Recent advances in inoculant technology and prospects for the future

Soil Bid.

Pergamon

0038-0717(94)00097-2

RECENT

Biochem. Vol. 21, No. 415, pp. 683-697, 1995 Copyright 0 1995 ElsevierScienceLtd Printed in Great Britain. All rights reserved 003%0717/95 $9.50 + 0.00

ADVANCES IN INOCULANT TECHNOLOGY AND PROSPECTS FOR THE FUTURE J. BROCKWELL’*

and P. J. BOTTOMLEY’

‘CSIRO Division of Plant Industry, G.P.O. Box 1600, Canberra, ACT 2601, Australia and ZDepartment of Microbiology and Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331-3804, U.S.A. Summary-Although no accurate information is available, it seems clear that world-wide production of legume inoculant is static or in decline. This is a paradoxical situation in a world where improved strategies for augmenting biological N, fixation have the potential to contribute enormous returns in the quality and quantity of food for humans and domestic animals. Here we examine some of the reasons. Peat or related materials of biological origin have been used as carriers for legume inoculants for more than 90yr. Little progress has been made with alternative carriers that might enhance the numerical quality of inoculants. The traditional means of delivery of inoculant into the soil is by seed inoculation. There are now other methods of delivery which are both practical and ecologically sound. The most efficient strategy for many situations would be to breed or select promiscuous lines of legume with enhanced capacity to fix N with populations of rhizobia resident in the soil. This strategy would bypass the act of legume inoculation. These and related matters are discussed in the paper.

INTRODUCTION

Estimates of global N2 fixation are in the order of 175 million tonnes yr-’ (Burns and Hardy, 1975). Legume N2 fixation accounted for, perhaps, 40% of that figure. Since then estimates of global N2 fixation have been augmented because of higher levels attributed to marine fixation (Bunt, 1988). Values estimated for various legume crops and pasture plants are often impressive. Using a r5N technique, Bergersen et al. (1985) have calculated that a crop of soybeans fixed 234 kg N ha-‘. Values quoted by Burns and Hardy (1975) for other legumes include 208, 105-200 and 169 kg N ha-’ yr-r, respectively, for lucerne, clover and lupin. There are also reports of low rates of legume N, fixation in the field, frequently attributed to legume production under stressful conditions resulting in poor nodulation (numerous citations in texts and reviews from Fred et al., 1932 to Bottomley, 1992). Inadequate nodulation of legumes is not always manifest in poor yield because the plant compensates by increased uptake of soil N (Herridge et al., 1984). Such exploitation of the reserves of soil N is not sustainable. Plant and soil scientists have a responsibility to devise strategies for legume cultivation that optimize N, fixation, conserve soil N and, indeed, augment the pool of soil N for the benefit of rotational non-leguminous crops. For soybeans, this objective is within reach. Data presented by Brockwell et al. (1985) suggest that optimum nodulation *Author for correspondence.

and N, fixation are functions of early colonization of the plant rhizospheres by Bradyrhizobium japonicum. Other information obtained in our laboratories indicates that the extent of rhizosphere colonization and, therefore, N, fixation can be increased by method of inoculation (e.g. Brockwell et al., 1989) manipulation of rhizobia (Almendras and Bottomley, 1987, 1988; Brockwell et al., 1987), soil nitrate (Herridge et al., 1984) soil moisture (Brockwell et al., 1988b), and by choice of soybean cultivar (Brockwell et al., 1988a). There is reason for confidence that, by using species with the abilities to nodulate vigorously and to fix N efficiently combined with simple, sensible agronomic strategies for cultivation of grain legumes, it is feasible to grow crops that are both highly productive of seed and contributory to reserves of soil N. In this review we discuss inoculant technology and how inoculants and inoculant delivery into the soil might be enhanced to optimize persistence in soils. In addition, naturally occurring populations of rhizobia are considered in relation to their inherent value as “natural inocula” for legumes and to their effects on the introduction of new strains. INOCULATION

Used judiciously where needed and performed properly, legume inoculation is a significant agency for improving crop productivity and soil fertility. The need for inoculation

Although rhizobia (Rhizobium, Bradyrhizobium, Azorhizobium, etc.) are as widely distributed as 683

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legumes themselves, there are many soils strains suitable for an introduced legume soils where rhizobia are few or absent. In the question “Is it necessary to inoculate?” Allen (1961) listed four indicators which the use of inoculant preparations:

devoid of and some answer to Allen and warranted

?? the

absence of the same or a symbiotically related legume in the immediate past history of the land ?? poor nodulation when the same crop was grown on the land previously ?? when the legume follows a non-leguminous crop in a rotation ?? in land reclamation undertakings.

Roughley and Brockwell (1987), with a mandate restricted to grain legumes, placed emphasis on previous land management and responded to the question with queries of their own: how specific is the legume in its rhizobial requirements? what is the likelihood of effective rhizobia spreading from volunteer legumes? has the legume been sown before and for how many seasons was it grown continuously? how long since it was last sown and, in the interim, were conditions likely to favour survival of the rhizobia? Inoculation, of course, is not required if sufficient effective rhizobia are already present in the soil. In the absence of simple indicators of the need for inoculation, many farmers inoculate as a form of insurance. Otherwise, recourse is necessary to an experimental approach combining agronomic and microbiological expertise. Appropriate field experiments (Bell and Nutman, 1971; Brockwell, 1971; Date, 1977) can provide an accurate prognosis of the need to inoculate and a detailed diagnosis of the reasons for failure of inoculation, nodulation or Nz fixation (Date, 1982). However, field experiments take at least several months to complete. An expeditious (28 days) microbiological assay for estimating the NJ-fixing capacity of rhizobia resident in soil was presented by Brockwell et al. (1988a). When combined with a serial-dilution, plant-infection technique for counting rhizobia (Brockwell, 1963; Vincent, 1970), this assay is a reliable guide to the need for inoculation in the field. A related procedure (Thies et al., 1991) makes it possible to predict the likely success of introducing inoculant rhizobia into the soil by considering indices of the size of resident rhizobial population and of the N status of the soil. Inoculant technology Vincent (1970), Burton (1976, 1982) Date and Roughley (1977) Brockwell (1977) and Thompson (1980) are major texts on the principles and practice of inoculant preparation and use. In one area of inoculant technology, these contributions present

