Academic literature on the topic 'BIOTIC ELICITORS'

Aim. The usage of biotic elicitors for elicitation of defense responses may induce plant disease resistance and prevent increased environmental pollution by pesticides. Hydrogen peroxide (HP) is a signal molecule for photosynthetic status and for stomatal movements, and systemic acquired resistance to pathogens in plants proposed to be dependent on H2O2. It is shown in our previous research that biotic elicitors influence on H2O2 content in plants. Kojic acid inhibits tyrosinase activity in melanin synthesis. In plant-pathogen interaction melanin plays role for filaments growth of fungal agent. The aim of research was to analyze in field trials effect of kojic acid with additional donor NO treatment of winter wheat under biotic stress. Methods. Content of endogenous H2O2 was measured in kojic acid and NO donor treated wheat plants (cv. Legenda Myronivska) during different ontogenesis phases. The extent of morphometric parameters and yield structure were analyzed. Results. The data obtained suggest that kojic acid and donor NO decreased the HP content in wheat leaves and increased the grain number and yield. Conclusions. Kojic acid with donor NO is effective combination and could be used as biotic elicitor. Keywords: winter wheat, kojic acid, NO, biotic elicitors, induced resistance, Triticum aestivum L., Septoria tritici Rob.et Desm.

Several biotic and abiotic clicitors of the hypersensitive reaction in wheat leaves are described. The elicitors induce enhanced activity of phenylalanine ammonia-lyase with subsequent lignifica­tion, visible as a yellow autofluorescence. The deposited material stains positively with phloroglucinol. DEAE-dextran, epoxystearic acid and chitosan are strong elicitors, while the glucans tested have no activity.A biotic elicitor (genuine Pgt-elicitor) was isolated from the germ tube walls of uredospores of Puccinia graminis f. sp. tritici. The high molecular weight, water soluble elicitor is heat stable and unaffected by mild acid and mild alkaline treatments. It seems to be a glycoprotein with the carbohydrate moiety bearing the active residues, as indicated by periodate sensitivity and pro­tease stability. The carbohydrate moiety consists mainly of glucose with some galactose and mannose.Upon ultrafiltration and gel chromatography elicitor activity was associated with fractions of molecular weight of more than 100 kD.When injected into the intercellular space of primary wheat leaves, the elicitor induces lignifica­tion, preceded by an increase in phenylalanine ammonia-lyase activity.

Panax vietnamensis Ha et Grushv. is an endemic ginseng of Vietnam which has highly nutraceutical, medicinal and comercial values. Many its special saponins provide resistance to stress, disease and exhaustion. In this study, the biotic elicitors (yeast extract and chitosan) at concentrations from 50 – 200 mg/l and abiotic elicitors (jasmonic acid, abscisic acid and salicylic acid) at concentrations from 50 – 200 µl/l were used to evaluate the possibility of increasing secondary root’s biomass and saponin in Panax vietnamensis adventitious root cultures. This cultures were maintained on Innova 2100 shaker shaker plantform at a speed of 100 rpm, and its modified MS medium (NH4+/NO3-: 7.19/18.5 mM/mM) were supplemented with 7 mg/l IBA, 0.5 mg/l BA and 3% sucrose. Results after 56 days of cultured showed that most elicitors significantly reduced dry biomass of secondary roots as compared to control (exception the addition of 50 – 100 mg/l yeast extract or 50 µl/l salicylic acid). However, all elicitors increased saponin-accumulation, and abiotic elicitor is more effective saponin-accumulation than those biotic elicitors. Jasmonic acid obviously gave the best results; with total amount of 3 saponins at 150 µl/l, Rg1 at 100 µl/l, Rb1 at 50 µl/l and MR2 at 200 µl/l and Rg1 (3.22%), significantly higher than other concentration of elicitor. However, for accumulating saponins and developing secondary root, 150 mg/l YE is the most real effective in all elicitors (0.88 mg).

Abstract - Isoflavone content in soybean seeds was enhanced by the elicitor-mediated approach under field conditions through the floral application of abiotic elicitors-salicylic acid, methyl jasmonate and biotic elicitors-Aspergillus niger and Rhizopus oligosporus. Among isoflavones, daidzein and glycitein were found to be highly responsive to elicitors, with an increase of 53.7% and 78.7%, respectively as compared to control. Highest total isoflavone content (1276.4 mg g-1 of seeds) was observed upon the administration of 0.1 mMsalicylic acid, which is 92.7% higher than in control. This study would be valuable for augmentation of the isoflavone content in soybean seeds in field grown plants for better nutraceutical potential.

An antibiotic-resistant and multidrug-resistant (MDR) issue open the role of researchers to continue to search for natural potential as a source of new antimicrobials. One of the potential fungi isolates that can produce antimicrobial active compounds from Indonesian tropical peat soils is Penicillium sp. LBKURCC34. In this study, the production of antimicrobial compounds from local isolates was carried out by batch fermentation method in liquid media with the addition of biotic elicitors to increase the extraction activity and yield. This study aims to optimize the results based on the time the elicitor is added. Staphylococcus aureus was used as a biotic elicitor, which was added on days 2, 3, and 4 in the production of antibiotics by fermentation incubation of 6-14 days. The antibiotic production media was extracted with ethyl acetate and evaporated. The antimicrobial test was carried out by the disk diffusion method against pathogenic bacteria Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Staphylococcus epidermidis using three crude extract contents (19; 38; and 57 µg/disc). Amoxicillin® was used as a positive control (10 µL/disc). The results showed that the addition of S. aureus biotic elicitor extended the log phase growth of the fungus Penicillium sp. LBKURCC34. The optimum condition of production was obtained by adding initiator treatment on the 3rd day for 14 days incubation with the highest yield and could inhibit the growth of all pathogenic microbes.

