Discovery of <Emphasis Type="Italic">Ophiostoma tsotsi</Emphasis> on <Emphasis Type="Italic">Eucalyptus</Emphasis> wood chips in China

Ophiostoma species such as O. quercus are the most frequent causal agents of sapstain of freshly felled hardwood timber and pulpwood. Many species are regarded as economically important agents of wood degradation. The aim of this study was to identify a collection of Ophiostoma isolates, resembling O. quercus, found on stained Eucalyptus pulpwood chips in China. DNA sequences of the internal transcribed spacer regions, including the 5.8S region, of the ribosomal DNA, and parts of the β-tubulin and elongation factor-1α genes, revealed that the isolates were not O. quercus. Surprisingly, they represented O. tsotsi, a wound-infesting fungus recently described from hardwoods in Africa. In addition, sequence data from an isolate from agarwood in Vietnam, identified in a previous study as belonging to an unknown Pesotum species, were also shown to represent O. tsotsi. A high level of genetic variability was observed among isolates of both O. quercus and O. tsotsi. This was unexpected and suggests that both species have been present in Asia for a significant amount of time.     

h-TBP: an approach based on intron-length polymorphism for the rapid isolation and characterization of the multiple members of the β-tubulin gene family in <Emphasis Type="Italic">Camelina sativa</Emphasis> (L.) Crantz

We have developed a new version of the cTBP (combinatorial tubulin-based polymorphism) method, a previously described approach based on intron-length polymorphism (ILP), to rapidly characterize the β-tubulin gene family of Camelina sativa (L.) Crantz, a plant species of importance for oil production but still largely unexplored at genomic level. The method, named h-TBP, allows the rapid cloning of the β-tubulin genomic sequences that encompass the two introns, invariantly present at fixed positions within the coding region of the vast majority of the plant species. The β-tubulin sequences cloned by h-TBP also comprise part of exon1 and exon3 and the whole sequence of exon2. The h-TBP method has then been used to isolate, clone and characterize the β-tubulin gene family of C. sativa, composed of at least 20 different β-tubulin isotypes, named CsTUB1 through CsTUB20. The relatively high number of β-tubulin genes has been further substantiated by Southern-blot analysis. Comparison of the β-tubulin exon sequences of C. sativa with those of Arabidopsis thaliana, the closest relative among crucifers, defines distinct groups of putative orthologous genes, identified by a UPMGA cluster analysis. Analysis of the C. sativa β-tubulin intron sequences reveals some molecular features that can provide the first hints for the understanding of intron plasticity and evolution. From a more immediate perspective, these data provide the first substantial contribution to the characterization of the largely unexplored genome of C. sativa, and the tools for assisting programmes of breeding and selection of the most productive plants.     

<Emphasis Type="Italic">Ophiostoma tsotsi</Emphasis> sp. nov., A Wound-infesting Fungus of Hardwood Trees in Africa

Polymorphic sequence-characterised marker assays from a recent diversity study on the Ascomycete fungus Ophiostoma quercus reported that some isolates from Africa were genetically distinct from O. quercus. In the present study, these African isolates were compared with authentic O. quercus isolates by evaluating morphological characters, growth in culture, mating compatibility and DNA sequence data. The isolates from Africa were morphologically similar to O. quercus, presenting Pesotum and Sporothrix synanamorphs in culture. Phylogenetic analyses of the ribosomal internal transcribed spacer regions 1 and 2, β-tubulin and translation elongation factor 1-α gene regions confirmed that the African group represents a distinct species within the hardwood lineage of the O. piceae complex, closely related to O. ulmi and O. himal-ulmi. Mating studies between O. quercus and the African isolates showed that isolates mated predominantly with those of their own group, although there were rare cases of fertile crosses between the groups. Isolates residing in the African lineage are described here as a new species, O. tsotsi sp. nov.     

Phylogenetic analysis of the Glomeromycota by partial β-tubulin gene sequences

The 3’ end of the β-tubulin gene was amplified from 50 isolates of 45 species in Glomeromycota. The analyses included a representative selection of all families except Pacisporaceae and Geosiphonaceae. Phylogenetic analyses excluded three intron regions at the same relative positions in all species due to sequence and length polymorphisms. The β-tubulin gene phylogeny was similar to the 18S rRNA gene phylogeny at the family and species level, but it was not concordant at the order level. Species in Gigasporaceae and Glomeraceae grouped together but without statistical support. Paralogous sequences in Glomus species likely contributed to phylogenetic ambiguity. Trees generated using different fungal phyla as out-groups yielded a concordant topology. Family relationships within the Glomeromycota did not change regardless if the third codon position was included or excluded from the analysis. Multiple clones from three isolates of Scutellospora heterogama yielded divergent sequences. However, phylogenetic patterns suggested that only a single copy of the β-tubulin gene was present, with variation attributed to intraspecific sequence divergence.     

