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Thesis (Ph.D)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Copper oxychloride is a fungicide that is extensively used in vineyards in the Western Cape to treat and prevent fungal diseases. It is however not clear what the effects are on soil organisms, which play an important role in soil fertility, in South African soils. There is paucity of data linking results obtained in the laboratory to effects observed in the field, which will only become useful if a clear relation can be demonstrated. The aims of this study were to: ~ Determine the effects of copper oxychloride on field populations of earthworms and simultaneously monitor lysosomal membrane stability, measured as neutral red retention time (NRRT). ~ Validate experimental field studies by doing inventories of earthworm populations in long-term sprayed vineyards. ~ Determine the LC50 of copper oxychloride and simultaneously measuring NRRT, and linking them to the experimental field studies. ~ Conduct bioassays, burrowing activity- and soil-avoidance experiments to investigate their relations to earthworm population responses in the experimental field studies. Earthworms were sampled by hand-sorting in the field tests on treated and untreated field plots in the Western- (October 1998 - July 1999) and Northern Cape (April 1998 - October 1999). Soil samples and worms were analysed for copper contents and coelomocytes of live earthworms were used to conduct the neutral red retention assays. Acute toxicity tests were conducted over a period of 28 days during which the earthworms (Eisenia fetiday were exposed to different concentrations of copper oxychloride. Change in biomass and mortality were measured as endpoints, as well as NRRT. Bioassays, burrowing activity and soil-avoidance were conducted by exposing Aporrectodea caliginosa to grassland- and vineyard soil as well as grassland soil spiked with 60 J.1g.g-1copper in the form of copper oxychloride. Growth and mortality were recorded in the bioassays as well as copper concentrations In earthworm body tissues and substrates used over a period of28 days. Burrowing activity and soil-avoidance were determined by measuring the length of tunnels burrowed by A. caliginosa in soil profiles over a period of 4 days under different exposure regimes. Results from the field tests showed that spraying of copper oxychloride had a negative effect on earthworm populations at the prescribed application rates. NRR T in earthworms from the exposure plots was significantly (p<0.05) lower after just one spraying application. It was concluded that spraying copper oxychloride at prescribed application rates caused a decrease in field populations of earthworms and that NRRT was an early and reliable biomarker since it was indicative of later effects observed at the population level. Results obtained from the field inventory of earthworms in vineyards at Nietvoorbij, Robertson end Worcester confirmed data from the two field studies. The calculated LC50 of 882.78 I1g.g-1 for copper oxychloride and 519.40 I1g.g-1 for copper was ecologically relevant if a safety factor of 10 was applied. NRRT which manifested earlier than effects on biomass change in the acute toxicity tests, were significant when viewed against the background of responses of field populations of earthworms. From the bioassay experiments it was found that A. caliginosa exposed to copper oxychloride spiked soil had significantly (p<0.05) higher weight loss and mortality than those in grassland- and vineyard soil. This indicated that changes in biomass and mortality were indicative of population responses in the field and can be considered as ecologically relevant. Burrowing activity of A. caliginosa was significantly (p<0.05) lower in vineyard and copper oxychloride spiked soil than in grassland soil. Similarly in the soil avoidance experiments it was found that A. caliginosa avoided vineyard- and copper oxychloride