CN107130035A - The PCR quick determination methods of Citrus Huanglongbing pathogen bacterium - Google Patents
The PCR quick determination methods of Citrus Huanglongbing pathogen bacterium Download PDFInfo
- Publication number
- CN107130035A CN107130035A CN201710392814.8A CN201710392814A CN107130035A CN 107130035 A CN107130035 A CN 107130035A CN 201710392814 A CN201710392814 A CN 201710392814A CN 107130035 A CN107130035 A CN 107130035A
- Authority
- CN
- China
- Prior art keywords
- primer
- citrus huanglongbing
- huanglongbing pathogen
- pcr
- pathogen bacterium
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
It is that performing PCR amplification, last electroresis appraisal are entered to testing sample with Citrus Huanglongbing pathogen bacterium specific primer the invention discloses a kind of PCR quick determination methods of Citrus Huanglongbing pathogen bacterium；The specific primer is the nucleotide sequence of the gyrase B subunit genes (gyrase subunit B) based on Citrus Huanglongbing pathogen bacterium.This is different from other detection techniques to primer used in this detection technique, can be used for quick and accurately detects Citrus Huanglongbing pathogen.
Description
Technical field
The present invention relates to a kind of detection method of Citrus Huanglongbing pathogen bacterium, espespecially a kind of detection Citrus Huanglongbing pathogen bacterium
（Candidatus Liberibacter spp.）PCR（PCR）Method.
Background technology
Citrus Huanglongbing pathogen（Candidatus Liberibacter spp.）It is most destructive citrus in world wide
Nearly 50 countries and regions citruses of one of disease, at present just serious threat Asia, Africa, Oceania, South America and North America
The development of industry.China is occurred in the area such as Guangdong, Guangxi report, and with global warming, diaphorina citri is survived the winter
Area boundary is moved northward, and there is the trend constantly expanded in the epidemic-stricken area of Citrus Huanglongbing pathogen.Huanglong germ is, by insect Vector transmission, to be confined to
The gramnegative bacterium of bast, fix tentatively for Candidatus liberibacter category (Candidatus Liberibacter) 3 kinds, point
Not Wei Asia kind (Ca. L. asiaticus), Africa plant (Ca. L. africanus) and America kind (Ca. L. americanus), whereinCa. L. asiaticusWorldwide it is distributed the most extensively, causes Citrus Industry loss the most
Seriously, also it is this kind in the Huanglong pathogen of distribution in China.Citrus Huanglongbing pathogen causes plant strain growth to be obstructed, branch yellow, and it is most
The foundation that the typical mottled type yellow of symptom blade and " red nose fruit " can diagnose as preliminary field.
The current germ has been listed in China and entered the territory and internal quarantine harmful organism.The U.S., Brazil, Argentina, soil
Ear its, Iran, Spain, many countries and regions in more than 30, the whole world such as Italy, be all classified as quarantine harmful organisms, it is right
Citrus Huanglongbing pathogen bacterium quarantine, and forbid it to export and input.
In commercially producing, Citrus Huanglongbing pathogen is closely that diaphorina citri takes food generation with the main path of middle propagation
's.In addition, the human activity such as grafting in orchard management maintenance is also beneficial to propagation of the Citrus Huanglongbing pathogen between plant.Its is remote
Propagation then merchandise to realize by the main transport by seedling, scion, stock.Therefore, fast and efficiently citrus Huanglong is set up
Pathogen detection technology, is the important foundation for preventing and treating Citrus Huanglongbing pathogen.At present, Citrus Huanglongbing pathogen bacterium is detected by round pcr, mainly
It is the specific OI1/OI2 filtered out according to Citrus Huanglongbing pathogen 16S rRNA regions（OI1：5’-
GCGCGTATTTTACGAGCGCA-3’；OI2：5 '-GCCTCGCGAGTTCGCAACCCAT-3 ', PCR primer size is 1160bp
Left and right）（Sandrine Jagoueix, Joseph Marie Bové, Monique Garnier, PCR detection of
the two«Candidatus»liberobacter species associated with greening disease of
citrus, Molecular and Cellular Probes, Volume 10, Issue 1, 1996, Pages 43-50,
ISSN 0890-8508,）.Although Citrus Huanglongbing pathogen bacterium detection method is with higher specificity, sensitivity not enough goes out often
Existing false negative, have impact on the accuracy of detection.
