Ated Grasses. 1
This dissertation has been microfilmed exactly as received 66-13~715
WHITNEY~ Arthur Sheldon~ 1933- NITROGEN FIXATION BY THREE TROPICAL FORAGE LEGUMES AND THE UTILIZATION OF LEGUME-FIXED NITROGEN BY THEIR ASSOCI ATED GRASSES.
University of Hawaii~ Ph.D.~ 1966 Agriculture~ plant culture
University Microfilms, Inc., Ann Arbor, Michigan NITROOEN FIXATION BY TIlREE TROPICAL FORAGE LEGUMES
AND TIlE UTILIZATION OF LEGUME-FIXED NITRCX3EN
BY TIlEIR ASSOCIATED GRASSES
A TIlESIS SUBMITTED TO TIlE GRADUATE SQIOOL OF THE
UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR TIlE DEGREE OF
DOCTOR OF PHILOSOPHY
IN SOIL SCIENCE
JANUARY 1966
By Arthur Sheldon Whitney
Thesis Committee: Yoshinori Kanehiro, Chairman Bruce J. Cooil Robert L. Fox Leslie D. Swindale Goro Uehara ii PREFACE
The subject matter discussed'on the following pages is one that has challenged the writer for a number of years, and has been the object of much enquiry by him in Asia as well as nearer home. He is deeply grateful for the financial assistance provided by the East-West Center during his residence at the University of Hawaii, and to the personnel of the Hawaii Agricultural Experiment Station who made facilities and materials available for his research program. Thanks are especially due Dr. G. Donald Sherman for his assistance during the planning and ini tiating of this investigation. iii ABSTRACT
Three tropical legumes, Desmodium intortum, Desmodium~ and
Centrosema pubescens, were grown alone and in combination with napier grass (Pennisetum purpureum) and pangola grass (Digitaria decumbens) in fresh volcanic cinders under continuously moist climate on the Island of Hawaii. Q. intortum gave high yields of both dry matter (ca. 17,000 pounds per acre) and nitrogen (ca. 300 pounds per acre) in a 12-month period, and transferred small but significant amounts of nitrogen to its associated grasses. D. canum yields were low under these conditions, and the nitrogen yields of grasses associated with this legume were depressed. f. pubescens in pure stand was intermediate in yield of dry matter, but equalled!L. intortum in nitrogen yield. However, when com bined with grasses, the dry matter and N yields of this legume were reduced by one-half. Transfer of nitrogen to the grasses by f. pubescens was noted only when a 6-month growing period was allowed. The total fixation of nitrogen from the atmosphere during the test period averaged 340 pounds per acre for Q. intortum, 82 pounds per acre for Q. canum, and 156 pounds per acre for f. pUbescens. Of the total nitrogen fixed by Q. intortum, 5% or less was transferred to the associated grasses; but with f. pUbescens, transfer amounted to 11% of the nitrogen fixed in one instance. Transfer due to the release of nitrogen from roots of these legumes was evaluated by circulating nutrient solution through the root systems of plants growing in cinders in the glasshouse. The roots equilibrated with only trace amounts of solution nitrogen, but marked increases in the levels of ammonium and amino nitrogen occurred immediately after iv defoliation. When the root systems of nitrogen-starved pangola plants were included in the perfusion systems, significant transfer of nitro gen occurred from the more vigorous legume plants', especially following defoliation. Of the nitrogen mobilized in the legume roots in the 3 week period after defoliation, the proportions transferred ranged from slightly over 1% for g. canum to 9% for the more vigorous Q. intortum plant. Transfer of nitrogen through the leaching of nitrogen from legume leaves was studied by shaking intact leaves of varying ages in distilled water. The amounts extracted were small, between 0.4% and 0.7% of the total leaf nitrogen. Extractable amino nitrogen tended to be relatively high in rapidly expanding leaves, yellowing leaves, and shaded leaves. Leaf fall accounted for significant nitrogen losses from Q. intor
!Ym and ~. pubescens in situations where leaf senescence equalled the rate of production of new leaves. Under these conditions, the dead leaves from these legumes supplied nitrogen equivalent to over 1.2 pounds per acre per week. This pathway could thus account for appreciable transfer if long growing periods were allowed. The combined action of these three pathways provides an adequate explanation for the nitrogen transfer observed in the field. A number of ways in which transfer by these means would be affected by manage ment and by soil and weather conditions are discussed. v
TABLE OF COOTENTS
PREFACE...... ii
ABSTRACT ••••••••••••••••••••••••••••••••••••••••••••••••••••••••.• iii
LIST OF TABLES ••••••••••••••••••• 0 ••••••••••••••••••••••••••••••• vi ... LIST OF ILLUSTRATIOOS •••••••••••••••••••••••••••••••••••••••••••• • VJ.1J. INTRODUCTIOO ...... 1 REVIEW OF LITERATURE ••••••••••••••••••.••••••••••••••••••••••••••• 3
SMALL PLOT EXPERIMENT Materials and Methods • ••••••••••••••••••••• e.•••••••••••••••• 16 Results ...... 25 ROOT PERFUSIOO EXPERIMfNT Materials and Methods · - . 62 Results •••••••••••••••••••••••••••••••••• oo ••••••••• ~.e ••••• 70
LEAF NITROOfN EXPERIMENT
Materials and Methods • ••••••••••••••••• lit •••••••••••••••••••• 81 Results ...... 82 DISCUSS100 ...... 91 SUMMARY AND CCNCLUSlOOS ...... 103 APPENDIX •••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 108 LITERATURE CITED ...... 109 vi
LIST OF TABLES
TABLE I. TOTAL DRY MATTER YIELDS FOR GRASSES, LEGUMES, AND MIXTURES ••••••••••••••••••••••••••••••••••••••••••••• 30
TABLE II. DRY MATTER YIELDS OF LEGUMES, ALCNE AND IN ASSOCIATION WITH GRASSES ••••••••••••••••••••••••••••••••••••••••• 34
TABLE III. DRY MATTER YIELDS OF GRASSES, ALONE AND IN ASSOCIATlOO WITH LEGUMES •••••••••••••••••••••••••••••••••••••• •• 35
TABLE IV. ANALYSIS OF VARIANCE OF DRY MATTER YIELDS •••••••••••• 36
TABLE V. DRY MATTER PRODUCTlOO PER WEEK BY lWO GRASSES AND THREE LEGUMES •••••••••••••••••••••••••••••••••••••••• 39
TABLE VI. PERCa-JTAGE OF NITROGEN IN TOP GROWTH OF GRASSES AND LEGUMES ••••••••••••••••••••••••••••••••••••••••••••••• 40
TABLE VII. TOTAL NITROGffi YIELDS FOR GRASSES, LEGUMES, AND MIXTURES ••••••••••••••••••••••••••••••••••••••••••••• 43
TABLE VIII. NITROGa-J YIELDS OF LEGUMES, ALOOE AND IN ASSOCIATION WITH GRASSES ••••••••••••••••••••••••••••••••••••••••• 44
TABLE IX. NITROG~ YIELDS OF GRASSES, ALOO E AND IN ASSOCIATIOO WITH LEGUMES ••••••••••••••••••••••••••••••••••••••••• 46
TABLE X. ANALYSIS OF VARIANCE OF NITROGa-J YIELDS •••••••••••••• 47
TABLE XI. NITROGEN YIELD PER WEEK BY THREE LEGUMES, AVERAGE OF THREE TREATMa-JTS ••••••••••••••••••••••••••••••••••••• 48
TABLE XII. RATIOS OF ROOT NI TOP N FOR GRASS AND LEGUME SPECIES •• 50
TABLE XIII. NITROGa-J ())NTAINED IN THE ROOTS OF GRASSES AND LEGUMES 52
TABLE XIV. NITROGffi ())NTAINED ~ THE ROOTS OF GRASSES, LEGUMES, AND MIXTURES ••••••••••••••••••••••••••••••••••••••••• 53
TABLE XV. LEGUME CONTRIBUTION TO TOTAL N YIELDS •••••••••••••••• 55
TABLE XVI. LEGUME N ())NTRIBUTION PER WEEK TO YIELDS OF TOP GROWTH ••••••••••••••••••••••••••••••••••••••••••••••• 56
TABLE XVII. LEGUME N COO'TRIBUTION REFLECTED IN THE TOTAL ROOT N LEVELS •••••••••••••••••••••••••••••••••••••••••••••• 56
TABLE XVIII. NITROGa-J RELEASED TO PEROOLATE IN CERTAIN LEGUME PLOTS •••••••••••••••••••••••••••••••••••••••••••••••• 60 vii
TABLE IXX. LEGUME AND GRASS YIELDS FROM PERFUSED GINDER CULTURE IN lHE GLASSHOUSE •••~...... 71 TABLE XX. NITROGEN CXlNSTlTUENTS EXTRACTED FROM DIFFERENT SERIES OF LEAF SAMPLES ••••••••••••••••••.••••••••••••••••••• 89 TABLE XXI. ESTIMATED TRANSFER OF NITROGEN FROM LEGUMES TO ASSO CIATED GRASSES BY THREE DIFFERENT PATHWAYS ••••••••••••IOI viii
LIST OF ILLUSTRATIONS
FIGURE 1. LAYOUT FOR LEGUME-NITROGEN EXPERIMENT, WAIAKEA, HAWAII •• 17
FIGURE 2. ROOT SAMPLING CYLINDER FOR SMALL PLOTS ••••••••••••••••• 21
FIGURE 3. NAPIER ROOTS PROLIFERATING IN AND AROUND CENTRO NODULES •••••••••••••••••••••••••••••••••••••••••••••••• 26
FIGURE 4. DRY MATTER YIELDS PER ACRE OF GRASSES, LEGUMES, AND
MIXTURES •••••••••••••••••••••••••• So •••••••••••••••••• •• 31
FIGURE 5. NITROOEN YIELDS PER ACRE OF GRASSES, LEGUMES, AND MIXTURES ••••••••••••••••••••••••••••••••••••••••••••••• 42
FIGURE 6. DIAGRAM OF PERFUSION SUBSYSTEM •••••••••••••••••••••••• 63
FIGURE 7. LEGUME ROOTS IN APRIL •••••••••••••••••••••••••••••••••• 73
FIGURE 8. LEGUME ROOTS IN JULy ••••••••••••••••••••••••••••••••••• 74
FIGURE 9. NITROGEN LEVELS IN SOLUTIOOS AFTER PERFUSING LEGUME ROOTS •••••••••••••••••• •• ••••••••••••••••••••••••••• •• 76
FIGURE 10. VIEW OF GRASSES GROWN IN SERIES WITH LEGUMES. CCN- CLUSIOO OF FIRST THREE WEEK PERIOD ••••••••••••••••••••• 78
FIGURE H. VIEW OF GRASSES GROvtJ IN SERIES WITH LEGUMES. CCN- CLUSIOO OF SECOND THREE WEEK PERIOD •••••••••••••••••••• 79
FIGURE 12. TOTAL NITROGEN AND EXTRACTABLE NITROGEN IN KAIMI LEAVES OF DIFFERENT AGES •••••••••••••••••••••••••••••••• 83
FIGURE 13. TOTAL NITROGEN AND EXTRACTABLE NITROGEN IN CENTRO LEAVES OF DIFFERENT AGES ••••••••••••••••••••••••••••••• 84
FIGURE 14. TOTAL NITROGEN AND EXTRACTABLE NITROGEN IN INTORTUM LEAVES OF DIFFERENT AGES ••••••••••••••••••••••••••••••• 85
FIGURE 15. TOTAL NITROGEN AND EXTRACTABLE NITROGEN IN LEAVES OF THREE LEGUME SPECIES ••••••••••••••••••••••••••••••••••• 87
FIGURE 16. SUMMARY OF WEATHER roJDITIOOS AT WAIAKEA FARM, ISLAND OF HAWAII, 1962-1964 •••••••••••••••••••••••••••••••••••108 INTRODUCTION
In many parts of the tropics, pasture improvement provides a potentially important way of increasing the production of protein food stuffs. In Hawaii, most grasslands have been planted to introduced grasses, but increased beef yields and higher production efficiency can still be expected through pasture improvement wherever rainfall is adequate. Although ranching is the third largest source of agricultural income in the state, most pastures presently receive little or no fertilizer and have no significant legume component. As increasing land and labor costs bring greater pressures for better utilization of grazing lands, the adoption of more intensive pasture management practices will become an economic necessity. Most of the land presently relegated to grazing elsewhere in the tropics is unimproved and consists primarily of seasonal and low-yielding grasses. Leguminous browse plants may be found in these areas, but their contributions per unit area are generally low. The production of forage by such pastures is thus poor in terms of both quantity and quality. In wetter areas, nitrogen fertilization usually results in large increases in dry matter yields and also raises the protein content of the forage. However, the application of fertilizer nitrogen to such areas is sometimes uneconomical or impractical, especially since the responses obtained are very short-lived. The possibility of providing nitrogen to tropical pastures by establishing an effectively nodulated herbaceous legume in the sward provides an attractive alternative. Fortunately among the large variety 2 of tropical forage legumes availabl~ some are adapted to the low pH, calcium and phosphorous status of the soils found extensively in tropi cal areas suited for grazing. A few of the more promising herbaceous legumes have undergone evaluation and improvement, especially in Australia; and these appear to be capable of growing vigorously and fixing significant quantities of nitrogen under tropical grassland con ditions. Some of these are in use or are being evaluated in Hawaii, but their capacity for fixing atmospheric nitrogen has not been deter mined. Also, the extent to which these legumes can transfer nitrogen to the grass component of a mixed sward, as well as contribute high protein forage themselves, is largely unknown. The present studies represent an attempt to evaluate these factors for three legumes which are adapted to the humid tropical areas of Hawaii. One of these legumes (Desmodium canum) is a widespread road side and pasture plant which is low growing but has been thought to contribute to the protein yields of pastures. Another (Q. intortum) is a semi-viny plant widely adapted in Hawaii and known to be high yielding, but it is not widely grown due to its strict management requirements. The third (Centrosema pUbescens) has a viny habit and is a promising recent introduction from Australia. The mechanisms by which these legumes might transfer nitrogen to an associated grass are also of interest, and three different pathways of nitrogen transfer were thus evaluated for each of the above species. With a better understanding of these pathways and how they vary in importance for different species under various climatic and management conditions, it should be possible to improve procedures for species selection and pasture management so as to favour nitrogen transfer. 3 REVIEW OF LITERATURE
The capacity of legumes in association with the appropriate rhizobia strain to fix atmospheric nitrogen has long been appreciated as a means of increasing forage protein yields (Prianishnikov, 49). Many estimates of the extent of N fixation by temperate forage legumes have been published, but relatively few data are available for tropical species. Henzell, in Queensland, Australia, compared four tropical legumes (18) with white clover and alfalfa in sand culture in the green house, and found the temperate legumes only slightly superior in their ability to fix atmospheric nitrogen. In Ceylon, Fernando (17) reported gains in beef production of 150 pounds per acre in 260 days where centro (Centrosema pubescens) was included in a Brachiaria brizantha sward. The gains were attributed to a 50% increase in dry matter yield and over twice the percent protein in the forage. Similar results were obtained with alyce clover (Alysicarpus vaginalis) and tropical kudzu (Pueraria phaseoloides). Several studies have included centro mixtures. Watson (73) measured the N obtained in centro tops, and in leachate from the soil, and estimated that 210 pounds of N were fixed in 5 months. Moore (36) reported a 5 year experiment in Nigeria in which centro in combination with stargrass (Cynodon plectostachyum) yielded 560 pounds of N per acre more than the grass grown alone, or an average of 115 pounds of N fixed per year. In Taiwan, Luh (29) reported data showing no benefit from adding centro to pangola grass (Digitaria decumbens). Desmodium intortum and Glycine javanica with this grass greatly increased total protein yields over grass alone, but G. javanica almost disappeared from the mixture in less than one year. 4 D. intortum has been tested in Hawaii. Moomaw and Takahashi (35) measured the yield of an intortum-pangola pasture 3 months after the start of "production". Results were very variable, but both dry matter and protein yields were related to the percent legume in the mixture. The highest, at 30% legume, yielded 5,360 pounds of dry matter and 452 pounds of protein per acre. Younge, Plucknett and Rotar (80) reported a ·number of experi ments involving this and other legume species. At Waimanalo, Oahu, intortum averaged over 8,500 pounds of dry matter and 1,350 pounds of protein per acre per year over a three year period. Spanish clover (Q. sandwicense) and tropical kudzu yields were nearly as high. In another test on bauxitic soil, intortum and pangola grass yielded over 13,000 pounds of dry matter per acre per year. On a Humic Ferruginous Latosol where this mixture contained about 35% legumes, moderate gains of about 200 pounds per acre per year of beef were obtained. Kaimi clover (Q. canum) was also studied at this location. In mixture with pangola, yields of 3,000-6,000 pounds per acre per year were obtained, with the legume providing 2-20% of the dry matter. The highest yields were associated with treatments receiving high levels of lime and ferti lizer (N not included). Younge (79) reported one other experiment in which intortum and pangola grass were grown on bauxitic soil under high fertilization. One year after the original complete fertilizer treatment, dry matter yields of about 13,000 pounds per acre per year were obtained. Extra potash fertilizer resulted in a 30% increase in yield, due to a brief stimulation of the potassium-sensitive pangola grass. Nitrogen ferti lizer did not further increase yields, but seriously depressed the 5 legumes for a short time. The percentage of legume in the mixture was favored by later cutting, due to the slower recovery of intortum. No protein yields were reported, but the lack of response to N fertilizer indicates that in the absence of applied N most of the harvested protein resulted from N fixed by the legume. Legumes have been shown to benefit grass yields in a number of experiments. This effect has been noted particularly with white clover. Sears (53) reviewed a number of New Zealand experiments and concluded that a large percentage of clover-fixed N was apparently transferred to the grass. This amounted to 55 pounds out of 230 pounds per acre fixed annually at one location, and 140 pounds out of 500 pounds per acre fixed by pedigreed white clover at another location. The amount of N transferred was reduced by longer intervals between cuttings or by application of N fertilizer. Walker, Orchiston and Adams (71) treated data from a large number of grass-legume experiments, including Sears' above, and developed rough correlations based on average root:top ratios, N mineralization rates and a number of other factors. They then calculated from these correlations the approximate amounts of N transferred underground from the clovers to the grasses in various experiments. They concluded that transference ranged from very little to about one-half of the total N fixed. Application of fertilizer N or the return of animal manures, however, changed the relative amounts transferred. An equation relating the N yield of grass in a mixture to soil, fertilizer and legume sources was developed. For a ryegrass (Lolium sp.) white clover (Trifolium repens) mixture, approximately two thirds of both legume N and ferti lizer N was recovered in the grass. Since later work by Walker (69) 6 with N15 showed that the legume component absorbs essentially none of the N mineralized by the soil, the soil-N factor for any