Identi®cation of tolerance to soil acidity in inoculant strains ofRhizobium leguminosarum bv. trifolii

Elizabeth L.J. Watkin*, Graham W. O'Hara, John G. Howieson, Andrew R. Glenn 1

Centre For Rhizobium Studies, School of Biological Sciences and Biotechnology, Division of Science and Engineering, Murdoch University,

Murdoch, WA 6150, Australia

Received 14 September 1999; received in revised form 8 February 2000; accepted 23 February 2000

Abstract

The acid-soil tolerance of six strains (WU95, NA3001, WSM409, TA1, NA3025 and NA3039) of Rhizobium leguminosarumbv. trifolii was assessed in a three-year cross-row ®eld experiment in an acid sandy soil of pH 4.2. Strains WSM409, NA3039

and WU95 were more acid-soil tolerant than strains NA3025, TA1 and NA3001. Strains WSM409 and NA3039 colonised andpersisted in acid-soil to a greater degree than strains TA1 and NA3001. The data from this study clearly identi®ed strainWSM409 as a strain with outstanding potential for improving the production of clovers on acid soils. 7 2000 Elsevier Science

Ltd. All rights reserved.

Keywords: Soil acidity; Acid tolerance; Rhizobium leguminosarum bv. trifolii

1. Introduction

Agricultural systems have been developed in Austra-

lia based on the rotation of annual pasture legumes

with cereal crops using a range of management prac-

tices such as ley and phase farming strategies (Cocks

et al., 1980; Ewing et al., 1992; Reeve and Ewing,

1993). In these systems, the pasture legume is inocu-

lated with an appropriate strain of root nodule bac-

teria during the year of establishment (Brockwell et al.,

1995). In subsequent years, the successful regeneration

of the legume pasture will depend on the survival of

the inoculant strain in the soil in the absence of the

legume host (Howieson, 1995a). In the Mediterranean

environment of southern Australia, this requires the

persistence of the root nodule bacteria in naturally

acidic-soils over the hot, dry summer (Chatel and Par-

ker, 1973a).

The failure of N2-®xing Rhizobium-legume symbioses

in acid soils is a signi®cant problem a�ecting agricul-

tural production in many areas of the world (Coventry

and Evans, 1989; Wright and Zeto, 1991). Sustainable

production of legume pastures on acid soils can be

severely limited by poor growth and survival of root

nodule bacteria (Robson and Loneragan, 1970), which

die when exposed to acid conditions (O'Hara and

Glenn, 1994). Subterranean clover (Trifolium subterra-

neum ) is the most important and widely sown pasture

legume in southern Australia. Acid-tolerant strains of

clover root nodule bacteria would be bene®cial in

improving clover pasture production (Slattery et al.,

1992).

Studies on the e�ects of low pH on root nodule bac-

teria have focused largely on attempts to select acid-

tolerant strains for use as inoculants (Graham et al.,

1982; Thornton and Davey, 1983b; Howieson et al.,

1988). Much of the work has used growth on agar

plates (Brom®eld and Gareth Jones, 1980; Lindstrom

and Myllyniemi, 1987; Richardson and Simpson, 1989)

Soil Biology & Biochemistry 32 (2000) 1393±1403

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.

PII: S0038-0717(00 )00057 -2

www.elsevier.com/locate/soilbio

* Corresponding author. Tel.: +61-08-9360-2439; fax: +61-08-

9360-6486.

E-mail address: ewatkin@central.murdoch.edu.au (E.L.J. Wat-

kin).1 Present address: O�ce of the Pro Vice Chancellor (Research),

University Of Tasmania, Hobart, Tasmania, Australia.

and in broth culture (Thornton and Davey, 1983a;Wood and Cooper, 1985; Richardson and Simpson,1989) though there are some data from ®eld exper-iments (Thornon and Davey, 1984; Wood et al., 1984;Lindstrom and Myllyniemi, 1987). Howieson andEwing, (1986) used a cross-row technique in a longterm ®eld experiment to study acid-soil tolerance inSinorhizobium spp. The cross-row procedure involvessowing rows of inoculated seed in the ®rst year of theexperiment, and then, in subsequent years, sowinguninoculated seed in rows at right angles to the orig-inal row. This technique assesses the persistence ofstrains in the soil at the site of inoculation and alsodetermines their ability to colonise the soil away fromthe point of initial inoculation.

The ability of inoculum strains of root nodule bac-teria to colonise a soil in the absence of the hostlegume is important in situations where it is advan-tageous to introduce the root nodule bacteria separatefrom the host legume seed (Howieson, 1995b). Thesesituations may arise when fungicides are used on thelegume seed (Stovold and Evans, 1980; Evans et al.,

1986), or when the legume is sown in conditions thatare harmful to the survival of the bacteria. Therefore,an understanding of the saprophytic competence ofinoculum strains is important in devising inoculationstrategies to overcome these problems (Howieson,1995a).

In this study, we have used the cross-row techniquein a three-year ®eld experiment to examine the per-formance in an acid-soil (pH 4.2) of six selected strainsof Rhizobium leguminosarum bv. trifolii, hereafterreferred to as R. trifolii. Persistence and colonisationwas assessed over the three years of the experiment fol-lowing the introduction of strains into the acid-soileither as seed-coated peat inoculum, or as peat addeddirectly to the soil in the absence of the host plant.

