Charcoal rot of soybean is an important disease affecting soybean. Charcoal rot is caused by the ubiquitous soilborne fungus Macrophomina phaseolina (Tassi) Goid. The fungus infects over 500 plant species. Although charcoal rot occurs primarily under drought-like conditions, it has also been reported in high-yield irrigated soybean environments. Symptoms of charcoal rot on soybean include wilting, stunting, and premature death. Management options to reduce charcoal rot-associated losses are limited. The objective of this research was to reduce M. phaseolina colonization of soybean by supplementing with secondary nutrients, specifically calcium and magnesium; therefore, reducing the concentration of the associated toxin, and determine if isolates of M. phaseolina from non-soybean hosts are pathogenic on soybean and rotational hosts. Between 2014 and 2016, non-irrigated, M. phaseolina-inoculated field and field replicate greenhouse experiments evaluated applications of 1,120 kg/ha of Ca and Mg alone and in combination at pre-plant, at-plant, and pre-plant followed by at-plant compared with an inoculated and non-inoculated. Disease ratings were visually assessed from roots collected at R3, R5, R7, and R8. Colony forming units (CFU) were used to quantify fungal colonization in root tissue. Gas chromatography mass spectrometry was used to quantify the concentration of botryodiplodin, a toxin previously associated with charcoal rot from root tissue. An application of calcium applied pre-plant provided the greatest numerical reduction, 39.7%, in toxin concentration and also reduced disease severity by 1.0% and CFUs by 15.4% when compared to the inoculated control in a moderately resistant cultivar. Although numerical benefits were observed, these data do not support applications of Ca and Mg as a charcoal rot management option. Numerical differences were observed between rotational hosts and isolates in pathogenicity studies. The greatest numerical reduction of 34% in soybean dry plant weight was observed with the corn isolate when compared to the non-inoculated control. Macrophomina isolates from corn had up to an 8% greater colonization in soybean than corn or cotton plants. Although no significance was observed between isolates with regard to dry plant weight and colonization, colonization occurred regardless of treatment; suggesting crop rotation should not be a stand-alone charcoal rot management option.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Thesis (doctoral)--Universität, Göttingen, 1999.

No description available.

Master of Science / Department of Plant Pathology / Christopher R. Little / Christopher R. Little / Charcoal rot, caused by Macrophomina phaseolina, is the most important soybean disease in Kansas. Several strategies have been recommended to control this disease including crop rotation, lower plant densities, biological control, plant resistance and tolerance, and fungicide application. However, those techniques have not been completely effective and the information concerning soil texture, irrigation and micronutrient fertility (particularly manganese) upon charcoal rot disease severity and the pathogen population is limited. The objective of this study was to determine key factors that affect the biology of M. phaseolina and charcoal rot processes under laboratory, greenhouse and field conditions. M. phaseolina microsclerotia were produced from PDA pure isolate and infested Japanese millet in the laboratory and characterized by different techniques such as serial dilutions in semi selective media with the aim to produce quality inoculum to reliably infect soybean seedling roots under greenhouse conditions; production of inoculum by infesting Japanese millet was the most efficient method. Root colonization and root infection of soybean seedlings was assessed through the use of M. phaseolina inoculum under controlled conditions in the greenhouse. Root infection by M. phaseolina and microsclerotia longevity in soil is determined by environmental factors such as soil moisture content, soil texture and source of inoculum. The objective of the greenhouse study was to determine the impact of these variables on seedling root infection at the V1 and V2 development stages. Artificial soils with different textures were infested; M. phaseolina microsclerotia and soybean seedlings were exposed to different soil moisture contents including pot saturation, pot (field) capacity, and permanent wilting point. Soil populations and levels of root colonization for the stages were assessed by estimating CFUs and root length. Results indicate that soil texture has a significant impact upon root morphology and root length. Root populations of M. phaseolina were significantly higher in sandy soil textures and lower in the fine-textured soils, suggesting an impact of soil water holding capacity in the root infection process. The effect of water stress on seedling root colonization by M. phaseolina indicates that early infection may be more important than previously thought. A field study was also conducted to determine the effect of the aforementioned variables in a 2-year field experiment conducted at two Kansas locations. Pathogen colonization was iii assessed by measuring colony-forming units (CFUs) from ground root tissue at R2-R4 (post-flowering/early pod development) and R8 (maturity) stages. Soil populations (pre-planting and post-harvest) of M. phaseolina, yield parameters, and plant characteristics were obtained. Results indicated that there are complex relationships between soil physiochemical properties (pH, NPK content, exchangeable cations, and organic matter) and soil texture (sand, soil, and clay composition), which may mitigate disease severity and pathogen levels in host tissue. Results also indicated that in natural M. phaseolina-infested soils, cropping history and soil texture play an important role in charcoal rot processes and influence the levels of pathogen soil populations, root colonization at maturity and, more importantly, soybean yield.

