CN106544276A - Flight Mutagenesis obtain thick wall Pu Keniya bacterium mutant strain and its preparation and the application in Biological control - Google Patents
Flight Mutagenesis obtain thick wall Pu Keniya bacterium mutant strain and its preparation and the application in Biological control Download PDFInfo
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Abstract
The invention discloses Flight Mutagenesis obtain thick wall Pu Keniya bacterium mutant strain and its preparation and the application in Biological control.Thick wall Pu Keniya bacterium are carried No. 8 airships of divine boat and carry out Flight Mutagenesis by the present invention, with the original strain that do not carry as control, the thick wall Pu Keniya bacterium Flight Mutagenesis bacterial strain that preliminary screening is obtained salt tolerance and the resistance to benomyl are carried out including Biological Detection such as colony growth rate, dry mycelial weight, sporulation quantity and pathogenicities and have been determined, final screening obtains one plant and produces spore amount height, the mutant Pc m 38 strong to the pathogenecity of root-knot nematode egg.Invention further provides the biological prevention and control agent prepared with the Flight Mutagenesis bacterial strain Pc m 38 and its application in preventing and treating root-knot nematode.
Description
Technical field
The present invention relates to thick wall Pu Keniya bacterium (Pochonia chamydosporia) Flight Mutagenesis mutant, especially relates to
And thickness wall Pu Keniya bacterium Flight Mutagenesis mutant Pc-m-38 and biological prevention and control agent prepared therefrom, the invention further relates to should
Application of the mutant in Biological control root-knot nematode, belongs to screening and its application of thick wall Pu Keniya bacterium mutants.
Background technology
Thick wall Pu Keniya bacterium (Pochonia chamydosporia=Verticillium chlamydosporium)
Category Deuteromycotina (Deuterom ycotina) Hyphomycetes (Hyphomycetes), is a kind of important to bite Plant nematode funguses
(Lin Maosong, Shen Suwen. heavy wall Verticillium preventing and treating Meloidogyne incognita First Report of Studies [J]. Biological control is circulated a notice of, and 1994,10 (1):
7-10), Meloidogyne incognita (Meloidogyne incognita), peanut root-knot nematode can effectively be prevented and treated
(Wang comes the plant nematodes such as (Meloidogyne arenaria), soybean cyst nematode Heterodera glycines (Heterodera glycines)
To send out, Yang Baojun, close Wen Gang etc. Paecilomyces lilacinus and heavy wall Verticillium preventing and treating Meloidogyne incognita [J], Sichuan Agricultural University is learned
Report, 1998,16 (2):231-233；Liu Chang. parasitic and preventive and therapeutic effect of the Verticillium chlamydosporium V10 bacterial strains to Meloidogyne incognita. it is wide
Western agricultural sciences, 2004,35 (2)：135-137.).Thick wall Pu Keniya bacterium mainly by entozoic mode parasitize ovum and
In female adult body, make nematicide dead by amount reproduction, which is to plant nematode ovum and the parasitism of female adult, right under field conditions (factors)
The control of plant nematode plays an important role, and is one of the biocontrol fungi of most potential Development volue (Qiu Weifan, Gao Ren
Perseverance, Liu Xingzhong. the Biological control brief introduction [J] of root-knot nematode. Guizhou Agriculture College's journal, 1996,15 (2):51-55.), thick wall is general
Can Buddhist nun Asia bacterium have wider adaptability, it is easy to cultivate, and with parasitic various plants nematicide, thus field control can be conducive to
Application.Meanwhile, which substitutes chemical pesticide as nematicide biocontrol microorganisms and has begun to popularization and application in some countries, and achieves one
A little achievements.But due to the complexity of soil ecosystem, particularly soil fungistasis, cause the application of biocontrol microorganisms to be limited
System.
Breeding by Space Mutagenesis technology is creating specific mutagenesis gene money as a kind of effective mutagenic breeding new technique
Important effect is shown in terms of source and cultivation New Crop Varieties.It has been reported that Chinese Shenzhou 8 spacecraft carries Monochamus alternatus Hope
Detached beauveria bassiana on larva, has filtered out parasite killing speed, and the high mutagenic strain of pathogenicity is brown in Stand control pine
Anoplophorae seu Aprionae have very big application potential (Wang Xizhuo, Wang Laifa, Ma Jianwei, Guo Minwei, Liu Hongjian, Dong Guangping. Monochamus alternatus Hope ball spore
The screening [J] of the high virulence Flight Mutagenesis bacterial strain of muscardine. insecticide journal, 2014, (11):1299-1305.)；Meanwhile, divine boat eight
After number Spaceship Carrying Plant nematode biocontrol microorganisms Paecilomyces lilacinus Shandong bacterial strain, mutagenic strain biological characteristicses and original strain are also made
There is different degrees of differentiation, filtered out the preponderant strainses that positive variation by a relatively large margin occurs in terms of growth characteristics and pathogenicity
Strain (Wang Yuan, Wang Laifa, Wang Xizhuo, Zhu Tianhui. the biological effect [J] of spaceship-carried Paecilomyces lilacinus. nuclear agricultural science report,
2014,(11):1933-1940.).Thus illustrate, the aberration rate of Flight Mutagenesis is high, and variation type enriches, and beneficial variation increases,
More chances are provided for selection-breeding strain excellent, the strain excellent for meeting Production requirement and strong stress resistance for selection-breeding provides new
Approach.Breeding by Space Mutagenesis technology is used as a kind of effective mutagenic breeding new technique, Flight Mutagenesis the same with other mutagenic breedings
The detection of various character is must pass through after process, the target variety of specific variation is obtained with the effect and screening that judge Flight Mutagenesis
(agriculture is to group, Zhang Zehua, Hu Pan, Gao Song, Zhang Lisheng. biological effect [J] of the Flight Mutagenesis to insect pathogenic fungus. bacteriology
Report, 2006,25 (4):674-681.).
