Identi®cation of tolerance to soil acidity in inoculant strains of
Rhizobium leguminosarum
bv.
trifolii
Elizabeth L.J. Watkin*, Graham W. O'Hara, John G. Howieson, Andrew R. Glenn
1Centre 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 leguminosarum
bv.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 and persisted in acid-soil to a greater degree than strains TA1 and NA3001. The data from this study clearly identi®ed strain WSM409 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 leguminosarumbv.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 aecting 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 eects 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)
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* Corresponding author. Tel.: 9360-2439; fax: +61-08-9360-6486.
E-mail address: [email protected] (E.L.J. Wat-kin).
1
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 and Ewing, (1986) used a cross-row technique in a long term ®eld experiment to study acid-soil tolerance in Sinorhizobium spp. The cross-row procedure involves sowing rows of inoculated seed in the ®rst year of the experiment, and then, in subsequent years, sowing uninoculated seed in rows at right angles to the orig-inal row. This technique assesses the persistence of strains in the soil at the site of inoculation and also determines their ability to colonise the soil away from the point of initial inoculation.
The ability of inoculum strains of root nodule bac-teria to colonise a soil in the absence of the host legume is important in situations where it is advan-tageous to introduce the root nodule bacteria separate from the host legume seed (Howieson, 1995b). These situations may arise when fungicides are used on the legume seed (Stovold and Evans, 1980; Evans et al.,
1986), or when the legume is sown in conditions that are harmful to the survival of the bacteria. Therefore, an understanding of the saprophytic competence of inoculum strains is important in devising inoculation strategies to overcome these problems (Howieson, 1995a).
In this study, we have used the cross-row technique in a three-year ®eld experiment to examine the per-formance in an acid-soil (pH 4.2) of six selected strains of Rhizobium leguminosarum bv. trifolii, hereafter referred to as R. trifolii. Persistence and colonisation was assessed over the three years of the experiment fol-lowing the introduction of strains into the acid-soil either as seed-coated peat inoculum, or as peat added directly 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 of Howieson and Ewing (1986) to evaluate acid tolerance
in strains ofSinorhizobium spp. (Fig. 1(a) and (b)). Six strains of R. trifolii (Table 1), plus an uninoculated control treatment, in factorial combination with host Trifolium subterraneum (cv. Dalkeith), plus a nil host treatment, in quadruplicate.
2.2. Site preparation
The experiment was carried out on the Dryland Research Institute, Merredin, Western Australia (318
28' 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 growing season between May and October. The yearly rainfall during the experiment was 405 mm in 1992, 266 mm in 1993, and 154 mm in 1994. The site had been cleared of the original vegetation of acacia and wodgil in 1965, and cultivated in a cereal/voluntary pasture rotation until 1982. Wheat/lupin rotation was sown during 1983±1990; the lupin was inoculated with Bra-dyrhizobium sp. (Lupini) strain WU425. During 1991 the site was under voluntary pasture. There is no record 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-Uc 4.21) with an organic carbon content of 0.9%, total nitrogen 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 ingredient 450 g Glyphosphate/l) to kill weeds, then cultivated to a depth of 2 cm.
2.3. Preparation of seed and inoculum
Rhizobial inocula were prepared using cultures of the six strains ofR. trifolii grown at 288C in Tryptone Yeast (TY) broth (Beringer, 1974) to late exponential phase (OD600 approx. 1.0). Twenty ®ve ml of culture
of each strain was aseptically injected into separate bags 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 was prepared by injecting 25 ml of sterile TY broth into a 50 g bag of sterile peat. The inoculated and control peats were thoroughly mixed by hand manipulation and incubated at 288C for 10 days. The inocula were then stored at 48C. Viable counts were performed on the stored, inoculated peats one week before use in the ®eld experiment, and bags with similar numbers of rhi-zobia (2±5.5108 cells gÿ1peat) were used to inocu-late sterile seeds.
Seeds of T. subterraneum were surface sterilised by immersion (5 s) in 95% (v/v) ethanol followed by 3 min 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 ¯ow cabinet. Sterile dry seeds were immediately inoculated and lime-pelleted by ®rst mixing 1 g of 2% (w/v) meth-ocel adhesive solution with 0.5 g of the appropriate peat, and then adding 50 g of the appropriate peat, and then adding 50 g of clover seed and 35 g of ®nely ground lime (Brockwell et al., 1982; Howieson and Ewing, 1986). The seed was mixed aseptically until uni-formly coated with peat and lime. Inoculated seed was stored at 48C overnight and then sown.
