Four species of acacia of Auatralian origin are associated with the wattle industry in South Africa. Black wattle, Acacia mollissima willd., is the most important of these and is today almost exclusively cultivated. The tree grows successfully only in a limited area of rich soil and high rainfall and is easily affected by adverse conditions caused by insect pests, frost damage and drought. Expansion of the area under cultivation is therefore, not feasible, although the world demand for vegetable extracts far exceeds the available supply. The remaining species such as green (Acacia decurrrens willd.) and silver wattles (Acacia dealbata Link.) possess many desirable characteristics which resist such adverse factors. Their barks, however, give reddish extracts, which are considered unsuitable for tannery usage because of the red colour they impart to the leather. Hybridisation studies, involving the crossing of green and silver wattle with the black, and aimed at produc1ng progenies containing many of the desirable characterlstics of the parent plants, are thus a natural result and have been in progress for a considerable period. Summary, p. i.

Proanthocyanidins (PAs), also known as condensed tannins (CTs), are oligomers or polymers of flavan-3-ols. They have a very important role in plant-environment interactions, such as defense against herbivory and pathogens. They may also be important for light stress tolerance. In poplar, PAs can make up as much as 30% of the leaf dry weight. The synthesis of PAs in poplar was demonstrated to be inducible by both abiotic and biotic stresses. The B-ring hydroxylation pattern of flavan-3-ols directly affects the structure of PAs, and many studies have shown that B-ring hydroxylation of PAs is associated with their biological functions, including effects on leaf litter decomposition rate and anti-herbivore activity. Anthocyanins are very important colour pigments in plants, and share the intermediate leucoanthocyanidin with PAs. The role of anthocyanins in plant pollination, light stress tolerance, and seed dispersal has been well studied. A change in B-ring hydroxylation pattern can modify the colour of anthocyanins dramatically and also change their biological function. Flavonoid 3’-hydroxylase and flavonoid 3’, 5’-hydroxylase (F3’H and F3’5’H) are the two enzymes involved in determining the B-ring hydroxylation pattern of both PAs and anthocyanins. The objective of this study is to characterize the possible role of flavonoid 3’, 5’-hydroxylase in PA and anthocyanin biosynthesis in poplar. A candidate F3’5’H was identified in the Populus trichocarpa genome database based on previous expression profile experiments, and called PtF3’5’H1. The predicted protein shares high sequence similarity with previously characterized F3’5’H proteins from other plants. To test the function of PtF3’5’H1 directly, transgenic hybrid poplar plants overexpressing PtF3’5’H1 were generated. Preliminary LC-MS analysis showed that the hydroxylation pattern of the PA in the transgenic poplars was not significantly modified. Likewise, overexpression of PtF3’5’H1 in poplar did not change the overall amount of PAs. These results suggest that overexpression of PtF3’5’H1 in poplar is not sufficient to modify the B-ring hydroxylation pattern of PA, and that additional factors may be required. By contrast, the transgenic PtF3’5’H1 overexpressing poplar did show an alteration in anthocyanin profile. In leaves of transgenic poplars, several putative delphinidin derivatives were observed at greater levels than in the wild type, indicating that PtF3’5’H1 participates in the anthocyanin production in poplar. However, transiently introducing PtF3’5’H1 into Nicotiana benthamiana had no effect on the anthocyanin profile in this plant. I conclude that PtF3’5’H1 is very likely to be involved in the anthocyanin synthesis in poplar, while the function of PtF3’5’H1 in poplar PA synthesis has yet to be demonstrated. / Graduate

