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Fusarium head blight (FHB) is one of the most economically important diseases of small grains and continues to impact crops when environmental conditions are favorable to Gibberella zeae (Fusarium graminearum), the causal agent of the disease. Corn residues are considered to be primary sources of inoculum for epidemics of FHB. Therefore, knowledge of the movement of Gibberella zeae from a local source of infested corn residue is critical to the management of FHB in wheat and barley. Previous research made significant progress in defining the spatial dissemination of inoculum sources of G. zeae within agricultural fields, but was unable to clearly distinguish between within-field and background sources. Using amplified fragment length polymorphism, released clones of G. zeae were tracked within wheat and barley fields. This strategy allowed the distinction between the contributions of released clones to FHB, compared to that of background inocula. Corn residue infested with clones of G. zeae was placed into small replicated plots in winter wheat fields in New York and Virginia in 2007 and 2008 and wheat spikes were collected at 0, 3, 6, and ≥24 m from the inoculum sources. Recovery of released clones decreased an average of 90% between 3 and 6 m from inoculum sources. Various amounts of corn residue infested with a single clone of G. zeae were placed into small replicated plots in winter wheat and barley fields in Virginia from 2008 to 2010. The use of minimal or conventional tillage and a moderately resistant cultivar of wheat or barley may reduce the contribution of within-field inocula to FHB; however, environmental conditions play an important role in the effectiveness of these management strategies. With the increase of corn production due to incentives for ethanol-based fuel, overwintering sites for G. zeae on corn residue are likely to increase. Our work contributes to an increased understanding of the influence of overwintered corn residue to FHB which will also direct future research on how to reduce the inoculum potential from within-field sources. / Ph. D.

Fusarium head blight (FHB), caused by Fusarium graminearum Schwabe, is a devastating fungal disease that affects barley production worldwide through damaged kernels, low yields, and production of mycotoxins. The most effective means of control is through the use of FHB resistant cultivars that are developed through gene pyramiding and incorporation of various sources of resistance. Resistance identified in winter barley cultivar Eve has been identified in Virginia Tech's Small Grains Breeding Program. The objectives of this study are to validate resistance and identify additional resistance QTL, and identify tightly linked and diagnostic markers for use in MAS programs. A population of F5:7 RILs derived from the cross Eve/Doyce were evaluated for FHB resistance. FHB incidence (Inc), FHB severity (Sev), Fusarium damaged kernels (FDK), and deoxynivalenol (DON) were assessed over 2014-15 and 2015-16 at Blacksburg, VA, Mount Holly, VA, Kinston, NC, and Lexington, KY. A QTL on chromosome 6H associated with FHB Sev, FDK, and DON was identified. The QTL accounted for as high as 14.5% of the phenotypic variation for DON accumulation, 8.3% for FHB Sev, and 11.87% to 17.63% for FDK. The QTL marker region spans 56.5 to 66.6 cM with flanking markers SCRI_RS_147342 and Bmag0613. QTL for morphological traits, heading date and height, were found in the same region as the FHB resistance traits. Diagnostic SNP makers associated with the FHB resistance QTL identified can be used for FHB resistance identification in MAS breeding programs to incorporate and pyramid resistance QTL and genes into other plant material. / Master of Science

