The drop plate method as an alternative for Azospirillum spp. viable cell enumeration within the consensus protocol of the REDCAI network.

Quality evaluation of commercial inoculants is essential to warrant an adequate crop response to inoculation within a biosecurity framework. In this sense, this work is aimed at standardizing and validating the drop plate method for the enumeration of Azospirillum viable cells as an alternative to the spread plate technique, which is currently proposed in the consensus protocol of the REDCAI network. Between 14 and 25 private and public laboratories participated in three independent trials. We obtained consistent and robust results that allowed to confirm that both techniques are equivalent, concluding that the drop plate method is an alternative enumeration technique that is adequate to be included in the abovementioned consensus protocol.

Cytokinin production by Pseudomonas fluorescens G20-18 determines biocontrol activity against Pseudomonas syringae in Arabidopsis.

Plant beneficial microbes mediate biocontrol of diseases by interfering with pathogens or via strengthening the host. Although phytohormones, including cytokinins, are known to regulate plant development and physiology as well as plant immunity, their production by microorganisms has not been considered as a biocontrol mechanism. Here we identify the ability of Pseudomonas fluorescens G20-18 to efficiently control P. syringae infection in Arabidopsis, allowing maintenance of tissue integrity and ultimately biomass yield. Microbial cytokinin production was identified as a key determinant for this biocontrol effect on the hemibiotrophic bacterial pathogen. While cytokinin-deficient loss-of-function mutants of G20-18 exhibit impaired biocontrol, functional complementation with cytokinin biosynthetic genes restores cytokinin-mediated biocontrol, which is correlated with differential cytokinin levels in planta. Arabidopsis mutant analyses revealed the necessity of functional plant cytokinin perception and salicylic acid-dependent defence signalling for this biocontrol mechanism. These results demonstrate microbial cytokinin production as a novel microbe-based, hormone-mediated concept of biocontrol. This mechanism provides a basis to potentially develop novel, integrated plant protection strategies combining promotion of growth, a favourable physiological status and activation of fine-tuned direct defence and abiotic stress resilience.

Physiological and biochemical characterization of Azospirillum brasilense strains commonly used as plant growth-promoting rhizobacteria.

Azospirillum is a plant growth-promoting rhizobacteria (PGPR) genus vastly studied and utilized as agriculture inoculants. Isolation of new strains under different environmental conditions allows the access to the genetic diversity and improves the success of inoculation procedures. Historically, the isolation of this genus has been performed by the use of some traditional culture media. In this work we characterized the physiology and biochemistry of five different A. brasilense strains, commonly used as cereal inoculants. The aim of this work is to contribute to pose into revision some concepts concerning the most used protocols to isolate and characterize this bacterium. We characterized their growth in different traditional and non-traditional culture media, evaluated some PGPR mechanisms and characterized their profiles of fatty acid methyl esters and carbon-source utilization. This work shows, for the first time, differences in both profiles, and ACC deaminase activity of A. brasilense strains. Also, we show unexpected results obtained in some of the evaluated culture media. Results obtained here and an exhaustive knowledge revision revealed that it is not appropriate to conclude about bacterial species without analyzing several strains. Also, it is necessary to continue developing studies and laboratory techniques to improve the isolation and characterization protocols.

Azospirillum and arbuscular mycorrhizal colonization enhance rice growth and physiological traits under well-watered and drought conditions.

The response of rice plants to inoculation with an arbuscular mycorrhizal (AM) fungus, Azospirillum brasilense, or combination of both microorganisms, was assayed under well-watered or drought stress conditions. Water deficit treatment was imposed by reducing the amount of water added, but AM plants, with a significantly higher biomass, received the same amount of water as non-AM plants, with a poor biomass. Thus, the water stress treatment was more severe for AM plants than for non-AM plants. The results showed that AM colonization significantly enhanced rice growth under both water conditions, although the greatest rice development was reached in plants dually inoculated under well-watered conditions. Water level did not affect the efficiency of photosystem II, but both AM and A. brasilense inoculations increased this value. AM colonization increased stomatal conductance, particularly when associated with A. brasilense, which enhanced this parameter by 80% under drought conditions and by 35% under well-watered conditions as compared to single AM plants. Exposure of AM rice to drought stress decreased the high levels of glutathione that AM plants exhibited under well-watered conditions, while drought had no effect on the ascorbate content. The decrease of glutathione content in AM plants under drought stress conditions led to enhance lipid peroxidation. On the other hand, inoculation with the AM fungus itself increased ascorbate and proline as protective compounds to cope with the harmful effects of water limitation. Inoculation with A. brasilense also enhanced ascorbate accumulation, reaching a similar level as in AM plants. These results showed that, in spite of the fact that drought stress imposed by AM treatments was considerably more severe than non-AM treatments, rice plants benefited not only from the AM symbiosis but also from A. brasilense root colonization, regardless of the watering level. However, the beneficial effects of A. brasilense on most of the physiological and biochemical traits of rice plants were only clearly visible when the plants were mycorrhized. This microbial consortium was effective for rice plants as an acceptable and ecofriendly technology to improve plant performance and development.