WO2014109629A1

WO2014109629A1 - A method for identifying a ganoderma-infected palm tree

Info

Publication number: WO2014109629A1
Application number: PCT/MY2013/000187
Authority: WO
Inventors: Ooi Eng Keong Tony; Daniela Jeffery Daim Leona; Ithnin Nalisha; Li Sim Ho; Bin Mohd Yusof@Hassan HIRZUN; A/L K. Kulaveerasingam Harikrishna; Nazir Basiran Mohd.
Original assignee: Sime Darby Malaysia Berhad
Priority date: 2013-01-14
Filing date: 2013-10-24
Publication date: 2014-07-17
Also published as: MY170201A; SG11201504781SA

Abstract

This invention relates to a method for identifying a Ganoderma-infected palm tree. The method comprises isolating protein from a sample derived from leaves of a palm tree; determining protein profile of the sample by subjecting the proteins from the sample to gel electrophoresis on a 2-dimensional gel; and comparing the protein profile of the sample to a protein profile obtainable from a normal non-Ganoderma- infected palm tree, wherein a difference in the protein profile of the sample as compared to the protein profile of the normal non-Ganoderma-infected palm tree indicates that the palm tree is infected with Ganoderma disease.

Description

A METHOD FOR IDENTIFYING A GANODERMA-MFECTED PALM TREE

FIELD OF THE INVENTION

The present invention relates to a method for identifying a Ganocerma-infected palm tree. More particularly, the present invention relates to a method for identifying a Ganoc/erma-infected palm tree using sample derived from leaves of a palm tree and applying difference gel electrophoresis (DIGE) technique to the sample.

BACKGROUND OF THE INVENTION

Proteomics is the study of the protein complement of the genome. It can be defined as the large-scale study of proteins in terms of their expression levels, post- translational modifications and interactions with other molecules to obtain a global view of cellular processes at the protein level.

The field of plant proteomics is just at the very beginning and lags several years as compared to proteomics of unicellular prokaryotes and mammalian eukaryotes. There is no doubt that the understanding of protein expression in plants will contribute to crop improvement.

Basal stem rot (BSR), which is caused by Ganoderma sp, is the most serious disease which affects mature palm trees. Ganoderma come from the fungi kingdom and are basidiomycetes which cause the rotting of hardwoods of palm trees by decomposing lignin, as well as cellulose and related polysaccharides. Ganoderma species which cause stem rot (or butt rot) disease include, but are not limited to, Ganoderma boninense, Ganoderma lucidium (prominent in temperate region), Ganoderma miniatocinctum, Ganoderma zonatum and Ganoderma tornatum. Of particular interest is Ganoderma boninense, which is a main threat to palm trees, such as oil palms in the Southeast Asia region. The disease involves degradation in the base or butt of the palm trees. AH palm trees are susceptible to Ganoderma basal stem rot. Currently, no palm trees are resistant to this disease. The abovementioned problem has been known for generations as search for solutions has been considerably hindered by natural restrictions. These restrictions include slow disease progress where the disease does not show any visual or physical symptoms until the oil palms are at least 7 to 15 years old. Though many researches on Ganoderma infection have been carried out over the years, basic understanding at the molecular level of Ganoderma infection has not been st!udied. Most of the studies looked at the problem from an applied point of view and this has had limited results. Some studies looked at detecting Ganoderma disease using roots of palm tree. This method however requires digging into the soil to harvest the roots for analysis. This method can cause adverse harm to the palm tree especially if digging is not carried out with care.

Consequently, there is a need to provide a method that seeks to detect Ganoderma- infected palm trees at an early stage without causing any adverse harm to the palm trees or to address at least one of the problems described hereinabove, or at least to provide an alternative.

SUMMARY OF THE INVENTION The above and other problems are solved and an advance in the art is made by providing a method for identifying a Ganocferma-infected palm tree in accordance with this invention. It is an advantage that the method in accordance with this invention allows early detection of Ganoderma disease in a palm tree without causing any adverse harm to the palm tree. A second advantage of this invention is that with the early detection, actions can be taken early to prevent basal stem rot disease in palm tree or to cure the palm tree of the disease.

In accordance with an embodiment of this invention, a method for identifying a Ganoderma-infected palm tree is provided. The method comprises the steps of isolating proteins from a sample derived from leaves of a palm tree; determining protein profile of the sample by subjecting the proteins from the sample to ge! electrophoresis on a 2-dimensional gel; and comparing the protein profile of the sample to protein profile obtainable from a normal non-Ganoc erma-infected paim tree, wherein a difference in protein profile of the sample as compared to the protein profile of the normal non-Ganocferma-infected palm tree indicates that the palm tree is infected with Ganoderma disease.

In accordance with some embodiments of this invention, the palm tree is selected from the group comprising oil palm, coconut palm, date palm, sago palm, nipa palm betelnut (Acreca catechu) and ornamental palms. In accordance with an embodiment of this invention, the palm tree is an oil palm.

In accordance with some embodiments of this invention, the sample comprises a palm tree extract or a palm tree fraction obtainable from the leaves of the palm jree.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other features and advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings:

Figure 1 illustrates how artificial infection is carried out to obtain Ganoderma-infected seedlings.