As biological pesticide, although thick wall Pu Keniya bacterium have chemical pesticide not with other Plant nematode biocontrol microorganisms are the same
Analogous plurality of advantages, but while there is also poor growth and affected by environment larger, prevention effect it is unstable and itself
The shortcomings of resistance is poor, it is therefore necessary to Flight Mutagenesis are carried out to nematicide biocontrol fungi thickness wall Pu Keniya bacterium, screening is produced
Spore amount is high, nematicide ability is strong, have the mutagenic strain of fine resistance to pesticide so as to preferably play preventing and treating Plant nematode
Effect.
The content of the invention
The main object of the present invention is that thick wall Pu Keniya bacterium (Pochonia chamydosporia) are carried out space flight to lure
Become the mutant that one plant of speed of growth block, the excellent performance strong to the pathogenecity of root-knot nematode egg are obtained to screening；
It is another object of the present invention to the mutant of the excellent performance for being obtained to be applied to the Biological control of root-knot nematode.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
The thick wall Pu Keniya bacterium of culture activation are carried out spaceship-carried (Shenzhou 8) by the present invention；Flight Mutagenesis are completed
Afterwards, using the thick wall Pu Keniya bacterium original strains that do not carry as control, compare Flight Mutagenesis bacterial strain and become in colonial morphology, pigment
The variability of the aspects such as change, spore shape, the speed of growth, sporulation quantity, pathogenicity, salt tolerance and benomyl resistance.The present invention sends out
Thick wall Pu Keniya bacterium are generated into abundant character variation Jing after Flight Mutagenesis now.In general, Flight Mutagenesis are to growth
Many trait expressions such as speed, sporulation quantity, pathogenicity, salt tolerance and benomyl resistance have gone out different variation trends and width
Degree.The speed of growth, sporulation quantity and pathogenicity etc. are the most important indexs for screening biocontrol strains, and Flight Mutagenesis make these characteristics
Show Different Variation to tend to and amplitude, it was demonstrated that Flight Mutagenesis be non-fixed point, popularity, it is positive and negative amphitropic, and lure
Become that site is more, induced mutation rate high, caused by institute organism variation have physiological variation to be likely to heritable variation (agriculture is opened to group
Damp China, Hu Pan, Gao Song, Zhang Lisheng. biological effect [J] of the Flight Mutagenesis to insect pathogenic fungus. fungus journal, 2006,25
(4)：674～681).
Obtain for screening that sporulation quantity high pathogenecity to root-knot nematode egg is significantly increased, good salt tolerance is suitable for
The mutant of biological prevention and control, the present invention obtain many plants thick wall Pu Keniya bacterium Flight Mutagenesis bacterial strains of screening have been carried out form,
The Biological Detection such as pigment, mycelia and spore shape, colony growth rate, dry mycelial weight, sporulation quantity and pathogenicity is simultaneously determined
The salt tolerance and the resistance to benomyl of each Flight Mutagenesis bacterial strain：
Bacterial strain Jing after Flight Mutagenesis occurs in that the different variations tended to amplitude in growth rate, and variance analyses show
Show, mutant strain Pc-m-38 is very fast in the growth rate of front 9d, the significant difference (P between original strain Pc<0.05).
During Flight Mutagenesis strain culturing, dry mycelial weight occurs in that positive and negative two direction variation, wherein, mutant Pc-m-38's
Sporulation quantity is obviously improved compared with original strain Pc.
Thick wall Pu Keniya bacterium Flight Mutagenesis bacterial strains generate significantly variation to the pathogenicity of Meloidogyne incognita.Mutation
Bacterial strain Pc-m-38 is to the parasitic rate of Meloidogyne incognita ovum compared with original strain for the parasitic rate of Meloidogyne incognita ovum has significantly
Lifted；Variance analyses show that Pc-m-38 is to significant difference (P between the parasitic rate and original strain of root-knot nematode egg<0.05),
Its higher parasitic rate has great importance for field biological and ecological methods to prevent plant disease, pests, and erosion practice.
