CN106350464A - Microbial agent for preventing and treating cumquat huanglongbing and preparation method thereof - Google Patents
Microbial agent for preventing and treating cumquat huanglongbing and preparation method thereof Download PDFInfo
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Abstract
The invention provides a microbial agent for preventing and treating cumquat huanglongbing and a preparation method thereof, and particularly provides a microbial agent for preventing huanglongbing of cumquat, citrus shantianju and citrus and a preparation method thereof. The microbial agent is formed by fermenting and then mixing bacteria like Gulake streptomyces, Kawuer streptomyces, bacillus psychrosaccharolyticus, nissl bacillus and tufted trichoderma proportionally. Procreation and propagation of gram negative bacteria are inhibited by means of root irrigation and foliage spray, bacteria are used to treat bacteria, and harmful pathogenic bacteria in soil are killed, so that tree root systems are more developed, tree cell tubes are smooth, yellow leaves of fruit trees are turned green, and healthy new treetops are enabled to grow; the microbial agent generates insecticidal antibiotics like flavensomycin and inhibits or damages development process of eubactericera and eggs of other disease-transmitting insect (like citrus aphid), so that adult rate is reduced, orchard pest occurrence quantity is reduced, transmission media of huanglongbing are cut off, and effects on preventing and controlling citrus huanglongbing are realized.
Description
Technical field
The present invention relates to technical field of microbe application, in particular to a kind of prevention and control Fructus Fortunellae Margaritae, Sha Tianju, Citrus are yellow
Microbial bacterial agent of dragon disease and preparation method thereof.
Background technology
Citrus (comprising Fructus Fortunellae Margaritae, Sha Tianju, common Citrus) are the most fruit of world wide production, have had more than 4000 in China
The cultivation history in year.China's citriculture areas in 2009 and yield all exceed Brazil, the U.S., occupy first place in the world, but in Citrus chachiensis Hort.
While Fructus Citri tangerinae industry develops rapidly, yellow twig has become and has endangered destructive disease the most serious on Orange Producing.Early in 18 generation
Record the middle period, just there is relevant report in India.
Reinking has carried out reported first, since over half a century, Citrus the twenties in 19th century to South China of China
Yellow twig seriously constrains Guangdong, Guangxi, the development of Fujian 3 provinces and regions Citrus Industry.Late 1970s, Sichuan Province Xichang ground
Area and Dukou, Ganzhou area and Yunnan Province obtain some areas and also successively find Citrus Huanglongbing pathogen, to 80 years 20th century
For mid-term, Citrus Huanglongbing pathogen in the Citrus producing region extensive widespread in China Guangdong, Guangxi, Fujian, Hainan and Taiwan, and in Zhejiang
River, Guizhou, Hunan occur in succession, and 19 provinces of China's citriculture, existing 11 of area are endangered, and it is total that injured area accounts for Citrus
More than the 80% of cultivated area, yield accounts for the 85% about of total output.SaoPaulo State,Brazils in 2004 and U.S. Fo Luoli in 2005
Dazhou City's yellow twig occurs in succession, causes the fear of Citrus practitioner.So far, yellow twig has been distributed widely in Asia, Africa, ocean
More than the 40 of continent, South America and North America country.Yellow twig also known as yellow tip disease, yellow blight, Fructus Canarii disease, are the important diseases of Citrus
Evil, for international, internal quarantine object.
There is the stress that 11 producing regions receive yellow twig in 17 Citrus producing regions of China, and wide as Citrus major production areas
The ground such as east, Guangxi, Fujian, Yunnan, Hainan are particularly acute.The classical symptom of morbidity Citrus plant is " the yellow tip ", the mottled Huang of blade
Change, fruit diminishes, form unworthy " Fructus Canarii " or " coppernose fruit ", morbidity later stage root system necrosis, tree vigo(u)r fails, leads to fall
Leaf, shedding, plant death etc..When disease severe epidemic, often make the total destruction in several years of large stretch of orangery, to Orange Producing
Threaten greatly.
The cause of disease of yellow twig is to parasitize the gram negative bacteria of phloem, can bark tissue, leaf, flower, root and
Detect in fruit.Gram negative bacteria route of transmission is extensively it is very difficult to be killed.Fruit tree once infects this pathogenic bacteria, is
Avoid other fruit trees infected, control the propagation of yellow twig and spread, people can only excavate or surgery disease tree, lose huge.
Therefore, in the daily management of fruit tree, the prevention and control carrying out Citrus Huanglongbing pathogen become the most important thing.
At present, the preventions for Citrus Huanglongbing pathogen mainly have two aspects: one is the defeated of cut-out pathogenic bacteria from root
Enter, mainly check on nursery stock, strengthen seedling-wood breeding management, exclude sick seedling in time, from anosis seedling growth and scion.Two
From the communication media of cut-out pathogenic bacteria, the main method preventing and treating wood louse using chemistry, physics.It can be seen that, for Citrus in prior art
It is fewer that the communication media of yellow twig is paid close attention to, and mainly or on preventing and treating wood louse, and has no the prevention and control to other communication medias
Means.
Marquis appoint the phase etc. disclose in the patent No. 2015104457788 a kind of microbial manure of prevention and control Citrus Huanglongbing pathogen and
Its preparation method.The microbial manure being provided includes following components: Paecilomyces lilacinus, bacillus licheniformis, trichoderma and have
Machine matter;In described microbial manure, the viable bacteria content of described Paecilomyces lilacinus is not less than 0. 2 hundred million/gram, described lichens bud pole
The viable bacteria content of bacterium is not less than 0. 1 hundred million/gram, the viable bacteria content of described trichoderma is not less than 0. 05 hundred million/gram.In the present invention, light
Purple Paecilomyces varioti, lichens bud pole bacterium, three kinds of strain collective effects of trichoderma, suppression or the growth course destroying line eggs, thus
Reduce nematicide adult rate, reduce orchard nematicide basis generating capacity, the communication media of cut-out yellow twig, reach prevention and control Citrus Huanglongbing pathogen
Effect.Main or a kind of biological organic bacterial manure, thalline content is low, and consumption is big, in-convenience in use.Liang little Wen etc. is in patent
A kind of microbial composite bacteria and preparation method thereof and its answering in terms of preventing and treating Citrus Huanglongbing pathogen is disclosed in 2016100006863
With.Described microbial composite bacteria, including Ke Liben series bacillus and bacillus subtilises, described Ke Liben series bacillus
Biological deposits to number be cgmcc n0.7996, microbial composite bacteria can be specifically microbe composite bacterial liquid or microorganism is multiple
Close microbial inoculum, pathogen can be suppressed, but there is no the function of parasite killing it is impossible to the communication media of cut-out yellow twig, reach prevention and control Citrus
The effect of yellow twig.
