Timothy C. Todd

Transgenic soybeans expressing siRNAs specific to a major sperm protein gene suppress Heterodera glycines reproduction

The soybean cyst nematode (SCN), Heterodera glycines, is the major disease-causing agent limiting soybean production in the USA. The current management strategy to reduce yield loss by SCN involves the deployment of resistant soybean cultivars and rotation to non-host crops. Although this management scheme has shown some success, continued yearly yield loss estimates demonstrate the limitations of these techniques. As a result, new control strategies are needed to complement the existing methods. Reported here is a novel method of SCN control that utilises RNA interference (RNAi). Transgenic soybeans were generated following transformation with an RNAi expression vector containing inverted repeats of a cDNA clone of the major sperm protein (MSP) gene from H. glycines. The accumulation of MSP-specific short interfering RNA (siRNA) molecules were detected by northern blot analysis of transgenic soybeans. T0 plants displaying MSP siRNA accumulation were deployed in a bioassay to evaluate the effects of MSP interfering molecules on H. glycines reproduction. Bioassay data has shown up to a 68% reduction in eggs g-1 root tissue, demonstrating that MSPi transgenic plants significantly reduced the reproductive potential of H. glycines. An additional bioassay evaluating progeny nematodes for maintenance of reproductive suppression indicated that progeny were also impaired in their ability to successfully reproduce, as demonstrated by a 75% reduction in eggs g-1 root tissue. The results of this study demonstrate the efficacy of an RNAi-based strategy for control of the soybean cyst nematode. In addition, these results may have important implications for the control of other plant parasitic nematodes.

Effects of management practices on nematode community structure in tallgrass prairie

Abstract The effects of burning, mowing, and nitrogen (N) and phosphorus (P) fertilization on the trophic structure of a tallgrass prairie nematode community were examined in a long-term field experiment established in 1986. Nematode densities and trophic composition were determined in October of 1987, 1989, and 1994 following 2, 4, and 9 years of treatment, respectively. Total populations of obligate herbivores tended to increase with annual burning and N fertilization but responses by individual taxa were relative to the structure and composition of vegetation as determined by management practice. In contrast, mowing resulted in consistent reductions in herbivore densities. The family Tylenchidae , with species classified as both root and fungal feeders, exhibited short-term decreases in numbers due to burning and long-term increases in numbers due to mowing. Treatment responses by this group were more consistent with the behavior of known fungal-feeding than root-feeding nematodes. Microbivore densities increased with burning and N fertilization, while numbers of omnivores and predators declined with chronic N additions. The effects of P fertilization on nematode population densities varied with management practice, primarily N fertilization. Canonical discriminant analysis differentiated trophic groups based on their responses to mowing and P-fertilization. Measurements of the trophic composition of the nematode community, particularly the relative abundance of individual herbivore taxa and the proportion of fungivores to microbivores, were good indicators of ecosystem responses to management practices.

Caenorhabditis elegans genomic response to soil bacteria predicts environment-specific genetic effects on life history traits.

With the post-genomic era came a dramatic increase in high-throughput technologies, of which transcriptional profiling by microarrays was one of the most popular. One application of this technology is to identify genes that are differentially expressed in response to different environmental conditions. These experiments are constructed under the assumption that the differentially expressed genes are functionally important in the environment where they are induced. However, whether differential expression is predictive of functional importance has yet to be tested. Here we have addressed this expectation by employing Caenorhabditis elegans as a model for the interaction of native soil nematode taxa and soil bacteria. Using transcriptional profiling, we identified candidate genes regulated in response to different bacteria isolated in association with grassland nematodes or from grassland soils. Many of the regulated candidate genes are predicted to affect metabolism and innate immunity suggesting similar genes could influence nematode community dynamics in natural systems. Using mutations that inactivate 21 of the identified genes, we showed that most contribute to lifespan and/or fitness in a given bacterial environment. Although these bacteria may not be natural food sources for C. elegans, we show that changes in food source, as can occur in environmental disturbance, can have a large effect on gene expression, with important consequences for fitness. Moreover, we used regression analysis to demonstrate that for many genes the degree of differential gene expression between two bacterial environments predicted the magnitude of the effect of the loss of gene function on life history traits in those environments.