O principal impacto ambiental do processo de beneficiamento da casca de coco verde é a geração de resíduo líquido com elevada carga orgânica. Este trabalho teve como objetivo aplicar o sistema convencional de lodos ativados para o tratamento biológico do líquido gerado no beneficiamento da casca de coco verde, além de utilizar o teste de taxa de consumo de oxigênio (TCO) para avaliar a toxicidade do efluente ao lodo aeróbio. O reator de escala laboratorial foi inoculado com lodo proveniente de uma estação de tratamento de esgoto em escala plena. Inicialmente o sistema foi alimentado com efluente sintético contendo sacarose e nutrientes para posterior adição progressiva do resíduo líquido proveniente da reciclagem da casca de coco verde. Para o ensaio de respirometria, as composições testadas foram 25, 50, 75 e 100% de resíduo líquido em termos de carga orgânica e o teste se baseou na medição manual da TCO ao longo do tempo. Para cada composição de efluente se verificou o valor máximo de TCO, o nível de toxicidade devido ao líquido da casca de coco verde (LCCV) e a indicação de inibição permanente. O parâmetro usado nos cálculos de toxicidade e de inibição permanente foi a taxa de consumo de oxigênio específica (TCOe) máxima. Para o monitoramento do sistema de fluxo contínuo, foram empregados os parâmetros pH, índice volumétrico de lodo (IVL), oxigênio dissolvido (OD), demanda química de oxigênio (DQO), taninos totais e sólidos suspensos. O reator foi operado com carga orgânica volumétrica (COV) de 0,8 a 2,9 gDQO/L.d e tempo de detenção hidráulica (TDH) de 26,7 d. Devido à presença natural de leveduras no resíduo líquido, os testes de respirometria foram realizados com o efluente tanto esterilizado quanto in natura. Como os resultados preliminares de respirometria não foram conclusivos, optou-se por começar a adaptação do reator de fluxo contínuo com a menor porcentagem testada: 25% de resíduo líquido. Nos testes adicionais de respirometria não houve toxicidade ao lodo e a TCOe foi diretamente proporcional à concentração de resíduo líquido. A remoção média de DQO obtida durante a operação (LCCV a 100%) foi de 81% com amostra filtrada e 82% com amostra bruta. Houve diminuição significativa da concentração de taninos, de 5332 para 1206 mg/L, em média. No início da operação ocorreu o decaimento da concentração de oxigênio dissolvido (OD) no reator para valores menores que 1 mg/L em decorrência do aumento da concentração de biomassa, mas esta situação não afetou a remoção de matéria orgânica e nem a sedimentabilidade do lodo. A remoção de matéria orgânica obtida com o tratamento em escala laboratorial do LCCV mostra que o sistema convencional de lodos ativados pode ser aplicado como alternativa no tratamento biológico dos resíduos líquidos do beneficiamento da casca de coco verde, porém a demanda elevada de oxigênio pode ser um entrave na operação em escala plena. / The main disadvantage of coconut recycling is the highly pollutant wastewater generated during the process. The objectives of this study were to treat the coconut husk liquor (CHL) using a conventional activated sludge system (AS) and to use the oxygen uptake rate (OUR) test for determining the initial effluent composition of reactor feeding and for wastewater toxicity assessment. The laboratory-scale reactor was inoculated with sludge from a full-scale sewage treatment plant. Firtly the system was fed with synthetic effluent containing saccarose and nutrients which was substituted by CHL. For the OUR test the substrate compositions were 25, 50, 75 and 100% of CHL. For each substrate composition, it was verified the maximum OUR value, the toxicity level and permanent inhibition caused by CHL. The maximal specific oxygen uptake rate (SOUR) was the parameter applied for the toxicity assessment and permanent inhibition calculation. The lab-scale reactor was monitored on the basis of pH, sludge volume index, dissolved oxygen, chemical oxygen demand (COD), total tannins and suspended solids. The respirometry assays were carried out with sterilized and natural effluent because of yeast contamination in CRW. Because of the lack of conclusive results in the first respirometry test, it was chosen the lower composition for reactor adaptation beginning: CRW 25%. In the additional OUR tests it was not detected toxicity and the SOUR values were as higher as CRW concentration. After initializing the reactor adaptation with CRW 25%, the percentage changed to 30% and than increased by 10% each 2 or 3 days until the operation beginning. The mean COD removal values were 81% (filtered sample) and 82% (raw sample). The mean tannin concentration decreased from 5332 to 1206 mg/L. The organic removal and sludge settleability were not affected by the DO concentration decrease to less than 1 mg/L due to biomass increase. The organic removal obtained in the lab-scale AS treatment shows that this system can be an alternative for CHL biological treatment, even though the high oxygen demand could be a disadvantage at full-scale treatment.

