CN101434945A - Nematophagous fungi infectious extracellular serine proteinase crystal morphology - Google Patents
Nematophagous fungi infectious extracellular serine proteinase crystal morphology Download PDFInfo
- Publication number
- CN101434945A CN101434945A CNA200810058981XA CN200810058981A CN101434945A CN 101434945 A CN101434945 A CN 101434945A CN A200810058981X A CNA200810058981X A CN A200810058981XA CN 200810058981 A CN200810058981 A CN 200810058981A CN 101434945 A CN101434945 A CN 101434945A
- Authority
- CN
- China
- Prior art keywords
- protease
- enzyme
- proteolytic enzyme
- diffraction
- serine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to an extracellular serine protease crystal structure with nematophagous fungi infectivity and belongs to the fields of molecular biology and applied microbiology. The invention implements liquid cultivation with Lecanicillium psalliotae YMF1.00112 and Paecilomyces lilacinus M-14, fractional precipitation of ammonium sulphate with a fermented liquid, and dissolution of protease-containing active component with buffer solution, and then obtains raw enzyme solution. Cation exchange chromatography and molecular sieve purification of the raw enzyme solution are implemented. After purified enzyme is obtained, concentration processing is implemented so as to obtain an enzyme solution with higher concentration. Hanging drop vapor diffused crystallization of the enzyme solution with higher concentration is implemented. The crystallization conditions are filtered with a reagent kit. Enough diffraction pictures of obtained crystal are collected through diffraction on an X-ray machine. The diffraction pictures are processed with HKL2000, protein structure is analyzed and optimized with software O, ccp4 and the like. The invention discloses the extracellular serine protease crystal structure for the first time and provides foundation for improving the hydrolysis activity of protease and the infectivity of nematophagous fungi.
Description
Technical field:
The present invention relates to the Nematophagous fungi infectivity extracellular serine proteinase crystalline structure of determining with X-ray crystalline diffraction method, belong to molecular biology and applied microbiology field.
Background technology:
Nematode is the linear many cells protozoon of a class, is distributed widely in seawater, fresh water and the soil, and wherein a part can parasitize in plant or the animal body.The serious plant disease that plant nematode can cause the diversified economy crop causes enormous economic loss.Wherein the most serious nematode of harm comprises root knot nematode (Meloidogyne spp.), Cyst nematode (Heterodera spp.) and pine wood nematode (Bursaphelenchus xylophilus) etc.Though the applied chemistry agricultural chemicals has good prevention effect to pathogenic nematode, but chemical pesticide causes environmental pollution, pesticide residue and nematode to be easy to generate shortcomings such as drug resistance easily, so research wireworm-killing biologic resource and exploitation biological mematocide receive publicity day by day.
Nematophagous fungi is meant parasitism, seizure, grows surely and poisons the quasi-microorganism of nematode, and they play important effect in the biological control of the cause of disease insect of plant and nematode.Wherein few spore nodal plexus spore (Arthrobotrys oligospora), thick wall spore Pu Keniya bacterium (Pochonia chlamydospora) and Paecilomyces lilacinus Nematophagous fungis such as (Paecilomyces lilacinus) can be secreted extracellular enzyme and participate in degraded and infection processs (2 to the nematode body wall in infecting the process of nematode, 6,7).The infectivity extracellular enzymes such as proteolytic enzyme that studies show that to Nematophagous fungi play important effect (3-5) in infecting the process of nematode.
Yet these Nematophagous fungis produce under field conditions (factors) and the concentration of excretory extracellular enzyme is low-down (1～2 μ g/L) (7), are a kind of approach that improves nematode biological prevention and control agent preventive effect so improve the expression level of Nematophagous fungi extracellular protease.The research work of Ahman etc. (1) shows that the few spore nodal plexus spore mutant strain that contains multiple copied extracellular protease (PII) encoding gene that the applied molecular biology means make up not only can make biocontrol strain secrete more extracellular enzyme, and the prey ability of biocontrol strain also obtained great enhancing, and this also provides thinking and method preferably for the genetic modification of Nematophagous fungi.
