A indução de resistência envolve a ativação de mecanismos de defesa latentes existentes nas plantas em resposta ao tratamento com agentes bióticos ou abióticos. As plantas apresentam sistema de defesa induzível, com a finalidade de economizar energia. Desse modo, a resistência induzida em condições naturais representará custo apenas na presença do patógeno. Porém, plantas que investem seus recursos para se defenderem na ausência de patógenos arcarão com custos que refletirão na produtividade, uma vez que as alterações metabólicas que levam a resistência apresentam custo adaptativo associado, o qual pode pesar mais do que o benefício. O efeito negativo na produtividade ocorre principalmente onde indutores químicos são utilizados repetidas vezes ou em doses mais elevadas. Assim, em alguns casos podemos estar caminhando sobre uma estreita linha entre custo e benefício, onde a cura pode ser tão ruim quanto a própria doença. Neste trabalho foram conduzidos experimentos objetivando verificar alterações bioquímicas e fisiológicas, correlacionando-as com parâmetros de produção do feijoeiro entre a indução mediada por acibenzolar-S-metil (ASM), indutor químico, e, a mediada por Bacillus cereus, indutor biológico, antes da chegada do patógeno. Para tanto, foram avaliados plantas de feijão, induzidas por esses dois indutores e desafiadas com Xanthomonas axonopodis pv. phaseoli, para constatar a ocorrência do fenômeno da indução de resistência. Na ausência do patógeno, foram avaliados os parâmetros fisiológicos respiração e fotossíntese, determinada a atividade de enzimas envolvidas no processo de defesa como peroxidase, quitinase, β-1,3-glucanase, fenilalanina amônia-liase e polifenoloxidase e a atividade de enzimas envolvidas no catabolismo como proteases, a síntese de compostos do metabolismo secundário como fenóis totais e lignina, a síntese de compostos do metabolismo primário como proteínas e açúcares redutores. Também se avaliou o crescimento das plantas, a produtividade e parâmetros de produção e alguns parâmetros de qualidade dos grãos. Observou-se a ocorrência da indução de resistência em função da aplicação dos dois indutores utilizados, porém para o indutor ASM a indução de resistência estava associada a aumentos na atividade de peroxidase, quitinase, β-1,3-glucanase e proteases, aumento da síntese de lignina e redução no teor de fenóis, aumentos no teor de proteínas solúveis e de açúcares redutores nas folhas, redução do crescimento e da produtividade, aumento do teor de proteína dos grãos e redução do teor de amido nestes. Já o B. cereus apenas ocasionou aumento na atividade de peroxidase de forma atenuada e tendeu a aumentar a atividade de proteases, e reduzir o teor de proteínas nas folhas sem interferir no crescimento ou na produtividade, mas reduziu o teor de proteína dos grãos, mas aumentou o teor de amido nestes. Portanto, o indutor B. cereus , aparentemente alterou muito pouco o metabolismo do feijoeiro, sem interferir na produtividade e melhorando a qualidade da produção, enquanto que o indutor abiótico ASM alterou muito mais seu metabolismo, gerando um custo metabólico e redirecionando os fotoassimilados para investir em defesas, a custo da redução da produtividade. / The induction of systemic resistance involves the activation of latent resistance mechanisms in plants against pathogens in response to the treatment with biotic or abiotic agents. The plants present latent defense system that can be activated with the goal of saving energy. Thus, the induced resistance under natural conditions will represent cost only in the pathogen presence. In this way, plants that invest their resources to defend themselves in the absence of the pathogen will pay off with costs that will reflect in productivity, since the metabolic changes that led to resistance have associated fitness cost which could outweigh the benefit. The negative effects on plant productivity usually occur when chemical inducers are used repeatedly or in higher doses, mainly in the absence of the pathogen. Thus, we can say that in some cases we can be walking on a fine line between cost and benefit, where the cure may be as bad as the disease itself. In this work, experiments were carry out to verify biochemical and physiologic alterations, correlating them with production parameters of bean plants treated with acibenzolar-S-methyl (ASM), chemical inducer, or Bacillus cereus , biological inducer, before the pathogen arrival. Initially, bean plants were evaluated for induced resistance against Xanthomonas axonopodis pv. phaseoli when treated with the two inducers. In