Dissertations / Theses on the topic 'Induced systemic resistance'

Biochar is the solid, carbon-rich by-product obtained from pyrolysis. It offers the prospect of long-term carbon sequestration and soil conditioning with agronomic benefits, often referred to as the ‘biochar effect‘. These multiple direct or indirect changes in the soil plant interface have also been associated with the control of plant diseases by influencing the host’s systemic induced resistance. The biological impact of biochar on the phytopathology of a major cereal grain has not yet been investigated. The most damaging foliar disease of barley in the UK is Rhynchosporium leaf blotch caused by the hemibiotrophic fungal pathogen Rhynchosporium commune. The aim of this project was to evaluate biochar performance and effectiveness as a disease control agent in the barley – R. commune – pathosystem, and attempted to identify underlying mechanisms by which biochar may function in the interaction between barley and the causal pathogen. Therefore, a unique set of nine slow pyrolysis biochars were established along a 350 to 800°C pyrolysis temperature gradient, with eight of these made from pelleted softwoods and a single one made from Miscanthus straw. A comprehensive biochar quality assessment was undertaken and established that the biochars differed in their chemical composition, which largely depended on production parameters, predominantly temperature (P < 0.05). The analysis proposed that biochar 9, made from Miscanthus at 800°C, showed added value as a soil conditioner over softwood biochars, due to higher pH, mineral ash and macronutrient recoveries, which pointed towards a possible liming potential. Regardless of the feedstock, biochars pyrolysed above 600°C indicated potential use for carbon sequestration purposes, due to higher carbon stability. Short-term controlled bioassays showed significant restricted growth of R. commune mycelium on defined medium to direct (1.0% w/w) and indirect volatile exposure from certain biochars (P < 0.001). The findings suggested a synergistic effect of the softwood biochars acidic nature and presence of fungicidal compounds, with observed inhibition of 100% attributed to re-condensation of tarry vapours onto biochar surfaces during pyrolysis. Qualitative biochar volatile organic compound analysis was conducted and identified biocide active phenolic and organic acid compounds, similar to those commonly found in smoke, bio-oils or wood vinegars. These findings proposed possible application for mitigation of inoculum pressure in field-grown barley, but the toxic nature of volatiles raised concerns over risks to human and environmental health, as also evidenced by detrimental barley growth effects. Subsequent controlled in vivo and in planta experiments revealed significant (P < 0.05) symptomatic barley leaf blotch reduction effects of up to 100%, following 5% (w/w) application of biochars 4, 5, 8 and 9. Barley plants transcriptional changes in ISR-dependent LOX2 and SAR-dependent PR1-b expression in planta verified systemic induced resistance as mechanisms behind the significant disease suppression of barley plants grown in soil amended with biochar 5 and 8. Disease reduction and biochar mediated induced resistance was attributed to either low concentrations of phytotoxic compounds, a direct toxicity effect from fungicidal compounds or indirect promotion of beneficial microbes. The results provided evidence, that in the case of the studied pathosystem, there is potential for biochar with specific characteristics to be considered as a soil amendment, offering not only carbon sequestration, but also possible improved disease resistance.

Plants have evolved an efficient mechanism to defend themselves against pathogens. Many biotic and abiotic agents have been shown to induce defense mechanism in plants. Acibenzolar-S-Methyl (ASM) is a commercially available chemical inducer of local and systemic resistance (SAR) response in plants. ASM functioning at molecular level is mostly unclear. This research was designed to investigate the mechanism of ASM action in plants. It was hypothesized that SABP2, a plant protein, plays an important role in ASM-mediated defense signaling. Biochemical studies were performed to test the interaction between SABP2 and ASM. Transgenic SABP2-silenced tobacco plants were used to determine the role of SABP2 in SAR induced by ASM. The expression of PR-1 proteins was used as a marker for SAR induction. Results showed that SABP2 converts ASM into acibenzolar that induces the expression of PR-1 proteins and develops the SAR response in ASM-treated plants.

Most rice protection methods have currently used toxic chemicals to control pathogens and pests, which leads to environmental pollution. Systemic acquired resistance (SAR) taking advantage of natural defence reaction of plants could be proposed as an alternative, ecologically friendly approach for plant protection. Its application into rice production could minimize the chemicals quantity used and could contribute to the decrease of environmental pollution and the development of sustainable agriculture. The research was conducted to select the most effective chemical and suitable method to improve the health of rice plants infected by grassy stunt disease in net-house of Can Tho University. SAR chemicals were used at very low concentrations (in mM). Results showed that the height of rice plants treated with SAR chemicals was higher than that of plants untreated. Besides, the number of diseased plants was reduced and the ratio of firm grain and yield increased when plants were applied by SAR. Among the used substances, oxalic acid provided the best systemic acquired resistance. With oxalic acid, seed soaking was better than seed coating in systemic acquired resistance against rice grassy stunt disease
Hầu hết các phương pháp sản xuất lúa hiện nay đều sử dụng các hóa chất độc hại trong việc phòng trừ bệnh và côn trùng gây hại, nên dẫn đến ô nhiễm môi trường. Kích thích tính kháng lưu dẫn giúp kích hoạt cơ chế tự nhiên kháng bệnh của cây có thể là giải pháp bảo vệ thực vật thay thế an toàn với môi trường. Việc ứng dụng tiến bộ này vào trong sản xuất lúa có thể làm giảm lượng hóa chất sử dụng, đóng góp vào việc giảm thiểu ô nhiễm môi trường và sự phát triển của một nền nông nghiệp bền vững. Nghiên cứu đã được thực hiện tại nhà lưới trường Đại học Cần Thơ để tuyển chọn hóa chất và phương pháp sử dụng hóa chất để tăng cường sức khỏe giúp cây lúa vượt qua bệnh vàng lùn. Hóa chất kích kháng được sử dụng ở một nồng độ rất thấp (đơn vị là mM). Kết quả cho thấy chiều cao cây lúa khi xử lý chất kích kháng tốt hơn so đối chứng không xử lý. Bên cạnh đó, số cây lúa nhiễm bệnh giảm, tỉ lệ hạt chắc và năng suất tăng khi cây lúa được xử lý với chất kích kháng. Trong số các chất kích kháng đã sử dụng, acid oxalic cho hiệu quả vượt trội. Với chất acid oxalic, phương pháp ngâm hạt cho hiệu quả kích kháng tốt hơn phương pháp áo hạt

Wheat is the most economically important crop forming one quarter of Australian farm production. The wheat industry is severely affected by diseases, with fungal pathogens causing the most important economic losses in Australia. The application of fungicides and chemicals can control crop diseases to a certain extent, however, it is expensive and public concern for the environment has led to alternative methods of disease control to be sought, including the use of microorganisms as biological control agents. Microorganisms are abundant in the soil adjacent to plant roots (rhizosphere) and within healthy plant tissue (endophytic) and a proportion possess plant growth promotion and disease resistance properties. Actinobacteria are gram-positive, filamentous bacteria capable of secondary metabolite production such as antibiotics and antifungal compounds. A number of the biologically active endophytes belonging to the Actinobacteria phylum were isolated in our laboratory. A number of these isolates were capable of suppressing the wheat fungal pathogens Rhizoctonia solani, Pythium sp. and Gaeumannomyces graminis var. tritici, both in vitro and in planta indicating the potential for the actinobacteria to be used as biocontrol agents. The aim of this research was to investigate the molecular mechanisms underlying this plant-microbe interaction. The indigenous microbial populations present in the rhizosphere and endophytic environment are critical to plant health and disruptions of these populations are detrimental. The culture-independent technique Terminal Restriction Fragment Length Polymorphism (T-RFLP) was used to characterise the endophytic actinobacteria population of wheat roots under different conditions. Soils which support a higher number of indigenous microorganisms result in wheat roots with higher endophytic actinobacterial diversity and level of colonisation. Sequencing of 16S rRNA gene clones, obtained using the same actinobacteria-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity, and identified a number of Mycobacterium and Streptomyces species. It was found that the endophytic actinobacterial population of the wheat plants contained a higher diversity of endophytic actinobacteria than reported previously, and that this diversity varied significantly among different field soils. The endophytic actinobacteria have previously been shown to protect wheat from disease and enhance growth when coated onto the seed before sowing. As the endophytes isolated were recognised as potential biocontrol agents, the impact on the indigenous endophytic microbial population was investigated. Utilising the T-RFLP technique it was established that the use of a commercial microbial inoculant, containing a large number of soil bacterial and fungal strains applied to the soil, disrupts the indigenous endophyte population present in the wheat roots. The hypothesis is that non-indigenous microbes proliferate and dominate in the soil preventing a number of endophytic-competent actinobacterial genera from access to the seed and ultimately endophytic colonisation of the wheat roots. This dramatically reduces diversity of endophytes and level of colonisation. In contrast the use of a single endophytic actinobacteria endophyte inoculant results in a 3-fold increase in colonisation by the added inoculant, but does not significantly affect this indigenous population. Colonisation of healthy plant tissues with fungal endophytes has been shown to improve the competitive fitness with enhanced tolerance to abiotic and biotic stress and improved resistance to pathogens and herbivores. In this study the fungal endophyte population of wheat plants grown in four different soils was analysed using partial sequencing of 18S rRNA gene sequences. Sequence anlaysis of clones revealed a diverse range of fungal endophytes. In this diverse range of fungal endophytes a number sequences were highly similar to those of previously known fungal phytopathogens. A number of sequences detected were similar to fungal species previously identified in soil or plant material but not as endophytes. The remaining sequences were similar to fungal species without a known relationship with plants. Plants have developed an inducible mechanism of defence against pathogens. In addition to local responses plants have developed a mechanism to protect uninfected tissue through a signal that spreads systemically inducing changes in gene expression. In the model plant Arabidopsis thaliana activation of the Systemic Acquired Resistance (SAR) pathway and the Jasmonate (JA)/Ethylene (ET) pathway is characterised by the production of pathogenesis-related (PR) and antimicrobial proteins resulting in systemic pathogen resistance. Endophytic actinobacteria, isolated from healthy wheat roots in our laboratory, have been shown to enhance disease resistance to multiple pathogens in wheat when coated onto the seed before sowing. Real Time RT-PCR was used to determine if key genes in the SAR and JA/ET pathways were induced in response to inoculation with endophytic actinobacteria. Inoculation of wild-type Arabidopsis thaliana with selected strains of endophytic actinobacteria was able to �prime� the defence pathways by inducing low level expression of SAR and JA/ET genes. Upon pathogen infection the defence-genes are strongly up-regulated and the endophyte coated plants had significantly higher expression of these genes compared to un-inoculated plants. Resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora was mediated by the JA/ET pathway whereas the fungal pathogen Fusarium oxysporum triggered primarily the SAR pathway. Further analysis of the endophytic actinobacteria-mediated resistance was performed using the Streptomyces sp. EN27 and Arabidopsis defence-compromised mutants. It was found that resistance to E. carotovora subsp. carotovora mediated by Streptomyces sp. EN27 occurred via a NPR1-independent pathway and required salicylic acid whereas the jasmonic acid and ethylene signalling molecules were not essential. In contrast resistance to F. oxysporum mediated by Streptomyces sp. EN27 occurred via a NPR1-dependent pathway but also required salicylic acid and was JA- and ET-independent. This research demonstrated that inoculating wheat with endophytic actinobacteria does not disrupt the indigenous endophytic population and may be inducing systemic resistance by activating defence pathways which lead to the expression of antimicrobial genes and resistance to a broad range of pathogens.

