, and uniformly mixing to obtain the compound microbial fertilizer for tomatoes.

In the embodiment of the application, the addition amount of the humic acid and the biochar is 1 wt% of the weight of the wormcast.

In the embodiment of the application, the adding amount of the calcium amino acid and the magnesium amino acid is 1 wt% of the weight of the wormcast, the adding amount of the phosphate fertilizer is 3 wt% of the weight of the wormcast, and the adding amount of the potassium fertilizer is 2 wt% of the weight of the wormcast.

In a fourth aspect, the embodiment of the application also discloses the application of the bacillus subtilis in the first aspect, the composite microbial fertilizer in the second aspect, or the composite microbial fertilizer prepared by the preparation method in the third aspect in tomato seedling cultivation.

In a fifth aspect, the present application also discloses the application of the bacillus subtilis related to the first aspect in resisting Fusarium oxysporum (Fusarium oxysporum.f.sp.cucmrium) or pseudomonas solanacearum (Pseudo-monas solanacearum).

Compared with the prior art, the embodiment of the application has at least the following beneficial effects:

the bacillus subtilis is derived from wormcast, can generate antagonistic action on fusarium oxysporum and pseudomonas solanacearum, and plays a key role in tomato disease resistance; meanwhile, the strain is transplanted in wormcast again for propagation to form a special dominant strain, so that the quality and the quantity of the strain are improved; the chelated medium-amount Mg and Ca elements and a proper amount of phosphorus and potassium elements are added to prepare the compound microbial fertilizer, and the compound microbial fertilizer can help tomatoes to resist diseases such as blight, bacterial wilt and the like, can provide nutrient components required by the growth of the tomatoes and promote the growth of the tomatoes.

Drawings

FIG. 1 is a diagram showing the bacteriostatic effect of bacteria No.1 on pathogenic bacteria of tomato wilt disease provided in the example of the present application; the left picture is a pathogenic bacteria plate of the blight, and the right picture is a culture plate (the middle is pathogenic bacteria plaque) for the bacteria and the pathogenic bacteria of the blight.

FIG. 2 is a diagram showing the bacteriostatic effect of bacteria # 2 on pathogenic bacteria of tomato wilt disease according to the embodiment of the present application; the left picture is a pathogenic bacteria plate of the blight, and the right picture is a coculture plate of the bacteria and the pathogenic bacteria of the blight (the middle is pathogenic bacteria plaque).

FIG. 3 shows the bacteriostatic effect of the first bacterium on pathogenic bacteria of ralstonia solanacearum; the right picture is a bacterial pathogen plate of ralstonia solanacearum, and the left picture is a bacterial and ralstonia solanacearum co-culture plate (pathogenic bacterial plaque in the middle)

FIG. 4 shows the fusion effect of fungus and pathogenic bacteria of ralstonia solanacearum provided in the present application; the left panel is a bacterial wilt pathogen plate, and the right panel is a bacterial wilt pathogen cocultivation plate.

FIG. 5 shows the antagonistic effect of the compound microbial fertilizer prepared by QK-1 bacteria on tomato blight provided by the examples of the application; the left picture is a pathogenic bacteria flat plate of the blight, and the right picture is the pathogenic bacteria flat plate of the blight coated by the compound microbial fertilizer.

FIG. 6 shows the antagonistic effect of the compound microbial fertilizer prepared from commercial bacteria on tomato wilt disease; the left picture is a pathogenic bacteria flat plate of the blight, and the right picture is the pathogenic bacteria flat plate of the blight coated by the compound microbial fertilizer.

FIG. 7 shows the growth of tomato seedlings in CK group provided in the examples of the present application.

FIG. 8 shows the growth of tomato seedlings in the disease-causing group provided in the examples of the present application.

