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,:
,

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212
1:. <‘OKNli’l, (‘. O’I’I’O, Ci. I<lNAClI~O.
I
I
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6. I~IEht, Y. DOXIhIERGUE~
1. - 18TRODUCTlON
A numbcr of tropical Austrnlian acacia species are successfully used in uid
and scn+;G.l ;I~~;LS whcrc lhcy providc net only iircwood but also fend for livesto&
ancl somclimcs sccottdary usclitl products such as gunis (c>. g. ,4. stwgol). Acaci;ts
gencrally thrive in soils of low ferlility nnd are renowned for their tolerance to drouzht.
‘Uecausc of Ihcir associalion with rhizobia acacias fis nitrogen and contribute to the
itnprovcmcnt ol‘ Lhc soi1 nitrogcn st;tIus. T~~US acacias nppear to be protnising tt-em
l’or rcl8restnlion p~~rposcs in Lltc adverse cnvironmentnl conditions that prcvsil
in Wcstcrn Al’rica. Among Ilicsc trccs, rlc’trc%l holoscw?~cw, :i smsll trec nntke to Xus-
tralia, bas becn d is considered as an interesting species because it is fast growinp.
I Iowcvcr, ils NJixing poknliul lus ncvcr bccn cstimnled. The put-pose ofthe prcscur
study wits to MC;tsttrC this polcntiitl, by compnring the uptake of nitrogen by nndulated
(inoculukx~) aiid non-noclulntcd (unitioculalcd) piants prown i n fiel&sirnulatinp
conditions illld usitig sitiittllaticouslg t\\vO t5N tneIhods (the direct isotopic mcthod
and thc A vuluc tncthod) itnd the d~ifference method.
A ) EXIVXIMMTAI. I)ICSICiN
‘1%~ cxpcrinlerlt WIIS co~~dt~ctc~l il1 19S2-1983 ut thr OIISTOhl Bel Air Station in D&ur, Senepl.
on sixlccn I tn3 concrctc containers Iorming 1 111~ microplots. Thc soi1 used was Bel Air soil, a typical
sandy, ncnlral (pl 1 7.0) soi1 (ps;tmmcIiL; bkxxucul:lr namc: Dior) with C and N contents of 0.3 and
0,025 % rcspcctivcly. Avttilablc 1’ (OMEN PI ~1.. 1954) ws relatively high (25 ppm) for the soi1 type.
Thc soi1 wtts carclùlly htmoycttirctl, introduccd into the concrete containers and finaily fumigatd
willi mclhyl broiiiitlc.
Ob/triniig Acaçin holoscri0x swtlliu,qs. - To obtnin rcgttlnr germination the seeds erre prc-
lrcalctl wilh conccnlralcd sttlfuric asitl for 30 min. UermiiitIted secdlings \\vere planted in 5 1 25 cm
polycthylçnc potxhcs conlaining stcrilc Ilcl Air soi1 with a Iaycr of 100 ml stcrile vçrmkulite at thc
top.
Iircwtrlu/iotr pr~mdtir~*.
I’ll~~Ctilillitlll with Rlfizohirrrrr \\V;IS performcd hy pouring 1 ml ofs kxlturc
ofstrain ORS841 a1 LIK hxsc ol’cach scctlling at planting time. ORS811 hsd been previously shown to
bc ont of thc mosl cllfclivc slrains in ttssocittl ion with i-L~~~~itr /to/o.~ri~~~ grown in Dior soi1 (CORSEI‘
& DWM, 1982). Inoculation with G/omts UIO.S.CWP ws performcd hy applying about 1 8 (frcsh weighr\\
of Stlclall gras rools iltfcclcd vil II (;lo~/t~.~ ~I’IOSS~W on thc root si stcm of thc secùlinp LtI plantin$
timc.
ïT~r~ts~~/tr~rli~r~, .-- PI:mts wcrc raiscd in thc po~~chcs for 4.5 months nnd then transplantrd
into
thc microplots wilh l’our planls pcr microplot. ht th:it time their total N content WPS ca. 30 mg K
pltttlt -1. Jus1 bclorc lronsple~~lation, ail thc plots hacl reccived PK as K-HPO, at the rate of 17 g m-‘.
‘ficYr//rwrr/.>.