Isolation and sequence analysis of a β-tubulin gene from arbuscular mycorrhizal fungi

A full-length β-tubulin gene has been cloned and sequenced from Gigaspora gigantea and Glomus clarum, two arbuscular mycorrhizal fungi (AMF) species in the phylum Glomeromyota. The gene in both species is organized into five exons and four introns. Both genes are 94.9% similar and encode a 447 amino acid protein. In comparison with other fungal groups, the amino acid sequence is most similar to that of fungi in the Chytridiomycota. The codon usage of the gene in both AMF species is broad and biased in favor of an A or a T in the third position. The four introns varied in length from 87 to 168 bp for G. gigantea and from 90 to 136 bp for G. clarum. Of all fungi in which full-length sequences have been published, only AMF do not have an intron before codon 174. The introns positioned at codons 174 and 257 in AMF match the position of different introns in β-tubulin genes of some Zygomycete, Basidiomycete, and Ascomycete fungi. The 5′ and 3′ splice site consensus sequences are similar to those found in introns of most fungi. Sequence analysis from single-strand conformation polymorphism analysis confirmed the presence of two β-tubulin gene copies in G. clarum, but only one copy was evident in G. gigantea based on Southern hybridization analysis.     

Ophiostomatoid fungi associated with the northern spruce engraver, <Emphasis Type="Italic">Ips perturbatus</Emphasis>, in western Canada

A number of ophiostomatoid fungi were isolated from the spruce-infesting bark beetle, Ips perturbatus and its galleries collected from felled spruce trees and logs in northern BC and the Yukon Territory. Isolates were identified to species using morphological characteristics, nuclear ribosomal DNA and partial β-tubulin gene sequences. Thirteen morphological and phylogenetic species were identified among the isolates. Leptographium fruticetum, Leptographium abietinum, Ophiostoma bicolor, Ophiostoma manitobense, O. piceaperdum, and eight undescribed species of the genus Ophiostoma and the anamorph genera Leptographium, Hyalorhinocladiella, Ambrosiella and Graphium. A number of these species, i.e. L. fruticetum, Hyalorhinocladiella sp. 2, O. bicolor and O. manitobense, were isolated repeatedly from I. perturbatus, while others, i.e. Graphium sp. 1 and O. piceaperdum, seemed to be␣sporadic associates. Among all the isolates, L. fruticetum had the highest relative dominance in this survey. A high frequency of occurrence of this species with the beetle may indicate a specific relationship between the two partners.     

In trangenic rice, α- and β-tubulin regulatory sequences control GUS amount and distribution through intron mediated enhancement and intron dependent spatial expression

The genomic upstream sequence of the rice tubulin gene OsTub6 has been cloned, sequenced and characterized. The 5′UTR sequence is interrupted by a 446 bp long leader intron. This feature is shared with two other rice β-tubulin genes (OsTub4 and OsTub1) that, together with OsTub6, group in the same clade in the evolutionary phylogenetic tree of plant β-tubulins. Similarly to OsTub4, the leader intron of OsTub6 is capable of sustaining intron mediated enhancement (IME) of gene expression, in transient expression assays. A general picture is drawn for three rice α-tubulin and two rice β-tubulin genes in which the first intron of the coding sequence for the formers and the intron present in the 5′UTR for the latters, are important elements for controlling gene expression. We used OsTua2:GUS, OsTua3:GUS, OsTub4:GUS and OsTub6:GUS chimeric constructs to investigate the in vivo pattern of beta-glucuronidase (GUS) expression in transgenic rice plants. The influence of the regulatory introns on expression patterns was evaluated for two of them, OsTua2 and OsTub4. We have thus characterized distinct patterns of expression attributable to each tubulin isotype and we have shown that the presence of the regulatory intron can greatly influence both the amount and the actual site of expression. We propose the term Intron Dependent Spatial Expression (IDSE) to highlight this latter effect. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High polymorphism and resolution in targeted fingerprinting with combined β-tubulin introns

Tubulin-Based-Polymorphism (TBP) was originally introduced as a novel method for assaying genetic diversity in plants. TBP is based on polymorphism resulting from the PCR-mediated amplification of the first intron in the coding region of the β-tubulin gene family. Although, the method was successful in genetic assessment of some plant species and varieties, it suffered from low number of molecular markers due to limited variation in the first intron of β-tubulin gene family. We have now rectified this limitation by introducing the second intron of the β-tubulin genes as a valuable source of molecular markers. We show that the combined use of the two introns substantially increases the number of molecular markers and results in a reliable assessment of species/varieties relationships. After a preliminary validation on Brassica, this new combinatorial method was tested on species of Eleusine and Arachis. For both, reliable assessment of species relationships were obtained that were consistent with recently published studies resulting from more elaborated methods including DNA sequencing. Combinatorial TBP is a reliable, reproducible, simple, fast, and easy to score method that is very useful for breeding programs and species and variety assessments.     

