Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani
TL;DR: Results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance.
Abstract: Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to evaluate the genetic diversity of 45 isolates within and between anastomosis groups (AGs) in Rhizoctonia solani. The 5.8s rDNA sequence was completely conserved across all the AGs examined, whereas the ITS rDNA sequence was found to be highly variable among isolates. The sequence homology in the ITS regions was above 96% for isolates of the same subgroup, 66-100% for isolates of different subgroups within an AG, and 55-96% for isolates of different AGs. In neighbor-joining trees based on distances derived from ITS-5.8s rDNA sequences, subgroups IA, IB and IC within AG-1 and subgroups HG-I and HG-II within AG-4 were placed on statistically significant branches as assessed by bootstrap analysis. These results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance.
Citations
More filters
TL;DR: The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in the authors' libraries relative to other eukaryotes suggest that the abundance and diversity of fungiIn forest soils may be much higher than previously hypothesized.
Abstract: Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized.
914 citations
TL;DR: These studies show that asexual or sexual reproductive morphology does not necessarily correlate with clonal or recombining reproductive behavior, and that fungi with all types of reproductive morphologies and behaviors can be accommodated by a phylogenetic species concept.
Abstract: Phylogenetic and population genetic methods that compare nucleic acid variation are being used to identify species and populations of pathogenic fungi and determine how they reproduce in nature. These studies show that asexual or sexual reproductive morphology does not necessarily correlate with clonal or recombining reproductive behavior, and that fungi with all types of reproductive morphologies and behaviors can be accommodated by a phylogenetic species concept. Although approximately one fifth of described fungi have been thought to be asexual and clonal, recent studies have shown that they are also recombining. Whether a particular pathogen reproduces clonally or by recombination depends on factors relating to its biology and its distribution in space and time. Knowing the identity of species and populations and their reproductive modes, while taking a broad view of pathogen behavior in space and time, should enhance the ability of pathologists to control pathogens and even predict their behavior.
519 citations
Cites background from "Sequence variation of the rDNA ITS ..."
...solanithat correlate with phylogenetic species defined by analysis of nuclear large subunit rDNA and ITS variation (102, 162)....
[...]
TL;DR: The development of molecular techniques has provided a new range of tools that can provide clear insights into specific interactions and activities in soil environments and the combination of broad spectrum polymerase chain reaction (PCR) detection, coupled with single strand conformation polymorphisms (SSCP) or denaturing gradient gel electrophoresis (DGGE), can give more accurate answers to fundamental questions on ecosystem diversity.
Abstract: The great majority of the 80 000+ fungal species so far named and described are likely to occur in the soil environment at some stage in their life-cycle. Fungi therefore have many different functions in soils, which include both active roles, such as the degradation of dead plant material, or inactive roles where propagules are present in the soil as resting states. Current knowledge of fungal diversity in soil is based largely on observations of fruiting bodies present in an environment, or from cultures obtained from soil isolation exercises. Both of these approaches have serious limitations for the detection of the true diversity in any chosen environment. An organism that exists only in a mycelial form in the soil is unlikely to be identified from direct observation if a fruiting body is not formed. Therefore, classical observation through direct microscopy will give a greatly reduced measure of the true diversity in the environment. Culturing fungi from soil isolations will only result in the detection of those propagules that are able to grow and sporulate on the isolation medium used. This again will lead to a greatly reduced measure of diversity, as at the present time only about 17% of the known fungal species can be successfully grown in culture. The recovery of a culture from soil also does not distinguish whether the fungus was an active part of the original ecosystem or present in an inactive resting state. The development of molecular techniques has provided a new range of tools that can provide clear insights into specific interactions and activities in soil environments. The combination of broad spectrum polymerase chain reaction (PCR) detection, coupled with single strand conformation polymorphisms (SSCP) or denaturing gradient gel electrophoresis (DGGE), can give more accurate answers to fundamental questions on ecosystem diversity. This technique does not however distinguish between active and resting stages, and in order to interpret results accurately, some a priori knowledge of the ecology and function of the organisms is required.
302 citations
TL;DR: A primer able to amplify the internal transcribed spacers of the ribosomal DNA (rDNA), having enhanced specificity for ascomycetes, was identified by reviewing fungal ribosomic DNA sequences deposited in GenBank.
270 citations
TL;DR: Comparing anastomosis reactions, rDNA-internal transcribed spacer (ITS) sequences, and virulence of isolates representing Rhizoctonia solani AG-BI and six subsets of anastsomosis group (AG)-2 found grouping based on virulence does not conform to established grouping patterns within AG-2 and does not seem useful as a group-defining criterion.