Figure 2 shows images of a 2-dimensional gel electrophoresis separation of proteins in GeM containing samples H2 (not infected with Ganoderma at month 2), M2 (infected with Ganoderma boninense PER71 at month 2) and an overlay image of both samples.

Figure 3 shows images of a 2-dimensional gel electrophoresis separation of p'roteins i in Gel_2 containing samples H3 (not infected with Ganoderma at month 3), M3 (infected with Ganoderma boninense PER71 at month 3) and an overelay image of both samples.

Figure 4 shows images of a 2-dimensional gel electrophoresis separation of proteins in Gel_3 containing samples H4 (not infected with Ganoderma at month 4), M4 (infected with Ganoderma boninense PER71 at month 4) and an interna! overlay image of both samples. Figure 5 shows images of a 2-dimensional gel electrophoresis separation of proteins in Gel_4 containing samples H5 (not infected with Ganoderma at month 5), M5 (infected with Ganoderma boninense PER71 at month 5) and an overlay irrjage of both samples.

Figure 6 shows images of a 2-dimensional gel electrophoresis separation of proteins in Gel_5 containing samples H6 (not infected with Ganoderma at month 6), M6 (infected with Ganoderma boninense PER71 at month 6) and an overlay image of both samples.

Figure 7 shows images of a 2-dimensional gel electrophoresis separation of proteins in Gel_6 containing samples H7 (not infected with Ganoderma at month 7), M7 (infected with Ganoderma boninense PER71 at month 7) and an overelay irriage of both samples.

Figure 8 shows images of a 2-dimensional gel electrophoresis separation of proteins in Gel_7 containing samples E2 (infected with Ganoderma boninense SDG55 at month 2), E3 (infected with Ganoderma boninense SDG55 at month 3) and an overlay image of both samples.

Figure 9 shows images of a 2-dimensional gel electrophoresis separation of proteins in Gel_8 containing samples E4 (infected with Ganoderma boninense SDG55 at month 4), E5 (infected with Ganoderma boninense SDG55 at month 5) and an overlay image of both samples.

Figure 10 shows images of a 2-dimensional gel electrophoresis separation of proteins in Gel_9 containing samples E6 (infected with Ganoderma boninense SDG55 at month 6), E7 (infected with Ganoderma boninense SDG55 at month 7) and an overlay image of both samples.

Figure 1 1 is an exploded image of an overlay of samples H2/M2 for GeM .

Figure 12 is an exploded image of an overlay of samples H3/ 3 for Gei_2 Figure 13 is an exploded image of an overlay of samples H4/M4 for Gel_3.

Figure 14 is an exploded image of an overlay of samples H5/M5 for Gel_4.

Figure 15 is an exploded image of an overlay of samples H6/M6 for Gel_5.

Figure 16 is an exploded image of an overlay of samples H7/M7 for Gel_6.

Figure 17 is an exploded image of an overlay of samples E2/E3 for Gel_7.

Figure 18 is an exploded image of an overlay of samples E4/E5 for Gel_8.

Figure 19 is an exploded image of an overlay of samples E6/E7 for Gel_9.

Figure 20 shows a representative image of a 2-dimensional DIGE gel produced using sample H6 vs 6 (where H6 is a non-Ganoderma infected sample at month 6; M6 is a Ganoderma PER71 infected sample at month 6). The DIGE gel image shown in Figure 20 has been cropped to illustrate only protein spots from pH 4 to 9. The numbers shown on the image correspond to the spot numbers listed in Table 1

Figure 21 is a pie chart showing the functions of the differentially expressed proteins during Ganoderma infection.

DESCRIPTION OF THE INVENTION

The present invention relates to a method for identifying a Ganoderma-infected palm tree. More particularly, the present invention relates to a method for identifying a Ganoderma-infected palm tree using sample derived from leaves of the palm tree and applying difference gel electrophoresis (DIGE) technique to the sample.

The method comprises first isolating proteins from a sample derived from leaves of the pa!m tree. The proteins from the sample can be isolated by a known method described in Wang et al. (2006), "A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis", Electrophoresis 27:2782-2786 ί or using any suitable method known in the art.

After the proteins are isolated from the sample, the proteins are subjected to gel electrophoresis on a 2-dimensional gel to determine the protein profile of the sample. The protein profile of the sample is then compared with the protein profile of a normal non-Ganoderma-infected palm tree. A difference in the protein profiles of the sample and that of the normal non-Ganoderma-infected palm tree indicates that the palm tree is infected with Ganoderma disease.

The Ganoderma species which causes the Ganoderma disease may include, but is not limited to, Ganoderma boninense, Ganoderma lucidium (prominent in temperate region), Ganoderma miniatocinctum, Ganoderma zonatum and Ganoderma tornatum. In one embodiment of the present invention, the Ganoderma species that causes the Ganoderma disease is Ganoderma boninense.