Phenotypic and genotypic characterization of hulled and hulless winter barley (Hordeum vulgare L.) is necessary for improvement using traditional and molecular breeding techniques.  Identification of genomic regions conferring resistance to Fusarium head blight (caused by Fusarium graminearum), leaf rust (caused by Puccinia hordei G. Otth), powdery mildew [caused by Blumeria graminis (DC.) E.O. Speer f. sp. hordei Em. Marchal], net blotch (caused by Pyrenophora teres) and spot blotch [caused by Cochliobolus sativus (Ito & Kuribayashi) Drechsler ex Dastur] will greatly aid in breeding for improved resistance.  Determining factors that contribute to yield differences between hulled and hulless genotypes, and identification of markers associated with yield and yield related traits will greatly aid in improvement of hulled and hulless genotypes.  The hulled cultivar Nomini, hulless cultivar Eve, and hulless line VA06H-48 were consistently resistant to Fusarium head blight (FHB) and had low deoxynivalenol (DON) accumulation.  Screening with molecular markers on chromosomes 2H and 6H for FHB and DON identified quantitative trait loci (QTL) which may confer resistance in Virginia Tech germplasm.  Evaluation of hulled and hulless full-sibs from four populations indicated  that  grain volume weight and protein concentration were significantly (P d 0.05) higher for hulless genotypes, while seedling emergence and grain ash concentration were significantly (P d 0.05) higher for hulled genotypes.  In linear regression analysis, none of the assessed traits explained yield variation in all populations and environments.  Identification of hulless genotypes having yield potentials similar to those of their hulled sibs should be possible after adjusting for hull weight.  A genome wide association study was used to identify chromosome regions governing traits of importance in six-rowed winter barley germplasm and to identify single nucleotide polymorphisms (SNPs) markers for use in a marker-assisted breeding program. Significant SNPs associated with previously described QTL or genes were identified for heading date, test weight, yield, grain protein, polyphenol oxidase activity, and resistance to leaf rust, powdery mildew, net blotch, and spot blotch.  Novel QTL also were identified for agronomic, quality, and disease resistance traits.  These SNP-trait associations provide the opportunity to directly select for QTL contributing to multiple traits in breeding programs. / Ph. D.

Fusarium Head Blight (FHB) is a devastating fungal disease of wheat (Triticum aestivum L.) and results in significant economic losses due to reductions in grain yield and the accumulation of mycotoxins, such as deoxynivalenol (DON) and nivalenol (NIV). As a result, breeding programs have been working to identify resistance genes in wheat varieties known to be resistant to FHB. Some of the major quantitative trait loci (QTL) for FHB resistance identified to date have been from exotic sources such as 'Sumai3' and the Chinese landrace Wangshuibai, and native resistance has been identified in North American cultivars such as Ernie and Truman which are being used in breeding programs. This study was conducted to characterize and map QTL for resistance to FHB in the soft red winter wheat cultivar Jamestown and to identify tightly linked DNA markers associated with those QTL so that marker-assisted selection (MAS) can be used in pyramiding these and other known QTL into elite backgrounds. Types of resistance assessed in this study include: Type I (resistance to initial infection; incidence), Type II (resistance to spread in wheat spike; severity), and decreases in mycotoxin accumulation (DON) and percentage of Fusarium damaged kernels (FDK). A population composed of 186 F5:7 recombinant-inbred lines (RILs) from the cross Pioneer Brand '25R47'/Jamestown were used to evaluate these traits in six environments (MD, NC, and VA in 2011 and 2012). This study identified a QTL for resistance to DON accumulation and FHB severity on the wheat chromosome 1B. The QTL accounted for 12.7% to 13.3% of the phenotypic variation in DON accumulation and 26.1% of the phenotypic variation in FHB severity. The most diagnostic marker for the QTL on chromosome 1B associated with resistance to FHB severity and DON accumulation is Xwmc500.6 located 7.2 cM from the QTL peak and flanked by markers Xwmc500.7 and Xgwm273.2 (28.2 cM interval). / Master of Science

