CN105483053A - Microbial compound bacteria for preventing and treating citrusyellowshoot and preparation method and application thereof - Google Patents
Microbial compound bacteria for preventing and treating citrusyellowshoot and preparation method and application thereof Download PDFInfo
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- CN105483053A CN105483053A CN201610000686.3A CN201610000686A CN105483053A CN 105483053 A CN105483053 A CN 105483053A CN 201610000686 A CN201610000686 A CN 201610000686A CN 105483053 A CN105483053 A CN 105483053A
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Abstract
The invention provides microbial compound bacteria, a preparation method thereof and application of the microbial compound bacteria to prevention and treatment of citrusyellowshoot. The microbial compound bacteria comprise Cribben paenibacillus and bacillus subtilis, the biological preservation serial number of the Cribben paenibacillus is CGMCC NO.7996, and the microbial compound bacteria can be specifically microbial compound bacterium liquid or a microbial compound bacterium agent. The preparation method of the microbial compound bacteria includes the specific steps that the Cribben paenibacillus and the bacillus subtilis are mixed in proportion after being fermented. According to the microbial compound bacteria for preventing and treating citrusyellowshoot and the preparation method and application thereof, procreation and propagation of gram negative bacteria are inhibited in a root filling and leaf surface spraying mode, 'bacteria are controlled through bacteria', harmful bacteria in soil are killed, soil quality is optimized, root and stem phloem sieve tubes can be dredged, the problem of nutrient substance conveying barrier is solved, diseased or infected plant nutrients are recovered, growth of new roots is promoted, tree roots are more developed, tree tubular cells are smoother, yellow leaves of fruit trees are turned green, and healthy new tips grow.
Description
Technical field
The present invention relates to field of biological control, particularly a kind of microbial composite bacteria preventing and treating Citrus Huanglongbing pathogen and its preparation method and application.
Background technology
Oranges and tangerines are the maximum fruit of world wide production, have had the cultivation history of more than 4000 year in China.China's citriculture areas in 2009 and output all exceed Brazil, the U.S., occupy first place in the world in position, but while Citrus Industry develops rapidly, and yellow twig has become on Orange Producing and endangers destructive disease the most serious.As far back as the mid-eighteenth century, just there is relevant report in India.Reinking carries out reported first the twenties in 19th century to South China of China, and since over half a century, Citrus Huanglongbing pathogen seriously constrains the development of Guangdong, Guangxi, provinces and regions, Fujian 3 Citrus Industry.Late 1970s, Xichang Region, Sichuan Province and Dukou, area, Ganzhou and Yunnan Province obtain some areas and also successively find Citrus Huanglongbing pathogen, to 20th century the mid-80, Citrus Huanglongbing pathogen in China Guangdong, Guangxi, Fujian, Hainan and Taiwan oranges and tangerines producing region extensive widespread, and in Zhejiang, Guizhou, Hunan occur in succession, 19 provinces of China's citriculture, existing 11 of district are endangered, injured area accounts for more than 80% of the total cultivated area of oranges and tangerines, and output accounts for about 85% of ultimate production.SaoPaulo State,Brazils in 2004 and Fla. yellow twig in 2005 occur in succession, cause the fear of oranges and tangerines practitioner.So far, yellow twig has been distributed widely in more than 40 countries of Asia, Africa, Oceania, South America and North America.
The young sprout of yellow twig main harm oranges and tangerines, tender leaf, flower and fruit.There is the yellow of minority young sprout in the initial stage, be commonly called as " inserting golden flower ", " chicken head is yellow " in dark green tree crown.There are even yellow, mottled type yellow and nutritional deficiency type yellow three kinds of symptom types in blade.Mottled yellow blade and " red nose fruit " are the symptoms of most allusion quotation, can as the Main Basis of this disease of Morphological Identification.When field disease tree investigation, the childhood of non-result sets with mottled type yellow blade for diagnosis basis, and bearing-age tree is using " red nose fruit " as diagnosis basis.
