SEARCH

SEARCH BY CITATION

Keywords:

  • DNA fingerprinting;
  • Fusarium;
  • genetic diversity;
  • palm wilt;
  • Phoenix canariensis;
  • vegetative compatibility

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fusarium oxysporum f.sp. canariensis causes vascular wilt disease of Phoenix canariensis, the Canary Island date palm. Seventy-two isolates of this fungus were obtained from diverse geographic locations including France, Japan, Italy, the Canary Islands, and California, Florida and Nevada, USA. The isolates were tested for vegetative compatibility and for similarities based on mitochondrial DNA (mtDNA), single-copy sequences and repetitive DNA (pEY10) polymorphisms. Seventy-one percent of the isolates belonged to a single vegetative compatibility group (VCG 0240), and four closely related mitochondrial RFLP patterns were found. A subset of the isolates was further tested for single-copy RFLPs and repetitive DNA fingerprints. Only four single-copy RFLP haplotypes were found among 25 representative isolates of F. oxysporum f.sp. canariensis tested, using nine polymorphic single-locus probe/enzyme combinations. Finally, 32 different pEY10 DNA fingerprints were found out of 57 isolates examined. Overall the results indicate that F. oxysporum f.sp. canariensis is a single lineage with a low to moderate level of genetic diversity.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The soilborne fungus Fusarium oxysporum contains many host-specialized, pathogenic forms or formae speciales that cause vascular wilt and/or root rot disease in particular plant species. Fusarium oxysporum f.sp. canariensis ( Mercier & Louvet, 1973) causes a severe vascular disease known as Fusarium wilt on the Canary Island date palm (Phoenix canariensis). Fusarium wilt is currently threatening these ornamental palms in landscapes and nurseries in many parts of the world. Molecular genetic studies indicate that there are different patterns of diversity exhibited by various formae speciales of F. oxysporum ( Kistler, 1997). Some have few multilocus haplotypes based on single-locus RFLP data, have similar or identical mitochondrial RFLP haplotypes, and/or belong to a single vegetative compatibility group (VCG). This simple pattern of diversity is found in F. oxysporum f.spp. albedinis ( Tantaoui & Fernandez, 1993; Tantaoui et al., 1996 ; Fernandez et al., 1997 ) and conglutinans ( Bosland & Williams, 1987; Kistler et al., 1987 ), and is consistent with a population structure resulting from the clonal expansion of a single, successful pathogenic genotype. Other formae speciales have patterns of diversity that also are consistent with clonality, but are characterized by two or more distinct lineages within a forma specialis, as reflected in multiple VCGs, several diverse mitochondrial haplotypes, and relatively larger numbers of multilocus haplotypes defined by RFLPs or randomly amplified polymorphic DNAs (RAPDs). This pattern is exemplified by F. oxysporum f.spp. cubense ( Bentley et al., 1995 ; Koenig et al., 1997 ; O'Donnell et al., 1998 ), melonis ( Jacobson & Gordon, 1988, 1990) and lycopersici ( Elias & Schneider, 1991; Elias et al., 1993 ). Distance measurements between lineages within these formae speciales indicate that they may be very closely related or quite distant. This research is the first to examine the relationships of isolates within F. oxysporum f.sp. canariensis with respect to vegetative compatibility and molecular characteristics. It is still to be determined whether this forma specialis consists of a single lineage, like F. oxysporum f.spp. albedinis and conglutinans, or whether it is made up of multiple lineages like F. oxysporum f.spp. cubense, melonis and lycopersici. If F. oxysporum f.sp. canariensis is a single lineage, then detection by a single diagnostic method such as that developed by Plyler et al. (1999 ) would be practical. This information could also influence strategies for deployment of disease resistance and disease management.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fungal isolates