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

Resumo: Objetivou-se o ajuste de metodologia e seleção de genótipos de guandu para resistência a Macrophomina phaseolina a partir de material obtido pela Embrapa Pecuária Sudeste, e verificar o desenvolvimento micelial e esporulação do fungo em meios de cultura. O trabalho foi conduzido em casa de vegetação na UNESP/Jaboticabal no período de agosto de 2004 a dezembro de 2005. Para o ajuste de metodologia e seleção de genótipos resistentes ao fungo as sementes foram submetidas a escarificação com lixa d'água e inoculação artificial através do método de exposição das mesmas ao patógeno por diferentes períodos, que variaram de O a 72 horas. Foram avaliadas porcentagem de plantas sobreviventes e massa fresca. Já para o crescimento micelial e esporulação do fungo foi utilizado o método de sobreposição de discos de diferentes hospedeiros no meio de cultura. A escarificação das sementes contribuiu para a penetração do fungo nas mesmas o período de 24h de exposição das sementes ao fungo são suficientes para detectar diferenças no grau de resistência dos genótipos. Os genótipos mais resistentes são g167-97, g124-95, g27-94, g40-95, g154-95, g127-97 e g9m-97, e os mais suscetíveis são g48-95, g123-95, g8-95, g168-99 e g1m-95. A sobreposição de discos foliares de guandu em meio BDA e folha de papel de filtro em meio sojinha proporcionam um incremento na esporulação de M. phaseolina. / Abstract: This work had the objective of determining the best schedule for artificial inoculation and select pigeon pea genotypes resistant to Macrophomina phaseolina in material obtained by Embrapa Pecuária Sudeste, and verify the mycelial growth and sporulation of the fungi in middle of culture. The work were carried in greenhouse at the UNESP/Jaboticabal, from August 2004 to December 2005. For the methodology and selection adjustment of resistant genotypes to the fungi the seeds were submitted scarified with water sandpaper and artificial inoculation the seeds were the contact method to fungi for different periods, which varied from O to 72 hours. They were evaluated percentage of surviving plants and fresh mass. For the mycelial growth and sporulation of the fungi was used the superposition of disks method of different hosts in the middle of culture. The scarified of the seeds contributed for penetration of the fungi at the seeds; the period of 24h of contact of the seeds to the fungi enough to detect differences in the resistance degree ofthe genotypes. The genotypes g167-97, g124-95, 927-94, g40-95, g154-95, g127-97 and g9m-97 were found to be the most resistant and most susceptible were g48-95, g123-95, g8-95, g168-99 and g1m-95. The treatment with superposition of the leaf disks of pigeon pea in BDA and disks of filter paper in middle of soybean extract were the treatments that provided better sporulation levei in the conditions of that experiment were half. / Orientador: Rita de Cássia Panizzi / Coorientador: Rodolfo Godoy / Banca: Antonio de Goes / Banca: Patrícia Menezes Santos / Mestre

