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Applied and Environmental Microbiology, December 2005, p. 8466-8471, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8466-8471.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Gibberella xylarioides Sensu Lato from Coffea canephora: a New Mating Population in the Gibberella fujikuroi Species Complex

Pascale C. E. Lepoint,^1* Françoise T. J. Munaut,² and Henri M. M. Maraite^1,2

Unité de Phytopathologie, Université catholique de Louvain, Croix du Sud 2/3, B-1348 Louvain-la-Neuve, Belgium,¹ Mycothèque de l'Université catholique de Louvain, Belgian Co-ordinated Collection of Micro-organisms, Unité de Microbiologie, Croix du Sud 3/6, B-1348 Louvain-la-Neuve, Belgium²

Received 27 January 2005/ Accepted 7 September 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gibberella xylarioides Heim & Saccas (presumed anamorph, Fusarium xylarioides Steyaert) is the causal agent of coffee wilt disease, an economically important tracheomycosis in Africa. In vitro crosses carried out with Congolese, Ugandan, and Tanzanian single-ascospore/conidial isolates originating from diseased Coffea canephora/excelsa demonstrated a heterothallic mating system, controlled by a single locus with two alleles, MAT-1 and MAT-2. Compatible isolates produced fertile perithecia within 2 to 8 weeks after mating. Mating type (MAT) was characterized by PCR with primer pairs previously developed for the Gibberella fujikuroi species complex (GFC) and for Fusarium oxysporum. All strains analyzed were morphologically identical and corresponded to Booth's description of the "female" F. xylarioides strain. Based on crossing results and MAT-2/translation elongation 1- (tef) sequence data, G. xylarioides, as currently understood, is demonstrated to encompass at least three "groups": G. xylarioides sensu strictu Ia, defined hitherto by two "historical" West African strains originating from the severe 1930s to 1950s epidemic (CBS 25852 and CBS 74979); G. xylarioides sensu strictu Ib, defined by two "historical" Central African lowland strains (DSMZ 62457 and ATCC 15664); and G. xylarioides sensu lato II, containing Congolese, Ugandan, and Tanzanian C. canephora/excelsa isolates. Infertility of crosses between the coffee wilt pathogen and known GFC mating populations demonstrates that G. xylarioides sensu lato constitutes a new biological species within the G. fujikuroi complex. MUCL 44532/MUCL 43887 and MUCL 35223/MUCL 44549 are proposed as G. xylarioides sensu lato II MAT-1/MAT-2 reference mating type tester strains.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Coffee wilt disease (CWD) was first noticed around 1927 in Oubangui-Chari (Central African Republic [CAR]) on Coffea excelsa (27). Between 1937 and 1939 the disease spread to C. canephora and C. liberica in Cameroon, Guinea, Côte d'Ivoire, and the Democratic Republic of Congo (DRC), where up to 40% of plantations were infected (8, 11, 12, 19, 26, 28). Since then, CWD has reemerged on C. canephora/excelsa in portions of the DRC (mid-1980s), affecting up to 90% of plantations (6), and more recently (1993) in Uganda (7), and the Lake Victoria region of Tanzania.

Gibberella xylarioides sensu strictu Heim & Saccas (1950) (supposed anamorph, Fusarium xylarioides Steyaert [1948]) was determined as the causal agent of the severe coffee tracheomycosis or carbunculariosis, more commonly known as CWD, reported in the 1930s-50s epidemic. On the basis of a probable initial misidentification as F. oxysporum (8) and F. oxysporum f. xylarioides (5), the pathogen was thought to be a saprophyte endemic in intertropical African soils invading coffee bushes through wounds. Gibberella xylarioides was considered by Booth (3) as heterothallic, with sex-linked morphological characteristics. "Female" strains produced highly curved, 0-3-septate conidia, and masses of small bluish-black stromata, some of which represented perithecial initials. "Male" strains had a slimy appearance due to the presence of pionnote sporodochia containing long, thin, 5-7-septate conidia. Perithecia, occurring frequently in nature (3, 8, 11, 12, 19, 26, 28), were produced in vitro if the correct mating types were brought together under suitable conditions (3). However, representative mating type strains were never deposited in a culture collection and crossing conditions were not specified. As a consequence, von Blittersdorff and Kranz (31) were unable to repeat Booth's in vitro production of the teleomorph and the "male" strain was in fact reidentified as F. stilboides (20, 31) and more recently as belonging to the "Lateritium clade" (9).

