CN111763634A - Biocontrol microbial inoculum for preventing and treating potato black shank and preparation method thereof - Google Patents
Biocontrol microbial inoculum for preventing and treating potato black shank and preparation method thereof Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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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
- 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
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- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
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- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/60—Biocides or preservatives, e.g. disinfectants, pesticides or herbicides; Pest repellants or attractants
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Abstract
The invention belongs to the technical field of microorganism application. Biocontrol microbial inoculum for controlling potato black shank, wherein each gram of microbial inoculum contains bacillusThe content of viable bacteria is more than or equal to 1 × 108The bacillus is a compound bacterium of bacillus pumilus, bacillus megaterium, bacillus amyloliquefaciens, bacillus licheniformis and bacillus mucilaginosus. The biocontrol microbial inoculum has good biocontrol effect and growth promotion effect, and can effectively prevent and control the potato black shank.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and relates to a biocontrol microbial inoculum for preventing and treating potato black shank and a preparation method thereof.
Background
Potato is the fourth largest crop following rice, wheat, corn. China is the first big country of potato production. However, bacterial diseases seriously affect the yield and quality of potatoes. Among them potato phytophthora parasitica is one of the most common bacterial diseases. Potato black shank is a serious disease caused by pectobacterium, can occur at the base of the stem and on the tuber of a potato plant, and mainly infects vascular bundle tissues. The disease can occur from the germination of the seed potato to the later growth stage, and the seedling stage is most abundant. The disease is mainly transmitted through the seed potato with the disease and soil, the disease is early, the propagation speed is high, the prevention and the control are difficult, and the yield and the quality of the potato are seriously influenced.
At present, the potato black shank is mainly controlled by chemical agents with wide sterilization and quick response, and the application amount of chemical pesticides is continuously increased due to easy generation of drug resistance of pathogenic bacteria, so that serious pesticide residues and damage to the biological structure of soil are caused. The use of pesticides and chemical fertilizers is reduced, and the soil ecosystem can be fundamentally restored by applying the microbial fertilizer. The application of the microbial fertilizer can improve the utilization rate of the fertilizer, reduce the occurrence of crop diseases, improve the agricultural ecological environment, and have irreplaceable effects on the aspects of improving the quality of crop products, food safety and the like. Researches show that the bacillus has antagonistic action with several common plant pathogenic bacteria and can effectively inhibit the growth of the pathogenic bacteria; in the production of biofertilizers, the high stress resistance of bacillus provides advantages over bacteria in other microbial fertilizers in terms of fermentation, transport and survival in the harsh environment of the fertilizer in the field. However, the current systematic research on the influence of the microbial ecological agent compounded by various bacteria on the plant micro-ecological environment and the soil enzyme activity and the relation between the microbial ecological agent and the disease control is relatively less.
Disclosure of Invention
The invention aims to solve the technical problem of providing the biological control microbial inoculum for preventing and controlling the potato black shank, which has good biological control effect and growth promotion effect and can effectively prevent and control the potato black shank.
The technical scheme of the invention is as follows:
a biocontrol microbial inoculum for preventing and treating potato black shank has a viable bacillus content of more than or equal to 1 × 10 per gram of microbial inoculum8The bacillus is a compound bacterium of bacillus pumilus, bacillus megaterium, bacillus amyloliquefaciens, bacillus licheniformis and bacillus mucilaginosus.
Furthermore, the ratio of the colony forming effective viable Count (CFU) of the bacillus pumilus, the bacillus megaterium, the bacillus amyloliquefaciens, the bacillus licheniformis and the bacillus mucilaginosus is (2.3-5.2): (2.3-6.0): (2.0-5.0): (2.0-5.5): (2.5-5.5).