the absence of the pathogen, it was evaluated the physiological parameters respiration and photosynthesis and the activity of enzymes involved in the defense as peroxidase, chitinase, β-1,3-glucanase, phenylalanine ammonia-lyase and polifenoloxidase and activity of enzymes involved in the catabolism as proteases, and the synthesis of compounds of the secondary metabolism as phenols and lignin, and the synthesis of compounds of the primary metabolism as proteins and sugars. The growth of the plants was evaluated as well as their productivity and production parameters. Some quality parameters of the grains were also evaluated. The occurrence of the resistance induced in the bean plants against the pathogens was observed for the two inducers. However, for the ASM the resistance induced was associated to increases in peroxidase, chitinase and β-1,3-glucanase activities, increase in the protease activity, increase in lignin synthesis and reduction in the phenol content, increase in soluble proteins and sugar content in the leaves, reduction of the growth and productivity, increasing the protein and reducing the starch content of the grains. The B. cereus only increased peroxidase activity in a lower way and showed a tendency to increase protease activity, and to reduce the protein content in the leaves without interfering in the growth or in the productivity, but it reduced the protein content and it increased the starch content of the grains. Therefore, the biotic inducer, B. cereus altered a minimum the metabolism of the bean plant, without interfering in the productivity and improving the quality of the production, while the abiotic inducer ASM altered its metabolism, generating a metabolic cost and consuming the plant photosyntathes to invest in defenses, causing a reduction in the productivity.

The biological control of plant pests with beneficial microbes has become increasingly important over the last decades. Soil microbes such as fungi and bacteria colonise the roots of plants and promote their growth. Some beneficial microbes can trigger a weak plant defence response that enhances the immune response of the plant at subsequent pathogen attacks and therefore increase the resistance of the plant to other invaders. This mechanism is called “priming”. While biocontrol agents are applied against a variety of plant pests fundamental knowledge of the molecular mechanisms of plant-microbe interactions is still lacking. Especially molecular studies on the role of resistance genes in the interaction of plants with beneficial endophytic fungi are rare. In this study it was investigated how the fungal biocontrol agent Acremonium alternatum affects the development of the clubroot pathogen Plasmodiophora brassicae within the plant host Arabidopsis thaliana. Clubroot is a devastating disease in crop plants such as cabbage and rapeseed and causes abnormal root growth that leads to so called “club roots”. P. brassicae develops within the plant roots and forms resting spores that are very durable and stay infective in soils for up to 2 decades. The control of clubroot by chemical means is difficult and the disease continues to spread on all continents and was also found in Saxony, Germany in recent years. In 2 preliminary studies the co-inoculation of clubroot plants with the fungus A. alternatum resulted in reduced clubroot symptoms in Chinese cabbage and Arabidopsis. It was therefore hypothesised that A. alternatum induces resistance mechanisms in the plant and thus enhances immunity. The focus of this study was to test this hypothesis by carrying out expression analyses on root tissue of infected Arabidopsis plants. For this the plants were inoculated with spores of P. brassicae and A. alternatum before RNA was extracted from the roots, followed by cDNA synthesis and quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR). A microarray of root tissue of infected Arabidopsis plants was carried out to depict the events at the stage of initial root hair infection with the clubroot pathogen. The findings from the gene expression analyses were verified for 2 genes with Arabidopsis mutants that are defective in the respective gene and with 2 overexpressor lines. Clubroot symptoms were assessed by rating the root galls according to their stage of development. The overall plant health was further evaluated by recording the developmental stage of the plants (generative vs. vegetative), stem lengths and plant biomass. In addition, 2 local varieties of the economically important crop plant rapeseed (Brassica napus var. Ability and var. Visby) were investigated