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Rhizobial Inoculants for Legume CropsNewton Z. Lupwayi a , George W. Clayton b & Wendall A. Rice aa Agriculture & Agri-Food Canada Beaverlodge Research Farm, Box29, Beaverlodge, Alberta, Canada, TOH 0C0b Lacombe Research Centre, 6000 C & E Calgary Trail, Lacombe,Alberta, Canada, T4L 1W1Published online: 24 Sep 2008.

To cite this article: Newton Z. Lupwayi , George W. Clayton & Wendall A. Rice (2006) RhizobialInoculants for Legume Crops, Journal of Crop Improvement, 15:2, 289-321, DOI: 10.1300/J411v15n02_09

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Rhizobial Inoculants for Legume Crops

Newton Z. LupwayiGeorge W. Clayton

Wendall A. Rice

SUMMARY. Legumes are an important source of protein for humansand livestock. Legumes have also been used for soil improvement forcenturies because of their N and non-N rotational benefits to non-le-gume crops. The N benefits include N2 fixation and mineralization, spar-ing of soil inorganic N, and reduced immobilization of soil inorganic N.The non-N benefits include breaking pest cycles, improvement of soilstructure, and the nutritional and disease-control effects of endophyticrhizobia. Therefore, optimizing the legume-Rhizobium symbiosis is im-portant, and it can be done by selecting or modifying either (or both)symbiotic partner(s) for desirable traits related to N2 fixation. Rhizobiumstrains can be selected or genetically modified for traits like N2 fixationpotential, nodulation competitiveness, persistence in soil, compatibilitywith inoculant carriers, and tolerance to environmental stress factors.Legume genotypes can also be selected, bred or genetically modified forN2 fixation potential, restricted or preferential nodulation, and toleranceto nitrate and environmental stress factors. When choosing prospective

Newton Z. Lupwayi and Wendall A. Rice are affiliated with the Agriculture &Agri-Food Canada Beaverlodge Research Farm, Box 29, Beaverlodge, Alberta, Can-ada T0H 0C0.

George W. Clayton is affiliated with the Lacombe Research Centre, 6000 C & ECalgary Trail, Lacombe, Alberta, Canada T4L 1W1.

Address correspondence to: Newton Z. Lupwayi at the above address (E-mail:LupwayiN@AGR.GC.CA).

[Haworth co-indexing entry note]: “Rhizobial Inoculants for Legume Crops.” Lupwayi, Newton Z.,George W. Clayton, and Wendall A. Rice. Co-published simultaneously in Journal of Crop Improvement(Food Products Press, an imprint of The Haworth Press, Inc.) Vol. 15, No. 2 (#30), 2005, pp. 289-321; and:Enhancing the Efficiency of Nitrogen Utilization in Plants (ed: Sham S. Goyal, Rudolf Tischner, and AmarjitS. Basra) Food Products Press, an imprint of The Haworth Press, Inc., 2005, pp. 289-321. Single or multiplecopies of this article are available for a fee from The Haworth Document Delivery Service [1-800-HAWORTH,9:00 a.m. - 5:00 p.m. (EST). E-mail address: docdelivery@haworthpress.com].

Available online at http://www.haworthpress.com/web/JCRIP© 2005 by The Haworth Press, Inc. All rights reserved.

doi:10.1300/J411v15n02_09 289

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strains or legume genotypes for a particular environment, time and re-sources can be saved by realizing that the most adaptable rhizobia or le-gume genotypes are usually those isolated from similar environments.Inoculant delivery methods also affect N2 fixation. Soil inoculation, par-ticularly with granular inoculants, seems to be often better and neverworse than seed inoculation for initiating nodulation and N2 fixation.Use of pre-inoculated seeds eliminates the seed inoculation operation,but Rhizobium numbers in pre-inoculated seeds tend to be lower thanthose in traditional inoculant products. Therefore, the time saved by us-ing pre-inoculated seeds should be weighed against the possibility thatcrop yields may be lower if insufficient Rhizobium numbers are deliv-ered. Until tools for genetic modification of rhizobia or legumes to suitspecific requirements are commonly used, N2 fixation can be enhancedby adopting practices like choosing the best combinations of Rhizobiumstrains and legume genotypes, the best inoculant formulation and deliv-ery methods, optimum inoculation rates, and providing favourable grow-ing conditions for the crop. [Article copies available for a fee from The HaworthDocument Delivery Service: 1-800-HAWORTH. E-mail address: <docdelivery@haworthpress.com> Website: <http://www.HaworthPress.com> © 2005 by TheHaworth Press, Inc. All rights reserved.]

KEYWORDS. Inoculant delivery, inoculant formulation, legume geno-type selection, N2 fixation, Rhizobium strain screening, rotational bene-fits

INTRODUCTION

Of all nutrients, N is required in the greatest amounts for plantgrowth. Therefore, fertilizer N inputs constitute the greatest expense oncrop nutrition in agriculture. Smil (1999) has prepared a global accountof N flows in current food production (Table 1). It shows that the contri-bution of biological nitrogen fixation (BNF) (33 Tg N yr�1) is secondonly to that of synthetic fertilizers (80 Tg N yr�1), and that BNF contrib-utes 19% of all N available to world crops (169 Tg N yr�1). These BNFcontributions are average figures that encompass both developed anddeveloping countries. However, the relative contribution of BNF in de-veloping countries is much higher because of low fertilizer use. Moreimportant is the qualitative contribution of BNF. Biologically fixed N ismuch less susceptible to losses in soil, and so more of it stays in thesoil-plant system to be eventually used by crops. In contrast, losses of Nfrom applied fertilizers contribute to a variety of undesirable impacts

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that include high nitrate levels in ground and surface waters (Ondersteijnet al., 2002) and ammonia deposition on forests and grasslands.

The objective of this chapter is to review the importance of legume Nin agriculture and how this N input can be optimized through inoculation.

HISTORY OF LEGUME CROP INOCULATION

Early Accounts of the Soil-Restorative Capacity of Legumes

Apart from supplying food or feed, legumes have been used for soilimprovement for centuries. Some of the early accounts of their soil-re-storative capacity include the following, by Theophrastus (370-285

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TABLE 1. Mean N flows in global food production (data from Smil, 1999).(1 Tg = 1012 g)

N flow Amount (Tg yr�1)

Input

Synthetic fertilizer 80

Biological fixation 33

Atmospheric deposition 20

Animal manures 18

Crop residues 12

Irrigation water 4

Seeds 2

Total 169

Output

Harvested crops 60

Crop residues 25

Total 85

Losses

Denitrification and ammonia volatilization 33

Leaching 17

Soil erosion 20

Losses from tops of plants 10

Total 80

Balance +4

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B.C.): “Beans are not a burdensome crop to the ground: they even seemto manure it, because the plant is of loose growth and rots easily” (Fredet al., 1932). In 1731, Jethro Tull described a well-established practiceof including clover (Trifolium spp.), sanfoin (Onobrychis viciiafolia)and lucerne (Medicago sativa) in crop rotations in England, where prof-its were greater on farms on which it was practiced than on other farms(Aulie, 1970; Bergesen, 1980). The practice spread to continental Eu-rope, resulting in a 40-50% increase in food production in the eigh-teenth century (Bergesen, 1980). However, it was not until late in thenineteenth century that the basis for legume soil improvement wasestablished.

