submitted in partial fulfilment
of the requirements for the Degree
Doctor of Philosophy
University of Canterbury
PIATERIALS AND METHODS
MORPHOLOGY AND DEVELOPMENT OF OOASE
AND BROOM PLANTS
IV.ACETYLENE REDLCTION BY E>CISED GORSE AND
Materials and Methods
METABOLISM AND TRANSLDCA nON OF FtXED NITROr:;EN
WITIiIN CIlRSE AND ffiOOM
A.Free amino acids in xyl ern sap and of
soluble nitrogen pool of root nodules
Free amino acids of sap
Free amino acids of nodules
8. 15N labelling studies
Metabolism of fixed 15N2
Translocation of fixed nitrogen
VI. EFFECT OF TEMPERA TURE AND LIG-iT ON rnOWTH,
NITROfIN ASSIMILAnON BY OORSE AND ffiOOM
Materials and methods
Results and discussion
VII. EFFECT OF PHOSPI-V\TE AND COMBINED NITROGEN ON rnOWTH
AND NITROfIN FIXATION BY OORSE AND ffiOOM
VIII. OXYGEN REQUIREMENTS AND THERMODYNAMICS OF SYMBIOTIC
NITROGEN FIXATION.THEORETICAL DISCUSSION.87
IX. OORON REQUIREMENTS FOR rnOWTH AND NITROfIN FIYATION
IN OORSE AND ffiOOM
~~terials and methods
Results and discussion
TRANSFER OF NITROGEN FROM OORSE AND EflOOM
GENERAL DIOCUSSlON. COT\CLUSIONS.
A C K N O V ~ E D G E M E N T S
TAB-ES, FIGURES, PlATES
A series of glasshouse and laboratory experiments was carried out
to enable comparison of two woody perennial legumes, gorse and broom, with
other legumes, nodulated non-legumes and other biological nitrogen fixing
80th species had distinct juvenile phases in which broom closely
resembled herbaceous species in appearance, but adult plants of both species
bore little resemblance to each other or to other legume species. Nodule
development was similar to that of other legumes, but mature nodules exhibited
structural adaptations to longevitymeristematic activity, a well
developed vascular system and numerous cytoplasmic granul es in cortical cells.
cant nued for muchonger
excision than has been observed in other legumes. In all experiments, broom
nodules exhibited higher rates of acetylene reduction and nitrogen fixation
than did gorse nodules.
The first detectable product of nitrogen fixation in excised nodules -
was rapidly incorporated into
~ - a m i n o
groups and another
unidentified fraction. The principle free amino acid in nodules and sap was
asparagine.Its preponderance incr88sed as plants aged.
Whole nodulated plants and excised nodules of both species exhibited
a relatively low temperature optimum for growth and nitrogen fixation (22oC).
They were very sensitive to elevated
Results indicated that
gorse and broom have relatively low light requirements.
When aeration was sufficient, combined nitrogen had little effect on
growth of nodulated plants.Nodulation in both species was reduced by
High levels ( 100 mg11) of nitrate
increasing amounts of combined nitrogen.
and ammonia caused consiQerable inhibition of nitrogen function.
80th species showed large respnses to phosphate, but were 6ble to grow
and fix nitrogen when supplied with low amounts of phosphate.
Boron deficiency reduced ni trogen fixation.Noduliltion was increased to
compenlule for this.
Considerable amounts of nitrogen can be contributed to the ecosystem in
gorse end broom litter. Direct transfer between gorse or broom and ~
radiate is likely to be
s n ~ l l
and may be masked by competition for other
These findings are discussed with respect to the use of gorse and
broom to overcome nitrogen deficiency in reafforestation on the Moutere
Gravels, in Nelson, N.l.
A band of soil formations, termed the "Moutere Gravels" occurs over an
area of some 315,000 acres in Central Waimea County, Nelson Province. N.Z.
At the time of European settlement. in the mid-nineteenth century. vegetation
on these soils was mainly leptospermum, the fern Pteridium. and grasses, with
pockets of NothofnpuS in gullies (Stone and Will. 1965j Appleton and Slow,1966).
After failure of farming and orcharding on many of these soils in the 1920's and
1930'S large areas were planted in forests, mainly Pinus radiata.
Growth of the first rotation (15-30 years) on the best forest sites on
the Moutere Gravels
considered to be similar to that on other good sites
in New Zealand and poor sites were not considered to be extensive.
