Doctor of Philosophy / Department of Plant Pathology / Christopher R. Little / Charcoal rot, caused by the necrotrophic fungus, Macrophomina phaseolina (Tassi) Goid., is an important disease in sorghum (Sorghum bicolor (L.) Moench). The molecular interactions between sorghum and M. phaseolina are poorly understood. In this study, a large-scale RNA-Seq experiment and four follow-up functional experiments were conducted to understand the molecular basis of charcoal rot resistance and/or susceptibility in sorghum. In the first experiment, stalk mRNA was extracted from charcoal-rot-resistant (SC599) and susceptible (Tx7000) genotypes and subjected to RNA sequencing. Upon M. phaseolina inoculation, 8560 genes were differentially expressed between the two genotypes, out of which 2053 were components of 200 known metabolic pathways. Many of these pathways were significantly up-regulated in the susceptible genotype and are thought to contribute to enhanced pathogen nutrition and virulence, impeded host basal immunity, and reactive oxygen (ROS) and nitrogen species (RNS)-mediated host cell death. The paradoxical hormonal regulation observed in pathogen-inoculated Tx7000 was characterized by strongly upregulated salicylic acid and down-regulated jasmonic acid pathways. These findings provided useful insights into induced host susceptibility in response to this necrotrophic fungus at the whole-genome scale. The second experiment was conducted to investigate the dynamics of host oxidative stress under pathogen infection. Results showed M. phaseolina’s ability to significantly increase the ROS and RNS content of two charcoal-rot-susceptible genotypes, Tx7000 and BTx3042. Over-accumulation of nitric oxide (NO) in stalk tissues in the pathogen-inoculated susceptible genotypes was confirmed using a NO-specific fluorescent probe and confocal microscopy. Significantly increased malondialdehyde content confirmed the enhanced oxidative stress experienced by the susceptible genotypes after pathogen inoculation. These findings suggested the contribution of oxidative stress-associated induced cell death on charcoal rot susceptibility under infection. In the third functional experiment, the behavior of the sorghum antioxidant system after pathogen inoculation was investigated. M. phaseolina significantly increased the glutathione s-transferase (GST), glutathione peroxidase (GPX), glutathione reductase (GR), and peroxidase activities of the susceptible genotypes (Tx7000, BTx3042) but not in the resistant genotypes (SC599, SC35). Increased activities of these enzymes in susceptible genotypes may contribute to reduced oxidative stress thus lowering charcoal rot susceptibility. The fourth functional experiment was designed to quantify induced host-derived cell wall degrading enzymes (CWDEs) using crude enzyme mixtures from stalks. A gel diffusion assay revealed significantly increased pectinesterase activity in pathogen-inoculated Tx7000 and BTx3042 while significantly increased polygalacturonase activity was determined by absorbance. Fluorimetric determination of cell extracts revealed significantly increased cellulose degrading enzyme activity in M. phaseolina-inoculated Tx7000 and BTx3042. These findings revealed the pathogen’s ability to promote charcoal rot susceptibility in grain sorghum through induced host CWDEs. The last functional study was designed to profile the stalk tissue lipidome of Tx7000 and SC599 after M. phaseolina inoculation using automated direct infusion electrospray ionization-triple quadrupole mass spectrometry (ESI-MS/MS). M. phaseolina significantly decreased the phytosterol, phosphatidylserine, and ox-lipid contents in Tx7000 while significantly increasing stigmasterol:sitosterol ratio. Except for ox-lipid content, none of the above was significantly affected in resistant SC599. Results suggested the lethal impacts of M. phaseolina inoculation on plastid- and cell- membrane integrity and the lipid-based signaling capacity of Tx7000. Findings shed light on the host lipid classes that contribute to induced charcoal rot susceptibility in grain sorghum.