Improve Nematophagous fungi at present and infect the copy number that active method only limits to utilize genetically engineered raising infectivity serine protease gene, increase expression amount and realize, but the structure of serine protease is not carried out careful research.
By literature search, find open report with content same document of the present invention.
Reference:
1.Ahman，J.，T.Johansson，M.Olsson，P.J.Punt，C.A.van?den?Hondel，and?A.Tunlid.2002.Improving?the?pathogenicity?of?a?nematode-trapping?fungus?by?geneticengineering?of?a?subtilisin?with?nematotoxic?activity.Appl?Environ?Microbiol68：3408-15.
2.Bonants，P.J.，P.F.Fitters，H.Thijs，E.den?Belder，C.Waalwijk，and?J.W.Henfling.1995.A?basic?serine?protease?from?Paecilomyces?lilacinus?with?biologicalactivity?against?Meloidogyne?hapla?eggs.Microbiology?141(Pt4)：775-84.
3.Gillespie?J?P，B.R.，Charnley?A?K.1998.Role?of?cuticle?degrading?proteases?in?thevirulence?of?Metarhizium?spp.for?the?desert?locust，schistocerca?gregaria.J?InvertebrPathol?71：128-137.
4.H，M.1996.Role?of?microbial?proteases?in?pathogenesis.Microbiol?Immunol40：685-693.
5.Huang，X.，N.Zhao，and?K.Zhang.2004.Extracellular?enzymes?serving?asvirulence?factors?in?nematophagous?fungi?involved?in?infection?of?the?host.ResMicrobiol?155：811-6.
6.Segers，R.，T.M.Butt，B.R.Kerry，and?J.F.Peberdy.1994.The?nematophagousfungus?Verticillium?chlamydosporium?produces?a?chymoelastase-like?protease?whichhydrolyses?host?nematode?proteins?in?situ.Microbiology?140(Pt?10)：2715-23.
7.Tunlid，A.，S.Rosen，B.Ek，and?L.Rask.1994.Purification?andcharacterization?ofan?extracellular?serine?protease?from?the?nematode-trapping?fungus?Arthrobotrysoligospora.Microbiology?140(Pt?7)：1687-95.
8. Wang's housekeeping, the bright .2000. protein of model technical manual. Science Press.
Summary of the invention:
The objective of the invention is to method, determine the crystalline structure of Nematophagous fungi infectivity extracellular serine proteinase by the X-crystalline diffraction.
The present invention is by tool setting spore Verticillium (Lecanicillium psalliotae, YMF1.00112), preserving number CGMCCNo.1312, and Paecilomyces lilacinus (Paecilomyces lilacinus, M-14), preservation CGMCC No 0241 is fermentation culture in inducing product enzyme liquid nutrient medium PL-4, makes fungi secrete a large amount of serine proteases in fermented liquid.Then fermented liquid is carried out ammonium sulfate precipitation (saltouing), the AKTA (TM) that is deposited in that protease activity is arranged (GE) is carried out cation-exchange chromatography and molecular sieve (gel column) purifying on the purifying instrument.Purity by SDS-PAGE check proteolytic enzyme.Obtaining electrophoresis pure (〉 95%) proteolytic enzyme after (Millipore USA) is concentrated to higher concentration with it with the ultrafiltration and concentration pipe.Using hanging drop gas phase diffusion method to carry out crystal then cultivates.The crystallization condition screening reagent box of Hampton (USA) company is used in the screening of crystallization condition.After obtaining good screening conditions, promptly under this condition, cultivate albumin crystal.The crystal diffraction on roentgen machine that obtains.Use HKL2000, O, softwares such as ccp4 are handled, and finally obtain the three-dimensional structure of this proteolytic enzyme.
The present invention is achieved in that
Fungi liquid is cultivated, and fermented liquid is carried out ammonium sulfate precipitation.Tool protease activity part with the damping fluid dissolving, is promptly obtained crude enzyme liquid.(TM carries out cation-exchange chromatography and molecular sieve purification on GE) at protein purification instrument AKTA with crude enzyme liquid.By SDS-PAGE check purity.Obtain pure enzyme and carry out concentration later on to obtain the enzyme solution of higher concentration.The enzyme solution of high density is carried out the crystallization of hanging drop gas phase diffusion.Crystallization condition screens by test kit.The crystal that obtains diffraction and collect abundant diffraction picture on roentgen machine.Handle the diffraction picture with HKL2000, and use O, softwares such as ccp4 are resolved and the optimization protein structure.