Content of the invention
The first object of the present invention be to provide one kind can parasite killing again can be antibacterial, also there is the anti-of root mark growth-promoting functions
Control the microbial bacterial agent of Fructus Fortunellae Margaritae yellow twig.The proportioning raw materials of this microbial bacterial agent are: Gu Lake streptomycete fermentation liquid 10-30 part,
Ka Wuer streptomycete fermentation liquid 20-40 part, cold solution sugar fermentation of bacillus liquid 10-30 part, Nissl fermentation of bacillus liquid 10-30
Part, tuft Trichoderma spp. fermentation liquid 10-30 part.In described microbial bacterial agent, Gu Lake streptomycete, Ka Wuer streptomycete, cold solution sugar
Bacillus cereuss, Nissl bacillus cereuss and five kinds of bacterium of tuft Trichoderma spp. are the energy symbiosis that screening obtains from more than 500 plants of microbial strains
Coexist, the antibiotic such as Ka Wuer chain of the growth course of multiple suppression wood louses and other insect vector (as Fructus Citri tangerinae aphid) worm's ovum can be produced
Mycete produces flavensomycin, a kind of antibiotic of parasite killing, thus reducing its adult rate, reducing orchard pest basis generating capacity, cutting
The communication media of disconnected yellow twig, reaches the effect of prevention and control Citrus Huanglongbing pathogen；On the other hand, multiple antagonism Gram-negatives can be produced
The antibiotics of antibacterial such as Gu Lake streptomycete produces ancient cured mycin, a kind of antibiotics of anti-gram negative bacteria, and energy is preferably
Restrain the pathogen of yellow twig.
The second object of the present invention is to provide a kind of described microbial bacterial agent of microbial control Fructus Fortunellae Margaritae yellow twig
Preparation method, the method step is simple it is ensured that the drug effect of microbial bacterial agent projects.In addition, this 5 plants of bacterial strains being used are all can
It is directly isolated to obtain from soil, all there are root mark growth-promoting functions.
In order to realize the above-mentioned purpose of the present invention, spy employs the following technical solutions: a kind of micro- life of preventing and treating Fructus Fortunellae Margaritae yellow twig
The preparation method of thing microbial inoculum, comprises the following steps:
Step one: the preparation of Gu Lake streptomycete fermentation liquid
Take out Gu Lake streptomyces species preservation pipe, draw flat board with Gause I solid medium and recovered, 30 DEG C of cultures 7
My god.Under flat board, the streak inoculation of picking single bacterium colony, equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is being cultivated
Cultivate 6-8 days for 30 DEG C in case, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution that a large amount of spore powders can use 500 milliliters
Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, as Gu Lake streptomycete seed liquor；
Fermentation: by the Gu Lake streptomycete seed liquor of above-mentioned preparation with 1% inoculum concentration be seeded to equipped with sterilizing 600l Gu cured
In the 1000l fermentation tank of section's streptomycete fermentation culture medium, 28-32 DEG C of temperature control, open and stir 200r/min, first 24 hours, ventilation
For per minute for 200l air, 24-36 hour, ventilation is 400l, and after 36 hours, ventilation is 600l, and 40-48 is little for culture
When, treat that thalline content reaches 60g/l, you can stop tank, that is, obtain Gu Lake streptomycete fermentation liquid；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05
Gram, magnesium sulfate: 0.5 gram, sodium chloride: 0.5 gram, ferrous sulfate: 0.01 gram, agar: 20 grams, 1000ml supplied by distilled water,
Ph7.2～7.4；
Wherein, described Gu Lake streptomycete fermentation culture medium: bean cake 2%, corn starch 2%, stone powder 1%, soy molasses 2%, sulfur
Sour manganese 0.004%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.05%, the condition of each medium sterilization is: 0.10-0.15mpa, 121
DEG C sterilizing 30 minutes.
Step 2: the preparation of Ka Wuer streptomycete fermentation liquid
Take out Ka Wuer streptomyces species preservation pipe, draw flat board with Gause I solid medium and recovered, 30 DEG C of cultures 7
My god.Under flat board, the streak inoculation of picking single bacterium colony, equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is being cultivated
Cultivate 6-8 days for 30 DEG C in case, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution that a large amount of spore powders can use 500 milliliters
Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, as Ka Wuer streptomycete seed liquor；
Fermentation: the Ka Wuer streptomycete seed liquor of above-mentioned preparation is seeded to the card 5 of the 600l equipped with sterilizing with 1% inoculum concentration
In the 1000l fermentation tank of your streptomycete fermentation culture medium, 28-30 DEG C of temperature control, first 18 hours, ventilation was per minute empty for 300l
Gas, 18-32 hour, ventilation is 500l, and after 32 hours, ventilation is 700l, opens stirring 200r/min, cultivates 40-48 hour,
Treat that thalline content reaches 80g/l, you can stop tank, that is, obtain Ka Wuer streptomycete fermentation liquid；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05
Gram, magnesium sulfate: 0.5 gram, sodium chloride: 0.5 gram, ferrous sulfate: 0.01 gram, agar: 20 grams, 1000ml supplied by distilled water,
Ph7.2～7.4；
Wherein, described Ka Wuer streptomycete fermentation culture medium: bean cake 3%, cottonseed meal 1%, corn starch 2%, stone powder 1%, soy sugars
Honey 3%, manganese sulfate 0.002%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.02%, the condition of each medium sterilization is: 0.10-
0.15mpa, 121 DEG C sterilize 30 minutes.
