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Physiological and Molecular Plant Pathology 74 (2010) 287e294

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Physiological and Molecular Plant Pathology

journal homepage: www.elsevier .com/locate/pmpp

Mechanisms of date palm resistance to Bayoud disease: Currentstate of knowledge and research prospects

Cherkaoui El ModafarLaboratory of Biotechnology Valorisation and Protection of Agro-Resources, Agro Biotech L02B005, University of Cadi Ayyad, Faculty of Science and Technology Gueliz, P.O. Box 549,Marrakech 40 000, Morocco

a r t i c l e i n f o

Article history:Accepted 30 June 2010

Keywords:Date palmFusarium oxysporum f. sp. albedinisResistanceHost defense mechanisms

E-mail addresses: elmodafar@fstg-marrakech.ac.m

0885-5765/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.pmpp.2010.06.008

a b s t r a c t

The Bayoud disease, caused by Fusarium oxysporum f. sp. albedinis (Foa), represents a major limitingfactor of date palm culture in Morocco and constitutes a serious threat to the date palm plantations inAlgeria and all other countries. Efficient disease prevention requires the development of resistantcultivars. In Morocco, among the cultivars listed, only six appear to be resistant to Bayoud disease, butthey produce poor quality fruit. Thus, the Moroccan program of date palm genetic improvement is basedon directed crossing between resistant cultivars and susceptible cultivars with good date quality traits toselect resistant genotypes producing high quality fruits. In addition to the separation of the resistance toBayoud disease and quality of the fruits characters, this breeding program is really complex due to thesex separation in the date palm, the duration of juvenile phase which is very long, and the lifespan of thedate palm which requires a durable polygenic resistance. Then, the selected genotypes must be of femalesex, of good date quality, and possess effective defense mechanisms against the pathogen. Moreover, theselection of the date palm resistance must necessarily take into account the mechanisms of pathogenaggressiveness. In this review, we will present and discuss studies developed on the Bayoud disease ofthe date palm, particularly on the disease control, the biochemical and molecular markers of resistance,the program of date palm genetic improvement of resistance, the Foa pathogenicity factors, and hostdefense mechanisms. It will also highlight the recent studies that showed that differential behaviour ofthe resistant and susceptible cultivars was not related to a difference of induction of the defensemechanisms, but to the suppression of their elicitation in the susceptible cultivars.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Bayoud disease, caused by Fusarium oxysporum f. sp. albedinis(Foa), appeared for the first time in 1870 in the Draa valley in thesouth of Morocco [1]. It was described as the most importantdisease of date palm [2]. The pathogen invades the plant throughthe roots [3] producing foliar withering, and leading to the death ofthe date palm tree [4]. Foa produces typical micro- and macro-conidia, as well as chlamydospores, allowing the pathogen tosurvive under adverse environmental conditions [4]. During thiscentury, Foa has been responsible for the destruction of two-thirdsof Moroccan palm plantations (more than 10 million trees) causingconsiderable economic, ecological and social damage [2,5]. Indeed,the incidence Bayoud causes not only reduction in the productionof dates, the principal food of humans and animals in the desert, but

a, elmodafar@ucam.ac.ma.

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also an imbalance of the oases ecosystem (desertification, disap-pearance of the subjacent cultures: cereal, fodder and vegetablescultures and fruit trees). Consequently, the dense Moroccan palmplantations were transformed in one century into clearings.Morocco which was previously a date exporter has now to importdates to satisfy its own internal consumption. Similarly, the Bayouddisease destroyed more than 3 million trees in Algeria [6]. TheTunisian palm plantations, 56% of which are made up of the Deglet-Nour cultivar, are currently only protected by prophylactic methods[7,8]. This disease was also found in Argentina [9] and constitutesa serious threat to the palm plantations of other countries.

Among the 223 cultivars listed in Morocco, only six appear to beresistant to the Bayoud disease, but they produce poor quality fruit[10]. Genetic resistance represents currently the only effectivemeans to control this disease. The Moroccan program of date palmgenetic improvement is based on directed crossing between Foaresistant and susceptible cultivars of good date quality, in order toselect genotypes combining the two characteristics [2]. This reviewrepresents a critical synthesis of themajor research approaches and

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achievements on date palm resistance to Bayoud disease anddiscussions will be developed and future prospects will beconsidered so that research could progress successfully.

