Dissertations / Theses on the topic 'Plant diagnosis'

Operators can become confused while diagnosing faults in process plant while in operation. This may prevent remedial actions being taken before hazardous consequences can occur. The work in this thesis proposes a method to aid plant operators in systematically finding the causes of any fault in the process plant. A computer aided fault diagnosis package has been developed for use on the widely available IBM PC compatible microcomputer. The program displays a coloured diagram of a fault tree on the VDU of the microcomputer, so that the operator can see the link between the fault and its causes. The consequences of the fault and the causes of the fault are also shown to provide a warning of what may happen if the fault is not remedied. The cause and effect data needed by the package are obtained from a hazard and operability (HAZOP) study on the process plant. The result of the HAZOP study is recorded as cause and symptom equations which are translated into a data structure and stored in the computer as a file for the package to access. Probability values are assigned to the events that constitute the basic causes of any deviation. From these probability values, the a priori probabilities of occurrence of other events are evaluated. A top-down recursive algorithm, called TDRA, for evaluating the probability of every event in a fault tree has been developed. From the a priori probabilities, the conditional probabilities of the causes of the fault are then evaluated using Bayes' conditional probability theorem. The posteriori probability values could then be used by the operators to check in an orderly manner the cause of the fault. The package has been tested using the results of a HAZOP study on a pilot distillation plant. The results from the test show how easy it is to trace the chain of events that leads to the primary cause of a fault. This method could be applied in a real process environment.

The development of computer based tools, to assist process plant operators in their task of fault/alarm diagnosis, has received much attention over the last twenty five years. More recently, with the emergence of Artificial Intelligence (AI) technology, the research activity in this subject area has heightened. As a result, there are a great variety of fault diagnosis methodologies, using many different approaches to represent the fault propagation behaviour of process plant. These range in complexity from steady state quantitative models to more abstract definitions of the relationships between process alarms. Unfortunately, very few of the techniques have been tried and tested on process plant and even fewer have been judged to be commercial successes. One of the outstanding problems still remains the time and effort required to understand and model the fault propagation behaviour of each considered process. This thesis describes the development of an experimental knowledge based system (KBS) to diagnose process plant faults, as indicated by process variable alarms. In an attempt to minimise the modelling effort, the KBS has been designed to infer diagnoses using a fault tree representation of the process behaviour, generated using an existing fault tree synthesis package (FAULTFINDER). The process is described to FAULTFINDER as a configuration of unit models, derived from a standard model library or by tailoring existing models. The resultant alarm diagnosis methodology appears to work well for hard (non-rectifying) faults, but is likely to be less robust when attempting to diagnose intermittent faults and transient behaviour. The synthesised fault trees were found to contain the bulk of the information required for the diagnostic task, however, this needed to be augmented with extra information in certain circumstances.

Eutypa dieback of grapevines, caused by Eutypa lata, is a major cause of reduced longevity in vineyards worldwide. The fungus grows in the woody tissue of infected vines, producing translocatable toxins that cause foliar symptoms of the disease. By the time foliar symptoms are evident the pathogen may have become well established in the vine. One aim of this study was to develop DNA markers to allow rapid reliable identification of E. lata and to detect the pathogen in infected wood. The second aim was to analyse secondary metabolite production by E. lata in order to gain information on the compounds responsible for the foliar symptoms of the disease and to identify metabolites which could be used as markers to detect the early stages of the disease prior to the expression of foliar symptoms. In addition, genetic variation of the pathogen was assessed using RFLP and RAPD analysis. Two techniques were used to develop DNA markers; first, SCAR markers derived from RAPD fragments were developed and, second, an E. lata genomic DNA library was constructed, from which DNA fragments specific to E. lata were identified. These markers were used in either PCR- or Southern hybridisation-based assays to detect the pathogen in infected wood. PCR-based detection of the pathogen in infected wood was prone to inhibition by phenolic compounds, however, Southern hybridisation techniques were capable of detecting E. lata in wood. Genetic variation among 38 isolates of E. lata was assessed using six randomly selected clones from the genomic DNA library. A subset of 11 isolates was subjected to RAPD analysis using 10 random primers. Considerable genetic diversity, in terms of RFLP and RAPD profiles, was observed among isolates. There was no apparent correlation between grouping of isolates following neighbour joining analysis and either host species or geographic origin of isolates. The RAPD and RFLP profiles of two isolates differed significantly from the majority of the other isolates. These isolates, which were morphologically similar to all other isolates, were subsequently found not to be E. lata. Secondary metabolite production of 11 isolates was analysed by HPLC following growth on a range of media. A wider range of secondary metabolites was detected in E. lata than has previously been reported. Two of the secondary metabolites, eutypine and an unidentified compound with a retention time of 19.6 min, were produced by eight of nine isolates of E. lata. Neither of the non-E. lata isolates produced these compounds. It was concluded that the remaining isolate of E. lata may have lost the ability to produce these compounds following storage. Whilst a wider range of isolates needs to be screened before a candidate marker can be selected, these results suggest that certain compounds are present in the majority of E. lata isolates and, hence, may prove suitable markers for the detection of the pathogen prior to the expression of foliar symptoms. The molecular probes developed in this study will allow the rapid and reliable identification and detection of E. lata in grapevine cane or wood. These probes also have the potential to be used as a research tool to gather information on the epidemiology of the disease and to assess the efficacy of potential control agents against E. lata. Suitable control measures could then be applied to vines which have been shown by the use of chemical markers to have latent infection. Used in combination, therefore, the DNA and biochemical markers could facilitate improved management of eutypa dieback.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2003.

Knowledge-Based Systems (KBSs) represent a relatively new programming approach and methodology that has evolved and is still evolving as an important sub-area of Artificial Intelligence (AI) research. The most prevalent application of KBSs, which emerged in recent times, has been various types of diagnosis and troubleshooting. KBS has an important role to play, particularly in fault diagnosis of process plants, which involve lot of challenges starting from commonly occurring malfunctions to rarely occurring emergency situations. The KBS approach is promising for this domain as it captures efficient problem-solving of experts, guides the human operator in rapid fault detection, explains the line of reasoning to the human operator, and supports modification and refinement of the process knowledge as experience is gained. However, most of the current KBSs in process plants are built on expert knowledge compiled in the form of production rules. These systems lack flexibility due to their process-specific nature and are unreliable when faced with unanticipated faults. Although attempts have been made to integrate knowledge based on experience and 'deep' process knowledge to overcome this lack of flexibility, very little work has been reported to make the diagnostic system flexible and usable for various plant configurations. In this thesis, we propose a hybrid knowledge framework which includes both process-specific and process-common knowledge of the structure and behavior of the domain, and a process-independent diagnostic mechanism based on causal and qualitative reasoning. This framework is flexible and allows a unified design methodology for fault diagnosis of process plants. The process-specific knowledge includes experiential knowledge about commonly occurring faults, behavioral knowledge about causal interactions among process-dependent variables, and structural knowledge about components' description and connectivity. The process-common knowledge comprises template models of various types of components commonly present in any process plant, constraints and confluences based on mass and energy balances between parameters across components. The process behavioral knowledge is qualitatively represented in the form of Signed Digraph (SDG), which is converted into a set of rules (SDGrules), added with control premises for the purpose of diagnostic reasoning. Frame-objects are used to represent the structural knowledge, while rules are used to capture experiential knowledge about common faults. An interface program viz., Knowledge Acquisition Interface (KAI) aids acquisition and conversion of (i) behavioral knowledge into a set of SDG-rules and (ii) structural knowledge and experience-based heuristic rules into a set of facts. The Diagnostic Mechanism is based on a steady state model of the process and is composed of three consecutive phases for locating a fault. The first phase is Malfunction Block Identification (MBT), which locates a malfunctioning subsystem or Malfunction Block (MB) that is responsible for causing the process malfunction. It is based on alarm data whenever violation of process parameters occurs. Once the suspected MB is identified, the second phase viz., Malfunction Parameter Identification (MPI) is invoked t o locate parameters which indicate the prime cause(s) of the fault in that MB. This is achieved by correlating various instrumentation data through causal relationships described by the SDG-rules of that MB. Finally, Malfunctioning Component Identification (MCI) phase is invoked to locate the malfunctioning component. MCI phase uses the malfunction parameter (s) obtained from previous phase and experiential and structural knowledge of that MA for this purpose. The Diagnostic Mechanism is process-independent and, therefore, is capable of adapting to various types of plant configurations. Since, the Knowledge Base and the Diagnostic Mechanism are separate, modification of either of them can be done independently. The Diagnostic Mechanism is potentially capable of investigating symptoms that have multiple or unrelated origins. It also provides explanation facility for justifying the line of diagnostic reasoning to the human operator.

Water desalination is one approach to force water scarcity. One of the processes used for desalination is reverse osmosis. Like other systems, a reverse osmosis plant is susceptible to faults. A fault can lead to a loss of efficiency, or if the fault is severe to a total breakdown. Appropriate measures can minimize the impact of faults, but this requires in time fault detection. The following thesis shows a proposal for an online fault diagnosis system of a reverse osmosis plant. For the model-based approach, a mathematical model of a reverse osmosis plant has been developed. The model contains a new approach for modeling the interaction between the high-pressure pump, the brine valve, and the membrane module. Furthermore, six faults considered for fault diagnosis have been modeled. Two of the faults are plant faults: The leakage of the feed stream and membrane fouling. The other four faults are sensor or actuator malfunctions. The fault diagnosis system is developed via structural analysis, a graph-based approach to determine a mathematical model’s overdetermined systems of equations. With the structural analysis, 73 fault-driven minimal structurally overdetermined (FMSO) sets have been determined. The results show that all six faults are detectable. However, two faults are not isolable. Five of the FMSO sets have been chosen to deduce the residuals used for online fault detection and isolation. The simulations demonstrate that the calculated residuals are appropriate to detect and isolate the faults. If one assumes that only the considered faults occur, it is possible to determine some faults’ magnitude.

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Tese (Doutoramento)
IPEN/T
The University of Tennessee

Detection of the shaft crack in a rotating machine is one of the most challenging problems in equipment predictive maintenance. In the available literature, various crack detection methods have been applied to study the dynamic behaviour of a cracked shaft. This study sought to attempt a vibration-based method. Elastic shafts with three different types of transverse cracks, including experimentally-induced fatigue crack, welded shaft crack, and wire-cut crack, were fabricated, and used to analyse the bending stiffness and frequency response in the vertical direction. The results from the cracked shafts were compared with that of an intact shaft. Bending stiffness of different shafts was measured as a function of rotation angle of the shafts. Among the three different crack types, the bending stiffness of the fatigue crack shaft showed a typical breathing behaviour, which was consistent with the previous theoretical results. The welded shaft crack also demonstrated opening and closing characteristics, but the stiffness was found to be much lower compared with that of a fatigue cracked shaft. As for the wire-cut crack, no breathing mechanism was observed for any rotational angle, due to the big width of the gap. Therefore, it is concluded that the fatigue induced crack is the most accurate method to evaluate the vibration characteristics of cracked shafts. Our results also indicated that existing switching model and harmonic models cannot describe the periodic stiffness of a transverse shaft crack accurately. Modal analysis was carried out on the intact shaft, as well as the three types of cracked shafts. Frequency responses in the X-axis direction were obtained. The correlation between the bending stiffness and the resonant frequency was examined, and it was experimentally proved that the decrease in resonant frequency was almost proportional to the reduction in the stiffness. Also, the amplitude of vibration response was found to be amplified by the crack element. The cause and implications of these results were analysed, and they are expected to deepen our understanding of crack diagnosis using vibration method.

