Genetic variability in symbiotic nitrogen fixation within and between provenances of two Casuarina species using the 15N-labeling methods

Soil Bid. Bkwkm. Vol. 22. No. 4. pp. 539-547. 1990 Printed in Great Britain. All rightsreset-d

0038-07 I7,90 53.00 + 0.w Copyright D 1990 Pergamon Press pk

GENETIC VARIABILITY IN SYMBIOTIC NITROGEN FIXATION WITHIN AND BETWEEN PROVENANCES OF TWO CASCJARINA SPECIES USING THE “N-LABELING METHODS N. SANGISGA,’ G.

D.

BOWEN~

and S. K.

A. DANSO’

‘Soil Science Unit and ?Soil Fertility. Irrigation and Crop Production Section of the Joint FAOilAEA Division International Atomic Energy Agency, P.O. Box 100, I400 Vienna, Austria

(Accepred IS October 1989) Summary-Differences in the nitrogen-fixing abilities of provenances of Caruorima equiseri/olia and C. cunninghamiama were assessed in pot experiments. Three methods. the “N isotope dilution. the A value and total N difference were used to measure N, fixed. There was a good agreement between the A value and isotope dilution methods for measuring Nr fixed. The total N difference method gave unreliable values, with large coefficients of variation. There were significant differences in the proportions and amounts of Nz fixed in the two Casuorina species with C. equisetijoliaderiving on the average 63% or 45 mg N plant-’ from atmospheric Nr fixation. compared to 43% or 22 mg N plant-’ by C. cunninghumirma. Nitrogen fixation also varied substantially within provenances of each species with the percentage of N derived from atmospheric Nr fixation (% Ndfa) ranging from I4 to 76% for the C. cunninghamho provenances and from 25 to 75% within C. c~quixfifoliu (equivalent to 2-25 mg N plant-’ and 4 - 29 mg N plant-’ for the two species, respectively). Growth of C. rqui.wfi/i,lia and C. runninghumiana increased with either inoculation with Fronkiu or N fertilizer addition. but marked differences developed between these N treatments with time Growth of inoculated plants was more variable (CV = 38%) than that of plants dependent on soil N of fertilizer N. This variation in the growth of the inoculated plants was thus due to the large ditTercnces in the N,-fixing abilities than to intrinsic growth differences.

The potential of Cusuurinu. actinorhizal plant spccics as multi-purpose tree crops is recognized worldwide. Cusuarinu is widely used in tropical and subtropical countries for dune stabilization, establishment of shelterbelts. production of fuelwood and in agroforestry (National Academy of Sciences, 1984). The outstanding ability of Casuarinu species to form symbiotic N,-fixing associations with Frankiu enables them to thrive in N-deficient soils. and making them desirable in agroforestry systems. Enhancing this capacity would greatly increase their usefulness. The microbial partner in the actinorhizal-Frunkiu symbiosis has received considerable attention (Coyne. 1983; Diem er al.. 1983; Zhang and Torrey. Reddell er 01.. 1986). with considerably less research on the existing variability within the host-plant as an approach for deriving greater benefits from Nr fixation (Dommergues. 1987). Attempts to select superior genotypes. using different host-Frunkiu pairings demonstrated that diffcrenccs in Nz fixation can be due largely to plant gcnotypic differences (Simon er al.. 1985). Sougoufara er ul. (1987, 1990) have shown significant diffcrenccs between clones of C. equiseri/oliu in growth. nodulation and N: fixation. Only a few provenances wcrc. howcvcr. screcncd and thus only a limited gene pool was exploited. Also, although there are indications that the potential of other Cusuurinu species such as C. cunninghamiuno is high for agroforestry, the available information is still very scanty. 539

With the wide geographical and ecological divcrsity in the genus Cusuarinu (Bond, 1983). the existing gcnctic variability could be great, and exploiting this would be most useful in N,-fixation improvement programmes. We have investigated genetic variation in the N,-fixing abilities of I I provenances of C. eyuisetijoliu and I I provenances C. cunninghamiana. and assessed the suitability of some common methods for measuring Nz fixed, as well as some plant variables that could be used in the initial screening of provenances for differences in N, fixation. MATERIALS

AND METHODS

The experiment was held in a glasshouse at the International Atomic Energy Agency (IAEA), Seibersdorf Laboratory, Austria. Mean day and night temperatures were 28’ and 2O’C; the light intensity was ca 10,000 lux for a I2 h photoperiod and the relative humidity varied between 60 and 70% (daynight amplitude). Soil characteristics and preparation