Concrete steps of the present invention are as follows
1, the cultivation of fungi
Fungi is gone down to posterity with the PDA flat board by the PDA slant preservation.A small amount of mycelia is inserted liquid product enzyme PL-4 substratum, and (potato 100g/L boils and added gelatin 1g/L, glucose 1g/L, ammonium sulfate 1g/L, potassium primary phosphate 2g/L, sal epsom 0.5g/L, ferrous sulfate 0.001g/L in 25 minutes; Packing 200mL in every 500mL triangular flask) in, on shaking table 28 ℃, 150rpm cultivates.Cultivate and collected nutrient solution in 6 days later on.The mensuration that enzyme is lived adopts Folin-phenol method (8).
2, the purifying of extracellular protease
Fermented product is removed mycelia with 4 layers of filtered through gauze, collect supernatant liquor.The solid ammonium sulfate (final concentration 5-85%) that adds different mass is saltoutd, and centrifugal 12 minutes of 8500rpm abandons supernatant, with phosphate buffered saline buffer (10mM PBS, Ph6.0) dissolution precipitation of fermented liquid 1/20 volume.Survey the protease activity of each solution example.
Sample 45um filtering with microporous membrane with protease activity.Be added on the AKTA protein purification instrument and use 10mMPBS, on the Resource15S cation seperation column that the pH6.0 balance is crossed.With the 10mM PBS that contains 1M NaCl, pH6.0 buffer solution for gradient elution.Collect different elution peaks, survey the proteolytic enzyme enzyme respectively and live.The SDS-PAGE electrophoresis detection.
The elutriant that will have protease activity concentrates the back and goes up molecular sieve (Superdex7526/60), and damping fluid is 50mM PBSpH7.0, adds 0.15M NaCl.Collect different elution peaks, survey protease activity respectively.The SDS-PAGE electrophoresis detection.
3, the crystallization of proteolytic enzyme
Obtain electrophoretically pure proteolytic enzyme sample by above purification step.This protein solution is added Amicon (TM, Millipore) ultrafiltration and concentration pipe.4000rpm is centrifugal, up to the volume that reaches expection.Use the Brandford staining to determine protein concentration.Use Hampton crystallization test kit to carry out the screening of crystallization condition.Protease concentration is 5mg/ml, and 1ul albumen and 1ul pond liquid are mixed a little on the cover glass of silication, and back-off is sealed on the groove that contains 200ul pond liquid, carries out the crystallization of hanging drop gas phase diffusion at 16 ℃.Specifically see the test kit specification sheets.Observed crystallization every 8 hours.Under producing the crystalline condition to protein concentration and pond liquid in the concentration of each material carry out gradient optimizing to obtain albumen crystallization preferably.
4, X-ray diffraction and structure elucidation
After collecting the X ray diffracting data of proteolytic enzyme, at first use (comprising Mosflm, D*trek etc.) software such as HKL2000 that previous step is collected the diffraction data that obtains in rapid and handle, obtain complete data file; Secondly, use Phaser, Molrep etc. in CNS or the CCP4 routine package, utilize the method for molecular replacement (Molecular Replacement), obtain the fine three dimensional structure of protease P L646 and inhibitor.
Reported first of the present invention the crystalline structure of Nematophagous fungi infectivity extracellular serine proteinase, for the hydrolytic activity that improves proteolytic enzyme and the infection ability of Nematophagous fungi are laid a good foundation.
Description of drawings:
Fig. 1 is the ribbon model of protease P L646 crystalline structure.Protease P L646 comprises 6 α spirals as seen from the figure, 7 strands of parallel sheets, two 2 strands of antiparallel sheets.The binding site of having showed inhibitor among the figure simultaneously.
