P u b l i s h i n g

Australian Journal of Soil ResearchCSIRO PublishingPO Box 1139 (150 Oxford St)Collingwood, Vic. 3066, Australia

Telephone: +61 3 9662 7628Fax: +61 3 9662 7611Email: publishing.sr@csiro.au

Published by CSIRO Publishing for CSIRO and the Australian Academy of Science

w w w . p u b l i s h . c s i r o . a u / j o u r n a l s / a j s r

All enquiries and manuscripts should be directed to:

Volume 40, 2002© CSIRO 2002

Australian Journalof Soil Research

An international journal for the publication oforiginal research into all aspects of soil science

Aust. J. Soil Res., 2002, 40, 1319–1329

© CSIRO 2002 0004-9573/02/080131910.1071/SR01052

Diversity and symbiotic effectiveness of Rhizobium leguminosarum bv. trifolii isolates from pasture soils in south-western Australia

M. T. CollinsA, J. E. ThiesAB, and L. K. AbbottAC

ACentre for Legumes in Mediterranean Agriculture (CLIMA) and School of Earth and Geographical Sciences, Faculty of Natural and Agricultural Sciences, The University of Western Australia,Crawley, WA 6009, Australia.

BDepartment of Crop and Soil Sciences, Cornell University, Ithaca, NY 14853, USA.CCorresponding author; email: labbott@cyllene.uwa.edu.au

Abstract

The abundance of the Australian inoculant strain of Rhizobium leguminosarum bv. trifolii for subterraneumclover (WU95) and the diversity of naturalised rhizobia were assessed in 3 subterranean clover pastures inthe Albany region of south-western Western Australia. Most probable number, enzyme linkedimmunosorbent assay (ELISA), and polymerase chain reaction (PCR) techniques were used. A putativestrain similar to inoculant strain WU96 was uncommon at one site (South Stirling) and not isolated at 2other sites. Randomly amplified polymorphic DNA (RAPD) PCR fingerprinting using the RPO1 primeridentified 45 different profiles amongst the 208 isolates examined. RAPD-PCR fingerprinting using theprimers RPO4 and RPO5 confirmed most groupings based on RPO1 fingerprint patterns and revealedfurther genetic diversity within some groups. Overall, 54 putative strains were defined by RAPD-PCRfingerprint profiles across the 3 sites. Subterranean clover rhizobia at the Manypeaks and Mount Shadforthsites were dominated by isolates with 1 or 2 RPO1 RAPD profiles at 2 sampling times, while the populationat South Stirling was much more diverse. The symbiotic effectiveness of 11 rhizobial isolates, representingthe major RPO1 RAPD profile groups within naturalised rhizobial populations, were compared in potculture with those of the 2 commercial inoculant strains for subterranean clover, WU95 and TA1, on 3cultivars. Differences in effectiveness among 3 of the 11 isolates were observed in comparison to both thecommercial strains and other naturalised isolates. The nitrogen fixing effectiveness of 8 isolatesrepresenting different subgroups from one RP01 group was not the same. The use of all 3 primers increasedthe precision in defining putative strains of Rhizobium leguminosarum bv. trifolii, and although naturalisedrhizobia from these pastures are saprophytically competent, their dominance in nodules does not appear tobe linked to symbiotic effectiveness.

Additional keywords: subterranean clover, rhizobia, diversity, MPN, PCR, ELISA.

SR01052Diver si ty of r hizobi aM. T. Coll inset al .

Introduction

In south-western Australia, subterranean clover (Trifolium subterraneum L.) has been usedas a pasture legume for many years (Chatel and Parker 1973; Chatel et al. 1973). Assubterranean clover is not native to Australia, there are typically no native rhizobia presentin uncultivated field soil that are compatible with this species. Consequently, new standsrequire inoculation to establish the symbiosis. Once established, subterranean cloverpastures are infrequently re-inoculated and farmers generally rely on naturalised rhizobiafor clover nodulation and N2 fixation. Thus, the size of the resident rhizobial populationsand their competitiveness and effectiveness are important for the nitrogen (N) nutrition ofthese pastures (Gibson et al. 1976; Howieson 1995). A survey of the symbioticperformance of legumes in annual pastures in the lower south-western region of WesternAustralia showed that almost a third of sites had low levels (<50%) of plant N derived fromthe atmosphere (Sanford et al. 1993). An assessment of the combined effectiveness ofrhizobia in soil suspensions from 9 of these sites demonstrated the potential to select moreeffective stains for use as inoculants (Unkovich and Pate 1998).