Step 3, the preparation of cold solution sugar fermentation of bacillus liquid
Take out the sugared bacillus cereuss preservation pipe of cold solution, draw flat board respectively with lb solid medium and recovered, cultivate 48 hours for 30 DEG C.
Under flat board, picking single bacterium colony is seeded to equipped with lb solid medium, cultivates 48 hours, use 3000ml in 30 DEG C of incubators
Sterilized water, by the lawn eluting in three Fructus Solani melongenae bottles, is seeded to the 500l equipped with the sugared fermentation of bacillus culture medium of the cold solution of 300l and sends out
In fermentation tank, open stirring 120r/min, ventilation is 200l/min within first 12 hours, after 12 hours, ventilation is 320l/min, 30 DEG C
Culture 26-36 hour, treats that total spore content is not less than 6,000,000,000 cfu/ml, you can as the sugared fermentation of bacillus liquid of cold solution；
Wherein, described lb solid medium: yeast extract 5.0 g, peptone 10.0, nacl10.0 g, agar 20 g, water
1000ml, ph7.2；
Wherein, described cold solution sugar fermentation of bacillus culture medium: glucose 8g/l, corn starch 30 g/l, bean cake powder 40 g/
L, magnesium sulfate 0.4 g/l, manganese sulfate 0.5 g/l, potassium dihydrogen phosphate 0.5 g/l, ph7.0；The condition of each medium sterilization is:
0.10-0.15mpa, 121 DEG C sterilize 30 minutes.
Step 4, the preparation of Nissl fermentation of bacillus liquid
Take out Nissl bacillus cereuss preservation pipe, draw flat board respectively with nitrogen-free solid medium and recovered, cultivate 72 hours for 30 DEG C.
Under flat board, picking single bacterium colony is seeded to equipped with nitrogen-free solid medium, cultivates 72 hours in 30 DEG C of incubators, uses
3000ml sterilized water is seeded to the lawn eluting in three Fructus Solani melongenae bottles equipped with 300l Nissl fermentation of bacillus culture medium
In 500l fermentation tank, open stirring 120r/min, ventilation is 100l/min within first 8 hours, after 8-24 hour, ventilation is 200l/
Min, after 24 hours, ventilation is 300l/min, 30 DEG C of culture 32-48 hours, treats that total spore content is not less than 3,000,000,000 cfu/
Ml, you can as Nissl fermentation of bacillus liquid；
Wherein, described nitrogen-free solid medium: sucrose 10g, dipotassium hydrogen phosphate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.2g, sulphuric acid
Calcium 0.1g, Calcium Carbonate 5g, agar 18g, distilled water 1000ml, ph7.2；
Wherein, described Nissl fermentation of bacillus culture medium: molasses 20g/l, corn starch 10 g/l, cottonseed meal powder 40 g/l, sulfur
Sour magnesium 0.4 g/l, manganese sulfate 0.6g/l, potassium dihydrogen phosphate 0.6 g/l, Calcium Carbonate 10 g/l, ph7.0；Each medium sterilization
Condition is: 0.10-0.15mpa, and 121 DEG C sterilize 30 minutes.
Step 5, the preparation of tuft Trichoderma spp. fermentation liquid
Take out tuft Trichoderma spp. strain preservation pipe, draw flat board with pda solid medium and recovered, cultivate 7 days for 30 DEG C.Under flat board
Picking single bacterium colony streak inoculation, equipped with the Fructus Solani melongenae bottle of 150 milliliters of pda solid mediums, cultivates 6-8 for 30 DEG C in incubator
My god, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution eluting that a large amount of spore powders can use 500 milliliters, adjust spore dense
Spend for 0.05 hundred million cfu/ml, as tuft Trichoderma spp. seed liquor；
Fermentation: the tuft Trichoderma spp. seed liquor of above-mentioned preparation is seeded to equipped with 300l tuft Trichoderma spp. fermentation culture with 1% inoculum concentration
In the 500l fermentation tank of base, open stirring 200r/min, ventilation is 120l/min within first 8 hours, and after 8-24 hour, ventilation is
240l/min, after 24 hours, ventilation is 360l/min, 30 DEG C of culture 32-48 hours, treats that thalline content reaches 100g/l, that is,
Tank can be stopped, you can as tuft Trichoderma spp. fermentation liquid；
Wherein, described pda solid medium: Rhizoma Solani tuber osi 200g, sucrose 20g, water 1000ml, agar 20g；
Wherein, described tuft Trichoderma spp. fermentation medium: rapeseed cake 2%, cottonseed meal 2%, corn starch 2%, stone powder 1%, soy molasses 3%,
Manganese sulfate 0.001%, dipotassium hydrogen phosphate 0.04%, magnesium sulfate 0.02%, the condition of each medium sterilization is: 0.10-0.15mpa,
121 DEG C sterilize 30 minutes.
Step 6, mixed fermentation liquid
The bacterium solution of first five step preparation is pressed Gu Lake streptomycete fermentation liquid 10-30 part, Ka Wuer streptomycete fermentation liquid 20-40
Part, cold solution sugar fermentation of bacillus liquid 10-30 part, Nissl fermentation of bacillus liquid 10-30 part, tuft Trichoderma spp. fermentation liquid 10-30
Part mixes, that is, obtain preventing and treating the microbial bacterial agent of Fructus Fortunellae Margaritae yellow twig.
Wherein, this microbial bacterial agent preventing and treating Fructus Fortunellae Margaritae yellow twig method be foliage-spray 1-2l/ mu, foliage-spray dilute
The multiple released is 100-200 times, pouring root simultaneously, and consumption is 50-200ml/ fruit tree；The extension rate of pouring root is 100-500 times；
The sugared bacillus cereuss of cold solution and Nissl bacillus cereuss are contained, bacillus cereuss are that a kind of comparatively ideal bio-feritlizer adds in the present invention
Plus bacterial strain, have that strong stress resistance, nutrition is simple, reproduction speed is fast, living bacteria count amount is high, stable performance, and Storage period length etc. is excellent
Gesture and become study hotspot in recent years.Additionally, most strains have diseases prevention, growth-promoting, phosphorus decomposing, potassium decomposing, nitrogen fixation, for sending out
The exhibition ecological agriculture, has important practical significance.