2. Materials and methods

2.1. Experimental design

The experimental design was adapted from that ofHowieson and Ewing (1986) to evaluate acid tolerance

Fig. 1. Sowing and harvesting regimes. Plots sown in the ®rst year with (a) T. subterraneum inoculated with R. trifolii (± � � ±) or with (b) R. trifo-

lii in the absence of a host (± ± ±). All plots sown with uninoculated T. subterraneum (ÐÐ) in the second and third years. Plants harvested in

the ®rst, second, and third years ( . . . . . . ) 8 weeks after sowing.

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±14031394

in strains of Sinorhizobium spp. (Fig. 1(a) and (b)). Sixstrains of R. trifolii (Table 1), plus an uninoculatedcontrol treatment, in factorial combination with hostTrifolium subterraneum (cv. Dalkeith), plus a nil hosttreatment, in quadruplicate.

2.2. Site preparation

The experiment was carried out on the DrylandResearch Institute, Merredin, Western Australia (31828 ' S, 1188 18 ' E). This area has a Mediterranean cli-mate with an average annual rainfall of 315 mm. Sev-enty percent of the rain falls during the growingseason between May and October. The yearly rainfallduring the experiment was 405 mm in 1992, 266 mmin 1993, and 154 mm in 1994. The site had beencleared of the original vegetation of acacia and wodgilin 1965, and cultivated in a cereal/voluntary pasturerotation until 1982. Wheat/lupin rotation was sownduring 1983±1990; the lupin was inoculated with Bra-dyrhizobium sp. (Lupini ) strain WU425. During 1991the site was under voluntary pasture. There is norecord of the site being sown to inoculated sub-clover.The soil is a combination of Wodgil sand (Northcote:Uc 5.22) and Ulva gravelly sand (Northcote: KS-Uc4.21) with an organic carbon content of 0.9%, totalnitrogen of 0.055% and pH 4.2 (1:5 in 0.01 M CaCl2).The site was sprayed two weeks prior to sowing the ex-periment with 1l/ha Roundup C.T. (active ingredient450 g Glyphosphate/l) to kill weeds, then cultivated toa depth of 2 cm.

2.3. Preparation of seed and inoculum

Rhizobial inocula were prepared using cultures ofthe six strains of R. trifolii grown at 288C in TryptoneYeast (TY) broth (Beringer, 1974) to late exponentialphase (OD600 approx. 1.0). Twenty ®ve ml of cultureof each strain was aseptically injected into separatebags of sterile peat (50 g, Biocare Technology, NSW,

Australia) to give a ®nal concentration of approx. 1 �109 cells gÿ1 peat. An uninoculated control peat wasprepared by injecting 25 ml of sterile TY broth into a50 g bag of sterile peat. The inoculated and controlpeats were thoroughly mixed by hand manipulationand incubated at 288C for 10 days. The inocula werethen stored at 48C. Viable counts were performed onthe stored, inoculated peats one week before use in the®eld experiment, and bags with similar numbers of rhi-zobia (2±5.5 � 108 cells gÿ1 peat) were used to inocu-late sterile seeds.

Seeds of T. subterraneum were surface sterilised byimmersion (5 s) in 95% (v/v) ethanol followed by 3min treatment with acidi®ed 0.01% (w/v) HgCl2 (Vin-cent, 1970). After six washes in sterile deionised water,the seeds were air-dried aseptically in a laminar ¯owcabinet. Sterile dry seeds were immediately inoculatedand lime-pelleted by ®rst mixing 1 g of 2% (w/v) meth-ocel adhesive solution with 0.5 g of the appropriatepeat, and then adding 50 g of the appropriate peat,and then adding 50 g of clover seed and 35 g of ®nelyground lime (Brockwell et al., 1982; Howieson andEwing, 1986). The seed was mixed aseptically until uni-formly coated with peat and lime. Inoculated seed wasstored at 48C overnight and then sown.

2.4. Sowing

2.4.1. Plots sown with inoculated seed in year oneThe experiment was sown on 8th June 1992 in ®ne

sunny conditions. Each treatment was sown in twoparallel rows, 2 m long and 0.5 m apart at a rate ofapproximately 1 g of inoculated clover seed per meter(Fig. 1(a)). Each plot was separated by a 1 m borderto minimise movement of inoculum strains betweenplots. All plots were hand fertilised at the time of sow-ing with superphosphate equivalent to 200 kg haÿ1.

