Giberella fujikuroi species complex isolated from maize and wheat in Iran: distribution, molecular identification and fumonisin B1 in vitro biosynthesis.

BACKGROUND: Contamination of food and agricultural crops by Fusarium species is a major concern of food spoilage and a potential public health hazard. In the present study, natural contamination of maize and wheat samples from main cultivation areas of Iran by Fusarium species belonging to the Giberella fujikuroi species complex was evaluated, with special attention to the ability of the isolates to produce fumonisin B1 (FB1 ). RESULTS: A total of 55 Fusarium isolates were obtained from 27/32 maize samples (84.4%) and 11/15 wheat samples (73.3%). They were identified as F. verticillioides (47.3%), F. proliferatum (47.3%), F. fujikuroi (1.8%), F. nygamai (1.8%) and F. redolens (1.8%) by sequence analysis of translation elongation factor 1-α (TEF1-α). Twenty-two of 55 Fusarium isolates belonging to F. proliferatum (23.6%), F. verticillioides (14.5%) and F. fujikuroi (1.8%) produced FB1 in the concentration range 230.4-9565.0 µg mL(-1) . The dendrogram resulting from the TEF1-α profile showed that the genotypes were divided into clusters I, II and III, of which cluster III contained only F. redolens, its first report from Iran. CONCLUSION: On the basis of in vitro FB1 biosynthesis of the analyzed strains, the high degree of contamination of maize and wheat with Fusarium strains reported here should be considered as a potential public health threat, because a meaningful number of the isolates were found to produce hazardous levels of carcinogenic FB1 .

Gibberella moniliformis AH13 with antitumor activity, an endophytic fungus strain producing triolein isolated from Adlay (Coix lacryma-jobi: poaceae).

In this study, the isolation of an endophytic fungus from the leaves of the medicinal herb adlay (Coix lacryma-jobi L. var. ma-yuen Stapf) is reported for the first time. The fungus produced Triolein (trioleoylglycerol), a major constituent of triacylglycerols (TAGs) of adlay, in rice medium under shake-flask and bench-scale fermentation conditions. The fungus was identified as Gibberella moniliformis (Fusarium verticillioides) by its morphology and authenticated by ITS analysis (ITS1 and ITS2 regions and the intervening 5.8S rDNA region). Triolein was identified by HPLC-ELSD coupled with APCI-MS and confirmed through comparison with authentic standard. The concentration of triolein produced by G. moniliformis AH13 reached 2.536 ± 0.006 mg/g dry weight of mycelium. Moreover, the EtOAc extract of G. moniliformis AH13 showed strong antitumor activity against four types of tumor cells (A549, HCT116, MDA-MB-231, and SW1990). These results suggest that G. moniliformis AH13 in adlay has significant scientific and industrial potential to meet the pharmaceutical demands and sustainable energy requirements for TAGs in a cost-effective, easily accessible, and reproducible way and is also a potential novel source of natural antitumor bioactive agents.

Genome distribution and validation of novel microsatellite markers of Fusarium verticillioides and their transferability to other Fusarium species.

Improved population studies in the fungus Fusarium verticillioides require the development of reliable microsatellite markers. Here we report a set of ten microsatellite loci that can be used for genetic diversity analyses in F. verticillioides, and are equally applicable to other fungi, especially those belonging to the Gibberella fujikuroi clade.

Detection and dynamics of different carbendazim-resistance conferring ß-tubulin variants of Gibberella zeae collected from infected wheat heads and rice stubble in China.

BACKGROUND: Carbendazim has been used in the control of Fusarium head blight (FHB) for more than 30 years in China. Thus, carbendazim-resistant (Car(R) ) populations of Gibberella zeae have developed in some areas. In this study, 9341 G. zeae isolates were collected from the ten main wheat-producing regions of China in the period from 2008 to 2012, and sensitivity to carbendazim was detected. RESULTS: A high frequency of Car(R) isolates was observed in Zhejiang and Jiangsu provinces. Car(R) isolates were recovered from Anhui and Henan provinces in 2009 and 2012, respectively, but were not detected in the other six regions. Available (F167Y, E198Q and F200Y) and newly developed (E198L and E198K) allele-specific PCR assays were used to genotype field Car(R) isolates. The ß-tubulin variants harbouring point mutation F167Y or E198Q accounted for >95% in Car(R) populations. Quantitative allele-specific real-time PCR assays were developed to determine the frequencies of five different ß-tubulin variants present in populations of perithecia sampled from rice stubble. CONCLUSION: Car(R) populations of G. zeae develop rapidly under the selection pressure of carbendazim. Real-time PCR assays detecting the resistance frequencies in populations of perithecia would provide useful information for FHB control and management of resistance.

