CN104818216B - One plant is used to prevent and treat tomato and the Paecilomyces lilacinus of grape root knot nematode disease evil - Google Patents
One plant is used to prevent and treat tomato and the Paecilomyces lilacinus of grape root knot nematode disease evil Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/79—Paecilomyces
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
Abstract
It is used to prevent and treat tomato and the Paecilomyces lilacinus of grape root knot nematode disease the invention discloses one plant.Paecilomyces lilacinus provided by the present invention is the Paecilomyces lilacinus YT08, and it is CGMCC No. 10026 in the numbering of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation date is on November 26th, 2014.Paecilomyces lilacinus YT08 CGMCC No. 10026 provided by the invention have strong prevention effect to tomato and grape root-knot nematode, can be developed into the novel biocontrol agents for agriculture root knot nematode disease.
Description
Technical field
It is used to prevent and treat tomato and the Paecilomyces lilacinus of grape root knot nematode disease evil the present invention relates to one plant（Paecilomyces lilacinus）, belong to microbial technology field.
Background technology
Nemas is one of four big pathogenic organisms of crops, vegetables, fruit tree, forest, flowers and medicinal material plantation, its
Harm is only second to fungi, more than bacterium and virus.Plant nematode causes the loss about 100,000,000,000 that world agriculture produces every year
Dollar, wherein a kind of plant nematode for endangering maximum is root-knot nematode.
The preventing and treating of plant nematode mainly has chemical nematicides, cultivation technique, cultivates resistant variety and biological pesticide.
Due to root-knot nematode generally survive in soil and plant roots in, the still chemical pesticide without special efficacy, can only be controlled in a short time at present
Nematodiasis processed occurs, therefore chemical pesticide is difficult to control its harm, and due to potential environmental problem, is related to human and animal
Health, the use of chemical nematicides are very restricted.In addition cultivation technique（Such as crop rotation）Although, cultivate resistant variety etc.
Certain preventive and therapeutic effect can be played, but limitation is larger in actual application.Therefore, as safe and effective preventing and treating
The application of the method for root-knot nematode disease, biological prevention and its formulation products in plant nematode diseases preventing and treating just seems outstanding
To be important.
Paecilomyces lilacinus belongs to Deuteromycotina, and clump stalk embraces mesh, Cong Gengbao sections, paecilomyces, can parasitize nematode extensively
Ovum, insecticidal effect is efficient, has the characteristics that safe efficient, the lasting period is long, it is considered to be the most nematode biological and ecological methods to prevent plant disease, pests, and erosion of application prospect
Fungi.Paecilomyces lilacinus in addition to nematode effect is killed, can with parasitic Semiptera, Homoptera, Isoptera and coleoptera and
The insects such as Lepidoptera, to phytopathogen（Such as corn southern leaf blight, wheat scab, cucumber anthracnose, cotton wilt and water
Bakanae disease of rice etc.）With antagonism.The secondary metabolites of Paecilomyces lilacinus also have certain physiological potency, such as produce similar Yin
Indolylbutyric acid product, promote root system of plant and plant strain growth, there is facilitation to Seed Germination and growth, moreover it is possible to produce a variety of enzymes
And there is certain chemical pesticide degradation effect.
Paecilomyces lilacinus has infecting potential to root-knot nematode, but due to the difference of bion characteristic, causes different next
Source Paecilomyces lilacinus is variant to the prevention effect of root-knot nematode, it is therefore desirable to which constantly separation has higher cause a disease to root-knot nematode
The bacterial strain of property.
The content of the invention
It is used to prevent and treat tomato and the Paecilomyces lilacinus of grape root knot nematode disease evil the invention provides one plant, to tomato and Portugal
Grape root knot nematode disease has strong pathogenic, can be developed into the novel biocontrol agents for agriculture root knot nematode disease.
It is Paecilomyces lilacinus that one plant, which is used to prevent and treat tomato and the Paecilomyces lilacinus of grape root knot nematode disease evil, in the present invention
（Paecilomyces lilacinus）YT08, it is in China Committee for Culture Collection of Microorganisms's common micro-organisms center
Numbering of registering on the books is CGMCC No. 10026, and the preservation time is on November 26th, 2014.
Paecilomyces lilacinus YT08 of the present invention is isolated from Yantai City sea-farming sea-tangle epidermis, and the bacterial strain is trained in separation
Support and cultivated on base, bacterium colony initial stage is white and mycelia densification, as incubation time extends, gradually becomes lavender, bacterium colony and training
Foster base is completely embedded, and is not easy picking.The bacterial strain produces conidium, and spore is oval, and conidiophore is in broom shape.