Bacterial strain Jing after Flight Mutagenesis occurs in that different types of variation to salt tolerance.First type is with salinity
Rising growth rate accelerate, slow down with the rising growth rate of salinity after reaching certain value, the salt of its most suitable growth is dense
Spend for 0.10-0.15mol/L.Second type is to slow down with the rising growth rate of salinity.Variance analyses show, Pc-
M-38 is to significant difference (P between the toleration and original strain Pc of hypersaline environment (0.50mol/L)<, and can be in high salt 0.05)
Stably grow under environment.
Finally, the present invention from many plants of space flight mutants which hads screening obtain one plant of excellent performance suitable for biological prevention and control
Mutant Pc-m-38, the mutant growth rate block, produce spore amount it is high, can under hypersaline environment stably growth, to root knot line
The pathogenecity of worm's ovum is significantly increased, therefore, mutant Pc-m-38 as biocontrol agent root-knot nematode control and application
It is upper that there is important prospect.
Thick wall Pu Keniya bacterium (Pochonia chamydosporia) boat body mutagenic mutant Pc-m-38 is carried by the present invention
Preservation is handed over, its microbial preservation number is：CGMCC No.12512；Classification And Nomenclature：Thick wall Pu Keniya bacterium Pochonia
chamydosporia；The preservation time：On June 14th, 2016；Depositary institution is：China Committee for Culture Collection of Microorganisms
Common micro-organisms center；Preservation address is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research
Institute.
Invention further provides a kind of biological prevention and control agent of preventing and treating root-knot nematode, including：The thick wall of effective dose in preventing and treating
The spore powder or fermentation liquid and carrier of Pu Keniya bacterium (Pochonia chamydosporia) mutant Pc-m-38.
Used as reference, those skilled in the art refer to following methods and prepare thick wall Pu Keniya bacterium (Pochonia
Chamydosporia) mutant Pc-m-38 spore powders：
(1) thick wall Pu Keniya bacterium mutant Pc-m-38 fermentation liquids are prepared；(2) from thick wall Pu Keniya bacterium mutant Pc-
Spore product is reclaimed in m-38 fermentation liquids；(3) the reclaimed thick wall Pu Keniya bacterium mutant Pc-m-38 tunnings of drying,
Obtain final product spore powder.
Those skilled in the art can prepare thick wall Pu Keniya bacterium according to the various methods disclosed in document
Fermentation liquid；For example, using third stage culture method, i.e.,：Seed culture, secondary liquid amplification culture and liquid fermentation and culture, prepare
Obtain thick wall Pu Keniya bacterium space flight mutant Pc-m-38 fermentation liquids；Wherein, in liquid fermentation and culture, can be using fermentation
Tank is fermented or fermentation culture is carried out by the way of shake-flask culture；Culture medium and its condition of culture used in third stage culture is
Disclose in the literature, as reference, wherein, the constituent of described liquid fermentation medium includes carbon source and nitrogen source；It is described
Carbon source include but is not limited to glucose, sucrose, maltose, soluble starch or Semen Maydis powder in any one or more.It is described
Nitrogen source include but is not limited to sodium nitrate, ammonium sulfate, peptone or analysis for soybean powder in any one or more.
The present invention further provides a kind of method of the thick wall Pu Keniya bacterium biological prevention and control agents for preparing preventing and treating root-knot nematode, should
Method is comprised the following steps：By the Pc-m-38 spore powders or fermentation liquid and carrier of thick wall Pu Keniya bacterium Flight Mutagenesis mutant
Mix, stir, pulverize and sieve, prepare corresponding microorganism formulation, for example, it may be granular preparation, powder
Agent, wettable powder or microcapsule microbial agent etc..
Wherein, described carrier can be kieselguhr, Kaolin, wood flour, activated carbon, turf, agricultural crop straw, drying
Farm manure etc.；Additionally, can also add adjuvant or/and auxiliary agent in the thick wall Pu Keniya bacteria microorganism preparations of the present invention；Described
Adjuvant can be crab shell powder or chitin etc.；Described auxiliary agent can be wetting agent, dispersant or stabilizer etc..
Description of the drawings
Fig. 1 is bacterium colony front, the back side and the side morphotype of Flight Mutagenesis thickness wall Pu Keniya bacterium；
Fig. 2 is the spore shape of Flight Mutagenesis thickness wall Pu Keniya bacterium；
Fig. 3 is the block diagram of the colony growth rate of Flight Mutagenesis thickness wall Pu Keniya bacterium；
Fig. 4 is the block diagram of Flight Mutagenesis thickness wall Pu Keniya bacteria strain dry mycelial weights；
Fig. 5 is the block diagram of the sporulation quantity of Flight Mutagenesis thickness wall Pu Keniya bacterium.
