SG189821A1 - Markers for ganoderma disease diagnosis and a use of thereof - Google Patents
Markers for ganoderma disease diagnosis and a use of thereof Download PDFInfo
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- 241000222336 Ganoderma Species 0.000 title claims abstract description 39
- 201000010099 disease Diseases 0.000 title claims abstract description 37
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 37
- 238000003745 diagnosis Methods 0.000 title claims abstract description 14
- 241000512897 Elaeis Species 0.000 claims abstract description 26
- 241001133760 Acoelorraphe Species 0.000 claims abstract description 20
- 239000012634 fragment Substances 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000003550 marker Substances 0.000 claims abstract description 14
- 239000000203 mixture Substances 0.000 claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 238000000137 annealing Methods 0.000 claims abstract description 4
- 239000011541 reaction mixture Substances 0.000 claims abstract description 3
- 235000001950 Elaeis guineensis Nutrition 0.000 description 14
- 239000000872 buffer Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
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- 241000196324 Embryophyta Species 0.000 description 5
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 5
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 4
- 208000035240 Disease Resistance Diseases 0.000 description 3
- 241000401653 Ganoderma orbiforme Species 0.000 description 3
- 108010006785 Taq Polymerase Proteins 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- OSBLTNPMIGYQGY-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;boric acid Chemical compound OB(O)O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O OSBLTNPMIGYQGY-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 241000233788 Arecaceae Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical class [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 244000060011 Cocos nucifera Species 0.000 description 1
- 235000013162 Cocos nucifera Nutrition 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 240000003133 Elaeis guineensis Species 0.000 description 1
- 241001475335 Ganoderma sp. Species 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- 235000019482 Palm oil Nutrition 0.000 description 1
- 239000008051 TBE buffer Substances 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 239000012677 causal agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002540 palm oil Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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- 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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
Abstract MARKERS FOR GANODERMA DISEASE DIAGNOSIS AND A USE OF THEREOFThe invention relates to a marker comprising a sequence as set forth in SEQ. ID 1 or a functional fragment thereof to diagnose Ganoderma disease in palm plants in particular oil palm plants. The invention also relates to a marker comprising a sequence as set forth in SEQ. ID 2 or a functional fragment thereof to diagnose Ganoderma disease in palm plants in particular oil palm plants. The invention also provides the use of one or a combination thereof of markers comprising a sequence as set forth in SEQ. ID 1 or functional fragment thereof and markers comprising a sequence as set forth in SEQ. ID 2 or functional fragment thereof to diagnose Ganoderma disease in palm plants in particular oil palm plants. The invention also provides a method of diagnosing Ganoderma disease in palm plants in particular oil palm plants wherein the method includes the steps of (a) preparing a PCR reaction mixture with selected markers for detecting Ganoderma disease and (b) running a PCR process with the mixture from step (a) with an annealing temperature between the range of 50 °C to 61 °C. The markers used is one or a combination thereof of markers comprising a sequence as set forth in SEQ. ID 1 or functional fragment thereof and markers comprising a sequence as set forth in SEQ. ID 2 or functional fragment thereof and wherein the diagnosis of a palm plant infected or has a risk of being infected by the Ganoderma disease is determined by the absence of gene of interest.FIGs. 1, 2, 3 and 4
Description
MARKERS FOR GANODERMA DISEASE DIAGNOSIS AND A USE OF THEREOF
The invention relates to markers for detecting of Ganoderma disease palm plants in particular oil palm plants, a use of the markers and the method for detecting the disease using the markers.
Oil palm plantation has been fighting against basal stem rot (BSR) disease known to be caused by the fungal species Ganoderma boninense since early 1980 when it was first reported.
The disease can result in the death of more than 80% of the plants by the time they are half-way through their normal economic life and losses reaching 30% have quite frequently occurred.
The Ganoderma Basal Stem Rot (BSR) disease is caused by the fungus Ganoderma. It is lethal and incurable. If infected old oil palm trunks are left to rot on the ground, various fruiting bodies of Ganoderma will thrive and eventually infect other nearby plants. Ganoderma Basal
Stem Rot (BSR) is a major threat to oil palm cultivation and palm oil production in Malaysia.
It is difficult to control the spread of basal stem rot fungus disease. In oil palms, the main causal agent of this disease is the fungus Ganoderma species. This major agricultural problem particularly found in oil palm and coconut plantations.
