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. First Report of Fusarium subglutinans f. sp. pini on Pine Seedlings in South Africa . A. VILJOEN, Department of Microbiology and Biochemistry, University of the. Mango (Mangifera indica L.) floral malformation caused by the fungal pathogen Fusarium subglutinans. Healthy mango inflorescence. Photographs courtesy.

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This species complex includes F. Differentiating these forms of F. Our objective was to develop a technique to differentiate isolates of F. 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. This fungus can be spread by both infected seedlings and seed 1 The management of F.

Three of these mating populations, B, E, and H F. Strains of Fusarium isolated from pineapple F. These assays are time-consuming and labor-intensive and do not always yield clear-cut answers. Because of the technical difficulties associated with mitochondrial RFLP and the low repeatability of RAPD data, we do not consider these techniques useful for diagnostic purposes.

Two different copies of the ITS2 region were identified in the same isolate within some of the species in Fusarium section Liseola 2537and a reliable diagnostic technique based on these sequences could not be developed. We used an alternative fusariu, of the genome, the histone H3 gene, to distinguish Fusariu.

Histone genes encode histone proteins, which are subglytinans major constituents of chromatin 16 The gene encoding the H3 protein is well conserved, especially at the amino acid level 1231and subglutihans presence of introns enhances its value in taxonomic and phylogenetic studies of closely related organisms 8 Although the histone H4 gene also has these characteristics, it is generally too highly conserved subgluginans be suitable for evolutionary studies Our objectives in this study were i to sequence the histone H3 gene from various strains in the G.

We examined 42 Fusarium isolates, including F. To test the efficacy of the PCR-RFLP technique for use as a species diagnostic technique see belowwe tested subgljtinans strains of the H mating population identified by Britz et al. These strains were reassorted and then encoded so that the assays were done in a blind manner. Host and origin of the different Fusarium isolates from the G. We performed phenol-isoamyl alcohol-chloroform Nucleic acids were precipitated by adding 0. This protocol is a variation of the one developed by Murray and Thompson These primers were constructed to flank at least one intron and amplify approximately bp of the Neurospora crassa histone H3 gene.


Each PCR mixture contained 1 mM deoxynucleotide triphosphates 0. Sequence Navigator version 1. Bootstrap analyses were based on subglutlnans, replications. Fusarium oxysporum MRC was used as an outgroup. Crosses were scored as positive when ascospores were observed exuding from perithecia. Nucleic acids were visualized with a UV transilluminator nm. Subglutinnas test the efficacy of the PCR-RFLP technique described here, histone H3 gene PCR products from 60 strains representing mating population H and 80 strains representing mating populations A to F were amplified, digested, and electrophoresed as described above.

The Fusarium histone H3 gene fragments ranged from to subglufinans in length and contained two introns intron 1 and intron 2 whose positions within the sequences were conserved. Usbglutinans 1 was 83 bp long for strains from mating population H, F. Intron 2 was 57 bp long for all of the isolates, except for F. The coding regions of the Fusarium histone H3 genes were highly conserved, and we observed no deletions or insertions.

We detected no differences in amino acid sequence, and coding sequence variation within the Fusarium genes was generally limited to the third position within the codon. The Fusarium histone H3 amino acid sequence differed from that of N.

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Phylogenetic analysis with PAUP 4. This tree was comprised of two distinct clades. Clade 1 included isolates from mating populations H and E as well as isolates of F. Phylogram generated with histone H3 gene sequence data from the isolates included in this study by use of PAUP 4. Bootstrap values based on 1, replications are indicated as percentages.

Bold letters in parentheses refer to the G. This dendrogram is fusarrium to F. The length of the tree was steps, and the values for homoplasy index and the retention index were 0. Two subgroups made up clade 1 Fig. The first subgroup included F. The second subgroup included F. Clade 2 was subdivided into two smaller subgroups, one of which included isolates from mating populations B, C, and D as well as the F.

Three of the F.

The remaining four isolates did not cross with one another or either of the tester strains. From the restriction enzyme profiles, we constructed restriction maps for all host-specific groups fusariuj F.

Lanes M, bp ladder 1, 1,,, and bp ; lane A, mating population Subglutinams lane B, mating population B F. Restriction maps of the histone H3 genes from the different isolates of Fusariumgenerated with restriction enzymes Dde I and Cfo I. The Fusarium introns are indicated as boxes, and the exons are indicated as horizontal lines. Bold letters refer to the G.

An arrow indicates a Cfo I restriction site, and a vertical line indicates a Dde I restriction site. Exon and fragment sizes are indicated in base pairs. All 60 mating population H strains were positively identified as F.

We identified none of the strains from the collection subbglutinans Yan et al.


Differentiation of Fusarium subglutinans f. sp. pini by Histone Gene Sequence Data

In this study, we were able to distinguish F. Seven outbreaks of root rot in South African nurseries have been correctly diagnosed as being caused by F. We thus have confidence that this technique is robust and can be used with a high degree of certainty. Phylogenetic analyses with the Fusarium histone H3 gene sequence data ffusarium a phylogram Fig. First, mating populations C and D form a closely related group in all cases. There are, however, two fuzarium differences between DNA-based phylogenies and those based on isozymes.

With isozymes, Huss subglutibans al. The DNA-based phylogenies 26 ; this studyhowever, indicate that mating population G is most closely related to mating populations A and F and that these three mating populations form a distinct cluster separate from both mating populations C and D.

Also, in contrast to the results from the isozyme study 15 both DNA-based phylogenies 26 ; this study indicate ffusarium mating populations C and D subglutnans most closely related to mating population B. Although some of the F. Since some individuals from both of these groups could cross with one of the mating type E tester strains, we do not believe that the second cluster of isolates from ffusarium represents a separate mating population.

This similarity suggests an equivalence of F. The latter clade also includes F. Moreover, rapid identification of fungi such as the pitch canker pathogen is now possible with a PCR-RFLP technique based on the histone H3 gene sequence.

National Center for Biotechnology InformationU. Journal List Appl Environ Microbiol v. Wingfield1 T. Coutinho1 M. Wingfield1 and W. Author information Article notes Copyright and License information Disclaimer. Received Feb 9; Accepted May This article has been cited by other articles in PMC. Abstract Fusarium subglutinans f. Open in a separate window.

The proposed synonyms for F. Occurrence of fungi inside dusarium pine seeds produced in seed orchards in the United States. Pitch canker of slash pine seedlings: Activity of restriction enzymes in a PCR mix. Pitch canker disease in California: Genetic diversity in California and Florida populations of the pitch canker fungus Fusarium subglutinans f.

Primer sets developed to amplify conserved genes from filamentous ascomycetes are useful in differentiating Fusarium species associated with conifer. Sequence, organization and expression of the core histone genes of Aspergillus nidulans. Gerlach W, Nirenberg H I. The genus Fusarium —a pictorial atlas. Development subglurinans primer sets designed for use with PCR to amplify conserved genes from filamentous ascomycetes. Pitch canker, a new disease of some southern pines.

Mating groups in Fusarium moniliforme.