MARCELLA VIANA DE SOUSA TRANSMISSION, COLONIZATION AND MOLECULAR DETECTION OF Fusarium oxysporum f. sp. phaseoli IN COMMON BEAN SEEDS LAVRAS – MG 2013 MARCELLA VIANA DE SOUSA TRANSMISSION, COLONIZATION AND MOLECULAR DETECTION OF Fusarium oxysporum f. sp. phaseoli IN COMMON BEAN SEEDS Tese apresentada à Universidade Federal de Lavras, como parte das exigências do Programa de Pós- Graduação em Agronomia, área de concentração em Fitopatologia, para a obtenção do título de Doutor. Orientador Dr. José da Cruz Machado Coorientador Dr. Gary P. Munkvold LAVRAS - MG 2013 Sousa, Marcella Viana de. Transmission, colonization and molecular detection of Fusarium oxysporum f. sp. phaseoli in common bean seeds / Marcella Viana de Sousa. – Lavras : UFLA, 2014. 119 p. : il. Tese (doutorado) – Universidade Federal de Lavras, 2013. Orientador: José da Cruz Machado. Bibliografia. 1. Fusarium wilt. 2. Green fluorescent protein. 3. Real-time PCR. 4. Seed pathology. 5. Phaseolus vulgaris. I. Universidade Federal de Lavras. II. Título. CDD – 632.43 Ficha Catalográfica Elaborada pela Coordenadoria de Produtos e Serviços da Biblioteca Universitária da UFLA MARCELLA VIANA DE SOUSA TRANSMISSION, COLONIZATION AND MOLECULAR DETECTION OF Fusarium oxysporum f. sp. phaseoli IN COMMON BEAN SEEDS Tese apresentada à Universidade Federal de Lavras, como parte das exigências do Programa de Pós- Graduação em Agronomia, área de concentração em Fitopatologia, para a obtenção do título de Doutor. APROVADA em 27 de novembro de 2013. Dr. José Otávio Machado Menten USP/ESALQ Dra. Patrícia Gomes Cardoso UFLA Dra. Antônia dos Reis Figueira UFLA Dr. Eduardo Alves UFLA Dr. José da Cruz Machado Orientador Dr. Gary P. Munkvold Coorientador LAVRAS - MG 2013 Aos meus pais, Luiza e Vico (in memoriam), que foram os meus primeiros professores ao me ensinarem as primeiras palavras; o verdadeiro valor e significado da vida, do amor, da família e do trabalho; ao me incentivarem a sempre seguir em frente para alcançar meus objetivos. DEDICO Aos meus irmãos, Vicente Sérgio, Luiz Otávio, Lourenço e Ana Luiza, pelo apoio incondicional, amizade, carinho e incentivo; Às minhas sobrinhas que tanto amo, Giovana, Vivian e Mariana, por serem parte de minha vida, e minha constante renovação de energia através de um sorriso e um abraço apertado; Ao meu noivo Adriano, pelo amor concedido, pela presença sempre constante, pelo grande e importante apoio e sugestões, em todas as fases de meu doutorado. OFEREÇO AGRADECIMENTOS À Deus, pela constante presença em minha vida. À Universidade Federal de Lavras, em especial ao Departamento de Fitopatologia, pelo alto nível de ensinamentos e qualificação de seus discentes, aos quais sou grata pela oportunidade de obtenção do meu título de Doutora. À CAPES, por conceder a bolsa de doutorado no país e no exterior. Ao CNPq/MAPA, pelo apoio financeiro ao projeto de pesquisa. Ao meu orientador, Dr. José da Cruz Machado, pela oportunidade, ensinamentos, aconselhamentos, amizade e confiança inegáveis, desde a iniciação científica. Ao meu coorientador, Dr. Gary P. Munkvold, pela oportunidade de se obter a experiência internacional, pela confiança, apoio e sugestões, durante e após a minha estada na Iowa State University. Aos membros da banca examinadora: Dr. José Otávio Machado Menten, Dra. Patrícia Gomes Cardoso, Dra. Antônia dos Reis Figueira, Dr. Eduardo Alves, por me honrarem com sua participação, assim como pelas críticas e valiosas contribuições. Ao Dr. Murillo Lobo Jr., pela contribuição no meu trabalho de pesquisa. Às empresas fornecedoras de sementes de feijão: Grupo Farroupilha, Embrapa Arroz e Feijão, Departamento de Biologia/UFLA, Harris Moran, Seed Savers Exchange, pela confiança em nosso trabalho de pesquisa. A todos os pesquisadores, professores e amigos que nos ajudaram a obter os isolados de Fusarium oxysporum f. sp. phaseoli, em especial, Dr. Ludwig Pfening, Heloisa Moraes, Margarida Itto, Howard Schwartz e Gary Munkvold. Aos amigos do LAPS: Carla Correa, Iara Eleutéria, Carol Siqueira, Ursula Abreu, Mirella Almeida , Luana Botelho, Luiza Nunes, Maria Salustiano, Angela Santos, Rayana Martins, Mirian Salgado, Ellen Barrocas, Bruno Moretti, Willian Zancan, Cristiano Sousa, pelo auxílio e contribuição para a concretização desta tese, companheirismo e bons momentos compartilhados ao longo dos anos. A todos os professores do Departamento de Fitopatologia, por me ensinarem a ser uma profissional capacitada a enfrentar o mercado de trabalho. Aos funcionários do DFP, pelo apoio no decorrer do curso. A todos os colegas de pós-graduação, pelos trabalhos desenvolvidos em grupo e pelo agradável convívio. Às grandes amizades, em especial a Maira Oliveira, Amanda Correa, Isabel Guedes, Margaret Ellis e David Cruz, por minimizarem a distância do dia- a-dia sempre com um e-mail, telefonema, mensagem ou skype. As pessoas que entraram em minha vida, ocupando o lado esquerdo do peito, Danielle Figueiredo, Roberta Machado, Ana Paiva, Anselmo Custódio, Ana Paula Andreotti, Angelo Andreotti, Ana Clara Andreotti, Alexandre Custódio, Melyssa Freitas, Lorenzo Custódio, Renato Carvalho e Rafaela Sâmia, pelo carinho, amizade, apoio, conversas e momentos felizes e agradáveis proporcionados. A todos os autores citados que, somente através de seus esforços e dedicação na geração dos manuscritos, puderam suportar esta pesquisa. A todos aqueles que, de alguma forma, contribuíram para a concretização desta tese, assim como para meu crescimento pessoal e profissional. MUITO OBRIGADA! “ Progress is impossible without change, and those who cannot change their minds cannot change anything”. George Bernard Shaw BIOGRAFIA Marcella Viana de Sousa, filha de Luiza C. Viana de Sousa e de Octávio de Sousa Filho, nasceu em 21 de fevereiro de 1981, na cidade de Lavras (MG). Concluiu o primeiro e segundo grau no Instituto Presbiteriano Gammon, em dezembro de 1998, na cidade de Lavras (MG). Em agosto de 1999, ingressou no curso de Agronomia na Universidade Federal de Lavras (MG), obtendo o título de Engenheira Agrônoma em janeiro de 2004. Nesse período, desenvolveu trabalhos na Epamig e no Laboratório de Patologia de Sementes. Iniciou o curso de Mestrado em Agronomia/Fitopatologia na Universidade Federal de Lavras, em Lavras (MG), em março de 2004, concluindo-o em fevereiro de 2006. Trabalhou na Usina Bunge Itapagipe, em Itapagipe/MG como supervisora de produção industrial de açúcar e etanol no período de agosto de 2006 à setembro de 2008. Em março de 2010, ingressou no curso de Doutorado em Agronomia/Fitopatologia, na Universidade Federal de Lavras (MG). Durante o doutoramento, no período de agosto de 2012 a agosto de 2013, realizou o doutorado sanduíche na Iowa State University. Em dezembro de 2013 defendeu sua tese, obtendo o título de Doutora em Ciências. GENERAL ABSTRACT The common bean (Phaseolus vulgaris L.) is a crop of great economic and social importance in Brazil and one of the basic diets of the Brazilian population. Several diseases occur in this crop, causing yield losses and/or decreases in seed quality, such as Fusarium wilt, caused by Fusarium oxysporum f. sp. phaseoli (Fop). This organism can be spread by seeds and it is classified as a Regulated Non-Quarantine Pest in Brazil. The current known methods for its detection and identification in seeds are blotter tests and semi-selective agar medium, followed by a pathogenicity test. The goals in this work were to extend knowledge on seed transmission of Fop in common bean as well as to investigate the close interaction between that fungus in infected seeds through GFP technique associated to scanning electron microscopy and to establish a protocol for detection of this fungus in seeds by real time PCR (qPCR). In paper 1, transmission rates of the pathogen from artificially and naturally Fop- contaminated seeds to emerged plants were tested. Two strains of Fop, two genotypes of bean, two environment temperatures and four inoculum potentials were used in the experiments with artificially inoculated seeds, in order to assess the symptomatic and asymptomatic plants. The frequence of symptomatic plants was lower than 5% but the transmission rates of those plants were 100%. The transmission rates of asymptomatic plants were 57% and 49.7% for BRSMG Majestoso and Ouro Negro, respectively. In respect to comparison between temperatures, the rates were 54.4% at 20 ºC and 52.3% at 25 ºC. For Fop strains, the transmission rates were 83.6% and 94.2% for FOP005 and FOP014. The mean rate at P3 was 64.4% and 58% at P1. From the assays with naturally Fop- contaminated seeds, transmission rates were lower than those determined for inoculated seeds, ranging from 8.1% to 16.7%. In paper 2, Fop was transformed by green fluorescent proteins (GFP) containing the resistance gene of hygromycin-B. Seed infection by the transformed Fop was visualized in the embryo, including the plumule, and in the endosperm. A large amount of fluorescent mycelium was observed externally on bean seedling roots, which presented vascular discoloration, which is the typical symptom of Fusarium wilt disease. In paper 3, the results of the experiments on molecular detection of Fop in common bean seed samples showed that the specific primers and probe