Specific embodiment
Embodiments of the present invention will be described in more by the following example, it should be understood that the embodiment is only model
Example property, any restriction is not constituted to the scope of the present invention.It will be understood by those skilled in the art that without departing from the present invention
Spirit and scope under the details of technical solution of the present invention and form can be modified or replaced, but these modification or replace
Each fall within protection scope of the present invention.
The preparation of embodiment thickness wall Pu Keniya bacterium wettable powders
First, the preparation of thick wall Pu Keniya bacterium spore powders
(1), the preparation of fermentation liquid
1st, after by thick wall Pu Keniya bacterium Flight Mutagenesis mutant actication of culture, carry out shake-flask seed culture；
The composition (by mass percentage) of shake-flask seed culture medium：
Glucose 1.5-2%
Sodium nitrate 0.4-0.6%
Ammonium sulfate 0.2-0.3%
Analysis for soybean powder 0.2-0.3%
Dipotassium hydrogen phosphate 0.1-0.2%
Magnesium sulfate 0.1-0.2%
Water surplus
Liquid fermentation condition：Load seed culture medium 200ml in 500ml triangular flasks, be inoculated with after autoclaving it is thick wall is general can
Buddhist nun Asia bacterium spore suspension, inoculum concentration is 1.5-3%, is placed in 24-28 DEG C, shaking table 150-200r/min, cultivates 18-36h.
2nd, secondary liquid amplification culture (by mass percentage):
The culture medium composition of secondary liquid amplification culture：
Glucose 3-5%
Sodium nitrate 0.4-0.6%
Ammonium sulfate 0.2-0.3%
Analysis for soybean powder 0.2-0.3%
Dipotassium hydrogen phosphate 0.1-0.2%
Magnesium sulfate 0.1-0.2%
Water surplus
Above-mentioned secondary liquid amplification culture base is sterilized in fermentation tank in place；
Liquid fermentation condition：25-28 DEG C of fermentation temperature, tank pressure 0.6-0.8bar, its oxygen-supply quantity can be 100-300L/h,
Rotating speed 150-250rpm, initial ph value 5.5-6.0.
3rd, liquid fermentation and culture：
The culture medium composition of liquid fermentation and culture：
Glucose 10-18%
Sodium nitrate 0.2-0.3%
Ammonium sulfate 0.1-0.15%
Analysis for soybean powder 0.1-0.15%
Dipotassium hydrogen phosphate 0.1-0.2%
Magnesium sulfate 0.1-0.2%
Rhizoma Solani tuber osi leachate (20%) 10-30%
Water surplus
Aforesaid liquid culture medium is sterilized in fermentation tank in place；
Liquid fermentation condition：Fermentation temperature is 28-30 DEG C, and its oxygen-supply quantity can be 100-300L/h, front 24h, ventilation
Control was controlled in 250-300L/h in 100-150L/h, later stage；Initial pH：5.0-6.0；Rotating speed：150-200r/min；Tank pressure：
0.6-0.8Mpa, fermentation period are controlled in 6d, and general microscopy spore concentration reaches 109Individual/more than ml.
(2), reclaim from fermentation liquid and be dried spore product
Fermentation liquid is centrifuged, centrifugal condition is：Relative centrifugal force(RCF) is 4000g, and centrifugation time is 40min, is sent out per 100 milliliters
Flocculant 2.0g is added in zymotic fluid；Supernatant is abandoned, by the water content into product of drying in the shade after the diatomite support absorption of precipitation bacterium slurry
Less than 10%, thick wall Pu Keniya bacterium spore powders are obtained final product.
2nd, the preparation of wettable powder
Each component is weighed by following percent mass proportionings：By the spore powder 85% of above-mentioned preparation, kieselguhr 11%, wetting agent
PEG 2%, dispersant wood sodium 2%；325 mesh sieves will be crushed after spore powder and kieselguhr mix homogeneously；By the product after crushing
Uniformly levigate with airflow milling afterwards with wetting agent and dispersant, mix homogeneously is obtained final product.
The screening of 1 thickness wall Pu Keniya bacterium of test example boat body mutagenic mutant Pc-m-38 and the determination test of mutagenic effect
1 material and method
1.1 biomaterial
Strains tested thickness wall Pu Keniya bacterium (P.chamydosporia), from the U.S., is for preventing and treating root-knot nematode
Production bacterial strain, culture presevation is in RES INST OF FOREST ECOLOGY ENV.After Flight Mutagenesis ground return,
It is in the 4 DEG C of preservations of the present inventor's laboratory, standby.Meloidogyne incognita (M.incognita) for trying is ground by China Forest science
Study carefully institute's forest ecological environment to be provided with Protective strategy.
1.2 it is spaceship-carried
The mycelia block that activation 7d is cultivated with PDA is transferred to aseptic EP pipes (1.5mL) 2 to manage, sealing.Wherein a pipe is carried out
Spaceship-carried (Shenzhou 8), another pipe are stored in 4 DEG C of refrigerators as control material.