The soil, from Seibersdorf, Lower Austria, classified as a Typic Eutrocrepts (calcareous. clay loam with abundant gravel in the top layer, pH 8.3; total N, 0.3%. extractable P, 55.8 fig g-l, humus, 6.7%) was air-dried, sieved (2 mm), mixed with sand (I : I) and then transferred to plastic pots (5 kg pot-‘). It was kept moist by watering with deionized water to approximately field capacity. A basal fertilization consisting of 50 mg K kg-’ soil as muriate of potash

540

N. SANGlNG.4el al.

and I ml of a combination of micronutrient solution (B O.OS%; Mg 0.05%; Zn 0.005%; MO 0.005% and Cu 0.002%) kg-’ of soil was applied in all pots before planting. Seed sources, germination

and transplanling

Seeds of C. equisetifolia and C. cunninghamiana provenances (Table I), obtained from CSIRO, Australia were surface-sterilized with 96% (v/v) ethanol for 1 min and then with 3% (w/v) sodium hypochlorite for 10 min. They were rinsed several times in sterile water and germinated in sterile quartz sand. When I month old, inoculated or uninoculated seedlings were planted into 5 kg pots filled with a mixture of quartz sand and soil (1: I). After 18 weeks, they were transplanted into 10 kg pots with 4 plants pot -1. Frankia

srrains. cultivation

and inocuiulion

Strains of Frankia used for inoculum were ORS 002 1001 isolated from C. equiseti/olia in Thailand (Diem et al., 1983). HFP 020203 from C. cunninghamiana in USA (Zhang et al., 1984). These had been shown to be effective for the respective species. They wcrc cultured in liquid media for 4 weeks, using either pyruvatc-BAP medium modified by Murry ~1 al. (1984) or DPM medium (Baker and O’Kccfe, 1984). The inoculum was prepared by macerating the cells with a glass tissue grinder and washing them by ccntrifugation in two changes of stcrilc distilled water. The washed cells wcrc rcsuspcndcd in stcrilc water for inoculation at a ccl1 density of ca 5 mg protein ml -I. The Frankia strains were inoculated onto seedling (average 2.5 cm in shoot height and 3-4 cm in root length) rcmovcd from the tray. Batches of four seedlings were placed in sterile Petri dishes containing 20 ml of the inoculum suspension for I hr. and were then transplanted into one pot. Two weeks later, seedlings were again inoculated near the roots with the washed and homogenized suspension of the respective Frankiu. Experinrenrul

design and treulments

Treafmetrfs. (a) Plant species and provenances: 11 provenances of C. equisetifoliu and I I provenances of C. cunninghomiuna were used (Table I). (b) Nitrogen treatments: three N treatments were used for each species provenance studied: (i) ambient soil N plus 20 kg N ha-’ and inoculation with Frankia. (ii) ambient soil N plus 20 kg N ha-‘, but with no inoculation and (iii) ambient soil N in uninoculated pots, with 100 mg N kg-’ soil applied. For the 20 kg N ha-’ rate, IO atom% 15N excess KNO, was applied in solution to inoculated and control pots whilst for the 100 mgN kg-’ rate, the 15N enrichment was 2 atom% “N excess. Both rates 2 weeks after transplanting the were applied seedlings. These treatments will henceforth be described as inoculated, uninoculated and N fertilized respectively, since the main purpose of adding the 20 kg N ha-’ rate was to label the soil rather than to fertilize it. There were three replicate pots per treatment. for each of the two test species provenances. They were

Genetic variability for NL fixed by Casuarinrr arranged regularly

randomly within blocks and watered with deionized water to field capacity.

Harrests The seedlings were harvested at 18 and 36 weeks after transplanting and roots and shoots were separated. Soil was gently washed from the roots and nodules were collected and counted. The above ground parts and nodules were oven-dried for 72 hr at 70X, then weighed.

Analytical melhods Total N for the different plant provenance was determined on the Automatic Nitrogen Analyzer 1500 Carlo Erba and the N isotope-ratio analyses were measured on a VG-Isogas mass spectrometer (Fiedler and Proksch. 1975). The isotope dilution equation of Fried and Middelboe (1977) was used to calculate Nz fixed and to derive N uptake from soil. Values of N2 fixed in the different Casuafimz provenances were determined using as a control the corresponding uninoculated and non-nodulating Cusuafina provenances.

