This thesis describes fundamental biological characteristics of soybean. Main topic of the thesis is finding resistence of twelve soybean varieties to bacterial blight. Virulence of four Pseudomonas syringae pv. glycinea isolates was tested in laboratory conditions to find isolates with the highest virulence, which were then used for testing of selected soybean varieties resistance to this patogen. The highest virulence was found in Šumperk 112 isolate and Žabčice isolate. These isolates were used for testing of soybean varieties resistance. The lowest resistance was found in Moravians variety, low resistance was found in Laurentiana variety and Bru-nensis variety. High resistance was found in Gallec variety and Chinese NM3 variety.

No description available.

<p>&nbsp;Bakteriozno su&scaron;enje tre&scaron;nje (Prunus avium L.) poslednjih nekoliko godina u mladim zasadima i plantažama tre&scaron;nje predstavlja značajan problem u proizvodnji ove voćne vrste. Simptomi bolesti se ispoljavaju u vidu su&scaron;enja grana, grančica ili celih stabala, &scaron;to se uglavnom zapaža na mestima rezidbe i oko pupoljaka, sa uočljivim promena boje tkiva kore, koje puca i nastaju rak rane. U periodu od 2012 &ndash; 2015 godine izvr&scaron;en je monitoring zdravstvenog stanja tre&scaron;nje kojim je obuhvaćeno nekoliko plantaža i manjih zasada tre&scaron;nje iz vi&scaron;e lokaliteta na području AP Vojvodine i centralne Srbije (Ritopek). Mlade voćke su očigledno najugroženije, jer smo na osnovu praćenja zdravstvenog stanja u vi&scaron;e lokaliteta i zasadima različite starosti, pojavu bakterioznog su&scaron;enja u jačem ili slabijem intenzitetu, konstatovali samo u mladim zasadima (do 3 godine starosti &ndash; Selenča, Gornji Tavankut, Donji Tavankut, Ljutovo, Mikićevo i Kanjiža). Izolacijama na strandardne hranljive podloge, iz prikupljenih obolelih uzoraka tre&scaron;nje, kao i sa zdravih pupoljaka i listova tre&scaron;nje (epifitna populacija), dobijeni su brojni izolati bakterija P. syringae pvs. od kojih je za dalja ispitivanja odabrano 155 izolata. Identifikikacija dobijenih izolata je izvr&scaron;ena je na osnovu fenotipskih i genotipskih metoda. Na osnovu LOPAT testova izolati pripadaju Ia grupi fluorescentnih vrsta Pseudomonas syringae. Prema GATTa testovima utvrđene su dve grupe izolata u okviru vrste P. syringae: I grupa (G+A+T&ndash;Ta&ndash;) i II grupa (G&ndash;A&ndash;T+Ta+). Dodatni testovi su potvrdili GATTa testove, na osnovu kojih je zaključeno da su&scaron;enje mladih stabala tre&scaron;nje prouzrokuju dve grupe bakterije P. s. pv. syringae (I grupa) i P. s. pv. morsprunorum rasa 1 (II grupa). Među ispitivanim izolatima nije bilo odstupanja u pogledu fenotipskih karakteristika u okviru iste grupe, osim sposobnosti stvaranja siringomicina pojedinih izolata I grupe (pv. syringae). Proverom patogenosti na raznim test biljkama i biljci domaćinu utvrđene su razlike, ali i određene sličnosti između izolata I i II grupe. Jasne razlike između grupa izolata utvrđene su pri inokulaciji zelenih plodova tre&scaron;nje, vi&scaron;nje, ringlova i kru&scaron;ke, paradajza, paprike i mahuna boranije. Pri inokulaciji odvojenih listova jorgovana izolati I grupe (pv. syringae), kao i većina izolata II grupe (pv. morsprunorum rasa 1) su pozitivno reagovali, &scaron;to ukazuje na heterogenost populacije bakterije P. s. pv. morsprunorum rasa 1. Pri inokulaciji sejanaca voćnih podloga (divlja tre&scaron;nja, magriva, divlja &scaron;ljiva, divlja kru&scaron;ka) svi izolati pv. syringae su prouzrokovali karakteristične patolo&scaron;ke promene na podlogama svih voćnih vrsta, a izolati pv. morsprunorum rase 1 takođe na svim vrstama, osim na sejancima divlje &scaron;ljive. Ovi rezultati ukazuju da je &scaron;irenje bakterija moguće i putem podloga koje takođe mogu biti zaražene. Inokulacijama dvogodi&scaron;njih grančica tre&scaron;nje u periodu mirovanja zaključeno je da su svi izolati pv. syringae i morprunorum rasa 1 podjednako patogeni na svim sortama tre&scaron;nje (Burlat, Summit, Hedelfigenska i Germerzdorfska). Najveća dužina nekroze najče&scaron;će je zabeležena na sortama Burlat i Summit u kombinaciji sa izolatima I grupe (pv. syringae) u pojedinim slučajevima i sa izolatima II grupe (pv. morsprunorum rasa 1), a najmanja uglavnom kod sorti Germerzdorfska i Hedelfigenska sa izolatima II grupe (pv. morsprunorum rasa 1). Identifikacija izolata KBNS71 &ndash; 84 (Gornji Tavankut) i KBNS85 &ndash; 94 (Selenča) na bazi MLST kori&scaron;ćenjem gena gyrB, rpoD, gapA i gltA, jasno je pokazala prisustvo dva patovara P. s. pv. syringae i P. s. pv. morsprunorum rasa 1. Pri poređenju sa sojevima H &ndash; 1, V &ndash; 85, V &ndash; 88 (vi&scaron;nja) i V &ndash; 109 (tre&scaron;nja) utvrđene su značajne razlike i postojanje genetskog diverziteta populacije ovih patogena. Simultana detekcija gena syrB i syrD utvrđena je kod 70 izolata I grupe (pv. syringae), a samo SyrB kod 9 izolata iste grupe (pv. syringae). Gen za sintezu koronatina detektovan je kod svih 76 izolata II grupe (pv. morsprunorum rasa 1). Rep &ndash; PCR metodom ustanovljene su značajne razlike (58%) između I i II grupe izolata (pv. syringae i pv. morsprunorum rasa 1). Ispitivani izolati sa tre&scaron;nje