2. Pathogenicity and genetic diversity of Foa

In addition to the date palm (Phoenix dactylifera), Foa alsoattacks the Canary Island palm (Phoenix canariensis) and otherspecies of palmaceae [11]. Other plant-hosts may serve as symp-tomless carriers of Foa such as Henna (Lawsonia inermis), alfalfa(Medicago spp.), and clover (Trifolium ssp.) [11,12]. Various aggres-siveness levels were observed among the isolates obtained fromthe date palm and those isolated from symptomless carriers, suchas the soil and the Canary Island palm [13e15]. In addition, thestrains isolated from the rachis were found to be generally moreaggressive than those isolated from the roots [2,16]. However, theprecise evaluation of pathogen aggressiveness encounters meth-odological difficulties, particularly in the determination of the plantgrowth stage susceptible to the pathogen and the great heteroge-neity of the seedlings, because they have been obtained from seedsand not from identical in vitro clones. Moreover, the reducednumber of date palm cultivars presenting differential behavioursdoes not allow completed studies on virulence to determine theoccurrence of physiological races. In order to overcome several ofthese difficulties, studies were developed using molecular markersto identify the isolates and to analyse their genetic polymorphism.Thus, several molecular approaches (RAPD, RFLP, AFLP, SSR, etc.)were developed and the first analysis distinguished the specialform “albedinis” from non-pathogenic (saprophytes in the soil) andpathogenic forms of F. oxysporum from other plants [17e19].

The restriction profiles of mitochondrial DNA showed that Foaisolates from Morocco and Algeria constitute one RFLP group andone RAPD group [17,20,21] although they present differentphenotypic characters [22]. In addition, it was shown that thegenome of F. oxysporum contains several copies of the transposableelement Fot1. The activity of these transposable elements canexplain part of the genetic variation observed in F. oxysporumspecies [23]. The study of Foa genetic diversity was deepened by theuse of the transposable element Fot1 like repeated and dispersedgenomic probe [21]. This molecular marker allowed the distinctionof various profiles of Foa populations, but without any relationshipwith their pathogenicity or geographic origin [5,21,24]. Molecularfingerprinting results were exploited to develop a specific test fordetection of Foa isolates amplified by polymerase chain reaction(PCR). The presence of the transposable element Fot1 in F. oxy-sporum species and the conservation of some copies in the specialforms suggested that Fot1 could play a role in the pathogenicity ofFoa [23,25]. The transposable element Fot1 exists in several copiesin Foa, whereas it is either rare or absent in non-pathogenic andother pathogenic special forms of Fusarium [5,18,21]. The analysis ofall the genetic fingerprinting of the Moroccan and Algerian Foaisolates allowed the identification of four DNA bands which remainpreserved and can thus have a role in the determination of thespecial form “albedinis” [25]. The insertion sites of two DNA bandswere cloned and sequenced to determine the oligonucleotidesequences to be used as PCR primers in order to develop a sensitivediagnostic tool for Foa detection. Two couples of primer pairs weredefined: BIO3-FOA1 and FOA28-TL3 [25]. The primer pair BIO3-FOA1 led to the identification of 95% of Foa isolates and the secondprimer pair FOA28-TL3 identified 99% of the isolates. However, noamplification was obtained by the two sets of primers in theisolates of others pathogenic and non-pathogenic Fusarim species(Fernandez et al., 1998). Similar results were obtained with Foaisolates of Algerian origin [15,24,26].

3. Control of Bayoud disease

3.1. Prophylactic control

Prophylactic treatments don’t present a viable mitigation optionin Morocco since all the palm plantations have been contaminatedby Foa [2]. Similarly, the palm plantations of the Western and thecentral regions of Algeria are infected by Foa [27]. However, theTunisian palm plantations are not infected by Foa and are currentlyprotected almost exclusively by prophylactic methods [8].

