An investigation of ethylene inhibition of growth in etiolated Avena sativa coleoptiles /

Growth rates of etiolated Avena sativa coleoptiles in pH 7.0 buffered medium are stimulated in a synergistic manner by IAA and 320 ~l/l carbon dioxide. The suggestion that carbon dioxide stimulated growth involves dark fixation is supported by the ability of 1 mM malate to replace carbon dioxide, with neither factor able to stimulate growth in the presence of the other (Bown, Dymock and Aung, 1974). The regulation of Avena coleoptile growth by ethylene has been investigated in the light of this data and the well documented antagonism between carbon dioxide and ethylene in the regulation of developmental processes. The influence of various permutations of ethylene, IAA, carbon dioxide and malate on the rates of growth, l4c-bicarbonate incorporation, l4C-bicarbonate fixation, and malate decarboxylation have been investigated. In the presence of 320 ~l/l carbon dioxide, 10.8 ~l/l ethylene inhibited growth both in the absence and presence of 20 ~M IAA with inhibition times, of 8-10 and 12-13 minutes respectively. In contrast ethylene inhibition of growth was not significant in the absence of growth stimulation by CO2 or 1 mM malate, and the normal growth increases in response to CO2 and malate were blocked by the simultaneous application of ethylene. The rates of incorporation and dark fixation of l4C-bicerbonate were not measurably. influenced by ethylene, IAA or malate, either prior to or during the changes in growth ,ates induced by these agents. The data does not support the hypothesis that ethylene inhibition of growth results from an inhibition of dark fixation, but suggests that ethylene may inhibit a process which is subsequent to fixation.

Measurement of growth in the lichen Rhizocarpon geographicum using a new photographic technique

Lichenologists and users of lichenometry have long used calipers or photogrammetry to measure the growth of crustose lichens. Now, digital photography and popular computer software provide methodological alternatives. This thesis developed and tested a new methodology for tracking change and growth of the lichen, Rhizocarpon geographicum. Adobe Photoshop CS3 Extended software and a photographic time series (1996,2003,2006 and 2007) were used to measure thallus diameter, area, prothallus width and areolae area in 115 small R. geographicum thalli (0.53-1049.88 mm2 ). Measures of 8 diameters per thallus showed that change in diameter was highly variable and is a weak index of growth. Thallus area was a reliable measure of growth (power correlation, R2 = 0.89). Rapid, highly irregular growth occurred in small thalli «30 mm2 ), and steady, uniform growth occurred in larger thalli (>30 mm2 ). This new methodology is tedious but can potentially generate accurate and precise measures for even the tiniest of lichens.

The role of learning and growth hormone-releasing factor in the control of protein intake in rats /

Both learning and basic biological mechanisms have been shown to play a role in the control of protein int^e. It has previously been shown that rats can adapt their dietary selection patterns successfully in the face of changing macronutrient requirements and availability. In particular, it has been demonstrated that when access to dietary protein is restricted for a period of time, rats selectively increase their consumption of a proteincontaining diet when it becomes available. Furthermore, it has been shown that animals are able to associate various orosensory cues with a food's nutrient content. In addition to the role that learning plays in food intake, there are also various biological mechanisms that have been shown to be involved in the control of feeding behaviour. Numerous studies have documented that various hormones and neurotransmitter substances mediate food intake. One such hormone is growth hormone-releasing factor (GRF), a peptide that induces the release of growth hormone (GH) from the anterior pituitary gland. Recent research by Vaccarino and Dickson ( 1 994) suggests that GRF may stimulate food intake by acting as a neurotransmitter in the suprachiasmatic nucleus (SCN) and the adjacent medial preoptic area (MPOA). In particular, when GRF is injected directly into the SCN/MPOA, it has been shown to selectively enhance the intake of protein in both fooddeprived and sated rats. Thus, GRF may play a role in activating protein consumption generally, and when animals have a need for protein, GRF may serve to trigger proteinseeking behaviour. Although researchers have separately examined the role of learning and the central mechanisms involved in the control of protein selection, no one has yet attempted to bring together these two lines of study. Thus, the purpose of this study is to join these two parallel lines of research in order to further our understanding of mechanisms controlling protein selection. In order to ascertain the combined effects that GRF and learning have on protein intake several hypothesis were examined. One major hypothesis was that rats would successfully alter their dietary selection patterns in response to protein restriction. It was speculated that rats kept on a nutritionally complete maintenance diet (NCMD) would consume equal amount of the intermittently presented high protein conditioning diet (HPCD) and protein-free conditioning diet (PFCD). However, it was hypothesized that rats kept on a protein-free maintenance diet (PFMD) would selectively increase their intake of the HPCD. Another hypothesis was that rats would learn to associate a distinct marker flavour with the nutritional content of the diets. If an animal is able to make the association between a marker flavour and the nutrient content of the food, then it is hypothesized that they will consume more of a mixed diet (equal portion HPCD and PFCD) with the marker flavour that was previously paired with the HPCD (Mixednp-f) when kept on the PFMD. In addition, it was hypothesized that intracranial injection of GRF into the SCN/MPOA would result in a selective increase in HPCD as well as Mixednp-t consumption. Results demonstrated that rats did in fact selectively increase their consumption of the flavoured HPCD and Mixednp-f when kept on the NCMD. These findings indicate that the rats successfully learned about the nutrient content of the conditioning diets and were able to associate a distinct marker flavour with the nutrient content of the diets. However, the results failed to support previous findings that GRF increases protein intake. In contrast, the administration of GRF significantly reduced consumption of HPCD during the first hour of testing as compared to the no injection condition. In addition, no differences in the intake of the HPCD were found between the GRF and vehicle condition. Because GRF did not selectively increase HPCD consumption, it was not surprising that GRF also did not increase MixedHP-rintake. What was interesting was that administration of GRF and vehicle did not reduc^Mixednp-f consumption as it had decreased HPCD consumption.

