CN108277167B - Method for biologically preventing and treating phytophthora capsici leonian - Google Patents
Method for biologically preventing and treating phytophthora capsici leonian Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a method for biologically preventing and controlling phytophthora capsici, which comprises the following steps: adding the biological agent into water of 200 times of the biological agent by weight, adding glucose of 2-3 times of the biological agent by weight and urea of 1-2 times of the biological agent by weight, stirring for 3min at 200rpm, activating for 3-5h at 28-30 ℃, immersing the pepper seedlings into the liquid for 30-60min, taking out the pepper seedlings, and planting the pepper seedlings into soil. The method is environment-friendly and pollution-free, and can effectively prevent phytophthora capsici.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for biologically preventing and treating phytophthora capsici.
Background
Phytophthora (Phytophthora), Peronosporales (Peronosporales) pathogenic bacteria, lower sporangium peduncles and hyphae have no obvious difference, have high difference, sporangium is lemon-shaped or oval, have papilla at the top, cause potato late blight, taro epidemic disease and the like. Hyphae are not isolated and have multiple cores at first, many branches are acute-angled, and are often constricted at the branches, are 2-10 μm thick, and some of them can produce haustoria, sometimes form large hypha bulges. The sporangium stem is not greatly different from hypha to be obviously differentiated, and is irregularly branched, coaxially branched or emerges from the inner layer of the sporangium. The sporangium has large morphological change, generally being oval, inverted pear-shaped and elliptical, and also having irregular shape and large size change; mastoid, hemimastoid or mastoid-free on the top; typically, they are alone born on the sporocyst stalk, occasionally internationally; shedding or non-shedding after maturation, with long (> 20 μm), medium (5-20 μm) or short (< 5 μm) stalks; can directly germinate to produce germ tubes or indirectly germinate to produce zoospores. Zoospores are oval or broad bean-shaped, and have the shape of a cell wall formed after the flagella are dormant, and the zoospores are spherical and are called resting spores. Chlamydospores are mostly spherical, thin-walled or thick-walled, colorless to dark-colored, terminal or intergrown. Spherical or nearly spherical, funnel-shaped, smooth or textured wall, colorless, yellow to brown. The male organs are different in size and shape, colorless, and are either enclosed (male-piercing) or laterally born. Oospore is spherical, thick-walled or thin-walled, colorless to light-colored, full or not full.
Pepper production is affected by a variety of pests, with phytophthora capsici being one of the major soil-borne pests. The phytophthora capsici is damaged at the green part and the root of the ground of the full growth period of the capsicum, the base part of a stem which is ill in the seedling period is in a dark green water stain shape and is soft rot or lodging, namely, the base part of the stem which is ill in the seedling period is black brown, seedlings wither and die, the root and stem tissues of wooden young stems are rotten, stems and leaves are withered rapidly, and seedlings are withered. The basal part of the main stem and the branch of the adult plant are damaged, less brown spots are generated at first, then the adult plant rapidly expands to the periphery, including the whole stem, and the diseased part is brown at the later stage and has obvious boundary line with the healthy tissue. The infected roots of the plants at the roots are reduced, the lateral roots are light brown or dark brown, and the plants are rotten at the later stage. The whole plant of the main stem or the root is dead, the lateral branches are damaged, and the branches on the lateral branches are dead. The leaves are soaked in water and spread inwards from the edge, and then are light brown. The disease of the floral organs is manifested as browning, soft rot and abscission. Fruits usually attack pedicles, and are watery spots in early stage, soft and rotten when wet, and the boundary between diseases and health is obvious. If the environment is suitable, the environment is rapidly expanded outwards, which leads to fruit rot.