1320 M. T. Collins et al.

One of our aims was to determine if strains WU95 or TA1 of Rhizobium leguminosarumbv. trifolii were present at 3 sites across an annual rainfall gradient of nearly 1200 mm to<600 mm. TA1 had been used prior to WU95 as an inoculant for subterranean clover inWestern Australia (D. Pulseford, pers. comm.). None of the sites had been regularlyinoculated. In addition, we assessed seasonal population size and strain diversity andeffectiveness at each site. We used molecular techniques to detect the presence of inoculantand background strains of rhizobia (Thies et al. 2001).

Three pasture sites were selected across the rainfall isotherms investigated by Sanfordet al. (1993). The abundance of rhizobia at the 3 study sites was low but highly effective atSouth Stirling, high but ineffective at Manypeaks, and high and highly effective at theMount Shadforth site (Unkovich and Pate 1998). We examined rhizobial diversity at thesesites using the enzyme linked immunosorbent assay (ELISA) technique with antisera to theinoculant strain WU95 to determine the presence of this inoculant strain and the randomlyamplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique ofRichardson et al. (1995) with the directed primer RPO1, and random primers RPO4 andRPO5. The RAPD-PCR technique was used by Hebb et al. (1998) to follow the fate ofinoculant strains in the field and was found to be a rapid and reliable method forcharacterisation at the strain level. This technique has also been successfully used todemonstrate a high level of diversity among isolates of Rhizobium leguminosarum bv.viceae (Handley et al. 1998).

In addition to the seasonal flux in population size and diversity, we compared thenitrogen fixing effectiveness of isolates representing some of the major RAPD-PCR groups(defined by the primer RPO1) against the inoculant strains WU95 and TA1. Twoexperiments were carried out to determine whether these groups represented effectivenessgroups with potential as inoculant strains in this region.

Materials and methods

Study sites were located in the Albany region of lower south-western Australia at South Stirling(400–600 mm annual rainfall), Manypeaks (600–800 mm annual rainfall), and Mount Shadforth(1000–1200 mm annual rainfall). Each site had been cleared for over 30 years. They were acid soils withno recent inoculation history (Table 1). At each site, 30 soil cores 2 by 10 cm were taken within a 30 by30 m grid. Soil cores were combined into 2 replicate samples per site, sieved (2 mm), and mixed. Particlesize analysis was determined (McArthur 1991) and soil pH was measured in 0.01 M CaCl2. Soil rhizobialpopulations were enumerated at 3-monthly intervals over 1 year (August 1993–August 1994) in thecomposite soil samples from each site using Trifolium subterraneum cv. Trikkala and T. subterraneumcv. Dalkeith as MPN bait plants (Somasegaran and Hoben 1985).

Rhizobial isolation

Nodules were collected for isolation of rhizobia from high dilution MPN tubes at 2 sampling times (May1994 and August 1994) and a random selection was collected from subterranean clover in the field inAugust 1994. The May soil sampling occurred before the commencement of autumn rains when fewsubterranean clover plants were present. High dilution MPN tubes were chosen as a source of isolates toobtain representatives of the major components of each population. Rhizobia were isolated from field plantsand MPN tubes (Somasegaran and Hoben 1985) after nodules were surface sterilised in 5% sodiumhypochlorite for 3 min. Pure cultures were authenticated on axenically grown T. subterraneum cv. Trikkalain agar slopes. Isolates were stored at 4°C on yeast mannitol agar (YMA) slopes. All isolates were freshlysubcultured prior to characterisation by ELISA and RAPD-PCR.

ELISA

ELISA was carried out using polyclonal antisera to WU95 and isolates grown on YMA slopes accordingto the technique of McInnes (2002). Colour development was measured in a Titertek Multiskan Plus

Diversity of rhizobia 1321

microplate reader (Flow Laboratories, North Ryde, NSW). Culture collection isolates of strains WU95 andWU425 were used as positive and negative controls, respectively. ELISA was performed using cell wallpreparations at a minimum OD capable of completely blocking all antigen-binding sites in the wells(determined experimentally to be OD >0.9) and WU95-specific rabbit antisera.