SIulisticul analyses Data were analyzed statistically using the computer programme statistical package (Mohan and Plane, 1985). Two-way analyses of variance were made for each provenance and N trcatmcnt combination to dctcrminc trcalmcnt clfccts. SCpiWiltC one-way analyscs of analyses of variance wcrc made for each N treatment to test the effect of plant provenances. When a significant ff < 0.05) treatment effect was found, an LSD was calculated to compare treatment mtans. Student’s I test was used within treatments to examine differences among the lsN-dilution, A value and N-diti’crencc methods of estimating symbiotic N, fixation. RESULTS

of C. equiseti/olia and C. cunninghamiana affected by provenances and N treatments. Inoculation with Frnnkia and N fertilizer application increased mean shoot dry weight of both C. equise~i~~ia and C. ~unningha~tiuna relative to the uninoculated controls (Fig. I). These differences continued over 36 weeks (Fig. I) and were still evident at 48 weeks after planting for C. cunninghumiana (data not presented). Significant differences in plant growth bctwecn inoculated and N-fertilized Cusuafina occurred with time. At 18 WAP, growth of N-fertilized C. equiser~~~~iaplants was signi~can[iy higher than the inoculated plant but the reverse results occurred at 36 WAP. With C. cunninghamiuna. N-fertilized plants maintained a significantly (P = 0.05) higher growth at both I8 and 36 WAP than either the uninoculated or inoculated plants. Growth of plant provenances differed signi~cantly within each species and a significant interaction between them and the source and amount of N in soil was found. A[ 18 WAP, growth of uninoculated C. equiserifoliu provenances varied from 0.9 I to I.24 g plant-’ with a coefficient of variation (CV) of 14% compared to I I % for provenances of C. cunningGrowth

was significantly

541

humianu. There

was less variability (CV = 8%) in growth when the different provenances of both spe ties were fertilized with N than when plants were inoculated with Ffunkiu. Growth of the inoculated plants varied between 1.22 and 3. IO g plant-’ for C. equiset~~oliaand between 1.32 and 3.68 g plant-’ for C. cunninghamiana (Figs 2 and 3) with CVs of 23 and 53%. respectively. The N-fertilized and Frankin-inoculated treatments significantly increased total N in shoots compared to the uninoculated control plants (Fig. 4). Inoculated and N-fertilized plants of C. cunninghamianu contained similar amounts of total N at both harvests. in contrast to inoculated plants of C. equisefifihu which accumulated more N than the N-fertilized plants (I5 and 43% greater at 18 and 36 WAP, respectively). There were significant differences in total amounts of N among the inoculated provenances within each species (Figs 2 and 3). Ranking of provenances often differed with harvest. The I8 WAP data in Table 2 show that within C. equisetifoliu three provenances, 1, 3 and 4 ranked low in percentage of relative effectivcness (total N of inoculated plants expressed as a percentage of N-fertilized controls), while two provenances. IOand I5 ranked the highest. Provenance IO maintained a high symbiotic cffcctiveness at 36 WAP with total N being almost 97 and 74% greater than for the uninoculated or N-fcrtilitcd plants, rcspcctivcly. Two inoculated provenances, 9 and 6, which had intermcdiatc total N at 18 WAP, were very effective at 36 WAP with rclativc cfli.ctivencss slightly higher than that of provenance IO. Although provenances I and 3 each gave a relative effectiveness of cu 20% over the N treatments, they consistently ranked low throughout this experiment. while provenancc 5, which ranked high at I8 WAP had a compardbie low elTectivencss index to provcnanccs I and 3 at 36 WAP. With C. ~u~~~i~ghumiuffu(Fig. 2) only three provenances. 2, 3 and I I showed a consistent relative effectiveness of over 100% at 18 and 36 WAP, while five provenances, 5,6,7, 8 and IO consistently ranked below 100%. Three provenances. I. 4 and 9. which were poor in total N at I8 WAP, ranked the highest at 36 WAP,

~odulu~i#n and nirrogen fixarivn No nodules developed on the uninoculated ptants, whereas all inoculated plants were nodulated. The number of nodules ranged from 3 to 25 plant-’ for C. equiserqolia provenances and from 2 to 8 for C. cunni~gh~zi#~u (Table 3). In general there was a significant and negative correlation between the number of nodules and their specific weight (r = -0.95). The proportions and amounts of N2 fixed by provenances of the two species are presented in Figs 5 and 6. The proportion of N derived from N: fixation (% Ndfa) in C. equisetgolia at 18 WAP averaged 61% and was 65% at 36 WAP, with C. cunninghamiunu deriving 42 and 45% during the two corresponding periods. The equivalent amounts of Nz fixed were, 19 and 45 mg plant-’ for C. eyuiserifoliu. and 10 and 22 mg plant-’ for C. cunninghamiunu. The proportions and amounts of N, fixed differed among the provenances within each species. The