Palm tree as herein referred to includes, but is not limited to, oil palm, coconut balm, date palm, sago palm, nipa palm, beteinut (Acreca catechu) and ornamental p'alms. In some embodiments of the present invention, the sample may comprise a palm tree extract or a palm tree fraction obtainable from the leaves of a palm tree.

In the present invention, key plant proteins which are related to fungal infection responses in palm tree can be identified by analyzing proteomes at various infection stages. For example, proteins can be isolated from samples derived from palm tree and analyzed across several time points, such as in a monthly or bimonthly manner.

A palm tree is determined to be infected with Ganoderma disease when the protein profile of the sample from the palm tree includes two or more proteins selected from the group consisting of the proteins as shown in Table 1 below.

Table 1

[Elaeis guineensis]

218,224, 60S ribosomal protein L7-iike

gi 1192912978 Metabolism protein synthesis 225 protein [Elaeis guineensis]

ABC transporter B family

integral membrane member 15 OS=Arabidopsis

334 AB15B ARATH Metabolism proteins involved in thaliana GN=ABCB15 PE=1

diverse cellular processes SV=1

ABC transporter C family integral membrane

56 member 2 OS=Arabidopsis AB2C ARATH Metabolism proteins involved in thaliana GN=ABCC2 PE=1 SV=2 diverse cellular processes abscisic stress ripening over-expressed! under

267 gi 1270064305 Stress response

[Musa ABB G oup] water/salt stress acetyl-CoA carboxylase

57, 78 gi 1209168853 Metabolism biosynthesis of fatty acids

[Elaeis guineensis]

170, 172 actine [Elaeis guineensis] gi 1 8527433 Cell structure housekeeping

AGAMOUS-like MADS box

203,

transcription factor gi 173852969 Metabolism metabolic pathway/KEGG 206,207

[Elaeis guineensis]

catalyse mainly the aldo/keto reductase family reduction of carbonyl protein groups or carbon-carbon

220 gi 1297848944 Metabolism

[Arabidopsis lyrata subsp. double bonds of a wide lyrata] variety of substrates

including steroids reactive oxygen gene

290, ascorbate peroxidase, cytosolic Disease

gi 1192912966 network; cellular signaling;92, 293 [Elaeis guineensis] /defense

PR-9

136, ATP synthase beta subunit nergy - electro gi 1192910868 Energy

12, 335 [Elaeis guineensis] transport

ATP-dependent clp protease

28, 36 gi 1307136002 Energy chloroplast function

[Cucumis melo su bsp.]

301,

307, carbonic anhydrase 3 [Flaveria

308, vaginata] & carbonic i 1326582932,

Energy carbohydrate metabolism 316, anhydrase [Triticum turgidum Ί 290875537

317, subsp. durum x Secale cereale]

20, 321

CBL-interacting protein kinase

302 4 05=Oryza sativa subsp. CIPK4 ORYSJ Disease/defense signaling

japonica GN=CIPK4 PE=2 SV=2

chlorophyll A/B binding

287 protein, putative gi 1255557387 Energy light-harvest g

[Ricinus communis]

chloroplast manganese

260 stabilizing protein gi 1313586398 Energy PS II

[Solanum tuberosum]

catalyze the isomerization of peptidyl— prolyl bonds, play a variety of important roles in infection,

Housekeeping

including facilitating host

357 cyclophilin [Elaeis guineensis] gi 1192910744 disease/

penetration and defense

colonization and activating pathogen effector proteins within the host cytoplasm electron transport chain

277, cytochrome c oxidase subunit 11

ei l 109892850 Energy transmembrane protein37, 370 PS17, putative

complex I

96, 98 disulfide isomerase 2 precu rsor gi 1192912964 Cell structure protein foldi g

134,

138,

139,

141,

143,

144,

155,

161,

181,

183,

193,

196,

276,

281,

297,

339,

341,

343,

348,

371,

375,

377,

380,

serine-glyoxylate transferase; participates in

73 168 aminotransferase, putative gi 12754849 Metabolism glycine, serine and

[Fritillaria agrestis] threonine metabolism stearoyl-Acyl-carrier protein i

74 186 gi 11785862 Metabolism fatty acid biosynthesis desaturase [Elaeis guineensis]

328,

329, thioredoxin peroxidase Disease/

75 gi 1192910848 PR-9

330, [Elaeis guineensis] defense

331, 332

T-protein of the glycine

185, phosphoresp ratory

76 decarboxylase complex gi 1407475 Metabolism

188, 189 pathwa /

[Pisum sativum]

reductive and cjxidative pentose phosphate

64, 70, gi | 14787119,

77 transketolase Metabolism pathways - synthesis of 72, 73 gi 175140229

sugar phosphate intermediates translation elongation factor G, protein synthesis; peptide

78 44 gi 1 255537029 Cell structure

putative [Ricinus communis] elongation

carbohydrate metabolism, triose phosphate isomerase

balance of metabolic

79 286, 288 cytosolic isoform gi 1 192910674 Metabolism

fluxes in plant primary [Elaeis guineensis]

metabolism vitamin-bl2 independent

methionine synthase, 5- photosysnthesis and

80 49 methyltetrahydropteroyltriglut gi 1224131686 Metabolism

respiration; methylation amate-homocysteine

[Papulus trichocarpa]