contaminated soil. It is therefore concluded that burrowing activity and soil avoidance were ecologically relevant endpoints since they corresponded with population responses in the field. The study thus revealed that the long-term usage of copper oxychloride could have negative effects on earthworm populations. The spraying of copper oxychloride can have important implications on the sustainable use of agricultural soils since earthworms and other soil organisms play such an important role in soil fertility. The use of biomarkers and other ecotoxicological indicators can provide an early warning that soil organisms are under environmental stress. / AFRIKAANSE OPSOMMING: Die fungisied koperoksichloried word wyd gebruik in die Wes-Kaap om swamsiektes in wingerde te beheer en te voorkom. Dit is egter nie bekend wat die effek daarvan op Suid Afrikaanse grondbiota, wat 'n belangrike rol speel in grondvrugbaarheid, is nie. Daar is ook 'n tekort aan inligting wat die resultate van laboratoriumondersoeke in verband bring met veldstudies. Die doelstellings van die studie was om: ~ Die effek van koperoksichloried op erdwurmpopulasies in die veld te ondersoek en terselfdertyd membraanstabiliteit, as moontlike biomerker, gemeet as neutraal rooi retensietye (NRRT), te monitor. ~ Die geldigheid van eksperimentele veldstudies te toets deur ook grondanalises te doen in wingerde wat oor langtermyn met koperoksichloried bespuit is. ~ Die LC50 van koperoksichloried vir erdwurms te bepaal en terselfdertyd NRR T te meet asook om dié gegewens in verband te bring met die resultate van seisoenale veldstudies oor die uitwerking op erdwurmpopulasies. ~ Bio-evaluerings ("bioassays"), tonnelaktiwiteit- en vermydingseksperimente te onderneem en die verband tussen die toksiteitstoetse en populasieresponse, soos waargeneem in die veld, te ondersoek. Erdwurms is versamel deur handsortering tydens die veldtoetse in die Wes- (Oktober 1998 - Julie 1999) en Noord-Kaap (April 1998 - Oktober 1999) op kontrole en bespuite persele. Grondmonsters en erdwurms is spektrofotometries geanaliseer om koperinhoude te bepaal. Die selomosiete van lewende wurms is gebruik om NRR T te bepaal. Akute toksisiteitstoetse is uitgevoer oor 'n tydperk van 28 dae waartydens Eisenia fetida blootgestel is aan verskillende koperoksichloried konsentrasies. Veranderinge in biomassa en mortaliteit is bepaal asook NRRT. Bioevaluerings ("bioassays"), tonnelaktiwiteit- en vermydingseksperimente IS uitgevoer deur Aporrectodea caliginosa bloot te stel aan grasveld- en wingerdgrond asook grasveldgrond wat met koperoksichloried gekontamineer is. Groei en mortalitiet is bepaal in die "bioassays" asook koperkonsentrasies in die grond en erdwurm liggaamsweefsels oor 'n tydperk van 28 dae. Tonnelaktiwiteit en grondvermyding is bepaal deur die lengte van tonnels wat deur A. caliginosa gegrawe is te meet oor 'n tydperk van vier dae vir die verskillende blootgestelde groepe. Die resultate het aangedui dat koperoksichloriedbespuiting 'n negatiewe invloed het op erdwurmpopulasies teen die voorgeskrewe toedieningsprogram. NRRT in erdwurms van die blootstellingperseel, was beduidend (p<0.05) laer na 'n enkele bespuiting. Daar is verder bevind dat NRR T 'n betroubare en vroeë biomerker is, aangesien dit 'n aanduiding gegee het van latere effekte wat op populasievlak na vore getree het. Veldopnames in Nietvoorbij, Robertson en Worcester het die geldigheid van data verkry uit die veldstudies ondersteun. Die berekende LC50 van 882.78 ug.g" vir koperoksichloried en 519.40 ug.g" VIr koper was ekologies relevant indien 'n veiligheidsfaktor van 10 toegepas is. NRRT se ekologiese relevansie is bevestig deur dit te vergelyk met response wat in die veldtoetse waargeneem is. Deur bioevalueringseksperimente is bevind dat gewigsverlies en mortaliteit van A. caliginosa beduidend hoër was in koperoksichloried gekontamineerde grond as in die grasveld- (kontrole) en wingerdgronde. Veranderinge in biomassa en mortalitiet was