Isolation and characterization of the female sex pheromone of the sugar beet cyst nematode, heterodera schachtii, and an analysis of male precopulatory behaviour /

The sugar beet cyst nematode, Heterodera schachtii, is a major agricultural pest. The disruption of the mating behaviour of this plant parasite in the field may provide a means of biological control, and a subsequent increase in crop yield. The H. schachtii female sex pheromone, which attracts homospecific males, was collected in an aqueous medium and isolated using high performance liquid chromatography. Characterization of the male-attractive material revealed that it was heat stable and water soluble. The aqueous medium conditioned by female H. schachtii was found to be biologically active and stimulated male behaviour in a concentration dependent manner. The activity of the crude pheromone was specific to males of H. schachtii and did not attract second stage juveniles. Results indicated that vanillic acid, a putative nematode pheromone, is not an active component of the H. schachtii sex pheromone. Male H. schachtii exhibited stylet thrusting, a poorly understood behaviour of the male, upon exposure to the female sex pheromone. This behaviour appeared to be associated with mate-finding and was used as a novel indicator of biological activity in bioassays. Serotonin, thought to be involved in the neural control of copulatory behaviour in nematodes, stimulated stylet thrusting. However, the relationship between stylet thrusting induced by the sex pheromone and stylet thrusting induced by serotonin is not clear. Extracellular electrical activity was recorded fi-om the anterior region of H. schachtii males during stylet thrusting, and appeared to be associated with this behaviour. The isolation of the female sex pheromone of H. schachtii may, ultimately, lead to the structural identification and synthesis of the active substance for use in a novel biological control strategy.

Meloidogne incognita (nematode) parasitism of Lycopersicon esculentum (tomato) plants : Ethylene action in susceptible and resistant host responses

Involvement of ethylene in the etiology of tomato plants (Lycopersicon esculentum) infected with the root-knot nematode (Meloidogyne incognita) was investigated. Endogenous root concentrations of ethylene were not significantly different in uninfected resistant var. Anahu and susceptible var. Vendor plants. Exposure of resistant plants to high doses of infectious nematode larvae did not affect root ethylene concentrations during the subsequent 30 day period. The possibility that ethylene may be involved in the mechanism of resistance is therefore not supported by these experiments. In no experiments did ethylene concentrations in roots of susceptible plants increase significantly subsequent to ~ incognita infestation. This result is not consistent with the hypothesis in the literature which suggests that increased ethylene production accompanies gall formation. Growth of susceptible tomato plants was affected by ~ incognita infestation such that root weights increased (due to galling), stem heights decreased and top weights increased. The possibility that alterations in stem growth resulted from increased production of 'stress' ethylene is discussed. Growth of resistant plants was unaffected by exposure to high doses of ~ incognita and galls were never detected on the roots of these plants. Root ethane concentrations generally varied in parallel with root ethylene concentrations although ethane concentrations were without exception greater. In 4 of 6 experiments conducted ethane/ethylene ratios increased significantly with time. These results are discussed in the light of published data on the relationship between ethane and ethylene synthesis. The term infested is used throughout this thesis in reference to plants whose root systems had been exposed to nematodes and does not distinguish between the susceptible and resistant response.

CENTRAL NORTH ATLANTIC PALEOCEANOGRAPHY DURING THE MIDDLE PLEISTOCENE (ca. 726–603 ka): INSIGHTS FROM THE DINOFLAGELLATE CYST RECORD

Integrated Ocean Drilling Program (IODP) Site U1313, located at the northern boundary of the subtropical gyre in the central North Atlantic, lies within the southern part of the ice-rafted debris belt. Seventy-three palynological samples were studied from an uninterrupted interval ca. 726–603 ka (upper Marine Isotope Stage [MIS] 18 through lower MIS 15) to resolve conflicting paleoceanographic interpretations. Glacial stages were characterized by high productivity surface waters reflecting a southward shift of the Arctic Front. Sea surface salinities (SSSs) and sea surface temperatures (SSTs) were obtained by transfer functions using the Modern Analogue Technique. The lowest SSTs of 9ºC (±1.3) and 10ºC (±1.3) were recorded in glacial MIS 16 and MIS 18 respectively. However, these reconstructions are influenced by abundant heterotrophic taxa and may reflect elevated nutrient levels rather than lowered temperatures. Reworked palynomorphs uniquely indicate a Cretaceous as well as Paleozoic provenance for the first Heinrich-like events.

