LU503373B1

LU503373B1 - Compound microbial inoculum, preparation method and application thereof

Info

Publication number: LU503373B1
Application number: LU503373A
Authority: LU
Inventors: Xiaohu Wang; Liqin Zhou
Original assignee: Guangxi Acad Sciences
Priority date: 2023-01-19
Filing date: 2023-01-19
Publication date: 2023-07-19

Abstract

The invention relates to the technical field of microorganisms, in particular to a compound microbial inoculum, a preparation method and application thereof. The compound microbial agent of the invention comprises Bacillus velezensis wxh1 and soil biocontrol bacterium Bacillus subtilis wxh2, which are selected by the applicant from the soil around the banana root. The identification shows that both strain wxh1 and strain wxh2 can inhibit the growth of Fusarium oxysporum Foc1 and Foc4, and can effectively control banana Fusarium wilt and speed up the bearing fruit. Adding these two microbial agents into organic fertilizer can improve the disease resistance of banana to Fusarium wilt and reduce the biodiversity of infected soil, so they are biocontrol strains that can speed up the bearing fruit and control banana Fusarium wilt.

Description

DESCRIPTION LU503373

COMPOUND MICROBIAL INOCULUM, PREPARATION METHOD AND

APPLICATION THEREOF

TECHNICAL FIELD

The invention relates to the technical field of microorganisms, in particular to a compound microbial inoculum, a preparation method and application thereof.

BACKGROUND

Fusarium oxysporum f. sp. Cubense is one of the pathogenic bacteria causing plant

Fusarium wilt, and banana Fusarium wilt is the most harmful disease in the current banana planting process. The occurrence of banana Fusarium wilt seriously affects the growth and yield of banana plants. At present, chemical control method is widely used for Fusarium wilt, that is, chemical agents are used to treat Fusarium wilt. However, due to the rapid variation of microorganisms, it is often necessary to change chemical agents irregularly. Of course, at present, microbial agents are also used to control banana

Fusarium wilt, such as some endophytic fungi with parasitic and disease-resistant functions or Bacillus with antibacterial functions from some bacterial secretions. With the further research on microorganisms, more and more microbial strains have been found to be effective in inhibiting Fusarium wilt. However, for the field of biological control, obtaining more disease-resistant microbial rich germplasm banks is the first step of biological control at present. Therefore, technicians in this field have been constantly screening new strains with good disease resistance for later biological control. In the actual application process of biocontrol bacteria, the technicians in this field also found that the inhibitory effect of some strains on Fusarium oxysporum decreased with the increase of application time. Therefore, it is more necessary to screen new strains and use different strains alternately.

SUMMARY LU503373

In view of the above, it is necessary to provide a new strain that can inhibit the growth of Fusarium oxysporum, improve the resistance of banana to banana Fusarium wilt, speed up the bearing fruit and reduce the incidence of banana Fusarium wilt, and its application.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

Compound microbial agent, which comprises strains of Bacillus velezensis wxh1 of banana stem and/or soil biocontrol bacteria Bacillus subtilis wxh2; the banana stem

Bacillus velezensis wxh1 has a preservation number of GDMCC NO.61657 and is preserved in Guangdong Microbial Culture Collection Center, the address of which is the 5th floor, Building 59, No.100 Xianlie Middle Road, Guangzhou, and the preservation date is May 11th, 2021. The soil biocontrol bacterium Bacillus subtilis wxh2 has a preservation number of GDMCC NO.61658 and is preserved in Guangdong Microbial

Culture Collection Center, the address of which is the 5th floor, Building 59, No.100

Xianlie Middle Road, Guangzhou, and the preservation date is May 11th, 2021.

Further, the composite microbial agent also comprises saccharomycetes.

The invention also comprises a leavening agent containing the compound microbial inoculum; in the composite microbial agent, the strain Bacillus velezensis wxh1, soil biocontrol bacterium Bacillus subtilis wxh2 and saccharomycetes are mixed according to the mass ratio of 1-2:1-2:1-3.

The invention also comprises an organic fertilizer containing the compound microbial inoculum.

