This dissertation has been

microfilmed exactly as received 66-13~715

WHITNEY~Arthur Sheldon~ 1933-NITROGEN FIXATION BY THREE TROPICALFORAGE LEGUMES AND THE UTILIZATION OFLEGUME-FIXED NITROGEN BY THEIR ASSOCI­ATED GRASSES.

University of Hawaii~ Ph.D.~ 1966Agriculture~ 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, ChairmanBruce J. CooilRobert L. FoxLeslie D. SwindaleGoro 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 MethodsResults

• ••••••••••••••••••••• e.•••••••••••••••• 1625

•••••••••••••••••••••••••••••••••• oo ••••••••• ~.e •••••

ROOT PERFUSIOOMaterialsResults

EXPERIMfNTand Methods · - . 62

70

.....................................................LEAF NITROOfN EXPERIMENT

Materials and MethodsResults

• ••••••••••••••••• lit •••••••••••••••••••• 8182

DISCUSS100 ........................................................ 91

SUMMARY AND CCNCLUSlOOS ........................................... 103

APPENDIX •••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 108

LITERATURE CITED .................................................. 109

vi

LIST OF TABLES

TABLE I.

TABLE II.

TABLE III.

TABLE IV.

TABLE V.

TABLE VI.

TABLE VII.

TOTAL DRY MATTER YIELDS FOR GRASSES, LEGUMES, ANDMIXTURES ••••••••••••••••••••••••••••••••••••••••••••• 30

DRY MATTER YIELDS OF LEGUMES, ALCNE AND IN ASSOCIATIONWITH GRASSES ••••••••••••••••••••••••••••••••••••••••• 34

DRY MATTER YIELDS OF GRASSES, ALONE AND IN ASSOCIATlOOWITH LEGUMES • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •• 35

ANALYSIS OF VARIANCE OF DRY MATTER YIELDS •••••••••••• 36

DRY MATTER PRODUCTlOO PER WEEK BY lWO GRASSES ANDTHREE LEGUMES •••••••••••••••••••••••••••••••••••••••• 39

PERCa-JTAGE OF NITROGEN IN TOP GROWTH OF GRASSES ANDLEGUMES ••••••••••••••••••••••••••••••••••••••••••••••• 40

TOTAL NITROGffi YIELDS FOR GRASSES, LEGUMES, ANDMIXTURES ••••••••••••••••••••••••••••••••••••••••••••• 43

TABLE VIII. NITROGa-J YIELDS OF LEGUMES, ALOOE AND IN ASSOCIATIONWITH GRASSES ••••••••••••••••••••••••••••••••••••••••• 44

TABLE IX. NITROG~ YIELDS OF GRASSES, ALOO E AND IN ASSOCIATIOOWITH LEGUMES ••••••••••••••••••••••••••••••••••••••••• 46

TABLE X. ANALYSIS OF VARIANCE OF NITROGa-J YIELDS •••••••••••••• 47

TABLE XI. NITROGEN YIELD PER WEEK BY THREE LEGUMES, AVERAGE OFTHREE 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.

TABLE XVI.

LEGUME CONTRIBUTION TO TOTAL N YIELDS •••••••••••••••• 55

LEGUME N ())NTRIBUTION PER WEEK TO YIELDS OF TOPGROWTH ••••••••••••••••••••••••••••••••••••••••••••••• 56

TABLE XVII. LEGUME N COO'TRIBUTION REFLECTED IN THE TOTAL ROOTN LEVELS•••••••••••••••••••••••••••••••••••••••••••••• 56

TABLE XVIII. NITROGa-J RELEASED TO PEROOLATE IN CERTAIN LEGUMEPLOTS •••••••••••••••••••••••••••••••••••••••••••••••• 60

TABLE IXX.

TABLE XX.

TABLE XXI.

vii

LEGUME AND GRASS YIELDS FROM PERFUSED GINDER CULTUREIN lHE GLASSHOUSE •••~.................................71

NITROGEN CXlNSTlTUENTS EXTRACTED FROM DIFFERENT SERIESOF LEAF SAMPLES ••••••••••••••••••.••••••••••••••••••• 89

ESTIMATED TRANSFER OF NITROGEN FROM LEGUMES TO ASSO­CIATED 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 CENTRONODULES •••••••••••••••••••••••••••••••••••••••••••••••• 26

FIGURE 4. DRY MATTER YIELDS PER ACRE OF GRASSES, LEGUMES, ANDMIXTURES •••••••••••••••••••••••••• So • • • • • • • • • • • • • • • • • • •• 31

FIGURE 5. NITROOEN YIELDS PER ACRE OF GRASSES, LEGUMES, ANDMIXTURES ••••••••••••••••••••••••••••••••••••••••••••••• 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 LEGUMEROOTS • • • • • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • • • • • • • • • • • • • •• 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 KAIMILEAVES OF DIFFERENT AGES•••••••••••••••••••••••••••••••• 83

FIGURE 13. TOTAL NITROGEN AND EXTRACTABLE NITROGEN IN CENTROLEAVES OF DIFFERENT AGES ••••••••••••••••••••••••••••••• 84

FIGURE 14. TOTAL NITROGEN AND EXTRACTABLE NITROGEN IN INTORTUMLEAVES OF DIFFERENT AGES ••••••••••••••••••••••••••••••• 85

FIGURE 15. TOTAL NITROGEN AND EXTRACTABLE NITROGEN IN LEAVES OFTHREE LEGUME SPECIES ••••••••••••••••••••••••••••••••••• 87

FIGURE 16. SUMMARY OF WEATHER roJDITIOOS AT WAIAKEA FARM, ISLANDOF 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 680 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

No-Grass

Replication I

Pangola Napier Pangola

Replication II

No-Grass Napier.. ,., r ...