Sexual reproduction in heterothallic filamentous ascomycetes is controlled by a single mating type (MAT) gene with two functional alleles/idiomorphs. The MAT-1 idiomorph contains three open reading frames (ORFs), one of which (MAT-1-1) encodes a protein with a motif called the alpha-box, while the MAT-2 idiomorph contains a single ORF (MAT-2-1) encoding a regulatory protein with a DNA-binding domain of the high-mobility-group (HMG) type (4). The conservation of certain amino acids in these regions could enable the PCR amplification of the as-yet-undescribed G. xylarioides MAT-1/MAT-2 alleles using previously developed G. fujikuroi species and F. oxysporum primer pairs (30).

In addition to mating type, mating success in heterothallic fungi is also influenced by an isolate's ability to produce the required sexual structures. One of the parents must be "female fertile," i.e., capable of producing perithecia, and the other parent must be "male fertile," i.e., capable of fertilizing the female structure. Self-sterile hermaphroditic individuals can function as either the male or female parent in a cross.

The objectives of the present study were (i) to establish the heterothallic nature of the pathogen, (ii) to test Booth's hypothesis of sex-linked morphological dimorphism, and (iii) to determine whether cryptic speciation has occurred within the recent C. canephora/excelsa pathogen population and/or between existent and historical pathogen populations. Our working hypothesis was that G. xylarioides strains isolated from C. canephora/excelsa trees of diverse geographic origins produce the teleomorph when opposite mating types are paired under in vitro conditions. We tested here the utility of the biological and phylogenetic species concept in the G. xylarioides complex, allowing us to improve our knowledge of the reproduction mode and diversity of this important fungus.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fungal isolates and culturing conditions.
Accession numbers, geographical origins, and host substrates are listed for the isolates studied here (Table 1). Single conidia and ascospore-derived F. xylarioides/G. xylarioides strains were obtained from stem samples of diseased C. canephora trees recently collected in the DRC and Uganda. Ugandan and Tanzanian isolates not carrying a MUCL number were donated by CABI and CIRAD. Five "historical" strains associated with coffee wilt symptoms were obtained from international culture collections. An in-depth study of fertility and mating type was carried out on strains and MAT and translation elongation factor 1- (tef) genes were sequenced. The MAT/tef accession numbers are indicated in Table 1. Cultures were routinely grown on synthetic low-nutrient agar (SNA) (10) and incubated at 25 ± 2°C with a 12-h photoperiod under light banks of cool white fluorescent lights (General Electric 35099 F36W/33) and black lights (Philips TLD 36W/80) in a 3:2 ratio spaced 15 cm apart and 40 cm from the petri dishes. Correspondence with the morphological characteristics described for F. xylarioides (3, 5, 19, 26, 28), as well as with Fusarium sp./Lateritium clade (strain ATCC 36325), was established for strains after 7 days with conidia produced on SNA plates. All strains with a MUCL prefix are stored for long-term conservation on SNA slants, in lyophilized form and by cryopreservation (–130°C), at the Mycothèque de l'Université Catholique de Louvain (BCCM/MUCL) culture collection.

