Botrytis cinerea est un champignon nécrotrophe et polyphage capable de provoquer la pourriture grise sur plusieurs centaines d’espèces végétales. Les pertes engendrées par cette maladie sont importantes à travers le monde notamment sur des espèces économiquement importantes comme la tomate, la fraise ou encore la vigne. Parmi les facteurs de virulence identifiés chez B. cinerea se trouvent deux toxines non-hôte spécifiques. Il s'agit du sesquiterpène botrydial et du polycétide acide botcinique. Même si leur rôle redondant dans la nécrotrophie a été démontré, les mécanismes qui gouvernent l’expression des clusters responsables de leur synthèse (respectivement BOT et BOA) restent inconnus. Dans ce contexte, l’objectif de mon projet de thèse était de caractériser les différents mécanismes qui régulent le métabolisme secondaire chez B. cinerea. Je me suis particulièrement intéressé aux clusters BOT et BOA ainsi qu’à un troisième cluster (PKS7) qui, d’après le phénotype d’un mutant d’insertion (ADN-T), pourrait être impliqué dans la nécrotrophie. Grâce à la disponibilité du nouvel assemblage du génome de la souche modèle B05.10, un gène candidat codant un facteur de transcription (FT) putatif a pu être identifié à proximité du cluster BOT. La caractérisation de ce gène a permis de démontrer le rôle majeur de la protéine (BcBot6) dans l’activation des gènes Bcbot et la production subséquente de botrydial. De même, la caractérisation de Bcboa13, un gène codant un FT putatif présent au sein du cluster BOA, a permis de démontrer le rôle de régulateur positif de BcBoa13 envers les gènes Bcboa. A la différence des clusters BOT et BOA, le cluster PKS7 ne contient pas de gène codant pour un potentiel FT spécifique. Afin de confirmer le rôle du métabolite putatif produit par ce cluster et d’identifier sa structure chimique, l’inactivation du gène clé codant une PKS-NRPS (Bcpks7) a été réalisée et des analyses métaboliques ont été initiées. Finalement, la présence au sein des clusters BOT et BOA de nombreux transposons ayant subi des mutations de type RIP (Repeat-Induced Point mutations) ainsi que la position sub-télomérique des clusters BOA et PKS7 nous a amené à nous intéresser au rôle de la structure chromatinienne dans la régulation de ces clusters. Dans ce cadre, trois mutants délétés dans des gènes codant des modificateurs chromatiniens putatifs (les histones méthyltransférases BcDim-5 et BcKmt6 et la protéine hétérochromatinienne BcHp1) ont été générés. L’expression des gènes clés des clusters BOT, BOA et PKS7 chez les mutants Bcdim-5, Bchp1 et Bckmt6 suggère que différents mécanismes chromatiniens interviennent pour le contrôle des clusters BOT et BOA d’une part et du cluster PKS7 d’autre part. L’ensemble des résultats obtenus pendant cette thèse apporte une contribution majeure à la compréhension des mécanismes de régulation spécifiques mais aussi de ceux en lien avec la structure chromatinienne de la production de métabolites phytotoxiques impliqués dans la nécrotrophie chez B. cinerea. / Botrytis cinerea is a necrotrophic polyphagous fungus able to induce the gray mold disease on hundreds of plant species. The resulting losses are important worldwide notably on economically important crops such as tomato, strawberry or grapevine. Among the virulence factors identified in B. cinerea stand two non-host specific toxins: the sesquiterpene botrydial and the polyketide botcinic acid. Although their redundant role in necrotrophy has been shown, the mechanisms governing the clusters responsible for their synthesis (respectively BOT and BOA) remain unknown. In this context, the aim of my PhD project was to characterize the different mechanisms that regulate secondary metabolism in B. cinerea. I particularly focused on BOT and BOA clusters as well as on a third one (PKS7) which, according to the phenotype of an insertion-based mutant (T-DNA), could be involved in necrotrophy. Thanks to the newly assembled genome of the B05.10 wild type strain, a candidate gene encoding a putative transcription factor (TF) could be identified near the BOT cluster. The characterization of this gene allowed pointing out the major role of the protein (BcBot6) in the activation of Bcbot genes and in the subsequent botrydial production. Similarly, the characterization of Bcboa13, a putative TF-encoding gene present into the BOA cluster, allowed demonstrating the positive regulatory role of BcBoa13 on Bcboa genes. Unlike the BOT and BOA clusters, the PKS7 one does not contain any putative TF-encoding gene. In order to confirm the role of the putative metabolite produced by this cluster and to identify its chemical structure, the inactivation of the PKS-NRPS key enzyme-encoding gene (Bcpks7) was conducted and metabolic analyses were initiated. Finally, the presence of many RIP(Repeat-Induced Point mutations)-inactivated transposons within BOT and BOA clusters as well as the subtelomeric location of BOA and PKS7 clusters raised our interest about the role of chromatin structure on those clusters regulation. In this context, three mutants inactivated into putative chromatin modifiers encoding-genes (the histone methyltransferases BcDim-5 and BcKmt6 and the heterochromatin protein BcHp1) were generated. The expression analysis of the key genes of the BOT, BOA and PKS clusters suggests different chromatin-based mechanisms that intervene for the BOT and BOA cluster on one side and on the PKS7 cluster on another. Altogether, the results generated during this PhD project are a major contribution to the comprehension of pathway-specific as well as chromatin-based mechanisms that regulate the production of necrotrophy-involved phytotoxins by B. cinerea.

