Haruhisa Suga

Molecular Characterization of the Fusarium graminearum Species Complex in Japan

Members of the Fusarium graminearum species complex are important cereal pathogens worldwide and belong to one of at least nine phylogenetically distinct species. We examined 298 strains of the F. graminearum species complex collected from wheat or barley in Japan to determine the species and trichothecene chemotype. Phylogenetic analyses and species-diagnostic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLPs) revealed the presence and differential distribution of F. graminearum sensu stricto (s. str.) and F. asiaticum in Japan. F. graminearum s. str. is predominant in the north, especially in the Hokkaido area, while F. asiaticum is predominant in southern regions. In the Tohoku area, these species co-occurred. Trichothecene chemotyping of all strains by multiplex PCR revealed significantly different chemotype compositions of these species. All 50 strains of F. graminearum s. str. were of a 15- or 3-acetyl deoxynivalenol type, while 173 (70%) out of 246 strains of F. asiaticum were of a nivalenol type. The possibility of gene flow between the two species was investigated by use of 15 PCR-RFLP markers developed in this study. However, no obvious hybrids were detected from 98 strains examined, including strains collected from regions where both species co-occur.

One fungus, one name

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice.

Chromosome Complement of the Fungal Plant Pathogen Fusarium graminearum Based on Genetic and Physical Mapping and Cytological Observations

A genetic map of the filamentous fungus Fusarium graminearum (teleomorph: Gibberella zeae) was constructed to both validate and augment the draft whole-genome sequence assembly of strain PH-1. A mapping population was created from a cross between mutants of the sequenced strain (PH-1, NRRL 31084, originally isolated from Michigan) and a field strain from Minnesota (00-676, NRRL 34097). A total of 111 ascospore progeny were analyzed for segregation at 235 loci. Genetic markers consisted of sequence-tagged sites, primarily detected as dCAPS or CAPS (n = 131) and VNTRs (n = 31), in addition to AFLPs (n = 66) and 7 other markers. While most markers exhibited Mendelian inheritance, segregation distortion was observed for 25 predominantly clustered markers. A linkage map was generated using the Kosambi mapping function, using a LOD threshold value of 3.5. Nine linkage groups were detected, covering 1234 cM and anchoring 99.83% of the draft sequence assembly. The nine linkage groups and the 22 anchored scaffolds from the sequence assembly could be assembled into four chromosomes, leaving only five smaller scaffolds (59,630 bp total) of the nuclear DNA unanchored. A chromosome number of four was confirmed by cytological karyotyping. Further analysis of the genetic map data identified variation in recombination rate in different genomic regions that often spanned several hundred kilobases.

One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use.

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice.

Refined PCR protocol for detection of plant pathogens in soil

Abstract A polymerase chain reaction method to detect soil-borne plant pathogens such as Pythium ultimum, Plasmodiophora brassicae, and Verticillium dahliae in soil was developed and used with a range of soil textures. DNA extraction was based on lyses with alkali buffer (pH 9.0) containing sodium dodecyl sulfate, extraction with benzyl chloride, and collection by ethanol precipitation. Four modified protocols – purification of extracted DNA from soil, independent addition of glass beads or skim milk to the extraction buffer, and addition of bovine serum albumin (BSA) to the PCR reaction mixture – were evaluated to enhance the sensitivity of detection. The effectiveness varied for three tested pathogens, but none of the four protocols negatively affected PCR detection. DNA purification was essential for detecting the three tested pathogens. Physical disruption with glass beads and the addition of BSA to the PCR reaction mixture were necessary for detecting V. dahliae, and the addition of skim milk was needed for Pl. brassicae. Additions of BSA and skim milk enhanced the detection of P. ultimum. The developed protocol seems applicable to the range of soil textures that are naturally inhabited by these three pathogens. By integrating multiple protocols to enhance sensitivity, PCR can be used to detect various soil microorganisms.

Phylogenetic relationships of Pythium species based on ITS and 5.8S sequences of the ribosomal DNA

The sequences of ITS regions in 30 species and two groups of the genusPythium were resolved. In the phylogenetic trees, the species were generally divided into two clusters, referred to here as the F and S groups. The species in the two groups correspond in terms of their sporangial morphology, with the F group being filamentous/lobulate and the S group being spherical. Genetic divergence within the F group was lower than that within the S group. Other morphological characteristics such as oogonial structure and sexual nature appeared to be unrelated to the groupings in these trees. An alignment analysis revealed common sequences to all the species and arrangements specific to each F or S group. It was found that the ITS region was a good target in designing species-specific primers for the identification and detection ofPythium species. In the tree based on 5.8S rDNA sequences, oomycetes are distantly related to other fungi but separated from algae in Chromista.

Intraspecific DNA polymorphisms of Pythium irregulare

Forty-seven isolates of Pythium Irregulare from different hosts and geographic origins were compared from molecular, morphological and physiological viewpoints. They were divided into four groups (I-IV) based on ITS-RFLP analysis and RAPD analysis. Groups I and II included 32 and eight isolates, respectively, collected from diverse hosts and geographic origins, and groups III and IV comprised seven isolates derived from sugar beet and sugar beet field soil. Group I had smaller oogonia and oospores than did the other three groups. In groups I and II, a significantly higher percentage of the oogonia produced multiple projections compared to groups III and IV which occasionally produced one projection. The growth rate of the four groups was similar at 5-30°C. At 33°, many isolates of group I grew rapidly but most of the isolates of other groups grew slowly, and at 35°, the former grew but the latter did not. In phylogenetic analysis based on sequences of the ITS region, four groups of P. irregulare were included in one cluster with P. sylvaticum. Groups I-II and III-IV clustered more tightly in the same branch, respectively. The genetic divergence between I-II and III-IV was higher than between each group (I-II and III-IV) and P. sylvaticum, indicating that groups I-II and III-IV may represent two different species.

