Preinoculation of seed is a convenient alternative method to inoculating seed on-farm. With preinoculation, a range of plant-growth and protection agents, polymer adhesives, colour pigments or dyes, and powder materials may be incorporated into an inoculant adhesive-slurry prior to seed coating. However, our recent point-of-sale surveys support findings of previous studies that survival of rhizobia on preinoculated seed is variable and can be poor. We focussed our research, both in the laboratory and at commercial facilities, on some of the factors that may contribute to poor survival of rhizobia on preinoculated seed. We found that rhizobial survival was affected by water quality; filtration improved cell survival but was not equal to distilled water. We also found that polymers affected cell survival differently for each rhizobial strain, and that slowing the desiccation rate reduced the cell rate of decline. Although fewer in cell number, older inoculant afforded more protection for survival of rhizobial cells. There is a need to test each ingredient and stage in the seed-coating process for compatibility to determine the best practices to promote rhizobial survival on seed. Failure to act on these factors prolongs the status quo of the findings from recent retail surveys.

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During 1999–2003, 293 samples of preinoculated and custom-inoculated lucerne, subterranean clover, white clover, red clover and miscellaneous species (mainly other clovers) were sourced from commercial outlets and assessed for numbers of rhizobia, seed pellet pH and toxicity, and nodulation in a ‘grow-out’ test. Average rhizobial counts were 8400/seed for preinoculated lucerne, 1380/seed for subterranean clover and <100/seed for white and red clovers and for the miscellaneous species. These counts compared poorly with the average counts of 35 100/seed, 13 800/seed and 10 000/seed for freshly-inoculated lucerne, subterranean clover and white clover, respectively. Thus, overall pass rates of the preinoculated seed were reasonable for lucerne (73%), marginal for subterranean clover (32%) and very low for white clover (3%), red clover (4%) and the miscellaneous species (0%). The ‘grow-out’ tests for nodulation were positively correlated with rhizobial numbers on seed, confirming the use of plate counting of rhizobia to assess quality of pre- and custom-inoculated seed. Many of the seed pellets were toxic to the 2 clover rhizobial strains tested, although the toxicity did not affect numbers of rhizobia on the seed. In light of these results and other data on rhizobial survival on seed, we suggest the current Australian standards for rhizobial numbers on pasture legume seed at the time of sale of 500/seed (very small-seeded legumes with seed numbers >750 000/kg) and 1000/seed (other larger-seeded species, seed numbers <750 000/kg) remain in place. We recommend shelf lives be restricted to 6 months for preinoculated lucerne and the annual medics, to 6 weeks for preinoculated subterranean clover, and to 2 weeks for white clover, red clover and other miscellaneous species. In the long-term, new products and procedures will hopefully enhance the numbers and survival of rhizobia on seed such that the needs of both manufacturers and customers are satisfied.

The effect of rice culture on changes in the number of a strain of soybean root-nodule bacteria, (Bradyrhizobium japonicum CB 1809), already established in the soil by growing inoculated soybean crops, was investigated in transitional red-brown earth soils at two sites in south-western New South Wales. At the first site, 5.5 years elapsed between the harvest of the last of four successive crops of soybean and the sowing of the next. In this period three crops of rice and one crop of triticale were sown and in the intervals between these crops, and after the crop of triticale, the land was fallowed. Before sowing the first rice crop, the number of Bradyrhizobium japonicum was 1.32 x 105 g-1 soil. The respective numbers of bradyrhizobia after the first, second and third rice crops were 4.52 × 104, 1.26 x 104 and 6.40 x 102 g-1 soil. In the following two years the population remained constant. Thus sufficient bradyrhizobia survived in soil to nodulate and allow N2-fixation by the succeeding soybean crop. At the second site, numbers of bradyrhizobia declined during a rice crop, but the decline was less than when the soil was fallowed (400-fold cf. 2200-fold). Multiplication of bradyrhizobia was rapid in the rhizosphere of soybean seedlings sown without inoculation in the rice bays. At 16 days after sowing, their numbers were not significantly different (p < 0.05) from those in plots where rice had not been sown. Nodulation of soybeans was greatest in plots where rice had not been grown, but yield and grain nitrogen were not significantly different (p < 0.05). Our results indicate that flooding soil has a deleterious effect on the survival of bradyrhizobia but, under the conditions of the experiments, sufficient B. japonicum strain CB 1809 survived to provide good nodulation after three crops of rice covering a total period of 5.5 years between crops of soybean.

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Following numerous reports of nodulation failures in pigeonpea [Cajanus cajan (L.) Millsp.] crops in New South Wales, a series of experiments was conducted in glasshouses and at 6 locations in the field. When inoculated seed was grown in moist vermiculite or in sand beds in the glasshouse, pigeonpea nodulated, and fixed N2, normally; but at 3 sites in the field, we could detect neither nodulation nor N2 fixation, despite adequate inoculation or a population of suitable rhizobia in the soil. At another site there was only sporadic occurrence of effective nodules. Nitrogen was fixed at 2 of the 3 field sites on acid soils, but at 1 site it appeared that nodulation was due to a naturally occurring population of soil rhizobia and not to the inoculant. When comparisons were made, pigeonpea was invariably inferior to symbiotically related legumes, cowpea and adzuki bean, in nodulation and N2 fixation. This inferiority was associated with substantially poorer rhizobial colonisation of pigeonpea rhizospheres. The experimental findings confirmed the anecdotal evidence that pigeonpea is an erratically nodulating grain legume on neutral and alkaline soils.

Extension of the current 12-month expiry of rhizobial inoculants in Australia to 18 months would have commercial benefits for the manufacturers and resellers. The dilemma, however, is that numbers of rhizobia in the inoculants decline over time and individual cells may lose efficacy. The research undertaken in this study shows the effect of lupin and chickpea inoculant age (i.e. 0, 6, 12, 15 and 18 months old) on numbers of rhizobia, rhizobial cell characteristics and efficacy. For the latter, assessments included colony size on plates, survival on inoculated beads, and infectiveness and effectiveness in field experiments at 3 sites. Assessment of commercially produced inoculants at the Australian Legume Inoculants Research Unit (ALIRU) laboratory indicated that, on average, chickpea and lupin inoculants had counts of about log10 9.6 when fresh, delivering >log10 6 rhizobia/seed. At 12 months, the average counts had fallen to log10 9.4, delivering slightly less than log10 6 rhizobia/seed. By 18 months, average counts were log10 9.3, delivering log10 5.9 rhizobia/seed. The lines of best fit indicated decline rates of 0.0005 log10 units/day. We found no evidence that the rhizobia in the older inoculants (i.e. >12 months old) had lost any ability to grow on nutrient agar, survive on inoculated beads, and nodulate and fix nitrogen with the host plant. While the chickpea and lupin inoculants produced currently in Australia are as efficacious after 18 months of storage at 4°C as they are when fresh, efficacy of other inoculant types may fall below acceptable levels at <12 months.