Thesis (MScAgric)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Knowledge of the presence of Botrytis cinerea in morphological parts of bunches and leaves of grapevine would help to find a reliable, sensitive, and specific assay to verify the actual occurrence of latent infection, and to plan strategies for the effective control of B. cinerea bunch rot. The aim of this study was (i) to determine natural B. cinerea infection at specific sites in leaves and bunches of grapevine at different phenological stages, and (ii) to determine resistance in the morphological parts to disease expression. Bunches and leaves of the wine grape cultivar Merlot and the table grape cultivar Dauphine, were collected at pea size, bunch closure and harvest from five vineyards in the Stellenbosch and De Dooms regions respectively. The material was divided into two groups and sealed in polythene bags. The bags were lined with wet paper towels to establish high relative humidity. Leaves and bunches incubated in one group of bags were first treated with paraquat in order to terminate active host responses. These treatments provided conditions that facilitated disease expression under two host resistance levels by different inocula during the period of moist incubation. Disease expression was positively identified by lesion development, and the formation of sporulating colonies of B. cinerea at a potential infection site. Sites in leaves were the blades and petioles. Sites in bunch parts were rachises, laterals and pedicels, and on berries sites were the pedicel-end, cheek and style-end. In Dauphine, the various sites were at all stages classified as resistant to moderately resistant. However, at pea size and bunch closure, in spite of their resistance, nearly all the sites carried high to very high inoculum levels. The only exception was the berry cheek, which carried intermediate inoculum levels at pea size, and low inoculum levels at bunch closure. In nearly all sites, inoculum levels were lower at harvest. The decrease was the most prominent in petioles, rachises, laterals, pedicels and the pedicel-end of the berry. All these sites carried intermediate to low inoculum levels at harvest. In Merlot, sites constantly exibited a resistant reaction, except for the pedicel and pedicel-end of the berry, which changed from resistant at the early developmental stages to susceptible at harvest. Inoculum levels decreased during the season in the rachises and laterals, but were constantly high during the season in the pedicel and pedicel-end of the berry. According to this pattern of natural occurrence, B. cinerea fruit rot in these vineyards was not caused by colonisation of the pistil, and subsequent latency in the style end of grape berries. However, fruit rot was primarily caused by colonisation of the pedicel, and subsequent latency in the pedicel or pedicel-end of the berry. These findings furthermore support the hypothesis of increased host resistance during development, but also indicate that in the Western Cape province, inoculum in vineyards is abundant during the early part of the season, and less abundant later in the season. More information is therefore needed on the behaviour of the different types of B. cinerea inocula on the different morphological parts of grapevine to validate the pathway described for natural B. cinerea infection in vineyards. The penetration and disease expression at the different morphological parts of bunches of two grape cultivars (Dauphine and Merlot) under conditions simulating natural infection by airborne conidia was therefore investigated. The two cultivars did not differ in resistance of the berry cheek, which was at all stages classified as resistant. However, in Dauphine, latent inoculum levels in berry cheeks declined from intermediate at pea size to low at the following stages, whereas in Merlot, levels were intermediate during pea size and at harvest. Some differences between cultivars were found in the resistance of the structural bunch parts, and of their latent inoculum levels. In Dauphine, the rachis reacted susceptible at pea size, and was classified moderately resistant later in the season. Laterals and pedicels were moderate resistant at pea size, and resistant at later stages. Inoculum levels in rachises, laterals and pedicels were high at pea size, but intermediate at bunch closure and at harvest. The finding that B. cinerea infected and naturally occurred more commonly in the tissues of immature than mature bunches, that the structural parts of the bunch carried more B. cinerea than the berry cheek, and that these infections may be more important in B. cinerea bunch rot than infection of the cheek or the style end, suggest that emphasis should be placed on the disease reaction of the pedicel and related parts of immature bunches rather than on the berry. The resistanc-e reaction of leaf blades, petioles, internodes and inflorescences on cuttings, compared to those on older shoots from the vineyard were therefore investigated. In the case of vinelets, leaf blades, petioles, internodes and inflorescences were all classified susceptible to highly susceptible. The different parts furthermore all carried very high latent inoculum levels. In vineyard shoots the petioles and inflorescences showed resistance, and carried intermediate to latent inoculum levels. This finding suggests that leaf blades are not appropriate parts for studying the behaviour of inoculum of B. cinerea and host responses in grape bunches. In stead, petioles and inflorescences of vineyard shoots should be used for this purpose. / AFRIKAANSE OPSOMMING: WEERSTAND TEEN BOTRYTIS CINEREA IN MORFOLOGIESE DELE VAN BLARE EN TROSSE VAN WINGERD Kennis oor die teenwoordigheid van Botrytis cinerea in morfologiese dele van wingerd word benodig vir die ontwerp van 'n betroubare, sensitiewe en spesifieke toets vir die bevestiging van latente infeksies, en vir die implementering van strategieë vir die effektiewe beheer van B. cinerea-vrot. Die doel van hierdie studie was om (i) natuurlike B. cinerea infeksie by spesifieke areas in blare en trosse van wingerd te bepaal, en (ii) om weerstand teen siekte-uitdrukking in hierdie morfologiese dele vas te stel. Trosse en blare van die wyndruif kultivar Merlot en die tafeldruif kultivar Dauphine, is by ertjiekorrel, tros-toemaak en oes in vyf wingerde in die Stellenbosch- en De Doomsomgewing, onderskeidelik, versamel. Die materiaal is in twee groepe verdeel en in polietileen sakkies verseël. Die