Characterization of on-target generated tryptic peptides from Giberella zeae conidia spore proteins by means of matrix-assisted laser desorption/ionization mass spectrometry.

Traditionally characterization of microbial proteins is performed by a complex sequence of steps with the final step to be either Edman sequencing or mass spectrometry, which generally takes several weeks or months to be complete. In this work, we proposed a strategy for the characterization of tryptic peptides derived from Giberella zeae (anamorph: Fusarium graminearum) proteins in parallel to intact cell mass spectrometry (ICMS) in which no complicated and time-consuming steps were needed. Experimentally, after a simple washing treatment of the spores, the aliquots of the intact G. zeae macro conidia spores solution, were deposited two times onto one MALDI (matrix-assisted laser desorption ionization) mass spectrometry (MS) target (two spots). One spot was used for ICMS and the second spot was subject to a brief on-target digestion with bead-immobilized or non-immobilized trypsin. Subsequently, one spot was analyzed immediately by MALDI MS in the linear mode (ICMS) whereas the second spot containing the digested material was investigated by MALDI MS in the reflectron mode ("peptide mass fingerprint") followed by protonated peptide selection for MS/MS (post source decay (PSD) fragment ion) analysis. Based on the formed fragment ions of selected tryptic peptides a complete or partial amino acid sequence was generated by manual de novo sequencing. These sequence data were used for homology search for protein identification. Finally four different peptides of varying abundances have been identified successfully allowing the verification that our desorbed/ionized surface compounds were indeed derived from proteins. The presence of three different proteins could be found unambiguously. Interestingly, one of these proteins is belonging to the ribosomal superfamily which indicates that not only surface-associated proteins were digested. This strategy minimized the amount of time and labor required for obtaining deeper information on spore preparations within the nowadays widely used ICMS approach.

Isolation and characterization of Gibberella intermedia CA3-1, a novel and versatile nitrilase-producing fungus.

Nitrilase-mediated biocatalysis has attracted substantial attention for its application in carboxylic acid production in recent years. In the present study, the fungus CA3-1 was isolated and identified as Gibberella intermedia based on its morphology, its 18S ribosomal DNA (rDNA), and internal transcribed spacer (ITS) sequences. The enzymatic properties of G. intermedia resting cells were determined, and the optimum activity was achieved at 40 °C with pH 7.6. The half-lives of the nitrilase at 30, 40, and 50 °C were 231.1, 72.9, and 6.4 h, respectively. This Gibberella nitrilase showed a wide substrate spectrum with high specificity for heterocyclic and aliphatic nitriles. It remained extremely active in 5% propanol. The presence of Ag(+), Hg(2+), and excess substrate inhibited the nitrilase activity, whereas Fe(2+), Mn(2+), and Li(+) improved enzyme activity. 3-Cyanopyridine (50 mM) was hydrolyzed into nicotinic acid within 30 min, whereas only <5% of nicotinamide was detected. The results show that this fungal nitrilase is a promising candidate for commercial application in nicotinic acid production.

Fusarium tupiense sp. nov., a member of the Gibberella fujikuroi complex that causes mango malformation in Brazil.

Fusarium tupiense, the main causal agent of mango malformation in Brazil, is described through a combination of morphological, biological and molecular markers. This new species belongs to the Gibberella fujikuroi species complex (GFSC) and has an anamorph morphologically similar to Fusarium mangiferae and F. sterilihyphosum. F. tupiense can be differentiated from other species in the G. fujikuroi species complex on the basis of sexual crosses, amplified fragment length polymorphism (AFLP) markers and partial sequences of the tef1 and tub2 genes. Female fertility for field isolates of F. tupiense appears to be low. PCR with primers specific for the mating type (MAT) alleles and sexual crosses identified this species as heterothallic with two idiomorphs. Female-fertile tester strains were developed for the identification of field strains of this species through sexual crosses.