important differences of approach. Burton’s methods involve the use of non-sterile peat carriers for preparation of inoculants. On the other hand, both Date and Roughley and Thompson indicate a strong preference for a sterile carrier. Indeed, Date and Roughley (1977) state that inoculants prepared with non-sterile peat may contain IOO-fold fewer rhizobia than those made with sterilized peat and that, because mortality of rhizobia increased in unsterilized peat, the difference increases during storage. Production Commercial production of legume inoculants commenced in 1895 in the U.S.A. and the U.K. In 1993 they are produced in many countries on all continents. Most are prepared in powdered, organic carriers such as peat which remains the favoured base particularly when rendered sterile by y-irradiation (Roughley and Vincent, 1967). Nevertheless, the search for alternative carrier materials continues particularly in regions without natural deposits of peat. Dommergues et al. (1979) reported that an inoculant, in which B. japonicum was entrapped in polyacrylamide gel, survived and nodulated soybeans as well as a peat-based inoculant. The concept has been extended to the use of other polymers (Jung et al., 1982). Good survival depends upon the maintenance of moisture in the gels. Jawson et al. (I 989) used several cellulose gels as inoculant carriers and reported excellent survival of B. japonicum and good nodulation of soybeans in sand culture and in the field where there was a resident population of competing strains. Inoculation of seed with fluid gels lends itself to fluid drilling for sowing grain legumes. Kremer and Peterson (1982) prepared inoculants, by resuspending lyophilized cultures of rhizobia in vegetable oil, that survived on seed as well as or better than peat cultures and performed well in field trials (Kremer and Peterson, 1983a). Hoben et al. (1991) reported favourably on oils as adhesives for seed inoculation. Thompson (1980) presented an imposing list of inoculant carriers that have been considered potentially useful and that list has been augmented since (e.g. Crawford and Berryhill, 1983; Sparrow and Ham, 1983; Chao and Alexander, 1984; Philip and Jauhri, 1984; El Shafie and El Hussein, 1991; Figueiredo et al., 1992). Thompson (1983) Williams (1984) and Smith (1992) have given practical guides to production, quality control and use of legume inoculants. Usually, inoculants are prepared by adding fermenter-grown broth containing a large population of rhizobia to powdered carrier followed by a period of incubation (e.g. Roughley and Pulsford, 1982). Somasegaran and Halliday (1982) and Somasegaran (I 985) considered this practice unnecessary; populations of rhizobia that developed in their peat cultures reached similarly high levels within 7 days of inoculation with broths of rhizobia varying between I x IO’ and I x IO* bacteria ml-‘. Standard

Inoculant technology broths contain growth factors, usually supplied as yeast extract, a C source and minerals. However, Bissonette et al. (1986) successfully used unsupplemented whey for growing R. meliloti. Graham-Weiss et al. (1987) proposed direct fermentation on nutrient-supplemented vermiculite for production of bacterial inoculants. An Australian company has recently been experimenting with the production of broth inoculant. Although details of the process are “commercial in confidence”, it is believed that a sterile fermentation liquor is used as a nutrient broth for fermenter cultivation of rhizobia to a population of > 1 x lo9 cells ml-‘. An analogous system has recently been described by Lie et al. (1992). This broth, usually packaged in dispenser bottles, can be used as a seed inoculant or for delivery directly into the seed bed. Preliminary experiments indicate that this form of broth inoculant has good storage characteristics in the bottle or on the seed, adheres tenaciously to the seed coat without the need for adhesive, can be delivered into the seed bed with spray inoculation equipment, and gives rise to nodulation and N, fixation as good as can be obtained with peat inoculant. The process appears more apt for slow-growing rhizobia such as B. japonicum and Bradyrhizobium sp. (Lupinus) than for fast growers, but the reason for this is not known. Application

of inoculant

A prime aim of legume inoculation is to maximize survival of inoculant during the period between its introduction to the soil and the development of a legume rhizosphere which it can colonize. The literature attests to the significance of high rates of inoculation in achieving this objective and optimizing subsequent nodulation and N, fixation (e.g. Weaver and Frederick, 1974; Smith et al., 1980; Nambiar et al., 1983; La Favre and Eaglesham, 1984; Lowther and Littlejohn, 1984; Bergersen et al., 1985; Wedderburn, 1986; Brockwell et al., 1987; Berg et al., 1988; Somasegaran et al., 1988). Application of inoculant to the seed surface prior to sowing is the traditional, most commonly used and easiest means of inoculation, although viability of the rhizobia is subject to the hazards of drying (Salema et al., 1982), fertilizer contact (Kremer et al., 1982), seed coat toxicity (Materon and Weaver, 1984), incompatible pesticidal and mineral additives (Skipper et al., 1980; Gault and Brockwell, 1980) and inimical soil factors (Mahler and Wollum, 1982; Kremer and Peterson, 1983b). Proposals to extend the life expectancy of rhizobia on seed including curing (storing at 20-27’C for up to 4 weeks) inoculants before use (Burton, 1976; Materon and Weaver, 1985) and suspending cultures in alginate gel rather than sucrose before application to seed (Rawsthorne and Summerfield, 1984) have not been adopted by the industry. There are numerous adhesives suitable for attaching inoculant to seed (e.g. Brockwell, 1962; Elegba and Rennie, 1984; Hoben

