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NODULATION STUDIES IN

1. THE SYNOHRONIZATION OF HOST AND SYMBIOTIC DEVELOPMENT IN THE FIELD , ARVENSE L.

By J. S. PATE*

[Manu8cript received December 20, 1957]

Summary Symbiotic development of two varieties of field pea (Pi8um arven8e L.) was studied by periodic sampling from populations of field· grown material. Various features of effective were found to be characteristically synchronized with host life cycles. Nodule initiation was complete at the 7·leaf stage of plant development. Peak values in total nodule numbers and nodule/plant weight ratio occurred at this stage. Later, the regular decline in nodule numbers was offset by large in· creases in the size and fixation efficiency of remaining nodules. Well-defined pigment changes occurred in nodule populations. Numbers of young (white) nodules developed during precocious expansion in the 1-3-leaf stages. Haemoglobin formation was rapid in all nodules. The first-developed nodules on primary were pigmented (red) just before cotyledon reserves were exhausted. Maxima in total red nodules in both varieties were attained in the 6-8-leaf stages. The subsequent active (red) life of nodules varied from 8 to 80 days. Senescent (green) nodules accumulated on roots following extensive haemo­ destruction in the nodules of maturing . Average nodule fresh weight increased some 30-50-fold during plant growth. Comparisons of the average sizes of green and red nodule-population samples revealed a progressive elimination of smaller nodules throughout plant development. Nodule nitrogen and haemoglobin concentrations increased together to peak values in mid-vegetative stages. The maximum in total red-nodule nitrogen which occurred just before flowering, and some 7 -leaf units before a maximum in total plant nitrogen, reflected a general increase in nodule fixation efficiency as host plants matured. Approximately 30 per cent. of the nitrogen in the red nodule was removed as nodules turned green in early senility. Nitrogen returns from this source were estimated at less than 3 per cent. of the fixation benefit from healthy (red) nodule activity. High nodule-plant nitrogen transfer rates were recorded from red nodules from the 5-leaf stage until plant flowering.

1. INTRODUOTION Early observations of symbiotic in the annual led to the conclusion that legumes were rich in nitrogen at maturity through concerted nodule utilization in flowering and fruiting stages of host plant development. It was tacitly assumed that fixed nitrogen was stored in nodules and later released in host digestion of aging bacterial tissues (see monograph by Fred, Baldwin, and McCoy 1932).

* School, University of Sydney. NODULATION STUDIES IN LEGUMES. I 367

G. Bond (1936) was prominent among the first authors to question any sub­ stantial host plant benefit through nodule emptying. In. the plant he described a regular and immediate transfer of fixation products from nodule to host plant in stages of plant growth where there were no signs of any degenerative changes in nodule populations. Wilson and Umbreit (1937) recognized three important phases in soybean symbiosis. First, a short initial phase when a large proportion of fixed nitrogen was retained in the nodules. Then, a longer phase, extending over most of vegeta­ tive growth, when a large-scale transfer of fixation products took place giving logarithmic increases in plant growth and nitrogen content. Finally, a phase when fixation slowed down, nodule to plant transfer was highest, and nodule to plant weight ratios decreased. Further studies by the Wisconsin school established a close interdependence of symbiotic nitrogen fixation and carbon assimilation processes in legume develop­ ment (see Wilson 1940). The discovery of nodule haemoglobin as an integral part of the fixation mechanism has led to considerable expansion of earlier concepts of symbiosis. Virtanen and co-workers found a positive correlation between nitrogen fixation rates and nodule haemoglobin concentrations in a number of legume associations. For the pea plant Virtanen et al. (1947) and Virtanen, Erkama, and Linkola (1947) showed that nodule haemoglobin development paralleled the course of fixation; that the commencement of fixation was attendant upon pigment formation in young nodules; and that the cessation of nodule fixation activity in fruiting plants was associated with widespread pigment destruction in senescent nodules. This relation­ ship between pigment changes and nodule fixation activity has been noted for various legumes by other authors, e.g. Jordan and Garrard (1951), Nowotny­ Mieczynska (1952), and Heumann (1952). The aim of this series' of investigations is to provide further information on the general pattern of symbiotic development in legumes. The present paper on nodulation of the field pea outlines various aspects of the initiation and functional life of root nodule populations in an effort to determine the role of the individual nodule in plant nitrogen economy. Further papers will attempt to evaluate the much neglected topics of environment and host determination of legume nodule activity and longevity. II. MATERIALS AND METHODS (a) Plant Material Symbiotic development in the field pea was studied by periodic sampling from two series of sowings: 1954 Series.-Pisum arvense L., var. New ·Zealand maple pea-summer sowing, growing period June 11, 1954, to August 30, 1954. 1955 Series.-Pisum arvense L., var. Black-eyed Susan-summer sowing, growing period May 25, 1955, to August 28, 1955. Both varieties were grown in 60 by 40-ft plots of red subsoil sand at C~erry­ valley, Belfast, Northern Ireland. The ground was chosen for its light texture and 368 J. S. PATE low plant nutrient content. A balanced nitrogen-free mineral fertilizer applied to the plots before each sowing created conditions suitable for maximum symbiotic development. Row cultivation was practised to facilitate extraction of nodulated roots. Approximately 10,000 seeds were sown in each season to give plant popu­ lations well in excess of predicted numerical demands in sampling. A seed-applied rhizobial supplement was given to each series since the subsoil of the plots was found to be markedly deficient in indigenous Pisum-group . Inoculants were derived from a stock culture originally isolated from an effective nodule of maple pea. A satisfactory set of effective nodules was obtained. in both seasons allowing for uniform symbiotic development in both varieties.