Fig. 2 is inhibitor and protease P L646. bonded fine structure: tetrapeptide (Ala-Ala-Pro-Val) forms hydrogen bond with proteolytic enzyme.The C end carbon atom of the terminal carbon of inhibitor and Val forms covalent linkage with the Histidine of catalytic center and serine residue respectively.
Embodiment:
Below be embodiments of the invention, but content of the present invention is not limited thereto.
Embodiment one: the structure elucidation of cutter spore Verticillium Lecanicillium psalliotae extracellular serine proteinase
With the cutter spore Verticillium cultivated on the PDA flat board (Lecanicillium psalliotae, YMF1.00112), the inoculation of preserving number CGMCC No.1312 is in producing the enzyme substratum, 28 ℃, the 150rpm shaking table was cultivated 6 days.Filtering fermentating liquid, the supernatant liquor ammonium sulfate precipitation.The part that protease activity will be arranged is carried out purifying with the dissolving of 10mM PBS (pH6.0) damping fluid by cation-exchange chromatography.The elutriant that will have enzyme to live is further purified by molecular sieve.Obtain electrophoretically pure proteolytic enzyme Ver112, carry out crystallization after concentrating and obtain protease crystals.Crystal is used HKL2000 by the X-ray diffraction, and software processes such as ccp4 obtain the three-dimensional structure of this proteolytic enzyme.
Embodiment two: the structure elucidation of Paecilomyces lilacinus Paecilomyces lilacinus extracellular serine proteinase
With the Paecilomyces lilacinus cultivated on the PDA flat board (Paecilomyces lilacinus, M-14), the inoculation of preservation CGMCC No0241. is in producing the enzyme substratum, 28 ℃, the 150rpm shaking table was cultivated 6 days.Filtering fermentating liquid, the supernatant liquor ammonium sulfate precipitation.The part of protease activity is dissolved with 10mMPBS (pH6.0) damping fluid, carry out purifying by cation-exchange chromatography.The elutriant that will have enzyme to live is further purified by molecular sieve.Obtain electrophoretically pure protease P L646, carry out crystallization after concentrating and obtain protease crystals.Crystal is used HKL2000 by the X-ray diffraction, and software processes such as ccp4 obtain the three-dimensional structure of this proteolytic enzyme.
The crystalline structure of the outer alkaline serine enzyme of the Nematophagous fungi born of the same parents that above-mentioned embodiment obtains is characterized by: resolving power reaches respectively behind two kinds of outer alkaline serine protease Ver112 of Nematophagous fungi born of the same parents and the PL646 crystalline diffraction
With
Two proteolytic enzyme all are globular proteins, have 6 α spirals, 7 strands of parallel sheets, and two 2 strands of antiparallel sheets are formed, and Ver112 has one 3/10 spiral in addition.Two proteolytic enzyme all contain 5 halfcystines, wherein form two disulfide linkage for four.Two proteolytic enzyme all contain a calcium binding site, can improve the thermostability of proteolytic enzyme in conjunction with calcium ion.
The active centre of the outer alkaline serine protease of the Nematophagous fungi born of the same parents that above-mentioned embodiment obtains is characterized by: by having determined the substrate binding pocket of proteolytic enzyme behind the diffraction to the complex crystallization of PL646 and proteinase inhibitor (methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone) thereof.Two peptide chains of the tetrapeptide fragment of inhibitor and substrate land are connected to form 3 strands of antiparallel pleated sheets by hydrogen bond.Its C end forms covalent linkage with the Serine and the histidine residues of catalytic center.4 substrate binding pockets have been determined by this structure, S1-S4, four amino-acid residue P1-P4 of bound substrates respectively.The peptide bond of P1 residue C end is interrupted by nucleophillic attack by the Sauerstoffatom of the Serine side chain of catalytic center, thus catalysis the hydrolysis reaction of protein substrate.
Table one protease crystals parameter diffraction data
aValues?in?parentheses?correspond?to?the?highest?resolution?shell.
bR
merge＝Σ
hkl∑
i|I(hkl)
i-<I(hkl)>|/∑
hkiΣ
iI(hkl)
i，where<I(hkl)>is?the?mean?intensity?of?theobservations?I(hkl)
i?of?reflection?hkl.