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et al., 1991). Tenacity is an important characteristic of adhesives to ensure that inoculant is not lost from the seed during handling and passage through sowing machinery. Naturally, inoculant adhesive must be free from any preservative that might diminish the viability of rhizobia. Preinoculation of legume seed, i.e. inoculation with rhizobia before sale, excited the interest of inoculant manufacturers, seed merchants and research microbiologists in the 195Os, 1960s and 1970s (Thompson et al., 1975). Inoculation in conjunction with seed coating and impregnation of seed with broth inoculant were usual processes. Generally the product was disappointing (e.g. Quackenbush et al., 1961; Brockwell et al., 1975; Schall et al., 1975) because of the poor survival of rhizobia. A variant of preinoculation is “custom inoculation”, i.e. inoculation of seed for sowing within, at most, 10 days of treatment. The numerical quality of this product was sometimes erratic (Brockwell and Roughley, 1967) but it has found favor with many farmers, especially in New Zealand. Although it seemed to attract little attention in 1993, reliable preinoculation has a great deal to offer and remains a tantalizing concept. There are some situations where seed application of rhizobia may be an inefficient means of inoculation, e.g. with seed dressed with pesticide incompatible with rhizobia; for inoculation for broad-acre sowing of crop legumes with high seeding rates; for seeds such as peanuts which are too fragile for seed-surface inoculation (Brockwell, 1982). Preparations and procedures for inoculant application directly into the seed bed are now in vogue, viz. solid inoculant (“soil implant”) (Scudder, 1975; Barkdoll et al., 1983; Hegde and Brahmaprakash, 1992) and liquid inoculant (Schiffman and Alper, 1968; Hely et al., 1980). These methods are often better and never worse than conventional seed inoculation for initiating nodulation and N2 fixation (e.g. Brockwell et al., 1980; Muldoon et al., 1980; Hale, 1981; Chamber, 1983; Jensen, 1987; Danso et al., 1990a; Rice and Olsen, 1992). Solid inoculant may be introduced into the seed bed through an insecticide attachment to the seed drill. Liquid inoculation requires an inoculant tank, a pump, a manifold, and capillary tubes to deliver the liquid into the seed bed beside and beneath the seed (Brockwell, 1982). Liquid inoculation equipment is inexpensive to fabricate and the method is widely used in Australian agriculture and also in the U.S.A. Australian operators prepare liquid inoculant as a suspension of peat culture in water. Frozen concentrated broth cultures have been used in the U.S.A. (Gault, 1980). Liquid inoculant prepared from peat must be free of fibre which could block nozzles, and mineral grit which might damage pumps. Inoculant has been successfully introduced into rhizobia-free soil under irrigation-water-run inoculation (Ciafardini and Barbieri, 1987). Gault et al. (1994) examined the feasibility of using water-run inoculation for furrow-irrigated soybeans. Successful

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nodulation and N, fixation appeared to depend on the infiltration characteristics of the soil. Inclusion of the inoculant with furrow irrigation was inferior to conventional methods on a poorly-structured redbrown earth where rhizobia had to percolate laterally a distance of ca. 18cm to reach the seed. On the other hand, distribution of inoculant via flood irrigation on a gray clay where water needed to infiltrate vertically only 5 cm to sowing depth, induced an effective symbiosis. Post-emergence inoculation of un-nodulated legume stands is sometimes effective (Rogers ef al.. 1982; Boonkerd et al.. 1984; Atkin-Smith et al., 1986: Danso et al., 1990a, b) and sometimes not (Gault et al., 1984). Success depends on environmental conditions at the time of treatment. Legume inoculant retained its viability in hydroseeding mixes (fertilizer. lime, mulch, seed, water), used for revegetation of overburden from surface mining, provided the blend had a pH > 6.0 (Brown et al., 1983). Smith (I 992) has reviewed many aspects of inoculant application and delivery. He saw as a challenge for the future, the provision of improved inoculant carriers that supported high populations of rhizobia, of inoculants with extended shelf-like which gave the rhizobia protection against environmental stresses. and of systems which delivered inoculant to the seed or into the soil in a convenient and cost-effective manner. He makes no mention of preinoculation. hoculant

qualit?

control

Evaluation of inoculant quality by enumeration of viable rhizobia is an accurate index of inoculating potential (Hiltbold et al., 1980). Numerical considerations are of such significance in determining quality of inoculant and inoculant products and their success in the field that the necessity for quality control systems, exemplified by Roughley et al. (1983), is widely recognized. Some countries (e.g. Canada, Uruguay) have regulatory authorities supported by appropriate legislation; in others (e.g. Australia. India, New Zealand, South Africa) inoculant manufacturers participate voluntarily in quality control schemes (Thompson, 1983). In the U.S.A., regulatory control has not been considered necessary since the 1940s. However, the results of independent tests published from time to time (e.g. Schall et al.. 1975; Skipper ef al., 1980; Vincent and Smith, 1982; Olsen et al., 1995) indicate that substantial proportions of the inoculants examined appear unsatisfactory for farmer use because of low populations of rhizobia or high numbers of microbial contaminants. Despite nearly 100yr of experience, it is unfortunately true that most of the inoculant produced in the world today is of relatively poor quality (e.g. Olsen et nl., 1995) and that. frankly, some of it is extremely bad. Even good quality inoculants are often not used to the best advantage. We venture to say that 90% of all inoculant has no practical effect whatsoever on the productivity of the legumes for which it is used or on