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TABLE 1. Recent and historical isolates analyzed in this study

Standard G. fujikuroi mating populations mating-type tester strains from the Fungal Genetics Stock Center (FGSC; University of Kansas Medical Center, Kansas City, Kans.) (18), were used for the cross-fertility study: FGSC 7600 (G. moniliformis, MAT-1), FGSC 7603 (G. moniliformis, MAT-2), FGSC 7611 (G. sacchari, MAT-1), FGSC 7610 (G. sacchari, MAT-2), FGSC 8931 (G. fujikuroi, MAT-1), FGSC 8932 (G. fujikuroi, MAT-2), FGSC 7615 (G. intermedia, MAT-1), FGSC 7614 (G. intermedia, MAT-2), FGSC 7616 (G. subglutinans, MAT-1), FGSC 7617 (G. subglutinans, MAT-2), FGSC 7057 (G. thapsina, MAT-1), FGSC 7056 (G. thapsina, MAT-2), FGSC 8934 (G. nygamai, MAT-1), FGSC 8933 (G. nygamai, MAT-2), FGSC 9022 (G. circinata, MAT-1), FGSC 9023 (G. circinata, MAT-2), FGSC 8910 (G. konza, MAT-1), and FGSC 8911 (G. konza, MAT-2).

Sexual compatibility tests and mating type.
Crosses were made on carrot agar (CA), modified carrot agar (MCA), and coffee twig agar (CTA). CA (14) was prepared with 200 g of carrots per liter of media. MCA was prepared by using only 50 g of carrots and then filtering the juice through a fine cheesecloth. For the preparation of CTA plates, C. canephora twigs (2 cm in diameter) were cut into 2- to 3-cm-long segments. Segments were split lengthwise, and half of their bark was removed. These segments were autoclaved at 121°C for 30 min on each of two consecutive days before being embedded into 2% water agar with the cambium side exposed above the agar surface.

Crosses were set up in triplicates on at least two separate occasions. Diallel CA/MCA crosses were carried out according to the protocol established for F. moniliforme (14) except that the male parent was inoculated onto a SNA plate and a Tween 20 (Merck, Munich, Germany) water solution (25 µl of Tween 20 per 100 ml of distilled water) was used. Each female parent was self-inoculated ("selfed") to test for potential homothallism and/or contamination. Cultures were incubated at 25 ± 2°C with a 12:12 h light-dark cycle. Crosses on CTA plates were tested by placing 4-mm mycelial plugs of the isolates on either side of the twig and incubating the plates at 25°C for 2 weeks in total darkness until the colonies had intermingled. Plates were placed in the light and examined weekly for perithecial development for up to 12 weeks. Crosses were scored as positive when ascospores were observed oozing out of perithecia in at least two different confrontations. Tests for interfertility between the 18 MP (A-I) tester strains of the GFC and F. xylarioides/G. xylarioides isolates were carried out in triplicates on CA.

DNA extraction, amplification and nucleotide sequencing.
Isolates were grown in the dark at 25°C for 3 days in a 2% malt extract broth medium (Duchefa, Haarlem, The Netherlands) on a rotary shaker (100 rpm). Mycelium was harvested by centrifugation (2,250 x g, 4°C, 15 min), and the pellets were lyophilized. Fungal DNA was extracted from 30-mg mycelium samples by using a procedure based on the method of Lee et al. (15), and crude nucleic acids were precipitated with a double volume of absolute ethanol and kept 1 h at –80°C before dissolving the pellet in 100 µl of a sterile water solution. A second purification was carried out on DNA samples using the GeneClean III kit (Q-Biogene, Carlsbad, CA) according to the manufacturer's recommendations before being quantified with an Eppendorf Bio Photometer (Eppendorf, Hamburg, Germany). Purified DNA and dilutions were kept at –20°C. PCR amplification of the tef 1- gene and sequencing of the amplicon was performed with primer pair ef1/ef2 (22). Previously described degenerate F. oxysporum primers F1/F2 (2) and F. oxysporum-specific PCR primer pairs GfHMG1/GfHMG2 (13) and Gfmat1a/1b and Gfmat2c/2d (27) were used to amplify and sequence parts of the G. xylarioides MAT gene. Primers were suspended in ultra pure water (W4502; Sigma, Steinheim, Germany) and stock solutions (100 µM) stored at –20°C. MAT and tef PCRs were performed with Taq DNA polymerase recombinant (Invitrogen Life Technologies, Carlsbad, CA) in an Eppendorf Mastercycle thermocycler (Eppendorf). PCR mixtures and amplification conditions for the chosen primer pairs were identical to those described previously (2, 13, 22, 27). PCR products were purified with a QIAQuick PCR purification kit 250 (QIAGEN, Inc., Hilden, Germany) according to the manufacturer's recommendations. Sequencing reactions were performed by using a CEQ DTCS Quick Start kit (Beckman Coulter, Inc., Fullerton, CA), and nucleotide sequence chromatograms were obtained with a CEQ 2000 XL capillary automated sequencer (Beckman Coulter). Sequences were assembled and corrected manually using the Sequencher 4.1 program (Gene Code Corp., Ann Arbor, MI). MAT and tef sequences were deposited in the EMBL database (Table 1).