1.3 screening mutant
Thick wall Pu Keniya bacterium Jing after Flight Mutagenesis process and original strain are respectively prepared into spore suspension, spore is diluted
Sub- concentration is to 1.0 × 103Cfu/mL, (adds 0.1mL spore suspension per ware) on the PDA plate of even spread to a diameter of 9cm,
After 25 DEG C of constant temperature culture 6d-9d, observation single bacterium colony size, form and positive and negative color are picked out brighter with original strain difference
Aobvious single bacterium colony bacterial strain；Original number is " Pc ", and remaining strain number is " Pc-m-No. ".
The detection of 1.4 Flight Mutagenesis effects
Using the thick wall Pu Keniya bacterium original strains that do not carry as control, compare Flight Mutagenesis bacterial strain colonial morphology,
The variability of the aspects such as pigment change, spore shape, the speed of growth, sporulation quantity, pathogenicity, salt tolerance and benomyl resistance.
1.4.1 the form of bacterium colony and pigment change observation
From on the PDA plate for having grown, 3 pieces of bacterium colony is cut with the card punch of a diameter of 5mm and contain 0.5 ‰ tweens in 5mL
In solution, fully vibrate, make spore suspension.2.5 μ L spore suspension are accessed in 9cm culture dishs (15mL culture medium) central authorities
(spore concentration is 1 × 106Cfu/mL), it is placed in 25 DEG C of incubators and cultivates, each bacterial strain is repeated 3 times.
1.4.2 the observation of spore shape
By spore suspension, (spore concentration is 1 × 106Cfu/mL) drop in and on microscope slide, make interim slide, and micro-
Microscopic observation spore shape.
1.4.3 colony growth rate is determined
Full spore suspension is dipped in the filter paper of a diameter of 5mm of bacterium of having gone out, and (spore concentration is 1 × 106Cfu/mL) it is placed on
PDA culture medium flat board central authorities, are placed in 25 DEG C of incubators and cultivate, and colony diameter is surveyed per 3d with crossing method, until bacterial strain is covered with
Stop determining during whole ware.Original strain is control, is repeated 3 times.
1.4.4 dry mycelial weight is determined
The bottled 50mL PD culture medium of 150mL tapers, (spore concentration is 1 × 10 to draw 200 μ L6Cfu/mL) spore suspension
In every bottle of culture medium, constant-temperature table temperature is 25 DEG C to liquid, and rotating speed is 120r/min.After culture 96h, will be all of in triangular flask
Bacterium solution is poured in the centrifuge tube of 50mL, 5000r/min centrifugation 3min, pours out supernatant, precipitation is poured on filter paper, 50 DEG C of bakings of baking oven
Do to constant weight, weigh.
1.4.5 sporulation quantity is determined
Beat 3 pieces of truffle for taking a diameter of 5mm with card punch in bacterium colony central authorities to the midpoint at edge, be put into 5mL and tell containing 0.5 ‰
In the solution of temperature, fully vibrate, spore content is determined with blood counting chamber (25 × 16 lattice), if 3 repetitions.
1.4.6 Pathogenic Tests
Booth gathers old complaint, and old complaint is cleaned, and is cut into the segment of 0.5cm, loads in 500mL triangular flasks, pours 200mL into
1% liquor natrii hypochloritises, shake after sealing 3min, 200 mesh sieve of rapid mistake, then 500 mesh sieve of rapid mistake, with distilled water repeatedly
The ovum that flushing is stayed in 500 mesh sieve, is finally collected in aseptic small beaker with aseptic water washing, basis of microscopic observation, is adjusted
Worm's ovum concentration is saved to 1000/mL.Add 100 μ L ovum suspensions in the culture dish of diameter 6cm, and add 2mL 1.0 ×
106The mutant spore suspension of individual/mL, wild type Pc are compareed.Each mutant strain is respectively repeated 3 times, 25 DEG C of culture 5d, system
The parasitic number of meter ovum and not parasitic number, and calculate parasitic rate of the bacterial strain spore to Meloidogyne incognita worm's ovum.
1.4.7 Salt resistant test
Prepare and be respectively 0.05mol/L containing NaCl, 0.10mol/L, 0.15mol/L, 0.20mol/L, 0.25mol/L and
The PDA culture medium of 0.50mol/L.Full Flight Mutagenesis bacteria strain spore suspension (spore is dipped in the filter paper of a diameter of 5mm of bacterium of having gone out
Sub- concentration is 1 × 106Cfu/mL the central authorities of the PDA culture medium containing NaCl of above variable concentrations) are placed on, are placed in 25 DEG C of incubators and are trained
Support, colony diameter, and calculated growth speed are measured after 9d.