I:otir lrCiltIlll!IlIS willi livir rsplications cxh wcrt’ uscd BS f0ll~~~S:
. ‘I’rcatmc~ll U.?. llr~inocula~cd Irccs. ;ipplic;ilion ol‘l”N-l;~hellcd fcrtilizcr at thc rate of2 g K m *
as a solution 0l’(~~Ntl.,),S0,,
with 10.05 aton yi tBN esccss.
. ‘I‘rc;iLmcnl UIO. Unitltkwlatcd lrcc’s, application o f 13N-labelled ferrilizsr at rhe mtc o f
10 g N m L as a solution ol’(i~N14.,)~S0, with 1 .W atom “, *A N CSCCSS.
. ‘I‘l’callllcnL f\\>.?. ‘l‘rces inoctilaccil only \\vith Rlti:obitrttr ORSS41 ; application of ‘SS-lakllcd
fcrtiliwr as in trcatmcnl iJ2.
. Trcatmcnl KGZ. Trccs inoculntctl with Rhixhirtur ORSS4l sud G/OWU srussrnr; application
of ~“N-labcllctl I‘crlilizcr as in lrcolmcnl U2.

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N-free nutricnt solution upplicd at the rate of 1 I per microplot every 2 weeks. Throughout their
growth, thc plants wcrc curcfully irrigated to nvoid uny moisture stress. Plants were haruested
6.5 months after transpi~~ntotiou 10 thc microplots.
S~rwpli~rg. - - LWVCS (‘~~hyllodcs).
branchlcts (diamctcr i; 4 mm) stcms nnd branches {dinmeter
. . 4 mm) wcrc sa~~~plctl scpaiatcly. I:or ~;I~II indiviJu;ll plot thc Icavcs. branchletsstcmsand branches
of thc four trccs wçrc pooled together, tbcn drieSti to u constant weight tlt 60-70° C. The totality of
cacb fraction was grountl inlo 100 /ri11 powdcr. :Samplcs of cnch powdered fraction \\vere annlyzed
for total N and ‘“N (GAUTIIIIX ~‘1 cri., 198.5).
TRC roots wcrc S(;linctl according to III~ msthod of I’IIII.I.II>S & H:\\\\-sL\\K (1970). tut into 5 mm
scgmcnls 2nd olxcrvc~l unilcr ;I dissesliny mkroscopc to determine thc pcrccntnge of infecled roots
(früqucncy).
c’) Mi~llol,s OI ASSI:SSMI NI‘ 111‘ N2 I~IS,\\rION
I:~I ilnaling Ns Iisalion was ;Iccomplishcd 11): using simlllt;mcousl!’ thrcc mrthods. ail of them
based on thc comparison of N,-lising and non-N,-lising plants: direct isotope dilution merhod.
A valtic nicti~od ailil dilli9cncc incthod.
Whcn using thc tlirccl isotope dilution mcthod (I~KEMNFK, 1977; VOSE c*t d., 1952; RES~IE
& RENN~E, I983), the 15N-labcllcd fcrtilizcr was applied nt tbc same rate to both N,-fising(treatment R2
md K62) and non-N,-lixillp plants (trcatmcnl LF).
Whcn tising lhc A valuc mcthod (FIuE~) c‘k ~KIEKI-IART. 1975: FKIED & hllDDLEBOE, 1977) the
I”N-labcllctl li’rtilkx WXG applicd at a low rate to tbr N,-tising trt’cs (trcatments RI und RG-‘)
but at a normal (‘highcr) rate to thc non-N,-lising trccs (trcatmcnt URI). The A value method involves
the assumption that, whcn confrontcd witb ditfcrent sources of N. the plant uptake is directly pro-
portionnl to thc nmount of N ;Ivailablc in c;~ch source. prnvided that this nmount is meusured in the
samc unit, tlesignateJ A. ‘l’his unit is csprssscd as fcrtilircr N cquivalcnt (as g fcrtilizer N plant-
or m .B in otir cxpcrimcnt):
“A, N dcrivcd from fcrlilizcr
‘x, N dcrivcd from NI fixation (~3
YO N derived from soi1
-
-
A valuc 01’ I’crlilizcl
il ValtIc OT NC IiSiitiOll
A value of soi1
‘I’hc basiç assiimplioi~ of lhc ililYcrcn~e mcthod is that thc tixing and non-lixing plants assimilate
idcntical amonnts of mincral (soi1 1 fcrtilkcr) nitrogcn. T~US thc quantity of NI fised is meosured
by the dill’crcncç bclwccn thc total N content of inoculated (No-fising) planrs (trentments RI snd
RG2) and lhc total N conlcnl of uninoculatcd (non-N,-fisinp) plants grolving with the samc Iow
;tpplic;tlion of N IcrliliLcr (Ircalmcnl U2).
