PCR sequencing analysis to test the presence of the inoculants in the nodules
Creators: name: Tampakaki Anastasia P., Efstathiadou Evdoxia
Publisher: name: Zenodo
Year: 2018

description: <strong>Assessment of nodule occupancy (competitiveness)</strong> Nodule occupancy (by rhizobia), is an important characteristic when assessing the efficacy of specific rhizobia strain(s) applied to legume seeds or seedlings. Questions can be raised, and all concern the possibility that the strain which was applied did not nodule the legume in question. This possibility might arise due to various factors, such as the strain applied failed to: survive on the seed or root surface; or, compete with native strains already resident in the soil. Therefore, it is important that nitrogen fixing nodules of the host plant are assessed for the identity of the rhizobia which have occupied them. Identification of the strain or strains within nodules can be determined by isolation and molecular characterisation of the rhizobia from the nodules of the plant which arose from the inoculated seed. In this way, the approach aims to satisfy Koch’s Postulates, a set four criteria to test scientifically a causative relationship between a microbe and its function, which in this case is its capacity to form a symbiotic association. Standard approaches to characterise isolated rhizobia strains may rely on phenotypic, proteomic and genotyping. Examples of these methods include antibiotic resistance, ELIZA, MALDI-TOF, PCR-fingerprinting and all have limitations such as insufficient discrimination, cross-reactions, expense and or labour-intensiveness. A more specific approach for discriminating inoculant strains from those present in soil or used as competitors is the tagging of inoculant strains with marker genes. Marker genes encode fluorescent proteins or enzymes that cleave chromogenic substrates into coloured products following oxidation. Several marker genes have been used so far to prepare tagged bacteria (<em>e.g.</em> cat; celB; gusA; lacZ; gfp and derivatives, lux, nptII, and phoA). The introduction of marker genes into rhizobia can be performed either by mini-transposon mutagenesis or by plasmids. The main advantage of these approaches is the generation of stable tagged bacteria, which can be grown without antibiotic selection. However, they are laborious procedures unsuitable to use with many strains and which require prior knowledge of genome of the target organism. Bacteria tagged with different marker genes can be discriminated with relative ease and high certainty in co-inoculation or strain competition studies. However, due to EU and national legal restrictions the use of such genetically engineered bacteria in field studies is not allowed. In addition, there are questions regarding the possibility that such engineering incurs a metabolic cost to the engineer organism, and therefore any differences in its symbiotic performance may not reflect the true performance of that strain in field. Therefore, assessments of nodule occupancy are usually achieved with molecular tools using ‘finger-printing’ techniques such as ERIC-, BOX- or REP-PCR techniques whose products are resolved by gel-electrophoresis to generate specific banding patterns or “finger-prints”. Also, strains may be discriminated by ‘sequencing’ of specific gene fragments generate as PCR products from application of gene-specific primers. Here we describe the methods used to: carry out inoculation trials – to test the competitive efficiency of different rhizobia strains to infect a host plant in ex situ environment. how to isolate rhizobia from the nodules which are formed; how to test the nodules (or strains) which are isolated to identify which strain was more competitive i.e. occupied the nodule to the greatest (or sole) extent The DNA template for the PCR is prepared from pure rhizobial cultures according to the procedure described by Howieson and Dilworth, 2016.
description: The information presented here has been thoroughly researched and is believed to be accurate and correct. However, the authors cannot be held legally responsible for any errors. There are no warranties, expressed or implied, made with respect to the information provided. The authors will not be liable for any direct, indirect, special, incidental or consequential damages arising out of the use or inability to use the content of this publication.