sakkies is met klam papierdoekies uitgevoer om sodoende hoë relatiewe humiditeit te verseker. Blare en trosse wat in die een groep geïnkubeer is, is eers met paraquat behandel om aktiewe gasheerreaksies te beëindig. Hierdie behandelings het toestande geskep wat gedurende die periode van vogtige inkubasie gunstig was vir siekteontwikkeling deur verskillende inokula by twee gasheer-weerstandsvlakke. Siekteuitdrukking is positief geïdentifiseer deur letsel-ontwikkeling en die vorming van sporuierende kolonies van B. cinerea by 'n potensiële infeksie-area. Dele waarop in die blare gekonsentreer is, was die blaarskyf en -steel. In die trosse was die dele die rachis, lateraal en korrelsteel, en op korrels was dit die korrelsteel-end, wang en styl-end. In Dauphine is die verskillende dele tydens al die fenologiese stadia as weerstandbiedend tot matig weerstandbiedend geklassifiseer. Die verskillende dele her egter, ten spyte van hul weerstandbiedendheid, hoë tot baie hoë inokulumvlakke by ertjiekorrel- en tros-toemaakstadium gedra. Die enigste uitsondering was die korrelwang, wat 'n middelmatige inokulumvlak by ertjiekorrel, en 'n lae inokulumvlak by tros-toemaak, gedra het. Die inokulumvlakke was in byna al die dele laer by oes. Die afname in inokulumvlakke was die prominentste in die blaarstele, rachi, laterale, korreisteie en die korrelsteel-end van die korrel. Al hierdie dele het 'n middelmatige tot lae inokulumvlak by oes gehad. In Merlot was die dele konstant weerstandbiedend, behalwe vir die korrelsteel en die korrelsteel-end van die korrel, wat gewissel het van weerstandbiedend by die vroeë ontwikkelingstadia, tot vatbaar by oes. lnokulumvlakke in die rachis en lateraal het gedurende die seisoen afgeneem; maar was deur die seisoen konstant hoog in die korrelsteel en korrelsteel-end van die korrel. Volgens die patroon van natuurlike voorkoms, word B. cinerea-vrot in hierdie wingerde nie deur kolonisasie van die stamper, en die daaropvolgende latensie in die styl-end van die korrels, veroorsaak nie. Vrot word egter primêr deur kolonisasie van die korrelsteel, en die daaropvolgende latensie in die korrelsteel of korrelsteel-end van die korrel, veroorsaak. Hierdie bevindinge ondersteun die hipotese van toenemende gasheerweerstand gedurende ontwikkeling, en dui ook daarop dat inokulumvlakke in wingerde in die Wes-Kaap provinsie volop is gedurende die eerste deel van die seisoen, en minder volop is later in die seisoen. Meer inligting word dus benodig aangaande die gedrag van die verskillende inokulum tipes van B. cinerea op die verskillende morfologiese dele van wingerd, ten einde die infeksieweg vir natuurlike B. cinerea infeksie in wingerde te bevestig. Die vestiging van latente infeksies in die verskillende morfologiese dele van trosse van twee kultivars (Dauphine en Merlot), onder toestande wat natuurlike infeksie deur luggedraagde konidia simuleer, is dus ondersoek. Die twee kultivars se weerstand in die korrelwang het nie verskil nie en is by alle fenologiese stadia as weerstandbiedend geklassifiseer. Die latente inokulumvlakke in die korrelwang van Dauphine het egter van middelmatig by ertjiekorrel, tot laag in die daaropvolgende stadia afgeneem, terwyl die vlakke in Merlot middelmatig by ertjiekorrel en oes was. Verskille tussen die twee kultivars is gevind ten opsigte van die weerstand in die trosdele, asook hulle latente inokulumvlakke. Die rachis van Dauphine was by ertjiekorrel vatbaar, en matig weerstandbiedend later in die seisoen. Die lateraal en korrelsteel was matig weerstandbiedend by ertjiekorrel en weerstandbiedend by latere stadia. lnokulumvlakke in rachi, laterale en korreisteie was hoog by ertjiekorrel, maar middelmatig by tros-toemaak en oes. Die bevindinge dat B. cinerea natuurlik meer algemeen in die weefsel van onvolwasse trosse voorgekom en laasgenoemde meer algemeen geïnfekteer het, dat B. cinerea se voorkoms hoër was in die morfologiese dele van die tros as in die korrelwang, en dat hierdie infeksies van groter belang in B. cinerea-vrot mag wees as infeksie van die wang of styl-end, dui daarop dat klem gelê moet word op die siektereaksie van die strukturele dele van onvolwasse trosse, eerder as van die korrel. Die weerstand van blaarskywe, blaarstele, internodes en blomtrossies van steggies, in vergelyking met die op ouer lote in wingerde, is dus ondersoek. Blaarskywe, blaarstele, internodes en blomtrossies van steggies is almal as vatbaar tot hoogs vatbaar geklassifiseer. Die verskillende dele het verder ook almal baie hoë latente inokulumvlakke gedra. By die ouer lote van wingerde het die blaarstele en blomtrossies weerstandbiedend vertoon, en middelmatige latente inokulumvlakke gedra. Hierdie bevindinge dui daarop dat blaarskywe nie die ideale morfologiese deel is vir gedragstudies van B. cinerea in druiwetrosse nie. Blaarstele en blomtrossies van ouer lote moet eerder vir die doel gebruik word.

Thesis (MScAgric)--University of Stellenbosch / ENGLISH ABSTRACT: An understanding of the infection pathways of Botrytis cinerea in grape bunches will help to combat this devastating pathogen of grape. Many studies have been done to determine the possible infection pathways of B. cinerea. Most of these studies made use of artificial inoculations that deposit groups of conidia on the plant surface. The deposition of clusters of conidia is not a common phenomenon in nature. The aim of this study was to investigate the infection pathways of (i) naturally- as well as (ii) artificially inoculated B. cinerea conidia during all the phenological stages of three wine grape cultivars, and to compare the (iii) pathogenicity and virulence, on grape and nectarine fruit, of isolates obtained from different host plants. In the natural infection study the occurrence of Botrytis cinerea and subsequent disease expression at different positions in bunches of wine grapes (cultivars Chenin Blanc, Shiraz and Chardonnay) was determined from 1999 to 2001. Different techniques were used to detect viable inoculum at different positions (rachises, laterals, pedicels, and the peicel end, cheek and style end of berries) in bunches. Isolations were made on Kerssies' B. cinerea selective medium, or bunches were used untreated, or treated with paraquat. Paraquat was used to terminate host resistance and to promote the development of the pathogen from the tissues. The material was used untreated to detect the pathogen on the surface, or were surface-sterilized to detect mycelia (latent infection) in the tissue. In the artificial