2.4. Sowing
2.4.1. Plots sown with inoculated seed in year one The experiment was sown on 8th June 1992 in ®ne sunny conditions. Each treatment was sown in two parallel rows, 2 m long and 0.5 m apart at a rate of approximately 1 g of inoculated clover seed per meter (Fig. 1(a)). Each plot was separated by a 1 m border to minimise movement of inoculum strains between plots. 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 ofRhizobium leguminosarumbv.trifoliiused 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 fromT. subterraneumon acid-soil in Sardinia. Minimum pH (agar) for growth, 4.5 (Watkin et al., 1997)
JG Howieson Centre forRhizobium Studies
WU95 Commercial inoculum forT. subterraneum. Minimum pH (agar) for growth, 4.6 (Watkin et al., 1997)
weeks after the ®rst rain and again after a further week. The plots were fertilised at the time of sowing with superphosphate at a rate of 150 kg haÿ1.
In the second year, all undisturbed portions of the original rows were resown with surface-sterilised clover seeds on 4th June 1993. In addition, the right-hand row of each plot had three 0.5 m rows sown at right angles to, and intersecting to the right of the original row (Fig. 1(a)).
In the third year, the plots were resown with sur-face-sterilised clover seeds on 23rd July 1994 in the top 1 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-hand row. This was done to minimise contamination between plots due to site disturbance in the second year.
2.4.2. Plots inoculated without host in the ®rst year In the ®rst year, those plots that were not to be sown to clover had the peat inoculant of each strain sprinkled along the two 2 m rows and covered with soil (Fig. 1(b)). In the second year these rows were sown to clover as described above. In the third year, the total 2 m of the left-hand row was resown with surface sterilised clover seed with four 0.5 m rows sown 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 year In the ®rst year the plants in the lower 1 m of the left-hand row of each plot were harvested 8 weeks after sowing (Fig. 1(a)) to a depth sucient to recover the total root system. The remaining plants in each plot were left undisturbed.
In the second year, eight weeks after sowing, the plants were harvested in the original right-hand row and in the three cross rows (Fig. 1(a)) in the following four 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, the plants were harvested in the original top 1 m of the left-hand row and the two cross rows (Fig. 1(a)), with sampling regions as in the second year.
2.5.2. Plots inoculated without host plant
In the ®rst year there was no harvest (Fig. 1(b)). In the second year the plants were harvested as described above for plots sown with inoculated seed in the ®rst year (Fig. 1(b)).
In the third year, the original left-hand row and two cross rows were harvested in the four sampling regions
described above, except that the top and bottom 1 m sections were harvested separately (Fig. 1(b)).
2.6. Assessment of nodulation, yield and nitrogen
At each harvest, all plants from each sampling region within a plot (in total approx. 200 plants) were pooled, 20 plants were selected randomly and then assessed for nodulation using a scoring system adapted from 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 nodulation scores for the 20 plants were averaged. Up to 25 nodules were picked from each of the 20 plants (i.e., to a maximum of 500 nodules); these were stored at 48C over granulated CaCO3 until the resident bacteria
could be typed.
The tops of the 20 plants were removed, bulked and dried at 708C for 48 h and the dry weights measured. Dry tops were milled ®nely and the total nitrogen was determined using Kjeldahl digestion (Dalal et al., 1984), and ammonia determined by the method of Searle (1984).
2.7. Nodule typing
Nodule isolates were typed using pH sensitivity and intrinsic antibiotic resistance patterns (Josey et al., 1979), and con®rmed using polymerase chain reaction with 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 then thoroughly washed at least six times with sterile deio-nised water (Vincent, 1970). Each nodule was crushed in a small drop of sterile deionised water with a sterile orange stick and the suspension patched onto TY agar. After 3±4 days incubation at 288C colonies were respotted onto TY master plates and incubated for 3 days at 288C. The master plates were then replica pla-ted onto minimal medium (Watkin et al., 1997) at pH 4.35, 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 mg
mlÿ1). A TY plate was included at the end of each run
to ensure adequate replication. Fifty nodules were typed for each plot.
The identi®cation of the nodule isolates obtained from the pH and antibiotic resistance pro®les were con®rmed using polymerase chain reaction ampli®ca-tion pro®les (Richardson et al., 1995). The primer RPO1, a directed primer based on a reiterated Rhizo-bium nif promoter consensus element, was obtained from BRESATEC and used following the protocols of Richardson et al. (1995).
identi®ed by nodule morphology in the the second and the third years.