RAPD-PCR

DNA templates were prepared by suspending 1 inoculation loop-full of 3-day-old YMA culture in 50 µL ofsterile Milli-Q water in a 1.5-mL Eppendorf tube. Tubes were subjected to 3 cycles of repeated heating(100°C) and cooling (0°C) in water baths. Cell debris was removed by centrifugation (14000 rpm, 2 min)and the supernatant used in subsequent amplification reactions. RAPD-PCR amplifications using the RP01primer (a 20-base oligonucleotide primer corresponding to a conserved nif H gene promoter region, RPO4and RPO5, both 10-base random primers) were performed (Richardson et al. 1995) with the omission ofgelatin from the reaction mixture. RPO1 was used at 75 ng/10 µL reaction and RPO4 and RPO5 at150 ng/10 µL reaction (concentration based on manufacturers claims). Amplification products wereseparated on 3% agarose gels, stained with ethidium bromide, visualised on a UV lightbox, andphotographed with Polaroid 660 film. The RPO1 RAPD-PCR profiles generated were then used to groupthe isolates according to their gel banding patterns. The RPO1 RAPD-PCR groups were confirmed byrepeating (in parallel) the amplification of isolates with similar gel banding patterns. RPO1 groups wererun in parallel with the random primers RPO4 and RPO5. RPO4 and RPO5 groups were again confirmedby repeating amplifications in parallel. Differences based on minor banding differences (faint bandsdifferent only) were not considered to represent different strains.

Effectiveness testing

Two effectiveness tests were performed. Rhizobial isolates were randomly selected for testing from withineach of 11 RPO1 RAPD-PCR groups. The groups were chosen as representative of the major populationconstituents on the basis that each constituted at least 10% of the isolates from at least 1 site at 1 samplingtime (Table 2). Where a group occurred at more than 1 sampling time or site, an isolate was selected fromthe sampling time or site with the largest number of isolates from that group. Isolates MP3.3, SS1.2, MPTrik-7.1, SS Dal-5.2, MTS2-7.2, MTS2-9.2, SS1-3.3, SS2-3.1, SS2-4.2, SS2-6.1, and SS2-7.1 were testedin parallel with the 2 inoculant strains, WU95 and TA1. WU95 is the current commercial inoculant in WAfor subterranean clover and TA1 was the inoculant prior to 1972.

The 11 putative rhizobial strains and 2 commercial inoculant strains were examined for their ability tonodulate and fix nitrogen with 3 subterranean clover cultivars (Dalkeith, Trikkala, and Gosse) (Expt 1).There were 3 replicates of each host/strain combination, along with uninoculated and N-fertilised controls.Pots were arranged in randomised blocks in a daylight phytotron maintained at 25°C/20°C (day/nighttemperatures).

In the subsequent effectiveness test (Expt 2), isolates were selected from 2 RPO1 groups tested in thefirst experiment that had either very poor or very good nitrogen fixing effectiveness. This was done todetermine if RPO1 groups were linked with the nitrogen fixing effectiveness of the bacteria. The mosteffective RPO1 group (26) contained 84 isolates. Two isolates from each of 4 RPO4/RPO5 subgroupswithin this group were selected for further testing (MP1-8.3 and MTS2-7.2; MP1-4.2 and MTS1-7.3; MP2.1 and MTS 2.4; and MTS 4.1 and MTS Trik-5.2). The least effective group (7) contained only 2 isolates

Table 1. Site information in south-western Australia (Albany Region)Based on Unkovich and Pate (1998)

Site Soil pH (CaCl2)

MPN Annual rainfall 1992 (mm)

%Ndfa Sept. 1992

Site inoculation and cropping history

South Stirling 4.6 00404 0600 99 Cleared and probably inoculated in 1954, pasture, infrequently cropped, reinoculated early 1970s

Manypeaks 5.0 13500 0650 63 Permanent pasture, never inoculated

Mount Shadforth 4.9 70600 1200 94 Permanent pasture, unknown inoculation history

1322 M. T. Collins et al.

(SS2-3.1 and SS2-3.2) and both had the same RAPD-PCR gel profiles using RPO4 and RPO5. All isolateswere examined in parallel with the inoculant strains WU95 and TA1 for their ability to nodulate and fixnitrogen with 1 subterranean clover cultivar (Trikkala). There were 3 replicates of each host/straincombination, uninoculated and N-fertilised controls. Pots were arranged in randomised blocks in a daylightphytotron maintained at 25°C/20°C (day/night temperatures).

