CN106520595B - A kind of arthrobacterium and its application in terms of biological control bacterial wilt of tomato - Google Patents
A kind of arthrobacterium and its application in terms of biological control bacterial wilt of tomato Download PDFInfo
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Abstract
The invention discloses a kind of arthrobacterium and its applications in terms of biological control bacterial wilt of tomato.Wherein the 16SrRNA of arthrobacterium has nucleotide sequence shown in SEQ ID No.1.The invention also discloses the preparation methods of the screening technique of arthrobacterium and antagonism bacterial wilt of tomato microbial inoculum and antagonism bacterial wilt of tomato seedling medium.The present invention passes through to Antagonistic Strains Against Tomato Bacterial Wilt separation screening and its efficiency test, separate the bacterial strain for having antagonism to bacterial wilt of tomato, after first passing through culture breeding, the microbial inoculum product is made, using the mushroom slag after edible fungus culturing by being configured to seedling medium by a certain percentage with other substrates after fermentation maturity, biocontrol agent is directly organically combined with seedling medium, it has abundant available resources, it is low in cost, many advantages, such as Ralstonia solanacearum can be avoided secondary environmental pollution is prevented and treated using it, it is to solve one effective approach of soil-borne disease bacterial wilt of tomato.
Description
Technical field
The present invention relates to bacterial wilt of tomato biological and ecological methods to prevent plant disease, pests, and erosion technical field more particularly to a kind of arthrobacterium (Arthrobacter
Sp.), antagonism bacterial wilt of tomato microbial inoculum, antagonism bacterial wilt of tomato seedling medium and the application in terms of control of plant bacterial wilt.
Background technique
Bacterial wilt of tomato (Bacterial wilt of tomato) is a kind of by green withered Raul Salmonella (Ralstonia
Solanacearum bacillary soil-borne disease caused by), mostly in the torrid zone, semi-tropical countries and regions, such as: South America, Japan,
India, Philippine, the ground such as China Guangdong, Zhejiang, Fujian, Jiangsu, Hubei, Sichuan, Chongqing occur.The disease can cause the big face of plant
Product wilting is dead, and general field disease incidence is 10%~15%, and grave illness field disease incidence is up to 80%~98.5%, and tomato is caused to produce
Degradation is measured, is resulted in significant economic losses.R.solanacearum is a kind of soil inhabitant, can be in no host's situation
Under soil in survive for a long time.R.solanacearum mainly invades plant from root system, can also invade simultaneously from stem's wound
It is directly entered conduit system, mass propagation and generates metabolite wherein, In Water Transportation In Plants is hindered, plant is caused to wither
It is listless.
Currently, lacking effective chemical agent for bacterial wilt of tomato, Ralstonia solanacearum is also easy to produce anti-medicine to chemical pesticide in addition
Property, and largely will cause environmental pollution and production cost rising using chemical pesticide.Biological control crop disease has can benefit
With it is resourceful, low in cost, can be avoided secondary environmental pollution, it is considered to be solve one promising way of soil-borne disease
Diameter.The important component that biological control is biological control is carried out using pathogen antagonistic microbe.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of arthrobacterium (Arthrobacter sp.).
The invention solves another technical problem be to provide the screening technique of arthrobacterium a kind of.
The invention solves there are one technical problems to be to provide the preparation method of antagonism bacterial wilt of tomato microbial inoculum a kind of.
The present invention technical problem also to be solved is to provide a kind of preparation side of antagonism bacterial wilt of tomato seedling medium
Method.
For arthrobacterium, the technical solution adopted by the present invention is that, which is preserved in China typical culture collection
The heart, deposit number are CCTCC No.M2016421, its 16SrRNA has nucleotide sequence shown in SEQ ID No.1.