The cold solution sugar bacillus cereuss containing in the present invention, cold solution sugar bacillus cereuss can be internal or native in crop surface, plant
Some secondary metabolites must be secreted to plant rhizosphere while flourish, it is possible to increase the suction to nutrient for the plant in earth
Receive, stimulate the comprehensive function such as plant strain growth and suppression pathogenic bacteria.The sugared bacillus cereuss of cold solution can controlling plant diseases come from it can
Produce multiple antibiotic substance, including multiple compounds such as lipopeptid class, peptides, phospholipid, many alkenes, amino acidses and nucleic acids,
These antibacterial mass-energy suppress the normal growth of funguses, antibacterial, virus and bacterium substance etc..Cold solution sugar bacillus cereuss are in natural growth
The lipopeptide antibiotic producing with the fermentation culture later stage is its most important antibiotic substance.Lipopeptide antibiotic includes her withered grass bacterium
Plain (iturins), general leather plain (fengycins) and Surfactin (surfactin), wherein iturin and general leather element tool
There is very strong antifungal activity, and Surfactin has very high inhibitory activity to virus, tumor, mycoplasma；
For a long time, though root nodule bacteria and azotobacter etc. have stronger nitrogen fixing capacity, resistance is low, not shelf-stable, no
Commercialization beneficial to China's bio-azotobacter fertilizer and promoting the use of.Obtain one plant in Japan's separation first from hin in 1958 to have
After the bud pole bacterium of high nitrogenase activity, in soil, the newfound bud pole bacterium with nitrogen fixation gets more and more.There is fixed nitrogen
Effect
Bacillus cereuss are added in bio-feritlizer, can make that bio-feritlizer is acidproof, salt tolerant, high temperature resistant and high pressure resistant, are biological nitrogen fixation
The research and development of fertilizer, promotion and application will play an important role.Think that the nitrogen fixing capacity of aerobic bud pole bacterium is higher in the world at present
For fixed nitrogen bud pole bacterium, the Nissl bacillus cereuss in the present invention have stronger nitrogen fixation, and have obvious root mark rush
Raw effect.
Tuft Trichoderma spp. is contained, tuft Trichoderma spp. creates a series of pathogen hyphal cells during superparasitism in the present invention
The hydrolytic enzyme of wall.Trichoderma, when invading or penetrating host's hyphal cell, creates chitinase (chitinases), glucosan
((proteinases), lipase etc. be for enzyme, cellulase (cellulases), xylanase (xylanaes) and protease
Row hydrolase, to clear up the cell wall of pathogen.They are mostly induced by polysaccharide and fungal cell wall, and are subject to metabolic degradation
Thing, as checking of high concentration glucose.In these cell wall degrading enzymes, chitinase and glucanase are acknowledged as affecting
The important factor of biocontrol fungi superparasitism ability, and there is synergism.Chitinase includes restriction endonuclease and excision enzyme two class, its
Middle excision enzyme is the general enzyme of n mono- acetylglucosamine (n-acetylglucosaminidase) and chitobiose enzyme
(chitobiosidase).This two fermentoid has strong hydrolysis to the cell wall of plant pathogenic fungi, thus suppressing cause of disease
Bacterium spore germination simultaneously causes mycelia and embraces clearing up of son, and has synergism between this two fermentoid, also have simultaneously with
The synergistic effect of the bio-control factors such as antibacterial and antibacterial.Many trichoderma strains produce volatility or nonvolatile antibiotics
Class material, such as trichodermin (trichoderlnin), gliotoxin (gliotoxin), viridin (viridin), antibacterial peptide
((peptideantibiotic) etc..The producible multiple antibiotics of tuft Trichoderma spp., these materials include antibiotic and some
Enzyme.The chemical property of these antibiotics is different, includes pentanone, octanone, class mushroom, polypeptide and amino acid derivativges etc.
Several big class.These metabolite can destroy hyphal cell wall, so that intracellular matter is exosmosed, cause the primary of Rhizoctonia Solani
Matter is condensed, and mycelia fracture is disintegrated, and pathogen bacterium colony is had different degrees of
Inhibitory action, due to the species of antibiotics, the difference of chemical property and model of action, pathogen is often difficult to develop
Drug resistance.
Streptomycete is the major microorganisms source producing antibiotic.The antibiosiss of streptomycete are embodied in secondary metabolite
I.e. antibiotic suppression direct to pathogen and lethal effect.The impact to pathogen cell and macromolecular substances synthesis for the antibiotic
Show the synthesis of suppression protein, nucleic acid and plasma membrane, or the metabolic system of interference pathogen, thus suppressing its growth.Right
The research acting on protein synthesis system in antibiotic is more, the Gu Lake streptomycete in the present invention, can produce ancient cured mycin,
A kind of anti-gram negative bacteria and the antibiotics of antiviral；Can there is superpower suppression yellow twig pathogen；In the present invention
Ka Wuer streptomycete, display slight antibacterial activity, produce flavensomycin, a kind of anti-gram negative bacteria and parasite killing
Antibiotic；Can suppress or destroy wood louse and the growth course of other insect vector (as Fructus Citri tangerinae aphid) worm's ovums, the effectively evil such as killing wood louse
Worm.