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Cordia americana (L.) Gottschling & J.S.Mill. (guajuvira) is a forest species widely distributed in Brazil with importance in its economics and environment. Therefore, there is a great demand of research in seed technology for this species, as for the physiological and sanitary quality of seeds and, mainly, for the association of Fusarium spp. The objectives of this study were to determine the physiological maturity and seed collecting system to be more favorable to sanitary and physiological quality of the C. americana seeds and evaluate the pathogenicity of Fusarium sp. associated with them, morphological and molecularly characterizing the pathogen on the species level. The phenology of this species was closely observed during the development stages and, after the maturation onset of its fruits, they were collected in the plant and its soil. After the picking, it was determined the moisture content of the seeds, the period of maximum dry weight mass accumulation, maximum germination and vigor, besides, sanitary quality ratings. Through the transmission tests, it was found to occur Fusarium spp. causing disease symptoms in seedlings. After obtaining four isolates of the fungus, they were inoculated in seeds to prove its pathogenicity. The morphological characterization was performed using an identification key for the genus Fusarium and, for the molecular identification, genomic regions ITS were sequenced with its elongation factor 1-α. The results may conclude that physiological maturation in C. americana seeds occurs at 52 days after anthesis and the species F. acuminatum and F. verticillioides were pathogenic to species, causing damping-off in pre and post emergence. / Cordia americana (L.) Gottschling & J. S. Mill. (guajuvira) é uma espécie florestal amplamente distribuída no território brasileiro e que apresenta importância econômica e ambiental. Portanto, existe uma grande demanda por pesquisas na área de tecnologia de sementes para essa espécie, quanto à qualidade fisiológica e sanitária de suas sementes e, principalmente pela associação de Fusarium spp. Os objetivos do presente trabalho consistiram em determinar o ponto de maturação fisiológica e o sistema de coleta de sementes mais favorável à qualidade fisiológica e sanitária de sementes de C. americana, além de avaliar a patogenicidade de Fusarium sp. associado a sementes, caracterizando de forma morfológica e molecular o patógeno em nível de espécie. A fenologia da espécie foi acompanhada durante os estágios de desenvolvimento e, após o início da maturação dos frutos, foram realizadas coletas desses diretamente na planta e no solo. Após as coletas, foi determinado o teor de umidade das sementes, o período de máximo acúmulo de matéria seca, máxima germinação e vigor, além de avaliações da qualidade sanitária. Através dos testes de transmissão, foi verificada a ocorrência de Fusarium spp., causando sintomas de doença nas plântulas. Foram obtidos quatro isolados do fungo que foram inoculados nas sementes para provar a sua patogenicidade. A caracterização morfológica foi realizada através de uma chave de identificação para o gênero Fusarium e, para a identificação molecular, foram sequenciadas as regiões genômicas ITS e fator de elongação 1- α. Os resultados obtidos permitem inferir que o ponto de maturidade fisiológica nas sementes de C. americana ocorre aos 52 dias após antese e que as espécies F. acuminatum e F. verticillioides foram patogênicas a espécie, causando tombamento em pré e pós-emergência.

Made available in DSpace on 2014-07-29T15:16:28Z (GMT). No. of bitstreams: 1 Marcelo Ramada -.pdf: 3086154 bytes, checksum: d8d600492d75495c63dd916423b6e86b (MD5) Previous issue date: 2010-03-31 / Trichoderma harzianum is a saprophytic fungus, known for its potential as a biological control agent of different phytopathogens that causes losses in crops. Its action is based on different mechanisms like volatile and non-volatile antibiotics production, competition for nutrient and space, production of hydrolytic enzymes and mycoparasitism. Fusarium solani is the agent of bean dry root rot, and is responsible for significant losses. This work sought to analyze and identify secreted proteins from T. harzianum grown in the presence of F. solani cell wall, in an attempt to obtain new insights on biocontrol.In the dual culture test, T. harzianum proved to be a potent antagonist of F. solani. Some differences in the secreted proteins profile and final medium pH were observed when T. harzianum was grown in TLE medium containing glucose and in TLE and Minimum medium containing the cell wall of the phytopathogen. 