NODULATION STUDIES IN LEGUMES 1. THE SYNOHRONIZATION OF HOST AND SYMBIOTIC DEVELOPMENT IN THE FIELD PEA, PISUM 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 symbiosis were found to be characteristically synchronized with host plant 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 root expansion in the 1-3-leaf stages. Haemoglobin formation was rapid in all nodules. The first-developed nodules on primary roots were pigmented (red) just before cotyledon nitrogen 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 globin destruction in the nodules of maturing plants. 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 nitrogen fixation in the annual legume 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). * Botany 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 soybean 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 rhizobia. 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 soil-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 'bacteria 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 ammonia 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