Legume seed inoculation technology—a review

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Abstract

Inoculation of legume seed is an efficient and convenient way of introducing effective rhizobia to soil and subsequently the rhizosphere of legumes. However, its full potential is yet to be realised. Following widespread crop failures, the manufacture of high quality inoculants revolutionised legume technology in Australia in the 1960s. Many improvements to inoculants and the advent of an inoculant control service ensured that quality was optimised and maintained. Minimum standards for the number of rhizobia per seed were set after consideration of several factors including seed size and loss of viability during inoculation. Despite manufacturers' recommendations for storage and application of inoculants, there is a distinct lack of control over the inoculation process; hence the full potential of high quality products may not always be achieved. The efficacy of inoculation varies depending on several factors, all of which affect the number of viable rhizobia available for infection of legume roots. Increased numbers of viable rhizobia per seed by application of inoculant above the commercially recommended rate, results in a continued linear increase in nodulation and yield. Several studies have reported yield increases of up to 25%. However, applying higher quantities of inoculant is uneconomical and technically difficult. Alternatively, higher numbers of viable rhizobia per seed may be achieved by improving survival during seed inoculation. Despite recognition of the factors affecting survival of rhizobia on seed and a substantial demand for commercially pre-inoculated legume seed, poor survival is still a major concern. Desiccation, temperature and seed coat toxicity all influence survival of rhizobia on seed. Their adverse effects may be ameliorated by selecting tolerant rhizobial strains and legume seed cultivars with low toxicity or artificially, by the use of additives in the seed coating. The accumulation of the desiccant protectant trehalose in strains of rhizobia, may result in better survival under desiccation stress. Similarly, the accumulation of exopolysaccharide (EPS) may act as a barrier reducing excessive water loss. Polymeric adhesives such as gum arabic, methyl cellulose and polyvinyl pyrollidone (PVP) have improved survival. However, studies of additives used in inoculation have been ad hoc and little of their mode of action is understood. A better understanding of the mechanisms involved in the protection of rhizobia from adverse conditions will assist in defining the optimum conditions for seed inoculation and storage to ensure a higher quality product for farmers at the time of sowing.

Introduction

We review the literature relating to legume inoculation technology and propose research areas to improve the results of legume seed inoculation. In 1887, Hellriegel and Wilfarth showed in their classical experiments that N2 fixation was associated with nodulated legume roots and was essentially the role of the infective agents of nodules. These agents varied in their ability to nodulate different groups of plants. One year later, Beijerinck isolated and described the root nodule bacteria and by 1896 the definition of the ‘cross inoculation groups’ was in practical use (Fred et al., 1932). Such groups of legumes (usually closely related taxonomically) are nodulated by the same species of Rhizobium. It became common knowledge that there could be a need to inoculate legumes that had not been grown in a particular soil or for some years. This was particularly the case in Australia, where the legumes cultivated for agriculture were all introduced (Davidson and Davidson, 1993). Scientists later recognised there was a much more specific relationship between bacterial strains and legume-hosts in terms of infectiveness (the ability to nodulate) and effectiveness (the ability to fix N2). To improve pasture establishment in Australia and to support an ever-growing grazing industry, research was initiated from the 1930s at several institutions to isolate effective strains and determine their relationships with specific hosts. The aim of inoculation is to provide sufficient numbers of viable effective rhizobia to induce rapid colonisation of the rhizosphere allowing nodulation to take place as soon as possible after germination and produce optimum yields (Thompson, 1988, Catroux, 1991).

Section snippets

Production and quality control of legume inoculants

Legume seed is commonly inoculated with peat cultures of rhizobia. Their commercial production in Australia began in 1953 using finely milled peat as the bacterial carrier (Davidson and Davidson, 1993). The quality of inoculants was improved, following widespread nodulation failures, by the amelioration of five main factors affecting survival in peat (Roughley and Vincent, 1967). Firstly, the origin of the peat was shown to be important. Survival of clover, lucerne and cowpea rhizobia varied

Evidence of poor survival on seed and its impact on legume yield

Death of all species of rhizobia on inoculated seed occurs rapidly, particularly when environmental conditions are unfavourable (Bowen and Kennedy, 1959, Marshall, 1964, Diatloff, 1967, Brockwell et al., 1987). Early in the 20th century, researchers recognised the problem of poor survival of rhizobia on legume seed, and its partial amelioration through low temperature storage and the use of additives (Fred et al., 1932). Inoculation techniques were usually assessed in terms of resulting

The production of compatible solutes or osmoprotectants

Under osmotic stress, a balance between internal and external water potentials can be reached if the cells accumulate compatible solutes or osmoprotectants. These include potassium ions, glutamate, glutamine, proline, quaternary amines (glycine betaine) and the sugars trehalose, sucrose and glucosylglycerol. Compatible solutes help maintain the stability of proteins during osmotic stress via a ‘preferential exclusion mechanism’. Here the solute is held at a finite distance from the protein

Sugars, amino acids and sugar alcohols

In early studies on the freeze-drying of bacteria, the nature of the suspending media was identified as an important aid to survival (Heller, 1941, Annear, 1956, Annear, 1962, Vincent, 1958). Extensive research has been carried out on the use of bacterial nutrients as suspending agents for freeze-drying and storage of cells (Heller, 1941, Appleman and Sears, 1944, Annear, 1956, Annear, 1962, Redway and Lapage, 1974, Dye, 1982). Heller (1941) investigated the protective effects of crystalline

Properties of polymers required to improve survival of rhizobia on seed

Certain properties of polymers can be identified as having a beneficial effect on survival. Polymers should logically be non-toxic and free from preservatives that may be harmful to bacteria. A complex chemical nature would be advantageous so faster-growing antagonists in the soil could not rapidly utilise the polymeric coating and out-compete the rhizobia. The polymer should also be dispersable in water to allow release of rhizobia from the polymer matrix upon wetting and their subsequent

Conclusions

The currently known methods of inoculation limit the benefits of high quality legume inoculants produced in Australia. Research undertaken to date has identified factors that affect survival of rhizobia on legume seed and observed improvements in survival when various additives were used. However, the interpretation of data from this research has been difficult and often speculative due to the complex nature of the coated seed environment and the disparate nature of the additives being

Acknowledgements

We gratefully acknowledge the support of the Grains Research and Development Corporation, Key Centre for Polymer Colloids (University of Sydney), Australian Research Council, Bio-Care Technology, Council of Grain Grower Organisations, Ballard Seeds and the Centre for Rhizobium Studies (Murdoch University).

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