In the second and third years of the experiment,regenerative growth was killed with Glyphosphate two

Table 1

Strains of Rhizobium leguminosarum bv. trifolii used in this study

Strain Origin and characteristics Source

NA3001 Acid media tolerant (Richardson and Simpson, 1989) isolated in New South

Wales. Minimum pH (agar) for growth, 4.3 (Watkin et al., 1997)

G. Gemell, AIRCS

NA3025 Isolated from acid-soil in the central tablelands of New South Wales. Minimum

pH (agar) for growth, 4.6 (Watkin et al., 1997)

G. Gemell, AIRCS

NA3039 Isolated from acid-soil in the central tablelands of New South Wales. Minimum

pH (agar) for growth, 4.6 (Watkin et al., 1997)

G. Gemell, AIRCS

TA1 Commercial inoculum for white clover. Acid sensitive (Richardson and

Simpson, 1989). Minimum pH (agar) for growth, 4.6 (Watkin et al., 1997)

Murdoch University collection

WSM409 Isolated from T. subterraneum on acid-soil in Sardinia. Minimum pH (agar) for

growth, 4.5 (Watkin et al., 1997)

JG Howieson Centre for Rhizobium Studies

WU95 Commercial inoculum for T. subterraneum. Minimum pH (agar) for growth, 4.6

(Watkin et al., 1997)

Murdoch University collection

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±1403 1395

weeks after the ®rst rain and again after a furtherweek. The plots were fertilised at the time of sowingwith superphosphate at a rate of 150 kg haÿ1.

In the second year, all undisturbed portions of theoriginal rows were resown with surface-sterilised cloverseeds on 4th June 1993. In addition, the right-handrow of each plot had three 0.5 m rows sown at rightangles to, and intersecting to the right of the originalrow (Fig. 1(a)).

In the third year, the plots were resown with sur-face-sterilised clover seeds on 23rd July 1994 in the top1 m of the original left-hand row (Fig. 1(a)). In ad-dition two 0.5 m rows were sown at right angles to,and intersecting, to the left of the original left-handrow. This was done to minimise contaminationbetween plots due to site disturbance in the secondyear.

2.4.2. Plots inoculated without host in the ®rst yearIn the ®rst year, those plots that were not to be

sown to clover had the peat inoculant of each strainsprinkled along the two 2 m rows and covered withsoil (Fig. 1(b)). In the second year these rows weresown to clover as described above. In the third year,the total 2 m of the left-hand row was resown withsurface sterilised clover seed with four 0.5 m rowssown at right angles and to the left of the original row(Fig. 1(b)).

2.5. Harvesting

2.5.1. Plots sown with inoculated seed in ®rst yearIn the ®rst year the plants in the lower 1 m of the

left-hand row of each plot were harvested 8 weeksafter sowing (Fig. 1(a)) to a depth su�cient to recoverthe total root system. The remaining plants in eachplot were left undisturbed.

In the second year, eight weeks after sowing, theplants were harvested in the original right-hand rowand in the three cross rows (Fig. 1(a)) in the followingfour sampling regions: (1). the original right-hand row,(2). cross rows, 1±10 cm from the original row, (3).cross rows, 11±20 cm from the original row, and (4).cross rows, 21±50 cm from the original row.

In the third year, eight weeks after sowing, theplants were harvested in the original top 1 m of theleft-hand row and the two cross rows (Fig. 1(a)), withsampling regions as in the second year.

2.5.2. Plots inoculated without host plantIn the ®rst year there was no harvest (Fig. 1(b)). In

the second year the plants were harvested as describedabove for plots sown with inoculated seed in the ®rstyear (Fig. 1(b)).

In the third year, the original left-hand row and twocross rows were harvested in the four sampling regions

described above, except that the top and bottom 1 msections were harvested separately (Fig. 1(b)).

2.6. Assessment of nodulation, yield and nitrogen

At each harvest, all plants from each samplingregion within a plot (in total approx. 200 plants) werepooled, 20 plants were selected randomly and thenassessed for nodulation using a scoring system adaptedfrom that of Brockwell et al. (1982). This scoring sys-tem ranks nodulation for size of nodules as well as lo-cation of nodules on the root system. The nodulationscores for the 20 plants were averaged. Up to 25nodules were picked from each of the 20 plants (i.e., toa maximum of 500 nodules); these were stored at 48Cover granulated CaCO3 until the resident bacteriacould be typed.

The tops of the 20 plants were removed, bulked anddried at 708C for 48 h and the dry weights measured.Dry tops were milled ®nely and the total nitrogen wasdetermined using Kjeldahl digestion (Dalal et al.,1984), and ammonia determined by the method ofSearle (1984).

2.7. Nodule typing

Nodule isolates were typed using pH sensitivity andintrinsic antibiotic resistance patterns (Josey et al.,1979), and con®rmed using polymerase chain reactionwith directed primers (Richardson et al., 1995).Nodules were surface-sterilised by immersion in aqu-eous acidi®ed HgCl2 (0.01% w/v) for 0.5±2 min,depending on nodule size. Sterilised nodules were thenthoroughly washed at least six times with sterile deio-nised water (Vincent, 1970). Each nodule was crushedin a small drop of sterile deionised water with a sterileorange stick and the suspension patched onto TYagar. After 3±4 days incubation at 288C colonies wererespotted onto TY master plates and incubated for 3days at 288C. The master plates were then replica pla-ted onto minimal medium (Watkin et al., 1997) at pH4.35, 4.45, 4.70 and 7.0, or TY agar containing chlor-amphenicol (20 mg mlÿ1), or kanamycin (20 mg mlÿ1),or gentamycin (1.0 mg mlÿ1), or spectinomycin (20 mgmlÿ1). A TY plate was included at the end of each runto ensure adequate replication. Fifty nodules weretyped for each plot.