used as part of the qPCR protocol in this study were viable to detect Fop in infected seeds with high sensitivity, at 0.25% of Fop incidence. TaqMan assays provided more reliable, sensitive, effective and quicker results than SYBR Green assays, which confirm previous reports for other pathosystems. Analysis of naturally Fop- contaminated seeds by qPCR correlated with results of the blotter test but further studies are needed to optimize sampling and subsampling of seed health testing using PCR-based assays. Key-words: Fusarium wilt, green fluorescent protein, seed pathology, real-time PCR, Phaseolus vulgaris. RESUMO GERAL O feijoeiro (Phaseolus vulgaris L.) é uma cultura de grande importância econômica e social no Brasil por ser uma das dietas básicas da população brasileira. Diversas doenças ocorrem nessa cultura, causando perdas na produção e/ou reduzindo a qualidade das sementes, como é o caso da murcha de fusarium, cujo agente etiológico é Fusarium oxysporum f. sp. phaseoli (Fop). Esse organismo pode ser disseminado por sementes e é classificado como Praga Não Quarentenária Regulamentada no Brasil. Os métodos conhecidos para sua detecção e identificação em sementes são o blotter test e o meio semiseletivo, ambos seguidos pelo teste de patogenicidade. Objetivou-se, neste estudo, ampliar os conhecimentos na transmissão de Fop por sementes de feijão, assim como investigar a interação entre Fop, em sementes infectadas por meio da técnica de GFP, associada à microscopia eletrônica de varredura, além de estabelecer um protocolo para sua detecção em sementes de feijão por PCR, em tempo real (qPCR). No artigo 1, as taxas de transmissão do patógeno foram testadas, a partir de sementes artificialmente e naturalmente associadas ao Fop para plantas emergidas. Dois isolados de Fop, duas cultivares de feijoeiro, duas temperaturas e quatro potenciais de inóculo foram utilizados nos testes a partir de sementes artificialmente inoculadas, com a finalidade de avaliar as plantas sintomáticas e assintomáticas. A frequência de plantas sintomáticas foi menor do que 5%, com taxas de transmissão de 100%. As taxas de transmissão de plantas assintomáticas foram 57% e 49,7%, para BRSMG Majestoso e Ouro Negro, respectivamente. Em relação às temperaturas, as taxas foram 54,4% à 20 ºC e 52,3% à 25 ºC. Para isolados de Fop, as taxas de transmissão foram 83,6% e 94,2% para FOP005 e FOP014. A taxa média no P3 foi 64,4% e, 58% no P1. A partir dos ensaios com sementes naturalmente associadas ao Fop, as taxas de transmissão foram menores do que aquelas determinadas a partir de sementes inoculadas, variando de 8,1% a 16,7%. No artigo 2, Fop foi transformado pela inserção do gene que expressa as proteínas fluorescentes verdes (GFP) e contém o gene de resistência à higromicina-B. A infecção das sementes pelo Fop transformado foi visualizada no embrião das sementes, na plúmula, e no endosperma. Grande quantidade de micélio fluorescente foi observado externamente nas raízes das plântulas de feijão, as quais apresentaram escurecimento vascular, sintoma típico da murcha de fusarium. No artigo 3, os resultados dos experimentos sobre detecção molecular de Fop, em lotes de sementes de feijão, mostraram que primers e sonda específicos, usados como parte do protocolo de qPCR neste estudo, foram viáveis para detectar Fop em sementes infectadas, com alta sensibilidade, a 0,25% de incidência do patógeno em sementes. Os ensaios com TaqMan forneceram resultados mais confiáveis, sensíveis, eficientes e rápidos do que aqueles com SYBR Green, confirmando os relatos anteriores para outros patossistemas. As análises de qPCR, a partir de sementes naturalmente associadas ao Fop, correlacionaram com os resultados obtidos em blotter test, mas estudos adicionais são necessários para otimizar a amostragem e subamostragem, nos testes de sanidade baseados em PCR. Palavras-chave: murcha de fusarium, proteína fluorescente verde, patologia de sementes, PCR em tempo real, Phaseolus vulgaris. SUMMARY CHAPTER 1................................................................................................. 15 1 GENERAL INTRODUCTION................................................................. 15 2 LITERATURE REVIEW ......................................................................... 17 2.1 Economic importance of Phaseolus vulgaris L. ........................................ 17 2.2 General aspects of Fusarium wilt disease in common bean ....................... 18 2.3 Morphological and genetic aspects of Fusarium oxysporum f. sp. phaseoli19 2.4 Interaction and transmission of Fusarium oxysporum f. sp. phaseoli in common bean seeds........................................................................................ 22 2.5 Use of molecular techniques in seed health test for fungal detection.......... 24 REFERENCES............................................................................................. 30 CHAPTER 2 - PAPERS............................................................................... 40 Paper 1. Transmission of Fusarium oxysporum f. sp. phaseoli from seed to emerging plants of common bean ................................................................ 40 ABSTRACT ...................................................................................................41 RESUMO .......................................................................................................42 INTRODUCTION ..........................................................................................43 MATERIALS AND METHODS.....................................................................45 RESULTS.......................................................................................................50 DISCUSSION.................................................................................................57 ACKNOWLEDGEMENTS.............................................................................60 REFERENCES ...............................................................................................61 Paper 2. Studies of the association of Fusarium oxysporum f. sp. phaseoli in common bean seeds ...................................................................................... 64 ABSTRACT ...................................................................................................65 RESUMO .......................................................................................................66 INTRODUCTION ..........................................................................................67 MATERIALS AND METHODS.....................................................................68 RESULTS AND DISCUSSION......................................................................73 CONCLUSIONS.............................................................................................80 ACKNOWLEDGEMENTS.............................................................................80 REFERENCES ...............................................................................................81 Paper 3. Detection of Fusarium oxysporum f. sp. phaseoli in common bean seeds by real-time PCR assays ..................................................................... 84 ABSTRACT ...................................................................................................85 RESUMO .......................................................................................................86 INTRODUCTION ..........................................................................................87 MATERIALS AND METHODS.....................................................................90 RESULTS..................................................................................................... 100 DISCUSSION............................................................................................... 105 ACKNOWLEDGEMENTS........................................................................... 109 REFERENCES ............................................................................................. 110 FINAL COMMENTS................................................................................. 116 APPENDIX................................................................................................. 