The preparation method of the biocontrol microbial inoculum for preventing and treating potato black shank comprises the following steps:
a. inoculating activated bacillus mucilaginosus into a silicate liquid culture medium, inoculating activated bacillus pumilus, bacillus megaterium, bacillus amyloliquefaciens and bacillus licheniformis into an LB liquid culture medium, wherein the inoculation amount is 5-8%, and culturing for 12-24h under the conditions that the temperature is 35-37 ℃, the stirring speed is 110-120r/min, so as to prepare a seed solution;
b. and fully mixing the dried and sieved carrier with the seed solution according to the proportion of 10-20ml:200-300g, culturing for 36-48h at normal temperature by taking 5wt% of sodium chloride, 2.5wt% of sucrose and 1.25wt% of citric acid as protective agents, and drying after the pH value is less than 4, wherein the drying temperature is 45-60 ℃, thus obtaining the biocontrol microbial inoculum.
Further, the silicate liquid culture medium and the LB liquid culture medium comprise 10wt% of a carbon source and 5wt% of a nitrogen source, and an orthogonal experiment is designed by taking the viable count as an index to optimize the carbon source and the nitrogen source of the culture medium.
Further, the carbon source is brown sugar, and the nitrogen source is 3wt% of bean flour and 2wt% of milk powder.
Further, the carrier is at least one of bean flour and diatomite, and is wet, loose and non-caking.
The invention has the following beneficial effects:
the bacillus pumilus, the bacillus megaterium, the bacillus amyloliquefaciens, the bacillus licheniformis and the bacillus mucilaginosus selected by the invention have excellent nitrogen fixation and phosphate solubilizing effects, and meanwhile, the bacillus licheniformis and the bacillus mucilaginosus have obvious potassium solubilizing effect, so that the biocontrol bactericide has good growth promoting effect, the potato yield is improved, and the growth promoting and yield increasing effect is more obvious when the biocontrol bactericide is mixed with a chemical fertilizer for use. The composite bacillus can improve the activity of defensive enzyme of potato leaves, improve the activity of soil urease and sucrase and improve soil fertility, thereby improving the disease resistance of plants and promoting the growth of the plants.
The bacillus pumilus, the bacillus megaterium and the bacillus licheniformis in the biocontrol agent have obvious antagonistic effect on the phytophthora parasitica. The composite bacillus is dominant in space site competition, can quickly propagate and colonize at different parts of plants, and effectively prevents colonization and infection of pathogenic bacteria, thereby achieving the effects of inhibiting bacteria and controlling diseases. The compound spore fungicide enables the structure of a soil microbial flora to change, improves the species abundance of the soil microorganisms in the rhizosphere of the potatoes, increases the proportion of soil actinomycetes, and is beneficial to enhancing the biocontrol capability of plants.
Drawings
FIG. 1 is a graph of the dynamics of potato yield and leaf defense enzyme activity in various groups in a field trial;
FIG. 2 is a graph showing the dynamic changes of urease and sucrase activities in rhizosphere soil of various groups of potatoes in a field test;
FIG. 3 is a dynamic change diagram of active flora in rhizosphere soil of various groups of potatoes in a field test;
FIG. 4 is a plot of gate (Phylum) level annotation for each treatment group in a high throughput sequencing analysis;
FIG. 5 is a Weighted UniFrac PCoA ranking chart in a high throughput sequencing assay.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples, which are only preferred embodiments of the present invention and are not intended to limit the present invention.
Examples
A biocontrol microbial inoculum for preventing and treating potato black shank has a viable bacillus content of 1 × 10 per gram of microbial inoculum8The bacillus is a compound bacterium of bacillus pumilus, bacillus megaterium, bacillus amyloliquefaciens, bacillus licheniformis and bacillus mucilaginosus; wherein the ratio of the colony forming effective viable Count (CFU) of the bacillus pumilus, the bacillus megaterium, the bacillus amyloliquefaciens, the bacillus licheniformis and the bacillus mucilaginosus is 2.3:2.3:2.0:5.5: 5.5.