Paecilomyces lilacinus YT08 of the present invention is insensitive to chloramphenicol, and nutritional requirement is low, raw on brewex's grains solid medium
It is long good, and can normally be cultivated at 20 DEG C -35 DEG C.
Paecilomyces lilacinus YT08 of the present invention has strong prevention effect to tomato and grape root knot nematode disease, right in pot experiment
The prevention effect of tomato root-knot eelworm is 89.69%, and the prevention effect to grape root-knot nematode is 93.42%.
Paecilomyces lilacinus YT08 of the present invention belongs to paecilomyces（Paecilomyces）Paecilomyces lilacinus kind
（Paecilomyces lilacinus）.
Biomaterial information：
Classification And Nomenclature：Paecilomyces lilacinus（Paecilomyces lilacinus）.
Strain number：YT08.
Preservation mechanism：China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Preservation mechanism is referred to as：CGMCC.
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Preservation date：On November 26th, 2014.
Collection is registered on the books numbering：CGMCC No. 10026.
Brief description of the drawings
Fig. 1 is cultural characteristics of the Paecilomyces lilacinus YT08 on isolation medium；Fig. 2 is the micro- of Paecilomyces lilacinus YT08
Mirror morphologic observation feature.
Embodiment
The present invention is described further with reference to specific embodiment, these embodiments are used to understanding rather than limiting this
Invention.
One plant is used to prevent and treat tomato and the Paecilomyces lilacinus of grape root knot nematode disease evil, and strain is named as YT08, classification life
Name：Paecilomyces lilacinus（Paecilomyces lilacinus）, its deposit number：CGMCC No. 10026, depositary institution：In
State's Microbiological Culture Collection administration committee common micro-organisms center, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, preservation date：On November 26th, 2014.
Embodiment 1：Bacterial strain YT08 separation screening
Paecilomyces lilacinus YT08 is isolated in the sea-tangle epidermis by the Yantai City's sea-farming of Shandong Province of China province.Separation screening
Follow the steps below：By the sea-tangle sample of collection according to 1:100 ratio adds sterilized water dilution, is shaken in 28 DEG C of constant temperature
Shake 30 minutes, be coated with superclean bench after diluting step by step on screening and culturing medium flat board, 28 DEG C of constant-temperature table in bed
To single bacterium colony is grown, picking single bacterium colony carries out repeatedly line on isolation medium flat board and isolated and purified, through microscope within 4-6 days
Microscopy is rose-bengal solid slope culture medium of after pure culture, transferring, and 28 DEG C of constant-temperature table culture is placed at 4 DEG C for 4-6 days to be protected
Deposit, as Paecilomyces lilacinus YT08.
Screening and culturing medium component used is as follows：The g/L of ammonium sulfate 10, the g/L of dipotassium hydrogen phosphate 2, the g/L of sodium chloride 15, seven water
5.8,121 DEG C of the g/L of magnesium sulfate 1, the g/L of colloid chitosan 100, agar 20 g/L, pH sterilizes 20 minutes.
Isolation medium component used is as follows：The g/L of glucose 30, the g/L of sodium nitrate 2, the g/L of dipotassium hydrogen phosphate 1, sulphur
The sour g/L of iron 0.01, the g/L of potassium chloride 0.5, the g/L of epsom salt 0.5, the g/L of colloid chitosan 6, agar 20 g/L, pH
6.5,121 DEG C sterilize 20 minutes.
The rose bengal medium component of culture presevation is as follows：The g/L of peptone 5, the g/L of glucose 10, di(2-ethylhexyl)phosphate
The g/L of hydrogen potassium 1, the g/L of epsom salt 0.5, the g/L of agar powder 20, the g/L of rose-bengal 0.0133, the g/ of chloramphenicol 0.1
6.5,121 DEG C of L, pH sterilizes 20 minutes.
Embodiment 2：Bacterial strain YT08 identification of bacteria
1. bacterial strain YT08 morphological feature：By the bacterial strain YT08 streak inoculations of above-mentioned separation screening on isolation medium,
28 DEG C of constant-temperature table culture, routine observation colony growth state.Bacterial strain is inoculated with another isolation medium using rubbing method,
Simultaneously by the cover glass after sterilizing（1cm×1cm）Oblique cutting enters in culture medium, is placed in cultivate 6 days in 28 DEG C of constant incubator and treats
After mycelia is climbed on cover glass, mycelia and spore shape that cover glass is placed in optical microphotograph Microscopic observation bacterial strain YT08 are taken out.
