Shiv Hiremath

Cloning and complete sequence characterization of two gypsy moth aminopeptidase-N cDNAs, including the receptor for Bacillus thuringiensis Cry1Ac toxin.

The complete cDNAs corresponding to two distinct gypsy moth (Lymantria dispar) larval gut aminopeptidases, APN1 and lambda APN2, were cloned and sequenced. The 3.4 kilobasepair cDNA of APN1 which encodes a 1017 amino acid prepro-protein corresponds to the previously-identified gypsy moth APN (APN-1) that specifically binds the Cry1Ac delta-endotoxin of Bacillus thuringiensis. Analysis of the primary structure of APN1 revealed a cluster of five potential N-linked glycosylation sites near the N-terminus and a C-terminal sequence characteristic of a putative glycosylphosphatidyl-inositol (GPI) anchor signal sequence. The cDNA of APN1 encodes the N-terminal peptide sequence and nine internal sequences obtained from the purified brush border membrane vesicle Cry1Ac receptor by protein sequencing. The lambda APN2 cDNA encodes a shorter protein with 51% similarity to APN1 that also appears to have a GPI anchor signal sequence. Expression of the APN1 cDNA in a baculovirus vector was confirmed by immunoblotting.

STRUCTURE OF THE GYPSY MOTH VITELLOGENIN GENE

Genomic clones containing the vitellogenin (Vg) gene from the gypsy moth were isolated from two genomic libraries and characterized. The nucleotide sequence of a 16,132 bp region of the gypsy moth genome was determined which included a 3,666 bp region upstream from the transcription initiation site and 499 bp region downstream from the transcribed region. Primer extension analysis was performed to identify the transcription initiation site. Gene sequence confirmed the sequence of VgmRNA recently reported [Hiremath and Lehtoma, J. Insect Biochem. Mol. Biol. (1997) 27:27-35] and indicated that the gypsy moth Vg gene contains seven exons interrupted by six introns. Sequence analysis of the promoter region revealed presence of several motifs associated with sex-specific and developmentally regulated genes in other systems. The nucleotide sequence comparison analyses showed that the gypsy moth Vg gene had considerably similarity with the Bombyx mori Vg gene but not with those from Anthonomous grandis and Aedes aegypti.

Soil preparation methods promoting ectomycorrhizal colonization and American chestnut Castanea dentata establishment in coal mine restoration

Summary 1. The objective of this research was to evaluate soil subsurface methods that may aid in seedling establishment and encourage root colonization from a diverse group of ectomycorrhizal (ECM) fungi during restoration projects. 2. American chestnut Castanea dentata Marsh. Borkh. and backcrossed chestnuts seedlings were planted on a reclaimed coal mine site in central Ohio, USA. Roots from chestnut seedlings planted in the plots that were cross-ripped, plowed and disked, or a combination of treatments were sampled for ECM fungi and compared with control plots. The presence and identification of native ECM were determined by fungal DNA sequencing of the internaltranscribed (ITS) region. 3. After two growing seasons, mechanical soil treatments resulted in seedlings with significantly more ECM species when compared to seedlings grown in the control plots (P <0 0001). A nonmetric multidimensional scaling ordination followed by a permutational MANOVA confirmed significant dissimilarities in community composition between the control and mechanically treated plots (F =0 24, P =0 015). 4. Ectomycorrhizal root colonization was significantly higher on the chestnut seedlings sampled from the mechanically treated plots when compared to the control plots (F =1 063, P <0 0001). Differences did not exist among the three mechanical treatments. There was also a significant increase in above-ground seedling growth in the plots that were treated with a surface soil method (F =1 572, P <0 0001). It is not clear whether ECM activity was the driver of plant growth; regardless, both are strong indicators of healthy tree establishment. 5. Synthesis and applications. This study illustrates that the use of soil subsurface methods increased ectomycorrhizal (ECM) activity and seedling growth. Employing methods that encourage the root colonization by beneficial ECM and promote healthy seedling establishment may aid the long-term survival of chestnuts in restoration projects. This can be applied to other hardwood seedlings used in reforestation in soils compacted after anthropogenic disturbances.