(b) Sampling Procedure Fifty plant samples were randomly selected from the plots at 3-7-day intervals in the life cycles of the host plants. This rather large sample unit was necessary for the construction of reliable nodulation histories from -grown legume sowings, and involved a 0·5-5 per cent. standard error in host or symbiotic characters. Extreme care was exercised in the lifting and washing of plant root systems. Diseased, chlorotic, or otherwise atypical plants were rejected from samples.

(c) Recordings from Samples The following quantities were recorded for each sample: (i) Age.-Age of the sample in days from sowing. (ii) Developmental Age (expressed as average number of expanded leaves per plant).-Leaf age is of special value in gauging progress towards maturity in different seasons of growth. (iii) Nodule Number.-All nodules visible to the naked eye were recorded in population counts on primary and secondary root systems of the plants of the sample. (iv) Nodule Oolour.-Three colour classes were recognized in nodule counts: White (W-type) Nodules.-Small young nodules with cream-coloured contents, marking sites of recent infections on root systems. Red (R-type) Nodules.-Bacteroid-filled nodules with haemoglobin­ pigmented bacterial tissues. Green (G-type) Nodules.-Senescent nodules showing haemoglobin decomposition into bile-type pigment. Nodules remain green for a short period before entry by foreign ' causes extensive necrosis of internal tissues. A nodule was classed as G-type once more than two-thirds of its proximal bacterial tissues had turned green. Brown, disintegrating nodules were ignored in all nodule counts. (v) Nodule Weight and Tissue Analyses.-Nodules were removed by a razor stroke parallel to and touching the root surface. Representatives of the W +R and the G colour classes were separated, and both of these colour samples were weigh~d and subjected to Kjeldahl digestion. Determination of total nitrogen from aliquots of nodule or host plant tissue followed the conventional semi-micro- NODULATION STUDIES IN LEGUMES. I 369

Kjeldahl method, using a Markham steam-distillation outfit for recovery from digests. Portions of the 1954 series W +R samples were analysed for nodule haemoglobin. Nodule haemoglobin was extracted as pyridine haemochromogen (see Virtanen et al. 1947; Virtanen, Erkama, and Linkola 1947) and the reduced haemo­ chromogen estimated spectrophotometrically at 555mf-t. The latter absorption maximum gives least interference from the green pigment (see Hartree 1955). (vi) Host Plant Weight and Analysis.-Average fresh weights of individual plant organs were determined for each sample. In the 1955 series, plant tops and roots, cotyledons, fruits, and flowers were subjected to Kjeldahl analysis for total nitrogen. Nitrogen analyses were confined to cotyledon and nodule tissues in the 1954 series. III. RESULTS Various aspects of the symbiotic development of the field pea are summarized in the figures and table accompanying the text.

(a) Nodule Number (Figs. l(a), l(b)) Extensive and rapid infections of primary and secondary root systems result in sharp peaks in total nodule numbers in both varieties in the 6-8-leaf stages. A subsequent regular decline in nodule numbers occurs in late vegetative and repro­ ductive development. In the 1954 series some 20-40 per cent. of nodule populations persisted on roots until the end of the plant life cycle. The 1955 season allowed a more normal ripening of fruits, and this series recorded a higher efficiency of nodule utilization. The two varieties show marked differences in primary root, but not in secondary root, nodule numbers. In other trial sowings it was found that primary root nodule intensities in a legume species varied considerably with soil and prevalent light and temperature conditions, and consequently the observed differences are not taken to represent varietal differences in rhizobial acceptance. An analysis of this aspect of symbiosis in various members of the Pisum cross-inoculation group will form the basis of a further paper in this series.