Environmental Diagnosis of Process Plants by Life Cycle Techniques
Haydée A. Yrigoyen González
El objetivo de la investigación es desarrollar una herramienta que relacione aspectos de
simulación, evaluación ambiental y análisis de sensibilidad. Para lo cual se estableció una
metodología que consta de cinco niveles: Simulación de proceso, Inventario, evaluación de
impactos ambientales, análisis económico y análisis de sensibilidad.
La metodología describe las variables relacionadas con el proceso, así como los
impactos asociados a cada una de sus etapas y la viabilidad económica del proceso, e
identifica las etapas de proceso con el mayor impacto ambiental (mediante el análisis de
sensibilidad).
Para la simulación de procesos se empleó el simulador ASPEN Hysys®. El inventario,
la evaluación de impactos y el análisis económico se lleva a cabo en hojas de cálculo de forma
automática.
La obtención del inventario de efectos ambientales y la evaluación de los
correspondientes impactos se realizan siguiendo la metodología de ciclo de vida, por lo que se
consideran las cargas ambientales asociadas a las materias primas, la generación de
electricidad y utilidades. Para obtener el inventario se construyó una base de datos que
contiene la información ambiental asociada a varios procesos industriales que se relacionan
indirectamente al proceso bajo estudio. Similarmente, se incluyó una base de datos con los
factores de caracterización de las categorías de impacto más importantes.
La validación de la metodología y de la herramienta desarrollada se ha llevado a cabo
mediante tres procesos industriales: polietileno de baja densidad (LDPE), óxido de etileno (EO)
y biodiesel. Para cada proceso se han evaluado diferentes configuraciones para poder
determinar cual de ellas es la mejor opción desde el punto de vista ambiental y económico.
En el caso del LDPE, el cambio de configuración se ha enfocado en el origen de la
electricidad, la cual puede ser proveniente de la Red Nacional Española o de una unidad de
cogeneración. Los resultados indican que la mejor configuración corresponde al proceso que
emplea electricidad proveniente de la unidad de cogeneración, puesto que se obtiene vapor
como sub-producto y se evitan las emisiones asociadas a la generación de electricidad, lo que
se refleja en una importante reducción de los impactos ambientales asociados.
En el segundo proceso analizado, referente a la producción de oxido de etileno, se han
evaluado cuatro configuraciones, empleando aire u oxígeno como materia prima y electricidad
de la Red Española o produciéndola mediante cogeneración. En relación al origen de la
electricidad, al emplear la cogeneración, el comportamiento ambiental del proceso mejora
considerablemente. En cuanto a la importancia de la materia prima empleada, al utilizar
oxígeno se obtiene un mejor rendimiento en la etapa de reacción, con lo cual se compensa los
costes asociados a la materia prima con la productividad del proceso.
Finalmente, se ha llevado a cabo la evaluación del proceso de producción de biodiesel,
se comparó el comportamiento ambiental del proceso empleando un catalizador ácido y un
catalizador básico. En el proceso ácido se generan menores impactos ambientales. De forma
similar, ésta configuración tiene un mejor perfil económico ya que los costes asociados a la
producción son menores y no se requiere ninguna unidad de pretratamiento (necesaria en el
proceso alcalino).
Mediante la herramienta desarrollada, la información inicial puede modificarse en
cualquier momento con el fin de obtener los valores correspondientes a nuevas condiciones.
Uno de los aspectos más importantes es el que la herramienta se adapta fácilmente con el
mínimo de variaciones. Las bases de datos que se incluyen en las hojas de cálculo pueden ser
actualizadas por el usuario o ajustarse a las necesidades específicas de cada proceso. Todo el
análisis se lleva a cabo de forma automática, una vez introducida la información inicial del
proceso e información económica.

Environmental Diagnosis of Process Plants by Life Cycle Techniques
Haydée A. Yrigoyen González
The objective of this work is to develop a tool that integrates simulation, environmental
assessment and sensitivity analysis aspects. To support this tool, a methodology consisting of
five levels was established. These are: process simulation, Inventory, environmental impacts
assessment, economic analysis and sensitivity analysis.
The developed methodology describes the variables related to the process, as well as
the impacts associated to each stages, the economic viability of the process, and the process
stages with the highest environmental impact (by means of the sensitivity analysis).
ASPEN Hysys® is the chosen software for the simulation of processes. The inventory,
impact assessment and the economic analysis are automatically obtained in spreadsheets, by
means of macros execution.
The inventory and the impacts assessment are performed following the Life Cycle
methodology. Therefore, the environmental loads of the raw materials, electricity generation and
utilities are considered. In order to generate the inventory, a data base was constructed; it
contains the environmental information associated to industrial processes that are indirectly
related to the process under study. Similarly, a data base with the characterization factors of the
most important impact categories was included in the tool.
The validation of the methodology and the developed tool has been accomplished by
their application to three industrial processes: low density polyethylene (LDPE), ethylene oxide
(EO) and biodiesel production. Different configurations have been evaluated for each process to
determine the best option from the environmental and economic point of view.
For the LDPE process, the configuration change has focused in the origin of the
electricity, which can be supplied by the Spanish National Network or a cogeneration unit.
Based on our results, the best configuration corresponds to the process employing electricity by
cogeneration, since steam is obtained as by-product and the emissions associated to the
electricity generation are eliminated. These facts are reflected in an important reduction of the
overall impacts associated to this process.
In the second analyzed process, referring to the production of ethylene oxide, four
configurations have been evaluated: using air or oxygen as raw material and electricity from the
Spanish Network or produced by cogeneration. Related to the origin of the electricity, using
cogeneration, a better environmental profile is obtained. On the other hand, the oxygen as raw
material is better than air due to the best yield of ethylene oxide in the reaction stage. Due to
the better selectivity of the oxygen in the reaction, the costs of O2 as raw material are
compensated by high production.
Finally, the process evaluation of the biodiesel production has been carried out. In this
case, an acid and a basic catalyst were compared. The best configuration corresponds to the
process using an acid catalyst. In the acid process lower environmental impacts are generated.
Furthermore, this configuration has a better economical profile since the costs associated to the
production are smaller and a pre-treatment unit is not required, as in the alkaline process.
The initial information can be modified at any time to obtain the profile associated to the
new conditions by means of the developed tool. Also, the tool can be adapted to any process in
an easy way. The included database can be updated or adjusted by the user at any time to
personalize them to the specific necessities of each process. Once the initial information is
introduced, the analysis is executed automatically.
The developed tool is able to make the simulation, its environmental diagnosis,
economic evaluation and the sensitivity analysis of any industrial process, introducing the initial
operation conditions.

The grape industry in Oklahoma was valued at $98 million in 2010. In 2015, symptoms resembling Grapevine Leafroll disease were observed, but Grapevine Leafroll-associated Viruses were not detected using enzyme-linked immunosorbent assay (ELISA). A 2-year Cooperative Agricultural Pest Survey was initiated to determine the etiology of the red leaf symptoms in Oklahoma vineyards. In 2016, a total of 121 symptomatic grapevines from 13 counties were sampled and 96 symptomatic grapevines from 14 counties were sampled in 2017. Each sample was tested for Grapevine Red Blotch Virus (GRBV), Xylella fastidiosa (Pierce’s Disease), and ‘Candidatus Phytoplasma spp,’ by polymerase chain reaction (PCR). ELISA was used to test for Grapevine Leafroll associated Virus (GLRaV) strains 1,3 and 4 strains. Rotbrenner, caused by Pseudopezicula traceiphila, (2017 only), can be found in xylem from petioles and the xylem was examined morphologically for signs of fungal structures. In 2016, GRBV was detected in 38% of 121 symptomatic samples, GLRaV-1 and -3 were detected in 16%, GLRaV 4 strains were detected in 2%, and X. fastidiosa was detected in 2%. There were no detections of ‘Ca Phytoplasma spp’ in 2016 or 2017. In 2017, GRBV was detected in 34% of the 96 samples, GLRaV-1 and -3 were detected in 17%, GLRaV 4 strains were detected in 3%, and X. fastidiosa was detected in 3%. Rotbrenner was not detected in any of the samples in 2017. The findings of this survey provide information to Oklahoma grape growers and extension personnel about the cause of red leaf diseases affecting grapevines so that appropriate management strategies can be implemented in the near future.

Production plants used in modern process industry must produce products that meet stringent environmental, quality and profitability constraints. In such integrated plants, non-linearity and strong process dynamic interactions among process units complicate root-cause diagnosis of plant-wide disturbances because disturbances may propagate to units at some distance away from the primary source of the upset. Similarly, implemented advanced process control strategies, backup and recovery systems, use of recycle streams and heat integration may hamper detection and diagnostic efforts. It is important to track down the root-cause of a plant-wide disturbance because once corrective action is taken at the source, secondary propagated effects can be quickly eliminated with minimum effort and reduced down time with the resultant positive impact on process efficiency, productivity and profitability. In order to diagnose the root-cause of disturbances that manifest plant-wide, it is crucial to incorporate and utilize knowledge about the overall process topology or interrelated physical structure of the plant, such as is contained in Piping and Instrumentation Diagrams (P&IDs). Traditionally, process control engineers have intuitively referred to the physical structure of the plant by visual inspection and manual tracing of fault propagation paths within the process structures, such as the process drawings on printed P&IDs, in order to make logical conclusions based on the results from data-driven analysis. This manual approach, however, is prone to various sources of errors and can quickly become complicated in real processes. The aim of this thesis, therefore, is to establish innovative techniques for the electronic capture and manipulation of process schematic information from large plants such as refineries in order to provide an automated means of diagnosing plant-wide performance problems. This report also describes the design and implementation of a computer application program that integrates: (i) process connectivity and directionality information from intelligent P&IDs (ii) results from data-driven cause-and-effect analysis of process measurements and (iii) process know-how to aid process control engineers and plant operators gain process insight. This work explored process intelligent P&IDs, created with AVEVA® P&ID, a Computer Aided Design (CAD) tool, and exported as an ISO 15926 compliant platform and vendor independent text-based XML description of the plant. The XML output was processed by a software tool developed in Microsoft® .NET environment in this research project to computationally generate connectivity matrix that shows plant items and their connections. The connectivity matrix produced can be exported to Excel® spreadsheet application as a basis for other application and has served as precursor to other research work. The final version of the developed software tool links statistical results of cause-and-effect analysis of process data with the connectivity matrix to simplify and gain insights into the cause and effect analysis using the connectivity information. Process knowhow and understanding is incorporated to generate logical conclusions. The thesis presents a case study in an atmospheric crude heating unit as an illustrative example to drive home key concepts and also describes an industrial case study involving refinery operations. In the industrial case study, in addition to confirming the root-cause candidate, the developed software tool was set the task to determine the physical sequence of fault propagation path within the plant. This was then compared with the hypothesis about disturbance propagation sequence generated by pure data-driven method. The results show a high degree of overlap which helps to validate statistical data-driven technique and easily identify any spurious results from the data-driven multivariable analysis. This significantly increase control engineers confidence in data-driven method being used for root-cause diagnosis. The thesis concludes with a discussion of the approach and presents ideas for further development of the methods.