For the screening technique of arthrobacterium, the technical solution adopted by the present invention is that, comprising the following steps:
(1) casein peptone agar medium is prepared by formula as below, and is sterilized 25 minutes using damp and hot 115.0 DEG C of high pressure,
Obtain bacterium slant medium；
The casein peptone agar medium is by glucose 5.0g, casein hydrolysate 1.0g, bacterium grade peptone
10.0g, 15.0~20.0g of agar and distilled water 1000ml composition, and the pH value for the preceding casein peptone agar medium that sterilizes is 7.0
~7.2；
(2) preparation of soil dilution liquid
A) 10g soil is weighed, 90ml sterile saline is added, vibrates 15 minutes, obtains 10-1Dilution soil liquid A；
B) dilution soil liquid A 1ml is drawn, 9ml sterile saline is added, obtains 10-2Dilution soil liquid B；
C) dilution soil liquid B 1ml is drawn, 9ml sterile saline is added, obtains 10-3Dilution soil liquid C；
D) dilution soil liquid C 1ml is drawn, 9ml sterile saline is added, obtains 10-4Dilution soil liquid D；
E) dilution soil liquid D 1ml is drawn, 9ml sterile saline is added, obtains 10-5Dilution soil liquid E；
F) dilution soil liquid E 1ml is drawn, 9ml sterile saline is added, obtains 10-6Dilution soil liquid F；
(3) dilution soil liquid C, dilution soil liquid D, dilution soil liquid E 0.2ml are drawn respectively, are added to junket egg
It is coated culture in white peptone agar medium plate, and is cultivated 2~3 days at 25~28 DEG C；
(4) it selects the culture dish of 20~200 clump counts to carry out picking colony and carries out inclined-plane culture, condition of culture is 25~28
It is cultivated 2~3 days at DEG C；
(5) the isolated arthrobacterium.
For the preparation method of antagonism bacterial wilt of tomato microbial inoculum, the technical solution adopted by the present invention is that, comprising the following steps:
(1) inorganic salts calcium, magnesium, phosphorus is added with casein peptone culture medium in the arthrobacterium screened such as claim 2
Carry out liquid shake-flask fermentation, 25~35 DEG C of cultures, 120~220rpm of revolving speed；Finally obtain strain；
(2) liquid parent species, the original of the liquid fermentation tank are made with the obtained strain inoculation liquid fermentation tank of step (1)
Material is that wheat bran, rapeseed cake and beancake powder difference by weight percentage 1%~5%: 0~3%: 0~3% and inorganic salts calcium, magnesium, phosphorus stir
Mix mixing, amount of water is 99~89%, after high pressure sterilization, when fermentor fluid temperature is cooled to 35 DEG C or less, then press 1.0~
10% inoculum concentration is inoculated in the culture medium of liquid fermentation tank in a manner of sterile working, and liquid fermentation tank remains
The positive pressure of 0.01MPa or more, 25~35 DEG C of ventilations are cultivated 24~48 hours, and fermentation bacterium number is up to 108Cfu/ml or more, it is sterile
It is packaged into microbial inoculum.
Preferably, the inorganic salts dosage that casein peptone culture medium is added in step (1) are as follows: 0.9~1.5g/l of calcium, magnesium
0.5~1.3g/l, 0.8~2.5g/l of phosphorus；
The inorganic salts dosage being added in fermentor described in step (2) are as follows: 0.9~1.5g/l of calcium, 0.5~1.3g/l of magnesium,
0.8~2.5g/l of phosphorus.
For the preparation method of antagonism bacterial wilt of tomato seedling medium, the technical solution adopted by the present invention is that, including it is following
Step:
(1) sawdust 20%, cotton seed hulls 70% and auxiliary material 10% is taken to be configured to mushroom culture medium and upper by mass percentage
Face cultivated mushroom, then by the particle that the mushroom culture medium after cultivated mushroom is crushed to 2~3cm be mushroom slag；The auxiliary material
By wheat bran, lime, gypsum, 8:1:1 is formulated by mass percentage；
(2) harmless treatment of mushroom slag: nitrogen source is added in the mushroom slag with compost principle, carbon-nitrogen ratio is adjusted, adds water tune
Whole mushroom slag moisture content is 55~65%, by 2kg/m3Compost fermenting agent is added, heap is done, by 55~65 DEG C of pyroprocess
It 30~35 days, is mixed therebetween by 3~5 turnings, obtains the decomposed material of mushroom slag；And keep the decomposed material water content of mushroom slag 55~
60%；
(3) preparation of antagonism bacterial wilt of tomato seedling medium: the decomposed material of mushroom slag, turf, vermiculite and perlite are pressed respectively
The ratio uniform of percent by volume 45%, 35%, 13%, 7% mixes, by 3kg/m3Antagonism obtained by claim 3 is added
Bacterial wilt of tomato microbial inoculum is simultaneously uniformly mixed, then the moisture content for adjusting matrix with tap water makes in matrix to 30%~40% containing short of money
Antibacterial number is up to 107Cfu/g or more obtains antagonism bacterial wilt of tomato seedling medium.