(b) Nodule Colour (Figs. 2(a), 2(b); 3(a), 3(b)) The four graphs depicting fluctuations in the numbers of W, R, and G nodules over the host cycle provide conclusive evidence of ordered pigmentation sequences in nodule populations. (i) Root Infection (W curves).-Nodule initiation on a particular root system is related to the preceding growth activity of that system, since infection in both varieties was consistently limited to -invested portions of the roots. The early peaks in total W nodule numbers may be correlated with precocious root expansion over the 1-3-leaf stages. A similar relationship between root growth and nodule inception has been noted by the author in annual species of , Lathyrus, , Trifolium, , , and Ornithopu8. These species all exhibited root hair-invasion patterns, and showed remarkably early development of the nodule­ bearing parts of their primary and secondary root systems. 370 J. S. PATE

(ii) Nodule Activity (R curves}.-Pigmentation of nodules follows a strict aging sequence on all root' systems, where W-type nodules are consistently nearer root apices than their older, pigmented companions. Thus, inspection of the ascending portions of Wand R curves gives a fairly reliable picture of pigmentation rates on the various root systems. A time lapse of 1-6 days occurs between a nodule being first observed on a root system and its being recorded as showing visible

JUNE JULY AUGUST

2 z.6 3-S 4·3 5-3 6-8 '·7 9-01()09 13·814-, 15-8 18-4 20-3 21-8 LEAF AGE ) I I I I I I I I

FLOWERING 60 r (a)

150

~ 40 s>mM ~ r~=AC ROOT ~30 .J '"j 0 ~ 20 f .,co~~. 10 PRIMARY ROOT ..-----~-~------6_

0 10 20 30 40 50 60 70 80 PLANT AGE (DAYS)

MAY, JUNE JULY AUGUST

2 2·, 3-1 » 4-9 509 6-9 8-510-512-6 13-7 15-9 ,,.a 21·4 23-1 23-8 LEAF AGE I I I I I I I I I I I I

FLOWERING ~rM FRUITING 70 ~ Z~ •~~ i~ .J ~~ ~w m PRIMARY...... '--.-- ROOT ...... ~ __

0 m 20 30 40 50 60 70 80 90 PLANT AGE (DAYS) Fig. I.-P. arvense-nodulation: nodule numbers on primary, secondary, and complete root systems. (a) 1954 series, var. New Zealand maple pea; (b) 1955 series, var. Black.eyed Susan. haemoglobin colour. In both series first-formed nodules on a root system are slower to develop pigment than those arising from later infections. This is thought to be largely a seasonal effect since, in comparisons of pigmentation rates in V icia sativa L. as a winter and as a summer annual, later-formed nodules on a root system coloured more slowly than their older companions in autumal conditions, but more quickly in spring conditions (Pate, unpublished data). NODULATION STUDIES IN LEGUMES. I 371

(iii) Nodule Senescence (G curves}.-The active life of nodules in the sets of effective nodules of the two series is of very variable duration, healthy haemoglobin­ pigmented nodules remaining on a root system for anything from 8 to 80 days. The total root system maxima in G nodule numbers are attained at plant flowering. It is to be expected that senescent nodules will accumulate in late vegetative development.

JUNE JULY AUGUST

2206 3-54-3 5-3 &8 7-7 900 10·9 13-814·' Is-a 18·4 20-3 2\-8 LEAF AGE" " I I I I I "FLOWERING (a) \: ~ 30 ~ ~ ~ 20 -' ::> o /J~~~ 10 oZ

70 80 o 10 20 30 40 50 60 PLANT AGE (DAYS)

MAY, JUNE JULY AUGUST

I 2 2-73-1 3-9 4-9 509 &9 &5 la's 12-6 1307 1509 17·8 21-4 2301 23-8 LEAF AGE I I I I I I I I I I I I

FLOWERING 60 (b) FRUITING 50 1'\ ~ 40 « -' 30 ~W -' ) \ 5 ~ 20 w/~L '> 10 I ".~

80 90 o 10 20 30 40 50 60 70 PLANT AGE (DAYS) Fig. 2.-P. arvense-nodulation: nodule .colour. complete root system. W, young white nodules; R, red actively fixing nodules; G, green senescent nodules. (a) 1954 series; (b) 1955 series. (c) Nitrogen Levels in Plant Tissues (Figs. 4(a), 4(b)) Nitrogen concentrations in plant parts are expressed on a fresh weight basis. (i) Nodules.-As the nodule population becomes increasingly active in fixation nitrogen levels in the W +R samples rise. A significant drop ~ nodule nitrogen level is noted in mid-vegetative growth for the 1954 series,. but not for the 1955 series. A post-flowering decrease in red-nodule nitrogen levels is noted in both varieties. 372 J. S. PATE

Parallel increases in concentrations of nodule haemoglobin and nodule nitrogen are noted for the W +R sample of the 1954 series. Maxima in both quantities are attained early in host development.