3.2. Chemical control

Since Foa is a soil-borne fungus and a vascular pathogen, chem-ical treatments using fungicides appeared constraining, expensive,and ineffective [28]. Foa produces chlamydospores that allow thepathogen to survive under adverse environmental conditions [4],and can survive in the plant vessels and at great soil depths [29,30].Although chemical control was considered through the globalapproach taking account of the plantesoilepathogen complex,soil-borne and vascular pathogens are difficult to control evenwith systemic fungicides [28]. Moreover, these chemical productsconstitute a potential source of pollution for the fragile oasesecosystem and can exert a selective pressure on the Foa populationsleading to the possible selection of pathogenpopulations resistant tothe fungicides.

3.3. Biological control

In order to exploit the good date quality traits of some suscep-tible cultivars (particularly Mejhoul and Boufeggous cultivars)which are threatened with disappearance, research on soilssuppressive to Bayoud disease constitutes an alternative which wasthe subject of many studies [31,32]. The suppressive nature of somesoils was related to a microbial antagonism against Foa by bacteria,particularly various species of Pseudomonas [33] and Bacillus [34],actinomycetes [35,36], and fungal species in particular Aspergillus,Penicillium [34] and saprophytic species of Fusarium [32]. Thus, theamendment of palm plantation soils by these antagonistic micro-organisms to Foa was suggested as a biological control [37]. Theuse of mycorrhizal fungi represents another approach of biologicalcontrol. Thus, the mycorrhization of date palm seedlings byarbuscular mycorrhizal fungi, in particular by species of Glomus,reduced disease severity [38,39]. By checking the receptivity of thepalm plantation soils to the mycorrhizal fungi, this approach couldconstitute an effective alternative not only to protect the palmtree against Bayoud disease, but also for the improvement ofmineral nutrition and tolerance to drought. However, these bio-logical approaches to control by antagonistic micro-organisms andmycorrhizal fungi remain far from practical application in thisparticular ecosystem, where the introduction of these micro-organisms could lead to their ecological rejection.

3.4. Selection of resistant genotypes of date palm

The selection and/or creation of genetically resistant cloneswould constitute the most effective solution to safeguard palmplantations against Bayoud disease. Basic work lasting 25 yearscharacterized the behaviour of the various cultivars of the datepalm to Foa [10]. The use of vitroplants led to the control of theinoculation technique and use of appropriate inoculum concen-trations [40,41], two fundamental variables whose optimizationplays a significant role in determining the level of resistance ofa specific cultivar. However, one of the major problems in theselection of resistant genotypes to Bayoud disease resides in the

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fact that the resistant cultivars do not produce good date quality[10]. So, the Moroccan program of date palm genetic improvementrelies mainly on directed crossing, between resistant cultivars andsusceptible cultivars of good date quality, in order to select geno-types combining the two characteristics [2]. The selection of thedate palm trees resistant to Foa has been based on experimentalinoculation of seedlings and the observation of foliar witheringsymptoms [2,41]. Although this method made it possible to char-acterize resistant clones [2,42], it remains time-consuming andarbitrary and cannot guarantee that the plant will be resistant atthe adult stage. Indeed, this selection test is generally carried out onyoung seedlings and the inoculum concentration represents anarbitrary variable in the discrimination of the behaviour of theseedlings. Consequently, a genotype not expressing the foliarwithering symptoms, with a determined inoculum concentrationand at a specific developmental stage, can still develop them witha higher concentration and/or a more advanced stage of thedevelopment of the date palm. Moreover, this selection test doesnot make it possible to guarantee the durability of the selectedresistance. It may be that exploited resistance is monogenic andthus easily surmountable by the appearance of new virulent racesof Foa.