Bacterial strain YT08 is white in Initial stage of culture colony colour, and color is gradually deepened after extending with incubation time, is produced
Colony colour is shown as lavender after spore.Fig. 1 show the spy of the form after bacterial strain YT08 is cultivated 6 days on isolation medium
Sign, it is circular that bacterium colony is presented crowned, bacterium colony is completely embedded with culture medium, is not easy picking, mycelia densification and to external diffusion, color
For lavender, white is around shown slightly, does not secrete soluble pigment.The bacterial strain YT08 morphological features observed under microscope are shown in Fig. 2,
Its conidiophore is in broom shape, and spore is oval, and bunchiness is in the chain of not branch, and substrate mycelium is in light brown.
2. bacterial strain YT08 18S rDNA sequence analyses
Bacterial strain YT08 STb gene is extracted, using fungi 18S rDNA universal primer（NS1-5’-
GTAGTCATATGCTTGTCTC-3 ', NS6-5 '-GCATCACAGACCTGTTATTGCCTC-3 '）Enter performing PCR amplification, PCR primer
Through agarose gel electrophoresis, the DNA purpose bands as needed for the cutting of PCR primer electrophoresis result, reclaimed using gel reclaims kit
After deliver company sequencing.
Obtained strains YT08 18S rDNA sequences are sequenced as shown in sequence table, compares and analyzes by NCBI Blast, should
Sequence and Paecilomyces lilacinus in GenBank（Paecilomyces lilacinus）Multiple isolates（As JF824691.1,
AB124670.1、AY526475.2、AB084157.1、AB104884.1、AB023945.1、EF638694.1、AB103380.1、
AF548079.1 etc.）Sequence similarity be up to 100%.
By combining morphological feature and 18S rDNA sequence signatures on bacterial strain YT08, bacterial strain YT08 can be accredited as light
Purple Paecilomyces varioti（Paecilomyces lilacinus）.
Embodiment 3：Paecilomyces lilacinus YT08 fermented and cultured
1. strain activation and culture
Add 100ml strain activation and culture base in per 500ml conical flasks, 121 DEG C of steam sterilizings 20 minutes.Used
Strain activation and culture base forms（g/L）：Glycerine 10, soy peptone 15, MnSO42, the powdered chitosan of 85% deacetylation
8, NaCl 6, CaCl21, MgSO43, FeSO40.5, initial pH 7.
Choose a fritter lawn from solid slope strain, be directly accessed the strain activation and culture base of liquid, at 28 DEG C and
Shaking table shaking culture 48 hours under 200rpm, to growing up to shallow thick white mycelium.
2. solid fermentation culture
Solid fermentation culture medium：Dry beer vinasse 3kg, sucrose 20g, soy peptone 60g, the powder shell of 85% deacetylation
Glycan 10g, NaCl 30g, MgSO46g, MnSO44g, 7 liters are settled to running water.Compound method：First with water by NaCl,
MgSO4、MnSO4, sucrose and soy peptone dissolving, stir, then mixed with other materials, pH is natural.Above-mentioned solid is sent out
Ferment culture medium is fitted into 10L solid-state fermentation tanks, steam sterilizing 20 minutes at 118 DEG C.
According to 10%（Percent by volume）Inoculum concentration, Paecilomyces lilacinus YT08 seed culture product is poured into solid fermentation
In culture medium, starting mechanical agitation makes strain be mixed with fermentation medium.The static culture at 28 DEG C, nothing was passed through every 3 hours
Bacterium air 1 time.Mycelia is covered with to culture medium top layer after cultivating 3 days, mechanical agitation is again started up and smashes all substances in fermentation tank
Broken mixing, the static culture at 28 DEG C were passed through filtrated air every 5 hours 1 time, continued after culture 4 days to covering with culture medium
Fermentation is terminated after lavender spore.