with qRT-PCR and by recording the disease parameters just described. A second goal of this study was to assess the general biocontrol potential of the yet relatively unknown endophyte A. alternatum in terms of enzymatic activity and competitive behaviour against other phytopathogenic fungi. The potential of this fungus for the use in integrative pest management was investigated. The results presented here are novel findings for this fungus and have not been studied before. The microarray from Arabidopsis roots revealed that the clubroot pathogen P. brassicae suppresses its recognition by pathogen receptors of the plant and thus prevents the host to induce resistance mechanisms. The fungus A. alternatum boosted the level of the pathogen recognition-related genes BAK1 and FLS2 and thus helped to establish early plant defence responses. PCR analyses confirmed that these early responses led to salicylic acid-dependent resistance in the plants which was maintained for several days as shown by elevated levels of the PATHOGENESIS-RELATED gene PR1. Marker genes for an alternative resistance pathway that is mediated over the plant signals jasmonate and ethylene were not activated in Arabidopsis. The co-inoculation of Arabidopsis plants with the endophyte A. alternatum resulted in a significant reduction of clubroot symptoms by up to 24%. In rapeseed the reduction of disease symptoms was 19% and 28% when the plants were treated with a crude cell wall extract of A. alternatum before inoculation with the clubroot pathogen. PCR analyses from Arabidopsis showed a strong response of pathogen recognition genes to the cell wall extract and spores of the endophytic fungus. In rapeseed all of the investigated pathogen recognition genes were upregulated after the endophyte treatment but not with the clubroot pathogen. Together with the PCR results from the microarray these findings suggest that A. alternatum primes its host plant and enhances the resistance of the plant towards P. brassicae. In addition, the fungus increased biomass, stem lengths and survival rates of clubroot-infected plants. In vitro tests revealed that the endophyte can solubilise phosphate and is not very competitive against other phytopathogenic fungi such as Aspergillus or Fusarium which is likely an effect of the relatively slow growth of the endophyte on agar plates. From this study it can be concluded that i) the fungus Acremonium alternatum induces resistance mechanisms in Arabidopsis and 2 Brassica napus cultivars and facilitates the recognition of the clubroot pathogen Plasmodiophora brassicae; ii) that Arabidopsis and Brassica react differently to this beneficial microbe, a fact that has been observed for Plasmodiophora and other microorganisms as well; iii) living spores are not necessary for clubroot biocontrol in rapeseed as a crude cell wall extract reduces symptoms more efficiently. Overall the endophyte A. alternatum is a very promising candidate for the use in integrative pest management in plant strengtheners or as biocontrol agent. / Die biologische Kontrolle von Pflanzenkrankheiten gewinnt zunehmend an Bedeutung. Bodenbewohnende Mikroben wie Pilze oder Bakterien kolonisieren die Wurzeln von Pflanzen und fördern deren Wachstum. Einige dieser förderlichen Mikroben aktivieren eine schwache Abwehrreaktion in der Pflanze die sich verstärkt bei einer weiteren Infektion mit einem Krankheitserreger. Dieser Mechanismus, den man “Priming” nennt, führt zu einer verbesserten Resistenz der Pflanze gegenüber Pflanzenpathogenen. Obwohl natürliche Schädlingsbekämpfer bereits gegen eine Vielzahl an Krankheiten eingesetzt werden, weiss man über grundsätzliche molekulare Mechanismen dieser Pflanzen-Mikroben-Interaktionen nur wenig. Besonders die Rolle von Resistenzgenen ist bisher wenig erforscht, welche bei der Beziehung zwischen Pilzen und Pflanzen eine Rolle spielen. In der hier vorliegenden Arbeit wurde untersucht, wie der endophytische Pilz Acremonium alternatum die Entwicklung des Krankheitserregers Plasmodiophora brassicae in der Pflanze Arabidopsis thaliana beeinflusst. Die Kohlhernie, ausgelöst von P. brassicae, ist eine verheerende Krankheit die u. a. bei Kohl und Raps auftritt und Wurzelgallen, so genannte “Hernien”, hervorruft. Der Krankheitserreger entwickelt sich im Wurzelsystem der Pflanze und bildet Dauersporen, die bis zu 20 Jahre lang im Boden infektiös überdauern können. Ein Eindämmen der Krankheit mit Pflanzenschutzmitteln ist durch den komplexen Lebenslauf des Erregers sehr schwierig, das führte zu einer weltweiten Verbreitung der Kohlhernie. Auch in Sachsen