Phylogenetic analysis.
Similarity searches were done against the GenBank/EMBL databases by using the BLASTN 2.2.9 program (1). Based on the results, sequences were aligned by using CLUSTAL W 1.82 (24) with files containing available DNA sequences representing known mating populations within the G. fujikuroi species complex (27) (for MAT-2-1), part of the African clade (21) (for tef), and the Lateritium clade sensu Geiser (9) (for tef analysis of historical strain ATCC 36325). The phylogenetic relationship between the coffee wilt pathogen and other closely related Fusarium spp. was inferred from the maximum-parsimony analysis of aligned sequences by using the PAUP* version 4.0b10 phylogenetics package (29). Heuristic searches were performed by using random sequence addition with a tree-bisection-reconnection branch-swapping algorithm. Confidence in the branching points was established by performing 1,000 bootstrap replicates using maximum-parsimony as the criterion and random sequence addition. For the F. xylarioides analysis, trees were generated for MAT-2-1 and tef using, respectively F. oxysporum AB011378 and AF160312 sequences as outgroups. F. xylarioides AJ539581 was used as the outgroup in the ATCC 36325 tef analysis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sexual reproduction by G. xylarioides.
The perfect state was not produced when strains were selfed on any of the crossing media used. Based on CTA crosses, strains could be divided into two mating groups, MAT-1 and MAT-2, confirmed with PCR assays. The MAT-1/MAT-2 ratio was 6:9 in isolates originating from the DRC, 5:4 in isolates from Uganda, and 2:3 in the Tanzanian collection. Strains could be divided into either female fertile, male fertile, or hermaphrodite classes (Table 1). Ascospore-derived strains examined were hermaphrodites.

Perithecia oozing ascospores (Fig. 1) appeared on CA/MCA 2 to 6 weeks after spermatization, whereas on CTA mature perithecia were observed 4 to 8 weeks after inoculation. To confirm outcrossing potential in G. xylarioides, MAT-PCR was used to analyze nine ascospore progeny from each of the three following crosses, CAB003 x OUG008, CAB003 x MUCL 44549, and MUCL 46056 x MUCL 14186. MAT-1/MAT-2 ratios were, respectively, of 4:5, 7:2, and 5:4. Based on the abundance of perithecia (>30) formed in CTA pairings, hermaphroditic strains MUCL 44532/MUCL 43887 and MUCL 35223/MUCL 44549 were chosen as MAT-1/MAT-2 C. canephora reference mating type tester pairs and have been deposited at the FGSC (Table 1).

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FIG. 1. Purple-black G. xylarioides sensu lato perithecia producing an orange cirrhus of ascospores on a carrot agar plate. Scale bar, 500 µm.

Crosses between C. canephora tester strains and the historical isolates did not produce fertile perithecia when opposite mating types were paired. Warty bodies presenting external perithecial characteristics contained either unidentified or asci-like structures enclosing "pseudo-microascospores" in DSMZ 62457 x MUCL 44532, ATCC 15664 x MUCL 44536, and CBS 74979 x MUCL 44532 crosses. Perithecial initials were observed in the DSMZ 62457 x ATCC 15664 crosses. The MP A, B, D, E, and F control crosses all produced mature perithecia under G. xylarioides crossing conditions (25 ± 2°C with a 12-h photoperiod), while MP C, G, H, and I produced only protoperithecia under these conditions. No intercrossing was observed between G. xylarioides and any of the tested mating populations in the GFC.

