Foi feita uma revisão bibliográfia sobre a aplicação da amostragem seqüencial em manejos de pragas. A metodologia para o desenvolvimento de planos de amostragem seqüencial, foi baseada na teoria de Wald. Verificou-se que a distribuição binomial ajustou-se aos dados de presença-ausência da cochonilha-branca Aulacaspis tubercularis na cultura da manga, obtidos em duas propriedades localizadas na região de Jaboticabal, interior do estado de São Paulo. Seis planos de amostragem seqüencial foram elaborados para o monitoramento dessa praga, três com níveis de erros iguais a 0; 20 e outros três com níveis de erros iguais a 0; 10. Dentre esses, foram elaborados: dois planos para a amostragem em ramos durante o período vegetativo, dois para a amostragem em ramos durante o período de frutificação e outros dois planos para a amostragem em frutos. Nos planos de amostragem seqüencial para ramos no período vegetativo foram utilizados níveis de segurança iguais a 0; 35 e níveis de dano econômico iguais a 0; 50. Já no período de frutificação, houve um rigor maior quanto ao uso desses níveis, devido à presença de frutos. Foram utilizados níveis de segurança iguais a 0; 20 para ramos e 0; 05 para frutos, e níveis de dano econômico iguais a 0; 30 e 0; 15 para ramos e frutos, respectivamente. Foram construídas tabelas para o processo de amostragem em campo, facilitando assim, o trabalho do amostrador. Com o uso dessas tabelas o produtor poderá decidir rapidamente se há, ou não, a necessidade de se realizar o controle dessa praga. / A bibliographical revision on the application of the sequential sampling in management of pests was made. The methodology for the development of plans of sequential sampling, was based on the theory of Wald. It was verified that the binomial distribution adjusted to data of presence-absence of the mango scale Aulacaspis tubercularis on mango crop, gotten in two properties located at the region of Jaboticabal, interior of the state of São Paulo. Six plans of sequential sampling was elaborated for the monitoring of this pest, three with errors levels equal to 0; 20 and others three with errors levels equals to 0; 10. Amongst these, they was elaborated: two plans for the sampling in branches during the vegetative period, two for the sampling in branches during the period of fructification and others two plans for the sampling in fruits. In the plans of sequential sampling for branches in the vegetative period security level equal to 0; 35 and economic damage level equal to 0; 50 had been used. Already in longer period of fructification, had a bigger severity how much to the use of these levels, due to presence of fruits. Security levels equals to 0; 20 for branches and 0; 05 for fruits, and economic damage levels equals to 0; 30 and 0; 15 for branches and fruits, respectively, had been used. Tables for the sampling process in field, thus facilitating, the sampler's work was constructed. With the use of these tables the producer will be able to decide quickly has itself, or not, the necessity of if carrying through the control from this pest.

Foi feita uma revisão bibliográfia sobre a aplicação da amostragem seqüencial em manejos de pragas. A metodologia para o desenvolvimento de planos de amostragem seqüencial, foi baseada na teoria de Wald. Verificou-se que a distribuição binomial ajustou-se aos dados de presença-ausência da cochonilha-branca Aulacaspis tubercularis na cultura da manga, obtidos em duas propriedades localizadas na região de Jaboticabal, interior do estado de São Paulo. Seis planos de amostragem seqüencial foram elaborados para o monitoramento dessa praga, três com níveis de erros iguais a 0; 20 e outros três com níveis de erros iguais a 0; 10. Dentre esses, foram elaborados: dois planos para a amostragem em ramos durante o período vegetativo, dois para a amostragem em ramos durante o período de frutificação e outros dois planos para a amostragem em frutos. Nos planos de amostragem seqüencial para ramos no período vegetativo foram utilizados níveis de segurança iguais a 0; 35 e níveis de dano econômico iguais a 0; 50. Já no período de frutificação, houve um rigor maior quanto ao uso desses níveis, devido à presença de frutos. Foram utilizados níveis de segurança iguais a 0; 20 para ramos e 0; 05 para frutos, e níveis de dano econômico iguais a 0; 30 e 0; 15 para ramos e frutos, respectivamente. Foram construídas tabelas para o processo de amostragem em campo, facilitando assim, o trabalho do amostrador. Com o uso dessas tabelas o produtor poderá decidir rapidamente se há, ou não, a necessidade de se realizar o controle dessa praga. / A bibliographical revision on the application of the sequential sampling in management of pests was made. The methodology for the development of plans of sequential sampling, was based on the theory of Wald. It was verified that the binomial distribution adjusted to data of presence-absence of the mango scale Aulacaspis tubercularis on mango crop, gotten in two properties located at the region of Jaboticabal, interior of the state of São Paulo. Six plans of sequential sampling was elaborated for the monitoring of this pest, three with errors levels equal to 0; 20 and others three with errors levels equals to 0; 10. Amongst these, they was elaborated: two plans for the sampling in branches during the vegetative period, two for the sampling in branches during the period of fructification and others two plans for the sampling in fruits. In the plans of sequential sampling for branches in the vegetative period security level equal to 0; 35 and economic damage level equal to 0; 50 had been used. Already in longer period of fructification, had a bigger severity how much to the use of these levels, due to presence of fruits. Security levels equals to 0; 20 for branches and 0; 05 for fruits, and economic damage levels equals to 0; 30 and 0; 15 for branches and fruits, respectively, had been used. Tables for the sampling process in field, thus facilitating, the sampler's work was constructed. With the use of these tables the producer will be able to decide quickly has itself, or not, the necessity of if carrying through the control from this pest.

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.