Susan V. Diehl

Effects of Zn and Cd accumulation on structural and physiological characteristics of barley plants

The objectives of this study were to identify the structural changes caused by Zn and Cd accumulation in shoots and roots of barley (Hordeum vulgare) plants; and to correlate metal accumulation with anatomical, physiological and morphological changes. Potted plants were exposed to metal treatments of Zn and Cd for 19 and 16 d respectively. Leaves, stems and roots were harvested to identify structural changes and analyze metal accumulation. Barley effectively accumulated Zn (up to 11283 mg kg-1) and Cd (up to 584 mg kg-1) in the shoots. Microscopic structural changes, such as a decrease in intercellular spaces, breakdown of vascular bundles, and shrinkage of palisade and epidermal cells, occurred in leaves, stems and roots of plants treated with high concentrations of Zn. Zinc accumulation also resulted in a significant decrease in water content, fresh weight, dry weight and plant height. Cadmium only caused structural changes in roots at the higher concentrations. Barley plants were able to accumulate significant amounts of Zn and Cd without exhibiting symptoms of phytotoxicity when the metal concentrations were relatively low.

Spectral reflectance and leaf internal structure changes of barley plants due to phytoextraction of zinc and cadmium

The focus of our research is to seek spectral signatures that indicate the impact and content of heavy metals in the leaves and canopies of living plants during the process of phytoremediation. Potted plants of barley (Hordeum vulgare) were grown for 5–6 weeks before being subjected to metal treatments of Zn and Cd. Diffuse reflectance spectra (350–2500 nm) of the plant canopies were collected daily using a portable spectroradiometer throughout the treatment period. Foliar structural changes of Zn‐treated plants included a decrease in intercellular space, palisade and epidermal cell size while Cd‐treated plants displayed fewer structural changes in leaf. Spectral analysis revealed that the band ratios at 1110 nm to that at 810 nm might be used as an indicator of the accumulation of certain metals in plant shoots. Normalized Difference Vegetation Index (NDVI) and leaf‐water‐content indices examined as part of our spectral analysis were not able to distinguish plants treated with different metals. Our ratio index R1110/R810, on the other hand, correlates closely with the magnitude of leaf structural changes. This study suggests that the infrared reflectance spectrum (800–1300 nm) of plant canopy might provide a non‐intrusive monitoring method for the physiological status of plants grown on heavy metal contaminated soil.

Effect of Phytoaccumulation of Arsenic and Chromium on Structural and Ultrastructural Changes of Brake Fern (Pteris vittata)

Structural and ultrastructural changes caused by bioaccumulation of As and Cr in brake fern (Pteris vittata), a known arsenic hyperaccumulator, were investigated. Potted plants of brake fern were exposed to metal treatments of As and Cr for three weeks. Leaf, stem and root samples were collected periodically and fixed for LM (Light Microscopy), SEM (Scanning Electron Microscopy) and TEM (Transmission Electron Microscopy) to evaluate anatomical changes. The fresh weights, dry weights, RWC (Relative Water Content) and plant heights were obtained before the brake fern plants were harvested for metal accumulation analysis. The As accumulated mainly in the shoots while Cr accumulated mainly in the roots of the metal-treated plants. Significant changes in the ferns physical characters, including fresh weight, dry weight, RWC, and plant height were observed for only Cr-treated plants but not for As-treated plants. Microscopic studies reveal the Cr accumulation resulted in dehydration and collapse of internal structure of leaves and cellular breakdown of roots. The As-treated plants showed no significant structural changes in leaves, stems and roots compared to control plants. Clotted depositions were observed in roots and stems of plant groups treated with highest concentration of Cr and As when compared to control (T0) group. Our study indicates that Cr has a profound impact on physiology and structure of fern plants. The accumulation of Cr resulted in decrease in growth rate, total biomass and RWC. We believe that brake fern plants can uptake, translocate and sequester As because it caused no significant structural changes in leaves, stems and roots of the plants.

Monitoring the effects of arsenic and chromium accumulation in Chinese brake fern (Pteris vittata)