1.4.8 benomyl resistance screening
(benomyl specification is 50% wettable powder, river to prepare the PDA culture medium of final concentration of 30 μ g/mL benomyls
The rich biochemical industry limited company products of Su Lan).Full Flight Mutagenesis bacteria strain spore is dipped in the filter paper of a diameter of 5mm of bacterium of having gone out
(spore concentration is 1 × 10 to fullness over the chest during pregnancy supernatant liquid6Cfu/mL the PDA culture medium central authorities of final concentration of 30 μ g/mL benomyls) are placed on, are placed in
Cultivate in 25 DEG C of incubators, colony diameter, and calculated growth speed are measured after 9d.
1.4.9 data process＆analysis
Data are processed using Microsoft Excel softwares.One factor analysis of variance is carried out using SPSS 19.0
(One-way ANOPC-M-A), with Duncan methods to different strains multiple comparisons (P=0.05), test data is using average
The form of value plus-minus standard deviation (mean ± SD, n=3) is represented.
2 results and analysis
Impact of 2.1 Flight Mutagenesis to colonial morphology and pigment change
Flight Mutagenesis thickness wall Pu Keniya bacterium are obtained after stable character for successive transfer culture by 3-4, and observation finds that bacterium colony has
Obvious Morphological Differentiation (Fig. 1), can according to the front of bacterium colony, the back side and side form and color change will break up bacterial strain be divided into I,
IIth, III, IV type, 4 type：I types colonial morphology is similar to original strain Pc, the white down-like in bacterium colony front, and bacterium colony back is
Faint yellow, bacterium colony side accounts for the 37.9% of Flight Mutagenesis bacterial strain in smooth dome-shaped, totally 11 plants of I types bacterium colony；II type bacterial strain bacterium colony
Front is white down-like, bacterium colony central concave and has fold, back side color for faint yellow compared with I moldeed depth, totally 14 plants of II type bacterium colony,
Account for the 48.3% of Flight Mutagenesis bacterial strain；III type bacterium colony front is white down-like, bacterium colony central concave corrugationless, and back side color is
Faint yellow, totally 3 plants of III type bacterium colony is Pc-m-13, Pc-m-26 and Pc-m-54 respectively, accounts for the 10.3% of Flight Mutagenesis bacterial strain；Ⅳ
Type bacterium colony front is white down-like, and bacterium colony center is white raised and has a fold, and back color is yellow, and IV type bacterium colony only has
One bacterial strain Pc-m-9, accounts for the 3.4% of Flight Mutagenesis bacterial strain.
Impact of 2.2 Flight Mutagenesis to spore shape
By (100 μm) observations of microscope, the conidium form of Flight Mutagenesis thickness wall Pu Keniya bacterium is spherical or near
It is spherical, it is transparent smooth, four types (I, II, III, IV) with original strain Pc no significant differences.
Impact of 2.3 Flight Mutagenesis to colony growth rate
It has been observed that the speed of growth becomes after the speed of growth of Flight Mutagenesis thickness wall Pu Keniya bacterium is about 1cm/3d, but 15d
Slowly, bacterium colony needs about 20d just cover with whole ware.The colony diameter of culture 15d includes Pc- more than or equal to the bacterial strain of original strain Pc
M-10 has 10 plants interior, accounts for the 34.5% of mutant strain；Bacterial strain less than original strain Pc has 19 plants, accounts for mutant strain
65.5%.Variance analyses show that mutant strain Pc-m-38 is very fast in the growth rate of front 9d, the difference between original strain Pc
Significantly (P<0.05), growth rate gradually slack-off (table 1, Fig. 3) afterwards.
The colony growth rate of 1 Flight Mutagenesis of table thickness wall Pu Keniya bacterium
Table 1 Varieties of colony growth rate of aerospace mutant strains
of Pochonia chamydosporia /cm
Note：In table, data are mean+SD, and after data, * is represented significant difference (P compared with original strain Pc
<0.05)。Note:Data in the table is mean±SD,data marked*represents significant
difference(P<0.05).
Impact of 2.4 Flight Mutagenesis to dry mycelial weight
During Flight Mutagenesis strain culturing, dry mycelial weight occurs in that positive and negative two direction variation (Fig. 4).With original strain Pc phases
Than what dry mycelial weight was improved has 13 plants, accounts for the 44.8% of mutant strain；What dry mycelial weight was reduced has 16 plants, accounts for mutant strain
55.2%.Wherein, the dry mycelial weight of Pc-m-26 is most heavy, is 0.2776g；The minimum mutagenic strain of dry weight is Pc-m-27, and which is done
Weight is 0.1559g.Including the dry mycelial weight (P not notable with original strain difference of some mutant strains including Pc-m-38>
0.05)。
Impact of 2.5 Flight Mutagenesis to sporulation quantity
Result of the test is shown in Table 2 and Fig. 5.Bacterial strain Jing after Flight Mutagenesis occurs in that the different variations (table 2) tended to amplitude.