Aim. The usage of biotic elicitors for elicitation of defense responses may induce plant disease resistance and prevent increased environmental pollution by pesticides. Hydrogen peroxide is a well-known signal molecule for photosynthetic status and for stomatal movements, and systemic acquired resistance to pathogens in plants proposed to be dependent on H2O2. The aim of research was to analyze in field trials the effect of oxalic, ferulic and kojic acid on H2O2 content and winter wheat resistance against Septoria tritici. Methods. Content of endogenous H2O2 was measured in elicitor treated and inoculated by S. tritici wheat plants (cv. Oberig) during different ontogenesis phases. The extent of disease development, morphometric parameters and yield structure were analyzed. Results. It is shown that the lowest level of hydrogen peroxide in leaves at the necrotrophic stage of pathogen infection was after oxalic acid treatment, and the highest – after koijc acid influence. Conclusions. The data obtained suggest that elicitors induced defense responses in winter wheat against S. tritici and hydrogen peroxide content is an important and valuable parameter. Keywords: biotic elicitors, hydrogen peroxide, induced resistance, Triticum aestivum L., Septoria tritici Rob et Desm.

The in vitro cell cultures derived from the grapevine (Vitis vinifera L.) have been used for the production of stilbenes treated with different biotic and abiotic elicitors. The red-grape cultivar Váh has been elicited by natural cellulose from Trichoderma viride, the cell wall homogenate from Fusarium oxysporum and synthetic jasmonates. The sodium-orthovanadate, known as an inhibitor of hypersensitive necrotic response in treated plant cells able to enhance production and release of secondary metabolite into the cultivation medium, was used as an abiotic elicitor. Growth of cells and the content of phenolic compounds trans-resveratrol, trans-piceid, δ-viniferin, and ɛ-viniferin, were analyzed in grapevine cells treated by individual elicitors. The highest accumulation of analyzed individual stilbenes, except of trans-piceid has been observed after treatment with the cell wall homogenate from F. oxysporum. Maximum production of trans-resveratrol, δ- and ɛ-viniferins was triggered by treatment with cellulase from T. viride. The accumulation of trans-piceid in cell cultures elicited by this cellulase revealed exactly the opposite effect, with almost three times higher production of trans-resveratrol than that of trans-piceid. This study suggested that both used fungal elicitors can enhance production more effectively than commonly used jasmonates.

Elicitors are stressors that activate secondary pathways that lead to the increased production of bioactive molecules in plants. Different elicitors including the fungus Aspergillus niger (0.2 g/L), methyl jasmonate (MeJA, 100 µM/L), and silver nanoparticles (1 µg/L) were added, individually and in combination, in a hydroponic medium. The application of these elicitors in hydroponic culture significantly increased the concentration of photosynthetic pigments and total phenolic contents. The treatment with MeJA (methyl jasmonate) (100 µM/L) and the co-treatment of MeJA and AgNPs (silver nanoparticles) (100 µM/L + 1 µg/L) exhibited the highest chlorophyll a (29 µg g−1 FW) and chlorophyll b (33.6 µg g−1 FW) contents, respectively. The elicitor MeJA (100 µM/L) gave a substantial rise in chlorophyll a and b and total chlorophyll contents. Likewise, a significant rise in carotenoid contents (9 µg/g FW) was also observed when subjected to meJA (100 µM/L). For the phenolic content, the treatment with meJA (100 µM/L) proved to be very effective. Nevertheless, the highest production (431 µg/g FW) was observed when treated with AgNPs (1 µg/L). The treatments with various elicitors in this study had a significant effect on flavonoid and lignin content. The highest concentration of flavonoids and lignin was observed when MeJA (100 mM) was used as an elicitor, following a 72-h treatment period. Hence, for different plant metabolites, the treatment with meJA (100 µM/L) and a co-treatment of MeJA and AgNPs (100 µM/L + 1 µg/L) under prolonged exposure times of 120–144 h proved to be the most promising in the accretion of valuable bioactive molecules. The study opens new insights into the use of these elicitors, individually or in combination, by using different concentrations and compositions.

Aim: To study the enhancement of solasodine content using elicitors such as NaCl, pectin, salicylic acid and yeast extract in cell suspension cultures of Solanum incanum, Solanum nigrum, Solanum surattense and Solanum villosum. Methodology: In-vitro callus induction from leaf explants was carried out on MS media supplemented with auxin, 2, 4- Dichlorophenoxyacetic acid (2.0 mg l-1). MS liquid medium supplemented with 2, 4-D (2.0 mg l-1) and varied concentrations of different elicitors were used for cell suspension culture. Results: The elicitor NaCl (150 mM) indicated maximum increment in solasodine production in three Solanum species studied. Salicylic acid with 75 μM resulted in considerable elevation in solasodine content in Solanum spp. Response to elicitation by pectin was high at different concentrations for different species of Solanum. Biotic elicitor yeast extract at 3.0 g l-1 concentration considerably increased solasodine production in S. nigrum and S. villosum. Solanum villosum exhibited best results in terms of solasodine concentration enhancement in response to pectin and yeast extract elicitors whereas Solanum incanum responded best to pectin followed by NaCl. Interpretation: The current results indicated that NaCl, pectin, salicylic acid and yeast extract can be effectively applied as influential elicitors for the enhancing the production of solasodine in cell suspension culture of different Solanum species. Key words: Callus, Cell suspension culture, Elicitors, Solanum, Solasodine, Steroidal alkaloids