Fungos fitopatogênicos veiculados por sementes de soja podem causar sérios prejuízos à cultura, bem como danos diretos reduzindo o poder germinativo, vigor e emergência das sementes. A detecção e identificação precisa de fitopatógenos é uma das estratégias mais importantes para iniciar as medidas preventivas ou curativas no controle de doenças de plantas. Para se evitar a introdução e propagação de patógenos em áreas onde ainda não ocorrem, uma atenção especial deve ser tomada na detecção de agentes patogênicos em sementes. Os métodos tradicionalmente utilizados para detectar e identificar fungos em sementes são, muitas vezes, demorados, laboriosos e exigem um conhecimento extenso de taxonomia clássica. Métodos moleculares têm sido utilizados para detectar, identificar e quantificar uma longa lista de fungos fitopatogênicos. A técnica da reação em cadeia da polimerase em tempo real (qPCR) é atualmente considerada a mais eficiente para a detecção de fitopatógenos, não exigindo conhecimentos taxonômicos especializados para interpretar seus resultados. Considerando a importância e as implicações da diagnose rápida e bem sucedida de agentes patogênicos em sementes de soja, este estudo teve como objetivo estabelecer uma metodologia para elevar a eficiência de detecção dos fungos fitopatogênicos Sclerotinia sclerotiorum, Colletotrichum truncatum, Phomopsis spp. e Corynespora cassiicola, encontrados com maior frequência em sementes de soja, por meio de qPCR. Iniciadores e sondas de hidrólise TaqMan® foram projetados para os diferentes patógenos e avaliados quanto à sua especificidade e sensibilidade. Curvas padrões, baseando-se em diluições seriadas dos DNAs alvos de iniciadores e sondas específicas, foram estabelecidas para a quantificação desses patógenos. Amostras de sementes de soja naturalmente infectadas, provenientes dos Estados de Goiás, Minas Gerais e Paraná, foram submetidas a testes de detecção por meio de qPCR e métodos tradicionais, para fins de comparação. De todos os iniciadores e sondas projetados, apenas os de Phomopsis spp. e S. sclerotiorum apresentaram-se específicos e sensíveis, viabilizando sua utilização na detecção desses patógenos. Por meio dos testes tradicionais de detecção, Phomopsis spp. apresentou incidência máxima de 2,75% em uma amostra de Minas Gerais e S. sclerotiorum não foi detectado em nenhuma das amostras avaliadas. O método de qPCR proporcionou a detecção de Phomopsis spp. em todas as amostras testadas, alcançando o nível de incidência máximo de 6,75%. em amostra de Minas Gerais. S. sclerotiorum não foi detectado em nenhuma das amostras avaliadas pelo método de qPCR. Comparando-se os métodos de detecção testados, a qPCR foi mais sensível na detecção de Phomopsis spp. em sementes de soja.
Seed-born pathogenic fungi in soybean can cause serious damage to the crop, as well as direct damage by reducing seeds germination, vigor and emergence. The detection and accurate identification of plant pathogens is one of the most important strategies to initiate preventive and curative measures in the management of plant diseases. Particular attention should be taken in the detection of pathogens in seeds in order to avoid introduction and spread of pathogens in areas where they do not occur. The traditionally used methods for detection and identification of seed-born pathogenic fungi are often time consuming, laborious and require extensive knowledge of classical taxonomy. Molecular methods have been used to detect, identify and quantify a long list of plant pathogenic fungi. Quantitative real time polymerase chain reaction (qPCR) is currently considered the most efficient technique for the detection of pathogens because it does not require specialized taxonomical knowledge to interpret its results. Given the importance and implications of rapid and successful diagnosis of seed-born pathogenic fungi in soybean, this study aimed to establish a qPCR methodology to increase the detection efficiency of plant pathogenic fungi Sclerotinia sclerotiorum, Colletotrichum truncatum, Phomopsis spp. and Corynespora cassiicola, the most commonly occurring seed-born pathogenic fungi. Primers and TaqMan® hydrolysis probes were designed for these four pathogens and tested for specificity and sensitivity. Standard curves were established to quantify these pathogens, based on serial dilutions of the target DNA and specific primers and probes. Samples of naturally infected soybean seed from the states of Goiás, Minas Gerais and Paraná were subjected to detection tests using qPCR and traditional methods, for comparison purposes. From all primers and probes designed, only those for Phomopsis spp. and S. sclerotiorum showed up specificity and sensitivity, enabling their use to detect of these pathogens. Detection by traditional tests, resulted in a maximum Phomopsis spp. incidence of 2.75% in a sample from Minas Gerais and S. sclerotiorum was not detected in any of the samples. The detection and quantification of these pathogens by qPCR revealed the presence of Phomopsis spp. in all tested samples, the highest incidence level of 6.75% in a sample from Minas Gerais. S. sclerotiorum was not detected in any sample assessed by qPCR method. In comparison with traditional methods, qPCR was more sensitive in detecting Phomopsis spp. in soybean seeds.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior
Plant virus identification and characterization can be achived by several methods based in the biological, morphological, cytological, serological and molecular virus properties. The molecular properties have been used with frequency for vÃrus identification and characterization and the reverse transcription polymerase chain reaction (RT-PCR) has constituted an efficient and precise method for researches with RNA plant viruses. On the other hand, the enzyme-linked immunosorbent assay (ELISA) is the most used serological method for virus detection in plant tissues. A techncal innovation developed for plant virus identification represents a great technological development and support for plant virus research. A new approach involving virus particle immune precipitation to be used for RNA amplification by RT-PCR named IP-RT-PCR was sucessefuly used for amplification of RNA fragments from five virus species from the genera Comovirus, Cucumovirus, Potyvirus and Sobemovirus. Considering that the immune biological Companies have developed several DAS-ELISA kits, but neither of them produce and commercialize PTA-ELISA kits, a simple and practical PTA-ELISA kit was developed and validated for plant virus detection. The second part of the research had the objective to study, comparatively, the biological, serological and molecular properties of four plant virus isolates obtained from naturally infected Passiflora edulis (PWV-PET and PWV-GUA) and from naturally infected Vigna unguiculata (CABMV-BV and CABMV-FOR) with the objective to elucidate the identity of the causal agent of passion fruit woodiness in Brazil. In host range studies onle Canavalia ensiformes and Macroptilium lathyroides were infected by virus isolates obtained from cowpea and from passion fruit. The isolate PWV-GUA was purified from systemically infected M. lathyroides plants and the virus purified preparation (18.24 mg of virus.ml-1) was used for rabitt immunization for polyclonal antiserum production, which showed a title of 1:128,000 in PTA-ELISA. The electrophoresis analysis of the purified virus showed a unique capsidial protein with 34 kDA. Plant virus interaction studies in C. ensiformis indicated unilateral cross protection between PWV-GUA and CABMV-FOR. On the other hand, the isolate PWV-PET did not cross protect passion fruit plants against PWV-GUA. Filogenetic analysis of nucleotiode sequencies from cDNA fragments corresponding to coat protein (CP) genes amplified by IP-RT-PCR from the genomic virus isolates compared with virus sequencies from the Genbank grouped according to the host specifities. Based on the biological, serological and mainly molecular results, the virus isolates studied were classified into two biotypes: Biotype CABMC-C (Cowpea) to include isolates obtained from cowpea that do not infect passion fruit, and biotype CABMV-P (Passion fruit) to include the virus isolates responsible for the passion fruit woodiness in Brazil.
A identificaÃÃo e a caracterizaÃÃo de vÃrus de planta podem ser realizadas por vÃrios mÃtodos envolvendo propriedades morfolÃgicas, biolÃgicas, citolÃgicas, moleculares e sorolÃgicas. As tÃcnicas moleculares tÃm sido usadas com frequencia para identificaÃÃo e caracterizaÃÃo de vÃrus, e a tÃcnica de âreverse transcription polymerase chain reactionâ (RT-PCR) tem se constituÃdo em mÃtodo eficiente e preciso para pesquisas com vÃrus de planta com genoma de RNA. De outra parte, a tÃcnica de enzyme-linked immunosorbent assay (ELISA) constitui o mÃtodo sorolÃgico mais usado para detecÃÃo de vÃrus em tecidos vegetais. Uma inovaÃÃo tecnolÃgica desenvolvida nesta pesquisa para diagnose de vÃrus de planta representa grande avanÃo tecnolÃgico e suporte para pesquisa em virologia vegetal. A inovaÃÃo envolvendo a imunoprecipitaÃÃo (IP) de partÃculas de vÃrus para uso na RT-PCR denominada de IP-RT-PCR foi usada com sucesso para amplificaÃÃo de fragmentos de RNA de cinco espÃcies de vÃrus dos gÃneros Comovirus, Cucumovirus, Potyvirus e Sobemovirus. Considerando que kits de DAS-ELISA tÃm sido produzidos e comercializados por companhias de imunobiologicos, mas nenhuma companhia produz kits de PTA-ELISA, um kit simples e prÃtico de PTA-ELISA foi desenvolvido e validado para detecÃÃo de vÃrus de planta. A segunda etapa da pesquisa teve como objetivo estudar as propriedades biolÃgicas, sorolÃgicas e moleculares de isolados de vÃrus do gÃnero Potyvirus obtidos de maracujazeiro (Passiflora edulis) (PWV-PET e PWV-GUA) e isolados de Cowpea aphid-borne mosaic virus (CABMV-FOR e CABMV-BV) obtidos de feijoeiro caupi (Vigna unguiculata), visando elucidar a identidade do agente causal do endurecimento dos frutos do maracujazeiro no Brasil. Em estudos de gama de plantas hospedeiras, somente Canavalia ensiformis e Macroptilium lathyroides foram infetadas por isolados obtidos de maracujazeiro e de feijoeiro caupi. O PWV-GUA foi purificado a partir de plantas de M. lathyroides sistemicamente infetadas e a preparaÃÃo viral purificada (18,24 mg de vÃrus.ml-1) foi usada para imunizaÃÃo de coelho com a produÃÃo de antissoro policlonal com tÃtulo de 1:128.000 em PTA-ELISA. AnÃlise eletroforÃtica da preparaÃÃo viral purificada revelou uma Ãnica proteÃna capisidial com peso molecular de 34 kDa. Experimentos de interaÃÃo entre os isolados virais em C. ensiformis indicaram proteÃÃo unilateral entre PWV-GUA e CABMV-FOR. De outra parte, o isolado PWV-PET nÃo protegeu plantas de maracujazeiro contra a super infecÃÃo de PWV-GUA. AnÃlises filogenÃticas das seqÃÃncias dos fragmentos de cDNA correspondentes Ãs capas protÃicas (CP), amplificados a partir dos genomas dos isolados virais de maracujazeiro e de feijoeiro caupi por IP-RT-PCR, agruparam-se com as seqÃÃncias de isolados virais de referidas culturas depositadas no GenBank, apresentando um agrupamento em funÃÃo da especificidade de hospedeiros. Com base nos resultados dos estudos biolÃgicos, sorolÃgicos e, sobretudo moleculares, os isolados virais estudados foram classificados em dois biÃtipos: BiÃtipo CABMV-C (Cowpea) incluindo os isolados obtidos de feijoeiro caupi e biÃtipo CABMV-P (Passion fruit) para incluir os isolados responsÃveis pelo endurecimento dos frutos do maracujazeiro no Brasil.

Kyoto University (京都大学)
0048
新制・課程博士
博士(エネルギー科学)
甲第12618号
エネ博第136号
新制||エネ||34(附属図書館)
UT51-2006-S626
京都大学大学院エネルギー科学研究科エネルギー社会・環境科学専攻
(主査)教授 手塚 哲央, 教授 三島 嘉一郎, 助教授 下田 宏
学位規則第4条第1項該当

O cultivar Piatã de Brachiaria brizantha é uma gramínea forrageira de lançamento recente para uso nas pastagens brasileiras e a pesquisa relativa à nutrição mineral deste capim é escassa. O nitrogênio é o principal responsável pelo aumento da produtividade em plantas forrageiras, porém a associação com o enxofre pode influir nas respostas das plantas forrageiras à adubação. Mediante a combinação de doses de nitrogênio com doses de enxofre, objetivou-se estudar as características morfogênicas, nutricionais e produtivas do capim-piatã cultivado em solução nutritiva, empregando-se a sílica como substrato. O experimento foi realizado em casa de vegetação e estudou cinco doses de nitrogênio (2; 9; 16; 23 e 30 mmol L-1) e cinco doses de enxofre (0,1; 0,7; 1,3; 1,9 e 2,5 mmol L-1), em esquema fatorial 52 fracionado, em delineamento de blocos casualizados, com quatro repetições. As plantas foram submetidas a dois cortes, sendo o primeiro aos 44 dias após o transplantio e o segundo 25 dias após o primeiro corte. A interação doses de nitrogênio x doses de enxofre foi significativa para o número total de folhas e perfilhos, taxa de aparecimento de folhas e filocrono, área foliar, massa seca da parte aérea e das raízes, valor SPAD, comprimento e superfície radiculares totais, concentração de nitrogênio e de enxofre nos tecidos foliares e radiculares, concentração de nitrato e relação nitrogênio total:enxofre total (Nt:St) em pelo menos um dos dois ciclos de crescimento do capim-piatã. As mais elevadas doses de nitrogênio e de enxofre maximizaram o crescimento e desenvolvimento da parte aérea e do sistema radicular das plantas. Doses elevadas de nitrogênio diminuíram o comprimento radicular específico e a relação enxofre-sulfato:enxofre total (S-SO4:St) nas folhas diagnósticas do capim, além de aumentar a atividade da enzima redutase do nitrato e a concentração de amônio nesses tecidos. A dose de enxofre de aproximadamente 1,3 mmol L-1 proporcionou o maior comprimento e superfície radiculares específicos, enquanto a dose mais alta de enxofre (2,5 mmol L-1) maximizou a concentração de enxofre-sulfato e a relação S-SO4:St nas folhas diagnósticas das plantas. Correlações significativas e positivas foram constatadas entre a concentração de nitrogênio total, valor SPAD e a concentração de enxofre total das folhas diagnósticas e a produção de massa seca das plantas, nos dois cortes do capim-piatã. A concentração de nitrogênio nas folhas diagnósticas do capim-piatã e o valor SPAD servem como bons parâmetros para a avaliação do estado nutricional das plantas em relação ao nitrogênio, enquanto para a avaliação do estado nutricional do enxofre nas folhas diagnósticas do capim-piatã, a concentração de enxofre total e a relação Nt:St devem ser tomadas em conjunto.
Piatã cultivar of Brachiaria brizantha is a recently released forage grass to be used in brazilian pastures and research on mineral nutrition of this grass is scarce. Nitrogen is the main nutrient to increase forage productivity, but the association with sulphur can influence plant response to fertilization. Through the combination of nitrogen rates with sulphur rates, the objective was to study the morphological, nutritional and productive characteristics of Piatã palissadegrass grown in nutrient solution, by using ground quartz as substrate. The experiment was conducted in greenhouse and five rates of nitrogen (2, 9, 16, 23 and 30 mmol L-1) were combined with five rates of sulphur (0.1, 0.7, 1.3, 1.9 and 2.5 mmol L-1) in a 52 fractionated factorial set in randomized block design, with four replications. Plants were harvested two times, the first harvest was done 44 days after transplanting and the second one was 25 days after the first harvest. The interaction nitrogen rates x sulphur rates was significant for the total number of leaves and tillers, leaf appearance rate, phyllochron, leaf area, dry mass of shoots and roots, SPAD value, root length and root surface, nitrogen and sulphur concentrations in the diagnostic leaves and roots, nitrate concentration and total nitrogen:total sulphur ratio in at least one of the two growth cycles of Piatã palissadegrass. High nitrogen and sulphur rates maximized growth of shoots and roots of the plants. High nitrogen rates resulted in low specific root length and sulfate-sulphur:total sulphur ratio (SSO4: St) in diagnostic leaves, in addition to increasing the activity of the nitrate reductase and ammonium concentration in diagnostic tissue. Sulphur rate about 1.3 mmol L-1 provided the highest specific root length and surface, while the highest sulphur rates (2.5 mmol L-1) maximized sulfate-sulphur concentration and S-SO4:St ratio in diagnostic leaves. There was a significant and positive correlation between the concentration of total nitrogen, SPAD value and total sulphur concentration in diagnostic leaves with plant dry matter production, in both harvests of Piatã palissadegrass. Nitrogen concentration and SPAD value in diagnostic leaves of Piatã palissadegrass can be used as good parameters for assessing the nutritional status of plants in relation to nitrogen, while for the assessment of sulphur nutrition status in diagnostic leaves of Piatã palissadegrass, the concentration of total sulphur and total nitrogen:total sulphur ratio should be taken together.

