e}ective nitrogen!_xing associations with host!plants from that site showed signi_cant variation in 11 host ... less than one!tenth the size of the best combinations[.
Journal of Applied Ecology 0888\ 25\ 287Ð397
Variation in the effectiveness of symbiotic associations between native rhizobia and temperate Australian Acacia] within!species interactions J[J[ BURDON\ A[H[ GIBSON\ SUZETTE D[ SEARLE \ M[J[ WOODS and J[ BROCKWELL Centre for Plant Biodiversity Research\ CSIRO Plant Industry\ GPO Box 0599\ Canberra\ ACT 1590\ Australia^ and CSIRO Forestry and Forest Products\ PO Box E3997\ Kingston\ ACT 1593\ Australia
Summary 0[ The ability of di}erent rhizobial isolates collected from any one site to establish e}ective nitrogen!_xing associations with host!plants from that site showed signi_cant variation in 11 host Acacia species and nearly all of 56 populations[ The average Acacia hostÐRhizobium strain combination was only about 69) e}ective[ Many combinations were far poorer^ in a few cases the worst combination resulted in plants less than one!tenth the size of the best combinations[ 1[ The ability of rhizobial isolates to form e}ective symbiotic interactions showed marked host population and rhizobial!isolate e}ects in a study of eight\ four and nine populations of A[ dealbata\ A[ implexa and A[ mearnsii[ A more complete trial involved three populations of each of A[ dealbata\ A[ implexa\ A[ irrorata\ A[ mearnsii and A[ melanoxylon[ These were inoculated with a range of rhizobial isolates previously shown to be highly\ moderately or weakly successful in forming an e}ective associ! ation[ Evidence of marked host population and rhizobial origin e}ects was found but there was very little evidence of isolateÐhost population interaction e}ects[ 2[ The general lack of host populationÐrhizobial origin interaction e}ects suggests that rhizobial strains selected as highly e}ective for an Acacia species growing in a particular population will generally perform well symbiotically with that species in other populations[ This will make their practical application as inoculants in reveg! etation and forestry situations much easier[ 3[ Signi_cant host!based variability in the ability to form e}ective symbiotic inter! actions was detected in comparisons of half!sib families of A[ dealbata\ A[ mearnsii and A[ melanoxylon[ In the case of A[ dealbata\ the interaction between half!sib family lines and rhizobial isolates was complex\ with {locally| derived isolates performing better than {foreign| ones[ There were also signi_cant interaction e}ects[ In A[ mearnsii\ on the other hand\ the only signi_cant di}erences were detected between the response of di}erent half!sib families to the same rhizobial isolate[ The occurrence of host! based variability indicates that in Acacia breeding programmes attention should be given to the possibility of inadvertent selection a}ecting these relationships[ Key!words] Bradyrhizobium\ nodulation\ Rhizobium\ symbiotic e}ectiveness[ Journal of Applied Ecology "0888# 25\ 287Ð397 Introduction Two characteristic features of the majority of Aus! tralian ecosystems are the low fertility of their soils
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287
Correspondence] Dr J[J[ Burdon "fax 50 1 51354138^ e!mail Jeremy[BurdonÝpi[csiro[au#[ In this paper\ the term rhizobia is used to describe Acacia root!nodule bacteria of the bacterial genera Rhizobium and Bradyrhizobium
and the diversity of legume species that they support[ In these circumstances\ plantÐmicrobial associations that help circumvent nutrient de_ciencies are poten! tially of considerable importance in determining the species and structural diversity of individual eco! systems "Allen + Allen 0870^ Read 0882#[ One group of such associations occurs between the very large numbers of species of Acacia found in Australia and their rhizobial symbionts[ Acacias are a dominant
288 Australian native rhizobiaÐAcacia associations
Þ 0888 British Ecological Society\ Journal of Applied Ecology\ 25\ 287Ð397
component of many Australian ecosystems\ whether this is measured in terms of their structural position\ numbers or overall biomass[ In these situations\ the interaction they form with root!nodule bacteria can be responsible for substantial levels of nitrogen _x! ation "N1 _xation# "Hingston\ Malajczuk + Grove 0871^ Langkamp\ Farnell + Dalling 0871^ Hansen et al[ 0876#[ Indeed\ in plantations\ N1 _xation may be as high as 199 kg haÐ0 "Sherry 0860#[ Despite the dominance of Acacia in Australia\ and the growing world!wide importance of the genus in plantation and farm forestry\ surprisingly little is known about the speci_city of the symbiotic relationship between aca! cias and rhizobia[ Using limited numbers of rhizobial isolates\ often collected from diverse habitats and geographical regions\ a high degree of Acacia hostÐrhizobial strain speci_city for both nodule formation and the e}ec! tiveness of the symbiotic relationship has been shown within "Dart\ Umali!Garcia + Almendras 0880^ Sun\ Simpson + Sands 0880# and among "Roughley 0876^ Barnet + Catt 0880# a range of Acacia species[ While such studies have provided a preliminary picture of the broader host range of individual rhizobial isolates\ the diversity of the rhizobialÐhost interaction occur! ring at the individual population level "i[e[ the extent of variation in symbiotic e}ectiveness\ de_ned here as the capacity to _x nitrogen following nodule for! mation^ Chanway\ Turkington + Holl 0880#\ and how this may di}er from one population to another within the one host species\ is still unclear[ The study reported here was part of a larger project aimed at assessing the potential of temperate Aus! tralian Acacia species for use in a range of plantation and farm forestry situations in Australia\ China and Vietnam\ where rapid growth is essential[ Because of the accumulated evidence of the central importance of the rhizobialÐhost interaction in the nitrogen "N# economy and growth of Acacia species\ the study aimed at assessing three speci_c aspects of the inter! action between Acacia hosts and their rhizobial sym! bionts[ These were] "i# the extent of variation in the ability of rhizobial isolates to form symbiotically e}ective associations with their host species of origin collected from their site of origin^ and "ii# the extent to which {local| site measures "the relative per! formance of di}erent rhizobial isolates on their {home| Acacia species# of symbiotic e}ectiveness are pre! dictive of the e}ectiveness of rhizobial isolates when associated with di}erent populations of the same host species[ Finally\ looking at these interactions from the host side of the interaction\ we assessed "iii# the contribution host!based genetic variation might make in determining the e}ectiveness of any given rhi! zobialÐhost!plant combination[
