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Physiological and Molecular Plant Pathology 74 (2010) 287e294

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Physiological and Molecular Plant Pathology


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Mechanisms of date palm resistance to Bayoud disease: Current state of knowledge and research prospects
Cherkaoui El Modafar
Laboratory 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 palm Fusarium oxysporum f. sp. albedinis Resistance Host defense mechanisms

a b s t r a c t
The Bayoud disease, caused by Fusarium oxysporum f. sp. albedinis (Foa), represents a major limiting factor of date palm culture in Morocco and constitutes a serious threat to the date palm plantations in Algeria and all other countries. Efcient disease prevention requires the development of resistant cultivars. In Morocco, among the cultivars listed, only six appear to be resistant to Bayoud disease, but they produce poor quality fruit. Thus, the Moroccan program of date palm genetic improvement is based on directed crossing between resistant cultivars and susceptible cultivars with good date quality traits to select resistant genotypes producing high quality fruits. In addition to the separation of the resistance to Bayoud disease and quality of the fruits characters, this breeding program is really complex due to the sex separation in the date palm, the duration of juvenile phase which is very long, and the lifespan of the date palm which requires a durable polygenic resistance. Then, the selected genotypes must be of female sex, of good date quality, and possess effective defense mechanisms against the pathogen. Moreover, the selection of the date palm resistance must necessarily take into account the mechanisms of pathogen aggressiveness. In this review, we will present and discuss studies developed on the Bayoud disease of the 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 host defense mechanisms. It will also highlight the recent studies that showed that differential behaviour of the resistant and susceptible cultivars was not related to a difference of induction of the defense mechanisms, 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 rst time in 1870 in the Draa valley in the south of Morocco [1]. It was described as the most important disease of date palm [2]. The pathogen invades the plant through the roots [3] producing foliar withering, and leading to the death of the date palm tree [4]. Foa produces typical micro- and macroconidia, as well as chlamydospores, allowing the pathogen to survive under adverse environmental conditions [4]. During this century, Foa has been responsible for the destruction of two-thirds of Moroccan palm plantations (more than 10 million trees) causing considerable economic, ecological and social damage [2,5]. Indeed, the incidence Bayoud causes not only reduction in the production of dates, the principal food of humans and animals in the desert, but

E-mail addresses: elmodafar@fstg-marrakech.ac.ma, elmodafar@ucam.ac.ma. 0885-5765/$ e see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2010.06.008

also an imbalance of the oases ecosystem (desertication, disappearance of the subjacent cultures: cereal, fodder and vegetables cultures and fruit trees). Consequently, the dense Moroccan palm plantations were transformed in one century into clearings. Morocco which was previously a date exporter has now to import dates to satisfy its own internal consumption. Similarly, the Bayoud disease destroyed more than 3 million trees in Algeria [6]. The Tunisian palm plantations, 56% of which are made up of the DegletNour cultivar, are currently only protected by prophylactic methods [7,8]. This disease was also found in Argentina [9] and constitutes a serious threat to the palm plantations of other countries. Among the 223 cultivars listed in Morocco, only six appear to be resistant to the Bayoud disease, but they produce poor quality fruit [10]. Genetic resistance represents currently the only effective means to control this disease. The Moroccan program of date palm genetic improvement is based on directed crossing between Foa resistant and susceptible cultivars of good date quality, in order to select genotypes combining the two characteristics [2]. This review represents a critical synthesis of the major research approaches and