N. SANCISGA ef al

C.cunninghamiono 4

i z Z a

EA

Inoculatd

BBIBI

N fartfiizu

3

a2

1

0 16

36

Week8 afta

16

36

planting

Fig. I. Effect of Frankia inoculation and N fertilizer on shoot dry weight (p plant-‘) and C. cunninghamiana.

%Ndfa ranged from 14 to 76 within C. cunninghaminncr. There was a significant correlation between % or total N: fixed and total N (R = 0.95; 0.98). Provcnances 2 and 3, which had the highest %Ndfa, also

0

accumulated

the greatest total N from fixation (Fig. 6) and had the highest symbiotic-effectiveness (Table 2). Provenance 5 which had a markedly low symbiotic-cffectivcncss index (Table 2) also had low

1 9 101

1234567

of C. equisefijolia

1234567691011

Provenancea

100

0

t

18 WAP

1254567891011

36

WAP

1254567691011

Fig. 2. Shoot dry weight and total N at I8 and 36 WAP of I I provenances of C. equisefijolia inoculated with a mixture of Fronkia strains.

Genetic

variability

for NZ fixed by Crrruarinu

36 WAP

18 WAP c !u

4

01

4 n

LSD

5X

n

5 6 7 6 9 1011

1 2 3 4 5 6 7 6 9 1011

Pravanancoa

100

18 WAP

t

36 WAP

”

1 2 3 4 5 6 7 6 9 1011

1 2 3 4

5

6 7 6 9 1011

Ravanancar Fig. 3. Shoot dry weight and total N at 18 and 36 WAP of II provenances of C. cunn@+n~icma with a mixture of Frunkk strains.

%Ndfa and total N, fixed (Fig. 6). The three variablcs thcrcfore matched closely in their rankings. Within C. eyuisrtijbliu, provenances 6 and IO had the

C.squisetifolia

inoculated

highest %Ndfa (73 and 76%). highest total N, fixed (Fig. 5) and ranked high in symbiotic-eNecGvencss index (Table 2). In the lowest range of fixation in

C.cunninghamiana

60

Con&o1 lnoculotsd N fa-tlliza

k -0 P

60

Z

40

20

0

18

56

16

56

Wseka after planting Fig. J. Effect of Frankia inoculation

and N fertilizer

on total

C. cunninglrunrianu.

N (mg plant-‘)

of C. equisrrfi)~ia and

fable 1. Symbmtic cffcccweners’ index (%) of different plant provenances of C. rqurserr~ol~oand C. ~nnrnghamiunu at I8 and 36 WAP C. eyuurtltbiiu Provcnancts I

I8 WAP 58

9

; 4 5 6 7 g 9 IO II

104 94 72 IS4 130 II? 144 120 171 IOO

Symbiotic effectweness index =

C. cunnmghumwtu

36 WAP

I8 WAP

36 WAP

126 128 115 I41 I17 181 129 135 178 175 IS7

5’_ 138 I32 42 6 70 76 50 87 65 121

Is4 104 I41 121 71 78 77 87 116 82 100

toul N of inoculated plants _I__ x 100. louI N of N ferulitcd planrs

C. eqz~iset~~~~u,the matching between %Ndfa and total N: fixed was not clear cut. However. generally. provenances 1 and I I were the lowest in these two variables. There was a good agreement (P = 0.05) between the isotope dilution and the A value mthod for estimating %Ndfa by the two Cmunrbur species; the coefficient for variation was < 20% and the Student’s I test for values obtained by the two methods was not significant (Table 4). The total N difference method gave larger errors and cocfhcients of variation. and even some negative values of N, fixed. Howcvcr, good agreement frcqucntly existed between the isotope methods and the N diffcrcncc method.

I)I.sCL’ss1oN . .

