Ras2 is important for growth and pathogenicity in Fusarium circinatum.

In this study, we investigated to possible role of Ras2 in Fusarium circinatum- a fungus that causes pine pitch canker disease on many different pine species and has a wide geographic distribution. This protein is encoded by the RAS2 gene and has been shown to control growth and pathogenicity in a number of fungi in a mitogen-activated protein kinase- and/or cyclic adenosyl monophosphate pathway-dependent manner. The aim was therefore to characterize the phenotypes of RAS2 gene knockout and complementation mutants of F. circinatum. These mutants were generated by transforming protoplasts of the fungus with suitable split-marker constructs. The mutant strains, together with the wild type strain, were used in growth studies as well as pathogenicity assays on Pinus patula seedlings. Results showed that the knockout mutant strain produced significantly smaller lesions compared to the complementation mutant and wild type strains. Growth studies also showed significantly smaller colonies and delayed conidial germination in the knockout mutant strain compared to the complement mutant and wild type strains. Interestingly, the knockout mutant strain produced more macroconidia than the wild type strain. Collectively, these results showed that Ras2 plays an important role in both growth and pathogenicity of F. circinatum. Future studies will seek to determine the pathway(s) through which Ras2 controls these traits in F. circinatum.

Saprophytic and pathogenic fungi in the Ceratocystidaceae differ in their ability to metabolize plant-derived sucrose.

BACKGROUND: Proteins in the Glycoside Hydrolase family 32 (GH32) are carbohydrate-active enzymes known as invertases that hydrolyse the glycosidic bonds of complex saccharides. Fungi rely on these enzymes to gain access to and utilize plant-derived sucrose. In fungi, GH32 invertase genes are found in higher copy numbers in the genomes of pathogens when compared to closely related saprophytes, suggesting an association between invertases and ecological strategy. The aim of this study was to investigate the distribution and evolution of GH32 invertases in the Ceratocystidaceae using a comparative genomics approach. This fungal family provides an interesting model to study the evolution of these genes, because it includes economically important pathogenic species such as Ceratocystis fimbriata, C. manginecans and C. albifundus, as well as saprophytic species such as Huntiella moniliformis, H. omanensis and H. savannae. RESULTS: The publicly available Ceratocystidaceae genome sequences, as well as the H. savannae genome sequenced here, allowed for the identification of novel GH32-like sequences. The de novo assembly of the H. savannae draft genome consisted of 28.54 megabases that coded for 7 687 putative genes of which one represented a GH32 family member. The number of GH32 gene family members appeared to be related to the ecological adaptations of these fungi. The pathogenic Ceratocystis species all contained two GH32 family genes (a putative cell wall and a putative vacuolar invertase), while the saprophytic Huntiella species had only one of these genes (a putative cell wall invertase). Further analysis showed that the evolution of the GH32 gene family in the Ceratocystidaceae involved transposable element-based retro-transposition and translocation. As an example, the activity of a Fot5-like element likely facilitated the assembly of the genomic regions harbouring the GH32 family genes in Ceratocystis. CONCLUSIONS: This study provides insight into the evolutionary history of the GH32 gene family in Ceratocystidaceae. Our findings suggest that transposable elements shaped the evolution of the GH32 gene family, which in turn determines the sucrolytic activities and related ecological strategies of the Ceratocystidaceae species that harbour them. The study also provides insights into the role of carbohydrate-active enzymes in plant-fungal interactions and adds to our understanding of the evolution of these enzymes and their role in the life style of these fungi.

Novel taxa in the Fusarium fujikuroi species complex from Pinus spp.

The pitch canker pathogen Fusarium circinatum has caused devastation to Pinus spp. in natural forests and non-natives in commercially managed plantations. This has drawn attention to the potential importance of Fusarium species as pathogens of forest trees. In this study, we explored the diversity of Fusarium species associated with diseased Pinus patula, P. tecunumanii, P. kesiya and P. maximinoi in Colombian plantations and nurseries. Plants displaying symptoms associated with a F. circinatum-like infection (i.e., stem cankers and branch die-back on trees in plantations and root or collar rot of seedlings) were sampled. A total of 57 isolates were collected and characterised based on DNA sequence data for the translation elongation factor 1-α and ß-tubulin gene regions. Phylogenetic analyses of these data allowed for the identification of more than 10 Fusarium species. These included F. circinatum, F. oxysporum, species within the Fusarium solani species complex and seven novel species in the Fusarium fujikuroi species complex (formerly the Gibberella fujikuroi species complex), five of which are described here as new. Selected isolates of the new species were tested for their pathogenicity on Pinus patula and compared with that of F. circinatum. Of these, F. marasasianum, F. parvisorum and F. sororula displayed levels of pathogenicity to P. patula that were comparable with that of F. circinatum. These apparently emerging pathogens thus pose a significant risk to forestry in Colombia and other parts of the world.