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

3. Control of Bayoud disease 3.1. Prophylactic control Prophylactic treatments dont present a viable mitigation option in Morocco since all the palm plantations have been contaminated by Foa [2]. Similarly, the palm plantations of the Western and the central regions of Algeria are infected by Foa [27]. However, the Tunisian palm plantations are not infected by Foa and are currently protected almost exclusively by prophylactic methods [8]. 3.2. Chemical control Since Foa is a soil-borne fungus and a vascular pathogen, chemical treatments using fungicides appeared constraining, expensive, and ineffective [28]. Foa produces chlamydospores that allow the pathogen 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 global approach taking account of the plantesoilepathogen complex, soil-borne and vascular pathogens are difcult to control even with systemic fungicides [28]. Moreover, these chemical products constitute a potential source of pollution for the fragile oases ecosystem and can exert a selective pressure on the Foa populations leading to the possible selection of pathogen populations resistant to the fungicides. 3.3. Biological control In order to exploit the good date quality traits of some susceptible cultivars (particularly Mejhoul and Boufeggous cultivars) which are threatened with disappearance, research on soils suppressive to Bayoud disease constitutes an alternative which was the subject of many studies [31,32]. The suppressive nature of some soils 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, the amendment of palm plantation soils by these antagonistic microorganisms to Foa was suggested as a biological control [37]. The use of mycorrhizal fungi represents another approach of biological control. Thus, the mycorrhization of date palm seedlings by arbuscular mycorrhizal fungi, in particular by species of Glomus, reduced disease severity [38,39]. By checking the receptivity of the palm plantation soils to the mycorrhizal fungi, this approach could constitute an effective alternative not only to protect the palm tree against Bayoud disease, but also for the improvement of mineral nutrition and tolerance to drought. However, these biological approaches to control by antagonistic micro-organisms and mycorrhizal fungi remain far from practical application in this particular ecosystem, where the introduction of these microorganisms could lead to their ecological rejection. 3.4. Selection of resistant genotypes of date palm The selection and/or creation of genetically resistant clones would constitute the most effective solution to safeguard palm plantations against Bayoud disease. Basic work lasting 25 years characterized the behaviour of the various cultivars of the date palm to Foa [10]. The use of vitroplants led to the control of the inoculation technique and use of appropriate inoculum concentrations [40,41], two fundamental variables whose optimization plays a signicant role in determining the level of resistance of a specic cultivar. However, one of the major problems in the selection of resistant genotypes to Bayoud disease resides in the

2. Pathogenicity and genetic diversity of Foa In addition to the date palm (Phoenix dactylifera), Foa also attacks the Canary Island palm (Phoenix canariensis) and other species of palmaceae [11]. Other plant-hosts may serve as symptomless carriers of Foa such as Henna (Lawsonia inermis), alfalfa (Medicago spp.), and clover (Trifolium ssp.) [11,12]. Various aggressiveness levels were observed among the isolates obtained from the date palm and those isolated from symptomless carriers, such as the soil and the Canary Island palm [13e15]. In addition, the strains isolated from the rachis were found to be generally more aggressive than those isolated from the roots [2,16]. However, the precise evaluation of pathogen aggressiveness encounters methodological difculties, particularly in the determination of the plant growth stage susceptible to the pathogen and the great heterogeneity of the seedlings, because they have been obtained from seeds and not from identical in vitro clones. Moreover, the reduced number of date palm cultivars presenting differential behaviours does not allow completed studies on virulence to determine the occurrence of physiological races. In order to overcome several of these difculties, studies were developed using molecular markers to identify the isolates and to analyse their genetic polymorphism. Thus, several molecular approaches (RAPD, RFLP, AFLP, SSR, etc.) were developed and the rst analysis distinguished the special form albedinis from non-pathogenic (saprophytes in the soil) and pathogenic forms of F. oxysporum from other plants [17e19]. The restriction proles of mitochondrial DNA showed that Foa isolates from Morocco and Algeria constitute one RFLP group and one RAPD group [17,20,21] although they present different phenotypic characters [22]. In addition, it was shown that the genome of F. oxysporum contains several copies of the transposable element Fot1. The activity of these transposable elements can explain part of the genetic variation observed in F. oxysporum species [23]. The study of Foa genetic diversity was deepened by the use of the transposable element Fot1 like repeated and dispersed genomic probe [21]. This molecular marker allowed the distinction of various proles of Foa populations, but without any relationship with their pathogenicity or geographic origin [5,21,24]. Molecular ngerprinting results were exploited to develop a specic test for detection of Foa isolates amplied by polymerase chain reaction (PCR). The presence of the transposable element Fot1 in F. oxysporum species and the conservation of some copies in the special forms suggested that Fot1 could play a role in the pathogenicity of Foa [23,25]. The transposable element Fot1 exists in several copies in Foa, whereas it is either rare or absent in non-pathogenic and other pathogenic special forms of Fusarium [5,18,21]. The analysis of all the genetic ngerprinting of the Moroccan and Algerian Foa isolates allowed the identication of four DNA bands which remain preserved and can thus have a role in the determination of the special form albedinis [25]. The insertion sites of two DNA bands were cloned and sequenced to determine the oligonucleotide sequences to be used as PCR primers in order to develop a sensitive diagnostic tool for Foa detection. Two couples of primer pairs were dened: BIO3-FOA1 and FOA28-TL3 [25]. The primer pair BIO3FOA1 led to the identication of 95% of Foa isolates and the second primer pair FOA28-TL3 identied 99% of the isolates. However, no amplication was obtained by the two sets of primers in the isolates of others pathogenic and non-pathogenic Fusarim species (Fernandez et al., 1998). Similar results were obtained with Foa isolates of Algerian origin [15,24,26].