Cucumber root rot caused by Pythium aphanidermatum can be suppressed by introduced plant growth-promoting rhizobacteria (PGPR). Preliminary experiments clarified that this root disease could be suppressed by strains of Pseudomonas aureofaciens, P. corrugata, and P. fluorescens. To determine whether the mechanism was a systemic resistance induced by PGPR, a split root technique was employed on greenhouse cucumbers grown in soilless substrates. On the split roots, bacteria which were introduced into one side of the root were completely separated from pathogen challenged-inoculated roots-on the other side of the roots. Results from the series of experiments conducted with this design demonstrated that (i) the resistance against root rot induced by PGPR was systemic, (ii) germination of P. aphanidermatum zoospores was reduced in extracts from bacterized roots compared to non-treated control, and (iii) spread of Pythium mycelia was delayed and zoospore germination was inhibited on the distant induced root, compared to the non-bacterized control. Furthermore, enzyme analysis indicated that phenylalanine ammonia lyase, peroxidase and polyphenoloxidase increased on cucumber roots two days after they were bacterized with Pseudomonas strains 13 or 63--28. When the bacterized roots were challenged with P. aphanidermatum, these plant defense enzymes increased as the symptoms appeared, but this accumulation of enzymes was not any higher on roots induced with each of the Pseudomonas strains compared to the Pythium inoculated control. This enzyme stimulation was also systemically induced by PGPR or P. aphanidermatum on cucumber roots. The patterns of iso-peroxidase induced with the PGPR and P. aphanidermatum treatments were different. High levels of salicylic acid (SA) accumulated in bacteria-induced roots, as well as in pathogen-infected roots, which suggests that SA may be associated with cucumber resistance response. But exogenous application of SA did not induce any systemi

Plants use several strategies to defend themselves against microbial pathogens. These include basal resistance, which is induced in response to pathogen encoded effector proteins, and resistance (R) protein-mediated resistance that is activated upon direct or indirect recognition of pathogen encoded avirulence protein(s). The activation of Rmediated signaling is often associated with generation of a signal, which, upon its translocation to the distal uninfected parts, confers broad-spectrum immunity against related or unrelated pathogens. This phenomenon known as systemic acquired resistance (SAR) is one of the well-established forms of induced defense response. However, the molecular mechanism underlying SAR remains largely unknown. Induction of plant defense is often associated with a fitness cost, likely because it involves reprogramming of the energy-providing metabolic pathways. Glycerol metabolism is one such pathway that feeds into primary metabolism, including lipid biosynthesis. In this study, I evaluated the role of glycerol-3-phosphate (G3P) in host-pathogen interaction. Inoculation with the hemibiotrophic fungal pathogen Colletotrichum higginsianum led to increased accumulation of G3P in wild-type plants. Mutants impaired in biosynthesis of G3P showed enhanced susceptibility, suggesting a correlation between G3P levels and basal defense. Conversely, increased biosynthesis of G3P correlated with enhanced resistance. The Arabidopsis genome encodes one copy of glycerol kinase (GK), which catalyzes phosphorylation of glycerol to G3P, and five copies of G3P dehydrogenase (G3Pdh), which catalyze reduction of dihydroxyacetone phosphate to G3P. Analysis of plants mutated in various G3Pdh's showed that plastidal lipid biosynthesis was only dependent on the GLY1 isoform but the pathogen induced G3P pool required the function of GLY1 and two other G3Pdh isoforms. Interestingly, compromised G3P biosynthesis in GK and G3Pdh mutants also compromised SAR, which was restored when G3P was provided exogenously. Detailed biochemical analysis showed that G3P was transported to distal tissues and that this process was dependent on a lipid transfer protein, DIR1. Together, these results show that G3P plays an important role in both basal- and induced-defense responses.

Au cours de cette thèse, un isolat de sol de désert, Saccharothrix algeriensis NRRL B-24137, a été évalué pour ses propriétés bioactives contre le champignon phytopathogène Botrytis cinerea, pour sa colonization sur Vitis vinifera L., et Arabidopsis thaliana ainsi qu’en vue d’étudier les méchanismes de résistance systémique induite (ISR) contre B. cinerea. Les résultats obtenus nous ont permis premièrement de montrer que Sa. algeriensis NRRL B-24137 peut présenter des activités antifongiques contre B. cinerea et que des métabolites peuvent être responsables de cette activité antifongique. Bien que ces métabolites soient encore en cours d’étude et que cette étude mérite d’être approfondie, nous avons démontré ensuite les propriétés de colonisation de l’isolat du sol du désert chez la vigne. Les résultats ont permis de montrer que la souche peut former des populations rhizosphèriques ainsi que des sous-populations endophytiques chez des plants de vigne (Cabernet Sauvignon sur porte-greffe 44-53 M) à des étapes précoces de colonisation. Puis nous avons démontré que la souche bénéfique peut induire une résistance systémique contre B. cinerea. Bien que les mécanismes impliqués ne soient pas encore compris, des parties préliminaires de ces travaux démontrent que les expressions de gènes responsables de la production de glucanase, chitinase ainsi qu’un inhibiteur de polygalacturonase ne semblent pas potentialisés pendant le phénomène de résistance systémique. Enfin nous avons démontré l’interaction entre Sa. algeriensis NRRL B-24137 et Arabidopsis thaliana qui résulte dans une association intime dûe également à colonisation rhizosphèrique et endophytique de la plante modèle. La souche bénéfique peut églement induire un phénomène de résistance systémique sur A. thaliana contre B. cinerea et les analyses de plantes mutées ont permis de determiner des parties des mécanismes impliqués dans l’ISR aini que des nouveaux mécanismes impliqués qui peuvent être induits par des microbes bénéfiques
In this thesis, the desert soil isolate, Saccharothrix algeriensis NRRL B-24137, has been evaluated for its bioactive properties towards the phytopathogenic fungus Botrytis cinerea, for its colonization of Vitis vinifera L., and Arabidopsis thaliana as well as to study the mechanisms of induced systemic resistance (ISR) towards B. cinerea. The results obtained allowed us firstly to show that Sa. algeriensis NRRL B-24137 can exhibit strong antifungal properties towards B. cinerea and that some metabolites can be responsible of this antifungal activity. Although these metabolites are still under consideration and that this study needs further works, we have demonstrated then the colonization properties of the desert soil isolate with grapevine plants. The results showed that the strain can form rhizospheric as well as endophytic subpopulations with grapevine plants (Cabernet Sauvignon cultivar graffed on 44-53 M rootstock) at early step of colonization. Then we have demonstrated that the beneficial strain could induce a systemic resistance towards B. cinerea. Although the mechanisms are not yet well understood, preliminary parts of this work demonstrated that the genes responsible of glucanase production, chitinase as well as inhibitor of polygalacturonase activity do not seems to be primed during the systemic resistance phenomenon. Finally we demonstrated that the interaction between Sa. algeriensis NRRL B-24137 and Arabidopsis thaliana plants results in a close association due also to a rhizo- and endophytic colonization of the model plant. The beneficial strain can also induce a systemic resistance in A. thaliana towards B. cinerea and analyzes of plant mutants have allowed to determine parts of the mechanisms involved in ISR as well as new mechanisms that could be trigerred by beneficial microbes