Fig. 9 shows the growth of tomato seedlings in the treatment groups provided in the examples of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

1. Isolated culture of wormcast bacteria

Taking 10g of fresh earthworm cast from an earthworm breeding bed, placing in a 250mL triangular flask, adding 90mL of sterilized water, shaking for 30min at 25 ℃ and 180rpm on a shaking instrument, standing for 30min, taking 1mL of supernatant, adding into a test tube containing 9mL of sterilized water, diluting by 10⁶～10⁸Taking 1mL of the culture medium per degree of gradient, placing the culture medium into a sterilized culture dish, pouring a beef extract peptone solid culture medium which is heated, dissolved and cooled to about 50 ℃ into a flat plate, shaking the culture medium uniformly, placing the culture medium for complete solidification, placing the culture medium into a constant-temperature incubator at 25 ℃ for culture, culturing 5 flat plates per degree of gradient for 48 hours, picking out bacteria with different forms in the flat plate, performing streak purification culture for 2-3 times to form purified single bacterial colonies, obtaining 4 (respectively ((r)) single bacteria with different forms altogether, and performing pathogenic bacteria disease resistance test on the 4 purified single bacteria.

2. Tomato blight pathogenic bacterium flat plate antagonistic test

The pathogenic bacteria source is as follows: the scientific name Fusarium oxysporum f.sp.Lycopersici Snyder et Hansen called Fusarium oxysporum solani (abbreviated as FOL) purchased from Ming boat organism, Cathaya, Cathayensis, under the accession number BMZ 099230.

FOL flat plate: inoculating FOL pathogenic bacteria to a PDA (PDA plate) plate, culturing until bacterial plaque reaches 5cm, and taking the bacterial plaque as an FOL plate for later use;

test bacteria plate: respectively carrying out streak culture on the bacteria of the first step, the second step, the third step and the fourth step on a beef extract peptone culture medium for 48 hours until bacterial plaque grows to form 4 test bacteria plates of the bacteria of the first step, the second step, the third step and the fourth step for later use;

the test method comprises the following steps: a0.8 cm puncher is used for taking bacterial plaque of about 0.8cm of an FOL flat plate to be placed on a potato culture medium flat plate, meanwhile, 4 bacterial plaques of 0.8cm of test bacteria are respectively placed at four corners of the FOL bacterial plaque, the diameters of pathogenic bacteria are measured after 1 week, and the bacteriostasis rate is calculated.

The bacteriostatic rate (%) is (blank plaque diameter-pathogen plaque diameter)/blank plaque diameter × 100%.

As shown in FIGS. 1 and 2, firstly, the strain has antagonistic action against pathogenic bacteria of tomato blight, and secondly, the strain has no antagonistic action.

As shown in Table 1, the bacteria (i) and (ii) have antagonistic action against pathogenic bacteria of tomato wilt, and the inhibition rates were 77.42% and 72.76%, respectively. In table 1, "-" indicates that the test bacteria were fused with the PS strain, and the inhibition zone diameter and inhibition rate thereof could not be measured.

TABLE 1

3. Tomato bacterial wilt antagonism test

As the results of the above examples show that only the bacteria (i) and (ii) have antagonistic action against pathogenic bacteria of tomato wilt, the present application further performs antagonistic test against pathogenic bacteria of tomato wilt using the strains (i) and (ii) having antagonistic action against pathogenic bacteria of tomato wilt.

The tomato bacterial wilt pathogen is pseudomonas solanacearum, and has the scientific name: pseudo-monas solllamacearum (Smith) Smith, abbreviated PS, purchased from manboat organisms under the designation B82437.

PS plate: inoculating PS pathogenic bacteria to a beef extract peptone solid plate, and culturing until bacterial plaque reaches 1cm for later use;

test bacteria plate: respectively carrying out streak culture on the first bacterium and the fourth bacterium on a beef extract peptone culture medium for 48 hours until bacterial plaque reaches 1cm for later use;