Prior art adopts the method for integrated control to control this disease usually: one is strict Plant Quarantine, checks on from nursery stock; Two is cultivate and promote virus-free nursery stock; Three is excavate diseased plant in time, strengthens orchard management; Four is prevent and treat diaphorina citri by physics and chemistry agricultural chemicals mode, cuts off contagium.But these methods " are cured the symptoms, not the disease ", do not deal with problems from cause of disease depths, currently available technology lacks the feasible method that effectively directly can reduce or eliminate cause of disease in diseased plant.
Summary of the invention:
In view of this, the object of the present invention is to provide a kind of can microbial composite bacteria directly reducing or eliminating cause of disease in Citrus Huanglongbing pathogen diseased plant and its preparation method and application effectively.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of microbial composite bacteria, comprise Ke Liben series bacillus and subtilis, the biological deposits of described Ke Liben series bacillus is numbered CGMCCNO.7996, and described microbial composite bacteria is microbe composite bacterial liquid or complex microbial inoculum.
Preferably, in described microbe composite bacterial liquid, the volume ratio of Ke Liben series bacillus and subtilis is (20 ~ 60): (5 ~ 8);
Preferably, in described microbe composite bacterial liquid, the total viable count of Ke Liben series bacillus is 50 ~ 10,000,000,000/ml, and the total viable count of subtilis is 50 ~ 10,000,000,000/ml.
Preferably, in described complex microbial inoculum, the weight ratio of Ke Liben series bacillus and subtilis is (5 ~ 80): (10 ~ 80).
Preferably, in described complex microbial inoculum, the total viable count of Ke Liben series bacillus is 50 ~ 20,000,000,000/g, and the total viable count of subtilis is 60 ~ 15,000,000,000/g.
Present invention also offers a kind of preparation method of microbe composite bacterial liquid, comprise the following steps:
A1) fermentation of Ke Liben series bacillus, described Ke Liben series bacillus fermentation condition is: liquid nutrient medium, temperature is 29 ~ 30 DEG C, incubation time is 48 ~ 120 hours, ferment after 72 ~ 96 hours and detected fermented liquid total viable count every 2 ~ 4 hours, stop fermentation when total viable count reaches 50 ~ 10,000,000,000/ml, obtain Ke Liben series bacillus fermented liquid;
A2) fermentation of subtilis, the fermentation condition of described subtilis seed is: liquid nutrient medium, temperature is 29 ~ 30 DEG C, 28 ~ 48 hours time, ferment and after 28 ~ 32 hours, detected the total viable count of fermented liquid every 2 ~ 4 hours, stop when total viable count reaches 50 ~ 10,000,000,000/ml fermentation to obtain fermentation of bacillus subtilis liquid;
Above-mentioned two kinds of fermented liquids aseptically, are (20 ~ 60): the ratio of (50 ~ 80) is mixed to get microbe composite bacterial liquid by the a3) mixing of fermented liquid by volume;
Step a1) and step a2) between limit without time sequence.
Present invention also offers a kind of preparation method of complex microbial inoculum, comprise the following steps:
B1) fermentation of Ke Liben series bacillus, described Ke Liben series bacillus fermentation condition is: temperature is 29 ~ 30 DEG C, incubation time is 48 ~ 120 hours, ferment after 72 ~ 96 hours and detected fermented liquid total viable count every 2 ~ 4 hours, stop fermentation when total viable count reaches 50 ~ 10,000,000,000/ml, obtain Ke Liben series bacillus fermented liquid;
B2) fermentation of subtilis, the fermentation condition of described subtilis seed is: temperature is 29 ~ 30 DEG C, incubation time 28 ~ 48 hours, ferment and after 28 ~ 32 hours, detected the total viable count of fermented liquid every 2 ~ 4 hours, stop when total viable count reaches 50 ~ 10,000,000,000/ml fermentation to obtain fermentation of bacillus subtilis liquid;
B3) mixing of fermented liquid, aseptically, the ratio being 1: 0.5 ~ 1.5 by volume by above-mentioned two kinds of fermented liquids is mixed to get microbe composite bacterial liquid;
B4) drying of microbe composite bacterial liquid, by step b3) in obtain complex microbial inoculum after the microbe composite bacterial liquid drying that obtains;
Described step b1) and step b2) between limit without time sequence.
