Sergey Bulat

UP-PCR analysis and ITS1 ribotyping of strains of Trichoderma and Gliocladium

Universally primed PCR (UP-PCR) fingerprinting combined with UP-PCR product cross hybridization, and ITS1 ribotyping were used to study the genetic relatedness of strains of Trichoderma and Gliocladium for two purposes: (1) to evaluate the ability of the methods to discriminate closely related strains and as tools to group strains which is necessary to facilitate; (2) identification of markers for development of specific detection assays for selected strains. Included among the strains were one T. harzianum, two T. virens, and one G. roseum that had been selected previously for their antagonistic ability against soil-borne phytopathogens. Similarity among strains, found by cross dot blot hybridization using UP-PCR amplification products, was used to group them into 15 genetic entities. ITS1 ribotyping of the strains was performed by digestion of the PCR amplified rDNA spacer region and electrophoresis of the products. The differences obtained from ribotyping as well as the differences in mobility of the intact spacer region were used for grouping of the strains. The UP-PCR hybridization groups and the ITS1 based groups proved to be consistent, but the resolution of the UP-PCR based approach was superior. The results demonstrate that the combination of UP-PCR and ribotyping can aid in clarifying species distinction in Trichoderma and Gliocladium and has the potential to become a valuable tool for studies of diversity and genetic structure of populations of these fungi. Furthermore, identification of single strains by the specific UP-PCR fingerprint seems feasible.

DNA signature of thermophilic bacteria from the aged accretion ice of Lake Vostok, Antarctica: implications for searching for life in extreme icy environments

We have used 16S ribosomal genes to estimate the bacterial contents of Lake Vostok accretion ice samples at 3551 m and 3607 m, both containing sediment inclusions and formed 20000–15000 yr ago. Decontamination proved to be a critical issue, and we used stringent ice chemistry-based procedures and comprehensive biological controls in order to restrain contamination. As a result, up to now we have only recognized one 16S rDNA bacterial phylotype with confident relevance to the lake environment. It was found in one sample at 3607 m depth and represents the extant thermophilic facultative chemolithoautotroph Hydrogenophilus thermoluteolus of beta- Proteobacteria , and until now had only been found in hot springs. No confident findings were detected in the sample at 3551 m, and all other phylotypes revealed (a total of 16 phylotypes, 336 clones including controls) are presumed to be contaminants. It seems that the Lake Vostok accretion ice is actually microbe-free, indicating that the water body should also be hosting a highly sparse life. The message of thermophilic bacteria suggests that a geothermal system exists beneath the cold water body of Lake Vostok, what is supported by the geological setting, the long-term seismotectonic evidence from 4 He degassing and the ‘ 18 O shift’ of the Vostok accretion ice. The seismotectonic activity that seems to operate in deep faults beneath the lake could sustain thermophilic chemolithoautotrophic microbial communities. Such a primary production scenario for Lake Vostok may have relevance for icy planets and the approaches used for estimating microbial contents in accretion ice are clearly relevant for searching for extraterrestrial life.

Identification of a Universally Primed-PCR-Derived Sequence-Characterized Amplified Region Marker for an Antagonistic Strain of Clonostachys rosea and Development of a Strain-Specific PCR Detection Assay

ABSTRACT We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.

Molecular, morphological and phylogenetic analysis of the Fusarium avenaceum/F. arthrosporioides/F. tricinctum species complex – a polyphasic approach

