Field of Invention

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.

Background of Invention

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.

Summary of Invention 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.

Brief Description of the Figures

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 1 170 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 1 170 to 1230 base pairs. Brief Description of the Invention

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.

NBS profiling was performed according to van der Linden et a/. (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 μΙ of [γ- ³³ Ρ] 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 μΙ 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 μΙ 10X PCR buffer with 15 mM of MgCI ₂ ; 4.00 μΙ [10 mM] dNTP; 5.00 μΙ [10 mM] (Y- ³³ P)ATP-labeled NBS primer; 2.00 μΙ [10 Mm] Adapter primer; and 0.80 μΙ HotStar Taq polymerase [5 U/μΙ; Qiagen]. The final volume was adjusted to 200 μΙ with sterile distilled water.

Polyacrylamide Gel Electrophoresis (PAGE) was used to separate the [γ- Ρ] 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 μΙ 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 μΙ of ammonium persulfate (APS) (25%, w/v; 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 μΙ of TE buffer respectively. The solution was then heated to 100 ^° C for 5 minutes using a thermomixer (Thermomixer Compact, Eppendorf). Then, 5 μΙ 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 μΙ 10X PCR buffer with 15 mM of MgCI ₂ ; 0.8 μΙ [10 mM] dNTP; 0.8 μΙ [10 mM] NBS-specific primer; 0.8 μΙ [10 Mm] Adapter primer; and 0.1 μΙ HotStar Taq polymerase [5 U/μΙ; Qiagen]. The final volume was adjusted to 50 μΙ 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. QIAquick 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 μΙ of buffer EB (10 mM Tris-CI, 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 μΙ 10X PCR buffer with 15 mM of MgCI ₂ ; 0.8 μΙ [10 mM] dNTP; 0.8 μΙ [10 mM] NBS-specific primer; 0.8 μΙ [10 Mm] Adapter primer; and 0.1 μΙ HotStar Taq polymerase [5 U/μΙ; Qiagen]. The final volume was adjusted to 50 μΙ 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 sees, 72 ^° C for 2 mins for 35 cycles; and final extension of 72 ^° C for 20 mins and holding at A°C. Table 1 represents the primer sequences for the primers (markers). Table 1 : Sequences for the FP1 and FP2 primers (markers).

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 1030 - 1060 bp and 1 170 - 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

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.