En horticulture, les effets négatifs du déficit hydrique (WD) sur le rendement peuvent être en partie compensés par des effets positifs sur la qualité des fruits et la stimulation des défenses de la plante face à d’autres stress. L'objectif de la thèse était d'explorer un certain nombre de pistes permettant d'évaluer les effets positifs et négatifs du WD, et d'appréhender les facteurs de variations. Après avoir établi un état des connaissances actuelles des effets du WD sur la qualité des fruits, plusieurs questions ont été abordées: 1) quels sont les effets de WD appliqués à des stades précis du développement de la plante ou du fruit, 2) existe-t-il une mémoire du stress en cas d’alternance de période de WD et de récupération, 3) les défenses de la plante sont-elles stimulées par un WD lors d'une infection par un pathogène, et enfin 4) quelle est la variabilité génétique associée aux réponses au WD au travers des objectifs précédents. Pour cela, plusieurs expérimentations ont été réalisées en serres et en phytotrons. Les parents de la population MAGIC de la tomate (ayant une forte variabilité allélique) ont été étudiés, ainsi que deux tomates commerciales (pour l’étude de l’interaction WD – Botrytis cinerea). Les mécanismes de réponses induits lors du WD ont été caractérisés aux stades végétatif et reproductif à l’échelle de la plante, puis lors des phases de développement du fruit (division cellulaire, expansion cellulaire et maturation). Les stades végétatif et reproductif se distinguent par les mécanismes de gestion du stress oxydatif engendré par le WD. Les plantes végétatives seraient plus affectées au niveau des photosystèmes II alors que les plantes reproductives modulent plutôt leur besoin en eau et sont plus sensibles à l’approche des photosystèmes I. L’impact du WD sur la qualité des fruits diffère en fonction de leurs phases de développement. De forts effets génotypes ont été observés avec une amélioration de la qualité dite gustative (par rapport aux teneurs en sucres et acides) pour un génotype dont les caractéristiques initiales étaient plus faibles que le second génotype testé. L’alternance de périodes de WD et de récupérations constitue un bon compromis entre économie d’eau et adaptation des plantes (via une bonne gestion du stress oxydatif). Cette alternance de stress a des effets neutres à positifs sur les teneurs en sucres des fruits en fonction des génotypes et des réponses très variables pour les teneurs en acides organiques, caroténoïdes et acide ascorbique en fonction des génotypes. L’interaction entre un WD et B. cinerea génère une diminution du ratio C/N associé à une stimulation des défenses de la plante. Cependant l'interaction favorise le développement du pathogène dans les tiges. Cet effet délétère de l'interaction semble lié aux régulations hormonales mises en jeu avec la stimulation de l'ABA (par le WD) et de la voie SA (par B. cinerea et indirectement par le WD) qui sont antagonistes aux voies JA/ET de réponse aux pathogènes nécrotrophes. Toutefois, un effet positif a été observé pour les infections sur feuilles détachées qui constituent un instantané des défenses de la plante, et ce, uniquement pour les plantes déjà infectées sur tige. Ces résultats suscitent de nouvelles interrogations sur la stimulation des défenses de la plante. Ainsi, le WD permet une augmentation du pool de sucres solubles, sans impact notable sur le rendement, avec des effets variables sur les composés secondaires. Le WD permet également dans une certaine mesure de stimuler les défenses de la plante mais de nouvelles recherches sont nécessaires. Enfin, la variabilité génétique des réponses à l’échelle de la plante et du fruit est importante et interagit avec le WD. / In horticulture, yield losses associated to water deficit (WD) could be compensated by positive effects on fruit quality and stimulation of plant to others stresses. The aim of the thesis was to explore a number of questions to assess the positive and negative effects of the WD, and to understand factors variations after setting a current knowledge of the effects of WD quality fruit. The questions were: 1) to characterize the effects of WD applied at specific stages of development of plant or fruit, 2) to understand the stress memory effect by alternating period of WD and recovery, 3) to test the stimulation of plant defences by WD during an infection by a pathogen, and 4) to study the genetic variability associated with responses to WD through the previous objectives. For this, several experiments were conducted in greenhouses and growth chambers. The parents of the MAGIC population of tomato (with rich allelic variability) were studied, as well as two commercial tomatoes (for the study of the interaction between WD and Botrytis cinerea). Plant response mechanisms induced during a WD were characterized in both plant vegetative and reproductive stages, and then during the fruit development stages (cell division, cell expansion and maturation). The plant vegetative and reproductive stages are distinguished by different mechanisms of oxidative stress management generated by WD. Vegetative plants are more affected at the photosystem II while reproductive plants rather modulate their water needs and responses at the photosystem I. The impact of WD on fruit quality differs according to fruit developmental stages. Strong genotypes effects were observed with an important increase in fruit taste quality (according to the concentrations in sugars and acids) for one genotype, which had poorer initial fruit quality than the second. Alternating periods of WD and recovery is a good compromise between water saving and plant adaptation (through proper management of oxidative stress). This alternation of stress has neutral to positive effects on fruit sugar content according to the genotypes and highly variable responses for contents of organic acids, carotenoids and ascorbic acid according to the genotypes. The interaction between a WD and B. cinerea generates a decrease of C/N ratio that can be associated to a stimulation of the pool of plant defence. However, the interaction between B. Cinerea and this WD promote pathogen development in stems. This deleterious effect of interaction seems attributable to hormonal regulation with the stimulation of the ABA pathway (by WD) and the SA pathway (by B. cinerea), which are antagonistic to the JA / ET pathway involved in response to necrotroph pathogens. However, a positive effect was observed for infections in detached leaves of infected plants, detached leaves represent a snapshot of plant defences and these results raise new questions. Overall, these data provide that WD improve fruit taste (sugars and acids) without yield losses but had variables effects on health promoting compounds. WD could permit the stimulation of plant defence but more research is needed. To finish, the genetic variability induced important variability in plant and fruit responses and interact with WD.