Phylogenetic analysis of the phytopathogenic fungus Fusarium solani based on the rDNA-ITS region

The nucleotide sequence of the internal transcribed spacer region of the ribosomal RNA gene (rDNA-ITS) was obtained for strains belonging to 10 special forms (f. spp.) of the phytopathogenic fungus Fusarium solani (teleomorph Haematonectria haematococca). Phylogenetic analysis of nucleotide sequences of the rDNA-ITS region showed that most clusters were composed of strains belonging to the same special form. F. solani f. sp. eumartii and f. sp. phaseoli were excluded from the cluster composed of all other special forms. In order to use the molecular data to identify each special form, several restriction enzymes specific for each special form were selected from the mapping data. The digestion pattern of the rDNA-ITS region with these restriction enzymes was able to distinguish F. solani f. spp. phaseoli, cucurbitae race 1, batatas, piperis, eumartii, and xanthoxyli. Although f. spp. mori, robiniae and pisi were not differentiated by restriction analysis, they were separated in the phylogenetic tree.

Characterization of a New Subgroup of Rhizoctonia solani Anastomosis Group 1 (AG-1-ID), Causal Agent of a Necrotic Leaf Spot on Coffee.

ABSTRACT A new foliar disease on coffee leaves was observed in Mindanao, Philippines, in 1996. The symptoms appeared as large circular or irregularly shaped necrotic areas with small circular necrotic spots (1 mm or less in diameter) usually found around the periphery of the large necrotic areas. Rhizoctonia solani was consistently isolated from these diseased coffee leaves. Isolates obtained were multinucleate (3 to 12 nuclei per hyphal cell), had an optimum temperature for hyphal growth at 25 degrees C, prototrophic for thiamine, and anastomosed with tester isolates belonging to R. solani anastomosis group 1 (AG-1). Mature cultures on potato dextrose agar (PDA) were light to dark brown. Sclerotia, light brown to brown, were formed on the surface of PDA and covered the whole mature colony culture. Individual sclerotia often aggregated into large clumps (3 to 8 mm in diameter) and their color was brown to dark brown. In pathogenicity tests, isolates from coffee caused necrotic symptoms on coffee leaves, whereas isolates of AG-1-IA (not isolated from coffee), 1-IB, and 1-IC did not. The results of analyses of restriction fragment length polymorphism of ribosomal DNA internal transcribed spacer, random amplified polymorphism DNA, and fatty acid profiles showed that R. solani isolates from coffee are a population of AG-1 different from AG-1-IA, 1-IB, and 1-IC. These results suggest that R. solani isolates from coffee represent a new subgroup distinct from AG-1-IA, 1-IB, and 1-IC. A new subgroup ID (AG-1-ID) is proposed.

Differentiation of Rhizoctonia AG-D isolates from turfgrass into subgroups I and II based on rDNA and RAPD analyses

Binucleate Rhizoctonia anastomosis group (AG) D is the cause of rhizoctonia-patch and elephant-footprint diseases of zoysiagrass, and winter-patch disease of bentgrass. Rhizoctonia AG-D is also known as the causal pathogen of other diseases such as sharp-eye-spot of cereals, foot-rot of cereals and winter-stem-rot of mat rush. Isolates of AG-D have been divided into the two subgroups AG-D (I) and AG-D (II), based on the results of cultural characteristics and pathogenicity tests. Isolates obtained from zoysiagrass exhibiting symptoms of rhizoctonia-patch disease, from bentgrass with winter-patch disease, from wheat with foot-rot disease, and from mat rush with winter-stem-rot disease were reported to belong to subgroup AG-D (I). On the other hand, isolates obtained from zoysiagrass with elephant-footprint disease were assigned to subgroup AG-D (II). To confirm the existence of these two subgroups in AG-D, the genetic structure of AG-D isolates from turfgrass and other crops was compared. RFLP analysis of the ITS region from rDNA after digestion with the restriction enzymes EcoRI, HaeIII, HhaI, HinfI, and MboI separated AG-D isolates into two groups corresponding to AG-D (I) and AG-D (II). Furthermore, other AGs except AG-Q (AGs-A, Ba, Bb, C, E, F, G, I, K, L, O, P, and R. solani AG1-IC) did not have the same patterns that were seen for the two AG-D subgroups. AG-Q isolates from bentgrass showed the same patterns as AG-D (I). The results of the RAPD analysis also revealed the existence of two groups that corresponded to AG-D (I) and AG-D (II). These analyses revealed that Rhizoctonia AG-D isolates from turfgrass could be divided into two subgroups consistent with those based on cultural characteristics and pathogenicity. In addition, isolates of foot-rot disease of wheat and isolates of winter-stem-rot disease of mat rush whose cultural characteristics were the same as those of AG-D (I) also showed similar RFLP and RAPD patterns to those of AG-D (I) isolates from turfgrass.