inoculation study, bunches of wine grapes (cultivars Chenin Blanc, Chardonnay and Shiraz) at pea size, bunch closure, and harvest were dusted with dry conidia of Botrytis cinerea in a settling tower and incubated for 24 h at high relative humidity (±93%). Following incubation, the bunches were divided in two groups. The one group was surface-sterilised in 70% ethanol for 5 s, the other group was left untreated. Bunches of the sterile group, and from the untreated group were used for isolation. From each bunch rachis segments, laterals, pedicels and berry skin segments (from the pedicel-end and cheek) were removed. The sections were placed in Petri dishes on Kerssies' B. cinerea selective medium and on a water agar medium supplemented with paraquat, and incubated at 22°C under diurnal light. Occupation by the pathogen was positively identified by the formation of sporulating colonies of B. cinerea on the different tissues. Lastly, in the virulence and pathogenicity experiment on grape and nectarine fruit Botrytis cinerea isolates, which were obtained from different host plants, were compared by simulating natural infection. Cold-stored fruit, considered highly susceptible to B. cinerea were therefore inoculated with single, airborne conidia of the pathogen. Different tests were conducted to assess surface penetration and lesion formation. Isolations were made from fruit skins on Kerssies' B. cinerea selective medium. Nectarine fruit were treated with paraquat, and grape berries were frozen for 1 h at -12°C. Paraquat and freezing were used to terminate host resistance and to promote the development of the pathogen from the tissues. In the natural infection studies B. cinerea occurred in a consistent pattern in bunches of the three cultivars. B. cinerea consistently developed from the tissue of the rachis, laterals, pedicel and pedicel-end, but not from the berry cheek. The rachis, lateral and pedicel contained much higher levels of B. cinerea than any position on the berry. Furthermore, the pathogen consistenly occurred at relatively high levels on the rachises throughout the season. Collectively, the data showed that in the Western Cape province, B. cinerea occured more regularly in wine grape bunches during the early part of the season, than later in the season. The data of the artificial studies confirmed the findings made with the natural infection studies. In these experiments the pathogen resided more often on the structural bunch parts than on the berries. Overall, the isolation studies revealed that conidia occurred predominantly on the rachis. The incidence of B. cinerea was furthermore constantly high in the inner bunch after each inoculation, and in bunches of different maturities. The data therefore indicated that, when available, conidia penetrated loose and tight clustered bunches in a similar way. Finally, in the virulence and pathogenicity experiments the results showed clearly that no host specialisation exists in the B. cinerea isolates used in this study. From these studies it is clear that in the Western Cape province B. cinerea occurs more readily in the inner structural parts of the bunches and more so during the earlier parts of the season. These findings should be considered when planning and implementing disease control programmes. / AFRIKAANSE OPSOMMING: INFEKSIEWEË VAN BOTRYTIS CINEREA OP GESELEKTEERDE WYNDRUIF KULTIVARS Indiepte kennis van die infeksieweë van Botrytis cinerea op druiwetrosse word benodig vir die beheer van dié vernietigende patogeen van druiwe. Vele studies is al gedoen om die moontlike infeksieweë van die swam op druiwe trosse te ondersoek. Die meeste van die studies het gebruik gemaak van kunsmatige inokulasie tegnieke waar die konidia van die swam in groepe op die korreloppervlak gedeponeer is. In die natuur is dit 'n rare verskynsel dat konidia in groepe op die korreloppervlak land. Die doel van die studie was om die infeksieweë van B. cinerea op drie wyndruif kultivars te ondersoek wat (i) natuurlik- en (ii) kunsmatig geïnokuleer is met konidia gedurende al die fenologiese stadia, en om die (iii) virulensie en patogenisisteit van isolate wat van verskillende gashere verkry is, op druiwe en nektariens te vergelyk. In die natuurlik-geïnokuleerde druiwe is die voorkoms van B. cinerea en die gevolglike siektevoorkoms op verkillende posisies in trosse van wyndruiwe (Chenin Blanc, Chardonnay, Shiraz) gedurende 1999 tot 2001 bepaal. Verskillende tegnieke is gebruik om lewensvatbare inokulum by verskillende posisies (ragis, lateraal, pedisel en pedisel-end van die korrel) in die tros waar te neem. Isolasies is op Kerssies' B. cinerea selektiewe medium gemaak, of trosse is onbehandeld gebruik, of behandel met paraquat. Paraquat is gebruik om die gasheer se natuurlike weerstand te verlaag en om die ontwikkeling van die patogeen te bevorder. Die plantmateriaal is onbehandeld gelaat om die patogeen op die oppervlak waar te neem, of die oppervlak is gesteriliseer om die latente myselium in die weefsel waar te neem. In die kunsmatige inokulasiestudies is trosse, van wyndruiwe (Chenin Blanc, Chardonnay, Shiraz), geïnokuleer met droë spore, van B. cinerea, in 'n inokulasietoring en die plantmateriaal is dan geinkubeer vir 24 h by 'n hoë relatiewe humiditeit (93%). Na die inkubasie proses is die trosse in twee groepe verdeel. Die een groep druiwe het oppervlak sterilisasie ondergaan in 70% etanol vir 5 s, en die ander groep was onbehandeld gelaat. Trosse van die onbehandelde en gesteriliseerde groep druiwe is gebruik vir isolasies. Vanuit elke tros is daar segmente van die ragis, laterale, pediselle en korrels (van die pedisel-end en wang gedeeltes) geïsoleer. Die segmente is in Petri bakkies met Kerssies' B. cinerea selektiewe medium en op water agar medium, wat paraquat bevat het, geïsoleer en geïnkubeer onder 'n 12 h dagligperiode teen 22°C. Die patogeen is positief geïdentifiseer deur sporuierende kolonies op die onderskeie weefseltipes. Laastens, in die virulensie- en patogenisiteitsproewe op druiwe en nektariens is verskillende isolate van B. cinerea, verkry vanaf verskillende gasheerplante, vergelyk deur natuurlike inokulasie toestande na te boots. Koue opgebergde vrugte, wat beskou word as hoogs vatbaar vir die infeksie van B. cinerea, is geïnokuleer met droë, enkel luggedraagde spore van die patogeen. Verskillende toetse is gedoen om die oppervlak penetrerende en letselvormende vermoëns van die onderskeie isolate te toets. Isolasies is van die skille van die vrugte gemaak en op