CN115141059B - Microbial fertilizer for preventing and treating red root disease of rubber tree and preparation method thereof - Google Patents
Microbial fertilizer for preventing and treating red root disease of rubber tree and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a microbial fertilizer for preventing and treating rubber tree red root diseases and a preparation method thereof. The microbial fertilizer consists of the following components in parts by weight: 5-15 parts of microorganism bacteria, 70-90 parts of a mixture of livestock and poultry waste and agricultural waste, and 5-15 parts of an organic fertilizer component; wherein, the weight part ratio of the livestock and poultry waste to the agricultural waste is 59-70: 30 to 41. The following criteria are met: the effective viable count is more than or equal to 6.53 multiplied by 10 8 CFU·g ‑1 The method comprises the steps of carrying out a first treatment on the surface of the The spore number is more than or equal to 5.97X10 8 CFU·g ‑1 The method comprises the steps of carrying out a first treatment on the surface of the The germination index of seeds is more than or equal to 80 percent; N+P 2 O 5 +K 2 The O content is more than or equal to 6.13 percent, and the organic matters are more than or equal to 54.12 percent. The microbial fertilizer can increase the organic matter content of soil and has obvious soil improvement effect; the composition is mixed with triazole preparations, so that the use amount of chemical agents is reduced, the occurrence and spread of rubber tree red root diseases are more effectively inhibited, and the curative effect is obvious.
Description
Technical Field
The invention relates to the technical field of microbial fertilizers, in particular to a microbial fertilizer for preventing and treating red root diseases of rubber trees and a preparation method thereof.
Background
Natural rubber is an important raw material for economic development, industrial production and national defense construction, is an important strategic resource, and depends on import for more than 75% each year. The root disease is one of three diseases which endanger the production of natural rubber, and commonly occurs in all glue-planting areas of the world, and the occurrence of the glue-planting areas in Yunnan and Hainan is most serious in China. The disease is mainly transmitted through root system contact and can be caused by infection of various fungi; the root is harmed, the root rot is caused, and the plant is finally withered and dead. In recent years, due to low price of rubber, the phenomenon that farmers discard pipes in rubber gardens (trees) is common, so that the occurrence of rubber tree root diseases is more serious, plant crowns are sparse, terminal buds cannot be extracted or the buds are uneven, and leaves lose water, turn yellow, become small, roll and matt; many dried branches, necrotic root system and dead plants. The red root disease of the rubber tree is the first root disease affecting the natural rubber production in China, has high morbidity, quick transmission, high mortality and wide host range, and if the infected root disease tree is not prevented and treated in time, more than 90% of the disease trees die within one year and two years, and becomes a great threat to the development of the natural rubber industry.
Tridemorph is commonly used for root irrigation to prevent and treat rubber tree red root diseases in the current production, but a series of problems of high medicament cost, drug resistance of pathogenic bacteria, reduced prevention effect, ecological safety to rubber forests and the like exist in single application of the bactericide for a long time. Compared with other methods, the biological control technology has the characteristics of safety, sustainability, environmental friendliness and the like, and is widely applied. The research and development of comprehensive prevention and control measures combined with biological prevention and control technology and the like has important significance for the healthy development of the natural rubber industry.
There has been studied screening of microorganisms having iron-philic elements from the roots of rubber trees and having antagonism against red root pathogens of rubber trees, for example, chinese patent No. ZL201110042863.1 issued for 4 months and 16 days in 2014 "an antagonizing bacterium (Bacilus subtilis Czk 1) capable of producing siderophores to control plant diseases".
The livestock manure contains abundant crude proteins, amino acids, minerals, trace elements, vitamins and the like, and simultaneously contains harmful substances such as heavy metals, antibiotics, pathogenic microorganisms, parasites, harmful gas hydrogen sulfide and the like, and if the livestock manure is abandoned at any place, the environment pollution and epidemic disease transmission can be caused, and a large amount of nutrient sources are caused and the waste is greatly caused. In addition, agricultural wastes (such as straw, fungus chaff and the like) are not utilized in time, so that pollution and resource waste are caused to the environment, and sustainable development of agriculture in China is restricted. The chicken manure, fungus chaff, coconut chaff and other agricultural wastes are piled and fermented at high temperature to prepare the biological fungus fertilizer which is then applied to the soil, so that the problem of agricultural organic wastes can be effectively solved, and the use amount and the input of chemical fertilizers are reduced. Therefore, agricultural organic waste is used as a basic raw material, is fermented at high temperature to form high-quality organic fertilizer, and is used as a carrier of soil beneficial microorganisms to prepare the microbial fertilizer with definite functions, so that the microbial fertilizer has important application value.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a microbial fertilizer for preventing and treating rubber tree red root disease and a preparation method thereof, so as to solve the problems in rubber production.
The above object of the present invention is achieved by the following technical solutions:
the microbial fertilizer for preventing and treating the red root disease of the rubber tree is prepared from the following components in parts by weight: 5-15 parts of microorganism bacteria, 70-90 parts of a mixture of livestock and poultry waste and agricultural waste, and 5-15 parts of an organic fertilizer component; wherein, the weight part ratio of the livestock and poultry waste to the agricultural waste is 59-70: 30 to 41.
Preferably, the microbial fertilizer consists of the following components in parts by weight: 10 parts of microorganism bacteria, 80 parts of a mixture of livestock and poultry waste and agricultural waste, and 10 parts of an organic fertilizer component; wherein, the weight portion ratio of the livestock and poultry waste to the agricultural waste is 62:38.
the livestock and poultry waste is chicken manure which is poultry waste.
The agricultural waste comprises at least one of corn straw, fungus chaff and coconut chaff; preferably fungus chaff, such as needle mushroom chaff.