Differences in morphology, ITS, IGS, mtSSU and β-tubulin sequences and UP-PCR hybridization were compared between morphologically identified F. avenaceum, F. arthrosporioides, F. anguioides, F. tricinctum, F. graminum and F. acuminatum strains. According to the combined β-tubulin, IGS and ITS tree, the strains of the Fusarium avenaceum/F. arthrosporioides/F. tricinctum species complex species can be divided into seven clusters supported by bootstrap values higher than 50%. The two main groups of European F. avenaceum , which cannot be distinguished by morphology, were separated in the tree based on β-tubulin sequences and less clearly in trees based on IGS and ITS sequences. MtSSU sequences were identical in all F. avenaceum and F. tricinctum strains studied. The European F. avenaceum strains of main group II had identical β-tubulin sequences with one American F. avenaceum strain and four European F. arthrosporioides strains, while F. avenaceum strains of main group I were closely related to two European F. arthrosporioides strains and to one Japanese F. anguioides strain. According to the combined β-tubulin/IGS/ITS sequence tree, European F. arthrosporioides strains were divided into four groups; F. tricinctum strains formed a wellsupported cluster, in which two European clusters were separated from one African isolate. In the IGS sequence tree two European F. acuminatum strains together with one American F. acuminatum strain formed a cluster, which was separate from another American F. acuminatum strain. The F. acuminatum cluster was nested within the large F. tricinctum cluster together with one F. reticulatum strain in the combined IGS/β-tubulin tree. Several strains may be intermediates between the F. avenaceum/F. arthrosporioides/F. anguioides and F. tricinctum clusters and represent their own species. These results are partially supported by the results of UP-PCR hybridization analysis. Thus the molecular results may be helpful in future revision in the taxonomy of these species.

Delineation of Trichoderma harzianum into two different genotypic groups by a highly robust fingerprinting method, UP-PCR, and UP-PCR product cross-hybridization

Strains of Trichoderma harzianum possess biocontrol capabilities. As background for identification of strain-specific markers for monitoring strains of interest, the relationship of strains designated as T. harzianum , including representatives of three biological forms Th1, Th2 and Th3, were analysed by Universally Primed PCR, UP-PCR, using UP and random primers. Cross dot blot hybridization of UP-PCR products generated with either of two different UP primers or a random primer showed unequivocal differences among strains. Using this approach, T. harzianum strains were distributed into two different genotypic groups. One of the T. harzianum groups included forms Th1 and Th2 while the other group accounted for the Th3 form. The relatedness of strains of each group was estimated by UPGMA analysis based on markers revealed with three primers. It was found that both genotypic groups are heterogeneous, and Th2 form strains definitely cluster together with those of Th1. Division of the three biological forms of T. harzianum into two groups was also supported by rDNA-ITS1 analysis, where Sau 3A digestion of the amplified ITS1 region gave a restriction fragment profile specific for each genotypic group. Two strains with known biocontrol capabilities were found to relate to the genotypic group containing Th1 and Th2 forms and, based on variation within this group, to belong to a homogeneous group of form Th1 strains. The robustness and reliability of UP-PCR fingerprinting were demonstrated by obtaining identical banding profiles using different conditions for PCR in different laboratories.

An International Plan for Antarctic Subglacial Lake Exploration

Discovery of at least 100 subglacial lakes beneath the vast East Antarctic Ice Sheet has focused international attention on the challenges presented by the way we conduct science in such unique and inhospitable settings in an atmosphere of increasingly stringent environmental concerns. Exploration of subglacial environments will require careful and detailed planning, organization, and international cooperation. To this end, the Scientific Committee on Antarctic Research (SCAR) convened an international Group of Specialists (Subglacial Antarctic Lake Exploration Group of Specialists—SALEGOS) to develop a detailed assessment of the needs and critical milestones to be accomplished during the implementation of a subglacial exploration and research program. This paper surveys the progress and recommendations made by SALEGOS since its inception regarding the current state of knowledge of subglacial environments, technological needs and challenges, international management, the portfolio of scientific projects, and “clean” requirements for entry, observatory deployment, and sample retrieval.