Pueraria tuberosa contains a wide range of bioactive compounds, including polyphenols, alkaloids, and phytosterols, which make it valuable to the pharmaceutical and food industries. Elicitor compounds trigger the defense mechanisms in plants and are widely used to increase the yield of bioactive molecules in in vitro cultures. The present study was conducted to evaluate the effects of different concentrations of biotic elicitors such as yeast extract (YE), pectin (PEC), and alginate (ALG) on growth, antioxidant activity, and metabolite accumulation in in vitro propagated shoots of P. tuberosa. The elicitors applied to shoot cultures of P. tuberosa significantly increased biomass (shoot number, fresh weight, and dry weight), and metabolites such as protein, carbohydrates, chlorophyll, total phenol (TP), and total flavonoid (TF) contents, as well as antioxidant activity compared to untreated control. Biomass, TP, and TF contents, as well as antioxidant activity, were most significant in cultures treated with 100 mg/L PEC. In contrast, chlorophyll, protein, and carbohydrate increased most in cultures treated with 200 mg/L ALG. Application of 100 mg/L of PEC led to the accumulation of high amounts of isoflavonoids including puerarin (220.69 μg/g), daidzin (2935.55 μg/g), genistin (5612 μg/g), daidzein (479.81 μg/g), and biochanin-A (111.511 μg/g) as analyzed by high-performance liquid chromatography (HPLC). Total isoflavonoids content of 100 mg/L PEC treated shoots was obtained as 9359.56 μg/g, 1.68-fold higher than in vitro propagated shoots without elicitors (5573.13 μg/g) and 2.77-fold higher than shoots of the mother plant (3380.17 μg/g). The elicitor concentrations were optimized as 200 mg/L YE, 100 mg/L PEC, and 200 mg/L ALG. Overall, this study showed that the application of different biotic elicitors resulted in better growth, antioxidant activity, and accumulation of metabolites in P. tuberosa, which could lead to obtaining phytopharmaceutical advantages in the future.

Tomato (Lycopersicon esculentum Mill., Solanum lycopersicon L.) is one of the most popular vegetable throughout the world, and the importance of its cultivation is threatened by a wide array of pathogens. In the last twenty years this plant has been successfully used as a model plant to investigate the induction of defense pathways after exposure to fungal, bacterial and abiotic molecules, showing triggering of different mechanisms of resistance. Understanding these mechanisms in order to improve crop protection is a main goal for Plant Pathology. The aim of this study was to search for general or race-specific molecules able to determine in Solanum lycopersicon immune responses attributable to the main systems of plant defense: non-host, host-specific and induced resistance. Exopolysaccharides extracted by three fungal species (Aureobasidium pullulans, Cryphonectria parasitica and Epicoccum purpurascens), were able to induce transcription of pathogenesis-related (PR) proteins and accumulation of enzymes related to defense in tomato plants cv Money Maker,using the chemical inducer Bion® as a positive control. During the thesis, several Pseudomonas spp. strains were also isolated and tested for their antimicrobial activity and ability to produce antibiotics. Using as a positive control jasmonic acid, one of the selected strain was shown to induce a form of systemic resistance in tomato. Transcription of PRs and reduction of disease severity against the leaf pathogen Pseduomonas syringae pv. tomato was determined in tomato plants cv Money Maker and cv Perfect Peel, ensuring no direct contact between the selected rhizobacteria and the aerial part of the plant. To conclude this work, race-specific resistance of tomato against the leaf mold Cladosporium fulvum is also deepened, describing the project followed at the Phytopathology Laboratory of Wageningen (NL) in 2007, dealing with localization of a specific R-Avr interaction in transfected tomato protoplast cultures through fluorescence microscopy.

Tomato (Lycopersicon esculentum Mill., Solanum lycopersicon L.) is one of the most popular vegetable throughout the world, and the importance of its cultivation is threatened by a wide array of pathogens. In the last twenty years this plant has been successfully used as a model plant to investigate the induction of defense pathways after exposure to fungal, bacterial and abiotic molecules, showing triggering of different mechanisms of resistance. Understanding these mechanisms in order to improve crop protection is a main goal for Plant Pathology. The aim of this study was to search for general or race-specific molecules able to determine in Solanum lycopersicon immune responses attributable to the main systems of plant defense: non-host, host-specific and induced resistance. Exopolysaccharides extracted by three fungal species (Aureobasidium pullulans, Cryphonectria parasitica and Epicoccum purpurascens), were able to induce transcription of pathogenesis-related (PR) proteins and accumulation of enzymes related to defense in tomato plants cv Money Maker,using the chemical inducer Bion® as a positive control. During the thesis, several Pseudomonas spp. strains were also isolated and tested for their antimicrobial activity and ability to produce antibiotics. Using as a positive control jasmonic acid, one of the selected strain was shown to induce a form of systemic resistance in tomato. Transcription of PRs and reduction of disease severity against the leaf pathogen Pseduomonas syringae pv. tomato was determined in tomato plants cv Money Maker and cv Perfect Peel, ensuring no direct contact between the selected rhizobacteria and the aerial part of the plant. To conclude this work, race-specific resistance of tomato against the leaf mold Cladosporium fulvum is also deepened, describing the project followed at the Phytopathology Laboratory of Wageningen (NL) in 2007, dealing with localization of a specific R-Avr interaction in transfected tomato protoplast cultures through fluorescence microscopy.