Dentre os diversos fatores que afetam a produção e qualidade de frutos em plantas cítricas, os fatores fitossanitários destacam-se pela grande importância. Entre as diversas doenças que afetam a cultura dos citros, a Clorose Variegada dos Citros (CVC) é uma das mais severas e destrutivas, provocando tanto perdas em produção quanto em qualidade. Pelo fato desta doença ser causada por uma bactéria que se aloja no xilema, toda a dinâmica da água e nutrientes é afetada, resultando em uma série de sintomas visuais em diferentes graus. Buscou-se avaliar o estado nutricional de plantas cítricas sem sintomas vis uais de CVC e, com sintomas em diferentes níveis, através do Critério de Faixas de Suficiência, tradicionalmente utilizado na Citricultura Paulista e, através do Sistema Integrado de Diagnose e Recomendação, denominado pela sigla DRIS. O estudo foi realizado em pomares comerciais de laranjeira 'Pera' enxertada em limão 'Cravo', com sete anos de idade, no município de Araraquara – SP. Foram selecionadas em dois talhões, plantas com três níveis de CVC e plantas sem sintomas visuais, determinando-se os teores dos macronutrientes (N, P, K, Ca, Mg, S) e dos micronutrientes (B, Cu, Fe, Mn e Zn) em folhas de ramos frutíferos, bem como produção de frutos de cada planta. A interpretação através do método DRIS, envolveu a utilização de um "software" aplicado especialmente para a cultura dos citros pelo Instituto Agronômico de Campinas - IAC, onde foi utilizado o método de cálculo proposto por Jones (1981). O DRIS foi eficiente, possibilitando a ordenação dos nutrientes, e, indicando deficiência nutricional para alguns nutrientes considerados adequados ou excessivos pelo Critério de Faixas de Suficiência. Independentemente da presença de sintomas de CVC, todas as plantas apresentaram baixos teores de Ca, resultando em diagnose de deficiência pelos dois critérios de interpretação; de modo geral, houve uma redução nos teores foliares de N e P, em plantas com sintomas mais severos. Com relação aos teores dos demais nutrientes não houve um padrão de decréscimo ou aumento em função da CVC. O "software" DRIS aplicado especialmente para a cultura dos citros pelo IAC, com a população de referência por ele utilizada, deve ainda sofrer ajustes, de acordo com as condições e objetivos do trabalho para melhor correlação com produtividade.
Among all factors which affect the yield and fruit quality from citric plants, pests and diseases are of outstanding importance. There are several pests and diseases wich affect the citrus crop. The citrus variegated chlorosis (CVC) is one of the most severe and destructive diseases affecting both yield and quality. Because this disease is caused by a xylem bacteria, the water and nutrients dinamics is affected resulting in a series of visual simptons of different intensities. There objective of this work was to evaluate the nutritional status os citrus plants without visual simptons of CVC and, with different levels of simptons of this disease. The Sufficiency Range Criteria, traditionally used in the state of São Paulo and the Diagnosis and Recommendation Integrated System (DRIS) were the methods used for leaf analysis interpretation. The study was carried out in commercial orchards of 'Pêra' sweet orange budded on 'Cravo' lemom, seven years old, in the region of Araraquara – SP. Plants with three levels of CVC and plants without visual simptons were selected from two plots. Concentrations of macronutrients (N, P, K, Ca, Mg, S) and micronutrients (B, Cu, Fe, Mn, Zn) were determined on leaves of fruit branches. Fruit production from each plant was measured. The analysis through the DRIS method involved the utilization of a software developed specially for the citrus crop by the Instituto Agronômico de Campinas – IAC, where the method proposed by Jones (1981) was used. The results showed that the DRIS was efficient in ranking the nutrients and indicating the nutritional defficiency for some nutrients considered adequate or excessive by the Sufficiency Range Criteria. Regardless the presence of CVC simptons, all plants show low foliar concentrations of Ca, wich resulted in a diagnose of defficiency by the two criteria of interpretation. There was a foliar concentration reduction of N and P, in plants with more severe simptoms. With respect to the foliar concentration of the other nutrients there was not observed a standard decrease or increase due to CVC. The local DRIS-Citros IAC software norms utilized should be adjusted according to the work conditions and objectives to a improve yield correlation with plant nutrition.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination.
AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies. Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde. Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie materiaal word meestal geënt op onderstokmateriaal waarvan die virus status onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n noodsaaklike beheermeganisme vir virus-infeksie. Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut konstruk bevat. ‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3 infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat v getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het. Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante wat teenwoordig is in Suid-Afrika nie. Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus weerstandbiedendheids studies. Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.

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The aim of this study was to establish norms of the Diagnosis and Recommendation Integrated System (DRIS) in the culture of banana (Musa sp) for the subgroup Cavendish. We used 107 leaf samples from plants on farms in the towns of Andirá and Rancho Alegre, both located in Northern Paraná State, cultivars Nanicão and Grande Naine , with and without irrigation, aged 2 to 10 years. Chemical analysis determined the content of leaf 11 nutrients N, P, K, Ca, Mg, S, B, Cu, Fe, Mn and Zn. DRIS norms have been established for less than average productivity of 75 t ha-1, where it was found differences between the patterns of levels of nutrients and DRIS norms developed in this study. It was recommended the need for reprogramming of the quantities of fertilizer to be used according to the results obtained for the N, Ca, Mg, S, Fe and Zn contents presenting below the pre-established standards tracks.
O objetivo do presente trabalho foi estabelecer normas do Sistema Integrado de Diagnose e Recomendação (DRIS) na cultura da banana (Musa sp) para o sub grupo Cavendish. Foram utilizadas 107 amostras foliares de plantas em lavouras situadas nos municípios de Andirá e Rancho Alegre, ambos localizados na Região Norte do Estado do Paraná, cultivares Nanicão e Grande Naine , com e sem irrigação, idade variando entre 2 a 10 anos. As análises químicas foliares determinaram os teores de 11 nutrientes: N, P, K, Ca, Mg, S, B, Cu, Fe, Mn e Zn. Normas DRIS foram estabelecidas para produtividade média igual ou superior a 75 t ha-1, onde constatou-se divergências entre os teores padrões dos nutrientes e os teores das normas DRIS estabelecidas neste estudo. Recomendou-se a necessidade de reprogramação das quantidades a serem utilizadas de fertilizantes de acordo com os resultados obtidos, para N, Ca, Mg, S, Fe, e Zn que apresentaram índices abaixo das faixas padrões pré estabelecidas.

A crescente demanda pela melhoria operacional dos processos aliada ao desenvolvimento da tecnologia da informação tornam a utilização de controladores preditivos baseados em modelos (MPC) uma prática comum na indústria. Estes controladores estimam, a partir dos dados de planta e de um modelo do processo, uma sequência de ações de controle que levam as variáveis ao valor desejado de forma otimizada. Dessa forma, dentre os parâmetros de configuração de um MPC, a baixa qualidade do modelo é, indiscutivelmente, a mais importante fonte de degradação de seu desempenho. Este trabalho propõe uma série de metodologias para a avaliação da qualidade do modelo do controlador preditivo, as quais consideram sua velocidade em malha fechada. Tais metodologias são baseadas na filtragem dos erros de simulação a partir função nominal de sensibilidade, e possuem a capacidade de informar o impacto dos problemas de modelagem no desempenho do sistema, além de localizar as variáveis controladas que estão com tais problemas e se os mesmos são provenientes de uma discrepância no modelo ou de um distúrbio não medido. As técnicas ainda possuem a vantagem de serem independentes do setpoint, o que as torna flexível de também serem utilizadas em controladores nos quais as variáveis são controladas por faixas. A abordagem proposta foi testada em dois estudos de caso simulados, sendo eles: a Fracionadora de Óleo Pesado da Shell e a Planta de Quatro tanques Cilíndricos. As técnicas também foram avaliadas em dados de processo da Unidade de Coqueamento Retardado de uma refinaria. Os resultados indicam que as mesmas apresentam resultados coerentes, corroborando seu elevado potencial de aplicação industrial.
The growing demand for operational improvement and the development of information technology make the use of model predictive controllers (MPCs) a common practice in industry. This kind of controller uses past plant data and a process model to estimate a sequence of control actions to lead the variables to a desired value following an optimal policy. Thus, the model quality is the most important source of MPC performance degradation. This work proposes a series of methods to investigate the controller model quality taking into account its closed loop performance. The methods are based on filtering the simulation errors using the nominal sensitivity function. They are capable detect the impact of modeling problems in the controller performance, and also to locate the controlled variables that have such problems and if it is caused by a model-plant mismatch or unmeasured disturbance. The techniques have the advantage to be setpoint independent, making them flexible to be also used in MPCs with controlled variables working by range. The proposed approach was tested in two simulated case studies The Shell Heavy Oil Fractionator Process and The Quadruple-tanks Process. The methods are also evaluated in process data of the Delayed Coking Unit of a Brazilian refinery. Results indicate that the method is technically coherent and has high potential of industrial application.