The organic fertilizer component is soybean powder.
The microorganism is bacillus subtilis Czk1 which is preserved in China general microbiological culture Collection center, and the preservation unit address: no. 1 and No. 3 of Beijing city, the morning sun area, the Beichen xi lu,the preservation number of the China academy of sciences microbiological culture collection is CGMCC No.4408. The bacterial concentration of the seed culture solution of Bacillus subtilis Czk1 was 0.5X10 8 CFU/mL～2.5×10 8 CFU/mL, preferably 1X 10 8 CFU/mL。
The microbial fertilizer prepared by the invention meets the following indexes: the effective viable count is more than or equal to 6.53 multiplied by 10 8 CFU·g -1 The spore number is more than or equal to 5.97X10 8 CFU·g -1 The germination index of seeds is more than or equal to 80 percent, and N+P 2 O 5 +K 2 The O content is more than or equal to 6.13 percent, and the organic matters are more than or equal to 54.12 percent.
The microbial fertilizer for preventing and treating rubber tree red root disease and the preparation method thereof are prepared by the following steps:
(1) Preparing seed liquid: transferring the saved Bacilus subtilis Czk strain to an LB culture medium plate, and culturing for 48-72 h at 37 ℃; selecting single colony with fixed form on the flat plate, culturing in LB liquid medium containing 50-100 mL at 37 ℃ and 180r/min for 12-16 h to obtain Bacilus subtilis Czk seed liquid; the bacterial concentration was 0.5X10 8 CFU/mL～2.5×10 8 CFU/mL；
(2) Crushing: crushing livestock and poultry waste and agricultural waste into 20-mesh particles;
(3) Mixing: mixing livestock and poultry waste and agricultural waste according to the weight part ratio, and uniformly stirring; intermittently sterilizing for two times at 121 ℃ for 30min to obtain a mixture of livestock and poultry waste and agricultural waste;
(4) Fermentation: according to the weight portion ratio, 5 to 10 parts of Czk1 seed liquid obtained in the step (1) is added into the mixture of the livestock and poultry waste and the agricultural waste obtained in the step (3), 5 to 10 parts of soybean powder is added, the mixture is uniformly mixed, the water content of the material is adjusted to 40 to 50 percent, and the mixture is subjected to standing fermentation for 7d at 37 ℃ for 72h and is turned over once; obtaining the microbial organic fertilizer.
The germination index of the seeds of the fermented fertilizer is more than or equal to 80 percent.
Another object of the invention is: provides an application of a microbial fertilizer for preventing and treating rubber tree red root disease.
Compared with the prior art, the invention provides the microbial fertilizer for preventing and treating the red root disease of the rubber tree and the preparation method thereof, and the microbial fertilizer has the following beneficial effects:
the bacillus subtilis microbial fertilizer based on the decomposed chicken manure as a main raw material fully utilizes crop wastes such as livestock manure, straw, coconut chaff and fungus chaff, can realize the recycling of resources, reduces the harm of the manure to the cultivation environment, and accords with the development concept of the recycling agriculture and the development requirement of the green agriculture; simultaneously meets the nutrition requirement of microbial fermentation and the nutrient supplement of the organic fertilizer; the microbial fertilizer produced by the invention has the function of regulating soil fertility, and bacillus subtilis can also play a role through various growth promoting and biocontrol mechanisms, so that the effects of promoting growth, improving crop disease resistance and preventing and treating rubber tree red root diseases are achieved.
Drawings
FIG. 1 is a graph showing the effect of the addition of fungus chaff on the fermentation of Czk1 in chicken manure compost
FIG. 2 is the effect of straw addition on Czk1 fermentation in chicken manure compost.
FIG. 3 is a graph showing the effect of coconut coir addition on the fermentation of Czk1 in chicken manure compost.
FIG. 4 shows the relative germination rates under different bio-organic fertilizer leaches.
FIG. 5 is a graph showing the effect of inoculum size on Czk1 fermentation in mushroom bran chicken manure compost
FIG. 6 shows the effect of soybean meal addition on Czk1 fermentation in mushroom bran chicken manure compost
FIG. 7 is a graph showing the effect of inoculum size on Czk1 fermentation in straw chicken manure compost
FIG. 8 influence of soybean flour addition on Czk1 fermentation in straw chicken manure compost
FIG. 9 influence of straw addition on fermentation of Czk1 in mushroom bran chicken manure compost
FIG. 10 influence of the addition of the fungus chaff on the fermentation of Czk1 in straw chicken manure compost
Detailed Description
The technical solutions of the present invention will be clearly and completely described below by means of examples, and it is obvious that the described examples are only some, but not all, examples of the present invention. All other technical solutions, based on the embodiments of the present invention, which a person skilled in the art would obtain without making any inventive effort, fall within the scope of the present invention.
Example 1 optimized screening of biological bacterial Fertilizer Components of the invention
1. Preparation of microbial inoculum
Bacillus subtilis Bacilus subtilis Czk (CN 102643760B is disclosed as bacillus subtilis) Czk1 which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 4408) is a biological control microorganism which is screened, separated and identified from rubber rhizosphere and preserved in the laboratory and has the functions of promoting growth, antagonizing and enhancing plant disease resistance, has good antagonism on various root diseases of rubber trees, and Czk1 can secrete various lipopeptides antibacterial substances such as surfactant, iturin, fender and the like, can generate 33 volatile substances such as hydrocarbon, amine, alcohol, alkene, phenol and the like, and can well cooperate with chemical bactericides and inhibit the pathogen of the rubber tree root diseases. The preparation method of the seed liquid of the bacillus subtilis Czk1 comprises the following steps:
inoculating Bacillus subtilis Czk1 into LB liquid medium (peptone 10g, yeast extract 5g, naCl 5g, adding water to 1000mL, adjusting pH to 7.0.) under aseptic condition at 37deg.C and 180r/min for 12 hr, and culturing to obtain final concentration of Czk1 bacterial liquid of about 1×10 8 CFU/mL is Bacilus subtilis Czk seed liquid.