The identi®cation of the nodule isolates obtainedfrom the pH and antibiotic resistance pro®les werecon®rmed using polymerase chain reaction ampli®ca-tion pro®les (Richardson et al., 1995). The primerRPO1, a directed primer based on a reiterated Rhizo-bium nif promoter consensus element, was obtainedfrom BRESATEC and used following the protocols ofRichardson et al. (1995).

Strain NA3025, which forms large numbers of dis-tinctive, extremely small (1 mm dia.) nodules, was

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±14031396

identi®ed by nodule morphology in the the second andthe third years.

2.8. Dual nodule occupancy

For each plot, the exudates from ten sterilisednodules were streaked onto TY agar and incubated at288C for 3±4 days. Ten colonies from each nodulewere patched onto TY plates and incubated for afurther 3±4 days. These were then replica plated fortyping as described above.

2.9. Numbers of R. trifolii in the soil

The most probable number (MPN) of indigenousstrains of R. trifolii at the ®eld site prior to sowing in1992, and the population level of the six inoculantstrains of R. trifolii in the ®eld plot at the time of sow-ing in the second and third years, were determinedusing the plant infection test of Brockwell (1982)except screwcap tissue culture tubes (11 � 4 cm dia.)were used instead of test tubes. In the second year, rhi-zobial numbers in the soil at the point of original in-oculation were estimated at depths 0±2 cm and 2±5cm. In the third year, the 0±5 cm pro®les were bulked.The minimum level of detection for this assay is 11rhizobia gÿ1 soil with 95% con®dence limits (Woomeret al., 1988).

2.10. Presence of background R. trifolii

Samples of the top 5 cm of soil were collectedduring April 1992, prior to the break of season fromthree locations at the ®eld site and stored for 2 daysprior to mixing, air drying and sieving. The soil wasthen placed into 3 kg pots and watered to ®eld ca-pacity (13.5% w/v) with sterile, deionised water. Sur-face sterilised seeds of T. subterraneum cv. Dalkeithwere imbibed in sterile, deionised water for 4 h andgerminated on water agar at 258C for 48 h in the dark.Six germinated seeds were sown at a depth of 1 cm ineach pot, covered with soil and a 1 cm layer of sterilevermiculite. The pots were maintained in a root cool-ing tank at 21218C and watered to ®eld capacity withsterile water through watering tubes on every secondday. The plants were harvested at 6 weeks and rootsexamined for the presence of nodules.

3. Results and discussion

3.1. Background population of R. trifolii

No naturalised strains of R. trifolii were detected inthe soil samples collected from the ®eld site prior tosowing the experiment in 1992 i.e. nodules did notform on the roots of any of the plants used for theMPN estimation. In addition, none of the plants

grown in potted soil collected from the site were nodu-lated after six weeks growth in the glasshouse.

In the ®rst year of the experiment, 5% of cloverplants grown in the uninoculated plots were ine�ec-tively nodulated by naturalised strains of R. trifolii.This nodulation consisted of one or two white (ine�ec-tive), large nodules being present on the extreme lat-eral roots. Using intrinsic antibiotic resistance andgrowth on low pH agar plates, ®ve di�erent strainswere identi®ed from these isolates. Plants sown in thesecond and third years in uninoculated soil, not pre-viously sown with clover, were not nodulated by natur-alised strains.

Naturalised populations of R. trifolii are present inmany soils in the agricultural regions of south-westAustralia (Parker, 1962; Chatel and Parker, 1973b).Population density, e�ectiveness, and competitive abil-ity have been identi®ed as the primary characteristicsof indigenous rhizobial populations that a�ect inocu-lation responses (Thies et al., 1991). Naturalised popu-lations of root nodule bacteria can signi®cantly limitresponses to inoculation as long as the population con-tains some e�ective strains (Singleton and Tavares,1986).

The background population of R. trifolii in the soilat the site of this study comprised ®ve ine�ectivestrains with very poor competitive ability. Thesestrains were only detected in the uninoculated controlplants grown during the ®rst season of the experimentand were not detected in the subsequent seasons. Per-haps the dry, below average rainfall in 1993 and 1994reduced their limited capacity to nodulate the uninocu-lated clover. Clearly the background populations ofnaturalised strains of R. trifolii were not a signi®cantfactor in the performance of the inoculum strains inthis experiment. From this data, it would seem reason-able to propose that there was little competition forinoculum strains from the naturalised strains and as aconsequence the inoculant strains were able to easilynodulate the clover in the ®rst year of the experiment.

3.2. Movement of inoculum strains between plots

In the second year of the experiment, there was pro-li®c nodulation of sub-clover plants grown in plotswhich had previously been sown to an uninoculatedhost in the ®rst year (Table 2, Nil treatment). Typingof the isolates from these nodules consistently showedthat this nodulation was entirely due to the presence inthe uninoculated plots of the inoculant strains fromimmediately adjacent plots. The majority of isolatesfrom nodules in uninoculated plots were the strainthat had been inoculated into the adjacent plot on theeast side. This indicates that a consistent pattern ofmovement occurred mostly in an east to west direc-tion, possibly caused by the strong easterly winds in

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±1403 1397

summer moving dry soil from plot to plot. The factthat there was no evidence for movement of strains toplots not previously sown with sub-clover (Table 3,Nil treatment) may indicate that the presence of senes-cent plant material above ground may trap the drydust and enhance colonisation of the soil. In addition,presence of senescing roots of sub-clover may havepromoted the establishment of the rhizobia in theseplots.