La vigne est une culture pérenne sensible à de nombreux bioagresseurs et sur laquelle il est nécessaire de réaliser de nombreux traitements pesticides, susceptibles de causer des problèmes environnementaux, de santé humaine et d’apparition de résistance au sein des populations de bioagresseurs. Aujourd’hui, il est indispensable de développer des stratégies nouvelles de lutte contre les bioagresseurs, plus raisonnées mais permettant de conserver une viticulture compétitive. L’utilisation de produits de biocontrôle semble, en ce sens, être une approche prometteuse permettant d’allier agriculture durable et intensive.Deux écoproduits généralistes à fort potentiel ont été identifiés, comme possédant des actions intéressantes sur les principales maladies cryptogamiques de la vigne. Le premier est un extrait naturel de plante, sans action fongicide directe mais capable de stimuler efficacement et de façon systémique les défenses de la plante. Le second, quant à lui, est un microorganisme qui possède une forte action antagoniste fongicide, mais aussi la capacité à stimuler les défenses de plante. Dans un premier temps, des études en conditions contrôlées ont mis en évidence l’efficacité des deux actifs pour inhiber le développement de diverses souches d’Erysiphe necator, Plasmopara viticola, Botrytis cinerea et Botryosphaeriaceae. En parallèle, des expérimentations au vignoble, ont confirmé le fort potentiel de ces produits de biocontrôle, avec des bonnes efficacités, particulièrement stable avec l’extrait naturel. Ces différentes études nous ont permis d’identifier et d’élaborer des stratégies d’utilisation pour ces deux produits de biocontrôle. / Grapevine is a perennial crop sensitive to many fungal pathogens that require numerous pesticide treatments. However, its uses lead to environmental, human health and fungicide resistance problems. Developing sustainable pest management strategies while keeping a good wine quality is of major importance. In this sense, the use of bio-pesticides products seems to be a promising approach to combine sustainable and intensive agriculture.Two generalist bio-pesticides of great potential have been preliminary identified, forits actions on major fungal diseases of grapevine. The first one is a natural plant extract, with no direct fungicide action but able to systemically stimulate plant defenses. The second one is a microorganism showing strong antagonist fungicide actions, and important ability to stimulate plant defenses. First, the studies conducted in controlled conditions have demonstrated the effectiveness of both products in the suppression of various isolates of Erysiphe necator, Plasmopara viticola, Botrytis cinerea and Botryosphaeriaceae. In parallel,the good efficiencies of these products have been confirmed during vineyard assays. This was especially well demonstrated for the natural extract. As a whole, these studies confirm thepotential of these two products as promising bio-pesticides, of which the strategy of application have been further defined.

La pourriture grise, causée par le champignon Botrytis cinerea, est l'une des principales maladies aériennes fongiques sur diverses cultures d’importance agronomique. La diversité génétique de B. cinerea est très forte et la capacité rapide d’adaptation de ce champignon à une pression sélective est également avérée. Ce champignon est ainsi capable de développer des résistances à une grande variété de composés fongicides de synthèse ou d'origine naturelle. Des méthodes alternatives de lutte ont de ce fait été développées ces dernières années : divers agents de lutte biologique (ALB) présentant différents modes d’actions ont été identifiés et pour certains d’entre eux commercialisés pour contrôler B. cinerea. Cependant la durabilité de la lutte biologique est un domaine encore très peu étudié. La perte d'efficacité d'un ALB pourrait résulter de la préexistence d’isolats moins sensibles de pathogènes dans les populations naturelles et/ou de la capacité de l’agent pathogène à produire, sous une pression de sélection continue exercée par l’ALB, des mutants ayant une sensibilité réduite. L'objectif global de la présente étude est d'évaluer le risque potentiel de perte d'efficacité de la lutte biologique vis-à-vis de B. cinerea. Dans cette étude, les efforts ont été concentrés sur la pyrrolnitrine, un antibiotique produit par divers ALBs, dont certains sont efficaces contre B. cinerea. Les objectifs spécifiques de l'étude étaient (i) d’évaluer la diversité de la sensibilité à la pyrrolnitrine au sein de la population naturelle de B. cinerea, (ii) d'estimer le risque de perte d'efficacité des ALBs produisant la pyrrolnitrine due à la pression de sélection exercée par la pyrrolnitrine et (iii) d'étudier le mécanisme de résistance à la pyrrolnitrine chez B. cinerea. Parmi 204 isolats de B. cinerea, une gamme importante de sensibilité à la pyrrolnitrine a été observée, avec un facteur de résistance de 8,4 entre l’isolat le plus sensible et l'isolat le moins sensible. La production de 20 générations successives pour 4 isolats de B. cinerea, sur des doses croissantes de pyrrolnitrine, a abouti au développement de mutants avec des niveaux élevés de résistance à l'antibiotique, et à une réduction in vitro de la sensibilité à la bactérie productrice de pyrrolnitrine Pseudomonas chlororaphis PhZ24. La comparaison entre les mutants résistants à la pyrrolnitrine et leurs parents sensibles pour la croissance mycélienne, la sporulation et l'agressivité sur plantes a révélé que la résistance à la pyrrolnitrine est associée à un fort coût adaptatif. Des observations cytohistologiques sur tomates ont confirmé que l’isolat sensible à la pyrrolnitrine attaque le pétiole rapidement et envahit la tige, alors que le mutant résistant à la pyrrolnitrine ne s'étend pas au-delà du pétiole. De plus, ce dernier mutant forme un mycélium anormal et des cellules ressemblant à des chlamydospores. Les résultats ont d'autre part révélé que les mutants de B. cinerea résistants à la pyrrolnitrine sont résistants au fongicide iprodione, suggérant ainsi qu'une pression exercée par la pyrrolnitrine sur le champignon conduit à une résistance au fongicide. Réciproquement, la production de générations successives sur iprodione conduit à une résistance à l'antibiotique. Afin d'étudier les déterminants moléculaires de la résistance de B. cinerea à la pyrrolnitrine, le gène histidine kinase Bos1, impliqué entre autres dans la résistance aux fongicides chez B. cinerea a été séquencé chez les souches sensibles et les mutants résistants. La comparaison des séquences a mis en évidence des mutations ponctuelles différentes chez les mutants de B. cinerea obtenus sur la pyrrolnitrine et ceux obtenus sur l'iprodione. De plus, les résistances à la pyrrolnitrine et à l'iprodione ne sont pas systématiquement associées à une mutation ponctuelle dans le gène Bos1. Enfin, aucune modification n'a été détectée dans la taille des allèles de neuf locus microsatellites quelle que soit la pression sélective exercée et quelle que soit le phénotype du mutant produit. Cette étude montre qu'un champignon pathogène des plantes est capable de développer progressivement une moindre sensibilité à un agent de lutte biologique mais que cette moindre sensibilité est associée à une forte perte de fitness / Gray mould, caused by Botrytis cinerea, is a severe disease on a wide range of crops. Disease control generally relies on chemicals, although biological control strategies have been intensively studied over the last decades. This pathogen can withstand a wide variety of fungitoxic compounds including fungicides and natural molecules. This capacity to adapt to different stress might, potentially, compromise the durability of biological control methods. The global purpose of that work was to estimate the potential of B. cinerea to overcome the efficacy of biological control agents. Knowledge on the potential development of resistance to biological control agents can help to devise or improve resistance management strategies. In this work, efforts have been