ABSTRACT The objective of this work was to evaluate the ability of Trichoderma asperellum isolates and their culture filtrates to control Lasiodiplodia theobromae in vivo and to promote the growth of pecan seedlings. Two experiments were carried out, both in a greenhouse. In the first experiment, the aerial part of the plants was sprayed by biological control treatments and, 14 days later, the pathogen was inoculated into the stem, through injury. In the second experiment, the substrate was moistened with biological control treatments and 14 days later the pathogen was inoculated following the same methodology as the first experiment. The following parameters were evaluated: length of the aerial part, stem diameter, fresh mass of the aerial part and the root, dry mass of the aerial part and the root, incidence and severity of the disease. The comparison of means was performed using the Skott-Knott test at a 5% probability of error, using the SISVAR software. In both experiments, the greatest responses were obtained in the growth parameters evaluated in the treatments with the culture filtrates and with the spore suspension in relation to the treatment inoculated with the pathogen without the use of biological control treatments. These treatments also reduced the incidence and severity of stem canker in pecan seedlings, and the best treatment varied with the form of application. The isolates of T. asperellum and the culture filtrates stimulated the initial growth of the plants, decreasing the incidence and severity of stem canker in pecan plants, regardless of the form of application.

RESUMO O objetivo deste trabalho foi avaliar a ação de isolados de Trichoderma asperellum e de seus filtrados de cultura no controle de Lasiodiplodia theobromae in vivo e na promoção do crescimento de mudas de nogueira-pecã. Foram desenvolvidos dois experimentos, ambos em casa de vegetação. No primeiro experimento, a parte aérea das plantas foi aspergida pelos tratamentos de controle biológico e 14 dias após foi inoculado o patógeno no caule, por meio de ferimento. No segundo experimento, o substrato foi umedecido com os tratamentos de controle biológico e 14 dias após foi feita a inoculação do patógeno seguindo mesma metodologia do primeiro experimento. Foram avaliados os seguintes parâmetros: comprimento da parte aérea, diâmetro do caule, massa fresca da parte aérea e da raiz, massa seca da parte aérea e da raiz, incidência e severidade da doença. A comparação das médias foi realizada pelo teste de Skott-Knott a 5% de probabilidade de erro, com o software SISVAR. Nos dois experimentos, obtiveram-se as maiores respostas nos parâmetros de crescimento avaliados nos tratamentos com os filtrados de cultura e com a suspensão de esporos, em relação ao tratamento inoculado com o patógeno sem o uso dos tratamentos de controle biológico. Esses tratamentos também reduziram a incidência e a severidade do cancro do tronco em mudas de nogueira-pecã, sendo que o melhor tratamento variou com a forma de aplicação. Os isolados de T. asperellum e os filtrados de cultura estimularam o crescimento inicial das plantas, diminuíram a incidência e a severidade do cancro do tronco nas plantas de nogueira-pecã, independente da forma de aplicação.

Resumo Nas regiões Centro-Oeste e Sul do Brasil, a cultura da erva-mate (Ilex paraguariensis) constitui-se uma importante fonte de extrativismo florestal, além de diversificar a renda, principalmente na agricultura familiar. A erva-mate, como outras espécies florestais, é infectada por diversos patógenos, como o gênero Fusarium, causador da podridão-de-raízes. Os patógenos possuem ampla variabilidade genética, mesmo dentro de um único gênero e dentro de uma única espécie, resultado de adaptações ao meio em que vivem. Este estudo teve como objetivo avaliar o sequenciamento das regiões do genoma ITS e β-tubulina na identificação em nível de espécie dos isolados de Fusarium spp. patogênicos à erva-mate, e identificar a formação de grupos de compatibilidade vegetativa (VCG) entre os isolados de Fusarium spp. patogênicos à erva-mate. O sequenciamento das regiões ITS e β-tubulina consolidou a separação entre os sete isolados patogênicos em F. solani e F. oxysporum. Para esses isolados, a técnica para obtenção de mutantes nit foi adequada para a classificação fenotípica, fundamentais nos testes de VCG para espécies de Fusarium, sendo obtidos cinco VCG.

Abstract In the central-western and southern regions of Brazil, the cultivation of yerba mate (Ilex paraguariensis) is considered an important source of forest extraction, besides providing an alternative income, especially for families. The yerba mate, like other forest species, is infected by several pathogens, such as fungi of the genus Fusarium, causing root rot. Pathogens have wide genetic variability, even within a single genus and species, resulting from adaptations to their environments. This study aimed at characterizing molecularly isolated Fusarium spp. through the sequencing of the ITS and β-tubulin genome regions, identifying the species level as well as the formation of vegetative compatibility groups (VCG) among these isolates of the root rot pathogen in yerba mate. The sequencing of the ITS and β-tubulin regions, defined the separation between the seven pathogenic isolates of F. solani and F. oxysporum. For the same isolates, the technique to obtain nit mutants was suitable for the phenotypic classification, obtaining five VCGs for species of Fusarium, root rot in yerba mate.