aanduidend van populasieresponse soos waargeneem in die veldstudies en kan dus as ekologies relevante eindpunte beskou word. Tonnelaktiwiteit van A. caliginosa was beduidend (p<0.05) laer in wingerd- en koperoksichloried gekontamineerde grond as in grasveldgrond. Dieselfde is gevind in die grondvermydingstoetse waar A. caliginosa wingerd- en koperoksichloried gekontamineerde grond vermy het. Dit kan dus afgelei dat tonnelaktiwiteit en grondvermyding ook ekologies bruikbare eindpunte is aangesien dit verband hou met populasieresponse soos waargeneem in die veldstudies. Hierdie studie het getoon dat die herhaalde gebruik van koperoksichloried 'n nadelige invloed kan hê op erdwurmbevolkings. In die lig van die belangrike rol wat erdwurms en ander grondorganismes speel in grondvrugbaarheid kan die oormatige gebruik van hierdie fungisied ernstige implikasies inhou vir volhoubare benutting van landbougronde. Die gebruik van biomerkers en ander ekotoksikologiese eindpunte kan egter as vroeë waarskuwingsmetode dien dat die grondorganismes onder omgewingstres verkeer.

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Fruit trees are exposed to various factors that can adversely affect the production of quality fruit. These factors can directly affect the fruit and the health of the tree and can be classified according to their biotic or abiotic nature, such as pathogenic attacks and drought stress respectively. One of the cultural practices used commercially to address these stresses is the application of fungicides and bactericides. The fruit production industry is under severe pressure from consumers, retailers and environmentalists, locally and internationally, to reduce chemical applications to fruit and fruit trees. The use of natural plant defence elicitor compounds and nutrients offer a potential alternative to fungicide and bactericide sprays and may also increase fruit quality and size as result of a reduction of plant stress. Trials were conducted to evaluate the efficiency of natural plant defence elicitors i.e. salicylic acid (SA) and flavonoids, in addition to mineral nutrients and bactericide/fungicides, on peach (incidence of Xanthomonas infection), plum (induced drought stress and Mg/Mn deficiencies) and apple (Mg/Mn deficiencies) fruit and trees against specified biotic or abiotic stress factors. Trial on Prunus persica cv. ‘Sandvliet’were conducted over two seasons (2008/2009 and 2011/2012) on a commercial site, Protea Farm, in the Worcester area in the Western Cape Province. During the 2008/2009 season the SA (AlexinTM, AlexiboostTM) containing treatments were applied first at 75% petal drop at concentrations of 125 and 250 ml. 100 L-1. The copper (StCu, Cu)-containing treatment was applied at 50% petal drop, while dichlorophen (XanbacTM) treatments were applied at fruit set at concentrations of 150, 300 and 200 ml. 100 L-1. The flavonoid (CroplifeTM) treatment was applied at the start of petal drop at a concentration of 150 ml.100 L-1. During the 2011/2012 season, a new flavonoid (CropbiolifeTM) treatment, as well as potassium (K-MaxTM) treatment, were incorporated into the trial and applied at concentrations of 150 and 500 ml. 100 L-1 respectively. Additionally a SA (AlexinTM) and dichlorophen (XanbacTM) treatments that performed well during the first season, were incorporated into the second season with application times and rates similar to the first season’s protocol. In addition to fruit size and quality measurements, the percentage Xanthomonas infection was determined on the leaves and fruit of the experimental trees. The SA (AlexinTM) containing treatment significantly reduced the incidence of Xanthomonas infection on leaves and fruit compared to the control in the first season. However, results varied between the two seasons, as no significant difference from the control could be obtained in the following season. The AlexinTM treatments also significantly increased the fruit size and quality. The flavonoid (CropbiolifeTM) and K (K-MaxTM) containing treatments similarly reduced the Xanthomonas infection on leaves and fruit, as well as increasing the fruit size and quality in the second season. The dichlorophen (XanbacTM) containing treatment recorded varying results as it significantly reduced the Xanthomonas infection on the fruit only in the second season. The plum trials were conducted over the 2011/2012 season on ‘Laetitia’ and ‘Songold’ plum trees, Welgevallen Experimental Farm, Stellenbosch University. Three SA (AlexinTM, AlexSal and RezistTM) containing foliar treatments were applied on the ‘Laetitia’ trees. Only two SA (AlexinTM, AlexSal) containing foliar treatments were applied on the ‘Songold’ trees. Additionally, a foliar treatment containing only K, Ca, Mg and B, was applied in both the ‘Laetitia’ and ‘Songold’ trials. All the treatments were first applied at 75% petal drop, at the same concentration of 250 ml. 100 L-1. Additionally to fruit size and quality, the mineral nutrient content of the leaves and fruit was determined. The ascorbic acid and glutathione content was determined in fruit at harvest and again after storage. None of the treatments had a positive effect on the parameters measured, except the SA (AlexinTM) containing treatments which increased the titratible acidity (TA) in both at harvest and after storage. The treatments also did not alleviate the induced stress compared to the control. The apple and plum tree trials were conducted over the 2011/2012 season in a semi-closed greenhouse, at the Welgevallen Experimental Farm, Stellenbosch. Magnesium (Mg) and Manganese (Mn) deficiencies were induced in one-year-old ‘Royal Beaut’ apple and ‘Laetitia’ plum trees planted in 10 L nursery bags, by omitting these nutrients from a standard Long Ashton soil application. Foliar treatments of Mg (MagMaxTM) and Mn (ManMaxTM) containing sprays were subsequently applied at concentrations of 250 and 75 ml. 100 L-1 respectively, after deficiency symptoms for these nutrients were visually observed. Mineral nutrient analysis of the leaves were analysed on the 13th of February for the plums and 30th of March 2012, for the apples. The Mn (ManMaxTM) containing treatment successfully overcame the Mn induced deficiency. The Mg (MagMaxTM) containing treatment did not overcome the induced Mg deficiency and was probably due to the deficient nitrogen levels in the plants, caused by an error in the initial Long Ashton nutrient solution formulation. In conclusion AlexinTM, K-MaxTM and CropbiolifeTM have shown their ability to decrease Xanthomonas infection in peaches. Additionally to their positive effect on fruit size and quality on the peaches. SA was not able to overcome the induced stress on plums, but had a positive effect on the fruit quality and size. The ManMaxTM been proven to overcome the induced Mn deficiency, while MagMaxTM was unsuccessful to overcome the Mg deficiency. / AFRIKAANSE OPSOMMING: Vrugtebome word blootgestel aan verskeie faktore wat die produksie van kwaliteit vrugte nadelig kan beïnvloed. Hierdie faktore kan ‘n direkte invloed hê op die vrugte en op die gesondheid van die boom en kan geklassifiseer word op grond van hulle biotiese of abiotiese natuur, soos patogeen infeksie en droogte stres onderskeidelik. Van die produksie praktyke wat gebruik word sluit in die toepassing van verskillende swamdoders en bakterisiede. Die vrugtebedryf is onder geweldige druk van verbruikers, die kleinhandel en omgewingsbewustes om die toediening van chemikalieë aan vrugte en vrugtebome te verminder. Die gebruik van natuurlike plant verdediging stimulerende verbindings en nutriënte, bied 'n moontlike alternatief tot die spuit van swamdoders en bakterisiede, en kan ook moontlik ʼn bydrae maak tot verbeterde vrugkwaliteit en -grootte. Proewe is uitgevoer om die effektiwiteit van die natuurlike plant verdediging stimulante, salisielsuur (SA) en flavonoïede, addisioneel tot verskillende voedingstowwe en bakterieële / swamdoders op perske, pruim en appels teen Xanthomonas infeksie, droogte stres en Mg / Mn tekorte as biotiese en abiotiese stres faktore onderskeidelik te evalueer. Die Prunus persica ‘Sandvliet’ proewe is oor twee seisoene (2008/2009 en 2011/2012) op 'n kommersiële perseel, Protea Farm, in die Worcester-area in die Wes-Kaap Provinsie, uitgevoer. Gedurende die 2008/2009 seisoen is die SA (AlexinTM, AlexiboostTM) bevattende behandelings eers toegedien by 75% blomblaarval teen konsentrasies 125 en 250 ml. 100 L-1. Die koper (StCu, Cu) bevattende behandeling is toegedien by 50% blomblaarval, terwyl die dichlorofen (XanbacTM) bevattende behandelings toegedien is by vrugset, teen konsentrasies van 150, 300 en 200 ml. 100 L-1. Die flavonoïde (CroplifeTM) behandeling is toegedien by die begin van blomblaarval teen 'n konsentrasie van 150 ml. 100 L-1. Gedurende die 2011/2012 seisoen was 'n nuwe flavonoïd (CropbiolifeTM) en ‘n kalium (K-MaxTM) behandeling toegevoeg tot die eksperiment, met ʼn toediening teen konsentrasies van onderskeidelik 150 en 500 ml. 100 L-1. Daarbenewens is die SA (AlexinTM) en dichlorofen (XanbacTM) behandeling van die 2008/2009 seisoen herhaal teen dieselfde konsentrasies en toedieningstye soos in die protokol van die eerste seisoen. Behalwe vir die bepaling van vruggrootte en –kwaliteit, is die persentasie Xanthomonas infeksie op blare en vrugte ook bepaal. Die SA (AlexinTM) bevattende behandeling het die voorkoms van Xanthomonas infeksie op die blare en vrugte betekenisvol verminder in vergelyking met die kontrole. Resultate het egter gewissel in die daaropvolgende seisoen en geen beduidende verskille tussen die behandelings is waargeneem nie. Hierdie SA-bevattende behandelings het ook tot ‘n toename in vruggrootte en -kwaliteit gelei. Die flavonoïde bevattende behandelings, (CropbiolifeTM) en K (K-MaxTM), het soortgelyke afnames in Xanthomonas infeksie op die blare en vrugte in die tweede seisoen getoon, sowel as ‘n toename in vruggrootte en -kwaliteit. Die dichlorofen (XanbacTM) bevattende behandeling het variërende resultate getoon aangesien dit slegs tot ‘n beduidende afname in Xanthomonas infeksie op die blare en vrugte in die tweede seisoen kon lei. Pruim proewe is uitgevoer in die 2011/2012 seisoen op ‘Laetitia’ en ‘Songold’ pruimbome te Welgevallen Proefplaas, Universiteit van Stellenbosch. Drie SA (AlexinTM, AlexSal en RezistTM) bevattende blaar behandelings is toegedien op die ‘Laetitia’ bome. Slegs twee SA (AlexinTM, AlexSal) blaar behandelinge is toegedien op die ‘Songold’ bome. ʼn Verdere K, Ca, Mg en B blaar behandeling is ook toegedien in beide die ‘Laetitia’ en ‘Songold’ proewe. Al die behandelings se eerste toediening het saamgeval met 75% blomblaarval, teen dieselfde konsentrasie van 250 ml. 100 L-1. Addisioneel tot vruggrootte en –kwaliteit, is die mineraal element inhoud van die blare en vrugte bepaal. Die askorbiensuur en glutatioon inhoud is bepaal in die vrugte met oes asook na opberging. Geen behandeling het 'n positiewe uitwerking op die parameters wat gemeet is getoon nie, behalwe een van die SA (AlexinTM) bevattende behandelings wat die titreerbare sure (TS) verhoog het in beide kultivars. Die behandelings kon ook nie die geïnduseerde stres verlig in vergelyking met die kontrole nie. Die appel- en pruim proewe is uitgevoer gedurende die 2011/2012 seisoen in 'n semi-geslote glashuis te Welgevallen Proefplaas, Universiteit van Stellenbosch. Magnesium (Mg) en Mangaan (Mn) tekorte is geïnduseer in een-jaar-oue ‘Royal Beaut’ appel en ‘Laetitia’ pruim bome, aangeplant in 10L kwekerysakke, deur dié elemente uit ʼn toediening van standaard Long-Ashton voedingsoplossing aan die grond weg te laat. Mg (MagMaxTM) en Mn (ManMaxTM) bevattende blaarspuite is daarna toegepas teen onderskeidelik konsentrasies van 250 en 75 ml. 100 L-1. ʼn Minerale analise van die blare is uitgevoer op 13 Februarie, op die pruime en 30 Maart 2012, op die appels. Die Mn (ManMaxTM) bevattend behandeling het die