119 15 CHAPTER 1 1 GENERAL INTRODUCTION Common bean (Phaseolus vulgaris L.) is a legume that provides dietary proteins and plays an important role in human nutrition. Brazil is the largest producer of this crop and has been affected by several diseases, such as Fusarium wilt, caused by the necrotrophic fungus Fusarium oxysporum f. sp. phaseoli (Fop). This disease has attracted special interest in the last years due to the increasing dissemination of the pathogen associated with the higher degree of mechanization in the fields, successive plantings in the same area and for producing more than one cycle per year (PEREIRA; RAMALHO; ABREU, 2009). Fop can be spread over long distance by association with seeds (TOLEDO- SOUZA et al., 2012) and has been listed as a Regulated Non-Quarantine Pest in Brazil. As a risk pathogen the tolerance level of zero in 400 seed samples submitted to laboratories has been proposed for it in certification programs in order to avoid the inoculum spreading. Thus, studies of seed-to-plant transmission of the pathogen, influence of external biotic and abiotic factors on seed transmission, dynamic of seed infection and its colonization should be well known. Pathogen detection on seeds is performed by traditional incubation methods which involve plating seeds by blotter method, observation of morphological structures by microscopy and symptoms in plants by pathogenicity test, if necessary. These methods are labor-intensive and time- consuming. The incubation diagnostic procedure is rather questionable as to the occurrence of saprophytic strains of F. oxysporum on diseased common bean plants, which are morphologically identical to F. oxysporum f. sp. phaseoli (ALVES-SANTOS et al., 2002a). In order to establish a protocol for seed testing 16 to detect that pathogen, PCR-based methods which are faster, more reliable and accurate should be tested. In this work, studies were performed with the objectives of elucidating the transmission rate, colonization and detection of Fop in common bean seeds. The aim in the first study was to calculate the seed-to-emerged plant Fop transmission rate and its occurrence in different plant tissues in order to understand the seed-pathogen interaction, seed transmission mechanisms and the consequence of the fungus inoculum present on seeds. In the second study, the aim was to closely follow the seed infection and colonization process through GFP markers and scanning electron microscopy. The third work was proposed to establish a faster, efficient and reliable PCR-based protocol of seed health testing to be used in routine analysis for detection of such a pathogen. 17 2 LITERATURE REVIEW 2.1 Economic importance of Phaseolus vulgaris L. Common bean is the third most important food legume crop worldwide (SCHWARTZ et al., 2005). It is widely consumed throughout the world and is considered a good source of protein (23%), complex carbohydrates, dietary fiber and some vitamins and minerals (CAMPOS-VEGA et al., 2013). Common bean is the most widely grown of the four species belonging to the genus Phaseolus. It is widely cultivated in North, South, and Central America, Africa, Asia and throughout Europe (SCHWARTZ et al., 2005). Myanmar, India and Brazil are the largest Phaseolus-producing nations in the world; however, Myanmar and India produce large quantities of Vigna beans while Brazil is the largest producer of common bean (FOOD AND AGRICULTURAL ORGANIZATION OF THE UNITED NATIONS - FAO, 2012) with an annual production of 2,899,000 tons (ANUÁRIO…, 2013). Brazil also remains the most important country for consumption of beans in the world, followed by Mexico (FAO, 2012). These two countries are nearly self-sufficient in the crop, but bean imports can be essential to supplement periodic production shortfalls. The largest producing state in Brazil is Paraná with 23.37% of the national production, followed by Minas Gerais with 22.17% (ANUÁRIO…, 2013), where the black and brown beans are the favorite among the consumers. For several communities in Brazil, the common bean stands for the major economical activity and alternative for many jobs. The common bean crop (Phaseolus vulgaris L.) is cultivated in a large number of farms with variable sizes and farming systems in Brazil. The plant undergoes four distinct developmental stages during its life cycle that ranges from 65 to 100 days. The time period required to complete each stage varies 18 among cultivars and is influenced by environmental factors (SCHWARTZ et al., 2005). The irrigation system used in some fields in Brazil as well as inadequate management practices are causes for the occurrence of many diseases in common bean, such as Fusarium wilt. According to literature, infected seeds are the major source for spreading the pathogen over long distances (SANTOS et al., 1996; SCHWARTZ et al., 2005). 2.2 General aspects of Fusarium wilt disease in common bean Fusarium wilt was originally discovered in dry beans in California in 1928, and later found in large areas of the United States, Brazil, Colombia, Peru, Costa Rica, Italy, Spain, Greece, the Netherlands and Central Africa (ALVES- SANTOS et al., 1999; BURUCHARA; CAMACHO, 2000; CRAMER et al., 2003). The disease is considered important in Brazil due the lack of crop rotation, intensive cultivation of common bean per year and the intensive movement of machines and implements between fields (PEREIRA; RAMALHO; ABREU, 2009). Fusarium wilt is caused by Fusarium oxysporum Schlechtend.:Fr. f. sp. phaseoli J. B. Kendrick & W. C. Snyder (Fop). Symptoms of the disease occur only on Phaseolus spp. but the pathogen is able to colonize the roots of other plants, particularly legumes, and produce chlamydospores without causing symptoms or disease. Infection of Phaseolus beans by Fop occurs through roots and hypocotyls, most commonly through wounds. Initial symptoms are yellowing and premature senescence of the lower leaves. The chlorotic symptoms progress up the plant until all leaves are bright yellow, followed by wilting and discoloration of foliage. If plants are infected when young, they remain stunted. The vascular tissues usually become reddish brown, often 19 extending beyond the second node (SCHWARTZ et al., 2005). Severe infections can kill the whole plant within a few weeks due the presence and activities of the pathogen in the xylem vessels of the plant. Only when the infected plant is killed by the disease do these fungi move into other tissues and sporulate at or near the surface of the dead plant (AGRIOS, 2005). The optimum temperature for disease development is 20 °C. Extremes of soil moisture levels do not appear to be required for the disease to occur but can influence disease severity. Soil compaction and poor drainage also appear to aggravate disease severity (SCHWARTZ et al., 2005). In general, little information is available on management of Fusarium wilt. Resistance to Fop is usually race specific (ALVES-SANTOS et al., 2002b), conferred by single to multiple genes from different races of beans, that have been incorporated with conventional breeding and molecular techniques into various bean cultivars (BRICK et al., 2006; CROSS et al., 2000; FALL et al., 2001; PEREIRA et al., 2013; PEREIRA; RAMALHO; ABREU, 2009; RIBEIRO; HAGEDORN, 1979a; RONQUILLO-LÓPEZ; GRAU; NIENHUIS, 2010; SALA et al., 2006; SALGADO; SCHWARTZ; BRICK, 1995). As a consequence, correct identification of the local race is essential for the choice of resistant cultivars (ALVES-SANTOS et al., 2002b). Crop rotations associated with the use of healthy seeds are important measures to reduce levels of inoculum in soil (TOLEDO-SOUZA et al., 2012). Chemical seed treatment (MACHADO, 1986) and reduction of soil compaction may also be useful in the control of Fusarium wilt (JENSEN; KURLE; PERCICH, 2004). 2.3 Morphological and genetic aspects of Fusarium oxysporum f. sp. phaseoli According to Leslie and Summerell (2006), Fusarium oxysporum has been defined by morphology as an asexual reproductive structure and was placed in 20 the section Elegans by Wollenweber and Reinking in 1935. The fungus typically has hyaline, nonseptate chlamydospores (2-4 x 6-15 µm) and macroconidia formed in pale orange, usually abundant, sporodochia. The macroconidia are short to medium in length, falcate to almost straight, thin walled and usually 3- septate. The apical cell is short and is slightly hooked in some isolates. The basal cell is notched or foot-shaped. Macroconidia are formed from monophialides on branched conidiophores in sporodochia and to a lesser extent from monophialides on hyphae. Microconidia usually are 0-septate, may be oval, elliptical or reniform (kidney-shaped), and are formed abundantly in false heads on short monophialides. Maximum mycelial growth occurs on culture medium at 28 ºC (SCHWARTZ et al., 2005). The pathogen is a common, widespread fungus found in soil, being spread over long distance by infected seeds (MBOFUNG; PRYOR, 2010; TOLEDO- SOUZA et al., 2012). The pathogen inhabits soil and can survive as chlamydospores in the absence of its hosts. Soil pH changes result in a transcription factor that activates alkaline-expressed genes and inhibits acid- expressed genes and thereby affect fungal cell growth, development, and possibly pathogenicity (AGRIOS, 2005). Pathogenic variability has been analyzed in Fop by the specific pathogenic interaction of the fungus with a set of differential cultivars (WOO et al., 1996) and, so far, seven races have been described. The classification seems to be related to the geographic origin, as race 2 includes isolates from Brazil (RIBEIRO; HAGEDORN, 1979b), race 3 includes isolates from Colombia (SALGADO; SCHWARTZ; BRICK, 1995), race 4 includes one isolate from Colorado, USA (SALGADO; SCHWARTZ, 1993) and race 5 includes isolates from Greece (WOO et al., 1996). Races 6 and 7 were characterized in Spain (ALVES-SANTOS et al., 2002b). However, the results of pathogenicity and race characterization using the CIAT (Centro Internacional de Agricultura Tropical) 21 differential cultivars system of Fop isolates from Spain and Greece indicated that isolates classified in the same race were not homogeneous with