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fact that the resistant cultivars do not produce good date quality [10]. So, the Moroccan program of date palm genetic improvement relies mainly on directed crossing, between resistant cultivars and susceptible cultivars of good date quality, in order to select genotypes combining the two characteristics [2]. The selection of the date palm trees resistant to Foa has been based on experimental inoculation of seedlings and the observation of foliar withering symptoms [2,41]. Although this method made it possible to characterize resistant clones [2,42], it remains time-consuming and arbitrary and cannot guarantee that the plant will be resistant at the adult stage. Indeed, this selection test is generally carried out on young seedlings and the inoculum concentration represents an arbitrary variable in the discrimination of the behaviour of the seedlings. Consequently, a genotype not expressing the foliar withering symptoms, with a determined inoculum concentration and at a specic developmental stage, can still develop them with a higher concentration and/or a more advanced stage of the development of the date palm. Moreover, this selection test does not make it possible to guarantee the durability of the selected resistance. It may be that exploited resistance is monogenic and thus easily surmountable by the appearance of new virulent races of Foa. 4. Biochemical markers of date palm resistance Many studies sought markers able to distinguish the resistant and susceptible cultivars to Bayoud disease. Enzymatic polymorphism was used for the study of several enzymatic systems particularly of peroxidases [43e45], esterases, oxidases [46], hydrolases [47], and transferases [47,48] which have been proposed as biochemical markers for the selection of resistant genotypes. However, these studies were mainly carried out in the leaves which are not the target organ of Foa and not located in the sites of penetration and colonization of pathogen, in particular the roots and the rachis. Moreover, the studied enzymes are involved in various physiological processes and could present quantitative and qualitative 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, these biochemical markers could also be used for varietal identication [45,51], response to somatic embryogenesis [52,53], sex identication [54,55], date fruit quality [56] (Booij et al., 1995) and characterization of resistance to Bayoud disease [43,44]. However, the required genotypes must possess effective defense mechanisms directly involved in resistance expression, and not only enzymatic markers which could be correlated positively or negatively with several other physiological processes. Moreover, the concept of markers is difcult to associate with a very complex physiological phenomenon such as resistance, which depends not only on the defense mechanisms of the host-plant, but also on the pathogenicity factors of the parasite, and the environment. 5. Molecular markers of date palm resistance Several works sought to identify cytoplasmic and nuclear molecular markers of date palm resistance to Bayoud disease. Various mitochondrial plasmid-like DNAs were identied and characterized in the date palm [57]. A study undertaken on six date palm cultivars showed that the size of these plasmids was associated with resistance/susceptibility to Foa. In particular, a plasmid R (1346 Pb) was shown to exist only in the resistant cultivars and a plasmid S (1454 Pb) appeared to exist only in susceptible cultivars [58,59]. However, the widening of these studies to include several other cultivars did not show a correlation between the presence of