Preferably, step b4) described in drying be spraying dry, spray-dired temperature out is 90 ~ 95 DEG C, and temperature in is 145 ~ 150 DEG C,
Preferably, described spray-dired pan feeding speed 1000 ~ 1500ml/h.
Preferably, step 4) described in spraying dry need add protective material, described protective material is skim-milk or sucrose, and protectant concentration is 15 ~ 25%.
Present invention also offers the application of described microbial composite bacteria in control Citrus Huanglongbing pathogen, the application method of described microbial composite bacteria is that root is filled with or spraying; Described filling is specially: every strain tree use 0.5 ~ 1kg composite bacteria, waters at root 15-25 centimetre place; Described spraying is specially: every strain tree uses composite bacteria 0.5 ~ 1kg, after being watered 3 ~ 5kg, and spraying blade.
Know-why of the present invention: Ke Liben series bacillus produces the antimicrobial substances such as purine analogue during the fermentation, shape dominant microflora in soil, suppresses the growth of harmful bacteria; In process of growth, produce a large amount of exocellular polysaccharides and small molecular protein simultaneously, to be formed " microbial film " in the field planting of the fruit tree tip of a root, and the intercellular spaces accumulation outside root vascular cylinder, " microbial film " of generation can accelerate the process that fruit tree absorbs nutritive substance greatly.Subtilis carries out growth and breeding by nutrient competition thus the mode occupying living space stops the growth of phytopathogen; the highly dense protective membrane of one deck can be formed rapidly at plant surface; make pathogenic bacteria can not get living space, thus cover crop endanger from pathogenic bacteria.
Beneficial effect of the present invention: microbial composite bacteria of the present invention can prevent and treat Citrus Huanglongbing pathogen effectively, by suppressing the reproduction procreation of the gram negative bacterium of oranges and tangerines diseased plant root and leaf portion, reach " fungus treatment ", the object of kill harmful bacterium, use microbial composite bacteria of the present invention that the yellow leaf of mandarin tree can be made to turn green, yellow transference cure, the Citrus Huanglongbing pathogen sickness rate of test group is starkly lower than conventional processing, 35.6% ~ 67.5% is reduced to by the sickness rate of conventional processing 75.1%, sickness rate is the highest compared with conventional processing reduces 52%, there is the effect of preventing and treating yellow twig significantly, the output comparatively the highest increase of conventional processing 90 kgs/acre of test group, stimulation ratio is up to 30%, there is significant effect of increasing production.
Accompanying drawing explanation
Fig. 1 is the prevention effect histogram of the trees sickness rate that the embodiment of the present invention 3 obtains;
Fig. 2 is the tree yield statistics histogram that the embodiment of the present invention 3 obtains.
Embodiment:
The invention provides a kind of microbial composite bacteria, comprise Ke Liben series bacillus and subtilis, the biological deposits of described Ke Liben series bacillus is numbered CGMCCNO.7996.
Microbial composite bacteria provided by the invention comprises subtilis, and the source of the present invention to described subtilis is not particularly limited, and adopts the common commercial goods of subtilis well known to those skilled in the art.
In the present invention, described microbial composite bacteria is preferably microbe composite bacterial liquid.When provided by the invention be microbe composite bacterial liquid time, in described microbe composite bacterial liquid, the volume ratio of Ke Liben series bacillus and subtilis is preferably (20 ~ 60): (50 ~ 80), are more preferably (30 ~ 50); (55 ~ 75), most preferably are (35 ~ 45): (60 ~ 70).In described microbe composite bacterial liquid, the total viable count of Ke Liben series bacillus is preferably 50 ~ 10,000,000,000/ml, is more preferably 80 ~ 10,000,000,000/ml, most preferably is 85 ~ 9,500,000,000/ml; In described microbe composite bacterial liquid, the total viable count of subtilis is preferably 50 ~ 10,000,000,000/ml, is more preferably 70 ~ 9,500,000,000/ml, most preferably is 80 ~ 9,000,000,000/ml.