u okviru pv. syringae nisu ispoljili međusobne razlike, ali se razlikuju od sojeva sa drugih lokaliteta i ranije izolovanih sa istog domaćina (V &ndash; 109 i T6), kao i od sojeva sa drugih domaćina &ndash; vi&scaron;nje (V &ndash; 85) i uljane tikve (Tk21) do 37%. Razlike među izolatima pv. morsprunorum rase 1 iznosile su manje od 5%, a 24% u odnosu na soj CFBP2119 istog patogenog varijeteta. Rep &ndash; PCR analiza ukazala je na nizak nivo heterogenosti ispitivanih izolata u okviru istog patogenog varijeteta. RAPD metoda, kori&scaron;ćenjem većeg broja prajmera, bila je uspe&scaron;nija za poređenje ispitivanih izolata od rep &ndash; PCR. Od testiranih 11 prajmera, 4 (SPH1, DJP17, DJ15, DJ16) su selektovana za dalji rad na osnovu razlika među izolatima unutar patogenih varijeteta. Kumulativna RAPD analiza pokazala je da između ispitivanih izolata pv. syringae postoje razlike do 24%, a 41% u poređenju sa sojem KFB0103, dok su kod izolata pv. morsprunorum rase 1 razlike iznosile do 15%, a 36% u odnosu na soj</p><p>4<br />CFBP2119. Dobijeni rezultati RAPD analize ukazuju da u okviru populacije obe grupe ispitivanih izolata postoji određena heterogenost, ali je genetski diverzitet izraženiji kod pv. syringae. Proučavanjem epidemilogije ovih patogena u poljskim uslovima inokulacijom jednogodi&scaron;njih grana / mladara sortama Burlat, Germerzdorfska, Hedelfigenska i Droganova žuta, zaključeno je da tre&scaron;nja u na&scaron;im agroekolo&scaron;kim uslovima ranije postaje osetljiva (oktobar) prema P. s. pv. morsprunorum rasa 1 u odnosu na pv. syringae. Prvi pozitivni rezultati pri inokulaciji sojevima pv. syringae utvrđeni su pri inokulaciji u novembru. U pogledu dužine nekroze najuspe&scaron;nije su bile novembarske inokulacije (najduže nekroze; 2,17 &ndash; 3,35 cm), uspe&scaron;ne su bile i januarske i martovske inokulacije, ali je dužina nekroze bila sve manja, respektivno. Generalno najduže nekroze su ostvarene kod sorte Burlat, a najkraće kod sorte Germerzdorfska. Sve inokulacije urađene u periodu vegetacije su bile negativne. Inokulacijama dvo &ndash; trogodi&scaron;njih grana na sorti Summit prve uspe&scaron;ne inokulacije (oba patovara) su ostvarene tek u novembru (oktobarske su bile negativne), kada je utvrđena i veća agresivnost patovara syringae. Pri inokulacijama u januaru dužina nekroze je bila manja, a martovska je bila negativna. Sve inokulacije vr&scaron;ene u periodu od bubrenja pupoljaka do opadanja li&scaron;ća takođe su bile negativne. Ispitivanjem osetljivosti sotrimenta tre&scaron;nje i pojedinih sorti vi&scaron;nje zaključeno je da su prema oba patovara (syringae i morsprunorum rasa 1) najosetljivije sorte tre&scaron;nje Katalin, Linda, Summit, New Star i Burlat, srednje osetljive su sorte vi&scaron;nje Erdi Botermo i sorte tre&scaron;nje Droganova žuta, CarmCarmen, Germerzdorfska i Rana od Noara, a slabo osetljive sorte vi&scaron;nje &Scaron;panska i Ujfeheti firto&scaron; i sorta tre&scaron;nje Rita.</p> / <p>Bacterial die back (canker) of sweet cherry (Prunus avium L.) in young orchards and sweet cherry plantations in the past few years has been a significant problem in the production of this fruit species. Symptoms of the disease were manifested in the form of drying branches, twigs or whole trees, which were mainly observed in places of pruning or around the buds, bark changes a color, cracks and cankers has formed. In the period 2012 - 2015 monitoring of the health status of sweet cherries was carried out covering several plantations and smaller orchards of sweet cherries in several localities in Vojvodina and central Serbia (Ritopek). Young fruit trees are obviously the most susceptible, based on monitoring of the health status in many localities and plantations of different ages, the occurrence of bacterial canker in a stronger or weaker intensity was found only in young plantations (up to 3 years old - Selenča, Gornji Tavankut, Donji Tavankut, Ljutovo, Mikićevo and Kanjiža). From collected diseased samples of sweet cherries, as well as healthy buds and leaves of sweet cherry (epiphytic population) isolations on standard nutrient medium, were obtained numerous isolates of P. syringae pvs. and for further investigations was selected 155 isolates. Identification of isolates was performed on the basis of phenotypic and genotypic methods. Based on LOPAT tests isolates belonging to Ia group fluorescent Pseudomonas syringae. According to GATTa tests two groups of P. syringae isolates were identified, I group (G+A+T-Ta-) and II group (G-A-T+Ta+). Additional tests confirmed the GATT tests, on the basis which it was concluded that the drying of young sweet cherry trees caused P. s. pv. syringae (I group) and P. s. pv. morsprunorum race 1 (II group). Among the tested isolates was not exceptions in phenotypic characteristics within the same group, except for the ability to produced syringomycine for some isolates of I groups (pv. syringae). In pathogenicity tests on various plants and host plant were observed differences, but also and some certain similarity between isolates of I and II groups. Clear differences between the groups of isolates were determined in the inoculations of green fruit of sweet cherry, sour cherry, cherry