The Influence of Inoculated and Native Ectomycorrhizal Fungi on Morphology, Physiology and Survival of American Chestnut

The objective of this study was to evaluate the influence of five different species of ectomycorrhizal (ECM) fungi on root colonization of native fungi on putatively blight resistant chestnut hybrids (Castanea dentata x C. mollissima) in a reclaimed mine site in central Ohio. The five species were Hebeloma crustuliniforme, Laccaria bicolor, Scleroderma polyrhizum, Amanita rubescens, and Suillus luteus. We used a combination of DNA sequencing of the ITS region and phylogenetic analyses to indentify fungi found on roots after 12 and 18 months in the field. Non-metric multidimensional scaling (NMDS) ordinations were used to determine if ECM community composition was influenced by the fungal inoculum used. The results of this study demonstrated that the selected ECM species do not persist on chestnut after one year in the field. In addition, these selected ECM species did not impede natural root colonization of native fungi or influence ECM community composition after two growing seasons. Although these species did not persist in the field, the presence of ECM inoculum (with the exception of Amanita) greatly contributed to the survival of hybrid chestnut seedlings. Therefore, introduced inoculum that was present in the very early stages of outplanting had persisting effects with regard to seedling establishment in the field, even if the original inoculum did not persist. ECM fungi native to the area colonized chestnuts resulting in increased growth rates. These native assemblages may contain species better able to form functional mycorrhizas under these environmental extremes. Therefore, the conservation of these species may be necessary to facilitate long-term survival of deciduous tree species historically native to these lands. Additional Keywords: root colonization of fungi, chestnut restoration.

AILANTHUS ALTISSIMA INTERFERS WITH BENEFICIAL SYMBIONTS AND NEGETIVELY IMPACTS OAK REGENERATION 1

The invasion of Tree-of-Heaven (Ailanthus altissima) has been documented in disturbed landscapes leading to biodiversity loss and degradation of ecosystem function. Ailanthus interferes with the restoration of native species by its aggressive growth habit, alteration of nutrient cycles, and allelopathic chemical production. Recent studies suggest that allelopathy has a negative effect on the growth of red oak (Quercus rubra), possibly by interfering with the symbiosis of beneficial ectomycorrhizal fungi (ECM). This fungal symbiont is essential for healthy tree growth and the unavailability of these fungi may impede the success of seedling regeneration. This study investigated the effects of Ailanthus on biomass production and ectomycorrhizal fungal (ECM) colonization of red oak (Q. rubra) seedlings on a reclaimed coal mine site in eastern Ohio. Six plots were designated in an existing riparian buffer zone in a wetland at The Wilds Conservation Center in Muskingum County. Three of the plots were in an area where mature Ailanthus was present. The other three plots were located in the same riparian zone that was without Ailanthus. Naturally regenerating two- year-old red oak seedlings were selected for study (10 seedlings per plot, 60 seedlings total). The oak seedlings were sampled for biomass (g) and ECM root colonization. Two-year-old oak seedlings growing among mature Tree-of- Heaven produced significantly less biomass, specifically in root production, than the oaks growing without the invasive tree (P = 0.02). There was a decrease in ECM colonization (P = 0.001) and a shift in ECM community composition in plots where the Tree-of-Heaven was present (P = 0.0004). The increase in root biomass and ECM colonization may aid in the plants competitive ability for belowground resources, important for reestablishment. These data suggest that areas impacted by the invasion of Tree-of-Heaven may require restoration with plant species less reliant on ECM colonization when planting in soils immediately following invasive species removal.