Plant growth conditions

Plants were grown in free-draining plastic pots (1.1 L) that had been surface disinfected by soaking in 70%ethanol and drying. Sterile paper towels were inserted aseptically in the base of the pots to prevent loss ofsoil mix and pots were filled with a steamed soil mixture (1:2, river sand/yellow sand). The soil mixturewas leached 3 times with hot sterile distilled water to remove any residual N and allowed to drain. Surfacedisinfected (70% ethanol) subsurface watering tubes (25 by 150 mm) were inserted in pots and capped.After sowing, pots were covered in alkathane beads (soaked in 70% ethanol and air-dried in the laminar flowovernight) and watered with a sterile incomplete nutrient solution (minus N). Sterile incomplete plantnutrient solution (Howieson et al. 1995) and fertiliser N solutions 5 g/L KNO3 were applied twice weeklyto appropriate pots. Pots were watered every second day with sterile deionised water.

Preparation of inoculum

The selected rhizobial isolates and 2 inoculum strains (WU95 and TA1) were streaked over 4 YMA platesand incubated at 28°C for 3–5 days, until confluent growth had occurred. For each of the isolates, thebacteria from 4 plates were suspended in 60 mL of sterile 0.001 M phosphate buffer (pH 7.2). Each vial was

Table 2. Origins of rhizobial isolates examined for symbiotic effectiveness in Expts 1 and 2F indicates field isolate

Isolate Site of origin RPO1 profile

RPO4 profile

RPO5 profile

Putative strain

Expt 1

MP3.3 Manypeaks (F) 03 03c 03d 11SS1.2 South Stirling (F) 41 41a 41a 41MP Trik-7.1 Manypeaks 31 16a 16a 31SS Dal-5.2 South Stirling 14 03a 03a 14MTS2-7.2 Mount Shadforth 26 26a 26a 26MTS2-9.2 Mount Shadforth 36 36a 36a 36SS1-3.3 South Stirling 01 01a 01a 1 (WU95)SS2-3.1 South Stirling 07 07a 07a 07SS2-4.2 South Stirling 02 02a 2a 02SS2-6.1 South Stirling 09 09a 09a 09SS2-7.1 South Stirling 11 04a 04a 11WU95 Inoculant strain 01 01a 01a 01TA1 Inoculant strain 39 39a 39a 39

Expt 2

SS2-3.1 South Stirling 07 07a 07a 07SS2-3.2 South Stirling 07 07a 07a 07MP1-8.3 Manypeaks 26 26a 26a 26MTS2-7.2 Mount Shadforth 26 26a 26a 26MP1-4.2 Manypeaks 26 26a 26a 26MTS1-7.3 Mount Shadforth 26 26a 26a 26MP 2.1 Manypeaks (F) 26 26b 26b 26MTS 2.4 Mount Shadforth (F) 26 26b 26b 26MTS 4.1 Mount Shadforth (F) 26 26c 26c 26MTS Trik-5.2 Mount Shadforth 26 26c 26c 26WU95 Inoculant strain 01 01a 01a 01TA1 Inoculant strain 39 39a 39a 39

Diversity of rhizobia 1323

mixed by inversion until suspensions were homogeneous then stored at 4°C prior to use. Bacterialsuspensions were prepared and used on the day of seed sowing.

Sowing and harvesting procedures

Seeds were washed for 30 s in 100% ethanol, 30 s in 70% ethanol, surfaced sterilised for 5 min by shakingin 6.25% sodium hypochlorite, and rinsed 6 times in sterile deionised water. Seeds were incubated on wateragar plates (1% agar) at 20°C for 2 days. Six seeds with approximately 1 cm radicles were planted in eachpot and each seed was inoculated with 1 mL of the appropriate bacterial suspension. Plants were thinned to3 plants/pot after 14 days. Stringent aseptic handling techniques resulted in uninoculated controlsremaining nodule free. Plants were harvested after 42 days. Shoots, roots and nodules were separated,weighed and dried at 70°C for 2 days, then cooled and reweighed.