Mestrado em Biomedicina Molecular
Fatty liver disease is simultaneously a cause and a consequence of type 2 diabetes. Hepatic lipid metabolism is altered in obese patients, causing insulin resistance. More, inhibition of insulin signaling may also affect hepatic lipid metabolism, causing a feedback that may lead to hepatic steatosis, common in such patients. In this work, we intended to assess the role of glycation (methylglyoxal-induced) in the hepatic lipid metabolism of high-fat diet-fed rats, using lipidomic approaches and magnetic resonance imaging, which identify hepatic lipid species, including phospholipids (PL), triglycerids (TG), diacylglycerols (DAG) and fatty acids (FA). Wistar rats were maintained during 4 months with methylglyoxal (MG) supplementation (100mg/Kg/day) (MG group), a high-fat diet rich in TG (HFD group) or both (HFDMG group) and compared with controls feeding a standard diet (n=6/ group). Lipidomic approaches, namely liquid chromatography - mass spectrometry (LC-MS) and gas chromatography (GC) were used to determine liver composition in PL, TG and FA. Non-invasive 1H nuclear magnetic resonance (NMR) spectroscopy (9 Tesla) of liver tissues in vivo was used to determine lipid species, such as TG and DAG. The total and phosphorylated levels of the mediators of the insulin receptor pathway and lipid oxidation were determined by western blotting. High-fat diet-fed (HFD) rats showed increased body weight in relation to controls, but this effect was partially inhibited by MG supplementation (HFDMG group). Moreover, HFDMG group showed increased plasma free fatty acid levels, hyperinsulinemia, insulin resistance and glucose intolerance. In liver, lipidomic techniques and 1H NMR showed increased fat mass in the liver of HFD and HFDMG rats. HFD rats, but not HFDMG, showed increased total levels of the 18:1 fatty acid (common in high-fat diets). Despite no differences were observed for HFD group, HFDMG rats showed decreased fraction of unsaturated lipids and increased fraction of saturated lipids. This difference was obtained due to a decrease in monounsaturated FA. Regarding lipid esterification, HFDMG group showed lower percentage of esterified glycerol carbons, suggesting an increased concentration of DAG in relation to TG. In accordance, this group showed higher fatty acids/glycerol ratio, suggesting increased liver non-esterified fatty acid levels. Western Blotting analyses showed decreased activation of insulin pathway, especially HFDMG group, as well as decreased activation of the insulin receptor in HFDMG group. Data suggest that glycation changes lipid metabolism in a context of hyperlipidemia, possibly contributing to hepatic lipotoxicity and to accelerate progression of insulin resistance and fatty liver disease.
O fígado gordo é simultaneamente uma causa e consequência da diabetes mellitus tipo 2. O metabolismo lipidico-hepático (MLH) encontra-se alterado em obesos, causando insulino-resistência. A diminuição da sinalização da via da insulina pode igualmente afetar o MLH, estimulando o desenvolvimento de esteatose hepática, comum nos doentes. Neste trabalho, pretende-se analisar o papel da glicação (induzida por metilglioxal) no MLH em ratos com dieta gorda, através de técnicas de lipidómica e ressonância magnética, para identificar as espécies lipídicas hepáticas, tais como fosfolípidos (FL), triglicéridos (TG), diacilgliceróis (DAG) e ácidos gordos (AG). O modelo animal usado foi o rato Wistar, mantido nos últimos 4 meses, antes de completar 1 ano de idade, com metilglioxal (100mg/Kg/dia) (grupo MG), com dieta gorda rica em TG (grupo HFD) ou com ambas (grupo HFDMG) e comparados com os controlos com dieta normal (n=12/grupo). As técnicas de lipidómica usadas foram cromatografia líquida com espetrometria de massa e cromatografia gasosa para determinar a composição hepática de PL, TG e AG. Usou-se também espectroscopia (9 Tesla), não invasiva, de ressonância magnética nuclear 1H (NMR) nos ratos vivos para determinar os TG e DAG hepáticos. Os mediadores proteicos totais e fosforilados da via da insulina e da oxidação lipídica no fígado também foram analisados por western blot. Os ratos, com dieta gorda (HFD), aumentaram o peso corporal, mas o efeito foi parcialmente inibido pelo metilglioxal (HFDMG). Além disso, o grupo HFDMG apresenta um aumento dos ácidos gordos livres no plasma, hiperinsulinemia, insulino-resistência e intolerância à glicose. No fígado, as técnicas de lipidómica e NMR mostraram um aumento da massa gorda no fígado nos grupos HFD e HFDMG, mas apenas no grupo HFD se verifica o aumento do AG 18:1 (comum na dieta). Apesar de não haver diferença significativa no grupo HFD, o grupo HFDMG apresenta uma diminuição dos AG insaturados e aumento dos saturados; isto deve-se à diminuição dos monoinsaturados neste grupo. Quanto à esterificação dos glicerolípidos, o grupo HFDMG apresenta uma menor percentagem da total esterificação dos gliceróis, sugerindo o aumento dos DAG, em relação aos TG. Também, este grupo apresenta um ratio AG/glicerol aumentado, ou seja, com aumento de AG não esterificados. A análise por western blot mostrou uma diminuição da via do receptor da insulina especialmente no grupo HFDMG. Em suma, estes resultados sugerem que a glicação causa alterações do metabolismo lipidico-hepático num contexto de hiperlipidemia, contribuindo possivelmente para a lipotoxicidade hepática, progressão acelerada de insulino-resistência e patologia do fígado gordo.

Food safety has become a worldwide priority in public health. Among the most important food threats, mycotoxin contamination in agricultural products represents a past, daily and incoming problem to solve. Mycotoxins are chemical compounds produced by a large variety of Fungi with very different hosts and climatic needs. In European countries, climatic conditions lead to the development of different mycotoxigenic fungi. Climate conditions of Mediterranean basin promote the development, among all, of Fusarium verticillioides in maize and Aspergillus carbonarius in grape, responsible for the production of fumonisins and ochratoxins respectively. Treatments with many synthetic chemical compounds have been proved to reduce the contamination levels but economical, ecological and nutritional aspects in public opinion, lead to attempt new strategies, as biological control. The state of art for mycotoxins production and regulation is very diversified according to the importance of economical threat, so while fumonisins are well studied, the knowledge on ochratoxins presents many gaps. In the present work, molecular approaches as enzymatic activity, Real-Time PCR and cDNA-AFLP have been used to provide useful knowledge and strategies to biological control of mycotoxigenic fungi. First, the ability of Trichoderma harzianum T22 to induce resistance in maize against Fusarium verticillioides was evaluated, pointing out the decrease of fumonisin accumulation in field trials. The second section improved the importance of reactive oxygen species for initiation and modulation of fumonisin biosynthesis in F. verticillioides supporting the hypothesis that plant stresses promote fumonisins accumulation in kernels. Finally, in the latter part, 119 differentially expressed sequences by Aspergillus carbonarius gave an important contribution to the understanding of OTA biosynthesis suggesting, according to the up- and down-regulation patterns, a possible model for OTA biosynthesis and regulation.
La sicurezza alimentare è oggigiorno considerata una priorità a livello mondiale. Tra le principali minacce legate al settore alimentare, le micotossine rappresentano una problematica da contenere e risolvere. Le micotossine sono sostanze chimiche prodotte dal metabolismo secondario di alcuni funghi con ospiti e necessità climatiche molto diversi tra loro. Diverse condizioni climatiche portano alla sviluppo di funghi capaci di produrre diverse micotossine e per i paesi affacciati sul bacino mediterraneo, due tra i funghi micotossigeni più presenti risultano essere Fusarium verticillioides in mais e Aspergillus carbonarius sull’uva, responsabili dell’accumulo rispettivamente di fumonisine e ocratossina A. I trattamenti effettuati con composti chimici hanno dato prova di essere parzialmente efficaci nel ridurre i livelli di contaminazione, ciononostante i vari aspetti economici, ecologici e di salubrità dovrebbero condurre allo sviluppo di nuove strategie a basso impatto come il controllo biologico. Le conoscenze finora ottenute sulla produzione e la regolazione delle micotossine sono molto diversificate in base all’importanza economica che queste rivestono, mentre le fumonisine sono ben studiate, le informazioni riguardanti le ocratossine sono ancora lacunose. Nel presente lavoro diversi approcci molecolari quali studi di attività enzimatica, Real-Time PCR e cDNA-AFLP sono stati utilizzati per fornire elementi e strategie utili al controllo biologico dei funghi micotossigeni. Nella prima parte del presente lavoro è stata valutata la capacità di Trichoderma harzianum T22 di indurre resistenza in mais nei confronti di F. verticillioides evidenziando in particolar modo l’abbattimento del contenuto di fumonisine nella granella raccolta. La seconda parte è focalizzata sui cambiamenti indotti dalle specie reattive dell’ossigeno su F. verticillioides, confermando anche per questo fungo l’importanza dei ROS nell’attivazione e la modulazione della biosintesi delle fumonisine. Tali risultati supportano l’ipotesi che l’accumulo di fumonisine sia fortemente influenzato dalle situazioni di stress percepite dalla pianta. Infine, lo studio svolto sulle sequenze differentemente espresse di A. carbonarius rappresenta un importante contributo alla comprensione della pathway biosintetica suggerendo, in accordo con i diversi pattern di espressione, un possibile modello di regolazione e biosintesi dell’ocratossina A.

