Entire ITS Region Research Articles

Nucleoli, rRNA genes and ITS region in Posidonia oceanica (L.) Delile.

The maximum number of nucleoli was counted in interphase nuclei of Posidonia oceanica, and a restriction pattern of nuclear rDNA was obtained after digestion with four restriction endonucleases and Southern hybridization. P. oceanica has only one type of ribosomal gene whose size was estimated to be 18.5 kbp long. The nucleotide sequence of the entire ITS region was also determined by direct sequencing of PCR amplified DNA fragments. The sequence of the ITS region was aligned with those of homologous regions of other monocots available in literature, and phylogenetic trees were obtained.

Molecular phylogeny of the Leptosphaeria maculans-L. biglobosa species complex

Leptosphaeria maculans (anamorph Phoma lingam), the ascomycete causing stem canker of crucifers, is a species complex that can be separated into at least seven distinct subgroups using a combination of biochemical and molecular criteria. In the present study sequences of the entire ITS region, including the 5.8S rDNA, of 38 isolates representing the seven subgroups, along with specimens from culture collections, were analysed, compared to those of closely related Leptosphaperia species, and the phylogeny inferred using parsimony and distance analyses. A well-supported clade encompassed all isolates of the seven subgroups along with L. conferta, a known saprobe of dried crucifer stems. The L. maculans isolates were further separated into two well-supported clades corresponding to L. maculans s. str. and the recently named L. biglobosa. Parsimony and distance analyses further separated groups within both species, usually corresponding to specific host plants or geographic origin, e.g. L. maculans 'brassicae' from cultivated Brassica, L. maculans 'lepidii'. from Lepidium sp., L. biglobosa 'brassicae', from various Brassica species, L. biglobosa 'thlaspii' from Thlaspi arvense, L. biglobosa 'erysimii' from Erysimum sp., and L. biglobosa 'canadensis' mostly found in central Canada. The oldest L. maculans specimens maintained in international collections clustered with either L. maculans 'brassicae', L. biglobosa 'brassicae', or a still different group closely related to L. biglobosa 'thlaspii'. The evolutionary relationships between the seven infraspecific groups are discussed in terms of phytopathological relevance and species isolation linked with specific life cycle, geographic isolation or host specificity.

A new plant pathogenic sterile white basidiomycete from Australia

A sterile white fungus was isolated from the healthy looking roots of buffalo grass (Stenotaphrum secundatum) grown on cleared bush land in Perth, Western Australia. The fungal strain was pathogenic on 12 plant species screened under the greenhouse conditions. The clamp connections and dolipore septa indicated that the isolate was a Basidiomycete. Mycelial features, growth rate at different temperatures, as well as pathogenicity patterns of this sterile white basidiomycete (SWB) were distinctly different from those of a strain with a similar morphology, ATCC 28344, previously described as a pathogen in Florida and Georgia (USA). All attempts to induce sporulation failed. The isolates were also compared using the nucleotide sequence analysis of the ribosomal DNA array. Approximately 1 kbp of the 5′ end of the large subunit ribosomal RNA gene, complete sequences of the small subunit ribosomal RNA gene and the entire ITS region (including ITS1, ITS2 and 5.8S gene) were sequenced for the purpose. The obtained sequences were compared with the homologous regions of other genera of Agaricales available in GenBank. Relatively low sequence similarities between the American and Australian strains, as well as the phylogenetic analysis of the studied regions has suggested that these two fungi belong to different genera. Interesting results were achieved in the case of the large subunit ribosomal DNA since this region has been widely studied for taxonomy of Basidiomycetes. The Australian strain 3034 appeared to be closely related to the genus Campanella and the American SWB was identified as belonging to the genus Marasmius, possibly to M. graminum. Our data suggest that the Australian strain is a novel pathogen, and is different from the American SWB isolates described to date.

Molecular phylogeny of Leptosphaeria and Phaeosphaeria

The objectives of this study were to determine the phylogenetic relationships of species of Leptosphaeria and Phaeosphaeria and evaluate the phylogenetic significance of morphological characters of the teleomorph, anamorph, and host. Sequences of the entire ITS region, including the 5.8S rDNA, of 59 isolates representing 54 species were analyzed and the phylogeny inferred using parsimony and distance analyses. Isolates grouped into three well-supported clades. The results of this study support the separation of Phaeosphaeria from Leptosphaeria sensu stricto. Leptosphaeria bicolor and the morphologically similar Leptosphaeria taiwanensis formed a separate, well-supported clade. We conclude that peridial wall morphology, anamorph characteristics, and to a lesser extent host, are phylogenetically significant at the generic level. Ascospore and conidial morphology are taxonomically useful at the species level.

Molecular identification of Colletotrichum gloeosporioides causing yam anthracnose in Nigeria

Four forms of Colletotrichum representing three distinct virulence phenotypes were found associated with foliar anthracnose of yam in Nigeria: the highly virulent (= severity of disease) slow‐growing grey (SGG); the moderately virulent fast‐growing salmon (FGS); the weakly virulent fast‐growing grey (FGG); and the moderately virulent fast‐growing olive (FGO) morphotype. Isolates of the four forms were identified as C. gloeosporioides, based on morphology. The reaction of monoconidial cultures on casein hydrolysis medium (CHM), PCR‐RFLP and sequence analysis of the internal transcribed spacer region of the ribosomal DNA (ITS1‐5·8S‐ITS2) were used to establish the identity of the yam anthracnose pathogen(s). All yam isolates were distinguished from C. acutatum by the absence of protease activity on CHM. On ITS PCR and enzymatic digestion of PCR products, all FGS, FGO and SGG isolates produced RFLP patterns identical to those of C. gloeosporioides reference isolates, while FGG isolates revealed unique ITS RFLP banding patterns. Sequence analysis of the ITS1 region and of the entire ITS region revealed that SGG, FGS and FGO isolates were highly similar (98–99% nucleotide identity) and showed 97–100% identity to C. gloeosporioides. Less than 93% similarity of these fungal isolates to reference C. acutatum and C. lindemuthianum isolates was observed. The molecular study confirmed that foliar anthracnose of yam is caused by C. gloeosporioides. While a high similarity was found among most C. gloeosporioides fungi from yam, isolates of the FGG form did not cluster with any previously described Colletotrichum species, and probably represent a distinct species.

