The tobacco cyst nematode (TCN), Globodera tabacum solanacearum [(Miller and Gray, 1972) Behrens 1975] Stone 1983, is one of the most economically important pests of flue-cured tobacco (Nicotiana tabacum L.) in Virginia. Although TCN has been reported from other countries, the geographical distribution of G. t. solanacearum within the United States is limited to Virginia, North Carolina, and one county in Maryland. Approximately 30% of the tobacco acreage in Virginia is infested; average yield reduction is 15%, but complete crop failure can occur. The objectives of this research were to examine intraspecific variability within G. t. solanacearum and to evaluate the relative adaptability of G. t. solanacearum on a resistant (NC567) and a susceptible (K326) flue-cured tobacco cultivar. Nineteen geographic isolates of G. t. solanacearum, one isolate each of G. t. virginiae and the Mexican cyst nematode (G. "mexicana"), two isolates of G. t. tabacum, and five Heterodera species were characterized by DNA fingerprinting using the RAPD-PCR technique. Reproducible differences in fragment patterns allowed similar differentiation of the isolates and species with each primer. Hierarchical cluster analysis was performed to illustrate the relatedness between nematode isolates and species. In contrast to reports in the literature, we found a Miller isolate of G. "mexicana" to cluster more closely with G. t. solanacearum than with G. t. tabacum or G. t. virginiae. Although no pathotype differences have been found within G. t. solanacearum, the average Jaccard's similarity index among isolates of G. t. solanacearum was 74%, representing greater variation than that observed across different pathotypes of the closely related potato cyst nematode, Globodera pallida. This result suggests that the emergence of resistance-breaking biotypes is more likely than previous research suggests. If a new pathotype is reported, a RAPD marker associated with virulence against a specific host resistance gene could prove to be a valuable tool in population diagnosis, resistance screening, and overall TCN management. One isolate of G. t. solanacearum was cultured on a resistant (NC567) and a susceptible (K326) flue-cured tobacco cultivar over five generations. Variable TCN reproduction was observed on both cultivars over each generation. This variability in reproduction could be attributed to differences among generations in the time interval between inoculation and cyst extraction, temperature, possible diapause effects, and/or daylength. Ninety-eight cysts were produced in the fifth and final generation compared to the 14 to 50 cysts produced during each of the previous four generations. Increased reproduction on the resistant variety suggests that increased virulence might be selected, but research involving additional generations would need to be carried out in order to conclude whether or not TCN virulence is being selected. / Master of Science

Comparative study of homologous structures in closely related species allows the identification of changes in gene regulatory mechanisms and their impact on the evolution of developmental processes. Nematodes, the invertebrate roundworms, are well suited for such studies, especially the Caenorhabditis briggsae and its famous cousin C. elegans. These two worms diverged from a common ancestor roughly 30 million years ago, yet appear morphologically almost identical. My Ph.D. thesis has focused on a set of nuclear factors in C. briggsae that negatively regulate cell proliferation to generate the hermaphrodite-specific mating and egg-laying organ, i.e., vulva. To this end, I have taken a two-pronged approach: one, developing resources to facilitate genetic and genomic studies in this species, and two, characterizing the roles of a novel class of genes and known repressors of vulval development. My work has uncovered substantial differences in the underlying genetic networks that regulate vulva formation in C. briggsae and C. elegans. / Thesis / Candidate in Philosophy

Plant-parasitic nematodes represent a substantial constraint on global food security by reducing the yield potential of all major crops, including soybean (Glycine max L.). The soybean cyst nematode (SCN) (Heterodera glycines I.) is widely distributed across all soybean production areas of the US, and is the major yield-limiting factor, especially in the Midwestern US. Double cropping (DC) is defined as producing more than one crop on the same parcel of land in a single growing season. Compared to conventional single annual crops, DC provides many advantages, including improving soil health, enhanced nutrient provisioning to plants, improvement of soil physical properties, control of erosion, decrease in tillage requirements, and enhanced profitability. In some double-cropping systems, soybean is planted following winter wheat (Triticum aestivum L.), and several reports suggest the potential of wheat to suppress SCN populations. Field trials were conducted from 2017 to 2018 to investigate the effect of wheat on SCN populations in double-cropping soybean. Nine fields with three levels of initial SCN populations (low, moderate, and high) were selected in Illinois. Wheat was planted in strips alternating with strips-maintained weed-free and under fallow over winter and early spring. Soybean was planted in all strips after the wheat harvest. Soybean cyst nematode egg densities were acquired at four time points: wheat