2. Influence of different raw materials and proportions on secondary solid fermentation of bacillus subtilis Czk1
2.1 detection of Properties of four materials
A large number of experiments show that the decomposed chicken manure has the best fermentation and activity effects on bacillus subtilis Czk1, and meanwhile, corn stalks, fungus chaff and coconut chaff are mainly used for chicken manure composting (primary fermentation), but few research reports are provided in secondary fermentation production of bio-organic fertilizers. Therefore, the effect of improving the carbon nitrogen ratio, the pH and the functional bacteria of the fertilizer is still unclear; secondly, as the waste such as straw and the like is not completely degraded, whether the prepared fertilizer inhibits seed germination is unclear; finally, the temperature, the water content and other factors in the secondary fermentation process of the bio-organic fertilizer restrict the propagation of the functional bacteria. Therefore, the bacillus subtilis Czk1 is taken as a research object, the proportion of different wastes (coconut chaff, fungus chaff and straw) and decomposed chicken manure and the influence of various condition parameters on the total quantity of viable bacteria and the number of spores of the fermented Czk1 are researched, and the optimal effect is obtained.
The invention selects four raw materials as follows: the decomposed chicken manure is purchased from a rich agricultural supermarket, the flammulina velutipes fungus chaff (hereinafter referred to as fungus chaff) is purchased from a biological technology limited company of Liyun gang Bei Sen, the coconut chaff is purchased from an agricultural company of Tropical agricultural academy of China, and the corn stalk powder is purchased from a deep processing company of Linfeng agricultural products.
Physical and chemical property detection of four raw materials: pH and conductivity (EC): uniformly mixing the completely fermented fertilizer sample with deionized water in a ratio of 1:10, vibrating for 2 hours by a horizontal shaking table, standing for 30min, and detecting by a pH meter and a conductivity meter.
The biological organic fertilizer is digested by concentrated sulfuric acid-hydrogen peroxide, and the total nitrogen content is measured by adopting a Kjeldahl nitrogen determination method; measuring the total phosphorus content by a vanadium-molybdenum Huang Bise method; measuring the total potassium content by flame photometry; the organic matter adopts an elemental analyzer. The physicochemical properties of the four raw materials are shown in Table 1, the pH values of the four raw materials are different, the pH value of the decomposed chicken manure is the highest, and the pH value of the coconut coir is the lowest. The conductivity of the four raw materials is different, and the highest is straw and the lowest is coconut coir. The total nitrogen, total phosphorus, total potassium and the highest organic content are respectively coconut chaff, chicken manure and straw, and the lowest is respectively mushroom chaff, straw, mushroom chaff and chicken manure. The carbon-nitrogen ratio of the fungus chaff is the highest and the lowest is the decomposed chicken manure. The total content of nitrogen, phosphorus and potassium in the decomposed chicken manure is higher than that of other three auxiliary materials.
TABLE 1 basic physicochemical Properties of the four raw materials
Note that: different letters in the same column of values indicate significant differences at the P < 0.05 level.
2.2 influence of different proportions of fungus chaff and chicken manure on solid fermentation fungus amount
Adopting 250mL triangular flask solid fermentation, taking decomposed chicken manure as a base fertilizer, wherein the proportion of fungus chaff is 0%, 33%, 38%, 41% and 100%, respectively. The raw materials are crushed and sieved by a 20-mesh sieve, 40g of organic fertilizers with different proportions are filled in a triangular flask, and the materials are intermittently sterilized twice at 121 ℃ for 30 min. Sterile water was adjusted to 40% moisture content and each treatment was repeated 3 times. Inoculating Bacilus subtilis Czk seed liquid 5%, standing in a 30 ℃ incubator, fermenting for 7d, turning over and throwing once for 24h, and carrying out the next screening.
10g of the biological organic fertilizer is added into a triangular flask containing 10 glass beads and 90mL of sterile water by adopting a sterile water serial dilution plate coating counting method, 180r/min is oscillated for 30min, 1mL of diluent is added into 9mL of sterile water, serial dilution is carried out according to the diluent, finally 100ul of plates of proper diluent are coated, and the living bacteria number is measured by overnight culture counting in a 37 ℃ incubator. The spore amount of the suspension was measured by the same method after 20min of water bath treatment at 80 ℃.
As can be seen from fig. 1, the number of viable bacteria and the number of spores increased with the addition of the fungus chaff, but the number of viable bacteria and the number of spores after fermentation decreased with the addition of the fungus chaff exceeding 41%. Therefore, the ratio of the addition of the fungus chaff was 38%, and the amount of the fungus and the number of spores were all the maximum in this state.
2.3 influence of different proportions of straw chicken manure on Czk1 secondary solid fermentation
Corn stalk is added into decomposed chicken manure in 0%, 33%, 38%, 41% and 100% separately, and the mixture is left to ferment in a 30 deg.c incubator for 7d and once turned over for 24 h. Other conditions are the same as in step 2.2.
As can be seen from fig. 2, the number of viable bacteria and spores after fermentation increases with the increase of the straw addition amount, but the number of viable bacteria and spores after fermentation decreases with the increase of the straw addition amount exceeding 41%. Therefore, the proportion of straw suitable for addition during fermentation is 38%, and the number of viable bacteria and the number of spores reach the highest value at the addition amount.