Relevance of prokaryotic subspecies in the age of genomics.

The availability of multiple gene sequences, and in particular full genome sequence data, for microbial strains has changed how taxonomists delineate subspecies belonging to the Archaea and Bacteria. Well-defined phylogenetic lineages that share higher genome similarity values compared to the widely used species thresholds are often described as subspecies, despite clear evidence of genetic isolation between them. These well-defined lineages, reflecting notable genetic isolation of the core genome represent more recently evolved, unique and sui generis evolutionary units. Because they bear all of the hallmarks of species, most contemporary subspecies likely represent species in their own right. Although there is considerable value in defining intraspecies variation (e.g., pathovar, serovar and symbiovar), the discriminating properties of such units are mostly encoded on accessory subgenomic compartments. We therefore argue that the taxonomic category of subspecies has become irrelevant and propose that its use should be discontinued. This will minimize inconsistencies related to the subjective nature of species-subspecies distinctions. Formal recognition of biologically relevant variation within species based on the accessory genome information will have practical significance in fields such as clinical, industrial and agricultural microbiology.

Gene expression associated with vegetative incompatibility in Amylostereum areolatum.

In filamentous fungi, vegetative compatibility among individuals of the same species is determined by the genes encoded at the heterokaryon incompatibility (het) loci. The hyphae of genetically similar individuals that share the same allelic specificities at their het loci are able to fuse and intermingle, while different allelic specificities at the het loci result in cell death of the interacting hyphae. In this study, suppression subtractive hybridization (SSH) followed by pyrosequencing and quantitative reverse transcription PCR were used to identify genes that are selectively expressed when vegetatively incompatible individuals of Amylostereum areolatum interact. The SSH library contained genes associated with various cellular processes, including cell-cell adhesion, stress and defence responses, as well as cell death. Some of the transcripts encoded proteins that were previously implicated in the stress and defence responses associated with vegetative incompatibility. Other transcripts encoded proteins known to be associated with programmed cell death, but have not previously been linked with vegetative incompatibility. Results of this study have considerably increased our knowledge of the processes underlying vegetative incompatibility in Basidiomycetes in general and A. areolatum in particular.

Deciphering the effect of FUB1 disruption on fusaric acid production and pathogenicity in Fusarium circinatum.

Fusarium circinatum is an important pathogen of pine trees. However, little is known regarding the molecular processes underlying its pathogenesis. We explored the potential role of the phytotoxin fusaric acid (FA) in the pathogenicity of the fungus. FA is produced by products of the FUB biosynthesis gene cluster, containing FUB1-12. Of these, FUB1 encodes the core polyketide synthase, which we disrupted. We used the resulting mutant strain to investigate whether FUB1 and FA production play a role in the virulence of F. circinatum on pine. Our results showed that FA production was abolished both in vitro and in planta. However, bikaverin production was increased in the knockout mutant. FUB1 disruption also corresponded with downregulation of a F. circinatum homologue of LaeA, a master transcriptional regulator of secondary metabolism. Lesion lengths produced by the FUB1 knockout mutant on inoculated Pinus patula seedlings were significantly smaller than those produced by the wild type strain. Collectively, these results show that FUB1 plays a role in FA production in F. circinatum, and that this gene contributes to the aggressiveness of F. circinatum on P. patula. This study will contribute to the limited knowledge we have about the molecular basis of pathogenicity in this fungus.

Genetic linkage map for Amylostereum areolatum reveals an association between vegetative growth and sexual and self-recognition.

Amylostereum areolatum is a filamentous fungus that grows through tip extension, branching and hyphal fusion. In the homokaryotic phase, the hyphae of different individuals are capable of fusing followed by heterokaryon formation, only if they have dissimilar allelic specificities at their mating-type (mat) loci. In turn, hyphal fusion between heterokaryons persists only when they share the same alleles at all of their heterokaryon incompatibility (het) loci. In this study we present the first genetic linkage map for A. areolatum, onto which the mat and het loci, as well as quantitative trait loci (QTLs) for mycelial growth rate are mapped. The recognition loci (mat-A and het-A) are positioned near QTLs associated with mycelial growth, suggesting that the genetic determinants influencing recognition and growth rate in A. areolatum are closely associated. This was confirmed when isolates associated with specific mat and het loci displayed significantly different mycelial growth rates. Although the link between growth and sexual recognition has previously been observed in other fungi, this is the first time that an association between growth and self-recognition has been shown.

Evolutionary relationships among the Fusarium oxysporum f. sp. cubense vegetative compatibility groups.