4. Biochemical markers of date palm resistance

Many studies sought markers able to distinguish the resistantand susceptible cultivars to Bayoud disease. Enzymatic poly-morphism was used for the study of several enzymatic systemsparticularly of peroxidases [43e45], esterases, oxidases [46],hydrolases [47], and transferases [47,48] which have been proposedas biochemical markers for the selection of resistant genotypes.However, these studies weremainly carried out in the leaves whichare not the target organ of Foa and not located in the sites ofpenetration and colonization of pathogen, in particular the rootsand the rachis. Moreover, the studied enzymes are involved invarious physiological processes and could present quantitative andqualitative variations depending on plant developmental stage [49]and in response to various nutritional and environmental factors[50] generally not controlled in these studies. In addition, thesebiochemical markers could also be used for varietal identification[45,51], response to somatic embryogenesis [52,53], sex identifi-cation [54,55], date fruit quality [56] (Booij et al., 1995) and char-acterization of resistance to Bayoud disease [43,44]. However, therequired genotypes must possess effective defense mechanismsdirectly involved in resistance expression, and not only enzymaticmarkers which could be correlated positively or negatively withseveral other physiological processes. Moreover, the concept ofmarkers is difficult to associate with a very complex physiologicalphenomenon such as resistance, which depends not only on thedefense mechanisms of the host-plant, but also on the pathoge-nicity factors of the parasite, and the environment.

5. Molecular markers of date palm resistance

Several works sought to identify cytoplasmic and nuclearmolecular markers of date palm resistance to Bayoud disease.Various mitochondrial plasmid-like DNAs were identified andcharacterized in the date palm [57]. A study undertaken on six datepalm cultivars showed that the size of these plasmids was associ-ated with resistance/susceptibility to Foa. In particular, a plasmid R(1346 Pb) was shown to exist only in the resistant cultivars anda plasmid S (1454 Pb) appeared to exist only in susceptible cultivars[58,59]. However, the widening of these studies to include severalother cultivars did not show a correlation between the presence of

mitochondrial plasmid-like DNAs and date palm resistance toBayoud disease [60e62].

Several studies were undertaken on the use of nuclear molec-ular markers, in particular RAPD, SSR and ISSR markers, for theidentification of resistant cultivars [63e67], the analysis of geneticdiversity of date palm [68e71] and the relationship with theresistance to Foa [66,72,73]. However, these molecular markers didnot allow workers to distinguish between the resistant andsusceptible cultivars [66,72,73].

Although molecular markers generally reflect the structure ofthe nuclear and mitochondrial genome and can assist the geneticimprovement program for the selection of resistant genotypesto Bayoud disease, the guarantee of effective resistance resides ina solid knowledge of the defense mechanisms directly involved inthe resistance of the host and the pathogenicity factors responsiblefor pathogen aggressiveness.

6. Defense mechanisms and date palm resistance

6.1. Induction of tyloses in vessel cells

The induction of tyloses in plant vessel cells infected by vascularpathogens, representing one of the typical mechanisms of defenseto vascular diseases [74], was identified in the date palm [75].Tyloses are vesicular expansions of the adjacent parenchyma cellsand play an important role in the mechanical resistance of theplants infected by vascular pathogens [74,76]. In the date palminfected by Foa, the accumulation of polymerized flavans in the rootvessels was highlighted by both histological [38] and biochemical[77] approaches. The accumulation of these phenolic compounds inthe date palm vessels could be the result of tylose disintegration.Indeed, tyloses can contain antifungal substances, in particularphenolic compounds [78]. While disintegrating, the tyloses pourtheir phenolic contents in side the vessels directly in contact withthe pathogen [76]. A positive relationship was established betweenthe restriction of the pathogen development in the plant vessels,the increase in the flavan-3-ols contents, their polymerization toproanthocyanidins and their mobilization in the tyloses towardsthe vessel cells [76,78].

6.2. Induction of pathogenesis-related proteins

Pathogenesis-related proteins (PR-proteins) are monomericproteins of low molecular weight having a great stability undervery acidic pH and a strong resistance to proteolysis [79]. These PR-proteins are defense molecules synthesized by the plants inresponse to pathogen infection [80]. Chitinases and poly-galacturonases were identified in date palm roots in response toFoa [81]. Although the involvement of PR-proteins in resistance isnot yet established in the date palmeFoa interaction, these proteinshave a serologic homology with those of corn whose role inchemical defense has been clearly established [82].