In the present invention, described microbial composite bacteria is preferably complex microbial inoculum.When provided by the invention be complex microbial inoculum time, in described complex microbial inoculum, the weight ratio of Ke Liben series bacillus and subtilis is preferably (5 ~ 80): (10 ~ 80), be more preferably (40 ~ 60): (45 ~ 65), most preferably are 50: 55.In described complex microbial inoculum, the total viable count of Ke Liben series bacillus is preferably 50 ~ 20,000,000,000/g, is more preferably 100 ~ 15,000,000,000/g, most preferably is 12,500,000,000/g; In described complex microbial inoculum, the total viable count of subtilis is preferably 60 ~ 15,000,000,000/g, is more preferably 80 ~ 13,000,000,000/g, most preferably is 10,000,000,000/g.
When provided by the invention be microbe composite bacterial liquid time, the invention provides a kind of preparation method of microbe composite bacterial liquid, comprise the following steps:
A1) fermentation of Ke Liben series bacillus;
A2) fermentation of subtilis;
A3) mixing of fermented liquid;
Step a1) and step a2) between limit without time sequence.
Ke Liben series bacillus ferments by the present invention, obtains Ke Liben series bacillus fermented liquid.In the present invention, what described Ke Liben series bacillus fermentation was concrete is:
Ke Liben series bacillus bacterial classification is carried out activation culture;
Strain inoculation after described activation culture is carried out liquid fermenting in liquid nutrient medium, obtains Ke Liben series bacillus fermented liquid.
The Ke Liben series bacillus strain inoculation of preservation is preferably carried out activation culture by the present invention in nutrient agar.The container of the present invention to described activation culture does not have special restriction, adopts activation culture container well known to those skilled in the art, as adopted eggplant type bottle.In the present invention, the temperature of described activation culture is preferably 29 ~ 30 DEG C; The time of described activation culture is preferably 8 ~ 12h, is more preferably 10h.
In the present invention, described nutrient agar preferably includes following component: peptone 10 parts, 3 parts, extractum carnis powder, 5 parts, sodium-chlor, 15 parts, agar.Described nutrient agar final pH is 7.1 ~ 7.5.
After completing described activation culture, the Ke Liben series bacillus after activation culture is inoculated in liquid nutrient medium and carries out liquid culture by the present invention.In the present invention, Ke Liben series bacillus is inoculated after the preferred sterilizing of described liquid nutrient medium.In the present invention, described sterilizing is preferably high-temperature sterilization, and the temperature of described sterilizing is preferably 120 ~ 130 DEG C, is more preferably 121 DEG C; The time of described sterilizing is preferably 30-45min, is more preferably 38min.
The container of the present invention to described liquid fermenting does not have special restriction, adopts the equipment of fermentation well known to those skilled in the art, as being fermentor tank.In the present invention, the temperature of described liquid fermenting is preferably 29 ~ 30 DEG C, is more preferably 30 DEG C; The pH value of described liquid fermenting is preferably 6.8-7.0, is more preferably 6.9; The pressure of described liquid fermenting is preferably 0.08 ~ 0.12MPa, is more preferably 0.09 ~ 0.11MpPa; Air flow in described liquid fermenting process is preferably 0.8 ~ 1.2V/Vmin, is more preferably 1V/Vmin; The described liquid fermenting time is preferably 48-120 hour, is more preferably 72 ~ 84 hours;
The present invention preferably detected fermentation total viable count every 2 ~ 4 hours at described liquid fermenting after 72 ~ 96 hours, to determine the time point stopping fermentation; Preferably, stop fermentation when total viable count reaches 50 ~ 10,000,000,000/ml, preferred, stop fermentation when total viable count reaches 80 ~ 10,000,000,000/ml, obtain Ke Liben series bacillus fermented liquid.The present invention does not have special restriction to the method that described fermentation total viable count detects, and adopts conventional method of counting well known to those skilled in the art, such as microscopic count, plate count, mensuration cell weight etc.