the conservation of N in the soils in which they are grown. Of course, some good inoculants are produced and some of those are used properly in situations where they are needed for N, fixation and the conservation of soil N. In these circumstances, legume inoculation may be the most cost-effective of all agricultural practices. Too often, unfortunately, the reverse is true and many farmers, even scientists, in the developed and developing worlds, see little value in the practice of inoculation. We are pessimistic about the prospects for the inoculant industry and its capacity for large-scale production of high quality inoculants. Existing inoculants of highest quality tend to be those produced by small factories under the umbrella of a quality control authority. Within this context, Hoben and Somasegaran (1992) and Somasegaran et al. (1992) have described smalland medium-scale fermenters for production of legume inoculants. Measurement

of success

Major criteria for evaluating success of inoculation are extent of nodulation, proportion of nodules occupied by the inoculant strain, and indices of plant response which may include the total amount of N fixed, the proportion of plant N due to N, fixation, and dry matter production. These subjects are dealt with comprehensively in Vincent (1970), Bergersen (1980) Somasegaran and Hoben (1985) and Peoples et al. (1989). Suffice to say that even the most symbiotically promiscuous legume (e.g. mungbean) growing in a soil which contains a large population of suitable rhizobia will always respond to inoculation provided that the rate of application is sufficiently high. The response may only be transitory (an improvement in early nodulation) and of no economic consequence, and the rate of inoculation needed to obtain that response may be absurdly high. There is, of course, no need for inoculation in these circumstances. Schemes for visual rating the extent of nodulation (e.g. Corbin et al., 1977; Sykes et al., 1988) appear statistically sound and are less onerous than counting nodules or measuring nodule mass. Immunological and drug-resistance procedures remain reliable means for evaluating nodule occupancy but there is an increasing use of molecular techniques (Welsh and McClelland, 1990; Williams et nl., 1990; Harrison et al., 1992) which can be simple, expeditious and accurate (Richardson et al., 1995). By and large, most strain identification markers remain stable in soil for several years at least (Lindstrom et a/., 1990). Plant response as N fixed is now routinely measured using lSN techniques (Shearer and Kohl, 1986; Ledgard and Peoples, 1988). Using natural abundance of “N, Bergersen et al. (1985) and Doughton et a/. (1993) demonstrated substantial accretion of fixed N in the soil following cropping with properly-inoculated soybeans and chickpeas. The relative abundance of the products of N? fixation, as ureides (McClure et a/.,

Inoculant technology 1980; Herridge, 1984; Streeter, 1985) or as amides (Peoples et al., 1987), is regularly used to provide a reliable measure of the N,-fixing status of leguminous grain crops.

MANIPULATION

OF RHIZOBIAL POPULATIONS

Naturally-occurring populations are significant factors determining the establishment of inoculant strains in the field. It is germane to consider, therefore, how rhizobial populations in soil can be manipulated to influence, with or without effectual inoculation, legume nodulation, N, fixation and productivity. New populations The root-nodule bacteria are very widespread as a result of the natural distribution of the legumes and the cultivation of leguminous crops and pastures. However, there are many soils still devoid of strains of rhizobia for particular crops; e.g. soybean rhizobia do not occur naturally in Australia (Diatloff and Brockwell, 1976). Establishment of new populations in these situations is achievable with relative ease. The essential ecological niche is provided by growing a host legume and the bacteria are supplied by inoculation. Provided that edaphic conditions are suitable for the healthy growth of the host and that sufficient of the inoculant survives until a rhizosphere is available for colonization, infection of the root and noduIation will occur as a matter of course. The nodule itself represents an environment akin to pure culture and, within it, great multiplication of the rhizobia occurs. When, subsequently, nodule break-down takes place, large numbers of viable cells are released into the soil (e.g. KuykendaII et af., 1982) where they constitute a potent source of infection for subsequent crops and usually become a persistent component of the soil microflora. Naturally-occurring

populations

Most naturalized populations of a species of rhizobia contain a number of components. For instance, several serogroups, characterized by the use of surface somatic antigens, are known to exist within naturalized populations of B. juponicum in the U.S.A. (e.g. Damirgi et nl., 1967; Keyser et al., 1984; Kamicker and Brill, 1986). Those serogroups can be sub-divided into serotypes (e.g. Gibson et al., 1971; Schmidt et al., 1986). Still further differentiation can be obtained using growth response to antibiotics and energy sources and especially with SDS-PAGE (protein profile developed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis) (Dughri and Bottomley, 1984; Demezas and Bottomley, 1984; Hickey et al., 1987) with RFLP (restriction fragment length polymorphism) (Young and Wexler, 1988; Demezas et al., 1991) or with PCR (polymerase chain