establishment, post-wheat/pre-soybean, mid-soybean (R1 growth stage or beginning of flowering), and post-soybean harvest. Wheat strips reduced SCN egg densities compared with fallow strips at the R1 stage (−31.8%) and after soybean harvest (−32.7%). Field locations with noted SCN suppression were selected for a metagenomics study. The structure of fungal communities differed significantly between DC and fallow plots at soybean planting and after harvest (P<0.001). Fungal populations were affected by location at all sampling times and by treatments before planting and after soybean harvest. Several enriched fungal and bacterial taxa in wheat plots, including Mortierella, Exophiala, Conocybe, Rhizobacter spp., and others, were previously reported to parasitize SCN and other plant-parasitic nematodes. Knowing that phytocompounds with potential nematicidal activity are released via the root system of plants, we implemented a gas chromatography–mass spectrometry (GC-MS) pipeline to investigate the profile of phytochemicals in soil samples collected from these fields and reviewed the potential nematicidal activity of compounds with higher concentration in double cropping fields. A total of 51 compounds were detected with the GSMS analysis, 8 with unknown identification. Several compounds, including multiple fatty acids, had greater relative peak areas in double-cropping samples compared to fallow. This multi-approach study provides a better understanding of the mechanisms governing wheat's effect on SCN populations. Rather than a single mechanism, the suppression of SCN in soybean fields double-cropped with winter wheat is potentially linked to enriched microbial communities, increased populations of beneficial organisms, and higher concentrations of phytochemicals with nematicidal activity. This is the first study implementing metagenomics and GCMS to characterize soil microbial and chemical profiles in soybean fields double-cropped with winter wheat. Finally, a set of studies were conducted to evaluate the effect of two seed-applied succinate dehydrogenase inhibitors (SDHI) compounds, fluopyram and pydiflumetofen, on SCN population densities, plant injury, and plant growth. Next-generation sequencing was later employed to identify transcriptomic shifts in gene expression profiles of soybean seedlings treated with fluopyram and pydiflumetofen. Fluopyram reduced both SCN egg and cyst counts in comparison to pydiflumetofen and the untreated control. RNA expression patterns of seed treatments clustered by sampling time (5 DAP vs 10 DAP). Multiple DEGs identified in soybean seedlings treated with fluopyram are reported to be linked to systemic resistance, suggesting a role of systemic resistance on the suppression of SCN by fluopyram. The non-target inhibition of soybean succinate dehydrogenase genes by fluopyram may be the origin of the phytotoxicity symptoms observed and potentially the cause of the systemic resistance activation reported in the current study. To our knowledge, this is the first report of systemic resistance being activated by fluopyram in addition to the suppression of soybean succinate dehydrogenase (SDH) and ubiquinone oxidoreductase genes. This work helps to elucidate the mechanisms of suppression of SCN by fluopyram

<p> Caenorhabditis elegans (C. elegans) is a model organism which is known for its transparent body, small body size, high reproductivity and short lifecycle. Several important genes and signal transduction pathways are well conserved in C. elegans. lin//, a LIM homeobox family member, plays a crucial role in the development of the vulva in C. elegans. LIM homeobox genes are a subgroup of Homeobox family that play fundemental role in animal development. In C. elegans lin-If mutant animals fail to form a functional vulva and vulval-uterine connection and consequently exhibit egg-laying defective phenotype. The cell lineage and marker gene expression studies have shown that lin-// is required for the patterning of all primary and secondary lineage vulval cells. lin- II also functions in the nervous system. </p> <p> lin-// expression is mainly observed in the developing vulval cells and in the pi cells which are involved in the formation of vulval-uterine connection. lin-If expression is also seen in VCs and in some of the head and tail neurons. The completed genome sequences of closely related species in Caenorhabditis genus serve as a power tool to do systematic comparative studies. The lin-If regulatory sequences from these species have been compared along with the expression patterns. </p> <p> We looked at the regulation of lin-// in closely related nematode species like C. elegans, C. briggsae, C. remanei and Caenorhabditis n species. </p> <p> Consistent with this. expression of lin-11 is observed in the developing vulval cells. We are interested in understanding evolutionary changes in the regulation and function of lin-II in reproductive system </p> <p> /in-11 is a LIM homeodomain family member which IS involved in several developmental events. lin-11 role is documented in the thermoregulatory circuit specifying AIY interneuron, in chemosensory neurons like A W A and olfactory neurons A WS. During vulval development lin-II expression is dynamically expressed in subset of secondary lineage cells and is broadly expressed in all the cells indicating its role in cell identity and cell fusion of the vulval cells. lin-II is also required for the formation of vulval uterine connection which is