Effect of Bt Corn for Corn Rootworm Control on Nontarget Soil Microarthropods and Nematodes

Abstract The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of corn in Kansas. Planting Bt corn hybrids resistant to this pest is being tested as a method to control the larval stage of corn rootworms. These hybrids express Cry3Bb1 toxin and are expected to only directly impact chrysomelids and possibly related taxa. Soil samples were examined to evaluate the effect of Bt corn for corn rootworm control on soil microarthropods and nematodes in Kansas in 2000 and 2001. Soil samples from soil close to Bt corn and to its isoline were taken on three occasions (early, mid, and late season) from eight locations in 2000 and three locations in 2001. Soil mites and Collembola were extracted using a modified Tullgren high-gradient extractor. Nematodes were extracted using a centrifugal-flotation procedure. In general, numbers of soil mites (Prostigmata, Mesostigmata, and Oribatei), Collembola, and nematodes were similar in soil planted with Bt corn and soil planted with its isoline.

Macroinvertebrates in North American tallgrass prairie soils: effects of fire, mowing, and fertilization on density and biomass

The responses of tallgrass prairie plant communities and ecosystem processes to fire and grazing are well characterized. However, responses of invertebrate consumer groups, and particularly soil-dwelling organisms, to these disturbances are not well known. At Konza Prairie Biological Station, we sampled soil macroinvertebrates in 1994 and 1999 as part of a long-term experiment designed to examine the effects and interactions of annual fire, mowing, and fertilization (N and P) on prairie soil communities and processes. For nearly all taxa, in both years, responses were characterized by significant treatment interactions, but some general patterns were evident. Introduced European earthworms (Aporrectodea spp. and Octolasion spp.) were most abundant in plots where fire was excluded, and the proportion of the total earthworm community consisting of introduced earthworms was greater in unburned, unmowed, and fertilized plots. Nymphs of two Cicada genera were collected (Cicadetta spp. and Tibicen spp.). Cicadetta nymphs were more abundant in burned plots, but mowing reduced their abundance. Tibicen nymphs were collected almost exclusively from unburned plots. Treatment effects on herbivorous beetle larvae (Scarabaeidae, Elateridae, and Curculionidae) were variable, but nutrient additions (N or P) usually resulted in greater densities, whereas mowing usually resulted in lower densities. Our results suggest that departures from historical disturbance regimes (i.e. frequent fire and grazing) may render soils more susceptible to increased numbers of European earthworms, and that interactions between fire, aboveground biomass removal, and vegetation responses affect the structure and composition of invertebrate communities in tallgrass prairie soils.

Relatedness of Macrophomina phaseolina isolates from tallgrass prairie, maize, soybean and sorghum

Agricultural and wild ecosystems may interact through shared pathogens such as Macrophomina phaseolina, a generalist clonal fungus with more than 284 plant hosts that is likely to become more important under climate change scenarios of increased heat and drought stress. To evaluate the degree of subdivision in populations of M. phaseolina in Kansas agriculture and wildlands, we compared 143 isolates from maize fields adjacent to tallgrass prairie, nearby sorghum fields, widely dispersed soybean fields and isolates from eight plant species in tallgrass prairie. Isolate growth phenotypes were evaluated on a medium containing chlorate. Genetic characteristics were analysed based on amplified fragment length polymorphisms and the sequence of the rDNA‐internal transcribed spacer (ITS) region. The average genetic similarity was 58% among isolates in the tallgrass prairie, 71% in the maize fields, 75% in the sorghum fields and 80% in the dispersed soybean fields. The isolates were divided into four clusters: one containing most of the isolates from maize and soybean, two others containing isolates from wild plants and sorghum, and a fourth containing a single isolate recovered from Solidago canadensis in the tallgrass prairie. Most of the sorghum isolates had the dense phenotype on media containing chlorate, while those from other hosts had either feathery or restricted phenotypes. These results suggest that the tallgrass prairie supports a more diverse population of M. phaseolina per area than do any of the crop species. Subpopulations show incomplete specialization by host. These results also suggest that inoculum produced in agriculture may influence tallgrass prairie communities, and conversely that different pathogen subpopulations in tallgrass prairie can interact there to generate ‘hybrids’ with novel genetic profiles and pathogenic capabilities.

Long-term nitrogen amendment alters the diversity and assemblage of soil bacterial communities in tallgrass prairie.