33 proteins from 18 different genes were identified. The protein Sm1 was identified and secreted in all growth conditions. Many hydrolytic enzymes related to mycoparasitism, like endochitinases, β-1,3, β-1,6-glucanases, proteases and α-1,3-glicanases, were identified and among the proteases, a hypothetical metalocarboxipeptidase and a serine subtilisin-like never described for T. harzianum. Some enzymes with unkown roles in the mycoparasitism, such as β-1,3-glucanosyltransferase, secreted only in MM growth medium supernatant, were also identified. / Trichoderma harzianum é um fungo saprofítico, conhecido devido ao seu grande potencial como agente de controle biológico de diferentes fitopatógenos. Sua ação é baseada em diferentes mecanismos como a produção de antibióticos voláteis e não-voláteis, competição por espaço e nutrientes, produção de enzimas hidrolíticas e o micoparasitismo. Fusarium solani causa a podridão radicular seca no feijoeiro, resultando em perdas significativas. Este trabalho buscou analisar e identificar as proteínas secretadas por T. harzianum quando crescido na presença de parede celular de F. solani, em uma tentativa de obter novas informações sobre o biocontrole. Nos testes de pareamento, T. harzianum provou ser um potente antagonista de F. solani. Foi observada uma variação tanto no perfil de proteínas secretadas quanto no pH final, quando T. harzianum foi crescido em meio TLE contendo glicose e em meio TLE e Mínimo (M.M.) contendo parede celular do fitopatógeno. No total, 33 proteínas de 18 genes diferentes foram identificadas neste trabalho. A proteína Sm1 foi identificada e secretada em todas as condições de crescimento. Várias enzimas hidrolíticas relacionadas ao micoparasitismo, como endoquitinases, β-1,3, β-1,6-glicanases, proteases e α- 1,3-glicanases, foram identificadas e dentre as proteases, uma hipotética metalocarboxipeptidase e uma serina subtilisin-like nunca descritas em T. harzianum. Foram identificadas, também, enzimas que não possuem função conhecida no micoparasitismo, como a β-1,3-glicanosiltransferase, presente somente no sobrenadante do crescimento realizado em MM.

La fusariose des épis des céréales est due à un champignon pathogène (Fusarium spp.) qui entraine non seulement une perte directe de rendement en interrompant le remplissage des grains, mais également une perte indirecte par la production de mycotoxines, responsables de perturbations de procédés industriels et d'intoxications alimentaires. Parmi les différents moyens de lutte, la création de variétés résistantes semble la stratégie la plus efficace et la plus durable. Plusieurs variétés exotiques ont ainsi été identifiées comme résistantes mais présentent la particularité d'être difficilement cultivables sous nos latitudes. L'obtention de variétés résistantes adaptées à nos climats passe donc par une meilleure connaissance des mécanismes de résistance disponibles. La détection de QTL (Quantitative Trait Loci) chez le blé tendre a déjà permis de localiser certains des facteurs génétiques impliqués dans des mécanismes de résistance. Cependant l'utilisation de ces résultats en amélioration des plantes se heurte à deux contraintes majeures : la position des QTL reste le plus souvent imprécise et ces QTL, dérivant de populations biparentales, ne représentent qu'une fraction de la diversité génétique potentiellement intéressante pour ce caractère. L'objectif de la thèse était d'étudier la résistance du blé à la fusariose en réalisant une approche de génétique d'association. L'utilisation de génotypes faiblement apparentés et d'une densité élevée de marqueurs a permis de détecter des associations entre le polymorphisme génétique et des variations phénotypiques. Après avoir réalisé une synthèse sur le pathogène et sur l'approche par génétique d'association, le premier travail a été de réaliser une carte génétique fiable afin de pouvoir localiser les associations mises en évidence. Un panel de 195 variétés de blé tendre élites a été évalué pour leur résistance globale à la fusariose, pour leur résistance à la progression des symptômes et pour leur résistance à l'accumulation des mycotoxines. Des notations morphologiques (précocité, taille, aristation, nombre d'épillets par épi et extrusion des anthères) ont également été réalisées afin d'évaluer leur influence dans l'infestation. Ces variétés ont été génotypées avec 3016 marqueurs SNP, 200 marqueurs SSR et 1400 