Claims (1)
1, a kind of Nematophagous fungi infectivity extracellular serine proteinase crystalline structure is characterized in that the crystalline structure of serine protease obtains through following steps:
A. fungi is gone down to posterity with the PDA flat board by the PDA slant preservation, and a small amount of mycelia is inserted liquid produce in the enzyme substratum, on shaking table 28 ℃, the 150rpm cultivation; Cultivate and collected nutrient solution in 6 days later on;
B. filter and collect supernatant liquor, ammonium sulfate precipitation; (sourece15S cation seperation column and gel column superdex75 26/60) carries out purifying by AKTA protein purification instrument, obtains electrophoretically pure proteolytic enzyme;
C. detect the inhibition activity of peptide inhibitor methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone to protease P L646; .
D. the electrophoresis pure protein enzyme sample that obtains by above purification step carries out the meteorological diffusion process of hanging drop and carries out crystallization;
E. collect X ray diffracting data with the precession method: after collecting the X ray diffracting data of proteolytic enzyme, at first use HKL2000, comprise Mosflm, D*trek software collects the diffraction data that obtains to previous step in rapid and handle, and obtains complete data file; Re-use Phaser, Molrep software in CNS or the CCP4 routine package, utilize the molecular replacement method of Molecular Replacement, obtain the fine three dimensional structure of protease P L646 and inhibitor; Wherein:
The overall structure of Nematophagous fungi extracellular serine enzyme is characterized by: resolving power reaches 1.6 respectively behind two kinds of Nematophagous fungi extracellular serine proteinase Ver112 and the PL646 crystalline diffraction
With 2.1
Two proteolytic enzyme all are globular proteins, have 6 α spirals, 7 strands of parallel sheets, and two 2 strands of antiparallel sheets are formed, and Ver112 has one 3/10 spiral in addition; Two proteolytic enzyme all contain 5 halfcystines, wherein form two disulfide linkage for four, all contain a calcium binding site, can improve the thermostability of proteolytic enzyme in conjunction with calcium ion;
The active centre of the outer alkaline serine protease of Nematophagous fungi born of the same parents is characterized by: by having determined the substrate binding pocket of proteolytic enzyme behind the diffraction to the complex crystallization of PL646 and proteinase inhibitor (methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone) thereof; Two peptide chains of the tetrapeptide fragment of inhibitor and substrate land are connected to form 3 strands of antiparallel pleated sheets by hydrogen bond; Its C end forms covalent linkage with the Serine and the histidine residues of catalytic center; 4 substrate binding pockets have been determined by this structure, S1-S4, four amino-acid residue P1-P4 of bound substrates respectively; The peptide bond of P1 residue C end is interrupted by nucleophillic attack by the Sauerstoffatom of the Serine side chain of catalytic center, thus catalysis the hydrolysis reaction of protein substrate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200810058981XA CN101434945A (en) | 2008-09-27 | 2008-09-27 | Nematophagous fungi infectious extracellular serine proteinase crystal morphology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200810058981XA CN101434945A (en) | 2008-09-27 | 2008-09-27 | Nematophagous fungi infectious extracellular serine proteinase crystal morphology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101434945A true CN101434945A (en) | 2009-05-20 |
Family
ID=40709584
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA200810058981XA Pending CN101434945A (en) | 2008-09-27 | 2008-09-27 | Nematophagous fungi infectious extracellular serine proteinase crystal morphology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101434945A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757974A (en) * | 2012-06-05 | 2012-10-31 | 陕西省微生物研究所 | Novel preparation method for recombinant human epidermal growth factor |
| CN101717762B (en) * | 2009-12-15 | 2013-07-24 | 中国水产科学研究院黄海水产研究所 | Novel low temperature alkaline protease MP crystal of marine bacteria |
