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Call number: LD2668 .T4 1979 J65 / Master of Science

Thesis (M.S.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references.

Thesis--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references.

International trade in plants, especially with potting substrates, is recognised as the main pathway of plant pathogen dissemination on a global scale. In the last 20 years, the wide use of internet commerce has become common in the nursery sector and, due to the nature of online sales, may be aggravating this risk. Oomycetes in the genera Phytophthora, Pythium and Phytopythium, cause a range of important plant diseases, responsible for serious economic and biological losses. This research focused on the detection of Oomycetes in imported potted ornamental plants in the UK and The Netherlands, including internet sales and asymptomatic plants. Isolation techniques and molecular protocols were developed to quantify pathogen load in ornamental plants, using TaqMan PCR and Next Generation Sequencing (NGS) to assess Oomycete diversity using a multi-locus approach. Survival of Phytophthora cinnamomi and Fusarium verticillioides was estimated in two commercial potting mixes used in ornamental plant production. Oomycetes were detected in all samples analysed with the NGS approach, with 38 Phytophthora spp. and 48 Pythium/Phytopythium spp. identified. Phytophthora ramorum, P. alni subsp. alni and P. cryptogea were common. TaqMan PCR quantification showed high numbers of Oomycetes in all samples, especially in substrates, followed by roots and baiting waters. During sampling by isolation, Pythium kashmirense was recovered from Viburnum plicatum, the first record of this species in the UK. The survival experiment showed that Fusarium verticillioides remained viable after 17 months, whereas Phytophthora cinnamomi was viable up to 7 months after inoculation. This work clearly demonstrated the widespread presence of Oomycete pathogens in the plants for planting pathway. Moreover, the protocols developed and findings of this work contribute greatly to the understanding of the potential for pathogens to spread in the international horticultural trade and may help to improve plant biosecurity protocols in the UK and Europe.

To evaluate the effects of stock plant nutrition and propagation medium moisture content on stem cutting propagation, two separate studies were conducted. In one study, stem cuttings of Juniperus horizontalis Moench ‘Wiltonii’, Rhododendron (Lindl.) Planch ‘Hino-Crimson’, and Ilex crenata ‘Helleri’, were propagated in 1 peat : 1 perlite (v/v) at 125 %, 250 %, 375 %, 500 %, and 625 % moisture. Stem cutting survival and rooting, midday xylem water potential, and basal water uptake all generally increased with increasing medium moisture level. Incidence of cutting basal rot was not directly related to medium moisture level, but was related to species and growth stage of the stock plant. Basal water uptake by cuttings was highest during the first few days after insertion and thereafter decreased until root emergence. Propagation was most successful in the wettest medium (625 %). In a second study, containerized stock plants of Ilex crenata Thunb. ‘Rotundifolia’ were liquid-fertilized with 25, 50, 100, 200, and 300 mg N · liter⁻¹ in two forms (100 % NH₄NO₃ or 50 % Urea + 50 % NH₄NO₃) in a factorial treatment design. Percent rooting of stem cuttings decreased linearly with fertilizer rate. Leaf and stem percent N increased from suboptimal to excessive levels with fertilizer rate. Total nonstructural carbohydrates (TNC) levels increased in leaves and remained constant in stems with increasing fertilizer rate. Stem cutting percent moisture was highly correlated with fertilizer rate. The form of N applied made no statistical difference in these trends. The decrease in percent rooting with increasing fertilizer rate was attributed to increases in shoot growth activity. / Master of Science

published_or_final_version / Architecture / Master / Master of Architecture

Thesis (Ph. D.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references.