This PhD project aims to study the interactions between endophytic bacteria and phytoplasmas associated with grapevines to clarify the role of endophytic bacteria in grapevine yellows (GY) recovery and discuss the possibility to apply these bacteria for GY management. Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. To identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Library analysis of a PCR-amplified 16S rDNA fragment showed best sequence matches with Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Nucleotide sequences of 16S rDNA from bacteria isolated through cultivation showed best sequence matches with Curtobacterium, Stenotrophomonas, Methylobacterium, Pectobacterium, Enterobacter, Brevundimonas, Agrobacterium, Brevibacillus, Staphylococcus, Sphingomonas, Acaricomes, and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Furthermore, composition and structure of endophytic bacterial community were examined in healthy, phytoplasma-diseased and recovered grapevine plants. LH-PCR of total DNA from grapevine leaves was used to generate amplicon profiles that were analyzed with univariate and multivariate statistical methods. Jaccard analyses highlighted that microbial diversity and structure is different in healthy, diseased and recovered grapevine plants. Multivariate analyses confirmed this trend and showed that three LH-PCR peaks determined the variation in microbial composition. Furthermore, LH-PCR database were used to monitor the distribution of bacterial endophytes in total DNAs from analyzed plants. Bacterial community associated with healthy plants was characterized by a greater richness (higher number of LH-PCR peaks) than that present in diseased and recovered plants. Observed low bacterial richness and different microbial composition in infected and recovered plants suggest that phytoplasma infection could directly and/or indirectly restructure bacterial community selecting endophytic strains that are able to elicit plant defense response. Moreover, we investigated the influence of Flavescence dorée phytoplasmas (FDp) on endophytic bacterial community by studying the seasonal fluctuation of bacterial species associated with healthy, FDp-diseased and recovered plants during phytoplasma infection process. Statistical analysis indicated that, before phytoplasma titre inside diseased plant tissues becomes detectable, endophytic bacterial community is similar to that associated with healthy plants and differs from that associated with recovered plants. Moreover, it seems that a change in microbial composition could be determined when phytoplasmas start to replicate. LH-PCR database showed a seasonal fluctuation of some bacteria identified in the analyzed grapevines; these fluctuations are also related to the presence/absence of the pathogen. On the basis of these evidences we hypothesized that phytoplasma replication determines a change in microbial composition selecting few endophytes that could induce recovery mediated by priming response. Finally, in order to study in which way phytoplasmas interact with endophytes and host plant we developed and tested two different microscopy techniques. Fluorescence in situ hybridisation (FISH) allowed to localise, as expected, phytoplasmas in Catharanthus roseus L. (G. Don) phloem tissues, and also to visualize endophytes in phloem, xylem and parenchyma in the same leaf tissues. On the other hand, immune-confocal laser scanning microscopy was applied for demonstrating that SAP11 (a phytoplasma effector protein) can be unloaded from the phloem by itself. These techniques will be used in combination for clarifying if phytopasmas co-localise with ISR bacterial-inducers and for studying proteins involved in phytoplasmas-endophytes-host cross-talk.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The massive use of chemicals to control plant diseases cause numerous negative impacts on agricultural production system on non-target organisms, consumers and farmers. Therefore, the use of biological control organisms such as Trichoderma harzianum and Bacillus subtilis, able to act on the control of plant pathogens and / or induce resistance in plants is characterized as an important and with great potential yet to be explored tool. The objective of this study was to identify metabolites produced by these organisms grown in liquid medium and test the use of filtered culture and of living organisms, applied via seed or leaf in the control of anthracnose in beans. Biological control organisms T. harzianum and B. subtilis were grown in liquid culture medium for 96 h and 12 h photoperiod. After incubation, the media were filtered through Millipore membrane extraction of the bacterial cells and spores. Metabolites were extracted with four organic solvents, ethanol, methanol, ethyl acetate and hexane and subjected to gas chromatography and mass spectrometry and the chromatograms generated for each organism and solvent used. The evaluation of the induction of defense responses in plants was held in cultivating Minuano with susceptibility reaction to the pathogen Colletotrichm lindemuthianum. The bean plants were evaluated for disease index, area under the disease progress curve and changes in peroxidase activity, β-1,3-glucanase and IAA - oxidase, as well as certain fresh and dry weight of plants in two stages, before and after inoculation of the challenging pathogen. The chromatographic identification of compounds from culture filtrates showed that both test organisms produce fatty acids and oxylipins in liquid medium with antifungal action and inducing defense responses, respectively. In the assessments performed in bean plants, both T. harzianum as B. subtilis reduced the severity and progress of the disease, and produced significant increases in the activities of peroxidase isozymes and β-1,3-glucanase after inoculation of the pathogen, indicating the activation of induced resistance. The induction of responses did not affect the dry mass of the plants, which shows no energy expenditure that could affect plant growth. The best answers to T. harzianum are observed in foliar applications, either spore suspension or culture filtrate, as for B. subtilis, the best answers are observed in the foliar application of culture filtrate. For ease of production, culture filtrates of Trichoderma harzianum and Bacillus subtilis can give bioproducts for agricultural use.
O uso massivo de produtos químicos para controle de doenças em plantas causa inúmeros impactos negativos no sistema de produção agrícola, sobre organismos não alvo, consumidores, agricultores e o ambiente. Diante disso, a utilização de organismos de controle biológico, como Trichoderma harzianum e Bacillus subtilis, capazes de agir no controle de fitopatógenos e/ou induzir resistência em plantas, se caracteriza como uma ferramenta importante e com muito potencial ainda a ser explorado. O objetivo deste trabalho foi identificar metabólitos produzidos por estes organismos cultivados em meio líquido e testar a utilização destes filtrados de cultura, bem como dos organismos vivos, aplicados via semente ou foliar, no controle da antracnose em feijoeiro. Os organismos de controle biológico T. harzianum e B. subtilis foram cultivados em meio de cultura líquido por 96h e fotoperíodo de 12h. Após a incubação, os meios foram filtrados em membrana milipore para extração dos esporos e das células bacterianas. Os metabólitos foram extraídos com quatro solventes orgânicos, etanol, metanol, acetato de etila e hexano e submetidos à cromatografia gasosa e espectrofotometria de massa e gerados cromatogramas para cada organismo e solvente utilizado. A avaliação da indução de respostas de defesa em plantas foi realizada na cultivar Minuano, com reação de suscetibilidade ao patógeno Colletotrichum lindemuthianum. As plantas de feijão foram avaliadas quanto ao índice de doença, área abaixo da curva de progresso da doença e às alterações nas atividades de peroxidase, β-1,3-glucanase e AIA oxidase, assim como determinada a massa fresca e seca das plantas em dois momentos, antes e após a inoculação do patógeno desafiante. A identificação cromatográfica dos compostos presentes nos filtrados de cultura demonstrou que ambos os organismos testados produzem ácidos graxos e oxilipinas em meio líquido, com ação antifúngica e indutoras de respostas de defesa, respectivamente. Nas avaliações realizadas em plantas de feijão, tanto T. harzianum quanto B. subtilis, reduziram a severidade e o progresso da doença, assim como produziram acréscimos significativos nas atividades das isoenzimas peroxidase e β-1,3-glucanase após a inoculação do patógeno, indicando a ativação da resistência induzida. As respostas de indução não comprometeram o acúmulo de massa seca das plantas, o que demonstra não haver gasto energético capaz de comprometer o crescimento das plantas. As melhores respostas para T. harzianum são observadas nas aplicações foliares, seja de suspensão de esporos ou filtrado de cultura, já para B. subtilis, as melhores respostas são observadas na aplicação foliar do filtrado de cultura. Pela facilidade de produção, filtrados de cultura de Trichoderma harzianum e Bacillus subtilis podem originar bioprodutos para uso agrícola.

Il est actuellement estimé qu’environ 13% du vignoble français est improductif suite aux pathologies affectant le bois des ceps, la principale d’entre elles étant l’esca. Parmi les moyens de lutte mis en œuvre, le biocontrôle, via l’utilisation d’un oomycète, Pythium oligandrum, est actuellement développé pour protéger les plants de vigne contre un agent pathogène pionnier de l’esca, Phaeomoniella chlamydospora. La sélection de souches de P. oligandrum, isolées du vignoble, et produisant in vitro des quantités importantes d’une protéine élicitrice, l’oligandrine, des systèmes de défense des végétaux a d’abord été réalisée. Trois essais en serre ont montré qu’une réduction significative (40 à 50%) des nécroses dues P. chlamydospora était observée après application d’inocula de l’oomycète sur les racines des plants de vigne pied-francs Au niveau de la tige, le niveau d’expression de 22 gènes impliqués dans les mécanismes de défenses de Vitis vinifera a été mesuré par PCR quantitative et des réponses spécifiques du végétal ont été observées selon les traitements. Six gènes (protéines PR, voie des phenylpropanoïdes, oxylipines et le système d’oxydo-réduction) ont été fortement induits lorsque les plants ont été pré-inoculés par P. oligandrum puis infectés par P. chlamydospora. Afin de mettre en évidence les mécanismes spécifiques mis en place lors de cette interaction tripartite, l'analyse de la réponse transcriptomique globale de la vigne (par microarray et RNAseq), au niveau de la tige, a été réalisée chez ces plants qui manifestent une résistance induite systémique (ISR). Plusieurs gènes impliqués dans la synthèse de l’éthylène et des jasmonates sont fortement induits, chez les plants pré-traités par l’oomycète puis infectés par l’agent pathogène. Plusieurs facteurs de transcription régulant ces voies de signalisation sont également fortement induits. Suite à l’analyse des populations de messagers (mRNA) de P. chlamydospora, il a été observé que les niveaux d’expression de gènes impliqués dans la synthèse des métabolites secondaires, des facteurs de transcription impliquées dans la régulation de différentes voies chez les champignons et certaines Carbohydrates Actives enZymes étaient modulés en présence de P. oligandrum au niveau racinaire. Ces résultats montrent que la colonisation du végétal par l’oomycète, même à distance de P. chlamydospora, induit un stress indirect important chez celui-ci. Afin d’optimiser l’implantation de cet agent de biocontrôle en pépinière et au vignoble, l’aptitude de P. oligandrum à coloniser les racines de plants de vignes greffés et à les protéger contre P. chlamydospora a été étudiée. Trois portes-greffes (SO4, 3309 et 101-14) greffés sur des cépages (Cabernet Sauvignon et Sauvignon Blanc) ont été inoculés ou non par P. oligandrum. L’oomycète s’implantait sur les différents systèmes racinaires, mais en proportion variable selon les associations cépage/porte-greffe utilisées. Les analyses par empreintes moléculaires (Single Strand Conformation Polymorphism) ont montré que des microflores fongiques et bactériennes complexes et diversifiées colonisaient les feuilles et les racines, mais que l’introduction de P. oligandrum sur la plante n’induisait pas de bouleversements directs ou indirects notables au niveau de ces microflores indigènes. Une protection des jeunes plants de vigne greffés (SO4 + Cabernet Sauvignon) semble être induite par P. oligandrum contre l’agent pathogène, P. chlamydospora
Approximately 13% of French vineyards are currently considered unproductive due to trunk diseases, mainly Esca, a particularly destructive disease that affects grapevines worldwide. Accordingly, biological control of a pathogen implicated in Esca, Phaeomoniella chlamydospora, was developed using the oomycete, Pythium oligandrum. The selection of P. oligandrum strains, isolated from vineyards, which produced in vitro large quantities of oligandrin, an elicitin-like protein inducing plant defences, was carried out. Three greenhouse assays showed that the necroses caused by P. chlamydospora were significantly reduced (40 to 50%) when P. oligandrum colonized the root system of vine cuttings. At stem level, the expression of a set of 22 genes involved in Vitis vinifera defence mechanisms was measured by quantitative PCR. Depending on the treatments employed, significant differences in grapevine responses were observed. Six of the genes (PR proteins, phenyl-propanoid pathway, oxylipins and the oxydo-reduction system) were strongly induced in plants pre-treated with P. oligandrum, and subsequently infected by P. chlamydospora. In order to characterize the mechanisms occurring during this tri-partite interaction, the global transcriptomic grapevine responses at stem level were analysed, using microarray and RNAseq, in plants in which induced systemic resistance (ISR) had taken place. Several genes involved in ethylene and jasmonate biosynthesis were strongly induced in plants that were pre-treated with P. oligandrum, and subsequently infected by P. chlamydospora. The transcription factors involved in the regulation of these signalisation pathways were also induced. Analysis of the P. chlamydospora RNA messenger (mRNA), showed that certain genes involved in secondary metabolite synthesis, transcription factors implicated in pathway regulations, and certain Carbohydrate Active enZymes, were modulated, when P. oligandrum colonised the roots. These results demonstrated that root inoculation with P. oligandrum induced indirect stress on P. chlamydospora responses. In order to promote P. oligandrum implantation in nurseries and vineyards, the capacity of this biocontrol agent to colonize the roots of grafted-plants, and to protect them against P. chlamydospora attacks, was studied. Three rootstocks (SO4, 3309 and 101-14), grafted on two scion varieties (Cabernet Sauvignon and Sauvignon Blanc), were inoculated or not with P. oligandrum. Depending on the particular scion/rootstock associations, the oomycete colonized the various root systems differently. Single Strand Conformation Polymorphism (SSCP) analyses revealed complex and diverse fungal and bacterial communities in both the rhizosphere and the phyllosphere. These microflora, which were organ-dependent, were not direcly or indirectly affected by the root inoculation of P. oligandrum. Protection of grafted vines (SO4 + Cabernet Sauvignon) was probably induced by P. oligandrum against the pathogen, P. chlamydospora