Results

The South Stirling soil contained almost no fine particulate material (approx. 1% combinedclay and silt) but Manypeaks and Mount Shadforth contained approximately 10% and 19%total fine particulate material. A decrease in the number of rhizobia in South Stirling soiloccurred during the summer and autumn (Fig. 1). The putative strain similar to inoculantstrain WU96 was present at South Stirling (Table 2) as a low percentage of noduleoccupants but was not isolated at the other sites. No difference in nodule occupancy by theputative strain similar to inoculant strain WU96 was found between nodules on plantsgrown in the MPN tubes or the field.

Rhizobial isolates were grouped according to visual similarities in RPO1 RAPD-PCRgel profiles. Subsequently, the groupings based on the primer RPO1 were compared withthose obtained by RAPD-PCR using the random primers RPO4 and RPO5. Isolates withsimilar gel banding patterns for each primer were regarded as belonging to the sameRAPD-PCR group giving a total of 54 putative strains from the 3 populations. Fewer strainswere present in nodules of field-grown plants at the August sampling than were detectedwithin the nodules of plants inoculated with the highest dilution from the MPN tubes

Sampling date

Aug.'93 Nov.'93 Feb.'94 May'94 Aug.'94

Rhizobial numbers at three sites

2.5

3

3.5

4

4.5

5

5.5

6

6.5

Num

ber

(log)

per

g s

oil

Fig. 1. Seasonal variation in abundance of Rhizobium leguminosarum bv. trifolii (no. per g of soil) inthree pasture soils in south-western Australia.

1324 M. T. Collins et al.

(Table 3). The strains present in nodules of field-grown plants were generally not the sameas those from MPN tubes with the exception of Groups 1, 2, 13, 14, 19, and 41 from SouthStirling, Group 3 from Manypeaks, and Groups 26 and 52 from Mount Shadforth (Table 4).

The highest diversity of rhizobia was observed at South Stirling, with 34 groupsdetected. The lowest diversity of gel profile groups occurred at the sites of greatest rainfallwhere 12 groups were detected among isolates from both Manypeaks and Mount Shadforth(Table 4). The populations at the latter 2 sites were dominated by 1 or 2 groups at eachsampling time (Table 4). At Manypeaks, the dominant RAPD-PCR group changed withsampling time and method. In May, Group 26 (76% of isolates) dominated the sampledpopulation, while in August, isolates in Group 3 (70%) were dominant in field nodules andGroups 3 (40%) and 31 (30%) dominated nodules formed in the high dilution MPN tubes.Isolates within Group 26 remained present amongst the August nodules taken from thefield but were a reduced proportion of the total (15%). Similarly, Group 3 was present innodules collected in May as a reduced proportion (11%). Group 31 appeared only amongstthe isolates from MPN tube nodules from the August sampling. The isolates from MountShadforth were dominated by one PCR profile at each sampling time, Group 26. This grouprepresented 72% of the sampled population in May and 80% of field isolates and 70% ofMPN tube isolates in August. The RAPD-PCR profile of WU95 was assigned to Group 1and was detected as a low proportion of isolates from the South Stirling population,confirming the ELISA results. TA1 (Group 39) was found only at Mount Shadforth as 5%of August isolates from MPN tubes.

The effectiveness of each isolate in increasing growth of subterranean clover wasgenerally similar for all 3 subterranean clover cultivars (Fig. 2a–c) but there were severalexceptions. For the cultivar Dalkeith, isolates MTS2-7.2, SS2-4.2, and MP3.3 were all moreeffective than WU95; and isolates SS2-6.1 and SS2-3.1 were ineffective. For Trikkala,isolates MTS2-7.2, MP3.3, SS1.2, and SS Dal-5.2 were more effective than WU95; andSS2-3.1, SS2-6.1, and SS2-7.1 were ineffective. For Gosse, all isolates were of similareffectiveness to WU95.

Within-group comparison of effectiveness showed that both isolates from RPO1 Group7, SS2-3.1 and SS2-3.2, were ineffective with Trikkala (Fig. 3). All isolates from the fourRPO4/RPO5 subgroups within RPO1 Group 26 were effective with Trikkala. The poorestperforming isolates were MTS 4.1 and MTS Trik-5.2; both of these isolates were from thesame RPO4/RPO5 subgroup.