respect to virulence (ALVES-SANTOS et al., 2002b; ZANOTTI et al., 2006). Besides the pathogenic population, F. oxysporum is commonly isolated from asymptomatic roots of crop plants (GORDON; MARTIN, 1997). Saprophytic strains of F. oxysporum on diseased common bean plants are morphologically identical to Fop. Because of this, the use of molecular marker techniques, as the analysis of random amplified polymorphic DNA (RAPD), can facilitate the identification of the population (ZANOTTI et al., 2006). Langin, Capy and Daboussi (1995) reported that the presence of transposable element impala in nonpathogenic isolates is able to genetically distinguish them from pathogenic ones. Previous studies reported that highly virulent strains of Fop were able to kill the host in two weeks and weakly virulent strains caused only a lower degree of damage (ALVES-SANTOS et al., 2002a). These findings suggested a differential expression of the transcription factor genes involved in virulence, such as Fusarium transcription factor 1. The gene ftf1encodes a transcription factor containing a Zn(II)2-Cys6 binuclear cluster DNA-binding motif and it is present as a multiple copy gene in highly virulent strains of Fop (RAMOS et al., 2007). The corresponding gene was confirmed to possess the STE and C2H2 domains, characteristic of the fungal Ste12 transcription factor family of proteins (GARCÍA-SÁNCHEZ et al., 2010; VEGA-BARTOL et al., 2011). Homologs of Ste12 identified in several pathogenic fungi have been shown to be involved in pathogenicity (DENG; ALLEN; NUSS, 2007; PARK et al., 2004; TSUJI et al., 2003). 22 2.4 Interaction and transmission of Fusarium oxysporum f. sp. phaseoli in common bean seeds Infected seed is one of the causes for the introduction of Fop into common bean fields or production areas. The presence of the pathogen does not assure however its transmission to plants due to interferences from several factors related with the host, environment and pathogen (MACHADO; POZZA, 2005). Once introduced, the pathogen can survive in soil over extended periods, on crop residues and nonhost crops, and by forming chlamydospores (HAEGI et al., 2013). The pathogen transmission from seed to seedling/plant is influenced by several factors such as inoculum potential and position in seed, environment, time of infection, host resistance and soilborne microbiota (MACHADO; POZZA, 2005). Velicheti and Sinclair (1991) observed that hyphae of F. oxysporum grew over the seed surface, in the hilar region, and seed coat of soybean. Sharma (1992 apud SINGH; MATHUR, 2004) observed that the hyphae of F. oxysporum were confined to the soybean seed coat and the hilar stellate parenchyma in asymptomatic seeds and in all layers of the seed coat, stellate parenchyma in symptomatic seeds with weak infection. In moderately infected seeds, inter and intracellular mycelium occurred in all components. Heavy colonization was found in the seed coat with mycelial mat in the parenchymatous region. In cotyledons the infection was more on the abaxial than the adaxial surface. Infection in the embryonal axis was rare. In heavily infected seeds, aggregation of mycelium occurs in different components including the embryonal axis. In general, pathogen-seed interaction and transmission to plants can negatively affect the yield and seed quality, resulting in considerable economic 23 losses. For F. oxysporum-common bean seeds interaction results in reduced seed germination, reduced vigor (PRYOR; GILBERTSON, 2001), decreased pre- emergence and post-emergence (DUTHIE; HALL, 1987), stunted growth (SCHWARTZ et al., 2005) or a combination of symptoms. The infection mechanisms involved in the interaction between seeds and plant pathogenic fungi vary according to seed structure and the nature of the pathogens. Seed with a large embryo, such as seeds of legumes, may be often expected to carry the infection of fungi in the embryo (NEERGAARD, 1977). Several methodologies are applied to analyze these aspects, making microscopic techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), and fluorescence microscopy, the tools that complement other methodologies, allowing the observation from bio-images of the changes produced. The studies of transmission mechanisms of pathogenic fungi from seeds to plants have been developed by incubation of host tissues in sterile culture medium. After incubation, observations are performed in the microscopy to identify the typical structures of the pathogens (DUTHIE; HALL, 1987; GÖRE et al., 2011; SANTOS et al., 1996; SHAH; BERGSTROM, 2000). For example, 42.8% of Fop was transmitted from common bean seed to seedlings (SANTOS et al., 1996) but very little is known about the mechanisms involved. Other techniques to monitor seed transmission of pathogens are structural analyses by using electron microscopy (WANG; MAULE, 1994), molecular markers as heavy phosphorus (BAHAR; KRITZMAN; BURDMAN, 2009) or through biochemical tests (MICHENER; PATAKY; WHITE, 2002). Currently, green fluorescent protein (GFP) has shown to be useful for studying plant-fungus and fungus-fungus interaction in vivo. Lagopodi et al. (2002) visualized in details the tomato root colonization and infection processes by gfp-labeled F. oxysporum f. sp. radices-lycopersici. Sarroco et al. (2007) 24 followed the colonization of carnation roots by F. oxysporum f. sp. dianthi transformed with GFP and red fuorescent protein (DsRedFP), observing that the hyphae were confined within the vascular cylinder by the endodermal cells beginning from the zone of differentiation of vascular tissues, and were able to grow inside vessels. Vallad and Subbarao (2008) compared the infection and colonization steps of resistant and susceptible lettuce roots by gfp-tagged Verticillium dahliae. Other studies have demonstrated the transmission of the GFP-labeled F. verticillioides from inoculated maize seed to plants under different temperatures (MURILLO-WILLIAMS; MUNKVOLD, 2008; WILKE et al., 2007). The results of both studies indicated that, if maize seed is infected by F. verticillioides, seed transmission is common and symptomless systemic infection can be initiated under a broad range of temperatures. 2.5 Use of molecular techniques in seed health test for fungal detection Plant pathogens that causes seed discoloration or are visibly evident as mycelium or as fruiting structures, have a greater chance to be detected and subsequently discarded (ELMER, 2001). However, most pathogens infest the seeds and use them as a vehicle for transportation of the inoculum over long distances (ELMER, 2001). There are many types of associations between seed pathogens and their hosts. Agarwal and Sinclair (1997) reported that pathogens associated with seeds are more localized on seed coats. This external contamination is also considered as infestation of seeds. The infection of the seeds is observed when the pathogen is internally localized on seeds, colonizing them. Long distance movement and establishment of disease in foreign regions are favored by asymptomatic colonization of the seed by the pathogen (ELMER, 2001). So, accurate seed health testing for seedborne pathogens is an important 25 tool of disease management for reducing the chances of disease spread and for being helpful in decision making regarding the appropriate use of seed treatment, and other practical application in seed certification programs (MBOFUNG; PRYOR, 2010). Seed health testing is an essential management tool for the control of seedborne and seed-transmitted pathogens and continues to be an important activity for their regulation and control through phytosanitary certification and quarantine programs in domestic and international seed trade (MORRISON, 1999). F. oxysporum f. sp. phaseoli has been proposed in Brazil as ‘Regulated Non-Quarantine Pest’ and its tolerance level in common bean seed samples is zero from a total of 400 analyzed seeds in a routine test, according to National Program of Seed Health Quality Control. Conventional methods recommended for detection of Fop in common bean seeds are the blotter test and plating seeds in semi-selective medium supplemented with PCNB (ALBORCH; BRAGULAT; CABAÑES, 2010; BRASIL, 2009; SOUSA, 2006). These methods are not able to differenciate formae speciales, races or saprophytic isolates that are morphologically identical to pathogenic isolates. In this case, pathogenicity test should be performed to obtain reliable informations about the pathogen occurrence in each seed lot. In addition, those methods are time- consuming, labor intensive, require skilled personnel, and are not suited for the rapid and high-throughput type of testing, as demanded in screening commercial seed (ALVES-SANTOS et al., 2002a; MBOFUNG; PRYOR, 2010). The low level of sensitivity and specificity of the current biological methods are obstacles for their adoption in routine analysis (MBOFUNG; PRYOR, 2010). Therefore the development of a reliable, rapid and sensitive diagnostic method that allows for the detection and quantification of Fop in common bean seeds is essential. The polymerase chain reaction (PCR) assay has been used 26 widely as a diagnostic method, as it allows for detection of extremely small quantities of