Seventy-two isolates of F. oxysporum f.sp. canariensis were used to measure genetic diversity ( Table 1). These included 13 isolates (three from the Canary Islands, six from France, three from Japan and one from California, USA) that were confirmed to be F. oxysporum f.sp. canariensis via pathogenicity tests (J. Louvet, INRA, Dijon, France, personal communication). The remaining isolates used in this study came from tissues of Phoenix canariensis showing symptoms, which were sent to the Florida Extension Plant Disease Clinic in Gainesville from locations in Florida, California, and Nevada, or to the Universita' di Torino from Sicily. These isolates were identified by primers that detected the authentic palm pathogens and by mitochondrial RFLP profiles that were similar to the original pathogen strains ( Plyler et al., 1999 ). Fusarium oxysporum f.sp. cubense isolate F9127, f.sp. raphani isolate 699, f.sp. elaeidis isolate EQ and f.sp. elaeidis isolate Dem1 were used as outgroups. All DNA was extracted according to the methods discussed previously, and single spores of the isolates were stored in 50% glycerol at −80°C ( Plyler et al., 1999 ).

Table 1. Fusarium oxysporum f.sp. canariensis isolates and outgroups listed by geographic origin, vegetative compatibility to VCG 0240 testers, mitochondrial haplotype, RFLP multilocus haplotype and pEY10 fingerprint F. oxysporum f.sp. canariensis isolates from:athe collection of Diana Fernandez, IRD, Montpellier, France;bthe collection of Gary W. Simone, Florida Extension Plant Disease Clinic, Gainesville, FL, USA;cQuirico Migheli, Universita di Torino, Italy.*The abbreviation Co. represents the county in Florida from which the isolates were obtained.†RFLP fingerprints were generated with HindIII (H) or with EcoRV (E) using the polymorphic probes 31, 91 and 106, represented in this order within the haplotype: H or E (91, −, −, −, 31, 106, −, −, −).‡Two isolates of F. oxysporum f.sp. elaeidis from Ecuador were used for analysis. EQ was tested with the mitochondrial probe but not with single-copy probes. Dem1 was tested with single-copy probes but not the mitochondrial probe.Thumbnail image of

Vegetative compatibility determination

Single-spore isolates of F. oxysporum f.sp. canariensis were removed from −80°C and placed onto PDA. After 2–5 days' incubation at room temperature, a 1 cm2 block of mycelium was removed from the margin of the actively growing culture and placed onto PDA containing 30 g L−1 potassium chlorate. Chlorate-resistant sectors would appear from 5 to 9 days as an area of wild-type growth ( Puhalla, 1985).

A 1 cm2 block of mycelium from the chlorate-resistant mutants was placed onto basal medium, which consisted of 30 g sucrose, 1 g KH2PO4, 0.5 g MgSO4.7H2O, 0.5 g KCl, 10 mg FeSO4, 20 g laboratory grade agar, 0.2 mL trace element solution (5 g citric acid, 5 g ZnSO4.7H2O, 1 g Fe(NH4)2(SO4)2.6H2O, 0.25 g CuSO4.5H2O, 50 mg MnSO4.H2O, 50 mg H3BO3, 50 mg NaMoO4.H2O per 95 mL water), amended with 2 g NaNO3 in 1 L water to make minimal medium. Mutants unable to use nitrate appeared as a layer of thin mycelial growth ( Correll et al., 1987 ).

To identify hypoxanthine nonutilizing mutants (nitM), a 1 cm2 block of mycelium from the minimal medium was placed on hypoxanthine media containing 0.2 g hypoxanthine L−1 added to the basal medium ( Correll et al., 1987 ). Two mutants, derived from isolates 94-871 and 95-F051F, were identified by their inability to use hypoxanthine as a sole nitrogen source and appeared as a thin layer of mycelial growth on this medium. These two mutants were used as nitM testers for vegetative compatibility pairings.