There are several advantages in using leaves or leaf tissues of palm tree to determine if a palm tree is infected with Ganoderma disease in the method of the present invention. First of all, the present invention relies on the systemic responses, in terms of protein profiles, of palm trees in determining if the palm trees are infected with the Ganoderma disease. Regardless of which root branch is infected with the Ganoderma disease, infection can easily be detected from the leaves of the palm tree by applying DIGE technique to proteins isolated from samples derived from the leaves of the palm tree to determine the protein profile of the palm tree. A difference in the protein profile of the samples and that of a normal non-Ganocferma-infected palm tree indicates that the palm tree is infected with the Ganoderma disease. One may not accurately detect that the palm tree is infected with the Ganoderma disease if root tissues are used as the root that is used for the detection may not be th one that is infected with the disease.

Another advantage of the present invention is that leaves grow on the aerial part of palm tree and they are therefore easily accessible. Collecting samples from leaves of palm tree for analysis will not cause any adverse harm or injury to the palmj tree. Roots of palm tree, on the other hand, are hidden underground, in the soil. To harvest roots for analysis, one needs to dig into the soil to harvest the roots. Digging into the soil can sometimes cause adverse harm or injury to the roots especially if digging is not carried out with care.

Leaves of palm tree are present in abundance. Samples from leaves of palm tree can therefore easily be obtained and this allows regular inspection of the palm tree to be carried to determine if the palm tree is infected with the Ganoderma disease, j This in turn allows early detection of the Ganoderma disease.

Furthermore, roots are often contaminated with soil and microbes which may hjinder accurate determination of the disease. Leaves, on the other hand, are relatively clean and this allows more accurate analysis to take place. The use of leaves allows easy-to-use diagnostic kits to be developed for the detection of Ganoderma disease.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Ganoderma strain isolation and identification To infect oil palm seedlings with Ganoderma disease, two strains of Ganoderma boninense were used as inocula. They were PER71 (a strain originated from United Plantations, Perak and is obtainable from the Pathology Lab, Malaysian Palm Oil Board) and SDG55 (a strain isolated by Plant Protection Unit, Sime Darby _{ R&D Centre).

Inoculum cubes were prepared by culturing the fungal pathogen on surfaces of 6 x 6 x 6 cm rubber wood cubes based on the method described in Idris et al. (2006), "Technique for inoculation of oil palm geminated seeds with Ganoderma", MPOB Information Series No. 321 , pp. 4, June 2006. The cubes of dried rubber wood were washed and put into polypropylene bags containing 60 ml of 5.6% malt extract agar. The bags were sealed prior to autoclaving for 1 hour at 121°C. Upon cooling down, the cubes were inoculated with Ganoderma boninense and then placed in the dark at room temperature for 60 days. Fully colonized cubes were used as inoculum cubjes.

Preparation of Non-Ganoderma infected and infected samples of oil palms

Germinated seeds of oil palm used in this example were of commercial DxP standard cross, obtained from Sime Darby Seeds & Agricultural Services Sdn. Bhd., Banting, Malaysia.

Ganoderma-infected seedlings were produced by artificial infection (see Figure 1 ). Here, the inoculum cubes, fully colonized by the pathogen, were placed in the middle of a 30 x 38 cm black polythene bag filled with unsterilized top soil. Germinated seeds were then planted 4 to 5 cm above the inoculum cubes. For control where

infected seedlings were needed, the seeds were planted! as described but with the absence of inoculum cubes. All the seedlings were maintained with regular watering and manuring as prescribed by Sime Darby Good Agriculture Practice (Sime Darby Plantation, 2001 ), but with no application of any fungicide or pesticide throughout the whole period of growing the seedlings. To minimize jaest attack, netting was put up as a physical barrier around and above the nursery where the oil palms were kept. Leaf samples from the seedlings were collected monthly, beginning from the second month to the seventh month after the seeds were planted, for analysis. It is believed that infection, via the roots, has taken place by the second month under this mode of artificial infection; supported by the presence of disease symptom like foliar yellowing and browning, as described in (dris et al. (2006).

In this Example, leaves of 10 different seedlings per treatment were pooled add their protein extracted for Difference Gel Electrophoresis (DIGE) analysis. 2-Dimensional DIGE experiment

A modified protein extraction method published by Wang et al. (2006) was used to isolate total leaf protein. Subsequently, the extracted protein samples were re- i suspended in a 2-dimensional cell lysis buffer (30 mM Tris-HCI, pH 8.8, containing 7 M urea, 2 M thiourea and 4% CHAPS). The mixture was sonicated at 4°C, followed by shaking for 30 minutes at room temperature. The samples were then centrifuged for 30 minutes at 14,000 rpm and the resulting supernatant was collected. Protein concentration of the supernatant fraction was measured using Bio-Rad protein assay method as described in Bradford M.M. (1976), "A dye-binding method for the determination of microgram quantities of protein", Anal. Biochem, 72:248-54.