Fusarium oxysporum f. sp. cubense, the causal agent of fusarium wilt of banana (Musa spp.), is one of the most destructive strains of the vascular wilt fungus F. oxysporum. Genetic relatedness among and within vegetative compatibility groups (VCGs) of F. oxysporum f. sp. cubense was studied by sequencing two nuclear and two mitochondrial DNA regions in a collection of 70 F. oxysporum isolates that include representatives of 20 VCGs of F. oxysporum f. sp. cubense, other formae speciales, and nonpathogens. To determine the ability of F. oxysporum f. sp. cubense to sexually recombine, crosses were made between isolates of opposite mating types. Phylogenetic analysis separated the F. oxysporum isolates into two clades and eight lineages. Phylogenetic relationships between F. oxysporum f. sp. cubense and other formae speciales of F. oxysporum and the relationships among VCGs and races of F. oxysporum f. sp. cubense clearly showed that F. oxysporum f. sp. cubense's ability to cause disease on banana has emerged multiple times, independently, and that the ability to cause disease to a specific banana cultivar is also a polyphyletic trait. These analyses further suggest that both coevolution with the host and horizontal gene transfer may have played important roles in the evolutionary history of the pathogen. All examined isolates harbored one of the two mating-type idiomorphs, but never both, which suggests a heterothallic mating system should sexual reproduction occur. Although, no sexual structures were observed, some lineages of F. oxysporum f. sp. cubense harbored MAT-1 and MAT-2 isolates, suggesting a potential that these lineages have a sexual origin that might be more recent than initially anticipated.

Agrobacterium-mediated transformation of Ceratocystis albifundus.

Functional association between genomic loci and specific biological traits remains lacking in many fungi, including the African tree pathogen Ceratocystis albifundus. This is mainly because of the absence of suitable transformation systems for allowing genetic manipulation of this and other fungi. Here, we present an optimized protocol for Agrobacterium tumefaciens-mediated transformation of C. albifundus. Strain AGL-1 of A. tumefaciens and four binary T-DNA vectors (conferring hygromycin B or geneticin resistance and/or expressing the green fluorescent protein [GFP]) were used for transforming germinated conidia of three isolates of C. albifundus. Stable expression of these T-DNA-encoded traits was confirmed through sequential sub-culturing of fungal transformants on selective and non-selective media and by using PCR and sequence analysis. Single-copy integration of the respective T-DNAs into the genomes of these fungi was confirmed using Southern hybridization analysis. The range of experimental parameters determined and optimised included: (i) concentrations of hygromycin B and geneticin required for inhibiting growth of the wild type fungus and (ii) the dependence of transformation on acetosyringone for inducing the bacterium's virulence genes, as well as (iii) the duration of fungus-bacterium co-cultivation periods and (iv) the concentrations of fungal conidia and bacterial cells used for the latter. The system developed in this study is stable with a high-efficiency, yielding up to 400 transformants per 106 conidia. This is the first report of a transformation protocol for C. albifundus and its availability will be invaluable for functional studies in this important fungus.

IMA Genome-F 11: Draft genome sequences of Fusarium xylarioides, Teratosphaeria gauchensis and T. zuluensis and genome annotation for Ceratocystis fimbriata.

Draft genomes of the fungal species Fusarium xylarioides, Teratosphaeria gauchensis and T. zuluensis are presented. In addition an annotation of the genome of Ceratocystis fimbriata is presented. Overall these genomes provide a valuable resource for understanding the molecular processes underlying pathogenicity and potential management strategies of these economically important fungi.

Mango malformation disease and the associated fusarium species.

ABSTRACT Mango malformation disease (MMD) occurs in Asia, Africa, and the Americas and was first reported in India in 1891. The vegetative form of MMD was first reproduced in 1966 with Fusarium moniliforme and the floral form with isolates of F. moniliforme var. subglutinans from both vegetative shoots and floral tissue. The fungi were subsequently recognized as F. subglutinans. In 2002, a new species, F. mangiferae, was established based on nuclear and mitochondrial DNA sequences; it included strains of F. subglutinans from Egypt, Florida, Israel, Malaysia, and South Africa, some of which had been shown to cause MMD by artificial inoculation. At least three additional taxa have been associated with MMD: F. sterilihyphosum from Brazil and South Africa, and Fusarium sp. nov. and F. proliferatum (teleomorph: Gibberella intermedia) from Malaysia. To date, Koch's postulates have not been completed with them. In the future, gene sequencing will be essential to identify the Fusarium spp. that are associated with MMD. Work remains to be done on the morphology, sexual compatibility, pathogenicity, and toxigenicity of these taxa.

Current status of the taxonomic position of Fusarium oxysporum formae specialis cubense within the Fusarium oxysporum complex.