the test method comprises the following steps: the test is divided into a control group and a treatment group, and only PS bacterial plaques are inoculated on a test bacterial plate of the control group; in the treatment group, 1 PS plaque was placed on 1 test bacterial plate, and 4 (r) plaques or (r) plaques were inoculated around the plate. The specific method comprises the steps of respectively taking PS bacterial plaques, bacterial plaques and bacterial plaques, respectively placing 1 PS bacterial plaque in the middle of each 1 beef extract peptone flat plate by using a 0.8cm puncher, placing 4 bacterial test bacterial flat plates, namely the bacterial test bacterial plaques or 4 bacterial test bacterial plaques at four corners of each 1 PS bacterial plaque, placing the inoculated bacterial test bacterial plaques into a constant-temperature incubator at 25 ℃ for culture and observation for 7 days, respectively measuring the diameters of the PS bacterial plaques, calculating the bacteriostasis rate by referring to the formula of the embodiment, and processing 5 flat plates each.

TABLE 2

As can be seen from FIGS. 3-4 and Table 2, only the bacteria had inhibitory effects on pathogenic bacteria of ralstonia solanacearum and the bacteria had no inhibitory effects at all and fused with the pathogenic bacteria. In Table 2, "-" indicates that the test bacteria were fused with the PS strain, and the inhibition zone diameter and inhibition rate thereof could not be measured.

4. Identification of bacterial strains

Therefore, 16SrDNA sequence analysis and identification are carried out on the bacteria of (i).

Extracting DNA from a thallus sample by adopting a silicon substrate adsorption column method, carrying out PCR amplification on the extracted DNA by adopting 2 xTaq Master Mix (Sammerfying), wherein the PCR amplification system is 2 xTaq Master Mix 25 mu L, bac _16s _27f (shown as SEQ ID NO. 1) 10 mu M1 mu L, bac _16s _1492r (shown as SEQ ID NO. 2) 10 mu M1 mu L and 1 mu L gDNA, and adding ddH₂O to 50 mu L, and carrying out one-generation sequencing (bidirectional sequencing) on the amplification product; the amplification conditions are 94 ℃ for 5min, 95 ℃ for 15s, 50 ℃ for 20s, 72 ℃ for 40s, and 40 cycles; and subjected to sequencing analysis by Shanghai Shen biomedical science and technology Co. Sequencing resultsSubmitting GenBank of NCBI website, logging in BLAST program for comparison, constructing a strain phylogenetic tree by MEGA6.0 software, and setting the self-development times as 1000 by NJ algorithm; the strain is identified as Bacillus subtilis by combining with a common bacteria identification manual, is named as Bacillus subtilis QK-1, and has a 16SrDNA sequence shown as SEQ ID NO. 3.

5. Fermentation test of wormcast planted in QK-1 field

The test was divided into control group, treatment 1 group and treatment 2 group.

The control group was fermented only with sterilized wormcast. Treatment 1 group commercial Bacillus subtilis (Guangzhou Lvhui Biotech Co., Ltd.) was inoculated into sterilized wormcast and fermented. And (2) inoculating the screened QK-1 into sterilized wormcast for fermentation.

The preparation method of the QK-1 bacterium comprises the following steps: transferring the strain subjected to solid culture into a beef extract peptone liquid culture medium for liquid culture for 48 hours, centrifuging at the rotating speed of 5000rpm for 5min, pouring out supernatant, collecting the centrifuged bacterial liquid for later use, and simultaneously measuring the concentration of the bacterial liquid by adopting a dilution plate method.

And (3) fermentation process: weighing 1Kg of wormcast per treatment, putting into a 1L beaker, sealing with paper, putting into a sterilization pot, sterilizing at 121 deg.C for 30min, and taking out the wormcast from the sterilization pot. When the sterilized wormcast is cooled to the normal temperature, 0.5g of commercial bacteria (the number of added bacteria is 0.5 hundred million/g) and humic acid (chemical Limited of chemical industry, Lobster, China and Hainan province) accounting for 1% of the weight of the wormcast are uniformly stirred and then added into the sterilized wormcast to serve as a treatment group 1, 0.78g of QK-1 bacteria (the number of added bacteria is 0.5 hundred million/g) are added into the humic acid accounting for 1% of the weight of the wormcast to be uniformly stirred and then added into the sterilized wormcast to serve as a treatment group 2, and the sterilized wormcast serves as a control group. The three treatments are put into an incubator to be cultured for 48 hours at 25 ℃, and the number of the bacillus subtilis is detected for each treated air-dried sample by a dilution plate method.