Materials and methods SEED COLLECTIONS
Collections of seed from a total of 56 populations of
11 species of Acacia were made across south!eastern Australia in the period 0882Ð84 "Table 0#[ These spec! ies all have the potential to grow to form small! to medium!sized trees\ often but not exclusively as part of the secondary canopy layer of eucalypt!dominated forests[ All species possess an open pollination system\ with seed resulting from both sel_ng and out!crossing events[ Each population consisted of a pure stand of a single Acacia species[ For multiple!sampled species "represented by three or more populations#\ collection sites were spread across the distribution range "Table 0#[ At each population\ a bulk sample of seed was made by combining ripe pods from a minimum of 09 trees well spaced through the population[ In addition\ at a number of populations\ seed from indi! vidual trees "subsequently called half!sib families# was collected and bagged separately[ Collected seed was returned to the Australian Tree Seed Centre "CSIRO Forestry and Forest Products\ Canberra\ Australia#\ cleaned and fumigated with CO1 to kill any insects\ and stored in air!tight containers at room temperature until required[ RHIZOBIAL COLLECTIONS
Following the collection of seed\ the rhizobial popu! lation occurring at each site was sampled in two ways[ First\ roots of a number of plants were excavated and\ where possible\ bacterial nodules were collected from Acacia trees "0 nodule per tree^ 2Ð7 mm in length# until at least 09 nodules were found[ Secondly\ two samples of soil "c[ 39 g# were collected from around the roots of a random sample of 09 trees[ The soil samples were air!dried\ and the nodules were stored over silica gel before being returned to the laboratory for processing[ Where su.cient nodules could not be collected in the _eld\ additional samples were generated by isolation from the soil samples[ This was achieved by making a suspension of 09 g of _eld!collected soil in 89 ml of sterile Jensen|s nitrogen!free seedling nutrient solution "containing per litre 9=94 g of K1HPO3\ NaCl and MgSO3=6H1O\ 9=14 g CaHPO3\ 9=994 g FeCl2\ and 0 ml of a trace element solution#[ After shaking vig! orously for 29 min\ 0 ml of this suspension was used to inoculate enclosed test tubes containing sterilely grown seedlings of the Acacia species occurring at the site of origin of the soil sample[ Nodules were harvested 5 weeks after inoculation[ RHIZOBIAL ISOLATION AND IDENTIFICATION
Dried nodules were reconstituted overnight by immer! sion in sterile water and then surface sterilized in 9=0) HgCl1 for c[ 34 seconds\ thoroughly washed in sterile distilled water to remove the mercury\ and _nally cru! shed individually with a sterile glass rod[ The resultant liquid was streaked onto 0=7) yeast mannitol agar "YMA^ Vincent 0869# plates\ which were then incu! bated at 17 >C for 2Ð4 days[ After this time\ all plates were carefully examined and an isolate picked from a
399 J[J[ Burdon et al[
Table 0[ Variation in symbiotic e}ectiveness within 56 populations of 11 Acacia host species inoculated with rhizobial strains obtained from the same sites as where the hosts were collected[ "A full listing of the location of all sites is available from the senior author#[ E}ectiveness is scaled by expressing growth performance of individual strainÐhost combinations as a percentage of that of the best combination[ With the exception of one population of A[ mearnsii\ signi_cant variation was detected in all populations Population performance
E}ectiveness within single strainÐhost population combinations ranged from the performance of the least e}ective com! bination "data in this column# to the performance of the best combination " 099)#[ $Mean shoot dry weight for all strainÐhost combinations divided by that of the best strain[
Þ 0888 British Ecological Society\ Journal of Applied Ecology\ 25\ 287Ð397
single bacterial colony[ Where cultures were con! taminated\ a single colony was repeatedly replated until a pure culture was obtained[ Cultures were then stored on YMA slopes at 3 >C and in 19) gly! cerol:79) yeast mannitol broth at Ð79 >C until required[ Isolates were authenticated as strains of rhizobia by inoculating sterilely grown seedlings of Siratro ðMac! roptilium atropurpureum "Mocino + Sesse ex DC[# UrbanŁ\ a universally promiscuous host[ The seedlings were grown singly in Thornton "0879# tubes "049 × 14 mm# containing washed vermiculite moist! ened with N!free Jensen|s solution\ and plugged with a short length of polyurethane foam[ When 2 days old\ the seedlings were inoculated with a heavy sus! pension of the bacterial cells in Jensen|s solution[ There were two seedling tubes per isolate[ After 01 weeks of growth in a louvre!shaded glasshouse "Hely 0848#\ the nodulating capabilities of the isolates were assessed by visual inspection of the root system of inoculated plants[ Isolates that failed to produce at least one nodule were considered non!rhizobial and discarded[
RHIZOBIAÐ ACACIA SYMBIOTIC EFFECTIVENESS TRIALS