The invention also includes the application of the compound microbial agent in the preparation of organic fertilizer.

The invention also includes the application of the compound microbial agent in inhibiting the growth of Fusarium oxysporum, preventing and treating banana Fusarium wilt, accelerating the bearing fruit and/or reducing the biodiversity of soil affected by banana Fusarium wilt.

The invention also includes a preparation method of the organic fertilizer, whid#/503373 comprises the following steps: weighing the raw materials according to the following mass percentage: 40% of cassava residue, 30% of cow dung, 20% of fallen leaves and 10% of tree branches, fully mixing and crushing; then, adding the leavening agent according to the inoculation amount of 5% of the total mass of the base material (the leavening agent consists of the strain Bacillus velezensis wxh1, the soil biocontrol bacterium Bacillus subtilis wxh2 and Angel Saccharomycetes according to the mass ratio of 1:1:1); composting at room temperature for 21 days to obtain organic fertilizer.

The invention also includes a fertilization method for preventing and controlling the incidence of banana Fusarium wilt, which comprises the following steps: applying the organic fertilizer to the root of banana at the four-leaf stage according to the fertilization amount of 20g/ plant; spreading the organic fertilizer in a radius of 1m with banana plants as the center.

The invention has the following beneficial effects:

The compound microbial agent of the invention comprises Bacillus velezensis wxh1 and soil biocontrol bacterium Bacillus subtilis wxh2, which are selected by the applicant from the soil around the banana root. The identification shows that both strain wxh1 and strain wxh2 can inhibit the growth of Fusarium oxysporum, and can effectively control banana Fusarium wilt and speed up the bearing fruit. Adding the above two microbial agents into organic fertilizer can improve the disease resistance of banana to Fusarium wilt and reduce the biodiversity of infected soil, which is a biocontrol strain that can speed up the bearing fruit and control banana Fusarium wilt.

BRIEF DESCRIPTION OF THE FIGURES LU503373

Fig. 1 shows the morphology of wxh1 strain of the present invention on a plate;

Fig. 2 shows the morphology of wxh2 strain of the present invention on a plate;

Fig. 3- Fig. 4 are phylogenetic tree diagrams of wxh1 and wxh2 strains of the present invention;

Fig. 5 shows the plate antagonistic experiment of strains wxh1 and Fusarium oxysporum, in which: (1) the plate antagonistic experiment of wxh1 and Fusarium oxysporum (Foc1); (2) the plate antagonistic experiment diagram of wxh1 and Fusarium oxysporum (Foc4); A is the control group which is not inoculated with strain wxh1 plate, and B is the experimental group which is inoculated with strain wxh1 plate;

Fig. 6 shows the plate antagonistic experiment of strain wxh2 and Fusarium oxysporum, in which: (1) the plate antagonistic experiment of wxh2 and Fusarium oxysporum (Foc1); (2) the plate antagonistic experiment diagram of wxh2 and Fusarium oxysporum (Foc4); A is the control group which is not inoculated with strain wxh1 plate, and B is the experimental group which is inoculated with strain wxh1 plate;

Fig. 7 shows the growth inhibition results of strains wxh1 and wxh2 on Fusarium oxysporum (Foc1);

Fig. 8 shows the growth inhibition results of Fusarium oxysporum (Foc4) by strains wxh1 and wxh2;

Fig. 9 shows the results of inhibition of metabolites of strains wxh1 and wxh2 on

Fusarium oxysporum (Foc1) by orifice plate experiment;

Fig. 10 shows the results of the inhibition of metabolites of strains wxh1 and wxh2 on Fusarium oxysporum (Foc4) by orifice plate experiment;

Fig. 11 shows the inhibition results of metabolites of strains wxh1 and wxh2 on

Fusarium oxysporum (Foc1);

Fig. 12 shows the inhibition results of metabolites of strains wxh1 and wxh2 on

Fusarium oxysporum (Foc4);

Fig. 13 shows the results of banana planting in the field with the application 68503373 organic fertilizer in the experimental group, in which (1) is a banana plant; (2) Anatomical map of banana stem: (3) Picture of banana leaves;