mitochondrial plasmid-like DNAs and date palm resistance to Bayoud disease [60e62]. Several studies were undertaken on the use of nuclear molecular markers, in particular RAPD, SSR and ISSR markers, for the identication of resistant cultivars [63e67], the analysis of genetic diversity of date palm [68e71] and the relationship with the resistance to Foa [66,72,73]. However, these molecular markers did not allow workers to distinguish between the resistant and susceptible cultivars [66,72,73]. Although molecular markers generally reect the structure of the nuclear and mitochondrial genome and can assist the genetic improvement program for the selection of resistant genotypes to Bayoud disease, the guarantee of effective resistance resides in a solid knowledge of the defense mechanisms directly involved in the resistance of the host and the pathogenicity factors responsible for 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 vascular pathogens, representing one of the typical mechanisms of defense to vascular diseases [74], was identied in the date palm [75]. Tyloses are vesicular expansions of the adjacent parenchyma cells and play an important role in the mechanical resistance of the plants infected by vascular pathogens [74,76]. In the date palm infected by Foa, the accumulation of polymerized avans in the root vessels was highlighted by both histological [38] and biochemical [77] approaches. The accumulation of these phenolic compounds in the date palm vessels could be the result of tylose disintegration. Indeed, tyloses can contain antifungal substances, in particular phenolic compounds [78]. While disintegrating, the tyloses pour their phenolic contents in side the vessels directly in contact with the pathogen [76]. A positive relationship was established between the restriction of the pathogen development in the plant vessels, the increase in the avan-3-ols contents, their polymerization to proanthocyanidins and their mobilization in the tyloses towards the vessel cells [76,78]. 6.2. Induction of pathogenesis-related proteins Pathogenesis-related proteins (PR-proteins) are monomeric proteins of low molecular weight having a great stability under very acidic pH and a strong resistance to proteolysis [79]. These PRproteins are defense molecules synthesized by the plants in response to pathogen infection [80]. Chitinases and polygalacturonases were identied in date palm roots in response to Foa [81]. Although the involvement of PR-proteins in resistance is not yet established in the date palmeFoa interaction, these proteins have a serologic homology with those of corn whose role in chemical defense has been clearly established [82]. 6.3. Accumulation of polyamines and phenolamides Investigating the potential role of polyamines in the chemical defense of date palm to Foa, it is reported that in vitro fungitoxicity tests showed that these molecules did not have a fungitoxic effect [83] and could even stimulate fungal growth [84]. Inhibitors of polyamine biosynthesis were found to inhibit the growth of Foa [84]. Although these results suggested that polyamines were required for Foa development, it was suggested that these molecules might affect defense mechanisms of date palm to Bayoud disease [83]. On the other hand, the accumulation of molecules associated with phenolamides was identied in the rachis naturally

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infected by Foa [85]. The treatment of date palm seedlings with exogenic polyamines resulted in an increase in phenolamide content and mortality in infected and non-infected seedlings [83]. It appears that phenolamide accumulation could be a consequence of stress related to the toxicity of polyamines. The accumulation of phenolamides in the rachis [85] would be rather a consequence of stress associated with drying and death of the palms (leaves and rachis), rather than mechanisms of host defense. The accumulation of phenolamides in the necrosed callus of date palm [86] conrms this hypothesis. Indeed, the rachis used in these studies came from date palm trees at the drying stage, whereas it is known that defense reactions must be active at earlier stages in pathogenesis and particularly before symptoms development. 6.4. Accumulation of caffeoylshikimic acid and post-inhibitins Resistant cultivars were characterized by a rhizospheric activity of Foa weaker than that of susceptible cultivars [87,88]. This difference was related to a differential effect of the root exudates of the two types of cultivars [89,90]. Root exudates of resistant cultivars inhibited conidial germination of the various isolates of Foa whereas those of susceptible cultivars led rather to a stimulation of germination [90,91]. Thorough biochemical analysis carried out in date palm roots showed that caffeoylshikimic acid is the major phenolic compound in the roots [92,93] and represents the main fungitoxic compound against Foa [94]. The binding of caffeoylshikimic acid by polyvinylpyrrolidone (PVP) [95] greatly decreases the fungitoxicity of the root extract [94]. In addition, caffeoylshikimic acid contents increase after infection in resistant cultivars while those of susceptible cultivars do not differ signicantly from those obtained from non-infected plants [77]. The caffeoylshikimic acid accumulating in the resistant cultivars are suggested to inhibit growth and conidiogenesis of Foa, whereas the much lower concentrations in susceptible cultivars have no effect [94]. Similarly, caffeoylshikimic acid inhibits both directly and indirectly the activity and production of cell-wall degrading enzymes of Foa [96]. It generates hydrolysis products (particularly caffeic acid) and oxidation products (quinones) which inhibit the activity and production of polygalacturonases, pectinemethylesterases, polygalacturaonate trans-eliminases, cellulases and proteases [96]. The caffeoylshikimic acid was then associated with an inhibitin, and caffeic acid and the quinones were associated with post-inhibitins. These results suggest that Foa produces esterases hydrolysing the caffeoylshikimic into caffeic acid and shikimic acid and tyrosinases oxidizing the caffeoylshikimic acid and the caffeic acid in quinones [96]. These oxidation products of phenolic compounds are very toxic against micro-organisms and play an important role in the inactivation of pathogen enzymes [97e99]. However, the caffeoylshikimic acid contents accumulated in resistant cultivars do not completely inhibit conidial germination, mycelium growth [94], and the activity and production of the cell-wall degrading enzymes by Foa [96]. The role played by the caffeoylshikimic acid in date palm defense against Bayoud disease is not sufcient to explain the level of resistance observed on plants suggesting the involvement of other constitutive or induced defense mechanisms. 6.5. Induction of phytoalexin biosynthesis The inoculation of date palm roots with Foa induces the accumulation of phytoalexins identied as coumarin derivatives, propyl-7-aesculetin and hydroxy-5-propyl-7-aesculetin [100,101]. The time course of phytoalexin accumulation clearly distinguishes resistant and susceptible cultivars [101]. Thus, during the rst infection phase (5e30 days), phytoalexin accumulation is rapid and reaches much higher contents in the resistant cultivars, with