Studies on oxamyl : analytical method development and investigation of fate in peach seedlings and corn seeds

A high performance liquid chromatographic method employing two columns connected in series and separated~y·a.switching valve has been developed for the analysis of the insecticide/ nematicide oxamyl (methyl-N' ,N'-dimethyl-N-[(methylcarbamoyl) oxy]-l-thiooxarnimidate) and two of its metabolites. A variation of this method involving two reverse phase columns was employed to monitor the persistence and translocation of oxamyl in treated peach seedlings. It was possible to simultaneously analyse for oxamyl and its corresponding oxime (methyl-N',N'-dimethyl-N-hydroxy-l-thiooxamimidate}, a major metabolite of oxamyl in plants, without prior cleanup of the samples. The method allowed detection of 0.058 pg oxamyl and 0.035 p.g oxime. On treated peach leaves oxamyl was found to dissipate rapidly during the first two-week period, followed by a period of slow decomposition. Movement of oxamyl or its oxime did not occur in detectable quantities to untreated leaves or to the root or soil. A second variation of the method which employed a size exclusion column as·the first column and a reverse phase column as the second was used to monitor the degradation of oxamyl in treated, planted corn seeds and was suitable for simultaneous analysis of oxamyl, its oxime and dimethylcyanoformamide (DMCF), a metabolite of oxamyl. The method allowed detection of 0.02 pg oxamyl, 0.02 p.g oxime and 0.005 pg DMCF. Oxamyl was found to persist for a period of 5 - 6 weeks, which is long enough to permit oxamyl seedtreatment to be considered as a potential means of protecting young corn plants from nematode attack. Decomposition was found to be more rapid in unsterilized soil than in sterililized soil. DMCF was found to have a nematostatic effect at high concentrations ( 2,OOOpprn), but at lower concentrations no effect on nematode mobility was observed. Oxamyl, on the other hand, was found to reduce the mobility of nematodes at concentrations down to 4 ppm.

The determination of sinigrin in Brassica, and investigations into the use of allyl-isothiocyanate as a nematicide

The goal of this thesis was to study factors related to the development of Brassica juncea as a sustainable nematicide. Brassica juncea is characterized by the glycoside (glucosinolate) sinigrin. Various methods were developed for the determination of sinigrin in Brassica juncea tissue extracts. Sinigrin concentrations in plant tissues at various stages of growth were monitored. Sinigrin enzymatically breaks down into allylisothiocyanate (AITC). AITC is unstable in aqueous solution and degradation was studied in water and in soil. Finally, the toxicity of AITC against the root-lesion nematode (Pratylenchus penetrans) was determined. A method was developed to extract sinigrin from whole Brassica j uncea tissues. The optimal time of extraction wi th boiling phosphate buffer (0.7mM, pH=6.38) and methanol/water (70:30 v/v) solutions were both 25 minutes. Methanol/water extracted 13% greater amount of sinigrin than phosphate buffer solution. Degradation of sinigrin in boiling phosphate buffer solution (0.13%/minute) was similar to the loss of sinigrin during the extraction procedure. The loss of sinigrin from boiling methanol/water was estimated to be O.Ol%/minute. Brassica juncea extract clean up was accomplished by an ion-pair solid phase extraction (SPE) method. The recovery of sinigrin was 92.6% and coextractive impurities were not detected in the cleaned up extract. Several high performance liquid chromatography (HPLC) methods were developed for the determination of sinigrin. All the developed methods employed an isocratic mobile phase system wi th a low concentration of phosphate buffer solution, ammonium acetate solution or an ion-pair reagent solution. A step gradient system was also developed. The method involved preconditioning the analytical column with phosphate buffer solution and then switching the mobile phase to 100% water after sample injection.Sinigrin and benzyl-glucosinolate were both studied by HPLC particle beam negative chemical ionization mass spectrometry (HPLCPB- NCI-MS). Comparison of the mass spectra revealed the presence of fragments arising from the ~hioglucose moiety and glucosinolate side-chain. Variation in the slnlgrin concentration within Brassica juncea plants was studied (Domo and Cutlass cuItivars). The sinigrin concentration in the top three leaves was studied during growth of each cultivar. For Cutlass, the minimum (200~100~g/g) and maximum (1300~200~g/g) concentrations were observed at the third and seventh week after planting, respectively. For Domo, the minimum (190~70~g/g) and maximum (1100~400~g/g) concentrations were observed at the fourth and eighth week after planting, respectively. The highest sinigrin concentration was observed in flower tissues 2050±90~g/g and 2300±100~g/g for Cutlass and Domo cultivars, respectively. Physical properties of AITC were studied. The solubility of AITC in water was determined to be approximately 1290~g/ml at 24°C. An HPLC method was developed for the separation of degradation compounds from aqueous AITC sample solutions. Some of the degradation compounds identified have not been reported in the literature: allyl-thiourea, allyl-thiocyanate and diallyl-sulfide. In water, AITC degradation to' diallyl-thiourea was favored at basic pH (9.07) and degradation to diallyl-sulfide was favored at acidic pH (4 . 97). It wap necessary to amend the aqueous AITC sample solution with acetonitrile ?efore injection into the HPLC system. The acetonitrile amendment considerably improved AITC recovery and the reproducibility of the results. The half-life of aqueous AITC degradation at room temperature did not follow first-order kinetics. Beginning with a 1084~g/ml solution, the half-life was 633 hours. Wi th an ini tial AITC concentration of 335~g/ml the half-life was 865 hours. At 35°C the half-life AITC was 76+4 hours essentially independent of the iiisolution pH over the range of pH=4.97 to 9.07 (1000~g/ml). AITC degradation was also studied in soil at 35°C; after 24 hours approximately 75% of the initial AITC addition was unrecoverable by water extraction. The ECso of aqueous AITC against the root-lesion nematode (Pratylenchus penetrans) was determined to be approximately 20~g/ml at one hour exposure of the nematode to the test solution. The toxicological study was also performed with a myrosinase treated Brassica juncea extract. Myrosinase treatment of the Brassica juncea extract gave nearly quantitative conversion of sinigrin into AITC. The myrosinase treated extract was of the same efficacy as an aqueous AITC solution of equivalent concentration. The work of this thesis was focused upon understanding parameters relevant to the development of Brassica juncea as a sustainable nematicide. The broad range of experiments were undertaken in support of a research priority at Agriculture and Agri-Food Canada.