Il primo step del progetto di ricerca si è incentrato sulla coltivazione delle piantine di pomodoro (Solanum lycopersicum L.). le piantine sono state fatte crescere in un ambiente controllato per poter mantenere delle condizioni standard ed evitare di sottoporre a stress abiotici. Al raggiungimento della quarta foglia vera le piante sono state trattate con diversi agenti stressogeni. Come prevedeva il progetto di ricerca i trattamenti si sono incentrati su: - crescita in assenza totale di azoto; - crescita in azoto 20 Mm; - crescita in azoto 30 Mm; - trattamento con chitosano 10 gel in acido acetico; - trattamento con 1 m acido acetico (controllo negativo per il chitosano); - trattamento con fitosanitario a base di traizolici e strobilurine, quilt xcell (Syngenta) a base di azoxystrobin 141.4 g/l+ propioconazole 122.4 g/l; - trattamento con fitosanitario a base di triazolici, opinion ecna (Adama) propioconazolo 250 g/l; - trattamento con acido salicilico 0.01 mm; - trattamento con poliamine 0.1 mm; - trattamento con benzothiadiazole 137 mg/l; - trattamento con Trichoderma; - trattamento con Microrizza; - non trattato, il controllo negativo per le analisi. A seguito dei trattamenti le piante sono state sottoposte ad harvest dopo 5 giorni. Il campionamento si è basato su peso fresco e secco delle piante, raccolta di 3-5 foglie per palco. I campioni sono stati così stoccati a -18°c. Le foglie sono state poi triturate con azoto liquido di modo da mantenere intatti i metaboliti secondari presenti. Lo step successivo vede l’estrazione dei metaboliti con un protocollo di estrazione con metanolo 80%+ acido formico 1%, omogenizzazione con ultraturex, centrifugazione, filtrazione e stoccaggio a -18°C. I campioni sono stati elaborati con un untarget LC-MS/MS. L’analisi statistica dei dati raccolti è stata realizzata basandosi su HCA (hierarchical combined tree on treatment), PCA, OPLS-DA, volcano analysis e l’utilizzo combinato del database plant metabolic network, keeg pathway. Dall’analisi dei dati è emerso che, i trattamenti in presenza di un eccesso di azoto hanno portato ad una upregolazione degli alkaloids di difesa, anotcianine, flavonoidi e fitoalexine. Mentre, le piante cresciute in carenza di azoto hanno dimostrato una attivazione differenziale degli stessi pathway e maggiori condizioni di stress. Per quanto riguarda le piante di pomodoro incubate con Trichoderma hanno evidenziato una upregolazione dei pathway coinvolti nella detossificazione di composti tossici per la pianta, up regolazione anche nella produzione degli alkaloidi di difesa, della famiglia delle antocianine e fitoalexine. Una up regolazione è stata anche evidenziata nella produzione di fitosteroli. Mentre, le piante incubate con mychorrhiza non evidenziano una attivazione dei pahtway di detossificazione cellulare ma, una up regolazione nella produzione di isoflavonodi e antocianine nella forma glucosilata. Mentre, le piante trattate con controllo positivo, chitosano, una mixture di poliamine o acido sialicidico hanno mostrato come non vi sia una risposta diretta volta ad aumentarne la biomassa. Invece, ad essere aumentata in tutte le tesi è la produzione di metaboliti della classe dei pheylpropanoids ed il profilo lipidico. Tutti i cambiamenti sembrano indirizzare la pianta verso una più efficiente risposta allo stress. In ultimo, le piante sottoposte all’azione dei pesticidi hanno mostrato una diminuzione della crescita combinata con un aumento dell’attività dei processi di detossificazione ed anche in questo caso è stata riscontrata una maggiore presenza dei compositi della classe dei phenylpropanoids coinvolti in processi di difesa. In conclusione, tutti i trattamenti si sono dimostrati efficienti nell’indurre cambiamenti metabolici tali da aumentare la resistenza delle piante a condizioni avverse.
Plants are sessile organisms and therefore, they are subject to different sources of abiotic and biotic stresses. Example of abiotic stresses includes radiation, salinity, floods, drought, extremes in temperature and heavy metals. Unlike vertebrates, plants lack mobile immune cells and an adaptive immune system therefore, they have evolved different strategies to perceive and respond to the stress. Unlike vertebrates, plants lack mobile immune cells and an adaptive immune system therefore, they have evolved different strategies to perceive and respond to the stress. The first layer of plant defense systems are physical barriers, the cuticle and the cell wall, that deny access to a wide range of microbes but, also reduce water loss and protect against UV radiation. In addition to these non- specific defense mechanisms, plants have evolved a sophisticated immune response activated by the perception of highly conserved molecular features of different classes of bacterial and fungal pathogens, referred to as microbe/pathogen- associated molecular patters (M/PAMPS). This results in the activation of a defense response referred to as M/PAMPS- trigged immunity (M/PTI). Despite the activation of this line of defense, some pathogens have evolved strategies to suppress M/PTI. To overcome this infection strategy, plant have evolved specialized immune receptors encoded by resistance (r) genes (R proteins) that recognize these pathogen- specific effectors, thereby leading to an amplified secondary immune response known as effector- trigged immunity (ETI). ETI is characterized by the induction of localized programmed cell death (PCD) (referred to as the hypersensitive response or HR) in order to limit the spread of the infection, activation of defence gene expression and, induction of systemic acquired resistance (SAR) to conferring broad spectrum resistance in plants. SAR increases plant defence not only at point of infection but from whole plant. The systemic plant resistance can also be mediated by beneficial microbes living in the rhizosphere, like bacteria and fungi, this kind of plant resistance is known as induced systemic resistance (ISR). ISR is associated with enhanced ability, the so- called “priming”, to resist to stress conditions. Pricing is a mechanism that does not involve a direct activation of plant defense machinery but, it is an improved of perception and/ or amplification of defense. Priming is an adaptive, low- cost defensive measure because defense responses are only, slightly and transiently, activated by a given priming stimulus. Following the perceptions of a second stress signal (triggering stimulus), defense responses are deployed in a faster, stronger, and/or more sustained manner. Priming can involve various layers of induced defense mechanisms that are active during different levels of plant- pathogen interactions. To better understand the intracellular pathways activated upon the priming phase, molecular studies of priming strategy have been performed. These studies have recorded chromatin changes and the accumulation of mRNA of genes with a signaling role in defense, of signaling proteins and plant recognition receptors (PRRS), metabolites, and other molecular components supporting a faster, stronger, and more sustained response to a triggering stress. However, the complete elucidation of molecular pathways activated upon the perception of primed stimulus is not truly clear therefore, further studies are required. The goal of this work is to investigate on molecular mechanism of priming in the induction of ISR in plants. Metabolomics is a new field of studies that able to detect and measure all the small- molecules, metabolites, present in a given moment into a biological system. Therefore, metabolomics can be the molecular tool to detect all the changes that occur in the plant cells upon the exposure to the pricing agent and it is the perfect tool to link the metabolic change in the cell to the phenotype. To this purpose, tomato (Solanum lycopersicum L.) has been selected as model plant due to its economic interest and because of its diverse secondary metabolism. Tomato plants were grown devoid of chemical or microbiological treatments until growth stage of 9 or more leaves on main shoot unfolded and treated with different priming elicitors: Muscular mycorrhizal fungi (AMF), Trichoderma spp., benzothiadizole as positive control, triazole fungicide, a combination of strobiulurin and triazole fungicide, chitosan molecule and acetic acid (since the chitosan compound is soluble in acid medium therefore, an additional control is required), salicylic acid, polyamine mixture and in presence on nitrogen deficiency and nitrogen surplus. For plants grown under nitrogen deficiency and nitrogen surplus a different grown medium was required to avoid interference, these plants were grown in coconut coir. Tomato plants were harvested after 15 days treatments with chemical compounds and Trichoderma spp. And after 30 days for AMF inoculation. For plants grown under nitrogen deficiency/ surplus the harvest was made at growth stage of first flower bud visible. After biomass of leaves was determined together with extraction of metabolites for UHPLC/qTOF-MS analysis to investigate on molecular pathways. The study demonstrated as plants inoculated with either Arbuscular mycorrhizal fungi (AMF) or Trichoderma spp. Showed a positive effect on plant growth increasing their biomass index. The same beneficial effect on plant growth was observed in plants grown with a nitrogen surplus. While, the biomass index was not increased when plants were treated with benzothiadizole, chitosan, polyamines, salicylic acid or two pesticides, one containing only triazole and second one containing a combination of triazole and strobilurin. Notwithstanding, a broad molecular cell re-programming was also observed to include some common responses between thesis. In particular, the phenylpropanoid biosynthetic pathway was strongly elicited, with the production of defense phenolics like coumarins, bis-noryangonin, anthocyanins, and their glycosylated form in tomato under biotic stress. While, under abiotic stress (benzothiadizole, nitrogen deficiency, nitrogen surplus, chitosan, polyamines, salicylic acid, triazole compounds and a combination of triazole and strobilurin) there was an over expression of quercetin, terpenoid, amide derivate and, also anthocyanins. Another important aspect was the remodeling of membrane lipids and the production of sphingolipids as signal molecules. Under abiotic stress the sterol/phospholipid ratio increased with increasing of membrane rigidity, changes in membrane permeability and activation of stress response to abiotic factors. While, in presence of nutritional alteration (both in deficiency and surplus) the membrane composition changed decreasing the sterol to phospholipid ratio increasing in membrane fluidity probably in one case to boost nutrient uptake and in second one to avoid an intoxication due to a high amount of nitrogen in the cell. At same time, the shaping of phytohormone profiles resulted in the accumulation of auxins, cytokinins, and jasmonate under biotic stress. While, under abiotic stress there was an increasing in gibberellin and cytokinins to boost pant defenses. The treatments with pesticides lead to an increasing in brassinosteroids involved in detoxification pathways. To conclude, the establishment of symbiosis between plant and AMF and Trichoderma impacted several plant secondary metabolism processes in a fashion that supports both plant growth promotion and immunity. While the stress induced by abiotic factors were demonstrated to active similar cellular re- programming. Even if treatments do not increase plant growth, they were efficiently to increase plant survival to future stresses.