2.8. Dual nodule occupancy
For each plot, the exudates from ten sterilised nodules were streaked onto TY agar and incubated at 288C for 3±4 days. Ten colonies from each nodule were patched onto TY plates and incubated for a further 3±4 days. These were then replica plated for typing as described above.
2.9. Numbers of R. trifolii in the soil
The most probable number (MPN) of indigenous strains of R. trifolii at the ®eld site prior to sowing in 1992, and the population level of the six inoculant strains ofR. trifolii in the ®eld plot at the time of sow-ing in the second and third years, were determined using the plant infection test of Brockwell (1982) except screwcap tissue culture tubes (114 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±5 cm. In the third year, the 0±5 cm pro®les were bulked. The minimum level of detection for this assay is 11 rhizobia gÿ1 soil with 95% con®dence limits (Woomer et al., 1988).
2.10. Presence of background R. trifolii
Samples of the top 5 cm of soil were collected during April 1992, prior to the break of season from three locations at the ®eld site and stored for 2 days prior to mixing, air drying and sieving. The soil was then 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. Dalkeith were imbibed in sterile, deionised water for 4 h and germinated on water agar at 258C for 48 h in the dark. Six germinated seeds were sown at a depth of 1 cm in each pot, covered with soil and a 1 cm layer of sterile vermiculite. The pots were maintained in a root cool-ing tank at 21218C and watered to ®eld capacity with sterile water through watering tubes on every second day. The plants were harvested at 6 weeks and roots examined for the presence of nodules.
3. Results and discussion
3.1. Background population of R. trifolii
No naturalised strains of R. trifolii were detected in the soil samples collected from the ®eld site prior to sowing the experiment in 1992 i.e. nodules did not form on the roots of any of the plants used for the MPN 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 clover plants grown in the uninoculated plots were ineec-tively nodulated by naturalised strains of R. trifolii. This nodulation consisted of one or two white (ineec-tive), large nodules being present on the extreme lat-eral roots. Using intrinsic antibiotic resistance and growth on low pH agar plates, ®ve dierent strains were identi®ed from these isolates. Plants sown in the second 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 in many soils in the agricultural regions of south-west Australia (Parker, 1962; Chatel and Parker, 1973b). Population density, eectiveness, and competitive abil-ity have been identi®ed as the primary characteristics of indigenous rhizobial populations that aect inocu-lation responses (Thies et al., 1991). Naturalised popu-lations of root nodule bacteria can signi®cantly limit responses to inoculation as long as the population con-tains some eective strains (Singleton and Tavares, 1986).
The background population of R. trifolii in the soil at the site of this study comprised ®ve ineective strains with very poor competitive ability. These strains were only detected in the uninoculated control plants grown during the ®rst season of the experiment and were not detected in the subsequent seasons. Per-haps the dry, below average rainfall in 1993 and 1994 reduced their limited capacity to nodulate the uninocu-lated clover. Clearly the background populations of naturalised strains of R. trifolii were not a signi®cant factor in the performance of the inoculum strains in this experiment. From this data, it would seem reason-able to propose that there was little competition for inoculum strains from the naturalised strains and as a consequence the inoculant strains were able to easily nodulate the clover in the ®rst year of the experiment.
3.2. Movement of inoculum strains between plots
summer moving dry soil from plot to plot. The fact that there was no evidence for movement of strains to plots 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 dry dust and enhance colonisation of the soil. In addition, presence of senescing roots of sub-clover may have promoted the establishment of the rhizobia in these plots.