Anthropogenic changes are altering the environmental conditions and the biota of ecosystems worldwide. In many temperate grasslands, such as North American tallgrass prairie, these changes include alteration in historically important disturbance regimes (e.g., frequency of fires) and enhanced availability of potentially limiting nutrients, particularly nitrogen. Such anthropogenically-driven changes in the environment are known to elicit substantial changes in plant and consumer communities aboveground, but much less is known about their effects on soil microbial communities. Due to the high diversity of soil microbes and methodological challenges associated with assessing microbial community composition, relatively few studies have addressed specific taxonomic changes underlying microbial community-level responses to different fire regimes or nutrient amendments in tallgrass prairie. We used deep sequencing of the V3 region of the 16S rRNA gene to explore the effects of contrasting fire regimes and nutrient enrichment on soil bacterial communities in a long-term (20 yrs) experiment in native tallgrass prairie in the eastern Central Plains. We focused on responses to nutrient amendments coupled with two extreme fire regimes (annual prescribed spring burning and complete fire exclusion). The dominant bacterial phyla identified were Proteobacteria, Verrucomicrobia, Bacteriodetes, Acidobacteria, Firmicutes, and Actinobacteria and made up 80% of all taxa quantified. Chronic nitrogen enrichment significantly impacted bacterial community diversity and community structure varied according to nitrogen treatment, but not phosphorus enrichment or fire regime. We also found significant responses of individual bacterial groups including Nitrospira and Gammaproteobacteria to long-term nitrogen enrichment. Our results show that soil nitrogen enrichment can significantly alter bacterial community diversity, structure, and individual taxa abundance, which have important implications for both managed and natural grassland ecosystems.

Biotechnological application of functional genomics towards plant-parasitic nematode control.

Plant-parasitic nematodes are primary biotic factors limiting the crop production. Current nematode control strategies include nematicides, crop rotation and resistant cultivars, but each has serious limitations. RNA interference (RNAi) represents a major breakthrough in the application of functional genomics for plant-parasitic nematode control. RNAi-induced suppression of numerous genes essential for nematode development, reproduction or parasitism has been demonstrated, highlighting the considerable potential for using this strategy to control damaging pest populations. In an effort to find more suitable and effective gene targets for silencing, researchers are employing functional genomics methodologies, including genome sequencing and transcriptome profiling. Microarrays have been used for studying the interactions between nematodes and plant roots and to measure both plants and nematodes transcripts. Furthermore, laser capture microdissection has been applied for the precise dissection of nematode feeding sites (syncytia) to allow the study of gene expression specifically in syncytia. In the near future, small RNA sequencing techniques will provide more direct information for elucidating small RNA regulatory mechanisms in plants and specific gene silencing using artificial microRNAs should further improve the potential of targeted gene silencing as a strategy for nematode management.

Molecular approach for assessing responses of microbial-feeding nematodes to burning and chronic nitrogen enrichment in a native grassland

A substantial proportion of the primary productivity in grassland ecosystems is allocated belowground, sustaining an abundant and diverse community of microbes and soil invertebrates. These belowground communities drive many important ecosystem functions and are responsive to a variety of environmental changes. Nematodes, an abundant and diverse component of grassland soil communities, are particularly responsive to altered environmental conditions, such as those associated with reduced fire frequency and nitrogen enrichment, with the most consistent responses displayed by microbial‐feeding nematodes. However, much of the available research characterizing nematode responses to environmental change has been carried out at the taxonomic level of family or by broad trophic categories (e.g. fungivores, bacterivores). The extent to which differential responses to environmental change occurs at the genus level or below is unclear. Therefore, the objective of this study was to use molecular methods to quantify the response of microbial‐feeding nematodes, at the lowest levels of taxonomic resolution, to nitrogen enrichment and changes in fire frequency. Using sequencing and quantitative polymerase chain reaction (PCR) probes for the 18S ribosomal RNA gene and the ITS1 region, we identified 19 microbial‐feeding nematode taxa across four families. When nematodes were sampled across treatments, we found that some nematode taxa within a family responded similarly to nitrogen and burning treatments, while other taxa within the same family respond quite differently. Additionally, although nematodes from different families on average responded differently to nitrogen enrichment and burning, similar responses were seen in nematode taxa that span three taxonomic families. Thus, if nematodes are to be used as indicators of environmental change, care should be taken to assess the response at the lowest taxonomic level possible.

Soybean Cyst Nematode Reproduction in the North Central United States

An experiment was conducted in Heterodera glycines-infested fields in 40 north central U.S. environments (21 sites in 1994 and 19 sites in 1995) to assess reproduction of this nematode. Two resistant and two susceptible soybean cultivars from each of the maturity groups (MG) I through IV were grown at each site in 6.1 m by 4 row plots. Soil samples were collected from each plot at planting and harvest and processed at Iowa State University to determine H. glycines initial (Pi) and final (Pf) population densities as eggs per 100 cm3 of soil. Overall, reproduction (Pf/Pi) of H. glycines on susceptible cultivars in all MG was similar. Reproduction was higher on MG III and IV susceptible cultivars than on those in MG I and II. Resistant MG I and II cultivars reduced nematode population densities more consistently than those in MG III and IV. Reproduction of the nematode was similar among sites within the same maturity zone (MZ), defined as the areas of best adaptation of the corresponding MG. Nonetheless, careful monitoring of nematode population densities is necessary to assess changes that occur over time in individual fields.