To test for vegetative compatibility, a 1 cm2 block of mycelium from one of the nitM testers was placed in the centre of a plate containing minimal medium. A 1 cm2 block of chlorate-resistant mutants or nitrate-nonutilizing mutants derived from three other isolates were placed adjacent to the testers and allowed to grow together. Heterokaryon formation, which appeared as an area of dense, wild-type growth, occurred between 3 and 14 days. All isolates were tested against both hypoxanthine mutants (94-871 and 95-F051F). If an isolate formed a heterokaryon with either or both of these mutants, it was considered to be in VCG 0240, as assigned by the VCG numbering coordinator ( Katan, 1999). If one of the nit mutants in the plate paired with an adjacent nit mutant, these were considered vegetatively compatible as well. If one of the two adjacent nit mutants had paired with the tester, all three isolates were considered to be within VCG 0240. Isolates that formed heterokaryons were, by definition, in the same vegetative compatibility group.

Mitochondrial haplotypes

DNA was extracted according to the methods described previously ( Plyler, 1997). Approximately 1 μg of total DNA from all of the F. oxysporum f.sp. canariensis isolates and from 20 other isolates of F. oxysporum was digested with HaeIII, loaded onto 0.7% agarose gels and run between 19 and 22 h at 33 V. The gels were prepared for Southern blotting on a Nytran nylon membrane (Schleicher and Schuell, Keene, NH, USA) using capillary transfer methods essentially as described previously ( Sambrook et al., 1989 ). The membrane was exposed to UV light (254 nm) to immobilize the DNA.

For the hybridization probe, mitochondrial DNA (15 μg) was extracted from F. oxysporum f.sp. cubense isolate 3S1 ( Koenig et al., 1997 ). The DNA was digested with HindIII, labelled, hybridized to the membrane at 65°C overnight, and detected on autoradiography film according to the manufacturer's protocol (Dupont NEN ‘Renaissance’, E. I. Du Pont de Nemours and Co., Boston, MA, USA).

Single-copy RFLPs

A subset of 25 isolates were chosen for RFLP analysis so that each geographic location of the isolates was represented ( Table 1). DNA was extracted as previously described from single-spore cultures of each isolate. Approximately 1 μg of total DNA was digested with either HindIII, XbaI, EcoRI, DraI or EcoRV for 7–24 h at 37°C. The digested DNA was run on 0.7% agarose gels for 19–22 h at 31–34 V. The DNA was transferred by Southern blotting to Zeta Probe nylon membranes with 0.4 m NaOH. The DNA was fixed by heating at 80°C for 30 min

Seventeen clones out of a plasmid library of total DNA from isolate 95-913 were screened against the digested DNAs for polymorphisms. The clones were labelled by nick translation with 32P (Gibco BRL Life Technologies, Gaithersburg, MD, USA). Hybridization of the clones to the membrane was made at 65°C according to the methods described previously ( Plyler, 1997). After hybridization, the membranes were exposed to autoradiography film at −80°C for 1–2 days depending on the strength of the signal.

DNA fingerprinting

Approximately 1 μg of total DNA from each of the F. oxysporum f.sp. canariensis isolates and from the outgroup isolates was digested with HindIII, loaded onto 0.7% agarose gels and run at 33 V for 18–21 h. The gels were prepared for Southern blotting using the alkaline capillary transfer methods previously mentioned for single-copy RFLPs.

The fingerprinting probe used in this study, pEY10, was obtained from a genomic library of DNA from F. oxysporum f.sp. conglutinans ( Kistler et al., 1991 ) and corresponds to an open reading frame with a predicted amino-acid sequence having 51% identity to the putative transposase from Magnaporthe grisea repetitive element Pot3 (R.E. Pettway and H.C. Kistler, personal communication). DNA from pEY10 was labelled by nick translation (Gibco BRL). Hybridization of the probe to the membrane and detection of the signal was performed as described in the methods for single-copy RFLPs.