particular soil is a constant; and the equation for the experiment mentioned thus became: Grass N = 36 + 0.67 (Legume N + Fertilizer N). A later experiment (Walker, Adams and Orchiston, 70) using N15 in the greenhouse showed no evidence of underground transfer of N from white clover to associated ryegrass plants in 3k months. However, the clover took up much less soil N than the grass when the two were grown in mixture. The extent to which legumes use soil N has also been studied by other investigators. McAuliffe, et ale (32) reported that Ladino white clover and alfalfa seedlings obtained over 40% of their assimi lated N from the soil during the first ten weeks of growth. Older Ladino clover plants in mixture with fescue (Festuca sp.), however, fixed 65% of the N present in the clover tops. Transference of clover N was not studied. Shishchenko (55) found that red clover obtained 20% of its assimilated N from fertilizer sources during the first year, but only 0.2-1.7% the second year. Herriott and Wells (19) found that white clover apparently trans ferred about 50% of its fixed N to ryegrass regardless of N fertili zation. With orchard grass (Dactylis glomerata), about 33% was trans ferred, but when N fertilizer was added, this percentage was greatly reduced. Nishimura, Saito and Kijima (40) found a beneficial effect of vetch (Vicia sp.) on the N content of associated grasses, especially during later periods of growth. Peterson and Bendixen (45) presented data which corroborated Walker's equation. Ladino clover in mixture 7 with orchard grass was able to completely replace up to 160 pounds of N per year. Nitrogen recovery in the orchard grass tops was linearly related to the sum of clover N and fertilizer N present, amounting to an average recovery of 72%. Cowling (11) grew white clover with orchard grass and reported that the clover raised the N-yield of the grass by 60-100 pounds of N per acre per year. Dry matter yields of the mixture equalled that of grass alone plus 160 pounds of N per year. No response to N fertilizer was observed for the mixture, how ever. He later analyzed similar results (Cowling, et sl., 12) according to Walker's equation. The correlations obtained were sig nificant but variable due to negative correlations between the amounts of clover N and fertilizer N. A similar negative relationship between white clover N and ferti lizer N was found for yields of clover mixtures by Holliday and Willman (20). A small positive correlation existed between clover N and the N present in simulated animal returns, however. Nitrogen fixation by the clover was enhanced in the presence of a grass. Castle and Reid (10) evaluated white clover in mixture with either ryegrass or orchard gra.ss and found that management practices which maintained 30% clovers in the mixtures gave maximum returns. Grazing gave smaller yields but resulted in more transference of N to the grass than cutting treatments. Less work has been done on the benefit to grass yields obtained from tropical legumes. Several investigators have found that legumes in mixture with grasses have resulted in little or no differences in grass dry matter yields, but that grass protein yields were increased due to higher protein contents in the grasses. Vicente-Chandler, 8
Caro-Costas and Figarella (62) reported this for kudzu, molasses grass (Melinis minutiflora) mixtures in Puerto Rico. Moore (37) in East Africa found that the N content of stargrass increased from 1.8 to 2.4% when associated with centro. Fernando (17) found that both centro and kudzu resulted in marked increases in the percent of protein in the associated Brachiaria brizantha in Ceylon. Centro and stylo (Stylosanthes gracilis) were -bot~ active in increasing the N yields of associated grasses in Nigeria (McIlroy, 33). In other cases, however, only small amounts of N were made avail able to the grass. In Younge's experiment (79) mentioned above, in spite of extensive N fixation by intortum, apparently very little of the fixed N was available to the associated grass. Seeger (54) obtained only small and occasional benefits to corn from a number of associated short term legumes in 18 trials in tropical Africa. Other wise, combined yields showed an advantage for the presence of legume only with respect to non-utilization of soil N by the legume. Henzell (18) found that transference from two tropical legumes (Indigofera spicata and Desmodium uncinatum) to associated paspa1um grass was higher during the second year than the first, but this amounted to less than 1.7% of the N fixed by D. uncinatum and less than 0.6% of that fixed by the indigo. The pathways by which nitrogen can be transferred from legume to grasses have received scattered attention. Much interest has centered on the possibility of direct excretion of nitrogenous compounds by legume roots. Although this was postulated over fifty years ago (Lyon and Bizzell, 30; Lipman, 26), it remained- for Virtanen (65, 67) to show conclusively in a series of well designed experiments that peas 9 could excrete such compounds under Finnish conditions. He identified these compounds as aspartic acid, which was an efficient N source for legumes, and ~-alanine, which was an efficient N source for a number of grasses. Wilson (75), however, was unable to obtain similar excretion from identical experiments in Wisconsin unless light intensity, temperature and day length were governed within certain critical limits. On the basis of this and other experiments, Wilson and Wyss (76, 78), con cluded that (a) long days and low temperatures favored excretion, but that substrate and strains of legume and rhizobia were also inter related, and (b) excretion takes place only when fixation exceeds the formation of new protein tissue. Virtanen (66) amplified these con clusions slightly on the basis of his review of published reports on this problem and placed particular stress upon the carbohydrate: nitrogen ratio as the governing factor in nitrogen fixation and excretion of nitrogen by legumes. The relationship between carbohydrate levels and N excretion was also corroborated by Wilson (75) who reported that increasing the supply of carbohydrates by supplying additional 002 resulted in increased N fixation, even in the presence of excess nitrate ~n the media. A similar effect was achieved by Kalanis (24) who fed sucrose to legume plants growing under high N fertilization. Steward and Street (57) also concluded that, in general, protein synthesis and decomposition are intimately related to carbohydrate synthesis. In another early experiment using sand culture (Thornton and Nicol, 59), Italian ryegrass in association with alfalfa had 250% as much N after 18 weeks as grass grown alone under similar conditions. 10 More recently Baitulin (3) has shown that this may be primarily due to root effects. He observed Dactylis glomerata root systems in the presence of alfalfa roots in sand culture, with or without a glass partition between the plants. Grass roots were better only when the partition was not present. The roots of peas and soybean were shown by Katznelson, Rouatt and Payne (25) to exude amino acids in sand culture. Exudation was
more a~ the seedling stage than at flowering and was much greater when the plants were allowed to wilt briefly and then remoistened than under continuously moist conditions. The amino acids thus liberated were all found to be utilizable by soil micro-organisms. Rovira (52) studied the exudation of amino acids from the roots of subterranean clover (Trifolium subterraneum) seedlings over a period of 4 weeks •.. Exudation was greater for the second half of the period and was favored by higher light intensities and higher tem peratures. Higher temperatures particularly increased the exudation of asparagine, but up to 17 amino acids in all were detected. Butler and Bathurst (8) found, however, that the roots of inocu lated clover plants growing in aerated nutrient solutions released only minute quantities of ammonia and amino acids, regardless of light and temperature conditions or the presence of absorbents in the solu tions. The excretion of ninhydrin-positive substances has also been observed for the roots of germinating broad beans (Pearson and Parkinson, 44). Excretion occurred only in a limited region near the root tip which was characterized by high proteolytic activity. 11 Several experiments showing increases in soil N after growing legumes have been recorded. In early work in Hawaii, Thompson (58) found small but consistent increases in soil N after the growth of a large variety of legumes in pots. She concluded that this may have been due to excretion, but was just as likely the result of decomposing roots and nodules. Several Indian workers have also cited evidence of excretion by legumes. Acharya, Jain and Jha (1) observed 40% higher soil N in plots where phosphorus-fertilized berseem (Trifolium alexandrinum) had been included in the rotation for ten years. Where phosphate was omitted, only 17% more N was obtained. They concluded that excretion probably accounted for the bulk of the N contribution. Biswas and Das (5) found larger amounts and a larger variety of amino acids in soil under berseem than in fallow soil. Large increases in soil N after a three year experiment involving guar (Cyamopis psoralioides) were also observed by Rewari, Sen and Pandey (51), and they estimated that root excretion amounted to 8.1-20.9% of the N fixed by the legume. However, some of the above studies may partly reflect the activity of soil micro-organisms capable of excreting amino acids. Becker and Schmidt (4) found in the rooting region of various plants, 27 bacteria, 10 fungi and 2 actinomycetes which could excrete amino acids. Fewer isolates were obtained at the immediate root surface. Transfer of nitrogen by way of decomposing legume roots and nodules has also been proposed (e.g. 8, 58, 71, 75). This pathway was investigated for three temperate legumes by Butler, Greenwood and Soper (9). A rapid turnover of roots and nodules was observed under 12 recurrent defoliation. With each defoliation, the death of some older roots and nodules and the development of new ones were observed, es pecially in the case of white clover. Roots of birdsfoot trefoil (Lotus uliginosus) were also lost rapidly under defoliation but regrowth was small. The 1055 of red clover roots was slower, but there was also little regrowth after defoliation. Shading also induced the death of root and nodule tissue; under shading, little or no regrowth of new roots was observed. Nodule decay was always associated with root decay on the observed portion of the root system, and both phe nomena were related to treatments which reduced the carbohydrate supply of the plant. The results of this study are consistent with the observations of Fergus (16) thirty years ago that nitrogen tended to accumulate in pastures during periods of increasing clover vigor and then to be released during periods of clover decline. Turnover of nodule tissue in the vetch and pea was observed by Pate (43) to be related to flowering, leaf production, and accumulation of leaf nitrogen, with marked decreases in nodule numbers and weights occurring at the'time of flowering. Bowen (7), however, found no effect of flowering on nodulation and nodule senescence in centro. These events were related instead to the occurrence of new vegetative growth. Frequent defoliation resulted in the loss of two-thirds of the root weight. Seeger (54) found some benefit to corn by decomposing roots and nodules of older annual legumes. The contribution of N by the root portion of legume plants is also indicated by results obtained by Watson and Lapins (72). They found a constant accumulation of N in 13 the soil under subterranean clover whether or not the herbage or animal residues were returned. Losses from legume leaves have received very little attention as a possible pathway of nitrogen transfer. The leaching of numerous cations has been shown for the leaves of several plants by Tukey, Tukey, and Wittwer (60). Losses were higher with older leaves of non waxy varieties. Stenlid (56) also indicated that leaching losses were greater from older leaves and from the upper leaf surfaces. Wittwer and Teubner (77) concluded that in most instances, losses of ions from leached leaves could be accounted for by simple diffusion of exchange able ions. This was enhanced where large quantities of ions were present outside the plasma membrane or when the retentive ability of the cells had been impaired. No reports dealing with the loss of nitrogen by legume leaves have been seen by this writer to date. However, there are a number of recent reports dealing with the amino acid levels in legume plants which are related to this problem. Thus higher levels of free amino acids were found in inoculated than non-inoculated plants by Aseeva and Kirillova (2), Ebertova (15), and Dinchev (13). In addition to higher levels of aspartic acid, histidine and methionine appeared after the start of symbiotic nitrogen fixation (Aseeva and Kirillova). Increasing levels of asparagine, glutamic acid and alanine were also observed in soybean plants by Ebertova. However, Dinchev reported th:t most of the increase in free amino acids due to nodulation of bean plants was found in the roots and stems. Increases in free amino acid levels were also obtained with ammonium or nitrate fertilization of temperate legumes. Uziak and 14
Koter (61) found that ammonium N favored high glutamic acid and aspara gine while nitrate N favored high glutamic and aspartic acid contents. Free amino acids decreased with age in N-fertilized plants, but gradu ally increased in unfertilized inoculated plants. Pleshkov (47), however, found that while N-fertilizers affected free amino acid con- tents, they had little influence on plant protein composition. Phos phate and potash fertilization was found by MacGregor, Tashovitch and Martin (31) to have no marked affect on the amino acid composition of alfalfa even though higher yields were obtained. Amino N consti tuted 2.35-2.70% of the plant dry weight; ammonium N, on the other hand was only 0.34-0.47% of the plant weight. The release of amino N by a legume plant, either by excretion, leaching of leaf N, or decomposition of dead leaves, roots and nodules, creates a situation where free N is momentarily present in the sub strate. These may be reabsorbed by plant roots. In addition to
Virtanen's work with aspartic acid and ~-alanine mentioned previously, Ratner, et ale (50) found that corn and sunflowers could grow on amino acids, including glycine, aspartic acid, glutamic acid and arginine as a sole source of N, but that none of these was as effective as in organic N sources. The sap of plants grown on amino acids also had a higher content of ammonia. If the released amino compounds are not immediately reabsorbed by either legume or grass roots, however, losses by deamination may occur. Losses of amino acids in nutrient solution were observed by Moreau (38). Conversion of glycine, serine and alanine to ammonia was promoted by the addition of a soil suspension or a carbon source such as sodium pyruvate. Loginow (28), however, found humic acids more effective in 15
promoting deamination, while glucose counteracted this effect, probably due to changes in the oxidation-reduction conditions. Vlassak (68) studied a large number of amino acids and was able to categorize them into three groups: (a) glycine group, which is rapidly converted to ammonia and thence to nitrate; (b) d, I-methionine group, which slowly ammonifies but does not nitrify appreciably; and (c) I-leucine group, which undergoes nitrification after ten days of incubation. While the initial inorganic products of deamination are still good N sources for plants and would not o~dinarily be lost from a dense root system (Holmes and Aldrich, 21; Pfaff, 46), the presence of an amino acid such as glutamic acid in the soil was found by Wheeler (74) to promote denitrification. The loss of N was higher where added nitrate or nitrites were present than where deamination was the sole source of ammonia and nitrate. 16 SMALL PLOT EXPERIMENT
Materials and Methods A. Field. A small plot experiment to evaluate the nitrogen economy of three legumes and two grasses, alone and in all combi nations, was conducted at the Waiakea Farm of the East Hawaii Branch Station, Hawaii Agricultural Experiment Station. This farm is situated at 800 feet elevation and receives an average of approximately 150 inches of rainfall per year with relatively good distribution throughout the year. The mean annual temperature is 71.50 with mean monthly temperatures ranging from 730 in JUly-Sept. to 68 0 in Jan. Mar. Detailed weather information for the farm is included in the Appendix. Two series of 13 bottomless frames, each 4 feet wide, 8 feet long and 20 inches deep, and with the long sides of adjacent frames in common, were constructed of wood and placed on a graded sloping surface. Two similar series were made with frame dimensions of 4 feet by 4 feet and 20 inches deep. These were arranged so as to give four replicates and designated as shown in Figure 1, with the smaller plots adjacent to a trench 3-4 feet deep on the downslope side. Each of the frames was supplied with a shallow protective layer of sugar cane bagasse across the bottom and then lined with two layers of 6-mil polyethylene film. A water-tight drain connection was fitted to the downslope end of the double lining and connected to a one-half inch plastic hose to direct excess water away from the plot. In the case of the smaller plots, these hoses led to epoxy-coated 55-gallon collection drums situated directly downslope from each plot in the trench. The Replication I Replication II
No-Grass Pangola Napier Pangola No-Grass Napier .. ,. #' , r ... f Y "" UOJ K No C In C No K In ~ K No In C K In C No C No K In ~ No C In K
Replication III Replication IV
No-Grass Pangola Napier ___..JA A F Y"" "'\ r_---JA.----_1 III In I No IK KIC No I In I K I In
KEY: In: Desmodium intortum ("intortum"). K: Desmodium canum (Kaimi clover). c: Centrosema~scens. No: No legume.