‘I’hc prcparaliori ol’s~~nplch ;III~ emission spcclroiuclcr nniilysis \\\\crr\\ pcrformrd in our Isb0r3-
tory according lo thc mcthotls dcscribcd b y FIEDLER & I’RoESTtt (197% und ~~ARTIN et ut. (1981).
‘1%~ spcclromclcr LIS~~ wiis il GSI nianufaclrrrcd
by Supra (l).
III. - KESU LTS
Table 1 shows lhnl inoculnting Acoci~7 lroloscv?cw with Rhixhim (treatment RI)
increased height by 36 y,:, dry weight by 43 7; and total N content by 40 7; in compa-
rison with uninoculatcd plants which had received the sme low amount (7 g N rn-‘)
(‘) Xopru, 6X, roc I’iorrc-J«igllc~~tI>i.
1: 9X.70 Bois-Colombes.
Vol. 6 (20), Il” 2 - 19x5

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214
1:. U)ltNI:‘l‘, (‘. Oll-0. G. RlNAUI>O, 1.1. ‘3. I-IlEhl,
Y
.

DOLIRLERGUES
of N (Lrcnlmcnl VI). Thc addition of 10 g N m-’ to the uninoculated plnnts (trot-
ment UIO) signilicantl~ increascd thcir growth (height, dry weight) and N content
in comparison with LttlttIocttltttcd plants which hnd received only 2 g N nl-;L (treat-
ment U2) but this incrensc ws signiticnntl~ lower thon thnt resulting from inocula-
tion with Rlrizobi~~r (treatment I(2), whtch indicates that inoculation improved
tltc plant growth signilfcunlly mort than the application of the higher Ievel of N fer-
tilizcr (treatmcnl UIO).
III gc~~ct~l (IIC duaI itloculittiotl (I h'hi:obiruu + G'/or~r~~ nrtusmt~) did net signitk
canlly aIl’cc1 thc growlh chnractcrislics and thc N content of the plant compare4
to Lhc inoculation Will1 b!/rirohiru!/ doilc. Howcvc~*, the m~corrtlizu~ infection 3ppetlred
to inducc ;t signilicnnt dccrcasc in the N ,‘,l, of tllc pl1yllodes of Acacir1 flolostw?Y~l?.
b ) ~~i~Il:<‘I‘ O N NOl)lll.h’l‘lON ANI) IINl~Ohl\\‘~‘Ol~l~tll~,\\l. lN1~tiC’1’I~)N
In trcalmcnt fi2 aiid KG2 (plants inocitlated with Rhi:ohitrni), nodule wei=htS
wcrc vcry similnr (tablc II). III trcatmcnt lJ2 and UlO(uninocul3ted plants) some rhizo-
bial conlnminatioti occurrcd, probably with native Rlri:ohim strains brought b‘,
watcr or dusI, hiil this type 01’ cont:tminaliott w;ts limitcd siucr only four planb
werc li~nd to bcar ~~odulcs: throc pllittlts out of 16 in treatmrnt 15’1, ;md only WR
pluilt oit1 of 16 in Lrcatnictit Uld. ‘fhc cont;tititiatioti \\V:\\s prObilbl~ late sinte tlie

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nodules on contnn~inn~cd plants wcrc 1cw and s~nall. By contrnst, contamination of
plants uninoculntcd with Glonr~ts ws more widespread: a11 the control plants (treat-
ment R2) WCI’C infcclcd by cndomycorlhi;?ill fiingi, but the infection frequency wns
relativcly low (10.8 y,) COlIliX~lTd to the infection frequency of inoculated plants
(37.2 y<), which suggcsls 11 rclalivcly Iütc inlkction of the control plots. Control plants
contaminatcd with Xhizohi/orr or endomycorïhiznc \\~XC not discarded but pooled
with the non-contaminated ones.