Molecular identification of Fusarium species in Gibberella fujikuroi species complex from rice, sugarcane and maize from Peninsular Malaysia.

The objective of this study was to identify Fusarium species in the Gibberella fujikuroi species complex from rice, sugarcane and maize as most of the Fusarium species in the species complex are found on the three crops. Isolates used were collected from the field and obtained from culture collection. The Fusarium isolates were initially sorted based on morphology and identifications confirmed based on the DNA sequence of the translation elongation factor 1-α (TEF-1α) gene. Based on the closest match of BLAST analysis, five species were recovered, namely, F. sacchari, F. fujikuroi, F. proliferatum, F. andiyazi and F. verticillioides. This is the first report regarding F. andiyazi from rice in Malaysia and Southeast Asia. The phylogenetic tree generated by using the neighbor joining method showed that isolates from the same species were grouped in the same clade. The present study indicated that Fusarium species in the G. fujikuroi species complex are widespread in rice, sugarcane and maize in Peninsular Malaysia. The findings also suggest that the use of morphological characters for identification of Fusarium species in the G. fujikuroi species complex from the three crops will lead to incorrect species designation.

Trichothecene profiling and population genetic analysis of Gibberella zeae from barley in North Dakota and Minnesota.

Gibberella zeae, the principal cause of Fusarium head blight (FHB) of barley, contaminates grains with several mycotoxins, which creates a serious problem for the malting barley industry in the United States, China, and Europe. However, limited studies have been conducted on the trichothecene profiles and population genetic structure of G. zeae isolates collected from barley in the United States. Trichothecene biosynthesis gene (TRI)-based polymerase chain reaction (PCR) assays and 10 variable number tandem repeat (VNTR) markers were used to determine the genetic diversity and compare the trichothecene profiles of an older population (n = 115 isolates) of G. zeae collected in 1997 to 2000 with a newer population (n = 147 isolates) collected in 2008. Samples were from across the major barley-growing regions in North Dakota and Minnesota. The results of TRI-based PCR assays were further validated using a subset of 32 and 28 isolates of G. zeae by sequence analysis and gas chromatography, respectively. TRI-based PCR assays revealed that all the G. zeae isolates in both populations had markers for deoxynivalenol (DON), and the frequencies of isolates with a 3-acetyldeoxynivalenol (3-ADON) marker in the newer population were ≈11-fold higher than those among isolates in the older population. G. zeae populations from barley in the Midwest of the United States showed no spatial structure, and all the isolates were solidly in clade 7 of G. zeae, which is quite different from other barley-growing areas of world, where multiple species of G. zeae are commonly found in close proximity and display spatial structure. VNTR analysis showed high gene diversity (H = 0.82 to 0.83) and genotypic diversity but low linkage disequilibrium (LD = 0.02 to 0.07) in both populations. Low genetic differentiation (F(ST) = 0.013) and high gene flow (Nm = 36.84) was observed between the two populations and among subpopulations within the same population (Nm = 12.77 to 29.97), suggesting that temporal and spatial variations had little influence on population differentiation in the Upper Midwest. Similarly, low F(ST) (0.02) was observed between 3-ADON and 15-acetyldeoxynivalenol populations, indicating minor influence of the chemotype of G. zeae isolates on population subdivision, although there was a rapid increase in the frequencies of isolates with the 3-ADON marker in the Upper Midwest between the older collection made in 1997 to 2000 and the newer collection made in 2008. This study provides information to barley-breeding programs for their selection of isolates of G. zeae for evaluating barley genotypes for resistance to FHB and DON accumulation.

Gibberella musae (Fusarium musae) sp. nov., a recently discovered species from banana is sister to F. verticillioides.