A preparation method of a biocontrol microbial inoculum for preventing and treating potato black shank comprises the following steps:
a. respectively coating bacillus pumilus, bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis, bacillus megaterium and bacillus mucilaginosus on an LB solid culture medium by a filter paper sheet method for performing antagonistic test of bacteria, and setting a blank control group to show that no antagonistic action exists among the bacillus;
b. inoculating activated bacillus mucilaginosus into a silicate liquid culture medium, inoculating activated bacillus pumilus, bacillus megaterium, bacillus amyloliquefaciens and bacillus licheniformis into an LB liquid culture medium, wherein the inoculation amount is 5-8%, and culturing for 12-24h under the conditions that the temperature is 35-37 ℃, the stirring speed is 110-120r/min, so as to prepare a seed solution; wherein the silicate liquid culture medium and the LB liquid culture medium comprise 10wt% of a carbon source and 5wt% of a nitrogen source, and an orthogonal experiment is designed by taking the viable count as an index to optimize the carbon source and the nitrogen source of the culture medium; the carbon source is brown sugar, and the nitrogen source is 3wt% of bean flour and 2wt% of milk powder.
c. And (3) fully mixing the dried and sieved soybean powder carrier with the seed liquid according to the proportion of 10-20ml:200-300g, culturing for 36-48h at normal temperature by taking 5wt% of sodium chloride, 2.5wt% of sucrose and 1.25wt% of citric acid as protective agents, and drying after the pH value is less than 4, wherein the drying temperature is 45-60 ℃, thus obtaining the biocontrol microbial inoculum.
First, field test
The soil physical and chemical indexes of the field to be tested are as follows:
| soil information | pH | Organic matter (g/Kg) | Basic hydrolyzed nitrogen (mg/kg) | Available phosphorus (mg/kg) | Quick-acting potassium (mg/kg) |
| | 5.72 | 117.48 | 123.20 | 32.43 | 56.92 |
| Field 2 | 5.90 | 120.47 | 137.24 | 29.61 | 77.05 |
The potato variety to be tested is Dutch No. 7, 60 potatoes with similar growth vigor are respectively selected from field 1 and field 2, 120 potatoes are divided into 6 groups, 20 potatoes are respectively selected from each group, 10 potatoes are respectively selected from field 1 and field 2, and a parallel sample n =3 is detected. Comparing the fertilizer efficiency of the biocontrol microbial inoculum (FH), the Gexi Tianzhu fertilizer (GT fertilizer) and the Lvdao No. 1 fertilizer (LS), the fertilizer application scheme is shown in the following table, the application amount of the biocontrol microbial inoculum is 20ml per plant, and other fertilizers are applied according to the formula.
1. Potato yield assay
The yield of each group of 20 potatoes is measured in the harvest period, the growth promotion effect of the biocontrol microbial inoculum is evaluated by taking the yield of each potato as an index, the test result is shown in figure 1 (A), the yield of a composite spore microbial inoculum group (FH) is remarkably higher than that of a blank control group (BC), and the yield of the composite spore microbial inoculum and a fertilizer application Group (GF) is higher, so that the biocontrol microbial inoculum has the growth promotion effect on the growth of the potatoes, and is matched with a fertilizer for use, and the yield promotion effect on the potatoes is more remarkable.
2. Potato leaf defensive enzyme activity assay
The method is characterized in that the last fertilization of potatoes (from a seedling stage to a bud stage) is taken as a starting point, 3 plants of each group are randomly taken for detection, the activities of Peroxidase (POD), superoxide dismutase (SOD), Catalase (CAT), polyphenol oxidase (PPO) and Phenylalanine Ammonia Lyase (PAL) of potato leaves are respectively measured 6d after fertilization, the influence of different fertilizers on the potato defensive enzyme activity is compared through SPSS analysis (n = 3), the test results are shown in the following table and a figure 1 (B-F), and compared with a blank control group (BC), the experimental groups (FH and GF) added with biocontrol bacteria have very significant difference in the activities of Peroxidase (POD) and Phenylalanine Ammonia Lyase (PAL) and have significant difference in the activity of polyphenol oxidase (PPO). When the biocontrol microbial inoculum is matched with a fertilizer, the activities of polyphenol oxidase (PPO) and Phenylalanine Ammonia Lyase (PAL) are obviously higher than those of a blank control group.
