A simple method to evaluate the number of bradyrhizobia on soybean seeds and its implication on inoculant quality control

Claudio Penna,Gabriel Gutkind,Florencia Olivieri,Rosana Massa,Fabricio Cassán

doi:10.1186/2191-0855-1-21

Claudio Penna, Gabriel Gutkind + Show 3 more

Open Access

https://doi.org/10.1186/2191-0855-1-21

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Abstract

Soybean seeds are non-sterile and their bacterial population interferes with the enumeration of beneficial bacteria, making it difficult to assess survival under different conditions. Within this context, the principal aims of this work were: (1) to improve a selective media for the enumeration of B. japonicum recovered from inoculated soybean seeds; (2) to establish the most representative mathematical function for B. japonicum mortality on soybean seeds after inoculation; (3) to evaluate if environmental or physiological conditions modify B. japonicum mortality on soybean seeds; and (4) to create a new protocol for quality control of soybean inoculants. We successfully evaluated the combination of pentachloronitrobenzene and vancomycin added to the yeast-mannitol medium to inhibit most fungi and Gram-positive soybean microbiota, thus producing reliable counts of B. japonicum from inoculated soybean seeds. Percentages of recovery and survival factors were obtained and used to construct a two-phase exponential decay non-linear regression function. High temperature and desiccation decreased these parameters, while the optimization of temperature and the use of osmoprotective compounds with inoculants increased them. The use of this protocol minimized heterogeneity between experiments and may be considered more reliable than the simple expression of direct colony count of bacteria recovered from seeds.

Highlights

Soybeans [Glycine max L. (Merr.)] are one of the major crops in grain-producing countries worldwide
Formulation and evaluation of the selective medium Based on the vancomycin minimal inhibitory concentrations obtained for all evaluated strains summarized in Table 1, it was decided to add 1 mg.l-1 of vancomycin to the P-Yeast Extract-Mannitol (YEM) medium
Considering the whole population of samples analyzed during the experiments, which were mostly obtained from different soybean cultivars and regions of Argentina, it was found that the PVYEM medium showed ability to prevent the growth of Gram-positive microbiota and fungi when compared with the YEM base media, and this fact improved the efficiency of bradyrhizobial count on soybean seeds

Summary

Introduction

Soybeans [Glycine max L. (Merr.)] are one of the major crops in grain-producing countries worldwide. (Merr.)] are one of the major crops in grain-producing countries worldwide. Improved crop productivity has been associated with different practices, such as the incorporation of agronomical, physical, chemical or biological technologies. Inoculation with symbiotic nitrogen-fixing Bradyrhizobiaceae bacteria has become a simple and effective way to significantly improve soybean. Inoculant improvement has been associated to more effective methods to evaluate the quality of bacterial formulations (inoculants) (Tong and Sadowsky, 1994), as well as those related with the isolation, identification and enumeration of bacteria in carriers, seeds, soil and plants. The enumeration of B. japonicum could be considered the most essential method used for defining the soybean inoculants quality, even before the evaluation of activity under experimental field conditions

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