Orientador: Wagner Bettiol
Banca: Antonio Carlos Maringoni
Banca: Carlos Gilberto Raetano
Banca: Regina Maria Villas Boas de Campos Leite
Banca: Marcelo Augusto Boechat Morandi
Resumo: O mercado de produtos de controle biológico, principalmente os que possuem isolados de Bacillus spp. em sua formulação, tem aumentado significativamente, devido às exigências de diminuição do uso de agrotóxicos e maior sustentabilidade do segmento agrícola. Assim, o presente trabalho teve por objetivos: 1- Avaliar o potencial de bactérias do gênero Bacillus no controle de diferentes doenças de plantas; 2- Selecionar isolados de Bacillus spp. promotores de crescimento da soja e antagonistas a Sclerotinia sclerotiorium; 3- Avaliar o potencial de um produto comercial à base de B. subtilis QST 713 e outro à base de óleo de café torrado para compor um programa de manejo da ferrugem-asiática da soja (Phakopsora pachyrhizi) em aplicação sequencial ou alternada com um fungicida. Inicialmente foi avaliado o efeito de B. subtilis, B. firmus e B. amyloliquefaciens no controle dos patógenos Erysiphe diffusa, Podosphaera xanthii, Sclerotinia sclerotiorum, P. pachyrhizi, Fusarium solani, Fusarium oxysporum f. sp. phaseoli, Colletotrichum lindemuthianum e Corynespora cassiicola em condições controladas. Neste estudo, nenhum dos isolados controlou Erysiphe difusa e Podosphaera xanthii. Em folhas destacadas, todos os isolados reduziram o diâmetro da lesão de S. sclerotiorum e a severidade de P. pachyrhizi, sendo mais efetivos com aplicação preventiva. B. subtilis, B. firmus e B. amyloliquefaciens também inibiram o desenvolvimento de F. oxysporum f. sp. phaseoli, C. cassiicola e C. lindemuthia... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The market of products for biological control, especially formulations using Bacillus spp., has increased significantly, due to the requirements for the reduction of the use of agrochemicals in crop protection and sustainability of the agricultural sector. The objectives of this thesis were: 1- To evaluate the potential of Bacillus in plant disease control; 2- To select isolates of Bacillus spp. for growth promotion of soybean and antagonists to Sclerotinia sclerotiorium; 3- To evaluate the potential of a commercial product based on B. subtilis QST 713 and another product based on roasted coffee oil in order to compose a management program for asian soybean rust (Phakopsora pachyrhizi) in sequential or alternating application with a fungicide. Initially, the effect of B. subtilis, B. firmus and B. amyloliquefaciens on the control of pathogens Erysiphe diffusa, Podosphaera xanthii, Sclerotinia sclerotiorum, P. pachyrhizi, Fusarium solani, Fusarium oxysporum f. sp. phaseoli, Colletotrichum lindemuthianum and Corynespora cassiicola was evaluated under controlled conditions. In this study, Bacillus isolates did not controlled Erysiphe diffusa and Podosphaera xanthii. All isolates were effective in reducing the diameter of the S. sclerotiorum lesion and the severity of P. pachyrhizi, being more effective by preventive application in detached leaf. B. subtilis, B. firmus and B. amyloliquefaciens inhibited the mycelial growth of F. oxysporum f. sp. phaseoli, C. cassiicola and C. lindemuthianum ...
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The cultivation of irrigated common bean is an important economic activity for the city of Cristalina, Goias. The proper management of manure promotes the production of safer, contributing to the economic and environmental sustainability. The Diagnosis and Recommendation Integrated System (DRIS) was developed to provide a diagnosis of the nutritional status of cultivated plants, helping the decision on fertilizer recommendation for crops, being based on the balance of essential nutrients to plants. However, this study aimed to establish reference standards DRIS and assess the nutritional status of irrigated common bean grown in the municipality of Cristalina, GO. In this, there were 86 samples of soil samples, 86 samples from leaf samples and 82 samples of common bean yield in 2009/2010. With the data organized into a database. The database was analyzed from two aspects known as the traditional methods of Critical Levels and Concentration Ranges, and also by the method of DRIS. The attributes B, S, MO and T were considered insufficient, while nutrients Cu, Fe, Mn, Zn, K and P were considered excessive in the soil. Fe, Zn and Mo were the nutrients in the leaves with smaller amplitudes located within the range considered appropriate, nutrients S, Ca and Cu were considered insufficient, and the P, N, and K have been considered excessive in the plant, comparing the criteria established. The DRIS method was more sensitive to diagnose nutritional problems, especially for micronutrients
O cultivo do feijoeiro comum irrigado representa uma importante atividade econômica para o município de Cristalina em Goiás. O manejo adequado da adubação propicia a produção de alimentos mais seguros, contribuindo com a sustentabilidade econômica e ambiental. O Sistema Integrado de Diagnose e Recomendação (DRIS) foi desenvolvido para fornecer um diagnóstico do estado nutricional de plantas cultivadas, auxiliando na decisão sobre a recomendação de adubação para culturas agrícolas, sendo baseado no balanço dos nutrientes essenciais aos vegetais. Este trabalho teve por objetivo estabelecer as normas de referência DRIS e avaliar o estado nutricional do feijoeiro comum irrigado cultivado no município de Cristalina, GO. Foram realizadas 86 coletas de amostras de solo, 86 coletas de amostras de folhas e 82 amostras de produtividade do feijoeiro comum na safra 2009/2010. Com os dados obtidos organizou-se um banco de dados. O banco de dados foi analisado sob duas vertentes, o método tradicional conhecido como métodos dos Níveis Críticos ou Faixas de Concentração, e também, pela metodologia do DRIS. Os atributos B, S, MO e T foram considerados deficientes, enquanto que os nutrientes Cu, Fe, Mn, Zn, K e P foram considerados excessivos no solo. O Fe, Zn e Mo foram os nutrientes nas folhas com menores amplitudes, situadas nas faixas consideradas adequadas; os nutrientes S, Ca e Cu foram considerados deficientes, e o P, N, e K foram considerados excessivos na planta, comparando aos critérios estabelecidos. O método DRIS apresentou maior sensibilidade para diagnosticar problemas nutricionais, especialmente, para micronutrientes.

Cette thèse traite de l’analyse et de la synthèse de multimodèles pour la simplification de modèles, l’estimation d’état et le diagnostic des systèmes non linéaires caractérisés par une ou plusieurs échelles de temps. Ces travaux visent, dans un premier temps, à développer une procédure systématique de transformation d’un système non-linéaire en le récrivant sous une forme multimodèle, en évitant quelques inconvénients majeurs : la transformation est réalisée sans perte d’information, le choix de différents points de fonctionnement n’est plus nécessaire, le choix de variables de prémisse est réalisé d’une façon systématique. De plus, la méthode offre le choix entre différents multimodèles. Ce degré de liberté sera utilisé pour faciliter les études de contrôlabilité, d’observabilité et d’analyse de stabilité. Dans un deuxième temps, l’obtention de la forme à perturbations singulières d’un système non linéaire est proposée, en éliminant quelques contraintes structurelles et en rendant l’identification et la séparation des échelles de temps indépendante de la structure du modèle. Dans un troisième temps, la synthèse de plusieurs observateurs robustes vis-à-vis des perturbations, des erreurs de modélisation et des entrées inconnues a été réalisée afin dereconstruire l’état et l’entrée inconnue du système. La difficulté de cette étude provient du fait que le multimodèle utilisé dépend de variables de prémisse non mesurables, situation qui n’est pas intensivement étudiée, alors qu’elle est naturellement issue de l’approche par transformation système non linéaire!multimodèle. Ensuite, le diagnostic de défauts de systèmes est réalisé au moyen de bancs d’observateur à entrées inconnues permettant la génération et la structuration de résidus indicateurs de défauts. Finalement, tous les travaux proposés sont appliqués au modèle d’une station d’´epuration, Activated Sludge Model No.1, qui est largement utilisé dans le domaine du traitement des eaux usées
This thesis deals with analysis and synthesis of multiple model structures for model simplification, state estimation and diagnosis of nonlinear systems represented by one or several time-scales. This work aims, at first, to develop a systematic procedure to transform a nonlinear system into a multiple model form, by avoiding some major drawbacks : the transformation causes no information loss, the choice of the different operating points is no more necessary, the choice of the premise variables is realized in a more systematic way. Furthermore, the method gives the possibility of choosing between different multiplemodel structures. This degree of freedom will be used to ease the controllability, observ-ability, stability analysis studies. Secondly, the derivation of a singularly perturbed form for a multiple time scale non linear system is proposed, by eliminating some structuralconstraints and by making the identification and the separation of the time-scales independent to the model structure. Thirdly, the robust observer synthesis with respect to perturbations, modeling errors and unknown inputs are presented for state and unknowninput estimation. The difficulty of these studies comes from the fact that the multiple model depends on unmeasurable premise variables, this case being not intensively studied, whereas it results naturally from the method of transformation nonlinear system - multiple model. Afterward, fault diagnosis is performed using banks of observer to generate andstructure residual signals. Finally, this works are applied to a model of wastewater treatment plant, Activated Sludge Model No.1 (ASM1) that is largely used in the concerned fiel

A adubação equilibrada com nitrogênio e enxofre pode alterar a nutrição e produção das gramíneas forrageiras. Mediante as aplicações de combinações de doses de nitrogênio com doses de enxofre, objetivou-se avaliar os efeitos: a) nas frações desses nutrientes no solo e correlacionar com as quantidades acumuladas desses nutrientes pela Brachiaria brizantha cv. Marandu; b) nas variáveis de crescimento, concentração total e formas inorgânicas de nitrogênio e enxofre e relação N:S nas raízes da gramínea forrageira; c) no consumo e eficiência do uso da água pela gramínea; d) nas variáveis estruturais, produção de massa seca e na quantidade acumulada desses nutrientes no capim-marandu e, e) na diagnose nutricional de nitrogênio e enxofre no capim, buscando determinar os níveis críticos para esses nutrientes. Dois experimentos simultâneos foram conduzidos em casa de vegetação, um com e outro sem o capim-marandu, no período de setembro a dezembro de 2008, utilizando um solo classificado como Neossolo Quartzarênico. Cinco doses de nitrogênio (0; 100; 200; 300 e 400 mg dm-3) foram combinadas com cinco doses de enxofre (0; 10; 20; 30 e 40 mg dm-3) em um fatorial 52 fracionado, com quatro repetições. No experimento sem plantas verificou-se o efeito da adubação com doses de nitrogênio e enxofre nas frações desses nutrientes no solo. A adubação com nitrogênio aumentou as frações inorgânicas de nitrato e amônio, nitrogênio total hidrolisável, nitrogênio amoniacal hidrolisável, aminoaçúcar e nitrogênio não identificado. A aplicação de doses de enxofre incrementou enxofre-éster e o enxofre residual do solo. No experimento com plantas observou-se que o fornecimento de doses de nitrogênio com doses de enxofre aumentou a massa seca de raízes, as concentrações de enxofre, nitrato e sulfato nas raízes, enquanto o comprimento e a superfície radicular, concentrações de nitrogênio e de amônio e relação N:S no sistema radicular responderam significativamente às doses de nitrogênio. Ocorreu aumento no consumo e na eficiência do uso da água pelo capim com adubação combinada de doses de nitrogênio com doses de enxofre. Número de folhas, número de perfilhos, área foliar, massa seca da parte aérea e quantidade acumulada de nutrientes pela parte aérea do capim responderam significativamente à adubação nitrogenada no primeiro corte das plantas, mas houve a necessidade de adicionar o enxofre na adubação com nitrogênio para maximizar essas variáveis nos dois crescimentos seguintes do capim. Apenas a adubação com nitrogênio foi significativa no primeiro período de crescimento, enquanto as combinações de doses de nitrogênio com doses de enxofre foram significativas no segundo e terceiro períodos de crescimento para o valor SPAD, concentrações de nitrogênio e enxofre e relação N:S nas folhas diagnósticas do capim. Correlações significativas entre a concentração de nitrogênio nas folhas diagnósticas e o valor SPAD foram observadas para os três períodos de crescimento do capim. A falta de resposta ao enxofre no primeiro período de crescimento da gramínea mostrou a baixa demanda por esse nutriente nessa fase. Os benefícios da adubação nitrogenada foram maximizados pelo suprimento conjunto com enxofre nos dois períodos subseqüentes de crescimento do capim-marandu.
Nitrogen and sulphur balanced fertilization may change mineral nutrition and yield of forage grasses. Based on the supply of combinations of nitrogen and sulphur rates, the objectives were to evaluate the effects: a) in these nutrient fractions in the soil and to correlate this fractions with nitrogen and sulphur content in Brachiaria brizantha; b) the combination of nitrogen rates with sulphur rates on the root growth characteristics, in nitrogen and sulphur, total concentration and inorganic forms and N:S ratio in roots of the forage grass; c) on water consumption and efficient use by Marandu palisadegrass; d) on structural characteristics, aboveground forage yield and nutrient content in the grass and e) on nutritional diagnosis of these nutrients in the forage, trying to obtain the critical levels of these nutrients. Two experiments were carried out at the same time under greenhouse conditions, one with and one without the Marandu palisadegrass from September to December 2008, and the soil was an Entisol. Five nitrogen rates (0; 100; 200; 300 and 400 mg dm-3) were combined with five sulphur rates (0; 10; 20; 30 and 40 mg dm-3) in fractionated 52 factorial, with four replications. In the experiment without plants, the nitrogen and sulphur fertilization changed these nutrients fractions in the soil. Nitrogen fertilization increased inorganic fractions of nitrate and ammonium, hydrolyzable total nitrogen, hydrolyzable ammonium, amino sugars and unidentified nitrogen. Application of sulphur rates increased estersulphur and residual sulphur in the soil. In the experiment with plants, supply of both nitrogen and sulphur increased root dry matter production, total sulphur, nitrate and sulphate-sulphur concentrations, while for total root length and root surface area, total nitrogen and ammonium concentrations and N:S only ratio nitrogen rates had significant responses. Nitrogen and sulphur rates increased the water consumption and efficiency of use by grass. Numbers of leaves, number of tillers, leaf area, shoot dry masss and nitrogen content in the shoots of forage grass showed significant responses to nitrogen fertilization in the first growth period but it was necessary to add sulphur in the fertilization to maximize these variables for following two growth period of the grass. Only nitrogen fertilization was significant in the first growth period, while the combinations of nitrogen and sulphur rates were significant for the SPAD value, concentrations of nitrogen and sulphur and N:S ratio in diagnostic leaves of the grass. Significant correlation was observed for nitrogen concentration in diagnostic leaves and SPAD value the three growth periods. Sulphur demand by Marandu palissadegrass was low in the first growth period, but the nitrogen fertilization benefits were maximized by adding sulphur in the fertilization of this grass.

