Call number: LD2668 .T4 1965 S623 / Master of Science

Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Phytophthora cactorum (Lebert & Cohn) Schrot. is the primary cause of crown, collar and root rot diseases of apple (Malus domestica Borkh.) trees worldwide. This pathogen is most destructive in commercial apple orchards under waterlogged soil conditions and has recently been identified as causing serious disease in some South African apple orchards. Crown, collar and root diseases are difficult to control because of their unpredictability and catastrophic nature. The use of resistant cultivars and rootstocks is economical and environmentally considerate. Therefore the need to develop screening techniques that will enable the selection of desirable disease resistant traits as part of an apple-breeding program in South Africa was identified. The work undertaken in this study was aimed at optimizing different techniques to test resistance. Using two direct inoculation techniques (excised stem and intact stem) the aggressiveness of lO isolates of P. cactorum on apple rootstocks was determined. The susceptibilities of five apple rootstocks were also compared. Results have shown isolate by rootstock interaction which means isolate aggressiveness was influenced by rootstocks tested. The selectivity of isolates suggests that there may be several strains of the pathogen. Population studies of the pathogen might contribute valuable information that could lead to better interpretation of results. Rootstock susceptibility was monitored in vitro throughout the season by inoculating at monthly intervals for 26-months. It was observed that during winter, rootstock susceptibility was low compared to high susceptibility during summer. These results have revealed new information regarding changes in the relative resistance of the different rootstocks over the growing season, e.g. the susceptibility pattern of rootstock MMl06 occurred 1 to -2 months later than that of other rootstocks. This finding has important implications on the way in which resistance test results are interpreted, and emphasizes the importance of not relying on point sampling. Furthermore, useful information has been acquired regarding the epidemiology of the disease with regard to "windows of susceptibility". The phenomenon of a phase shift in susceptibility of different rootstocks needs to be tested on a broader scale to assess whether it has any practical application on resistance testing. Although different inoculation techniques are applied in breeding programs, up to now there is no consensus on which technique works best for seedling selections. Since large numbers of individuals must be tested to improve the chances of detecting resistant genotypes, mass inoculations of young seedlings is a rapid way of identifying resistant individuals. Two different screening methods were tested during this study. Using the sand-bran technique, seedlings were transplanted onto inoculated soil and the root mass was used as a measure of resistance. In a second method zoospore inoculum was applied to seedlings growing in a sand:bark mixture at different concentrations and the seedlings were subjected either to water drenching or not. In both trials the aggressiveness of isolates differed significantly from each other and only higher inoculum concentrations were effective in causing disease. The age of seedlings used in tests emerged as an important factor. Seedlings under five-months-old should not be used. Drenching inoculated seedlings enhanced disease development but the production of sufficiently high numbers of zoospores was a laborious task. Thus, it is recommended that the sand-bran inoculum technique be tested with the drenching treatment for mass selection. In conclusion this study confirms the importance of both choice of isolate and choice of inoculation intervals in determining susceptibility of rootstocks to infection. In spite of the fact that stem inoculation bioassays have limited resemblance to natural disease situations, these bioassays are useful for obtaining an indication as to whether genotypes have a degree of resistance and merit further testing. For this reason refinement of the stem inoculation bioassay is worthwhile pursuing. With regard to seedling trials, both the sand-bran and the zoospore technique appear promising but refinement of these techniques is necessary in order to present a more practical way of testing large volumes of seedlings. / AFRIKAANSE OPSOMMING: Evaluering van inokulasietegnieke om weerstand teen Phytophthora cactorum in appels te evalueer: Phytophthora cactorum (Lebert & Cohn) Schrot. is die primêre oorsaak van kroon-, kraag en wortelvrot van appelbome (Malus domestica Borkh.). Dit is die mees verwoestende patogeen in kommersiële appelboorde waar daar versuipte toestande grond voorkom. P. cactorum is onlangs identifiseer as die patogeen wat ernstige kroon- en kraag-verotting in Suid Afrikaanse appelboorde veroorsaak. Kroon-, kraag- en wortelvrot is moeilik om te beheer as gevolg van die onvoorspelbaarheid en rampspoedige aard van die siekte. Die gebruik van kultivars en onderstamme wat weerstandbiedend is teen siektes en plae is omgewingsvriendelik en is ekonomies van belang, dus het die behoefte ontstaan om inokulasietegnieke te ontwikkelom weerstandige saailinge te identifiseer en te selekteer as deel van 'n appelteelprogram in Suid Afrika. Die doelwit van hierdie studie is om verskillende inokulasietegnieke te toets en te verfyn om weerstand in appelsaailinge te identifiseer. Deur gebruik te maak van twee inokulasietegnieke (die afgesnyde loot- en intakte loot tegniek), is die relatiewe aggressiwiteit van 10 isolate van P. cactorum en die vatbaarheid van vyf appelonderstamme ondersoek. Resultate het aangetoon dat die aggressiwiteit van die isolate gevarieer het na aanleiding van die onderstam wat getoets is. Die selektiwiteit van die isolate is 'n aanduiding dat daar moontlik verskeie rasse van die patogeen voorkom. Toekomstige studies op die populasiestruktuur van P. cactorum sal 'n belangrike bydrae maak tot die interpretasie van resultate oor weerstand en weerstandsteling. Die vatbaarheid van onderstamme was ook in in vitro proewe ondersoek deur maandelikse inokulasies toe te pas oor 'n tydperk van 26 maande. Dit is opgemerk dat die onderstamvatbaarheid gedurende die winter laag was in vergelyking met die somer. Nie al die onderstamme het dieselfe gereageer gedurende verskillende toetstye nie. Hierdie resultate toon aan dat die relatiewe weerstand van verskillende onderstamme oor die groeiseisoen verskil, byvoorbeeld die vatbare reaksie van die onderstam 'l\.1MI06' het een tot twee maande later voorgekom in vergelyking met ander onderstamme wat getoets is. Hierdie bevinding het belangrike implikasies op die interpretasie van weerstandstoetsing en beklemtoon die moontlike tekortkominge in enkelproefwaarnemings. Bruikbare inligting ten opsigte van die epidemiologie van die siekte is versamel wat beskryf kan word in terme van vensters van vatbaarheid wat verskil van onderstam tot onderstam. Verdere ondersoeke in die verband word aanbeveel. Hoewel verskeie inokulasietegnieke bestaan om jong saailinge vir weerstand te toets, is daar tot op hierdie stadium nog nie ooreenstemming oor die beste tegniek wat toegepas moet word om saailingseleksie te doen nie. Omdat groot getalle saailinge getoets moet tydens die seleksieproses sal massa-inokulasie van saailinge die aangewese metode wees. Twee verskillende inokulasie tegnieke is getoets in die studie. Deur gebruik te maak van die sandsemel tegniek, is saailinge geplant in geinfesteerde plantmedium, waartydens die wortelmassa van saailinge gebruik is om die reaksie op infeksie te kwantifiseer. Die soëspoor inokulasietegniek was toegepas op saailinge wat in 'n sand en basmengsel geplant is teen verskillende inokulurnkonsentrasies. 'n Waterverdrenkingsbehandeling is ook getoets. In albei hierdie proewe het die aggressiwiteit van die isolate van mekaar verskil. Slegs die hoër inokulumkonsentrasies was effektief in die ontwikkeling van die siekte. Die ouderdom van saailinge is ook uitgewys as 'n belangrike faktor wat 'n rol speel in weerstandstoetsing. Saailinge jonger as 5 maande word nie aanbeveel vir hierdie toetse nie. Verdrenking van saailinge het die voorkoms van die siekte verhoog, maar die produksie van groot getalle soëspore was 'n beperkende faktor in die uitvoering van die proef Dit word aanbeveel dat die sand-semel inokulasietegniek verder evalueer moet word onder verskeie toestande, onder andere deur dit met verdrenkinghte kombineer. Die belang van die keuse van isolaat en inokulasiedatum in bepaling van relatiewe weerstand van onderstamme teen P. cactorum is tydens die studie bevestig. Afgesien van die beperking van die staminokulasietegnieke in soverre dit verwyderd is van natuurlike infeksie, word die tegnieke aanbeveel om 'n indikasie te kry van die relatiewe weerstand van onderstamme. Beide die sand-semel en soëspoor tegnieke kan gebruik word om weerstandige saailinge te identifiseer, maar tegniese verfyning van hierdie tegnieke is nodig om saailinge in massa te evalueer.

Apple scab is one of the greatest apple management problems throughout the world Much work has been done on cultivars resistant to Venturia inaequalis (Cke.) Wint., but few have been a commercial success. This frequently leaves fungicides as the only control method used. As Quebec growers select new cultivars for planting, more information is needed on their relative susceptibility for efficient scab control. In this light, 21 cultivars common to central and eastern Canada, were examined for their relative susceptibility using several components of partial resistance; disease severity, incubation period, latent period, lesion size, and conidial production. The cultivars used were Cortland, Early Geneva, Empire, Golden Delicious, Golden Russet, Idared, Jersey Mac, Jonagold, Jonamac, Lobo, Lodi, Summerland McIntosh, Mutsu (Crispin), Northern Spy, Paulared, Red Cortland, Red Delicious, Royal Gala, Spartan, Sunrise, and Vista Bella. A final ranking of the cultivars and selection of partial resistance components was done using the principal components analysis. (Abstract shortened by UMI.)

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Crown gall disease is an agricultural problem caused by the soil-borne bacterium, Agrobacterium tumefaciens. A. tumefaciens oncogenes cause transformed plant cells to overproduce the hormones, auxin and cytokinin. High hormone levels cause unorganized plant cell growth resulting in a gall. Control of crown gall disease is difficult because after plant cell transformation has occurred, the bacterium is no longer required for the disease to progress. Apple trees engineered to express double-stranded RNA of two A. tumefaciens oncogenes, ipt and iaaM, silenced the expression of the wild-type oncogenes and prevented crown gall disease. Only the iaaM oncogene was targeted for posttranscriptional gene silencing (PTGS) as measured by biological assays and by quantitative reverse-transcription polymerase chain reaction (q-RTPCR) on transgenic tissue. However, if the translation initiation sequence of the iaaM construction was eliminated, gall formation was not prevented, indicating that translatable RNA initiates silencing whereas untranslatable RNA does not. Other data indicate that the Arabidopsis thaliana micro-RNA pathway gene is involved in A. tumefaciens-mediated tumorigenesis. A. thaliana plants with a mutation in HEN1, a gene required for micro-RNA maturation, demonstrated a tenfold reduction in tumorigenesis upon A. tumefaciens infection compared to wild-type. The same plant line showed no difference in T-DNA transfer and nuclear uptake. / Graduation date: 2006

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Apple (Malus x domestica Borkh.) is an important horticultural crop worldwide and in the Western Cape. The income generated from apple and other deciduous fruit production amounts to approximately 25% of the gross total value of horticultural production in the Western Cape. Unfortunately diseases and pests adversely affect fruit production in this region. Woolly apple aphids (Eriosoma lanigerum L. (Hausmann» have a significant effect on the apple industry in the Western Cape. Damage caused is two-fold, occurring aerially and terrestrially. Insects colonise the plants, feeding off the phloem sap. Aphid infestation around the root system results in repeated infestation of the foliage as it serves as a reservoir of aphids. In extreme cases, the apple cores are also infested, thus affecting the sale of apples. In 1962, Northern Spy was identified as a woolly apple aphid resistant rootstock and has since then formed the basis for traditional rootstock breeding programmes. The Er1 gene in Northern Spy confers resistance. According to one report, the natural resistance of Northern Spy was overcome in South Africa in 1968, but this was not confirmed in an independent study. The main objectives of this study was to firstly identify molecular markers more closely linked to the woolly apple aphid resistance gene, Er1, than existing markers, by applying AFLP technology to selected seedlings, identified to be resistant by conventional phenotyping. If identified, these markers can be incorporated into existing breeding programmes. Secondly, previously identified RAPD and SCAR markers were tested to determine their applicability in local populations for use in breeding programmes. Ultimately the segregation of the Er1 gene in South African populations can be determined if tightly linked markers are identified. Three families were derived from crosses of each of three resistant genotypes, namely Northern Spy, Rootstock 5 and Russian Seedling and a susceptible cultivar, Braeburn. For the three successive years of the study, each resistant genotype was allowed to cross-pollinate in isolation with the susceptible parent. Two hand-pollinated families, Russian Seedling x Liberty and Russian Seedling x Northern Spy, were also included in the study. The amplified fragment length polymorphism (AFLP) technique was used in an attempt to identify markers in the resistant and susceptible seedlings. No markers were identified using this technique. Known sequence characterised amplified regions (SCAR) and random amplified polymorphic DNA (RAPD) markers were used due to their suitability in marker-assisted selection for woolly apple aphid resistance. Varying results were obtained with these markers and no conclusive information was acquired with regard to the segregation of the Er] gene in any of these rootstocks and crosses. This underlines the need for the development of markers that can readily be applied in local breeding programmes. The identification and integration of such markers will greatly benefit the local and world wide apple industries. / AFRIKAANSE OPSOMMING: Appels (Malus x domestica Borkh.) is wêreldwyd en in die Wes-Kaap 'n belangrike landbougewas. Inkomste gegenereer deur appels en ander sagtevrugte vorm bykans 25% van die bruto inkomste uit vrugte in die Wes-Kaap. Siektes en insekpeste verlaag egter die produksie van vrugte in hierdie streek. Appelbloedluise (Eriosoma lanigerum L. (Hausmann» het 'n groot invloed op appelproduksie in die Wes-Kaap. Skade word bogronds en ondergronds aangerig. Insekte koloniseer die plant en leef op floeëmsap. Besmetting van die wortels lei tot herhaalde besmetting van bogrondse dele aangesien die insekte aanteelop die wortels. In uiterste gevalle word die vrugte geaffekteer, wat vrug-verkope beïnvloed. 'Northern Spy' is in 1962 geïdentifiseer as 'n onderstam met natuurlike weerstand teen appelbloedluis en het vir lank die basis gevorm vir tradisionele telingsprogramme. Weerstand word verleen deur die Erf geen. Volgens een verslag is die natuurlike weerstand van Northern Spy egter in 1968 in Suid-Afrika oorkom, maar dit is nog nie in 'n onafhanklike studie bevestig word nie. Die hoof doelstellings van hierdie studie was om eerstens deur middel van die AFLP tegniek molekulêre merkers te identifiseer wat nouer gekoppel is aan die appelbloedluis weerstandsgeen, En, as bestaande merkers. Hierdie tegniek is toegepas op saailinge wat deur konvensionele fenotipering geselekteer is. Indien merkers suksesvol geïdentifiseer is, kan dit in bestaande telingsprogramme geïntegreer word. Tweedens is bestaande RAPD en SCAR merkers ook getoets om hul toepaslikheid te bepaal vir gebruik in plaaslike teelprogramme. Oplaas sal die segregasie van die Erf geen in Suid- Afrikaanse populasies ook deur middel van nou gekoppelde merkers bepaal kan word. Kruisings van elk van die drie weerstandbiedende genotipes, naamlik 'Northern Spy', 'Rootstock 5' en 'Russian Seedling', en die vatbare kultivar, 'Braeburn' , het drie families daargestel. Elke weerstandbiedende genotipe is toegelaat om in isolasie te kruisbestuif met die vatbare ouer. Twee hand-bestuifde families, 'Russian Seedling' x 'Liberty' en 'Russian Seedling' x 'Northern Spy', is in 'n latere stadium van die studie ingesluit. Die AFLP tegniek is gebruik vir die identifikasie van polimorfiese merkers tussen vatbare en weerstandbiedende populasies. Geen merkers is egter geïdentifiseer nie. Bestaande SCAR en RAPD merkers is vervolgens gebruik om te bepaal of hulle geskik is vir gebruik in merker-bemiddelde seleksie vir appelbloedluis weerstand. Wisselende resultate is verkry ten opsigte van amplifikasie, herhaalbaarheid van resultate was swak en geen onweerlegbare bewyse oor die segregasie van die Erfgeen is bekom nie. Dit beklemtoon die noodsaaklikheid om merkers wat geredelik in plaaslike teelprogramme toegepas kan word, te ontwikkel. Die identifikasie en integrasie van sulke merkers sal die plaaslike en wêreld-wye appel industrieë aansienlik bevoordeel.