After clear felling of areas of forests, and hauling tree lengths over
slash remaining after trimming, dense natural tree regeneration wasailowedto
occur. In many areas. growth of this regeneration has been shown to pe poor
(Stone and Will. 1965).By comparing these second rotation trees with stumps
remaining from the first generation. Whyte (1966) was able to demonstrate that
height growth stagnated longer in the second rotation on all but the best sites.
Later growth was generally comparable with first crops.
This lag represents a loss of apprC?ximately three years in the early
life of the tree.On very poor soils (for example on ridges), there was no
sign of the later recovery of second crop growth, even twelve years after est-
ablishment. Trlis and the lack of early thinning resulted, on these very poor
sites, in 12 Yl?.ar old crops producing only 30% of the OOs<;\l area of first rot-
ation trees, and only 7e/o of the height. The decline was generally transi,t:ory
lasting up to 5 - 8 years, though in the worst areas it was considerably more
persistent (Whyte, 1966;Whyte et aI, 1968).
Early indications were that this productivity
was due to defic-
iency of nitrogen and phosphate (Stone and Will, 1965j Jackson, 1967).Appleton
and Slow (1966) were able to obtain responses to nitrogen in yellowing Pinus
radiata on these soils.This treatment, however, accentuated both phosphate
deficiency and seasonal die back of young growing tips, the latter a character-
istic of boron deficiency (Appleton and Slow, lac.cit.: Proctor, 1957).
Use of nitrogen, .phosphorus and boron fertili zers can overcome these
deficiencies to a large extent, but in some soils, magnesium deficiency requires
treatment.However, there are some drawbacks to the use of nitrogenous fer-
tilizers in these forests:-
the stimulatory effect is only' transitory and declines by the third
year of treatment (Richardson, 1965;Will, 1965),
.2.economics of the use of such fertilizers in New Zealand are doubtful.
Responses of forest trees to nitrogen fertilizers have been reported in
other countriesj in Corsican Pine in Scotlanq (Miller, 1965), in Red Pine seed
production in Canada (Cayford and Jarvis, 1957) and in Kauri Pine in Australia
(Richards and Sevege, 1958).Ricrlards and 8evege
( l o c . c i t ~ ) also observed a
depression in tree growth when phosphate was applied without nitrogen. Such a
response was also noted bY,APpleton and Slow. (1956) in Nelson forests.The use
of fertilizers in forests was reviewed by Stewart and Svain (1965) who reported
responses of Norway Spruce to nitrogen fertilizers in Finland and Florida,and
by Baule and Fricker (1970) in Germany, who noted responses by several species
to a variety of fertilizers.According to Hewitt (1966), macro-nutrient
deficiencies are quite common in forest tree species and in nurseries, although
it appears that the importance of these deficiencies (especially nitrogen,
phosphorus and potassium) was only then being fully realized.
The widespread use of legumes as sources of nitrogen in New Zealand
pastures, suggests the possibility of using suitable legume species to over-
come nitrogen deficiencies in forests.Very little a t t ~ n t i Q n ,
been paid to legumes other than those useq in agriculture (see Norris, 1965)
J.S. and [.-",RT,
The influence of inoculum stl",-dn find tiM'! of f'ppl it;iltion of
o:mi1onium nitrdtB on ::.ymbiutic resprmse.
Nodul,tiLln studil?:.l in legumes.
PATE, .J.S. and mEIG.
activities of the nodulated root of the legume.
19G4.Rhythmic fluctuations in the synthutic
Plant Goil U.
PATE,J.S••MLKER. J. and II,iALlACE,
I1. ThE: significance
e bleeding sap.
compounds in the shoot system of Pisum 11rvense l.
of amino acids and amides r
PATE. J.S. and w\LLACE,
the root system of the field pea (Pisurn arvemm L.).
W.1964. Movement of assimilated nitrogen from
PATE, J.S., W\LLACE. W. Bnd van DIE.
in the examination of the circulation of nitrogenous substan:::es in
R. and PARKINSON.
ninhydrin positivB substances by broad
D. 1961.The sites of excretion of
PILAND. J.R•• IRELAND.
The importance of boron in legume seed production in the South.
Soil Sci •
C.F. and RElSENM'::UA,
of nitrogen fixation.
Rolevant aspects or the physiological chemistry
A nutritional disorder il"l Pines.,Commonw.F'or.Rev.
clover and lucerne.
D.N. 1951.Nitrogen in the life of
Kramer Business Service Inc.
p l ~ n t s .
Restriction of infection threads in nodulatiol"l of
Aust.J.8iol.Sci. 11. 155-161.
II. Influence of inoculation
the nodulation of Alnus glutinosa and its importance to our unoer-
standing of the mechanism of infection.