Alicaligenes faecalis : identification and study of its antagonistic properties against Botrytis cinerea

A Gram negative aerobic flagellated bacterium with fungal growth inhibitory properties was isolated from a culture of Trichoderma harzianum. According to its cultural characteristics and biochemical properties it was identified as a strain of Alcaligenes (aeca/is Castellani and Chalmers. Antisera prepared in Balbc mice injected with live and heat-killed bacterial cells gave strong reactions with the homologous immunogen and with ATCC 15554, the type strain of A. taeca/is, but not with Escherichia coli or Enterobacter aerogens in immunoprecipitation and dot immunobinding assays. Growth of Botrytis cinerea Pers. and several other fungi was significantly affected when co-cultured with A. taeca/is on solid media. Its detrimental effect on germination and growth of B. cinerea has been found to be associated with antifungal substances produced by the bacterium and released into the growth medium. A biotest for the antibiotic substances, based on their inhibitory effect on germination of B. cinerea conidia, was developed. This biotest was used to study the properties of these substances, the conditions in which they are produced, and to monitor the steps of their separation during extraction procedures. It has been found that at least two substances could be involved in the antagonistic interaction. One of these is a basic volatile substance and has been identified as ammonia. The other substance is a nonvolatile, dialysable, heat stable, polar compound released into the growth medium. After separation of growth medium samples by Sephadex G-10 column chromatography a single peak with a molecular weight below 700 Daltons exhibited inhibitory activity. From its behaviour in electrophoretic separation in agarose gels it seems that this is a neutral or slightly positively charged.

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.

The effects of cultural conditions and parasitism on the lipid composition of choanephora cucubitarum (Berk. Rav.) thaxter /|nJohn M. Deven. -- 260 St. Catharines [Ont. : s. n.],

The fatty acid composition of the total, neutral, sterol, free fatty acid and polar-lipid fractions in the mycelium of Choanephora cucurbitarum was determined. The major fatty acids in all lipid fractions were palmitic, oleic, linoleic and y-linolenic acid. Different lipid fractions did not show any particular preference for any individual fatty acid; however, the degree of unsaturation was different in various lipid fractions. Addition of glutamic acid to the malt-yeast extract medium resulted in the biosynthesis of a number of long-chain fatty acids beyond y-linolenic acid. These fatty acids, e.g. C22~1' C24:0 and C26=Q were never observed to be present in the fungus when grown on a malt-yeast extract medium without glutamic acid. Furthermore, thin-layer chromatographic analysis showed a larger and denser spot of diphosphatidyl glycerol from the mycelium grown on the glutamic acid medium than from the control mycelium. Various cultural conditions such as temperature, age, pH, light and carbon:nitrogen ratio in the growth medium used in this study did not alter the qualitative profile of fatty acids normally present in the organism. Neither did these conditions stimulate the production of further long-chain fatty acids (C20 - C26) beyond y-linolenic acid as observed in growth media containing glutamic acid. These cultural conditions influenced the degree of unsaturation, this being due mainly to changes in the concentration of y-linolenic acid. The fatty acid pattern of the lipid fractions though the same qualitatively, differed quantitatively due to the variation in the y-linolenic acid content under different cultural conditions. The degree of unsaturation of various lipid fractions decreased with increases in temperature, light intensity and pH, but within each treatment the same pattern of decreasing degree of unsaturation with increasing age was observed. The cultural conditions, used in this study, are also known to influence the degree and rate of development of the parasite, Piptocephalis virginiana. A direct correlation was observed between the levels of y-linolenic acid in C. cucurbitarum during the early stages of growth (24 h) and the degree of parasitism of P. virginiana. The amount of y-linolenic acid present in the host mycelium was found to be unrelated to either the dry weight of the mycelium or to the total lipid contents. K. virginiana is confined to host species which produce y-linolenic acid in their mycelium. The lipid profile of the host, C. cucurbitarum, did not show a significant qualitative or quantitative change in the lipid profile as a result of infection by the parasite, P. virginiana,e However, an increase in the total lipid was observed in the infected host mycelium. The significance of these results is discussed.