The present invention has the advantage that and beneficial effect: prevents and treats in the present invention in the microbial bacterial agent of Fructus Fortunellae Margaritae yellow twig containing Gu
Cured section streptomycete, Ka Wuer streptomycete, cold solution sugar bacillus cereuss, Nissl bacillus cereuss and tuft Trichoderma spp., five kinds of strains can pass through
Competition, hyperparasitism, antibiosiss, bacteriolysiss, toxic protein and induction of resistance etc. carry out short of money to phytopathogen
Anti-, thus killing or reducing pathogen, play the effect of diseases prevention；In addition, may also include in its Biocontrol Mechanism: (1) is in adverse circumstance
In, under the such as dry early, stress of nutrient, improve patience by strengthening the growth of root system and plant.(2) can induce plant to pathogenic bacteria
Resistance.(3) increase the dissolubility of nutritional labeling in soil, and promote its absorption.(4) make the enzymatic inactivation of pathogen.Therefore, many bacterium
The plyability planting mixing Biocontrol Mechanism further increases its biological and ecological methods to prevent plant disease, pests, and erosion application effect；As Gu Lake streptomycete can produce ancient cured mycin, one
Plant the antibiotics of anti-gram negative bacteria and antiviral；Ka Wuer streptomycete, antibacterial activity that display is slight, produce yellow matter
Mycin, the antibiotic of a kind of anti-gram negative bacteria and parasite killing；On the one hand produce suppression or destruction wood louse and pass sick elder brother with other
The growth course of worm (as Fructus Citri tangerinae aphid) worm's ovum, thus reducing its adult rate, reduces orchard pest basis generating capacity, cut-out yellow twig
Communication media, reaches the effect of prevention and control Citrus Huanglongbing pathogen.
On the other hand, the Gu Lake streptomycete screened, Ka Wuer streptomycete, cold solution sugar bacillus cereuss, Nissl spore bar
Bacterium and five kinds of strains of tuft Trichoderma spp. can produce the antibiotics suppressing gram negative bacteria, can preferably restrain yellow twig
Pathogen, thus directly acting on yellow twig pathogen, reaches the effect preventing and treating yellow twig；
And, the Gu Lake streptomycete of the present invention, Ka Wuer streptomycete, cold solution sugar bacillus cereuss, Nissl bacillus cereuss and tuft
Five kinds of strain combinations of Trichoderma spp., are from 500 multiple beneficial bacterium, obtain through careful screening, they can symbiotic co-existence, be blended in
The strain combination of disease prevention growth-promoting effect can be mutually reinforcing together.
Specific embodiment
Embodiment 1
A kind of microbial bacterial agent of preventing and treating Fructus Fortunellae Margaritae yellow twig, the proportioning raw materials of this microbial bacterial agent are: Gu Lake streptomycete fermentation
20 parts of liquid, 30 parts of Ka Wuer streptomycete fermentation liquid, cold solution 20 parts of fermentation of bacillus liquid of sugar, Nissl fermentation of bacillus liquid 20
Part, 20 parts of tuft Trichoderma spp. fermentation liquid；
In order to achieve the above object, a kind of preparation method of the microbial bacterial agent of preventing and treating Fructus Fortunellae Margaritae yellow twig, comprises the following steps:
Step one: the preparation of Gu Lake streptomycete fermentation liquid
Take out Gu Lake streptomyces species preservation pipe, draw flat board with Gause I solid medium and recovered, 30 DEG C of cultures 7
My god.Under flat board, the streak inoculation of picking single bacterium colony, equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is being cultivated
Cultivate 7 days for 30 DEG C in case, treat that lawn covers with Fructus Solani melongenae bottle, produce a large amount of spore powders and can use 500 milliliters of physiological saline solution to wash
De-, regulation spore concentration is 0.1 hundred million cfu/ml, as Gu Lake streptomycete seed liquor；
Fermentation: by the Gu Lake streptomycete seed liquor of above-mentioned preparation with 1% inoculum concentration be seeded to equipped with sterilizing 600l Gu cured
In the 1000l fermentation tank of section's streptomycete fermentation culture medium, 28-32 DEG C of temperature control, open and stir 200r/min, first 24 hours, ventilation
For per minute for 200l air, 24-36 hour, ventilation is 400l, and after 36 hours, ventilation is 600l, cultivates 42 hours, inspection
Survey thalline content and reach 65g/l, stop tank, that is, obtain Gu Lake streptomycete fermentation liquid；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05,
Magnesium sulfate: 05, sodium chloride: 05, ferrous sulfate: 0 01 grams, agar: 20 grams, distilled water supplies 1000ml, ph7 2～7 4；
Wherein, described Gu Lake streptomycete fermentation culture medium: bean cake 2%, corn starch 2%, stone powder 1%, soy molasses 2%, sulfur
Sour manganese 0.004%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.05%, the condition of each medium sterilization is: 0.10-0.15mpa, 121
DEG C sterilizing 30 minutes.
Step 2: the preparation of Ka Wuer streptomycete fermentation liquid
Take out Ka Wuer streptomyces species preservation pipe, draw flat board with Gause I solid medium and recovered, 30 DEG C of cultures 7
My god.Under flat board, the streak inoculation of picking single bacterium colony, equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is being cultivated
Cultivate 6-8 days for 30 DEG C in case, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution that a large amount of spore powders can use 500 milliliters
Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, as Ka Wuer streptomycete seed liquor；
Fermentation: the Ka Wuer streptomycete seed liquor of above-mentioned preparation is seeded to the card 5 of the 600l equipped with sterilizing with 1% inoculum concentration
In the 1000l fermentation tank of your streptomycete fermentation culture medium, 28-30 DEG C of temperature control, first 18 hours, ventilation was per minute empty for 300l
Gas, 18-32 hour, ventilation is 500l, and after 32 hours, ventilation is 700l, opens stirring 200r/min, cultivates 44 hours, inspection
Survey thalline content is 82g/l, stops tank, that is, obtains Ka Wuer streptomycete fermentation liquid；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05
Gram, magnesium sulfate: 05 grams, sodium chloride: 05 grams, ferrous sulfate: 0 01 grams, agar: 20 grams, distilled water supplies 1000ml, ph7
2～7 4；
Wherein, described Ka Wuer streptomycete fermentation culture medium: bean cake 3%, cottonseed meal 1%, corn starch 2%, stone powder 1%, soy sugars
Honey 3%, manganese sulfate 0.002%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.02%, the condition of each medium sterilization is: 0.10-
0.15mpa, 121 DEG C sterilize 30 minutes.
Step 3, the preparation of cold solution sugar fermentation of bacillus liquid
Take out the sugared bacillus cereuss preservation pipe of cold solution, draw flat board respectively with lb solid medium and recovered, cultivate 48 hours for 30 DEG C.
