RESUMEN
Inoculant plant-growth-promoting bacteria are emerging as an important component of sustainable agriculture. There is a need to develop inexpensive methods for enumerating these organisms after their application in the field, to better understand their survival and impacts on yields. Immunoblotting is one potential method to measure viable cells, but the high cost of the conventionally used nylon membranes makes this method prohibitive. In this study, less expensive alternative materials such as filter papers, glossy photo papers, and transparencies for the purpose of colony immunoblotting were evaluated and the best substance was chosen for further studies. Whatman filter paper No. 541 combined with a 0.01 mol·L(-1) H(2)SO(4) rinsing step gave similar results to nylon membranes but <20% of the overall cost of the original colony immunoblotting assay. The application of the modified immunoblot method was tested on nonsterile clay soil samples that were spiked with high numbers (>10(7) CFU·g(-1)) of the plant-growth-promoting bacteria Pseudomonas fluorescens , Azospirillum brasilense , or Rhizobium leguminosarum . The modified protocol allowed the identification and recovery of over 50% of the inoculated cells of all three strains, amidst a background of the native soil microflora. Subsequently, the survival of P. fluorescens was successfully monitored for several months after application to field-grown rice at Jerilderie, New South Wales, Australia, thus validating the procedure.
Asunto(s)
Immunoblotting/instrumentación , Plantas/microbiología , Microbiología del Suelo , Azospirillum brasilense/fisiología , Immunoblotting/economía , Nueva Gales del Sur , Nylons , Oryza/microbiología , Pseudomonas fluorescens/fisiología , Reproducibilidad de los Resultados , Rhizobium/fisiologíaRESUMEN
Nitrogen-fixing bacteria were isolated from the rhizosphere of different crops of Korea. A total of 16 isolates were selected and characterized. Thirteen of the isolates produced characteristics similar to those of the reference strains of Azospirillum, and the remaining 3 isolates were found to be Enterobacter spp. The isolates could be categorized into 3 groups based on their ARDRA patterns, and the first 2 groups comprised Azospirillum brasilense and Azospirillum lipoferum. The acetylene reduction activity (ARA) of these isolates was determined for free cultures and in association with wheat roots. There was no correlation between pure culture and plant-associated nitrogenase activity of the different strains. The isolates that showed higher nitrogenase activities in association with wheat roots in each group were selected and sequenced. Isolates of Azospirillum brasilense CW301, Azospirillum brasilense CW903, and Azospirillum lipoferum CW1503 were selected to study colonization in association with wheat roots. We observed higher expression of beta-galactosidase activity in A. brasilense strains than in A. lipoferum strains, which could be attributed to their higher population in association with wheat roots. All strains tested colonized and exhibited the strongest beta-galactosidase activity at the sites of lateral roots emergence.
Asunto(s)
Azospirillum/enzimología , Azospirillum/crecimiento & desarrollo , Productos Agrícolas/microbiología , Nitrogenasa/metabolismo , Raíces de Plantas/microbiología , Triticum/microbiología , Acetileno/metabolismo , Fusión Artificial Génica , Azospirillum/clasificación , Azospirillum/aislamiento & purificación , Técnicas de Tipificación Bacteriana , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Enterobacter/clasificación , Enterobacter/crecimiento & desarrollo , Enterobacter/aislamiento & purificación , Expresión Génica , Genes Reporteros , Corea (Geográfico) , Oxidación-Reducción , Análisis de Secuencia de ADN , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
This paper is an overview of a non-specific local early induced resistance (EIR) mechanism, distinct from the incompatible-specific hypersensitive reaction (HR). We have shown that the local induced resistance (LIR) described earlier is not a single and uniform response to pathogen infection, because an early (EIR) and a late form can be distinguished. EIR operates from 3-6 h post-inoculation (hpi) until about 20 hpi, and is inhibited by a short heat-shock or the eukaryotic protein synthesis inhibitor, cycloheximide. In contrast, LIR, which corresponds to the induced resistance forms discovered earlier, requires more time (about 24 h) and intensive illumination to develop, and is effective for a longer period. EIR develops parallel with HR and is sometimes able to prevent it when the induction time of HR is longer than the time required for the development of EIR. It seems that EIR inhibits the metabolism of bacteria and the activity of hrp genes which otherwise are required for the induction of HR. In a compatible host-pathogen relationship the effect of EIR fails to take place. The rapid development of EIR is greatly influenced by temperature and the physiological state of the plant. EIR activates the accumulation of hydrogen peroxide at the bacterial attachment, expressing new peroxidase isoenzymes in the initiated plant tissue. It seems that this is a native general local defence mechanism which can localise foreign organisms even at the penetration site.