Several strains of Fusarium isolated from banana were identified previously as F. verticillioides (Sacc.) Nirenberg but described as unable to produce fumonisin. Here we report biochemical and morphological evidence, as well as multilocus phylogenetic analyses based on elongation factor (EF-1α), calmodulin, ß-tubulin, and the second largest subunit of RNA polymerase II (RPB2) sequences, indicating that these isolates represent a unique lineage in the Gibberella fujikuroi species complex related to but distinct from F. verticillioides. Together with previous results of molecular studies, as well as with results of metabolite analyses, crossing experiments, pathogenicity tests and morphological characterization, these new data indicate that these strains isolated from banana represent a new species, Gibberella musae Van Hove et al. sp. nov. (anamorph: Fusarium musae Van Hove et al. sp. nov.), which is described herein.

Fusarium ananatum sp. nov. in the Gibberella fujikuroi species complex from pineapples with fruit rot in South Africa.

Pineapple (Ananas comosus) is native to South America and widely planted as a fruit crop in the tropics and sub-tropics. This plant is susceptible to a number of fungal diseases of which the most severe is fusariosis. The disease is caused by Fusarium guttiforme and occurs only in South and Central America. The occurrence of a similar disease on pineapples in South Africa has prompted a re-evaluation of the Fusarium sp. associated with pineapple fruit rot. Phylogenetic relationships of isolates from pineapples collected in Brazil and South Africa were assessed based on sequence data for the translation elongation factor-1-α, histone H3 and ß-tubulin gene regions. Analyses showed that the South African isolates represent a species distinct from Brazilian isolates. The South African isolates are characterised by a concentration of aerial mycelium at the centres of the colonies, different to the Brazilian isolates that have an even distribution of aerial mycelium. Both phylogenetic and morphological data show that the disease on pineapple in South Africa is caused by a new Fusarium species described here as F. ananatum sp. nov.

Fusarium spp. associated with rice Bakanae: ecology, genetic diversity, pathogenicity and toxigenicity.

African and Asian populations of Fusarium spp. (Gibberella fujikuroi species complex) associated with Bakanae of rice (Oryzae sativa L.) were isolated from seeds and characterized with respect to ecology, phylogenetics, pathogenicity and mycotoxin production. Independent of the origin, Fusarium spp. were detected in the different rice seed samples with infection rate ranges that varied from 0.25% to 9%. Four Fusaria (F. andiyazi, F. fujikuroi, F. proliferatum and F. verticillioides) were found associated with Bakanae of rice. While three of the Fusaria were found in both African and Asian seed samples, F. fujikuroi was only detected in seed samples from Asia. Phylogenetic studies showed a broad genetic variation among the strains that were distributed into four different genetic clades. Pathogenicity tests showed that all strains reduced seed germination and possessed varying ability to cause symptoms of Bakanae on rice, some species (i.e. F. fujikuroi) being more pathogenic than others. The ability to produce fumonisins (FB(1) and FB(2)) and gibberellin A3 in vitro also differed according to the Fusarium species. While fumonisins were produced by most of the strains of F. verticillioides and F. proliferatum, gibberellin A3 was only produced by F. fujikuroi. Neither fumonisin nor gibberellin was synthesized by most of the strains of F. andiyazi. These findings provide new information on the variation within the G. fujikuroi species complex associated with rice seed and Bakanae disease.

Dominance of Group 2 and fusaproliferin production by Fusarium subglutinans from Iowa maize.