Fig. 14 shows the results of banana planting in the field after applying organic fertilizer in the control group, in which (1) is banana plant; (2) Anatomical map of banana stem; (3) Picture of banana leaves;

Fig. 15 is a graph showing the effect of applying compound microbial agents to ferment organic fertilizer on banana morbidity;

Fig. 16 is a graph showing the effect of organic fertilizer fermentation with compound microbial agents on banana yield;

Fig. 17- Fig. 18 are the results of Alpha diversity analysis of soil microorganisms; Fig. 17 shows: Alpha diversity chao1 type; Fig. 18 shows shannon type in Alpha diversity;

Fig. 19- Fig. 20 are the results of horizontal species abundance and Venn diagram of soil microflora; Fig. 19 shows: species abundance diversity at phylum level; Fig. 20 shows the species abundance of genus level;

Fig. 21 shows the results of Beta diversity analysis of soil microorganisms;

Fig. 22 shows the results of species abundance of soil microbial diversity at species level;

Fig. 23 shows the results of species composition analysis of three groups of samples at the level of soil microbial phylum;

Fig. 24 is the result diagram of the phylogenetic tree of soil microbial diversity at the taxonomic level;

Fig. 25- fig. 27 are the results of the network diagram of species correlation of soil microbial diversity;

Fig. 28 shows the results of phenotypic analysis of soil microorganism BugBase; Fig.

A shows the pathogenicity prediction at phylum level, fig. B shows the species level, fig.

C shows the trend of pathogenicity prediction, and fig. D shows the threshold of species diversity.

Fig. 29 is the result diagram of the statistics of hanging fruit;

Fig. 30 is the result of observation by flat-plate confrontation microscope.

DESCRIPTION OF THE INVENTION LU503373

All features disclosed in this specification, or steps in all methods or processes disclosed, can be combined in any way except mutually exclusive features and/or steps.

Unless otherwise stated, any feature disclosed in this specification (including any appended claims and abstract) is only one example in a series of equivalent or similar features.

Example 1:

In this example, two strains of Bacillus which can control banana Fusarium wilt were screened out, namely:

Bacillus velezensis wxh1, an endophytic bacterium of banana, whose preservation number is GDMCC NO.61657, is preserved in Guangdong Microbial Culture Collection

Center, the address of which is the 5th floor, Building 59, No.100 Xianlie Middle Road,

Guangzhou, and the preservation date is May 11th, 2021.

The soil biocontrol bacterium Bacillus subtilis wxh2, whose preservation number is

GDMCC NO.61658, is deposited in Guangdong Microbial Culture Collection Center, the address of which is the 5th floor, Building 59, No.100 Xianlie Middle Road, Guangzhou, and the preservation date is May 11th, 2021.

The above-mentioned strains were collected from banana rhizosphere soil in banana planting experimental farm in 2019.

Morphological classification and molecular biological identification of microorganisms in this example are as follows: 1. Morphological classification of strains:

Strain wxh1 was inoculated on LB medium, and the morphological characteristics of the colony were observed. As shown in Fig. 1, the colony was white on the plate, with smooth edge and concave center, and the single colony was spherical. Strain wxh2 was inoculated on LB medium, and the morphological characteristics of the colony were observed. As shown in Fig. 2, the colony was white on the plate, the edge of the colony was smooth, and the single colony was spherical.

2. Molecular biological identification LU503373

Sequencing and verifying the above strains: using universal primers: 5'-CCTACGGGAGGCAGCAG-3' and 5-ATTACCGCGGCGCTGCTGG-3' to amplify the 16S rRNA gene sequence of the strains, and then compare it with EzBioCloud database and BLASTn database in GenBank to construct the phylogenetic tree of the two strains: as shown in fig. 3- fig. 4, both strain wxh1 and strain wxh2 are 99.7% similar to Bacillus sp., but they are different, in which strain wxh1 and endophyte Bacillus velezensis cluster into one cluster, while strain wxh2 and endophyte Bacillus subtilis cluster into one cluster.

Therefore, the above two strains belong to the genus Bacillus, but they are different species.