The objectives of this study were (i) to investigate the feasibility of using spectral reflectance for monitoring As and Cr accumulation in Chinese brake fern (Pteris vitatta), and (ii) to search for spectral indices sensitive to structural changes caused by metal accumulation during the process of phytoremediation. Potted Chinese brake fern plants were exposed to As (100 and 300 ppm) and Cr (300 and 600 ppm) treatments for 22 days. The plants were then harvested and analysed for metal accumulation. Diffuse reflectance spectra (350–2500 nm) of the plant canopies were collected regularly throughout the metal treatment period using a portable spectroradiometer. Leaf reflectance is governed by leaf surface properties, internal structure, and foliar pigments and biochemical components. Leaf samples were collected and analysed for structural changes through microscopic observations. Our microscopic studies on changes of leaf structure provide insight into the physical changes that are remotely detected as changes in reflectance, and may permit extrapolation of these results to other plant species. Cr accumulation resulted in a decrease in biomass, relative water content (RWC), and changes in the internal structure of the leaf. The structural and spectral results show significant changes in Cr‐treated plants while the changes were minimal in As‐treated plants compared to untreated plants. Our spectral analysis revealed that a unique ratio index R 1110/R 810 can be used to monitor structural changes in plants due to accumulation of Cr. This index distinguished Cr‐treated plants from untreated and As‐treated plants. The Normalized Difference Vegetative Index (NDVI) distinguished stressed plants, but NDVI cannot distinguish Cr‐stressed plants from As‐stressed plants. Our results show that brake fern can accumulate significant amounts of Cr in shoots (2108 mg kg−1 dry weight), but it is not a hyperaccumulator for Cr because much higher Cr accumulation was found in roots (7686 mg kg−1 dry weight). This study suggests that the infrared reflectance spectrum (800–1300 nm) of plant canopy may provide a non‐intrusive monitoring method to access the physiological status of plants grown in heavy metal‐contaminated soil.

Short-Read Sequencing for Genomic Analysis of the Brown Rot Fungus Fibroporia radiculosa

ABSTRACT The feasibility of short-read sequencing for genomic analysis was demonstrated for Fibroporia radiculosa, a copper-tolerant fungus that causes brown rot decay of wood. The effect of read quality on genomic assembly was assessed by filtering Illumina GAIIx reads from a single run of a paired-end library (75-nucleotide read length and 300-bp fragment size) at three different stringency levels and then assembling each data set with Velvet. A simple approach was devised to determine which filter stringency was “best.” Venn diagrams identified the regions containing reads that were used in an assembly but were of a low-enough quality to be removed by a filter. By plotting base quality histograms of reads in this region, we judged whether a filter was too stringent or not stringent enough. Our best assembly had a genome size of 33.6 Mb, an N50 of 65.8 kb for a k-mer of 51, and a maximum contig length of 347 kb. Using GeneMark, 9,262 genes were predicted. TargetP and SignalP analyses showed that among the 1,213 genes with secreted products, 986 had motifs for signal peptides and 227 had motifs for signal anchors. Blast2GO analysis provided functional annotation for 5,407 genes. We identified 29 genes with putative roles in copper tolerance and 73 genes for lignocellulose degradation. A search for homologs of these 102 genes showed that F. radiculosa exhibited more similarity to Postia placenta than Serpula lacrymans. Notable differences were found, however, and their involvements in copper tolerance and wood decay are discussed.

Gene Expression Analysis of Copper Tolerance and Wood Decay in the Brown Rot Fungus Fibroporia radiculosa

ABSTRACT High-throughput transcriptomics was used to identify Fibroporia radiculosa genes that were differentially regulated during colonization of wood treated with a copper-based preservative. The transcriptome was profiled at two time points while the fungus was growing on wood treated with micronized copper quat (MCQ). A total of 917 transcripts were differentially expressed. Fifty-eight of these genes were more highly expressed when the MCQ was protecting the wood from strength loss and had putative functions related to oxalate production/degradation, laccase activity, quinone biosynthesis, pectin degradation, ATP production, cytochrome P450 activity, signal transduction, and transcriptional regulation. Sixty-one genes were more highly expressed when the MCQ lost its effectiveness (>50% strength loss) and had functions related to oxalate degradation; cytochrome P450 activity; H2O2 production and degradation; degradation of cellulose, hemicellulose, and pectin; hexose transport; membrane glycerophospholipid metabolism; and cell wall chemistry. Ten of these differentially regulated genes were quantified by reverse transcriptase PCR for a more in-depth study (4 time points on wood with or without MCQ treatment). Our results showed that MCQ induced higher than normal levels of expression for four genes (putative annotations for isocitrate lyase, glyoxylate dehydrogenase, laccase, and oxalate decarboxylase 1), while four other genes (putative annotations for oxalate decarboxylase 2, aryl alcohol oxidase, glycoside hydrolase 5, and glycoside hydrolase 10) were repressed. The significance of these results is that we have identified several genes that appear to be coregulated, with putative functions related to copper tolerance and/or wood decay.