maximum accumulation on the 15th day (2.36 mmol g1 FW). In the second phase (30e60 days), a reversal occurs and susceptible cultivars accumulate higher contents reaching a maximum on the 40th day, (2.96 mmol g1 FW) coinciding with generalized root necrosis and foliar withering on the 15th day after inoculation (2.36 mmol g1 FW) [101]. The phytoalexin concentrations accumulated in resistant cultivars at the 15th day after inoculation (2.36 mmol g1 FW) strongly reduced conidial germination by 17e86%, germ tube growth, conidiogenesis, and mycelium growth of Foa. However, the concentrations accumulated in susceptible cultivars (0.55 mmol g1 FW) had only a weak inhibitory effect (16e27%). The role of phytoalexins in date palm resistance to Bayoud disease is then related to the rapidity and intensity of their accumulation at the fungitoxic concentrations during the initial infection stage. The phytoalexins do not seem to be efcacious in date palm when they are induced at more advanced stages, even if they are produced at larges quantities. Although the phytoalexins appear to support the defense of the resistant cultivars, they do not play the major role in date palm resistance to Bayoud disease [101]. Indeed, the phytoalexins concentrations accumulated in resistant cultivars only have a biostatic effect and they do not completely suppress Foa growth. 6.6. Cell-wall reinforcement by cell wall-bound phenols and lignication The studies undertaken on the interaction between the cell-wall degrading enzymes produced by Foa (polygalacturonases, polygalacturonate trans-eliminases, pectinemethylesterases, and cellulases) and date palm cell walls show a relationship between the susceptibility of root cell walls and the susceptibility of cultivars to Bayoud disease [102]. The cell walls of resistant cultivars are more resistant than those of susceptible cultivars to the action of pathogens hydrolytic enzymes. This differential susceptibility of cell walls in resistant and susceptible cultivars to Foa is related to the involvement of two constitutive defense mechanisms in cell walls of the resistant cultivars [102,103]. The rst, a mechanical mechanism involving lignin and cell wall-bound phenolic acid, intervenes in the initial cell-wall degradation stages to limit the action of the pectinolytic and cellulolytic enzymes of the pathogen on the cell wall of resistant cultivars. A second chemical mechanism intervenes at a more advanced stage to inhibit the production of cell-wall degrading enzymes by Foa. This inhibition is related, at least in part, to the intervention of cell wall-bound phenolic acid [103]. These cell wall-bound phenolics, identied as p-hydroxybenzoic, p-coumaric, ferulic and sinapic acids [104], inhibit mycelium growth and cell-wall degrading enzyme production by Foa [103]. The degree of inhibition of these cell wall-bound phenols depends on the concentration and structure of the phenolic compounds particularly methoxylation and hydroxylation. Methoxylation increases inhibition, whereas hydroxylation decreases it [103]. The cell wall-bound phenol concentrations accumulated in roots of resistant cultivars greatly reduce Foa growth and production of hydrolytic enzymes, whereas the concentrations found in susceptible cultivars have no signicant effect. The resistance of date palm cell walls to Foa pectinolytic and cellulolytic enzymes is related to the intervention of constitutive cell wall-bound phenols and lignin, which constitute a component of a mechanical defense and to the inhibiting effects of cell wallbound phenols on the mycelium growth and the production of pathogen hydrolytic enzymes constituting a second component of a chemical defense. In addition, the inoculation of date palm roots with Foa results in important modications in cell wall-bound phenols and lignin contents, which allow distinction of the different cultivars according to their resistance/susceptibility to

