Applicability of the 16S-23S rDNA internal spacer for PCR detection of the phytostimulatory PGPR inoculant Azospirillum lipoferum CRT1 in field soil

Aims: To assess the applicability of the 16S-23S rDNA internal spacer regions (ISR) as targets for PCR detection of Azospirillum ssp. and the phytostimulatory plant growth-promoting rhizobacteria seed inoculant Azospirillum lipoferum CRT1 in soil. Methods and Results: Primer sets were designed after sequence analysis of the ISR of A. lipoferum CRT1 and Azospirillum brasilense Sp245. The primers fAZO/rAZO targeting the Azospirillum genus successfully yielded PCR amplicons (400-550 bp) from Azospirillum strains but also from certain non-Azospirillum strains in vitro, therefore they were not appropriate to monitor indigenous Azospirillum soil populations. The primers fCRT1/rCRT1 targeting A. lipoferum CRT1 generated a single 249-bp PCR product but could also amplify other strains from the same species. However, with DNA extracts from the rhizosphere of field-grown maize, both fAZO/rAZO and fCRT1/rCRT1 primer sets could be used to evidence strain CRT1 in inoculated plants by nested PCR, after a first ISR amplification with universal ribosomal primers. In soil, a 7-log dynamic range of detection (102-108 CFU g-1 soil) was obtained. Conclusions: The PCR primers targeting 16S-23S rDNA ISR sequences enabled detection of the inoculant A. lipoferum CRT1 in field soil. Significance and Impact of the Study: Convenient methods to monitor Azospirillum phytostimulators in the soil are lacking. The PCR protocols designed based on ISR sequences will be useful for detection of the crop inoculant A. lipoferum CRT1 under field conditions

Assessment of SCAR markers to design real-time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize

International audienceAims: To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real-time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP-154 and CFN-535 in the rhizosphere. Methods and Results: Primers were designed based on strain-specific SCAR markers and were screened for successful amplification of target strain and absence of cross-reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real-time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 · 103 (for UAP-154) and 4 · 104 CFU g)1 (for CFN-535) in the maize rhizosphere. Inoculant quantification was effective from 104 to 108 CFU g)1 soil. Conclusion: BOX-based SCAR markers were useful to find primers for strainspecific real-time PCR quantification of each A. brasilense inoculant in the maize rhizosphere. Significance and Impact of the Study: Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation-independent monitoring methods were lacking. The real-time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP-154 and CFN-535

The role of the antimicrobial compound 2, 4-diacetylphloroglucinol in the impact of biocontrol Pseudomonas fluorescens F113 on Azospirillum brasilense phytostimulators

International audiencePseudomonads producing the antimicrobial metabolite 2,4-diacetylphloroglucinol (Phl) can control soil-borne phytopathogens, but their impact on other plant-beneficial bacteria remains poorly documented. Here, the effects of synthetic Phl and Phl+ Pseudomonas fluorescens F113 on Azospirillum brasilense phytostimulators were investigated. Most A. brasilense strains were moderately sensitive to Phl. In vitro, Phl induced accumulation of carotenoids and poly-β-hydroxybutyrate-like granules, cytoplasmic membrane damage and growth inhibition in A. brasilense Cd. Experiments with P. fluorescens F113 and a Phl- mutant indicated that Phl production ability contributed to in vitro growth inhibition of A. brasilense Cd and Sp245. Under gnotobiotic conditions, each of the three strains, P. fluorescens F113 and A. brasilense Cd and Sp245, stimulated wheat growth. Co-inoculation of A. brasilense Sp245 and Pseudomonas resulted in the same level of phytostimulation as in single inoculations, whereas it abolished phytostimulation when A. brasilense Cd was used. Pseudomonas Phl production ability resulted in lower Azospirillum cell numbers per root system (based on colony counts) and restricted microscale root colonization of neighbouring Azospirillum cells (based on confocal microscopy), regardless of the A. brasilense strain used. Therefore, this work establishes that Phl+ pseudomonads have the potential to interfere with A. brasilense phytostimulators on roots and with their plant growth promotion capacity

Didactic Strategies Stop The Development Of Critical Thinking In University Students

La educación debe evolucionar para afrontar los retos que plantea el futuro, en la actualidad vivimos en un mundo globalizado que comparte grandes cantidades de información en tiempo real, el problema radica en que no toda la información es fiable, por consiguiente, es necesario generar estrategias efectivas en las aulas, buscando que el estudiante genere independencia de pensamiento para que desarrolle criterios propios de pensamiento crítico, lo anterior permite que el estudiante adquiera adquirir las destrezas para generar habilidades de pensamiento superior que lo lleven a la independencia intelectual.Education must evolve to meet the challenges of the future, we live in a globalized world that shares large amounts of information in real time, the problem is that not all information is reliable, therefore it is necessary to generate strategies Effective in the classroom, seeking the student to generate independence of thought to develop criteria critical thinking, the previous allows the student to acquire the skills to generate higher thinking skills that lead to intellectual independence

In Situ Localization and Strain-Specific Quantification of Azospirillum and Other Diazotrophic Plant Growth-Promoting Rhizobacteria Using Antibodies and Molecular Probes

A central issue in the understanding of the interaction and symbiotic function of diazotrophic bacteria with non-leguminous crop plants is detailed knowledge about the localization of the associated diazotrophic bacteria within the plant, their in situ activities in the plant-associated niches, and strain-specific quantification of inoculated bacteria. In addition to the colonization of rhizosphere soil and the rhizoplane, it has become apparent that an endophytic location of a diazotroph would provide it with a higher potential to interact more closely with the plant, particularly with respect to increasing the availability of carbon and energy nutrients derived from the plant, as well as the possibility, in return, of improving the transfer of bacterial-derived metabolites to the plant. Detailed localization of bacteria was successfully performed using fluorescence labeled ribosome-directed oligonucleotide probes in the fluorescence in situ hybridization (FISH) approach coupled to the use of confocal laser scanning microscopy (CLSM), and via immunolocalization with specific antibodies using transmission electron microscopy (TEM). Furthermore, the fate of inoculated bacteria could be traced by using specifically marked strains by applying the genes for the green or red fluorescent protein (GFP, RFP) and β-glucuronidase (GUS). Strain-specific quantification approaches for inoculants based on quantitative PCR using sequence characterized amplified regions (SCARs) and other genomic marker sequences have been developed and successfully applied. In this chapter major achievements and existing obstacles using these high resolution approaches to analyze bacteria in situ are presented together with some basic protocols