marqueurs DArT. Préalablement à la réalisation des tests d'association, l'étude de la structure a mis en évidence trois origines géographiques (française, anglaise et allemande) des variétés du panel. De même, l'étude du déséquilibre de liaison a montré que celui-ci était conservé sur une distance comprise entre 2 et 6 cM. L'étude d'association réalisée sur les notations d'infestation a permis d'identifier plusieurs zones associées, dont plusieurs sont colocalisées avec des Meta-QTL et/ou avec des zones identifiées avec les notations morphologiques. La densification en marqueurs de 6 zones associées avec les différentes notations d'infestation, localisées sur les chromosomes 1A, 1D, 2A, 2B, 5A et 7A, ont permis de confirmer ces régions, de restreindre fortement l'intervalle d'intérêt et de mettre en évidence de potentiels gènes candidats. Cette analyse a ainsi permis de mieux comprendre les mécanismes génétiques et morphologiques impliqués dans la résistance à la fusariose des épis. La décomposition en trois partie de cette résistance (résistance globale, résistance à la progression des symptômes, et résistance à l'accumulation des mycotoxines) montre l'existence de mécanismes génétiques indépendants et donc complémentaire à prendre en compte dans une stratégie de création de variétés résistantes à la fusariose des épis. / Fusarium head blight (FHB), caused by the fungal pathogen Fusarium spp., is a major cereals disease that not only cause direct yield losses, by interrupting grain filling, but also indirect quality losses by the production of mycotoxins, responsible of industrial processes disturbance and food poisoning. Most wheat breeding programs in FHB-affected areas of the world have been screening germplasm to identify sources of improved tolerance. Unfortunately, these sources of genetic resistance are often of exotic origin and not adapted to West European growing conditions. The selection of adapted varieties with improved tolerance therefore needs a better characterization of resistance mechanisms. Several QTL for FHB resistance in wheat have been identified in European germplasm, but the use of these information in marker-assisted selection is constrained by the precision of the QTL and the low diversity tested by using biparental population.The aim of the present study is to use association mapping, also called linkage disequilibrium mapping, to identify loci involved in the resistance to FHB. This method refers to the analysis of statistical associations between genotypes determined in a collection of individuals, and the phenotypes of the same individuals. A dense genetic map was compiled to localize precisely the association results. Resistance to F. graminearum was studied in a panel of 195 elite wheat varieties by the evaluation of three components: resistance to global infection, resistance to symptom progression and resistance to accumulation of mycotoxins. Morphological factors (plant height, heading date, awnedness, spikelets per ears, anther extrusion), known to influence resistance to FHB, were also recorded. All the varieties have been genotyped with around 3300 SNP markers, 200 SSR markers and 1400 DArT markers. We first investigated the structure of the panel, which could generate bias in the estimate of allele effects, if not included explicitly in the association models. We showed that the structure is based on geographical origin (French, German and UK). Study of the linkage disequilibrium (LD) showed an extent of LD between 2 and 6 cM. Results of association studies permitted to identify several loci for each of the evaluated components of resistance. Some of these loci colocalized with the results of the MetaQTL analysis and/or with loci associated with morphological traits. We selected more specifically 6 loci, located on chromosome 1A, 1D, 2A, 2B, 5A and 7A. Marker saturation of the regions, allowed to confirm the genome wide association results and to increase the accuracy of the loci of interest. This analysis allowed to better understand the many factors that influenced FHB resistance, whether genetic or morphological. Results show that the genetic mechanisms are independent between the three components and therefore, information obtained for each component are to be used complementary to create varieties with increased resistance to FHB.