I-' -.J FIGURE 1. LAYOUT FOR LEGUME-NITROGEN EXPERIMENT, WAIAKEA, HAWAII. 18
drain outlets were covered with several layers of glass wool before filling the plots to a depth of 18 inches with andesitic pumice cinders from the 1961 Kapoho eruption area. The cinders were clean, undecomposed, and virtually free of available fixed nitrogen. Par ticle sizes ranged from silt to large gravel, with the approximately 1-2 cm sizes dominating the physical characteristics of the material. Rock and soil were banked against the outside of the frames and covered with more cinders to provide a border area outside the plots. The cinders, both inside and outside the plots, were finally levelled and thoroughly tamped to a uniform firmness. Legume seeds of kaimi clover (Oesmodium canum L9mel-l Schinz & Thellung), intortum (Q. intortum LMill.-7 Urb., Hawaii introduction no. 4247), and centro (Centrosema pUbescens, Benth., C.S.I.R.O. of Australia selection) were scarified by abrading between two sheets of sandpaper, then moistened and inoculated with a commercial peat culture. Type "E" rhizobia was used for the Desmodium species and a specific Centrosema selection was used for the centro. Sowing was done on July 13, "1962 immediately after inoculation. The seeds were broadcast and covered by light raking and tamping. Fertilizer was also broadcast on the plots to supply the following: 100 lb. of P and 240 lb. of Ca par acre as single superphosphate, 100 lb. of K and 100 lb. of Mg per acre as "Sulpomag", 200 lb. per acre of K as sulfate of potash, 5 lb. of Mn, 5 lb. of Zn and 5 lb. of Cu per acre as the sulfates, 0.2 lb. per acre of Mo as Mo03, 0.1 lb. per acre of Co as
CoC1 2, 3 lb. per acre of borax and 20 lb. per acre of iron chelate. Irrigation was provided after the fertilizer application and subse quently as required to maintain the cinders in a moist condition. 19 Reseeding of the legumes was carried out July 31, 1962 in plots or parts of plots where poor stands were observed. Collection of percolate from the smaller plots was also begun on this date. Five ml of toluene were added to each drum to retard biological N losses, and the drums were covered loosely with insulating board. Sampling was done whenever the drums approached capacity by measuring the amount of leachate in each drum and then withdrawing approximately 500 ml for analysis before emptying the drum. The samples were air freighted to Honolulu where they were immediately analy~edfor nitro gen as described later. The two grasses were planted on Sept. 6-7, 1962 as follows: fresh stems of napier grass (Pennistum purpureum) and pangola grass (Digitaria decumbens) were cut into segments of one or two nodes each, soaked briefly in a solution of complete fertilizer plus a rooting hormone formulation, and sprigged into the appropriate plots, including border areas and portions of the buffer plots separating the napier plantings from other treatments. The cuttings were arranged in three rows per plot with approximately 6 inches between napier plants and 4 inches between pangola plants. A commercial fertilizer mixture providing 15 lb. of nitrate Nand 34 lb. of K per acre was then broad cast on all plots. Subsequent light dressings of fertilizer were applied as follows: Oct. 23, 1962; 10 lb. of P and 24 lb. of Ca per acre as super phosphate, 10 lb. of Mg and 10 lb. of K per acre as "Sulpomag", 20 lb. of K per acre as sulfate of potash, and 5 lb. per acre of N as urea. Dec. 31, 1962; 50 lb. of P and 120 lb. of Ca per acre as super phosphate plus all trace elements except iron and cobalt at one-half the original rate of application. 20 Feb. 6, 1963. An initial harvest showed that establishment was still incomplete, and nitrogen was thus again included in the ferti lizer mixture as follows: 20 lb. per acre of N as ammonium sulfate, 45 lb. of P and 110 lb. of Ca per acre as superphosphate, 5 lb. of K and 5.5 lb. per acre of Mg as "sulpbmag" and trace elements (except Fe and Co) at one-half the original rate. June 18, 1963; 50 lb. per acre of P as treble superphosphate, 50 lb. per acre of K as sulfate of potash, and trace elements (except Co) at one-quarter of the original application rate. July 21, 1963; same as on June 18 above. Observations were recorded at intervals on the top growth obtained, and when maximum vegetative growth of anyone species seemed apparent, the plots were cut by hand to approximately 4-5 cm of the substrate surface. The entire area of the smaller plots and the lower half of the larger plots were harvested, the grass and legume components separated, and the green material dried to constant weight at 70oC. In addition, a small sample of tops and roots was taken in the upper half of each large plot in an area which appeared to be representative of
the vegetation in the lower half~ A sharpened bottomless steel cylinder, 14 inches in diameter and 18 inches deep was placed over the vegetation and driven through it and into the cinder substrate to the full depth
of the plot (Figure 2). This sample was then moved intact onto a sheet steel plate and lifted onto a small canvas for manual separation of grass tops, grass roots, legume tops, legume roots and cinder substrate. Root growth and behavior were studied at this time, and in the case of the initial harvest, representative entire plants from each treatment were photographed. As it was impossible to completely separate the 21
FIGURE 2. ROOT SAMPLING CYLINDER FOR SMALL PLOTS. 22
roots from their attached cinders, a root-cinder mixture was obtained
for each species in each plot. These were then dried and ground ~ toto for subsequent nitrogen analysis. The tops from each species and a sample of the well-mixed cinder substrate were also dried and ground separately. The percent N in the tops provided a basis for computing the N yield of the plot as a whole, and the ratio of root N:top N in
the small sample was applied to the plot N yie~d to estimate the root nitrogen of the entire plot. In the case of napier, however, a better estimate was obtained by mUltiplying the sample root N by the factor, plot area/sample area, due to the unevenly distributed topgrowth and relatively uniform distribution of napier roots throughout the.plot. Finally, all border areas and non-harvested plot portions were cut back to the same level. Harvests were made of all plots on May 28, 1963 and August 6, 1963; of the large plots (including root samples) on Feb. 5-6, 1964; and of the small plots on July 14, 1964. Napier yields from the Feb., 1964 harvest of plots 23-26 gave erroneously high results because the roots of some plants had escaped the plot volume. These values were subse quently multiplied by a correction factor (plot-bound yield7total yield) determined for each plot at a later date. B. Analytical. Plant and cinder samples were analyzed for nitro gen by the modified Kjeldahl procedure described by Jackson (23). Nitrate was not included, since it was found to be low in the test species. Topgrowth subsamples were 0.50 and 2.00 g for legume and grass materials, respectively. It was necessary to mix each root-cinder sample thoroughly with an electric propeller-type mixer and then with draw several cores from the entire depth of the material with a 1 cm 23 corkborer tube in order to obtain representative subsamples. Sub samples of 10 or 20 g, depending on the root:cinder proportions, were obtained in this way for analysis. Thirty g subsamples of cinder sub strate were weighed out similarly. The leachate samples were distilled with MgO and Devarda's alloy
(5 g per 400 ml) into Erlenmeyer flasks marked at 250 ml and containing
O.l~ 30 ml of H2S04• Distillation was stopped when 200 ml or more had been distilled over. Each flask was then made up to volume, and an aliquot was transferred to a 100 ml volumetric flask. This solu tion was Nesslerized by the addition of 4 ml of Nessler's reagent and distilled water to volume. The yellow color was read after 20 minutes with a Klett-Summerson colorimeter, using a blue filter with maximum transmission at 4~0 ~. c. Statistical. Inasmuch as no recognizable source of variation could be attributed to either blocks or replications, the experiment was treated as a completely random design with eleven treatments and four replications. However, only two observations per treatment were available for the later harvests and for evaluating %N data and root N:top N ratios. Also, the lack of any consistent relationship between top yields and root N:top N ratios permitted the estimation of root N levels for only the two replications actually sampled. The different number of replications harvested on the four occa sions reduced the sensitivity of the statistical tests for differences among those harvests which 'differed as to numbers of observations. However, this was not serious since these differences were quite large. The rather doubtful assumption that two replications gave as good an estimate of the true means as did four replications was required in 24 this treatment of the data, but this was advantageous in that it per mitted evaluating treatment by harvest interactions and treatment differences that were consistent over several harvests. Where treat ment by harvest interactions were significant, the data from individual harvests were then analyzed separately. In all cases, harvests were considered to be random effects while treatments were evaluated as a factorial arrangement of fixed grass and legume effects. In the case of total yields (for mixtures or species grown singly), non-orthogonality was encountered in separating treatment differences into grass and legume effects, with the grass by legume interaction term occasionally being negative. Although grasses and legumes, as fixed effects, were tested against the error mean square, apparent sig nificance was occasionally encountered where no differences among means existed as determined by Duncan'? new multiple range test (14). Some bias was also introduced into the ranges used for comparing these means, but this is believed to be relatively small. The desired information on grass by legume interactions was obtained by studying the yields of the grass and legume components separately. Grass yields and legume yields analyzed separately were orthogonal; but in the case of legume yields, the error mean squares for the high yielding and low yielding species were quite different, indicating populations of unequal variance. The confidence levels for comparisons between treatments containing different legume species are thus probably lower than the 5% level indicated. Also, in some cases, it was advan tageous to use the within-kaimi mean square to detect differences in kaimi yields. 25 Results A. Field Observations. The initial sowing of legumes was largely damaged by salt burning during emergence due to the high concentrations of fertilizer salts at the substrate surface. Centro was less affected than the other two species. Excess salts were removed by leaching before reseeding, however, and acceptable stands of healthy plants were
obtained for all three species. Ho~ever, growth of intortum was better near the center of the plots, and centro stands continued to be rela tively sparse despite several replantings involving both seeds and seedlings. The grass species developed excellent uniform stands• .An initial harvest was made on Feb. 4-6, 1963 of all species except pangola and kaimi, which were still quite small. In addition, top and root samples were taken from two replications, and repre sentative plants were photographed. Root growth was extensive in relation to top growth. Except for the smaller kaimi and pangola plants, roots extended to the plot bottoms where they tended to develop a mat. Napier tended to develop a very extensive fibrous root system, concentrated primarily in the top 4-6 inches of cinders. These roots were very active at the substrate surface wherever shade was adequate and quickly proliferated into any dead legume leaves present. In addition, napier roots were found to be actively proliferating into decomposing nodules of centro (Figure 3). This was not observed with pangola or with the nodules of intortum or kaimi, but the latter may have been due to greater difficulty in detection since these species have much smaller and darker nodules than does centro. Pangola roots were apparently more thinly and evenly distributed throughout the substrate and were not as active at the substrate surface 26
. I ,.\ "." ...... !.:..~~'"' .. .''.: ..-.:l .~.. ~ "~.~ .
",,:, "': . ;' - c.· ...... ; -., " !
FIGURE 3. NAPIER ROOTS PROLIFERATING IN AND AROUND CENTRO NODULES. 27 or around legume roots. The three legumes all had highly-branched root systems which were well distributed throughout the plot volume.
Centro and intortum were observed to be well nodulated and kaimi , moderately nodulated. Kaimi topgrowth was compact while intortum was characterized by vigorous spreading semi-viny stems, and centro was of the viny, climbing type. Since establishment was still in- complete, all yields were low, in the range of 300-1300 pounds per acre of dry matter and 10-35 pounds per acre of nitrogen. Centro and intortum yields were quite variable. Kaimi, although not harvested, was observed to be much denser, taller, and more vigorous in asso- ciation with napier than alone or with pangola. There was no evidence of any effect of legumes on napier yields.
Regrowth was observed April 13, 1963 at which time centro and intortum both had numerous stems 3-4 feet or longer in length. Ground cover was poor in the centro plots due to inadequate stand, especially where it grew in association with either grass. Intortum growth tended to be concentrated in the middle part of the plot where it developed a vigorous mass of vegetation about two feet high. Both napier and pangola were noticeably taller and greener in association with intortum. Otherwise, the grasses exhibited symptoms of moderate
N deficiency.
A complete harvest on May 28, 1963 yielded data on all species and combinations. At this time, napier was 4-5 feet tall, and intortum growth was at about the maximum which could easily be confined within the plot boundaries. Also, intortum was affected by a disease which resulted in extensive leaf yellowing and necrosis. 28
Depending on the degree of self-shading, prostrate stems of all three legumes were observed to be rooting at the nodes. Pangola stems were spreading and rooting vigorously. The behavior of the subsurface root systems was the same as observed at the initial harvest, except for the more extensive development, especially by pangola and kaimi roots. Regrowth was observed on June 20, 1963. Pangola appeared greener and more vigorous where intortum growth was strong, but no other differences were obvious. Napier, however, appeared somewhat greener and taller in mixture with centro than alone or with kaimi and was markedly better in the intortum plots. Some phosphorous deficiency symptoms were also evident on napier plants, except in the intortum mixture. Regrowth of kaimi was very slow, with most plants still only two inches or less in height. A second complete harvest was obtained August 6, 1963. All three legumes were growing vigorously, with the intortum vegetation reaching 2-3 feet in height. Kaimi plants gave fair ground cover and had numerous upright flowering stems 8-24 inches high. Centro was inter- mediate, fonning a dense mat of vegetation 6-10 inches thick when grown alone and markedly less when grown in association with grasses. In a few cases, the stands of kaimi and centro were insufficient to give good ground cover. Pangola was small and very nitrogen deficient except in the intor- tum mixture where it was noticeably taller, greener, leafier and with more flowering stems than the control. Total pangola vegetation was still small, however, probably due to competition with the intortum ~- for light. The pangola in the centro and some of the kaimi plots 29 appeared to be intermediate between the intortum and the control treat ments. Napier showed a similar apparent legume effect. Plants ranged from very yellow and less than two feet high in the control plot to yellow-green and 4-4t feet high in association with intortum. Within the napier plots the relative vigor of legume plants, especially centro and intortum, which were situated immediately adjacent to nap~er clumps was much greater than those further removed. Kaimi continued to appear larger, darker green and generally more vigorous in association with napier than when alone or mixed with pangola. A subsequent harvest of two of the four replications was taken in the author's absence on Feb. 5-6, 1964. No visual observations were recorded. Observations at the time of the final harvest (July 14, 1964), however, were in accord with those made earlier.. In addition, it was noticed that a few intortum plants were volunteering in the napier plots and that this took place only in the center of existing napier clumps. B. Dry Matter Yields. The total dry weight yields obtained from each of the eleven treatments are presented in Table I, and the mean squares from the associated analysis of variance are shown in Table IV. In addition, Figure 4 shows the dry matter yields obtained over a twelve-month period, calculating by summing the yields from the May, August and February harvests. Since the treatment by harvest interaction was significant, the individual harvests were analyzed separately. Most treatment differ ences tended to be consistent over the four harvests, however, and the Duncan's multiple range test thus provided a relatively sensitive ~ . determination of differences between treatment means for all harvests. 30
TABLE I. TOTAL DRY MATTER YIELDS FOR GRASSES, LEGUMES, AND MIXTURES. Treatments Dry matter yields, pounds per acre May August Feb. JUly 1963t 1963t 1964:1: 1964:1: Average
A. Individual treatment effects.*
Pangola Alone 2810 be 250 a 1660 ab 1980 ab 1670 a Napier Alone 2610 be 430 ab 1120 a 480 a 1160 a Kaimi Alone 690 a 1050 abc 1410 a 2350 ab 1370 a Kaimi + Pangola 2150 ab 700 ab 2300 ab 2450 ab 1900 a Kaimi + Napier 4010 cd 1960 cd 2950 ab 3670 abc 3150 b Centro Alone 2180 ab 2320 d 6470 bed 2010 abc 3440 b Centro + Pango1a 2820 be 1570 bed 4790 abc 3890 abc 3270 b Centro + Napier 3090 be 1520 bed 4730 abc 2410 ab 2930 b Intortum Alone 3740 d 4410 e 10500 e 5750 bed 6100 c Intortum + Pango1a 4760 d 4200 e 12160 e 6610 cd 6930 cd Intortum + Napier 7060 e 4850 e 8840 cde 8360 d 7280 d Overall Average 3260 2110 5170 3640 3560 B. Means for grass effects.* No Grass 2200 a 2590 a 6130 a 3650 a 3640 a Pango1a 3140 b 1680 b 5230 a 3730 a 3440 a Napier 4220 c 2190 ab 4410 a 3230 a 3630 a C. Means for legume effects.* No Legume 2710 a 340 a 1390 a 1230 a 1420 a Kaimi 2290 a 1240 b 2220 a 2820 a 2140 b Centro 2690 a 1800 b 5330 b 3040 a 3220 c Intortum 5180 b 4490 c 10500 c 6910 b 6770 d
* Means in the s~me column followed by the same letter are not signifi cantly different at the 5% level. t Means of four observations per harvest.
:I: Means of two observations per harvest.
L_ • 31
~ PANGOLA ~ 20.000 NAPIER R LEGUMES POUNDS 15.000' DRY MATTER PER 10.000 YEAR 5.000
GRASS KAIMI CENTRO INTORTUM ALONE
FIGURE 4. DRY MATTER YIELDS PER ACRE OF GRASSES, LEGUMES AND MIXTURES. 32
However, differences among treatment means for individual harvests were less easy to detect, especially in the later harvests where a larger mean square and fewer degrees of freedom resulted from having only two observations per treatment. In addition to the legume effects mentioned later, several treat ment differences are apparent in Table I A. Kaimi alone and kaimi plus pangola yields did not significantly exceed, on the average, yields of the grasses alone, but kaimi plus napier yields were higher than the other kaimi treatments. Also, intortum plus napier outyielded intor tum alone. On an individual harvest basis these differences could only be detected statistically in the May harvest, but the trends were quite consistent in each case. Some seasonal effects are also apparent, but these will be mentioned later in connection with the yields of individual species. Because of non-orthogonality in the treatment of grasses and legumes as factorial components of treatment effects, significant differences among grass treatments (over all harvests) are indicated in Table IV A, whereas a Duncan'S test shows that no differences are present (Table I B). However, significant differences among grass treatments do occur at the May and August harvests. The high grass yields in the May harvest probably reflect the earlier nitrogen ferti lization, since this effect is lost by the August harvest. All differences among total dry matter yields for the four legume effects, were significant when averaged over all harvests and grass associations. Kaimi treatments yielded 50% more than grasses alone (llno legume"), but this was significant only at the August harvest. Yields of centro treatments were intermediate, averaging over twice 33 as much as grasses alone and 50% more than the kaimi treatments. When the individual harvests are treated separately, the advantage of centro over kaimi was significant only in the February harvest, but its advan- tage over grasses alone was significant for both the August and Febru- ary harvests. The intortum treatments consistently averaged nearly twice as much as the other two legumes and except for the May harvest '-/ yielded 5-12 times as much as did grasses alone. On a twelve-month basis, the average dry matter yields in pounds per acre for the four legume effects were: no legume (grasses alone), 4,400; kaimi, 5,750; centro, 9,820; and intortum, 20,170, with standard error =± 1,350 pounds per acre. This represents a very broad range, from 2.2 to over 10 tons of dry matter per acre per year. Yields of the legume components and grass components are shown separately in Tables II and III, and the associated ANOVA mean squares are included in Table IV. Most of the variation among legume yields was due to differences among harvests and the three legume species themselves. Companion grasses, however, significantly affected the yields of kaimi and centro. Kaimi yielded consistently lower in association with pangola and higher in association with napier than when grown alone. The differences, however, were significant only for all harvests combined. The yields of centro, on the other hand, were consistently depressed by the presence of either grass to approximately half of the centro-alone production. The sum of these two different grass by legume interactions accounts for the significantly higher yield obtained for the legume alone treatments ("no grass"), when averaged over the three legume species (Table 2 B). 34 TABLE II. DRY MATTER YIELDS OF LEGUMES, ALONE AND IN ASSOCIATION WITH GRASSES.