Phytophthora capsici is caused by the flagellata subphylum fungus Phytophthora capsici. The pathogenic bacteria mainly take oospores and thick tapestry spores as main infection sources to live through the winter on diseased residues or soil and seeds, wherein the diseased residues in the soil have high bacteria carrying rate. Under proper conditions, the germs after overwintering are transmitted to the stem base or plants near the ground through rainwater splashing or irrigation water, so that the diseases are caused. Therefore, phytophthora capsici becomes a devastating disease with short disease period and rapid epidemic speed. High temperature, high humidity, large number of rainfall days and large rainfall, and is beneficial to the occurrence of diseases. The existing control method mainly comprises the following steps: agricultural control, chemical control, and biological control. The agricultural prevention and control mainly comprises reasonable stubble ploughing and scientific planting. The reasonable stubble ploughing is a considerable cultivation measure in agricultural production, and the reasonable cultivation system and layout are very beneficial to the growth of the pepper. Chemical control includes soil application and field application; however, chemical drugs are likely to remain and cause environmental pollution, and the more serious and poorer the chemical control effect due to the generation of antagonistic bacteria. Antagonistic microorganisms are mainly adopted for biological prevention and treatment, so that the biological prevention and treatment agent is environment-friendly and cannot cause pollution. In the prior art, single microbial agents are more common, such as streptomyces, bacillus subtilis and the like, but a single bacterial strain often has the defects of low control effect, slow drug effect and the like, and a plurality of research institutes have conducted research on compound biocontrol agents at present. The compound biocontrol preparation generally refers to a preparation comprising more than two microorganisms, and the selection of strains in the compound biocontrol preparation is more critical and difficult; if the selection is careless, the strains can play antagonistic roles instead. The technical problem to be solved is to develop a method for preventing and controlling the phytophthora capsici leonian by using a compound biocontrol agent.
Disclosure of Invention
In order to overcome the defects of poor biological control effect, residue in chemical control and the like in the prior art, the invention provides a method for biologically controlling phytophthora capsici.
The invention is realized by the following technical scheme:
a method for biologically controlling phytophthora capsici leonian comprises the following steps: adding the biological agent into water of 200 times of the biological agent by weight, adding glucose of 2-3 times of the biological agent by weight and urea of 1-2 times of the biological agent by weight, stirring for 3min at 200rpm, activating for 3-5h at 28-30 ℃, immersing the pepper seedlings into the liquid for 30-60min, taking out the pepper seedlings, and planting the pepper seedlings into soil.
In particular, the amount of the solvent to be used,
the biological agent comprises myceliophthora, trichoderma koningii, bacillus subtilis and rhodopseudomonas palustris.
In particular, the amount of the solvent to be used,
the biological agent is prepared by the following process:
1) inoculating the hyphomycete on a YPDA culture medium for culturing to obtain a single colony; selecting single colony, inoculating to YPD liquid culture medium, culturing at 30 deg.C for 24 hr, and collecting Trichosporon sinensis seed liquid;
inoculating Trichoderma koningii to a PDA culture medium for culture by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a PDA liquid culture medium, and performing shake cultivation at 30 ℃ and 200rpm for 36h to obtain Trichoderma koningii seed liquid for later use;
firstly, inoculating trichoderma koningii seed liquid into a fermentation culture medium according to the inoculation amount of 10%, culturing for 6 hours at 30 ℃, then inoculating the filamentous spore yeast seed liquid into the fermentation culture medium according to the inoculation amount of 5%, and continuously culturing for 18 hours at 30 ℃ to obtain trichoderma koningii-filamentous spore yeast mixed fermentation liquid; the fermentation medium comprises the following components: 16g/L of corn straw powder, 10g/L of soybean meal, 5g/L of ammonium chloride, 2g/L of dipotassium phosphate, 1g/L of monopotassium phosphate, 0.5g/L of sodium chloride, 0.1g/L of magnesium sulfate and 0.01g/L of ferrous sulfate;
2) performing streak culture on rhodopseudomonas palustris on an LB (Langerhans) plate to obtain a single colony; selecting a single colony to be inoculated into a seed culture medium to be cultured to a logarithmic growth phase, wherein the culture conditions are as follows: at 30-32 ℃, illuminating 3000-; then inoculating the strain into a fermentation culture medium according to the inoculation amount of 10 percent, and culturing for 24 hours at the temperature of 30-32 ℃ to obtain rhodopseudomonas palustris fermentation liquor; the seed culture medium and the fermentation culture medium comprise the following components: 8g/L glucose, 5g/L yeast extract, 1g/L ammonium sulfate, 0.2g/L potassium dihydrogen phosphate, 0.2g/L dipotassium hydrogen phosphate, 0.1g/L sodium bicarbonate and 0.01g/L magnesium sulfate;
3) carrying out streak culture on bacillus subtilis on a beef extract peptone agar culture medium to obtain a single colony; selecting single colony, inoculating to seed culture medium, shake culturing at 30 deg.C and 200rpm to logarithmic phase, and making into seed solution; then inoculating the bacillus subtilis into a fermentation medium according to the inoculation amount of 8%, and culturing for 36h at 30 ℃ to obtain bacillus subtilis fermentation liquor; the seed culture medium and the fermentation culture medium comprise the following components: 10g/L glucose, 5g/L yeast extract, 3g/L ammonium sulfate, 1g/L uremia, 0.1g/L monopotassium phosphate, 0.1g/L dipotassium phosphate, 0.02g/L calcium chloride and 0.01g/L ferrous sulfate;
4) uniformly mixing Trichoderma koningii-myceliophthora yeast mixed fermentation liquor, Rhodopseudomonas palustris fermentation liquor and Bacillus subtilis fermentation liquor according to the volume ratio of 2-3:1-2:1-2, and then carrying out vacuum freeze drying to obtain the bacterial powder.