Preferably, mushroom slag is the mushroom culture medium planted after mushroom or needle mushroom.
In addition, application of the above-mentioned arthrobacterium in terms of control of plant bacterial wilt.
The beneficial effects of the present invention are:
Arthrobacterium of the present invention (Arthrobacter sp.) is selected from nature directional separation, passes through plate antagonistic experiment
It has been shown that, which has the good ability for inhibiting Ralstonia solanacearum growth, while the bacterial strain can be in heavy metal tolerance cadmium ion
(200ug/ml) illustrates that the bacterial strain can survive in cadmium ion contaminated soil.
The present invention lacks effective chemical agent for bacterial wilt of tomato, and medication easily causes Ralstonia solanacearum to generate anti-medicine repeatedly
The problem of property.By the way that Antagonistic Strains Against Tomato Bacterial Wilt separation screening and its efficiency test, separating has bacterial wilt of tomato
The bacterial strain of antagonism is made the microbial inoculum product, passes through fermentation using the mushroom slag after edible fungus culturing after first passing through culture breeding
After decomposed and other substrates are configured to seedling medium by a certain percentage, and biocontrol agent has directly organically been combined with seedling medium
Come, it has abundant available resources, low in cost, use
Biomaterial preservation
Arthrobacterium (Arthrobacter sp.) SJN-5 (hereinafter referred to as SJN-5 bacterial strain), the bacterium in April, 2014 extremely
In October, from Anhui Province various regions, pedotheque is isolated in aforementioned manners.It is identified through sequencing, ultimate analysis determines, which is
Arthrobacterium (Arthrobacter sp.).The new strains are on the August 10th, 2016 of Wuhan in the Wuhan University of Wuhan City, Hubei Province
University collection China typical culture collection center (CCTCC) preservation, deposit number are CCTCC NO.M2016421.
Specific embodiment
Embodiment 1: the acquisition of arthrobacterium (Arthrobacter sp.) SJN-5 bacterial strain
Present embodiments provide a kind of bacterial strain using pathogen antagonistic microbe control of plant bacterial wilt, specifically pole
Bacterium (Arthrobacter sp.) SJN-5.2014~2015 years experimenters are from Guichi, Anhui, Mount Huang, Shucheng County, Shexian County, Jixi etc.
Different regions acquire the pedotheque on continuous cropping tomato ground, and using bacteria culture media, using isolation by dilution method, therefrom directional separation is each out
More than totally 100 plants of class bacterium etc., and bacterial strain purifying is carried out, and then inclined-plane and shaking flask are passed through under 28~30 DEG C of condition of culture to bacterial strain
It cultivates and is measured in a variety of seedling medium effect of inoculation, be screened out from it the arthrobacterium SJN-5 of antagonism ralstonia solanacearum of tomato.
1 culture medium
Casein peptone agar medium: glucose 5.0g, casein hydrolysate 1.0g, bacterium grade peptone 10.0g, agar
15.0~20.0g, distilled water 1000ml, pH value are 7.0~7.2.It is sterilized 25 minutes using damp and hot 115.0 DEG C of high pressure.
2 test procedures
The preparation of 2.1 culture mediums
The casein peptone agar medium as described in 1 is prepared, bacterium slant medium is prepared.
The preparation of 2.2 soil dilution liquid
(1) 10g pedotheque is weighed, is added in 90ml sterile water 250ml triangular flask, vibrates 15 minutes, obtains 10-1It is dilute
Release soil liquid A；
(2) dilution soil liquid A 1ml is drawn, is added in 9ml sterile water 50ml triangular flask, obtains 10-2Dilution soil
Solution B；
(3) dilution soil liquid B 1ml is drawn, is added in 9ml sterile water 50ml triangular flask, obtains 10-3Dilution soil
Solution C；
(4) dilution soil liquid C 1ml is drawn, is added in 9ml sterile water 50ml triangular flask, obtains 10-4Dilution soil
Solution D；
(5) dilution soil liquid D 1ml is drawn, is added in 9ml sterile water 50ml triangular flask, obtains 10-5Dilution soil
Solution E；
(6) dilution soil liquid E 1ml is drawn, is added in 9ml sterile water 50ml triangular flask, obtains 10-6Dilution soil
Solution F.