16S rRNA sequences using than wide, but also gradually appear current at its deficiency, and 16S rDNA are present in
In all bacterial chromosomal genes, 16S rRNA gene orders are more larger than the information content entrained by gyrB gene order, and length is fitted
In, but its resolution ratio is relatively low.Accordingly, it would be desirable to find more effective detection method.
The content of the invention
Its remarkable advantage of yrB genes is that gene evolution rate is larger, and base replacement rate is higher, in mutually bacteroid detection and mirror
Fixed aspect is more more advantageous than 16S rRNA genes.Citrus Huanglongbing pathogen gyrB genes are searched for containing 2460 nucleotides in NCBI
To 18 Citrus Huanglongbing pathogen gyrB genes series, compared by DNAman and find some conservative regions, devise a pair of specificity
Primer is between sequence 1953-2414pb.Concluded in an experiment by the band of gel electrophoresis, gyrB primers are than 16SrRNA more
Plus it is sensitive.
Therefore, the technical problems to be solved by the invention are：For above-mentioned the deficiencies in the prior art, there is provided a kind of energy is accurate
Identify the PCR quick determination methods of Citrus Huanglongbing pathogen.Pair of primers used in this method is the rush based on Citrus Huanglongbing pathogen bacterium
The nucleotide sequence of enzyme B subunit genes (gyrase subunit B) is revolved, this is different to primer used in this detection technique
In other detection techniques.
In order to solve the above-mentioned technical problem, the technical solution adopted in the present invention is：A kind of PCR detections of Citrus Huanglongbing pathogen
Method, this method is to enter performing PCR amplification, last electroresis appraisal to testing sample with the specific primer of Citrus Huanglongbing pathogen bacterium.Should
Specific primer is：
Forward primer（gyr1）：5’-TCTCGCTTCTAAATTGGC -3’
Reverse primer（gyr2）：5’-GGCTCTACTTCATCACCC -3’
The design of above-mentioned specific primer is gyrase B subunit genes (the gyrase subunit based on Citrus Huanglongbing pathogen bacterium
B nucleotide sequence), PCR primer size is 461bp or so.The present invention is to be measured with Citrus Huanglongbing pathogen bacterium specific primer
Sample enters performing PCR amplification, last electroresis appraisal；The specific primer is the gyrase B subunits base based on Citrus Huanglongbing pathogen bacterium
Because of the nucleotide sequence of (gyrase subunit B).This is different from other detection skills to primer used in this detection technique
Art, can be used for quick and accurately detects Citrus Huanglongbing pathogen.
Brief description of the drawings
The specific primer of Fig. 1 present invention makees PCR electrophoresis result.
In figure：Electrophoresis band from left to right is followed successively by：M- Marker ,+- positive control（From the fixed sun in Yizhang County
Property bacterium）, -- negative control（Clear water）, 1-6 be diseased plant（Positive bacteria from Jiangyong County to determine）；Electrophoresis band from top to bottom according to
It is secondary to be：The DNA profiling amount of Citrus Huanglongbing pathogen bacterium is 10 times, 100 times, 1000 times of dilution.
Fig. 2 OI1/OI2 specific primers make PCR electrophoresis result figure.
In figure：Electrophoresis band from left to right is followed successively by：M- Marker ,+- positive control（From the fixed sun in Yizhang County
Property bacterium）, -- negative control（Clear water）, 1-6 be diseased plant（Positive bacteria from Jiangyong County to determine）；Electrophoresis band from top to bottom according to
It is secondary to be：The DNA10 of Citrus Huanglongbing pathogen bacterium1、102、103Dilute again.
Embodiment
First, design of primers：
The present inventor utilizes the conservative of prokaryotes evolutionary process more control sequences, is searched for from NCBI and downloads Citrus Huanglongbing pathogen
Bacterium（Candidatus Liberibacter spp）Gyrase B subunit genes (gyrase subunit B) nucleotides
Sequence（AP014595.1）, some candidate drugs are obtained with Premier softwares, finally by screening, pair of primers are designed：
Forward primer（gyr1）：5’-TCTCGCTTCTAAATTGGC -3’
Reverse primer（gyr2）：5’-GGCTCTACTTCATCACCC -3’
Primer OI1/OI2 and primer gyr1/gyr2 are retrieved through BLAST respectively, 1 and table 2 is see the table below, as a result shows, with primer
OI1/OI2 and primer gyr1/gyr2 sequence 100% are covered and 100% identical is entirely the nucleotides sequence of Citrus Huanglongbing pathogen bacterium
Row.