N, lis:lt ion (3 hy .4ctwh /tul<rsrrictw
inoculatcd uvith
----_------
Mçthuds
Rlrkobirrttl
Rhizobiwrt -t Ghws
_ . _. _ ..” .
. _..._... -.... “.- .__--..- -._ ..-. -..~
Dircçt isotope tlilulion. . . . . . . . , . . . . . . . . .
1.35 4 0.26
1.14 t 0.16
,4 VÜJUC................................
I .?6 2. 0.17
1.05 T 0.1s
Diffcrcncc..............................
1.12 3,: 0.45
0 . 9 7 $0.6?
(1) Mcnn valuc I çonlidcnec inlcrval (1’
0.05).
(2) N, fixation rclalivc Lu thc pcriud or 6.5 I~IOII~~IS tbllubing tr~uisplar~taliu~i.
Vol. 6 (20), II” 2 I 1985

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216
1:. (‘0RNI:l. C . OI‘TO, (;. RINAIII~O. 1 1 . Ci. DII:hI. Y . DOMR<ERGIIES
Direct isotope dilution
A value
-----4Je
/
-
.
T4--
0
d
26.2%
23-
5
4.1%
?
f 2-
76.8i6
67.7.q
l-
o-
IV. - CONCI,lEION
I‘hc only sigttilicnnt dii~crcttcc in thc elTcct of tnxtnw~t R.? and RC;) ~~1s that
lhc N x, COII~CII~ OI’ phyllodc~ \\\\Jus l~~\\ver i n pl:tuts duitlly inoculated. OU~: should
also nolc ;I IWK~ ~OWIK~S ;t dcct~~e i n total N content (table 1) itttd N, frssrion
(table IV) in plants dunlly inoculated. One of us (F. C.) has already observed this
tlcprcssivc cllccl ol‘cltllo~~~ycl)~~lti~itl
ittl’cclil~u OII Nr tis:ttion. SUC~ illl c’tTcct h:ts bt~~n
rcporlcd by I3ihlliN’l’liKIo CI (~1. (1%) in soybeatt. SA~IT~-I C! DAR (1%‘) in .\\larkwgo
safitu ancl VAN KESSIX Jt I<OSKOS~;I (I!N) in Irr,yn .jirtiwi/ bvhen the soi1 content in
ttvailablc 1’ is rclalivcly high. Tlw dccrcasc in N2 tisarion obsrrved hrrc ws probahl>
duc 10 ttll cxccssivc absorption of 1’ by niycorrhizal plants, high Ievels of P bzing
knowrt 10 inhibil uitïogcnitsc oclivily (~IHSON 1’1 ai., 1976). The content in ayailable
of the soi1 used in our experiment was 25 ppm, a figure which indicates a relativel~
high Icvel of I-’ for A. ho/tw~~icwr, In such conditions endomycorrhizal inf&tion enhan-
cd 1’ absorplion SO LhitI Iltc nulriliott ol’ the host attd N, tisittiott \\yere significantly
afTectctl.

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for inoculatcd plants thc pcrccntnge of plant N derived from soi1 \\V~S in thç
range of 61.8-67.7 y< :~cc»rdillg 10 the n~~thoct of :lssessment ~nd the treatmcnt
(lig. 1). This high contribtition of soi1 N 10 thc pl:lllt nutrition C;m only partiallj
be cxplained by thc rclativcly high content of availnblc N in the soil; it cari probably
be mostly attributcd to thc low N-lixing potuntiul of rl~acitr ldosericea, which will
be discussctl in thc following sccticin.
Thc pcrccntnpc of plant N dcrivcd from ft’rtilizcr was 4.1-5.9 0; in the case
of (11~ luwcr npplicalion 01’ N I’crlili/.cr ;III~ 73.7 “,; in tlic c;lsc of Lhe highcr opplicatiou
of N fcrtilizcr (Iig. 1). ‘I‘his small contribution of N fcrtilizcr to the plant N nutrition
rcsultcd I’wm thc low pcrccnlagc of utilization of lhc fcrtilizer. in relation to the
losscs lliroiigll clcnilrilic;llioll, volntilizalion and possihly Icnchinp.