Discovery of Nitrogen Fixation

In the nineteenth century, the quest for the value of legumes in croprotations was full of controversies, centred mainly on experimental de-sign and analytical methodology. They have been described in detail byAulie (1970) and well summarised by Bergesen (1980). The followingis a brief summary taken from these two reports.

In 1841, Jean-Baptiste Boussignault published results which indi-cated that the superior nutritive quality of legumes and their benefits tosoil were due to their N content. In a 5-year forage legume-based rota-tion, he found that legume plants accumulated more N than that addedin manure and wondered whether the excess N had been acquired fromthe air. But Justus von Liebig, a very reputable organic chemist, con-tended that plants acquired their N from ammonia, which was releasedto the atmosphere through decomposition and returned to the soil in rainwater and directly absorbed from the air by plant leaves. In the 1850s,Boussignault, Sir John Laws and Dr Joseph Gilbert, and James ThomasWay all published data that discounted the theory that atmospheric am-monia and nitrate could supply plant N requirements.

From 1850-1856, two contrasting views were presented before theFrench Academy about whether plants could utilize the molecular N ofthe air. Georges Ville concluded from controlled experiments in enclosedsystems that plants could not utilize molecular N, but Boussignault con-cluded that they could and that Ville’s experimental design and tech-nique was flawed. The Academy took Ville’s view. At Rothamsted,Laws and Gibert evaluated, in enclosed systems, the two opposingviews and in a paper in 1861, they too concluded that plants could notutilize atmospheric N. All these experiments were well designed andexecuted, but they were misleading because the then unknown micro-

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organisms that fixed N had been carefully removed in efforts to removeall forms of combined N from the air, water, soil and inner surfaces ofthe apparatus.

No progress was made until the 1880s, when W.O. Atwater in Con-necticut conducted N balance experiments with potted peas (Pisumsativum) in a glasshouse and reported that the peas had acquired more Nthan the amount supplied. Although he was aware that the possibility ofpeas assimilating molecular N was contrary to prevailing views then,Atwater continued to make the point in 1885 and 1886. Meanwhile, af-ter three years of experimentation, H. Hellriegel and H. Wilfarth in Ger-many reasoned that peas probably acquired their excess N throughbacteria dropping onto the sterilized pots from the air, thereby infectingthe roots to produce nodules, which enabled the plants to utilize free N2from the air. They tested this hypothesis by deliberately infecting steril-ized pots of peas with extracts of garden soil. In 1886, they presentedtheir results at a meeting chaired by Joseph Gilbert: peas grown in ster-ile sand in a sterile environment grew poorly without added combinedN, but peas which had received soil extract grew vigorously and growthwas correlated with nodulation. Laws and Gilbert remained scepticaluntil they completed their own evaluations of the new experiments.Their experiments were thorough, backed by comprehensive N analy-ses and observation of nodule development, “and were as conclusive inproving the nitrogen fixing ability of nodulated legumes . . . as had beenthe earlier negative experiments” (Bergesen, 1980). Legume noduleshad been studied microscopically as early as 1858 by Lachmann andwere considered to contain micro organisms, but no scientist beforeHellriegel and Wilfarth had noted the presence or absence of nodules intheir experiments (Bergesen, 1980).

First Isolation of Rhizobium and Sale of Pure Cultures

M.W. Beijerinck was the first to isolate Rhizobium from nodules in1888 (Fred et al., 1932). (In this chapter, the terms Rhizobium orrhizobia are used collectively for the genera Rhizobium, Bradyrhizo-bium, Sinorhizobium, Mesorhizobium, Allorhizobium, and Azorhizo-bium, unless specified otherwise.) This paved the way for artificialinoculation to replace the “soil transfer” method (Fred et al., 1932).Nobbe and Hintner patented the first commercialized pure (agar) cul-ture inoculants in both England and the United States in 1895 under thename Nitragin (van Kessel and Hartley, 2000; Date, 2001).

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LEGUME-RHIZOBIUM INTERACTIONS

Recognition Between Rhizobia and Legumes

The formation of nodules in legumes is species-specific, resulting incross-inoculation groups, i.e., only certain rhizobial strains infect par-ticular legume hosts,. The basis of this specificity has been investigatedfor many years. The lectin hypothesis was advanced in the 1970s(Bohlool and Schmidt, 1974; Dazzo and Hubbell, 1975). In this hypoth-esis, host plant lectins, which are carbohydrate-binding proteins, inter-act selectively with rhizobial cell surface carbohydrates and serve asdeterminants of recognition or host specificity. A lectin cross-bindingmodel was proposed in which the lectin of a particular legume linkscross-reactive antigens of the rhizobia with those of the plant. Althoughthe discovery of nod genes and Nod factors (see below) later showedthat the latter are a major determinant of host specificity, it is stillthought that lectins play a role in mediating specificity, perhaps (a) asglue that holds the rhizobia to a root hair site until Nod factors are trans-mitted, or (b) by transmitting signals to either or both symbiotic partners(Hirsch, 1999).

It is now believed (Long, 1996; Broughton and Perret; 1999; Luytenand Vanderleyden, 2000; Cullimore et al., 2001) that the initiation ofthe legume-Rhizobium symbiosis involves molecular communicationbetween the partners. The root exudates of legumes contain phenoliccompounds, mainly flavanoids that induce rhizobia to express a set ofgenes, known as nod genes that are essential for nodulation and hostrange. These genes encode enzymes that are involved in the synthesisand secretion of host-specific Nod factors. These factors, which arelipo-chito-oligosaccharides, signal back to the plant and induce defor-mation of the root hair that results in infection and formation of the rootnodule. Structural variations in Nod factors, which are controlled by theproducts of nod genes, are the determinants of host specificity, i.e., en-zymes encoded by host-specific nod genes modify the core molecule ofNod factor and impose specificity.

Infection and Nodule Initiation

When homologous rhizobia attach to legume root hairs and secreteNod factors, early root-hair responses include periodic oscillations inintercellular calcium, followed by root-hair curling, which traps therhizobia between root hair cell walls (Gage and Margolin, 2000; Hirsch

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et al., 2001). The root cell wall invaginates, resulting in the formation ofan internal tubular structure called the infection thread that contains themultiplying rhizobia. Cortical cell division starts simultaneously, prob-ably due to expression of plant nodulin genes, giving rise to noduleprimordium and meristem. The infection thread extends toward the di-viding cells of the nodule primordium and, after it enters the cortex, itbranches extensively and releases the rhizobia into the nodule cells. Therhizobia are then budded off into the cell cytoplasm, where they remainsurrounded by a peribacteroid membrane. This membrane (a) providesphysical protection against host cell defence reactions, and (b) controlsnutrient exchange between the symbiotic partners. When they get intothe cytoplasm of the nodule cells, the rhizobia differentiate into bacte-roids and begin synthesizing the enzyme (nitrogenase) and other proteinsrequired for nitrogen fixation.