focused on the antibiotic pyrrolnitrin produced by various bacteria described as potential biological control agents against B. cinerea. The specific objectives of the study were (i) to evaluate the diversity in susceptibility to pyrrolnitrin among natural population of B. cinerea, (ii) to estimate the risk of loss of efficacy of pyrrolnitrinproducing biological control agent due to selection pressure exerted by pyrrolnitrin and (iii) to study the mechanism of resistance to pyrrolnitrin in B. cinerea. An important range of sensitivity to pyrrolnitrin with an 8.4-fold difference in EC50 values between the most sensitive and the least sensitive isolates was observed within the 204 isolates tested. The production of 20 generations, for 4 isolates of B. cinerea, on increasing doses of pyrrolnitrin, resulted in the development of mutants of B. cinerea with high levels of resistance to the antibiotic and a reduced sensitivity in vitro to the pyrrolnitrin-producing Pseudomonas chlororaphis PhZ24. Comparison of the pyrrolnitrin-resistant mutants and their sensitive parent isolates for mycelial growth, sporulation and aggressiveness on plant tissues revealed that the high level of resistance to pyrrolnitrin has resulted in a high fitness cost. Additional cytohistological investigations revealed that while the sensitive isolate spread throughout the petiole and rapidly invaded the stem via the abscission zone, the pyrrolnitrinresistant mutant failed to extend beyond petiole to invade the stem. Moreover, the pyrrolnitrin-resistant mutant formed abnormal mycelium and chlamydospore-like cells. The comparison of resistance to pyrrolnitrin and to the iprodione fungicide in B. cinerea revealed that fungicide pressure exerted on the fungus is able to build-up resistance to pyrrolnitrin. Comparison of sequences of the osmosensing class III histidine kinase encoding gene bos1 revealed different mutations in pyrrolnitrin- and iprodione-resistant mutants. However, resistance to pyrrolnitrin and to iprodione was not systematically associated with a point mutation in the Bos1 gene. Finally, no changes were observed in the allele size at nine microsatellite loci whatever the four selective pressure endured by the fungus despite their phenotypic changes. This study provides evidence that a fungal plant pathogen is able to gradually build-up resistance to an antibiotic produced by a biocontrol agent

In Karpfen sächsischer Karpfenteichwirtschaften spielen u.a. die Zestoden Neogryporhynchus cheilancristrotus und Paradilepis scolecina und der Trematode Diplostomum pseudospathaceum eine Rolle (KAPPE 2004, PIETROCK et al. 2006, KAPPE & SEIFERT unveröff.). Weitere Fischarten (Schleie, Plötze, Regenbogenforelle) sind mit den Trematoden Diplostomum sp., Hysteromorpha triloba, Posthodiplostomum cuticola infiziert (PIETROCK et al. 2006). Mit dieser Arbeit sollte der Versuch unternommen werden, die Bedeutung Fisch fressender Vögel als Vektor Fisch pathogener Helminthen in Sachsen aufzuklären. Dafür wurden im Zeitraum von Juni 2003 bis Dezember 2004 97 Kormorane (Phalacrocorax carbo) und 46 Graureiher (Ardea cinerea) aus dem Einzugsgebiet sächsischer Teichwirtschaften auf ihren Befall mit Endohelminthen untersucht. Im Winter 2004 konnte zusätzlich ein Silberreiher (Casmerodius albus) und eine Silbermöwe (Larus argentatus) in die Untersuchungen einbezogen werden. Während des Untersuchungszeitraumes konnten so insgesamt 29 Parasitengattungen bzw. -arten nachgewiesen werden, denen z.T. kommerziell genutzte Süßwasserfische als Zwischenwirte oder paratenische Wirte dienen. Die Parasiten des Kormorans umfassten 15 verschiedene Parasitenarten. Dabei handelte es sich um die beiden Zestoden Ligula intestinalis und Paradilepis scolecina, die sechs Nematoden Baruscapillaria spp., Contracaecum rudolphii, Cyathostoma microspiculum, Desmidocercella incognita, Eucoleus contortus und Syncuaria squamata und die sieben Trematoden Clinostomum complanatum, Holostephanus dubinini, Hysteromorpha triloba, Metorchis xanthosomus, Paryphostomum radiatum, Petasiger exaeretus und P. phalacrocoracis. Außerdem wurden die zwei Kratzer Corynosoma strumosum und Polymorphus sp. nachgewiesen. Im Graureiher konnten dagegen nur neun Helminthenarten nachgewiesen werden. So der Zestode Neogryporhynchus cheilancristrotus, die sechs Trematoden Apharyngostrigea cornu, Bilharziella polonica, Diplostomum sp., Echinoasmus belocephalus, Posthodiplostomum cuticola und Tylodelphis excavata und die drei Nematoden Avioserpens galliardi, Desmidocercella incognita und Desmidocercella numidica. In der Silbermöwe konnten der Zestode Echinocotyle verschureni, die vier Trematoden Apophallus mühlingi, Diplostomum pseudospathaceum, Renicola lari und Metorchis xanthosomus sowie die drei Nematoden Desmidocercella incognita, Cosmocephalus obvelatus und Paracuaria tridentata nachgewiesen werden. Die parasitologischen Befunde des Silberreihers beschränkten sich auf Apharyngostrigea sp., Desmidocercella incognita und Desmidocercella numidica. Eine Beeinflussung der Helminthenfauna von Karpfen durch Kormoran und Reiher als auch umgekehrt konnte anhand der eigenen Untersuchungen in sächsischen Teichwirtschaften nicht sicher nachgewiesen werden. / In Saxonian carp ponds the cestodes Neogryporhynchus cheilancristrotus and Paradilepis scolecina and the trematode Diplostomum pseudospathaceum occur amongst others in carps (KAPPE 2004, PIETROCK et al. 2006, KAPPE & SEIFERT unpublished). Further fish species (tench, roach, rainbow trout) are infected with the trematodes Diplostomum sp., Hysteromorpha triloba and Posthodiplostomum cuticola (PIETROCK et al. 2006). This work was performed to analyse the importance of fish eating birds as vectors of fish pathogenic helminths. For this purpose 97 common cormorants (Phalacrocorax carbo) and 46 grey herons (Ardea cinerea) were examined on their infestation with endohelminths during the term of June 2003 to December 2004. All these birds came from the vicinity of Saxonian fish ponds. In winter 2004 additionally an egret (Casmerodius albus) and an hering gull (Larus argentatus) were included into the investigations. Altogether 29 parasite species and genus respectively could be detected. Some of these endohelminths use fresh water fishes as intermediate or paratenic hosts. The cormorants harboured 15 different parasite species and genus respectively: the two cestodes Ligula intestinalis and Paradilepis scolecina, the six nematodes Baruscapillaria carbonis, Contracaecum rudolphii, Cyathostoma microspiculum, Desmidocercella incognita, Eucoleus contortus and Syncuaria squamata and seven trematodes the Clinostomum complanatum, Holostephanus dubinini, Hysteromorpha triloba, Metorchis xanthosomus, Paryphostomum radiatum, Petasiger exaeretus and P. phalacrocoracis. In addition the two acanthocephales Corynosoma strumosum and Polymorphus sp. were detected. In the grey heron nine helminth species/genus could be detected: the cestode Neogryporhynchus cheilancristrotus, the six trematodes Apharyngostrigea cornu, Bilharziella polonica, Diplostomum sp., Echinoasmus belocephalus, Posthodiplostomum cuticola and Tylodelphis excavata and the three nematodes Avioserpens galliardi, Desmidocercella incognita and Desmidocercella numidica. In the hering gull the cestode Echinocotyle verschureni, the four trematodes Apophallus muehlingi, Diplostomum pseudospathaceum, Renicola lari and Metorchis xanthosomus as well as the three nematodes Desmidocercella incognita, Cosmocephalus obvelatus and Paracuaria tridentata were detected. Merely three endohelminths could be demonstrated in the investigated egret: Apharyngostrigea sp., Desmidocercella incognita and D. numidica. Six of the 29 endohelminth species and genus respectively were found in this examination to infest both fish and birds in Saxonian fish ponds. Nevertheless the helminth fauna of the inestigated birds, in particular of cormorants and herons did not make a definite impact on the carps´ infestation with endohelminths. As well as the fish helminth fauna were not unequivocally linked to those of the birds.

