CN104630081A - Yeast-like symbiote and use thereof - Google Patents
Yeast-like symbiote and use thereof Download PDFInfo
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Abstract
The invention discloses a yeast-like symbiote and use thereof. The yeast-like symbiote is obtained through being separated from a fat body of laodelphax striatellus by an in-vitro culture technology, is named Pichia anomala SP2014 through microbial taxonomic identification, has the collection number of CCTCC No: M 2014534, and is collected at the CCTCC (China Center for Type Culture Collection) at Oct. 17, 2014. Shown by research and analysis on the sensitivity of the yeast-like symbiote to different microbicides, two kinds of microbicides, namely Antracol and Nativo, with obvious inhibitory effects to P. anomala SP2014 are sprayed to wheat seedlings at the concentration of 800mg/L, the death rate reaches 46.7% and 63.3% respectively after the wheat seedlings are eaten by laodelphax striatellus, the death rate is remarkably higher than that of control, and the weight is remarkably lower than that of a control group. Proven by fluorescent quantitative detection results, the amount of yeast-like symbiotes in laodelphax striatellus bodies is remarkably reduced after the microbicides are used. The yeast-like symbiote can serve as an indication marker for monitoring vitality of laodelphax striatellus or be used for screening or producing pesticides for preventing and treating laodelphax striatellus.
Description
Technical field
The present invention relates to technical field of biological control, relate to the separation of a strain Yeast-like symbionts and the exploration of control small brown rice planthopper novel method.
Technical background
Small brown rice planthopper
laodelphax striatellusbelong to Hemiptera (Hemiptera) Delphacidae (Delphacidae), a kind of important Agricultural pests, inhale hazard rice, barley, wheat etc. divided by thorn and make beyond the region of objective existence, also propagate the virus diseases such as stripe disease, black streaked dwarf virus of rice and maize rough dwarf virus, cause crop yield loss huge (Ding Jinhua and Su Jianya, 2001).The current control to small brown rice planthopper is mainly based on Utilization of pesticides.But due to long-term a large amount of and use of insecticide unreasonably, current small brown rice planthopper is caused to create resistance (Wang Yanhua etc. in various degree to a lot of sterilant, 2010), people then continue to increase Utilization of pesticides dosage for improving insecticidal effect, formation vicious cycle like this, not only plant hopper is difficult to be prevented and treated, and creates serious harm to environment and non-target organism simultaneously.Therefore find new controlling way, the use effectively reducing chemical insecticide is the key of small brown rice planthopper control.
Research finds Yeast-like symbionts (yeast-like symbiotes, YLS) be present in the fungal component in plant hopper stomach fat body, have been reported the amino acid metabolism (Koyama of it and brown paddy plant hopper, 1985), nutrition provides closely bound up (Lee and Hou, 1987), to growth, the growth of brown paddy plant hopper, breed, cause the aspects such as harmful property change and to play an important role (Lv Zhongxian etc., 2001a; B).Studies have found that the small brown rice planthopper after pyroprocessing, the fungal component quantity in body obviously reduces, and finally causes the resistance of small brown rice planthopper to weaken (Zhang Xiaojie etc., 2008).Sterilant compared by sterilant, and to have toxicity low, the features such as target is strong, therefore, the collaborative use of sterilant and sterilant will become a kind of new way of preventing and treating plant hopper, when not affecting insecticidal effect and even improving insecticidal effect, the use of chemical insecticide will be greatly reduced.CN 103918701A, CN 103918702 A, CN 103918703 A, disclose mixing formula preparation and the preparation method of the brown paddy plant hopper control being directed to Yeast-like symbionts, but the screening of sterilant also lacks theoretical foundation, random factor is very large, the consumption of sterilant is higher in addition, may cause ecological impact.And owing to lacking theoretical foundation, brown paddy plant hopper control lacks directive function to the control of small brown rice planthopper.
Specify the diversity of small brown rice planthopper parachorium bacterium, and the isolated culture of fungal component and the research of physiological property can be small brown rice planthopper " antibacterial insect protected " and the selection of sterilant provides foundation.Chen Fajun etc. (2006) tentatively think to there is multiple YLS in small brown rice planthopper body by freezing microtome section and microscopic examination; This study group proves there is YLS, as Noda bacterium in small brown rice planthopper body by Molecular Detection biology techniques early stage
hypomyces chrysospermuswith Pichia guilliermondii bacterium
pichia guilliermondiiexistence (Bai Xu etc., 2010).Because people are difficult to the microenvironment in simulated hexapod body, therefore the isolated culture of YLS is very difficult.Kusumi etc. (1979) once isolated culture obtain a few strain small brown rice planthopper YLS, but have no and carry out precise Identification and characteristic research to it.
