CN115500194B - Method for preventing lily wilt by utilizing interaction of earthworms and bacillus bailii YFB3-1 - Google Patents
Method for preventing lily wilt by utilizing interaction of earthworms and bacillus bailii YFB3-1 Download PDFInfo
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- CN115500194B CN115500194B CN202211313248.4A CN202211313248A CN115500194B CN 115500194 B CN115500194 B CN 115500194B CN 202211313248 A CN202211313248 A CN 202211313248A CN 115500194 B CN115500194 B CN 115500194B
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
Abstract
The invention belongs to the technical field of soil-borne disease prevention and control, and particularly relates to a method for preventing lily wilt by utilizing interaction of earthworms and bacillus bailii YFB3-1. The method comprises the following steps: (1) Firstly, putting soil in an empty basin, loosening the soil, and then applying chemical fertilizers; (2) Planting lily bulbs in the pot after the fertilizer is applied in the step (1), then covering soil, and spraying YFB3-1 bacterial liquid; (3) Putting earthworms on the table Shi Niufen on the basin treated in the step (2), and covering the surface with straw; (4) Spraying YFB3-1 bacterial liquid after the lily seedlings in the basin in the step (3) are developed. The invention improves the relative abundance of Flavobacteriales, pseudomonadales, and pseudomonades can generate antibacterial secondary metabolites, can effectively inhibit the occurrence of soil-borne diseases and reduce the occurrence of continuous cropping lily fusarium wilt diseases; the invention can promote the reproduction of Bacilles, maximize the abundance of Bacilles, further inhibit the occurrence of soil-borne diseases and reduce the occurrence of continuous cropping lily fusarium wilt diseases.
Description
Technical Field
The invention belongs to the technical field of soil-borne disease prevention and control, and particularly relates to a method for preventing lily wilt by utilizing interaction of earthworms and bacillus bailii YFB3-1.
Background
The lily has economic values of medical use, eating, ornamental use and the like, but the occurrence of diseases is more and more severe, and the problem that the occurrence of lily wilt becomes a hot spot in cultivation is relieved.
The lily wilt is also called lily stem rot and root rot, is a disease caused by soil-borne bacteria, and has higher incidence rate in lily, especially in the large-area cultivation area of Longya lily. When the disease is popular, the plant disease susceptibility rate reaches 91.42%, the disease index is 88.50, and the yield is reduced by more than 46% on average. The lily wilt disease is serious in the middle and later stages of lily growth, and the first disease part is between the stem and the soil surface. After the plant is infected, the plant grows slowly, the plant cannot transport nutrients upwards, the leaves turn yellow and gradually wilt from bottom to top, the vascular bundles of the later-stage plant are necrotized and attached with white dots, and the root system grows slowly. Pink or pink mold covers the affected area when wet.
In order to effectively prevent and treat lily wilt, growers mainly treat soil in a physical, chemical and biological mode, wherein physical methods include lime, ammonium bicarbonate and the like, chemical methods include trifloxystrobin, chlorothalonil, thiram, carbendazim, mancozeb and the like, and biological methods include biocontrol bacteria, biological organic fertilizers, wormcast and the like. The treatment methods are beneficial to improving the microbial community structure of continuous cropping soil, improving limiting factors which are unfavorable for crop growth and yield, and mainly adjusting the transformation of fungal soil into bacterial soil.
The biocontrol bacteria have the characteristics of disease resistance, growth promotion, strong stress resistance, environmental friendliness and the like, and are one of the hot spots for developing biocontrol researches for scientific researchers. At present, researches on the release of continuous cropping obstacle by the biocontrol microbial inoculum are mainly focused on the effects of a flat plate counter test and a potting test, and few products capable of relieving the continuous cropping obstacle of crops are applied to a field, and the main reason is that the biocontrol microbial inoculum is difficult to colonize under the influence of the field environment, so that the control effect of the biocontrol microbial inoculum is often inferior to that of the application of a chemical bactericide.
At present, a method is needed in the market, which can effectively utilize biocontrol bacteria to prevent and treat the fusarium wilt of lily, and achieves the effect of reducing the morbidity and the disease index of continuous cropping lily fusarium wilt.