Preferably, the first and second electrodes are formed of a metal,
the Trichoderma koningii is ATCC 66766; the filamentous spore yeast is ATCC 201110; the rhodopseudomonas palustris is ATCC 17001; the Bacillus subtilis is ATCC 6633.
Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:
the biological agent is adopted to prevent and treat phytophthora capsici, the problems of environmental pollution, drug residue and the like caused by chemical prevention and treatment are avoided, the pollution-free production of crops is facilitated, and farmers can avoid or reduce other measures for preventing and treating phytophthora capsici and improving stress resistance and growth promotion, so that the burden of planting is relieved for the farmers, the quality and the yield of products are improved, and the agricultural income is increased.
The trichoderma koningii and the filamentous spore yeast have no antagonistic action and can symbiotic with each other, the trichoderma koningii can generate cellulase, and the straw cellulose is decomposed into a carbon source which can be utilized by the filamentous spore yeast, so that the filamentous spore yeast grows by utilizing the carbon source generated by decomposing the agricultural waste straw powder, and the fermentation cost is reduced; inoculating Trichoderma koningii first for 6 hr and inoculating saccharomycete; the saccharomycetes can promote the growth of trichoderma koningii and greatly improve the enzyme yield of the trichoderma, so that the ability of resisting botrytis cinerea is improved. The rhodopseudomonas palustris and the bacillus subtilis can secrete various antibacterial substances and can compete with pathogenic bacteria for nutrition and space; the compound microbial preparation provided by the invention adopts four biocontrol strains, can realize synergistic symbiosis, is reasonable in compatibility and free from antagonism, greatly improves the phytophthora control effect, is harmless to human and livestock, harmless to crops, environment-friendly, relatively simple in preparation process, convenient and fast in use method and wide in application prospect.
Drawings
FIG. 1: the influence of different biological agents on the disease index of phytophthora capsici;
FIG. 2: influence of different biological agents on the phytophthora capsici prevention rate;
FIG. 3: effect of different biological agents on pepper biomass.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the present application will be clearly and completely described below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for biologically controlling phytophthora capsici leonian comprises the following steps: adding biological agent into 100 times of water, adding glucose 2 times of biological agent and urea 1 times of biological agent, stirring at 200rpm for 3min, activating at 30 deg.C for 3 hr, soaking the young pepper in the above liquid for 30min, taking out, and planting in soil.
The biological agent comprises myceliophthora, trichoderma koningii, bacillus subtilis and rhodopseudomonas palustris.