In the present invention, liquid nutrient medium in described Ke Liben series bacillus fermenting process preferably includes following component: sucrose 1-50 weight part, SODIUMNITRATE 0.1-5.0 weight part, potassium primary phosphate 1.0-10 weight part, magnesium sulfate 0.1-1.0 weight part, Repone K 0.1-2.0 weight part, ferric sulfate 0.01-1.0 weight part.
Fermentation of bacillus subtilis of the present invention is specially:
The activation of bacterial classification;
Strain inoculation after activation is carried out liquid fermenting in liquid nutrient medium, obtains Ke Liben series bacillus fermented liquid.
The Bacillus subtilis strain bought preferably is inoculated in nutrient agar and carries out activation culture by the present invention.The container of the present invention to described activation culture does not have special restriction, adopts activation culture container well known to those skilled in the art, as adopted eggplant type bottle.In the present invention, the temperature of described activation culture is preferably 29 ~ 30 DEG C; The time of described activation culture is preferably 8 ~ 12h, is more preferably 10h.
In the present invention, described nutrient agar preferably includes following component: peptone 10 parts, 3 parts, extractum carnis powder, 5 parts, sodium-chlor, 15 parts, agar; Described nutrient agar final pH is 7.1 ~ 7.5.
After completing described activation culture, the subtilis after activation culture is inoculated in liquid nutrient medium and carries out liquid culture by the present invention.In the present invention, Ke Liben series bacillus is inoculated after the preferred sterilizing of described liquid nutrient medium; Described sterilizing is preferably high-temperature sterilization, and the temperature of described sterilizing is preferably 120 ~ 130 DEG C, is more preferably 125 DEG C; The time of described sterilizing is preferably 30-45min, is more preferably 40min.
The container of the present invention to described liquid fermenting does not have special restriction, adopts the equipment of fermentation well known to those skilled in the art, as being fermentor tank.In the present invention, the temperature of described liquid fermenting is preferably 29 ~ 30 DEG C, is more preferably 30 DEG C; The pH value of described liquid fermenting is preferably 6.8-7.0, is more preferably 6.9; The pressure of described liquid fermenting is preferably 0.05 ~ 0.1MPa, is more preferably 0.07 ~ 0.08MpPa; Air flow in described liquid fermenting process is preferably 0.8 ~ 1.2V/Vmin, is more preferably 1V/Vmin; The described liquid fermenting time is preferably 28 ~ 48 hours, is more preferably 36 ~ 44 hours;
The present invention preferably detected fermentation total viable count every 2 ~ 4 hours at described liquid fermenting after 28 ~ 32 hours, preferably, stop fermentation when total viable count reaches 50 ~ 10,000,000,000/ml, preferred, stop fermentation when total viable count reaches 85 ~ 9,500,000,000/ml, obtain fermentation of bacillus subtilis liquid.The present invention does not have special restriction to the method that described fermentation total viable count detects, and adopts conventional method of counting well known to those skilled in the art, such as microscopic count, plate count, mensuration cell weight etc.
In the present invention, liquid nutrient medium in described fermentation of bacillus subtilis process preferably includes following component: peptone 0.01-0.1 weight part, yeast powder 0.01-0.1 weight part, sucrose 0.001-0.050 weight part, ammonium sulfate 0.001-0.007 weight part, Trisodium Citrate 0.001-0.01 weight part, potassium primary phosphate 0.001-0.01 weight part, magnesium sulfate 0.0001-0.001 weight part, ferric sulfate 0.00001-0.0001 weight part, manganous sulfate 0.01-0.1 weight part.