plum and pears, tomatoes, peppers and green bean pods. In the case of inoculation of separate lilac leaves isolates of I group (pv. syringae) and most isolates of II group (pv. morsprunorum race 1) reactions were positive, what indicating the heterogeneity of the population of P. s. pv. morsprunorum race 1. In the inoculation of fruit rootstock seedlings (wild cherry, Magriva, wild plum, wild pear) all isolates pv. syringae caused the characteristic pathological changes on the all fruit species, isolates of pv. morsprunorum race 1 also except on the seedlings of wild plum. These results suggest that the spreading of bacteria is possibly through the rootstock that can also be infected. Inoculations of two &ndash; years old branches of sweet cherry during dormancy, was concluded that all isolates pv. syringae and morprunorum race 1 were equally pathogenic in all sweet cherry cultivars (Burlat, Summit, Hedelfigen and Germersdorf). The longest length of necrosis usually was observed on the cultivars Burlat and Summit in combination with isolates of I groups (pv. syringae), in some cases with isolates of II group (pv. morsprunorum race 1), and the lowest mainly in cultivars Germersdorf and Hedelfigen with isolates of II group (pv. morsprunorum race 1). Identification of isolates KBNS71 - 84 (GornjiTavankut) and KBNS85 - 94 (Selenča) based on MLST using genes gyrB, rpoD, gapA and gltA genes clearly showed the presence of two patovars P. s. pv. syringae and P. s. pv. morsprunorum race 1. Comparison with strains H - 1, V - 85 V - 88 (sour cherry) and V - 109 (sweet cherry) showed significant differences and the existence of genetic diversity in the population of these pathogens. Simultaneous detection of syrB and syrD gene was found in 70 isolates of I group (pv. syringae) and only syrB gene in 9 isolates of the same group (pv. syringae). The gene for coronatine synthesis was detected in all 76 isolates of II group (pv. morsprunorum race 1). Rep - PCR method detected significant differences (58%) between isolates of I and II groups (pv. syringae and pv. morsprunorum race 1). The tested isolates from sweet cherry within pv. syringae did not show differences between them, but they were different from the strains from other locations and previously isolated from the same host (V - 109 and T6), as well as strains from other hosts - cherry (V - 85) and pumpkin (Tk21) to 37 %. The differences between isolates pv. morsprunorum race 1 were less than 5% and 24% compared to the same pathovar strain CFBP2119. Rep - PCR analysis indicated a low level of heterogeneity of isolates within the same pathovar. RAPD method using a large number of primers were more successful to compare isolates than rep - PCR. Among 11 tested primers, 4 (SPH1, DJP17, DJ15, DJ16) were selected for further work on the basis of the difference between isolates within same pathovar. Cumulative RAPD analysis showed up to 24% differences among tested isolates of pv. syringae and 41% compared to the strain KFB0103, while among isolates pv. morsprunorum race 1 differences were 15% and 36% compared to the strain CFBP2119. The results of RAPD analysis indicate that a certain heterogeneity<br />7<br />exists in the population of both tested groups of isolates, but genetic diversity is more pronounced among isolates of pv. syringae. Studying the epidemiology of this pathogen in field conditions, by inoculating one &ndash; year old branches / or shoots sweet cherry cultivars Burlat, Germersdorf, Hedelfigen and Droganova žuta, it was concluded that the sweet cherry in our agroecological conditions becoming sensitive (October) to P. s. pv. morsprunorum race 1 before in relation to the pv. syringae. The first positive results of inoculations with strains pv. syringae were determined in November. Regarding the length of necrosis most successful were inoculation in the November (necrosis longest; 2.17 to 3.35 cm), inoculations also were successful in the January and the March, but the length of necrosis was smaller, respectively. Generally longest necrosis were observed in the cultivar Burlat, and the shortest in cultivar Germersdorf. All inoculations carried out in the period of vegetation were negative. Inoculations of two &ndash; three &ndash; years old branches of the cultivar Summit, first successful inoculations (for both pathovar) were observed only in November (October was negative), when a greater aggressiveness of pathovar syringae were determined. In inoculations in January length of necrosis was smaller, and in March was negative. All inoculations carried out in the period from buds swelling to leaf falling were also negative. Investigation susceptibility of sweet cherry and some sour cherry cultivars was concluded that against to both pathovars (syringae and morsprunorum race 1) the most susceptible were cultivars of sweet cherry Katalin, Linda, Summit, New Star and Burlat, medium susceptible were cultivar of sour cherry Erdi Botermo and sweet cherry cultivars Droganova žuta, Carmen, Germersdorf and Rana od Noara and low susceptible cultivars of sour cherry &Scaron;panska and Ujfeheti firto&scaron; and cultivar of sweet cherry Rita.</p>