O principal impacto ambiental do processo de beneficiamento da casca de coco verde é a geração de resíduo líquido com elevada carga orgânica. Este trabalho teve como objetivo aplicar o sistema convencional de lodos ativados para o tratamento biológico do líquido gerado no beneficiamento da casca de coco verde, além de utilizar o teste de taxa de consumo de oxigênio (TCO) para avaliar a toxicidade do efluente ao lodo aeróbio. O reator de escala laboratorial foi inoculado com lodo proveniente de uma estação de tratamento de esgoto em escala plena. Inicialmente o sistema foi alimentado com efluente sintético contendo sacarose e nutrientes para posterior adição progressiva do resíduo líquido proveniente da reciclagem da casca de coco verde. Para o ensaio de respirometria, as composições testadas foram 25, 50, 75 e 100% de resíduo líquido em termos de carga orgânica e o teste se baseou na medição manual da TCO ao longo do tempo. Para cada composição de efluente se verificou o valor máximo de TCO, o nível de toxicidade devido ao líquido da casca de coco verde (LCCV) e a indicação de inibição permanente. O parâmetro usado nos cálculos de toxicidade e de inibição permanente foi a taxa de consumo de oxigênio específica (TCOe) máxima. Para o monitoramento do sistema de fluxo contínuo, foram empregados os parâmetros pH, índice volumétrico de lodo (IVL), oxigênio dissolvido (OD), demanda química de oxigênio (DQO), taninos totais e sólidos suspensos. O reator foi operado com carga orgânica volumétrica (COV) de 0,8 a 2,9 gDQO/L.d e tempo de detenção hidráulica (TDH) de 26,7 d. Devido à presença natural de leveduras no resíduo líquido, os testes de respirometria foram realizados com o efluente tanto esterilizado quanto in natura. Como os resultados preliminares de respirometria não foram conclusivos, optou-se por começar a adaptação do reator de fluxo contínuo com a menor porcentagem testada: 25% de resíduo líquido. Nos testes adicionais de respirometria não houve toxicidade ao lodo e a TCOe foi diretamente proporcional à concentração de resíduo líquido. A remoção média de DQO obtida durante a operação (LCCV a 100%) foi de 81% com amostra filtrada e 82% com amostra bruta. Houve diminuição significativa da concentração de taninos, de 5332 para 1206 mg/L, em média. No início da operação ocorreu o decaimento da concentração de oxigênio dissolvido (OD) no reator para valores menores que 1 mg/L em decorrência do aumento da concentração de biomassa, mas esta situação não afetou a remoção de matéria orgânica e nem a sedimentabilidade do lodo. A remoção de matéria orgânica obtida com o tratamento em escala laboratorial do LCCV mostra que o sistema convencional de lodos ativados pode ser aplicado como alternativa no tratamento biológico dos resíduos líquidos do beneficiamento da casca de coco verde, porém a demanda elevada de oxigênio pode ser um entrave na operação em escala plena. / The main disadvantage of coconut recycling is the highly pollutant wastewater generated during the process. The objectives of this study were to treat the coconut husk liquor (CHL) using a conventional activated sludge system (AS) and to use the oxygen uptake rate (OUR) test for determining the initial effluent composition of reactor feeding and for wastewater toxicity assessment. The laboratory-scale reactor was inoculated with sludge from a full-scale sewage treatment plant. Firtly the system was fed with synthetic effluent containing saccarose and nutrients which was substituted by CHL. For the OUR test the substrate compositions were 25, 50, 75 and 100% of CHL. For each substrate composition, it was verified the maximum OUR value, the toxicity level and permanent inhibition caused by CHL. The maximal specific oxygen uptake rate (SOUR) was the parameter applied for the toxicity assessment and permanent inhibition calculation. The lab-scale reactor was monitored on the basis of pH, sludge volume index, dissolved oxygen, chemical oxygen demand (COD), total tannins and suspended solids. The respirometry assays were carried out with sterilized and natural effluent because of yeast contamination in CRW. Because of the lack of conclusive results in the first respirometry test, it was chosen the lower composition for reactor adaptation beginning: CRW 25%. In the additional OUR tests it was not detected toxicity and the SOUR values were as higher as CRW concentration. After initializing the reactor adaptation with CRW 25%, the percentage changed to 30% and than increased by 10% each 2 or 3 days until the operation beginning. The mean COD removal values were 81% (filtered sample) and 82% (raw sample). The mean tannin concentration decreased from 5332 to 1206 mg/L. The organic removal and sludge settleability were not affected by the DO concentration decrease to less than 1 mg/L due to biomass increase. The organic removal obtained in the lab-scale AS treatment shows that this system can be an alternative for CHL biological treatment, even though the high oxygen demand could be a disadvantage at full-scale treatment.