Disclosure of Invention
In view of the above disadvantages, the present invention provides a method for preventing lily wilt by utilizing the interaction of earthworm and Bacillus bailii YFB3-1. The invention improves the relative abundance of Flavobacteriales, pseudomonadales, and simultaneously the antibacterial secondary metabolite produced by pseudomonades can effectively inhibit the occurrence of soil-borne diseases and reduce the occurrence of continuous cropping lily fusarium wilt diseases; the invention can promote the reproduction of Bacilles, maximize the abundance of Bacilles, further inhibit the occurrence of soil-borne diseases and reduce the occurrence of continuous cropping lily fusarium wilt diseases.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for preventing lily wilt by utilizing interaction of earthworms and bacillus belicus YFB3-1, comprising the following steps:
(1) Firstly, loosening soil, and then applying chemical fertilizer;
(2) Planting lily bulbs after applying the fertilizer in the step (1), covering soil, and spraying YFB3-1 bacterial liquid;
(3) After the treatment in the step (2), table Shi Niufen, then putting earthworms and covering the surface with straw;
(4) Spraying YFB3-1 bacterial liquid after the lily seedlings in the step (3).
Preferably, the preservation number of the bacillus belicus YFB3-1 in the China center for type culture collection is CCTCC M20221140, and the preservation organization address is: eight paths of 299 in Wuchang district of Wuhan, hubei province, date of preservation: 2022, 7, 21, classification name: bacillus velezensis YFB 3-3.
Preferably, the fertilizer in the step (1) is a mixture of urea, superphosphate and potassium sulfate, and the application amount of the urea is 20-30g/m 2 The application amount of the superphosphate is 140-240g/m 2 The application amount of the potassium sulfate is 50-100g/m 2 。
Preferably, the planting row spacing of the lily bulbs in the step (2) is 0.15-0.25m; the thickness of the covered soil is 0.01-0.02m; the spraying quantity of the YFB3-1 bacterial liquid is 20-50mL/m 2 The thallus concentration is 1-10×10 8 CFU/mL。
Preferably, in the step (3), the earthworms are Eisenia foetida Daping II, and the adding amount of the earthworms is 1-3 earthworms per lily; the application amount of the cow dung is 2-2.5kg/m 2 。
Preferably, the YFB3-1 bacterial liquid in the step (4) is sprayed in an amount of 20-50ml/m 2 The thallus concentration is 1-10×10 8 CFU/mL。
Compared with the prior art, the invention has the following beneficial effects:
(1) The interaction of the earthworm activity and bacillus beliensis YFB3-1 improves the relative abundance of Flavobacteriales, pseudomonadales, is more beneficial to promoting the propagation of beneficial rhizosphere microorganisms, improves the effective phosphorus content of the continuously-cropping lily, increases the soil fertility, promotes the growth robustness and the stress resistance of the lily, and meanwhile, the antibacterial secondary metabolite produced by Pseudomonadales can effectively inhibit the occurrence of soil-borne diseases and reduce the occurrence of continuous cropping lily fusarium wilt.
(2) Earthworm activity combined with YFB3-1 spraying promotes the proliferation of Bacilles, maximizing Bacilles abundance. Meanwhile, bacillus beleiensis belongs to one species under bacillus, so that the interaction relationship between earthworms and bacillus beleiensis YFB3-1 is stronger, the occurrence of soil-borne diseases is further inhibited, and the occurrence of continuous cropping lily fusarium wilt diseases is reduced.
(3) The interaction of earthworm and bacillus belay YFB3-1 can reduce cost investment and increase the yield of continuous cropping lily.
Drawings
FIG. 1 is a schematic diagram showing a method for preventing lily wilt by utilizing interaction of earthworm and Bacillus bailii YFB3-1 in the present invention;
FIG. 2 is a diagram of example 1 for analyzing earthworm and Bacillus bailii YFB3-1 treated specific high abundance species based on LEfSe;
FIG. 3 is a graph of comparative example 1 for analyzing specific high abundance species of conventional fertilizers and cow dung based on LEfSe;
FIG. 4 is a graph of comparative example 2 for analyzing earthworm-treated specific high abundance species based on LEfSe;
FIG. 5 is a graph of comparative example 3 treatment specific high abundance species from Bacillus bailii YFB3-1 based on LEfSe analysis;
FIG. 6 is a graph showing the difference in relative abundance of Bacillus species from different treatments.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise specified, both chemical reagents and materials in the present invention are purchased through a market route or synthesized from raw materials purchased through a market route.
The Eisenia foetida big flat No. 2 is derived from North Hunan earthworm culture Limited company in Hunan province; the preservation number of the bacillus belicus YFB3-1 in the China center for type culture collection is CCTCC M20221140, and the preservation date is as follows: 2022, 7, 21, classification name: bacillus velezensis YFB3-1, deposit institution address: no. 299 of the Wuchang district of Wuhan, hubei province; the lily variety is lilium candidum; the cow dung is sourced from cow farms of long sand agricultural environment observation research stations of subtropical agricultural ecological institute of China academy of sciences, and has the following basic physicochemical properties: pH 8.87, SOC 345.94g/kg, TN 14.85g/kg, P 2 O 5 6.58g/kg，K 2 O9.76 g/kg, water content 76.42%,the carbon-nitrogen ratio is 23.30.