To aid downstream analysis, an internal standard (IS) was prepared, by mixing equal amount of protein from each sample. Each internal standard prepared was included in the 2-dimensional DIGE experiment.

CyDye iabeSiing

For each sample, 30 g of protein was mixed with 1 .0 μΙ of diluted CyDye, and the mixture was kept in the dark on ice for 30 minutes. Samples from each pair (e.g. not . infected and infected samples) were labelled as Cy3 and Cy5 respectively, while the internal standard was labelled as Cy2, The labelling reaction was carrying out by adding 1 .0 μΙ of 10 mM Lysine to each sample, and incubating each sample in the dark on ice for additional 15 minutes. The 3 labelled samples were then mixed together. Two times of the 2-D sample buffer (8 M urea, 4% CHAPS, 20 mg/rni DTT, 2% pharmalytes and trace amount of bromophenol blue), 100 μΙ of Destreak solution and rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mg/ml DT|T, 1 % pharmalytes and trace amount of bromophenot blue) were added to the labelling mix to make a total volume of 250 μΙ. The resulting mixture was thoroughly mixed and spun down prior to loading the labelled samples onto immobilized pH gradient gel (IPG) strips housed in a strip holder.

Setup of 2-dimensional DIGE analytical gels

DIGE gels were designed to contain the appropriate sample pairings in order to facilitate gel analysis in the later part of the experiment. A total of 9 DIGE gels were produced with the sample pairings indicated below:

Gel 1 : H2, M2, IS

Gel 2: H3, M3, IS

Gel 3: H4, 4, IS

Gel 4: H5, M5, IS

Gel 5: H6, M6, IS

Gel 6: H7, 7, IS

Gel 7: E2, E3, IS

Gel 8: E4, E5, IS

Gel 9: E6, E7, IS wherein,

H represents

infected samples (control);

M represents samples infected with Ganoderma boninense PER71 ;

E represents samples infected with Ganoderma boninense SDG55; and

IS represents the internal standard which was made up of a pool of all the 18 sanhples included in this analysis. The numbers indicate months after seed planting. internal Standard (IS) internal Standard (IS) is used to match and normalize protein patterns across different DIGE gels, thereby negating the problem of inter-gel variation. The iS allows accurate quantification of differences between samples with an associated statistical significance. Quantitative comparisons of protein between samples are made on the relative change of each protein spot to its own in-gel internal standard.

IEF (isoelectric focusing) and SDS-PAGE (polyacrylamide gel electrophoresis)

The labelled samples prepared above were then loaded onto pH 3 to 10 IPG strips. Isoelectric focusing (IEF) was run following the protocol provided by Amersham Biosciences in GE Healthcare (2004), "2-D electrophoresis: principle and methods, pp. 43-72. Upon finishing the IEF, the IPG strips were incubated in a freshly prepared equilibration buffer 1 (50 mM Tris-HCI, pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS, trace amount of bromophenol blue and 10 mg/ml DTT) for 15 minutes^' with gentle shaking. The strips were then rinsed in another freshly prepared equilibration buffer 2 (50 mM Tris-HCI, pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS, trace amount of bromophenol blue and 45 mg/ml DTT) for 10 minutes with gentle shaking. Following that, the IPG strips were rinsed in a SDS-gel running buffer before the strips were transferred into 12% SDS-gels. The SDS-gels were run at 15°C until the dye front of the strips ran out of the gels.

Image scan and data analysis

Gel images were scanned immediately following the SDS-PAGE using Typhoon '^"RIO (Amersham Biosciences). The scanned images were then analyzed by Image Quant software (version 6.0, Amersham Biosciences), followed by DeCyder™ 2D software version 6.5 (Amersham Biosciences).

The gel images of the 9 analytical gels were as shown in Figures 2 to 10 respectively.

RESULTS

2-Dimensaonal D!GE Analytical Gels results

The 9 analytical gels, produced from 18 samples, were analyzed to detect the differentia! expression of the proteins in following pairs: (1) M2 vs.. H2

(2) M3 vs. H3

(3) M5 vs. H5

(4) M6 vs. H6

(5) M7 vs. H7

(6) E2 vs. H2

(7) E3 vs. H3

(8) E4 vs. H4

(9) E5 vs. H5

(10) E6 vs. H6

(1 1) E7 vs. H7

Figure 20 is a representative image of a 2-dimensional DIGE gel produced using sample H6 vs M6 (where H6 is a non-Ganoderma infected sample at month 6; i i6 is a Ganoderma PER71 infected sample at month 6). The DIGE gel image shown in Figure 20 has been cropped to illustrate only protein spots from pH 4 to 9.

Collectively, the number of differentially expressed proteins found in these pair-wise analyses was 382, after being narrowed down to those with expression ratio greater than 1.5 folds change (see Table I below). Sequentially, the corresponding protein spots in their respective gels were cross-checked by eye to ensure they were of good quality/resolution spots with no artificial streaks or not an artifact. All were not found to be artifacts. Of the screened spots, 210 were subsequently selected for identification via mass spectrometry.