2.4 influence of different proportions of coconut Cocois and chicken manure on secondary solid fermentation of Czk1
Adding 0%, 33%, 38%, 41% and 100% of coconut husk into decomposed chicken manure, standing at 30deg.C in incubator, fermenting for 7d, and turning over once for 24 h. Other conditions are the same as in step 2.2.
As can be seen from FIG. 3, the amount of coconut husk added is 33% and the number of viable bacteria and spores is the highest at this added amount. However, the addition of coconut coir does not significantly promote the increase of the bacterial load and the spore load compared with the fungus chaff and the straw, so that the addition of coconut coir is not suitable for fermenting the bio-organic fertilizer.
3. Influence of Czk1 bio-organic fertilizer on seed germination rate
Test materials: cucumber seeds (boyou green melon king 308F 1) are purchased through the local farmer market; bacilus subtilis Czk1 the microbial fertilizer is prepared from 2.2-2.4.
Test site: laboratory of the national academy of sciences of tropical agriculture, environmental and economic crop disease research group of the plant protection institute.
And taking a single-factor test organic fertilizer sample to measure the germination index of the seeds so as to show the material decomposition degree and the toxicity of the organic fertilizer. The specific method comprises the following steps: scalding cucumber seeds with hot water at 60 ℃ for 10min, sterilizing with 2% sodium hypochlorite for 3min, and flushing with distilled water for 3-4 times. The cucumber seeds are coated with red color, the coating is usually washed off through the processes, partial red coated seeds are often damaged, and damaged seeds are picked out and discarded. Two pieces of sterilizing filter paper are filled in glass culture dishes (9 cm), and 20 seeds are uniformly placed in each culture dish. The fertilizer with measured pH and conductivity (EC) was filtered, 5mL of the filtrate was added, and the culture was performed in the dark at 28℃for 48 hours, and the germination rate and root length were measured, and each treatment was repeated 3 times. Distilled water was used as a control. The germination index of the seeds is calculated according to the following formula: germination index (GI,%) = (sample germination rate x average root length)/(control germination rate x average root length) ×100.
The research results of the relative germination rates of the leaching solutions of the Czk1 bio-organic fertilizers prepared from different composting raw materials in the graph 4 and the table 2 show that after 48 hours, the relative germination rates of the leaching solutions of different bio-organic fertilizers on cucumber seeds are not less than 80 percent except for the treatment that the straw addition proportion is 100 percent. The relative germination rate of the treatment with the straw adding proportion of 100 percent is only 62.87 percent, which is unfavorable for the germination of seeds.
Table 2.Bacilus subtilis Czk1 Effect of microbial fertilizers on cucumber seed germination
And (3) selecting agricultural waste fungus chaff with good promoting effect on the fungus amount for a next test according to the test results of the straw, fungus chaff and coconut chaff added in different proportions in the step (2-3).
4. Influence of fermentation conditions on secondary solid fermentation of Czk1 in mushroom bran chicken manure compost
4.1 Effect of Czk1 inoculum size on fermentation in Mushroom bran chicken manure
Fungus chaff and chicken manure in a proportion of 38:62 parts by weight of the components are mixed and stirred uniformly; sterilizing at 121deg.C for 30min for two times to obtain the mixture of chicken manure and fungus chaff.
Inoculating Czk1 seed liquid inoculum sizes of 5%, 10% and 15% of the total raw materials into the fungus chaff chicken manure mixture respectively, adjusting the water content to 40%, repeating each treatment for 3 times, turning over and throwing once every 24 hours, and standing and fermenting in a 30 ℃ incubator for 7d.
As can be seen from FIG. 5, the amount of bacteria and the amount of spores increased with the increase of the inoculum size, but the amount of bacteria and the amount of spores decreased when the inoculum size was 15%. Therefore, the most suitable inoculation amount in the fungus chaff chicken manure is 10%.
4.2 influence of the added amount of the soybean meal on the fermentation of Czk1 in the fungus chaff chicken manure
Fungus chaff and chicken manure in a proportion of 38:62 parts by weight of the components are mixed and stirred uniformly; sterilizing at 121deg.C for 30min for two times to obtain the mixture of chicken manure and fungus chaff.
Inoculating Czk1 seed liquid into the fungus chaff chicken manure mixture according to the inoculation amount of 10% of the total amount of the raw materials, setting the addition amount of soybean powder to be 0%, 5% and 10%, adjusting the water content to be 40%, repeating each treatment for 3 times, turning over and throwing once every 24 hours, and standing and fermenting for 7d in a 30 ℃ incubator.
As can be seen from FIG. 6, the number of viable bacteria and the number of spores increased with the increase in the amount added. Therefore, the optimal soybean meal addition amount in the mushroom bran chicken manure compost is 10 percent.
4.3Czk1 fermentation condition optimization in fungus chaff chicken manure compost
Fungus chaff and chicken manure in a proportion of 38:62 parts by weight of the components are mixed and stirred uniformly; sterilizing at 121deg.C for 30min for two times to obtain the mixture of chicken manure and fungus chaff.
Solid fermentation orthogonal test: the Czk1 seed liquid is inoculated into a fungus chaff chicken manure mixture with an inoculation amount of 10 percent of the total weight of the fertilizer raw material, a proper amount of soybean powder is added, different temperatures (20 ℃,30 ℃,37 ℃) and water contents (30%, 40% and 50%) are detected respectively, and the turning times (1 time/24 h, 1 time/48 h and 1 time 72 h) influence on the total bacterial body amount is achieved. Each treatment was set to 3 replicates.