The Citrus Huanglongbing pathogen bacterium primer OI1/OI2 of table 1 BLAST results
The Citrus Huanglongbing pathogen bacterium primer gyr1/gyr2 of table 2 BLAST results
Thus, searched in NCBICandidatus Liberibacter,Find gryB genes and download, at DNAMAN
Reason and comparison, find its conservative region, and the primer of design is designed and assessed using primer5.）, design a pair of specificity
Primer：
Forward primer（gyr1）：5’-TCTCGCTTCTAAATTGGC -3’
Reverse primer（gyr2）：5’-GGCTCTACTTCATCACCC -3’
Above-mentioned primer is by giving birth to work bioengineering（Shanghai）Co., Ltd synthesizes, it is contemplated that PCR primer is 461 or so bases.
2nd, PCR processes：
Enter performing PCR to Citrus Huanglongbing pathogen bacterium with above-mentioned gyr1/gyr2 and OI1/OI2 primer pairs to detect, PCR reaction systems are 20 μ
L, is specifically shown in table 3 below：
Each main component of PCR reaction systems of table 3 and consumption
Gyr1/gyr2 primer reaction conditions：94 DEG C of pre-degeneration 5min, then 95 DEG C are denatured 30 sec, 55 DEG C of 30 sec of annealing,
72 DEG C of extension 30sec, carry out 35 circulations, last 72 DEG C re-extend 10 min on this condition.
OI1/OI2 primer reaction conditions：94 DEG C of pre-degeneration 5min, then 95 DEG C are denatured 30 sec, 58 DEG C of annealing 30
Sec, 72 DEG C of extension 1min, carries out 35 circulations, last 72 DEG C re-extend 10 min on this condition.
The 1.0% agarose glue that will be cooled to 50-60 DEG C is poured into glue plate, and comb, Ran Houxiang are transferred to after glue solidifies completely
1 × TBE dilution buffers are added in groove and did not had offset plate upper surface just to liquid level.
The μ l of reaction product 5 and 2 μ l Loading Buffer and SYBR Green nucleic acid gel dye liquors are taken by 3:6:3 mixing,
Take 10 μ l mixed liquors to carry out electrophoresis to be detected, standard molecule is used as using 2000 bp DNA Ladder.Using voltage 110V,
Electrophoresis time 30min, is observed with ultraviolet gel imaging system, as a result amplifies 461 bp band, be consistent with expected size.
Compared with prior art, it is an advantage of the invention that：Primer pair used in this method is turning for Citrus Huanglongbing pathogen bacterium
The nucleotide sequence of gyrase B subunit genes (gyrase subunit B), the primer pair is different from other detection techniques,
It can be used for quick detection Citrus Huanglongbing pathogen.
Application Example
With the specific primer in the present invention to from Hunan Province Jiangyong County Yun Shan towns and day supply and marketing cooperative（E 111°15′37″；N
25°15′54″）Citrus Huanglongbing pathogen bacterial strain on the pathogen that is separated to enter performing PCR, to the DNA of fixed 6 positive germs
Carry out 101、102、103Dilution, while entering performing PCR amplification, amplifies the band of 461 bases.See Fig. 1, as a result detect 101
The band of the 1-6 positive bacterias of dilution is all consistent with positive control band；102The band of the 1-6 positive bacterias of dilution all with sun
Property control it is consistent；103Dilution 1,2,3,5, No. 6 positive bacterias it is consistent with positive control, the feature and negative control of No. 4 positive bacterias
Unanimously.Illustrate the present invention this be special and high sensitivity to Citrus Huanglongbing pathogen bacterium to specific primer, can quick detection
Go out Citrus Huanglongbing pathogen.