Several di}erent e}ectiveness trials were undertaken to examine aspects of the interaction between rhizobia and the various Acacia species[ In all these trials\ the appropriate host seed was taken from store\ dried for 2Ð3 h in a desiccator\ surface sterilized by immersion in concentrated sulphuric acid for 29 min\ washed 09 times with sterile distilled water\ placed in sterile moist river sand in covered plastic boxes\ and incubated at 14 >C for a week[ Germinated seeds were planted\ singly\ in 7!cm diameter tubes containing a mixture of sterilized\ washed vermiculite and river sand "0 ] 0\ v]v#[ The seedlings were inoculated 1 days later[ Inoculum of each rhizobia strain was prepared by streaking the culture onto YMA slopes and incubating at 17 >C for 4Ð6 days[ After that period\ 09 ml of sterile N!free Jensen|s solution was added to the slope and the bacterial growth gently rubbed o}[ The resulting suspension was decanted into a sterile bottle\ vortexed to disperse the bacterial cells and then checked under a microscope to ensure the presence of at least 0499 bacterial cells mlÐ0[
390 Australian native rhizobiaÐAcacia associations
Except where a deliberate comparison was made between the e}ectiveness of soil!derived and nodule! derived rhizobia isolates\ preference was given in all e}ectiveness trials to bacterial isolates derived orig! inally from nodules collected from acacias growing in the _eld[ Seedlings were inoculated with the appropriate rhi! zobial culture by pipetting 0 ml of the inoculum liquid onto the base of the hypocotyl[ To reduce the possi! bilities of cross!contamination\ the bottoms of the pots were then placed in individual plastic bags and the soil surface covered with a thick layer of plastic beads[ Plants were harvested after 01Ð05 weeks of growth in a glasshouse at 07Ð13 >C and under ambient light conditions[ During that time plants were watered two to three times per week with UV!sterilized Jen! sen|s N!free solution[ At harvest\ each plant was cut at ground level and weighed immediately^ these tops were then bagged and dried at 59 >C for 0 week\ after which dry weights were recorded[ Dry weight data were used in all analyses[ The root system was not weighed[ However\ as each plant was harvested\ the root system was examined to determine whether or not nodules were present[ If no nodules were present\ the data from that replicate were discarded[ Through! out this paper\ relative symbiotic e}ectiveness is de_ned as the growth performance "dry matter pro! duction# of any particular rhizobial isolateÐhost line combination\ expressed as a fraction of the per! formance of the best rhizobial isolateÐhost line com! bination involving the same host population in the same trial[ Highly e}ective rhizobial isolates were ones that supported dry weight production in the associ! ated host which was within 19) of the maximum for that trial^ moderately e}ective isolates resulted in dry weight production in the range of 49Ð69) of the maximum\ while hosts nodulated by ine}ective iso! lates showed growth that was less than 39) of the maximum[ A series of seven experiments was carried out[
Within!host population variation in the ability of iso! lates to form effective symbiotic associations
Þ 0888 British Ecological Society\ Journal of Applied Ecology\ 25\ 287Ð397
This involved testing 09 isolates of rhizobia collected from each Acacia population against bulk seed col! lected from that population "with 09 replicates of each isolate#[ Because of the very large number of species and populations tested "11 species^ 56 populations#\ this assessment was split across three separate experi! ments[ Data for hosts collected from single popu! lations were subjected to a simple analysis of variance^ those for hosts sampled from multiple populations were subjected to two!way ANOVA[ Where appro! priate\ within!site comparisons were made of the rela! tive e}ectiveness of rhizobial strains isolated directly from _eld!collected nodules and those {trapped| from _eld!collected soil[
Among!host population variation in the ability of iso! lates to form effective symbiotic associations Two separate experiments were conducted to deter! mine "i# the relative response of Acacia species to {local| and {foreign| " non!local# rhizobial isolates\ and "ii# the consistency of the relative performance of the rhizobia[ The _rst experiment involved three species of Acacia "A[ dealbata\ A[ implexa and A[ mearnsii# from eight\ four and nine populations\ respectively\ inoculated with single rhizobial strains from four\ four and _ve of those sites\ respectively[ The resultant data were analysed separately for each Acacia species using a two!way ANOVA[ The rhizobia used in this experiment were selected at random from among the isolates that had performed well in the within!site assessments\ with each coming from di}erent populations^ the Acacia populations were selected to cover the geographical distribution range available[ All combinations were replicated 09 times[ The second experiment was more detailed\ involv! ing reciprocal inoculations among three populations of each of _ve species of Acacia "namely A[ dealbata\ A[ implexa\ A[ irrorata\ A[ mearnsii and A[ melano! xylon#[ Hosts from each population were inoculated with a total of 01 rhizobial isolates "four from each site# that had previously been shown to be {highly e}ective| "two isolates#\ {moderately e}ective| "one# or {ine}ective| "one# in forming an association with the Acacia host[ Highly e}ective isolates were chosen from among the two best performing isolates^ ine}ective ones from the two worst performing isolates^ and moderately e}ective isolates from among the three performing closest to the mean[ The performance of highly e}ective and moderately e}ective isolates was not always statistically di}erent[ However\ the per! formance of ine}ective isolates was always sig! ni_cantly poorer than that of the highly e}ective isolates[ All treatments were replicated 09 times[ For each Acacia species\ the resultant data were subjected to an ANOVA with rhizobial isolate nested within origin "local vs[ foreign#[