As can be seen from Table 3, the optimal fermentation process in the compost of the fungus chaff and chicken manure is that the culture temperature is 37 ℃, the water content of the material is 40%, the turning time is that the turning is carried out once every 72 hours, and the viable count is 8.63 multiplied by 10 8 CFU·g -1 The number of spores is 8.37 multiplied by 10 8 CFU·g -1 。
As can be seen from the analysis of the limit in Table 4, the main factor affecting the fermentation was the culture temperature, the limit was 3.9, the moisture was the next, and the turning time was the last. In the three conditions of temperature, the bacterial load of fermentation is directly proportional with the increase of temperature. The difference in the medium and low water contents is larger than the difference in the medium and high water contents among the three conditions of the water contents. In the turning time factor, every 72h of turning is larger than that of 24h of turning.
As can be seen from the extremely poor analysis in Table 4, the main factors affecting the fermentation of spores are the culture temperature, the moisture, and finally the turnover time. In the three conditions of temperature, the bacterial load of fermentation is directly proportional with the increase of temperature. The difference in the medium and low water contents is larger than the difference in the medium and high water contents among the three conditions of the water contents. In the turning time factor, the turning of 72h is larger than that of 24h and 48h, but the turning difference is smaller than that of 48 h.
TABLE 3 orthogonal test results of fermentation condition optimization of Czk1 in mushroom bran chicken manure compost
TABLE 4 evaluation of orthogonal test results of fermentation condition optimization of Czk1 in mushroom bran chicken manure compost
Note that: b (B) 1 、B 2 、B 3 The average value of the viable count after fermentation at 3 levels of culture temperature, water content, and number of turnovers is represented, respectively.
5. Influence of fermentation conditions on secondary solid fermentation of Czk1 in straw chicken manure compost
5.1 Effect of Czk1 inoculum size on fermentation in straw chicken manure
Corn stalk and chicken manure in a proportion of 38:62 parts by weight of the components are mixed and stirred uniformly; and (3) intermittently sterilizing for two times at 121 ℃ for 30min to obtain the straw chicken manure mixture.
Czk1 is inoculated into straw chicken manure with inoculum sizes of 5%, 10% and 15%, and the straw chicken manure is subjected to standing fermentation for 7d at 30 ℃ in an incubator and is turned over once for 24 h. As can be seen from FIG. 7, the number of viable bacteria and the number of spores increased with the increase of the inoculum size, but the number of viable bacteria and the number of spores decreased when the inoculum size was 15%. Therefore, the optimal inoculation amount in the straw chicken manure compost is 10 percent.
5.2 influence of the added amount of the soybean meal on the fermentation of Czk1 in straw chicken manure
Corn stalk and chicken manure in a proportion of 38:62 parts by weight of the components are mixed and stirred uniformly; and (3) intermittently sterilizing for two times at 121 ℃ for 30min to obtain the straw chicken manure mixture.
Inoculating Czk1 seed liquid into the straw chicken manure mixture according to the inoculation amount of 10% of the total amount of the raw materials, respectively adding soybean powder of 0%, 5% and 10% into the straw chicken manure compost, and standing and fermenting for 7d at 30 ℃ in an incubator, and turning over and throwing once for 24 h. As can be seen from FIG. 8, the number of viable bacteria and the number of spores increased with the increase in the amount added. But the addition amount of 5% is not much different from that of 10%. Therefore, according to the economic principle, the optimal soybean meal addition amount in the straw chicken manure compost is 5 percent.
5.3 orthogonal optimization of Czk1 fermentation conditions in straw chicken manure compost
Fungus chaff and chicken manure in a proportion of 38:62 parts by weight of the components are mixed and stirred uniformly; sterilizing at 121deg.C for 30min for two times to obtain the mixture of chicken manure and fungus chaff.
Solid fermentation orthogonal test: inoculating 10% of the total weight of the fertilizer raw material into a straw chicken manure mixture, adding 5% of soybean powder, respectively detecting different temperatures (20 ℃,30 ℃,37 ℃) and water contents (30%, 40% and 50%), and turning the total amount of the bacterial cells (1 time/24 h, 1 time/48 h and 1 time 72 h). Each treatment was set to 3 replicates.
As shown in Table 5, the optimal fermentation process in the compost of the fungus chaff and chicken manure is that the culture temperature is 37 ℃, the water content of the material is 40%, the turning time is that the turning is carried out once every 72 hours, and the number of viable bacteria after fermentation is 6.53 multiplied by 10 8 CFU·g -1 Spore number 5.97X10 8 CFU·g -1 。
As can be seen from the analysis of the limit in Table 6, the main factor affecting fermentation was the culture temperature, the limit was 2.81, the moisture was the next, and the turning time was the last. In the three conditions of temperature, the bacterial load of fermentation is directly proportional with the increase of temperature. The difference in the medium and low water contents is larger than the difference in the medium and high water contents among the three conditions of the water contents. In the turning time factor, every 72h of turning is larger than that of 24h of turning.
As can be seen from the extremely poor analysis in Table 6, the main factors affecting the fermentation of spores are the culture temperature, the moisture, and finally the turnover time. In the three conditions of temperature, the bacterial load of fermentation is directly proportional with the increase of temperature. The difference in the medium and low water contents is larger than the difference in the medium and high water contents among the three conditions of the water contents. In the turning time factor, the turning of 72h is larger than that of 24h and 48h, but the turning difference is smaller than that of 48 h.
TABLE 5 orthogonal test results of optimization of fermentation conditions of Czk1 in straw chicken manure compost
TABLE 6 evaluation of orthogonal test results of fermentation condition optimization of Czk1 in straw chicken manure compost
Note that: b (B) 1 、B 2 、B 3 The average value of the viable count after fermentation at 3 levels of culture temperature, water content, and number of turnovers is represented, respectively.