Fusarium oxysporum is an asexual fungal species that includes human and animal pathogens and a diverse range of nonpathogens. Pathogenic and nonpathogenic strains of this species can be distinguished from each other with pathogenicity tests, but not with morphological analysis or sexual compatibility studies. Substantial genetic diversity among isolates has led to the realization that F. oxysporum represents a complex of cryptic species. F. oxysporum f. sp cubense (Foc), causal agent of Fusarium wilt of banana, is one of the more than 150 plant pathogenic forms of F. oxysporum. Multi-gene phylogenetic studies of Foc revealed at least eight phylogenetic lineages, a finding that was supported by random amplified polymorphic DNAs, restriction fragment length polymorphisms and amplified fragment length polymorphisms. Most of these lineages consist of isolates in closely related vegetative compatibility groups, some of which possess opposite mating type alleles, MAT-1 and MAT-2; thus, the evolutionary history of this fungus may have included recent sexual reproduction. The ability to cause disease on all or some of the current race differential cultivars has evolved convergently in the taxon, as members of some races appear in different phylogenetic lineages. Therefore, various factors including co-evolution the plant host and horizontal gene transfer are thought to have shaped the evolutionary history of Foc. This review discusses the evolution of Foc as a model formae specialis in F. oxysporum in relation to recent research findings involving DNA-based studies.

Differentiation of Fusarium subglutinans f. sp. pini by histone gene sequence data.

Fusarium subglutinans f. sp. pini (= F. circinatum) is a pathogen of pine and is one of eight mating populations (i.e., biological species) in the Gibberella fujikuroi species complex. This species complex includes F. thapsinum, F. moniliforme (= F. verticillioides), F. nygamai, and F. proliferatum, as well as F. subglutinans associated with sugarcane, maize, mango, and pineapple. Differentiating these forms of F. subglutinans usually requires pathogenicity tests, which are often time-consuming and inconclusive. Our objective was to develop a technique to differentiate isolates of F. subglutinans f. sp. pini from other isolates identified as F. subglutinans. We sequenced the histone H3 gene from a representative set of Fusarium isolates. The H3 gene sequence was conserved and contained two introns in all the isolates studied. From both the intron and the exon sequence data, we developed a PCR-restriction fragment length polymorphism technique that reliably distinguishes F. subglutinans f. sp. pini from the other biological species in the G. fujikuroi species complex.

Gibberella fujikuroi mating population E is associated with maize and teosinte.

Summary Isolates of Fusarium subglutinans mating population E are usually found on maize. This fungus forms part of the so-called Gibberella fujikuroi species complex. Previously, F. subglutinans has been associated with two additional mating populations (B and H) and a variety of plant hosts. This was mainly due to a lack of diagnostic morphological characters, but the use of DNA sequence information showed that the strains making up mating populations B, E and H, as well as those associated with the different plant hosts, represent separate species. Recently, another putative mating population has been reported on the wild teosinte relatives of maize. Based on sexual compatibility studies, these isolates were apparently closely related to the pitch canker fungus, F. subglutinans f. sp. pini (= F. circinatum;G. fujikuroi mating population H). The aim of the current study was to determine whether the population of F. subglutinans from teosinte constitutes a new or an existing lineage within the G. fujikuroi complex. For this purpose, portions of the mitochondrial small subunit ribosomal DNA, calmodulin and beta-tubulin genes from the fungi were sequenced. Phylogenetic analyses and comparison with sequences from public domain databases indicated that the F. subglutinans isolates from teosinte are most closely related to strains of G. fujikuroi mating population E. These results were confirmed using sexual compatibility studies. The putative mating population from the wild relatives of maize therefore forms part of the existing E-mating population and does not constitute a new lineage in the G. fujikuroi species complex.

PCR-based identification of MAT-1 and MAT-2 in the Gibberella fujikuroi species complex.

All sexually fertile strains in the Gibberella fujikuroi species complex are heterothallic, with individual mating types conferred by the broadly conserved ascomycete idiomorphs MAT-1 and MAT-2. We sequenced both alleles from all eight mating populations, developed a multiplex PCR technique to distinguish these idiomorphs, and tested it with representative strains from all eight biological species and 22 additional species or phylogenetic lineages from this species complex. In most cases, either an approximately 800-bp fragment from MAT-2 or an approximately 200-bp fragment from MAT-1 is amplified. The amplified fragments cosegregate with mating type, as defined by sexual cross-fertility, in a cross of Fusarium moniliforme (Fusarium verticillioides). Neither of the primer pairs amplify fragments from Fusarium species such as Fusarium graminearum, Fusarium pseudograminearum, and Fusarium culmorum, which have, or are expected to have, Gibberella sexual stages but are thought to be relatively distant from the species in the G. fujikuroi species complex. Our results suggest that MAT allele sequences are useful indicators of phylogenetic relatedness in these and other Fusarium species.