Lack of diversity in MAT-1.
A degenerate F. oxysporum primer pair (2) and a specific Fusarium primer pair (27) could be used to amplify a 400- and a 300-bp band, respectively, for recently collected MAT-1 strains and for historical strain ATCC 15664. The resulting 329-bp sequences (AJ876531 to AJ876534, AM072512 to AM072514, AM072517, and AM072521) were 100% identical and 96% similar to the F. nygamai (MP G) MAT-1-1 gene (AF236763) and 92% similar to the F. oxysporum f. sp. niveum MAT-1-1 gene (AY040736.1).

Diversity at MAT-2 level.
Recent F. xylarioides/G. xylarioides strains and historical isolates CBS 74979, CBS 25852, and DSMZ 62457 produced a single amplification product of 200 and 850 bp using specific primer pairs (13, 27). The resulting 627-bp nucleotide sequences from recent C. canephora strains (AJ876535 to AJ876541, AM072515, AM072516, and AM072518) were 100% identical to each other and 94% similar to both F. oxysporum f. radici-lycopersici (AB011378.1) and to F. nygamai (MP G, AF236771). CBS strains 25852 (AM072519) and 74979 (AM072520) have identical sequences but differ from the historical G. xylarioides isolate DSMZ 62457 (AM072522) and the recent C. canephora isolates by 4 and 6 bp, respectively.

Diversity at tef level.
All strains had an 800-bp fragment amplified when the ef1/ef2 primer pair was used; historical strain ATCC 36325 produced a slightly larger fragment. Four sequence patterns were identified. The first group contained the previously described (9) historical isolates CBS 25852 and 74979 that differed at 14 of 633 sites (2.2%) from recent F. xylarioides strains, confirming available sequences for these strains (AY707136 and AY707120). The second group, consisting in historical isolates DSMZ 62457 (AM072542) and ATCC 15664 (AM072541), differed at 20 of 633 sites (3.2%) from recent F. xylarioides isolates and at 6 of 633 sites (0.9%) from the historical CBS isolates forming the first group. The third and quantitatively most important group consisted in all recent MAT-1/MAT-2 C. canephora isolates originating from the DRC, Uganda and Tanzania (AM072523 to AM072540) and was 100% similar to previously sequenced F. xylarioides strains deposited by Munaut (AJ539579 to AJ539582) and Geiser et al. (9) (AY70719 and AY707121 to AY707135). BLAST searches of the 667-bp ATCC 36325 historical isolate tef sequence presented the highest similarity (93%) to Lateritium clade I sensu Geiser strain L-376 (from coffee seed in Brazil).

Phylogenetic comparison with related Fusarium spp.
Partial MAT-1 and MAT-2 sequences obtained from recent and historical F. xylarioides/G. xylarioides isolates were compared to available G. fujikuroi MP sequences (A to H, AF236757 to AF236772). The resulting phylogenetic trees are very similar to those of Steenkamp et al. (27) with the exception of the placement of F. xylarioides. MAT-1-1 sequences from the recent and historical DSMZ isolates could not be associated to a particular portion of the phylogenetic tree with any statistical confidence (data not shown). MAT-2-1 sequences of recent and historical isolates (Fig. 2A) form a strongly supported clade (100%) and group as a sister clade (83% bootstrap value) with those from F. verticilloides (MP A), F. nygamai (MP G), and F. thapsinum (MP F) of the previously described "African" clade (9, 21). The F. xylarioides/G. xylarioides clade is composed of three subclades (or "alleles"): all recent C. canephora isolates from DRC, Uganda, and Tanzania form a well-supported clade (group II), while historical isolate DSMZ 62457 and CBS isolates 25852 and 74979 form two distinct clades, respectively, called "historical" groups Ib and Ia.

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FIG. 2. Maximum-parsimony phylograms based on partial MAT-2 (27) (A) and tef (21) (B) gene sequences of species belonging to the G. fujikuroi species complex including representative coffee wilt isolates (in gray) from Coffea canephora/excelsa and historical strains. Trees were generated with PAUP* v.4.0 b10 (29) with F. oxysporum as outgroups. Bootstrap values based on 1,000 replications are indicated in percentages above the internodes when replication frequencies exceed 50%. African (Af), Asian (As), and American (Am) clades sensu O'Donnell (21) are shown. MP, mating population.

