Mark T. Windham

Two-year field analysis of reduced recalcitrance transgenic switchgrass.

Switchgrass (Panicum virgatum L.) is a leading candidate for a dedicated lignocellulosic biofuel feedstock owing to its high biomass production, wide adaptation and low agronomic input requirements. Lignin in cell walls of switchgrass, and other lignocellulosic feedstocks, severely limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars and subsequently biofuels. Low-lignin transgenic switchgrass plants produced by the down-regulation of caffeic acid O-methyltransferase (COMT), a lignin biosynthetic enzyme, were analysed in the field for two growing seasons. COMT transcript abundance, lignin content and the syringyl/guaiacyl lignin monomer ratio were consistently lower in the COMT-down-regulated plants throughout the duration of the field trial. In general, analyses with fully established plants harvested during the second growing season produced results that were similar to those observed in previous greenhouse studies with these plants. Sugar release was improved by up to 34% and ethanol yield by up to 28% in the transgenic lines relative to controls. Additionally, these results were obtained using senesced plant material harvested at the end of the growing season, compared with the young, green tissue that was used in the greenhouse experiments. Another important finding was that transgenic plants were not more susceptible to rust (Puccinia emaculata). The results of this study suggest that lignin down-regulation in switchgrass can confer real-world improvements in biofuel yield without negative consequences to biomass yield or disease susceptibility.

DNA amplification fingerprinting provides evidence that Discula destructiva, the cause of dogwood anthracnose in North America, is an introduced pathogen

DNA amplification fingerprinting was used to characterize 28 isolates of Discula destructiva and three isolates of an undescribed species of Discula. These filamentous fungi cause anthracnose of various species of dogwood (Cornus). Isolates were obtained from throughout the disease range in the eastern and western United States and western Canada and DNA

Plant Pathology Concepts and Laboratory Exercises

Introductory Concepts Plant Pathology and Historical Perspectives, M.T. Windham and A.S. Windham What Is A Disease? M.T. Windham and A.S. Windham Introduction to the Groups of Plant Pathogens, M.T. Windham Groups of Plant Pathogens Plant Pathogenic Viruses, M.A.C. Langham Mechanical Inoculation of Plant Viruses, M.A.C. Langham Plant-Parasitic Nematodes, J.P. Noe Pathogenicity and Isolation of Plant Parasitic Nematodes, J.P. Noe Plant Pathogenic Fungi and Fungal-like Organisms, A. Brooks Gould Slime Molds and Zoosporic Fungi, S.E. Mozley-Standridge, D. Porter, and M.A. Cubeta Laboratory Exercises with Zoosporic Plant Pathogens, M.A. Cubeta, D. Porter, and S.E. Mozley-Standridge Archiascomycete and Hemiascomycete Pathogens, M. L. Daughtrey, K. T. Hodge, and N. Shishkoff The Powdery Mildews, M.L. Daughtrey, K.T. Hodge, and N. Shishkoff Ascomycota: Pyrenomycetes, Discomycetes, and Loculoascomycetes, K.J. Curry and R.E. Baird Deuteromycota: An Artificial Assemblage of Asexually Reproducing Fungi, R.E. Baird Laboratory Exercises with Selected Asexually Reproducing Fungi, R.E. Baird Smut and Rust Diseases, L.J. Littlefield, Y.H. Li, and D. Hensley Basidiomycota: Fleshy Mushrooms and Other Important and Symbiotic Associations, R.E. Baird Oomycota: The Fungi-like Organisms, R.N. Trigiano, M.H. Ament, and K.H. Lamour Laboratory Exercises with the Oomycetes, R.N. Trigiano, R. E. Baird, and S. N. Jeffers Soilborne Plant Pathogens, B.H. Ownley and D. Michael Benson Laboratory Exercises with Soilborne Plant Pathogens, D M. Benson and B.H. Ownley Parasitic Seed Plants, Protozoa, Algae, and Mosses, M.T. Windham and A.S. Windham Abiotic Diseases, A.S. Windham and M.T. Windham Molecular Tools for Studying Plant Pathogens Molecular Tools for Studying Plant Pathogens, T.A. Rinehart, X.W. Wang, and R.N. Trigiano Molecular Techniques Used for Studying Systematics and Phylogeny of Plant Pathogens, R.N. Trigiano, M.H. Ament, S.L. Finley, R.E. DeVries, N.R. Rowland, and G. Caetano-Anolles Plant-Pathogen Interactions Plant-Fungal Interactions at the Molecular Level, R.B. Ferreira, S. Monteiro, R. Freitas, C.N. Santos, Z. Chen, L.M. Batista, J. Duarte, A. Borges, and A.R. Teixeira Testing Blad, a Potent Antifungal Polypeptide, S. Monteiro and R. Boavida Ferreira Detecting and Measuring Extracellular Enzymes Produced by Fungi and Bacteria, R.N. Trigiano and M.H. Ament Host Defenses: A Physical and Physiological Approach, K.D. Gwinn, S.E. Greene, J.F. Green, and D. Trently Disruption of Plant Function, M.B. Riley Epidemiology and Disease Control Plant Disease Epidemiology, K.L. Bowen Host Resistance, J.K. Pataky and M.L. Carson Cultural Control of Plant Diseases, G. Moorman and K.D. Gwinn Chemical Control of Plant Diseases, A.S. Windham and M.T. Windham Biological Control of Plant Pathogens, B.H. Ownley and M.T. Windham Integrated Pest Management, C.A. Hollier and D.E. Hershman Plant Disease Diagnosis, J.M. Mullen Diagnostic Techniques and Media Preparation, J.M. Mullen Special Topics In Vitro Plant Pathology, J. Subramanian and D.J. Gray Proper Use of Compound and Stereo Microscopes, D.T. Webb Appendix 1: Careers In Plant Pathology, A.S. Windham and M.T. Windham Glossary Index