6.3. Accumulation of polyamines and phenolamides

Investigating the potential role of polyamines in the chemicaldefense of date palm to Foa, it is reported that in vitro fungitoxicitytests showed that these molecules did not have a fungitoxic effect[83] and could even stimulate fungal growth [84]. Inhibitors ofpolyamine biosynthesis were found to inhibit the growth of Foa[84]. Although these results suggested that polyamines wererequired for Foa development, it was suggested that these mole-cules might affect defense mechanisms of date palm to Bayouddisease [83]. On the other hand, the accumulation of moleculesassociated with phenolamides was identified in the rachis naturally

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infected by Foa [85]. The treatment of date palm seedlings withexogenic polyamines resulted in an increase in phenolamidecontent and mortality in infected and non-infected seedlings [83].It appears that phenolamide accumulation could be a consequenceof stress related to the toxicity of polyamines. The accumulation ofphenolamides in the rachis [85] would be rather a consequence ofstress associated with drying and death of the palms (leaves andrachis), rather than mechanisms of host defense. The accumulationof phenolamides in the necrosed callus of date palm [86] confirmsthis hypothesis. Indeed, the rachis used in these studies came fromdate palm trees at the drying stage, whereas it is known thatdefense reactions must be active at earlier stages in pathogenesisand particularly before symptoms development.

6.4. Accumulation of caffeoylshikimic acid and post-inhibitins

Resistant cultivars were characterized by a rhizospheric activityof Foa weaker than that of susceptible cultivars [87,88]. Thisdifference was related to a differential effect of the root exudates ofthe two types of cultivars [89,90]. Root exudates of resistant culti-vars inhibited conidial germination of the various isolates of Foawhereas those of susceptible cultivars led rather to a stimulation ofgermination [90,91]. Thorough biochemical analysis carried out indate palm roots showed that caffeoylshikimic acid is the majorphenolic compound in the roots [92,93] and represents the mainfungitoxic compound against Foa [94]. The binding of caffeoyl-shikimic acid by polyvinylpyrrolidone (PVP) [95] greatly decreasesthe fungitoxicity of the root extract [94]. In addition, caffeoyl-shikimic acid contents increase after infection in resistant cultivarswhile those of susceptible cultivars do not differ significantly fromthose obtained from non-infected plants [77]. The caffeoylshikimicacid accumulating in the resistant cultivars are suggested to inhibitgrowth and conidiogenesis of Foa, whereas the much lowerconcentrations in susceptible cultivars have no effect [94]. Simi-larly, caffeoylshikimic acid inhibits both directly and indirectly theactivity and production of cell-wall degrading enzymes of Foa [96].It generates hydrolysis products (particularly caffeic acid) andoxidation products (quinones) which inhibit the activity andproduction of polygalacturonases, pectinemethylesterases, poly-galacturaonate trans-eliminases, cellulases and proteases [96]. Thecaffeoylshikimic acid was then associated with an inhibitin, andcaffeic acid and the quinones were associated with post-inhibitins.These results suggest that Foa produces esterases hydrolysing thecaffeoylshikimic into caffeic acid and shikimic acid and tyrosinasesoxidizing the caffeoylshikimic acid and the caffeic acid in quinones[96]. These oxidation products of phenolic compounds are verytoxic against micro-organisms and play an important role in theinactivation of pathogen enzymes [97e99]. However, the caf-feoylshikimic acid contents accumulated in resistant cultivars donot completely inhibit conidial germination, mycelium growth [94],and the activity and production of the cell-wall degrading enzymesby Foa [96]. The role played by the caffeoylshikimic acid in datepalm defense against Bayoud disease is not sufficient to explain thelevel of resistance observed on plants suggesting the involvementof other constitutive or induced defense mechanisms.

6.5. Induction of phytoalexin biosynthesis

The inoculation of date palm roots with Foa induces the accu-mulation of phytoalexins identified as coumarin derivatives,propyl-7-aesculetin and hydroxy-5-propyl-7-aesculetin [100,101].The time course of phytoalexin accumulation clearly distinguishesresistant and susceptible cultivars [101]. Thus, during the firstinfection phase (5e30 days), phytoalexin accumulation is rapid andreaches much higher contents in the resistant cultivars, with