K No C In C No K In

~K No In C

Replication III

f#'

Y "" UOJ

K In C No C No K In

~No C In K

Replication IV

No-Grass Pangola___..JA AF Y"" "'\

III

KEY: In: Desmodium intortum ("intortum").K: Desmodium canum (Kaimi clover).c: Centrosema~scens.

No: No legume.

Napier_---JA.----_r 1

In I No I K K I C No I In I K I In

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

CoC12, 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

FIGURE 2. ROOT SAMPLING CYLINDER FOR SMALL PLOTS.

21

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

30 ml of O.l~ 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

. I,.\ "

-".

.......... !.:.. '"' ..

'.: ..-.: ~~.' l .~..~ "~.~ .",,:, "': . ;'

- c.· .......... ;., " !

26

FIGURE 3. NAPIER ROOTS PROLIFERATINGIN 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 acreMay August Feb. JUly1963t 1963t 1964:1: 1964:1: Average

A. Individual treatment effects.*

Pangola Alone 2810 be 250 a 1660 ab 1980 ab 1670 aNapier Alone 2610 be 430 ab 1120 a 480 a 1160 a

Kaimi Alone 690 a 1050 abc 1410 a 2350 ab 1370 aKaimi + Pangola 2150 ab 700 ab 2300 ab 2450 ab 1900 aKaimi + Napier 4010 cd 1960 cd 2950 ab 3670 abc 3150 b

Centro Alone 2180 ab 2320 d 6470 bed 2010 abc 3440 bCentro + Pango1a 2820 be 1570 bed 4790 abc 3890 abc 3270 bCentro + Napier 3090 be 1520 bed 4730 abc 2410 ab 2930 b

Intortum Alone 3740 d 4410 e 10500 e 5750 bed 6100 cIntortum + Pango1a 4760 d 4200 e 12160 e 6610 cd 6930 cdIntortum + 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 aPango1a 3140 b 1680 b 5230 a 3730 a 3440 aNapier 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 aKaimi 2290 a 1240 b 2220 a 2820 a 2140 bCentro 2690 a 1800 b 5330 b 3040 a 3220 cIntortum 5180 b 4490 c 10500 c 6910 b 6770 d

* Means in the s~me column followed by the same letter are not signifi­cantly different at the 5% level.

t Means of four observations per harvest.

:I: Means of two observations per harvest.

L_ •

POUNDSDRY

MATTERPER

YEAR

20.000

15.000'

10.000

5.000

~ PANGOLA

~ NAPIER

R LEGUMES

GRASS KAIMIALONE

CENTRO INTORTUM

31

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 ANDIN ASSOCIATION WITH GRASSES.

Treatments Dry matter yields, pounds per acreMay August Feb. JUly1963t 1963t 1964:1: 1964:1: Average

A. Individual treatment effects.*

Kaimi Alone 690 ab 1050 a 1410 a 2350 ab 1370 bKaimi w!Pango1a 340 a 490 a 1310 a 1480 a 910 aKaimi w/Napier 1990 be 1310 ab 1990 a 3160 a 2110 c

Centro Alone 2180 bc 2320 cd 6470 ab 2870 ab 3460 dCentro w!Pango1a 750 ab 1010 a 2990 a 2080 ab 1710 beCentro w/Napier 830 ab 900 a 2710 a 1260 abc 1430 be

Intortum Alone 3740 de 4410 e 10500 b 5750 bc 6100 eIntortum w!Pango1a 2930 cd 3320 de 10540 b 4610 abc 5350 eIntortum w/Napier 5030 e 3460 e 6190 ab 6830 c 5380 e

Ave. for Legumetreatment 2050 2030 4900 3380 3090

B. Means for gr.ass effects.*

No Grass 2200 ab 2590 b 6130 a 3650 a 3640 bPango1a 1340 a 1610 a 4950 a 2720 a 2660 aNapier 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 aCentro 1250 a 1410 a 4060 a 2070 a 2200 bIntortum 3900 b 3730 b 9080 b 5730 b 5610 c

* Means in the same column followed by the same letter are not sig­nificantly 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 ANDIN ASSOCIATION WITH LEGUMES.

Treatments Dry matter yields, pounds per acreMay August Feb. July1963t 19631' 1964:1= 1964:1: Average

A. Individual treatment effeets.*

Pangola Alone 2810 a 250 a 1660 ab 1980 a 1670 ePangola w/Kaimi 1810 a 210 a 1000 a 970 a 1000 aPangola w/Centro 2070 a 560 b 1810 abc 1810 a 1560 bePangola w/lntortum 1820 a 880 e 1610 ab 2030 a 1590 be

Napier Alone 2610 a 430 ab 1120 ab 480 a 1160 abNapier w/Kaimi 2020 a 650 be 950 a 520 a 1040 aNapier w/Centro 2260 a 620 be 2020 e 1150 b 1510 beNapier w/lntortum 2030 a 1390 d 2650 e 1530 b 1900 e

Ave. for Grasstreatments 2180 620 1600 1310 1430

B. Means for grass effeets.*

Pango1a 2130 a 470 a 1520 a 1700 a 1460 aNapier 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 bKaimi 1920 a 430 be 980 a 740 a 1020 aCentro 2170 a 590 e 1910 be 1480 a 1540 beIntortum 2180 a 1130 d 2130 e 1780 a 1750 e

* Means in the same column followed by the same letter are not sig­nificantly 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 SWIaresMay 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 3,566Harvests 3 1,309,038**T X H 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**T X H 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 590Compan. Leg. 3 30,979 28,015** 30,438** 21,589 42,952**G X L 3 2,293 2,420 11,702 6,662 13,337Harvests 3 367,015**T X H 21 15,463*Error 12,777 961 3,955 17,338 7,813