Data analysis

The pEY10 fingerprints were obtained for 57 F. oxysporum f.sp. canariensis isolates, 23 of which were also tested with the single-copy RFLP probes. A matrix of polymorphic restriction sites was created for these 23 isolates, with the exception of 84-Cala and 96-1613, and entered into the matrix ( Fig. 1). A migration position on the gel was determined for each band detected using this probe. The presence or absence of a band at each migration position was scored 1 or 0, respectively. Genetic distance was determined using the heuristic search option in paup*4.0d64 ( Swofford, 1998) for the pEY10 fingerprint data ( Fig. 2). Fusarium oxysporum f.spp. cubense and raphani were used as outgroups.

image

Figure 1 Matrix of band positions for pEY10 fingerprint. 1. , presence of band; 0, absence of band.

Download figure to PowerPoint

image

Figure 2. Unrooted dendrogram of pEY10 fingerprint data. Branches show the genetic distance as determined by paup*4.0d64. Numbers at the ends of branches represent the pEY10 fingerprint designation given in Table 1.

Download figure to PowerPoint

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

VCG determination

Approximately 71% of isolates (49 of 69) formed heterokaryons with 94-871 and/or 95-F051F nitM testers, or with a nit mutant that had paired with one of the testers. Nit tester mutants of 94-871 or 95-F051F did not pair with each other, but could be linked by the nit mutant of isolate 95-513 that paired with both of them. Isolates that paired included isolates from every country represented in this study. All isolates that formed heterokaryons with the testers or that could be linked to the testers were considered a single vegetative compatibility group (VCG 0240). Among F. oxysporum f.sp. canariensis isolates unable to pair with either VCG 0240 tester, isolates 95-514C and 95-1005 formed heterokaryons with each other, as did isolates 96-1672 and 87-Guil2.

The remaining isolates did not form heterokaryons with any other isolate of F. oxysporum f.sp. canariensis. These included three isolates from California, five from Florida (three from Lake County, one from Hillsborough County and one from Sarasota County), two of the three isolates from Japan, one of the two isolates from Sicily, four isolates from France, and one of the three isolates from the Canary Islands. Nit mutants of these isolates were not further characterized.

Mitochondrial haplotypes

Four mitochondrial haplotypes were observed by hybridization of mitochondrial DNA to DNA from isolates of F. oxysporum f.sp. canariensis digested with HaeIII ( Fig. 3). The haplotypes were very similar and appeared to represent all four combinations of two length mutations. One of the mutations appeared as an insertion/deletion (indel) in the largest HaeIII fragment (7 or 5.5 kb) of the F. oxysporum f.sp. canariensis mitochondrial genome. The other indel resulted in the presence or absence of an approximately 750 bp band after HaeIII digestion.

image

Figure 3 Mitochondrial haplotypes generated from total F. oxysporum f.sp. canariensis DNA digested with HaeIII and probed with mitochondrial DNA from F. oxysporum f.sp. cubense isolate 3. S1. Letters A–D correspond to the mitochondrial haplotype designation shown in Table 1.

Download figure to PowerPoint

The two most commonly occurring haplotypes (A, B) each corresponded to 43.1% of the isolates ( Table 1). The less common haplotypes (C, D) represent profiles for 11 and 2.8% of the isolates, respectively ( Table 1).

Mitochondrial haplotype did not strictly correlate with the geographic origins of the isolates. All haplotypes were found among the isolates from Florida. Only the two most common haplotypes were found among isolates from California. The three isolates from Japan had haplotypes B and D, whereas all the isolates from France and the Canary Islands had mitochondrial haplotype A. The 20 isolates of F. oxysporum tested from hosts other than P. canariensis all produced banding patterns different from those of the four haplotypes found for F. oxysporum f.sp. canariensis (data not shown).