C) N2 I;IXA’I’ION
‘I‘al)lc IV inclicatcs Lli:rl, wlialcvcr incthod \\\\‘a used, thc‘ cstiniations of NI tkl-
(ion wcrc roUgllly Illc S~IIllC. ‘I‘hc ;~m»unt of NS tiscd by the young .-lcocin I~oln.~ri~~~
at tr:~nsplaiit~itit,n liinc (whcii plants wcrc 4.5 months old) \\Y;lS lcss thW 30 lllg N,
plant-’ sincc thcir total N c011tcn1 WIS < 30 mg N z at that time. This amount \\vas
negligible (1 ‘x0) compared with the amount of N- lixed by the 1 I-month-old plants
so tli:~t it ciill bc ;ISSLIIINX~ that Iignrcs in tehle l\\7 ;I~L\\ rcl;\\tcd to NI fixation for the
6.5 mont11 pcriocl Iollowing tr:lnsl~lantaltion.
II’ WC :~ssumc thnt N2 Iixulion rcmaincd constant during the year following
trnnsplantution, which is a conscrvntivc prediction, \\ve cari admit that cach tret
lixed about 2.2 g N, yenr-‘. Extrapolation of this fisure to 1 ha basis would deprnd
011 thc trcc clcnsity 01’ thc pl:iiil;rtion. I II scnli-nrid conditions, \\vliere .-it*rlrici Irolo-
,y(jricrcr is plantctl witlc-spaccd, NJ Iis:ltion during thc Iirst ~c:lrs of plantation \\~ould
be only 4 or I I kg N2 ha- ’ ycar .’ for pli~~~t~~tion densitics of?,000 or 5,000 trees ha”’
rcspcçlively.
It is intcrcsting to co~r~parc this :II~~LII~~ of N,-tiscd h! ,4 . /toloscritva \\vith thr
arnount OI’ N2 lixccl by CC~~rrtr~Urrr cnc///i.scB/j/i)/icr OI’ SilmC agc und ~IU\\YII in identical
conditions at thc Sttlljc tilnc (GAUIIIIIX CI rrl., I9S5). N2 Jisation by .4c~it1 I~O/OSW~CW>
as estilnutccl in thc prcscnt cxpcrimcnt \\\\a I .3 or 1.2 p N, treç-’ accordin-, to the
mcthod ofcstin~nlio~l (direct isotope dilution or A vnluc). In thc rspcrimcnt dcscribcd
by GAIITIIIIX PI tri. ( 19115) NJ lisnlion by rrc~rlcr~ina c~~rk~~t~/i~lic~
ux to two three times
highcr (3.3-2.3 g N2 trcc”’ ‘). ‘I’hc tlill’crcncc in NI Iisution betnwn thc tree species
did no1 appcar to hc rcl:iLcd t« tlic dry wcight of thc plants or of thcir photosynthetic
orgtms which ww co~~lpart~blc (in thc COS~ of Acnc~ia I~o/~se~i~ca; the average weipht
o f ptlyllodcs was 3 3 0 g trcc~- ’ : in 111~ ci\\sc of C’U.YI/C~~~M~ r~/~~i,~~t[fi~/icl
fhC \\~çight of
clntlodcs W;IS 260 g Ira~.-‘). Nz lixotion clcurly :lppcared to bc relatrd to the nodule
dry wcight which wx ~nuch higher in C’a~~rr~i~u trecs (17 g nodules tree-‘) thnn in
Acucicl(4 g ~~oclulcs trcc-‘). T~U! rcliItiol1 bet\\vccn the N,-fising :tbilitv nnd the nodule
wcight is l)robably net lillcilr bccnusc the specilic Nz-fsing activity if acacia nodules
is known 10 bc highcr thun that ol’casuarinn nodules (unpublished datn). In conclusion
evcn if WC acknowlcdgc thnt thc mc~hods of asscssment uscd are liable to criticism
( WrrrY, I983), tlic prcscnt sludy suggcsls tliat .A<ïrcic~ Mosc~ri~i~a is not a yery effectke
Nz-fixing tree.
Vol. 6 (20), II” 2 - 19x5

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