Community analysis of preservative-treated southern pine (Pinus spp.) using terminal restriction fragment length polymorphism (T-RFLP) analysis. Part 2: Bacteria field study

Abstract The effects of chlorothalonil (CTN), butylated hydroxytoluene (BHT), and ammoniacal copper quat (ACQ-C) on the fungal community on southern yellow pine (SYP) were assessed using terminal restriction fragment length polymorphism (T-RFLP) analysis over 15 months. Field stakes, treated with 0.25 and 0.37% ACQ-C, 0.1 and 0.25% CTN, 2% BHT alone, 0.1 and 0.25% CTN combined with 2% BHT, and untreated controls, were installed in two field sites in Mississippi. Stakes were sampled at 90-day intervals and rated for decay damage. Fungal DNA was extracted and amplified by non-specific (total fungi) and specific (Basidiomycete) primers and processes for T-RFLP. a-Diversity (richness and diversity) and b-diversity (similarity between communities) were calculated by means of T-RFLP data. The presence of wood preservatives slowed the initial colonization of field stakes by total fungi, resulting in lower richness and diversity that increased over time; however, preservatives increased the richness and diversity of Basidiomycetes. The b-diversity of treated samples was less similar in the early stages of exposure (3–9 months), but coalesced over time into equilibrium communities that were similar to communities on untreated controls. Basidiomycete species compositions were different among treated samples while control communities shared more than 75% of their species. Correlations were found between depletion of 0.1% CTN and increasing fungal diversity, but no other significant correlations were found.

Effects of Composting On Removal of Nitrogen, Phosphorus, and Potassium from Sawdust Amended with Chicken Litter

A six month study evaluated composting affects on nitrogen (N), phosphorus (P), and potassium (K) leaching from hardwood and furniture sawdust amended with 20% and 30% chicken litter. Toxicity Characteristic Leaching Procedure (TCLP) was obtained from substrates. Moisture was provided through precipitation, and the substrate was aerated once or twice per week, depending on the amount of rainfall. Samples were collected at 45 day intervals for analysis. A significant reduction in N and K concentrations occurred for all amended compost by day 180. Hardwood substrate matured quicker than furniture compost based on a radish seed germination test. Toxicity reduction occurred in every treatment by day 180. Chicken litter control showed the best overall weight loss; however, its toxicity levels were still much higher on day 180.

Effect of Cercosporella rubi on blackberry floral bud development

Rosette, caused by the fungus Cercosporella rubi, is an important blackberry disease in the southeastern United States. This disease severely reduces fruit production, and its management has been erratic due to a limited understanding of the host-pathogen relationship. In this study, we expand on previous histological investigations of the development of C. rubi on blackberry at tissue and cellular levels from floral bud initiation through senescence of the flower. Symptomatic and asymptomatic floral buds were examined with light microscopy and both scanning and transmission electron microscopy (SEM and TEM, respectively). Fungal development on the surface of floral buds was examined with SEM. Previous light microscope histological studies were unclear about whether C. rubi penetrated host tissue. With TEM, we demonstrated an intimate association between fungal and host cells with no penetration prior to death of the host tissue. C. rubi was present on symptomatic floral buds before development and through senescence. No morphological differences were seen between healthy and diseased floral buds ≤5.0 mm in diameter other than the presence of C. rubi. Necrosis was observed in symptomatic buds at the 6.0-mm-diameter stage and progressed through floral senescence.

A Molecular Clone and Culture Inventory of the Root Fungal Community Associated with Eastern Hemlock in Great Smoky Mountains National Park

Abstract Below- and above-ground microbial communities are expected to dramatically change following the loss of healthy Tsuga canadensis (Eastern Hemlock) ecosystems due to the effects of Adelges tsugae (Hemlock Woolly Adelgid [HWA]) infestation. In 2006, few healthy Eastern Hemlock stands remained in Great Smoky Mountains National Park (GRSM), and we sought to obtain baseline soil microbial data from these undisturbed areas to better understand them. This study surveyed root fungal communities (e.g., mycorrhizae) using molecular cloning and cultural methods at 2 remaining healthy Eastern Hemlock stands in GRSM. We sampled roots from 40 trees representing 2 age classes (<75 and >150 years old) and 2 elevations (486 m and 1158 m) to obtain molecular clones and isolated cultures. Molecular clones from the ITS region indicated the presence of 124 taxa in 23 genera. The dominant genera were Piloderma (8.2%), Russula (6.8%), Tomentella (4.0%), Meliniomyces (3.4%), and Clavulina (2.9%). Ninety-seven of the taxa identified occurred at <1.0% relative abundance. The most frequent fungal species were Meliniomyces variabilis (22.6%), Russula granulata (16.1%), Phialocephala fortinii (9.7%), and Cladophialophora chaetospira (16.1%). Species richness, diversity, and evenness for total fungi, ectomycorrhizal species, and saprobic species were similar at different elevations and stand ages. Using ITS sequence data, we identified 9 species from 1800 fungal isolates from Eastern Hemlock root tissue, the most common of which were Trichoderma harzianum (24.4%), Trichoderma viride (24.4%), Trichoderma asperellum (17.1%), and Ilyonectria radicicola (12.2%). Species richness, diversity, and evenness were not significantly different across locations for the molecular data. Baseline data for the fungal taxa identified in the study may be important for understanding ecological changes that can occur post-HWA damage and HWA-caused stand losses. These data could also be used to support Eastern Hemlock reforestation efforts within current or former habitats in GRSM and surrounding ecosystems.