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Bayoud disease [104]. Thus, the post-infectional response of metabolism of parietal phenols and lignin occurs early and is strong in the resistant cultivars, whereas it is late and weak in the susceptible cultivars. The cell wall-bound phenols accumulate in resistant cultivars at fungitoxic concentrations during the rst infection stages. The rapidity and intensity of cell wall-bound phenol accumulation and the lignication process in the roots in response to Foa infection are also determinants of the resistance of the 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, postinhibitins, avanes, lignin) depend on the phenylalanine ammoniaelyase activity (PAL) [105,106], the key enzyme of phenylpropanoid metabolism [97]. It was shown that the differential induction of the defense mechanisms in the resistant and susceptible cultivars in response to infection by Foa was related to a difference in PAL activity [107]. Post-infectional PAL activity in roots the resistant cultivars was faster and higher than in susceptible cultivars. The induction of these defense mechanisms occurs early and intensely in resistant cultivars, whereas it occurs late and weakly in susceptible cultivars. A slow response would give the pathogen time to grow abundantly and produce toxins [108e111] interfering with the regulation of different physiological functions in the plant, thus causing the typical symptoms of the disease. The post-infectional induction of PAL activity was related to a carbohydrate elicitor localized in the mycelial wall of Foa [107]. However, the culture ltrate was not a signicant elicitor activity of PAL, contrary to studies which reported an increase in the total contents of soluble phenols in the date palm callus in response to culture ltrate from Foa [112,113]. Although simplied models (callus and cell culture) have been used to study plantepathogen interactions [114e118], it is difcult to extrapolate the response of cells cultivated in vitro on articial medium to that of the date palm placed in its real environment. Indeed, in the case of a vascular disease such as Bayoud disease, Foa preferentially colonizes the vessels and the true hostepathogen interaction is established when the fungus reaches the vascular system of the host-plant [74]. On the other hand, phenolic metabolism is very sensitive to various mechanical [119,120], nutritional [121,122], drought [123], and environmental [124,125] types of stress. Moreover, the phenolic composition of the callus is very different from that of the date palm tree. The increase in the phenol content of the date palm callus in response to the treatment by the culture ltrate of Foa [112] could be the consequence of a stress rather than a defense reaction since the culture ltrate does not elicitor defense mechanisms in the plant [107]. However, particularly in the case of the vascular diseases, the cell remains better adapted for the in vitro creation and selection of the resistant genotypes to pathogen, but not to the study of the defense mechanisms of the host-plant [126e128]. Furthermore, although infection induced differential responses in resistant and susceptible cultivars, the carbohydrate elicitor from the mycelial wall induced an identical PAL response in all date palm cultivars [107]. The differences in PAL induction in response to Bayoud disease and to a carbohydrate elicitor of Foa is related to suppression of the PAL induction in the susceptible cultivars by a soluble proteinaceous suppressor, produced constitutively by the pathogen [129]. Thus, the differential induction of defense mechanisms in resistant and susceptible cultivars does not relate to the degree of PAL elicitation [107], but seems to be related to the suppression of PAL gene expression by the pathogen suppressor. In susceptible cultivars, PAL gene expression would be suppressed by