Infection without formation of infection threads has been observed inArachis spp. and Stylosanthes spp. (Boogerd and van Rossum, 1997),but most legumes are infected via infection threads.

Nodule Development and Nitrogen Fixation

Depending on the host plant, two types of nodules are distinguishablebased on whether nodules contain a persistent meristem or not: inde-terminate (elongate) and determinate (spherical) types (Luyten andVanderleyden, 2000). The indeterminate nodule type originates fromthe nodule primordium in the inner cortex of the root. It has a persistentmeristem generating new cells that continue to be infected, resulting inelongated, club-shaped nodules. Determinate nodules are initiated inthe outer cortex. Cell division stops during development, and cell ex-pansion results in spherical nodules. The development of indeterminatenodules will be described briefly.

The meristem, i.e., the apex of the nodule, is a region (termed Zone I)of actively dividing cells and does not contain rhizobia. The meristem isactive throughout the lifetime of the nodule and grows outward, givingrise to cells that are filled with invading rhizobia. This is the infectionzone (Zone II) where bacteria are released into root cells via infectionthreads. These bacteria are surrounded by the peribacteroid membrane.In successive zones of the nodule from the tip to the point of attachmentto the plant root (Zone IV), the rhizobia are in different stages of differ-entiation, with N2 fixation occurring in Zone III after the bacteria differ-entiate fully into bacteroids. Zone IV is the senescence zone for bothsymbiotic partners and the number of rhizobia decreases. The genes in-

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volved in these developmental stages of rhizobia are described byLuyten and Vanderleyden (2000).

The overall N2 fixation process, which includes reduction of N2 andassociated reduction of H+, can be represented as (Paul and Clark,1989):

N2 + 16 ATP + 8 e� + 10 H+ = 2 NH4+ + H2 + 16 ADP + Pi

The exact equation is difficult to establish under normal conditions be-cause of variability in the amount of ATP hydrolysed per electron pairtransferred and in the ratio of N2 to H+ reduced. The process is mediatedby nitrogenase enzyme (a complex of two iron-sulphur proteins, one ofwhich also contains molybdenum). The equation shows that the N2 fixa-tion process requires a lot of energy (ATP) from the host legume’sphotosynthate, but some of this energy is “wasted” on reduction of H+ toH2. Some rhizobia have the ability to recycle some of this lost energy.

CONTRIBUTION OF LEGUMES IN CROP ROTATIONS

The nitrogen fixed by legumes benefits the legumes themselvesdirectly, thereby (a) reducing fertilizer N inputs in agriculture, and(b) providing protein-rich food or feed. But other crops grown in rota-tion (or intercropped) with legumes also benefit indirectly from legumeN, i.e., they take up inorganic N released into the soil after decomposi-tion of legume plant parts. Besides the N benefits, legumes impartnon-N benefits to other crops in rotations. These benefits include break-ing of disease cycles and improvement of soil structure.

Nitrogen Contributions

The N contributions of legumes vary with legume type or how the le-gumes are used in cropping systems. Thus, because grain legume seedis harvested and taken off the farm, grain legumes contribute less N inrotations than legumes that are used as green manure.

Nitrogen Fixation and Mineralization

The amounts of N2 fixed by grain legumes range from 0 to 450 kgha�1 (Unkovich and Pate, 2000). On the lower end of this scale arechickpeas (Cicer arietum) (0-141 kg ha�1), common beans (Phaseolus

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vulgaris) (0-165 kg ha�1) and lentil (Lens spp.) (5-191 kg ha�1), and onthe upper end are lupin (Lupinus spp.) (19-327 kg ha�1), Faba bean(Vicia faba) (12-330 kg ha�1) and soybean (Glycine max) (0-450 kgha�1). Most of this N is in the grain, hence the high protein content ofgrain legumes, and removed from the farm at harvest. This removal of Nthrough grain harvest means that (a) little N is returned to the soil withcrop residues, e.g., 3-166 kg N ha�1 in tropical agriculture (Giller et al.,1997), and (b) the crop residues have wide C:N, lignin: N and poly-phenol: N ratios and therefore decompose slowly, resulting in the littleN they contain being released even more slowly or immobilized by thedecomposing microflora. For example, field peas cut at flowering stagehad an average C:N ration of 20 and lignin:N ratio of 2, but the residuesafter grain harvest had a C:N ratio of 63 and a lignin:N ratio of 14(Lupwayi et al. 2004b). However, 15N studies have shown that a suc-ceeding crop can recover 2-26% of the N applied through grain legumeresidues (Mohr et al., 1998; Giller et al., 1997; Fillery, 2001).

Annual forage and pasture legumes fix 2-238 kg N ha�1 and peren-nial legumes fix 4-291 kg N ha�1 yr�1 (Peoples and Baldock, 2001),providing protein-rich feed for livestock. Most of these legumes are cutfor hay, but they are also grazed. In intensively managed pastures,60-70% of the legume N is ingested by grazing livestock, but most of itis returned through urine (Fillery, 2001). Export of N in milk can be sig-nificant (80 kg N ha�1 yr�1), but exports through meat and wool areinsignificant. Non-leguminous companion species in pastures, e.g.,perennial ryegrass, are important sinks for inorganic N produced fromdecomposition of legume residues and livestock excreta. A succeedingcrop recovers 4-29% of the N contained in pasture legumes (Fillery,2001). In a trial in Canada, alfalfa (Medicago sativa) fixed up to 466 kgN ha�1 yr�1 and had net soil N balances of 84, 148 and 137 kg N ha�1 inthe first, second, and third years of the stand, respectively (Kelner et al.,1997). But some of the positive net N balances for deep-rooted legumeslike alfalfa and agro forestry legumes (see below) are due to recyclingdeep-leached nitrate (Entz et al., 2001).

Green manure and cover crop legumes fix up to 280 kg N ha�1 intropical agriculture (Giller et al., 1997). Since there is no N removalthrough grain, hay or milk, green manure legumes return more N to thesoil than grain or pasture legumes, e.g., 12-307 kg N ha�1 in the tropics,with residual effects of 3-90 kg N ha�1 in fertilizer equivalents (Giller etal., 1997).

Agro forestry legume trees and shrubs fix 0-377 kg N ha�1 yr�1.Prunings of these legumes input 10-500 kg N ha�1 yr�1 to the soil, and

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N mineralization results in fertilizer equivalents of 43-183 kg N ha�1

yr�1 (Giller et al., 1997; Lupwayi and Haque, 1999).