Fusarium subglutinans (teleomorph Gibberella subglutinans, member of the Gibberella fujikuroi complex) is an important toxigenic pathogen of maize. Recently, two cryptic species (Groups 1 and 2) have been described within F. subglutinans, but little is known about the occurrence of the two groups in North America or their relative capacities to produce mycotoxins. In this study, 58 F. subglutinans strains from kernels of maize grown in Iowa, USA, were evaluated for cryptic speciation and production of the mycotoxins fusaproliferin and beauvericin. Fifty-six of the 58 strains (97%) belonged to Group 2, and two strains belonged to Group 1, based on restricted fragment length polymorphisms derived from amplification of histone H3 and beta-tubulin gene fragments. Fifty-four Group 2 strains and both Group 1 strains produced fusaproliferin at concentrations ranging from 12 to 3000 microg g(-1) of solid maize culture. None of the F. subglutinans strains from Iowa produced beauvericin at detectable amounts, although most F. subglutinans strains from Europe and elsewhere are beauvericin producers. These results indicate that F. subglutinans strains infecting maize kernels in Iowa belong almost exclusively to Group 2 and that they have a high potential for fusaproliferin production; furthermore, the results confirm an association between Group 2 genotypes and lack of beauvericin production. This is the first report characterizing the phylogenetic groups of F. subglutinans occurring in Iowa; the predominance of Group 2 suggests that populations of the fungus in Iowa and Europe remain isolated from each other. Fusaproliferin contamination of grain appears to be a risk wherever F. subglutinans occurs, but beauvericin contamination from F. subglutinans is associated only with Group 1.

Genetic relationships among populations of Gibberella zeae from barley, wheat, potato, and sugar beet in the upper Midwest of the United States.

Gibberella zeae, a causal agent of Fusarium head blight (FHB) in wheat and barley, is one of the most economically harmful pathogens of cereals in the United States. In recent years, the known host range of G. zeae has also expanded to noncereal crops. However, there is a lack of information on the population genetic structure of G. zeae associated with noncereal crops and across wheat cultivars. To test the hypothesis that G. zeae populations sampled from barley, wheat, potato, and sugar beet in the Upper Midwest of the United States are not mixtures of species or G. zeae clades, we analyzed sequence data of G. zeae, and confirmed that all populations studied were present in the same clade of G. zeae. Ten variable number tandem repeat (VNTR) markers were used to determine the genetic structure of G. zeae from the four crop populations. To examine the effect of wheat cultivars on the pathogen populations, 227 strains were sampled from 10 subpopulations according to wheat cultivar types. The VNTR markers also were used to analyze the genetic structure of these subpopulations. In all populations, gene (H = 0.453 to 0.612) and genotype diversity (GD = or >0.984) were high. There was little or no indication of linkage disequilibrium (LD) in all G. zeae populations and subpopulations. In addition, high gene flow (Nm) values were observed between cereal and noncereal populations (Nm = 10.69) and between FHB resistant and susceptible wheat cultivar subpopulations (Nm = 16.072), suggesting low population differentiation of G. zeae in this region. Analysis of molecular variance also revealed high genetic variation (>80%) among individuals within populations and subpopulations. However, low genetic variation (<5%) was observed between cereal and noncereal populations and between resistant and susceptible wheat subpopulations. Overall, these results suggest that the populations or subpopulations are likely a single large population of G. zeae affecting crops in the upper Midwest of the United States.

Sexual compatibility in Fusarium pseudograminearum (Gibberella coronicola).

Numerous pathogenic Fusarium species have well-characterized sexual cycles, whereas others, including the crown rot fungus F. pseudograminearum, do not. We conducted studies to elucidate the potential frequency and nature of sexual reproduction in field populations of F. pseudograminearum and developed tester strains for controlled crossings under laboratory conditions. Studies on the role of sexual recombination in the life cycle of F. pseudograminearum revealed apparently low levels of female fertility under controlled laboratory conditions, despite the observation of naturally occurring perithecia of the teleomorph Gibberella coronicola at two field sites. Female fertility levels were experimentally increased to produce female fertile tester strains using four generations of single and multi-stage crossings between sibling progeny derived from fertile laboratory crosses between field isolates collected in northeastern Australia. The production of reliable female fertile tester strains has potential applications for the construction of biological species boundaries, elucidation of the physical characters of reproductive structures, and the generation of genetic diversity via sexual recombination in F. pseudograminearum. As such, the current study is a significant advancement in the understanding of G. coronicola, allowing for future characterisation of various biological, epidemiological, and genetic parameters.

Inter- and intra-specific genetic variation in Fusarium.