JUNE JULY AUGUST

22-6 )-54-3 5'3 6-8 7·' 9{)1(}9 1306 141 15>8 18·4 Z0-3 21-8 LEAF AGE I J I I I 1 I I I I I I ,

:\ FLOWERING Ca) ,:" \,, 30 f \ Rl ,: ~' , '' , '' ... ~.--~ \ z , ,. .J .." 20 ! :." ...... \, ;;;- : \ ~ :J ! .---.'-'~"" o o z j • G, ''.._-...... _ .0 W j ~., ,./ \\

/'; .---. ,/ \./ R """~""'--- __ I to ____ ,i'-.. __...... _""- -. 1 .~ G -----A ,,' '. ~ ------o '0 20 30 40 50 60 70 80 PLANT AGE (DAYS)

MAY. JUNE JULY AUGUST 1 2: H 3-1 3-9 4-9 5-9 &9 &5 I(}S 12-61H 15-9 \7·8 21-4 23-1 Zl-B LEAF AGE r I I I I I I I I I I I I I I

FLOWERING FRUITING 40 f Cbl r- ...... : \ ; \ Rl

30 ! '& ... i ' . Z R f , ..".J .-,i , 111 20 --W ! ...... '\... ~-.-'\ .J :J w. : , 0 I ' '\....~ , 0 ,: \ , z , . ' If AI' .. ' " .. \ '0 \:,/,.~. _/-..~: "-'~;.'..';:::.::-.- ... -----. iX .. >..:~- G, "-._.__ .... ~ ..

o '0 20 30 40 50 60 70 80 90 .00 PLANT AGE (OA YS) Fig. 3.-P. arvense-nodulation: nodule colour, primary and secondary root systems. W, R, and G symbols as in Figure 2. W, R, and G curves: primary root system; WI, R 1, Gl curves: secondary root system. (a) 1954 series; (b) 1955 series.

G-nodule nitrogen concentrations are about 60-80 per cent. of current values for W +R nodules. As the nodule does not change in shape or turgidity while pigment destruction is proceeding it can be assumed that approximately one-third of the total nitrogen in the nodule is removed in early senility. NODULATION STUDIES IN LEGUMES. I 373

(ii) Host Plant Tissues.--Cotyledon nitrogen levels decrease as nitrogen is removed in seedling growth. The minimum value for plant nitrogen co~centration coincides with the transition period between cotyledon and nodule nitrogen nutrition. Nitrogen levels rise to a pre-flowering maximum associated with the initiation of flowering primordia. "

JUNE JULY -' AUGUST

2'02.63-54-35-3 6-S H 900 109 13-814·' 15-81&4 21>3 21-8 LEAF AGE " ! , I I I I I' I Iii ~ 24 FLOWERING :l RED NODULE HAEMATIN 300 m~ 8z w 250 g C :l 20 CI w m 200 0 0: j: ~ ~ ISO ~ ~ ~ 16 J: J: w ,A.-~ 100 w III ..0: ? RED NODULE NITROGEN J W SO CI 12 5 ~ Z . ~----. o w ~~ GREEN NODULES oCI e \J ~ 0: :I: I- III COTYLEDONS Z J:" CI 4 ""---" ~ (a) ~

o 10 20 30 40 !SO 60 70 eo PLANT AGE (DAYS)

MAY I JUNE JULY AUGUST

, 2 2·73-1 3-9 409 5-9 &9 &5 I(}SI2-6 13-7 15-9 17-8 21-4 23-1 2308 LEAF AGE I I I I I I I I I I I I J I , t'"L.uvvERING 12

~ 10 m j: ~ e w ..0: CI 6 Z Id CI o 4 REST OF PLANT 0: !: z CI 2 ~ (b)

o 10 w ~ ~"~ ~ ~ 00 ~ ~ PLANT AGE (DAYS) Fig. 4.-P. arvense-nodulation: nitrogen concentrations in nodule and host plant tissues. (a) 1954 series (also with red­ nodule haemoglobin levels); (b) 1955 series.