Freshwater dinoflagellates as proxies of cultural eutrophication: a case study from Crawford Lake, Ontario

Crawford Lake, Ontario, provides an ideal natural laboratory to study the response of freshwater dinoflagellates to cultural eutrophication. The anoxic bottom waters that result from meromixis in this small (2.4 ha) but deep (24 m) lake preserve varved sediments that host an exceptional fossil record. These annual layers provide dates for human activity (agriculture and land disturbance) around the lake over the last millennium by both Iroquoian village farmers (ca. A.D. 1268-1486) and Canadian farmers beginning ~A.D. 1883. The well established separate intervals of human activity around Crawford Lake, together with an abundance of available data from other fossil groups, allow us to further investigate the potential use of the cyst of freshwater dinoflagellates in studies of eutrophication. Cyst morphotypes observed have been assigned as Peridinium willei Huitfeldt-Kaas, Peridinium wisconsinense Eddy and Peridinium volzii Lemmermann and Parvodinium inconspicuum (Lemmermann) Carty. The latter two cyst-theca relationships were determined by culturing and by the exceptional preservation of thecae of P. inconspicuum in varves deposited at times of anthropogenic reductions in dissolved oxygen.

Palynology and foraminiferal geochemistry of the Lower Pleistocene Olduvai Subchron (ca. 1.8 Ma) in DSDP Hole 603C, western North Atlantic

Marine palynology and benthic and planktonic foraminiferal geochemistry are combined to reveal long- and short-term (Milankovitch-scale) paleoceanographic changes across the upper half of the Olduvai Subchron (ca. 1.86--1.77 Ma, lower Pleistocene) in DSDP Hole 603C from the lower New Jersey continental rise. Planktonic foraminiferal Mg/Ca ratios reveal annual sea-surface temperatures between 14.5° and 25°C, whereas modern values vary between 16° and 20°e. Despite evidence of downslope transport in much of the studied interval, dinoflagellate cyst and acritarch assemblages appear to reflect fluctuating temperate to subtropical water masses. These assemblages comprise both neritic and oceanic species, and are marked by a transition upsection from warm conditions, dominated by Lingulodinium machaerophorum, Polysphaeridium zoharyi and Cymatiosphaera? invaginata, to cooler conditions dominated by Filisphaera filifera. Combining dinoflagellate cyst proxies with planktonic foraminiferal geochemistry allows downslope transport events to be recognized during glacial episodes, and events dominated by intensified bottom-water circulation during interglacial episodes. Sixtytwo in-situ dinoflagellate cyst and acritarch taxa were recorded including several not previously described.

The Cenozoic Gonyaulacacean Dinoflagellate Genera Operculodinium Wall, 1967 and Protoceratium Bergh, 1881 and Their Phylogenetic Relationships

The palynology of Ocean Drilling Program Site 1007, leeward of the present Bahamas Bank, provides insights into upper Oligocene–lower Pleistocene dinoflagellate cyst associations in the tropical Americas. These associations are reviewed along with the sedimentary paleoenvironment to provide context for a morphological study of the cystdefined dinoflagellate Operculodinium bahamense and its comparison with the thecadefined dinoflagellate Protoceratium reticulatum which produces a cyst assignable to the cyst-defined genus Operculodinium. Detailed reconstructions of the tabulation in both species reveal strong similarities, having a sexiform hyposomal tabulation and L-type or modified L-type ventral organization. Protoceratium reticulatum has dextral torsion of the hypotheca, requiring assignation of the genus to the subfamily Cribroperidinioideae, whereas Operculodinium bahamense has neutral torsion requiring assignation to the subfamily Leptodinioideae. Results either imply polyphyletic origins for the genus Operculodinium or that combinations of ventral organization and torsion cannot always be applied rigidly to subdivide the family Gonyaulacaceae.