Characterization of a Single Clonal Lineage of Fusarium oxysporum f. sp. albedinis Causing Bayoud Disease of Date Palm in Morocco Abdelaziz Tantaoui, Mohamed Ouinten, Jean-Paul Geiger, and Diana Fernandez ist author: Laboratoire de Phylopathotogie, Institut National de Ja Recherche Agronomique (INRA), B.P. $33 Marrakech, Morocco; sec- fond, third, and fourth authors: Laboratoire de Phytopathologie, Institut Frangais de Recherche Scientifique pour le Développement en Coopération (ORSTOM), B.P., $045, 34032 Montpellier Cedex 1, France. A. Tantaoui was supported by the Institut Frangais de Recherche Scientifique pour le Développement en Coops ion (ORSTOM), We thank our colleagues who provided us with molecular probes and H. C. Kistler (University of Florida), T. Langin (Institut de génétique et microbiologic, University of Paris XI), and M. Raymond (Institut des sciences de l'évolution, University of Montpellier Il, France) for reviewing the manuscript. ‘Accepted for publication 23 April 1996, ABSTRACT A,,Ouinten, M., Geiger, J-P., and Fernandez, D. 1996, Charac- ion of a single clonal lineage of Fusarium oxysporum f. sp. albed nis causing Bayoud discase of date palm in Morocco, Phytopathology 86; 787-192, Bayou, the Fusarium wilt of date palm, was first detected in southern Morocco (Draa Valley) after which it spread to most of the Moroccan palm groves. To assess whether the epidemic results fom the spread of @ single virulent clone, 42 isolates of Fusarium oxysporum f. sp. albedinis were collected from several cultivars of wilted palms at different loca: tions in Morocco; two isolates were included from Algeria where the disease also occurs. The isolates were tested for vegetative compatibility zroup (VCO), restriction fragment length polymorphism (RFLP), and random amplified polymorphic DNA (RAPD). No polymorphism was observed either in RFLP studies on mitochondrial DNA o in RAPD analysis, and all strains belonged to a single VCG (0170). Sequences hhomologous to the DNA transposable element Fotl were found in the genome of the F oxysporum f. sp. albedinis strains, Repetitive DNA, Patterns were produced when EcoRI-digested DNA of the isolates was ‘probed with Fotl; 23 distinct hybridization patterns were established among. the 44 isolates. OF these patterns, 4 accounted for more than 50% ofthe isolates, 1 was found twice, and 18 were represented by a single isolate cach. Common hybridization patterns were found in the Moroccan palm stoves surveyed; the two Algerian isolates had a pattern that also’ was found in the Draa Valley. Cluster analysis grouped most of the Foxy. sporum {. sp. albedinis strains at a genetic distance of 0.11. Such close genetic relationships between the isolates provides evidence that Moree can E oxysporum sp, albedinis populations may belong to a single lonal lineage that originated in Moracean palm groves and eventually reached the Algerian oases, Additional keywords: genetic diversity, Phoenix dactyifera, population ‘Stueture. Bayoud, the fungal vascular wilt of date palm (Phoenix dac- 1plifera L.) caused by Fusarium oxysporum Schlechtend.:Fe. . sp, albedinis (Killian & Maite) Gordon, is widely distributed in all date palm-growing areas of Morocco and in the western and cen- tral parts of Algeria. ‘The disease first appeared before 1870 in the Draa Valley, a large palm grove that extends for 300 km in south- em Morocco. Epidemiological data (33) show new occurrences of | the disease both in the western and eastern parts of Morocco a few years late; the oases of westem Algeria were reached as carly as 1898. It has been estimated that more than 10 million tees (rep- resenting two-thirds of the Moroccan palm grove) have been killed, threatening both the economic resources of the local human