Summary of the invention
In order to overcome the deficiencies in the prior art, the invention provides strain Yeast-like symbionts and a purposes.
First object is that first passage vitro culture is separated Yeast-like symbionts-Pichia anomala in small brown rice planthopper body.
Second motivated Pichia anomala of the present invention is as biomarker.
3rd object of the present invention is the sensitivity experiments by Pichia anomala physio-biochemical characteristics and different sterilant thereof, and screening significantly can suppress Pichia anomala, thus reduces the sterilant of small brown rice planthopper activity.
4th order of the present invention provides a kind of new tool and the new medicament that improve small brown rice planthopper prevention effect.
The object of the invention is to be achieved by following technique means:
One strain Yeast-like symbionts, bacterial strain SP2014 Classification And Nomenclature be Pichia anomala (
pichia anomala) SP2014, be preserved in China typical culture collection center on October 31st, 2014, deposit number is: CCTCC NO:M 2014534, address is: Wuhan, China Wuhan University, postcode: 430072, this bacterial strain is to the safe and sound life of sterilant, take enemy steady responsive, when safe and sound raw concentration is 400 mg/L, abnormal candiyeast can not grow, when taking the steady concentration of enemy to reach 800 mg/L, abnormal candiyeast can not grow, and the inhibition of silver-colored force method and Shi Jiale is not obvious, when both concentration reach 5000 mg/L respectively, abnormal candiyeast still can grow.
Described abnormal candiyeast is separated by vitro culture and obtains in the body of small brown rice planthopper.
Described Process of in vitro is as follows: aseptically, dissects small brown rice planthopper belly and collects fatty body, be spread evenly across on YPD solid medium, be positioned in 28 ° of C constant incubators and cultivate.
A purposes for described Yeast-like symbionts, as the cue mark thing of monitoring small brown rice planthopper vigor.
A purposes for described Yeast-like symbionts, prevents and treats the purposes of small brown rice planthopper sterilant as screening or initiative.
For preventing and treating a hybrid medicine for small brown rice planthopper, comprise the sterilant for Pichia anomala and the sterilant for small brown rice planthopper, described sterilant is selected from safe and sound life, takes enemy steady, and sterilant is Provado.
Described sterilant is mixed with takes the steady water dispersible granules of enemy, and safe and sound raw wettable powder, described Provado is imidacloprid water dispersible granule.
Described takes the steady water dispersible granules of enemy, and effective constituent mass concentration is 75%, and wherein oxime bacterium ester accounts for 25%, and tebuconazole accounts for 50%; By imidacloprid water dispersible granule with take the steady water dispersible granules of enemy to dissolve with distilled water respectively, mixing, being made into final concentration is that 0.4g/L takes the steady and 0.004g/L Provado mixing formula of enemy.
beneficial effect of the present invention:
The present invention screens a strain Yeast-like symbionts Pichia anomala by vitro culture in small brown rice planthopper body, using this bacterium as indicator microoraganism, screening has obvious inhibiting sterilant to it, this bactericide synergetic sterilant is used control small brown rice planthopper, not only reduce the use of chemical insecticide, improve prevention effect simultaneously.
Accompanying drawing explanation
Fig. 1 is the slat chain conveyor figure of Abnormal pichia anomala of the present invention;
Fig. 2 is NS1/NS8 amplified production electrophorogram;
Fig. 3 is systematic evolution tree;
Fig. 4 is pcr amplification DGGE collection of illustrative plates.
Embodiment
embodiment 1:(
pichia anomalain-vitro separation and qualification)
Small brown rice planthopper raises in growth cabinet (temperature 26 ± 1 DEG C, relative humidity 80 ± 5%, light application time 16L:8D), more than Herba Hordei Vulgaris continuously rearing 50 generation after periphery rice field, Hangzhou gathers.Get the female worm of small brown rice planthopper 10 just sprouted wings, the alcohol surface sterilization with 75% 3 times, each 3min, rinsed with sterile water 3 times, each 3min, takes its stomach fat body at Bechtop and collects.The fatty body gathered is added YPD respectively, and in PDA, YM, Candia Elective Agar substratum, coating evenly, is positioned over and observes after cultivating 48h in 28 DEG C of constant incubators and to record result.On the substratum grown there being bacterium, with transfering loop picking list bacterium colony, join in corresponding liquid nutrient medium, 28 DEG C, 24h cultivated by 180r/min shaking table.Repeat 3 times.