Compare with original strain Pc, what sporulation quantity was improved there are 24 plants, accounts for the 82.8% of mutant strain；What sporulation quantity was reduced has 5 plants, accounts for prominent
Become the 17.2% of bacterial strain.Variance analyses show there is no significant difference between the sporulation quantity and original strain of Pc-m-38.
The sporulation quantity of 2 Flight Mutagenesis of table thickness wall Pu Keniya bacterium
Table 2 Sporulation of aerospace mutant strains of Pochonia
chamydosporia /(×106cfu·mL-1)
Note：In table, data are mean+SD, and after data, * is represented significant difference (P compared with original strain Pc
<0.05)。Note:Data in the table is mean±SD,data marked*represents significant
difference(P<0.05).
Impact of 2.6 Flight Mutagenesis to pathogenicity
Result of the test is shown in Table 3.Thick wall Pu Keniya bacterium Flight Mutagenesis bacterial strains are generated to the pathogenicity of Meloidogyne incognita
Significantly make a variation.Compared to original strain Pc, there are 20 plants to the bacterial strain that the parasitic rate of Meloidogyne incognita ovum is improved, account for mutant bacteria
The 69.0% of strain；The bacterial strain that parasitic rate is reduced has 9 plants, accounts for the 31.0% of mutant strain.Parasitic rate plus variant rate is up to
12.67%, minus variant rate is up to -15.67%, and variation amplitude is between -15.67% to 12.67%.Pc-m-38 is to root knot
The parasitic rate of line eggs has than original strain and is obviously lifted, and reaches, and variance analyses show, Pc-m-38 is to root knot line
Significant difference (P between the parasitic rate and original strain of worm's ovum<0.05).
3 Flight Mutagenesis of table thickness parasitic rate of the wall Pu Keniya bacterium to Meloidogyne incognita ovum
Table 3 Parasitic rate of aerospace mutant strains of Pochonia
Chamydosporia against Meloidogyne incognitaeggs/%
Note：In table, data are mean+SD, and after data, * is represented significant difference (P compared with original strain Pc
<0.05)。Note:Data in the table is mean±SD,data marked*represents significant
difference(P<0.05).
Impact of 2.7 Flight Mutagenesis to salt tolerance
Result of the test is shown in Table 4.Bacterial strain Jing after Flight Mutagenesis occurs in that different types of variation to salt tolerance.First species
Type is to accelerate with the rising growth rate of salinity, as the rising growth rate of salinity slows down after reaching certain value, its
The salinity of the most suitable growth is 0.10-0.15mol/L, and totally 17 plants of such mutant strain accounts for 58.6%, the Pc-m- of mutant strain
38 and original strain Pc fall within this type.Second type is to slow down with the rising growth rate of salinity；Such
Totally 9 plants of mutant strain, accounts for the 31.0% of mutant strain.There are 3 plant mutant bacterial strains to be not admitted to both the above type in addition, be respectively
Pc-m-30, Pc-m-39 and Pc-m-51, account for the 10.3% of mutant strain.Variance analyses show that Pc-m-38 is to hypersaline environment
(0.50mol/L) significant difference (P between toleration and original strain Pc<0.05) can grow, and stably under hypersaline environment.
The Salt resistant test of 4 Flight Mutagenesis of table thickness wall Pu Keniya bacterium
Table 4 Salt tolerance of aerospace mutant strains of Pochonia
chamydosporia/cm
Note：In table, data are mean+SD, and after data, * is represented significant difference (P compared with original strain Pc
<0.05)。Note:Data in the table is mean±SD,data marked*represents significant
difference(P<0.05).
Experimental example 1 prevents and treats Pericarpium Zanthoxyli by wettable powder prepared by thick wall Pu Keniya bacterium Flight Mutagenesis mutant Pc-m-38
The field test of root-knot nematode
1 test material and method
1.1 for examination microbial inoculum
Test microbial inoculum：Wettable powder (the embodiment prepared with thick wall Pu Keniya bacterium Flight Mutagenesis mutant Pc-m-38
1 prepares)；
Control microbial inoculum：The wettable powder prepared with thick wall Pu Keniya bacterium original strains (not carrying out Flight Mutagenesis) (is pressed
It is prepared according to the method for embodiment 1).
1.2 experiment crops
The Chinese pricklyash of Pericarpium Zanthoxyli growing area plantation.
1.3 experimental design
Experiment place is located at that nematicide worm amount is big and uniform Pericarpium Zanthoxyli growing area, and root-knot nematode second instar larvae insect population is investigated before medicine
In every 100g soil 20-30 heads.
Experiment is divided into 3 groups, tests 1 group, tests 2 groups and clear water matched group, and specific experiment design is as follows：
Test 1 group of wettable powder prepared using embodiment 1 to be processed；
Compare 2 groups of wettable powders prepared using comparative examples to be processed.