6. Optimization research of biological organic fertilizer formula
6.1 influence of straw addition on fermentation of Czk1 in fungus chaff chicken manure
Respectively adding 0%, 10%, 20%, 30% and 40% straw into the fungus chaff chicken manure, standing and fermenting in an incubator at 37 ℃ for 7d, and turning over and throwing for 72 h. As can be seen from fig. 9, the number of viable bacteria and spores after fermentation also decreased with increasing straw addition. Therefore, the straw is not suitable to be added in the mushroom bran chicken manure.
6.2 influence of the addition of the fungus chaff on the fermentation of Czk1 in straw chicken manure
Respectively adding 0%, 10%, 20%, 30% and 40% fungus chaff into straw chicken manure compost, standing and fermenting in an incubator at 37 ℃ for 7d, and turning over and throwing for 72 h. As can be seen from fig. 10, the amount of bacteria and spores after fermentation increased with the addition of the fungus chaff, but when the amount of the fungus chaff added exceeded 30%, the amount of bacteria and spores after fermentation decreased. Therefore, the ratio of the bacterial bran suitable for addition during fermentation was 30%, and the number of viable bacteria and the number of spores were all maximized at this addition amount.
7. Physicochemical properties of Czk1 fermented bioorganic fertilizer
As can be seen from Table 7, the biological organic fertilizers with the highest total nitrogen, total phosphorus, total potassium and organic matters are chicken manure fungus chaff biological fertilizers. The total nutrient of the three types of biological organic fertilizers is above 6%, and the requirements of the biological organic fertilizers are completely met.
TABLE 7 basic physicochemical Properties of three bioorganic fertilizers
Note that: different letters in the same column of values indicate significant differences at the P < 0.05 level.
8. Discussion of the invention
Temperature, inoculum size, moisture content, aeration condition, pH, carbon nitrogen ratio, etc. are important factors affecting solid fermentation. In this context, the carbon-nitrogen ratio of the four raw materials is greatly different, the carbon-nitrogen ratio, the water content, the ventilation condition and the like can be changed by adding auxiliary materials, and the bacterial bran, the straw and the soybean meal are added in this context to find that the bacterial fermentation of Czk1 is greatly influenced, but the carbon-nitrogen ratio of the final fertilizer product is not more than 20, and is somewhat different from 25 mentioned in certain papers.
The germination index of the seeds is used to determine the toxicity of the organic fertilizer to the plant and thus determine the degree of maturity of the organic fertilizer, typically with a GI greater than 60%, considered substantially mature, greater than 80%, considered fully mature.
The research result of the inventor shows that the solid formula of the bacillus subtilis Czk1 fermentation is optimal to be fungus chaff and straw, the fungus chaff is added to the decomposed chicken manure respectively, the adding effect of the fungus chaff is more than that of the straw, the promoting effect of the coconut chaff on the fermentation is smaller, the germination index of the seeds treated by the straw is 60%, the germination index of the seeds treated by the other seeds is more than 80%, and the untreated straw has a certain inhibiting effect on the germination of the seeds.
The optimal parameters of the bacillus subtilis Czk1 in the solid fermentation of the straw chicken manure compost are as follows: 10% of inoculation amount, 5% of soybean meal addition amount, 37 ℃ of culture temperature, 40% of water content and 72% of culture temperatureTurning over and throwing once, under the condition, the number of viable bacteria after fermentation is 6.53 multiplied by 10 8 CFU·g -1 Spore number 5.97X10 8 CFU·g -1 . Under the prescription, 30% of fungus chaff is added, the number of viable bacteria can reach 8.70X10 8 CFU·g -1 The number of spores is 6.90×10 8 CFU·g -1 . The indexes of total nitrogen, total phosphorus, total potassium, organic matters and the like are lower than those of the chicken manure fungus chaff biological organic fertilizer, and the total nutrient and organic matter content of the chicken manure fungus chaff biological organic fertilizer are the highest.
Therefore, the optimal parameters of the solid fermentation of bacillus subtilis Czk1 in the fungus chaff chicken manure compost are as follows: 10% of inoculation amount, 10% of soybean meal addition amount, 37 ℃ of culture temperature, 40% of water content and one turning and throwing every 72 hours, under the condition, the number of viable bacteria after fermentation is 8.63 multiplied by 10 8 CFU·g -1 Spore count 8.37X10 8 CFU·g -1 。
Example 2: the invention relates to a microbial fertilizer and a preparation method thereof
1. Preparation of biocontrol bacterium fermentation liquid
(1) Activating strains: transferring Bacilus subtilis Czk1 strain stored on 4 ℃ inclined plane to an LB culture medium plate, and culturing for 48 hours at 37 ℃;
(2) Preparing seed bacterial liquid: selecting single colony with fixed form on plate, culturing in 250mL triangular flask containing 100mL LB liquid medium at 37deg.C under 180r/min for 12 hr to obtain Bacilus subtilis Czk seed solution with bacterial content of 1×10 8 CFU/mL。
(3) Biocontrol bacterium fermentation liquid: inoculating Bacilus subtilis Czk seed liquid into a fermentation bottle for fermentation culture to obtain biocontrol bacterium fermentation liquid.
2. Decomposition of raw materials
(1) Crushing raw materials of chicken manure, fungus chaff, straw and coconut chaff into particles with 20 meshes;
(2) Blending according to weight, and taking 59-70 parts by weight of chicken manure and agricultural waste (mushroom bran or straw) crushed in the step (1): mixing in a proportion of 30-41, sterilizing at 121 ℃ for 30min twice, and killing parasites, weed seeds and pathogenic bacteria by using high temperature. Obtaining the mixture of chicken manure and agricultural waste.