Example 2:

In this example, the antagonistic effects of strain wxh1 and strain wxh2 on Fusarium oxysporum f sp. cubense were studied.

Previous studies have found that banana Fusarium wilt is caused by Fusarium oxysporum f. sp. Cubense. In order to study the inhibitory effect of the strain on Fusarium oxysporum f. sp. Cubense, this embodiment carried out the antagonism experiment between Foc1 and Foc4, as follows: (1) Preparation of pathogenic bacteria suspension: add agar medium with pathogenic bacteria into PDB liquid medium, and culture at 28°C and 170rpm for 7 days.

After the culture, filter the suspension through four layers of gauze, and then adjust the final concentration to 10x10° cfu/ml with distilled water for later use. (2) Inoculating the suspension of the above pathogenic bacteria to PDA culture medium, in which the control group only inoculated the pathogenic bacteria of the new strain of the application; in the experimental group, pathogenic bacteria were inoculated on one side of PDA culture medium, and corresponding screened strains: wxh1 and/or wxh2 were inoculated on the opposite side for antagonistic experiment.

The results are shown in Fig. 5, which shows the plate antagonistic experiment of strains wxh1 and Fusarium oxysporum, in which: (1) the plate antagonistic experiment of wxh1 and Fusarium oxysporum (Foc1); (2) the plate antagonistic experiment diagram of wxh1 and Fusarium oxysporum (Foc4); A is the control group which is not inoculated with strain wxh1 plate, and B is the experimental group which is inoculated with straltJ503373 wxh1 plate;

As can be seen from the above two figures, the control plate is basically covered with hyphae, while the two plates in the experimental group have obvious inhibitory effects on Fusarium oxysporum.

Results The results are shown in Figure 30. It can be seen that the hyphae of the control group in the figure grow strongly, while the experimental groups wxh1 and wxh2 show the phenomena of hyphae growth inhibition and dissolved vacuolation, which further shows that the two strains wxh1 and wxh2 in the experimental group have obvious inhibitory effects on Fusarium oxysporum.

In this embodiment, the growth inhibition curves of the two groups of experiments are also calculated, and the calculation formula is (corresponding to the calculation formula of the following experimental results): the inhibition effect of pathogen growth is estimated: P=D or p = d; P= inhibition of pathogen growth (cm); D=diameter of pathogen growth in the control panel (mm); D=growth diameter of pathogen tested (mm).

According to the experimental results in fig. 7-8, strain wxh1 and strain wxh2 have obvious inhibition on Fusarium oxysporum Foc1 after 10 days, among which strain wxh1 and strain wxh2 have obvious inhibition on Fusarium oxysporum Foc4 after 10 days.

Example 3:

In this example, the antagonistic effects of metabolites of strain wxh1 and strain wxh2 on Fusarium oxysporum were studied.

Mainly adopts the following experimental steps: (1) Preparation of secondary metabolites of strain wxh1 and strain wxh2: Strain wxh1 and strain wxh2 were inoculated into PDA liquid culture medium respectively, the inoculation amount was 1%, and the culture time was 12h. After the culture, the culture solution was centrifuged, the liquid was taken, and the secondary metabolites weté/503373 obtained after sterile filtration with 0.22um. (2) Preparation of pathogenic bacteria suspension: the preparation method is the same as step (1) of Example 2. (3) The secondary metabolites prepared in step (1) are added into PDA culture medium according to the addition amount of O (Control group), 20%, 40%, 60%, 80% and 100% of the volume of PDA culture medium, and then the PDA culture medium containing the secondary metabolites in step (1) is subpackaged into 24-hole plates, with 4 holes in each concentration gradient. After that, the pathogenic bacteria in step (2) are inoculated into plates containing the cell extracts with different concentrations and incubated for 6 days.

The experimental results are as follows:

Fig. 9 shows the inhibition results of wxh1 and wxh2 metabolites on Fusarium oxysporum Foc1. It can be seen from the figure that when the addition of wxh1 or wxh2 metabolites reaches 40%, it begins to inhibit the growth of Fusarium oxysporum Foc1: when the concentration reaches 60%, wxh1 or wxh2 metabolites completely inhibit the growth of mycelium.