3.3. Persistence of inoculant strains in the presence of the host plant
In the ®rst year of the experiment, all inoculated plants had abundant nodules on their crown roots. Typing of the isolates from these nodules using intrin-sic antibiotic resistance and pH sensitivity pro®les showed that all the nodules produced in an inoculated plot were formed by the inoculum strain (data not shown). The persistence of the inoculant strains in the
soil at the site of inoculation was assessed over a three year period in two ways. Firstly, by MPN counts of populations of inoculum strains in soil at the point of original inoculation collected at the time of sowing in second and third years. Secondly, by examining the nodulation of plants sown in the second and third years in the original inoculated row. The percentage of plants nodulated by inoculant strains in years sub-sequent to initial inoculation and sowing is generally considered 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 in the second year of the experiment strains NA3039, NA3025 and WSM409 were present in the soil at more than 103 cells/g of soil in the top 0±2 cm and at a depth of 2±5 cm. Strain TA1 was present at 102cells/g of soil in the surface horizon and could not be detected at 2±5 cm depth. Strain NA3001 was just detectable 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 ofRhizobium legumino-sarumbvtrifoliiand 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)
aValues are the means of four measurements with standard deviations in parenthesis. Values in each column followed by the same letter are
not signi®cantly dierent (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 ofRhizobium legumino-sarumbvtrifoliiinoculated 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
but was present at 2104cells/g soil at 2±5 cm. Strain WU95 was not detected in soil from plots inoculated with this strain in the ®rst year. There were clear dierences between the strains in their capacity to maintain populations in the soil during the long, dry summers.
WSM409 and NA3025 were the only strains present in detectable numbers in soil collected at the time of sowing in the third year of the experiment at 26 and 11 cells/g soil. The third year of the experiment was delayed by a late break of season and sown six weeks later in the year than in the two previous seasons. The surviving inoculant bacteria were, therefore, subjected to a longer period of desiccation stress in the third year and this may have been a signi®cant factor redu-cing the populations of inoculant strains present in the soil. It might be anticipated that low populations of surviving inoculant may reduce early eective nodula-tion of regenerating clover under these condinodula-tions. The detectable populations of WSM409 and NA3025 indi-cate that these strains were present in greater numbers in the soil after a period of considerable desiccation and the acid-soil tolerance of these two strains. R. tri-folii was not detected in uninoculated soil collected at the time of sowing in the second and third years of the experiment.
Typing of isolates from nodules, formed on plants sown in inoculated plots, showed that the nodulation of these plants in the second year was almost entirely due to the inoculant strain. The percentage occupancy of nodules from inoculated plots due to the inoculant strain ranged from 74% for strain NA3001 to 97% for strain WU95. Low levels of dual nodule occupancy were observed in the second year of the experiment ranging from 1.3% for strain NA3001 to 6.7% for strain NA3039. The co-occupant strain was not ident-i®ed in these cases. Thus, the nodules formed on plants sown in the second year in plots inoculated in the ®rst year were predominantly formed by the strain added to the soil in the ®rst year. The inoculum strains domi-nated the nodulation of clover plants in inoculated plots in all three years of the experiment.
In the second year, the plots inoculated with strains WSM409 and NA3039 had a signi®cantly higher per-centage of plants nodulated in the original row than those plots inoculated with strains WU95 and NA3001 (Table 2). The lower nodule scores for strains TA1 and NA3001 in the second year (Table 2) were a result of nodules only being formed by these two strains on the lateral roots. Strains WSM409, WU95, NA3025 and NA3039 all produced high nodule scores (Table 2) as a consequence of the nodules being positioned on the crown and upper lateral roots.
In the third year, strain WU95 nodulated a signi®-cantly higher proportion of plants in the original inoculated 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 WU95 in the plots sown each year to sub-clover. These three strains clearly have the capacity to stongly colonise the acid-soil at the site of inoculation and persist from year to year in long term pastures. The poorer per-formance of strains NA3001, and TA1 in persisting at the site of inoculation indicates that these two strains may 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 be either a consequence of its ineectiveness 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 host plant
The survival of strains in the acid-soil in the absence of their host plant was examined by inoculating plots in the ®rst year with peat inoculum alone. Sterilised clover seeds were subsequently sown in the inoculated rows in the second and third years, and the nodulation of these plants was used to assess the persistence of the inoculum. In this situation, strains WSM409, WU95 and NA3039 nodulated a signi®cantly higher percentage of plants in the second year at the site of inoculation than the other strains (Table 3); they had also signi®cantly greater nodule scores than strains NA3001 and TA1 in the second year (Table 3). In these plots, strains WSM409, WU95 and NA3039 formed medium sized nodules (2±5 mm dia.) on both crown and lateral roots (early nodulation possibly in-dicative of a larger population of these strains). In contrast, strains NA3001 and TA1 produced nodules only on lateral roots (late nodulation indicative of a small population). The high nodule scores recorded for strain NA3025 were due to the formation of numerous very small, white nodules (1 mm dia.) on the crown region of the roots.
In the third year, after the absence of a host plant in the ®rst year and the presence of clover in the second year, strains TA1 and NA3001 produced a signi®cantly lower percentage of nodulated plants (Table 3). Strains WU95 and NA3039, produced crown nodules, whereas the other strains only formed nodules on lateral roots.