Error d.f. 24 24 8 8 64 w0'

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 slowL-

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 78Napier 139 77 65 ~ 80Ave. for grasses 136 62 62 57 79

Kaimi 63 95 60 101 80Centro 79 141 156 83 116Intortum 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 aPango1a w/Kaimi .37 .49 .37 .41 aPango1a w/Centro .36 .53 .42 .44 aPangola w/Intortum .62 .61 " .58 .60 b

Average .42 .53 .45 .46

Napier Alone .43 .61 .40 .48Napier w/Kaimi .41 .60 .40 .47Napier w/Centro .41 .68 .36 .48Napier w/Intortum ~ .56 .41 -d§

Average .43 .61 .39 .48

Kaimi Alone 2.02 2.33 1.38 1.91Kaimi w/Pango1a 1.84 2.42 1.33 1.86Kaimi 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 2.49Centro w/Pango1a 3.14 2.80 b 1.34 2.43Centro 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 2.06Intortum w/Pangola 2.19 2.30 1.10 1.86Intortum wjNapier 2.55 2.33 1.49 2.12

Average 2.41 2.32 1.31 2.01

Ave: 'Grasses .425 .57 .42 .47Ave: Legumes 2.43 2.48 1.48 2.13Overall Average 1.44 1.58 .96 1.30

ANOVA mean squares

Source Grasses Legumesd.f. Pango1a Napier d.f. Kaimi Centro Intortum

Companion spp. 3 .0518** .0001 2 .0045 .0783 .1086Harvest 2 .0264* .1085** 2 1.6488** 2.3796* 2.2263**S X H 6 .0043 .0039 4 .0159 .1216 .0218Error 12 .0022 .0029 9 .0345 .1284 .0686

* Means of two observations per treatment per harvest. Letters followonly 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,

~ PANGOLA

300 ~ NAPIER

n LEGUMES'I

~. : I

200POUNDS N

PERYEAR

100

42

GRASS KAIMIALONE

CENTRO INTORTUM

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 acreMay August February

1963t 1963t 1964:1: Average

A. Individual treatment effects.*

Pangola Alone 9.0 a 1.3 a 6.7 a 5.7 aNapier 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 abKaimi + Pangola 13.2 a 13.0 ab 22.0 a 16.1 aKaimi + 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 dCentro + Pangola 30.1 a 32.0 b 60.4 abc 40.8 cCentro + 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 eIntortum + Pangola 82.0 c 82.4 cd 147.5 d 104.0 eIntortum + Napier 130.0 d 88.8 cd 91.4 bcd 103.4 e

B. Means for grass effects.*

No GrassPangolaNapier

56.0 b33.7 a55.6 b

64.9 b32.2 a37.9 a

90.5 b49.2 a45.7 a

70.5 b41.7 a37.0 a

C. Means for legume effects.*

No LegumeKaimiCentroIntortum

10.1 a24.3 ab43.1 b

100.7 c

2.0 a24.6 b41.2 c91.2 d

5.5 a24.0 a77.1 b

125.5 c

5.9 a24.3 b53.8 c

105.8 d

D. ANOVA mean squares

Source

TreatmentsGrassesLegumesGXLHarvestsT X HError

Error d.f.

d.f.

103252

20

May1963

174.93**167.68**479.27**

neg.

15.3833

August1963

142.76**111.85**415.19**

neg.

7.7633

February1964

152.61**98.16*

433.82**5.67

21.1011

AllHarvests

426.95**197.29**

1,286.60**15.0780.17*21.6812.93

77

* 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.

44

TABLE VIII. NITROOEN YIELDS OF LEGUMES, ALONE ANDIN ASSOCIATION WITH GRASSES.

Treatments N yields, pounds per acreMay August Feb.1963t 1963t 1964:1: Average

A. Individual treatment effects.*

Kaimi Alone 13.1 a 25.3 b 19.3 a 19.3 aKaimi w/Pangola 6.5 a 11.9 a 17.9 a 12.1 aKaimi 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 bCentro w!Pangola 22.4 a 29.2 b 53.0 ab 34.9 aCentro 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 cIntortum w!Pango1a 70.7 bc 77.0 cd 138.1 c 95.3 bcIntortum w/Napier 121.2 d 80.3 cd 81.1 ab 94.2 bc

Average for legumetreatments 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 aCentro 37.3 a 39.0 b 72.0 b 49.6 bIntortum 94.0 b 86.5 c 118.9 c 99.8 c

C. Means for grass effects.*

No GrassPango1aNapier

56.0 b33.2 a61.2 b

64.9 b36.0 a44.3 a

90.5 a69.7 a52.1 a

70.5 b47.4 a52.5 a

§ D~ffers significantly from the other two kaimi treatments whentested by within-kaimi error terms.

* Means in the same column followed by the same letter are not sig­nificantly 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.

TABLE IX. NITROGEN YIELDS OF GRASSES, ALONE ANDIN ASSOCIATION WITH LEGUMES.