Thesis (MScAgric)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Various studies revealed that Botrytis cinerea, the causal pathogen of Botrytis bunch rot, is mostly associated with pedicels, rachises, laterals and berry bases, and not with berry skins as previously understood. Provided that sufficient coverage of inner bunch parts was achieved, laboratory studies have shown that fungicides can effectively reduce the amount of B. cinerea at the various positions in bunches, and prevent infection and symptom expression at all growth stages. The same efficacy was, however, not achieved with the same fungicides when using conventional spraying methods in vineyards. Poor disease control on fruit and leaves in vineyards is attributed to inappropriate timing of fungicide applications and/or insufficient coverage of susceptible tissue. Previously, spray coverage evaluations in South Africa were based on the use of water-sensitive cards. A variety of other methods have been used to assess spray coverage in vineyards, but none of these methods could assess spray deposits on a very small, three-dimensional area of interest such as the susceptible grape bunch parts. The methods were furthermore dependent on human objectivity, which lacks quantitative measuring and speed of measurement. Suitable technology to determine spray coverage on susceptible bunch parts is, therefore, not available. The aim of this study was to develop a protocol to visualise and quantify spray deposits in grape bunches, specifically on the inner bunch parts and to use the protocol to determine the effect of different levels of spray cover on artificially inoculated B. cinerea grape bunches, in order to facilitate future determination of minimum effective coverage levels for effective B. cinerea control. A spray coverage assessment protocol using fluorometry, photomicrography and digital image analyses was developed to measure spray coverage on susceptible grape bunch parts. Among several fluorescent pigments tested, a yellow fluorescent pigment (SARDI Fluorescent Pigment) from Australia was selected on the basis of its small particle size (2.45 - 4.90 μm). Bunches were sprayed at pea size and bunch closure with different volumes of a mixture of fenhexamid and the yellow fluorescent pigment. Sprayed parts from bunches were illuminated under black light (UV-A light in the 365 nm region) and visualised under a stereo microscope at 20 x magnification. Photos of the berry skin, pedicel and rachis were taken with a digital camera (Nikon DMX 1200). Image analysis of photos was done with Image- Pro Discovery version 4.5 for Windows (Media Cybernetics) software. The total area of deposited pigment in selected areas of interest (AOI) was calculated. The percentage area covered was subsequently calculated for each AOI. Good correlation was evident between the parameters, sum of objects and percentage area covered. Bunch parts at pea size generally had higher coverage values than at bunch closure. Spray applications earlier in the season would therefore result in higher and more effective spray coverage of the susceptible bunch parts. Similar deposition trends were observed on the inner bunch parts (pedicel and rachis). These were, however, significantly different from berry skins, which had significantly higher levels of spray deposits than the inner bunch parts. The variance component analysis indicated that the highest variance was observed for berries and bunches, and substantially less for image readings. For the same accuracy, means for percentage coverage values of at least 10 bunches per treatment (1 part per bunch and 3 readings per part) will be sufficient. In order to determine the biological efficacy of different levels of spray coverage on B. cinerea incidence on grape bunches, bunches were sprayed at pea size and bunch closure with different volumes of a mixture of fenhexamid and a yellow fluorescent pigment and the percentage fluorescent pigment coverage on pedicels was determine. Bunches were subsequently dusted with dry airborne conidia of B. cinerea in a settling tower and incubated for 24 h at high relative humidity (98%). Infection was determined by estimating the amount of B. cinerea infections occurring on sprayed bunch parts with isolations on to paraquat and Kerssies mediums. Linear regressions for the part x stage combinations of percentage B. cinerea incidence on different bunch parts were fitted on mean coverage levels. An increase in spray cover caused linear reductions in levels of B. cinerea on susceptible bunch parts. Higher B. cinerea incidences were recorded at pea size. Furthermore, higher B. cinerea incidences were found on paraquat medium for both stages, than on Kerrsies medium. The information gathered from this study will be used to facilitate future determination of minimum effective coverage levels for effective B. cinerea control in grape bunches. In these validation experiments, the results clearly showed that the protocol can be used to determine the effect of different levels of spray coverage on B. cinerea incidence and that an increase in spray coverage will decrease B. cinerea incidence. The information gathered from this study will be used to facilitate future determination of minimum effective coverage levels for effective B. cinerea control in grape bunches and subsequently be used as benchmarks to evaluate spray application in vineyards. / AFRIKAANSE OPSOMMING: Vaalvrot by wingerde word veroorsaak deur Botrytis cinerea. Verskeie studies het getoon/gewys dat die oorsaaklike patogeen meestal geassosieer word met die pedisel, ragis, laterale en die korrelbasis, en nie met die korrelskil soos voorheen beweer nie. Laboratorium studies het getoon dat swamdoders wel effektief is om B. cinerea by alle trosdele te verminder en simptoomontwikkeling te voorkom tydens alle groeistadia, mits die binne-trosdele voldoende spuit bedekking ontvang het. Dieselfde effektiwiteit is egter nie gevind in wingerde met konvensionele spuittegnieke nie. Onvoldoende siektebeheer van vrugte en blare van wingerde kan toegeskryf word aan verkeerde spuit skedulering en/of swak spuitbedekking van vatbare gasheerweefsel. Evaluering van spuitbedekking is voorheen in Suid Afrika deur middel van water-sensitiewe papier gedoen. Verskeie ander metodes is al gebruik om spuitbedekking te evalueer in wingerde, maar nie een van hierdie metodes kan gebruik word om spuitbedekking op ’n baie klein, drie-dimensionele oppervlak, soos die vatbare trosdele, te evalueer nie. Verder was die tegnieke afhanklik van menslike objektiwiteit, en gevolglik ontbreek kwantitatiewe meting en metingspoed. Daar is dus nie geskikte tegnologie vir die evaluering van spuitbedekking op vatbare trosdele nie. Die doel van hierdie studie was die ontwikkeling van ‘n protokol vir die visualisering en kwantifisering van spuitbedekking op spesifiek die binne-tros