Example 1
A method for preventing lily wilt by utilizing interaction of earthworms and bacillus belicus YFB3-1, comprising the following steps:
(1) Firstly, putting soil into an empty basin with the length, width and height dimensions of 0.40m multiplied by 0.30m multiplied by 0.16m, loosening the soil, and then uniformly applying 3.0g of urea, 22.7g of calcium superphosphate and 9.1g of potassium sulfate;
(2) Planting lily bulbs in the pot with the fertilizer applied in the step (1), wherein the spacing between planting rows is 0.15m, then covering a layer of soil with the spacing of 0.01m, and spraying the lily bulbs with the concentration of 1 multiplied by 10 8 CFU/mL bacillus bailii YFB3-1 bacterial liquid with spraying amount of 30mL/m 2 ；
(3) Applying 0.27kg of cow dung on the basin treated in the step (2), then putting 1 Eisenia foetida Daping No. two on each lily bulb planting position, and covering the surface with straw;
(4) Spraying the lily in the basin of the step (3) to a concentration of 1 multiplied by 10 after emergence of seedlings 8 CFU/mL bacillus bailii YFB3-1 bacterial liquid with spraying amount of 30mL/m 2 。
Comparative example 1
A method for preventing lily wilt, comprising the steps of:
(1) Firstly, putting soil into an empty basin with the length, width and height dimensions of 0.40m multiplied by 0.30m multiplied by 0.16m, loosening the soil, and then uniformly applying 3.0g of urea, 22.7g of calcium superphosphate and 9.1g of potassium sulfate;
(2) Planting lily bulbs in the pot after the fertilizer is applied in the step (1), wherein the spacing between planting rows is 0.15m, and then covering a layer of soil with the spacing of 0.01 m;
(3) Applying 0.27kg of cow dung to the basin treated in the step (2), and covering the surface with straw;
the difference between the comparative example and the example is that the comparative example is not provided with Eisenia foetida No. two and is not sprayed with Bacillus belicus YFB3-1 bacterial liquid.
Comparative example 2
A method for preventing lily wilt, comprising the steps of:
(1) Firstly, putting soil into an empty basin with the length, width and height dimensions of 0.40m multiplied by 0.30m multiplied by 0.16m, loosening the soil, and then uniformly applying 3.0g of urea, 22.7g of calcium superphosphate and 9.1g of potassium sulfate;
(2) Planting lily bulbs in the pot after the fertilizer is applied in the step (1), wherein the spacing between planting rows is 0.15m, and then covering a layer of soil with the spacing of 0.01 m;
(3) And (3) applying 0.27kg of cow dung on the basin treated in the step (2), then putting 1 Eisenia foetida Daping No. two on each lily bulb planting position, and covering the surface with straw.
The difference between the comparative example and the example is that the comparative example does not spray Bacillus bailii YFB3-1 bacterial liquid.
Comparative example 3
A method for preventing lily wilt, comprising the steps of:
(1) Firstly, putting soil into an empty basin with the length, width and height dimensions of 0.40m multiplied by 0.30m multiplied by 0.16m, loosening the soil, and then uniformly applying 3.0g of urea, 22.7g of calcium superphosphate and 9.1g of potassium sulfate;
(2) Planting lily bulbs in the pot with the fertilizer applied in the step (1), wherein the spacing between planting rows is 0.15m, then covering a layer of soil with the spacing of 0.01m, and spraying the lily bulbs with the concentration of 1 multiplied by 10 8 CFU/mL bacillus bailii YFB3-1 bacterial liquid with spraying amount of 30mL/m 2 ；
(3) Applying 0.27kg of cow dung to the basin treated in the step (2), and covering the surface with straw;
(4) Spraying the lily in the basin of the step (3) to a concentration of 1 multiplied by 10 after emergence of seedlings 8 CFU/mL bacillus bailii YFB3-1 bacterial liquid with spraying amount of 30mL/m 2 。
The difference between the comparative example and the example is that the comparative example does not put Eisenia foetida Daping No. two.
The basic physicochemical properties of the plough layer soil at the beginning of the test are: pH value is 5.42, organic matter is 20.57g/kg, total nitrogen is 1.72g/kg, total phosphorus is 0.91g/kg, total potassium is 21.24g/kg, alkaline hydrolysis nitrogen is 89.73mg/kg, available phosphorus is 77.68mg/kg, and quick-acting potassium is 92.54mg/kg.