Pyrolusite Process ® to Remove Acid Mine Drainage Contaminants from Kimble Creek in Ohio: A Pilot Study

The Kimble Creek abandoned coal mine site, located on Wayne National Forest in southeastern Ohio, is among several abandoned coal mine sites that have been responsible for the acid mine drainage (AMD) polluting ground and surface water. Materials released by AMD include iron, aluminum, manganese, other hazardous substances, and acidity that are harmful to aquatic life. We tested at the Kimble Creek site the Pyrolusite Process®, a microbiological water treatment system that employs metal-oxidizing bacteria adsorbed on limestone rocks. The pilot treatment plant constructed was inoculated with a mixture (five strains) of metal-oxidizing bacteria. Water was examined at regular intervals for pH, concentrations of metals (Fe, Al, Mn, Mg, Zn, Ca), dissolved oxygen, temperature, conductivity, hardiness, sulfate and chloride contents, acidity, alkalinity, and dissolved solids. Results showed that the Prylosite Process consistently increased the pH of effluent water to above-neutral levels.

THE EFFICIENCY OF INTRODUCED PISOLITHUS TINCTORIUS INOCULUM ON BACKCROSSED CHESTNUT GERMINATION AND SURVIVAL

American chestnut was eliminated as a canopy tree from the Appalachian region of North America with the introduction of chestnut blight in the early 1900s. Breeding programs initiated in the 1980s have produced seedling lines that display the pure American morphology with potential resistance to chestnut blight. More work is required to assess their field performance in field sites representative of their native range. This study used American (Castanea dentata) and backcrossed hybrid chestnuts (C. dentata × C. mollissima) on an abandoned coal mine in southeastern Ohio. Half of the seeds were planted with ectomycorrhizal fungus (ECM) Pisolithus tinctorius (Pt) as a granular inoculum. Germination, survival, percent ECM colonization, and ECM community were assessed. In addition, soil data such as pH, cation exchange capacity, nutrients, texture, temperature, moisture, and organic matter were evaluated to determine their influence on ECM. After the first and second growing season, germination and survival were not influenced by the Pt inoculum or chestnut genotypes. In addition, ECM root colonization, fungal community composition, and host response were similar between seedling types, regardless of the inoculum added. This indicated: 1) backcrossed breeding produces seedlings similar to pure American chestnuts with regard to growth, establishment, and fungal symbionts, and 2) the granular inoculum had no influence on ECM colonization in this field site. Introducing spore inoculum to a field site greatly deficient in nutrients and organic matter is not an efficient method to ensure ECM symbiosis. However, natural colonization by native ECM fungi, though limited, did result in larger chestnut seedlings. Cation exchange capacity significantly contributed to the percent of root colonization of ECM fungi on chestnut. Determination of planting protocols and factors that influence ECM root colonization will be useful for in future mine restoration projects using backcrossed American chestnut as a restoration tree. Additional

Identification of a small heat-shock protein associated with a ras-mediated signaling pathway in ectomycorrhizal symbiosis

Initiation, development, and establishment of a functional ectomycorrhiza involve a series of biochemical events mediated by a number of genes from the fungus as well as the host plant. We have identified a heat shock protein gene from Laccaria bicolor (Lbhsp) that appears to play a role in these events. The size and characteristics of Lbhsp suggest that it belongs to the family of small heat-shock proteins described in the literature. Nucleotide sequencing of an almost full length cDNA indicated that the Lbhsp mRNA is about 611 nucleotides long and codes for a single protein of ~ 17 kDa. Isolation and characterization of the Lbhsp gene showed that it was made up of three exons separated by two small introns. Southern analysis suggested that the L. bicolor genome contains at least two copies of the Lbhsp gene. Temporal expression analyses revealed that the gene is expressed within 4 to 12 hours after interaction with red pine roots. The yeast two-hybrid studies showed that the Lbhsp was closely associated with the ras gene (Lbras) described earlier. The data suggest that Lbhsp may play a supporting role in ras-mediated mycorrhizal signaling pathways during various stages of ectomycorrhizal development.