popu- lations and the ecological maintenance of the oasis (reviewed in 25). Currently, disease control methods in Algeria and Morocco primarily invoive preventitive measures, including strict phytosan- itary mules at borders of date palm-growing countties that remain free of Bayoud, Attempts to characterize and identify & oxysporum f. sp. albe- dinis isolates showed that the pathogen vary in colony morphol- ‘ogy and pathogenicity (9,10). No races within F oxysporum f. sp. Corresponding autor: D. Fernandez; E-mail adres: femande@ortomsio.et Publication no, P-1096-0528.01R '© 1986 The Amorcan Prytopathologial Society albedinis have been identified to date, mainly due to problems in performing pathogenicity tests because no well-defined differen- {ial series of host genotypes is available. Differentiation between E oxysporum f. sp. albedinis and some formac speciales of F ‘oxysporum was made by conducting vegetative compatibility analyses (8). In preliminary study, we showed that F oxysporum £. sp. albedinis could be differentiated from nonpathogenic F. oxy- sporum isolated from date palm roots or soil by vegetative com- patibility, mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP), or random amplified polymorphic DNA (RAPD) tests (16,31,32). These analyses also suggested that litle ‘genetic variability might exist in the forma specials albedinis. ‘The objective of the current study was to test the hypothesis of the unique origin of F oxysporum f. sp. albedinis isolates as a result of the invasion of the Moroccan oasis by a single virulent clone. Fourty-two F oxysporum f. sp. albedinis isolates were col- lected from several date palm cultivars in Moroccan palm grove ‘Two isolates collected in Algeria also were included for compa son. We used vegetative compatibility group (VCG) genetic mar- kers as well as molecular markers (RAPD and RFLP) to investi- ate the genetic relatedness of the F oxysporum f. sp. albedinis isolates. Both the nuclear and the mitochondrial genome were ‘examined, and repetitive hybridization patterns were established with the DNA transposable clement Fotl cloned from F. oxyspor- lum (. sp. melonis (7). A preliminary report on this research has been published (15). Vol. 88, No. 7, 1996 787MATERIALS AND METHODS Isolates. The geographic location, year of isolation, and host origin of the 44 isolates of F oxysporum f. sp. albedinis examined ‘are listed in Table 1, Nonpathogenic F oxysporum strain A1S, isolated from roots of a wilted date palm (16,31,32) was added to serve as an outgroup in Fotl hybridization-pattern analysis (Table 2), These isolates are part ofthe Institut National de la Recherche ‘Agronomique (INRA) fungal collection in Marrakech and have all bboen checked for pathogenicity on date palms, Fourteen locations were sampled in the Moroccan palm groves, Isolates collected during 1991 in Zagora (Dra Valley) were sampled at the INRA experimental station where date palms resulting from genetic crosses are tested for resistance to Bayoud. Hyphal mass isolates were obtained from the rachis of diseased palms by standard tech- iques (25). Al cultures were single spored and were maintained as mycelia on potato dextrose agar slants for short-term storage or as chlamydospores in sand for long-term storage. ‘TABLE 1, Feserium oxysporum fs, albeini slates tested and their comes ponding Fol fingerprint a dterined in this study Foil Dae pai Geographic Year of hybridization Isolate ost ogi ssoaion pater ® Toute ‘cola ar) Fo halt, ‘ckoria wa Plo 1s25 Kat ‘aromiate wom PLL 1519 Kat ‘Arcomiate wn iss ihe ‘sir 12 Ph ta att Biot 192 7 Bowslikhane Hort 12 139 Bourar [tOuctah PS vs hal Itehae Po vai Male reap P23 A Maou! Meskuziz P20 tus halt Osozag0r PB i Bourse m vis » 6 titel P M45 SairLayslate