Phenolic diversity in the defence reaction of the oil palm against vascular wilt disease

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1 AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA ISSN Print: , ISSN Online: , ScienceHuβ, Phenolic diversity in the defence reaction of the oil palm against vascular wilt disease Diabaté 1 Sékou, Aké 2 Séverin, Kouamé 3 Kouamé Roger, Coulibaly 4 Ouolo Arsène, N'guessan Walet Pierre CNRA Laboratoire Central de Biotechnologies, Tel:(225) FAX: Corresponding Authors: sekou_diabate2002@yahoo.fr. COTE D IVOIRE 2 UFR Biosciences, Université de Cocody 22 BP 582 Abidjan 22 COTE D IVOIRE 3 CNRA Station de Recherche de La Mé 13 B.P. 989 Abidjan 13 COTE D IVOIRE 4 CNRA Station d Expérimentation et de Production B.P. 8 DABOU COTE D IVOIRE. ABSTRACT The dosage of the rooting and pseudobulb extracts of oil palm, after infection by the wilt disease pathogenic agent has revealed the presence of hydrophilic and lipophilic phenolic compounds in the tolerant crosses and clones. The analysis of the absorption spectrums of these compounds has shown that they belonged to five phenolic families: the cinnamic glycosylated derivatives, the flavonones, the chlorogenic esters, the flavones and the flavonols. This phenolic diversity has conferred the oil palm a strong tolerance to wilt disease. Keywords: Oil palm wilt disease - Tolerant - Phenol - Spectrum INTRODUCTION The vascular wilt disease of the oil palm is a disease having a cryptogamic origin, provoked by a soil fungi, Fusarium oxysporum f. sp elaeidis, which enters the plant trough the roots, migrates into the xylem and which is able to cause a more or less quick blight of the infected palm tree (Franqueville et Diabate, 1995). Two types of the disease exist on the fruitbearing palm tree: the typical or acute type (figure 1) which is characterized by a blight of the low leaves, the rachis breaks into approximately one third from the basis, the leaves hang alongside the stipe. The death occurs three to four months after the first symptoms (Diabate, 2008). This type is predominant after planting on forest. The chronic type (figure 2) is found especially in the savannah. The infected palm trees do not die but their production is reduced or null. The dry leaves fall then the palm tree emits two or three spears that open slowly. The stipe shrinks and takes the aspect of a pencil point (Diabate,2008). The methods of fight have been preventive ones and located essentially at the level of the choice of vegetable equipment after inoculation of the pathogenic agent and symptom observations at the pre-nursery stage. Since 1985, the research on the physiological mechanisms of the oil palm resistance have started in order to complete the genetic fight (Diabate,1985). Many works about the defence reaction of the oil palm against the wilt disease have highlighted the role of phenolic compounds in the roots and pseudobulbs (Taquet, 1985; Diabaté, 1985, 1990; Traoré, 2002). The tolerant crosses or clones of palm trees have shown more toxic phenol than the sensitive crosses or clones (Diabaté, 1992). What remains is to know to which molecule families belongs this phenolic group. In response, the absorption spectrums of the phenolic compounds resulting from alcoholic extracts of roots and pseudobulbs will be analyzed. The expected results of these works should permit to identify the tolerance markers to the oil palm wilt disease from the phenolic families. MATERIAL AND METHODS Biological material: It is composed of plants resulting from the "in vitro" vegetative multiplication called known wilt disease index clones (I). Clones Wilt disease Index (I) LMC LMC LMC LMC LMC The clone is tolerant when the wilt disease tolerance index (I) is inferior to 100. Inoculation: The chosen method is the one which consists in soaking the roots into the liquid inoculum of Fusarium oxysporum f. sp. elaeidis (pathogenic