681

reaction) techniques (Richardson et al., 1995). Likewise, mixed populations of other species commonly occur: R. meliloti differentiated by intrinsic antibiotic resistance (Jenkins and Bottomley, 1985), by phage sensitivity (Thurman and Bromfield, 1988), and by N,-fixing capacity (Lowther et al., 1987); R. leguminosarum bv. trifolii by SDS-PAGE (Demezas and Bottomley, 1984) and serologically (Dughri and Bottomley, 1984); R. leguminosarum bv. viciae detected by serotyping (Mahler and Bezdicek, 1980). Several factors have been defined as determinants of the field distribution of the various components of mixed rhizobial populations: the host (Dughri and Bottomley, 1984; Thurman and Bromfield, 1988; Cregan and Keyser, 1988); soil type (Ham et al., 1971); acidity (Dughri and Bottomley, 1983); applications of lime and phosphate (Dughri and BottomIey, 1983; Almendras and Bottomley, 1987, 1988). Demezas and Bottomley (1987) warn that results from experiments in axenic environments may not mimic those obtained in the field. In addition, the composition of naturalized populations of rhizobia in the field may vary over very short distances (Bromfield et al., 1986). The characteristics of root-nodule bacteria of most practical significance are those which lead to effective N, fixation. The various components of mixed populations of rhizobia frequently express different levels of effectiveness. Soil acidity appears to be an important determinant of N,-fixing capacity in field populations of R. leguminosarum bv. trifolii, ineffectiveness being more common in soil of low pH (e.g. Jones and Burrows, 1969; Brockwell and Roughley, 1984). Considerable variation exists in N,-fixing capacity within field populations of R. meliloti (e.g. Bottomley and Jenkins, 1983) and R. leguminosarum bv. trifolii (e.g. Hagedorn et al., 1983). Sometimes, sharp differences in the level of effectiveness may occur between field sampling sites separated by as little as 10 cm (Gibson et al., 1975). In this context, Wollum and Cassel (1984) caution against sampling regimes biased towards one particular area of a field. This type of diversity in N,-fixing capacity has the advantage that different rhizobial requirements of different legumes may be satisfied by different components of the same population (e.g. Robinson, 1969; Brockwell and Katznelson, 1976; Bromfield, 1984). Great variation also occurs in the numerical size of field populations of rhizobia. The presence, continuing or periodic, of an appropriate host is a major determinant (Hiltbold et al., 1985; Rupela et al., 1987) but soil acidity (Rice et al., 1977), seasonal effects and depth of sampling (Rupela et al., 1987), and soil texture and density of gramineaceous herbage (Brockwell and HeIy, 1962) are other factors. Brockwell and Robinson (1970) considered that environmental factors had no regular influence on Rhizobium occurrence except in so far as they affected the occurrence of leguminous vegetation.

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and P. J. Bottomley

Manipulation of naturally-occurring populations The magnitude of naturally-occurring populations of rhizobia, reported in a number of the papers already cited, often reaches 1.0 x IO4 gg’ of soil, sometimes as high as I .O x IO’gg ‘, and in one case more than I .O x IO7g-’ A population of I .O x IO4 rhizobia g _’ is equivalent to 1.5 x lOi ha-’ to a depth of IO cm. With subterranean clover, inoculated at I .O x IO4 rhizobia seed- ’ and sown at a rate of IO kg ha-‘, the number of bacteria introduced into the soil is approximately 6.0 x IO” ha- ‘. If the naturally-occurring population is 1.O x I O4g- ’of soil and assuming that all the inoculant remains viable, the introduced strain is outnumbered 250: I by resident rhizobia. Despite advantages of its strategic placement and whatever competitive superiority the inoculant might have, it would be optimistic to expect it to be more than transiently successful in forming nodules and persisting in the soil. The situation is exacerbated by the frequency of poor quality inoculants, alluded to earlier, and by mortality of inoculant immediately following its introduction into the soil (e.g. Bowen and Kennedy, 1959; Brockwell et al., 1987). Numerous studies bear testimony to the futility of inoculation in soils containing large resident populations of rhizobia (e.g. Johnson et al., 1965: Ham et al., 1971; Weaver and Frederick, 1974; Brockwell et al., 1987). It seems sensible, therefore, in soils where there are large numbers of naturallyoccurring rhizobia, to ignore inoculation altogether and instead attempt strategically to manipulate those components of the rhizobial population which have the potential to maximize N, fixation.

genes (reviewed by Rolfe and Gresshoff, 1988). These phenomena have implications for an understanding of the processes involved in competition between rhizobia in nodule formation. In general, it appears that the more effective strains are more competitive or, in other words, the host legume exercises a selective preference for the more effective component of a mixed population (e.g. Robinson, 1969). However, there are many exceptions. Indeed, in elegant experiments with effective strains of R. meliloti and their ineffective mutants, Amarger (198 1) demonstrated that competitive success in forming nodules was a characteristic of each parent strain, was independent of level of effectiveness, and was retained during mutation from effectiveness to ineffectiveness. In laboratory and glasshouse experiments, successful manipulation of competitiveness has been achieved by utilizing fungicide-resistant mutants as inocula for seed treated with fungicide active against competing strains (Odeyemi and Alexander, 1977; Jones and Giddens, 1984). The genera1 principle lends itself to other developments for manipulating soil bacteria. Weight of evidence indicates clearly that the best way to establish a new strain of rhizobia amongst a resident population is to apply a heavy rate of effective, persistent inoculum placed strategically close to that point in the soil where the legume roots will first accept infections. In selecting strains suitable for inoculants, the pragmatic approach is to presume that the strain which performs best in the field is the most desirable strain for field inoculation (Brockunderlies the well et al., 1982). This presumption importance of strain testing in field situations.