Test in 5 kinds of substratum used in isolated culture, only have YPD substratum to be separated and obtain a strain Yeast-like symbionts (Fig. 1), this bacterial strain is creamy white, smooth surface with aromatic odour.All can grow for 2 ~ 12 times at pH, optimal pH is 7.The assimilation of 12 kinds of carbon sources the results are shown in Table 1, with yeast classification handbook (Kurtzman
et al., 2011) in the description of Abnormal pichia anomala basically identical.
The carbon assimilation result of table 1 Abnormal pichia anomala on nitrogen basic medium
| Carbon source | Growing state | Carbon source | Growing state |
| L-arabinose | - | D-rhamnosyl | - |
| D-Fructose | ++ | D-ribose | - |
| D-Glucose | ++ | D-wood sugar | - |
| D-semi-lactosi | - | D-sucrose | + |
| Lactose | - | Starch | - |
| N.F,USP MANNITOL | +++ | Glycerine | +++ |
+++: raised growth; ++: more growth; +: grow on a small quantity;-: do not grow or substantially do not grow.
Extract this strain chromosome DNA, using the DNA of said extracted as template, amplification the primer is fungi 18S rDNA universal primer NS1/NS8.Amplification system is 50 μ l, comprising LATaq DNA polysaccharase (5 U/ μ l) 0.5 μ l, 10 × LA buffer(containing Mg
2+) 5 μ l, dNTP (2.5m mol/L) 4 μ l, each 1 μ l of primer (10mmol/L), DNA profiling 2 μ l, adds ddH
2o polishing to 50 μ l.PCR response procedures is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, and 55 DEG C of annealing 30s, 72 DEG C extend 1min, 35 circulations.After having increased, get 5 μ l PCR primer and detect with 1% agarose gel electrophoresis.Adopt 18S rDNA universal primer NS1/NS8 amplification to obtain the sequence (GenBank no. KJ659884) (Fig. 2) that is about 1766 bp, by homology analysis contrast find with
pichia anomala18S rRNA sequence (GenBank no. X58054) has high consistence (99%), as can be seen from systematic evolution tree (Fig. 3), this strain YLS that we are separated to
pichia anomalabe classified as a class.
With the chromosomal DNA of this bacterial strain for template, design two primers, pcr amplification 18S rDNA, design of primers is as follows:
NS1：5’-GTAGTCATATGCTTGTCTC-3’（SEQ ID NO：1）；
NS8：5’-TCCGCAGGTTCACCTACGGA-3’（SEQ ID NO：2）。
embodiment 2: DGGE(
pichia anomalachecking)
Get the female adult worm 100 of just sprouting wings, the alcohol surface sterilization of 75% 3 times, each 3min, rinsed with sterile water 3 each 3min.Take stomach fat body, 75% alcohol disinfecting, rinsed with sterile water, then be the percoll of 30% with final concentration, the centrifugal 10min of 7000 r/min, removes upper strata foreign protein and fat, and lower sediment final concentration is 75% percoll, 10000 r/min, ultracentrifugation 20min, the floss of 75% layer is Yeast-like symbionts, extracts genome with pastoris genomic dna test kit.
First round PCR: with said extracted genome for template, amplification 18Sr DNA-5.8Sr DNA-ITS sequence, amplimer is universal primer NS1/ITS4, amplification system and condition the same.Nest-type PRC: do template after being diluted by PCR primer 1:100, amplimer is universal primer NFGC/NR, ITS3GC/ITS4, increase 18Sr DNA respectively, one section in ITS sequence, in order to improve the detection efficiency of DGGE, add the GC clip of 40 bases at 5 ' end of upstream primer.Amplification system is with first round PCR, and amplification condition is 94 DEG C of denaturation 5 min, 94 DEG C of sex change 30 s, and 55 DEG C of annealing 30 s, 72 DEG C extend 30 s, 30 circulations.After having increased, get 5 μ l PCR primer and detect with 1.5% agarose gel electrophoresis.