46

Treatments N yields, pounds per acreMay August Feb.1963t 1963t 1964* Average

A. Individual treatment effects.*

Pangola AlonePangola w/KaimiPangola w/CentroPangola w/Intortum

Napier AloneNapier w/KaimiNapier w/CentroNapier w/Intortum

Average for grasstreatments

9.0 a6.7 a7.7 a

11.3 b

11.2 a8.7 a9.2 a8.8 a

9.1

1.3 ab1.1 a2.8 be5.4 d

2.6 abc4.0 cd3.8 c8.5 e

3.7

6.7 ab4.1 a7.3 ab9.4 c

4.4 a3.7 a7.9 b

10.3 b

6.7

5.6 ab3.8 a5.9 b8.7 cd

6.1 b5.5 b7.1 be9.2 d

6.5

B. Means for grass effects.*

PangolaNapier

8.6 a9.6 a

2.6 a4.7 b

6.8 a6.5 a

6.1 a7.0 b

C. Means for legume effects.*

No LegumesKaimiCentroIntortum

10.1 a7.7 a8.7 a

10.0 a

2.0 a2.5 a3.3 a6.9 b

5.5 ab3.9 a7.6 b9.8 be

5.9 b4.6 a6.5 b8.9 c

* Means in the same column followed by the same letter arenot 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 SquaresMay August Feb. All1963 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 9.74Error 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**G X L 3 1.99** .06 .07 .54**Harvests 2 6.68**T X H 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 weekMay 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 CombinationsMay1963

Root N:Top N ratio*

Pangola AlonePangola w/KaimiPangola w/CentroPangola w/lntortum

Average

Napier AloneNapier w/KaimiNapier vi/CentroNapier vi/lntortum

Average

Kaimi AloneKaimi w!PangolaKaimi w/Napier

Average

Centro AloneCentro w!Pango1aCentro vi/Napier

Average

Intortum AloneIntortum w/PangolaIntortum vi;Napier

Average

Average GrassesAverage Legumes

.61

.76

.61

.16

.54

.531.291.191.261.07

.44

.37

.47

.41

.ll

.14

.14

.13

.18

.23

.09

.17

.80

.24

5.342.741.87b..§Q3.19

5.553.843.452.233.77

.25

.45

.65- .45

.52

.42~

.49

3.48.54

.32

.31

.85

.33

.45

.45

.95

.80

.95-:79

2.39.71.93

1.34

.14

.16

.18-:T6

.21

.40....:.19

.34

.62

.61

2.09 b1.27 a1.11 a1.10 a1.36

2.182.031.811.481.87

1.18 b.54 a.73 a

-:BI

.17

.25

.32--:25

.30

.35

.34--:33

1.61.46

ANOVA mean squares for individual species.

Source Grassesd.f. Pangola Napier d.f.

Leguines

Companion spp.HarvestS X HError

326

12

1.57617.602*

2.094*.492

.52521.665**1.793

.753

2249

.668**1.385

.519**

.038

.0409

.1871*

.0208

.0217

.0045

.1602*

.0195

.0107

* Means of two observations per harvest. Letters follow only meansof 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 acreMay August Feb.1963 1963 1964 Average

A. Grass Component*

Pangola Alone 6.0 6.4 2.5 ab 4.9 aPangola w/Kaimi 6.4 3.1 1.1 a 3.5 aPangola wjCentro 5.8 4.7 5.9 d 5.4 aPangola 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 bNapier w/Kaimi 10.5 12.7 3.8 bcd 9.0 bNapier wjCentro 12.1 12.2 5.1 cd 9.8 bNapier 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 cdKaimi w/Pangola 2.8 8.4 12.2 a 7.8 aKaimi 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 bCentro w!Pangola 4.6 15.7 8.7 a 9.7 aCentro 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 eIntortum wjPangola 15.3 32.1 26.0 b 24.5 dIntortum 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 thesame letter are not significantly different.

TABLE XIV. NITROGEN CCNTAINED IN THE ROOTS OF GRASSES,LEGUMES, AND MIXTURES.

Species Combinations Total root N" pounds per acreMay August Feb.1963 1963 1964 Average

Pangola Alone 6.0 6.4 2.5 4.9Napier Alone 7.9 ~ -L.2 ~

Average 6.9 11.1 2.2 6.7

Kaimi Alone 7.1 16.5 45.8 23.1Kaimi + Pangola 9.2 11.5 13.3 11.3Kaimi + Napier ~ 29.0 29.1 26.7

Average 12.8 19.0 29.0 20.2

Centro Alone 9.4 21.2 14.7 15.1Centro + Pangola 10.3 20.4 14.6 15.2Centro + Napier 14.8 ~ 14.9 ll:.§.

Average 11.5 21.7 14.7 16.0

Intortum Alone 13.4 45.1 52.5 37.0Intortum + Pangola 17.3 43.1 29.0 29.9Intortum + Napier ~ 12:1. 47.0 .~

Average 17.5 54.4 42.8 38.3

Ave: Legume Alone 10.0 27.6 37.7 25.1Legume + Pangola 12.3 25.0 19.0 18.8Legume + Napier 19.6 42.6 30.3 30.8

,Overall Average 12.7 28.0 23.9 21.5

53

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 acreTop Growth

May August Feb. Annual Root Overall1963 1963 1964 Total Component Total

Kaimi Alone 3.0 23.3 13.7 40.0 43.6 83.6Kaimi w/Pangola 4.2 11.7 15.3 31.2 10.8 42.0Kaimi 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 12.5 241.6Centro w!Pangola 21.7 30.7 53.7 106.1 12.1 118.2Centro 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 50.3 362.7Intortum w!Pangola 73.0 81.1 140.8 294.9 26.6 321.5Intortum 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

U1U1

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 1.08Kaimi w/Pangola .26 1.17 .59 .67Kaimi w/Napier 2.21 3.30 1.02 &1.§Average .89 2.27 .71 1.29

Centro Alone 3.43 6.50 4.20 4.71Centro w!Pangola 1.31 .3.07 2.06 2.15Centro 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 6.70Intortum w!Pangola 4.56 8.ll 5.41 6.03Intortum w/Napier 7.42 8.62 3.35 6.46Average 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 acreMay August Feb.1963 1963 1964

Kaimi Alone .2 5.5 43.6Kaimi w/Pangola 3.2 5.1 10.8Kaimi w/Napier 14.1 13.2 27.2

Average ~ ~ ~

Centro Alone 2.5 10.1 12.5Centro w/Pango1a 4.3 14.0 12.1Centro w/Napier 6.9 7.8 13.0

Average 4:6 10:6 12.5

Intortum Alone 6.5 34.0 50.3Intortum w!Pangola 11.3 36.7 26.6Intortum 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)

C1'o

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.