Therefore, the biocontrol microbial inoculum (FH group) can obviously improve the activity of the Peroxidase (POD) of the potato leaves and is beneficial to maintaining the metabolic balance of active oxygen in plants. The GF group used by the biocontrol microbial inoculum and the GT fertilizer has obviously improved polyphenol oxidase (PPO) activity, can convert phenolic substances into lignin, phytoalexin and the like so as to enhance the plant resistance, and can effectively resist the infection of pathogens.
3. Potato soil urease and sucrase activity assay
And taking the last fertilization of the potatoes (from the seedling stage to the bud stage) as a starting point, randomly taking 3 plants from 5 plants in each group for detection, and respectively measuring the urease activity and the sucrase activity in the rhizosphere soil of the potatoes at 3d, 6d, 9d and 12d after fertilization.
(1) The results of the urease activity test are shown in FIG. 2. As shown in fig. 2(a), at the 3d, the soil urease activity of the biocontrol agent FH group is the highest and is significantly higher than that of the blank control group (BC); and 9d, the urease of GF groups which are applied by the biocontrol microbial inoculum and GT fertilizer is the highest, and then the urease activity of each group is greatly reduced. The urease activities of the biocontrol microbial inoculum (FH group) and the microbial inoculum and fertilizer matched application (GF group) in the whole treatment period are higher than those of the blank control group.
Soil urease is a key enzyme for converting nitrogen in soil, and the activity of the urease is used for representing the nitrogen condition of the soil and evaluating the soil fertility. The application of the biocontrol microbial inoculum is beneficial to improving the activity of soil urease in the early stage, and the urease activity of the potato plants treated by the biocontrol microbial inoculum reaches a peak value after 6-9 days of spraying treatment.
(2) The results of the sucrase activity tests are shown in the following table and fig. 2(B), and the urease activities of the biocontrol microbial inoculum (FH group) and the microbial inoculum and fertilizer co-application (GF group) were all higher than those of the placebo group (BC group) during the whole treatment period.
The sucrase can hydrolyze sucrose in soil into glucose and fructose which are easy to be absorbed and utilized by organisms, can increase soluble nutrient substances in soil, and is one of important indexes for representing the biochemical activity of rhizosphere soil. Therefore, the application of the biocontrol microbial inoculum is beneficial to improving the activity of soil sucrase, and the biocontrol microbial inoculum has better effect when being matched with GT fertilizer.
Second, potting test
1. Determination of physiologically active flora at potato rhizosphere
Selecting 30 potatoes with similar growth vigor in the greenhouse, dividing the potatoes into 6 groups, and dividing each group into 5 potatoes, wherein the fertilization and germ application schemes are shown in the following table:
wherein the viable bacteria content of the bacillus in each gram of the composite microbial inoculum is 1 × 108CFU, the bacillus is a composite bacterium of bacillus pumilus, bacillus megaterium and bacillus licheniformis, the ratio of the colony forming effective viable Count (CFU) of the bacillus pumilus, the bacillus megaterium and the bacillus licheniformis is 4.3:3.3:7.5, and the preparation method of the composite bacterium agent is the same as that in the embodiment; the compound microbial inoculum and the application method of the biological control microbial inoculum comprise the following steps: 0.2L of the fertilizer is applied to each plant, and the plant is diluted by 1:200 and irrigated into roots; 500 muL of germs (about OD value = 1.0) are taken, and the germs are diluted to 50ml for carrying out the stem injury and root irrigation on the potatoes.