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2018-04-19T16:19:33Z No. of bitstreams: 1 texto completo.pdf: 2766887 bytes, checksum: 6f8f01cd7e538b366f3e29fab1e9c82c (MD5) / Made available in DSpace on 2018-04-19T16:19:33Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2766887 bytes, checksum: 6f8f01cd7e538b366f3e29fab1e9c82c (MD5) Previous issue date: 2017-07-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O Brasil é o segundo produtor mundial de soja. Porém, estresses bióticos e abióticos tem limitado a expansão da produtividade. Nosso grupo de pesquisa do LBMP, BIOAGRO-UFV, tem observado que plantas transgênicas superexpressando BiP (Binding protein), chaperona com atividade associada à via UPR (unfolded protein response) e à modulação de eventos de PCD (programmed cell death), são mais tolerantes à seca pela manutenção da homeostase celular e retardo do acionamento da PCD. Dos fitopatógenos, a Pseudomonas syringae pv. tomato provoca reação de hipersensibilidade na soja, uma vez que a interação planta-bactéria é incompatível. Neste trabalho, foi caracterizado o perfil metabólico dos genótipos transgênico superexpressando BiP (C9) e selvagem (WT) por GC-MS. Foram também avaliados por LC-MS, a abundância de fito-hormônios e alguns metabólitos secundários alvos em resposta à interação de soja com P. s. pv. tomato para determinar alterações metabólicas nestes genótipos relacionadas com o fenótipo de morte celular e reações de hipersensibilidade. O acúmulo de aminoácidos, açúcares e ácidos orgânicos pode estar relacionado ao controle de danos ocasionado pelo estresse biótico, sendo menor em C9 devido à capacidade de BiP em manter a homeostase celular. Compostos como di- hidroesfingosina (DHS) e gama-aminobutirato (GABA) podem estar envolvidos com mecanismos de PCD em células visando restringir a colonização do tecido foliar por bactérias. Além disso, observou-se uma tendência para o aumento de ácido salicílico (SA) e ácido jasmônico (JA), embora os níveis de ácido abscísico (ABA) em C9 tenham sido menores, indicando o papel antagonista do ABA na via de sinalização mediada por SA/JA e síntese de fitoalexinas ao longo da infecção bacteriana. A considerável importância do SA e JA, que disparam vias de sinalização de controle microbiano, em C9 possivelmente está no controle negativo da PCD por BiP, que impede a contenção do patógeno por esta via. As concentrações de metabólitos secundários, por sua vez, sofreram o maior incremento ao longo do tempo, sobretudo daidzeína e genisteína, sendo maior em C9. Este acréscimo pode estar associado com as propriedades antimicrobianas das isoflavonas. Por isso, em plantas C9, a superexpressão de BiP atrasa a via de PCD, impedindo a contenção da colonização bacteriana em tempo hábil, sendo a ação antimicrobiana o mecanismo de resposta predominante em C9, ao passo que em WT predomina a via de PCD. / Brazil is the world's second largest soybean's producer. However, biotic and abiotic stresses have limited productivity expansion. Our research group at the LBMP, BIOAGRO-UFV, has observed that transgenic plants overexpressing BiP (Binding protein), chaperone with activity associated with pathway UPR (unfolded protein response) and modulation of PCD (programmed cell death) events, are more tolerant to drought by the maintenance of cellular homeostasis and retarding the activation of PCD. Plant pathogens, Pseudomonas syringae pv. tomato provokes hypersensitivity reaction in soybean, since it is an incompatible interaction. In this work, the metabolic profile of the transgenic genotypes overexpressing BiP (C9) and wild type (WT) by GC-MS. We also evaluated by LC-MS, the abundance of phytohormones and some target secondary metabolites in response to the interaction of soybean with P. s. pv. tomato to determine metabolic changes in these genotypes related to cell death phenotype and hypersensitivity reactions. The accumulation of amino acids, sugars and organic acids may be related to damage control caused by biotic stress, being lower in C9 due to BiP's ability to maintain cellular homeostasis. Compounds such as dihydrosphingosine (DHS) and gamma aminobutyric acid (GABA) may be involved with PCD mechanisms in cells with the aim of to restrain the colonization of foliar tissue by bacteria. Furthermore, there was a tendency for increased salicylic acid (SA) and jasmonic acid (JA), although abscisic acid (ABA) levels have been lower in C9 plants, indicating the ABA antagonist role in the SA-mediated or JA-mediated signaling pathway and phytoalexin synthesis throughout the bacterial infection. The considerable importance of SA and JA, which trigger pathways of microbial control signaling, in C9 is possibly in the negative PCD control by BiP, which prevents the containment of the pathogen for this pathway. Concentrations of secondary metabolites suffered the greatest increase over time, especially daidzein and genistein, being higher in C9 plants. This increase may be associated with the antimicrobial properties of isoflavones. Therefore, in C9 plants, the overexpression of BiP delays the PCD pathway, preventing bacterial colonization in a timely manner, and the antimicrobial action is the predominant response mechanism in C9, whereas in WT, the PCD pathway predominates.