Strawberries are considered an important crop in California where in 2018 it was in the top 5 valued fruit and vegetable commodities valued at $2.84 billion accounting for 88% of the total U.S. production. Strawberry production can be severely impacted by soilborne pathogens that can affect strawberry roots, crowns and leaves which can result in plant mortality. As much as 50 to 60% mortality can occur in one field. Pathogens responsible for such losses include Colletotrichum acutatum (syn.C. nymphaeae), Macrophomina phaseolina and Verticillium dahliae. With the phaseout of methyl bromide, host resistance and an understanding of host-pathogen interactions can play an important role in control of these diseases. A two-year study was conducted in order to evaluate host resistance of anthracnose in 105 cultivars and elite breeding lines developed by six strawberry breeding programs. Cultivars and elite breeding lines were inoculated using three local isolates in both years. All breeding programs provided genotypes that had a wide range of anthracnose susceptibility ranging from 0 to 100% mortality during both years. In both years an average of 78% of all the plant mortality occurred by 1 January. From the 105 cultivars and elite breeding lines, 30 cultivars were common to both years. Of these 30 cultivars, nine of them differed in their disease susceptibility between experiments by more than 20%. This suggests that several years of field evaluation may be necessary to determine susceptibility to anthracnose. Popular cultivars that represent the spectrum of susceptibility are Monterey (susceptible), Festival (moderately resistant), and Sensation (resistant). A second study was conducted toevaluate pathogen colonization of resistant and susceptible strawberry cultivars, testing interactions among crown and root plant tissue and two sampling timings. These cultivars were challenged with two soilborne pathogens, Macrophomina phaseolinaand Verticillium dahliae,over two years. Existing qPCR protocols for M. phaseolina and V. dahliae were used in order to quantify how much pathogen DNA was detected in crown and root samples. For the 2016-2017 V. dahliae trial there were significant effects for cultivar. Cultivar Benicia had significantly higher pathogen DNA compared to resistant cultivars Marquis, UC-12 and Camino Real. Susceptible cultivar BG 1975 had significantly less pathogen DNA compared to resistant cultivars San Andreas and Petaluma. In the 2017-2018 V. dahliaetrial pathogen DNA amount was not significantly different based on cultivar, plant part colonization, or the sampling period. In the 2017-2018 M. phaseolina trial all three of the fixed factors, cultivars, plant part colonization and sampling period were statistically significant. Cultivar ‘Sweet Ann’ had a significantly higher level of M. phaseolinaDNA in the early vs. the late sampling.

The fungus Macrophomina phaseolina (Tassi) Goidanich (Mp) is the causal agent of charcoal rot in soybean and infects over 500 plant species worldwide. Mp produces various mycotoxins and is suspected of utilizing a toxin-mediated process to penetrate host tissue. Identification and evaluation of secondary metabolites produced by Mp will further elucidate the pathogenesis mechanisms used by the fungus. Mp cultures isolated from soybean were evaluated for phytotoxicity in a hydroponic soybean bioassay and chemically analyzed by LC-MS/MS. All Mp cultures at two dilutions induced phytotoxicity symptoms including chlorosis, necrosis, wilting, stunting, and death. Analysis identified 13 unreported secondary metabolites including mellein, a compound with various biological activities. The phytotoxicity of mellein was evaluated against soybean seedlings in hydroponic culture, and symptoms of wilting and stunting were observed at levels above 40 MUg/L. Observations suggest that mellein does not directly contribute to the phytotoxic effects of Mp cultures.