Single-copy RFLPs

Polymorphisms were detected using 9 of 17 probes in DNA of F. oxysporum isolates, including three strains from other hosts. Of these nine, only three enzyme/probe combinations (HindIII/probe 91, HindIII/probe 106 and EcoRV/probe 31) revealed polymorphisms among DNAs from F. oxysporum f.sp. canariensis isolates. Using EcoRV/probe 31, only two haplotypes were observed within F. oxysporum f.sp. canariensis ( Table 1). With HindIII, probes 91 and 106 each produced polymorphisms and together resulted in four haplotypes ( Table 1).

The most common haplotype with EcoRV (111111111) was found in combination with all four mitochondrial haplotypes. The second single-locus haplotype (111121111) was found in only one mitochondrial haplotype and for only two F. oxysporum f.sp. canariensis isolates, 95-F050F and 95-F051F ( Table 1). The most commonly occurring single-locus haplotype with HindIII was found for isolates that contained all four mitochondrial haplotypes and were from a diversity of locations. The isolates from the Canary Islands produced a distinctive single-locus haplotype.

All multilocus single-copy haplotypes for the three isolates of F. oxysporum from other hosts were different from those of F. oxysporum f.sp. canariensis and from each other. As anticipated from previous work ( O'Donnell et al., 1998 ; Plyler et al., 1999 ), F. oxysporum f.sp. cubense isolate F9127 had a haplotype very similar to that of the F. oxysporum f.sp. canariensis isolates. Using HindIII in combination with the nine probes, isolate F9127 shared alleles with F. oxysporum f.sp. canariensis at all loci, but had a unique combination of alleles. The isolates Dem1 and 699 produced haplotypes that were different for almost every probe/enzyme combination. These outgroups rarely shared alleles with the F. oxysporum f.sp. canariensis isolates.

DNA fingerprinting

DNA fingerprints for 57 F. oxysporum f.sp. canariensis isolates were obtained with the probe pEY10. Thirty-two fingerprint patterns were found for the 57 isolates. The fingerprint patterns appear to align with the geographic origins of the isolates in some cases. For instance, all isolates from Cannes, France have the same fingerprint. On the other hand, isolates from the same geographic location did not always share the same DNA fingerprint. For example, isolate 84-207 from Kagoshima, Japan had a different DNA fingerprint from isolates 84-305 and 84-104 also from Kagoshima. Isolate 84-207 also had a different mitochondrial haplotype from that of the latter isolates.

The ability to form heterokaryons did not correlate with pEY10 fingerprint patterns. Most isolates with identical DNA fingerprint patterns also formed heterokaryons with either nitM 94-871 or nitM 95-F051 tester isolates. However, all but six isolates with unique pEY10 fingerprint patterns were also vegetatively compatible with the nitM testers.

While isolates that shared pEY10 DNA fingerprint patterns also shared single-copy multilocus RFLP haplotypes ( Table 1), isolates with different pEY10 fingerprints also sometimes shared multilocus RFLP haplotypes. For example, among the eight isolates having RFLP haplotype 211112111 created by HindIII, four different DNA fingerprinting patterns were discerned. The isolates of F. oxysporum from other hosts displayed pEY10 fingerprinting patterns unlike those for F. oxysporum f.sp. canariensis isolates.

The fingerprints from a group of isolates from Florida (95-913, 95-1000 to 95-1007, 95-513 and 95-514C) are similar to those of isolates from Sicily (Sicily 1 and Sicily 2), France (84-01a, 84-01b, 84-P1, 84-P2 and 84-02b), and Japan (84-207). Many of the isolates from Florida did not share the same fingerprint. Fourteen individual DNA fingerprints were detected in the 32 isolates of F. oxysporum f.sp. canariensis from Florida.