Treatments Dry matter yields, pounds per acre May August Feb. JUly 1963t 1963t 1964:1: 1964:1: Average A. Individual treatment effects.*
Kaimi Alone 690 ab 1050 a 1410 a 2350 ab 1370 b Kaimi w!Pango1a 340 a 490 a 1310 a 1480 a 910 a Kaimi w/Napier 1990 be 1310 ab 1990 a 3160 a 2110 c Centro Alone 2180 bc 2320 cd 6470 ab 2870 ab 3460 d Centro w!Pango1a 750 ab 1010 a 2990 a 2080 ab 1710 be Centro w/Napier 830 ab 900 a 2710 a 1260 abc 1430 be Intortum Alone 3740 de 4410 e 10500 b 5750 bc 6100 e Intortum w!Pango1a 2930 cd 3320 de 10540 b 4610 abc 5350 e Intortum w/Napier 5030 e 3460 e 6190 ab 6830 c 5380 e Ave. for Legume treatment 2050 2030 4900 3380 3090 B. Means for gr.ass effects.*
No Grass 2200 ab 2590 b 6130 a 3650 a 3640 b Pango1a 1340 a 1610 a 4950 a 2720 a 2660 a Napier 2620 b 1890 a 3630 a 3750 a 2970 a C. Means for legume effects.*
Kaimi 1010 a 950 a 1570 a 2330 a 1460 a Centro 1250 a 1410 a 4060 a 2070 a 2200 b Intortum 3900 b 3730 b 9080 b 5730 b 5610 c
* Means in the same column followed by the same letter are not sig nificantly different at the 5% level. t Means of four observations per harvest.
:f: Means of two observations per harvest. 35
TABLE III. DRY MATTER YIELDS OF GRASSES, ALONE AND IN ASSOCIATION WITH LEGUMES. Treatments Dry matter yields, pounds per acre May August Feb. July 1963t 19631' 1964:1= 1964:1: Average
A. Individual treatment effeets.* Pangola Alone 2810 a 250 a 1660 ab 1980 a 1670 e Pangola w/Kaimi 1810 a 210 a 1000 a 970 a 1000 a Pangola w/Centro 2070 a 560 b 1810 abc 1810 a 1560 be Pangola w/lntortum 1820 a 880 e 1610 ab 2030 a 1590 be Napier Alone 2610 a 430 ab 1120 ab 480 a 1160 ab Napier w/Kaimi 2020 a 650 be 950 a 520 a 1040 a Napier w/Centro 2260 a 620 be 2020 e 1150 b 1510 be Napier w/lntortum 2030 a 1390 d 2650 e 1530 b 1900 e Ave. for Grass treatments 2180 620 1600 1310 1430
B. Means for grass effeets.* Pango1a 2130 a 470 a 1520 a 1700 a 1460 a Napier 2230· a 770 b 1690 a 920 a 1400 a
C. Means for legume effeets.* No Legume 2710 b 340 a 1390 ab 1230 a 1420 b Kaimi 1920 a 430 be 980 a 740 a 1020 a Centro 2170 a 590 e 1910 be 1480 a 1540 be Intortum 2180 a 1130 d 2130 e 1780 a 1750 e
* Means in the same column followed by the same letter are not sig nificantly different at the 5% level. t Means of four observations per harvest. ; Means of two observations per harvest. TABLE IV. ANALYSIS OF VARIANCE OF DRY MATTER YIELDS.
Source d.f. Mean SWIares May 1963 Aug. 1963 Feb. 1964 July 1964 All Harvests
A. Total dry matter Treatment 10 303,337** 302.798** . 826,250** 297,930* 1',307,278** Grasses 2 382,611** 81,653* 141,509 558 493,469** Legumes 3 574,147** 954,134** 2,448,851** 864,730** 4,022,671** GXL 5 109,142* 455 126,585 76,799 3,566 Harvests 3 1,309,038** TXH 30 141,329** Error 30,435 15,621 114,552 77,430 41,253 Error d.f. 33 33 11 11 88 B. Legume dry matter Treatments 8 284,299** 217,242** 759,154* 209,507 1,116,829** Legume Spp. 2 855,725** 740,574** 2,437,099** 695,319** 3,950,261** Compan. Grass 2 141,238* 86,035** 260,257 53,698 233,885** LXG 4 70,092 21,179 169,631 44,506 139,085* Harvests 3 1,166,826** TXH 24 117,791** Error 31,510 14,901 142,411 66,917 43,570 Error d.f. 27 27 i 9 9 72
C. Grass dry matter Treatments 7 14,579 15,816** 18,631 21,709 24,208** Grass Spp. 1 2,245 19,404** 3,997 67,211 590 Compan. Leg. 3 30,979 28,015** 30,438** 21,589 42,952** GXL 3 2,293 2,420 11,702 6,662 13,337 Harvests 3 367,015** TXH 21 15,463* Error 12,777 961 3,955 17,338 7,813 w Error d.f. 24 24 8 8 64 0' 37
The legume effects generally followed those observed for total dry matter yields. Intortum consistently yielded much higher than the other two legumes. When averaged over all harvests, the superiority of intortum was significant for all combinations. Centro was again intermediate. Apparently, most of centro's advantage over kaimi was recorded at the February harvest, even though the difference was not significant when tested against the error term for this harvest. Based on the sum of the yields for the May, August and February harvests, the average twelve-month dry matter yields for the three legumes were kaimi, 3,500; centro, 6,700; and intortum, 16,710 pounds per acre. The. dry matter yields of the grass components (Table III) show no differences between the overall yields of the two grasses, except at the August harvest where napier proved significantly superior to pangola. However, there were several major effects of the companion legumes upon grass yields. Pangola yields were depressed, on the average, by the presence of kaimi in the mixture. Conversely~ napier yields were significantly higher where intortum was present. Centro also resulted in increased napier yields in the February and JUly harvests. And in the third harvest, both centro and intortum benefitted pangola yields. Considering the two grasses together, yields were depressed by all three legumes at the first harvest, apparently due to competition for nitrogen remaining from earlier fertilizations. However, the average yields of subsequent harvests show a significant benefit to the grasses from centro and intortum, with intortum providing a greater stimulus than centro. On the other hand, a significant depression in grass yields 38 by kaimi is indicated by the combined data from all four harvests; and on this basis, the only significant benefit to grass yields was that given by intortum. Seasonal effects on the dry matter yields of the grasses and legumes studied may be best evaluated by comparing their average weekly production. These were computed by dividing harvest weights by the number of weeks intervening since the previous harvest. These are presented in Table V. After the first harvest, the average grass yields were remarkably uniform, averaging 60 pounds per acre per week. However, this seemed to be the mean of two different trends, namely a summer depression and spring maximum in pangola yields plUS an opposite trend in napier yields. Kaimi appeared to have a summer maximum in dry matter production. Low yields during the first period may have been a result of slow L- establishment, in which case the maximum growth period would probably include the spring months as well. Centro and intortum both produced less in the first and last periods, indicating a probable depression in yield during the spring. C. Nitrogen Yields. The percentage of nitrogen in the top growth was estimated from the sample means shown in Table VI. For a particular harvest and species, the percentage of N was quite constant, except (a) in the case of pangola, where N levels were consistently higher in the presence of intortum, and (b) centro, where the percent N in the presence of napier was lower at the August harvest. Thus, with these two exceptions, the average percent N for a particular species at a particular harvest was applied to the dry matter yields of that species in all treatments and replications. TABLE V. DRY MATTER PROOOCTIOO PER WEEK BY TWO GRASSES AND THREE LEGUMES.
Species Weekly dry matter production, pounds per acre. Feb.-May May-Aug. Aug. 1963- Feb.-July Average 1963 1963 Feb. 1964 1964 All Periods Pangola 133 47 58 74 78 Napier 139 77 65 ~ 80 Ave. for grasses 136 62 62 57 79 Kaimi 63 95 60 101 80 Centro 79 141 156 83 116 Intortum 244 373 ~ 248 304 Ave. for legumes -128 -157 188 146 166
VJ \.() 40 TABLE VI. PERCENTAGE OF NITROGEN IN TOP GROWTH OF GRASSES AND LEGUMES. Species Combinations Percent N in tops* May August Feb. 1963 1963 1964 Average Pango1a Alone .32 .48 .42 .41 a Pango1a w/Kaimi .37 .49 .37 .41 a Pango1a w/Centro .36 .53 .42 .44 a Pangola w/Intortum .62 .61 " .58 .60 b Average .42 .53 .45 .46 Napier Alone .43 .61 .40 .48 Napier w/Kaimi .41 .60 .40 .47 Napier w/Centro .41 .68 .36 .48 Napier w/Intortum ~ .56 .41 -d§ Average .43 .61 .39 .48 Kaimi Alone 2.02 2.33 1.38 1.91 Kaimi w/Pango1a 1.84 2.42 1.33 1.86 Kaimi w/Napier 1.85 2.49 1.39 1.91 Average 1.90 2.41 1.365 1.89 Centro Alone 2.80 2.98 b 1.69 2.49 Centro w/Pango1a 3.14 2.80 b 1.34 2.43 Centro wjNapier 3.00 2.31 a 1.30 2.20 Average 2.98 2.30 1.775 2.48 Intortum Alone 2.48 2.34 i.35 2.06 Intortum w/Pangola 2.19 2.30 1.10 1.86 Intortum wjNapier 2.55 2.33 1.49 2.12 Average 2.41 2.32 1.31 2.01 Ave: 'Grasses .425 .57 .42 .47 Ave: Legumes 2.43 2.48 1.48 2.13 Overall Average 1.44 1.58 .96 1.30
ANOVA mean squares Source Grasses Legumes d.f. Pango1a Napier d.f. Kaimi Centro Intortum Companion spp. 3 .0518** .0001 2 .0045 .0783 .1086 Harvest 2 .0264* .1085** 2 1.6488** 2.3796* 2.2263** SXH 6 .0043 .0039 4 .0159 .1216 .0218 Error 12 .0022 .0029 9 .0345 .1284 .0686
* Means of two observations per treatment per harvest. Letters follow only means of sets found to contain significant differences. 41
Nitrogen levels in the two grasses were consistently less than one third of those observed for the legumes. Of the three legumes, kaimi tended to be the lowest in N content except at the August harvest, following relatively vigorous growth of this species. Percent N was highest at the August harvest for all species except intortum which was slightly higher in nitrogen at the May harvest. The lower percent N generally found at the February harvest was probably due to the greater maturity of the vegetation harvested on that occasion. The total N yields from all eleven treatments are shown in Figure 5. These data together with their associated analysis of variance are also presented in Table VII. The yields tended to follow the same pattern observed for dry matter, except that grass yields formed a much lower proportion of the total. Thus the higher yields observed for kaimi plus napier or centro alone, as compared to other mixtures of these two legumes,reflect more fully the differences in N yields of the legume component. Also, there was a distinct decrease in N yield associated with legume-grass mixtures compared to legumes alone ("no grass"), but the only differences between the two grass species were at the May harvest where napier treatments yielded more than pangola treatments. The superiority of intortum is indicated by the fact that all three intortum treatments yielded significantly more nitrogen, averaged over all harvests, than any of the other treatments. The average N yield for all intortum treatments was nearly double that of centro treatments and over four. times that of kaimi treatments. Grasses alone, on the other hand, averaged only about one-fourth the nitrogen of the low yielding kaimi. The three harvests comprise exactly one year of growth, 42
~ PANGOLA
300 ~ NAPIER n LEGUMES 'I ~. : I 200 POUNDS N PER YEAR 100
GRASS KAIMI CENTRO INTORTUM ALONE
FIGURE 5. NITROOEN YIELDS PER ACRE OF GRASSES, LEGUMES, AND MIXTURES. 43 TABLE VII. TOTAL NITROOEN YIELDS FOR GRASSES, LEGUMES, AND MIXTURES.'
Treatments N yields, pounds per acre May August February 1963t 1963t 1964:1: Average A. Individual treatment effects.* Pangola Alone 9.0 a 1.3 a 6.7 a 5.7 a Napier Alone 11.2 a 2.6 a 4.4 a 6.1 a Kaimi Alone 13.1 a 25.3 ab 19.2 a 19.2 ab Kaimi + Pangola 13.2 a 13.0 ab 22.0 a 16.1 a Kaimi + Napier 46.6 ab 35.6 b 30.9 ab 37.7 bc Centro Alone 64.9 bc 67.0 c 114.8 cd 82.2 d Centro + Pangola 30.1 a 32.0 b 60.4 abc 40.8 c Centro + Napier 34.4 ab 24.6 ab 56.0 abc 38.3 bc Intortum Alone 90.1 c 102.3 d 137.6 d 110.0 e Intortum + Pangola 82.0 c 82.4 cd 147.5 d 104.0 e Intortum + Napier 130.0 d 88.8 cd 91.4 bcd 103.4 e B. Means for grass effects.* No Grass 56.0 b 64.9 b 90.5 b 70.5 b Pangola 33.7 a 32.2 a 49.2 a 41.7 a Napier 55.6 b 37.9 a 45.7 a 37.0 a C. Means for legume effects.* No Legume 10.1 a 2.0 a 5.5 a 5.9 a Kaimi 24.3 ab 24.6 b 24.0 a 24.3 b Centro 43.1 b 41.2 c 77.1 b 53.8 c Intortum 100.7 c 91.2 d 125.5 c 105.8 d D. ANOVA mean squares Source d.f. May August February All 1963 1963 1964 Harvests
Treatments 10 174.93** 142.76** 152.61** 426.95** Grasses 3 167.68** 111.85** 98.16* 197.29** Legumes 2 479.27** 415.19** 433.82** 1,286.60** GXL 5 neg. neg. 5.67 15.07 Harvests 2 80.17* TXH 20 21.68 Error 15.38 7.76 21.10 12.93 Error d.f. 33 33 11 77
* Means in the same column followed by the same letter are not sig nificantly different. t Means of four observations per harvest. * Means of two observations per harvest. 44
TABLE VIII. NITROOEN YIELDS OF LEGUMES, ALONE AND IN ASSOCIATION WITH GRASSES. Treatments N yields, pounds per acre May August Feb. 1963t 1963t 1964:1: Average A. Individual treatment effects.* Kaimi Alone 13.1 a 25.3 b 19.3 a 19.3 a Kaimi w/Pangola 6.5 a 11.9 a 17.9 a 12.1 a Kaimi w/Napier 37.9 ab§ 31.7 b 27.2 a 32.2 a§ Centro Alone 64.9 bc 67.0 c 114.8 bc 82.2 b Centro w!Pangola 22.4 a 29.2 b 53.0 ab 34.9 a Centro w/Napier 24.7 a 20.8 ab 48.1 ab 31.2 a Intortum Alone 90.1 cd 102.3 d 137.6 c 110. a c Intortum w!Pango1a 70.7 bc 77.0 cd 138.1 c 95.3 bc Intortum w/Napier 121.2 d 80.3 cd 81.1 ab 94.2 bc Average for legume treatments 50.1 49.5 70.9 56.8 B. Means for legume effects.* Kaimi T9~-f-a -_. 23.0 a 21.8 a 21.2 a Centro 37.3 a 39.0 b 72.0 b 49.6 b Intortum 94.0 b 86.5 c 118.9 c 99.8 c C. Means for grass effects.* No Grass 56.0 b 64.9 b 90.5 a 70.5 b Pango1a 33.2 a 36.0 a 69.7 a 47.4 a Napier 61.2 b 44.3 a 52.1 a 52.5 a
§ D~ffers significantly from the other two kaimi treatments when tested by within-kaimi error terms. * Means in the same column followed by the same letter are not sig nificantly different. t Means of four observations per harvest.
:I: Means of two observations per harvest. 45 and thus the harvest totals estimate the annual nitrogen yield for the various treatments. The mean annual production for the four legume effects are: no-legume 18; kaimi 73; centro 162; and intortum 317 pounds of N per acre per year. This is equivalent to nearly 2,000 pounds of crude protein per acre per year for the intortum treatments. The nitrogen yields of the legume components are shown in Table VIII (ANOVA , see Table X). The largest differences are again asso ciated with legume species, with centro consistently yielding approxi mately twice as much N as kaimi, and intortum yielding twice as much as centro, except at the February harvest where intortum yields averaged less than double the centro yields. Only one grass effect was seen for the three legume species in general, i.e. reduced legume yields in the presence of either grass, except in the case of napier treatments at the first harvest. How ever, there were several within-legume effects of grasses. Kaimi N yields averaged significantly higher in kaimi-napier combination than when grown alone or with pangola. Centro yielded significantly more in pure stand than in mixture with either grass. Intortum associated with napier yielded more than intortum associated with pangola in the first harvest; but by August, intortum with napier was significantly lower yielding than either of the other two intortum treatments. Table IX shows the N yields of the napier and pangola components in different treatments, and the ANOVA mean squares are included in Table X. The data indicate that intortum significantly increased the N yield of both grasses. With pangola, this was significant for each of the three harvests. With napier the benefit from intortum was observed in the August and February harvests but not in the first harvest. 46
TABLE IX. NITROGEN YIELDS OF GRASSES, ALONE AND IN ASSOCIATION WITH LEGUMES. Treatments N yields, pounds per acre May August Feb. 1963t 1963t 1964* Average A. Individual treatment effects.*
Pangola Alone 9.0 a 1.3 ab 6.7 ab 5.6 ab Pangola w/Kaimi 6.7 a 1.1 a 4.1 a 3.8 a Pangola w/Centro 7.7 a 2.8 be 7.3 ab 5.9 b Pangola w/Intortum 11.3 b 5.4 d 9.4 c 8.7 cd
Napier Alone 11.2 a 2.6 abc 4.4 a 6.1 b Napier w/Kaimi 8.7 a 4.0 cd 3.7 a 5.5 b Napier w/Centro 9.2 a 3.8 c 7.9 b 7.1 be Napier w/Intortum 8.8 a 8.5 e 10.3 b 9.2 d Average for grass treatments 9.1 3.7 6.7 6.5 B. Means for grass effects.*
Pangola 8.6 a 2.6 a 6.8 a 6.1 a Napier 9.6 a 4.7 b 6.5 a 7.0 b C. Means for legume effects.*
No Legumes 10.1 a 2.0 a 5.5 ab 5.9 b Kaimi 7.7 a 2.5 a 3.9 a 4.6 a Centro 8.7 a 3.3 a 7.6 b 6.5 b Intortum 10.0 a 6.9 b 9.8 be 8.9 c
* Means in the same column followed by the same letter are not significantly different. t Means of four observations per harvest. * Means of two observations per harvest. 47
TABLE X. ANALYSIS OF VARIANCE OF NITROOEN YIELDS Source d.f. Mean Squares May August Feb. All 1963 1963 1964 Harvests A. Legume nitrogen yields. Treatments 8 168.60** 116.29** 133.52* 367.70** Legumes 2 507.08** 365.08** 396.12** 1,212.99** Grasses 2 74.58* 61.28** 61.72 110.88** GXL 4 46.37 80.68** 38.11 73.41** Harvests 2 81.45** TXH 16 9.74 Error 19.15 8.98 26.14 19.76 Error d.f. 27 27 9 63 B. Grass nitrogen yields.