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Made available in DSpace on 2017-07-10T17:36:49Z (GMT). No. of bitstreams: 1 2013_Laline_Broetto.pdf: 1586357 bytes, checksum: 1ec5db5ff1f6efea61394595db367816 (MD5) Previous issue date: 2013-03-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Charcoal rot caused by Macrophomina phaseolina is a disease that occurs in a large number of plant species and besides not being economically controlled by fungicides, no genetic resistance has been found. On the other hand, there is a growing awareness of the proper handling of pesticides or of the search for new and environmentally safer technologies for handling of plant diseases. Therefore this study aimed to select isolates of Trichoderma sp. for biological control of M. phaseolina and to evaluate their mechanisms of antagonism, as well as its effect as growth promoter of common bean. Seventeen isolates of Trichoderma sp. were used, in two in vitro tests: direct confrontation with the pathogen and production of volatile compounds. After the in vitro tests, six isolates that showed increased aggressiveness to the pathogen were selected and tested in vivo for effectiveness in controlling M. phaseolina and capacity of promoting common bean growth. The isolates TI1, TM3, TM4, TLB2, TLB3, TLB9, TLB15 and TLB17 showed antagonism against M. phaseolina in direct confrontation, and the isolates TM2, TLB2, TLB12, TLB15 and TLB17 reduced the mycelial growth of the pathogen in the volatile compounds test. Regarding microsclerotia of the pathogen, all the isolates were capable of reducing their production. The isolates TI1, TM3, TLB3, TLB12 and TLB17 were good growth promoters of common bean and the isolates TI1, TM3, TLB2, TLB3, TLB12 and TLB17 increased production per plant, especially the isolate TLB12. Regarding the protection of common bean against M. phaseolina, the isolates TLB2, TLB3, TLB12 and TLB17 were efficient. Fungi of the genus Trichoderma show effectiveness in controlling M. phaseolina and stimulate growth and increase the production of common bean / Podridão de carvão causada por Macrophomina phaseolina é uma doença que ocorre em muitas espécies de plantas e, além de não ser controlada economicamente por fungicidas, nenhuma resistência genética foi encontrada. Por outro lado, existe uma crescente conscientização no manejo correto do uso de defensivos agrícolas ou na busca por novas tecnologias de manejo de doenças de plantas ambientalmente mais seguros. Sendo assim, este trabalho teve como objetivo selecionar isolados de Trichoderma sp. para controle biológico de M. phaseolina e avaliar seus mecanismos de antagonismo, bem como, seu efeito como promotor de crescimento do feijoeiro. Foram utilizados dezessete isolados de Trichoderma sp., em dois testes in vitro: confronto direto com o patógeno e produção de compostos voláteis. Após os testes in vitro, seis isolados que apresentaram maior agressividade ao patógeno foram selecionados e testados in vivo quanto à eficiência no controle de M. phaseolina e capacidade de promoção do crescimento do feijoeiro. Os isolados TI1, TM3, TM4, TLB2, TLB3, TLB9, TLB15 e TLB17 apresentaram antagonismo contra M. phaseolina no confronto direto, sendo que os isolados TM2, TLB2, TLB12, TLB15 e TLB17 reduziram o crescimento micelial do patógeno no teste dos compostos voláteis. Em relação aos microescleródios do patógeno, todos os isolados foram capazes de reduzir sua produção. Os isolados TI1, TM3, TLB3, TLB12 e TLB17 foram bons promotores de crescimento do feijoeiro, e os isolados TI1, TM3, TLB2, TLB3, TLB12 e TLB17 aumentaram a produção de grãos por planta, com destaque para o isolado TLB12. Em relação à proteção do feijoeiro contra M. phaseolina, os isolados TLB2, TLB3, TLB12 e TLB17 foram eficientes. Fungos do gênero Trichoderma apresentam eficiência no controle de M. phaseolina e estimulam o crescimento e aumento da produção do feijoeiro

Apesar da espécie Vigna unguiculata (L.) Walp. ser bastante rústica e estar adaptada às condições adversas de clima e solo brasileiros, seu rendimento é muito baixo. Diversas causas têm sido levantadas para explicar esse comportamento; entre elas destacam-se as doenças fúngicas, sobretudo aquelas cujos patógenos são transmitidos por sementes, em especial a podridão cinzenta do caule, causada por Macrophomina phaseolina (Tassi) Goid. A abordagem analítica desse patossistema revelou alguns problemas emergentes. Entre eles, destacam-se: a) desconhecimento da qualidade sanitária das sementes de caupi, utilizadas para semeadura; b) desunifomidade na metodologia usada para detectar os patógenos presentes nas semente; c) dificuldade na esporulação do patógeno, máxime de alguns isolados reticentes em esporular em meios artificiais de cultivo, cujo comportamento dificulta os trabalhos de seleção de genótipos resistentes; d) carência de medidas de controle do patógeno, que empreguem