Our data. similar to those of Cauthicr cv 01. (19115) show that Custrtrrindl spccics derived on the average 43-M% of their N from symbiotic N2 fixation. These values arc also similar to those of Gliri~i;iiff .~e~iu~; and Leuc~ew Ieu~ocep~lu~f~ grown under fairly similar conditions (N. Sanginga. G. D. Bowen and S. K. A. Danso, unpublished data). Thus Cusrturino species could be considered as efficient in fixing atmospheric N: as some of the commonly-grown leguminous trees. This N,-fixation capacity offers a great advantage where agroforestry is the farming system of choice and where soil restoration or conservation is the major concern.

C. rq~i.~~li~J~iu

Comparable iarge variations among provenances of C. equiserublia or C. cunninghumiana in their N,-fixing abilities and growth response to inoculation found in this study have already been reported. For exampfe. Fleming et al. (1987) found differences of up to 30% between different symbiont-provenance pairings in C. cunninghamiana under glasshouse conditions. Also. the studies of Sougoufara et al. (1987) revealed significant differences in the N--fixing abilities between three clones of C. equisefifo(io inoculated with the same Frunkia strain. In our study, the correlation between nodulation (number and nodule mass) and N2 fixed by the different CuJtcurina provenances was not significant, in contrast to the strong and positive correlation (r = 0.96) between N: fixed (% and total) and the total plant N. Growth of inoculated plants was more variable than that of the N fertilized but uninoculated plants. This indicates that there was little difference in the potential for growth in these provenances under non-limiting conditions for N, i.e. the heterogeneity in growth of inoculated plants was due to the great ditrerences in the NJixing abilities of these provenances. Thus under N-dclicicnt conditions, the ability of a provenance of Cmccrrint~ to fix N2 will be crucial in determining its growth. Comparisons of N: fixed may be based on % or total N: fixed. Differences in total N: fixed may howcvcr be based on dilfercnccs in total N yields, which have somctimcs given misleading rankings of the N,-t’xation abilities of various legumes [c.g. Rcddcl et ni. (1986)]. Yield-indcpcndcnt estimates of Nz fixed, such as %Ndfa or ~~t(Jnl% “N enri~hlnents which have been used to compars N,-fixation abilities [e.g. Hardarson cl ul. (19X4); Ruschel et ui. (I979)j may therefore romctimcs give diRerent rankings from those obtained using total N2 fixed, as obtained in our study. For example, although provenance 8 of C. eyuiurr~@i,(iu derived a greater proportion of its N from Nz fixation than provenan~c 9, for total Nz fixed, the trend was reversed, because of the higher yield of provenance 9. Another factor that influenced the rankings made was the time or age at which the plants wcrc harvcstcd. Thus even though the initiation of substantial N, fixation may be delayed in some legumes, it may increase rapidly to eventually meet the plant’s N requirement. This is illustrated by the initial poorer growth of the inoculated C. cunninghumiana than the

Provcnances

Number (No. plam ’ )

~Ma55 (mg nodule ’ )

I 2 3 4 5 6 7 H Y IO II LSD $0,

3 3 9 n 9 7 IO IO IO 9 2.5 i?

4-l 54 II 19 21 29 20 ?I 23 22 5 38

C. c~~ni~~hu~i~~ff .-_-._Mas5 (mg nodult ‘)

Kumbcr (No. plant ‘1 4 7 2 7

37 IV x I9

I

42

6 7 n 7 2 2 ss

22 22 20 23 22 77 42

Genetic variability

for N: lixed by C’u.suurina

545

60 0 P n

40

12

3

4

5

6

7

6

9 1011

1 2 3 4 5 6 7 6 9 1011

Provenances

80

,

60

t

0 1 LSD 5% 2

;c

l8 wAf’

2 a.

Tl :: F 2

20

E

400

L 1 2 3 4 5 6 7 6 9 1011

1 2 3 4 5 6 7 6 9 1011

Provenancea Fig.

N-fcrtilizrd

5. Gcnotypic

plants

variation

in N-lixation at 18 and 36 WAP of I I provcnanccs of C. equi.wfi/oliu inoculated with a mixture of Frunkiu strains.

at 18. but not at 36 WAP.