Ganoderma species infects from the roots and gradually spread to the bole of the stem, causing dry rot, thus preventing nutrients from being absorbed by the plant tissues. The palm will gradually lose its ability to produce fruits and eventually collapse. One feature of
Ganoderma sp. is the persistence of the pathogens in infected woody tissues and soil borne debris allowing the disease to reappear in oil palm estates which are doing their second or even third replanting. It is critical to conserve the present oil palm land by controlling the spread of the disease.
The invention relates to a marker comprising a sequence as set forth in SEQ. ID 1 or a functional fragment thereof to diagnose Ganoderma disease in palm plants in particular oil palm plants. The invention also relates to a marker comprising a sequence as set forth in SEQ. ID 2 or a functional fragment thereof to diagnose Ganoderma disease in palm plants in particular oil palm plants.
The invention also provides the use of one or a combination thereof of markers comprising a sequence as set forth in SEQ. ID 1 or functional fragment thereof and markers comprising a sequence as set forth in SEQ. ID 2 or functional fragment thereof to diagnose
Ganoderma disease in palm plants in particular oil palm plants.
The invention also provides a method of diagnosing Ganoderma disease in palm plants in particular oil palm plants wherein the method includes the steps of (a) preparing a PCR reaction mixture with selected markers for detecting Ganoderma disease and (b) running a PCR process with the mixture from step (a) with an annealing temperature between the range of 50 °C to 61 °C. The markers used is one or a combination thereof of markers comprising a sequence as set forth in SEQ. ID 1 or functional fragment thereof and markers comprising a sequence as set forth in SEQ. ID 2 or functional fragment thereof and wherein the diagnosis of a palm plant infected or has a risk of being infected by the Ganoderma disease is determined by the absence of gene of interest.
FIG. 1 relates to the PCR product result when marker FP 1 was tested on a susceptible population RT1. The marker is able to amplify PCR products with sizes between the range 465 to 480 base pairs.
FIG. 2 relates to the PCR product result when both markers FP2 were tested on a susceptible population RT1. The markers able to amplify PCR products with sizes between the range 1030 to 1060 base pairs or 1170 to 1230 base pairs.
FIG. 3 relates to the PCR product result when marker FP 1 was tested on a susceptible population RT 4. The marker is able to amplify PCR products with sizes between the range 465 to 480 base pairs.
FIG. 4 relates to the PCR product result when both markers FP2 were tested on a susceptible population RT4. The markers able to amplify PCR products with sizes between the range 1030 to 1060 base pairs or also 1170 to 1230 base pairs.
The present invention provides marker which is able to diagnose Ganoderma disease in palm plants in particular oil palm plants by determining the presence of the disease resistant gene. Hereinafter, this specification will describe the present invention according to the preferred embodiments of the present invention. However, it is to be understood that limiting the description to the preferred embodiments of the invention is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications and equivalents without departing from the scope of the appended claims.
The invention’s aims include the developing of the markers which will be used to amplify the targeted gene which is resistant to Ganoderma disease. The detection of the resistant gene will be determined by the presence or absence of PCR products which correlates to the infectious status of the tested sample.
Germinated seedlings of oil palm (Elaeis guineensis) (Dura X Pisifera) solely from Felda
Agricultural Services Sdn Bhd (FASSB) were used in the nursery trial. Pathogenic Ganoderma boninense strain was provided and an artificial inoculation of Ganoderma boninense was carried out. This was conducted onto germinated seedlings according to Idris, A.S. et al. (2006) in “Technique for inoculation of oil palm germinated seeds with Ganoderma”. MPOB Information
Series. MPOB TT No. 314. An artificial shade was also set up to maintain ambient temperature below 35°C as described by Breton et al. (2006) to enhance the manifestation of mycelium. All the seedlings were watered twice a day and fertilizers were applied according to standard plantation practice.
Total DNA was extracted using a modified CTAB method as described by Seng and
Faridah (2006) in "DNA extraction from mature oil palm leaves." Journal of Oil Palm Research. 18: 219-224. 5 NBS profiling was performed according to van der Linden et al. (2004) in “Efficient targeting of plant disease resistance loci using NBS profiling”; Theoretical of Applied Genetics; 109: 384-393; with modifications. Approximately 250 ng of total DNA was digested with restriction enzyme. Twenty units of restriction enzyme were used to digest total DNA for minimum of 5 hours at 37 °C. During this digestion process, 40 units of ligase were also added together with 1.5 pmol of long and short arm adaptor. The reaction was then inactivated to ensure all restriction enzyme and ligase does not interfere with the following processes.