When DNA fingerprinting data were used for distance analysis, dendrograms showed that outgroups F. oxysporum f.sp. raphani (699) and F. oxysporum f.sp. cubense (F9127) were distinct from F. oxysporum f.sp. canariensis isolates. The F. oxysporum f.sp. canariensis isolates were separated only by a small genetic distance.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Several lines of evidence suggest that F. oxysporum f.sp. canariensis consists of a single clonal lineage with a moderate level of genetic diversification within that lineage. The first line of evidence, vegetative compatibility testing, indicates that there is a single major VCG (0240) within this forma specialis, with 71% of the isolates tested belonging to this VCG. Isolates that did not belong to VCG 0240 were nevertheless very similar to, or even indistinguishable from, authentic VCG 0240 isolates based on other genetic criteria ( Table 1). However, it is likely that other VCGs exist in F. oxysporum f.sp. canariensis, as suggested by the fact that isolates 95-514C and 95-1005, as well as isolates 96-1672 and 87-Guil2, formed heterokaryons with each other but not with the nitM testers of VCG 0240. These VCGs might have arisen by mutation at one or more loci for vegetative compatibility.

Comparisons of the VCG information with the other genetic markers used in this study also revealed that all F. oxysporum f.sp. canariensis isolates were similar. Only four closely related mitochondrial haplotypes were described for the 72 isolates of F. oxysporum f.sp. canariensis examined. Isolates from each of the four mitochondrial haplotypes were found to belong to VCG 0240. In other formae speciales of F. oxysporum, mtDNA haplotypes also show strong correlation with vegetative compatibility groups ( Jacobson & Gordon, 1990; Gordon & Okamoto, 1992a, b; Tantaouiet al., 1996 ). The mtDNA profiles for F. oxysporum f.sp. canariensis contained all four combinations of two length mutations. These combinations of alleles could have resulted from mitochondrial recombination between isolates, or alternatively could have arisen through independent mutations occurring at high frequency at particular sites on the mitochondrial chromosome.

Single-copy haplotype data also support a moderate level of genetic diversity with the probes tested in this study. Upon examination of the single-copy RFLP haplotypes, it was found that typing with the enzyme/probe combinations HindIII/91 and HindIII/106 can be used to distinguish isolates from the Canary Islands. In fact, the single-copy haplotype of the Canary Island isolates (111112111) differed from all other single-copy haplotypes by only a single step ( Fig. 4). No other haplotype has a similar, one-step relationship to all other haplotypes. For this reason, it is suspected that isolates from the Canary Islands may represent the ancestral form of F. oxysporum f.sp. canariensis. This would be consistent with the idea that the pathogen arose from the centre of origin of the host species. Additional information from other polymorphic single-copy loci could be informative for supporting or refuting this hypothesis.

image

Figure 4. Relationship of the F. oxysporum f.sp. canariensis haplotypes defined by single-copy RFLPs.

Download figure to PowerPoint

According to mitochondrial haplotypes and single-copy RFLPs, F. oxysporum f.sp. canariensis appears to be more genetically diverse than F. oxysporum f.sp. albedinis, the cause of Bayoud disease on Phoenix dactylifera. Previously it was found that a collection of isolates spanning the entire geographic range of F. oxysporum f.sp. albedinis all belonged to a single VCG, and had identical mitochondrial DNA RFLP patterns and RAPD profiles ( Tantaoui et al., 1996 ). Only DNA fingerprinting using the transposable element FotI from F. oxysporum was able to differentiate the isolates, yielding 23 DNA fingerprint patterns among the 44 isolates examined. Conversely, F. oxysporum f.sp. canariensis appears less diverse than f.sp. cubense, which has been shown to have multiple evolutionary origins. F. oxysporum f.sp. cubense has at least 16 VCGs with multiple mtDNA haplotypes and at least 10 widely divergent clonal lineages ( Koenig et al., 1997 ).