Tata Pls rGy —Bouty Tina ” 1 Kal ‘iezoaline Ps 12 Bowsks ‘inzoutine Ps 2 Agu agora » 9 ‘Augrao ‘agora Ps OTK takin Zagora Pio 30 Boule Zagora is u ‘Aacgz00 Zagora Ps » ‘Abardane Zagora ” BR Bari Zagora Ps Bz Boufg-omosss Zagora Ps Boufgemosia Zagora ris PA Boufgcmossa Zagora ris & Zagora Pu cu INRA station Zagora) is cr INRA station Zager) Ps ci INRA station @Zagor) Pio a@ INRA station Zager) ple o INRA station Zager) Ps ce INRA station Zspee) PIs cs INRA station (Zapor) Ps 3 INRA station (Zagora) Pio cr INRA station (Zagora) pis ce INRA station Zagor) PIT o INRA station Zagora) PIs cio INRA sation (Zagora) Pr ALL Alig Agena Ps DN __DeglatNour Algeria PS Runes present dite palm caltivar designations except for Khalt he common tame for spontanctsly growing date palm tres, for which ealtva eanot be ‘seas, Male (a male tree for wbih eultvar cannot be assessed) and a bers (progeny from gone roses besween dat palm eulivas) DNA patent ave sted in Table 2. 788 PHYTOPATHOLOGY ‘VCG analysis. VCGs were determined according to Puhalla (29) and Correll et al. (5) with spontaneous nitrate nonutilizing (ut) mutants, Genomic DNA extraction. Fungal mycelium was cultivated in '500-ml flasks containing 200 ml of GYP medium (glucose 2%, yeast extract 0.59%, and peptone 0.5%) for 5 days at 25°C. The mycelium was harvested by filtration, washed with sterile distilled ‘water, and lyophilized for 48 h. Total DNA extraction was per- formed by a miniprep procedure (24), Restriction endonuclease digestion, electrophoresis, and blot- ting, For each isolatc, approximately 3 to 5 wg of total genomic DNA was digested with 20 to 30 units of the restriction enzymes (Boehringer Mannheim, Meylan, France) with the addition of 3 mM spermidine per reaction for 6 to 16 h at 37°C. Restriction ragments were separated by electrophoresis in 0.8 or 1.2% agarose gels in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8,0) at 1 V cm? ovemight. Gels stained with ethidium bromide (0.5 jig mI") were photographed on a UV transilluminator. DNA fragments were blotted onto NylonN membranes (Amersham, Les Ulli, France) by alkaline vacuum transfer (TE 80 TransVac, Hoefer Scientific Instruments, San Francisco). Labeling of probes and hybridization conditions. Two miDNA probes obtained from F. oxysporum f. sp. conglutinans (20) were used for mtDNA RELP analysis: the whole purified 52-kb mol- cule and pUFI-14 (6.1-kb EcoRI-fragment in pUC119). Probe Fotl was a F oxysporum f, sp. melonis 4.6-kb EcoRI-fragment containing the 1.9-kb Fotl sequence (7). DNA probes were la- beled with =P-dCTP by random-priming according to the manu facturer’s specifications (Megaprime kit, Amersham) and hybridized to membrane-bound DNA fragments in standard hybridization buffer (Amersham) for at least 16 h at 65°C. Blots were washed twice with 2x SSC (1x SSC is 0.15 M sodium chloride plus 0.015 sodium citrate, pH 7.0) and 0.1% sodium dodecyl sulfate (SDS) for 20 min at room temperature, once with 1x SSC and 0.1% SDS for 20 min at 65°C, and twice with 0.5X SSC for 20 min at 65°C, Hybridized membranes were exposed to X-ray film (Amersham) at -80°C with intensifying screens. mtDNA RELP analysis. Previously (32), we found that the com- bination Hinf/pUF1-14 allowed detection of mDNA length vari- ations between F oxysporum f. sp. albedinis and other F. oxyspor- lum isolates, and the combination Belll/whole purified mtDNA. gave the most readable mtDNA hybridization patterns in F oxy- sporum f. sp. albedinis. In addition, the combination BgliV/whole purified mDNA revealed the highest number of restriction site polymorphisms in F oxysporum f. sp. vasinfectum (14). Based on these results, we chose the enzyme/probe combinations BellV/whole purified mtDNA and Hinfl/pUF1-14 to test the F oxysporum