Development and characterization of simple sequence repeats for flowering dogwood (Cornus florida L.)

Abundant, codominant simple sequence repeats (SSRs) markers can be used for constructing genetic linkage maps and in marker-assisted breeding programs. Enrichment methods for SSR motifs were optimized with the ultimate aim of developing numerous loci in flowering dogwood (C. florida L.) genome. Small insert libraries using four motifs (GT, CT, TGG, and AAC) were constructed with C. florida ‘Cherokee Brave’ deoxyribonucleic acid (DNA). Colony polymerase chain reaction (PCR) of 2,208 selected clones with three primers we reported previously indicated that 47% or 1,034 of the clones harbored one of the four targeted SSR motifs. Sequencing the putative positive clones confirmed that nearly 99% (1,021 of 1,034) of them contained the desired motifs. Of the 871 unique SSR loci, 617 were dinucleotide repeats (70.8%), and 254 were trinucleotide or longer repeats (29.2%). In total, 379 SSR loci had perfect structure, 237 had interrupted, and 255 had compound structure. Primer pairs were designed from 351 unique sequences. The ability of the 351 SSR primer pairs to amplify specific loci was evaluated with genomic DNA of ‘Appalachian Spring’ and ‘Cherokee Brave’. Of these primers, 311 successfully amplified product(s) with ‘Cherokee Brave’ DNA, 21 produced weak or faint products, and 19 did not amplify any products. Additionally, 218 of the 311 primers pairs revealed polymorphisms between the two cultivars, and 20 out of 218 primers detected an average of 13.7 alleles from 38 selected Cornus species and hybrids. These SSR loci constitute a valuable resource of ideal markers for both genetic linkage mapping and gene tagging of flowering dogwood.

Associations Between Causal Agents of the Beech Bark Disease Complex [Cryptococcus fagisuga (Homoptera: Cryptococcidae) and Nectria spp.] in the Great Smoky Mountains National Park

Abstract American beech, Fagus grandifolia Ehrhart, is currently threatened by the insect-mediated disease complex known as beech bark disease. The organisms (beech scale, Cryptococcus fagisuga Lindinger, and two pathogenic fungi, Nectria galligena Bresadola and Nectria coccinea variety faginata Lohman, Watson, and Ayers) associated with beech bark disease were assessed using a qualitative rating system and correlated with other biotic and abiotic factors in 10 permanent plots in the Great Smoky Mountains National Park from spring 1994 through spring 1997. Tree mortality, as well as the presence of another scale species [Xylococculus betulae (Pergande)], also were documented and analyzed. During this study, incidence of C. fagisuga and overall tree mortality increased (55.6–87.9 and 16.0–26.8%, respectively). A forward stepwise logistic regression model selected average overall ratings of C. fagisuga, sum of presence of X. betulae, average presence of. X. betulae, average overall ratings of Nectria spp., maximum presence of X. betulae, sum of south ratings of C. fagisuga, average south ratings of C. fagisuga, and diameter at breast height (dbh) of the tree as significant variables that best explained mortality of American beech. Pearson correlation analysis showed significant associations between ratings of Nectria spp. and C. fagisuga from both the previous season and previous year during fall 1996 and spring 1997. Moderate correlations between elevation, as well as aspect, and north, south, and overall ratings of C. fagisuga from fall 1995 to spring 1997 were documented. The significant association between presence of C. fagisuga and infection by Nectria spp. suggests that control of C. fagisuga could decrease occurrence and slow the spread of beech bark disease. Unless appropriate control and/or management of the disease complex is identified and implemented, mortality of American beech is expected to increase throughout most areas in the Great Smoky Mountains.

Patterns of evolution in Discula fungi and the origin of dogwood anthracnose in North America, studied using arbitrarily amplified and ribosomal DNA.