Thesis (MScAgric)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Phaeomoniella chlamydospora is the main causal organism of Petri disease, which causes severe decline and dieback of young grapevines (1-7 years old) and also predisposes the wood for infection by other pathogens. Knowledge about the epidemiology and especially inoculum sources of this disease is imperative for subsequent development of management strategies. Through isolation studies it was shown that Pa. chlamydospora is mainly distributed through infected propagation material in South Africa. However, the infection pathways and inoculum sources in grapevine nurseries are still unclear. The only existing method to detect this pathogen in various media is by means of isolation onto artificial growth media. This has proven to be problematic since this fungus is extremely slow growing (up to 4 weeks from isolation to identification) and its cultures are often over-grown by co-isolated fungi and bacteria before it can be identified. The aim of this study was (i) to develop a protocol for the molecular detection of Pa. chlamydospora in grapevine wood, and (ii) to use this protocol along with others, to test different samples (water, soil, rootstock and scion cuttings and callusing medium) collected from nurseries in South Africa at different nursery stages for the presence of Pa. chlamydospora. A protocol was developed and validated for the molecular detection of Pa. chlamydospora in grapevine wood. Firstly, several previously published protocols were used to develop a cost-effective and time-efficient DNA extraction method from rootstock pieces of potted grapevines. Subsequently, PCR amplification using species-specific primers (Pch1 and Pch2) was found to be sensitive enough to detect as little as 1 pg of Pa. chlamydospora genomic DNA from grapevine wood. The protocol was validated using various grapevine material from 3 different rootstock cultivars (101-14 Mgt, Ramsey and Richter 99) collected from each of 3 different nurseries, including grapevines that were subjected to hot water treatment. The basal end of the rootstock was parallel analysed for Pa. chlamydospora using isolations onto artificial medium and molecular detection. The identity of PCR products obtained from a subset of samples, that only tested positive for Pa. chlamydospora based on molecular detection, was confirmed to be Pa. chlamydospora specific through restriction digestion with AatII. Molecular detection was found to be considerably more sensitive than isolations, detecting Pa. chlamydospora from samples with positive as well as negative isolations. On average, the molecular technique detected Pa. chlamydospora in 80.9% of the samples, whereas only 24.1% of the samples tested positive for Pa. chlamydospora by means of isolations. Pa. chlamydospora was not isolated from hot water treated samples. The results confirm the importance of hot water treatment for proactive management of Petri disease in grapevine nurseries. However, Pa. chlamydospora DNA was molecularly detected in hot water treated samples in frequencies similar to that detected in non-hot water treated samples. As expected, the DNA in hot water treated plants was not destroyed and could be detected by the developed molecular detection protocol. This is an important consideration when using molecular detection for disease diagnosis or pathogen detection and shows that these methods should be used in conjunction with other diagnostic tools. Most importantly, the DNA extraction protocol was shown to be 10 to 15 times cheaper than commercial DNA extraction kits. Preliminary studies showed that the aforementioned molecular detection technique was not specific and sensitive enough for detection of Pa. chlamydospora in soil and water (unpublished data). Therefore, a one-tube nested-PCR technique was optimised for detecting Pa. chlamydospora in DNA extracted from soil, water, callusing medium and grapevine wood. Rootstock cane sections and soil samples were taking from the mother blocks from several nurseries. Water samples were collected from hydration and fungicide tanks during pre-storage and grafting. Scion and rootstock cuttings were also collected during grafting and soil were collected from the nursery beds prior to planting. The one-tube nested-PCR was sensitive enough to detect as little as 1 fg of Pa. chlamydospora genomic DNA from water and 10 fg from wood, callusing medium and soil. PCR analyses of the different nursery samples revealed the presence of several putative Pa. chlamydospora specific bands (360 bp). Subsequent sequence analyses and/or restriction enzyme digestions of all 360 bp PCR bands confirmed that all bands were Pa. chlamydospora