| CN108779449A (en) * | 2016-02-06 | 2018-11-09 | 诺维信公司 | Polypeptide with proteinase activity and encode its polynucleotides |
-
2008
- 2008-09-27 CN CNA200810058981XA patent/CN101434945A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717762B (en) * | 2009-12-15 | 2013-07-24 | 中国水产科学研究院黄海水产研究所 | Novel low temperature alkaline protease MP crystal of marine bacteria |
| CN102757974A (en) * | 2012-06-05 | 2012-10-31 | 陕西省微生物研究所 | Novel preparation method for recombinant human epidermal growth factor |
| CN102757974B (en) * | 2012-06-05 | 2014-07-23 | 陕西省微生物研究所 | Novel preparation method for recombinant human epidermal growth factor |
| CN108779449A (en) * | 2016-02-06 | 2018-11-09 | 诺维信公司 | Polypeptide with proteinase activity and encode its polynucleotides |
| US11236317B2 (en) | 2016-02-06 | 2022-02-01 | Novozymes A/S | Polypeptides having protease activity and polynucleotides encoding same |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Raven et al. | The evolution of silicification in diatoms: inescapable sinking and sinking as escape? | |
| Vohnik | Ericoid mycorrhizal symbiosis: theoretical background and methods for its comprehensive investigation | |
| IJdo et al. | Methods for large-scale production of AM fungi: past, present, and future | |
| Kim et al. | Biological control of apple anthracnose by Paenibacillus polymyxa APEC128, an antagonistic rhizobacterium | |
| Baker et al. | Actinorhizal symbioses | |
| Liang et al. | The crystal structures of two cuticle–degrading proteases from nematophagous fungi and their contribution to infection against nematodes | |
| Sandoval-Denis et al. | New Fusarium species from the Kruger national park, south Africa | |
| El-Mehalawy et al. | Influence of maize root colonization by the rhizosphere actinomycetes and yeast fungi on plant growth and on the biological control of late wilt disease | |
| CN108949584A (en) | A kind of Aspergillus terreus bacterial strain that salt resistance is alkaline-resisting, its ITS sequence and application | |
| Lee et al. | Role of lytic enzymes secreted by Lysobacter capsici YS1215 in the control of root-knot nematode of tomato plants | |
| CN111040974A (en) | Banana endophytic Bacillus belgii and application thereof | |
| CN103160442A (en) | Paecilomyceslilacinus strain having strong pathogenicity for diaphorina citri | |
| Abdelaziz et al. | Pathogenicity of three entomopathogenic fungi, to the aphid species, Metopolophium dirhodum (Walker)(Hemiptera: Aphididae), and their Alkaline protease activities | |
| Ayuningrum et al. | Screening of actinobacteria-producing amylolytic enzyme in sediment from Litopenaeus vannamei (Boone, 1931) ponds in Rembang District, Central Java, Indonesia | |
| CN101434945A (en) | Nematophagous fungi infectious extracellular serine proteinase crystal morphology | |
| CN108192831A (en) | A kind of Penicillium notatum and its application in aromatic compound of degrading | |
| Kobayashi et al. | Nature of suppression of Rhizoctonia solani in Hawaiian soils | |
| Linderman et al. | Survival of Phytophthora ramorum compared to other species of Phytophthora in potting media components, compost, and soil | |
| Velavan et al. | Occurrence of Metarhizium spp. isolated from forest samples in South India and their potential in biological control of banana stem weevil Odoiporus longicollis Oliver | |
| Fravel et al. | Effect of temperature, soil type, and matric potential on proliferation and survival of Fusarium oxysporum f. sp. erythroxyli from Erythroxylum coca | |
| Shivani et al. | Extracellular enzymatic profile of fungal deteriogens of historical palace of Ujjain | |
| Panneerselvam et al. | Larvicidal potential of Skermanella sp. against rice leaf folder (Cnaphalocrosis medinalis Guenee) and pink stem borer (Sesamia inferens Walker) | |
| Adetunji et al. | Effect of wild and mutant strain of Lasiodiplodia pseudotheobromae mass produced on rice bran as a potential bioherbicide agents for weeds under solid state fermentation | |
| Grishechkina et al. | Search for natural isolates of Bacillus thuringiensis for development of ecologically friendly biologicals | |
| Sutherland et al. | Evaluation of oospore hyperparasites for the control of Phytophthora crown rot of pepper |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20090520 |