Sparing Soil Nitrogen

It has been argued that since part of their N requirement is met by N2fixation, legumes utilize less of the available soil N than cereals, thereby“sparing” or “conserving” inorganic N for the intercrop or followingcrop (Chalk et al. 1993; Herridge et al., 1995). In a study conductedacross the southern cereal belt of Australia, Evans et al. (1991) con-cluded that spared N contributed more to the average N benefits of 40and 33 kg ha�1 for lupin and field pea (Pisum sativum), respectively,than N released from the legume residues. Herridge et al. (1995) esti-mated that nitrate spared by chickpea ranged from 6 to 31 kg ha�1 at dif-ferent sites and in different treatments in their study. However, Nsparing is not universal since legumes sometimes take up as much oreven more soil inorganic N than comparable non-legume crops (Unkovichand Pate, 2000).

Less Immobilization of Nitrate During Decomposition

Green and Blackmer (1995) have proposed another explanation forthe N benefits observed in legume-cereal rotations. They observed thatboth corn (Zea mays) and soybean residues resulted in a period of net Nimmobilization followed by net N mineralization. However, becausesoybean produces less residue than corn, the immobilization period isshorter and less N is immobilized during soybean residue decomposi-tion than during corn residue decomposition. Therefore, a crop grownafter soybean will need less fertilizer N than the same crop grown aftercorn.

Non-Nitrogen Contributions

Often, the yields of non-leguminous crops grown after legume cropsare greater than what can be explained by the legume contribution of Nto the soil/plant system. The non-N benefit of a legume in a legume-based rotation is the portion of the yield increase that cannot be ac-counted for by addition of fertilizer N. This benefit, which was respon-sible for 91% of the yield advantage of wheat (Triticum aestivum) in awheat-pea rotation in Saskatchewan (Stevenson and van Kessel, 1996),

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is attributable to several mechanisms, including the ones describedbelow.

Breaking Pest Cycles

Growing the same crop continuously on the same piece of land fos-ters build-up of pests (pathogens, insects and weeds) that the crop is sus-ceptible to. Rotating crops with non-host crops interrupts the pest cycle.For cereals, rotations with legumes are particularly attractive becausethe legumes are also likely to contribute N to the soil/plant system.

Stevenson and van Kessel (1996) found that the severity of wheatleaf diseases was 3.1 units less (on a 0-11 scale) in a pea-wheat rotationthan in continuous wheat. Bailey et al. (2001) found similar results.Oswald and Ransom (2001) reported that less Striga, which is a para-sitic weed that affects cereal crops in Africa, emerged if maize (Zeamays) was grown after pigeon pea (Cajanus cajan). Blackshaw et al.(1994) reported less downy brome populations in lentil-based and cano-la-based wheat rotations than in continuous wheat, but they noted thatsome of these differences were due to the herbicides used on the legumecrops. Stevenson and van Kessel (1996) attributed 21% of the non-Nbenefit to reduced grassy weed infestation in the pea-wheat rotation.These effects of legumes on pests do not always occur, e.g., opposite ef-fects on Pythium spp. have been reported (Pankhurst et al., 1995).

Soil Structure Improvement

Farmers usually observe that, after growing some legumes, soils areeasier to work than after growing other crops, and this is related to soilstructure. In Australia, the order of effects of legumes on soil aggrega-tion was observed to be lupin > lentil > canola (Brassica napus) > fieldpea = linseed (Linum usitatissimum) > barley (Hordeum vulgare) (Chanand Heenan, 1991). Further experiments (Chan and Heenan, 1996) re-vealed that lupin was effective in aggregate formation as well as aggre-gate stabilization, whereas field pea was effective only in aggregateformation with little stabilization. These differences are due to differentabilities of plant roots to (a) stabilize soil structure through mechanismslike root and hyphal enmeshment and production of stabilizing exudatesin the rhizosphere, or (b) destabilize soil structure by processes likephysical fragmentation of soil aggregates when roots penetrate the soil,and by production of organic acids that modify soil pH, redox potential,etc.

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Rhizobia as Plant Growth-Promoting Bacteria

Endophytic Rhizobium leguminosarum bv. trifolii has recently beenisolated from rice (Oryza sativa) (Yanni et al. 1997). Since then,rhizobia have been isolated from other non-leguminous crops, e.g.,Azorhizobium caulinodans from rice and wheat (Webster et al., 1997),R. etli bv. phaseoli from maize (Gutierrez-Zamora and Martinez-Romero,2001) and R. leguminosarum bv. viceae from barley, wheat and canola(Lupwayi et al., 2004a). Therefore, the rhizobial life cycle includes(endo) colonization of roots of non-legume crops. Although theserhizobia have been found to be beneficial to non-legume crops (Biswas2000a), there is no conclusive evidence that the benefits involve symbi-otic nitrogen fixation (James 2000, Yanni et al. 2001). They seem to actas plant growth-promoting (PGP) microorganisms that change the physi-ology and morphology of inoculated roots to favour uptake of N andother nutrients (Biswas et al. 2000b). Other evidence points to increasein photosynthesis, production of phytohormones, phosphate solubili-zation, bacteriocin production and inhibition of fungal growth (Yanni etal., 2001; Peng et al., 2002). Therefore, inoculation of non-legumecrops with rhizobia can benefit the crops through improved nutritionand biological pest control (Lupwayi and Clayton, 2004).

OPTIMIZING LEGUME-RHIZOBIUM INTERACTIONS

The nitrogen fixation potential of the legume-Rhizobium symbiosisis affected by factors about the Rhizobium strain, the legume genotypeand the environment. Therefore, for effective legume-Rhizobium com-bination in a given environment, both Rhizobium strains and legume ge-notypes need to be screened (or modified) for N2 fixation potential inthat environment. However, usually both the legume genotype and theenvironment are fixed, and the task becomes selection of Rhizobiumstrains for a particular host and environment.

Selecting and Modifying Rhizobium Strains

The criteria used for strain screening include ability to form effectiveN2-fixing nodules, ability to compete with indigenous strains for nodu-lation, growth and survival ability in inoculant carriers, tolerance to en-vironmental stresses and persistence in the soil. However, in practice,

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due mainly to the cost of strain evaluation, effectiveness is the maincriterion.

Effectiveness

Strain screening using aseptic growth systems is usually the firststep, especially when a large number of strains have to be screened. Thestrains with high N2 fixation potential are then further tested in pottedsoil or intact soil cores, and eventually verified in the field. Methods forquantifying N2 fixation include acetylene reduction, N-difference, xy-lem ureide assay and 15N-based methods (Elkan, 1987; Beck et al.,1993; Somasegaran and Hoben, 1994). Choosing an appropriate methodis a major problem because each method has unique advantages andlimitations that range from inaccuracy to prohibitive cost. Indirect indi-cators of N2 fixation, e.g., biomass N or dry matter yields, especially un-der low soil N levels, have also been used.

Knowledge gained from understanding legume-Rhizobium commu-nication during nodule-initiation has been used to enhance nodulationand N2 fixation. Thus, addition of nod gene-inducing flavanoids torhizobial inoculants has been shown to increase nodulation even in thefield, but crop yield response has yet to be demonstrated (Begum et al.,2001).