,r::. - _.--

1 1

lb

63

FIGURE 6. DIAGRAM OF PERFUSION SUBSYSTEM FOR PERFUSION OF LEGUMEROOTS (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 CINDERCULTURE IN ruE GLASSHOUSE ..

Harvest Legume PlantsDate 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 36.36N content (%) 2.98 3.77 2.87 3.41 3.52 3.35N 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 35.95N content (%) 4.16 3.93 3.45 3.43 3.17 2.92N 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 21.90N content (%) 3.64 3.97 3.64 2.96 2.91 2.00N yield (g) .23 .14 .40 .42 .46 .44

June 18: D.M. yield (g) 5.70 4.00 12.40 16.90 20.45 39.20N content (%) 3.52 3.71 3.21 3.29 3.25 3.35N 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 7.19N content (%) 4.29 4.23 4.31 4.03 3.33 3.01N 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 7.30N 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 22.40N uptake (mg)* 1.60 .58 4.52 4.03 4.74 21.39

% of mobile legumeN 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, firstperiod, 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.

150 i. AMINO N::'. NH4 " N

NO.-N

100

KAIMI-I

"

...

700Cl KAIMI- 2 150

100

76

p.g NFROMROOTS

1£0 NFROMROOTS

1£0 NFROMROOTS

50

150

2

100

~

177

~II.m CENTRO-I

INTORTUM-I

2 f4 6

CENTRO-2

50

150

100

50

150

100

50

TIME IN WEEKS TIME IN WEEKS

FIGURE 9. NITROGEN LEVELS IN SOLUTIOOS AFTER PERFUSINGLEGUME 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

FIGURE 10. VIEW OF GRASSES GROWN IN SERIESWITII LEGUMES (FOREGROUND). CONCLUSION OF

FIRST TIIREE-WEEK PERIOD.

78

~rr·.4?'*A{*%;¥A; ~·;;·"·§#~~0T;A~¥!;1Yi'-S7;&'tst~~~m:.t:!,-.,~/YJ4,k'~'\.

·\;j'·:~.>:~G~i

FIGURE 11. VIEW OF GRASSES GROWN IN SERIESWITH LEGUMES (FOREGROUND). cc:NCLUSIOO

OF SECOND THREE-WEEK SERIES.

79

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

A. Bright sunlight.

200

... g Nextracted

per 8leaves 100

~ amino N_

NH4

N_N03 N

40

mgtotal Nper 8

leaves20

B. Light shade.

40

mgtotal Nper 8

20 leaves

200

""g Nextracted

per "8leaves 100

c. Moderate shade

40

mgtotal Nper 8

leaves20

200

...g Nextracted

.per 8leaves 100

2 4 6 8 10 12 14

Approximate leaf age in weeks.

FIGURE 12. TOTAL NITROOEN AND EXTRACTABLE NITROGmIN KAIMI LEAVES OF DIFFERENT AGES.

84

A. Bright sunlight.A---4 amino N"......;..0 NH4 N-N03 N

JIg Nextracted

per 6leaves

mgtotal N

per 620 leaves

B. Bright sunlight.

c. Bright sunlight.

mgtotal N

per 6leaves

20100

200

~g Nextracted

per 6leaves

"'g Nextracted

per 6leaves

40

mgtotal N

per 6leaves

20

2 4 6 8 10 12 14

Approximate leaf age in weeks.

FIGURE 13. TOTAL NITROOEN AND EXTRACTABLE NITROGENIN CENTRO LEAVES OF DIFFERENT AGES.

A. Bright sunlight.

200

..g Nextractedper 4leaves

100

B. Moderate shade.

~ amino N~NH4N

-N0

3N

40

mgtotal N

per 4leaves

20

85

Io/g Nextracted

per 4leaves

c. Partiallyself-shaded.

200

100

40

mgtotal N

per 4leaves

20

Jig Nextracted

per 4leaves

40

mgtotal N

per 420 leaves

2 4 10 12 1'1

Approximate leaf age in weeks

FIGURE 14. TOTAL NITROOEN AND EXTRACTABLE NITROGENIN 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

of shade was thus observed. However, it was noted that under severe

self-shading, the shaded leaves yellowed and died, regardless of age.

1_

A. Kaimi

flog Nextracted

per 8leaves

B. Centro

HNextracted

per 6leaves

C. Intortum

II-g Nextracted

per 4leaves

200

100

200

200

100

_ amino N

---- NH4 N••••••••• N03 N

""

~Shade '"

j sun '"

~ -----------~~........._---- ,~L:. _-_ _ _._ __ -

--- .

2 4 6 8 10 12 14

Approximate leaf age in weeks.

mgtotal N

per 8leaves

mgtotal Nper 6

leaves20

mgtotal Nper 4

leaves20

87

FIGURE 15. TOTAL NITROGEN AND EXTRACTABLE NITROOEN INLEAVES 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.

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 DIFFERENTSERIES 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 .27Series B .02 .21 .15 .38Series C .05 .21 .42 .68

Weighted Ave. :03 :T9 .21 .44

Centro, Series A .02 .21 .23 .46Series B .01 .24 .10 .35Series C .03 .30 .16 .49

Weighted Ave. .02 :25 .15 :42

Intortum, Series A .05 .14 ---.44 .64Series B .07 .14 .58 .79Series 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.

L _

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 FROMLEGUMES TO ASSOCIATED GRASSES BY

THREE DIFFERENT PATHWAYS.

Legumes N Transferred, pounds per acreSpecies From roots From leaves From leaf-fall

(per cutting) (per storm) (per week)

Kaimi < 0.1 <O~l 0.2

Centro 0.1 0.15 1.2

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.