Design of primers is as follows:
ITS4：5’-TCCTCCGCTTATTGATATGC-3’（SEQ ID NO：3）；
NFGC： 5’- GCGGTAATTCCAGCTCCAA-3’（SEQ ID NO：4）；
NR： 5’-CCCCGACTGTCCCTATTAATC-3’（SEQ ID NO：5）；
ITS3GC：5’-CATCGATGAAGAACGCAGC-3’（SEQ ID NO：6）；
5'GC-clip sequence is: CGCCCGCCGCGCCCGCGCCAGCCGCCGCGCCCGCCGCG(SEQ ID NO:7); .
DGGE profiling results shows, with ITS3GC – ITS4 and NFGC – NR for inner primer amplification obtains 10 specific bands (Fig. 4) altogether, clip size about 298 ~ 377 bp, wherein both comprised the Noda bacterium reported, Pichia guilliermondii, candiyeast, also comprises the fungi that some can not be cultivated, and the Abnormal pichia anomala that this experiment isolated culture obtains is in table 2.Thus demonstrate the existence of Abnormal pichia anomala in small brown rice planthopper body.
The qualification of table 2 DGGE specific band
| Sequence length (bp) | Primer | The most seemingly sequence species and accession number | Nucleotide sequence identity (%) |
| 298 | NFGC– NR | Pichia anomala (EF427893) | 99 |
| 305 | NFGC– NR | Candida (HQ916867) | 99 |
| 308 | NFGC– NR | Hypomyces chrysospermus (AF267232) | 99 |
| 373 | ITS3GC– ITS4 | Pichia anomala (AF218991) | 99 |
| 377 | ITS3GC– ITS4 | Pichia guilliermondii (KF468192) | 99 |
| 335 | ITS3GC– ITS4 | uncultured fungus (KF800290) | 99 |
embodiment 3: pichia anomalathe sensitivity analysis of different agricultural chemicals
This experiment is for examination agricultural chemicals: this experiment is all purchased from Bayer Hangzhou crop science company limited for examination agricultural chemicals, comprise: (effective constituent mass concentration is 75% to take the steady water dispersible granules of enemy, wherein oxime bacterium ester accounts for 25%, tebuconazole accounts for 50%), safe and sound raw wettable powder (effective constituent zinc 1,2-propylene bisdithiocarbamate mass concentration is 70%), imidacloprid water dispersible granule (effective constituent mass concentration is 70%), fluopicolide+propamocarb suspension agent (effective constituent 687.5 g/L), the happy 40% phonetic mould amine suspension agent of Shi Jia.What table 3 was listed is under different concns sterilant, and in small brown rice planthopper body, separation obtains
p. anomalagrowing state on solid medium.Safe and sound raw inhibition is the most obvious, when concentration reaches 400mg/L
p. anomalacan not grow, secondly be take enemy steady, can not grow when concentration reaches 800mg/L, and the inhibition of fluopicolide+propamocarb and Shi Jiale is more weak.
The different sterilant of table 3 is to the inhibiting experimental result of Pichia anomala
embodiment 4:different agricultural chemicals is on the impact of small brown rice planthopper and YLS thereof
According to the susceptibility results of Pichia anomala to different agricultural chemicals, we choose and suppress stronger safe and sound life to Pichia anomala, take enemy steady, and the more weak Shi Jiale of inhibition tests, with clear water sprinkling for negative control, Provado sprays for positive control.
Provado is prepared by minimum recommended concentration 40 mg/L, and all the other sterilant adopt 400 mg/L, 800 mg/L two concentration experiment.Experiment is carried out in 18 × 180 mm glass test tubees, puts into the Herba Hordei Vulgaris that agricultural chemicals was sprayed in 2 strains in each test tube, and adds 10 female adult worm of just having sprouted wings, and seals with double gauze.Be positioned in growth cabinet and raise, with water sprinkling for blank, often organize 3 repetitions.In 1-5 d, every day is added up the mortality ratio of small brown rice planthopper in every pipe.5th, the worm that residue in every pipe lived was weighed statistics, averages.Afterwards, each process is got 10 worms alive and is extracted YLS genome in body, and dissect after collecting its fatty body and directly extract with pastoris genomic dna extraction test kit, the absolute fluorescence for YLS determines the experiment of PCR amount.