Treatments 7 .28 .63** .34** .76** Grasses 1 .19 .95** .01 .73** Legumes 3 .30 1.10** .72** 1.48** GXL 3 1.99** .06 .07 .54** Harvests 2 6.68** TXH 14 .25* Error .21 .03 .06 .11 Error d.f. 24 24 8 56 48 A significant gain in napier N yield was also obtained with centro in the final harvest. On the other hand, a depression of grass N yields in the presence of kaimi clover was observed. This effect appeared consistently in both grasses but was significant only in the combined means, averaged over all harvests. The two grass species were also significantly different in N yield, with napier outyielding pangola, particularly at the August harvest. The increases in N yield due to intortum were also reflected in different ways, i.e. pangola increases were primarily due to higher percentages of N in the tissue, while napier responded mainly by in- creasing dry matter yields at a constant percent nitrogen. The increased N yields due to association with intortum amounted to slightly over 9 pounds (± 2 pounds) of N per acre per year for both grasses. The benefit from centro to napier at the last harvest was about 3.3 pounds (± 1.1 pound) after a 6 month growing period. Transfer of nitrogen from legumes to grasses was thus very limited. The average nitrogen yield per week was computed for each of the three legumes (Table XI) in order to estimate seasonal and cutting
TABLE XI. NITROGEN YIELD PER WEEK BY THREE LEGUMES, AVERAGE OF THREE TREAlMENTS.
Species N yields, pounds per acre per week May August Feb. 1963 1963 1964 Average Kaimi 1.19 2.29 .82 1.44 Centro 1.40 3.89 2.77 3.00 Intortum 3.87 8.65 4.57 5.70 Average 3.14 4.94 2.72 3.38 49
frequency factors in the nitrogen production of these species. These two factors are confounded, but it can be noted that the highest N yield per week was obtained in the May-August period for all three species. The production during the February-May period and again during the longer August-February period was markedly less. The latter decrease was less severe in centro than in the other two legumes, however. The differences among legume species reflect in general the yield differences noted above. D. Root Nitrogen Levels. Root N:top N ratios from 14 inch di ameter cores supplied information as to plot root nitrogen yields, except in the case of napier where the sample root N was mUltiplied by an area factor to estimate plot root N. A plot root N:top N ratio was then recovered for this species also for c~mparison with the four other species. These data are presented in Table XII and show a pronounced increase in the ratio values between the May and August harvests. This was followed by a marked decrease except for kaimi which kept on increasing. In general, the grasses maintained a higher pro portion of their total plant nitrogen in the roots than did the legumes, with the root N:top N ratio for napier consistently being the higher of the two grasses. A number of within-species differences occurred which were not consistent over more than one harvest and could not be easily related to differences in top growth, but which usually were consistent over both replications sampled. thus, no attempt to extrapolate the sample ratio beyond the plot actually sampled was made, and attention was directed to the amounts of root nitrogen present rather than to any relationships to top yields. 50 TABLE XII. RATIOS OF ROOT N:TOP N FOR GRASS AND LEGUME SPECIES .. Species Combinations Root N:Top N ratio* May 1963
Pangola Alone .61 5.34 .32 2.09 b Pangola w/Kaimi .76 2.74 .31 1.27 a Pangola w/Centro .61 1.87 .85 1.11 a Pangola w/lntortum .16 b..§Q .33 1.10 a Average .54 3.19 .45 1.36
Napier Alone .53 5.55 .45 2.18 Napier w/Kaimi 1.29 3.84 .95 2.03 Napier vi/Centro 1.19 3.45 .80 1.81 Napier vi/lntortum 1.26 2.23 .95 1.48 Average 1.07 3.77 -:79 1.87
Kaimi Alone .44 2.39 1.18 b Kaimi w!Pangola .37 .71 .54 a Kaimi w/Napier .47 .93 .73 a Average .41 1.34 -:BI
Centro Alone .ll .25 .14 .17 Centro w!Pango1a .14 .45 .16 .25 Centro vi/Napier .14 .65 .18 .32 Average .13 - .45 -:T6 --:25 Intortum Alone .18 .52 .21 .30 Intortum w/Pangola .23 .42 .40 .35 Intortum vi;Napier .09 ~ ....:.19 .34 Average .17 .49 .34 --:33
Average Grasses .80 3.48 .62 1.61 Average Legumes .24 .54 .61 .46 ANOVA mean squares for individual species. Source Grasses Leguines d.f. Pangola Napier d.f.
Companion spp. 3 1.576 .525 2 .668** .0409 .0045 Harvest 2 17.602* 21.665** 2 1.385 .1871* .1602* SXH 6 2.094* 1.793 4 .519** .0208 .0195 Error 12 .492 .753 9 .038 .0217 .0107
* Means of two observations per harvest. Letters follow only means of sets found to contain significant differences. 51
Table XIII shows the computed root N contents for each species in various combinations. Averaged over all harvests, several significant differences were apparent. Root N levels were significantly higher for napier than for pangola in all treatments. There were no signifi cant legume effects in pangola, but intortum had a favorable effect on the root N maintained by napier. Considering the two grasses to gether, however, grass roots at the final harvest averaged signifi cantly higher in both the centro and intortum mixtures than in the grass alone treatments. Root N declined markedly in both grasses after the August harvest, with the greatest losses occurring in the pangola with intortum, napier alone, and napier with kaimi treat ments. Among the legumes, kaimi root N levels were significantly less in association with pangola than in the kaimi alone or kaimi with napier treatments. Centro root N was seriously depressed by the ad mixture of either grass. Intortum maintained a significantly higher level of root N in all treatments than was attained by any of the other legumes except at the final harvest, where intortum root N in the pangola mixture was lower than kaimi root N in the pure kaimi plots. Although kaimi root N was still increasing, all the other species had attained a maximum by the August harvest. And with the exception of intortum, the amount of nitrogen present in the roots of legumes as well as grasses was surprisingly low, averaging less than 20 pounds per acre for most treatments. The calculated total (grass plus legume) root N levels for the eleven treatments are presented in Table XIV. These data were not analyzed statistically; but at the final harvest, 52 TABLE XIII. NITROGEN CONTAINED IN THE ROOTS OF GRASSES AND LEGUMES. Species Combinations Root N, pounds per acre May August Feb. 1963 1963 1964 Average A. Grass Component* Pangola Alone 6.0 6.4 2.5 ab 4.9 a Pangola w/Kaimi 6.4 3.1 1.1 a 3.5 a Pangola wjCentro 5.8 4.7 5.9 d 5.4 a Pangola w/lntortum 2.0 1l.0 3.0 abc 5.4 a Average 5.1 a 6.3 a 3.1 4.8 Napier Alone 7.9 15.8 1.9 ab 8.5 b Napier w/Kaimi 10.5 12.7 3.8 bcd 9.0 b Napier wjCentro 12.1 12.2 5.1 cd 9.8 b Napier w/lntortum 12.9 1.2& 10.3 e 13.4 c Average 10.9 b 14.4 b 5.3 10.1 Ave. for grasses 8.0 10.3 4.2 7.5 B. Legume Component* Kaimi Alone 7.1 16.5 45.8 d 23.1 cd Kaimi w/Pangola 2.8 8.4 12.2 a 7.8 a Kaimi w/Napier 11.5 16.4 ~b 17.7 bc Average 7.1 a 13.8 a 27.8 16.2 Centro Alone 9.4 21.2 14.7 a 15.1 b Centro w!Pangola 4.6 15.7 8.7 a 9.7 a Centro w/Napier 2.7 11.5 9.8 a 8.0 a Average 5.6 a 16.1 a 11.1 10.9 Intortum Alone 13.4 45.1 52.5 d 37.0 e Intortum wjPangola 15.3 32.1 26.0 b 24.5 d Intortum w/Napier 9.0 58.2 ~c 34.7 e Average 12.6 b 45.2 b 38.4 32.0 Ave. for legumes 8.4 25.0 25.5 19.7
* Means of two observations per harvest. Means followed by the same letter are not significantly different. 53
TABLE XIV. NITROGEN CCNTAINED IN THE ROOTS OF GRASSES, LEGUMES, AND MIXTURES. Species Combinations Total root N" pounds per acre May August Feb. 1963 1963 1964 Average
Pangola Alone 6.0 6.4 2.5 4.9 Napier Alone 7.9 ~ -L.2 ~ Average 6.9 11.1 2.2 6.7 Kaimi Alone 7.1 16.5 45.8 23.1 Kaimi + Pangola 9.2 11.5 13.3 11.3 Kaimi + Napier ~ 29.0 29.1 26.7 Average 12.8 19.0 29.0 20.2
Centro Alone 9.4 21.2 14.7 15.1 Centro + Pangola 10.3 20.4 14.6 15.2 Centro + Napier 14.8 ~ 14.9 ll:.§. Average 11.5 21.7 14.7 16.0 Intortum Alone 13.4 45.1 52.5 37.0 Intortum + Pangola 17.3 43.1 29.0 29.9 Intortum + Napier ~ 12:1. 47.0 .~ Average 17.5 54.4 42.8 38.3 Ave: Legume Alone 10.0 27.6 37.7 25.1 Legume + Pangola 12.3 25.0 19.0 18.8 Legume + Napier 19.6 42.6 30.3 30.8 ,Overall Average 12.7 28.0 23.9 21.5 54 a wide range of from two to nearly fifty pounds of root N per acre in the various treatments was apparent. Except for kaimi alone at the last harvest, high root N levels were mostly associated with treat ments containing intortum. Within the legume classes, napier combi nations also generally resulted in higher total root N. Centro treat ments for anyone harvest, however, were remarkably uniform, indicating the probable presence of some equilibrium between the root development of centro and its associated grasses. A few cinder substrate samples representing the expected extremes in N levels were analyzed, but the amount of N present was insigni ficantly small and these analyses were thus discontinued. E. Legume N Contribution. The amount of nitrogen contributed by the legume to the forage yield of each mixture was calculated by sub tracting the N yield of the appropriate grass in pure stand from the total N yield for that mixture. In the case of legumes alone, the N yields were corrected by subtracting the average of the two grasses grown alone. The first three columns of Table XV show the amounts of nitrogen contributed to forage N yields for the three harvests. Most treat ment differences were relatively consistent for the three harvests, but it is apparent that kaimi N fixation got off to a much faster start in the napier plots than in the kaimi alone or kaimi with pangola treat ments. And while the presence of napier seemed to enhance the nitrogen contribution of intortum at first, the opposite apparently held true at the final harvest. Some information as to the seasonal factor is supplied by com puting the N contribution per week (Table XVI). A summer maximum was TABLE Y01 .. LEGUME CaJTRIBUTIOO TO TOTAL N YIELDS ..
Species Combinations N suppljed, pounds per acre Top Growth May August Feb. Annual Root Overall 1963 1963 1964 Total Component Total Kaimi Alone 3.0 23.3 13.7 40.0 43.6 83.6 Kaimi w/Pangola 4.2 11.7 15.3 31.2 10.8 42.0 Kaimi w,INapier 35.4 33.0 26.5 94.9 27.1 121.9 Average 14.2 22.7 18.5 55.4 27.2 82.5
Centro Alone 54.8 65.0 109.3 229.1 12.5 241.6 Centro w!Pangola 21.7 30.7 53.7 106.1 12.1 118.2 Centro vi/Napier 23.2 22.0 51.6 96.8 13.0 109.8 Average 33.2 39.2 71.6 144.0 12.5 156.5
Intortum Alone 80.0 100.3 132.1 312.4 50.3 362.7 Intortum w!Pangola 73.0 81.1 140.8 294.9 26.6 321.5 Intortum w,INapier 118.8 86.2 87.0 292.0 45.1 337.1 Average 90.6 89.2 120.0 299.8 40.6 340.4
U1 U1 56
TABLE XVI. LEGUME N COOTRIBUTION PER WEEK TO YIELDS OF TOP GROWTH. Species Combinations N contribution, pounds per acre per week. Feb.-May May-Aug. Aug. 1963- Average 1963 1963 Feb. 1964 all periods Kaimi Alone .19 2.33 .53 1.08 Kaimi w/Pangola .26 1.17 .59 .67 Kaimi w/Napier 2.21 3.30 1.02 &1.§ Average .89 2.27 .71 1.29 Centro Alone 3.43 6.50 4.20 4.71 Centro w!Pangola 1.31 .3.07 2.06 2.15 Centro w;Napier 1.45 2.20 b.22 J&§ Average 2.08 3.92 2.75 2.91 Intortum Alone 5.00 10.03 5.08 6.70 Intortum w!Pangola 4.56 8.ll 5.41 6.03 Intortum w/Napier 7.42 8.62 3.35 6.46 Average 5.66 8.92 4:6T 6:40 recorded for all species in all combinations with·grasses. This was most pronounced in kaimi; least pronounced in centro. A portion of the legume.. N contribution was also reflected in the root N levels. This was estimated in the same way as for forage N yields above, and the results were compiled in Table XVII. The extent
TABLE XVII. LEGUME N CGJTRIBUTlOO REFLECTED IN THE TOTAL ROOT N LEVELS. Species Combinations N contribution to roots, pounds per acre May August Feb. 1963 1963 1964 Kaimi Alone .2 5.5 43.6 Kaimi w/Pangola 3.2 5.1 10.8 Kaimi w/Napier 14.1 13.2 27.2 Average ~ ~ ~ Centro Alone 2.5 10.1 12.5 Centro w/Pango1a 4.3 14.0 12.1 Centro w/Napier 6.9 7.8 13.0 Average 4:6 10:6 12.5 Intortum Alone 6.5 34.0 50.3 Intortum w!Pangola 11.3 36.7 26.6 Intortum w;Napier 14.0 59.3 45.1 Average TO:6 43:3 40.6 57 to which legumes affected root N yields was small at the time of the May harvest. Intortum showed a considerable effect on root N levels by the August harvest. Kaimi contributions were low but still in creasing at the final harvest; in pure stand, it accumulated espe cially high levels of root N during the fall and winter. Centro had a relatively high but increasing effect throughout. The sum of the N contributions to top growth for all three harvests plus the N contribution reflected in root N at the final harvest provided a useful estimate of the total annual N contribution (or N fixation) for each legume. It is biased upwards only slightly by the assumption that no root N contribution had occurred before the start of the 12-month period. These estimates are presented in the last column of Table xv. Al though, they were not analyzed statistically, the main differences followed closely those judged statistically significant in dry matter and N yield comparisons. Thus kaimi clover contributed much more N to top growth yields in association with napier than otherwise, but it was partly offset by the high root N accumulated by kaimi alone. Centro fixed over twice as much N in pure stand as it did in asso ciation with either grass. Nitrogen fixation by intortum was quite high and apparently unaffected by grass treatments. The approximately 340 pounds of N per acre per year fixed by intortum was double the average N supplied by centro and four times the N from kaimi. The best of the kaimi and centro treatments (i.e. kaimi plus napier, and centro alone) were well above the averages for these two species, but were still significantly inferior to intortum~ 58
F. N released to Percolate. Percolate collected in the drums below replications III and IV was measured and sampled at intervals ranging from 7-40 days depending on rainfall and atmospheric condi tions. Eventually, leaks developed in several plots, resulting in the incomplete recovery of percolate. A semi-quantitative approach was then adopted and the samples analyzed were considered to represent approximately the same volume of percolate for all plots and collec tions. After allowing for analytical error, a further allowance was made for nitrogen contributed by rainfall as evidenced by the presence of nitrogen in samples from the check plots. A few plots apparently released some nitrogen in the first two months after planting the legumes, but the amounts were small and irregular and were disregarded as resulting from decomposing seeds, inoculum and other contaminants. Following the application of nitrate fertilizer, recovery of the added N in the percolate was quantitative in plots having very little plant growth and nearly quantitative in . the other plots. A much smaller proportion of the fertilizer N was recovered following subsequent additions of ammonium or urea ferti lizers, especially in plots having vigorous vegetative growth. Data which might indicate nitrogen release by the test plants was thus not obtained until March, 1963. Analyses continued until August 22, 1963, a period which bracketed both the May 28 and Aug. 6 harvests. A second series of analyses on the legume alone and legume plus pangola plots followed the July 14, 1964 harvest. Weekly samples taken over a 5 week period were analyzed for nitrate N by the phenoldisul phonic acid method. However, collections from some of the plots were not obtained following periods of light rainfall. 59
The results from both series are presented in Table XVIII. No evidence of N releaseJby grasses alone or by mixtures containing napier was obtained and these are omitted from the table. The first series showed only two questionable instances of N release by kaimi, both in April 1963 and in the amount of-approxi mately 0.4 pound of N per acre. However, the second series indicated that trace amounts of N were released in three of the four plots tested at 4-5 weeks after harvest. In replication IV centro alone showed a small (about 1.1 pound per acre) but definite release of N immediately following both the May 28, 1963 and Aug. 5, 1963 harvests. A release of trace amounts of N after the May harvest was indicated for replication III also. Following the July 1964 harvest, N release by centro alone was observed in the second week in replication III. Information from the other weeks and from replication IV were lacking, due to missing samples in this treatment. However, centro with pangola exhibited N release in the second, third and fourth weeks after cutting. The highest release occurred during the fourth week when the equivalent of approxi mately 0.15 pound of N per acre was measured in the percolate. Intortum was shown to release relatively large amounts of N (approximately 3.2 and 2.0 pounds per acre for the two replications) immediately after the May 28, 1963 harvest. Two intortum treatments also appeared to release traces of N on separate occasions in April. In the August 1964 series relatively large amounts of N were released by one intortum-alone plot, during all five weeks. This amounted to approximately 1.0 pound of N per acre in the first week and a high of 2.0 pounds of N per acre in the fourth week. Much smatter release was TABLE XVIII. NITROGrN RELEASED TO PERCOLATE IN CERTAIN LEGUME PLOTS. Species Combinations Repl. N released. pounds per acre. 1963 Series 1964 Series Kaimi Alone III April 15, tr.; July 22, tr. Through 5th wk., tr. IV None Through 4th wk., tr. Kaimi + Pangola III None 4th wk., tr. IV None None Centro Alone III july 14, tr.? 2nd wk., tr. IV April 15, tr.; June 14, 1.1; Aug. 23, 1.1 (no samples) Centro + Pangola III None 2-4th wks., tr. IV None 2-4th wks., tr. Intortum Alone III June 14, 3.2 2-4th wks., tr. IV April 15, tr.; June 14, 2.0 5 wks.: 1.0, 0.2, tr., 2.0, 0.9 Intortum + Pangola III April 24, tr. 2nd wk., tr. IV None 4th wk., tr. (no earlier samples)
oC1' 61 also observed in the other replication during the same period. There . was some evidence of a release of trace amounts of N by the intortum plus pangola treatments, in the second week in one replication and the fourth week in the other, but complete sets of samples were lacking for these plots. 62 ROOT PERFUSION EXPERIMENT
Materials and Methods A. Cultural. Eight pyrex 4000 ml percolator tubes, six inches inside diameter at the top and with the sides sloping inward to a rounded bottom equipped with an outlet, were installed in a wooden frame in the glasshouse. Figure 6 illustrates diagrammatically one of these tubes as well as some of the operations described' below. The sides of the frame were then enclosed with heavy corrugated card board to prevent the entrance of sunlight, and the outlet of each tube was connected to a one-quart Mason jar through a flexible connec tion. Pumice cinders from the same source as in the previous experiment were prepared by screening to select particles in the 6 mesh to 30 mesh size range, followed by thorough washing with tap water. Cinders were placed in the tubes to within one-half inch of the top, compacted by light tamping, and washed by leaching the filled tubes twice with distilled water. Each percolator was then washed with 800 ml of the nutrient solution. Finally, 500 ml of nutrient solution was then added and allowed to remain in the percolator tube-reservoir system. The nitrogen-free nutrient solution was made up in distilled water according to the directions of Norris (42) in batches of 18 liters. The pH was adjusted to 6.0 at which point the CaS04 completely dissolved. The basic trace element and iron solutions were made up separately and used for succeeding batches as well, but it was nec essary to replace the ferrous sulfate-citric acid solution after about six months. 63
,r::. - --_.