wurden in den letzten Jahren Fälle von Kohlhernie gemeldet. Wie 2 Studien zeigen, führt die Ko-Inokulation von Kohlhernie-erkrankten Pflanzen mit A. alternatum zu einer Verringerung der Symptome in Chinakohl und Arabidopsis. Es wurde daher die Hypothese aufgestellt, dass der Pilz Resistenzmechanismen in der Pflanze anschaltet und damit ihre Immunität erhöht. Um diese Hypothese zu testen, wurden in der hier vorliegenden Studie Genexpressionsanalysen an infizierten Arabidopsiswurzeln durchgeführt. Dafür wurden die Pflanzen zunächst mit Sporen des Kohlhernieerregers und des Pilzes inokuliert, es wurde RNA aus den Wurzeln extrahiert, in cDNA umgeschrieben und diese mittels quantitativer Reverse-Transkriptase-Polymerasenkettenreaktion (RT-qPCR) untersucht. Ein Microarray von Wurzeln infizierter Pflanzen wurde durchgeführt um die Ereignisse abzubilden, die sich zeitnah nach der Infektion in den Wurzeln abspielen. Die Ergebnisse der Genexpressionsanalysen wurden dann an Arabidopsismutanten, die einen Gendefekt im jeweiligen Gen haben, und an Überexprimierer-Pflanzen verifiziert. Kohlherniesymptome an Pflanzen wurden durch eine Kategorisierung der Schadsymptome erfasst. Die allgemeine Pflanzengesundheit sowie der Entwicklungsstand der Pflanze, Stengellängen und das Frischgewicht wurden bestimmt. Zusätzlich wurden 2 Rapssorten, die in Sachsen angebaut werden, untersucht im Hinblick auf die Krankheitsenwicklung und die Reguation von Abwehrgenen. Ein weiteres Ziel dieser Arbeit war es das Biokontrollpotential des bisher schlecht untersuchten Pilzes A. alternatum zu bestimmen. Dazu wurde in vitro die Enzymaktivität des Pilzes getestet sowie seine Konkurrenzfähigkeit gegenüber anderen pflanzenpathogenen Pilzen. Das Potential des Pilzes für die Anwendung im integrierten Pflanzenschutz wurde getestet. Die hier präsentieren Ergebnisse stellen neue Erkenntnisse dar, die für diesen Pilz noch nie untersucht wurden. Der Microarray von Arabidopsiswurzeln zeigte, dass der Kohlhernieerregers die Erkennung durch die Pflanze verhindert und damit Abwehrmechanismen verhindert. Der Pilz A. alternatum förderte die Aktivität der pflanzlichen Erkennungsrezeptoren FLS2 und BAK1 und setzte damit die Erkennung von P. brassicae in Gang. PCR-Analysen ergaben, dass diese früh induzierten Abwehrmechanismen zu einer systemischen Resistenz in der Pflanze führte durch die Aktivierung des Pathogenese-relevanten Gens PR1. Genmarker, die die Aktivität eines alternativen, von Jasmonat und Ethylen vermittelten Abwehrweges anzeigen, waren nicht ativiert. Die Ko-Inokulation von Arabidopsis mit dem Endophyten führte zu einer signifikanten Reduktion der Krankheitssymptome um 24%. In Raps betrug die Reduktion 19% und 24% wenn die Pflanzen vor der Kohlhernie-Infektion mit einem Zellwandextrakt des Pilzes behandelt wurden. Mittels PCR konnte gezeigt werden, dass Gene für das Erkennen von Pathogenen in der Wurzel von Arabidopsis auf den Zellwandextrakt und Sporen des Pilzes reagieren. In Raps wurden alle der untersuchten Erkennungsgene aufreguliert nach der Infektion mit A. alternatum, nicht jedoch bei der Infektion mit P. brassicae. Zusammenfassend lässt sich sagen, dass der endophytische Pilz A. alternatum die Wirtspflanze auf eine folgende Infektion vorbereitet (Priming) und systemische Abwehr-mechanismen in der Pflanze induziert, wenn diese mit Kohlhernie infiziert ist. Außerdem treibt der Pilz das Sprosswachstum voran, erhöht die Biomasse und fördert das Überleben von Kohlhernie-infizierten Pflanzen. In vitro-Tests ergaben, dass der Endophyt Kalziumphosphat löslich machen kann und wenig kompetitiv gegenüber Pflanzenpathogenen wie Aspergillus oder Fusarium ist. Dies ist vermutlich mit dem langsameren Wachstum des Endophyten im Gegensatz zu den anderen Pilzen zu erklären. Aus den Ergebnissen dieser Arbeit lassen sich folgende Schlüsse ziehen: i) der endophytische Pilz Acremonium alternatum induziert Resistenzmechanismen in Arabidopsis und Raps und und fördert die Erkennung des Kohlhernieerregers Plasmodiophora brassicae; ii) Arabidopsis und Raps reagieren unterschiedlich auf diesen förderlichen Pilz, ein solcher Unterschied wurde bereits für Plasmodiophora und andere Mikroben beschrieben; iii) lebende Sporen des Pilzes sind nicht notwendig um Krankheitssymptome der Kohlhernie in Raps zu verringern, ein Zellwandextrakt von A. alternatum ist dafür besser geeignet. Ganz allgemein lässt sich sagen, dass der endophytische Pilz Acremonium alternatum ein sehr vielversprechender Kandidat ist für den Einsatz im integrierten Pflanzenschutz in Pflanzenstärkungsmitteln oder als Biokontrollorganismus.

