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

Date Published: July 19, 2011

Publisher: Springer

Author(s): Claudio Penna, Rosana Massa, Florencia Olivieri, Gabriel Gutkind, Fabricio Cassán.


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.

Partial Text

Soybeans [Glycine max L. (Merr.)] are one of the major crops in grain-producing countries worldwide. Particularly in Argentina, production for 2010 has been estimated in about 50.0 million tons, which represents 7 percent less than the record of 54.0 million tons covering a production area of 18.0 million hectares (USDA, 2010). Improved crop productivity has been associated with different practices, such as the incorporation of agronomical, physical, chemical or biological technologies. The use of mineral fertilizers has been subordinated to biological fertilization, principally due to its economical and environmental advantages (Adesemoye and Kloepper, 2009). Inoculation with symbiotic nitrogen-fixing Bradyrhizobiaceae bacteria has become a simple and effective way to significantly improve soybean yield and productivity. Within the genus Bradyrhizobium, B. japonicum is the best example of successful symbiotic fixation under very large scale field conditions. Therefore, B. japonicum has been successfully incorporated as the active principle of soybean inoculants in Argentina, Brazil, Paraguay, USA, Canada and other producing countries worldwide in the last 30 years.

The inoculation of plants with beneficial bacteria can be traced back for centuries (Bashan, 1998). By the end of the 19th century, the practice of mixing “naturally inoculated” soil with seeds became a recommended method of legume inoculation in the USA (Smith, 1992). A decade later, the first patent for plant inoculation with Rhizobium sp was registered. (Nobbe and Hiltner, 1896). The practice of soybean inoculation with Bradyrhizobium sp. became common and economically recommended in many producer countries. In Argentina and many South American countries, soybeans [Glycine max (Merr.) L.] are commonly not fertilized but only inoculated with nitrogen. In 2010, Argentina, Bolivia, Paraguay and Uruguay produced more than 20 million hectares of soybeans, almost 16 million of which (more than 80%) were inoculated with products generated by more than 100 companies with common market.

MPN: most probable number; CFU: colony forming units; YEM: Yeast Extract-Mannitol; PCNB: pentachloronitrobenzene; MIC: minimal inhibitory concentration; RFP: recovery factor percentage; SFP: survival factor percentage; PGPB: plant growth promoting bacteria

The authors declare that they have no competing interests.




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