Host!based genetic variability in the ability to form effective associations Variation in this aspect of the symbiotic interaction was assessed in two separate experiments[ The _rst measured the growth response of 09 half!sib "open! pollinated# families "09 replicates per family# from one population of each of A[ dealbata "S07#\ A[ mearnsii "S0# and A[ melanoxylon "S17#[ Each Acacia species was inoculated only with rhizobial isolates collected from that species at that site\ with three such isolates being used[ These isolates represented single examples of {highly e}ective|\ {moderately e}ective| and {ine}ective| isolates identi_ed in earlier within!popu! lation trials[ A second experiment involved half!sib families from
391 J[J[ Burdon et al[
two additional single populations of A[ dealbata "S05# and A[ mearnsii "S15#[ In this instance\ the growth response of 09 half!sib families was examined in response to inoculation by three local "collected from the same population as the hosts# and three {foreign| rhizobial isolates "collected from the same host species growing at a di}erent site#[ Each triplet of isolates was again selected to represent the symbiotic classi! _cations of {highly e}ective|\ {moderately e}ective| and {ine}ective|[ In both experiments\ each half!sib family was replicated 09 times[
Results WITHIN!POPULATION VARIATION IN EFFECTIVENESS
Þ 0888 British Ecological Society\ Journal of Applied Ecology\ 25\ 287Ð397
A comparison of the symbiotic e}ectiveness "as mea! sured by dry weight of host!plants# of individual rhi! zobial strains isolated from _eld!collected nodules and those trapped from soil collected from under Acacia trees at the same site showed considerable variation[ In some instances there was no di}erence in the per! formance of any particular pair of rhizobialÐhost combinations^ in others signi_cant di}erences fav! ouring either source of isolates were detected "data not shown#[ However\ overall there was a clear tend! ency for isolates derived from _eld!collected soil to perform better than strains isolated from _eld!col! lected nodules[ The reason for this di}erence is obscure[ Unconscious "non!random# sampling of larger "and perhaps more e}ective# nodules from the trap plants used to obtain rhizobia from _eld!collected soil may have been a factor[ Where there were sig! ni_cant di}erences between the e}ectiveness of rhi! zobia obtained by the two modes of isolation\ results have been analysed separately[ With the exception of one population of A[ mearnsii\ marked and highly signi_cant within!popu! lation variation in seedling growth occurred as a result of inoculation with the di}erent strains of rhizobia isolated from those populations "Table 0#[ In contrast\ there was evidence in only one host species\ for which three or more populations were sampled "A[ dealbata#\ of a signi_cant di}erence in the mean performance of rhizobia isolates taken from di}erent sites "data not shown#[ In several instances\ the symbiotic e}ectiveness " growth performance of an individual strainÐhost com! bination as a percentage of that of the best com! bination# of the least e}ective rhizobialÐhost com! bination was less than 19) of that of the most e}ective combination "Fig[ 0 and Table 0#[ In general though\ the mean e}ectiveness of rhizobial isolates was substantially greater than the mean of the two extreme performances "Table 0#[ In the _ve species for which data were collected for four or more popu! lations\ the distribution of symbiotic e}ectiveness values showed considerable variability among host
Fig[ 0[ Representative examples of the variability in the e}ectiveness of the symbiotic relationship "measured as mean shoot weight# formed between bulk samples of seed collected from single populations of two Acacia hosts "A[ dealbata and A[ melanoxylon# and each of 09 rhizobial strains obtained from the same sites as where the hosts were collected[
species "Fig[ 1#[ Thus\ while the distribution of per! centage e}ectiveness values for all hostÐisolate dis! tributions was skewed towards the high end\ marked di}erences occurred between the distributions detected in A[ implexa\ A[ irrorata and A[ mearnsii on the one hand\ and in A[ dealbata and A[ melanoxylon on the other[ For the latter two species the e}ec! tiveness of individual hostÐisolate combinations was much more evenly distributed over the range between highly e}ective and completely ine}ective[
AMONG!POPULATION VARIATION IN EFFECTIVENESS
The _rst of the two trials investigating among!popu! lation variation in the symbiotic e}ectiveness "as mea! sured by dry weight production# of rhizobial isolates detected signi_cant variation in the mean response of individual host populations to all isolates "local and foreign# and in the ability of isolates to form e}ective associations across Acacia hosts from all populations "Table 1#[ However\ in only one of the three host spec! ies assessed "A[ implexa# was there evidence of a di}erential populationÐisolate interaction e}ect^ in the interactions involving the other two Acacia species "A[ dealbata and A[ mearnsii# expression of e}ec! tiveness by the di}erent rhizobia did not change sig! ni_cantly in response to changes in the population of origin of the host[ The substantial e}ect of host population\ the markedly lesser e}ect of rhizobial iso! late\ and the absence of signi_cant host populationÐ isolate interaction is illustrated for A[ dealbata in Fig[ 2[ The second among!population assessment of vari! ation was a more detailed trial involving reciprocal inoculation of rhizobial isolates of varying e}ec! tiveness on to each of _ve species of Acacia "A[ deal! bata\ A[ implexa\ A[ irrorata\ A[ mearnsii and A[ mel! anoxylon# from three di}erent populations of origin[ Each Acacia species was treated separately and sig!