Symbiotic nitrogen fixation in non-leguminous
density and 'external fectors onplants.
Annu.Rev.Plant Physiol. la.
M. and ,RAGGIO.
1952. Root nodules.
N. and TORREY.
The nodulation of excised
RENE.E. and SHIVE,
relationships in plant nutrition.
upon the decline of productivity under forest mnnoculture.
B.N. and BEVEGE,
1967a.Factors which may have a bearing
ocono'flY of Grtifi,;tul
perennial legumes and c:anifuou5 tree species.
B.N. i,,"d H;c.VE.£l.
1 ~ t , 7 h .
fhe prOdlJr:t.!vity l'nd nitrog(;n
[ j ' , ~ O S ) l 5 t G m S c:orr.pri'5:lll(] wlriou3 c:::Jn)cin'ltions of
' \ ! J : ; ~ . J .
pine on a Interitic ponzolie soil in Southern rJucans) and.
f1.N. and 8[\}ECF.0.1.196iL
Nutrh'nt rl1quiremfrnl..1 O' k;\uri
The influence of' combined nitroge::n on nodulalinn and ni troge, fixation
D.C. and G\RRAAD, E.H. 1957.
A h i , z o ! ) i ~ ! meli:lati [)..3ngeard.GaQ.J.Pll'lnt Eei. £' 205-214.
sulphur fertilization .on 1he growth and distl'ibution of dry m,,,tter,
nitrogen, phosphorlJS and sulohur in Townsville stylo.
h u m i l i ~ l .Aust.J.Agric.Res.~
P.,I. and .JONES,R.K.1972. The effect of phosphorus and
( S t y l 2 . ~ ~ t h e s
phosphorus during early growth.
R.G. and KH1TON,D.J.
rne uptake and utili<.ation of
R h i Z O ~ J l u m
number's in the
~ ' h i z . c . s p h e r e s of red clover
and paspalum in relation ta soil trc.ltme:1t and the numbe!'s of bacteria
and fungi.Aust.J.Agric. Res.
their inFII)Elnca on soil
ROVIRA, A.D.1965.Plant root exudatos and
Proc. lnt.Symp.LJnili.Calif. ~ 1 S : l i e r l eV'
W . C ~ f ' i n y d e r .
rna phyl1osphere. rrr.
N1 trogen fixation in the
RUSCHEL, A.P. and DOBfREINfR,
bacteria in tne rhizospnere of gramineous forage plants.
Gassl. Congr. £' 1103-110?
SAMUELS, G. and LANDRAU,
boron on the nodulation, yield and protein content of tropical kudzu.
P ~ J r .
1953. Influence of nitrogen calcium and
.Trace elements in agriCulture,
SCHOLLHORN, R. and BURRIS,
inhibitor of nitrogen fixation.
A.S.1957.Acetylene as a competitive
Proc.Nat. Acad••Sci. U.S.A.
1958. Factors effecting the uptake of nitrogen by
Nutr1tion of the legumes.
Proe.Unit.Nott. 5th Easter Sch.Agric.Sci.
effectivoness in mutant strains of rhizobium by acetylene reduction
relative to other criteria of nitrogen fixation.
(.A., EVANS,H.J. and DAWSON,M.D. 19'70.Evaluation of
Plant Soil ~,
amino acids in
B U ' ~ N ~ "
l f ~ 0 u r n o nooelles.
:l, study wi..th p\"tpf!r chrom..~ t.ogrof1hy of the
Bot. [i..,l. ill,
of shade on growth of
ro~n5villel ~ : c r r . ~
( ~ 3 t . t l o s a n t h e 5 humili..::! H.8.K.). Queens.
, J . A ~ j r i c . A m i n . S G i .~,
Biology and ecology of symbiotic associations of
non-leguminous plants.Prac.Roy.Soc.Lond. 8.
SILVER,W.S. and M(\GUE,
terrestrial enviionment in field conditions.
T.1970. Assessment of nitrogen fixation in
NaturE (Lond.) ~,
th.O., Lincoln College, University of Cilntcr.b'Jry,
N ~ t r ' O q G l l fiX3tion by ro:::t n:J.:.'ulc5 of C::Jriaria.
N . Z ~
1965.The transference of nitrogen from pasture
legumes to an a:;;sociated grass under severel systems of management
in pot cuItures. Aust.J .Agrlc.Res.
SKIPPER, E.G. 1922.
on Hindhead Common.
F . ~ c l o g y
of gorse (~)
with special referencs
J.£col. 1£, 24-52. to the growth
SKDK, J. 1958.'rhe role of boron in the plant cell.