3. Preparation of microbial fertilizer
Adding soybean powder into the mixture of chicken manure and agricultural waste, inoculating the mass ratio, adding Czk1 seed bacterial liquid into the mixture, uniformly mixing, regulating the water content to 40-50%, standing and fermenting in a 37 ℃ incubator for 7d, turning over and throwing once for 72h, so that the effective viable count content of biocontrol bacteria is not less than 1 multiplied by 10 8 CFU.g-1 to obtain the microbial organic fertilizer. The proportion of the added parts by weight is that soybean meal: czk1 seed bacterial liquid: mixture of chicken manure and agricultural waste = 5-10: 5-10: 80-90.
The effective viable count content of the bacillus subtilis microbial fertilizer obtained by the method is not less than 1 multiplied by 10 8 CFU·g -1 The method comprises the steps of carrying out a first treatment on the surface of the The total nitrogen content is more than or equal to 1.6%, the total phosphorus content is more than or equal to 2.41%, the total potassium content is more than or equal to 2.12%, and the organic matter content is more than or equal to 54.12%.
Example 3: influence of microbial fertilizer on red root disease of rubber tree
Test agent: the microbial fertilizer for preventing and treating rubber tree red root disease prepared by the invention. The preparation method comprises the following steps:
1. preparation of biocontrol bacterium fermentation liquid
(1) Activating strains: transferring Bacilus subtilis Czk1 strain stored on 4 ℃ inclined plane to an LB culture medium plate, and culturing for 48 hours at 37 ℃;
(2) Preparing seed bacterial liquid: selecting single colony with fixed form on plate, culturing in 250mL triangular flask containing 100mL LB liquid medium at 37deg.C under 180r/min for 12 hr to obtain Bacilus subtilis Czk seed solution with bacterial content of 1×10 8 CFU/mL。
(3) Biocontrol bacterium fermentation liquid: inoculating Bacilus subtilis Czk seed liquid into a fermentation bottle for fermentation culture to obtain biocontrol bacterium fermentation liquid.
2. Decomposition of raw materials
(1) Crushing raw materials of chicken manure, fungus chaff, straw and coconut chaff into particles with 20 meshes;
(2) Blending according to weight, and taking the chicken manure and agricultural waste (flammulina velutipes fungus chaff) crushed in the step (1) according to the weight part ratio of 62:38, and sterilizing at 121deg.C for 30min twice, and killing parasite, weed seed and pathogenic bacteria at high temperature. Obtaining the mixture of chicken manure and agricultural waste.
3. Preparation of microbial fertilizer
Adding soybean powder into the mixture of chicken manure and agricultural waste, inoculating the mass ratio, adding Czk1 seed bacterial liquid into the mixture, uniformly mixing, regulating the water content to 40%, and standing and fermenting at 37 ℃ in an incubator for 7d,72h to turn over once, so that the effective viable count content of biocontrol bacteria is not less than 1 multiplied by 10 8 CFU.g-1 to obtain the microbial organic fertilizer. The proportion of the added parts by weight is that soybean meal: czk1 seed bacterial liquid: mixture of chicken manure and agricultural waste = 10:10:80.
test crop: rubber tree, age: for 20 years; control object: red root disease of rubber tree.
Test site: the Miao nationality in the Miao nationality of Hainan province newly enters the farm red star team.
Field test: the test set up 5 treatments, each treatment selected 6 rubberis red root disease plants. Treatment 1, control group, applying organic fertilizer (biological bacterial fertilizer fermentation substrate of example 2); treatment II, applying the microbial fertilizer of the invention; treatment III, carrying out treatment by applying a 100-time liquid 25% propiconazole-tebuconazole preparation; treatment IV, the microbial fertilizer of the invention is applied to a 25% propiconazole-tebuconazole preparation which is 200 times liquid for treatment; treatment v, blank with no drug and fertilizer applied. Each treatment was repeated 3 times. The ditching ring application method is adopted, biological bacterial fertilizer is evenly spread into the soil around the roots, the amount of the applied biological bacterial fertilizer is 1.5 kg/plant, and the propiconazole-tebuconazole preparation is used for root irrigation. Red root disease plants were administered 2 times in total for a time interval of 60d. The disease grade of red root disease was investigated before each treatment application and 90d after the second application, and the control effect of each treatment was calculated.
Disease index = Σ (number of individual plants grade)/(number of total plants highest grade investigated) ×100.
Control effect (%) = {1- (pre-treatment area drug disease index x post-treatment area drug disease index)/(post-treatment area drug disease index x pre-treatment area drug disease index) } ×100.
Test results show that after 3 months of treatment, the control group (applying the organic fertilizer) has a control effect of 48.96%, the test group independently applying the microbial fertilizer has a control effect of 63.47%, and the test group added with the microbial fertilizer and bactericide combination has a control effect of 79.68%.
Table 8 effect of microbial fertilizer on controlling red root disease of rubber tree
| Treatment of | Control effect (%) |
| Treatment I (control group) | 48.96 |
| Process II | 63.47 |
| Process III | 66.88 |
| Process IV | 79.68 |
| Treatment V (blank control) | 0 |
The microbial fertilizer prepared by the invention can effectively inhibit the spread of red root disease of rubber trees by being applied in combination with triazole preparations, and has better control effect on the disease.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent material change and modification made to the above examples according to the technical matter of the present invention still fall within the scope of the technical scheme of the present invention.