3.3. Persistence of inoculant strains in the presence ofthe host plant

In the ®rst year of the experiment, all inoculatedplants had abundant nodules on their crown roots.Typing of the isolates from these nodules using intrin-sic antibiotic resistance and pH sensitivity pro®lesshowed that all the nodules produced in an inoculatedplot were formed by the inoculum strain (data notshown). The persistence of the inoculant strains in the

soil at the site of inoculation was assessed over a threeyear period in two ways. Firstly, by MPN counts ofpopulations of inoculum strains in soil at the point oforiginal inoculation collected at the time of sowing insecond and third years. Secondly, by examining thenodulation of plants sown in the second and thirdyears in the original inoculated row. The percentage ofplants nodulated by inoculant strains in years sub-sequent to initial inoculation and sowing is generallyconsidered a measure of persistence at the site of in-oculation (Chatel and Greenwood, 1973; Howieson,1995a).

The MPN data show that at the time of sowing inthe second year of the experiment strains NA3039,NA3025 and WSM409 were present in the soil at morethan 103 cells/g of soil in the top 0±2 cm and at adepth of 2±5 cm. Strain TA1 was present at 102 cells/gof soil in the surface horizon and could not bedetected at 2±5 cm depth. Strain NA3001 was justdetectable in the top 0±2 cm of soil at 11 cells/g of soil

Table 2

The percentage of plants nodulated and nodule score in the original row in the second and third year for the six strains of Rhizobium legumino-

sarum bv trifolii and the uninoculated controla

Inoculant strain Second year Third year

Percentage of plants nodulated Nodule score Percentage of plants nodulated Nodule score

NA3001 78.8a (22.1) 3.7b (1.4) 71.7abc (27.9) 4.5ab (0.6)

NA3025 87.5ab (18.9) 6.6a (3.6) 72.0abc (30.4) 5.2b (1.7)

NA3039 98.8b (2.5) 5.8a (1.4) 77.5ab (33.0) 5.8b (1.6)

TA1 86.7ab (5.8) 2.3c (2.1) 58.8c (33.3) 3.5a (1.4)

WSM409 97.5b (5.0) 5.6a (1.3) 70.6abc (24.0) 4.7ab (0.5)

WU95 78.8a (18.9) 5.8a (1.3) 86.7a (12.6) 4.5ab (0.7)

Nil 96.3b (4.8) 5.9a (0.3) 53.3c (18.9) 4.5ab (0.7)

a Values are the means of four measurements with standard deviations in parenthesis. Values in each column followed by the same letter are

not signi®cantly di�erent (P = 0.05). Analyses of variance were performed on the arcsin transformation of percentage of plants nodulated raw

data and on the square root transformation of nodule score raw data.

Table 3

The percentage of plants nodulated and nodule score in the original row in the second and third year for the six strains of Rhizobium legumino-

sarum bv trifolii inoculated into the soil in the ®rst year in the absence of a host and the uninoculated controla

Inoculant strain Second year Third year

Percentage of plants nodulated Nodule score Percentage of plants nodulated Nodule score

NA3001 25.0a (28.6) 1.3a (1.1) 15.8c (13.6) 2.3ab (1.7)

NA3025 60.0c (39.4) 8.0c (4.8) 63.7b (24.4) 3.4ab (0.4)

NA3039 91.3b (17.5) 5.0b (1.8) 80.8ab (20.1) 4.4bc (0.5)

TA1 33.8a (37.7) 1.9a (2.6) 33.6c (32.1) 2.7ab (1.4)

WSM409 92.5b (11.9) 4.8b (1.0) 84.7a (11.7) 3.5ab (1.4)

WU95 95.0b (10.0) 6.1bc (2.6) 77.5ab (27.2) 5.8c (0.8)

Nil 1.3d (2.5) 1.0a (1.0) 9.1c (12.9) 4.0abc (4.0)

a Values are the means of four measurements with standard deviations in parenthesis. Values in each column followed by the same letter are

not signi®cantly di�erent (P = 0.05). Analyses of variance were performed on the arcsin transformation of percentage of plants nodulated raw

data and on the squareroot transformation of nodule score raw data.

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±14031398

but was present at 2� 104 cells/g soil at 2±5 cm. StrainWU95 was not detected in soil from plots inoculatedwith this strain in the ®rst year. There were cleardi�erences between the strains in their capacity tomaintain populations in the soil during the long, drysummers.

WSM409 and NA3025 were the only strains presentin detectable numbers in soil collected at the time ofsowing in the third year of the experiment at 26 and11 cells/g soil. The third year of the experiment wasdelayed by a late break of season and sown six weekslater in the year than in the two previous seasons. Thesurviving inoculant bacteria were, therefore, subjectedto a longer period of desiccation stress in the thirdyear and this may have been a signi®cant factor redu-cing the populations of inoculant strains present in thesoil. It might be anticipated that low populations ofsurviving inoculant may reduce early e�ective nodula-tion of regenerating clover under these conditions. Thedetectable populations of WSM409 and NA3025 indi-cate that these strains were present in greater numbersin the soil after a period of considerable desiccationand the acid-soil tolerance of these two strains. R. tri-folii was not detected in uninoculated soil collected atthe time of sowing in the second and third years of theexperiment.