Academic Press Inc., New York.
SLDGER, C.1969.Symoiotic effectiveness and nitrogen fixation in
C. and SILVER,
symbiotic nitrogen fixing bacteria.
P.S.1967.Biological redl'ctions catalysed
Bacteriol. Proc. (1967)112.
The effect of combined nitrogen on nodulation and
S.Afr.J. S c ~ . §?,
e f f e c ~
roots of Phaseolus vulgaris.
M.C., CLARKE, B. and ffiOBI3£LAAA,
of whole plants
Theof temperature on nodulation
SMALL, J.G.C. and JOFFE,
genus Trifolium with special reference to the South African species.
II.The influence of root temperature on growth, nodulation and
symbiotic nitrogen fixation.S.Afr.pgric.Sci. 11, 41-56.
A.S. 1968.Physiological studies on the
SMALL,J.G.C. and LEONARD,
labelled photosynthate in nOdulated legumes as influenced
,by nitrate nitrogen.Am.J.8ot. §2, 187-194.
D.A.1969.Translocation of 14C-
SMITH,W.H. and PETERSON,
cen'bohydrate fraction of root exudate or red clover (Trifolium
e r a t e r 1 ~ L.) on Fusarium spp. isolated from clover root Bnd rhizosphere.
J.L.1960.The influence of the
populations of Tr'ifnl ium
H.W. and ffi,(,CSK!'\'I. A.Q.
r e p e n ~
L. in rC'3DOnsE' to mineral nutrients.
Differences betwoen mllural
1966.A compcu'ison of tho Pl"'loc;phate
nutrition of Australian heath plants and introduced economic plants.
Prolonged reduction of acetylene by detar.h8d soybean
T. Effects on the physiology of detached nodules.
New.Phytol. 2£, 9-17.
rheiffects of water stress on nitrogen-fixing root
SPRENT, J.L. 1972a.The effects of water stress on nitroger-
fixing root ncdules•. II. Effects on the fine stncture of (ietached
soybean nodules. New Phytol. 2!,
The effects of water stress on nitrogen-fixing
Effects on whole plants of ~
Sl'EVAAT. H. and S'lAIN,
fertilizers in forestry.
Reviewing the scientific use of
STEVART, W.D.P.1953a. The effect of combined nitrogen on growth and
nodule aevelopment of Myrica and Casuarina.
W.O.P. 1963b. The effect of available nitrate and ammonium
nitrogen on the growth of two nitrogen fixing blue-green algae.
Nitrogen fixation in plants. The Althone Press,
W.O.P.1969.Biological and ecological aspects of nitrogen
fixation by free-living micro-organisms.
STEMRT, W.O.P. and EIONO,
on fixation of elemental nitrogen in ~
347 - 359.
1961. The effect of ammonium nitrogen
and Myrica. Plant Soil ~,
In situ stUdies on nitrogen fixation using the acetylene reduction
technique. . Proc.Nat.Acad.SCi. U.S.A.
STEMRT. W.O.P., FITZGERALD,
Acetylene reduction by nitrogen fixing blue-green algae.
G.P. and BURRIS,A.H.1968.
STEVAAT.W.O.P., M/l.GUE. T•• FITZG::AALD,
Nitrogenase activity in Wisconsin lakes of differing degrees of
G.P. and ALJRAIS.A.H.1971.
New Phytol. 2£, 497-509.
E.L. and FISHER,
An effect of conifers on available
srClNE, E.l. ond WILL,
generation radiata pir,H in New Lealand.
soil relationships in
Conf. OregrJn State UnivlH'sity.
University Press, CorvalliB, Oregon.
G.M. 1965.Ni trogen df,flclnncy in second
: ~ D r t ~ l America.Proc.2nu N.r..m 8 r ; " F o r t ~ : : ; t
ed. C.T.YoungborfJ, Oreaon State
fixing enzyme system in AzotObacter vinelandii.
25 - 31.
G.W. and WILSON, P.W.
Formation of the nitroger. -
of the plant and its root exudate.
N.S. and VA5l\NTHA,
vitro and nitrate effect on the amino acid Gomposition
P. 1965.Nodulation of Trifolium
TAWltKA,H.1967a. Boron absorption by crop plants as affected by
other nutrients of the medium. Soil Sci.Plant NutI'. 21, 41-44.
8Qron absorption by plant roots.