Genetic modification of rhizobia to increase the efficiency of N2 fixationhas been attempted. An example is hydrogen uptake (Hup). Rhizobiaevolve large amounts of hydrogen as a by-product of N2 fixation (seethe N2 fixation equation), and this is a source of inefficiency that was es-timated in one study (Monza et al., 1997) to correspond to 38-69% of to-tal nitrogenase activity. A limited number of Rhizobium strains possessa Hup system that recycles the hydrogen released. Genes that regulatethe Hup system have been identified and efforts are underway to trans-fer Hup+ capability to Hup-strains of Rhizobium (Brito et al., 2002).

Competitiveness

The term competitiveness describes the ability of two or more strainsto grow, survive and form nodules in the same environment (Date,1988). Strains may compete for factors like carbon and micro-site loca-tion. Competition among strains of rhizobia for nodulation of legumesis a major practical problem that frequently results in the highly effec-tive N2-fixing strains being worthless in the field because they areout-competed by the better-adapted, but usually less effective, indige-

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nous strains. Therefore, where large populations of indigenous rhizobiafor a particular legume are established, it is necessary to include nodu-lation competitiveness as a criterion in strain screening, even if it meansaccepting a strain with lower N-fixing ability.

Evaluation of competitive ability involves quantifying the number ofnodules formed by a particular strain in the presence of one or moreother strains. Thus, rhizobia are isolated from individual nodules andthe strain(s) occupying each nodule identified by DNA-based, sero-logical, antibiotic resistance and other methods described in severalmanuals (Elkan, 1987; Beck et al., 1993; Somasegaran and Hoben, 1994).

Besides selecting for inherently competitive strains, several strategiesfor overcoming the competition problem have been attempted, includ-ing repeated mass inoculation (Martensson, 1990) and pre-treatment ofrhizobia with nod gene-inducing flavanoids from the host (Pan andSmith, 2000). Genetic modification of Rhizobium to increase its com-petitive ability is promising. For example, production of the antibiotictrifolitoxin (TFX) by R. leguminosarum bv. trifolii has been shown toconfer a competitive advantage for nodulation (Maier and Triplett,1996). Genes for TFX production and resistance have been cloned andinserted into strains of Rhizobium that are known to improve legumeyield, and field studies showed that TFX-producing strains of R. etli oc-cupied at least 20% more nodules than non-TFX producing strains(Robleto et al., 1998).

Persistence

Producers usually ask whether they should re-inoculate their legumecrop if they grew the legume on the same piece of land two or moreyears previously, e.g., in crop rotations. It depends on the persistence ofthe Rhizobium strains used previously, and persistence may also dependon environmental factors. Strain persistence is the ability of rhizobia tolive as a continuing member of the soil microflora even in the absence ofits host legume, and it can be evaluated only if it is possible to enumer-ate and identify strains at the same time. Ideally, the whole rhizobialpopulation should be isolated from the soil, enumerated and the teststrains identified. Without selective media for rhizobia, this is a bigtask. Usually, the soil rhizobial population is enumerated using themost-probable-number plant-infection method, and the nodules formedare typed for occupancy by the strain in question. An easier, althoughless accurate method of assessing strain persistence is to introduce amarked strain into soil at (or soil collected from) the site, plant a test le-

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gume, evaluate total nodulation and nodule occupancy, then calculate aplant-reinfection index (Lupwayi et al., 1997). The reason that thismethod is less accurate is that a strain may persist in the soil but fail tore-infect plants in a competitive environment. However, the index inte-grates survival traits with those of re-colonisation and renewed nod-ulation, which are more useful characteristics of an inoculant strain thanpersistence per se.

Tolerance to Environmental Stress Factors

The major environmental factors that will affect Rhizobium survivalin the soil include high soil temperature (tropical regions), low soil tem-perature (temperate regions), soil acidity, Al toxicity, and low soilmoisture. Strains of Rhizobium tolerant to these soil conditions havebeen selected (Rice, 1982; Hungria et al., 1993; Graham et al., 1994;Issa and Wood, 1995). The molecular basis for tolerance of Rhizobiumto some of these constraints is now being defined, and this will enableuse of biotechnological approaches to overcoming the constraints. Thus,Dilworth et al. (2001) have identified (a) genes essential for growth atlow pH, and (b) acid-responsive (induced) genes, which are not them-selves critical to growth at low pH. Based on the way these genes func-tion, Dilworth et al. (2001) suggest ways to improve laboratory selectionof acid-tolerant rhizobia, e.g., getting candidate isolates from acid soilson which legumes nodulate, and using properly-buffered low-pH, low-Ca selection media (preferably liquid).

Compatibility with Inoculant Carriers

A Rhizobium strain that multiplies poorly in broth, or one with poorsurvival in peat or other inoculant carriers, cannot produce a high-qual-ity inoculant. However, compatibility with inoculant carriers is usuallyone of the last factors considered in rhizobial strain selection.

Selecting, Breeding and Modifying Legume Cultivars

Herridge and Rose (2000) suggested three general strategies for en-hancing legume N2 fixation through breeding: (a) maximising legumebiomass and seed yield, (b) enhancing symbiotic nitrate tolerance, and(c) optimising legume nodulation through specific nodulation traits likemass and duration, and selective or promiscuous nodulation depending

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on the circumstances. However, breeding for enhanced N2 fixation willrarely be successful if done in isolation of other traits, like disease resis-tance, that are critical to commercial acceptance and use.

N2 Fixation Potential

Legume nodulation and N2 fixation have been improved both by(a) indirect selection for seed and biological yield under N-limiting con-ditions (Pazdernik et al., 1997), and (b) by direct selection for nodulemass, nitrogenase activity and xylem ureide content (Bliss, 1993). Useof DNA markers in genotype selection is a new tool that could facilitateselection of high N2-fixing legume genotypes (Graham and Vance,2000).

Restricted or Preferential Nodulation

One approach to overcoming the problem of competition betweeninoculant and indigenous strains of rhizobia is to find or develop legumegenotypes that restrict nodulation by the indigenous ineffective rhiz-obia. In the Midwestern soybean regions, strains of Bradyrhizobiumjaponicum serogroup 123 are some of the most dominant indigenousrhizobia. Soybean genotypes that restrict nodulation by this serogroup,but show effective symbiosis with inoculant strains, have been identi-fied (Cregan and Keyser, 1986) and the trait has been bred into othergenotypes.

The converse approach to restricted nodulation is preferential nodu-lation with a desirable rhizobial strain, in the presence of indigenousstrains. Rosas et al. (1998) identified common bean (Phaseolus vul-garis) genotypes that preferentially nodulated with KIM5s, a very ef-fective strain of R. etli. If genes responsible for preferential nodulationare identified in these genotypes, this approach could be more widelyapplicable than restricted nodulation.