<Emphasis Type="Italic">Aurifilum,</Emphasis> a new fungal genus in the Cryphonectriaceae from <Emphasis Type="Italic">Terminalia</Emphasis> species in Cameroon

Native Terminalia spp. in West Africa provide a popular source of construction timber as well as medical, spiritual and social benefits to rural populations. Very little is, however, known regarding the diseases that affect these trees. During an investigation into possible diseases of Terminalia spp. in Cameroon, orange to yellow fungal fruiting structures, resembling those of fungi in the Cryphonectriaceae, were commonly observed on the bark of native Terminalia ivorensis, and on dead branches of non-native Terminalia mantaly. In this study the fungus was identified based on morphological features as well as DNA sequence data (ITS and β-tubulin) and its pathogenicity was tested on T. mantaly seedlings. Our results showed that isolates of this fungus represent a previously undescribed genus in the Cryphonectriaceae, which we describe as Aurifilum marmelostoma gen. et sp. nov. Pathogenicity tests revealed that A. marmelostoma is pathogenic on T. mantaly. These tests, and the association of A. marmelostoma with disease symptoms on T. ivorensis, suggest that the fungus is a pathogen of this important tree.     

Analysis of beta-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products

The anti-cancer drug taxol binds to β-tubulin in assembled microtubules and causes cell cycle arrest in animal cells; in contrast, in fungi, the effect of taxol varies. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (IC₅₀ greater than 11.7 μM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC₅₀ 0.1 μM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding β-tubulin from both P. microspora and P. ultimum. The deduced amino acid sequence of β-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of β-tubulin from P. ultimum is very similar to those from Oomycetes and non-fungal organisms, many of which are sensitive to taxol. To examine the interaction between taxol and fungal microtubules, binding studies were performed with fungal cells, using [³H]taxol. The labeled taxol was found to bind specifically to P. ultimum, but not to P. microspora. In addition, the amount of [³H]taxol specifically bound to P. ultimum was reduced by the microtubule-depolymerizing drug thiabendazole, in a dose-dependent manner. These results suggest efficient binding of taxol to microtubules in P. ultimum, but not in P. microspora, and are consistent with the differential taxol sensitivity of these two organisms. Finally a comparison of previously characterized taxol binding sites in various β-tubulin sequences showed that β-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but β-tubulins of resistant organisms, including P. microspora, contain Asn or Gln at this position, suggesting an important role for residue 219 in the interaction between taxol and β-tubulin. Received: 16 March 1999 / Accepted: 21 August 1999     

An auxin-inducible gene from loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis> L.) is differentially expressed in mature and juvenile-phase shoots and encodes a putative transmembrane protein

We have isolated a gene from loblolly pine, 5NG4, that is highly and specifically induced by auxin in juvenile loblolly pine shoots prior to adventitious root formation, but substantially down-regulated in physiologically mature shoots that are adventitious rooting incompetent. 5NG4 was highly auxin-induced in roots, stems and hypocotyls, organs that can form either lateral or adventitious roots following an auxin treatment, but was not induced to the same level in needles and cotyledons, organs that do not form roots. The deduced amino acid sequence shows homology to the MtN21 nodulin gene from Medicago truncatula. The expression pattern of 5NG4 and its homology to a protein from Medicago involved in a root-related process suggest a possible role for this gene in adventitious root formation. Homology searches also identified similar proteins in Arabidopsis thaliana and Oryza sativa. High conservation across these evolutionarily distant species suggests essential functions in plant growth and development. A 38-member family of genes homologous to 5NG4 was identified in the A. thaliana genome. The physiological significance of this redundancy is most likely associated with functional divergence and/or expression specificity of the different family members. The exact biochemical function of the gene is still unknown, but sequence and structure predictions and 5NG4::GFP fusion protein localizations indicate it is a transmembrane protein with a possible transport function.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations ABA Abscisic acid - BA Benzylaminopurine - EST Expressed sequence tag - NAA 1-Naphthaleneacetic acid - GFP Green fluorescent protein - ORF Open reading frame     