Fig. 10 shows the inhibition results of wxh1 and wxh2 metabolites on Fusarium oxysporum Foc4. It can be seen from the figure that when the addition of wxh1 metabolites reaches 40%, it begins to inhibit the growth of Fusarium oxysporum Foc4.

When the addition of wxh2 metabolites reaches 60% concentration, wxh2 metabolites inhibit the growth of Fusarium oxysporum Foc4. In addition, the inhibition effect of wxh1 on Foc4 is stronger than that of wxh1. When it reaches 60% concentration, wxh2 metabolites inhibit the growth of Fusarium oxysporum FOCA4.

Fig. 11 is a graph showing the inhibitory effect of metabolites of strains wxh1 and wxh2 on Fusarium oxysporum (Foc1). From the data, it can be seen that the inhibitory ability of secondary metabolites of wxh1 on Fusarium oxysporum (Foc1) is greater than that of secondary metabolites of wxh2.

Fig. 12 shows the inhibition results of metabolites of strains wxh1 and wxh2 on

Fusarium oxysporum (Foc4). From the data, it can be seen that the inhibition ability of secondary metabolites of wxh1 on Fusarium oxysporum (Foc4) is slightly greater thdn/503373 that of secondary metabolites of wxh2.

Example 4:

In this example, the above-mentioned strain wxh2 and strain wxh1 were used to prepare microbial agents to inhibit banana Fusarium wilt.

In this example, a compound microbial agent is used: soil biocontrol bacterium

Bacillus subtilis wxh2, banana stalk Bacillus velezensis wxh1 and saccharomycetes are prepared into bacterial powder and then mixed according to the mass ratio of 1-2:1-2:1-3.

Among them, saccharomycetes is purchased from Angel Company, and its model is high-temperature resistant and high-activity yeast.

The preparation method of the organic fertilizer in the experimental group of this embodiment is as follows: each raw material is weighed according to the following mass percentage: 40% of cassava residue, 30% of cow dung, 20% of fallen leaves (optional banana leaves) and 10% of branches (optional Chinese fir branches), fully mixed and crushed, and then the above mixed bacterial powder is added according to 5% of the total mass of the base material; composting at room temperature for 21 days to obtain organic fertilizer.

The preparation method of the organic fertilizer of the control group in this embodiment is: 40% cassava residue, 30% cow dung, 20% fallen leaves (optional banana leaves) and 10% branches (optional Chinese fir branches), which are fully mixed and then crushed; Without adding compound bacterial powder, the organic fertilizer can be obtained by composting at room temperature for 21 days.

After the organic fertilizer is prepared, the field experiment is conducted, and the field experiment is divided into an experimental group and a control group:

Experimental group: applying organic fertilizer in the experimental group according to the fertilization rate of 20g/ plant;

Control group: applying organic fertilizer in control group according to the fertilization rate of 20g/ plant;

Other planting management methods adopt conventional management methods.

After 120 days of planting management, it was observed that:

As shown in fig. 13, the banana with the organic fertilizer of the experimental grougJ503373 is shown in the picture. As shown in fig. 14, the banana to which the organic fertilizer of the control group has been applied. It can be seen from figs. 13-14 that the banana leaves in fig. 14(1) show symptoms of yellowing, stunting and withering, while those in fig. 13(1) do not appear.

Fig. 14(2), (3) Black stripes appeared on the stems and leaves, and the root rotted, and the longitudinal sections through the stems showed severely necrotic stem tissue, which was consistent with the report of Fusarium oxysporum wilt phenotype. Therefore, the strain of this application can effectively control banana Fusarium wilt in field application. The applicant uses the following formula to calculate the incidence of banana Fusarium wilt:

Formula for calculating the incidence of banana Fusarium wilt: the incidence of banana Fusarium wilt is estimated: P=A/B; A=the number of banana cases; B=banana planting tree experimental group;

As shown in fig. 15, the incidence rate of the control group (control group) fermented without compound microbial agents was 36%; the incidence rate of the experimental group fermented with compound microbial agents was 5.6%, which was significantly lower than that of the control group.