Typing of isolates from nodules, formed on plantssown in inoculated plots, showed that the nodulationof these plants in the second year was almost entirelydue to the inoculant strain. The percentage occupancyof nodules from inoculated plots due to the inoculantstrain ranged from 74% for strain NA3001 to 97% forstrain WU95. Low levels of dual nodule occupancywere observed in the second year of the experimentranging from 1.3% for strain NA3001 to 6.7% forstrain NA3039. The co-occupant strain was not ident-i®ed in these cases. Thus, the nodules formed on plantssown in the second year in plots inoculated in the ®rstyear were predominantly formed by the strain addedto the soil in the ®rst year. The inoculum strains domi-nated the nodulation of clover plants in inoculatedplots in all three years of the experiment.

In the second year, the plots inoculated with strainsWSM409 and NA3039 had a signi®cantly higher per-centage of plants nodulated in the original row thanthose plots inoculated with strains WU95 and NA3001(Table 2). The lower nodule scores for strains TA1and NA3001 in the second year (Table 2) were a resultof nodules only being formed by these two strains onthe lateral roots. Strains WSM409, WU95, NA3025and NA3039 all produced high nodule scores (Table 2)as a consequence of the nodules being positioned onthe crown and upper lateral roots.

In the third year, strain WU95 nodulated a signi®-cantly higher proportion of plants in the originalinoculated row than TA1 (Table 2). Strains NA3039

and NA3025 had higher nodule scores than strain TA1(Table 2).

Strains WSM409 and NA3039 performed as well as,or better than, the commercial inoculant strain WU95in the plots sown each year to sub-clover. These threestrains clearly have the capacity to stongly colonise theacid-soil at the site of inoculation and persist fromyear to year in long term pastures. The poorer per-formance of strains NA3001, and TA1 in persisting atthe site of inoculation indicates that these two strainsmay be impaired in their capacity to colonise this acid-soil from the site of inoculation. For TA1, this may in-dicate acid-soil sensitivity and for NA3001 this may beeither a consequence of its ine�ectiveness or an indi-cation of acid-soil sensitivity in this strain that is toler-ant of acidity in the laboratory.

3.4. Persistence of inoculant strains without the hostplant

The survival of strains in the acid-soil in the absenceof their host plant was examined by inoculating plotsin the ®rst year with peat inoculum alone. Sterilisedclover seeds were subsequently sown in the inoculatedrows in the second and third years, and the nodulationof these plants was used to assess the persistence ofthe inoculum. In this situation, strains WSM409,WU95 and NA3039 nodulated a signi®cantly higherpercentage of plants in the second year at the site ofinoculation than the other strains (Table 3); they hadalso signi®cantly greater nodule scores than strainsNA3001 and TA1 in the second year (Table 3). Inthese plots, strains WSM409, WU95 and NA3039formed medium sized nodules (2±5 mm dia.) on bothcrown and lateral roots (early nodulation possibly in-dicative of a larger population of these strains). Incontrast, strains NA3001 and TA1 produced nodulesonly on lateral roots (late nodulation indicative of asmall population). The high nodule scores recorded forstrain NA3025 were due to the formation of numerousvery small, white nodules (1 mm dia.) on the crownregion of the roots.

In the third year, after the absence of a host plant inthe ®rst year and the presence of clover in the secondyear, strains TA1 and NA3001 produced a signi®cantlylower percentage of nodulated plants (Table 3). StrainsWU95 and NA3039, produced crown nodules, whereasthe other strains only formed nodules on lateral roots.

The six strains R. trifolii di�ered in their persistencein the acid-soil for two years without clover being pre-sent. In this situation, the level of persistence shownby each strain was similar to that shown by the strainwhen the host plant was absent for only one year.Strains WSM409, WU95, NA3039, and NA3025 nodu-lated a signi®cantly greater percentage of plants thanstrains NA3001 and TA1 (Table 4). Strains WU95,

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±1403 1399

WSM409, and NA3025 produced crown nodules andstrains NA3001, NA3039, and TA1 formed nodulesonly on the lateral roots. Strain WU95 had a signi®-cantly larger nodule score than strains NA3001,NA3039, and TA1 (Table 4).

Previously, Gemell and Roughley (1993) found thatstrain NA3001 persisted poorly in an acid-soil inNSW, and they attributed the poor survival of thisstrain to competition from indigenous strains. In thestudy reported here, the poor performance of strainNA3001 was clearly not related to strong competitionfrom the background strains in the soil at the time ofestablishing this experiment. In addition, the presenceof other inoculant strains in plots in the second andthird years of the experiment did not provide strongcompetition for strain NA3001 because 75±85% ofnodules formed on plants sown in plots inoculatedwith NA3001 contained this strain. Thus, the poor per-formance of strain NA3001 in this Western Australiansoil was more likely to have been due to its poor survi-val. The same strain has been reported to persist in anacid-soil in Victoria (Slattery pers. com.), indicatingthat di�erences in background rhizobial populationsand soil characteristics between sites can a�ect strainsurvival.