La fusariose des épis est l’une de maladies les plus dommageables des céréales tempérées et est principalement causée par le champignon toxinogène Fusarium graminearum (Fg). Ces dix dernières années, de nombreuses études ont rapporté l’induction transcriptionnelle de gènes de la plante codant pour des cytochromes P450 (P450) en réponse l’infection par Fg. Les P450s constituent une famille enzymatique impliquée dans de nombreuses voies métaboliques, certaines avec des intérêts potentiels dans la résistance face aux maladies. Nous avons utilisé la petite graminée modèle Brachypodium distachyon (Bd) pour caractériser fonctionnellement le premier gène codant pour un P450 induit chez la plante au cours de la fusariose des épis par l’utilisation de lignées altérées dans la séquence ou l’expression du gène Bradi1g75310 codant le P450 BdCYP711A29. Nous avons montré qu’en plus d’être un facteur de sensibilité à la maladie, le gène Bradi1g75310 est impliqué dans une voie de biosynthèse hormonale chez Bd, celle des strigolactones (SLs). En effet, en plus de complémenter génétiquement les phénotypes aériens de la lignée mutante max1-1 d’Arabidopsis thaliana altérée dans le gène homologue MAX1 (AtCYP711A1), une lignée de Bd surexprimant Bradi1g75310 (lignée OE) exsude davantage d’orobanchol, une SL spécifique, que la lignée sauvage ou mutante. Une analyse préliminaire de l’impact direct de l’orobanchol sur la croissance de Fg semble indiquer une activation des étapes précoces du développement du champignon (germination) qui pourrait être à l’origine de l’induction plus rapide de gènes de défenses observée chez une lignée OE de Bradi1g75310. Nous avons également montré que les 4 paralogues de Bradi1g75310 chez Bd, qui codent également pour des CYP711A, sont tous capable de complémenter la lignée max1-1 et avons généré du matériel végétal fondamental pour la poursuite de l’étude de la diversification des SLs chez la plante monocotylédone modèle Bd. Au global, ce projet constitue une première étape dans la caractérisation de l’implication des P450 dans la réponse de la plante face à l’infection par Fg en plus de donner de nouveaux indices concernant le rôle des SLs dans les interactions plante-pathogène. Les résultats obtenus au cours de ce travail de thèse pourront permettre l’amélioration de caractères tant développementaux que de résistance à la fusariose chez les céréales cultivées. / Fusarium Head Blight (FHB) is one of the most important diseases of temperate cereals and is mostly caused by the toxin producing-fungus Fusarium graminearum (Fg). This last decade, several studies reported the transcriptional activation of cereal cytochrome P450-encoding genes (P450s) in response to Fg infection. P450s constitute an enzymatic family participating in very diverse metabolic pathways with potential interest for disease resistance. We used the model temperate cereal Brachypodium distachyon (Bd) to functionally characterize the first FHB-induced P450- encoding gene using Bd lines altered in the locus or gene expression of the Bradi1g75310 gene encoding the BdCYP711A29 P450. We showed that in addition to be a plant susceptibility factor towards the disease, the Bradi1g75310 gene is involved in the hormonal biosynthetic pathway of strigolactones (SLs) in Bd. Indeed, in addition to genetically complement the shoot phenotypes of the Arabidopsis thaliana mutant line for the homologous gene MAX1 (AtCYP711A1, max1-1 line), a Bd linewhich overexpresses the Bradi1g75310 gene (OE) exudes more orobanchol, a specific SL, compared to wild-type or mutant lines. Preliminary analysis of the direct impact of orobanchol on Fg growth suggests an activation of early fungal development (germination) likely to induce faster induction of defense-related genes during FHB, observed in Bradi1g75310 OE line. We showed that the four paralogs of Bradi1g75310 encoding BdCYP711A P450s are all able to genetically complement max1-1 line and provide important plant material for studying SLs diversification in the model monocot B. distachyon. Overall, this project constitutes a first step in the characterization of P450s involvement in plant response towards Fg infection in addition to give new evidences about the role of SLs in plant-pathogen interactions. Results obtained during this Ph.D. project will allow the improvement of both developmental and FHB-related traits in cereal crops.