RAPD-PCR, isozyme, rDNA RFLP and rDNA sequence analyses in identification of Finnish Fusarium oxysporum isolates

Twenty-seven Fusarium oxysporum isolates were studied by RAPD-PCR and isozyme analyses. Thirteen isolates were from barley and the rest from different hosts, most of which were dicotyledonous plants. All isolates could be distinguished from each other by RAPD-PCR analysis, and clustered into seven groups in NJ and parsimony consensus trees. Isozyme analysis detected polymorphism in five of the six enzymes and the isolates could be divided into 26 different electrophoretic groups. Five groups were supported by high branch support and bootstrap values in the approximate support tree of combined RAPD-PCR and isozyme data. These five groups were found also in NJ and parsimony consensus trees. The matrices from RAPD-PCR and isozyme data proved to be incongruent, but they did not totally disprove each other. Some correlation was found between geographical origin and phylogenetic relationships of isolates collected from barley. Representatives of the main clades of phylogenetic trees, were further studied by rDNA RFLP and rDNA sequence analyses, together with isolates of other Fusarium species. Isolates of F. oxysporum and F. avenaceum formed distinct groups in the phylogenetic analyses, except for two isolates of F. oxysporum which were grouped with isolates of F. redolens.

Quantification of Dissolved Organic Carbon at Very Low Levels in Natural Ice Samples by a UV-Induced Oxidation Method

The study of chemical impurities trapped in solid precipitation and accumulated in polar ice sheets and high-elevation, midlatitude cold glaciers over the last several hundreds of years provides a unique way to reconstruct our changing atmosphere from the preindustrial era to the present day. Numerous ice core studies of inorganic species have already evaluated the effects of growing anthropogenic emissions of SO(2) or NO(x) on the chemical composition of the atmosphere in various regions of the world. While it was recently shown that organic species dominate the atmospheric aerosol mass, the contribution of anthropogenic emissions to their budget remains poorly understood. The study of organics in ice is at the infancy stage, and it still is difficult to draw a consistent picture of the organic content of polar ice from sparse available data. A UV oxidation method and IR quantification of CO(2) was optimized to obtain measurements of dissolved organic carbon content as low as a few ppbC. Stringent working conditions were defined to prevent contamination during the cleaning of ice. Measurements in various ice cores corresponding to preindustrial times revealed dissolved organic carbon content of less than 10 ppbC in Antarctica and up to 75 ppbC in alpine ice.

Exploring subglacial Antarctic lake environments

While subglacial lakes have been suspected, and speculated about, for more than 50 years, recent analyses of historical and new data have shown that liquid water environments are common beneath the vast Antarctic Ice Sheet. Airborne radar surveys have now documented more than 145 subglacial lakes, the largest being Lake Vostok located 4 km beneath the vast East Antarctic Ice Sheet (Figure 1). The public and scientists alike have been intrigued by the possibility that these environments harbor life in conditions not previously studied on our planet. Planning for the exploration and study of these unique environments has focused international attention on the challenges presented by the way science is conducted in such settings while providing for environmental protection and stewardship. Exploration of subglacial environments will require careful and detailed planning, adoption of environmental protocols, and international cooperation.

Microbiology of the subglacial Lake Vostok: first results of borehole-frozen lake water analysis and prospects for searching for lake inhabitants.

This article examines the question of the possible existence of microbial life inhabiting the subglacial Lake Vostok buried beneath a 4 km thick Antarctic ice sheet. It represents the results of analysis of the only available frozen lake water samples obtained upon the first lake entry and subsequent re-coring the water frozen within the borehole. For comparison, results obtained by earlier molecular microbiological studies of accretion ice are included in this study, with the focus on thermophiles and an unknown bacterial phylotype. A description of two Lake Vostok penetrations is presented for the first time from the point of view of possible clean water sampling. Finally, the results of current studies of Lake Vostok frozen water samples are presented, with the focus on the discovery of another unknown bacterial phylotype w123-10 distantly related to the above-mentioned unknown phylotype AF532061 detected in Vostok accretion ice, both successfully passing all possible controls for contamination. The use of clean-room facilities and the establishment of a contaminant library are considered to be prerequisites for research on microorganisms from Lake Vostok. It seems that not yet recorded microbial life could exist within the Lake Vostok water body. In conclusion, the prospects for searching for lake inhabitants are expressed with the intention to sample the lake water as cleanly as possible in order to make sure that further results will be robust.