(d) Nitrogen Oontent of Plant Tissues, 1955 Series (Figs. 5(a), 5(b)) Figure 5(a) outlines the nitrogen economy of the young seedling. The coty­ ledon source is depleted towards the end of the 3-leaf stage. A small residue of unavailable nitrogen, amounting to less than 20 per cent. of the initial seed reserves, is lost as cotyledons are sloughed off the seedling axis. 374 J. S. PATE

Total plant nitrogen remains fairly constant over early seedling stages, but rises sharply once nodules turn red and commence fixation. There is no evidence of a check in plant development through a nitrogen hunger period following exhaustion of cotyledon reserves.

2" 3-1 3'9 4·9 LEAF ..,.GE I I I I

NODULES WITH 48 68 71 80 HAEMOGLOBIN (0/0) I I I I

(a) 161- 141- ) 12 1- «Z i 10 z w J 8 TOTALP~// 0 "0: 1- Z / 6 ---/Tops + ROOTS "::< .---- 4 COTYLEDONS "---0 21- "" NODULES

0---0 ~, I I I I l 0 4 8 12 16 20 24 28 32 SEEDLING AGE (DAYS)

MAY, JUNE JULY__ AUGUST

1 2 2·73·' 3-9 4-9 509 6·9 &5 1M 12-6 1:}7 15-9 17·8 2:1-4 2).1 23-8 LEAF AGE I I I I I J

FLOWERING SOO r • FRUITING (bY

400 RED NODULE:.-.!\

1- Xl00j Z « r1.. 300 Z --w 0" / ~ zoo z "::< GREE:·~ 100 / NOOUmx 100_ ~ /'/ / ~~~ + FRUITS ;..--"'-'" 60 70 80 90 0 10 ZO 40 50 100 PLANT AGE (DAYS) Fig. 5.-P. arven8e-nodulation: nitrogen content of nodule and host plant tissues (1955 series): (a) seedling nitrogen economy; (b) nitrogen contents of host plant and nodules over the complete life cycle.

Plant red-nodule nitrogen increases rapidly and shows a well-marked peak slightly before flowering. Green-nodule nitrogen represents a much smaller proportion NODULATION STUDIES IN LEGUMES. I 375 of total plant nitrogen. The significance of nitrogen withdrawal in nodule aging is considered in Section IV. . Total plant nitrogen increases in a sigmoid fashion with a maximum in late flowering stages some 2-3 weeks (7-leaf units) later than the nodule nitrogen maximum. This lag between nitrogen accumulation in symbiotic organs and in the complete plant can be explained on the assumption that nodules become more efficient in fixation as they mature.

(e) Average Nodule Size (Fig. 6) Remarkably similar sets of curves are obtained for the two series: W +R Sample.-Average nodule size (fresh weight) increases slowly in early plant growth since new nodules are being constantly added to plant root systems.

FLOWERING 32 ------. 28 ..- __ '!'..:!:.R 1955 9 W+R 1957 , -- 5 24 o / -~ Z --- ,I ~ ~ 20 w ,,.r) G >OM N iii 16 ' ~ .. .-... -... :l ,: .~-.-. G 1955 8 12 Z .! ' /') ,- w "~ 8 . l' " w /// // ·.... --1 «> 4 ' ..... ~ '"'J...... ,. ....".-,._--~y'o-__ .:

o 10 20 30 40 ~ ro M 00 ~ ~ PLANT AGE (DAYS) Fig. 6.-P. arvense-nodulation: average nodule size. W +R, young white+red actively fixing sample; G, senescent green nodule sample. 1954 series, var. New Zealand maple pea; 1955 series, Val. Black-eyed Susan.

Once nodule initiation ceases existing nodules grow rapidly, average size being increased some 30-50-fold in later plant growth. Nodules may show active meri· stematic apices almost until the death of the adult plant. Mature nodules are of smaller average size in the variety Black-eyed Susan. G Sample.-The first samples of green nodules are of greater average size than their active contemporaries since they represent the more mature portions of a population composed of many small recently developed nodules. At all stages of later development green nodules are of much smaller size than members of the current red· nodule population. This must be interpreted as a progressive elimination of the smaller nodules on root systems throughout host plant development. 376 J. S. PATli1

(f) Nodules Weight/Total Plant Weight Ratio (Fig. 7) This ratio may be considered as an index of the relative significance of the symbiotic organs at a particular stage in the plant life cycle. The ratio is low in early vegetative growth, rising to a peak value of 4-5 per cent. of the plant weight during the 4-6-leaf stages in both varieties. The ratio is progressively lowered in later development, until at plant maturity less than O· 5 per cent. of the total plant fresh weight is comprised of nodule tissue.