Kerssies' B. cinerea selektiewe medium geplaas. Die nektarienvrugte is met paraquat behandel en die druifkorrels is gevries vir 1 h teen -12°C. Paraquat en bevriesing is gebruik om die gasheer se weerstand te verlaag en om die ontwikkeling van die patogeen te bevorder. In die natuurlik-geïnokuleerde studies het B. cinerea 'n konstante patroon getoon in die trosse van die drie verskillende wyndruif kultivars. B. cinerea het konstant ontwikkel uit die ragis, laterale, pedisel en pedisel-end, maar selde uit die korrelwang. Die ragis, lateral en pedisel dele het baie hoër vlakke van van die swam bevat as enige deel op die korrel. Die patogeen het ook konstant volop deur die hele seisoen op die ragis voorgekom. Gesamentlik wys die data dat, B. cinerea in wyndruiwe, in die Wes Kaap provinsie, meer geredelik vroeër in die seisoen voorkom, eerder as later. Data van die kunsmatige inokulasiestudies het die bevindinge van die natuurlike inokulasiestudies tot 'n groot mate bevestig. In dié studies het die patogeen meer geredelik die strukturele dele van die tros, eerder as op die korrels, bewoon. Oor die algemeen het die isolasieproewe gewys dat die konidia meer op die ragis voorkom as op enige ander deel. Die voorkoms van B. cinerea was ook oor die algemeen baie hoër in die strukturele dele van die tros, as op die korrel self. Die verskynsel het onder trosse van verskillende ontwikkelingsvlakke voorgekom. Die data het dus ook gewys dat konidia, wanner dit beskikbaar is, minder- sowel as meer kompakte trosse op 'n soortgelyke manier penetreer. Laastens, in die virulensie en patogenisiteitseksperimente het die resultate duidelik gewys dat daar geen gasheer spesifieke gedrag onder B. cinerea isolate is nie. In die studies het dit duidelik na vore gekom dat, B. cinerea meer geredelik in die strukturele binne dele van die wyndruif tros, in die Wes Kaap provinsie voorkom. En so ook eerder aan die begin van die seisoen, as later in die seisoen. Dié kennis moet in aanmerking geneem word by die beplanning en implementering van siektebeheerprogramme.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The raffinose family of oligosaccharides (RFOs) are α1,6-galactosyl extensions of sucrose (Suc-Galn) unique to the plant kingdom. Their biosynthesis is mediated via α1,6-galactosyltransferases which catalyse the formation of raffinose (Raf, Suc-Gal1), stachyose (Sta, Suc-Gal2) and higher oligomers (Suc-Galn, n ≥ 13) in a stepwise manner. RFOs are well known for their historical roles as phloem translocates and general carbon storage reserves. In recent years their physiological roles have expanded to include potential functions in global plant stress-responses, where correlative mass increases are associated with abiotic stresses such as desiccation, salinity and low temperatures and, to a lesser extent biotic stress (pathogen infection). This study focused on (i) the functional characterisation of a putatively annotated stachyose synthase from Arabidopsis seeds (RS4, At4g01970), (ii) dissection of the proposed functional role of the RFO precursor galactinol in biotic stress tolerance using the Arabidopsis/Botrytis cinerea pathosystem and, (iii) an attempt to engineer long-chain RFOs into Arabidopsis by constitutive over-expression of the unique RFO chain elongation enzyme galactan:galactan galactosyltransferase (ArGGT) from Ajuga reptans. In Arabidopsis Raf is the only RFO known to accumulate in leaves, strictly during conditions of abiotic stress. However, seeds accumulate substantial amounts of both Raf and Sta. While RFO physiology in Arabidopsis leaves and roots is quite well characterised, little is known about the RFO physiology in the seeds. Apart from a single enzyme being described to partially contribute to seed Raf accumulation (RS5, At5g40390), no other RFO biosynthetic genes are known. In this work we functionally characterised an α1,6-galactosyltransferase putatively annotated as a stachyose synthase (RS4, At4g01970) in the Arabidopsis database. Using two insertion mutants (atrs4-1 and 4-2) we demonstrated Sta deficiency in mature seeds. A double mutant with the recently characterised RS5, shown to partially be responsible for Raf accumulation in mature seeds was completely deficient in seed RFOs. This provided the first hint that RS4 could potentially also be involved in Raf biosynthesis. Seed specific expression of RS4 was deregulated by constitutive over-expression in wild-type (Col-0) and the atrs5 mutant background (RS and Raf deficient). Both Raf and Sta unusually accumulated in Col-0 leaves over-expressing RS4, under normal growth conditions. Further, leaf crude extracts from atrs5 insertion mutants (RS and Raf deficient) over-expressing RS4 showed enzyme activities for both RS and SS, in vitro. Collectively our findings have physiologically characterised RS4 as a RFO synthase responsible for Sta and, partially Raf (along with RS5) accumulation during Arabidopsis seed development. The galactosyl donor in RFO biosynthesis, galactinol (Gol) has recently been implicated in biotic stress signalling (pathogen response) in cucumber, tobacco and Arabidopsis. Those studies focused exclusively on Gol in their experimental approaches using both over-expression (tobacco, Arabidopsis) and loss-of-function (Arabidopsis) strategies. However, they did not address the invariable accumulation of Raf that is routinely obtained from such over-expression strategies. We therefore investigated if Raf could play a functional role in induced systemic resistance (ISR), a well-studied mechanism employed by plants to combat necrotrophic pathogens such as Botrytis cinerea. To this end we looked to the RS5 mutant backgrounds (Raf deficient but Gol hyper-accumulating) reasoning that the Gol accumulating mutants should be resistant to B. cinerea (as previously described for transgenic over-expression of GolS1 isoforms in tobacco and Arabidopsis). Such findings would then preclude a role for Raf, since the system would be Raf deficient. Surprisingly, two independent T-DNA insertion mutants for RS5 (atrs5-1 and 5-2) were equally hypersensitive to B. cinerea infection as two independent T-DNA insertion mutants for GolS1 (atgols1-1 and 1-2). The hyper-sensitivity of the GolS1 mutant background has previously been demonstrated. The RS5 mutant backgrounds accumulate substantial amounts of Gol, comparable to those reported for transgenic plants (tobacco and Arabidopsis) where pathogen resistance was reported. Further, during the course of our investigations we discovered