L'induction du métabolisme secondaire fait partie du système de défense des plantes lors d’une attaque par un microorganisme pathogène. Les propriétés antimicrobiennes ou de signalisation des métabolites secondaires (MS) peuvent être régulées par un processus majeur et efficace, la glycosylation. Cette réaction est catalysée par les glycosyltransférases (UGTs) qui assurent le transfert d’une molécule de sucre sur un MS. Mon travail de thèse a porté sur l’étude de trois UGTs d’Arabidopsis thaliana (Arabidopsis), UGT76E12 d’une part, et UGT73B3 et UGT73B5 d’autre part, et a montré que 1) UGT76E12 est impliquée dans la mise en place de la résistance basale à la souche virulente de la bactérie Pseudomonas syringae pv. tomato, Pst DC3000, et participe au dialogue entre les voies de signalisation hormonale de l’acide jasmonique (JA) et de l’acide salicylique (SA). Des analyses par clustering hiérarchique ont révélé une co-expression forte entre le gène UGT76E12, et deux gènes codant une terpène synthase (TPS4) et un cytochrome P450 (CYP82G1), impliqués dans la biosynthèse d’un diterpène, le TMTT ((E,E)-4,8,12-triméthyletridéca-1,3,7,11-tetraène), suggérant un rôle pour UGT76E12 dans la régulation de la voie de biosynthèse des terpènes ; 2) UGT73B3 et UGT73B5 sont impliquées dans la mise en place de la résistance spécifique d’Arabidopsis à la souche avirulente Pst DC3000-AvrRpm1, notamment par la régulation de MS intervenant dans le maintien de l’état redox cellulaire au cours de la réaction d’hypersensibilité (HR). Ces deux gènes appartiennent à la classe des « SA-early induced-genes » et présentent un profil co-expression avec le gène TOLB-related et deux gènes codant des glutathion S-transférases (GSTU7 et GSTU24) impliqués dans la réponse au stress oxydant et la détoxication des MS. Nos résultats suggèrent un rôle pour UGT73B3 et UGT73B5 dans la glycosylation de métabolites oxydants associés à la mise en place de la HR. / Plant secondary metabolism induction is part of an integrated defense system after pathogen infection. Signalling and antimicrobial properties of secondary metabolites (SM) can be regulated by an efficient process named, glycosylation. This reaction is ensured by glycosyltransferases (UGTs) which catalyze the transfer of a sugar moiety on a SM. My PhD work was divided in two parts, the first on UGT76E12 and the second part on UGT73B3 and UGT73B5, three UGTs of Arabidopsis thaliana (Arabidopsis) and it demonstrated that 1) UGT76E12 is involved in basal resistance establishment during plant challenge with the virulent strain of Pseudomonas syringae pv. tomato (Pst DC3000), and participates to the hormonal crosstalk between jasmonic acid (JA) and salicylic acid (SA). Clustering analyses revealed a strong co-expression between UGT76E12 and two genes encoding a terpene synthase (TPS4) and a cytochrome P450 (CYP82G1), both involved in the biosynthesis of a diterpene named TMTT ((E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene), which suggests a role for UGT76E12 in terpene biosynthesis pathway regulation. 2) UGT73B5 and UGT73B3 are involved in the establishment of Arabidopsis specific resistance against the avirulent strain of Pst DC3000-AvrRpm1, especially in the regulation of MS playing a role in redox homeostasis status during the hypersensitive response (HR). These two genes are part of SA-early induced-genes and showed co-expression patterns with the TOLB-related gene and two genes encoding glutathione S-transferases (GSTU7 and GSTU24) involved in oxidative stress responses and in MS detoxification. Our results suggest a role for UGT73B3 and UGT73B5 in the glycosylation of oxidant MS associated with HR establishment.

Atualmente, o HLB &eacute; considerado a principal doen&ccedil;a que acomete as plantas c&iacute;tricas. Diante desse fator, pesquisas por cultivares resistentes a esta doen&ccedil;a s&atilde;o necess&aacute;rias. A transforma&ccedil;&atilde;o gen&eacute;tica via Agrobacterium, juntamente com o uso de plantas modelos, tem sido uma alternativa para verifica&ccedil;&atilde;o do funcionamento dos genes em resposta a pat&oacute;genos, isto porque as plantas modelos possuem como caracter&iacute;stica ciclo de vida curto e alto poder de regenera&ccedil;&atilde;o. Assim sendo, objetivou-se com o presente trabalho, a transforma&ccedil;&atilde;o gen&eacute;tica via Agrobacterium tumefaciens, de tomate Micro-Tom (Solanum lycopersicum L.) e de laranja doce, com os genes que codificam as prote&iacute;nas PR-8 e CDR-1, isolados a partir de Citrus simensis. Os cotil&eacute;dones provenientes de sementes germinadas in vitro de tomate Micro-Tom foram utilizados como fonte de explante para os experimentos de transforma&ccedil;&atilde;o gen&eacute;tica com os genes PR-8 e CDR-1. Esses explantes foram subcultivados at&eacute; o aparecimento de brotos regenerantes e posteriormente plantas transg&ecirc;nicas, as quais foram aclimatizadas e levadas a casa-de-vegeta&ccedil;&atilde;o. A transgenia foi confirmada por PCR e o n&uacute;mero de inser&ccedil;&otilde;es do gene por Southern blot. As plantas foram cultivadas at&eacute; a obten&ccedil;&atilde;o da gera&ccedil;&atilde;o T1. Simultaneamente, foram realizados experimentos de transforma&ccedil;&atilde;o gen&eacute;tica em segmentos de epic&oacute;tilo, provenientes de sementes de laranja &lsquo;Hamlin&rsquo; germinadas in vitro, com o gene CDR-1, a fim de se obter plantas transg&ecirc;nicas e sua caracteriza&ccedil;&atilde;o. Paralelamente, foi realizada a constru&ccedil;&atilde;o da curva padr&atilde;o pela an&aacute;lise de qPCR para identifica&ccedil;&atilde;o de Pseudomonas syringae pv. tomato. Foram obtidas treze plantas transg&ecirc;nicas de tomate Micro-Tom com o gene PR-8 e tr&ecirc;s com o gene CDR-1. As efici&ecirc;ncias de transforma&ccedil;&atilde;o foram em torno de 0,38 a 1,98%. Tr&ecirc;s plantas de tomate Micro-Tom transg&ecirc;nicas com o gene PR-8 foram caracterizadas por Southern blot e o n&uacute;mero de inser&ccedil;&otilde;es variou de 1 a 3. Dezenove plantas transg&ecirc;nicas de laranja &lsquo;Hamlin&rsquo; com o gene CDR-1 foram obtidas atrav&eacute;s dos experimentos de transforma&ccedil;&atilde;o gen&eacute;tica. A efici&ecirc;ncia de transforma&ccedil;&atilde;o foi de 2,06 a 5,96%. Dessas, apenas uma foi caracterizada por Southern blot apresentando 1 n&uacute;mero de c&oacute;pia do DNA no genoma da planta. / HLB is currently considered the main disease affecting citrus plants. Given this factor, research for cultivars resistant to this disease is needed. Genetic transformation via Agrobacterium with the use of model plants has been an alternative for checking the gene function in response to pathogens, because these model plants have as characteristic a short life cycle and high power of regeneration. Therefore, the aim of this work was to produce transgenic plants, via Agrobacterium tumefaciens, of Micro-Tom tomato (Solanum lycopersicum L.), and sweet orange, with the genes encoding the PR-8 and CDR-1 proteins isolated from Citrus sinensis. The cotyledons from in vitro germinated Micro-Tom tomato seeds were used as explants source for genetic transformation experiments with PR-8 and CDR-1 genes. These explants were subcultured until the appearance of regenerating shoots and after transgenic plants, which were acclimatized and taken to a greenhouse. The transgenic plants were confirmed by PCR and the number of gene insertions by Southern blot. The plants were grown until T1 generation was obtained. Simultaneously genetic transformation experiments were performed with epicotyl segments from \'Hamlin\' sweet orange seeds germinated in vitro with CDR-1 gene in order to obtain transgenic plants and their characterization. Simultaneously, the standard curve construction was performed by qPCR analysis for identification of Pseudomonas syringae pv. tomato. Thirteen transgenic plants of Micro-Tom tomato with PR-8 gene and three with CDR-1 gene were obtained. The transformation efficiencies were around 0,38 to 1,98%. Three transgenic plants of Micro-Tom tomato with PR-8 gene were characterized by southern blot, and the number of inserts ranged from 1 to 3. Nineteen transgenic \'Hamlin\' sweet orange plants with CDR-1 gene were obtained through genetic transformation experiments, and the transformation efficiency was 2,06 to 5,96%. One plant was characterized, by Southern blot and has one DNA copy number in the plant genome.