The separation of 2.3 bacteriums
(7) dilution soil liquid C, dilution soil liquid D, dilution each 0.2ml of soil liquid E are drawn respectively, are added to junket
It is coated culture in peptone agar medium plate, and is cultivated 2~3 days at 28-30 DEG C.
The step of above-mentioned bacterium separation, is repeated 3 times, synthesis result, bacterium.The way is more in order to obtain
Bacterial strain, at the same verify each experiment screening obtain bacterial strain whether quantity identical or gap size, final choice screening effect
Good and strain activity is higher to be used as aimed strain.
(8) it selects the culture dish of 20~200 clump counts to carry out picking colony and carries out inclined-plane culture, condition of culture is 28~30
It is cultivated 2~3 days at DEG C；
A pedotheque more than 2.4
(9) 10 pedotheques are taken altogether, and each pedotheque repeats above-mentioned 2.1~2.3 step.
2.5 result
More than 100 plants of isolated bacterium according to the above method.
Embodiment 2: plate antagonistic effect of the isolated bacterium to bacterial wilt of tomato eggplant Ralstonia solanacarum
1. culture medium
1.1 solid medium
The preparation of ralstonia solanacearum culture medium: potassium dihydrogen phosphate 0.2g, dipotassium hydrogen phosphate 0.8g, yeast extract 5.0g, junket
Protolysate 1.0g, glucose 5.0g, agar 15.0g, distilled water 1000ml, pH 7.0~7.2.It is damp and hot using high pressure
115.0 DEG C sterilize 25 minutes.
The preparation of casein peptone agar medium: with the casein peptone agar medium in embodiment 1.
1.2 fluid nutrient medium
Using casein peptone agar medium but agar is not added, and arthrobacterium and bacterial wilt opportunistic pathogen can be in the liquid
It is grown on body culture medium.
2, test procedure
The preparation of 2.1 culture mediums
Ralstonia solanacearum culture medium and fluid nutrient medium are prepared respectively by above-mentioned formula, prepare pathogen eggplant Ralstonia solanacarum
Inclined-plane；
Casein peptone agar medium and fluid nutrient medium are prepared respectively by above-mentioned formula, prepare bacterium inclined-plane.
2.2 plate antagonistic effects
2.2.1 actication of culture
Ralstonia solanacearum of tomato and bacterium carry out actication of culture: ralstonia solanacearum of tomato and separation of bacterial are switched to casein
Peptone agar medium inclined-plane, is cultivated 16~28 hours by 28~30 DEG C, spare.
2.2.2 preparing ralstonia solanacearum plate
Will activation ralstonia solanacearum access fluid nutrient medium in carry out shaking flask culture, 25~28 DEG C, 180 revs/min.Every other day with
Ralstonia solanacearum culture medium inverted plate, (dilute bacterial wilt opportunistic pathogen concentration is addition 0.2ml in each plate after waiting culture mediums to solidify
103~104Cells/ml) ralstonia solanacearum fermentation liquid (culture about 36 hours), even spread, 25~28 DEG C of overnight incubations are spare.
2.2.3 preparing bacterial liquid fermentation liquid
After ralstonia solanacearum liquid medium within shaking flask culture 2 days, activation separation of bacterial transfer again in fluid nutrient medium into
Row shaking flask culture, it is 25~28 DEG C, 180 revs/min, spare every other day (culture about 36 hours).
2.2.4 plate antagonistic effect
Separation of bacterial fermentation liquid is dipped with sterilizing filter paper piece (diameter 9mm) and is put into ralstonia solanacearum plate, and each plate puts 3
The different antagonistic bacteriums of strain, 3 repetitions.20 hours or so observation experiments as a result, mainly investigate inhibition zone whether there is or not and size.