In addition to the above incidence, the applicant also counted and calculated the yield and bearing fruit time of the two groups of experiments; the results are as follows: (1) After statistical calculation of plant yield in two seasons, the results are shown in

Fig. 16. The yield of the experimental group with compound microbial inoculum fermentation is 23kg, and that of the control group without compound microbial inoculum fermentation is 23kg. Therefore, strain wxh1 and strain wxh2 did not affect banana yield. (2) After the statistical calculation of two seasons’ bearing fruit time, it was found that the bearing fruit time of the experimental group fermented with compound microbial inoculum was earlier than that of the control group fermented without compound microbial inoculum, and the difference was significant: as shown in Fig. 29, the bearing fruit rate of the control group from March to December was 23%, while that of the experimental group was 69%. Therefore, the application of organic fertilizer containing compound microbial agents of strain wxh2 and strain wxh1 can significantly increase tH&J503373 bearing fruit rate of banana.

Among them, the bearing fruit rate is calculated as follows: bearing fruit index = > (value of bearing fruit plants)/ (sum of experimental plants);

Example 5:

This example studies the effect of compound microbial agents on soil microbial diversity:

In order to study the changes of soil microbial community structure and composition caused by wxh1 and wxh2 spores, we divided the soil into three categories to study soil diversity, including healthy group (health group), disease group (disease group) and biological control group (bsrs).

Take three soil samples from each group, sequence the data and calculate the results of Alpha diversity analysis of soil microorganisms, as shown in Fig. 17- Fig. 18. chao reveals that there is no obvious difference in bacterial alpha diversity (Fig. 17), but shannon shows that there are significant differences in species diversity among the three soil types. In biocontrol strains, the a diversity produced by bacterial communities is significantly lower than that of healthy groups (Fig. 18). The community composition of each sample in the biological control group and the healthy group was counted at the flora level. The most important OTU, which belongs to Bacteroides, is rich in soil regulated by health group and biological control group. The relative abundance of fungi is high, while the relative abundance of y-Proteus is low in the soil regulated by biological control group (Fig. 19). Comparing the results of horizontal disease group with those of healthy group and biological control group, it is found that there is a great difference between them. The bacterial species in the disease group (346) and the healthy group (3321) are more than those in the biological control group (104), indicating that the diversity of soil microbial species in the biological control group is relatively low (Fig. 20).

In addition, the importance of using the same method to evaluate unique species characteristics. In the healthy group (17), there are more unique bacteria OTU than the diseased soil (12), while the strains in the biological control group regulate the soil (9) (Fig. 20). Among the five banana species with the highest abundance levels, the soil microbial abundance of banana Fusarium wilt is low (Fig. 23). However, the results bE503373 principal coordinate analysis (PCA) and Bray-Curtis distance show that there is no significant difference in bacterial communities among the three soil types (Fig. 22). The structure and composition of soil microbial community are related to the low incidence of banana Fusarium wilt.

Fig. 24 is a multi-sample classification tree diagram; the tree diagram of multi-sample classification in the figure compares the sequence abundance differences of different groups or samples on the branches, and the differences are represented by the color pie chart. The larger the pie chart area, the greater the sequence abundance at the branch, and different colors represent different groups or samples. The larger the sector area of color, the more sequences of corresponding groups or samples on this branch than other groups or samples.

The correlation between species was calculated and statistically tested. Based on

Python, the coexpression analysis network diagram of Bsrs was drawn. The results are shown in Fig. 25- Fig. 27. There are more nodes and links in the microbial characteristic network of BSRS. Further understanding of the characteristic network of bacteria shows a high average degree. Some genera, such as the biocontrol strains of /deonella_sp,

Haliangium, tepidum, Labilithrix luteolaC, Sphingomonas_sp, Tepidisphaera mucosa and Candidatus_alborgensis have strong correlation, high relative abundance and bacterial network in soil. There are more nodes and links in the healthy soil characteristic network, including Areca, Andromeda, Flavobacterium, Monas Terry, Vibrio Chlorella,

Bacillus westermani, Aeromonas, Chujiibacter soli and Nitrospira japonica. The characteristic network of diseases has high intimacy among the characteristic networks of diseases and insect pests such as Ideonella_sp, sphingosine, Flavisolibacter, ginsengiterrae, saccharomycetes, Gay bacteria, Longimicrobium terrae,

Mucilaginibacter_sp saburae, etc.