Sampling for three times before applying bacteria at 5d, 9d and 15d, randomly taking 3 plants from each group of 5 plants for detection, obtaining 3 parts of rhizosphere soil by a five-point sampling method, and preparing soil suspension to 10 parts by a 10-time dilution method-6. Selecting proper dilution and respectively inoculating the selected dilution with a nutrient agar culture medium, an improved Gao's No. 1 culture medium, a PDA culture medium, a nitrogen-free culture medium and a peptone ammoniation culture medium, culturing at 28 ℃ for 1-3d, and then counting bacterial colonies of bacteria, actinomycetes, fungi, azotobacter and ammoniated bacteria, wherein a test result is shown in figure 3, biocontrol bacteria and phytophthora parasitica are applied to disease prevention groups (DPA and DPB) firstly, and then, biocontrol bacteria are applied to disease treatment groups (DCA and DCB), and then, biocontrol bacteria are applied to disease infection groups (DG), only the phytophthora parasitica is applied to disease infection groups (DG), and the biocontrol bacteria are not applied, so that the active bacteria number of the disease prevention groups, disease treatment groups and disease infection groups is decreased, the actinomycetes is increased, the ammoniated bacteria are increased and decreased firstly, and are different from the blank control group (BC) to a certain extent, but the difference is not obvious.
The application early stage of the composite spore fungicide increases the quantity of the actinomycetes, and can stimulate the response reaction of the actinomycetes to pathophysiology. The actinomycetes have important function in biological prevention and control of plant diseases. The number of the composite spore fungicide for the azotobacter and the ammonifying bacteria is increased, but the increase is not obvious enough.
2. High throughput sequencing analysis of soil microbial community structure
Selecting 10 potatoes with similar growth vigor in a greenhouse, dividing the potatoes into two groups, wherein each group comprises 5 potatoes, and the fertilization and germ application schemes are shown in the following table:
| time of day | 1d | 3d | 5d | 7d | 9d | 12d | 15d |
| Experimental group GF (5 strains) | Examples | GT fertilizer | Examples | GT fertilizer | Examples | Applying germs after sampling | Sampling |
| Control group GT (5 strain) | Clean water | GT fertilizer | Clean water | GT fertilizer | Clean water | Applying germs after sampling | Sampling |
GF (GT fertilizer and the biocontrol microbial inoculum of the invention) is selected in an experimental group, the GT fertilizer is used for strengthening seedlings, and the phytophthora parasitica is applied after the biocontrol microbial inoculum is alternately applied for 3 times. Wherein, the biocontrol microbial inoculum is diluted by 1:200, and the root of each plant is irrigated by 0.5L; 500 muL of germs (about OD value = 1.0) are taken, and the germs are diluted to 50ml for carrying out the stem injury and root irrigation on the potatoes. The control Group (GT) is not applied with the composite micro-ecological preparation, but only applied with GT fertilizer for strengthening the seedlings and irrigated with clear water.
（1）Effect sequence
Selecting an experimental group GF, respectively and randomly taking 3 parallel soil samples before and after the phytophthora parasitica is applied, extracting total DNA of potato rhizosphere soil by using an improved CTAB method, and purifying the DNA by using a gel recovery kit. The V4-V5 hypervariable region of the gene was amplified with the universal primers 515F and 909R. Purifying by using a SanPrep DNA gel recovery kit after agarose gel electrophoresis, mixing the obtained DNAs in equal amount, and performing machine sequencing. Soil DNA samples were sequenced with Illumina Hiseq PE 250. And after the machine is off, splicing the Merged sequences to obtain an Effect sequence. Sequencing work was performed by Guangdong Meilikang Biotechnology Ltd. The number of sequences (Effect sequences) per sample after processing the preliminarily obtained Merged sequences by splicing is shown in the following table:
(2) OTU clustering analysis and annotation
OTU clustering analysis and annotation are carried out on the Effect sequence, Reads with the sequence similarity of more than 97% are classified into a class of Operational Taxonomic Units (OTUs) by Uprease, and the OTUs result of each sample is shown in the following table:
the PecQ group was lower at phylum, class level than the PecH group and BC group; at the level of orders, families, genera and OTUs PecQ group > PecH group > BC group, with slightly higher numbers of OTUs in PecQ group than PecH group. The application of the biocontrol microbial inoculum can reduce the species of harmful microbe phyla, while the application of the phytophthora parasitica (PecH) can cause the plants to have pathological effects to a certain extent, so that the species of microbes at the phyla level are increased. Therefore, the application of the biocontrol microbial inoculum is beneficial to improving the structure of soil flora and improving the abundance of soil microbial species.