Dans leur environnement, les plantes sont fréquemment soumises à des attaques de microorganismes pathogènes. Pour leur faire face, elles mettent en place des mécanismes de défense activés suite à la détection du microorganisme via des motifs moléculaires ou IPs (Invasion Patterns). Les rhamnolipides (RLs) sont des molécules glycolipidiques amphiphiles produites par des bactéries des genres Pseudomonas et Burkholderia. Ces molécules sont capables d’induire, au niveau foliaire chez différentes plantes, une résistance locale contre des microorganismes phytopathogènes. L’acide 3-hydroxydécanoïque (3-OH-C10:0), le constituant de base de la partie lipidique des RLs, active aussi une réponse immune dans la partie aérienne de la plante Arabidopsis thaliana. Cette réponse immune est déclenchée suite à sa perception par le récepteur kinase S-lectine LORE. Les travaux menés au cours ce projet de thèse ont permis de mettre en évidence que le 3-OH-C10:0 est perçu au niveau racinaire par LORE, conduisant à l’activation d’une réponse immune innée et à la mise en place d’une résistance systémique (ISR) efficace contre le champignon nécrotrophe Botrytis cinerea. D’autre part, ces travaux ont révélés que les RLs sont aussi perçus au niveau racinaire et activent une ISR contre B. cinerea n’impliquant pas le récepteur LORE. L’ensemble de ces résultats montrent que les RLs ainsi que le 3-OH-C10:0, sont deux IPs reconnus par A. thaliana au niveau racinaire via des mécanismes indépendants et tous deux conduisant à l’activation d’une résistance systémique
In their environment, plants are frequently challenged by pathogenic microorganisms. Plants are able to trigger an innate immune response to fight against the infection. This immune response is activated after perception of the microorganisms through Invasion Patterns (IPs). Rhamnolipids (RLs) are amphiphilic glycolipidics molecules produced by some bacterial species including Pseudomonas and Burkholderia. RLs are able to induce an immune response in the aerial part of several plant which is effective against phytopathogens. 3-hydroxydecanoic acid (3-OH-C10:0), a lipid building block from RLs, is known to trigger Arabidopsis thaliana immune responses in leaves after its perception by the bulb-type lectin receptor kinase LORE. In the present work, we showed that the 3-OH-C10:0 is also sensed by roots through LORE, triggering local immune responses and a systemic induced resistance (ISR) effective against the necrotrophic fungus Botrytis cinerea. In addition, this work revealed that RLs are also recognized by root cells, activating a LORE-independent ISR against B. cinerea. This work shows that RLs and 3-OH-C10:0 are different IPs independently recognized by A. thaliana roots but both inducing a systemic resistance in plants

It is a known fact that the whole agriculture system is suffering from the diseases caused by plant pathogens, affecting negatively the crop yield production, food security, biodiversity, agricultural ecosystem and hence agricultural economy. In many countries, the containment strategies of plant pathogens are still depending on chemical pesticides that cause adverse effects in the long term. According to the implementations reinforced by European council 2009/128/EC, biocontrol strategies are considered as the most profound and integrated approach for sustainable disease management. Defining biocontrol in terms of plant pathology, it is the purposeful utilization of beneficial microbes, or its molecules, to suppress phytopathogens’ ability to colonize or induce symptoms in the host. In spite of their lesser shelf-life and unreliability as compared to conventional pesticides, their targeted biological interaction with the phytopathogens reduces the possibility of affecting non-target organisms, environment and the development of resistance in the pathogen. In this context, exploitation of bacterial endophytes has gained much attention during the past decades. Endophytic plant growth promoting bacteria (ePGPBs) mediate their biocontrol efficacy by targeting species through a multitude of direct or indirect biological interactions, often employing both modes of action, such as plant growth promotion, host’s resistance induction, allelochemicals secretion, and nutrients and niche competition. Another strategy that has gained popularity is the exogenous application of double stranded RNA (dsRNA), which is considered as the key trigger molecule of RNA interreference (RNAi), a post-transcriptional gene silencing mechanism, and has been shown to provide protection without the need for integration of dsRNA-expressing constructs as transgenes. In the present doctoral thesis, the above-mentioned biocontrol strategies were adapted, utilizing “ePGPBs as microbial inoculants” and “exogenously applied dsRNA as RNAi based natural product”, against several phytopathogens belonging to different families of viruses and fungi. Regarding ePGBPs as microbial inoculants, the objective of this study was to extend our understanding of five endophytic bacterial strains; Pantoea agglomerans (255-7), Pseudomonas syringae (260-02), Lysinibacillus fusiformis (S4C11), Paraburkholderia fungorum (R8), Paenibacillus pasadenensis (R16); that have shown a promising result in previous studies. In the present doctoral study, these strains were tested in planta to evaluate their role in providing plant growth promotion and broad-spectrum protection against two target pathosystems (viruses and fungi) that might have direct, indirect or simultaneous effects, proceeded with two following aims: (Aim 1) action against viruses: Cymbidium ringspot virus (CymRSV), Cucumber mosaic virus (CMV), Potato virus X (PVX), and Potato virus Y (PVY) on Nicotiana benthamiana plants, comparing their effects with those of three chitosan-based products, which are known to induce resistance in plants; and (Aim 2) action against fungal pathogens: Rhizoctonia solani, Pythium ultimum and Botrytis cinerea on Lactuca sativa plants, comparing their effects with Bacillus amyloliquefaciens strain (CC2) and Trichoderma spp. based product, under controlled conditions. To test the priming efficacy of ePGPBs against target viruses, several phenotypic parameters were observed along with the evaluation of three plant defense related genes (EDS1, PR2B and NPR1) on Nicotiana benthamiana plants. Interestingly, the symptoms reduction was successfully registered against CymRSV and CMV with increased heights of the plants. Some of the treatments were shown correlation between severity of symptoms and the virus concentration in the plants. Furthermore, the molecular interaction indicated the involvement of a salicylic acid (SA) mediated defense pathway as evidenced by the increased expression levels of EDS1 gene in strains R16 and 260-02. Whereas, strain S4C11 showed downregulation of PR2B gene, suggesting that SA-independent pathways could be involved. These findings opened queries regarding the duration of the protective effect, host-plant- pathogen interaction, and epidemiological implications of the use of similar biocontrol strains, that reduce the symptoms but not the concentration of virus in the host. To test the ePGPBs role against target fungi (pre- and post-harvest stage), several experiments were conducted including phenotypic paraments, gene expression analysis (PR1, PAL, ThlP3, ERF1 and ACCS1), microbiota analysis in bulk soil, rhizosphere, and root associated with Lactuca sativa in the presence or absence of the inoculants, and nutritional quality parameters at time of harvest and during shelf-life of Romaine lettuce. The results were accompanied in terms of symptoms reduction by strain R16 (P. ultimum, R. solani, B. cinerea) and strain 260-02 (R. solani, B. cinerea); % seed germination by strains R16, 260-02, 255-7, S4C11 in some healthy and R. solani infected lettuce varieties; inhibition of R. solani population in soil and rhizosphere soil by strains R16, 260-02 and 255-7. Furthermore, composition of the bacterial microbiota was radically different in the rhizosphere and the root endosphere among treatments, while the bulk soil formed a single cluster regardless of treatment, indicating that the use of these treatments did not have an ecological impact outside of the plant. Also, these strains were able to contribute to the maintenance of nutritional quality indexes of lettuce at harvest and during storage. All the obtained results indicated that these strains were involved directly (via antibiosis) and indirectly (via SA or ET/JA) in the observed reduction of symptoms. Particularly, strain R16 upregulated both PAL and ACCS1 gene in R. solani infected L. sativa (suggesting co-activation of SA- and JA/ ET mediated ISR resistance); strain 260-02 upregulated PAL gene in R. solani infected romaine lettuce and showed higher levels of ascorbic acid (AsA) production in B. cinerea infected romaine lettuce (suggesting the activation of SA- and AsA-mediated antioxidant resistance); and strain 255-7 triggered PAL and ThlP3 gene up-stream expression levels indicating SA mediated pathways in R. solani infected romaine lettuce. These findings affirmed the previous conclusions and added valuable pieces of information regarding the traits these ePGPB carried, most importantly, in individuating different mode of action of the different strains in different host plants with or without the presence of pathogen. Regarding the second approach, non-transgenic strategy was employed to induce resistance against Tomato Aspermy Virus (TAV) in N. benthamiana plants. DsRNA molecules for coat protein (CP) gene was produced by a two-step PCR assay followed by in vitro transcription and purification and was exogenously applied. The implementation of CP-derived dsRNA TAV was not successful in reducing observed symptoms (mosaics, blisters, crinkling, leaf distortion, and systemic vein clearing), regardless of treatments or days of post inoculation. Only a slight difference was found in plant heights indicating that the treatment managed to reduce stunted growth of the plant at dilution 01:10 (6 and 12 dpi). The reasons could involve inappropriate concentration of dsRNA inoculum. Therefore, future studies will be conducted to optimize in vitro dsRNA molecules production to obtain higher concentrations or more specific sequences, and more suitable viral genes. Both strategies have shown interesting outcomes and gave us the future direction which will help us in designing the adequate trials (in planta or semi-field) for the disease management and diseases control through the application of ePGPBs as a microbial inoculants and dsRNA-based product individually or in combination.