A identificação de constituições genéticas de feijão (Phaseolus vulgaris L.) tolerantes à deficiência hídrica (DH) é de grande importância para os programas de melhoramento genético. O objetivo deste trabalho foi caracterizar genótipos de feijão em resposta à DH no estádio reprodutivo por meio de análises fisiológicas, agronômicas e moleculares. O experimento foi conduzido em casa de vegetação, em delineamento de blocos ao acaso com quatro repetições, utilizando 10 cultivares: ANFC 9, ANFP 110, BRS Esplendor, BRSMG Realce, IPR Siriri, IPR Tangará, IPR Tuiuiú, IPR Uirapuru, IAC Imperador e IAC Milênio em duas condições de irrigação: plantas irrigadas durante todo o ciclo, e plantas submetidas a supressão de irrigação no estádio reprodutivo (R7), até o solo atingir 16% da capacidade de campo, quando a irrigação foi reestabelecida. Nos últimos quatro dias de estresse foram analisadas as trocas gasosas. No último dia de estresse foi analisado percentual de estômatos fechados na face abaxial das folhas durante o dia, e coletadas plantas para as seguintes análises: massa fresca e massa seca das folhas, hastes e legumes, e conteúdo de prolina em folhas e raízes. Na maturidade fisiológica foi realizada a colheita e avaliados os componentes do rendimento e estimada a produtividade de grãos. Adicionalmente, visando identificar polimorfismo sequencias gênicas a promotoras relacionadas com a tolerância à DH, foram testados sete pares de primers no grupo de genótipos estudado. O índice de suscetibilidade à seca (ISS) variou de 0,65 a 1,10 no grupo de genótipos, sendo os menores valores observados para IAC Imperador (0,65) e BRS Esplendor (0,87) indicando a capacidade destes dois genótipos em manter a produtividade de grãos na condição de estresse. Todos os genótipos avaliados apresentaram redução nos componentes do rendimento quando estressados pela DH. IAC Imperador (43,4%) e BRS Esplendor (60,6%) apresentaram as menores reduções na produtividade e mantiveram cerca de 50% dos estômatos fechados em todos os horários analisados no último dia de DH. IAC Imperador apresentou maior eficiência no uso da água e taxa de assimilação de CO2 na DH. IPR Tuiuiú, IPR Tangará e IAC Imperador apresentaram as maiores concentrações de prolina nas raízes. Na condição de DH, houve forte correlação positiva significativa entre percentual de estômatos fechados com número de grãos por legume (0,696) e massa fresca da folha (0,731), sendo o percentual máximo de estômatos fechados 73,71% em DH. O caractere acúmulo de prolina na raiz foi o que mais contribuiu para a divergência entre os genótipos na DH, porém, nem sempre os genótipos que mais acumularam prolina foram os mais tolerantes. Os polimorfismos no DNA de sequências codificadoras e promotoras dos fatores de transcrição estudados neste experimento não discriminaram os genótipos tolerantes dos sensíveis à DH.
The identification of genotypes for drought tolerance has a great importance in breeding programs. The aim of this study was to characterize genotypes of beans in response to drought tolerance in different reproductive stages through physiologic, agronomic and molecular analysis. The experiment was conducted in greenhouse, using a randomized block design with four replicates; 10 cultivars: ANFC 9, ANFP 110, BRS Esplendor, BRSMG Realce, IPR Siriri, IPR Tangará, IPR Tuiuiu, IPR Uirapuru, IAC Imperador and IAC Milênio under two conditions of irrigation: plants irrigated during their entire life cycle, and plants under irrigation suppression in the reproductive stage (R7) until 16% of field capacity, when the irrigation was restored. In the last four days of stress, the gas exchanges were analyzed, and in the last day of stress was analyzed the percentage of closed stomata in the abaxial surface of the leaves, collected in different times of the day (9h, 12h, 15h and 18h). Additionally, plant samples were collected for the following analysis: fresh and dry mass of leaves, stems and legumes, and proline content in leaves and roots. The plants were harvested at the physiological maturity and the yield components and grain yield were determined. In addition, in order to identify polymorphisms in the sequences of promoters and genes related to drought, seven pairs of primers were tested on the group of genotypes. The drought susceptibility indexes (ISS) ranged from 0.65 to 1.10 in the group of genotypes, which the lowest values observed were for IAC Imperador (0.65) and BRS Esplendor (0.87), indicating the ability of these two genotypes to maintain grain yield under water stress condition. All genotypes showed reduction in yield components under water stress. IAC Imperador (43.4%) and BRS Esplendor (60.6%) had the lowest reductions in productivity and kept about 50% of the stomata closed during all the different times evaluated at last day of irrigation suppression. IAC Imperador showed greater water use efficiency and CO2 assimilation rate under drought stress. IPR Tuiuiú, IPR Tangará and IAC Imperador had the highest proline concentrations in the roots. Under water stress condition, there was a strong positive correlation (0.696) between the percentage of stomata closed with the number of grains per plant (0.696) and the fresh mass of leaves (0.731), the maximum percentage of stomata closed 73.71% in water stress. The accumulation of proline in the root was the character that most contributed to the divergence between the genotypes under water deficit, but not always the genotypes that have accumulated more proline were the most tolerant. The polymorphisms in DNA of coding and promoting sequences of transcription factors studied in this experiment did not discriminate tolerant genotypes from the sensitive ones to water stress.

La bacteriose a e. Chrysanthemi pv. Dianthicola (echr) facteur limitant de la production du dahlia est transmise par la multiplication vegetative. Afin de proposer une methode de diagnostic plus precise que la detection visuelle, les methodes immunoenzymatiques ont ete etudiees et adaptees pour la detection d'echr dans les tissus du dahlia. La methode das-elisa (double antibody sandwich) est evaluee par rapport aux methodes de diagnostic de reference (isolement et immunofluorescence). Son utilisation, pour l'analyse sanitaire du materiel de propagation vis-a-vis d'echr seul et associe eventuellemnt a la mosaique du dahlia (damy), est etudiee en vue d'une selection sanitaire. Les etudes effectuees pour optimiser les reactifs, pour determiner les parametres pouvant modifier la reaction antigene-anticorps

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Aeronautics and Astronautics, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 193-197).
Over-subscription, that is, being assigned too many tasks or requirements that are too demanding, is commonly encountered in temporal planning problems. As human beings, we often want to do more than we can, ask for things that may not be available, while underestimating how long it takes to perform each task. It is often difficult for us to detect the causes of failure in such situations and then find resolutions that are effective. We can greatly benefit from tools that assist us by looking out for these plan failures, by identifying their root causes, and by proposing preferred resolutions to these failures that lead to feasible plans. In recent literature, several approaches have been developed to resolve such oversubscribed problems, which are often framed as over-constrained scheduling, configuration design or optimal planning problems. Most of them take an all-or-nothing approach, in which over-subscription is resolved through suspending constraints or dropping goals. While helpful, in real-world scenarios, we often want to preserve our plan goals as much possible. As human beings, we know that slightly weakening the requirements of a travel plan, or replacing one of its destinations with an alternative one is often sufficient to resolve an over-subscription problem, no matter if the requirement being weakened is the duration of a deep-sea survey being planned for, or the restaurant cuisine for a dinner date. The goal of this thesis is to develop domain independent relaxation algorithms that perform this type of slight weakening of constraints, which we will formalize as continuous relaxation, and to embody them in a computational aid, Uhura, that performs tasks akin to an experienced travel agent or ocean scientists. In over-subscribed situations, Uhura helps us diagnose the causes of failure, suggests alternative plans, and collaborates with us in order to resolve conflicting requirements in the most preferred way. Most importantly, the algorithms underlying Uhura supports the weakening, instead of suspending, of constraints and variable domains in a temporally flexible plan. The contribution of this thesis is two-fold. First, we developed an algorithmic framework, called Best-first Conflict-Directed Relaxation (BCDR), for performing plan relaxation. Second, we use the BCDR framework to perform relaxation for several different families of plan representations involving different types of constraints. These include temporal constraints, chance constraints and variable domain constraints, and we incorporate several specialized conflict detection and resolution algorithms in support of the continuous weakening of them. The key idea behind BCDR's approach to continuous relaxation is to generalize the concepts of discrete conflicts and relaxations, first introduced by the model-based diagnosis community, to hybrid conflicts and relaxations, which denote minimal inconsistencies and minimal relaxations to both discrete and continuous relaxable constraints. In addition, we present the design and implementation of Uhura, the integrated plan advisory system that incorporates BCDR for resolving over-subscribed temporal plans. Uhura can efficiently produce a relaxed plan for the user to support multiple, interrelated constraints and activities. We have applied Uhura to different types of plans to illustrate the practical generality of our approach, which includes deepsea exploration, job-shop scheduling and transit system management. Results from the computational experiments we performed also show that BCDR is one to two orders of magnitude faster than existing algorithms that build on state-of-the-art numerical solvers, making it an effective approach for many large-scale plans in the aforementioned domains.
by Peng Yu.
Ph. D.

Objetivo: avaliar a relação entre o tipo facial e as variações da inclinação do Plano Oclusal e do ângulo ANB em indivíduos com oclusão normal. Material e Método: a amostra foi composta por 98 telerradiografias em norma lateral de 98 indivíduos (47 homens e 51 mulheres) com idade média de 15,2 anos (desvio padrão de 1,4 anos), com oclusão normal e perfil harmonioso. As radiografias foram digitalizadas em escala 1:1 eo programa Nemoceph (Nemotec® - Espanha), foi usado para a análise cefalométrica, que incluiu as cinco variáveis que segundo Ricketts definem o padrão facial, a relação sagital (SNA, SNB, ANB, N.S.Ba, SN.Go-Me, ANB individualizado, AP-BP e Wits)e ainclinação do Plano Oclusal (PLO.Linha S-N, PLO.PF, PLO.PP, PLO.XiPm e PLO.PM). Os dados foram analisados estatisticamente com um nível de significância de 5%. Resultados: acomposição dos tipos faciais foi: 11% dólicofaciais, 39% mesofaciais e 50% braquifaciais. Os valores médios e intervalo de confiança de 95% das variáveis que avaliaram a inclinação do plano oclusal, para os tipos faciais dólico, meso e braqui foramrespectivamente: PLO.SN: 21,7° (± 2,14°), 17,0° (± 1,10°) e 15,1° (± 1,43°); PLO.PF: 13,5° (±1,77)°, 10° (SD 1,01°) and 8,1° (± 1,25°); PLO.PP: 12,2° (±1,99°), 9.1° (± 1,31°) e 8,1° (± 1,21°); PLO.Xi-Pm: 20,5° (± 1,76°), 19,2° (± 0,96°) e 16,4° (± 1,12°); PLO.PM: 17,5° (± 2,02°), 15,3° (± 1,27°) e 12,1° (± 1,19°). Os valores médios das variáveis que avaliam a relação sagital maxilomandibular respectivamente foram: ANB: 3,8° (± 0,98°), 2,5° (± 0,62°) e1,3° (± 0,53°); distância AP-BP: 7,3mm (± 2,19mm), 3,4mm (± 0,94mm) e 1,9mm (± 0,78mm); ANB Individualizado: 4,7° (± 0,70°), 4,4° (± 0,43°) e 3,7° (± 0,39°); Wits: 1,8mm (± 1,27mm), -2,4mm (± 0,83mm) e -3,5mm (± 0,92mm). Conclusões: Tanto as variáveis que avaliam a inclinação do Plano Oclusal como as variáveis que avaliam a relação sagital maxilo-mandibular ANB e AP-BP, caracterizam-se diferencialmente segundo o tipo facial, observando-se um decréscimo dos valores do tipo dólicofacial para o braquifacial.
Objective: evaluate the relation between the facialtype and the inclination of the occlusal plane and ANB angle in individuals with normal occlusion.Material and Methods: the sample was composed of 98 radiographs, in lateral norm, of 98 individuals (47 men, 51 women), with an average age of 15.2 years (standard deviation of 1.4 years), normal occlusion and harmonic profile.The radiographs were digitalized in a 1:1 scale and the software Nemoceph (Nemotec® - Spain) was used for the cephalometric analysis, which included Rickettsfive variables thatdetermine the facial type, the saggital relation (SNA, SNB, ANB, N.S.Ba, SN.Go-Me, ANB individualized, AP-BP and Wits) and theinclination of the occlusal plane (OPL.S-N, OPL.PF, OPL.PP, OPL.XiPmand OPL.PM). The data was analyzed statistically with a significance level of 5%.Results: the composition of the sample by facial type was: 11% dolichofacial, 39% mesofacial e 50% braquifacial. The averagevalues and 95% confidence interval of the variables that measured the inclination of the occlusal plane, for the dolichofacial, mesofacial and braquifacialgroups were,respectively: OPL.SN: 21.7° (± 2.14°), 17.0° (± 1.10°) and 15.1° (± 1.43°); OPL.PF: 13.5° (±1.77)°, 10° (SD 1.01°) and 8.1° (± 1.25°); OPL.PP: 12.2° (± 1.99°), 9.1° (± 1.31°) and 8.1° (± 1.21°); OPL.Xi-Pm: 20.5° (± 1.76°), 19.2° (± 0.96°) and 16.4° (± 1.12°); OPL.PM: 17.5° (± 2.02°), 15.3° (± 1.27°) and 12.1° (± 1.19°). The average values and 95% confidence interval of the variables that measure the maxilar-mandibular saggital relation were respectively: ANB: 3.8° (± 0.98°), 2.5° (± 0.62°) and1.3° (± 0.53°);distance AP-BP: 7.3mm (± 2.19mm), 3.4mm (± 0.94mm)and 1.9mm (± 0.78mm); ANB Individualized: 4,7° (± 0.70°), 4.4° (± 0.43°) and 3.7° (± 0.39°); Wits: 1.8mm (± 1.27mm), -2.4mm (± 0.83mm)and -3.5mm (± 0.92mm). Conclusion: Both variables that measure the inclination of the occlusal plane and the variables that measure the ANB and AP-BP maxillomandibularsaggital relation have different characteristics according to the facial type, being observed a decrease in values from the dolichofacial type to the brachifacial type.