maximum accumulation on the 15th day (2.36 mmol g�1 FW). In thesecond phase (30e60 days), a reversal occurs and susceptiblecultivars accumulate higher contents reaching a maximum on the40th day, (2.96 mmol g�1 FW) coinciding with generalized rootnecrosis and foliar withering on the 15th day after inoculation(2.36 mmol g�1 FW) [101]. The phytoalexin concentrations accu-mulated in resistant cultivars at the 15th day after inoculation(2.36 mmol g�1 FW) strongly reduced conidial germination by17e86%, germ tube growth, conidiogenesis, and mycelium growthof Foa. However, the concentrations accumulated in susceptiblecultivars (0.55 mmol g�1 FW) had only a weak inhibitory effect(16e27%). The role of phytoalexins in date palm resistance toBayoud disease is then related to the rapidity and intensity of theiraccumulation at the fungitoxic concentrations during the initialinfection stage. The phytoalexins do not seem to be efficacious indate palm when they are induced at more advanced stages, even ifthey are produced at larges quantities. Although the phytoalexinsappear to support the defense of the resistant cultivars, they do notplay the major role in date palm resistance to Bayoud disease [101].Indeed, the phytoalexins concentrations accumulated in resistantcultivars only have a biostatic effect and they do not completelysuppress Foa growth.

6.6. Cell-wall reinforcement by cell wall-boundphenols and lignification

The studies undertaken on the interaction between the cell-walldegrading enzymes produced by Foa (polygalacturonases, poly-galacturonate trans-eliminases, pectinemethylesterases, andcellulases) and date palm cell walls show a relationship betweenthe susceptibility of root cell walls and the susceptibility of cultivarsto Bayoud disease [102]. The cell walls of resistant cultivars aremore resistant than those of susceptible cultivars to the action ofpathogen’s hydrolytic enzymes. This differential susceptibility ofcell walls in resistant and susceptible cultivars to Foa is related tothe involvement of two constitutive defense mechanisms in cellwalls of the resistant cultivars [102,103]. The first, a mechanicalmechanism involving lignin and cell wall-bound phenolic acid,intervenes in the initial cell-wall degradation stages to limit theaction of the pectinolytic and cellulolytic enzymes of the pathogenon the cell wall of resistant cultivars. A second chemical mechanismintervenes at a more advanced stage to inhibit the production ofcell-wall degrading enzymes by Foa. This inhibition is related, atleast in part, to the intervention of cell wall-bound phenolic acid[103]. These cell wall-bound phenolics, identified as p-hydrox-ybenzoic, p-coumaric, ferulic and sinapic acids [104], inhibitmycelium growth and cell-wall degrading enzyme production byFoa [103]. The degree of inhibition of these cell wall-bound phenolsdepends on the concentration and structure of the phenoliccompounds particularly methoxylation and hydroxylation.Methoxylation increases inhibition, whereas hydroxylationdecreases it [103]. The cell wall-bound phenol concentrationsaccumulated in roots of resistant cultivars greatly reduce Foagrowth and production of hydrolytic enzymes, whereas theconcentrations found in susceptible cultivars have no significanteffect. The resistance of date palm cell walls to Foa pectinolytic andcellulolytic enzymes is related to the intervention of constitutivecell wall-bound phenols and lignin, which constitute a componentof a mechanical defense and to the inhibiting effects of cell wall-bound phenols on the mycelium growth and the production ofpathogen hydrolytic enzymes constituting a second component ofa chemical defense. In addition, the inoculation of date palm rootswith Foa results in important modifications in cell wall-boundphenols and lignin contents, which allow distinction of thedifferent cultivars according to their resistance/susceptibility to

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Bayoud disease [104]. Thus, the post-infectional response ofmetabolism of parietal phenols and lignin occurs early and is strongin the resistant cultivars, whereas it is late and weak in thesusceptible cultivars. The cell wall-bound phenols accumulate inresistant cultivars at fungitoxic concentrations during the firstinfection stages. The rapidity and intensity of cell wall-boundphenol accumulation and the lignification process in the roots inresponse to Foa infection are also determinants of the resistance ofthe date palm to Bayoud disease.

6.7. Elicitation and suppression of defense mechanism induction

The principal mechanisms of defense induced in the date palm(caffeoylshikimic acid, cell wall-bound phenols, phytoalexins, post-inhibitins, flavanes, lignin) depend on the phenylalanine ammo-niaelyase activity (PAL) [105,106], the key enzyme of phenyl-propanoid metabolism [97]. It was shown that the differentialinduction of the defense mechanisms in the resistant and suscep-tible cultivars in response to infection by Foa was related toa difference in PAL activity [107]. Post-infectional PAL activity inroots the resistant cultivars was faster and higher than in suscep-tible cultivars. The induction of these defense mechanisms occursearly and intensely in resistant cultivars, whereas it occurs late andweakly in susceptible cultivars. A slow response would give thepathogen time to grow abundantly and produce toxins [108e111]interfering with the regulation of different physiological functionsin the plant, thus causing the typical symptoms of the disease.