WSM409, and NA3025 produced crown nodules and strains NA3001, NA3039, and TA1 formed nodules only 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 that strain NA3001 persisted poorly in an acid-soil in NSW, and they attributed the poor survival of this strain to competition from indigenous strains. In the study reported here, the poor performance of strain NA3001 was clearly not related to strong competition from the background strains in the soil at the time of establishing this experiment. In addition, the presence of other inoculant strains in plots in the second and third years of the experiment did not provide strong competition for strain NA3001 because 75±85% of nodules formed on plants sown in plots inoculated with NA3001 contained this strain. Thus, the poor per-formance of strain NA3001 in this Western Australian soil was more likely to have been due to its poor survi-val. The same strain has been reported to persist in an acid-soil in Victoria (Slattery pers. com.), indicating that dierences in background rhizobial populations and soil characteristics between sites can aect strain survival.
3.5. Colonisation of acid-soil by R. trifolii
Colonisation of soils by the inoculum strain of root nodule bacteria is essential for the establishment of productive pastures because of the need for eective nodulation of plants located at points distant from the site of inoculation. The colonisation of the acid-soil by the six inoculant strains was assessed in the second and third years of the experiment by observing the nodulation of clover plants sown in cross rows at dis-tances from the original inoculated row. There were considerable dierences between strains in their ability to 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 ofRhizobium leguminosarumbv. tri-foliiand 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
assisted the survival of plants during the water stress that 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 to survive and colonise the acid-soil was the situation where plots were inoculated in the ®rst year in the absence 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 plants nodulated at 1±10 cm than strains TA1, NA3001 and NA3025 (Table 6). Plants nodulated by NA3039 had a signi®cantly higher nodule score than NA3001, NA3025, TA1, and WSM409, (Table 6), with nodules formed on the crown and lateral roots. Strains TA1 and NA3001 had signi®cantly lower nodule scores
than the other strains (Table 6) with nodules only forming on lateral roots. In the 11±20 cm region strain NA3039 had signi®cantly better nodulation than strains NA3001, NA3025, and TA1 (Table 6) and strains TA1 and NA3001 had the lowest nodule scores (Table 6).
The data from the third year of the experiment could not be statistically analysed because the poor survival of plants resulted in large amounts of missing data. However, some plants did survive and nodulate in 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 NA3039 nodulated 30% or more of plants in the 1±10 cm sampling region (Fig. 2). In the plots, where the host was absent in the second and third years strains NA3039, WSM409, and WU95 produced more than 50% plant nodulation at 1±10 cm (Fig. 2), whilst in the 21±50 cm region only strains WSM409 and NA3039 produced any nodulation at all (data not shown).
3.7. Eectiveness of inoculant strains
As a measure of eectiveness 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 with WU95, WSM409, and TA1 produced similar nitrogen concentrations. However, when expressed as nitrogen accumulation per plant, those plants inoculated with WSM409 had the highest amount followed by WU95 and then plants inoculated with TA1. Plants inocu-lated with NA3025 contained a lower percentage of nitrogen and lower amount of nitrogen per plant than the uninoculated controls (Table 7).
Acid-soil-tolerant and acid-soil sensitive strains ofR. trifolii have been identi®ed using a ®eld experiment based 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 leguminosarumbvtrifoliiand 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)
aValues are the means of four measurements with standard deviations in parenthesis. Values in each column followed by the same letter are
not signi®cantly dierent (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 ofRhizobium leguminosarum bvtrifolii 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
ability of the strains to persist and colonise the soil. Strains WSM409 and NA3039 were better colonisers of the acid-soil (pH 4.2) than the commercial inoculum strain WU95, and the two acid tolerant strains were more eective in the ®eld than WU95. The data from this study and from work determining the broad host range of WSM409 (Howieson et al., 2000) provides clear support for the selection of WSM409 as an acid-soil tolerant inoculum strain for a range of Trifolium spp. to replace WU95. The use of WSM409 should
increase the productivity of clover-based pastures on acidic-sandy soils in southern Australia. The character-isation described here of acid-soil sensitive and acid soil-tolerant strains of R. trifoliiprovides resources for laboratory-based investigations on the basis of acid-soil tolerance inR. trifolii.
Acknowledgements
We gratefully acknowledge research support from the Grains Research and Development Corporation.
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