the passage to lay eggs in the hermaphrodite. linllloss of function hermaphrodites have change in the axis of the secondary lineage cells during vulval development, uterine Jt cell migration defect, defects in the AIY, A W A and A WS interneurons resulting in egg-laying defect and protruding vulva and neuronal defects and reduced mating efficiency. </p> <p> The expression pattern of lin-If in closely related species is highly similar but not identical. From the sequence comparison of lin-If regulatory sequences a 1 kb conserved block of sequences have been identified which includes the regulatory sequences responsible for the expression of lin-If in vulva and Jt cells. We propose that cisregulatory elements controlling lin-If gene expression are slowly evolving though there is no change in the function which indicates that lin-If plays critical role during the development of the vulva and other tissues. </p> / Thesis / Master of Science (MSc)

Evolutionary variations during vulval development in C. elegans and its related nematode species are well analyzed. The formation of C. elegans vulva involves many complex cell-cell interactions that are mediated through well conserved EGF/EGFR/RAS, LIN12/Notch and WNT signaling pathways. These pathways specify distinct cell fates of the six epidermal vulval precursor cells (VPCs), P(3-8).p. pry-1/Axin in C. elegans is identified as a part of destruction box complex that mediates (beta)-catenin degradation and is known to negatively regulate canonical Wnt signaling pathway during its development. I focused on the genetic analysis of pry-1 I Axin function in C. briggsae, sister species to C. elegans, to study inter-species comparisons of vulva formation. Three alleles, lin(sy5353), lin(sy5411) and lin(sy5270) were genetically mapped to LG I using standard genetic and in del mapping techniques. Interestingly, a unique simultaneous Multi vulva and Vulvaless (Muv-Vul) phenotype was observed during vulva formation in Cbr-pry-1 alleles, resulting from the varying induction potentials of the VPCs along the A/P axis, compared to Cel-pry-1 animals. In order to analyze these phenotypic differences between Cel-pry-1 and Cbr-pry-1 in greater detail, I dissected vulval development in sy5353 animals. VPC competence analysis was done through cell lineages and ablations studies, while the C. briggsae vulval cell fate markers were used for cell fate specification analysis. Cell ablations revealed that P7.p and P8.p in Cbr-pry-1 animals exhibited non-competence towards anchor cell signaling. Additionally, gonad-independent inductions was observed in P(3-8).p cells and they adopted 2° cell fate specifications. Using RNAi approach, Cbr-pry-1 interactions with other vulval pathway genes were dissected and it was observed that Cbr-lin-12 is involved in VPC competence of P7.p and Cbr-pop-1 exhibited different regulatory levels during vulval development compared to C. elegans. Thus, it can be inferred that the mechanisms of vulva formation in C. briggsae has evolved through changes in the competence of VPCs. / Thesis / Master of Science (MS)

Entomopathogenic nematodes (EPN) (Heterorhabditis bacteriophora and H. megidis) and entomopathogenic fungi (EPF) (Metarhizium anisopliae and Beauveria bassiana) were evaluated for efficacy against masked chafer white grub, Cyclocephala spp., under laboratory and greenhouse conditions, as well as their efficacy against various grub stages in the field.  Under both laboratory and greenhouse conditions, additive interactions were found between EPN and EPF in their combined application against Cyclocephala spp., except a few observations that showed antagonism or synergism.  Significantly greater control occurred from the combination of a nematode and a fungus compared with a fungus alone, but not compared with a nematode alone.  The combined effect did not differ significantly for nematode and fungi applied simultaneously or at different times.  EPF had no significant impact on EPN infection and production of infective juveniles (IJs) in grub carcasses.  Nematodes alone or in combination with fungi were comparable to the insecticide Merit 75 WP (imidacloprid) against 3rd instar Cyclocephala spp in the greenhouse. Efficacy of EPF and EPN varied dramatically between field sites and conditions; EPN and EPF applied alone or in combination were less effective than Merit 75 WP in >50% field trials, but some EPN + EPF treatments were more effective than the insecticide in reducing grub numbers.  EPN and EPF showed better potential than insecticides for providing extended control of white grubs in the subsequent generation.  In addition, the sub-lethal effects of EPF on southern masked chafer, C. lurida, were investigated.  Neither M. anisopliae nor B. bassiana had a sub-lethal effect on grub weight gain, adult longevity, oviposition, pupation and eclosion.  Finally, interaction between H. bacteriophora and M. anisopliae was examined to determine the potential of the nematode in improving fungal distribution in soil.  H. bacteriophora enhanced fungal distribution in sandy loam soil without grass thatch, but not in sandy soil with thatch.  In both soil types, soil depths significantly affected nematode and fungal distribution. In water profile, M. anisopliae conidia germinated hyphae that attached to sheath of H. bacteriophora IJs, which molted to detach from the fungus.  IJs mortality and virulence were not affected by the presence of M. anisopliae. / Ph. D.

Potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are economically important pests of potato (Solanum tuberosum) crops in potato growing regions worldwide. Integrated management is under threat, with effective nematicides increasingly being withdrawn on environmental and health grounds. Alternative strategies are urgently needed and trap cropping could be one of them. The non-tuber-bearing Solanum sisymbriifolium is regarded as an effective trap crop for PCN with strong hatching ability and immunity to PCN infection and has been used in the UK and The Netherlands. However, its mode of action is unknown. In order to shed light on the mode of action so that a novel control strategy could be identified, the interactions between G. pallida and S. sisymbriifolium were investigated using in vitro bioassays. In choice assays, G. pallida J2s were equally attracted to the roots of S. sisymbriifolium and to those of S. tuberosum. However, potato root diffusate (PRD), which is routinely used to induce PCN hatch, failed to attract G. pallida J2s in chemotaxis bioassays, indicating hatching factors (HFs) and soluble compounds present in PRD are not involved in attraction of G. pallida J2s to potato roots. The J2s invaded the roots of S. sisymbriifolium in large numbers but failed to develop further. To facilitate continuous observation of nematode development, a novel in vitro method was devised with the use of Pluronic F-127, which requires no sterilisation, and the life cycle of G. pallida was successfully observed in S. tuberosum roots. Quantitative real-time polymerase chain reaction analyses of defence related genes of S. tuberosum and S. sisymbriifolium infected with G. pallida revealed up-regulation of the chitinase gene (ChtC 2.1) at 3 days post inoculation in S. sisymbriifolium but not in S. tuberosum. Electrospray ionisation-mass spectrometry analyses of root exudate extracts of the two Solanum species and subsequent bioassay-guided fractionation showed that the HF of S. sisymbriifolium differs from that of S. tuberosum. Previously, attention had been solely paid to the hatching ability of the root exudate of S. sisymbriifolium, but this study revealed for the first time that the aerial part extract possesses a significant hatching ability.