Fungicide application is now the most efficacious method for controlling plant diseases caused by oomycetes and fungi. As legislation is limiting and reducing their use, it is strongly stimulating studies for the identification of additional and environmentally friendly approaches in the control of their associated diseases. Among these, systemic acquired resistance (SAR) offers the prospect of long-lasting, broad-spectrum disease control through activation of the resistance defence machinery of the plant itself. Plant activators are products employed in crop protection able to elicit SAR. Therefore, they may trigger the plant own defence response against pathogen attacks, mainly stimulating mechanisms such as the biosynthesis of phytoalexins, plant secondary metabolites with a broad spectrum biological activity. In this study, two plant defence inducers, benzothiadiazole (BTH) and chitosan (CHT), deserving particular attention because of their efficacy and low toxicity, have been used. CHT is a natural and low-cost polymer (from the waste products of the crustacean carapace), obtained by chitin deacetylation. Its effectiveness is higher when molecular weight is between 10 and 100 kD and the deacetylation degree range is from 80 to 90 percent. BTH [benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester] is a synthetic compound and a functional analogue of salicylic acid, a plant hormone-like compound deeply involved in resistance against pathogens. Two different phytoiatric campaigns were planned, in 2009 and 2010, on two red grapevine (Vitis vinifera L.) varieties cultivated in experimental vineyards located at distinct sites: Groppello, an autochthonous cultivar of Lombardia, at Raffa di Puegnago (Brescia), and Merlot at Conegliano Veneto (Treviso). Open field treatments carried out on Groppello grapevines were: i) 0.03% (w/v) CHT (76 kDa molecular weight and 85% deacetylation degree), ii) 0.03% CHT in combination with 150 g hL-1 copper hydroxide (CHT/Cu) and iii) 0.3 mM BTH; whereas, on Merlot, besides CHT and CHT/Cu, BTH were replaced by 300 g hL-1 potassium phosphites. Untreated vines were used as negative control, while plants treated with conventional fungicides (penconazole and methyldinocap) were the positive control. In both field surveys, the trial was set up as a complete randomized block design in 4 replications, with 10 vines (a parcel) per treatment in each block. Plants were sprayed approximately every 10 days, according to the meteorological conditions, from the beginning of grape susceptibility to fungal diseases until the complete véraison. The phytosanitary status of vineyards was assessed weekly on leaves and bunches, by visual inspections, though both sites were not particularly predisposed to severe fungal or other epidemics. The epidemiological evaluations were performed on bunches alone and infection indexes were calculated. All the treatments were effective in controlling fungal infections (downy mildew, Plasmopara viticola and powdery mildew, Erysiphe necator), in terms of disease incidence (I %), disease severity (S %) and infection degree (ID %), though these indexes were low in untreated control grapevines, particularly in 2010. Sampling was scheduled at two phenological phases: pre-véraison and 100% véraison; bunches were randomly collected from plants during the morning and stored at -20 °C until analyses. Berry tissues were separated into skin, flesh and seeds, powdered and extracted. Then, extracts were tested to evaluate their melatonin levels [by ultra performance liquid chromatography (UPLC)-MS/MS], total polyphenols (TP, by Folin-Ciocalteau assay) and antiradical activity [by DPPH, 2,2-diphenyl-1-pycryl hydrazyl and ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay]. Groppello and Merlot experimental wines were produced, by standard microvinification techniques, in the Centro Vitivinicolo Provinciale of Brescia and Centro di Ricerca per la Viticoltura of Conegliano (TV), respectively, and stored at 4 °C in the dark until analyses. Microvinificates were produced from grapes treated with both elicitors and conventional fungicides (controls), and the following analyses, besides those also conducted for all berry tissues, were carried out: tryptophan, serotonin and melatonin detection (by UPLC-MS/MS); content of cis and trans resveratrol (by UPLC-MS/MS); levels of mycotoxins (manly ochratoxin A, OTA, by UPLC-MS/MS). In general, berry tissues treated with elicitors and the corresponding experimental wines showed higher levels of melatonin and polyphenols, as well as a higher antiradical activity than samples treated with conventional fungicides. High concentrations of tryptophan were detected in all samples, contrary to serotonin, which was not detected. The most effective elictors were CHT/Cu and CHT. In all wines, the level of OTA was below the allowable threshold of 2 ng/mL. To the best of our knowledge, these results represent the first data on the effects of agrochemicals on the melatonin content of red wine, and the presence of melatonin was reported, for the first time, in berry seeds and flash, after its previously detection in skin. Furthermore, the level of the indolamine in berry tissues varies according to the phenological stage, resulting more abundant in seed at pre-véraison and in skin at véraison. The good agreement between the data obtained in 2009 and 2010 for Groppello cultivar, and, in 2009, between Groppello and Merlot varieties cultivated in different geographical areas, suggests that, in general, plant activators may improve some qualitative/healthy treats ascribed to red wine, though their efficacy in controlling grapevine fungal diseases should be better ascertained. Finally, the role of melatonin, a powerful antioxidant, in grapevine physiology is still somewhat obscure. It is possible that, at pre-véraison, when skin anthocyanic pigmentation is still lacking, this compound may defend berry from damage due to photooxidation or UV radiation, whereas, at véraison, the indolamine may protect the germ tissues, particularly rich in storage lipids and membranes and vulnerable to oxidative damage. In conclusion, the possibility of enhancing the pharmaconutritional potential of grape/wine with phytosanitary treatments should be further tajen into account.

L’association d’un trouble respiratoire au cours du sommeil (TRS) chez des patients présentant une cardiopathie ischémique est un facteur de mauvais pronostic. Au cours des TRS, les patients souffrent d’épisodes de désaturation-resaturation aussi appelés hypoxie intermittente (HI) et décrite comme l’une des caractéristiques des TRS la plus délétère sur le système cardiovasculaire. Les mécanismes induits par l’HI et responsables des complications cardiovasculaires ne sont pas totalement explicités et nécessitent d’être affinés afin de proposer de nouvelles stratégies thérapeutiques. Aujourd’hui, le traitement de référence des apnées du sommeil consiste à porter un support pressionnel pendant le sommeil. Seulement l’efficacité en regard de la morbi-mortalité cardiovasculaire est discutée.L’objectif de cette thèse était d’étudier l’impact de l’HI sur le développement de la cardiopathie ischémique et les mécanismes impliqués, avec un focus particulier sur l’activité sympathique.Nous avons utilisé un modèle d’ischémie coronaire permanente chez le rat, puis nous avons exposé les animaux à 12 semaines d’HI. Nous avons montré que l’HI précipite le remodelage (hypertrophie, fibrose, apoptose) et la dysfonction contractile cardiaque (fraction d’éjection). D’un point de vue mécanistique, nous avons mis en évidence une augmentation précoce et soutenue de l’activité sympathique, associée, dès 6 semaines d’exposition à l’HI, à une diminution de la réserve adrénergique cardiaque (moindres réponses à un agoniste β-adrénergique sur cardiomyocytes isolés).L’inhibition de l’activité sympathique cardiaque par l’utilisation d’un modèle chirurgical de sympathectomie cardiaque spécifique, par ablation des ganglions moyen cervicale et stellaire gauche, nous a permis de mettre en évidence que l’activité sympathique induite par l’HI est en partie responsable de l’aggravation de la cardiopathie ischémique. En effet, la dénervation sympathique cardiaque rétablie la réserve adrénergique, diminue le remodelage cardiaque (hypertrophie, apoptose) et améliore la fonction cardiaque (fraction d’éjection).Ces résultats de thèse mettent en évidence l’importance de diagnostiquer précocement les TRS chez les patients présentant une cardiopathie ischémique afin de prendre en charge l’hyperactivité sympathique comme facteur de risque
Sleep disordered breathing (SDB) is a factor of poor prognosis in patients suffering from ischemic cardiomyopathy. During sleep, SDB patients exhibit repetitive apneas causing chronic blood desaturation-resaturation, currently named intermittent hypoxia (IH). IH appears to be the most detrimental feature of SDB for the cardiovascular system; however, involved mechanisms are not fully understood. Thus, it seems necessary to determine them, in order to propose novel therapeutic strategies. Indeed, whereas positive airway pressure remains the gold standard treatment, the efficacy in regard of the cardiovascular mortality is not clear.The aim of the present thesis was to assess the impact of IH on ischemic cardiomyopathy and the mechanisms involved, with a particular focus on sympathetic activity.For that purpose, we used a model of ischemic cardiomyopathy induced by a permanent ligature of the left coronary artery in rats. Rats were then exposed to IH until 12 weeks. IH exposure induced cardiac remodeling (hypertrophy, fibrosis and apoptosis) and contractile dysfunction (ejection fraction). Mechanistically, IH exposure quickly raised sympathetic activity, which was associated with a decrease in cardiac adrenergic reserve (isolated cardiomyocyte response to adrenergic agonist decreases) after 6 weeks IH exposure. Cardiac sympathetic activity inhibition by surgical ablation of left middle cervical and stellate ganglions restored adrenergic reserve, limited cardiac remodeling (hypertrophy, apoptosis) and preserved cardiac function (ejection fraction) in our IH model.These thesis results highlight the importance to diagnose SDB after myocardial infarction and to take care about sympathetic hyperactivity as a major risk factor

M.Sc.
Wheat is one of the most extensively grown small grain crops in Southern Africa. It is a staple food source used by humans, animals and other living organisms around the globe (Feldman, 2001). One of the major threats of wheat production in South Africa is the Russian wheat aphid (RWA), (Kurdjumov), (Diuraphis noxia, Homoptera: Aphididae) (Brooks et al., 1994; Du Toit & Walters, 1984). The RWA was first described as a pest in South Africa in 1978 and in the US in 1986. Thereafter it was rapidly reported as becoming a major pest of cereals in these countries (Brooks et al., 1994; Du Toit & Walters 1984). A short life span, asexual reproduction, the development of resistance towards insecticides and rapid colonisation of the host plant are all factors that have made the management of the RWA a difficult task (Dogimont et al., 2010; Hein et al., 1998). As a result of complications such as these, host plant resistance is the most viable option to counter the RWA (Dogimont et al., 2010). The method of feeding of the RWA is detrimental to the plant as they insert their stylets into, and feed primarily from, the phloem sieve elements. This damages the plants through nutrient drainage (Dixon, 1985; Klingler et al., 2009) and results in a variety of symptoms, the most common being chlorosis, necrosis, wilting, stunting, curling of the leaves (which provides the pest with a sheltered environment protected from predators and pesticides), misshapen or nonappearance of new growth, and localised cell death at the site of aphid feeding. The RWA elicits an increase in essential amino acids in the phloem sap, by triggering the breakdown of proteins in infested wheat leaves (Burd & Burton, 1992; Du Toit, 1986; Haley et al., 2004; Ma et al., 1998; Miller et al., 2001; Walters et al., 1980). It also reduces transport of labelled tracers (amino acids) from the feeding site to the roots and other sinks in the plant. This increases the nutrient concentration at the site of aphid feeding by increasing the import of resources from other sites in the plant, mobilising local resources and blocking their export to other organs. The damage of the foliar tissue that occurs as a result of the RWA feeding is thought to play a role in the pest’s ability to increase the nutritional quality of the host plant (Botha et al., 2006; Goggin, 2007; Shea et al., 2000).