In November of 1984 and 1985, Euphorbia lathyris was planted into a field naturally infested with Macrophomina phaseolina located at the Campbell Avenue Farm in Tucson, Arizona. Plants without foliar symptoms and rhizosphere soil were sampled regularly from emergence until the following May or June. Soil rhizosphere populations ranged from 0.7-3.0 cfu/g soil in 1985 to 8.0-24.1 cfu/g soil in 1986, and did not change significantly over either growing season (P > 0.05). Both the incidence of disease and the number of infection sites per cm of root increased significantly (P < 0.05) over each growing season and were not related to rhizosphere soil populations of M. phaseolina (P > 0.05). The distribution of infection sites along the tap root over both growing seasons remained the same in that most were located in the top 0-7 cm of tap root. Infected E. lathyris without apparent symptoms were subjected to low-water and high-temperature stress treatments in growth chambers. Root infection was not found to be dependent upon any stress. Lesion development was significantly dependent upon the imposition of any stress treatment, and further root colonization was significantly dependent upon low-water stress (P < 0.05). M. phaseolina was consistently recovered from asymptomatic roots. A consistently lower leaf water potential was measured on infected E. lathyris than from non-infected controls when no stress treatment was applied. Polyclonal antisera made against hyphae and microsclerotia of M. phaseolina was not successful in detecting this pathogen in E. lathyris by I-ELISA. Antisera applied to fresh thin sections of infected plant tissue was effective in staining hyphae of M. phaseolina when used with a second antibody conjugated to fluorescence isothiocyanate or to an enzyme (to which a substrate was added to "stain" hyphae).

Resumo: O objetivo dessa pesquisa foi verificar a influência de Macrophomina phaseolina, Colletotrichum dematium f. truncata e Colletotrichum lindemuthianum na qualidade fisiológica de sementes de feijoeiro, em especial nos resultados do teste de condutividade elétrica. Foram utilizadas sementes das cultivares Carioca e FT Nobre. As sementes foram infectadas com os fungos em meio de cultura BDA sem e com restrição hídrica (acrescido de manitol a -1,0 MPa). Nos tratamentos testemunhas foram utilizados os mesmos meio de cultura, porém, sem a presença dos fungos. Para cada tratamento as sementes foram sobrepostas nos meios de cultura por 16 horas para M. phaseolina e por 48 horas para o caso de C. dematium f. truncata e C. lindemuthianum. Após secagem natural foram realizados testes de sanidade, germinação em areia e de vigor, como, índice de velocidade de emergência, peso da matéria seca da plântula, teste de frio, teste de envelhecimento acelerado e condutividade elétrica. Também foram realizadas análises na água de embebição das sementes no teste de condutividade elétrica, determinando-se as concentrações de potássio, cálcio e magnésio, assim como nos meios de cultura utilizados com e sem a sobreposição de sementes, para verificação da hipótese de consumo de nutrientes das sementes pelos fungos...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this research was to verify the influence of Macrophomina phaseolina, Colletotrichum dematium f. truncata and Colletotrichum lindemuthianum in the physiological quality of bean seeds, special in the results of electrical conductivity test. There were used seeds from Carioca and FT Nobre cultivars. The seeds were artificially inoculated with fungi M. phaseolina, C. dematium f. truncata and C. lindemuthianum in BDA medium culture with and without hydric restriction (it was added -1,0 MPa manitol). In the control treatment there was used the same medium culture without the presence of the fungi. For each treatment the seeds were placed on the medium culture for 16 hours for M. phaseolina and 48 hours for C. dematium f. truncata and C. lindemuthianum. After a nature dry, seeds were evaluated, by the blotter test, sand germination and vigor tests: speed of emergence index, seedlings dry weigh, cold, accelerated aging and electrical conductivity tests. Analyses from the imbibition water of the seeds in the electrical conductivity test, were also done a measuring of concentrations of potassium, calcium and magnesium, as well as in the medium culture used with and without the seeds, to verify the hypothesis of seeds nutrients consumption by the fungi...(Complete abstract, click electronic address below) / Orientadora: Rita de Cássia Panizzi / Coorientador: Nelson Moreira de Carvalho / Banca: Margarete Camargo / Banca: Juliana Altafin Galli / Mestre

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.