Plumbago zeylanica (chitraka) is the most prominent herbal plant which is used as a medication to cure skin problems and infections such as ringworm, dermatitis, sores, acne and scabies. Traditionally all parts of Plumbago zeylanica have been utilized as a medication against many diseases. In Ayurveda chitraka is described as tumour- negating, appetizer, anti-anorexic and pain-reliever due to the presence of several important bioactive compounds such as alkaloid, tannins, phenols and naphthoquinones like plumbagin which is the most active constituent of this herb. Due to extensive use of this plant as a potent medicinal herb, micropropagation is necessary for higher biomass and plumbagin production. Since resistance to antibiotic against several microbes has become a critical issue in the whole world. So, to conquer this difficulty, identification and discovery new herbal drugs are necessary. In the current study, three elicitors namely yeast extract, malt extract and chitosan were used to enhance the plumbagin synthesis in Plumbago zeylanica as well as analysis of its antioxidant and antibacterial activity. Shoot culture of Plumbago zeylanica was performed in MS media which was supplemented with 200µl BAP and 150mg/l elicitors and then incubated for several weeks. Application of three biotic elicitors enhance the plumbagin production significantly. Phytochemical analysis of compound was carried out by UV-visible analysis which exhibited the presence of total phenol and tannin. DPPH free radical scavenging activity of all accession was performed to check their antioxidant potential. Cultures were also analysed for their antibacterial potential against E,coli and S.aureus. Results were expected that application of elicitors in Plumbago zeylanica shoot culture enhance the plumbagin production as well as other bioactive compounds.