(3) Analysis of Community Structure and species abundance between groups at phylum and genus taxonomy level
Figure 4 shows the portal (Phylum) level annotation for each treatment group. The species abundances of the three groups of PecQ group, PecH group and BC group are basically the same in the top 10 gates, but the species abundances of each group are different. Compared with a blank control group (BC), the embodiment is that the two experimental groups of the compound bacillus agent before (PecQ group) and after (PecH) the black shank bacterium application are added, and the highest abundance is respectively proteobacteria, actinomycetemcomitans, acidibacterium and bacteroidetes; the difference between a PecQ group and a BC group in an actinomycete phylum is very obvious, the difference between a PecH group and the BC group is obvious, and the experiment groups show that the abundance is reduced; the PecH group and the BC group in the bacteroidetes are obviously different, and the abundance of the experimental group is increased; in addition, there were differences between the experimental groups (PecQ, PecH) and the BC group in proteobacteria, firmicutes, and between the PecH and BC groups in acidophyla, pythium and blastomyces. The phylum firmicutes, acidibacterium and proteobacteria are dominant in rhizosphere microorganisms, the genus of phytophthora nigra is in the phylum proteobacteria, and the biocontrol bacillus belongs to the phylum firmicutes. Therefore, the biocontrol microbial inoculum disclosed by the invention is beneficial to improving the relative abundance of microorganisms such as rhizosphere soil proteobacteria, acidobacter and closterium of the potato potted plant, reducing the relative abundance of microorganisms such as actinomycetes and firmicutes, maintaining the dynamic stability of a soil microbial flora after black shank treatment, and playing an important role in the prevention and control of the black shank, the soil material circulation and the ecological environment construction process.
Dominant Genus statistics with significant differences at the Genus (Genus) level for the PecQ, PecH and BC groups are shown in the following table:
the microorganism of BC group, PecQ group and PecH group has 1187 types in total, wherein the content of the microorganism is more than 26 types, and the black shank bacterium (Pebacter spp.) is not detected in each treatment group, which indicates that the applied black shank bacterium does not become the dominant genus of rhizosphere soil microorganism. The dominant genera shared by the BC group and the PecQ group and the PecH group areArthrobacter(Arthrobacter), SAGMA-X,Gaiellaceae(Actinomyces),Rhodoplanes(genus rhodobacter),Candidatus Nitrososphaera(genus Ammoxalactophora),Gemmataceae(genus Bacillus), MND1, Koribacteraceae. Pronuclei of BC group, PecQ group and PecH groupThe dominant genera of microbial communities vary widely in species and composition. Compared with BC group, the PecQ group and PecH group have increased dominant bacteria, Actinomycetes and rhodobacter become main dominant bacteria, and the content of Arthrobacter and SAGMA-X (Spongyloidea) is reduced significantly. The PecH group has reduced content of actinomyces and SBla14 compared with the PecQ group; bacillus (A), (B), (C) and (C)Bacillus) The relative abundance is improved by nearly 2 times, and the strain becomes a dominant genus; rhodobacter is the most dominant bacterium. The phytophthora parasitica is applied to a certain extent to enable the plants to have pathological effects, so that the abundance of actinomyces species is reduced; but also stimulates the abundance of the bacillus species to increase, and improves the resistance to the phytophthora parasitica to a certain extent. Therefore, the biocontrol microbial inoculum is beneficial to improving the abundance of actinomyces, rhodobacter and archaea ammoniagenes, improving the disease resistance of potatoes and playing a role in inhibiting the planting of the phytophthora parasitica.