The lily is planted in the first year, the lily is planted in the next year about 9 months and 26 days from the first year, 4 lily plants are planted in each pot, each treatment is repeated 3 times, and a total of 60 pots are planted in each 5 pots. The second year of lily maturity (around 7 months and 16 days each year) is investigated for the incidence rate and disease index of lily wilt, and lily is harvested on 7 months and 28 days. The incidence and disease index of lily wilt are measured in a grading manner according to the incidence degree of lily wilt by taking plants as units, 16 plants are repeatedly measured every time, and the total number of plants and the number of plants of each stage of disease are recorded and measured. The survey grading criteria are as follows: the level 0 is that the plant stalk is normal, and the whole plant has no disease leaves; the level 1 is that the number of yellowing or purple leaves at the bottom of a disease plant is not more than 25% of the number of leaves of the whole plant, the top of a stem becomes light purple, and a heart leaf is slightly bent to one side; 2. the plant is characterized in that the leaf number of the leaf withering or withering at the bottom of the plant accounts for 25-50% of the whole plant, and the upper part of the stem becomes purple and is obviously bent; the level 3 is that the withered leaves of the plant exceed 50%, the middle and upper parts of the stems become purple and are severely bent; the leaves of the whole plant of grade 4 are withered or the whole plant is withered, and the stem basal vascular bundle turns brown. Wherein:
morbidity = number of diseased plants/total number of plants x 100%;
disease index = Σ (number of individual disease plants x number of relative stages)/(total number of plants x number of highest stages) ×100.
1 pot in each repetition is randomly extracted in the vigorous long-term lily (5 months and 10 days in 2020), soil is taken out by a cutting ring at the middle position between two lily plants, the porosity and the volume weight of the soil are measured by a cutting ring method, and then 4 lily plants are taken out from one pot in each repetition by a small shovel. Slightly shaking lily to remove non-rhizosphere soil, and then taking rhizosphere soil on a lily stem root system to divide into three parts: one part is used for measuring the bacterial, fungal, actinomycete and Fusarium quantity of soil by a dilution coating plate method; measuring the physical and chemical property related indexes of the soil after natural air drying: the pH is measured by a PHS-3C pH meter according to the soil-water ratio of 1:5, soil organic matters are measured by a sulfuric acid-potassium dichromate oxidation method, alkaline hydrolysis nitrogen is measured by an alkaline hydrolysis diffusion method, active phosphorus is measured by a sodium bicarbonate extraction-molybdenum-antimony colorimetry, and quick-acting potassium is measured by an ammonium acetate leaching-flame photometry method; and (3) packaging the materials with a sterilizing centrifuge tube, quick-freezing the materials in an ultralow temperature refrigerator at the temperature of minus 80 ℃, and then delivering the materials to Shanghai Meiji biological medicine technology Co., ltd by utilizing an ice box for gene sequencing.
The plant height and stem thickness of Lily in continuous cropping were measured for a vigorous period (25 days of 5 months in 2020). Plant height and stem thickness were measured using tape measure and vernier caliper, respectively. The incidence and disease index of lily wilt at maturity (7.19 days in 2020) were investigated and harvested at 30 days in 7 months and yield was determined.
The test data results are shown in tables 1-9:
TABLE 1 Effect of Lumbricus and YFB3-1 on continuous cropping Lily growth and yield
Note that: data are the average of three replicates. Lowercase letters indicate that the differences between the different treatments are significant (P < 0.05)
As is clear from Table 1, the plant height and stem thickness of lily are the best as in example 1 in vigorous periods. The highest yield of lily of example 1 was 1567g/m 2 Significantly higher than other treatments. Therefore, the earthworm activity combines with the YFB3-1 microbial inoculum spraying, and has the best effect of promoting the growth and increasing the yield of the continuous cropping lily.
TABLE 2 influence of Lumbricus and YFB3-1 on physicochemical properties of Lily soil in continuous cropping
Note that: data are the average of three replicates. Lowercase letters indicate that the differences between the different treatments are significant (P < 0.05)
From Table 2, it can be seen that the alkaline hydrolysis nitrogen, available phosphorus and quick-acting potassium content are all significantly higher than the other treatments, with the examples being the largest. Therefore, the earthworm activity combined with the YFB3-1 spraying can effectively activate the availability of soil nutrients (alkaline hydrolysis nitrogen, available phosphorus and available potassium content), and the activating effect on the nutrients is more obvious.