碩士
國立臺北科技大學
化學工程研究所
101
Ginkgo biloba is a medicinal plant of the Ginkgoaceae family often used in traditional Chinese medicine to soothe coughs and shortness of breath. Modern pharmacology experiments have also confirmed that G. biloba extracts possess numerous components with medicinal properties, with the compound Ginkgolide B (GB) receiving the most attention. This compound protects against the formation of blood clots or thrombus and platelet clots, and possesses anti-aging and therapeutic qualities regarding metastasis. Consequently, market demand for GB is significant, and mass production of GB using suspension cultures from G. biloba calluses is a necessity. However, G. biloba suspension cultures generally provide a minimal yield of GB, and secondary metabolites are mostly byproducts of the plant defense mechanisms. Consequently, organic (e.g., yeast extract and chitosan) and inorganic (e.g., salicylic acid and methyl jasmonate) elicitors are used to activate defense mechanisms, thereby increasing the production of GB. This study used H2O2 concentration (an indicator of the strength of the defense mechanisms) and cell survival rates as indicators to determine the effects of different elicitors on GB production. The experimental results showed that methyl jasmonate and salicylic acid produced the best results. Their defense mechanism strengths were 4.1 times and 3.9 times stronger than those of the control group, with extracellular GB production increasing 5.0 times and 3.6 times, respectively. Although elicitors inhibit cell viability, 80% of the cells nevertheless survived. Furthermore, although yeast extract and chitosan increase the defense mechanism strength by 3.5 times and 4.4 times, respectively, extracellular GB production only increased 3.1 times. In addition, 80% of the cells survived the application of yeast extract; however, this figure dropped significantly to 70% when chitosan was applied. The initial experimental results demonstrated that elicitors effectively activate defense mechanisms in G. biloba; however, because elicitors inhibit cells, selecting inorganic elicitors that damage cells less and yield greater GB is preferred and more appropriate.

Plants are sessile organisms in environments inhabited by living beings potentially dangerous to them (pathogens or phytophages) (biotic stress); moreover, they are subjected to stress caused by non-living factors depending on climate conditions (abiotic stress). For these reasons, they had to evolve specific mechanisms to detect and consequently act against complex stress combinations. In the present work, the effect of biotic and abiotic stress on Hypericum perforatum roots was evaluated, administering chitosan oligosaccharides (COS), methyl jasmonate (MeJA), salicylic acid (SA), hydrogen peroxide (H2O2), cadmium (Cd) and arsenic (As). This project had both applicative and basic purposes: biotic elicitors may be used in order to stimulate secondary bioactive metabolite biosynthesis for drug production and to elucidate the influence of shoot/root interaction on elicitor perception in H. perforatum; the treatment with toxic metals could help in understanding the processes that occurs when H. perforatum grows on polluted soils.

Hyacinth bean also known as Indian bean is multipurpose legume crops consumed both as food by humans and as forage by animals. Being a rich source of protein, it also produces distinct secondary metabolites such as flavonoids, phenols and tyrosinase which not only help strengthened plant’s own innate immunity against abiotic/biotrophic attackers but also play important therapeutic role in the treatment of various chronic diseases. However, despite its immense therapeutic and nutritional attributes in strengthening food, nutrition and therapeutic security in many developing countries, it is still considered as an “orphan crop” for unravelling its genetic potential and underlying molecular mechanisms for enhancing secondary metabolite production. Several lines of literatures have well documented the use of OMICS based techniques and biotic and abiotic elicitors for stimulating secondary metabolite production particularly in model as well as in few economically important crops. However, only limited reports have described their application for stimulating secondary metabolite production in underutilised crops. Therefore, the present chapter will decipher different dimensions of multi-omics tools and their integration with other conventional techniques (biotic and abiotic elicitors) for unlocking hidden genetic potential of hyacinth bean for elevating the production of secondary metabolites having pharmaceutical and therapeutic application. Additionally, the study will also provide valuable insights about how these advance OMICS tools can be successfully exploited for accelerating functional genomics and breeding research for unravelling their hidden pharmaceutical and therapeutic potential thereby ensuring food and therapeutic security for the betterment of mankind.

Tea is one of the most popular beverages consumed across the world and is also considered a major cash crop in countries with a moderately hot and humid climate. Tea is produced from the leaves of woody, perennial, and monoculture crop tea plants. The tea leaves being the source of production the foliar diseases which may be caused by a variety of bacteria, fungi, and other pests have serious impacts on production. The blister blight disease is one such serious foliar tea disease caused by the obligate biotrophic fungus Exobasidium vexans. E. vexans, belonging to the phylum basidiomycete primarily infects the young succulent harvestable tea leaves and results in ~40% yield crop loss. It reportedly alters the critical biochemical characteristics of tea such as catechin, flavonoid, phenol, as well as the aroma in severely affected plants. The disease is managed, so far, by administering high doses of copper-based chemical fungicides. Although alternate approaches such as the use of biocontrol agents, biotic and abiotic elicitors for inducing systemic acquired resistance, and transgenic resistant varieties have been tested, they are far from being adopted worldwide. As the research on blister blight disease is chiefly focussed towards the evaluation of defense responses in tea plants, during infection very little is yet known about the pathogenesis and the factors contributing to the disease. The purpose of this chapter is to explore blister blight disease and to highlight the current challenges involved in understanding the pathogen and pathogenic mechanism that could significantly contribute to better disease management.