Cotton seedling disease complex is caused by a number of pathogens inducing similar symptoms and can lead to thin, uneven stands that grow slowly and yield poorly. Preliminary work indicated that a set of high tannin cotton lines developed and released in 1989 by Texas A&M AgriLife Research, (Smith et al., 1990a, Smith et al., 1990b, Schuster et al., 1990) may possess increased resistance to disease. This evidence, along with additional studies that show a clear role of tannin in disease resistance, suggest that these high tannin cotton lines may prove useful in breeding for increased resistance to cotton seedling disease complex. High tannin cotton lines were screened for their resistance to Rhizoctonia solani and Pythium aphanidermatum. While no high tannin germplasm line was more resistant to R. solani than the resistant control, Tamcot SP 21, the potential for significant gains from selection was demonstrated. Fifteen high tannin lines expressed resistance to P. aphanidermatum equal to the resistant control, Tamcot Sphinx (El-Zik and Thaxton, 1996). This resistance was not shown to be correlated with tannin content, though it is still unclear whether or not this lack of correlation is real or due to limitations in measuring tannin in infected seedlings.

Xanthomonas axonopodis pv. malvacearum(Smith), Pythium aphanidermatum(Edson) and Rhizoctonia solani(Kuhn) have all been shown to cause significant yield losses in cotton. Previous work has demonstrated that a set of high tannin cotton germplasm lines developed and released in 1989 by Texas A&M AgriLife Research may possess resistance to these three diseases. In this research, the usefulness of these high tannin lines in breeding for resistance to these pathogens as well as the role of tannin in conferring this resistance were examined. The high tannin lines were screened for their resistance to Xanthomonas axonopodis pv. malvacearum and five highly resistant lines were subjected to generation means and diallel analysis in order to determine the relative importance of different types of gene action in conferring resistance as well as which lines would be most useful in breeding for resistance. The effect of selection for R. solani resistance was measured and selected lines were subjected to diallel analysis. Lines showing elevated resistant to P. aphanidermatum were subjected to a single cycle of selection and the effect of selection was measured. Lines possessing high degrees of P. aphanidermatum resistance were subjected to generation means and diallel analysis. The importance of tannin content in conferring resistance to X. axonopodis and R. solani was also measured. Four high tannin lines were found to be not different from Tamcot Sphinx in their resistance to Xam. Generation means analysis for Xam resistance showed that in the five high tannin lines tested, additive gene effects were of the greatest importance. No correlation between tannin content and Xam resistance was detected. Under the conditions of this study tannin content was also shown to be unimportant in R. solani resistance. Two rounds of recurrent phenotypic selection were effective in increasing the resistance of selected HT lines to R. solani. One cycle of phenotypic selection for P. aphanidermatum resistance was found to produce significant improvements in seven of the most resistant HT lines. Generation means analysis for P. aphanidermatum resistance showed that in the HT lines tested, additive genetic effects were by far the most important.