Limit without time sequence between the fermentation of Ke Liben series bacillus and fermentation of bacillus subtilis step in the present invention, first carry out the fermentation of Ke Liben series bacillus, and then carry out the fermentation of subtilis or first can carry out the fermentation of subtilis, carry out the fermentation of Ke Liben series bacillus again, then or the fermentation of Ke Liben series bacillus and subtilis carry out simultaneously.
After completing the fermentation of described Ke Liben series bacillus and fermentation of bacillus subtilis, the present invention aseptically, be preferably (20 ~ 60) by volume by described Ke Liben series bacillus fermented liquid and fermentation of bacillus subtilis liquid: the ratio mixing of (50 ~ 80), preferred is (35 ~ 45) by volume: the ratio of (60 ~ 70) is mixed to get microbe composite bacterial liquid; Described aseptic condition is realized by ultraviolet Bechtop or aseptic workplace.
When the present invention is to provide a kind of complex microbial inoculum, present invention also offers a kind of preparation method of complex microbial inoculum, comprising the following steps:
Microbe composite bacterial liquid is prepared according to technique scheme;
Described microbe composite bacterial liquid is dry, obtain complex microbial inoculum.
The present invention prepares microbe composite bacterial liquid according to technique scheme, does not repeat them here.
In the present invention, described drying is preferably spraying dry; Spray-dired temperature out is preferably 90 ~ 95 DEG C, is more preferably 93 DEG C; Temperature in is preferably 145 ~ 150 DEG C, is more preferably 148 DEG C; Described spray-dired pan feeding speed is preferably 1000 ~ 1500ml/h, is more preferably 1200ml/h;
The present invention preferably adds protective material in described spray-dired process, with the survival rate ensureing that microbial composite bacteria is higher in spray-dired process.In the present invention, described protective material is preferably skim-milk or sucrose.The present invention preferably adopts the protective material of solution state, and the mass concentration of described protective material solution is preferably 15 ~ 25%, is more preferably 20%.
Present invention also offers the application of described microbial composite bacteria in control Citrus Huanglongbing pathogen, the application method of described microbial composite bacteria is that root is filled with or spraying; Fill with for described and be preferably specially described microbial composite bacteria is watered at plant root 15-25 centimetre place, preferredly water at root 18 ~ 22 centimeters place; Every strain tree is preferred uses 0.5 ~ 1kg composite bacteria, preferred every strain tree use 0.8 ~ 0.9kg; Described spraying, every strain tree is preferred uses composite bacteria 0.5 ~ 1kg, after being watered 3 ~ 5kg, spraying blade, preferred, every strain tree uses composite bacteria 0.8 ~ 0.9kg, after being watered 4 ~ 4.5kg, spraying blade.
Below in conjunction with embodiment, microbial composite bacteria provided by the invention is described in detail, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
The preparation of Ke Liben series bacillus fermented liquid, to be seeded in the liquid culture after 121 DEG C of sterilizing 35min after Ke Liben series bacillus enlarged culturing, described liquid culture based component is sucrose 30 weight part, SODIUMNITRATE 1.5 weight part, potassium primary phosphate 1.0 weight part, magnesium sulfate 0.5 weight part, Repone K 0.5 weight part, ferric sulfate 0.01 weight part.In fermentation cylinder for fermentation, fermentation condition: temperature is 30 DEG C, pH value is 6.9, tank internal pressure 0.1MPa, fermentation air flow maintain 1v/vmin, 76 hours time, ferment every the total viable count of the 2 hours detection of the method by microscopic inspection fermented liquids after 72 hours, stop fermentation obtaining Ke Liben series bacillus fermented liquid when total viable count reaches 9,600,000,000/ml.