2007-2009 metais buvo tiriamas auksinio bulvinio nematodo ir blyškiojo bulvinio nematodo paplitimas Vilniaus regione. Tyrimai buvo atliekami Vilniaus regiono bulvių augintojų, dekoratyvinių, sodo ir miško augalų augintojų ir šiltnamių kontroliuojamuose ūkiuose. Iš vieno hektaro buvo imami 4 dirvožemio pavyzdžiai, o iš bulvių sėklininkystės ūkių ir ūkių, kuriuose auginama dauginimui skirta sodinamoji medžiaga, buvo imami 8 pavyzdžiai. Valstybinės augalų apsaugos tarnybos Fitosanitarinių tyrimų laboratorijoje pavyzdžiai buvo ištirti naudojant centrifugavimo Schuiling‘o centrifugos metodą. Apibendrinus 2007-2009 metų tyrimų duomenis nustatyta, kad buvo ištirtas dirvožemis 428-iuose kontroliuojamuose ūkiuose, kurių bendras tirtų laukų plotas buvo 877,51 ha. Iš viso buvo paimta 4343 pavyzdžiai. Iš kurių 652 buvo užkrėsti auksiniu bulviniu nematodu. Globodera pallida nebuvo identifikuota. Daugiausiai užkrėstų pavyzdžių nustatyta Švenčionių ir Trakų rajonuose (37,5 ir 33,6 % nuo tirtų tame rajone pavyzdžių). Mažiausiai užkrėstas Ukmergės ir Širvintų rajonų (12,03 ir 11,69 % nuo tirtų tame rajone pavyzdžių) dirvožemis. / The research of the spread of Globodera rostochiensis (Wollenweber) Behrens and Globodera pallida (Stone) Behrens was conducted in the Vilnius region in 2007 - 2009. It was done by potato cultivators of the Vilnius region, by cultivators of decorative, garden and forest plants as well as by controlled greenhouse farms. During the research 4 samples were taken from each 1 hectare and 8 samples from potato seed farms as well as farms where planting material was cultivated for the reproduction purposes. Each sample was tested in the Phytosanitary Research Laboratory of the State Plant Protection Service by using the Schuiling centrifuge method. When summarising the research data of 2007 - 2009 it was found out that the soil test was conducted in 428 controlled farms with the total tested field area of 877.51 ha. 4343 samples were taken in total, 652 of which were contaminated with the Golden Nematode. Pale cyst Nematode was not identifyed. The highest number of contaminated samples was found in Švenčionių and Trakų districts (37.5 % and 33.6 % of the tested samples respectively). Meanwhile the least contaminated soil was in Ukmergės and Širvintų districts (12.03% and 11.69% of the tested samples respectively).

Master of Science / Department of Statistics / Leigh Murray / The southern root-knot nematode (SRKN) and the weedy perennials, yellow nutsedge (YNS) and purple nutsedge (PNS) are simultaneously occurring pests in the irrigated agricultural soils of southern New Mexico. Previous research has very well characterized SRKN, YNS and PNS as a mutually-beneficial pest complex and has revealed their enhanced population growth and survival when they occur together. The density of nutsedge in a field could be used as a predictor of SRKN juveniles in the soil. In addition to SRKN, which is the most harmful of the plant parasitic nematodes, in southern New Mexico, other species or categories of nematodes could be identified and counted. Some of them are not as damaging to the plant as SRKN, and some of them may be essential for soil health. The nematode species could be grouped into categories according to trophic level (what nematodes eat) and herbivore feeding behavior (how herbivore nematodes eat). Subsequently, three ratios of counts were calculated for trophic level and for feeding behavior level to investigate the soil nematode community structure. These proportions were modeled as functions of the weed hosts YNS and PNS by generalized linear regression models using the logit link function and three probability distributions: the Binomial, Zero Inflated Binomial (ZIB) and Binomial Hurdle (BH). The latter two were used to account for potential high proportions of zeros in the data. The SAS NLMIXED procedure was used to fit models for each of the six sampling dates (May, July and September) over the two years of the alfalfa study. General results showed that the Binomial pmf generally provided the best fit, indicating lower zero-inflation than expected. Importance of YNS and PNS predictors varied over time and the different ratios. Specific results illustrate the differences in estimated probabilities between Binomial, ZIB and BH distributions as YNS counts increase for two selected ratios.