Clear water matched group：(medicament for not applying any preventing and treating nematicide) is processed using clear water, is not made anti-nematicide and is processed；
Each processes 30 repetitions (point three row, 10 plants of Chinese pricklyashes of each column), respectively processes random alignment.
April 15 carried out microbial inoculum plot experiment on 10th to May, pushs root surface 10cm thickness, radius aside in Chinese pricklyash surrounding and is
The soil layer of 50cm, by 1 milliliter of opportunistic pathogen agent (test microbial inoculum and control microbial inoculum consumption all same, be 1 milliliter of opportunistic pathogen agent/
Strain) microbial inoculum after dilution is uniformly sprayed in the hole dug using spray pattern after dilute with water, make microbial inoculum be distributed in rhizosphere,
Make again to push soil reset aside.Second instar larvae number in Chinese pricklyash root knot index (disease index) and soil is investigated after dispenser 90d, is evaluated
Prevention effect.
Root knot index (disease index) computational methods：Every process Radix Zanthoxyli Bungeani is dug out, and investigation index is classified according to root-knot nematode
Investigated, root knot severity point 0-10 levels, grade scale is with reference to (Benjamin D, Grover C B such as Benjamin
J.Comparison of compatible and incompatible response of potato to Meloidogyne
Incognita.Journal of Nematology, 1987,19:218-221).Root knot formula of index is as follows：
Larva reduces percentage rate (with respect to decline rate) computational methods：Per cell geotome (2cm × H20cm) from root of the crop
The soil sample that (0-20cm depths) gathers 5 points is enclosed, after fully mixing, 100g centrifugation floating partition method (Karssen G.The is taken
Plant Parasitic Nematode Genus Meloidogyne Goldi,(Tylenchida)in Europe[M]
.Gent:Drukkeru Modern,1982:Second instar larvae in soil sample is separated 5-24.), it is heat-killed rear solid with 4% formalin
It is fixed, count under inverted microscope, calculate root-knot nematode second instar larvae number and larva reduction percentage rate in 100g soil samples (relative to subtract
Move back rate).Computing formula is as follows：
It is as follows with respect to prevention effect computing formula：
2 experimental results
Experimental result is shown in Table 5.
5 thick preventing and treating of the wall Pu Keniya bacterium Flight Mutagenesis mutant Pc-m-38 wettable powders to Pericarpium Zanthoxyli root-knot nematode of table
Effect experimental
From the test data of table 5, test microbial inoculum is (with thick wall Pu Keniya bacterium Flight Mutagenesis mutant Pc-m-38 systems
Standby wettable powder) to the relative prevention effect of root-knot nematode than control microbial inoculum (with thick wall Pu Keniya bacterium original strain systems
Standby wettable powder) 25.00 percentage points have been higher by, the larva reduction percentage rate for testing microbial inoculum is higher by than control microbial inoculum
21.3 percentage points.The result of the test of field control root-knot nematode shows, thick wall Pu Keniya bacterium Flight Mutagenesis mutant Pc-m-
38 to be much better than original strain for the Biological control effect of root-knot nematode for the Biological control effect of root-knot nematode.
Claims (10)
1. one plant thick wall Pu Keniya bacterium (Pochonia chamydosporia) Flight Mutagenesis mutant Pc-m-38, its feature
It is that its microbial preservation number is：CGMCC No.12512.
2. the thick wall Pu Keniya bacterium Flight Mutagenesis mutant Pc-m-10 described in claim 1 preventing and treating plant insect in should
With.
3. according to the application described in claim 2, it is characterised in that：Described plant insect is nematicide.
4. according to the application described in claim 3, it is characterised in that：Described nematicide is root-knot nematode.
5. it is a kind of preventing and treating plant insect biological prevention and control agent, it is characterised in that include：Thick wall Pu Keniya described in claim 1
The spore powder or fermentation liquid and carrier of bacterium Flight Mutagenesis mutant Pc-m-38.
6. according to the biological prevention and control agent described in claim 5, it is characterised in that：Described carrier be kieselguhr, Kaolin, wood flour,
The farm manure of activated carbon, turf, agricultural crop straw or drying.
7. according to the biological prevention and control agent described in claim 5, it is characterised in that：Also contain adjuvant or auxiliary agent.
8. according to the biological prevention and control agent described in claim 7, it is characterised in that：Described adjuvant is crab shell powder or chitin；It is described
Auxiliary agent be wetting agent, dispersant or stabilizer.
9. according to the biological prevention and control agent described in claim 5, it is characterised in that：Described plant insect is root-knot nematode.
10. a kind of method for preparing biological prevention and control agent described in claim 5, comprises the following steps：(1) cultivate described in claim 1
Thick wall Pu Keniya bacterium Flight Mutagenesis mutant Pc-m-38, obtain spore powder or fermentation liquid；(2) by the spore powder for being obtained
Or fermentation liquid and carrier are mixed, and are stirred, and are pulverized and sieved, and obtain biological prevention and control agent.
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