2 agent of wilt disease) initiated by the CNRA s laboratory of palm tree phytopathology. The clones are dug up in order to provoke injuries at the level of the roots; afterwards the rooting system is washed into water, and then soaked into the liquid inoculum for 10 minutes. Afterwards the planting stocks are planted out in the loam before sampling. Incubation periods: After 48 hours of incubation following the F.O.E inoculation, each genotype is sacrificed by giving out a bunch of roots on one hand and a bunch of pseudobulbs (of leaves) on the other hand: these two organs are weighed and preserved into the cold. Extraction: The roots and the pseudobulbs are crushed into the liquid nitrogen (-160 C) and taken back into 40 ml of ethanol-water (70: 30) in the presence of an antioxidant, the sodium metabisulfite. The extraction of phenol is carried out by maceration of the tissues through vacuum boiling during 30 mn. After refrigeration the mixture is filtered and dried into the Rotavapor (double boiler) through vacuum at 40 C and taken back later into 4 ml of ethanol-water (70-30). Then the extract obtained is preserved into the cold. High pressure liquid chromatography (HPLC) analysis: The elution is done per gradient. The stationary phase is constituted by silica microspheres on which are grafted carbonated residues. The mobile phase is an aqueous solvent (solvent A = Water- Acetic acid ), which is enriched progressively during organic stage (solvent B = Acetonitrile-water-acetic acid ) according to a programmable gradient (Diabaté et al, 2009). The detection of phenolic compounds is done through spectrophotometry U.V visible between 280 and 350 nm. Structural analysis programs: From roots and pseudobulbs extracts, the objective was to isolate by semi-preparative HPLC, the main polyphenolic compounds of these two organs, in order to know the molecular structures. The chromatographic column used is a reverse-phase C18 ultrabase 250 mm (life sciences international) one. The detection was done by scanning of 250 to 350 nm. The identification of the oil palm defence phenolic compounds has caused the use of the spectrophotometry U.V. visible (Harbonne, 1967, Marbry et al, 1970, Markham, 1982), The coupling of high performance liquid chromatography (HPLC) with a spectrophotometric multidetector with diode battens which permitted to obtain the spectrum U.V. visible of each elution peak. RESULTS Comparison of the chromatographic profiles HPLC of the roots and pseudobulb of oil palm clones: The chromatographic profiles in the figures 3 and 4 have revealed 15 peaks or peak groups characterising the phenolic compounds resulting from the roots and pseudobulbs extracts. A qualitative comparison of the two organs has revealed two rather different metabolisms on a global basis: almost all the chromatographic information of the pseudobulbs was obtained in 53 mn meanwhile the roots have reached the chromatographic peak n 5. The phenolic metabolism of the pseudobulbs gave way then to the accumulation of products which are more hydrophilic than lipophilic contrary to the roots. Chromatographic profiles Analysis of pseudobulbs and roots phenolic extracts of clones infected by F.O.E. : The figures 5 to 9 have shown the spectrophotometric plotting obtained on the pseudobulbs and roots extracts. Thus five spectrums belonging to five phenolic chemical families were identified: Plotting A: cinnamic glycosylated derivatives Plotting B: flavonones, that is to say the precursors of types D and E Plotting C: representatives of the chlorogenic esters family Plotting D: flavones Plotting E: flavonols The structural families were positioned on the whole of the profile HPLC of an inoculated pseudobulb extract read at 3 different wavelengths: 280, 320 and 350 nm (figure 10). The reading at 280 nm has privileged the left part of the profile that is to say the most hydrophilic compounds while the reading at 350 nm has optimized the right part of the profile that is to say the most lipophilic phenolic compounds. This profile has revealed by order of elution the most hydrophilic compounds towards the most lipophilic ones: Structural family (A) of cinnamic derivatives; Structural family (D) of flavones: these two families were clearly detected at 280 nm; structural Family (B) of flavones; The representatives of the chlorogenic derivatives family (C) have had a diffuse distribution on the profile. 408