3, result
3 plants of the preferable bacterium of antagonistic effect is obtained by plate antagonistic effect, bacterial strain SJN-5 is exactly wherein one plant.Experiment
0.5ml Ralstonia solanacarum fermentation liquid is applied on plate for elder generation, then bacterial strain SJN-5 fermentation liquid is dipped with filter paper and is placed on plate
On, while cultivating and obtaining.
By observation pole bacterial strain SJN-5 to the antagonistic effect of Ralstonia solanacarum, it can be seen that inhibition zone is obvious, explanation
The bacterial strain SJN-5 speed of growth is fast, and diffusivity is strong, has significantly antagonism to eggplant Ralstonia solanacarum.
Arthrobacterium (Arthrobacter sp.) SJN-5 (hereinafter referred to as SJN-5 bacterial strain), the bacterium in April, 2014 extremely
It is isolated in aforementioned manners from Anhui Province various regions October.It is sequenced and identifies through fruit, ultimate analysis determines, which is arthrobacterium
(Arthrobacter sp.).The new strains were on August 10th, the 2016 Wuhan University guarantors in the Wuhan University of Wuhan City, Hubei Province
Hiding center China typical culture collection center (CCTCC) preservation, deposit number are CCTCC NO.M2016421.
Embodiment 3: the preparation of the drip irrigation of bacterial strain SJN-5
The microbial bacterial agent with antagonism bacterial wilt of tomato of the present embodiment is by arthrobacterium (Arthrobacter sp.)
SJN-5 bacterial strain is cultivated, is bred.
Specific operation process is as follows: arthrobacterium SJN-5 be added based on casein peptone culture medium inorganic salts calcium 0.9~
1.5g/l, 0.5~1.3g/l of magnesium, 0.8~2.5g/l of phosphorus carry out liquid shake-flask fermentation, and 25~35 DEG C are cultivated, and revolving speed 120~
220rpm.Liquid parent species are made as strain inoculation liquid fermentation tank, liquid fermentation tank raw material is wheat bran, rapeseed cake and soya-bean cake
Powder by weight percentage 1%~5%: 0~3%: 0~3%: 0~3%) and a small amount of inorganic salts (0.9~1.5g/l of calcium, magnesium
0.5~1.3g/l, 0.8~2.5g/l of phosphorus), amount of water is 99~89%, after high pressure sterilization, is cooled to fermentor fluid temperature
It at 35 DEG C or less, then is inoculated in a manner of sterile working in the culture medium of liquid fermentation tank by 1.0~10% inoculum concentration, liquid
Fermentor remains the positive pressure of 0.01MPa or more, and 25~35 DEG C of ventilations are cultivated 24~48 hours, and fermentation bacterium number is up to 108cfu/
Ml or more, at microbial inoculum, packaged form is packed or bottled for aseptic packaging.
Bacterium number in the microbial inoculum made of arthrobacterium SJN-5 is up to 108When cfu/ml, microbial inoculum is applied in seedling medium i.e.
It can achieve the effect of effectively preventing bacterial wilt of tomato, this 108Cfu/ml be as in fermenting agent bacterium number it is minimum critical
It is worth, more than this numerical value can achieve the effect of control of plant bacterial wilt, but because during specific test in microbial inoculum
Bacterium number of fermenting is it is not possible that unconfined increase, so bacterium number of fermenting in the specific application process is there are the upper limit, lower limit is sent out
Yeast-like fungi number is 108cfu/ml。
Embodiment 4: the preparation of antagonism bacterial wilt of tomato seedling medium
Mushroom slag is first made by the following method: sawdust 20%, cotton seed hulls 70% and auxiliary material 10% being taken to prepare by mass percentage
At mushroom culture medium, wherein by wheat bran, lime, gypsum, 8:1:1 is formulated auxiliary material by mass percentage.Again in mushroom culture medium
Mushroom or needle mushroom are planted above, and the mushroom culture medium after plantation mushroom or needle mushroom is crushed to the particle of 2~3cm i.e.
Obtain mushroom slag.