Cette thèse propose une approche hiérarchique du diagnostic des systèmes complexes par décomposition en sous-systèmes élémentaires de diagnostic puis agrégation de ceux-ci. Le chapitre 1 décomposition en sous-systèmes élémentaires de diagnostic puis agrégation de ceux-ci. Le chapitre 1 détaille la problématique du diagnostic en temps réel ainsi que celle des systèmes complexes. Il en propose une solution par décomposition hiérarchique et présente l'outil logiciel dialogs issu de ce concept. Il présente enfin un système complexe illustrant cette approche. La station d'épuration de Maxéville et son séchage thermique des boues. Les chapitres 2 et 3 abordent l'aspect modulaire de cette approche au travers de l'étape de génération de symptômes. Le chapitre 2 propose pour cela des méthodes de détection de défauts permettant, de générer des symptômes indépendants. Le chapitre 3 présente des techniques plus élaborées assurant également la localisation de défauts au travers d'ensemble de symptômes. La nécessité d'associer les résidus fournis par ces méthodes dans des niveaux de décision hierarchiquement supérieurs impose leur homogénéisation au moyen de tests de décision présentes chapitre 2. Le chapitre 4 développe les notions de hiérarchisation et d'agrégation de systèmes élémentaires au travers de deux méthodes de localisation de défauts, la décision structurée et la décision à objectifs multiples. Une troisième technique, la décision générique, permet de conclure quant à l'état du système complet

Phytoplasmas are wall-less, non-culturable, phloem-limited bacterial pathogens that belong to the Mollicutes. They cause many diseases in lots of plant species (wild and cultivated) belonging to many different plant families, resulting in significant losses in important crops, and economically damaging epidemics worldwide. Phytoplasmas infect major cultivated crops such as many annual crops, fruit trees, grapevines and palms, which makes control of these diseases a priority, and the first important step for management is efficient and effective phytoplasma diagnosis. Detection of phytoplasmas is difficult because of their irregular distribution within the diseased plants and low concentration inside infected plants. In the last two decades most research toward the detection of phytoplasmas has used nucleic acid-based techniques such as PCR, which is used to amplify regions of phytoplasma genomes existing inside infected plants. However, most routine diagnostics has moved from general PCR to real-time PCR, due to the improved sensitivity and reduced risk of contamination due to the use of a closed system for product detection. Also the method can be developed into a semi-quantitative method. In this project the aim has been to improve the specificity and reliability of phytoplasma diagnostic techniques by using primers that detect specific genes in phytoplasma genomes, and designing new universal primers for conventional and real-time PCR. This has involved developing new assays for 16Sr groups II, III, V, VI, XI, and XII, to facilitate analysis of changes in levels of different phytoplasmas in mixed infections. In addition, this work has involved the evaluation of LAMP (loop mediated isothermal amplification) diagnostic assays for different phytoplasma groups (I, II, III, V, VI, X, and XII) and also validating a rapid DNA extraction method and whether this is effective for all plant species (i.e. Madagascan periwinkle versus Napier grass and other grasses). The second main objective was to investigate the rate of evolution of phytoplasma genomes. For this, infected plants (of phytoplasma groups 16SrI, II, III, VI, and X) were grafted onto fresh plants at 3-4 month intervals throughout the project. Once the phytoplasma had re-established, DNA was extracted and a range of genes including 16S rRNA, secA, tuf, and rp were amplified and sequenced. The aim was to determine whether there was any evidence of genome evolution over time. However, the results suggested that in these genes at least, the rate of change due to point mutations and/or insertion of potential mobile elements (PMUs) was slow, with no sequence changes being detected over the three years of study.

La contribución que se pretende con esta tesis se refiere a la implantación de un sistema de diagnosis de fallos en plantas químicas completas integrado al sistema de supervisión, gestión y control de la producción.
El sistema de diagnosis de fallos que se presenta consiste en una combinación de un sistema de reconocimiento de patrones basado en redes neuronales artificiales y un sistema de inferencia basado en la lógica difusa. La información necesaria para desarrollar el sistema de diagnosis incluye los datos históricos, un análisis de riesgo y operabilidad y un modelo de la planta química. La entrada al sistema son las mediciones directas o indirectas de la planta y la salida consiste en una señal para cada fallo (0: no fallo; 1: fallo). Primero se definen los fallos posibles. La red neuronal se entrena con datos históricos de fallos ocurridos en el pasado, con el objeto de reconocer los patrones respectivos. En el caso de que no se posean los datos históricos de alguno de los fallos, por ejemplo porque nunca hayan ocurrido, se obtienen los patrones mediante la simulación, usando el modelo de la planta. El sistema de lógica difusa contiene un conjunto de reglas si-entonces que pueden ser de dos tipos: las basadas en el conocimiento de la planta, mediante el análisis de riesgo o la experiencia con la simulación, y las basadas en la experiencia con el uso de la red neuronal, previamente entrenada. Otro aspecto novedoso es la posibilidad de entrenar la red neuronal con "características" extraídas de las variables medidas mediante su pre-procesamiento con wavelets. Esta variante permite obtener un alto rendimiento del sistema de diagnosis en plantas químicas discontinuas y continuas complejas. Para optimizar los parámetros de los componentes del sistema de diagnosis se propone un índice de rendimiento. Además, se utiliza el índice de rendimiento para comparar el sistema propuesto con otros métodos.
Las señales dadas por el sistema de diagnosis pueden ser usadas por el sistema de programación de la producción para actualizar el plan de la manera más efectiva, por el sistema de control para actuar en forma automática y por los operadores de planta como soporte para la toma de decisiones. Se han sentado las bases para la traducción de la salida del sistema de diagnosis para su utilización por los demás niveles del soporte informático. Se usa una estrategia basada en el análisis de riesgo y operabilidad de la planta.
El sistema propuesto es consecuencia de sucesivas mejoras, al trabajar con diferentes casos de estudio. Los escenarios académicos corresponden a una planta química continua con una corriente de reciclo y un reactor discontinuo. Los casos a escala de planta piloto corresponden a escenarios construidos en la UPC: un reactor de gasificación, un reactor semicontinuo y una planta discontinua multipropósito. Los escenarios industriales corresponden a dos refinerías de azúcar y a un sector de una planta petroquímica.
El sistema propuesto muestra ser ventajoso respecto a otros métodos tanto en cuanto a la rapidez de diagnosis como en cuanto a su capacidad para aislar los fallos. La simplicidad del desarrollo y la flexible estrategia de implementación del sistema propuesto auguran un futuro promisorio a la tecnología presentada. Nuevas líneas de investigación se pueden emprender mediante el desarrollo de un sistema de gestión de las alarmas. Otro aspecto importante es la posibilidad de participar en la estandarización de las interfaces del programa de diagnosis.
The pretended contribution of this thesis deals with the implementation of a fault diagnosis system in chemical plants integrated to the monitoring, management and control system. The proposed fault diagnosis system consists in a combination of a pattern recognition approach based on artificial neural networks and an inference system based on fuzzy logic. The information needed to develop the fault diagnosis system includes the historical data, the hazard and operability study and the model of the chemical plant. The inputs to the system are the direct or indirect measurements from the plant and the output consists in a signal for each fault (0: no fault; 1: fault). First, the possible faults are defined. The artificial neural network is trained with historical data of faults occurred in the past, with the aim of recognising the respective patterns. In the case that the corresponding historical data are not available, for example due to the no occurrence of the fault, the patterns are obtained through simulation, using the plant model. The fuzzy logic system contains a set of if-then rules that can be of two types: those based in the process knowledge, by the hazard analysis or by the experience with simulation, and those based on the experience with the use of an artificial neural network, previously trained. Other novel aspect is the possibility of artificial neural network training by using signals features that are extracted by its pre-processing using wavelets. This alternative allows a higher fault diagnosis system performance in batch and complex continuous chemical plants. In order to optimise the parameters of the components of the fault diagnosis system, a performance index is proposed. The performance index is also utilised to compare the proposed fault diagnosis system against other methods.
The signals provided by the fault diagnosis system can be used by the scheduling system to update the schedule in the most effective way, by the control system to take automated control actions and by plant's operators as support for decision-making. The basis of the translation of the system output, for its utilisation at other levels in the information system, has been settled.
The proposed strategy is based on the hazard and operability analysis.
The proposed system is the result of successive improvements, by working with different case studies. The academic scenarios correspond to a continuous chemical plant with a recycle stream and a batch reactor. The pilot plant scale cases correspond to scenarios built at UPC: a reactor gasifier, a fed-batch reactor and a multipurpose batch chemical plant. The industrial scenarios correspond to two sugar refineries and a sector of a petrochemical plant.
The proposed system shows to be advantageous with respect to other methods in relation to the fastness of the diagnosis and also its capacity to isolate faults. The simplicity of the development and the flexible strategy of implementation of the proposed fault diagnosis system give a promising future to the presented technology. New research lines can be considered by developing the alarm handling system. Other important aspect is the possibility of the participation in the standardisation of the interfaces of the fault diagnosis program.

Phytoplasmas are plant pathogens that are small prokaryotic, wall-less bacteria causing significant diseases in hundreds of plant species globally. They have been named as distinct ‘Candidatus phytoplasma’ species and belong to the Mollicutes. These microorganisms are non-cultivable plant pathogens (obligate), so their diagnoses are primarily achieved by molecular techniques. In infected plants, they inhabit phloem tissues (sieve cells) and they are transmitted between plants by insects (phloem feeders) such as those in the orders Cicadellidae, Psyllidae and Fulgoridae. Phytoplasmas have extremely small genomes and also have very low levels of the nucleotides cytosine and guanine. Polymerase Chain Reaction (PCR) has been developed and employed for investigating phytoplasmas in their hosts, and such techniques have been advanced through the use of nested PCR and real-time PCR to improve the diagnosis of phytoplasmas with low titre in their different hosts, and also to be able to identify the different taxonomic groups of the pathogens. In addition, real-time PCR can be used to quantify phytoplasmas in the infected plants or insects. The purpose of this project was to concentrate initially on 16S rRNA, leucyl tRNA synthetase and secA based PCR assays and test these on periwinkle plants containing phytoplasmas from six taxonomic groups, 16SrI, II, III, V, VI and X. Work also focussed on transmitting the six phytoplasma strains between periwinkle plants and other host plants such as Napier grass, oil palm, and tomato plants through dodder shoots. Cuttings were taken from mother plants, that are all from the same periwinkle genetic background, to enable the propagation of new plants containing isolates from the six groups. These were treated with different potential control strategies, and conventional and real-time PCR and (Loop-mediated isothermal amplification) LAMP were used to test the levels of phytoplasma in these plants following treatments to determine the efficacy of the different control treatments. In more detail, the control strategies being tested included altered nitrogen fertilization regimes, treatment with salicylic acid, and UV-C pulse treatments. As a consequence, phytoplasma titre of different phylogenetic groups were affected by the application of different nitrogen fertilizers and different amounts, and this showed the potential for use of various nitrogen sources as a control method for phytoplasma diseases. Furthermore, this research has shown that UV-C light can be applied to phytoplasma-infected plants to enhance the resistance characteristics of plants against phytoplasma and reduce phytoplasma titre within diseased plants. Through applying different control approaches against phytoplasma infected plants, it has been shown that low concentrations of salicylic acid application had an optimal significant effect on phytoplasma quantity and reduced infection on diseased periwinkle plants. However, a high concentration of the same solution produced a negative effect and phytotoxicity on periwinkle plants.

