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.

Techn. Universiẗat, Diss., 2005--München.

VARELA, Anna Lídia Nunes. Ação de galactomanana sulfatada isolada de Adenanthera pavonina na resposta bioquímica de defesa do feijão-de-corda [Vigna unguiculata (L.) Walp.] desafiado pelo fungo Colletotrichum gloeosporioides. 2012. 96 f. Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza-CE, 2012. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-08T13:35:57Z No. of bitstreams: 1 2012_dis_alnvarela.pdf: 2177919 bytes, checksum: b7898740c28944706d715c2b6f0eb897 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:17:13Z (GMT) No. of bitstreams: 1 2012_dis_alnvarela.pdf: 2177919 bytes, checksum: b7898740c28944706d715c2b6f0eb897 (MD5) / Made available in DSpace on 2016-08-02T20:17:13Z (GMT). No. of bitstreams: 1 2012_dis_alnvarela.pdf: 2177919 bytes, checksum: b7898740c28944706d715c2b6f0eb897 (MD5) Previous issue date: 2012 / Cowpea [Vigna unguiculata (L.) Walp.] is a food of great commercial importance, being the main food source in some regions such as northeastern Brazil. Unfortunately, cowpea is subject to attack by various pathogens and pests, causing considerable losses in productivity. To reduce these losses, there are several chemical methods that are widely used, but the indiscriminate use of fungicides has caused damage to the environment and living beings. In search of alternative environmentally friendly, a promising option would be the induction of defense mechanisms of the plant itself through elicitor molecules. Given the above, this study aimed to elicit defense responses in cowpea through the use of galactomannan isolated from Adenanthera pavonina and induce resistance to Colletotrichum gloeosporioides. Twelve days after germination, cowpea plants (Vigna unguiculata) were treated with A. pavonina galactomannan (at concentrations of 100 mg/L and 200 mg/L) in the absence or presence of the fungus C. gloeosporioides (inoculated 6 hours after treatment with carbohydrate) for 6, 12, 24 and 48 h were carried out on biochemical analysis of protein extracts obtained from primary leaves. For enzymes POX and APX a significant increase occurred after 12 h of treatment with the galactomannan. With respect to antifungal activity of the enzymes and βGLU CHI, the plants treated with the galactomannan in general, showed an increase above 50% compared to control. Macroscopic analysis revealed that the leaves treated with 200 mg/L of galactomannan and infected with C. gloeosporioides showed necrotic lesions visibly reduced compared to control. Furthermore, the galactomannan was unable to inhibit the germination of spores and vegetative growth of the fungus C. gloeosporioides in vitro. The results of the present study indicate that galactomannan isolated from A. pavonina can increase crop tolerance of cowpea (V. unguiculata) against the fungus C. gloeosporioides. / O feijão caupi [Vigna unguiculata (L.) Walp.] é um alimento de grande importância comercial, sendo a principal fonte alimentícia em algumas regiões, como no Nordeste brasileiro. Infelizmente, o feijão-de-corda está sujeito ao ataque de vários patógenos e pestes, acarretando perdas consideráveis em sua produtividade. Para diminuir essas perdas, existem diversos métodos químicos que são largamente utilizados, porém o uso indiscriminado de fungicidas tem causado danos ao meio ambiente e aos seres vivos. Na busca de alternativas ecologicamente saudáveis, uma opção promissora seria a indução de mecanismos de defesa da própria planta através de moléculas elicitoras. Diante do exposto, o presente trabalho teve como objetivo elicitar respostas de defesa em feijão-de-corda através do uso de galactomanana sulfatada isolada de Adenanthera pavonina e induzir a resistência ao fungo Colletotrichum gloeosporioides. Doze dias após germinação, plantas de feijão-de-corda (Vigna unguiculata) foram tratadas com galactomana de A. pavonina (nas concentrações de 100 mg/L e 200 mg/L), na ausência ou presença do fungo C. gloeosporioides (inoculado após 6 h do tratamento com carboidrato) por 6, 12, 24 e 48 h, tendo sido realizadas as análises bioquímicas nos extratos protéicos obtidos das folhas primárias. Para as enzimas POX e APX ocorreu um significativo aumento a partir de 12 h do tratamento com a galactomanana. Em relação a atividade das enzimas antifungicas βGLU e CHI, as plantas tratadas com a galactomanana, no geral, apresentaram um aumento acima de 50% em relação ao controle. Análises macroscópicas revelaram que as folhas tratadas com 200 mg/L da galactomanana e infectadas com o C. gloeosporioides apresentaram lesões necróticas visivelmente reduzidas em relação ao controle. Ademais, a galactomana não foi capaz de inibir a germinação de esporos e crescimento vegetativo do fungo C. gloeosporioides in vitro. A partir dos resultados obtidos neste trabalho, conclui-se que, a galactomanana isolada de A. pavonina é capaz de aumentar a tolerância da cultura de feijão-de-corda (V. unguiculata) contra o fungo C. gloeosporioides.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Tereza Ošťádalová Supervisor: Doc. PharmDr. Lenka Tůmová, CSc. Title of diploma thesis: The selenium effect on secondary metabolites production in in vitro cultures of medicinal plants - II. Key words: callus, suspension culture, abiotic elicitor, selenium, Fagopyrum In vitro plant cultures usually produce only a small amount of secondary metabolites. The method of elicitation is one of the options how to increase the production of these substances. The effect of selenium as abiotic elicitor on rutin production in callus and suspension culture of Fagopyrum esculentum Moench., variety Pyra was observed in this study. The release of rutin into the nutrient medium was studied as well. The cultivation was performed in Murashige and Skoog (MS) nutrient medium which was enriched with 1 mg/l of 2,4-dichlorophenoxyacetic acid. The solution of selenium in three different concentrations (c1 = 9.012.10-3 mol/l, c2 = 9.012.10-4 mol/l, c3 = 9.012.10-5 mol/l) was used. The samples were taken after 6, 12, 24, 48, 72 and 168 hours of elicitor treatment. The rutin content was determined by high-performance liquid chromatography (HPLC). The elicitation led to increasing of rutin amount in callus and also in suspension...