The post-infectional induction of PAL activity was related toa carbohydrate elicitor localized in the mycelial wall of Foa [107].However, the culture filtrate was not a significant elicitor activity ofPAL, contrary to studies which reported an increase in the totalcontents of soluble phenols in the date palm callus in response toculture filtrate from Foa [112,113]. Although simplified models(callus and cell culture) have been used to study plantepathogeninteractions [114e118], it is difficult to extrapolate the response ofcells cultivated in vitro on artificial medium to that of the date palmplaced in its real environment. Indeed, in the case of a vasculardisease such as Bayoud disease, Foa preferentially colonizes thevessels and the true hostepathogen interaction is establishedwhenthe fungus reaches the vascular system of the host-plant [74]. Onthe other hand, phenolic metabolism is very sensitive to variousmechanical [119,120], nutritional [121,122], drought [123], andenvironmental [124,125] types of stress. Moreover, the phenoliccomposition of the callus is very different from that of the datepalm tree. The increase in the phenol content of the date palmcallus in response to the treatment by the culture filtrate of Foa[112] could be the consequence of a stress rather than a defensereaction since the culture filtrate does not elicitor defense mecha-nisms in the plant [107]. However, particularly in the case of thevascular diseases, the cell remains better adapted for the in vitrocreation and selection of the resistant genotypes to pathogen, butnot to the study of the defense mechanisms of the host-plant[126e128].

Furthermore, although infection induced differential responsesin resistant and susceptible cultivars, the carbohydrate elicitor fromthemycelial wall induced an identical PAL response in all date palmcultivars [107]. The differences in PAL induction in response toBayoud disease and to a carbohydrate elicitor of Foa is related tosuppression of the PAL induction in the susceptible cultivars bya soluble proteinaceous suppressor, produced constitutively by thepathogen [129]. Thus, the differential induction of defense mech-anisms in resistant and susceptible cultivars does not relate to thedegree of PAL elicitation [107], but seems to be related to thesuppression of PAL gene expression by the pathogen suppressor. Insusceptible cultivars, PAL gene expression would be suppressed by

Foa, whereas in the resistant cultivars, the suppression would beabsent or substantially reduced [129]. PAL suppression by Foaseems to constitute a primary factor determining date palmsusceptibility to Bayoud disease. These results suggest that speci-ficity in the date palmeFoa interaction was not explained bya difference in defense mechanism elicitation, but by a difference inthe degree of suppression of the mechanisms. Early suppressorproduction by the pathogen, in particular by germinating conidia,may predispose plants to pathogen colonization before penetration[105,129]. The characterization of the PAL induction suppressorrepresents an important focus for future work.

7. Conclusion and research prospects

Date palm resistance to Bayoud disease is related to multifac-torial defense mechanisms, some of which are constitutive (e.g.,caffeoylshikimic acid, post-inhibitins, lignin, cell wall-boundphenols, flavans), and others are induced de novo (e.g., phyto-alexins, PR-proteins, tyloses). Depending on their role in the host-plant defense strategy, these mechanisms can be categorized in totwo types:

� Mechanical mechanisms (lignin, cell wall-bound phenols,tyloses) which limit the action of the cell-wall degradingenzymes of Foa on the cell wall of date palm root cells;

� Chemical mechanisms (phytoalexins, caffeoylshikimic acid,post-inhibitins, flavans, cell wall-bound phenols, PR-proteins,)which have been shown to inhibit Foa growth as well as theactivity and production of its pectinolytic, cellulolytic, andproteolytic enzymes.