Under flat board, picking single bacterium colony is seeded to equipped with lb solid medium, cultivates 48 hours, use 3000ml in 30 DEG C of incubators
Sterilized water, by the lawn eluting in three Fructus Solani melongenae bottles, is seeded to the 500l equipped with the sugared fermentation of bacillus culture medium of the cold solution of 300l and sends out
In fermentation tank, open stirring 120r/min, ventilation is 200l/min within first 12 hours, after 12 hours, ventilation is 320l/min, 30 DEG C
Culture 30 hours, the total spore content of detection is 6,500,000,000 cfu/ml, that is, as the sugared fermentation of bacillus liquid of cold solution；
Wherein, described lb solid medium: yeast extract 5.0 g, peptone 10.0, nacl10.0 g, agar 20 g, water
1000ml, ph7.2；
Wherein, described cold solution sugar fermentation of bacillus culture medium: glucose 8g/l, corn starch 30 g/l, bean cake powder 40 g/
L, magnesium sulfate 0.4 g/l, manganese sulfate 0.5 g/l, potassium dihydrogen phosphate 0.5 g/l, ph7.0.The condition of each medium sterilization is:
0.10-0.15mpa, 121 DEG C sterilize 30 minutes.
Step 4, the preparation of Nissl fermentation of bacillus liquid
Take out Nissl bacillus cereuss preservation pipe, draw flat board respectively with nitrogen-free solid medium and recovered, cultivate 72 hours for 30 DEG C.
Under flat board, picking single bacterium colony is seeded to equipped with nitrogen-free solid medium, cultivates 72 hours in 30 DEG C of incubators, uses
3000ml sterilized water is seeded to the lawn eluting in three Fructus Solani melongenae bottles equipped with 300l Nissl fermentation of bacillus culture medium
In 500l fermentation tank, open stirring 120r/min, ventilation is 100l/min within first 8 hours, after 8-24 hour, ventilation is 200l/
Min, after 24 hours, ventilation is 300l/min, cultivates 38 hours for 30 DEG C, the total spore content of detection is 3,100,000,000 cfu/ml, that is, make
For Nissl fermentation of bacillus liquid；
Wherein, described nitrogen-free solid medium: sucrose 10g, dipotassium hydrogen phosphate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.2g, sulphuric acid
Calcium 0.1g, Calcium Carbonate 5g, agar 18g, distilled water 1000ml, ph7.2；
Wherein, described Nissl fermentation of bacillus culture medium: molasses 20g/l, corn starch 10 g/l, cottonseed meal powder 40 g/l, sulfur
Sour magnesium 0.4 g/l, manganese sulfate 0.6g/l, potassium dihydrogen phosphate 0.6 g/l, Calcium Carbonate 10 g/l, ph7.0.Each medium sterilization
Condition is: 0.10-0.15mpa, and 121 DEG C sterilize 30 minutes.
Step 5, the preparation of tuft Trichoderma spp. fermentation liquid
Take out tuft Trichoderma spp. strain preservation pipe, draw flat board with pda solid medium and recovered, cultivate 7 days for 30 DEG C.Under flat board
Picking single bacterium colony streak inoculation, equipped with the Fructus Solani melongenae bottle of 150 milliliters of pda solid mediums, cultivates 6-8 for 30 DEG C in incubator
My god, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution eluting that a large amount of spore powders can use 500 milliliters, adjust spore dense
Spend for 0.05 hundred million cfu/ml, as tuft Trichoderma spp. seed liquor；
Fermentation: the tuft Trichoderma spp. seed liquor of above-mentioned preparation is seeded to equipped with 300l tuft Trichoderma spp. fermentation culture with 1% inoculum concentration
In the 500l fermentation tank of base, open stirring 200r/min, ventilation is 120l/min within first 8 hours, and after 8-24 hour, ventilation is
240l/min, after 24 hours, ventilation is 360l/min, cultivates 39 hours for 30 DEG C, detection thalline content is 110g/l, stops tank,
Can be used as tuft Trichoderma spp. fermentation liquid；
Wherein, described pda solid medium: Rhizoma Solani tuber osi 200g, sucrose 20g, water 1000ml, agar 20g；
Wherein, described tuft Trichoderma spp. fermentation medium: rapeseed cake 2%, cottonseed meal 2%, corn starch 2%, stone powder 1%, soy molasses 3%,
Manganese sulfate 0.001%, dipotassium hydrogen phosphate 0.04%, magnesium sulfate 0.02%, the condition of each medium sterilization is: 0.10-0.15mpa,
121 DEG C sterilize 30 minutes.
Step 6, mixed fermentation liquid
The bacterium solution of first five step preparation is pressed 20 parts of Gu Lake streptomycete fermentation liquid, 30 parts of Ka Wuer streptomycete fermentation liquid, cold
Sugared 20 parts of the fermentation of bacillus liquid of solution, 20 parts of Nissl fermentation of bacillus liquid, the 20 parts of mixings of tuft Trichoderma spp. fermentation liquid, that is, prevented
Control the microbial bacterial agent of Fructus Fortunellae Margaritae yellow twig.
Embodiment 2
Microbial composite bacteria of the present invention detects to the prevention effect of Fructus Fortunellae Margaritae yellow twig, is tested on March 1st, 2015
Carry out on March 1st, 2016 in Jiangmen City of Guangdong Province Xinhui District fruit industry base, the morbidity of pilot region yellow twig is heavier,
Sickness rate has substantially achieved 65%；Carried out by the way of pouring root and foliar spray.It is divided into two groups of process, test group: 100
Tree, the strain of pouring root l00ml/, foliage-spray 2l/ mu, all dilute 100 times of process；Matched group: 100 trees, 20% good year winter cream (fourth
Carbosulfan) 1500-2000 times, 800-1000 times of beta-cypermethrin；Medicinal 99% mineral oil (green grain husk) 150- is selected after fruit picking
1500 times of 200 times of+48% chlopyrifos；Every month is at least using once；
Result of the test: prevention effect is shown in Table 1, and the yellow twig sickness rate of test group is significantly lower than conventional treatment, by conventional treatment
52.9% sickness rate is reduced to 15.2%, and sickness rate highest reduces 71.3% compared with conventional treatment, has significantly anti-Yellow River harnessing
The effect of dragon disease.Effect of increasing production is also obvious as described in Table 1, and it is public that yield increases to 2510 by processed conventionally 1782 kilograms
Jin, the more conventional highest that processes of the yield of test group increases by 728 kilograms, and rate of growth is up to 40.85%, has significant effect of increasing production.