In order to determine whether the characteristics of soil bacterial community can be used as a method to predict the phenotype of potential pathogens, Bugbase first standardized OTU through the predicted 16S copy number, and then predicted the microbial phenotype using the provided pre-calculated files. Estimate the relative trait abundance in the whole coverage threshold range (0 to 1, in increments of 0.01) of ead{/503373 sample in the biological data set. BUG selects the highest coverage threshold (0.48, 0.59, 0.55) in all samples for each function in the user data (Figure 28A), and generates a taxon contribution graph of the relative richness of the population with characteristic traits. The results of potential pathogenicity show that the relative abundance of diseased bacterial communities is significantly higher than that of healthy soil samples, while biological control strains can regulate the soil (Figure 28B), such as

G_Burkholderia G_Candidatus G_Solibater G_Klebsiella Marensis and tenuis (Fig. 28B).

In addition, at Phylum level, the relative relative abundance of bacteria in pathogenic bacteria is also significantly higher, and the relative abundance of bacterial communities is also significantly higher (Fig. 28C).

To sum up, the application of the strain can effectively reduce the biodiversity of the diseased soil.

To sum up, the compound microbial inoculum of the present invention, including

Bacillus velezensis wxh1 and soil biocontrol bacterium Bacillus subtilis wxh2, can inhibit the growth of Fusarium oxysporum, effectively control banana Fusarium wilt, speed up banana bearing fruit, improve banana disease resistance to Fusarium wilt and reduce the biodiversity of infected soil, and is a biocontrol strain with good performance.

The above-mentioned examples only show several embodiments of the present invention, and their descriptions are more specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be pointed out that for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can be made, all of which belong to the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the appended claims.

Claims

CLAIMS LU503373

1. A compound microbial agent, characterized by comprising strains Bacillus velezensis wxh1 of banana stem and/or soil biocontrol bacteria Bacillus subtilis wxh2; the Bacillus velezensis wxh1 of banana stem has the preservation number of GDMCC

NO.61657; the preservation number of the soil biocontrol bacterium Bacillus subtilis wxh2 is GDMCC NO.61658.

2. The compound microbial agent according to claim 1, characterized by further comprising saccharomycetes.

3. A leavening agent containing the compound microbial agent according to claim 1 or 2, characterized in that the leavening agent is prepared by mixing strains of Bacillus velezensis wxh1, soil biocontrol bacteria Bacillus subtilis wxh2 and saccharomycetes in a mass ratio of 1-2:1-2:1-3.

4. An organic fertilizer containing the compound microbial agent according to claim 1 or

2.

5. Application of the compound microbial agent according to claim 1 or 2 in the preparation of organic fertilizer.

6. Application of the compound microbial agent according to claim 1 or 2 in inhibiting the growth of Fusarium oxysporum, preventing and controlling banana Fusarium wilt, speeding up the bearing fruit of banana and/or reducing the biodiversity of soil affected by banana Fusarium wilt.

7. A preparation method of organic fertilizer according to claim 4, characterized thy503373 weighing the raw materials according to the following mass percentages: 40% cassava residue, 30% cow dung, 20% leaves and 10% tree branches, fully mixing and crushing; then adding the leavening agent according to claim 3 according to the inoculation amount of 5% of the total mass of the base material; composting at room temperature for 21 days to obtain organic fertilizer.

8. A fertilization method for controlling the incidence of banana Fusarium wilt, characterized by applying the organic fertilizer according to claim 4 or 7 to the root of banana at the four-leaf stage according to the fertilization amount of 20g/ plant; spreading the organic fertilizer in a radius of 1m with banana plants as the center.