specific target DNA (LEE; TEWARI; TURKINGTON, 2002). Diagnostic methods based on polymerase chain reaction (PCR) have high analytical sensitivity to discriminate between different strains of fungi and have been used to detect a number of form speciales within the F. oxysporum complex (ALVES-SANTOS et al., 2002a; ATTITALLA et al., 2004; CHIOCCHETTI et al., 2001; DITA et al., 2010; MBOFUNG; PRYOR, 2010; SILVA; JULIATTI ; JULIATTI, 2007; ZHANG et al., 2005). In addition to high sensitivity and specificity, PCR-based methods have the advantage to process a large number of samples within a short period of time and can be conveniently applied to commercial seed testing and certification. The method also detects all pathogen inoculum present both within the seed and on the seed surface (GLYNN; EDWARDS, 2010). However the high levels of polysaccharides and phenolic compounds frequently present in seed may affect the efficiency of DNA extraction, and the presence of PCR inhibitors can negatively impact successful amplification of the recovered DNA (DE BOER et al., 1995; MA; MICHAILIDES, 2007; MURILLO; CAVALLARIN; SAN SEGUNDO, 1998; ZOUWEN et al., 2002). In literature several reports provide the potentiality of using molecular techiques in seed health testing. A successful example was the use of specific primers and PCR to identify Tilletia indica on wheat seed through the washing extraction method, separating T. indica from T. barclayana and other Tilletia spp (SMITH et al., 1996). Other reports of PCR detection and quantification were Rhynchosporium secalis in asymptomatic wheat seedlots (LEE; TEWARI; TURKINGTON, 2002) and F. oxysporum f.sp. lactucae in commercial lettuce seedlots (MBOFUNG; PRYOR, 2010). The results of both works demonstrated the potential of the PCR assays as an alternative seed health testing method. 27 Diagnostic methods based on polymerase chain reaction (PCR) have high analytical sensitivity (MBOFUNG; PRYOR, 2010) and have been developed along immunoassays and nondestructive seed health tests, such as ultrasound, optical and infrared analyses, and biopsis (the removal and analysis of tissue cores from seeds) (MUNKVOLD, 2009). According to Munkvold (2009), the PCR-based methods for detecting pathogens in seeds have begun to be implemented in the vegetable seed industry and in some official seed testing laboratories for quality control, but this process has been slow in international seed testing programs. The author still described some reasons for the slow adoption: costs, technical expertise, poor quality DNA, PCR inhibitors from seed extracts (leading to false negatives), remnant DNA from nonviable pathogen propagules (potential for false positives) and sample sizes. Several strategies have been developed to overcome the technical impediments. For improving the DNA quality, commercial DNA extraction kits have been recommended but they are not able to remove all inhibitors in some specific cases (MA; MICHAILIDES, 2007). For attenuating the effects of PCR inhibitors, various techniques have been suggested by Ma and Michailides (2007), like the use of commercial DNA extraction kits combined with additions of amplification facilitators in DNA extraction and PCR reaction buffers. The magnetic capture hybridization (MCH) is another procedure that can concentrate target DNA and separate it from inhibitory compounds and nontarget DNA, increasing sensitivity of the PCR (HA et al., 2009; MUNKVOLD, 2009; WALCOTT; GITAITIS; LANGSTON JUNIOR, 2004). Some approaches to ensuring that PCR is detecting DNA from viable pathogen cells are the use of BIO-PCR coupled with nested-PCR, flow cytometry or propidium monoazide. BIO-PCR involves propagation of putative pathogen propagules on a culture medium and subsequent PCR (MUNKVOLD, 2009). Nested-PCR is also used to enhance the sensitivity and specificity of the detection (ROBÈNE- 28 SOUSTRADE et al., 2010). The use of propidium monoazide can selectively remove free DNA from dead cells (MUNKVOLD, 2009). Some conflicts exist about sample sizes and more studies need to be developed and refined about this topic. The introduction of real-time PCR technology, which is characterized by the inclusion of a fluorescent reporter molecule in each reaction that yields increased fluorescence with an increasing amount of product DNA, has improved and simplified methods for PCR-based quantification. The quantification of a pathogen upon its detection and identification is an important aspect as it can estimate potential risks regarding disease development, spread of the inoculum, and economic losses. Apart from this potential, it provides the information required to make appropriate management decisions (LIEVENS; THOMMA, 2005). According to Mumford et al. (2006), there are ranges of alternative real- time detection chemistries which are used for the plant pathogens detection, including SYBR Green, TaqMan® and FRET. SYBR Green is a method based on DNA-intercalating dyes. Methods based on separation between the reporter and quencher dyes by cleaving of labeled-probe (TaqMan®) results in an increase of fluorescence, which is related to the amount of amplified product. Methods using fluorescent resonance energy transfer (FRET) probes, where spatial separation between the reporter and quencher dyes is achieved through a loss of complex secondary structure due to probe binding, as ‘Molecular Beacons’and ‘Scorpion primers’. Several studies have been done to analyse the real-time PCR as a tool for seed health tests through detection and quantification of pathogenic fungi in different plants and seeds. Bilodeau et al. (2007) used three different reporter technologies, TaqMan, SYBR Green and Molecular Beacons to differentiate Phytophthora ramorum from 65 Phytophthora spp. and to detect the target DNA in infected plants of different hosts. The chemistries were P. ramorum- 29 detectable but TaqMan seemed to be more sensitive. Chilvers et al. 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Abstracts… Wageningen: ISTA, 2002. p. 53. 40 CHAPTER 2 - PAPERS Paper 1 Transmission of Fusarium oxysporum f. sp. phaseoli from seed to emerging plants of common bean Transmissão de Fusarium oxysporum f. sp. phaseoli de sementes para plantas emergidas de feijão Prepared according to Seed Science and Technology guidelines Marcella Viana de Sousaa, José da Cruz Machadoa aUniversidade Federal de Lavras, Departamento de Fitopatologia, Caixa Postal 3037, 37200-000, Lavras, MG, Brasil. 41 ABSTRACT Fusarium oxysporum f. sp. phaseoli (Fop) causes severe losses in common bean fields and the pathogen can be seed-transmitted and disseminated over long distances. The objective in this work was to estimate the seed-to-emerged plant transmission rates of Fop in relation to different factors under controlled conditions. The work was conducted using artificially and naturally Fop- contaminated seeds. For seed inoculation by osmotic technique two cultivars (BRSMG Majestoso and Ouro Negro), two temperatures (20 and 25 ºC), two strains (FOP005 and FOP014) and four inoculum potentials (P0, P1, P2 and P3) were used. For naturally Fop-contaminated seed trials three common bean cultivars (Horizonte, Cometa and Valente) in three seed sizes (small, medium and large) at two temperatures (20 and 25 ºC) were used. The transmission rates from artificially and naturally contaminated seeds were 100% in symptomatic plants although the number of emerged symptomatic plants was lower than 5%. Asymptomatic plants contaminated with Fop were observed in high frequencies on different plant tissues, especially on hypocotyl and main root, confirmed by pathogenic Fop-positive bands in conventional PCR. A steep decline of the fungus recovery was observed in the cotyledons and first node tissues. From these results it was observed that the transmission rates of Fop from infected bean seeds to emerged symptomatic and asymptomatic plants is quite high and has to be considered as an important factor in the disease management. Key-words: Fusarium wilt, Phaseolus vulgaris, seed infection, seed pathology. 42 RESUMO Fusarium oxysporum f. sp. phaseoli (Fop) é responsável por perdas severas em campos de produção de feijoeiro, sendo o patógeno transmitido e disseminado por sementes a longas distâncias. Objetivou-se, neste estudo, estimar as taxas de transmissão de Fop das sementes para as plantas emergidas, em relação a diferentes fatores sob condições controladas. O trabalho foi conduzido utilizando-se sementes artificialmente e naturalmente associadas ao Fop. Para a inoculação das sementes através da técnica de condicionamento osmótico foram utilizadas duas cultivares (BRSMG Majestoso e Ouro Negro), duas temperaturas (20 e 25 ºC), dois isolados (FOP005 e FOP014) e quatro potenciais de inóculo (P0, P1, P2 e P3). Para ensaios com sementes naturalmente associadas ao Fop foram utilizadas três cultivares de feijão (Horizonte, Cometa e Valente), em três tamanhos de peneiras (pequeno, médio e grande) e em duas temperaturas (20 e 25 ºC). As taxas de transmissão de sementes artificialmente e naturalmente associadas ao Fop foram 100% para plantas sintomáticas, apesar do número de plantas sintomáticas emergidas ser menor do que 5%. Plantas assintomáticas e associadas ao Fop foram observadas em alta frequência, em diferentes tecidos da planta, especialmente no hipocótilo e raiz principal, confirmado por bandas positivas de Fop patogênico, em PCR convencional. Grande redução do crescimento fúngico foi observado nos cotilédones e inserção dos cotilédones. A partir destes resultados foi observado que as taxas de transmissão de Fop de sementes de feijão infectadas para plantas emergidas sintomáticas e assintomáticas foram elevadas e devem ser consideradas como um fator importante no manejo de doenças. Palavras-chave: murcha de fusarium, Phaseolus vulgaris, infecção de sementes, patologia de sementes. 