Chitosan (poly[β-(1-4)-2-amino-2-deoxy-d-glucopyranose]) is a non-toxic and biocompatible cationic polysaccharide produced by partial deacetylation of chitin isolated from naturally occurring crustacean shells. Its low solubility limits its application, improving the solubility by reducing the molecular weight, increases its wide application in food, agriculture, pharmaceutical and other technical applications. Low molecular weight chitosan, acts as a potent biotic elicitor, induce plant defense responses, activating different pathways that increase the crop resistance to diseases. Antimicrobial activity of chitosan inversely proportional to its molecular weight. Chitosan degradation has many techniques, ultrasound, electron beam plasma, solution plasma, cavitation, mechanical, microwave, photo irradiated and chemical. Chemical depolymerization can be affected utilizing alkalis (NaOH, KOH), sodium nitrite, sodium hypochlorite, hydrogen peroxide etc. In our study we used chemical method to reduce molecular weight of chitosan, utilizing sodium nitrite at various concentrations. During depolymerization its impact on purity of chitosan was studied. Depolymerized chitosan molecular weights were ascertained by intrinsic viscosity method, its purity was measured by UV-Vis method.

In this project it was found that the activation of the mechanism of resistance in avocado fruits to Colletotrichum gloeosporioides depends on the increase of the level of the preformed antifungal diene. This increase is regulated by the synthesis of the flavonoid epicatechin present in the fruit peel. Epicatechin is an inhibitor of the enzyme lipoxygenase whose activity catalyze the breakdown of the antifungal diene. Increase in epicatechin concentration inhibits the breakdown of the antifungal compound and since the compound is continuously synthesized, both combined processes result in the increase of the antifungal level. Biotic and abiotic elicitors affecting the mechanism of resistance, all activate the synthesis of epicatechin. As abiotic elicitors were tested wounding, ethylene and CO2 treatments. As biotic elicitors were tested challenge inoculation with C. gloeosporioides, Colletotrichum magna (a non pathogen of avocado) and also non pathogenic strain of C. magna. In all the cases activation of the key enzymes of the phenylpropanoic pathway is followed by an enhance in the level of epicatechin and the antifungal diene. In order to determine the level of regulation by the different elicitors of the mechanism, the genes encoding for key enzymes of the phenylpropanoic pathway were cloned and it was found that the different elicitors regulate the expression of those genes at a translational level. Modulation of the mechanism of resistance could also be done by activation of lipoxygenase gene expression. For this purpose lipoxygenase from avocado was cloned and its over-expression, under the effect of methyl jasmonate, determined.

The most important pathological factor limiting fruit life after harvest in subtropical fruits are quiescent infections of anthracnose caused by Colletotrichum gloeosporioides. Prusky and Keen elucidated the mechanism of resistance in avocado fruits to quiescent infections of C. gloeosporioides and determined that the major biocide involved is the preformed compound,1-acetoxy-2-hydroxy-4-oxo-heneicosa-13, 15 diene. Two possibilities exist for maintaining fungitoxic levels of antifungal compounds in the tissue of ripening fruits: (i). Prevention of catabolism (ii). Induction of synthesis. Previous work has demonstrated that increased fruit susceptibility after fruit harvest occurs through diene catabolism mediated by oxidation of the antifungal compound by the enzyme lipoxygenase. Levels of a non-specific inhibitor, epicatechin, in turn, regulate activity of lipoxygenase, present in the peel of unripe but not ripe fruit. In this proposal, we examined the possibility of exploiting induced synthesis of the antifungal compound for the study of the synthetic pathway. The general objective of the present research was to study the mechanism of biosynthesis of natural antifungal compounds in order to regulate the process of resistance to postharvest diseases in ripening avocado fruits. The specific objectives of the research were: 1. To localize synthesis of the antifungal diene and modulate the process by biotic or a biotic elicitors. 2. To determine the relation between synthesis of the diene and accumulation in the peel and fruit resistance to decay 3. To characterize the biosynthetic pathway and the diene and the genes involved. The analysis of the antifungal compounds in avocado resulted in the detection of a new antifungal compound (E, Z, Z)-1-acetoxy-2-hydroxy-4-oxo- heneicosa-5, 12,15-triene. This new compound was shown to inhibit spore germination of C. gloeosporioides similarly as the antifungal diene. We had localized one of the biosynthetic places of these antifungal compounds in specialized idioblast cells (oil cells) in the mesocarp that can be easily enhanced by elicitors as ethylene. Results have also suggested that the antifungal compounds can be "exported" from the mesocarp to the pericarp where its main activity takes place. The search for the biosynthesis of antifungal compounds and the genes involved took two directions i. direct search for specific genes involved in the synthesis of the diene and ii. Indirect selection of genes using the differential display library. We have cloned , The most important pathological factor limiting fruit life after harvest in subtropical fruits are quiescent infections of anthracnose caused by Colletotrichum gloeosporioides. Prusky and Keen elucidated the mechanism of resistance in avocado fruits to quiescent infections of C. gloeosporioides and determined that the major biocide involved is the preformed compound,1-acetoxy-2-hydroxy-4-oxo-heneicosa-13, 15 diene. Two possibilities exist for maintaining fungitoxic levels of antifungal compounds in the tissue of ripening fruits: (i). Prevention of catabolism (ii). Induction of synthesis. Previous work has demonstrated that increased fruit susceptibility after fruit harvest occurs through diene catabolism mediated by oxidation of the antifungal compound by the enzyme lipoxygenase. Levels of a non-specific inhibitor, epicatechin, in turn, regulate activity of lipoxygenase, present in the peel of unripe but not ripe fruit. In this proposal, we examined the possibility of exploiting induced synthesis of the antifungal compound for the study of the synthetic pathway. The general objective of the present research was to study the mechanism of biosynthesis of natural antifungal compounds in order to regulate the process of resistance to postharvest diseases in ripening avocado fruits. The specific objectives of the research were: 1. To localize synthesis of the antifungal diene and modulate the process by biotic or a biotic elicitors. 2. To determine the relation between synthesis of the diene and accumulation in the peel and fruit resistance to decay 3. To characterize the biosynthetic pathway and the diene and the genes involved. The analysis of the antifungal compounds in avocado resulted in the detection of a new antifungal compound (E, Z, Z)-1-acetoxy-2-hydroxy-4-oxo- heneicosa-5, 12,15-triene. This new compound was shown to inhibit spore germination of C. gloeosporioides similarly as the antifungal diene. We had localized one of the biosynthetic places of these antifungal compounds in specialized idioblast cells (oil cells) in the mesocarp that can be easily enhanced by elicitors as ethylene. Results have also suggested that the antifungal compounds can be "exported" from the mesocarp to the pericarp where its main activity takes place. The search for the biosynthesis of antifungal compounds and the genes involved took two directions i. direct search for specific genes involved in the synthesis of the diene and ii. Indirect selection of genes using the differential display library. We have cloned D9 and D12 desaturase, a protein kinase and a elongase that their transcriptional activation is significantly enhanced during the enhanced synthesis of the antifungal diene. Although we are far away from a complete elucidation of the synthesis of the antifungal compound we have stepped forward determining some of the key steps that might be involved in its synthesis.