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O feijÃo caupi [Vigna unguiculata (L.) Walp.] à um alimento de grande importÃncia comercial, sendo a principal fonte alimentÃcia em algumas regiÃes, como no Nordeste brasileiro. Infelizmente, o feijÃo-de-corda està sujeito ao ataque de vÃrios patÃgenos e pestes, acarretando perdas considerÃveis em sua produtividade. Para diminuir essas perdas, existem diversos mÃtodos quÃmicos que sÃo largamente utilizados, porÃm o uso indiscriminado de fungicidas tem causado danos ao meio ambiente e aos seres vivos. Na busca de alternativas ecologicamente saudÃveis, uma opÃÃo promissora seria a induÃÃo de mecanismos de defesa da prÃpria planta atravÃs de molÃculas elicitoras. Diante do exposto, o presente trabalho teve como objetivo elicitar respostas de defesa em feijÃo-de-corda atravÃs do uso de galactomanana sulfatada isolada de Adenanthera pavonina e induzir a resistÃncia ao fungo Colletotrichum gloeosporioides. Doze dias apÃs germinaÃÃo, plantas de feijÃo-de-corda (Vigna unguiculata) foram tratadas com galactomana de A. pavonina (nas concentraÃÃes de 100 mg/L e 200 mg/L), na ausÃncia ou presenÃa do fungo C. gloeosporioides (inoculado apÃs 6 h do tratamento com carboidrato) por 6, 12, 24 e 48 h, tendo sido realizadas as anÃlises bioquÃmicas nos extratos protÃicos obtidos das folhas primÃrias. Para as enzimas POX e APX ocorreu um significativo aumento a partir de 12 h do tratamento com a galactomanana. Em relaÃÃo a atividade das enzimas antifungicas βGLU e CHI, as plantas tratadas com a galactomanana, no geral, apresentaram um aumento acima de 50% em relaÃÃo ao controle. AnÃlises macroscÃpicas revelaram que as folhas tratadas com 200 mg/L da galactomanana e infectadas com o C. gloeosporioides apresentaram lesÃes necrÃticas visivelmente reduzidas em relaÃÃo ao controle. Ademais, a galactomana nÃo foi capaz de inibir a germinaÃÃo de esporos e crescimento vegetativo do fungo C. gloeosporioides in vitro. A partir dos resultados obtidos neste trabalho, conclui-se que, a galactomanana isolada de A. pavonina à capaz de aumentar a tolerÃncia da cultura de feijÃo-de-corda (V. unguiculata) contra o fungo C. gloeosporioides. / Cowpea [Vigna unguiculata (L.) Walp.] is a food of great commercial importance, being the main food source in some regions such as northeastern Brazil. Unfortunately, cowpea is subject to attack by various pathogens and pests, causing considerable losses in productivity. To reduce these losses, there are several chemical methods that are widely used, but the indiscriminate use of fungicides has caused damage to the environment and living beings. In search of alternative environmentally friendly, a promising option would be the induction of defense mechanisms of the plant itself through elicitor molecules. Given the above, this study aimed to elicit defense responses in cowpea through the use of galactomannan isolated from Adenanthera pavonina and induce resistance to Colletotrichum gloeosporioides. Twelve days after germination, cowpea plants (Vigna unguiculata) were treated with A. pavonina galactomannan (at concentrations of 100 mg/L and 200 mg/L) in the absence or presence of the fungus C. gloeosporioides (inoculated 6 hours after treatment with carbohydrate) for 6, 12, 24 and 48 h were carried out on biochemical analysis of protein extracts obtained from primary leaves. For enzymes POX and APX a significant increase occurred after 12 h of treatment with the galactomannan. With respect to antifungal activity of the enzymes and βGLU CHI, the plants treated with the galactomannan in general, showed an increase above 50% compared to control. Macroscopic analysis revealed that the leaves treated with 200 mg/L of galactomannan and infected with C. gloeosporioides showed necrotic lesions visibly reduced compared to control. Furthermore, the galactomannan was unable to inhibit the germination of spores and vegetative growth of the fungus C. gloeosporioides in vitro. The results of the present study indicate that galactomannan isolated from A. pavonina can increase crop tolerance of cowpea (V. unguiculata) against the fungus C. gloeosporioides.