Specifically, the biological agent is prepared according to the following process:
inoculating the hyphomycete on a YPDA culture medium for culturing to obtain a single colony; selecting a single colony, inoculating the single colony on an YPD liquid culture medium for culture, and culturing at 30 ℃ for 24 hours to obtain a seed solution for later use;
the YPD medium formula comprises: 20g of glucose, 20g of peptone and 10g of yeast extract powder, and supplementing distilled water to 1000 mL;
the YPDA culture medium formula comprises: 20g of glucose, 20g of peptone, 10g of yeast extract powder and 20g of agar, and supplementing distilled water to 1000 mL;
inoculating Trichoderma koningii to a PDA culture medium for culture by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a PDA liquid culture medium for culturing, and performing shake culture at 30 ℃ and 200rpm for 36 hours to obtain a seed solution for later use;
firstly, inoculating Trichoderma koningii seed liquid according to the inoculation amount of 10% (the inoculation density is 1 multiplied by 10)7CFU/ml) was inoculated into a fermentation medium, cultured at 30 ℃ for 6 hours, and then a filamentous yeast seed solution was inoculated in an amount of 5% (inoculation density of 1X 10)7CFU/ml) is inoculated into a fermentation medium, and the mixture is continuously cultured for 18 hours at the temperature of 30 ℃ to obtain Trichoderma koningii-myceliophthora mixed fermentation liquor; the fermentation medium comprises the following components: 16g/L of corn straw powder, 10g/L of soybean meal, 5g/L of ammonium chloride, 2g/L of dipotassium phosphate, 1g/L of monopotassium phosphate, 0.5g/L of sodium chloride, 0.1g/L of magnesium sulfate and 0.01g/L of ferrous sulfate;
performing streak culture on rhodopseudomonas palustris on an LB (Langerhans) plate to obtain a single colony; selecting a single colony to be inoculated into a seed culture medium to be cultured to a logarithmic growth phase, wherein the culture conditions are as follows: at 30-32 ℃, illuminating 3000-; then according to the inoculation amount of 10% (the inoculation density is 3 multiplied by 10)7CFU/ml) is inoculated into a fermentation medium, and the fermentation medium is cultured for 24 hours at the temperature of 30-32 ℃ to obtain rhodopseudomonas palustris fermentation liquor; the seed culture medium and the fermentation culture medium comprise the following components: 8g/L glucose, 5g/L yeast extract, 1g/L ammonium sulfate, 0.2g/L potassium dihydrogen phosphate, 0.2g/L dipotassium hydrogen phosphate, 0.1g/L sodium bicarbonate and 0.01g/L magnesium sulfate;
carrying out streak culture on bacillus subtilis on a beef extract peptone agar culture medium to obtain a single colony; picking single colony to be inoculated on a seed culture medium, and inoculating the single colony on the seed culture medium at 30 ℃ and 200rpCulturing the seeds by a shaker to logarithmic growth phase to prepare seed liquid; then according to the inoculation amount of 8% (the inoculation density is 5X 10)6CFU/ml) is inoculated into a fermentation medium, and the mixture is cultured for 36 hours at the temperature of 30 ℃ to obtain bacillus subtilis fermentation liquor; the seed culture medium and the fermentation culture medium comprise the following components: 10g/L glucose, 5g/L yeast extract, 3g/L ammonium sulfate, 1g/L uremia, 0.1g/L monopotassium phosphate, 0.1g/L dipotassium phosphate, 0.02g/L calcium chloride and 0.01g/L ferrous sulfate;
uniformly mixing Trichoderma koningii-myceliophthora yeast mixed fermentation liquor, Rhodopseudomonas palustris fermentation liquor and Bacillus subtilis fermentation liquor according to the volume ratio of 2:1:1, and then carrying out vacuum freeze drying to obtain the strain powder.
The Trichoderma koningii is ATCC 66766; the filamentous spore yeast is ATCC 201110; the rhodopseudomonas palustris is ATCC 17001; the Bacillus subtilis is ATCC 6633.
Example 2
A method for biologically controlling phytophthora capsici leonian comprises the following steps: adding the biological agent into 200 times of water, adding glucose 3 times of the biological agent and urea 2 times of the biological agent, stirring at 200rpm for 3min, activating at 28 deg.C for 5h, soaking the pepper seedling in the above liquid for 60min, taking out the pepper seedling, and planting in soil.
The biological agent comprises myceliophthora, trichoderma koningii, bacillus subtilis and rhodopseudomonas palustris.