The spot selection criteria for mass spectrometric identification practiced in this example were based on (i) the visibility of the spots on the DIGE gels; (ii) the projein expression ratio greater than 1 .5 folds change; and (iii) the occurrence of protein isoform. Candidate spots that were not clearly visible on the DIGE gel (and having lower than 1 .5 folds change in expression), as well as being redundant (its isoform has already been chosen), were excluded for identification work.

2-dimensionai DiGE preparative gels for spot picking 13 000187

Gels used for spot picking are termed as preparative gels. The preparative gel was run after differentially abundant spots were identified from analytical gels. For spot identification work, Gel 5 (with H6, M6, IS samples) was reproduced as prepajrative DIGE gel for spot picking as it contains all the 382 differentially expressed spots.|

To have sufficient protein sample for identification purposes, 3 preparative DIGEE gels were prepared for spot picking and the spots pooled. These gels were used fori spot- picking all the 210 protein spots of interest. The spots picked were sent for jmass spectrometry identification via MALDI-ToF ToF.

Identification assignment of differentially expressed protein spots

Of the 210 spots sent for mass spectrometric identification, 200 spots were positively matched based on peptide mass finger-printing spectra to existing proteins from public domains (NCBI non-redundant database) while 10 were matches of no confidence (protein score C.I. (confident index) % lower than 80% is regarded as no confidence). From the 200 positive matched ID's, repetitive identities assigned to different spots for 120 proteins were removed, leaving behind 80 unique pj-otein matches or ID's (see Tables 1 and 2). Of the 40 proteins with repetitive assignments, majority were of photosynthesis related proteins like ribulose-1 ,5-bisphosphate carboxylase/oxygenase large subunit and oxygen evolving enhancer proteins.

In the present invention, 80 protein leads (see Table 1 ) have been identified to be associated to the occurrence of ganodermatosis in oil palm seedlings.

The 80 proteins identified were grouped based on their molecular functions (see Table 1). Of these, 1 proteins were found to be uncharacterized. The annotated proteins were found to be involved in photosynthesis, protein modifications, transport, energy, signal transduction, metabolisms, stress responses and defenses (Figure 20).

Apart from the 80 proteins detected, more differentially expressed proteins are expected, knowing that disease manifestation is a complex interaction between the pathogen and its host, possibly involving multiple intermediary metabolic pathways. In this example, protein samples were focused on broad pH range IEF strips, i.e. between pH 3 and 10. Therefore, resolution of some proteins could be comprojnised and the protein spots could possibly be masked by proteins of higher abundance.

Of the 80 proteins detected, ginkbilobin is of particular interest because of its antifungal property (Wang et al. (2000), "Ginkbilobin, a novel antifungal protein from Ginkgo biloba seeds with sequence similarity to embryo-abundant protein", Biochemical and Biophysical Research Communications 279:407-11).

In this study, ginkbilobin was up-regulated 1.9 folds in seedlings 6 months after the inoculation of Ganoderma (see expression profile of Spot 271 in Table 2). Although the up-regulation observed was not greater than 2 folds, the significance of this protein in oil palm defense against Ganoderma still warrants a further investigation.

In this study, cyclophilin, a ubiquitous housekeeping protein, was expressed 2.8 folds higher in infected seedlings at month 5 post infection (see Spot 357 in Tab le 2). Godoy et al. (2000). "Expression of a Solanum tuberosum cyclophilin gejne is regulated by fungal infection and abiotic stress conditions", Plant Sci. 152:123-34 observed a higher cyclophilin RNA transcripts in tuber tissues (compared to other tissues) of Solanum tuberosum during the infection of fungus Fusarium solani. Cylophilin, which catalyzes the isomerization of peptidyl— prolyl bonds, known to accelerate folding of certain proteins and can function as a chaperone (Freskgard et al. (1992), "Isomerase and chaperone activity of prolyl isomerase in the folding of carbonic anhydrase", Science 258: 466-68; Gething et al. (1992), "Protein folding in the cell", Nature 355:33-35; Schmid et al. (1993), "Prolyl isomerases: role in protein folding", Adv. Protein Chem. 44: 25-66). It has also been indicated to play a variety of important roles in infection, including facilitating host penetration, as well as host colonization (Gan et al. (2009), "Characterization of cyclophilin-encoding genes in Phytophthora" , ol. Genet. Genomics 281 : 565-78). Furthermore, the activation of pathogen effector proteins within the host cytoplasm is attributed to this protein (Gan et al. , 2009). Taking all these together, the up-regulation of oil palm cyclophilin during infection is intriguing, as if the host is encouraging the invading pathogen.

The differential regulation of proteins in non-infected and infected oil paim ailuded to the possibility of using non-pathogen related proteins as markers in differentiating a diseased palm from a healthy one. Building on this thought, antibodies for the proteins identified are obtained for further validation work via immuno-detectiojn in a larger sample size, including samples from the fields. The protein leads identified in the present invention can also be used as markers in screening for oil palms which are resistant or tolerant to Ganoderma. As plant infection takes place, it would onset a mechanism called as systemic acquired resistance (Colditz et al. (2007), "Plant proteomics upon fungal attack", Plant Proteomics, Samaj, J. and Thelen, J. (eds.). pp. 283-309. Springer). During this mechanism, plants would up-regulated defense related to biochemical compounds, may it be in the form of metabolites or polypeptides, to counter the invading pathogen systemically. Proteins found differentially expressed in this study, especially the up- regulated ones, would probably have an important role in systemic acquired resistance; making them a potential markers for selecting tolerant palms. The capability in identifying disease tolerant palms would be invaluable to oil palm breeding program and industry.