IV. DISCUSSION Figure 8 summarizes the relationship between host plant development (as gauged by leaf production and nitrogen accumulation) and the various represen­ tations of symbiosis studied. Remarkably similar sequences of attainment of symbiotic maxima are recorded for the two varieties. The Black-eyed Susan variety

~ 5 ~ FLOWERING ,.: i: 4 I- (\ Z , \ , \ , \ ".J 1955 / ', \ .~ .. 3 RIES1/ , ___ ', "- .J "I-o SE /.-' "v '\'" I- 2 / .,' '\ l- i: ',:\:,~~, ~-...... ~ ~ 1 I .J :J : --- o "'-.---.. --. o // z _.' o 10 20 30 40 50 60 70 80 90 100 PLANT AGE (DAYS) Fig. 7.-P. arvense-nodulation: fluctuations in the ratio nodules weight/total plant weight, 1954 and 1955 series. flowers some two leaf units earlier than New Zealand maple pea and shows com-' parable earliness in symbiotic development on a leaf age basis. It would be interesting to know whether early-flowering varieties show more precocious nodulation sequences than late-flowering varieties of the same species. Similarly, would photo­ periodic or vernalization induction of earlier flowering be anticipated by a general hastening of symbiotic development in earlier stages of vegetative development? Each feature of symbiosis studied adds its own information to the general pattern of integration of host and rhizobial activities, and it is possible to construct the following phasic analysis of symbiotic development for the field pea (see also Fig. 8).

(a) Phase I-Seedling Growth (1-3-1eaf stages) Cotyledon reserves are the major nitrogenous source in this phase. The essential nodule-bearing portions of the root system are expanded, and large numbers of young nodules accumulate on roots. The ordered infection sequences on primary and secondary roots offer evidence of root hair-invasion patterns in the field pea, NODULATION STUD1ES !N LEGUMES. 1 377 as have been recorded for other members of the subtribe Vicieae (L. Bond 1948; Nutman 1956). In the middle of the seedling phase the first-formed nodules of the primary root become haemoglobin-pigmented. The end of the phase shows a minimum in plant nitrogen concentration. In fixation studies of and Gicer in Indian conditions, Raju (1939) described critical lag periods between sowing and nodu­ lation, and between nodule formation and subsequent fixation. These periods were found to be more prolonged and accentuated in poor light conditions or inefficient t z 0 &jz - 0:0 !l:::< t:: :0 Z U «U LEAF PRODUCTION -

1954 SERIES H NEW ZEALAND MAPLE PEA

A C F J B D E G K L M N o I I LEAF AGE In A C D H G M N o B E I K L 1955 SERIES BLACK- EYED SUSAN

PHASE I PHASE II P .... ASE III -+--.... J:( ..... Fig. S.-Diagram summarizing the synchronization of host and symbiotic development in two varieties of field pea, P. arvense. Various representations of the nodulation cycle are related to host plant leaf age and nitrogen accumulation. A, maximum primary-root white nodules; B, first nodule turns red; 0, 90 per cent. of cotyledon nitrogen exhausted; D, maximum secondary-roots white nodules; E, maximum primary-root red nodules; F, maximum haemo­ globin concentrations in red nodules (1954 series); G, maximum nitrogen concentration in red nodules; H, maximum total-roots nodules; I, maximum total-roots red nodules; J, maximum nodules/plant weight ratio; K, maximum secondary-roots red nodules; L, nodule initiation ceases; M, 50 per cent. of red nodules destroyed; N, maximum in red-nodule nitrogen/plant; 0, maximum in total plant nitrogen. symbiosis. There is no evidence here of any such nitrogen hunger period in the transition period between cotyledon and nodule nitrogen nutrition despite the fact that the varieties were grown in soil abnormally low in combined forms of nitrogen. This would question the real value of the local Irish practice of giving nitrate supplements to legumes at sowing. Although added inorganic nitrogen might benefit seedling growth it might endanger subsequent fixation by suppressing nodule development. Fm:ther work is obviously required on this important aspect of nodulation. 378 J. S. PATE

(b) Phase II-Early Vegetative Growth (3-8-leaf stages) This phase of development witnesses maxima in total nodule numbers, in total red nodules, and also in the ratio nodule weight/plant weight. Peak values in red-nodule haemoglobin and nitrogen concentration also occur, fixation efficiency increases, and nodule to plant transfer ratio increases. Pigmentation of nodules is completed, and some green nodules are recorded on roots.

