John D. Castello

Development of a multiplex immunocapture RT-PCR assay for detection and differentiation of tomato and tobacco mosaic tobamoviruses.

Immunocapture (IC) RT-PCR assays were developed for detection of tomato (ToMV) and tobacco mosaic (TMV) tobamoviruses in spruce and pine extracts. When purified viruses were diluted in root or needle extracts of virus-free conifer seedlings, both IC-RT-PCR assays detected their respective target viruses at concentrations of 10-100 fg ml(-1). This compared to ELISA detection sensitivities of 1 ng ml(-1). Primers were designed from regions of high sequence diversity. Specificity of all primer pairs was confirmed by sequencing of PCR products. PCR distinguished more reliably between the two viruses than ELISA. Moreover, a multiplex IC-RT-PCR assay for the simultaneous detection and differentiation of TMV and ToMV was developed. When root extracts were seeded with both viruses simultaneously, the multiplex assay detected each virus at concentrations of 1-10 pg ml(-1). Six TMV and 18 ToMV isolates from various hosts, water samples and a soil sample were amplified and differentiated by multiplex IC-RT-PCR. No amplifications were observed against pepper mild mottle and ribgrass mosaic tobamoviruses and against six viruses belonging to other taxonomic groups.

Life in ancient ice

List of Figures ix List of Tables xiii Contributors xv Acknowledgments xix CHAPTER 1: Introduction by John D. Castello and Scott O. Rogers 1 CHAPTER 2: Recommendations for Elimination of Contaminants and Authentication of Isolates in Ancient Ice Cores by Scott O. Rogers, Li-Jun Ma, Yinghao Zhao, Vincent Theraisnathan, Seung-Geuk Shin, Gang Zhang, Catherine M. Catranis, William T. Starmer, and John D. Castello 5 CHAPTER 3: Perennial Antarctic Lake Ice: A Refuge for Cyanobacteria in an Extreme Environment by John C. Priscu, Edward E. Adams, Hans W. Paerl, Christian H. Fritsen, John E. Dore, John T. Lisle, Craig F. Wolf, and Jill A. Mikucki 22 CHAPTER 4: The Growth of Prokaryotes in Antarctic Sea Ice: Implications for Ancient Ice Communities by David S. Nichols 50 CHAPTER 5: Frozen in Time: The Diatom Record in Ice Cores from Remote Drilling Sites on the Antarctic Ice Sheets by Davida E. Kellogg and Thomas B. Kellogg 69 CHAPTER 6: The Nature and Likely Sources of Biogenic Particles Found in Ancient Ice from Antarctica by Raymond Sambrotto and Lloyd Burckle 94 CHAPTER 7: Microbial Life below the Freezing Point within Permafrost by Elizaveta Rivkina, Kayastas Laurinavichyus, and David A. Gilichinsky 106 CHAPTER 8: Yeasts Isolated from Ancient Permafrost 118 by Rushaniya N. Faizutdinova, Nataliya E. Suzina, Vitalyi I. Duda, Lada E. Petrovskaya, and David A. Gilichinsky 118 CHAPTER 9: Fungi in Ancient Permafrost Sediments of the Arctic and Antarctic Regions by Nataliya E. Ivanushkina, Galina A. Kochkina, and Svetlana M. Ozerskaya 127 CHAPTER 10: Viable Phototrophs: Cyanobacteria and Green Algae from the Permafrost Darkness by Tatiana A. Vishnivetskaya, Ludmila G. Erokhina, Elena V. Spirina, Anastasia V. Shatilovich, Elena A. Vorobyova, Alexander I. Tsapin, and David A. Gilichinsky 140 CHAPTER 11: The Significance and Implications of the Discovery of Filamentous Fungi in Glacial Ice by Li-Jun Ma, Catherine M. Catranis, William T. Starmer, and Scott O. Rogers 159 CHAPTER 12: Yeasts in the Genus Rhodotorula Recovered from the Greenland Ice Sheet by William T. Starmer, Jack W. Fell, Catherine M. Catranis, Virginia Aberdeen, Li-Jun Ma, Shuang Zhou, and Scott O. Rogers 181 CHAPTER 13: Plant and Bacterial Viruses in the Greenland Ice Sheet by John D. Castello, Scott O. Rogers, James E. Smith, William T. Starmer, and Yinghao Zhao 196 CHAPTER 14: Viral Pathogens of Humans Likely to Be Preserved in Natural Ice by Dany Shoham 208 CHAPTER 15: Classification of Bacteria from Polar and Nonpolar Glacial Ice by Brent C. Christner, Ellen Mosley-Thompson, Lonnie G. Thompson, and John N. Reeve 227 CHAPTER 16: Common Features of Microorganisms in Ancient Layers of the Antarctic Ice Sheet by S.S. Abyzov, M.N. Poglazova, J.N. Mitskevich, and M.V. Ivanov 240 CHAPTER 17: Comparative Biological Analyses of Accretion Ice from Subglacial Lake Vostok by Robin Bell, Michael Studinger, Anahita Tikku, and John D. Castello 251 CHAPTER 18: Search for Microbes and Biogenic Compounds in Polar Ice Using Fluorescence by Ryan Bay, Nathan Bramall, and P. Buford Price 268 CHAPTER 19: Living Cells in Permafrost as Models for Astrobiology Research by Elena A. Vorobyova, V.S. Soina, A.G. Mamukelashvili, A. Bolshakova, I.V. Yaminsky, and A.L. Mulyukin 277 CHAPTER 20: A Synopsis of the Past, an Evaluation of the Current, and a Glance toward the Future by John D. Castello and Scott O. Rogers 289 Index 301