f. sp. ‘lbedinis isolates inthis study. Fott hybridization-pattern analysis. DNA from the 44 F ox- sporum f. sp. albedinis isolates and from nonpathogenic Foxy ‘sporum strain A15 was digested with EcoRI and hybridized to the Fotl probe. Fotl-hybridizing restriction fragments were scored either as present (1) or absent (0); unique band patterns were identified with specific pattem numbers. Bands of the same elec- trophoretic mobility were scored as identical. Analyses were based ‘on Jaceard's distance (18), which measures the proportion of com- ‘mon discrete data (C) between the isolates: d= 1 ~ C/(2N~ C), in ‘which W is the total number of different bands in the two com- pared hybridization patterns. A dendrogram was derived from the stance matrix by the the unweighted paired-group method, arith- ‘metic mean (UPGMA) algorithm (30) contained in the computer program package Phylip 3.4 (developed by J. Felsenstein, Depart ‘ment of Geneties, University of Washington, Seattle). RAPD assay. The primers used were random 10-base oligomers kit F, Operon Technologies, Alameda, CA), We selected three RAPD primers, OPFO4 (5°GGTGATCAGG3 ), OPFI2 (5“ACG- GTACCAG3}), and OPFI3 (5'GGCTGCAGAAS)) from a set of 20 previously analyzed (16) because these primers differentiatedF oxysporum f. sp. albedinis trom F. oxysporum, and they pro- ‘duced bright, reproducible bands. Amplification reactions were performed as described previously (2). After the reaction, either 20 pl of the amplification products was separated by electrophor- exis on 1.4% agarose gels stained with ethidium bromide and then photographed under UV light, or 10 lof the amplification prod- cts was separated by electrophoresis on 7% polyacrylamide gels and stained with silver nitrate. RAPD assays were performed at least twice foreach isolate with each primer to ensure that ampli- fication patterns were reproducible. RESULTS Vegetative compatibility tests. For each isolate, anit mutant was generated and paired with a NitM{ mutant tester from the four VCGs identified previously (31). Complementation tests showed that the 44 isolates were all vegetatively compatible and belonged to the group formerly designated VCGI (31), which, henceforth, we will call oxysporum f sp. albedinis group YCGO17O, to follow the designation firs used by Puhalla (29) and others (12,1934), ‘mtDNA. RELP analysis. No polymorphisms were deleted among the 44 isolates with the two enzyme/probe combinations tested Gig. 1), Fourteen Bgill-resrition fragments (with estimated sizes of 13, 125, 78, 42, 38, 3.6, 24, 22, 1.5, 13, 12, 1.1, 09, and 0.5 kb, respectively) hybridized with the whole purified mDNA probe, and four HinfL-restriction fragments (with estimated sizes 0f 1.25, 08, 0.6, and 0.4 kb, respectively) hybridized with the PUFI-14 probe. The length of oxysporum. sp. albedinis mxDNA, estimated to be 55 kb (32), was confirmed RAPD analysis. Initially, polymerase chain reaction (PCR) am- plificaton products were separated by electrophoresis in 1.4% ag arose gels. Depending on the primer tested, three to four DNA bands were obtained for cach isolate, and no polymorphism was detected among isolates within our collection. To enhance separ- ation and detection ofthe RAPD fragments, amplification products also were resolved by polyacrylamide gel electrophoresis and stained With silver nitrate. Five to eight amplified DNA fragments were identified foreach isolate, and again, no polymorphism was found (Fig. 2) Fotl hybridization-pattern analysis. For F oxysporum sp. albedinis isolates, an average of 20 EcoRI fragments per isolate, ranging from 0.8 to 15 kb, hybridized with the Fot probe. A total of 27 distinct restriction fragments were obtained among the 44 isolates, allowing detection of 23 DNA hybridization