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Thesis (MScAgric)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Stone fruit is susceptible to chilling injury and intermittent warming has been shown to alleviate chilling injury during cold storage. A dual temperature storage regime was developed in South Africa for plums based on the principles of intermittent warming. The regime consists of an initial period at -0.5°C, a variable duration warming period at 7.5°C, followed by -0.5°C. Refrigerated integral containers were designed to maintain product temperature and not to reduce product temperature, per se. Considering that dual temperature shipment requires significant refrigeration and effective distribution of cool air to remove sensible- and respiratory heat, the capacity of integral containers to ship plums successfully at dual temperature is questioned. The objectives of this study were, firstly, to analyse pulp temperature data and possibly identify different temperature zones within containers shipping plums at dual temperature. Secondly, to understand the underlying processes differentiating the temperature zones and thirdly, to determine the effect of container performance on fruit quality. Three processes were identified as important characteristics of pulp temperature data sets recorded during dual temperature shipping, namely cooling down, heating up and over heating in the container. The order of importance differed according to the cultivar shipped and the container’s performance. Three temperature zones were identified in dual temperature containers, where the average pulp temperature, time to heat up and time to cool down for each temperature zone increased along the length, across the width from the left to the right and up the container system. The variable temperature conditions were possibly due to a variation in delivery air temperature, poor airflow and the effect of increased respiration and, therefore, production of vital heat by the fruit. The cooling down process was identified as the most important process discriminating the temperature zones. With the exception of ‘Fortune’, variable temperature conditions found within integral containers shipping plums at dual temperature had a significant influence on the fruit firmness post-shipment, where deterioration levels increased from the front to the door end of the container due to an increase in pulp temperature. However, it was also shown that fruit firmness prior to shipment could have a determining effect on differences found. It could not be proven that variable temperature conditions resulted in significantly higher levels of internal defects within the integral container. Temperature zones could not be identified within refrigerated integral containers shipping plums at single temperature, suggesting that the containers are able to maintain the temperature well throughout the container area. A constant 2°C storage temperature could possibly replace the commercial dual temperature regime in the case of ‘Pioneer’ plums due to improved fruit firmness, similar colour development to the control and less sensible heat produced in the container resulting in a more stable container environment. However, unacceptably high levels of shrivel and internal browning were found. / AFRIKAANSE OPSOMMING: Steenvrugte is vatbaar vir koueskade en dit is bewys dat periodieke verwarming gedurende koelopberging koueskade kan verlig. Die dubbeltemperatuur opbergingsregime is in Suid- Afrika ontwikkel vir pruime en is gebaseer op die beginsels van periodieke verwarming. Die regime bestaan uit ‘n inisiële periode by -0.5°C, ‘n variërende periode by 7.5°C, gevolg deur -0.5°C. Verkoelde integrale houers is ontwerp om produktemperature te handhaaf en nie soseer om produktemperatuur te verlaag nie. Die kapasiteit van integrale houers om pruime suksesvol teen dubbeltemperatuur te verskeep word dus bevraagteken, in ag geneem dat dubbeltemperatuurverskeping betekenisvolle verkoeling en effektiewe verspreiding van koue lug vereis om die waarneembare- en respiratoriese hitte te verwyder. Die doelwitte van die studie was eerstens om die pulptemperatuurdata te analiseer en moontlik verskillende temperatuursones binne houers wat pruime teen dubbeltemperatuur verskeep te identifiseer. Tweedens, om die onderliggende prosesse wat die temperatuursones van mekaar onderskei te verstaan, en derdens om die effek van die houer se werkverrigting op vrugkwaliteit te bepaal. Drie prosesse is geïdentifiseer as belangrike eienskappe van pulptemperatuur datastelle aangeteken gedurende dubbeltemperatuurverskeping, naamlik afkoeling, opwarming en oorverhitting wat binne die houer plaasvind. Die volgorde van belangrikheid het gevarieer afhangende van die kultivar verskeep en die houer se werkverrigting. Drie temperatuursones is geïdentifiseer binne integrale houers wat pruime teen dubbeltemperatuur verskeep, waar die gemiddelde pulptemperatuur, die opwarmingstyd en die afkoelingstyd vir elke temperatuursone in die lengte, oor die wydte van links na regs en van onder na bo in die houersisteem toegeneem het. Die variërende temperatuur toestande kan moontlik toegeskryf word aan ‘n variasie in leweringstemperatuur, swak lugvloei en die effek van toenemende respirasie, en dus die produksie van hitte vrygestel deur die vrugte. Die afkoelingsproses is geïdentifiseer as die belangrikste proses wat die temperatuursones van mekaar onderskei. Behalwe in die geval van ‘Fortune, het variërende temperatuurtoestande in integrale houers wat pruime teen dubbeltemperatuur verskeep ‘n betekenisvolle invloed op die vrugfermheid na verskeping gehad, waar vrugveroudering toegeneem het van voor in die houer na die deur van die houer as gevolg van ‘n toename in pulptemperatuur. Daar is egter bewys dat die vrugfermheid voor verskeping ook ‘n bepalende effek kon hê op die fermheidsverskille. Dit kon nie bewys word dat die variërende temperatuurtoestande betekenisvol hoër vlakke van interne defekte binne die integrale houer veroorsaak het nie. Temperatuursones kon nie geïdentifiseer word binne verkoelde integrale houers wat pruime teen enkeltemperatuur verskeep het nie, wat dus impliseer dat die houers daartoe instaat is om temperatuur goed te onderhou binne die houer. ‘n Konstante 2°C opbergingstemperatuur kan moontlik die kommersiële dubbeltemperatuurregime vervang in die geval van ‘Pioneer’ pruime as gevolg van verbeterde vrugfermheid, soortgelyke kleurontwikkeling as die dubbeltemperatuurregime en minder hitte geproduseer binne die houer deur die pruime, wat ‘n meer stabiele houeromgewing veroorsaak. Onaanvaarbare hoë vlakke van verrimpeling en interne verbruining is egter gevind.

The aim of this research was to develop a new harvest maturity index for highly coloured stone fruit that is not affected by seasonal or climatic factors. The judgement of commercial maturity is difficult with some cultivars of stone fruit because they develop intense skin pigmentation several days before they are ripe. Studies of the changes of the physiochemical and physiological parameters associated with ripening confirmed that no single measurement is suitable for the assessment of harvest maturity in the Japanese type plum cultivars(Prunus salicina Lindl). Two distinct patterns of ripening behaviour were identified. The cool storage (0 degrees Centigrade) responses of fruit harvested at three stages of maturity were examined. Total soluble proteins were extracted and separated from fruit during maturation and ripening using 2-dimensional gel electrophoresis. At least three proteins were first detected in fruit at optimum commercial harvest maturity. Since Gulfruby, Beauty and Rubyred (Japanese type plum cultivars) accumulated similar concentrations of aminocyclopropane-1-carboxylic acid(ACC), it was suggested that the suppressed-climacteric phenotype is the result of an impaired ability of the fruit to convert this compound to ethylene / Doctor of Philosophy (PhD) (Ph.D.)

Among several early-season insect pests threatening apple production in eastern North America, plum curculio (PC), Conotrachelus nenuphar Herbst (Coleoptera: Curculionidae), is one of the most devastating pests of pome and stone fruits. Historically, several methods have been used to monitor and control plum curculio in apple orchards. The odor-baited trap tree approach has proven to serve as an effective monitoring and attract-and-kill system for plum curculio. The synergistic lure consisting of benzaldehyde (a plant volatile) and grandisoic acid (the PC aggregation pheromone) is used in this approach. However, the high cost of the lure and the degradation of benzaldehyde by UV light and heat have resulted in limited grower adoption. The major goals of this study were (1) to evaluate under field conditions, the attractiveness of methyl salicylate (MES) to PC, and to determine if it can replace the benzaldehyde (BEN), and (2) to test whether we can manipulate insect pest behavior through host plant preference by grafting selected perimeter- row trees with multiple cultivars. My results indicated that (1) methyl salicylate in combination with grandisoic acid was as attractive to PC as the standard synergistic lure composed of benzaldehyde and grandisoic acid, therefore MES can be used as a replacement for BEN, (2) trees grafted with six cultivars were more attractive to PC and concentrated more PC fruit injuries, compared to non-grafted trees, and (3) ‘Wickson’ and ‘Red Astrachan’ received numerically more PC injuries while ‘Dabinett’ received the lowest level of injury compared with other cultivars. These results highlight that a semiochemical-based strategy is a low-cost and grower-friendly approach to monitoring and controlling the PC population.

Plum curculio, Conotrachelus nenuphar (Herbst), is an endemic pest of stone and pome crops of the eastern United States. Two morphologically identical strains of plum curculio have been described and documented in Virgina: a univoltine strain and a multivoltine strain. Because of the cryptic coloring and behaviours of the plum curculio adults, monitoring in orchards is difficult and often ineffective. RAPD-PCR assay was an effective method for separation of the geographical strains. Of the tested primers four, OPE 01, OPE 03, OPE 04, and OPE 07, gave 21 amplimers that are useful for distinguishing individuals from the univoltine and multivoltine populations. Gene targeted PCR revealed the presence of Wolbachia in both populations. Analysis of the wsp gene sequence showed the univoltine population of plum curculio is associated with a strain of Wolbachia in supergroup B, most closely related to a strain identified from Perithemis tenera (Say) (Odonata). The multivoltine populations of plum curculio are associated with strains of Wolbachia which are in supergroup A, and most closely related to Wolbachia strains associated with Dacus destillatoria, Bactrocera sp., and Callosobruchus chinensis Linn. Three different trap designs baited with grandisoic acid, plant volatiles, and a combination of pheromone and plant volatiles were tested. In 1999, significantly more plum curculios were captured with Tedders traps baited with grandisoic acid and unbaited control in traps baited with limonene, plum essence or ethyl isovalerate. In 2000, Circle traps baited with plum essence, sour cherry essence and grandisoic acid yielded no results. In 2001, branch mimic traps yielded no significant differences among three release rates of a blend of benzaldehyde, ethyl isovalerate, trans-2-hexenal, and limonene; although, there was a significant interaction between the pheromone and the host plant volatiles. Overall however, all three of the traps were not effective. / Master of Science

La maladie de la Sharka est due à un virus de quarantaine, le Plum Pox Virus (PPV), infectant les arbres fruitiers du genre Prunus. Il est nécessaire de trouver des moyens de lutte, telle que la sélection de plantes résistantes. Or chez ces espèces, les sources de résistance sont à l’heure actuelle en nombre limité, voire inexistantes. Il a été montré, au laboratoire, que ce virus est capable d’infecter Arabidopsis thaliana et qu’il existe chez cette espèce une grande diversité de réponse à l’infection. En effet nous avons pu observer que les accessions St-0 et JEA avais un comportement résistant, alors que l'accession Cvi-1 été partiellement résistante. Deux méthodes d’inoculation ont été comparées: une inoculation mécanique à partir de feuilles de Nicotiana benthamiana inoculées avec pICPPVnkGFP et une inoculation par agro-infection à partir d’une souche Agrobacterium tumesfasciens contenant l’isolat viral pBINPPVnkGFP. L'emploi de ces deux méthodes d'inoculation nous a permis de mettre en évidence une variabilité de la réponse au PPV en fonction de la méthode utilisée. En conséquence, cette étude visait donc à identifier le ou les facteur(s) de la plante hôte impliqué(s) dans l'infection virale. L'agro-infection de populations recombinantes (F2 et RIL), de lignées multi-parentales ainsi que l'emploi de la génétique d'association a mis en évidence chez St-0 ainsi que dans plusieurs accession distinct (sept) un locus majeur sur le groupe de liaison 3, appelé sha3. Il apparait indispensable dans le mouvement longue distance du PPV. De plus l'utilisation de la génétique d'association a permis d'initier la cartographie fine de sha3 et de réduire considérablement le nombre de gènes candidats. Un criblage de mutants a été initié afin de déterminer le ou les gènes candidats contrôlant le phénotype Sha3. Après inoculation mécanique, l’analyse d'une population recombinante a mis en évidence la présence d’un locus majeur, distinct de sha3 et positionné au milieu du bras long du groupe de liaison 1. Ce locus co-localise avec rpv1, locus identifié précédemment dans la descendance Cvi x Ler (Sicard, Loudet et al. 2008). Ce même locus a été également confirmé à la fois dans une population multi-parentale et par une approche de génétique d'association. Un gène candidat est actuellement en cours de validation au laboratoire. Une étude visant à décomposer le mécanisme de résistance porté par l’accession JEA a été mise en place. Dans ce cas, il apparait que la propagation du virus est inhibée dans les feuilles de la rosette mais pas dans les tissus floraux. Ainsi, la résistance/sensibilité au PPV chez JEA est fortement conditionnée par les stades physiologiques de la plante hôte. Des travaux complémentaires seront indispensables afin de décrire plus finement ce mécanisme de résistance très particulier. Au terme de cette thèse, nous nous attendons à ce que l’identification de ces nouveaux gènes de résistance chez Arabidopsis permette, après transfert, d’accroître la diversité des sources de résistance à la Sharka chez les arbres fruitiers. / The Plum Pox Virus (PPV) infects Prunus species (stone fruit) and is the causal agent of the Sharka disease. This disease is vastly devastating for fruit and plant productivity and quality. Its cost reaches 10 billions of euros over the last 30 years. Breeding programs have been carried out with the aim to implement resistant cultivars but the number of sources of resistance in Prunus species is rather limited. It has been shown in the laboratory that this virus is able to infect Arabidopsis thaliana with a wide range of response to infection. Indeed, we observed that accessions St-0 and JEA had a resistant behavior, while accession Cvi-1 was partially resistant. Two inoculation methods were compared: mechanical inoculation from Nicotiana benthamiana leaves inoculated with pICPPVnkGFP and agro-inoculation infection from an Agrobacterium strain containing the viral isolate tumesfasciens pBINPPVnkGFP. The use of these two methods of inoculation allows us to highlight variability in the response to PPV depending on the method used. This study aims to identify the factor (s) of the host (s) involved in viral infection. Agro-infection of recombinant populations (F2 and RIL), multi-parental lines and the use of genetic association demonstrate in St-0 and several distinct accessions (seven) a major locus on linkage group 3, called sha3. It appears essential in the long-distance movement of PPV. Use of association genetics helped initiate the fine mapping of sha3 and significantly reduce the number of candidate genes. Screening of mutants was initiated to determine the gene controlling the phenotype Sha3. After mechanical inoculation, the analysis of a recombinant population revealed the presence of a major locus positioned in the middle of the long arm of linkage group 1. This locus co-localizes with rpv1, previously identified in Cvi x Ler offspring (Sicard, Loudet et al. 2008). The same locus was also confirmed with a multi-parental population and by a genetic association approach. A candidate gene is currently being validated in the laboratory. The study of the resistance mechanism carried by the accession JEA was initiated. In this case, it appears that the spread of the virus is inhibited in basal leaves but not in floral stem. The resistance / susceptibility to PPV in JEA appear to be strongly influenced by the physiological stages of the host plant. Further work will be necessary to describe more precisely this resistance mechanism very special. At the end of this thesis, we expect that the identification of these new resistance genes in Arabidopsis allows, after transfer, to increase the diversity of sources of resistance to plum pox virus in fruit trees.

6 pp. / Training and pruning newly-planted deciduous fruit trees is one of the most important steps in developing trees with a strong framework (scaffold branches). Trees with a good framework of branches can support heavy crops without limb breakage and will help to bring the young tree into production at an early age. Selection and arrangement of these branches determines the type of development and growth in later years. The goal of pruning and training is to balance vegetative and fruiting wood growth.

Thesis (PhD)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Bacterial canker of stone fruit, caused by Pseudomonas syringae pv. syringae, is one of the most destructive crop diseases in South Africa. Chemical control has failed completely and effective long-term management strategies will have to rely on the breeding of resistant host trees. To assist in such breeding programmes, investigations into the molecular basis of the interaction between P. s. pv. syringae and stone fruit trees have been undertaken in collaboration with the ARC-Fruit, Wine and Vine Research Institute in Stellenbosch. The aim of this dissertation was to clone and identify genes that are involved in interaction between the bacterial canker pathogen and stone fruit trees. In the first part of the study, the harpin encoding gene of a local strain of the pathogen, P. s. pv. syringae NV, was amplified in a polymerase chain reaction (PCR) strategy with primers based on the hrpAZB sequences of the bean pathogen, P. s. pv. syringae 61. Sequencing of this hrpZpssNvgene revealed a high degree of homology (96%) between the harpin encoding genes and harpin proteins of the two strains. The hrpZpssNvgene was subsequently cloned into the pMAL-c2 expression vector and expressed in Escherichia co/i. This system was used for the production of purified, biologically active, recombinant HrpZpSSNV protein. In the second part of the study, differential display (DD) technology was used to identify genes that are induced in stone fruit trees in response to P. s. pv. syringae and/or its harpin elicitor. For this purpose, actively growing shoots of two Prunus sa/icina cultivars, the moderately resistant cv. 'Laetitia' and the highly susceptible cv. 'Songold' were treated with recombinant harpinpssNvprotein or live P. s. pv. syringae NV bacteria. An untreated control and wounding control was included in the experiment. Total RNA was isolated for comparative mRNA analysis 24 hours after treatment. DD profiles were generated with fifteen primer combinations. Eight candidate bands were re-amplified, cloned and sequenced. Reverse transcription PCR was employed to verify the expression patterns of the cloned bands in the original RNA sample set. Two bands, DDc and DD4 were shown to be differentially expressed between treatments and/or cultivars, while no differences in the expression levels of the remaining six bands (DDa, DDe, DD3, DD5, DD6 and DD7) were observed. BLAST similarity searches yielded significant matches for DDe, DD4 and DD7 with plant defense-related genes. / AFRIKAANSE OPSOMMING: Bakteriese kanker van steenvrugte, wat deur Pseudomonas syringae pv. syringae veroorsaak word, is een van die mees verwoestende siektes van landbougewasse in Suid-Afrika. Chemiese beheermaatreëls het geheel en al misluk en effektiewe langtermyn beheerstrategieë sal op die teling van weerstandbiedende gasheerbome moet staatmaak. Ondersoeke na die molekulêre basis van die interaksie tussen P. s. pv. syringae en steenvrugbome is in samewerking met die LNR-Vrugte-, Wyn- en Wingerdnavorsingsinstituut in Stellenbosch van stapel gestuur om tot sulke telingsprogramme by te dra. Die doelwit van hierdie proefskrif was om gene wat betrokke is in die interaksie tussen die bakteriese kanker patogeen en steenvrugbome te kloneer en te identifiseer. In die eerste gedeelte van die studie is die harpien-koderende geen van 'n plaaslike ras van die patogeen, P. s. pv. syringae NV, geamplifiseer in 'n polimerase kettingreaksie (PKR)-strategie met peilers wat op die hrpAZB-geenopeenvolgings van die boontjiepatogeen, P. s. pv. syringae 61, gebaseer is. Volgordebepaling van hierdie hrpZpssNv-geen het 'n hoë vlak van homologie (96%) tussen die harpien-koderende gene en harpien proteïene van die twee rasse getoon. Die hrpZpssNv-geen is vervolgens in die uitdrukkingsvektor pMAL-c2 gekloneer en uitgedruk in Escherichia coli. Hierdie sisteem is vir die produksie van suiwer, biologies-aktiewe, rekombinante HrpZpssNv-proteingebruik. In die tweede gedeelte van die studie is die differensiaalvertoon (DD) tegniek gebruik om gene te identifiseer wat deur P. s. pv. syringae en/of sy harpien elisitar in steenvrugbome geïnduseer word. Vir hierdie doel is aktief-groeiende lote van twee Prunus sa/icina kultivars, die matig weerstandbiedende kv. 'Laetitia' en die hoogs vatbare kv. 'Songold', met rekombinante harpienpssNvproteïen of lewende P. s. pv. syringae NV bakterieë behandel. 'n Onbehandelde- en verwondingskontrole is in die eksperiment ingesluit. Totale RNA is 24 uur na behandeling vir vergelykende mRNA-analise geïsoleer. DD-profiele is met vyftien peilerkombinasies gegenereer. Agt kandidaatbande is geheramplifiseer en gekloneer, waarna hul DNA-opeenvolgings bepaal is. Trutranskriptase-PKR is gebruik om die ekspressiepatrone van die gekloneerde bande in die oorspronklike RNA monsters na te gaan. Daar is vasgestel dat twee van die bande, DDc en DD4, differensieel tussen kultivars en/of behandelings uitgedruk is, terwyl geen verskille in die ekspressievlakke van die oorblywende ses bande (DDa, DOe, 003, DOS, 006 en DO7) waargeneem is nie. BLAST-soektogte het betekenisvolle ooreenkomste vir DDe, DD4 en DD7 met plant weerstandsgeassosieerde gene opgelewer.

Thesis (PhD(Agric))--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Postharvest decay of stone fruit in the Western Cape province of South Africa is caused primarily by Botrytis cinerea (grey mould) and Monilinia laxa (brown rot). Little is known about the relative importance and seasonal occurrence of the two pathogens in nectarine and plum orchards, the mode of penetration of fruits by M laxa, latency and subsequent disease expression by the latter pathogen. These aspects were investigated in this study. By sampling from the Unifruco Quality Evaluation Scheme and from 11 stone fruit orchards, observations were made over a 3-year period of the occurrence of grey mould and brown rot in the major stone fruit regions. Botrytis cinerea was found to be the most important pathogen causing blossom blight and postharvest decay on stone fruit. The pathogen was most prominent on early- and mid-season culti~ars. Brown rot was exclusively caused by M laxa and no evidence was found that M fructicoZa had been introduced into the region. Monilina laxa was most prominent on the later maturing cultivars. Botrytis cinerea blossom infection did not contribute directly to postharvest decay. Both surface inoculum and latent infection consistently occurred on fruit in each orchard, although at fluctuating levels. Disease expression on developing fruit was not governed by the amount of B. cinerea occurring on fruit surfaces, but by the ability of fruit to resist disease expression. The amount of B. cinerea on fruits was generally higher during spring than during summer. Monilinia laxa occurred sporadically on the blossoms of late-maturing cultivars. Immature fruit were generally pathogen-free and disease expression occurred on maturing fruit only. These findings suggest that conidia of M laxa are generally produced in orchards when fruits are approaching maturity and can penetrate and infect maturing fruit only. The behaviour of airborne M laxa conidia was subsequently studied on nectarine (cultivar Flamekist) and plum (cultivar Laetitia) fruit. For these studies, an inoculation method that simulates natural infection by airborne conidia was used. Fruit at pit hardening, 2 wk before harvest, harvest stage and after cold storage (nectarines 4 wk at -o.soC followed by 1 wk at 23°C at ±56% RH; plums 10 days at .....().5°C,18 days at 7.5°C followed by 1 wk at 23°C at ±56% RH) were dusted with dry conidia of M laxa in a settling tower. The fruits were incubated for periods ranging from 3 to 48 h at high relative humidity (2':93%, humid fruit) or covered with a film of water (wet fruit). Behaviour of the solitary conidia was examined with an epifluorescence microscope on skin segments stained in a differential stain containing fluorescein diacetate, aniline blue and blankophor. The ability of solitary conidia to colonise the fruit surface, penetrate fruit skins and to induce disease expression was determined by using a differential set of tests. For these tests, fruit were surface-sterilised (30 s in 70% ethanol) or left Unsterile. From each group, fruit were selected for isolation (skin segment test), immersed in a 3% paraquat solution (paraquat-treated fruit test) or left untreated (sound fruit test). 1be findings demonstrated that solitary conidia of M laxa behaved consistently on plum and nectarine fruit surfaces: appressorium formation and direct penetration was not observed on any of the fruit surfaces and germ tubes penetrated fruit predominantly through stomata, lenticels and microfissures in the fruit skin. The monitoring of airborne conidia revealed subtle effects of the fruits on the behaviour of solitary germlings, which could not be seen when using conidial suspensions. On both fruit types, no deleterious effect was seen on conidial and germling survival when fruit were kept humid at pit hardening, 2 wk before harvest and harvest. However, conidial and germling survival were drastically reduced by prolonged wet incubation of fruits. The findings on disease expression in the skin segment, paraquat-treated fruit and sound fruit tests clearly showed that the skin of both nectarine and plum fruits were not penetrated at the pit hardening stage, latent infections were not established and fruitsreacted resistant to disease expression. These facets on both fruit types were furthermore unaffected by wetness. The barrier capacity of the fruit skin of the two stone fruit types however differed drastically later in the season. On nectarine, fruit skins were more readily penetrated and disease expression became more pronounced when fruit approached maturity. Penetration and disease expression on ripening nectarine fruit were furthermore greatly influenced by wetness. Maturing plum fruit, on the other hand, did not display the drastic change in the barrier capacity of fruit skins as observed on nectarine. The influence of wetness on infection and disease expression was also less pronounced than on nectarine. In fact, plum fruit remained asymptomatic in the sound fruit test after inoculation and humid incubation at the 2 wk before harvest stage, harvest stage and after cold storage. Plum fruit at these stages only developed disease after a prolonged period (~12 h) of wet incubation. The paraquat fruit test revealed that these fruits became more susceptible to latent infection, but they were not as susceptible as nectarine. Collectively, these findings indicate that M. laxa fruit rot epidemics on plum and nectarine are driven by inoculum levels on fruit approaching maturity and by weather conditions prevailing during the preharvest and harvest period. However, the barrier capacity of plum skins is considerably more effective than that of nectarine fruit. Wounds would therefore play an important role in the epidemiology of M. laxa on plum fruit. Infection of fresh wounds by airborne M. laxa conidia, and by conidia and germlings that have established on fruits, was therefore investigated. Plum fruit (cultivar Laetitia) at pit hardening, 2 wk before harvest, harvest stage and after cold storage were dusted with dry conidia of M. laxa in a settling tower.- Infection of rionwounded fruit and of fresh wounds by \ the airborne conidia on dry, humid and wet plum fruit surfaces, and by conidia and germlings that have been established on fruits under the wetness regimes was then investigated. Nonwounded immature and mature fruit remained mostly asymptomatic, whereas nonwounded cold stored fruit decayed readily. Wounding drastically increased infection by airborne conidia. Immature fruits were less susceptible to wound infection by the airborne conidia than mature fruits. Conidia dispersed freshly were more successful in infecting fresh wounds than conidia that were deposited, or germlings that established, on fruit surfaces 4 days prior to wounding. This decrease in infectivity was especially pronounced on humid and even more on wet incubated fruit. This study clearly showed that in order to reduce. the incidence of brown rot, inoculum levels on fruit approaching maturity should be reduced by sanitation practices and fungicide applications. Furthermore, it is essential to protect fruits, especially. near-mature fruits, from being wounded. / AFRIKAANSE OPSOMMING: EPIDEMIOLOGIE VAN MONILINIA LAXA OP NEKTARIEN EN PRUIM: INFEKSIE VAN VRUGTE DEUR KONIDIA OPSOMMING Naoesverrotting van steenvrugte in die Wes-Kaap provinsie van Suid-Afrika word hoofsaaklik veroorsaak deur Botrytis cinerea (vaalvrot) en Monilinia laxa (bruinvrot). Min is bekend oor die relatiewe belang en seisoenale voorkoms van hierdie patogene in nektarienen pruimboorde, asook oor die infeksieweg, latensie en daaropvolgende siekte-uitdrukking van M laxa. Hierdie aspekte is in dié studie nagevors. \ \ Monsters IS oor 'n 3-jaar periode van die Unifruco Kwaliteitsevalueringskema, en ook van 11 steenvrugboorde verkry. Die voorkoms van vaalvrot en bruinvrot in die hoof steenvrugareas is so bepaal. Botrytis cinerea was die belangrikste patogeen wat betref bloeiselversenging en naoesverrotting. Verder was hierdie patogeen ook meer prominent op die vroeë- en middel-seisoen kultivars. Bruinvrot is uitsluitlik deur M Iaxa veroorsaak en geen aanduiding omtrent die moontlike voorkoms van M fructicola in Suid-Afrika is waargeneem nie. Monilinia laxa was meer prominent op die laat-seisoen kultivars. Botrytis cinerea bloeiselinfeksie het nie direk bygedra tot naoesverrotting nie. Beide oppervlakkige inokulum en latente infeksie het deurgaans, maar wel teen wisselende hoeveelhede, op vrugte in die onderskeie boorde voorgekom. Siekte-uitdrukking op ontwikkelende vrugte is egter nie beinvloed deur die hoeveelheid B. cinerea op die vrug nie, maar eerder deur die vermoë van die vrug om siekte-uitdrukking te onderdruk. Die hoeveelheid B. cinerea op vrugte was verder hoër gedurende lente as gedurende somer. Monilinia laxa het slegs sporadies op die bloeisels van laat-seisoen kultivars voorgekom. Groen vrugte was in die algemeen vry van die patogeen en siekte-uitdrukking het slegs op ryp vrugte plaasgevind. Hierdie bevindinge dui daarop dat M laxa in boorde hoofsaaklik op ryper vrugte geproduseer word. Hierdie swam infekteer ook net ryp vrugte. Die gedrag van luggedraagde M laxa conidia is bestudeer op nektarien- (kultivar Flamekist) en pruimvrugte (kultivar Laetitia). 'n Inokulasie-metode wat natuurlike infeksie deur luggedraagde konidia simuleer, is vir hierdie studies gebruik. Vrugte van die pitverharding-, twee weke voor oes-, oesstadium, asook koud-opgebergde vrugte (nektariene, 4 weke by -o.soe gevolg met 1 week by 23°C en ±56% RH; pruime, 10 dae by -O.5°e, 18 dae by 7.Soe gevolg deur 1 week by 23°C en ±56% RH), is met droë konidia in 'n inokulasietoring geïnokuleer. Die vrugte is vir periodes wat gewissel het van 3 tot 48 h geïnkubeer by hoë relatiewe humiditeit (~93% RH, vogtige vrugte), of dit is bedek met'n film water (nat vrugte). Die gedrag van die enkelspore (konidia) op die vrugoppervlak is met 'n epifluorisensiemikroskoop bestudeer. Skilsegmente is gekleur in 'n kleurstof, bevattende fluorisein diasetaat, analien-blou en blankofor. Die vermoë van die enkelspore om die vrugoppervlak te koloniseer, te penetreer en om siekte-uitdrukking te induseer, is met 'n differensiële stel toetse bepaal. Vir hierdie toetse is die vrugte oppervlakkig gesteriliseer (30 s in 70% etanol), of nie-steriel gelaat. In elke groep is vrugte geneem vir isolasie (skilsegment-to\~ts), of gedoop in "n 3% parakwat-oplossing (parakwat vrugtoets), of\, onbehandeld gelaat (onbehandelde vrugtoets ). Die. bevindinge het op die soortgelyke gedrag van M laxa enkelspore op die verskillende vrugsoorte gedui: appressoria en direkte penetrasie is nie waargeneem nie, en kiembuise het die vrugte hoofsaaklik deur huidmondjies, lentiselle en mikro-krakies .in die vrugskil gepenetreer. Deur luggedraagde spore te bestudeer, is sekere subtiele effekte van die vrug op die gedrag van enkelspore op die vrugoppervlak waargeneem. Op beide vrugtipes is geen nadelige effek op konidiurn- en kiembuisoorlewing opgemerk wanneer die vrugte onder hoë vogtoestande geïnkubeer is. Konidiurn- en kiembuisoorlewing is egter drasties verlaag hoe langer die vrugte onder nat toestande geïnkubeer is. Die bevindinge van die skilsegment-, parakwat en onbehandelde vrugtoetse het duidelik daarop gewys dat die vrugskil van nektarien en pruim nie gepenetreer is tydens die pitverhardingstadium nie, latente infeksies is nie gevorm nie, en die vrugte was bestand teen siekte-uitdrukking. Hierdie fasette op beide vrugtipes is ook nie beinvloed deur inkubasie-natheid nie. Die beskermingskapasiteit van die vrugskil van hierdie steenvrugtipes het egter drasties verskil later in die seisoen. Nektarien-vrugskille is meer geredelik gepenetreer en siekte-uitdrukking het toegeneem met rypwording. Penetrasie en siekteuitdrukking is verder in 'n groot mate deur inkubasie-natheid bevoordeel. Rypwordende pruime het egter nie so In drasties verandering in die beskermingskapasiteit van die vrugskil getoon nie. Die invloed van inkubasie-natheid op infeksie en siekte-uitdrukking was ook minder opsigtelik as op nektarien. Pruimvrugte van die twee weke voor oes-, oesstadium, en , koud-opgebergde pruime, wat onder hoë vog geïnkubeer is, het simptoomloos in die onbehandelde vrugtoets gebly. Vrugte van hierdie stadia het slegs simptome ontwikkel na periodes van langer as 12 h onder nat toestande. Die parakwat-behandelde vrugtoets het egter gewys dat die pruimvrugte meer vatbaar vir latente infeksies raak, maar steeds nie so vatbaar soos die nektarienvrugte nie. Gesamentlik dui hierdie bevindinge daarop <41tM laxa bruinvrot epidemies op pruim en nektarien afhanklik is van inokulumvlakke op rypwordende vrugte, asook die weerstoestande gedurende die vooroes- en oesstadia. Die beskermingskapasiteit van pruim vrugskille was egter aansienlik meer effektief as dié van nektarien vrugte. Wonde op vrugte sal dus 'n groter rol speel in die epidemiologic van M laxa op pruim. Infeksie van vars wonde deur luggedraagde M laxa konidia, en deur konidia en kiembuise wat reeds op die vrugoppervlak gevestig is, is gevolglik bestudeer. Pruimvrugte (kultivar Laetitia) van die pitverharding-, twee weke voor oes-, oesstadium, asook koud- \ \ opgebergde vrugte is in 'n inokulasie-toring geïnokuleer met droë M laxa konidia. .Infeksie , , van nie-gewonde vrugte en van vars wonde deur luggedraagde konidia op droë, vogtige en nat pruim vrugoppervlaktes, asook deur konidia en kiembuise wat reeds op die vrugoppervlak onder hierdie toestande gevestig is, is bepaal. Nie-gewonde groen tot ryp vrugte het meestal simptoomloos gebly, terwyl koud-opgebergde ryp vrugte wel verrot het. Wonde .het die hoeveelheid infeksie deur luggedraagde spore drasties vermeerder. Konidia wat geïnokuleer is op vrugte met vars wonde, was meer in staat om hierdie wonde te infekteer as konidia en kiembuise wat 4 dae voor wonding gevestig is. Hierdie afname in infektiwiteit was meer sigbaar op die vogtige, maar veral die nat vrugte. Hierdie studie het duidelik gewys dat inokulumvlakke op rypwordende vrugte verlaag moet word deur sanitasie-praktyke en fungisiedtoedienings. Dit is verder belangrik om vrugte, veral rypwordende vrugte, teen wonding te beskerm.

<p>Cilj ove doktorske disertacije je bio da se oceni mogućnost upotrebe tri sorte domaće &scaron;ljive, različitih epoha sazrevanja (Čačanska rana, Čačanska lepotica i Požegača), kao sirovina za proizvodnju voćnog vina. Utvrđeni mehanički sastav plodova i hemijske karakteristike kljuka i soka ispitivanih sorti &scaron;ljive ukazuju da se čačanska lepotica i Požegača mogu smatrati boljim sirovinama za proizvodnju voćnog vina u odnosu na sortu Čačanska rana. Vr&scaron;ena je optimizacija uslova alkoholne fermentacije (temperature, vrednosti pH, trajanja fermentacije i doze enzimskog preparata), u sklopu koje je, takođe, ispitana i upotreba različitih pektolitičkih enzima za tretman kljuka i ocenjen uticaj upotrebe različitih sojeva kvasaca, kao proizvodnih mikroorganizama, na kvalitet vina od &scaron;ljive. Utvrđeno je da, među ispitanim proizvodnim organizmima, kvasac Spriferm (S. cerevisiae) daje vino od &scaron;ljive najboljeg kvaliteta. Postupkom numeričke optimizacije dobijene su sledeće vrednosti procesnih parametara fermentacije vina od &scaron;ljive: temperatura 25 &deg;C, vrednost pH 3,5 i doza pektolitičkog enzima 0,5 g/100 kg. Pri navedenim uslovima dobijeni fitovani modeli predviđaju prinos etanola od 7,5% v/v, prinos glicerola od 5g/l, prinos vina od 48% (48 ml vina na 100 g kljuka) i formiranje 710 mg/l metanola. Karakterizacija proizvedenog vina od &scaron;ljive podrazumevala je određivanje sadržaja najvažnijih sastojaka: alkohola, kiselina, mineralnih materija, fenolnih i aromatičnih jedinjenja, kao i ocenu njegovih funkcionalnih karakteristika (antiradikalske, antimikrobne i antiproliferativne aktivnosti). Na kraju, ocenjena je mogućnost smanjenja produkcije metanola u vinu od &scaron;ljive primenom različitih fizičko-hemijskih tretmana kljuka. Utvrđena je značajno veća efikasnost postupaka koji uključuju neki vid toplotnog tretmana kljuka u odnosu na postupke koji podrazumevaju upotrebu određenog enolo&scaron;kog sredstva.</p> / <p>The aim of this PhD thesis was to assess the possibility of using three native plum varieties, with different ripening periods (Ĉaĉanska rana, Ĉaĉanska lepotica and Poņegaĉa), as raw material for the production of fruit wines. Determined mechanical composition and chemical characteristics of fruit pomace and juice indicate that the Ĉaĉanska lepotica and Poņegaĉa are considered as better raw materials for the production of fruit wine compared to Ĉaĉanska rana. Optimization of fermentation conditions (temperature, pH, the duration of fermentation and the dose of pectolytic enzyme) was conducted. This step also included investigation of the different pectolytic enzymes use for the treatment of pomace and evaluated the impact of using different yeast strains, as well as the effect of different production microorganisms on the plum wine quality. It was found that, among the tested production microorganisms, Spriferm (S. cerevisiae) yeast gives the plum wine of best quality. Numerical optimization procedure resulted with the following values of the process parameters of plum wine fermentation: temperature 25 &deg;C, pH value 3.5 and pectolytic enzyme dose of 0.5 g/100 kg. Under these conditions the obtained fitted models predict the ethanol yield of 7.5% v/v, glycerol yield 5 g/l, the wine yield of 48% (48 ml from 100 g of pomace) and the formation of 710 mg/l of methanol. Characterization of the produced plum wines included the determination of the most important ingredients: alcohol, acids, minerals, phenolic and aromatic compounds, as well as evaluation of their functional characteristics (antiradical, antimicrobial and antiproliferative activities). Finally, the possibility of reducing the methanol production in plum wine was estimated by applying different physico-chemical treatments of the pomace. Significantly higher efficiency of procedures that involve some form of heat treatment of pomace, compared to treatments which involve the use of certain oenological means, was observed.</p>