Fermentation starts mechanical stirring device after terminating, and culture is broken up into mixing, is taken out from discharging opening, is put into shady and cool ventilation
Place dries, and produces Paecilomyces lilacinus YT08 solid pharmaceutical preparation.Using dilution spread method step by step, examined on rose bengal medium
The viable count for surveying Paecilomyces lilacinus YT08 in discovery solid pharmaceutical preparation is 4.7 × 109 cfu/g。
Embodiment 4：Parasitics of the Paecilomyces lilacinus YT08 to tomato root-knot eelworm ovum
If treatment group and control group, 3 repetitions of every group of setting（Culture dish）.2mL ovum grains are added in sterile petri dish to hang
Separately add 2mL Paecilomyces lilacinus YT08 spore suspensions, each culture of control group in each culture dish of supernatant liquid, wherein treatment group
Separately add 2mL sterilized waters in ware.After being cultivated 8 days at 28 DEG C, egg hatching rate and percentage of egg parasitism are calculated according to the following formula：
Paecilomyces lilacinus YT08 the results are shown in Table 1 to the parasitics of tomato root-knot eelworm ovum.
Parasitics of the Paecilomyces lilacinus YT08 of table 1 spore suspension to tomato root-knot eelworm ovum
Tomato root-knot eelworm ovum access approaches used are as follows：Gather the root of root-knot nematode diseased plant tomato in warmhouse booth
System, is rinsed well with water first, is added after being cut into segment in liquor natrii hypochloritis, after sealing is shaken 3 minutes, first crosses 200 mesh sieves
And with distilled water flushing, after 500 mesh sieves, after being rinsed repeatedly with distilled water, sterile small beaker is collected in, and adjust and make suspension
Ovum grain is 50/mL.
The preparation method of Paecilomyces lilacinus YT08 used spore suspension is as follows：Paecilomyces lilacinus YT08 streak inoculations in
On rose bengal medium, after being cultivated 7 days at 28 DEG C, Paecilomyces lilacinus YT08 spore is washed out with sterilized water, microscope inspection
Spore concentration is calculated, is configured to concentration as 107Cfu/mL spore suspension.
Embodiment 5：Parasitics of the Paecilomyces lilacinus YT08 to grape root-knot nematode egg
If treatment group and control group, 3 repetitions of every group of setting（Culture dish）.2mL ovum grains are added in sterile petri dish to hang
Separately add 2mL Paecilomyces lilacinus YT08 spore suspensions, each culture of control group in each culture dish of supernatant liquid, wherein treatment group
Separately add 2mL sterilized waters in ware.After being cultivated 8 days at 28 DEG C, egg hatching rate and percentage of egg parasitism are calculated according to the following formula：
Paecilomyces lilacinus YT08 the results are shown in Table 2 to the parasitics of grape root-knot nematode egg.
Parasitics of the Paecilomyces lilacinus YT08 of table 2 spore suspension to grape root-knot nematode egg
Grape root-knot nematode egg access approaches used are as follows：The root system of root-knot nematode diseased plant snake resource is gathered, it is first
First rinsed well with water, be cut into after segment and add in liquor natrii hypochloritis, after sealing is shaken 3 minutes, first cross 200 mesh sieves and with steaming
Distilled water is rinsed, and after 500 mesh sieves, after being rinsed repeatedly with distilled water, is collected in sterile small beaker, and adjusting makes the suspension ovum grain be
50/mL.
The preparation method of Paecilomyces lilacinus YT08 used spore suspension is as follows：Paecilomyces lilacinus YT08 streak inoculations in
On rose bengal medium, after being cultivated 7 days at 28 DEG C, Paecilomyces lilacinus YT08 spore is washed out with sterilized water, microscope inspection
Spore concentration is calculated, is configured to concentration as 107Cfu/mL spore suspension.
Embodiment 6：The pot experiment of Paecilomyces lilacinus YT08 preventing and treating tomato root-knot eelworm diseases
Cultivation matrix is according to 9 without sick soil, organic fertilizer and sand:1:3 ratio mixed preparings, cultivation matrix used is after testing
Exist without nematode.The tomato seedling of picking 12cm or so length is cultivated in 30 × 30cm flowerpot, per 1 plant of basin, totally 60 plants.
200 2 age tomato root-knot eelworms are applied in the cultivation matrix of 60 above-mentioned basin tomato seedlings, wherein to apply 10g again pale purple for 30 basins
Paecilomyces varioti YT08 solid pharmaceutical preparations（Making step is shown in embodiment 3）, in addition 30 basins apply 10g distiller's dried grains again as blank control.Often
Reason is regulated after 60 days, calculates prevention effect.