Nitrate Tolerance

Inorganic nitrogen, particularly nitrate, is known to inhibit moststages leading to nodulation and N2 fixation. This can be a problem inagricultural soils where nitrate levels can be very high, e.g., afterdrought years when fertilizer N is not well utilized. Several approacheshave been attempted to overcome this problem. Betts and Herridge(1987) screened and identified soybean genotypes capable of nodu-lation and nitrogen fixation under high nitrate levels. Secondly, muta-

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genesis has been used to develop super-nodulating mutants of soybean,field pea and common bean that formed up to 40 times the number ofnodules as the parent in the absence or presence of nitrate, apparentlydue to defective auto regulation of nodulation (Carroll et al., 1985). Un-fortunately, super-nodulation occurs at the expense of crop growth andyield. Thus, field studies showed that yields of super-nodulating mutantsoybeans were reduced by 20-41% compared to the parent cultivar (Wuand Harper, 1991; Pracht et al., 1994). However, super-modulatorshave been reported to increase the yield of a following cereal crop (Songet al., 1995), presumably due to reduced uptake of soil nitrate by super-modulators or due to mineralization of the greater quantities of N fromtheir residues (because of low N harvest indices, increased plant N con-centration and the high nodule weights) (Herridge and Rose, 2000). Thethird approach that has been considered is selection of mutants with re-duced ability to utilize nitrate, i.e., with reduced nitrate reductase activ-ity (Nelson et al., 1983), but it has not been successful.

Tolerance to Environmental Stress Factors

Environmental stress factors that affect legume growth include highair temperature, soil acidity and the related problems of Al toxicity andP deficiency, and low soil moisture. Soil acidity is the biggest problemon a global scale, and its management is increasingly dependent on useof acid-tolerant legume cultivars and rhizobia, with soil liming limitedto inactivating the toxicity of Al and Mn (Graham and Vance, 2000).Such management of soil acidity has enabled the Brazilian Cerrado re-gion, a savanna region that constitutes 25% of Brazilian land, to pro-duce legume crops.

Biotechnological solutions to soil acidity and associated problemsfor legumes are now being explored (Vance, 2001). Exudation of citrateand malate from plant roots has been shown to solubilize unavailablesoil P sources. Transgenic alfalfa plants modified to over-express theenzyme malate dehydrogenase have shown increased aluminum toler-ance (Tesfaye et al., 2001), P accumulation and N2 fixation (Vance,2001).

Inoculant Delivery

Seed or Soil Inoculation

The objective of legume inoculation is to provide maximum numberof viable, infective rhizobia in the developing rhizosphere to facilitateoptimum nodulation of the root system. The importance of high rates of

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inoculation to achieve this objective has been demonstrated (Brockwellet al. 1995). Seed inoculation is a common method of applying Rhizo-bium spp. to legumes. Direct application of peat powder inoculants toseed was the most common method until an easier method of seed appli-cation was introduced with liquid inoculants. However, there are sev-eral situations in which seed application of rhizobia may be inefficient,including those where: (a) pre-emergence diseases make it necessary touse seed treatments which are toxic to rhizobia, (b) inoculation of sub-stantial quantities of large-seeded legumes is a major task that limitshow quickly seeding can be completed, (c) some seeds are too fragile tobe inoculated and over-handling can cause reduced germination andemergence, (d) the seed surface limits the number of rhizobia that canbe applied, a common problem when it is necessary to apply large num-bers of an introduced strain to compete with naturally occurring rhizo-bia, (e) the seed coat of some species may contain materials toxic torhizobia, (f) environmental stresses may contribute to increased die-offon the seed, and (g) seeding is delayed because of inclement weather orequipment breakdown and re-inoculation is necessary. In these situa-tions, soil inoculation, i.e., direct application of inoculant to the soil inthe vicinity of the legume seed, would be more effective than seedinoculation.

Granular forms of inoculants are commonly used in soil inoculation,but liquid formulations have also been used. Brockwell et al. (1995) re-viewed the literature on delivery systems, and concluded that “thesemethods (soil inoculation) are often better and never worse than theconventional seed inoculation for initiating nodulation and N2 fixation.”Other field studies in which crop response to different inoculation meth-ods and formulations were compared are listed in Table 2. In five studiescomparing soil inoculation using granular formulations with seed-ap-plied peat inoculant, crop response to soil inoculation was greater thanseed-applied inoculant. In four studies comparing seed-applied peatinoculant with soil or seed-applied liquid inoculant, there was no differ-ence in crop response to the two methods of inoculation. In one study,grain yields were greater with seed applied peat than with seed-appliedliquid. In another study, crown and total nodulation, and dry matter wasgreater with seed-applied peat than soil-applied liquid, but lateral nodu-lation was greater with soil-applied liquid than with seed-applied peat.In eight studies comparing granular (soil), peat (seed) and liquid (seedor soil) inoculant delivery methods, crop response to granular (soil) wasgenerally greater than with seed-applied peat or liquid.

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Applied Aspects 307

TABLE 2. Crop responses to peat (seed), granular (soil), and liquid (seed orsoil) inoculant delivery methods in field experiments.

Crop Inoculant delivery methods compared Crop response Reference

(a) Peat vs. granular

Faba bean Peat (4.2 g kg�1 seed) vs.granular (33 gkg�1 seed in soil).

Granular > peat in nodulation,nitrogenase activity and plantDM.

Dean andClark (1977).

Soybean Peat (0.24 kg ha�1) vs. granular (32 kgha�1).

Granular > peat in nodulenumber and weight, N fixedand grain yield.

Muldoon et al.(1980).

Soybean Peat vs. granular (no rates given). Granular > peat in grain yieldand grain protein.

Dubetz et al.(1983).

Soybean Peat (1.5 mg seed�1) vs. granular (0.55g m�1row).

Granular > peat in noduleweight and grain yield.

Bezdicek et al.(1978).

Field pea Peat (8-50 � 104 cells seed�1) vs.granular (8-50 � 104 cells seed�1 insoil).

Granular > peat in nodulationand plant DM.

Brockwell etal. (1980).

Lupin Peat (8-50 � 104 cells seed�1) vs.granular (8-50 � 104 cells seed�1 insoil).

Granular > peat in nodulationand plant N.

Brockwell etal. (1980).

(b) Peat vs. liquid

Lentil Peat (3 g kg�1 seed) vs. liquid on seed(4 mL kg�1 seed).

No differences in nodulationand grain yield.

Hynes et al.(1995).

Field pea Peat (3 g kg seed�1) vs. liquid on seed(4 mL kg�1 seed).

No differences in nodulationand grain yield.

Hynes et al.(1995).

Soybean Peat (2 � 105 cells seed�1) vs. liquidon seed (5 � 104 to 5 � 105 cellsseed�1).

Peat > liquid in nodulation andgrain yields.

Burton andCurley (1965).

Soybean Peat (107 cells seed�1) vs. liquid in soil(5 � 108 cells m�1 row).

Peat > liquid in crownnodulation and total nodule DM,but liquid > peat in lateral rootnodulation. Lateral root nodules> crown nodules in N2 fixed.

Hardarson etal. (1989).