392 Australian native rhizobiaÐAcacia associations
Fig[ 1[ Mean symbiotic e}ectiveness "measured in terms of dry weight per plant# of all isolates of rhizobia collected from _ve species of Acacia "A[ dealbata\ A[ implexa\ A[ irrorata\ A[ mearnsii and A[ melanoxylon# for which data were assembled for four or more populationÐrhizobial interactions[ For each populationÐrhizobia set "7Ð09 combinations#\ e}ectiveness was assessed as percentage of the best combination[ The total number of comparisons involved is shown in parentheses[
Table 1[ The range and statistical signi_cance of variation "host population\ rhizobial isolate\ host populationÐrhizobial interaction# in the symbiotic e}ectiveness ðmeasured in terms of host!plant growth "dry weight#Ł of selected rhizobial isolates on Acacia species from which they were isolated[ Isolates\ each from a di}erent population\ were selected on the basis of high e}ectiveness in within!population trials[ P!values are based on a two!way ANOVA with 09 individual plants scored per host population ðtotal sample size 09 × "number of host populations# × "number of rhizobial isolates#Ł Source of variation
Þ 0888 British Ecological Society\ Journal of Applied Ecology\ 25\ 287Ð397
Host
Number of host Number of populations rhizobial isolates
Population "range in g plantÐ0#
Isolate "range in g plantÐ0#
Population × isolate
A[ dealbata A[ implexa A[ mearnsii
7 3 8
"9=7Ð3=2# P ³ 9=990 "1=5Ð2=8# P ³ 9=990 "0=6Ð3=7# P ³ 9=990
"1=1Ð2=4# P ³ 9=990 "1=8Ð2=7# P ³ 9=990 "2=0Ð3=6# P ³ 9=990
NS P 9=993 NS
3 3 4
ni_cant variation was again detected in the mean response of individual host populations to all isolates "Table 2#[ However\ for three of the _ve species "A[ dealbata\ A[ implexa and A[ mearnsii# there was no signi_cant di}erence in the performance of isolates with di}erent site origins when compared across all sites[ This result is perhaps not surprising given that the isolates chosen from each site ranged in symbiotic ability from highly e}ective to ine}ective[ This is illus! trated in Fig[ 3 for interactions involving A[ irrorata[
Similarly\ with the exception of A[ dealbata\ there was no evidence of a host populationÐrhizobial origin interaction e}ect[ As part of a wider phylogenetic survey of Australian native rhizobial communities\ B[ Lafay and J[J[ Bur! don "unpublished data# examined a subset of 007 of the strains isolated here using a partial 05S ribosomal DNA gene sequence that identi_ed a series of genomic species[ Full details of this experimental protocol are given elsewhere "Lafay + Burdon 0887#[ This
393 J[J[ Burdon et al[
Fig[ 2[ Di}erences in the growth response of eight populations of A[ dealbata "S22\ S07\ S35\ S26\ S12\ S34\ S08\ S36# to each of four isolates of rhizobia collected from host sites S22\ S26\ S35 and S36\ respectively[ Di}erences between host populations were signi_cant "P ³ 9=90#[ Neither isolates nor populationÐisolate interaction showed any signi_cant di}erences "P × 9=94#[
Table 2[ The range and statistical signi_cance of variation "host population\ rhizobial isolate\ host populationÐrhizobial interaction# in the symbiotic e}ectiveness ðmeasured in terms of host!plant growth "dry weight#Ł of selected rhizobial isolates on Acacia species from which they were isolated[ For each population four isolates were selected on the basis of within! population trials as {highly e}ective| "two#\ {moderately e}ective| "one# and {ine}ective| "one#[ P!values are based on a two! way ANOVA\ with 09 individual plants scored per host population ðtotal sample size 09 × "number of host popu! lations# × "number of rhizobial isolates#Ł Source of variation
Host
Number of host Number of populations rhizobial isolates
"9=87Ð0=36# P ³ 9=990 "0=81Ð1=54# P ³ 9=990 "9=47Ð0=92# P ³ 9=990 "0=04Ð1=15# P ³ 9=990 "9=37Ð0=31# P ³ 9=990
"0=96Ð0=19# NS "1=07Ð1=27# NS "9=54Ð9=79# P 9=992 "0=49Ð0=47# NS "9=68Ð0=97# P ³ 9=990
P ³ 9=990 NS NS NS NS
01 01 01 01 01
approach detected eight di}erent genomic species of Bradyrhizobium and one of Rhizobium[ At sites where more than two individuals of two or more genomic species were found\ comparison of symbiotic e}ec! tiveness detected no signi_cant di}erences associated with genomic identity ðe[g[ at A[ mearnsii population 29\ two genomic species "A and H# were detected^ performance means 2 SE A 2=35 2 9=13 g plantÐ0^ H 2=03 2 9=11 g plantÐ0^ t 9=88\ P × 9=94#[ Þ 0888 British Ecological Society\ Journal of Applied Ecology\ 25\ 287Ð397
HOST!BASED GENETIC VARIABILITY IN FORMATION OF EFFECTIVE ASSOCIATIONS
An initial assessment trial examined the genetic basis of the growth response of A[ dealbata\ A[ mearnsii and
A[ melanoxylon to three of their own rhizobial isolates previously shown to range from ine}ective to highly e}ective[ For each species\ at least one of the three combinations showed a signi_cant level of host!based variation "P ³ 9=90# for ability to form an e}ective association "Table 3#[ In the case of A[ mearnsii both of the better performing isolates showed a signi_cant "P ³ 9=94# interaction with host genotype\ while in A[ dealbata and A[ melanoxylon signi_cant interactions only occurred with the least e}ective isolate[ However\ the most dramatic e}ects were seen among those com! binations where the interaction between ine}ective isolate 0790 and the A[ dealbata half!sib family SDS 0182 produced plants with a dry weight more than 09 times greater than the mean of the interaction of the
394 Australian native rhizobiaÐAcacia associations