specific, except for five bands obtained from callusing media and one band from water. Considering only Pa. chlamydospora specific PCR bands, the molecular detection technique revealed the presence of Pa. chlamydospora in 25% of rootstock cane sections and 17% of the soil samples collected from mother blocks, 42% of rootstock cuttings collected during grafting, 16% of scion cuttings, 40% of water samples collected after the 12- hour pre-storage hydration period, 67% of water samples collected during grafting and 8% of the callusing medium samples. These media should therefore be considered as potential inoculum sources or infection points of the pathogen during the nursery stages. The results furthermore confirmed previous findings that Pa. chlamydospora is mainly distributed through infected rootstock canes and cuttings. Infected scion cuttings were also shown to be potential carriers of the pathogen. Management strategies should include wound protection of rootstock mother plants, eradicating this pathogen from rootstock-cuttings (e.g. hot water treatment), biological or chemical amendments in the hydration water and callusing medium and wound protection from soil borne infections. / AFRIKAANSE OPSOMMING: Phaeomoniella chlamydospora is die hoof veroorsakende organisme van Petri se siekte wat lei tot die agteruitgang en terugsterwing van jong wingerdplante (1-7 jaar oud) en veroorsaak verhoogde vatbaarheid van hout vir infeksie deur ander patogene. Kennis oor die epidemiologie en veral die inokulumbronne van die siekte is noodsaaklik vir die daaropvolgende ontwikkeling van beheerstrategieë. Isolasies het getoon dat Pa. chlamydospora meestal versprei deur middel van geïnfekteerde voortplantingsmateriaal in Suid-Afrika. Die infeksieweë en inokulumbronne in wingerdkwekerye is egter steeds onbekend. Die enigste bestaande metode vir die opsporing van die patogeen, in verskeie mediums, is deur middel van isolasie op kunsmatige groeimediums. Dit is egter gevind om problematies te wees aangesien die swam uiters stadig groei (dit vat tot 4 weke vanaf isolasie tot identifikasie) en die kulture is telkens oorgroei deur ander organismes voordat identifikasie kan plaasvind. Die doel van die studie was (i) om ‘n protokol te ontwikkel vir die molekulêre opsporing van Pa. chlamydospora in wingerdhout, en (ii) om die protokol te gebruik, saam met ander, om verskillende monsters (water, grond, onderstok- en bostok-ente en kallusmedium) te toets, wat versamel is van kwekerye in Suid- Afrika, tydens verskillende kwekerystadiums, vir die teenwoordigheid van Pa. chlamydospora. ‘n Protokol is ontwikkel en geverifieer vir die molekulêre opsporing van Pa. chlamydospora in wingerdhout. Eerstens is verskeie protokols wat voorheen gepubliseer is, is as grondslag gebruik vir die ontwikkeling van ‘n ekonomiese en tydbesparende DNA ekstraksie protokol. Hierna is PKR (polimerase ketting reaksie) amplifikasie met spesie-spesifieke inleiers (Pch1 en Pch2) gevind om sensitief genoeg te wees om so min as 1 pg van Pa. chlamydospora genomiese DNA van wingerdhout op te spoor. Die protokol is geverifieer deur verskeie wingerdhoutmateriaal van 3 verskillende onderstokkultivars (101-14 Mgt, Ramsey en Richter 99) te gebruik, wat elk versamel is van 3 verskillende kwekerye. ‘n Aantal van die wingerstokke is ook onderwerp aan warmwaterbehandeling. Die basale kant van die onderstok is parallel geanaliseer vir Pa. chlamydospora deur gebruik te maak van isolasies op kunsmatige groeimedium asook molekulêre opsporing. Die identiteit van ‘n submonster van PKR produkte van verskeie monsters, wat slegs positief getoets het vir Pa. chlamydospora met die molekulêre opsporing, is bevestig om Pa. chlamydospora spesifiek te wees. Dit is gedoen deur middel van restriksie ensiem analise met AatII. Molekulêre opsporing is gevind om aansienlik meer sensitief te wees as isolasies, deurdat Pa. chlamydospora opgespoor is van positiewe sowel as negatiewe isolasies. Die molekulêre tegniek het Pa. chlamydospora in ‘n gemiddeld van 80.9% van die monsters opgespoor, terwyl slegs ‘n gemiddeld van 24.1% van die monsters postief getoets het vir Pa. chlamydospora, deur middel van isolasies. Pa. chlamydospora is nie geïsoleer van die monsters wat warmwaterbehandeling ondergaan het nie. Die resultate bevestig hoe belangrik warmwaterbehandeling is vir die proaktiewe beheer van Petri se siekte in wingerdkwekerye. Pa. chlamydospora DNA is met die molekulêre tegniek opgespoor, in warmwaterbehandelde monsters, in getalle wat ooreenstemmend is met die van niewarmwaterbehandelde monsters. Soos verwag, is DNA in warmwaterbehandelde plante nie vernietig nie en kon dit telke male opgespoor word deur die ontwikkelde molekulêre opsporing