práticas naturais, como uso de sementes sadias, de indutores de resistência e de cultivares resistentes, de fácil uso e passível de adoção por parte dos produtores. Na estruturação da matriz lógica do presente estudo, referidos problemas foram transformados em objetivos. Os trabalhos foram conduzidos no Departamento de Entomologia, Fitopatologia e Zoologia Agrícola da ESALQ/USP, em Piracicaba-SP. Os resultados indicaram o teste de sanidade de sementes de caupi empregando o método do papel de filtro com restrição hídrica utilizando NaCl a -0,8Mpa, como o mais adequado para detecção dos fungos presentes nas sementes de caupi, especialmente M. phaseolina. A análise sanitária das amostras de sementes originadas de vários estados brasileiros revelou que, em 62% das amostras analisadas, o fungo M. phaseolina estava presente, sendo as amostras originadas do estado da Paraíba, Piauí, Pará e Bahia as que apresentaram maiores níveis de incidência do patógeno. Os melhores níveis de esporulação do patógeno foram conseguidos com a combinação de sobreposição de discos de folhas de trigo ao meio BDA, com temperatura de 25oC. Quanto à identificação de indutores de resistência, capazes de controlar M. phaseolina, os resultados revelaram que o acibenzolar-S-metil (ASM) foi o mais eficiente, quando comparado com quitosana e com um produto silicatado derivado de rocha micronizada (PSiM), apresentando um controle residual por mais de 40 dias após a semeadura. A maior eficiência verificada pelo ASM ocorreu devido a sua capacidade de ativar mecanismos bioquímicos de defesa, configurando-se em efetivo ativador da resistência induzida nas plantas de caupi, por atuar na cinética de importantes enzimas relacionadas à defesa, como a fenilalanina amônia-liase, peroxidase e quitinase. Quanto à reação de cultivares de caupi à doença, foi possível verificar razoável nível de resistência de algumas cultivares, tendo sido consideradas resistentes Mulato, Guariba e Maratauã. / Notwithstanding the specie Vigna unguiculata (L.) Walp is sufficiently rustic and adapted to the adverse conditions of the Brazilian soil and climate, its improvement is very low. Many causes have been raised in order to explain such behavior; among them the fungal diseases stand out, over all those whose pathogens are transmitted by the seeds especially the charcoal rot disease caused by Macrophomina phaseolina (Tassi) Goid. The analytical approach of such pathosystem has revealed some emerging problems. Among them, it stands out: a) the ignorance of the sanitary quality of the cowpea seeds used for sowing; b) the non-uniformity in the used methodology in order to detect the pathogens, which are present in the seed; c) the difficult in pathogen sporulation, principally of some isolated reticent in forming spores in cultivation artificial environments whose behavior hampers the selection works of the resistant genotypes; d) lack of pathogen control measures, which utilize natural practices, such as the use of healthy seeds, resistance inducers and resistant cultivars of easy utilization and liable to adoption by the producers. In structuring the logical matrix of this study, such problems were transformed into objectives. The works were conducted at the Entomology, Phytopathology and Agricultural Zoology Departments of ESALQ/USP, in Piracicaba-SP. The results have pointed out the sanity test of the cowpea seeds through the method of filter paper with hydric restriction using NaCI - 0,8Mpa, as the most suitable for detecting the current fungus in cowpea seeds, especially M. phaseolina. The sanitary analysis of the seeds samples originated from several Brazilian states has revealed that in 63% of the analyzed samples, the fungus M. phaseolina was present, and the samples originated from the states of Paraíba, Piauí, Pará and Bahia were those that have presented higher incident levels of pathogen. The best levels of sporulation were obtained with the combination of the superposition of wheat leaves disks in the middle of BDA in 25ºC. As to the identification of the resistance inducers, capable of controlling the M. phaseolina, the results have revealed that the acinbezolar-S-methyl (ASM) was more efficient when compared to chitosan and with a silicate product originated from micronized rock (PsiM), presenting a residual control for more than 40 days after the sowing. The greatest efficiency ascertained by ASM has occurred due to its capacity of activate the defense biochemistries mechanisms, forming itself in an activator effect of the induced resistance in cowpea plants because it acts in the kinetic of important enzymes related to the defense, such as the phenylalanine ammonia-lyase, peroxidase and chitinase. As to the cowpea cultivars reaction to the disease, it was possible to ascertain a reasonable resistance level of some cultivars, and BR 14 Mulato, Guariba e Maratauã were considered as resistant.