None of the three pathways of N transfer which were investigated

could completely account for all the nitrogen transfer which was found

to occur under small-plot conditions. An adequate explanation, how­

ever, is provided by these three processes acting in combination. A

number of ways in which management and soil and weather conditions

would influence the factors involved in nitrogen transferred by these

means are briefly discussed.

108

APPENDIX

JUly1964

Jan.JUly1963

80

75

Mean 70Monthly

Temp.(Of) 65

60

55

400

MeanDaily 350

Radiation2gm cal/cm300

250

30

MonthlyRainfa1l 2O(inches)

10

0 NO DATAJUly Jan.

1962

FIGURE 16. SUMMARY OF WEATIiER aJNDITIONS ATWAIAKEA FARM, ISLAND OF HAWAII, 1962-1964.

109

LITERATURE CITED

1. Acharya, C. N., Jain, S. P. and Jha, J. Studies on the building

up of soil fertility by the phosphatic fertilization of legumes.

Influence of growing berseem on the nitrogen content of the soil.

I· Ind. Soc. §2i! §£i. 1:55-64. 1953.

2. Aseeva, I. V. and Kirillova, N. F. LAction of soil-dwelling

bacteria on free amino acid content of Leguminosae. (In Russian)-7.

Nauch. ~. Vyssh. Shkoly, BioI. Nauki No.1, 139-144. 1960.

(Soils and ~. Abstr. 14:3426).

3. Baitulin, I. O. LEffect of blue lucerne on the conditions of nitro­

gen nutrition of grasses grown with it. (In Russian)-7. Vestn.

Akad.~ Kazakh. SSR 17 (No.1) 53-59. 1961.

Abstr. 14:2206).

(Soils &Fert.--

4. Becker, G. E. and Schmidt, E. L. Excretion of amino acids by soil

and rhizosphere isolates. ~. f!2£. 1960: 29. 1960. (Soils!

~. Abstr. 23:1542).

5. Biswas, T. D. and Das, N. B. Amino acids in soils growing berseem.

I. !lli!. Soc.· §2i! Sci. 5:31-37, 1957. (~. Abstr. 51, 13288).

6. Block, R. J. and K. W. Weiss. Amino Acid Handbook. pp. 120-124.

Springfield, Ill. 1956.

7. Bowen, G. D. Field studies on nodulation and growth of Centrosema

pubescens Benth. gao 1. Agric. Sci. 16:253-265. 1959.

8. Butler, G. W. and Bathurst, N. O. The underground transference of

nitrogen from clover to associated grass. Proc. Seventh 1n!.

Grassld. Congo Session 4, No. 14: 1·2 pp. 1956.

110 .

9. Butler, G. W., Greenwood, R. M. and Soper, K. Effects of shading

and defoliation on the turnover of root and nodule tissue of plants

of Trifolium repens, Trifolium pratense and Lotus uliginosis.

~.~. 1. Agric. Res. 2:415-426. 1959.

10. Castle, M. E. and Reid, D. Nitrogen and herbage production. 1.

Brit. Grassld. Soc. 18:1-6. 1963.

11. Cowling, D. W. The effect of white clover and nitrogenous ferti­

lizer on the production of a sward. 1.~. Grassld. Soc. 16:

281-290. 1961.

12. Cowling, D. W., Green, J. O. and Green, S. M. The effect of white

clover and nitrogenous fertilizer on the production of a sward.

III. Statistical interpretation of their relative contributions.

~. Brit. Grassld. §2£. 19:419-424. 1964.

13. Dinchev, D. ~ature 'of the nitrogen fixed by nodule bacteria of

Phaseolus. (In Bulgarian)-7. Izv. Tsentral. Nauch.-izsled. Inst.

Pochvoznan. Agrotekh. "Pushkarov". 4:155-165. 1962. (Soils ~

~. Abstr. 26:1835).

14. Duncan, D. B. Multiple range and multiple f tests. Biometrics

11:1-42. 1955.

15. Ebertova, H. LAffiino acids in the nodules, aerial parts and roots

of soybean in symbiosis with Rhizobium japonicum. (In Czech. >-7.Sborn. ~. ~. Zemed.~. 33:1371-1382. 1960. (Soils ~

Fert. Abstr. 14:445).

16. Fergus, E. N. The place of legumes in pasture production. 1.

Amer. Soc. Agron. 27:367-373. 1935.

17. Fernando, G. W. E. Preliminary studies on the associated growth of

grass and legumes. Trop. Agr'st. 117:167-177. 1961.

III

18. Henzell, E. F. Nitrogen fixation and transfer by some tropical

and temperate pasture legumes in sand culture. Aust. 1. Expt.

Agr. Animal Husb. 2:132-140. 1962.

19. Herriott, J. B. D. and Wells, D. Clover nitrogen and sward

productivity. 1.~. Grassld. Soc. 15:63-69. 1960.

20. Holliday, R. and Willman, D. The effect of white clover, ferti­

lizer nitrogen and simulated animal returns on yield of grass­

land herbage. 1. Brit. Grass1d. Soc. 17:206-213. 1962.

21. Holmes, W. and Aldrich, D. T. A. Nitrogenous fertilizers and the

production of crops and grass. £!2£.~. Soc. 16:9-14. 1957.

22. Hosaka, E. Y. Pango1a and colonial guinea grass management-­

additional notes. Hawaii~. Circular 353. 1956.

23. Jackson, M. L. Soil Chemical Analysis. pp. 187-190. Englewood

Cliffs, N.J. 1958.

24. Ka1anis, A. The effect of nitrogen on the relationship of root­

nodule bacteria and legumes. Trudy~. ~.-kh.~. 12:29-42.