43 INTRODUCTION Fusarium oxysporum Schlechtend:Fr. f. sp. phaseoli J. B. Kendrick & W. C. Snyder (Fop) is the causal agent of Fusarium wilt of common bean (Phaseolus vulgaris L.), which is distributed worldwide (Alves-Santos et al., 2002a) as a soil inhabitant, being able to survive in the form of chlamydospores or in infected seeds (Schwartz et al., 2005). Symptoms of the disease are characterized by chlorosis of leaves, necrosis of the vascular system and general wilt and death of the colonized plant (Vega- Bartol et al., 2011). Highly virulent strains are able to kill common bean plants in about two weeks (Alves-Santos et al., 2002a). The optimum temperature for Fop development is 28 °C but for disease development is 20 °C (Schwartz et al., 2005). The most efficient and viable management practices for Fusarium wilt control are the use of healthy seeds (Santos et al., 1996) and use of resistant cultivars (Pereira et al., 2009). No information was found in literature about resistance related to seed transmission of Fop in common bean seeds. A study by Pereira et al. (2013) indicated that Fop structures were observed in the xylem vessels of resistant cultivar of P. vulgaris, although no disease symptom was observed in plants. These plants become an important inoculum source in the field but the question as to whether seed from resistant cultivars contaminated with Fop inoculum is able to transmit it to seedling continues to be misunderstood. Santos et al. (1996) showed that infected common bean seeds with incidence of 14% by F. oxysporum, transmitted the pathogen to plants in a high percentage (42.8%). Some aspects such as genotype, environmental conditions and pathogenicity/ virulence of the fungus were not reported in their study. 44 Although Fusarium wilt is one of the most important diseases in common bean in Brazil (Paula Júnior et al., 2006), little information is known about seed- Fop interaction as well as the plant-to-plant transmission rate in field conditions. This work was proposed with the objective to determine the rates of seed transmission of Fop in common bean. This kind of information is essential for establishing health standars for that pathosystem which are of great interest in Seed Certification Programs. 45 MATERIALS AND METHODS Origins of Fop strains, common bean cultivars and seed inoculation procedures. For this experiment two Fop strains, two cultivars of common bean, two temperatures and four inoculum potentials were used following the model proposed for other pathosystems (Botelho et al., 2013). The two Fop strains, FOP005 and FOP014 were obtained from the Mycological Collection of Seed Pathology Laboratory, Lavras, MG, Brazil and from Agronomic Institute of Campinas, Campinas, SP, Brazil, respectively. These strains were identified as F. oxysporum on synthetic-nutrient-agar (SNA) and potato-dextrose-agar (PDA; Difco Laboratories, Plymouth, MN), according to Leslie & Summerell (2006). Single spore isolates were prepared and maintained on PDA. The pathogenicity was tested according to Alves-Santos et al. (2002a); FOP005 was identified as a highly virulent pathogen and FOP014 as a weakly virulent pathogen (data not shown). Fop was confirmed using specific primers to conventional PCR from Alves-Santos et al. (2002b). The two Phaseolus vulgaris cultivars used, BRSMG Majestoso and Ouro Negro, are recommended for planting in the State of Minas Gerais (Paula Júnior et al. 2010). The absence of F. oxysporum in seeds was initially confirmed by blotter test (Brasil, 2009) and conventional PCR, using the primer set published by Alves-Santos et al. (2002b). For inoculation of seeds, bulks of 1,000 seeds of each cultivar were inoculated through osmotic technique described by Sousa et al. (2008). Through that technique, seeds were surface disinfected by soaking for one minute in a 1% NaHClO solution followed by drying on a sterile filter paper in a laminar flow hood. Both isolates FOP005 and FOP014 were cultured for five days on potato- dextrose-agar (PDA) medium at 22 °C. Macro and microconidia were harvested by washing the surface of a culture with 10 mL of sterile distilled water. The 46 resulting suspensions were diluted with sterile water to obtain a final concentration of 106 spores mL-1 (counts adjusted with a hemacytometer). Inoculum suspensions were sprayed on seeds and on PDA supplemented by mannitol with osmotic potential adjusted to -1.0 MPa, according to software SPMM (Michel & Radcliffe, 1995). Seeds were kept at 20 °C, 12h photoperiod, for four incubation periods, 0, 36, 72 and 96h of the exposure seeds to Fop colonies. The different incubation periods were considered as different inoculum potentials (P0, P1, P2 and P3, respectively) of the pathogen. Artificially inoculated seeds were then removed and air dried overnight under a hood. Negative controls were prepared for each cultivar and incubation period using PDA supplemented by mannitol (-1.0 MPa) with absence of Fop. Sowing artificially Fop-contaminated seeds to evaluate seed-to-plant transmission rate under controlled conditions. To assess the incidence of seed infection by Fop, 100 inoculated seeds and 100 non-inoculated seeds, without surface disinfestation, were placed on blotter test moistened with PDB supplemented with 1 ppm of PCNB (Sousa, 2006). The seeds were kept in incubation room at 20 °C, 12 h photoperiod, for seven days. The incidence of seeds with Fop was recorded. For estimating the potential rate of seed-to-plant transmission, the experiment was displayed in three blocks with 20 treatments (2 cultivars x 2 Fop strains x 4 inoculum potentials + 4 negative controls). Sixty seeds of each treatment were individually sowed in 300 mL-plastic cups, containing soil: sand: compost substrate (Tropstrato HA Hortaliças) in equal proportion (by volume). The cups were arranged in randomized blocks in two growth chambers with temperatures adjusted to 20 and 25 °C. Light was supplied in each shelf by three horizontally oriented cool white fluorescent bulbs (NSK T10 40W 6500K FL40T10-6 60Hz). 47 Final evaluations were made on emerged plants of 25 days-old by counting the stand and looking at typical symptoms of Fusarium wilt. Plants were considered symptomatic when they presented yellowing, wilt and/or vascular discoloration. Those plants were counted and expressed as a percentage of the total number of emerged plants in each treatment. The asymptomatic plants were also removed from the cups and 4-cm fragments of the main root, hypocotyl and first node were cut for examination. The cotyledons were collected from 10-15 day-old plants when they fell from the plants. Every tissue from each plant was surface disinfestated with 70% ethanol for 2 min, 1% sodium hypochlorite solution for 2 min, and sterile distilled water for 2 min and placed in PDA medium supplemented with 1 ppm PCNB. The materials were kept in an incubator with 12h photoperiod for seven days at 20 °C, and microscopically examined for morphological structures of F. oxysporum. Plant fragments colonized by F. oxysporum were scored as positive transmission, and conventional PCR were performed to confirm the identity of pathogenic Fop. Symptomatic plants as well as each plant fragments of asymptomatic plants with or without presence of F. oxysporum growth were frozen in liquid nitrogen and ground into a fine powder. DNA extractions were performed using Wizard® Genomic DNA Purification Kit (Promega, Madison, WI) according to the manufacturer’s protocol. Amplifications were performed in thermocycler (Multigene TC 9600-G; Labnet International Inc.; software V3.3.4C), primer set from Alves-Santos et al. (2002b) (B310: 5’-CAGCCATTCATGGATGACATAACGAATTTC-3’ and A280: 5’-TATACCGGACGGGCGTAGTGACGATG-3’), and the components were added following the protocol of Platinum® Taq DNA polymerase (Invitrogen, Carlsbad, CA). The amplification conditions were as follows: a denaturation step for 5 min at 94 °C, followed by 40 amplification cycles consisting of 1 min at 94 °C, 1 min at 65 °C, and 2 min at 72 °C. A final 48 extension step was performed for 5 min at 72 °C. Samples of the PCR products were run on 1.2% agarose gels with GelRed® (Biotium) in 1x Tris-borate-EDTA buffer, and DNA was visualized by L-Pix HE (Loccus Biotecnologia, Cotia, SP). Sowing naturally Fop-contaminated seeds. Seeds from three cultivars of Phaseolus vulgaris L., Cometa, Horizonte and Valente, obtained in 2010 from Fop-infected fields of Embrapa (Rice and Bean) Arroz e Feijão, Santo Antônio de Goiás, GO, Brazil were used in this study. The Fop-susceptible cultivars were separated in sieve sizes during seed processing, and three different seed sizes, small, medium and large, were used. Seed infection incidence by F. oxysporum was determined by assaying 400 seeds without surface disinfestation on blotter test modified for addition of 1 ppm of PCNB. Seeds were placed on PDA modified by addition of manittol -1.0 MPa, calculated by software SPMM (Michel & Radcliffe, 1995) for three days. Seeds with Fusarium sp. growth were collected and sown individually in 300 mL- plastic cups containing 1 part soil: 1 part sand: 1 part of organic compound (by volume). The experimental unit was 60 plastic cups, with three blocks arranged in randomized blocks in each of two growth rooms, at 20 and 25 °C. The plants developed until 25 days after sowing. The collecting period, types of fragments of plant with plating procedures on culture medium and analysis by conventional PCR were performed as the same for artificially Fop-contaminated seeds. Symptomatic or asymptomatic plants that showed at least one fragment with Fop-mycelial growth, which was positive through conventional PCR, were counted as Fop-positive transmission in each treatment. The rates of transmission of Fop from seeds to plants were estimated by dividing the number of Fop-positive transmission by number of sowed seed and by multiplying by 100. 