Genetic distance data derived from pEY10 fingerprints yielded 32 different fingerprints for 57 isolates of F. oxysporum f.sp. canariensis. The fingerprint patterns are consistent with the idea that pEY10 identifies clonally derived isolates. All isolates with identical pEY10 fingerprint patterns also have the same mitochondrial and single-copy multilocus haplotype. These isolates almost certainly were clones and were often isolated from the same geographical region. Such clones were identified in isolates from California, Florida, Japan, Sicily, France and the Canary Islands ( Table 1). On the other hand, DNA fingerprint patterns were sometimes shared by isolates from different geographic locations, suggesting the dispersal of clones. For example, isolates 95-513, 95-514C, 95-913, 95-1000 to 95-1004, 95-1006 and 95-1007 all have the DNA fingerprint profile number 13, yet were isolated from either Manatee County or Lake County, Florida. These counties are separated in distance by about 80 km.

Genetic distance data indicate that isolates from Florida are, in many cases, more closely related to isolates from other parts of the world than they are to each other. For example, Florida isolate 95-1714 (fingerprint pattern 14 in Fig. 2) is most closely related to isolate 70-MercIV (fingerprint pattern 6 in Fig. 2) from France (distance = 0.03), while it is separated from Florida isolates in fingerprint pattern 13 ( Fig. 2) by a genetic distance of 0.284. In another example, isolate 94-861 (fingerprint pattern 11) from Hillsborough County, Florida is separated from California isolates 96-08, 96-13 and 96-14 (fingerprint pattern 2) by a distance of 0.059. On the other hand, the distance between 94–861 and the Florida isolates in fingerprint pattern 13 is 0.284. In these instances, the isolates from Florida are more closely related to isolates from distant areas than to each other. As the presence of the fungus in Florida was not documented prior to 1994, it is unlikely that the Florida isolates have evolved these levels of differences within the state; these observations appear to support the idea that F. oxysporum f.sp. canariensis arrived in the state through multiple, independent introductions. Transport of contaminated vegetative or seed palm material would probably be a method of introduction to Florida.