Abstract Pecan cultivation in Brazil has gained increasingly economic importance in the southern part of the country. The objective of this work was to identify the pathogen that causes root rot in Carya illinoinensis seedlings in the nursery, through morphological, molecular and pathogenicity tests. Fungi from the genus Fusarium found in symptomatic roots of pecan seedlings were isolated in potato dextrose agar (PDA) culture medium and purified. Morphological characterization was performed in PDA and carnation leaf agar (CLA) media. For the molecular characterization, the genomic segments ITS (internal transcribed spaces), β-tub (beta-tubulin), and tef1-α (elongation fact 1-alpha) were sequenced. The pathogenicity test was performed on healthy seedlings in the greenhouse. The combination of morphological and molecular characters was fundamental for identification of the species, showing that the most suitable genes for identification were ITS and tef1-α. The causative agent of root rot in C. illinoinensis seedlings in the nursery was identified as Fusarium oxysporum.

RESUMO A erva-mate (Ilex paraguariensis) tem grande importância socioeconômica na agricultura familiar do Sul do Brasil. A principal doença da erva-mate é a podridão-de-raízes causada por Fusarium spp. Pouco se sabe a respeito da fisiologia deste patógeno, principalmente quanto à agressividade associada à produção de enzimas extracelulares. Neste trabalho, avaliou-se a agressividade de isolados de F. oxysporum e F. solani patogênicos à erva-mate e determinou-se as atividades das enzimas extracelulares catalase, lacase, celulase, caseinase, amilase, protease, lipase e pectinases produzidas por Fusarium spp. em meio de cultura. Foram utilizados seis isolados de F. solani e um isolado de F. oxysporum patogênicos à erva-mate. O isolado de F. oxysporum mostrou-se menos agressivo em relação aos demais isolados de F. solani. Todos os isolados de Fusarium spp. produziram, de maneira semiquantitativa, as enzimas extracelulares catalase, lacase, celulase, caseinase, amilase, protease, lipase e pectinases (poligalacturonase e pectato liase). Entretanto, a quantidade produzida de cada enzima foi significativamente diferente entre os isolados. Com exceção das enzimas lacase e poligalacturonase o isolado M7C1 de F. solani exibiu as maiores médias do índice enzimático e foi também o responsável pelo maior percentual de morte de mudas de erva-mate.

ABSTRACT The yerba-mate (Ilex paraguariensis) has great socioeconomic importance on family farming in Southerm Brazil. One of the main yerba-mate disease is root rotting, caused by Fusarium spp. Little is known about the pathogen physiology, especially regarding the aggressiveness associated with the production of extracellular enzymes. On this work, the aggressiveness of isolates of F. oxysporum and F. solani pathogenic to yerba-mate was evaluated and it was determined the activities of extracellular enzymes catalase, laccase, cellulase, caseinase, amylase, protease, lipase and pectinases produced by Fusarium spp. in culture medium. Six isolates of F. solani and one isolate of F. oxysporum pathogenic to yerba-mate were used. The F. oxysporum isolate proved to be less aggressive in relation to the other F. solani isolates. All isolates of Fusarium spp. produced, on a semiquantitative manner, the extracellular enzymes catalase, laccase, cellulase, caseinase, amylase, protease, lipase and pectinases (polygalacturonase and pectate lyase). However, the quantity produced for each enzyme was significantly different among the isolates. With the exception of the laccase and polygalacturonase enzymes, the M7C1 isolate showed the highest enzymatic index and was also responsible for the highest percentage of yerba-mate seedlings death.

ABSTRACT The cultivation of yerba-mate (Ilex paraguariensis) has been intensified in the State of Rio Grande do Sul, as result of the valuation of the price paid to the producer. However, studies related to Fusarium pathosystem vs. yerba-mate are still insufficient, so that many producers have faced significant losses in herbal production due to the occurrence of the roots rotting. Considering the above, the objectives of this study were: a) morphologically characterize the Fusarium spp. isolates collected in sick plants of yerba-mate; b) select efficient morphological characters on separating the Fusarium spp. isolates in similarity groups; c) identify the Fusarium spp. isolates to species level through the sequencing of the genomic region of elongation factor 1-α (TEF-1α); and d) testing the pathogenicity of the Fusarium spp. isolates selected based on the morphological characterization. For such, collections for the pathogen isolates were made in five cities and the collection points were georeferenced. Thirty-nine isolates were morphologically characterized by variables as mycelial growth, sporulation, macroconidia length and width, microconid format, colonies pigmentation and formation of chlamydospore, and tested for pathogenicity by substrate inoculation. The variables used in the morphological characterization were efficient in discriminating the isolates in seven groups, especially length of macronidia associated to sporulation. Seven pathogenic isolates of Fusarium spp. were identified, also through molecular sequencing of the TEF-1α gene region. The sequencing of the TEF-1α region consolidated the identification of pathogenic isolates as F. solani and F. oxysporum.