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.
Bacterial counts on non-selective media are a routine quality control procedure for defining a basic threshold in inoculant quality control. Those inoculants that do not fulfill the requested bacterial numbers are discarded in compliance with different regulations (country dependent). These counts are easily performed, even by small, non-specialized microbiology laboratories when inoculants only contain the desired rhizobial population, that is, when inoculants are formulated in a sterile carrier. However, most inoculants are finally applied on non-sterile material like seeds or even directly on the soil. Once there, rhizobial enumeration is a non-reproducible task, due to the presence of Gram-positive bacteria and fungi in variable numbers that may interfere with their direct growth, as in the case of fast growers, or by the synthesis and secretion of toxic compounds, which make comparisons among different formulations for on-seed survival very difficult to achieve, thus delaying better formulation developments.
Some selective agents have been previously used for selective enumeration of Rhizobium and Bradyrhizobium sp. from soils and non-sterile inoculants. Selective agents included antibiotics, heavy metals, dyes and metabolic inhibitors (Danso et al, 1973; Graham, 1969; Thompson, 1967; Van Schreven, 1970). Pattison and Skinner (1974) reported a formulation that contained pentachloronitrobenzene (PCNB), brilliant green (BG), sodium azide, crystal violet, and penicillin. Gómez et al. (1995) proposed two selective media for the enumeration of B. japonicum from soybean inoculants (at that time mostly using non-sterile peat as carrier) containing tetracycline, rifampicin and chloramphenicol to control bacteria and cycloheximide and pimafucin to control fungal contaminants. In our study, most fungal contamination could be prevented by using previously reported PCNB concentrations on the YEM base medium (P-YEM). However, the presence of large mucoid colonies of some gram-positive bacilli after one week incubation prevented in most cases the differentiation and enumeration of typical rhizobium colonies. Our use of the combined PV-YEM medium allowed a significant improvement in the efficiency of on-seed rhizobial enumeration as it effectively prevented the growth of Gram-positive bacteria and fungi (Figure 1).
As already stated, one of the major problems following soybean inoculation is fast bacterial death (Salema et al., 1982; Streeter, 2003). The seed storage temperature after inoculation is empirically considered the most important parameter related to rhizobial survival after seed treatment (Vriezen et al., 2005, 2006; Kremer and Peterson 1983). Moreover, temperature also directly affects the inoculated population desiccation rate generating a second stress condition (Streeter 2003 and 2007). Salinity and seed coat toxicity stress (Deaker et al., 2004) have a strong influence on bacterial survival; however, temperature and the consequent desiccation rate have been the major predicted cause of bacteria injury on seeds. Nevertheless, only few studies have involved the actual analysis of seed survival, perhaps due to contamination problems (Vincent et al., 1962; Bushby and Marshall, 1977; Jansen van Rensburg and Strijdom, 1980; Mary et al., 1986; Cliquet and Catroux, 2001, Kosanke et al., 1992; Vriezen et al., 2006). According to our results, seed storage after inoculation should be recommended at a temperature below 30°C and preferably below 20°C. We propose a survival factor percentage (SFP) defined as a single time parameter resulting from the number of bacteria recovered from seeds after the mortality function reached the plateau for decay, which in some way means when the survival bacterial population stabilized on seeds after inoculation. In our experiments, SFPs were defined 4 hours after inoculation. However, for research and development purposes, we suggest standardizing SFPs at 30°C, as these may reduce the time required for product (or product variation) analysis.
Any plant growth promoting bacteria (PGPB), including rhizobial survival on the seed surface, is usually lower than on solid carriers such as peat, vermiculite or alginate (Bashan et al., 2002) due to the lack of protection against desiccation, and/or toxic compounds present on the seed coat (Burton 1972, 1968). Even if an increase of provided B. japonicum cells improves the nodulation process, plant nitrogen assimilation and grain yield in laboratory conditions (Papakosta, 1992), bacterial physiological state and its resistance to environmental stress may also be critical for its survival on seed and field conditions (Streeter 2007). Roughley et al. (1993) reported a high mortality of Bradyrhizobium sp. after the inoculation of lupine seeds, decreasing by a factor of 10 after one hour, of one hundred after four hours and more than one thousand after one day on the seeds. Similar results were presented by Catroux et al. (1996) in B. japonicum inoculated soybean seeds. The microorganism population recovery from inoculated soybean seeds (as a whole) may be extremely heterogeneous when expressed as cfu.seed-1, depending on the two components, inoculants and seeds. In the case of inoculants, the major variation sources are the formulation, the presence of some osmoprotectant (e.g. sucrose or carboximethylcellulose), the number of viable microorganisms per volume unit and the recommended doses. In the case of seeds, normally the genotype, the size and the total surface of each grain, storage conditions, aw, integrity of teguments and mechanical damage can be easily mentioned. Due to all these complex variables, comparison of inoculation treatments (that is, a stated seed A with a stated inoculant A vs another seed B with an inoculant B combination) is frequently misleading.
Our use of relative values like RFPs or SFPs helps dissect the simplest questions, keeping others under the control state, and may even help compare experiments separated in time (Table 3, Figure 2).
The primary benefit of bacterial biological nitrogen (N2) fixation in grain and forage legumes is the enhancement of yield potentials decreasing the use of exogenous fertilizers. This ability had resulted in commercial legume inoculant production facilities worldwide (Brockwell and Bottomley, 1995). Unfortunately, the widespread availability of legume and other related inoculants had not resulted efficiently equal at level of development of quality control standards protocols. Many countries, including Australia, the Netherlands, Rwanda, Thailand and Russia (Smith, 1992; Marufu et al., 1995) have legislated requirements for minimum populations of target organisms and contaminants per weight unit of inoculants. Canada and France have legislated on product quality by defining the number of viable Rhizobium/Bradyrhizobium required (provided) per seed, or a rate of application, for a full range of crops. The United States and United Kingdom leave the issue on product quality and rates of application at the manufacturer's discretion (Smith, 1992). In Argentina, Brazil and other South American countries actual regulations are insufficient to put pressure on local companies to improve the quality of their products. The lack of internationally accepted regulations for legume inoculant quality and usage parameters has led, on many occasions to inadequate inoculant performance and to the subsequent abandonment of their use. To reverse this trend, a complementary method for soybean inoculant quality control was herein proposed. We improved a selective media for the enumeration of B. japonicum from inoculated soybean seeds, established the most representative mathematical function for B. japonicum mortality on soybean seeds after inoculation and generated Recovery and Survival Factor Percentages as novel tools for inoculant quality control.