Foa, whereas in the resistant cultivars, the suppression would be absent or substantially reduced [129]. PAL suppression by Foa seems to constitute a primary factor determining date palm susceptibility to Bayoud disease. These results suggest that specicity in the date palmeFoa interaction was not explained by a difference in defense mechanism elicitation, but by a difference in the degree of suppression of the mechanisms. Early suppressor production by the pathogen, in particular by germinating conidia, may predispose plants to pathogen colonization before penetration [105,129]. The characterization of the PAL induction suppressor represents an important focus for future work. 7. Conclusion and research prospects Date palm resistance to Bayoud disease is related to multifactorial defense mechanisms, some of which are constitutive (e.g., caffeoylshikimic acid, post-inhibitins, lignin, cell wall-bound phenols, avans), and others are induced de novo (e.g., phytoalexins, PR-proteins, tyloses). Depending on their role in the hostplant defense strategy, these mechanisms can be categorized in to two types:  Mechanical mechanisms (lignin, cell wall-bound phenols, tyloses) which limit the action of the cell-wall degrading enzymes of Foa on the cell wall of date palm root cells;  Chemical mechanisms (phytoalexins, caffeoylshikimic acid, post-inhibitins, avans, cell wall-bound phenols, PR-proteins,) which have been shown to inhibit Foa growth as well as the activity and production of its pectinolytic, cellulolytic, and proteolytic enzymes. Date palm resistance to Bayoud disease also depends on pathogenicity factors of Foa which can be distinguished as follows:  Cell-wall degrading enzymes which allow penetration and host tissue colonization by Foa;  Carbohydrate elicitor which induces identical defense mechanisms in resistant and susceptible cultivars;  A proteinaceous suppressor that suppresses the elicitation of defense mechanisms in susceptible cultivars;  Toxins involved in the development of disease symptoms. Furthermore, in the absence of direct studies on the complex of genes involved in the expression of the Foa pathogenic capacity, genomic analysis by molecular markers shows that the Moroccan and Algerian isolates of Foa are genetically very similar and seem to have the same clonal origin. Overall, these results (selection of resistant genotypes, molecular characterization of the determinism of sex and date quality, host defense mechanisms and fungal pathogenicity factors) would certainly contribute to the development of a more effective resistance of date palm to Bayoud disease. However, any improvement due to only one component of defense could be surmounted by new pathogen populations likely to appear by selective pressure. Indeed, the principal defense mechanisms of the date palm are under the control of a key enzyme, PAL. It was demonstrated that the Foa can suppress PAL induction, and lead consequently, to the suppression of defense mechanisms in the host-plant. Any improvement in PAL induction is likely to exert a selective pressure during the coevolution of the plantepathogen interaction leading to the appearance of new Foa isolates able to suppress the expression of genes coding for PAL. Bayoud disease symptoms were thus observed in some cultivars (IKL, SLY and BFGM) which are known for their resistance to Foa [10,17]. Consequently, although resistant genotypes were obtained by directed crossings and mass

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selection, the improvement program for Bayoud resistance must necessarily take into account the mechanisms of pathogen aggressiveness determined by its aptitude to produce pathogenicity factors, which could overcome the selected resistance. These ndings demonstrate the urgent need to acquire complementary elements in order to better manage the genetic strategy for improving durable resistance in date palm. The continuation of studies of molecular markers related to the sex determinism and date quality [130,131] and the thorough knowledge of the defense mechanisms involved in date palm resistance and the pathogenicity factors of Foa, would make it possible to assist the selection program. The association of a multitude of defense components must be taken into account to reinforce the resistance level of the host-plant and to avoid thus their suppression by the pathogen. Integrated genetic and biotechnological approaches can be considered for the improvement of date palm genetic resistance to Bayoud disease. The use of callus and cell culture could be combined with an in vitro selection approach. Foa produces toxins involved in the expression of disease symptoms [111], and these toxins were tested for the selection of seedlings resistant to Foa [72]. It is then conceivable to seek a resistance against pathogen toxins by exposing the callus or the cell suspensions to the action of these toxins. This approach would make mass selection possible very quickly. However, in addition to the difculties encountered in date palm for the regeneration of the plants from cells and callus [27,132,133], these approaches would have a practical interest only if the toxins produced by Foa are the primary determinants of the symptoms of the Bayoud disease. The characterization of Foa toxins and their biological activity will undoubtedly have a fundamental and applied interest. If the toxins proved to be major determinants of the symptoms of Bayoud disease, the creation of transgenic plants with detoxication genes of toxins can be realized. Indeed, the genetic transformation of plants in order to detoxify toxins strongly involved in pathogenesis has been successful [128,134,135]. Therefore, it is probable that this approach would useful, particularly if combined with the techniques of genetic transformation and regeneration of date palm vitroplants [27,133,136].

Acknowledgements This manuscript was revised by Dr. Albert Sasson (International Adviser in agriculture and biotechnology, senior consultant of United Nations and UNESCO, member of the Hassan II Academy for Science and Technology and the Royal Institute of Strategic StudiesMorocco, author of several books in biotechnology), Dr. Abdellah Oihabi (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 of Biotechnology, Morocco), Pr. Lahcen Hassani (Director of Doctoral Centre of the Cadi Ayyad University, Morocco) and Dr. Jean-Pierre Pros (Montpellier SupAgro, France).

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