1 1 b l
FIGURE 6. DIAGRAM OF PERFUSION SUBSYSTEM FOR PERFUSION OF LEGUME ROOTS (a), AND FOR PERFUSION OF PANGOLA AND LEGUME ROOTS IN SERIES (b). 64
Seeds of the same legumes as used in the small plot experiment were scarified and inoculated as described previously. Each legume was planted in two percolator tubes on Sept. 2, 1964 by manually placing approximately eight seeds per tube at a depth of 1 cm in the cinders toward the middle of the surface area. Two unplanted tubes were left as controls. The tubes were arranged in two replications
and the top oi each tube was covered with aluminum foil except imme diately over the seeded area. The percolated nutrient solution was poured over twice a day for a time and distilled water was added as required to maintain the initial volume of solution. At weekly intervals, the tubes were allowed to drain overnight, and the solu tions then removed and taken to the laboratory for analysis. At the same time, 500 ml of fresh nutrient solution was added to the top of each tube. Kaimi was replanted on Sept. 12, 1964 and the less vigorous seed lings of the other species were removed. Additional inoculum was also added to the surfaces of the appropriate tubes. Each tube was subse quently thinned to one plant per tube. Centro plants from both replications were cut back on Dec. 1, 1964 and the roots exposed at the sides of the tube were photographed. The procedure of manually pouring over percolate was replaced
Jan. 27, 1965 with a perfusion system (Figure 6a) modelled after that of Morrill and Dawson (39). A high capacity diaphram-type aquarium pump supplied air to a manifold from which air was supplied to each perfusion subsystem through one-inch sections of 0.5 mm capillary tubing and lengths of tygon tubing. Each air line joined a syphon from the collection jar at a point approximately 30 inches below the bottom of 65
the jar. From there a delivery tube led to the top of the percolatQr. The flow of solution through each subsystem was regulated by a one inch length of 1.0 mm capillary tubing. The tubing was protected by black paper from the sun over most of its length in order to minimize the growth of algae. The apparatus was allowed to run continuously except that each week it was turned off the evening before the nutrient solution was changed. Daily attention was required, however, to replace evapotranspiration losses and to check for plugging of the capillary tubing by root or algal particles. Solutions which had been perfused through the root-cinder media for one week were taken to the laboratory for nitrate analysis by the modified method described later. Legume top growth was harvested from all tubes at intervals of 5-6 weeks, and dry matter and nitrogen yields were determined as described for the previous experiment. Following the first cutting, and weekly thereafter, the perfused nutrient solution was also analyzed for ammonia and amino nitrogen as described below. Roots and nodules visible through the tube walls were photographed after each harvest. Early in April, additional percolator tubes were constructed by cutting standard 5 pint clear glass reagent bottles in cross section so that slightly more than half of the volume remained in the neck portion. The inverted neck was fitted with a rubber stopper and glass tubing outlet. This was then covered with several layers of glass wool and the vessel filled to within one inch of the top with washed and screened cinders. The cinders were tamped and levelled and then covered with I cm of finely ground cinders to provide a water retentive layer. This in turn was covered by 1 cm of the screened cinders, forming 66 a layer through which water could easily distribute, but from which evaporation would be minimal. The filled tubes were washed with several volumes of distilled water and then with nutrient solution similar to the legume solution except that the calcium sulfate was replaced by its molar equivalent of calcium nitrate. Fresh stems of pangola were cut into segments of one node each and selected for uniformity. Six cuttings were sprigged into each tube so that the node was at the same level as the fine cinder layer. The planted tubes were placed in a darkened rack and arranged so that all the outlets led into a common carboy containing the nutrient solution. A small laboratory pump equipped with a timer, cycled nutrient solution to the tops of the tubes for a short period every 30 minutes. The plants were allowed to gradually deplete the nitrogen in the solution for 3 weeks. They were then cut back to within 3 cm of the surface and a nitrogen-free nutrient solution was substituted for the original mixture. The plants were cut again three weeks later on May 10. The regrowth subsequent to this cutting was uniformly yellow-green. The root systems, however, were extensive and healthy in appearance as judged by many roots along the walls of the tube. The plants were allowed to grow until May 28 at which time growth had virtually ceased due to depletion of all available nitrogen sources. At this date, the plants were again cut back to within 3 cm of the surface, and each grass tube was placed in series with a legume plant by locating it above and to the side of a legume percolator tube. The appropriate outlet from the perfusion apparatus was then directed to the top of the grass tube, and the grass tube outlet then discharged 67
onto the legume tube (see Figure 6b). The perfusion system was allowed to operate for three weeks, following which both legumes and grasses, including grasses in series with legume-free tubes, were cut and ana lyzed for nitrogen yield. The system was then operated for a second 3-week period immediately after this harvest. This was concluded by a final harvest of both grass and legume regrowth. The nutrient solu tion was discarded and replaced weekly during the six weeks of opera tion. B. Analytical. Analysis of the perfusate for nitrate N w.as done initially by evaporating 200 ml of solution to dryness on the steam
bath, adding 2 ml of phenoldisulfonic acid, 14 ml of approx. 6 ~ KOH, and distilled water to a volume of 50 mI. Resulting precipitates and off-colors required numerous modifications to this method. The procedure finally adopted was as follows: The nutrient solution was changed to a chloride-free solution by replacing the KGl with K2S04 (3.13 gm per 18 1.). The solution to be analyzed was made up to 500 ml volume with distilled water and then made strongly alkaline by the addition of a few drops of concentrated
NaOH. The resulting GaS04 precipitate was allowed to settle out over night. Two hundred ml of solution were then evaporated by adding 50 ml at a time to 8 cm evaporating dishes. During evaporation of the final 50 ml portion, any organic matter in the dish contents was digested by the addition of 1 ml of micro-analysis grade H202 and covering with a watch glass while heating on the steam bath for two hours. The solution was then evaporated to dryness and allowed to remain on the steam bath for an additional half hour to destroy any residual hydrogen peroxide. 68 The dish was then cooled, and 2 ml of phenoldisulfonic acid were added quickly with swirling. Lumps of residue were broken up with a stirring rod, using a separate rod for each dish. After 10 minutes, approximately 20 ml of distilled water were added with stirring, rinsing the stirring rod with the final portion. The solution and residue were then transferred to a 50 ml centrifuge tube with a minimum of washing and centrifuged at full speed (ca. 3100 rpm) for 15 minutes in a clinical-type centrifuge. The supernatant solution was decanted into a 50 ml volumetric flask. To this was added water to bring the volume to approximately 35 ml, 11 ml of approximately 6 H NH40H, and distilled water to volume. The flask contents were mixed by shaking, and the yellow color was read after 10-15 minutes with a Klett-Summerson
colorimeter using a blue filter with maximum transmission at 420~. The colors were evaluated against a standard prepared by adding known amounts of 5 ppm KN03 to 500 ml samples of fresh nutrient solution and carrying them through the entire procedure. A linear relationship between ab sorbance vaiues and final concentrations of nitrate N was found for the range 0-0.8 ppm N. By these means, undesirable precipitates and off colors were largely avoided, but occasional brownish tinges still occurred in the perfusates from certain plants. Perfusate samples were also analyzed for ninhydrin-positive (amino) N and ammonium N beginning March 3, 1965. The amino acid analysis followed the method described by Block and Weiss (6) except that the final diluent used was 60% (v/v) ethanol rather than propanol. In practice, it was possible to store solutions of both the citrate buffer (refrigerated) and ninhydrin-methyl cellosolve (brown bottle), and either make up the stannous chloride as needed or store it under mineral oil 69 for no longer than two weeks. The three components were then mixed in the proper proportions just before use. One ml samples of perfusate were used without pretreatment, as they were not appreciably buffered in the pH range required. The final color was read with a Klett Summerson colorimeter using a green filter with maximum transmission
at 540~. Readings were evaluated by comparison against ~-glutamic acid standards, prepared by dissolving the salt in fresh nutrient solution at concentrations of 0-2.8 ppm of amino nitrogen (0.0-0.2 ~ moles of the acid). Since ammonium N was also partially determined by this method, it was necessary to make corrections based on the ammonium N present in the sample. Ammonium standards were found to give readings which, based on glutamic acid standards, were equivalent to 84% of the ammonium N present. Each apparent amino N concentration was thus corrected by subtracting the product: 0.84 times the concentration of ammonium N in that sample (as determined below). Ammonium N was initially determined by steam distilling 100 ml
of solution with MgO into a ~ boric acid-mixed indicator solution and titrating with 0.005 [HCl. This method, though accurate enough for slightly larger amounts of nitrogen, was not satisfactory for the low ammonium levels encountered. It was thus modified as follows. One hundred ml of solution were distilled with MgO in a micro-distil lation apparatus as before, but the distillate was collected in a 50 ml centrifuge cup marked at 32 ml and containing 3 ml of 0.17 [ HCl. Distillation was stopped when a total of 32 ml was obtained, and the tube contents were transferred to a 50 ml volumetric flask with a minimum of washing. The contents were then Nesslerized by Middleton's 70 modified method (32), adjusted for larger aliquots by adding 5 ml per flask of the following reagents: (a) 0.2% (w/v) gum arabic prepared as directed, (b) modified Nessler's reagent containing 15 g
HgI2 and 20 g KI per liter, and (c) 3.40 ~ NaOH. This method yielded precise results over the range 0-1.5 ppm N as ammonia. Higher con centrations often resulted in turbidities. Turbidities were also encountered whenever traces of organic solvents, such as those used in the amino determination, were present in the air, on the glass ware, or dissolved in the reagents. The yellow colors from Ness lerized samples or standard NH4Cl solutions were read with a Klett Summerson colorimeter with a blue filter with maximum transmission at 420 ~. Plant samples were dried at 70oC, weighed, ground, and analyzed for total N as described for the previous experiment.
Results A. Legume Yields. Establishment and early growth were slow due to inadequate water in the root zone during the first few months. Growth improved, however, as the roots developed throughout the sub strate, and was optimum after installation of the continuous perfusion apparatus. Dry matter and N yields for the six plants are shown in Table IXX A. The data show large differences in the vigor, both among the three species, and between the plants within each species, especially kaimi and intortum. These two species are known to be very hetero geneous, and the results provide some indication as to the behavior of the different plant types encountered. Centro yields were 71
TABLE IXX .. LEGUME AND GRASS YIELDS FROM PERFUSED CINDER CULTURE IN ruE GLASSHOUSE .. Harvest Legume Plants Date Kaimi Centro Intortum ! 2 1 2 1 2 A. Legume Yields
Feb. 24: D.M. yield (g) 27.78 6.01 9.60 14.93 11.09 36.36 N content (%) 2.98 3.77 2.87 3.41 3.52 3.35 N yield (g) .81 .23 .28 .51 .39 1.22 April 8: D.M. yield (g) 7.50 2.95 8.15 9.40 13.00 35.95 N content (%) 4.16 3.93 3.45 3.43 3.17 2.92 N yield (g) .31 .12 .28 .32 .41 1.05 May 14: D.M. yield (g) 6.35 3.45 11.00 14.05 15.85 21.90 N content (%) 3.64 3.97 3.64 2.96 2.91 2.00 N yield (g) .23 .14 .40 .42 .46 .44 June 18: D.M. yield (g) 5.70 4.00 12.40 16.90 20.45 39.20 N content (%) 3.52 3.71 3.21 3.29 3.25 3.35 N yield (g) .20 .15 .40 .56 .67 1.31 July 9: D.M. yield (g) 2.17 1.32 3.22 4.77 5.59 7.19 N content (%) 4.29 4.23 4.31 4.03 3.33 3.01 N yield (g) .09 .06 .14 .19 .19 .22 B. Yields of grasses in series culture with legumes. June 18: N yield (mg) 2.20 2.61 3.64 3.66 2.57 7.30 N uptake (mg)* 0 .28 1.31 1.33 .24 4.97 July 9: N yield (mg) 2.61 1.59 5.53 5.04 5.75 22.40 N uptake (mg)* 1.60 .58 4.52 4.03 4.74 21.39 % of mobile legume N transferred 1.71% 1.11% 3.16% 2.06% 2.49% 9.00%
*N yield less the average N yields of two checks (2.33 mg N, first period, and 1.01 mg N, second period). 72 relatively lower than the others at first harvest since these plants were cut back 3 months earlier. The large discrepancy between the yields of the two kaimi plants appeared to be related in part to differences in their rate of initial establishment since the differ ences decreased somewhat with time. Plant no. 1, however, possessed visibly larger leaves, longer internodes, and a more extensive root system than the second plant. No striking morphological features distinguished the two centro plants, but the intortum plants differed markedly in respect to their plant coloring, the less vigorous (no. 1) plant being characterized by green stems and the other by reddish stems. The first plant was also very slow in establishment, and the extension of its root system into the lower part of the tube was greatly retarded. The second plant, on the other hand, quickly de veloped a very extensive and heavily nodulated root system throughout the entire cinder mass. This plant, with its larger nutrient requirements, apparently was the most seriously affected by the iron deficiency that developed briefly 3 weeks prior to the May 14 harvest. This was reflected both in a lighter green leaf color and in the lower percentage of N in the harvested vegetation. The other plants were affected to a lesser degree, and recovered quickly when the deficiency was corrected. The development of the roots of several plants is shown in the sequences of photographs presented in Figures 7 and 8. The large nodules of centro are especially evident. The photographs show that root development and nodulation were in progress during the experimental period; and that most plants had healthy well-nodulated root systems when the perfusate analysis was begun. 73
. l'- 74
co• 75 B. Release of Root N. Results of the weekly analysis of per fusate are shown in Figure 9. Only nitrate data are available for the first three weeks. Levels of nitrate N were small in all cases, exceeding 25 ~g per plant in only one instance. There was no dis cernable pattern to the nitrate levels, but within a particular species, a higher average level of nitrate occurred in perfusates from the more vigorous of the two plants. Ammonium nitrogen occurred, with two exceptions, only in samples taken within two weeks after defoliation. Of the two exceptions, one sample was taken on the third week following unsually high rates of N release and the other was taken on the date of harvest. Rather high levels of ammonium were observed following the April harvest in perfusates from one kaimi, one centro, and both intortum plants, and again following the May harvest in the perfusate from one kaimi plant and both centro plants. Ninhydrin-positive (amino) N also was present primarily in samples taken on the date of harvest or within two weeks afterward. Commonly the highest amino N levels were attained on the second week after defoliation. In one instance, following unusually high rates of N release in the first 2 weeks, significant amounts of amino acids were found on the third week. On the 12th week, both intortum plants and the more vigorous kaimi plant released amino acids plus somewhat larger amounts of nitrates than in the weeks immediately preceding and following. This coincided with the onset of iron deficiency symptoms in the plant tops. When this deficiency was corrected, no nitrogen whatsoever was found in any of the perfusates the following week. 76
700 KAIMI-I KAIMI- 2 150 150 i. AMINO N Cl ::'. NH 4 " N NO.-N " ... 100 100 p.g N FROM ROOTS 50 50
2 f4 6
177 II. 150 ~ m CENTRO-I CENTRO-2
100
1£0 N FROM ROOTS 50
2
150 150 100 INTORTUM-I ~
100
1£0 N FROM ROOTS 50
TIME IN WEEKS TIME IN WEEKS
FIGURE 9. NITROGEN LEVELS IN SOLUTIOOS AFTER PERFUSING LEGUME ROOTS. (VERTICAL ARROWS ALOOG HORIZOOTAL AXES INDICATE HARVEST DATES.) 77 Extrapolation to field conditions of the release of N by legume roots to a perfusing solution requires several questionable assumptions. However, if a kaimi plant of the size grown here is assumed to require
two square feet of area in the field, each 21,000 ~g of nitrogen released is equivalent to one pound per acre. The observed maximum of
706 ~g per plant, would then correspond to approximately 0.033 pound of N per acre. Likewise, if four square feet per plant are allowed for centro or intortum, a maximum release of approximately 0.044 pound of N per acre by intortum was observed following the April harvest. The above figures are of doubtful value, though, mainly because they represent only the amounts of N present in equilibrium situations, in which the legume roots had continuous opportunity to recover any N released to the solutions. However, when the roots of N-starved pangola plants were placed in series with the legume roots, it was possible to estimate the extent to which N would be released to a con tinuously deficient system. Information on this was obtained during two 3-week periods; the first was a period of normal vegetative growth ending June 18, and the second included the period of regrowth ending July 9. At the start of the first period, low equilibrium levels of N in the solutions had already been attained as determined by the ana lyses described previously. The grass yields from both periods are entered in Table IXX B, and the legume yields are included in Table IXX A. The extent of N transfer is also clearly visible in photographs of the plants taken at the conclusion of each period (Figures 10 and 11). Little or no N transfer from kaimi to pangola was measured during the first period. The more vigorous of the intortum plants supplied 78
FIGURE 10. VIEW OF GRASSES GROWN IN SERIES WITII LEGUMES (FOREGROUND). CONCLUSION OF FIRST TIIREE-WEEK PERIOD. 79
~rr·.4?'*A{*%;¥A; ~·;;·"·§#~~0T;A~¥!;1Yi'-S7;&'tst~~~m:.t:!,-.,~/YJ4,k'~'\.