Competition rhizobia

introduced

Competition between different components of naturalized populations of rhizobia

In several of the papers cited above, there has been explicit or implicit recognition of numerical considerations related to success in nodule formation, interstrain competition and persistence. Thus, where naturally-occurring rhizobia are few or absent, introduction of new strains by inoculation of seed or soil is normally successful provided some prudence is exercised. On the other hand, where large populations of rhizobia occur, inoculation is invariably futile. It is at the intermediate levels of naturalized rhizobial populations, say between IO and 1000 g-’ of soil, that competition between naturalized and introduced rhizobia for nodule formation is of practical concern, and only then if the naturalized population, or a substantial component of it, is poorly effective for the target legume. Hundreds of papers have dealt with strain competition and the subject has been amply reviewed by Trinick (1985) and Dowling and Broughton (1986). Bacteria of the Rhizobiaceae possess a suite of nod genes which are involved in root infection and nodule formation (Dowling and Broughton. 1986). Flavonoid compounds of host origin activate these

Selective enrichment of particular components of naturalized populations of rhizobia may be the only way to influence legume nodulation in field situations where ineffective or poorly-effective populations are so large that conventional inoculation procedures are ineffectual. This proposition has been little addressed. Renwick and Jones (1986) showed that increasing amounts of lime significantly influenced the relative proportions of nodules formed on white clover by two inoculum strains applied at equal rates of inoculation. Similar observations for lime and phosphate have been made by Almendras and Bottomley (1987). Work by Demezas and Bottomley (1986a. 1987) suggested that different selective preferences may be exercised by different cultivars or species of clover. They cautioned, however, that nodule formation by competing strains is not necessarily an index of proportional representation in the rhizosphere and that the incidence of dual (or multiple) occupancy of nodules by more than one strain makes results difficult to interpret (Demezas and Bottomley, 1986b). Work done at Beltsville has led to an ingenious

between

naturalized

and

Inoculant technology proposal for the replacement of resident rhizobia by introduced strains. Fast-growing soybean rhizobia (R. fiedii) of Chinese origin are more effective for Chinese lines of soybean, including Glycine soja, than for North American cultivars (Dowdle and Bohlool, 1985). Strains of B. japonicum serogroup 123, which dominate in many soils in the U.S.A. and which are often regarded as less than fully effective, rank poorly in the nodulation of soybean germplasm from China (Keyser and Cregan, 1987). This observation led Cregan and Keyser (1988) to suggest a means of replacing strains resident in soil with introduced strains: lines of G. soja inoculated with strains of R. fredii highly effective for G. max should be grown in soil occupied by B. japonicum; the G. soja would be selectively nodulated by R. fredii; when those nodules disintegrated and released their rhizobial contents (e.g. Kuykendall et al., 1982), the soil populations would be dominated by R. fredii. This intriguing proposition remains to be tested. Sometimes legume breeders have a choice of selecting symbiotic promiscuity or symbiotic specificity, e.g. in soybean breeding. Problems of competition between inoculant strain and naturalized rhizobia would be alleviated were this choice resolved in favor of promiscuity. In addition, plant breeding to improve N, fixation is feasible (Heichel, 1982; Mytton et al., 1984). The principle of limiting factors states that “the level of crop production can be no higher than that allowed by the maximum limiting factor”. The principle is especially applicable to legume inoculation. Whatever advances are possible through improvements in inoculants and delivery systems, they will not be realized in the absence of sound agronomic practice. If a leguminous crop or pasture has a low yield potential because of disease, nutrient deficiency, weed competition, insect infestation or hostile edaphic factors, high-quality inoculant will be of little help. Only healthy legumes free of environmental stress can fully express their potential for N, fixation. PROGNOSTlCATlON

The legume inoculant industry has made, and continues to make, an enormous contribution to the Earth’s capacity to feed and clothe its peoples. We do not believe that the industry can sustain itself on the proposition that “inoculation is good insurance”. Where inoculant is unnecessary or no good, its application is a waste of time and money. To claim otherwise will ultimately affect the industry’s credibility. Its future, in our opinion, depends on improving inoculant quality-both numerical and competitive, on targeting specific legumes including new crops and on being available in an easily used form. The last major advance in inoculant quality was generated by the work of Roughley and Vincent (1967). They demonstrated that a IO-fold improve-

689

ment in numerical quality from the use of peat carrier sterilized by irradiation. Commercial inoculants prepared in this way contain ca. 2.0 x IO9 rhizobia g-’ of peat. A population of this magnitude occupies ~0.13% of the total volume of the inoculant. Surely it is not overly ambitious to aim for a further IO-fold increase. The desirability of this was demonstrated by Hume and Blair (1992) who found a curvilinearly upward response in soybean nodulation and seed yield to increasing rates of inoculation, and by Roughley et al. (1993) who made similar findings with lupin. Griffith and Roughley (1992) and Griffith et al. (1992) have suggested that some advance might be made through the control of moisture contents in peat culture and correct use of packaging films. The oxygen and nutrient requirements of rhizobia in peat culture remain to be properly addressed. The stage of growth of the fermenter-grown rhizobia when it is added to the peat carrier may be a significant factor determining the final population of the peat inoculant. Strain selection will have an important role in maintaining and enhancing inoculant quality. There is scope to improve the N,-fixing capacity of inoculant strains by mutagenesis and genetic engineering (Paau, 1991). Previously neglected sources in Nature will be explored for superior rhizobia exemplified by the successful discovery of acid tolerant Medicago spp and R. meliloti strains by Howieson and Ewing (1986, 1989) and Bounejmate and Robson (1992). In this context East Asia and the tropics, for instance, may well yield useful strains for North Americantype soybeans (La Favre et al., 1991; Thompson et al., 1991; Ravuri and Hume, 1992). Studies will continue on strain competition and the ability of inoculants to compete successfully with naturalized rhizobia for nodule sites. Use will be made of selective hosts to give competitive advantage to particular strains (Cregan and Keyser, 1988). The ability to persist as a permanent component of soil microflora (Date, 1991) and to maintain symbiotic characteristics (Brunei et al., 1988) will remain objectives in inoculant strain selection. Research into synergistic effects on N, fixation by rhizobia accompanied by other organisms is not new. The subject of dual inoculation of white clover with Rhizobium and vesicular-arbuscular (VA) mycorrhizal fungi was raised by Smith and Daft (1978). For a period, it was considered that additional benefit from mycorrhizae accrued only under conditions of low fertility where the fungal hyphae scavenged plant nutrients otherwise unavailable to the host legume. Now it is recognized that co-inoculation may have a wider application (Thiagarajan er al., 1992; Mahdi and Atabani, 1992) perhaps even to commercial inoculants (Rice et al., 1995). Other microorganisms as well (e.g. Penicillum bilaji-Downey and van Kessel, 1990; Pseudomonas Jluorescens-Nishijima et al., 1988) in association with rhizobia have been reported to enhance legume nodulation. Rice et al.