Comparing embodiment
With this OI1/OI2 specific primers to from Hunan Province Jiangyong County Yun Shan towns and day supply and marketing cooperative（E111°15′37″；N25°
15′54″）Citrus Huanglongbing pathogen bacterial strain on the pathogen that is separated to enter performing PCR, the DNA of fixed 6 positive germs is carried out
101、102、103Dilution, while entering performing PCR amplification, amplifies the band of 1161 bases.See Fig. 2, as a result detect 101Dilution
1,2,3,4, the bands of No. 6 positive bacterias it is all consistent with positive control band, the feature of No. 5 positive bacterias is consistent with negative control；
102Dilution 1,2,4, the bands of No. 6 positive bacterias it is all consistent with positive control, 3, the feature of No. 5 positive bacterias and negative control one
Cause；103Dilution 1,2, No. 4 positive bacterias run out of 1161pb band ,+, the feature of 3,5, No. 6 positive bacterias and negative control one
Cause.Illustrating OI1/OI2, this has special to specific primer to Citrus Huanglongbing pathogen bacterium, but quick detection goes out Citrus Huanglongbing pathogen
Sensitivity is not high, false negative occurs in the majority, it is impossible to accurately detects Citrus Huanglongbing pathogen.
Claims (4)
1. a kind of PCR quick determination methods of Citrus Huanglongbing pathogen bacterium, it is characterised in that：This method is special with Citrus Huanglongbing pathogen bacterium
Property primer pair testing sample enter performing PCR amplification, last electroresis appraisal；The specific primer is：
Forward primer（gyr1）：5’-TCTCGCTTCTAAATTGGC -3’
Reverse primer（gyr2）：5’-GGCTCTACTTCATCACCC -3’.
2. the PCR quick determination methods of Citrus Huanglongbing pathogen bacterium as claimed in claim 1, it is characterised in that：
Each main component of PCR reaction systems and consumption are as follows：
3. the PCR quick determination methods of Citrus Huanglongbing pathogen bacterium as claimed in claim 2, it is characterised in that：
Gyr1/gyr2 primer reaction conditions：94 DEG C of pre-degeneration 5min, then 95 DEG C are denatured 30 sec, 55 DEG C of 30 sec of annealing,
72 DEG C of extension 30sec, carry out 35 circulations, last 72 DEG C re-extend 10 min on this condition.
4. a kind of primer pair of the PCR quick detections of Citrus Huanglongbing pathogen bacterium, the primer is：
Forward primer（gyr1）：5’-TCTCGCTTCTAAATTGGC -3’
Reverse primer（gyr2）：5’-GGCTCTACTTCATCACCC -3’.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710392814.8A CN107130035A (en) | 2017-05-27 | 2017-05-27 | The PCR quick determination methods of Citrus Huanglongbing pathogen bacterium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710392814.8A CN107130035A (en) | 2017-05-27 | 2017-05-27 | The PCR quick determination methods of Citrus Huanglongbing pathogen bacterium |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107130035A true CN107130035A (en) | 2017-09-05 |
Family
ID=59733436
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710392814.8A Pending CN107130035A (en) | 2017-05-27 | 2017-05-27 | The PCR quick determination methods of Citrus Huanglongbing pathogen bacterium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107130035A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109828019A (en) * | 2019-02-21 | 2019-05-31 | 南昌大学 | The method that electron spray extraction ionization mass spectrometry quickly detects Citrus Huanglongbing pathogen |
| CN112195223A (en) * | 2020-09-30 | 2021-01-08 | 北京农业智能装备技术研究中心 | Rapid detection method for citrus huanglongbing |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525943A (en) * | 2013-11-01 | 2014-01-22 | 广西壮族自治区农业科学院园艺研究所 | Method for PCR detection of pathogen of citrus greening disease |
| CN103820561A (en) * | 2014-03-10 | 2014-05-28 | 广西壮族自治区农业科学院园艺研究所 | Nested-PCR (polymerase chain reaction) amplification detection system and application of citrus yellow shoot Candidatus Liberobacter asiaticus |
| CN105695626A (en) * | 2016-01-25 | 2016-06-22 | 湖南农业大学 | RT-PCR (Reverse Transcription-Polymerase Chain Reaction) primer and method for detecting pepper vein yellows virus (PeVYV) |
-
2017
- 2017-05-27 CN CN201710392814.8A patent/CN107130035A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525943A (en) * | 2013-11-01 | 2014-01-22 | 广西壮族自治区农业科学院园艺研究所 | Method for PCR detection of pathogen of citrus greening disease |
| CN103820561A (en) * | 2014-03-10 | 2014-05-28 | 广西壮族自治区农业科学院园艺研究所 | Nested-PCR (polymerase chain reaction) amplification detection system and application of citrus yellow shoot Candidatus Liberobacter asiaticus |