Mn-geïnduseerde tekort verlig. Die Mg (MagMaxTM) bevattende behandeling het nie die geïnduseerde Mg-tekort verlig nie. Dit is moontlik toe te skryf aan die stikstof tekort in die plante wat te wyte was aan ʼn foutiewe Long Ashton voedingsoplossing formulasie wat aanvanklik toegedien is. Ten slotte het AlexinTM, K-MaxTM en CropbiolifeTM getoon dat hul die vermoë het om Xanthomonas infeksie te verminder, asook om vruggrootte en kwaliteit in perskes te verbeter. SA was nie in staat om die geïnduseerde stres op pruime te oorkom nie, maar het 'n positiewe uitwerking op die vruggroote en kwaliteit gehad. ManMaxTM het getoon dat dit ʼn geïnduseerde, visuele Mn tekort kan oorkom, terwyl MagMaxTM onsuksesvol was om die Mg-tekort te oorkom

Thesis (PhD(Agric)(Botany and Zoology))--University of Stellenbosch, 2001. / Copper oxychloride (Cu2CI(OH)3) is a broad-spectrum fungicide, intensively sprayed in many South African vineyards and orchards. It is necessary to find accurate and effective methods of monitoring the effects of this fungicide on the biota of the agricultural environment. The use of biomarkers may be a possible method to employ for this purpose. This study investigated the effects of copper, as a result of copper oxychloride exposure, on the biology of the common garden snail Helix aspersa, as welI as a number of cellular responses to exposure to the fungicide. The possible use ofthese responses as biomarkers was also investigated. Two groups of snails were exposed to 80 and 240 tJg g-! copper oxychloride respectively, for six weeks. A third group served as control. On a weekly basis, body mass, number of eggs produced, neutral red retention (NNR) times of haemocytic lysosomes, and body copper concentrations were determined for each individual. At the end of the experiment, the digestive glands, ovotestes and hermaphrodite ducts of a number of snails were prepared for histological analysis. The following parameters were investigated: tubule area, epithelium height and area in the digestive gland, spermatozoan area in the vesicula seminalis and ovotestis, as wen as oocyte numbers in the ovotestis. To test the validity of the laboratory results, a field survey was conducted. Snails were colIected from an uncontaminated vineyard and on two occasions from a contaminated vineyard in the Western Cape. The same cellular responses were investigated as in the laboratory study. The results showed that growth, egg production and hatching success in Helix aspersa were affected by experimental exposure to copper oxychloride. In both the laboratory study and field survey, copper in the body of H. aspersa was shown to be compartmentalized and the digestive gland was the most important site of copper accumulation. NNR times of haemocytic Iysosomes were shown to be affected by copper oxychloride exposure, already during the first week of exposure. A time evolution of copper accumulation and lysosomal damage existed. Epithelium height and area of digestive gland tubules, and spermatozoan and oocyte densities in the ovotestis, were also affected by copper oxychloride exposure and the concomitant copper burdens in the respective organs. Through the field survey it was ascertained that these histopathological changes were largely dependent on exposure time. It was concluded that lysosomal response of H. aspersa haemocytes, as measured by the NNR time assay, could be considered a useful biomarker of copper oxychloride exposure, since it provides an early warning of stress induced by this fungicide. Changes in digestive gland epithelium cells, and gametes in the ovotestis, can also possibly serve as biomarkers of copper oxychloride exposure. However, these can not serve as an early warning. All of the cellular responses identified in the present study can be used in combination with other cellular and physiological parameters and toxicological endpoints in order to improve the reliability and accuracy of interpretations regarding cause and effect.