1963. (Soils! Fert. Abstr. 28:298).

25. Katzne1son, H., Rouatt, J. W. and Payne, T. M. B. The liberation

of amino acids and reducing compounds by plant roots. Plant and

Soil. 7:35-48. 1955.

26. Lipman, J. G. The associative growth of legumes and non-legumes.

lie Jersey Sta. Bull. 253. 1912.

27. Little, S., Vicente-Chandler, J. and Abruna, F. Yield and protein

content of irrigated napier grass, guinea grass and pango1a grass

as affected by nitrogen fertilization. Agron. 1. 51:111-113.

1959.

112

28. Loginow, W. The influence of humus substances on the process of

deamination. Roczn. Glebozn. 14 Supple 39-44. 1964. (Soils

~ Fert. Abstr. 28:781).·

29. Luh, T. H. and Ream, H. W. Grassland potential and current develop-

ment on Taiwan. Chinese-Amero JCRR Animal Ind. Series Bull. No.6.

1961.

30. Lyon, T. L. and Bizzell, J. A. A heretofore unnoted benefit from

the growth of legumes. ti. y. Expt. Sta •.Bull. No. 294:365-374.

1911.

31. MacGregor, J. M., Taskovitch, L. and Martin, W. P. Effect of

phosphate and potash on the amino acid content of alfalfa. Agron.

~. 53:215-216. 1961.

32. McAUliffe, C., Chamblee, D. S., Uribe-Arango, H., .et al. Influ-

ence of inorganic nitrogen on nitrogen fixation by legumes as

revealed' by N15. Agron. ~. 50:334-337. 1958.

33. McIlroy, R. J. Grassland improvement and utilization in Nigeria.

Outlook 2U Agric. 3:174-179. 1962.

34. Middleton, K. R. New Nessler reagent and its use in the direct

Nesslerization of Kjeldahl digests. 1. 8PP!. Chern. 10:281-286.

1960.

35. Moomaw, J. C. ao~ Takahashi, M. Initial response of pangola grass-

giant Spanish clover. pasture to -lime and fertilizer. Hawaii Farm

Sci. 7(2):4-5. 1958.

36. Moore, A. W. Symbiotic nitrogen fixation in a grazed tropical

grass-legume pasture. Nature (Lond.) 185:638. 1960.;

37. Moore, A. W. The influence of a legume on soil fertility under a

grazed tropical pasture. Emp.~. Exp. Agric. 30:239-248. 1962.

113

38. Moreau, R. Sur la destruction de quelques acides amines par la

microflore du sol. f.E. Acad. Sci. Paris. 252:154-156. 1961.

(Soils ~ Fert. Abstr. 24:1396).

39. Morrill, L. G. and Dawson, J. E. An improved percolation system.

~ Sci. Soc. Amer. Proc. 28:710-711. 1964.

40. Nishimura, S., Saito, Y., and Kijima, K. L!he effect of legumes

as nitrogen suppliers to associated Italian ryegrass. (In

Japanese)J. ~. Crop Sci. So.£. Japan. 28:377-379. 1960.

(Soils ~~. Abstr. 24:4~4).

41. Nordfeldt, S., Iwanaga, I., Tom, A. K. S. and Henke, L. A. Studies

of napier grass. Hawaii Agric. Expt. Sta. l!£h. Bull. No. 12.

1951.

42. Norris, D. O. Techniques used in work with Rhizobium. in~

concepts ~ methods·in sub-tropical pasture research. (Common­

wealth Bureau of Pastures and Field Crops Bulletin No. 47). p. 88.

Hurley. 1964.

43. Pate, J. S. Nodulation studies in legumes. II. The influence of

various environmental factors on symbiotic expression in the vetch

(Vicia sativa L.) and other legumes. Aust. 1. BioI. Sci. 11:496­

515. 1958.

44. Pearson, R. and Parkinson, D. The sites of excretion of ninhydrin­

positive substances by broad bean seedlings. Plant and §2il. 13:

391-396. 1961.

45. Peterson, M. L. and Bendixen, L. E. Plant competition in relation­

ship to nitrogen economy. Agron. 1. 53:45-49. 1961.

46. Pfaff, C. LThe behavior of ni~rogen in soil during long term

114

lysimetex txials. (In German)-7. Z. Ackex-u. PflBau. 117:77-

99. 1963. (Soils &Fert. Abstr. 26:1615).

47. Pleshkov, B. P. LEffects of nutrient conditions on the content

of free amino acids and the amino-acid content of proteins of

agricultural plants. (In Russian)-7. Izv. Timiryazev. ~.-Kh.

Akad. No.3, 141-151. 1964. (Soils ~~. Abstr. 27:3630).

48. Prevatt, R. W. and Engibous, J. C. Effect of rate and frequency

of nitrogen application in yield and quality of Pangola grass.

§£!! Crop §£!. Fla. Proc. 19:179-183. 1959.

49. Prianishnikov, D. N. Nitrogen in~ Life of Plants. Chapter 5.

Madison, (Transl. from Russian by S. A. Wilde, 1951).

50. Ratner, E.: I., Kolosov, I. I., Ukhina, S. F., Dobrokhotova, I. N.

and Kazuto, o. N. LJhe utilization by plants of amino acids as

the source of nitrogen. (In Russian)-7. Izvest. Akad. Nauk.

S.S.S.R. ~. ~. No.6, 46-82. 1956. (Soils ~~. Abstr.

201976).

51. Rewari, R. B., Sen, A. and Pandey, S. L. Excretion of N from the

roots of guar bean, Cyamopsis psoxalioides.

5:237-245. 1957.

J. Ind. Soc. Soil Sci.

52. Rovira, A. D. Root excretions in relation to rhizosphere effect.

IV. Influence of plant species, age of plant, light, temperature

and calcium nutrition on exudation. Plant and Soil. 11:53. 1959.