DNA studies in the Galerina marginata complex

The distinctiveness of the European Galerina marginata and the American G. autumnalis is tested with analyses of DNA sequences from the internal transcribed spacer (ITS-2) of the nuclear ribosomal repeat and RFLP analysis of the entire ITS region. European and North American material from six taxa described in Galerina subgenus Naucoriopsis are included along with out-group material of six taxa representing two other subgenera of Galerina. The results do not indicate any genetic difference between species by Smith & Singer (1964) referred to the two ‘stirpes’ Marginata and Autumnalis of Naucoriopsis, i.e. G. autumnalis, G. marginata, G. oregonensis, G. pseudomycenopsis, G. unicolor, and G. venenata and all except G. pseudomycenopsis are considered later synonyms of G. marginata. Galerina badipes (syn. G. cedretorum var. bispora of ‘stirps’ Cedretorum is well supported as a distinct species. Subgenus Naucoriopsis appears as a distinct infrageneric unit well separated from the infrageneric units represented by the out-group species.

A comparison of Bulinus africanus group species (Planorbidae; Gastropoda) by use of the internal transcribed spacer 1 region combined by morphological and anatomical characters

The morphological and anatomical characters applied to determine species identity in the Bulinus africanus group species are insufficient to unambiguously discriminate between arbitrary species of given populations. In order to solve this problem, four snail populations from Kenya have been investigated morphologically and anatomically, and the species status compared with the result of molecular methods. We have amplified the entire ITS region and found that the investigated populations showed intra-specific genetic polymorphism, thereby giving the taxa an identity which were indistinct when the region was cut with restriction enzymes. Instead, an amplification of the sub-region ITS 1 revealed an unambiguous identification, because the amplification revealed only one single fragment. We also found that the observed heterogeneity of the entire ITS region could be confined to the sub-region ITS 2. Furthermore, the micro-sculpture of the shell and penis to preputium proportion, which are normally applied as morphological characters, might be considered as inadequate, because of the lack of a significant difference in those characters between the two well established species, namely B. africanus and B. nasutus in the Kisumu area. Instead, these two taxa were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to be one single species with a highly variable morphology. This result was further confirmed by random amplified polymorphic DNA (RAPD) data and the mitochondrial cytochome oxidase subunit I (COI).

Molecular Phylogenetic Studies of Brassica,Rorippa,Arabidopsis and Allied Genera Based on the Internal Transcribed Spacer Region of 18S–25S rDNA

The phylogenetic relationships of nine genera in four tribes of the family Brassicaceae were estimated from the sequences of the internal transcribed spacer region (ITS) of the 18S–25S nuclear ribosomal DNA. The entire ITS region of 16 accessions belonging to 10 species of seven genera was sequenced. Eight published sequences of Brassicaceae were also used. A total of 27 sequences were included in this study; four of them were found to be pseudogenes. Both the neighbor-joining and the parsimony trees suggest that the nine genera can be divided into three groups: (1) Arabidopsis,Cardaminopsis,Capsella, and Lepidium; (2) Rorippa and Cardamine; and (3) Brassica, Sinapis, and Raphanus. In contradiction to the proposal that Cardaminopsis and Arabidopsis be put into an expanded tribe Arabideae, our data show that these two genera are more closely related to Capsella and Lepidium (tribe Lepidieae) than to Rorippa and Cardamine (tribe Arabideae). Further, our data show that within the tribe Brassiceae, Raphanus is more closely related to B. nigra than to the B. oleracea/B. rapa clade. This result is in agreement with the nuclear data obtained in several studies, but is in conflict with the RFLP data of mitochondrial and chloroplast DNA. As pointed out by previous authors, it is possible that Raphanus is a hybrid between the B. nigra and B. oleracea/B. rapa lineages with the latter as the maternal parent.

Origins of domestication and polyploidy in oca (Oxalis tuberosa: Oxalidaceae): nrDNA ITS data

As part of a study aimed at elucidating the origins of the octoploid tuber crop "oca," Oxalis tuberosa, DNA sequences of the internal trancribed spacer of nuclear ribosomal DNA (nrDNA ITS) were determined for oca and several wild Oxalis species, mostly from Bolivia. Phylogenetic analysis of these data supports a group of these species as being close relatives of oca, in agreement with morphology and cytology, but at odds with traditional infrageneric taxonomy. Variation in ITS sequences within this group is quite low (0-7 substitutions in the entire ITS region), contrasting with the highly divergent (unalignable in some cases) sequences within the genus overall. Some groups of morphologically differentiated species were found to have identical sequences, notably a group that includes oca, wild populations of Oxalis that bear small tubers, and several other clearly distinct species. The presence of a second, minor sequence type in at least some oca accessions suggests a possible contribution from a second genome donor, also from within this same species group. ITS data lack sufficient variation to elucidate the origins of oca precisely, but have identified a pool of candidate species and so can be used as a tool to screen yet unsampled species for possible progenitors.