Orientador: Adriana Zanin Kronka / Banca: Danila Comelis Bertolin / Banca: Marileia Regina Ferreira / Banca: Martha Maria Passador / Banca: Silvia Renata Siciliano Wilcken / Resumo: O feijoeiro comum (Phaseolus vulgaris L.) está sujeito à ocorrência de doenças, cujos agentes causais podem ser transportados e transmitidos via sementes, além de comprometer o potencial fisiológico destas. Dentre os patógenos que influenciam negativamente, se presentes nas sementes de feijão, está o fungo Macrophomina phaseolina (Tassi) Goid., que pode ser transmitido às plantas, causando a podridão cinzenta da haste do feijoeiro, uma doença com sérios impactos na cultura. Apesar do tratamento químico de sementes ser uma prática eficiente no controle de patógenos, o emprego de substâncias naturais com ação fungicida e de antagonistas de fitopatógenos surge como uma opção de manejo sustentável. Diante do exposto, o objetivo desta pesquisa foi avaliar o efeito do tratamento de sementes de feijão com óleos essenciais (Capítulo I) e antagonistas (Capítulo II) sobre a incidência do fungo M. phaseolina e o potencial fisiológico das sementes tratadas. No primeiro capítulo, inicialmente, investigou-se a ação in vitro dos óleos essenciais de cravo da índia, pimenta preta e gerânio sobre o crescimento micelial e a esporulação do fungo. Dois tratamentos testemunha foram incluídos: fungicida tiofanato metílico + fluazinam e BDA puro. Verificou-se inibição total do crescimento micelial pelos óleos essenciais de cravo da índia e pimenta preta, com ação semelhante ao fungicida. O óleo essencial de gerânio não apresentou efeito inibitório satisfatório sobre o fungo. Os óleos essenciais de c... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Common bean crop (Phaseolus vulgaris L.) is subject to diseases occurrence, whose causal agents can be transported and transmitted through seeds, besides compromising their physiological potential. Macrophomina phaseolina (Tassi) Goid. is one of the pathogens that negatively influences this potential, if present in common bean seeds. This fungus can be transmitted to the plants, causing charcoal rot of common bean, a disease with serious impacts on this crop. Although the chemical treatment of seeds is an efficient practice in the pathogens control, the use of natural substances with fungicidal action and antagonists of phytopathogens appears as a sustainable management option. This research aimed to evaluate the effect of common bean seeds treatment with essential oils (Chapter I) as well as antagonists (Chapter II) on the incidence of M. phaseolina and the physiological potential of the treated seeds. In the first chapter, the in vitro action of clove, black pepper and geranium essential oils on mycelial growth and fungal sporulation was investigated. Two control treatments were included: methyl thiophanate + fluazinam fungicide and pure potato-dextrose-agar (PDA). Total inhibition of mycelial growth was verified by clove and black pepper essential oils, which showed a fungicide-like action. The geranium essential oil had no satisfactory inhibitory effect on the fungus. Clove and black pepper essential oils were used in the common bean seeds treatment in order to determine their potential for M. phaseolina control and possible effects on the physiological quality of treated seeds. The essential oils treatments were compared with the methyl thiophanate + fluazinam fungicide one. Inoculated and uninoculated seeds were also evaluated. The essential oils reduced the fungus incidence in the seeds, however, clove essential oil caused a germination reduction. In spite of this, there was no negative ... / Doutor

Apesar da espécie Vigna unguiculata (L.) Walp. ser bastante rústica e estar adaptada às condições adversas de clima e solo brasileiros, seu rendimento é muito baixo. Diversas causas têm sido levantadas para explicar esse comportamento; entre elas destacam-se as doenças fúngicas, sobretudo aquelas cujos patógenos são transmitidos por sementes, em especial a podridão cinzenta do caule, causada por Macrophomina phaseolina (Tassi) Goid. A abordagem analítica desse patossistema revelou alguns problemas emergentes. Entre eles, destacam-se: a) desconhecimento da qualidade sanitária das sementes de caupi, utilizadas para semeadura; b) desunifomidade na metodologia usada para detectar os patógenos presentes nas semente; c) dificuldade na esporulação do patógeno, máxime de alguns isolados reticentes em esporular em meios artificiais de cultivo, cujo comportamento dificulta os trabalhos de seleção de genótipos resistentes; d) carência de medidas de controle do patógeno, que empreguem práticas naturais, como uso de sementes sadias, de indutores de resistência e de cultivares resistentes, de fácil uso e passível de