博士
國立臺灣大學
植物病理與微生物學研究所
97
Application of Bacillus cereus C1L suspension in the rhizosphere of Lilium formosanum and lily cv. Star Gazer to reduce gray mold severity has been demonstrated. In this dissertation, the mechanism of induced systemic resistance (ISR) was approached to figure out the signaling pathway involved. Firstly, differentially expressed genes in strain C1L-treated L. formosanum were identified to be LfGRP1, LfMT1, and LfPsbR, by the use of suppression subtractive hybridization method. When L. formosanum was treated with strain C1L, LfMT1 was up-regulated. However, LfGRP1 and LfPsbR were down-regulated. The expression patterns of the 3 homologues in oriental lily cv. Star Gazer were identical to those of LfGRP1, LfMT1 and LfPsbR. When using ethylene and methyl jasmonate to treat lily cv. Star Gazer, LsGRP1 and LsPsbR were up-regulated. On the contrary, these genes were down-regulated by the treatment of abscissic acid (ABA), indicating that ABA signaling pathway is involved in the systemic resistance induced by strain C1L. When ‘Stra Gazer’ lily plants were treated with strain C1L 24 hours before inoculation with B. elliptica, accumulation of hydrogen peroxide and cell death in the leaves was suppressed as compared to those exhibited in the plant singly inoculated with B. elliptica. The peroxidase activity was not increased in the leaves inoculated with B. elliptica post strain C1L treatment, unlike that inoculated with B. elliptica singly. On the other hand, strain C1L induced early expression of LpGlu1 (β-1,3-glucanase) in response to B. elliptica infection post strain C1L treatment as compared to that inoculated with B. elliptica singly. In order to dissect the mechanism of ISR activated by strain C1L, cDNA-AFLP was analyzed and 104 cDNA clones representative of differentially expressed genes of lily were obtained. Expressions of selected genes were analyzed by semi-quantitative reverse-transcription polymerase chain reaction. The results showed that strain C1L could induce systemic resistance via calcium ion signaling pathway and ABA signaling pathway, followed by anti-oxidative response. Additionally, strain C1L induced expression of glutamate synthase of lily which might decrease cellular toxicity caused by B. elliptica and therefore reduced the disease severity.

The biocontrol fungus Trichoderma virens is an avirulent symbiont with the ability to control plant disease by the production of antibiotic compounds, induction of plant resistance to pathogens, and mycoparasitism of other fungi. In this document, the analysis of a putative terpene biosynthesis gene cluster (vir cluster) in T. virens is described. The vir cluster contains genes coding for four putative cytochrome P450s, an oxidoreductase, MFS transporter, and a terpene cyclase. To determine the function of this cluster in secondary metabolism biosynthesis, a strain of T. virens with a deletion of the putative cyclase, vir4, was constructed. Deletion mutants were deficient in the synthesis of sesquiterpene volatiles and complementation of vir4 restored this loss in transformants, albeit at a lower level of production. An analysis of phenotypic characteristics between mutant and wild-type strains did not identify any differences when the strain interacted with other fungi, bacteria, or Arabidopsis seedlings. Paralogs of the gene encoding the elicitor SM1 were examined as genetic sources for potential elicitors to induce systemic resistance in plants. A search of the T. virens genome revealed the presence of three paralogs of sm1. One paralog, sm3, was found to be expressed when grown in association with plant roots and in still-culture. The Pichia pastoris protein expression system was used to generate sufficient quantities of SM3 to allow characterization of its function. The purified protein from the yeast system (picSM3) was shown to be glycosylated and to increase expression of a plant defense gene in maize seedlings. Mutant strains in which sm3 was either deleted or over-expressed were constructed to further explore the potential of sm3 as an elicitor of ISR. The differential production of SM1 and SM3 by these strains suggested that SM1 and SM3 may be co-regulated and native SM3 may be glycosylated. To further understand the role of a putative glycosylation site as a mechanism to prevent dimerization and subsequent elicitor activity, a point mutation was created in a sm1 deletion strain. Analysis of the behavior of the protein demonstrates that the putative glycosylation site is not involved in protein aggregation and deletion of this site does not prevent the protein from testing positive for glycosylation. We propose that SM1is not glycosylated but instead may interact with an oligosaccharide or other small molecule. However, the mechanism of dimerization in SM1 remains unknown.

碩士
國立臺灣大學
農業化學研究所
105
During agricultural practice, chemical fertilizers and pesticides are often applied to enhance crop yield and prevent losses caused by pests and pathogens. However, environmental issues derived from utilization of such chemicals raise concerns about further damage to our ecosystem, and alternative means with similar effects should be developed to lessen the problem. Use of plant growth-promoting rhizobacteria (PGPR) with ability to promote plant growth directly or indirectly becomes a good strategy incorporated into the integrated pest management (IPM) program. In our studies, two soil habitants, Pseudomonas putida A7 and Streptomyces coelicolor strain M145, were proved to be plant growth promoting rhizobacteria, which not only promote plant growth but also induce resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 in Arabidopsis thaliana. Further studies showed that P. putida A7 can induce resistance against Pst DC3000 in the Arabidopsis mutant lines, jar1, pad4 and npr1. By means of semiquantitative RT-PCR, we also found that higher amount and faster expression of the salicylic acid (SA) maker genes PR1 and PR5 were observed in P. putida A7-pretreated Arabidopsis compared to the water-treated control after Pst DC3000 infection. Stronger and faster responses upon Pst DC3000 infection in the A7-pretreated Arabidopsis were also found in the expression of genes involved in jasmonic acid (JA) and ethylene (ET) signaling pathways, such as LOX2, HEL and GST2. These data indicate that SA and JA/ET signaling pathways are involved in P. putida A7-induced systemic resistance to Pst DC3000 in A. thaliana. On the other hand, S. coelicolor M145-treated Arabidopsis mutant lines, jar1, npr1 and etr1, lost their ability to trigger systemic resistance to Pst DC3000. Futhermore, the expressions of PR1 and PR5 as well as PDF1.2, LOX2, HEL and GST2 were also stronger and faster after Pst DC3000 infection. Taking togather, we demonstrated P. putida A7 and S. coelicolor M145 probably can use different mechanisms to affect signaling pathways involving SA, JA and ET, to induce systemic resistance against Pst DC3000 in A. thaliana.

Cultivars of Agrostis stolonifera showed weak and strong responsiveness to the systemic acquired resistance (SAR) activator, benzothiadiazole (BTH), or the induced systemic resistance (ISR) activator, 2R, 3R-butanediol (BD). Next Generation RNA sequencing was used to identify 2163 putative transcripts with increased expression in BTH versus water-treated A. stolonifera. Among three BTH-induced genes, AsASP-2 and AsHIR-1 were induced faster, while AsLOX-1 had stronger transient induction, in one out of two strongly BTH-responsive cultivars. Three ISR-responsive genes, AsGNS-5, AsOPR-4 and AsAOS-1, showed no greater induction or priming in the strongly versus weakly BD-responsive cultivars. Cultivars of A. stolonifera vary significantly in their response to defense activators, however this is not consistently related to defense gene expression. To quantify disease severity, chlorophyll fluorescence imaging of the maximum quantum efficiency of photosystem II (Fv/Fm) was tested on Nicotiana benthamiana infected with Colletotrichum orbiculare. Leaf areas of healthy, non-necrotic affected and necrotic tissue could be individually quantified, which demonstrated that BD delayed symptom development by approx. 24-hour and reduced non-necrotic affected tissue compared to controls. Chlorophyll fluorescence imaging can quantify and reveal novel features about induced disease resistance.

碩士
臺北市立大學
運動科學研究所
107
Purpose: To investigate the effect of systemic hypoxia-reoxygenation or -hyperoxia preconditioning on aerobic exercise performance and acute heavy resistance exercise-induced muscle damage in athletes. Methods: In study I, 11 male athletes are recruited in single-blind, randomized- controlled crossover rotated trial, supplied either control (CON) (21% O2) or hypoxia- hyperoxia (10- 99% O2) (HH) (5 min hypoxia- 5 min hyperoxia a circle for 6 circles). After 30 min, an acute heavy resistance exercise (AHRET) was performed. Heart rates variability (HRV), muscle hemodynamic, inflammation, oxidative stress and muscle strength are determined before intervention, immediately, 24 and 48 hours after exercise. In study II, 9 male athletes are supplied normoxia or three modes of hypoxia-reoxygenation or -hyperoxia (10-21, 60 or 99% O2) (HR, HHI, HH). Hemodynamic changes of muscle and brain are measured during intervention, following by a maximal aerobic capacity (VO2max) test 30 minutes after intervention. Result: In study I, there are significantly lower creatine kinase and myoglobin in HH than that in CON 24 (p<0.05; p<0.05) and 48 hours (p=0.08; p=0.05) after AHRET. There is significantly higher IL-6 (p<0.05) in HH than that in CON 24 and 48 hours after AHRET. No significant difference in other markers between two groups. In study II, there are significantly higher VO2max (p<0.05) and exercise time (p<0.05) in HH group. Conclusion: Systemic hypoxia-hyperoxia preconditioning could reduce exercise-induced muscle damage and enhance aerobic exercise performance. These effects may be associated with the increase of inflammatory marker and the decrease in body anabolism.

碩士
國防醫學院
生理學研究所
100
Obesity induced adipose tissue inflammation and systemic insulin resistance is associated with the secretion and functional properties of inflammatory chemokines and cytokines, such as the well-established MCP-1/CCR2 chemokine system. Recent studies showed that the RANTES/CCR5 system could also participate in the complex process of adipose tissue inflammation and insulin resistance. In this study, CCR5 and CCL5 deficient mice were fed either with normal diet or high fat diet, which are then experimented to study the mechanism of the involvement of CCR5 and CCL5 in adipose tissue inflammation and systemic insulin resistance. The results show that, under normal diet, CCR5 KO mice and CCL5 KO mice both had no significant differences in energy balance. CCR5 KO mice showed more improved insulin sensitivity, whereas CCL5 KO mice worsen. And for systemic and adipose tissue inflammatory response, CCR5 KO mice showed an attenuated inflammatory response. On the contrary, CCL5 KO mice had a more deleterious condition. And last, CCR5 KO mice showed an overall improved adipogenesis, whereas, CCL KO mice was worsen The results indicate that, under different diets, CCR5 and CCL5 have collaborative and separate roles in regulating the progress of adipose tissue inflammation and adipose tissue adipocyte differentiation. CCR5 showed dominant influence in promoting adipose tissue inflammation and the infiltration of immune cells, and has a role in down regulating adipocyte proliferation and differentiation, which leads to systemic insulin resistance. On the contrary, CCL5 plays a vital role in the regulation of adipose tissue inflammation, which probably works as a balance between chemokines and cytokines such as MCP-1 in an inflammatory response. On the other hand, CCL5 may play a role in adipose tissue lipogenesis, which may affect ectopic fat accumulation, in which leads to insulin resistance. This study suggests that CCR5/CCL5 pathway could regulate high fat induced adipose tissue inflammation and systemic insulin resistance.