Genetic variation occurs at all levels across the genus Fusarium. In some cases such variation has been used to define species, and in others to describe populations or lineages. When amplified fragment length polymorphisms (AFLPs) are evaluated, strains in different species usually share at least 60% of the fragments and those in different species 40% of the fragments, or less, with isolates sharing between 40 and 60% of the fragments in an indeterminant situation. This gray area also is reflected in morphological characters, usually indistinguishable, and cross-fertility, usually some cross-fertility but often not as fertile as are strains that are more closely related. In terms of DNA sequence, the genes used for species diagnostics often have not been tested on large numbers of strains. For example, the TRI101 gene of F. graminearum contains at least 25 single nucleotide polymorphisms (SNPs) from 36 strains and yielded 17 alleles that have been proposed as a means to subdivide this species into at least nine. However these subdivisions fare poorly as more strains are analyzed, with the number of alleles increasing to >40 when approximately 500 strains from Korea and South America are sequenced. Some of the newly identified alleles cannot be correctly assigned to one of the nine subdivisions based on the proposed diagnostic SNPs. Before SNPs are proposed as characters to define species, it is important to verify their specificity based on a sufficiently large sample and to evaluate the genetic variation present in terms of an independent measure of genetic relationships. Only in such a manner can names that are meaningful in the context of trade and quarantine regulations be developed.

Evidence for recombination and segregation of virulence to pine in a hybrid cross between Gibberella circinata and G. subglutinans.

Two species associated with the Gibberella fujikuroi species complex, G. circinata (the cause of pitch canker in pines) and G. subglutinans (avirulent on pine), were found to have limited interfertility in hybrid crosses. MAT idiomorphs, polymorphisms in the histone H3 gene, vegetative compatibility, and virulence phenotypes were used to verify recombination. The MAT idiomorphs appeared to be assorting independently, but the histone H3 haplotype was disproportionately represented by that of the G. subglutinans parent. Ninety-eight percent (45/46) of the progeny tested were vegetatively incompatible with both parents. All F(1) progeny were avirulent to pine, but a wide range of virulence was restored through a backcross to the virulent parent (G. circinata). Attempts at hybrid crosses using other isolate combinations were rarely successful (1/26). This limited interfertility supports retention of G. circinata and G. subglutinans as separate species, but offers opportunities to characterize the inheritance of virulence to pine.

Gibberella xylarioides sensu lato from Coffea canephora: a new mating population in the Gibberella fujikuroi species complex.

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-alpha (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.

Description of Gibberella sacchari and neotypification of its anamorph Fusarium sacchari.

We described the teleomorph of Fusarium sacchari as Gibberella sacchari, sp. nov. This species can be separated from other species of Gibberella on the basis of the longer, narrower ascospores found in G. sacchari and by sexual cross fertility. Female-fertile mating type tester strains were developed that can be used for making sexual crosses with this heterothallic fungus under laboratory conditions. The anamorph, Fusarium sacchari, was neotypified.

Gibberella xylarioides (anamorph: Fusarium xylarioides), a causative agent of coffee wilt disease in Africa, is a previously unrecognized member of the G. fujikuroi species complex.

Tracheomycosis or coffee wilt has emerged as a major disease of robusta coffee in Uganda in the past 10 years. Coffee wilt historically has been associated with Fusarium xylarioides Steyaert (teleomorph Gibberella xylarioides Heim and Sacc.), a species that has been classified as a member of Fusarium section Lateritium. We investigated the molecular phylogenetics of fusarial coffee wilt isolates by generating partial DNA sequences from two protein coding regions, translation elongation factor 1-alpha and beta-tubulin, in 36 isolates previously identified as F. xylarioides and related fusaria from coffee and other woody hosts, as well as from 12 isolates associated with a current coffee wilt outbreak in Uganda. These isolates fell into two morphologically and phylogenetically distinct groups. The first group was found to represent previously unidentified members of the Gibberella fujikuroi species complex (GFC), a clade that replaces the artificial Fusarium section Liseola. This group of isolates fit the original description of F. xylarioides, thus connecting it to the GFC. The second group, which was diverse in its morphology and DNA sequences, comprised four distinct lineages related to Fusarium lateritium. Our finding of unrelated species associated with coffee wilt disease has important implications regarding its epidemiology, etiology and control.