To mushroom slag carry out harmless treatment obtain the decomposed material of mushroom slag: with compost fermentation principle by mushroom slag be added 0.8~
1.2kg/m3For urea as nitrogen source, adding water adjustment mushroom slag water content is between 55~65%, by 2kg/m3Compost fermenting is added
Microbial inoculum is cooked heap, the high 100~150cm of heap, 200~300cm of diameter, by pyroprocess 30~35 days of 55~65 DEG C, at it
Between by 3~5 times turning mix, obtain the decomposed material of mushroom slag, and keep the decomposed material water content of mushroom slag 55~60%.Mushroom slag warp
After crossing biofermentation pyrolytic, potential cause of disease miscellaneous bacteria, harmful organism and noxious material are killed or are eliminated in mushroom slag, are reached
To the effect of the decomposed material physicochemical character stable homogeneous of mushroom slag.
The preparation of antagonism bacterial wilt of tomato seedling medium: the decomposed material of mushroom slag, turf, vermiculite and perlite are pressed into volume
45%, 35%, 13%, 7% ratio is prepared matrix and is uniformly mixed, then green with the obtained antagonism tomato of claim 3
Blight microbial inoculum, by 3kg/m3It is uniformly mixed, and the moisture content for adjusting matrix with tap water makes in matrix to 30%~40% containing short of money
Antibacterial SJN-5 number is up to 107Cfu/g or more can be packed.Bacterium number containing antagonism is up to 10 in matrix7The numberical range lower limit of cfu/g or more
It is 107Cfu/g, the upper limit be also due to specifically testing process existing for, explanation is same as above.
The basic physical and chemical of seedling medium after preparation is shown in Table 1:
Table 1:
| Total porosity | 60~82.6 |
| Aeration porosity | 14~18.5 |
| Water holding hole | 55~66 |
| Bulk density (g/cm3) | 0.25 |
| Conductivity (ms/cm) | 0.19~0.22 |
| Full nitrogen (%) | 0.28~0.33 |
| Available nitrogen (g/kg) | 2.1 |
| Rapid available phosphorus (g/kg) | 5.5 |
| Available potassium (g/kg) | 13.4 |
| Organic carbon (%) | 22~36 |
| PH value | 6.3~7.0 |
Tomato seedling experimental observation is carried out by the antagonism bacterial wilt of tomato seedling medium of the present embodiment configuration to show, preparation
Seedling medium is better than the seedling medium of conventional formulation.
Embodiment 5: the mushroom slag seedling medium of the antagonism bacterial wilt of tomato containing SJN-5 microbial inoculum is withered to tomato blueness in potting
The test of sick biocontrol effect
Steps are as follows for specific experiment:
1. nursery
Antagonistic Fungi seedling medium are as follows: by the decomposed material of mushroom slag, turf, vermiculite and perlite by volume 45%, 35%, 13%,
7% ratio is prepared matrix and is uniformly mixed, and the obtained antagonism bacterial wilt of tomato microbial inoculum of claim 3 is then used, by 3kg/
m3It is uniformly mixed, and the moisture content for adjusting matrix with tap water reaches the number of SJN-5 containing Antagonistic Fungi in matrix to 30%~40%
107Cfu/g or more, it is spare.
Routine control matrix are as follows: by the decomposed material of mushroom slag, turf, vermiculite and perlite by percent by volume 45%, 35%,
13%, 7% ratio is prepared matrix and is uniformly mixed, and the moisture content of tap water adjusting matrix is spare to 30%~40%.
2. sowing
It is respectively charged into two kinds of matrix of the above-mentioned preparation of 3/4ths high left and right in seedling culture hole plate (50 hole) every cave, uses hand
Light pressure, is then sown into 1~2 tomato seeds, then covered with matrix, and cover hole tray with plastic film, prevents moisture in every cave
Evaporation, it is spare.
3. emergence
Ready sowing hole tray is put into greenhouse, 25 DEG C of temperature, maintain day and night the temperature difference at 10 DEG C or more.About 4
It emerges within~5 days, takes plastic film after emergence off, culture is spare to 3~4 true leaves (about 3~4 weeks) in hole tray.
4. ralstonia solanacearum Lei Er Salmonella fermentation liquid prepares
Then Ralstonia solanacearum activation connects liquid submerged culture for every plant, 25~28 DEG C, 180 revs/min, cultivates about 36
Hour, it is spare.