Evidence of Influenza A Virus RNA in Siberian Lake Ice

ABSTRACT Influenza A virus infects a large proportion of the human population annually, sometimes leading to the deaths of millions. The biotic cycles of infection are well characterized in the literature, including in studies of populations of humans, poultry, swine, and migratory waterfowl. However, there are few studies of abiotic reservoirs for this virus. Here, we report the preservation of influenza A virus genes in ice and water from high-latitude lakes that are visited by large numbers of migratory birds. The lakes are along the migratory flight paths of birds flying into Asia, North America, Europe, and Africa. The data suggest that influenza A virus, deposited as the birds begin their autumn migration, can be preserved in lake ice. As birds return in the spring, the ice melts, releasing the viruses. Therefore, temporal gene flow is facilitated between the viruses shed during the previous year and the viruses newly acquired by birds during winter months spent in the south. Above the Arctic Circle, the cycles of entrapment in the ice and release by melting can be variable in length, because some ice persists for several years, decades, or longer. This type of temporal gene flow might be a feature common to viruses that can survive entrapment in environmental ice and snow.

Detection of tomato mosaic tobamovirus RNA in ancient glacial ice

Abstract Tomato mosaic tobamovirus is a very stable plant virus with a wide host range, which has been detected in plants, soil, water, and clouds. Because of its stability and prevalence in the environment, we hypothesized that it might be preserved in ancient ice. We detected tomato mosaic tobamovirus RNA by reverse-transcription polymerase chain reaction amplification in glacial ice subcores <500 to approximately 140,000 years old from drill sites in Greenland. Subcores that contained multiple tomato mosaic tobamovirus genotypes suggest diverse atmospheric origins of the virus, whereas those containing tomato mosaic tobamovirus sequences nearly identical to contemporary ones suggest that recent tomato mosaic tobamovirus populations have an extended age structure. Detection of tomato mosaic tobamovirus in ice raises the possibilities that stable viruses of humans and other hosts might be preserved there, and that entrapped ancient viable viruses may be continually or intermittently released into the modern environment.

Comparisons of protocols for decontamination of environmental ice samples for biological and molecular examinations

ABSTRACT Drilling and laboratory manipulations of glacial ice cores introduce contemporary microbes and biomolecules onto the cores. We report herein a systematic comparative study of several decontamination protocols. Only treatment with 5% sodium hypochlorite eliminated all external contaminating microbes and nucleic acids while maintaining the integrity of those within the cores.

Quantitative, Nondestructive Assessment of Beech Scale (Hemiptera: Cryptococcidae) Density Using Digital Image Analysis of Wax Masses

ABSTRACT Beech scale, Cryptococcus fagisuga Lindinger, is a non-native invasive insect associated with beech bark disease. A quantitative method of measuring viable scale density at the levels of the individual tree and localized bark patches was developed. Bark patches (10 cm2) were removed at 0, 1, and 2 m above the ground and at the four cardinal directions from 13 trees in northern New York and 12 trees in northern Michigan. Digital photographs of each patch were made, and the wax mass area was measured from two random 1-cm2 subsamples on each bark patch using image analysis software. Viable scale insects were counted after removing the wax under a dissecting microscope. Separate regression analyses at the whole tree level for the New York and Michigan sites each showed a strong positive relationship of wax mass area with the number of underlying viable scale insects. The relationships for the New York and Michigan data were not significantly different from each other, and when pooling data from the two sites, there was still a significant positive relationship between wax mass area and the number of scale insects. The relationships between viable scale insects and wax mass area were different at the 0-, 1-, and 2-m sampling heights but do not seem to affect the relationship. This method does not disrupt the insect or its interactions with the host tree.

Forest health : an integrated perspective

Preface Part I. Forest Health and Mortality: 1. The past as key to the future: a new perspective on forest health S. A. Teale and J. D. Castello 2. Mortality: the essence of a healthy forest L. Zhang, B. D. Rubin and P. D. Manion 3. How do we do it, and what does it mean?: forest health case studies J. D. Castello, S. A. Teale and J. A. Cale Part II. Forest Health and its Ecological Components: 4. Regulators and terminators: the importance of biotic factors to a healthy forest S. A. Teale and J. D. Castello 5. Alien invasions: the effects of introduced species on forest structure and function D. Parry and S. A. Teale 6. Out of sight, underground: forest health, edaphic factors, and mycorrhizae R. D. Briggs and T. R. Horton 7. Earth, wind, and fire: abiotic factors and the impacts of global environmental change on forest health J. E. Lundquist, A. E. Camp, M. L. Tyrrell, S. J. Seybold, P. Cannon and D. J. Lodge Part III. Forest Health and the Human Dimension: 8. Silviculture, forest management, and forest health: an axe does not a forester make C. A. Nowak, R. H. Germain and A. P. Drew 9. Biodiversity, conservation, and sustainable timber harvest: can we have it all? S. P. Campbell, D. A. Patrick and J. P. Gibbs 10. Seeing the forest for the trees: forest health monitoring M. Fierke, D. Nowak and R. Hofstetter 11. What did we learn, and where does it leave us?: concluding thoughts J. D. Castello and S. A. Teale Appendix A. Microsoft Excel instructions for Chapter 2 Appendix B. Microsoft Excel instructions for Chapter 3 Appendix C. Glossary of terms Index.