The use of biological control agents (BCAs) in the past has shown limited success as its application has often been done incorrectly, and in addition, management practices are rarely altered to incorporate BCAs. Criteria for the correct application of BCAs have been devised as part of the research, and companies selling these products may use the said criteria. Such application will ensure the correct BCAs are used and, more specifically, used under the correct conditions. The powdery mildew (PM) fungus is often seen to develop resistance to synthetic fungicides and, therefore, alternative control measures are required. BCAs as an alternative pose less risk to the environment, workers and the consumer. A pre-trial has been conducted with a range of BCAs to see if they can control powdery mildew (PM) in a greenhouse environment on hydroponically grown cucumber (Cucumis sativus L.) plants using the variety Baccara that has only a moderate tolerance to PM. The BCAs have been compared to the control (synthetic fungicide: Bravo). Comparative work includes Coyier's model, which has been modified and adapted for these trials to determine the percentage of leaf area covered by the PM infection. Furthermore, the number of fruit harvested per treatment, kilogram yield, total mass of yield and average fruit mass is also used to determine the efficacy of the BCAs as these factors have economic significance to commercial growers. The pre-trial showed promise until the fertigation computer failed, resulting in a nutrient shortage and imbalance, confirming that BCAs alone cannot control PM. Synthetic fungicides were applied until control of PM and plant nutrition was regained. BCAs were re-introduced and used until the end of crop production. The confirmation from the pre-trial has led to the inclusion of silicon in conjunction with the BCAs in the two subsequent trials (Trials 1 & 2). Silicon was applied with the BCAs as a foliar spray on a weekly basis. In trials 1 and 2, the cucumber variety, Palladium, with a high genetic tolerance to PM is used, as this variety is suited to form part of the holistic approach used for trials 1 and 2. Trial 1 showed that treatment A, containing Streptomyces griseovirdis and Streptomyces aureofaciens, had the highest yield. Both of these are bacterial BCAs and demonstrated their adaptability to varied climatic conditions, notably when low humidity was experienced. In treatment B, Trichoderma harzianum strains, Rifai and Uppington, show the slowest rate of PM development. In trials 1 and 2, the best actual PM control was obtained by two fungal based BCAs (Trial 1, treatment C was Ampelomyces quisqualis) and (Trial 2, treatment B was Trichoderma harzianum strains, Rifai and Uppington), showing that fungal BCAs have a place for this application, but the growth-enhancing properties of bacterial based BCAs make economic sense and would make them attractive to growers. Treatment A (Streptomyces spp.) had the most number of fruit for the entire growing period and the best overall yield (kg yield) again. Two of the BCA / silicon treatments have marginally better PM control compared to that of the control (E) treatment, although not statistically significant. Treatment E (control) has the highest average fruit mass in this instance but does not have the highest yield (kg yield) when compared to treatments A and B, possibly due to the growth-enhancing properties of most of these BCAs. Therefore, most of these BCA treatments give fairly inconsistent results that vary possibly according to season, humidity and temperature, making it difficult to predict their efficacy. Using combinations or weekly alternations of these BCAs with extremes of climatic adaptation will probably be the most reliable method of obtaining consistent results. Bacterial BCAs are shown to have lower humidity requirements and produce the most consistent results in terms of fruit number, yield and fruit mass and a combination of bacterial and fungal based BCAs would possibly be the best as this would control PM and yet still have the growth enhancing properties from the bacterial based BCAs. From the research, it can be said that some BCAs in trials 1 and 2 produce results similar to that of the control in terms of percentage leaf area covered by PM and some are shown to have improved yields. Results produced from certain BCA treatments are thus equal to the control; yet provide an environmentally friendly alternative to synthetic fungicides. Silicon is listed as a beneficial element rather than an essential element; however, literature claims it to be highly effective in treating PM in cucurbits. Results from trials 1 and 2 show that control of PM is possible in most cases, when a holistic approach is used. This approach includes a cucumber variety with a high PM tolerance, optimum nutrition, cultural practices and silicon in combination with the BCAs. A complete change of management practices is necessary to implement such a BCA program. / Agriculture, Animal Health & Human Ecology / M. Tech. (Nature Conservation)