Atualmente, o HLB &eacute; considerado a principal doen&ccedil;a que acomete as plantas c&iacute;tricas. Diante desse fator, pesquisas por cultivares resistentes a esta doen&ccedil;a s&atilde;o necess&aacute;rias. A transforma&ccedil;&atilde;o gen&eacute;tica via Agrobacterium, juntamente com o uso de plantas modelos, tem sido uma alternativa para verifica&ccedil;&atilde;o do funcionamento dos genes em resposta a pat&oacute;genos, isto porque as plantas modelos possuem como caracter&iacute;stica ciclo de vida curto e alto poder de regenera&ccedil;&atilde;o. Assim sendo, objetivou-se com o presente trabalho, a transforma&ccedil;&atilde;o gen&eacute;tica via Agrobacterium tumefaciens, de tomate Micro-Tom (Solanum lycopersicum L.) e de laranja doce, com os genes que codificam as prote&iacute;nas PR-8 e CDR-1, isolados a partir de Citrus simensis. Os cotil&eacute;dones provenientes de sementes germinadas in vitro de tomate Micro-Tom foram utilizados como fonte de explante para os experimentos de transforma&ccedil;&atilde;o gen&eacute;tica com os genes PR-8 e CDR-1. Esses explantes foram subcultivados at&eacute; o aparecimento de brotos regenerantes e posteriormente plantas transg&ecirc;nicas, as quais foram aclimatizadas e levadas a casa-de-vegeta&ccedil;&atilde;o. A transgenia foi confirmada por PCR e o n&uacute;mero de inser&ccedil;&otilde;es do gene por Southern blot. As plantas foram cultivadas at&eacute; a obten&ccedil;&atilde;o da gera&ccedil;&atilde;o T1. Simultaneamente, foram realizados experimentos de transforma&ccedil;&atilde;o gen&eacute;tica em segmentos de epic&oacute;tilo, provenientes de sementes de laranja &lsquo;Hamlin&rsquo; germinadas in vitro, com o gene CDR-1, a fim de se obter plantas transg&ecirc;nicas e sua caracteriza&ccedil;&atilde;o. Paralelamente, foi realizada a constru&ccedil;&atilde;o da curva padr&atilde;o pela an&aacute;lise de qPCR para identifica&ccedil;&atilde;o de Pseudomonas syringae pv. tomato. Foram obtidas treze plantas transg&ecirc;nicas de tomate Micro-Tom com o gene PR-8 e tr&ecirc;s com o gene CDR-1. As efici&ecirc;ncias de transforma&ccedil;&atilde;o foram em torno de 0,38 a 1,98%. Tr&ecirc;s plantas de tomate Micro-Tom transg&ecirc;nicas com o gene PR-8 foram caracterizadas por Southern blot e o n&uacute;mero de inser&ccedil;&otilde;es variou de 1 a 3. Dezenove plantas transg&ecirc;nicas de laranja &lsquo;Hamlin&rsquo; com o gene CDR-1 foram obtidas atrav&eacute;s dos experimentos de transforma&ccedil;&atilde;o gen&eacute;tica. A efici&ecirc;ncia de transforma&ccedil;&atilde;o foi de 2,06 a 5,96%. Dessas, apenas uma foi caracterizada por Southern blot apresentando 1 n&uacute;mero de c&oacute;pia do DNA no genoma da planta. / HLB is currently considered the main disease affecting citrus plants. Given this factor, research for cultivars resistant to this disease is needed. Genetic transformation via Agrobacterium with the use of model plants has been an alternative for checking the gene function in response to pathogens, because these model plants have as characteristic a short life cycle and high power of regeneration. Therefore, the aim of this work was to produce transgenic plants, via Agrobacterium tumefaciens, of Micro-Tom tomato (Solanum lycopersicum L.), and sweet orange, with the genes encoding the PR-8 and CDR-1 proteins isolated from Citrus sinensis. The cotyledons from in vitro germinated Micro-Tom tomato seeds were used as explants source for genetic transformation experiments with PR-8 and CDR-1 genes. These explants were subcultured until the appearance of regenerating shoots and after transgenic plants, which were acclimatized and taken to a greenhouse. The transgenic plants were confirmed by PCR and the number of gene insertions by Southern blot. The plants were grown until T1 generation was obtained. Simultaneously genetic transformation experiments were performed with epicotyl segments from \'Hamlin\' sweet orange seeds germinated in vitro with CDR-1 gene in order to obtain transgenic plants and their characterization. Simultaneously, the standard curve construction was performed by qPCR analysis for identification of Pseudomonas syringae pv. tomato. Thirteen transgenic plants of Micro-Tom tomato with PR-8 gene and three with CDR-1 gene were obtained. The transformation efficiencies were around 0,38 to 1,98%. Three transgenic plants of Micro-Tom tomato with PR-8 gene were characterized by southern blot, and the number of inserts ranged from 1 to 3. Nineteen transgenic \'Hamlin\' sweet orange plants with CDR-1 gene were obtained through genetic transformation experiments, and the transformation efficiency was 2,06 to 5,96%. One plant was characterized, by Southern blot and has one DNA copy number in the plant genome.