patterns (Fig. 3; Table 2). Only 7 of 27 fragments were common to all isolates tested, and the repetitive DNA patterns differed from each other by one to six bands, For F oxysporum strain ALS, only four EcoRL fragments hybridized with the Fot1 probe (Fable 2). Cluster anal ysis showed that all F oxysporum f. sp. albedinis isolates were ‘ery similar and formed a very tight cluster (Fig. 4). The isolates wwete grouped at a genetic distance of 0.11, except one isolate (1525, displaying pattern PIT) that clustered at 0.17. F axyspor- tum sain ALS clustered at 0.46 (Fig. 4) The high similarity de- tected among the 23 hybridization pattems indicates tha the 44 F axysporum {. sp. albedinis isolates belong 0 a single genetic group. Among the 23 hybridization patterns, 4 paterns (P2, P3, P10, and PIS) accounted for more than 50% ofthe isolates, and 18 were represented by single isolates (Table 2). Common hyp dization pattems (P2, P8, and P10) were found in the Moroccan locations. The two Algerian isolates exhibited a patter (PS) also found in Zagora (Draa Valley) (Table 1), DISCUSSION ‘The aim of this study was to test whether the F oxysporum f. sp.albedinis isolates causing Bayoud disease on date palm in Mor- cco originate from a unique virulent clone that spread throughout the oasis, ‘We used a large set of genetic and molecular markers to reveal polymorphisms in F oxysporum. All the F oxysporum f. sp. al Dedinis isolates grouped ina single VCG (0170) and displayed the same mtDNA resrition patterns and RAPD profiles. Sequences homologous to the DNA transposable element Fotl (7) were found in the genome of the F oxysporum f. sp. albedinis strains, revealing 23 closely related repetitive DNA patterns among the 44 isolates (Figs. 3 and 4). VCG, RAPD, and RFLP analyses have proved useful in detcting polymorphisms among pathogenic and nonpathogenic F oxysporum isolates and have increased our knowledge of the population biology of F oxysporum (24,12 14,17,19,21,23,26-28,34), Our analyses clearly showed that the E oxysporum . sp. albedinis isolates tested belong to a single genetic group. The high level of genetic similarity found among "TABLE 2. Fot! hybridization patterns (presence [1Vabsence [0] of each EcoRI fragment) obtained among 44 Fusarium oxysporum f. sp. albedinis isolates and E oxysporum sain A15 (outgroup) Patter No.of Noof m0. Haplorype fragments isolates Pr Trorrirortritooo1ti1too01r1torri v P2 Pritrrorritooorritrrortrroriya 2» 7 P3 Pritrrotrtiroorrtritrortroria & 1 Pa Pirtrirootr+trooooortriortroira wo 1 Ps Peitrrortrirooorrirtriorerrita mw 8 Pritororttrooorrit ortrortr 2 1 Perio Prooottritirititrorra 2 1 Protrrirotrirtrrooortritariororrord 2% 2 Pert rrorttrooorortriortrorrd a 1 Pettrrorttroootrritrrorororrd 9 4 FLoorroorttooorritirootrtioooo 3s 1 Prtrrrorrirooorrirororororrid 2» 1 Pri trorootrtroootTtTitirirottiroridi 2% 1 Protrroririrooor+rior+rorortriara 2% I Prottrrorrirooorritrrrororrirgri 5 Prooo1rotirt 1 ooOoOTTIE1eOot1HiO1II B 1 ProOtrtrioortroootrtrititotortooo t Pettrrorrtroootrrrorrotrrirorrd 3 t Pri tororriroootrortririorrirorirs 2% ' P20 Protrrrotrirororririrootriorrd 4 ' Pat Petrororriroootrititorororii 2 ' pa Prit?rotrrtroootrortriorriroerri 4 ' P23 Pri trtorrrrroorrirtrtrorriroriia 2% Ougrup 9 OL OOHTOOOFTOOOOOXOOCOO OOOO 4 Vol. 86, No. 7, 1996 789the isolates is consistent with the hypothesis that all F oxysporum £, sp. albedinis isolates belong to a unique clonal lineage and that the present Moroccan populations may derive from a single clone. Th addition, isolates from Morocco and Algeria had the same ‘multilocus genotype, and identical Fot! hybridization pattems were found in distinct palm groves (Table 1). Additional data obtained by the same techniques on a large sampling of Algerian