3.5. Colonisation of acid-soil by R. trifolii

Colonisation of soils by the inoculum strain of rootnodule bacteria is essential for the establishment ofproductive pastures because of the need for e�ectivenodulation of plants located at points distant from thesite of inoculation. The colonisation of the acid-soil bythe six inoculant strains was assessed in the secondand third years of the experiment by observing thenodulation of clover plants sown in cross rows at dis-tances from the original inoculated row. There wereconsiderable di�erences between strains in their abilityto colonise the acid-soil and nodulate plants at a dis-tance from the original site of inoculation. Strains

WSM409, NA3039, and WU95 were the most success-

ful strains in colonising the acid-soil and strains TA1

and NA3001 were the poorest colonisers. In the second

year of the experiment, strains NA3039 and WU95

nodulated a signi®cantly greater percentage of plants

than strains NA3001, NA3025 and TA1 in the zones

at distances of 1±10 cm and 11±20 cm from the orig-

inal inoculated row (Table 5). At 21±50 cm, strain

NA3039 nodulated a signi®cantly higher percentage of

plants than strains TA1, NA3001, and NA3025

(Table 5). Strains WU95, NA3039, and WSM409 pro-

duced signi®cantly higher numbers of nodules at 21-50

cm than strains TA1, NA3001 and NA3025 (Table 5).

Plants were poorly nodulated when sown in cross rows

at distances greater than 11 cm from the original

inoculated row on plots inoculated with strains TA1,

NA3001 and NA3025 (Table 5).

The results from the third year of the experiment

were compromised by an extremely dry year with only

154 mm of rainfall compared to an average annual

rainfall of 315 mm. This resulted in very poor estab-

lishment and survival of clover sown in this season. As

a consequence, statistical analyses could not be per-

formed on results because of the missing data. How-

ever, observations of the pattern of nodulation in the

cross rows provided evidence for the survival of strains

in this extremely dry season. All strains except

NA3025, were able to nodulate plants at 1±10 cm

(Fig. 2); strains NA3001, WSM409, and NA3039

nodulated plants at 11±20 cm (data not shown) and

strain WSM409 was the only strain to nodulate plants

sown at a distance of 21±50 cm from the original

inoculated row. This strongly indicates that this strain

was the best at colonising the acid-soil at a substantial

distance from the point of inoculation. The success of

WSM409 in the third year of the experiment may have

also been a result of the ability of this strain to rapidly

nodulate young roots. Early nodulation results in

more productive growth of roots and may have

Table 4

The percentage of plants nodulated and nodule score in the third year in the original row for the six strains of Rhizobium leguminosarum bv. tri-

folii and the uninoculated control inoculated into the soil in the ®rst year in the absence of a host. Host also absent in the second yeara

Strains Percentage of plants nodulated Nodule score Number of replicates

NA3001 28.7b (33.6) 3.1a (1.9) 4

NA3025 76.0a (25.7) 4.3ab (1.5) 3

NA3039 69.3a (21.8) 4.1a (1.0) 3

TA1 43.9b (26.4) 2.9a (2.6) 3

WSM409 78.4a (32.6) 4.5ab (0.4) 4

WU95 77.4a (21.8) 5.9b (2.0) 3

Nil NG NG

a Values are the means of four measurements with standard deviations in parentheses. NG = plants did not grow in any of the four replicates.

Values in each column followed by the same letter are not signi®cantly di�erent (P = 0.05). Analyses of variance were performed on the arcsin

transformation of percentage of plants nodulated raw data and on the squareroot transformation of nodule score raw data.

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±14031400

assisted the survival of plants during the water stressthat occurred later in the third year.

3.6. Colonisation of the soil in the absence of a host

A more extreme test of the ability of R. trifolii tosurvive and colonise the acid-soil was the situationwhere plots were inoculated in the ®rst year in theabsence of the host and then sown with sterilised clo-ver in the second year. Plots containing strains WU95,WSM409, and NA3039 had signi®cantly more plantsnodulated at 1±10 cm than strains TA1, NA3001 andNA3025 (Table 6). Plants nodulated by NA3039 had asigni®cantly higher nodule score than NA3001,NA3025, TA1, and WSM409, (Table 6), with nodulesformed on the crown and lateral roots. Strains TA1and NA3001 had signi®cantly lower nodule scores

than the other strains (Table 6) with nodules onlyforming on lateral roots. In the 11±20 cm region strainNA3039 had signi®cantly better nodulation thanstrains NA3001, NA3025, and TA1 (Table 6) andstrains TA1 and NA3001 had the lowest nodule scores(Table 6).

The data from the third year of the experimentcould not be statistically analysed because the poorsurvival of plants resulted in large amounts of missingdata. However, some plants did survive and nodulatein the cross rows at a distance from the original row.In plots where the host was absent in the ®rst year,only strains WSM409, WU95, NA3025, and NA3039nodulated 30% or more of plants in the 1±10 cmsampling region (Fig. 2). In the plots, where the hostwas absent in the second and third years strainsNA3039, WSM409, and WU95 produced more than50% plant nodulation at 1±10 cm (Fig. 2), whilst inthe 21±50 cm region only strains WSM409 andNA3039 produced any nodulation at all (data notshown).