Discussion

The abundance of Rhizobium leguminosarum bv. trifolii in 3 pastures in south-westernAustralia was adequate for nodulation throughout the year according to previous estimates

Table 3. Percentage of nodule isolates at each site identified as the strain WU95 by ELISA

Source of isolates indicated as either from MPN nodules or nodules collected from roots of field plants. Numbers in brackets indicate the number of isolates analysed

Site May 1994 August 1994MPN nodules MPN nodules Field nodules

South Stirling 10 (20) 5 (20) 5 (20)Manypeaks 00 (37) 0 (20) 0 (20)Mount Shadforth 00 (32) 0 (20) 0 (19)

Diversity of rhizobia 1325

Table 4. Number of isolates of each putative rhizobial strain identified at each field site in nodules formed on plants grown in MPN tubes (May,

August) and in field-collected nodules (August)Group 1 corresponded to WU95 by ELISA and RAPD PCR, and Group 39

corresponded to TAI by RAPD PCR. Sites were South Stirling (SS), Manypeaks (MP), and Mount Shadforth (MTS)

RAPD May August Augustgroup (MPN nodules) (MPN nodules) (field nodules)

SS MP MTS SS MP MTS SS MP MTS

01 02 01 0102 03 01 0103 04 08 01 01 1404 0205 0106 0107 0208 0109 0210 0111 03 0112 01 0213 01 0214 01 0215 0116 0317 0118 0119 01 0120 0121 0122 0123 0124 0125 01 0126 28 23 14 03 1627 0128 0129 0430 0231 0632 0133 0134 0135 0336 0437 0138 0139 0140 0241 02 0342 0143 0144 0245 0146 0147 0148 0149 0150 0151 0152 01 0153 0154 01

Total no. 20 37 32 19 20 20 19 20 20No. types 11 06 05 18 05 07 14 04 04

1326 M. T. Collins et al.

Gossel.s.d. = 0.032

Sho

ot d

ry w

eigh

t (g/

plan

t)

0.250

0.200

0.150

0.100

0.050

0.000

Isolate

+N −N 1 2 3 4 5 6 7 8 9 C1 C210 11

Trikkalal.s.d. = 0.032

Dalkeithl.s.d. = 0.032

0.250

0.200

0.150

0.100

0.050

0.000

0.250

0.200

0.150

0.100

0.050

0.000

(c)

(b)

(a)

Fig. 2. Symbiotic effectiveness (measured as shoot dry weight per plant) ofeleven rhizobial isolates representing major RPO1 RAPD profile groups isolatedfrom naturalised rhizobial populations of three pasture soils in south-westernAustralia compared with the two commercial inoculant strains, WU95 and TA1, onthree cultivars of subterranean clover: (a) Dalkeith, (b) Trikkala, and (c) Gosse.(P < 0.05, Fischer’s l.s.d.)

Diversity of rhizobia 1327

made for these root nodule bacteria in eastern Australia (Richardson and Simpson 1988;Slattery and Coventry 1993). The marked decrease in numbers of rhizobia at South Stirlingduring the autumn corresponded with that observed in East Gippsland, Victoria(Richardson and Simpson 1988), and in north-eastern Victoria for soils with more acidconditions (Slattery and Coventry 1993).

Based on the ELISA assay, the inoculant strain for subterranean clover, WU95, waspresent as a low proportion of isolates at 1 of the 3 sites (South Stirling) and absent at theother sites. The RAPD-PCR fingerprinting technique (Richardson et al. 1995) allowed usto distinguish 54 putative strains amongst the isolates across the 3 sites. Patternscorresponding to WU95 and TA1 were rare.

Differences in diversity of rhizobia isolated from subterranean clover pastures fromdifferent sites illustrate the potential complexity of the community of root nodule bacteriain soil and the unknown background into which inoculant strains are now introduced as hasbeen demonstrated for Trifolium leguminosarum bv. viciae (Handley et al. 1998). Thenumber of different RAPD-PCR profiles among the population of Rhizobiumleguminosarum bv. trifolii was greater at South Stirling than at the other sites. No straindominated this population at either sampling time. In contrast, the Mount Shadforthpopulation was dominated by one RAPD-PCR profile group at both sampling times,whereas the Manypeaks population was dominated by rhizobia of different RAPD-PCRprofile groups at each sampling time.

Considering that these soils were devoid of clover rhizobia prior to clover introduction,it is interesting to contemplate the potential source(s) of the diversity now observed in thesesoils. Strain variation may arise from migration of new strains carried on seed, farmimplements or dust, recombination events between introduced strains and non-symbioticrhizobia in soil (Sullivan et al. 1995, 1996), or clonal selection (Maynard-Smith et al. 1993).