3 Structural family (E) of flavonols: these last 3 families have been clearly identified at 350 nm. DISCUSSION The active defence reaction results in the formation of mechanical and biochemical barriers. Concerning the biochemical barriers that we have studied, two categories of substances are implied; the pre-existing substances to the infection which contents, in parasite-bearing cells increase or don t change (Santiago et al, 2005), and the elaborated phytoalexins after the infection as it is the case in our results. The phenolic and terpenic compounds are the main pre-existing barriers to the infection, nevertheless compounds having higher molecular weight such as lignins, subérin and wall proteins rich in hydroxyprolin (HRPG) can intervene, particularly in the vascular diseases. The research on the phytoalexins gave rise to important works and several groups of inhibiting compounds are currently known in several botanical families. The inhibiting compounds having an in vitro toxic action against the micro-organics have been identified in great numbers in the dicotyledons and in the oil palm (Diabaté et al, 1992). In the framework of the parenchyma parasites and mainly in the legumes, there is an accumulation of isoflavonoids, kieviton in Phaseolus vulgaris (fabaceae) and pterocarpanes (Smith et al, 1973), pisatin in Pisium sativum (fabaceae) (Sutherland et al, 1980). All the same, in the shrubby plants, the response of the host to the infection can correspond to a synthesis (de novo) of phytoalexin; it is the case of the borbonol in the avocado tree. In the poaceae, avenalumins (Mayama et al, 1982) and triticens have been highlighted. The phytoalexins contribution has been also was described in the case of vascular disease: in the segments of tomato stem infected by Verticillium albo atrum (Deuteromycetes), the resistance is associated to an accumulation of rishitin as well as to that of phenolic compounds (Elgersma, 1980). In the case of the oil palm, our results have shown that in reaction to Fusarium oxysporum f. sp. elaeidis, the oil palm accumulated hydrophilic and lipophilic phenolic compounds in the roots and the infected pseudobulbs. They are a namely the flavones cinnamic derivatives, the flavonones, the chlorogenic and flavonols derivatives. These phenols, according to the classification of Haslam (1975), belong to the secondary flavonic compounds called flavonoids. Many functions are attributed to them in the resistance mechanisms of the plants (Maclure, 1975). Thus some flavones and flavonols have shown an oestrogen activity that would allow the plants to defend themselves against certain animals (Harbonne, 1977). Other flavonoids have also revealed insecticide properties like the rotenone tested by Elliger et al, (1980) that has shown an activity marked by the development of Heliotis zea caterpillars (destructive Lepidoptera of the corn). As well as the isochlorogenic acid which has shown inhibiting properties on the growth of Sclerotinia sclerotiorum (ascomycetes) in the sunflower, the biocide activity of the flavonoids, alkaloids and saponins extracted from the fruits of Capsicum fruitescens L. (Solanaceae) was used for the destruction of eggs and adults of Bemisia tabaci (aleurode) infesting tomatoes plants, Solanum lycopersicum (solanaceae) in Morocco (Aziz et al, 2005). In the oil palm, the flavonic molecules seem to act in different ways: indeed the core samplings that we have made in the stipe of fruit-bearing palm trees in production have revealed the presence of the fungi pathogen (brown fibres) in certain trees without demonstration of wilt disease (Diabaté, personal communication). The cultivation of these tissues on agar environments in laboratory indicated that the fungi was either living, or dead in the stipe of the palm tree. These results show that the phenol that intervene in the defence reaction of the oil palm possess two great properties: In the case when the fungi is living in the stipe of the palm tree without harming its development and its cluster production, the phenols would exercise on the fungi a fongistatic action conferring to the palm tree a tolerance to the disease; If case the fungi is noted as dead in the stipe of the palm tree, the phenols would as a fungicide on the fungi conferring to the palm tree a total resistance. Our results are therefore in favour of the fact that the majority of flavonoid phenolic compounds identified in the oil palm would play the role of an antibiotic, particularly phytoalexins which contribution is effective in the presence of the Fusarium oxysporum f. sp. elaeidis in order to confer the oil palm, a strong tolerance to vascular wilt disease. 409

4 Beginning of symptoms End of symptoms Fig 1 : Typical or acute symptoms of wilt disease in the oil palm at the station of Dabou (DIABATE, 2000) Beginning of symptoms End of symptoms Fig 2 : Chronic symptoms of wilt disease in the oil palm at the station of Dabou (DIABATE, 2000) 410

5 Fig 3: HPLC Profile of a phenolic extract of oil palm pseudobulbs detected at A=280 nm ; B= 320 nm ; C= 340 nm Legend: h= height of peaks λ= Wavelength Fig 4: HPLC Profile of a phenolic extract of oil palm root detected at A=280 nm ; B= 320 nm ; C= 340 nm 411 Legend : h= height of peaks λ= Wavelength

6 Fig 5: Spectrum of glycosylated cinnamic derivatives resulting from phenolic extracts of oil palm roots and pseudobulbs Fig 6 : Spectrum of flavonones resulting from phenolic extracts of oil palm roots and pseudobulbs Fig 7 : Spectrum of chlorogenic esters resulting from phenolic extracts of oil palm roots and pseudobulbs 412

7 Fig 8 : Spectrum of flavones resulting from phenolic extracts of oil palm roots and pseudobulbs Fig 9 : Spectrum of flavonols resulting from phenolic extracts of oil palm roots and pseudobulbs 413

8 Fig 10 : Repartition of phenolic molecular types on an HPLC profile of phenolic extracts of oil palm pseudobulbs at three wavelengths : 280, 320 et 350 nm Légende : h : height of peaks 414