49 Data analysis. For both experiments, analyses of variance were made on transmission rate of symptomatic and asymptomatic plants from artificially and naturally Fop-contaminated seeds as well as the percentage of Fop occurrence in infected cotyledons, first node, hypocotyl and main root. A square-root transformation was used to equalize variances in the occurrence of the fungus in each plant fragment from naturally Fop-contaminated seeds. Analyses were conducted with the general linear model (GLM) procedure of SAS ver. 9.1 (SAS Institute Inc., Cary, NC, USA). Least significant difference (LSD) tests at the 0.05 level were calculated to compare means of cultivars and temperature, and Tukey test was used to compare means of isolates. Regression models were calculated to compare periods of exposition of seeds to Fop (inoculum potentials). 50 RESULTS Artificially Fop-contaminated seeds. The initial germination percentages of common bean seeds of both cultivars used in this study, BRSMG Majestoso and Ouro Negro were 97% and 95%, respectively. The results of the botter test and conventional PCR showed that both common bean seed lots were Fop-free. After artificial seed infection by the osmotic technique, the incidence of Fop in inoculated seeds was 100%. The influence of the biotic (cultivar, pathogen virulence, inoculum potential) and abiotic factors (temperature) were evaluated individually because no statistical differences were found by the interaction between them (Appendix 1). Typical symptoms of Fusarium wilt in emerged plants of common bean at 25 °C were observed in 3.9% of the plants (Fig. 1A), 1.6% for BRSMG Majestoso and 2.3% for Ouro Negro (Fig. 1B) and 4.2% for highly-virulent strain (FOP005) compared to 2.3% of the weakly-virulent one (FOP014) (Fig. 1C). However, all symptomatic plants presented high Fop incidence in culture medium with a 100% transmission rate in these plants. Most incidence of the pathogen in emerged plants was found in asymptomatic plants (Fig. 1 A, B, C), with transmission rates ranging from 54.4% at 20 ºC to 52.3% at 25 ºC (Fig. 1A); 49.7% to 57% for Ouro Negro and BRSMG Majestoso (Fig. 1B); 83.6% to 94.2% for FOP005 and FOP014 (Fig. 1C), respectively. Regression equations were obtained for potential transmission rates and seed- Fop contact period (inoculum potential). Linear equation was used relating that the potential transmission rate of symptomatic plants increased by 0.0273 for each 1 hour in the period of seed-Fop contact (Fig. 2A). The coefficient of determination was 0.78 and not significant. Polynomial equations showed maximum potential transmission rate for asymptomatic plants related to periods 51 of Fop exposure around 71 h, with significant coefficient of determination (0.97) (Fig. 2A). Figure 1. Seed-to-plant transmission rate (%) of Fusarium oxysporum f. sp. phaseoli and emerged plants (%) in inoculated common bean (Phaseolus vulgaris L.) seeds, with symptomatic and asymptomatic 52 plants: A) in two temperatures (20 and 25 °C); B) in two cultivars (BRSMG Majestoso and Ouro Negro); C) with two Fop isolates (FOP 005 and FOP 014) and no Fop (negative control) Figure 2. Inoculated common bean (Phaseolus vulgaris L.) seeds by Fusarium oxysporum f. sp. phaseoli in different seed-Fop contact periods 53 (inoculum potentials): 0h (P0), 36h (P1), 72h (P2) and 96h (P3). A) Seed-to-plant transmission rate (%) with symptomatic and asymptomatic plants; B) Emerged plants at 25 days after planting The population of symptomatic plants ranged from 1.9% (P1) to 3.2% (P3) whereas asymptomatic plants with presence of pathogenic Fop ranged from 56.2% to 61.3% (Fig. 2A). The emerged plants from inoculated seeds showed variable decrease according to the inoculum potentials used in this study (Fig. 2B). The highest frequence of isolation of Fop occurred on hypocotyl followed by main root, cotyledons and first node of asymptomatic plants (Fig. 3). Recoveries of Fop from hypocotyl were 55.8, 57.6% for 20 and 25 °C (Fig. 3A), 56.7% for both cultivars (Fig. 3B), 0, 93, 96% for non-inoculated seeds, FOP005 and FOP014 (Fig. 3C), and 0, 62.4, 65.3, 61.4 % for P0, P1, P2 and P3 (Fig. 3D). Figure 3. Fusarium oxysporum f. sp. phaseoli recovery frequency (%) in four different fragments (hypocotyl, main root, cotyledons and first node) 54 of 25-days old asymptomatic common bean plants (Phaseolus vulgaris L.) assessed, A) in two temperatures, 20 and 25 °C; B) in two cultivars, BRSMG Majestoso and Ouro Negro; C) with two Fop isolates, FOP 005, FOP 014 and no Fop (negative control); and D) in different seed-Fop contact period/inoculum potentials 0 h (P0), 36 h (P1), 72 h (P2) and 96 h (P3) Naturally Fop-contaminated seeds. A low proportion of symptomatic plants, lower than 1.5%, were observed at 20 and 25 °C, in all cultivars and seed sizes (Fig. 4). However, for the results of conventional PCR, all symptomatic plants were pathogenic Fop-positive corresponding to a100% transmission rate in these plants. The results of conventional PCR also showed that Fop as non-pathogenic F. oxysporum were present in asymptomatic plants. The colonies were similar in culture medium but they were genetically distinguishable. Fop was found in asymptomatic plants, having 13.0% and 10.4% transmission rate at 20 and 25 °C, respectively; for cultivars Valente, Cometa and Horizonte the transmission rates were 8.1%, 10.3% and 16.7%, respectively; 14.7%, 9.7% and 10.6% for small, medium and large sizes of common bean seeds, respectively (Fig. 4). Fop was recovered from each plant fragment with great incidence in the hypocotyl followed by main root and a steep decline of the fungus recovery in the cotyledons and first node (Fig. 5). 55 Figure 4. Transmission rate (%) of Fusarium oxysporum f. sp. phaseoli from common bean (Phaseolus vulgaris L.) seeds harvested in infected fields seeds to emerged symptomatic and asymptomatic plants: A) in two temperatures, 20 and 25 °C; B) in three cultivars, Valente, Cometa and Horizonte; C) with three seed sizes, small, medium and large, obtained by seed processing 56 Figure 5. Fusarium oxysporum f. sp. phaseoli recovery frequency (%) in four different fragments (hypocotyl, main root, cotyledons and first node) of 25-days old asymptomatic common bean plants (Phaseolus vulgaris L.) assessed, A) in two temperatures, 20 and 25 °C; B) in three cultivars, Valente, Cometa and Horizonte; C) with three seed sizes, small, medium and large, obtained by seed processing 57 DISCUSSION The seed-to-plant transmission rates, as calculated in this work, provided an estimate about the risk of using Fop-contaminated/infected seeds of common bean. The results indicate that by the individual analysis of the factors used in this study (Appendix 1), like temperature, host genotype, pathogen population and its biomass (inoculum potential) on seeds can affect the normal development of plants and the beginning of the disease process. In literature, information on that kind of interaction is found for some pathosystems such as those involving F. verticillioides in maize (Wilke et al., 2007), Verticillium dahliae in cotton (Göre et al. 2011), F. graminearum in winter wheat (Duthie & Hall, 1987). In those pathosystems a close relationship was also seen between seed infection and effects of the pathogens on initial plant development. The estimated rate of seed-to-plant transmission of Fop in artificially inoculated seeds was higher than rates that occurred for naturally Fop- contaminated seeds. The fungus biomass present on inoculated seeds may be higher which explains this difference. Plants incubated at 20 °C did not show Fusarium wilt symptoms at 25-days after sowing, and only a low percentage of symptomatic plants were observed at 25 °C (Fig. 1A). The influence of temperature on Fusarium wilt development in common bean has not been well characterized and understood. Ribeiro & Hagedorn (1979) reported that a low temperature, around 20 °C, is the optimum for this disease development. Other authors mentioned that high temperature associated to high moisture is the favorable environmental condition to cause Fusarium wilt disease in common bean (Pastor-Corrales & Abawi, 1987; Buruchara & Camacho, 2000; Pereira et al., 2011). The results of this work indicate that, if seed is infected with Fop, seed transmission can be initiated under a broad range of temperature conditions. 