Linear and exponential PCR was carried out using NBS primers described in van der
Linden et al. (2004), PCR reaction was performed on a Thermal Cycler (MJ Research) without modifying the annealing temperature and PCR condition.
In this labeling process, 0.1 pl of [y-**P] ATP was incorporated into 1 pmol of NBS primer with 0.1 unit of T4-Polynucleotide Kinase. The final volume was then adjusted to 0.5 pl which is adequate for one labelling-reaction. The mixture was incubated overnight at 37 °C and the mixture T4-polynucleotide kinase activity was inactivated at 70 “C for 10 minutes.
After the incubation was terminated, a master mix for ten reactions was prepared and the following reagents were mixed in a clean microcentrifuge tube (20.00 ul 10X PCR buffer with 15 mM of MgCly; 4.00 pl [10 mM] dNTP; 5.00 pl [10 mM] (y-**P)ATP-labeled NBS primer; 2.00 pl [10 Mm] Adapter primer; and 0.80 pl HotStar Taq polymerase [5 U/ul; Qiagen]. The final volume was adjusted to 200 pl with sterile distilled water.
Polyacrylamide Gel Electrophoresis (PAGE) was used to separate the [y-**P] ATP- labeled PCR products. A 6% PAGE gel was prepared by adding 30 ml of 30% acrylamide:bis at 19:1 ratio, 15 ml of 10X TBE buffer, 63g urea and 150 pl of TEMED (BIO-RAD). The final volume was adjusted to 150 ml with deionised water to allow dissolution of urea. The solution was then de-gassed using a vacuum pump for at least ten minutes. The solution was kept at 4 °C for longer term storage (not more than 1 month). In order to cast the 6% PAGE, approximately 100 ml of PAGE solution was mixed with 500 pl of ammonium persulfate (APS) (25%, wiv; BIO-RAD) and the solution was swirled gently before being pumped into the cast.
The comb was fixed on top of the Sequi-gel (BIO-RAD) to position the well.
All markers identified were carefully excised from the PAGE and suspended with 40 pl of
TE buffer respectively. The solution was then heated to 100 °C for 5 minutes using a thermomixer (Thermomixer Compact, Eppendorf). Then, 5 pl of the solution was used as a template for re-amplification of the marker for sequencing. The following master mix for one reactions was prepared; 5.00 ul 10X PCR buffer with 15 mM of MgCl; 0.8 pl [10 mM] dNTP; 0.8 pl [10 mM] NBS-specific primer; 0.8 ul [10 Mm] Adapter primer; and 0.1 pl HotStar Taq polymerase [5 U/ul; Qiagen). The final volume was adjusted to 50 pl with sterile distilled water.
The same PCR condition mentioned above was used.
The amplified PCR product was extracted using QIAquick Gel Extraction. Kit (QIAGEN,
Germany). The PCR product was separated on 1% (w/v) agarose gel and excised with a sterile scalpel. The gel slice then weighs in a colorless tube. Buffer QG (3 volumes) was added to 1 volume of gel and then incubate at 50 °C for 10 minute to help dissolve the gel. QlAquick spin column was placed in a 2 ml collection tube and the sample was added into the column and spin for 1 minute. The flow-through was discarded and 0.75 ml of buffer PE was added into the column to wash and then centrifuge for 1 minute. The flow-through was discarded and the column was centrifuge for another 1 minute to remove residual ethanol from buffer PE. Then, the QIAquick column was placed into a clean 1.5 ml centrifuge tube. 50 pi of buffer EB (10 mM
Tris-Cl, pH 8.5) was added to the center of the column membrane and centrifuge for 1 minute at maximum speed.
Two sets of PCR primers (markers), FP1 with the sequence as forth in SEQ. ID 1 and
FP2 with the sequence as set forth in SEQ. ID2, were designed based on the NBS-LRR disease resistance protein homologue flanking the restriction site where the DNA was digested earlier.
The following master mix for one reactions was prepared; 5.00 ul 10X PCR buffer with 15 mM of
MgCl; 0.8 ul [10 mM] dNTP; 0.8 pl [10 mM] NBS-specific primer; 0.8 pl [10 Mm] Adapter primer; and 0.1 ul HotStar Taq polymerase [5 U/ul; Qiagen]. The final volume was adjusted to 50 pl with sterile distilled water. The following PCR cycle was carried out: 95 °C for 15 mins, 95 °C for 30 s, range from 50-65 °C for 1 min 40 secs, 72 °C for 2 mins for 35 cycles; and final extension of 72 °C for 20 mins and holding at 4°C. Table 1 represents the primer sequences for the primers (markers).