For C. the results indicate that the early high rate of N2 fixation was maintained throughout the duration of the cxpcriment. Thus for some provenances or species, diffcrcnt conclusions could bc reached on the N,-fixation potential. depending on the duration of the cxpcrimcnts. Since trees are normally perennial. studies of longer duration may therefore be prefcrablc if possible. Similar to thr results of Talbot et ul. (1982). Hardarson er ul. (1984) and Ruschcl et al. (1979), the isotopic methods were more sensitive than the total N ditkrence method for assessing differences in %Ndfa. Also, in agreement with the report of Rcnnic (1981) in which hc obtained negative values of N2 fixed in N,-fixing plants using the total N-ditkcnce method, WC occasionally obtained negative Nr fixed using this method, but not when the isotope methods were used. The only times when the nitrogen difkencc and isotope methods gave similar results were, as indicated by Rennie (1983). when both fixing and reference crops had similar % fertilizer-utilization cfficicncy. A further advantage with the isotope methods was the higher precision in their estimates (CV c 20%) compared with a CV >40% for the N-difference method. A good correlation was found

equi.seti/idirr on the

other

hand.

between the total N in plants and the isotopic measurements (%Ndfa and N, fixed) in inoculated plants. This suggests that the total N of inoculated plants could be used for assessing differences in N, fixation during the initial screening for N,-fixation diffcrenccs. especially from a large collection (Danso. 198.5). This will represent a substantial reduction in costs and materials. Our results have demonstrated that the genetic variability in NJixing abilities of Cusuurina is high, and that N, fixation by this species may be significantly improved in any given cnvironmcnt by screening a large collection of diffcrcnt host genotypes for high symbiotic performance with inoculated Frankiu. This promising approach has however been given little attention relative to the microbial symbiont. Selecting superior plant genotypes should substantially increase yield of these trees to be established in N-deficient soils. The data also indicate that although on the average, C. equisetijoliu was superior than C. cunningharniuna in Nr fixation, Nr fixed in a few of the C. ctmnb~ghamiuna provenances was similar to the avcragc Nr fixed in C. equiseti/o/iu. This supports the suggestion for the screening of genotypes, even for species suspected to be poor N2 fixers.

N. SASGWGA et al.

546

60

18 WA/’

36 WAP

18 WAP

36 WAP

,

1 2 3 4 5 6 7 6 9 1011

t 2 3 4 5 6 7 8 9 1011

Rwsnances Fig. 6. Genotypic variation in N-fixation at IH and 36 WAP of II provenances of C. cunninghumiunu inoculated with a mixture of Frunkiu strains. Table 4. Precision of “N isoto~ methods and N belancc measurements uf N: fixccf and pcrccntagc N in she phnr de&cd from the atmosphere [%N~f~) as indicated by the analysis of variance, the SEM mJ cocttkicnt of varialmn for means only %Ndh

N: fixed

C. equi.v&&

ID AV

&. cunn~n~~~~lmiunu

DM ID ;:

Mcilns

SE

cv (%)

Means

SE

cv (Q/o)

32’ 24b 31’ 16’ IC 16’

x 4 7 R 4 7

26 I5 24 37 35 49

61b 6Q 79’ 44c 41’ 374

II t1 28 13 14 25

IR 18 35 28 33 66

ID = Irotopc dduuon. AV = A VDIUC. DM - Difference melhod

AcX-no~i~~ge~~eats_We thank MS Helga Axmann and the analytical staff of the Soil Science Unit. IAEA Seibersdorf Laboratory, for technical assistance and MS Mehrnaz Tadjbakhsh for typing the manuscript and for secretarial services and Dr G. Hardarson for helpful suggestions. We also thank Drs D. D. Baker. Y. Dommergues and H. G. Diem for providing the Frunkiu strains and CSfRO. Australia for providing seeds used in this experiment.

REFERENCES Baker D. and O’Keefc 0. (1984) A modified sucrose fractionation proccdurc for the isolation of frankiae from

actinorhi~~ root nodules and soil samples. PIunr unrf Soil ‘78, 23-18. Bond G. (1983) Taxonomy and distribution of non-legume nitrogen-fixing systems. In Biological Nifrogen Fixurion in Forest Ecosysrems: Foundufions und Applicurions (J. C. Gordon and C. T. Wheeler, Eds). pp. 55-87. Martinus NijhotT/Dr W. Junk Publishers. Coyne P. D. (1983) Specificity between Cusuurinu species and root nodule organisms. In Cusuctrina Ecology, Nunugemenr und Uiili;otion (5.1. Midgley. J. W. Turnbuft and R. D. Johnston. Eds). pp. ?OS-210. CSIRO. Melbourne. Danso S. K. A. (1975) Methods of estimating biological nitrogen fixation. In Biological Nilrogen Fixation in Africa (H. Ssali and S. 0. Keya, Eds), pp. 224-244. Rhi:obium Mircen. Nairobi.

Genetic

variability

for N, fixed by Cusuarina

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