5. potting
Using the serious plot soil of tomato planting base bacterial wilt morbidity, every basin (320 × 260mm of specification) weighs 3.0
Kilogram soil, be uniformly mixed 2% organic fertilizer, be fitted into basin.It is moved in all basins and cuts out tomato seedling band root matrix transplanting, every basin
5 plants of tomato seedlings of middle transplanting, appropriate moisturizing as needed.This test sets two controls and a processing, 5 basins of each processing, every basin 5
Young plant is uniformly distributed.Control 1: any microbial inoculum is not added with control matrix seedling when transplanting；Control 2: 20ml blueness is only poured for every plant when transplanting
Withered bacterium dilutes fermentation liquid (105cfu/ml)；Antagonistic Fungi matrix treatments: every plant of root when being transplanted when transplanting with Antagonistic Fungi substrate seedling
First pour 20ml Ralstonia solanacearum dilution fermentation liquid (105Cfu/ml), the strain number of record morbidity daily.
6. interpretation of result
By potted plant experiment the results show that the tomato plant for not being inoculated with pathogen does not occur disease plant；It is inoculated with bacterial wilt
Opportunistic pathogen disease incidence reaches 60% or more；Delay tomato blueness withered using the processing with antibacterial substrate seedling that SJN-5 bacterial strain is handled
The incidence of disease is reduced to 35% by the generation of disease, illustrates that the seedling medium cultivates processing to bacterial wilt of tomato with anti-
Effect.Consider amount of survival of the bacterial strain in substrate seedling simultaneously, it is proposed that using with bacterial strain SJN-5 connect antibacterial substrate seedling into
When row bacterial wilt prevents and treats, until tomato seedling is grown to 20~25cm high, and the benefit bacterial strain SJN-5 preparation before colonizing, simultaneously
In conjunction with plantation precautionary measures, reinforcing effect.
It is inoculated with the microbial inoculum SJN-5 of antagonism ralstonia solanacearum using mushroom slag as the seedling medium that primary raw material is prepared, cultivates tomato and educates
Transplanting Test is carried out after seedling, compared with not connecing Antagonistic Fungi and being only inoculated with the control of ralstonia solanacearum, bacterial wilt disease incidence is reduced to 35%
Hereinafter, and occurrent time postponement 10 days or more.
To sum up, the present embodiment has the effect that
First is that being applied to the production of seedling medium using the pole bacteria agent of antagonism bacterial wilt of tomato, sent out using solid dung
The form of ferment improves the utilization rate of production mushroom waste, reduces the production cost of nursery, then compound to be configured to antagonism
The seedling medium of soil-borne disease bacterial wilt of tomato.
Second is that reducing the secondary pollution of environment by the harmless treatment of the agricultural wastes such as mushroom slag, there is preferable warp
The effect of Ji, prevention and treatment environmental pollution and protection soil-borne disease bacterial wilt of tomato.
The present embodiment screening obtains the pole bacterial strain with antagonism bacterial wilt opportunistic pathogen, and can become the waste of Mushroom production
To recycle resource, compounding forms the commodity seedling medium with antagonism soil-borne disease, which is suitable for scale agricultural production,
Agricultural benefit can be improved.
The embodiments of the present invention described above are not intended to limit the scope of the present invention.It is any in the present invention
Spirit and principle within made modifications, equivalent substitutions and improvements etc., should be included in claim protection model of the invention
Within enclosing.
Claims (6)
1. a kind of arthrobacterium is preserved in China typical culture collection center, deposit number is CCTCC No.M2016421,
It is characterized in that, the 16SrRNA of the arthrobacterium has nucleotide sequence shown in SEQ ID No.1.