TABLE 3

As can be seen from Table 3, a small amount of bacteria exist in the sterilized wormcast, the sterilized wormcast does not reach a completely sterile state, and after the commercial bacteria and the QK-1 bacteria are added, the bacteria can be propagated in the wormcast in a large quantity, but the propagation quantity of the bacillus subtilis QK-1 screened from the wormcast is obviously higher than that of the commercial bacteria in the wormcast, so that the planting effect of the bacillus subtilis screened from the wormcast is better than that of the commercial bacteria in the wormcast, and the dominant bacteria can be better formed in the wormcast.

6. Composite microbial fertilizer flat plate countermeasure test

On the basis, the embodiment of the application also provides a compound microbial fertilizer for tomatoes, which is prepared from QK-1. The composite microbial fertilizer comprises QK-1 with the concentration of not less than 0.2 hundred million/g, organic matter with the concentration of not less than 20 wt% and N-P with the concentration of not less than 8 wt%₂O₅-K₂O, 1-2 wt% of medium trace elements, 2-5 wt% of CaO and 1-2 wt% of MgO. Wherein "wt%" means weight percentage.

Specifically, the preparation method of the compound microbial fertilizer comprises the following steps:

QK-1 was cultured in liquid medium, the content of the liquid medium:

the culture medium used for liquid culture is an industrial culture medium: 1 wt% of glucose, 2.5 wt% of soybean meal, 0.8 wt% of corn flour, 0.003 wt% of manganese sulfate, 0.5 wt% of magnesium sulfate, 0.052 wt% of sodium dihydrogen phosphate and 0.233 wt% of disodium hydrogen phosphate, adjusting the pH value to 7, placing the mixture in a 100L fermentation tank, wherein the culture medium accounts for 60% of the tank volume, inoculating QK-1 strain after online sterilization, wherein the inoculation amount is 5 wt% of the liquid culture medium, the fermentation temperature is 37 ℃, the ventilation amount is 1v/v.min, the pH value is controlled in the fermentation process to 7, sampling and detecting the fermentation liquor by using a viable count method, stopping fermentation when the viable count in the fermentation liquor is not less than 7 wt%, stopping fermentation when the spore rate reaches 90%, collecting the fermentation liquor, and centrifugally removing the supernatant to obtain concentrated standby bacterial liquid.

Mixing humic acid and biochar (Hongyuan water purification material Co., Ltd.) with the above bacterial liquid, adding into wormcast, and culturing for 48h to make the strain fix-planted in wormcast to obtain wormcast culture; and adding amino acid calcium, amino acid magnesium, phosphate fertilizer and potash fertilizer into the culture, and uniformly mixing to obtain the compound microbial fertilizer for tomatoes.

Wherein, the addition amount of humic acid and biochar is 1 wt% of the weight of wormcast; the adding amount of amino acid calcium (Xian Yang Xun Biotechnology Co., Ltd.) and amino acid magnesium (Xian Yang Xun Biotechnology Co., Ltd.) are 1 wt% of the wormcast, the adding amount of phosphate fertilizer (phosphorus ammonium, Shandong Qizhi chemical new material Co., Ltd.) is 3 wt% of the wormcast, and the adding amount of potassium fertilizer (potassium sulfate, Zheng Zhou Zhongzhi chemical product Co., Ltd.) is 2 wt% of the wormcast.

Therefore, the embodiment of the application also utilizes the compound microbial fertilizer to carry out a tomato blight plate resistance test, and the specific test is as follows:

(1) the test was divided into control group, treatment 1 group and treatment 2 group. Experiments FOL plate and test plate were prepared separately.

(2) FOL flat plate: culturing FOL bacteria with PDA solid culture medium until bacterial plaque reaches above 5 cm.