Table 2

^ j m r q -— o w

«V oi ^■* oi co

co -— o o

co

co co co m c° ^ ° co co r— c o

r— r— r— r- r— r— c m

" oi

T

VDAC_PE gi|19291067 gi|25555738 gi|19291067 gi|19291296 gi|19291296 gi|19291296

FBK69_ARA gi|218210

gi|3265829 CIPK4 ORY gi|813159 gi|1113403

GUN18_OR gi|2555476

PUR6_VIG gi|2555476 gi|2555476 gi|2555514 gi|1929107 gi|1929109 gi|776808 gi|13117 ] gi|152301 gi|1956338 gi|1956338 DCL1_OR gi|115472

PS17 PIN

Appearance # of gels the spots can be detected

P value Student t test

Av. Ratio: M / H, E / H Positive value means increased ratio

Negative value means decreased ratio

The results above demonstrate that the technique of protein analysis using DIGE can effectively differentiate non-Ganoderma infected oil palm from Ganoderma nfecjted oil palm. The above is a description of the subject matter the inventors regard as the invention and is believed that others can and will design alternative processes that include this invention based on the above disclosure.

Claims

1. A method for identifying a Ganoderma-infected palm tree, comprising:

isolating proteins from a sample derived from leaves of a palm tree;

determining protein profile of the sample by subjecting the proteins from the sample to gel electrophoresis on a 2-dimensional gel; and

comparing the protein profile of the sample to a protein profile obtainable^ from

Ganoderma disease.

2. The method according to claim 1 , wherein the palm tree is infected; with

I

Ganoderma disease when the protein profile of the sample includes two or more proteins having an energy function, wherein the proteins are selected from the group consisting of ATP synthase beta subunit (ID No. gi|192910868); ATP-dependent dp protease (ID No. gi|307136002); carbonic anhydrase 3 and carbonic anhydrase (ID No. gi|326582932 and, gi|290875537); chlorophyll A/B binding protein, putative (ID No. gi|255557387); chloroplast manganese stabilizing protein (ID No. gi|313586398); cytochrome c oxidase subunit II PS17, putative (ID No. gi|109892850); ferredoxin- NADP reductase, putative (ID No. gi|255538962); glyceraldehyde 3-phosphate dehydrogenase (ID No. gi|824002 5); NADH-cytochrome b5 reductase-like protein OS=Arabidopsis thaliana GN=At5g20080 PE=1 SV=2 (ID No. NCB5R_ARATH); oxygen evolving enhancer protein 1 [Litchi chinensis] (ID No. gi|326467059); oxygen evolving enhancer protein 2 (ID No. gi|813 593, gi|297826499 and gi|1 1 134035); oxygen-evolving enhancer protein 3-1 [Zea mays] (ID No. gi|195633817); peroxisomal glycolate oxidase (ID No. gij 167962794); phosphoenolpyruvate carboxylase (ID No. gi|7768089); Photosystem I reaction center subunit II, chloroplast (ID No. gi|255551453); Photosystem I reaction center subunit IV, chloroplastic, Short=PSI-E; AltName: Full=P (ID No. gi|131 176); Photosystem II 23 kDa polypeptide (ID No. gi|19898); protochlorophyllide reductase A, chloroplastic; Short=PCR A; AltName: Full=NADPH- protochlorophyllide oxidoreductase A (ID No. gi|129708); PsbP (id No. gi|25992740); . and ribuiose-1 ,5-bisphosphate carboxylase/oxygenase (ID I No. gi| 1 155871 7, gi|76574195 gi|134104 and gi|7960277).

3. The method according to claim 1 , wherein the palm tree is infected with Ganoderma disease when the protein profile of the sample includes two or more proteins having a metabolism function, wherein the proteins are selected from the group consisting of 24 kDa seed maturation protein (ID No. gi|192910858); 40S ribosomal protein S5 (ID No. gi| 192910844); 60S ribosomal protein L7-like protein (ID No. gi|192912978); ABC transporter B family member 15 OS=Arabidopsis thaliana GN=ABCB15 PE=1 SV=1 (ID No. AB 5 B_AR ATH ) ; ABC transporter C family member 2 OS=Arabidopsis thaliana GN=ABCC2 PE=1 SV=2 (ID No. AB2C_ARATH); cetyl-CoA carboxylase (ID No. gi|209168853); AG A O US-like MADSi box transcription factor (ID No. gi|73852969); aldo/keto reductase family protein (ID No. gi|297848944); E3 ubiquitin-protein ligase SINA-like 6, putative (ID I No.