Condensed tannins (CT) play an integral role in terrestrial nutrient cycling. Despite being the fourth most abundant terrestrial biochemical product, the regulatory processes of tannin production in plants and their subsequent ecological influences are not completely understood. The defining characteristic of CT’s is their affinity for proteins, though they willingly interact with minerals, carbohydrates, and other polyphenolic compounds. Previous tannin investigations in agriculture have centred on bioactivity related to ruminant digestive physiology and pathology. These studies have revealed that CT’s have the potential to increase liveweight gain, wool production, and sheep ovulation rates, prevent pasture bloat, reduce enteric and stockpiled manure greenhouse gas (GHG) emissions, and control gastrointestinal parasites. The present study has explored how tannin-containing resident plant material and applied manure derived from tannin-containing beef cattle diets influences tame forage biomass yield, proximate analysis, and tannin production, as well as residual fall soil nutrient status, in southern Manitoba. To test these hypotheses, we conducted an experiment over two growing seasons (2007-2008) where in the fall of 2007, both tannin-derived (sainfoin, Onobrychis viciifolia) and non-tannin (alfalfa, Medicago sativa) composted beef manures were applied at a rate of 44.8 t/ha in a randomized split plot fashion, including a manure-free control, on a four repetition randomized complete block design which included both tannin-containing and non-tannin containing forage monocultures and mixtures. Plant samples were harvested in two cuts, and soil measurements were taken in the fall of both years. It was found that across all sainfoin treatments, manure origin did not have a significant effect on plant CT concentration, which was extremely variable within treatments and cuts. Over four harvest dates, sainfoin CT concentrations ranged from 14.1 g/kg to 91.9 g/kg in monoculture plots, and 16.6 g/kg to 123 g/kg when grown in a mixture with meadow brome. In most cases, the presence of either manure type did not significantly affect soil nutrient status or forage yield, though results were similarly variable. Using a stepwise regression which included all soil and plant measurements across all cuts and treatments, it was found that NDF (R2 = 0.548) and plant phosphorus (R2 = 0.126) were the only significant model contributors to tannin concentration in sainfoin at P < 0.15. These findings suggest that nutrient effects of beef cattle manure are not realized in either plant or soil in the year following application, and consequently, that tannin agronomy requires longer-term analysis.

A study was conducted to determine if sainfoin, a condensed tannin (CT) containing legume, fed to beef cattle as hay or silage during a western Canadian winter would result in a reduction in methane (CH4) emissions without compromising animal performance. Forty yearling beef steers were fed four diets in a factorial design consisting of two legume forage species (sainfoin or alfalfa) and two preservation methods (silage or hay) over 15 weeks (wks). For each sample wk, animal weight, 24-h CH4 expiration and rumen fluid samples were obtained. Rumen methanogens were characterized using terminal restriction fragment length polymorphisms analysis. Specific bacteria were quantified with real-time polymerase chain reaction analysis. Sainfoin silage (SS) and sainfoin hay (SH) contained 11.9 and 10.5 mg g-1 of CT respectively and supported an acceptable growth rate for backgrounding steers. A decline (P < 0.05) in enteric CH4 formation could only be detected from SH-fed animals compared to alfalfa hay (AH) fed animals when CH4 was expressed as L d-1 or L kg BW-1. The rumen archaeal community structure of experimental animals remained stable regardless of diet type or sample wk. Structural carbohydrate-fermenting bacteria were suppressed in silage diets. Methanogens were less abundant in the rumen fluid samples of steers fed SS but not SH.