(3) Test plate: the compound microbial fertilizer produced by QK-1 provided by the embodiment of the application and sterile water are subjected to constant temperature oscillation at a temperature of 25 ℃ for 30min according to a ratio of 1:10, standing is performed for 30min after oscillation is finished, 1mL of leaching liquor is taken and mixed into a PDA solid culture medium to be gelled, shaking is performed uniformly, and gelling is performed to obtain a sample flat plate for treating 1 group. The sample plates of treatment 2 were processed according to the above method except that the composite microbial fertilizer prepared according to the above method using commercial bacteria was used to prepare the sample plates of treatment 2. In contrast, the control group used PDA plate function test plates.

(4) Bacterial plaques with the diameter of 0.8cm are prepared by an FOL flat plate through a puncher and are respectively placed on sample flat plates of the treatment group 1, the treatment group 2 and the control group, after the sample flat plates are continuously cultured for 5 days, the inhibition zones and the inhibition rates of commercial bacteria and QK-1 bacteria on pathogenic bacteria FOL are respectively measured according to the method of the embodiment.

TABLE 4

As shown in fig. 5, 6 and table 4, the effect of the compound microbial fertilizer prepared by using QK-1 bacteria is higher than the bacteriostasis rate of the compound microbial fertilizer prepared by using commercial bacteria, and can reach more than 75%, and is higher than the bacteriostasis rate of the commercial bacteria compound microbial fertilizer by 8.39%. Therefore, the compound microbial fertilizer prepared from the QK-1 bacteria provided by the embodiment of the application has more excellent effect of inhibiting pathogenic bacteria of tomato blight.

7. Tomato seedling blight resistance test

In order to enable the compound microbial fertilizer to act with tomato seedlings together with pathogenic bacteria, the compound microbial fertilizer is mixed into sterile water according to the weight ratio of 1:5, aerobic leaching is carried out for 30min, the mixture is sieved by a 100-mesh sieve, and the number of bacteria in the leaching solution is detected to be 0.4 hundred million/g, so that the obtained product is used as the fertilizer for treating the compound microbial fertilizer for experiments.

Tomato seedlings: each treatment planted 12 pots, 3 plants per pot, and 36 plants per treatment.

The experiment was divided into control, disease causing and treatment groups. Tomato seedlings of the control group were not fertilized with FOL, which was not inoculated with pathogenic bacteria. And (3) inoculating FOL pathogenic bacteria to the treated tomato seedlings, and simultaneously applying a compound microbial fertilizer. Tomato seedlings of pathogenic bacteria are only inoculated with FOL pathogenic bacteria and are not fertilized with compound microbial fertilizer. Wherein, the compound microbial fertilizer is prepared by the above embodiment.

The experimental process is as follows: tomato seedlings of the control group were directly established. The tomato seedlings of the disease causing group and the treatment group are pulled out from the matrix and put into the matrix with the concentration of 10⁶Soaking each/mL FOL bacterial liquid for 20min and then planting. After the tomato seedlings are planted, 30mL of clean water is respectively poured into each pot of the disease-causing group and the control group, the prepared fertilizer solution is added into the treatment group, and 30mL of the solution is applied into each pot. The experiment is performed in 5 monthsThe number of dead tomato seedlings in each group can be counted 7 times from the beginning of 24 days to the end of 23 days of 6 months, and the mortality rate is counted.

TABLE 5 tomato mortality at seedling stage

As can be seen from FIGS. 7-9 and Table 5, tomato seedlings established themselves with a mortality rate of 22.22%, whereas tomato seedlings in the diseased group after FOL inoculation were found to have a mortality rate of 55.56%. If the composite microbial fertilizer is applied to the treatment group at the same time of causing diseases, the tomato mortality is remarkably reduced to be equivalent to that of the control group, so that the composite microbial fertilizer provided by the embodiment of the application has the effect of resisting the pathogenic death of FOL on tomatoes, and the tomato seedling mortality is effectively reduced.