·\;j'·:~.>:~G~i
FIGURE 11. VIEW OF GRASSES GROWN IN SERIES WITH LEGUMES (FOREGROUND). cc:NCLUSIOO OF SECOND THREE-WEEK SERIES. 80 nearly 5 mg of N to the associated grass, but N transfer by the other intortum plant was negligible. The two centro plants were intermediate. However, immediately after defoliation of the legumes, larger amounts of N were supplied by the roots of all legume plants tested, ranging from approximately .6 mg to over 20 mg per plant. Kaimi roots supplied the least amount of N while centro was intermediate and intortum the highest. Large differences between the two kaimi plants and the two intortum plants were also observed, with the less vigorous intortum plant providing only slightly more N than the centro plants. Visual observation of the pangola plants indicated that a large propor tion of the N transferred to the grasses was made available in the first week. The total amount of N mobilized by the legume roots during this latter period was estimated by the sum of the N harvested in the legume tops plus that in the associated grass tops. The percent of this mobile N which was transferred to the grass was computed for each plant (bottom line, Table IXX). Among the kaimi and intortum plants, the more vigorous plant in each species released a larger proportion of its mobilized root N than did the less vigorous plant. Thus a maximum of 9% of the mobile N was supplied to the associated grass by intortum plant no. 2 during this period. In the two centro plants, the order seemed to be reversed. The slower-growing plant supplied as much or more N 'to the grass as the faster plant, resulting in a higher percentage transfer by the slower plant. 81
LEAF NITROGEN EXPERIMENT
Materials and Methods Plants of kaimi, centro and 'intortum were grown in 5 gallon con tainers filled with soil. The containers were filled with fertile Waimanalo silty clay soil and sown with 10 seeds of the appropriate legume, scarified and inoculated as described for the small plot experiment. Eight containers were planted to each species on Aug. 17, 1964. The plants were allowed to emerge' under regular irrigation out of-doors. The containers were then moved to the glasshouse and thinned to 3 plants per container on Sept. 21, 1964. Water was added at the soil surface to prevent leaching of leaf nitrogen. Intortum and centro were trained upwards on cylindrical frameworks. New leaves, in which the leaflets were completely unfolded, were tagged at weekly intervals. On two occasions, it was necessary to spray the plants with malathion to control mites (especially on centro), cottony-cushion scale (especially on intortum), and grasshoppers. Leaf samples of different ages were harvested from plants in individual containers in December 1964 and January 1965, after the oldest leaves began senescing. These samples were used in the develop ment of analytical procedures. The plants were cut back on Jan. 22, _1965, and tagging of leaves on the new growth was re-initiated on Feb. 1, 1965. Leaf analysis was begun again in mid-April, 1965, but ammonium N was not included in samples harvested prior to May 11, 1965. Leaf harvesting was done by plucking leaves of similar age as determined by their position on the stem with respect to tagged leaves of a known age. Eight leaves per age group (in the case of kaimi) 82 were plucked from the plants in a selected container at each harvest. For the other species, six leaves of centro or four leaves of intortum were taken. The leaflets were immediately removed from the leaf stalk and placed into a 125 ml Erlenmeyer flask containing 75 ml of distilled water. All flasks comprising one complete age series of leaflets were then shaken simultaneously on a reciprocating-type laboratory shaker for 20 minutes. The resulting solutions were immediately filtered through S &S white ribbon paper and analyzed for N constituents the same day. Ammonium N was determined by steam distillation and Nessleriza tion of the distillate as described for percolate in the preceding experiment, except that only 50 ml of sample was introduced into the still. The sample was distilled for 4 minutes to obtain ~pproximately 29 ml of distillate. Nitrate N was then determined in the same sample by introducing 1.0 gm of finely ground (60 mesh or finer) Devarda's alloy into the sample solution and distilling for another 4 minutes into a second centrifuge tUbe containing 3 ml of 0.17 ~ H2S04• This was Nesslerized as in the ammonium determination and evaluated against an NH4CI standard. Ninhydrin-positive (amino) N was determined as described previously for percolate, and evaluated against glutamic acid standards prepared in distilled water. Corrections for ammonium N were made as before.
Results The total nitrogen present in leaf samples harvested from the three legume species is shown in Figures 12-14. These figures also show the amounts of different nitrogen constituents extracted from these samples by distilled water extraction of intact leaflets. Data from 83
~ amino N _ 4 A. Bright sunlight. NH N _N0 N 3
200 40 mg ... g N total N extracted per 8 per 8 leaves leaves 100 20
B. Light shade.
200 40
""g N mg extracted total N per "8 per 8 leaves 100 20 leaves
c. Moderate shade
200 40 mg ... g N total N extracted per 8 .per 8 leaves leaves 100 20
2 4 6 8 10 12 14 Approximate leaf age in weeks.
FIGURE 12. TOTAL NITROOEN AND EXTRACTABLE NITROGm IN KAIMI LEAVES OF DIFFERENT AGES. 84
A---4 amino N A. Bright sunlight. "...... ;..0 NH4 N -N03 N
mg JIg N total N extracted per 6 per 6 20 leaves leaves
B. Bright sunlight.
200 mg ~g N total N extracted per 6 per 6 leaves leaves 100 20
c. Bright sunlight.
40 mg "'g N total N extracted per 6 per 6 leaves leaves 20
2 4 6 8 10 12 14 Approximate leaf age in weeks.
FIGURE 13. TOTAL NITROOEN AND EXTRACTABLE NITROGEN IN CENTRO LEAVES OF DIFFERENT AGES. 85
A. Bright sunlight. ~ amino N ~NH4N N0 N - 3
200 40
..g N mg extracted total N per 4 per 4 leaves leaves 100 20
B. Moderate shade.
200 40
mg Io/g N total N extracted per 4 per 4 leaves leaves 100 20
c. Partially self-shaded.
40
mg Jig N total N extracted per 4 per 4 leaves 20 leaves
2 4 10 12 1'1 Approximate leaf age in weeks
FIGURE 14. TOTAL NITROOEN AND EXTRACTABLE NITROGEN IN INTORTUM LEAVES OF DIFFERENT AGES. 86 three series of samples which were analyzed for all three forms of extractable N are included. In addition, freehand curves which summarize the data obtained for each species are shown in Figure 15. Certain plants received somewhat less sunlight than others due to their location with respect to greenhouse structures or large centro and intortum plants. Some of the centro and intortum plants were also affected by self-shading along part of the stem length as a result of newer growth covering portions of the older growth. The three age series shown in Figure 12 for kaimi thus represent plants from three levels of light intensity. Total N reaches a maxi mum at about three to four weeks, and appears to decrease more rapidly with increasing age where some shade is present. Most of the decrease in total N, however, is related to the smaller leaves which the plants put out in the early stages of regrowth. Nitrate N was very low throughout, but as much as 25 ~g per eight leaves was extracted from older shade leaves. Extractable ammonium N was stable at about 40-60
~g per eight leaves for all ages and degrees of shade, except that
80 ~g was obtained from mature shade leaves. Amino N levels appeared to be very shade dependent. For leaves in bright sunlight, less than
30 ~g was extractable from the green leaves. However, under light shade, a range of 30-50 ~g per eight leaves (with one exception) was obtained for full-sized green leaves. Under moderate shade, extractable amino
N was in all cases more than 80 ~g per eight leaves and after five weeks was in the range of 150-190 ~g. The centro plants used were all in direct sunlight, and no effect 1_ of shade was thus observed. However, it was noted that under severe self-shading, the shaded leaves yellowed and died, regardless of age. 87
_ amino N A. Kaimi ---- NH4 N ••••••••• N0 N 3 200 mg flog N " total N extracted " per 8 per 8 leaves leaves ~Shade '" 100 sun '" ~...... _----j ------~~, L:. _-_ _ _._ __ ~-
B. Centro
200 HN mg extracted total N per 6 per 6 leaves leaves 20
--- . C. Intortum
200 II-g N mg extracted total N per 4 per 4 leaves leaves 100 20
2 4 6 8 10 12 14 Approximate leaf age in weeks.
FIGURE 15. TOTAL NITROGEN AND EXTRACTABLE NITROOEN IN LEAVES OF THREE LEGUME SPECIES. SUMMARY OF THREE AGE SERIES PER SPECIES. ~- . 88
These leaves were as high or higher in extractable N constituents as older senescing leaves. In many respects the pattern of total and extractable N from the three series (Figure 13) are very similar. Maximum total N was attained by the third to fourth week, and remained high until the ninth or tenth week, when it abruptly dropped within a few weeks from 30-40 mg to less than 10 mg per six leaves. Extractable nitrate N was extremely low in all except the rapidly expanding young
leaves where it went up to about 30 ~g per six leaves. Ammonium N
was relatively stable throughout, with about 50-70 ~g per six leaves being obtained consistently. The amounts of amino N extracted from these leaves were relatively low except for a short peak during the rapid expansion of the young leaves. During this interval, over 150
~g per six leaves could be extracted, but the peak period must have been very short since it did not appear at all in series B. In the case of intortum (Figure 14), the effects of both general shade and partial self-shading were observed. Under moderate shade, the maximum total N per four leaves was somewhat lower than for sun leaves, but it is possible that this is partly the result of genetic variation among plants. Also, where shading was present, the final decrease in total N of aging leaves was later and more abrupt. Ex tractable nitrate N was low (5-30 ~g per four leaves) throughout and appeared to be affected very little by shade or leaf age. Under moderate shade, however, it appeared to be slightly higher in very young and in yellowing leaves. Ammonium N was somewhat higher than nitrate N, but was likewise relatively unaffected by shade or leaf age. The amount of amino N extracted from the leaves, however, was closely related to the degree of shading experienced by the particular leaves 89 in question. Under bright sunlight, amino N was high in both the expanding young leaves and older yellowing leaves, but low (about 50-85 ~9 per four leaves) in full-sized green leaves. Where the leaves were shaded, however, extractable amino N levels increased to
120 ~g per four leaves or higher, depending on the degree of shading. As the leaves turned yellow, amino N levels increased again. In Figure l4C, no data was obtained for abscissing yellow leavea. L _ The amounts of the different forms of N e~tracted, summed over all the samples in each age series are shown in Table XX as percent- ages of the total leaf N for the series. Thus, from 0.27-0.68% of
TABLE XX. NITROGEN CONSTITUENTS EXTRACTED FROM DIFFERENT SERIES OF LEAF SAMPLES. Species and Series N extracted per series. percent of total N. N03-N NH4-N Amino N Total Kaimi, Series A .03 .17 .07 .27 Series B .02 .21 .15 .38 Series C .05 .21 .42 .68 Weighted Ave. :03 :T9 .21 .44 Centro, Series A .02 .21 .23 .46 Series B .01 .24 .10 .35 Series C .03 .30 .16 .49 Weighted Ave. .02 :25 .15 :42
Intortum, Series A .05 .14 ---.44 .64 Series B .07 .14 .58 .79 Series C .05 .ill. .43 .59 Weighted Ave. .06 .13 .48 .66 the total leaf N was extracted from kaimi leaves, depending on the degree of shading. Most of the variation was a reflection of different levels of extractable amino N. On the average, the percentage of amino N extracted was only slightly more than the ammonium N. 90
In centro, however, extractable ammonium N averaged higher than amino N. The total percent of N extracted in the three forms was about the same in both kaimi and centro, i.e., slightly more than 0.4%. A large proportion of the N extracted from intortum leaves was in the amino form, amounting to nearly 0.5% of the total leaf N. The total extractable N averaged 0.66% for this species. For each .species, recently abscissed leaflets were also collected and analyzed for total and extractable N. The N present in these leaves, expressed as a fraction of the nitrogen in the corresponding series of living leaves, is as follows: kaimi, 1.72%; centro, 3.20%; and intortum, 1.16%. In each case, the total N remaining in one dead leaf is thus seen to be much higher than the water-extractable N in all of the living leaves. About 5-7% of the N present in the intact abscissed leaves was found to be water extractable. During the period of growth sampled, the approximate weekly leaf production by the three species was determined with the following results: kaimi, 0.8-1.0; centro, 1.0-1.2; and intortum, 1.5-1.8 leaves/week. After reaching maturity, it may be expected that vegetative plants similar to those sampled will also lose dead leaves at the same average rates as above. On this basis, the percentages of total leaf N being lost each week through leaf fall would amount to 1.4-1.7% for kaimi, 3.2-3.8% for centro, and 1.7-2.1% for intortum. 91
DISCUSSION
A. Small Plot Yields. The dry matter and N yields of legumes in the small plot experiment compare very favorably with those reported elsewhere (Moore, 36, Younge, ~ al., 80) for plants growing in soil. No good explanation is available for the improved growth of kaimi with napier. However, this phenomenon may be due to complementary root effects since it was also observed that (a) centro and intortum plants grew better adjacent to napier clumps and (b) intortum tended to volunteer in the vicinity of existing napier clumps. The depression in yields of centro when grown with either of the grasses was also unexpected. Whether this was due to some adverse effect upon the rhizobia symbiosis or upon moisture and nutrient competition between the legume and grass plants is not known, but a factor limiting the stand or root development of this legume seemed to be involved. Thus, centro stands and root N tended to be somewhat depressed where grass plants were present in the same area, although this effect was rather variable. Grass yields of dry matter and N were extremely low due to con tinuous nitrogen deficiency. The extent of the N stress was also shown by the yellow color and the low percent N in the grass tops. The highest percentage of N for pangola is equivalent to only 3.3% protein as compared to the range of 5-12% reported by Hosaka (22). Also the maximum for napier amounted to 3.9% protein which is con siderably lower than the range of 5.2-9.9% protein measured by Younge and Ripperton (81) in four tests, or the 4.6-9.8% protein measured by Nordfeldt et ale (41) for napier of different ages. The increase in 92
percent N for pangola grass associated with intortum is in accord, however, with several reports (27, 48, 63) showing increasing protein levels due to nitrogen fertilization. Similar increases have been reported for napier (Vicente-Chandler, Silva and Figarella, 64), but the only napier responses obtained here were consistently in the form of increasing dry matter yields. Apparently these two grasses utilized N contributed by the legume in different ways, probably due to the degree of shading experienced. The lower growing pangola was severely shaded by the associated intortum and was thus limited in its carbohydrate production; whereas the taller napier had more than adequate sunlight, and growth was limited only by the amount of N available for the formation of new protein tissue. Napier also accumulated more root N when grown with intortum, but the root N:top N ratios indicated that this was primarily a reflection of the increased top yields. At the final harvest, napier with intortum had 8.4 ± 1.5 pounds of root N per acre more than napier alone. The annual con- tribution of N by intortum to napier forage yields was an additional 9.4 pounds per acre. The sum of the increases in napier top Nand root N, or 17.8 pounds per acre, estimates the transfer of N from intortum to napier. Thus slightly more than 5% of the computed annual N fixation by intortum was apparently transferred to napier under these conditions. Since intortum did not significantly alter pangola root N levels, transfer of nitrogen from intortum to this grass was reflected only in the forage N yields. This amounted to 9.1 pounds of N transferred per acre, or 2.8% of the estimated N fixation of 322 pounds per acre. A smaller benefit to napier yields was obtained from centro at , 93 the winter harvest. Of the N apparently transferred to the grass over a six month period, an estimated 3.3 pounds were recovered in the grass tops and 3.2 pounds in the grass roots. The total transfer of 6.5 pounds of N per acre was thus about 11% of the-56.8 pounds of Nfixed by centro in combination with napier during this period. The data suggest a similar effect of centro on the root N levels of pan gola over the same period, although no differences in the respective N yields of grass tops were observed. This apparent contribution of approximately 3.4 pounds of N per acre to the pangola roots was 6.7% of the 57.1 pounds of N fixed by the centro in this mixture. Kaimi had no effect on the root N levels of the grasses. How ever, both dry matter and N yields of grasses associated with kaimi were significantly depressed, indicating that this legume is highly efficient in scavenging and retaining N. The results thus do not support the supposition which has been advanced by Younge ~ ale (80) that kaimi has the ability to transfer fixed nitrogen to the grass component of a mixed sward. On the other hand, the proportions of fixed nitrogen which were transferred by intortum and by centro (on one occasion) to their associated grasses is higher than that observed by Henzell (18) for two tropical species, including Desmodium uncinatum which is closely related to intortum. Henzell's reasons for the small transfer he measured in the glasshouse probably explain this dis crepancy in part. The glasshouse plants were exposed to fewer checks in their growth due to defoliation, shading, wilting, pests, diseases, etc. and thus less death and decay of roots and nodules occurred. Also, leaching of nutrients from the foliage or from dead leaves was excluded from his measurements. 94