58 The two genotypes exhibited asymptomatic plants with presence of the pathogenic Fop (Fig. 1B). The resistance of both cultivars used in the present work was not able to avoid the presence of pathogen biomass on seeds with their consequent infections, moving to plants showing or not showing typical symptoms. In relation to virulence of Fop strains, a higher transmission rate was observed for a highly virulent isolate, FOP005 (Fig. 1C), causing death of the young plants with visible sporulation of the fungus at the surface of the dead plants in symptomatic plants. Weakly virulent isolate (FOP014) was able to cause some yellowing symptoms in leaves but not wilting and death during the experimental period. Both were seed-trasmitted at 83.55 and 94.24% to asymptomatic plants (Fig. 1C); more studies are required to understand if they can start an epidemy if some changes occur, like favorable environmental conditions. The relation between transmission rate and inoculum potential (inoculum biomass) of Fop (Fig. 2A) was not explained by linear regression. The high inoculum potential in seeds (P3) probably caused the seed rot leading to the pre- emergence death of seeds. Thus, that migh be the cause of the low number of asymptomatic emerged plants (Fig. 2B) with infection by Fop. The higher occurrence of Fop in the present work was observed in the hypocotyl fragments for all treatments followed by the main root (Fig. 3) differing from a previous study from Wilke et al. (2007) who observed recovery of transformant F. verticillioides at V2 stage ranging from 60 to 80% in stems of maize plants and more than 90% in roots. Some factors may explain these differences, such as the inoculation method used for both studies, the differences between species of Fusarium and the germination type for each family of plants (Poaceae and Fabaceae). The occurrence of Fop in the cotyledons and first nodes can be associated to infected cotyledons remain in Fabaceae plants until the first 59 true leaves are unfolded, facilitating the stem infection. After that, the cotyledons eventually fall off and continue on the soil, being an important source of inoculum dissemination. Rates of seed-to-plant transmission of Fop observed in this study for naturally infected seeds are lower than the rate reported by Santos et al. (1996). The results in this work indicate that transmission rates of Fop in common bean, ranging from 10.6% to 18.1%, were temperature, genotype and seed-size dependents (Fig. 4). Santos et al. (1996) observed a 42.8% rate of transmission of F. oxysporum from 14% of contaminated seeds. Probably differences in methodology used in both studies may be the cause of those conflicting results. The highest transmission rate in this study was observed for the cultivar Horizonte small-seeded size at 20 ºC. All symptomatic plants resulting from naturally Fop-contaminated seeds died and the fungus sporulated on their external surface. For asymptomatic plants, pathogenic Fop was recovered on all assessed plant fragments, confirmed by conventional PCR, with variable incidences (Fig. 5), according to the incidences observed for artificially infected seeds. In this work the results indicated that Fop-contaminated common bean seeds are able to transmit the pathogen with observation of symptomatic and asymptomatic plants. Seed-transmitted Fop was shown to be dependent on the biomass and position of the inoculum in seeds, virulence of the pathogen as well as on some intrinsic characteristics of the host (resistance and seed size). Usually, the symptomatic emerged plants were lower than 4% with 100% of Fop transmission in these plants. Asymptomatic plants with the presence of Fop reached 94% at the highest values of inoculum biomass present on seeds. This information is of interest for establishing tolerance levels in seed certification programs and for improving the seed production and trade process. The establishment of phytosanitary standards is required, especially for Regulated 60 Non-Quarantine Pests due the great concern involving the seed movement and pathogen spreading. The results of this work confirm the huge importance of understanding the interaction between Fop and seeds of common bean while maintaining the management of this disease in practice in sight and the clear need of additional studies in this line of research. ACKNOWLEDGEMENTS The authors express their thanks to CNPq/MAPA and FAPEMIG for funding and supporting this research; CAPES for the scholarship provided to the first author; L.H. Pfenning and M.F. Itto for providing some of Fop isolates; Department of Biology/UFLA (Lavras, MG, Brazil) and EMBRAPA (Santo Antônio de Goiás, GO, Brazil) for the seed samples used in this work. 61 REFERENCES Alves-Santos, F.M., Cordeiro-Rodrigues, L., Sayagués, J.M., Martín- Domínguez, R., García-Benavides, P., Crespo, M.C., Díaz-Mínguez, J.M. and Eslava, A.P. (2002a). 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Plant Disease, 91, 1109-1115. 64 Paper 2 Studies of the association of Fusarium oxysporum f. sp. phaseoli in common bean seeds Estudos da associação de Fusarium oxysporum f. sp. phaseoli em sementes de feijão Prepared according to Scientia Agricola Journal guidelines Marcella Viana de Sousaa, José da Cruz Machadoa, Luana da Silva Botelhob, Eduardo Alvesa aUniversidade Federal de Lavras, Departamento de Fitopatologia, Caixa Postal 3037, 37200-000, Lavras, MG, Brasil; bInstituto Federal do Norte de Minas Gerais, Campus Universitário, Rodovia MG 202, CEP 38680-000, - Arinos, MG - Brasil. 65 ABSTRACT Green fluorescent protein (GFP) has been used as a marker for studying the colonization of different crops by plant pathogens. In order to follow the colonization process by Fusarium oxysporum f. sp. phaseoli (Fop) in common bean seeds (Phaseolus vulgaris), that marker was tested using one pathogenic strain of Fop which was GFP-labeled following protocols described in literature. The transformant was tested in PDA containing hygromycin-B (300 µg mL-1) and conventional PCR. The transformation of the pathogen can be considered successful by taking into account the high proportion of fungal colonies formed on PDA with hygromycin-B. Bean seeds, cv. Uirapuru, were surface-disinfected, dried in a hood overnight and inoculated with the transformed fungus using the osmotic technique. The pathogen was detectable in whole embryonic axis, including the plumule, and on endosperm of the common bean seeds, confirming the infection process. GFP-tagged mycelium was externally observed in the roots and hypocotyl of the plants. Vascular discolorations were well developed in advance of the Fop-infection. These results show that the pathogen was able to colonize both external and internal tissues of infected seeds as well as external and vascular tissues of the resulting plants. Key-words: Fluorescence microscopy, Fusarium wilt, Phaseolus vulgaris, seed- pathogen interaction. 66 RESUMO A proteína fluorescente verde (GFP) tem sido utilizada como um marcador nos estudos de colonização de fitopatógenos, em diferentes culturas. Com a finalidade de acompanhar o processo de colonização de Fusarium oxysporum f. sp. phaseoli (Fop), em sementes de feijão (Phaseolus vulgaris), aquela técnica foi testada usando um isolado patogênico de Fop, que foi marcado por GFP, seguindo protocolos descritos em literatura. O transformante foi testado em BDA, contendo higromicina-B (300 µg mL-1) e PCR convencional. A transformação do patógeno foi considerada bem sucedida, tendo-se em vista a alta proporção de colônias fúngicas formadas em meio BDA, com higromicina- B. Sementes de feijão, cv. Uirapuru, foram desinfestadas superficialmente, secas em câmara de fluxo overnight e inoculadas com o transformante pela técnica de condicionamento osmótico, descrita em literatura. O patógeno foi detectado em todo o eixo embrionário, incluindo a plúmula, e no endosperma de sementes de feijão, confirmando o processo de infecção nessa interação. O micélio marcado por GFP foi observado externamente nas raízes e hipocótilo das plantas. Escurecimento vascular foi bem desenvolvido com o avanço da infecção por Fop. Esses resultados indicaram que o patógeno foi capaz de colonizar ambos os tecidos internos e externos de sementes infectada, assim como tecidos externos e vasos das plantas resultantes. Palavras-chave: microscopia de fluorescência, murcha de fusarium, Phaseolus vulgaris, interação semente-patógeno. 67 INTRODUCTION Fusarium wilt is one of the most important diseases in common bean crop, caused by the fungus Fusarium oxysporum Schlechtend.: Fr. f. sp. phaseoli J. B. Kendrick & W. C. Snyder (Fop). The pathogen inhabits soil in the form of chlamydospores and may also infest seeds (Schwartz