The goals of the present research proposal were to elucidate the physiological and molecular basis of the regulation of stilbene metabolism in grape, against the background of (i) grape metabolic network behavior in response to drought and of (ii) varietal diversity. The specific objectives included the study of the physiology of the response of different grape cultivars to continuous WD; the characterization of the differences and commonalities of gene network topology associated with WD in berry skin across varieties; the study of the metabolic response of developing berries to continuous WD with specific attention to the stilbene compounds; the integration analysis of the omics data generated; the study of isolated drought-associated stress factors on the regulation of stilbene biosynthesis in plantaand in vitro. Background to the topic Grape quality has a complex relationship with water input. Regulated water deficit (WD) is known to improve wine grapes by reducing the vine growth (without affecting fruit yield) and boosting sugar content (Keller et al. 2008). On the other hand, irregular rainfall during the summer can lead to drought-associated damage of fruit developmental process and alter fruit metabolism (Downey et al., 2006; Tarara et al., 2008; Chalmers et al., 792). In areas undergoing desertification, WD is associated with high temperatures. This WD/high temperature synergism can limit the areas of grape cultivation and can damage yields and fruit quality. Grapes and wine are the major source of stilbenes in human nutrition, and multiple stilbene-derived compounds, including isomers, polymers and glycosylated forms, have also been characterized in grapes (Jeandet et al., 2002; Halls and Yu, 2008). Heterologous expression of stilbenesynthase (STS) in a variety of plants has led to an enhanced resistance to pathogens, but in others the association has not been proven (Kobayashi et al., 2000; Soleas et al., 1995). Tomato transgenic plants harboring a grape STS had increased levels of resveratrol, ascorbate, and glutathione at the expense of the anthocyanin pathways (Giovinazzo et al. 2005), further emphasizing the intermingled relation among secondary metabolic pathways. Stilbenes are are induced in green and fleshy parts of the berries by biotic and abiotic elicitors (Chong et al., 2009). As is the case for other classes of secondary metabolites, the biosynthesis of stilbenes is not very well understood, but it is known to be under tight spatial and temporal control, which limits the availability of these compounds from plant sources. Only very few studies have attempted to analyze the effects of different environmental components on stilbene accumulation (Jeandet et al., 1995; Martinez-Ortega et al., 2000). Targeted analyses have generally shown higher levels of resveratrol in the grape skin (induced), in seeded varieties, in varieties of wine grapes, and in dark-skinned varieties (Gatto et al., 2008; summarized by Bavaresco et al., 2009). Yet, the effect of the grape variety and the rootstock on stilbene metabolism has not yet been thoroughly investigated (Bavaresco et al., 2009). The study identified a link between vine hydraulic behavior and physiology of stress with the leaf metabolism, which the PIs believe can eventually lead to the modifications identified in the developing berries that interested the polyphenol metabolism and its regulation during development and under stress. Implications are discussed below.

The overall objective of this project was to develop a conceptual framework for enhancing root colonization by beneficial bacteria. To accomplish this aim we tested the hypothesis that production and excretion of the plant phytoalexin medicarpin can be used for creation of a special niche along the legume roots, where beneficial microorganism, such as rhizobium, will have a selective advantage. On the Israeli side it was shown that higher medicarpin levels are exuded following the application of Rhizobium meliloti to the rhizosphere but the specific biochemical pathway governing medicarpin production was not induced significantly enough to support a constant production and excretion of this molecule to the rhizosphere. Furthermore, pathogenic bacteria and chemical elicitors were found to induce higher levels of this phytoalexin and it became important to test its natural abundance in field grown plants. On the US side, the occurrence of flavonoids and nucleosides in agricultural soils has been evaluated and biologically significant quantities of these molecules were identified. A more virulent Agrobacterium tumefaciens strain was isolated from alfalfa (Medicago sativa L.) which forms tumors on a wide range of plant species. This isolate contains genes that increase competitive colonization abilities on roots by reducing the accumulation of alfalfa isoflavonoids in the bacterial cells. Following gene tagging efforts the US lab found that mutation in the bacterial efflux pump operons of this isolate reduced its competitive abilities. This results support our original hypothesis that detoxification activity of isoflavenoids molecules, based on bacterial gene(s), is an important selection mechanism in the rhizosphere. In addition, we focused on biotin as a regulatory element in the rhizosphere to support growth of some rhizosphere microorganisms and designed a bacterial gene construct carrying the biotin-binding protein, streptavidin. Expressing this gene in tobacco roots did not affect the biotin level but its expression in alfalfa was lethal. In conclusion, the collaborative combination of basic and applied approaches toward the understanding of rhizosphere activity yielded new knowledge related to the colonization of roots by beneficial microorganisms in the presence of biological active molecules exuded from the plant roots.