dele en om die protokol dan te gebruik om die effek van verskillende vlakke van spuitbedekking op B. cinereageinokuleerde druiwetrosse te bepaal, Protokol vir evaluasie van spuitbedekking op vatbare druifdele is ontwikkel deur gebruik te maak van fluorometrie, fotomikrografie en digitale beeldanalise. Van die verskillende fluoresensie pigmente wat getoets is, is ‘n geel flouresensie pigment (SARDI Flourescent Pigment) van Australië gekies op grond van sy klein partikelgrootte (2.45 - 4.90 μm). Druiwetrosse is gespuit tydens ertjie- en trostoemaakstadia met verskillende volumes van ’n mengsel van fenheksamied en die geel fluorosensie pigment. Die gespuite druifdele is dan verlig onder swartlig buise (UV-A lig in die 365 nm spektrum) en gevisualiseer deur ’n stereo mikroskoop by 20x vergroting. Foto’s van die korrelskil, pedisel en ragis is met ‘n digitale kamera (Nikon DMX 1200) geneem. Beeldanalise is gedoen met ImagePro Discovery weergawe 4.5 vir Windows (Media Cybernetics) sagteware. Die totale area neerslag van die pigment is in geselekteerde areas bereken. Die presentasie area bedek is bereken vir elkeen van hierdie areas. Goeie korrelasie is gevind tussen die parameters aantal fluoresserende partikels en die persentasie bedekte area. Trosdele tydens ertjie-stadium het in die algemeen hoër waardes gehad as by trostoemaak. Dit blyk dus dat spuittoediening vroeg in die seisoen meer effektief sal wees vir die bedekking van vatbare trosdele. Soortgelyke bedekkings patrone is gevind by die binne trosdele (pedisel en ragis). Dit het egter betekenisvol verskil van die korrelskil, wat betekenisvol meer spuitbedekking as die binne trosdele gehad het. ’n Variasie komponent analise het getoon dat die meeste variasie gevind is tussen korrels en trosse, en heelwat minder vir die beeld analise lesings. Om dieselfde akkuraatheid te behou, is ten minste 10 trosse per behandeling (1 deel per tros en 3 lesings per deel) nodig. Vir die bepaling van biologiese effektiwiteit van verskillende vlakke van spuitbedekking op B. cinerea voorkoms op druiwe, is druiwe gespuit tydens ertjie- en trostoemaak-stadia met verskillende volumes van ’n mengsel van fenheksamied en die geel fluorosensie pigment. Die persentasie fluoresensie pigment is bepaal op die pedisels. Trosse is vervolgens geinokuleer met droë luggedraagde konidia van B. cinerea in ’n inokulasietoring en geïnkubeer vir 24 h by hoë relatiewe humiditeit (98%). Die voorkoms van B. cinerea infeksie op gespuite tros dele is bepaal deur middel van isolasies op paraquat en Kerssies medium. Liniêre regressies vir trosdeel x stadium kombinasies van persentasie B. cinerea voorkoms op verskillende trosdele is gepas vir gemiddelde bedekkings waardes. ’n Verhoging in spuit bedekking het ‘n liniêre vermindering van B. cinerea voorkoms op vatbare trosdele veroorsaak. Verder is hoër vlakke van B. cinerea op paraquat medium as op Kerssies medium vir beide die groeistadia gevind. Die kennis wat verkry is uit hierdie studie sal gebruik word om minimum effektiewe spuitbedekkingsvlakke vir die beheer van B. cinerea op druiwetrosse te bepaal.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: In the field of agriculture, plant pathogens are a major concern because of the severe damage these organisms cause to crops yearly. Fundamental studies regarding plant pathogens and their modes of action made it possible for researchers in the field of molecular biology to investigate pathogens further on a molecular level. Botrytis cinerea, has been used to great effect as a model system to investigate various aspects regarding pathogenesis, also on a molecular level. Molecular research done on B. cinerea over the last few years has shown that the endopolygalacturonases (EPGs) of this fungus are key role players in pathogenesis. This hydrolytic enzyme family of six members, encoded by the Bcpg1-6 genes, are important in breaking down the complex cell wall polymers of host plants, enabling the fungus to penetrate its host sufficiently. It has been shown that both BcPG1 and 2 are crucial for virulence of B. cinerea. A leucine-rich repeat inhibitor protein situated in the cell wall of various plant species, the polygalacturonase-inhibiting protein (PGIP), has been proven to interact with and inhibit EPGs, and thus the necrotic actions of B. cinerea. From literature it was clear that specific data regarding individual interactions of fungal EPGs with PGIPs are lacking currently. Furthermore, most experiments regarding the effects of EPG as well as interaction and inhibition studies of EPGs and PGIPs, rely on in vitro methods, without the possibility to contextualize the results on an in vivo or in planta level. The scope of this study was to specifically address the issues of individual EPG:PGIP interactions and the use of possible in vivo methodology by using EPGs from a highly virulent South African strain of B. cinerea and the grapevine VvPGIP1 that has been previously isolated in our laboratory. This PGIP, originally isolated from Vitis vinifera cv Pinotage, has been shown to inhibit a crude EPG extract from this strain with great efficiency. The approach taken relied on heterologous over-expression of the individual Bcpg genes and the isolation of pure and active enzymes to evaluate the inhibition of the EPGs with VvPGIP1. The genes were all successfully over-expressed in Saccharomyces cerevisiae with a strong and inducible promoter, but active enzyme preparations have been obtained only for the encoding Bcpg2 gene, as measured with an agarose diffusion assay. The in vitro PGIP inhibition assay is also based on the agarose diffusion assay and relies on activity of the EPGs to visualize the inhibiting effect of the PGIP being tested. The active EPG2, however, was not inhibited by VvPGIP1 when tested with this assay. The EPG encoding genes from B. cinerea were transiently over-expressed also in Nicotiana benthamiana by using the Agrobacterium-infiltration technique. Transgene expression was confirmed by Northern blot analysis and EPG-related symptoms were observed five to eight days post-infiltration. Differential symptoms appeared with the various EPGs, providing some evidence that the symptoms were not random events due to the infiltration or a hypersensitive response. Moreover, the symptoms observed for EPG2 was similar to those that were reported recently by another group on the same host. In spite of the expression data and the clear symptoms that developed, active preparations, as measured with the agarose diffusion plate asay, could only be obtained for EPG2 again. In our search for a possible in vivo method to detect and quantify EPG activity and inhibition by PGIPs, we tested and evaluated a technique based on chlorophyll fluorescence to detect the effect of EPGs on the rate of photosynthesis. Our results showed that the over-expression of these genes reduced the rate of