| CN105695626A (en) * | 2016-01-25 | 2016-06-22 | 湖南农业大学 | RT-PCR (Reverse Transcription-Polymerase Chain Reaction) primer and method for detecting pepper vein yellows virus (PeVYV) |
Non-Patent Citations (3)
| Title |
|---|
| JACQUELINE MORRIS ET AL.: "Novel ‘Candidatus Liberibacter’ species identified in the Australian eggplant psyllid, Acizzia solanicola", 《MICROBIAL BIOTECHNOLOGY》 * |
| 卢乃会等: "柑橘黄龙病分子生物学检测方法研究进展", 《现代农业科技》 * |
| 程保平等: "以omp基因为靶标的3种检测方法对柑橘黄龙病菌检测效果比较", 《广东农业科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109828019A (en) * | 2019-02-21 | 2019-05-31 | 南昌大学 | The method that electron spray extraction ionization mass spectrometry quickly detects Citrus Huanglongbing pathogen |
| CN112195223A (en) * | 2020-09-30 | 2021-01-08 | 北京农业智能装备技术研究中心 | Rapid detection method for citrus huanglongbing |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Feng et al. | Bacillus asahii comes to the fore in organic manure fertilized alkaline soils | |
| Janezic et al. | Highly divergent Clostridium difficile strains isolated from the environment | |
| Das et al. | Diversity of fungi, bacteria, and actinomycetes on leaves decomposing in a stream | |
| Knauth et al. | Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice | |
| Chu et al. | Community structure of ammonia-oxidizing bacteria under long-term application of mineral fertilizer and organic manure in a sandy loam soil | |
| Cardinale et al. | Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities | |
| Waite et al. | Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA | |
| Senechkin et al. | Isolation and partial characterization of bacterial strains on low organic carbon medium from soils fertilized with different organic amendments | |
| Zul et al. | Effects of plant biomass, plant diversity, and water content on bacterial communities in soil lysimeters: implications for the determinants of bacterial diversity | |
| Bougoure et al. | Assemblages of ericoid mycorrhizal and other root‐associated fungi from Epacris pulchella (Ericaceae) as determined by culturing and direct DNA extraction from roots | |
| Foong et al. | Metagenomic analyses of the dominant bacterial community in the fildes peninsula, king george Island (South Shetland Islands) | |
| Filteau et al. | Seasonal and regional diversity of maple sap microbiota revealed using community PCR fingerprinting and 16S rRNA gene clone libraries | |
| Ezeokoli et al. | PCR-denaturing gradient gel electrophoresis analysis of microbial community in soy-daddawa, a Nigerian fermented soybean (Glycine max (L.) Merr.) condiment | |
| Shimano et al. | Application of nested PCR-DGGE (denaturing gradient gel electrophoresis) for the analysis of ciliate communities in soils | |
| Solieri et al. | Development of a sequence-characterized amplified region marker-targeted quantitative PCR assay for strain-specific detection of Oenococcus oeni during wine malolactic fermentation | |
| Dent et al. | Molecular profiling of microbial communities associated with seeds of Beta vulgaris subsp. vulgaris (sugar beet) | |
| Adewumi et al. | Genome subtyping of autochthonous Bacillus species isolated from Iru, a fermented Parkia biglobosa seed | |
| Hong et al. | Culture‐based and denaturing gradient gel electrophoresis analysis of the bacterial community from chungkookjang, a traditional Korean fermented soybean food | |
| CN107130035A (en) | The PCR quick determination methods of Citrus Huanglongbing pathogen bacterium | |
| Xu et al. | Molecular-based environmental risk assessment of three varieties of genetically engineered cows | |
| Chang et al. | Quantitative real time PCR assays for the enumeration of Saccharomyces cerevisiae and the Saccharomyces sensu stricto complex in human feces | |
| Lee et al. | Characterization of Erwinia chrysanthemi, the soft‐rot pathogen of white‐flowered calla lily, based on pathogenicity and PCR‐RFLP and PFGE analyses | |
| Rasolofo et al. | Assessment of the bacterial diversity of treated and untreated milk during cold storage by T-RFLP and PCR-DGGE methods | |
| Husni et al. | Current classification of the bacillus pumilus group species, the rubber-pathogenic bacteria causing trunk bulges disease in Malaysia as assessed by MLSA and multi rep-PCR approaches | |
| Huang et al. | Use of novel specific primers targeted to pheS and tuf gene for species and subspecies identification and differentiation of the Bacillus subtilis subsp. subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |