Certains microorganismes qui coexistent sur la vigne, peuvent avoir des effets bénéfiques sur la qualité du vin alors que d'autres peuvent être à l'origine de déviations organoleptiques. Dans la dernière décennie, dans diverses régions viticoles de France, plusieurs odeurs de moisi ou de terre ont été mises en évidence. La (-)-géosmine a été considérée comme étant le principal composé responsable de ce défaut. Des moisissures comme Botrytis cinerea et d’autres appartenant au genre Penicillium ont été souvent isolées à partir des raisins présentant l’odeur « moisi-terreuse ». Les effets de cette molécule sur la qualité des vins a motivé notre étude sur la caractérisation des moisissures responsables de ce défaut organoleptique. A partir d'échantillons prélevés en 2007 en Bourgogne, on a identifié, par des méthodes morphologiques et moléculaires, les moisissures présentes sur les raisins, Une souche de Penicilium expansum (25.03) et deux souches de Botrytis cinerea (BC1 et BC2) ont été sélectionnées. Sur baies de raisin, la validation d’un modèle prédictif des effets combinés de la température et de l'activité de l’eau, sur la croissance des champignons, a pu être mise en oeuvre. Elle a montré, sur de larges gammes de T°C et d’aw, que les modèles cardinaux avec inflexion peuvent être validés sur les produits agro-alimentaires en utilisant le gamma concept. L’étude de l’effet du cuivre sur le taux de croissance radiale et le temps de latence des moisissures, a été entreprise afin de mieux comprendre les mécanismes de résistance au cuivre des champignons et d’en déduire des résultats pour une meilleure efficacité des fongicides. Les moisissures testées ont montré une grande tolérance au cuivre, jusqu’à 4,7 mM pour P. expansum et jusqu’à 8, 2 mM et 7,3 mM respectivement pour B. cinerea, BC1 et BC2. L’étude des effets combinés des facteurs environnementaux et nutritionnels (T°C, CO2; Cu+2) sur la production de géosmine par P. expansum, a conduit à définir les conditions minimisant la production de géosmine. Ainsi, on a pu déterminer que le cuivre (composant actif de nombreux fongicides) est un facteur clé dans la production de géosmine par P. expansum. / Some microorganisms that co-exist on the grapevine may have beneficial effects on the quality of wine whereas others may be at the origin of organoleptic deviations. In the last decade, several mouldy or earthy odors have been highlighted in various wine regions from France. (-)-geosmin was found to be the major compound responsible for this deviation, along with Botrytis cinerea and fungi belonging to the genus Penicillium, since they were frequently isolated from “earthy-musty” odor grapes. The extent of damage on the quality of wines, motivated our study on the caracterisation of grape rot fungi. First of all, the microflora of grapes from Burgundy vineyards was identified (morphological and molecular methods), from samples prelevated in 2007. A Penicilium expansum strain (25.03) and two Botrytis cinerea strains (BC1 and BC2) were chosen for further experiments. The validation of a predictive model for the combined effect of temperature and water activity, on the growth of fungi on grape berries, demonstrated that cardinal models with inflexion can be validated on agri-food products, over a wide range of T°C and aw, using the gamma concept. Further we were focused on the influence of copper on the lag time and radial growth rate of moulds in order to better understand copper resistance mechanisms of the fungi and the efficacity of fongicides. The moulds tested showed a great copper tolerance, 4.7 mM for P. expansum and 8.2 and 7.3 mM for B. cinerea strains, BC1 and BC2 respectively. These results motivated our study on the influence of environmental and nutritional factors (T°C, CO2; Cu2+), using a Doehlert matrix, on geosmin production of the fungi tested. Copper (the active component of the “Bordeaux mixture”) showed to be a key factor in the increase of geosmin production by P. expansum.

La pourriture bleue causée par Penicillium expansum est l'une des maladies les plus dommageables des fruits pomaceae (pommes et poires). Outre des dégâts directs, cette maladie pose un problème de santé publique car l'agent pathogène produit des mycotoxines nocives pour l'homme et les animaux dont la plus sérieuse est la patuline. La croissance du champignon pathogène et la production de patuline requièrent des conditions physico-chimiques particulières. Les informations existantes à ce propos demeurent cependant modestes et insuffisantes pour envisager de développer des moyens de lutte contre l'apparition du champignon. Par ailleurs, la patuline reste avec l'ochratoxine A, les seules toxines dont la voie de biosynthèse n'a pas encore été complètement établie, tant sur le plan chimique que moléculaire. Cette étude apporte dans un premier temps des données complémentaires sur les facteurs physico-chimiques (température, pH….) qui conditionnent la croissance de P. expansum de même que sa capacité à produire la patuline. La connaissance de ces besoins et de ces conditions conduit en pratique à lutter et contrôler la contamination par la patuline tout le long de la chaine alimentaire. Dans un deuxième temps, cette thèse apporte des améliorations spectaculaires sur le plan fondamental, en termes d'élucidation de la voie de biosynthèse de la patuline. Le cluster des gènes impliqués dans la biosynthèse de cette mycotoxine chez l'espèce la plus préoccupante P. expansum a été entièrement identifié et caractérisé. Pour lever encore plus le voile sur la biosynthèse de cette mycotoxine, la caractérisation du facteur de régulation spécifique de cette voie (patL) a été également établie. Une perturbation de ce gène a provoqué une incapacité de production de patuline et une sévère diminution de l'expression des gènes Pat. De même, grâce à ce mutant déficient, il a été montré que la patuline pourrait agir comme facteur de virulence lors du développement de la moisissure dans les pommes. La caractérisation de la dernière étape de la voie de biosynthèse de la patuline a ensuite été entreprise par mutagenèse dirigée du gène patE du cluster de la patuline, chez la même espèce. Ce dernier code pour une Glucose méthanol Choline (GMC) oxydoréductase responsable de la conversion de l'ascladiol en patuline. L'ascladiol est également une molécule clé de la dégradation de la patuline par diverses espèces bactériennes ou de levures et plus particulièrement lors de la fermentation alcoolique. La non-toxicité de l'ascladiol accumulé chez le mutant ∆patE a été démontrée sur une lignée cellulaire intestinale humaine (Caco-2), suggérant que la patuline perd sa toxicité avec l'ouverture du deuxième cycle. Finalement, un système de détection et de quantification de P. expansum par PCR en temps réel a été développé en ciblant un gène hautement spécifique de la voie de biosynthèse de la patuline, patF. Cette approche préventive nous a ainsi permis d'avoir une estimation rapide de la contamination en patuline dans les pommes à partir de la quantification d'ADN de P. expansum. En conclusion, l'ensemble de ces travaux qui s'inscrivent dans le cadre de la gestion du risque « patuline » dans la filière fruit a permis d'amener des réponses tant sur le plan fondamental que sur le plan appliqué avec le séquençage du cluster, le développement d'un outil de diagnostic et la démonstration que l'ascladiol ne présentait aucune cytotoxicité. / Among diseases affecting apples, blue mold caused by Penicillium expansum is a major concern causing yield and quality losses due to the production of mycotoxins, of which patulin is the most alarming one. This mycotoxin was proven to be harmful for humans and animals. The pathogen growth and the patulin production occur under specific physico-chemical conditions (temperature, pH…). However, the description of these conditions in literature remains largely insufficient for the development of strategies to fight the development of the fungus. Furthermore, patulin remains, along with ochratoxin A, the only toxins for which the biosynthetic pathway is not fully established yet at both chemical and molecular levels. Firstly, this study provides supplementary data on the physico-chemical factors that modulate P. expansum growth and its ability to produce patulin. The acquaintance of these conditions leads, in practice, to the control of the patulin contamination along the food chain. Secondly, significant improvements were brought on the fundamental level, especially by elucidating the patulin biosynthetic pathway. The cluster of genes involved in the biosynthesis of this mycotoxin was fully identified and characterized in the species of greatest concern P. expansum. In order to reveal additional info on the biosynthesis of this mycotoxin, the specific factor of the pathway (patL) was characterized. The disruption of this gene has led to failure in patulin production and an important decrease in Pat genes expression. Furthermore, pathogenesis studies, using this same deficient strain showed that patulin potentially acts as a virulence factor during P. expansum development on apples. The last step of the patulin biosynthetic pathway was later characterized by site-directed mutagenesis of the patE gene in the same species. This gene encodes a Glucose Methanol Choline (GMC) oxidoreductase that is responsible for the conversion of ascladiol to patulin. Ascladiol is not only the last intermediate in the patulin pathway but also the main product of patulin degradation during the alcoholic fermentation of apple juice. The non-toxicity of ascladiol accumulated by the ΔpatE strain was proved against the human Caco-2 cell line. Finally a Real time PCR assay was developed to specifically detect and quantify P. expansum. This was done by targeting a highly specific gene from the patulin gene cluster in P. expansum, patF. This predictive approach allowed the quick estimation of the patulin content via the quantification of the P. expansum DNA in apples. To conclude, this thesis is part of the patulin's risk management study in the fruit sector; it provides significant improvements on both fundamental and practical levels. These advances are mainly characterized by the sequencing of the patulin gene cluster, the development of a molecular diagnostic tool and the demonstration of the non-cytotoxicity of ascladiol.

Parmi les maladies affectant les pommes, la moisissure bleue causée par Penicillium expansum est une préoccupation majeure. Elle cause des pertes de rendement et de qualité dues également à la production de mycotoxines telles que la patuline (PAT) et la citrinine (CIT). La PAT est la plus alarmante en raison de ses propriétés cytotoxiques, génotoxiques et immunosuppressives.L'Union européenne (UE) a établi des réglementations spécifiques pour protéger la santé des consommateurs et des niveaux maximaux de 50 g / kg sont fixés pour les jus de fruits et les produits dérivés, 25 g / kg pour les purées de pommes et les compotes et 10 g / kg pour les aliments destinés aux bébés et aux jeunes enfants. En dépit de ces mesures, la PAT continue à être présente dans les aliments et / ou les boissons commerciaux, dépassant parfois les limites maximales. Des recherches supplémentaires sont par conséquent nécessaires pour minimiser la contamination des produits alimentaires par cette mycotoxine et son champignon producteur. Bien que la plupart des études sur P. expansum soient essentiellement centrées sur la PAT, le génomede ce champignon présente d'autres clusters de métabolites secondaires (SM) prédits dont certains peuvent être associés à des métabolites potentiellement toxiques. Afin de contrôler la synthèse de SM, l'étude des facteurs de transcription globaux régulant leur production est essentielle. Dans une première partie, le gène veA, appartenant à la famille des protéines du complexe velvet, a été caractérisé et son impact sur le développement du champignon, sa virulence et son métabolisme secondaire a été élucidé. La délétion de ce gène a conduit à une réduction de la production de PAT et de CIT et à une diminution de l'expression de leurs clusters de gènes. VeA a également un impact global sur le métabolisme secondaire, puisque 15 des 35gènes de structure présentent une régulation différentielle sur les milieux testés. Dans une deuxième partie, l’influence de l’acide ascorbique (AA) sur la concentration de PAT dans le jus de pomme trouble a été étudiée à la fois en laboratoire et en milieu semi-industriel. Une méthodologie analytique séparant la PAT et d'autres composés générés au cours de la réaction aété optimisée. Les conditions optimales d'action de l’AA sur la PAT ont été analysées. De plus,nous avons identifié des produits de dégradation moins toxiques que la PAT et résultant du traitement par l’AA. Pour conclure, cette thèse se rattache à la gestion des risques de la PAT dans le secteur des fruits ; elle apporte des connaissances et des améliorations significatives tant sur le plan fondamental que sur le plan pratique. Ces avancées résident principalement dans la description d'une souche mutée de P. expansum moins toxique que celle naturellement retrouvée dans la nature, et décrivant un additif alimentaire améliorant les qualités de nombreux produits transformés et diminuant la concentration de PAT en générant des composés moins toxiques. / Among diseases affecting apples, blue mould caused by Penicillium expansum is a major concern. It causes yield and quality losses, as well as food safety issues due to the production of mycotoxins such as patulin (PAT) and citrinin (CIT). PAT is the most worrying one and has cytotoxic, genotoxic and immunosuppressive properties. The European Union (EU) has established specific regulations to protect the consumer’s health and maximum levels of PAT of 5 g/kg is set for fruit juices and derived products, 25 g/kg for apple purees and compotes and 10g/kg for food intended for babies and young children. However, PAT is still found in commercial food and/or beverage products, sometimes exceeding the maximum limits and more research is needed to minimize contamination of food products by this mycotoxin and its fungus. Even though most studies on P. expansum have focused on PAT itself, the genome of this fungus exhibits other predicted secondary metabolite (SM) clusters, some of which may be associated with potentially toxic metabolites. In order to control the synthesis of SMs, the study of global transcription factors regulating their production is essential. In a first part, the veA gene, belonging to the velvet family, was characterised and its impact on the development of the fungus, its virulence and its secondary metabolism was elucidated. The disruption of this gene led to the failure in PAT and CIT production and a decrease in the expression of their gene cluster. It also revealed a global impact on the secondary metabolism, as 15 of 35 backbone genes showed differential regulation on the media tested. In a second part, the influence of ascorbic acid (AA) on the concentration of PAT in cloudy apple juice was studied on both lab and semi-industrial scale. An analytical methodology separating PAT and other compounds generated during the reaction was optimized. Optimal conditions of action of AA on PAT were studied. In addition, degradation products less toxic than PAT and resulting from AA treatment were identified. To conclude, this thesis is part of the risk management of PAT in the fruit sector; it provides significant improvements at both fundamental and practical levels. These advances are mainly characterized by the description of a mutated strain of P. expansum that is less toxic than that naturally occurring in nature, and the description of a food additive that improves numerous products qualities and affects PAT concentration, thusgenerating less toxic compounds

La domestication est un modèle idéal pour étudier les processus évolutifs car elle implique des événements d'adaptation récents avec une sélection forte. Les champignons sont de bons organismes modèles pour étudier la domestication et plus généralement l’adaptation, grâce à leurs petits génomes et leur facilité de manipulation. Ils sont utilisés depuis longtemps pour la transformation alimentaire, par exemple P. camemberti et P. roqueforti pour la fabrication du fromage, et la levure Saccharomyces pour la fermentation du vin et de la bière. Chez ces champignons, des caractéristiques bénéfiques ont été acquises pour la transformation alimentaire, et les transferts horizontaux de gènes se sont révélés être un moyen essentiel d’adaptation rapide dans l’environnement alimentaire. Ici, j’ai étudié principalement l’adaptation de deux espèces de Penicillium relativement distantes phylogénétiquement - P. nalgiovense et P. salamii, toutes deux utilisées pour la maturation de la viande séchée. J’ai étudié si ces champignons ont été domestiqués, c’est-à-dire si les populations alimentaires se sont adaptées à l’environnement de la viande séchée, et s’il y a eu une différenciation génétique par rapport à d’autres populations; j’ai aussi recherché si des traces génomiques d’adaptation pouvaient être détectées. En analysant des génomes complets, j’ai trouvé peu de diversité génétique et de structure de population chez P. salamii et encore moins chez P. nalgiovense. Des expériences ont montré que les populations de P. salamii et P. nalgiovense provenant de viande séchée présentaient des taux de protéolyse et de lipolyse plus faibles et des couleurs différentes de celles des populations de viande non séchée. De plus, nous avons trouvé des transferts de gènes horizontaux partagés par P. salamii et P. nalgiovense et absents chez d’autres espèces de Penicillium. En résumé, ces résultats indiquent une évolution convergente et une adaptation des populations de P. salamii et P. nalgiovense à la viande séchée. J'ai également étudié les conséquences de la domestication chez le champignon utilisé pour la production de fromages bleus, P. roqueforti, montrant une faible fertilité des souches fromagères par rapport aux souches non fromagères. Les résultats de la thèse soulignent donc l'importance des transferts de gènes horizontaux pour une adaptation rapide chez les champignons et renforcent l'idée que les champignons domestiqués pour la production de nourriture sont de bons modèles pour étudier l'adaptation et l'évolution. / Domestication is an ideal model to study evolutionary processes due to the recent adaptation events and strong selection it implies. Fungi in particular are good model organisms to study domestication and more generally adaptation, with their small genomes and experimental tractability. Fungi have been used for food production, e.g., P. camemberti and P. roqueforti for cheesemaking, and Saccharomyces yeast for wine and beer fermentation. In these fungi, beneficial traits have been acquired for food production, and horizontal gene transfers (HGTs) have been shown to be a major way to rapid adaptation in food environment. Here, I mainly studied the adaptation of food Penicillium fungi using two distantly related Penicillium species - P. nalgiovense and P. salamii, both used for dry-cured meat maturation, to assess whether these fungi have been domesticated, i.e., whether food populations adapted to the dry-cured meat environment, whether were genetically differentiated from other populations, and whether we could find genomic footprints of adaptive events. Using genome sequencing, we found little diversity and population structure in P. salamii and even less in P. nalgiovense. Experiments showed that both P. salamii and P. nalgiovense dry-cured meat populations had lower proteolysis and lipolysis rates and different colors from non-dry-cured meat populations. Furthermore, we found HGTs shared by P. salamii and P. nalgiovense while lacking in other Penicillium species. Altogether, these results indicate convergence evolution and adaptation in P. salamii and P. nalgiovense dry-cured meat populations, as was previously found in cheese Penicillium fungi. I also studied the consequences of domestication in the blue cheese fungus P. roqueforti, showing lower fertility of cheese strains compared to non-cheese strains. The results of the thesis thus point out the importance of HGTs for rapid adaptation in fungi and reinforce the view that fungi are ideal models to study adaptation and evolution.

Thesis (MSc (Plant Pathology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Dry core rot (DCR) and wet core rot (WCR) are among some of the most important postharvest diseases of apples in South Africa. Mouldy core (MC) is also a symptom associated with the core region of apples, but it is not of economical importance since apple tissue surrounding the core region is not affected as is the case with DCR and WCR. The incidence of core rots in harvested fruits can be as high as 12%, but in general ranges from 3 to 8%. Infections and losses can also occur during fruit handling in pack houses and during storage. Additionally, yield losses also occur prior to harvest within orchards due to premature fruit drop of core rot affected fruits. The incidence of core rot diseases in apples differ among apple cultivars, with most Red Delicious varieties being susceptible to the development of core rots, whereas core rots have rarely been reported in other cultivars such as Granny Smith. The etiology and epidemiology of WCR and DCR are poorly understood. Although many fungal genera have been associated with the diseases, small-spored Alternaria species are mainly associated with DCR, whereas Penicillium species including P. roquefortii, P. expansum and P. funiculosum have mainly been associated with WCR. Dry core rot infections have long been known to occur pre-harvest, whereas WCR is primarily known as a post-harvest disease where infections take place during fruit handling in pack houses. Recently, Tarsonemus mites have also been indicated as being a potential role player in the etiology of core rot diseases. The mites have been hypothesised to carry pathogen spores into the core region of apples, and they may also possibly cause small wounds that facilitate pathogen entry. In South Africa, apple growers have recently reported WCR as being present prior to harvest, which has not been reported previously. Therefore, the first aim of the study was to investigate the incidence, as well as the causal agent/s of pre-harvest WCR. The incidence of WCR ranged from 0% to 1.7% in eleven orchards, and was in general lower than that of DCR (0.4% to 6%). Isolation studies from eight internal positions in WCR apples showed that Penicillium was the predominant fungal genus in most of the positions, including the lesion area. Morphological and molecular characterisation of Penicillium isolates from WCR showed that P. 2 ramulosum prov. nom. was the main species isolated from lesions, as well as other isolation positions. However, this species was also the main species isolated from DCR, MC and asymptomatic apples. Penicillium expansum was only isolated at low frequencies from WCR and DCR apples. Other Pencillium species that were occasionally isolated included P. glabrum, P. chloroloma, P. chermisinum and a putative new species with closest affinity to P. dendriticum (P. species aff. dendriticum) on a DNA nucleotide sequence basis. Pathogenicity and virulence studies using three different inoculation methods showed that P. expansum was the most virulent species, followed by P. species aff. dendriticum. The P. ramulosum prov. nom. isolates varied in their virulence, but were all considered to have low virulence. The role of Tarsonemus mites in the etiology and epidemiology of core rot diseases is poorly understood, and therefore the second aim of the study was to investigate some of these aspects. The specific aims of the study were to (1) investigate the ecology of Tarsonemus mites in Red Delicious and Granny Smith orchards during different apple developmental stages, (2) determine if there is a significant association of Tarsonemus mites with diseased (WCR and DCR) fruits and (3) determine if potential core rot pathogenic fungi are associated with the mites. Tarsonemus mites were found in all of the investigated apple developmental stages (buds, blossoms, 4cm diameter fruit, mature fruit and mummies), having the highest incidence in mummies and mature fruits from Red Delicious and Granny Smith orchards. In Red Delicious fruits the Tarsonemus mites were found within the core and/or calyx tube, whereas in Granny Smith fruits the mites were restricted to the calyx tube. In Red Delicious fruits there was a significant association between dry core rot as well as total core rot (wet- and dry-core rot) with the presence of mites in the core, as well as total mites (mites in core and calyx tubes). Fungal isolation studies from the Tarsonemus mites showed that they carried potential core rot fungal pathogens within the genera Penicillium and Alternaria. The Penicillium species isolated from the mites included two of the most virulent WCR species, P. expansum and P. species aff. dendriticum. / AFRIKAANSE OPSOMMING: Droë kernvrot and nat kernvrot is van die belangrikste na-oes siektes van appels in Suid- Afrika. Beskimmelde kern word ook met die kern van appels geassosieer, maar hierdie toestand is egter nie van ekonomiese belang nie, aangesien die weefsel rondom die kern nie geaffekteer word soos in die geval van nat- en droë kernvrot nie. Die voorkoms van kernvrot in vrugte na oes, kan vlakke van tot 12% bereik, maar oor die algemeen is die voorkoms tussen 3 en 8%. Infeksie en verliese kan ook voorkom gedurende die hantering en verpakking van vrugte in pakhuise en gedurende storing. Addisionele verliese in opbrengs kan ook voor-oes voorkom in boorde. Dit is te wyte aan voortydige vrugval van appels wat besmet is met kernvrot. Die voorkoms van kernvrot by appels verskil tussen kultivars. Meeste van die “Red Delicious” variëteite is vatbaar vir die ontwikkeling van kernvrot. Die toestand is egter skaars by ander kultivars soos Granny Smith. Die etiologie en epidemiologie van nat- en droë kernvrot word nie goed verstaan nie. ‘n Groot aantal swamgenera is al met kernvrot geassosieer. Klein-spoor Alternaria spesies word hoofsaaklik met droë kernvrot geassosieer en Penicillium spesies, insluitende P. roquefortii, P. expansum en P. funiculosum, word meestal met nat kernvrot geassosieer. Dit is lank reeds bekend dat droë kernvrot as voor-oes siekte kan voorkom, maar nat kernvrot is algemeen bekend as na-oes siekte waar infeksie tydens vrughantering en verpakking plaasvind. Daar is onlangs aangedui dat Tarsonemus myte potensiële rolspelers in die etiologie van kernvrot is. Hipoteties is die myte in staat om spore van die patogene in die kern van die appels in te dra, asook om klein wonde te veroorsaak wat infeksie deur patogene vergemaklik. In Suid-Afrika is nat kernvrot wat voor-oes in die boorde ontstaan onlangs deur boere aangemeld; hierdie toestand is nog nie op ‘n vorige geleentheid aangemeld nie. Die eerste doelwit van hierdie studie was dus om die voorkoms en veroorsakende organisme/s van voor-oes nat kernvrot te ondersoek. Die voorkoms van nat kernvrot was tussen 0 en 1.7% in elf boorde en was oor die algemeen laer as die voorkoms van droë kernvrot (0.4 tot 6%). Isolasiestudies uit agt interne posisies van nat kernvrot appels het getoon dat Penicillium die dominante swamgenus in die meeste posisies was, insluitend die letsels. Morfologiese en molekulêre karakterisering van 4 Penicillium isolate uit nat kernvrot letsels het aangedui dat P. ramulosum prov. nom. die spesie is wat die meeste geïsoleer is vanuit die letsels, asook ander isolasie posisies. Dié spesie was egter ook die mees algemene spesie wat uit nat- en droë kernvrot, asimptomatiese appels en appels wat slegs swamgroei in die kern gehad het, geïsoleer is. Penicillium expansum was ook in lae getalle uit nat- en droë kernvrotletsels geïsoleer. Ander Penicillium spesies wat ook soms geïsoleer is, sluit P. glabrum, P. chloroloma, P. chermisinum, asook ‘n moontlik nuwe spesie wat op DNA volgorde basis die naaste aan P. dendriticum (P. spesie aff. dendriticum) is. Studies wat patogenesiteit en virulensie van die isolate ondersoek het, is ook uitgevoer deur gebruik te maak van drie verskillende inokulasie metodes. Die studies het aangedui dat P. expansum die mees virulente spesie is, gevolg deur P. spesie aff. dendriticum. Die P. ramulosum prov. nom. isolate het variasie in virulensie getoon maar is oor die algemeen aanvaar om minder virulent te wees. Die rol van Tarsonemus myte in die etiologie en epidemiologie van kernvrot word nie goed verstaan nie en dus was die tweede doelwit van die studie om sommige van dié aspekte te ondersoek. Die spesifieke doelwitte was (1) om die ekologie van die Tarsonemus myte in “Red Delicious” en Granny Smith boorde tydens verskillende ontwikkelingstadiums van die appels te ondersoek, (2) om te bepaal of daar ‘n betekenisvolle assosiasie van Tarsonemus myte met siek (nat- en droë kernvrot) vrugte bestaan en (3) om te bepaal of potensiële kernvrot patogeniese swamme geassosieer is met die myte. Tarsonemus myte is gevind in al die ontwikkelingstadiums (knoppies, bloeisels, 4 sentimeter deursnee vrugte, volwasse vrugte en mummies) van appels wat ondersoek is. Die hoogste voorkoms van myte was in die mummies en volwasse vrugte van “Red Delicious”, asook Granny Smith kultivars gevind. In “Red Delicious” vrugte is myte in die kern en/of kaliksbuis gevind, maar in die Granny Smith vrugte was die myte tot die kaliksbuis beperk. In “Red Delicious” vrugte was daar ‘n betekenisvolle assosiasie tussen droë kernvrot, asook totale kernvrot (nat en droë kernvrot) met die teenwoordigheid van myte in die kern, asook totale myte (myte in die kern en kaliksbuis). Swam isolasiestudies vanaf die Tarsonemus myte het aangetoon dat potensiële kernvrot swampatogene in die genera Penicillium en Alternaria wel by die myte teenwoordig was. Die Penicillium spesies wat vanaf die myte geïsoleer is het twee van die mees virulente nat kernvrot spesies ingesluit, nl. P. expansum en P. spesie aff. dendriticum.

Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study looked at the incidence, etiology and epidemiology of core rot of apples in orchards situated in the Western Cape, South Africa. Core rot is a post-harvest disease, with three symptoms, namely mouldy core (MC), dry core rot (DCR) and wet core rot (WCR). These symptoms are caused by various pathogenic fungi, including Alternaria and Penicillium. Although MC is not economically important, DCR and WCR are, as they affect the flesh of the fruit. Core rot occurs worldwide in susceptible apple cultivars such as ‘Starking’ and ‘Red Delicious’. These cultivars have a wider, open calyx tube which results in an open core area. In South Africa, core rot of apples are important post-harvest diseases and losses of between 5 and 12% occur in apple cultivars. An in depth literature search was done on core rot including literature on each core rot symptom, the genuses Alternaria and Penicillium, molecular identification and techniques, disease incidence and its economic importance, various inoculum sources, pathogenicity of core rot organisms and integrated management of core rot. This study included two research chapters, with seven objectives, namely, to 1, determine the incidence of core rot in apples from commercial orchards both pre- and post-harvest; 2, to identify the causal organisms associated with core rot symptoms; 3, to identify potential sources of inoculum of core rot pathogens and determine whether there is synergism between Alternaria and Tarsonemus mites associated with core rot; and 4, to determine whether the fungicide Bellis®, used a full bloom application, can be used to manage core rot in South Africa; 5, to identify the species of Alternaria and Penicillium sampled from core rot symptomatic fruit and inoculum sources (air, apple mummies and mites), using morphological and molecular methods; 6, to compare Penicillium species isolated from pre- and post-harvest WCR symptomatic fruit, using molecular species identification methods and 7, to compare and to select the most reliable pathogenicity test for use in future research. The total decay incidence for Ceres is considerably higher than the previous losses indicated in literature. Pre-harvest core rot, which was confirmed by previous studies, had a higher incidence of each core rot symptom than previously indicated. The two most frequently isolated causal organisms were Alternaria and Penicillium. Other organisms isolated and then identified from the symptoms were Fusarium, Cladosporium, Epicoccum, Ulocladium, Stemphylium, Phoma, Botryosphaeria, Botrytis, Trichoderma, Verticillium, Paecilomyces and Gliocladium. Three inoculum sources, air, mummies and mites, were regarded as potential sources of infection for core rot. During this study the sources of infection were verified and core rot causing organisms were isolated from these sources. Alternaria was isolated from air inoculum samples, but was not found on the other two sources. This dismissed the hypothesis that there was a possible synergism between Alternaria and Tarsonemus mites. Penicillium species were isolated from all three sources, more frequently from the mummies and mites. Bellis® was applied three times during the bloom period. The subsequent results showed a significant difference between the control and Bellis® treated treatments with the treated fruit having a significant higher incidence than the controlled fruit. No control was observed with this result and managing core rot with only Bellis® is not advisable. Alternaria species were identified using the following genetic loci, ITS, OPA1-3, 2-1 and 10-2 as well as endoPG. Isolates from pre- and post-harvest symptoms and air inoculum were identified using each of the genetic loci. Alternaria arborescens was one of the species that was identified. The other isolates obtained were A. alternata, A. tenuissima, A. gaisen, A. dumosa, A. turkisafria and A. perangusta. Separating combined species was not possible. Another molecular technique, ISSR, was used to identify Alternaria species. This technique, after multiple re-runs, did not give consistent results and species could not be identified. Penicillium species were identified using the genetic loci ITS for isolates collected from pre- and post-harvest symptoms and inoculum sources. Thirteen clades were identified, including the species P. ramulosum, P. sp. (aff. cecidicola), P. sp (aff. dendriticum), P. expansum, P. paneum, P. solitum, P. crustosum , P. brevicompactum, P. novae-zeelandiae, P. glabrum and P. rugulosum. Penicillium expansum and P. ramulosum had the highest distribution between the isolates. Pre- and post-harvest WCR isolates were identified using the partial beta-tubulin PCR-RFLP method, and comparing different banding-patterns. The species identified using this method were P. expansum, P. ramulosum, P. sp. (aff. cecidicola), P. sp (aff. dendriticum), P. rugulosum, P. chermesinum and P. glabrum. Penicillium ramulosum and P. expansum had the highest incidence with P. ramulosum occurring more frequently pre-harvest than post-harvest and P. expansum occurring more frequently post-harvest. Five methods, previously published, were compared to select the most reliable pathogenicity test. The methods included surface wounding of an apple with colonised toothpicks, surface wound inoculated with a pipette, inoculation of an open mesoderm core cavity, deep and non-wounding of apple fruit with colonised toothpicks. The surface wounding with a colonised toothpick gave the most reliable results and can be used in industry as a pathogenicity test for Alternaria in apples. This study contributed to our understanding on the incidence and etiology of core rot in the Western Cape as well as in identifying inoculum sources from where infection can take place in the orchard. The results for the fungicide trial were not as anticipated and more research is required on selecting fungicides for the control of core rot in South African orchards. Although molecular techniques reduce the time in identifying fungal species, it is costly and mistakes can occur due to contamination. Identification of species can be incorrect when using a Genbank as the sequence information may be incorrect. Molecular techniques, though a good tool in identifying species, should be combined with morphological characteristics to ensure more accurate results. / AFRIKAANSE OPSOMMING: Hierdie studie het gekyk na die insidensie, etiologie en epidemiologie van kernvrot in appels vanuit boorde in die Wes-Kaap, Suid-Afrika. Kernvrot is ‘n na-oes iekte, met drie simptome, naamlik beskimmelde kern, droë kernvrot en nat kernvrot. Hierdie simptome word veroorsaak deur verskillende patogeniese swamme, insluitend Alternaria en Penicillium. Alhoewel beskimmelde kern nie ekonomies belangrik is nie, is droë en nat kernvrot wel belangrik, omdat hulle die vrug se vlees affekteer. Kernvrot kom wêreldwyd voor in vatbare kultivars soos ‘Starking’ en ‘Red Delicious’. Hierdie kultivars het ‘n wye, oop kelkbuis wat ‘n oop kern area veroorsaak. In Suid-Afrika is kernvrot van appels ‘n belangrike na-oes siekte en verliese tussen 5 en 12% kom voor in appel kultivars. ‘n In diepte literatuurstudie is gedoen omtrent kernvrot, insluitend literatuur omtrent elke kernvrot simptoom, die genera Alternaria en Penicillium, molekulêre identifikasie en tegnieke, siekte insidensie en sy ekonomiese impakte, verskillende inokulum bronne, patogenisiteit van kernvrot organismes en die geïntegreerde bestuur van kernvrot. Hierdie studie sluit in twee navorsings hoofstukke met sewe doelwitte, naamlik om 1, te bepaal wat die insidensie van kernvrot in appels is vanuit kommersiële boorde vir beide voor en na-oes; 2, om veroorsakende organismses wat met kernvrot simptome geassosieër is te identifiseer; 3, om potensiële inokulum bronne van kernvrot patogene te identifiseer en te bepaal of daar ‘n sinergisme tussen Alternaria en Tarsonemus myte, wat geassosieër is met kernvrot, is; 4, om te bepaal of die fungisied Bellis®, gebruik as ‘n volblom toediening, gebruik kan word om kernvrot in Suid-Afrika te beheer; 5, om die Alternaria en Penicillium spesies wat uit simptomatiese kernvrot vrugte en inokulum bronne geïsoleer is te identifiseer; 6, om Penicillium spesies, wat uit voor en na-oes nat kernvrot simptome geïsoleer is, te vergelyk deur gebruik te maak van molekulêre spesies identifiserings metodes en 7, om die betroubaarste patogenisiteits toets te vergelyk en selekteer vir toekomstige gebruik. Die totale bederfde insidensie vir Ceres is heelwat hoër as die vorige verliese wat aangedui is in literatuur. Vooroes kernvrot, wat deur vorige studies bevestig is, het ‘n hoër insidensie vir elke kernvrot simptoom gehad as wat voorheen aangedui is. Die twee geïsoleerde veroorsakende organismes wat die meeste voorgekom het was Alternaria en Penicillium. Ander organismes wat geïsoleer en geïdentifiseer is vanuit die simptome was Fusarium, Cladosporium, Epicoccum, Ulocladium, Stemphylium, Phoma, Botryosphaeria, Botrytis, Trichoderma, Verticillium, Paecilomyces en Gliocladium. Drie inokulum bronne, lug, gemummifiseerde vrugte en myte, is geag as potensiële bronne van infeksie vir kernvrot. Gedurende hierdie studie is hierdie bronne bevestig en kernvrot veroorsakende organismes is uit die bronne geïsoleer. Alternaria is geïsoleer vanuit die lug inokulum monsters, maar is nie geïsoleer vanuit die ander twee bronne nie. Dus die hipotese dat daar ‘n sinergisme tussen Alternaria en Tarsonemus myte is, is verwerp. Penicillium spesies is geïsoleer vanuit al drie bronne, maar meer gereeld vanuit die gemummifiseerde vrugte en die myte. Bellis® is drie keer gedurende die bot toegedien. Die daaropvolgende resultate het ‘n betekenisvolle verskil tussen die kontrole en Bellis® beheerde behandelings getoon, met die behandelde vrugte wat ‘n betekenisvolle hoër insidensie gehad het as die kontrole vrugte. Geen beheer is waargeneem nie en beheer van kernvrot met net Bellis® word nie aanbeveel nie. Alternaria spesies is geïdentifiseer deur die volgende genetise lokusse, ITS, OPA1-3, 2-1 en 10-1, asook endoPG. Isolate van voor en na-oes simptome en lug inokulum is geïdentifiseer deur elk van die genetiese lokusse. Alternaria arborescens is een van die spesies wat geïdentifiseer is. Ander isolate wat verkry is, was A. alternata, A. tenuissima, A. gaisen, A. dumosa, A. turkisafria and A. perangusta. Om gekombineerde spesies te skei was nie moontlik nie. ‘n Ander molekulêre tegniek, ISSR, was gebruik om Alternaria spesies te identifiseer. Hierdie tegniek, na menigte probeerslae, het nie konsekwente resultate gegee nie en spesies kon nie hiermee geïdentifiseer word nie. Penicillium spesies, versamel vanuit voor en na-oes simptome en inokulum bronne, is geïdentifiseer deur die genetiese lokus ITS. Dertien ‘clades’ is geïdentifiseer, insluitend die spesies P. ramulosum, P. sp. (aff. cecidicola), P. sp (aff. dendriticum), P. expansum, P. paneum, P. solitum, P. crustosum , P. brevicompactum, P. novae-zeelandiae, P. glabrum en P. rugulosum. Penicillium expansum en P. ramulosum het die hoogste distribusie tussen die isolate. Voor en na-oes nat kernvrot isolate is geïdentifiseer deur die deels beta-tubulin PCR-RFLP metode, en verskillende band patrone te vergelyk. Die spesies geïdentifiseer deur hierdie metode is P. expansum, P. ramulosum, P. sp. (aff. cecidicola), P. sp (aff. dendriticum), P. rugulosum, P. chermesinum en P. glabrum. Penicillium ramulosum en P. expansum het die hoogste insidensie gehad met P. ramulosum wat meer dikwels vooroes voorkom en P. expansum wat meer dikwels na-oes voorkom. Vyf metodes, wat voorheen gepubliseer is, is vergelyk om die betroubaarste patogenisiteits toets te selekteer. Die metodes sluit in die oppervlak wond van ‘n appel met ‘n gekoloniseerde tandestokkie, oppervlak wond geïnokuleer met ‘n pipette, inokulasie van ‘n oop mesoderm kern area, diep besering en nie-besering van die appel met gekoloniseerde tandestokkies. Die oppervlak besering met ‘n gekoloniseerde tandestokkie het die betroubaarste resultate gegee en kan in die industrie gebruik word as ‘n patogenisiteits toets vir Alternaria in appels. Hierdie studie het bygedra tot ons kennis van die insidensie en etiologie van kernvrot in die Wes-Kaap sowel as die identifisering van die inokulum bronne, van waar die infeksie in die boord kan plaasvind. Die resultate vir die fungisied proef was nie wat ons verwag het nie en meer navorsing word benodig om fungisiede te selekteer vir die beheer van kernvrot in Suid-Afrikaanse boorde. Alhoewel molekulêre tegnieke die tyd verminder om ‘n swam spesie te identifiseer, is dit wel duur en foute kan voorkom as gevolg van kontaminasie. Identifikasie van spesies kan verkeerd wees indien Genbank gebruik is, omdat die informasie daar nie altyd korrek is nie. Molekulêre tegnieke, alhoewel ‘n goeie manier om spesies te identifiseer, moet gekombineer word met morfologiese karakter eienskappe om akurate resultate te verseker.

Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Please refer to full text for abstract / AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

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Made available in DSpace on 2015-04-22T22:12:44Z (GMT). No. of bitstreams: 1 DISSERTACAO - JOSY CALDAS DA SILVA.pdf: 387435 bytes, checksum: f7060f21d123d5cf685b7a0dc1bfdc5e (MD5) Previous issue date: 2008-02-20 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / Penicillium are anamorphics fungi economically important for producing composts for uses such as supplement or food products improvement and also are used in bioremediation for environments recovery. Therefore, this work had as objective to verify the purity, authenticity, viability and the antimicrobial activity of 60 cultures of Penicillium from the Amazon region preserved in distilled sterilized water and under mineral oil, deposited in DPUA Culture Collection. The revised microorganisms were reactivated in Czapek Yeast Extract Agar (CYA) and authenticated based on macro and micromorphological characteristics on Malt Extract Agar (MEA), Glicerol Nitrate Agar 25% (w/v) [G25N] and Czapek Yeast Extract Agar (CYA). After authentication, the cultures were submitted to antimicrobial activity by the Gelose Block Method. The antimicrobial activity was performed in selective culture medium at 25oC and 37oC against the following test microorganisms: Candida albicans DPUA 1340, Staphylococcus aureus CCT 1352, Escherichia coli CCT 0547 and Mycobacterium smegmatis PDUFPE-7. The antimicrobial activity was evaluated by measuring the halo of inhibition against the test microorganisms. For biocomposts detection by bioautography plugs of cultures of Penicillium selected by diffusion in agar were submitted to the extraction in Ethyl Acetate (6: 4, v/v) using as standard Itraconazol and Riphampicine. Among the examined cultures, 90.0% expressed viability, purity and taxonomically confirmed in accordance with specialized literature. From these, 46.66% demonstrated positive result in the antimicrobial activity for diffusion in agar and 25.0% in the bioautographic assay. From the results it has proved preservation methods efficiency in distilled sterilized water and under mineral oil, as well as the Penicillium species potential in the biocomposts production with antimicrobial activity. / Penicillium são fungos anamorfos economicamente importantes por produzirem compostos para uso como suplemento ou melhoramento de produtos alimentícios e, também são utilizados em biorremediação para recuperação de ambientes. Dessa forma, este trabalho teve como objetivo verificar a pureza, autenticidade, viabilidade e a atividade antimicrobiana de 60 culturas de Penicillium da região amazônica, preservadas sob água destilada esterilizada e óleo mineral, pertencentes ao acervo de Coleção de Cultura DPUA. Os microrganismos revisados foram reativados em Ágar Extrato de Levedura Czapek (CYA) e autenticados, com base nas características macro e micromorfológicas, em Ágar Extrato de Malte (MEA), Ágar Glicerol Nitrato 25% (p/v) [G25N] e Ágar Extrato de Levedura Czapek (CYA). Posterior a autenticação, as espécies foram submetidas à atividade antimicrobiana pelo Método do Bloco de Gelose. A atividade antimicrobiana foi realizada em meio de cultura seletivo, a 25 oC e 37oC, frente aos seguintes microrganismos-teste, Candida albicans DPUA 1340, Staphylococcus aureus CCT 1352, Escherichia coli CCT 0547 e Mycobacterium smegmatis PDUFPE-7. A atividade antimicrobiana foi avaliada medindo-se a área de inibição contra o microrganismo-teste. Para detecção de biocompostos por bioautograifa, fragmentos das culturas dos Penicillium selecionados por difusão em ágar foram submetidos à extração em Acetato de Etila (6:4, v/v) utilizando-se como padrão Itraconazol e Rifampicina. Entre as culturas examinadas, 90% expressaram viabilidade, pureza e taxonomicamente confirmadas de acordo com a literatura especializada. Dessas, 46,66% apresentaram resultado positivo na atividade antimicrobiana por difusão em ágar e 25% nos ensaios bioautográficos. A partir dos resultados comprovou-se a eficiência dos métodos de preservação em água destilada esterilizada e óleo mineral, assim como o potencial de espécies de Penicillium na produção de biocomposto com atividade antimicrobiana.

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-05-07T23:12:51Z No. of bitstreams: 1 131150001.pdf: 1709524 bytes, checksum: 7ab2f239e6dc021c9db84badef44c320 (MD5) / Made available in DSpace on 2015-05-07T23:12:51Z (GMT). No. of bitstreams: 1 131150001.pdf: 1709524 bytes, checksum: 7ab2f239e6dc021c9db84badef44c320 (MD5) Previous issue date: 2014-12-19 / Os fungos na Antártica ocupam nichos distintos e realizam diferentes interações, porém sua importância nestes nichos e interações ainda são pouco compreendidas. Interações entre fungos e musgos vêm sendo relatadas para a Antártica, um exemplo dessa interação fungo- musgo é a presença de fungos formadores de anéis sobre carpetes de musgos antárticos. Estes fungos formadores de sistemas de anéis concêntricos podem causar necroses e manchas amarelas e marrons sobre os carpetes de musgos. Contudo, devido à complexidade destes fungos, as informações sobre estes são fragmentadas não existindo ainda uma caracterização completa destes organismos. Assim, este estudo buscou identificar o isolado nomeado FIMA 665-5, encontrado em amostras do musgo Sanionia uncinata (Hedw.) Loesk. Além disso, caracterizar fisiológica, bioquimicamente e testar a presença de atividade patogênica do isolado. As coletas do material para o estudo foram realizadas na Expedição Antártica Brasileira no verão austral de 2012/2013. Para a caracterização fisiológica do isolado foram testados diferentes meios de cultura (BDA, Sabouraud e MEA) e diferentes temperaturas (- 6°C, 1°C, 5°C, 10°C, 20°C e 30°C), onde a velocidade de crescimento do isolado foi medida com o auxílio de um paquímetro a cada 24h, até que o crescimento de uma das colônias atingisse a borda da placa. Os resultados foram submetidos à ANOVA e teste de Tukey (p <0,05), utilizando o software Statistix 8. Para a caracterização bioquímica foram realizados ensaios enzimáticos para verificar a produção de pectinase, celulase, protease e amilase nas temperaturas de 10°C e 30°C. Para avaliar a atividade patogênica, foram realizados testes de patogenicidade química a partir do extrato obtido do isolado, bem como teste de confronto por disco em difusão. Através de métodos moleculares e filogenéticos o isolado foi identificado como pertencente ao gênero Penicillium Link, sendo um organismo psicrotrófico, com crescimento entre 1-30°C, tendo um crescimento micelial ótimo a 20°C. O meio de cultura onde ocorre às condições nutricionais mais favoráveis para o crescimento micelial do isolado é o meio BDA. No que se refere à patogenicidade, o isolado apresentou capacidade de inibir o crescimento in vitro e causar descoloração total nos gametófitos do musgo Physcomitrium acutifolium Broth. / Fungi in Antarctica occupy different niches and perform different interactions, but its importance in these niches and interactions are still poorly understood. Interactions between fungi and mosses have been reported to Antarctica an example of these interactions is the presence of fungi forming rings on of carpets Antarctic mosses. These fungi forming rings can cause necrosis and yellow to brown stains on the carpets of mosses. However, due to the complexity of these fungi information about these are fragmented and there is still no complete characterization of these organisms. This study aimed to identify the isolated named FIMA 665-5, found in samples of moss Sanionia uncinata (Hedw.) Loesk. Further, characterize physiological, biochemical and test the presence of pathogenic activity of isolated. The collections of the material for the study were performed at the Brazilian Antarctic Expedition in the austral summer of 2012/2013. Through taxonomic, molecular and phylogenetic methods the isolate was identified belonging to the genus Penicillium Link (1809). For physiological characterization of different culture media (PDA, MEA and Sabouraud) and different temperatures (-6°C, 1°C, 5°C, 10°C, 20°C and 30°C) were tested where the diameter colony was measured with a caliper every 24h, until the growth of a colony reached the edge of the plate. The results were submitted to ANOVA and Tukey test (p <0.05) using the Statistix 8. For the biochemical characterization were performed enzymatic assays with different culture media to verify the production of enzymes such as pectinase, cellulase, protease and amylase at temperatures of 10°C and 30°C. To evaluate the pathogenic activity chemical pathogenicity tests were performed obtained from the isolated extract and by confronting disk diffusion test. Through taxonomic methods, molecular and phylogenetic isolated was identified belonging to the genus Penicillium Link, one psychrotrophs organism, with growth between 1-30°C, with a great mycelial growth at 20°C. The culture medium which has more favorable nutritional conditions for mycelial growth of isolate is PDA medium. Regarding to pathogenicity, the isolate showed ability to inhibit the in vitro growth and cause complete discoloration in the gametophytes of moss Physcomitrium acutifolium Broth.