Abstract. The anthracnose epidemic caused by exotic filamentous fungi of the genus Discula threatens the future of the prized flowering (Cornus florida L.) and Pacific (C. nuttalli Aud.) dogwoods in North America. A cross-section of fungi that cause anthracnose in broadleaf temperate trees was characterized using DNA amplification fingerprinting, sequence and secondary structure analysis of the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA), and compatibility of hyphal anastomosis. ITS-inferred phylogenies rejected the null hypothesis of only one fungal lineage, by defining four monophyletic and well differentiated groups, corresponding to Discula sp., D. quercina, D. umbrinella and D. destructiva, with the last two species sharing a common and recent ancestor. In turn, they showed that the dogwood pathogen, D. destructiva, did not evolve directly from an indigenous population related to Discula sp. In this study, rDNA spacers that are generally considered important for protein synthesis but are selectively neutral, appeared functionally constrained and subject to selective sequence diversification. Results confirmed the high variability of D. umbrinella and the remarkable homogeneity and exotic nature of D. destructiva at the genetic level, clarified the taxonomy and phylogeny of Discula, and provided clues as to the origin and diversification of dogwood anthracnose-causing fungi.

Natural Occurrence of Microsphaera pulchra and Phyllactinia guttata on Two Cornus Species

Powdery mildew has become a common foliar disease of Cornus florida and other dogwood species in the eastern United States during the last several years. This study was conducted to determine the identity of powdery mildew fungi on C. florida and C. amomum. Ascocarps of Microsphaera pulchra and Phyllactinia guttata occurred singly and together on both C. florida and C. amomum leaves. M. pulchra ascocarps occurred at a higher density than P. guttata ascocarps on C. florida leaves, whereas P. guttata ascocarps occurred more frequently than M. pulchra ascocarps on C. amomum leaves. Histological studies, however, did not provide supplementary data of infection by the powdery mildew species that occurred less frequently on the leaves of each dogwood species. M. pulchra did not penetrate the cells of C. amomum, and likewise P. guttata did not enter through stomata of C. florida leaves. The presence of ascocarps of both species was not the result of infection of the dogwoods by both pathogens. The ascocarps of M. pulchra probably became airborne and then settled on the C. amomum leaves. Similarly, the ascocarps of P. guttata landed on C. florida leaves. These results emphasize the importance of correct pathogen identification using several criteria such as ascocarp morphology, host-pathogen relationships, distribution of the pathogen, conidial morphology, and histology.

Development and characterization of sixteen microsatellite loci for Geosmithia morbida, the causal agent of thousand canker disease in black walnut (Juglans nigra)

Sixteen polymorphic microsatellite loci were identified from the fungal pathogen Geosmithia morbida. Loci were characterized for 13 different isolates collected in 2010 from symptomatic black walnut trees in Tennessee. A total of 77 loci were tested and 16 of those were optimized, screened and selected for diversity studies of G. morbida. Number of alleles per locus ranged from 3 to 8. These microsatellite loci will be useful for rapid disease diagnostic, population genetic analyses on a global scale as well as further epidemiological studies of G. morbida.

Analysis of genetic diversity in flowering dogwood natural stands using microsatellites: the effects of dogwood anthracnose.

Flowering dogwood (Cornus florida L.) populations recently have experienced severe declines caused by dogwood anthracnose. Mortality has ranged from 48 to 98%, raising the concern that genetic diversity has been reduced significantly. Microsatellite data were used to evaluate the level and distribution of genetic variation throughout much of the native range of the tree. Genetic variation in areas affected by anthracnose was as high as or higher than areas without die-offs. We found evidence of four widespread, spatially contiguous genetic clusters. However, there was little relationship between geographic distance and genetic difference. These observations suggest that high dispersal rates and large effective population sizes have so far prevented rapid loss of genetic diversity. The effects of anthracnose on demography and community structure are likely to be far more consequential than short-term genetic effects.

Powdery Mildew of Dogwoods: Current Status and Future Prospects

Cornus is a large genus of trees and shrubs that are collectively referred to as dogwoods. Flowering dogwood (C. florida L.) and kousa dogwood (C. kousa (F. Buerger ex Miq.) Hance) and interspecific hybrids of these species are popular ornamental trees that are known for their showy bracts, red berries (drupes), and/or fall color. Other species that are commercially grown for specialty markets include the pagoda dogwood (C. alternifolia L.), giant dogwood (C. controversa Hemsl.), cornelian cherry (C. mas L.), Pacific dogwood (C. nuttallii Aud.), and redosier dogwood (C. sericea L.). The foliage of native species, such as flowering dogwood and pagoda dogwood, is high in calcium (12) in quantities above what is needed for skeletal growth of wildlife; it is the preferred browse material for lactating does in late spring while many other trees are still leafless (13,22). The berries of flowering dogwood have high oil content and provide mast for numerous species of migrant songbirds, wild turkeys, and large and small mammals (22). For many years, nurseries that produced flowering and kousa dogwoods had the luxury of working with relatively diseasefree crops. Disease management and control costs were minimal and estimated at approximately