Extraction of mangrove bark with 4.0% aqueous sodium sulfite and 0.4% aqueous sodium carbonate at 100° and 2 hours gives 24-26% yields compared with extraction by water at 70° for 2 hours which gives 21%. The hot water extracts are more acidic (pH 3.6) than is the sulfite extract (pH 5.6); both are reasonably reactive toward formaldehyde (Stiasny number 70.6 using water and 85.4 using aqueous sulfite-carbonate). The ¹³C NMR spectra of <I>R. mucronata </I>shows this tannin to have phloroglucinolic A-rings with hydroxy groups at C-5 and C-7 and pyrogallolic B-rings with hydroxy groups at C-3', C-4', and C-5'. The interflavanoid linkages are C-4→C-8 and C-4→C-6. The ¹³C NMR spectra also indicate the presence of a considerable amount of carbohydrate which is shown to be mainly rhamnose, glucose, arabinose, and uronic acids. Sulfitation of <I>R. mucronata </I>bark reduces the total carbohydrate and the rhamnose extracted but increases the amount of arabinose and uronic acids. The bark storage period has significant effects on the pH and the reactivity of the aqueous tannin solution. Barks stored for <4 weeks produce higher extraction yields than those stored for > 6 weeks and contain significantly larger amount of reactive tannin and have shorter gel times. The reactivity of bark extracts towards formaldehyde can be controlled either by limiting the duration of bark storage to 4 weeks or by maintaining the aqueous tannin solution at pH <10.0. The aqueous tannin solution from <I>R. mucronata </I>exhibited properties such as viscosity, solubility and tackiness which were superior to those from the <I>R. apiculata </I>extract while the mixed <I>R. mucronata-R. apiculata </I>aqueous tannin solution had properties in between these. These barks could be used singly or together as a source of tannin without the bond strength of the resulting glued joints being significantly affected. The "honeymoon" bonding technique improved the bond strengths of joints made using sulfited tannin but is suitable only for tannin solutions containing > 4% aqueous sodium hydroxide. The viscosity of sulfited tannin adhesives is influenced by (a) the amount of aqueous sodium hydroxide added to the aqueous tannin solution, and (b) the lapse time, i.e. the period between the addition of sodium hydroxide to the aqueous tannin solution and the addition of phenol-resorcinol-formaldehyde (PRF) resin, hardener, and paraformaldehyde. The present study shows that with the addition of 5% w/w sodium hydroxide and at a curing temperature of 40° the sulfited tannin extracts from the bark of mangrove trees can replace about 50% w/w of the PRF resin in cold-setting wood adhesives with the production of bond strengths comparable to those produced by 100% PRF resin.

Condensed tannins (CT) play an integral role in terrestrial nutrient cycling. Despite being the fourth most abundant terrestrial biochemical product, the regulatory processes of tannin production in plants and their subsequent ecological influences are not completely understood. The defining characteristic of CT’s is their affinity for proteins, though they willingly interact with minerals, carbohydrates, and other polyphenolic compounds. Previous tannin investigations in agriculture have centred on bioactivity related to ruminant digestive physiology and pathology. These studies have revealed that CT’s have the potential to increase liveweight gain, wool production, and sheep ovulation rates, prevent pasture bloat, reduce enteric and stockpiled manure greenhouse gas (GHG) emissions, and control gastrointestinal parasites. The present study has explored how tannin-containing resident plant material and applied manure derived from tannin-containing beef cattle diets influences tame forage biomass yield, proximate analysis, and tannin production, as well as residual fall soil nutrient status, in southern Manitoba. To test these hypotheses, we conducted an experiment over two growing seasons (2007-2008) where in the fall of 2007, both tannin-derived (sainfoin, Onobrychis viciifolia) and non-tannin (alfalfa, Medicago sativa) composted beef manures were applied at a rate of 44.8 t/ha in a randomized split plot fashion, including a manure-free control, on a four repetition randomized complete block design which included both tannin-containing and non-tannin containing forage monocultures and mixtures. Plant samples were harvested in two cuts, and soil measurements were taken in the fall of both years. It was found that across all sainfoin treatments, manure origin did not have a significant effect on plant CT concentration, which was extremely variable within treatments and cuts. Over four harvest dates, sainfoin CT concentrations ranged from 14.1 g/kg to 91.9 g/kg in monoculture plots, and 16.6 g/kg to 123 g/kg when grown in a mixture with meadow brome. In most cases, the presence of either manure type did not significantly affect soil nutrient status or forage yield, though results were similarly variable. Using a stepwise regression which included all soil and plant measurements across all cuts and treatments, it was found that NDF (R2 = 0.548) and plant phosphorus (R2 = 0.126) were the only significant model contributors to tannin concentration in sainfoin at P < 0.15. These findings suggest that nutrient effects of beef cattle manure are not realized in either plant or soil in the year following application, and consequently, that tannin agronomy requires longer-term analysis.