Fig[ 3[ Di}erences in the growth response of A[ irrorata from three populations "4\ 6 and 56# to rhizobial isolates previously identi_ed as capable of forming e}ective "isolates 494\ 497\ 693\ 695\ 5611\ 5614#\ moderately e}ective "426\ 609\ 5620# or ine}ective "416\ 691\ 5626# symbiotic interactions[
Table 3[ The range and statistical signi_cance of host!based variation in the establishment of e}ective nitrogen!_xing symbiotic associations between three Acacia species "09 half!sib families from each population# and associated rhizobial isolates previously identi_ed as highly e}ective\ moderately e}ective and ine}ective in within!population trials[ P!values are based on an ANOVA with 09 individual plants scored per half!sib familyÐrhizobial!isolate combination Range in growth "g plantÐ0# in response to bacterial isolates that were Host
Popu! lation
A[ dealbata S07 A[ mearnsii S0 A[ melanoxylon S17
Þ 0888 British Ecological Society\ Journal of Applied Ecology\ 25\ 287Ð397
Highly e}ective
Moderately e}ective
Ine}ective
"0=28Ð1=05# F 0=17^ NS "9=88Ð0=38# F 9=66^ NS "9=09Ð0=92# F 02=88^ P ³ 9=990 "0=49Ð2=10# F 2=15^ P 9=991 "0=36Ð2=16# F 7=94^ P ³ 9=990 "0=62Ð1=58# F 0=04^ NS "0=45Ð1=18# F 0=96^ NS "9=80Ð0=66# F 0=35^ NS "9=00Ð9=54# F 4=12^ P ³ 9=990
same rhizobial isolate and the nine other half!sib fam! ilies "Fig[ 4#[ A repeat assessment of the interaction between isolate 0790 and half!sib families 0180\ 0181 and 0182 con_rmed the signi_cantly greater per! formance of half!sib family 0182 "F1\16 2=24^ P 9=94#[ A second trial looked not only at the interaction among half!sib families of A[ dealbata and A[ mearnsii and three local isolates of rhizobia but also their com! parative response to three foreign rhizobial isolates[ As the half!sib families used in this trial were from di}erent populations to those used in the _rst host
genetic variation trial "above#\ the local rhizobial iso! lates of experiment 0 were used as the foreign isolates of experiment 1[ For both species there was again signi_cant di}erences among the half!sib families in their response to the di}erent isolates "P ³ 9=990#[ In the case of A[ dealbata\ local isolates performed signi_cantly better than foreign ones did "mean dry weight plantÐ0 0=32 and 0=02 g\ respectively#\ while the di}erent rhizobial isolates performed according to their prior classi_cation "Table 4#[ Furthermore\ there was a signi_cant interaction e}ect\ with the per! formance of di}erent half!sib families di}ering
395 J[J[ Burdon et al[
Fig[ 4[ Marked di}erences in the growth of half!sib families of A[ dealbata in the presence of e}ective "isolate 0716#\ moderately e}ective "0796# and ine}ective "0790# rhizobial isolates[
Table 4[ Sources of signi_cant host!based variation in the establishment of e}ective nitrogen!_xing symbiotic associations between 09 half!sib families from one population of each of A[ dealbata and A[ mearnsii\ and three local and three foreign rhizobial isolates previously identi_ed as highly e}ective\ moderately e}ective and ine}ective in within!population trials Acacia dealbata Source of variation
d[f[
F!value
P
d[f[
F!value
P
0[ Among half!sib families 1[ Among local and foreign isolates 2[ Among e}ectiveness classi_cations of isolates Interaction 1 with 0 Interaction 0 with 2
8 0 3 8 25
13=48 39=88 48=36 1=07 2=98
9=9990 9=9990 9=9990 9=9108 9=9990
8 0 3 8 25
4=45 1=72 9=27 9=03 9=71
9=9990 9=9818 9=7086 9=8874 9=6465
according to the foreign or local nature of the isolate[ In contrast\ for A[ mearnsii\ no signi_cant di}erences were found either between rhizobial origin "local vs[ foreign^ F 1=72^ P 9=982# or between the di}erent rhizobial isolates classi_ed according to prior assessed performance "highly e}ective\ moderately e}ective\ ine}ective^ F 9=27^ P 9=719# "Table 4#[ Þ 0888 British Ecological Society\ Journal of Applied Ecology\ 25\ 287Ð397
Acacia mearnsii
Discussion Two features\ the ability to form nodules "infectivity# and symbiotic e}ectiveness "capacity to _x nitrogen#\
are commonly used to assess the ecological and evol! utionary relationship between rhizobia and their hosts "Chanway\ Turkington + Holl 0880^ Brockwell 0887#[ The large number of rhizobial isolates assessed in this study all showed a capacity to induce nodule for! mation on the host!plants to which they were intro! duced[ However\ the symbiotic e}ectiveness of the resultant relationship showed considerable variability[ In virtually all assessments there was evidence of sig! ni_cant variation at the individual population level in the e}ectiveness of the relationship formed between plants derived from seed collected from trees in that
396 Australian native rhizobiaÐAcacia associations
Þ 0888 British Ecological Society\ Journal of Applied Ecology\ 25\ 287Ð397
population and the rhizobial strains isolated from the same site[ Indeed\ for most species the mean e}ec! tiveness of any randomly chosen isolate was 04Ð19) lower than that of the most e}ective line "Table 0#[ At the lower extreme of the e}ectiveness distribution "Table 0 and Figs 0 and 1# nodulation occurred\ but the relationship was ine}ective\ with dry matter pro! duction often being less than 09) of that of the most e}ective combinations[ In general\ the performance rank of rhizobial isolates\ as assessed by association with host seedlings from the site of common origin\ altered little when tested against host!plants from other populations[ Certainly there were highly signi_cant di}erences in the mean growth rate of plants from di}erent host populations "Fig[ 2#\ a feature consistent with genetic di}erences between host populations in general growth rates[ However\ in eight between!population comparisons involving _ve Acacia species\ only two "A[ dealbata and A[ implexa# showed an interaction e}ect\ indicating changes in the rank order of sym! biotic e}ectiveness of the inoculant strains[ Further! more\ while these species were involved in two tests "Tables 1 and 2# they showed a rank order change in one test only[ The consistency of the pattern of isolates maintaining a generally uniform symbiotic e}ec! tiveness capability in response to hosts from di}erent populations was reinforced by the way in which rhi! zobial isolates of all performance categories behaved similarly[ Thus\ for instance\ rhizobial isolates that proved to be either only moderately e}ective or ine}ective on home site plants of A[ irrorata\ formed similarly ine}ective associations with A[ irrorata plants from other populations "Fig[ 3#[ The practical implications of these results are gen! erally encouraging[ Acacia species are being used increasingly as sources of fuel and pulpwood in many parts of the world[ In addition\ they are often impor! tant in attempts to restore degraded natural areas\ farmland and mine sites[ Because highly e}ective strains selected from one site perform well in associ! ation with host lines from other sites\ it should be feasible to develop inoculants that will establish early e}ective nodulation in a range of seedlots collected widely across the native range of the Acacia species in question[ The application of such inoculants either to germinating nursery stock or directly into seedbeds in the _eld\ maximizes the chances of rapid growth of e}ectively nodulated seedlings and hence increased survivorship during the early establishment phase[ The symbiosis between rhizobia and their hosts is one in which genetic di}erences in the ability to develop and sustain a signi_cant relationship may occur at two levels Ð in the host and in the bacterium[ While di}erences observed among bacterial isolates from individual populations were the most signi_cant detected in this study\ the use of a series of half!sib families of three of the economically most important hosts "A[ dealbata\ A[ mearnsii and A[ melanoxylon#
showed the existence of genetically based di}erences within single host populations "Table 3 and Fig[ 4#[ In all three species\ the response to inoculation of some particular combination of bacterial isolate and half! sib family groupings di}ered from the response of other half!sib families inoculated with the same isolate[ Indeed\ the association between ine}ective iso! late 0790 and A[ dealbata half!sib family 0182 resulted in a greater than 09!fold increase in dry matter pro! duction\ such that the performance was similar to that of 0182 inoculated with e}ective "0716# or moderately e}ective isolates "0796# "Fig[ 4#[ It should be noted\ however\ that some of these di}erences may be caused by maternal di}erences among di}erent half!sib fam! ilies[ The more detailed assessment of host!based vari! ation in the establishment of e}ective N1!_xing associ! ations and their interaction with rhizobial variation "local and foreign highly e}ective\ moderately e}ective and ine}ective isolates# shows how variable these interactions may be[ Thus the growth of 09 half!sib families of A[ dealbata was a}ected by half!sib family identity\ the origin of the isolates "local or foreign# and their e}ectiveness classi_cation\ with a further level of complexity being introduced by signi_cant interactions between several of these characters "Table 4#[ In contrast\ the only signi_cant variation detected in the performance of A[ mearnsii was between half!sib families[ These complexities strongly suggest that\ in any Acacia breeding programme\ attention must be given to the e}ectiveness of sym! biotic relationships[ This could be done simply by periodic monitoring of the N1!_xing capacity of breeding material and rigorous culling of lines that exhibit symbiotic aberrancies[ The complex nature of natural rhizobial popu! lations was underscored by the frequent detection of a range of {genomic| species of rhizobia "identi_ed as taxa with di}erent base pair sequences of the 05S rDNA gene^ Lafay + Burdon 0887# occurring at indi! vidual sites\ although no correlation was detected between genomic identity and symbiotic e}ectiveness[ Similar population!level heterogeneity has been reported previously for both rhizobial isolates associ! ated with Acacia species "Barnet + Catt 0880# and also among strains isolated from a range of non!Acacia shrubby legumes "Lafay + Burdon 0887#[ What then is the source of the variation in symbiotic e}ectiveness found amongst rhizobial isolates< In particular\ what role do the ine}ective isolates play< Are they simply parasitic {cheaters| in an evolutionary game\ or are they strains that really {belong| on some other geno! type of the same host species or\ indeed\ on some di}erent legume species altogether< In very many communities in Australia a wide range of di}erent legume species may be found growing in close juxtaposition[ For example\ at the Ben Boyd and Lobs Hole sites studied by Lafay + Burdon "0887^ J[J[ Burdon\ personal observation# there were 00 and nine
397 J[J[ Burdon et al[
References