9 CONCLUSION The identification of major polyphenolic molecules in the roots and pseudobulbs of oil palm plants after inoculation by Fusarium oxysporum f. sp. Elaeidis has revealed to us the presence of hydrophilic and lipophilic phenolic compounds. Thus from hydrophilic compounds towards lipophilic compounds we have identified cinnamic derivatives, flavones detected at 280 nm, flavonones, chlorogenic derivatives, flavonones clearly detected at 350 nm. These phenolic compounds commonly called flavonoids intervene in the defence reaction of many cultivated plants and are signalled herein for the first time to the best of our knowledge and belief on his working equipment. REFERENCES Aziz B. ; Ahmed B. AD. et Abelali B Effets biocides des alcaloïdes, des saponines et des flavonoïdes extraits de Capsicum frutescens L. (Solanaceae) sur Bemisia tabaci (Gennadius) (Homoptera aleyrodidae). Biotechnol. Agron. Soc. Environ. 9 (4) Diabate S Etude comparative des réactions de défense contre la fusariose vasculaire. DEA de phytopathologie. Paris XI INA Paris-Grignon, 1-10 Diabate S. ; Taquet B. ; Renard J.L. ; Franqueville (DE) H. et REISER P Analyse en HPLC des substances produites par le palmier à huile au cours de l infection par le Fusarium oxysporum f. sp. elaeidis. Perspective pour la sélection. Oléagineux, 45 (2) Diabate S Contribution à l étude de la diversité phénolique dans la réaction de défense du palmier à huile (Elaeis guineensis Jacq) contre la fusariose vasculaire. Thèse de Doctorat de l Université de Cocody.UFR Biosciences. Abidjan. 155p. Diabate S. ; LEDEME P. ; FRANQUEVILLE (DE) H. et KOUAME B Effet d une souche avirulente de Fusarium oxysporum f. sp. elaeidis sur l expression de la fusariose et sur les propriétés fongitoxiques des extraits racinaires de palmier à huile. In. Interaction Plantes- Microorganismes. FIS Diabate S.; FRANQUEVILLE, H (De). ; ADON B.; COULYBALY O. A.; and AKE S The role of phenolic coumponds in the determination of wilt disease tolerance of oil palm (Elaeis guineensis JACQ).African Journal of Biotechnology 8 (21) Elgersma D. M Accumulation of rishitin in susceptible and resistant tomato plants after inoculation with Verticillium albo-atrum. Physiol. Plant. Pathol. 16, Elliger C. A. ;CHAN B.C. et WAISS A. C. JR Flavonoids as larval growth inhibitors. Naturwissenschafter, 67, Franqueville (De) H. ; DIABATE S La fusariose du palmier à huile en Afrique de l ouest. Plantation, Recherche, Développement. Juillet-aout Harborne J.B Comparative biochemistry of the flavonoids. Acad. Press. London. 383p. Harborne J. B Introduction to ecological biochemistry. Acad. Press, London.243 p. Haslam E Natural proanthocyanidins. In the flavonoids. Ed., Harborne J. B.; Mabry T.J. et CHAPMAN et HALL. London, Maclure J Physiology and functions of flavonoïds. In flavonoïds. Ed., HARBORNE J. B. ; MABRY T. J. et MABRY H. ; Chapmann et Hall. London, Marbry T.J; Markham K.R and Thomas M.B The systematic identification of flavonoids. Springer verlog, Berlin. 178p Markham K.R Techniques of flavonoids identification. Academic Press, London. 113p. Mayama S.; Matsuura Y. Iida H. ; and Tani T The role of avenaluminn in the resistance of ast to crown rust, Puccinia coronate f. sp. avenae. Physiol. Plant Pathol. 20, Santiago R.; Malvar R. A. ; Baamonde M.D Free phenols in maize pith and their relationship with resistance to Sesamia nonagrioides (Lepidoptera: Noctuidae) attak Journal of Economic entomology. 98, Smith, D. A.; Van Etten H. D. and Bateman D.F The principal antifungal component of substance isolated from Rhyzoctonia infected bean tissues. Physiol. Plant. Pathol. 3, Sutherland O.R.W.; Russel G.B.; Biggs D.R. and Lane G.A Insect feeding deterrent activity of phytoalexin isoflavonoïds. Biochem. Syst. Ecol. 8, Taquet B Les mécanismes physiologiques de la réaction de défense du palmier à huile contre la fusariose vasculaire. Application à la recherche de nouveaux moyens de lutte. Thèse de Doctorat 3è cycle. Paris p. Traore A Fusariose vasculaire du palmier à huile : étude épidémiologique en champ et microanalyse des composés de défense. Mémoire pour l obtention du Diplôme d Agronomie Approfondie (DAA). ESA Yamoussoukro. Juillet p 415