Sporisorium scitamineum is the causal agent of sugarcane smut disease. The fungus establishes a biotrophic interaction with sugarcane tissues, and unlike smut fungi of other monocot hosts, the primary meristem of sugarcane plants develops a whip-like structure instead of a tumour-like galls emerging from floral structures (tassels and ears). We examined (GFP)-tagged S. scitamineum infecting tissues of three sugarcane genotypes with distinct responses to smut (susceptible, intermediate resistant and resistant). Mating compatible haploid cells gfp-expressing were obtained by Agrobacterium tumefaciens-mediated transformation (ATMT) using the integrative vector pFAT-gfp. Regardless of the inoculation method (drop inoculation and hypodermal syringe inoculation), all genotypes were colonised by the fungus. GFP-tagged strains of opposite mating reaction were able to: (a) grow in vitro as fluorescent yeast-like cells; (b) generate infectious dikaryon; (c) penetrate sugarcane tissues; (d) colonise tissues by growing a filamentous network; and (e) form the characteristic highly branched hyphae within host cells. Fungal colonisation 160 DAI revealed an association of the fungus with vascular vessels disrupting their organisation in all three genotypes analysed. However, the resistant plants did not develop whips spanning the experiment time. The first whips emerged 76 DAI from plants of the susceptible genotype whereas for intermediate resistant plants whips were detected at 137 DAI. These whips were dissected and fluorescent sporogenesis and teliospore maturation were analysed. In vitro germination of recovered teliospores revealed after meiosis the formation of a three-celled hyphal filament, where the fourth cell was likely maintained in the teliospore coat. These cells showed independent segregation of the gfp marker, as a result of gfp insertions in different chromosomes of each compatible haploid strain. This work presents the complete fungal life cycle of GFP-marked S. scitamineum to study developmental stages in planta.
Sporisorium scitamineum is the fungus that causes sugarcane smut disease. Despite of the importance of sugarcane for Brazilian agribusiness and the persistence of the pathogen in most cropping areas, genetic variation studies are still missing for Brazilian isolates. In this study, sets of isolates were analyzed using two molecular markers (AFLP and telRFLP) and ITS sequencing. Twenty-two whips were collected from symptomatic plants in cultivated sugarcane fields of Brazil. A total of 41 haploid strains of compatible mating types were selected from individual teliospores and used for molecular genetic analyses. telRFLP and ITS analyses were expanded to six Argentine isolates, where the sugarcane smut was first recorded in America. Genetic relationship among strains suggests the human-mediated dispersal of S. scitamineum within the Brazilian territory and between the two neighboring countries. Two genetically distinct groups were defined by the combined analysis of AFLP and telRFLP. The opposite mating-type strains derived from single teliospores were clustered together into these main groups, but had not always identical haplotypes. telRFLP markers analyzed over two generations of selfing and controlled outcrossing confirmed the potential for emergence of new variants and occurrence of recombination, which are relevant events for evolution of virulence and environmental adaptation.
pela oportunidade de realizar o curso de Doutorado. À minha orientadora profª Drª Maria Carolina Quecine Verdi pela inspiração, acolhimento, compreensão, pragmatismo, bons conselhos e, sobretudo, por acreditar em nosso trabalho, mesmo em meio às mudanças. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) processo 140590/2017 e ao Programa de Aperfeiçoamento do Ensino (PAE-USP) pelas bolsas concedidas. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Código de Financiamento 001. Ao professor Dr. João Lucio de Azevedo pela agradável convivência, sabedoria, entusiasmo e bom humor. Ao professor Dr. Roberto Fristche Neto do Laboratório de Plantas Alógamas da ESALQ pela disponibilização das sementes de milho utilizadas nos experimentos de casa de vegetação e da estufa utilizada para secagem de amostras vegetais. À professora Dra. Claudia Barros Monteiro Vitorello pelo apoio e aconselhamento em momentos importantes e por ter disponibilizado equipamento para realização das análises de expressão gênica. Ao professor Dr. José Roberto Postali Parra e a querida técnica Sra Neide Graciano Zério e demais colegas do Laboratório de Biologia de Insetos da ESALQ por terem nos recebido tão bem e fornecido todo suporte para realização dos testes entomológicos. À professora Dra. Maria Lúcia Carneiro Vieira, do Laboratório de Biologia Celular e Molecular de Plantas da ESALQ, pela disponibilização das estufas utilizadas para experimento de casa de vegetação e para preparo de vidraria para extrações de RNA. Aos professores Dr. Fábio Tebaldi Nogueira, Dr. Marcio Castro Silva-Filho e minha orientadora, e colegas Carolina, Filipe, Gleicy, Paula, Renata e Raphael, pelos ensinamentos, conselhos, apoio e atitudes apreendidos durante os dois Estágios Supervisionados em Docência realizados na ESALQ. Ao professor Dr. Antonio Vargas de Oliveira Figueira e Dra. Flavia Bento pelo aprendizado, convivência, respeito e conselhos que levarei comigo da etapa vivida no CENA.
pelas risadas, pela paciência, pelas palavras de apoio, por todas as confraternizações, reuniões, conversas, conselhos e disposição em ajudar, mesmo que apenas para molhar as caninhas e coletar os teliósporos. Às colegas do Laboratório de Biologia Celular e Molecular de Plantas-ESALQ pelo apoio, convivência e afeto, especialmente Carmelice, Luciane, Carla, Alessandra, Cássia, Lourdes e Zirlane. À minha família sem a qual nada disso teria sido possível, por todo amor, entrega, compreensão, preces, carinho, paciência e incontáveis momentos de apoio e inspiração, em especial meus pais Antonio Jorge e Eliana, minha irmã Carolina, meus avós Antonio (Nio), Ana, Maria Clementina e Angelino (in memoriam), meus tios, tias, primos e primas. A todos aqueles que diretamente ou indiretamente contribuíram para a realização deste trabalho.
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