In 2011 was conducted experimental plantings on land ZF Mendelu in the Lednice for determining the relative resistance of selected varieties of apricots, and to determine the potential substances to enhance resistance to P. syringae pv. syringae. The varieties were inoculated with strains FN3, Lmg1247 and CCM4073. In 2013 and 2014 were observed selected symptoms on plants, which was determined the level of relative resistance of each variety. As resistant varieties were determined 'Kuresia', 'Orangered', 'Bergeval' and 'Bergarouge'. As moderately susceptible varieties were determined 'Tardicot', 'Leskora', 'Aurora', 'Harogem', 'Pinkcot' and 'Sylvercot' on rootstock 'Waxva'. As susceptible varieties were determined 'Goldrich', 'Kioto', 'Bergeron' and 'Sylvercot' on rootstock 'Rubira'.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-20T13:00:17Z No. of bitstreams: 1 texto completo.pdf: 327861 bytes, checksum: 10b3c4ac9c1fb002cc94c4da4da28d0e (MD5) / Made available in DSpace on 2017-04-20T13:00:17Z (GMT). No. of bitstreams: 1 texto completo.pdf: 327861 bytes, checksum: 10b3c4ac9c1fb002cc94c4da4da28d0e (MD5) Previous issue date: 2002-12-03 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A busca de alimentos produzidos sob um sistema de manejo menos agressivo ao meio ambiente vem sendo adotado por um número cada vez maior de produtores. Entretanto, apesar de existirem diversos benefícios na redução ou até eliminação do uso de defensivos, a grande diversidade de doenças em tomateiro (Lycopersicon esculentum Mill.), capazes de limitar a produção, torna necessária a busca por alternativas viáveis, eficientes e tecnicamente comprovadas. Dentre os organismos mais estudados, bactérias têm sido relatadas como agentes de biocontrole capazes de atuar por meio de mecanismos como antibiose, parasitismo, competição e indução de resistência. O presente trabalho teve como objetivos selecionar bactérias do filoplano do tomateiro, baseado em uma estratégia de seleção in vivo, verificando se há um método de isolamento que permita obter antagonistas eficientes no controle da pinta-preta, causada por Alternaria solani, requeima por Phytophthora infestans, mancha-bacteriana pequena por Pseudomonas syringae pv. tomato e mancha-bacteriana por Xanthomonas vesicatoria. Objetivou ainda estudar a possibilidade dos mecanismos de antibiose e indução de resistência serem responsáveis pelo controle destas doenças e se testes de antibiose in vitro são adequados como critério de seleção. Caracterizar aspectos biológicos dos antagonistas que podem otimizar sua aplicação como agente de biocontrole. Determinar a quais produtos antimicrobianos os isolados são insensíveis, visando fornecer subsídios para o desenvolvimento de meios semi-seletivos e estudos de dinâmica populacional. Verificar sua compatibilidade com antibióticos e fungicidas registrados para o controle de enfermidades do tomateiro, a fim de inseri-lo no sistema de manejo integrado e estudar a eficiência de antagonistas selecionados em condições de campo. Os resultados demonstram que, em folíolos mais jovens, os métodos de isolamento que visam obter bactérias da população total e da superfície do filoplano, foram os que permitiram obter a maioria dos antagonistas. O único obtido de folíolos mais velhos foi proveniente da população capaz de habitar sítios protegidos do filoplano e/ou resistir a fatores de estresse. Não se observou relação entre características biológicas dos antagonistas e dos patógenos testados. Nos testes de antibiose com os antagonistas selecionados, o isolado UFV-STB 6 foi capaz de produzir compostos voláteis e inibir a germinação de cistos de Phytophthora infestans, o que possivelmente deve estar envolvido no controle da requeima. O isolado UFV-IEA 6 produziu quitinase, havendo uma tendência em reduzir a taxa de crescimento de Alternaria solani por compostos voláteis. Ficou demonstrado que os testes de antibiose in vitro são inadequados como critério para seleção de agentes de biocontrole do filoplano de tomateiro. A caracterização dos melhores antagonistas demonstrou que três são bactérias Gram-positivas, em forma de bastonete, e uma Gram-negativa, pleiomórfica. Dentre as Gram-positivas todas são anaeróbias facultativas e uma forma endósporos. Nenhum antagonista foi capaz de causar reação de hipersensibilidade (HR) em fumo e produzir pigmento fluorescente in vitro. Os períodos de geração calculados a partir das curvas de crescimento revelaram que três isolados são capazes de se multiplicar rapidamente em meio de cultura, o que é uma característica desejável. Os resultados obtidos a partir dos antibiogramas, mostraram que existem antibióticos que podem ser utilizados para elaboração de meios semi-seletivos, adequados a cada antagonista e os testes de compatibilidade com antibióticos e fungicidas utilizados na cultura do tomateiro revelaram que os antagonistas podem ser expostos aos fungicidas benomyl, enxofre, dimetomorph e tiofanato-metílico. Verificou-se também a inadequação de se utilizarem compostos antimicrobianos em meio de cultura para isolamento de agentes bacterianos de controle biológico, uma vez que os antagonistas selecionados foram sensíveis à maioria dos produtos testados. Os testes com as enzimas indicadoras do estado de indução de resistência, β-1,3-glucanases, Fenilalanina amônia-liase (PAL), Peroxidases (PO), Polifenoloxidases (PPO) e Lipoxigenases (LOX), indicaram que o isolado UFV-IEA 6 foi capaz de promover aumento significativo na atividade das PO, evidenciando a possibilidade do antagonista agir como indutor de resistência. Esse parece ser o primeiro caso que se tem conhecimento de uma bactéria não fitopatogênica do filoplano induzindo resistência na mesma cultura de onde foi obtida. Os testes com os dois antagonistas em condições de campo demonstraram que UFV-STB 6 foi o mais eficiente em reduzir