Embodiment 2
The preparation of fermentation of bacillus subtilis liquid, to be seeded in the liquid culture after 121 DEG C of sterilizing 40min after subtilis enlarged culturing, described liquid culture based component is peptone 0.03 weight part, yeast powder 0.03 weight part, sucrose 0.025 weight part, ammonium sulfate 0.007 weight part, trisodium citrate 0.003 weight part, potassium primary phosphate 0.003 weight part, magnesium sulfate 0.0005 weight part, ferric sulfate 0.00005 weight part, manganous sulfate 0.01 weight part.In fermentation cylinder for fermentation, fermentation condition: temperature is 29 DEG C, pH value is 6.8, tank internal pressure 0.07MPa, fermentation air flow maintain 1v/vmin, 36 hours time, ferment after 28 hours and detected fermented liquid total viable count every 2 hours, stop fermentation obtaining fermentation of bacillus subtilis liquid when total viable count reaches 9,500,000,000/ml.
Embodiment 3
The preparation of complex microbial inoculum, by the fermentation of bacillus subtilis liquid obtained in the Ke Liben series bacillus fermented liquid obtained in embodiment 1 and embodiment 2, be mixed to get microbe composite bacterial liquid in the indoor ratio being 1: 0.83 in Ke Liben series bacillus fermented liquid and fermentation of bacillus subtilis liquid volume ratio of aseptic technique.The microbe composite bacterial liquid obtained is carried out spraying dry, and spray-dired temperature out is 94 DEG C, and temperature in is 148 DEG C, and pan feeding speed is 1200ml/h, adds the skim-milk of 21% as protective material, obtain complex microbial inoculum during spraying dry.
Embodiment 4
Microbial composite bacteria of the present invention detects the prevention effect of Citrus Huanglongbing pathogen, test and carried out in back of the body navel orange fruit industry base, high hill, Anyuan County, Ganzhou City on January 1st, 2014 to December 31, the morbidity of pilot region yellow twig is heavier, adopts the mode of filling with root and foliar spray to carry out.Be divided into 4 groups of process, root 1kg/ strain is filled with in process 1, root 1.5kg/ strain is filled with in process 2, and root 1kg/ strain+foliar spray 0.5kg/ strain is filled with in process 3, processes the clear water that 4 Routine control (ck) i.e. root fills with 1.5kg/ strain, root-irrigation method is described microbe composite bacterial liquid or clear water are watered at every strain trees ' root 15-25 centimetre place, fill with once every 30 days roots, the method for foliar spray is that every strain tree uses complex microbial inoculum 0.5kg, after being watered 4kg, spraying blade, every 30 days spraying blades once.
Prevention effect is shown in Fig. 1, the Citrus Huanglongbing pathogen sickness rate of process 1 ~ 3 is starkly lower than conventional processing, be reduced to 35.6% ~ 67.5% by the sickness rate of conventional processing 75.1%, sickness rate is the highest compared with conventional processing reduces 52%, has the effect of preventing and treating yellow twig significantly.
Effect of increasing production is shown in Fig. 2, and output is increased to 390 kgs/acre by processed conventionally 300 kgs/acre, and the output comparatively the highest increase of conventional processing 90 kgs/acre of test group, stimulation ratio, up to 30%, has significant effect of increasing production.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a microbial composite bacteria, is characterized in that, comprises Ke Liben series bacillus and subtilis, and the biological deposits of described Ke Liben series bacillus is numbered CGMCCNO.7996.
2. microbial composite bacteria according to claim 1, is characterized in that, described microbial composite bacteria is microbe composite bacterial liquid;
In described microbe composite bacterial liquid, the volume ratio of Ke Liben series bacillus and subtilis is (20 ~ 60): (50 ~ 80);
In described microbe composite bacterial liquid, the total viable count of Ke Liben series bacillus is 50 ~ 10,000,000,000/ml, and the total viable count of subtilis is 50 ~ 10,000,000,000/ml.