碩士
輔仁大學
生命科學系碩士班
101
Chrysanthemum (Chrysanthemum morifolium) is one of the world's three major cut flowers and one of the most important economical flowers in Taiwan. However, chrysanthemum has many diseases, for example, bacterial soft rot which vastly affects yield. This disease is caused by the gram-negative bacterium Pectobacterium chrysanthemi (Erwinia chrysanthemi), which infects host through wounds in young organs and resulted in extended necrosis of infected tissues. Using general pesticide can only reduce the rate of symptoms development but not to cure it. In order for plant to effectively fight against broad spectrum of pathogens and thrive in nature, the best strategy is to activate their de novo systemic resistant ability. Systemic acquired resistance (SAR) is an inducible defense response found in a large range of plant species when encountered biotic and abiotic stresses. Activation of NPR1 transcription factor will be triggered by SA accumulation, and resulted in cascade defense responses performed by pathogenesis-related (PR) gene products. The benzothiadiazole derivative acibenzolar-S-methyl (BTH), a well-known chemical activator of SAR response was used in this study to evaluate its ability to potentiate resistance in chrysanthemums. We found that 2.4mM is the optimal concentration to induce NPR1-like gene expression in chrysanthemum. Five days after treating with BTH, the glucanase and chitinase activity increase 2.6-fold and 19-fold, respectively, compared to untreated control. When challenged with P. chrysanthemi, the necrotic area of BTH-treated leaf was 7-fold smaller compared to untreated control. In addition, we have also found that if treated lower leaves of a plant with BTH in advance can enhance the resistance ability in the upper leaves. In summary, we conclude that the BTH can induce SAR resistance in chrysanthemums.

碩士
國立臺灣大學
植物病蟲害學系
85
systemic acquired resistance(SAR)is a defense system of plantevolved to protect themselves from pathogen attack. the resistance of spectrum SAR is broad including fungi, bacteria and virus. salicylic acid(SA)is known to be essnitial for the establishment of acquired resistance in uninfected tissues upon local induction with a necrotizing pathogen.Furthermore, the treatment of SA is able to induce disease resistance in many plant systems.In this study, the effect of SA to induce disease resistance in lily wereinvestigated. The results indicated that SA treatment induced systemic resistance of lily against Botrytis elliptica and Sclerotium rolfsii, but not to Phytophthora parasitica. Three consecutive treatments of SA enhanced the level of induced resistance. in different growth stages of lily, SA treatment,the endogenous SA level increased one day post SA-treatment and declined seven days after treatment. At the concentration no more than 5 mM, SA did notinhibit the conidial germination and mycelial growth of B. elliptica in vitro. Dot blot hybridization of total RNA of lily revealed that the expressionof four SAR-related genes, chalcon synthase, PR4, PR5, and 14-3-3 may be enhanced after the treatment of SA.

博士
國立中興大學
農藝學系所
101
Rice blast, caused by the fungus Magnaporthe oryzae, is one of the devastating epidemics in all rice-growing regions around the world. This fungus can attack rice plant at any growth stage and cause severe yield loss and grain quality reduction which becomes a serious economic and humanitarian issue. The studies of rice blast resistance are focused on screening and mapping resistant Pi locus. Most of cloned Pi loci are encoded NBS-LRR; indeed, that can recognize M. oryzae Avr proteins, introduce local hypersensitive response, and then transmit signal to induce defense response genes. The signal transduction of plant innate immune system is performed by post-translational modification, hence proteomic analysis is the best method and strategy to study rice blast resistance. The study and application of Pi loci is required to conform R gene’s behavior to follow gene-for-gene theory, and consequently it is difficult to improve rice blast resistance. Sodium azide mutant lines SA0009 and SA0169 carried broad-spectrum resistance to rice blast fungus are generated from high susceptible rice variety Tainung 67 (TNG67) and can be considered rice blast resistant near-isogenic lines (NILs) of TNG67 due to they are very similar from phenotype to genotype. In this study, the differentially expressed proteomic analysis between high resistant mutants and high susceptible wild type can easily discover rice blast resistant protein or involved protein under less background interference. In order to obtain disease responsive 2-DE protein spot, the positive control of moderate resistant and susceptible materials, the normalization of protein spot volume, and the expression profile analysis are used to study 523 2-DE protein spots. Besides 17 resistant and 12 susceptible protein spots are obtained, 53 protein spots with undesirable expression profile are chosen. Total 82 protein spots on different position collected from 4 different rice blast reactions are executed by protein mass spectrometric identification. Total 5338 identifier derived from 1967 SwissProt unique proteins are successfully indentified, and the amount of unique proteins is assigned as protein signal volume. The expression profile of normalized protein signal volume is estimated to find out differentially expressed protein including 14 resistant and 22 susceptible proteins. Resistant proteins F-box/LRR-repeat protein At4g14103, Auxin response factor 2, Ethylene-responsive transcription factor ESR1 and E3 ubiquitin-protein ligase ATL6 are involved in stress response and defense signal transduction. Susceptible proteins MLP-like protein 423 and Cysteine-rich repeat secretory protein 39 participate with disease susceptible and Serine/threonine-protein phosphatase PP2A-5 catalytic subunit及Protein SPA1-RELATED 4 are also involved in stress response and defense signal transduction. After annotated 1967 unique proteins, 199 biological functions of 10 catalogues are discovered including cell killing, cell death, response to stress, response to stimulus, signal transduction, gene expression, metabolic process, cellular process, cellular component organization, and unknown. Because protein spots with desirable expression profile contain more programmed cell death and stress response proteins involved in disease resistance, the expression profile of protein spot volume can directly indicate rice blast reaction, and furthermore rice blast resistant and/or susceptible proteins are discovered to be biomarker. According to the biological function and protein’s normalized expression level analysis, the list of resistant and susceptible proteins carried desirable expression profile is established and contains 145 resistant and 171 susceptible proteins which’s corresponding rice loci are 132 resistant and 63 susceptible. These 295 rice blast resistant and susceptible corresponding rice loci can be designed as resistance selection molecular marker of mutants SA0009 and SA0169 in breeding program and demonstrates that the resistance of mutants SA0009 and SA0169 is different to known Pi loci in accordance with proteomic-genomics integrative map. Based on all evidences, the resistance of mutants SA0009 and SA0169 is defensin, which is untypical R protein encoded NBS-LRR or LRR kinase, actively kills microorganism, has broad-spectrum resistance to different isolates and even pathogens, but does not trigger downstream defense gene, like PR gene. Finally, this study is first report about rice defensin carried rice blast resistance.

Avocados are one of the major food sources in tropical and subtropical regions and are an important horticultural crop in South Africa. Avocados are exported over long distances and may have storage times of up to 30 or more days at temperatures of about 5.5oC. This procedure increases the risk of poor fruit quality, including physiological disorders, early softening and postharvest disease incidence. A major component of the postharvest diseases is Anthracnose caused by Colletotrichum gloeosporioides. Anthracnose infects unripe fruit and once infected, the fungus remains dormant in the fruit until ripening begins. This leads to a problem for producers and packers, as the presence of the disease cannot be detected on the pack line, and fruit is not removed. Anthracnose control is normally done through pre-harvest treatment with copper-based fungicides. While effective such treatment needs to be repeated frequently, resulting in copper residues on the avocados. The study was conducted to investigate the effects of phosphoric acid and potassium silicate on known antifungal compounds and critical enzymes of the pathways elemental for systemic resistance inducers, so as to evaluate the potential for using them as alternatives to or in conjunction with, copper fungicides in the control of Anthracnose in avocado fruit. The study included storage temperature and time variations, to take account of the logistics in shipping avocado fruit to distant markets. Pre- and postharvest applications of phosphoric acid and potassium silicate were used, and after harvest, fruit were either ripened at room temperature (22oC) without storage or stored for 28 days at temperatures of 5.5oC or 2oC before analysis. Concentrations of phenolics, activity of the enzyme phenylalanine ammonia lyase (PAL) and a known antifungal diene were determined in the fruit exocarp. Pre-harvest treatments of phosphoric acid showed that the highest phenolic concentration was found in fruit harvested 14 days after application for fruit stored at room temperature. For fruit stored at 5.5°C it was seen that as fruit softened, phenolic concentrations increased compared with hard fruit immediately after storage, with the highest increase noted for fruit harvested 7 days after application. When comparing the three storage temperatures, phenolic concentrations were enhanced most when fruit was stored at 2°C. Postharvest treatments showed a significant increase in phenolic concentrations for potassium silicate treated fruit stored at room temperature and 2°C when determined immediately after storage. Fruit stored at 5.5°C showed an increase in phenolic concentrations as it became softer. When considering PAL enzyme activity, it was found that postharvest treatments of both potassium silicate and phosphoric acid influenced enzyme activity, with potassium silicate having greater effects. Similarly, an increase in PAL activity was noted in the pre-harvest phosphoric acid treatment harvested 14 days after application for fruit ripened immediately as well as fruit stored at 5.5°C. Fruit stored at 2°C showed the highest PAL activity for fruit harvested 7 days after application. No results were obtained in the analysis of antifungal compounds for both pre- and postharvest treatments. However, it is suggested that the antifungal diene could follow similar trends to those found for phenolics. It is concluded that applications of both phosphoric acid and potassium silicate do create changes in phenolic concentrations and the activity of the enzyme PAL which is involved in the synthesis of phenolic compounds known to possess antifungal properties. It is therefore possible that phosphoric acid and potassium silicate may be used as part of an integrated programme for Anthracnose control, and should be tested as potential alternatives for high volume copper-based fungicides.
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.