Tomato root-knot eelworm access approaches used are as follows：The root system of root-knot nematode diseased plant tomato in warmhouse booth is gathered,
Rinsed well with water, added after being cut into segment in liquor natrii hypochloritis first, after sealing is shaken 3 minutes, first crossed 200 mesh sieves and be used in combination
Distilled water flushing, after 500 mesh sieves, after being rinsed repeatedly with distilled water, it is collected in sterile small beaker.By the root-knot nematode egg of collection
Hatch 3 days at 26 DEG C, that is, obtain 2 instar larvaes of tomato root-knot eelworm.
Raw more major general's coincident with severity degree of condition according to root knot and are divided into 5 grades：It it is 0 grade without root knot, root knot accounts for full root system
1% -25% for 1 grade, root knot account for 26%-the 50% of full root system for 2 grades, root knot account for 51%-the 75% of full root system for 3 grades, root
Knot account for full root system 76% -100% for 4 grades.The calculation formula of disease index and prevention effect is as follows：
Results from pot experiment test is as shown in table 3, and the prevention effect of Paecilomyces lilacinus YT08 preventing and treating tomato root-knot eelworms is
89.69%.Root weight growth rate, plant height growth rate and the aerial part weight growth rate for the treatment of group tomato are all remarkably higher than control group.
Prevention effect of the reagent agent of table 3 to tomato root-knot eelworm
Embodiment 7：Paecilomyces lilacinus YT08 prevents and treats the pot experiment of grape root knot nematode disease
Cultivation matrix is according to 9 without sick soil, organic fertilizer and sand:2:2 ratio mixed preparings, cultivation matrix used is after testing
Exist without nematode.The snake resource seedling of picking 20cm or so length is cultivated in 30 × 30cm flowerpot, per 1 plant of basin,
Totally 40 plants.200 2 age grape root-knot nematodes are applied in the cultivation matrix of 40 above-mentioned basin grape seedlingses, wherein 20 basins apply again
10g Paecilomyces lilacinus YT08 solid pharmaceutical preparations（Making step is shown in embodiment 3）, in addition 20 basins apply 10g distiller's dried grains again as blank
Control.After Routine Management 90 days, prevention effect is calculated.
Grape root-knot nematode access approaches used are as follows：The root system of root-knot nematode diseased plant snake resource is gathered, first
Rinsed well with water, be cut into after segment and add in liquor natrii hypochloritis, after sealing is shaken 3 minutes, first cross 200 mesh sieves and with distilling
Water rinses, and after 500 mesh sieves, after being rinsed repeatedly with distilled water, is collected in sterile small beaker.By the root-knot nematode egg of collection in 26
Hatch 3 days at DEG C, that is, obtain 2 instar larvaes of snake resource root-knot nematode.
Raw more major general's coincident with severity degree of condition according to root knot and are divided into 5 grades：It it is 0 grade without root knot, root knot accounts for full root system
1% -25% for 1 grade, root knot account for 26%-the 50% of full root system for 2 grades, root knot account for 51%-the 75% of full root system for 3 grades, root
Knot account for full root system 76% -100% for 4 grades.The calculation formula of disease index and prevention effect is as follows：
Results from pot experiment test is as shown in table 4, and Paecilomyces lilacinus YT08 prevents and treats the prevention effect of snake resource root-knot nematode
For 93.42%.The heavy growth rate of the root for the treatment of group snake resource, plant height growth rate and aerial part weight growth rate are significantly high
In control group.
Prevention effect of the reagent agent of table 4 to snake resource root-knot nematode
Claims (1)
1. one plant is used to prevent and treat tomato and the Paecilomyces lilacinus of grape root knot nematode disease evil, it is characterised in that the bacterial strain is pale purple plan
Mould（Paecilomyces lilacinus）YT08, it is to separate to obtain from the sea-tangle epidermis of Yantai City sea-farming
, there is strong prevention effect to tomato and grape root knot root knot nematode disease, on November 26th, 2014 in China Microbiological bacterium
The common micro-organisms center registration preservation of the kind preservation committee, deposit number is CGMCC No. 10026.
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| Parasitism if meloidogyne spp. on grape and kiwifruit by the fungal egg parasites paecilomyces lilacinus and verticillium chlamydosporium;Mertens等;《Nematologica》;19931231;第39卷;第400-410页 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105316243B (en) * | 2015-11-23 | 2018-07-13 | 鲁东大学 | A kind of preparation method and applications of agricultural root-knot nematode compound biocontrol fungicide |
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| Publication number | Publication date |
|---|---|
| CN104818216A (en) | 2015-08-05 |
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