3.7. E�ectiveness of inoculant strains

As a measure of e�ectiveness of nitrogen ®xation,total nitrogen of the clover plants at the ®rst year har-vest, was expressed as percent nitrogen and mg nitro-gen per plant (Table 7). Clover plants inoculated withWU95, WSM409, and TA1 produced similar nitrogenconcentrations. However, when expressed as nitrogenaccumulation per plant, those plants inoculated withWSM409 had the highest amount followed by WU95and then plants inoculated with TA1. Plants inocu-lated with NA3025 contained a lower percentage ofnitrogen and lower amount of nitrogen per plant thanthe uninoculated controls (Table 7).

Acid-soil-tolerant and acid-soil sensitive strains of R.trifolii have been identi®ed using a ®eld experimentbased on the cross-row technique to determine the

Table 5

The percentage of plants nodulated and nodule score at 1±10, 11±20 and 21±50 cm from the original row in the second year for the six strains of

Rhizobium leguminosarum bv trifolii and the uninoculated controla

Inoculant strain Percentage of plants nodulated Nodule score

1±10 cm 11±20 cm 21±50 cm 1±10 cm 11±20 cm 21±50 cm

NA3001 53.8a (28.1) 36.3a (39.3) 25.0ab (26.5) 3.6ab (1.3) 1.6b (1.4) 1.9b (1.3)

NA3025 70.0ab (28.3) 41.2ab (27.8) 26.3ab (42.7) 2.7b (1.8) 3.1a (1.3) 1.5b (1.2)

NA3039 88.8c (9.5) 71.2c (24.6) 55.0c (37.4) 4.3a (1.8) 3.8a (2.3) 3.5a (2.1)

TA1 63.3ab (17.6) 33.3a (10.4) 20.0a (13.2) 3.1ab (0.9) 1.1b (0.6) 0.7b (0.2)

WSM409 77.5bc (11.9) 57.5bc (15.0) 38.8bc (23.2) 4.0ab (1.2) 3.7a (1.0) 3.1a (1.0)

WU95 93.8c (7.5) 72.5c (18.5) 41.3bc (19.3) 4.4a (1.3) 3.8a (1.4) 3.1a (1.6)

Nil 58.8a (16.0) 41.3ab (11.1) 17.5a (14.4) 3.0ab (0.4) 3.6a (0.9) 1.4b (0.5)

a Values are the means of four measurements with standard deviations in parenthesis. Values in each column followed by the same letter are

not signi®cantly di�erent (P = 0.05). Analyses of variance were performed on the arcsin transformation of percentage of plants nodulated raw

data and on the square root transformation of nodule score raw data.

Table 6

The percentage of plants nodulated and the nodule score at 1±10,

and 11±20 cm from the original row in the second year for the six

strains of Rhizobium leguminosarum bv trifolii and the uninoculated

control inoculated into the soil in the ®rst year in the absence of a

hosta

Inoculant Percentage of plants nodulated Nodule score

strain 1±10 cm 11±20 cm 1±10 cm 11±20 cm

NA3001 33.8bc (35.0) 13.8ab (24.3) 0.9a (0.8) 0.9a (1.2)

NA3025 40.0c (31.1) 11.3ab (13.2) 3.1b (2.2) 4.3cde (3.4)

NA3039 75.0d (28.0) 33.3d (30.8) 5.5c (2.0) 1.8bc (1.5)

TA1 16.3ab (2.5) 21.3abc (11.9) 1.4a (0.8) 1.3ab (1.1)

WSM409 61.3d (43.3) 30.0cd (14.7) 3.2b (2.8) 2.9cde (1.6)

WU95 62.5d (35.2) 25.5bcd (15.4) 3.9bc (1.5) 2.4bcd (1.4)

Nil 6.25a (7.5) 6.25a (6.3) 3.0b (0.4) 3.6cde (0.9)

a Values are the means of four measurements with standard devi-

ations in parenthesis. Values in each column followed by the same

letter are not signi®cantly di�erent (P = 0.05). Analyses of variance

were performed on the arcsin transformation of percentage of plants

nodulated raw data and on the squareroot transformation of nodule

score raw data.

E.L.J. Watkin et al. / Soil Biology & Biochemistry 32 (2000) 1393±1403 1401

ability of the strains to persist and colonise the soil.Strains WSM409 and NA3039 were better colonisersof the acid-soil (pH 4.2) than the commercial inoculumstrain WU95, and the two acid tolerant strains weremore e�ective in the ®eld than WU95. The data fromthis study and from work determining the broad hostrange of WSM409 (Howieson et al., 2000) providesclear support for the selection of WSM409 as an acid-soil tolerant inoculum strain for a range of Trifoliumspp. to replace WU95. The use of WSM409 should

increase the productivity of clover-based pastures onacidic-sandy soils in southern Australia. The character-isation described here of acid-soil sensitive and acidsoil-tolerant strains of R. trifolii provides resources forlaboratory-based investigations on the basis of acid-soil tolerance in R. trifolii.

Acknowledgements

We gratefully acknowledge research support fromthe Grains Research and Development Corporation.

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