TABLE 3 influence of Lumbricus and YFB3-1 on microorganism number in rhizosphere soil of Lily continuous cropping
Note that: data are the average of three replicates. Lowercase letters indicate that the differences between the different treatments are significant (P < 0.05)
As can be seen from Table 3, the bacterial, actinomycete and Bacillus amounts of the soil were all significantly higher than other treatments, with example 1 being the most abundant. The numbers of fungi and fusarium were the greatest in comparative example 1 and the least in example 1. Therefore, the combination of earthworm activity and YFB3-1 spraying is more beneficial to increasing the quantity of bacteria, actinomycetes and bacillus in rhizosphere soil and reducing the quantity of fungi and fusarium.
TABLE 4 influence of Lumbricus and YFB3-1 on the incidence and disease index of continuous cropping Lily wilt
| Project | Incidence of wilt disease | Index of disease condition |
| Example 1 | 46％ | 25 |
| Comparative example 1 | 73％ | 40 |
| Comparative example 2 | 69％ | 36 |
| Comparative example 3 | 56％ | 30 |
As can be seen from table 4, the incidence and index of disease for example 1 were both minimal and significantly lower than for the other treatments. Therefore, the combination of earthworm activity and YFB3-1 spraying is more beneficial to reducing the incidence rate and the disease index of continuous cropping lily wilt.
TABLE 5 microbial alpha diversity of Lily rhizosphere soil in continuous cropping
Ace index and Chao1 index are used for representing species richness, and the larger the parameters of the Ace index and the Chao1 index are, the higher the species richness is; shannon index is used to characterize biodiversity, the larger its parameters, the higher the biodiversity. As can be seen from Table 5, both Ace index and Chao1 index were the largest as in example 1, as analyzed by high throughput sequencing data. The inoculation of earthworms and the spraying of YFB3-1 microbial inoculum are more beneficial to improving the richness and diversity of bacterial community structure species of the rhizosphere soil of the continuous cropping lily. Meanwhile, the Coverage rate (Coverage) of each sample is 98.22% -98.49%, which shows that the probability of undetected gene sequences is very low, and the sequencing result can represent the real situation of continuous cropping lily stem root soil bacterial community under different treatment modes.
As can be seen from fig. 2-5, based on the effect magnitude linear discriminant analysis, the different treated shoot root soil bacteria were analyzed for a significant difference in target levels of the marker species. The relative abundance of seven species, namely, the threshold LDA of the linear discriminant analysis is set to be more than or equal to 4.0, and the sample in the aspects of the method of example 1 is significantly higher than that of other treatments at the department level Flavobacteriaceae, pseudomonadaceae, xanthobateraceae, the mesh level Flavobacteriales, pseudomonadales and the genus level Flavobacterium, pseudomonas.
In fig. 6, example 1 is denoted by T3, comparative example 1 is denoted by CK, comparative example 2 is denoted by T1, and comparative example 3 is denoted by T2. As can be seen from FIG. 6, the relative abundance of Bacilles in example 1 (T3) is maximized.
The foregoing is a further detailed description of the present invention in connection with specific embodiments, and it is not intended to limit the practice of the invention to such specific embodiments, but it will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and the invention is to be considered as falling within the scope of the invention.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (1)
1. A method for preventing lily wilt by utilizing interaction of earthworms and bacillus belicus YFB3-1, comprising the steps of:
(1) Firstly, loosening soil, and then applying chemical fertilizer;
(2) Planting lily bulbs after applying the fertilizer in the step (1), covering soil, and spraying YFB3-1 bacterial liquid;
(3) After the treatment in the step (2), table Shi Niufen, then putting earthworms and covering the surface with straw;
(4) Spraying YFB3-1 bacterial liquid after the lily seedlings in the step (3) emerge;
the preservation number of the bacillus belicus YFB3-1 in the China center for type culture collection is CCTCC M20221140, and the preservation organization address is that: no. 299 of the Wuchang district of Wuhan, hubei province;
the fertilizer in the step (1) is a mixture of urea, calcium superphosphate and potassium sulfate, and the application amount of the urea is 20-30g/m 2 The application amount of the superphosphate is 140-240g/m 2 The application amount of the potassium sulfate is 50-100g/m 2 ；
The planting row spacing of the lily bulbs in the step (2) is 0.15-0.25m; the thickness of the covered soil is 0.01-0.02m; the spraying quantity of the YFB3-1 bacterial liquid is 20-50mL/m 2 The thallus concentration is 1-10×10 8 CFU/mL；
The earthworms in the step (3)The earthworms are put into 1-3 lily plants for Eisenia foetida Daping II; the application amount of the cow dung is 2-2.5kg/m 2 ；
The spraying amount of the YFB3-1 bacterial liquid in the step (4) is 20-50ml/m 2 The thallus concentration is 1-10×10 8 CFU/mL。
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