Higher plants represent an important source of valuable substances, so called secondary metabolites, which can be obtained through explant cultures of plants. Elicitation is a method of increasing the secondary metabolites production. This study aims to evaluate the secondary metabolites production in Fagopyrum esculentum variety Spacinska cultures in vitro after abiotic elicitor treatment. The experiment was focused on alteration of rutin production in callus and suspension cultures of F. esculentum var. Spacinska after selenium application. Murashige and Skoog nutrient medium supplemented with 1 mg l-1 2,4-dichlorophenoxyacetic acid was used for the cultivation. Selenium solutions of various concentrations (c1 = 9.012×10-3 mol l-1 , c2 = 9.012×10-4 mol l-1 , c3 = 9.012×10-5 mol l-1 ) were affecting the cultures for 6, 12, 24, 48, 72 and 168 hours. The content of rutin was determined by HPLC. The release of secondary metabolites into the nutrient medium was studied as well. After elicitor application, the rutin production increased in both callus and suspension cultures. Higher levels of rutin content were detected in callus culture. The maximum rutin content (0.6 mg g-1 DW) was reached after 12 h of elicitor treatment of c2 concentration in callus culture. Concerning suspension culture, the...

The Effect of Abiotic Elicitors on the Content of Secondary Metabolites in In vitro Plant Cultures - II. The method of elicitation is used for the increased production of secondary metabolites in in vitro plant cultures. In this study, there the effect of pyridine derivate N-(5-chlorpyridin-2-yl)-4-ethylbenzamide in three concentration - 3.845.10-3 mol/l; 3.845.10-4 mol/l and 3.845.10-5 mol/l for the production of flavonolignans in callus and suspension cultures of Silybum marianum was tested. The evaluation of elicitation effect proceeded after 6, 24, 48, 72 a 168 hours against control samples without the elicitor treatment after 24 and 168 hours. In the same intervals the samples of culture medium were taken and assessed. The cells were cultivated on Murashige and Skoog medium with 10 mg/ml of α-naphtylacetic acid as growth regulator. The obtained and adjusted samples were analysed by the HPLC method. From the parts of silymarin complex were detected silychristin, silybin A, isosylibin A, isosilybin B. Flavonoid taxifolin wasn̕ t detected in any case. In callus culture, there was achieved a maximal production of all detected components of silymarin complex (30.508 μg/g DW) after 168 hours of elicitor treatment in concentration of 3.845.10-4 mol/l, it was about only substance - silychristin. The...