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(1995) suggest how preparation of a co-inoculant might be effected. Other options for increasing the occurrence of inoculant strains in nodules have been explored. Kishinevsky et al. (1992) used soil fumigation with methyl bromide. While most successful in achieving its objective, this procedure appears dangerous and uneconomical except for experimental purposes. At a molecular level, Cunningham et al. (1991) identified inhibitors of the n&Y expression in B. japonicum. The addition of an inhibitor significantly reduced nodulation by a sensitive strain and could be used to manipulate the competition between strains for soybean nodulation. However, the authors recognized limitations to the practical use of the methodology. Hashem and Angle (1988, 1990) demonstrated that phage introduced into the soil could influence which particular strain of B. juponicum dominated nodule formation on soybean. Basit et al. (1992) found that similar effects could be obtained by coating the seed with phage. It seems certain that this type of work will continue but its potential for the inoculant industry is uncertain. It may be that the way to increase Nz fixation in many situations is through the plant, not the rhizobia. Plant lines with increased capacity for nodulation (supernodulation) and NZ fixation, often in the presence of nitrate, have been identified from collections of germplasm (Herridge and Betts, 1988) and developed by mutagenesis (soybean-Carroll ef al., 1985; Phuseolus bean-Park and Buttery, 1988). The breeding of symbiotically promiscuous lines of soybean is a matter for serious consideration. These plants would not require inoculation because they would nodulate and fix N vigorously with the large diverse populations of B. japonicum that occur naturally in many tropical soils (e.g. Thompson et al., 1991). The work of Kipe-Nolt et al. (1992) suggests that certain wild accessions of Phaseolus bean have sufficient nodulation specificity to exploit in a breeding program to produce a strain-specific plant. Of course, examples of strain-specific legumes are well known and their potential for plant breeding well understood (Lie, 1978). Fobert et al. (1991), working in a glasshouse, exploited the specificity between a symbiotic gene in pea and a nodulation gene in R. kguminosarum bv. viciae to preempt competition from a Rhizobium strain in the soil against inoculant strains. The authors considered their results sufficiently encouraging to justify field experimentation. Legume inoculants will, indeed must. become easier to use. Shelf life of inoculants will be extended (e.g. Griffith and Roughley, 1992). They will be easier to apply, e.g. the new liquid inoculants alluded to earlier in this paper. The wider use of multi-strain inoculants will reduce the total number of inoculants available as well as the scope for user error. Somasegaran and Bohlool (1990) made an extensive comparison of multi-strain and single-strain inoculants. They found that in almost all cases the effec-

tiveness of a multi-strain inoculant exceeded or equalled the performance of the best strain in that inoculant. Paau (1989) also obtained excellent results with a multi-strain soybean inoculant fermented directly in the point-of-use container with a vermiculite carrier. Excellent extension programs prorhizobial inoculants are conducted in moting Thailand (Chanaseni and Kongngoen, 1992) and worldwide by NifTAL. The credibility of the inoculant industry will be well served by also publicizing those situations where inoculation is not required. Other perspectives on the potential for increasing biological Nz fixation, with special reference to soybean, were presented by Keyser and Li (1992). CONCLUDING

REMARKS

Today, the urgency of seeking alternatives to inorganic forms of fertilizer N is perceived to be less than at the time of the energy crises of the 1970s. Yet much of the nitrogenous fertilizer in current use is a subsidized by-product of fossil fuels. On the other hand, the volume of biological Nz fixation is not constrained by a finite resource. Plant and soil scientists have a responsibility to society to find ways and means of making biological N, fixation, in particular N2 fixation by legumes, an economically efficient substitute for fertilization of crops and pastures with inorganic N. It is not enough merely to maximize N, fixation itselc effectual means of efficiently utilizing fixed N are just as important. For instance, nitrification must occur before organic N of legume origin becomes available to plants. If abundant nitrification takes place at a time when plant growth is negligible and other sinks do not exist, as may happen in autumn in some Mediterranean-type climatic zones, nitrate may be lost from the upper soil profile and perhaps finds its way as a pollutant into waterways. This combination of events is probably uncommon but may occur on lime-deficient, legume-based annual pastures in Australia (Helyar, 1976; Helyar and Porter. 1989). Biological Nz fixation is seen as a solution to the pollutant effects of inorganic N fertilizer and, indeed, this would generally be true. Potential loss from the soil of the products of fixed N by denitrification to ammonia and nitrogen oxides also needs to be considered. Of the multiple benefits of legume growth the most well recognized is the ability of the legume to fix its own N supply. Of equal significance is the potential capacity of the legume to conserve and augment the pool of soil N. Augmentation occurs when the amount of soil N removed as animal product, hay and seed is less than the amount of fixed atmospheric N that remains behind in the legume trash. Pasture legumes utilized for forage fulfil this role effectively because only meat. milk or fiber are removed and much of the fixed N is returned to the ecosystem where it is available for rotational crops. This concept is the basis of ley farming where leguminous pastures

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Inoculant technology and cereal crops alternate. Rather more fixed N is removed from the system when the legume is harvested for hay and more still by grain legumes grown for their seed.