Abstract: Hyphal anastomosis reactions, rDNA-internal transcribed spacer (ITS) sequences, and virulence of isolates representing Rhizoctonia solani AG-BI and six subsets of anastomosis group (AG)-2 (-2-1, -2-2 IIIB, -2-2 IV, -2-2 LP, -2-3, and -2-4) were compared. AG-2-4 is a subset described for the first time in this report. Anastomosis reactions within AG-BI and the listed subsets of AG-2 were generally strong but, between subsets, ranged from strong to a very weak "bridging" -type reaction. Anastomosis reaction alone generally did not provide adequate evidence for placement of an isolate into a subset of AG-2. Anastomosis reactions between AG-BI and the original subsets of AG-2 (-2-1 and -2-2) are very strong; for this reason, we propose that it be included as a subset of AG-2 (designation AG-2 BI). Subsets -2-3 and -2-4 show very weak bridging-type anastomosis reactions with all other subsets of AG-2 and thus may be candidates for independent AG status. Grouping within AG-2 based on rDNA-ITS sequences was consistent with the abovementioned subsets. However, grouping based on virulence as measured herein does not conform to established grouping patterns within AG-2 and does not seem useful as a group-defining criterion. A broad range of damage was observed among members of the most virulent subsets (-2-1, -2-2 IIIB, -2-2 IV, and -2-4), whereas other subsets (-2 BI, -2-2 LP, and -2-3) were similar to one another in causing a minimal level of damage. Group-specific primer pairs for each of the seven subsets of AG-2 were designed based on the abovementioned rDNA-ITS sequences. Primer pairs proved dependable and subset specific in polymerase chain reaction amplifications of purified genomic DNA from 109 isolates of R. solani and two isolates of binucleate Rhizoctonia. These primers will provide a simple and useful method for subset-specific characterization within AG-2 if further critical evaluations confirm their specificity.
261 citations
References
More filters
TL;DR: The neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods for reconstructing phylogenetic trees from evolutionary distance data.
Abstract: A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
57,055 citations
"Sequence variation of the rDNA ITS ..." refers methods in this paper
...A tree showing the phylogenetic relatedness between isolates was constructed from distance matrix values by the neighbor-joining method ( Saitou and Nei 1987 ), using the computer software package CLUSTAL V. (Higgins et al. 1992)....
[...]
01 Jan 1990
26,241 citations
TL;DR: Some examples were worked out using reported globin sequences to show that synonymous substitutions occur at much higher rates than amino acid-altering substitutions in evolution.
Abstract: Some simple formulae were obtained which enable us to estimate evolutionary distances in terms of the number of nucleotide substitutions (and, also, the evolutionary rates when the divergence times are known). In comparing a pair of nucleotide sequences, we distinguish two types of differences; if homologous sites are occupied by different nucleotide bases but both are purines or both pyrimidines, the difference is called type I (or “transition” type), while, if one of the two is a purine and the other is a pyrimidine, the difference is called type II (or “transversion” type). Letting P and Q be respectively the fractions of nucleotide sites showing type I and type II differences between two sequences compared, then the evolutionary distance per site is K = — (1/2) ln {(1 — 2P — Q) }. The evolutionary rate per year is then given by k = K/(2T), where T is the time since the divergence of the two sequences. If only the third codon positions are compared, the synonymous component of the evolutionary base substitutions per site is estimated by K'S = — (1/2) ln (1 — 2P — Q). Also, formulae for standard errors were obtained. Some examples were worked out using reported globin sequences to show that synonymous substitutions occur at much higher rates than amino acid-altering substitutions in evolution.
26,016 citations
TL;DR: An algorithm was developed which facilitates the search for similarities between newly determined amino acid sequences and sequences already available in databases and increases sensitivity by giving high scores to those amino acid replacements which occur frequently in evolution.
Abstract: An algorithm was developed which facilitates the search for similarities between newly determined amino acid sequences and sequences already available in databases. Because of the algorithm's efficiency on many microcomputers, sensitive protein database searches may now become a routine procedure for molecular biologists. The method efficiently identifies regions of similar sequence and then scores the aligned identical and differing residues in those regions by means of an amino acid replacability matrix. This matrix increases sensitivity by giving high scores to those amino acid replacements which occur frequently in evolution. The algorithm has been implemented in a computer program designed to search protein databases very rapidly. For example, comparison of a 200-amino-acid sequence to the 500,000 residues in the National Biomedical Research Foundation library would take less than 2 minutes on a minicomputer, and less than 10 minutes on a microcomputer (IBM PC).
3,902 citations
"Sequence variation of the rDNA ITS ..." refers methods in this paper
...Percentage homology is calculated at different nucleotide sites (transition, transversion, or deletion), using the Lipman-Pearson algorithm ( Lipman and Pearson 1985 ) bacco had identical sequences, for both ITS regions....
[...]