that both AtGolS1 mutants also accumulated substantial amounts of both Gol and Raf under normal growing conditions. This was not reported in previous studies. Collectively our findings argue against a role for either Gol or Raf being responsible for the induction/signalling of ISR. However, we do not preclude that the RFO pathway is somehow involved, given the previous reports citing pathogen resistance when GolS1 genes are over-expressed. We are further investigating a potential role for the GolS transcript and/or protein being the component of the suggested signalling function in ISR. The unique enzyme from A. reptans (galactan:galactan galactosyltransferase, ArGGT) is able to catalyse the formation of higher oligomers in the RFO pathway without the use of Gol as a galactosyl donor but rather, using RFOs themselves as galactose donors and acceptors (Gol-independent biosynthesis). We constitutively over-expressed ArGGT in Arabidopsis as a way to engineer long-chain RFO accumulation to further dissect a role for them in improving freezing tolerance. To this end we have been unsuccessful in obtaining RFOs higher than Sta (which occurred in extremely low abundance) in the leaves. Since ArGGT would appear to show substrate preference for Sta, and Arabidopsis seeds accumulate substantial quantities of Sta, we further analysed the seed water soluble carbohydrate (WSC) profiles of three independent transgenic lines but detected no additional RFO oligomers beyond the normally accumulating Raf and Sta. We suggest further strategies to improve this approach (Chapter 4). Collectively this work represents case studies of RFOs in seed physiology, their abilities/requirement in biotic stress and the use of unique enzymes to engineer long-chain RFO accumulation using the Arabidopsis model. At the time of submission of this dissertation the following contributions have been made to the general scientific community: (i) Presentation of chapter 2 at the 26th International Conference for Arabidopsis Research (26th ICAR, 2015, Paris, France) and, (ii) Submission of chapter 2 as a manuscript presently under peer review for possible publication in Plant and Cell Physiology. / AFRIKAANSE OPSOMMING: Die raffinose familie van oligosakkariede (RFO) is α1,6-galactosyl uitbreidings van sukrose (Suc-Galn) uniek aan die plante koningryk. Hul biosintese word bemiddel deur α1,6-galactosyltransferases wat in 'n stapsgewyse manier die vorming van raffinose (Raf, Suc-Gal1), stachyose (Sta, Suc-Gal2) en hoër oligomere (Suc-Galn, n ≥ 13) kataliseer. RFOs is bekend vir hul historiese rol as floëem translokate en algemene koolstof reserwes. Meer onlangs was hul fisiologiese rolle uitgebrei om potensiële funksies te vervul in globale plant stres-reaksies, waar korrelatiewe massa toenames geassosieer word met abiotiese stresfaktore soos uitdroging, soutgehalte en lae temperature en tot 'n mindere mate biotiese stres (patogeen infeksie). Hierdie studie fokus op (i) die funksionele karakterisering van 'n tentatief ge-annoteerde stachyose sintase van Arabidopsis sade (RS4, At4g01970), (ii) disseksie van die voorgestelde funksionele rol van die RFO voorloper galactinol in biotiese stres verdraagsaamheid, met behulp van die Arabidopsis/Botrytis cinerea patogeen sisteem en (iii) 'n poging om 'n lang-ketting RFOs in Arabidopsis te inisieer deur konstitutiewe oor-uitdrukking van die unieke RFO ketting-verlengings ensiem galactan:galactan galactosyltransferase (ArGGT) afkomstig van Ajuga reptans. In Arabidopsis is Raf die enigste RFO bekend daarvoor om te versamel in die blare, ekslusief tydens toestande van abiotiese stres. Maar, sade versamel aansienlike konsentrasies van beide Raf en Sta. Terwyl RFO fisiologie in Arabidopsis (blare en wortels) baie goed gekenmerk is, is min bekend oor die RFO fisiologie in die saad. Afgesien van 'n enkele ensiem wat beskryf word om gedeeltelik by te dra tot Raf versameling (RS5, At5g40390), is geen ander RFO biosintetiese gene bekend in saad nie. In hierdie werk beskryf ons die funksionele karakterisering van ‘n α1,6-galactosyltransferase wat tenetatief ge-annoteer word as 'n stachyose sintase (RS4, At4g01970) in die Arabidopsis databasis. Met die gebruik van twee invoegings mutante (atrs4-1 en 4-2) het ons die verlies van Sta in volwasse sade gedemonstreer. RFOs was heeltemal absent in sade van 'n dubbele mutant met die onlangs gekarakteriseerde RS5 (verantwoordelik vir gedeeltelike Raf versameling in volwasse sade). Dit het die eerste aanduiding daargestel dat RS4 potensieel ook betrokke kan wees in Raf biosintese. Saad-spesifieke uitdrukking van RS4 was gedereguleer deur konstitutiewe oor-uitdrukking in wilde-tipe (Col-0) en die atrs5 mutant agtergrond (RS en Raf gebrekkig). Oor-uitdrukking van RS4 in Col-0 blare het gelei tot beide buitengewone Raf en Sta konsentrasies, onder normale groeitoestande. Verder, oor-uitdrukkingvan RS4 in atrs5 invoeg mutante (waar beide RS en Raf absent is) het in vitro ensiemaktiwiteite vir beide RS en SS getoon. Gesamentlik beskryf ons bevindinge die fisiologies karakterisering van RS4 as 'n RFO sintase, verantwoordelik vir Sta en gedeeltelik Raf (saam met RS5) sintese tydens Arabidopsis saad ontwikkeling. Die galactosyl skenker in RFO biosintese, galactinol (Gol), was onlangs beskryf om ‘n rol te speel in biotiese stres (patogeen reaksie) in komkommer, tabak en Arabidopsis. Daardie studies het uitsluitlik gefokus op Gol in hul eksperimentele benaderings deur die gebruik van beide oor-uitdrukking (tabak, Arabidopsis) en die verlies-van-funksie (Arabidopsis) strategieë. Maar hulle het nie die onveranderlike opeenhoping van Raf, wat gereeld verky word uit sulke oor-uitdrukking strategieë, aangespreek nie. Ons het dus ondersoek of daar 'n funksionele rol vir Raf in geïnduseerde sistemiese weerstand (ISR) kan wees. ISR is 'n goed-bestudeerde meganisme wat deur plante ge-implementeer word om nekrotrofiese patogene soos Botrytis cinerea te beveg. Vir hierdie doel het ons gekyk na die RS5 mutant agtergronde (absent in Raf, maar hiper-akkumulasie van Gol) met die redenasie dat die Gol akkumulerende mutante weerstandbiedig teen B. cinerea moet wees (soos voorheen beskryf vir transgeniese oor-uitdrukking van GolS1 in tabak en Arabidopsis). Sulke bevindings verhinder dan 'n rol vir Raf, aangesien die stelsel geen Raf akkumuleer nie. Verbasend, twee onafhanklike T-DNA invoeg mutante vir RS5 (atrs5-1 en 5-2) was ewe hiper-sensitief vir B. cinerea infeksie as twee onafhanklike T-DNA invoeg mutante vir GolS1 (atgols1-1 en 1-2). Die hiper-sensitiwiteit van die GolS1 mutant agtergrond was reeds voorheen gedemonstreer. Die RS5 mutant agtergronde versamel aansienlike konsentrasies van Gol, vergelykbaar met dié berig vir transgeniese plante (tabak en Arabidopsis) waar patogeen-weerstandbiedigheid aangemeld is. Verder, in die loop van ons ondersoeke het ons ontdek dat beide AtGolS1 mutante ook aansienlike konsentrasies van beide Gol en Raf onder normale groei-toestande akkumuleer. Dit was nie aangemeld in die vorige studies nie. Gesamentlik argumenteer ons bevindinge teen 'n rol vir óf Gol, of Raf, tydens die induksie van ISR. Alhoewel, ons elimineer nie ‘n rol vir die RFO padweg nie, gegewe dat oor-uitdrukking van GolS1 gene tydens patogeen-weerstandbiedigheid in vorige verslae verwysig was. Ons ondersoek verder 'n moontlike rol vir die aanwesigheid van die GolS transkrip en/of proteïen as ‘n moontlike komponent van die voorgestelde funksie in ISR. Die unieke ensiem van A. reptans (galactan:galactan galactosyltransferase, ArGGT) is in staat om die vorming van hoër oligomere in die RFO pad te kataliseer sonder die gebruik van Gol as 'n skenker galactosyl, maar eerder, met behulp van die RFO's hulself as galaktose skenkers en aanvaarders (Gol-onafhanklike biosintese). Ons het ArGGT konstitutief ooruitgedruk in Arabidopsis as 'n manier om 'n lang-ketting RFO akkumulasie daar te stel met die doel om 'n rol vir hulle in die verbetering van vriestoleransie verder te ontleed. Ons was tot dusver onsuksesvol in die verkryging van RFOs hoër as Sta in die blare (wat akkumuleer het in 'n baie lae konsentrasie). Sedert ArGGT ‘n affiniteit vir Sta as substraat toon, en Arabidopsis sade versamel aansienlike hoeveelhede Sta, het ons verder die saad water oplosbare koolhidraat (WSC) profiele van drie onafhanklike transgeniese lyne ontleed, maar bespeur geen bykomende RFO oligomere buite die normale Raf en Sta konsentrasie nie. Ons stel verdere strategieë voor om hierdie benadering (Hoofstuk 4) te verbeter. Gesamentlik verteenwoordig hierdie werk gevallestudies van RFOs in saadfisiologie, hul vermoëns/vereiste in biotiese stres en die gebruik van unieke ensieme om lang-ketting RFO akkumulasie daar te stel met behulp van die Arabidopsis model. Teen die tyd van die indiening van hierdie tesis was die volgende bydraes gemaak aan die algemene wetenskaplike gemeenskap: (i) Aanbieding van hoofstuk 2 op die 26ste Internasionale Konferensie vir Arabidopsis Navorsing (26ste ICAR, 2015, Parys, Frankryk), en (ii) indiening van hoofstuk 2 as 'n manuskrip tans onder nasiening vir moontlike publikasie in die joernaal ‘Plant and Cell Physiology’.

The fungus Botrytis cinerea is an opportunistic pathogen on a wide variety of crops, causing disease known as grey mould through infections via wounds or dead plant parts. Synthetic fungicides for controlling this disease are fast becoming ineffective due to the development of resistance. This, coupled with consumers world wide becomng increasingly conscious of potential environment and health problems associated with the build up of toxic chemicals, (particularly in food products), have resulted in pressure to reduce the use of chemical pesticide volumes as well as its residues. An emerging alternative to random synthesis is the study and exploitation of naturally occurring products with fungicidal properties. There have been reports on the uses of synthetic fungicides for the control of plant pathogenic fungi. When utilized in two-way mixtures, such fungicides may maintain or enhance the level of control of a pathogen at reduced rates for both components utilized in combinations, or alone at normal rates. For this study it was hypothesize that the addition of plant extracts may enhance the antifungal efficacy of the synthetic strobilurin fungicide, kresoxim-methyl against Botrytis cinerea.

The interest in production of fuel ethanol from lignocellulosic materials is continuouslyincreasing due to the urgency of finding non-food substrates for production of bio-fuels.Marabou (Dichrostachys cinerea) is one of the abundant lignocellulosic bio-resources in Cuba,and it could be useful to produce bio-ethanol. Pre-treatment is an important step to produceethanol from lignocellulosic materials since it allows the separation of cellulose, hemicelluloseand lignin, and activates cellulose towards enzymatic hydrolysis. During the past few years,organosolv methods have been reported for effective separation of the main components oflignocellulosic materials and improvement of the enzymatic hydrolysis of cellulose. By usingacetosolv method lignin is separated under mild conditions and many of the lignin properties arewell preserved.The present work was aimed to perform a chemical characterisation of marabou biomass and toevaluate acetosolv delignification of the material. In this work the content of moisture, ash,extractives, easy-to-hydrolyze polysaccharides, difficult-to-hydrolyze polysaccharides, andKlason lignin of marabou biomass were analyzed. Klason lignin of the marabou biomass was23.4% of the mass. Acetosolv delignification was performed at normal boiling temperature(NBT) and 121oC, using 50-50, 70-30 and 90-10 acetic acid – water mixtures with 10% of solidsload during 1h. Hydrochloric acid (0.2g / 100g of mixture) was used as catalyst. Thedelignification of marabou biomass was also evaluated for the combination of dilute acid prehydrolysis(DAPH) and acetosolv with the same reaction conditions. This investigation provedthat acetosolv pretreatment was effective for solubilizing lignin contained in marabou biomass.The degree of lignin solubilisation increased with increasing acetic acid concentration in thereaction mixture. Lignin removals above 80% were achieved consistently both at NBT and121oC with 90% acetic acid, while only around 44.6 and 6.8% of the initial lignin was removedusing, respectively, 70 and 50% acetic acid at 121oC. The effect of temperature ondelignification was only marginal when acetosolv was conducted with 90% acetic acid, but itwas remarkable for lower acetic acid concentrations. A two-fold decrease of lignin removal wasobserved for the NBT acetosolv compared with the process performed at 121oC using both 70and 50% acetic acid. The insertion of a DAPH step prior to acetosolv considerably improvedlignin removal using 70 and 50% acetic acid at both temperatures, but its effect on the processesusing 90% acetic acid was minimal. High lignin yields were achieved upon its precipitation fromacetosolv liquors.