We believe that it is feasible to meet the challenge of devising simple, sensible strategies for cultivating legumes that are both high yielding and significantly contributory to reserves of soil N. Some of the information we have presented suggests that N2 fixation can be enhanced by choice of cultivar, control of soil nitrate and soil moisture and, in particular, by manipulation of rhizobial inocula, either supplied as inoculant or already resident in the soil. We are confident that these data can be utilized to develop legume-based systems that optimize Nz fixation, conserve soil N and augment the pool of soil N for the benefit of rotational, non-leguminous crops. Our speculations about manipulation of rhizobia may have wider application. As agricultural biotechnology evolves, much greater use will be made of soil microorganisms both as inoculants and by manipulating them in the soil or the plant rhizosphere. Mycorrhizal fungi, Azolla and Anabaena in rice culture, benign organisms that compete for rhizosphere space with root pathogens thereby reducing the incidence of plant disease, organisms that dissolve forms of phosphate otherwise unavailable to plants, freeliving diazotrophs and cellulolytic organisms are all presently under-utilized. Ecological principles and practices that are appropriate for the manipulation of the rhizobia will quite likely prove suitable models for other soil microorganisms as well, e.g. Azospirillum (Fages, 1992). Despite the immense potential benefits to be had from applied microbial ecology, it is sobering to observe that this field of scientific endeavour still lags well behind other disciplines of N, fixation research. It is apt to quote from D. Gareth Jones (1991) who closed a presidential address to the Association of Applied Biologists with the words “the technology is now available to genetically modify Rhizobium in a number of useful ways and it still puzzles me why the eminent scientists who have captured the imagination of so many of us lesser mortals in developing such sophisticated techniques for molecular biology, have not seized the opportunity of achieving long-lasting fame and, perhaps, fortune by applying their obvious skills to practical advantage?‘.

Almendras A. S. and Bottomley P. J. (1988) Cation and phosphate influences on the nodulating characteristics of indigenous serogroups of Rhizobium trrfolii on soil grown Trijolium subterraneum L. Soil Biology & Biochemistry 20, 345-351. Amarger N. (1981) Competition for nodule formation between effective and ineffective strains of Rhizobium meliloti. Soil Biology & Biochemistry

13, 475-480.

Atkin-Smith R. E., Gault R. R. and Brockwell J. (1986) Effective rhizobia can be introduced, by surface application, into poorly-nodulated subterranean clover pasture. In Proceedings of the Eighth Australian Nitrogen Fixation Conference. (W. Wallace and S. E. Smith, Eds), AIAS Occasional Publication No. 25. pp. 151-152. Australian Institute of Agricultural Science, Adelaide. Barkdoll A. W., Sartain J. B. and Hubbell D. H. (1983) Effect of soil implanted granular and pellet Rhizobium inoculant in Phaseolus vulgaris L. in Honduras. Soil and Crop Science Society of Florida Proceedings 42, 184-189.

Basit H. A., Angle J. S., Salem S. and Gewaily E. M. (1992) Phage coating of soybean seed reduces nodulation by indigenous soil bradyrhizobia. Canadian Journal of Microbiology

38, 1264-1269.

Bell F. and Nutman P. S. (1971) Experiments on nitrogen fixation by nodulated lucerne. Plant and Soil special vol., 231-264. Berg R. K. Jr, Loynachan T. E., Zablotowicz R. N. and Lieberman M. T. (1988) Nodule occupancy by introduced Bradyrhizobium japonicum in Iowa soils. Agronomy

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Bergersen F. J. (Ed.) (1980) Methods for Evaluating Biological Nitrogen Fixation. Wiley, Chichester. Bergersen F. J., Turner G. L., Chase D. L., Gault R. R. and Brockwell J. (1985) The natural abundance of i5N in an irrigated soybean crop and its use for the calculation of nitrogen fixation. Australian Journal of Agricultural Research 36, 41 l-423.

Bissonette N., Lalande R. and Bordeleau L. M. (1986) Large-scale production of Rhizobium meliloti on whey. Applied and Environmental

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Boonkerd N., Arunsri C., Rungrattanakasin W. and Vasurat Y. (1984) EtTect of postemergence inoculation on field grown soybeans. In Advances in Nitrogen Fixation Research (C. Veeger and W. E. Newton, Eds). , D. _ 327. Nijhoff/Junk, The-Hague. Bottomley P. J. (1992) Ecology of Bradyrhizobium and Rhizobium. In Biological Nitrogen Fixation (G. Stacey, R. H. Burris and H. J. Evans, Eds), pp. 293-348. Chapman & Hall, New York. Bottomley P. J. and Jenkins M. B. (1983) Some characteristics of Rhizobium meliloti isolates from alfalfa fields in Oregon, Soil Science Society of America Journal 41, 1153-l 157.

Bounejmate M. and Robson A. D. (1992) Differential tolerance of genotypes of Medicago truncatula to low pH. Australian Journal of Agricultural

Research 43, 731-737.

Bowen G. D. and Kennedy M. (1959) Effect of high soil temperature on Rhizobium spp. Queensland Journal of Agricultural

Science 16, 177-197.

Brockwell J. (1962) Studies on seed pelleting as an aid to legume seed inoculation. I. Coating materials, adhesives, and methods of inoculation. Australian Journal of Agricultural Research 13, 638649.

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