Sous le terme de maladies mitochondriales, on désigne des maladies multi-systémiques ou à expression tissu-spécifique dues à un déficit de la phosphorylation oxydative qui est assurée par le fonctionnement de 5 complexes protéiques enzymatiques (chaîne respiratoire) parmi lesquelles 13 sous-unités sont codées par le génome mitochondrial, les autres par le génome nucléaire. Ces pathologies recouvrent donc en pratique des maladies génétiques par mutation de l’ADN mitochondrial (ADNmt) mais aussi des maladies génétiques à hérédité mendélienne classique. Dans les cytopathies mitochondriales liées à des mutations de gènes nucléaires, il existe deux sortes de gènes (i) à effet direct correspondant à des gènes codant pour les sous-unités protéiques de la chaîne respiratoire ou leur assemblage, et (ii) à effet indirect correspondant à des gènes codant pour des protéines impliquées dans le maintien et la réplication de l'ADN mitochondrial. Des mutations dans cette dernière classe de gènes peuvent s'accompagner d'anomalies quantitatives ou qualitatives de l'ADNmt : déplétion de l'ADNmt (réduction majeure du nombre de molécules d'ADNmt) et délétions multiples.Après des dosages enzymatiques de l’activité des complexes respiratoires mitochondriaux chez les patients, le ou les types de complexes altérés sont connus. Un grand nombre de gènes mutés responsables de pathologies mitochondriales ont été identifiés, tous codant des constituants des différents complexes de la chaîne respiratoire. Ces dernières années, le groupe d’Agnès Rötig (Hôpital Necker, Paris) a identifié de nouveaux gènes grâce à une approche gènes candidats ou grâce à des tours de génome de familles consanguines de patients qui permettent de délimiter une région chromosomique portant la mutation à l’état homozygote. La validation de l’effet délétère de la mutation identifiée se fait en général en utilisant des organismes modèles d’étude comme les cellules humaines en culture ou bien la levure. Cependant, il reste un grand nombre de cas où la mutation n’a pas pu être identifiée, soit parce que le déficit de tel ou tel complexe ne met pas en jeu un des composants connus de ce complexe ou bien plusieurs complexes de la chaîne respiratoire sont déficitaires mettant en jeu, dans un grand nombre de cas, le maintien de l’ADN mitochondrial pour lequel peu de gènes sont connus.Au laboratoire d’Orsay, nous disposons de deux organismes modèles d’étude, la levure S. cerevisiae et le nématode C. elegans. La levure S. cerevisiae est l’organisme modèle de choix pour étudier les fonctions mitochondriales grâce à ses caractéristiques comme la respiration facultative, mais aussi et surtout par la puissance de sa génétique et le fait que les mitochondries peuvent être transformées. Cependant de par sa respiration facultative et sa division clonale, elle ne se prête pas facilement à des études sur la stabilité de l’ADNmt. En effet, S. cerevisiae perd très facilement son ADNmt après inactivation d’un grand nombre de gènes impliqués dans pratiquement toutes les voies de la biogenèse mitochondriale. Cette levure ne peut donc pas être utilisée de façon simple pour l’étude de la transmission de l’ADN mitochondrial. C’est pourquoi nous nous sommes alors intéressés à l’autre organisme modèle développé au laboratoire, le nématode C. elegans dont ses caractéristiques en font un excellent modèle complémentaire à la levure. / Mitochondrial respiratory chain diseases of nuclear origin represent one of the major causes of metabolic disorders. These diseases are characterized by a huge clinical and genetic heterogeneity which is a major problem in identifying the disease causing gene. Although several gene mutations have already been found in some patients or families, the disease causing gene of the majority is yet to be determined. The overall structure and gene content of the mitochondrial genome and the proteins required for mtDNA transactions are largely conserved from yeast to human offering the opportunity to use animal models to understand the molecular basis of mitochondrial dysfunctions. To expand the number of human candidate genes of mitochondrial diseases involved in mtDNA maintenance, we have developed in this study, the nematode Caenorhabditis elegans as a model organism to identify new proteins involved in mtDNA maintenance by combining RNAi and ethidium bromide exposure. We have developed a large-scale screening method of genes required for mtDNA maintenance in the worm and initially indentified four new C. elegans genes (atad-3, dnj-10, polrmt, phi-37 and immt-1) involved in mtDNA stability. The human homologs of these genes (ATAD3, DNAJA3, POLRMT and ATP5A1) can be now considered as candidate genes for patients with quantitative mtDNA deficiencies. Using our screening design we have begun to screen all the C. elegans genes encoding mitochondrial proteins. Of the 721 estimated C. elegans mitochondrial genes homologous to human genes, we have tested 185 genes and found that 41 genes are required for the maintenance of the mitochondrial genome in post mitotic cells. These genes fall into three main functional categories of metabolism, protein synthesis and oxidative phosphorylation. Finally, in this study, we investigated the reversibility of mtDNA depletion with drugs to counteract POLG dificiency. Three molecules, Chlorhexidine, Resveratrol and Bezafibrate, have been tested to restore normal mtDNA content and worm life cycle. These experiments hold promise for future work using C. elegans as a pharmacological model for mitochondrial diseases.Altogether, the data generated in this work is a starting point for promising advances in the mitochondrial field, showing the relevance of the nematode as a model organism to study fundamental processes as well as human health research.