The phylogenetic position of <Emphasis Type="Italic">Obolarina dryophila</Emphasis> (Xylariales)

The inconspicuous inner-bark parasite Obolarina dryophila is reported from wood of Quercus petraea and as an endophyte of Salix alba. In addition, viable ascospores of O. dryophila have been found in the gut of the oak bark weevil Gasterocercus depressirostris, suggesting a possible dissemination mechanism for the fungus. A phylogenetic analysis based on three genes (nrDNA, actin, β-tubulin) placed Obolarina inside the genus Biscogniauxia as a close relative of the oak pathogens B. atropunctata and B. mediterranea.     

DNA barcoding of the fungal genus <Emphasis Type="Italic">Neonectria</Emphasis> and the discovery of two new species

To determine a suitable DNA barcode for the genus Neonectria, the internal transcribed spacer rDNA, β-tubulin, EF-1α, and RPB2 genes were selected as candidate markers. A total of 205 sequences from 19 species of the genus were analyzed. Intra- and inter-specific divergences and the ease of nucleotide sequence acquisition were treated as criteria to evaluate the feasibility of a DNA barcode. Our results indicated that any single gene among the candidate markers failed to serve as a successful barcode, while the combination of the partial EF-1α, and RPB2 genes recognized all species tested. We tentatively propose the combined partial EF-1α and RPB2 genes as a DNA barcode for the genus. During this study, two cryptic species were discovered, based on the combined data of morphology and DNA barcode information. We described and named these two new species N. ditissimopsis and N. microconidia.     

Mapped DNA probes from loblolly pine can be used for restriction fragment length polymorphism mapping in other conifers

A high-density genetic map based on restriction fragment length polymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm. Thirty complementary DNA and two genomic DNA probes from loblolly pine were hybridized to Southern blots containing DNA from five species of Pinus (P. elliottii, P. lambertiana, P. radiata, P. sylvestris, and P. taeda), one species from each of four other genera of Pinaceae (Abies concolor, Larix laricina, Picea abies, and Pseudotsuga menziesii), one species from each of three other families of Coniferales [Sequoia sempervirens (Taxodiaceae), Torreya californica (Taxaceae) and Calocedrus decurrens (Cupressaceae)], and to one angiosperm species (Populus nigra). Results showed that mapped DNA probes from lobolly pine will cross-hybridize to genomic DNA of other species of Pinus and some other genera of the Pinaceae. Only a small proportion of the probes hybridized to genomic DNA from three other families of the Coniferales and the one angiosperm examined. This study demonstrates that mapped DNA probes from loblolly pine can be used to construct RFLP maps for related species, thus enabling the opportunity for comparative genome mapping in conifers.     

Genetic diversity within and among populations of shortleaf pine (Pinus echinata Mill.) and loblolly pine (Pinus taeda L.)

Shortleaf pine (n = 93) and loblolly pine (n = 112) trees representing 22 seed sources or 16 physiographic populations were sampled from Southwide Southern Pine Seed Source Study plantings located in Oklahoma, Arkansas, and Mississippi. The sampled trees were grown from shortleaf pine and loblolly pine seeds formed in 1951 and 1952, prior to the start of intensive forest management across their native ranges. Amplification fragment length polymorphism (AFLP) markers were developed and used to study genetic diversity and its structure in these pine species. After screening 48 primer pairs, 17 and 21 pairs were selected that produced 794 and 647 AFLPs in shortleaf pine and loblolly pine, respectively. High-AFLP-based genetic diversity exists within shortleaf pine and loblolly pine, and most (84.73% in shortleaf pine; 87.69% in loblolly pine) of this diversity is maintained within physiographic populations. The high value of unbiased measures of genetic identity and low value of genetic distance for all pairwise comparisons indicates that the populations have similar genetic structures. For shortleaf pine, there was no significant correlation between geographic distance and genetic distance (r = 0.28), while for loblolly pine there was a weak but significant correlation (r = 0.51).     

Molecular analysis of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> isolated from Brazil nuts

Brazil nuts are an important export market in its main producing countries, including Brazil, Bolivia, and Peru. Approximately 30,000 tons of Brazil nuts are harvested each year. However, substantial nut contamination by Aspergillus section Flavi occurs with subsequent production of aflatoxins. In our study, Aspergillus section Flavi were isolated from Brazil nuts (Bertholletia excelsa), and identified by morphological and molecular means. We obtained 241 isolates from nut samples, 41% positive for aflatoxin production. Eighty-one isolates were selected for molecular investigation. Pairwise genetic distances among isolates and phylogenetic relationships were assessed. The following Aspergillus species were identified: A. flavus, A. caelatus, A. nomius, A. tamarii, A. bombycis, and A. arachidicola. Additionally, molecular profiles indicated a high level of nucleotide variation within β-tubulin and calmodulin gene sequences associated with high genetic divergence from RAPD data. Among the 81 isolates analyzed by molecular means, three of them were phylogenetically distinct from all other isolates representing the six species of section Flavi. A putative novel species was identified based on molecular profiles.     