Only isolates in Group 1 RAPD-PCR gel banding pattern responded positively to WU95antisera in the ELISA analyses. The consistency between the RAPD-PCR and ELISA

Trikkalal.s.d. = 0.032

Sho

ot d

ry w

eigh

t (g/

plan

t)

0.250

0.200

0.150

0.100

0.050

0.000

Isolate

+N -N 8 12 13 14 15 3 16 17 18 19 C1 C2

Fig. 3. Symbiotic effectiveness (measured as shoot dry weight per plant) of tenrhizobial isolates representing four RPO1 RAPD profile subgroups within oneeffective RPO1 RAPD profile group and both isolates from within anotherineffective group, isolated from naturalised rhizobial populations of three pasturesoils in south-western Australia, compared with the two commercial inoculantstrains, WU95 and TA1, on the subterranean clover cultivars Trikkala (P < 0.05,Fischer’s l.s.d.).

1328 M. T. Collins et al.

techniques confirmed that there was a low abundance of rhizobia in this soil populationcorresponding to the inoculant strain. This contrasts with the high level of recovery ofinoculant strains in recently inoculated legume pastures in eastern Australia (Hebb et al.1998). Isolates with the dominant RPO1 gel pattern (Group 26) present at the Maysampling from Manypeaks and both sampling times from Mount Shadforth were highlyeffective at increasing growth of subterranean clover in N-deficient soil. While this RPO1RAPD-PCR group was also present in the middle of the growing season (August) atManypeaks, 2 other groups (Groups 3 and 31) were dominant in nodules at that time. Group3 and Group 31 were similar in effectiveness. Group 31 was found only among isolates fromAugust sampling MPN tubes. Thus, the apparent population predominance by Group 31may be an artefact of isolating from MPN tubes rather than a reflection of the noduleoccupancy in the field soils. The poor N2 fixation in this pasture measured previously(Unkovich and Pate 1998) may be due to some other edaphic factor either suppressingnodulation or reducing the competitiveness of the effective strains that are present.

Further comparison of RAPD-PCR gel banding patterns using the primers RPO4 andRPO5 confirmed most of the RPO1 groupings but showed that some RPO1 groupscontained subgroups. The two isolates from the RPO1 RAPD-PCR Group 7 were extremelypoor N2 fixers, while a range of N2 fixing effectiveness occurred within the RPO1RAPD-PCR Group 26. Thus, the RPO1 groups did not define N2 fixing effectivenessgroups. However, only isolates from a subgroup with major gel band differences (isolatesMTS 4.1 and MTS Trik-5.2) had significantly different N2 fixing capacity to other RPO1RAPD-PCR Group 26 isolates tested.

Our effectiveness testing results indicated that saprophytically competent rhizobia werenot necessarily symbiotically effective. While the presence of symbiotically ineffectiverhizobia in nodules could account for the poor levels of plant N derived from theatmosphere in the previous pasture survey (Sanford et al. 1993), our research has shownthat this is not necessarily the case. At Manypeaks, the collections of isolates were biasedtoward poorly effective isolates in MPN tubes. Naturalised clover rhizobia populations havepreviously been found to consist primarily of ineffective or poorly effective strains (Holdingand King 1963; Bergersen 1970). There is evidence that the presence of the host can exerta selective effect and that clovers nodulated preferentially with effective symbionts in soilswith a mixed naturalised rhizobial population (Robinson 1969). Thus, effective strainscould eventually dominate naturalised populations, but the presence of ineffective strainsin the soil populations at South Stirling and Manypeaks indicates that effectiveness is notthe prime selective agent determining persistence in these subterranean clover pastures.The importance of genotype-specific selection between legumes and rhizobia is not wellunderstood but appeared to be unimportant for another Trifolium species (Hagen andHamrick 1996).

Acknowledgments

We thank Professor John Pate and Dr Murray Unkovitch for advice and discussion.

References

Bergersen FJ (1970) Some Australian studies relating to the long-term effects of the inoculation of legumeseeds. Plant and Soil 32, 727–736.

Chatel DL, Parker CA (1973) Survival of field-grown rhizobia over the dry summer period in WesternAustralia. Soil Biology and Biochemistry 5, 415–423.

Chatel DL, Shipton WA, Parker CA (1973) Establishment and persistence of Rhizobium trifolii in WesternAustralian soils. Soil Biology and Biochemistry 5, 815–824.