(c) Phase III-Late Vegetative and Reproductive Development (8-23-leaf stages) There is a regular decline in nodule numbers over phase III resulting in the loss of some 60 per cent. of red nodules by the commencement of flowering. This early destruction of nodule populations in the field pea is particularly interesting. Companion studies on vetch (Vicia sativa L.) nodulation showed a much later maximum in nodule numbers, with extensive nodule shedding delayed until flowering had commenced. Fred, Baldwin, and McCoy (1932) have recorded a similar nodule emptying in fruiting stages of large-seeded legumes. The early peak in nodule numbers in the field pea is undoubtedly associated with a completion of root develop­ ment well before plant flowering. However, this fails to account for the premature nodule aging noted in both series. In mixed nodule populations derived from several rhizobial forms one might expect an early loss of less-effective symbiotic units. Here a very variable .active life of 6-80 days is recorded from the symbiotic per­ formance of one effective . Possibly some host factor induces early nodule destruction. There is considerable evidence of internodular inhibitions in nodule initiation (see Nutman 1956) and similar interactions may obtain later in nodule life. Alternatively, competition for some nutrient factor, e.g. carbohydrate, might account for wide variations in nodule life span. This latter view is consistent with the observation that smaller nodules are progressively shed from roots at all stages of development. The pattern of nitrogen accumulation over phases II and III may be related to fixation activities of nodule populations in the following manner: (i) The Fixation Potential of the Nodule Population.-Plant red-nodule nitrogen is probably the most adequate expression of the fixation potential of the plant in that it is obviously related to the volume of active bacterial tissues (see Fig. 5(b)). Both G. Bond (1936) and Aprison and Burris (1952) describe fixation rates in terms of the nitrogenous contents of the nodule. (ii) The Apparent Fixation Efficiency of Red Nodules.-Fluctuations in apparent fixation intensity in red nodules are recorded in Table 1 (1955 series). Fixation rates are calculated as milligrams nitrogen fixed/gram red nodules/day, assuming that all nitrogen accumulated above cotyledon level is derived from nodule activity, and that there is no wide-scale excretion of fixation products from nodules to external medium. The validity of either assumption was not checked, but it is unlikely that excretion would occur in the rapid growth conditions in both seasons, and it was known that the subsoil of the plots was extremely low in combined forms of nitrogen. NODULATION STUDIES IN LEGUMES. I 379

Significant fixation activity is recorded from the 3-leaf stage until the beginning of fruiting, and shows a general increase trend of from 7 to 65 mg nitrogen fixed/g red nodules/day. The extremely high nodule activity on flowering plants accounts for the lag between maxima in weight of red nodules and total plant weight. There is some evidence of a final burst of activity prior to nodule destruction, but the nodule population at this stage is composed of large mature nodules with relatively

TABLE 1

THE RELATIONSHIP BETWEEN NODULE FIXATION ACTIVITY AND NITROGEN ACCUMULATION IN THE FIELD PEA, PISUM ARVENSE L. (1955 SEASON)

Apparent Nitrogen Nitrogen Retention . Increment Fixation Nodule (mg) (mg) of Fixed Plant in Total Intensity in to Released in Available Nitrogen Leaf Age Plant Red Nodules Plant Nodule through (mg) (stages) Nitrogen (mg N Transfer* Decom- Reduction in Persisting (mg) fixed/day/g position in RNt NoduleRi red nodules) (N)t

2,7- 3·1 1·2 7·2 81·6 - - 0·21 3·1- 3·9 2·7 8·2 83·6 - - 0·43 3·9- 4·9 5·0 10·5 90·0 - 0·72 4·9- 5·9 8·1 11·7 91·5 0·64§ - 0·90 5·9- 6·9 10·4 10·7 90·1 } - 1·18 6·9- 8·5 27·0 17·3 97·4 0·17 - 0·86 8·5-10·5 37·4 26·4 99·7 0·40 - 0·50 0·5-12·6 14·9 9·7 96·9 0·22 - 0·68 2·6-13·7 31·6 20·9 99·9 0·86 - 1·57 3·7-15·9 57·0 30·4 100+ 0·21 - } 15·9-17·8 32·7 18·5 100+ 1·17 '1 - 17·8-21·4 87·1 65·0 100+ 0·37 - 21·4-23·1 - - 100+ 0·27 J2.42 - 23·1-23·8 - - 100+ 0·61 ------Totals 315·1 4·92 2·42 7·05