isolates confirm the existence of a unique genetic group among F. oxy- sporum f. sp. albedinis isolates ({16], M. Ouinten, unpublished data). Bayoud was first described in the Draa Valley in southern Morocco (33); F oxysporum f. sp. albedinis is suspected to have been introduced along with contaminated date palm material in other locations and to have been successively transported from fone oasis to another (25,33). Our results are consistent with the 1234M kb M1234 Fig. 1. Mitochondtial DNA (rmtDNA) restriction paters of Fusarium ox)- sporun sp albedinis isolates. Left bybridization pattems ofthe whole pu fied mtDNA on Bglll-digested DNAS, Right hybridization paters of probe pUFI-14 on Minfligested DNAs. Lanes Ito 4, isolates OTK, Cl ALL, respectively. Lancs M, molecular weight marker (adiolabeled EcoRI. “Hindit-digested phage lambda DNA); the fragment size in kilobases is in- ‘icated in the mid. A B kb M1234 5 kb M12345 =a si 2.0 | 1.0 _] os Fig. 2. Random ampliied polymorphic DNA patteos generated from Fusar- fan oxysporum fp. albedinis isolates with primer OPF-12. Ay Agarose gel Stained with ethidium bromide; B, polyacrylamide gel stained with silver ni- teate Lanes 1105, isolates 133, OTK, Cl, 145, and ALL, rexpestivly. Lanes IM, molecular weight marker (EcoRt-Hindll-digested phage lambda DNA): the fragment size in kilobases is indicated othe left of A and B. 790 PHYTOPATHOLOGY scenario involving evolution in Morocco of a virulent clone and its subsequent geographic spread. ‘The special form albedinis, thus, presents a remarkable level of genetic homogeneity compared with those described in other F ‘oxysporum. In most of the special forms studied, several genetic ‘groups, which can be referred to as different clonal lineages, have been identified on the basis of VCG, RFLP, and RAPD analyses (2,12-14,19,26-28,34). To our knowledge, the existence of a uunigue clonal lineage within a special form is restricted to the eru- cifer-infecting formae speciales conglutinans, raphani, and matthioli G,20,21,23). These special forms were classified into the forma specialis conglutinans by Armstrong and Armstrong (1) because they share common host specificities. ‘The limited geographic arca of extension of Bayoud disease and the dispersal of F oxysporum f. sp. alhedinis to only two countries ‘could account for the lack of diversity in this special form but might not be the only explanation, There are several examples in pathogenic F. oxysporum populations in which genetic diversity is distributed on a small scale (13,14,19,26,27,34). Our results from E oxysporum. sp. albedinis can be compared with those obtained from another Arecaceae-attacking special form. F oxysporum f. sp. elaeidis, the oil palm (Elaeis guinensis) wilt pathogen is di tributed in Africa and America, and no races have been described so far (II). At least 10 genetic groups have been identified based ‘on VCG (11,12) and fingerprinting analysis with the F oxysporum sp. elaeidis cloned transposable element Palm (28). These clo- ral Tineages cocxist in at least two African countries (Ghana and Zaire), and two genetically distinct clones have been isolated from the same tree in Ghana. On the other hand, some clonal lineages ‘may be dominant on a regional or continental scale; analyses strongly suggest that the recently discovered American populations of F oxysporum f. sp. elaeidis were intraduced from Africa (11,12,28). ‘Whether several clones were the origin of Bayoud disease in the Dra Valley in Morocco and then lost by random genetic drift dur- ing the past 120 years is questionable and cannot be assessed easily. Loss of genotypes through genetic drift can be particularly important in strictly asexually reproducing populations. In the same ‘way, reduction