Die arbuskuläre Mykorrhiza (AM) ist eine mutualistische Symbiose, die die Phosphataufnahme und Pathogenresistenz von Pflanzen verbessern kann. In der vorliegenden Doktorarbeit wurde die Rolle der Protonen-pumpenden ATPase MtHA1 für die AM Symbiose in Medicago truncatula untersucht. In MtHA1 Mutanten konnten AM Pilze nur noch verkürzte Arbuskel ohne typische Verzweigungen ausbilden. Dies zeigte sich auch in Expressionsmustern von Genen, die für Proteine in verschiedenen Bereichen der periarbuskulären Membran kodieren. Außerdem waren AM Pilzbesiedelung, die verbesserte Nährstoffaufnahme und die Wachstumsförderung in MtHA1 mutierten Pflanzen reduziert. Die Mykorrhiza-induzierte Resistenz (MIR) wurde näher in M. truncatula Pflanzen untersucht, die von Aphanomyces euteiches infiziert waren, dem Erreger einer Wurzelfäule in Leguminosen. In einem geteilten Wurzelsystem, das eine hohe Expression von Verteidigungsgenen aufwies, unterdrückte ein AM Pilz diese Expression und erhöhte in Folge die Empfindlichkeit für das Pathogen. In Wurzeln von Topfkulturen dagegen konnte eine typische MIR beobachtet werden, die wahrscheinlich auf erhöhter Aktivität der Jasmonat/Ethylen-regulierten Verteidigungsantwort beruht, verursacht durch eine Unterdrückung der Salizylsäuresynthese. Im Ergebnis zeigt diese Arbeit die bedeutende Rolle des Gens MtHA1 für die Bildung und Funktion der arbuskelhaltigen Zellen. Die Mutation des Gens führt zur verminderten Arbuskelverzweigung, reduzierter Phosphataufnahme und Wachstumsförderung in der Mykorrhiza und schließlich zu einer geringeren Gesamtbesiedelung durch den AM Pilz. Genexpressionsanalysen weisen darauf hin, dass unterschiedliche Mechanismen den lokalen und systemischen Wechselwirkungen zwischen AM Pilzen und Pathogenen in der Wurzel zu Grunde liegen. Verschieden physiologische Zustände von geteilten Wurzelsystemen und Wurzeln in Topfkulturen erschweren allerdings einen direkten Vergleich der beiden experimentellen Ansätze. / Arbuscular mycorrhiza (AM) is a wide spread mutualistic symbiosis, which can improve phosphate acquisition and pathogen resistance of plants. In the current Ph.D. thesis the role of a proton pumping ATPase (MtHA1) for the AM symbiosis in Medicago truncatula was investigated. In MtHA1 mutant plants, different AM fungi only developed truncated arbuscules without forming typical hyphal branches, and this phenotype was mirrored by expression patterns of genes for proteins located in different areas of the periarbuscular membrane. AM fungal colonization, improved phosphate uptake and plant growth promotion were reduced in MtHA1 mutant plants. Mycorrhiza-induced resistance (MIR) and the nodule symbiosis were, however, not affected. MIR was further analyzed in the M. truncatula infected with Aphanomyces euteiches which causes a root-rot disease in legumes. In a split root system showing high levels of defense-gene expression, colonization of an AM fungus reduced this expression and in consequence increased susceptibility of the roots for the pathogen. In roots of pot cultures, however, a typical MIR was observed and could be based on the higher activity of jasmonate/ethylene-regulated defense responses due to suppression of salicylic acid biosynthesis. In conclusion, this work shows that the gene MtHA1 encoding a proton pumping-ATPase plays a critical role in the formation and function of arbuscule-containing cells. Expression of the mutated gene results in reduced formation of arbuscule branches. This in turn negatively influences mycorrhizal phosphate uptake, plant growth promotion and overall mycorrhizal colonization of the roots. Gene expression analyses indicate that different mechanisms underlay local and systemic interactions between the mycorrhizal fungus and the root pathogen. The different physiological stages of pot culture and split root system make a comparison of the two experimental approaches, however, difficult.