* Percentage increment in nitrogen in plants minus nodules/increment in total plant nitrogen. t See text for explanation of symbols and method of calculating N. i Expressed as red-nodule nitrogen increment plus nitrogen loss in nodule emptying. § Approximate value based on a loss of 20 nodules from roots over the 3·9-6·9 leaf stages (see Section IV). larger volumes of bacterial tissues. Furthermore, overall efficiency may increase with advancing season, and with the progressive loss of the smaller, possibly less active, members of the nodule complement. (iii) Apparent Nodule to Plant Transfer.-This is defined as percentage nitrogen increment in plant minus nodules/nitrogen increment in total plant. Values recorded in Table 1 show a progressive increase in transfer from 80 to 100 per cent. as nodule 380 J. S. PATE populations mature. Similar ranges of values have been noted for soybean symbiosis by G. Bond (1936) and Wilson and Umbreit (1937). (iv) Nitrogen Available to Host Plant or Returned to the Soil in Nodule Decom­ position.-No data are available for annual legumes on this much-debated aspect of nodule nutrition. Butler and Bathurst (1957) recently provided an estimate of 72lb nitrogen/acre/year available through nodule decomposition in New Zealand perennial and ryegrass swards. Their estimate corresponds to an annual release of 240 mg of nitrogen from a single white clover plant. The data on nodule-colour changes in the 1955 series enable a fairly accurate estimate to be made of nitrogen increments to plant or soil in nodule aging. Over the 3-7 -leaf stages, nodule turnover is difficult to assess as nodules are still being added to roots. Later, once nodule initiation ceases, nitrogen availabl!! from nodule shedding (N) can be calculated from the expression:

N = Rn X RN X Gw , where Rn = number of red nodules lost over a particular period, R N = current nitrogen concentration in red nodules, and Gw = current average weight of green nodules (the weight of the smaller members of red-nodule populations being eliminated from roots). Values for the quantity N over the age increments studied are included in Table l. Total nitrogen available in nodule decomposition is found to be 4· 92 mg/plant/season. The amount of this actually sequestered by the host plant is problematical. Even on the assumption that there is a 100 per cent. efficiency of nodule emptying, plant benefit over the life cycle from this source would be only 1 ·6 per cent. of the fixation returns from healthy nodules. A further source of plant nitrogen in nodule aging is through a progressive lowering of red-nodule nitrogen concentration in fruiting phases. This is calculated as 2· 42 mg/plant/season, giving a total release in nodule senescence of 7· 34 mg nitrogen/plant/season. The latter is equivalent to 2·4 per cent. of fixation returns from active nodules. The above data show that little benefit to associated could materialize from nitrogen returns to the soil in nodule emptying. Moreover, the extremely small quantities of nitrogen available from this source would be returned to the soil late in growth. Claims of nitrogenous benefit to companion cereals have been made for mixed sowings of maple pea and oats in Northern Ireland, and it is obvious that nitrogen transfer is from some other source than nodule disintegration. Decay of root tissues and active excretion of nitrogen from nodules are other possible sources of nitrogen benefit to a non-legume. If substantial benefit occurs early in legume and cereal development, it would be reasonable to assume that excretion was the major exchange mechanism in underground transference of nitrogen. (v) Retention of Nitrogen in Persisting Nodules.-A fraction of the nitrogen fixed is retained in persisting members of nodule populations allowing for the 30-50-fold increases in average nodule size observed over the nodulation cycle. The observed increment in red-nodule nitrogen over a specific period actually under­ estimates nitrogen retention as nodule nitrogen losses have occurred in nodule NODULATION STUDIES IN LEGUMES. I 381

destruction. Hence values in Table 1 are derived from the expression red-nodule nitrogen increment + nitrogen loss in nodule destruction. The values recorded show considerable retention of fixed nitrogen in persisting nodules from the 3-leaf stage right up to flowering. This period corresponds to stages of development recording rapid increases in average nodule size.

V. ACKNOWLEDGMENTS The work described in this paper forms part of a Ph.D. thesis and the author wishes to express his thanks to his supervisor, Professor J. Heslop-Harrison, Queens University, Belfast, for helpful suggestions and criticism. The author is also greatly indebted to Dr. R. N. Robertson, Plant Unit, Division of Food Preser­ vation and Transport, C.S.I.R.O., University of Sydney, and Professor J. M. Vincent, Faculty of Agriculture, University of Sydney, for valuable help and advice in the preparation of this paper.

VI. REFERENCES

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