Diversity of rhizobia 1329

http://www.publish.csiro.au/journals/ajsr

Gibson AH, Date RA, Ireland JA, Brockwell J (1976) A comparison of competitiveness and persistenceamongst five strains of Rhizobium trifolii. Soil Biology and Biochemistry 8, 395–401.

Hagen MJ, Hamrick JL (1996) Population level processes in Rhizobium leguminosarum bv. trifolii: The roleof founder effects. Molecular Ecology 5, 707–714.

Handley BA, Hedges AJ, Beringer JE (1998) Importance of host plants for detecting the populationdiversity of Rhizobium leguminosarum biovar viceae in soil. Soil Biology and Biochemistry 30,241–249.

Hebb DM, Richardson AE, Reid R, Brockwell J (1998) PCR as an ecological tool to determine theestablishment and persistence of Rhizobium strains introduced into the field as seed inoculant.Australian Journal of Agricultural Research 49, 923–934.

Holding AJ, King J (1963) The effectiveness of indigenous populations of Rhizobium trifolii in relation tosoil factors. Plant and Soil 18, 191–198.

Howieson JG (1995) Rhizobial persistence and its role in the development of sustainable agriculturalsystems in Mediterranean environments. Soil Biology and Biochemistry 27, 603–610.

Howieson JG, Loi A, Carr SJ (1995) Biserrula pelecinus L.—a legume pasture species with potential foracid, duplex soils which is nodulated by unique root-nodule bacteria. Australian Journal of AgriculturalResearch 46, 997–1009.

Maynard-Smith J, Smith NH, O’Rourke M, Spratt BG (1993) How clonal are bacteria? Proceedings ofNational Academy of Sciences USA 90, 4384–4388.

McArthur WM (1991) ‘Reference soils of south Western Australia.’ (Western Australia Department ofAgriculture: Perth, WA)

McInnes A (2002) Field populations of Bradyrhizobia associated with serradella. PhD thesis, TheUniversity of Western Australia.

Richardson AE, Simpson RJ (1988) Enumeration and distribution of Rhizobium trifolii under asubterranean clover-based pasture growing in an acid soil. Soil Biology and Biochemistry 20, 431–438.

Richardson AE, Viccars LA, Watson JM, Gibson AH (1995) Differentiation of Rhizobium strains using thepolymerase chain reaction with random and directed primers. Soil Biology and Biochemistry 27,515–524.

Robinson AC (1969) Host selection for effective Rhizobium trifolii by red clover and subterranean cloverin the field. Australian Journal of Agricultural Research 20, 1053–1060.

Sanford P, Pate JS, Unkovich MJ (1993) A survey of proportional dependence of subterranean clover andother pasture legumes on N2 fixation in south-west Australia utilizing 15N natural abundance. AustralianJournal of Agricultural Research 45, 165–181.

Slattery JE, Coventry DR (1993) Variation of soil populations of Rhizobium leguminosarum bv. trifolii andthe occurrence of inoculant rhizobia in nodules of subterranean clover after pasture renovation innorth-eastern Victoria. Soil Biology and Biochemistry 25, 1725–1730.

Somasegaran P, Hoben HJ (1985) ‘Methods in legume-Rhizobium technology.’ (University of HawaiiNifTAL Project: Paia, HI)

Sullivan JT, Eardly BD, van Berkum P, Ronson CW (1996) Four unnamed species of nonsymbiotic rhizobiaisolated from the rhizosphere of Lotus corniculatus. Applied and Environmental Microbiology 62,2818–2825.

Sullivan JT, Patrick HN, Lowther WL, Scott DB, Ronson CW (1995) Nodulating strains of Rhizobium lotiarise through chromosomal symbiotic gene transfer in the environment. Proceedings of NationalAcademy of Sciences USA 92, 8985–8989.

Thies JE, Holmes EM, Vachot A (2001) Application of molecular techniques to studies in Rhizobiumecology: a review. Australian Journal of Experimental Agriculture 41, 299–319.

Unkovich MJ, Pate JS (1998) Symbiotic effectiveness and tolerance to early season nitrate in indigenouspopulations of subterranean clover rhizobia from S.W. Australian pastures. Soil Biology andBiochemistry 30, 1435–1443.

Manuscript received 25 June 2001, accepted 15 July 2002