Specifically, the biological agent is prepared according to the following process:
inoculating the hyphomycete on a YPDA culture medium for culturing to obtain a single colony; selecting a single colony, inoculating the single colony on an YPD liquid culture medium for culture, and culturing at 30 ℃ for 24 hours to obtain a seed solution for later use;
the YPD medium formula comprises: 20g of glucose, 20g of peptone and 10g of yeast extract powder, and supplementing distilled water to 1000 mL;
the YPDA culture medium formula comprises: 20g of glucose, 20g of peptone, 10g of yeast extract powder and 20g of agar, and supplementing distilled water to 1000 mL;
inoculating Trichoderma koningii to a PDA culture medium for culture by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a PDA liquid culture medium for culturing, and performing shake culture at 30 ℃ and 200rpm for 36 hours to obtain a seed solution for later use;
firstly, inoculating Trichoderma koningii seed liquid according to the inoculation amount of 10% (the inoculation density is 1 multiplied by 10)7CFU/ml) was inoculated into a fermentation medium, cultured at 30 ℃ for 6 hours, and then a filamentous yeast seed solution was inoculated in an amount of 5% (inoculation density of 1X 10)7CFU/ml) is inoculated into a fermentation medium, and the mixture is continuously cultured for 18 hours at the temperature of 30 ℃ to obtain Trichoderma koningii-myceliophthora mixed fermentation liquor; the fermentation medium comprises the following components: 16g/L of corn straw powder, 10g/L of soybean meal, 5g/L of ammonium chloride, 2g/L of dipotassium phosphate, 1g/L of monopotassium phosphate, 0.5g/L of sodium chloride, 0.1g/L of magnesium sulfate and 0.01g/L of ferrous sulfate;
performing streak culture on rhodopseudomonas palustris on an LB (Langerhans) plate to obtain a single colony; selecting a single colony to be inoculated into a seed culture medium to be cultured to a logarithmic growth phase, wherein the culture conditions are as follows: at 30-32 ℃, illuminating 3000-; then according to the inoculation amount of 10% (the inoculation density is 3 multiplied by 10)7CFU/ml) is inoculated into a fermentation medium, and the fermentation medium is cultured for 24 hours at the temperature of 30-32 ℃ to obtain rhodopseudomonas palustris fermentation liquor; the seed culture medium and the fermentation culture medium comprise the following components: 8g/L glucose, 5g/L yeast extract, 1g/L ammonium sulfate, 0.2g/L potassium dihydrogen phosphate, 0.2g/L dipotassium hydrogen phosphate, 0.1g/L sodium bicarbonate and 0.01g/L magnesium sulfate;
carrying out streak culture on bacillus subtilis on a beef extract peptone agar culture medium to obtain a single colony; selecting single colony, inoculating to seed culture medium, shake culturing at 30 deg.C and 200rpm to logarithmic phase, and making into seed solution; then according to the inoculation amount of 8% (the inoculation density is 5X 10)6CFU/ml) is inoculated into a fermentation medium, and the mixture is cultured for 36 hours at the temperature of 30 ℃ to obtain bacillus subtilis fermentation liquor; the seed culture medium and the fermentation culture medium comprise the following components: 10g/L glucose, 5g/L yeast extract, 3g/L ammonium sulfate, 1g/L uremia, 0.1g/L monopotassium phosphate, 0.1g/L dipotassium phosphate, 0.02g/L calcium chloride and 0.01g/L ferrous sulfate;
uniformly mixing Trichoderma koningii-myceliophthora yeast mixed fermentation liquor, Rhodopseudomonas palustris fermentation liquor and Bacillus subtilis fermentation liquor according to the volume ratio of 3:2:2, and then carrying out vacuum freeze drying to obtain the strain powder.
The Trichoderma koningii is ATCC 66766; the filamentous spore yeast is ATCC 201110; the rhodopseudomonas palustris is ATCC 17001; the Bacillus subtilis is ATCC 6633.
Example 3
The invention has the effect of preventing and treating phytophthora capsici
Biological agent group: experimental groups: example 1; control group 1: the same procedure as in example 1 was repeated except that only the mixed fermentation broth of Trichoderma koningii and Trichosporon mentagroides was used; control group 2: the same procedure as in example 1 was repeated except that Rhodopseudomonas palustris fermentation broth and Bacillus subtilis fermentation broth were used; control group 3: separately fermenting trichoderma koningii and myceliophthora, and mixing the four kinds of fermentation liquor, the rest is the same as the example 1; blank control: equal amount of clear water.