The aim of this work was to investigate the effect of pyrazine derivative, 1-octyl-3-(pyrazin-2- yl)urea, as an abiotic elicitor on the production of flavonoid rutin in in vitro cultures of Fagopyrum esculentum Moench., Cultivar Bamby. Suspension and callus cultures were cultivated on Murashige and Skoog nutrient medium (MS) with the addition of 2,4-dichlorophenoxyacetic acid (2,4-D) as growth regulator at a concentration of 1 mg/l. The elicitor solution was added to the cultures at three concentrations: c1 (100,0 mg/100 ml), c2 (10,0 mg/100 ml) and c3 (1,0 mg/100 ml). The elicitor was monitored at six time intervals: 6, 12, 24, 48, 72 and 168 hours. To control samples 1 ml of ethanol 96% was added instead of elicitor solution and samples were collected after 24 and 168 hours. Samples were taken at given time intervals and dried. Subsequently, the rutin content was monitored by HPLC. The rutin release into the nutrient medium was also tested. During the experiment on the callus cultures no statistically significant increase in rutin production after elicitor treatment was observed. But elicitor increased rutin production in suspension cultures after treatment in all tested concentrations. The calluses always released rutin into the nutrient medium. The rutin content in the media ranged from 53,4 to...

Verticillium wilt, caused by the soil-borne Verticillium dahliae Klebahn is a serious problem in the production of sunflower worldwide. To date, information on sunflower resistance to Verticillium spp. is very scarce, although it is critical for an effective management of this pathogen. In this study, two highly aggressive (Vd1396-9 and Vd1398-21) and two weakly aggressive V. dahliae isolates (Vs06-07 and Vs06-14) were used to inoculate moderately resistant (IS6111) and susceptible (IS8048) sunflower hybrids. VdNEP (V. dahliae necrosis and ethylene-inducing protein), an elicitor from V. dahliae, was also used to infiltrate sunflower plants. Our results indicate that VdNEP has a dual role in the interaction between sunflower and V. dahliae. VdNEP acted not only as a pathogenicity factor on sunflower by inducing wilting symptoms such as chlorosis, necrosis and vascular discoloration, but also as an elicitor triggering defense responses of the host. VdNEP induced the hypersensitive cell death in Nicotiana benthamiana leaves and sunflower cotyledons. Moreover, VdNEP activated the production of reactive oxygen species and the accumulation of fluorescent compounds in sunflower leaves. Pathogenesis-related genes (Ha-PR-3, and Ha-PR-5), two defensin genes (Ha-PDF and Ha-CUA1) and genes encoding Ha-ACO, Ha-CHOX, Ha-GST and Ha-SCO were up-regulated by VdNEP, suggesting that multiple signaling pathways are involved in this interaction. Two SA-related genes (Ha-PAL and Ha-NML1) were slightly suppressed after infiltration with VdNEP, suggesting a possible involvement of VdNEP in affecting sunflower defenses.

Verticillium wilt, caused by the soil-borne Verticillium dahliae Klebahn is a serious problem in the production of sunflower worldwide. To date, information on sunflower resistance to Verticillium spp. is very scarce, although it is critical for an effective management of this pathogen. In this study, two highly aggressive (Vd1396-9 and Vd1398-21) and two weakly aggressive V. dahliae isolates (Vs06-07 and Vs06-14) were used to inoculate moderately resistant (IS6111) and susceptible (IS8048) sunflower hybrids. VdNEP (V. dahliae necrosis and ethylene-inducing protein), an elicitor from V. dahliae, was also used to infiltrate sunflower plants. Our results indicate that VdNEP has a dual role in the interaction between sunflower and V. dahliae. VdNEP acted not only as a pathogenicity factor on sunflower by inducing wilting symptoms such as chlorosis, necrosis and vascular discoloration, but also as an elicitor triggering defense responses of the host. VdNEP induced the hypersensitive cell death in Nicotiana benthamiana leaves and sunflower cotyledons. Moreover, VdNEP activated the production of reactive oxygen species and the accumulation of fluorescent compounds in sunflower leaves. Pathogenesis-related genes (Ha-PR-3, and Ha-PR-5), two defensin genes (Ha-PDF and Ha-CUA1) and genes encoding Ha-ACO, Ha-CHOX, Ha-GST and Ha-SCO were up-regulated by VdNEP, suggesting that multiple signaling pathways are involved in this interaction. Two SA-related genes (Ha-PAL and Ha-NML1) were slightly suppressed after infiltration with VdNEP, suggesting a possible involvement of VdNEP in affecting sunflower defenses.