a severidade da requeima no terços médio e superior das plantas, enquanto UFV-IEA 6, somente no terço superior. Houve tendência na redução do progresso da septoriose por UFV-STB 6 e capacidade em diminuir o número de frutos com sintomas de requeima. Os resultados demonstram o potencial de uso dos agentes de biocontrole selecionados para as doenças da parte aérea de tomateiro estudadas. / Farmers are increasingly adapting environmentally less aggressive management systems for food production and there are many benefits in reducing or even eliminating pesticide use. Due to a large number of production limiting diseases on tomato (Lycopersicon esculentum), it is desirable to find technically viable and proven alternatives for their control. Among the microorganisms, bacteria have been reported as biocontrol agents capable of acting through antibiosis, parasitism, competition and induced resistance. The present study aimed at selecting the tomato phylloplane bacteria, based on in vivo isolation strategy, and to determine if this method permits obtaining efficient antagonists to control Alternaria solani leaf spot, Phytophthora infestans blight, small bacterial leaf spot caused by Pseudomonas syringe pv tomato, and bacterial leaf spot caused by Xanthomonas vesicatoria. The study also aimed at determining mechanisms of action, such as antibiosis and induced resistance, involved in disease control, and to determine if the antibiosis tests are sufficient selection criteria. The study also included biological characterization of the antagonists that may optimize their use. To help develop selective or semi-selective media for population dynamic studies, insensitivity of selected isolates to some antimicrobial compounds was also determined. The compatibility of select antagonists with antibiotics and fungicides registered for control of tomato diseases was elucidated so that the antagonist can be inserted in the integrated management. Field studies were done to determine the efficiency of select antagonists. The isolation method that obtain total bacterial population from the phylloplane of the young leaflets permitted obtaining maximum number of antagonists. The only one isolate obtained from the older leaflets originated from the population capable of inhabiting protected sites of phylloplane and/or that resist stress factors. There was no relation between biological characteristics of the antagonists and of the pathogen tested. In the antibiosis testes, the isolate UFV-STB 6 produced volatile compounds that inhibited germination of P. infestance cyst and may be involved in the control of blight. The isolate UFV-IEA 6 produced chitinase and showed a tendency to reduce A. solani growth by the volatile compounds. In vitro antibiosis testes were inadequate criteria to select biocontrol agents from tomato phylloplane. The characterization of promising antagonists showed that three were Gram positive bacilli and one was gram negative pleiomorphic bacteria. Among the Gram positives all were facultative anaerobes and the one formed endospores. None of the antagonists caused hypersensitive reaction (HR) in tobacco, and did not produce fluorescent pigment in vitro. The generation period calculated from the growth curve revealed that three isolates are capable of multiplying rapidly in the culture media, which is a desirable characteristic. The antibiograms showed that there are antibiotics that can be used for elaboration of semi-selective media for each of antagonists and the compatibility testes with antibiotics and fungicides used on tomato crop revealed that the antagonists can be exposed to fungicides such as benomyl, sulfur, dimethomorph and thiophante-methyl. Many antimicrobial compounds were inhibitory to the selecte antagonists in culture media used for isolation of bacterial biocontrol agents, therefore were inadequate for use in selective media. The analysis of enzymes involved in induced resistance, like -1,3-gluconase, phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO) and lipoxigenase (LOX), showed that the isolate UFV-IEA 6 was capable of increasing PO activity, showing the possibility of being a resistance inducer . This appears to be the first case of a non-pathogenic phylloplane bacterium inducing resistance in a plant of origin. The field testes with two antagonists, UFV-STB 6 was more efficient in reducing the blight severity in the middle and upper third of the plant, while UFV-IEA 6 only in the upper third. The latter isolate also showed a tendency for reducing the Septoria leaf spot progress and the number of fruits with the blight symptoms. The results showed these isolates have the potential of use to control tomato diseases of aerial parts. / Tese importada do Alexandria

The aim of this diploma thesis is determination of the influence of deficient nutrition in combination of Pseudomonas syringae pv. tomato infection on tomato (Solanum lycopersicum L.) growth and development. In hydroponic cultivation experiment, tomato variety Darinka was cultivated in solutions without N, P, K, Ca, Mg and in variant with all nutrition (control variant). The dry matter of root and stem was weighed and root length, density and electrical capacitance were set. The content of nutrition in plants was also assessed. The level of infection was determined in 1st term, the highest level had plants with insufficient of Ca and P. High reduction of root and stem dry matter was found in Ca, N, P and Mg deficient plants. The low root electrical capacitance, length and density was determined in plants with Ca, N a P deficiency within infected group and in Ca and N deficient plants within noninfected group. Bacterial infection caused the reduction of all observing parameters in contrast to the plants from noninfected group. The antagonism and synergism between the elements were also showed.