The observed activity of napier roots in the vicinity of decaying leaves and decaying centro nodules did not appear to be especially significant in terms of explaining the yield differences between the two grasses, or in the response of napier to different legumes. How ever, these phenomena did shed some insight into the mechanisms involved in the transfer observed for both centro and intortum in association with these grasses. The two suggested routes, i.e. through decomposing roots and nodules or through decomposing legume leaves may both be involved. These will be discussed later in more detail, but the fact that N transfer from centro to the grasses occurred only during the longest growing period suggests that for this species, decomposing leaves provided the more important means of transfer. Several significant seasonal trends were noted, but some of these are confounded with different lengths of growing period. Thus the low percentage of nitrogen in tops harvested at the February cutting reflects, - at least in part, the stemmy nature of the forage. This may have also affected the calculated values for nitrogen yield and nitrogen con tribution per week for the legume species since the summer harvest which gave the highest values also represented the shortest growing period. Nevertheless, a summer maximum for nitrogen fixation seems to be clearly indicated. Centro showed less seasonal variation than the other two species, but this may have been partly due to the sparse stands. A longer time would thus have been required for complete cover of the ground by this species, so that the equilibrium between new growth and senescence was achieved later in the growing period. Seasonal changes in the root N:top N ratios were related to several factors. Most of the increases in grass root N:top N ratios noted at 95 the August harvest are the result of low top N yields accompanied by rather constant root N levels. However, by the following February, much of the root N appears to have been utilized for top growth, resulting in low root N:top N ratios. The legumes, on the other hand, tended to accumulate root N during the summer months with the result that root N:top N ratios for the August harvest were higher. In addition, kaimi continued to accumulate root N during the Aug.-Feb. period, probably due to the longer period of uninterrupted vegetative growth. The low root N:top N ratios and the associated low levels of root N maintained by pangola indicated the value of this grass as a sensitive indicator of available soil N. This characteristic was subsequently utilized in the perfusion experiment discussed below. B. Pathways of Nitrogen Transfer. The limited data from analysis of percolate obtained from certain of the small plots provided some indications of a variable but occasionally significant release of nitrogen to the substrate by legume roots immediately after defoliation of the plants, especially centro and intortum. This phenomenon was clearly confirmed in the root perfusion experiment. While the amounts of nitrogenous compounds equilibrating with perfusates were small and somewhat variable, a peak in the con centrations of both ammonium and amino N after one or more of the harvests was found for each species, indicating relatively higher concentrations of mobile N in the root systems at this time. This is probably the result of a relatively constant rate of proteolytic activity in the roots, but a temporarily curtailed N requirement for growth. Proteolysis may be stimulated at this time also, but no 96 information on this point was obtained. The fact that ammonium N appeared in the perfusing solutions only after defoliation and was of short duration suggests that this compound accumulated in the roots when growth was interrupted. This may be ex plained either on the basis of accumulating products of continued symbiotic N fixation, or by proteolysis and subsequent hydrolysis of proteinaceous plant materials, or both may be involved. Deamination of the amino compounds present in soil solutions, has been shown to be rapid (28, 38, 68, 74), and some conversion'of the amino compounds liberated by legume roots probably did occur, especially when larger amounts were released. The work of Pfaff (46) with lysi meters, however, indicates that ammonia produced in this way as well as any direct loss of ammonium or nitrate ions by the roots would rarely be lost from the plant-soil system if a dense root system were present. The amounts of N which the legumes released in excess of plant requirements during this time were estimated by the uptake of pangola plants in series-culture. The grasses, by maintaining solution N levels at the constantly low leve~,.promoted movement of excess mobile N out of the legume roots, and this N was then accumulated by the grass much as it would under field conditions. The data indicate that the release of nitrogenous compounds immediately after defoliation can be a small but significant pathway of N transfer, depending on legume species and the vigor of the individual plants. The N released by even the more vigorous centro and intortum plants is not enough however to explain completely the N transfer in the plots unless stand densities considerably higher than four square feet per plant are assumed. It is therefore doubtful that this pathway of transfer accounted for more 97 than one-third of the total transfer which occurred in the small plots. The more vigorous intortum plant and the centro plants also released small amounts of N during a three-week period of vegetative growth. If this process were to be continuous over a long period of time, it also could account for a significant transfer of N. The occurrence of higher concentrations of nitrate and amino N in perfusates sampled after a short period of iron deficiency suggests that excess mobile N is also present in legume root systems after growth is checked by factors other than defoliation. In addition to shading (Butler, ~ al., 9), probably wilting, mechanical damage, cold damage, and the effects of pests and diseases could affect the equilibrium between N mobilization and utilization such that excess nitrogen would be liberated by the root systems into the substrate. The larger amounts of nitrogen removed from the legume roots when the solution concentrations were maintained at low levels by the in clusion of a grass in the system indicates that the movement of nitro genous compounds out of the root is largely due to mass action. There fore, if the soil solution under field conditions was already high in these constituents due to fertilization or the mineralization of organic matter, transfer from legumes to grasses by this pathway would probably be considerably reduced. The differences between individual plants of the same species were striking in the case of kaimi and intortum. The amounts of N supplied by their respective root systems was directly related to the relative vigor of the plants. The more vigorous plants not only utilized more N themselves in rapid regrowth, but released a larger percentage of the total mobilized N to the perfusion system than their less vigorous 98
counterparts. This may be due to a larger root surface for mass action, larger root carbohydrate or protein reserves, faster rates of N fixation or proteolysis, or any combination of the above factors. Apparently, however, the factors responsible for legume vigor and rapid regrowth were also responsible for the larger amounts of N available for transfer. The picture was slightly different in the case of centro where the two plants lost similar amounts of N to associated grasses, but one plant supplied somewhat more N to its regrowing tops than the other. This probably was the result of small differences in the root surface area available for extraction of N compounds, accompanied by larger differences in the amounts of N mobilized by the two plants. The results of the leaf nitrogen experiment show that very small proportions of the leaf N of the three legumes tested could be removed by distilled water extraction. The percentages of leaf N leached from very young or from old yellowing leaves were higher, but the low N content of these leaves largely counterbalanced the higher percentage extracted. However, levels of extractable N, especially amino N, reached a high peak in young expanding centro leaves and also in yellowing intortum leaves. These stages coincided with either rapid gains or losses in the content of total N in these leaves, and are probably due to high concentrations of mobile N. Amino N was also the only leaf N constituent which was markedly affected by shading. Shaded leaves from intortum stems contained levels of extractable amino N double those of leaves from the same stems which were exposed to bright sunlight, or from stems of plants grown in a sunny location. More amino N was also extracted from kaimi plants 99 receiving moderate shade, but this was more evident in the older leaves where self-shading added to the general shade present. No affect of self-shading on older leaves was noted for plants in light shade or bright sunlight, however. Extractable amino N was also relatively higher in intortum leaves than in leaves of kaimi or centro. Since the leaves of intortum were softer and more succulent, this may have been caused in part by more extensive crushing of the leaf tissue during extraction.' Amino N was lowest in centro leaves, which were thinner and tougher than intortum leaves, but seemed to be more succulent than leaves of kaimi. Both kaimi and centro contained relatively high levels of extractable ammonium N, however. The rather uniformly low levels of extractable nitrate in leaves of all three species indicate that losses of N in this form are insignificant. For the three forms of N combined, the significance of N losses from legume leaves by the action of water is still slight. The largest percentage of leaf N was lost ~y intortum, i.e. less than 0.7%. Younge, et al. (80) state that the leaf portions of kaimi and intortum contain 60-80% of the N present in the forage. This would amount to an average of 70 pounds of leaf N per acre of mature intortum plants, based on the small plot yield data. Assuming that a heavy rain would remove the same amounts of nitrogen from the foliage as did the distilled water extraction, less than 0.5 pound of N would be washed from the intortum leaves per storm. The amounts leached from foliage of the other two species would be correspondingly lower. Although nitrogen lost by this means may be significant in areas of frequent heavy rains, it still does not entirely account for the nitrogen transfer observed in the small plots. 100
The amounts of nitrogen lost from legume plants by leaf-fall provides a third pathway of N transfer. The amounts of leaf N returned to the root-soil system by this means was shown to be significant for plants which are allowed to grow beyond the point where leaf fall begins to match leaf production. Significant losses would also be expected where heavy leaf fall due to other causes such as drought, pest damage, etc. occurred. This pathway seems especially important for centro, and would account for the fact mentioned earlier that transfer from centro to either grass was noted only after a prolonged growing period. Adopting the average forage N yields obtained from the small plots, and assuming that leaf N amounted to 70% of the N in the harvested forage, it is possible to account for weekly N losses of 0.2 pounds per acre for kaimi, 1.2 pounds per acre for centro and 1.3 pounds per acre for intortum. At this rate, a large portion of the transfer from centro and intortum plants in the small plots could be accounted for by several weeks of leaf fall at the maximum equi librium rate. In the case of centro, the average N yield and thus the estimated losses from leaf fall are lower if the legume-alone treat ment is excluded. However, this is partly compensated for by the fact that more than 70% of the total forage N of this species can probably be assumed to be present as leaf N. An appraisal of the relative importance of the three pathways studied was made possible by compiling the various estimates of transfer into Table XXI. Three things are clearly apparent from these data: (a) transfer of N by kaimi was small by all three pathways, (b) transfer from centro plants probably occurs mainly as a result of leaf-fall, and (c) intortum has a high capacity for transferring N by all three pathways. 101
TABLE XXI. ESTIMATED THANSFER OF NITROOFN FROM LEGUMES TO ASSOCIATED GRASSES BY THREE DIFFERENT PATHWAYS. Legumes N Transferred, pounds per acre Species From roots From leaves From leaf-fall (per cutting) (per storm) (per week)
Kaimi < 0.1 Intortum 0.1-0.5 0.5 1.3 In actual field practice, however, transfer of N from legumes to grasses by the three pathways studied will be greatly affected by management. On the basis of the results discussed above, a number of statements on this problem are possible. 1. The selection of legume species is important. Also, more vigorous strains of Desmodium spp. not oru¥ give higher protein yields, but liberate more N from their roots than weaker strains. The selection of a companion grass may also affect protein yields, especially in the case of kaimi mixtures. 2. The proportion of grasses to legumes may have several effects: (a) total protein yields increase with an increasing proportion of legumes, (b) shading of the legume by excessive grass growth could result in more N transfer to the grass due to leaching of N (especially amino N) from the shaded legume leaves, but this would probably be counteracted by (c) the interception of rainfall by the grass leaves. If the shading is severe, the resulting curb in legume growth may also induce transfer due to losses of N from the legume roots. 3. Defoliation by cutting or grazing has important implications for N transfer. Severe defoliation causes N to be released from the 102 roots and nodules of vigorous legume plants. Also, even light defo liation will tend to increase the proportion of very young leaves which, especially in the case of centro, are more susceptible to leaching losses and are exposed to the direct action of rainfall. Lack of defoliation for a long period, on the other hand, will result in more self-shading of older leaves (allowing more amino N to be leached from these leaves) and will allow leaf fall to reach signi ficant proportions. 4. Grazing will not only cause the defoliation effects already mentioned, but will result in the trampling injury of much vegetation not actually ingested. Leaves killed by trampling would be expected to supply much more N to the root-substrate complex than leaves which senesced normally. Torn vegetation would probably be susceptible to greater leaching losses than intact leaves. Return of animal manures also provides a very important pathway of N transfer, but this aspect is outside the scope of this study. 5. Soil and climatic conditions. The frequency of rainstorms will directly affect the losses of N due to the leaching of legume leaves. In addition, the depth of rooting of the different species, as determined by soil and water conditions will determine in part the relative advantage of the grasses and legumes in recovering N liberated by the legume, whether from the foliage or from the roots and nodules. SUMMP.rlY AND CONCLUSIOOS A small plot experiment designed to evaluate the capacity of three tropical forage legumes to fix nitrogen and to supply nitrogen to two associated grasses was established under continuously moist climate near Hilo, Island of Hawaii. Kaimi clover (Desmodium canum), centro (Centrosema pUbescens), and intortum (~. intortum) were grown alone, and together with pangola grass (Digitaria decumbens) and napier grass (Pennisetum purpureum) in all combinations. The two grasses were also grown alone. Plantings were made in 1962 in 4'x4' and 4'x8' plots which were lined with polyethylene film and filled with fresh volcanic cinders. Dry matter and N yields of the forage as well as root N levels were determined at each of three harvests. One additional harvest, in which only dry matter yields were measured, was taken later. Due to the severe N stress on the grasses throughout the experi ment, a large proportion of the dry matter yields and most of the N yields were harvested in the legume component. The grasses thus averaged about 4,400 pounds of dry matter per acre over a twelve month period, while the legumes yielded on the average: kaimi, 3,530; centro, 6,720; and intortum, 16,710 pounds per acre during the same period. The results indicated that intortum fixed approximately 340 pounds of N in 12 months, based on both forage N yields and root N, and that about 17.8 pounds of this, or 5%, w~re transferred to napier growing in association with it, and about 9 pounds, or 2.7% of, fixation, were transferred to associated pangola. The high nitrogen-fixing capacity 104 of this legume would seem to justify its broader utilization in tropical pastures, even though different and more rigid management practices were required. Centro fixed approximately 240 pounds of N per acre when grown alone, but ~n association with grasses, fixation dropped to a little over 110 pounds per acre on the average. Some transfer of N from ' centro to the grasses was noted during the longest (6 months) growing period, amounting to 6.5 pounds per acre to napier (11% of the N fixed) and 3.4 pounds per acre to pangola (6.7% of fixation). Although this legume is very promising as a nitrogen-fixer, its behavior in grass mixtures needs further study. Nitrogen fixation by kaimi was low, averaging about 82 pounds per acre per twelve months, with considerable variation between treatments. Thus, only 42 pounds of N were fixed by kaimi mixed with pangola, but in the napier mixture, 122 pounds of N were fixed. No evidence of nitrogen transfer from kaimi to either grass was obtained; on the contrary, yields of grasses associated with this legume were depressed. The value of this legume in supplying nitrogen to tropical pastures is therefore questionable. Root nitrogen was low for all species except intortum, which at the last harvest had nearly 40 pounds of N per acre in its sub-aerial portions. At the same time, the grasses averaged less than 5 pounds of root N per acre, but some contributions to grass root N levels by associated centro and intortum plants were noted. The nitrogen transfer which occurred was small but significant. Information as to the pathways by which transfer occurred was obtained from analyses of percolate obtained from certain plots, and also from 105 the results of two experiments conducted in the glasshouse. In the first of these studies, losses of N from legume roots were estimated for plants grown in percolator tubes containing inert cinders and perfused with nitrogen-free nutrient solution. In the second study, the amounts of nitrogen which could be.washed from legume leaves of different ages were estimated by distilled water extractions. Losses from leaf-fall were also evaluated. That legume roots release measurable quantities of nitrogen to the substrate was shown both by the percolate analyses from the small plots and the perfusate analyses from individual plants. The latter clearly showed a marked rise in levels of ammonium and amino nitrogen immedia~e1y after defoliation. A small rise in amino N levels also occurred concurrently with iron-deficiency symptoms. Nitrate N was relatively low at all times. The amounts of nitrogen which equi1ib- rated with the perfusing solution indicated the relative concentrations of mobile nitrogenous materials in the roots, but could not quanti- tative1y estimate the N available for transfer. When the roots of N- starved pango1a plants were placed in series-culture with the legumes, however, much larger amounts of N moved into the continuously N-defi- cient solution and were taken up by the grass. During normal vegetative growth, little or no N was transferred from legume to grass roots. However, after defoliation, measurable amounts of N were supplied to the grasses by all of the legume plants. The kaimi plants transferred less than 2% of the small amounts of nitrogen mobilized in their roots for regrowth. Centro plants mobilized over twice as much N, and 2.0- 3.2% of it was transferred to the grass. The two intortum plants were ~ both active in supplying N for regrowth and transfer, but the proportion 106 transferred was much higher for the more vigorous plant. This plant transferred over 20 mg of N or 9% of the total N mobilized during the three week period following defoliation. At an assumed stand of 11,000 plants per acre, this would amount to approximately 0.5 lbs·. transferred per acre by intortum and less than one-fourth of this amount for the other two species teste9, even if a stand of 33,000 plants per acre were assumed in the case of kaimi. The amounts of N extracted from living leaves were small. Ex tractable amino N tended to be high in intortum leaves, in shaded leaves, and in yellowing leaves. Centro leaves were moderately high in ammonium N, and expanding young leaves of this legume were also high in extractable amino N. Kaimi leaves were fairly high in extractable ammonium N and, where shaded, high in amino N. The combined totals for all forms of extractable N comprised only 0.4-0.7% of the total leaf N, or an estimated maximum of 0.5 pounds per acre. Leaching losses from green leaves were thus considered to be of some signi ficance, particularly where heavy rains were frequent; but this type of loss probably accounted for only a small portion of the transfer observed in the plot experiment. Leaf fall accounted for somewhat larger losses of N from those plants in which leaf senescence equalled the rate of production of new leaves. Under these conditions, and assuming that 70% of the average nitrogen yield obtained in the plots was in the leaves, estimated weekly losses due to leaf fall for the three legumes were: kaimi, 0.2; centro, 1.2; and intortum, 1.3 pounds of N per acre per week •. Where plants are allowed to reach this equilibrium situation and then continue growing for a time, or where heavy leaf-fall occurred due to other 107 causes, losses by this means would be substantial. 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