(4) Alpha-diversity index
The α -diversity index (Shannon index, PD index, Chao1 index, Simpson index) results are shown in the following table:
the α -diversity index PecQ group > PecH group > BC group, and the α -diversity indices of the PecQ group and PecH group were not significantly different. Therefore, the application of the biocontrol microbial inoculum can effectively improve the diversity of rhizosphere soil microorganisms of the potato pot culture soil, and the diversity of the soil microorganisms is kept relatively constant after the potato pot culture is treated by the phytophthora parasitica.
(5) PCoA sequencing analysis
The Weighted UniFrac PCoA ranking graph is shown in FIG. 5, and Weighted UniFrac PCoA reflects the degree of similarity in species composition from group to group: the BC group is far away from the PecQ group and the PecH group, which shows that the species composition difference of the BC group is the largest as compared with the PecQ group and the PecH group, and the difference of the two groups is smaller; further, as can be seen from fig. 5, for the first principal component having the smallest contribution value, the BC group is spaced farther apart, and the PecQ group and the PecH group are spaced relatively closer together. Therefore, the species composition structure difference is large before the biocontrol microbial inoculum is applied, and the biocontrol microbial inoculum has important effects on maintaining the stable structure of the soil microbial community after the biocontrol microbial inoculum is applied and maintaining the stable structure of the soil microbial community after the phytophthora parasitica treatment.
Claims (6)
1. The biocontrol microbial inoculum for preventing and treating potato black shank is characterized in that the viable bacteria content of bacillus in each gram of microbial inoculum is more than or equal to 1 × 108The bacillus is a compound bacterium of bacillus pumilus, bacillus megaterium, bacillus amyloliquefaciens, bacillus licheniformis and bacillus mucilaginosus.
2. The biocontrol microbial inoculum for controlling potato black shank as in claim 1, wherein the ratio of the colony forming effective viable Count (CFU) of the bacillus pumilus, the bacillus megaterium, the bacillus amyloliquefaciens, the bacillus licheniformis and the bacillus mucilaginosus is (2.3-5.2): (2.3-6.0): (2.0-5.0): (2.0-5.5): (2.5-5.5).
3. The preparation method of the biocontrol microbial inoculum for controlling the potato black shank as the claim 1 is characterized by comprising the following steps:
a. inoculating activated bacillus mucilaginosus into a silicate liquid culture medium, inoculating activated bacillus pumilus, bacillus megaterium, bacillus amyloliquefaciens and bacillus licheniformis into an LB liquid culture medium, wherein the inoculation amount is 5-8%, and culturing for 12-24h under the conditions that the temperature is 35-37 ℃, the stirring speed is 110-120r/min, so as to prepare a seed solution;
b. and fully mixing the dried and sieved carrier with the seed solution according to the proportion of 10-20ml:200-300g, culturing for 36-48h at normal temperature by taking 5wt% of sodium chloride, 2.5wt% of sucrose and 1.25wt% of citric acid as protective agents, and drying after the pH value is less than 4, wherein the drying temperature is 45-60 ℃, thus obtaining the biocontrol microbial inoculum.
4. The preparation method of the biocontrol microbial inoculum for controlling potato blackleg as claimed in claim 3, wherein the silicate liquid culture medium and LB liquid culture medium comprise 10wt% of carbon source and 5wt% of nitrogen source.
5. The preparation method of the biocontrol microbial inoculum for controlling potato blackleg as claimed in claim 4, wherein the carbon source is brown sugar, and the nitrogen source is 3wt% of bean flour and 2wt% of milk powder.
6. The preparation method of the biocontrol microbial inoculum for controlling potato blackleg as claimed in claim 3, wherein the carrier is at least one of soybean flour and diatomite.
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