Bibliography: leaves 193-224.

Intact strawberry fruit did not produce detectable t-2-H which is derived from -linolenic acid (18:3). However, in response to wounding by gentle bruising, strawberry fruit emitted t-2-H with the largest quantity produced within 10 min following injury. The level of total lipid 18:3 in the fruit increased two-fold in response to wounding whereas free 18:3 declined slightly (about 30%). At 10 min following wounding, fruit exhibited a 25% increase in 13-lipoxygenase (LOX) activity, which leads to the production of 13-hydroperoxyoctadecatrienoic acid (13-HPOT) from 18:3. The activity of hydroperoxide lyase (HPL), which catalyzes formation of cis-3-hexenal (c-3-H), the precursor of t-2-H, from 13-HPOT, increased two-fold at 10 min after wounding. Thus, within 15 min after wounding, free 18:3 substrate availability and the activity of two key enzymes, LOX and HPL, changed in a manner consistent with increased t-2-H biosynthesis. The site and mode of interaction of C6 aldehydes with Botrytis cinerea, a common pathogen of many plant species, was characterized using radiolabeled six carbon (C6) aldehydes, including c-3-H and t-2-H. An approximately 25% molar conversion of 18:3 to C6 aldehydes was obtained by enzymatic manipulation with LOX and HPL extracts. Following exposure of Botrytis cultures to radiolabeled aldehydes, radiolabeled aldehydes were recovered in protein fractions, but not in the lipid fraction. They were incorporated into conidia at a 20-fold higher level than mycelia (per mg fresh weight). About 95% of the radiolabeled aldehyde was recovered in proteins on the surface (wash protein) of the fungal tissue, while 5% was from protein in internal tissue (cell wall and membrane and cytosol). Supplementing radiolabeled aldehydes with nonradiolabled C6 aldehydes to increase the vapor phase concentration affected distribution of radiolabel in each protein fraction. The t-2-H at both 5.4 and 85.6 mol affected protein expression patterns, changing the intensity of expression in over one third of all proteins. Both up- and down-regulation of specific proteins were observed. Though five proteins of interest were analyzed, their identities were not determined. However, the data indicate a clear effect of t-2-H on protein expression in Botrytis cinerea.

La microflore des raisins est importante d'un point de vue technologique car elle conditionne en partie la qualité du vin. Or, la diversité des flores fongiques présentes sur baies de raisin ainsi que leur potentiel de contamination du produit final ne sont pas encore pleinement connus. Dans ce cadre, la caractérisation des flores fongiques cultivables présentes sur baies de raisin a été réalisée par PCR ITS-RFLP. 41 espèces de moisissures différentes sur les 43 étudiées appartenant à 11 genres différents ont été caractérisées de façon fiable. Seules les espèces Penicillium thomii et Penicillium glabrum ont présenté le même profil. Ainsi 96.3% des souches étudiées ont été caractérisées avec au maximum 4 enzymes de restriction et 41.5% des souches ont pu l'être avec seulement 2 enzymes de restriction. Ces résultats ont permis d'enrichir les bases de données, moyennement pourvues en séquences ITS caractéristiques de genres ou d'espèces de moisissures présentes sur baies de raisin. De plus, une étude exhaustive des moisissures présentes sur baies de raisin en Bourgogne a permis, par PCR ITS-RFLP, d'identifier 199 souches au niveau de l'espèce et ce quelque soit le genre. Penicillium spinulosum est l'espèce majoritaire isolée pour le millésime 2008 en Bourgogne. Parallèlement, la quantification de Botrytis cinerea, choisi comme micro-organisme modèle, a été réalisée par qPCR. La technique qPCR décrite dans ce travail présente (i) une bonne sensibilté avec une limite de détection de 6.4 pg d'ADN correspondant à 540 spores, (ii) l'originalité de travailler en échantillons naturellement contaminés et la fiabilité d'utiliser un standard interne. L'évaluation de l'efficacité de différentes stratégies de traitements anti-Botrytis a confirmé l'importance de la prophylaxie (effeuillage) dans la lutte contre Botrytis cinerea.

The fungus Botrytis cinerea is an opportunistic pathogen on a wide variety of crops, causing disease known as grey mould through infections via wounds or dead plant parts. Synthetic fungicides for controlling this disease are fast becoming ineffective due to the development of resistance. This, coupled with consumers world wide becomng increasingly conscious of potential environment and health problems associated with the build up of toxic chemicals, (particularly in food products), have resulted in pressure to reduce the use of chemical pesticide volumes as well as its residues. An emerging alternative to random synthesis is the study and exploitation of naturally occurring products with fungicidal properties. There have been reports on the uses of synthetic fungicides for the control of plant pathogenic fungi. When utilized in two-way mixtures, such fungicides may maintain or enhance the level of control of a pathogen at reduced rates for both components utilized in combinations, or alone at normal rates. For this study it was hypothesize that the addition of plant extracts may enhance the antifungal efficacy of the synthetic strobilurin fungicide, kresoxim-methyl against Botrytis cinerea.