(c) Granular vs. peat vs. liquid

Alfalfa Granular (5 or 50 kg ha�1) vs. peat (10g kg seed�1) vs. liquid in soil (330 Lha�1)

Granular > peat & liquid innodule number, nodule weightand nodule occupancy.

Rice andOlsen (1992)

Soybean Granular (2.5 kg ha�1) vs. peat (6 gkg�1 seed) vs. liquid in soil (4.3-5.7 Lha�1)

Granular > liquid & peat innodule DM.

Chambers(1983).

Soybean Granular (8-50 � 104 cells seed�1) vs.peat (8-50 � 104 cells seed�1) vs. liquidin soil (8-50 � 104 cells seed�1)

Granular & liquid > peat innodulation and plant DM whenfungicides applied to seeds.

Brockwell etal. (1980).

Field pea Granular (6 � 1011 cells ha�1) vs. peat(1 � 105 cells seed�1) vs. liquid onseed (1 � 105 cells seed�1)

Granular & peat > liquid innodule number and occupancy.No differences in grain yields.

Hynes et al.(2001).

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These studies support the conclusion reached by Brockwell et al.(1995). Soil inoculation with granular inoculants, in particular, seems tobe often better but never worse than seed inoculation, as recent resultson field peas in Alberta confirm (Table 3). The advantage of using gran-ular inoculants is especially pronounced under soil stress conditionslike soil acidity, moisture stress or cool, wet soils. In greenhouse stud-ies, only granular inoculant was effective in establishing nodules at soilpH 4.4, but granular and seed-inoculated powdered peat inoculantswere effective at pH 5.4, and all three formulations (granular, powderedpeat seed-inoculated liquid) were effective at pH 6.6 (Rice et al.,2000a). Under moisture stress, granular inoculant applied to the soilwas much more effective in maintaining field pea yield than seed-ap-plied inoculants (Miller et al., 2002). Under cool, wet conditions in thespring, the rhizobial population in pea rhizosphere continued to increasewhen granular inoculant was used, whereas the populations declined fora period of time before recovering with the seed-applied liquid inocu-lant (Hynes et al., 2001). In a study conducted in Alberta, Canada,McKenzie et al. (2001) reported that field pea yield was significantly in-creased by soil-applied granular inoculant in 41% of the trials with anaverage yield increase of 14% compared to an un-inoculated check.Yield response was 29% if soil NO3-N was less than 20 kg ha�1 and10% if soil NO3-N was more than 20 kg ha�1.

308 Enhancing the Efficiency of Nitrogen Utilization in Plants

TABLE 2 (continued)

Crop Inoculant delivery methods compared Crop response Reference

Field pea Granular (25 kg ha�1) vs. peat (3 gkg�1 seed) vs. liquid on seed (4 mLkg�1 seed)

Granular > peat > liquid =uninoculated check in nodulenumber, N accumulation andN2 fixation.

Clayton et al.(2004a)

Field pea Granular (25 kg ha�1) vs. peat (3 gkg�1 seed) vs. liquid on seed (4 mLkg�1 seed)

Granular resulted in 17, 50 and56% higher pea grain yield thanpeat, liquid and uninoculatedcheck, respectively.

Clayton et al.(2004b)

Chickpea Granular (1 � 1011 cells ha�1) vs. peat(1� 105 cells seed�1) vs. liquid on seed(1 � 105 cells seed�1)

Peat & liquid > granular incrown nodulation, but granular> others in lateral rootnodulation. Granular & peat >liquid in N2 fixation. Granular >peat & liquid in grain yields.

Kyei-Boahenet al. (2002).

Chickpea Granular (8-50 � 104 cells seed�1) vs.peat (8-50 � 104 cells seed�1) vs. liquidin soil (8-50 � 104 cells seed�1)

Granular and liquid > peat innodulation and plant DM whenfungicides applied to seeds.

Brockwell et al.(1980).

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The practical application of soil inoculation delivery systems isdependent on the cost:production ratio of soil and seed applicationmethods. The rates (kg ha�1) of application of soil-applied granularinoculants reported in the studies shown in Table 2 varied from 2.5 to 50kg inoculant ha�1. The rates used in the other studies, which were re-ported as g inoculant kg�1 seed, g inoculant�1 row, rhizobia cellsseed�1, and rhizobia cells ha�1, probably also fall within the range re-ported on the kg ha�1 basis. In order to optimise N2 fixation and yieldfor a specific crop in a given agro-climatic region, more detailed infor-mation on granular inoculant rates and other factors related to this man-agement strategy compared with other inoculation methods is required.An example of the kind of agronomic information required is given herefor field pea production in western Canada.

Granular Inoculant Rate and Placement

The rate of inoculation is important because it directly affects thecost:benefit ratio, and the placement of granular inoculant relative to theseed will affect nodulation. Table 4 shows that field peas did not re-spond further to inoculation rates above 2.5 kg ha�1. Thus, grain yieldwas 49, 47, 49, and 49% higher than the un-inoculated control with 2.5,5, 7.5 and 10 kg ha�1, respectively. Placement of granular inoculant inthe seed row resulted in greater pea biomass and biomass N at the flat

Applied Aspects 309

TABLE 3. Relative field pea yields (% of uninoculated control at 0 N) from soilinoculation (granular), seed inoculation (peat powder and liquid) and unin-oculated at various N rates. The yield of uninoculated control at 0 N was 2320kg ha�1.

Nitrogen rate (kg ha�1)

Formulation 0 20 40 80 Mean

Granular 176 188 185 185 183

Peat powder 162 165 173 147 162

Liquid 144 115 138 129 132

Unincoulated 100 100 91 88 95

Mean 145 142 147 137

Adapted from Clayton et al. (2004b).

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pod growth stage than when the granular inoculant was placed in a bandbeside and below the seed, or surrounding the seed when the seed andinoculant were distributed below the soil surface with a sweep (Ta-ble 4). The placement effect at the flat pod stage did not carry over toseed yield, seed N or total (seed + straw) N at harvest. Thus, pea yieldwas 48, 44 and 54% greater than the un-inoculated control when granu-lar inoculant was applied in the band, in the seed row or with a sweep,respectively. Therefore, granular inoculant applied at 2.5 kg ha�1 in aband, with seed, or applied in a sweep type opener, may be adequate forhigh pea yield. However, farm scale trials showed that peas under envi-ronmental stresses, e.g., low soil pH or dry soil conditions, could benefitfrom higher application rates (up to 10 kg ha�1).

Addition of N fertilizer to Boost N2 Fixation

Whether “starter” or additional N is required to boost or supplementN2 fixation is another issue worth addressing. Figure 1 shows that appli-cation of N fertilizer reduced the number of N-fixing (pink) nodules,particularly with granular and peat powder inoculant formulations. Ad-dition of 20 to 80 kg N ha�1 did not increase pea grain yield (Table 4),presumably because N fertilizer simply replaced N2 fixation. In an Al-berta study, McKenzie et al. (2001) reported that application of N fertil-

310 Enhancing the Efficiency of Nitrogen Utilization in Plants

TABLE 4. Field pea biomass and biomass N at flat pod stage and seed yield,seed N content and total (seed + straw) N content at maturity. The yield of theuninoculated control was 2930 kg ha�1.