Date palm resistance to Bayoud disease also depends on path-ogenicity factors of Foa which can be distinguished as follows:

� Cell-wall degrading enzymeswhich allow penetration and hosttissue colonization by Foa;

� Carbohydrate elicitor which induces identical defense mecha-nisms in resistant and susceptible cultivars;

� A proteinaceous suppressor that suppresses the elicitation ofdefense mechanisms in susceptible cultivars;

� Toxins involved in the development of disease symptoms.

Furthermore, in the absence of direct studies on the complex ofgenes involved in the expression of the Foa pathogenic capacity,genomic analysis by molecular markers shows that the Moroccanand Algerian isolates of Foa are genetically very similar and seem tohave the same clonal origin.

Overall, these results (selection of resistant genotypes, molec-ular characterization of the determinism of sex and date quality,host defense mechanisms and fungal pathogenicity factors) wouldcertainly contribute to the development of a more effective resis-tance of date palm to Bayoud disease. However, any improvementdue to only one component of defense could be surmounted bynew pathogen populations likely to appear by selective pressure.Indeed, the principal defense mechanisms of the date palm areunder the control of a key enzyme, PAL. It was demonstrated thatthe Foa can suppress PAL induction, and lead consequently, to thesuppression of defense mechanisms in the host-plant. Anyimprovement in PAL induction is likely to exert a selective pressureduring the coevolution of the plantepathogen interaction leadingto the appearance of new Foa isolates able to suppress theexpression of genes coding for PAL. Bayoud disease symptoms werethus observed in some cultivars (IKL, SLY and BFGM) which areknown for their resistance to Foa [10,17]. Consequently, althoughresistant genotypes were obtained by directed crossings and mass

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selection, the improvement program for Bayoud resistance mustnecessarily take into account the mechanisms of pathogenaggressiveness determined by its aptitude to produce pathoge-nicity factors, which could overcome the selected resistance. Thesefindings demonstrate the urgent need to acquire complementaryelements in order to better manage the genetic strategy forimproving durable resistance in date palm. The continuation ofstudies of molecular markers related to the sex determinism anddate quality [130,131] and the thorough knowledge of the defensemechanisms involved in date palm resistance and the pathoge-nicity factors of Foa, would make it possible to assist the selectionprogram. The association of a multitude of defense componentsmust be taken into account to reinforce the resistance level of thehost-plant and to avoid thus their suppression by the pathogen.

Integrated genetic and biotechnological approaches can beconsidered for the improvement of date palm genetic resistance toBayoud disease. The use of callus and cell culture could becombined with an in vitro selection approach. Foa produces toxinsinvolved in the expression of disease symptoms [111], and thesetoxins were tested for the selection of seedlings resistant to Foa[72]. It is then conceivable to seek a resistance against pathogentoxins by exposing the callus or the cell suspensions to the action ofthese toxins. This approach would make mass selection possiblevery quickly. However, in addition to the difficulties encountered indate palm for the regeneration of the plants from cells and callus[27,132,133], these approaches would have a practical interest onlyif the toxins produced by Foa are the primary determinants of thesymptoms of the Bayoud disease. The characterization of Foa toxinsand their biological activity will undoubtedly have a fundamentaland applied interest. If the toxins proved to be major determinantsof the symptoms of Bayoud disease, the creation of transgenicplants with detoxification genes of toxins can be realized. Indeed,the genetic transformation of plants in order to detoxify toxinsstrongly involved in pathogenesis has been successful[128,134,135]. Therefore, it is probable that this approach woulduseful, particularly if combined with the techniques of genetictransformation and regeneration of date palm vitroplants[27,133,136].

Acknowledgements

This manuscript was revised by Dr. Albert Sasson (InternationalAdviser in agriculture and biotechnology, senior consultant ofUnited Nations and UNESCO, member of the Hassan II Academy forScience and Technology and the Royal Institute of Strategic Studies-Morocco, author of several books in biotechnology), Dr. AbdellahOihabi (Director of FOA Program, KSA), Dr. Rachid Serraj (IRRI,Philippines), Pr. Josiane Courtois (University of Picardie Jules Verne,France), Pr. Abdelkarim Filali (Director of National pole ofBiotechnology, Morocco), Pr. Lahcen Hassani (Director of DoctoralCentre of the Cadi Ayyad University, Morocco) and Dr. Jean-PierrePéros (Montpellier SupAgro, France).

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