Plant S:Jil gz,
TAWltKA, H., Y"'..TAZA'IA, M. and IYER,
elements in nursery soils ·in Wisconsin•
TANNER,J.W. and ANDERSON,
inorganic nitrogen in root nodulation.
Soil Sci.Plant NutI'.
Supply of trace
An external effect of
Nature (Lond.) 198,-
TARRANT, R.F. and TRAPPE,
the forest environment.
J.M. 1971. The role of Alnus in improving
Plant Soil spec.vol.,335=348.
TAYLOR,R.C.1969.Standard common names for weeds in New Zealand.
The N.Z.Weed and Pest Control Soc.(Inc.). Sigma Print Ltd.,Petone,N.Z.
growth produced by the addition of sodium nitrate to lucerne in sand
culture. J.Agric. Sci.
H.G. and NICOL, H. 1935.Reduction in nodule numbers and
Ecology of leaf-surface microorganism.
C.H.Dickinson. Academic Press, London.
Leaching of substances from plants.
Sept. 1970.eds. T.F.Preece and
TUKEY, H.B.Jr.1970b. The leaChing of substances from plants.
TURNER,N.A. and AEOGEWELL,
separation of amino acids by thin-layer chromatography.
R.J. 1966.A mixed layer for
VANCURA, V.1964. Root exudates of plants. I. Analysis of root
exudates of barley and wheat in their initial phases of growth.
Plant Soil ~,
Acetylene reduction in relation to levels of phosphate and fixed
nitrogen in Green Bey.
L.N., DANA,B., EMERICH, D. and BURRIS,R.H.1972.
New phytol. 21, 1097-1105.
compounds on nodulation and growth of Trifolium eratense L. in water
culture.Plant Soil ~,
and SMALL,J.G.C.1972.Effect of iron
Symbiotic 5[Jeci fie i ty.
in sand dune.
nodule bacteria of leguminous plants.
on the effect of the air content of the medium on
and function of the nodule.
A.I. end von HC.LJSEN,S.
I n v e ! > t i g " t i . c , , ~ un the j·o,:,t
i n v e & t 1 ~ j < 1 t i C o ( 1 ' "
with legume bacteria and plants.
nitrogenous compounds extractable from the root nOfJules of legumes.
A.I., von Hl\USEN.
S. and KARSmOM,
XII. Utilization by rJon-ll?g,;rnes,of the
Fixation of molecular nitrogen by excised nodules of alder.
A.I., MOISIO,T., ALLISON,
R.M. and BURRIS,
nitrogen by nodules excised from illuminated and darkened pea plants.
Acte.Chem.SCand. 2, 184-186.
A.I., MOISIO, T. and BURRIS,R.H. 1955.Fixation of
Acetylene reduction assay for nitrogen fixation
Non-symbiotic nitrogen fixation in the rhizosphere of several
tropical non-legumes. Rev. Ecol. 8iol. Soc. §, 367-373.
G. and DOl\f'/tERGUES,
enzymatic activity in soybean and sunflower.
G.S.19726.Influence of ammonium and nitrate nutrition on
Plant Physio!. 49.
pyridine and adenine nucleoticles of soybean and sunflower.
G.5.1972b.Influence of a~nonium and nitrate nutrition on the
WHEELER, C.T.1971. The causation of the diurnal changes in nitrogen
fixation in nodules of ~
G.T. and BONO, G.
1970. The free amino
~cids of non-legume
Phytochemistry 2, 705-708.
A.G.P.1966.Differences in growt:h#
A.G.P., WILL, G.M. and MEAD.
disorders in Pinus radiate in Nelson.
K.T. and BAHKUIS,
selecting effective strains of Rhizobia.
J.A. 1957.Chromatography as a means of
Plant Soil §. 254-262.
WILL, G.M. 1966.
lines of subterranean clover.
J.D.1972. Effects of root temperature on growth of four
WILSON,P.W. 1940. Tho biochemistry of symbiotic nitrogen fixation.
University Wisconsin Pross, Madison, Wisconsin.
WILSON,P.W. and FRED,
II.The P02 function.
Mechanism of symbiotic nitrogen
WYSS,O. and WILSON,
nitrogen by legumes.
Factors influencing exudation of
YAO, P.K. and VINCENT,
root hair 'curling factor' of Rhizobium spp.
J.M. 1969.Host specificity in the
accumulation in red alder stands in West Oregon.
J. and NEWTON, M.1971.Litterfall and litter
Plant Soil ~,
ZELITCH, I.WILSON, P.W. and BURRIS,
Plant Physiol. gr, 1-8.
in soybean root nodules
acid composition and distribution of 15N
supplied with 15N.