Detection and Partial Characterization of Tenuiviruses from Black Spruce

Filamentous viral ribonucleoproteins (RNPs) 12 to 16 nm in diameter and 100 to 1,260 nm in length, and characteristic of the genus Tenuivirus, were detected by transmission electron microscopy in purified extracts of needles collected from two mature, asymptomatic black spruce (Picea mariana) trees in New York, but not in extracts of needles from nursery seedlings. Purified RNPs from one tree had a buoyant density in CsCl = 1.39 g/cm3 and an A 260/280 = 1.436. Four ssRNA segments of 1.3, 2.1, 2.3, and 3.5 kb, but not the 8- to 9-kb fragment characteristic of most tenuiviruses, were detected in purified RNA extracts. Amplification products of the expected size were observed when RNA extracts from the two spruce trees and Maize stripe tenuivirus (MStpV), but not from tobacco, Chenopodium quinoa, or spruce seedlings were subjected to reverse transcription-polymerase chain reaction (RT-PCR) using primers to the p3 open reading frame (ORF) of MStpV vRNA 3. However, only MStpV amplified when primers to the nucleocapsid ORF (pc3 ORF on vcRNA 3) were used. Similarly, only MStpV amplified by immunocapture polymerase chain reaction (PCR) using antiserum to MStpV and primers to the p3 ORF. Sequence comparisons suggest that two distinct tenuiviruses occur in black spruce, one more closely related to MStpV than the other. One of these tenuiviruses was detected in one of 10 additional black spruce trees tested, but not in trees of six other coniferous species sampled in the Adirondack Mountains of New York.

Chapter 3. Perennial Antarctic Lake Ice: A Refuge for Cyanobacteria in an Extreme Environment

John C. Priscu, Edward E. Adams, Hans W. Paerl, Christian H. Fritsen, John E. Dore, John T. Lisle, Craig F. Wolf and Jill A. Mikucki Addresses: John C. Priscu, Craig F. Wolf, Jill A. Mikucki. Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, Montana 59717 Edward E. Adams. Department of Civil Engineering, Montana State University, Bozeman, Montana 59717 Hans W. Paerl, Institute of Marine Sciences, University of North Carolina at Chapel Hill, 3431 Arendell Street, Morehead City, North Carolina 28557 Christian H. Fritsen, Division of Earth and Ecosystem Science, Desert Research Institute, 2215 Raggio Parkway, Reno Nevada 89512 John E. Dore, SOEST, Department of Oceanography, University of Hawaii, 1000 Pope Road, Honolulu, Hawaii 96822 John T. Lisle, U.S.Geological Survey, Center for Coastal & Regional Marine Studies 600 4th Street-South, St. Petersburg, FL 33701

Chapter 15. Classification of Bacteria from Polar and Nonpolar Glacial Ice

SNOWFALL ACCUMULATES as glacial ice at both poles and globally at high altitudes in nonpolar regions. Archived chronologically within these glaciers are samples of the atmospheric constituents at the time of snow deposition, including particulates of inorganic and biological origin deposited originally on the surface of the snow, often by attachment to snowflakes. Studies of ice cores have established past climate changes and geological events, both globally and regionally, but rarely have these results been correlated with the insects, plant fragments, seeds, pollen grains , fungal spores, and bacteria, that also are present, and very few attempts have been made to determine the diversity and longevity of viable species entombed in such glacial ice. Fungi, algae, protists, bacteria, and viruses have been detected and recovered from polar ice cores (1, 2, 3, 10, 15, 41), but there are very few similar reports describing the microorganisms preserved in nonpolar glacial ice of different age and from different locations. Fortunately, for such studies, we have access to ice cores archived at the Byrd Polar Research Center (BPRC) at the Ohio State University. These ice cores have been collected over many years from globally distributed sites, and many have already been subjected to extensive physical and chemical analyses. These, therefore, provide the opportunity to isolate and to characterize microorganisms from glacial ice formed at defined dates, under known climate conditions, at geographically very different locations (Figure 15.1). To avoid problems of surface contamination, we constructed an ice core sampling system that melts the ice and collects only the resulting interior core meltwater. Here we review the results of bacterial isolations from meltwater generated using this system from the interiors of nonpolar and polar glacial ice cores of different vintage, and from Lake Vostok accretion ice ( 12, 14 ).