Dans ce memoire est presentee une methode de diagnostic par spectroscopie optique en emission applicable aux plasmas presentant un plan de symetrie. Cette methode consiste a determiner les parametres locaux du plasma (coefficient d'emission volumique, temperature, concentration electronique) a partir des intensites integrees des raies atomiques, ioniques et du continuum, ainsi qu'a partir des profils spectraux d'intensite des raies atomiques neutres. Cette methode evite l'utilisation de methodes de tomographie telles que la procedure d'inversion d'abel dans le cas d'un plasma a symetrie axiale. Dans le premier chapitre, sont exposes le formalisme mathematique de la methode ainsi que les resultats des simulations numeriques de l'equation de transfert radiatif. Des relations algebriques sont etablies entre l'intensite des raies atomiques ou ioniques ou du continuum et le coefficient d'emission volumique dans le plan de symetrie du plasma. La temperature dans le plan de symetrie est exprimee en fonction des rapports des intensites de raies atomiques ou ioniques ou du rayonnement du continuum. La concentration electronique est determinee a partir du deplacement et de l'elargissement stark des profils spectraux des intensites des raies. Dans le deuxieme chapitre, l'auteur effectue sur un plasma d'arc transfere a symetrie axiale une verification experimentale ainsi qu'une verification numerique de la methode developpee. Dans le troisieme chapitre, la methode est appliquee au diagnostic de plasmas a symetrie plane, generes par un systeme de torches jumelles bipolaires. Ont ainsi ete etudies des plasmas d'argon pur, d'argon + oxygene, d'argon + oxygene + particules d'oxyde de titane (tio#2) et d'argon + oxygene + particules d'oxyde de titane (tio#2) + particules d'alumine (al#2o#3)

The need to apply modern signal processing and analysis techniques to enhance the performance of process instrumentation systems has been identified as one of the priority areas for research and development in process instrumentation and process control. This enhancement of performance can be in the form of extracting additional information from flow sensors beyond the customary requirements of the basic process measurement, that is, flow rate. In conjunction with, and within the expert systems approach, an enhanced flowmeter can, for example, be utilised for condition monitoring purposes and, for diagnostic engineering management and optimisation of process plant operations. This thesis demonstrates the new importance of flow measurement signals from the point of view of extracting additional information which include: - (i) the basic process measurement value (ii) a quality or validity index associated with the basic measurement value, (iii) any other information which can be used to characterise the operational status of the plant and associated instrumentation. The signal processing tasks involve spectral analysis and spectrum estimation, system identification and parametric time series modelling techniques. Qualitative signatures which have been identified for different flowmeters operating under a wide variety of conditions in different process flow rigs are described. The utilisation of the results towards enhancing the performance the of the process instrumentation system is emphasised and demonstrated throughout the thesis.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1989.
Science hard copy bound in 2 v.
Includes bibliographical references (leaves 300-307).
by Francis Eric Finch.
Ph.D.

Os efeitos conjugados das mudanças tecnológicas, das teorias de gestão flexível das empresas e da globalização dos mercados e da informação forçosamente resultaram em alterações profundas na esfera do trabalho humano. O presente estudo tem como objetivo descrever o atual plano de formação da Sines Tecnopolo, identificando, de forma clara, o plano e, com a ajuda da análise do diagnóstico de necessidades de formação, propor um ajuste no plano de formação, para que a academia supere as fragilidades e lacunas nele encontradas, (no plano atual.) Assim, de modo a atingir os objetivos delineados, foi utilizado, no presente relatório, como princípio organizador, o método de investigação-ação, projetado em três etapas: conhecer, investigar e identificar; Diagnosis of training needs assessment in an organizational context ABSTRACT: About the combined effects of the technological changes, the theories of flexible management of enterprises and the globalization of the markets, and the information led to big changes in the human word field. This study aims to describe the actual plan of building Sines Tecnopolo, identifying clearly the plan and, with the help of the analysing of building necessities diagnosis supporting an agreement about the building plan, such that the academy overcomes its weaknesses and gaps (in the actual plan). Like that, to achieve the planned objectives, A method of investigation-action (was used in this report as a main organizer) which was done in three steps: understanding, investigating and identifying.

Nematode identification by classical methods is a highly skilled undertaking, in which trained taxonomists examine samples microscopically and identify nematodes using keys based on morphological details. The accuracy of diagnosis depends considerably on the skill of the taxonomist. More recently, molecular diagnostic techniques have been developed to identify such nematodes, and the focus of this thesis is the development and application of new approaches to nematode diagnostics. The species studied included root lesion nematodes (RLNs, Pratylenchus spp.), cyst nematodes (CNs Heterodera and Globodera spp.), and the pine wood nematode (PWN, Bursaphelenchus xylophilus). Six populations of four species of RLNs isolated from wheat and sorghum plants were maintained on carrot pieces in vitro for the work. Similarly, seventeen populations of five species of CNs were also studied: in some cases for biosecurity species (eg for soybean and potato cyst nematodes, and PWN) the materials had either to be studied overseas or extracts obtained from overseas for analysis. For nucleic acid based diagnostics, an ITS-based PCR approach was used to identify different species of RLNs and study sequence differences within and between different species and populations. Phylogenetic trees were constructed to compare the data generated in this thesis with those of published sequences for the nematodes studied. The results obtained showed that there were relatively small differences in sequence between different populations of a individual species, but significantly greater differences between species. Comparing ITS sequences of different RLN species, that of P. neglectus was 58% similar to that of P. thornei, and compared to that of P. penetrans and P. zeae, the similarity was 60% and 53% respectively. Similarly, comparing ITS sequences, P. penetrans was more closely related to P. neglectus (60%) than to P. thornei (59%) and P. zeae (58-59%). For CNs, for the genus Heterodera, the similarity in ITS sequence of H. schachtii and H. glycines was 97-98%. When comparing H. schachtii and cereal cyst nematodes (CCNs), the similarity in ITS sequences was less, at 77-78% for H. avenae. H. glycines, a biosecurity listed pathogen, is not present in Australia and DNA from a Japanese population was obtained and sequenced. Based on the close sequence similarity of ITS regions (97-98%) between H. schachtii and H. glycines, it is suggested that H. schachtii can be use as a model for detecting future incursions of H. glycines. Of the three species of CCNs studied (H. avenae, H. latipons and H. filipjevi), the two species, H. latipons and H. filipjevi, are also not present in Australia, and so published sequence data for these species was used for comparisons. ITS sequences of Australian H. avenae populations were compared to those of H. latipons and H. filipjevi: which were 86-88% and 94-96% similar to those of H. avenae. In the absence of the two biosecurity pathogens, H. avenae can be used as a suitable model to develop methods to detect the other two CCN species. For the genus Globodera, a comparison of ITS sequences of G. rostochiensis (potato cyst nematode) populations from New Zealand and Japan differed by 2% and 3% from the consensus world collection of G. rostochiensis ITS sequences. These differences might reflect different routes of introduction of potato into Japan and New Zealand. It was also noted that, in some cases, PCR analysis can lead to mis-identification. For example, in this study, a RLN from Queensland was identified by classical taxonomy as P. zeae, but when DNA sequencing was undertaken for this population, the resulting ITS sequence obtained was more similar to database sequences identified as P. bolivianus than to P. zeae. Such differences may result from initial mis-identification of the original sample, or from sequencing error. Protein profiling was used as an alternative approach to ITS-based PCR identification of plant nematodes. This involves separating nematode proteins using Matrix Assisted Laser Desorption Ionisation Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to generate diagnostic protein profiles. Protein profiling by MALDI-TOF MS was developed as a novel, rapid (<2hr) approach to identify plant-parasitic nematodes. Methods were developed to extract and analyse protein spectra by MALDI-TOF MS to identify nematode species of local and biosecurity concern. Protein profiles were generated for H. glycines, G. rostochiensis, H. schachtii and H. avenae, and for the RLNs, P. neglectus, P. zeae, P. thornei and P. penetrans. Diagnostic species-specific protein peaks were identified in the profiles for each species. The results obtained show that protein profiling using MALDI-TOF MS is a valid and rapid method for identifying plant nematodes, and the database provided here represents the most comprehensive resource for protein-based diagnostics of plant nematodes. Two dimensional protein gel electrophoresis (2DE) was also assessed as a tool to analyse RLN and CN proteins in more detail. Nematode proteins were extracted and separated in a protein extraction buffer first by isoelectric point (pH range 3-10, 16.5% polyacrylamide gel) , followed by size separation using SDS gel electrophoresis, and gels stained with silver nitrate. The protein spots in the second dimension were recorded using a high resolution gel scanner. Ninety three distinct protein spots were found for Pratylenchus spp. and 89 distinct spots for Heterodera spp. Differences in the protein profiles between populations of one species, and between different species, were identified readily using ‘Progenesis SameSpots’ software. Thirteen protein spots for RLNs and nine spots for CNs were further analysed and characterised. From these, two significant proteins were identified as useful biomarkers that were present in two different populations of P. neglectus and one significant biomarker was identified present in P. penetrans. Similarly, specific differences in protein profiles were found within populations of H. schachtii species which provided biomarkers that identified the different populations. For H. schachtii and H. avenae, two distinct protein spots were chosen as potential species specific biomarkers since they were present in three H. schachtii populations, and three species-specific biomarkers were chosen as specific to H. avenae. Individual protein biomarkers were excised and sequenced after trypsin digestion to release peptides from the gel. The m/z ratio of the peptides fragments were then analyses by MALDI-TOF MS, and the pattern of fragmented peptides was then compared using blastP with those recorded on Mascot and NCBI databases to identify proteins from which the peptides were derived. Identified proteins included RutC family protein C23G10.2, major sperm protein, probable arginine kinase, ATP synthase subunit alpha-mitochondrial, glyceraldehyde-3-phosphate dehydrogenase 2 and vacuolar H atpase protein 8, protein C14C10.2b, Y20F4.3 transcript:Y20F4.3, protein 19C07, arabinogalactan endo-1,4- β -galactosidase 2, heat shock 70 kDa protein C, annexin 4F01, β-1, 4-endoglucanase 1 precursor, aldolase, cathepsin L and pectate lyase 1. Although it is not necessary to identify the function of diagnostic proteins, such additional information is useful. The value of identified species-specific proteins is their potential be use to develop antibody-based diagnostic tests, such as Lateral Flow Devices (LFDs), in which antibodies raised against specific biomarker proteins can be developed to provide a rapid field-based method for nematode identification. Another nucleic acid based approach was also investigated, which also made use of ITS sequence data – termed anti-primer quantitative PCR (aQPCR) technology, using a qPCR equipment platform. In this approach an additional ‘anti-primer’ was added to fluorescent QPCR reactions (specific primers labeled with FAM, Cy5 or TET), which binds to and quenches unbound fluorescent label. This approach decreases background fluorescence and so increases accuracy of the diagnostic test, and was developed to provide a multiplex high-throughput assay for nematode diagnostics. With different fluorescence labels to tag different primers specific to different RLN species (P. neglectus, P. penetrans and P. thornei), the results obtained successfully differentiated these three species in a multiplex system, in a total reaction time of 2.5 hours. For this approach, the number of detectable species depends on the number of different fluorescent channels the qPCR machine can detect. A further diagnostic procedure was also developed to try to increase the number of samples that could be analysed at one time. This procedure was termed ‘Multiplex anti-primer denaturation PCR’ (MAD PCR). It was derived by combining aQPCR technology and ‘auto-sticky’ PCR. This approach makes use of ‘C3 linkers’ (or ‘blocks’) in primers, which prevent further PCR copying of the strand in which they occur, resulting in an overhang. With C3 linkers inserted at different positions in different primers, the qPCR was developed into a multiplex high-throughput assay. Different C3 positioning on the primer results in different melt temperatures in a melt analysis after PCR. The difference in melt temperatures enabled differentiation between different C3 inserted primers. To extend this technology, different fluorescent labels (such as FAM) were combined with different C3 positions for primers specific to different species. After PCR, melt analysis was done to differentiate the species by the different temperatures at which melting of dsDNA occurred, as followed by changes in fluorescence with changing temperature. In the work undertaken C3 blocks were incorporated in different primers to detect three different RLNs, ie a triplex system. The analysis of up to 12 species per qPCR could be done with the system as developed here, but with further refinement, and incorporating additional fluorescence labels with the ‘triplex’ system, 18 species could be detected using 6 channels. As for other soil-borne disease agents, nematodes are usually extracted from plant tissue or soil samples for detection. The time taken to extract them is usually several days. In the final Results chapter extraction of DNA from nematode infected soil samples was combined with molecular identification, and developed as a potential package for a rapid nematode diagnostics in a field situation. To do this, a rapid isolation method was developed and termed ‘DNA isolation rapid technique from soil’- ‘DIRT(s)’. This involved extraction of DNA from nematode-infected soil samples using a customised blender (time taken about 2 min) and DNA capture column, followed by elution from the column and aQPCR analysis: the whole procedure took only 4 hours. Using the DIRT(s) technique and aQPCR technology, three different RLN species (P. neglectus, P. thornei and P. penetrans) infecting wheat plants were successfully identified after DIRT(s) extraction from soil using aQPCR in a multiplex assay. The results are discussed in relation to current techniques used for nematode diagnostics. It is suggested that both protein-based and the novel PCR-based technologies (aQPCR, MAD PCR), and the new soil extraction method (DIRT(s)), can be developed to provide useful new approaches to detect and diagnose plant nematodes in biosecurity applications.