Table 1: Sequences for the FP1 and FP2 primers (markers). :
SEQ. ID 1 FP1 — Forward TGCCTCTAGCTATTGTGGCC
FP1 — Reverse CCTCTGCCACCTCTTCCTTT
SEQ. ID 2 FP2 — Forward CGTGTTCTGGAGCTGCGGCC
FP2 — Reverse GCCGAAGGCCTGACAGAGAAC
The expected range of PCR product size for FP1 is 465 - 480 base pairs (bp).
Meanwhile, FP2 is able to amplify 2 different PCR products or combination of size ranging from
S 1030 - 1060 bp and 1170 — 1230 bp on all oil palm tested samples. With reference to FIG. 1,
FIG. 2, FIG. 3 and FIG. 4, the correlation between the presence and absence of the PCR product and the phenotypic observation is represented in Table 2.
Table 2: The correlation between the absences of PCR product with phenotypic observation ee ewe] rn me
Ganoderma
Sample ID FP1 FP2 FP2 infection status (465-480 bp) | (1030 — 1060 bp) | (1170 — 1230 bp) co ce [
Based on the data observed, the absence of this disease resistance protein homologue represented by both primer sets (FP1 and FP2) is able reflect the significant of Ganoderma infection incidence based on the phenotypic data. Out of the 10 samples tested and results shown and with the exception of sample GS66 and GS96 which did not showed any correlation between phenotypic results and the absence of the PCR products, both sets of primers (markers) can provide up to 80% accuracy on the Ganoderma susceptibility and tolerance.
Although oil palm is described in this invention as the preferred embodiment, it is understood that other plants under the palm family may be applicable.
Claims (10)
1. A marker comprising a sequence as set forth in SEQ. ID 1 or a functional fragment thereof for diagnosis of Ganoderma disease in palm plants.
2. A marker comprising a sequence as set forth in SEQ. ID 2 or functional fragment thereof for diagnosis of Ganoderma disease in palm plants.
3. The markers as claimed in claims 1 to 2 wherein the markers are used for diagnosis of Ganoderma disease in oil palm plants.
4. A use of a marker comprising a sequence as set forth in SEQ. ID 1 or a functional fragment thereof for diagnosis of Ganoderma disease in palm plants.
5. A use of a marker comprising a sequence as set forth in SEQ. ID 2 or functional fragment thereof for diagnosis of Ganoderma disease in palm plants.
6. A use of one or a combination thereof of markers comprising a sequence as set forth in
SEQ. ID 1 or functional fragment thereof and markers comprising a sequence as set forth in
SEQ. ID 2 or functional fragment thereof for diagnosis of Ganoderma disease in palm plants.
7. The use as claimed in claims 4 to 6 wherein the markers are used for diagnosis of Ganoderma disease in oil palm plants.
8. A method of diagnosing Ganoderma disease in a palm plant wherein the method includes the steps of:-
a. preparing a PCR reaction mixture with selected markers for detecting Ganoderma disease; and b. running a PCR process with the mixture from step (a) with an annealing temperature between the range of 50 °C to 61 °C; wherein the markers used is one or a combination thereof of markers comprising a sequence as set forth in SEQ. ID 1 or functional fragment thereof and markers comprising a sequence as set forth in SEQ. ID 2 or functional fragment thereof; and wherein the diagnosis of a palm plant infected or has a risk of being infected by the Ganoderma disease is determined by the absence of gene of interest.
9. The method as claimed in claim 8 wherein the PCR products has a size or a combination thereof ranging from 465 to 480 base pairs, 1030 to 1060 base pairs and 1170 to 1230 base pairs.
10. The method as claimed in claims 8 to 9 wherein the method is for the diagnosis of Ganoderma disease in oil palm plants.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/MY2011/000228 WO2013066144A1 (en) | 2011-10-31 | 2011-10-31 | Markers for ganoderma disease diagnosis and a use of thereof |
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| MY189736A (en) | 2017-12-14 | 2022-02-28 | Acgt Sdn Bhd | Methods for detecting pathogenicity of ganoderma sp. |
| KR102147412B1 (en) * | 2019-02-20 | 2020-08-25 | 씨제이제일제당 주식회사 | A composition for detecting Ganoderma microorganism and diagnosing basal stem rot and a method using the same |
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| CN101365794A (en) * | 2005-08-12 | 2009-02-11 | 巴斯福植物科学有限公司 | Nucleic acid sequences encoding proteins associated with abiotic stress response and plant cells and plants with increased tolerance to environmental stress |
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