Table 1 microbial bacterial agent is to the preventing and treating of yellow twig and effect of increasing production
| Group | Sickness rate (%) | Preventive effect (%) | Yield (kg) | Rate of growth (%) |
| Test group | 15.2 | 71.3 | 2510 | 40.85 |
| Matched group | 52.9 | 1782 |
The above is only the preferred embodiment of the present invention it is noted that coming for those skilled in the art
Say, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (5)
1. a kind of microbial bacterial agent of preventing and treating Fructus Fortunellae Margaritae yellow twig is it is characterised in that the proportioning raw materials of this microbial bacterial agent are: ancient cured
Section's streptomycete fermentation liquid 10-30 part, Ka Wuer streptomycete fermentation liquid 20-40 part, cold solution sugar fermentation of bacillus liquid 10-30 part,
Nissl fermentation of bacillus liquid 10-30 part, tuft Trichoderma spp. fermentation liquid 10-30 part.
2. a kind of microbial bacterial agent of preventing and treating Fructus Fortunellae Margaritae yellow twig according to claim 1 is it is characterised in that its preparation side
Method, comprises the following steps:
Step one: the preparation of Gu Lake streptomycete fermentation liquid
Take out Gu Lake streptomyces species preservation pipe, draw flat board with Gause I solid medium and recovered, 30 DEG C of cultures 7
My god, picking single bacterium colony streak inoculation under flat board, equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is being cultivated
Cultivate 6-8 days for 30 DEG C in case, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution that a large amount of spore powders can use 500 milliliters
Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, as Gu Lake streptomycete seed liquor；
Fermentation: by the Gu Lake streptomycete seed liquor of above-mentioned preparation with 1% inoculum concentration be seeded to equipped with sterilizing 600l Gu cured
In the 1000l fermentation tank of section's streptomycete fermentation culture medium, 28-32 DEG C of temperature control, open and stir 200r/min, first 24 hours, ventilation
For per minute for 200l air, 24-36 hour, ventilation is 400l, and after 36 hours, ventilation is 600l, and 40-48 is little for culture
When, treat that thalline content reaches 60g/l, you can stop tank, that is, obtain Gu Lake streptomycete fermentation liquid；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05
Gram, magnesium sulfate: 0.5 gram, sodium chloride: 0.5 gram, ferrous sulfate: 0.01 gram, agar: 20 grams, 1000ml supplied by distilled water,
Ph7.2～7.4；
Wherein, described Gu Lake streptomycete fermentation culture medium: bean cake 2%, corn starch 2%, stone powder 1%, soy molasses 2%, sulfur
Sour manganese 0.004%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.05%, the condition of each medium sterilization is: 0.10-0.15mpa, 121
DEG C sterilizing 30 minutes；
Step 2: the preparation of Ka Wuer streptomycete fermentation liquid
Take out Ka Wuer streptomyces species preservation pipe, draw flat board with Gause I solid medium and recovered, 30 DEG C of cultures 7
My god, picking single bacterium colony streak inoculation under flat board, equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is being cultivated
Cultivate 6-8 days for 30 DEG C in case, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution that a large amount of spore powders can use 500 milliliters
Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, as Ka Wuer streptomycete seed liquor；
Fermentation: the Ka Wuer streptomycete seed liquor of above-mentioned preparation is seeded to the card 5 of the 600l equipped with sterilizing with 1% inoculum concentration
In the 1000l fermentation tank of your streptomycete fermentation culture medium, 28-30 DEG C of temperature control, first 18 hours, ventilation was per minute empty for 300l
Gas, 18-32 hour, ventilation is 500l, and after 32 hours, ventilation is 700l, opens stirring 200r/min, cultivates 40-48 hour,
Treat that thalline content reaches 80g/l, you can stop tank, that is, obtain Ka Wuer streptomycete fermentation liquid；
Wherein, described Gause I solid medium is identical with the Gause I solid medium in step one；
Wherein, described Ka Wuer streptomycete fermentation culture medium: bean cake 3%, cottonseed meal 1%, corn starch 2%, stone powder 1%, soy sugars
Honey 3%, manganese sulfate 0.002%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.02%, the condition of each medium sterilization is: 0.10-
0.15mpa, 121 DEG C sterilize 30 minutes；
Step 3, the preparation of cold solution sugar fermentation of bacillus liquid
Take out the sugared bacillus cereuss preservation pipe of cold solution, draw flat board respectively with lb solid medium and recovered, cultivate 48 hours for 30 DEG C,
Under flat board, picking single bacterium colony is seeded to equipped with lb solid medium, cultivates 48 hours, use 3000ml in 30 DEG C of incubators
Sterilized water, by the lawn eluting in three Fructus Solani melongenae bottles, is seeded to the 500l equipped with the sugared fermentation of bacillus culture medium of the cold solution of 300l and sends out
In fermentation tank, open stirring 120r/min, ventilation is 200l/min within first 12 hours, after 12 hours, ventilation is 320l/min, 30 DEG C
Culture 26-36 hour, treats that total spore content is not less than 6,000,000,000 cfu/ml, you can as the sugared fermentation of bacillus liquid of cold solution；
Wherein, described lb culture medium: yeast extract 5.0 g, peptone 10.0, nacl10.0g, agar g, water 1000ml,
Ph7.2,；
Wherein, described cold solution sugar fermentation of bacillus culture medium: glucose 8g/l, corn starch 30 g/l, bean cake powder 40 g/
L, magnesium sulfate 0.4 g/l, manganese sulfate 0.5 g/l, potassium dihydrogen phosphate 0.5 g/l, ph7.0；The condition of each medium sterilization is:
0.10-0.15mpa, 121 DEG C sterilize 30 minutes；
Step 4, the preparation of Nissl fermentation of bacillus liquid
Take out Nissl bacillus cereuss preservation pipe, draw flat board respectively with nitrogen-free solid medium and recovered, cultivate 72 hours for 30 DEG C,
Under flat board, picking single bacterium colony is seeded to equipped with nitrogen-free solid medium, cultivates 72 hours in 30 DEG C of incubators, uses
3000ml sterilized water is seeded to the lawn eluting in three Fructus Solani melongenae bottles equipped with 300l Nissl fermentation of bacillus culture medium
In 500l fermentation tank, open stirring 120r/min, ventilation is 100l/min within first 8 hours, after 8-24 hour, ventilation is 200l/
Min, after 24 hours, ventilation is 300l/min, 30 DEG C of culture 32-48 hours, treats that total spore content is not less than 3,000,000,000 cfu/
Ml, you can as Nissl fermentation of bacillus liquid；
Wherein, described nitrogen-free solid medium: sucrose 10g, dipotassium hydrogen phosphate 0.2g, magnesium sulfate 0.2g, sodium chloride 0.2g, sulphuric acid
Calcium 0.1g, Calcium Carbonate 5g, 18g, distilled water 1000ml, ph7.2；
Wherein, described Nissl fermentation of bacillus culture medium: molasses 20g/l, corn starch 10 g/l, cottonseed meal powder 40 g/l, sulfur
Sour magnesium 0.4 g/l, manganese sulfate 0.6g/l, potassium dihydrogen phosphate 0.6 g/l, Calcium Carbonate 10 g/l, ph7.0；Each medium sterilization
Condition is: 0.10-0.15mpa, and 121 DEG C sterilize 30 minutes；
Step 5, the preparation of tuft Trichoderma spp. fermentation liquid
Take out tuft Trichoderma spp. strain preservation pipe, draw flat board with pda solid medium and recovered, cultivate 7 days, under flat board for 30 DEG C
Picking single bacterium colony streak inoculation, equipped with the Fructus Solani melongenae bottle of 150 milliliters of pda solid mediums, cultivates 6-8 for 30 DEG C in incubator
My god, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution eluting that a large amount of spore powders can use 500 milliliters, adjust spore dense
Spend for 0.05 hundred million cfu/ml, as tuft Trichoderma spp. seed liquor；
Fermentation: the tuft Trichoderma spp. seed liquor of above-mentioned preparation is seeded to equipped with 300l tuft Trichoderma spp. fermentation culture with 1% inoculum concentration
In the 500l fermentation tank of base, open stirring 200r/min, ventilation is 120l/min within first 8 hours, and after 8-24 hour, ventilation is
240l/min, after 24 hours, ventilation is 360l/min, 30 DEG C of culture 32-48 hours, treats that thalline content reaches 100g/l, that is,
Tank can be stopped, you can as tuft Trichoderma spp. fermentation liquid；
Wherein, described pda solid medium: Rhizoma Solani tuber osi 200g, sucrose 20g, water 1000ml, agar 20g；
Wherein, described tuft Trichoderma spp. fermentation medium: rapeseed cake 2%, cottonseed meal 2%, corn starch 2%, stone powder 1%, soy molasses 3%,
Manganese sulfate 0.001%, dipotassium hydrogen phosphate 0.04%, magnesium sulfate 0.02%, the condition of each medium sterilization is: 0.10-0.15mpa,
121 DEG C sterilize 30 minutes；
Step 6, mixed fermentation liquid
The bacterium solution of first five step preparation is pressed Gu Lake streptomycete fermentation liquid 10-30 part, Ka Wuer streptomycete fermentation liquid 20-40
Part, cold solution sugar fermentation of bacillus liquid 10-30 part, Nissl fermentation of bacillus liquid 10-30 part, tuft Trichoderma spp. fermentation liquid 10-30
Part mixes, that is, obtain preventing and treating the microbial bacterial agent of Fructus Fortunellae Margaritae yellow twig.
3. a kind of microbial bacterial agent of preventing and treating Fructus Fortunellae Margaritae yellow twig according to claim 1 is it is characterised in that this microbial bacteria
Agent is foliage-spray each 1-2l/ mu in the method for preventing and treating Fructus Fortunellae Margaritae yellow twig, pouring root simultaneously, and consumption is 50-200ml/ fruit tree；
Monthly using once to twice.
4. according to claim 3 a kind of preventing and treating Fructus Fortunellae Margaritae yellow twig microbial bacterial agent using method it is characterised in that
The multiple of the dilution of foliage-spray is 100-200 times.
5. according to claim 3 a kind of preventing and treating Fructus Fortunellae Margaritae yellow twig microbial bacterial agent using method it is characterised in that
The extension rate of pouring root is 100-500 times.
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| CN107027492A (en) * | 2017-05-10 | 2017-08-11 | 徐建斌 | A kind of ecology planting method for preventing and treating yellow twig |
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| CN108220185A (en) * | 2017-12-19 | 2018-06-29 | 华中农业大学 | A kind of application of biological control agent in citrus treelet yellow twig cause of disease is removed |
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| CN105061098A (en) * | 2015-07-27 | 2015-11-18 | 侯期任 | Microbial fertilizer for preventing and controlling citrus Huanglongbing, and preparation method thereof |
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| CN107384841A (en) * | 2017-09-15 | 2017-11-24 | 延边大学 | A kind of microbial inoculum prevented and treated lepidoptera pest and preparation method thereof |
| CN108064891A (en) * | 2017-12-01 | 2018-05-25 | 钟山县德福农产品有限公司 | The pesticide control of Citrus Huanglongbing pathogen |
| CN108220185A (en) * | 2017-12-19 | 2018-06-29 | 华中农业大学 | A kind of application of biological control agent in citrus treelet yellow twig cause of disease is removed |
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| CN108934714A (en) * | 2018-07-31 | 2018-12-07 | 和平县维康农业科技有限公司 | A kind of citrus yellow shoot disease preventing control method |
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Application publication date: 20170125 |