electrons flowing through photosystem II, indicating metabolic stress occurring in the plant. We used the same technique to evaluate possible interaction between VvPGIP1 respectively with BcPG1 and 2 and found that the co-expressing of the Vvpgip1 gene caused protection of the infiltrated tissue, indicating inhibition of EPG1 and 2 by VvPGIP1. For EPG2, the observed interaction and possible inhibition by VvPGIP1 is the first report to our knowledge of an interaction between this specific EPG2 and a PGIP. Moreover, to further elucidate the in planta interaction between VvPGIP1 and the EPGs from the South African B. cinerea strain, we tested for possible interactions by making use of a plant two-hybrid fusion assay, but the results are inconclusive at this stage. Previous studies in our laboratory have shown that several natural mutations exist between PGIP encoding genes from different V. vinifera cultivars. Based on this finding and the fact that these natural mutations could result in changes with regard to EPG inhibition and ultimately disease susceptibility, we isolated an additional 37 PGIP encoding genes from various grapevine genotypes, some of which are known for their resistance to pathogens. Combined, these results make a valuable contribution to understand plant pathogen interactions, specifically in this case by modeling the interactions of pathogen and plant derived proteins. The possibility to use in vivo methods such as chlorophyll fluorescence to follow these interactions on an in planta level, provides exciting possibilities to strenghten and contextualize in vitro results. / AFRIKAANSE OPSOMMING: Plantpatogene organismes veroorsaak jaarliks erge skade aan landbougewasse en word dus as ’n ernstige probleem in die landbousektor beskou. Diepgaande studies wat handel oor plantpatogene en hul metodes van infeksie het dit vir molekulêre bioloë moontlik gemaak om patogene nou ook op molekulêre vlak verder te bestudeer. Botrytis cinerea is baie effektief as modelsisteem gebruik om verskeie aspekte van patogenese verder te bestudeer, ook op ‘n molekulêre vlak. Molekulêre navorsing op B. cinerea, het getoon dat die endopoligalakturonases (EPGs) van dié swam kernrolbelangrik in patogenese is. Hierdie sesledige hidrolitiese ensiemfamilie word gekodeer deur die Bcpg1-6 gene en is belangrik vir die afbraak van die komplekse selwandpolimere van plantgashere, om suksesvolle gasheerpenetrasie te veroorsaak. Daar is aangetoon dat beide BcPG1 en 2 essensieël vir virulensie van die patogeen is. ’n Leusienryke-herhalings inhibitorproteïen wat in die selwand van verskeie plantspesies voorkom, die poligalakturonase-inhiberende proteïen (PGIP), het interaksie met en inhibeer EPGs en gevolglik ook die nekrotiserende aksies van B. cinerea. Uit die literatuur is dit duidelik dat spesifieke inligting aangaande individuele interaksies van fungiese EPGs met PGIPs tans nog ontbreek. Verder word daar op in vitro metodologie staatgemaak wannneer die effekte van EPGs asook die interaksie en inhibisie met PGIPs bestudeer word, sonder om die konteks van die in vivo- of in planta-omgewing in ag te neem. Die fokus van hierdie studie was om aspekte van individuele EPG:PGIP interaksies, asook die moontlike gebruik van in vivo metodologie te bestudeer deur EPGs, afkomstig van ’n hoogs virulente Suid-Afrikaanse ras van B. cinerea en die wingerd VvPGIP1, wat vroeër in ons laboratorium geïsoleer is, te gebrruik. Hierdie PGIP wat uit Vitis vinifera cv Pinotage geïsoleer is, inhibeer ’n kru EPG-ekstrak van bogenoemde ras baie effektief. Die benadering wat gevolg is het op die ooruitdrukking van die individuele Bcpg-gene in heteroloë sisteme staatgemaak en die gevolglike isolering van suiwer en aktiewe ensieme om EPG-inhibisie deur VvPGIP1 te beoordeel. Al die gene is suksesvol in Saccharomyces cerevisiae ooruitgedruk onder ’n sterk induseerbare promotor, maar volgens ’n agarose-diffundeerbare toets kon aktiewe ensiempreparate slegs vir die enkoderende Bcpg2 verkry word. Die in vitro PGIP-inhibisie toets is ook op die gemelde toets gebasseer en vereis EPG-aktiwiteit om die inhiberende effek van die PGIP, te visualiseer. Die aktiewe EPG2 is egter nie deur VvPGIP1 geïnhibeer met die aanleg van hierdie toets nie. Die EPG-enkoderende gene van B. cinerea is ook tydelik in Nicotiana benthamiana ooruitgedruk deur gebruik te maak van ’n Agrobacterium-infiltrasietegniek. Transgeenuitdrukking kon met die Noordelike kladtegniek bevestig word en EPG-verwante simptome is vyf tot agt dae na infiltrasie waargeneem. Verskillende simptome vir die verskillende EPGs is waargeneem, wat aanduidend is dat die simptome nie lukrake gevolge van die infiltrasies, of ’n hipersensitiewe respons is nie. Verder kon die simptome wat EPG2 vertoon het, gekorreleer word met dié wat onlangs deur ’n ander groep op dieselfde gasheer waargeneem is. Ten spyte van die ekspressiedata en die waargenome simptome, kon aktiewe ensiempreparate op die agarose-diffundeerbare toets, weereens slegs vir EPG2 waargeneem word. ’n Metode wat gebasseer is op chlorofilfluoressensie is getoets en geëvalueer as ’n moontlike in vivo metode om EPG aktiwiteit en inhibisie deur PGIPs waar te neem en te kwantifiseer. Die resultate het bevestig dat die ooruitdrukking van hierdie gene die elektronvloeitempo deur fotosisteem II verminder het wat ’n aanduiding is dat metaboliese stres in die plant heers. Dieselfde tegniek is gebruik om die moontlike interaksies tussen BcPG1 en 2 en VvPGIP1 te bestudeer en het aangetoon dat die mede-uitdrukking van die Vvpgip1-geen aanleiding gee tot ’n beskermende effek van die geinfiltreerde weefsel, wat aanduidend is van inhibisie van EPG1 en 2 deur VvPGIP1. In die geval van EPG2 is hierdie interaksie en moontlike inhibisie met ’n PGIP die eerste waarneming in die verband. In ’n verdere poging om die in planta-interaksie tussen VvPGIP1 en die EPGs van die Suid-Afrikaanse B. cinerea ras uit te klaar, is ’n plantgebasseerde twee-hibriede toets aangelê, maar geen klinkklare resultate kon verkry word nie. Vorige werk het bevestig dat verskeie natuurlike mutasies in PGIP-enkoderende gene, afkomstig van verskillende V. vinifera kultivars, voorkom. Hierdie resultaat en die feit dat hierdie mutasies verskille in EPG inhibisie en uiteindelik vatbaarheid vir siektes kan beïnvloed, het aanleiding gegee tot die isolering van ’n verdere 37 PGIP-enkoderende gene uit ‘n verskeidenheid druifplantgenotipes, sommige waarvan juis bekend vir hul weerstand teen patogene is. Die gekombineerde resultate wat in dié studie verkry is, maak ’n waardevolle bydrae tot die verstaan van plant-patogeeninteraksies, spesifiek met die modelering van interaksies van patogeen- en plantgebasseerde proteïene. Die moontlikheid om in vivo-metodes soos chlorofilfluoressensie te gebruik in in planta-analises, is besonder bemoedigend om in vitro-resultate te versterk en ook in konteks te plaas.