2. a kind of preparation method of antagonism bacterial wilt of tomato microbial inoculum, which comprises the following steps: (1) by claim 1
In arthrobacterium with casein peptone culture medium be added inorganic salts calcium, magnesium, phosphorus carry out liquid shake-flask fermentation, 25~35 DEG C culture, turn
120~220rpm of speed；Finally obtain strain；
(2) liquid parent species are made with the obtained strain inoculation liquid fermentation tank of step (1), the raw material of the liquid fermentation tank is
Wheat bran, rapeseed cake and beancake powder difference by weight percentage 1%~5%: 0~3%: 0~3% and inorganic salts calcium, magnesium, phosphorus stir mixed
It closes, amount of water is 99~89%, after high pressure sterilization, when fermentor fluid temperature is cooled to 35 DEG C or less, then presses 1.0~10%
Inoculum concentration be inoculated in a manner of sterile working in the culture medium of liquid fermentation tank, liquid fermentation tank remain 0.01MPa with
On positive pressure, 25~35 DEG C of ventilations cultivate 24~48 hours, and fermentation bacterium number is up to 108Cfu/ml or more, aseptic packaging is at bacterium
Agent.
3. the preparation method of antagonism bacterial wilt of tomato microbial inoculum according to claim 2, which is characterized in that described in step (1)
The inorganic salts dosage that casein peptone culture medium is added are as follows: 0.9~1.5g/l of calcium, 0.5~1.3g/l of magnesium, 0.8~2.5g/l of phosphorus；Step
Suddenly the inorganic salts dosage being added in fermentor described in (2) are as follows: 0.9~1.5g/l of calcium, 0.5~1.3g/l of magnesium, phosphorus 0.8~
2.5g/l。
4. a kind of preparation method of antagonism bacterial wilt of tomato seedling medium, which comprises the following steps:
(1) mushroom slag makes: sawdust 20%, cotton seed hulls 70% and auxiliary material 10% being taken to be configured to mushroom culture medium simultaneously by mass percentage
It is mushroom slag in cultivated mushroom above, then by the particle that the mushroom culture medium after cultivated mushroom is crushed to 2~3cm；It is described
By wheat bran, lime, gypsum, 8:1:1 is formulated auxiliary material by mass percentage；
(2) harmless treatment of mushroom slag: nitrogen source is added in the mushroom slag with compost principle, carbon-nitrogen ratio is adjusted, water is added to adjust mushroom
Slag moisture content is 55~65%, by 2kg/m3Be added compost fermenting agent, do heap, by 55~65 DEG C of pyroprocesses 30~
It 35 days, is mixed therebetween by 3~5 turnings, obtains the decomposed material of mushroom slag；And keep the decomposed material water content of mushroom slag 55~
60%；
(3) the decomposed material of mushroom slag, turf, vermiculite and perlite the preparation of antagonism bacterial wilt of tomato seedling medium: are pressed into volume respectively
The ratio uniform of percentage 45%, 35%, 13%, 7% mixes, by 3kg/m3Antagonism tomato obtained by claim 3 is added
Bacterial wilt microbial inoculum is simultaneously uniformly mixed, then the moisture content for adjusting matrix with tap water makes in matrix to 30%~40% containing Antagonistic Fungi
It counts up to 107Cfu/g or more obtains antagonism bacterial wilt of tomato seedling medium.
5. preparation method as claimed in claim 4, which is characterized in that the mushroom slag is the mushroom planted after mushroom or needle mushroom
Culture medium.
6. application of the arthrobacterium as described in claim 1 in terms of control of plant bacterial wilt.
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| CN110627537A (en) * | 2018-06-21 | 2019-12-31 | 福建三炬生物科技股份有限公司 | Multifunctional microbial fertilizer and preparation method thereof |
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| CN1616358A (en) * | 2003-11-13 | 2005-05-18 | 栾兴社 | Method for preparing biological flocculant from arthrobacter |
| CN101914449A (en) * | 2010-02-08 | 2010-12-15 | 浙江省农业科学院 | Fungal inoculant for controlling bacterial wilt of tomato and preparation method thereof |
| CN102643763A (en) * | 2012-01-10 | 2012-08-22 | 浙江省农业科学院 | Tomato bacterial wilt antagonist and application thereof |
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| CN1616358A (en) * | 2003-11-13 | 2005-05-18 | 栾兴社 | Method for preparing biological flocculant from arthrobacter |
| CN101914449A (en) * | 2010-02-08 | 2010-12-15 | 浙江省农业科学院 | Fungal inoculant for controlling bacterial wilt of tomato and preparation method thereof |
| CN102643763A (en) * | 2012-01-10 | 2012-08-22 | 浙江省农业科学院 | Tomato bacterial wilt antagonist and application thereof |
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