3. microbial composite bacteria according to claim 1, is characterized in that, described microbial composite bacteria is complex microbial inoculum;
In described complex microbial inoculum, the weight ratio of Ke Liben series bacillus and subtilis is (5 ~ 80): (10 ~ 80);
In described complex microbial inoculum, the total viable count of Ke Liben series bacillus is 50 ~ 20,000,000,000/g, and the total viable count of subtilis is 60 ~ 15,000,000,000/g.
4. the preparation method of microbial composite bacteria according to claim 2, comprises the following steps:
A1) fermentation of Ke Liben series bacillus, described Ke Liben series bacillus fermentation condition is: temperature is 29 ~ 30 DEG C, incubation time is 48 ~ 120 hours, ferment after 72 ~ 96 hours and detected fermented liquid total viable count every 2 ~ 4 hours, stop fermentation when total viable count reaches 50 ~ 10,000,000,000/ml, obtain Ke Liben series bacillus fermented liquid;
A2) fermentation of subtilis, the fermentation condition of described subtilis seed is: temperature is 29 ~ 30 DEG C, incubation time 28 ~ 48 hours, ferment and after 28 ~ 32 hours, detected the total viable count of fermented liquid every 2 ~ 4 hours, stop fermentation when total viable count reaches 50 ~ 10,000,000,000/ml, obtain fermentation of bacillus subtilis liquid;
A3) aseptically, be (20 ~ 60) by volume by described Ke Liben series bacillus fermented liquid and fermentation of bacillus subtilis liquid: the ratio of (50 ~ 80) is mixed to get microbe composite bacterial liquid;
Step a1) and step a2) between limit without time sequence.
5. the preparation method of microbial composite bacteria according to claim 3, comprises the following steps:
B1) fermentation of Ke Liben series bacillus, described Ke Liben series bacillus fermentation condition is: temperature is 29 ~ 30 DEG C, incubation time is 48 ~ 120 hours, ferment after 72 ~ 96 hours and detected fermented liquid total viable count every 2 ~ 4 hours, stop fermentation when total viable count reaches 50 ~ 10,000,000,000/ml, obtain Ke Liben series bacillus fermented liquid;
B2) fermentation of subtilis, the fermentation condition of described subtilis seed is: temperature is 29 ~ 30 DEG C, incubation time 28 ~ 48 hours, ferment and after 28 ~ 32 hours, detected the total viable count of fermented liquid every 2 ~ 4 hours, stop when total viable count reaches 50 ~ 10,000,000,000/ml fermentation to obtain fermentation of bacillus subtilis liquid;
B3) aseptically, the ratio being 1: 0.5 ~ 1.5 by volume by described Ke Liben series bacillus fermented liquid and fermentation of bacillus subtilis liquid is mixed to get microbe composite bacterial liquid;
B4) by described step b3) in the microbe composite bacterial liquid that obtains dry, obtain complex microbial inoculum;
Described step b1) and step b2) between limit without time sequence.
6. the preparation method of complex microbial inoculum according to claim 5, is characterized in that, step b4) described in drying be spraying dry, spray-dired temperature out is 90 ~ 95 DEG C, and temperature in is 145 ~ 150 DEG C.
7. the preparation method of complex microbial inoculum according to claim 6, is characterized in that, described spray-dired pan feeding speed is 1000 ~ 1500ml/h.
8. the preparation method of complex microbial inoculum according to claim 6, adds protective material in described spray-drying process, and described protective material is skim-milk or sucrose, and protectant concentration is 15 ~ 25%.
9. the application of the microbial composite bacteria that described in the microbial composite bacteria described in claims 1 to 3 any one or claim 4 ~ 8 any one, preparation method obtains in control Citrus Huanglongbing pathogen.
10. the application of microbial composite bacteria according to claim 9, is characterized in that, the application method of described microbial composite bacteria is that root is filled with or spraying;
Described filling is specially: every strain tree use 0.5 ~ 1kg microbial composite bacteria, waters at root 15-25 centimetre place;
Described spraying is specially: every strain tree uses composite bacteria 0.5 ~ 1kg, after being watered 3 ~ 5kg, and spraying blade.
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