The procedure was as in example 1.
Soil pretreatment: after solid culture of phytophthora capsici for 5 days, inoculating the phytophthora capsici into a V8 liquid culture medium, culturing for 5 days at 25 ℃, pouring out the liquid culture medium, weighing hyphae, and inoculating the hyphae into soil of each cell according to the amount of 8 g/kg of soil.
The treatment method comprises the following steps: the hot pepper test field is divided into five cells, and each cell corresponds to each group; after being treated according to the mode of example 1 (different biological agents), the pepper seedlings are transplanted to each cell; sampling after 21 days, counting the disease degree, and calculating the disease index; the total fresh weight of the plants and the fresh weight of the roots were also examined.
Detection standard: the degree of phytophthora blight is classified as grade 6. 0 level is that the root system is not attacked; grade 1 is that the incidence of root system is less than or equal to 30 percent and the leaves are normal; grade 2 is 30%, the incidence rate of roots is less than or equal to 60%, and the leaves are normal; grade 3 is 60% < the root disease incidence is less than or equal to 80%; the leaves turn yellow, the incidence rate of roots is more than 80% in 4 grades, the leaves wither, the whole plants die in 5 grades, and the leaves are dry. The disease index and the prevention and treatment effect are calculated according to the following formula, wherein the disease index is [ (disease-grade plant number multiplied by representative value)/(total plant number multiplied by highest disease-grade representative value) ] multiplied by 100. The control rate is [ (control disease index-treatment disease index)/control disease index ] × 100%.
As shown in fig. 1-2, the experimental group has the highest control rate on phytophthora capsici, which is obviously higher than that of the control group 1-2 and also higher than that of the control group 3 in a separate fermentation mode, which indicates that trichoderma serrulatum and trichoderma koningii can be fermented together, trichoderma is firstly inoculated to produce cellulase and decompose cellulose into carbon sources which can be used by the trichoderma serrulatum, so that the subsequently inoculated trichoderma serrulatum grows by using the carbon sources produced by the agricultural waste straw powder, the fermentation cost is reduced, the yeast can promote the growth of trichoderma koningii in turn, the enzyme yield of the trichoderma koningii is greatly improved, and the capability of antagonizing botrytis cinerea is improved. The plants absorb and utilize mineral nutrients from soil by root systems, and maintain the growth and the propagation of the plants, and the more developed the root systems, the better the plant growth; the plant represents the biomass of the plant and can be used as an important index for evaluating the yield and quality of the fruit; as shown in FIG. 3, the biomass of the experimental group and the control groups 1-3 is obviously higher than that of the blank control group, wherein the two indexes of the total fresh weight of the selected plants and the fresh weight of roots are obviously higher than that of the blank control group, and the biomass weight gain of the experimental group is the most obvious.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (1)
1. A method for biologically controlling phytophthora capsici leonian comprises the following steps: adding a biological agent into water of which the weight is 200 times that of 100-fold, adding glucose of which the weight is 2-3 times that of the biological agent and urea of which the weight is 1-2 times that of the biological agent, stirring for 3min at 200rpm, then placing at 28-30 ℃ for activation for 3-5h, then immersing the pepper seedlings into the liquid for 30-60min, taking out the pepper seedlings, and planting the pepper seedlings into soil;
the biological agent is prepared by the following process:
1) inoculating the hyphomycete on a YPDA culture medium for culturing to obtain a single colony; selecting single colony, inoculating to YPD liquid culture medium, culturing at 30 deg.C for 24 hr, and collecting Trichosporon sinensis seed liquid;