To 3 kinds of YLS:Noda bacterium in small brown rice planthopper body (
hypomyces chrysospermus), Pichia guilliermondii (
pichia guilliermondii), Pichia anomala (
pichia anomala) measure.Special fluorescent primer is devised for 3 kinds of bacterium by Primer Primers 5 and Oligo7 software, be respectively HF/HR, MF/MR, PF/PR, and with the genome of fungal component in female adult worm of just sprouting wings for template, carry out regular-PCR, screen fluorescent primer and determine the optimum annealing temperature carrying out qPCR.By increasing, the PCR primer obtained is connected on pMD18-T carrier.
Standard plasmid is carried out 10 times of gradient dilutions, each standard substance dilute 6 gradients, and with the logarithmic value of extension rate for X-coordinate, Ct value is ordinate zou, obtain the typical curve of three plasmid standards respectively.With the fungal component genome extracted for template, amplification system is as follows: SYBR Green mix 12.5 μ l, template 2 μ l, and each 0.5 μ l of primer, adds ddH
2o polishing to 25 μ l.Response procedures is: 94 DEG C of denaturation 1min, 94 DEG C of sex change 10 s, X DEG C of annealing 30 s, 40 circulations.Wherein annealing temperature X is 57 DEG C for Noda bacterium, is 56 DEG C for Pichia guilliermondii, is 55 DEG C for Pichia anomala.
Design of primers is as follows:
HF：5’-TCCCTCTGTGGAACCCCAT -3’ （SEQ ID NO：8）；
HR：5’-GGCGGTCCTAGAAACCAACA-3’（SEQ ID NO：9）；
MF：5’-TAGTCGGACTAGGCGTTTG-3’（SEQ ID NO：10）；
MR：5’-GTTTGGTTGTTGTAAGGCCGG-3’（SEQ ID NO：11）；
PF：5’-GTCAAGGGTTAACTTGAAATATTGAC-3’（SEQ ID NO：12）；
PR：5’-TTAGTTTATTGTTGTTAAGCCGA-3’（SEQ ID NO：13）。
Experimental result is as shown in table 4.Can find out that the lethality of pesticide imidacloprid to small brown rice planthopper reaches the most by force 95% by mortality ratio, and according to two kinds of sterilant 70% zinc 1,2-propylene bisdithiocarbamates that Pichia anomala screens, 50% tebuconazole+25% oxime bacterium ester is also particularly evident to the suppression of small brown rice planthopper, rising small brown rice planthopper mortality ratio along with concentration rises, under the 800 mg/L concentration that coexist, 70% zinc 1,2-propylene bisdithiocarbamate, the inhibition of 50% tebuconazole+25% oxime bacterium ester is apparently higher than 40% phonetic mould amine, and this and Pichia anomala susceptibility results are basically identical; In the small brown rice planthopper of survival, 800 mg/L70% zinc 1,2-propylene bisdithiocarbamates, the small brown rice planthopper of 50% tebuconazole+25% oxime bacterium ester process also also exists significant difference with other experimental group in body weight, YLS quantity in experimental group small brown rice planthopper body is all starkly lower than control group, and the quantity of YLS is negative correlation Long-term change trend in the mortality ratio of small brown rice planthopper, body weight and its body, and 40% more weak phonetic mould amine is suppressed to Pichia anomala, also not obvious to the action effect of small brown rice planthopper.Above result shows that sterilant can weaken the activity of small brown rice planthopper by the YLS in effect small brown rice planthopper body, thus produces certain influence to the survival of small brown rice planthopper.
The different agricultural chemicals of table 4 is on the impact of small brown rice planthopper
embodiment 5:mixing formula formulation is applied in control small brown rice planthopper
(1) by imidacloprid water dispersible granule, safe and sound raw wettable powder and take enemy steady water dispersible granules dissolve with distilled water respectively, be made into that final concentration is the safe and sound life of 0.4g/L respectively, 0.4g/L takes the steady and 0.004g/L Provado preparation of enemy, be mixed with different mixing pharmaceutical formulation.
(2) with clear water sprinkling for negative control, spray wheat seeding with mixed preparation of preparation in (1), spraying 0.04g/L Provado is positive control.Require that liquid Pcnten-1 yne-4 is even, coverage effect is good, and amount of liquid medicine is suitable, does not produce loss and is advisable, naturally dry with wetting target surface.