TABLE 6

Table 6 shows the criteria for grading the onset of tomato seedling stage and the disease index for grading calculation. As can be seen from Table 6, the disease index of the disease-causing group is 27.12% higher than that of the fertilizer group 1, which indicates that the application of the compound microbial fertilizer can alleviate the disease symptoms and alleviate the influence of the diseases on the growth of the tomatoes.

The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.

Sequence listing

<110> institute of soil and fertilizer for plant protection of academy of agricultural sciences of Hubei province

<120> composite microbial fertilizer for bacillus subtilis and tomato, preparation method and application thereof

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aggttacctc accgacttcg ggtgttacaa actctcgtgg tgtgacgggc ggtgtgtaca 60

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acgcagtcga gttgcagact gcgatccgaa ctgagaacag atttgtggga ttggcttaac 180

ctcgcggttt cgctgccctt tgttctgtcc attgtagcac gtgtgtagcc caggtcataa 240

ggggcatgat gatttgacgt catccccacc ttcctccggt ttgtcaccgg cagtcacctt 300

agagtgccca actgaatgct ggcaactaag atcaagggtt gcgctcgttg cgggacttaa 360

cccaacatct cacgacacga gctgacgaca accatgcacc acctgtcact ctgcccccga 420

aggggacgtc ctatctctag gattgtcaga ggatgtcaag acctggtaag gttcttcgcg 480

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ggggcggaaa ccccctaaca cttagcactc atcgtttacg gcgtggacta ccagggtatc 660

taatcctgtt cgctccccac gctttcgctc ctcagcgtca gttacagacc agagagtcgc 720

cttcgccact ggtgttcctc cacatctcta cgcatttcac cgctacacgt ggaattccac 780

tctcctcttc tgcactcaag ttccccagtt tccaatgacc ctccccggtt gagccggggg 840

ctttcacatc agacttaaga aaccgcctgc gagcccttta cgcccaataa ttccggacaa 900

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aggtaccgtc aaggtaccgc cctattcgaa cggtacttgt tcttccctaa caacagagct 1020

ttacgatccg aaaaccttca tcactcacgc ggcgttgctc cgtcagactt tcgtccattg 1080

cggaagattc cctactgctg cctcccgtag gagtctgggc cgtgtctcag tcccagtgtg 1140

gccgatcacc ctctcaggtc ggctacgcat cgttgccttg gtgagccgtt acctcaccaa 1200

ctagctaatg cgccgcgggt ccatctgtaa gtggtagccg aagccacctt ttatgtttga 1260

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Claims

1. The Bacillus subtilis is preserved in China center for type culture collection, the preservation number is CCTCC No. M2021996, the Bacillus subtilis QK-1 is named by classification, the preservation date is 2021 year 08 month 06, and the preservation address is eight-path Lopa mountain in Wuchang district, Wuhan city, Hubei province.

2. A compound microbial fertilizer for tomatoes, comprising the bacillus subtilis of claim 1 at a concentration of not less than 0.2 hundred million/g, not less than 20 wt% of organic matter, not less than 8 wt% of N-P₂O₅-K₂O, 1-2 wt% of medium trace elements, 2-5 wt% of CaO and 1-2 wt% of MgO.

3. The preparation method of the compound microbial fertilizer as claimed in claim 2, which is characterized by comprising the following steps:

4. The method according to claim 3, wherein the amount of humic acid and the biochar is 1 wt% of the weight of the wormcast.

5. The preparation method according to claim 3, wherein the amount of the calcium amino acid and the magnesium amino acid is 1 wt% of the wormcast, the amount of the phosphate fertilizer is 3 wt% of the wormcast, and the amount of the potassium fertilizer is 2 wt% of the wormcast.

6. The use of the bacillus subtilis of claim 1, the composite microbial fertilizer of claim 2 or the composite microbial fertilizer prepared by the preparation method of claim 3 in tomato cultivation.

7. Use of the Bacillus subtilis of claim 1 against Fusarium oxysporum (Fusarium oxysporum.f.sp.cucmium) or Pseudomonas solanacearum (Pseudomonas solanacearum).