adoção por parte dos produtores. Na estruturação da matriz lógica do presente estudo, referidos problemas foram transformados em objetivos. Os trabalhos foram conduzidos no Departamento de Entomologia, Fitopatologia e Zoologia Agrícola da ESALQ/USP, em Piracicaba-SP. Os resultados indicaram o teste de sanidade de sementes de caupi empregando o método do papel de filtro com restrição hídrica utilizando NaCl a –0,8Mpa, como o mais adequado para detecção dos fungos presentes nas sementes de caupi, especialmente M. phaseolina. A análise sanitária das amostras de sementes originadas de vários estados brasileiros revelou que, em 62% das amostras analisadas, o fungo M. phaseolina estava presente, sendo as amostras originadas do estado da Paraíba, Piauí, Pará e Bahia as que apresentaram maiores níveis de incidência do patógeno. Os melhores níveis de esporulação do patógeno foram conseguidos com a combinação de sobreposição de discos de folhas de trigo ao meio BDA, com temperatura de 25oC. Quanto à identificação de indutores de resistência, capazes de controlar M. phaseolina, os resultados revelaram que o acibenzolar-S-metil (ASM) foi o mais eficiente, quando comparado com quitosana e com um produto silicatado derivado de rocha micronizada (PSiM), apresentando um controle residual por mais de 40 dias após a semeadura. A maior eficiência verificada pelo ASM ocorreu devido a sua capacidade de ativar mecanismos bioquímicos de defesa, configurando-se em efetivo ativador da resistência induzida nas plantas de caupi, por atuar na cinética de importantes enzimas relacionadas à defesa, como a fenilalanina amônia-liase, peroxidase e quitinase. Quanto à reação de cultivares de caupi à doença, foi possível verificar razoável nível de resistência de algumas cultivares, tendo sido consideradas resistentes Mulato, Guariba e Maratauã. / Notwithstanding the specie Vigna unguiculata (L.) Walp is sufficiently rustic and adapted to the adverse conditions of the Brazilian soil and climate, its improvement is very low. Many causes have been raised in order to explain such behavior; among them the fungal diseases stand out, over all those whose pathogens are transmitted by the seeds especially the charcoal rot disease caused by Macrophomina phaseolina (Tassi) Goid. The analytical approach of such pathosystem has revealed some emerging problems. Among them, it stands out: a) the ignorance of the sanitary quality of the cowpea seeds used for sowing; b) the non-uniformity in the used methodology in order to detect the pathogens, which are present in the seed; c) the difficult in pathogen sporulation, principally of some isolated reticent in forming spores in cultivation artificial environments whose behavior hampers the selection works of the resistant genotypes; d) lack of pathogen control measures, which utilize natural practices, such as the use of healthy seeds, resistance inducers and resistant cultivars of easy utilization and liable to adoption by the producers. In structuring the logical matrix of this study, such problems were transformed into objectives. The works were conducted at the Entomology, Phytopathology and Agricultural Zoology Departments of ESALQ/USP, in Piracicaba-SP. The results have pointed out the sanity test of the cowpea seeds through the method of filter paper with hydric restriction using NaCI – 0,8Mpa, as the most suitable for detecting the current fungus in cowpea seeds, especially M. phaseolina. The sanitary analysis of the seeds samples originated from several Brazilian states has revealed that in 63% of the analyzed samples, the fungus M. phaseolina was present, and the samples originated from the states of Paraíba, Piauí, Pará and Bahia were those that have presented higher incident levels of pathogen. The best levels of sporulation were obtained with the combination of the superposition of wheat leaves disks in the middle of BDA in 25ºC. As to the identification of the resistance inducers, capable of controlling the M. phaseolina, the results have revealed that the acinbezolar-S-methyl (ASM) was more efficient when compared to chitosan and with a silicate product originated from micronized rock (PsiM), presenting a residual control for more than 40 days after the sowing. The greatest efficiency ascertained by ASM has occurred due to its capacity of activate the defense biochemistries mechanisms, forming itself in an activator effect of the induced resistance in cowpea plants because it acts in the kinetic of important enzymes related to the defense, such as the phenylalanine ammonia-lyase, peroxidase and chitinase. As to the cowpea cultivars reaction to the disease, it was possible to ascertain a reasonable resistance level of some cultivars, and BR 14 Mulato, Guariba e Maratauã were considered as resistant.