(3) wheat seeding after process in (2) is placed on 18 × 180 mm glass test tubees injecting nutrient solution, in each test tube, puts into 2 strain wheat seedings, and add 10 female adult worm of just having sprouted wings, seal with double gauze.Be positioned in growth cabinet and raise, often organize 3 repetitions.The mortality ratio of small brown rice planthopper in every pipe was added up in 5th day.
Experimental result is as shown in table 5, and bactericide synergetic sterilant uses the mixing formula preparation of preparation while reducing Utilization of pesticides concentration, effectively improve the prevention effect of small brown rice planthopper.
table 5 mixing formula preparation is to small brown rice planthopper prevention effect
| Reagent agent | Mortality ratio (%) |
| The safe and sound life of 0.2g/L+0.004g/L Provado | 78.4 |
| The safe and sound life of 0.4g/L+0.004g/L Provado | 90.2 |
| 0.2g/L takes the steady+0.004g/L Provado of enemy | 85.1 |
| 0.4g/L takes the steady+0.004g/L Provado of enemy | 98.4 |
| 0.004 g/L Provado | 61.2 |
| 0.04 g/L Provado | 95.0 |
| Clear water | 0 |
SEQUENCE LISTING
The <110> China Measures Institute
<120> Yeast-like symbionts and purposes
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Claims (8)
1. a strain Yeast-like symbionts, is characterized in that: bacterial strain SP2014 Classification And Nomenclature be Pichia anomala (
pichia anomala) SP2014, be preserved in China typical culture collection center on October 31st, 2014, deposit number is: CCTCC NO:M 2014534, this bacterial strain is to the safe and sound life of sterilant, by the steady sensitivity of enemy, when safe and sound raw concentration is 400 mg/L, abnormal candiyeast can not grow, when taking the steady concentration of enemy to reach 800 mg/L, abnormal candiyeast can not grow, and the inhibition of silver-colored force method and Shi Jiale is not obvious, when both concentration reach 5000 mg/L respectively, abnormal candiyeast still can grow.
2. Yeast-like symbionts as claimed in claim 1, is characterized in that: described abnormal candiyeast is separated by vitro culture and obtains in the body of small brown rice planthopper.
3. Yeast-like symbionts as claimed in claim 2, it is characterized in that: described Process of in vitro is as follows: aseptically, dissect small brown rice planthopper belly and collect fatty body, be spread evenly across on YPD solid medium, be positioned in 28 ° of C constant incubators and cultivate.
4. a purposes for Yeast-like symbionts as claimed in claim 1, is characterized in that: as the cue mark thing of monitoring small brown rice planthopper vigor.
5. a purposes for Yeast-like symbionts as claimed in claim 1, is characterized in that: the purposes of preventing and treating small brown rice planthopper sterilant as screening or initiative.
6. for preventing and treating a hybrid medicine for small brown rice planthopper, it is characterized in that: comprise the sterilant for Pichia anomala and the sterilant for small brown rice planthopper, described sterilant is selected from safe and sound life, takes enemy steady, and sterilant is Provado.
7. hybrid medicine as claimed in claim 6, is characterized in that: described sterilant is mixed with takes the steady water dispersible granules of enemy, and safe and sound raw wettable powder, described Provado is imidacloprid water dispersible granule.
8. hybrid medicine as claimed in claim 7, is characterized in that: described takes the steady water dispersible granules of enemy, and effective constituent mass concentration is 75%, and wherein oxime bacterium ester accounts for 25%, and tebuconazole accounts for 50%; By imidacloprid water dispersible granule with take the steady water dispersible granules of enemy to dissolve with distilled water respectively, mixing, being made into final concentration is that 0.4g/L takes the steady and 0.004g/L Provado mixing formula of enemy.
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| CN105462910A (en) * | 2015-12-16 | 2016-04-06 | 中国计量学院 | Method for carrying out in-vitro study on absorption for foreign particles by insect ovary |
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| CN103468798A (en) * | 2013-08-17 | 2013-12-25 | 中国计量学院 | Detecting method for yeast-like-symbiotes in vivo of brown planthopper in different instars |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105462910A (en) * | 2015-12-16 | 2016-04-06 | 中国计量学院 | Method for carrying out in-vitro study on absorption for foreign particles by insect ovary |
| CN105462910B (en) * | 2015-12-16 | 2019-03-01 | 中国计量大学 | A method of it studying insect ovary in vitro and absorbs extraneous particles |
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