Barnet\ Y[M[ + Catt\ P[C[ "0880# Distribution and charac! teristics of root!nodule bacteria isolated from Australian Acacia spp[ Plant and Soil\ 024\ 098Ð019[ Brockwell\ J[ "0887# Matching rhizobia and temperate spec! ies of Acacia[ Recent Developments in Acacia Planting "eds J[W[ Turnbull\ H[R[ Crompton + K[ Pinyopusarerk#\ pp[ 153Ð162[ ACIAR\ Canberra\ Australia[ Chanway\ C[P[\ Turkington\ R[ + Holl\ F[B[ "0880# Eco! logical implications of speci_city between plants and rhi! zosphere micro!organisms[ Advances in Ecological Research\ 10\ 010Ð058[ Dart\ P[\ Umali!Garcia\ M[ + Almendras\ A[ "0880# Role of symbiotic associations in nutrition of tropical acacias[ Advances in Tropical Acacia Research "ed[ J[W[ Turnbull#\ pp[ 02Ð08[ ACIAR\ Canberra\ Australia[ Gibson\ A[H[ "0853# Genetic control of strain!speci_c ine}ective nodulation in Trifolium subterraneum[ Aus! tralian Journal of Agricultural Research\ 04\ 26Ð38[ Hansen\ A[P[\ Pate\ J[S[\ Hansen\ A[ + Bell\ D[T[ "0876# Nitrogen economy of post!_re stands of shrub legumes in Jarrah "Eucalyptus marginata Donn ex Sm[# forest in S[W[ Australia[ Journal of Experimental Botany\ 27\ 15Ð30[ Hely\ F[W[ "0848# Adjustable shading to modify glasshouse temperatures[ Journal of Agricultural Engineering Research\ 3\ 022Ð027[ Hingston\ F[J[\ Malajczuk\ N[ + Grove\ T[S[ "0871# Acety! lene reduction "N _xation# by Jarrah forest legumes fol! lowing _re and phosphate application[ Journal of Applied Ecology\ 08\ 520Ð534[ Lafay\ B[ + Burdon\ J[J[ "0887# Molecular diversity of rhi! zobia occurring on native shrubby legumes in south!east! ern Australia[ Applied and Environmental Microbiology\ 53\ 2878Ð2886[ Langkamp\ P[J[\ Farnell\ G[K[ + Dalling\ M[J[ "0871# Nutri! ent cycling in a stand of Acacia holosericea A[ Cunn[ Ex G[ Don[ I[ Measurements of precipitation interception\ seasonal acetylene reduction\ plant growth and nitrogen requirement[ Australian Journal of Botany\ 29\ 76Ð095[ Mytton\ L[R[ "0864# Plant genotype × Rhizobium strain interactions in white clover[ Annals of Applied Biology\ 79\ 092Ð096[ Mytton\ L[R[\ El!Sherbeeny\ M[H[ + Lawes\ D[A[ "0866# Symbiotic variability in Vicia faba[ III[ Genetic e}ects of host plant\ Rhizobium strain and of hostÐstrain inter! action[ Euphytica\ 15\ 674Ð680[ Read\ D[J[ "0882# PlantÐmicrobe mutualisms and com! munity structure[ Biodiversity and Ecosystem Function "eds E[!D[ Schultze + H[A[ Mooney#\ pp[ 070Ð198[ Springer! Verlag\ Berlin\ Germany[ Roughley\ R[J[ "0876# Acacias and their root!nodule bacteria[ Australian Acacias in Developing Countries "ed[ J[W[ Turnbull#\ pp[ 34Ð38[ ACIAR\ Canberra\ Australia[ Sherry\ S[P[ "0860# The Black Wattle[ University of Natal Press\ Pietermaritzburg[ Sun\ J[\ Simpson\ R[J[ + Sands\ R[ "0880# Studies with seed! lings of provenances in Acacia mangium[ Ninth Australian Nitrogen Fixation Conference "eds A[E[ Richardson + M[B[ Peoples#\ pp[ 007Ð008[ Australian Society for Nitro! gen Fixation\ Canberra\ Australia[ Thornton\ H[G[ "0879# The early development of the root nodule of lucerne "Medicago sativa L[#[ Annals of Botany\ London\ 33\ 274Ð281[ Vincent\ J[M[ "0869# A Manual for the Practical Study of the Root!Nodule Bacteria[ Handbook no[ 00[ IBP\ Oxford\ UK[
Allen\ O[N[ + Allen\ E[K[ "0870# The Leguminosae[ A Source Book of Characteristics\ Uses\ and Nodulation[ University of Wisconsin Press\ Madison\ WI[
Received 12 February 0887^ revision received 11 February 0888
legume species\ respectively[ At Ben Boyd these cov! ered six genera and two tribes of the Papilionideae\ while at Lobs Hole diversity was even greater\ cover! ing six genera in three tribes of the Papilionideae plus two Acacia species[ If each such species exerted di}er! ent selective pressures on the rhizobial population fav! ouring the evolution of strains that are particularly e}ective in forming N1!_xing associations with them! selves\ then highly diverse plant and bacterial com! munities could result[ Under this scenario\ the highly e}ective rhizobial isolates detected in this study might represent isolates that have a specialized relationship with the Acacia species from which they were isolated\ while isolates that are less e}ective or ine}ective may more usually be associated with other legume species[ Several other explanations for the diversity in sym! biotic e}ectiveness are also possible[ Thus there may be a trade!o} between symbiotic e}ectiveness and host range such that\ in the bacteria\ wider host ranges "either intraspeci_c or interspeci_c# are linked with lower levels of e}ectiveness on any one host[ Additional complexities are potentially introduced by the fate of free!living rhizobia in the soil where selec! tive pressures are likely to be very di}erent from those involved in nodule formation[ Earlier work with agricultural legumeÐrhizobial interactions "Gibson 0853^ Mytton 0864^ Mytton\ El! Sherbeeny + Lawes 0866^ Chanway\ Turkington + Holl 0880#\ and the work presented here\ clearly shows the complex nature of the genetic interaction to which both components contribute[ Thus marked di}erences in the symbiotic e}ectiveness of normally highly ine}ective strains among di}erent individual trees within a single natural stand suggest the action of a few genes only "Fig[ 4#\ while the almost continuous distribution of e}ectiveness outcomes in the broader within!population comparisons "Fig[ 0# suggests that at least some aspects of the association may re~ect the action of many genes[
Acknowledgements This study was supported by a grant from the Aus! tralian Centre for International Agricultural Research "project number 8116#[ The work was begun by A[H[ Gibson who sadly died approximately halfway through the project[ The authors are grateful for the assistance and understanding of Mr Alan Brown "CSIRO!Forestry and Forest Products# and Dr John Fryer "ACIAR# during those di.cult times[ We are grateful to Dr P[H[ Thrall for his assistance and advice\ and to Mr W[J[ Muller for his assistance in the analysis of the data[
Þ 0888 British Ecological Society\ Journal of Applied Ecology\ 25\ 287Ð397