i

SINL6_ARATH); elongation factor Tu (ID No. gi|225456880); fructose-bisphosbhate aldolase (ID No. gi|255557204 and gi|300681519); Geminivirus Rep-interacting motor protein OS=Arabidopsis thaliana GN=GRIMP PE=1 SV=T (ID No. GRIMP_ARATH); glutamine synthetase, cytosolic (ID No. gi|113171384); IAA type protein (ID No. gi|50404477); isopentenyl-diphosphate Delta-isomerase I OS= Camptotheca acuminata GN=IPI1 PE=2 SV=1 (ID No. IDI _CAMAC); P1 B-ATPase heavy-jnetal transporter [Oryza sativa Japonica Group] (ID No. gi|315623028); pentatricopeptide, putative, expressed [Oryza sativa Japonica Group] / Pentatricopeptide repeat- containing protein At4g38010

thaliana GN=PCMP-E45 PE=2 SV=1 (ID No. At4g38010 and gi| 108864632); peptidyl-prolyl cis-trans isomerase, putative

phosphate isomerase cytosolic isoform (ID No. gi| 1929 0674); and vitamint-b12 independent methionine synthase, 5-methyltetrahydropteroyltrig!utart ate- homocysteine (!D No. gi{224131686).

4. The method according to claim 1 , wherein the palm tree is infected with Ganoderma disease when the protein profile of the sample includes two or more proteins having a stress response function, wherein the proteins are selected from the group consisting of abscisic stress ripening (ID No. gi|270064305); heat shock protein 70 or 90 (ID No. gi|238054071 , gi|255570990, gi|2827002, gi|6969970 and gi| 108862740); and phosphoglycerate kinase (ID No. gi|226530482 and gi|332591479).

5. The method according to claim 1 , wherein the palm tree is infected with Ganoderma disease when the protein profile of the sample includes two or¹ more proteins having a disease/defense function, wherein the proteins are selected from the group consisting of ascorbate peroxidase, cytosolic (ID No. gij 192912966); CBL- interacting protein kinase 4 OS=Oryza sativa subsp. japonica GN=CIPK4 PE=2 SV=2 (ID No. CIPK4_ORYSJ); endoglucanase 18 OS=Oryza sativa subsp. Japonica GN=Os06g0715300 PE=2 SV=1 (ID No. GUN18_ORYSJ); endoribonuclease jDicer homolog 1 OS=Oryza sativa subsp. japonica GN=DCL1 PE=3 SV=1 (ID No. DCL1_ORYSJ); ginkbilobin-1 (ID No. GNK1_GINBI); isoflavone reductase, putative (ID No. gi|255543713); peroxiredoxin (ID No. gi|192910922); peroxisome type ascorbate peroxidase (ID No. gi|192910808); pterocarpan reductase-like protein (ID No. gi| 169639232); thioredoxin peroxidase (ID No. gi|192910848); and cyclophilin (ID No. gi|192910744).

6. The method according to claim 1 , wherein the palm tree is infected j with Ganoderma disease when the protein profile of the sample includes two or more proteins having a cell structure function, wherein the proteins are selected from the group consisting of actine (ID No. gi|48527433); disulfide isomerase 2 precursor (ID No. gi|192912964); F-box/kelch-repeat protein (ID No. gi| 15230186); fiber protein Fb2 (ID No. gi|192910804); and translation elongation factor G, putative (ID No. gi|255537029);.

7. The method according to claim 1 , wherein the palm tree is infected with

Ganoderma disease when the protein profile of the sample includes two or more proteins selected from the group consisting of Micromonas pusilla CCMP1545 (ID No. gi|303275936); Oryza sativa Japonica Group (ID No. gi|22202781 ); Physcomitrella patens subsp. patens (ID No. gi|168034023); Populus trichocarpa (ip No. gi|224140861); Vitis vinifera (ID No. gi|225466832); Zea mays (ID No. gi|219363037); Selaginella moellendorffii (ID No. gi|302803987); VITISV_010874 (ID No. gi| 147843505) VITISV_023080 (ID No. gi| 147766924); LOC100284083 (ID No. gi|226530191) Os04g0234600 (ID No. gi|1 15457386); Os07g0544800 (ID No. gi| 115472625) porin, outer plastidial membrane protein OS=Pisum sativum GN=POR1 PE=1 SV=2 (ID No. VDAC_PEA); and Ras-related protein RIC1 (ID No. gi|192910784).

8. The method according to claim 1 , wherein the palm tree is infected with Ganoderma disease when the protein profile of the sample includes two or more proteins as claimed in any of claims 2 to 7.

9. The method according to claim 1 , wherein the palm tree is selected from the group consisting of oil palm, coconut palm, date palm, sago palm, nipa palm, betelnut

i

(Acreca catechu) and ornamental palms.

The method according to claim 9, wherein the palm tree is an oil palm.

11 . The method according to claim 1 or 9, wherein the sample comprises a palm tree extract or a palm tree fraction obtainable from the leaves of the palm tree.

12. The method according to claim 1 , wherein the protein profile of the sarhple comprises ginkbilobin and cyclophilin.

13. The method according to claim 1 or 10, wherein the Ganoderma diseasfe is caused by presence of Ganoderma boninense.