Placement Biomassat flat pod

Biomass Nat flat pod

Seed Yield(kg ha�1)

Seed N(kg ha�1)

Seed + straw N(kg ha�1)

Band 3800 81 4360 111 161

Seed 4570 96 4240 106 158

Sweep 3510 77 4520 107 173

S.E. 220 6 150 5 4

Rate

2.5 3970 84 4370 111 161

5 4210 91 4330 110 162

7.5 4010 83 4390 114 174

10 3640 80 4400 111 160

S.E. 300 7 180 5 5

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izer (20, 40 or 60 kg N ha�1) increased pea yield in 28% of 58 trialsconducted, and the average increase was 9%. When spring soil NO3-Nto 30 cm was less than 20 kg ha�1, application of N fertilizer increasedpea yield in 33% of the trials by an average of 11% (n = 12). Applicationof starter N provided modest, infrequent benefits. However, there areconditions when starter N may be required, e.g., when crops are seededin relatively cold soils where nodulation is likely to be delayed due toslow multiplication of rhizobia.

Granular Inoculant/Fertilizer Mixtures

Delivery of granular peat can be difficult in some situations and pro-ducers have considered applying mixtures of granular inoculant withfertilizer. The effect of such a practice on the survival of rhizobia andresponse of peas to inoculation yields were examined. In laboratorystudies, rhizobia survival declined rapidly within 3 h of blending theinoculant with mono-ammonium phosphate (11-51-0-0 N-P2O5-K2O-SO4) or ammonium sulphate (21-0-0-24) fertilizers (Figure 2). Coatingthe ammonium sulphate resulted in some protection of rhizobia for anadditional 3 h before rapid decline.

Applied Aspects 311

S.E. = 2.7

N Rate (kg/ha)

Pin

kN

odul

eN

umbe

r

40

35

30

25

20

15

10

5

00 20 40 60 80

FIGURE 1. Interactions between inoculant formulation [uninoculated (�), seed-applied liquid (�), seed-applied peat powder (�) and granular (�) formulation]and N rate on nodule number at flatpod stage of pea growth. Adapted fromClayton et al. (2004a).

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However, in field studies, mixing granular inoculant with mono-am-monium phosphate fertilizer for up to 6 h before application did not af-fect pea yield significantly in comparison with yield when the twoproducts had not been mixed (0 h) (Figure 3). One of the reasons isprobably because Rhizobium populations recover sufficiently in the soilto affect nodulation, if a good quality inoculant (at least 109 cells g�1) isused. For example, granular soil-applied inoculants in Canada are legis-lated (Fertilizers Act) to supply at least 1011 rhizobia per hectare. At anapplication rate of 5 kg ha�1, the inoculant needs to contain 2 � 107

rhizobia g�1 to meet the Fertilizer Act standard. In Figure 2, theinoculants contain at least 107 rhizobia g�1 up to about 12 h after mixingwith fertilizer even though the original Rhizobium numbers in theinoculants were less than 109 cells g�1. Therefore, good-quality granu-lar inoculant can probably be mixed with fertilizer and applied together,but the mixture should preferably be applied on the same day.

Pre-Inoculated Seeds

Use of pre-inoculated (coated) seeds eliminates the seed inoculationoperation, thereby giving producers more time to seed. In addition,

312 Enhancing the Efficiency of Nitrogen Utilization in Plants

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1FIGURE 2. Rhizobium survival when graunular inoculant (GI) was blendedwith ammonium sulphate (AS), coated ammonium sulphate (CAS) and mono-ammonium phosphate (MAP) fertilizers.

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lime, micronutrients and other seed treatments can be added to the seedcoatings, thereby alleviating the effects of stress conditions in soil onrhizobia. Thompson and Stout (1992) found no significant differencesin nodulation and DM yield between alfalfa seed pre-inoculated withthree commercial lime seed coatings and traditional seed inoculation.Rice et al. (2001a) found that alfalfa seed inoculated just before plantinghad more rhizobia at planting time than pre-inoculated seed, but the twoinoculation methods were not significantly different in their effective-ness. In field peas, there was indication that pre-inoculated seed may besuperior to traditional seed inoculation in acid soils (Rice et al., 2001b),but grain yields in the acid soils were so low that the advantage ofpre-inoculated seeds did not have significant value. In any case, Rhizo-bium counts on pre-inoculated pea seeds were about 1, 000 cells seed�1

less than counts on freshly-inoculated seeds. In the Canadian legumeinoculant testing program, Rhizobium numbers in pre-inoculated seedswere always lower than those in traditional inoculant products from1978 to 1998 (Lupwayi et al. 2000). In Australia and New Zealand, useof pre-inoculated seeds has produced erratic results (Brockwell et al.1995). Therefore, the time saved when pre-inoculated seeds are used

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should be weighed against the possibility that crop yields may be lowerbecause insufficient Rhizobium numbers are delivered.

Co-Inoculation of Rhizobia with Other PlantGrowth-Promoting (PGP) Microorganisms

Co-inoculation of rhizobia with P-solubilising fungi, arbuscular my-corrhizal fungi (AMF) and PGP bacteria is another approach that hasbeen used to enhance N2 fixation. Rice et al. (2000b) observed that dualinoculation of alfalfa with Sinorhizobium meliloti and Penicillium bilaii,a P-solubilising fungus, increased nodule number and occupancy, butresulted in only small increases in hay yield and total N and P concen-trations of hay compared with inoculation with S. meliloti alone. Therewas little response of alfalfa to P fertilizer at the sites, and this maypartly explain the results. Co-inoculation of rhizobia with AMF hasshown not only synergistic interactions in legume N and P nutrition(Xavier and Germida, 2002), but also biological control of Fusariumroot rot (Dar et al., 1997). However, most studies with AMF have notbeen conducted in the field because of problems in mass production ofinoculant. Probably the most studied PGP micro organism is Azo-spirillum spp. (Okon and Labandera, 1994), and co-inoculation withrhizobia has increased nodulation and N2 fixation (Tchebotsar et al.,1998), but results from field studies on PGP bacteria are inconsistent(Pan et al., 2002).

CONCLUDING REMARKS

Strains of rhizobia can be selected or modified for effectiveness,competitiveness, persistence, etc, but testing many strains for all desir-able characteristics may not be realistic. The same applies to legume ge-notypes. When choosing prospective strains or legume genotypes for aparticular environment, time and resources can be saved by realizingthat the most adaptable rhizobia or legume genotypes are usually thoseisolated from similar environments. Tools for genetic modification ofrhizobia or legumes to suit specific requirements may eventually becommonly used, but in the meantime, N2 fixation can be enhanced byadopting practices like choosing the best combinations of Rhizobiumstrains and legume genotypes, the best inoculant formulation and deliv-ery methods, optimum inoculation rates, and providing favorable grow-ing conditions for the crop.

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