in host population genetic diversity by the dramatic disappearance of some highly susceptible date palm cultivars could! have led 10 the reduction of genetic diversity in the actual pathogen population. In this study, the F oxysporum f. sp. al- bedinis isolates were collected from several date palm cultivars as ‘well as from trees resulting from genetic crosses at the INRA. experimental station in Zagora, We could not find particular asso- ciations between the Fot! hybridization patterns and the date palm cultivars from which the isolates were collected, including the highly ‘Bayoud-susceptible BoulTegous (Boufg) and Deglat Nour cultivars bo 1234 85678 9n Rw e ig. 3 Ft hybvidization patems obtained with 14 Fusarium oxyeporan sp. albedinis isolates (anes I trough 14). The fragment size is indicated the teeSeveral questions need to be addressed. First, the possible oc- currence of races in F oxysporum f. sp. albedinis and the patho- genic status of the isolates have to be determined. The genetic similarity detected by molecular markers or VCG does not neces- sarily mean there is no variation with respect to pathogenicity. In several other special forms of F oxysporum, isolates belonging to distinct races belong to the same VCG or RFLP or RAPD group (4,13,17,19,27), Thus, we cannot exclude the possible existence of races in F. oxysporum f. sp. albedinis based on the current results, Second, will this genetic homogeneity be maintained overtime? We have shown that the genome of the fungus contains several sequences homologous to the DNA transposable element Fot], which is known to be active in F. oxysporum (7). The high number of Fotl hybridization patterns in & oxysporum f. sp. albedinis strains (23 pattems versus 44 isolates) compared to the strict ge- netic homogeneity observed with other analyses (VCG, mtDNA, RFLP, and RAPD) suggests that some copies of Fotl may still be able to move or that some recombination events may cecur during ‘mitosis or via a parasexual process between vegetatively compat ible strains (6,22). If new Fott patterns can be generated by sev- eral molecular mechanisms associated with the presence of active transposable elements in the genome, then it would be possible for local genetic diversification of the fungus to occur. We have shown that the Fotl hybridization pattems make it possible to analyze F ‘oxysporum f. sp. albedinis populations on a local scale (oasis), enotic distance os 04 0st pu outgroup Fig. 4. Dendiogram showing the genetic relationships among Fusarium oxy: sporam sp. albedins isolates and nonpathogenic F oxysporum isolate AIS outgroup. Cluster analysis was performed bythe unweiphtedpaied- group ‘method with arithmetic mean, and! genetic distances were obtained based on Jaccar's similarity coefficient of 27 individual DNA bands produced by hybridization with the Fotl probe. For convenience, isolates are represented by their eomresponding hybedization-pattem numbers (Table 2). ‘Tracking genotypes would be useful to detect genetic changes over time and would allow testing for F oxysporum f, sp. albedinis Population differentiation in the oases of Moracco and Algeti. LITERATURE CITED 1. Anmstrong. G.M..and Armstrong J.K. L981. Formae specials and races ‘of Fusarium oxysporum causing wilt diseases, Pages 391-399 ins Fusar- ‘wm: Diseases, Biology and Taxonomy. PE. Nelson, T. A. Toussoun, and R.J. Cook, eds. The Pennsylvania State University Pres, University Pat, 2. Assighetse. K. B., Femander, D,, Dubois, M.P, and Geiger, 3. P1994, Differentiation of Fisarium oxysporum f 3p. vasnfectam races on cotton by random amplified polymarphie DNA (RAPD) analysis, Phytopathol ony 84:622-626, 4, Bosland, P.W,, and Williams, PH. 1987. 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