The same three clades can be differentiated with the tef sequences (Fig. 2B), with "historical" isolate ATCC 15664 joining DSMZ 62457 in its placement in group Ib within the African clade of the GFC. However, the F. xylarioides/G. xylarioides isolates do not form a monophyletic clade as in the MAT-2 phylogeny. Historical isolates nest together and form two well-supported sister "clades" (72%), while recent isolates form a well-supported clade but of uncertain relationship. F. udum, F. phyllophilum, and Fusarium sp. strain NRRL 26064 could not be placed unambiguously in the tree. Historical isolate ATCC 36325 can be placed either basally to the Lateritium clades I/II or to Lateritium clade II (Fig. 3).

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FIG. 3. Maximum-parsimony phylogram based on tef gene sequences of Lateritium clade fusaria (9) including historical coffee wilt isolate ATCC 36325 from Coffea excelsa (in gray). The figure was generated with PAUP* v.4.0 b10 (29) with F. xylarioides strain MUCL 43889 (AJ539581) as an outgroup. Bootstrap values, based on 1,000 replications, are indicated in percentages above the internodes when replication frequencies exceed 70%.

Top Abstract Introduction Materials and Methods Results Discussion References

 
G. xylarioides, a heterothallic fungus corresponding to Booth's "female" strain.
Results show that recent isolates of the G. xylarioides complex represent a heterothallic dimictic fungus. Single ascospore/conidia-derived hermaphroditic strains MUCL 44532/MUCL 43887 and MUCL 35223/MUCL 44549 were selected as MAT-1/MAT-2 reference mating type tester pairs. Both mating types are present in an approximately 1:1 ratio in the different regions sampled, which was expected since the perfect stage was frequently observed in situ. Analysis of progeny from three fertile crosses confirmed that the coffee wilt pathogen can outcross.

Strains of opposite mating type are morphologically identical and similar to those previously described as F. xylarioides "female" strains (3), which contradicts Booth's claim of sex-linked morphological characteristics and the existence of a morphologically distinct "male" strain in this species. Through carrot agar diallel crosses we identified hermaphroditic, male-fertile only, and female-fertile only (isolate OUG 008) strains within our CW isolates.

Among other G. fujikuroi MPs, Leslie and Klein (17) reported that, on a worldwide basis, the percentage of hermaphroditic strains ranges from 10 to 50%. Leslie (16) suggested that the loss of female functions is relatively common in these organisms because perithecium formation can be interrupted by mutation at any one of the many genes involved in the process. The observation of a female-fertile only strain (OUG 008) within isolates from C. canephora was unexpected since it is generally expected that all strains are capable of acting as the male parent (fertilizing agent) but only a subset are capable of acting as the female parent. Moreover, this female-fertile only strain, like all of the strains in the present study, produces abundant conidia, suggesting that a more detailed study of this strain is warranted to identify the genetic basis for its low/nonexistent male fertility observed.

Diversity within the historical and actual G. xylarioides population.
A recent publication (9) hypothesized that the usual concept of F. xylarioides/G. xylarioides could encompass cryptic species. This suggestion resulted from the discovery that two groups had different tef alleles. The larger group was composed of isolates from recently wilted C. canephora and C. arabica trees in Uganda and Ethiopia, while the second group consisted of two "historical" strains (1950s to 1960s) originating from Guinea (CBS 74979) and Côte d'Ivoire (CBS 25852). On the basis of this observation, a possible cryptic speciation event was suggested to separate Eastern and Western African strains.