53. Sears, P. D. Pasture growth and soil fertility. VII. General

discussion of the experimental results and their application to

farming practice in New Zealand. ~.~.!.. Sci. Technol., ~. ~.

35:221-236. 1953.

54. Seeger, J. R. Le probleme de l'excretion d'azote par les

115

legumineuses. Premieres recherches en region equatoriale. Plant

and Soil. 14:277-288. 1961. (Soils!~. Abstr. 24:3255).

55. Shishchenko, S. V. LThe nitrogen utilization of mineral ferti-

lizers by the leguminous and graminaceous components of a mixed

sowing. (In Russian)-7. Khimizatsiya Sel. ~. Bashkir. 3:9-

11. 1964. (Soils! Fert. Abstr. 28:577).

56. Stenlid, G. Encyclopedia of Plant Physiology. 4:615-637. Berlin,

1958. (Cited by Wittwer and Teubner, 77).

57. Steward, F. C. and Street, H. E. The nitrogenous constituents of•

plants. ~. Rev. Biochem. 16:471-502. 1947.

58. Thompson, A. R. Chemical studies of the efficiency of legumes as

green manures in Hawaii. Hawaii Agric. Expt. Sta. Bull. No. 43.

1917.

59. Thornton, H. G. and Nicol, H. Further evidence upon the nitrogen

uptake of grass grown with lucerne. 1. Agric. Sci. 24:540-543.

1934.

60. Tukey, H. B., Jr., Tukey, H. B. and Wittwer, S. H. Loss of

nutrients by foliar leaching as determined by radioisotopes. f!2£.

~. ~. Hort.~. 71:496-506. 1958.

61. Uziak, Z. and Koter, Z. Llnvestigations on the free amino acids

of leguminous plants given different forms of nitrogen. (In Polish)-7.

Ann. Univ. M. Curie Sk10dowska. l7E 1962:51-65. 1963. (Soils

! Fert. Abstr. 27:1814).

62. Vicente-Chandler, J., Caro-Costas, R. and Figarella, J. The effects

of two heights of cutting and three fertility levels on the yield,

protein content and species composition of a tropical kudzu and

molasses grass pasture. Agron. J. 45:397-400. 1953.

116

63. Vicente-Chandler, J., Figarella, J., and Silva, S. Effects of

nitrogen fertilization and frequency of cutting on the yield and

composition of. pangola grass in Puerto Rico. J. Agric. ~.

Puerto Rico. 45:37-45. 1961.

64. Vicente-Chandler, J., Silva, S. and Figarella, J. Effects of

nitrogen fertilization and frequency of cutting on the yield and

composition of napier grass in Puerto Rico. J. Agric. Univ.

Puerto Rico. 43:215-227. 1959.

65. Virtanen, A. I. Cattle Fodder and Human Nutrition. Cambridge,

1938.

66. Virtanen, A. I. The biology and chemistry of nitrogen fixation

by legume bacteria. Biol.~. 22:239-400. 1947.

67. Virtanen, A. I., von Hausen, S. and Laine, T. Investigations on

the root nodule bacteria of leguminous plants. XX. Excretion

of nitrogen in associated cultures of legumes and non-legumes.

1. Agric.~. 27:584-610. 1937.

68. Vlassak, K. ~Ammonification and nitrification of amino acids.

(In Flemish)-7. Agricultura Louvain. 12:427-447. 1964. (Soils

~~. Abstr. 28:1035).

69. Walker, T. W. Sulfur responses on pastures in Australia and New

Zealand. Soils ~~. 18:185-187. 1955.

70. Walker, T. W., Adams, A. F. R. and Orchiston, H. D. Fate of labeled

nitrate and ammonium nitrogen when applied to grass and clover

grown separately and together. 22il Sci. 81:339-351. 1956.

71. Walker, T. W., Orchiston, H. D. and Adams, A. F. R. The nitrogen

economy of grass legume associations. 1. Brit. Grass1d. Soc. 9:

249-274. 1954.

117

72. Watson, E. R. and Lapins, P. The influence of subterranean clover

pastures on soil fertility. II. The effects of certain manage­

ment systems. Aust. 1. Agric. Res. 15:885-894. 1964.

73. Watson, G. A. Nitrogen fixation by Centrosema pubescens. 1. Rub.

Res. Inst. Malaya. 15:168-174. 1957.

74. Wheeler, B. E. J. The conversion of amino acids in soils. II.

Denitrification in percolated soils. Plant and Soil. 19:219­

232. 1963.

75. Wilson, P. W. The Biochemistry of Symbiotic Nitrogen Fixation.

Madison, 1940.

76. Wilson, P. W. and Wyss, O. Mixed cropping and the excretion of

nitrogen by leguminous plants. Soil Sci. Soc. Amer. Proc. 2:

289-297. 1937.

77. Wittwer, S. H. and Teubner, F. G. Foliar absorption of mineral

nutrients. Ann. Rev. Plant Physiol. 10:13-32. 1959.

78. Wyss, O. and Wilson, P. W. Factors influencing excretion of

nitrogen by legumes. Soil §£i. 52:15-29. 1941.

79. Younge, O. R. Pangolagrass requires high potassium fertilization.

Hawaii £!!m Sci. 10(2):3-5. 1961.

80. Younge, o. R., Plucknett, D. L. and Rotar, P. P. Culture and

yield performance of Desmodium intortum and Q. canum in Hawaii.

Hawaii Agric. Expt. Sta. Tech. Bull. 59. 1964.

81. Younge, O. R. and Ripperton, J. C. Nitrogen fertilization of

pasture and forage grasses in Hawaii. Hawaii Agric. Expt. Sta.

Bull. 124. 47. 1960.