Whole‐exome targeted sequencing of the uncharacterized pine genome

The large genome size of many species hinders the development and application of genomic tools to study them. For instance, loblolly pine (Pinus taeda L.), an ecologically and economically important conifer, has a large and yet uncharacterized genome of 21.7 Gbp. To characterize the pine genome, we performed exome capture and sequencing of 14 729 genes derived from an assembly of expressed sequence tags. Efficiency of sequence capture was evaluated and shown to be similar across samples with increasing levels of complexity, including haploid cDNA, haploid genomic DNA and diploid genomic DNA. However, this efficiency was severely reduced for probes that overlapped multiple exons, presumably because intron sequences hindered probe:exon hybridizations. Such regions could not be entirely avoided during probe design, because of the lack of a reference sequence. To improve the throughput and reduce the cost of sequence capture, a method to multiplex the analysis of up to eight samples was developed. Sequence data showed that multiplexed capture was reproducible among 24 haploid samples, and can be applied for high‐throughput analysis of targeted genes in large populations. Captured sequences were de novo assembled, resulting in 11 396 expanded and annotated gene models, significantly improving the knowledge about the pine gene space. Interspecific capture was also evaluated with over 98% of all probes designed from P. taeda that were efficient in sequence capture, were also suitable for analysis of the related species Pinus elliottii Engelm.     

Protein adaptation to low temperatures: a comparative study of α-tubulin sequences in mesophilic and psychrophilic algae

The α-tubulin genes from two psychrophilic algae belonging to the genus Chloromonas (here named ANT1 and ANT3) have been isolated and sequenced. The genes ant1 and ant3 contain 4 and 2 introns, respectively. The coding DNA sequences are 90% identical but the degree of isology is very high at the polypeptide level (more than 97% strict identities). The ANT1 and ANT3 α-tubulin amino acid sequences were compared to the corresponding sequence of the mesophilic alga Chlamydomonas reinhardtii. Of the 15 substitutions detected in ANT1 and/or ANT3, 5 are common to both psychrophilic algae. The recorded substitutions have been analyzed in terms of cold adaptation on the basis of the available three-dimensional structure of the α,β-tubulin heterodimer from pig brain. Most of these are subtle changes, but two substitutions, M268V and A295V occurring in the region of interdimer contacts, could be of great significance for the cold stability of Antarctic algae microtubules due to the fact that the entropic control of microtubule assembly is particularly high in cold adaptes species. Received: December 24, 1998 / Accepted: April 2, 1999     

Cloning and sequence analysis of the plasmid DNA found inRhizoctonia solani AG-2-2 LP isolate and its potential use for fungal detection

A plasmid DNA (PE-42 plasmid) obtained fromRhizoctonia solani AG-2-2 LP isolate PE-42, the causal agent of large patch disease of zoysiagrass (Zoysia spp.), was partially cloned. Sequence analyses of the 1.2-kb and 0.2-kb cloned fragments revealed that the nucleotide sequence of the 0.2-kb fragment was similar to that of the 5′ region of the 1.2-kb fragment (pSH4). Southern hybridization analysis of total DNA of a large patch isolate using the 1.2-kb fragment as a probe showed two bands differing slightly in size. These results indicated that the PE-42 plasmid consisted of at least two components having similar nucleotide sequences with different sizes. The nucleotide sequence of the pSH4 fragment showed no significant homology with known DNA sequences. The pSH4 fragment hybridized to all of the 22 large patch isolates tested, but not to other subgroup isolates in AG-2-2, other anastomosis groups ofR. solani, or other pathogens of zoysiagrass. These results indicated that the pSH4 fragment can be used as a specific probe to detect the large patch fungus. The detection limit for the large patch fungus using the pSH4 fragment as a probe was 0.1 μg of the total DNA of the fungus, which was significantly higher than those for other fungi. However, with improvement of the detection sensitivity and simplification of the detection procedure, the pSH4 fragment has potential for use in molecular diagnosis of the large patch disease of zoysiagrass. Contribution No. 140 from the Laboratory of Plant Pathology, Mie University.