Thesis (PhD(Agric))--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Despite adherence to fungicide spray schedules and label recommendations, table and wine grape producers invariably suffer crop losses when environmental conditions are conducive to fruit and foliar pathogens. Registered fungicides are effective and poor control is often attributed to: 1) improper spray timing, 2) reduced sensitivity to fungicides in the pathogen populations, and 3) poor spray deposition. Spray timing, management of fungicide resistance and the epidemiology of Botrytis cinerea have been thoroughly researched under South African conditions on grape crops. However, limited research regarding spray deposition exists in South Africa, probably due to a lack of proper spray deposition assessment protocols. To determine minimum spray deposition quantity and quality levels needed for effective B. cinerea control, bunches and leaves of table (Waltham Cross) and wine grapes (Chenin blanc) were sprayed at various stages using different volumes with a precision spray gun. A deposition assessment protocol using fluorometry, photomicrography and digital image analyses was improved. Deposition values correlated favourably with Botrytis infection. Increasing spray volume increased spray deposition; however, at a certain point, deposition quality remained constant and B. cinerea infections did not decrease significantly with increasing spray volume, indicating the importance of both spray deposition quantity and quality. Fluorescent pigment area that effected 75% control of B. cinerea infection (FPC75 values) was calculated for leaves, pedicels and receptacles at different growth stages. The FPC75 values obtained in this study can be used as benchmarks to evaluate future spray application. In order to study the optimisation of spray deposition with existing application technology (air blast and air shear sprayers) in commercial vineyards, spray deposition quantity and quality values were assessed from leaves and structural bunch parts of wine (Chenin blanc) and table grapes (Waltham Cross) and compared with FPC75 values. Spray trials were conducted at different growth stages at current best-practice recommendations, and with a range of spray volumes but with spray mixture concentration amended accordingly (i.e. fixed dosage per hectare). Spray trails indicated that deposition levels following current best-practice spray application were sub-optimal to control B. cinerea infections on bunches and leaves. Deposition values between air blast and air shear sprayers were generally similar. The air blast sprayer resulted in higher deposition levels with diluted spraying and increased spray volume; however, when dosage per hectare was kept constant, no significant differences were calculated between spray volumes (250-1000 L/ha), indicating that this sprayer can as effectively but more efficiently be used at lower spray volume. The air shear were not as efficient at higher spray volumes (>500 L/ha), but was superior at low volume concentrate application (≈250 L/ha at 4× concentration). This study clearly demonstrated the efficacy and cost-saving potential in optimising spray application with respect to application technology. / AFRIKAANSE OPSOMMING: Wingerdprodusente kan oesverliese ondervind indien omgewingstoestande bevorderlik is vir swampatogene. Siektes word onvoldoende beheer ten spyte van die nakoming van korrekte swamdoder aanbevelings. Geregistreerde swamdoders is effektief, mits die vatbare plantdele voldoende spuitbedekking ontvang. Onvoldoende siekte beheer kan gewoonlik toegeskryf word aan: 1) verkeerde spuit tydsberekening, 2) vermindere sensitiwiteit in patogeen-populasies teen swamdoders, en 3) swak spuitbedekking. Spuit tydsberekening, die bestuur van weerstand teen swamdoders en die epidemiologie van Botrytis cinerea is deeglik onder Suid-Afrikaanse toestande nagevors. Nietemin is daar beperkte navorsing oor spuitbedekking, waarskynlik weens 'n gebrek aan behoorlike spuitbedekking assesseringsprotokol. Om te bepaal hoeveel spuitbedekking (% area bedek deur fluoresserende pigment) nodig is om 75% van B. cinerea infeksies (FPC75 waardes) op vatbare wingerddele te beheer, is druiwetrosse en blare van tafel- en wyndruiwe (Waltham Cross en Chenin blanc, onderskeidelik) op verskillende groei stadiums en spuitvolumes in die laboratorium gespuit. ‘n Assesseringsprotokol van spuitbedekking op vatbare druifdele en blare is ontwikkel deur gebruik te maak van fluorometrie, fotomikrografie en digitale beeldanalise. Spuitbedekking het goed met Botrytis infeksies gekorreleer. Toenemende spuitvolume het bedekking laat toeneem, maar egter net tot 'n sekere punt, waar die kwantiteit van die bedekking nog toegeneem het, maar die kwaliteit van bedekking en B. cinerea infeksies nie beduidend toegeneem het nie. Dit is ‘n aanduiding van die belangrikheid van beide die kwantiteit en kwaliteit van spuitbedekking. Die FPC75 waardes wat in hierdie studie verkry is, kan as drempelwaardes om toekomstige spuittoediening te evalueer, gebruik word. Ten einde spuitbedekking met bestaande tegnologie (druk en waaierpomp spuitmasjiene) te optimiseer, is kommersiële wyn- en tafeldruiwe (Chenin blanc en Waltham Cross, onderskeidelik), volgens huidige spuit aanbevelings vir wingerde tydens verskillende groeistadiums en met ‘n reeks van verskillende spuitvolumes gespuit. Die konsentrasie van die spuitmengsel is dienooreenkomstig gewysig, i.t.v. ‘n vaste dosis per hektaar ongeag die spuitvolume. Bedekkingswaardes is met FPC75 waardes vergelyk en het aangedui dat kommersiële spuit aanbevelings aan produsente sal lei tot sub-optimale beheer van B. cinerea op beide blare en druiwetrosse. In die algemeen was bedekkingswaardes vir beide druk- en waaierpomp spuitmasjiene soortgelyk. Vir die waaierpomp teen verskillende spuitvolumes en aanbevole konsentrasie het ‘n toename in spuitvolumes tot hoër beddekingswaardes gelei, maar indien die dosis per hektaar van die spuitmengsel konstant behou is, is geen betekenisvolle verskille tussen spuitvolumes (250-1000 L/ha) voorspel nie. Hierdie dui aan dat die waaierpomp net so doeltreffend, maar meer effektief teen laer spuitvolumes gebruik kan word. Die drukpomp was nie so doeltreffend teen hoër spuitvolumes (> 500 L/ha) nie, maar was aansienlik beter by lae volume konsentraat toediening (≈ 250 L/ha op 4 × konsentrasie). Die studie toon duidelik die doeltreffendheid en moontlike kostebesparing moontlikhede deur bespuiting relatief tot bespuitingstegnologie te optimiseer. / Department of Plant Pathology, National Research Foundation, THRIP, Deciduous Fruit Producers’ Trust, Winetech, Bayer, BASF, Dow Agrosciences, DuPont, Syngenta, Nexus, Terason, UAP and Wenkem for financial assistance

The trade-off between gametes and soma is central to life-history evolution. Oosorption has been proposed as a mechanism that can mediate this trade-off. When conditions are not conducive to successful reproduction, females are expected to be able to recoup nutrients from unfertilized oocytes and reinvest them into the somatic processes that promote survival and hence future reproduction. Although positive correlations between oocyte degradation and lifespan have been documented in oviparous insects, the adaptive significance of this process in species with more complex reproductive biology has not been explored. Oocyte degradation via apoptosis (programmed cell death) occurs in response to enforced virginity in females of the ovoviviparous cockroach, Nauphoeta cinerea. Observed apoptosis may represent oosorption, however, an alternative but not mutually exclusive argument is that oocyte apoptosis may represent oocyte ageing and clearance in order to maintain reproductive synchrony. The aim of this thesis was to test the hypothesis that the function of oocyte apoptosis is oosorption in N. cinerea. I found that in addition to enforced virginity, starvation induces oocyte apoptosis. However, the life history outcome following one form of stress is the opposite of the other. Hence, the functional role of oocyte apoptosis appears to be different depending on whether apoptosis is induced through starvation or age. Following a period of starvation-induced apoptosis females exhibit the increase in survival and future reproduction predicted by oosorption. Whereas, following a period of age-induced apoptosis females suffer fecundity and longevity cuts. However, age-induced apoptosis does not appear to simply be cellular ageing and clearance. In conjugation with age-induced apoptosis, ovariole number declines whilst the size of surviving oocytes increases. Hence, it appears that resources from sacrificed oocytes are being recycled into the survivors, and that this reinvestment in current reproduction trade-offs with future reproductive capacity. My thesis shows the importance of studying proximal mechanisms alongside more traditional measures of life history, as the relationship between isolated biological levels is not always clear.