protokol. Dit is ‘n belangrike feit wat in ag geneem moet word wanneer molekulêre opsporing gebruik word in siekte diagnose en opsporing van patogene en dit is ‘n aanduiding dat die metodes gebruik moet word in samewerking met ander diagnostiese tegnieke. Die DNA ekstraksie protokol het getoon om tot en met 10 tot 15 kere goedkoper te wees as kommersiële DNA ekstraksie pakkette. Voorlopige studies het getoon dat die bogenoemde molekulêre opsporings tegniek nie spesifiek en sensitief genoeg is vir die opsporing van Pa. chlamydospora uit grond en water nie (ongepubliseerde data). Daarom is ‘n enkel-buis geneste-PKR tegniek geoptimiseer vir die opsporing van Pa. chlamydospora DNA wat geëkstraheer is vanaf grond, water, kallusmedium en wingerdhout. Dele van onderstokke en grond monsters is geneem vanaf moederblokke van verskeie kwekerye. Gedurende die voor-opberging en enting periodes is watermonsters versamel vanaf hidrasie en fungisied tenke. Bostok- en onderstokente is ook versamel gedurende enting en grond is versamel vanaf kwekerybeddens net voor uitplanting. Die enkelbuis geneste-PKR was sensitief genoeg om so min as 1 fg van Pa. chlamydospora genomiese DNA vanaf water en 10 fg vanaf hout, kallusmedium en grond op te spoor. PKR analise van die verskillende kwekerymonsters het getoon dat daar ‘n teenwoordigheid is van verskeie putatiewe Pa. chlamydospora spesifieke bande (360 bp). Daaropvolgende analise deur middel van DNA volgordebepaling en restriksie ensiem analise het bevestig dat al die 360 bp PKR bande wel Pa. chlamydospora spesifiek is, behalwe vir vyf bande wat verkry is vanaf kallusmedium en een band verkry vanaf water. As slegs Pa. chlamydospora spesifieke bande in ag geneem word, is daar met molekulêre opsporing die teenwoordigheid van Pa. chlamydospora gevind in 25% van die onderstokke, 17 % van die grond versamel vanaf moederblokke, 42% van die onderstokente versamel tydens enting, 16% van die bostokente, 40% van die watermonsters versamel voor die 12-uur hidrasie periode, 67% van die watermonsters versamel gedurende enting en 8% van die kallusmediummonsters. Hierdie mediums moet dus beskou word as potensiële inokulumbronne of infeksiepunte van die patogeen gedurende die kwekerystadiums. Die resultate bevestig ook verdere bevindinge wat aandui dat Pa. chlamydospora meestal versprei word deur geïnfekteerde onderstokke en ente. Geïnfekteerde bostokente is ook aangedui om potensiële draers van die patogeen te wees. Beheerstrategieë moet dus wondbeskerming van onderstok moederplante insluit, asook uitwissing van die patogeen vanaf onderstokente (bv. warmwaterbehandeling), toediening van biologiese of chemiese stowwe in die hidrasie water en kallusmedium en wondbeskerming teen grondgedraagde infeksies.

Thesis (MScAgric)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Grey mould, caused by Botrytis cinerea, is the most important foliar disease of rooibos seedlings. Although the disease is primarily controlled with applications of fungicides, the improvement of cultural methods of disease management should lessen this dependence on chemical control. Such improvements would, however, not be possible without knowledge of the inoculum sources and dispersal of the pathogen. The aim of this study was to investigate the inoculum ecology of B. cinerea in rooibos nurseries in order to identify primary sources of inoculum and to improve the environmentally friendly management of the disease. The study was conducted in four nurseries over two production seasons (March to July 2003 and 2004). Levels of airborne inoculum of B. cinerea were monitored on a monthly basis inside and around the nurseries with spore traps. Samples of plant material and organic debris were taken in the corresponding areas to determine the incidence of plant material infected by the pathogen and the incidences of grey mould in the nurseries were recorded. Low numbers of B. cinerea colonies were observed on the spore traps. Similar levels of airborne inoculum were observed inside and around the nurseries. The incidence of plant material yielding B. cinerea was higher outside the nurseries than inside, indicating the importance of such materials as potential sources of inoculum. Since patterns of airborne inoculum observed in this study confirmed reports of the local dispersal of B. cinerea, the removal of possible hosts outside the nurseries could aid in the management of grey mould in rooibos nurseries. Resistance to dicarboximide fungicides is a genetically stable trait in B. cinerea, and therefore has the potential to be used as a phenotypic marker. This marker can be used to gain knowledge on the dispersal of B. cinerea inoculum inside and outside rooibos nurseries. Isolates of B. cinerea collected from the air and from plant material in and around four rooibos nurseries were assessed for resistance to iprodione at 1 and 3 μg/ml a.i. Some of the isolates showed resistance to iprodione at 1 μg/ml a.i. However, none of the isolates showed resistance at 3 μg/ml a.i. iprodione. The initial incidence of dicarboximide-resistance at the nurseries was slightly higher than expected. As the season progressed, the incidence of iprodione-resistant isolates decreased towards May, after which an increase was observed towards July. A relatively high percentage of isolates collected outside the nurseries was found to be dicarboximide-resistant. Two of the nurseries had a significant higher incidence of resistant isolates on plant material collected inside, than on plant material collected outside the nursery. However, when looking at resistance levels of airborne isolates, no significant differences were found in the incidence of resistant isolates sampled inside and outside the four nurseries. The data indicated the importance of organic debris and seed-borne infections in the survival and dispersal of dicarboximide-resistant isolates of the pathogen. With the current emphasis on organic agriculture the knowledge gained in this study presents valuable possibilities of improving the cultural management of grey mould in rooibos nurseries. / AFRIKAANSE OPSOMMING: Vaalvrot, veroorsaak deur Botrytis cinerea, is die belangrikste bo-grondse siekte van rooibossaailinge. Alhoewel die beheer van die siekte hoofsaaklik op die gebruik van fungisiede berus, behoort die verbetering van verbouingspraktyke hierdie afhanklikheid van chemiese beheer te verminder. Sulke verbeteringe sal egter slegs moontlik wees indien voldoende kennis van die inokulumbronne en verspreiding van die patogeen beskikbaar is. Die doel van hierdie ondersoek was om die inokulum ekologie van B. cinerea in rooibos kwekerye te ondersoek sodat primêre inokulumbronne opgespoor en omgewingsvriendelike siektebestuurspraktyke verbeter kan word. Die ondersoek is in vier kwekerye oor twee produksie seisoene (Maart tot Julie 2003 en 2004) uitgevoer. Vlakke van luggedraagde inokulum van B. cinerea is op ’n maandelikse basis met behulp van spoorvangers binne en buite die kwekerye gemonitor. Monsters van plantmateriaal en organiese materiaal is in ooreenstemmende areas geneem om die voorkoms van B. cinerea geïnfekteerde plantmateriaal vas te stel en die voorkoms van vaalvrot in die kwekerye is aangeteken. Min B. cinerea kolonies is op die spoorvangers waargeneem. Soortgelyke vlakke van luggedraagde inokulum is binne en buite die kwekerye waargeneem. Die hoër voorkoms van B. cinerea geïnfekteerde plantmateriaal buite die kwekerye as binne, dui op die belang van sulke materiaal as potensiële inokulumbronne. Aangesien die patrone van luggedraagde inokulum, soos waargeneem in hierdie ondersoek, ander berigte van B. cinerea se beperkte verspreidingsvermoë bevestig, kan die verwydering van moontlike alternatiewe gashere buite die kwekerye die bestuur van die siekte binne die kwekerye verbeter. Weerstand teen dikarboksimied fungisiede is ’n geneties-stabiele kenmerk in B. cinerea en het daarom potensiaal om as ’n fenotipiese merker gebruik te word. Hierdie merker kan gebruik word om kennis aangaande die verspreiding van B. cinerea in en om rooibos kwekerye in te samel. Botrytis cinerea isolate in lug en op plantmateriaal in en om vier rooibos kwekerye is gedurende 2003 en 2004 versamel. Die isolate is vir weerstandbiedendheid teen iprodioon by konsentrasies van 1 en 3 μg/ml aktiewe bestandeel (a.b.) getoets. Isolate met weerstand teen 1 μg/ml a.b. iprodioon is waargeneem, maar nie teen 3 μg/ml nie. Die aanvanklike voorkoms van dikarboksimiedweerstand by die kwekerye was hoër as verwag. Hierdie vlak het egter gedaal met die verloop van die seisoen tot in Mei, waarna ’n toename tot in Julie waargeneem is. Die persentasie dikarboksimied-weerstandbiedende isolate buite die kwekerye was relatief hoog. In twee van die kwekerye was die voorkoms van weerstandbiedende isolate op plantmateriaal in die kwekerye betekenisvol hoër as op plantmateriaal buite die kwekerye. Daar was egter geen betekenisvolle verskille in die voorkoms van luggedraagde weerstandbiedende isolate nie, ongeag van die kwekery of posisie. Die data dui op die belang van organiese materiaal en saadgedraagde infeksies in die oorlewing en verspreiding van dikarboksimied-weerstandbiedende isolate van die patogeen. Met die huidige klem op organiese landbou bied die inligting wat in hierdie ondersoek versamel is moontlike praktyke wat geïmplementeer kan word om die beheer van vaalvrot in kwekerye met behulp van verbouingspraktyke te verbeter.