The use of biological control agents (BCAs) in the past has shown limited success as its application has often been done incorrectly, and in addition, management practices are rarely altered to incorporate BCAs. Criteria for the correct application of BCAs have been devised as part of the research, and companies selling these products may use the said criteria. Such application will ensure the correct BCAs are used and, more specifically, used under the correct conditions. The powdery mildew (PM) fungus is often seen to develop resistance to synthetic fungicides and, therefore, alternative control measures are required. BCAs as an alternative pose less risk to the environment, workers and the consumer. A pre-trial has been conducted with a range of BCAs to see if they can control powdery mildew (PM) in a greenhouse environment on hydroponically grown cucumber (Cucumis sativus L.) plants using the variety Baccara that has only a moderate tolerance to PM. The BCAs have been compared to the control (synthetic fungicide: Bravo). Comparative work includes Coyier's model, which has been modified and adapted for these trials to determine the percentage of leaf area covered by the PM infection. Furthermore, the number of fruit harvested per treatment, kilogram yield, total mass of yield and average fruit mass is also used to determine the efficacy of the BCAs as these factors have economic significance to commercial growers. The pre-trial showed promise until the fertigation computer failed, resulting in a nutrient shortage and imbalance, confirming that BCAs alone cannot control PM. Synthetic fungicides were applied until control of PM and plant nutrition was regained. BCAs were re-introduced and used until the end of crop production. The confirmation from the pre-trial has led to the inclusion of silicon in conjunction with the BCAs in the two subsequent trials (Trials 1 & 2). Silicon was applied with the BCAs as a foliar spray on a weekly basis. In trials 1 and 2, the cucumber variety, Palladium, with a high genetic tolerance to PM is used, as this variety is suited to form part of the holistic approach used for trials 1 and 2. Trial 1 showed that treatment A, containing Streptomyces griseovirdis and Streptomyces aureofaciens, had the highest yield. Both of these are bacterial BCAs and demonstrated their adaptability to varied climatic conditions, notably when low humidity was experienced. In treatment B, Trichoderma harzianum strains, Rifai and Uppington, show the slowest rate of PM development. In trials 1 and 2, the best actual PM control was obtained by two fungal based BCAs (Trial 1, treatment C was Ampelomyces quisqualis) and (Trial 2, treatment B was Trichoderma harzianum strains, Rifai and Uppington), showing that fungal BCAs have a place for this application, but the growth-enhancing properties of bacterial based BCAs make economic sense and would make them attractive to growers. Treatment A (Streptomyces spp.) had the most number of fruit for the entire growing period and the best overall yield (kg yield) again. Two of the BCA / silicon treatments have marginally better PM control compared to that of the control (E) treatment, although not statistically significant. Treatment E (control) has the highest average fruit mass in this instance but does not have the highest yield (kg yield) when compared to treatments A and B, possibly due to the growth-enhancing properties of most of these BCAs. Therefore, most of these BCA treatments give fairly inconsistent results that vary possibly according to season, humidity and temperature, making it difficult to predict their efficacy. Using combinations or weekly alternations of these BCAs with extremes of climatic adaptation will probably be the most reliable method of obtaining consistent results. Bacterial BCAs are shown to have lower humidity requirements and produce the most consistent results in terms of fruit number, yield and fruit mass and a combination of bacterial and fungal based BCAs would possibly be the best as this would control PM and yet still have the growth enhancing properties from the bacterial based BCAs. From the research, it can be said that some BCAs in trials 1 and 2 produce results similar to that of the control in terms of percentage leaf area covered by PM and some are shown to have improved yields. Results produced from certain BCA treatments are thus equal to the control; yet provide an environmentally friendly alternative to synthetic fungicides. Silicon is listed as a beneficial element rather than an essential element; however, literature claims it to be highly effective in treating PM in cucurbits. Results from trials 1 and 2 show that control of PM is possible in most cases, when a holistic approach is used. This approach includes a cucumber variety with a high PM tolerance, optimum nutrition, cultural practices and silicon in combination with the BCAs. A complete change of management practices is necessary to implement such a BCA program. / Agriculture, Animal Health and Human Ecology / M. Tech. (Nature Conservation)