Greenhouse and laboratory experiments in 2011 determined that Clonostachys rosea can effectively prevent Botrytis cinerea infection in Vaccinium angustifolium blossoms. In vitro testing demonstrated that C. rosea germination was not significantly affected by the presence of Switch®, but was by either Pristine® or Maestro®. Field experiments completed during the summer of 2010 and 2011 indicated that the dispenser designs tested had no significant effects on Bombus impatiens foraging behaviours, aside from hive-activity. There was also no difference in the quantity of C. rosea applied by each to bees, the distribution of product in the field, or for blossoms exposed to bees from each dispenser to resist infection by B. cinerea. However, B. cinerea prevalence in blossoms from both treatments was significantly different from the control, with infection reduced by 10-20%. Technical issues with dispensers currently appear to be the limiting factor for application within commercial wild blueberry production.

Waxflower (Chamelaucium spp. and hybrids) is the singlemost important Australian export cut-flower. The major problem in waxflower trading is flower abscission after harvest. While several factors are involved, ethylene production resulting from preharvest infection with the fungus Botrytis cinerea is the most important cause. The general objectives of this study were to investigate the biology of Botrytis infecting waxflower flowers and potential elicitation of host defence against this pathogen. Effects of anti-ethylene and S-carvone treatments on Botrytis-induced flower abscission were also evaluated. Infection of flowers by Botrytis was studied on two waxflower cvs. Mullering Brook and My Sweet Sixteen using light and electron microscopy. Conidial germination and protoappressorial formation occurred within 8 h post-inoculation (hpi). Infection of most floral organs, including petals, anthers and filaments, stigma, and hypanthium, was within 24 hpi. Infection cushions on stamen bases were formed at 36 hpi by saprophytic hyphae that originated from anthers. This infection route probably gives rise to the typical tan-coloured Botrytis symptoms that appear to radiate from this part of the flower. Subcuticular hyphae were present at very high density near stamen bases. They evidently resulted at multiple penetrations from single infection cushions. Flower abscission occurred at 72 hpi. At this time, floral tube tissues remained uninfected. This temporal pattern infers the possible transmission of a signal (e.g. ethylene) upon Botrytis infection (6–36 hpi) that intiates a defence response of shedding infected flowers (72 hpi). Susceptibility of waxflower before and after harvest to B. cinerea under various environmental conditions (laboratory, greenhouse, and field) was investigated. Flowers, either on plants or on cut stems showed similar susceptibility to B. cinerea and abscised under cool temperatures (~20 ºC) and high humidity (>95% RH) conditions following infection. Compared to cv. Mullering Brook, cv. My Sweet Sixteen was somewhat more resistant to B. cinerea infection under field conditions. Constitutive and inducible antifungal compounds in waxflower flower tissues were screened in cvs. CWA Pink, Stephan’s Delight, Mullering Brook and My Sweet Sixteen using thin layer chromatography bioassays with isolates of B. cinerea and Alternaria alternata (pathogenic) and Cladosporium cladosporioides (non-pathogenic). Common inhibition zone observed at Rf 0.28–0.38, 0.46–0.56 and 0.67–0.76 contained phenolic compounds. There were at least five (cv. Mullering Brook) and one (cv. My Sweet Sixteen) inducible antifungal phenolic compounds as judged by increases in inhibition area as a result of B. cinerea infection and methyl jasmonate treatment. The total areas of B. cinerea- and MeJA-induced inhibition zones were approximately 2.0- and 2.5-folds greater, respectively, than zones from control flowers. Preharvest sprays of three different known host plant defence elicitors, methyl jasmonate (MeJA), benzothiadiazole (BTH), and silicon (Si), were applied to waxflower cvs. Mullering Brook and My Sweet Sixteen plants. BTH or Si sprays generally had no significant effect on postharvest Botrytis severity on either cultivar. MeJA sprays did not reduce B. cinerea on cv. Mullering Brook. MeJA slightly suppressed B. cinerea on cv. My Sweet Sixteen at 500 and 750 µM. Overall, field applications of these host plant defence elicitor chemicals as spray treatments had little effect on vase life, water uptake and relative fresh weight of the cut sprigs. Moreover, they did not appreciably suppress B. cinerea or associated postharvest floral abscission. The efficacy of combined elicitor treatments and combined pre- and postharvest MeJA treatments were assessed. Preharvest foliar applications of MeJA (1000 µM; 2 or 4 times), MeJA (1000 µM) combined with BTH (150 mg/L), and MeJA combined with Si (1500 mg SiO2/L) generally did not suppress postharvest B. cinerea development and flower abscission from harvested sprigs. A pre- plus post-harvest 1000 µM MeJA spray treatment consistently but only slightly suppressed B. cinerea infection on flowers from both pot- and field-grown plants. Pre- and post-harvest MeJA treatments reduced B. cinerea development, but increased flower abscission. Combined MeJA and anti-ethylene treatments were then screened for potential to suppress B. cinerea while preventing flower abscission. However, the combined MeJA and 1-MCP treatment reduced neither Botrytis disease nor flower abscission on sprigs from pot- and field-grown plants. The combined MeJA and STS treatment reduced disease severity for up to 6 days on sprigs harvested from pot-grown plants but tended to increase Botrytis severity on sprigs from field-grown plants 6 days after inoculation. Antifungal effects of the essential oil S-carvone against B. cinerea germination and mycelial growth were demonstrated in vitro. Inhibition increased with increasing S-carvone concentrations from 0.64 mM to 5.08 mM. However, in planta, S-carvone concentrations in this range did not affect either Botrytis disease levels or flower abscission on cut waxflower flowers.