Claims (8)
1. A microbial fertilizer for preventing and treating red root disease of rubber tree is characterized in that: the microbial fertilizer is prepared from the following components in parts by weight: 5-15 parts of microorganism bacteria, 70-90 parts of a mixture of livestock and poultry waste and agricultural waste, and 5-15 parts of an organic fertilizer component; the weight ratio of the livestock and poultry waste to the agricultural waste is 59-70: 30-41 parts;
the microorganism is bacillus subtilisBacillus subtilis) Seed culture solution of Czk1, the bacillus subtilis @Bacillus subtilis) Czk1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.4408; bacillus subtilis @Bacillus subtilis) The bacterial concentration of the seed culture solution of Czk1 was 0.5X10 8 CFU/mL~2.5×10 8 CFU/mL; the agricultural waste is fungus chaff; the livestock and poultry waste is chicken manure; the organic fertilizer component is soybean powder;
the preparation method of the microbial fertilizer for preventing and treating the red root disease of the rubber tree comprises the following steps:
(1) Preserving bacillus subtilisBacillus subtilis) Inoculating Czk1 strain to an LB culture medium plate, and culturing at 37 ℃ for 48-72 h; selecting single colony with fixed form on the flat plate, culturing in LB liquid medium containing 50-100 mL at 37 ℃ under 180r/min for 12-16 h to obtain bacillus subtilis @Bacillus subtilis) Czk1 seed solution with a bacterial concentration of 0.5X10 8 CFU/mL~2.5×10 8 CFU/mL；
(2) Mixing livestock and poultry waste and agricultural waste according to the weight part ratio, and uniformly stirring; 121. intermittently sterilizing for two times at the temperature of 30min to obtain a mixture of livestock and poultry waste and agricultural waste;
(3) According to the weight portion ratio, the Czk1 seed liquid obtained in the step (1) is added into the mixture of the livestock and poultry waste and the agricultural waste obtained in the step (2) according to the adding amount of 5-10 weight portions, 5-10 weight portions of soybean powder is added, the mixture is uniformly mixed, the water content of the material is adjusted to 40-50%, the mixture is subjected to standing fermentation at 37 ℃ for 7 days, and the mixture is turned over and thrown once for 72 hours; obtaining the microbial fertilizer; the raw material livestock and poultry waste and agricultural waste are crushed into 20-mesh particles.
2. The microbial fertilizer for preventing and treating red root disease of rubber tree according to claim 1, wherein: the bacterial concentration of the seed culture solution of bacillus subtilis (Bacillus subtilis) Czk1 is 1 multiplied by 10 8 CFU/mL。
3. The microbial fertilizer for preventing and treating red root disease of rubber trees according to claim 1, which is characterized in that: the agricultural waste is flammulina velutipes fungus chaff.
4. The microbial fertilizer for preventing and treating red root disease of rubber tree according to claim 1, wherein: the microbial fertilizer consists of the following components in parts by weight: 10 parts of microorganism bacteria, 80 parts of a mixture of livestock and poultry waste and agricultural waste, and 10 parts of an organic fertilizer component; wherein, the weight portion ratio of the livestock and poultry waste to the agricultural waste is 62:38.
5. the microbial fertilizer for preventing and treating red root disease of rubber tree according to claim 1, wherein: the microbial fertilizer meets the following indexes: the effective viable count is more than or equal to 6.53 multiplied by 10 8 CFU·g -1 The method comprises the steps of carrying out a first treatment on the surface of the The spore number is more than or equal to 5.97X10 8 CFU·g -1 The method comprises the steps of carrying out a first treatment on the surface of the The germination index of seeds is more than or equal to 80 percent; N+P 2 O 5 + K 2 The O content is more than or equal to 6.13 percent, and the organic matters are more than or equal to 54.12 percent.
6. The method for preparing the microbial fertilizer for preventing and treating rubber tree red root disease according to claim 1-5, comprising the following steps:
(1) Preserving bacillus subtilisBacillus subtilis) Inoculating Czk1 strain to an LB culture medium plate, and culturing at 37 ℃ for 48-72 h; selecting single colony with fixed form on the plate, culturing in LB liquid medium containing 50-100 mL at 37 ℃ under 180r/min for 12-16h to obtain bacillus subtilisBacillus subtilis) Czk1 seed solution with a bacterial concentration of 0.5X10 8 CFU/mL~2.5×10 8 CFU/mL；
(2) According to any one of the weight parts ratio of claim 1-5, mixing livestock and poultry waste and agricultural waste, and uniformly stirring; 121. intermittently sterilizing for two times at the temperature of 30min to obtain a mixture of livestock and poultry waste and agricultural waste;
(3) According to any one of the weight portions of the components in the step 1 to 5, the Czk1 seed liquid obtained in the step 1 is added into the mixture of the livestock and poultry waste and the agricultural waste obtained in the step 2 according to the adding amount of 5 to 10 weight portions, 5 to 10 weight portions of soybean powder is added, the mixture is uniformly mixed, the water content of the material is adjusted to 40 to 50 percent, the mixture is subjected to standing fermentation at 37 ℃ for 7 days, and the mixture is turned over and thrown once for 72 hours; obtaining the microbial fertilizer; the raw material livestock and poultry waste and agricultural waste are crushed into 20-mesh particles.
7. Use of the microbial fertilizer according to any one of claims 1 to 5 for controlling rubberish root diseases of rubber trees.
8. The use according to claim 7, characterized in that: in the application, the microbial fertilizer and the triazole preparation are applied in a matched manner, the microbial fertilizer and the triazole preparation are mixed and treated by 200-time diluent, and the microbial fertilizer and the triazole preparation are applied for 2 times by sprinkling or root irrigation at intervals of 60 days.
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