In the present study, recent ascospore-derived isolates from the DRC corresponded consistently morphologically, sexually, and molecularly (MAT and tef sequence data) with recent F. xylarioides isolates from the DRC, Uganda, and Tanzania. This confirms that the perfect stage observed in recent outbreaks corresponds to the anamorph isolated in the DRC, Uganda, and Tanzania since 1968. However, these isolates differ from "historical" isolates in their sexual compatibility and in tef and MAT-2-1 sequences, differences that are consistent with cryptic speciation. We have been unable to access the F. xylarioides (28) type material, so the connection between historical G. xylarioides and historical F. xylarioides cannot be proven. For the moment it remains unclear whether recent epidemics observed on C. canephora and those reported in the 1950s and 1960s from Central Africa are due to the same pathogen or whether they constitute different cryptic species. The apparent sterility of crosses between these two groups is being evaluated in more detail and may enable us to understand some of the mechanisms underlying mating and fertility and to better characterize pathogen diversity. Historical strains may have limited sexual cross fertility with any strain due to long-term storage under less-than-ideal conditions, making that it essential to increase the number of "historical" strains studied. However, perithecial initials were observed in historical isolate DSMZ 62457 cultures and selfings, implying potential female fertility. We recommend that the term G. xylarioides sensu strictu refer to all that are strains molecularly identical to (and sexually compatible with) historical CBS strains 25852 and 74979 (group Ia) and DSMZ 62457 and ATCC 15664 (group Ib), whereas the term G. xylarioides sensu lato (group II) would also include strains that are molecularly identical to (and sexually compatible with) recently isolated C. canephora/C. excelsa strains.

Within the "F. xylarioides" strains received from international culture collections, strain ATCC 36325 had clearly different conidial characteristics, resembling F. lateritium sensu lato, and had a strong phylogenetic connection to the "Lateritium clade" sensu Geiser (9). This observation further confirms that historically strains belonging to this clade were incorrectly identified as F. xylarioides "male" strains, by authors such as Booth, most likely due to their frequent coisolation from wilted samples.

G. xylarioides sensu lato is a biological species of the GFC.
The MAT sequences obtained with previously described primer pairs were 94 to 96% similar to those already available for species in the GFC as well as for F. oxysporum. MAT-2 sequence similarities combined with tef sequencing results are consistent with the recent placement of the G. xylarioides groups in the "African" clade of the G. fujikuroi species complex rather than in Fusarium section Lateritium (9), suggesting the eventual use of the MAT loci for phylogenetics in heterothallic fungi (23, 27). The inability of G. xylarioides strains to cross with known GFC MP mating-type tester strains leads us to suggest that G. xylarioides sensu lato is the 11th biological species in the GFC, i.e., MP K, after the recently described G. konza (32) and G. gaditjirrii sp. nov. (25). The status of the historical strains as a biological species within the GFC is unclear since the perfect state has not been obtained in in vitro conditions.

In conclusion, the identification of at least three distinguishable groups of strains within G. xylarioides populations, their placement within the G. fujikuroi species complex, and the ability to reliably cross strains of interest should enable genetic analysis of critical traits such as pathogenicity, and mycotoxin production that were not previously possible. The frequent recovery of perithecia under field conditions suggests that new genotypes for multigenic traits can be formed much more readily in this species than in other species of the G. fujikuroi species complex.

 
This study was supported by a grant from the Université catholique de Louvain (Louvain, Belgium) within the framework of the INCO- COWIDI ICA4-CT-2001-10006 European Community-funded project. We acknowledge the financial support received from the Belgian Federal Science Policy Office (contracts BCCM C2/10/007 and C3/10/003).

We thank A. Kalonji and P. Tshilenge for providing diseased coffee tree samples, M. Rutherford (CABI) and D. Bieysse (CIRAD) for supplying Ugandan and Tanzanian isolates (all except MUCL 43887 and MUCL 43889), and C. Decock for helpful insights in the interpretation of results and preparing the final manuscript.

 
* Corresponding author. Mailing address: Unité de Phytopathologie, Université catholique de Louvain, Croix du Sud 2/3, B-1348 Louvain-la-Neuve, Belgium. Phone: (32)10473751. Fax: (32)10478697. E-mail: lepoint{at}fymy.ucl.ac.be.

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Applied and Environmental Microbiology, December 2005, p. 8466-8471, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8466-8471.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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