inoculating Trichoderma koningii to a PDA culture medium for culture by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a PDA liquid culture medium, and performing shake cultivation at 30 ℃ and 200rpm for 36h to obtain Trichoderma koningii seed liquid for later use;
firstly, inoculating trichoderma koningii seed liquid into a fermentation culture medium according to the inoculation amount of 10%, culturing for 6 hours at 30 ℃, then inoculating the filamentous spore yeast seed liquid into the fermentation culture medium according to the inoculation amount of 5%, and continuously culturing for 18 hours at 30 ℃ to obtain trichoderma koningii-filamentous spore yeast mixed fermentation liquid; the fermentation medium comprises the following components: 16g/L of corn straw powder, 10g/L of soybean meal, 5g/L of ammonium chloride, 2g/L of dipotassium phosphate, 1g/L of monopotassium phosphate, 0.5g/L of sodium chloride, 0.1g/L of magnesium sulfate and 0.01g/L of ferrous sulfate;
2) performing streak culture on rhodopseudomonas palustris on an LB (Langerhans) plate to obtain a single colony; selecting a single colony to be inoculated into a seed culture medium to be cultured to a logarithmic growth phase, wherein the culture conditions are as follows: at 30-32 ℃, illuminating 3000-; then inoculating the strain into a fermentation culture medium according to the inoculation amount of 10 percent, and culturing for 24 hours at the temperature of 30-32 ℃ to obtain rhodopseudomonas palustris fermentation liquor; the seed culture medium and the fermentation culture medium comprise the following components: 8g/L glucose, 5g/L yeast extract, 1g/L ammonium sulfate, 0.2g/L potassium dihydrogen phosphate, 0.2g/L dipotassium hydrogen phosphate, 0.1g/L sodium bicarbonate and 0.01g/L magnesium sulfate;
3) carrying out streak culture on bacillus subtilis on a beef extract peptone agar culture medium to obtain a single colony; selecting single colony, inoculating to seed culture medium, shake culturing at 30 deg.C and 200rpm to logarithmic phase, and making into seed solution; then inoculating the bacillus subtilis into a fermentation medium according to the inoculation amount of 8%, and culturing for 36h at 30 ℃ to obtain bacillus subtilis fermentation liquor; the seed culture medium and the fermentation culture medium comprise the following components: 10g/L glucose, 5g/L yeast extract, 3g/L ammonium sulfate, 1g/L uremia, 0.1g/L monopotassium phosphate, 0.1g/L dipotassium phosphate, 0.02g/L calcium chloride and 0.01g/L ferrous sulfate;
4) uniformly mixing Trichoderma koningii-myceliophthora yeast mixed fermentation liquor, Rhodopseudomonas palustris fermentation liquor and Bacillus subtilis fermentation liquor according to the volume ratio of 2-3:1-2:1-2, and then carrying out vacuum freeze drying to obtain bacterial powder;
the Trichoderma koningii is ATCC 66766; the filamentous spore yeast is ATCC 201110; the rhodopseudomonas palustris is ATCC 17001; the Bacillus subtilis is ATCC 6633.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1106217A (en) * | 1994-09-24 | 1995-08-09 | 王厚德 | Prodn. technique and prepn. of combined soil forage |
| CN101381680A (en) * | 2008-10-15 | 2009-03-11 | 中国农业大学 | Microbial preparation prepared by fermentation of stalk hydrolysate and use thereof |
| CN102515891A (en) * | 2012-01-09 | 2012-06-27 | 信息产业电子第十一设计研究院科技工程股份有限公司 | Microbial fertilizer containing bacillus subtilis |
| JP2013201996A (en) * | 2012-03-29 | 2013-10-07 | Taiho Kk | Water-soluble royal jelly and method for producing the same |
-
2018
- 2018-04-02 CN CN201810280270.0A patent/CN108277167B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1106217A (en) * | 1994-09-24 | 1995-08-09 | 王厚德 | Prodn. technique and prepn. of combined soil forage |
| CN101381680A (en) * | 2008-10-15 | 2009-03-11 | 中国农业大学 | Microbial preparation prepared by fermentation of stalk hydrolysate and use thereof |
| CN102515891A (en) * | 2012-01-09 | 2012-06-27 | 信息产业电子第十一设计研究院科技工程股份有限公司 | Microbial fertilizer containing bacillus subtilis |
| JP2013201996A (en) * | 2012-03-29 | 2013-10-07 | Taiho Kk | Water-soluble royal jelly and method for producing the same |
Non-Patent Citations (1)
| Title |
|---|
| 酶制剂在玉米秸秆双菌种固态发酵生产微生物油脂中的应用;李新社等;《中南大学学报(自然科学版)》;20110626(第06期);21-24 * |
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