In summary, the data presented here support the idea that F. oxysporum f.sp. canariensis is derived from a single lineage. However, the pathogen has diversified in various regions of the world and has probably also been introduced into new areas through international trade. Recently developed diagnostic systems for the pathogen ( Plyler et al., 1999 ) will aid in the early detection of the fungus on live plants, and facilitate efforts for exclusion of the pathogen and production of pathogen-free transplant materials.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors would like to thank Dr Brian Bowen and Dr Gerry Benny for their reviews of this research. Our sincere appreciation goes to David L. Swofford (Smithsonian Institution) for permission to publish results obtained with paup*4.0d64. We thank the Florida Extension Plant Disease Clinic for isolation of F. oxysporum f.sp. canariensis and thank those who donated other DNAs. We also thank Ms Ulla Benny for her technical advice.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    Bentley S, Pegg KG, Dale JL, 1995. Genetic variation among a world-wide collection of isolates of Fusarium oxysporum f.sp. cubense analyzed by RAPD-PCR fingerprinting. Mycological Research 99, 1378 84.
  • 2
    Bosland PW & Williams PH, 1987. An evaluation of Fusarium oxysporum from crucifers based on pathogenicity, isozyme polymorphism, vegetative compatibility, and geographic origin. Canadian Journal of Botany 65, 2067 73.
  • 3
    Correll JC, Klittich CJR, Leslie JF, 1987. Nitrate non-utilizing mutants of Fusarium oxysporum and their use in vegetative compatibility tests. Phytopathology 77, 1640 6.
  • 4
    Elias KS & Schneider RW, 1991. Vegetative compatibility groups in Fusarium oxysporum f.sp. lycopersici. Phytopathology 81, 159 62.
  • 5
    Elias KS, Zamir D, Lichtman-Pleban T, Katan T, 1993. Population structure of Fusarium oxysporum f.sp. lycopersici: restriction fragment length polymorphisms provide genetic evidence that vegetative compatibility group is an indicator of evolutionary origin . Molecular Plant–Microbe Interactions 6, 565 72.
  • 6
    Fernandez D, Ouinten M, Tantaoui A, Geiger J-P, 1997. Molecular records of micro-evolution within the Algerian population of Fusarium oxysporum f.sp. albedinis during its spread to new oases. European Journal of Plant Pathology 103, 485 90.
  • 7
    Gordon TR & Okamoto D, 1992 a. Population structure and the relationship between pathogenic and nonpathogenic strains of Fusarium oxysporum. Phytopathology 82, 73 7.
  • 8
    Gordon TR & Okamoto D, 1992 b. Variation in mitochondrial DNA among vegetatively compatible isolates of Fusarium oxysporum. Experimental Mycology 16, 245 50.
  • 9
    Jacobson DJ & Gordon TR, 1988. Vegetative compatibility and self-incompatibility within Fusarium oxysporum f.sp. melonis. Phytopathology 78, 668 72.
  • 10
    Jacobson DJ & Gordon TR, 1990. Variability of mitochondrial DNA as an indicator of relationships between populations of Fusarium oxysporum f.sp. melonis. Mycological Research 86, 734 44.
  • 11
    Katan T, 1999. Current status of vegetative compatibility groups in Fusarium oxysporum. Phytoparasitica 27, 51 64.
  • 12
    Kistler HC, 1997. Genetic diversity in the plant-pathogenic fungus Fusarium oxysporum. Phytopathology 87, 474 9.
  • 13
    Kistler HC, Bosland PW, Benny U, Leong S, Williams PH, 1987. Relatedness of strains of Fusarium oxysporum from crucifers measured by examination of mitochondrial and ribosomal DNA. Phytopathology 77, 1289 93.
  • 14
    Kistler HC, Momol EA, Benny U, 1991. Repetitive genomic sequences for determining relatedness among strains of Fusarium oxysporum. Phytopathology 81, 331 6.
  • 15
    Koenig RL, Ploetz RC, Kistler HC, 1997. Fusarium oxysporum f.sp. cubense consists of a small number of divergent and globally distributed clonal lineages. Phytopathology 87, 915 23.
  • 16
    Mercier S & Louvet J, 1973. Recherches sur les fusarioses. X. Use fusariose vasculaeri (Fusarium oxysporum) du palmier des Canaries (Phoenix canariensis) . Annales de Phytopathologie 5, 203 11.
  • 17
    O'donnell K, Kistler HC, Cigelnik E, Ploetz RC, 1998. Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. Proceedings of the National Academy of Science, USA 95, 2044 9.
  • 18
    Plyler TR, 1997. Genetic diversity studies on Fusarium oxysporum f.sp. canariensis and the development of a polymerase chain reaction technique for its detection. MSc thesis. Gainesville, FL: University of Florida.
  • 19
    Plyler TR, Simone GW, Fernandez D, Kistler HC, 1999. Rapid detection of the Fusarium oxysporum lineage containing the Canary Island Date Palm wilt pathogen. Phytopathology 89, 407 13.
  • 20
    Puhalla JE, 1985. Classification of strains of Fusarium oxysporum on the basis of vegetative compatibility. Canadian Journal of Botany 63, 179 83.
  • 21
    Sambrook J, Fritsch EF, Maniatis T, 1989. Molecular Cloning. A Laboratory Manual (2nd edn) Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
  • 22
    Swofford DL, 1998. Phylogenetic Analysis using Parsimony. 4.0d64 pre-beta-test version. Sunderland, MA: Sinauer.
  • 23
    Tantaoui A & Fernandez D, 1993. Comparaison entre Fusarium oxysporum f.sp. albedinis et Fusarium oxysporum des sols de palmeraies par l'étude du polymorphisme de longueur des fragments de restriction (RFLP) . Phytopathologia Mediterranea 32, 235 44.
  • 24
    Tantaoui A, Oiunten M, Geiger J-P, Fernandez D, 1996. Characterization of a single clonal lineage of Fusarium oxysporum f.sp. albedinis causing Bayoud disease of date palm in Morocco. Phytopathology 86, 787 92.